After Human Poisoning in Croatia (Adriatic Sea)

Toxicon 79 (2014) 28–36

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Toxicon

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PSP toxins proﬁle in ascidian Microcosmus vulgaris (Heller, 1877) after human poisoning in Croatia (Adriatic Sea)

Romana Roje-Busatto, Ivana Ujevic* Institute of Oceanography and Fisheries, Setaliste I. Mestrovica 63, 21000 Split, P.O. Box 500, Croatia article info abstract

Article history: Toxins known to cause Paralytic Shellfish Poisoning (PSP) syndrome in humans that can Received 2 October 2013 have serious economic consequences for aquaculture were determined in ascidians of the Received in revised form 30 December 2013 genus Microcosmus. Significant concentrations of toxins were confirmed in all tested Accepted 31 December 2013 samples collected from the western coast of Istria Peninsula (Adriatic Sea, Croatia) when Available online 10 January 2014 six people were poisoned following the consumption of fresh ascidians. Several species of bivalves that were under continuous monitoring had not accumulated PSP toxins although Keywords: they were exposed to the same environmental conditions over the survey period. In the Paralytic Shellfish Poisoning Ascidian present study, HPLC-FLD with pre-column oxidation of PSP toxins has been carried out to fi Microcosmus provide evidence for the rst human intoxication due to consumption of PSP toxic as- Adriatic Sea cidians (Microcosmus vulgaris, Heller, 1877) harvested from the Adriatic Sea. Qualitative Human intoxication analysis established the presence of six PSP toxins: saxitoxin (STX), decarbamoylsaxitoxin HPLC-FLD (dcSTX), gonyautoxins 2 and 3 (GTX2,3), decarbamoylgonyautoxins 2 and 3 (dcGTX2,3), gonyautoxin 5 (GTX5) and N-sulfocarbamoylgonyautoxins 1 and 2 (C1,2), while quantitative analysis suggested STX and GTX2,3 as dominant toxin types and the ones that contribute the most to the overall toxicity of these samples with concentrations near the regulatory limit. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction in wild and aquacultured organisms. PSP toxins occur naturally in cyanobacteria, that can exist as plankton Research on toxicity in different marine food cycles (Pomati et al., 2000), marine dinoflagellates (Harada et al., contributes to clarifying the role of Paralytic Shellfish 1982) and some heterotrophic bacteria (Gallacher and Poisoning (PSP) toxins in the natural marine environment Smith, 1999). Nevertheless, marine biotoxins affecting and their toxic effect on marine organisms and humans filter-feeding organisms increase human health risks (Deeds et al., 2008; Vale, 2008). Detrimental changes in the (García et al., 2004; Yen et al., 2004; Ujevic et al., 2012)as marine environment possibly synergistically caused by worldwide toxic events caused by harmful phytoplankton global climate changes, increased pollution and eutrophi- blooms result in accumulation of phytoplankton toxins in cation (enhanced utilization of coastal waters for aquacul- shellfish and other aquatic organisms (Deeds et al., 2008). ture, as well as agricultural, urban and industrial run offs), Ecological consequences of these events include marine tourism expansion, increased shipping activity (ballast trophic structure alterations where filter-feeders function waters) and overfishing are affecting biochemical processes as vectors for transport of phycotoxins through the food web to higher level consumers such as fish, dolphins, whales and sea birds, often with resultant animal deaths

* Corresponding author. (Anderson and White, 1992; Anderson et al., 2002; E-mail addresses: [email protected] (R. Roje-Busatto), [email protected] (I. Landsberg, 2002; Deeds et al., 2008; Abbott et al., 2009) Ujevic). and subsequent poisoning of humans (Shumway, 1990;

García et al., 2004). Chen and Chou (1998) found evidence decarbamoylgonyautoxins 1–4 (dcGTX1–4) and decar- for food chain transmission of PSP toxins from dinoflagel- bamoylneosaxitoxin (dcNEO); in the deoxydecarbamoyl late Alexandrium minutum to carnivorous gastropod group there are deoxydecarbamoylsaxitoxin (doSTX), through a filter-feeding bivalve, while White (1979, 1980) deoxydecarbamoylneosaxitoxin (doNEO) and deoxy- concluded that Atlantic herring (Clupea harengus hare- decarbamoylgonyautoxin 1–3 (doGTX1–3) toxin types ngus) accumulated PSP toxins through ingestion of ptero- (Lagos and Andrinolo, 2000; FAO, 2004). Specific toxicities pods which had grazed on Alexandrium tamarense in the of each analogue within the PSP group originate from Bay of Fundy (Canada) that resulted in massive fish kills. different structures of the toxins. STX is the most potent Hallegraeff (1993) mentioned that there were 2000 cases of within the group, while the toxicity of other congeners is Paralytic Shellfish Poisoning with 15% human mortality compared with pure STX and expressed as STX equivalents reported every year throughout the world, while Relox and (Shimizu, 1987). Understanding of biotoxin conversions in Bajarias (2003) noted 2122 PSP cases with 117 human marine organisms is critically important since toxins of low deaths between 1983 and 2002 in the Philippines. In potency in dinoflagellate cells can be converted to highly Alaska, between 1973 and 2008 at least 204 people were potent toxins through bacterial, enzymatic or pH mediated affected by PSP (Trainer, 2002), but Gessner and Schloss activity (Shimizu and Yoshioka, 1981; Sullivan et al., 1983), (1996) argue that this number probably under represents thereby affecting the net toxicity. the number of people impacted by a factor of 10 or even 30 due to under-reporting or misdiagnosis. 1.2. The occurrence of PSP toxic species and shellfish toxicity Microcosmus is a solitary ascidian, a sedentary animal in the Adriatic Sea that remains attached for its lifetime to a hard surface on the sea floor. Water enters through the incurrent and exits In the spring of 1994, an A. minutum bloom caused the through the excurrent siphon. Suspended material first recorded PSP toxicity in the Italian north-west Adriatic (plankton, algal spores, bacteria and stirred-up detritus) in region (Honsell et al., 1996). There was also suspected PSP the water is caught in the pharynx by mucus made from the toxicity presence in the central part of the eastern Adriatic endostyle and transported to digestive tract. Since ascid- Sea (Kastela Bay) in 1994 through 1996, based on phyto- ians mostly prefer protected seabed areas with good water plankton cell numbers (Orhanovic et al., 1996; Marasovic circulation (Carballo, 2000; Goodbody, 2000)itistobe et al., 1998; Pavela-Vrancic and Marasovic, 2004). More expected to be easily found at shellfish farms. Most of the recently, Ujevic et al. (2012) recorded and quantified PSP ascidians are found to accumulate heavy metals (Philp shellfish toxicity for the first time in Croatian shellfish et al., 2003), but there is also evidence of high levels of farms with a few samples exceeding the regulatory limit set PSP toxin accumulation in ascidians that persists for a long by the European Commission. That first PSP occurring period of time (Sekiguchi et al., 2001). Ascidians, especially period was represented mainly by STX and GTX2,3 in the species Microcosmus sabatieri (Roule, 1885) and Micro- farmed mussels (Mytilus galloprovincialis) from the north- cosmus vulgaris (Heller, 1877) are considered a delicacy in ern Adriatic Sea during winter and spring of 2009 (from North America, Japan, Korea, Chile, Peru, New Zealand, and January through April). Besides this period of toxicity, a also in Spain, Italy and France. Common names for M. vul- sporadic case of dcSTX occurring in very low concentrations garis are: ‘grooved sea squirt’ or ‘sand violet’ (English), (below 35 mgkg 1) was in late 2008 detected in shellfish ‘violet de sable’ (French), ‘boniato de mar’ (Spanish) and from the southern Adriatic Sea (Ujevic et al., 2012). For ‘limone di mare’ or ‘uovo di mare’ (Italian). Microcosmus has Mediterranean waters, there are more PSP toxic dinofla- a high nutritional value and is a good source of vitamins, gellate occurrences recorded than reported PSP contami- minerals (especially iodine) and long chain n-3 poly- nated organisms (Ciminiello et al., 2000; Vila et al., 2001; unsaturated fatty acids (PUFA) that have hypolipidemic, Lilly et al., 2002; Montresor et al., 2004). antihypertensive, anti-inflammatory, antithrombotic and On December 16, 2008 there were six recorded cases of antiarrhythmic properties (Stamatis et al., 2008; Stamatis human poisoning (one woman and five men from Rovinj, et al., 2007). López-Rivera et al. (2009) found the pres- Croatia) after consumption of fresh ascidians caught in ence of Domoic acid (causative toxin of Amnesic Shellfish fishing nets. Traditionally in this region the ascidians are Poisoning) in the ascidian Pyura chilensis (Molina, 1782) in eaten raw and are considered a delicacy. Thirty minutes the south of Chile and call for the inclusion of these or- after consumption, all six individuals reported the same ganisms in sanitation programs for marine toxins. symptoms of dizziness, vomiting, weakness in the legs and blurred vision. They were admitted to the emergency room 1.1. Paralytic Shellfish Poisoning toxins and within a few hours all the symptoms and clinical problems had disappeared (Basic-Palkovic, 2008). According to EFSA (2009) at least 30 compounds belong to the Paralytic Shellfish Toxin family. These can be divided 2. Materials and methods into four groups based on their side chain chemical structure. The most toxic carbamate group comprises saxitoxin 2.1. Study area and sampling frequency (STX), neosaxitoxin (NEO), gonyautoxins 1–4 (GTX1–4); the least toxic, N-sulfocarbamoyl group consists of gonyautoxin During December 2008 through May 2009, 15 samples 5 and 6 (GTX5,6) and N-sulfocarbamoylgonyautoxin 1–4 of the ascidian M. vulgaris (grooved sea squirt) were (C1–4); the decarbamoyl group with intermediate toxicity sampled at shellfish harvesting stations S1, 2 and 3 along includes decarbamoylsaxitoxin (dcSTX), the West Istrian coast (northern Adriatic Sea; Fig. 1) that 30 R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36

Fig. 1. Geographic locations of sampling stations S1, 2 and 3 on the Croatian coast (eastern part of the Adriatic Sea). Stations are located along the West Istrian Peninsula. were suspected to be PSP toxic when six people were of each sample were weighed into 50 mL polypropylene poisoned after their consumption (Table 1). Sampling was centrifuge tubes, extracted, vortexed, heated in a water carried out according to the field weather conditions. bath (100 C) for 5 min, remixed and centrifuged for 40 min at 4500 rpm (3600 g;20C). The supernatant 2.2. HPLC-FLD method was carefully decanted into 15 mL polypropylene tubes. The pellet was re-extracted, vortexed and centrifuged High Performance Liquid Chromatography with Fluo- again. Two supernatants were pooled and diluted to a final rescence Detection (HPLC-FLD) method (2005.06 AOAC; volume of 10 mL with DI water. Tubes were stored at 4 C Lawrence et al., 2005) includes a pre-chromatographic until analysed. oxidation of PSP toxins with hydrogen peroxide and peri- The purification procedure was based on Lawrence et al. odate solution as oxidants. (2005). Varian, Bond Elut C-18 (500 mg/6 mL) SPE cartridges (Varian, USA) were appropriately conditioned with 2.3. Chemicals, reagents and standards methanol and DI water, and used to produce a C-18 cleaned extract that should contain all PSP toxins. 1 mL of sample Reagents and chemicals used were of HPLC grade ob- extract was run through an SPE C-18 cartridge, the eluate tained from Sigma-Aldrich, and the following solutions collected, then the cartridge was eluted with DI water, the were prepared in deionized (DI) water: glacial acetic acid eluates combined, pH corrected to 6.5 and volume adjusted (1%, 0.1 mM and 0.1 M aq), acetonitrile (5% aq), ammonium to 4 mL. Aliquots were submitted to oxidation with acetate (0.01 M aq), ammonium formate (0.1 M and 0.3 M peroxide and periodate oxidants prior to HPLC analyses. aq), disodium hydrogen phosphate (Na2HPO4, 0.3 M aq), Second column purification was carried out on extracts hydrogen peroxide (10% aq), methanol, sodium chloride with N-1-hydroxylated PSP toxins after C-18 cleanup. Var- (0.05 M and 0.3 M aq), sodium hydroxide (0.2 M and 1 M aq), ian Bond Elut CBA (500 mg/3 mL) ion exchange cartridges periodic acid (0.03 M aq). Mobile phases consisted of (A) (Varian, USA) were conditioned by washing with aqueous 0.1 M ammonium formate and (B) 0.1 M ammonium formate ammonium acetate and a 2 mL aliquot of C-18 purified in 5% acetonitrile, and 0.1 M acetic acid was used to adjust pH extract was added to each cartridge and the eluate to 6.0. Certified standard calibration solutions for analysis of collected. After every addition of the adequate eluent, DI PSP toxins were obtained from the Certified Reference Ma- water was run through the cartridge and volumes adjusted. terials Program (CRMP) of the Institute for Marine Biosci- First eluted was fraction No. 1 that, if all present, should ence (IMB), National Research Council of Canada (Halifax, contain C1-4 toxins. The same CBA cartridge was treated Canada) and included NRC CRM: STX-e, dcSTX, NEO-c, again with 0.05 M NaCl and this tube was assumed to dcNEO-b, GTX1,4-c, GTX2,3-c, dcGTX2,3-b, GTX5-b and C1,2. contain fraction No. 2 with toxins: GTX1–6 and dcGTX2,3. Additional elution with 0.3 M NaCl resulted in fraction No. 3 2.4. Extraction and cleanup that should contain toxins STX, dcSTX, NEO and dcNEO. A matrix modifier was prepared from PSP-free ascidian Ascidians (Fig. 2a) were washed externally, opened, soft soft tissue extract. After the SPE C-18 purification, as pre- tissue (Fig. 2b) removed and subsequently homogenized viously described, pH was adjusted to 6.5, filtered and left (100 g) in a glass blender and frozen (20 C) until to precipitate co-extracted material for two days. Periodate required for PSP analysis. Paralytic toxins were extracted and peroxide oxidation of the matrix was done to ensure following the Association of Official Analytical Chemists the absence of toxins before being used for periodate Official Method (AOAC) 2005.06. Aliquots (5.00 0.10 g) analysis of the samples, as it enhances the fluorescent R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36 31

Table 1 STX ¼ 1.1, dcSTX ¼ 1.9, GTX2,3 ¼ 2.1, dcGTX2,3 ¼ 1.1, Sampling dates of ascidian M. vulgaris (/ sampling not performed, þ GTX5 ¼ 0.4 and C1,2 ¼ 4.2 mgkg 1. sampling was performed). Date S1 S2 S3 3. Results 17 Dec’08 / / þ 29 Dec’08 / þþ3.1. Toxin profile 26 Jan’09 / / þ 09 Feb’09 / / þ 22 Feb’09 þ / þ In the present study, ascidian samples were subjected to 09 Mar’09 þþþHPLC-FLD analysis in order to detect the presence of the 30 Mar’09 / þ / following PSP toxin types: STX, dcSTX, NEO, dcNEO, GTX1,4, 06 Apr’09 þþþGTX2,3, dcGTX2,3, GTX5 and C1,2. 25 May’09 / þ / As evident from Fig. 3, S1 had the most diverse profile of all the stations considered in this study. There were STX, dcSTX, GTX2,3, dcGTX2,3, GTX5 and C1,2 toxins present intensity of periodate oxidation products of N-1-hydrox- during this period, with GTX2,3 and dcGTX2,3 as the most ylated PSP toxins (Lawrence et al., 2005). An aliquot of the abundant toxins in the samples. GTX2,3 exhibited the extract mixed with the ascidian matrix modifier was ana- highest concentration per sample (655.72 mgkg1), fol- lysed without oxidation to ensure the absence of naturally lowed by GTX5 with 585.21 mgkg 1. DcGTX2,3 was detec- fluorescent compounds. Aliquots of all fractions were ted in February and April with concentrations of oxidized by peroxide (hydrogen peroxide) and periodate 389.18 mgkg 1 and 348.60 mgkg 1, respectively. The lowest (periodic acid, ammonium formate and disodium hydrogen detected concentrations were those of dcSTX, within the phosphate) solutions for HPLC pre-column analysis. range of 17.30–78.11 mgkg 1. Oxidative conversion of PSP toxins to fluorescent de- Fig. 4 shows the total PSP toxicity found in ascidians at rivatives is necessary in order to perform HPLC analysis. station S1 calculated by summing the toxicity contribution Five point calibration curves were obtained by pre-column of each quantified toxin (mg STX eq. kg 1). The major peroxide and periodate oxidations of four standard PSP contributor to PSP toxicity on this station was GTX2,3, as toxin mixes (Ujevic et al., 2012). the least toxic representative of the most toxic carbamate group. DcGTX2,3 was the second toxicity contributor, as it is 2.5. HPLC toxin analysis of intermediate toxicity, but was in high concentrations during this period. GTX5 has an extremely low toxicity The PSP ascidian extracts were analysed using a Varian factor, and yet it contributed to the overall toxicity because ProStar 230 analytical solvent system coupled with a of its high concentration in one sample. C1,2 was detected ProStar 363 fluorescence detector (ex. 340 nm; em. only in April at a concentration of 326.20 mgkg 1, and as it 390 nm), ProStar 410 autosampler and ProStar 500 column is an N-sulfocarbamoyl toxin, it contributed very little to valve module. Separation of toxin oxidation products was the total sample toxicity. A sample from February 2009 carried on reversed-phase C18 column (Restek, Pinnacle II reached (806.95 mg STX eq. kg 1), the regulatory limit of 150 4.6 mm, 5 mm particle size) protected by a guard 800 mg STX eq. kg 1. cartridge Pinnacle II C18, 20 4.0 mm (Varian, SAD). Col- During the same period, ascidian samples from station umn temperature was kept at 30 C, run time 15.00 min S2 showed PSP toxicity with STX, dcSTX, GTX2,3 and C1,2 and partial loopfill volume set to 25 mL for peroxide and toxins present (Figs. 5 and 6). STX was by far the most 50 mL for periodate oxidized samples. Data collection and dominant over other toxins found in the analysed samples, result treatment were performed with the Varian Star with sample concentrations of 193.85 mgkg1, Chromatography software. The PSP toxin profile of ascidian 197.73 mgkg1 and 549.51 mgkg1, in December 2008, samples was identified by comparing with chromatograms March and April 2009, respectively. DcSTX was present in of standard solutions. all the analysed samples from this station, but in low con- As the fluorescence is compound dependent it requires centrations, from an almost undetectable 9.90 mgkg 1– individual toxin calibration and Toxicity Equivalence Fac- 63.32 mgkg 1, at the end of March when it was the only tors (TEFs) to calculate the total mg STX dihydrochloride toxin detected in the sample. There was only one episode of equivalents per 100 g of sample. Specific toxicity factors of GTX2,3 presence at a concentration of 427.40 mgkg1 PSP toxins by EFSA (2009) were used for calculations. Ac- during March 2009, when it was found in combination with cording to Lawrence’s pre-column HPLC-FLD method, STX and dcSTX. C1,2 was detected only once at this station toxins that co-elute are determined together (GTX1 and (353.85 mgkg1). Fig. 6 shows STX as the dominant GTX4; GTX2 and GTX3; dcGTX2 and dcGTX3; C1 and C2) by contributor to the overall toxicity on this station, as it has choosing the highest toxicity factor of the two co-eluted the highest toxicity factor and was detected in high con- compounds. Total PSP toxicity was calculated by summing centrations during the investigated period. the toxicity contribution of each quantified toxin expressed At station S3, STX and GTX2,3 were the two dominating in terms of mg STX eq./100 g or mg STX eq. kg 1. Limit of derivatives with concentration ranges of 345.17– detection (LOD) was determined based on a 3:1 signal-to- 653.17 mgkg1 and 150.96–373.69 mgkg1, respectively noise ratio, by comparing measured signals from samples (Fig. 7). With their relatively high concentrations and very spiked with known low concentrations of PSP toxin stan- high toxicity factors, the overall toxicity of the station was dards with those of blank samples. Detection limits were: approaching the regulatory limit in this period (Fig. 8). The 32 R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36

Fig. 2. Ascidian Microcosmus vulgaris; whole body (a) and soft tissue (b). highest total toxicities for this station were exhibited in As shown in Figs. 3–8, STX, dcSTX, GTX2,3, dcGTX2,3, samples collected at the end of January and beginning of GTX5 and C1,2 represent the ascidian PSP toxin profile February 2009. DcSTX was always in relatively low con- determined in samples from the West Istrian coast (Adri- centrations (9.21–96.50 mgkg 1). GTX5 was found in only atic Sea, Croatia) at stations S1, 2 and 3 during the period one ascidian sample from this station in mid-December from December 2008 through May 2009. These toxin types 2008, with a concentration of 41.94 mgkg 1, in addition were found in different combinations in analysed samples, to dcSTX (9.21 mgkg 1) and GTX2,3 (249.77 mgkg 1) that while toxins GTX1,4, NEO and dcNEO were not detected represented the ascidian toxin profile and the amount of during this period. There is also a possibility of undetected toxins the day after contaminated ascidians caused the presence of C3,4 and GTX6 toxins since their standard so- moderate poisoning of six people. C1,2 was recorded only lutions were not commercially available at the time of in April 2009 (258.57 mgkg 1) in combination with dcSTX conducting this study. Ascidian samples harvested at sta- (96.50 mgkg 1). Generally, as GTX5 and C1,2 are N-sulfo- tion S3 the day after reported human poisoning and ana- carbamoyl toxins and were in low concentrations, their lysed by HPLC-FLD pre-chromatographic oxidation method toxicity contribution was negligible. exhibited toxicity (167.26 mg STX eq. kg 1) five times lower than the maximum permitted level according to EU and 4. Discussion Croatian legislatives (800 mg STX eq. kg 1). Moreover, on the same harvesting locations and during the same period The presence of PSP toxins in the northern part of the several shellfish species were tested by Official Method Croatian Adriatic Sea, where toxic Alexandrium species 959.08 (AOAC, 1990) for the presence of Paralytic Shellfish were only sporadically present (Boni et al., 1992; Honsell Poisoning (PSP) toxins, as a part of a continuous monitoring et al., 1996; Ujevic et al., 2012), was for the first time programme, and the results showed that these toxins were revealed in the ascidian M. vulgaris. Samples of M. vulgaris not found in natural populations of the following filtering soft tissue from the three stations along the west coast of bivalves: proteus scallop (Flexopecten proteus), Mediterra- Istrian peninsula (Fig. 1) were analysed by AOAC Official nean scallop (Pecten jacobaeus) and European flat oyster Method 2005.06 (Lawrence et al., 2005) after six people (Ostrea edulis), from stations S1, 2 and 3, respectively experienced dizziness, vomiting, weakness in the legs and (Ujevic et al., 2012). These results may be compared to vision problems as a consequence of ingesting raw ascid- those of Sekiguchi et al. (2001) where ascidians (Hal- ians caught in fishing nets (Basic-Palkovic, 2008). ocynthia roretzi) accumulated about two times more toxins

) S1 -1 700 600 500 400 300 200 100 0 Toxin concentration (µg kg Toxin concentration (µg 22.02. 22.02. 09.03. 06.04. 22.02. 09.03. 06.04. 22.02. 06.04. 22.02. 06.04. STX dcSTX GTX2,3 dcGTX2,3 GTX5 C1,2

Fig. 3. Toxin concentrations in ascidian samples at station S1 (West Istrian area) during 2009. R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36 33

stx dcstx gtx2,3 gtx5 c1,2 dcgtx2.3 stx dcstx gtx2,3 c1,2

25. May 06.Apr 06.04. Apr

09.Mar 30.Mar 2009

Sampling data 09. Mar Sampling data 22.Feb 29. Dec. 2008 2009 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 Toxin concentration (µg STX eq. kg-1) Toxin concentration (µg STX eq. kg-1)

1 Fig. 4. Total PSP toxicity (mg STX eq. kg 1) in ascidian samples at station S1. Fig. 6. Total PSP toxicity (mg STX eq. kg ) in ascidian samples at station S2.

than other bivalves (scallop, mussel, oyster, short-necked A. minutum and A. tamarense have already been re- clam), although they ingested equal amounts of A. tamar- ported in coastal areas of the Adriatic Sea (Boni et al., 1992; ense. When compared to the paper of Ujevic et al. (2012) Honsell et al., 1996; Ujevic et al., 2012) as potential causa- fi fi that revealed the rst PSP toxin pro le for mussels M. tive organisms of PSP in shellfish. It should be mentioned galloprovincialis from the Croatian part of the Adriatic Sea, that Xie et al. (2013) and Chou et al. (2004) found GTX1–4 and found mussels from the West Istrian coast that were to be the only PSP toxins present, while Chang et al. (1997) affected during the same period from January through April discovered NeoSTX to be the dominant one, but also STX 2009 by STX, GTX2,3 and dcSTX, our analysis of ascidians and GTX1–4inA. minutum profiles. In the study of Asakawa fi showed a more diverse toxin pro le than the mussels, but et al. (1995), GTX4 and C2 are identified as major contrib- toxins were in lower concentrations. A small content of N- utors to A. tamarense toxin profile from Hiroshima Bay in sulfocarbamoyl (C1,2 and GTX5) and decarbamoyl Japan. Kwong et al. (2006) recorded a higher content of (dcGTX2,3) toxins was observed in ascidian, but not in carbamate toxins in mussels, although they ingested di- mussel tissue (Ujevic et al., 2012). C1,2 was found in April noflagellates (Alexandrium fundyense) that had N-sulfo- 2009 at all three stations (S1, 2 and 3) along the West carbamoyl as a more dominant group in their toxin profile. m 1 Istrian coast in concentrations of 326.21 gkg, Although we do not suspect A. fundyense to be the causative m 1 m 1 353.85 gkg and 258.57 gkg , respectively. GTX5 was organism for presently reported PSP toxicity, this could be detected only twice in all the analysed samples with con- one of the possible explanations for the ascidian PSP profile m 1 m 1 centrations of 41.94 gkg at S3 and 585.21 gkg at S1, revealed in this study where the major toxins were carba- in December 2008 and February 2009, respectively. These mates (STX and GTX2,3) detected in more than half of are the N-sulfocarbamoyl toxins that make very low contaminated ascidian samples (Fig. 9). However, the same contribution to the total sample toxicity because of their culture strains of phytoplankton may exhibit different PSP extremely low toxicity factor 0.01, according to EFSA profiles across both local and regional-scale spatial range, (2009). Decarbamoyl dcGTX2,3 was only evident in two which complicates the comparison (Ichimi et al., 2002). ascidian samples from station S1 (February and April) with Ciminiello et al. (1995) documented that mussels (M. gal- relatively small contribution to the overall toxicity (relative loprovincialis) from the north-west Adriatic Sea contain low toxicity factors of 0.2 and 0.4; EFSA, 2009; Fig. 8.), while levels of GTX2,3 from A. minutum, Pistocchi et al. (2012) dcSTX was detected in almost all the samples with con- came to the same conclusion and Abouabdellah et al. m 1 centrations not exceeding 96.50 gkg . The most domi- (2008) concluded the same for mussels from southern nant toxins at all three stations were GTX2,3 and STX, with Atlantic coasts of Morocco, while Alvarez et al. (2009) noted m 1 concentration ranges from 150.96 to 655.72 gkg and two PSP episodes in Chile with mostly STX and GTX2,3 – m 1 123.85 653.17 gkg , respectively, that contributed the toxins present in shellfish tissue. However, it has to be most to the overall toxicity of the ascidian samples. noted that bivalves also become toxic by ingesting Gym- nodinium,aswellasPyrodinium dinoflagellates. Bivalves contaminated by Gymnodinium reveal profiles with N-sulfocarbamoyl and decarbamoyl toxins: dcGTX2,3, C1,2, S2 dcSTX and GTX5 (Rodrigues et al., 2012). 700 Information regarding ascidian toxicity is scarce. As ) 600 ascidians are suspension-feeding organisms that ingest 500 considerable amounts of plankton, suspended organic 400 matter and particles mostly in the 0.5–2 mm size range 300 (Riisgård and Larsen, 2000; Tatián et al., 2002) there is a

200 risk of accumulating high levels of toxins in their tissues. ﬂ 100 In addition, toxic dino agellates can form resting cysts

Toxin concentration (µgToxin concentration kg that, some authors argue, might contain a ten to thousand 0 29.12. 09.03. 06.04. 29.12. 09.03. 30.03. 06.04. 25.05. 09.03. 06.04. fold higher saxitoxin concentration than the mobile cells STX dcSTX GTX2,3 C1,2 (White and Lewis, 1982). These cells sink to the bottom of Fig. 5. Toxin concentrations in ascidian samples at station S2 (West Istrian the sea and accumulate there until beneﬁcial growth area) during 2008–2009 sampling period. conditions appear. Hamer et al. (2001) also considered a 34 R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36

S3 700 )

-1 600 500 400 300 200 100

Toxin concentration (µg (µg Toxinkg concentration 0 26.01.09.02.09.03. 17.12.29.12.09.02.22.02.06.04. 17.12.26.01.22.02.09.03. 17.12. 06.04. STX dcSTX GTX2,3 GTX5 C1,2

Fig. 7. Toxin concentrations in ascidian samples at station S3 (West Istrian area) during 2008–2009 sampling period. Note: 17th December 2008, is the day after human poisoning. hazard of importing toxic cysts or vegetative cells of alien 5. Conclusions organisms via ship ballast waters. Recorded toxicity during the December to May period of this study is a logical PSP-contaminated ascidians from the West Istrian area extension of enhanced mixing and resuspension of benthic of the Adriatic Sea revealed complex toxin profiles organic matter that ensures higher nutrient supply during including the most potent carbamates STX and GTX2,3, less the winter months. The majority of PSP toxin contami- potent decarbamoyls dcGTX2,3 and dcSTX, and the least nated ascidian samples were from the January to April toxic N-sulfocarbamoyl group represented by GTX5 and period which is in agreement with periodicity found for C1,2. Six people suffered mild symptoms of PSP, as dizzi- the Mediterranean coast (Taleb et al., 2001), although ness, vomiting, weakness in the legs and vision problems Mons et al. (1998) argue that dinoflagellates develop at after eating fresh ascidians. Even though they are currently relatively high temperatures and abundant sunlight, not commercially significant, ascidians are a valuable therefore in Europe cases of human intoxication and additional source of seafood. Investigations of biotoxin mortality occur mainly between May and November. Po- occurrences in different marine organisms contributes to tency of PSP toxins accumulated in filter feeding organ- clarifying their role and fate in the marine food web, as isms is well demonstrated in the example of lethal there may be variability of toxin composition and their poisoning of two fishermen in Chile, in 2002, three to four metabolic transformation in different organisms producing hours after consumption of contaminated ribbed mussels. modified forms of the toxins and possibly increasing their These mussels reached a toxicity of 8575 mg of STX eq. toxicity. There may be expected to be an increased number kg 1 in shellfish meat measured by mouse bioassay of toxic episodes throughout the world as red tide phyto- (García et al., 2004). Undoubtedly, very important is sur- plankton can be stimulated by burgeoning coastal pollution veillance of PSP toxins in lower levels of marine food webs and aquaculture activity in coastal waters that can amplify that are the vectors of PSP toxin transmission to higher nutrient enrichment. Nevertheless, many red tide species consumers and ultimately human populations. Our form resting stages, spores or cysts that can persist for a investigation revealed a new vector of PSP toxins in the long time and may be transported by the ballast water of Adriatic Sea, suggesting that consumption of contami- ships or dispersed within a region by currents, upwellings, nated ascidians poses an additional route for causing tides and storms. Possible spreading of Paralytic Shellfish shellfish poisoning in consumers.

100 Mean Mean±SD Min-Max 80 stx dcstx gtx2,3 gtx5 c1,2 60 06.Apr 09.Mar 22.Feb 40

09.Feb Percentage (%) 26.Jan 20

Sampling data Sampling 29.Dec

200817.Dec 2009 0 0 100 200 300 400 500 600 700 800 STX GTX2,3 C1,2 dcGTX2,3 GTX5 dcSTX Toxin concentration (µg STX eq. kg-1) Fig. 9. Mass percentage of PSP toxins in ascidian tissue according to all Fig. 8. Total PSP toxicity (mg STX eq. kg 1) in ascidian samples from station obtained results, STX (n ¼ 7), GTX2,3 (n ¼ 8), C1,2 (n ¼ 3), dcGTX2,3 (n ¼ 2), S3. GTX5 (n ¼ 2) and dcSTX (n ¼ 13). R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36 35

Poisoning to other regions of the Adriatic Sea may have Protist 150, 245–255. fi serious health and economic consequences. García, C., Bravo, M.C., Lagosa, M., Lagos, N., 2004. Paralytic shell sh poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords. Toxicon 43, 149–158. Acknowledgements Gessner, B.D., Schloss, M.S., 1996. A population-based study of paralytic shellfish poisoning in Alaska. Alsk. Med. 38 (2), 54–58. Goodbody, I., 2000. Diversity and distribution of ascidians (Tunicata) at Erasmus Mundus Master of Science in Marine Biodi- the Pelican Cays, Belize. Atoll Res. Bull. 480, 302–326. versity and Conservation scholarship and the Croatian Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79–99. Ministry of Science, Education and Sports of the Republic of Hamer, J.P., Lucas, I.A.N., McCollin, T.A., 2001. Harmful dinoflagellate Croatia (through the grant 001-0010501-0848) supported resting cysts in ships’ ballast tank sediments: potential for intro- this study. duction into English and Welsh waters. Phycologia 40, 246–255. Harada, T., Oshima, Y., Yasumoto, T., 1982. Structures of two paralytic shellfish toxins, gonyautoxins V and VI, isolated from a tropical Conflict of interest dinoflagellate, Pyrodinium bahamense var. Compressa. Agric. Biol. Chem. 46, 1861–1864. The authors declare that there are no conflict of Honsell, G., Polleti, R., Pompei, M., Sidari, L., Milandri, A., Casadei, C., Viviani, R., 1996. Alexandrium minutum Halim and PSP contamination interests. in the northern Adriatic Sea (Mediterranean Sea). In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (Eds.), Harmful and Toxic Algal Blooms. UNESCO, Paris, France, pp. 77–80. References Ichimi, K., Suzuki, T., Ito, A., 2002. Variety of PSP toxin profiles in various culture strains of Alexandrium tamarense and change of toxin profile Abbott, J.P., Flewelling, L.J., Landsberg, J.H., 2009. Saxitoxin monitoring in in natural A. tamarense population. J. Exp. Mar. Biol. Ecol. 273 (1), 51– three species of Florida puffer fish. Harmful Algae 8, 343–348. 60. Abouabdellah, R., Taleb, H., Bennouna, A., Erler, K., Chafik, A., Moukrim, A., Kwong, R.W.M., Wang, W.X., Lam, P.K.S., Yu, P.K.N., 2006. The uptake, 2008. Paralytic shellfish poisoning toxin profile of mussels Perna distribution and elimination of paralytic shellfish toxins in mussels perna from southern Atlantic coasts of Morocco. Toxicon 51 (5), 780– and fish exposed to toxic dinoflagellates. Aquat. Toxicol. 80, 82–91. 786. Lagos, N., Andrinolo, D., 2000. Paralytic shellfish poisoning (PSP): toxi- Alvarez, G., Uribe, E., Avalos, P., Marino, C., Blanco, J., 2009. First identi- cology and kinetics. In: Botana, L.M. (Ed.), Seafood and Freshwater fication of azaspiracide and spirolides in Mesodesma donacium and Toxins: Mode of Action, Pharmacology and Physiology. Marcel Dekker Mulinia edulis from Northern Chile. Toxicon 55 (2–3), 638–641. Inc., New York, pp. 203–215. Anderson, D.M., White, A.W., 1992. Marine biotoxins at the top of the food Landsberg, J.H., 2002. The effects of harmful algal blooms on aquatic or- chain. Oceanus 35 (3), 55–61. ganisms. Rev. Fish. Sci. 10, 113–390. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal Lawrence, J.F., Niedzwiadek, B., Menard, C., 2005. Quantitative determi- blooms and eutrophication: nutrient sources, composition, and con- nation of paralytic shellfish poisoning toxins in shellfish using pre- sequences. Estuaries 25 (4b), 704–726. chromatographic oxidation and liquid chromatography with AOAC, 1990. Paralytic shellfish poison. Biological method. Final action. In: fluorescence detection: collaborative study. J. AOAC Int. 88, 1714– Hellrich, K. (Ed.), Official Methods of Analysis, 15th ed. Association of 1732. Official Analytical Chemists, Arlington, Virginia, USA, pp. 881–882. Lilly, E.L., Kulis, D.M., Gentien, P., Anderson, D.M., 2002. Paralytic shellfish Sec 959.08. poisoning toxins in France linked to a human-introduced strain of Asakawa, M., Miyazawa, K., Takayama, H., Noguchi, T., 1995. Dinoflagellate Alexandrium catenella from the western Pacific: evidence from DNA Alexandrium tamarense as the source of paralytic shellfish poison and toxin analysis. J. Plankton Res. 24, 443–452. (PSP) contained in bivalves from Hiroshima Bay, Hiroshima Prefec- López-Rivera, A., Pinto, M., Insinilla, A., Suárez Isla, B., Uribe, E., ture, Japan. Toxicon 33 (5), 691–697. Alvarez, G., Lehane, M., Furey, A., James, K.J., 2009. The occurrence of Basic-Palkovic, D., 2008. Sestero Rovinjaca otrovalo se morskim jajima. domoic acid linked to a toxic diatom bloom in a new potential vector: http://www.rovinj.hr/rovinj/press/56 (accessed 10.04.13.). the tunicate Pyura chilensis (piure). Toxicon 54 (6), 754–762. Boni, L., Mancini, L., Meandri, A., Poletti, R., Pompei, M., Viviani, R., 1992. Marasovic, I., Nincevic, Z., Pavela-Vrancic, M., Orhanovic, S., 1998. A survey First cases of diarrhoetic shellfish poisoning in the Northern Adriatic of shellfish toxicity in the central Adriatic Sea. J. Mar. Biol. Assoc. U. K. Sea. In: Vollenweider, R.A., Marchetti, R., Viviani, R. (Eds.), Marine 78, 745–754. Coastal Eutrophication. Elsevier, Amsterdam, pp. 419–426. Mons, M.N., van Egmond, H.P., Speijers, G.J.A., 1998. Paralytic Shellfish Carballo, J.L., 2000. Larval ecology of an ascidian tropical population in a Poisoning; a Review, vol. 47. National Institute of Public Health and Mediterranean enclosed ecosystem. Mar. Ecol. Prog. Ser. 195, 159–167. the Environment, Bilthoven, The Netherlands. Chang, F.H., Anderson, D.M., Kulis, D.M., Till, D.G., 1997. Toxin production Montresor, M., John, U., Beran, A., Medlin, L.K., 2004. Alexandrium tamu- of Alexandrium minutum (Dinophyceae) from the bay of plenty, New tum sp. nov. (Dinophyceae): a new nontoxic species in the genus Zealand. Toxicon 35, 393–409. Alexandrium. J. Phycol. 40, 398–411. Chen, C.Y., Chou, H.N., 1998. Transmission of the paralytic shellfish Orhanovic, S., Nincevic, Z., Marasovic, I., Pavela-Vrancic, M., 1996. Phyto- poisoning toxins, from dinoflagellate to gastropod. Toxicon 36, 515– plankton toxins in the central Adriatic Sea. Croat. Chem. Acta 69, 291– 522. 303. Chou, H.N., Chen, Y.M., Chen, C.Y., 2004. Variety of PSP toxins in four Pavela-Vrancic, M., Marasovic, I., 2004. Paralytic shellfish poisoning (PSP) culture strains of Alexandrium minutum collected from southern in the central Adriatic Sea. Croat. Chem. Acta 77 (4), 627–631. Taiwan. Toxicon 43 (3), 337–340. Philp, R.B., Leung, F.Y., Bradley, C., 2003. A comparison of the metal Ciminiello, P., Fattorusso, E., Magno, S., Oshima, Y., Poletti, R., Viviani, R., content of some benthic species from coastal waters of the Florida Yasumoto, T., 1995. Determination of PSP toxins in mussels from the Panhandle using high-resolution inductively coupled plasma mass Adriatic Sea. Mar. Pollut. Bull. 30, 733–735. spectrometry (ICP-MS) analysis. Arch. Environ. Contam. Toxicol. 44, Ciminiello, P., Fattorusso, E., Fiorino, M., Montresor, M., 2000. Saxitoxin 218–233. and neosaxitoxin as toxic principles of Alexandrium andersoni Pistocchi, R., Guerrini, F., Pezzolesi, L., Riccardi, M., Vanucci, S., (Dinophyceae) from the Gulf of Naples, Italy. Toxicon 38, 1871–1877. Ciminiello, P., Dell’Aversano, C., Forino, M., Fattorusso, E., Deeds, J.R., Landsberg, J.H., Etheridge, S.M., Pitcher, G.C., Longan, S.W., Tartaglione, L., Milandri, A., Pompei, M., Cangini, M., Pigozzi, S., 2008. Non-traditional vectors for paralytic shellfish poisoning. Mar. Riccardi, E., 2012. Toxin levels and profiles in microalgae from the Drugs 6, 308–348. North-Western Adriatic sea – 15 years of studies on cultured species. EFSA, 2009. Scientific opinion of the panel on contaminants in the food Mar. Drugs 10, 140–162. chain on a request from the European commission on marine bio- Pomati, F., Sacchi, S., Rossetti, C., Giovannardi, S., Onodera, H., Oshima, Y., toxins in shellfish – saxitoxin group. EFSA J. 1019, 1–76. Neilan, B.A., 2000. The freshwater cyanobacterium Planktothrix sp. FAO, 2004. Marine Biotoxins. In: FAO Food and Nutrition Paper, vol. 80. FP1: molecular identification and detection of paralytic shellfish Food and Agriculture Organization of the United Nations, Rome, poisoning toxins. J. Phycol. 36, 553–562. p. 278. Relox Jr., R.J., Bajarias, F.A., 2003. Harmful algal blooms (HABs) in the Gallacher, S., Smith, E.A., 1999. Bacteria and paralytic shellfish toxins. Philippines. In: Furuya, K., Fukuyo, Y. (Eds.), Extended Abstracts of 36 R. Roje-Busatto, I. Ujevic / Toxicon 79 (2014) 28–36

Workshop on Red Tide Monitoring in Asian Coastal Waters. The Tatián, M., Sahade, R., Kowalke, J., Kivatinitz, S.C., Esnal, G.B., 2002. Food University of Tokyo, Tokyo, pp. 65–68. availability and gut contents in the ascidian Cnemidocarpa verrucosa Riisgård, H.U., Larsen, P.S., 2000. Comparative ecophysiology of active at Potter Cove, Antactica. Polar Biol. 25, 58–64. zoobenthic filter feeding, essence of current knowledge. J. Sea Res. 44, Trainer, V.L., 2002. Harmful algal blooms on the U.S. west coast. In: 169–193. Taylor, F.J.R., Trainer, V.L. (Eds.), Harmful Algal Blooms in the PICES Rodrigues, S.M., de Carvalho, M., Mestre, T., Ferreira, J.J., Coelho, M., Region of the North Pacific. PICES, Sidney, B.C., Canada, pp. 89–103. Peralta, R., Vale, P., 2012. Paralytic shellfish poisoning due to ingestion PICES scientific report no. 23. of Gymnodinium catenatum contaminated cockles – application of Ujevic, I., Roje, R., Nincevic-Gladan, Z., Marasovic, I., 2012. First report of the AOAC HPLC official method. Toxicon 59, 558–566. Paralytic Shellfish Poisoning (PSP) in mussels (Mytilus gallopro- Sekiguchi, K., Sato, S., Kaga, S., Ogata, T., Kodama, M., 2001. Accumulation vincialis) from eastern Adriatic Sea (Croatia). Food Control 25, 285– of paralytic shellfish poisoning toxins in bivalves and an ascidian fed 291. on Alexandrium tamarense cells. Fish. Sci. 67, 301–305. Vale, P., 2008. Fate of benzoate paralytic shellfish poisoning toxins from Shimizu, I., 1987. Dinoflagellate toxins. In: Taylor, F.J.R. (Ed.), The Biology Gymnodinium catenatum in shellfish and fish detected by pre-column of Dinoflagellates. Blackwell Scientific Publications, Oxford, pp. 282– oxidation and liquid chromatography with fluorescence detection. J. 315. Chromatogr. A 1190, 191–197. Shimizu, S.E., Yoshioka, M., 1981. Transformation of paralytic shellfish Vila, M., Garcés, E., Masó, M., Camp, J., 2001. Is the distribution of the toxic toxins as demonstrated in scallop homogenates. Science 212, 547– dinoflagellate Alexandrium catenella expanding along the NW Medi- 549. terranean coast? Mar. Ecol. Prog. Ser. 222, 73–83. Shumway, S.E., 1990. A review of the effects of algal blooms on shellfish White, A.W., 1979. Dinoflagellate toxins in phytoplankton and and aquaculture. J. World Aquac. Soc. 21, 65–104. zooplankton fractions during a bloom of Gonyaulax excavatum. In: Stamatis, N., Monios, G., Stergiou, D., Vafidis, D., 2007. Evaluation of some Taylor, D.L., Seliger, H.H. (Eds.), Toxic Dinoflagellate Blooms: Proc. 2nd quality parameters in spinialo of sea squirt (Microcosmus sabatieri) Int. Conf. Elsevier, pp. 381–384. from Kalymnos Island – Greece. In: Proceedings of 5th International White, A.W., 1980. Recurrence of kills of Atlantic herring (Clupea harengus Congress on Food Technology, “Nikolaos Germanos Congress Centre” harengus) caused by dinoflagellate toxins transferred through her- of HELEXPO, Thessaloniki, Greece, vol. 3, pp. 624–631. bivorous zooplankton. Can. J. Fish. Aquat. Sci. 37, 2262–2265. Stamatis, N., Arkoudelos, J., Vafidis, D., 2008. Differences in chemical, White, A.W., Lewis, C.M., 1982. Resting cysts of the toxic, red tide dino- microbial and sensory quality parameters of the marinated ascidia flagellate Gonyaulax excavata in Bay of Fundy sediments. Can. J. Fish. Microcosmus sabatieri Roule, 1885 during storage at 6 C under Aquat. Sci. 39, 1185–1194. vacuum conditions. Int. J. Food Sci. Technol. 43, 1705–1713. Xie, W., Liu, X., Yang, X., Zhang, C., Bian, Z., 2013. Accumulation and Sullivan, J.J., Iwaoka, W.T., Liston, J., 1983. Enzymatic transformation of depuration of paralytic shellfish poisoning toxins in the oyster Ostrea PSP toxins in the littleneck clam (Protothaca staminea). Biochem. rivularis Gould – Chitosan facilitates the toxin depuration. Food Biophys. Res. Commun. 114, 465–472. Control 30, 446–452. Taleb, H., Vale, P., Jaime, E., Blaghen, M., 2001. Study of paralytic shellfish Yen, I.C., Rojas de Astudillo, L., Soler, J.F., La Barbera-Sanchez, A., 2004. poisoning toxin profile in shellfish from the Mediterranean shore of Paralytic shellfish poisoning toxins in green mussels (Perna viridis) Morocco. Toxicon 39 (12), 1855–1861. form the Gulf of Paria, Trinidad. Toxicon 44, 743–747.