A study of paralytic and diarrhetic shellfish toxins in green-lipped mussels, perna viridis (L), collected from fish culture zones in Hong Kong

Abstract

Incidents of human poisoning are occurring more frequently worldwide due to the consumption of seafood products containing paralytic shellfish toxins (PSTs) and diarrhetic shellfish toxins (DSTs). PSTs are responsible for the neurotoxic syndrome commonly referred to as paralytic shellfish poisoning (PSP), which can cause death as a result of neuromuscular (respiratory) paralysis, while DSTs are responsible for diarrhetic shellfish poisoning (DSP), which is a gastrointestinal illness that can cause diarrhea, nausea, vomiting, cramps, headache and abdominal pain. Suspension-feeding bivalves are known to accumulate the toxins through the consumption of toxin-laden dinoflagellates, thus shellfish are one of the principal vectors of the toxins. In order to effectively assess the risks to seafood consumers from contaminated shellfish products, green-lipped mussels (Perna viridis) were regularly collected from each of seven fish culture zones (FCZs) in Hong Kong between January 2002 and April 2003, and analyzed for the relevant PSTs. Spatial and temporal variations in mussel toxin levels from various FCZs were investigated and correlated with the densities of relevant toxin-producing algal species. The mussels were divided into two parts, the hepatopancreas (HP) and the remaining tissues (RT) and analyzed separately for PSTs by high performance liquid chromatography (HPLC) with fluorescence detection following post-column derivatization. Mussel samples collected from various FCZs in February and April in both 2002 and 2003 had higher levels of N-sulfocarbamoyl-II-hydroxysulfate toxins (C1 and C2 toxins) (up to 1.51 and 0.62 μg/g HP wet wt., respectively) as compared to all other sampling months in 2002 and 2003. The relatively high PST levels may be a result of a recent exposure to Alexandrium tamarense and Gymnodinium catenaturn. Gonyautoxin I (GTX1) and gonyautoxin IV (GTX4) were not detected in any of the samples. The levels of decarbamoyl-gonyautoxin II (dcGTX2) and decarbamoyl-gonyautoxin III (dcGTX3) toxins (up to 0.16 and 0.32 μg/g HP wet wt., respectively) were relatively high in samples collected between April and August in 2002 and 2003. The formation of decarbamoyl derivatives may have occurred via hydrolysis of N-sulfocarbamoyl toxins at neutral pH in the shellfish. The levels of gonyautoxin II (GTX2) (up to 0.015μg/g HP wet wt.), gonyautoxin III (GTX3) (up to 0.028 μg/g HP wet wt.), neosaxitoxin (neoSTX) (up to 0.044 μg/g HP wet wt.) and saxitoxin (STX) (0.0067 μg/g HP wet wt.) toxins were relatively high in samples collected between June and December in 2002. None of the samples analyzed contained toxins exceeding the public health safety threshold (80 μg STXeq/100 g wet wt. of mussel tissue) for mussel consumption in Hong Kong. In conjunction with this research, P. viridis originating from mainland China were purchased from three different Hong Kong markets in July 2002 and January 2003. Mussel samples were analyzed for PSTs. Only STX (between 0.0029 and 0.0046 μg/g RT wet wt.) was detected in the July 2003 samples. C1 (up to 0.054 μg/g HP wet wt.), C2 (up to 0.095 μg/g RT wet wt.), dcGTX3 (up to 0.0088 μg/g RT wet wt.), GTX3 (up to 0.0013 μg/g HP wet wt.) and STX (between 0.0032 μg/g RT wet wt. and 0.013 μg/g HP wet wt.) were detected in January 2003 samples, respectively and overall the toxicity of the mussels was below the safety level of PSP For the analysis of okadaic acid (OA) in mussels, the HPLC method of Lee et al. (1987) was modified by incorporating a proteolytic digestion step (by proteinase K). Percentage recovery of OA in spiked HP samples was initially optimized by varying concentrations of proteinase K during the proteolytic digestion step. Results suggest that addition of proteolytic enzyme does, to some degree, enhance the extraction efficiency, giving higher percentage recoveries (~10% increasing). A relatively high percentage (78%) of OA was recovered from spiked samples after the addition of 1.08 mg proteinase K. This high recovery of OA was without having to resort to the use of excessive amounts (re: 4.31 mg proteinase K) of the enzyme. Thus, 1.08 mg proteinase K was used in all further validation analysis involving field samples. To validate the modified extraction procedure (i.e. addition of a proteolytic digestion step), OA levels in field collected HP tissues were determined both with and without the use of proteinase K. Samples were homogenized and pooled with subsamples selected for analysis. OA levels after incubation with proteinase K (306.20 ± 38.45 ng/g wet wt.) were 2.46 times higher than samples incubated without proteinase K (124.4 ± 40.28 ng/g wet wt.; two-sample t test: t = 10.32; df = 8; P < 0.0001). For HP subsamples from individual field-collected mussels, the results indicated that the additional digestion step can enhance OA extraction (and subsequently detection) by 3.07 times (two sample paired t test: P < 0.01). Results of this study also suggest that the enhancement of OA extraction by proteinase K is more prominent in field-collected mussel samples as compared to spiked samples. Despite the mussels being incubated for 24 h with OA in an "aging" step, the OA may be more readily released/extracted from the tissue, even in the absence of proteinase K, when compared to field samples in which OA is likely to be more tightly bound. Similar to the PSTs analysis, OA was extracted from HP mussels samples collected from FCZs in February, May, August and November 2002. Spatial and temporal variations in mussel toxin levels from various FCZs were investigated, and correlated with the densities of relevant toxin-producing algal species. The highest level of OA in mussel HP samples was 1160 ng/g HP wet wt. In terms of overall OA concentrations in mussel tissues, the levels were between 70.0 and 131 ng/g wet wt. of tissue, which did not exceed the regulatory level of DSP for safe human consumption (200 ng/g wet wt. of mussel tissue). The health risks of chronic exposure to DSTs should, however, be further examined due to the genotoxicity and tumour-promoting activity of OA.