Pinnatoxin G is responsible for atypical toxicity in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from Ingril, a French Mediterranean lagoon

Highlights

• HRMS confirmed Vulcanodinium rugosum (IFR-VRU-01) from France produces PnTX-G.

• The nAChR binding assay showed that synthetic and natural PnTX-G have similar activity.

• PnTX-G explains positive MBA results observed since 2006 in shellfish from Ingril.

• PnTX-G higher in mussels than in clams & highest reported so far (>1200 μg kg⁻¹).

Abstract

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay.

The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 μg kg⁻¹ in whole flesh, with a maximum in September 2010 of around 1200 μg kg⁻¹. These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 μg kg⁻¹ in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell⁻¹). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

Introduction

Pinnatoxins (PnTXs) had initially been isolated from Pinna muricata collected in Japan (Chou et al., 1996a, 1996b; Takada et al., 2001a; Uemura et al., 1995), the same genus of mollusc associated in the early 1990s with a Pinna attenuata poisoning in China. Metabolism pathways were subsequently postulated by Selwood et al. (2010) to explain biotransformation of the algal metabolites PnTXs -E, -F and -G into shellfish metabolites PnTXs A, B, C and D and pteriatoxins A, B and C, initially reported by Hao et al. (2006) and Takada et al. (2001b). The biological source of pinnatoxins had been unknown until a PnTX-producing dinoflagellate was discovered in New Zealand in 2010 (Rhodes et al., 2010). Subsequently, this organism was taxonomically identified as a previously undescribed dinoflagellate, Vulcanodinium rugosum (Nézan and Chomérat, 2011). A strain of this species was also recently isolated from Japanese (Smith et al., 2011) and from Chinese waters (Zeng et al., 2012).

In addition to the abovementioned reports on PnTXs from South East Asia, Australia and New Zealand, PnTXs have recently also been reported from Europe, specifically Norway (Miles et al., 2010; Rundberget et al., 2011), and from North America (McCarron et al., 2012). PnTXs can thus be considered to be fairly widespread. Maximum concentrations so far have been reported to be below 110 μg kg⁻¹ whole shellfish flesh in Canada (McCarron et al., 2012), below 120 μg kg⁻¹ in Norway (Rundberget et al., 2011) and below 200 μg kg⁻¹ in New Zealand (McNabb et al., 2012). These low levels appear very much in line with those typically found for spirolides: a relatively extensive data set with 1801 shellfish samples from France, Italy and the Netherlands showed a 95th percentile of 8.9 μg kg⁻¹ and a maximum of 105 μg kg⁻¹ for the sum of total spirolides over the 7-year period from 2002 to 2008 (EFSA, 2010).

Pinnatoxins exhibit fast-acting toxicity when injected intraperitoneally into mice (Munday et al., 2012), like many other toxins from the cyclic imine group of compounds (EFSA, 2010; Munday, 2008). The high intrinsic toxicity of pinnatoxins is indicated by low LD₅₀s i.p. in mice, namely 57, 13 and 48 μg kg⁻¹ bodyweight for PnTXs-E, -F and -G, respectively (Munday et al., 2012). In addition, the uptake from the gastro-intestinal tract has also been shown in mice, both for voluntary feeding and classical gavage exposure routes (Munday et al., 2012). These authors also report that there is only a comparatively small differential between the i.p. and the per os routes of exposure, i.e. a factor of about 3 for PnTX-G, and hence the toxic potential of PnTXs may constitute a risk to consumers of contaminated shellfish.

Only one poisoning event had ever been linked to the bivalve genus Pinna (Zheng et al. (1990) cited in Selwood et al. (2010)). Even so, the putative initial poisoning event in China may well not have been caused by pinnatoxins, as the presence of this toxin group was not demonstrated in this first event, and potential contamination with pathogenic vibrio had also been reported from that area. No acute poisoning events have been reported in direct relation to contamination with PnTXs before or since their chemical characterisation in 1995. Possible confusion may arise from the fact that PnTX-G, one of the main algal metabolites, has the same chemical sum formula (C₄₂H₆₃NO₇) as spirolide B and 13-desmethylspirolide D (Fig. 1), two other representatives from the cyclic imines group of fast acting toxins. Even though spirolides are produced by a different genus of dinoflagellate, i.e. Alexandrium ostenfeldii (Cembella et al., 1999) and Alexandrium peruvianum (Borkman et al., 2012; Touzet et al., 2008), they accumulate in shellfish and due to the absence of sufficient reference calibration standards may thus have been confounded with PnTX-G. However, no acute poisonings have been associated with this toxin group either. Unlike spirolides, PnTXs are chemically rather stable compounds: they resist alkaline hydrolysis in aqueous methanol, at 76 °C for 40 min (Rundberget et al., 2011) and aqueous HCl (pH 1.5) at 40 °C for 24 h (Jackson et al., 2012). Due to this unusual stability, and their high oral toxicity, PnTXs may be a significant threat to shellfish consumers.

Unfortunately, there is a general lack of information on occurrence for a large number of toxin groups on one hand, and epidemiological information on the other. The lack of information on toxin occurrence has been to some extent overcome by the use of the lipophilic mouse bioassay, also leading to criticism of this test, suggesting its potential for “false” positives. Several comparisons of chemical analyses using liquid chromatography coupled to mass spectrometry (LC-MS) or to tandem mass spectrometry (LC-MS/MS) with the mouse bioassay (MBA) have been conducted as part of routine shellfish safety surveillance programs, e.g. in Ireland and France (Belin et al., 2009; Clarke et al., 2006). In France, the mouse bioassay protocol for lipophilic toxins is based on the EU harmonised protocol, using a 24 h observation period and dichloro-methane as solvent for the partitioning clean-up (Yanagi et al., 1989; Yasumoto et al., 1984). The French study classified unexplained MBA positives as atypical toxicity (Belin et al., 2009). In this 6-year study (2003–2008), over 1000 shellfish samples were analysed using both LC-MS/MS and MBA, with over 25% of the MBA positives being not explained by chemical analysis.

This large percentage of unexplained MBA results prompted us to investigate several monitoring sites further. Here, we report the findings concerning Ingril Lagoon, with PnTX-G identified as the main source of MBA positives observed since 2006 in this lagoon. Particular attention is given to the levels and profiles of PnTXs in mussels and clams, the two main shellfish species occurring naturally in this lagoon. PnTX-G levels in cultures of the toxin-producing organism are reported, as well as results from a functional assay based on the mode of action of the toxin on the Torpedo nicotinic acetylcholine receptor (nAChR) (Aráoz et al., 2012).