Seasonal occurrence of toxic phytoplankton and phycotoxins at a mussel aquaculture site in Nova Scotia, Canada.

A three-year field study at a mussel (Mytilus edulis) aquaculture site in Ship Harbour, Nova Scotia, Canada was carried out between 2004 and 2006 to detect toxic phytoplankton species and dissolved lipophilic phycotoxins and domoic acid. A combination of plankton monitoring and solid phase adsorption toxin tracking (SPATT) techniques were used. Net tow and pipe phytoplankton samples were taken weekly to determine the abundance of potentially toxic species and SPATT samplers were deployed weekly for phycotoxin analysis. Mussels were also collected for toxin analysis in 2005. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the samples for spirolides (SPXs), pectenotoxins (PTXs), okadaic acid group toxins (OA, DTXs) and domoic acid (DA). Phycotoxins were detected with SPATT samplers beginning from the time of deployment until after the producing organisms were no longer observed in pipe samples. Seasonal changes in toxin composition occurred over the sampling period and were related to changes in cell concentrations of Alexandrium Halim, Dinophysis Ehrenberg and Pseudo-nitzschia (Hasle) Hasle. Spirolides peaked in late spring and early summer, followed by DA in mid-July. Okadaic acid, DTX1 and PTXs occurred throughout the field season but peaked in late summer. Concentrations of some phycotoxins detected in SPATT samplers deployed within the area where mussels were suspended on lines were lower than in those deployed outside the mussel farm. The SPATT samplers provided a useful tool to detect the presence of phycotoxins and to establish trends in their appearance in the Ship Harbour estuary.

Three decades of Canadian marine harmful algal events: Phytoplankton and phycotoxins of concern to human and ecosystem health.

Spatial and temporal trends of marine harmful algal events in Canada over the last three decades were examined using data from the Harmful Algal Event Database (HAEDAT). This database contains the most complete record of algal blooms, phycotoxins and shellfish harvesting area closures in Canada since 1987. This 30-year review of 593 Canadian HAEDAT records from 1988 to 2017, together with other Canadian data and publications, shows that recurring harmful algal events have been widespread throughout both the Atlantic and Pacific coastal regions. The 367 paralytic shellfish toxin (PST) reports revealed annual and frequent recurrence throughout both the Atlantic and Pacific regions, including multi-year PST events in the Bay of Fundy, the Estuary and Gulf of St. Lawrence and the Strait of Georgia. The 70 amnesic shellfish toxin (AST) records revealed no recognizable trend, as these events were usually area specific and did not recur annually. The increasing frequency of diarrhetic shellfish toxin (DST) events over the period of this review, in total 59 records, can be at least partially explained by increased sampling effort. Marine species mortalities caused by harmful algae (including diatoms, dictyochophytes, dinoflagellates, and raphidophytes), were a common occurrence in the Pacific region (87 reports), but have been reported much less frequently in the Atlantic region (10 reports). Notable Canadian records contained in HAEDAT include the first detection worldwide of amnesic shellfish poisoning (ASP), attributed to the production of domoic acid (an AST) by a diatom (Pseudo-nitzschia multiseries) in Prince Edward Island in 1987. The first proven case of diarrhetic shellfish poisoning (DSP) in Canada and North America was recorded in 1990, and the first closures of shellfish harvesting due to DST (associated with the presence of Dinophysis norvegica) occurred in Nova Scotia in 1992, followed by closures in Newfoundland and Labrador in 1993. In 2008, mass mortalities of fishes, birds and mammals in the St. Lawrence Estuary were caused by Alexandrium catenella and high levels of PST. During 2015, the Pacific coast experienced a large algal bloom that extended from California to Alaska. It resulted in the closure of several shellfish harvesting areas in British Columbia due to AST, produced by Pseudo-nitzschia australis. Data from the Canadian Arctic coast is not included in HAEDAT. However, because of the emerging importance of climate change and increased vessel traffic in the Arctic, information on the occurrence of harmful algal species (pelagic and sympagic = sea ice-associated) in that region was compiled from relevant literature and data. The results suggest that these taxa may be more widespread than previously thought in the Canadian Arctic. Information in HAEDAT was not always robust or complete enough to provide conclusions about temporal trends. Compilation of spatial and temporal information from HAEDAT and other records is nevertheless important for evaluating the potential role of harmful algae as a stressor on Canadian marine ecosystems, and will support the next step: developing a knowledge gap analysis that will establish research priorities for determining their consequences on human and ecosystem health.

Isolation and Characterization of [DLeu1]microcystin-LY from Microcystis aeruginosa CPCC-464.

[D-Leu1]MC-LY (1) ([M + H]+m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MCLR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC-HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC-HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC-MS/MS screening methods.

Production of domoic acid from large-scale cultures of Pseudo-nitzschia multiseries: A feasibility study.

The commercial demand for domoic acid (DA), the phycotoxin responsible for Amnesic Shellfish Poisoning, is currently met by extraction from a diminishing supply of stockpiled contaminated mussels (Mytilus edulis). As this supply becomes scarce, a more reliable source is needed. Purification of the toxin from an algal source would be easier and more economical than from shellfish tissue if algal growth and yield of toxin were maximized. This project was initiated to determine if DA could be produced using large-scale semi-continuous algal cultures, which should reduce labour and shorten the time required for biomass production. Pseudo-nitzschia multiseries was grown in 300-L fibreglass photobioreactors called a Brite-Box™. The effect of temperature and nutrient depletion on the yield of DA by P. multiseries was examined. A decline in maximum cell number without a substantial increase in cellular DA was associated with increased temperature. Maximum total cellular DA (8.8 pg cell-1) was achieved at 20 °C. Semi-continuous culture of P. multiseries is accompanied by increasing amounts of DA lost to the medium. The process was deemed to be feasible for growing P. multiseries but methods to recover this extracellular DA are necessary for this process to be economical.

Screening of cyclic imine and paralytic shellfish toxins in isolates of the genus Alexandrium (Dinophyceae) from Atlantic Canada.

The dinoflagellate genus Alexandrium Halim has frequently been associated with harmful algal blooms. Although a number of species from this genus are known to produce paralytic shellfish toxins (PST) and/or cyclic imines (CI), studies on comprehensive toxin profiling using techniques capable of detecting the full range of PST and CI analogues are limited. Isolates of Alexandrium spp. from Atlantic Canada were analyzed by targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS). Results showed a number of distinct profiles and wide ranging cell quotas of PST and spirolides (SPX) in both A. catenella (Whedon & Kofoid) Balech and A. ostenfedii (Paulsen) Balech & Tangen. The concentration of PST in A. catenella ranged from 0.0029 to 54 fmol cell-1 with the major components being C2 and GTX4. In addition, putative PST metabolites were confirmed for the first time in A. catenella by high resolution MS/MS. By comparison, A. ostenfeldii isolates showed much lower concentrations of PST (

Identification, growth and toxicity assessment of Coolia Meunier (Dinophyceae) from Nova Scotia, Canada.

Benthic dinoflagellates of the toxigenic genus Coolia Meunier (Dinophyceae) are known to have a global distribution in both tropical and temperate waters. The type species, C. monotis, has been reported from the Mediterranean Sea, the NE Atlantic and from Rhode Island, USA in the NW Atlantic, whereas other species in the genus have been reported from tropical locations. Coolia cells were observed in algal drift samples collected at seven sites in Nova Scotia, Canada. Clonal isolates were established from four of these locations and identified with light and scanning electron microscopy, then confirmed with genetic sequencing to be C. monotis. This is the first record of this species in Nova Scotia. The isolates were established and incubated at 18 °C under a 14:10 L:D photoperiod with an approximate photon flux density of 50-60 µmol m-2 s-1. Growth experiments using an isolate from Johnston Harbour (CMJH) were carried out at temperatures ranging from 5 to 30 °C under the same photoperiod with an approximate photon flux density of 45-50 µmol m-2 s-1. Cells tolerated temperatures from 5 to 25 °C with optimum growth and mucilage aggregate production between 15 and 20 °C. Methanol extracts of this isolate examined by Liquid Chromatography-Mass Spectrometry (LC-MS) did not show the presence of the previously reported cooliatoxin. Toxic effects were assayed using two zebrafish bioassays, the Fish Embryo Toxicity (FET) assay and the General Behaviour and Toxicity (GBT) assay. The results of this study demonstrate a lack of toxicity in C. monotis from Nova Scotia, as has been reported for other genetically-confirmed isolates of this species. Conditions in which cell growth that could potentially degrade water quality and provide substrate and dispersal mechanisms for other harmful microorganisms via mucilage production are indicated.

Characterization of a Dispiroketal Spirolide Subclass from Alexandrium ostenfeldii.

A new subclass of spirolide marine toxins, represented by spirolides H (1) and I (2), were isolated from the marine dinoflagellate Alexandrium ostenfeldii. Spirolides H and I are structurally distinct from other spirolides in that they contain a 5:6 dispiroketal ring system rather than the trispiroketal ring system characteristic of previously isolated spirolides. The structures were assigned using a combination of spectrometric and spectroscopic techniques. Previously isolated spirolides containing a cyclic imine moiety showed toxicity in the mouse bioassay. Spirolide H contains this cyclic imine moiety but does not show toxicity in the mouse assay, suggesting that the presence of the cyclic imine moiety is not the only structural requirement for toxicity.

Spirolides isolated from Danish strains of the toxigenic dinoflagellate Alexandrium ostenfeldii.

Using LC/MS methodology, spirolides were detected in two clonal isolates of Alexandrium ostenfeldii isolated from Limfjorden, Denmark. Examination of the LC/MS profiles of extracts from these Danish cultures revealed the presence of two dominant peaks representing two previously unidentified spirolide components and one minor peak identified as the previously reported desmethyl spirolide C (1). Culturing of these clonal strains, LF 37 and LF 38, of A. ostenfeldii resulted in the accumulation of sufficient cell biomass to allow for the isolation and structure elucidation of two new spirolides, 13,19-didesmethylspirolide C (2) and spirolide G (3). While 2 was found to differ from 1 only in that it contained one less methyl group, 3 was the first spirolide to be isolated that contained a 5:6:6-trispiroketal ring system. The effect of this new feature on the toxicity of 3 relative to other spirolides is presently being pursued.