Chemical Diversity, Origin, and Analysis of Phycotoxins - American

Review pubs.acs.org/jnp

Chemical Diversity, Origin, and Analysis of Phycotoxins Silas Anselm Rasmussen,† Aaron John Christian Andersen,† Nikolaj Gedsted Andersen,§ Kristian Fog Nielsen,† Per Juel Hansen,§ and Thomas Ostenfeld Larsen*,† †

Department of Systems Biology, Technical University of Denmark, Søltofts Plads 221, Kongens Lyngby, Denmark Marine Biological Section, Department of Biology, Copenhagen University, Strandpromenaden 5, Helsingør, Denmark

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ABSTRACT: Microalgae, particularly those from the lineage Dinoflagellata, are very well-known for their ability to produce phycotoxins that may accumulate in the marine food chain and eventually cause poisoning in humans. This includes toxins accumulating in shellfish, such as saxitoxin, okadaic acid, yessotoxins, azaspiracids, brevetoxins, and pinnatoxins. Other toxins, such as ciguatoxins and maitotoxins, accumulate in fish, where, as is the case for the latter compounds, they can be metabolized to even more toxic metabolites. On the other hand, much less is known about the chemical nature of compounds that are toxic to fish, the so-called ichthyotoxins. Despite numerous reports of algal blooms causing massive fish kills worldwide, only a few types of compounds, such as the karlotoxins, have been proven to be true ichthyotoxins. This review will highlight marine microalgae as the source of some of the most complex natural compounds known to mankind, with chemical structures that show no resemblance to what has been characterized from plants, fungi, or bacteria. In addition, it will summarize algal species known to be related to fish-killing blooms, but from which ichthyotoxins are yet to be characterized.

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INTRODUCTION Microalgae, such as dinoflagellates, constitute an important part of marine ecosystems, where they live in fierce competition with other marine prokaryotes and eukaryotes. As part of their survival strategies they often produce bioactive natural products that play important roles in marine chemical ecology.1,2 This includes signaling molecules, allowing different organisms to sense each other, as has recently been demonstrated for predator−prey interactions between microalgae and copepods,3 where the copepod lipid, copepodamide A, was shown elegantly to induce the production of saxitoxin in Alexandrium minutum. Given the right growth conditions, many microalgae can grow to extremely high cell densities, eventually turning into harmful algal blooms (HAB) with potentially severe impacts on human health, tourism, wild fish stocks, and aquaculture. Some microalgal compounds (phycotoxins) have been shown to be very toxic to humans, which are likely secondary effects considering that microalgae and humans have not interfered through evolution. The most studied phycotoxins are by far the compounds that accumulate in shellfish, for example, saxitoxin, which is one of the toxins responsible for paralytic shellfish poisoning (PSP). Mass occurrences of certain algal species also regularly lead to fish mortality as well as cause mortality to other aquatic organisms, i.e., bacteria, other algae, and protozoan and metazoan grazers. While the metazoan grazers, and sometimes also protozoan grazers, are likely to be affected by ingesting the toxic algae, the effect on fish, bacteria, and algae is suspected to be caused by phycotoxins that have been transferred by direct © XXXX American Chemical Society and American Society of Pharmacognosy

contact with the cell or upon lysis of the algal cell, whereby the phycotoxin is released to the surrounding water. Different types of toxic compounds have been suggested as the causative toxins, but in most cases the toxins have not been properly tested on live juvenile or adult fish, often due to lack of sufficient amounts of the toxins, and thus proper dose− response curves are lacking. In reality, this means that many of the suggested ichthyotoxins may not really be the toxins that kill the fish. At the end of this review, five examples of algal species will be given that are well-known to be implicated in fish kills during algal blooms, but need to be further investigated in order to clarify the chemical nature of the related phycotoxins. First, some of the most famous microalgal phycotoxins will be reviewed, with particular focus on their structural diversity, including both toxins that are related to human health and fish-killing toxins. The compounds have been divided into five major structural groups following the groupings recently presented by Anderson, Cembella, and Hallegraeff:4 (a) tetrahydropurines, (b) secondary amines, (c) macrocyclic imines, (d) linear and macrocyclic polyethers, and (e) ladder-frame polyethers. Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: December 2, 2015

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DOI: 10.1021/acs.jnatprod.5b01066 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Secondary Amines. Several species within the diatom genus Pseudonitzschia produce a kainoid analogue named domoic acid (24).24 This is a small secondary amine containing three carboxylic acids that accumulates in shellfish feeding on these diatoms and, due to its neurotoxicity, results in amnesic shellfish poisoning upon consumption of contaminated seafood. Domoic acid also causes problems in marine mammals, causing death of, for example, the Minke whale,25 while no effect of domoic acid is observed on fish.26 Azaspiracids (25−28) are a group of toxins incorporating a secondary amine in a six-membered ring, a carboxylic acid, and three spiro-assemblies into their structures (Chart 2). The first

MICROALGAL PHYCOTOXINS Tetrahydropurines. The tetrahydropurine group of metabolites are perhaps the most well studied of all algal toxins due to their potent toxicity toward humans.5 The first of the tetrahydropurine group of toxins to be discovered was saxitoxin (1), and this together with its many analogues (2−23) (Chart 1) are the principal metabolites responsible for PSP, a Chart 1. Structurally Elucidated Saxitoxin Analogues Isolated from Microalgaea

Chart 2. Structures of Secondary Amine-Containing Compounds

a

Abbreviations: STX, saxitoxin; GTX, gonyautoxin; dc, decarbomoylated; do, deoxydecarbomoylated.

condition that is induced by the potent neurotoxicity of these compounds as sodium ion channel blockers.6 Due to their adverse effects, these metabolites are known collectively as paralytic shellfish toxins (PST). The tetrahydropurine group has been associated predominantly with a number of dinoflagellate species, specifically from the genera Alexandrium, Gymnodinium, and Pyrodinium.7−11 It has also been shown that cyanobacteria,12 as well as bacteria cultivated from toxic dinoflagellates,13 can produce these toxins. Due to the ability of these metabolites to accumulate in higher marine organisms, PST have been isolated from various organisms, such as the puffer fish Takif ugu poecilonotus and T. vermicularis,14 marine snails such as Turbo marmoratus and T. argyrostoma,15 and various shellfish including saxitoxin (1) from the butter clam, Saxidomus gigantean.16 Since saxitoxin (1) was first structurally elucidated17−19 more than 50 derivatives, including analogues arising from metabolic conversion within higher organisms, have been identified.20 Although these metabolites are potent neurotoxins, and many species that produce these toxins have been associated with large fish kill events during HAB, the component or mechanism responsible for fish mortality is yet to be identified. It has been demonstrated that PST composition and concentration do not correlate with allelopathy,21,22 which may suggest that the metabolites or mechanism responsible for ichthyotoxicity is yet to be discovered. A recent review specifically detailing saxitoxin-related metabolites and their properties has been prepared by Cusick and Sayler.23

azaspiracid 1 (25) was isolated by Satake et al.27 from Irish mussels, the structure of which was later revised by Nicolauo et al.28 The exact origin of the toxin was, however, unknown for some time. Finally, in 2009, the azaspiracids were shown to be produced by the dinoflagellate Azadinium spinosum.26 Several species of Azadinium have now been shown to produce these toxins,30,31 and a number of congeners have been identified differing in the methylation and hydroxylation pattern; however, the spiro-polyether backbone remains the same for all congeners32 (Chart 2). The mode-of-action of azaspiracids is currently unknown.32 Macrocyclic Imines. The macrocyclic imine group consists of four classes of toxins, each having a macrocyclic scaffold containing an imine functional group (Chart 3). They are all fast-acting neurotoxins when injected into mice; however, no toxicity toward humans due to these compounds has been reported.33 The first isolated algal imine, prorocentrolide (29), was described by Torigoe et al.34 from the dinoflagellate Prorocentrum lima. Another imine-containing compound, gymnodimine (30), was isolated from Gymnodinium sp. and shown to be toxic in a mouse assay (ip LD50 450 μg kg−1).35 The absolute stereochemistry of gymnodimine was later reported by Munro and Blunt using X-ray crystallography.36 B



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Chart 3. Structures of Macrocyclic Imines

Chart 4. Structures of Linear and Macrocyclic Polyethers

About the same time, and independently, the two novel compounds pinnatoxin (31)37 and spirolides (32, 33)38 were both isolated from shellfish. Both compounds incorporate a seven-membered cyclic imine together with three spiro-fused cyclic ethers. The main difference between pinnatoxin and spirolides is the incorporation of an additional fused bicyclic ether into pinnatoxin. Spirolides are now known to be produced by the dinoflagellate A. ostenfeldii 39 and A. peruvianum,40 while pinnatoxins have been found to be produced by Vulcanodinium rugosum.41 The spirolides and gymnodimines have been found to bind to muscarinic acetylcholine receptors (mAChRs). However, recent work suggests that the cause of toxicity arises from antagonist interaction with nicotinic acetylcholine receptors (nAChRs).42 Recently, Bourne et al.43 showed by X-ray crystallography that pinnatoxins A and G bind strongly to muscle-type α12βγδ nAChRs. Linear and Macrocyclic Polyethers. This group consists of some of the largest carbon−carbon backbone toxins known, and unlike the ladder-frame polyethers (see below), they have little or no fused cyclic ethers (Chart 4 and Chart 5). Palytoxin (34), the second largest nonpeptide phycotoxin next to maitoxin (55) (vide infra), was isolated by Moore and Scheuer44 and later characterized through extensive degradation studies.45 Although palytoxin (34) was not isolated from a marine algal species, a 3,26-bisdesmethyldeoxypalytoxin has been isolated from the benthic alga Ostreopsis siamensis and named ostreocin D.46 Ciminiello et al.47 showed recently that the Mediterranean dinoflagellate Ostreopsis cf. ovata produces a palytoxin congener named ovatoxin-a (35) together with a smaller amount of palytoxin. The carbon−carbon backbone was identical, but it showed four additional hydroxy groups, at C-14, C-44, C-46, and C-64. Upon isolation of ovatoxin-a (35), mouse lethality was assessed, with the amount needed for mouse mortality determined to be less than 7.0 μg kg−1.47 Another superchain molecule is amphidinol 1, a compound that was originally isolated and characterized from the symbiotic dinoflagellate Amphidinium klebsii by Satake et al.48

Amphidinol 1 was found to exhibit both hemolytic and antifungal activity. Later, the absolute configuration of the congener amphidinol-3 (36) was reported by Murata et al.,49 using a J-based configurational method combined with degradation studies. Several congeners have later been isolated, namely, for example, amphidinols 2−18, carteraol-E, 50 lingshuiols A−C,51 karatungiols A and B,52 symbiopolyol,53 and luteophanols A−D,54 which were all isolated from Amphidinium spp. All congeners share a common molecular motif, consisting of two tetrahydropyran rings, linked together by an acyclic C6 chain with an exomethylene group, and two side chains, one polar and the other apolar. The differences between the congeners are in the length and substitutions of the side chains. The apolar side chain seems to be the most conserved, being either C16, C17, or C18. In addition, all amphidinols contain a triene motif in this side chain. The polar side arm shows more diversity, spanning from C23 to C37, and in the cases of amphidinol 2, amphidinol 11, and carteraol E, a third tetrahydropyran ring is incorporated. Carteraol E was also found to be ichthyotoxic (LD50 0.28 μM, 392 μg/L), but the authors did not state the experimental conditions.50 The amphidinols have also been found to exhibit anti-diatom properties with a minimal effective concentration between 0.1 and 1 mg/L.55 The mode of action was found to be the disruption of biomembranes by the apolar side chain penetrating the lipid bilayer.56,57 Interestingly, a structurally similar molecule, karlotoxin 2 (37), was isolated from the ichthyotoxic dinoflagellate Karlodinium venef icum, and this was the first report that a genus other than Amphidinium can produce this C



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Chart 5. Algal Toxins Belonging to the Linear Superchain Polyethers

class of compounds. However, unlike the amphidinols, karlotoxin 2 has been shown to exhibit ichthyotoxicity toward fish larvae and has been found during a HAB, making karlotoxin 2 the only toxin that has been directly linked to fish kills.58 Karlotoxins also introduced chlorination and sulfate to this group of compounds.59 To date, 15 karlotoxin congeners have been described, and these differ mostly in the polar side chain.59,60 In structure−activity relationship investigations, the length of the apolar arm has been shown to correlate with increased toxicity;60 it has also been demonstrated that hemolytic activity is significantly augmented with the incorporation of sulfonation.61 Ichthyotoxicity of karlotoxins is via pore-forming disruption of membranes containing cholesterol, resulting in swelling and ultimately osmotic lysis of target cells. Observations of the impact of karlotoxins on marine fish include bass,62 cod,63 and sheepshead minnow juveniles.64 Studies have indicated that karlotoxins can kill fish at ecological relevant concentrations. The LC50 value was around 0.5 μg mL−1 for zebrafish larvae and juveniles as well sheepshead minnow larvae.64,65

Yet another phycotoxin belonging to this group is okadaic acid (38), which was originally isolated from a black marine sponge, Halichondria okadoi, by Tachibana et al.,66 but later found to be produced by the dinoflagellates Prorocentrum lima67 and Dinophysis spp.68 Okadaic acid (38) and its analogues dinophysistoxins (39, 40) consist of spiro-fused tetrahydrofuran rings and a spiro tetrahydrofuran-dioxadecalin unit as well as a terminal carboxylic acid. These toxins cause diarrheic shellfish poisoning (DSP) upon human ingestion of contaminated shellfish. The mode of action of okadaic acid and denophysistoxin has been found to be inhibition of protein phosphatase activity.69 Pectenotoxin 1 (41) was isolated from Japanese scallops Patinopecten yessoensis by Yasumoto et al.,70 originally believed to cause diarrhetic shellfish poisoning. The algal species Dinophysis fortii was linked to the production of pectenotoxin,70 and several other pectenotoxins (42, 43) have been detected in Dinophysis spp.71 Pectenotoxins have been shown to be hepatotoxic to mice. Goniodomin A (44), a macrolide with one spiro assembly, was isolated from the dinoflagellate Alexandrium pseudogonyaulax by Murakami et al.,72 as it showed antifungal activity. Its D



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Chart 6. Structures of the Group of Ladder-Frame Polyethers

absolute configuration was later assigned73 and is believed to be structurally related to goniodomin,72 an antibiotic substance isolated from Alexandrium sp. 20 years previously.74 Goniodomin A has been found to affect the cytoskeletal reorganization through the conformational change of actin.75 Two other Alexandrium species, A. hiranoi and A. monilatum, have been shown to produce goniodomin A.76,77 Ladder-Frame Polyethers. This group of compounds contains some of the largest nonpeptide biotoxins that have been isolated, and their structures are highly complex (Chart 6 and Chart 7). The first toxin that was isolated and characterized was brevetoxin 2 (48)78 from the red-tide-forming dinoflagellate Karenia brevis. Brevetoxins (brevetoxin 1−10) can be divided into a type A and type B backbone. The type A (45− 47) brevetoxins consist of 10 fused cyclic ethers with five- to nine-membered rings,79 whereas type B brevetoxins (48−50) consist of 11 fused cyclic ethers ranging in size from six- to eight-membered rings.78,79 The mode of action of brevetoxins is the blocking of the voltage-sensitive sodium channels (VSSC), explaining the behavior of these compounds as potent neurotoxins.79 Some structurally similar compounds are called

ciguatoxins (51, 52), which are also lipophilic, and have been shown to interact with the same VSSC as brevetoxins, also rendering them highly potent neurotoxins. These compounds are found in contaminated fish upon bioaccumulation and cause ciguatera fish poisoning.80,81 Ciguatioxin-1 originally isolated from a morey eel (51) has been detected in the algae Gambierdiscus polysiensis,82 while ciguatoxin-4b (52) was isolated from Gambierdiscus toxicus.83 A structurally related compound to brevetoxins and ciguatoxins is yessotoxin (53), a toxin originally isolated and characterized from the scallop Patinopectin yessoensis by Murata et al.84 followed by determination of its absolute configuration by Takahashi et al.85 Yessotoxin was initially believed to cause DSP; however, no protein phosphatase inhibition, as is the case of okadaic acid, has been found.86 Yessotoxin is lethal to mice when administered ip, but not by oral exposure. The exact mode of action is not fully understood, although some studies suggest that it targets cardiac muscle cells.87 Yessotoxins have been found to be produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera. E



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Chart 7. Continued Structures of the Supersized Ladder-like Polyethers

neuroplasma cells as observed for ciguatoxin and brevetoxin,93 and their mode of action is still not known. Members of another subgroup of these ladder-frame polyethers are called supersized molecules and include maitotoxin (57), the prymnesins (58, 59), and brevisulcenalF (56) (Chart 7). Very recently, a novel compound, brevisulcenal-F (MW = 2076 Da) (59), was isolated in a Japanese−New Zealand joint venture from the dinoflagellate Karenia brevisulcata.94,95 Brevisulcenal-F (56) (MW = 2054 Da) was isolated and elucidated structurally based on a fish kill event in Auckland, New Zealand. Brevisulcenal-F exhibits the longest unbroken ladder of 17 fused polyether rings. It also contains a 2-methyl-2-butanal side chain as observed in gymnocins A and B. Brecisulcenal-F exhibits potent mouse

Gymnocins A (54) and B (55) were isolated by Satake et al.,88,89 in an attempt to isolate the ichthyotoxic principle from the dinoflagellate Karenia mikimotoi. Their absolute configurations were later assigned,90 and both compounds contain a 2-methyl-2-butanal side chain but differ in having 14 and 15 successive polyether rings, respectively. Gymnocins A and B exhibit cytotoxic activity in a P338 murine leukemia cell assay with IC50 values of 1.3 and 1.7 μM,89,91 respectively, and an SAR study has shown that an α,β-unsaturated aldehyde moiety is of high importance for the resultant in vitro activity.92 Interestingly, when a mixture of gymnocins A and B was tested toward the freshwater fish Tanichthys albonubes, they showed very little activity (250-fold decrease compared to brevetoxin).88 The gymnocins do not induce a Ca2+ influx in F


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Alexandrium (=Gonyaulax) tamarense (dinoflagellate) Karenia mikimotoi (dinoflagellate)

a

G

karlotoxins (Deeds et al. 2006,64 Van Wagoner et al. 2008,58 Peng et al. 2010165)

verified ichthyotoxin(s)

suggested ichthyotoxin(s) GAT toxins (Henrikson et al. 2010)110 prymnesins 1 and 2 (Igarashi et al. 1996)98,145 hemolysin 1 (Kozakai et al. 1982)109 various fatty acids (Bertin et al. 2012)111 reactive oxygen species (ROS) (Marshall et al. 2002) brevetoxins (Kahn et al. 1995, 1996154) free fatty acids (FFA) (Marshall et al. 2003, Dorentes-Aranda et al. 2015124) high molecular weight compound suggested as lytic compound (Ma et al. 2010140) free fatty acids (Parris et al. 1994, 1998,157,158 Fossat et al. 1999159) gymnodimine (Seki et al. 199635) gymnocin A (Satake et al. 200288) gymnocin B (Satake et al. 200589) NA

Tangen 1977,143 Ottway 1979,161 Cross and Southgate 1980,162 Dahl et al. 1982,163 Roberts et al. 1983, Takayama and Adachi 1986164

Seki et al. 1996,35 Blasco 1996160

Deeds et al. 2002,128 Kempton et al. 2002,128 Fensin 2004166

Mortensen 1985,137 Cembella et al. 2002156

Ogata and Kodama 1986155

Deeds et al. 2006,64 Mooney et al. 201065

Subrahmanyan 1954,148 Tseng et al. 1993,149 Tomas 1998,150 Tiffany et al. 2001,151 Jugnu and Kripa 2009,152 Tomas 1998,150 Lewitus et al. 2008153

Lee et al. 2003,146 Shen et al. 2011147

ichthyotoxic bloom(s) Otterstroem and Nielsen 1939,108 James and Delacruz 1989,107 Kaartvedt et al. 1991106

laboratory trial(s) Otterstroem and Nielsen 1939,108 Blossom et al. 2014112

Toxins are verified as ichthyotoxins only if proper dose−response relationships have been presented, indicating that the toxin is toxic at ecological relevant concentrations. NA = not applicable.

Karlodinium venef icum (dinoflagellate)

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Chattonella marina (raphidophyte)

none

none

Prymnesium parvum (haptophyte)

ichthyotoxic algal species

Table 1. Five Selected Species of Marine Microalgae Which Have Been Associated with Fish Kills in Nature, the Suggested, Verified Ichthyotoxins, and References to Laboratory Trials and Natural Ichthyotoxic Bloomsa

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Chattonella marina. This species is one out of five species of the raphidophyte genus Chattonella, which all are known for their implication in fish kills in Nature and in aquaculture. Chattonella species can be found worldwide in tropical, subtropical, and temperate regions, but fatal blooms are most abundant in subtropical and tropical coastal waters.114 The fishkilling mechanisms are still unclear, but suffocation is always the ultimate cause of fish mortality. Physical clogging of gills by Chattonella cells and mucus excretion has earlier been proposed as the cause of fish kills.115 Gill damage due to polyunsaturated fatty acids as hemolytic substances has also been suspected as a cause of fish mortality.116,117 Since some of these species produce brevetoxins (neurotoxins), they have also been suggested as the compounds responsible for the fish mortality.118−120 However, recent evidence points toward reactive oxygen species (ROS, e.g., superoxides) as the responsible agents for the gill tissue injury and mucus production that leads to the death of fish.121−123 A recent study by Dorantes-Aranda et al.124investigated the toxicity of several harmful algal species using a rainbow trout gill cell-based assay. This showed that ROS and polyunsaturated fatty acids (PUFA) did not play a significant role in the toxicity of many of the different harmful species. Only in the case of Chatonella marina could some of its toxicity be attributed the role of ROS and PUFAs. Karlodinium venef icum. This is one out of at least 11 species of the dinoflagellate genus Karlodinium. Previously, K. venef icum has been called Gyrodinium estuariale, Gymnodinium galatheanum, Gymnodinium venef icum, and K. micrum, all of which are now synonymous with K. venef icum.125 K. venef icum is a cosmopolitan species,126,127 and it has been responsible for numerous fish kills in temperate estuaries;59,128−131 karlotoxins have been linked to the observed ichthyotoxicity. Karlodinium armiger and Karlodinium australe are the only other species besides K. venef icum to have been implicated in fish kills in Nature.59,132 The toxins of K. armiger are unknown at present. Alexandrium tamarense. This is one of >30 marine species of the dinoflagellate genus Alexandrium, which has a cosmopolitan distribution.133 The genus is mostly known for the production of saxitoxin (1) and its many derivatives.133 Some species also produce goniodomin A (44) and spirolides (32, 33). In addition, A. tamarense and other Alexandrium species occasionally cause fish kills in coastal areas,134−137 and the compounds responsible for these are largely unexplored. Lytic compounds, which cause cell lysis in other algae and blood cells, have been reported, and they seem to be unrelated to the saxitoxins.138,139 Preliminary characterization of the lytic toxins from A. tamarense140 has indicated that these compounds are macromolecular or large aggregates (>5 kDa) and also that they are neither proteinaceous nor primarily polysaccharide derived. Whether or not these compounds are associated with fish kills is presently unknown. Karenia mikimotoi. This species (formerly also known as Gyrodinium/Gymnodinium aureolum or Gymnodinium nagasakiense) is one out of a total of at present 12 species of this dinoflagellate genus. At least 10 of these species have been implicated in fish kills in Nature or in aquaculture.141 K. mikimotoi is a cosmopolitan species commonly found in temperate to tropical neritic waters. Blooms have been reported from Australia, Denmark, Ireland, Japan, Korea, Norway, and Scotland.142,143 These blooms have resulted in devastating marine life mortalities, yet the toxin mechanism and chemical principles responsible are poorly understood. No reported human illnesses have resulted from consumption of fish or

lethality and P338 cytotoxicity; however, data on fish toxicity are still lacking. Maitotoxin (56) (MW = 3426 Da) is the largest nonpeptide biotoxin discovered so far. The complex structure of maitotoxin was elucidated by the Yasumoto group by extensive chemical degradation and NMR spectroscopy-based investigations.96 Maitotoxin is produced by the dinoflagellate Gambierdiscus toxicus and is believed to modulate the calcium influx in cells. The toxin also accumulates in fish, causing ciguatera fish poisoning. The last group of supersized toxins are prymnesin-1 (58) (MW = 2261 Da) and prymnesin-2 (59) (MW = 1967 Da), which have been isolated from the haptophyte Prymnesium parvum. Interestingly, at the time of discovery this was the first report of a polyether compound that has not been isolated from a dinoflagellate; later another study suggested that brevetoxins accumulate in the raphidophyte Chatonella marina.97 Prymnesins were isolated and elucidated structurally by Igarashi et al. (1996).98 These compounds are the first glycosylated algal natural products as well as the first conjugated with alkyne bonds. The relative and absolute configurations of the primary amine have also been revised and determined, respectively, using chemical synthesis.99−102 Prymnesins have been shown to kill the freshwater fish Tanichthys albonubes at 3 nM in laboratory experiments.103 In addition, their hemolytic activity is reduced upon the addition of lipids, suggesting a direct action on membranes.103 Chemically Poorly Described Algal Species Causing Fish Killing during Blooms. As discussed above, most of the algal toxins described so far have been isolated due to their toxicity toward humans. With the increasing importance of marine fish farming worldwide, and the fact that blooms of ichthyotoxic microalgae are a recurring phenomenon in coastal and river waters, an increased focus has now been turned toward some of the algal species that are major fish killers, but which no or only sparse chemistry has been performed. A novel assay for the assessment of ichthyotoxicity using an in vitro rainbow trout gill cell line assay has been suggested.104 This is interesting; however, no studies have so far proven that this assay is indeed a proxy for ichthyotoxicity. In the following paragraph the status of five of the most important ichthyotoxic species is reviewed. Table 1 lists the verified and suggested ichthyotoxins together with laboratory trials as well as reported blooms for the five selected species. Prymnesium parvum. This haptophyte alga has been known to be responsible for fish kills for more than a century.105 Blooms occur in marine waters,106 brackish waters,107 and freshwaters.108 It was discovered early on that an exotoxin released from the algal cells was involved in the fish-killing mechanism.108 A number of chemical substances have over time been suggested as being those responsible for fish mortality, including hemolysin 1,109 prymnesins 1 (57) and 2 (58),98 GAT toxins,110 and fatty acid amides.111 Recently, GAT toxins and the fatty acid amides have been ruled out as the main toxins, either because the suggested toxins are not produced in sufficient quantities (GAT toxins) or simply because they are plastic contaminants (e.g., oleamides).112 Furthermore, a mechanical effect of the alga has been suggested as the responsible mode of action.113 Despite the highly ichthyotoxic activity (3 nM) of prymnesins, it still remains to be established whether these compounds are indeed the toxic principles that accumulate and show deleterious activities during toxic blooms. H



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shellfish from bloom-affected areas.144 Research indicates that this species produces hemolytic and ichthyotoxic substances (Hallegraeff, 1991).142 Gymnodimine and gymnocins have been isolated from K. mikimotoi, but data confirming that these compounds have fish-killing effects at environmental realistic concentrations are lacking. Analysis of Phycotoxin Toxins. The structural diversity of phycotoxins has traditionally called for different analytical approaches. For many years, quality control of shellfish before consumption has been assessed by a mouse lethality bioassay, but this approach has now been taken over by LC-MS/MSbased methods.167 This is due to animal ethical reasons, but also due to the many unexplained positive results that have often been observed when using the mouse assay, resulting in high economical losses.168 Another challenge in the detection and quantification of many of these phycotoxins is the lack of certified analytical standards.47 The large range of polarity of the different types of algal toxins dictates that different modes of separation are needed for their separation by LC. Thus, hydrophilic interaction chromatography (HILIC) is required for, for example, domoic acid and PSP toxins,168 especially since ion-pair reagents are not compatible with MS detection, unless the instrument is dedicated to only one polarity. The lipophilic toxins are separated by reversed-phase LC, which is the most MS-friendly separation mode (low buffer concentrations), but also provides the best separation power and thereby sharpest chromatographic peaks. Mass spectrometry wise, the trend is going toward high-resolution mass spectrometry (HRMS), rather than MS/MS.168−171 This is partly due to the improved sensitivity of HRMS instruments, now fulfilling the sensitivity requirements for many analytes. HRMS is often also more sensitive for masses above 1500 Da than quadrupole-based instruments, which for some simpler models have cutoffs down to 1200−1500 Da. More importantly, LC-HRMS allows for retrospective data analysis, making rescreening of old data-files for elemental compositions and fragment ions for new toxins possible, which is important in this field due to the still many unexplored toxins.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +45 45 25 26 32 (T. O. Larsen). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Danish Council for Strategic Research for granting the project “HABFISH” (Project No. 0603-00449B). DEDICATION Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.

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