UNIALGAL AND BACTERIA-FREE CUUTURES OF G. BREVIS 



477 



of tlic experiment, were kept in a nonaerated 

 aquarium since this species survives well without 

 aeration. 



Five of the seven different test materials 

 included in this study were tested in duplicate. 

 These materials consisted of two different unialgal 

 G. hreins cultures (containers 1 and 2, 3 and 4) ; of 

 Gymnodinium sp. (containers 5 and 6) ; Proro- 

 cenfrum sp. (containers 7 and 8) ; and of culture 

 medium from bottle 3 (containers 9 and 10). 

 Also included was a filtrate prepared by passing 

 1 liter of G. brevis culture through a millipore 

 membrane (container 11) and the residues retained 

 with this membrane eluted in 1 liter of culture 

 medium from bottle 3 (container 12). The 

 millipore membrane (HA) retains particles as 

 small as 0.5 micron. Each of the 12 containers 

 (2-liter beakers) received approximately 1 liter 

 of test material that was not aerated. 



Before adding the fish, bacterial samples were 

 taken from two containers of G. brevis culture 

 (1 and 2) and one container of culture medium (9). 

 These containers were arbitrarily selected in 

 order to compare the bacterial counts in some of 

 the containers before the fish were added with 

 those counts  obtained after death of the test 

 fish. The samples for bacterial counts were 

 refrigerated from 3 to 3^3 hours before the prepa- 

 ration of pour-plates. All containers with dino- 

 flagellates (1 through 8) as well as the filtrate of 



the G. brevis culture (contain": 11) were sampled 

 for counts of these organisms just before the 

 fish were added. These counts were completed 

 within 3 hours after collection of samples. 



One M. cephalus was placed in each of the 12 

 containers. Each M. cephalus was removed from 

 its container shortly after death and a C. variegatus 

 was added. The fish were not introduced simul- 

 taneously, since the M. cephalus were rather 

 large for the containers. 



Immediately after the M. cephalus died in the 

 containers (1, 2, and 9), that were initially 

 sampled for bacterial counts, these containers 

 were again sampled for such counts. Such 

 samples were also taken from one of each of the 

 remaining duplicate test materials (containers 

 3, 5, and 7) and from the millipore filtrate (con- 

 tainer 11) following death of the M. cephalus. 

 Pour-plates were prepared with these samples 

 after 4 to 6)^ hours' refrigeration, except that the 

 sample from container 7 was stored only l)i hours. 



The results (table 5) of this 27-hour experiment 

 show that all of the M. cephalus subjected to 

 either G. hrei^s culture or filtrate and residues of 

 such a culture died in less than an hour. The 

 death times varied from 14 to 53 minutes. M. 

 cephalus in the uninoculated culture medium lived 

 approximately 2^ and 3 hours. The bacterial 

 count of 1.8 million per ml. of this culture medium 

 (bottle 3) was higher than the 1.0 to 1.5 million 



Table 5. — Experiment 7: Effects of unialgal cultures of Gymnodinium brevis, Prorocentrum sp., and Gymnodinium 

 sp. on Mugil cephalus and Cyprinodon variegatus and effects of filtration on toxicity of unialgal cultures 



1 Time (hr.: niin.) rcquirod for fish to show first sipns of imbalance. 

 'Time (hr.: niia.) of ces.^ul inn of oporcuhir movomont. 

 'All sampU'S for dinoflapollatf counts were collected from the containers 

 just before introduction of the fish. 

 * Samples for the first bacterial count listed for each container were col- 



lected just before introduction of the M. cephalus: samples for the second 

 count, in parentheses, were taken immediately after the A/, cephalus died. 



s Death or distress; did not occur duriuR the 27-hour tost period. 



• Original supply of medium In reservoir (bottle 3) became exhausted and 

 It was renewed about 6 weeks prior to experiment. 



