MAP Technical Reports Series No. 106 UNEP

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Transplantation of mussels (Mytilus edulis) contaminated by okadaic acid (0A) from
a more toxic environment in the northern part of the Swedish west-coast to a less toxic
environment in the southern part showed a decrease in 0A content of 12 mg 0A/100 g mussel
meat per day (Haamer et al., 1990). Transplantation of less toxic mussels from the south to the
north did not show a rapid uptake of 0A. Toxic mussels from the north were reimmersed in two
basins. One of them contained ordinary sea water, and to the other boiled baker's yeast was
added. Decreases of 4-5 µg 0A/100 g mussel meat per day were observed. The 0A-data
showed a more consistent behaviour when boiled yeast added. Without yeast, decreases
alternated with increases.
7.2.2.7 Methods of analysis for DSP
The bioassay of all DSP components is based on the dose that provokes a fixed death
time in mice injected intraperitoneally with a toxic residue extracted from shellfish with acetone
(Yasumoto et al., 1984). The acetonic phase is evaporated and the residue resuspended in 4
ml of 1% Tween 60. Aliquots of 1 ml of this solution are injected i.p. into 18-20 Swiss albino
mice. This procedure is the official method in Japan and in several other countries. In France
toxicity is expressed differently from the official Japanese biological test. In Italy, the method
established by the Ministerial Decree dated 1.9.1990 (Ministero della Sanità, 1990c) is similar
to the official method used in France (Marcaillon-Le Baut and Masselin, 1990). The mice were
observed for 24 hr and positive tests consists in T < 5 hr. Other bioassay methods are: suckling
mouse bioassay (Hamano et al., 1985), rat bioassay (Kat, 1983), Tetrahymena test (Shiraki et
al., 1985).
The fluorometric determination of okadaic acid and DTX-1 has been developed using
HPLC (Lee et al., 1987). The bioassay and the HPLC method have not been studied
collaboratively, and no attempts have been made to study the scientific parameters, such as
precision, sensitivity and specificity. The intercalibration procedure is not appliable since at
present only okadaic acid and DTX-1 as reference material for the DSP components are
commercially available. A "second stage" analysis is the identification of okadaic acid and other
DSP toxins through 1 H NMR spectroscopy of fractions positive to official mouse lethality test,
obtained by repeated chromatographic separation (Fattorusso et al., 1992). Although it is a more
laborious test than fluorimetric HPLC assay (Lee et al., 1987), which has a similar scope, it has
two important advantages: it does not need any standard sample of the toxins, which may be
difficult to purchase for toxins other than okadaic acid and DTX-1 (Krogh, 1989); only literature
NMR data are required. In addition this method is not limited to known toxins, but can potentially
work with new ones, also allowing their structural elucidation, provided a large enough quantity
of toxic material can be obtained.
7.2.2.8 Human intoxication: clinical toxicology
Frequency of signs and symptoms of DSP in patients were as follows: diarrhoea
(92%), nausea (80%), vomiting (79%), abdominal pain (53%), and chill (10%). The incubation
period ranged from 30 min to several hours but seldom exceeded 12 hr. Around 70% of the
patients developed symptoms within 4 hr. Suffering may last for 3 days in severe cases but
leaves no after-effects (Yasumoto et al., 1978; Lucas, 1985). Thus no fatal case has ever been
recorded. The minimum amount of DSP required to induce disease in adults has been
estimated from analysis of left-over food to be 12 MU (Yasumoto et al., 1984). In Scandinavia
mussels associated with the outbreak contained approximatively 17 MU per 100 g (Underdal et
al., 1985). In an inventory of phytoplankton perturbation along the French coast in 1986 the higher
DSP levels in shellfish were 10.6 MU/100 g (Belin and Berthomé, 1988).