First report of contamination of the abalone Haliotis discus hannai by okadaic acid and yessotoxin.
ABSTRACT The presence of the lipophilic toxins okadaic acid (OA), dinophysistoxin-1, pectenotoxin-2, and yessotoxin (YTX) in the abalone Haliotis discus hannai collected between March 2009 and May 2010 was analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the mouse bioassay. Thirty abalone samples collected from 2 sites, Geumdang and Chungdo on the southwest coast of Korea, were submitted for toxin analysis. Lipophilic toxins OA and YTX were detected in the digestive gland of the abalone by LC-MS/MS, but no toxins were detected in the foot muscle. The highest toxin concentrations in the digestive glands of the abalone were 4.7 ng/g OA and 1.3 ng/g YTX. Okadaic acid and YTX were detected mainly during winter and spring. However, dinophysistoxin-I and pectenotoxin-2 were not detected in any of the samples by LC-MS/MS. Diarrhetic shellfish poisoning toxins were not detected in the digestive gland or the foot muscle by mouse bioassay. Although low levels of toxins were detected, this is the first report identifying lipophilic toxins in abalone.
KEY WORDS: diarrhetic shellfish poisoning, lipophilic toxin, okadaic acid, abalone, gastropod, Haliotis discus hannai
Contamination of marine animals with lipophilic toxins, such as those that cause diarrhetic shellfish poisoning (DSP), is a worldwide problem for public health and the fishery industry (FAO 2004, FAO/IOC/WHO 2004). Lipophilic toxins associated with DSP lead to severe gastrointestinal distress, diarrhea, nausea, vomiting, abdominal pain, and shivers, but are usually not fatal. Three groups of toxins with chemically different basic structures have been associated with DSP. The toxins of the okadaic acid (OA) group, such as OA and dinophysistoxins (DTXs), are considered to be responsible for diarrhea. Animal oral administration studies indicate that pectenotoxins (PTXs) and yessotoxins (YTXs) are much less potent toxins than OA analogs (Yasumoto et al. 1985, Aune et al. 2002, Dominguez et al. 2010). It has recently been proposed that YTXs should be excluded from the DSP toxins group because they do not induce diarrhea (Paz et al. 2008).
Lipophilic toxins such as OA, DTXs, PTXs, and YTXs are known to be produced by dinoflagellate species (Lee et al. 1989, FAO 2004, Garcia Camacho et al. 2007). These toxins can accumulate in filter-feeding bivalves such as mussels, oysters, and clams. Bivalves are the most common vectors of marine biotoxins, but they are not the sole vectors. Occasionally, marine biotoxins are also taken up by other organisms, including several carnivorous gastropods, at higher trophic levels in the food chain. Incidents of paralytic shellfish poisoning (PSP) resulting from the consumption of toxic carnivorous gastropods are reported frequently globally (Halstead & Schantz 1984, Shumway 1995, FAO 2004, Deeds et al. 2008). However, herbivorous gastropods have received relatively little attention in the context of public health. Recently, high levels of PSP toxins were found in abalone, a group of herbivorous gastropods (Nagashima et al. 1995, Bravo et al. 1999, Pitcher et al. 2001). Dowsett et al. (2011) has demonstrated PSP toxin accumulation in abalone fed a toxin-supplemented diet. It seems possible that, in addition to carnivorous gastropods, herbivorous gastropods also accumulate marine toxins. Thus, we assumed that abalone can accumulate lipophilic toxins because both PSP and DSP toxins are produced by phytoplankton. On the southeast coast of Korea, low concentrations of lipophilic toxins have been found in bivalves and Dinophysis spp., a major producer of DSP toxins (Kim et al. 2008, Kim et al. 2010, Lee et al. 2011). In the current study, the presence of the lipophilic toxins OA, dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and YTX in the abalone Haliotis discus hannai was investigated by mouse bioassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
MATERIALS AND METHODS
Distilled water was passed through a Milli-Q water purification system (Millipore, Bedford, MA). High-performance liquid chromatography (HPLC)-grade solvents (acetonitrile and methanol) and analytical-grade solvents (acetone, diethyl ether, and methanol) were obtained from Merck (Darmstadt, Germany). Mass spectrometry-grade reagents (formic acid and ammonium formate) and analytical-grade reagents (sodium chloride and Tween 60) were purchased from Fluka (Buchs, Germany), Sigma (St. Louis, MO), or Merck.
Standard solutions OA (14.3 [micro]g/mL), PTX2 (8.6 [micro]g/mL), and YTX (5.3 [micro]g/mL) were obtained, in methanol, from the Institute for Marine Biosciences of the National Research Council (Halifax, Nova Scotia, Canada). Dinophysistoxin-1 was purchased from Wako Chemicals (Osaka, Japan) and dissolved in HPLC-grade methanol. Multistandard solutions for calibration and quantification of toxins were prepared in HPLC-grade methanol at concentrations of 10, 25, 50 and 100 ng/mL.
Abalone (Haliotis discus hannai) were collected every month at 2 sites--Geumdang and Chungdo on the southwest coast of Korea--from March 2009 through to May 2010. These samples were transported to the laboratory on crushed ice. On receipt, samples were shucked immediately and dissected into foot muscle and digestive gland for toxin analysis. Each sample comprised more than 12 abalone collected from the same station. Samples were homogenized and stored at less than -20[degrees]C prior to toxin analysis. Surface water temperature and salinity were measured using a YSI 556 multiprobe system (Yellow Springs, OH).
Mouse bioassays were carried out according to the method of the Japanese Ministry of Health and Welfare (1981). Tissue homogenates of each sample were extracted 3 times with acetone. After removal of the acetone by evaporation, the aqueous suspension was extracted with diethyl ether. The ether solution was washed with distilled water and evaporated. The residue was suspended in 1.0 mL 1% Tween 60 saline solution/20 g. An aliquot (1.0 mL) of this solution, corresponding to 20 g of the tissue sample, was injected intraperitoneally into each of 3 mice. One mouse unit is defined as the minimum amount of toxin needed to kill 2 out of 3 mice within 24 h of injection.
Sample Treatment for LC-MS/MS
To remove matrix interference, we used the solid-phase extraction method of Suzuki et al. (2005) and Torgersen et al. (2005) with minor modifications. The dissected tissue samples were homogenized in 9 volumes of 90% methanol and the extracts were centrifuged at 5,000g for 5 min. An aliquot of the supernatant was passed through a 0.2-[micro]m membrane filter (Adventec, Tokyo, Japan). Sep-Pak Plus C18 cartridges (Waters, Milford, MA) were conditioned with 5 mL methanol and 3 mL water. After conditioning, 5 mL methanol extract was diluted with 5 mL 20% methanol and loaded onto a cartridge. The cartridges were washed with 6 mL 20% methanol and the toxins were eluted in 5 mL methanol. The methanol eluents were evaporated and the residues were dissolved in 500 [micro]L 90% methanol. The toxic solution was passed through a 0.45-[micro]m syringe filter (Sartorius RC4; Vivascience, Hanover, Germany) for direct injection into LC-MS/MS.
Liquid chromatography--tandem mass spectrometry analysis of toxins was performed by selective reaction monitoring (SRM) (Kim et al. 2010). The HPLC unit consisted of a Surveyor MS Pump Plus and a Surveyor AS Plus (Thermo Electron, Finnigan, San Jose, CA). A triple-quadrupole mass spectrometer (Thermo Electron Finnigan TSQ Quantum Discovery Max; Finnigan) was used for mass detection. For identification of toxins, SRM chromatograms were recorded in negative ionization mode for the transitions m/z 803.5 [right arrow] 255.5 (OA), m/z 817.5 [right arrow] 255.5 (DTXI), m/z 903.5 [right arrow] 137.5 (PTX2), and m/z 1141.5 [right arrow] 1061.5 (YTX), and calibration curves were constructed for all of the 4 parent compounds. These separations were performed on a Luna C18 (2) column (150 X 2.0 mm, 5 [micro]m; Phenomenex, Torrance, CA), after first being passed through a guard column cartridge (4.0 x 2.0mm; Phenomenex). Eluent A was water and eluent B was acetonitrile--water (95:5), both containing 2 mM ammonium formate and 50 mM formic acid (Quilliam 2003).
Selective reaction monitoring LC-MS/MS chromatograms for the OA and YTX standards and the cleaned-up methanol extract of the abalone digestive gland (4.7 ng/g OA and 1.3 ng/g YTX) are shown in Figure 1.
Chromatographic peaks for the toxins in the 90% methanolic extracts of abalone digestive gland were rarely distinguishable from the background level (data not shown). However, the background level from matrix interference was reduced significantly after solid-phase extraction. Retention times for OA (11.3 min) and YTX (12.8 min) obtained from the abalone digestive gland extract matched exactly those recorded for authentic toxin standards.
Table 1 shows the highest concentrations of lipophilic (DSP) toxins measured by LC-MS/MS and mouse bioassay in the foot muscle and digestive gland of abalone (Haliotis discus hannai) during the survey period. Thirty abalone samples were collected from 2 sites: Geumdang and Chungdo on the southwest coast of Korea. Okadaic acid and YTX, but not DTX1 or PTX2, were detected in the digestive gland by LC-MS/MS. The highest toxin concentrations in the digestive gland were 4.7 ng/g for OA and 1.3 ng/g for YTX. No toxins were detected in the foot muscle samples. There were some tissue differences in lipophilic toxin concentrations; however, no sample gave positive results for DSP toxin in the mouse bioassay.
Monthly variation in surface water temperature and salinity at each sampling station between March 2009 and May 2010 is shown in Figure 2A and B. The surface water temperature ranged from 9.3[degrees]C in January to 25.3[degrees]C in August, and surface salinity in the region varied from 31.3-33.4. The monthly variation of OA and YTX concentrations in digestive gland of the abalone is shown in Figure 2C and D. While OA and YTX were not detected in the digestive gland between March 2009 and September 2009, these lipophilic toxins were detected at varying levels between October 2009 and May 2010. Although low levels of toxins were detected, the concentrations of OA and YTX showed seasonal variation. The highest concentrations of lipophilic toxins were detected in April 2010 for OA (4.7 ng/g) and February 2010 for YTX (1.3/ng/g).
Certain carnivorous gastropods are known to accumulate PSP toxins that are produced by dinoflagellates, whereas abalone from South Africa and Spain have also been reported to contain PSP toxins (Bravo et al. 1999, Nagashima et al. 1995, Pitcher et al. 2001, Deeds et al. 2008). It is not unusual to find marine toxins in abalone because PSP toxins have been identified in the group previously. In the current study, we attempted to investigate seasonal variation in lipophilic toxin levels in Korean abalone (Haliotis discus hannai) by LC-MS/ MS and mouse bioassay.
[FIGURE 1 OMITTED]
The survey area, the southwest coast of Korea, has a temperate climate. Although, seasonal variation in the surface water temperature was observed at the sampling stations, the water temperature remained more than 9[degrees]C, even in winter. In contrast, salinity showed no remarkable seasonal variation (Fig. 2A, B). The lipophilic toxins OA and YTX were detected in some digestive gland samples from the abalone by LC-MS/MS. However, OA and YTX were not identified easily in 90% methanolic extracts of abalone digestive gland because of high background signal levels. After cleanup by solid-phase extraction, the chromatographic background signal, which reduces LC-MS/MS sensitivity and selectivity, was reduced significantly. This is the first study to identify lipophilic toxins in abalone. The lipophilic nature of OA and YTX may increase their potential to accumulate in lipid tissues in abalone, such as the digestive gland (Yasumoto et al. 1978). Lipophilic toxins were detected in abalone when the water temperature was more than 9.3[degrees]C (Fig. 2). In Korean coastal waters, a Dinophysis sp. that produces lipophilic toxins was detected at water temperatures ranging from 4-27[degrees]C (Kwak et al. 2001). Thus, lipophilic toxins may occur throughout the year in the survey area: Geumdang and Chungdo. Although OA and YTX were detected, the highest toxin concentrations in the 30 samples of digestive gland analyzed (4.7 ng/g for OA; 1.3 ng/g for YTX; Table 1) were much lower than the European regulatory limit for toxins (European Commission 2002). The maximum level of lipophilic toxins in gastropods allowed by the EU is 0.16 mg OA equivalents (sum of the levels of OA, DTXs, and PTXs) per kilogram of meat, and 1 mg of YTX equivalents per kilogram of meat. Thus, the abalone analyzed were deemed safe for human consumption.
Concentrations of OA and YTX in the digestive gland of the abalone peaked during winter and spring (Fig. 2C, D). In previous studies, we found significant seasonal variation in the concentrations of lipophilic toxins in bivalves obtained from the southeast coast of Korea, with toxicity typically peaking in spring and summer (Kim et al. 2010, Lee et al. 2011). Different seasonal fluctuation patterns of lipophilic toxins were observed in the bivalves from the southeast coast of Korea and the abalone in this study. This discrepancy may be caused by or associated with various factors, including different sampling conditions (collection time and region), as well as diet and feeding habits. The pathways by which abalone accumulate lipophilic toxins are unknown, but possible mechanisms include incidental ingestion of macroalgal surface-attached benthic organisms such as dinoflagellates and their cysts, which produce lipophilic toxins (Reguera et al. 2003, Bravo et al. 2010), and ingestion of zooplankton that have grazed on the toxic dinoflagellates (Kozlowsky-Suzuki et al. 2006).
[FIGURE 2 OMITTED]
Understanding the transport pathway for lipophilic toxins in abalone is an essential step toward improving and ensuring food safety, although no relationship between the accumulation of PSP toxins in abalone and the occurrence of toxic algal bloom has been reported (Bravo et al. 1999, Pitcher et al. 2001). The recently developed LC-MS/MS method is sufficiently sensitive for quantification of extremely low levels of lipophilic toxins, even in plankton cells (Puente et al. 2004, Fernandez et al. 2006, Suzuki et al. 2009, Suzuki & Quilliam 2011). Liquid chromatographytandem mass spectrometry may facilitate elucidation of the lipophilic toxin transport pathway in the abalone.
In conclusion, the lipophilic toxins OA and YTX were detected in the abalone Haliotis discus hannai, a herbivorous gastropod. The levels of OA and YTX in the digestive glands of the abalone were negligible, but showed seasonal variation. Further study is necessary to determine the transport pathway and source of the toxins in abalone.
This work was funded by a grant from the National Fisheries Research and Development Institute, Korea (RP-2012-FS-011).
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JI HOE KIM, (1) * KA JEONG LEE, (1) TOSHIYUKI SUZUKI, (2) JONG SOO MOK, (1) KEUNBAWI PARK, (3) JI YEONG KWON, (l) KWANG TAE SON (1) AND KI CHEOL SONG (1)
(1) Food Safety Research Division, National Fisheries Research & Development Institute, 216, Gijanghaeanro, Gijang-up, Gijang-gun, Busan 619-705, Korea; (2) Biochemistry and Food Technology Division, National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fukuura, Kanazawa, Yokohama 236-8648, Japan; (3) Southwest Sea Fisheries Research Institute, National Fisheries Research & Development Institute, 22, Sepodangmeorigil, Hwayang, Yeosu, Jeonnam 556-823, Republic of Korea
* Corresponding author. E-mail: email@example.com
TABLE 1. Highest concentrations of lipophilic toxins measured by LC-MS/ MS and mouse bioassay in the foot muscle and digestive glands of abalone (Haliotis discus hannai) collected at the southwest coast of Korea between March 2009 and May 2010. Concentration (ng/g) as Measured by LC-MS/MS DSP Toxin Level as Measured by Location Sample (n) OA DTXI PTX2 YTX MBA (MU/g) Geumdang Foot ND ND ND ND <0.05 muscle (15) Digestive 3.6 ND ND 1.3 <0.05 gland (15) Chungdo Foot ND ND ND ND <0.05 muscle (15) Digestive 4.7 ND ND 1.0 <0.05 gland (15) DSP, diarrhetic shellfish poisoning; DTX1, dinophysistoxin-1; LC-MS/ MS, liquid chromatography-tandem mass spectrometry; MBA, mouse bioassay; MU, mouse unit; ND, not detected; OA, okadaic acid; PTX2, pectenotoxin-2; YTX, yessotoxin.