The attempt to reduce the initial bacterial contamination of 

 the bacteria-free G. brevis cultures and the sterile culture medium 

 by the previously mentioned precautions was successful. The counts 

 for these materials varied from to 100 bacteria per ml. The 

 bacterial counts in the initially bacteria-free G. brevjs cultures 

 varied from 4,^00 to 30,000 per ml at the time the last fish died 

 in the container (3A to 2i hours later). The initial count in the 

 unialgal culture was 1.3 million bacteria per ml; when the last fish 

 died 2j hours later the count was 1,4 million per ml. At the end of 

 the test period the bacterial concentration was in excess of 80 

 million per ml in one container of initially sterile culture medium 

 (13), After about I4 hours the count was 25 to 50 million bacteria 



per ml in the other ctJirtBlner of culture medium (I4) and in excess 



of 80 million per ml in one container of sea water. 



The pH of the initially sterile culture medium in container I4 

 increased from 5,9 to 6,4 during the 11-hour period after the last 

 fish died. Additional fish were subjected to this material to test 

 its toxicity at the higher pH. 



Experiment 9a. Supplementary Toxicity Tests of Some Test Materials 



Previously used in Experiment 9 



Near the close of experiment 9, we conduct a supplementary 

 study to determine whether the initially sterile culture medium in 

 container I4 of experiment 9 was still toxic to small M, cephalus . 

 Five other containers originally a part of experiment 9, one 

 container of 85^ autoclaved sea water (15) and the four containers 

 of initially bacteria-free G. brevis culture (9, 10, 11, and 12), 

 were included in this study. The other containers of culture medium 

 (13) and sea water (16) were not used because this part of the experi- 

 ment was still in progress as they contained one or more live fish. 



Small mullet from the group used in experiment 9 served as test 

 fish. Four of these fish, which were maintained in 85^ autoclaved 

 sea water for 24 hours, were placed in each container. Only two 

 of the four containers of G, brevis culture were aerated in an 

 attempt to determine the effects of agitation and aeration on the 

 toxicity of G. brevis . The culture medium (container I4) and sea 

 water (container 15) were not aerated. 



The results (Table 9) show that G, brevis cultures were still 

 toxic to small mullet, but the fish survived well in the previously 

 toxic culture medium. The 16 fish in the G. brevis cultures 

 died between 8 and 125 minutes; 10 of them survived less than 

 1 hour. In the control materials (containers I4 and 15), how- 

 ever, only one of the eight fish failed to survive the 24-hour 

 test period; one fish in the sea water succumbed after 5-3/4 hours. 

 Although the experiment was discontinued after 24 hours, containers 



31 



