UNIALGAL AND BACTERIA-FREE CULTURES OF G. BREVIS 



493 



(diameter 18.5 cm.) consists of cellulose fibers. 

 Retention of the toxic substance by filter paper 

 may be due to greater adsorptive area and/or to 

 differences in physical and chemical properties. 

 Another possibility is that filtration by gravity 

 flow used with filter paper may result in the re- 

 tention of considerably more intact organisms 

 than in the case of millipore filtration under suc- 

 tion. Assuming that greater numbers of G. brevis 

 were broken up by millipore filtration, more toxin 

 might be released in such a case. However, our 

 preliminary studies showed no apparent increase 

 in the toxicity of G. brevis cultures in which the 

 organisms were cytolyzed by gentle heating. 



There are no indications that the fish kills 

 caused by 6. brevis result from the great masses 

 of this organism depleting the oxygen in the sea 

 water. Connell and Cross (1950) suggested that 

 anaerobic conditions created by the high bio- 

 chemical oxygen demand of an armored dinoflag- 

 ellate, Gonijaulaj, was the cause of mass mortality 

 of fish associated with discolored water in OfFatts 

 Bayou (Galveston Bay) during the summer of 

 1949. Gunter et al. (1948) concluded that the 

 1946-47 incidence of mass mortality of marine 

 animals on the west coast of Florida was not 

 associated with low oxygen. Oxygen deficiency 

 can be excluded as a factor in the death of the 

 fish in the G. brevis cultures that were aerated. 

 In experiment 8 (tables 6 and 7), the dissolved- 

 oxygen content of all aerated test materials ex- 

 ceeded 75-percent saturation and some were as 

 high as 90 percent. With continuous gentle 

 aeration one of the G. brevis cultures was about 90- 

 percent saturated, although the bacterial count 

 was 24 million per ml. at the time the dissolved- 

 oxygen content was determined. 



The results of an attempt to determine the 

 effects of aeration on the toxicity of bacteria-free 

 G. brevis cultures were contradictory. In experi- 

 ment 8, the fish — especially Mugil cephalus — 

 showed distress and died more rapidly in the 

 aliquot that was aerated (dissolved oxygen, 89- 

 percent saturation) than in the one that was not 

 aerated (dissolved oxj-gen, 45-percent saturation). 

 Tlie fish in another nonaerated aliquot (dissolved 

 oxygen, 57-percent saturation) in this experiment 

 showed distress much sooner than those in the 

 aerated culture (dissolved oxygen, 82-percent 

 saturation). In experiment 9a (table 9), M. 

 cephalus died somewhat faster in the nonaerated 



aliquot of a G. brevis culture than in the aerated 

 aliquot. It is apparent from these contradictory 

 results that the influence of such factors as aeration 

 cannot be evaluated until standardization of the 

 toxicity tests is more complete. 



Several factors probably influence the degree 

 of toxicity of G. brevis cultures. Aside from the 

 concentration of G. brevis other factors such as 

 the growth phase of culture, pH of culture during 

 growth and during test period, temperature and 

 salinity of test culture, size and number of test 

 fish, volume and degree of aeration of test culture, 

 and bacterial growth, are among those that must 

 be consider h1 in standardizing the toxicity tests. 

 Shilo and Aschner (1953) found that a number of 

 factors influenced the toxicity of Prymnesium 

 parimm cultures. The toxicity was decreased by 

 oxidizing agents, aeration, adsorbents including 

 pond-bottom soils, bacterial growth, and low pH 

 (below 7.5). They improved the standardization 

 of their toxicity tests b}' using a buffer to control 

 pH of the test culture and streptomycin to sup- 

 press bacterial growth. Furthermore, the test 

 cultures were not aerated. McLaughlin ^ work- 

 ing with the same organism reported that cul- 

 tures grown in an alkaline medium were more 

 toxic than those grown in an acid medium. P. 

 parvum cultures (grown in alkaline media), 

 rendered nontoxic by lowering the pH to 6.0, 

 regain their toxicity when made alkaline (Shilo 

 and Aschner, 1953; McLaughlin '). 



Some of the factors mentioned above may ac- 

 count for the variable death times obtained in 

 duplicate containers of G. brems cultures. Since 

 on some occasions the pattern of response in one of 

 the duplicate containers was different from that 

 in the other, we consider that factors other than 

 variations of the individual test fish were respon- 

 sible. For example, in experiment 6 (table 4) 

 the fish died in 9 and 16 minutes in one container 

 whereas death occurred after 1% and 2 hours' 

 exposure in another. Both containers (non- 

 aerated) held similar amounts of the same unialgal 

 culture. 



There are other anomalies that defy explanation 

 at present. One of these concerns the variation 

 in the response of fish subjected to cultures sup- 

 posedly grown under the same conditions, and 



> McLaughlin. John J. \. The physiology and nutritioniil requirements 

 of .some chrysomonads. tJ.'i pp. Thesis, Ph. D., New York University. 

 1956. 



