Algal toxins... you may take our lives, but you’ll never take our freedom! (Okay... very politically incorrect - apologies).

Harmful algal blooms have increased dramatically since the 1980s and considered a major contributor in marine mammal mortalities worldwide. Dinoflagellates from the genus Alexandrium and the diatoms from the genus Pseudo-nitzschia are two of the most important toxin-producing species in Scottish waters and have the ability to produce marine toxins such as domoic acid (DA; Pseudo-nitzschia) and saxitoxins (STX; Alexandrium).

In Scotland, there has been regular monitoring for DA in shellfish since 1998 and it has been detected in shellfish above the regulatory limit (20 mg DA/kg of shellfish meat) on many occasions. It can also be toxic to humans and is known as amnesic shellfish poisoning (ASP), which can cause neuronal degeneration and necrosis in specific parts of the brain. A case study of DA exposed California sea lions (Zalophus californianus) highlighted neurological signs such as ataxia, head weaving, seizures or coma (this has also been reported for bottlenose dolphins (Tursiops truncatus) in the northern Gulf of Mexico). Studies have suggested that pregnant Z. californianus may be greater exposed due to toxins in the amniotic fluid and that DA causes reproductive failure.

Paralytic Shellfish Poisoning (PSP) toxins (STX) pose the greatest concern for seals in Scotland and due to a lack of information on chronic PSP toxicity data; the European Food and Safety Authority established an oral acute reference dose of 0.5 µg STX equivalents/kg body weight in humans. Canids (thought to be evolutionarily and physiologically similar to seals) have an acute oral LD₅₀ dose of 180-200 µg STX/kg, and whilst humans have a minimum oral dose of 7-16 µg of STX/kg, there have been cases where human mortalities have been observed at oral doses reaching 500-12,400 µg STX/kg. PSP toxins bind to the voltage-gated sodium channels in the brain blocking the flow of ions across the cell membrane. This can inhibit nerve and muscle cells to send electrical signals, which prevents normal cellular function, and ultimately lead to paralysis. Respiratory arrest is the most severe symptom of PSP exposure and can be rapidly be followed by death. Many case studies in the past has indicated evidence of STX exposure; the North Atlantic right whale (Eubalaena glacialis) in 2001 where the population was failing to recover from decline, the Mediterranean monk seal (Monachus monachus) off western Sahara in 1997 and humpback whales (Megaptera novaeangliae) off Cape Cod Bay, USA in 2001.

Harbour seals (Phoca vitulina) are exposed to these neurotoxins via the food web, consuming contaminated prey such as fish or squid and having a direct effect on health and survival rates. Jensen et al., (2015) hypothesised that a possible factor for the rapid decline of P. vitulina since 2000 is the effect of marine toxins have on the seals. Jensen et al., (2015) investigated the exposure and health effects of DA and STXs in P. vitulina and examined accumulated toxin levels in different prey species to explore possible links between toxin concentrations and degree of exposure between the declining and stable harbour seal populations.

Urine, faeces and blood samples were collected from live-captured harbour seals between 2008 and 2013, from three different regions, the Northern Isles (including the north coast of mainland Scotland), the west coast and the east coast; with the aim to detect and quantify DA and PSP toxins. As well as live capture, faecal and urine samples were collected from dead stranded harbour seals and were stored until needed. Fish otoliths present in faeces were removed and identified, if possible, to species level. Fish were collected from local fishermen in the summer and autumn of 2012 and throughout the calendar year of 2013 (except Jan, Feb and Apr). To investigate the presence of DA, extracted samples were used directly for the ASP Enzyme-Linked Immunosorbent Assay method (ELISA; for overview and protocol see http://www.biosense.com/comweb.asp?articleno=192&segment=3). The ELISA assay has no cross-reactivity to non-toxic, structural analogues like kainic acid (a natural marine acid present in some seaweeds). Results were confirmed using an ultra-high performance liquid chromatography-tandem mass spectrometry method (see Braña-Magdalena et al., 2014), with results from both methods correlating well. Analysis of PSP toxins in extracts was carried out using High Performance Liquid Chromatography with post-column oxidation and fluorescence detection with white blood cells counted. For the quantification of cortisol (stress response hormone) in the plasma samples, a commercially available solid phase enzyme-linked immunosorbent assay (ELISA) kit was used. Generalised linear models were used to select the models that best fitted the data with toxin concentration (in urine or faeces) as the dependent factor and sex and region as the independent factors.

Results for DA exposure in P. vitulina revealed a temporal change before and after 2012; urine samples collected between 2008 and 2010 contained quantifiable amounts of DA, whilst in 2012 and 2013 only 43.6 % had DA levels above limit of quantification (0.025 µg/g). This was complimented with faecal samples from both live captured harbour seals and anonymous faecal samples for which during 2008-2010 revealed the highest levels of DA. This shift in DA contamination of harbour seals relates to the annual variability in Pseudo-nitzschia spp. blooms in Scotland from 2006 to 2013 (two major blooms occurred in 2008 and one in 2010). Harbour seals on the east coast of Scotland had over three times higher DA concentrations compared to those from the Northern Isles and the west coast.

Results for PSP toxification was less clear, highlighting a likely chronic rather than acute exposure, where long-term effects are not yet fully understood. Despite the fact that the east coast is the region with the greatest population decline, over half of the live captured harbour seals found exposed to PSP toxins (55.6 %) were captured on the west coast (May 2013). Active screening of PSP toxins in the live captured harbour seals first started in 2012; however, both 2012 and 2013 were years where harbour seals were found exposed to PSP toxins, suggesting PSP toxins could constitute a potential risk for the health of this species in Scotland. Only acute effects of PSP toxins have been reported in mammals such as seals, long term exposure to non-lethal doses of STX may in fact make them potentially become less susceptible to the effects through their consumption of contaminated fish prey such as plaice, dab, long rough dab, whiting, and cod.

The authors highlighted that there were many variables in this study that need to be looked into further; i.e., there is little information on how widespread Pseudo-nitzschia blooms are due to the limited phytoplankton-monitoring network. Furthermore, light microscopy identification cannot differentiate between toxic and non-toxic species of Pseudo-nitzschia.

Jensen, S.K., Lacaze, J.P., Hermann, G., Kershaw, J., Brownlow, A., Turner, A., & Hall, A. (2015) 'Detection and effects of harmful algal toxins in Scottish harbour seals and potential links to population decline' Toxicon. 97, 1-14.

Reference for liquid chromatography-tandem mass spectrometry method

Braña-Magdalena, A., Leāo-Martins, J.M., Glauner, T., & Gago-Martínez, A. (2014) 'Intralaboratory validation of a fast and sensitive UHPLC/MS/MS method with fast polarity switching for the analysis of lipophilic shellfish toxins' J. AOAC. Int. 97(2), 285-292.