Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)



Degree Granting Department


Major Professor

Professor Dean F. Martin


Mass mortalities of marine organisms in the Gulf of Mexico have been associated with blooms of the unarmored dinoflagellate, Gymnodinium breve. This investigation concerns the factors affecting the growth of G. breve and the properties of a toxin produced by the organism.

A modification of the Flask Test of the Provisional Algal Assay Procedure of the Joint Industry/Government Task Force on Eutrophication was used to determine the response of G. breve to various natural waters and enrichments. The growth-promoting potential of natural waters obtained from areas where outbreaks of G. breve frequently occur was greater than that of waters from other areas, and addition of municipal waste materials to natural waters increased the maximum cell population significantly. Although enrichment, individually, with inorganic nutrients (orthophosphate, nitrate, or ammonia) had relatively little effect on G. breve growth parameters, enrichment with combinations of orthophosphate, nitrate and ammonia equivalent to the concentrations attained by enrichment with municipal waste materials effected an almost identical increase in the maximum population. The enrichment experiments with combinations of nutrients indicated that at concentrations of orthophosphate typical of west Florida coastal waters, the maximum cell population was a linear function of the amount of annnonia added (up to 0.10 ppm NH3-N).

The addition of orthophosphate or detergent-phosphate to municipal wastes, presumably free of detergent phosphate, did not significantly increase the growth-promoting potential of the waste material. Enrichment of G. breve cultures with nitrilotriacetic acid (NTA) alone or in the presence of sewage had no biostimulatory effect at concentrations of NTA expected if NTA completely replaced phosphates in detergent products.

Soil extract fractions were as effective as sewage (1%, v/v) in increasing the maximum cell population; the substances in the 500-1,000 molecular-weight fraction (iron rich) were the most effective on a mg C-added basis. The growth constant was not affected by enrichment with low molecular-weight compounds but was significantly lowered by the addition of the high molecular-weight substances (>10,000).

The toxin produced by G. breve is an endotoxin which is water in-soluble and chloroform soluble. The toxic material was acid stable, but heat and base labile. Photo-inactivation of the toxin occurred during irradiation with ultraviolet, visible, or infrared light.

The crude toxic extract was fractionated by silica gel, LH-20 Sephadex gel and DEAE-cellulose column chromatography, and a 27-fold increase in the specific activity of the toxic material was achieved with recovery of 71% of the total activity. The acidic polysaccharide content of the toxic material increased 5-fold during the purification procedure.

Elemental analysis of the purified toxin indicated an empirical formula of C87H153O18S, and the infrared absorption spectrum indicated the presence of one or more carbonyl groups and the absence of hydroxyl or amine groups. The purified toxin had a maximum absorption at 265 mμ that was optically active. The reduced molar ellipticity at 265 mμ was 3.3 x 103 degrees cm2/decimole.

The effect of the toxin on the prothrombin time of a standard plasma was determined, and the anticoagulant activity of the toxic material was 1/6 that of carrageenan and 1/70 that of heparin. Available evidence suggests the anticoagulant properties of the toxic material are not responsible for the ichthyotoxicity.

Included in

Chemistry Commons