MAP Technical Reports Series No. 106 UNEP

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(c) Acute toxicity tests on mice using samples of water, phytoplankton and mussels
extracts
(i) Direct extraction of biotoxins from water and phytoplankton
The biotoxins can be water or fat soluble. The water soluble toxins can be in solution
as well as in the phytoplankton cells; the fat soluble toxins are found only in the cells or
in particulate matter. At first the concentration of the phytoplankton can be attained by
filtration using Millipore filters or centrifugation. In the case of the fat soluble toxins from
G. breve, direct extraction with ethyl ether (McFarren et al., 1965; Cummins et al.,
1968) from 2-16 litres of sea water makes it possible to demonstrate the presence of
the biotoxins by evaporating the ether extract and injecting the lipid fraction
intraperitoneally into mice of 19-23 g (Cummins et al., 1968). For a more general assay
of the toxicity in mice, it is possible to use either filter extracts or the residue after
centrifugation. However, to carry out more detailed studies on the chemical nature of
the toxin, it is necessary to use an appropriate plankton net to collect sufficient
quantities of phytoplankton for application of the techniques for extracting water-soluble
(AOAC, 1970) and fat soluble toxins (McFarren et al., 1965; Scheuer et al., 1967;
Bagnis et al., 1974).
(ii) Concentration of the biotoxins by Mytilus galloprovincialis or M. edulis.
Another very useful method for demonstrating or excluding the presence of biotoxins
of the PSP and NSP type in discoloured sea water is to filter the sea water in 100 litre
tanks containing healthy mussels over a period of several days until the water becomes
transparent or clean, and, if necessary, this can be repeated for a week (Viviani,
1977a). The biotoxins of the water soluble (AOAC, 1970) and fat soluble type (McFarren
et al., 1965) are extracted from the mussels and assayed in mice.
(d) Haemolysis test of extracts obtained from the algal waters or mussels on mouse
blood cells
Bioassay using fish usually requires relative large amounts of samples, as the test
materials should be dissolved in relatively large volumes of water to keep test fish. In
addition dose-survival time responses of fish are often fluctuant. In order to surmount
difficulty and use a rapid and sensitive bioassay method a haemolytic test for screening
ichthyotoxins this has been applied. In fact many ichthyotoxins such as those of
Prymnesium parvum (Shilo, 1967), Amphidinium carteri (Yasumoto et al., 1987),
Chrysochromulina polylepis (Yasumoto et al., 1990; Edvardsen et al., 1990),
Gyrodinium aureolum (Yasumoto et al., 1990) and maitotoxin of Gambierdiscus
toxicus (Nakajima et al., 1981) are potent haemolysins. Also mussels accumulate the
haemolysins. Mussels exposed to a C. polylepis bloom displayed a higher haemolytic
activity than non contaminated mussels (Yasumoto et al., 1990). The discoloured
water, red tide or mucilaginous aggregates could be screened for the presence of
bioactive compounds using haemolytic tests. To this end the extracts obtained from
the marine waters or mussels dissolved in chloroform were purified. After purification,
haemolytic tests were carried out using mouse blood.
6.1.6 Studies on ichthyotoxic components of phytoplancton in the Mediterranean sea
Acute toxicity tests on fish
Using acute toxicity tests on Mugil cephalus with samples of marine water and