Effect of steam cooking on distribution of paralytic shellfish toxins in different tissue compartments of scallops Patinopecten yessoensis

Abstract

This study investigated the effect of steam cooking on distribution of paralytic shellfish poisoning toxins (PSP-toxins) in scallops Patinopecten yessoensis. Toxins analysis by high-performance liquid chromatography showed that most of the PSP-toxins (>70%) were accumulated in viscera and adductor muscle of the raw scallops. Steam cooking induced significant loss (p < 0.05) of PSP-toxins from viscera (16%), adductor muscle (24%), gill and mantle (11%) while 32% of the toxins were retained inside viscera and adductor muscle. Overall, 51% of PSP-toxins leaked out from scallop tissues during steam cooking. However, there was no significant loss (p > 0.05) of PSP-toxins from gonad. Consumption of viscera was the most significant risk factor for causing PSP, while gonad and scallop soup were the second most significant. A rapid PSP test further verified detectable levels of PSP-toxins in all samples. However, possible interfering substance(s) in adductor muscles and gonads might potentially affect the results from this test.

Introduction

Paralytic shellfish poisoning (PSP) is one of the life-threatening biotoxin illnesses caused by the consumption of filter-feeding molluscan shellfish (clams, mussels, oysters, and scallops) that have been contaminated with potent hydrophilic neurotoxins produced by toxic dinoflagellates. Shellfish feeding on some toxigenic dinoflagellates such as Alexandrium spp., Gymnodinium spp., and Pyrodinium spp. may accumulate PSP-toxins in their tissues (Lehane, 2000). Due to the ability of these shellfish to tolerate high levels of toxins (Asp et al., 2004, Bricelj and Shumway, 1998, Cembella et al., 1993), they pose a significant public health risk worldwide. In addition, toxins-contaminated shellfish can lead to drastic economic losses to fisheries resources and shellfish businesses.

PSP-toxins, having a skeleton of 3,4,6-trialkyltetrahydropurine, are fast-acting neurotoxins that can inhibit transmission of nerve impulses by blocking voltage-gated sodium channels in nerves, skeletal, and cardiac muscle fibres, and can lead to death, due to respiratory paralysis (Lehane, 2000). There are more than 20 naturally-occurring derivatives of PSP-toxins (Lagos, 1998, Oshima, 1995). They can be grouped into three major structural sub-groups in descending order of toxicity (Lehane, 2000, Luckas et al., 2003): (1) carbamoyl toxins (e.g., saxitoxin (STX), neosaxitoxin (NEO), and gonyautoxins 1–4 (GTX1–4)), (2) decarbamoyl toxins (e.g., dcSTX, dcNEO, and dcGTX1–4), and (3) N-sulfocarbamoyl toxins (e.g., gonyautoxins 5–6 (GTX5–6 or B1–2) and C-toxins (C1–4)). A considerable emphasis has been placed on the highly toxic STX as a toxicity reference to other PSP-toxins. The human mortality rate of PSP is the highest (13%) among all marine biotoxins (Lagos, 1998). In general, the intake of about 120–180 μg of the STX may cause moderate human illness (Shunway, 1995) and a “tolerable” dose is estimated to be as little as 12 μg STXeq (Lehane, 2000). The joint FAO/WHO/IOC expert consultation has recently established a provisional acute reference dose (RfD) of 0.7 μg STXeq/kg bw (for an adult of body weight 60 kg) for STX, which is based on its lowest observed adverse effect level (LOAEL), 2 μg STXeq/kg bw (Toyofuku, 2006).

PSP is one of the most common seafood biotoxin illnesses in Hong Kong. Between 1997 and 2006, a total of 69 suspected shellfish poisoning cases affecting 145 people were recorded (Centre for Health Protection, 2007). Clinical symptoms and recent shellfish consumption history in the preceding 24 h are the main diagnostic criteria for determining PSP illness in patients. Since there is no validated diagnostic assay for PSP in patients, laboratory analysis on the implicated food can provide essential evidence to support the clinical features (Kao, 1993). For effective risk reduction to people suffering PSP and to prevent the marketing of PSP-contaminated shellfish, a comprehensive surveillance programme has been implemented for PSP-toxins in Hong Kong. The AOAC mouse bioassay (AOAC, 2000) is one of the most recommended methods used by many countries to detect and monitor the total toxicity of PSP-toxins in shellfish (Fernández and Cembella, 1995, Toyofuku, 2006). PSP toxicity is determined by intraperitoneal injection to mice with shellfish extracts (LD₅₀ in mice: 0.2 μg per 20 g mice) followed by monitoring the time of mice death as quantitative endpoint (AOAC, 2000). The method allows determination of the total toxicity of PSP-toxins in shellfish samples. It should be noted, however, that there are some disadvantages such as non-specificity and non-selectivity in using mouse bioassay (McNabb et al., 2005, Park et al., 1986).

In order to characterise the variations in toxins profiles and concentrations of PSP-toxins, post-column derivatisation with reversed-phase high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) is a well-documented and widely-used analytical method (Oshima, 1995). The toxins are oxidised into fluorescent derivatives after column separation. Owing to its high sensitivity and good toxins specificity (Luckas et al., 2003, Oshima, 1995), the method is particularly useful for metabolic and biotransformation studies of PSP-toxins in shellfish. The detection limit of this analytical technique is 5–30 pg per injection (Oshima, 1995). Therefore, it can be used to detect levels of PSP-toxins which are below the detection limit of mouse bioassay.

Recently, a commercial rapid enzyme-linked immunosorbent assay (ELISA), MIST Alert™ test kit (Jellett Biotek Ltd., Dartmouth, NS, Canada), is a qualitative method for rapid screening of PSP-toxin-contaminated bivalve shellfish samples (Jellett et al., 2002, Mackintosh et al., 2002). It is designed to determine PSP-contaminated shellfish in less than 20 min and has an average detection limit of 40 μg STXeq/100 g of shellfish tissues. The kit is sensitive to the carbamoyl toxins, dcSTX, B1 and C1–2 (Mackintosh et al., 2002, Asp et al., 2004); low toxicity N-sulfocarbamoyl toxins (i.e., B1 and C1–2) can be readily transformed to more potent analogues during consumption of shellfish. This is beneficial for evaluating the potential overall toxicity of shellfish sample (Jellett et al. 2002). A recently new version of PSP rapid kit, Jellett Rapid PSP Test (Jellett Rapid Testing Ltd., NS, Canada), is designed as initial rapid PSP screening tool for shellfish samples, with results available between 35 and 60 min. In addition, the current test kit may reduce the degree of subjectivity in the result interpretation by improving interpretation criteria of C-line and T-line. The kit is based on the same technology and the same lower detection limit as its predecessor, MIST Alert™ test kit (Oshiro, Pham, Csuti, Dodd, Inami, & Brenden, 2006). Application of the three detection methods may provide a comprehensive perspective (three-tiered approach), in both qualitative and quantitative approaches, for PSP-toxins study and detection in shellfish.

The objective of this study was to characterise the PSP-toxins levels and patterns in different tissue compartments of scallop (Patinopecten yessoensis), before and after steam cooking, one of the most common culinary practices. The information is crucial for estimating the risk of PSP through steam cooking and evaluating food safety measures on different tissue compartments of PSP-toxins-contaminated scallops. Specimens from raw and steamed samples were mainly analysed with the HPLC-FLD method and a commercially available antibody-based qualitative test kit, Jellett PSP Rapid Test. The available specimens were additionally examined by the AOAC mouse bioassay for toxicity verification.