Dose-dependent genetic diversity and biochemical adaptation in H. lacustris and D. salina under chemical mutagen

2.1 Strains and cultural conditions

Microalgal strains H. lacustris and D. salina were obtained from Algae Research and Supply (San Diego, CA, USA). To verify that the cultures were pure and axenic, they were examined morphologically using a Nikon Eclipse 90i microscope (Nikon Instruments Inc., Melville, NY, USA) and subsequently inoculated onto both solid and liquid F/2 medium. The microalgal strains were cultivated under controlled conditions at 26 ± 2°C, with a white fluorescent light source providing an irradiance of 35.4 µmol m⁻² s⁻¹ and a photoperiod of 12 h light/12 h dark.

2.2 Mutagenesis protocol

During the logarithmic growth phase, microalgal cells were harvested and resuspended in 10 mL of fresh F/2 medium, and the optical density (OD) was adjusted to 0.5 nm. Subsequently, the cultures were exposed to EtBr at final concentrations of 0, 5, 10, and 20 μg/mL as a chemical mutagenic agent. The tubes were then incubated under dark conditions at 28°C with continuous shaking for 24 h in order to minimize the photoactivation of cellular DNA repair pathways (Yamamoto et al., 2017). These concentrations were selected based on a thorough review of the literature e.g., (Caniago et al., 2015, Al-Ziadi 2017). Following the initial 24 h dark phase, the cultures were grown under the previously described optimal conditions, specifically at 26 ± 2°C under controlled environmental parameters, with illumination provided by white fluorescent lamps at an irradiance of 35.4 µmol m⁻² s⁻¹ and a photoperiod of 12 h light/12 h dark. A spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, Buckinghamshire, UK) was employed to measure the OD of the microalgal cultures at 680 nm daily.

2.3 DNA isolation and Random Amplified Polymorphic DNA (RAPD)-PCR protocol

A Retsch mixer mill MM200 (Retsch, Newtown, PA, USA) was used to homogenize 100 mg of fresh microalgal biomass for 2 min. Genomic DNA was subsequently extracted in accordance with the manufacturer’s instructions using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). After that RAPD-PCR reactions were carried out in a final volume of 25 μL, containing 2 μL of template DNA (27 ng/μL), 2.5 μL of 10× PCR buffer, 0.5 μL of 10 mM dNTP mix, 1 μL of random amplified polymorphic DNA (RAPD) primer (100 nmol; eight primers selected for producing distinct and reproducible banding patterns; Table 1), 0.75 μL of 50 mM MgCl₂, and 2 U of Platinum Taq DNA Polymerase (5 U/μL; Invitrogen, Carlsbad, CA, USA). The thermocycling program consisted of an initial denaturation at 94°C for 90 s, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 35°C for 30 s, and extension at 72°C for 60 s, with a final extension step at 72°C for 90 s. Then PCR products were separated on 1% borate agarose gels, stained with EtBr, and visualized under ultraviolet (UV) illumination. RAPD-PCR analyses were performed in triplicate using independent biological replicates. Technical replicates were included to assess the reproducibility of the amplification profiles, and only bands that were consistently observed across all replicates were considered for downstream genetic analyses. Amplicons were electrophoretically resolved alongside a 1 kb Plus DNA Ladder (Invitrogen, Carlsbad, CA, USA) as a molecular size marker. Electrophoresis was performed in 1% borax running buffer at 60 V for 18 h in a cold room maintained at 4°C.

2.4 Estimation of pigment content

To quantify the photosynthetic pigments in microalgae samples, the protocol of Moran and Porath (1980) was followed. During the exponential growth phase, 2 mL of each strain culture were harvested by centrifugation at 13,000 rpm. The resulting cell pellets were weighed and transferred to covered glass tubes, then resuspended in 5 mL of 80% (v/v) ice-cold acetone and incubated at 4°C for 24 h. Subsequently, the samples were centrifuged at 13,000 rpm for 5 min, and the supernatants were collected for further analysis. The estimation of pigments was performed following the established protocols by Metzner et al., (1965) and Pflanz and Zude (2008), while spectrophotometric measurements were measured at 480, 645, and 663 nm.

2.5 Estimation of soluble carbohydrates content

Two milliliters of microalgal biomass were extracted with 80% (v/v) ethanol by heating the samples to 90°C. After that, a single extraction step of 10 min at boiling temperature was applied to maximize the recovery of soluble carbohydrates. Following derivatization and resuspension of the extracts in dH₂O, the resulting supernatants were stored at -80°C prior to analysis to minimize carbohydrate degradation during storage. The concentration of soluble sugars was determined by capillary electrophoresis (CE) using a Beckman P/ACE System 5500 equipped with a diode-array detector (DAD) set to 270 nm. Quantification was carried out by external calibration with appropriate standards (glucose and sucrose). The composition and pH of the background electrolyte were optimized for carbohydrate separation in accordance with the manufacturer’s recommendations.

2.6 Estimation of organic acids content

Organic acids from microalgal strains were extracted using a solution supplemented with 0.3% (w/v) butylated hydroxyanisole (BHA), employed as an antioxidant preservative to minimize oxidative and degradative processes during sample preparation. High-performance liquid chromatography (HPLC) analysis was conducted on a SUPELCOGEL C-610H column (30 cm × 7.8 mm I.D., polystyrene–divinylbenzene resin in the hydrogen form, 6% crosslinked), coupled to a UV detector set at 210 nm for the chromatographic determination of organic acids. The mobile phase consisted of 0.1% (v/v) phosphoric acid, delivered isocratically at a flow rate of 0.5-0.6 mL/min. The column operates according to an ion-exclusion chromatography mechanism, which is particularly suitable for the separation of weakly ionizable organic acids. Quantification was performed by external calibration using appropriate standards, specifically oxalic, malic, succinic, citric, isobutyric, and fumaric acids. Optimal chromatographic resolution was obtained at ambient temperature (25°C), with a total analysis time of 30 min.

2.7 Estimation of amino acids content

Amino acids were extracted from 5 mL aliquots of microalgal cultures using 80% (v/v) aqueous ethanol. The extraction buffer contained the internal standard norvaline at a final concentration of 500 μM to compensate for matrix effects and analyte losses occurring during ethanol extraction, centrifugation, and subsequent sample preparation steps. Centrifugation was carried out at 20,000 rpm for 20 min at room temperature to separate the soluble, amino acid–containing supernatant from the insoluble pellet. The resulting pellet was subjected to biphasic liquid-liquid extraction with chloroform and water (both HPLC grade) to further partition lipids from residual polar metabolites. Specifically, the pellet was resuspended in chloroform to promote phase separation and facilitate the removal of lipophilic contaminants. After centrifugation, the aqueous phase (containing polar metabolites and residual amino acids) was collected and combined with the initial ethanolic supernatant, while the chloroform phase (containing lipids and other non-polar constituents) was discarded. The combined extracts were subsequently centrifuged and passed through Millipore microfilters (0.2 μm size) to remove particulate matter and to ensure sample clarity prior to chromatographic analysis. Amino acid separation was achieved by hydrophilic interaction liquid chromatography (HILIC) using an XBridge Premier BEH Amide column (2.1 mm × 50 mm, 1.7 μm particle size) on a Waters Acquity ultra-performance liquid chromatography (UPLC) platform coupled to a Xevo TQD triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The mobile phases consisted of 0.2% (v/v) formic acid in water (aqueous phase A) and 0.2% (v/v) formic acid in acetonitrile (organic phase B). A gradient elution program was applied to optimize the resolution of all targeted amino acids, including isomeric pairs such as isoleucine/leucine and phenylalanine/tyrosine. Detection was performed in multiple reaction monitoring mode using compound-specific cone voltages (29-39 V) and collision energies (15-35 V), which were established by direct infusion of individual amino acid standards. The total run time per sample was typically 10 min. Quantification was based on external calibration using a validated amino acid standard mixture containing glycine, alanine, isoleucine, leucine, methionine, valine, phenylalanine, glutamine, asparagine, threonine, serine, cysteine (monomeric form, not cystine), tyrosine, lysine, histidine, arginine, glutamic acid, and aspartic acid at concentrations 0.25 μmol/mL.

2.8 Estimation of fatty acids (FA)

Total lipids were extracted from each 100 mg of microalgal biomass using a solvent system optimized for exhaustive lipid recovery. A chloroform:methanol mixture (2:1, v/v) was employed at ambient temperature (25°C). Following extraction, phase separation was induced by the addition of distilled water, and the lower organic phase containing total lipids was collected and evaporated to dryness under a gentle stream of nitrogen. The extracted lipids were subjected to chemical derivatization to convert fatty acids (FA) into their corresponding fatty acid methyl esters (FAMEs) prior to gas chromatography-mass spectrometry (GC-MS) analysis. Derivatization was performed by acid-catalyzed transesterification using 3% (v/v) methanolic hydrochloric acid at 75°C for 2 h. After completion of the reaction, FAMEs were partitioned into n-hexane. The hexane phase was washed with distilled water, dried over anhydrous sodium sulfate, and concentrated under nitrogen for GC-MS analysis. GC-MS analyses were carried out using an Agilent 6890 gas chromatograph coupled to an Agilent 5975 quadrupole mass spectrometer (Hewlett-Packard/Agilent, USA). Chromatographic separation was achieved on an HP-5ms capillary column (Agilent J&W; 30 m × 0.25 mm internal diameter × 0.25 μm film thickness) with a 5% phenyl-methylpolysiloxane stationary phase. The oven temperature program was set to an initial temperature of 60°C (held for 2 min), increased at 10°C min⁻¹ to 300°C, and held for 10 min to ensure complete elution of long-chain FA (C20-C24). Helium was used as the carrier gas at a constant flow rate of 1.0 mL min⁻¹. Mass spectrometric detection was performed in electron ionization mode at 70 eV, with a mass scan range of m/z 50-550. FA methyl esters were identified by comparison of their retention times with those of authentic FAME standards and by matching mass spectral fragmentation patterns with entries in the NIST 14 mass spectral library (2014). Quantitative analysis was performed using external calibration curves generated from a validated FAME standard mixture containing myristic acid (C14:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), docosanoic acid (C22:0), tricosanoic acid (C23:0), pentacosanoic acid (C25:0), palmitoleic acid (C16:1), heptadecenoic acid (C17:1), oleic acid (C18:1), α-linolenic acid (C18:3n-3), linoleic acid (C18:2n-6), and eicosenoic acid (C20:1).

2.9 Statistical analysis

The gel electrophoresis procedure produced banding patterns that were subsequently subjected to detailed analysis. Each distinct band was systematically scored using a binary system, in which ‘1’ indicated the presence of a band and ‘0’ its absence, with scoring performed using Bio-Rad Quantity One software (version 4.6.2). Similarity among samples was quantified using Jaccard’s coefficient, generating a pairwise similarity matrix. This matrix was then used to construct a dendrogram to visualize inter-sample relationships, employing the unweighted pair group method with arithmetic mean (UPGMA) for cluster analysis. Additional experiments were conducted in triplicate under a completely randomized design to enhance reliability and reproducibility. Data are expressed as mean ± standard error, and graphical representations were generated using GraphPad Prism software (version 10). Statistical analyses were performed using one-way and two-way analyses of variance (ANOVA), followed by Tukey’s post hoc test, with significance thresholds set at p ≤ 0.05, 0.01, 0.001, and 0.0001.

3.1 Effect of EtBr on microalgal growth

The effects of EtBr on the proliferative dynamics of microalgae species H. lacustris and D. salina, are depicted in Fig. 1. The influence of increasing EtBr concentrations on the temporal growth profile of H. lacustris is shown in Fig. 1(a). Whereas at an intermediate EtBr concentration of 10 µg/ml, a relatively stable growth trajectory is maintained. In contrast, exposure to a lower concentration of 5 µg/ml induces fluctuations in the growth pattern, characterized by a pronounced recovery in cell proliferation on day 5, at which point the growth index reaches 0.63, compared with the baseline value of 0.49 on day 1. The control group displays a steady increase in growth over the 5-day period, attaining a growth index of 0.62 on the final day, up from 0.44 at the start of the experiment. While the impact of EtBr on the growth of D. salina, as presented in Fig. 1(b), indicates marked modulation of growth across the tested concentration range. At the highest EtBr concentration (20 µg/ml), the growth parameter is reduced by 20% compared to the control; at the intermediate concentration (10 µg/ml), a 15% decrease is observed; and at the lowest concentration (5 µg/ml), the reduction is limited to about 5%. In contrast, the control culture exhibited a continuous enhancement in growth over the experimental period, reaching a growth index of 0.83 on the final day, compared with an initial value of 0.55, corresponding to 1.5-fold increase.

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Fig. 1.

Growth assessment of microalgal strains exposed to varying concentrations of EtBr over a five-day period. (a) OD₆₈₀ measurements for H. lacustris. (b) OD₆₈₀ measurements for D. salina. Statistical significance is indicated as follows: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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3.2 Effect of EtBr on genetic material

The assessment of genomic DNA alterations induced by EtBr exposure using RAPD analysis is presented in (Supporting Data Fig. S1) and Table 2. In H. lacustris, treatment with 20 µg/ml EtBr resulted in a polymorphism rate of 6.45%, with primer OPF-06 failing to yield any detectable amplification products. A substantial increase in polymorphism to 35.48% was observed at 10 µg/ml EtBr, whereas exposure to 5 µg/ml EtBr produced a polymorphism rate of 8.87%. In contrast, D. salina exhibited no detectable RAPD bands at 20 µg/ml EtBr. At 10 µg/ml EtBr, a polymorphism rate of 16.85% was recorded, which further increased to 33.71% at 5 µg/ml EtBr. Using primer OPF-05, a polymorphism rate of 11.11% was detected at the highest EtBr concentration, whereas intermediate and low EtBr concentrations yielded higher polymorphism rates of 33.33% and 16.67%, respectively. The wild-type strain of H. lacustris exhibited a polymorphism rate of 5.56%, indicating that exposure to elevated EtBr concentrations is associated with increased genomic variability. In D. salina, none of the eight primers produced detectable bands at 20 µg/ml EtBr. At 10 µg/ml, a polymorphism rate of 16.85% was observed, which increased to 33.71% at 5 µg/ml EtBr. The average polymorphic information content (PIC) values for the eight primers employed are presented in Table 3. PIC values reflect the discriminatory capacity of primers to produce polymorphic bands suitable for genetic analysis. In H. lacustris mean PIC values varied among primers. Primer OPF-06 exhibited the lowest PIC value (0.27), followed by OPA-03 (0.33). The highest PIC value was observed for OPE-02 (0.34), with OPF-05 and OPH-08 showing similarly elevated PIC values of 0.38 and 0.34, respectively. Primers OPY-04 and OPS-11 displayed PIC averages of 0.36 and 0.40, respectively, indicating a relatively high level of informativeness. For D. salina, primer OPF-05 had a PIC average of 0.34, while OPA-05 and OPE-02 exhibited PIC values of 0.34 and 0.35, respectively. Primer OPS-11 recorded the highest PIC value (0.33), whereas OPA-03 showed a PIC of 0.30. Primers OPY-04 and OPH-08 had PIC averages of 0.29 and 0.32, respectively. Notably, OPF-06 displayed the lowest PIC value (0.34) among the primers tested in D. salina.

The evaluation of genetic similarity among the various EtBr-treated H. lacustris individuals, as presented in Fig. 2, consistently yields a value of 1.00 for self-comparisons, reflecting the expected genetic identity of an organism with itself. A clear trend is observed whereby genetic similarity decreases as the concentration of EtBr increases. For example, the genetic similarity between H. lacustris exposed to high (20 µg/ml) and medium (10 µg/ml) EtBr concentrations is 0.32, indicating a relatively low degree of genetic similarity between these treatment groups. This trend is consistent with the expectation that higher EtBr concentrations induce more extensive genetic alterations. These reduced similarity values indicate increased genetic divergence or modification, supporting the mutagenic potential of EtBr in H. lacustris. Similarly, for D. salina in Fig. 3, genetic similarity decreases as the concentration of EtBr increases. For instance, the genetic similarity between treatment groups of D. salina exposed to high (20 µg/ml) and medium (10 µg/ml) EtBr concentrations is 0.76, indicating reduced genetic similarity at the higher EtBr dose. These results demonstrate that the progressive decline in genetic similarity with increasing EtBr concentrations reflects a substantial impact on the genetic composition of D. salina. Lower genetic similarity values indicate greater genetic divergence or genomic alterations, supporting the mutagenic effect of EtBr on D. salina.

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Fig. 2.

Dendrogram illustrating genetic similarity and relationships among H. lacustris strains treated with EtBr.

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Fig. 3.

Dendrogram of the genetic similarity illustrating the genetic relationships among D. salina treated with EtBr.

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3.3 Effect of EtBr on strains pigments content

Analysis of the pigment composition data presented in Fig. 4 indicates distinct, concentration-dependent responses to EtBr in both microalgal species. In H. lacustris Fig. 4(a), exposure to the highest EtBr concentration (20 µg/mL) was associated with pronounced elevations in pigment content: chlorophyll a (Chl-a) reached 6.26 µg/mL, corresponding to a 72.92% increase relative to the control; chlorophyll b (Chl-b) reached 7.54 µg/mL, representing a 92.34% increase; and carotenoid content increased to 2.10 µg/mL, reflecting a 156% increase. At an intermediate EtBr concentration (10 µg/mL), H. lacustris exhibited Chl-a levels of 5.67 µg mL⁻¹ (56.62% increase), Chl-b levels of 7.56 µg/mL (92.85% increase), and carotenoid content of 3.86 µg/mL, representing a substantial 370.7% increase and indicating a markedly enhanced photoprotective and/or antioxidative response. At the lowest EtBr dose (5 µg/mL), Chl-a reached 7.70 µg/mL (112.7% increase) and Chl-b reached 9.60 µg/mL (144.89% increase). By contrast, D. salina (Fig. 4b) exhibited more moderate pigment responses at the corresponding EtBr concentrations. At 20 µg/mL EtBr, Chl-a reached 4.89 µg/mL (25.45% increase), Chl-b reached 7.47 µg/mL (30.73% increase), and carotenoid content increased to 0.29 µg/mL (44.2% increase). At the intermediate EtBr concentration (10 µg/mL), D. salina exhibited Chl-a levels of 4.69 µg/mL (24.45% increase), Chl-b levels of 7.95 µg/mL (32.7% increase), and carotenoid content of 0.70 µg/mL (106.69% increase), consistent with an upregulated protective pigment response. Under the lowest EtBr dose (5 µg/mL), Chl-a reached 3.45 µg/mL (17.98% increase), Chl-b reached 3.26 µg/mL (13.4% increase), and carotenoids increased to 0.97 µg/mL (147.84% increase), suggesting a particularly pronounced carotenoid-mediated defense under relatively mild EtBr-induced stress.

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Fig. 4.

Effects of varying concentrations of EtBr on pigment profiles of (a) H. lacustris and (b) D. salina. Statistical significance is indicated as follows: ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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3.4 Effect of EtBr on strains metabolic profile

3.4.1 Carbohydrate content

The impact of EtBr on carbohydrate profiles by quantifying glucose, fructose, and total soluble sugars (TSS) is presented in Fig. 5. Evaluating EtBr-induced changes in these components provides insight into the adaptive capacity and metabolic resilience of microalgae under stress conditions. In H. lacustris, exposure to a high EtBr concentration resulted in a reduction in glucose content to 0.81 mg/g, corresponding to a 9% decrease relative to the control. In contrast, fructose levels increased to 1.65 mg/g (19.39% increase), while total soluble sugars rose by 4.32% to 7.79 mg/g. At the intermediate EtBr concentration, glucose content declined to 0.86 mg/g (3.28% decrease) and fructose decreased to 1.28 mg/g (3.23% decrease), accompanied by a 10.86% reduction in total soluble sugars to 6.61 mg/g. Notably, exposure to the lowest EtBr dose produced an opposite trend, with glucose increasing by 2.87% to 0.91 mg/g, fructose by 13.94% to 1.56 mg/g, and total soluble sugars by 6.33% to 7.95 mg/g, exceeding control values. In D. salina, a high EtBr concentration induced a pronounced decline in glucose content by 49.58% to 0.40 mg/g. Conversely, fructose levels increased by 12% to 0.96 mg/g, while total soluble sugars decreased by 8.72% to 4.88 mg/g. At the medium EtBr dose, glucose content decreased by 31.67% to 0.55 mg/g, fructose declined by 37.98% to 0.53 mg/g, and total soluble sugars were reduced by 30.32% to 3.72 mg/g. In contrast, the lowest EtBr concentration resulted in a smaller reduction in glucose (12% decrease to 0.70 mg/g), alongside increases in fructose (20.93% increase to 1.04 mg/g) and total soluble sugars (8.23% increase to 5.78 mg/g) relative to the control.

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Fig. 5.

Effects of varying EtBr concentrations on fructose, glucose, and total soluble sugar contents (mg/g) in H. lacustris and D. salina. Bars represent mean ± SD. Groups not sharing a letter (A–I) are significantly different (p < 0.05).

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3.4.2 Organic acids content

Organic acids are key intermediates in cellular metabolism, playing essential roles in energy production, metabolic regulation, and cellular homeostasis across diverse life forms. Alterations in carbohydrate levels induced by EtBr treatment are therefore likely to affect intermediates of the tricarboxylic acid (TCA) cycle, including various organic acids. In addition to their metabolic functions, organic acids contribute to cellular stress-response mechanisms, enabling microorganisms to adapt to adverse environmental conditions. The effects of EtBr on organic acid levels were dose-dependent, as illustrated in Fig. 6. In H. lacustris, the total organic acid content decreased to 21.38 mg/g under a high EtBr concentration, corresponding to a 30% reduction relative to the control. At the medium EtBr dose, total organic acids declined to 24.95 mg/g, representing an 18.35% decrease. Similarly, exposure to the lowest EtBr concentration resulted in a reduction to 20.26 mg/g, indicating a 33.72% decrease compared to control values. A comparable but distinct response pattern was observed in D. salina. Total organic acid content decreased to 19.35 mg/g at the highest EtBr dose, reflecting a 19.2% reduction, and further declined by 20.4% at the medium EtBr concentration. In contrast, exposure to the lowest EtBr dose resulted in an atypical increase in total organic acids of 20.8% relative to the control. The observed fluctuations in organic acid concentrations indicate a complex and dynamic metabolic response of both species to EtBr exposure. Key organic acids, including malic, succinic, and citric acids central intermediates of the TCA cycle exhibited distinct response patterns, suggesting perturbations in energy metabolism and stress adaptation mechanisms. Collectively, the alterations in total organic acid content demonstrate that EtBr exposure exerts a substantial impact on central metabolic pathways in both microalgal strains.

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Fig. 6.

Depicts the impact of varied EtBr concentrations on the composition of organic acids (mg/g) in H. lacustris and D. salina.

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3.4.3 Amino acids profile

The effects of EtBr treatment on the amino acid composition of two microalgal strains were systematically investigated, and the resulting changes in amino acid content are presented in Fig. 7. In H. lacustris, pronounced alterations in amino acid profiles were observed in response to varying EtBr concentrations. The total amino acid content declined from 75.93 mg/g in the untreated control to 65.94 mg/g at the highest EtBr concentration, corresponding to a reduction of 13.15%. At the intermediate EtBr concentration, the total amino acid content unexpectedly increased by 19.2%, whereas exposure to the lowest EtBr dose resulted in an 8.27% decrease relative to the control. Several amino acids, including lysine, histidine, and arginine, exhibited an overall decreasing trend with increasing EtBr concentration, a pattern that was particularly evident when comparing high and low EtBr treatments. Similarly, glycine, alanine, and glutamic acid showed a consistent decline as EtBr levels increased, suggesting a disruption of amino acid metabolism under elevated EtBr exposure. In D. salina, the total amino acid content was markedly reduced at all EtBr concentrations tested. Exposure to a high EtBr concentration resulted in a 13.21% decrease to 87.49 mg/g, while the intermediate EtBr dose induced a more pronounced reduction of 27.65% to 72.97 mg/g. The lowest EtBr concentration also led to a notable decrease of 19.5%, yielding a total amino acid content of 81.14 mg/g. This response pattern, broadly consistent with that observed in H. lacustris, indicates a substantial impact of EtBr on amino acid pools across a range of exposure levels. Individual amino acids, including histidine, isoleucine, leucine, and methionine, displayed dose-dependent declines with increasing EtBr concentration, mirroring trends observed in H. lacustris. Moreover, glycine, alanine, and glutamic acid followed comparable downward trajectories in both microalgal strains.

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Fig. 7.

Heat map showing the effects of varying EtBr concentrations on amino acid composition (mg/g) in H. lacustris and D. salina.

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3.4.4 Saturated and unsaturated fatty acids profile

The analysis revealed substantial variations in FA profiles across different EtBr concentrations are shown in Fig. 8 and (Supporting Tables S1 and S2). In H. lacustris, exposure to a high EtBr concentration resulted in a reduction in total FA content of 22.78%, yielding 34.08 mg/g. Similarly, treatment with a medium EtBr concentration caused a decrease of 8.78%, corresponding to a total FA content of 40.27 mg/g. In D. salina, exposure to high EtBr concentrations induced a pronounced reduction in total FA content of 59.14%, resulting in a final value of 18.44 mg/g. At the medium EtBr concentration, total FA decreased by 15.9%, reaching 37.95 mg/g. In contrast, exposure to the lowest EtBr dose enhanced total FA accumulation in both species, leading to increases of 67.97% in H. lacustris and 76.8% in D. salina relative to the control. Saturated fatty acids (SFAs), particularly palmitic acid, constituted a major fraction of the FA profiles. In H. lacustris, palmitic acid content increased from 25.78 mg/g under high EtBr exposure to 30.97 mg/g and 56.64 mg/g under medium and low EtBr concentrations, respectively. In D. salina, palmitic acid content was 14.21 mg/g at the highest EtBr concentration, increased to 29.13 mg/g at the medium dose, and further rose to 62.44 mg/g under low EtBr exposure.

Table S1

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Table S2

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Fig. 8.

Shows the lipid profile for H. lacustris and D. salina under different doses from EtBr.

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3.5 Principal component analysis (PCA) results

A principal component analysis was performed to evaluate the metabolic differentiation among microalgal strains under different growth conditions (Fig. 9). The PCA biplot revealed distinct clustering patterns that separated the two microalgal species (H. lacustris and D. salina) along the first two principal components, which collectively explained 66.74% of the total variance (PC1: 34.45%; PC2: 32.29%). The analysis demonstrated clear separation between the two microalgal species independent of treatment concentrations. H. lacustris samples clustered primarily in the positive PC1 region (right side of the biplot) across all tested concentrations (0, 10, and 20 μg/mL). In contrast, D. salina samples clustered in the negative PC1 region, with samples at higher concentrations (10 and 20 μg/mL) showing greater negative displacement along PC1 compared to the control (0 μg/mL), indicating a dose-dependent metabolic response.

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Fig. 9.

Principal component analysis (PCA) of H. lacustris and D. salina under EtBr treatments.

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The loading vectors in the biplot identified the biochemical compounds contributing most strongly to the principal components. Compounds associated with the positive PC1 axis (characteristic of H. lacustris) included Chl-a, Chl-b, carotenoids (Cart.), and fructose, indicating that this species maintains elevated photosynthetic pigment content and carbohydrate reserves regardless of growth conditions. The negative PC1 axis (characteristic of D. salina) was dominated by organic acid metabolites, particularly citric acid, succinic acid, and total amino acids, suggesting that D. salina relies more heavily on organic acid metabolism and amino acid biosynthesis as primary metabolic strategies. Additionally, total unsaturated fatty acids (USFA) and total SFA loaded positively on PC1 but also showed intermediate positioning, indicating that both species synthesize these lipid classes but with quantitatively different profiles. Along the second principal component (PC2: 32.29% variance explained), the separation was primarily driven by metabolites associated with total amino acids (negative PC2 direction) and Chl-b (negative PC2), in contrast to citric acid and other organic acids (positive PC2 direction). This axis appears to capture secondary metabolic variations, potentially representing stress responses or nutrient availability effects.

4.1 Effect of EtBr on microalgae cell and genomic material

The findings of the present study advance our understanding of the effects of environmental contaminants and xenobiotic chemical compounds on the growth and physiological responses of microalgae. Overall, exposure to EtBr was associated with a reduction in microalgal growth. In the case of H. lacustris, the data indicated a subtle and non-linear response to varying EtBr concentrations. Notably, an intermediate concentration of 10 µg/ml appeared to maintain a relatively stable growth pattern. This observation is consistent with the presence of a threshold effect, whereby the alga can tolerate or potentially acclimate to a specific level of EtBr exposure without incurring substantial impairment of its reproductive capacity. The observed fluctuations in growth patterns at a lower dose of 5 µg/ml, followed by a subsequent increase in proliferation, could suggest the activation of a stress response or an adaptive mechanism inside the cells. This was previously verified by Zhang et al., (2024) when Chromochloris was subjected to cadmium as external stressor. While the growth of D. salina is reduced in a dose-dependent manner as the concentration of EtBr increases, indicating a clear susceptibility to the cytotoxic effects of EtBr. The observed growth modulation, characterized by a 20% decrease at the maximum dosage of 20 µg/ml, highlights the potential of EtBr to hinder cellular function, either by causing DNA damage or disrupting cellular replication pathways. The diminishing growth impact observed as EtBr concentrations decrease indicates a dose-response relationship, a key principle in toxicology that signifies the correlation between the intensity of the action and the concentration of the molecule.

For the genetic material, RAPD is a molecular biology approach employed for identifying genetic variants or mutations by amplifying genomic DNA using random primers (Cho et al., 1996). The induction of polymorphisms in H. lacustris by EtBr depends on its concentration, resulting in a significant rise in polymorphism rates at specific concentrations. The absence of identifiable bands at a dose of 20 µg/ml of EtBr for primer OPF-06, along with the high rates of polymorphism found at lower concentrations, indicates substantial genomic modifications. The absence of observable bands suggests that EtBr has the potential to induce significant DNA damage or alterations that hinder the binding of the primer and the amplification of the target sequences. The rise in polymorphism rates at a concentration of 10 µg/ml underscores the amplified mutagenic impact of EtBr at this level. This could be attributed to enhanced DNA intercalation, leading to replication mistakes or DNA breakage. On the other hand, D. salina has a distinct pattern of reaction when exposed to EtBr. The lack of observable bands at the highest dose of 20 µg/ml once again indicates substantial DNA damage. The escalating levels of polymorphism observed at lower concentrations, especially the significant surge at 5 µg/ml, highlight the susceptibility of D. salina’s genome to modifications generated by EtBr. The contrasting reaction observed in these two species highlights the species-specific effect of EtBr on microalgal DNA. The observed heterogeneity may be ascribed to disparities in the genomic areas amplified by each primer; wherein certain regions are more prone to EtBr-induced mutations. The heightened levels of polymorphism seen at specific doses indicate that EtBr has the potential to cause a diverse array of genomic modifications, ranging from individual nucleotide alterations to more substantial DNA damage (Au et al., 2003). On the other hand, the observed decrease in genetic similarity with an increase in EtBr concentration is a critical finding. For H. lacustris, the genetic similarity between groups subjected to high (20 µg/ml) and medium (10 µg/ml) doses of EtBr being at 0.32 suggests a substantial genetic divergence induced by the higher concentration of EtBr. This pattern indicates that elevated EtBr concentrations are likely lead to more significant genetic modifications, evidenced by the reduced similarity scores between treatment groups. Similarly, for D. salina the trend of decreasing genetic similarity with increasing EtBr dosage is evident. The genetic similarity score between treatment groups subjected to high (20 µg/ml) and medium (10 µg/ml) doses of EtBr being at 0.76 further illustrates the dose-dependent effect of EtBr on genetic makeup.

Investigating variations in pigment composition, specifically chlorophylls and carotenoids, which fulfill pivotal functions in photosynthetic energy conversion and in the mitigation of oxidative stress, respectively (Perez-Galvez et al., 2020). Their levels can serve as an indicator of microalgae’s response to environmental stresses, such as chemical contaminants like EtBr. The observed elevation in pigment concentrations in H. lacustris, regardless of the EtBr dosage, indicates a significant stress response. The significant elevations in Chl-a and Chl-b, as well as carotenoids, especially at high and medium EtBr concentrations, indicate an intensified defensive mechanism likely designed to alleviate EtBr-induced oxidative stress (Wu et al., 2025). The notable rise in carotenoid content, particularly at a moderate EtBr concentration (370.7% increase), suggests a substantial enhancement in defensive mechanisms, as carotenoids have a vital function in neutralizing reactive oxygen species and safeguarding the photosynthetic apparatus against photodamage (Zandi and Schnug 2022). The H. lacustris exhibited a significant increase in Chl-a and Chl-b levels, with a respective rise of 112.7% and 144.89%, when exposed to the lowest dose of EtBr. This increase can be attributed to a compensation mechanism, where the microalgae enhance pigment synthesis to counteract potential harm to the photosynthetic apparatus (Kana et al., 1997). This response also contributes to a wider stress response, which improves the efficiency of photosynthesis to counteract the harmful effects of exposure to EtBr. Conversely, D. salina demonstrates a distinct pattern of pigment reaction when exposed to EtBr. The levels of Chl-a and Chl-b and carotenoids show a relatively moderate increase compared to H. lacustris, indicating a better controlled stress response or a distinct mechanism for dealing with EtBr-induced stress. The carotenoid content shows a remarkable rise at lower concentrations of EtBr, with a 147.84% increase at the lowest dose. This suggests a strong enhancement of protective mechanisms against oxidative stress, similar to H. lacustris, but to a lesser degree. The varied responses observed between H. lacustris and D. salina can be attributed to fundamental disparities in their biology, stress response mechanisms, and pigment metabolism. Whereas EtBr is a DNA-intercalating fluorochrome and potent cytoplasmic mutagen, exerts multifaceted effects on the growth, metabolism, and genetic integrity of microalgae. Previous investigations have demonstrated that EtBr modulates cell division dynamics, perturbs chlorophyll biosynthesis, and alters mitochondrial DNA structure and function in Euglena gracilis (Nass and Ben-Shaul 1973, Hayashi and Ueda 1992). For instance, Nass and Ben-Shaul (1973) observed that EtBr inhibited cell division and chlorophyll formation, alongside causing changes in mitochondrial ultrastructure and a reduction in mitochondrial DNA content. Similarly, Smith-Johannsen et al., (1980) found that Ochromonas danica treated with EtBr showed a normal rate of cell division but had significantly less mitochondrial rRNA. While, Czechowska and van der Meer (2012) reported that EtBr caused disturbances in cellular energy generation in Pseudomonas fluorescens, as evidenced by increased fluorescence in live, dividing cells. Additionally, Paixao et al., (2009) demonstrated that Escherichia coli actively expels EtBr, a process that involves efflux pumps and a balance between the influx and efflux of EtBr in the cells. Previously it was verified that, EtBr proflavine, and mitomycin C display limited or negligible mutagenic properties, yet they exhibit a notable capacity for inactivation. This is likely due to their interaction with DNA, as proposed by Lerman (1964), which results in significant DNA inactivation by impeding its biosynthesis. The interaction between these mutagens and the phosphate groups on the external surface of DNA in Nostoc is likely governed by relatively weak electrostatic forces, as indicated by the observed bathochromic shift (displacement of the absorption maximum toward longer wavelengths) in the spectral profile (Tripathi and Kumar 1986). This hyperchromic shift in the UV region is presumably attributable to the reduction or absence of strong intercalative interactions mediated by van der Waals forces between the mutagen molecules and the DNA bases (Tripathi and Kumar 1986). Therefore, if we consider the in vitro interaction of a mutagen with DNA as reflective of its in vivo interaction, it can be inferred that the inactivating changes of mutagens may be due to electrostatic binding to DNA, thereby hindering replication and transcription. The limited mutagenic response could stem from an absence of strong intercalation binding, reducing the likelihood of additions or deletions at mutational hotspots (Freese and Freese 1966). The pronounced morphological disruption may be associated with aberrant protein levels or alterations in amino acid composition, potentially resulting from ethidium-mediated inhibition of lytic enzyme synthesis in Bacillus subtilis (Richmond 1959). Also, Rodrigues et al., (2011) study associated EtBr transport across the mycobacterial cell wall with antibiotic resistance, indicating that EtBr efflux is impeded when the molecule is intercalated into DNA and thus rendered less accessible to the bacterial efflux pump systems, the insertion of EtBr into DNA can induce errors by DNA polymerases, frequently resulting in frameshift mutations (Caniago et al., 2015). The kinetics of amino acid incorporation indicate a progressive decline in protein-synthesizing capacity, potentially attributable to the translation of a deteriorating, non-renewed pool of messenger RNA (Levinthal et al., 1962).

The impact of EtBr on genomic DNA extends beyond simple intercalative binding, as demonstrated by numerous studies that elucidate its diverse molecular interactions and downstream biological consequences. Whereas, this finding further highlighted the complexity of EtBr’s biological effects by demonstrating its interactions with genetic polymorphisms in biotransformation and DNA repair enzymes, which substantially modulate the extent of EtBr-induced genotoxicity (Godderis et al., 2006). These interactions highlight the nature of EtBr’s effects, which are contingent upon the specific genetic makeup of the organism. Furthermore, it has been reported that EtBr possesses the capacity to induce permanent and heritable alterations in mitochondrial DNA (Mahler and Bastos 1974). Additionally, EtBr’s propensity to penetrate cellular membranes and rapidly bind to the nucleolus, indicating a pervasive and immediate impact upon cellular entry (Maleszka 1994). The study of Sato et al., (1973) revealed differential effects of EtBr on mitochondrial DNA synthesis and structure in mammalian cells, highlighting its powerful cytoplasmic mutagenic effects in yeast cells and the instability of its effects on cytochrome au3 content in mammalian cells. Moreover, Hayashi and Harada (2007) demonstrated that EtBr intercalation leads to the reversible unwinding of double-stranded DNA, affecting a significant portion of the DNA molecule (57% of base pairs at 1 mM of EtBr). This alteration in DNA structure has profound implications for DNA stability and function. Moreover, Osterlund et al., (1982) provided evidence of EtBr’s inhibitory effect on restriction endonucleases, resulting in single-strand breaks in circular DNA molecules.

4.2 Effect of EtBr on the metabolic profile of cells

Carbohydrates are essential in microalgae for storing energy, forming structures, and serving as metabolic intermediates (Fettke and Fernie 2015). Additionally they are important indicators of the physiological condition and response to stress (Markou and Nerantzis 2013). Here the reaction of H. lacustris to different doses of EtBr demonstrates a complex relationship in carbohydrate metabolism. The observed decrease in glucose concentrations following exposure to elevated levels of EtBr, concomitant with an increase in fructose and total soluble sugar content, indicates a reprogramming of carbohydrate metabolism, potentially favoring fructose biosynthesis or accumulation as part of a stress-responsive mechanism. This metabolic shift could function as an adaptive strategy to maintain osmotic homeostasis, preserve cellular integrity, and/or optimize energy storage and availability under stress conditions (Gonzalez-Fernandez and Ballesteros 2012, Pancha et al., 2015). The increase in carbohydrate levels observed at the lowest EtBr dose, surpassing those of the control group, indicates a harmonic response. This means that exposure to modest levels of stress can have a positive effect, potentially by stimulating the pathways responsible for carbohydrate synthesis or accumulation. The marked decline in glucose concentrations observed in D. salina at elevated EtBr levels, accompanied by a modest increase in fructose but an overall reduction in total soluble sugars, indicates a more pronounced stress response relative to that exhibited by H. lacustris. The observed variation in fructose levels, characterized by an increase at both high and low doses of EtBr but a decrease at the medium dose, indicates the presence of intricate regulatory mechanisms in response to EtBr-induced stress. Elevated and moderate quantities of EtBr tend to cause harmful consequences, suggesting metabolic strain and possible toxicity.

In addition, organic acids constitute key intermediates in the TCA cycle. They fulfill dual roles by facilitating ATP generation during cellular respiration and participating in the regulation of metabolic processes and cellular stress responses (Arnold and Finley 2023). These compounds play an essential role in the biosynthesis of amino acids, FA, and a variety of other key metabolites, thereby influencing the overall physiological status and adaptive capacity of microorganisms (Yin et al., 2015). The reduction in total organic acid content in H. lacustris observed across the tested EtBr concentrations indicates a disruption of central metabolic processes, particularly the TCA cycle and its associated pathways. Exposure to the highest EtBr concentration caused 30% decline in organic acids, whereas the medium concentration produced a decrease of 18.35%. These results underscore a clear dose-dependent impact of EtBr on cellular metabolism. The concomitant decrease in energy-generation efficiency and alterations in metabolic fluxes, even at low EtBr concentrations, suggest a persistent stress effect on metabolic networks. In D. salina, exposure to varying EtBr doses elicited a distinct response pattern, characterized by reduced total organic acid content at high and medium doses, but an atypical increase at low dose. This pattern reflects the complex interplay between EtBr-induced stress and metabolic reprogramming. The decline in organic acids at elevated EtBr levels is consistent with impaired TCA cycle functionality or a metabolic shift toward alternative pathways, possibly to mitigate EtBr-induced oxidative stress or DNA damage. Fluctuations in the concentrations of key organic acids, including malic, succinic, and citric acids, further support the existence of complex metabolic adjustments to EtBr exposure. Beyond their central roles in energy metabolism, these organic acids contribute to osmoregulation, maintenance of intracellular pH homeostasis, and the biosynthesis of stress-related metabolites, thereby linking EtBr-induced metabolic perturbations to broader cellular stress responses (Sanderson and Selvendran 1965). The intricate patterns of reaction to EtBr stress indicate variations in enzyme activity within the TCA cycle, modifications in substrate availability, or adjustments in the cellular redox state. These factors have the potential to impact on the amounts of organic acids. Furthermore, amino acids being the fundamental constituents of proteins, play a critical role in diverse cellular processes, such as enzymatic activity, cellular structure, transportation systems, and as precursors for essential metabolites (Pratelli and Pilot 2014). Consequently, alterations in amino acid concentrations can have extensive consequences for cellular well-being, stress reaction, and overall metabolic equilibrium (Fafournoux et al., 2000). The observed fluctuations in amino acid levels in H. lacustris in response to varying EtBr concentrations highlight the compound’s impact on both protein biosynthesis and proteolysis. The 13.15% reduction in total amino acid content at higher EtBr concentrations suggests a disruption of amino acid biosynthetic pathways and/or an enhancement of their catabolic turnover, potentially arising from the direct interaction of EtBr with DNA and the translational machinery, or indirectly via EtBr-induced oxidative stress. Conversely, the 19.2% increase in total amino acids at an intermediate EtBr concentration reflect a stress-induced metabolic response, in which specific amino acids are synthesized in elevated quantities to counteract cellular stress, facilitate the repair of damaged macromolecules, or function as part of an adaptive mechanism aimed at maintaining cellular homeostasis (Paliwal et al., 2017). The continuous decrease in glycine, alanine, and glutamic acid levels with increasing EtBr concentrations highlights the extensive influence of EtBr on amino acid metabolism. The notable decrease in overall amino acid content seen in D. salina, regardless of the quantity of EtBr, underscores the impact of EtBr on cellular metabolism as a stressor. The observed patterns, showing reductions of 13.21% at high doses, 27.65% at medium doses, and 19.5% at low doses, clearly indicate a dose-dependent effect on amino acid levels. The observed decrease in levels of several amino acids, including histidine, isoleucine, leucine, and methionine, as the dosage of EtBr increases, is consistent with the patterns observed in H. lacustris. This similarity suggests that both species exhibit comparable metabolic vulnerabilities and/or responses to stress. The observed variations in amino acid profiles indicate alterations in nitrogen uptake dynamics or a reallocation of metabolic resources toward stress-mitigation pathways (Khan 2025).

FA also play critical roles in preserving cellular structural integrity, regulating membrane fluidity, and supporting overall cell function, as well as in mediating cellular adaptation to various stress conditions (Parveen and Patidar 2022). They are particularly important as constituents of cellular membranes and as molecules for storing energy (Yaqoob 2003). The decrease in overall FA content by 22.78% in H. lacustris at high EtBr concentrations indicates a disturbance in lipid production or an elevation in lipid breakdown. The marginal decrease of 8.78% observed at moderate EtBr concentrations suggests the presence of a threshold-like phenomenon, in which the microalgae are able to partially mitigate the stress induced by EtBr, thereby preserving a larger fraction of their FA pool. In contrast, the pronounced increase in total FA content at lower EtBr doses 67.97% in H. lacustris and an even more pronounced 76.8% in D. salina is indicative of a hormetic effect. Under such conditions, low EtBr concentrations stimulate lipid accumulation, potentially as part of a stress-responsive protective mechanism (Ding et al., 2024). The high proportion of SFA, particularly palmitic acid, in the overall FA profile, and its further elevation under EtBr-induced stress, especially at lower doses, underscores adaptive modifications in lipid metabolism. As a major component of cellular membranes, palmitic acid may be synthesized in increased amounts to enhance membrane stability and structural integrity under stress conditions (Chandra et al., 2015, Carta et al., 2017). The significant reduction of 59.14% in total FA content in D. salina at high concentrations of EtBr indicates a serious disturbance in lipid metabolism, potentially beyond the algae’s ability to handle the stress. Whereas Dunaliella spp. are very permeable to external substances because of the lack of a hard cell wall (Barbosa et al., 2023). This permeability significantly affects their osmoregulation, carbon metabolism, gene expression, photosynthetic rate, and growth in stressful situations (Woyda-Ploszczyca and Rybak 2021). Furthermore, Dunaliella sp. is renowned for its rapid growth rate and capacity to flourish in challenging environments characterized by elevated salt concentrations, intense light, and limited nitrogen availability (Silva et al., 2021). The magnitude of stress directly affects the rate at which D. salina grows, hence impacting the overall amount of light that the cell absorbs during a single division cycle (Hosseini Tafreshi and Shariati 2009). The growth and physiology of D. salina are impacted by drought stress induced through the use of polyethylene glycol (Tafvizi et al., 2020). Also, salinity stress in H. lacustris can lead to changes in membrane permeability, resulting in damage to the membrane and impacting its integrity, fluidity, and ion transport selectivity (Sirohi et al., 2022). H. lacustris experiences a deceleration in development and a decrease in cell motility when exposed to intense light, food shortage, or high salt concentration. This is accompanied by the accumulation of lipids and FA within the cells (Pang et al., 2019). The production of astaxanthin in H. lacustris is associated with the accumulation of cellular reserves in lipid droplets during periods of cellular stress (Shah et al., 2016).

4.3 Comparative analysis of the effects of different chemical mutagenic agents on microalgae

Previous studies as depicted in Table 4, employing chemical mutagens such as ethyl methanesulfonate (EMS), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and N-methyl-N-nitrosourea (NTG) have consistently reported significant enhancements in industrially valuable metabolites across a wide range of microalgal species. Among these outcomes, lipid enrichment has been the most frequently targeted trait. For example, N. salina achieved the highest reported lipid increase following EMS treatment, with a 1.95-fold enhancement (Beacham et al., 2015), closely followed by N. gaditana 1.8-fold (Cecchin et al., 2020). Comparable lipid improvements have also been documented in C. minutissima (1.56-fold), Desmodesmus sp. (1.29-fold), and C. vulgaris (1.23-fold), underscoring the effectiveness of chemical mutagenesis as a strain-improvement strategy. In addition to lipid accumulation, chemical mutagenesis has been widely exploited to enhance carotenoid production, a key objective for nutraceutical and biofortification applications. Notably, C. sorokiniana treated with MNNG exhibited a twofold increase in lutein content (Cordero et al., 2011), representing one of the highest carotenoid enhancements reported to date. Similarly, EMS treatment led to a 1.25-fold increase in carotenoids in D. salina (Jin et al., 2003), while MNNG exposure in C. zofingiensis stimulated the accumulation of multiple carotenoids, including zeaxanthin, β-carotene, and lutein (Huang et al., 2018). Furthermore, NTG treatment has been shown to dramatically improve biomass productivity, with Chlorella sp. exhibiting a 5.83-fold increase in biomass (Kuo et al., 2017), highlighting the capacity of chemical mutagens to enhance growth and metabolic output simultaneously. In contrast to these predominantly beneficial outcomes, our investigation of EtBr exposure in H. lacustris and D. salina revealed a distinct and dose-dependent metabolic response. While higher EtBr concentrations induced metabolic suppression and stress-associated declines in multiple biochemical parameters, low-dose EtBr exposure (5 µg/mL) elicited a pronounced hormetic response. Under these conditions, total FA content increased substantially, by 67.97% in H. lacustris and 76.8% in D. salina, paralleling or exceeding lipid enhancement levels reported for many conventional chemical mutagens. This hormetic effect suggests that low-intensity EtBr-induced genetic stress may activate compensatory metabolic pathways that promote lipid biosynthesis, consistent with mechanisms proposed for EMS- and MNNG-mediated strain improvement. Notably, both species also exhibited significant increases in photosynthetic pigments under EtBr treatment. H. lacustris displayed particularly striking carotenoid accumulation, with a 370.7% increase at 10 µg/mL EtBr, phenotypically resembling the enhanced carotenoid production reported in chemically mutagenized strains such as C. sorokiniana, D. salina, and C. zofingiensis. Collectively, these findings indicate that, when applied at sublethal concentrations, EtBr can induce metabolite enhancement patterns comparable to those achieved using classical chemical mutagens, while higher doses shift the response toward metabolic inhibition and cellular stress.