14 and 15 were set aside and observed for 4 more days. Two of the 

 fish in the culture medium died between 30 and 48 hours, and the 

 other two were alive after 5 days. The three fish remaining in the 

 sea water also lived through 5 days. There was some indication 

 that non-aerated G™ brevis cultures were more toxic than the 

 aerated ones. 



Discussion of Results of Experiments with Bacteria-free Cultures 



The results of the experiments with bacteria-free G. brevis 

 confirm that this organism produces a fish-killing substance(s) as 

 indicated by tests with unialgal cultures. Bacteria-free G. brevis 

 was toxic to the two species of fish tested ( Cyprinodon variegatus 

 and Mugil cephalus ) . The minimal "death time" for C. variegatus 

 was about 3a: hours. The mullet were more sensitive with minimum 

 "death times" as low as 15 minutes. Furthermore, small mullet 

 appear to be less sensitive than the large ones since the three 

 small fish in each container of G. brevis culture outlived the 

 large one. The concentration of G. brevis in these bacteria-free 

 cultures varied from 2.3 to 4.8 million organisms per liter. Such 

 concentrations are considerably less than the 10 to 60 million per 

 liter sometimes encountered in areas where dead of dying fish occur. 



There was good agreement between the results of experiments 

 8 and 9 with regard to the toxicity of bacteria-free G. brevis 

 cultures to small M. cephalus , which was the only species tested 

 in both experiments. Nevertheless, the small mullet survived much 

 better in the control materials in experiment 8 then in experiment 9. 

 In the latter experiment the small mullet died rapidly in one 

 particular batch of sterile culture medium (containers 13 and I4). 

 Large M. cephalus , however, survived much better in this batch of 

 medium. 



We attribute the early death of the small mullet in the culture 

 medium placed in containers 13 and I4 of experiment 9 to the 

 abnormally low pH of this particular batch of medium. Moreover, 

 the relatively poor survival of these fish in some of the other 

 control containers of experiment 9 was possibly due to their being 

 damaged by the vigorous thrashing about of the large mullet. The 

 length of some of the large test fish slightly exceeded the inside 

 diameter of the experimental containers. 



The low pH of the material in container I4 suggests that the 

 batch of medium placed in container I4 was abnormal before the 

 fish were added. Approximately I4 hours after experiment 9 was 

 begun the pH of the medium in container I4 was 5.9 and the 

 bacterial count was between 25 and 50 million per ml. Since the 

 pH of this material increased from 5.9 to 6.4 during the next 11 

 hours in spite of heavy bacterial growth, the initial pH of this 



33 



