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The Florida stone crab, Menippe mercenaria, is a major commercial fishery that occurs primarily along Florida's west coast, where harmful algal blooms of Karenia brevis frequently develop. To determine sublethal and lethal effects of K. brevis on M. mercenaria, we exposed sublegal stone crabs to three seawater treatments in laboratory conditions: no K. brevis (control), a low-toxin K. brevis strain (Wilson LT), and a toxic K. brevis (New Pass strain). Total food consumed, reflex impairment and survivorship of each crab was monitored throughout the nine-day experiment. Crabs in the toxic treatment consumed 67% less food. The probability of an individual losing a reflex significantly increased with time (days), and there was a 42% decrease in survivorship in the toxic treatment. This is the first study to demonstrate negative effects of K. brevis on the stone crab, presenting the critical need of further investigation to fully understand how red tide may impact sustainability of the fishery.

The bivalve mollusc, Crassostrea virginica, is frequently exposed to blooms of Karenia brevis along the west coast of Florida during periods of spawning and early larval development. A continuous 4-day exposure of gametes and 2-4 cell stage embryos of C. virginica to whole-cell and culture filtrate of K. brevis at 500 and 5000 cells mL(-1), was followed by a 4-day 'recovery' period. Larval growth, percent of normal, abnormal and dead larvae, and the presence of food in the larval gut were measured throughout the exposure period. Results suggest that negative effects mainly occur during embryogenesis and early development. Damage to feeding apparatus/gut may occur during embryonic development or exposure to toxins may act as a feeding deterrent on non-toxic algae. Following 2-h in vitro exposure of gametes, differences in oocyte and sperm cell parameters were investigated using flow cytometry. The reduced sperm viability in the whole-cell 5000 cells mL(-1) treatment suggests the involvement of extracellular brevetoxins (PbTx) and perhaps other harmful, uncharacterized compounds associated with the K. brevis cell membrane. The cumulative effects of reduced sperm viability, fertilization success, embryonic and larval survival, and the near-annual exposure to blooms of K. brevis could cause significant bottlenecks on oyster recruitment.

Brevetoxins in clams (Mercenaria sp.) exposed to recurring blooms of Karenia brevis in Sarasota Bay, FL, were studied over a three-year period. Brevetoxin profiles in toxic clams were generated by ELISA and LC-MS. Several brevetoxin metabolites, as identified by LC-MS, were major contributors to the composite brevetoxin response of ELISA. These were S-desoxyBTX-B2 (m/z 1018), BTX-B2 (m/z 1034), BTX-B5 (m/z 911), open A-ring BTX-B5 (m/z 929), and BTX-B1 (m/z 1018). Summed values of these metabolites were highly correlated (R2 = 0.9) with composite B-type brevetoxin measurements by ELISA. S-desoxyBTX-B2, BTX-B2, and BTX-B1 were the most persistent and detectable in shellfish for several months after dissipation of blooms. These metabolites were selected as LC-MS biomarkers of brevetoxin exposure and reflective of composite B-type brevetoxins in hard clam. ELISA and LC-MS values were moderately correlated with toxicity of the shellfish by mouse bioassay. ELISA and LC-MS methods offer rapid screening and confirmatory determination of brevetoxins, respectively, as well as toxicity assessment in clams exposed to K. brevis blooms.

The declining health of coral reefs is intensifying worldwide at an alarming rate due to the combined effects of land-based sources of pollution and climate change. Despite the persistent use of mosquito control pesticides in populated coastal areas, studies examining the survival and physiological impacts of early life-history stages of non-targeted marine organisms are limited. In order to better understand the combined effects of mosquito pesticides and rising sea surface temperatures, we exposed larvae from the coral Porites astreoides to selected concentrations of two major mosquito pesticide ingredients, naled and permethrin, and seawater elevated +3.5 °C. Following 18-20 h of exposure, larvae exposed to naled concentrations of 2.96 µg L(-1) or greater had significantly reduced survivorship compared to controls. These effects were not detected in the presence of permethrin or elevated temperature. Furthermore, larval settlement, post-settlement survival and zooxanthellae density were not impacted by any treatment. To evaluate the sub-lethal stress response of larvae, several oxidative stress endpoints were utilized. Biomarker responses to pesticide exposure were variable and contingent upon pesticide type as well as the specific biomarker being employed. In some cases, such as with protein carbonylation and catalase gene expression, the effects of naled exposure and temperature were interactive. In other cases pesticide exposure failed to induce any sub-lethal stress response. Overall, these results demonstrate that P. astreoides larvae have a moderate degree of resistance against short-term exposure to ecologically relevant concentrations of pesticides even in the presence of elevated temperature. In addition, this work highlights the importance of considering the complexity and differential responses encountered when examining the impacts of combined stressors that occur on varying spatial scales.

In 2005 and 2006, the central west Florida coast experienced two intense Karenia brevis red tide events lasting from February 2005 through December 2005 and August 2006 through December 2006. Strandings of sea turtles were increased in the study area with 318 turtles (n = 174, 2005; n = 144, 2006) stranding between 1 January 2005 and 31 December 2006 compared to the 12-yr average of 43 +/- 23 turtles. Live turtles (n = 61) admitted for rehabilitation showed clinical signs including unresponsiveness, paresis, and circling. Testing of biological fluids and tissues for the presence of brevetoxin activity by enzyme-linked immunosorbent assay found toxin present in 93% (52 of 56) of live stranded sea turtles, and 98% (42 of 43) of dead stranded sea turtles tested. Serial plasma samples were taken from several live sea turtles during rehabilitation and toxin was cleared from the blood within 5-80 days postadmit depending upon the species tested. Among dead animals the highest brevetoxin levels were found in feces, stomach contents, and liver. The lack of significant pathological findings in the majority of animals necropsied supports toxin-related mortality.

Harmful algal bloom events caused by the dinoflagellate Karenia brevis occurred along the central west Florida, USA, coast from February 2005 through December 2005 and from August 2006 through December 2006. During these events, from 4 February 2005 through 28 November 2006, live, debilitated seabirds admitted for rehabilitation showed clinical signs that included disorientation, inability to stand, ataxia, and seizures. Testing of blood, biologic fluids, and tissues for brevetoxin by enzyme-linked immunosorbent assay found toxin present in 69% (n=95) of rehabilitating seabirds. Twelve of the 19 species of birds had evidence of brevetoxin exposure. Commonly affected species included Double-crested Cormorants (Phalacrocorax auritus), Brown Pelicans (Pelecanus occidentalis), Great Blue Herons (Ardea herodias), and Common Loons (Gavia immer). Serial blood and fecal samples taken from several live seabirds during rehabilitation showed that brevetoxin was cleared within 5-10 days after being admitted to the rehabilitation facility, depending on the species tested. Among seabirds that died or were euthanized, the highest brevetoxin concentrations were found in bile, stomach contents, and liver. Most dead birds had no significant pathologic findings at necropsy, thereby supporting brevetoxin-related mortality.

Blooms of Karenia brevis (also called red tides) occur almost annually in the Gulf of Mexico. The health effects of the neurotoxins (i.e., brevetoxins) produced by this toxic dinoflagellate on marine turtles are poorly understood. Florida's Gulf Coast represents an important foraging and nesting area for a number of marine turtle species. Most studies investigating brevetoxin exposure in marine turtles thus far focus on dead and/or stranded individuals and rarely examine the effects in apparently "healthy" free-ranging individuals. From May-July 2014, one year after the last red tide bloom, we collected blood from nesting loggerhead sea turtles (Caretta caretta) on Casey Key, Florida USA. These organisms show both strong nesting and foraging site fidelity. The plasma was analyzed for brevetoxin concentrations in addition to a number of health and immune-related parameters in an effort to establish sublethal effects of this toxin. Lastly, from July-September 2014, we collected unhatched eggs and liver and yolk sacs from dead-in-nest hatchlings from nests laid by the sampled females and tested these samples for brevetoxin concentrations to determine maternal transfer and effects on reproductive success. Using a competitive enzyme-linked immunosorbent assay (ELISA), all plasma samples from nesting females tested positive for brevetoxin (reported as ng brevetoxin-3[PbTx-3] equivalents [eq]/mL) exposure (2.1-26.7ng PbTx-3eq/mL). Additionally, 100% of livers (1.4-13.3ng PbTx-3eq/mL) and yolk sacs (1.7-6.6ng PbTx-3eq/mL) from dead-in-nest hatchlings and 70% of eggs (

Blooms of the brevetoxin-producing dinoflagellate, Karenia brevis , are a recurrent and sometimes devastating phenomenon in the Gulf of Mexico. The eastern oyster, Crassostrea virginica , is exposed regularly to these blooms, yet little is known about the impacts of K. brevis upon this important species. The present study considered the effects of exposure to both a natural bloom and cultured K. brevis on the reproductive development of C. virginica . Oysters had been exposed to a bloom of K. brevis that occurred in Lee County, Florida, from September 2012 through May 2013, during a period of gametogenesis and gamete ripening. Ripe adult oysters were collected from this bloom-exposed site and from a site 200 miles north which was not exposed to any bloom. In addition, responses to two 10-day laboratory exposures of either unripe or ripe adult oysters to whole cells of K. brevis at high bloom concentrations (1000 and 5000 cells mL −1 ) were determined. Both field- and laboratory-exposed adult oysters accumulated PbTx (attaining ∼22 × 10 3 ng g −1 and 922 ng g −1 PbTx-3 equivalents in the laboratory and the field, respectively), and significant mucal, edematous, and inflammatory features, indicative of a defense response, were recorded in adult tissues in direct contact with K. brevis cells. Laboratory-exposed oysters also showed an increase in the total number of circulating hemocytes suggesting that: (1) new hemocytes may be moving to sites of tissue inflammation, or, (2) hemocytes are released into the circulatory system from inflamed tissues where they may be produced. The area of oyster tissue occupied by gonad (representative of reproductive effort) and reactive oxygen species production in the spermatozoa of oysters exposed to the natural bloom of K. brevis were significantly lower compared to oysters that were not exposed to K. brevis . Additionally, following 10-day exposure of ripe oysters, a significant, 46% reduction in the prevalence of individuals with ripe gametes was obtained in the 5000 cells mL −1 K. brevis treatment. Brevetoxin (PbTx) was recorded within the spermatozoa and oocytes of naturally exposed oysters and was estimated to be 18 and 26% of the adult PbTx load, respectively. Larvae derived from gametes containing PbTx showed significantly higher mortalities and attained a smaller larval size for the first 6 days post-fertilization. These negative effects on larval development may be due to the presence of PbTx in the lipid droplets of the oocytes, which is mobilized by the larvae during embryonic and lecithotrophic larval development. Provision of a non-contaminated food source to larvae however, appeared to mitigate the early negative effects of this neonatal PbTx exposure. Results herein show that adult eastern oysters and their offspring are susceptible to exposure to K. brevis. Caution should therefore be exercised when identifying oyster reef restoration areas and in efforts to establish aquaculture in areas prone to red tides.

Red tide aerosols containing aerosolized brevetoxins are produced during the red tide bloom and transported by wind to coastal areas of Florida. This study reports the characterization of Florida red tide aerosols in human volunteer studies, in which an asthma cohort spent 1h on Siesta Beach (Sarasota, Florida) during aerosolized red tide events and non-exposure periods. Aerosol concentrations, brevetoxin levels, and particle size distribution were measured. Hourly filter samples were taken and analyzed for brevetoxin and NaCl concentrations. In addition, the aerosol mass concentration was monitored in real time. The results indicated that during a non-exposure period in October 2004, no brevetoxin was detected in the water, resulting in non-detectable levels of brevetoxin in the aerosol. In March 2005, the time-averaged concentrations of brevetoxins in water samples were moderate, in the range of 5-10 microg/L, and the corresponding brevetoxin level of Florida red tide aerosol ranged between 21 and 39 ng/m(3). The temporal profiles of red tide aerosol concentration in terms of mass, NaCl, and brevetoxin were in good agreement, indicating that NaCl and brevetoxins are components of the red tide aerosol. By continuously monitoring the marine aerosol and wind direction at Siesta Beach, we observed that the marine aerosol concentration varied as the wind direction changed. The temporal profile of the Florida red tide aerosol during a sampling period could be explained generally with the variation of wind direction.

Florida red tides, caused by the dinoflagellate Karenia brevis, produce a series of neurotoxins, brevetoxins, which have historically had a negative impact on coastal communities along the South Florida coast. While much work has examined the effects of K. brevis blooms or exposure to brevetoxins on a diverse array of marine organisms, there have been no reports studying the impact of K. brevis on coral physiology. We provide evidence that short-term exposure of naturally occurring concentrations of K. brevis and their associated toxins can induce oxidative stress in the coral larvae of Porites astreoides. Larvae of P. astreoides were exposed to aliquots of intact K. brevis cells at naturally occurring concentrations (6 × 105 – 4 × 106 cells L−1) or cellular lysates (containing 5.4–15.0 μg L−1 brevetoxin) for 20 h and a variety of larval physiological biomarkers were measured. In the presence of bloom scale concentrations of K. brevis larval respiration of P. astreoides was inhibited. This was accompanied with an increase in lipid hydroperoxide content and catalase activity indicating oxidative damage and a subsequent antioxidant response were occurring. However, when larvae were exposed to intact cells of K. brevis or their sonicated, lysed exudates stress indices including zooxanthellae expulsion (bleaching), larval protein carbonylation, and superoxide dismutase activity were not significantly altered. Even though there was evidence for short-term oxidative stress in larvae that were exposed to K. brevis there was no change in survival or settlement rates of larvae of P. astreoides.

Florida red tides are a manifestation of the larger growing environmental issue of harmful algal blooms (HABs) (Erdner et al. 2008; Van Dolah 2000). HABs are blooms of micro-algae in aquatic environments, causing harm to humans and other animals, particularly by the production of potent natural toxins. Florida red tide is caused by the marine dinoflagellate Karenia brevis, which produces the potent neurotoxins brevetoxins. These toxins cause the death of millions of fish annually in the Gulf of Mexico, as well as morbidity and mortality among marine mammals and sea birds. Exposure to seafood contaminated by brevetoxins is associated with neurotoxic shellfish poisoning in humans, whereas the aerosols of the Florida red tide have been reported to cause respiratory irritation in persons recreating and working in coastal communities during active blooms with onshore winds (Backer et al. 2003, 2005; Fleming et al. 2005a, 2005b, 2007; Watkins et al. 2008; Zaias et al., in press). Our prior research demonstrated that persons with a physician diagnosis of asthma experience a statistically significant change in their respiratory symptoms and pulmonary function after a 1-hr visit to the beach during a documented Florida red tide; these changes are not seen when the same individuals visit the beach for 1 hr during a non-Florida red tide period (Fleming et al. 2007; Milian et al. 2007). As part of our research, at the same time we collected symptom and pulmonary function data before and after the beach exposure for each individual subject, we collected substantial ambient samples including water for K. brevis cell counts and brevetoxin concentrations; ambient aerosol samples for brevetoxin concentrations and aerosol particle size; and individual personal air samples for brevetoxin concentrations (Cheng et al. 2005a, 2005b, 2005c; Naar et al. 2002; Pierce et al. 2003). In the present study we investigated for the first time which of the meas ures of brevetoxin concentrations in ambient samples were most closely associated with health effects and whether there were dose–response relationships between these ambient measurements and health outcomes.

Brevetoxin uptake and elimination were examined in Eastern oyster ( Crassostrea virginica ) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100 g. Oysters were toxic by mouse bioassay at levels ≥20 MU/100 g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated ( r 2 = 0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well ( r 2 = 0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well ( r 2 = 0.65), and were weakly correlated with mouse bioassay ( r 2 = 0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.

Metabolic rates and thermal tolerances of three species of mobile symbiotic molluscs (two provannid gastropods Alviniconcha sp. and Ifremeria nautilei; and the mussel Bathymodiolus brevior) from Lau Basin hydrothermal vents were measured. Thermal tolerances of the two gastropods were similar and well in excess of temperatures observed in situ, although Alviniconcha sp. appeared to have slightly higher tolerances. B. brevior had lower thermal limits that could exclude it from central areas of active venting. High-pressure respirometry experiments revealed that Alviniconcha sp. has very high potential growth rates; its uptake of carbon for its endosymbionts being among the highest recorded. Uptake rates of metabolites in I. nautilei and B. brevior were an order of magnitude lower than those observed in Alviniconcha sp. Taken together, these thermal tolerances and metabolite uptake rates help to explain the zonation patterns seen in situ, where Alviniconcha sp. is found closest to the source of vent fluid, followed by I. nautilei, and then B. brevior.

This project was undertaken to provide information about the composition and fate of brevetoxins in concert with the multidisciplinary study, ECOHAB-FL, of Karenia brevis blooms in the Gulf of Mexico. Brevetoxin composition was provided for water samples collected during and in the absence of K. brevis blooms from November 1998, through September 2002. The identity and concentration of the most abundant brevetoxins were determined using high performance liquid chromatography with ultraviolet diode array detection (HPLC-DAD). The analytical methods changed in 2002 to the use of a mass spectrometer for brevetoxin identification and quantitation. The most abundant brevetoxins observed during blooms were PbTx-1, -2 and -3. PbTx-2 was the most abundant toxin observed in viable bloom situations with an abundance of K. brevis cells. Starting with the 2000 cruises, a distinction was made between intra-cellular toxins (inside viable K. brevis cells) and extra-cellular brevetoxins (dissolved brevetoxins outside of the cell). An important observation was the change in composition of the major brevetoxins from intra-cellular to extra-cellular toxins. The most abundant intra-cellular toxin was PbTx-2, whereas the most abundant brevetoxin recovered from the extra-cellular (dissolved) fraction in the water was PbTx-3. The abundance of PbTx-3 relative to PbTx-2 generally increased as a bloom aged, indicating the conversion of PbTx-2 to -3 as cells lysed, and the persistence of PbTx-3 in the water after cell death.

Brevetoxins and ciguatoxins are closely related potent marine neurotoxins. Although ciguatoxins accumulate in fish to levels that are dangerous for human consumption, live fish have not been considered as potential sources of brevetoxin exposure in humans. Here we show that, analogous to ciguatoxins, brevetoxins can accumulate in live fish by dietary transfer. We experimentally identify two pathways leading to brevetoxin-contaminated omnivorous and planktivorous fish. Fish fed with toxic shellfish and Karenia brevis cultures remained healthy and accumulated high brevetoxin levels in their tissues (up to 2675 ng g−1 in viscera and 1540 ng g−1 in muscle). Repeated collections of fish from St. Joseph Bay in the Florida panhandle reveal that accumulation of brevetoxins in healthy fish occurs in the wild. We observed that levels of brevetoxins in the muscle of fish at all trophic levels rise significantly, but not to dangerous levels, during a K. brevis bloom. Concentrations were highest in fish liver and stomach contents, and increased during and immediately following the bloom. The persistence of brevetoxins in the fish food web was followed for 1 year after the K. brevis bloom.

Bottlenose dolphins (Tursiops truncatus) face a variety of threats, including risk of exposure to brevetoxins produced by blooms of the harmful alga Karenia brevis. This study investigated brevetoxin exposure in a population of dolphins inhabiting Sarasota Bay, Florida, USA (27°N, 82°W), utilizing tissues from dolphins recovered between 1994 and 2003. Brevetoxin levels detected by ELISA in tissues, gastric samples and excreta from dolphin carcasses (n = 19) associated with K. brevis blooms were compared with with levels in carcasses (n = 16) associated with background K. brevis conditions. In the K. brevis-exposed set, 84% of dolphin carcasses recovered during K. brevis blooms had detectable brevetoxin levels, with values ranging between 7 and 2,896 ng PbTx-3 eq g−1. Over 50% of dolphin carcasses recovered during non-bloom conditions also tested positive by ELISA for brevetoxins, with concentrations ranging from 6 to 44 ng PbTx-3 eq g−1. Control samples from the east coast of Florida were negative by the ELISA. Results from this study establish baseline brevetoxin body burdens in a dolphin population frequently exposed to K. brevis blooms, and data indicate that dolphin carcasses not associated with large-scale mortality events can contain levels of brevetoxins comparable to carcasses stranding during such events.

Relaxation on conference travel restrictions for federal scientists and engineers forms part of the bill. Relaxation on conference travel restrictions for federal scientists and engineers forms part of the bill.

Paul Ryan has voiced support for basic science while opposing applied energy research. Paul Ryan has voiced support for basic science while opposing applied energy research.

A new bill would require NSF to show how each grant serves the national interest. A new bill would require NSF to show how each grant serves the national interest.

If confirmed, Cherry Murray will head DOE’s science office; Richard Buckius will serve as NSF deputy director.

Despite operational challenges, the ISS continues to have strong bipartisan support in Congress.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44 kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

With some reservations, lawmakers expressed bipartisan support for continued R&D funding. With some reservations, lawmakers expressed bipartisan support for continued R&D funding.

Studies examining impression management (IM) in self-report measures typically assume that impression management is either a 1) trait or 2) situational variable, which has led to often conflicting results (Stark, Chernyshenko, Chan, Lee and Drasgow. 2001). This study examined the item-level and scale-level responses on six empirically-derived facets of conscientiousness from the California Psychological Inventory (CPI) between high and low IM groups. Subjects (N = 6,220) were participants in a management assessment conducted by an external consulting firm. Subjects participated in the assessment as part of either 1) a selection or promotional process, or 2) a feedback and development process, and two specific occupational groups (sales/marketing and accounting/finance) were examined. Using the IRT-based DFIT framework (Raju, Van der Linden & Fleer, 1995), the item-level and scale-level differences were examined for the situational IM and traited IM approaches. The results indicated that relatively little DIF/DTF was present and that the differences between the two approaches to examining IM may not be as great as previously suggested.

The toxic dinoflagellate, Karenia brevis, produces a suite of polyether neurotoxins (brevetoxins, PbTx) that cause massive fish kills and neurotoxic shellfish poisoning. A unique characteristic of K. brevis blooms is the associated airborne (aerosolized) toxin component causing respiratory irritation to humans and other mammals. This study was undertaken in collaboration with human respiratory effects studies to establish the type and amount of brevetoxins to which beach-goers were exposed during a coastal harmful algal bloom (HAB). Concentrations of K. brevis cells were monitored in water, and brevetoxin concentrations were monitored in water and air over 3-day periods during a non-exposure control study (no HAB) and an exposure study (HAB event) along the Gulf Coast of Sarasota, FL, USA. The aerosol particle size distribution was also determined to assess brevetoxin aerosol deposition in the human respiratory system. During the non-exposure study, very low to background K. brevis cell counts were observed on day 1 and at background levels (

Harmful algal blooms (HABs) occur worldwide causing serious threat to marine life, and to public health through seafood-borne illnesses and exposure to toxin-containing marine aerosol. This study was undertaken to assess the ability of phosphatic clay to remove the toxic dinoflagellate, Karenia brevis , and the potent neurotoxins (brevetoxins) produced by this species. Results showed that the addition of an aqueous slurry of 0.75 g (dry weight) clay to 3 l of K. brevis culture, containing 5×10 6 and 10×10 6 cells/l, removed 97±4% of brevetoxins from the water column within 4 h after the addition of clay. Clay flocculation of extra-cellular brevetoxins, released from cells ruptured (lyzed) by ultrasonication, removed 70±10% of the toxins. Addition of the chemical flocculant, polyaluminum chloride (PAC), removed all of the extra-cellular toxins. A 14 day study was undertaken to observe the fate of brevetoxins associated with clay flocculation of viable K. brevis cells. At 24 h following the clay addition, 90±18% of the toxins were removed from the water column, along with 85±4% of the cells. The toxin content of clay diminished from 208±13 μg at Day 1, to 121±21 μg at Day 14, indicating that the phosphatic clay retained about 58% of the toxins throughout the 14-day period. These studies showed the utility of natural clay as a means of reducing adverse effects from HABs, including removal of dissolved toxins, in the water column, although considerable work clearly remains before this approach can be used on natural blooms in open waters.

This investigation was undertaken to determine whether the mosquito larvicide temephos (applied as Abate 4-E ®) created an environmental hazard for nontarget organisms in a southwest Florida salt marsh community. Field toxicity tests were performed during routine larvicide applications using larvae of salt marsh crabs (Aratus pesonii and Uca rapax) and adult Mysidopsis bahia. Laboratory toxicity tests were performed using survival through 48 and 96 h and through first molt as toxicity endpoints. Larvae of both species exhibited 96 h lowest observable effects concentration (LOEC) in the range of 15 to 20 μg/L temephos. First-molt LOEC ranged from 7 to 12 μg/L. No effect on M. bahia survival, growth, or fecundity was observed up to 40 μg/L temephos. Field studies of routine temephos application rates produced an expected environmental concentration (EEC) of 12 ± 10 μg/L in marsh water, causing an environmental hazard to crab larvae. Reduction of the application rate effectively reduced the EEC to 4 ± 3 μg/L, indicating no hazard for 48 and 96 h toxicity; however, a hazard was observed for survival through the first molt. This information was used to modify larviciding applications to effect a reduction of the environmental hazard to nontarget organisms in the salt marsh.

Reversed-phase liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was employed to analyze brevetoxin compounds associated with naturally occurring red tide blooms collected from Sarasota Bay, FL, USA. The LC-ESI-MS method utilizes a C18 microbore column with a mobile phase consisting of methanol-water (85:15, v/v), a flow-rate of 8 μl/min and a post-column split ratio of 3:1 (UV-absorbance detector-mass spectrometer). Three known brevetoxins Btx-2, Btx-1 and Btx-3 were detected at 60 μg/l, 10 μg/l and 5.7 μg/l levels, respectively, in the natrual red tide bloom samples. This distribution differed quantitatively from that found in red tide culture extract samples. Btx-9 was not detected either in natural red tide bloom extracts or in red tide culture extracts, possibly due to the instability of this compound. An unknown component with a molecular mass of 941 found in the natural bloom extract was postulated to have the structure of a reduced form (hydrogenation of two double bonds in the alkyl side chain) of Btx-5.

The harmful alga, Karenia brevis, produces a suite of polyether neurotoxins, brevetoxins or PbTx, that cause marine animal mortality and neurotoxic shellfish poisoning (NSP). A characteristic of K. brevis blooms is associated airborne toxins that result in severe respiratory problems. This study was undertaken to determine the composition of aerosolized brevetoxins and oxidative derivatives to which beachgoers are exposed during a K. brevis bloom. The suite of brevetoxins and derivatives in seawater is comprised of intra-cellular (IC) and extra-cellular (EC) compounds. We hypothesized that aerosolized compounds are generated primarily from EC, hydrophobic compounds in seawater by bubble-mediated transport. Thus the composition of aerosolized brevetoxins and derivatives, to which beachgoers are exposed, would reflect the EC composition of the source matrix (the local surf zone). Brevetoxins were extracted from water collected along the shore and from marine aerosols along Siesta Beach and Lido Beach in Sarasota, FL, USA, during K. brevis blooms. Water samples were further processed into IC and EC components. The primary brevetoxins observed in water and air included PbTx-1, -2, -3, -PbTx-2-carboxylic acid, and brevenal. Oxidation and/or hydrolysis products of PbTx-1, -2, -3 and -7 were also found in EC water and in aerosol, but not IC.

Florida red tides, an annual event off the west coast of Florida, are caused by the toxic dinoflagellate, Karenia brevis. K. brevis produces a suite of potent neurotoxins, brevetoxins, which kill fish, sea birds, and marine mammals, as well as sickening humans who consume contaminated shellfish. These toxins become part of the marine aerosol, and can also be inhaled by humans and other animals. Recent studies have demonstrated a significant increase in symptoms and decrease lung function in asthmatics after only one hour of beach exposure during an onshore Florida red tide bloom. This study constructed a transect line placing high volume air samplers to measure brevetoxins at sites beginning at the beach, moving approximately 6.4 km inland. One non-exposure and 2 exposure studies, each of 5 days duration, were conducted. No toxins were measured in the air during the non-exposure period. During the 2 exposure periods, the amount of brevetoxins varied considerably by site and by date. Nevertheless, brevetoxins were measured at least 4.2 kilometers from the beach and/or 1.6 km from the coastal shoreline. Therefore, populations sensitive to brevetoxins (such as asthmatics) need to know that leaving the beach may not discontinue their environmental exposure to brevetoxin aerosols.

The Florida red tide is a descriptive name for high concentrations of the harmful marine alga, Karenia brevis. Although most prevalent along the south-west Florida coast, periodic blooms have occurred throughout the entire US and Mexico Gulf coasts and the Atlantic coast to North Carolina. This dinoflagellate produces a suite of polyether neurotoxins, called brevetoxins, that cause severe impacts to natural resources, as well as public health. These naturally produced biotoxins may represent one of the most common chemical stressors impacting South Florida coastal and marine ecosystems. Impacts include massive fish kills, marine mammal, sea turtle and sea bird mortalities, benthic community die-off and public health effects from shellfish contamination and inhalation of air-borne toxins. The primary mode of action is binding to voltage-gated sodium channels causing depolarization of nerve cells, thus interfering with nerve transmission. Other effects include immune depression, bronchial constriction and haemolysis. Parent algal toxins are synthesized within the unicellular organism, others are produced as metabolic products. Recent studies into the composition of brevetoxins in cells, water, air and organisms have shown PbTx-2 to be the primary intracellular brevetoxin that is converted over time to PbTx-3 when the cells are ruptured, releasing extracellular brevetoxins into the environment. Brevetoxins become aerosolized by bubble-mediated transport of extracellular toxins, the composition of which varies depending on the composition in the source water. Bivalved molluscs rapidly accumulate brevetoxins as they filter feed on K. brevis cells. However, the parent algal toxins are rapidly metabolized to other compounds, some of which are responsible for neurotoxic shellfish poisoning (NSP). These results provide new insight into the distribution, persistence and impacts of red tide toxins to south-west Florida ecosystems.

Bottlenose dolphins (Tursiops truncatus) along the Gulf of Mexico are frequently exposed to blooms of the toxic alga, Karenia brevis, and brevetoxins associated with these blooms have been implicated in several dolphin mortality events. Studies on brevetoxin accumulation in dolphins have typically focused on analyses of carcasses from large-scale die-offs; however, data are scarce for brevetoxin loads in live individuals frequently exposed to K. hrevis blooms. This study investigated in vivo brevetoxin exposure in free-ranging bottlenose dolphins resident to Sarasota Bay, Florida, utilizing samples collected during health assessments performed during multiple K. brevis blooms occurring from 2003 to 2005. Brevetoxins were detected by ELISA and LC-MS in 63% of bottlenose dolphins sampled (n = 30) concurrently with a K. brevis bloom. Brevetoxins were present in urine and gastric samples at concentrations ranging from 2 to 9 ng PbTx-3 eq/g, and in feces at concentrations ranging from 45 to 231 ng PbTx-3 eq/g. Samples from individuals (n = 12) sampled during nonbloom conditions (≤1,coo cells/L) were negative for brevetoxin activity. Brevetoxin accumulation data from this study complement dolphin carcass and prey fish data from the same study area, and aid in evaluating impacts of harmful algal blooms on sentinel marine animal species along the west Florida coast.

Blooms of Karenia brevis produce brevetoxins which cause neurotoxic shellfish poisoning and respiratory symptoms in humans as well as harmful effects on sea life. To investigate potential effects of brevetoxins on immune system components, a monocyte cell line (U-937) was exposed in vitro to PbTx-2. U-937 cells metabolized PbTx-2 through cellular detoxification mechanisms, as evidenced by depletion of intracellular glutathione and formation of glutathione and cysteine conjugates. Total intracellular glutathione was significantly decreased in toxin-treated cells compared to control cells, as measured using an enzymatic recycling method. LC/MS was used to detect the following brevetoxin metabolites: a cysteine-PbTx-2 conjugate (m/z 1018) and two putative glutathione-PbTx-2 conjugates (m/z 1204 and 1222). During 3h incubation, glutathione conjugates were detectable as early as 1h and increased in concentration after 2 and 3h. A cysteine-PbTx-2 conjugate appeared after 2h and increased in concentration after 3h. Detectable levels of brevetoxin conjugates were present in response to toxin concentrations of 1muM. Depletion of intracellular glutathione and formation of brevetoxin metabolites, with changes in concentrations over time, suggest immune cells (U-937) have important cellular detoxification pathways for PbTx-2.

Blooms of the brevetoxin-producing dinoflagellate Karenia brevis have been linked to high mortality of bottlenose dolphins Tursiops truncatus on Florida's Gulf of Mexico coast. A clear understanding of trophic transfer of brevetoxin from its algal source up the food web to top predators is needed to assess exposure of affected dolphin populations. Prey fish constitute a means of accumu- lating and transferring brevetoxins and are potential vectors of brevetoxin to dolphins frequently exposed to K. brevis blooms. Here we report results of brevetoxin analyses of the primary fish species consumed by long-term resident bottlenose dolphins inhabiting Sarasota Bay, Florida. Fish collected during K. brevis blooms in 2003 to 2006 were analyzed by competitive enzyme-linked immuno- sorbent assay (ELISA) and had brevetoxin concentrations ranging from 4 to 10 844 ng PbTx-3 eq g -1 tissue. Receptor binding assay (RBA) and liquid chromatography-mass spectrometry (LC-MS) analy- sis confirmed toxicity and the presence of parent brevetoxins and known metabolites. Fish collected in the absence of K. brevis blooms tested positive for brevetoxin by ELISA and RBA, with concentra- tions up to 1500 ng PbTx-3 eq g -1 tissue. These findings implicate prey fish exposed to K. brevis blooms as brevetoxin vectors for their dolphin predators and provide a critical analysis of persistent brevetoxin loads in the food web of dolphins repeatedly exposed to Florida red tides.

Efficient extraction and accurate analysis of lipophilic brevetoxins (PbTxs), produced by the harmful algal bloom (HAB) species Karenia brevis, are essential when assessing the toxicological potential of this dinoflagellate. One of the most commonly used brevetoxin extraction methodologies employs C18 solid-phase extraction (SPE). In this study, C18 SPEC discs were tested for extraction of spiked PbTx-3 in seawater and naturally produced brevetoxins from K. brevis. Quantification of brevetoxin in the extracts was determined using four independent methods: receptor binding assay (RBA), radioimmunoassay (RIA), neuroblastoma (N2A) cytotoxicity assay, and liquid chromatography/mass spectrometry (LC/MS). In addition to quantification of the brevetoxin concentration, LC/MS analysis provided identification of individual congeners and each of their hydrolyzed products. SPEC disc extractions prepared from sonicated cultures of non-brevetoxin-producing Karenia mikimotoi cultures spiked with PbTx-3 yielded extraction efficiencies of 108, 99, and 125% as determined by the RBA, RIA, and N2A cytotoxicity assay, respectively. In SPEC disc extracts of brevetoxin-producing K. brevis (isolate SP3) cultures, LC/MS analysis yielded the highest total concentrations, possibly due to the concurrent detection of hydrolytic brevetoxin congeners that accounted for up to 20% of the congener profile. Relative to the brevetoxin concentration as determined by LC/MS, the RBA, RIA, and N2A cytotoxicity assay detected 73, 83, and 51% of the total brevetoxin concentration. Stability experiments demonstrated that brevetoxins remain stable on the SPEC discs for at least 30 days, making this extraction method suitable for shipboard collections.

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Several novel brevetoxin derivatives were isolated and identified in Karenia brevis cultures and natural blooms by using solid phase extraction (SPE) and LC/MS(MS) techniques. These analogs were more polar compared with previously described brevetoxins, and were poorly extractable by conventional non-polar solvent (chloroform) partitioning. Brevetoxin analogs were structurally confirmed as hydrolyzed (open A-ring) forms of brevetoxins PbTx-1, PbTx-7, PbTx-2, and PbTx-3, and of oxidized PbTx-1 and PbTx-2. Some of these open A-ring derivatives were in greater abundance than their non-hydrolyzed counterparts. All were in much greater abundance in bloom water filtrate compared with cell-rich fractions. Open A-ring compounds were cytotoxic in mouse neuroblastoma (N2a) cell assay. In the K. brevis bloom-exposed Eastern oyster, brevetoxin metabolites with opened A rings were identified (e.g., open-ring cysteine-PbTx conjugates), contributing to their overall toxin burden.

The physiological ecology of back-arc basin fauna is only just beginning to be studied. We expect that it will differ from that of other deep ocean vent sites to the extent that the habitats and faunas are different. The critical parameters for the habitats include (1) thermal environment, (2) temporal stability of conditions, (3) concentrations of toxic dissolved materials, and (4) availability of substrates for chemoautotrophy. Of particular interest with regard to chemistry is the question of the use of sulfide or another sulfur species as the primary substrate for chemoautotrophic symbioses or the use of methanotrophic symbionts in such symbioses. Issues of the toxicity of certain metals may also be important in the andesitic sites. The faunal compositions have been previously described, but the interactions among the faunal components are still largely unknown. The critical abilities and capacities of the major faunal components remain to be studied.

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Red tides in the Gulf of Mexico are formed by blooms of the dinoflagellate Karenia brevis, which produces brevetoxins (PbTx). Brevetoxins can be transferred from water to air in the wind-powered whitecapped waves during red tide episodes. Inhalation exposure to marine aerosol containing PbTx causes respiratory problems. A liquid chromatograph/ tandem mass spectrometric method was developed for the detection and quantitation of several PbTxs in ambient samples collected during red tide events. This method was complemented by a previously developed antibody assay that analyzes the entire class of PbTx compounds. The method showed good linearity, accuracy, and reproducibility, allowing quantitation of PbTx compounds in the 10 pg/m3 range. Air concentrations of PbTxs and brevenal for individual samples ranged from 0.01 to 80 ng/m3. The particle size showed a single mode with a mass median diameter between 6 and 10 microm, which was consistent for all of the PbTx species that were measured. Our results imply that individual PbTxs were from the same marine aerosol or from marine aerosol that was produced from the same process. The particle size indicated the likelihood of high deposition efficiency in the respiratory tract with the majority of aerosol deposited in the upper airways and small but not insignificant deposition in the lower airways.

Florida red tides annually occur in the Gulf of Mexico, resulting from blooms of the marine dinoflagellate Karenia brevis. K. brevis produces highly potent natural polyether toxins, known as brevetoxins, that activate voltage-sensitive sodium channels. In experimental animals, brevetoxins cause significant bronchoconstriction. A study of persons who visited the beach recreationally found a significant increase in self-reported respiratory symptoms after exposure to aerosolized Florida red tides. Anecdotal reports indicate that persons with underlying respiratory diseases may be particularly susceptible to adverse health effects from these aerosolized toxins. Fifty-nine persons with physician-diagnosed asthma were evaluated for 1 hr before and after going to the beach on days with and without Florida red tide. Study participants were evaluated with a brief symptom questionnaire, nose and throat swabs, and spirometry approved by the National Institute for Occupational Safety and Health. Environmental monitoring, water and air sampling (i.e., K. brevis, brevetoxins, and particulate size distribution), and personal monitoring (for toxins) were performed. Brevetoxin concentrations were measured by liquid chromatography mass spectrometry, high-performance liquid chromatography, and a newly developed brevetoxin enzyme-linked immunosorbent assay. Participants were significantly more likely to report respiratory symptoms after Florida red tide exposure. Participants demonstrated small but statistically significant decreases in forced expiratory volume in 1 sec, forced expiratory flow between 25 and 75%, and peak expiratory flow after exposure, particularly those regularly using asthma medications. Similar evaluation during nonexposure periods did not significantly differ. This is the first study to show objectively measurable adverse health effects from exposure to aerosolized Florida red tide toxins in persons with asthma. Future studies will examine the possible chronic effects of these toxins among persons with asthma and other chronic respiratory impairment.

The dinoflagellate responsible for the Florida red tide, Karenia brevis, (formerly, Gymnodinium breve) (Duagbjerg et al. 2001) produces a suite of polyether neurotoxins called brevetoxins (Poli et al. 1986; Shimizu et al. 1990; Baden et al. 1995). A unique characteristic of these harmful algal blooms is the associated airborne (aerosolized) toxin component (Pierce et al. 1889; 1990). Toxins are released into the water by excretion and as cells rupture increasing the amount of extra-cellular toxins as a bloom progresses (Pierce et al. 2001). These extra-cellular toxins undergo bubble-mediated transport to the sea surface where they are ejected into the air as jet drops from the bursting bubbles. With on-shore winds and breaking surf, the toxins become incorporated into marine aerosol causing severe respiratory irritation to humans and other mammals along the shore (Pierce 1986; Pierce et al. 1990). This study was undertaken in cooperation with a human effects study to assess the amount of aerosolized brevetoxin to which beach goers were exposed during a Karenia brevis (K brevis) harmful algal bloom that occurred at Jacksonville Beach on the Atlantic coast of Florida during October, 1999 (Backer et al. 2002).

Unexpected brevetoxin vectors may account for deaths long after or remote from an algal bloom.

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESMS) has been successfully applied to the separation and identification of brevetoxins associated with "red tide" algae. Brevetoxins are toxic polyethers produced by the marine dinoflagellate Gymnodinium breve. They are responsible for fish kills, and they pose certain health risks to humans. The LC-MS method employs reversed-phase microbore HPLC on a C18 column with a mobile phase consisting of 85:15 methanol/water, a flow rate of 8 microL/min, and a postcolumn split ratio of 3:1 (UV absorbance detector/mass spectrometer). A brevetoxin culture sample was found to contain at least six components, including two well-separated peaks corresponding to the brevetoxins PbTx-2 and PbTx-1, as well as several unknown compounds, including one with a molecular mass of 899 Da (possibly an isomer of PbTx-9). The brevetoxin molecules exhibited a high tendency to bind to alkali cations in positive ion ESMS. For standard PbTx-9, PbTx-2, and PbTx-1 brevetoxins analyzed on our LC-MS system, the detection limits (employing mass spectrometer scans of 100 m/z units) were determined to be less than 600 fmol, 1 pmol, and 50 fmol, respectively (S/N = 3); the total analysis time was about 35 min.

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra-filtration cell concentrator to separate viable cells containing intra-cell toxins from ambient water containing extra-cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C-18 bonded-phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C-18 column and an 85:15 methanol:water (1 ml min−1) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra-cell toxins with extra-cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.