482 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



Table 7. — Experiment 8: Dissolved oxygen, temperature, 

 salinity, and pH data for test materials 



[See table 6 for materials In containers] 



' Percentage of saturation in relation to !^ea water of the given temperature 

 and salinity in equilibrium with normal dry atmosphere. 



2 Temperature of material in container at time dissolved oxygen was 

 determined. 



' Values In parentheses were determined 7 to 7^2 hours after start of 

 experiment. 



' The approximate time that elapsed between start of experiment and 

 collection of samples for analyses. 



Only 1 of the 32 fish subjected to initially 

 bacteria-free cultures survived the 3lK-hour test 

 period; all except 3 (C. variegatus) died within 

 8 hours (table 6). The lone fish (C. variegatus) 

 surviving the experimental period succumbed 

 about 30 minutes later. On the contrary, only 

 1 of the 21 fish exposed to control materials of sea 

 water and initially sterile culture medium failed 

 to survive the test period. This fish (C. variegatus) 

 died after nearly 30 hours in culture medium. 

 The death times of M. cephalus, which varied 

 from Yi hour to 4% hours, were considerably less 

 than were those of the C. variegatus, 3K to nearly 

 32 hours. Fifty percent (16) of the test fish in 



the bacteria-free cultures died earlier than those 

 (4) in the unialgal culture; the death time in this 

 culture was about 1% hours for M. cephalus and 

 6)i and 6% hours for C. variegatus. The test fish 

 survived considerably longer in the nonaerated 

 than in the aerated G. hrevis culture in one 

 instance; in the other case the opposite occurred, 

 although the difference was less marked. 



The bacterial counts of the initially bacteria- 

 free G. hrevis cultures sampled before adding the 

 test fish varied from 250 to 20,000 per ml. The 

 rather high initial bacterial counts are attributed 

 to the prolonged standing (5 days) of the distilled 

 water in the experimental containers while the 

 aeration equipment was being tested and adjusted. 

 The counts obtained from these cultures following 

 the death of the last fish in the container (5 to 8 

 hours later) varied from 33,000 to 600,000 bacteria 

 per ml. A count of 24 million bacteria per ml. 

 was obtained after 31)^ hours from the initially 

 bacteria-free G. hrevis culture in which one fish 

 survived the test period. The first bacterial 

 counts for two containers of initially sterile culture 

 medium were 1,000 (container 5) and 25,000 (con- 

 tainer 11) per ml. When these containers were 

 sampled again near the end of the 3lK-hour test 

 period the bacterial count had increased to 21 

 million (container 5) and 7 million (container 11) 

 per ml. 



The results of experiment 8 show clearly that 

 bacteria-free G. hrevis cultures are toxic to fish. 

 Nevertheless, we desired to confirm these results 

 by using test materials with greatly reduced 

 initial and terminal bacterial counts. 



EXPERIMENT 9.— Effects of Bacteria- Free and Unialgal Gymnodinium brevis Cultures, and Effects of Filtration 

 on Toxicity 



The second study with bacteria-free G. brevis 

 differed somewhat from the first one (experiment 

 8). In experiment 9, the initial bacterial con- 

 tamination of test materials by containers and 

 aeration equipment was reduced, the effects of 

 two methods of filtration on the toxicity of 

 bacteria-free cultures were studied, and the sensi- 

 tivity of two size groups of mullet (Mugil cephalus) 

 to G. brevis cultures was compared. The experi- 

 mental setup was the same as for experiment 8 

 (fig. 1) except that more containers and a larger 

 air pump were used. 



We employed three precautions to reduce the 



initial bacterial contamination of the test ma- 

 terials. One precaution was to heat the experi- 

 mental containers (2-liter beakers) in a hot air 

 oven at 150°-160° C. for 2 hours and allow them 

 to cool overnight in the oven. The containers 

 were removed from the oven shortly before the 

 test materials were added. Secondly, the aera- 

 tion apparatus was autoclaved for 15 minutes at 

 15 pounds pressure. Before sterilization this ap- 

 paratus was assembled and packaged so that a 

 glass air-delivery tube could be inserted into each 

 of the 18 containers without handling the portion 

 that contacted the test materials. All air pumped 



