Metabolomics Approach to Reveal the Effects of Ocean Acidiﬁcation on the Toxicity of Harmful Microalgae: A Review of the Literature

: Climate change has been associated with intensiﬁed harmful algal blooms (HABs). Some harmful microalgae produce toxins that accumulate in food webs, adversely affecting the environment, public health and economy. Ocean acidiﬁcation (OA) is a major consequence of high anthropogenic CO 2 emissions. The carbon chemistry and pH of aquatic ecosystems have been signiﬁ-cantly altered as a result. The impacts of climate change on the metabolisms of microalgae, especially toxin biosynthesis, remain largely unknown. This hinders the optimization of HAB mitigation for changed climate conditions. To bridge this knowledge gap, previous studies on the effects of ocean acidiﬁcation on toxin biosynthesis in microalgae were reviewed. There was no solid conclusion for the toxicity change of saxitoxin-producing dinoﬂagellates from the genus Alexandrium after high CO 2 treatment. Increased domoic acid content was observed in the diatom Pseudo-nitzschia . The brevetoxin content of Karenia brevis remained largely unchanged. The underlying regulatory mechanisms that account for the different toxicity levels observed have not been elucidated. Metabolic ﬂux analysis is useful for investigating the carbon allocations of toxic microalgae under OA and revealing related metabolic pathways for toxin biosynthesis. Gaining knowledge of the responses of microalgae in high CO 2 conditions will allow the better risk assessment of HABs in the future.


Introduction
Harmful algal blooms (HABs) refer to the rapid growth of phytoplankton, including cyanobacteria, dinoflagellates, raphidophytes, haptophytes, and macroalgae, which exerts harmful effects on the environment, human health, and the economy [1]. High microalgal biomass accumulation during HABs can cause hypoxia, suffocating surrounding aquatic organisms [2]. Some HAB species can produce algal toxins that accumulate in aquatic food webs [3]. Apart from causing the massive death of marine mammals and fish [4], algal toxins can lead to intoxication in humans. Approximately 50,000 to 500,000 cases of intoxication with a 1.5% mortality rate have been reported annually due to the consumption of contaminated shellfish or fish [5]. The health costs associated with HABs are substantial, ranging from around USD 90 to USD 12,000 for digestive and respiratory illnesses with different severities [6]. The aquacultural industry is the most vulnerable when considering the direct economic losses caused by HABs. From the 1980s to the 2010s, the aquacultural industry in Korea lost a total of USD 121 million due to HAB-induced fish and shellfish deaths [7]. In addition to the aquacultural industry, tourism is adversely affected by HABs. It was reported that the monthly revenue of coastal lodgings and restaurants decreased Organization (FAO), the recommended okadaic equivalents for okadaic acid, DTX1, and DTX2 are 1.0, 1.0, and 0.5, respectively [35]. Diarrhea, nausea, vomiting, and abdominal pain are the common symptoms of DSP [36]. DSP is generally not life-threatening, and hospitalization is not required [37]. As potent inhibitors of serine/threonine phosphatases [38], okadaic acids show specifically high binding affinity to protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2) [39]. The gastrointestinal symptoms of DSP may be a result of inhibited intestinal PP activities by okadaic acids [40]. In addition, the inhibiting effect of okadaic acids may induce the hyperphosphorylation of proteins that regulate the sodium secretion of intestinal cells [41]. The release of sodium from cells disturbs the osmotic gradient balance and subsequently causes the passive loss of fluids, leading to diarrhea. Both Prorocentrum and Dynophysis have expanded their niches in recent years. P. minimum is the most studied Prorocentrum species and is widespread in the Black Sea, the Baltic Sea, Lake Nakanoumi, the Mexican coast, and the Philippines [42]. The expansion of Dynophysis was observed in the coastal areas of the United States, Canada, and South Africa [13]. Moreover, increased abundances of Prorocentrum and Dynophysis in the Northeast Atlantic were reported [43].

Amnesic Shellfish Poisoning
ASP is caused by the consumption of domoic acid (DA)-accumulated shellfish. Red algae such as Chondria armata and diatoms from the genera Pseudo-nitzschia and Nitzchia (N. navis-varingica and N. bizertensis) are the primary producers of DA [44,45]. DA, a tricarboxylic amino acid, is analogous to glutamic acid and kainic acid [46]. Several DA isomers (isodomic acid A-H, 5 epi-DA) have been identified [47]. With such structural similarity, Das can bind and activate three ionotropic glutamate receptor subtypes (N-methyl-D-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors) located on the dendrites of postsynaptic cells [48,49]. An influx of calcium ions (Ca 2+ ) into neurons is induced upon the activation of kainate and AMPA receptors [50], while an influx of both Ca 2+ and sodium ions (Na + ) is induced upon the activation of the NMDA receptor [51]. Desensitization is prevented by the low conformational mobility of DAs when docked to the receptors, leading to a continuous flow of cations to the postsynaptic cells [52]. DAs exert excitotoxic effects primarily through excess intracellular Ca 2+ , which triggers the mobilization of glutamate-containing vesicles towards the membrane surface, followed by the release of glutamate (Glu) into the synaptic cleft [53]. Excess Glu results in neurodegeneration and apoptosis [54,55]. Since neurons in the hippocampus, where the consolidation of memories takes place, are affected by DAs [56], symptoms such as short-term memory loss and anterograde amnesia may result [57]. Generally, the toxicity of DA isomers is lower than that of DA because of the lower binding affinity of DA isomers to glutamate receptors [58][59][60]. One of the major DA producers, Pseudo-nitzschia, has been detected in the Pacific Ocean and the Southern Ocean [61]. Expanded distributions of Pseudo-nitzschia and N. navis-varingica have been observed recently [44]. Several locations, including the Arctic, Angola, Singapore, Ukraine, and Pakistan, have recorded the presence of Pseudo-nitzschia for the first time, while N. navis-varingica has expanded to Malaysia, Australia, Indonesia, and the Philippines.

Neurotoxic Shellfish Poisoning
NSP is caused by the consumption of shellfish contaminated with brevetoxins (PbTx). PbTx are a set of cyclic polyether neurotoxins primarily produced by dinoflagellate Karenia brevis [62]. Besides K. brevis, recent research indicated PbTx production by raphidophyte Chattonella cf. verruculosa [63]. PbTx-1 and PbTx-2 are regarded as the parent molecules for other derivatives based on their different structural backbones. The toxicity of all identified natural derivatives is lower than that of the parent molecules [64]. PbTx bind specifically to receptor site five of the voltage-gated sodium channels in the nerves and muscles, with a preference for those located in the nerves [65,66]. The influx of Na + ions into the cells is induced upon the activation of the sodium channels. Prolonged membrane depolarization persistently initiates the generation of action potentials in nerves and muscles, causing symptoms such as paresthesia, vertigo, and malaise [67]. Blooms of K. brevis occur nearly annually along the southwestern coast of Florida, which is notoriously known as the "Florida red tide" [68]. The low incident rate of NSP may be credited to the successful monitoring program of constantly occurring red tides. Apart from Florida, where regular red tides take place, a few large outbreaks of NSP have been reported in New Zealand and North Carolina [69].

Azaspiracid Shellfish Poisoning
Azaspiracids (AZAs) are polyether phytotoxins produced by some dinoflagellate species from the genera Azadinium and Amphidoma and the causative toxin of AZP [70]. AZA was named based on its unique spiro ring assemblies [71]. To date, over 60 AZA analogues have been identified [72]. They are different in the degrees of methylation and/or the number of hydroxyl groups and carboxyl groups that they possess. However, among the numerous analogues, only AZA1, AZA2, and AZA3 levels are monitored for regulatory purposes [33]. The symptoms of AZP are similar to those of DSP, including diarrhea, vomiting, nausea, and stomach cramps [73]. A minimum dosage ranging from 23 to 86 µg/person of AZA can have observable adverse health effects [74]. Unlike other phytotoxins, the molecular target(s) of AZAs has not been fully understood. Although the blocking of the hERG (human ether-à-go-go related gene) potassium channel by AZA1-3 was demonstrated in a recent study, relatively high concentrations of AZAs were required [75]. Therefore, there may be other molecular targets that have not been identified. The presence of toxigenic Azadinium species has been reported worldwide, including in the North Atlantic [76,77], Eastern South Atlantic [78], Mediterranean [79], Western Pacific [80], Eastern North Pacific [81], and Eastern South Pacific [82]. On the other hand, the occurrence of toxigenic Amphidoma species was only recorded in the North Atlantic [77,83,84].

Ocean Acidification
Ocean acidification (OA) is one of the major aspects of climate change and has indeed been significantly influencing the aquatic environment, where toxigenic microalgae live. Carbon dioxide (CO 2 ) emissions have increased dramatically since the Industrial Revolution, primarily due to the combustion of fossil fuels. At present, the recorded atmospheric CO 2 level is 417 ppm [85], which has increased by nearly 50% compared to the pre-industrial level. The atmospheric CO 2 concentrations will continue to rise until the end of this century unless a very stringent CO 2 emissions trajectory is satisfied (Representative Concentration Pathway 2.6) [86]. A large amount of atmospheric CO 2 traps and prevents heat from escaping the planet, driving global warming and subsequent climate change. As a natural carbon sink, the ocean has absorbed around 48% of anthropogenic CO 2 since the Industrial Revolution [87]. Given the elevated atmospheric CO 2 levels, more CO 2 has dissolved in the ocean. The dissolution of atmospheric CO 2 produces carbonic acid (H 2 CO 3 ), which dissociates to form bicarbonate ions (HCO 3 − ) and hydrogen ions (H + ). HCO 3 − further dissociates into carbonate ions (CO 3 2− ) and H + . The rise in the concentration of H + in turn has acidified the ocean, resulting in OA. OA has altered the carbonate chemistry of the aquatic system. Excess H + released from the dissociation of dissolved CO 2 creates an imbalance in the carbonate equilibrium. The equilibrium is attained by the natural buffering capacity of the ocean. The free CO 3 2− in the ocean binds with the excess H + to produce more HCO 3− . As a result, the dissolved CO 2 and HCO 3− concentrations increase while the CO 3 2− levels decrease under OA. Since the decrease in CO 3 2− levels leads to a lower saturation state of CaCO 3 , OA is particularly detrimental to marine-calcifying organisms such as molluscs, coccolithophores, and corals [88].
OA (increased pCO 2 /decreased pH) alters the carbon chemistry of the aquatic environment and acidifies the water. Microalgae have shown great adaptability towards different abiotic stress factors [89,90]. Therefore, it is believed that microalgae will respond and acclimate to OA by regulating their metabolic activities, which may in turn affect their toxicity. However, the effects of OA on microalgal toxicity and the underlying mechanisms have not been well characterized. Apart from leading to shellfish poisoning in humans, several studies have indicated the bioaccumulation of algal toxins in predators of microalgae and the biomagnification of algal toxins through food webs, which cause disease or even death among marine organisms [91][92][93][94][95]. A recent study suggested that the bioavailability of STX would increase under global warming and ocean acidification [96]. Although the bioavailability of STX and other phycotoxins in high CO 2 conditions has not been fully examined, the possibility that more toxins can be accumulated along the food chain and harm the health of marine organisms and humans should not be neglected [96]. Predicting the toxicity of microalgae under OA is therefore important to evaluate the public health concerns and ecological impacts of intensified HABs in future high atmospheric CO 2 scenarios, which will provide insights for policymakers to improve the monitoring programs for HABs. Considering the above, this paper reviewed the effects of OA on the toxicity of microalgae, and the underlying mechanisms proposed.

Effects of OA on the Toxicity of Microalgae
No studies that investigated the effects of OA on the toxicity changes in microalgae that produce okadaic acid and azaspiracids could be found in the literature. Therefore, the scope of the review was limited to microalgae that synthesize STXs, DA, and PbTx. The experimental setups and significant findings of the reviewed studies are summarized in Table 1. period of culture. b Significant outcomes refer to the significant differences in the growth rates, carbon fixation rates, and/or the toxicity observed between the corresponding control and high CO 2 treatment. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001,**** = p ≤ 0.0001. c Seawater was manipulated once by CO 2 gas bubbling before cell inoculation.

STXs-Producing Microalgae
Dinoflagellates from the genus Alexandrium are the most studied STX producers. However, there is not yet any solid conclusion drawn for the toxicity change in Alexandrium at elevated pCO 2 . Increases in cellular toxin levels were detected in A. catenella, A. minutum, and A. fundyense after high CO 2 treatment [97][98][99]. It is believed that elevated pCO 2 will permit microalgae to perform photosynthesis when more substrates are available for their unsaturated carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) [108]. Dinoflagellates may be particularly sensitive to elevated pCO 2 as they possess type II Rubisco, which has a higher affinity with O 2 , a competitive inhibitor of CO 2 [109]. Since carbon is essential for the synthesis of biomolecules, including phycotoxins, increased production of fixed carbon at elevated pCO 2 may thus contribute to the higher toxicity of microalgae [99]. However, the carbon fixation rates of A. catenella, A. minutum, and A. fundyense after high CO 2 treatment were not measured to support this hypothesis.
Another plausible explanation for the enhanced production of STXs is the increased availability of STX precursors [98]. To initiate the synthesis of STX, arginine (Arg), methionine (Met), and acetate are required [110]. In the study performed by Lian, Li, He, Chen, and Yu [98], the concentrations of Arg and Met were quantified in A. minutum at elevated pCO 2 . It was found that the content of Arg and Met increased at the beginning of the experiment and then decreased gradually. The increase in the Arg level was related to the enhanced activity of argininosuccinate synthase, an enzyme responsible for a rate-limiting step in Arg synthesis. However, the mechanisms behind the rise in the Met level remained undetermined. Since the STX content increased after the decline in Arg and Met levels with a slight delay, it was suggested that elevated pCO 2 had indirectly promoted STX production by increasing Arg and Met supplies.
On the other hand, significant decreases in STX content were detected in both A. tamarense and A. ostenfeldii at elevated pCO 2 , resulting in lower toxicity [100,101]. Van de Waal, Eberlein, John, Wohlrab, and Rost [101] isolated the RNA of A. tamarense cultured at different pCO 2 (180, 380, 800, 1200 ppm) to perform microarray-based gene expression analysis. When comparing the 800 ppm group to the 380 ppm group (control), 1238 differentially expressed genes (DEGs) were found. Genes associated with amino acid transport and metabolism were downregulated, implying that fewer Arg and Met molecules were synthesized and available for STX biosynthesis at elevated pCO 2 .
The differences in the toxicity of Alexandrium species may be caused by genetic variations between species. The gene expression patterns of the toxic A. fundyense and A. tamarense strains revealed by Taroncher-Oldenburg and Anderson [111] indicated the high interspecific variations between them. In parallel with this previous finding, A. fundyense and A. tamarense responded differently under OA. While A. fundyense promoted STX production at elevated pCO 2 [97], A. tamarense inhibited STX production by reducing the synthesis of STX precursors [101]. The Alexandrium species are likely to regulate their metabolism differently under OA due to the interspecific variations in genetic expression, thus contributing to the toxicity differences.
Regarding the STX composition of Alexandrium, increased GTX1/4 is a common change observed in A. catenella and A. tamarense, although the toxin profiles of Alexandrium are highly varied [99,101,112]. According to the gene expression profile of A. tamarense, sulfur metabolism was differentially regulated under high CO 2 treatment [101]. The significant upregulation of a putative sxtN homologue encoding a sulfotransferase involved in synthesizing sulfated STXs was found [113][114][115][116]. In contrast, genes encoding sulfatases that are responsible for the hydrolysis of sulfate esters were downregulated. Together, they might promote the transformation of non-sulfated STXs to sulfated STXs while inhibiting the transformation of sulfated STXs to non-sulfated STXs. Moreover, the downregulation of genes encoding sulfite reductase was reported. Inhibition of assimilated sulfur into amino acids might result in more sulfur being allocated for synthesizing sulfated STXs [117,118]. In parallel with these, lower non-sulfated STX content but higher sulfated GTX1/4 and C1/2 content of A. tamarense at elevated pCO 2 were observed. Nevertheless, increases in both non-sulfated and sulfated STX levels were exhibited in A. catenella after high CO 2 treatment [99], suggesting that the toxin composition could be altered by different metabolic pathways besides sulfur metabolism.

DA-Producing Microalgae
The diatom Pseudo-nitzschia has been extensively studied compared to other DA producers. Generally, both laboratory and mesocosm studies revealed increased cellular DA content of Pseudo-nitzschia at elevated pCO 2 [102][103][104][105]. In line with the assumption that excess fixed carbon may be shunted for toxin biosynthesis in microalgae under OA [99], an increased growth rate, carbon fixation rate, and cellular DA content were reported in P. fraudulenta at elevated pCO 2 [103]. Nonetheless, there were no consistent findings of P. australis [104]. The cellular DA content of P. australis was largely unchanged despite the increased carbon fixation rate at elevated pCO 2 .
For DA-producing Pseudo-nitzchia, environmental pH may also play a role in toxin biosynthesis by affecting cellular enzymatic activities, metal speciation, and the composition of symbiotic bacteria [119]. The intracellular pH of diatoms may be modified or maintained by regulating the metabolism under acidification [120]. Although research on the intracellular pH change of Pseudo-nitzchia at elevated pCO 2 is lacking, the effects of acidification on DA biosynthesis should not be neglected. Lundholm, Hansen, and Kotaki [119] proposed that there might be an optimal pH for DA biosynthesis; however, it remains to be determined as an increased cellular DA level was detected under both low and high pH conditions in Pseudo-nitzschia [102,103,119,121]. The DA biosynthetic pathway has been modeled recently based on the transcriptome sequencing of P. multiseries [122]. The intracellular pH of Pseudo-nitzschia may be modified under acidification, which possibly affects the activities of putative DA biosynthetic (Dab) enzymes including terpene cyclase (dabA), hypothetical protein (dabB), alpha-ketoglutarate-dependent dioxygenase (dabC), and CYP450 (dabD), causing changes in cellular DA content.
The toxicity and availability of metals are likely to be affected by a decreased pH. For example, a rise in the concentrations of free copper (Cu 2+ ) and dissolved iron (Fe 3+ ) was expected at lower pH levels due to increased solubility [123]. While the Cu 2+ level is generally closely related to its toxicity in phytoplankton [124], Fe 3+ is one of the essential micronutrients for microalgal growth. Although the ecological and physiological roles of DA have not been well established, it was suggested that DA might be a trace metal chelator involved in iron acquisition and copper detoxification, given its similar structure to phytosiderophores [125]. Therefore, increases in Cu 2+ and Fe 3+ levels under an acidic environment may stimulate the DA production of Pseudo-nitzschia. However, both the cellular and dissolved DA content of P. multiseries remained largely unchanged after exposure to different copper levels [126]. On the other hand, a positive relationship between the iron concentration and total DA content in P. multiseries was indicated [127]. Thus, enhanced DA production at elevated pCO 2 may be mainly contributed by the increased concentration of Fe 3+ rather than Cu 2+ under acidification.
Symbiotic bacteria are related to DA biosynthesis in Pseudo-nitzschia. Xenic cultures of P. multiseries exhibited increased DA content when compared with axenic cultures [128]. While free-living bacteria were likely to be incapable of producing DA autonomously [129,130], it is difficult to obtain a conclusion for epiphytic bacteria due to the difficulties in isolating them from the diatoms. Research on the bacteria-phytoplankton interaction mechanisms for DA biosynthesis in Pseudo-nitzschia is lacking. However, it was proposed that the attached bacteria might exchange metabolites that are used for DA biosynthesis with Pseudo-nitzschia [130]. Since bacterial abundance and diversity were shown to be affected by a decreased environmental pH [131], symbiotic bacteria that promote DA biosynthesis may become more abundant at lower pH levels given the observation of increased cellular DA content in Pseudo-nitzschia.

PbTx-Producing Microalgae
Dinoflagellate K. brevis is the primary producer of PbTx and its response to OA has been studied. Elevated pCO 2 had no significant effect on the cellular PbTx content of K. brevis [106,107]. Although K. brevis shifted its inorganic carbon preference from HCO 3 − to CO 2 and the half-saturation constant (K 1/2 ) for CO 2 increased under high CO 2 treatment, the growth rate, carbon fixation rate, and cellular carbon composition remained largely unchanged [106]. The relative insensitivity of K. brevis to elevated pCO 2 was surprising as high CO 2 availability had been thought to be especially beneficial to dinoflagellates that possess inefficient type II Rubisco [109].

OA May Affect the Central Carbon Metabolism of Toxic Microalgae
Based on the above, OA can affect the toxicity of some species of dinoflagellate Alexandrium and diatom Pseudo-nitzschia. On the other hand, dinoflagellate K. brevis was shown to be somewhat more resistant to OA and its toxicity was largely unchanged. The way in which they regulate central carbon metabolism under OA is likely to contribute to the changes in toxicity or to help to achieve homeostasis. OA was shown to be beneficial to P. fraudulenta as it promoted the carbon fixation rate by increasing inorganic carbon availability [103]. The increased growth rate and cellular toxin content gave rise to the hypothesis that excess carbon not used for growth might be shunted for toxin biosynthesis [99]. However, the positive relationship between the carbon fixation rate and toxin level does not always apply, as observed in P. multiseries [104]. All biomolecules are carbonbased, so the excess fixed carbon is not necessarily transported for toxin production. The detailed routes of carbon reallocation for toxin biosynthesis have not been elucidated either. Therefore, investigating the central carbon metabolism of Alexandrium and Pseudo-nitzschia is important to validate the hypothesis. For K. brevis, the acquisition of inorganic carbon was shown to be affected by OA despite the nearly unchanged growth rate, carbon fixation rate, and toxin content [106]. Since central carbon metabolism is highly conserved across phylogeny [132], comparing the central carbon metabolism of relatively resistant K. brevis to that of other sensitive microalgae at elevated pCO 2 may help to explain the unique response of K. brevis to OA. A decreased pH was proposed to affect the toxicity of Pseudo-nitzschia, though the exact mechanisms had not been demonstrated [119]. Increased inorganic carbon availability and decreased pH may have combined effects on the regulation of central carbon metabolism in microalgae, hence affecting toxin biosynthesis. Considering the above, studying the carbon fluxes in Alexandrium, Pseudo-nitzschia, and K. brevis is essential to deepen our understanding of the effects of OA on microalgae.

Future Research Directions
Manipulating seawater to a range of increased pCO 2 levels for OA experiments is not a straightforward task due to the interdependency of ocean carbon system components [133]. To predict the microalgal responses under OA accurately, the seawater used for microalgae cultivation should resemble the ocean carbon chemistry in future high CO 2 scenarios as closely as possible. Multiple seawater manipulation methods with distinct advantages and disadvantages have been applied to OA experiments. Most of the reviewed studies adopted CO 2 gas bubbling to model OA (Table 1). CO 2 gas bubbling is an efficient seawater manipulation method that exactly mimics the carbon chemistry of the ocean in future high atmospheric CO 2 scenarios (increased DIC without altering A T ) [134]. In addition, it is easy to implement and it can maintain the initial conditions in the long term [135]. Thus, CO 2 gas bubbling was adopted by most of the studies to investigate the effects of OA on microalgae. However, the turbulence induced by bubbling may affect the growth of phytoplankton to a different extent, especially Alexandrium spp. [136]. Hence, the effects of turbulence became an uncontrolled confounding variable, which increased the difficulties in generating reproducible results for the microalgal adaptive response to OA [137]. To minimize the effects of turbulence, a dialysis bag with a 3 kDa molecular weight cut-off should be used to enclose the microalgae, so as to reduce the mechanical disturbance introduced by the aeration [135]. Unfortunately, none of the reviewed studies mentioned the uses of dialysis bags in their methodology. Besides CO 2 gas bubbling, mixing CO 2 -enriched water prior to cell inoculation, and the combined addition of HCl and Na 2 CO 3 /NaHCO 3 , are other seawater manipulation methods that also closely resemble the carbon chemistry of the ocean in future high atmospheric CO 2 conditions [134,135]. These methods can be effective and reliable alternatives to CO 2 gas bubbling without the mechanical disturbance to microalgae. Thus, they are especially useful for studying the effects of OA on microalgae with high sensitivity to turbulence. Regardless of the method used, it is noteworthy that the biological processes of microalgae, such as respiration and photosynthesis, will affect the carbon chemistry of seawater, especially when the biomass is high [138,139]. Thus, it would be preferable to monitor at least two carbon chemistry parameters of pH, A T , DIC, and pCO 2 , in addition to temperature and salinity, throughout the experiments [140].
With appropriate methods to model OA and monitor the carbon chemistry of the seawater, metabolomics is a useful technique to investigate the metabolic changes of the microalgae in response to OA. This will help to reveal the biological regulation of toxin biosynthesis in microalgae under OA. Metabolomics is the investigation of a complete set of small molecules with molecular weights lower than 1500 Da, also known as the metabolome, of biological samples [141]. It has been increasingly applied in algal toxin research with technological advancements [142,143]. Metabolic flux analysis (MFA) is an effective metabolomic tool that examines the turnover rate of metabolites by using stable isotope tracers such as 13 C, 2 H, and 15 N [144]. With the help of nuclear magnetic resonance (NMR) or mass spectrometry (MS), the isotope labeling patterns of intracellular metabolites are determined, which helps to construct a flux map, including reverse fluxes [145]. Compared to the measurement of metabolite levels, analyzing metabolic fluxes is more informative as it reveals the production and consumption rates of metabolites, which illustrate the biochemical events behind the changes in metabolite levels [146]. Based on the reviewed studies, it is hypothesized that the central carbon metabolism of toxic microalgae may be regulated differently under OA. The altered central carbon metabolism may then affect the toxicity of microalgae or relieve the stress brought by OA. Therefore, further studies on the central carbon metabolism of toxic microalgae are warranted. MFA has been applied for the investigation of photosynthesis and central carbon metabolism in microalgae, C 3 , and C 4 plants recently in 13 CO 2 pulse labeling, and the distinct carbon flux pattern of microalgae has been successfully identified [147]. In this light, carbon allocation in toxic microalgae can be investigated to reveal the metabolic pathways related to toxin biosynthesis and their regulation under OA by MFA in order to elucidate the underlying mechanisms of toxicity changes. When conducting MFA to study the effects of OA on the carbon allocations of microalgae, it is critical to note that CO 2 gas bubbling without the continuous control of the pH would alter the pCO 2 , DIC, and pH of the seawater at the same time. As a result, it is difficult to determine which confounding variable causes the observed effects. Although CO 2 gas bubbling successfully mimics the carbon chemistry of seawater under OA observed in reality, this method fails to decouple different variables in the carbon chemistry to study the individual effects of the variables on the carbon allocations of microalgae. Therefore, other methods or systems that can decouple different variables of the carbon chemistry of seawater under OA should also be adopted to investigate in detail the biological mechanisms of the changes in carbon allocation of microalgae under OA when using the metabolomic approach (Figure 1) [148]. Knowledge of the relationship between environmental drivers and HABs is of fundamental importance to optimize the current mitigation of HABs [149]. Under climate change, it is undoubted that the HAB dynamic has been altered [1]. However, the physiological responses of toxigenic microalgae in changed climate conditions have not been well documented. A precise linkage between environmental factors and microalgal toxicity has not been confirmed due to the diverse responses displayed by microalgae belonging to the same genus or even the same species, as in the case of the STX-producing dinoflagellate Alexandrium [97][98][99][100][101]150]. MFA of the central carbon metabolism of microalgae can reveal the metabolic pathways that are responsible for the toxicity changes under OA. The identification of differentially regulated pathways will facilitate the discovery of universal biomarkers for potentially more toxic microalgae in future elevated atmospheric CO2 scenarios. Given the diverse responses of microalgae to different environmental conditions, detecting universal biomarkers is a relatively efficient way to identify microalgae with increased toxicity under climate change. Monitoring and forecasting these microalgae can be prioritized to help establish early warning systems for the coastal tourism and aquaculture sectors, to minimize the economic losses caused by HABs [151][152][153]. Policymakers can guide the closure of beaches, fish farms, and shellfish-harvesting areas in an appropriate timeframe according to the early warning system, to protect public Knowledge of the relationship between environmental drivers and HABs is of fundamental importance to optimize the current mitigation of HABs [149]. Under climate change, it is undoubted that the HAB dynamic has been altered [1]. However, the physiological responses of toxigenic microalgae in changed climate conditions have not been well documented. A precise linkage between environmental factors and microalgal toxicity has not been confirmed due to the diverse responses displayed by microalgae belonging to the same genus or even the same species, as in the case of the STX-producing dinoflagellate Alexandrium [97][98][99][100][101]150]. MFA of the central carbon metabolism of microalgae can reveal the metabolic pathways that are responsible for the toxicity changes under OA. The identification of differentially regulated pathways will facilitate the discovery of universal biomarkers for potentially more toxic microalgae in future elevated atmospheric CO 2 scenarios. Given the diverse responses of microalgae to different environmental conditions, detecting universal biomarkers is a relatively efficient way to identify microalgae with increased toxicity under climate change. Monitoring and forecasting these microalgae can be prioritized to help establish early warning systems for the coastal tourism and aquaculture sectors, to minimize the economic losses caused by HABs [151][152][153]. Policymakers can guide the closure of beaches, fish farms, and shellfish-harvesting areas in an appropriate timeframe according to the early warning system, to protect public health while minimizing economic losses. Moreover, more research efforts can be implemented to understand the interactions between the potentially more toxic microalgae and other organisms in the food web under climate pressure. The findings can be used to assess the ecological risk of HABs in the future, which will aid policymakers to achieve a balance between environmental protection and economic development.