chloride filtrate was evaporated slowly in a 

 desiccator and yielded a light yellow viscous 

 semisolid that was highly toxic. 



The interfacial layer (substance I) was sep- 

 arated and the toxin was isolated by removal 

 of solvent. The layer was composed of bubbles 

 trapped in chloroform; normally, a large 

 amount of salt water was associated with it. 

 We washed this layer twice with 250-ml. por- 

 tions of dilute hydrochloric acid solution (pH 

 3.5-4.0) in a separatory funnel. The organic 

 layer was collected in long test tubes and al- 

 lowed to stand over silica gel for drying. Thus 

 purified, substance I was mostly free from 

 sodium chloride. When the organic material was 

 suflficiently dried, flaky dark-green suspended 

 matter was found in the test tubes. Contents of 

 the tubes were transferred to a flask, and the 

 volume was increased 25 percent with absolute 

 ethanol. The mixture was stirred 4 hours, 

 cooled, and filtered. The residue was not toxic. 

 The filtrate was cooled in an ice-water bath and 

 evaporated under reduced pressure. 



The residue (substance I) was dissolved in 

 ethanol and purified further. The ethanol solu- 

 tion was cooled in a Dry-Ice bath, stirred with 

 bone charcoal, and filtered. When the filtrate 

 was evaporated, the residue was a toxic, yellow- 

 ish, oily solid. 



THE TOXIN UNIT 



One Gymnodinium breve fish-kill unit was 

 defined arbitrarily as the amount of toxin per 

 50 ml. of sea water which under standard condi- 

 tions killed a 3.0- to 3.5-g. (6- to 7.5-cm.) long- 

 nose killifish, Fundulus similis, in 7 to 8 

 minutes. The standard conditions included 50 

 ml. of test solution in a 400-ml. beaker, con- 

 trolled temperature (23°±2° C), and appro- 

 priate salinity (32-33 p.p.t.) andpH (8.0-8.2). 



EFFECT OF TEMPERATURE 



The toxin (substance I in interfacial layer) 

 was maintained at room temperature, 23° ±2° 

 C, and periodically tested for potency. The 

 results, summarized in table 1, indicate that a 

 solution of the toxin is stable at room tempera- 

 ture for at least 96 hours. 



All toxicity was lost when the sample in 

 chloroform-water solution was heated to 110° C. 



Table l. — Temporal stability of crude toxin (substance 

 I) from G. breve at room temperature 



Volume Amount Length Approximate 



Time of of water of weight Death Fish-kill 



toxin > taken fish = of fish time unit 



Hours Drops Ml. Cm. G. Minutes 



2 60 3.8 1.1 5 1 



1 2 50 3.8 1.2 5-6 1 



4 2 GO 3.8 1.5 5-6 1 



8 2 75 3.8 2.0 5-6 1 



24 2 75 3.8 2.0 6-7 1 



30 2 100 6.4 3.0 6 1 



96 2 50 3.5 1.1 6-7 1 



' Control: two drops of chloroform in same volume of sea water. 

 No effect on fish within 120 minutes. 

 - Fundulus simitis. 



for 5 minutes, and the loss in weight of the 

 sample was 0.3887 g. or 97.5 percent. At this 

 temperature, volatilization of chloroform and 

 water would be expected. 



In addition, presumably these data indicate 

 either the volatility or the ease of decomposi- 

 tion of the toxin. Attempts to detect decomposi- 

 tion products with gas chromatography were 

 unsuccessful. An Aerograph vapor phase chro- 

 matography unit, equipped with Carbowax 400 

 column and a thermal detector, was used. Col- 

 umn temperature was maintained at 55° C. The 

 amount of toxin (substance I in ether) may 

 have been insufficient to detect. 



Attempts were made to measure the volatility 

 of the sample as follows: One fish was placed in 

 each of two beakers (under the conditions pre- 

 viously defined for the toxin unit). The two 

 beakers were placed in a closed system, and the 

 first beaker was treated with 1 fish kill unit of 

 toxin (substance I). Four minutes later a 

 stream of nitrogen was passed through solution 

 in the first beaker, forcing any evolved gases to 

 pass through the second beaker. The fish in the 

 first beaker died after 6 minutes; the one in the 

 second beaker was unaff'ected for 12 minutes, 

 after which time the experiment was discon- 

 tinued. We concluded that the toxin was not 

 volatile at atmospheric pressure at room tem- 

 perature. However, a loss of toxicity under 

 reduced pressure (when a rotary evaporator is 

 used) was amply indicated. 



ELEMENTAL QUALITATIVE 

 ANALYSIS 



Purified samples of toxin (substance I and 

 substance II) were fused with sodium and de- 

 composed with water, and the solution was 

 analyzed for nitrogen, sulfur, halogen (chlorine, 



CHEMICAL AND PHYSICAL PROPERTIES OF TWO TOXINS FROM RED-TIDE ORGANISM 



435 



