First Detection and Seasonal Variation of Lipophilic Toxins Okadaic Acid, Dinophysistoxin-1, and Yessotoxin in Korean Gastropods

2000

Journal of Food Protection, Vol. 75, No. 11, 2012, Pages 2000–2006 doi:10.4315/0362-028X.JFP-12-192 Copyright G, International Association for Food Protection

KA JEONG LEE,1 JONG SOO MOK,2 KI CHEOL SONG,2 HONGSIK YU,1 DOO SEOG LEE,1 JEE HYUNG JUNG,3 AND 1

JI HOE KIM * Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021

1Food Safety Research Division, National Fisheries Research & Development Institute, 408-1, Sirang-ri, Gijang-up, Gijang-gun, Busan 619-705, Republic of Korea; 2West Sea Fisheries Research Institute, National Fisheries Research & Development Institute, 14, Seonnyobawiro, Eulwangdong, Junggu, Incheon 400-420, Republic of Korea; and 3College of Pharmacy, Pusan National University, Geumjeong-gu, Busan 609-735, Republic of Korea

MS 12-192: Received 2 May 2012/Accepted 13 July 2012

ABSTRACT Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography–tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

The accumulation of lipophilic toxins in marine have been correlated with the consumption of carnivorous animals, which causes diarrhetic shellfish poisoning (3, 9) and herbivorous (1, 21) gastropods. The accumulation (DSP), is a worldwide public health problem (6, 7). and transport of marine toxins through food chains can Lipophilic toxins associated with DSP, such as okadaic cause vectorial intoxication of higher trophic-level consum- acid (OA) and dinophysistoxins (DTXs), lead to severe ers that do not feed directly on toxic phytoplankton. Studies gastrointestinal illness accompanied by diarrhea, nausea, have demonstrated marine toxin accumulation in carnivo- vomiting, abdominal pain, and shivering (35). OA and rous gastropods fed a toxic diet (2, 26). Our previous studies DTXs, along with other lipophilic toxins, i.e., pectenotoxins found Dinophysis spp. in seawater and low levels of (PTXs) and yessotoxins (YTXs), have been shown to be lipophilic toxins in bivalves off the southern coast of Korea produced by dinoflagellate species (6, 8, 18). These toxins (13, 15, 19). Carnivorous gastropods that prey on lipophilic accumulate in filter-feeding bivalves, such as mussels, toxin–containing bivalves may become vectors of these oysters, and clams, which are the most widely studied disease-causing toxins. Although little information is vectors of shellfish toxins causing illnesses, including DSP, available on DSP toxins in gastropods, we hypothesized paralytic shellfish poisoning (PSP), and amnesic shellfish that carnivorous and herbivorous gastropods may be vectors poisoning. Marine toxins have also been detected in various of these toxins because both PSP and DSP toxins are animals, such as crustaceans, gastropods, and fish (3, 6, 9, produced by phytoplankton. 28). In Korea, gastropods are commonly consumed as Little attention has been given to lipophilic toxins in seafood. Although no DSP incident caused by gastropods gastropods because these animals do not feed on phyto- has been reported, the postulated accumulation of DSP plankton (dinoflagellate species). However, PSP incidents toxins in these animals has the potential to become a public health problem. In this study, we monitored the occurrence * Author for correspondence. Tel: (z82)-51-720-2610; Fax: (z82)-51- of lipophilic toxins (OA, DTX1, PTX2, and YTX) in two 720-2619; E-mail: [email protected]. species of carnivorous gastropods (Neptunea cumingii and J. Food Prot., Vol. 75, No. 11 LIPOPHILIC TOXINS IN KOREAN GASTROPODS 2001

Rapana venosa) and a herbivorous species (Batillus negative ionization mode for the transitions m/z 803.5 R 255.5 cornutus) in Korea from 2009 to 2010. (OA), m/z 817.5 R 255.5 (DTX1), m/z 903.5 R 137.5 (PTX2), and m/z 1,141.5 R 1,061.5 (YTX), and calibration curves were MATERIALS AND METHODS constructed for all four parent compounds. Full scans were performed by fragmenting the precursor ions m/z 803.5 (OA), m/ Reagents. High-performance liquid chromatography (HPLC)– z 817.5 (DTX1), m/z 903.5 (PTX2), and m/z 1,141.5 (YTX) at 48, grade solvents (acetonitrile and methanol) and an analytical-grade 48, 60, and 60 eV collision energy, respectively. These separations solvent (methanol) were obtained from Merck (Darmstadt, Ger- were performed on a Luna C18(2) column (150 by 2.0 mm, 5 mm; many). Mass spectrometry–grade reagents (formic acid and Phenomenex, Torrance, CA) preceded by a guard column cartridge ammonium formate) were purchased from Fluka (Buchs, Germany) (4.0 by 2.0 mm; Phenomenex). Eluent A was water and B was or Sigma (St. Louis, MO). Distilled water was passed through a acetonitrile-water (95:5), with both containing 2 mM ammonium Milli-Q water purification system (Millipore, Bedford, MA) and formate and 50 mM formic acid. The analytical conditions have used for the preparation of HPLC mobile phases. been described in detail previously, and the same adjustments were used in the present study (13, 24). Standard toxins. Standard solutions of OA (14.3 mg/ml), Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021 PTX2 (8.6 mg/ml), and YTX (5.3 mg/ml) in methanol were RESULTS obtained from the Institute for Marine Biosciences of the Canadian National Research Council (Halifax, Nova Scotia, Canada). DTX1 The lipophilic toxin profiles obtained from gastropod was purchased from Wako Chemicals (Osaka, Japan) and dis- tissues are listed in Table 1. Eighty specimens of N. solved in HPLC-grade methanol. Multistandard solutions for toxin cumingii, 65 specimens of R. venosa, and 95 specimens of calibration and quantification were prepared in HPLC-grade B. cornutus were submitted for lipophilic toxin analyses. methanol at concentrations of 10, 25, 50, and 100 ng/ml. OA, DTX1, and YTX were detected in meat and digestive gland samples of all gastropod species, whereas no PTX2 Gastropod samples and preparation for selective reaction was found in any sample tested. The activity rates of each monitoring with LC-MS/MS. Two species of carnivorous gastropods (N. cumingii and R. venosa) and one herbivorous type of toxin found were species and organ specific. gastropod (B. cornutus) were collected between May 2009 and Generally, the activity rates of the lipophilic toxin–positive December 2010 at a fish market in Tongyeong, a city on the samples and mean toxin concentrations in each organ were southern coast of Korea, and transported to the laboratory in higher in the carnivorous gastropod species than in the crushed ice. When possible, five specimens per species were herbivorous species. Lipophilic toxins were frequently examined each month. Carnivorous gastropods (N. cumingii and R. detected in the digestive glands of the gastropods, whereas venosa) were separated into meat, salivary gland, and digestive no lipophilic toxin was detected in the salivary glands of gland portions for toxin analyses, and the herbivorous species (B. carnivorous gastropods. cornutus) was separated into meat and digestive gland portions. The percentages of gastropods containing any toxic | The dissected tissues were homogenized with a 9 volume of component (OA, DTX1, and/or YTX) in the digestive 90% methanol, and the extracts were centrifuged at 5,000 | g for glands were 43.8% for N. cumingii, 53.8% for R. venosa, 5 min. An aliquot of supernatant was passed through a 0.2-mm- pore-size membrane filter (Advantec, Tokyo, Japan) for direct and 5.3% for B. cornutus. The highest concentrations of injection into a liquid chromatography–tandem mass spectrometry OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the (LC-MS/MS) system. digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands Sample treatment for full scanning with LC-MS/MS. To of N. cumingii. identify lipophilic toxins, we used the solid-phase extraction The total ion chromatograms and full-scan LC-MS/MS method of Torgersen et al. (32) with minor modifications. Tissue spectra of the reference OA and YTX and 90% methanol samples (2 g) were extracted with 8 ml of 90% methanol, as extracts from the digestive glands of R. venosa (21.5 ng/g described above. The methanol extract was passed through a 0.2- OA) and N. cumingii (13.7 ng/g YTX) are shown in mm-pore-size membrane filter. Sep-Pak Plus C (Waters, Milford, 18 Figure 1. The retention times for OA and YTX correspond- MA) cartridges were conditioned with 5 ml of methanol and 3 ml of water. After conditioning, 5 ml of methanol extract was diluted ed to those obtained for standard toxins (Fig. 1A and 1B). with 5 ml of 20% methanol and loaded onto a cartridge. The The major ion fragment peaks were observed at m/z 803.5, cartridges were washed with 6 ml of 20% methanol, and the toxins 563.5, 255.4, and 151.1 for R. venosa and at m/z 1,141.5, were eluted with 5 ml of methanol. The methanol elutes were 1,061.5, and 855.5 for N. cumingii. The toxins in gastropods evaporated, and residues were dissolved in 500 mlof90% were easily identified as OA or YTX by comparison with methanol. The toxic solution was passed through a 0.45-mm the spectral data of reference toxins. syringe filter (Sartorius RC4, Vivascience, Hanover, Germany) for Seasonal variations in mean concentrations of OA plus direct injection into a full-scan LC-MS/MS system. DTX1 and YTX in each gastropod organ are shown in Figure 2. The concentrations of OA plus DTX1 in LC-MS/MS analysis. LC-MS/MS analysis of toxins was carnivorous gastropods showed seasonal variations, peaking performed according to the method in a previous report (13) by primarily in spring and summer. The predominant toxin using selective reaction monitoring (SRM) and full scanning. The HPLC unit consisted of a Surveyor MS Pump Plus and Surveyor components (Fig. 3) and annual variation in lipophilic toxin AS Plus (Thermo Electron Finnigan, San Jose, CA). A triple- concentrations in N. cumingii were similar to those observed quadrupole mass spectrometer (Thermo Electron Finnigan TSQ in R. venosa. The nearly negligible levels of lipophilic Quantum Discovery Max; Finnigan) was used for mass detection. toxins in the herbivorous gastropod species (B. cornutus) For determination of toxins, SRM chromatograms were recorded in were detected sporadically and showed no significant 2002 E TAL. ET LEE

TABLE 1. Rate of lipophilic toxin-positive samples and toxin concentrations in tissues of gastropods obtained at the Tongyeong fish market, Korea, during 2009 and 2010a OA DTX1 PTX2 YTX

Gastropod species Digestive Salivary Digestive Salivary Digestive Salivary Digestive Salivary (no. of samples) Meat gland gland Meat gland gland Meat gland gland Meat gland gland

Neptunea cumingii (80) % positive samples 12.5 43.8 0.0 25.0 31.3 0.0 0.0 0.0 0.0 43.8 50.0 0.0 Toxin concn (ng/g) Mean 0.4 1.6 ND 1.5 2.5 ND ND ND ND 0.4 2.0 ND SD 0.5 1.2 — 1.4 1.7 — — — 0.2 4.8 — Min ND ND ND ND ND ND ND ND ND ND ND ND Max 0.8 3.6 ND 3.2 5.0 ND ND ND ND 0.9 13.7 ND Rapana venosa (65) % positive samples 30.8 53.8 0.0 30.8 53.8 0.0 0.0 0.0 0.0 38.5 30.8 0.0 Toxin concn (ng/g) Mean 1.1 6.1 ND 0.8 2.2 ND ND ND ND 0.3 0.2 ND SD 1.3 7.0 — 0.8 3.0 — — — — 0.1 0.0 — Min ND ND ND ND ND ND ND ND ND ND ND ND Max 2.9 21.5 ND 1.8 8.4 ND ND ND ND 0.6 0.2 ND Batillus cornutus (95) % positive samples 15.8 5.3 — 10.5 10.5 — 0.0 0.0 — 36.8 68.4 — Toxin concn (ng/g) Mean 0.4 0.1 — 0.5 0.6 — ND ND — 0.2 0.4 — SD 0.2 0.0 — 0.1 0.5 — ND ND — 0.1 0.2 — Min ND ND — ND ND — ND ND — ND ND — Max 0.6 0.1 — 0.6 1.0 — ND ND — 0.3 0.7 — .Fo rt,Vl 5 o 11 No. 75, Vol. Prot., Food J.

a OA, okadaic acid; DTX1, dinophysistoxin-1; PTX2, pectenotoxin-2; YTX, yessotoxin; ND, not detected; —, not tested; Min, minimum; Max, maximum. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021 September 24 on guest by http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf from Downloaded J. Food Prot., Vol. 75, No. 11 LIPOPHILIC TOXINS IN KOREAN GASTROPODS 2003 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021

FIGURE 1. Chromatograms and fragment ion spectra obtained from a full-scan LC-MS/MS analysis of the digestive glands of gastropods obtained at the Tongyeong fish market, Korea, during 2009 and 2010. Retention times of peaks in the samples were the same as retention times for OA and YTX in the standard solution. A(1), OA in standard solution; A(2), OA in extract of sample (Rapana venosa); A(3), fragment ion spectra of m/z 803.5 in extract of sample; B(1), YTX in standard solution; B(2), YTX in extract of sample (Neptunea cumingii); B(3), fragment ion spectra of m/z 1,141.5 in extract of sample. seasonal variation. The predominant toxin components in were quantified using LC-MS/MS. OA and YTX were the gastropods varied according to feeding habits and year. initially identified by comparing chromatographic properties In carnivorous gastropods (N. cumingii and R. venosa), and spectral data with those of reference standard toxins. DTX1 was the predominant toxin in 2009, whereas OA was The MS/MS spectra of R. venosa and N. cumingii samples predominant in 2010. Although OA and DTX1 were the were essentially identical to those of the OA and YTX predominant toxins in carnivorous gastropods, rare excep- reference standards, respectively (31). The highest lipophilic tions did occur. In the herbivorous gastropod (B. cornutus), toxin concentrations found in 240 gastropod specimens YTX was detected more frequently than OA or DTX1 (21.5 ng of OA per g, 8.4 ng of DTX1 per g, and 13.7 ng of (Fig. 3). YTX per g) (Table 1) in this study are much lower than the European standard (4). The maximum level of lipophilic DISCUSSION toxins allowed in gastropods established by the European Marine biotoxins produced by phytoplankton are found Union (EU) was 0.16 mg of OA equivalents (OA plus DTX in gastropods (1, 3, 9, 10, 16, 21), but very little is known plus PTX) or 1 mg of YTX equivalents per kg of meat. about the anatomical distribution of lipophilic toxins in YTXs have been detected in shellfish from various areas of gastropods. We hypothesized that gastropods would accu- the world. Their presence in shellfish was detected due to mulate lipophilic toxins produced by dinoflagellate species, their acute toxicity in mice after intraperitoneal injection of perhaps by a mechanism of food-chain uptake accumula- lipophilic extracts. However, there are no reports of human tion. In this study, the anatomical distribution of lipophilic intoxications caused by YTXs (7). toxins OA, DTX1, PTX2, and YTX in Korean carnivorous Significant seasonal variation in the mean concentra- and herbivorous gastropods was monitored using LC-MS/ tions of OA plus DTX1 was observed in the carnivorous MS. As we predicted, the lipophilic toxins OA, DTX1, and gastropods N. cumingii and R. venosa (Fig. 2). In previous YTX were found in many gastropod specimens. Although studies, we detected only low concentrations of lipophilic gastropods have been considered to be potential vectors of toxins in bivalves such as oysters (Crassostrea gigas) and DSP (10), toxins were found previously in only one 100-g mussels (Mytilus galloprovincialis) from the southern coast sample of Neverita didyma containing 3.2 mg of OA (34). of Korea, but we found significant seasonal variation with In this study, lipophilic toxins in three species of gastropods peaks of toxicity in spring and summer (13, 19). Thus, the 2004 LEE ET AL. J. Food Prot., Vol. 75, No. 11 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021

FIGURE 2. Annual variation in monthly mean toxin concentrations in the digestive glands and meat of gastropods collected at the Tongyeong fish market, Korea, during 2009 and 2010. (A) Neptunea cumingii (digestive gland), (B) N. cumingii (meat), (C) Rapana venosa (digestive gland), (D) R. venosa (meat), (E) Batillus cornutus (digestive gland), (F) B. cornutus (meat). seasonal variation in OA plus DTX1 concentrations in YTX were previously found in Korean bivalves, but no carnivorous gastropods shows the same trend as that of PTXs were observed in gastropods in this study. Direct toxin concentrations in bivalves in Korean coastal waters. comparison of the lipophilic toxin profiles of gastropods and These results suggest that these two seasonal variation bivalves is difficult because they do not coexist spatially or patterns are related. However, the lipophilic toxin profiles of temporally. Considering the wide distribution of PTX2 gastropods and bivalves differed; OA, DTXs, PTXs, and producers, such as Dinophysis spp., off the southeastern

FIGURE 3. Maximum lipophilic toxin concentration detected each month in the digestive glands and meat of gastropods collected at the Tongyeong fish market, Korea, during 2009 and 2010. (A) Neptunea cumingii (digestive gland), (B) N. cumingii (meat), (C) Rapana venosa (digestive gland), (D) R. venosa (meat), (E) Batillus cornutus (digestive gland), (F) B. cornutus (meat). J. Food Prot., Vol. 75, No. 11 LIPOPHILIC TOXINS IN KOREAN GASTROPODS 2005 coast of Korea (13, 19), the lack of PTX2 detection in this method for lipophilic toxins was recently developed (31) study is an interesting result. PTX2 may be rapidly and may substantially improve the detection of lipophilic converted into one of its derivative acids in gastropods, as toxin accumulation in gastropods. has been shown in bivalves (29, 30). In conclusion, lipophilic toxins, including DSP toxins, The concentrations of lipophilic toxins in gastropods were found in Korean gastropods. The total amounts of OA varied according to feeding habitat; they were higher in and DTX1 in the digestive glands of carnivorous gastropods carnivorous than in herbivorous species (Fig. 3). These showed seasonal variations. The concentrations of lipophilic toxins also tended to concentrate in the digestive glands of toxins in gastropod tissues were lower than the regulatory carnivorous and herbivorous gastropods. These results safety limits of the EU. In previous studies, Korean bivalves suggest the presence of different lipophilic toxin uptake were also found to contain lipophilic toxins in concentrations routes depending on feeding habitat. Halstead and Schantz below the regulatory limit (13, 15, 19). Korean gastropods (9) reported that toxins in dinoflagellate species, such as can apparently be deemed safe for human consumption

PSP toxins found in gastropods, have been acquired as a considering DSP toxin contents. However, to protect Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/11/2000/1685139/0362-028x_jfp-12-192.pdf by guest on 24 September 2021 result of predation on toxic bivalve mollusks. A few authors consumers, it is prudent to regard gastropods as potential have reported a positive relationship between PSP toxin vectors of several kinds of marine biotoxins. Moreover, it is concentrations in bivalves and in carnivorous gastropod difficult to predict the levels of lipophilic toxins in tissues (2, 10). DSP toxins, such as OA and DTXs, are carnivorous gastropods in the area, where spatial and highly lipophilic substances, and their lipophilic character temporal variations of lipophilic toxins in bivalves occurred. may increase their potential to accumulate in lipid tissues, Thus, we recommend the implementation of comprehensive such as the digestive glands of marine organisms (25, 35). screening to ensure the safety of gastropod consumption. Carnivorous gastropods prey on bivalves, such as mussels, clams, scallops, and oysters; R. venosa has been reported to ACKNOWLEDGMENT consume about one bivalve per day (or 1.2 g wet weight per This work was funded by a grant from the National Fisheries day) (27). Accumulation of lipophilic toxins by filter- Research and Development Institute of Korea (RP-2012-FS-005). feeding shellfish is a well-known phenomenon in many countries, including Korea (13, 15, 19), Japan (35), The REFERENCES Netherlands (11, 12), Portugal (33), Canada (23), and New 1. 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