The influence of the toxin-producing dinoflagellate, Alexandrium catenella, on feeding, reproduction and toxin retention in Calanus helgolandicus.

Copepods of the genus Calanus dominate the biomass of pelagic ecosystems from the Mediterranean Sea up into the Arctic Ocean and form an important link between phytoplankton and higher trophic levels. Impacts from toxin-producing harmful algae (HA) have been recorded throughout this region over the last 50 years, with potentially negative effects on Calanus spp. populations and the ecosystem functions and services they provide. Here we examine how ingestion, egg-production and egg-viability in Calanus helgolandicus are affected by the relative abundance of the toxin-producing dinoflagellate Alexandrium catenella in their diet. Our four-day experiments demonstrate that the ingestion rate of C. helgolandicus declined significantly as the percentage of toxin-producing A. catenella within their diet increased, whereas egg production and egg viability were unaffected. Toxin profile concentrations for A. catenella are presented alongside body toxin-loads in C. helgolandicus after 4 days of feeding on these cells. The body toxin concentrations of C. helgolandicus were 3.6-356.6 pg STX diHCl eq. copepod-1, approximately 0.02-3.3 % of the toxins ingested. Our work suggests that the effects of exposure to A. catenella may be negligible in the short-term but could manifest if bloom conditions persist for longer than our experimental duration.

Toxins from harmful algae in fish from Scottish coastal waters.

Harmful algal bloom events are increasing in a number of water bodies around the world with significant economic impacts on the aquaculture, fishing and tourism industries. As well as their potential impacts on human health, toxin exposure from harmful algal blooms (HABs) has resulted in widespread morbidity and mortality in marine life, including top marine predators. There is therefore a need for an improved understanding of the trophic transfer, and persistence of toxins in marine food webs. For the first time, the concentrations of two toxin groups of commercial and environmental importance, domoic acid (DA) and saxitoxin (including Paralytic Shellfish Toxin (PST) analogues), were measured in the viscera of 40 different fish species caught in Scotland between February and November, 2012 to 2019. Overall, fish had higher concentrations of DA compared to PSTs, with a peak in the summer / autumn months. Whole fish concentrations were highest in pelagic species including Atlantic mackerel and herring, key forage fish for marine predators including seals, cetaceans and seabirds. The highest DA concentrations were measured along the east coast of Scotland and in Orkney. PSTs showed highest concentrations in early summer, consistent with phytoplankton bloom timings. The detection of multiple toxins in such a range of demersal, pelagic and benthic fish prey species suggests that both the fish, and by extension, piscivorous marine predators, experience multiple routes of toxin exposure. Risk assessment models to understand the impacts of exposure to HAB toxins on marine predators therefore need to consider how chronic, low-dose exposure to multiple toxins, as well as acute exposure during a bloom, could lead to potential long-term health effects ultimately contributing to mortalities. The potential synergistic, neurotoxic and physiological effects of long-term exposure to multiple toxins require investigation in order to appropriately assess the risks of HAB toxins to fish as well as their predators.

The competitive dynamics of toxic Alexandrium fundyense and non-toxic Alexandrium tamarense: The role of temperature.

The dinoflagellate Alexandrium produces paralytic shellfish poisoning toxins. The genus is globally distributed, with Scottish waters being of particular interest due to the co-occurrence of different species and strains. In Scottish waters, Alexandrium was historically thought to be dominated by the highly toxic (Group I) Alexandrium fundyense. However, the morphologically indistinguishable (Group III) Alexandrium tamarense has recently also been found to co-occur, raising important questions in relation to Alexandrium biogeography. To begin to address these, we investigated Alexandrium growth, yield and toxin production in a range of temperature conditions characteristics of present and potential future conditions, using a recently developed flow cytometry method that allowed, for the first time, simultaneous enumeration of the cryptic species in co-culture. Experiments were undertaken in a range of temperatures (12, 15, 18 and 21°C) in the phosphate (P) limiting conditions that promotes A. fundyense toxicity. Cell/biomass yield was greater for A. tamarense at all temperatures, with observed growth rates varying with temperature. Growth rather and yield were different in mono- and co-culture with the outcome of these interactions also being temperature dependent. For toxic A. fundyense, GTX-3, STX and NEO were the dominant analogues, but total toxicity, toxicity per cell and the number of, and relative proportion of, toxin analogues changed in relation to the onset of P limitation and also as a function of temperature, with the highest toxin concentrations per cell being observed at 12°C. Toxin concentrations were approximately double in P limited stationary phase compared to exponential growth. Toxin concentrations were lower in the co-cultures, indicating inhibition of production in the presence of non-toxic A. tamarense. The strong performance of A. tamarense is in co-culture at odds with the historical understanding that Scottish waters were dominated by A. fundyense and indicates that changes in water temperatures, and also potentially alleopathic interactions, will influence Alexandrium populations and hence the PSP toxicity threat to humans from shellfish.

Detection and effects of harmful algal toxins in Scottish harbour seals and potential links to population decline.

Over the past 15 years or so, several Scottish harbour seal (Phoca vitulina) populations have declined in abundance and several factors have been considered as possible causes, including toxins from harmful algae. Here we explore whether a link could be established between two groups of toxins, domoic acid (DA) and saxitoxins (STXs), and the decline in the harbour seal populations in Scotland. We document the first evidence that harbour seals are exposed to both DA and STXs from consuming contaminated fish. Both groups of toxins were found in urine and faeces sampled from live captured (n = 162) and stranded animals (n = 23) and in faecal samples collected from seal haul-out sites (n = 214) between 2008 and 2013. The proportion of positive samples and the toxins levels measured in the excreta were significantly higher in areas where harbour seal abundance is in decline. There is also evidence that DA has immunomodulatory effects in harbour seals, including lymphocytopenia and monocytosis. Scottish harbour seals are exposed to DA and STXs through contaminated prey at potentially lethal levels and with this evidence we suggest that exposure to these toxins are likely to be important factors driving the harbour seal decline in some regions of Scotland.

Halogenated persistent organic pollutants in relation to trophic level in deep sea fish.

The bioaccumulation of persistent organic pollutants (POPs) in deep sea fish from the Rockall fishing area was investigated. Predator and prey species were analysed for stable isotopes, fatty acids, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). δ(15)N indicated that black scabbard was at the highest trophic level and the prey the lowest. The fatty acid signatures indicated that black scabbard and black dogfish fed at a higher trophic level compared to the roundnose grenadier. PCBs and PBDEs were detected in the liver of all three predator species. PCB concentrations were significantly higher in the roundnose grenadier, possibly due to their longer life span. PCB concentrations were compared to OSPAR assessment criteria, concentrations were above background but below Environmental Assessment Criteria for all but one congener. PCB concentrations were below food safety levels in the flesh, but exceeded the limit for liver in the roundnose grenadier and black dogfish.

Optimization of hydrophilic interaction liquid chromatography/mass spectrometry and development of solid-phase extraction for the determination of paralytic shellfish poisoning toxins.

The combination of hydrophilic interaction liquid chromatography (HILIC) and liquid chromatography/mass spectrometry (LC/MS) for the determination of paralytic shellfish poisoning (PSP) toxins has been proposed for use in routine monitoring of shellfish. In this study, methods for the detection of multiple PSP toxins [saxitoxin (STX), neosaxitoxin (NEO), decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNEO), gonyautoxins 1-5 (GTX1, GTX2, GTX3, GTX4, GTX5), decarbamoyl gonyautoxins (dcGTX2 and dcGTX3), and the N-sulfocarbamoyl C toxins (C1 and C2)] were optimized using single (MS) and triple quadrupole (MS/MS) instruments. Chromatographic separation of the toxins was achieved by using a TSK-gel Amide-80 analytical column, although superior chromatography was observed through application of a ZIC-HILIC column. Preparative procedures used to clean up shellfish extracts and concentrate PSP toxins prior to analysis were investigated. The capacity of computationally designed polymeric (CDP) materials and HILIC solid-phase extraction (SPE) cartridges to retain highly polar PSP toxins was explored. Three CDP materials and 2 HILIC cartridges were assessed for the extraction of PSP toxins from aqueous solution. Screening of the CDPs showed that all tested polymers adsorbed PSP toxins. A variety of elution procedures were examined, with dilute 0.01% acetic acid providing optimum recovery from a CDP based on 2-(trifluoromethyl)acrylic acid as the monomer. ZIC-HILIC SPE cartridges were superior to the PolyLC equivalent, with recoveries ranging from 70 to 112% (ZIC-HILIC) and 0 to 90% (PolyLC) depending on the PSP toxin. It is proposed that optimized SPE and HILIC-MS methods can be applied for the quantitative determination of PSP toxins in shellfish.

Solid-phase extraction and liquid chromatography--mass spectrometry for the determination of free fatty acids in shellfish.

A novel analytical protocol for the determination of free fatty acids (FFAs; saturated, monounsaturated and polyunsaturated) in shellfish using electrospray ionisation and liquid chromatography-mass spectrometry (LC-MS) is described. Total lipids were extracted from four commercially important shellfish species using chloroform-methanol in a modification of the traditionally used Bligh and Dyer method. FFAs were recovered from lipidic shellfish extracts by solid-phase extraction (SPE) on an aminopropyl-silica column using a 98:2 v/v diethyl ether (DEE)-acetic acid solution. Ether extracts containing the FFAs were evaporated and reconstituted in 70:30 v/v methanol-chloroform before analysis by LC-MS. The limits of quantification (LOQs) of the method ranged from 60 to 560 microgg(-1) wet weight depending on the different FFAs determined with selected ion monitoring (SIM). Results demonstrate that LC-MS is well suited for identification and quantification of FFAs in shellfish and negates the use of sample derivatisation required in gas chromatographic analysis.

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins.

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.