SECTION vn 



Sununaiy of Brevetoxin Analysis 



Staff 



Southeast Fisheries Science Center 



75 Virginia Beach Drive 



Miami, FL 33149 



The mass mortality of bottlenose dolphins along the U.S. east coast during 1987-88 

 was believed to possibly have been caused by a naturally-occurring neurotoxin (Geraci, 1989; 

 Anderson and White, 1989). The suspected neurotoxin, brevetoxin, is produced by a toxic 

 dinoflagellate, G. breve. Because of the previous implication of brevetoxin as a cause of mass 

 mortalities of bottlenose dolphins, a total of 50 bottlenose dolphin liver samples were 

 analyzed for individual brevetoxins. Forty of the samples were from January-June, 1990, Gulf 

 of Mexico strandings; 10 were control samples. The control samples were included to 

 determine if the assay methods could accurately detect samples spiked with brevetoxin. The 

 brevetoxin analysis was conducted under contract; the contract report is presented in 

 Appendix VI. TTie results of the analysis are summarized and discussed below. 



Summary and Discussion 



Toxicity was determined by several methods: 1) fish bioassay - Gambusia affinis, fish 

 death at a fixed interval indicates toxin present but does not necessarily indicate brevetoxin; 

 2) HPLC separation of toxin fractions - HPLC separation provides a means to confirm or 

 deny the presence of brevetoxins in comparison to valid PbTx-standards; 3) 

 Radioimmunoassay provides a means to positively identify brevetoxin-like materials and is 

 sensitive to authentic PbTx-3. 



Following the first thin-layer chromatography (TLC) plate, 33 of the 50 samples were 

 found non-toxic in the fish bioassay and were not tested further. Of the remaining 17 

 samples that tested positive in at least one fraction of the first TLC plate, nine had multiple 

 toxic fractions. Of the 17 samples, 12 tested negative by fish bioassay foUowing the second 

 TLC plate. Of the five fractions found toxic after the second TLC separation, three were 

 judged to be in such limited quantity to preclude further TLC separation. The other two 

 retained toxicity after the third TLC separation. 



The three toxic fi"actions of limited quantity were judged to contain less than 5ug 

 toxin/total original sample by HPLC; this was presumed to be a negative result. The other 



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