Water bottle samples were collected from selected depths (variable with station) at 44 stations in the study area. Coulter counts in the 14 to 28 um size class were routinely determined for comparison with the microscope counts provided by other researchers on the cruise. At six stations, water samples were filtered through a 40 um Nytex mesh and centrifuged for 10 minutes at 1200 x g and then frozen to isolate Karenia brevis cells for subsequent analysis (pg cell-1) of cellular lipid, free amino acid, and protein. The biochemical determinations were performed within two weeks of collection at Kamykowski's laboratory at NCSU.

Water bottle samples were collected from selected depths (variable with station) at 14 stations in the study area. Coulter counts in the 14-28 um size class were routinely determined for comparison with the microscope counts. Water samples were filtered through 40 um Nytex mesh and centrifuged for 10 minutes at 1200 x g to isolate Karenia brevis. Pellets were frozen for subsequent analysis (pg cell-1) of total lipid, neutral lipid, free amino acids, protein and RNA. The biochemical determinations were performed within two weeks of collection at Kamykowski's NCSU laboratory. Aliquots were passed through 0.4 um filters; the filters were placed in 90% acetone for subsequent analysis of chlorophyll and the supernatant was analyzed for nitrate.

Harmful algal blooms of the toxic dinoflagellate, Karenia brevis, have caused massive fish kills in the Gulf of Mexico since the 1500's, with most occurrences on the west coast of Florida. In 1996, the list of states that have experiences natural resource, public health and economic impacts related to this organism expanded, with the addition of Alabama, Mississippi, and Louisiana, to include all the Gulf-coast states and North Carolina. Estimates of economic impacts to Florida and North Carolina from two moderate intensity blooms ranged from 15 to 25 million dollars respectively. The harmful impacts caused by K. brevis occur only when cell concentrations increase significantly above low background concentrations that are present year-round in the Eastern Gulf of Mexico. Once a bloom has developed offshore in typically oligotrophic waters, cell concentrations at the 105 level can be maintained for months. During 21 of the past 22 years, red tide blooms have been observed within the region between Tampa Bay and Charlotte Harbor. The key to understanding any HAB lies in knowing how one algal species has adapted and come to dominate in its particular realm of physical, biological and chemical conditions. Our ability to predict initiation, maintenance, and dispersal of blooms on the Florida shelf has been severely limited by the lack of a quantitative description, or model, of their population dynamics and the physical, biological and chemical regime in which they are embedded. The modeling components of this project will incorporate the quantitative description of blooms and their surrounding environment provided by the field and laboratory portions of this project. The field component will employ a set of annual process cruises.

The U.S. Environmental Protection Agency (EPA) National Coastal Condition Assessment (NCCA) is a nationwide survey of coastal and estuarine water quality. During the 2015 EPA NCCA, samples were collected at 732 sites for analysis of algal toxins and cyanotoxins at the Organic Geochemistry Research Laboratory (OGRL) at the U.S. Geological Survey Kansas Water Science Center (KSWSC) by liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS). Samples from the Atlantic, Gulf, and Pacific coasts of the conterminous U.S. were analyzed for anatoxin-a, cylindrospermopsin, domoic acid, dinophysistoxin-1, dinophysistoxin-2, gymnodimine, 10 microcystin congeners, nodularin, okadaic acid, pectenotoxin-2, and 13-desmethyl spirolide c.

The U.S. Environmental Protection Agency (EPA) National Coastal Condition Assessment (NCCA) is a nationwide survey of coastal and estuarine water quality. During the 2015 EPA NCCA, samples were collected at 732 sites for analysis of algal toxins and cyanotoxins at the Organic Geochemistry Research Laboratory (OGRL) at the U.S. Geological Survey Kansas Water Science Center (KSWSC) by liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS). Samples from the Atlantic, Gulf, and Pacific coasts of the conterminous U.S. were analyzed for anatoxin-a, cylindrospermopsin, domoic acid, dinophysistoxin-1, dinophysistoxin-2, gymnodimine, 10 microcystin congeners, nodularin, okadaic acid, pectenotoxin-2, and 13-desmethyl spirolide c.

The toxicity of sediments in Pensacola, Choctawhatchee, St. Andrew and Apalachicola Bays was determined as part of bioeffects assessments performed by NOAA's National Status and Trends Program. The objectives of the survey were to determine: (1) the spatial patterns in toxicity throughout each bay, (2) the spatial extent of toxicity throughout and among the bays, (3) the severity or degree of toxicity, and (4) the relationships between chemical contamination and toxicity. The survey was conducted over two years: Pensacola Bay and St. Andrew Bay were sampled in 1993; and Choctawhatchee Bay, Apalachicola Bay and Bayou Chico (a sub-basin of Pensacola Bay) were sampled during 1994. Surficial sediment samples were collected from 123 randomly-chosen locations throughout the five areas. Multiple toxicity tests were conducted on all samples, and chemical analyses were performed on 102 of the 123 samples. Toxicological tests were conducted to determine survival, reproductive success, morphological development, metabolic activity, and genotoxicity; all bays showed toxicity in at least some of the samples. Toxicity was most severe in Bayou Chico, an industrialized basin adjoining Pensacola Bay. Other developed bayous adjoining Pensacola Bay and the other bays also showed relatively severe toxicity. The main basins of the bays generally showed lower toxicity than the adjoining bayous. The different toxicity tests, however, indicated differences in severity, incidence, spatial patterns, and spatial extent in toxicity. The most sensitive test, a bioassay of metabolic activity of bioluminescent bacteria, indicated toxicity was pervasive throughout the entire study area. The least sensitive test, an acute bioassay performed with a benthic amphipod, indicated toxicity was restricted to a very small portion of the area. Causes of toxicity were not determined in the survey. However, mixtures of potentially toxic substances, including pesticides, petroleum constituents, trace metals, and ammonia, were associated statistically with the measures of toxicity. The concentrations of many substances were highest in Bayou Chico, where the most severe toxicity was observed. At these toxic sites, some of the substances had considerably elevated concentrations, often exceeding numerical guidelines or known toxicity thresholds. The relationships between toxicity and chemical concentrations differed among the bays and toxicity tests.

Interactions between bacteria and species of harmful and/or toxic algae are potentially important factors affecting both the population dynamics and toxicity of these algae. Recent reports of bacteria lethal to certain harmful algal bloom (HAB) species, coupled with a rapidly evolving interest in attempting to minimize the adverse effects of HABs through various prevention, control, and mitigation strategies, has focused attention on defining the role of algicidal bacteria in bloom termination. The aim of the present study was to determine if algicidal bacteria active against Karenia brevis, a dinoflagellate responsible for frequent and protracted red tides in the Gulf of Mexico, are present in the waters of the west Florida shelf. To date we have detected one algicidal bacterium from samples obtained during ECOHAB-Florida cruises and we are continuing this effort. A taxon-specific 16S rRNA probe was developed for a previously isolated algicidal strain, 41-DBG2, and we have begun to screen water samples for its presence using fluorescent in-situ hybridization. In addition, we have employed denaturing gradient gel electrophoresis (DGGE) to characterize the diversity of the ambient microbial assemblages and to visualize the band characteristic of strain 41-DBG2. This band can then be analyzed further by excising and sequencing. Our screening results to date have yielded negative results on all but one of the field samples tested, which was positive with the rRNA probe. These largely negative findings may not be unexpected due to the marked temporal and spatial separation of the original algicidal bacterial isolation and the water samples being screened. Future efforts will focus on isolating algicidal bacteria from the same bloom event as the samples for screening are obtained, thereby increasing the likelihood of detecting algicidal strains and permitting a more effective analysis of their population dynamics.

Chlorophyll a is a standard measure for phytoplankton biomass. Routinely, samples for extracted chlorophyll a values are filtered at sea, stored in liquid nitrogen, transported on dry ice and kept at -80 degrees Celsius until extracted. These values are used to examine the relationship between Karenia brevis cell counts, water column toxin levels and chlorophyll a pigment content.