In 1996, the EU-NRL group agreed during the first Meeting of the EU National Reference Laboratories on Marine Biotoxins and Analytical Methods and Toxicity Criteria, that the mouse bioassay with the technique established by Yasumoto et al. (1978), with an observation time of 24 hours is currently the preferred method for the detection of the acute toxicity of acetone soluble DSP toxins. Based on acute toxic effects, a tolerable level of DSP toxins, including non-diarrhoeic acetone soluble toxins, of 80-160 mg of OA eq/kg of whole shellfish meat or 20-40 MU/kg of whole shellfish meat was agreed for EU member countries (EU-NRL, 1996).
In March 2002, the European Commission laid down the following rules (EC, 2002a):
Maximum level of OA, DTXs and PTXs together, in edible tissues (whole body or any part edible separately) of molluscs, echinoderms, tunicates and marine gastropods shall be 160 mg OA equivalents/kg.
Maximum levels of YTXs in edible tissues (whole body or any part edible separately) of molluscs, echinoderms, tunicates and marine gastropods shall be 1 mg YTX equivalents/kg.
The mouse or the rat (not for
yessotoxin) bioassay are the preferred methods of analysis for the toxins
mentioned above. A series of analytical methods such as LC with fluorometric
detection, LC-MS, immunoassays and functional assays such as the phosphatase
inhibition assay can be used as alternative or complementary method to the
biological assays, provided that either alone or combined they can detect at
least the following analogues, that they are not less effective than the
biological methods and that their implementation provides an equivalent level of
public health protection;
OA and DTXs: an hydrolysis step may be required in order to detect the presence of DTX3
PTXs: PTX1 and PTX2
YTXs: YTX, 45 OH YTX, homo YTX, and 45 OH homo YTX.
When results of analyses demonstrate discrepancies between the different methods, the mouse bioassay should be considered as the reference method.
The Biotoxin Monitoring Programme in Ireland began in 1984 and was initially based on the screening of samples for the presence of DSP toxins by bioassays. In recent years, the detection of additional toxins, including DA and in particular the azaspiracids, has led to an increase in monitoring efforts and the programme now includes weekly shellfish testing using DSP mouse bioassay, LC-MS (okadaic acid, DTX2, azaspiracids) and LC (DA) as well as phytoplankton analysis. Regular reports of the results of sample analysis are sent to the regulatory authorities, health officials as well as the shellfish producers and processors. A Web-based information system is being developed to increase access to the information (McMahon et al., 2001)
Regulation is based on mouse bioassay. No further information (Fernández, 2000).
3.8.2 North America
Hallegraeff et al. (1995) reported that in Canada monitoring for Dinophysis spp. and Prorocentrum spp. is carried out, and that closure of fishery product harvesting areas takes place when DSP toxin levels in shellfish exceed tolerable levels (i.e. >0.2 mg/g meat = 5 MU/100 g meat) using the mouse bioassay; not official).
The United States of America
No DSP has yet been confirmed so there is no DSP monitoring. The primary agency responsible for seafood safety and marine biotoxins is the Food and Drug Administration (FDA). The National Marine Fisheries Service (NMFS) of the National Oceanic and Atmospheric Administration (NOAA) has several marine biotoxin programmes, primarily focused on fish and wildlife. In the area of domestic food safety, cooperative programmes between the FDA and individual states exist. The National Shellfish Sanitation Programme provides guidelines for these cooperative agreements. Internationally, the FDA sets up memoranda of understanding with various countries to regulate imported seafood products programmes (APEC, 1997).
3.8.3 Central and South America
Argentina has a national monitoring programme for mussel toxicity in each coastal province using regional laboratories and one fixed station in Mar del Plata (Ferrari, 2001).
Brazil had a pilot monitoring initiative during one year but does not have a national monitoring programme (Ferrari, 2001).
Two types of monitoring are conducted in Chile. The National Health Service is responsible for detecting toxicity using a bioassay at 40 stations using monthly samples. In addition, the Fisheries Research Institute monitors toxicity in conjunction with universities. These are programmes that include measures of phytoplankton to understand more than just toxicity. However, there are problems with the methods since at times both PSP and DSP occur. The Ministry of Health, through the Regional Health Service, is responsible for the closure of contaminated harvesting areas. When DSP bioassay is positive, shellfish are quarantined. The National Fish Service (NSF) is responsible for seafood for export. At present, NSF has a memorandum of understanding with the USA and the EU to permit shellfish to be exported. No regulations exist for imported shellfish, since that is presently not a big market (APEC, 1997). Up until 2001, PSP and DSP toxins have had the most severe public health and economic impact in Chile. Consequently, all natural fish beds from 44 °SL southwards were closed and nationwide monitoring programmes maintained (Suárez-Isla, 2001). Regulation is based on mouse bioassay (no further information) (Fernández, 2000).
Uruguay has a national monitoring programme on mussel toxicity and toxic phytoplankton (Ferrari, 2001). Regulation is based on mouse bioassay (no further information) (Fernández, 2000).
Regulation is based on mouse bioassay (no further information) (Fernández, 2000).
No regulatory monitoring programme for toxins in shellfish and no regulations for algal biotoxins in seafood products exist in China. A major project on red tides has been funded that will include regular monitoring in two areas, one in the north and one in the south of the country. This will include bi-weekly monitoring of both plankton and shellfish (APEC, 1997).
Monitoring involving both plankton and shellfish is carried out. Researchers from Prefectural Fisheries Experimental Stations in major shellfish areas periodically collect plankton samples and carry out cell counts of key Dinophysis species. Shellfish are collected and assayed at least monthly during key seasons. When low levels of toxin are detected, monitoring frequency is increased and more stations are sampled. Tolerance level for DSP toxins in bivalves is set at 5 MU/100 g whole meat detected by the mouse bioassay (~0.2 µg/g). Information on shellfish toxicity is distributed through a well-defined network connecting governmental agencies, fisheries co-operatives, fishermen, mass media and the general public. Three weeks of toxicity levels below quarantine limits result in a lift of the shellfish harvesting ban (APEC, 1997 and Hallegraeff et al., 1995).
The Republic of Korea
The National Fisheries Research and Development Institute (NFRDI) collects and examines plankton samples in key areas on a bi-weekly basis from February to October. Over two hundred stations are sampled. Tests are run for ASP, but also for PSP and DSP. However PSP and DSP are not serious problems. DSP toxins were determined by means of LC (APEC, 1997). Monitoring for Prorocentrum spp. is carried out and fishery product harvesting areas are closed at concentrations greater than 105 cells/litre. Furthermore, a tolerance limit for DSP toxins in shellfish of 5 MU per 100 g detected by the mouse bioassay is applied (Hallegraeff et al., 1995).
Regulation is based on the mouse bioassay (no further information) (Fernández, 2000).
Regulations for DSP recommend 16 to 20 µg OA eq/100 g shellfish meat. However, for 1995, it is stated that the maximum permitted levels for DSP were 20 to 60 µg OA/100 g shellfish meat or 2 µg OA/g hepatopancreas. It was not clear whether these figures were enforced values or recommended guidelines, and from where these figures were derived (Burgess and Shaw, 2001).
The New Zealand Biotoxin Monitoring Programme combines regular shellfish testing and phytoplankton monitoring. The regulatory level in shellfish is 20 µg OA eq/100 g of shellfish meat (Sim and Wilson, 1997). Currently shellfish testing involves mouse bioassay screen testing with confirmatory testing approved for OA and DTX1 (DSP ELISA Check Kit, PP2A, LC-MS), PTXs and YTXs (LC-MS) (Busby and Seamer, 2001).
A new Biotoxin Monitoring Programme providing data that is highly accurate, in a shorter time and without the use of mouse bioassays is being developed. This new programme will implement test methods based on LC-MS providing chemical analytical data in place of bioassay screen test results. The development and implementation of new test methods are in discussion including funding, method validation, testing regulations, availability of analytical standards, comparison to existing tests, type of instrumentation and international cooperation (McNabb and Holland, 2001).