THE RALEIGH REPORT 1998
Pfiesteria Research Needs and Management Actions

OVERVIEW
In response to the effects of recent outbreaks of a single-celled dinoflagellate known as Pfiesteria piscicida in North Carolina waters, Governor James B. Hunt, Jr. asked the Water Resources Research Institute to convene a panel of scientists to assess the state-of-the-science and establish research priorities on the causes, effects and management of Pfiesteria. With the assistance of the Governor’s Office, N.C. Department of Environment and Natural Resources staff, and other North Carolina researchers, the Institute identified a group of fourteen scientists from throughout North America for this task. These distinguished scientists represented a variety of disciplines associated with harmful algal blooms and their ecological and human effects.

On December 4-5, 1997, this scientific panel met in the Research Triangle Park to discuss the four major objectives of the workshop and develop a document to address these objectives. The objectives were:

• based on the state of the science, characterize the causes, consequences, and possible corrective measures for Pfiesteria growths in North Carolina coastal waters;
• based on current knowledge, assess the impact of proposed management actions on Pfiesteria;
• based on critical gaps in current knowledge, recommend a feasible research program focused on decision-sensitive scientific uncertainties; and
• based on current management efforts, consider short-term and long-term management issues.

The panel was provided twenty-five recent research reports from the research contacts a week in advance of the workshop. Panel members divided into three groups to address the topics of Pfiesteria ecology, fisheries and public health/management. A draft report was developed and sent to the research contacts for their review by the middle of December. These review comments were sent to the panel, and a final report was prepared and submitted to the Institute in early January 1998.

This final report titled The Raleigh Report 1998 - Pfiesteria Research Needs and Management Actions was released the week of January 19, 1998, as Special Report No. SRS-19 of The Water Resources Research Institute of The University of North Carolina. This report is an on-going effort by North Carolina to better understand harmful algae blooms and their impact on the fishing industry, public health and tourism.

There have been three major federal reports addressing harmful algal blooms:

• 1993, Marine Biotoxins and Harmful Algae: A National Plan;
• 1995, ECOHAB: The Ecology and Oceanography of Harmful Algal Blooms, A National Research Agenda and
• 1997, National Harmful Algal Blooms Research and Monitoring Strategy: An Initial Focus on Pfiesteria, Fish Lesions, Fish Kills, and Public Health.

At the state level, there have been two recent reports from Maryland as a result of the fishkill episodes on the Eastern Shore. These 1997 reports are

• Agriculture and Its Relationship to Toxic Dinoflagellates in the Chesapeake Bay and
• The Cambridge Consensus - Forum on Land-Based Pollution and Toxic Dinoflagellates in Chesapeake Bay.

The Raleigh Report 1998 - Pfiesteria Research Needs and Management Actions is a continuation of this effort to further define the progress and management needed to control harmful algae blooms.

Workshop Panel

Table of Contents

EXECUTIVE SUMMARY

INTRODUCTION

NUTRIENTS, PFIESTERIA AND FISH

• Identification
• Population Dynamics in an Ecosystem Context
• Toxins and Toxicity
• Influences Of and Interactions With Fish

PUBLIC HEALTH

• Epidemiology in North Carolina
• Food Safety

MANAGEMENT

• Water Quality and Fisheries
• Public Health and Education

APPENDICES

Appendix A: Reference Material

Appendix B: Panel Members

Appendix C: Cambridge Consensus Report

EXECUTIVE SUMMARY

Overview Harmful algal blooms (HABs) are of growing national concern due to indications that their frequency and magnitude have increased in recent decades in estuarine and coastal waters of the United States and throughout the world. HABs can have significant impact on the fishing industry and on tourism. Further, as just one symptom of the over-enrichment of coastal waters with nutrients, HABs have implications concerning economic growth and development. Of particular interest, outbreaks of Pfiesteria piscicida and Pfiesteria-like species (the Pf-complex) in North Carolina and other mid-Atlantic states have been associated with the development of fish lesions, fish kills, and health problems of fishermen and other individuals with frequent contact with affected waters.

As part of North Carolina’s response to these problems and related concerns, Governor James B. Hunt Jr. requested that The University of North Carolina Water Resources Research Institute host a workshop comprised of scientific experts representing a variety of disciplines. Discussion at the December 4-5, 1997 workshop focused on the status of current knowledge and on research needs for scientifically sound management of the environment, living resources, and public health. Major conclusions and recommendations developed by scientists at the workshop are presented below in three general areas: (1) management, (2) public health and education, and (3) research.

Management
There can be little question that decreases in nutrient loading (both organic and inorganic forms of nitrogen and phosphorus) will reduce eutrophication and, thereby, lower the risk of toxic outbreaks of Pfiesteria-like dinoflagellates, hypoxia, and fishkills. However, sufficient scientific information does not exist at present to quantitatively determine causal relationships, with confidence, between specific sources and rates of nutrient inputs (that can be attributed to particular land-use practices) and the occurrence of toxic events attributed to Pfiesteria piscicida or the Pfiesteria-like organisms.

While it may be difficult to justify targeting any specific land use activity, the evidence is overwhelming that nutrient enrichment arising from human activities in the watershed results in coastal eutrophication as expressed by declining water quality, hypoxia, outbreaks of toxic microorganisms, or fiskkills. Given current levels of understanding, attempts to manage nutrient loading should be considered for all major sources of both organic and inorganic forms of nitrogen and phosphorus. These include: (i) deforestation, (ii) human wastes, (iii) artificial fertilizers, (iv) the storage of animal wastes and their use as fertilizer, (v) urbanization, and (vi) atmospheric deposition.

Public Health and Education
The effects of P. piscicida, Pf-like organisms and their toxins on human health are unclear. There are reports that P. piscicida produces an airborne toxin(s) that causes symptoms such as short-term memory loss, and that contact with water where P. piscicida, Pf-like organisms, or fishkills have occurred, can result in the development of skin lesions. In addition, although there is no evidence that P. piscicida affects human health through the ingestion of seafood products, this possibility cannot be ruled out based on the information available.

There has been considerable discussion, publicity, and misinformation concerning the human health effects of P. piscicida and Pf-like organisms. Thus, high priority should be given to the preparation of educational material (perhaps in collaboration with agencies from other affected States and the Centers for Disease Control) to inform those from the fishing industry, healthcare professionals including state and public health departments, the tourist industry, seafood consumers and the media of P. piscicida and its biological effects. In addition, an observational and communication network should be established to provide continual monitoring for both P. piscicida and Pf-like organisms, the presence of toxins, fish with lesions and fishkills in support of epidemiological studies.

Research
It is recommended that high priority be placed on the development of reliable techniques for rapid identification of toxic species in the Pf-complex. Given that this is difficult, if not impossible, with conventional light microscopy, careful work with scanning electron microscopy should be coordinated with the development of molecular probes to detect and enumerate the various species in the complex.

It is recommended that the State of North Carolina work with federal agencies and the Bigelow Laboratory for Ocean Sciences (where the Culture Collection of Marine Phytoplankton, CCMP, is housed and maintained) to establish a national archive where characterized isolates can be maintained and distributed.

It is recommended that high priority be placed on the chemical characterization of the toxin(s) associated with P. piscicida and the Pf-complex, and the development of analytical methods for routine and rapid detection and measurement of the toxins in a variety of matrices.

It is recommended that reliable and objective measures of exposure and of human health effects be developed to make epidemiological studies more effective in assessing public health risks. This will be facilitated by knowledge of the chemical nature and toxicology of the toxic agents. There is a need for general epidemiological surveillance to detect any illness due to Pf-related toxins with rapid follow-up testing of any individuals discovered through this surveillance.

It is recommended that high priority be placed on research designed to elucidate the mechanisms by which environmental factors govern life cycle transformations, control the growth of each stage in the life cycle, and stimulate cells to become toxic.

It is recommended that a coherent and coordinated research strategy be implemented that incorporates an appropriate spectrum of well controlled small scale laboratory experiments, medium scale mesocosm experiments, full scale field studies, and model results.

It is recommended that biochemical indicators of stress be employed together with otolith-based studies of growth rate in impacted versus non-impacted areas. In addition, the pathways by which toxin(s) enter fish (through the epidermis, gills, or by ingesting the toxic zoospores) should be determined.

INTRODUCTION

Outbreaks of harmful blooms of microscopic algae and protozoa (HABs) in coastal waters are a possible symptom of coastal eutrophication and have important ecological, economic, and public health implications. Ecologically, they may signal changes in the structure and function of coastal ecosystems and, therefore, in their capacity to provide ecosystem services from the processing of nutrients and fisheries to their effectiveness in buffering coastal human populations from the effects of storms and climate change. HABs also have significant economic impacts on the fishing industry and tourism.

Since many of these organisms produce toxins that are transmitted to people via seafood products or as aerosols (e.g., ciguatera fish poisoning, paralytic shellfish poisoning, amnesic shellfish poisoning, neurotoxic shellfish poisoning, and diarrhetic shellfish poisoning), they also have important implications for public health. HABs are of growing national concern because of indications that their frequency and magnitude have been increasing in recent decades in coastal waters of the United States and throughout the world.

Recent outbreaks of Pfiesteria piscicida and Pfiesteria-like species (the Pf-complex) in the mid-Atlantic region of the United States, including coastal waters of North Carolina and Maryland, are the latest examples of this problem. These events are of particular concern because of potential linkages to local and regional land-use practices that over-enrich coastal waters with nutrients, and they have been implicated in the development of fish lesions, fish kills, and health problems of fishermen and other individuals with frequent contact to affected waters.

As a part of North Carolina’s response to these problems and related concerns, The University of North Carolina Water Resources Research Institute hosted a workshop to review the current scientific understanding of the problem, to assess proposed management actions, and to recommend priorities for future research as a basis of adaptive management. Given the time available (2 days) and the information provided (Appendix A), workshop participants agreed that the panel (Appendix B) would focus its efforts on the status of current knowledge and on research needs for scientifically sound management of the environment, living resources and public health. The problem was divided into three broad areas:

• (1) nutrients, Pfiesteria and fish;
• (2) water quality, fish and public health; and
• (3) management.

NUTRIENTS, PFIESTERIA AND FISH

The related problems of nutrient enrichment and toxic blooms in the coastal zone involve complex interactions between terrestrial and estuarine ecosystems, climate, and people. Cyclic and episodic occurrences of oxygen depletion, HABs, fish kills and illnesses in people (who have eaten seafood or been exposed to toxic blooms) have forced the scientific and management communities to address the complex and poorly understood problem of the effects of land-use practices in coastal watersheds on water quality and living resources in coastal estuarine and marine ecosystems. It is in this context that we address the state of current knowledge and research needs that should be dealt with to advance our understanding of the causes and consequences of Pfiesteria piscicida and Pfiesteria-like dinoflagellates (the Pf-complex). As concluded in the Cambridge Consensus (Appendix C), the following should be emphasized from the beginning:

There can be little question that decreases in nutrient loading (both organic and inorganic forms of N and P) will reduce eutrophication and, thereby, lower the risk and severity of toxic outbreaks of Pfiesteria-like dinoflagellates, hypoxia and fish kills. However, sufficient scientific information does not exist at present to quantitatively determine causal relationships, with confidence, between specific sources and rates of nutrient inputs (that can be attributed to particular land-use practices) and the occurrence of toxic events attributed to Pfiesteria piscicida or the Pfiesteria-like organisms.

This conclusion applies to coastal waters throughout the United States and is not unique to North Carolina.

Here we summarize our perspective of the current understanding of the effects of nutrient enrichment on P. piscicida and of the research that will be needed to establish with known certainty causal links between nutrient enrichment and the abundance and toxicity of P. piscicida. Our emphasis is on those aspects of the problem that must be understood as a means of providing a foundation upon which to build ecologically sound strategies for the management of the environment and living resources. We highlight at the onset that many of the findings reported to date are both new and outside current dogma. Because of their potential significance to our understanding of the structure and function of coastal ecosystems and the microbial populations that inhabit them, it is important that, in the tradition of science, these results are verified and that research be pursued to clarify their ramifications for the management of the environment and the living resources they support. Four issues were identified for analysis and discussion:

• (1) identification and the need for isolates of known identity as a resource for the research community;
• (2) population dynamics in an ecosystem context;
• (3) toxins and toxin production, their toxicity and mode of action; and
• (4) influences of and interactions with fish.

Identification

Taxonomy is never more important than in the characterization of a harmful species. It is essential for the application of various types of data. Indeed, given infraspecific genetic variability, it may be necessary to discriminate at an intimate level (race, clone, etc.). Many dinoflagellate species can be identified with a light microscope fairly readily. Unfortunately some of the smallest species (< 20 microns in length, including P. piscicida), are difficult to recognize and enumerate by this method. In part because of this, algal- and fish-bioassay techniques have been used by different investigators to establish the presence of P. piscicida and Pf-like taxa.

Problems of identification and the use of assays that have not been intercalibrated have led to a category referred to as “Pfiesteria-like organisms” (referred to here as Pf-like or the Pf-complex) because they are similar in size and morphology and can cause fish kills. This category consists of several disparate species in at least four relatively unrelated genera whose ecology is unlikely to be similar. For example, Gyrodinium galatheanum, one of the members of the Pf-like group, is permanently photosynthetic and lacks a substantial theca. Even though it is mixotrophic (capable of supplementing growth with organic and inorganic matter, i.e., phagotrophy), it can be grown and cultured exclusively on inorganic nutrients. Although it can cause fish kills (though the toxicology and mode of action are different) and superficially looks like P. piscicida, its ecology is quite different.

As a consequence of the complexity of the biology, the linkages between toxic species, environmental conditions that promote their growth and fish kills in the field can be equivocal. Thus, it is imperative to discriminate to the species level within this complex in order to accurately understand how the growth of these organisms is controlled by environmental factors in nature and the toxins they produce.

It is recommended that high priority should be placed on the development of reliable techniques for rapid identification of toxic species in the Pf-complex. Given that this is difficult, if not impossible, with conventional light microscopy, careful work with scanning electron microscopy should be coordinated with the development of molecular probes to detect and enumerate the various species in the complex.

The development of molecular probes and extrapolation of experimental results from laboratory-based studies to nature are dependent on the availability of cultured clones of known identity that have been isolated from single vegetative cells and cysts, and grown in defined media. There is a clear need for the establishment of a national facility to archive and distribute cultures of toxic forms of algae and protozoa of known identity isolated from coastal waters.

It is recommended that the State of North Carolina should work with federal agencies and the Bigelow Laboratory for Ocean Sciences (where the Culture Collection of Marine Phytoplankton, CCMP, is housed and maintained) to establish a national archive where characterized isolates can be maintained and distributed.

In this context, there is an immediate need to intercalibrate the algal- and fish-bioassay techniques used by different investigators to establish the presence of P. piscicida and Pf-like taxa. This will help to resolve the controversial issue of whether these techniques are equally specific to P. piscicida or whether they are sensitive to the presence of other species in the Pf-complex.

Central to the issue of identification is the development of molecular-based assays to identify life cycle stages of P. piscicida and Pf-like species. The panel was not made aware of current progress in the development of surface antibodies for the identification of P. piscicida nor of any research currently underway to develop ELISA-based assays for toxins. Given the multi-species nature of the Pf-complex and the elaborate life history of P. piscicida, the effectiveness of a single antibody approach is questionable. Sensitive, highly specific and cost-effective methods are needed. Molecular genetic approaches that target diagnostic nucleic acid markers could be used to screen and catalog available strains as well as provide a powerful tool to develop in situ detection and enumeration capabilities. In this regard, the panel recognized several research priorities that would benefit from this technology:

(i) Molecular systematic studies of available strains will provide essential information on the relationship of the members of this species complex and a critical foundation to evaluate and compare results of current and past investigations.

(ii) Although current work to characterize the gene encoding the 18S rRNA of P. piscicida will provide a useful detection method to the genus level, the development of sensitive field methods for rapid, species-specific identification and enumeration will probably require the development of isolate-specific nucleic acid probes to achieve the required specificity. Surface antibody assays may also be useful for identifying encysted and amoeboid forms in sediments.

(iii) Of critical importance is the development of sensitive methodologies that would provide the capabilities to assess and monitor all stages of the P. piscicida population. Research will be needed to develop nucleic acid based probes specific to each of the critical non-toxic and toxic stages of the P. piscicida life cycle. Such probes, in combination with sensitive enumeration methodologies, would provide a powerful tool to monitor changes in the abundance and distribution of each life cycle stage during bloom events. This is important for elucidation of the organism’s population dynamics, and will provide critical information needed for water quality management.

Population Dynamics in an Ecosystem Context

In addition to problems of identification, the challenge of understanding the environmental factors that control the growth, distribution and toxicity of P. piscicida is made all the more difficult by the organism’s life cycle and by its multiple modes of nutrition.

P. piscicida has a complex life cycle that includes benthic and pelagic stages, vegetative and cyst stages, and nontoxic and toxic stages of zoospores and amoebae. The nontoxic benthic amoeboid stage is apparently the predominant form under most circumstances. It is heterotrophic (utilizes organic matter to support growth) with phagotrophy (able to ingest or engulf solid particles of food) apparently being the predominate mode of nutrition. Nontoxic stages have also been reported to have osmotrophic (capable of living in a wide range of salinities) and mixotrophic capabilities and the species is capable of kleptochloroplastidy (adoption of algal and/or plant-like nutrition by retaining the chloroplasts that the zoospores obtain from algal prey), a behavior that may be of transitory importance during short periods following the collapse of phytoplankton blooms.

In the absence of fish, transformations among the various stages of P. piscicida appear to be influenced by the availability of microbial prey, especially small flagellates (e.g., cryptomonads). Thus, the effects of nutrient enrichment on P. piscicida can be complex because it is phagotrophic and not directly stimulated by inputs of inorganic nutrients, i.e., effects of nutrient enrichment in nature are mediated by the responses of prey species. P. piscicida can be lethal to fish for undefined periods when schooling fish are in the vicinity, and the development of toxic stages of P. piscicida appears to be stimulated by the presence of a chemical(s) released by fish and fish tissues, including blood.

The availability of inorganic phosphorus (P) can also stimulate the growth of toxic zoospores (TZs) indicating that other factors such as nutrient stimulation may also come into play. In addition to producing and releasing toxins, the development of TZs is significant to understanding the population dynamics of this organism because it is this stage that engages in sexual reproduction.

These observations provide a good foundation for continued studies of the mechanisms by which nutritional status and the availability of microflagellate food organisms, dissolved organic nutrients, and dissolved inorganic nutrients and fish, affect the growth rate of major stages, life cycle transformations among stages, and the development of toxicity.

It is recommended that high priority should be placed on research designed to elucidate the mechanisms by which environmental factors govern life cycle transformations, control the growth of each stage in the life cycle and stimulate cells to become toxic.

The complex life cycle and modes of nutrition that characterize P. piscicida and the potential significance of Pf-like organisms exacerbate the problem of extrapolating the results of controlled, small scale experiments in the laboratory to ecosystems in nature. Additional studies are needed to investigate the behavior of P. piscicida under natural conditions and in experimental ecosystems (mesocosms) that simulate, at known levels of complexity, the array of ecological processes that influence the growth, development and toxicity of P. piscicida in nature. The effects of predators (e.g. ciliates, rotifers) and the effects of other organisms (e.g., bacteria, other protozoa, microzooplankton) with which P. piscicida must compete for nutrient resources (bacteria, microalgae, dissolved organic compounds) should be evaluated under realistic environmental conditions. Turbulence may be a factor, and results in the literature suggest that P. piscicida is sensitive to turbulence. Generally speaking, turbulence is an important parameter of species succession in plankton communities, of predator-prey interactions and the distribution and availability of dissolved constituents including chemicals that cause excystment and fish kills. Thus in this case, turbulence intensity may be of fundamental importance and should be quantified as part of field studies and carefully controlled in laboratory experiments and mesocosm studies to mimic the range of intensities found in nature.

It is recommended that a coherent and coordinated research strategy be implemented that incorporates an appropriate spectrum of well controlled small scale laboratory experiments (most of the work reported to date falls into this category), medium scale mesocosm experiments (not as well controlled as small scale laboratory experiments, but more realistic), full scale field studies (lacking in control, but most realistic), and modeling (to synthesize results across scales and predict the probability of blooms and associated risk factors).

Toxins and Toxicity

The development of toxic stages of P. piscicida from North Carolina waters has been reported to occur in the presence of fish schools in nutrient rich, calm, shallow and poorly flushed estuarine environments over a temperature range of 12-33oC (with TZs occurring most frequently >25oC) and a salinity range of 0-35 practical salinity units (psu) (with TZ toxicity peaking at 15 psu). At low temperatures (<15oC), a lobose amoeboid stage becomes the most toxic form. At temperatures >15oC, TZs predominate. Often these environmental conditions (with the exception of the presence of fish) are those that promote the growth of a broad range of microbial species, most of which are non-toxic. The question of what specific environmental factors or cues may trigger the development of toxic stages of P. piscicida remains to be answered.

It is also unclear whether there are threshold effects and whether low (chronic) levels of toxin(s) are produced more or less continuously or just under the influence of the presence of (high) densities of fish. These problems and the uncertainty of the exact identity of P. piscicida and Pf-like organisms in the field further underscores the requirement that techniques for routine, rapid and positive identification of this organism, other Pf-like species, and the toxins they produce, must be developed as quickly as possible.

The chemical characterization of toxins associated with P. piscicida or the Pf-complex is pivotal to many associated studies that require a knowledge of their occurrence, distribution, and toxicology. Information on the current status of the chemical isolation and nature of the toxins is sparse, and no information was supplied to the panel, though it is believed that research to determine the chemical composition, structure and mode(s) of action of toxins produced by P. piscicida and Pf-like organisms has been hindered by the availability of P. piscicida biomass. Based on prior work with other marine toxins, all approaches to identify new toxins have employed a bioassay-guided fractionation process to purify and isolate the toxic agents. Because evidence is emerging that several species may exist, it is essential to rigorously identify the biological sources of the toxin(s). In addition, a common and relevant bioassay is also required to ensure the identification of the correct compounds, and indeed it is likely that a family of related compounds will be identified as the agents responsible for the toxicity of P. piscicida or the Pf-complex. Already there are anecdotal reports that two toxins exist (a water soluble compound and a lipid soluble compound), though the structural relationship, if any, between these compounds has not been reported. The stability of the toxins in the environment and during food processing is also not known.

Once identified, rapid, robust and sensitive analytical methods must be developed in order to detect the toxin(s) in a variety of matrices (e.g. fish and shellfish tissues, mud and water). As soon as possible thereafter, toxin standards should be made available to facilitate research on physiology and ecology of the organism and risks to public health.

It is recommended that a high priority be placed on the chemical characterization of the toxin(s) associated with P. piscicida and Pf-complex, and the development of analytical methods for routine and rapid detection and measurement of the toxins in a variety of matrices.

Following this goal, important issues that need to be addressed include:

(i) identification of the environmental factors that simulate and govern toxin production;

(ii) determination of the biochemical pathways of synthesis and of biosynthetic control mechanisms;

(iii) elucidation of the phylogenetic character and role of endosymbiotic bacteria that are known to be present in TZs;

(iv) evaluation of the susceptibility of finfish and shellfish to the toxins; and

(v) the effects and mode(s) of action of the toxins on humans.

Influences of and Interactions with Fish

Existing studies leave little doubt of a direct correlation between outbreaks of P. piscicida and Pf-like organisms and some sudden kills of certain fish species in the field. There is also little doubt that a wide variety of fish are susceptible to P. piscicida when cultured together at very high densities in captivity. Death and/or the appearance of characteristic lesions among cultured fish is, in fact, the primary evidence of toxin activity. The data suggest some form of chemical signal exuded from fish stimulates toxin production in the laboratory, but it has not been shown that this mechanism occurs in nature.

Because toxin cannot be measured directly, an unfortunate and unavoidable circularity exists in that the presence of fish in culture, fish tissue, or fish secretions/excreta is needed both to stimulate and detect toxin activity, hence the stimulus cannot be measured independently of its effect. The proposed triggering mechanism to initiate toxicity (fish, fish tissue or exudate), as observed in the laboratory, is not completely explanatory, as fish, including Atlantic menhaden, are present in the Neuse-Pamlico estuarine systems all year, albeit at reduced densities and biomass during the winter. Therefore, along with other environmental factors noted earlier in the report, fish densities may have threshold effects.

Beyond knowledge of the fish species affected, and the observation that menhaden is the species primarily affected in fish kill events, little is definitively known about the interplay between fish species and P. piscicida in the wild. Laboratory evidence exists to suggest there is a range of susceptibility for fish and shellfish populations in the wild.

The fish kill events that have occurred so far have been so localized that they probably have had little effect on fish population dynamics of coast-wide stocks as a whole; however, sub-lethal effects that influence growth and physiological condition of fish may have long term detrimental consequences that compromise the nursery function of estuaries. In addition, both the species observed in fish kills and those with lesions are not representative samples of all life stages and species comprising estuarine fish communities. Menhaden and certain epibenthic species (e.g., flounder) seem to be most often affected. Bay anchovies in the open water column and silversides near shore are two of the primary forms of finfish biomass in estuaries, but these species are not recorded among those affected.

Thus species appear to vary dramatically in their susceptibility to P. piscicida and Pf-like organisms in the field. For example, in many estuarine systems subject to anthropogenic nutrient enrichment, high phytoplankton biomass occurs most frequently at intermediate salinities (10-20 psu) where the water column is shallow and poorly flushed. There appears to be a correlation between phytoplankton biomass, the abundance of nanophytoflagellates (including cryptophytes), and the abundance of P. piscicida. These are also regions where fish that feed on phytoplankton, such as menhaden, congregate. It may be more than coincidental that menhaden, the primary species observed in fish kills, is the only affected species that filter feeds on and ingests phytoplankton and is also afflicted with lesions that frequently appear near the anus. The location of these lesions might reflect localized responses to the discharges of the toxic remains of ingested P. piscicida cells.

The studies provided to the panel did not address Pfiesteria ingestion as a mode of uptake by fish. It is recommended that rigorous studies be conducted on fish kill events, sublethal effects and the relative vulnerability of different species of fish to exposure to P. piscicida and the Pf-complex.

Quantitative field and laboratory studies (including studies of aquaculture systems which are also susceptible to catastrophic fish kills), are needed to determine the relative vulnerability of fish species and their developmental stages. This will help determine why fish such as menhaden are more susceptible than fish such as anchovies. Such studies should include comprehensive surveys of finfish abundance (larvae, juveniles, and adults) in conjunction with measurements of environmental parameters and the abundance of P. piscicida and Pf-like organisms (as discussed above) from apparently “healthy” areas and from fish-kill zones. They should also address the possibility that there may be chronic, sublethal effects and “ulcerative mycosis” events occurring at such high rates that their impacts may be of greater long term significance than the more dramatic, localized kills.

It is recommended that biochemical indicators of stress be employed together with otolith-based studies of growth rate in impacted versus non-impacted areas. Finally, the pathways by which toxin(s) enter fish (through the epidermis, gills, or by ingesting the toxic zoospores) should be determined.

PUBLIC HEALTH

The effects of P. piscicida, Pf-like organisms and their toxins on human health are unclear. There are reports that P. piscicida produces an airborne toxin(s) that causes symptoms such as short-term memory loss, and that contact with water where P. piscicida, Pf-like organisms, or fish-kills have occurred, can result in the development of skin lesions. Rapid, accurate and cost-effective techniques for the identification of the species involved and the toxins produced will lead to improved ability to establish the causal linkages between P. piscicida, toxin production, and human health effects. Although there is no evidence that P. piscicida affects human health through the ingestion of seafood products, this possibility cannot be ruled out based on the information available.

Epidemiology in North Carolina

To date, the epidemiological data in this area consist of two clinical series (one unpublished) and one cross-sectional study. The clinical series are reports of the symptoms with some objective clinical findings from persons who have been predominantly exposed to water with Pf-like organisms and/or fish kills through their occupational activities. In all these reports there is no consistent case definition, no consistent objective measure of human health effects, and no consistent objective measure of exposure.

In this regard, the utility of epidemiological studies will be greatly enhanced by the availability of reliable and objective measures of exposure and effect (both of which depend on knowledge of the chemical nature of the toxins involved, their vectors, and the mechanism by which they affect human health). For example, if neuropyschological testing becomes the objective measure of human health effects, standardization of these tests must be accomplished. The numbers of subjects must be significantly larger, there should be an evaluation of motivation and emotional concern, and confounding factors such as alcohol and contact with solvents need to be addressed. Without accurate exposure assessment, it is impossible to assemble exposed and unexposed subject groups. Therefore, prospective follow up studies of affected and/or exposed individuals (e.g. occupational groups) over time randomly sampled from exposed and unexposed populations can be performed in the future, but only when exposure markers are available.

It is recommended that reliable and objective measures of exposure and of human health effects must be developed to make epidemiological studies more effective in assessing public health risks. This will be facilitated by knowledge of the chemical nature and toxicology of the toxic agents. There is a need for general epidemiological surveillance to detect any illness due to Pf-related toxins with rapid follow-up testing of any individuals discovered through this surveillance.

In addition, given the public health implications of the toxins produced by P. piscicida and Pf-like organisms, appropriate animal models should be employed to help develop a consistent disease description and objective diagnostic tests. As discussed above, understanding and assessing potential human health risks associated with P. piscicida require that purified and chemically characterized toxins are available to conduct acute and chronic animal studies. In addition, pharmacological studies should be conducted to determine the mode of action of the toxin(s). Treatment modalities will follow the discovery and characterization of the toxin(s) and biomarkers of exposure.

Food Safety

Although a preliminary survey of fish for toxicity has found no significant risk, and there is no current evidence that the toxins produced by P. piscicida and Pf-like organisms are transmitted to humans via their consumption of seafood, this possibility cannot be completely ruled out at this time. The degree and nature of risk to humans from the consumption of finfish or shellfish needs to be established. This is especially important in the case of filter feeders such as menhaden and many species of shellfish, since there is the possibility that they may ingest and store (or concentrate) toxins produced by P. piscicida and Pf-like organisms. There is some evidence that blue crabs may be affected by P. piscicida and Pf-like organisms. Thus, the pathways by which finfish and shellfish are affected and the extent to which they store and concentrate Pf-toxins should be determined.

MANAGEMENT

Water Quality and Fisheries

Aspects of this problem concerned with the nutrition and the effects of nutrient availability on P. piscicida and Pf-like organisms are addressed above. The concern here is the extent to which the sources, pathways of input, and scales of input (rates, variability in time and space) are known in sufficient detail to demonstrate causal relationships between specific land-use activities (e.g., hog and chicken production and the quantitative fate of their waste products). Until this is done, it will be difficult to justify targeting any specific land-use activity for remediation even though the evidence is overwhelming that nutrient enrichment arising from human activities in the watershed is causing coastal eutrophication as expressed by declining water quality, hypoxia, outbreaks of toxic microorganisms, or fish kills. Given current levels of understanding, attempts to manage nutrient loading should be considered for all major sources of both organic and inorganic forms of nitrogen (N) and phosphorus (P). These include: (i) deforestation (releases stored nutrients and reduces nutrient retention efficiency), (ii) human wastes, (iii) artificial fertilizers, (iv) the storage of animal wastes and their use as fertilizer, (v) urbanization, and (vi) atmospheric deposition.

Public Health and Education

There has been considerable discussion, publicity and misinformation concerning the human health effects of P. piscicida and Pf-like organisms. In the future, educational efforts should be directed at a variety of target groups including seafood consumers, fishing industry, healthcare professionals, and the media. The information should be available in appropriate educational level format with up-to-date factual information. Evaluation studies of the effectiveness of these educational efforts should be performed in the various target populations.

Observational and communication networks should be established to provide continual monitoring for both P. piscicida and Pf-like organisms, the presence of toxins, fish with lesions and fish kills in support of epidemiological studies. An active surveillance system of human health events must be instituted when and where Pf-like events occur. To this end, a consistent case definition with objective measures of disease needs to be developed and publicized to the medical and public health community. Samples of seafood and human bodily fluids should be collected and stored pending the development of toxin assays.

It is recommended that, in collaboration with agencies from other affected States and the Centers for Disease Control (CDC), high priority should be given to the preparation of educational material to inform those from the fishing industry, healthcare professionals including state and public health departments, the tourist industry, seafood consumers and the media of P. piscicida and its biological effects.

APPENDIX A
Reference Material

The following list of current reports on Pfiesteria was provided to the panel members on or before the workshop of December 4-5, 1997. These reports were used by the panel to review the current state of the science and make their recommendations.

• Baden, D.G., L.E. Fleming, and J.A. Bean. 1995. Marine Toxins. Handbook of Clinical Neurology 21(65):141-175.

• Burkholder, JoAnn. 1997. Pfiesteria/Pfiesteria-like Identification. Memorandum provided directly to panel members reviewing her research efforts from 1991 to the present including a list of references. December 3, 1997.

• Burkholder, JoAnn M. 1997. “Pfiesteria piscicida and Other Toxic Pfiesteria-Like Dinoflagellates.” Limnology & Oceanography - Special Edition. In press.

• Burkholder, JoAnn M. and Howard B. Glasgow Jr. 1997. Pfiesteria piscicida and Pfiesteria-Like Dinoflagellates: Behavior, Impacts, and Environmental Controls. Limnology & Oceanography. In press.

• Burkholder, JoAnn M., H.B. Glasgow, and A.J. Lewitus. 1997. Physiological Ecology of Pfiesteria piscicida with General Comments on “Ambush-Predator” Dinoflagellates. Harmful Marine Phytoplankton. UNESCO, Paris, France. In press.

• Burkholder, J.M., H.B. Glasgow, and C.W. Hobbs. 1995. “Fish Kills Linked to a Toxic Ambush-Predator Dinoflagellate: Distribution and Environmental Conditions.” Mar. Ecol. Prog. Ser. 124:43-61.

• Burkholder, J.M., and H.B. Glasgow. 1995. “Interactions of a Toxic Estuarine Dinoflagellate with Microbial Predators and Prey.” Arch. Protistenkd. 145:177-188.

• Burkholder, J.M., E.J. Noga, C.H. Hobbs, and H.B. Glasgow Jr. 1992. “New ‘Phantom’ Dinoflagellate is the Causative Agent of Major Estuarine Fish Kills.” Nature 258:407-410.

• The Cambridge Consensus. 1997. Forum on Land-Based Pollution and Toxic Dinoflagellates in Chesapeake Bay. Center for Environmental Science, University of Maryland. Cambridge, MD.

• Ecology and Oceanography of Harmful Algal Blooms. 1997. An Interagency Research Program: Call for Proposals. National Center for Environmental Research and Quality Assurance. Office of Research and Development, Environmental Protection Agency. December 2, 1997.

• Fleming, L., J.A. Bean, and D.G. Baden. 1995. “Epidemology and Public Health.” In: Manual on Harmful Marine Microalgae. Manual and Guide No. 33. UNESCO, Paris, France.

• Glasgow Jr., H.B., J.M. Burkholder, D.E. Schmechel, P.A. Tester, and P.A. Rublee. 1995. “Insidious Effects of a Toxic Estuarine Dinoflagellate on Fish Survival and Human Health.” J. Toxicol. & Environ. Health. 46:501-522.

• Griffith, D., A. Shechter, K. Borre, and V. Kelly. 1997. An Exploratory Study of Potential Human Health Effects of Deteriorating Water Quality Among North Carolina Crabbers. Draft report submitted to the UNC Sea Grant College Program. Raleigh, NC.

• Levin, E.D., D.E. Schmechel, J.M. Burkholder, H.B. Glasgow Jr., N.J. Deamer-Melia, V.C. Moser, and G.J. Harry. 1997. Persisting Learning Deficits in Rats After Exposure to Pfiesteria piscicida. Environ. Health Perspectives. 105(12). In press.

• Lewitus, A.J. 1997. Cleptoplastidy in the Toxic Dinoflagellate Pfiesteria piscicida. Baruch Marine Field Laboratory, University of South Carolina, Georgetown, SC. Submitted to Nature.

• McClellan-Green, P.D., L.A. Jaykus, and D.P. Green. 1997. Consumer Health Risks Due to Incidental Exposure of Fish to Pfiesteria piscicida. Draft report submitted to UNC Sea Grant College Program. Raleigh, NC.

• Morris, J.G., P. Charache, L.M. Grattan, M.H. Lowitt, D. Oldach, T.M. Perl. 1997. Medical Evaluation of Persons with Exposure to Water Containing Pfiesteria or Pfiesteria-Like Dinoflagellates. Interim Report. University of Maryland School of Medicine. Baltimore, MD.

• National Harmful Algal Bloom Research and Monitoring Strategy: An Initial Focus on Pfiesteria, Fish Lesions, Fish Kills and Public Health. 1997. Prepared by: U.S. Depatment of the Interior, Centers for Disease Control and Prevention, U.S. Food and Drug Administration, U.S. Department of Agriculture, U.S. Environmental Protection Agency, National Oceanic and Atmospheric Administration, and National Institute for Environmental Health Sciences. November 10, 1997.

• Paerl, H.W. and J.L. Pinckney. 1997. Environmental Control of Pfiesteria Outbreaks: The Role of Anthropogenic Nutrient Loading. Draft report submitted to UNC Sea Grant College Program. Raleigh, NC.

• Prasad, S.M., B. Sherman, and P.R. Epstein. 1997. Measuring the Economic Damages of Pfiesteria piscicida. Center for Health and the Global Environment. Harvard University. Boston, MA.

• Ramsall, D. 1997. Overview of NOAA-NMFS Charleston Laboratory objectives concerning Pfiesteria toxins. NOAA Charleston Laboratory - Marine Toxins Program. Charleston, SC.

• Rublee, P.A. 1997. Gene Probe Development to Pfiesteria piscicida. A review of his current project. Also included are two thesis abstracts and an abstract from a draft report submitted to UNC-Water Resources Research Institute. Raleigh, NC.

• Steidinger, K.A., J.M. Burkholder, H.B. Glasgow Jr., C.W. Hobbs, J.K. Garrett, E.W. Truby, E.J. Noga, and S.A. Smith. 1996. “Pfiesteria piscicida Gen. Et. Sp. Nov. (Pfiesteriaceae Fam. Nov.), A New Toxic Dinoflagellate with a Complex Life Cycle and Behavior.” J. Phycol. 32:157-164.

• Steidinger, K.A., J.H. Landsberg, and E.W. Truby. 1997. Identification and Taxonomy of Pfiesteria piscicida, Pfiesteria species, and Morphologically Similar Species. Review of current work provided directly to panel by researchers. December 3, 1997.

• Toffer, K.L. E.L. Schaefer, H.B. Glasgow Jr., J.M. Burkholder, and P.A. Rublee. 1997. Ribosomal DNA from the Toxic Dinoflagellate Pfiesteria piscicida. In: Harmful Microalgae, UNESCO, Paris, France.

Dr. Dean W. Ahrenholz - National Oceanic and Atmospheric Administration

Dr. H. Kay Austin - U.S. Environmental Protection Agency

Dr. Edward J. Carpenter - State University of New York

Dr. S. Craig Cary - University of Delaware

Dr. David Conover - State University of New York

Dr. Jeffrey M. Farber - Health Canada

Dr. Lora E. Fleming - University of Miami School of Medicine

Dr. Sherwood Hall - Food and Drug Administration

Dr. Susan S. Kilham - Drexel University

Dr. Thomas C. Malone - University of Maryland

Dr. Sandra E. Shumway - University of Long Island

Dr. Theodore J. Smayda - University of Rhode Island

Dr. F.J.R. Taylor - University of British Columbia

Dr. Jeffrey L. Wright - National Research Council of Canada

Dr. JoAnn Burkholder, North Carolina State University

Dr. Larry Crowder, Duke University

Dr. Paul Epstein, Harvard University

Dr. Edward Levin, Duke University

Dr. Alan Lewitas, University of South Carolina

Dr. Patricia McClellan-Green, Duke University

Dr. Mike Mallin, University of North Carolina at Wilmington

Dr. Peter Moeller, National Oceanic and Atmospheric Administration

Dr. Ed Noga, North Carolina State University

Dr. Hans Paerl, University of North Carolina at Chapel Hill

Dr. Jay Pinckney, University of North Carolina at Chapel Hill

Dr. John Ramsdell, National Oceanic and Atmospheric Administration

Dr. Kathleen Rein, University of Miami

Dr. Parke Rublee, University of North Carolina at Greensboro

Dr. Francis Schmitz, University of Oklahoma

Dr. Karen Steidinger, Florida Department of Environmental Protection

Dr. Pat Tester, National Oceanic and Atmospheric Administration

Dr. Kevin Seller, National Oceanic and Atmospheric Administration

Dr. Don Boesch, University of Maryland

Richard Whisnant, N.C. Department of Environment and Natural Resources

Dr. Robert E. Holman, Water Resources Research Institute

Marion Smith, N. C. Department of Environment and Natural Resources

Jeri Gray, Water Resources Research Institute

Dr. Kenneth H. Reckhow, Water Resources Research Institute

Panel

Dr. Dean W. Ahrenholz is a research fisheries biologist with the National Oceanic and Atmospheric Administration. For the past 23 years he has characterized the population biology and performed stock assessment on Atlantic and Gulf menhaden.

Dr. H. Kay Austin is a microbial ecologist with the National Center for Environmental Assessment, part of the Environmental Protection Agency. Her expertise is in environmental assessment of ecosystems and their ecology. Dr. Austin’s recent work has been an assessment of the impact exotic shrimp viruses have on wild shrimp and other non-shrimp estuarine species.

Dr. Edward J. Carpenter is a professor in the Marine Science Research Center located at the State University of New York at Stony Brook. He is an aquatic ecologist with expertise concerning the biology and ecology of estuarine/marine phytoplankton. His main interest lies in nutrient uptake & cycling, species-specific growth rates, primary production and species growth rates in nature.

Dr. S. Craig Cary is a professor in the College of Marine Studies located at the University of Delaware. He is a molecular biologist with expertise in comparative physiology, biochemistry and ecology of marine microbial communities. His current efforts are the development of molecular based diagnostic techniques to better assess microbial community structure and function in marine systems.

Dr. David Conover is a professor in the Marine Science Research Center located at the State University of New York at Stony Brook. He is a fisheries biologist with expertise in fish management and fish life history. Currently, he is on sabbatical at Florida State University as the Mote Eminent Scholar Chair and Visiting Professor.

Dr. Jeffrey M. Farber is a microbiologist with Health Canada located in Ottawa. His expertise is in applied food microbiology, dealing with conditions leading to the survival, growth and toxin production of pathogenic bacteria in foods.

Dr. Lori E. Fleming is a professor in the Department of Epidemiology and Public Health at the University of Miami. She is an occupational/ environmental health physician, with expertise in clinical and epidemiological issues related to marine toxin diseases and human health.

Dr. Sherwood Hall is a marine chemist with the Food and Drug Administration. His expertise is in evaluating seafood toxins and verifying shellfish safety.

Dr. Susan S. Kilham is a professor in the Department of Biosciences and Biotechnology located at Drexel University. Her expertise is in phytoplankton ecology, physiology and microalgal nutritional ecology. Dr. Kilham’s recent research is determining a link between climate change and diatom sedimentary assemblages.

Dr. Thomas C. Malone is Professor and Director of the Horn Point Laboratory that is part of the University of Maryland. He is an aquatic ecologist with expertise in phytoplankton ecology and is current President of the American Society of Limnology and Oceanography.

Dr. Sandra E. Shumway is a professor in the Natural Sciences Division of Southampton College, part of Long Island University. She is an aquatic ecologist and her expertise is in the fields of invertebrate physiology, shellfish biology and impact of harmful algae on shellfish, public health and industry.

Dr. Theodore J. Smayda is a professor in the Graduate School of Oceanography located at the University of Rhode Island. He is a physiological ecologist with expertise in coastal phytoplankton dynamics, mesocosm systems and harmful algal bloom ecology.

Dr. F.J.R. Taylor is a professor in the Department of Earth, Ocean and Sciences located at the University of British Colombia. He is a phytoplankton taxonomist with expertise in the identification of harmful algal blooms and their ecology. Dr. Taylor is a Fellow of the Royal Society of Canada and President of the International Society for the Study of Harmful Algae.

Dr. Jeffrey L. Wright is a biochemist with the Institute of Marine Biosciences, part of the National Research Council of Canada located in Nova Scotia. His expertise is isolation, characterization and chemistry of new marine toxins and the genetics and biochemistry of toxin production in marine microbes. Dr. Wright was chair of the workshop panel.