XJNIALGAL AND BACTERIA-FREE' CUL/TURES OF G. BREVIS 



479 



aiui, in some cases, even exceeding those of the 

 toxic G. brevis. In fact, M. cephalus survived 

 considerably longer in the Prorocentrum culture 

 than in the control material (uninoculated culture 

 medium) in experiment 7 (table 5). Prorocentrum 

 possibly aided survival of the fish by liberating 

 oxygen since the test materials were not aerated. 

 Gymnodinium sp. was toxic to M. cephalus and C. 

 variegatus (table 5). In view of these results 



and since this organism in culture is morphologi- 

 cally similar to G. breins, we tentatively consider 

 the Galveston Gymnodirdum to be G. hrenis. The 

 Galveston Gymnodinium was observed only three 

 times (all within the same week) although samples 

 were collected from the lagoon three times weekly 

 during an 11-month period. The concentrations 

 in the lagoon samples varied from 1,000 to 60,000 

 organisms per liter. 



EXPERIMENTS WITH BACTERIA- FREE CULTURES OF GYMNODINIUM BREVIS 



Mass bacteria-free cultures of G. brevis were 

 established following the completion of experi- 

 ment 7. The two experiments, 8 and 9, to follow 

 were the first toxicity tests to be conducted with 

 pure cultures. The importance of these studies 

 lies in the fact that the observed effects can be 

 attributed to G. brevis with certainty since no 

 other organisms were present during the incuba- 

 tion period. Substantiation of the toxicity of 

 unialgal G. brevis with bacteria-free G. brevis 



should establish the existence of a cause-and- 

 effect relation between blooms of this organism 

 and associated mass mortalit}' of marine animals. 

 The experiments with bacteria-free cultures were 

 more refined in several respects than those with 

 unialgal cultures. In addition to aerating most 

 of the test materials, such factors as temperature, 

 dissolved oxygen, salinity, and pH were deter- 

 mined. 



EXPERIMENT 8. — Effects of Unialgal and Bacteria- Free Gymnodinium brevis Cultures 



The first mass bacteria-free cultures of G. brevis 

 were tested for toxicity to striped mullet (Mugil 

 cephalus) and variegated minnows (Cyprivodon 

 variegatus). (\ variegatus (1% to \%, in. long) 

 were maintained in aerated aquariums for 5 days 

 and M. cephalus (1 to 1)4 in. long) were brought 

 into the laboratory the night preceding the ex- 

 periment. One liter of distilled water was placed 

 in each 2-liter beaker, containers for the various 

 test materials, 5 daA's before commencing the 

 experiment so that the aeration equipment could 

 be tested and adjusted. The water was dis- 

 carded and the beakers received no further 

 treatment before introduction of the various 

 test materials. The material in 12 of the 14 

 containers received gentle aeration continuously 

 from a small aerator; the main air line from 

 the aerator was equipped with a nonabsorbent 

 cotton filter. To preclude possible excessive 

 oxygen demand by G. brevis, light was provided 

 continuously with two fluorescent lamps equipped 

 with two 18-inch, 15-watt daylight tubes. The 

 experimental setup is shown in figure 1. 



Six different bacteria-free cultures (containers 

 3, 4, 6, 7, 9, 10, 12, and 13) and four hatches of 

 sterile control material (containers 5, 8, 11, and 

 14), all of which had incubated for a month, were 



employed in this study (table 6). The control 

 material consisted of uninoculated culture medium 

 otherwise subjected to the same conditions as the 

 inoculated medium. The sterility of the G. brevis 

 cultures and control materials was established by 

 inoculating routine sterility-test media with sam- 

 ples withdrawn from the culture vessels shortly 

 before these materials were dispensed into the 

 experimental containers. Duplicate containers (3 

 and 4, 6 and 7) of two of the six bacteria-free 

 cultures were set up to compare the survival of 

 test fish in aerated (containers 3 and 6) and non- 

 aerated (containers 4 and 7) cultures. A year-old 

 unialgal G. brevis culture (container 1), which was 

 replenished with fresh medium about 4 days 

 previously, was used to compare the effects of 

 unialgal and bacteria-free cultures. Aged sea 

 water (container 2) served as control material for 

 the entire experiment. Tlie volume of test ma- 

 terial placed in each container varied from 750 to 

 1,000 ml. 



Before adding the fish, samples were taken from 

 four containers (3, 6, 9, and 12) of bacteria-free 

 G. breins culture and two containers (5 and 11) of 

 uninoculated culture medium for bacterial counts. 

 Also at this time the nine containers with G. brevis 

 were sampled for enumeration of this organism. 



