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## Chapter 3: Occurrence of **Cyanobacterial Harmful Algal Blooms Workgroup Report** 

Edited by Anthony Fristachi and James L Sinclair

Workgroup Co-Chairs:

James L Sinclair, Sherwood Hall

Workgroup Members 1 : Julie A Hambrook Berkman; Greg Boyer; JoAnn Burkholder; John Burns; Wayne Carmichael; Al DuFour; William Frazier; Steve L Morton; Eric O’Brien; Steven Walker



### Acknowledgements

Appreciation is given to all those w hose efforts made the production of this book possible. Thanks are due to the editors, Anthony Fristachi, US Environmental Protection Agency, Offi ce of Research and Development, Cincinnati, Ohio and Jim Sinclair, PhD, US Environmental Protection Agency. Thanks are also due to Dr . Jeffrey Johansen of John Carroll University for his review and revisions to the Section 4 discussion of the current status of taxonomy of cyanobacteria. Special thanks to the workshop co–cha irs: Jim Sinclair, PhD, US Environmental Protection Agency, Office Wa ter, Cincinnati, Ohio and Sherwood Hall, PhD, Food and Drug Administration, CFSAN, Laurel, Maryland, who co–managed the process of preparing the manuscript.

1 See Workgroup members and affiliations in Invited Participants section.

### Invited Participants

**Occurrence Workgroup**

**Workgroup Members**

Sherwood Hall, Co-chair US Food & Drug Administration shall@cfsan.fda.gov

301-210-2160 Jim Sinclair, Co-chair

USEPA Sinclair.james@epa.gov

Gregory L BoyerCollege of Environmental Science and Forestry State University of New York glboyer@esf.edu

315-470-6825 Julie Berkman US Geological Survey jberkman@usgs.gov

614-430-7730

JoAnn M Burkholder Center for Applied Aquatic Ecology,

NCSU joann_burkholder@ncsu.edu

919-515-3421 John Burns

PBS&J jwburns@pbsj.com

904-477-7723

Wayne W. Carmichael Wright State University wayne.carmichael@wright.edu

937-775-3173 Al DuFour

USEPA dufour.alfred@epa.gov

William Frazier City of High Point bill.frazier@highpointnc.gov

336-883-3410 Tony Fristachi

NCEA CIN fristachi.anthony@epa.gov

513-569-7144

Steve Morton

NOAA steve.morton@noaa.gov Eric O'Brien University of Iowa eobrien@igsb.uiowa.edu

319-560-6128

Steven Walker Nebraska Department of Environmental Quality steve.walker@nde q.state.ne.us
402-471-4227 Preface XV4 6 A

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. S i n c l a i r



### Introduction

Freshwater cyanobacteria periodically accumulate, or bloom, in water bodies across the United States (US). These blooms, also known as cyanobacterial harmful algal blooms (CHAB), can lead to a reduction in the number of individuals who engage in recreati onal activities in lakes and reservoirs, degrade aquatic habitats and potentia lly impact human health. In 1998, Congress passed the 1998 Harmful Algal Bloom and Hypoxia Researchand Control Act (HABHRCA) to address CHABs that impacted living marine resources, fish and shellfish harvests and recreational and service industries along US coastal waters. In 2004, as part of its reauthorization,HABHRCA requires federal agencies to assess CHABs to include freshwater and estuarine environments a nd develop plans to reduce the likelihood of CHAB formation and to mi tigate their damage (NOAA 2004). Many federal agencies recognize th e potential impacts of CHABs and share risk management responsibilities; an interagency task force was established and charged to prepare a scientific assessment of the causes, occurrence, effects and economic costs offreshwater. The United States Environmental Protection Agency (EPA) has included “cyanobacteria (blue– green algae), other freshwater algae, and their toxins” in its Contaminant Candidate List (CCL) as one of the microbial drinking water contaminants targeted for additional study, but it does not specify which toxins should be targeted for study (EPA 2005b). Based on toxicological, epidemiology and occurrence studies, the EPA Office of Ground Water and Drinking Water has restricted its efforts to 3 of the over 80 variants of cyanotoxins reported, recommending that Microcystin (MC) congeners LR, YR, RR and LA, Anatoxin–a (AA) and Cylindros permopsin (CY) be placed on the Unregulated Contaminant Monitoring Rule (UCMR) (EPA 2001). The EPA uses the UCMR program to collect data for contaminants suspected to be present in drinking water that do not have health–based standards set. This monitoring supplies information on the nature and size of populations exposed to cyanotoxins through tap water use.Various federal agencies are mandated to address CHABs and their impacts, which commonly have been managed on a case–by–case, somewhat fragmented basis. A new national US plan, the Harmful Algal Research and Response National Environmental Science Strategy (HARNESS), is “designed to facilitate coordination by highlighting and justifying the needs and priorities of the research and management communities and by suggesting strategies or approaches to address them” (HARNESS 2005). CHABs cross all four critical areas identified for harmful algal research and response: bloom ecology and dynamics; toxins and their effects; foodChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 47

webs and fisheries; and public health and socioeconomic impacts. HARNESS (2005) also noted that “research on freshwater harmful algalblooms [mostly formed by cyanobacteria] has lagged behind efforts to address marine harmful algal blooms [in general], and there is no comprehensive source of information on the occurrence and effects of freshwater harmful algal blooms in the US”. This is ironic, considering that CHABs have been documented to be highly responsive to nutrient pollution (Glibert and Burkholder 2006), and toxic to livestock, wildlife and humans in the US and worldwide (Chorus and Bartram 1999). This chapter will describe the occu rrence of bloom–forming cyanobacteria and their toxins. Cyanobacteria water blooms are defined here as the visible coloration of a water body due to the presence of suspended cells, filaments and/or colonies and, in some cases, subsequent surface scums (surface accumulations of cells resembling clotted mats or paint–like slicks). They are a common occurre nce in the US and throughout the world. Over 2000 species of cyanobact eria, many associated with nuisance blooms, are currently known (e.g. Komárek and Anagnostidis 1999, Komárek 2003). Freshwater HABs can assume many forms and whilemost are composed of prokaryotic cyanobacteria (blue–green algae), eukaryotic species may be present in blooms. While surface scums are commonly associated with blooms of Microcystis, blooms of other species such as Cylindrospermopsis in Ariz ona, Indiana and Iowa can be characterized by lower cell numbers events . Although not all blooms are characterized by high biomass events, high biomass blooms of toxic species/strains can have significant impacts on ecosystems by adding potentially large amounts of toxins (Glibert and Burkholder 2006). In addition, high–biomass blooms, whether of toxic or nontoxic species, also increase in oxygen demand during blooms and bloom decline, leading to localized hypoxia/anoxia and fish kills. These blooms can also cause habitat loss and food web changes. For example, surface blooms can block the light from reaching the benthos, leading to chan ges in attached plant communities and increased re–suspension of nutrients from the sediments. High–biomass blooms also commonly lead to economic loss through their negative impacts on recreation activities, and tourism and their production of substances that cause taste–and–odor problems and increased treatmentcosts in potable water supply plants. Blooms of all types can lead to a decreased quality of life by causing a negative perception of the state of health of the water body. Multiple interacting physical, chemical and biological factors lead to the formation of CHABs. Planktonic cyanobacteria are a natural component of the phytoplankton in most surface wa ters of the world. Toxigenic spe-4 8

cies (capable of producing toxins, although not all populations or strains do so) are all naturally occurring members of freshwater and sometimesbrackish water phytoplankton. The toxins that they produce can be compared with other naturally produced toxins (Carmichael 1992). Since the first report of toxic cyanobacteria in the late 19th century, all continents except Antarctica have reported toxi c blooms (Carmichael 1992). Toxicblooms can be especially problematic and are characterized by their production of hepatotoxins, neurotoxins and acute dermatotoxic compounds (Chorus and Bartram 1999). In the US, blooms of cyanobacteria have

been associated with the death of both wildlife and domestic animals(Carmichael 1998). To date, no human fatalities in the US can unambiguously be attributed to cyanobacterial t oxins, though in at least one instance, this connection has been made in the popular press (Behm 2003; Campbelland Sargent 2004). Human health effects within the US have primarily included gastrointestinal illness in Pe nnsylvania (Lippy and Erb 1976),

swimmers itch in Florida and Hawa ii (Williams et al. 2001) and skin rashes, nausea, and other gastrointes tinal disorders in Nebraska (Walker 2005). In Brazil, a gastroenteritis ep idemic was associated with the construction of the Itaparica Dam in Brazil (Teixeira et al. 1993), and the hospitalization of 140 children when supplied with drinking water from a reservoir containing a bloom of Cylindros permopsis raciborskii in Australia (Chorus and Bartram 1999). The use of cyanotoxin–contaminated water indialysis equipment in Brazil accounted for the only reported human fatalities (Carmichael et al. 2001a; Azevedo et al. 2002).



### Distribution of CHABs across the US

**Blooms in freshwater environments**

Freshwater CHABs are ubiquitous throu ghout the US and Canada (Fig. 1). Cyanobacteria perform many roles thatare vital for the health of ecosystems, especially as photosynthetic organisms, but they may also cause harm through either excessively dense growth or release of toxins or other harmful metabolites. CHABs span ha bitats ranging from smaller eutrophic

p r a i r i e p o n d s t o l a r g e r m o r e o l i g o t r o p h i c r e g i o n s o f t h e G r e a t L a k e s

. T h i s

a d d i s t r i b u t i o n o f p o t e n t i a l b r o a d d i s t r i b u t i o n i s e x p e c t e d g i v e n t h e c o n s i d e r a b l e d i v e r s i t y i n h a b i t a t s occupied by cyanobacteria and the widespre toxicity in the more common genera. A

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. S i n c l a i rChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 49

**Blooms in estuarine and marine environments**

While toxigenic cyanobacteria are generally associated with freshwater blooms, there is increasing recognition that , in certain regions of the world,

blooms in estuarine and marine enviro nments may also have an importantimpact on human health. The Baltic Sea and the Gulf of Finland often sustain massive blooms of toxic Nodularia spumigena that are easily visible in satellite images of the region (Sivonen et al. 1989). These blooms affect the natural biota and can spoil recreational opportunities on the coastline. Similarly, large blooms of benthic cyanobacterium Lyngbya majuscule in Moreton Bay, Australia (Albert et al. 2005), and off the Hawaiian Islands (Moikeha and Chu 1971; Moikeha et al. 1971) have caused skin, eye andrespiratory effects on recreational users of these waters. Not all detrimental effects are directly associated with toxins; for example, increased cyanobacterial biomass in Florida Ba y and other estuaries has been associated with sea grass die–offs and the decline of the adjacent coral reef systems (Williams et al. 2001).

Both Microcystis aeruginosa and Cylindrospermopsis raciborskii have only limited tolerance to salinity (< 2–7 ppt) and this may limit the growth of these common toxic species in brackish waters environments (Barron etal. 2002). Increasing salinity in oligohaline and mesohaline estuaries results in increased osmotic stress and aggregation of cyanobacterial cells (Sellner et al. 1988). Many cyanobacterial species rapidly drop out from phytoplankton assemblages as salinity concentrations exceed their thresholds and thus are limited to freshwate r tidal portions of these major rivers (Sellner et al. 1988). Nevertheless, increased freshwater inputs into rivers

from increased runoff dilute the estua rine environment and can promote blooms of these toxic cyanobacterial (Sellner et al. 1988). For example, Lehman et al. (2005) recently documented a large bloom of M. aeruginosa in the upper San Francisco Bay Estuary. The bloom was widespreadthroughout 180 km of waterways; microcystins were detected at all stations sampled, and were also found in zooplankton and clam tissues. Other species of toxigenic cyanobacteria readily bloom in full salinity

conditions and can dominate these environments. Non–toxic Aphanizomenon flos–aquae and toxic Nodularia spumigena form high biomass blooms in the Baltic Sea, and have increased in abundance (Sivonen et al. 1989; Finn et al. 2001). Anabaena ( Aphanizomenon ) aphanizomenoides occasionally has been reported to be abun dant in the Neuse Estuary in North Carolina (Lung and Paerl 1988).5 0

**Changes in the Distribution of Toxic Cyanobacteria**

In considering long–term trends of both the frequency of occurrence and distribution of cyanobacterial toxins in the US and Canada, it is important to recognize the remarkable advances th at increased interest and awareness that has brought to the discipline over the last few decades. Both the number of known cyanobacterial toxins and toxigenic species has dramatically

increased with the increased focus. Wh ile few, if any, states had monitoring programs for cyanobacterial toxins in the mid 1970’s in most cases, toxic cyanobacteria were identified as being responsible for livestock or wildlife fatalities. By 2005, a number of states had monitoring programs in place, resulting in an increase in the number of CHABs reported. It isdifficult to determine if this increasing number of reports represents an increased incidence of CHABS, or is simply a reflection of increasing awareness and monitoring efforts. Several geographical regions have re cently introduced “regional” surveys for the occurrence of cyanobact erial toxins. These regional studieshave documented that the occurrence of cyanobacterial toxins has expanded outside of the midwestern prairi e states (Fig. 3, see Color Plate 1). Studies in New York, Nebraska, New Hampshire and Florida have shownan increase in the abundance of cyanobacterial blooms. Sasner et al. studied 50 New Hampshire lakes and reported that all had detectable levels ofmicrocystins (Sasner Jr. et al. 1981). Particulate concentrations of microcystins in the bloom spanned over fo ur orders of magnitude and ranged from 0.8 to 31,470 ng/g -1 wet weight; concentrati ons were generally higher in the summer than in the spring. In a similar study of 180 New York lakes and rivers, over 50% of the samp les contained easily detectable levels of particulate microcystins in the water column, ranging from 0 to over

1000 μ g/L (Boyer et al. 2004). Results of a short–term occurrence study by Carmichael et al. (unpublished 2006 ) in the Pacific No rthwest states of Washington, Oregon and Northern California (Fig. 4, see Color Plate 1), for the years 2002–2005, show that producers of microcystin and anatoxin–a occur on regular basis. In warmer climates, both the intensity and duration of these bloom events increase. For example, cyan obacterial blooms are common in Florida lakes, rivers, streams and ponds; data has been collected on a limitedbasis since 1999. Approximately 20 bloom–forming cyanobacteria, including Microcystis

, Cylindrospermopsis

, Anabaena

, Aphanizomenon

, Lyngbya and Planktothrix , are found distributed throughout the state with cyanotoxins detected in surface wate rs and post–treated drinking water (Williams et al. 2001). A

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Microcystin concentration was most often detected within the 0.1 to 10

g L -1 range with a maximum concentration of 107 μ g L -1 ; detected in post–treated drinking water at a maximum concentration of ~10 μ g L -1
(Burns 2005). Anatoxin–A was not detected in most samples collected,

but a maximum concentration of 156 μ g L -1 was detected in one sample; detected in post–treated drinking water at ~10 μ g L -1 . Cylindrospermopsin

was detected between 10 and 100 μ g L -1 (Burns 2005). The maximum cylindrospermopsin concentration de tected in surface waters was 202 μ g L -1

. The maximum concentration of cylindrospermopsin detected in post– treated drinking water was ~100 μ g L –1 (Burns 2005). The first report of

c y l i n d r o s p e r m o p s i in North America is mapped in Fig. 5.

While there is some evidence fo r seasonal changes in CHABS occurrence (Graham et al. 2004), overall there is a paucity of data related to the temporal distribution of CHABs.

Do invasive species change the occurrence of toxic species? Zebra mussels and Lake Erie (Vanderploeg et al. 2001)

The Great Lakes and especially Lake Erie have experienced manyproblems with invasive species over the past 100 years. The dreissenid zebra mussel, D r e i s s e n a p o l y m o r p h a e s t a b l i s h e d i t s e l f i nLake Erie in the late 1980’s and by 1995 blooms of the toxic cyanobacterium, M i c r o c y s t i s a e r u g i n o s a h a d b e c o m e a p r o b l e m

. S t u d i e s done since the late 90’s have led to the conclusion that zebra mussel selectively filtrate toxic M i c r o c y s t i s a e r u g i n o s a

, p r o m o t i n g t o x i c
M

i c r o c y s t i s blooms. This has resulted in new efforts to understand

and control CHABs in the Great Lakes. s μ5 2

Fig. 1. Distribution of Documented CHAB outbreaks in North America. Unpublished data (Carmichael 2006). A

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. S i n c l a i rFig. 2.Countries Reporting One or More CHAB Outbreaks. Unpublished data (Carmichael 2006). Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 53Fig. 3. Trends in Microcystin Occurr ence in Midwestern Lakes. Taken from Graham et al. 2004. (See Color Plate 1).

Fig. 4. C y a n o t o x i n E v e n t s i n t h e Pacific Northwest (2001–2005). Taken from Carmichael 2006. (See Color Plate 1). 5 4 A

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. S i n c l a i rFig. 3 (Chapter 3). Trends in Microcystin Occurr ence in Midwestern Lakes. Taken from Graham et al. 2004. (See page 54).

Fig. 4 (Chapter 3). Cyanotoxin Events in the Pacific Northwest (2001–2005). Taken from Carmichael 2006. (See page 54). 

## Color Plate 1Fig. 5. Florida Distribution of Cylindrospermopsin. (Williams et al. 2001). Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 55

### Cyanotoxin Production

The taxonomy of cyanobacteria, often im precise, is currently under a state of revision. In general, cyanobacter ia cells may be unicellular or filaments, existing singularly or in colonies. Morphological features can be distinct or variable, which has lead to considerable uncertainty when usingthe botanical taxonomic system that is based on morphological characteristics. For example, an early guide by Geitler (1932) and others listed more than 2000 species of cyanobacteria. Drouet and Daily, in a series of monograms published in the 1950s and 1960s (e.g. Drouet and Daily 1956; l957; Drouet l968), revised this flora using only the simplest of markers to lump everything together in only 62 species. Other taxonomists such as Stanier (1971), Castenholz (1992) and others proposed a taxonomic scheme for cyanobacteria based not on the botanical system, but using the provisions of the International Code of Nomenclature for Bacteria(Lapage et al. 1992). This system uses axenic clonal cultures and morphology as well as modern molecular and biochemical techniques to definethe species. Despite the age of these proposals, only one taxon of cyanobacteria is currently described under the International Code of Nomenclature of Prokaryotes or ICNP (Euzeby 2004). Bergey’s Manual of Systematic Bacteriology contains only a few of the many described cyanobacterial genera, and no species. Recently, Castenholz (2001) reversed his earlierposition and stated “I believe that the current rules of Nomenclature (botanical and bacteriological) should be mostly disregarded” and indicated that he preferred a moratorium on naming any species or undertaking any taxonomic revisions for 50 years or more so that molecular data sets could accrue (Hoffmann 2005). The botanical system for cyanobacter ia has recently been revised usingtraditional morphological characters resulting in a major revision of species, especially for the non–heterocyst ous filamentous forms such as O s c i l

l a t o r i a (Komárek and Anagnostidis 1999, 2005). The revised system resurrects many of the older botanical names for genera. While cell structure and ultrastructure (thylakoids, cell wall formation, cellular dimensions) and molecular data have been congrue nt, sheath characteristics, which are critical in definition of both species and genera, are not always congruent with the molecular data. Unfortunately, ultrastructure, biochemical, and molecular studies, such as RAPD fingerprinting and 16S rRNA gene sequences are only available for a small portion of cyanobacterial taxa. 5 6 A

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. S i n c l a i rThe ecological diversity observed in cyanobacteria is grossly underestimated if one looks only at those taxa in culture collections (the taxa whichare the source of the molecular and biochemical data). In some cases, molecular sequencing produces similar phyl ogenies. However, it is also quite easy to generate quite distinct ph ylogenetic lineages, depending on the particular part of the genome or trait that was used to generate the phylogeny.Most field taxonomists still rely on the traditional morphology–based botanical approach because molecular data have not yet been available for many species (Burkholder 2002), although the use of molecular based techniques is rapidly increasing, especia lly in those species that lack orcontain confusing morphological features. Fortunately, toxic cyanobacteria have received concentrated attention, and so the taxonomy of these taxa may be among the first to be revised based on polyphasic character sets.Toxic cyanobacteria blooms are associated with a wide diversity of species and environments. Chorus and Bartram (1999) (1999), in their classic monogram for the World Health Organization, Toxic Cyanobacteria in

Water: a Guide to Their Public Health Consequences, Monitoring and Management, reviewed which species of cyanobacteria have been associated with the production of toxins based on observations and reports since the mid 1980’s. Since that time, there have numerous new observations, including new toxigenic species such as the unknown Stigonematales species responsible for avian vacuolar myelinopathy (AVM) and new toxins

s u c h a s of species. Phormidium sp., which produces microcystin, has been found in shallows of reservoirs. Microcys tin LA is reported to account for the protein phosphatase inhibitory activity of the terrestrial cyanobacterium, Hapalosiphon hibernicus . Other terrestrial isolates produce the toxin β

– methylamino alanine (BMAA). With the recent report of microcystins

from lichen–associated Nostoc , it becomes apparent that non–aquatic environments must also be considered wh en discussing cyanobacterial toxicity. Table 1 lists 22 genera and 42 species associated with toxin formation. Most of the data was obtained from planktonic environments, although there are a number of toxic benthic species. Where appropriate, we have updated the nomenclature to the taxonomic scheme of Komárek and Anagnostidis (Komárek and Anagnostidis 1999, 2005). –N–methylamino alanine (BMAA) produced by a wide number β Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 57Table 1.Major Toxigenic Cyanobacteria Species, their Toxins and Distribution.Organism Toxin Location ReferenceAnabaena spp.microcystinsanatoxin–aDenmark, Egypt, Finland,Germany, Ireland, Japan, USA. circinalismicrocystinssaxitoxinsFinland, France, AustraliaA. flos–aquaemicrocystinsanatoxin–aCanada, Finland, Norway,CanadaA. lemmermanniimicrocystinssaxitoxinsFinlandA. planktonicaanatoxin–a ItalyA. variabilisBMAA USAphanizomenon spp.anatoxin–a Finland, GermanyA. (flos–aquae) issatschenkoisaxitoxins USA. flos–aquaeanatoxin–a(s) CanadaA. gracileLPS endotoxins USA. lemmermanniisaxitoxinsanatoxin–a(s)Portugal, DenmarkA. ovalisporumcylindrospermopsin IsraelArthrospirafusiformismicrocystins Africa, SpainAnabaenopsis milleriimicrocystins Greece 5 8 A

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. S i n c l a i rOrganism Toxin Location ReferenceCylindrospermum spp.anatoxin–a FinlandCylindrospermospis raciborskii, C. phillipinensissaxitoxinscylindrospermopsinBMAABrazil, Australia, Hungary,US, AustraliaHaphalosiphon hibernicusmicrocystins USLyngbya spp.pahayokolide–a USL. majusculaBMAA Africa, Australia, US, Pacific Is.L. wollei (Plectonema wollei)Farlowe exGomontdermatotoxinssaxitoxinsUSMicrocystis spp.microcystinsanatoxin–aBMAAWorldwideM. aeruginosamicrocystins WorldwideM. viridismicrocystins JapanM. botrysmicrocystins DenmarkNodularia spumigenanodularins CanadaN. spumigenanodularins Australia, New Zealand,Baltic SeaNostoc spp.microcystins Finland, England and US Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 59Organism Toxin Location ReferenceBMAAOscillatoria spp.anatoxin–a Finland, Scotland, Irelandand USO. brevis (Phormidium breve) (Kützing exGomont) Agnostidis et KomárekLPS endotoxins SwitzerlandO. limosa Agardh ex Gomontmicrocystins USO. nigroviridis (O. nigro–viridis) Twaitesex. Gomont sensu Parakuttydermatotoxins USO. tenuisLPS endotoxins USPhormidium spp.microcystinsBMAAsaxitoxinsUS and ItalyPlanktothrixP. agardhiimicrocystins, anatoxin–aMicrocystinsBMAAUS, FinlandDenmark, Finland, Norway,IrelandP. flavosumanatoxin–a FranceP. formosa*homoanatoxin–aNorwayP. mougeotii* (P. isothrix) (Skuja)Komárek and Komarkovamicrocystins DenmarkPlectonema sp.BMAA Unknown originProchlorococcus marinusBMAA Sargasso Sea 6 0 A

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. S i n c l a i rOrganism Toxin Location ReferenceRaphidiopsis spp.R. curvatacylindrospermopsinanatoxin–a,R. mediterraneahomoanatoxin–aJapanSchizothrix calcicolaaplysiatoxinsLPS (nontoxic)Pacific Islands and USStigonematales sp.avm USSynechococcus sp.BMAA USTrichodesmium thiebautiiBMAAsaxitoxinSargasso SeaUmezakia natanscylindrospermopsin Japan*denotes a species designation that is considered invalid using the current classification scheme Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 61Cyanobacteria toxicity is very common in natural blooms with estimates of toxicity approaching as high as 50% (WHO 2003). In the late 1990s, in an attempt to systematically estimate the abundance of toxic cyanobacteria, approximately 200 strains of the Pasteur Collection of Cyanobacteria (PCC) were screened for microcystins using ELISA and HPLC–PDA techniques. G. Codd and R Rippka ( unpublished) confirmed that more than 20 species produce cyanobacterial toxins, of which 11 strains of Microcystis and 1 strain of Nostoc produced concentrations of more than 100

μ g microcystins per gram dry weight (Boyer 2006). Many of the strains in

the PCC have also been screened for the genetic potential for microcystin production using PCR techniques (Dittm ann and Borner 2005). Similar

results were also obtained from the NIVA culture collection in Norway, where a large percentage of the nearly 500 strains of cyanobacteria in the collection appeared to produce toxins or other bioactive substances (Skulberg 1984). Microcystin and ha rmful hepatotoxic peptides were produced by many, but not all strains of the cyanobacteria belonging to the

genera A n a b a e n a

, M i c r o c y s t i s

, P l a n k t o t h r i x and N o s t o c (Hisbergues et al. 2003). Toxigenic cyanobacteria are generally defined as species that have toxic strains (populations), which are capab le of producing neurotoxic, hepatotoxic or dermatotoxic compounds (Bur kholder and Glibert 2006). This term is important to note, since with in a given species of toxigenic cyanobacteria, there commonly occur both toxic and nontoxic strains (Chorus and Bartram 1999; Burkholder and Glib ert 2006). If one expands the definition of toxins to include bioactive peptides with protease inhibition activity such as the micropeptins, cyanope ptolins, microviridins, oscillapeptins, oscillamides, nostopeptins, aeruginosins, aeuginopeptins anabaenopeptilides, anabaenopepins and the cytotoxic compounds from marine origin, then the list of toxin–producing cyanobacteria species becomes quite large. Recently, the PCC were screened for the presence of non– ribosomal peptide synthetase (NRPS) genes, those genes responsible for the synthesis of many bioactive pe ptides such as the microcystins, microveridins and anabaenapeptins (Christiansen et al. 2001). This biosynthetic activity is widespread througho ut the cyanobacteria taxa, with NRPS activity found in over 75% of the 146 st rains tested. While the presence ofthis gene activity does not directly correlate to toxicity, it indicates the potential for production of bioactive peptides. The non–ribosomal peptide synthetases used for microcystin toxin biosynthesis in cyanobacteria allow for considerable structural diversity (Christiansen et al. 2001). More than 400 individual peptides have been identified, mostly from Anabaena

, Lyngbya

, Nostoc and Microcystis species (Krishnamurthy et al. 1986; 6 2 A

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. S i n c l a i rLuesch et al. 2000; Pluotno and Carmeli 2005). Many of these peptides have protease inhibition ac tivity and can lead to detrimental effects if consumed as part of the food web. In addition to the production of toxi ns, cyanobacteria have often been associated with the production of tast e and odor compounds in with drinking water; the two most are geosmin and 2–methylisoborneol (MIB), alt h o u g h a n u m b e r o f d i f f e r e n t h y d r o x y k e t o n e s a n d

also add to the off–flavors associat ed with freshwater cyanobacteria (Izaguirre and Taylor 2004). While these taste and odor compounds are not actually toxic, they are of concern to the consuming public when their drinking water has an “off” flavor, resulting in considerable monitoring and treatment costs to water supply pr oviders. Taste and odor issues are complex and need not be associated solely with the presence of cyanobacteria. Many actinomycetes bacteria such as Actinomyces and Streptomyces species, aquatic fungi such as Basidiobolus ranarum , and myxobacteria

such as Nannocystis exedens can also produce these compounds (Niemi et al. 1982). Table 2 illustrates the large number of species associated with the production of compounds that affect taste and odor. These species mayor may not be the same species reported to produce toxins. The biosynthetic pathways for taste and odor compounds are separate and unrelated to the toxin biosynthetic pathways (Carmichael 2001). – i o n o n e d e r i v a t i v e s c a n β Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 63Table 2.Major Cyanobacterial Taste and Odor Producing Species.OrganismCompoundAnabaena spp.A. circinalisA. laxaA. macrosporaA. scheremetieviA. spiroidesA. viguieriGeosminAphanizomenonflos–aquaeGeosminCalothrix sp.(C. parietina)Ketones, IononesFischerella muscicolaGeosminJaaginema geminatum(Oscillatoria geminata)(Menehgnin ex Gomont Komárek and AnagnostidisMIBLyngbya spp.L. aestuarii (Lyngbya aestuarii) Liebman ex GomontL. cryptovaginata (Planktothrix cryptovaginalt) (Škorbatov) Anagnostidis et KomárekMIBGeosminOscillatoria spp.O. chalybea (Phormidium chalybeum) (Mertens ex Gomont) Anagnostidiset KomárekO. raciborskii (Planktothrix raciborskii) (Woloszyńska) Anagnostidis et KomárekMIBO. agardii (Planktothrix agardhii) (Gomont) Anagnostidis et KomárekO. amoena (Phormidium amoenum) (Kützing 1843) Anagnostidis et KomárekO. amphibia (Geitlerinema amphibium) (Agardh ex Gomont) AnagnostidisO. bornetii (Tychonema bornetii) (Zuka) Anagnostidis et KomárekGeosmin 6 4 A

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. S i n c l a i rOrganismCompoundO. brevis (Phormidium breve) (Kützing ex Gomont) Anagnostidis et KomárekO. variabilis (Oscillatoria variabilis) (Rao)O. prolifica (Planktothrixprolifica ([Grevelle] Gomont)Anagnostidis et KomárekO. simplicissima (Phormidium simplicissimum) (Gomont) Anagnostidis et KomárekO. splendida (Geitlerinema splendidum) (Greville ex Gomont) AnagnostidisO. cortiana (Phormidium cortiamum) (Meneghini ex Gomont) Anagnostidiset KomárekO. curviceps Agardh ex GomontO. limosa Agardh ex GomontO. tenuis Agardh ex GomontGeosmin, MIBPhormidium sp.P. autumnale (Phormidium autumnale) [Agardh] Trevisan ex GomontP. calcicola (Phormidium calcicola) GardnerP. tenue (Phormidium tergestinum) (Kützing) Anagnostidis et KomárekGeosminGeosmin, MIBMIBPlectonema sp.P. notatum (Leptolyngbya notata) (Schmidle) Anagnostidis et KomárekKetones, IononesPseudanabaena catenataP. catenataP. limnetica (Oscillatoria limnetica) (Lemmermann) Komárek and AnagnostidisMIBRivularia spp.Ketones, IononesSchizothrix muelleri(Symplocastrum muelliri) (Nägeli ex Gomont) AnagnostidisGeosminSymploca muscorumGomont ex Gomont GeosminTolypothrix distortaKetones, Ionones Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 65

### Cyanotoxin Occurrence and Distribution

Cyanobacterial toxins fall into several diverse categories and are usually divided based on their biological activity (hepatotoxins versus neurotoxins)or chemical structure (peptide toxins versus alkaloids) (Table 3). This division is becoming more complicated as new toxins are being identified. For example, in 1988, there were 10 reported microcystins; as of 1998, there are over 60 different chemically identified microcystins (Chorus and Bartram 1999). In the sections belo w, we summarize the basic classes of cyanobacterial toxins, their source organisms and their biological effects.

**Microcystins**

As shown in Table 1, microcystins have been reported in Microcystis

, Anabaena

, Oscillatoria

, Planktothrix

, Nostoc

, Hapalosiphon and Anabaenopsis with new sources appearing yearly (Huisman and Hulot 2005).Microcystins (Fig. 6) contain 7 amino acids in their cyclic peptide ring system, are closely related to nodularin (F ig. 7) or nodulapeptins, which have5 amino acids in the ring system, as well as anabaenopeptins, aeruginopeptin and other bioactive peptides (Sivonen and Rapala 1998; Harada2004). Most of these peptides are hepatotoxins, though their LD–50 depends on the specific amino acids present. 6 6 A

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. S i n c l a i rAdda

Arginine MeAsp (iso) Leu D -Glu (iso) Methyl dehydroAla (Mdha)

D -Ala O NH

NH

O NH CH 3

CO 2 H H 3 C CO 2 H

CH 3 CH 3 N

NH O

O CH 3 O

NH O

NH NH 2

NH CH 3 CH 3 CH 2

OCH 3 CH 3

S S R

R R

S S

2 1

Fig. 6. The generic structure of a microcystin.

Variations occur primarily at positions 1 and 2. For example, microcystin–LR contains the amino acids leucine (L) and arginine (R) at positions 1 and 2 respectively; microcystin–RR has arginine at both positions.

Nodularins are similar with the five amino acids Adda– γ Glu–Mdhb– β MeAsp–Arg making up the core ring system (Harada et al. 1996). Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 67Table 3.Commonly observed cyanotoxins in US fresh, estuarine and marine waters. (adapted from Codd et al. 2005).Toxin Type and (# congeners) Mode of ActionHepatotoxinsMicrocystins Cyclic heptapaptides (>70) Protein phosphatase inhibitor, tumor promoterNodularins Cyclic penta–peptide (9) Protein phosphatase inhibitor,Cylindrospermopsin Guanidine alkaloid (3)Protein synthesis inhibitor, genotoxic and necrotic injury to liver andother organelles.NeurotoxinsAnatoxin–a azobicyclic alkaloid (5)Postsynaptic neuromuscular blocking agent and AcetylcholinesteraseagonistAnatoxin–a(S) Guanidine organophosphate(1) Acetylcholinesterase inhibitorSaxitoxin Carbamate alkaloid (>20) Sodium channel blockerΒ–methylamino alanineModified amino acidNeurodegenerative agentDermatotoxinsLyngbyatoxin–a Indole Alkaloid (1) Inflammatory agent and Protein Kinase C activatorAplysiatoxin Polyacetate Alkaloid (2) Inflammatory agent and Protein Kinase C activatorEndotoxinsLipopolysaccharides Lipopolysaccharides Gastrointestinal irritants 6 8 A

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. S i n c l a i rFig. 7. The generic structure of a Nodularin.

**Cylindrospermopsin**

**Anatoxin–a**

The neurotoxic cyanobacterial toxins consist of anatoxin, anatoxin–a(S), saxitoxin and related analogs. The most important of these compounds Separate from the hepatotoxic cyclic peptides is the hepatotoxic alkaloid cylindrospermopsin (Fig. 8a). Th is toxin is usually produced by Cylindrospermopsis raciborskii and an outbreak of this organism in the drinkingwater supply on Palm Island, Queensland Australia led to a severe outbreak of hepatoenteritis among the in habitants of the island (WHO 2002). Cylindrospermopsin has also been reported from Umezakia natans in Japan, Aphanizomenon ovalisporum i n I s r a e l ( S i v o n e n a n d J o n e s 1 9 9 9 )

, a n d Raphidiopsis Curuata in China (Li et al., 2001). It was generally assumed that the toxigenic cylindrospermopsin–forming species only bloomed in tropical and arid environments and Cylindrospermopsis raciborskii blooms are commonplace in the warmer Florida drinking water reservoirs. Increasing reports of C. raciborskii occurring in temperate zones of Europe and the U.S have been documented (Padisak 1997). Cylindrospermopsin– producing blooms can be both high a nd low–biomass events, leading to difficulties in identifying the source of this toxin. Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 69from an environmental health aspect is probably anatoxin–a (Fig. 8b) (Carmichael et al. 1975). Anatoxin–a was originally isolated from Anabaena flos–aquae (Devlin et al. 1977), but is also reported in A. planktonica

, Oscillatoria spp., Planktothrix spp., and Cylindrosperum . Homoanatoxin–a, a toxic homologue with a propyl group replacing the acetyl group, was isolated from Phormidium ( Oscillatoria ) formosa . Both are potent nicotinic agonists and act as neuromuscular blocking agents.

**Anatoxin–a(S)**

Anatoxin–a(S) (Fig. 8c) is a naturally occurring organophosphate, with no reported structural variants, produced by Anabaena flos–aquae strain NRC 525–17 and more recently by Anabaena lemmermannii (Henriksen et al. 1997; Onodera et al. 1997). It is distin ct from the neurotoxic anatoxin–a in both its chemical structure and biological mode of action (Mahmood and Carmichael 1986b). Anatoxin–a(S) act s as an acetyl–cholinesterase inhibitor and its name was derived from the fact that the intoxicated animals often suffered from extreme salivation (Matsunaga et al. 1989). Anatoxin– a(S) is commonly reported in the prai rie states of the US (Kenefick et al. 1992); this may lead to an underestimat e of its occurrence since, lacking a clear cyanobacterial source, death of livestock or pets from anatoxin–a(S) would likely be attributed to pesticide intoxication.

**Saxitoxins and related analogs**

Saxitoxins (STX) (Fig. 8d) are repres entative of a large toxin family referred to as the Paralytic Shellfish Poisoning (PSP) toxins. These include the N–1 hydroxysaxitoxin or neosaxitoxin (NeoSTX), 11–sulfate analogs of STX and NeoSTX called gonyautoxi ns (GTX) and 6 N21 sulfo derivatives of STX, neoSTX and GTX toxins B1, B2, C1 to C4 (Mahmood and Carmichael 1986a; Chorus and Bartram 1999). Not all of these 18 PSP analogs have been identified in marine dinoflagellates and shellfish. Thesetoxins are identical to those produced by some toxigenic marine dinoflagellates that accumulate in shellfish that feed on those algae (Anderson 1994). STX, neoSTX, C1, C2 and GTX ha ve all been reported in freshwater cyanobacteria including Aphanizomenon spp., (Pereira et al. 2004, Li and Carmichael, 2003, Li et al. 2000) Anabaena c i r c i n a l i s

, wollei and a Brazilian isolate of Cylindrospermopsis raciborskii (Lagos et al. 1999). L y n g b y a 7 0 A

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. S i n c l a i r(a) cylindrospermopsin (b) Anatoxin–a

(c) Anatoxin–a(S) (d) saxitoxin

Fig. 8a–d. Structures of the alka loid cyanobacterial toxins.

**–methylamino alanine (BMAA)**

A recently discovered cyanobacterial neurotoxin is non–protein amino acid β –N–methylamino–L–alanine (BMAA). BMAA, originally isolated from a Guam cycad, Cycas circinalis , has been implicated as the causative agent of amyotrophic lateral sclerosis or Pa rkinsonism dementia (Murch et al. 2004b). Symbiotic Nostoc species associated with the roots of cycads produce BMAA and it accumulates in the seeds of the plant. The indigenous Chamorro people of Guam feed on the flying foxes, which consume these seeds, and are subject to a classic biomagnification of the toxin up throughthe higher trophic levels (Cox et al. 2005). BMAA may also be incorporated into peptides or proteins, bot h increasing its biomagnification and acting as a reservoir to slowly releasing toxins upon peptide hydrolysis (Murch et al. 2004a).Cox et al. (2005) surveyed 30 different strains of cyanobacteria obtained from culture collections that spanned the entire spectrum of cyanobacterial morphology and taxonomy for the presence of BMAA. Although

BMAA is most common in Nostoc species, with 7 out of 10 isolates conβ Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 71taining high levels of either the free or protein–bound amino acid, it is ubiquitous in its distribution, being found in freshwater, marine, brackish and terrestrial environments. Only 1 of the 3 species tested, a freshwater Synechocystis species, lacked BMAA. Algal strains in culture often produce much lower levels of toxins than found in situ suggesting that there is potential for widespread human exposure to this toxin (Cox et al. 2005).BMAA was recently detected in the brains of nine Canadian Alzheimer patients, but was not found in the brains of 14 other Canadians that died from causes unrelated to neuro–degenerati on (Murch et al. 2004a, 2004b). Since cycads are not part of the normal Canadian flora, cyanobacteria may be the source of BMAA in these patients. Drinking water contaminated bycyanobacterial blooms has been suggested as a potential pathway for human exposure to BMAA and a topic of future data needs.

**Lyngbyatoxins, Aplysiatoxin and Debromaplysiatoxin.**

Lyngbyatoxins, contact irritants, are mainly marine water toxins but their

occurrence in freshwaters h as not been fully assessed. Lyngbya , an aggressive genus capable of widespread growth, has been found in freshwater supplies in the US. Lyngbya b l o o m s a r e c o m m o n i n F l o r i d a , f o r m i n g mats in freshwater springs and overgrowing some coastal reefs, where low

levels of Lyngbya –toxin–A, debromoaplysiatoxin and saxitoxin have been detected (Williams et al. 2001). More data are needed to determine its

health effects and occurrence in fresh water supplies. The inflammatory activity of Lyngbya is caused by aplysiatoxin and debromoaplysiatoxin, which are tumor promoters and protein kinase–C activators (Berry et al. 2004). Debromoaplysiatoxin along with other toxic compounds has also

been isolated from other members of the family Oscillato riaceae such as

Schizothrix calcicola and Oscillatoria nigroviridis (Chorus and Bartram 1999).

**Other Bioactive Compounds**

Many cyanobacteria including Microcystis

, Planktothrix and Anabaena species have the ability to produce cy clic and linear peptide protease inhibitors that are potentially toxic (Harada 2004). Examples of these compounds include the micropeptins, cyanopeptolins, microviridin, oscillapeptin, oscillamide, nostopeptin, aeruginosins, aeruginopeptins, anabaenaopeptilides, anabaenope ptins and circinamide (papain inhibitor). Two such compounds isolated from Microcystis aeruginosa , aeruginopept7 2 A

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. S i n c l a i rins and cyanopeptolins, are shown in Fig. 9

. The effects of these compounds on the toxicity of cyanotoxins is not well known.

aeruginopeptin 228–a

cyanopeptolin–a

Fig. 9. Structures of two bioactive peptides isolated from Microcystis aeruginosa.



### Impacts on Waterbody Health and Ecosystem Viability

The factors that lead to CHAB formation and induce toxin production are not well known (Chorus and Bart ram 1999). Although multiple studies have examined the empirical relations hip between environmental variables and toxin concentrations, few have me asured the many different variables necessary to compile a comprehensive data set for analysis, (Kneale and Howard 1997). Even fewer have analy zed the data based on sound statistical methods recommended by EPA and other agencies (EPA 2003). Temporal and spatial patterns of CHAB fo rmation are often complex, and the mechanisms of bloom progression are largely unexplained. Additionally, CHABs can include both toxic and non–toxic strains, and many strains can be present during CHABs in specific lakes (Vezie et al. 1998; Jacoby et al. 2000; Pawlik–Skowronska et al. 2004). A major source of variation intoxin concentration appears to be the strain–dependant influence of enviChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 73ronmental factors on toxin release (Rapala and Sivonen 1998; Vezie et al. 2002) and successive replacement of toxic and non–toxic species (Kardinal and Visser 2005). No single modeling study has attempted to predict the dynamics of toxin releases and/or toxin levels, reflecting a limited understanding of factors influencing toxin release. Several recent reviews (Paerl 1996; Soranno 1997; Carmichael et al.2001b; Saker and Griffiths 2001; Landsberg 2002; Codd et al. 2005; Huisman and Hulot 2005). provide a comprehensive discussion of individual environmental factors that may be r esponsible for CHABs and their correlated effects on ecosystem health. Physical factors include temperature, light availability and meteorological conditions. Hydrologic factors include alteration of water flow, turbidity and vertical mixing. Chemical factors include nutrient loading (principally nitrogen [N] and phosphorous [P]), pH changes and trace metals, such as copper, iron and zinc.

**Freshwater Habitats**

Most of the world’s population relies on surface freshwaters as their primary source for drinking water. The drinking water industry is constantlychallenged with surface water contaminants that must be removed to protect public health. Recent reports suggest that CHABs are an emerging issue in the US because of increased s ource–water nutrient pollution causingeutrophication. Although CHABs historically have been strongly correlated with nutrient over–enrichment in many freshwater lakes with low abiotic turbidity (e.g Schindler 1987, Whitton and Potts 2000), flowing waters can also develop planktonic CHABs in slowly flowing segments(e.g Pinckney et al. 1997). Intermediate systems – run–of–river impoundments or reservoirs – generally have faster flushing and higher abiotic turbidity from suspended sediment loads than natural lakes (Wetzel 2001). Inthese systems light, rather than nutrients, often is the most important resource limiting cyanobacteria growth (Cuker et al. 1990). In addition, because of the surface characteristics of their cells, cyanobacteria can be especially susceptible to being attracted to sediment particles (Avnimelech etal. 1982, Søballe and Threlkeld 1988, Burkholder 1992). When that occurs, they become too heavy to remain in the water column and settle out of the water column (Burkholder a nd Cuker 1991). Nevertheless, during intervals between episodic sediment loading events from land erosion and runoff, dense cyanobacteria blooms can develop in reservoir systems, and nutrients – both nitrogen and phosphorus – can become important in controlling their production (Touchette et al. (submitted), Cuker et al. 1990, Burkholder and Cuker 1991); Fig. 10. 7 4 A

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. S i n c l a i rMany eutrophic lakes and impoundmen ts (reservoirs) have documentedCHAB problems, for example: The Metropolitan Water District of Southern California (MWDSC) has a long history of algal problems, in the form of planktonic blooms and benthic prol iferations (Izaguirre 2005). The main concern is taste and odor, specifically the compounds geosmin and

MIB. All of the reservoirs listed be low have experienced algal blooms of one kind or another, including in some cases known toxigenic species. In addition, Lakes Mathews, Skinner, Perris and Diamond Valley have developed benthic mats that have resulted in severe off–flavor problems(Izaguirre 2005). MWDSC, is composed of 26 member agencies and supplies drinking water to about 18 million people in six counties in thecoastal plain of southern California; its two sources of water are the Colorado River and water from northern California delivered through the California aqueduct feeding into three re servoirs in Riverside County: Lake Mathews, Lake Skinner and Diamond Valley Lake (Izaguirre 2005). In North Carolina, a large, previously undetected, sewer spill that occurred over several months was positively correlated with Lyngbya blooms (Frazier 2006). As research progresses, new toxins from cyanobacteria in freshwater systems are being discovered (Codd et al. 2001; Huisman and Hulot 2005), as well as new potential roles in their impacts on food webs. For example, avian vacuolar myelopathy (AVM), a neurological disease in waterfowl and their predators, was first described in the mid–1990s, with unknown cause (Thomas et al. 1998). AVM causes characteristic lesions in the myelin of the brain and spinal cord. Affected birds have abnormal flying and swimming because their muscle coordi nation is compromised. The causative agent of AVM is associated with submersed aquatic vegetation such as the aquatic weed hydrilla (Birrenkott et al. 2004). At present, the prime “suspect” is a toxin producing, yet unnamed cyanobacterium within theorder Stigonematales that colonizes the leaves of hydrilla and other submersed plants (Wilde et al. 2005). Thus, the working hypothesis being examined is that waterfowl such as coots consume submersed vegetation covered with the toxic cyanobacteria, bioaccumulate the toxin(s), and are impaired in their ability to escape from predators. The affected waterfowl are caught and consumed by higher pred ators such as bald eagles, which then also develop AVM. Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 75050 100 150 200 25004080120D. Old(r2= 0.81)0 20 40 60 80020406080C. Mod.(r2= 0.48)

Chl a (μg/L)TP (μg/L) TP (μg/L)020406080100120140200 400 600 800 1000 1200020406080

Chl a (μg/L)A. Mod(r2= 0.65)TN (μg/L) TN (μg/L)0 1000 2000 3000 4000 5000B. Old(r2= 0.48)050 100 150 200 25004080120D. Old(r2= 0.81)0 20 40 60 80020406080C. Mod.(r2= 0.48)

Chl a (μg/L)TP (μg/L) TP (μg/L)020406080100120140200 400 600 800 1000 1200020406080

Chl a (μg/L)A. Mod(r2= 0.65)TN (μg/L) TN (μg/L)0 1000 2000 3000 4000 5000B. Old(r2= 0.48)Fig. 10.Relationship between chlorophyll a (chla)and total nitrogen (TN, upper panels) or total phosphorus (TP, lower panels).(A,C) moderate–age reservoirs (mod., Group 1, 20–30 years post–fill,n = 5) and (B, D) old reservoirs (Group 2, 60–85 years post–fill,n = 6) during summer months when cyanobacteria were dominant(Touchette et al. (submitted)). 7 6 A

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. S i n c l a i r**Estuarine Habitats**

Estuaries are comprised of high po pulation densities of microbes, plankton, benthic flora and fauna. These or ganisms tend to be highly vulnerableto human activities and a wide array of human impacts that can compromise their ecological integrity. Many of the factors responsible for CHABS

as well as the CHABs themselves affect estuarine habitat health. Anthropogenic stress in the form of increased nutrient input significantly impactsestuaries as well as other water bodies. Modeling studies suggest the importance of nutrient loading to wa ter quality and CHAB probability and severity. Nitrogen (N) and phosphorus (P) enrichment are considered cyanobacteria bloom precursors. Many cyanobacteria are nitrogen–fixers,making N a limiting factor in competition over other phytoplankton species (Burkholder 2002). Although nutrient influences are widely studiedfactor linked to stimulation of CHABs, the nutrient(s) that are most important in limiting cyanobacteria growth are still in debate, and likely depend upon the habitat and history of nutrition (e.g. Lenes et al. 2001; Glibert et al. 2005, 2006). Cyanobacteria blooms were noted in the Potomac River and the upper Chesapeake Bay during the 1950s and 1960s coincident with the invasion of water milfoil. Since 1985, cya nobacteria blooms have been documented in the tidal tributaries of Chesapeake Bay almost annually during summer months by the Maryland Department of Natural Resources (MD–DNR) long–term comprehensive water–quality monitoring program. During September 2000, an extensive late summer bloom of Microcystis on the Sassafras River, however, was among the first blooms tested for cyanotoxins in Maryland and results were positive for elevated concentrations of microc y s t i n

. T h e m i c r o c y s t i n l e v e l s ( 5 9 1

. 4

– 1 0 4 1 g g -1 dry wt) led to the Kent County Health Department closing a public beach in the bloom area for the remainder of the year, the first beach closure in the history of the state dueto detected levels of cyanotoxins (Tango et al. 2005). Dr. Harold Marshall, a renowned phytoplankton specialist in the Chesapeake Bay area, has also documented significant increases in cyanobacteria biomass, mostly as Microcystis aeruginosa , from long–term datasets in tributaries of lower Chesapeake Bay (Fig. 11). μ Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 77Fig. 11.Long–term datasets (1987–2005) show increasing cyanobacteria biomass in tributaries of lower Chesapeake Bay. 7 8 A

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. S i n c l a i r**Marine Habitats**

In comparison to fresh and estuarine waters, relatively little is known about harmful cyanobacteria in marine ecosy stems, except for their negative effects on coral reefs. For example, the toxigenic, benthic cyanobacterium

L

y n g b y a m a j u s c u l a p r o l i f e r a t e s i n w a r m m a r i n e w a t e r s e n r i c h e d w i t h n u

trients (especially phosphorus or iron) (e.g., Watkinson et al. 2005). Nicknamed “stinging seaweed” (Sims and Zandee Van Rilland 1981), L

. m a

j u s c u l a p r o d u c e s a n a r r a y o f t o x i n s t h a t a d v e r s e l y a f f e c t h u m a n h e a l t h (Osborne et al. 2001) and have been implicated as causes of tumors and other diseases in marine fauna (e.g. Arthur et al. 2006). It also has causedserious ecological impacts in smothering coral reefs and seagrass meadows. For example, a recently described bloom expanded to 8 km 2 of coverage within 55 days, and its dense accumulation (average biomass 210 grams dry weight · m –2 ) led to declines in beneficial seagrasses (Watkinson et al. 2005). As another example, the cyanobacterium P h o r

m i d i u m c o r a l l y t i c u m i s a m e m b e r o f a microbial consortium that causes

black band disease and death of corals (Richardson 2005).

Although cyanobacteria in many saltwater ecosystems have not been rigorously examined for toxicity, limite d available evidence suggests that cryptic toxigenic species can be abundant in these habitats. For example, a survey of the landlocked, brackish to hypersaline, Salton Sea revealed that 85% of all water samples tested from areas with high incidence of grebedeaths contained low but detectable levels of microcystins produced by picoplanktonic S y n e c h o c o c c u s a n d b e n t h i c O s c i l l a t o r i a (Carmichael and Li 2006). Moreover, the livers of affected grebes contained microcystins at potentially lethal levels.

Possible interactions between cya nobacteria and marine harmful alg a l b l o o m s : T r i c h o d e s m u m s p p

. a r e m a r i n e c y a n o b a c t e r i a t h a t f i xnitrogen gas into inorganic nitrogen forms that are used to make proteins not only by the cyanobacteria, but by other organisms as nitrogen leaks from the cyanobacteria cells. The impacts are currently being explored and are not necessarily bad, but the added available nitrogen has been hypothesized to help support blooms of harmful algae such as K a r e n i a b r e v i s , the toxic ‘red tide’ organism of theGulf of Mexico (Lenes et al. 2001, Glibert et al. 2006). This hypothesis is presently under debate (Hu et al. 2006). Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 79**Potential Chronic, Insidious Impacts on Human Health**

Present understanding about the effects of toxigenic cyanobacteria on human health are mostly limited to obvious, acute impacts, and thus do not include chronic, subtle or insidious impacts that are happening but not yet detected, and potential impacts where h azards exist in remote areas where health impacts have not yet been sustaine d. It is expected that the risk from cyanobacterial CHABs to drinking an d recreational waters will increase with increased stress and demand on those systems (Glibert and Burkholder 2006) (e.g. Fig. 12, see Color late a n d F i g

. 1 3

, s e e C o l o r

cluding the increase in population and its exposure to toxins, the requirement for new drinking water and recreational sources and the speed and extent with which effective manage ment strategies are implemented. It is expected that the demand for drinking water in the US will increasedramatically in the future. Although many utilities in the US use underground water supplies, many also use surface water and that proportion is expected to increase in many areas (e.g. Martin 2001). Increasing population has placed increased demand on surface water supplies, either through their anthropogenic inputs or through their increased demand for drinking water. Conventional water treatment app ears to be ineffective in removingcyanotoxins from drinking water (Hoeger et al. 2004, 2005); cyanobacterial cells lyse during filtration and chlorine does not appear to readily oxidize the resulting MC (Wannemacher Jr. et al. 1993; Hitzfeld et al. 2000). To control HABS, algaecides, especia lly copper sulfate, are commonly added to surface drinking water sour ces (Chorus and Bartram 1999), butthis leads to cell lysis and a substantial release of cyanotoxins into the water, as well as potential copper toxicity (Kenefick et al. 1993). Powdered and granular activated carbon adsorptio n, with reported removal efficiencies of greater than 90%, removes toxins as well as intact cells and appears

to be a more effective and reliable methods for removing cyanotoxins than Cl (Hamann et al. 1990; Donati et al. 1994). A similar increased in risk is also expected for recreational waters where, for humans, the primary route of exposure is oral from accidental or deliberate ingestion of recreational water, inhalation of aerosolized ce lls or from dermal contact. P 2Plate 2). The magnitude of the threat will depend on many variables in8 0 A

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. S i n c l a i rOCCURRENCE OF CYANOBACTERIAL BLOOMS IN THELAKE PONTCHARTRAIN ESTUARY, LOUISIANAToxic cyanobacterial blooms have been reported in Lake Pontchartrain, an oligohaline lake bordering New Orleans,Louisiana. Lake Pontchartrain is a tidally influenced estuary that connects to the Mississippi River via the Bonnet CarréSpillway and to the Gulf of Mexico by the Rigolets strait, Chef Menteur Pass, and Lake Borgne. It represents the secondlargest salt–water lake in the US (1630 km2), after the Great Salt Lake in Utah, and the largest lake in Louisiana.Toxic cyanobacterial blooms (e.g.,Microcystis&Anabaena)have occurred on Lake Pontchartrain following the discharge of water from the Mississippi River to the estuaryvia the Bonnet Carré Bonnet Spillway. The spillway was constructed to divert floodwater through the lake before it reaches the city of New Orleans. The floodgate between thespillway and the river is only opened during severe flood events, but water can seep into the spillway through the closedgate (Lane et al. 2001).The seeping river water leaks brown clouds of sediment and nutrients into Lake Pontchartrain.When the floodgates have been opened in the past, the influxof fresh, nutrient–rich water has been followed by fish killsand toxic cyanobacterial blooms.The 1997 opening of the Bonnet Carré Spillway is one ofthe better–documented events where toxic cyanobacterialbooms occurred on the lake following the release of water fromthe river to the lake (Dortch et al. 1998). The algalbloom became apparent over large parts of the lake during May and produced high levels of hepatotoxins, promptinghealth advisories by the Louisiana Department of Health and Hospitals.Fig. 12.Anabaena bloom on Lake Pontchartrain. Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 81Fig. 12.

N 5 km

~2.2 miles 

### Falls Lake, North Carolina

~ 1

6

m

i l e

s ~ 1

6

m

i l e

s

N 5 km

~2.2 miles 

### Falls Lake, North Carolina

~ 1

6

m

i l e

s ~ 1

6

m

i l e

s

Fig. 13. Map of Falls Lake, NC, indicating six sampling stations and the location of the intake for the water treatment plant of the capital city, Raleigh. is sustaining rapid human population growth and associated increased nutrient runoff into receiving waters. Fr om Burkholder (2006b). (See Color Plate 2). T

h e w a t e r s h e d ) Burns. See Color Plate 2). 8 2 A

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. S i n c l a i r

c o n t

. ( Anabaena bloom on Lake Pontchartrain (Photo courtesy of JohnFig. 12 (Chapter 3). Anabaena bloom on Lake Pontchartrain (Photo courtesy of John Burns. See page 8 2).

N 5 km

~2.2 miles 

### Falls Lake, North Carolina

~ 1

6

m

i l e

s ~ 1

6

m

i l e

s

N 5 km

~2.2 miles 

### Falls Lake, North Carolina

~ 1

6

m

i l e

s ~ 1

6

m

i l e

s

Fig. 13 (Chapter 3). Map of Falls Lake, NC, indicating six sampling stations and the location of the intake for the water treatment plant of the capital city, Raleigh. The watershed is sustaining rapid human population growth and associated increased nutrient runoff into receiving waters. Fr om Burkholder (2006b). (See page 8 2). 

## Color Plate 2Fig. 14. Total N, total P, chlorophyll a (indi cator of algal/cyanobacterial biomass), and total N : total P ratios in Falls Lake plotted against distance downstream from

the headwaters to the dam.

The last station (intake) is the location of the intake for the City of Raleigh water treatment plant. About 75% of the phytoplankton cells in this reservoir during summer months are cyanobac teria, including various toxigenic species based on data from 2002–2005 (Burkholder 2006). The lower end of the reservoir where the water treatment plant is located presently has good water quality, but there is concern that as nutrient loading to the reservoir continues to increase, water quality will become more degraded throughout the reservoir and cyanobacteria will be further stimulated.

**Frequency and Severity Ranking.**

Both our exposure and our knowledge follow the hierarchy of frequency
and severity 

### freshwater > estuarine (brackish) > marine

. While adverse impacts of cyanobacteria in eutrophic freshwaters and (less so) estuaries are known worldwide (Whitton and Potts 2000), impacts on marine ecosystems have only recently begun to be recognized. Thus, the indicated rankings may change as additional information becomes available. Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 83

### Data Needs

To improve our ability to measure the incidence and predict the occurrence of CHABS, we need to make full use of existing information that is currently available through a variety of sources. State and regional investigators have conducted a number of surveys of cyanotoxins that needs to be compiled into a readily accessible database. Information that has been collected on the factors related to bloom occurrence should be compiled so

that it can be related to toxin occurre nce. This includes land use data, nutrient concentration in water, and weather conditions. Information on robust analytical methods for cyanobacter ia and toxin quantification in environmental waters needs also needs to be compiled.

**Short—term Objectives**

**Research Needs**

There is currently a lack of agreement on species level taxonomy for cyanobacteria. Because of this lack of agreement, cyanobacteria can only be identified easily to the genus leve l, which means that information may be lost in surveys when only genera ar e identified. Additionally, changingtaxonomy can be a confounding factor for older surveys. Modern molecular techniques offer one solution to this problem but even these techniques require a consensus on the definition of toxigenic cyanobacterial species. Considerable information on cyanobact erial species and their toxins is

also available in non–traditional litera ture sources. A comprehensive literature survey, including the grey literature, is needed. Both planktonic and benthic cyanobacteria should be included in this survey. These and

newer references need to be review ed and summarized for occurrence of toxigenic species. A working bibliography on cyanobacteria compiled by the EPA can be found at http://www. epa.gov/safewater/ucmr/ucmr1/docs/ meetings_umcr1_cyanobacteria_references.doc. An improved understanding is needed of the environmental factors that are responsible for cyanobacterial grow th and bloom formation. Additionally, a better understanding is needed of when cyanobacteria do and do not produce toxins, and factors responsible for production of toxins. This information can be obtained from both field studies and supporting laboratory experiments. Perhaps the best information currently available is for Microcystis aeruginosa ; the genes involved in toxin production have been identified, and various environmenta l factors including light, nitrogen, 8 A

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4Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 85

phosphorus, and iron nutrient supplies have been shown to influence toxin production (Zurawell et al. 2005).Research is needed to determine the importance of very small (picoplanktonic) cyanobacterial species in the production of cyanobacterial toxins. Similar research is needed for many benthic species. Among the many questions that remain unanswere d are: how significant are levels of toxins produced by planktonic and benthic species? How does their toxin production vary over time as compared to historically recognized bloom species? What kinds of toxins ar e produced by these cyanobacteria? BMAA originating from cyanobacteria on the roots of cycads has been reported to be associated with Alzheimer's disease in patients who havebeen exposed to BMAA from the food chain. Additional studies have detected BMAA in cyanobacteria from a variety of geographical locations (Cox et al. 2005). Because of the seriousness of Alzheimer's disease, plus the apparent widespread distribution ofBMAA, research is needed to determine the occurrence of BMAA in wate r in the US, including the fate of this toxin during water treatment for drinking water production. The conventional wisdom for cyanobacteria is that cylindrospermopsin is released extracellularly from hea lthy cells, whereas other commonly occurring cyanotoxins are contained in healthy cells, and only released if cells are damaged or dying. Recent evidence suggests that this generalization may not hold in some situations; thus, research is needed to verify that how toxins typically are distributed in commonly occurring species. These

studies should use more than one strain of each species, because as mentioned, within a given species of cyanobacteria are many nontoxic as well as toxic strains (Burkholder and Glibert 2006).Research is also needed to determine whether cyanotoxins are accumulated in the tissues of fish and she llfish. These studies should be done for the most important cyanotoxins which fish and shellfish are likely to be exposed to in both natural bodies of water as well as in aquaculture (e.g.nodularin in some estuarine/marine environments). The exposure of people to cyanotoxins originating from spray or aerosols should be investigated.

**Analytical Needs**

There is an immediate need for rapid techniques for both screening andconfirmatory methods for the cyanotoxin analyses, including simple, inexpensive methods for rapid screening of samples during sampling of blooms. Different types of methods are needed for different applications and each must be targeted towards its end users. Several ELISA screening86

In addition to identifying the toxins, there is a need for additional tools targeted towards identifying a pote ntially toxigenic organism. Species–specific and toxin–specific molecular probes, microarrays and other technology are needed for studies to dete rmine the occurrence and distribution of potentially toxigenic cyanobacteria. To be most useful, these detection

methods should be cost–eff ective, rapid and reliable. Standardized methods for quantifyi ng cells and estimating biomass are

also needed. These methods should be developed in conjunction withtaxonomic identification methods, since identification of species may depend on characteristics that may be altered in some cell quantification schemes. When developed, these methods should be validated. Since cell count methods using micro scopy generally are tedious and labor–intensive, alternate, accurate methods to dete rmine cell density or biomass are needed, such as improved techniques to quantify photosynthetic pigments, total suspended solids or particulate organic carbon. Unfortunately, these methods at present all fall short of accurately estimating cyanobacteria biomass, and cannot provide information about toxicity or the dominant taxa involved. Improvements in technology are needed to rapidly track toxigenic species and formation of blooms. These technologies include remote sensing, remote–operated ve hicles (ROVs) and automated sampling stations located on vulnerable bodies of water (Glasgow et al. 2004).

**Monitoring Needs**

There is an immediate need for a na tion–wide survey(s) on the occurrence and distribution of toxigenic cyanobacteria and their toxins throughout the US. Some surveys of cyanobacterial t oxins have been c onducted; however, information on cyanotoxin occurrence is still fragmented and limited. Any information of cyanotoxin occurrence is useful, but in particular, national A

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methods are available for microcystins, but none are currently manufactured for cylindrospermopsin and other toxins anatoxin–a. On–line detectors would be useful for water utilitie s for continuous monitoring; instrumental methods are needed for use where quantitation and specificity are important. Methods including LC/MS and LC/MS/MS have been developed, but none have been accepted as validated US EPA methods or consensus organization methods. There is a need for standardized instrumental methods that can be run by commercial labs. Many of the instrumental methods for cyanotoxins are dependent on sample cleanup methods that remove interfering substances. To be successful, these methods must perform in a wide variety of water types, including those with high levels of total organic carbon.Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 87

surveys, consistently conducted, ar e needed to assess cyanotoxin occurrence on a larger scale. The US EPA is planning to conduct a national survey of drinking water for cyanotoxins, under its Unregulated Contaminant Monitoring Rule (UCMR) when the suite of analytical methods needed becomes available. Monitoring will occur for one year atapproximately 2,800 large public water systems (PWS) and a representative sample of 800 (out of 66,000) small PWS. Transient water systems are not required to monitor nor are systems that purchase 100% of their wa ter (EPA 2005a). The monitoringresults from these systems will be used to form an initial nationally representative occurrence assessment of the most commonly occurring cyanotoxins in the US. A similar su rvey is needed for ambient watersources, including recreational and agricultural water. Currently, no program exists for conducting this kind of survey, which should include both randomly selected locations as well as targeted locations where blooms historically have been reported, or are reported to be in progress. This design would assure that both typical occurrences and unusual bloom events

would be considered and could be usedto determine risk to exposed people or animals. In many locations in the US, cyanobacterial blooms are common events. Nevertheless, they can be sporadic in occurrence and difficult to predict. Even targeting locations that recen tly have experienced blooms may not

mean that blooms will occur in these areas in the future; on the other hand, in developing watersheds blooms ma y begin to develop in previously

“bloom–free” locations. Undetected or missed blooms would result in an underestimate of the risk of exposure. A central reporting website needs to be established to assist in documenting blooms. This website needs to be publicized to utility operators, state department of environmental quality officials and others who would be aware of blooms as they occur. These reports would allow researchers who have the ability to analyze water samples for cyanotoxins to sample thes e events while they are occurring.

**Long—term Objectives**

**Research Needs**

As a longer–term project, an easily accessed database (i.e. website) on the historic occurrence of toxigenic strain s is critically needed, including user– friendly summaries for the general citizenry. This could be incorporated

into a national database of toxin occu rrence data that can be routinely updated. Currently, there is no central location for cyanotoxin data produced88

by states or other organizations. Long–term monitoring needs to be done to determine how cyanotoxins occur over time. This monitoring should also be done over a wide geographical area to determine how cyanotoxins are distributed spatially and to establish baseline information for assessinghow blooms and cyanotoxin distribution are changing over time. This research would clarify how commonly blooms resulting in high concentrations of cyanotoxins occur, and how this is related to geographical location. It is well known th at not all cyanotoxins have been described, so the distribution of known cyanotoxins may be greater presently understood. Thus, studies are also needed to identify new cyanotoxins.

**Analytical Needs**

For continual monitoring and developmen t of research tools, there is a long–term need for a stable supply of toxic organisms and toxins. Toxic strains of many species commonly have lost their ability to make toxins over time in the highly artificial cond itions represented by laboratory cultures. To avoid the loss of these sources of toxins, culture collections are needed which can supply a continuing source of toxin–producing strains of cyanobacteria recently isolated from natu ral habitats. Similarly, the development of analytical methods and othe r studies have been hampered by lack of availability of analytical chem istry standards for cyanotoxins. Information on concentration or puri t y o f a v a i l a b l e s t a n d a r d s i s u n a v a i l a b l e

**Monitoring Needs**

For long–term priorities, the same typ es of surveys are needed that were

described for short–term needs. Th ese surveys would include sampling at locations with typical levels of cyanobacteria as well as targeted surveysthat sample blooms in progress. To respond to the appearance of cyanobacterial toxins in a manner that will minimize public health risk, rapid analyses are needed which will permit a response before drinking water

consumers or recreational water users ar e exposed to the toxins. Rapid assays or screening methods are needed for this purpose. Data on long–term occurrence of cyanotoxins and cyanoba cteria that produce the toxins are needed to assess how commonly cyanotoxins occur, at what levels, and whether there are any long–term changes in their occurrence. o r a m b i g u o u s

. M a n u f a c t u r e r s n e e d t o p r o d u c e l a r g e r s u p p l i e s o f a t l e a s t t h e

m o r e c o m m o n t o x i n s , a n d t o c e r t i f y t h e i r p u r i t y

. T h e s e t o x i n s s h o u l d b e made commercially available. A

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### Summary

**Distribution of CHABs across the US**

• Freshwater blooms are ubiquitous across the US and Canada.

• Most cyanobacterial blooms are in freshwater, but some are found in

estuarine or marine waters.

• More detections of cyanotoxins ha ve been reported recently, however, increased awareness had led to more monitoring programs.

• Blooms of cyanobacteria tend to be greater and last longer in warmer

climates.

• D e t e c t i o n s i n a m b i e n t w a t e r s h a v e b e e n o b s e r v e d t o v a r y f r o m n o

r m o p s i n

. D e t e c

-

• Few data are available on temporal changes in cyanotoxin occurrence.

**Cyanotoxin production**

• Determination of taxonomic groups which produce toxins has been hampered by uncertainty in the ta xonomic definitions for cyanobacteria.

• Table 1 lists 22 genera and 42 species of cyanobacteria associated with

toxin formation.

• Table 2 lists taste and odor–producing species of cyanobacteria. d e t e c t i o n t o 3 1

, 4 7 0 μ g / L f o r m i c r o c y s t i n , n o d e t e c t i o n t o 1 5 6 μ g / L f o r anatoxin–a, and no detection to 202 μg/L for cylindrospe

tions occurred in treated water but concentrations were lower.

• Table 3 lists the commonly produced ty pes of cyanotoxins in freshwater.

• Microcystin has been reported to be produced by M i c r o c y s t i s

, A n a b a e n a

, O s c i l l a t o r i a ( P l a n k t o t h r i x

)

, N o s t o c

, H a

p a l o s i p h o n a n d A n a

b a e n o p s i s

• Cylindrospermopsin is produced by C y l i n d r o s p e r m o p s i s r a c i b o r s k i i

, A

p h a n i z o m e n o n o v a l i s p o r u m

, and U z m e k i a n a t a n s

.

• Anatoxin–a is produced by A n a b a e n a f l o s

– a q u a e

, A

. p l a n k t o n i c a

, O s c i l

l a t o r i a s p e c i e s , a n d C y l i n d r o s p e r u m . Homoanatoxin–a, was isolated

from P h o r m i d i u m F o r m o s a .

.• Anatoxin–a(s) was reported from A. flos–aquae strain NRC 525–17 and more recently by A

. l e m m e r m a n n i i

• Saxitoxin and related toxins are made by A p h a n i z o m e n o n f l o s

– a q u a e

, A n a b a e n a c i r c i n a l i s

, L y n g b y a w o l l e i and a Brazilian isolate of C y l i n d r o

s p e r m o p s i s r a c i b o r s k

i i

• BMAA is produced by N o s t o c species and many others.

• Lyngbyatoxins aplysiatoxin and debromoaplysiatoxin are made by

L

y n g b y a , Oscillatoriaceae such as S c h i z o t h r i x c a l c i c o l a and O s c i l l a t o r i a

n i

g r o v i r i d i s

• Cyanobacteria also produce other bioactive compounds.

**Impacts on waterbody health and ecosystem viability**

• The factors that lead to bloom formation and toxin production are not well understood. Light, temperature, hydrological and chemical factors may be involved.

• In freshwater, blooms in Metropolitan Water District’s reservoirs have led to taste and odor problems. A sewage spill in North Carolina led to

a L y n g b y a bloom.

• In estuarine habitats including the upper Chesapeake Bay, cyanobacterial blooms occurred with an invasion of water milfoil. Cyanobacterial blooms with fish kills ha ve occurred in brackish Lake Ponchartrain, Louisana from freshwater or nutrient influxes.

• Ecological effects have occurred as a result of cyanobacterial blooms that are caused by effects other than toxin production.

**Data Needs**

• A consensus is needed for speci es level taxonomy for cyanobacteria.

• Existing information needs to be compiled into an accessible form.

• New surveys should be conducted (including UCMR monitoring of drinking water), especially for newl y emerging toxins in raw water and drinking water.

• A national monitoring program is needed for recreational water. .

.

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• Analytical methods are needed including screening methods, instrumental methods, and online and remote detection. Sample preparation methods and analytical chemistry standards are needed to support analytical methods.

• A better understanding is needed of what factors lead to bloom formation and cyanotoxin production.

• The relation between toxin retenti on and release for different toxins

needs to be clarified.

• Accumulation of cyanotoxins in the tissues of fish and shellfish needs study.

• A website is needed for reporting of cyanotoxin occurrence information.92



### Glossary

Akinete – thick–walled, often enlarged cell that can survive adverse conditions and form a new filament; often refractive under light microscopy because it is filled with food reserve (cya nophycean starch); also ofte n orangish in color from abundant, protective carotenoi d pigments. This specialized cell is found in some

members of the Order Nostocales.

Anaerobic – Without oxygen.

Anoxic – Referring to habitats with little or no oxygen, such as the bottom waters of many eutrophic lakes in summer

Avian vacuolar myelinopathy [AVM] – a recently discovered neurological disease affecting water birds and their predat ors, primarily American coots and bald eagles, in the southern US. AVM causes char acteristic lesions in the myelin of the brain and spinal cord.

Benthic – Living in or on the bottom of a body of water, or growing attached tovarious substrata including other organisms (e.g. the noxious aquatic weed, hydrilla), but not free–floating in the water column. Note: Many benthic cyanobacteria can be moved into the plankton by wind/waves or other disturbance, and can

thrive in the water column.

Brackish – Somewhat salty, as in brackish (estuarine) water.

Buoyant – Floats easily, and is less dense than water.

Chlorophyll a – The “universal plant pigment”, found in all organisms that undergo plant–like photosynthesis with production of oxygen. The concentration of this pigment is often used as an indicator or surrogate measure for algal biomass or production.

Cyanobacteria – Formerly (and sometimes still) called blue–green algae, cyanophytes or mixophytes (means “slime plants”). In present–day taxonomy, considered a type of bacteria (“blue bacteria”) because of their primitive cell structureand other features, although they have the “universal plant pigment” that is directly involved in “higher plant” photosynthesis, chlorophyll a

.

Estuary – A semi–enclosed body of water where saltwater mixes with freshwater. A

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Eutrophic – Referring to aquatic freshwater and/or estuarine/coastal ecosystems that are nutrient–enriched and highly productive.

Filamentous – Group of cells aligned in a row; very thin, threadlike

.

Freshwater – Aquatic ecosystem with salinity lower than 0.1 ppt (see below).

Genus (plural: genera) – A taxonomic group below family and above species

Habitats – The places where plants and animals live.

Harmful algal bloom – A proliferation of microscopi c or macroscopic algae that are capable of causing disease or deat h of humans or beneficial aquatic life through production of toxins, or that ad versely affect aquatic ecosystems through other mechanisms (e.g. by depleting oxygen needed to sustain fish, or by reducing light that is essential for growth of beneficial submersed plants).

Heterocyte (formerly heterocyst) – thick–walled cell, usually pale in color compared to the other cells, used for nitrogen fixation in some cyanobacteria. This specialized cell is found in some members of the Order Nostocales.

Heterotroph – An organism that depends on consumption of other organisms and organic material for energy.

Hypersaline – referring to water with salinity higher than average seawater salinity.

Indicator species – A species whose status provid es information on the overall health of the ecosystem and of other spec ies in that ecosystem. Indicator species reflect the quality and changes in environmental conditions as well as aspects of community composition.

Macrophyte – Usually referring to a rooted submersed aquatic plant.

Mesohaline – Moderately salty water (salinity range 5–18 ppt).

Mixotroph – An organism that uses both photosynthesis and particulate or dissolved organic substances made by other organisms for its energy. Many cyanobacteria are mixotrophs.

Oligohaline – Low salinity (0.5–5 ppt).94

Phytoplankton– Microscopic algae that live and reproduce suspended in the water column of freshwater, estuarine, and marine ecosystems. Phytoplankton form the base of the food web in many aquatic ecosystems.

Picoplankton – Very small phytoplankton in aquatic ecosystems, including cyanobacteria (diameter 0.2 – 2 μm).

Polyhaline – Referring to waters that are variable in salinity, often highly brackish water with a salinity of 18–30 ppt.

Ppt – Parts per thousand, a measure of salinity. For example, the salinity of the open ocean is generally about 35 ppt; freshwaters are < 0.1 ppt.

Reservoir (impoundment) – an artificial (man–made) lake, formed by damming a

river.

Specialized cell – akinete, heterocyte (see above).

Strain – A population within a given species of cyanobacteria or microscopic algae.

Terrestrial – Living or growing on land or in the soil.

Tidal freshwater – Freshwater (0–0.5 ppt) that is tidally influenced.

Toxigenic – potentially toxic; referring to a species of cyanobacteria that is known to have some toxic strains. (Note: toxigenic algae have both nontoxic and toxic strains.) A

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### References

Albert S, O'Neil JM, Udy JW, Ahern KS, O'Sullivan CM, Dennison WC (2005) Blooms of the Cyanobacterium Lyngbya M a j u s c u l a i n C o a s t a l Q u e e n s l a n d

, Australia: Disparate Sites, Common Factors. Mar Poll Bull. 51 428–37. Anderson DM (1994) Red Tides. Scientific American. August 52–58. Arthur KE, Limpus CJ, Roelfsema CM, Udy JW, Shaw GR (2006) A Bloom of Lyngbya Majuscula in Shoalwater Bay, Queensl and, Australia: An Important Feeding Ground for th e Green Turtle ( Chelonia Mydas ). Harmful Algae. 5

251–65. Azevedo SM, Carmichael WW, Jochimsen EM, Rinehart KL, Lau S, Shaw GR, Eaglesham GK (2002) Human Intoxication By Microcystins During Renal Dialysis Treatment in Caruaru—Brazil. Toxicology. 181–182 (661) 441–46.Barron S, Weber C, Marino R, Davidson E, Tomasky G, Howarth R (2002) Effects of Varying Salinity on Phytoplankton Growth in a Low–Salinity Coastal

Pond Under Two Nutrient Conditions. Biol Bull. 260–61. Behm D (2003) Coroner Cites Algae in Teen's Death – Experts Are Uncertain

About Toxin's Role. Milwaukee Journal Sentinel , 2/5/04 2003. Berry JP, Gantar M, Gawley RE, Wang ML, Rein KS (2004) Pharmacology and Toxicology of Pahayokolide a, a Bioactive Metabolite From a Freshwater Species of L y n g b y a I s o l a t e d From the Florida Everglades. Comp Biochem Physiol–C: Phamacol Toxicol. 139 231–38. Birrenkott AH, Wilde SB, Hains JJ, Fische r JR, Murphy TM, Hope CP, Parnell PG, Bowerman WW (2004) Establishing a Food–Chain Link Between Aquatic Plant Material and Avian Vacuolar Myelinopathy in Mallards. J Wildlife Dis. 40 485–92. Boyer G (2006) Personal Communication. Boyer GL, Watzin MC, Shambaugh AD, Satchwell MF, Rosen BH, Mihuc T (2004) The Occurrence of Cyanobacterial Toxins in Lake Champlain. In Lake Champlain: Partnerships and Research in the New Millennium , eds. Boyer, GL, Watzin, MC, Shambaugh, AD, Satchw ell, MF, Rosen, BH, and Mihuc, T,

New York: Kluwer Academic/Plenum Publ. 241–57. Burkholder JM (2006) Falls Lake, North Carolina – a Major Potable Water Supply Reservoir Poised for Increased Cyanobacteria Blooms. Lakeline. Burkholder JM, Cuker BE (1991) Response of Periphyton Communities to Clay and Phosphate Loading in a Shallow Reservoir. Journal of Phycology. 27

373–84. Burkholder JM, Glibert, PM (2006) In traspecific Variability: An Important Consideration in Forming Generalizations About Toxigenic Algal Species. South Africa Journal of Marine Science. (in press). Burkholder JM (2002) Cyanobacteria. In Encyclopedia of Environmental Microbiology , ed. Bitton, G, New York: Wiley Publishers. 952–81. Burns J (2005) Cyanohabs – the Florida Experience. International Symposium on Cyanobacterial Harmful Algal Blooms Durham, NC. 09/6–09/10.96

Campbell R, Sargent R (2004) Wisconsin Teen's Death a Wake–Up Call About Toxic Algae. The Orlando Sentinel, 5 March 2004 , 2004. Carmichael WW (2006) Personal Communication. 07/26. Carmichael WW (1992) A Status Report on Planktonic Cyanobacteria (Blue Green Algae) and Their Toxins . Cincinnati, OH: Environmental Monitoring Systems Laboratory, Office of Research and Development, US Environmental Protection Agency. Carmichael WW (1998) Algal Poisoning. In The Merck Veterinary Manual , ed. Aiello, S, Whitehouse Station, New Jersey: Merck & Co., Inc. 2022–23. Carmichael WW (2001) Assessment of Blue–Green Algal Toxins in Raw and Finished Drinking Water . Denver, Colorado: AWWA Research Foundation and

American Water Works Assoc. ed. Carmichael WW, Azevedo SMFO, An JS , Molica RJR, Jochimsen EM, Lau S, Rinehart KL, Shaw GR, Eaglesham GK (2001a) Human Fatalities From Cyanobacteria: Chemical and Biol ogical Evidence for Cyanotoxins. Environmental Health Perspectives. 109 (6448) 663–68. Carmichael WW, Biggs DF, Gorham PR (1975) Toxicology and Pharmacological

Action of Anabaena Flos–Aquae Toxin. Science. 187 (118) 542–44. Carmichael WW, Li RH (2006) Cyanob acteria Toxins in the Salton Sea. Saline Systems. doi:10.1186/1746–1448–2–5Carmichael WW, Sirenko LA, Klochenko PD, Shevchenko TF (2001b) A Comparative Assessment of the Toxicity of Algae and Cyanobacteria in Water

Bodies of Ukraine. Phycologia. 40 (10338) 15. Castenholz RW (1992) Spec ies Usage, Concept, and Ev olution in the Cyanobacteria (Blue–Green Algae). Phycology. 28 737–45.Castenholz RW (2001) Phylum Bx. Cyanobacteria. Oxygenic Photosynthetic Bacteria. In Bergey’s Manual of Systematic Bacteriology , eds. D.R., B, and R.W., C, New York: Springer. 473–599. Chorus I, Bartram J (1999) Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management . London: E & FN Spon. (ed.) Christiansen G, Dittmann E, Ordorika LV, Rippka R, Herdman M, Börner T(2001) Nonribosomal Peptide Synthetase Genes Occur in Most Cyanobacterial Genera as Evidenced By Their Distribution in Axenic Strains of the PCC. Archives of Microbiology. 176 (388) 452–58.Codd GA, Morrison LF, Metcalf JS (2005) Cyanobacterial Toxins: Risk Management for Health Protection. Toxicology and Applied Pharmacology. 203 264–

72. Codd GA, Metcalf JS, Ward CJ, Beattie KA, Bell SG, Kaya K, Poon GK (2001) Analysis of Cyanobacterial Toxins By Physicochemical and Biochemical

Methods. J Assoc Off Analyt Chem Int. 84 (307) 1626–35. Cox PA, Banack SA, Murch SJ, Rasmussen U, Tien G, Bidigare RR, Metcalf JS, Morrison LF, Codd GA, Bergman B (2005) Diverse Taxa of Cyanobacteria Produce Beta–N–Methylamino–L–Alanine, a Neurotoxic Amino Acid. Proc

Natl Acad Sci. 102 5074–78.

A

. F r i s t a c h i a n d J

. L

. S i n c l a i rChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 97

Cuker BE, Gama P, Burkholder JM (1990) Type of Suspended Clay Influences Lake Productivity and Phytoplankton Response to P Loading. Limnology and Oceanography. 35 830–39.Devlin JP, Edwards OE, Gorham PR, Hunter NR, Pike RK, Stavric B (1977) Anatoxin–a, a Toxic Alkaloid From Anabaena Flos–Aquae Nrc–44H. Can J

Chem. 55 (177) 1367–71. Dittmann E, Borner T (2005) Genetic Contributions to the Risk Assessment of Microcystin in the Environment. Toxicology and Applied Pharmacology. 203

192–200. Donati C, Drikas M, Hayes R, Newcombe G (1994) Microcystin–LR Adsorption By Powdered Activated Carbon. Water Research. 28 (5328) 1735–42. Dortch Q, Peterson J, Turner R (1998) Algal Bloom Resulting From the Opening of the Bonnet Carre Spillway in 1997. Fourth Bi–annual Basics of the Basin Symposium 28–29. Drouet F, Daily WA (1956) Revi sion of the Coccoid Myxophyceae. Butler University Botanical Studies. 12 1–218. Drouet F, Daily WA (l957 ) Revision of the Coccoid Myxophyceae: Additions and

Corrections. Trans Am Microsc Soc. 76 219–22. Drouet F (l968) Revision of the Classification of Oscillatoriaceae. Monograph of the Academy of Natural Sciences . Philadelphia 15. EPA (2001) Creating a Cyanotoxin Target List for the Unregulated Contaminant Monitoring Rule. Summary of Meeting Held in Cincinnati, Oh on May 17–18,

2001 . Washington, DC: Office of Water, US Environmental Protection Agency. URL: http://www.epa.gov/OGWDW/ucmr/pdf s/meeting_ucmr1_m ay2001.pdf. Accessed: 06/15/05. EPA (2003) Statistical Analysis Methods for Biological Assessment . Washington, DC: US Environmental Protection Agency. URL: http://www.epa.gov/owow/monitoring/tech/appdixe.html . Accessed: 12/16/05. EPA (2005b) Drinking Water Contaminant Candidate List 2 . Washington, DC: Office of Water, US Environmental Protection Agency. URL: http://www.epa.gov/safewater/ccl/ccl 2_list.html. Accessed: 3/15/05. EPA (2005a) Unregulated Contaminant Monitori ng Regulation (UCMR) for Public Water Systems Revisions . 40 CFR Part 141. Washington, DC: US Environmental Protection Agency. 49093–138. Euzeby J (2004) Validation of Publication of New Names and New Combinations Previously Effectively Published Outside of the Ijsem. International Journal of Systematic and Evolutionary Microbiology. 54 1–2.Finn T, Kononen K, Olsonen R, Wallström K (2001) The History of Cyanobacteria Blooms in the Baltic Sea. Ambio. 30 172–78. Frazier W (2006) Personal Communication. 07/11. Geitler L (1932) Cyanophyceae. In Rabenhorst’s Kryptogamenflora , Leipzig: Akad. Verlagsges. Glasgow HB, Burkholder JM, Reed RE, Le witus AJ, Kleinman JE (2004) Real– Time Remote Monitoring of Water Quality: A Review of Current Applica-98

tions, and Advancements in Sensor, Telemetry, and Computing Technologies. Journal of Experimental Marine Biology and Ecology. 300 409–48.Glibert PM, Burkholder JM (2006) The Complex Relationships Between Increasing Fertilization of the Earth, Coastal Eutrophication, and Hab Proliferation, Chapter 24. In The Ecology of Harmful Algae , eds. Granéli, E, and Turner, J, Springer–Verlag: New York. (in press). Graham JL, Jones JR, Jones SB, Downin g JA, Clevenger, TE (2004) Environmental Factors Influencing Microcystin Distribution and Concentration in the

Midwestern United States. Water Research. 38 4395–404. Hamann CL, McEwen JB, AG M (1990) Guide to Selection of Water Treatment

Processes. In Water Quality and Treatment – a Handbook of Community Water Supplies , ed. Pontius, FW, New York: American Water Works Association. 157–87.Harada K (2004) Production of Secondary Metabolites By Freshwater Cyanobacteria. Chemical & Pharmaceutical Bulletin. 52 889–99. Harada KI, Fujii K, Hyashi K, Suzuki M, Ikai Y, Oka H (1996) Application of D,L–Fdla Derivatization to Determinati on of Absolute Configuration of Constituent Amino Acids in Peptide By Advanced Marfey's Method. Tetrahed

Lett. 37 (1386) 3001–04. HARNESS (2005) Harmful Algal Research and Response: A National Environmental Science Strategy 2005–2015 . Edited by Ramsdell, JS, Anderson, DM, and Glibert, PM. Washington, DC: Ec ological Society of America. ed.Henriksen P, Carmichael WW, An JS, Moestrup Ø (1997) Detection of an Anatoxin–a(S)–Like Anticholinesterase in Natural Blooms and Cultures of Cyanobacteria/Blue–Green Algae From Danish Lakes and in the Stomach

Contents of Poisoned Birds. Toxicon. 35 (1111) 901–13. Hisbergues M, Christiansen G, Rouhiainen L, Sivonen K, Börner T (2003) PCR– Based Identification of Microcystin–Producing Genotypes of Different Cyanobacterial Genera. Archives of Microbiology. 180 (10438) 402–10. Hitzfeld BC, Hoeger SJ, Dietrich DR (2000) Cyanobacterial Toxins: Removal During Drinking Water Treatment, and Human Risk Assessment. Environmental Health Perspectives. 108 (6) 113–22.Hoeger SJ, Hitzfeld BC, Dietrich D (2005) Occurrence and Elimination of Cyanobacterial Toxins in Drinking Water Treatment Plants. Toxicology and Applied Pharmacology. 203 231–42. Hoeger SJ, Shaw G, Hitzfeld BC, Dietrich DR (2004) Occurrence and Elimination of Cyanobacterial Toxins in Two Australian Drinking Water Treatment

Plants. Toxicon. 43 (14248) 639–49. Hoffmann L (2005) Nomenclature of Cyanophyta/Cyanobacteria: Roundtable on

the Unification of the Nomenclature U nder the Botanical and Bacteriological

Codes. Algological Studies. 117 13–29. Hu C, Muller–Karger FE, Swarzenski PW (2006) Hurricanes, Submarine Groundwater Discharge, and Florida’s Red Tides. Geophysical Research Letters. 33

doi:10.1029/2005GL025449.

A

. F r i s t a c h i a n d J

. L

. S i n c l a i rChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 99

Huisman J, Hulot FD (2005) Population Dynamics of Harmful Cyanobacteria. In Harmful Cyanobacteria , Aquatic Ecology Series. eds. Huisman, J, Matthijs, HCP, and Visser, PM, Dordrecht, The Netherlands: Springer. 143–76. Izaguirre G (2005) Harmful Algal Blooms and Cyanotoxins in Metropolitan Water

District’s Reservoirs. International Symposium on Cyanobacterial Harmful Algal Blooms Durham, NC. 09/6–09/10.Izaguirre G, Taylor WD (2004) A Guide to Geosmin–and Mib–Producing Cyanobacteria in the United States. Water Science and Technology. 49 (14448) 19–

24. Jacoby JM, Collier DC, Welch EB, Hardy FJ, Crayton M (2000) Environmental

Factors Associated With a Toxic Bloom of Microcystis Aeruginosa. Can J Fish Aquat Sci. 57 231–40. Kardinal WEA, Visser PM (2005) Dynamics of Cyanobacterial Toxins. In Harmful Cyanobacteria , Aquatic Ecology Series. eds. Huisman, J, Matthijs, HCP, and Visser, PM, Dordrecht, The Netherlands: Springer. Kenefick SL, Hrudey SE, Peterson HG, Prepas EE (1993) Toxin Release From Microcystis Aeruginosa After Chemical Treatment. Water Science and Technology. 27 (1316) 433–40. Kenefick SL, Hrudey SE, Prepas EE, Motkosky N, Peterson HG (1992) Odorous Substances and Cyanobacterial Toxins in Prairie Drinking Water Sources. Water Science and Technology. 25 (1317) 147–54. Kneale PE, Howard A (1997) Statistical Analysis of Algal and Water Quality

Data. Hydrobiologial. 349 59–63. Komárek J (2003) Coccoid and Coloni al Cyanobacteria, Chapter 4. In Freshwater Algae of North America – Ecology and Classification , eds. Wehr, JD, and Sheath, RG, Academic Press: Boston. Komárek, J, Anagnostidis K (1999) Cyanoprokaryota. 1. Chroococcales. In Suesswasserflora Von Mittleeuropa 19/1 , Stuttgart: Fischer Verlag. 548. Komárek J, Anagnostidis K (2005) Cyanoprokaryota. 2. Oscillatoriales. In Suesswasserflora Von Mittleeuropa 19/2 , Stuttgart: Fischer Verlag. 759. Krishnamurthy T, Carmichael WW, Sarver EW (1986) Toxic Peptides From Freshwater Cyanobacteria (Blue–Green Al gae) I. Isolation, Purification and Characterization of Peptides From Microcystis Aeruginosa and Anabaena Flos–Aquae

. Toxicon. 24 (414) 865–73. Lagos N, Onodera H, Zagatto PA, Andrinolo D, Azevedo SMFO, Oshima, Y (1999) The First Evidence of Paralytic Shellfish Toxins in the Freshwater Cyanobacterium Cylindrospermposis Raciborskii, Isolated From Brazil. Toxicon. 37 (11858) 1359–73.Landsberg JH (2002) The Effects of Harmful Algal Blooms on Aquatic Organisms. Rev Fish Sci. 10 (505) 113–390. Lane RR, Day JW, Kemp PG, Demcheck DK (2001) The 1994 Experimental Opening of the Bonnet Carre Spillway to Divert Mississippi River Water Into Lake Pontchartrain, Louisiana. Ecological Engineering. 17 (4) 411–22. Lapage SP, Sneath PHA, Lessel EF (1992) International Code of Nomenclature of Bacteria and Statutes of the International Committee on Systematic Bacteriol-ogy and Statutes of the Bacteriolog: Bacteriological Code . Washington, DC:

ASM Press. ed.Lehman PW, Boyer G, Hall C, Waller S, Gehrts K (2005) Distribution and Toxicity of a New Colonial M i c r o c y

s t i

s A e r u g i n o s a B l o o m i n t h e S a n F r a n c i s c o Bay Estuary, California. Hydrobiologia. 541 87–99.

Lippy EC, Erb J (1976) Gastrointes tinal Illness At Sewickley, Pa. J Am Water

Works Assoc. 68 (462) 606–10. Luesch H, Yoshida WY, Moore RE, Paul VJ (2000) Apramides a–G, Novel Lipopeptides From the Marine Cyanobacterium Lyngbya Majuscula

. J Natural Products. 63 (544) 1106–12. Lung WS, Paerl, HW (1988) Modeling Blue Green Algal Blooms in the Lower

Neuse River. Water Research. 22 (9648) 895–905. Mahmood NA, Carmichael WW (1986a) Para lytic Shellfish Poisons Produced By the Freshwater Cyanobacterium Aphanizomenon Flos–Aquae Nh–5. Toxicon. 24 (480) 175–86. Mahmood NA, Carmichael WW (1986b) The Pharmacology of Anatoxin–a(S), a Neurotoxin Produced By th e Freshwater Cyanobacterium Anabaena Flos– Aquae Nrc 525–17. Toxicon. 24 (481) 425–34. Martin E (2001) Drying Up – North Carolina’s Unquenchable Thirst Could Lead to Something More Than a Drinking Problem. Business North Carolina. Oct

46–59. Matsunaga S, Moore RE, Niemczura WP, Carmichael WW (1989) Anatoxin–a(S),

a Potent Anticholinesterase From Anabaena Flos–Aquae

. Journal of the American Chemical Society. 111 (494) 8021–23. Moikeha SN, Chu GW (1971) Dermatitis–Producing Alga Lyngbya Majuscula Gomont in Hawaii. II. Biological Properties of the Toxic Factor. J Phycol. 7 (542) 8–13. Moikeha SN, Chu GW, Berger LR (1971) Dermatitis–Producing Alga Lyngbya Majuscula Gomont in Hawaii. I. Isolation and Chemical Characterization of

the Toxic Factor. J Phycol. 7 (543) 4–8. Murch SJ, Cox PA, Banack SA (2004a) A Mechanism for Slow Release of Biomagnified Cyanobacterial Neurotoxins and Neurodegenerative Disease in

Guam. Proc Natl Acad Sci. 101 12228–31. Murch SJ, Cox PA, Banack SA, Steele JC , Sacks OW (2004b) Occurrence of Beta–Methylamino–L–Alanine (BMAA) in Als/Pdc Patients From Guam.

Acta Neurol Scand. 110 267–69. 100 A

. F r i s t a c h i a n d J

. L

. S i n c l a i r

Li R, Carmichael WW, Liu Y, Watanabe MM (2000) Taxonomic Re-evaluation of Aphanizomenon flos-aquae N H

- 5 B a s e d U p o n M o r p h o l o g y a n d 1 6 S r R N A Gene Sequences. Hydrobiologia. 438(1) 99–105. Li RH, Carmichael WW, Brittain S, Eaglesham GK, Shaw GR, Liu Y, WatanabeMM (2001) First Report of the Cyanotoxins Cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curuata (Cyanobacteria). J Phycol . 37(6)

1121–1126. Li RH, Carmichael WW (2003) Morpholog ical and 16S rRNA Gene Evidence for Reclassification of the Paralytic Shellfish Toxin Producing Aphanizomenon flosaquae LMECYA 31 as Aphanizomenon issatschenkoli J Phycol . 39 814–818.Chapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 101

Osborne NJT, Webb PM, Shaw GR (2001) The Toxins of Lyngbya Majuscula and Their Human and Ecological Health Effects. Environ Int. 27 (328) 381–92. Padisak J (1997) Cylindrospermopsis Raciborskii ( W o l o s z y n s k a ) S e e n a y y a E t Subba Raju, an Expanding, Highly Adaptive Cyanobacterium: Worldwide Distribution and Review of Its Ecology. Arch Hydrobiol/Suppl. 107 (4)

Paerl HW (1996) Microscal e Physiological and Ecological Studies of Aquatic Cyanobacteria: Macroscale Implications. Microscopy Res Technique. 33 (3658) 47–72. Pawlik–Skowronska B, Skowronski T, Pirszel J, Adamczyk A (2004) RelationshipBetween Cyanobacterial Bloom Composition and Anatoxin–a and Microcystin Occurrence in the Eutrophic Dam Reservoir (Se Poland). Polish J Ecology. 52 479–90.

Pinckney JL, Millie DF, Vinyard BT, Paerl HW (1997) Environmental Controls of Phytoplankton Bloom Dynamics in the Neuse River Estuary, North Carolina,

USA. Canadian Journal of Fisheries and Aquatic Science. 54 2491–501. Pluotno A, Carmeli S (2005) Banyasin a and Banyasides a and B, Three Novel Modified Peptides From a Water Bloo m of the Cyanobacterium N o s t o c S p

. Tetrahed. 61 575–83. Rapala J, Sivonen K (1998) Assessment of Environmental Conditions That Favor Hepatotoxic and Neurotoxic Anabaena Spp. Strains Cultured Under Light Limitation At Different Temperatures. Microbial Ecol. 36 (8038) 181–92. Richardson LL (2005) Coral Dis eases: What is Really Known? Trends in Ecology

and Evolution. 13 438–43. Saker ML, Griffiths DJ (2001) Occurr ence of Blooms of the Cyanobacterium Cylindrospermopsis Raciborskii (Woloszynska) Seenaya Et Subba Raju in a North Queensland Domestic Water Supply. Mar Freshwater Res. 52 (320)

907–15. Sasner Jr. JJ Ikawa M, Foxall TL, Watson WH (1981) Studies on Aphantoxin

From Aphanizomenon Flos–Aquae in New Hampshire. In The Water Environment: Algal Toxins and Health , ed. Carmichael, WW, New York: Plenum

Press. 389–403. 563–93. Niemi RM, Knuth S, Lundstrom K (1982) Actinomycetes and Fungi in Surface

Waters and in Potable Water. Applied and Environmental Microbiology. 43 (599) 378–88. NOAA (2004) Harmful Algal Bloom and Hypoxia Research and Control Reauthorization . Public Law 108–456, 01/06. US Department of Commerce, National Oceanic and Atmospheric Administration.Onodera H, Oshima Y, Henriksen P, Yasumoto T (1997) Confirmation of Anatoxin–a(S), in the Cyanobacterium Anabaena Lemmermannii, as the Cause of

Bird Kills in Danish Lakes. Toxicon. 35 (4098) 1645–48.

Pereira P, Li R, Carmichael WW, Dias E, Franca S (2004) Taxonomy and Production of Paralytic Shellfish Toxins by the Freshwater Cyanobacterium Aphanizomenon gracile LMECYA40. E u r

o p e a n J o u r n a l o f P h y c o l o

g

y . 39(4) 361–369.102

Spumigena in the Baltic Sea and the Structure of the Toxin. Applied and Environmental Microbiology. 55 (855) 1990–95. Sivonen K, Rapala J (1998) Assessment of Environmental Conditions That Favor Hepatotoxic and Neurotoxic Anabaena Spp. Strains Cultured Under Light Limitations At Different Temperatures. Microbial Ecol. 36 (3538) 181–92. Skulberg OM (1984) Directory to Toxic Cyanophy te Literature From Norden

. Oslo: Norsk institutt for vannforskning (NIVA). ed. Soranno PA (1997) Factors Affecting the Timing of Surface Scums and Epilimnetic Blooms of Blue–Green Algae in a Eutrophic Lake. Can J Fish Aquat

Sci. 54 1965–75. Stanier RY, Kunisawa R, Mandel M, Cohen–Bazire G (1971) Purification and Properties of Unicellular Blue–Gr een Algae (Order Chroococcales). Bacteriol

Rev. 35 (906) 171–205.Tango P, Butler W, Michael B (2005) Cyanotoxins in the Tidewaters of Maryland’s Chesapeake Bay: The Maryland Experience. International Symposium on Cyanobacterial Harmful Algal Blooms Durham, NC. 09/6–09/10. Teixeira MDGLC, Costa MDCN, Carvalho VLPD, Pereira MDS, Hage E (1993) Gastroenteritis Epidemic in the Area of the Itaparica Dam, Bahia, Brazil. Bull Pan Am Health Org. 27 (11918) 244–53. Thomas NJ, Meteyer CU, Sileo L (1998) Epizootic Vacuolar Myelinopathy of the Central Nervous System of Bald Eagles (Haliaeetus Leucocephalus) and

American Coots (Fulica Americana)

. Vet Pathol. 35 (2838) 479–87. Touchette BW, Burkholder JM, Alexande r JL, Allen EH, James J, Britton, CH(submitted) Eutrophication and Cyanobacteria Blooms in Run–of–River Impoundments in North Carolina, USA. Lake and Reservoir Management.Vanderploeg HA, Liebig JR, Carmichael WW, Agy MA, Johengen TH, Fahnenstiel GL, Nalepa TF (2001) Zebra Mussel (Driessena Polymorpha) Selective

Filtration Promoted Toxic Microcystis Blooms in Saginaw Bay (Lake Huron)

and Lake Erie. Can J Fish Aquat Sci. 58 (6598) 1208–21. Vezie C, Brient L, Sivonen K, Bertru G, Lefeuvre J–C, Salkinoja–Salonen M (1998) Variation of Microcystin Cont ent of Cyanobacterial Blooms and Isolated Strain in Lake Grand–Lieu (France). Microbial Ecol. 35 (2978) 126–35. A

. F r i s t a c h i a n d J

. L

. S i n c l a i r

Schindler DW (1987) Determining Ecosystem Responses to Anthropogenic

Stress. Canadian Journal of Fisheries & Aquatic Sciences. 44 (Suppl. 1) 6–

25. Sellner KG, Lacouture RV, Parrish CR (1 988) Effects of Increasing Salinity on Cyanobacteria Bloom in th e Potomac River Estuary. Journal of Plankton Research. 10 (1) 49–61. Sims JK, Zandee Van Rilland RD (1981) Escharotic Stomatitis Caused By the "Stinging Seaweed" Microcoleus Lyngbyaceus (Formerly Lyngbya Majuscula): Case Report and Literature Review. Hawaii Med J. 40 (839) 243–48. Sivonen K, Jones G (1999) Cyanobacterial Toxins. In Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management , eds. Chorus, I, and Bartram, J, London: E & FN Spon. 41–111. Sivonen K, Kononen K, Carmichael WW, Dahlem AM, Rinehart KL, Kiviranta J, Niemelä SI (1989) Occurrence of the Hepatotoxic Cyanobacterium NodulariaChapter 3: Cyanobacterial Harmful Algal Blooms Workgroup Report 103

Whitton BA, Potts M (2000) The Ecology of Cyanobacteria: Their Diversity in Time and Space . Dordrecht; Boston: Kluwer Academic Publishers. ed. WHO (2002) Guidelines for Drinking Water Quality. Addendum: Microbiological Agents in Drinking Water . Geneva, Switzerland: World Health Organization.

142. WHO (2003) Algae and Cyanobacteria in Coastal and Estuarine Waters: Guidelines for Safe Recreational Water Envi ronments– Vol. 1 Coastal and Fresh

Waters . Geneva, Switzerland: World Health Organization. 128–35. Wilde SB, Murphy TM, Hope CP, Habrun SK, Kempton J, Birrenkott A, Wiley F, Bowerman WW, Lewitus AJ (2005) Avian Vacuolar Myelinopathy Linked to Exotic Aquatic Plants and a Novel Cyanobacterial Species. Environmental Toxicology. 20 348–53. Williams CD, Burns J, Chapman A, Flewel ling L, Pawlowicz M, Carmichael WW (2001) Assessment of Cyanotoxins in Florida's Lakes, Reservoirs, and Rivers

. Palatka, FL: St. Johns River Water Management District. Zurawell RW, Chen HR, Burke JM, Prep as EE (2005) Hepatotoxic Cyanobacteria: A Review of the Bi ological Importance of Microcystins in Freshwater

Environments. J Toxicol Environ Health–Part B. 8 1–37. Vezie C, Rapala J, Vaitomaa J, Seitsonen J, Sivonen K (2002) Effect of Nitrogen and Phosphorus on Growth of Toxic and Nontoxic Microcystis Strains and on Intracellular Microcystin Concentrations. Microbial Ecol. 43 (492) 443–54. Walker SR (2005) Nebraska Experience. International Symposium on Cyanobacterial Harmful Algal Blooms Durham, NC. 09/6–09/10. Wannemacher Jr. RW, Dinterman RE, Thompson WL, Schimdt MO, Burrows WD (1993) Treatment for Removal of Biotoxins From Drinking Water . Technical Report 9120. Frederick, MD: US Army Biomedical Research and Development Laboratory at Fort Detrick. Watkinson AJ, O’Neill JM, Dennison WC (2005) Ecophysiology of the Marine Cyanobacterium, Lyngbya Majuscula (Oscillatoriaceae) in Moreton Bay, Australia. Harmful Algae. 4 697–715. Wetzel RG (2001) Limnology . New York: Academic Press. 3 ed.