Inhibitory Effects of Hydrogen Peroxide on Prorocentrum donghaiense Lu Under Varying Light Conditions and Iron Ion Environments

Abstract

Light and antioxidant systems play a crucial role in the life activities of algal cells. This study investigates the algicidal efficacy of hydrogen peroxide (H₂O₂) against the harmful algal bloom (HAB)-forming dinoflagellate Prorocentrum donghaiense Lu, with a focus on the modulating roles of light conditions and iron ion environments. Within 180 min, dark-adapted cells showed 78% greater viability loss than light-exposed ones, and Fe₃O₄ nanoparticles synergistically enhanced H₂O₂ inhibition. Imaging and cytometry confirmed cell damage, including membrane rupture. Mechanistically, H₂O₂ penetrated cells, induced severe oxidative stress, suppressed photosynthesis, and compromised membrane integrity. Darkness likely exacerbated toxicity by depleting antioxidant reserves. This study elucidates an apoptosis-like pathway underlying H₂O₂-induced cell death and highlights the critical influence of ambient light on treatment efficiency. These findings reveal an apoptosis-like death pathway and highlight ambient light’s critical role, suggesting that optimized nighttime H₂O₂ application with nanomaterial synergists could improve HAB control strategies.

1. Introduction

Due to eutrophication, climate anomalies, altered ocean currents, and species invasion via ballast water discharge, HABs occur frequently in coastal waters worldwide, posing significant threats to marine fisheries, aquaculture, and ecological balance [1,2]. Prorocentrum donghaiense Lu (P. donghaiense Lu), a bloom-forming dinoflagellate prevalent in the East China Sea (especially near the Yangtze River estuary), causes annual HABs from April to August [3]. Between 2008 and 2022, 81 outbreaks covering 49,407 km² were recorded [4]. Furthermore, P. shikokuense and P. donghaiense should be regarded as junior synonyms of P. obtusidens [5]. During HABs, rapid proliferation of algae depletes dissolved oxygen, limiting growth and causing mortality of other marine organisms [6,7], which can lead to growth restriction and even death [8,9]. Additionally, daytime migration of HABs to surface waters reduces water transparency, preventing light-dependent photosynthesis in aquatic plants, which will impede the normal metabolic processes of aquatic organisms and even cause their death [10,11]. As a common non-toxic HAB, understanding P. donghaiense Lu’s inactivation is crucial. However, effective, economical, feasible, and environmentally safe HAB control remains challenging.

Chemical agents such as copper sulfate (CuSO₄), sodium hypochlorite (NaClO), hydrogen peroxide (H₂O₂), ozone (O₃) and modified clay exhibit algicidal properties. NaClO and CuSO₄ inactivated Cochlodinium polykrikoides with 72-h EC₅₀ values of 0.584 and 0.633 mg L⁻¹, respectively, inducing ROS generation and pigment degradation [12]. However, their environmental persistence and toxicity to non-target organisms limit their application. O₃ inactivated C. polykrikoides (3.0 × 10³ cells/mL) within 300 s via oxidative damage, but produces carcinogenic bromate and disinfection by-products (DBPs) [13]. Modified clay could control the algae population of Prorocentrum donghaiense (P. donghaiense) through indirect effects such as oxidative stress, while the maximum mortality rates reached 50% [14]. Various doses of ozone were used to pre-ozonate Microcystis aeruginosa and Anabaena flosaquae at concentrations of 2.5 × 10⁵ cells mL⁻¹ and 1.5 × 10⁶ cells mL⁻¹ [15].

For H₂O₂ inactivation of five marine species (Corophium volutator, Artemia salina, Brachionus plicatilis, Dunaliella teriolecta, and Skeletonema costatum), with H₂O₂ concentration of 145 mg/L and a exposure time of longer than 50 d, a 50% likelihood exists that 95% of the species will be affected [16]. Previous studies have used H₂O₂ to treat Scripsiella trochoidea (S. trochoidea), with the result that H₂O₂ (100 mg/L) exhibited 87.2% inactivation efficiency after 5 min treatment [17]. A concentration of 50 mg·L⁻¹ of H₂O₂ was brought into an entire creek system with a special device for optimal dispersal of the added H₂O₂ over 24 h, with the result that 99.8% Alexandrium ostenfeldii cells and pellicle cysts were inactivated and concentrations of PSP in the water were reduced below local regulatory levels of 15 μg·L⁻¹ [18]. A water harrow setup, dispersing 2 mg L⁻¹ (60 mu M) of H₂O₂ homogeneously into the entire water volume of the lake with a special dispersal device, inactivated cyanobacterial population by 99% within a few days [19]. Dispersal of 14 mg/L H₂O₂ reduced P. parvum biomass and prymnesin concentrations, with limited negative impact on other phytoplankton [20]. A key advantage of H₂O₂ is its lack of persistent chemical residues, though high doses and prolonged treatments increase operational costs.

In the context of algal inactivation technologies based on Advanced Oxidation Processes (AOPs), such as photocatalysis, the Fenton process, O₃/H₂O₂, and UV/H₂O₂, hydroxyl radicals (•OH) have been identified as the key agent responsible for efficient inactivation [21,22,23]. Beyond these methods, ferrate(VI) can serve as a pre-oxidant to enhance conventional Fe(II) coagulation, thereby improving the removal of Microcystis aeruginosa. For instance, one study applied 20 µM ferrate(VI) combined with 80 µM Fe(II), achieving effective coagulation enhancement. However, the dosage of ferrate(VI) requires careful optimization, as higher concentrations treating Microcystis aeruginosa at 2.0 × 10⁶ cells/mL can achieve an inactivation rate of 88.4% but may also cause significant damage to algal cells [24]. This confirms the potential cell disruption effect of high-dose ferrate, indicating that precise dosing control is crucial in practical algal removal applications.

On the other hand, ozonation also serves as an effective method for algal inactivation. Research shows that a relatively low ozone dose (0.2 mg·min/L) can inactivate Microcystis aeruginosa (2.5 × 10⁵ cells/mL) and Anabaena flosaquae (1.5 × 10⁶ cells/mL) [15]. However, this technique may introduce notable environmental trade-offs: post-ozonation, the formation of disinfection by-products (DBPs) increases significantly, with trihalomethanes (THM) and haloacetic acids (HAA) rising by 174% and 65%, respectively [15]. These results suggest that while ozonation effectively inactivates algae, it may concurrently elevate environmental risks, raising concerns about its overall environmental friendliness.

This study investigates the algicidal efficacy of H₂O₂ against the HAB-forming dinoflagellate P. donghaiense Lu, with a focus on the modulating roles of light conditions and iron ion environments. In this study, the process of H₂O₂ apoptosis of algal cells was simulated in different iron-containing environments to further understand the mechanism of H₂O₂ on marine algae, which provides a theoretical basis for understanding the self-extinction of HABs and emergency management methods for HABs.

2. Materials and Methods

2.1. Algae Cultivation

Prorocentrum donghaiense Lu was obtained from Xiamen University Algae Collection Center, which was collected from Yangtze River Estuary in the East China Sea and is a harmful alga known to cause red tide [25]. The alga has an average cell size of 15~25 μm long and 8~15 μm broad, which shows an oval shape.

P. donghaiense Lu were cultured in f/2 culture medium [26], f/2 culture medium with twice the concentration of iron ions and f/2 culture medium without iron ions at 22 °C under 2200 lax light intensity with a light–dark cycle of 12 h/12 h in an incubator (Safe, Shanghai, China). After 20 days of culturing, P. donghaiense were at the plateau phase of growth with a final cell yield of 6 × 10⁴ cells/mL. To simulate an algae bloom in high algae density, the algae cultured at the stationary phase of growth were diluted to achieve a final cell density of in total 1 × 10⁴ cells/mL using seawater from Xiamen Bay.

2.2. Experimental Procedures

Five experimental groups and one control (n = 3) were established:

(a) Control, growth in f/2 medium; (b) 1 mg L H₂O₂, (c) 1 mg L H₂O₂ + 4 mL Fe₃O₄ NPs (10–30 nm, 25% in H₂O), (d) 4 mL Fe₃O₄ NPs (10–30 nm, 25% in H₂O), (e) 1 mg L H₂O₂ in Fe-enriched f/2, (f) 1 mg L H₂O₂ in Fe-depleted f/2.

H₂O₂ and Fe₃O₄ NPs (Haohe, Xiamen, China) were added to algae samples. Treatments were applied to algae pre-adapted to dark or light conditions. To evaluate the influence of illumination on H₂O₂ inactivation of algae, after a 12-h dark culture period, one group was immediately treated with H₂O₂ under visible light, while the other group was exposed to H₂O₂ following 3 h of incubation under illumination. Samples were quenched with Na₂S₂O₃ (saturated concentration) (Haohe, Xiamen, China) post-exposure, shown in Figure S1.

2.3. Analytical Methods

2.3.1. TRO Concentration Determination

TRO concentration is the abbreviation of the total reactive oxidants, which include •OH, H₂O₂ and other active oxygen. The total reactive oxidant in the liquid was combined with potassium iodide, which react with N, N-diethyl-p-phenylenediamine (DPD, 1 g/L) (Haohe, Xiamen, China) to emit light at the wavelength of 525 nm. The absorbance of the samples was measured with an Agilent G6860A spectrophotometer (Agilent Instruments Ltd., Santa Clara, CA, USA) with the US EPA’s Standard Method 330.5 [27].

2.3.2. Counts and Identification of Algal Cellular Viability

Flow cytometry (Accuri C6, BD, Franklin Lakes, NJ, USA) was used for all fluorescence measurements and the activity of algae cells at a fixed wavelength of 488 nm. Channel FL1 (530 nm) collected SYTOX Green (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) fluorescence emitted by nonviable cells; simultaneously, channel FL3 (675 nm) collected red chlorophyll fluorescence emitted by viable cells. According to the fluorescence measurements, viable and nonviable cells could be distinguished. All the samples were collected, washed with phosphate-buffered saline (PBS) (Haohe, Xiamen, China) and then loaded for analysis. Meanwhile, algae were counted with flow cytometry.

2.3.3. Cell Surface Morphology Analysis

The morphology of Prorocentrum donghaiense Lu was detected with a scanning electron microscope (SEM, SU-70, Hitachi, Tokyo, Japan) to confirm cellular integrity. Prior to observation, the algal suspension was concentrated through centrifugation and then fixed overnight in a 2.5% glutaraldehyde solution (Haohe, Xiamen, China). Following fixation, the samples were rinsed with PBS and centrifuged again to further enrich the algal cells. These enriched cells were then adhered to coverslips with an approximate area of 25 mm². The adhered samples were fixed in 2.5% (v/v) glutaraldehyde (Haohe, Xiamen, China) overnight at 4 °C, and were gradually dehydrated with ethanol. The slides with samples were immersed in ethanol for gradient dehydration, dried using a critical point dryer (Leica EM CPD300, Wetzlar, Germany), and coated with platinum using a metal sputtering device (JEOL JFC-1600, Tokyo, Japan). Finally, the prepared samples were observed and photographed using a field emission scanning electron microscope.

2.3.4. Photosynthetic Parameters

Different chlorophyll fluorescence parameters are obtained by irradiating algae with different excitation lights, which can reflect the physiological state of the algal photosynthesis system.

The photosynthetic activity of the algae was detected by pulse amplitude modulation (PHYTO-PAM, Walz, Effeltrich, Germany). F_v/F_m is maximal photochemical efficiency, determined as:

F_v/F_m = (F_m − F₀)/F_m

(1)

The minimum fluorescence (F₀) was obtained after dark adaptation for 10 min and the maximum fluorescence (F_m) was obtained after the first saturation pulse.

F_v/F_m reflects algae maximal photochemical efficiency, which proves algal cell viability.

2.3.5. Measurement of Intracellular •OH and ROS

The intracellular •OH and ROS levels were quantified using fluorescent probes 3′-(p-hydroxyphenyl) fluorescein (HPF, Invitrogen, USA) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen, USA). For detection, 1.5 μL HPF (10 mM) or 15 μL DCFH-DA (1 mM) was immediately added to 1.5 mL of algae suspension, and incubated in darkness for 30 min. Subsequently, the algae were pelleted by centrifugation at 6000 rpm for 5 min, washed and resuspended in phosphate buffer (pH 7.4) to a final volume of 200 μL for flow cytometric analysis. Measurements were performed using a 488 nm excitation wavelength with emission detection at 530 nm. Data acquisition and analysis were conducted using the BD Accuri software platform (C6 version), with further processing performed in FlowJo V10.4.

3. Results

3.1. Cell Viabilities of Algae with H₂O₂ Exposure

H₂O₂ exposure caused a rapid decrease in viable algal cells to 3.0 × 10³ cells/mL within 60 min, followed by regrowth to 2.6 × 10³ cells/mL by 180 min. In the control group, the viable algal cell count increased to 1.2 × 10⁴ cells/mL after 180 min. Neither iron-excess nor iron-depleted treatments enhanced algae inhibition (Figure 1).

Figure 1. The effect of H₂O₂ on cell viabilities of the algae under the different exposure conditions. Notes: Prorocentrum donghaiense Lu after a 7-day batch culture with normal f/2 medium, iron-excess treatment (+Fe) and iron-depleted treatment (−Fe), exposed to H₂O₂ (1 mM). For all exposure conditions, the initial algae content was 1.0 × 10⁴ cells/mL.

Following light exposure, control group algal cells increased to 1.1 × 10⁴ cells/mL by 180 min. With H₂O₂ treatment, viable cells dropped to 8.0 × 10³ cells/mL at 90 min then recovered to 1.0 × 10⁴ cells/mL by 180 min. Under both iron-excess and iron-depleted conditions, inhibition was stronger, with final counts of 7.7 × 10³ and 7.9 × 10³ cells/mL, respectively. Viable algae decreased significantly over time (p < 0.05) (Figure S2).

3.2. Variation in Photosynthetic Activity of Algal Cells

H₂O₂ can diffuse into cells, causing structural damage to intracellular components [28]. In algal cells, H₂O₂ has the ability to cause oxidation damage to photosynthesis [29,30,31]. The F_v/F_m value is the maximal photochemical efficiency, an indicator of the underlying growth ability of algal cells [32]. The F_v/F_m values under the different exposure conditions are presented in Figure 2.

Figure 2. The effect of H₂O₂ on F_v/F_m of algal cells under the different exposure conditions.

After the dark phase, the initial F_v/F_m was 0.20 in the control and increased to 0.26 after 180 min illumination. H₂O₂ exposure initially raised F_v/F_m within 60 min, but then it sharply declined to 0.01 by 90 min. Both iron-excess and iron-depleted treatments reduced F_v/F_m to 0 after 180 min of H₂O₂ exposure. Fe₃O₄ nanoparticles interfered with the measurements, preventing data acquisition.

In the control group after light-phase H₂O₂ exposure, the initial F_v/F_m was 0.27 and declined to 0.18 after 180 min. Under iron-excess and iron-depleted conditions, the initial values were lower at 0.13 and 0.22, respectively. H₂O₂ exhibited a biphasic effect—transient photosynthetic stimulation followed by sustained inhibition after 60 min, which corresponded with the timing of significant cell inactivation shown in Figure 1.

3.3. Variation in Cell Morphology of Algae

Scanning electron microscopy and flow cytometry revealed significant H₂O₂-induced morphological changes (Figure 3). Control cells maintained a plump spindle shape, while after 180 min of H₂O₂ exposure, widespread cell shrinkage, rupture, and reduced side scatter were observed. Both iron-excess and iron-depleted conditions caused similar damage. Notably, iron-excess treatment produced flocculent substances, and Fe₃O₄ nanoparticles attached to cell surfaces, inducing lysis-indicating ROS generation and flocculation potential by the nanoparticles.

Figure 3. The effect of H₂O₂ on cell morphology of the algae under the different exposure conditions. Notes: (A1–E1) SEM images of the control algae, the H₂O₂ group algae, the Fe iron-treatment (+Fe) group algae, the iron-depleted treatment (−Fe) group algae and the nano-Fe₃O₄ group algae. (A2–E2) Flow cytometry results of the control algae, the H₂O₂ group algae, the Fe iron-treatment (+Fe) group algae, the iron-depleted treatment (−Fe) group algae and the nano-Fe₃O₄ group algae.

Flow cytometry analysis revealed two critical parameters: elevated forward scatter (FS) correlates with higher intracellular granularity, while increased side scatter (SS) reflects larger cell size. Compared to the control, H₂O₂-exposed cells exhibited decreased FS/SS profiles, indicating reduced cellular content and diminished cell volume. Notably, Fe₃O₄ nanoparticles caused severe signal interference during flow cytometric detection due to nanoparticle aggregation.

3.4. Accumulation of Intracellular Reactive Oxygen Species

For H₂O₂ exposure after the dark phase, the H₂O₂ exposure induced intracellular reactive oxygen species (ROS) and hydroxyl radical (•OH) to increase to 1.85 times and 1.47 times the control group after 120 min (Figure 4). Iron-excess treatment, iron-depleted treatment and nano-Fe₃O₄ treatment all had a promoting effect on intracellular ROS accumulation, but not on •OH accumulation. However, after 180 min, the intracellular ROS and •OH reduced to the lower level of the control group.

Figure 4. The effect of H₂O₂ on intracellular reactive oxygen species under the different exposure conditions. Notes: The mean fluorescein was detected from 1000 algal cells. The letters a, b, c, d and e indicate significant differences between groups, p < 0.05.

4. Discussion

4.1. Causes of Inhibition of Algal Activity

Dark-adapted algae showed rapid viability loss (78% within 180 min) with H₂O₂ exposure (Figure 1). Fe₃O₄ NPs enhanced this effect, while iron-altered media had minimal impact. Light-adapted cells exhibited higher resistance, with viability recovering after 180 min. According to the experimental results, iron-excess treatment, iron-depleted treatment and Fe₃O₄ nanoparticles had no outstanding effect on the inactivation effect of H₂O₂ on P. donghaiense Lu. However, Fe₃O₄ nanoparticles significantly potentiated the inhibitory effect of H₂O₂. Notably, when algae were exposed solely to Fe₃O₄ nanoparticles, growth was suppressed but the viable cell count remained stable. In addition, the results in this paper showed that the cells of P. donghaiense Lu were more sensitive to H₂O₂ after the dark phase, which conform with previous findings. Erbes et al. found that Chlamydomonas reinhardtii exposed to H₂O₂ in light for 180 min had no dramatic effects on cell viability, while exposure to H₂O₂ in darkness resulted in a clear dose-related decrease in cell viability [33]. Volpert et al. found that cells of Thalassiosira pseudonana in the dark exhibited higher sensitivity to oxidative stress than cells in light, and suggested that the dark-dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione that accumulated during the light period [34]. However, information on algae sensitivity to oxidative stress under dark conditions is very limited.

4.2. The Mechanisms Influencing the Photosynthetic System

Photosynthesis is a complex process that involves the conversion of light energy into chemical energy for cellular metabolism. The results of this research showed that the destruction of the photosynthetic system is an important condition for H₂O₂ to lead to cell death, which demonstrates greater sensitivity to H₂O₂ than cellular viability. In addition, previous studies have shown that H₂O₂ can influence photosynthesis at various levels. When algal cells are exposed to H₂O₂, H₂O₂ in the water can enter the cell through the water channel in the cell membrane, but only if enough H₂O₂ is accumulated to play a destructive role. This biphasic response suggests initial PSII stimulation followed by irreversible damage. At low concentrations, H₂O₂ can enhance the efficiency of photosystem II (PSII], the primary photosynthetic apparatus, by accelerating the electron transport rate and decreasing nonphotochemical quenching [1,14].

Additionally, H₂O₂ can inhibit photosynthesis by damaging PSII and other photosynthetic pigments like chlorophyll and carotenoids. The severity of inhibition depends on the duration and concentration of H₂O₂ treatment, as well as the algal species and growth conditions [29]. Chloroplasts showed a rate of H₂O₂ generation of 5 μmol·mg chlorophyll⁻¹·hr⁻¹ under illumination. In the cellular environment, the decomposition of H₂O₂, catalyzed by reduced metal ions (Fe²⁺), results in the formation of hydroxyl radicals, which can affect a variety of macromolecules, such as proteins, fatty acids, and nucleic acids. This leads to decreased photosynthetic efficiency, photoinhibition, and accumulation of ROS [32]. H₂O₂ concentrations ≥ 200 mg L⁻¹ negatively affected photosystem II (PSII) in coralline alga Lithothamnion soriferum immediately after exposure, which was observed through a significant decline in F_v/F_m [35]. Meanwhile, reaction in darkness enhances the inhibitory effect of H₂O₂ on algae. This is consistent with previous studies [36]. Exposure of Chlamydomonas reinhardtii cells to H₂O₂ in light for 2 h had no dramatic effects on cell viability, while exposure to H₂O₂ in darkness resulted in a clear dose-related decrease of cell viability [33]. Light can regulate the photosynthetic activity of algal cells and impact the intracellular redox state. Under light conditions, the active processes of photosynthesis and electron flow lead to a more reduced state in cells [37]. In contrast, under dark conditions, the absence of light-induced reactions causes a decline in electron flow and a shift towards a more oxidized state. This could be a reason why H₂O₂ is more likely to induce cell inactivation under dark conditions.

4.3. Causes of Changes in Algal Surface Structure

Numerous studies have shown that exposure of algae to oxidants can alter cellular morphology and even lead to cell lysis. Cell membrane damage was observed on K. mikimotoi and P. donghaiense surfaces post ClO₂ treatment [38]. The mechanism of ClO₂ inactivation is that ClO₂ attacks cell wall and plasma membrane glycoproteins, glycolipids, or certain amino acids [39]. Cell surface damage to M. aeruginosa was also shown via SEM after 5 min of treatment with UV/H₂O₂ treatment, which led to the release of IOM [40]. H₂O₂ is a relatively strong oxidant with an oxidation potential of 1.76 V, which can easily pass through cell membranes by diffusion. Inside the cells, H₂O₂ induces oxidative stress, which causes damage to proteins, lipids and nucleic acids, and thereby changes in cell morphology and compromised cell viability.

4.4. Mechanism of Algae Inhibition by Hydrogen Peroxide

H₂O₂ can penetrate the cell membrane and enter the cell, leading to an elevation of intracellular ROS levels. This can trigger oxidative stress and tissue damage [41]. Apoptosis is an important mechanism of H₂O₂ lethal algal cells. H₂O₂ could inhibit M. aeruginosa growth after 24~48 h of treatment, which induced several classic parameters associated with apoptosis in cells, such as membrane deformation, cytoplasmic vacuolation, chromatin condensation, DNA fragmentation and increasing caspase-3-like activity [42]. Although it has been found that H₂O₂ can cause algal cell death by inducing apoptosis-like pathways, it is generally believed that cell membrane rupture is the main mechanism of cell death by conventional oxidants. Moreover, H₂O₂ can induce reactions between the lipid substances and proteins present with intracellular iron ions, giving rise to •OH and other free radicals [43,44]. Numerous studies have demonstrated that the time required for H₂O₂ treatment to eliminate algal cells typically ranges from a few hours to several days [18]. This may depend on the accumulation of H₂O₂ within the algal cells (Figure 5).

Figure 5. Biological mechanism of H₂O₂ on Prorocentrum donghaiense Lu.

The oxidizing properties, rapid degradation, environmentally friendly degradation products (water and oxygen), and the fact that it can be produced electrochemically make H₂O₂ a promising candidate for algicide of HABs and ballast water [30]. Based on the results of this study, when using H₂O₂ as an algicide, the impact of light should be taken into consideration. Since algae produce H₂O₂ and other ROS during photosynthesis, they possess mechanisms to regulate intracellular oxidative stress. The data presented provide support for the idea that algae have evolved mechanisms that allow maintenance of a certain level of H₂O₂. H₂O₂ released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. Ice algae can efficiently photo-acclimate to increased irradiance by modifying their photosynthetic machinery and light-harvesting pigments over hours to days [45,46]. This regulatory effect of algal cells is related to photoprotection. Light, as a regulatory factor, will affect the redox state of cells [47,48,49].

5. Conclusions

HABs have appeared frequently and pose a significant threat to marine ecology and security. H₂O₂ is a potential algicide due to its eco-friendly nature, environmental protection qualities, and strong oxidizing properties. This investigation revealed an apoptosis-like mechanism underlying H₂O₂-induced cell death in algal cultures. H₂O₂ exposure in darkness induced a rapid 78% reduction in algal cell viability within 180 min, significantly exceeding the effect observed under light conditions. Fe₃O₄ nanoparticles synergistically enhanced H₂O₂ efficacy, achieving significant cell inactivation after 180 min. Mechanistically, H₂O₂, after penetrating algal cells, induced intracellular reactive oxygen species accumulation, triggered oxidative stress, suppressed photosynthetic activity, and altered cell membrane permeability. To deal with toxic HABs, this method can effectively prevent the leakage of toxins. This method is suitable for HABs with a relatively small outbreak area and where the toxic algae species are the dominant ones. It enables the HABs to gradually disappear. These effects were exacerbated under dark adaptation, likely due to depletion of antioxidant reserves and cessation of photoprotective processes. This study provides theoretical insights into optimizing H₂O₂-based algal mitigation strategies, highlighting nighttime application and nanomaterial synergism to improve cost-effectiveness and environmental safety.