FEATURE
What Is Causing Toxic Algal Blooms? New research is providing clues about possible links between Pfiesteria-like
organisms and pollution.
JANET PELLEY
I
n August 1997, 30,000 fish with open sores died in Maryland's Pocomoke River on the Chesapeake Bay. More alarmingly, fishermen, waterskiers, and field researchers who came into contact with the estuary's shallow, nutrient-rich waters became sick with symptoms that included memory loss, shortness of breath, and skin rashes. A panel of scientists advised state officials that the problems in the Pocomoke and two other rivers were caused by toxic dinoflagellates similar to Pfiesteria, which had been cited in fish kills in North Carolina. At least two of the organisms are new to science. The outbreak led to high-level attention from the Clinton administration, Congress, and governors of the mid-Adantic states and culminated in congressional hearings, $7 million in new research funds, and legislative proposals to regulate animal waste—the presumed source of nutrients that had stimulated growth of the organisms. However, scientists remain cautious in their assessment of what causes such harmful algal blooms. Pfiesteria-like organisms can assume several life forms, only some of which are toxic, and they have complex life cycles that are poorly understood. New analytical and monitoring technology aimed at elucidating the nature and ecology of the algae is under development, and already some clues about Pfiesteria and other harmful agal blooms and methods for their control sxe emerging. Most scientists agree that harmful algal blooms have increased in frequency, size, geographic extent, and species complexity across the globe. "What scientists don't agree about," said Don Anderson, a senior scientist at Woods Hole Oceanographic Institution, "are the causes of this expansion." Increased research interest in the field led to the formation in June 1997 of the International Society for the Study of Harmful Algae; and a new U.S. federal 2 6 A • JAN. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
program, Ecology and Oceanography of Harmful Algal Blooms (ECOHAB), has been launched by the National Oceanic Atmospheric Administration (NOAA), the Office of Naval Research, and the National Science Foundation to foster a national research agenda. Harmful algal blooms expand globally Maryland's Pfiesteria-like dinoflagellate outbreak heightened concern about toxic algae and boosted research funding, partly because of its proximity to the nation's capitol. However, Pfiesteria is just one of many harmful algae species whose blooms, referred to as "red tides" in marine and estuarine waters, have increased in size and frequency worldwide over the past 20 years. Two years ago, a bloom of the toxic dinoflagellate Gymnodinium breve killed 141 manatees on Florida's Gulf Coast. Gymnodinium blooms, whose brevetoxins cause neurotoxic shellfish poisoning, recur annually at the end of summer and precipitate economic losses of $18 million to $24 million per episode (i). In 1987, Canadian officials reported 3 deaths and more than 150 cases of acute poisoning in humans from ingesting Prince Edward Island mussels contaminated with domoic acid from the diatom Pseudo-nitzschia multiseries. Scientists coined a new term, amnesic shellfish poisoning, for the victims' permanent short-term memory loss, which had never been observed before (2). Fish kills since 1986 in the Pacific Northwest Losses to fish farmers total about $4 million to $5 million per year (n The single-celled algae that produce these toxic outbreaks consist of species of dinoflagellates, raphidophytes, and diatoms. There are about 85 toxic microalgae worldwide. Their toxins can poison through direct contact and ingestion or by passing through the food 0013-936X/98/0932-26AS15.00/0 © 1997 American Chemical Society
chain from fish and shellfish to carnivFIGURE 1 orous fish, marine mammals, and humans. Symptoms—not all associated Expansion of U.S. hazardous algal blooms with each syndrome—include shortThe number of coastal areas with major, recurring blooms of harmful algal species more than doubled between 1972 and 1995. ness of breath, dizziness, diarrhea, disorientation, and short-term memory loss. Harmful algal blooms are not a new phenomenon. They have occurred globally throughout human history and are recorded in the Bible and the fossil record. They have had a major effect on marine food webs by killing wild and farmed fish and producing neurotoxins that accumulate in shellfish. What is new about these phenomena is their explosion (Figure 1) in recent years (3). Worldwide, areas with outbreaks of paralytic shellfish poisoning more tiian doubled between 1970 and 1990 (2). Although many scientists have suggested that the recent explosion of blooms is due to nutrient pollution of coastal waters, not all of them agree about the causes of this expansion. One theory is that there are more scientists working in die field widi improved analytical tools. Another proposal is that Source: Adapted from Don Anderson, Woods Hole Oceanographic Institution, as shown at http://habsen/1. whoi/hab/regionalevents/habexpand.html, accessed Oct. 15,1997). toxic algae have been transported across oceans in the ballast water of ships or in shellfish stocks sold for aquaculture. Species may also known to science until 1991 when JoAnn Burkbe dispersed by oceanic currents. holder, a professor of aquatic botany at North Carolina State University, discovered it at afishkill in North Anderson noted that there is a direct correlation Carolina's Pamlico Estuary. This predatory, singlebetween coastal pollution and the number of harmcelled alga witii 24 different life stages is represenful algal blooms in certain locations (2, 4). Altative of a new family, genus, and species of dinothough it seems obvious that nutrient pollution of flagellates. Nontoxic llfe forms of Pfiesteria oo rther coastal waters could increase die production of toxic closely related taxa have been found in estuaries from algal species, Anderson said, "Most of us believe that Delaware to Alabama (5). these problems are very subde and difficult to prove." Pfiesteria piscicida is a heterotroph—it cannot make its own food through photosynthesis—and Pfiesteria blooms linked to nutrient pollution In the case of recent blooms of Pflesteria-\ike spe- feeds on other phytoplankton, bacteria, and zooplankton. Pfiesteria'^ nontoxic forms snclude cysts shat cies on the mid-Atlantic U.S. coast, however, scienremain dormant in sediments, amoebae, and flagtists in North Carolina, Maryland, and South Caroellated zoospores. However, unknown substances exlina are making a strong argument for nutrient creted by schools of fish can stimulate diese nonpollution as a major cause. Massive fish kills assotoxic stages to transform into toxic zoospores. The ciated with algal blooms have occurred along wazoospores release toxins that drug fish and cause tersheds that are heavily polluted by hog and chicken open, bleeding sores. Pfiesteria then feeds on fish tisfarms and municipal sewage and in aquaculture imsue and blood. Within hours of the fish kill, Pfiespoundments. Although circumstantial evidence imteria reverts to a nontoxic amobae or cyst form (5). plicates nutrient pollution as a cause of the blooms, a firm link has been difficult to establish because of "Burkholder's experiments in the lab provide the ephemeral and complex nature of Pfiesteriastrong evidence of nutrient stimulation of Pfieslike dinoflagellates. teria," said Alan Lewiius, an assistant professor at the University of South Carolina. Burkholder has demPfiesteria-like species have probably existed for millions of years, but Pfiesteria piscicida—the only onstrated stimulation of Pfiesteria growth by orcurrendy described Pfiesteria species—remained un- ganic and inorganic forms of nitrogen and phosphoJAN. 1, 1998/ENVIRONMENTAL SCIENCE & TECHNOLOGY/NEWS " 2 7 A
port the nutrient hypothesis." He cautioned, however, "Although the nutrient hypothesis is tempting, it hasn't been proven." Don Boesch, president of the University of Maryland's Center for Environmental Science, agreed: "There is enough evidence to indict, but not yet convict." Research receives technology boost Pfiesteria piscieida and look-alikes ara difficulu to study in the field and are even hard to identify under a scanning electron microscope. Moreover, toxic forms can disappear within a few hours after a fish kill. However, scientists say, field research on Pfiesteria and other harmful algae is on the brink of a breakthrough, thanks to biotechnology and remote sensing. Although momentum has been building in harmful algal bloom research, much more information is needed to manage fisheries, protect public health, and guide public policy, according to Anderson. Among the practical challenges facing researchers are understanding transport by ocean currents, conducting long-term monitoring to study the relationship between anthropogenic changes and blooms, and examining how harmful algae affect organisms other than shellfish. Some of the most serious information gaps in harmful algal bloom ecology stem from the fact thcit species sxe difficult to identify and monitor; and, until a few years ago, remote sensing was expensive and difficult to access quickly. Ideally, understanding the causes of harmful algal blooms will allow scientists to predict and possibly prevent them. "Predicting red tides is like predicting a tornado. We can only indicate the general time they may occur if the region has favorable conditions to start blooms," said Dick Barber, Smith Professor of Biological Oceanography at Duke University. Those conditions, said Barber, are a stable water column with high nutrient levels and a sufficient number of seed organisms to start a bloom. Harmful algal blooms do not always lend themselves to easy observation. The toxic alga may occur in an assemblage of many nontoxic algae, and common measurements cannot distinguish among the speThe life stages of harmful algal blooms can be complicated— cies. Many blooms, such as those of Pfiesteria, are difPfiesteria piscicida has 24 known life stages, only some of ficult to monitor because they can be short in durawhich are toxic, and can assume 3 major forms: cysts (top), tion, and the species can take on many different forms. amoebae (middle), and zoospores (above). (Courtesy JoAnn However, new molecular probes promise to solve these Burkholder, North Carolina State University) problems and help scientists understand what stimulates and maintains the blooms. rus (6, 7). Burkholder's working hypothesis is that Pftesteria-\ike species can nb eirectly stimulated db orParke Rublee, an associate professor of biology at ganic nutrients, occurring in human and animal wastes, the University of North Carolina, developed a DNA because the species feed on these nutrients. probe for Pfiesteria piscieida. The probe consisis of a primer—a short piece of DNA unique to Pfiesteria piLewitus said that while "there has been scant field scicida—mat ts llnked to a ffuorescent dye. The primer testing of nutrient stimulation," there is some circumstantial evidence for the hypothesis. To date, Pfiesteria- will bind only to Pfiesteria cells. When examined uncaused fish kills have all occurred in shallow, nutrient- der a fluorescent microscope, the Pfiesteria celll sfuoresce and are distinguishable from other species. rich estuaries in watersheds dominated by large hog and poultry operations and by municipal sewage. LeAccording to Rublee, "The goal of this molecular witus cited one of Burkholder's experiments that demtool is to identify which species has caused a fish kill onstrated large populations of Pflesteria-]ike species be- and to test the water and sediment to look for cyst low sewage outfalls on the Neuse River. beds. It can also be used to look at geographic distribution and genetic variation." Rublee said the probe According to Dan Terlizzi, a research scientist with is being field-tested now and should be routinely the Maryland Sea Grant program, "The data on oravailable next summer. Because they are fast, cheap, ganic nutrient levels in the Pocomoke River sup2 8 A • JAN. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
and accurate, the probes will enable rapid response monitoring. DNA probes have also been developed for several other algal organisms. Woods Hole's Anderson has developed an antibody probe for the toxic dinoflagellate Alexandrium tamarense. The antibody was obtained by injecting Alexandrium into mammals, then producing antibodies to specific proteins found on the algal cell wall. The mammalian antibodies are attached to very small magnets. Samples of plankton are treated with the antibody probe, which attaches only to Alexandrium. These labeled cells are separated from nonlabeled cells with a magnet. Anderson said antibody probes can also detect algal proteins that are made under different environmental conditions. For instance, he has used the probes in lab cultures to determine whether cells are growth-limited by nitrogen phosphorus, or iron. Another promising technique is the use of reporter gene assays for algal toxins. John Ramsdell, the division chief of the NOAA Marine Biotoxins Program in Charleston, S.C., collaborated with Burkholder, Howard Glasgow, and Peter Moeller to develop a reporter gene assay for one of Pfiesteria's water-soluble toxins. Ramsdell and his colleagues isolated a mammalian gene that is activated only in the presence of Pfiesteria's toxins. They llnked the gene to for luciferase, the enzyme that gives fireflies their bright flashes of light, and inserted it into cells. When the cells 3xe exposed to Pfiesteria's toxins, the mammalian gene is activated and turns on the gene that produces luciferase. Bright flashes of light emanating from the engineered cells tell scientists that Pfiesteria's toxins are present A different assay, the receptor binding assay, has been developed for four other algal toxins—brevetoxin, ciguatera toxin, domoic acid, and saxitoxin. This assay uses proteins that bind to specific algal toxins. A cell preparation containing the receptor proteins and isotopically labeled toxin yields a reduced radioactive signal in die presence of the toxins. It also enables researchers to sample blooms, determine which species is causing it, and detect toxins in human serum and urine. Another molecular tool to detect toxins is the enzyme-linked immunosorbent assay (ELISA). Antibodies to specific algal toxins are linked to enzymes diat change color in the presence of me toxin. The assay is sensitive to the type and amount of toxin, said Daniel Baden, the director of the National Institute for Environmental Health Science Marine and Freshwater Biomedical Sciences Center at the University of Miami. Baden is working on an ELISA assay for Pfiesteria's lipid-soluble toxin. Remote sensing of algal blooms On a larger scale, remote sensing, such as satellite imagery, allows scientists to study harmful algal blooms over larger spatial areas and shorter time scales than is possible by sampling from ships. Because remote sensing data were costly and difficult to receive quickly, NOAAs Coastwatch Program, initiated in 1993, makes this technology accessible and inexpensive for harmful algal bloom researchers (8).
Satellite images of sea-surface temperature, such as this North Carolina coastal image of a Gymnodinium breve bloom, enable scientists to identify water masses that contain the blooms and provide clues about how algal cells migrate in coastal waters. (Courtesy Pat Tester, National Marine Fisheries Service, Beaufort, N.C.)
In general, water masses affected by algal blooms are tracked with sea-surface temperature sensors or multispectral scanners to detect reflectance of chlorophyll a and other pigments. This technique has been used recently to study the sources of blooms in the Gulf of Maine and off the North Carolina coast (9). In November 1987, Gymnodinium breve bloomed off North Carolina's coast for the first recorded time. Before 1987, it had never appeared north of Florida. Pat Tester, a research scientist with the National Marine Fisheries Service in Beaufort, N.C., combined direct water sampling with data from weather buoys and satellite images of sea-surface temperature to discover that the algal cells rode the western edge of the Gulf Stream north from the tip of Florida and remained stranded off North Carolina's coast for 17 days (9). Control efforts underway Whereas U.S. scientists have examined the causes of harmful algal blooms, researchers in Japan, Korea, and China are experimenting in the field with ways to control the blooms. Application of clay to bloominfested waters appears to cause the harmful algae to fall out of the water column into the sediments without harming aquaculture operations. Laboratory experiments and small-scale field trials with clay have demonstrated algal removal efficiencies of 9599% (10). Experiments in China have shown that these efficiencies can be increased by combining clay with coagulants, such as polyhydroxy aluminium chloride {11). According to Anderson, in the summer of 1997, Korea applied clay to 100 square miles of water to stop a bloom of toxic Cochlodinium in an aquaculture area. When 60,000 tons of clay were applied in 1996, the fish farms had only 1% of the losses that had occurred in 1995 when no control methods were used during a bloom. JAN. 1, 1998 /ENVIRONMENTAL SCIENCE S TECHNOLOGY / NEWS " 2 9 A
Scientists unravel the complicated biology of harmful algae by using advanced diagnostic technologies and laboratory techniques. At Woods Hole, Don Anderson (left) and a student inspect a sequencing gel as they search for genes involved in algal toxin production. (Courtesy Tom Leming, Stennis Space Center, Stennis, Miss.)
of several toxic dinoflagellates without harming odier algal species. Doucette stated that these algicidal bacteria either excrete a biologically active compound into the water or adhere to algal cells and excrete enzymes that break down the cell wall. With support from ECOHAB, Doucette has begun to look for bacteria that stop or slow the growth of Gymnodinium breve. He noted mat for this control lsrategy to be successful, it would have to be combined with a strong monitoring and forecasting program. Harmful algal blooms can occupy hundreds, even thousands, of square miles in shelf coastal waters; but, said Doucette, "If harmful algal blooms can be controlled while they are contained in a small embayment, control techniques will be more successful." Barber said that California is one state that has had success in predicting blooms of the toxic diatom Pseudo-nitzschia. Every summer, an upwelllng current off the Pacific coast brings in cool, nutrient-rich water. These are ideal conditions for harmful algal blooms, except for one crucial factor: sunlight. California's foggy summers prevent Pseudo-nitzschia from blooming, but as soon as there are two to three days of sun on die coast, the state closes the shellfish fishery. Anderson said that forecasting harmful algal blooms is hampered in part by "archaic systems where people run along the shore and dig clams and extract toxins." He predicts that in 5-10 years, the current system of sampling buoys and satellite data will be greatiy improved by new automated systems. For instance, sampling buoys currentiy radio data to shore on temperature, salinity, and chlorophyll levels. According to Anderson, scientists are working on developing automated DNA probes for buoys that would also radio in data on algal species' composition and abundance.
References However, such aggressive treatment is controver(1) Woods Hole Oceanographic Institution, The Harmful Alsial in the United States, and scientists admit that the gae Page. The Ecology and Oceanography of Harmful Alprocess is poorly understood. According to Andergal Blooms: A National Research Agenda. Regional HAB son, our level of understanding of the mechanisms Phenomena in the United States, www.redtide.whoi.edu/ underlying harmful algal blooms is nowhere near hab/ (accessed September 1997). (2) Anderson, D. M. Sci. Am. 1994, 271(2), 52. what is needed to protect marine ecosystems and hu(3) Woods Hole Oceanographic Institution, The Harmful Alman health from an expanding array of toxic and gae Page. The Ecology and Oceanography of Harmful Alharmful algae. Recognizing this gap, in 1997, ECOgal Blooms: A National Research Agenda, www.redtide. whoi.edu/hab/ (accessed September 1997). HAB awarded its first round of grants, totaling $3 mil(4) Anderson, D. M. In Red Tides: Biology, Environmental Scilion, and will award $3 million in each of the followence, and Toxicology; Okaichi, T„ Anderson, D. M., ing four years. Nemoto, T., Eds.; Elsevier: New York, 1989; p. 11. (5) Burkholder, J. M.; Glasgow, H. B., Jr.; Hobbs, C. W. Mar. With support from the ECOHAB program, AnderEcol.: Prog. Ser. 1995, 124, 43. son is teaming up with Korean colleagues to explore (6) Burkholder, J. M.; Glasgow, H. B., Jr. Limnol. Oceanogr., techniques for controlling blooms. In laboratory exin press. periments, Anderson found that some clays will selec- (7) Burkholder, J. M.; Glasgow, H. B., Jr.; Springer, J. J.; Lewitis, A. J. Influence of Water-Column Nutrient Sources tively rrmove dinoffagellates. He ii slso ccnducttng reon Planktonic and Benthic Stages of the Toxic Estuasearch to determine the fate of the algal toxins once they rine Dinoflagellate Pfiesteria piscicida. Proceedings of the reach the sediments and what effect they have on orEighth International Conference on Harmful Algae, Vigo, ganisms there. U.S. acceptance of these control methSpain, June 25-29, 1997. (8) Keafer, B. A.; Anderson, O. M. In Toxic Phytoplankton ods hinges on demonstration mat they are safe for nonBlooms in the Sea; Smayda, T. J.; Shimizu, Y., Eds.; Elsevier target benthic and planktonic organisms. Science Publishing: Amsterdam, The Netherlands, 1997; Although application of clay has been the leadpp. 763-68. (9) Tester, P. A.; Stumpf, R. P.; Vukovich, F. H.; Fowler, P. K.; ing control agent used in Asia, Japanese and U.S. sciTurner, J. T. Limnol. Oceanogr. 1991, 36(5), 1053. entists are also experimenting with algicidal bacte(10) Shirota, A. Int. J. Aquacult. Fisheries Technoll.989,11 ,95. ria and viruses. Greg Doucette, a research biologist (11) Yu, Z.; Zou, J. Z.; Ma, X. Oceanol. Limnol. Sin. 1994, 25, 226. at the NOAA Marine Biotoxins Program in Charleston, S.C., reported that his Japanese colleagues have discovered bacteria that will kill or slow the growth Janet Pelley ii a contributing gditor o/ES&TT 3 0 A • JAN. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
