Harmful Algae

Published by Elsevier
Print ISSN: 1568-9883
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Data collected along the southwest coast of Florida between Tampa Bay and Sanibel Island on the abundance of the toxic dinoflagellate Karenia brevis from 1954 to 2002 were examined for spatial and temporal patterns. K. brevis was found to be approximately 20-fold more abundant within 5 km of the shoreline than 20-30 km offshore. Overall, K. brevis was approximately 13-18-fold more abundant in 1994-2002 than in 1954-1963. In 1954-1963, K. brevis occurred primarily in the fall months. In 1994-2002, it was more abundant not only in the fall, but also in the winter and spring months. It is hypothesized that greater nutrient availability in the ecosystem is the most likely cause of this increase in K. brevis biomass, and the large increase in the human population and its activities in South Florida over the past half century is a major factor.
 
Human exposure to Florida red tides formed by Karenia brevis, occurs from eating contaminated shellfish and inhaling aerosolized brevetoxins. Recent studies have documented acute symptom changes and pulmonary function responses after inhalation of the toxic aerosols, particularly among asthmatics. These findings suggest that there are increases in medical care facility visits for respiratory complaints and for exacerbations of underlying respiratory diseases associated with the occurrence of Florida red tides. This study examined whether the presence of a Florida red tide affected the rates of admission with a respiratory diagnosis to a hospital emergency room in Sarasota, FL. The rate of respiratory diagnoses admissions were compared for a 3-month time period when there was an onshore red tide in 2001 (red tide period) and during the same 3-month period in 2002 when no red tide bloom occurred (non-red tide period). There was no significant increase in the total number of respiratory admissions between the two time periods. However, there was a 19% increase in the rate of pneumonia cases diagnosed during the red tide period compared with the non-red tide period. We categorized home residence zip codes as coastal (within 1.6 km from the shore) or inland (> 1.6 km from shore). Compared with the non-red tide period, the coastal residents had a significantly higher (54%) rate of respiratory diagnoses admissions than during the red tide period. We then divided the diagnoses into subcategories (i.e. pneumonia, bronchitis, asthma, and upper airway disease). When compared with the non-red tide period, the coastal zip codes had increases in the rates of admission of each of the subcategories during the red tide period (i.e. 31, 56, 44, and 64%, respectively). This increase was not observed seen in the inland zip codes.
 
In the fall of 2000, there was a red tide episode in the Gulf of Mexico near Corpus Christi, TX. We sampled at the Marine Science Institute in Port Aransas on 25 October 2000. Between 25-27 October 2000, we sampled at the Texas State Aquarium (TSA) near Corpus Christi Bay. Two high-volume samplers were equipped with a filter and a five-stage impactor, respectively. Because the amounts of brevetoxin (PbTx) collected in the air samples were low, we developed a LC/MS technique to analyze the PbTxs. Personal exposure was estimated with a personal filter sampler placed in the lapel of field workers. Concentrations of PbTx-2 and -3 were detected in the samples taken at the TSA; however, PbTx was not detected in the samples from the Marine Science Institute. The concentration of PbTx-2 was between 1.5 and 5.9 ng m(-3) and much lower concentrations for PbTx-3. The ratio of PbTx-2 and -3 was 8.7 +/- 5.2. In the highest exposure period (26-27 October), PbTx-6 was also detected. No respiratory symptoms were reported at the Marine Science Institute, whereas at the TSA, symptoms including irritation in the nose and throat, and itchy skin were reported among seven field study workers. The PbTx concentrations estimated from both high-volume impactor and filter samplers were similar. The mass median aerodynamic diameters were between 7 and 9 mum (geometric standard deviation of 1.6), a relatively large size for inhaled ambient particles. Inhaled particles of this size would be predominantly deposited in the upper respiratory tract (nasal, oral, and pharyngeal area), and subsequent respiratory irritation could result from the presence of the particles themselves or from toxins associated with the particles. Information gained from these studies will aid in evaluations of the human risk associated with inhalation of red tide aerosols.
 
During two separate Karenia brevis red tide events, we measured the levels of brevetoxins in air and water samples, conducted personal interviews, and performed pulmonary function tests on people before and after they visited one of two Florida beaches. One hundred and twenty-nine people participated in the study, which we conducted during red tide events in Sarasota and Jacksonville, FL, USA. Exposure was categorized into three levels: low/no exposure, moderate exposure, and high exposure. Lower respiratory symptoms (e.g. wheezing) were reported by 8% of unexposed people, 11% of the moderately exposed people, and 28% of the highly exposed people. We performed nasal-pharyngeal swabs on people who experienced moderate or high exposure, and we found an inflammatory response in over 33% of these participants. We did not find any clinically significant changes in pulmonary function test results; however, the study population was small. In future epidemiologic studies, we plan to further investigate the human health impact of inhaled brevetoxins.
 
Inland transect: February 2005  
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.
 
Recent studies demonstrate that most cyanobacteria produce the neurotoxin beta-N-methylamino-L-alanine (BMAA) and that it can biomagnify in at least one terrestrial food chain. BMAA has been implicated as a significant environmental risk in the development of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). We examined several blooms of cyanobacteria in South Florida, and the BMAA content of resident animals, including species used as human food. A wide range of BMAA concentrations were found, ranging from below assay detection limits to approximately 7000 μg/g, a concentration associated with a potential long-term human health hazard.
 
The dinoflagellate genus Alexandrium is one of the major harmful algal bloom (HAB) genera with respect to the diversity, magnitude and consequences of blooms. The ability of Alexandrium to colonize multiple habitats and to persist over large regions through time is testimony to the adaptability and resilience of this group of species. Three different families of toxins, as well as an as yet incompletely characterized suite of allelochemicals are produced among Alexandrium species. Nutritional strategies are equally diverse, including the ability to utilize a range of inorganic and organic nutrient sources, and feeding by ingestion of other organisms. Many Alexandrium species have complex life histories that include sexuality and often, but not always, cyst formation, which is characteristic of a meroplanktonic life strategy and offers considerable ecological advantages. Due to the public health and ecosystem impacts of Alexandrium blooms, the genus has been extensively studied, and there exists a broad knowledge base that ranges from taxonomy and phylogeny through genomics and toxin biosynthesis to bloom dynamics and modeling. Here we present a review of the genus Alexandrium, focusing on the major toxic and otherwise harmful species.
 
Florida Harmful Algal Bloom Historical Database (2002 to 2006; data provided by the Florida Fish and Wildlife Conservation Commission's Fish and Wildlife Research Institute; Haverkamp et al., 2004). " Identified in situ samples to be considered " (see Figure 2) illustrating the sampling location for Karenia brevis: red tides (≥1.5×10 4 cells l −1 ; " □ " ) and non-blooms (<1.5×10 4 cells l −1 ; " × " ). The region of the Central West Florida Shelf (CWFS – south of 28.25°N close to Tampa Bay and north of 25.75°N near Cape Romano) is indicated.  
Flowchart representing the stringent quality control tests applied to all near-coincident satellitein situ pairs; adapted from Carvalho (2008) and Carvalho et al. (2010). Steps to identify in situ samples to be considered (light gray); satellite processing to identify the geophysical parameter library (dark gray); level-by-level of the match-up analysis performed to derive the valid cloud-free match-ups (white); and data removed from the analysis (black). As the water column depth was not specified for the in situ sampling within the Florida HAB Historical Database provided by the Florida Fish and Wildlife Conservation Commission's Fish and Wildlife Research Institute (Haverkamp et al., 2004), the Earth TOPOgraphy database at twominute gridded resolution (ETOPO2 – http://www.ngdc.noaa.gov/mgg/gdas/gd_designagrid.html#) was used. Refer to Figure 6.3 in Carvalho (2008) for graphical representation of effective penetration depth (EPD; Gordon and McCluney; 1975) and apparent optical depth (AOD; Bailey and Werdell, 2006). Inherent optical properties (IOPs) includes: a(λ), a ph (λ), a dg (λ), b(λ), b bp (λ). For additional and information see Section 2.  
Venn diagrams showing the structure of the two configurations of the Hybrid Scheme (Section 4), which is based on the detection frequency of the Empirical Approach and the Bio-optical Technique. a) The positive identifications of the 1 st configuration are represented by YELLOW and BLUE (when either or both algorithms correctly flagged known samples), while the positive identifications of the 2 nd configuration are represented only by the BLUE (both algorithms had to flag simultaneously known samples correctly). b, c) All valid match-ups (n=371=[145+226]) sampled along the Central West Florida Shelf during the Summer-Fall periods (2002 to 2006). b) Shown in RED are red tide samples (RT; ≥1.5×10 4 cells l −1 ; n=145= [12+90+14+29]). c) Shown in GREEN are non-bloom samples (NB; <1.5×10 4 cells l −1 ; n=226=[25+137+15+49]). See Tables 1 and 2 for the detection frequency of the two configurations of the Hybrid Scheme.
In a continuing effort to develop suitable methods for the surveillance of Harmful Algal Blooms (HABs) of Karenia brevis using satellite radiometers, a new multi-algorithm method was developed to explore whether improvements in the remote sensing detection of the Florida Red Tide was possible. A Hybrid Scheme was introduced that sequentially applies the optimized versions of two pre-existing satellite-based algorithms: an Empirical Approach (using water-leaving radiance as a function of chlorophyll concentration) and a Bio-optical Technique (using particulate backscatter along with chlorophyll concentration). The long-term evaluation of the new multi-algorithm method was performed using a multi-year MODIS dataset (2002 to 2006; during the boreal Summer-Fall periods - July to December) along the Central West Florida Shelf between 25.75°N and 28.25°N. Algorithm validation was done with in situ measurements of the abundances of K. brevis; cell counts ≥1.5×10(4) cells l(-1) defined a detectable HAB. Encouraging statistical results were derived when either or both algorithms correctly flagged known samples. The majority of the valid match-ups were correctly identified (~80% of both HABs and non-blooming conditions) and few false negatives or false positives were produced (~20% of each). Additionally, most of the HAB-positive identifications in the satellite data were indeed HAB samples (positive predictive value: ~70%) and those classified as HAB-negative were almost all non-bloom cases (negative predictive value: ~86%). These results demonstrate an excellent detection capability, on average ~10% more accurate than the individual algorithms used separately. Thus, the new Hybrid Scheme could become a powerful tool for environmental monitoring of K. brevis blooms, with valuable consequences including leading to the more rapid and efficient use of ships to make in situ measurements of HABs.
 
As an alternative to traditional, morphology-based methods, molecular techniques can provide detection of multiple species within the HAB community and, more widely, the phytoplankton community in a rapid, accurate and simultaneous qualitative analysis. These methods require detailed knowledge of the molecular diversity within taxa in order to design efficient specific primers and specific probes able to avoid cross-reaction with non-target sequences. Isolates from Florida coastal communities were sequence-analyzed and compared with the GenBank database. Almost 44% of the genotypes obtained did not match any sequence in GenBank, showing the existence of a large and still unexplored biodiversity among taxa. Based on these results and on the GenBank database, we designed 14 species-specific probes and 4 sets of specific primers. Multiple simultaneous detection was achieved with a bead array method based on the use of a flow cytometer and color-coded microspheres, which are conjugated to the developed probes. Following a parallel double PCR amplification, which employed universal primers in a singleplex reaction and a set of species-specific primers in multiplex, detection was performed in a cost effective and highly specific analysis. This multi-format assay, which required less than 4 h to complete from sample collection, can be expanded according to need. Up to 100 different species can be identified simultaneously in a single sample, which allows for additional use of this method in community analyses extended to all phytoplankton species. Our initial field trials, which were based on the 14 species-specific probes, showed the co-existence and dominance of two or more species of Karenia during toxic blooms in Florida waters.
 
Nutrients in Gulf of Maine nearshore waters. (Panel A) Concentrations of nitrate plus nitrite (NO 3 À + NO 2 À ) and silicate (Si(OH) 4 ) in the Kennebec and Androscoggin Rivers in Maine over the period 1 January 2001 to 1 May 2002 (data from Vorosmarty et al.,  (Panel B) Concentrations of NO 3 À + NO 2 À and Si(OH) 4 collected in Casco Bay at the Southern Maine Community College dock, South Portland, Maine (from Townsend et al.,  (Panel C) Mixing curves for NO 3 À + NO 2 À and Si(OH) 4 vs. salinity for Gulf of Maine samples collected in a high- (April–May 2000), medium- (June 2000) and low-runoff period (July 2001) (Townsend et al., 2005). Assuming a northern Gulf of Maine coast-wide freshwater source concentration of nutrients similar to the Kennebec–Androscoggin (which was 32 m M Si(OH) 4 and 12 m M NO 3 À + NO 2 À in April 2000; n = 49;  and coastal seawater concentrations of ca. 8 m M of Si(OH) 4 and ca. 8 m M 
(A) Concentrations of DIN in western Great South Bay, NY (Suffolk County Department of Health Services Station 250) from 1976 to 2000, before and after the onset of brown tides. (B) Concentrations of DON in Great Peconic Bay, NY (Suffolk County Department of Health Services Station 130) from 1996 to 2005, since the last occurrence of brown tide in this system.
Temporal changes in land use, river flow, fertilizer use, and river N concentration in the Chesapeake Bay watershed. From Kemp et al., 2005 with permission of the publisher. 
Coastal waters of the United States (U.S.) are subject to many of the major harmful algal bloom (HAB) poisoning syndromes and impacts. These include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), ciguatera fish poisoning (CFP) and various other HAB phenomena such as fish kills, loss of submerged vegetation, shellfish mortalities, and widespread marine mammal mortalities. Here, the occurrences of selected HABs in a selected set of regions are described in terms of their relationship to eutrophication, illustrating a range of responses. Evidence suggestive of changes in the frequency, extent or magnitude of HABs in these areas is explored in the context of the nutrient sources underlying those blooms, both natural and anthropogenic. In some regions of the U.S., the linkages between HABs and eutrophication are clear and well documented, whereas in others, information is limited, thereby highlighting important areas for further research.
 
Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize to effects of brevenal, a radioactive analog ([(3)H]-brevenol) was produced by reducing the side-chain terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A KD of 67 nM and Bmax of 7.1 pmol/mg protein were obtained for [(3)H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [(3)H]-brevenol binding with Ki values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol can inhibit brevetoxin binding, brevetoxin was completely ineffective at competition for [(3)H]-brevenol binding. After examining other site-specific compounds, it was determined that [(3)H]-brevenol binds to a site that is distinct from the other known sites including the brevetoxin site (site 5) although some interaction with site 5 is apparent.
 
Over the years, numerous outreach strategies by the science community, such as FAQ cards and website information, have been used to explain blooms of the toxic dinoflagellate, Karenia brevis that occur annually off the west coast of Florida to the impacted communities. Many state and federal agencies have turned to funded research groups for assistance in the development and testing of environmental outreach products. In the case of Florida red tide, the Fish and Wildlife Research Institute/Mote Marine Laboratory (MML) Cooperative Red Tide Agreement allowed MML to initiate a project aimed at developing innovative outreach products about Florida red tide. This project, which we coined "The Art of Red Tide Science," consisted of a team effort between scientists from MML and students from Ringling College of Art and Design. This successful outreach project focused on Florida red tide can be used as a model to develop similar outreach projects for equally complex ecological issues.
 
A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as "present" (</=1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.
 
Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.
 
Having demonstrated significant and persistent adverse changes in pulmonary function for asthmatics after 1 hour exposure to brevetoxins in Florida red tide (Karenia brevis bloom) aerosols, we assessed the possible longer term health effects in asthmatics from intermittent environmental exposure to brevetoxins over 7 years. 125 asthmatic subjects were assessed for their pulmonary function and reported symptoms before and after 1 hour of environmental exposure to Florida red tide aerosols for upto 11 studies over seven years. As a group, the asthmatics came to the studies with normal standardized percent predicted pulmonary function values. The 38 asthmatics who participated in only one exposure study were more reactive compared to the 36 asthmatics who participated in ≥4 exposure studies. The 36 asthmatics participating in ≥4 exposure studies demonstrated no significant change in their standardized percent predicted pre-exposure pulmonary function over the 7 years of the study. These results indicate that stable asthmatics living in areas with intermittent Florida red tides do not exhibit chronic respiratory effects from intermittent environmental exposure to aerosolized brevetoxins over a 7 year period.
 
Oxygen-based productivity and respiration rates were determined in West Florida coastal waters to evaluate the proportion of community respiration demands met by autotrophic production within a harmful algal bloom dominated by Karenia brevis. The field program was adaptive in that sampling during the 2006 bloom occurred where surveys by the Florida Wildlife Research Institute indicated locations with high cell abundances. Net community production (NCP) rates from light-dark bottle incubations during the bloom ranged from 10 to 42 µmole O2 L(-1) day(-1) with highest rates in bloom waters where abundances exceeded 10(5) cells L(-1). Community dark respiration (R) rates in dark bottles ranged from <10 to 70 µmole O2 L(-1) day(-1) over 24 h. Gross primary production derived from the sum of NCP and R varied from ca. 20 to 120 µmole O2 L(-1) day(-1). The proportion of GPP attributed to NCP varied with the magnitude of R during day and night periods. Most surface communities exhibited net autotrophic production (NCP > R) over 24 h, although heterotrophy (NCP < R) characterized the densest sample where K. brevis cell densities exceed 10(6) cells L(-1).
 
A bloom of Karenia brevis Davis developed in September 2007 near Jacksonville, Florida and subsequently progressed south through east Florida coastal waters and the Atlantic Intracoastal Waterway (ICW). Maximum cell abundances exceeded 10(6) cells L(-1) through October in the northern ICW between Jacksonville and the Indian River Lagoon. The bloom progressed further south during November, and terminated in December 2007 at densities of 10(4) cells L(-1) in the ICW south of Jupiter Inlet, Florida. Brevetoxins were subsequently sampled in sediments and seagrass epiphytes in July and August 2008 in the ICW. Sediment brevetoxins occurred at concentrations of 11-15 ng PbTx-3 equivalents (g dry wt sediment)(-1) in three of five basins in the northern ICW during summer 2008. Seagrass beds occur south of the Mosquito Lagoon in the ICW. Brevetoxins were detected in six of the nine seagrass beds sampled between the Mosquito Lagoon and Jupiter Inlet at concentrations of 6-18 ng (g dry wt epiphytes)(-1). The highest brevetoxins concentrations were found in sediments near Patrick Air Force Base at 89 ng (g dry wt sediment)(-1). In general, brevetoxins occurred in either seagrass epiphytes or sediments. Blades of the resident seagrass species have a maximum life span of less than six months, so it is postulated that brevetoxins could be transferred between epibenthic communities of individual blades in seagrass beds. The occurrence of brevetoxins in east Florida coast sediments and seagrass epiphytes up to eight months after bloom termination supports observations from the Florida west coast that brevetoxins can persist in marine ecosystems in the absence of sustained blooms. Furthermore, our observations show that brevetoxins can persist in sediments where seagrass communities are absent.
 
Twenty-four specimen of macroalgae were collected in nearshore waters of the island of Hawaii, identified, and maintained to examine how the epiphytic relationship between Gambierdiscus toxicus (isolate BIG12) varied among the macroalgal species. Gambierdiscus cells were introduced to petri dishes containing 100 g samples of each macroalgal host, which were examined at two, 16, 24, and every 24 to 72 hours thereafter, over a 29-day period. Gambierdiscus proliferated in the presence of some host species (e.g., Galaxaura marginata and Jania sp.), but grew little in the presence of other species (e.g., Portieria hornemannii). Gambierdiscus exhibited high survival rates (>99%) in the presence of Chaetomorpha sp., but died before the end of the experiment (after 21 days) with other host species (e.g., Dictyota and Microdictyon spp.). Gambierdiscus avoided contact with Portieria hornemannii, but averaged up to 30% attachment with other host species. The numbers of Gambierdiscus cells belonging to one of three classes (alive and attached; alive and unattached; and dead) were determined for each time point. The 24 algal hosts were grouped according to their commonalities relative to these three classes using a Bray-Curtis similarity index, similarity profile (SIMPROF) permutation tests, and multi-dimensional scaling (MDS) analysis (PRIMER 6). The resultant six groupings were used to construct different Gambierdiscus growth profiles for the different algal hosts. Group A is characterized by a preponderance of unattached cells and high mortality rates. Groups B, C, E, and F also displayed high proportions of unattached cells, but mortality either occurred later (Groups B and C) or rates were lower (Groups E and F). Group D had the highest proportion of attached cells. Group E contained three out of the four chlorophyte species, while Group F contained the majority of the rhodophytes. Over 50% of the species in Group F are considered to be palatable, whereas Groups A, B, and C are composed of species that exhibit chemical defenses against herbivory. The results of this study coupled with previous findings indicate that Gambierdiscus is not an obligate epiphyte; it can be free-swimming and found in the plankton. The conditions that lead to changes between epiphytic and planktonic stages need to be better studied in order to determine how they affect Gambierdiscus growth and physiology, connectivity and dispersion mechanisms, and toxin movement up into the foodweb.
 
The marine dinoflagellate, Karenia brevis, is responsible for Florida red tides. Brevetoxins, the neurotoxins produced by K. brevis blooms, can cause fish kills, contaminate shellfish, and lead to respiratory illness in humans. Although several studies have assessed different economic impacts from Florida red tide blooms, no studies to date have considered the impact on beach lifeguard work performance. Sarasota County experiences frequent Florida red tides and staffs lifeguards at its beaches 365 days a year. This study examined lifeguard attendance records during the time periods of March 1 to September 30 in 2004 (no bloom) and March 1 to September 30 in 2005 (bloom). The lifeguard attendance data demonstrated statistically significant absenteeism during a Florida red tide bloom. The potential economic costs resulting from red tide blooms were comprised of both lifeguard absenteeism and presenteeism. Our estimate of the costs of absenteeism due to the 2005 red tide in Sarasota County is about $3,000. On average, the capitalized costs of lifeguard absenteeism in Sarasota County may be on the order of $100,000 at Sarasota County beaches alone. When surveyed, lifeguards reported not only that they experienced adverse health effects of exposure to Florida red tide but also that their attentiveness and abilities to take preventative actions decrease when they worked during a bloom, implying presenteeism effects. The costs of presenteeism, which imply increased risks to beachgoers, arguably could exceed those of absenteeism by an order of magnitude. Due to the lack of data, however, we are unable to provide credible estimates of the costs of presenteeism or the potential increased risks to bathers.
 
Florida red tides are a natural phenomenon caused by dense aggregations of single cell or several species of unicellular organisms. Patches of discolored water, dead or dying fish, and respiratory irritants in the air often characterize these algal blooms. In humans, two distinct clinical entities, depending on the route of exposure, are associated with exposure to the Florida red tide toxins (particularly the brevetoxins). With the ingestion of brevetoxin-contaminated shellfish, neurotoxic shellfish poisoning (NSP) presents as a milder gastroenteritis with neurologic symptoms compared with other marine toxin diseases such as paralytic shellfish poisoning (PSP) or ciguatera fish poisoning. With the inhalation of the aerosolized red tide toxins (especially the brevetoxins) from the sea spray, respiratory irritation and possibly other health effects are reported in both humans and other mammals (Baden 1995, Fleming 1998a, Fleming 1998b, Fleming 1999a, Bossart 1998, Asai 1982, Eastaugh 1989, Pierce 1986, Music 1973, Temple 1995, Anderson 1994).This paper reviews the literature on the known and possible human health effects of exposure to the Florida red tides and their toxins. The review includes discussion of the red tide organisms and their toxins, as well as the effects of these toxins on both wild and laboratory animals as they relate to possible human health effects and exposures.
 
The west coast of Florida has annual blooms of the toxin-producing dinoflagellate, Karenia brevis with Sarasota, FL considered the epicenter for these blooms. Numerous outreach materials, including Frequently Asked Question (FAQ) cards, exhibits for local museums and aquaria, public beach signs, and numerous websites have been developed to disseminate information to the public about this natural hazard. In addition, during intense onshore blooms, a great deal of media attention, primarily via newspaper (print and web) and television, is focused on red tide. However to date, the only measure of effectiveness of these outreach methods has been counts of the number of people exposed to the information, e.g., visits to a website or number of FAQ cards distributed. No formal assessment has been conducted to determine if these materials meet their goal of informing the public about Florida red tide. Also, although local residents have the opinion that they are very knowledgeable about Florida red tide, this has not been verified empirically. This study addressed these issues by creating and administering an evaluation tool for the assessment of public knowledge about Florida red tide. A focus group of Florida red tide outreach developers assisted in the creation of the evaluation tool. The location of the evaluation was the west coast of Florida, in Sarasota County. The objective was to assess the knowledge of the general public about Florida red tide. This assessment identified gaps in public knowledge regarding Florida red tides and also identified what information sources people want to use to obtain information on Florida red tide. The results from this study can be used to develop more effective outreach materials on Florida red tide.
 
We investigated the dynamics and toxicity of cyanobacteria populations in Lake Agawam, a eutrophic New York lake, and concurrently conducted experiments to evaluate the contrasting effects of zooplankton (mesozooplankton and microzooplankton) grazing and nutrient loading on the abundance and toxin content of cyanobacteria populations. Molecular techniques were used to assess the presence and expression of the microcystin synthetase gene. Lake Agawam hosted dense blooms (>105 cells mL−1) of Microcystis sp. and Anabaena sp. with consistently elevated levels of microcystin (1.0–25 μg L−1) and lower levels of anatoxin-a (∼1.0 μg L−1 during late summer only). Polymerase chain reaction (PCR) analysis targeting the microcystin synthetase gene (mcyE) indicated that Microcystis sp., and not Anabaena sp., was responsible for microcystin production in this system. Moreover, reverse transcriptase PCR (RT-PCR) indicated that the Microcystis population expressed the mcyE gene during summer months, but that gene expression declined to undetectable levels during the fall as in situ cell densities in the lake declined. During summer, when there was elevated Microcystis densities (>8 × 104 cells mL−1) expressing mcyE, experimental Daphnia sp. enrichment did not alter algal biomass (100% of experiments; n = 6). However, during fall months when the mcyE gene expression was not detected and Microcystis densities were lower and declining (4 × 103 to 5 × 104 cells mL−1), zooplankton enrichment yielded significantly reduced (p < 0.05) cyanobacteria biomass relative to control treatments in most experiments (80%; n = 4). In contrast to mesozooplankton, microzooplankton actively grazed algal biomass at significant rates (1.2 ± 0.3 day−1) throughout the study. Microcystis and other cyanobacterial populations did not respond to nutrient amendments during early summer but experienced significantly increased growth rates and toxin concentrations (microcystin and anatoxin-a) during late summer and early fall nitrogen enrichment experiments. As such, the dominance of Microcystis sp. blooms during the summer was linked to nutrient replete conditions and the suppression of mesozooplankton (but not microzooplankton) grazing which itself appeared to be influenced by cellular toxin synthesis by Microcystis. The demise of the bloom was associated with N-limitation which reduced growth rates and toxin production by Microcystis and in turn may have permitted zooplankton to graze cells.
 
Temporal evolution of toxicity in mussel and cockle: (a) DSP in Aveiro lagoon in 2002; (b) DSP in Aveiro lagoon in 2005; (c) PSP in Aveiro lagoon in 2005/2006; (d) PSP in O  ́ bidos lagoon in 2006. Maximum toxin levels in mussels are indicated. 
(a) Maximum monthly cellular concentrations of Dinophysis spp. and (b) months where mussel harvest was closed at Aveiro lagoon during the period 1987-1993 (dark squares).
Average monthly maximum levels of biotoxins in bivalves from Aveiro lagoon (as presented in Fig. 3): (a) DSP in mussel; (b) PSP in mussel and ASP in cockle. All scales were set at 8 times the respective EU regulatory limits. In (a), DSP is overlaid with average rainfall for the period 1994– 2006. 
Relative distribution of toxin levels found in all the samples analysed between 2003 and 2006 by three categories: above regulatory level (POS), between 0.1 and 1.0 times the regulatory level (NEG), and residual levels lower than 0.1 times the regulatory level (ND). (a) DSP toxins; (b) DSP toxins only in Mytilus spp. and Donax spp.; (c) DSP toxins in the remaining species; (d) PSP toxins; (e) ASP toxins.
Foodborne outbreaks attributed to marine biotoxins were first reported in Portugal in 1946. A regular monitoring programme was implemented in 1986 for PSP, in 1987 for DSP and in 1996 for ASP. The gradual introduction of HPLC methodologies for DSP and PSP allowed a better understanding of toxin biotransformation by bivalves, supplying more selective and sensitive data than mouse bioassays. A comprehensive exposure assessment from DSP toxins in bivalves from the whole coast was only obtained more recently with the introduction of LC–MS methodology. Data on maximum toxin levels found, geographic distribution, seasonality of toxin families, and frequency of samples above current regulatory limits is presented in order to review the data available on exposure assessment after two decades of monitoring. Contamination with DSP toxins was more severe in estuarine and offshore bivalves from the NW and in offshore Donax spp. from the SW and south coasts. DSP toxins were recurrent every year mainly between late spring/early autumn. PSP toxins appeared intermittently in some years between 1986 and 2006, predominantly in autumn. Bivalves from the entire coast were severely contaminated, although bivalves from the NW coast were affected more often. ASP toxins appeared between spring and autumn around the entire coast, but toxin levels rarely exceeded the regulatory limit. Azaspiracids occurred in trace levels below the regulatory limit. Yessotoxins and pectenotoxins occurred in bivalves but have no known effects on the consumers. Several intoxication outbreaks attributed to PSP and DSP occurred during the two decades of the monitoring programme.
 
Harmful algal blooms (HABs) have posed a serious threat to the aquaculture and fisheries industries in recent years, especially in Asia. During 1998 there were several particularly serious blooms in the coastal waters of south China, which caused a serious damage to aquaculture. We report a massive dinoflagellate bloom near the mouth of Pearl River in November 1998 with analyses of data from both in situ sea water measurements and satellites. A multi-parameter environmental mapping system was used to obtain real-time measurements of water quality properties and wind data through the algal bloom area, which allow us to compare water measurements from inside and outside of the bloom areas. This bloom with high concentrations of algal cells was evident as a series of red colored parallel bands of surface water that were 100–300 m long and 10–30 m wide with a total area of about 20–30 km2 by visual. The algal density reached 3.8×107 cells l−1 and the surface chlorophyll-a (Chl-a) concentration was high. The algal species has been identified as Gymnodinium cf. catenatum Graham. The water column in the bloom area was stratified, where the surface temperature was 24–25 °C, the salinity was 18–20%, and the northern wind was about 3–4 m s−1 in the bloom area. The SeaWiFS image has shown high Chl-a area coinciding with the bloom area. The sea surface temperature (SST) image of the Pearl River estuary combined with the in situ measurements indicated that the bloom occurred along a mixing front between cooler lower salinity river water and warmer higher saline South China Sea (SCS) water.
 
A 3 year study (2000–2002) in Barnegat Bay-Little Egg Harbor (BB/LEH), New Jersey (USA), was conducted by the New Jersey Department of Environmental Protection, Division of Science Research and Technology (DSRT) in cooperation with several partners to assess brown tide blooms in coastal waters in NJ. Water samples were collected by boat and helicopter at coastal stations from 2000 to 2002 along with field measurements. Aureococcus anophagefferens were enumerated and associated environmental factors were analyzed. A. anophagefferens abundances were classified using the Brown Tide Bloom Index and mapped, along with salinity and temperature parameters, to their geo-referenced location using the ArcView GIS. The highest A. anophagefferens abundances (>106 cells ml−1), including category 3 blooms (≥200,000 cells ml−1) and category 2 blooms (≥35,000 to ≤200,000 cells ml−1), recurred during each of the 3 years of sampling and covered significant geographic areas of the estuary, especially in Little Egg Harbor. While category 3 blooms were generally associated with warmer water temperatures (>16 °C) and higher salinity (>25–26 ppt), these factors were not sufficient alone to explain the timing or distribution of A. anophagefferens blooms. There was no significant relationship between brown tide abundances and dissolved organic nitrogen measured in 2002 but this was consistent with other studies. Extended drought conditions, with corresponding low freshwater inputs and elevated bay water salinities, occurring during this time were conducive to blooms. A. anophagefferens abundances were well above the reported levels that have been reported to cause negative impacts on shellfish. It was shown that over 50% of the submerged aquatic vegetation (SAV) habitat located in Barnegat Bay/Little Egg Harbor was categorized as having a high frequency of category 2 or 3 blooms for all 3 years.
 
In August and September 2001, Kuwait Bay, a semi-enclosed embayment of the Arabian Gulf, experienced a massive fish kill involving over >2500 metric tons of wild mullet (Liza klunzingeri), due to the bacterium Streptococcus agalactiae. In the Bay, this event was preceded by a small fish kill (100–1000 dead fish per day) of gilthead sea bream (Sparus auratus) in aquaculture net pens associated with a bloom of the dinoflagellate Ceratium furca. Sea bream were found to be culture positive for S. agalactiae, but did not show any visible signs of disease. Unusually warm temperatures (up to 35 °C) and calm conditions prevailed during this period. As the wild fish kill progressed, various harmful algae were observed, including Gymnodinium catenatum, Gyrodinium impudicum, and Pyrodinium bahamense var. compressum. Cell numbers of G. catenatum and G. impudicum exceeded 106 l−1 in some locations. All fish tested below the limits of detection for paralytic shellfish poisoning (PSP) and brevetoxins. Clams (Circe callipyga) were positive for PSP but at levels below regulatory limits. Nutrient concentrations, both inorganic and organic, were highly variable with time and from site to site, reflecting inputs from sewage outfalls, the aquaculture operations, a high biomass of decomposing fish, and other sources. It is hypothesized that many factors contributed to the initial outbreak of the bacterial disease, including unusual warm and calm conditions. The same factors, as well as enriched nutrient conditions, also apparently were conducive to the subsequent HAB outbreaks. The detection of PSP, while below regulatory limits, warrants further monitoring to protect human health.
 
In spring 2002 there was a significant outbreak of harmful microalgal bloom (HAB) in Hauraki Gulf on the north-eastern coast of New Zealand. With the exception of only a few sites where there was also a build-up of Noctiluca scintillans, the outbreak was largely associated with an almost monospecific bloom of Karenia concordia. At the peak of this bloom, mortalities of fish and abalone were observed. In areas where Noctiluca cells were found dead they had consumed large numbers of K. concordia cells. Laboratory tests showed cell extracts of K. concordia to be haemolytic and cytotoxic and confirmed that this species was responsible for marine life mortality. Satellite sea surface temperature (SST) data, obtained prior to the mid-October 2002 toxic outbreaks in Hauraki Gulf, showed signs of strong, along-shelf upwelling and also cross-shelf advection of warm, offshore, subtropical water into the Gulf through Jellicoe Channel. Time-series ocean colour data retrieved from the same region showed build-up of very high chlorophyll a level in Hauraki Gulf, virtually in same areas where bloom proportions of K. concordia (up to 3.3 × 107 cells l−1) were recorded. The relationships of this massive bloom to contemporaneous, remotely sensed SST and ocean colour satellite data (SeaWiFS) during the cold phase of ENSO are discussed.
 
Map of the Arabian Gulf showing location of field sites for samples collected in December 2008 from the coastal waters of Ras al-Khimah, UAE, near the Strait of Hormuz. Star symbol ($) indicates sample collection site, triangles (~) indicate locations where fish kills and marine mammal mortalities were reported. Arrows indicate circulation patterns (from Reynolds, 1993).
SEM micrographs of Cochlodinium polykrikoides from the Arabian Gulf. Cells are oriented apically. (A) Two-celled chain enmeshed in a mucoid matrix; sulcus can be observed in the top cell, just below the cingulum in the anterior portion of the cell. (B) Two-celled chain showing transverse flagellum of each cell; the excavated cingulum of upper cell is clearly visible. (C) Two-celled chain following treatment to remove polysaccharide mucilage, revealing excavated cingulum of both cells. (D) Four-celled chain. Scale bars: 10 mm.
Light and fluorescent micrographs of Cochlodinium polykrikoides from the Arabian Gulf. Cells are oriented apically. (A) Dorsal view showing longitudinal flagellum. (B) Dorsal view of cell showing the rounded epitheca and bilobed hypotheca; nucleus (n) and stigma (s) are visible in the anterior of the cell. (C) Dorsal view showing the rodshaped chloroplasts (ch); transverse flagellum (f) is visible in the top cingulum. (D) Two-celled chain with nuclei (n) located in the anterior of the cell. (E) Fluorescent micrograph showing many rod-shaped chloroplasts. (F) Fluorescent micrograph of two-celled chain. Scale bars: 10 mm.
Harmful algal blooms (HABs) caused by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides Margalef are responsible for mass mortalities of wild and farmed fish worldwide, with catastrophic impacts to aquaculture and local economies. Here we report on the Cochlodinium species responsible for a severe and widespread HAB in the Arabian Gulf and Gulf of Oman that has lasted for more than eight months at this writing, killing thousands of tons of fish and limiting traditional fishery operations, damaging coral reefs, impacting coastal tourism, and forcing the closure of desalination plants in the region. To identify the causative organism, cultures were established from cells isolated along the Arabian Gulf shore of the United Arab Emirates. Taxonomic analyses using scanning and light microscopy, and partial analysis of the large subunit (LSU) ribosomal RNA (rRNA) gene confirmed the C. polykrikoides classification. rRNA gene sequences of C. polykrikoides isolates from the Arabian Gulf were identical to isolates from the northeastern USA, Puerto Rico, Mexico, and Malaysia, known as the “American/Malaysian” ribotype. To our knowledge, this is the first HAB event associated with C. polykrikoides in the Arabian Gulf or the Gulf of Oman. The sudden emergence of C. polykrikoides in these Gulfs coincides with an apparent global expansion of this taxon, as well as a recent increase in HAB impacts observed in this region. The mechanisms underlying this expansion require further investigation, and may include increased nutrient enrichment of coastal waters in the Arabian Gulf and Gulf of Oman from domestic and industrial inputs, natural meteorological and oceanographic forcings, and the recent introduction of this species through ballast water discharge. A pattern of subsequent recurrence of C. polykrikoides blooms following an initial outbreak has been observed in other parts of the world, suggesting that this species may become a persistent HAB problem in this region. As Arabian Gulf countries rely on desalination plants as the primary source of freshwater, the disruption of plant operations by recurring Cochlodinium blooms poses a serious threat to the drinking water supply in the region, and represents an unprecedented HAB impact.
 
Map of the study area showing the location of sampling transects and stations. Transect 1 was tracked from 7 to 10 February 2003 and transect 2 from 15 to 19 May 2003. 
Light (LM) and scanning electron (SEM) micrographs of Pseudo - nitzschia species first reported in Argentinean waters. (1–4) P. heimii : (1–3) SEM, internal valve views showing the cell shape, apical and central part details; (4) LM, colony in girdle view. (5–8) P. lineola : (5–7) SEM, internal valve views showing the cell shape, apical and central part details; (8) LM, colony in girdle view. (9–12) P. turgiduloides : (9–11) SEM, internal valve views showing the cell shape, apical and central part details; (12) LM, colony in girdle view. 
Number of stations where Pseudo-nitzschia species were recorded during the sampling period in the Argentine Sea
Distribution patterns of Pseudo - nitzschia species in surface shelf waters of the Argentine Sea. 
The distribution pattern of Pseudo-nitzschia species, associated phytoplankton flora and its relationships with main environmental factors were studied for the first time in continental shelf surface waters of the Argentine Sea (Southwestern Atlantic Ocean, 38–55°S). Both qualitative and quantitative samples, collected during summer and fall 2003, were examined using light and scanning electron microscopy. Results indicated that the genus Pseudo-nitzschia has a wide distribution along the studied area. It was present at low densities, with infrequent peak abundances and appeared most frequently as a minor component of the diatom populations that typically develop on the continental shelf of the Argentine Sea. Moreover, phytoplankton communities were numerically dominated by unidentified phytoflagellates (≤5 μm) throughout almost all samples analyzed. Eight Pseudo-nitzschia species were identified in our study: P. australis, P. fraudulenta, P. heimii, P. lineola, P. pungens, P. cf. subcurvata, P. turgidula and P. turgiduloides. Of these, P. heimii, P. lineola and P. turgiduloides are new records for the Argentine Sea. Their presence in the area is attributable to the influence of southerly cold water masses. Spatial and temporal variations of the environmental parameters recorded in the study area generally determined the distribution of Pseudo-nitzschia species. P. pungens and P. australis were widely distributed and reached high densities, especially in waters with elevated temperatures and salinities (around 15 °C, 33.8 psu) and low nutrients concentrations. On the other hand, P. heimii, P. lineola, P. turgidula and P. turgiduloides showed a more restricted distribution, with lower densities in relatively cold, less saline (8 °C, 32.45 psu) and nutrient-rich waters. From the Pseudo-nitzschia species found throughout this survey, P. australis, P. fraudulenta, P. pungens and P. turgidula are known as domoic acid (DA) producers around the world, but there is little information on the potential toxicity of these species in Argentina.
 
We measured the presence, viability and potential toxicity of cyanobacteria in ships’ ballast tanks during three domestic voyages through the North American Great Lakes. Using molecular methods, the toxin-producing forms of Microcystis and Anabaena were monitored in ballast water after ships’ ballast tanks were filled at their first port of call, and at subsequent ports as ships transited the Great Lakes. Microcystis was detected in ballast water at intermediate and final ports of call in all three experiments, but the presence of Anabaena was more variable, suggesting low abundance or patchy distribution in ballast tanks. Both species were detected in ballast water up to 11 days old. Detection of the microcystin synthetase gene, mcyE, in ballast tanks indicated entrained cells were capable of producing microcystin, and further analyses of RNA indicated the toxin was being expressed by Microcystis, even after 11 days in dark transit. These data demonstrate within-basin transport and delivery of planktonic harmful algal bloom (HAB) species to distant ports in the world's largest freshwater reservoir, with potential implications for drinking water quality. These implications are discussed with respect to management of microbial invasions and the fate of introduced phytoplankton in their receiving environment.
 
Toxic algal blooms are common world-wide and pose a serious problem to the aquaculture and fishing industries. Dinoflagellate species such as Karenia brevis, Karenia mikimotoi, Heterosigma akashiwo and Chatonella cf. antiqua are recognised toxic species implicated in various faunal mortalities. Toxic blooms of Karenia cristata were observed on the south coast of South Africa for the first time in 1988 and were responsible for mortalities of wild and farmed abalone. K. cristata and various other dinoflagellate species common along the South African coast, as well as K. mikimotoi (Isolation site: Norway, Univ. of Copenhagen) and K. brevis (Isolation site: Florida, BIGELOW), were tested for toxicity by means of a bioassay involving Artemia larvae as well as abalone larvae and spat. K. cristata, like K. brevis, contains an aerosol toxin; however, the toxin present in K. cristata has not yet been isolated and remains unknown. K. brevis was, therefore, used to determine which developmental phase of the bloom would affect abalone farms most, and whether ozone could be used as an effective mitigating agent. Of the 17 dinoflagellate species tested, K. cristata, Akashiwo sanguinea, K. mikimotoi and K. brevis pose the greatest threat to the abalone mariculture industry. K. brevis was most toxic during its exponential and stationary phases. Results suggest that ozone is an effective mitigation agent but its economic viability for use on abalone farms must still be investigated.
 
The exposure of marine mammals to the toxins associated with harmful algae can be lethal. Domoic acid (DA) is a biotoxin produced by the Pseudo-nitzschia group of diatoms many of which are now a common component of the Scottish phytoplankton community (Stobo et al., 2008). DA is a potent excitatory neurotoxin that has caused large-scale mortality of marine mammals. We found harbour seals (Phoca vitulina) in Scotland are exposed to DA. Low levels, likely from recent exposure, were measured in the faeces and urine of live captured adult animals (using a direct competitive enzyme linked immunosorbent assay) and exposure was highest during August–September 2008 (7/32 of the faecal (22%) and 11/29 (38%) of the urine samples were positive). Median concentrations in positive faeces and urine were 25 ng/g and 6 ng/ml respectively. One positive pregnant female was subsequently found dead with 10 ng/ml DA in her amniotic fluid but the contribution of DA exposure to the cause of death could not be established. However, the highest levels in the study were found in anonymous faecal samples collected in September 2009 on the east coast of Scotland (up to 397 ng/g). Further studies are urgently needed to determine the importance of DA exposure to the population dynamics of Scottish harbour seals in light of the recently reported major population declines.
 
Ballast water was sampled on 12 occasions before and after an exchange process carried out in regional seas in order to assess the efficiency of this type of ballast water management at reducing the abundance and diversity of phytoplankton. Although there was an overall reduction in the average abundance and number of taxa after exchange this was not consistent between tanks and voyages. On some occasions there were changes in species composition after exchange and, in some cases, there were increases in potentially harmful species after the exchange process. Factors such as the depth of the water during the exchange process, the season and the method of exchange influenced the efficacy of the exchange process. The variability in the results after exchange mean that this is unlikely to be a ballast water management method that would give consistent results and careful consideration would have to be given to the suitability of using this method in regional seas as a means of reducing the risk of introducing non-native species.
 
Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 102 to 103 cells g−1 fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.
 
The seasonal distribution of Prorocentrum lima within the epibiotic community associated with wild and cultured shellfish was studied at eight sites in northeastern USA coastal waters. This dinoflagellate produces toxins that can potentially accumulate in shellfish and lead to diarrhetic shellfish poisoning. Four sites in semi-sheltered environments provided sufficient data for an evaluation of population dynamics. The two southernmost populations (Point Judith Pond and Bluff Hill Cove in Rhode Island) displayed a double peak in abundance, one from March to June, with several thousands cells per g dry weight of collected epibiota, and a second one, sometimes minor, in autumn. At the two northern stations (New Meadows River and Clam Cove in Maine), P. lima populations also peaked twice but with a delay in timing. All four sites harbored filamentous seaweeds and aggregations of tube-forming or chains of diatoms, providing a favorable habitat for this epiphytic dinoflagellate.
 
Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (102 to 103 mL−1), but capable of forming blooms of 104 to 105 cells mL−1 under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421 μm3 per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability.
 
Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm3 m−2 were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml−1 (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion, possibly additionally benefiting from high nitrogen loads.Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp3]-MC-RR and [Asp3,Dhb7]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.
 
This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography–mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18–23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential.
 
Blooms of the dinoflagellate Alexandrium spp. increase in their frequency, toxicity and historical presence with increasing latitude from New Jersey (USA) to the Gaspé peninsula (Canada). Biogeographic variation in these blooms results in differential exposure of geographically separate copepod populations to toxic Alexandrium. We hypothesize that the ability of copepods to feed and reproduce on toxic Alexandrium should be higher in copepods from regions that are frequently exposed to toxic Alexandrium blooms. We tested this hypothesis with factorial common environment experiments in which female adults of the copepod Acartia hudsonica from five separate populations ranging from New Jersey to New Brunswick were fed toxic and non-toxic strains of Alexandrium, and the non-toxic flagellate Tetraselmis sp. Consistent with the hypothesis, when fed toxic Alexandrium we observed significantly higher ingestion and egg production rates in the copepods historically exposed to toxic Alexandrium blooms relative to copepods from regions in which Alexandrium is rare or absent. Such differences among copepod populations were not observed when copepods were fed non-toxic Alexandrium or Tetraselmis sp. These results were also supported by assays in which copepods from populations both historically exposed and naı̈ve to toxic Alexandrium blooms were fed mixtures of toxic Alexandrium and Tetraselmis sp. Two-week long experiments demonstrated that when copepods from populations naı̈ve to toxic Alexandrium were fed a toxic strain of Alexandrium they failed to acclimate, such that their ingestion rates remained low throughout the entire two-week period. The differences observed among populations suggest that local adaptation of populations of A. hudsonica from Massachusetts (USA) to New Brunswick (Canada) has occurred, such that some populations are resistant to toxic Alexandrium.
 
Monitoring of lipophilic marine toxins was carried out in three shellfish production sites on the West Coast of Ireland. The toxins were monitored using passive samplers (solid phase adsorption toxin tracking; SPATT) and toxin-free mussels that were replaced weekly in the selected sampling stations. The toxin profiles and concentrations obtained in the SPATT and in the transplanted mussels were compared with those observed in indigenous (native) mussels from each production site as well as with the phytoplankton that was detected in the water. Numerous lipophilic toxins were detected in the SPATT discs by ultra-performance liquid-chromatography–mass spectrometry/mass spectrometry (UPLC–MS/MS) and included okadaic acid (OA), dinophysistoxin-2 (DTX2), pectenotoxin-2 (PTX2), AZA1, AZA2, yessotoxin (YTX) and SPX-13-DesMe-C. The accumulation rate of toxins in the indigenous mussels and in the SPATT discs correlated well. Toxins were detected in all SPATT discs from all locations, even in the absence of toxin-producing phytoplankton, as observed previously by other research groups. It is quite clear from our data that the presence of okadaic acid in the water (even at high concentrations) did not induce toxicity in the transplanted mussels in the absence of phytoplankton. A severe toxic event of azaspiracids (AZAs) occurred in one of the sampling stations. The SPATT discs accumulated predominantly AZA1 and -2 suggesting that both toxins are biosynthesized by the AZA-producing organism. As opposed to the DSP event, the AZA event resulted in the contamination of the transplanted mussels for several consecutive weeks. This is the first study that reports the presence of YTX and SPX-13-DesMe-C in Irish waters. In our study conditions, the SPATT did not enable the forecasting of shellfish contamination as the increase in toxin concentration occurred at the same time in the shellfish and in the SPATT.
 
Among the wide range of toxins produced by cyanobacterial blooms, microcystins (MCs) are the most common and are known to accumulate in aquatic organisms. Freshwater gastropods are grazers and likely to ingest toxic cyanobacteria, particularly Planktothrix agardhii, one of the most common species in the northern hemisphere. The study examines (i) the ingestion of toxic P. agardhii by the prosobranch Potamopyrgus antipodarum, (ii) the kinetics of MC accumulation and depuration in snail tissues during and post-exposure, and (iii) the impact of MCs on their life traits (survival, growth and fecundity). We showed that P. antipodarum ingested 71% of cyanobacteria available during the first 24 h in the presence or not of non-toxic food, and accumulated 1.3% of ingested MCs during the 5-week intoxication period. Elimination of MCs was total after 3 weeks of depuration. A decrease of growth and fecundity was observed during the intoxication period, but it was reversible after the end of exposure. Results are discussed in terms of variation of the response between prosobranch and pulmonate gastropods to toxic cyanobacteria exposure, and the negative impact of toxic cyanobacteria on natural communities of freshwater gastropods.
 
At about 20-year intervals, since at least the 1860s, there have been accounts of the accumulation of large quantities of mucilage in the waters off the northern coast of the South Island of New Zealand. On a few occasions these events have been associated with harmful effects, such as the mass mortality of marine fauna and the impediment of fishing activities, though previous attempts to identify the causative organism(s) or the environmental conditions which promote its development have been unsuccessful. In January 2000, there was a moderate re-occurrence of mucilage accumulation in Tasman Bay that led to the identification of the primary origin of this material as the planktonic, thecate, dinoflagellate Gonyaulax hyalina (Ostenfeld et Schmidt). Macro-aggregate accumulation was occurring in the water column when dinoflagellate cell numbers were relatively low. Experimental work on cultures of this organism showed that the dinoflagellate produced transparent exo-polymers (TEPs) composed primarily of galactose and glucose monomers (44.8 and 29.6 mol%, respectively). Analysis of natural and cultured mucilage showed this had a high inorganic content (>60% (w/w)). A substantial proportion (40% (w/w)) of this inorganic fraction was composed of calcium that apparently played an important role in stabilizing the gel. Phosphorus was also enriched in the mucilage. It is hypothesized that the high inorganic N/P ratio observed in the water column at the time of mucilage formation was an effect rather than a cause of this event, which resulted in the scavenging of particulate material from the water column and enrichment of sediments and bottom waters with organic material and phosphorus.
 
Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean. Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations (Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l−1. M. galloprovincialis was proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.
 
Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO3− or NH4+) present also influenced P. minimum response to DOC; 10–20 μg ml−1 additions of fulvic acid had no effect upon growth rates in the presence of NH4+ but significantly increased growth rates in the presence of NO3−, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO3− assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O2 cell−1 h−1, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml−1. Low concentrations (5–10 μg ml−1) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml−1 resulted in a 30% decrease in O2 evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O2 cell−1 h−1 and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.
 
Most species belonging to the toxigenic genus Dinophysis have chloroplasts of cryptophyte origin. Whether these chloroplasts are temporarily sequestered from the prey, or permanently established under the control of the dinoflagellate is currently disputed. To investigate this, a culture of Dinophysis acuminata was established by feeding it the phototrophic ciliate Mesodinium rubrum (= Myrionecta rubra), which again was fed the cryptophyte Teleaulax amphioxeia. Molecular analysis comprising the nucleomorph LSU and two chloroplast markers (tufA gene and a fragment from the end of 16S rDNA to the beginning of 23S rDNA) resulted in identical sequences for the three organisms. Yet, transmission electron microscopy of the three organisms revealed that several chloroplast features separated D. acuminata from both T. amphioxeia and M. rubrum. The thylakoid arrangement, the number of membranes around the chloroplast as well as the position and the arrangement of the pyrenoids were strikingly different. Considering both molecular and ultrastructural evidence, our data indicated that the chloroplasts in D. acuminata are permanent chloroplasts originating within Teleaulax or another closely related cryptophyte genus. Electron microscopy also provided new information on the peduncle of D. acuminata, which is used in food uptake.
 
The clarification of the identity of the prominent Dinophysis morphotype in Greek coastal waters, characterized until now as D. cf. acuminata, has become a major challenge since the first DSP outbreak in the year 2000. So far, definite identification of the morphotype at the species level has been a taunting task, since both morphological and common molecular tools have proved fairly inefficient towards this direction. Recently, the mitochondrial gene region encoding for the cytochrome c oxidase subunit I (cox1) has been opted for its efficiency to discriminate at the species level within the Dinophysisacuminata complex. Using both ribosomal and mitochondrial genetic sequence data, we have shown that the species D. cf. acuminata described from Greek coastal waters is actually more related to D.ovum. The implications involved with present molecular tools in deciphering taxonomic relationships among species belonging to the genus Dinophysis are also discussed.
 
On the Atlantic coasts of Andalucía, chronic spring–summer (March–June) diarrhetic shellfish poisoning (DSP) outbreaks are associated with blooms of Dinophysis acuminata, Claparède and Lachmann. Artificial neural networks (ANNs) have been successfully used to model primary production and have recently been tested for the prediction of harmful algae blooms. In this study, we evaluated the performance of feed forward ANN models trained to predict D. acuminata blooms. ANN models were trained and tested using weekly data (5 previous weeks) of D. acuminata cell counts from eight stations of the Andalucía HAB monitoring programme in the coasts of Huelva between 1998 and 2004. Principal component analysis (PCA) were previously carried out to find out possible similarities within time series from each zone with the aim of reducing the number of areas to model. Our results show that ANN models with a low number of input variables are able to reproduce trends in D. acuminata population dynamics.
 
Blooms of Dinophysis spp. associated with lipophilic shellfish toxin outbreaks are common in Northwestern Iberia waters from spring to autumn. Blooms of Dinophysis acuta are very seasonal (late summer-early autumn); they start earlier in Northern Portuguese waters during the upwelling season, and reach their maximum values in the Galician Rías during downwelling events at the end of the upwelling season. There is controversy about whether sudden increases in cell concentrations in the rías result from cross-shelf transport of populations previously established in adjacent shelf waters, or are due to longshore transport that brings populations located off Portugal to the North. In 2005, record concentrations of D. acuta were observed in Portuguese waters (14 × 104 cell L−1) off Aveiro in early September, while concentrations off the Galician coast were very moderate (102–103 cell L−1). During the autumn transition from upwelling- to downwelling-favourable winds, D. acuta declined abruptly off Portugal while the annual maximum (up to 22 × 103 cell L−1) was found in the Galician Rías. A new approach was used that combined physical observations (SST, current measurements); weekly observations from monitoring programmes in Galicia and Portugal; weekly division rate (μmin) estimates of D. acuta in Ría de Vigo, together with monthly transects and additional ad hoc sampling in the Ría. During August and early September, division rates were high in Ría de Vigo but concentrations were low, whereas higher SST values in Portugal (a proxy for thermal stratification) seemed to promote the build up of high densities of D. acuta. During the last week of October and the first week of November, populations declined, whereas in Galicia, maximum concentrations were reached while division rate estimates were almost zero. Results presented here confirm that increased numbers in the Galician Rías are not due to intrinsic growth but to physically driven accumulation; a simple cell concentration budget calculated during the accumulation period suggests that the high net growth observed during downwelling, in the absence of cellular division, must be due to cells imported by longshore transport.
 
This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell−1) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell−1 in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.
 
Between February and March 2000, seven lethal dose assays (48hLD50) were performed during a Gyrodinium corsicum proliferation in Alfacs Bay (Spain), in order to determine the densities that produce acute lethal effects for the main finfish species cultivated in the area. When samples were tested immediately after collection, the 48hLD50 for Sparus aurata ranged between 1268 and 3136 cells ml−1, and NOEC between 366 and 2372 cells ml−1. These consistent LD50 values on different dates suggests the presence of a toxic compound associated with G. corsicum. A progressive increase in toxicity observed during the bloom indicates a possible influence of environmental factors on toxic activity. The relationships between cell densities of natural G. corsicum populations and mortalities of juvenile seabream (S. aurata) and seabass (Dicentrarchus labrax) are reported for use in the prevention of fish kills by this dinoflagellate.
 
Top-cited authors
Christopher J. Gobler
  • Stony Brook University
Donald M Anderson
  • Woods Hole Oceanographic Institution
P. M. Glibert
  • University of Maryland Center for Environmental Science
William P Cochlan
  • San Francisco State University
Timothy Walter Davis
  • Bowling Green State University