Characterization of polar brevetoxin derivatives isolated from Karenia brevis cultures and natural blooms.

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.     

Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).

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/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g 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.     

Effects of the dinoflagellate Karenia brevis on larval development in three species of bivalve mollusc from Florida.

The effects of Karenia brevis (Wilson clone) on larval survival and development of the northern quahog, Mercenaria mercenaria, eastern oyster, Crassostrea virginica and bay scallop, Argopecten irradians, were studied in the laboratory. Larvae were exposed to cultures of whole and lysed cells, with mean total brevetoxin concentrations of 53.8 and 68.9 microgL(-1), respectively. Survival of early (3-day-old) larvae was generally over 85% for all shellfish species at K. brevis densities of 100 cells ml(-1) or less, and not significantly different between whole and lysed culture. At 1000 cells ml(-1), survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of late (7-day-old) larvae in all three species was not significantly affected by K. brevis densities of 1000 cells ml(-1) or less. At 5000 cells ml(-1), however, survival was reduced to 37%, 26% and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1000 cells ml(-1). These results suggest that blooms of K. brevis, and particularly their associated brevetoxins, may have detrimental consequences for Florida's shellfisheries by disrupting critical larval processes. Special attention should be paid to blooms of K. brevis where these shellfish occur naturally or where aquaculture and restoration activities are either ongoing or planned.     

Use of two detection methods to discriminate ciguatoxins from brevetoxins: application to great barracuda from Florida Keys.

In Florida (USA), numerous cases of human ciguatera fish poisoning, as well as neurotoxic shellfish poisoning following consumption of local seafood products, have been reported. By using in parallel, the sodium channel receptor binding assay (RBA), and the ouabain/veratridine-dependent cytotoxicity assay (N2A assay), we established criteria to identify, detect, and quantify ciguatoxins in fish extracts, with a brevetoxin as internal standard. Results showed that the Caribbean ciguatoxin C-CTX-1 exhibited an 8-fold higher potency in the RBA than brevetoxins and, a 440 and 2300-fold higher potency in the N2A assay than PbTx-1 and PbTx-3, respectively. Moreover, a sensitivity comparison between assays revealed that the N2A assay was more sensitive (12-fold) for ciguatoxin analysis, whereas the RBA was more sensitive (3-24-fold) for brevetoxins analysis. Based on the relative potency between toxins and the opposite sensitivity of both assays we have used the RBA and the N2A assay to screen great barracuda (Sphyraena barracuda) collected from the Florida Keys for ciguatoxins and brevetoxins. Fish extract analysis showed a sodium channel-dependent activity consistent with the presence of ciguatoxins, and not brevetoxins. Among 40 barracudas analyzed, 60% contained ciguatoxin levels in their liver measurable by the N2A assay with the most toxic fish containing 2.1ppb C-CTX-1 equivalents.     

Uptake, tissue distribution, and excretion of brevetoxin-3 administered to mice by intratracheal instillation

Brevetoxins are a family of potent lipid-soluble neurotoxins produced by the dinoflagellate Karenia brevis, the organism responsible for Florida red tide. Brevetoxins aerosolized by surf and wind produce irritation of the eyes, nose, and throat in people on or near red tide-affected beaches. The effects of chronic exposures to brevetoxins on healthy and health-compromised individuals are not known. The purpose of this study was to investigate the pulmonary uptake, tissue distribution, and excretion of polyether brevetoxin-3 in mice, a rodent model for investigating the potential systemic adverse health effects associated with repeated brevetoxin inhalation. Male CBA/CaJ mice were administered [3H]brevetoxin-3 by intratracheal instillation. Groups of 3 mice were sacrificed immediately after instillation and at 0.5, 3, 6, 12, 24, 48, and 96 h postinstillation. Four additional mice were placed into metabolism cages for excreta collection up to 168 h postinstillation. Brevetoxin-3 distributed rapidly to all tissues, with the highest initial doses in the liver and gastrointestinal tract. Elimination half-times ranged from approximately 28 h for fat, heart, intestines, kidneys, liver, and muscle to approximately 90 h for brain and testes. The total dose to tissue ranged from 39 ng brevetoxin equivalents-h/g for testes to 406 ng brevetoxin equivalents-h/g for liver. Approximately 90% of excretion had occurred within 96 h, with 11 and 64% of the initial brevetoxin dose excreted in urine and feces, respectively. These results are consistent with earlier reports of rapid absorption and widespread tissue distribution of brevetoxins in rats.     

Intrinsic potency of synthetically prepared brevetoxin cysteine metabolites BTX-B2 and desoxyBTX-B2.

In mammals and shellfish, brevetoxins produced by the dinoflagellate Karenia brevis are rapidly metabolized to cysteine conjugates. These metabolites identified by mass spectrometry are produced in abundance in mammals and are potentially major bioactive products for intoxication. They are also abundant metabolites in shellfish where they are, in contrast to mammals, retained for prolonged periods, posing a potential threat to shellfish consumers. In this work, we analyze the intrinsic potency of the semi-synthetic cysteine brevetoxin sulfoxide (BTX-B2) and the cysteine brevetoxin (desoxyBTX-B2), each confirmed for purity by LC-MS and NMR techniques, on receptor site 5 of the voltage-gated sodium channels (VGSCs) in brain, heart and skeletal muscle. We show that both brevetoxin conjugates compete with the tritiated reduced parent brevetoxin ([(3)H]PbTx-3) in rat brain membrane preparations and in HEK cells expressing skeletal muscle or cardiac VGSC, albeit, with 8-16-fold lower affinity than the PbTx-3. On neuroblastoma cell assays we show a 3-fold reduction in cytotoxic potency for BTX-B2 relative to PbTx-3, and an 8-fold reduction for desoxyBTX-B2. In conclusion, the major transformation product of brevetoxin observed in diverse species through cysteine adduction and oxidation leads to metabolites with reduced potency on brain, skeletal muscle and heart cells.     

Characterization of Florida red tide aerosol and the temporal profile of aerosol concentration

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 1 h 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 μg/L, and the corresponding brevetoxin level of Florida red tide aerosol ranged between 21 and 39 ng/m³. 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.     

Studies of the effect of Psi-APONIN from Nannochloris sp. on the Florida red tide organism Karenia brevis.

Studies were conducted on the conditions under which the red tide organism, Karenia brevis (a.k.a., Gymnodinium breve), was treated with Nannochloris sp. The latter organism is known to produce cytolytic agents called Apparent Oceanic Naturally Occurring Cytolin (APONINs). Conventional wisdom might suggest that brevetoxins would be released upon destruction of the single-celled dinoflagellate K. brevis and that efforts to treat red tide outbreaks would lead to release of brevetoxins and enhanced toxicity toward marine species. Earlier studies described conditions by which K. brevis cells were converted to a non-motile form when cultures of K. brevis were treated with an isolate (Psi-APONIN) produced by Nannochloris sp. but when centrifuged only a small amount of the toxin was released. The present study confirms that the toxin is not released when the K. brevis is undisturbed, however, when the culture is stressed (stirred with a magnetic stirring bar for 24, 48, and 72h) toxin was released, and the toxicity could be measured using a Microtox analyzer. In the study, it was found that at as few as eighty cells of K. brevis produced a toxic effect of 20% as measured by the effect on Vibrio fischeri. Nannochloris sp. had no effect on the bacteria used in the Microtox analyzer, nor did interaction of Nannochloris sp. with K. brevis in the short term. This effect is presumed to be due to the production of Psi-APONIN and conversion of K. brevis to a non-motile or resting form.     

Lipophilic toxicity from the marine dinoflagellate Karenia brevisulcata: use of the brevetoxin neuroblastoma assay to assess toxin presence and concentration.

A method for assessing the presence and concentration of a major toxin produced by Karenia brevisulcata has been developed, based on the neuroblastoma assay for brevetoxins. This cytotoxicity assay, coupled with observations of the dose-response relationship and of changes in cellular morphology during the assay gave a characteristic toxin 'signature'. This signature was consistent whether the toxicity was assessed in extracts of shellfish, seawater samples, uni-algal cultures of K. brevisulcata, or in partially purified toxin samples. Using this method it was possible to confirm the presence of this toxicity during a mixed Karenia bloom, and infer that K. brevisulcata toxin (KBT) was the probable cause of the fish and shellfish mortality observed at that time. The neuroblastoma assay thus provides a solution to the problem of confirming the presence of KBT in suspected K. brevisulcata events, even in the absence of a known toxin structure.     

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.     

Brevetoxin metabolism and elimination in the Eastern oyster (Crassostrea virginica) after controlled exposures to Karenia brevis.

The metabolism and elimination of brevetoxins were examined in the Eastern oyster (Crassostrea virginica) following controlled exposures to Karenia brevis cultures in the laboratory. After a 2-day exposure period ( approximately 62 million cells/oyster), elimination of brevetoxins and their metabolites was monitored by using liquid chromatography/mass spectrometry (LC/MS). Composite toxin in oyster extracts was measured by in vitro assay (i.e. cytotoxicity, receptor binding, and ELISA). Of the parent algal toxins, PbTx-1 and PbTx-2 were not detectable by LC/MS in K. brevis-exposed oysters. PbTx-3 and PbTx-9, which are accumulated directly from K. brevis and through metabolic reduction of PbTx-2 in the oyster, were at levels initially (after exposure) of 0.74 and 0.49 microg equiv./g, respectively, and were eliminated largely within 2 weeks after dosing. PbTx-7 and PbTx-10, the reduced forms of PbTx-1, were non-detectable. Conjugative brevetoxin metabolites identified previously in field-exposed oysters were confirmed in the laboratory-exposed oysters. Cysteine conjugates of PbTx-1 and PbTx-2, and their sulfoxides, were in the highest abundance, as apparent in LC/MS ion traces, and were detectable for up to 6 months after dosing. Composite toxin measurements by in vitro assay also reflected persistence (up to 6 months) of brevetoxin residues in the oyster. Levels of cysteine conjugates, as determined by LC/MS, were well correlated with those of composite toxin, as measured by ELISA, throughout depuration. Composite toxin levels by cytotoxicity assay were well correlated with those by receptor binding assay. Cysteine-PbTx conjugates are useful LC/MS determinants of brevetoxin exposure and potential markers for composite toxin in the Eastern oyster.     

Ciguatoxins are potent ichthyotoxins.

The ciguatoxins are lipid soluble polyether compounds which have structural and biochemical features in common with the brevetoxins. Pure ciguatoxin-1, ciguatoxin-2 or brevetoxin-2 added to water containing Gambusia affinis induced similar signs, including pronounced opercular movement and uncoordinated swimming preceding death. The estimated LD50s (48 hr) to G. affinis for ciguatoxin-1, ciguatoxin-2 and brevetoxin-2 were 0.5, 2.1 and 10 nmoles/litre, respectively, indicating that the ciguatoxins were up to 20-fold more potent than the brevetoxins in this assay. Previous studies reveal that the ciguatoxins are more potent than the brevetoxins in both i.p. lethality to mammals and affinity for voltage-dependent sodium channels. However, relative to their affinity for the voltage-dependent sodium channel, brevetoxin-2 is 4-fold more potent to fish than the ciguatoxins, whereas the ciguatoxins are up to 11-fold more potent to mice than brevetoxin-2. This study found that only 3.4% of administered ciguatoxin-1 was accumulated by G. affinis. Ciguatoxin-1 may be biotransformed by G. affinis. The lethal effects of the ciguatoxins in fish may impose an upper limit on the levels of ciguatoxin carried by fish, which could contribute to the low incidence of human fatality associated with ciguatera.     

Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.     

Cellular metabolism of brevetoxin (PbTx-2) by a monocyte cell line (U-937)

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 3 h incubation, glutathione conjugates were detectable as early as 1 h and increased in concentration after 2 and 3 h. A cysteine-PbTx-2 conjugate appeared after 2 h and increased in concentration after 3 h. Detectable levels of brevetoxin conjugates were present in response to toxin concentrations of 1 μM. 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.     

The Effects of the Harmful Algal Bloom Species Karenia brevis on Survival of Red Porgy (Pagrus pagrus) Larvae

The harmful algal bloom species, Karenia brevis, forms annual, often intense blooms in the Gulf of Mexico, particularly along the west Florida shelf. Though the ability of K. brevis blooms to cause mass mortalities in juvenile fish are well documented, the direct effect of bloom concentrations on larval fish has not been studied extensively. To better understand the potential effect of K. brevis on larval fish survival, laboratory spawned red porgy (Pagrus pagrus) larvae from 4–26 days post-hatch were exposed to concentrations of K. brevis observed in the field for either 24 or 48 h. This species is representative of fish which spawn in regions of the Gulf of Mexico and whose larvae are epipelagic and may encounter K. brevis blooms. In this study, three different K. brevis strains varying in the amount of brevetoxin produced were tested. Larval survivorship was found to be inversely proportional to the amount of brevetoxin produced by each strain. The EC₅₀ value from the combined 24 h experiments was ~163,000 K. brevis cells L⁻¹, which corresponds to cell concentrations found in moderately dense blooms. Larval mortality also increased substantially in the 48 h versus 24 h exposure treatments. These findings indicate K. brevis blooms have the potential to contribute to natural mortality of fish larvae and further reduce inter-annual recruitment of fishery species whose stocks in the Gulf of Mexico may already be depleted.     

Inhalation toxicity of brevetoxin 3 in rats exposed for 5 days

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assesses possible adverse health effects associated with short-term inhalation exposure to brevetoxin 3. Male F344/Crl/Br rats were exposed to 500 microg brevetoxin 3/m3 by nose-only inhalation for 0.5 or 2 h/d for 5 consecutive days. Control rats were sham exposed for 2 h to vehicle. Calculated deposited brevetoxin doses were 8.3 and 33 microg/kg/d for the low- and high-dose groups, respectively. At the termination of exposures, only body weights of the high-dose group (Group B) were significantly below control values. By immunohistochemistry (IHC), small numbers of splenic and peribronchiolar lymphoid tissue macrophages stained positive for brevetoxin, while nasal mucosa, liver, and brain were IHC negative for brevetoxin. No gross or microscopic lesions were observed in any tissue examined. There was no biochemical evidence of cytotoxicity or inflammation in bronchoalveolar lavage fluid. Alveolar macrophages showed some evidence of activation following brevetoxin exposure. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by a >70% reduction in splenic plaque-forming cells in brevetoxin-exposed animals compared to controls. Results suggest that the immune system may be a target of toxicity following brevetoxin inhalation. Future studies will focus on identification of a no-effect level and mechanisms underlying brevetoxin-induced immune suppression.     

Detection of sodium channel activators by a rapid fluorimetric microplate assay

Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, time-consuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i). the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potential-dependent, and (ii). dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.     

Uptake, tissue distribution, and excretion of brevetoxin 3 administered to rats by intratracheal instillation.

Brevetoxins are cyclic polyether neurotoxins produced by the marine dinoflagellate Ptychodiscus brevis. Blooms of P. brevis (red tides) are toxic to fish, marine mammals, and humans. Humans exposed to seaspray aerosols containing brevetoxins may experience respiratory tract irritation. Because a major route of human exposure to brevetoxins is via the respiratory tract, the objective of this study was to examine the toxicokinetics of brevetoxin 3 (PbTx-3) administered to the lung by intratracheal instillation. Twenty-one male F344/Crl BR rats, 12 wk of age, were administered 3H-PbTx-3 (1 microCi, 6.6 microg PbTx-3/kg) by intratracheal instillation. Groups of 3 rats were sacrificed at 0.5, 3, 6, 24, 48, and 96 h after exposure, and tissues were collected. Three additional rats were placed in glass metabolism cages for collection of urine and feces over a 7-d period. PbTx-3-associated activity was cleared rapidly from the lung and distributed throughout the body, chiefly to the carcass, intestines, and liver. Blood, brain, and fat contained the lowest percentages of the administered dose. Although a majority of the PbTx-3 was cleared rapidly from lung, liver, and kidneys, approximately 20% of the initial concentration present in each organ was retained for 7 d. Concentrations of PbTx-3 in brain and fat were low, but remained relatively constant over time. Approximately twice as much PbTx-3-associated activity was excreted in feces than in urine, with the majority of excretion occurring within 48 h after instillation. The results of this study indicate that over 80% of the PbTx-3 is rapidly absorbed from the lung to the blood and distributed to all tissues. The tissues containing the greatest amount of PbTx-3-associated activity reflect the compound's site of deposition, storage compartment, and major route of metabolism and excretion. These results illustrate that brevetoxin exposure by the respiratory route results in systemic distribution of brevetoxin and suggest that the initial respiratory irritation and bronchoconstriction may only be a part of the overall toxicological consequences associated with brevetoxin inhalation.     

Foodweb transfer, accumulation, and depuration of microcystins, a cyanobacterial toxin, in pumpkinseed sunfish (Lepomis gibbosus).

Zooplankton accumulate microcystins (MC), a potent cyanobacteria toxin, and therefore may act as vectors of the toxin up the aquatic food web; however this transfer has not yet been quantified. In addition there is a lack of information regarding fish's ability to metabolize MC when administered a low dose over a longer period of time. We monitored MC concentrations in three levels of an aquatic food web: phytoplankton, zooplankton, and sunfish (Lepomis gibbosus). Bosmina appeared to be both a major accumulator of MC in zooplankton and the major vector of MC to sunfish. In an accumulation experiment, sunfish were brought into the laboratory and fed MC-rich zooplankton pellets (50 ng MC kg(-1)d(-1)) for 9 days. Zooplankton directly transferred MC to sunfish, resulting in liver and muscle tissue accumulation. However, after 6 days of accumulation fish significantly decreased concentrations in their liver and muscle tissue, indicating the induction of a detoxification and excretion pathway. Sunfish retained MC in their liver and muscle tissue, showing no significant changes in toxin concentration over 2 weeks of fasted depuration.