Determination of Paralytic Shellfish Toxins in Shellfish by Receptor Binding Assay: Collaborative Study

Abstract

A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with 3H saxitoxin (STX) diHCl for binding to voltage- gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (μg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr, based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1–93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r2) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r2 of 0.92. When samples were sorted according to increasing toxin concentration (μg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 μg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

Marine Biotoxins in History: Misuse and Mayhem

“…Through the efforts of many, this hurdle has been overcome, partly by recruiting companies with radiolabeling facilities colocated in the same county as the means of toxin supply removing the initial international transfer. This has made the radiolabeled STX available for research as well as for use in bioassays for monitoring toxicity Robertson et al 2006;Van Dolah et al 2012) to prevent toxic seafood from entering the supply chain.…”

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

“…the US FDA has established an action level for OA at 0.16 ppm, and it recommends "a commercially available immu-ing assay was carried out by van Dolah and coworkers (Van Dolah et al, 2012) and demonstrated an acceptable correlation with the MBA (slope of 1.64 and an r 2 = 0.84), and the HPlC detection method using precolumn oxidation (slope of 1.20 and an r 2 =0.92).…”

“…Nevertheless, there are several attempts to develop assays on the basis of putative physods involving neuroblastoma cells (louzao et al, 2003Manger et al, 2003), an approach with rhodamine and synaptoneurosomes (Nicholson et al, 2002), and a patch clamp method (Velez et al, 2001). the radioactive method for saxitoxin analysis utilizing brain homogenates was recently accepted as an AOAC validated method (Van Dolah et al, 2012) and may be used for high-throughput screening of environmental samples (Doucette et al, 1997;Ruberu et al, 2003). the lOD of the latter method is reported at 2 µg Stxequiv./kg shellfish tissue.…”

“…the lOD of the latter method is reported at 2 µg Stxequiv./kg shellfish tissue. The modified method was recently subjected to a validation procedure (Van Dolah et al, 2012). An action potential voltage-clamp technique was introduced which could be the next generation tool for development of functional assays also for marine biotoxins acting via ion channels (Szentandrassy et al, 2011).…”

Analysis on New Type of Classification Machine with Vibration for Scallops

“…Special shape materials such as scallops, which has no special classification devices at domestic and international [4]. A new classification device is recommended to meet the needs, which combines the advantages of spherical materials grading machine and makes up for its shortcomings [5]. According to the approximate diameter, scallops can be divided into four levels to facilitate the subsequent processing and marketing [6].…”