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.     

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.     

Immune function in Trachemys scripta following exposure to a predominant brevetoxin congener,PbTx-3, as a model for potential health impacts for sea turtles naturally exposed to brevetoxins

Ecotoxicology - Many species of marine life in southwestern Florida, including sea turtles, are impacted by blooms of the toxic dinoflagellate, Karenia brevis. Sublethal exposure to toxins produced...     

Brevetoxins, like ciguatoxins, are potent ichthyotoxic neurotoxins that accumulate in fish.

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.     

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.     

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.     

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.     

Changes in early alveolar particle clearance due to single and repeated nitrogen dioxide exposures in the rabbit

To better understand the potential health risks associated with short-term NO2 exposures, a study was conducted to examine the effects of single and repeated NO2 exposures on the clearance of inert tracer particles from the alveolar region of rabbit lungs. Single 2-h exposures to 0.3, 1.0, 3.0, and 10.0 ppm produced a concentration-related acceleration in alveolar particle clearance, which resulted in greater particle removal when compared to control. The greatest response was produced at the lower NO2 levels, where as much as 40% more particles were cleared when compared to control. Fewer particles were cleared following a 10.0-ppm NO2 exposure when compared to the lower NO2 levels, and there were indications from the clearance pattern that the higher level was beginning to slow clearance, although an actual retardation was not found. Repeated 14-d exposures (2 h/d) to 1.0 or 10.0 ppm NO2 produced a response similar to a single exposure at the same concentration, suggesting a certain degree of adaptation was produced after the initial exposures. Possible mechanisms for these differences in clearance patterns are discussed. The results of this study demonstrated altered alveolar clearance following short-term NO2 exposure at environmentally relevant concentrations; changes in this important host defense mechanism may be indicative of some underlying pathologic condition.     

Pharmacokinetics of ceftizoxime

Summary The kinetics of ceftizoxime, a newly developed cephalosporin, were evaluated in 6 healthy subjects, with respect to its excretory pathways especially by the biliary route. Total, renal and biliary clearance were determined at two different steady states. Steady state was achieved by constant intravenous infusion (604.1 mg/h) over 6 h after an initial loading dose (750 mg); 1.5 h after discontinuation of that infusion, a further infusion was commenced at a lower rate (284 mg/h) over 3 h, the second steady state being reached 0.5 to 1.0 h later.The drug was mainly excreted by the kidneys (56.7 to 92.9% of the dose). Biliary excretion, measured by the duodenal perfusion and marker dilution technique, was low (0.2 to 7.8% of the dose). Urinary and biliary excretion as well as total clearance were not dose-dependent. However, there was pronounced interindividual variation in total (35.2 to 236 ml/min) and renal clearance (10.6 to 208 ml/min), which could both be explained by varying interindividual urinary flow rates (mean flow rate: 0.99 ml/min to 3.14 ml/min).Intraindividual variation in renal clearance was less pronounced, but in the same subject changes in renal clearance were correlated with changes in urinary flow rate. From the varying renal clearance, which exceeded the glomerular filtration rate at high urinary flow rates and was below it at low urinary flow rates, it can be concluded that, in addition to glomerular filtration, tubular secretion and tubular reabsorption are involved in the renal excretion of ceftizoxime.The half-life calculated from two point estimates after discontinuation of the infusion at the higher rate tended to be longer in subjects with high total clearance (e. g. 1.4 h, clearance 223 ml/min) and shorter in subjects with low total clearance (e.g. 0.85 h, clearance 35.2 ml/min). From this it is concluded that the true half-life was not observed after discontinuation of the infusion.     

Differential tolerance to copper, but no evidence of population-level genetic differences in a widely-dispersing native barnacle

Despite many estuaries having high levels of metal pollution, species are found to persist in these stressful environments. Populations of estuarine invertebrates exposed to toxic concentrations of such metals may be under selection. However, in species with a wide-dispersal potential, any short-term results of localized selection may be counteracted by external recruitment from populations not under selection. The barnacle Amphibalanus variegatus is found in nearshore coastal environments as well as sheltered embayments and estuaries, including metal-impacted estuaries, from New South Wales, Australia to Western Australia. The fertilised eggs of A. variegatus are brooded internally and released as larvae (nauplii), which remain in the water-column for ~14 days before settling. Hence the species has a considerable dispersal capacity. The purpose of this study was to examine whether populations of A. variegatus from metal-impacted sites, displayed a greater tolerance to a toxicant (copper) than reference populations. Adult barnacles where collected from twenty sites within two metal-impacted and fourteen sites within two reference estuaries. Within 24 h, adults were induced to spawn and the offspring were exposed to copper in a laboratory assay. Larvae collected from the metal-impacted estuaries demonstrated a greater tolerance to copper compared to those from reference sites. To determine if selection/localised in the metal impacted sites was occurring, the genetic structure of populations at three sites was examined using an AFLP methodology. No evidence of unique population identity and or selection (outlier loci) was detected suggesting that: (1) the tolerance displayed in the assay was derived from acclimation during development; and/or (2) that the populations are open preventing the fixation of any unique alleles.     

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense.

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects. Likewise, exposure of eggs to standard STX toxin, as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above; no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae. The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem.     

The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal.     

Sedimentation Patterns of Toxin-Producing Microcystis Morphospecies in Freshwater Reservoirs

Understanding the annual cycle of Microcystis is essential for managing the blooms of this toxic cyanobacterium. The current work investigated the sedimentation of microcystin-producing Microcystis spp. in three reservoirs from Central Spain during the summer and autumn of 2006 and 2007. We confirmed remarkable settling fluxes during and after blooms ranging 10⁶–10⁹ cells m⁻² d⁻¹, which might represent 0.1%–7.6% of the organic matter settled. A comprehensive analysis of the Valmayor reservoir showed average Microcystis settling rates (0.04 d⁻¹) and velocities (0.7 m d⁻¹) that resembled toxin settling in the same reservoir and were above most reported elsewhere. M. aeruginosa settling rate was significantly higher than that of M. novacekii and M. flos-aquae. Despite the fact that colony sizes did not differ significantly in their average settling rates, we observed extremely high and low rates in large colonies (>5000 cells) and a greater influence of a drop in temperature on small colonies (<1000 cells). We found a 4–14 fold decrease in microcystin cell quota in settling Microcystis of the Cogotas and Valmayor reservoirs compared with pelagic populations, and the hypothetical causes of this are discussed. Our study provides novel data on Microcystis settling patterns in Mediterranean Europe and highlights the need for including morphological, chemotypical and physiological criteria to address the sedimentation of complex Microcystis populations.     

Kinetics of granulocytic and erythroid progenitor cells are affected differently by short-term,low-level benzene exposure

In previous work, we determined that granulocytic (CFU-GM) and erythroid (CFU-E) progenitor cell populations exhibited disparate responses to short-term benzene exposures. We now report on work investigating possible mechanisms for these observed disparate responses. Mice were exposed to either air or 10 ppm benzene for 6 h/d X 5 d. Immediately after the last exposure, mice were injected, i. v., with either saline or hydroxyurea (HU). The dose of HU was sufficient to kill hematopoietic cells in or near S-phase of the cell cycle and sufficient to synchronize the surviving populations of hematopoietic cells. Three days after benzene exposure, CFU-E numbers had declined to 50% of control values while CFU-GM numbers were equal to control values at this time. The benzene exposures were sufficient to double the percentage of CFU-E in S-phase but produced no such increase among CFU-GM. During 3 days of recovery from benzene exposure and HU treatment, the CFU-E population expanded 30-fold while the CFU-GM population expanded less than 3-fold. Following benzene exposure and HU treatment, both progenitor cells produced elevated numbers of their respective progeny. When CFU-E from benzene-exposed mice were cultured with varying concentrations of erythropoietin (EPO), the response at maximal EPO concentration was 66% of the response by control CFU-E. This strongly suggests that the CFU-E populations from benzene-exposed mice had been depleted of cells in or near S-phase. The results indicate that CFU-GM respond to low-level benzene exposure by increasing their rate of differentiation but not their rate of proliferation, while CFU-E respond by increasing both their rates of differentiation and proliferation. We speculate that it is the increase in CFU-E proliferation that renders these cells more susceptible to benzene than their granulocytic counterparts, especially those CFU-E at or near the S-phase of the cell cycle.     

Evidence for PSP in mussels in Trinidad.

Herein we present the first evidence for the presence of Paralytic Shellfish Poison (PSP) in Trinidadian waters. The toxin was found in a meat extract of the mussel, Perna viridis. PSP has not previously been demonstrated in the shellfish of Caribbean islands. The presence of PSP in Trinidad is therefore significant in that it presents an opportunity to better understand the dynamics of PSP and algal blooms in both a region and island environment not normally associated with PSP.P. viridis is not native to Trinidad, but rather originates from eastern Asia. It presented itself only recently in Trinidadian waters. Interestingly, shellfish consumption and algal blooms have had a long history of coexistence in Trinidad without any record of human intoxications. In this context, potential Public Health implications of finding PSP in a non-native shellfish species are briefly discussed.     

Marine toxic cyanobacteria: Diversity, environmental responses and hazards

Toxic cyanobacterial blooms have been a primary concern predominantly in the plankton of freshwater bodies. Recently, however, the toxicity of benthic cyanobacteria is increasingly attracting attention of the scientific community and environmental agencies. The occurrence of toxic strains in benthic cyanobacteria is intimately linked to our understanding of the diversity and ecological responses of these organisms under field conditions. To that effect, we are engaged in combined morphotypic and genotypic characterization (polyphasic) of benthic natural populations of cyanobacteria in tropical lagoons and coral reefs, with the objective to provide a reliable reference for further comparative work. The methods of identification based on phenotypic properties and those based on molecular tools for genotypic identification are correlated. The approach is based on identifying the occurrences of cyanobacterial benthic blooms, tested for purity and analyzed by application of molecular tools. The questions addressed include the distinction between marine and freshwater taxa, between populations in geographically separate regions as well as between their potential vs. expressed toxicity.     

Acute effects of an anatoxin-a producing cyanobacterium on juvenile fish-Cyprinus carpio L.

The worldwide increase of eutrophication in fresh water bodies has caused cyanobacterial blooms to be more frequent. Anatoxin-a is a potent neurotoxin known to be produced by several genera of cyanobacteria including Anabaena. In this work, we exposed juvenile carps to freeze-dried cells of a toxic strain of the cyanobacterium Anabaena sp. during a 4-day period. Two different cell density--10(5) and 10(7) cell ml(-1)--were assayed. Lethality and anatoxin-a concentration in the whole fish were determined. In the higher cell density, all fish died between 26 and 29 h after exposure to toxin, whereas in the 10(5) cell ml(-1) density no deaths were observed. Levels of anatoxin-a in the whole fish ranged between 0.031 microg g(-1)d.w. at the 10(5) cell ml(-1) concentration and 0.768 microg g(-1)d.w. at 10(7) cell ml(-1). Minor uptake of anatoxin-a occurred.     

Relationship between bioenergetics responses and organic pollutants in the giant mussel,Choromytilus chorus (Mollusca: Mytilidae)

Samples of Choromytilus chorus (giant mussel) were collected at three sampling stations exposed to different degrees of pollution along the south-central portion of the Chilean coast in spring 1998 and summer 1999. Measurements were carried out on clearance rate, absorption efficiency, and oxygen consumption of the mussels under controlled laboratory conditions, and related to analytical data on organic pollutants in their tissues. Scope for growth (SFG) was employed as a physiological index to evaluate stress produced by pollutants existing at each sampling site. Individuals from San Vicente bay (highly polluted) showed negative SFG values in spring (-4.6 J/h per g) and summer (-3.5 J/h per g). These results indicated severe stress related to the accumulation of toxic compounds in their tissues. Specimens from Corral bay (medium level of pollution) gave a SFG of 15.5 J/h per g in spring and 6.5 J/h per g in summer, while those from Yaldad bay (low pollution) presented an inverse situation was observed with SFG values of 6.2 J/h per g in spring which was lower than the summer value of 25.7 J/h per g. There was a significant negative correlation between the SFG of the different populations of C. chorus and the concentrations of organochlorines (OChs) and polynuclear aromatic hydrocarbons (PAHs) in their tissues.     

Mucociliary clearance from the lungs of rabbits following single and intermittent exposures to ozone

This study examined the effects of ozone (O3) inhalation on mucociliary clearance from the tracheobronchial tree. Rabbits were exposed for 2 h at 0.1, 0.25, and 0.6 ppm or for 2 h/d for 14 consecutive days at 0.25 and 0.6 ppm. Clearance was assessed by measuring the retention of radioactivity tagged, inert tracer particles inhaled immediately after the single 2-h exposure, or 24 h after 2, 7, or 14 of the daily exposures. Single exposures resulted in a concentration-related trend toward retarded particle clearance, with a statistically significant difference occurring after exposure to 0.6 ppm. Intermittent exposures produced no significant change in clearance rate, although there was a suggestion of retarded clearance at early time points at both 0.25 and 0.6 ppm.