Mitigation of Marine Dinoflagellates Using Hydrogen Peroxide (H2O2) Increases Toxicity towards Epithelial Gill Cells

Hydrogen peroxide (H2O2) has been shown to efficiently remove toxic microalgae from enclosed ballast waters and brackish lakes. In this study, in vitro experiments were conducted to assess the side effects of mitigating toxic and non-toxic dinoflagellates with H2O2. Five H2O2 concentrations (50 to 1000 ppm) were used to control the cell abundances of the toxic dinoflagellates Alexandrium catenella and Karenia selliformis and the non-toxic dinoflagellates Lepidodinium chlorophorum and Prorocentrum micans. Photosynthetic efficiency and staining dye measurements showed the high efficiency of H2O2 for mitigating all dinoflagellate species at only 50 ppm. In a bioassay carried out to test cytotoxicity using the cell line RTgill-W1, control experiments (only H2O2) showed cytotoxicity in a concentration- and time- (0 to 24 h) dependent manner. The toxic dinoflagellates, especially K. selliformis, showed basal cytotoxicity that increased with the application of hydrogen peroxide. Unexpectedly, the application of a low H2O2 concentration increased toxicity, even when mitigating non-toxic dinoflagellates. This study suggests that the fatty acid composition of toxic and non-toxic dinoflagellate species can yield toxic aldehyde cocktails after lipoperoxidation with H2O2 that can persist in water for days with different half-lives. Further studies are needed to understand the role of lipoperoxidation products as acute mediators of disease and death in aquatic environments.


Introduction
Some coastal regions of the world are experiencing an increase in harmful algal blooms (HABs), especially under the ongoing climate change scenario [1]. These HABs have exerted significant negative impacts on human and environmental health, tourism, economies and worldwide aquaculture production [2,3]. These deleterious impacts produced by HABs present an important challenge to the institutions responsible for the management of coastal resources, resulting in the implementations of a wide variety of mitigation strategies, among which the use of chemicals, such as hydrogen peroxide (H 2 O 2 ), stands out.
Hydrogen peroxide, along with ion superoxide (O 2 − ) and hydroxyl radicals (OH·), is a reactive oxygen species (ROS) that is intermediate in the four-electron reduction of oxygen to water [4]. Since the discovery of the ubiquitous antioxidant enzyme superoxide dismutase (SOD) [5], it has been well-recognized that all oxygen-metabolizing organisms Karenia selliformis (CREAN_KS02) were isolated from the Aysén Region in 2019 and 2018, respectively. The non-toxic dinoflagellates Lepidodinium chlorophorum (CREAN_LC01 strain) and Prorocentrum micans (CREAN_PM02) were isolated in 2020 from the Los Lagos Region from the localities of Yates and the Moraleda Channel [27], respectively. All cultures were maintained in the CREAN-IFOP algal collection in Puerto Montt, Chile, in L1 sterile medium at 15 • C and at a salinity of 33 under 100 µmol photon m −2 s −1 and a 18:6 h light:dark cycle.

Epithelial RTgill-W1 Cell Line
A detailed description of the method used for the gill cell line culturing is provided by Mardones et al. [28]. The RTgill-W1 cell line (CRL-2523) was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was maintained in the dark at 19 • C in 25

Culture Chronic and Acute Effect of H 2 O 2 on Toxic Dinoflagellates
To determine the effects of five different concentrations (50,100,200, 500 and 1000 ppm) of H 2 O 2 (30% v/v, Sigma-Aldrich, St. Louis, MO, USA) on the toxic dinoflagellates, the cells of A. catenella and K. selliformis (1000 mL −1 ) were inoculated in triplicate into sterile 50 mL flasks containing 40 mL of seawater with L1 culture medium. Each H 2 O 2 treatment was assessed using a crossed factorial design with two different salinities (25 and 33) and two different temperatures (12 and 18 • C). The experiment was carried out for 1 h, with samples taken immediately after exposure. The chronic effects of H 2 O 2 on the dinoflagellate cultures were determined instantly after sampling based on the photosynthetic efficiency (PE) of the cells, and acute effects were determined based on direct observations of dinoflagellate cell viability (DCV) under an inverted microscope.

Photosynthetic Efficiency (PE) Measurement
The physiological responses of the toxic dinoflagellates to the different H 2 O 2 concentrations were assessed by measuring the maximal photosynthetic efficiency (PE) (F v /F m ) using a fast repetition rate fluorometer (FRRf, Chelsea Technologies Group, London, UK). The PE is described by Van Kooten and Snel [29] as follows: where F o is the minimal fluorescence in light, F m is the maximal fluorescence in light, and F v is the variable fluorescence in light. The measuring protocol of the FRRf was set to an acquisition sequence of 20 saturation flashes in 1 min, 20 relaxation flashes and 10 m/s sleep time between acquisitions. The flash duration was 0.65 µs. This sampling protocol was found to better characterize the fluorescence response (i.e., saturation curve fitting) of the dinoflagellate cultures in preliminary tests. F v /F m was measured 1 h after using hydrogen peroxide in the control and treatment samples. The calculation of the dinoflagellate PE % of the control is described as follows: Dinoflagellate PE (% of control) = (PET/PEC) × 100 (2) where PET corresponds to the photosynthetic efficiency of the experimental treatments, and PEC corresponds to the photosynthetic efficiency of the control treatments.

Dinoflagellate Cell Viability (DCV) Test
The acute effect of H 2 O 2 on dinoflagellate cell viability was assessed using Neutral Red (NR) following Onji et al. [30]. NR (Sigma-Aldrich, St. Louis, MO, USA) is a cationic dye that is able to permeate the plasmatic membrane and accumulate in the lysosomes of viable cells. Viable cells turn red. To test cell viability, a filtered (0.22 µm) stock solution was prepared by dissolving NR in ethanol 95% at a final concentration of 1% m/v. Later, the stock solution was dissolved in seawater (1:5 v/v) at a final concentration of 0.2% m/v. The final solution was added to 2 mL of each sample at a ratio of 1:10 v/v and incubated for 15 min at room temperature and under light. A Sedgwick Rafter chamber was used for microalgae cell counting to estimate cell viability. The DCV was calculated as follows: where LCT corresponds to the number of live cells after the experimental treatments, and LCC corresponds to the number of live cells after the control treatments. To assess the persistence of hydrogen peroxide toxicity in seawater, the RTgill-W1 cell line was exposed to six H 2 O 2 concentrations (0, 50, 100, 200, 500 and 1000 ppm) for 1 h in the dark. The cytotoxic effects of all treatments were measured at 0, 4 and 24 h after the preparation of the dilutions. After exposure, gill cell viability was measured as described in Section 2.4.

Combined Effects of H 2 O 2 and Toxic and Non-Toxic Dinoflagellates on Gill Cells
To test the combined effects of six concentrations of H 2 O 2 (0, 50, 100, 200, 500 and 1000 ppm) and the toxic dinoflagellates A. catenella and K. selliformis and the non-toxic dinoflagellates P. micans and L. chlorophorum, the cells of the factorial combination of each culture (1000 mL −1 ) were separately inoculated into each H 2 O 2 treatment concentration. The toxic dinoflagellates were cultured at salinities of 25 and 33 and at 12 and 18 • C, and the non-toxic dinoflagellates were cultured at 15 • C at a salinity of 33. For comparison purposes in the experiment using the non-toxic dinoflagellates, the toxic dinoflagellate A. catenella (cultured under the same salinity and light conditions) was added to the experimental setup as a positive control treatment. All treatments were later filtrated (0.22 µm) and inoculated in quadruplicate in 96-well plates containing RTgill-W1 cells attached to the bottom of the wells, and they were kept for 1 h in the dark at 19 • C. After exposure, gill cell viability was measured as described in Section 2.4.

Gill Cell Viability Endpoint
The RTgill-W1 bioassay has been widely used to detect cytotoxic activity in marine waters [6]. The assay estimates cell viability based on fluorescent indicator dyes when exposed to toxicants expressed as a percentage of unexposed control. Briefly, gill cell viability was determined using the indicator dye AlamarBlue 5% (DAL1025, Invitrogen, Waltham, MA, USA) diluted in phosphate-buffered saline (PBS). One hundred µL of the AlamarBlue solution was added to all cell-seeded wells and incubated for 1 h in the dark. AlamarBlue (non-fluorescence resazurin) enters the cells and is converted to the fluorescence resorufin by cytoplasmatic, mitochondrial or microsomal oxidoreductases. A decline in AlamarBlue fluorescence indicates a reduction in cellular metabolism (low GCV). A microplate reader (FLUOstar Omega, BMG Labtech 415-2871, Ortenberg, Germany) was used to measure the fluorescence of the dye with excitation and emission filters of 540 and 590 nm, respectively. Gill cell viability (GCV) is shown as the response percentage of the experimental treatments relative to that of the control treatments (% of control).

Statistical Analysis
The effects of the hydrogen peroxide concentration, species, salinity, temperature and time of exposure on the response variables, namely, (1) gill cell viability (GCV), (2) dinoflagellate cell viability (DCV) and (3) photosynthetic efficiency (PE), were assessed using an analysis of variance (ANOVA). All factors were evaluated as discrete variables, except for "concentration", which was assessed as a continuous variable. The ANOVAs were performed in a generalized lineal model (GLM) framework fitting a negative binomial residual distribution model. For significant effects, a Tukey's HSD multiple comparison test was used. In each case, the null statistical hypotheses were rejected under a significance level (α) of 0.05. All analyses were performed using R software version 4.1.2 (R Core Team, 2017), and the GLM was fitted using the R package "lme4" version 1.1-31 [31].

Effect of H 2 O 2 Concentration, Species, Temperature and Salinity on PE, DCV and GCV
The PE and DCV showed low values, even when the H 2 O 2 concentrations were minimal. The highest PE (12 %) was observed at 50 ppm of H 2 O 2 , and the lowest PE (1.6%) was observed at 1000 ppm ( Figure 1). The maximum and minimum DCV measurements were also achieved at 50 and 100 ppm of H 2 O 2 , with values of 6.4 and 0%, respectively (Figures 1 and 2). The PE was significantly affected by the peroxide concentrations (negative correlation) and temperature (p < 0.05). Salinity and species did not show effects (p > 0.05). The DCV was significantly affected by the peroxide concentrations, temperature, species and salinity (p < 0.05). The GCV was significantly affected by the peroxide concentrations (negative correlation), temperature and species (p < 0.05) compared to the control (100% ± 3% cell viability). The toxic dinoflagellates K. selliformis and A. catenella severely affected the gill cells in the control treatment (without H 2 O 2 ), with GCV values lower than 50% (LC 50 ) in K. selliformis ( Figure 3). The addition of H 2 O 2 to the toxic dinoflagellate cultures increased the cytotoxicity to the gill cells, with GCV values of 0 and 7.7 at 1000 and 50 ppm, respectively. except for "concentration", which was assessed as a continuous variable. The ANOVAs were performed in a generalized lineal model (GLM) framework fitting a negative binomial residual distribution model. For significant effects, a Tukey's HSD multiple comparison test was used. In each case, the null statistical hypotheses were rejected under a significance level (α) of 0.05. All analyses were performed using R software version 4.1.2 (R Core Team, 2017), and the GLM was fitted using the R package "lme4" version 1.1-31 [31].

Effect of H2O2 Concentration, Species, Temperature and Salinity on PE, DCV and GCV
The PE and DCV showed low values, even when the H2O2 concentrations were minimal. The highest PE (12 %) was observed at 50 ppm of H2O2, and the lowest PE (1.6 %) was observed at 1000 ppm ( Figure 1). The maximum and minimum DCV measurements were also achieved at 50 and 100 ppm of H2O2, with values of 6.4 and 0%, respectively (Figures 1 and 2). The PE was significantly affected by the peroxide concentrations (negative correlation) and temperature (p < 0.05). Salinity and species did not show effects (p > 0.05). The DCV was significantly affected by the peroxide concentrations, temperature, species and salinity (p < 0.05). The GCV was significantly affected by the peroxide concentrations (negative correlation), temperature and species (p < 0.05) compared to the control (100% ± 3% cell viability). The toxic dinoflagellates K. selliformis and A. catenella severely affected the gill cells in the control treatment (without H2O2), with GCV values lower than 50% (LC50) in K. selliformis ( Figure 3). The addition of H2O2 to the toxic dinoflagellate cultures increased the cytotoxicity to the gill cells, with GCV values of 0 and 7.7 at 1000 and 50 ppm, respectively.

Effects of H2O2 Concentration and Time of Exposure on GCV
The peroxide concentrations and time of exposure significantly affected the GCV (p < 0.05). A posteriori Tukey test showed a negative response of the gill cells after 0 and 4 h of H2O2 exposure, with GCV values lower than 50% (LC50). However, after 24 h of treatment, viability was recovered at the lower peroxide concentrations, 50 and 200 ppm, with values of 80% and 75%, respectively. Gill cells exposed to treatments of 500 and 1000 ppm showed CGV values of 24% and 0%, respectively (Figure 4).

Effects of H 2 O 2 Concentration and Time of Exposure on GCV
The peroxide concentrations and time of exposure significantly affected the GCV (p < 0.05). A posteriori Tukey test showed a negative response of the gill cells after 0 and 4 h of H 2 O 2 exposure, with GCV values lower than 50% (LC 50 ). However, after 24 h of treatment, viability was recovered at the lower peroxide concentrations, 50 and 200 ppm, with values of 80% and 75%, respectively. Gill cells exposed to treatments of 500 and 1000 ppm showed CGV values of 24% and 0%, respectively (Figure 4).

Synergistic Effect of H2O2 and Toxic and Non-Toxic Dinoflagellates on GCV
The toxic dinoflagellates K. selliformis and A. catenella severely affected the gill cel in the control treatment (without H2O2), with GCV values lower than 50% (LC50) for K selliformis (Figure 4). The GCV was significantly affected by the peroxide concentration (negative correlation), temperature and species (p < 0.05). The addition of H2O2 to the tox

Synergistic Effect of H 2 O 2 and Toxic and Non-Toxic Dinoflagellates on GCV
The toxic dinoflagellates K. selliformis and A. catenella severely affected the gill cells in the control treatment (without H 2 O 2 ), with GCV values lower than 50% (LC 50 ) for K. selliformis ( Figure 4). The GCV was significantly affected by the peroxide concentrations (negative correlation), temperature and species (p < 0.05). The addition of H 2 O 2 to the toxic dinoflagellate cultures increased the cytotoxicity to the gill cells, with GCV values of 0 and 7.7 at 1000 and 50 ppm, respectively.
An increase in H 2 O 2 concentration resulted in significant attenuation of the GCV (p < 0.05), with GCV values of 75 % (control without peroxide) and 0% (1000 ppm peroxide). Despite the significant differences among species (p < 0.05), the addition of H 2 O 2 reduced the GCV below the LC 50 in both the toxic and non-toxic dinoflagellate treatments. After 24 h of exposure to H 2 O 2 and the dinoflagellates, the GCV remained as low as that at 0 h (p > 0.05) ( Figure 5).
An increase in H2O2 concentration resulted in significant attenuation of the GCV (p < 0.05), with GCV values of 75 % (control without peroxide) and 0% (1000 ppm peroxide). Despite the significant differences among species (p < 0.05), the addition of H2O2 reduced the GCV below the LC50 in both the toxic and non-toxic dinoflagellate treatments. After 24 h of exposure to H2O2 and the dinoflagellates, the GCV remained as low as that at 0 h (p > 0.05) ( Figure 5).

Discussion
The present study investigated the potential deleterious effect of H2O2 on epithelial gill cells as a proxy for environmental safety risk after its application in marine waters to cease toxic and non-toxic dinoflagellate blooms.

Mitigation of Dinoflagellates Using H2O2
This study shows that the mitigation of toxic armored (A. catenella) and unarmored (K. selliformis) dinoflagellates using hydrogen peroxide is an effective method, even when using doses as low as 50 ppm (Figures 1 and 2). These in vitro results are in line with those found by Burson et al. [12], where 50 ppm of H2O2 was efficient to mitigate an A. ostenfeldii bloom and its paralytic toxins in the brackish Ouwerkerkse Kreek, the Netherlands. Even a lower dose of 30 ppm has been found to be effective in mitigating the ichthyotoxic dinoflagellate Cochlodinium sp. in Japan [20]. A key question is whether abiotic variables, such as temperature and salinity, can affect the effectiveness of H2O2 in mitigating toxic

Discussion
The present study investigated the potential deleterious effect of H 2 O 2 on epithelial gill cells as a proxy for environmental safety risk after its application in marine waters to cease toxic and non-toxic dinoflagellate blooms.

Mitigation of Dinoflagellates Using H 2 O 2
This study shows that the mitigation of toxic armored (A. catenella) and unarmored (K. selliformis) dinoflagellates using hydrogen peroxide is an effective method, even when using doses as low as 50 ppm (Figures 1 and 2). These in vitro results are in line with those found by Burson et al. [12], where 50 ppm of H 2 O 2 was efficient to mitigate an A. ostenfeldii bloom and its paralytic toxins in the brackish Ouwerkerkse Kreek, the Netherlands. Even a lower dose of 30 ppm has been found to be effective in mitigating the ichthyotoxic dinoflagellate Cochlodinium sp. in Japan [20]. A key question is whether abiotic variables, such as temperature and salinity, can affect the effectiveness of H 2 O 2 in mitigating toxic dinoflagellates or other HAB groups. The experiments in this study showed that temperature influenced the effectiveness of H 2 O 2 on the PE response of the dinoflagellates (chronic effect), and both temperature and salinity affected dinoflagellate cell viability when exposed to hydrogen peroxide (acute effect). It has been shown that salinity and temperature interact with H 2 O 2 degradation in seawater [32], but these two variables can also alter microalgae cell permeability, affecting osmotic processes [33]. Thus, careful consideration must be taken when using H 2 O 2 for HAB mitigation in highly variable aquatic scenarios.
The Chilean dinoflagellates A. catenella and K. selliformis have been shown to be cytotoxic to the RTgill-W1 cell line [9,34]. One possible cytotoxic mechanism in both dinoflagellates is the synergistic reaction between long-chain PUFAs (>20 carbons) and ROS. However, both species produce other phycotoxins that can exert cytotoxic effects.
Although true phycotoxins were not measured in this study, previous studies carried out by our research group have shown that the paralytic toxin analogs produced by A. catenella have low cytotoxic effects, whereas the brevenal-like compounds produced by K. selliformis have proven to be extremely cytotoxic. Despite the higher toxicity of K. selliformis towards the gill cells, a strong synergistic effect between hydrogen peroxide and the dinoflagellates was observed. This effect was surprisingly strong for Alexandrium, where the gill cell viability dropped from 93 to 60% with Alexandrium only and from 93 to 0% with Alexandrium +50 ppm H 2 O 2 . This effect was also observed in previous studies, where lysed cells generated ROS, which increased toxicity towards gill cells [9]. Hydrogen peroxide, besides lysing dinoflagellate cells, (1) may oxidize some of the cellular components, probably yielding metabolites that boost Alexandrium toxicity, and/or (2) may interact with other toxic metabolites, such as paralytic shellfish toxins (PSTs). The oxidizing effect of H 2 O 2 on PSTs has not been addressed to date, but the termination of the A. ostenfeldii bloom reported by Burson et al. [12] indicated that the concentration of PSTs was reduced after treatment with H 2 O 2 .
This study shows that the toxic effect of H 2 O 2 on the gill cell line degrades rapidly after 24 h at concentrations lower than 200 ppm (Figure 4). Higher concentrations of >1000 ppm can persist longer in seawater. Some studies have reported half-lives of hydrogen peroxide ranging from hours to more than 7 days, with them being highly dependent on organic matter concentration [32,35]. Thus, the obvious concern rises: can peroxide trigger toxicity in non-toxic dinoflagellates? The experiments that followed included the non-toxic dinoflagellate species Lepidodinium chlorophorum and Prorocentrum micans, where H 2 O 2 triggered a toxic effect at levels comparable to those observed for A. catenella and K. selliformis when exposed to the oxidizer ( Figure 5). These results support the hypothesis that hydrogen peroxide can boost toxic metabolite generation when mitigating toxic and non-toxic dinoflagellate blooms.

Toxicity in Dinoflagellate Bloom Mitigation Using H 2 O 2
Reactive oxygen species, including H 2 O 2 , can affect virtually any organic molecule susceptible to oxidation. Thus, ROS have a systemic effect on cells, with membrane fatty acids being one of the main targets of oxidation. Polyunsaturated fatty acid (PUFA) lipoperoxidation is a well-studied mechanism of oxidative stress, and it has been extensively studied in the clinical context for the formation of metabolites that can act as toxic or signaling molecules [36,37]. Oxidation can occur at different points of the molecule depending on the location of reactive sites (i.e., double bonds and functional groups). Thus, PUFA lipoperoxidation yields a cocktail of molecules depending on the locations of the unsaturations. For instance, ω6 PUFAs, such as linoleic and arachidonic acids, yield mainly 4-hydroxy-2E-nonenal (4-HNE), whereas ω3 PUFAs, such as docosahexanoic (DHA) acid, yield mainly 4-hydroxy-2E-henenal (4-HHE) [38,39]. Similarly, the oxidative degradation of any PUFA with more than two methylene-interrupted double bonds can yield malondialdehyde (MDA) [40]. For instance, the fatty acid composition of the Chilean A. catenella is dominated by ω3 PUFAs, with DHA (22:6ω3), octadecapentanoic (18:5ω3), stearidonic (18:4ω3), alpha-linoleic (18:3ω3) and eicosapentaenoic (20:5ω3) acids accounting for more than 50% of the fatty acids [9]. Since all the dominant PUFAs in this dinoflagellate have methylene-interrupted double bonds, it is probable that 4-HHE and MDA are the main aldehydes generated as lipoperoxidation products.
Aldehydes react with the primary amines present in proteins or DNA. These covalent modifications impair the structure and function of biomolecules, which results in cytotoxicity and a loss of viability [37]. As membrane proteins are directly exposed to the cell environment, sensors and transporters (e.g., ions and glutamate) are two of the primary targets [36,41]. For instance, the transient receptor potential (TRP) cation channel superfamily that regulates several physiological processes is impaired by MDA, 4-HNE and 4-HHE [41]. Lysed A. catenella cells can impair osmoregulation via a net K + efflux resulting in gill cell death [9,28]. Even though osmoregulation in other species, such as the model Danio rerio (zebrafish), is well-described, there is still knowledge gaps regarding the transporters involved [42]. In Salmo salar (Atlantic salmon), TRP transporters are expressed in several tissues, including the blood, spleen, kidney and gills [43]; thus, the involvement of this mechanism in osmoregulation in the rainbow trout RTgill-W1 cell line cannot be discarded. In Figure 6, this study proposes a ROS-mediated toxicity mechanism for gill cells in response to H 2 O 2 usage in order to mitigate HABs. modifications impair the structure and function of biomolecules, which results in cytotoxicity and a loss of viability [37]. As membrane proteins are directly exposed to the cell environment, sensors and transporters (e.g., ions and glutamate) are two of the primary targets [36,41]. For instance, the transient receptor potential (TRP) cation channel superfamily that regulates several physiological processes is impaired by MDA, 4-HNE and 4-HHE [41]. Lysed A. catenella cells can impair osmoregulation via a net K + efflux resulting in gill cell death [9,28). Even though osmoregulation in other species, such as the model Danio rerio (zebrafish), is well-described, there is still knowledge gaps regarding the transporters involved [42]. In Salmo salar (Atlantic salmon), TRP transporters are expressed in several tissues, including the blood, spleen, kidney and gills [43]; thus, the involvement of this mechanism in osmoregulation in the rainbow trout RTgill-W1 cell line cannot be discarded. In Figure 6, this study proposes a ROS-mediated toxicity mechanism for gill cells in response to H2O2 usage in order to mitigate HABs. Aldehydes are generated naturally by the breakdown of PUFAs in algae. For instance, arachidonic acid is largely accumulated in the microalgae Lobosphaera incisa [44], the brown algal kelp Laminaria digitata [45] and the dinoflagellate Prorocentrum cordatum [46]. Thus, the presence of these species represents an important potential source of aldehydes. Nevertheless, they are part of the volatile compounds produced under different environmental scenarios and related to defense mechanisms, such as oxylipin pathways [47]. The mitigation of algal or cyanobacterial blooms with H2O2, where populations can Aldehydes are generated naturally by the breakdown of PUFAs in algae. For instance, arachidonic acid is largely accumulated in the microalgae Lobosphaera incisa [44], the brown algal kelp Laminaria digitata [45] and the dinoflagellate Prorocentrum cordatum [46]. Thus, the presence of these species represents an important potential source of aldehydes. Nevertheless, they are part of the volatile compounds produced under different environmental scenarios and related to defense mechanisms, such as oxylipin pathways [47]. The mitigation of algal or cyanobacterial blooms with H 2 O 2 , where populations can be as high as 10 9 -10 10 cells m −3 , may be an important source of toxic aldehydes that affect other species in the ecosystem. For example, the termination of blooms carried out in the Netherlands, the USA and Brazil had an effect on the whole microbial, phytoplankton, zooplankton and/or macroinvertebrate community composition [12,48,49]. One of these studies suggests that H 2 O 2 treatment may not be an ideal mitigation approach in high-biomass ecosystems [48]. Surprisingly, none of these studies measured the generated metabolites, such as toxic aldehydes, before and/or after treatment. The fact that toxic aldehydes can persist in the water for days [47] and that the production can be increased by environmental parameters, such as the presence of iron ions [50] or other transition metals in the presence of H 2 O 2 , drives the need for further studies on the underlying mechanisms that boost their production in aquatic environments.

Conclusions
In this study, we show that the use of hydrogen peroxide for mitigating dinoflagellates enhanced the cytotoxicity in toxic dinoflagellate species but also caused toxicity when used in non-toxic dinoflagellates. We suggest that the fatty acid composition of microalgal blooming species can yield aldehyde cocktails under the oxidative stress produced by H 2 O 2 . Overall, further studies are needed to better understand the mechanisms underlying ROS-mediated toxicity in aquatic environments.