JoAnn M. Burkholder

Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences.

Although algal blooms, including those considered toxic or harmful, can be natural phenomena, the nature of the global problem of harmful algal blooms (HABs) has expanded both in extent and its public perception over the last several decades. Of concern, especially for resource managers, is the potential relationship between HABs and the accelerated eutrophication of coastal waters from human activities. We address current insights into the relationships between HABs and eutrophication, focusing on sources of nutrients, known effects of nutrient loading and reduction, new understanding of pathways of nutrient acquisition among HAB species, and relationships between nutrients and toxic algae. Through specific, regional, and global examples of these various relationships, we offer both an assessment of the state of understanding, and the uncertainties that require future research efforts. The sources of nutrients potentially stimulating algal blooms include sewage, atmospheric deposition, groundwater flow, as well as agricultural and aquaculture runoff and discharge. On a global basis, strong correlations have been demonstrated between total phosphorus inputs and phytoplankton production in freshwaters, and between total nitrogen input and phytoplankton production in estuarine and marine waters. There are also numerous examples in geographic regions ranging from the largest and second largest U.S. mainland estuaries (Chesapeake Bay and the Albemarle-Pamlico Estuarine System), to the Inland Sea of Japan, the Black Sea, and Chinese coastal waters, where increases in nutrient loading have been linked with the development of large biomass blooms, leading to anoxia and even toxic or harmful impacts on fisheries resources, ecosystems, and human health or recreation. Many of these regions have witnessed reductions in phytoplankton biomass (as chlorophylla) or HAB incidence when nutrient controls were put in place. Shifts in species composition have often been attributed to changes in nutrient supply ratios, primarily N∶P or N∶Si. Recently this concept has been extended to include organic forms of nutrients, and an elevation in the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC∶DON) has been observed during several recent blooms. The physiological strategies by which different groups of species acquire their nutrients have become better understood, and alternate modes of nutrition such as heterotrophy and mixotrophy are now recognized as common among HAB species. Despite our increased understanding of the pathways by which nutrients are delivered to ecosystems and the pathways by which they are assimilated differentially by different groups of species, the relationships between nutrient delivery and the development of blooms and their potential toxicity or harmfulness remain poorly understood. Many factors such as algal species presence/abundance, degree of flushing or water exchange, weather conditions, and presence and abundance of grazers contribute to the success of a given species at a given point in time. Similar nutrient loads do not have the same impact in different environments or in the same environment at different points in time. Eutrophication is one of several mechanisms by which harmful algae appear to be increasing in extent and duration in many locations. Although important, it is not the only explanation for blooms or toxic outbreaks. Nutrient enrichment has been strongly linked to stimulation of some harmful species, but for others it has not been an apparent contributing factor. The overall effect of nutrient over-enrichment on harmful algal species is clearly species specific.

Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates

ABSTRACT Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far,Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteriaspecies, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugols solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.

Impacts of Waste from Concentrated Animal Feeding Operations on Water Quality.

Waste from agricultural livestock operations has been a long-standing concern with respect to contamination of water resources, particularly in terms of nutrient pollution. However, the recent growth of concentrated animal feeding operations (CAFOs) presents a greater risk to water quality because of both the increased volume of waste and to contaminants that may be present (e.g., antibiotics and other veterinary drugs) that may have both environmental and public health importance. Based on available data, generally accepted livestock waste management practices do not adequately or effectively protect water resources from contamination with excessive nutrients, microbial pathogens, and pharmaceuticals present in the waste. Impacts on surface water sources and wildlife have been documented in many agricultural areas in the United States. Potential impacts on human and environmental health from long-term inadvertent exposure to water contaminated with pharmaceuticals and other compounds are a growing public concern. This work-group, which is part of the Conference on Environmental Health Impacts of Concentrated Animal Feeding Operations: Anticipating Hazards—Searching for Solutions, identified needs for rigorous ecosystem monitoring in the vicinity of CAFOs and for improved characterization of major toxicants affecting the environment and human health. Last, there is a need to promote and enforce best practices to minimize inputs of nutrients and toxicants from CAFOs into freshwater and marine ecosystems.

Insidious effects of a toxic estuarine dinoflagellate on fish survival and human health

The estuarine dinoflagellate Pfiesteria piscicida gen. et sp. nov. produces exotoxin(s) that can be absorbed from water or fine aerosols. Culture filtrate (0.22 microns porosity filters, > 250 toxic flagellated cells/ml) induces formation of open ulcerative sores, hemorrhaging, and death of finfish and shellfish. Human exposure to aerosols from ichthyotoxic cultures (> or = 2000 cells/ml) has been associated with narcosis, respiratory distress with asthma-like symptoms, severe stomach cramping, nausea, vomiting, and eye irritation with reddening and blurred vision (hours to days); autonomic nervous system dysfunction [localized sweating, erratic heart beat (weeks)]; central nervous system dysfunction [sudden rages and personality change (hours to days), and reversible cognitive impairment and short-term memory loss (weeks)]; and chronic effects including asthma-like symptoms, exercise fatigue, and sensory symptoms (tingling or numbness in lips, hands, and feet; months to years). Elevated hepatic enzyme levels and high phosphorus excretion in one human exposure suggested hepatic and renal dysfunction (weeks); easy infection and low counts of several T-cell types may indicate immune system suppression (months to years). Pfiesteria piscicida is euryhaline and eurythermal, and in bioassays a nontoxic flagellated stage has increased under P enrichment (> or = 100 micrograms SRP/L), suggesting a stimulatory role of nutrients. Pfiesteria-like dinoflagellates have been tracked to fish kill sites in eutrophic estuaries from Delaware Bay through the Gulf Coast. Our data point to a critical need to characterize their chronic effects on human health as well as fish recruitment, disease resistance, and survival.

PFIESTERIA PISCICIDA GEN. ET SP. NOV. (PFIESTERIACEAE FAM. NOV.), A NEW TOXIC DINOFLAGELLATE WITH A COMPLEX LIFE CYCLE AND BEHAVIOR

The newly described toxic dinoflagellate Pfiesteria piscicida is a polymorphic and multiphasic species with flagellated, amoeboid, and cyst stages. The species is structurally a heterotroph; however, the flagellated stages can have cleptochloroplasts in large food vacuoles and can temporarily function as mixotrophs. The flagellated stage has a typical mesokaryotic nucleus, and the theca is composed of four membranes, two of which are vesicular and contain thin plates arranged in a Kofoidian series of Po, cp, X, 4′, 1a, 5″, 6c, 4s, 5″′, and 2″″. The plate tabulation is unlike that of any other armored dinoflagellate. Nodules often demark the suture lines underneath the outer membrane, but fixation protocols can influence the detection of plates. Amoeboid benthic stages can be filose to lobose, are thecate, and have a reticulate or spiculate appearance. Amoeboid stages have a eukaryotic nuclear profile and are phagocytic. Cyst stages include a small spherical stage with a honeycomb, reticulate surface and possibly another stage that is elongate and oval to spherical with chrysophyte‐like scales that can have long bracts. The species is placed in a new family, Pfiesteriaceae, and the order Dinamoebales is emended.

KLEPTOPLASTIDY IN THE TOXIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)

The ichthyotoxic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder has a complex life cycle with several heterotrophic flagellated and amoeboid stages. A prevalent flagellated form, the nontoxic zoospore stage, has a proficient grazing ability, especially on cryptophyte prey. Although P. piscicida zoospores lack the genetic capability to synthesize chloroplasts, they can obtain functional chloroplasts from algal prey (i.e. kleptoplastidy), as demonstrated here with a cryptophyte prey. Zoospores grown with Rhodomonas sp. Karsten CCMP757 (Cryptophyceae) grazed the cryptophyte population to minimal densities. After placing the cultures in near darkness where cryptophyte recovery was restricted and further prey ingestion did not occur, the time‐course patterns in growth, prey chloroplast content·zoospore−1, and prey nucleus content·zoospore−1 were followed. Ingested chloroplasts were selectively retained in the dinoflagellate, as indicated by the decline and, ultimately, near absence of cryptophyte nuclei in plastid‐containing zoospores. Chloroplasts retained inside P. piscicida cells for at least a week were photosynthetically active, as indicated by starch accumulation and microscope‐autoradiographic measurements of bicarbonate uptake. Recognition that P. piscicida can function as a phototroph broadens our perspective of the physiological ecology of the dinoflagellate because it suggests that, at least during part of its life cycle, P. piscicida’s growth and survival might be affected by photoregulation and nutritional control of photosynthesis.

Characterization of the affinity for nitrogen, uptake kinetics, and environmental relationships for Prorocentrum minimum in natural blooms and laboratory cultures

During the late spring and early summer of 1998, an extensive bloom of the dinoflagellate Prorocentrum minimum (>93% of phytoplankton cell density) developed in several tributaries of the Chesapeake Bay, USA. In January 1999, a bloom of mixed dinoflagellates (Heterocapsa rotundata, H. triquetra and P. minimum, with P. minimum forming 21% of total phytoplankton cells and 39% of the total biovolume) developed in the mesohaline Neuse Estuary, North Carolina, USA. During these blooms, experiments were carried out to characterize the nitrogen uptake kinetics of these assemblages with isotopic techniques. Four nitrogenous substrates (NO3−, NH4+, urea, and a mixed amino acids substrate) were used to determine uptake rate and substrate preference. Rates of nitrogen uptake were also measured in P. minimum cultures grown on varying growth nitrogen substrates. The calculated kinetic parameters determined for the P. minimum-dominated field assemblages and the cultures indicated a preference for NH4+. NH4+ was also the primary nitrogen source supporting the blooms. In addition, a high affinity for urea was also found, and urea contributed significantly to the Neuse Estuary bloom. Furthermore, results showed that the regulation of uptake for each of the substrates was different: strong positive relationships between affinity and temperature were found for NH4+ and amino acids, while a negative response was found for NO3−, and very little response to temperature was noted for urea. These differences suggest that a diversity of nitrogen uptake mechanisms may aid the development and maintenance of P. minimum blooms.

WATER QUALITY TRENDS AND MANAGEMENT IMPLICATIONS FROM A FIVE‐YEAR STUDY OF A EUTROPHIC ESTUARY

The Neuse River and Estuary, a major tributary of the second largest estuary on the United States mainland, historically has sustained excessive blooms of algae and toxic dinoflagellates, hypoxia, and fish kills. Previous attempts have been made to use short-term databases of 2–3 years, or data sets from infrequent (monthly) sampling, to assess whether nutrient inputs to the Neuse are increasing and supporting higher algal production. These previous efforts also have relied on single-point-determined flow velocity data, at upstream sites remote from the estuary, to estimate the volume of flow in quantifying nutrient loading to the estuary. We completed a five-year study of the Neuse, including a comparative inventory of nutrients to the watershed from point sources and from concentrated animal operations (CAOs) as recent nonpoint sources, as well as an intensive assessment of water quality over time in the mesohaline estuary. Estimates of nutrient loads were based on volume of flow data from shore-to-shor...

Overview and present status of the toxic Pfiesteria complex (Dinophyceae)

Abstract This paper reviews the Pfiesteria issue and Pfiesteria science and presents new information on variation in toxicity among Pfiesteria strains, culture effects on their toxicity, the trophic interactions of Pfiesteria spp ., and impacts on fish and mammals. We also assess Pfiesteria spp. impacts on fish in comparison to certain other estuarine dinoflagellates of similar appearance. Species of the toxic Pfiesteria complex (TPC) thus far include P. piscicida and P. shumwayae. These species share morphological and genetic similarities, and both have toxic strains that (1) show strong attraction to live fish;(2) exhibit toxicity that is triggered by live fish or their fresh tissues and excreta; and (3) produce toxin(s) that cause fish stress, disease and death under ecologically relevant conditions (the standardized fish bioassay process involves testing live Pfiesteria cells at similar densities to those encountered during Pfiesteria-related fish kill/disease events). Both Pfiesteria species also have a complex life cycle with multiple amoeboid, flagellated and cyst stages, several of which are ichthyotoxic. TPC species are eurythermal and euryhaline, with prey spanning the estuarine food web, from bacteria to mammalian tissues. They can be stimulated directly or indirectly by nitrogen and phosphorus enrichment. Toxic strains can be either actively or potentially toxic (the TOX-A and TOX-B functional types, respectively); in addition, c.40% of randomly isolated clones have been found to be benign [the noninducible or NON-IND functional type, which apparently lacks the ability to produce bioactive substances (toxins) that cause fish disease or death]. These functional types differ significantly in response to algal prey, predators, nutrients and fish. Moreover, as an apparent artifact of culture conditions, toxic strains generally lose their ability to cause fish death and disease and become NON-IND within weeks to months. At low cell densities, toxic strains can be causative agents of acute and/or chronic diffuse and focal lesions and of other fish diseases, as demonstrated in fish bioassays. A partially purified, water-soluble Pjiesteria toxin disrupts calcium metabolism in rat pituitary cells and mimics an adenosine triphosphate neurotransmitter that targets P2X7 purinoreceptors found predominantly on immune cells. Respiratory, visual, and neurological impacts have been sustained by people exposed to aerosols from fish-killing Pfiesteria cultures or to water and aerosols during estuarine fish kills associated with toxic Pfiesteria. Neurocognitive impacts from exposure to toxic Pfiesteria have been replicated experimentally in small mammals. Toxic strains of Pfiesteria species have been confirmed from mid-Atlantic and Gulf Coast estuaries in the United States and from northern Europe and New Zealand, indicating that these toxic dinoflagellates are cosmopolitan in distribution.

North and South Carolina coasts

Abstract This coastal region of North and South Carolina is a gently sloping plain, containing large riverine estuaries, sounds, lagoons, and salt marshes. The most striking feature is the large, enclosed sound known as the Albemarle–Pamlico Estuarine System, covering approximately 7530 km 2 . The coast also has numerous tidal creek estuaries ranging from 1 to 10 km in length. This coast has a rapidly growing population and greatly increasing point and non-point sources of pollution. Agriculture is important to the region, swine rearing notably increasing fourfold during the 1990s. Estuarine phytoplankton communities in North Carolina are well studied; the most important taxonomic groups are diatoms, dinoflagellates, cryptomonads and cyanobacteria. Several major poorly flushed estuaries are eutrophic due to nutrient inputs, and toxic dinoflagellates ( Pfiesteria spp) can reach high densities in nutrient-enriched areas. Fully marine waters are relatively oligotrophic. Southern species enter in subsurface intrusions, eddies, and occasional Gulf Stream rings, while cool water species enter with the flow of the Labrador Current to the Cape Hatteras region. The Carolinas have a low number of endemic macroalgae, but species diversity can be high in this transitional area, which represents the southernmost extension for some cold-adapted species and the northernmost extension of warm-adapted species. In North Carolina the dominant seagrass, Zostera marina , lies at its southernmost extension, while a second species, Halodule wrightii is at its northernmost extent. Widgeon-grass Ruppia maritima is common, growing in brackish water or low-salinity pools in salt marshes. Seagrass meadows are now much reduced, probably due to elevated nitrogen and increased sedimentation. In sounds, numerically dominant benthic taxa include bivalves, polychaetes and amphipods, many showing gradients in community type from mesohaline areas of the eastern shore to near marine salinities in western parts. The semi-enclosed sounds have extensive shellfisheries, especially of blue crab, northern quahogs, eastern oysters, and shrimp. Problems include contamination of some sediments with toxic substances, especially of metals and PCBs at sufficiently high levels to depress growth of some benthic macroinvertebrates. Numerous fish kills have been caused by toxic Pfiesteria outbreaks, and fish kills and habitat loss have been caused by episodic hypoxia and anoxia in rivers and estuaries. Oyster beds currently are in decline because of overharvesting, high siltation and suspended particulate loads, disease, hypoxia, and coastal development. Fisheries monitoring which began in the late 1970s shows greatest recorded landings in 1978–1982; since then, harvests have declined by about a half. Some management plans have been developed toward improving water quality and fisheries sustainability. Major challenges include; high coliform levels leading to closures of shellfish beds, a problem that has increased with urban development and increasing cover of watershed by impervious surfaces; high by-catch and heavy trawling activity; overfishing which has led to serious declines in many wild fish stocks; and eutrophication. Comprehensive plans limiting nutrient inputs are needed for all coastal rivers and estuaries, not only those that already exhibit problems. There is a critical need to improve management of non-point nutrient runoff through increased use of streamside vegetated buffers, preservation of remaining natural wetlands and construction of artificial wetlands. Improved treatment processes, based on strong incentive programmes, should also be mandated for present and future industrial-scale animal operations.