### Proceedings of Health Effects of Exposure to Cyanobacteria Toxins:

State of the Science

August 13-14, 2002

A product of the collaborative effo rts of the Florida Department of

Health, the Center for Disease Contr ol, Mote Marine Laboratory, the

University of South Florida, and leading scientific contributors that

presented at the co nference.

Editors: David Johnson, M.D., M. S., Raymond D. Harbi s on, Ph.D.

1Table of Contents

P a g e

Description of Monograph Paul C. Grivas, MPH 4

Panel 1: Harmful Algal Blooms in Florida Moderator: Karen Steidinger, PhD

Karen Steidinger, PhD Florida Fish/W ildlife Conservation 6 Comm ission - Florida’s Harmful Al gal Bloom Task Force: History and Focus

John Burns, MS Cyanobacteria and Their Toxins in 16 Florida Surf ace W a ters

Erin Bledso e, MS University o f Fl orida - T h e Distribution of 22 Potentially Toxic Cyanobacte r ia in Florida

Panel 2: National and Internation a l Perspective Moderator: Lora Flem ing, MS, PhD

Lorraine Backer, PhD, MPH Centers for Disease Control and Prevention– 37 A Brief Introduction

Ian Falconer, DSc Australia’s Cy anobacteria Experience 40

Michele Gid d ings, BSc Cyanobacteria Toxins: The Developm ent 43 and Evaluation of Methods to Determ ine Microcystin Levels in Canadian W a ter Supplies

Anthony Lloyd, MS Blue-Green Alg ae and T h eir Toxins – 65 Great Britain’s Perspective

Ian Falconer, DSc WHO Guidelines (Summary) 73

2Page

Panel 3: Agency Activ i ties Moderator: Bart Bibler, PE

Wayne Carm ichael, PhD W r ight State University – Laboratory 74 Methods for the Study of Har m ful Cyanobacteria W a terbloom s: “The CyanoHABs”

Jam e s Evans, PhD Florida Dep a rt m e nt of Health – 86 Cyanobacteria Toxins

Jam e s Sinclair, PhD US Environm ental Protection Agency – 89 Current EP A W o rk on Algal Toxins in Drinking Water

Richard Drew, MS Florida Depart m e nt of Environm ental 93 Protection – Summ ary of FDEP

Richard Clark, MS Florida Depart m e nt of Health – DOH 97 Aquatic Toxins Program

Bruce McLeod, MS Florida Manat ee County – Summ ary of 100 Water Treatm ent

3A Brief Intr oduction to : Health Effects of Exposure to Cyanobac teria T oxins: State of the Science by Paul C. Grivas, M.P.H.

The conference entitled “HealthEffects of Exposure to Cyan obacteria Toxins: State of the Science” took place on August 13 th to the 14 th , 2002 in Sarasota, F l orida. The conference was sponsored by the Florida Depart m e nt of Health (FDOH) in collaboration with the Centers for Disease Control and Pr evention (CDC). Funding for the conference was provided, in part, through a Coope rative Agreem ent (U50/CCU415409-05-5 Cooperative Agreem ent for Pfiesteria-Rel ated S u rveillance and Prevention).

Cyanobacteria and th eir potential impact on human health is an em erging public health issue that has received in creasing scientific interest resulting in new and ongoing research. While it has been substantiated th at cyanobacteria p r oduce tox i n s that are potentially capable of causing disease in hum ans, m a ny questions rem a in unanswered regarding the identification and quantification of cyanobact eria and cyanotoxin in the environm ent and to what degree cyanobacteria found in the environm ent translates into risks to public health.

The vision f o r this conference was to bring to g e th er le ading sc ientis ts and public health officials inv o lved with the em erging issues of cy anobacteria to share the latest inform ation and research findings. The confer en ce was a success in th is r e gard in that participating presenters included state, nationa l and internatio nal res earch ers and ag en cies involved with studying and addres sing issues regarding cyanob acteria and their toxins.

The presentations were organi zed into a fram ework consisti ng of three panels, the scope of each bein g specifically focused. The first and second pan e ls took p l ace on August 13, with a presentation at the Mo te Marine Aquarium following the second session. The last panel, along with a tour of a water treatm ent facility in Manatee County, Florida, too k place on the 14 th of August.

The initial panel of the conference addressed the issues associated with cyanobacterial and other algal bloom s in Florida. D r . Karen Steidinger, who is the Task Force Chair of the Florida Fish and W i ldlife Conservation Co mm ission at th e Florid a M a rine Res earch Institute, m o derated this first panel. Th e focus of the second panel encom p assed the nationa l and inte rnationa l pers pectives on cyanob acte r ial exp o sures. The moderator f o r this panel w a s Dr. Lora Flem ing, from the Un iversity of Miam i. Mr. Bart Bibler, a professional engineer, was the m oderator of th e third panel. This final panel addressed f e deral, s t ate and loca l ag ency activities, prim arilythe labo rato ry tes ting of water supplies, ass o ciated withcyanobacteria.

Dr. Alan Rowan and Dr. David Johnson, aff iliated with the Flor ida Departm e nt of Health, org anized and w e lcom ed the attend ees to the conf eren ce. They als o outlin ed th e schedule of events, including the order of presen ters for th e three panels.

4This m onograph is a compilation of the pr esenter m a nuscripts, figures and related inf o rm ational m a teria l s that repre s e n t the m a te rial pres ented at the conference. For o n e presentation, where the presenter did not pr ovide a m a nuscript, a brief summary of the presentation is provided.

The m a terial inform ation and opinions in th is m a nuscript are those presented by the individual presenters and doe s not necessarily re present the position of the FDOH, C D C or the University of South Florida. F u rthe rm ore, the individual presentations have not been edited or endorsed by the FDOH, CDC, or the University of South Florida. Technical editing and form atting of the m onogr a ph was provided. The scientific content and opinions presented herein are th ose of the individual presenter.

It is the intent that scientists, researchers, govern m e nt agencies and others will use this monograph as a source of recent research findi n g s and that it be availab l e for those who were unable to attend the conference.

Special than ks are due to Mote Marin e Laborat o r y, Sarasota Florida for their as sis t an ce in planning and organizing the m eeting facilities and to the University of South Florida for assistance in producing this m a nuscript.

5FLORIDA’ S HARMF U L ALGAL BLOOM TASK FORCE: HISTORY AND FOCUS

Karen A. Steidinger Florida Fish and W ildlif e Conservation Comm ission Florida M a rine Research Institute 100 Eighth Avenue, S.E. St. Petersburg, FL 33701

INTRODUCTION

Harmful Al gal Bloom s or HABs occur worldwide in m a rine and freshwater environm ents from Norwa y to New Zealand (1,2). They can be defined as above background concentrations of aquatic nonvascular plants that have harm ful effects on natural and hum a n resources. Bloom s can occur in the open ocean, bays and lagoons, and freshwater environm ents. Most of the harm ful a l gae are m i croscopic and photosynthetic with plant pigm ents such as chlorophyll and accessory pi gm ents. If the cell concentrations are high enough they can cause a visible di scoloration of the water.

Dinoflagellates, a group characte rized by two whip-like flagella, com p rise >70 percent of the known harmful m i croalgae. At least one toxic dinoflagellate is documented in the f o ssil reco rd back 50 m illion year s. There ar e >1 00 toxic HAB species w o rldwide. Most of these species produce potent toxins, e .g., neurotoxins, ichthyotoxins, hemolysins, cytotoxins, or reactive biocom pounds. HABs th at cause known public health im pacts in

m a rine waters, e.g., shellfish toxicity, are ro utinely m onitored as part of program s to reduce or prevent hum an exposure (IOC, website, 3).

In Florid a, there are abo u t 45 m a rine/estu a ri n e an d 15 freshwater toxic m i croalg ae tha t

can or cou l d cause an imal m o rtalitie s, anim al dis ease, and ev en hum an illness (4). In the

m a rine environm ent, the organism that causes almost annual coastal im pacts is a dinoflagellate known as Karenia brevis , a dinoflagellate that produces a potent neurotoxin. These events have been referred to a s Florida red tide.

Obviously, such living and hum a n resource im p acts result in econom ic impacts to local communities through reduced touris m and recreational activ ities, redu ced work attendance, and the resultant loss of revenue. Marine and estu arine HABs affect coasta l communitie s, but f r esh w ater HABs can af f ect both inland and coasta l c o mm unities because they can affect lakes, rivers, reservo i rs and potable water supplie s. In the case of cyanobacteria bloom s, particularly in surf ace water supplies th at are used for drink i ng water, tox i n s rele ased in to the water, if not rem o ved, can cause m ild to serious illness, even death. This has been docum ent e d for anim a l s such as cows and dogs as well as hum a ns (2,5). Hum a n death fr om cyanotoxin exposure is rare but documented in unusual circum stances such as contam inated water for dialysis in Brazil or drinking directly f r om a contam inated ditch in China. The cost of these HAB events, whether m a rine or f r eshwater, is not well ch arac ter i zed but can be in the m illion s and tens of m illions US D per episode depending on the exte nt and duration of the event. In the 1970s, two red tide

6outbreaks caused by K. brevis affected several west coas t coun ties f o r thre e to f i ve months and caused an es tim ated 15 to 20 m illion

- dollar USD im pact to th ose counties (4). Today, an econom ic report estimates th at from 1987 to 1992 the average annual cost for US m a rine HABs wa s 49 m illion USD (in 2000 dollars, 6).

In October of 1997, the then Secretary of the Florida Department of Environm ental Protection, Virginia W e therall, created an Ad Hoc Task Force on Harmful Algal Bloom s with the then Secretary of th e Florida Departm e nt of Hea lth, Dr. Jam e s Howell, as ViceChairm an. The Task Force was created to add r es s harm ful algal bloom s in Florida waters, particularly red tides caused by Karenia brevis and po tential f i sh k ill/dis ease events caused by Pfies t er ia pisc icida

. The concept for the Task Force followed a MidAtlantic Sta t es crisis with Pfies t eria fish kills an d a m a jor 1996 m a natee mortality in Florida due to an uncommon winter-spring K. brevis red tide. These two events in 1996 and 1997 provided the impetus for the task force. The Task Force orig inally consisted of > 50 representatives from state agencies, e .g., D E P, DOH, Gam e and Fresh W a ter Fish,
water m a nagem e nt districts, and the governor ’s office, state and private universities, private laboratories, and interested partie s. In 2000-2001, 35 m e mbers were reappointed and represented scientists, e ngineers, econom ists, citizens groups and governm e nt. The Task Force was origina l ly charg e d with id entifying existing inform ation and data on HABs in Florida, identifying data gaps, a nd recommending how the data gaps could be f illed by ne w research and m onitoring program s. The Task Force cr eate d a 15 m e mber Technical Advisory Group (TAG) to devel op a review and make recommendations for the whole T a sk Force to consider an d a pprove. The recommendations were m a de in March 1999 and presented to the HAB Task For ce (4). These were approved by the Task Force and finalized in June 1999 and subm itted to the Legis l ature by October 1, 1999 , a requirem e nt of Florida law.

Until Julyof 1999, the T a sk Force w a s an ad hoc body. After the 1999 legislative ses s ion it becam e a legislatively created Task Force with a m a ndate, Ch. 370.06092, F.S. The Task Force had four specific tasks and serves as an advisory body to the Director of the Florida M a rine Research Institute (F MRI): 1) determ ine the status and adequacy of existing inform ation, 2) develop research and m onitoring priorities including detection, prediction, m itigation, and contro l, 3 ) deve lop recommendations for governm e nt whereby they can create response/contingency plans fitting their specific need s and resources, and 4) m a ke recomm endations to FMRI by October 1, 1999. Another statute (Ch. 370.06093, F.S.) authorized FMRI to im ple m ent a c ooperative HAB program and spend authorized funds on specific m a rine and estuarine topics if State m onies are appropriated or if federal and private grants are received. State fun d s were appropriated in f i scal years 1998-1999 to 2000-2001 for Task Force activities. Between July 1998 and July 2001, the Task Force received three m illion dollars through the Florida Fish and W i ldlife Conservation Comm ission’s Flor ida Marine Res e arch Ins t itut e. In addition, Task Force projects received $270,000 from FM RI’s 2001-2 002 appropriation. Each fiscal year, the appropriated m onies were non-recurring which m eant th at each year th e monies had to be requested in the agency’s budget request and lobbied for by the agency and private groups interested in Task Force priorities. Other State HAB monies during this tim e period were appropriated for FMRI and Mote Marine Laboratory (MML), a private

7laboratory in Sarasota, Florida, through FMRI. All m onies were directed toward the data gaps identified in the 1999 Task Force report or toward supplem e nting a federal grant awarded to Florida s c ie ntists ca lled ECOHAB : Florida withFMRI as the coordinating entity.

The March 1999 “white paper” on Harmful Al ga l Bloom s in Florida was restricted to HABs that Task Force mem b ers identified as priorities, i.e., red tides caused by Karenia brevis , possible fish kills or disease events caused by Pfies t eria piscicida , ciguatera fish poisoning events due to Gambierdis cus tox i cus and other ciguatera dinoflagellates, bluegreen algae or cyanobacteria bloom s in freshw ater and estuaries, potential tum o r causing dinoflagellates such as P r orocentrum , and m acroalgal blooms caused by Caulerpa, Codium, Gr acilaria and other m a rine m acrophytes. Although Florida has m i croalgal species that cause diarrhei c shellfish poisoning and am nesic shellfish poisoning, no public health cases of these illnesses have beenr e ported to d a te and there f ore this is n o t a current problem although it still rem a ins a po tential problem . At the tim e of the 1999 report, the T a sk Force co nsidered red tides caused by K. brevis to be the m o st sign if ican t HAB f actor to im pact Florida in te r m s of natural resources, public health and econom i cs because of its long history of neurotoxic sh ellfish poisoning cases, aero s ol irritation to beachgoers, alm o st annual occurrences with thou sands of square kilom e ters affected, and duration of bloom s from one to 18 months.

RED TIDE

Florida red tides, one type of HAB, result in m o rtalities of aq uatic anim als such as f i s h, birds or even m a rine m a mmals. Massive fi sh kills have been docum ented since the 1800s off southwest Florida and were even m e ntioned in Spanish explorer diaries. Offshore reef fish or bottom fish, on the bottom or floating, were often the first causalities to be noticed by fisherm e n or di vers. Beached dead fish presen t esoteric problem s in a tourist based economy and offer a challenge for rem oval, particularly once red tide is transported in to bays and lagoons . In addition to living natural resources, Florida red tides can im pact hum ans in several ways.

Som e HABs can cause hu m a n illnes s or di scom fort th rough consum ption of toxic shellf ish (1, 2 ). Shellf is h such as oy sters, cl am s, m u ssels and other bivalves can filter out toxic m i croalgae as p r ey item s and bioaccum u late the tox i ns. Hum a ns in turn can becom e ill f r om toxic she llf ish m eats. In th e c a se of Florida re d tides, the hu m a n illnes s from consumption of bivalves that h a ve accum u lated th e toxin from K. br evis is ca lled Neurotoxic Shellfish Poisoning (NS P ). W ithother tox i c m i croalg ae, the shellf ish poisoning m a y have another nam e , like Paraly tic Shellfish Poisoning. Each type of shellfish poisoning has different symptom s

. Some cause death. Fortunately, NSP has not been associated with hum a n fatalities. Flor ida red tides also produ ce a to xic ae rosol that can irritate people’s eyes, nose, and throat if they are exposed to the aerosol at the beach or in an area where there is sea spray or a boat wake. Usually th e resp irato ry effects disappear within sev e ral hours after leaving th e b each or th e exposure area. Respiratory irr ita tion du e to tox i c particles airbo r ne in sea sp r a y is typ i ca l of som e Kar e nia species

8and has been reported for a Trichodesmium bloom in Brazil (2). Otherwise, respiratory irr ita tion is not comm only assoc i ate d with HABs.

The top eight Task Force priorities for red ti de data and informati on (4) were 1) predict onset and m ovem e nt of r e d tides in shelf wa ters, 2) develop rapid chem ical assays to replace the mouse bioassay, 3) develop econom i c im pact studies to evaluate losses by location or industry, 4) develop epidem iological studies to de term ine public health risks, 5) investigate existing technologies for dead fish cleanup, 6) investig ate applicability and efficacy of control and mitig ation m e thods, 7) de term ine the fate and effect of toxins in the m a rine environm ent, and 8) continue and enhance public infor m ation and outreach (4). Item s 3) thru 6) and 8) were funded w ith State funds and item s 1), 2) and 7) were funded with both federal and state funds. Since the initial funding sponsored by FMRI for Task Force initiatives, m o re federal funds have been m a de available for epidem iological and control studies through other sources.

About one-h alf of all Tas k Force dollars we re dev o ted to red tide projects. Additional state dollars for HAB res earch were appropr iated for FMRI and MML red tide studies. Task Force red tide projects funded included: supplem e ntal funds for ECOHAB: Florida cruises and research; eco nom i c im pacts caus e d by red tides, developm ent of new, rapid immuno-chem ical assay for brevetoxins, occupational exposure to red tide aerosol and its ef

f ects, a re d tides and p ublic he alth litera ture review, and removal of floating dead fish at sea. In addition to targeted Task Force funded projects, F M RI funded a business
outrea c h an d inf o rm ation projec t tha tre lied on th e Red Tide Allianc e and business community for input. T h e product of this venture can be seen at www.redtideonline.com

. Solutions to Avoid Red Tide (START), a gras sroots citizen group, conducted the project, which has b een extrem ely succes sful.

The legislative language that established th e Task Force and its duties specifically

m e ntioned the federal program called ECOHAB: Florida. T h is is a five-year federal, state, academ ic, and private laboratory partn e rs hip to stu dy the develop m ent of Florida red tides and be able to predict their occu rrence, move m e nt, and landfall through coupled biophysical models. ECOHAB: Fl orida consists of 23 princi pal investigators from 13 institutions in Florida, Michigan, Mississi ppi, North Carolina, South Carolina and New Jersey. The program is in the fifth and fi nal year. Task Force funds were used to supplem ent ship tim e and analysis of data for the coupled physical-biological 3D m ode l being developed by Drs. John W a lsh and Bob Weisberg of the University of South Florida based on their own studies and thos e of other PIs. T h e backbone of the ECOHAB: Florida p r og ram consists of three sampling strategies to gather data to develop the m odel, i.e., m onthly cruises onto the shelf in a closed hourglass configuration so that boundary co nditions of a contro l volum e of s eawater cou l d be identified (USF, Dr. Gabriel Vargo), a weekly transect out to 30 m iles during red tide season and at other tim es as well (MML, Dr. Gary Kirkp a tr ick), a nd a process cruise of several weeks during red tides in 1998, 1999, 2000, 2001 and 2002 (collaborating PIs).

9Although the ECOHAB studies are not com p lete, it is obvious that phys ical processes of
water m ovem e nt and physical concentration at fronts as well as nutri ents influences plant abundance both offshore and inshore. ECOH AB: Florida funded studies are evaluating physical processes and nutrients and their s ources in relation to coastal red tides. Nutrients can be organic or inorganic, m a jor such as nitrogen and phosphorus, m i nor such as the trace elem ent iron, and co m e from very diverse so urces, e.g., o ffshore upwelling, atm o spheric deposition, and terres trial river runoff and estuarine flux. Karenia brevis is light adapted, favored by colored dissolved organic m a tter near shore waters, and is able to utili ze organic N which has m a ny sources (W alsh and Vargo, pers. comm.). Different sources of nutrients coupl ed with phytoplankton gr owth strategies can influence w h at HABs occur when o nthe W e st Florid a shelf. For exam ple nitrate and silicate favor diatom bloom s as would be expected.

One of the theories for why Florida has K. brevis bloom s involves species adaptations and interactions in a physical and ch em ical m a trix on the wes tFlorid a shelf. This theo ry

can accoun t for m o st of the larg e red tides off the west coast o f Florida (7). The theory recognizes that Saharan dust contains iron and iron is deposited over the west coast during sto r m events when the dus t is carried f r o mSouth Af rica ac ross th e Atlantic to the Gulf of Mexico (7,8). T h ese dust events precede Trichodesm ium bloom s based on an analysis of the historical database. The Trichodesmium bloom s precede K. brevis bloom s, whi c h lag behind the cyanophyte bloom s. Trichodesmium is an atm o spheric nitrogen fixer but requires am ong other elem en ts, iron for its enzym e s. This cyanophyte or blue-green alga can occupy thousands of s quare kilom e ters of the Gulf of Mexico and can be very dense. The regenerated nutrients from these bloom s , particularly regenerated nitrogen, could fuel large red tides, as coul d decomposing dead fish from fish kills. Phosphorus and its im portance in Florida red tides are also being evaluated (Vargo, pers. comm.).

In addition to providing ECOHAB s upport fo r cruises, analyses, and m o deling, Task Force and other funds have supported the de velopm ent of an ELISA technique for the detection of brevetoxins in s eawater and shellfish (9). The end product of this test hopefully will be a rapid, accurate test kit th at can replace the mous e bioassay which is the current public health standard used for NSP. Although there are m a ny m o re red tide studies funded with state funds, particularly at F M RI and MML, one effort points out that gathering data is not enough. There has to be adequate storage, com p atibility and retrieval of data. Accurate m e tadata are am ong the critical n eeds. The coastal ocean s observing system for HABs in the Gulf of Mexico has the acronym HABSOS ( www.ncdda.noaa.gov/habsos ) and through a state-federal pa rtnership it has the capacity to store, in tegrate, a nd layer red tide data from the Gulf states and provide inform ation to the public and educators. The future of red tide and other HAB m onitoring and forecasting in Florida an d elsewhere is in au tom a ted ins t rum e ntation pack ages aboard various fixed and not fixed platform s such as buoys or underwater vehicles or even aboard sa tellites that tran sm it real time or near rea l tim e data to f acilities that can pa ck age it f o r ana l ys es. These monitor i ng ne tworks w ill f eed dir ectly to data rece iving and/o r

m a nage m e nt portals such as HABSOS.

10PFIESTERIA

In 1997, the Mid-Atlantic States were faced withan environ m ental scare from a dinoflagellate that was as sociated with fish k ills and fish lesions (10). The organism was called the “cell from hell” because it was reported to attack and kill fish. Although put with other HAB species, thes e dinoflagellates do not have their own chlorophyll like most other HAB species but they are capable of taking in pigm ented organism s as prey. At first the only described species was P. piscicida m eaning “fish killer”, later another species n a med P. shumwayae was discovered (11). Most re cently this second species has been described from Ne w Zealand, Australia, Norway, Japan and other locations (12,13).

There are other Pfiesteria appearing dinoflagellates in estu arine and m a rine waters th at have not been described yet, but m a y be inthis complex. Today, it is re co gnized th at Pfies t eria, p a rticular ly P

. shumwayae is widely distributed and is being studied from t h e aspect of being ichthyotoxic to fish. Different studies yiel d different results, yet all indicate P. shumwayae can kill fish via one or an other m echanism s (14, 15). In Pfiesteria there are varying degrees of t oxicity that are defined by whet her they can be induced to kill f i sh ; the r e ar e also n ontoxic s t ra ins. Finding such species in water o r sedim e nt

sam p les does not autom a tica lly m ean that th ere is a problem since th ey are widely distributed. If the area has bloom s of Pfies t eria and the strain is toxic, there could be a problem that is visualized in fish kills. On the other hand, Pfiesteria is often thought of as causing lesions in fish, part icular ly charac ter i stic les i ons near the caudal fin and the anal ven t . Recent ev iden ce suggests that a fungus causes th e lesions, which can be a prim ary pathogen (16, 17). Studies at FMRI are looking at this pat hogenetic pathway for induced lesions using the fungus Aphanomyces (Sosa and L a ndsberg, pers. comm .).

The Task Force id entif ie d nine pr iorities f o r Pfies t eria and Pfiesteria -like species (4), five of which are: 1) determ ine occurrence and distribution of PLS ( Pfiesteria -like species) in F l orida, 2) develop m o lecular prob es for iden tification and d i fferentiation o f PLS in Florida, 3) develop m o lecular probes for detection of PLS toxins or bioactive

com pounds in natural w a ters, 4) determ ine if PLS isolates are toxic or produce bioactive

com pounds that affect fish, and 5) determ ine the distribution of PLS in relation to fish disease o r f

i sh kill “hot s pots” and r e lations h i p to other env i ro nm ental f actors such as nutrients. T a rgeted funds for Task Force prio rities were directed to 2) and 4). Probes were developed for cryptoperidiniopsoids and P. piscicida and bioactive com pounds were detected in cryptoperidiniopsoid and in a P L S species k nown as “Lucy”. The latter produced a lipid-soluble ichthyotoxic substance.

CIGUATE RA

Ciguatera is known as tropical fish poisoning or TFP. It is associated with the

consum ption of tropical fish usually from reef areas. It can also be asso ciated with invertebrates such as gastropods. T h e sym p toms are sim ilar to neurotoxic shellfish poisoning h o wever, there is a very lo w hum a n mortality rate. There are o v er 175 kno wn hum a n symptom s from rashes to aching teeth to disorientation and para lysis (2 ). It is thought th at more than o n e toxin is involve d and that is what accounts for the m u ltitud e

11of sym p tomology. It is projected that there are 900 cases per year in Dade County alone (4).

The Task Force identified 10 priorities for ciguate ra research (4), five of which were: 1) develop accurate and rapid test s to detect ciguatera toxins in f i sh, 2) expand the testing network to docum e nt occurrence and extent of ciguatoxic fish, 3) develop a better monitoring system for recording and docum en ting confirm e d incidences of ciguatera poisoning, 4) increase awareness and training in the m e dical community for recognizing, docum enting, and treating sym p toms, and 5) de term ine the econom ic i mpacts, including loss of revenue and productivity due to illness an d trea tm ent. No ciguate r aprior i ties were funded. Dr. Lora Flem ing, a Universityof Miam i epidem iologist/physician and a Task Force m e mber, considers ciguatera a si gnificant public health problem in Florida that deserves investigation and research support.

TUMOR PROMOTING MICROALGAE

Bioactive compounds in dinoflagellates and cy anobacteria have been associated with
tum o rs in experim e ntal anim als (2). Since the causative organism s occur in Florid a, the Task Force identif ied f i v e prior i ties ( 4 ) for research on tum o r prom oting substances produced by HAB species: 1) expl ore potential role of biotoxins such as okadaic acid in
tum o r developm ent in se a turtles, either f o r dir e c tturm origen ic ef f ects or as suble t hal immunosuppressive factors, 2) determ ine fate and effects of toxins in food web, 3) conduct anim al exposure studies to determ ine effects of specific tum o r-prom o ting

com pounds, 4) isolate and m a intain potentia lly toxic species f o r toxin or bioactive

com pound i d entification, and 5) include potenti al species of concern in any statewide survey. Number 5) is being addressed in ev ent responses, but none of the priorities w e re funded for targeted Task Force funds.

MACROALGAE

Macroalgal bloom s in Florida can be extensiv e whether they occur in bays and lagoons or on the shelf, east co ast o r west coas t. These bloom s have had significant impact by altering habitat and affecting trophic pathways. The Task Force identif ied eight priorities (4), four of which are listed here. 1) Survey Codium populations out to 300 ft. depth in area of kno wn occurren ce for spatial and tem poral variability and determ i n e sources of drift algae on the east coast. 2) Survey invertebrate herb ivore populations and determine food sources and feeding rates. 3) Verify e nvironm ental regulators of growth, e.g., light,
tem p erature, and nutrients. 4) Determ ine nutrien t sources an d if there is a lag in

m acroalgal growth influenced by nutrient sour ce or storage. One project was funded to investigate the source of nitrogen for Codium and Caulerpa bloom s on the southeast coast and m ap the distribution of bloom for m ing m acroalgae.

CYANOBACTE R IA (BLU E-GREEN ALG A E)

The Task Force id entified cyanobacteria b l oom s in fresh and brackish water as an

em erging HAB problem in Florida and elsewher e. Bloom s occur in surface freshwaters

12that either are used for drinking water or are being considered for drinking water (Mr. John Burns and Dr. Chris W illiam s , pers. comm .) . The six T a sk Force priorities (4) were 1) determ ine the distribution of toxic and nontoxic strains, 2) develop epidem iological studies to determ ine public health risks, 3) develop econom i c im pact studies to evaluate losses by location or industry, 4) determ ine th e roles of nutrient enrichm e nt and m a nage d freshwater flow in blooms, 5) determ ine fate and effects of toxins in the food web, and 6) investigate control and mitig ation m e thods.

Exam ples of cyanobacteria projects funded with targeted Task Force funds were “Assessm ent of Cyanobacteria in Florida Lake s, Reservoirs, and Rivers”, “Cyanobacteria Autom a ted Detection Workshop”, “ Cylindrospermopsis Culture for Production of Cylindrosperm opsin”, and “Cyanobacteria Public Health Issues: Education and Epidem iologic Study. Many of these Task Fo rce projects will be discussed in this

m eeting. Som e of the results th at can be highlighted are 1) Microcystis

, Cylindrospermopsis , and Anabaena occur state-wide, 2) m i crocystin, cylindrospermopsin, and anatoxin-A have been found in surface waters, 3) m i crocystins have been found in finished drinking wate r, and 4) no hum an health problem s from cyanotoxins in finished drinking wate r have been docum ented in Florida.

In June 2001, the Task Force m e t to revi ew interim progress of Task Force funded projects. At that m eeting summaries prep ared by the principal investigators were discussed and questions were ra ised about the projects. Th e following were som e of the questions about the cyan obacteria projects.

• “W hen will standard s f o r toxins b e a v ailab l e ? ”

• “Can blooms be prevented or reduced? ”

• “W hat is the ef f ectivenes s of hom e f ilte rs on tap water f o r re moving toxins if present?”

• “W hat is the lag tim e between the blo o m and the s p ike at the water treatment plan t?”

• “W hat are ef f ective wate r tre a tm ents f o r toxins in drinking wa ter ? ”

• “If toxins can be detected, can they be rem oved? ”

For FY 2000-2001 cyanobacterial project recom m e nda tions, either specific projects were

recomm ended to be continued, e.g., survey s, epidem iology studies, and educational

m a terials or new tim e sensitiv e pro j ects, e.g., cu lture of Cylindrospermopsis for toxin standards and a workshop to discuss probes or sensors to detect cyanobacteria and quantify their toxins. The latter would be a ppropriate for natural waters as well as water
treatm ent plants. The workshop entitled “C yanotoxin Detection and Quantification and Instrum e ntation” laid out a pl an for initial collaboration and approaches. It looked at instrum e ntation and packaging of the instrum e ntation for deploym e nt into natura l wate r bodies or installation in treatm e nt plants.

13The three m illion do llars f o r Task Force project s to f ill in da ta gaps was su ccessf ul, but just as im portantly, project re sults were used to leverage for additional funds and other phases of research. Add ition a l funds were m a de available through other s ources to 1 )
prom ote chem ical or cell assays to replace m ouse bioassays, 2 ) inves tigate effects of red tide a e roso l on hum a ns, 3) inves tiga t e the poss i bility f o r cy an otoxins in dr inking wate r , 4) prom ote autom a tion of detection and quantitation m e thods for natural waters, aquaculture, and water treatm e nt plants, 5) prom ote state m onitoring program s , and 6)
prom ote new HAB studies not c onsidered in the first three rounds of Task Force funding by pursuing new grants. Lastl y, the Research Co ntracts Re view Comm ittee of the Task Force recomm ended that two workshops be he ld, one to address treatm ent m e thods for bloom s and cyanotoxins in dr inking water and the other to address public health and cyanobacteria. The Florida Departm e nt of Environm ental Protection held the first

m eeting recently and the second is this m ee ting you are attending toda y that is sponsored by the Florida Departm e nt of Health. These workshops and other Task F o rce deliberation s will lay th e groundwork for a rem a ining Task F o rce activ ity , that of proposing recommendations for state and lo cal governm e nts to consider in the developm ent of their res ponse/contingency plans.

ACKNOWLEDGEME NTS

Each Task Force m e m b e r , including the Tec hnical Advisory Group, is thanked for input into the preparation of the 1999 white paper. In addi tion, the Sarasota, Manatee, and Pinellas County legislative delegations are acknowledged for their support, particularly Senator McKay, Senator Carlton, an d Repres entative Bradley. Senator Ken Pruitt is especially th anked f o r his initia l vis i on in r ecogn izing that H A Bs are a statewide natural resource and public health issue.

REFE RE NCES

Steidinger, K. A. 2002. Red tides and othe r harmful algal bloom s , p. 2685-2693. In G. Bitton (Ed.) The Encyclo p edia of Environmental Microb iolog y . J. W iley & Sons, New York.

Landsberg, J. H. 2002. The effects of harmful algal bloom s on aquatic organism s. Rev. Fish. Sci . 10:113-390.

Hallegraeff, G. M., Anderson, D. M., Ce m b ella, A. D. (Eds.). 1995 . Manual on Harmful Marine Microalgae . IO C Manuals and Guides No. 33, UNESCO.

Steidinger, K. A., Landsberg, J. H., Tom a s, C. R. and Burns, J. W

. 1999. Harmful alg a l blooms in Florida. Unpublished tech nical report subm itte d to Florida’s Harmful Algal Bloom Task Force, Florida Marine Re search Institute, 63 pp. and appendices.

Falconer, I. (Ed.) Algal Toxins in Seafood and Drinking W a ter. Academ ic Press, London.

14Anderson, D. M., Hoagland, P., Kaoru, Y. and White, A. W

. 2000. Estimated Annual Economic Impacts from Harmful Algal Blooms (HABs ) in the United States, WH O I

2000-11, Woods Hole Sea Grant, Woods Hole.

Walsh, J. J. and Steidinger, K. A. 2001. Saharan dust and Florida red tides: The cyanophyte connection. J. Geophys. Res . 106:11,597-11,612.

Lenes, J. M., Darrow, B. P., Cattrall, C., He il, C., Vargo, G. A., Callahan, M., Byrne, R. H., Prospero, J. M., Bates, D. E., Fanning, K. A. and W a lsh, J. J. 2001. Iron fertilization and the Trichodesmium response on the W e st Florida shelf. Limnol. Oceanogr.

Naar, J., Bourdelais, A., Tom a s, C., K ubanek, J., W h itney, P. L., Flewelling, L., Steidinger, K., Lancaster, J. and Baden, D. G. 2002. A com p arative ELIS A to detect brevetoxins from Karenia brevis (form e rly Gymnodinium breve ) in s eawa t er, she llf ish

, and m a mma lian body fluid. Environ m ental Health Perspectives , 110:179-185.

Rubin, C., et al, Em erging areas of research reported during the CD C national conference

on Pfies t eria : from biology to public health. Environmenta l. Health Pers pectives, 109:633-637.

Glasgow, H. B., Burkholder, J. B., Morton, S.L., and Springer, J. 2001. A second species of ichthyotoxic Pfiesteria (Dinam oebales, Dinop hyceae). Phycologia 40:234-245.

Rhodes, L. L., Burkholder, J. M., G l asgow, H. B., Rublee, P. A., Allen, C., Ada m son, J. E. 2002. Pfiesteria shumwayae (Pfiesteriaceae) in New Zealand. NZ J. Mar. Freshw. Res. 36:621-630.

Jakobsen, K.S., Tengs, T., Vatne, A., Bowers, H. A., Oldach, D. W ., Burkholder, J. M., Glasgow, H. B., Rublee, P. A. and Klav eness, D. 2002. Discovery of the toxin dinoflagellate Pfies t er ia from northern European waters . Proc. Roy. Soc. Lond. (B) (in press).

Vogelbein, W. K., Lovko, V.J., Shields, J.D ., Reece, K.S., Mason, P.L., Haas, L.W ., and Walker, C.C. 2002. Pfies t eria shumw a yae kills fish by m i crop redation not exotoxin secretion. N a ture. 418: 967-970.

Law, M. 2001. Differential diagnosis of ulcerative lesions in fish. Environ. Health Perspect . 109:681-693.

Blazer, V. S, Lilley, J.H., Schill, W

. B., Kiryu, Y., Densm o re, C.L. Panyawachira, V. and Chinubut, S. 2002. Aphanomyces invadans on Atlantic Menhaden along the East Coast of the United S t ate s

. Journal of Aquatic Animal Health . 14: 1-10.

Kiryu, Y., Shields, J.D., Vogelbein, W.K., Zw erner, D.E., and Kator, H. 2002. Induction of skin ulcers in Atlantic m e nhaden by injection and aqueous exposure to the zoospores of Aphanomyces in vada ns

. Journal of Aquatic A n imal Health 14: 11-24.

15Cyanobacteria and their toxins in Florida Surface Waters John Burns Chris Williams Andre wChapman

Introductio n

Cyanobacteria have likely been an important ecological component of aquatic system s in Florida for thousands of years. Howeve r, increased eutrophic ation (e.g., nutrient enrichm e nt) of lakes, rivers, and estuaries ha s led to a growing tre nd in the incidence of cyanobacterial bloom s and the production of t oxic m e tabolites. Flor ida’s sub-tropical clim ate, prolonged drought, significant hydrologic m odifications, and continued urbanization have also contri bu ted to the increas ed occurren ce of toxic cyanobacterial bloom s.

Toxic cyano b acterial blo o m s in Florida were firs t record ed byW a yne Carm ichael (1992) in Lake Okeechobee (1987, 1989) and Lake Is tokpoga (1988). Dead cattle, signs of poisoning in laboratory m i ce, and contact irritation we re found associated with Anabaena and Micro c y

s tis bloom s. The toxin m i crocystin a nd an unidentified neurotoxin were attributed to the toxic effects found inFlorida lake sam p les. Although toxic cyanobacterial bloom s had becom e a m a jor c oncern throughout the world, and the W o rld Health Organization had set provisional guide lin es for the co nsum p tion of m i crocystinLR ( W HO 1998), little infor m ation on toxic cyanobacteria in Flor ida waters had been published since those first toxic events iden tified in 1992. Following formation of the Florida Harmful Algal Bloom Task Force (FHABTF) in 1997 (Steidinger et al. 1999), cyanobacteria were recognized as a group of potentially toxic species in Florida that had increased in distribution and abundance and possibly represen ted m a jor threats to water quality, ecosystem s, drinking water supplies, and public health. Moreover, the FHABTF found that the growing dependence on cyanobact erial bloom effected lakes and rivers for drinking water supply was a prim ary impetus for the evaluation of the distribution and potential toxicity of cyanobacterial bl oom s throughout the state (SJRW M D 2000).

Methods

During 1998, the FHABTF, Florida Marine Resear ch Institute, and St. Johns River Water Managem e nt District initiated a collaborative stu dy with the Florida Departm e nt of Health and W r ight State University to identify potential cyanobacterial toxins in Florida’s lakes, rivers, reservoirs, and estuaries. Sa m p les were collected and analyzed during 1999 and extended through 2000 to increase the unde rstanding of cyanobacterial toxins in
waters curre ntly utilized f o r drinking water or identified as a potential future drinking
water source.

Methods em ployed to identify alga taxa included m i croscopic exam ination and epifluorescence. Algal tox i ns were charact erized and quantified by enzym e linked immunosorbant assay (E LISA), protein phospha tase inhibition assa y (PPIA), HPLC-Fl,

16HPLC-UV, and LC/MS/MS. Mouse bioassays w e re used to characterize toxicity by intraperitoneal in jecti on of freeze-dried sam p le extracts in to ICR-Swiss male m i ce.

Results & Discussion

W ith the assistance of num erous state and lo cal agencies in 1999, a total of 167 sam p les were collected throughout Florida, eighty- eight of these samples, representing 75 individual w a ter bodies, were found to cont ain cyanotoxins. Appr oxim a tely 80% of the

sam p les containing cyanotoxins were found to be lethal to m i ce following intraperitoneal injection.

Seven genera of cyanobacteria were id entif ied f r o mwater samples co llec t e d. Microc ys tis (43.1%), Cylindrospermopsis (39.5%), and Anabaena (28.7%) were observed m o st frequently and in greatest concentration. Plankto thrix (13.8%), Aphanizomenon (7.2%), Coelosphaerium (3.6%) and Lyngbya (1.2%) were found less fr equently, but at tim es accounted f o r a significant proportion of the planktonic and m acroalgal species

com position. Aphanizomenon and Anabaenopsis were also found consistently during the 2000 survey. Cyanobacterial bloom s were common thr oughout the state, som e of whi c h for m ed continuous bloom s in eutrophic and hype reutrophic system s. Many of the water bodies affected by cyanobacterial bloom s were identified by water m a nagem e nt agencies as areas of current concern or were bei ng addressed by ongoing or proposed restoration effort s.

Algal toxins identified from bloom mate rial during the study included hepatotoxic

m i crocystins, neurotoxic anatoxin-A, and th e cytotoxic alkaloid cylindrosperm opsin. Subsequent identification of lyngbyatoxi n-A and debrom oaplysiatoxin were found associated w ith Lyngbya wollei bloom s collected from Florida springs.

Microcystins were the m o st comm only found t oxins in Florida waters, occurring in all 87

sam p les analyzed during 1999. During the 2000 survey, m i crocystins were detected in pre- and post-treated drinking water. Finish ed w a ter concentrations ranged from below detection levels to 12.5 μg L -1 . Microcystins are consideredthe m o st frequently found cyanobacterial toxins around the world. Over 60 structural variants of this cyclic peptide have been reported, causing considerable c oncern due to their high chem ical stability, high water solubility, en vironm ental persiste nce and exposure to hum ans in surface w a ter bodies. The W o rld Health Organization has se t a provisional consum ption lim it of 1 μg L -1 for m i crocystin-LR (WHO 1998). The m a mm a lian toxicity of m i crocystin occurs by active transport across mem b rane boundaries and is m e diated through binding to protein phosphatases (Runnegar et al. 1991; Falconer et al. 1992). A n alysis of protein phosphatase inhibition activity, an index of m i crocystin bi oactivity, was found to be positiv e in 4 4 (69%) of 6 4 Florida s a mples tes t ed . There is lim ited evidence of tum o r prom otion (Sato et al. 1984; Falconer 1991, Nishiwaki-Matsushim aet al. 1992, W a ng and Zhu 1996) and clastogenic dose-related increases in chromosom a l breakage by

m i crocystin (Repavich et al. 1990), but no m u tagenic evidence has been reported (Runnegar and Falconer 1982, Re pavich et al. 1990).

17Anatoxin-a is a potent neurotoxic alk a loid that has been frequ e ntly im plicated in animal and wildfowl poisonings (Ressom et al. 1994). It is considered a ni cotinic agonist that binds to neuronal nicotinic acetylcholine re cepto r s which lead s to depolarization and a block of electrical transm i ssion in the body (Soliakov et al . 1995). At sufficiently high doses (oral LD 50 = >5,000 μg kg -1 body weight), it can lead to paralysis, asphyxiation, and death (Carm i chael et al. 1975, Carm ichael 1997). The authors have suggested that unexplained bird and alligator m o rtality even ts during cyanobacteria l bloom s in Florida lakes m a y be due to exposure to neurotoxic compounds produced by species of Anabaena

, Aphanizomenon , and Cylindrospermopsis . Anatoxin-a was found in three finished water sam p les and in tissues from Blue Tilapia and one W h ite Pelican during surveys in 2000. Anatoxin-A was found in the gut and liver of a W h ite Pelican and in Blue Tilapia (0.51 to 43.3 μg g -1 ) and in finished drinking wate r (b elow d e tec t able lim its to 8.46 μg L -1 ). Prelim inary evidence suggests that Cylindrospermopsis raciborskii isolates from Florida lakes and rivers can produce anatoxin-a (Mark Aubel, CyanoLab; Wayne Carm ichael, W r ight State U n iversity).

All 1999 samples containing the organism Cylindrospermopsis were pos itiv e f o r the toxin cylindrosperm opsin. Nine (9) finished drinking water sam p les collected during the 2000 survey were positive for cylindrosperm ops in and ranged in concentration from 8.07 to 97.12 μg L -1 . Identification of the algal toxin cylindrospermopsin during this study
repres ented the f i rs t reco rd of this he patoto xic alkaloid in North Am erica. This toxin prim arily affects the liver, but extracts give n orally or injected in m i ce also induce pathological dam a ge to kidneys, spleen, t hym us, and heart (Hawkins et al. 1985, 1997). Cylindrosperm opsin is a potential important c ontam inant of drinking waters in Australia, Central Europe, South Am erica, and the Unite d States. The toxin was identified after 138 children and 10 adults w e re poisoned following a Cylindrospermopsis bloom and copper sulfate applications in a water supply reservoi r on Palm Island, Australia (Hawkins et al. 1985). Over 69% of the affected individuals required intravenous th erapy for electrolyte im balance, and the m o re severe cases fo r hypovolem ic and acidotic shock (Byth 1980). The oral toxicity or lethal dose of cylindr osperm opsin has been reported between 4.4 and 6.9 m g kg -1 mouse body weight with death occurri ng 2-3 days after treatm e nt (Hum pa ge and Falconer 2002). In experim e nts where cell free extract of Cylindrospermopsis was adm i nistered to m i ce in drinking water ove r 90 days, no pathological symptom s were recorded up to a m a xi m u m dose of 150 m g kg -1 day -1 (Shaw et al. 2001). Hum p age and Falconer (2002) suggest a Tolerable Da ily Intake and Guideline Value for cylindrospermopsin in drinking water of 1 μg L -1 based on an oral No Observed Adverse Effect Level of 30-μg kg -1 day -1 and a Lowest Observed Adverse Effect Level of 60-μ g kg -1 day -1

. Lyngbyatoxin-a and debrom oaplysiatoxi n have been identified from Lyngbya wollei an d L. majuscula sam p les collected from Florida springs and m a rine em baym ents, respectively (J. Burns, N. Osborne, and G. Shaw, unpublished data). Concentrations have ranged from below detec tion to approxim a tely 4.5 m g kg -1 . The aply siatox ins an d lyngbyatox i ns are cons idered derm a t otoxic alkaloids, caus i n g severe d e rm atitis am ong swimmers and other recreational users of water bodies that com e into direct contact with the organism (Mynderse et al. 1977, Fujiki et al. 1990). Aplysiatoxins are lethal to m i ce

18at a m i nim u m dose of 0.3 m g kg -1 (Moore 1977). Aplysiatoxins and lyngbyatoxins are also considered potent tumor prom ot ers and pr otein kinase C activators (Fujiki et al. 1990). Osborne et al. (2001) reviewed the hum an and ecological effects of Lyngbya majuscula b l oom s and reported acute contac t der m atitis in H a waii, Jap a n , and Austra lia. One potential death due to e xposure via ingestion of turtle m eat containing lyngbyatoxina was reported by Yasumoto (1998). Recent rep o rts from Ca mbodia (Decem ber 2002) include 140 illn esses, 10 0 individu als hospita lized, and 3 deaths following consum ption of m a rine turtle m eat suspected to co ntain Lyngbya toxins (Helen Rogers, Centers for Disease Con t rol & Prevention, Atlanta, GA). Se vere derm atitis has also b e en reported in Florida following recreational activities in waters supporting L y ngbya wollei bloom s in Florida’s springs (John B u rns, Ian Stewart and G. Shaw, unpublished data).

Summary

The occurrence of toxic cyanobacterial bloom s in Florida have becom e more prom inent following increased growth, declining groundw ater supplies, and identification of im paired surface waters as future drinking water sources. Cy anobacterial toxins hav e been identified in source waters used fo r drinking water supply and in post-treated drinking water during algal bloom events. Al gal toxin concentrati ons in post-treated drinking water have exceed ed existing and proposed W o rld Health Organization guidelines for the oral consum ption of m i cr ocystin and cylindrosperm opsin. Severe derm atitis h a s also be en reported by swimmers in Florida sp r i ngs where Lyngbya wolle i

m a ts have expanded following significant in creases in groundwater nitrate levels.

This information should be useful in developing appr opriate Total Max imum Daily Loads for impaired Florida waters that do not currently m eet their designated use. It could also support further efforts to charac terize potential ecologi cal and hum a n health risks due to toxic cy ano b acter ial bloom s . Identif ication of algal toxins in finished drinking water and reports of severe skinir ritation f

o llowing contact with toxic cyanobacteria should be utilized for jus tification and im ple m en tation of increased monitoring of potentially toxic cy an obacter ial bloom s by surface water m a nagers and water utilitie s . Epidem iologica l stud ies m ay also be required in Florida to assess potential hum a n health risks due to algal toxin consum ption at the tap and for those exposed to cyanotoxic bloom s during recreati onal users of lakes, springs and rivers.

References

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Carm ichael, W. W. 1997. The cyan otoxins. Adv. Botan. Res. 27:211-256.

Carm ichael, W. 1992. A Status Report on Pl anktonic Cyanobacteria (blue-green algae) and their Toxins. EPA/600/R-92/079.

Carm ichael, W.W., D. Bi ggs, and P. Gor m an. 1978. Toxicology and pharm acological action of Anabaena flos-aquae toxin. Science 187:542-544.

19Falconer, I. R. 1991. Tum o r prom otion and liver injury cau sed by oral consum ption of cyanobacteria. Environ. Toxi col. W a ter Qual., 6(2):177:184.

Falconer, I.R., A. Choic e , and W

. Hosja. 1992. Toxicity of edible m u ssels (Mytilus edulis) grow ing naturally in an estuary dur ing a w a ter bloom of the blue-green alga Nodularia spumigena . J. Environ. Toxicol. W a ter Qual. 7, 119-123.

Fujiki, H., M. Suganum a, M. Nakayasu, H. Hoshino, R.E. Moore, T. Sugim u ra. 1982. The third class of new tumor prom ot ers, polyacetates (d ebro moaplysiato x in and aplysiatoxin ) , can differentiate bio l o g ical act ions relevant to tum o r prom oters. Gann 73:495-497.

Fujiki, H., M. Suganum a, H. Suguri, S. Yosh izawa, K. Takagi, M. Nakayasu, M. Ojika, K. Ya m a da, T. Yasum o to, R.E. Moore, a nd T. Sugim u ra. 1990. New tumor prom ot ers from m a rine natural products. In: S. Hall and G. Strichartz (eds.) Marine Toxins: Origin, Structure and Molecular Pharm acology, Am er ican Che m ical Society, W a shington D.C., pp. 232-240.

Hawkins, P.R., M.T.C. Runnegar, A.R.B. Jackson, and I.R. Falconer. 1985. Severe hepatotoxicity caused by the tropica l cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (W oloszynska) S eenaya and S ubba Raju isolated from a dom e stic water supply reservoir. Appl . Environ. Microbiol. 50(5):1292-1295.

Hawkins, P.R., N.R., Chandrasena, G.J. J ones, A.R. Humpa g e, and I.R. Falconer. Isolation and toxicity of Cylindrospermopsis raciborskii from an ornamental lake. Toxicon 35:341-346.

Hum p age, A. R. and I. R. Falconer. 2002. Oral Toxicity of Cylindrospermopsin: No Observed Adverse Effect Level Determ inati on in Male Swiss Albino Mice. Cooperative Research Center for W a ter Quality and Treatm e nt, Salisbury, South Australia, Australia.

Moore, R.E. 1977. Toxins from bl ue-green algae. Bioscien. 27:797-802.

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. S., R.E. Moore, M. Kas h iwagi, T.R. Norton. 1977. Antileuk em ia activity in the Oscillato riaceae: isolati on of debrom oaplysiatoxin from Lyngbya. Science 196:53 8- 539.

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. Car m ichael, and H. Fujik i. 1992. Liver tumor prom otion by the cyanobacterial cyclic pep tide toxin m i croc ystin L R . J. Cancer Res. Clin. Oncol. 118(6):420-424.

Osborne, N.T., P. W e bb, and G.R. Shaw. 2001. The toxins of Lyngbya majuscula and their hum an and ecological health effect s. Environm ent In ternational 27:381-392.

20Repavich, W.M., W

. C. Sonzogni , J.H. Standridge, R.E. W e depohl, and L.F. Meisner. 1990. Cyanobacteria (blue-green algae) in W i sc onsin waters: acute a nd chronic toxicity. Water Res. 24(2):225-231. Ressom

, R., F.S. Soong, J. Fitz gerald, L. Turczynowicz, O. El Saadi, D. Roder, T. Maynard and I. Falconer. 1994. Health E ffects of Toxic Cyanobacteria (Blue-Green Algae). Australian National Health and Medi cal Research Council, Looking Glass Press.

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. Bur n s (1999). Harmful Al gal Bloom s in Florid a. Subm itted to Florida’ s Har m f u l Algal Bloom Task Force by th e Harmful Al gal Bloom Task Force T echnical A dvisory Group. Florida Marine Research Institute, St. Petersburg, Florida.

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21The Distrib u tion of Potentially Toxic Cyanobacteria in Florida Edw a rd J. Phlips Erin Bledsoe Mary Cichra Susan Bady lak

Jessica Fro s t Department of Fisheries and Aqua tic Sciences University of Florida Gainesville, Florida

The cyanobacteria, or blue-green algae, ar e the oldest group of algae on earth, dating back over 3 billion years, and are still one of the most widespread and abundant form s of photosynthetic organism s. The fact that a num ber of species in the group not only form bloom s but also produce powerful toxins, places them into a position of particular hum a n interest and concern. This conc e rn is particular ly acute in Florida, which is exceptionally blessed with an abundance of a quatic natural resources . There is perhaps no other state in the continen tal United States where the quality of life and economy is m o r e closely tied to the integrity and sustainability of these re sources. There is no doubt that the issue of potentially toxic cyanobacteria bloom s de serves the attention of research ers and

m a nagers throughout the state. In this paper we exam ine this issue from the perspective of our research activities around th e state over the past twenty years. During this tim e we have carried out long-term studies of plankt onic algae populations inthree m a jor m a rine ecosystem s (Florida Bay, Indian River Lagoon and the Suwannee Estuary) and five m a jor freshwater ecosystem s (Lake Okeechobee, the S t . Johns River, Lake Griffin, the Rainbow River and th e Suwannee River)(F igure 1). Despite the bro a d range of environm ental conditions represented by these seven ecosystem s, cyanobacteria play an im portant role in the structure and function of all seven. There are a num ber of factors that play a
prom inent role in the co ntrol of alg a l bl oom s in m a ny of these ecosystem s, including trophic state, water exch ange rates, light avai lability and g r owth-lim iting nutrients s t atus.

Algal Blooms and Trophic Status

Many of Florida’s lakes and rivers have anam ple supply of nutri ents to sustain high levels of phytoplankton biom ass. In wate r m a nagem e nt and limnological circles such lakes are called eutrophic, Greek for ‘well-fed’. One of the comm on publ ic

m i sconceptions is that eutrophic con d itions are a certain s i gn of the influence of human activity. Actually, eutrophic la kes and rivers can arise natu rally due to accum u lation of organic m a tter over tim e or edaphic factor s, lik e surface or ground water input from naturally nutrient-rich sedim e nts. These phe nomena, in part, expl ain the high frequency of eutrophic lakes in certain regions of cen tral F l orida, where phosphorus-rich sedim e nts are widespread, as indicated by the phosphate m i ning activities in the region. This does not im ply that hum ans do not play a m a jor ro le in prom oting algal blooms. There is substantia l e v idence that the explo s io n of hum a n developm ent in Florida over the past century has elevated the trophic status of m a ny lakes and rivers, in som e cases dram atically. W ith this elev ation in trophic status, the fre quency and intensity of algal

22bloom s has increased. In Florida, cyanobacter ia h a ve been ex ception a lly s u ccessful in taking advantage of the increase in bloom potential (Canfield et al. 1989).

The Distrib u tion of Potentia lly Toxic Algae in Florida

For m o st lakes and rivers in Florida, historic al records of algal com position are absent or
very sparse, m a king it almost im poss i ble to define the distribution of potentially toxic algae sp ecies in past y e ars. Even where sign if ican t data sets are pr esent, inf o rm ation on the tox i city of algal spec ies is largelyabsent. Therefore, in m o st cases it is only possible to discuss the distribu tio n of ‘potentially ’ t oxic algae species, wh ich curr e n tly in clude s the species listed in Table 1. The five cy anobacterial genera (gr oups) that contain the freshwater s p ecies of greates tconcern are Microcystis, Oscillatoria, Anabaena, Cylindrospermopsis and Aphanizomenon. There are also gro ups of cyanobacteria that for m blooms, but whose toxicity is not as well established, like Lyngbya and the widely occurring marine species Synechococcus

. Since the greatest risk associated with cyanobacterial toxins is found under bloom conditions, it is obvious that eutrophic a nd hypereutrophic lakes, rivers and coastal ecosystem s are the focus of m u ch attention. As expected, recent surveys of Florida indicate th at cyanobacteria are common features of ecosyst em s that exhibit elevated trophic status (Canfield et al. 1989 ). Even in lakes, rivers and estuaries subject to high levels of nutrient loading, the expression of that nutrient availability in the for m of algal bloom can be effected by other factors, like residence tim e , light lim itation or biom ass loss through grazing, senescence or disease. Th e influence of these factors is m a nifested in our observations of the seven aforem entioned ecosystem s.

Lak e s

Lake Okeechobee, Florida’s biggest lake and the second largest lake in the continental United States, has been the site of som e of th e most widely publicized algal bloom s in the country. Since intensive m onitoring of the lake began in the 1970’s, bloom s of the cyanobacteria Micro cys tis
, Anabaena

, Oscilla toria and Cylindrospermopsis have been observed on num e rous occasions, in m a ny cases covering ov er 100 square m iles of the lake. Most of the largest bloom s have beenconfined to a crescent-shaped region along the western side of the lake. The center and eastern part of the lake generally contain relatively low algal standing crops , due in large part to the low light availability caused by the re-suspension of muddy flocculent se dim e nts (Phlips et al. 1995, 1997). These observations dem onstrate that the distributi on of potentially toxi c alg a l sp ecies in structurally com p lex ecosystem s, lik e Lake Okeechobee, m a y not be uniform (Phlips et al. 1993). C y anobacteria bloom s in Lake Ok eech obee also ex hibit significant tem poral variation. T h e inten s ity and com position of bloo m s vary on both intra- and inter-annual tim e scales (Figure 2), reflecti ng changes in lake stage, nu trient lim itation and nutrient loading (Phlips et al. 1997).

In contrast to the spatial and tem poral hete rogeneity of blooms in Lake Okeechobee, in

som e lakes, bloom s of key species can persist for extended periods of time. For example,

23bloom conc entrations of Cylindrospermopsis in the hypereutrophic Lake Griffin have been observed to extend over the entire lake for periods in excess of a year (Figure 3). The ability of algae bloom s to persist for long periods of tim e in Florida is in part attributable to the sub-tro p ical clim ate expe rienced over the peninsula, which dim i nishes the seasonality of blooms so of ten se en in tem p erate latitudes. This does n o t m e an that
tem poral variations of clim ate are not im por tant in Florida. R ecent d r oug ht and flood periods have had a dram atic im pact on the eco logy of Florida’s aquatic environm ents. In Lake Griffin, drought periods have been char acterized by low water turnover rates and elevated algal standing crops. Conversely, flood periods, like the El Nino of 1997/98, can reduce phytoplankton concentrations for peri ods of tim e by i n creasing the rate water turnover and dilution of biom ass. This is a feature shared by m a ny lakes in Florida, which are of ten characterized by shallow depths and low water volum e.

From a broa der geographic perspective, there are few regions of Florida where bloom s of the m a jor species of alg a e outlined above cannot be found in a m u ltitude of nutrient-rich lakes. W h ile som e regions of Florida have a higher proportion of eutrophic and hypereutrophic lakes and rivers than others , recent m onitoring of over almost a 1000 Florida lakes indicates that a bout half are subject to at le ast periodic algal bloom s, if defined as chlorophyll a concentrations exceed ing 40 μ g / liter (L akewatch, 2003). There are also factors other than trophic stat us that encourage the predom inance of cyanobacteria over other algal groups in Florida. For exam ple, Anabaena, Cylindrospermopsis and Aphanizomenon all fall into a select category of photosynthetic organism s that can convert biologically unus able elem ental nitrogen (which com p rises 80% of the air) into the plant nutrient ammonia through a process known as nitrogen fixation. Since the growth of algae in m a ny of Florida’s lakes and ri vers is periodically lim ited by the supply of nitrogen fertilizer (like a mmonia) the ability of these organism s to carry out nitrog en fixation p l aces them at a dis tinct selectiv e advantag e over all other algae and p l ants, which are in capable of fixing nitrogen.

Another example of an advantageous feature shared by Microcystis, Oscillatoria, Anabaena, Cylindrospermopsis and Aphanizomenon is buoyancy regulation. Unlike

m a ny species of algae that depend on m i xing energy to stay afloat, these five cyanobacteria can adju st their positio n in th e water columnby inflating or deflating gas cham bers in their cells. This ability is a great adv a ntage in highly productive lak e s an d rivers where light available fo r photosynthesis can be restricted to only the top portion of the water co lumn. This attribute als o explains why bloom s of the aforementioned cyanobacteria are often o b served as the dreaded s u rface scum

.

Rivers

Many of Florida’s rivers are ch aracterized by high nutrient le vels, due to both natural and anthropogenic factors. However, these high nutrient levels do notalways lead to planktonic algae bloom s. Most often, this is due to the im pact of short residence tim e

. The Suwannee and Rainbow Rivers are good ex amples of the latter phenom e non. While both rivers contain sufficient concentrations of both nitrogen and phosphorus to sustain bloom conc entrations of planktonic algae, neith er does, due to the fact that water seldom

24stays in eith er system for m o re than a few days, preventing the accum u lation of planktonic biom ass.

The Suwannee River is a blackwater river, which origin ates in the Okeefenokee Swamp in southeastern Georgia before winding southward 394 km to the Gu lf of Mexico. The Suwannee River, with its tribu t ar ies the Alap aha, W ithlacoochee, and Santa Fe Rivers, drains 28,500 km 2 of southern Georgia and north- central Florida. Phosphorus-rich freshwater inflow from t h e upper portions of the watershed are augm ented by substantial nitrogen-rich groundwater contributions from num e rous sp rings along the riverbanks, giving the river the second highe st m ean annual discharge of any river in Florida. Despite thes e high nutrie n t leve ls, th e i r im p act is not generally realized as plankton production within th e river (Bledso e and Ph lips 2000), altho ugh accum u lations of benthic cyanobacteria, like the p o tentially to xic Lyngbya , can occur, particularly when the

norm a lly highly colored river water becom e s clearer due to drought conditions in the watershed. In the case of the Rainbow River, the shallow depths and crystal clear water provide the light necessary to support large popu lations of the potentially harmful benthic cyanobacterium

, Lyngbya (Phlips 1999). Blooms of Lyngbya have becom e a

m a nage m e nt issue in a num b er of Florida’ s river system s, like Crystal River.

In contrast to the high flow environm ents of the Suwannee and Rainbow Rivers, there are river s in Flo r ida whose g e ntle slope r e sults invery low flow rates and sufficient residence tim e to sustain planktonic algae bloom s . Mo st prom inent among these is the St. Johns River, which during drought years can actually f l ow backwards for significant periods of tim e due to tidal forces. Cyanobacteria bloom s in the St. Johns River can be both extensive an d intensive; reach ing chlorophyll a concentrations in excess of 100 μ g / liter (Phlips et al. 2000). Bloom s of all five of the potentially toxic algae g r ou ps outlin ed above have been observed in the river (C ichra and Phlips 2002). Som e tim es these bloom s follow a recurring pattern. F o r exam ple, bloom s of Cylindrospermopsis and Anabaena in the St. Johns River occur in the summ er, although the intensity of bloom s can vary from year to year (Figure 4). In contrast, bloom s of Microcystis in the s a m e river are less predictable, occurr ing at any tim e during the year.

Estuaries

The estuaries of Florida are often th e recip i ents o f the nutrien t load ing ex perien ced in their resp ective watersheds. Therefore, th e im pa ct of hum an developm ent on land is by

no m eans restricted to freshwa ter ecosystem s . By the sam e token, the impact of hum an developm ent on coastal ecosystem s extends beyond just the issue of nutrient loading. In restricted co astal ecosys t em s like Florida Bay and the Ind i an River Lagoo n, changes in water flow patterns can have as dramatic an effect on the dynam ics of algal bloom s as changes in nutrient loading.

In Florida Bay, a 1600 km 2 inner-shelf lagoon located just south of the Everglades and west of the Florida Keys, a network of ve ry shallow m ud banks located throughout the Bay restrict water exchange be tween inner regions of the bay and the Atlantic Ocean and Gulf of Mexico. One of the dom inant feat ures of phytoplank t on communities in Florid a

25Bay is the potentially harmfu l picoplanktonic cyanobacterium Synechococcus , which frequently reaches cell d e nsities in excess of 10 6 cells / m l in parts of the bay (Phlips et al. 1999). T h e relative dom i nance of Synechococcus during bloom events in Florida Bay is m a nifested by tem poral patterns of phytoplankton biovolum e at four s a m p ling sites (Figure 5) representative of eco logically distinct regions in the bay (Phlips et al. 1995b). The north central region of Florida Ba y is the focal point for bloom s of Synechococcus

, which spread south during the fall and winter due to shifts in wind direction. The physical restriction of tidal water exchange between the interior of the bay and the surrounding oceanic environm ent contribu te to the build up of cyanobacterial standing crop by low e ring the rates of water turnover. Prior to hum an developm ent, surface water in South Florida flowed from Lake Okeec hobee down through the Everglades and into Florida Bay. Extensive developm ent of S outh Florida has restricted this flow and exacerb a ted the conditio ns favorable for algal b l oom s in the bay.

In another one of Florida’s la rgest flow restricted estuarie s, the Indian River Lagoon, the picoplanktonic cyanobacterium Synechococcus is once again a prom inent com pone nt of planktonic algae bloom s (Phlips and Badyl ak 2002). Although dinoflagellates and diatom s are the dom inant bloom -form i ng gr oups in the lagoon overall, num e rous cyanobacterial bloom s, dom i nated by Synechococcus , have been observed in the m o st flow-restricted portions of th e lagoon, like the Banana River region (F igure 6). Bloom levels of another cyanobacteria genu s known to contain toxic species, i.e. Oscilla toria
, have also been observed in the lagoon. The la tter events app e ar to be asso ciated with periods of high rainfall and m a y be indicativ e of washout of freshwater algae into the lagoon, rather than in situ , production (Phlips, unpublished data).

On the west coast of Florida there are a num ber of m a jor river discharges that contain high concentrations of nutrients. These include the two rivers with the highest freshw ater discharg e in Florida, the Apalachico l a and th e Suwannee Rivers, both of which flow into the area of Florida known as the Big Bend. Th e Big Bend is noted as one of the m o st productive areas of Florida’s coastline and certainly the nutrient discharged by these rivers contributes to that productivity. There is, however, con cern that continued escalation of nutrient levels due to anth ropogenic activities m a y soon create serious environm ental prob lem s , sim ilar to th ose experienced in the Mississippi delta, like hypoxia and seagrass die-off. In the estuar ine region of the Suwannee River, bloom s of planktonic algae are common and are m o st often dom i nated by diatom s and dinoflagellates (Bledsoe and Phlips 2000, 2003). There are, however, periods in which

sm all spherical cyanob acteria dom inate th e plankton comm unity and on occasion reach bloom proportions. B l oom s of these sp ecies are most common during th e summ er, particularly during periods of low freshwater outf l ow (Figur e 7). The potential toxicity of these species is not known.

Control of Potentia llyToxic Algae Blooms

The ideal lo ng-term stra tegy f o r dealing withtox i c algae is to prevent o r reduce the occurrence of bloom s . The m o st fundam e ntal way of addressing this challenge is to reduce the availability of nutrients that suppor t the growth of algae. There are lakes,

26rivers and es tuaries in F l orida where such water m a nage m e nt efforts have or could be used to achieve this goal. The restoration of lakes, rivers and their watersheds is a m a jor on going activity in m a ny regions of Florida. The reduction of nutri ent a v ailab i lity h a s been pursued in several different ways, includ ing the control of exte rnal nutrient lo ad ing from the watershed and the binding or rem oval of nutrients within a system using chem ical treatm ent (e.g. alum ) or sedim e nt rem o val (e.g. dredging) (Cooke et al. 1993). In term s of ecosystem s subject to high leve ls of external nutrient loading, long-term im prove m e nts of conditions generally require a strategy for the reduction of external load.

There are of course ecos ystem s where it m a y be impractic al , too expensive or funda m e ntally im possible to elim inate the o ccurrence of potentially toxic algae blooms. In such cases it m a y be necessary to carefu lly weigh the r i sk a ssocia t ed with certain activities in the system a nd m a ke appropria te us e recomm endations o r req u irem ents. Unfortunately, there is current ly in suf f i cient inf o rm ation f o r most system s to m a ke a

m eaningful risk determ ination at the presen t tim e. To m a ke such a determ ination a

num b er of important pieces of inform ation a bout individual lakes and rivers need to be available to water m a nagers, including: (1) the distribution of pot entia lly toxic a l gal species in tim e and space (i.e. tem p oral and sp a tial pattern s ), (2) the po ten tial im pact of f u ture chang e s in th e char acter of the ecosystem and its waters hed on alga l communitie s, (3) the am ount and relative stre ng th of the specif i c tox i ns as sociated with bloom s of the strains of potentially tox i c al gae species presen t in the ecosys t em in questio n and (4) th e pathways and m echanism s by which critical biological elem ents of the ecosystem

, includ ing hum a ns, m a y be exposed to , accum u late or pass-on toxins to o t h e r organ i sms.

It is well known that dif f er ent strain s of potentia lly tox i c a l g ae spec ies c a n vary in toxicity. Som e of this vari ation is due to genetic differe nces between populations of algae, som e of the variation is due to the im pact of environm e n tal variables (e.g.
tem p erature, light, nutrient leve ls) and som e of the variation is due to the life cycle of the algae (e.g. young versus old bloom s). Therefor e, determ ining the risk associated with algal toxins in individual lakes, rivers or estuarie s require a well-designed and im ple m ented m onitoring program that can be used to gather essential inform ation.

For short-term rem e diation of problem s withalgal bloom s in sm aller system s, including

m a n- m a de ponds, trad itional alg aecide appl ications, like cop p er-b ased products, have been em ployed for control. Unfortunately, su ch applications can worsen the problem by accelerating the releas e o f toxins stored insid e alg a l cells. In dealing withtoxic algae bloom s it may be necessary to em ploy an alte rnative approach that rem o ves algae from the water columnwithout killing the cells. Research is underway to explore alternative

m e thods for treating bloom s , like flocculati on. Overall, post-bloom treatm e nt is an expensive alternative. In s ituations w h ere surface water is used for hum a n consum ption, such expense m a y be acceptab l e and a num ber of water treatm ent technologies for elim inating toxicity are inthe process of developm ent (Chorus and Bartram 1999).

27References :

Badylak, S. and E.J. Phlips. 2002. Spatia l and temporal patterns of phytoplankton

com position and abundance in the Indian River L a goon. J. Phycology. In preparation for subm ission.

Bledsoe, E. L. and E. J. Phlips. 2003. The relationship between phytoplankton biom ass, nutrient loading, and hydrodynam i cs in an inner-shelf estuary. Ophelia. In review.

Bledsoe, E.L. and E.J. Phlips. 2003. The Cons equences of Suwannee River Eutrophication for the Dyna m i cs of Algae in the River and A ssociated Estuary. Op helia. In rev i ew.

Canfield, D.E., E.J. Phlips and C. D u arte. 1989. Factors influencing the abundance of blue-green algae in Florida lakes. Canadian J. F i sheries Aquatic Sci. 46: 1232-1237.

Chorus, I. and J. Bartram. 1999. Toxic Cyanob acteria in Water: A guide to their public health consequences, m onitoring and m a nagem e nt. E & FN Spoon, London.

Cichra, M., S. Badylak, N. Henderson, B. H. Rue t er and E. J. Phlips. 1995. Phytoplankton community structure in the open water zone of a shallow subtropical lake (Lake Okeechobee, Flor ida). Arch. Hydrobi ol. Beih. Ergebn. Limnol. 45:157-175.

Cichra, M. and E.J. Phlips. 2002. Phytoplankton comm uni ties of the Lower St. Johns River. J. Plankton Research. In pr eparation f o r subm ission.

Cooke, G. D., E. B. W e l c h, S. A. Petersen and P. R. Newroth. 1993. Restoration and Managem e nt of Lakes and Reservoirs, 2 ndEdition. Lewis Publishers.

Phlips, E.J. 1999. Lyngbya bloom s in the Rainbow River. Final Report to the Southwest Florida W a ter Management District, Tam p a, Flor ida.

Phlips, E. J. 2002. Eutrophication and alg ae. In G. Bitton (Ed.), Encyclopedia of Environm ental Microbiology. John W iley & Sons, New York.

Phlips, E.J., F. J. Aldridge, P. Hansen, P. V. Zi m b a, J. Ihnat, M. Conroy and P. Ritter.1993. Spatial and tempor al variability of trophic state param e ters in a shallow subtropical lake (Lake Okeecho b ee, Florida, USA). Arch. Hydrobiol., 128:437-458.

Phlips, E. J., F. J. Aldridge, C. L. Schelske and T

. L. Crism a n. 1995. Relationship b e t w e e n l i g h t a v a i l ab i l i t y , c h l o r o p h y l l a and tr ipton in a large shallow sub

- trop ica l lake. Limnol. Oceanogr. 40:416-421.

28P h l i p s , E

. J

. , T

. C

. L y n c h a n d S

. B a dy l a k

. 1 9 9 5 b

. C h l o r o p h y l l a , tripton, color and light availability in a shallow tropical inner shelf lagoon, Florida B a y. Marine Ecology Progress Series 127:223-234.

Phlips, E. J., M. Cichra, K. Havens, C. Ha nlon, S. Badylak, B. Rueter and P. Hansen. 1997. Light versus nutrient lim itation of phytoplankton abundance and structure in a shallow subtropical lake. J. Plankton Research. 19:319-342.

Phlips, E. J., S. Badylak and T. C. Lynch. 1999. Bloom s of the picoplanktonic cyanobacterium Synechococcus in F l orida Bay. L i mnology and Oceanography 44:1166-1175.

Phlips, E. J., M. Cichra, F. J. Aldridge , J. Hendrickson, J. Jembeck and R. Brody. 2000. Light availability and varia tions in phytoplankton standi ng crops in a nutrient-rich blackwater river. Limnol ogy and Oceanography 45:916-929.

Phlips, E. J., S. Badylak and T. Grosskopf . 2002. Factors affecting the abundance and

com position of phytoplankton in a restri cted subtropical lagoon. Coastal, Estuarine an d Shelf Science, In Pres s

. Phlips, E.J., M. Cichra and J. Frost. 2002b. Algae bloom s in Lake Griffin. Final Report to the St. Johns River Water Manage m e nt District, Palatka, Florida.

29Table 1. Major groups of toxins, their prim ar y site of action and the algae groups that contain species and strains capable o f p r o d u c i n g t h e t o x i n s

. U n d e r l i n e in dicates the group(s) it is m o st commonly associated with. N o te th at the potential for toxin production is restricted to certa in strains within the algal gr oups and can vary depending on environm ental conditions (Aft er Chorus and Bartram 1999).

Toxin Site of Action Algal Group M i c r oc ys ti n He pa t o t oxin – liver damage Microcystis , Anabaena, Planktothr i x ( O

scilla toria )

, Hapalosiphon, Anabaenopsis N o s t o c Cylindrosperm opsin Hepatot oxin – liver and other Cylindrospermopsis

, associated organs Aphanizomenon Nodularin Hepatotoxin – liver Nodularia Lyngbyatoxin Derm atoxin – skin and GI-tract Lyngbya Aplysiatoxin Derm atoxin – skin Lyngbya Anatoxin Neurotoxin – nerve synapse Anabaena

, A p h a n i z o m e n o n , Planktoth rix( O

scillator i a) Saxitoxin Neurotoxin – nerve axons Anabaena

, A p h a n i z o m e n o n , Lyngbya, Cylindrospermopsis



### Suwannee Riv e r St. Johns Ri v e r

Lake Okeechobee Lake Griffin Indi an Ri v e r Lagoon

F l orida Bay Rainbo w Riv e r

Figure 1. L ong-term study of planktonic algae was conducted on three m a jor m a rine system s (Florida Bay, Indian River L a goon and Suwannee River Estuary) and five m a jor

30freshwater ecosystem s (Lake Okeechobee, th e S t . Johns River, Lake Griffin, Rainbow River and the Suwannee River) in Florida.

i g u r e 2

. T h e spatial and tem poral distribu tion of cyanobacter ia biom ass ( m illion μ m 3 / F

m l ) during a high lake stage year (first num b e r ) ( 1 9 8 8

- 1 9 8 9 ) a n d a l o w l a k e s t a g e y e a r (second number)(1989-1990)(Cichra et al. 1995 ).

31

### 0 5 10 15 20 25

7/

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000

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2/

26/ 2001

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001

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/2

0

0

1 10/ 24/ 2

001 12/ 23/ 2

001

2/

21/ 2002

4/

22/ 2002Biovolume,million um3/ ml 

## Lake Griffin

Figure 3. T w o key taxa of cyanobacteria, Cylindrospermopsis and Oscillatoria (solid line) played an im portant role in the ov erall phytoplankton biom ass (triangle)(m illion
μm 3 / m l ) in the Lake Griffin (P hlips, Cichra and Frost 2002).

32S u

m

m

e r 9

4

S

u

m

m

e r

9

5

S

u

m

m

e r 9

6

S u

m

m

e r 9

7

S

u

m

m

e r 9

8

S u

m

m

e r 9

9 30, 0 00CyanobacteriaAbundance, Cells / ml 20, 0 00

10, 0 00 Cylindrospermopsi s sp
.

Anabaena s pp. 10, 0 00

6, 000

2, 000

Figure 4. T h e tem poral distribution of two potentially tox i c form s of cyanobacteria in the St. Johns River (cells / m l ) from 1994 through 1999 (Cichra and Phlips 2002).

33Figure 5. P hytoplank t o nbiom ass (m illion μm 3 / m l ) in four distinct regions of Florida Bay. The dom i nant cyanobacteria, Synechococcus cf. elongatus , is indicated in black (Phlips et al.1999).

34

## 0 2 4 6 8 10 12Sep97Oct-97Nov-97Dec-97Jan-98Feb-98Mar-98Apr-98May-98Jun98Jul-98Aug-98Sep98Oct-98Nov-98Dec-98Jan-99Feb-99Mar-99Apr-99May-99Jun99Jul-99Aug-99millionμm3/ml



### Fl a g Cy a n o Di a t o m Cryp t o Di n o Eu g l e n a Ch l o r o 

# Indian Rive r La goon
Bana na Ri ver Re gion

Figure 6. P hytoplank t o nbiom ass (m illion μm 3 / m l ) in the Indian River Lagoon. Cyanobacteria (b lack) w a s dom inated by Synechococcus sp. (Badylak and Phlips 2002).

35

### < 10000

0.5 m illion

12 million 

## Su wa nn e e Ri ve r Es t u a r y

Cy anobacteri a Biovolum e, μ m 3 

### / ml

Figure 7. Picoplanktonic cyanobacteria biovolum e ( μ m 3 / ml) distribution in the Suwannee River estuary during peak biom ass observed during the summ erof 2000. Darker shading indicates increased bi om ass (Bledsoe and Phlips 2002).

36CDC—A B r ief Introduction Lorraine C. Backer, Ph D, MPH National Center for En vironmenta l Hea l th Centers for Disease Control and Prevention 1600 Clifton Road N.E. MS E-23 Atlanta, GA 30333 Phone: 404-498-1342 Fax: 404-498-1355

What is the Centers for Disease Control and Prevention (CDC)?

CDC’s m i ssion is to promote health and quality of life, to protecthealth and safety, provide credible inform a tion to enhance h ealth decisions, and pr om ote health through strong partnerships. CD C is the national focus for developing and applying disease prevention and control strategies, environm ental health, and health prom otion and education ac tivitie s. CDC’s goal is to im pr ove the health of the people of the United States; however, CDC does not m a ke regul ations, recomm end legislation, or im pose program s on our state partners.

CDC is an agency within the U.S. Departm e ntof Health and Hum a n Services andcompris 12 offices and centers, including the National Center for Environm ental Health (NCEH) (see Figure 1). The m i ssion of NCEH is to provide national leadership in preventing and controlling disease and death resulting from the inte ractions between peo p le and th eir environm ent. The NCEH m i ssion is im ple m en ted by working with state and local health agencies to identify, investigate, and prevent environm entally induced illnesses in people. W ith the exception of the National In stitu te f o r O ccupational Saf e ty and Health (NIOSH), CDC has a presence in a s t ate o r local health agency only at the specific request and consent of the agency.

CDC funding sources include c ongressional budget funds and other program s targeted to address a specific public health i ssue, such as the presence of Pfies t eria p i scic ida in eastern U.S. estuaries. C D C passes much of the funding it receives directly to states in the form of grants, coop erative ag reem ents , and other assistance to health agencies.

CDC a nd Harmful Algal Blooms

CDC has be en directly involved in HABs re search since the summer of 1997, when the discovery of Pfies t eria p iscicida in the tr ibutar ie s of the Chesapeake Baycreated concerns about hum an e xposure to the organism . CDC participated in the public health response to com p laints of hum a n illness associ ated with exp o sure to waters contain i ng the organism by sending investigators to assi st the state of Mary land in conducting their investigation. CDC hel d a num ber of work shops and program m eetings to encourage federal and state health agencies to collabora tively develop a plan to assess the public health im pa ct of environm ental exposure to th is organism . W ith the states, CDC defined

a num ber of public health resear ch n eeds: 1) Inv e stig ate Pfiesteria p isc ic ida , including

37the ecology and behavior of the organism

, 2) Characterize the t oxins elaborated by P. piscicida and other estuarine dinof la gella tes, 3 ) Characte riz e the rou t es o f hum a n exposure, such as derm al contact or inha lation of aerosolized toxins, and 4) Conduct clinical and epidem iologic studies to dete rm ine the hum an hea lth effects from exposure to the organism , si m ilar organism s, and any toxins they produce.

CDC received direct co ngression al funding to support a number of harmful algal bloom (HAB) research activities, in cluding cooperative agreem ents with six states (Delaware, Florida, North Carolina, Maryland, S outh Caro lina, and Virginia) to assess the public health im pact associated with the p r esence of Pfiesteria piscicida in e s tuar ine waters used for recreatio nal activities and comm ercial fishi ng. The states devel oped program s to: 1) Conduct epidem iologic studies, 2) Create public education material s, and 3) Develop biological m a rkers of exposure to P. piscicida toxins and their bi ological effects. Detailed descriptions of CDC and state he a lth age n cy activitie s associated with P. piscicida can be found in the Environm ental He alth Perspectives Supplem ent (2001)

com p rising papers presented at the C D C conference (Pfiester ia : From Biology to Public Health, Stone Mountain, Geor gia, October 18-20, 2000).

CDC has conducted ann u al site visits with each of the states involve d in the cooperative
agreem ents. These site visits have provide d opportunities to expand th e research initially focused on a single organism to the more gene ral issue of the im pact of HABs on public health. Fo r exam ple, the cooperative agreem ents have supported a num ber of addition a l activities, including th e investigation of exposure to aer osolized brevetoxins during red tide ev ents, a study of th e efficacy of hom e wa ter filters to remove cyanobacterial tox i ns from water, this workshop, and the hum a n h ealth im pact of exposure to waste products from confined anim al feeding operations (CAFOs).

Future Challenges for CDC

CDC has de fined a number of future challenges in public health. They are: 1) Im prove people’s health, 2) Prevent violence and unintentional injury, 3) Meet health and safety needs of the workforce, 4) Provide credible health inform ation, 5) Protect individuals from e m erging diseases, 6) Elim inate racial/e thn i c dispar ities, 7) Foster safe, healthy environm ents, and 10) Work with partners to im prove global health. Collaborative efforts with public health partners will enable CDC to address these future challenges.

References

Lorraine C. Backer, Lora E. Flem ing, Alan Rowan, Yung-Sung Cheng, Janet Benson, Richard H. Pierce, Julia Zaias, Judy Bean, Gregory D. Bossart, David Johnson, Raul Quim bo, and Daniel G. Baden. Recreational Exposure to Aerosolized Brevetoxins During Florid a Red Tide Events. Harmful Algae, 2, 2003:19-28

Pfies t eria : From Biology to Public Health. En vironm ental Health Perspectives, 109 (Suppl 5), 2001. 808 pp.

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39Australia’s Cyanobacteria Experience Ian Falconer, D.Sc.

The m o st recent research into tox i c cyanob acteria in Australia has had th ree m a in

em phases. These are the ecology of cyanobact eria and its im plications for m a nage m e nt of water bodies; the toxicity and m echanis m of ac tion of cylindrosperm opsin; and the bioaccum u lation of toxin s by plants and anim als used for human consum ption.

Bloom s of toxic cyanobacteria in Australia are largely s easo n al in occurrence. Som e water storages have successions of bloom s of different species , rela ted to water
tem p erature and residence tim e of wa ter in lake s or weir pools. Often these are of toxic species, and hence a hazard to dom estic anim als or hum an consum ers.

The species Anabaena circinalis is comm on in lakes, reservoi rs and rivers in Australia and is toxic as a result of synthesizing a seri es of saxitoxin derivativ es. T h ese are stab le to boiling and are not removed by conventional chlorination, flocculation, or rapid sand filtration water treatm ent. They can be rem oved by activ ated carbon and other m o re sophisticated water treatm ents.

The organism bloom s most successfully in tem p eratur e-stratified rivers o r lakes. Und e r these cond itions anoxia at the s e dim e nt surface releases pho sp hate, which is the lim itin g nutrient for this nitrogen-fixing species. Th e im plications for m a nage m e nt to m i ni m i ze cyanobacterial bloom s of this species include flowcontrol in rivers, setting water discharge rates to assist m i xing, and varying discharge heights (W ebster et al., 2000).

Other eco lo gical research has explored the tox i c Cylindrospermopsis raciborskii tha t commonly occurs in the tropical lakes and rivers of Northe rn Australia. Monitoring 47 storage reservoirs and weir pools showed C. raciborskii in 70%, with highest bloom density in late summ er/autumn. In 14 reserv o i rs for drinking water sto r ag e, the averag e cylindrospermopsin concentration was 3.4 μ g/L. Toxin concentrations of 1.0 μ g/L were reached at a cell concentration of 20,000 cells/m l, which was taken as the health trigger for m onitoring drinking wate r for toxicity. High C. raciborskii con centrations were associated w ith long water residence tim e, high pH, high temperature, high incident radiation and a therm a lly stratified water column (M cGregor and Fabbro, 2000).

Recent stud ies of cylindrosperm opsin have inves t igated th e to xin distribu tion in m i ce, and the path way of excretion. Most appears in th e urine, withsom e in the feces. The liver and kidney concentrate the toxin, but it o c c u rs widely in the tissues (Norris et al., 2001). Studies of possible carcinogenicity dem o nstrated 10% of cylindrosperm opsindosed m i ce developed tum o rs over 210 days wh ereas no controls did so (Falconer and Hum p age, 2001). Parallel studies usi ng a cultured hum a n white blood cell line dem onstrated that m i cronucleus form ation increased in the p r esence of cylindrospermopsin, indicating chromosom e breakage and lo ss of whole chrom o somes (Hum page et al., 2000a). A sub chronic oral toxicitytrial with cylindrosperm opsin has just been completed (Humpage and Falconer, 2003).

40The potential of m i crocystin, also an im portant toxic cyanobacter ial contam inant of drinking water, as a tum o r prom oter in the lo wer gut was demonstrated byan increase in pre-neoplastic colon crypts in m i ce when m i crocystin was supplied in drinking water (Hum page et al., 2000b).

As a result of m a jor water bloom s of Nodularia in a fish and shellfish harvesting area, analyses were done on tissues from c o mm erci al species. Sufficient toxin was found in tissues for s h ellfish and crustacean h a rvesting to be banned, and use of finfish restricted to ' c leaned' fish only (Van Bounder et al, 2001).

References

Falconer, I. R. and Humpage, A. R. ( 2001) Prelim inary evidence for In-Vivo tum o r initiation by oral adm i nistration of ex tracts of the Blue-Green Alga Cylindrospermopsis raciborskii containing the toxin cylindrosperm opsin. Environ m ental Toxicology 16, 192- 195.

Hum p age, A. R., Fenech, M., Thom as, P. and Falconer, I. R. (2000a) Micronucleus induction and chrom o som e loss in W I L2-NS c e lls exposed to the cyanobacterial toxin, cylindrospermopsin. Mutation Research 472, 155-161.

Hum p age, A. R., Hardy, S. J., Moore, E. J ., Froscio, S. M. and Falconer, I. R. (2000b) Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the m ouse colon. Journal of Toxicology and En vironmental Health, Part A 61, 155-165.

Hum p age, A.R. and Falconer, I.R. (2003) Or al toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino m i ce: Determ ination of No Observed Adverse Effect Level for deriving a Dr inking W a ter Guid eline Va lue. Environmental Toxicolo gy 18, 94-103.

McGregor, G. B. and Fa bbro, L. D. (2000) Dom i nance of Cylindrospermopsis raciborskii (Nostocales, Cyanoprokaryota) in Q u eensla nd tropical and subtr opical reservoirs: Im plications for m onitoring and m a na gem e nt. Lak e s and Reservoirs: Research and Management 5, 195-205.

Norris, R. G. L., Seawright, A. A., Shaw, G. R., Sm ith, M. J., Chriswell, R. K. and Moore, M. R. (2001) Distribution of 14 C cylindrosperm opsin in vivo in the m ouse. Environmental Toxicolo gy 16, 498.

Van Buynder, P. G., Oughtred, T., Kirby, B., Ph illips, S., Eaglesham

, G., Thom as, K. and Burch, M. (2001) Nodularin uptake by seafood during a cyan obacterial bloom

. Environmental Toxicolo gy 16, 468-471.

41Webster, I. T., Sherm a n, B. S., Bor m ans, M. and Jones, G. (2000) Managem e nt strategies for cyanobacterial bloom s in an i mpounded lowland river. Regulated R i vers: Research and Management 16, 513-525.

42CYANOBACTE R IAL TOXINS: T H E DE VE LOPMENT AND EVALUATION OF METHODS TO DET E R MINE MICRO CYS T I N LEVEL S IN CANADIAN WATER SUPPLIES MICHELE GIDDINGS, HEALTH CANADA TIM MACAUL AY, S A SKATCH EWAN HE ALTH ABDEL-IL AH SADIKI, HEALT H CANADA JOANNE S K ETCHEL L , SASK W A TE R

INTRODUCTION

In Canada, the provision of safe dri nking water dem a nds the cooperation of all
governm e nts, particularly at the federal and provincial/territorial le vels. Since the late 1960s, these governm e nts have worked together to develop guidelines to m a intain or im prove the quality of drinking water acr oss the country. Federal-provincial collabo ratio nin this area ensures citizen s hav e access to the highest quality water possible.

The Federal-Provincial-Territorial C o mm ittee on Drinking Water (form e rly Subcomm ittee) is m a de up of representative s from Health Canada' s W a ter Quality and Health Bureau, Environm ent Canada, a nd provincial and terr itorial governm e nt departm e nts responsible for water qualit y. The group m eets twice annually and via conference calls at oth e r tim es to discuss issues related to drinking water quality, including:

* new and on-going rese ar ch related to the m i crobiologica l qua lity of wate r ,

* new and on-going research related to risks from exposure to particular chem ical
contam inants which m a y be present inCanadian drinking water supplies,

* the developm ent of ne w, and revision of current, guidelines for contam inants found in Canadian drinking water supplies, and

* the publication of educati onal tools dealing with specific topics for the public, health practitioners, and dr inking water authorities.

One of the substances current ly under the Comm i ttee' s eval uation is Microcystin-LR, a potent toxin that affects the liver (hep atotox in ). T h is hepatotoxin is naturally produced by m a ny species of blue-green algae (cyanoba cteria), which grow in shallow, war m , slow-m oving or still bodies of water common throughout Cana da. Many rural farm -type water supplies used for dom e stic purposes and f o r liv estock watering are subjec t to repeated blue-green algal bloom s, partic ularly during the hot summer m o nths. W h ile most species of blue-green algae are capable of producing toxins, not all blue-green algal bloom s do. When present, the concentration of toxins varies dram atically within the body of water and over tim e . Microcystins are extrem ely stable and will persist for long periods once they are rel eased into a water supply.

43This presentation focuses on the developm ent of a Canadian drinki ng water guideline for Microcystin-LR. In order to develop this guideline, federal and provincial governm e nts have worked together to conduct surveys to as sess risk and to im prove available detection and testing m e thods. In particular, an anal ytical m e thod was deve loped and a prototype field test kit created. The a pplicability of the analytical m e thod and field test kit for routine m o nitoring will be discussed. The success and im plem entation of these two

m e thods will provid e a more accurate and cos t -effective approach to th e m a nage m e nt of risk as sociated with blue-green al gal toxins to hum a ns and livestock.

GUIDELINE DEVELOPMENT

Histo ry

The first drinking water standards in Canadawere developed in 1923. These standards dealt with th e bacte r io lo gical quality of drinking water on ships in the Gr eat Lakes an d Inland W a ters. In 1930 and 1937, they were ex tended to cover all comm on carriers and coastal shipping, and were adopted in 1954 as regulations under the National Health and Welfare Act. Until 1968 , federal and provincia l d r inking water author ities generally us ed the United S t ate s Public Health Serv ice Standards as the basis for water quality objectives in Canada. In 1968, the first co m p rehensive Canadian drinking water quality guidelines w e re published. These guidelines, referred to as the Ca nadian Drinking Water Standards and Objectives (H ealth and W e lfare Canada, 1969), were developed by a joint

comm ittee m a de up of the Canadian Public H ealth Association Drinking Water Standards Comm ittee and an Advisory Comm i ttee convene d by the Departm e nt of National Health and W e lfare (now Health Canada). A lthough the developm ent of the 1968 drinking water standards was conceived as a federal in itiative, it involved the active participation of representatives from provincial agencies.

Ten years later, the standa rds were updated, and the 1978 Guidelines for Canadian Drinking Water Quality (Health and Welfare Canada, 1979) were publis hed. In addition to including more pesticides and organic chem icals, the 1978 Guidelines updated the earlier recommendations for radi oa c tive subs tan ces. W ith th e exception of pesticides and phenols, the presence of orga nic chem icals in drinking wate r had largely been ignored before the 1970s. Then, in 1974, scientists discovered that trihalom ethanes (THMs), a group of chlorinated disinfection by-products (CDBPs), could be for m ed during the drinking water treatm e nt process. This discov ery awakened in teres tin the presence of organic chem icals in drinking water. Interestfocused first on the health significance of the THMs th em selves, and then on th e presen ce and possible health signif i cance of other organic chem icals in drinking water.

As with earlier efforts, a working group de veloped the 1978 Guidelines as a joint federalprovincial p r oject with m e mber s drawn from each province and territory

, as well as f r om the federal governm e nt. Health Canada provi ded the te chnica l secre t ar iat to the workin g group, which then repo rted through the Fe deral-P r ovincial Advisory Comm ittee on Environm ental and Occupational Health, now called the Comm ittee on Environm ental

44and Occupational Health (CEOH), to the C onference of Deputy Ministers of Health. Once its task was com p l e ted in 1978, this working group was disbanded.

However, interest in drinking water qualit y rem a ined, and in 1983 another working group under the s a m e Federal-Provincial C o mm ittee began updatin g the 1978 edition of the guidelines. In 1986, this working group was changed to a s t anding subco mmittee, an d in 2002 it was changed to the Federal-P r ovincia l-Territorial Co mmittee on Drinking Water (Comm ittee), which exists to this day.

Since the publication of the 1978 guidelines, further advances in analytical m e thods, particularly in the field of gas chromatogra phy/mass spectrom etry, have resulted in the detection of a large number of organic chem icals found in drinking water supplies that had previously gone unnoticed. Several hundre d such chem icals have now been reported in Canadian drinking water s upplies, and m a ny hundreds m o re have been found in other parts of the world. The concentr atio ns of m o st of these chem icals are v e ry low, and little or no data e x ists to asse ss their tox i c ity. Governm e nt and international agencies have responded by assessing the need for standa rds or guidelines for a wide variety of substances.

The sixth, and m o st recent, edition of the Guidelines for Canadian Drink i n g W a ter Quality (Health Canada, 1996), published in Sep t em ber 1996, lists guidelines for m o r e than 80 physical and chem ical pa rameters and for 78 natural orartificial radionuclides. As in the 1978 edition, explanatory paragr aphs concerning the guideline derivation process are an integral part of the booklet . The docum e nts used to support the adoption of a particular guideline valu e are also m a de available on request, and can be found on Health Canada' s water quality website ( h ttp://w ww.hc-sc.gc.ca/wate rqua lity or http://www.hc-sc.gc.ca/eauqualite ). In order to keep stakeh olders apprised of new or revised guidelines between pub lic ations of the Guidelines booklet, a Sum m a ry Table is prepared an d posted to the website o n ce each year.

It should b e em phasized that the guid e lines ar e no t enforceab le standards unless th ey are

em bodied in provincial or territorial legisla tion. All provinces a nd territories use the guidelines as the basis for assessing the qual ity of their drinking water supplies. Most provinces h a ve contro l o v er the op er ation of tr eatm e nt f acilities, inc l uding the issu ance of operating perm its.

The Comm it tee continues to be m a de up of re presentatives from all of the provinces and
terr ito rie s , a s well as f r o mHealth Canada and Environm ent Canada. Health Canada' s Water Quality and Health Bureau also continue s to provide th e scien tif ic e xpertise to the Comm ittee, as its technical secretariat. The Committee m e ets biannually, alternating between Ottawa and a city in one of the provi nces or territories; the latest m eeting was held in W i nnipeg, Man i toba in April, 2003. At each m eeting, m e m b ers review and revise the priority list of substances schedu led for assess m e nt. In recent year s, th e Comm ittee has worked to define the specific roles each level of governm e nt pl ays in the guideline developm ent proces s, an d to work to gether to ide n tif y nation a l prio rities w h ich tak e into

45account the needs of each jurisd ictio n. A detailed summary of the Comm ittee' s activities can be found on the water quality w e bsite.

Current Practices

Because pro v incial and territorial go vernm e nts are respon sib l e for im plem enting the drinking water guidelines and for providing sa fe drinking water to Canadians, m e m b er s of the Comm ittee are accountable for the eval uation and approval step s of the drinking water guidelines developm ent process. Each recommended guideline value, and it’s

accom p anying health ris k assessm ent, is ev aluated for its practicality and impact. Consultations carried out by the secretar iat include posting docum e nts for public comment on the water quality website. Regiona l consultations m a y be c a rried out by the provinces and territories. Through this consensus-based developm ent process, a guideline is established, and the asso ciated healthrisk as sess m e nt is m o dified to create a criteria summ ary that reflects the risk m a nagem e nt decisions involved in the guideline’s developm ent.

The process of developing drinking w a ter guidelines for m i crobiological, chem ical/ physical and radiological substa nces is based on risk m a nage ment concepts and involves several steps: identification, assessm en t, evaluation, approval, announcement and publication (Federal-Provincia l Subcomm ittee on Drinking Wa ter, 1998; Health Canada, 1995). This process is flexible by n a ture, inthat it m u st accommodate the diverse needs of various jurisdictions (i.e., provincial, terr itorial and federal) in order to be deem ed successful. Certain step s with in the developm ent process m a y be m odifie d in order to satisfy the needs of the jurisdictions i nvolved. T h e process used in developing a guideline, from the selection of the param e te rs to be evaluated, through to approval and publication, takes several years to complete.

The guideline developm ent process is broke n down into a num ber of specif i c actions,

com p leted by the various m e mbers of the Comm i ttee. In gen e ral term s, the secr etar ia t (the W a ter Quality and Heath Burea u, Saf e Environm ents Programm e , Healthy Environm ents and Consum er Safety Branch), representing the federal governm e nt, is responsible for providing scien tific expertise and advice to the provinces and territories;

com p iling national data on substances under re view; assessing health risks; and writing, revising and publishing the criteria summ ary docum ents and the guidelines booklet as needed. Th e secretariat also m a intains the wate r quality webs ite, which houses m a ny of the docum ents related to the guideline developm ent process.

The provincial and territorial representatives, on the other hand, are responsible for the inform ation for and about their own specifi c jurisdictions. This includes regulating drinking water quality th rough the ad option of en forceable regulations, g u idelines an d objectives; conducting m onitoring program s, an d setting-up and m a in taining distribution system s and treatm ent plants for drinking wa ter. The Drinkin g W a ter Committee, as a group, evaluates all available scie ntific and econom i c data to establish or revise drinking water guidelines through a risk m a nagem e nt pr ocess. In this way, the work conducted by

46the various levels of governm e nt complem e nt e ach other and duplic ation is kept to a mi n i mu m.

Drinking water guidelines are developed for substances that a ppear on th e Comm ittee's "drinking water substances priori ty list." In order to be pl aced on this list, and therefore qualify for evaluation, a substance m u st m eet at least one of the followi ng criteria: it must be frequently detected in m a ny Canadian dri nking water supplies, detected in frequently elevated con centration s , or there m u st be evid enc e (pre -asse ss m e nt review) tha tthe substance m a y cause adv e rse health effects. If the substance m eets the criteria, the Comm ittee and CEOH place it on a list, which is then approv ed. This final substan ce priority list can be f ound on Health Canada' s wa ter quality website. The evaluation of the substance b e gins once an evaluator b ecom e s av ailable. Due to lim ited resources and data
gaps, m a ny of the substances currently on the priority list are awaiting evaluation. W o rk is also coordinated with ot her standards-setting organizations, such as the U.S. Environm ental Protection Agency and the W o rld Health Organization, to m i ni m i ze duplication of research or assessm e nt.

The evaluator begins the ri sk assessm e nt by researchi ng the substance through an extensive literature and da ta search, including gather ing inform ation from other jurisdictions and partners. The provinces and territories becom e involved at this point by identifying whether exposure (m onitoring) data on this particular substance is available from their own jurisdictions, whether from ex isting, current or future sam p ling program s

. They also in dicate when a summ ary of thei r m o nitor i ng data will be av ailable. The results of both thes e searches are then used to determ ine whether a need r eally exis ts f o r a guideline on this particular substance.

If a need do es exis t, jurisdictions co ncer ned abo u t the subs tance initia te monitoring
program s to satisfy their own infor m ationa l needs (e.g., exposure estim ates). They collect and s u mm arize th eir m onitoring da ta for the substance and subm it their summaries to the secretariat for consider ation in assessing national exposure to the
contam inant. This step ensures the proce ss is consistent with the population health directions and priorities of provincial, territorial and federal governm e nts.

The evaluator obtains data on the effects of exposure to chem ical agents from toxicological studies of anim al species and in ep id em iological studies of hum a n populations, where such studies exist. Effects vary, depending upon the dosage, route of exposure (e.g., ingestion, inhalation or derm al), frequency or dur ation of exposure and the species, and the sex and age of the e xposed population. The effects of exposure to chem icals are generally classified in the following broad categories: organ-specific, neurological/behavioral, re productive, teratological and oncogenic/carcinogenic/ mutagenic. Effects m a y be brief or prolonged, reversible or irreversible, immediate or delayed, single or m u ltiple. In general, th e nature, num ber, s e verity, incidence and/or prevalence o f specific effects in a pop ulation ge nerally increase with increasing dose; this is comm onl y referred to as the dose-response relationship.

47For som e types of toxic effects resulting from exposure to chem icals, it is believed that a dose (th r esh o ld) ex ists b e low which adverse e ffects will not occur. For other types of toxic ef f ects , it is assum e d, but not p r oven, tha ts o m e probability of harm exists at any level of exposure (i.e., no threshold). Atpresent, the la tte r as sum p tion is genera lly considered to be appropriate only for carci nogenesis. For som e types of carcinogens, specifically those that induce tum o rs by particular m echanism s , such as prom otion, it is believ e d tha ta th reshold dose m a y exist be low which tum o rs will not o c c u r.

Different approaches are a dopted for the derivation of guidelines for com p ounds considered to be carcinogenic and probably carcinogenic, compounds considered to be possibly carcinogenic and those considered being probably not carcinogenic or for which data are inadequate for evaluation. Che m ical s are therefore classifi ed with respect to their potential carcinoge nicity into various groups on the basis of rigorous exam ination of the quantity, quality and nature of the results of availab l e tox i cological an d epidem iological studies. Che m icals classifi ed as carcinogenic often also induce toxic effects other than m a lignant tum o rs; for thes e substances, the guideline is derived on the basis of the approach that leads to th e m o st strin g ent valu e.

If the substance undergoing evaluation is f ound to be not carcinogenic to hum a ns, its

recomm ended drinking water guideline value is derived based on the application of an uncertainty f actor to acco unt for inter- a nd intraspecies varia tio n to a no-ob servedadverse-effect level observed in toxicological studies in which rats ingest the substance in drinking water daily for periods ranging from 90 days to two years.

Once the evaluator has com p iled the above data , he or she drafts a criteria summ ary on the substance, incorporating the health risk assessm e nt info r m ation, the overall environm ental exposure to the substance, th e fraction of its expos ure attributed to drinking water, existing analyt ical/treatm e nt techniques and capabilities, as well as a

recomm ended guideline value.

The f i rst r e v i ew of the dr af t criter ia s u mm ary is com p leted within th e W a ter Quality and Health Bureau; the evalu a tor defends the cla ssification of the subs tance and its proposed guideline value to a senior evaluator. The cr iteria summ ary i s then rev i sed to reflect the experience of the senior evaluator, w ho m u st be c o m p letely s a tisf i ed with the criter ia summary before it is forwarded for an external review.

The evaluator then send s the draf t cr ite ria summary to thr ee external or third party reviewers who have expert knowledge of the s ubstance. These third-party reviewers are from Canadian or Am erican universities, the U.S. Environm e n tal Protection Agency Drinking Water Program or a Member State of the W o rld Health Organization. Also included among the reviewers is a drinki ng water treatm ent specialist, often

recomm ended by the Canadian W a ter and W a stewater Association. These experts critically rev i ew the criteria summ ary in acco rdan ce with th e Canadian pu blished approach policy and respond to questions set out in a guide for peer review ers. Their review focuses on the scientific component of the summ ary.

48Once the th ird-party reviewers written comments are receiv e d, the evaluato r asses s es th e comments and considers any additional info rm ation iden tif ie d by the rev i ewers. The evalua tor th en revis e s th e criter ia summary and subm its it f o r a f i nal inte rnal r e view.

The criter ia summary is then dis t r i bu ted to the Comm ittee mem b ers for their review of the substance' s health risk assessm ent and the proposed guideline value. Provincial/territorial review a nd assessm ent of the criteria summary varies from a brief departm e ntal (interna l) review to detailed evaluations by an external agency or nongovernm e ntal organ i zation. W r itten comments from all Comm ittee m e mbers are forwarded to the secretariat for consideration by the evaluator.

Once the risk assessm ent phase winds down, the process of risk m a nage ment becom e s more prom i n ent. Each jurisdiction evaluates the feasibility of im ple m enting the

recomm ended guideline for the substance in drinking water by taking treatm ent costs and socio-econom i c factors into consideration. A revised criteria summary is then redrafted for public comment. This docum ent contains as much provincial exposure and econom i c inf o rm ation as is ava i lab l e at the time of printin g

. It is then m a de available to the pu blic for comm ent through the provincia l representatives and the se cretariat. T h e docum ents are also available throug h the water quality website for public comm ent during a specified period (usu ally 4 m onths).

Following the public review, all comments ar e again reviewed and summarized by the evalua tor an d presented to the Committee. Jur i sd ictions conc erned th at p e ople living in their area may be exposed to drinking wate r containing the substan ce at concentrations that ex ceed the recomm ended guideline value w ill estim a te the costs for treating d r ink i ng water to reduce the concentration of the substa nce. The costs of controlling exposure to the substance from sources other than drinki ng water m a y also be estim a ted in order to conf irm that m odif y ing water trea tment is in fact the m o st cost-eff ective way of reducing intake of the substance. Control of other ro utes of exposure m a y be identified as a more effective m e ans for redu cing the p a rticular he alth risk. The developm ent of these cost estim ates is the respon sibility of Committee m e mbers; the level of detail of the cost estim ates is left to their discretion.

Weighed against these costs are the benefits of reducing exposure to the substance via drinking water if such data ar e available. For example, th ere m a y be direct savings in health care costs that woul d otherwise be incurred from a specific health problem associated w ith th e substance, or indirect s a vings in the form of socio-econom i c benefits such as savings in sick leave and subsequentincreases in producti on. Any side benefits that are an outcom e of i mproved drinking wate r treatm ent to contro l the substance (e.g., the rem oval of other contam inants or the ex tension of the life of the water distribution system ) m a y be considered in a cos t -benefit analysis.

When the new or revised guideline for a pa rticular substance has been approved by the Comm ittee and then CEOH, the secretariat evalu a tor m a kes all th e requ ired revis i ons to the criteria s u mm ary in preparation f o r its pub lic a tion in the Guideline s f o r Canadian

49Drinking Water Quality Supporting Docum e nt ation. The final criteria summary is published in both official language s, and is m a de available on the water quality website.

The guideline value for the substance is incl uded in the summary table of drinking water guidelines found in the “Guidelines for Ca nadian Drinking Water Quality” supporting docum entation and is posted on the website. This table is updated annually, following each spring m eeting of the Comm ittee. Notice is also sen tou t to subsc r ib ers to the W a ter Quality and Health Bure au’s Lis tser v e/m a iling list. The guid e line is a l so included in the “Guidelines for Canadian Drinking Water Quality” booklet when updated.

Re-evaluation of existing guidelines is a cont inuous process. Alt hough the secretariat is charged with the respon sibility of id entifyi ng ou tdated gu idelines each y e ar when the Comm ittee list of substances is e s tab lishe d, any Comm ittee m e mber or interested party can identify an outdated guideline. T h e avai lability of new research, m onitoring data, analytical methodology or treatm ent process m ay prom pt a re-evaluation of an existing guideline.

Although its prim ary task was initially to deve lop drinking w a ter guid elines, the role of the Comm ittee has evo l v e d to dea l with a variety of issues concerning treatm ent technolog ies and the quality of drinking water. A partial lis t o f the Comm i ttee' s current priorities in the area of risk m a nage mentand guideline developm ent focuses both on the

m i crobiological quality of dr inking water and on chem ical substances or groups of substances from the drinking water disi nfection process: protozoa, chlorinated disinfection by-products (CDBPs), uranium

, a nd cyanobacterial toxins (Microcystin-L R). Furtherm ore, Health Canada provides tim ely advice to other governm e nt agencies and departm e nts, the provinces, the territorie s, and the public in response to em ergency situations, such as spills.

CYANOBACTE R IAL BLOOM S AND MICROCYSTI N-LR

Cyanobacteria, uni- or multi-cellular prokar yotes that contain chlorophyll, for m in shallow, warm, slow m o ving or still water. A mass of cyanobacteria in a body of water (comm only referred to as pond scum) is ca lled a bloom . Far from being a new phenom enon, cyanobacterial bloom s have beenaround for centuries. Cyanobacteria

som e ti m e s produce toxins that either affect th e nervous system (neuro toxins) or the liver (hepatotoxins). Cyanobacterial neurotoxins are extrem ely unstable, which m a kes the m difficult to isolate and study; cyanobacter ial hepatotoxins, on the other hand, are
extrem ely stable.

Most hepatotoxins produced and released by cyanobacteria are called m i crocystins because they were first isolat ed from a cyanobacterium called Microcystis aeruginosa

. Microcystins are the m o st comm on of the cyanobacterial toxins found in water, and probably the ones m o st often responsible for poisoning anim als and hum ans who come into con t act with toxic bloom s . Microcystins are extrem ely stable in water becaus e of their chem ical structur e, which m eans they can survive in both warm and cold water and

50can tolerate radical chan ges in water chem istry, including pH . So far, m o re than 50 different kinds of m i crocystins have been iso l ated and identified.

Regardless of the type of toxin, determ ini ng which cyanobacterial blooms are toxic and which are harm less is a com p licated task. Re searchers gen e rally ag ree that 30 to 50 per cent of cyan obacterial bloom s are harm less b ecause they con t ain on ly non-toxic species of freshwater cyanobacteria. Currently, toxicity can only be determ ined through laboratory testing. However, becau se cy anobacterial b l oom s a r e becom i ng m o re and more common in surface waters across Cana da, Health Canada and Comm ittee m e mbers f r om aff ected jurisd ic tio ns have in itiated a p r ojec t tha twill h e lp asce rta i n the toxic ity o f bloom s in a sim p le, accurate, and efficient m a nner. One of the project' s outcom e s is a sim p le field test and analytical m e thod that will allow regular and m o re cost-effective monitoring of this substance.

Canadian Drinking Water Gu ideline fo r Microcy stin -LR

Health Canada, as the techni cal secretariat to the Comm itt ee, has adopted a guideline value of 1.5 μ g/L for Microcystin-L R . It is expected th at th is value will be protective of effects from other m i crocystins since M-LR is the m o st common of the toxins found in Canadian drinking water sources. The fina l criteria summ ary docum ent for MicrocystinLR is posted on the water quality website. The value is believed to be conservative as it is based on lifetim e oral exposure. F o r clim a tic reasons, toxins are probably not present in Canadian water supplies m o re thanfour or five months per year.

Som e m unicipal trea tm ent pl ants are now required to m onitor for the pres ence of Microcystin-LR in their water supplies, especi ally if the source is prone to cyanobacterial bloom s. Be cause m a intaining drinking wate r quality in Canada is a provincial responsib ility, m onitorin g and treatment strate gies will vary b e tween prov inces. To help provincial authorities in deali ng with elevated levels of toxins found in a given water supply, the Comm ittee has also dev e loped a risk m a nagem e nt strategy (flow chart) as part of the guideline docum ent.

Cyanobacterial Blooms and Toxins in Canada

While cyanobacterial bloom s occur in all provinces, they are most commonly found in the sm all, shallow, slow-moving bodies of water comm on to the prairies (A lberta, Saskatchew an, and Manitoba). Rural prairi e tow n s are th e most affected as they generally rely on surface water from shallow, nutrient-rich lakes for drinking water, and dugouts for dom e stic purposes and f o r wate ring livestock. Cyanobacterial bloom s and their toxins do not appear in groundwater supplies.

In the summer of 1990, m i crocystin-LR was det ected in cyanobacterial bloom s on three shallow Alberta lakes used as sources for m unicipal drinking water (Kenefick et al., 1992). Microcystin-LR was present in concentrations up to 500 μ g/g of algal biom ass. More than 70% of m o re than 380 bloom biom ass sam p les taken from 19 lakes in Alberta between 1990 and 1992 showed detect able levels of the toxin (>1 μ g m i crocystin-LR/g of

51dry biom ass). Levels of m i crocystin-LR in Alberta lakes and dugout ponds, m easured using high-perform a nce liquid chromatography w ith ultraviolet detect ion, ranged from 4 to 605 μ g/g dry weight of biom ass or up to 1500 μ g/g.

In late August 1993, a large alg a l blo o m de veloped in Deaco n Reservoir, W i nnipeg' s

m a in storage f acility f o r

water f r om Shoal Lake. The water from this lake is generally considered to be of high quality and require s on lydisinf ection with chlo rin e prior to distribution and consumption. In an attem p t to control al gal density and taste and odor problem s, municipal officials isolated the re servoir and treated it wi th copper sulphate. However, this action raised the concern that if the algal bloom c ontained toxin-producing algae, signif i cant quantities of the toxins m ay have been released into the reservoir.

Sa m p ling determ ined that toxin-producing bl ue-green algae w e re not present in the Deacon Reservoir, but they were present in Shoal Lake. Analysis of water sam p les (using the protein phosphatase bioassay) indi cated that m i crocystin -LR was present in

sam p les collected from Shoal Lake and from within the distribution system, but was not present at detectable levels (>0.05 μ g/L) in sam p l e s from D eacon Reservoir. Maxim u m

m i crocystin-LR concentrations m easured in th e raw water of Shoal Lake and in treated tap water were approximately 0.45 μ g/L and 0.55 μ g/L, respectively. Subsequent monitoring during the fall peri od showed a steep decline inconcentrations, suggesting that higher m i crocystin-LR levels may ha ve been present ear lier in August 1993.

Because the weather during the summ er of 1993 was characterized by below-norm a l
tem p eratures and above-norm a l precipitation, con d itions no t u s ually suppo rtiv e of algal bloom for m ation, a concern was raised that higher levels of m i crocystin-LR could develop in S hoal Lake during the m o re usual, relatively hot, dry summ er s. As a result, Manitoba Environm ent, in cooperation with the City of W i nnipeg, continued to m o nitor for m i crocystin-LR in Winnipeg' s water supp ly. Microcystin-LR was detected at concentrations ranging from 0.1 to 0.5 μ g/L on six occasions between 199 4 and the en d of 1996.

Because Shoal Lake, a relatively nu trien t -poor w a ter supp ly, supported a toxic b l ue-g reen algal b l oom , Manitob aEnvironm ent becam e c oncerned that toxic bloom s m a y also be occurring in more nutrient-rich rural surface water supplies th roughout so uthern Manitoba. To address these concerns, a com p rehensive two-year study (1995 and 1996) was conducted on water quality in rural sou t hw estern Man ito ba surface water supp lies. In the first year of the st udy, m i crocystin-LR was found to be widely distributed in all water supply categories. Rural m unicipa l water supplies had a higher detection frequency (9 3%) than on -farm dom e stic/liv estock dugouts (57 %), showing that conventional treatm e nt m e thods m a y be only partially successful in rem oving the toxin. Mean concentrations ranged from 0.23 μ g/L in recreati onal sites to 0.35 μ g/L in dugouts used exclusively for liv estock. In th e sec ond year of the stud y, seven rural surface w a ter supplies were intensively sam p led form i crocystin-LR. The hepatotoxin was found throughout the entire sampling period (June th rough December 1996), som e tim es at levels exceeding 0.5 μ g/L, the "Em e rgency Health Advi sory Guideline" form ulated by Health Canada in respon se to the 1993 incident.

52Several incidents of suspected animal poisoni ngs have been associated w ith toxic algal bloom s in Manitoba lakes. In June 1995, hepatotoxin poisoning was suspected in the deaths of two calves watering directly from Pelican Lake, where a m a ssive bloom was associated w ith m i crocystin-LR c oncentrations ranging from 0.8 to 1.2 μ g/L. In m i dJune 1996, 16 cattle and a dog died after drinki ng water from a creek im poundm e nt near Balder. Although m i crocystin-LR concentra tions were relatively low (0.4 and 0.23

μ g/L), the neurotoxin-producing blue-green algal genus Anabaena dom i n ated th e alg a l

com position. In July 1996, three dogs died afte r drinking water from recreational beaches along Dauphin Lake. Again, water sam p les cont ained low levels of m i crocystin-LR (<0.1 to 0.1 μ g/L), but the dom inant algal speci es were neurotoxin-producing A nabaena species. "T oxic Algae Advisory" signs were po sted along Dauphin Lake beaches to warn residents about the risks associ ated with using the lake fo r drinking water, pet watering, or swimm i ng.

Treatment Technology and Management

Good control technol ogy m u st reflect:

* proper m a nagem e nt of the watershed a nd reservoir to prev ent algal g r owth

,

* an appropriate m onitoring program , and

* correct treatm ent technology for both the cyanobacteria and their toxins.

Managem e nt options are sim ilar to comm on t echniques used to control algal populations in reservoirs, but with a f e w exceptions. Fo r example, the use of chem ica l s that would lead to the d i sintegration (lysis) of cy anobact erial cells m u st be avoided to prevent the release of their toxins. In th e past, chem ical treatm ent with copper sulphate had been the most common technique used to control alga l bloom s in drinking water reservoirs. However, Kenefick et al. (1993) found that al gal bloom m a te rial from a Canadian prairie lake treated with copper sulpha te released m o st of its toxi ns during the first three days following treatm e nt. Their results indicated th at a 99% reduction inm i crocystin levels would take approxim a tely three weeks, sugge sting that copper su lphate should not be used to treat potentially toxic cyanob acterial bloom s in waters to be consum ed by humans within sev e r a l weeks f o llowing trea tm ent.

An appropriate m onitoring program is essentia l to the overall control of cyanobacteria and their toxins. At present, m o st munici pal water treatm e nt plants in Canada do not regular ly m onitor their water supplies for cyanobacterial toxins. However, because cyanobacteria have strong sm ells and tastes and interf ere with certain wa ter trea tm ent processes, most m unicipalities with a hi story of b l oom s monitor their surf ace water supplies for cyanobacteria.

Monitoring program s must take spatial and tem p or al variability in m i crocystin levels into consideration, as there may be large f l uctua tions in the lev e ls of cyanobacteria and their

53toxins resulting from the interplay of a va riety of physical, chem ical and biological factors. Kotak et al. (1995) studied th e patterns of occurrence of m i crocystin-LR (m easured as μ g/g biomass of Microcystis aeruginosa ) in three hypereutrophic hard water lakes in central Albe rta ov er th ree seasons. Microcystis aeruginosa was highly variable both tem porally and sp atially, with differences up to three orders of m a gnitude within each lake over one year , between years in an individua l lake and between lakes in a year. Seasonal changes in m i crocystin-LR concentration w e re pos itiv ely corre lated to the abundance and biom ass of the M. aeruginosa , total and total dissolved phosphorus concentration, pH, and chlorophyll. Th ere was a negative co rrelation between

m i crocystin-LR concentration and nitrate con centration and no correlation with water
tem p erature. Over a 24-hour period, the con centration of m i crocystin-L R was six tim e s lower at night than during the day.

The final step in controlling cyanobacteria and their toxins is the drinking water treatment process. Although conventional surf ace wate r treatm ent plants using coag ulation, clarification, and filtratio n ar e effective in rem oving cyanobacterial cells, the Manitoba study noted above (Jones, 1996) illustrated that conventional water treatment m e thods

m a y be only partially successful in rem ovi ng cyanobacterial toxins. This has been confirm e d by Lam b ert et al. (1996), who exa m ined the rem o val of m i crocystins from drinking water at two full-scal e treatm ent plants in Alberta th at em ployed coagulation

sedim e ntatio n, dual-m e dia filtration, and chlo rination com b ined with eith er granu l ar activated carbon or powdered activated carbon filtration. The two processes generally

rem oved m o re than 80% of the m i crocystin fr om raw water, particularly w h en the raw water concentrations were high; however , a residual concen tration of 0.05-0.2 μ g

m i crocystin-LR equivalents/L was observed at both treatm ent facilities. The data thus suggests that drinking water consumers could be chronically exposed to low levels of

m i crocystin-LR for the duration of cyanobact erial bloom s. As previously m e ntioned, Lam b ert et al. (1996) also found that chlo rination was unsuccessful in achieving any reduction in m i crocystins.

Development of an Analytical Method

In 1997/98, Health Canada was approached by Sa skatchewan Health to help develop an analytical method to detect and assess the toxicity of cyanobacterial bloom s in an affordable, efficient, reliable, and ro utine way. After discussion w ith th e Saskatch ewan Health Provincial Laboratory staff, it was concluded that the mostsuitable m e thod would use High Perform a nce Liquid Chromat ography w ith UV detection (HPLC-UV).

Am ong the several analytical m e thods usi ng HPLC-UV for microcystin analysis, a procedure published by HMSO (London, UK) in1998 was chosen for m o dification. This

m e thod is part of the series Methods for the Exam ination of Waters and Associated Materials, and was developed to detect and quantify dissolved m i crocystin-LR,

m i crocystin-RR, m i crocystin-YR and nodularin in raw and treated wate rs (extra-cellular toxins).

54The procedur e consists o f m u ltiple steps. Firs t, s a m p les are f iltered to re move algal c e lls. The filtrate is then ex tracted using a p r ec onditioned C18 phase extraction cartridge. Th e cartridge is washed with aque ous m e thanol before elution wi th m e thanolic tr if luoroac e tic acid (0.1%v/v). The acidified m e thanol is re duced to dryness; the re sidue is redissolved in m e thanol and again evaporated to dryness. The residue is taken up in 70 percent
aqueous m e thanol and centrifuged (if necessary ) to rem ove solid m a terial. A portion of the supern atant solu tion is analyzed by reversed-phase high perform a nce liquid chrom a tography with UV detection at 238 nm , using a photodiode array detector.

During the initial m e thod evaluation conducted by Health Canada, two major difficulties arose. The f i rst problem was the high b ackground and interferen ce peaks in the HPLC chrom a togram s. The second was the obtained detection lim it, com p aratively higher than reported in the published m e thod.

Multip le a t tem p ts were carr i ed out b y changing s e vera l of the procedur e p a ram e ters. The high background and interference p e aks disa ppeared after the cartridge co ndition i ng procedure was m odified by passi ng 5 m L of methanolic trif luoroacetic acid (0.1%v/v) through it at the very beginning of the condi tioning. The detection lim it was slightly im proved by increasing the cartridge eluti on volum e (4.0 mL instead of 3.0 m L of

m e thanolic trifluoroacetic acid (0.1%v/v). During the developm ent of the analytical

m e thod, duplicate sam p les were sent to a HealthCanada lab in Ottawa in addition to the University o f Alberta Protein Phosp h atase Bio a s s ay Servic e, the only lab o ratory in Canada that had the capacity to routinely quant ify blue-green algae t oxins in water. T h e per sam p le cost for testing via this me thod is high, therefore m a king this m e thod unsuitable for routine monitoring practices.

Using the modified analytical m e thod for a 500 mL water sample, m i crocystin-LR can be detected at levels as low as 0.3 μ g/L. The m i ni mum quantifiable lim it (MQL) is 0.8

μ g/L. The MQL for the three other m i crocys tins (-RR, -LA and -YR) is sim ilar (0.5-0.8

μ g/L). W o r k is continuing on refining of the m e thod.

Evaluation and Testing

During the evaluation and testing phase, sam p ling was carried out at various sites in several provinces in Canada. During 1998, 279 raw water samples were collected from different surface water sources (lakes, rivers, du gouts, etc.). These sam p les were tested using the p r otein phosp h atase m e thod; 28 sam p les tested po sitiv e (0.3 μ g/L to 14 μ g/ L)

. Thirty-two duplicate sam p les (random l y sel ected from the original 279 samples) were sent to the H ealth Canada lab for testing using the HPLC m e t hod. Unfortunately, none of these sam p les tes t ed pos itiv e. (Beca use the sam p les were s e lected rando m l y, there was no guarante e of hitting a positiv e sa mple).

During the 1999 surveys, 92 sam p les were co llected and analyzed using the protein phosphatase m e thod, and 49 randomly selected duplicate sam p les were sent to the Health Canada lab for testing using the HPLC m e thod. This tim e, 27 of the original 92 sam p les tested positive (0.3 μ g/L to 9.4 μ g/L), as did seven of the 49 random sa m p les.

55In both surveys, treated water sam p les were analyzed only if m i crocystins were detected in the raw water supply. The flow charts in Appendix A show the process used during the 1999 survey to determ ine which bodies of raw water to sam p le, when to test treated water, and when to notify the community and po tentially switch to an alternative water supply or adjust treatm e n t. Appendix A show s the process f o r water destined for hum a n

consum ption.

Survey Results

As m e ntioned above, no m i crocystins were dete cted in water sam p les tested using the HPLC method during the 1998 survey. In 1999, Microcystins -LR, -LA and -RR were detected in seven raw water sam p les (m ainly r ecreational sites) at concentrations ranging from 0.3 to 3.1 μ g/L. One sam p le contained both Mi crocystin-LR and -RR. None of the four screened m i crocystins were de te cted in tre a ted water s a mples.

Other unknown com pounds that coul d be part of the m i crocystin fam ily were detected in several sam p les. Attempts to identify such com p ounds by liquid chrom a tography-m a ss spectrom e try will be carried ou t during the summ er of 2000.

Development of a Field Test Kit

As m e ntioned earlier, the t ools available to governm e nts and the public to address concerns about the presence of toxins in cy anobacterial bloom s are extrem ely li m ited. The field test kit, based on Dr. Hol m es’ pr otein phosphatase m e thod, is being developed
to com p le ment the analytical m e thod as a qui ck, easy to use, readily available tool to determ ine whether cyanobacteria l toxins are present in an affected water supply. This tool will be useful for:

* municipal w a ter utilities and agencies re sponsible for drinking water quality to monitor supplies for toxins,

* agencies responsible for recreational water quality to m onitor surface water bodies used for recreational cont act purposes for toxins, and

* lives tock ow ners and ag ricultural agencies in dete rm ining the saf e ty of water f o r livestock consum ption.

The prototype field test kit is a non-radioactive assay whic h e xploits the a b ility of

m i crocystins to specifically inhibit the type-1 protein phosphatase (PP-1). T h is colorim e tric assay is reproducible, fast, a nd capable of handling a large num b er of

sam p les with little tra i ning or equip m ent. The kit a llows untrained indiv i duals to v i su ally confirm the presence or absence of toxin in a wa ter sam p le b y com p aring the colo r reaction against a contro l sam p le. The absence o f toxin resu lts in a clear y e llow colo r whereas in the presen ce of the toxin, the so lu tion becom e s colorless. Th is com p arison will a l so ena b le the indiv i dual pe rf or

m i ng the test to ge t an a pproxim a te idea of the

56am ount of toxin present in the sam p le (positive control is 1.5 μ g/L M-LR). The prototype kit was validated dur ing field trials in the summ er of 2000. The summer trials will a llow v a lida tion tes ting of varia b les such a s the ef f ects o f turbidity, the stab ility of the PP-1, and the shelf life and storage tim e for the PP-1.

Test kit results will only be used as an initia l indicator of the presen ce/absence of toxins. Final de term ination of water s a f e ty will b e based on toxin lev e l resu lts der i ved f r om a laboratory analytical m e thodology. Currently, authorities mustissue warnings regarding the use of water by hum ans, pets, or lives tock where cyanobacterial bloom s have developed. Where doubt exists about the safetyof the water, the public must avoid the use of a particular water supply for drinking or recreation. This type of blanket approach is unsatisfactory

Future Applica t ions

In addition to its significance for drinki ng and recreational water authorities, the developm ent of the field test kit is also im portant to livestock producers throughout the country, as the loss of any livestock can have serious financial im plications. This tool will ass i st livestock own e rs to m a nage thei r re sour ces by prov iding th em with a rap i d

m e thod to determ ine the safety of water used for their herds. Early detection of the toxins will not only result in decreasing th e chances of financial loss du e to lives tock deaths, bu t will a l so help elim inate any con cern s that m a y be held by liv e stock owne rs and buyers that m eat from cattle which have con s um ed water contam inated by blue-g reen algae is ta inted.

CONCL US ION

In conclusion, the quality of drinking water in C a nada, indeed the envy of m o st of the world, can be directly linked to a successful system of fe deral-provincial cooperation in assessing and m a naging health ri sk s associated w ith its drinking water sup p lies. Over the past twenty years, the federal, provincia l and territorial govern m e nts have worked together to develop and revise guidelines for substances found in Canadian water supplies
to m a intain and im prove water quality. This relationship has evolved to include m o re than just guideline developm ent, as can be seen by the current project to develop im proved tools to m onitor the presence of cy anobacte r ia l tox i ns (m icrocy stin-LR) in blue-green algae bloom s. The success and im plem entation of the analytical m e thod and the field test kit will pro v ide a m o re accurate and cost-effective approach to the

m a nage m e nt of risk associated with blue-g reen algal toxins to hum a ns and livestock.

References

Health and Welfare Canada ( 1969). Canadian Drinking W a te r Standards and Objectives, 1968. Prepared by the Joint Comm it tee on Drinking W a ter Standards of the Advisory Comm ittee on Public Health Engineering, Department of National Health and Welfare and The Canadian Public H ealth Association. Ottawa.

57Health and Welfare Canada (1979), Guidelin es for Canadian Drinking Water Quality, 1978. Supply and Services Canada, H48-10-1978. Health Canada (1996). Guidelines for Canadian Drinking Water Quality - Sixth Edition. Supply and Services Canada, H48-10/1996E.

Federal-Provincial Subcomm ittee on Drinki ng Water (1998). Canadian Drinking W a ter Guidelines. Developm ent Process. July1998

Health Canada (1995). G u idelines for Ca nadian Drinking W a ter Quality. S upporting Docum e ntation. Part I. Approach to the Derivation of Drinking W a ter Guidelines. Ottawa. Environm ental Health Directorate.

Health Canada (1993) G u idelines for Ca nadian Drinking W a ter Quality. S upporting Docum e ntation. Part II. Trihalom ethane s. Ottaw a . Environmental Health Directo r ate.

Health and Welfare Canada (1978). Guidelin es for Canadian Drinking Water Quality. Supporting Docum e ntation. Uranium. Ottawa . Environm ental Health D i rectorate.

Health Canada (1987). G u idelines for Ca nadian Drinking W a ter Quality. S upporting Docum e ntation. Part II. Ur anium

. Ot tawa. Environm ental Health Directorate.

Health Canada (in press). Uranium in Drinki ng Water. Docum e nt for Public Comm ent. Ottawa. Environm ental Health Directorate.

Health Canada (1998). G u idelines for Ca nadian Drinking W a ter Quality. S upporting Docum e ntation. Part IIA lum i num

. Ottawa . Environm ental Health Directorate.

Health Canada (1998). C y anobacterial Toxi ns - Microcystins in Drinking Water. Docum e nt for Public Comment. Ottawa. Environm ental Health Directorate. HMSO. (1998). The determ ination of m i croc ystin algal toxins in raw and treated waters by high perform a nce liquid chrom a tography. Methods for the Exam ination of W a ters and Asso ciated Materials. London, UK, 1998.

Hrudey, S.E., Lam b ert, T.W

. and Kenefick, S. L. (1994). Health risk assessm e nt of

m i crocystins in drinking water supplies. In: Toxic cyanobacteria a global perspective. March 28, 1994, Adelaide, S outh Australia. Australian Centre for Water Quality Research, Salisbury, Australia. pp.12.

Gurney, S. and Jones, G. (1997) Toxic alg ae occurrence and distri bution in Manitoba surface wate rs. Proceed ings of the Rura l W a ter Quality Symposium

, March 25- 26, 1997, Winnipeg, Manitoba.

Jones, G., Gurney, S. and Rocan, D. (1996) W a ter Quality/Toxic Algae Study Interim Report: Summ ary of the 1995 Field S eason Results. Manitoba Environm ent, June 4, 1996.

58Jones, G. (1996). Toxic algae study summary. Manitoba Departm e nt of Environm ent, February.

Kenefick, S.L., Hrudey, S.E., Peterson, H.G. and Prepas, E.E. (1993) . Toxin release from Microcystis aeruginosa after chem ical treatm e nt. Water Sci. T echnol., 27: 433-440.

Kenefick, S.L., Hrudey, S.E., Prepas, E.E ., Motkosky, N. and Peterson, H

. G. (1992). Odorous substances and cyanobacterial toxi ns in prairie drinking water sources. Water Sci. T echnol., 25: 147-154.

Kotak, B.G., Kenefick, S.L., Fritz, D.L., Rousseaux, C.G., Pr epas, E.E. and Hrudey, S.E. (1993). Occurrence and toxicological ev aluation of cyanobacterial toxins in Alberta lakes and farm dugout s. W a ter Res., 27(3): 495-506.

Kotak, B.G., Lam

, A. J-Y., Prepas, E.E., Kenefick, S.L. and Hrudey, S.E. (1995). Variability of the hepatotoxin m i crocys itn-LR in hypereutrophic drinking water lakes. J. Phycol., 31: 248-263.

Lam b ert, T.W., Hol m es, C.F.B. and Hrudey, S. E. (1996). Adsorption of m i crocystin-LR by activated carbon and rem oval in full-scale water treatm ent. Water Res., 30 (6): 1411-1422.



### Annex A: Stepw ise Protoc ol for Microcystin-LR in Water Supplies Used for Human Consumption

Preamble

Summ er conditions can lead to the growth of blue-green algae in bodies of water (generally sm aller or shallow lakes, rese rvoirs, sloughs or dugout s) throughout Canada. In m a ny cases, bloom s tend to recur within th e sam e bodies of water year after year. While m o st species of blue-g reen algae are capab le of producing nerve and liver toxins, not all blue-green algal bloom s produce toxins, and, when pr esent, the amount of toxins varies dram atically within the body of water and over tim e

. Analytical studies over the past few years in dugouts and other water s upplies in Manitoba, Sa skatchewan and the Peace River region of Alberta i ndicate that b l ue-g reen algal to xins are m u ch m o re common in rural water supplies than or iginally thought. A lthough there are few quantitative data availab l e, ther e are indications that these tox i ns m a y also be occurring in various water supplies in other provinces (e.g., O n tario, British Colum b ia, Quebec and Prince Edward Island).

The factors inducing the production of toxi ns by cyanobacteria are not well known. Laboratory studies dem o nstrate th at s o m e environ m ental factors, such as temperature, light, nitrogen concentrations, carbon availability (in the form of bicarbonate, carbonate

59and carbon dioxide), phosphate concentrations and pH, could be im portant. As toxin production varies greatly am ong diffe r ent strain s of the sam e species, genetic differences and m e tabolic processes m a y also be im por tant in the production of these secondary

m e tabolites. Studies hav e shown that the abil ity to produce tox i ns can vary tem porally and spatially at a particular site. Cyanob acterial toxins tend to be associated with cyanobacterial cells and m a y be m e mbrane bound or occur free within the cells. In laboratory studies, m o st of the toxin release o ccurs as cells ag e and die an d passively leak their cellular contents; som e active release of toxins can also occur from young, growing cells. Toxin levels do no t necess a rilycoincid e with m a xi m u m algal b i om ass; there can be significant variation in the am ount of t oxin per unit biom a ss of cyanobacteria over tim e, which is independent of changes in th e blue-green algal popul ation. Concentrations of m i crocystins were higher in blooms taken during the day than at night in one study, for

exam ple, and no significant difference in tox i n co ncentration s from cyanobacteria incubated for 24 hours at different depths in a reservoir was observ ed in another study. Microcy s tin s are r e la tive l y pers istent in the aquatic environm ent. Studies in Australia have shown that m i crocystin-LR was presen t up to 21 days following treatm ent of a Microcystin aeruginosa bloom with an algicide.

Recent stud ies have led to increasing con cern by governm e nt agencies an d the public regarding the safety of water supplies that m ay be potential sources of these toxins. The purpose of this annex an d the accom p anying flo w chart (s ee next page) is to provid e water purveyors and health and environm ent au th orities with a summ ary of the im portant factors that should be cons idered during bloom events and recommendations on actions that m a y be taken to address the issue.

60Step 1: Visually monitor for bloom formation As bloom s t e nd to recur in the sam e water supp lies, water bodies that have historically exhibited algal bloom s should be visually m onitored for bloom for m ation and hence toxin production during the peak season (usually late May to early October). Au thorities should visually m o nitor supplies for algal growth and conduct sam p ling during and after collapse of the bloom. As well, bodies of water that, due to variables such as size, water depth and nutrient content, m a y be susceptible to alga l bloom s should be considered for m onitoring
program s. Toxins m a y persist follow i ng the colla pse of bloom s, particularly in the late summer and early fall, when the onset of co lder temperatures a nd decrease in light intensity result in decreased rates of toxin de gradation. A visual bloom is identified by

61the appearance of “soupy” water. Colours can ra nge from grey or tan to blue-green or bright blue or reddish. The appearance of bl ooms m a y also be described as fine grass clippings or sm all clum ps.

Step 2: Sample raw 1 and treated su pplies for to xin (algal id entification ) Sa m p les of raw and treated supplies (if appli cable) should be taken at the sam e ti m e in order to save tim e and money. For laboratory analysis, raw water sam p les should be colle cted p r ior to any tre a tm ent, including f iltration; sam p les f r om the raw water tap at the treatm e nt plan t are acceptab l e if no pretr eatm e nt is applied. Sam p ling from a reservoir should be done as close to the inlet/shor e and/or the bloom form ation as possible. However, when possible, it is suggested that sam p les from se veral sites be pooled for the determ ination of toxicity, as cyanobacterial species/cell abundance and biom ass vary spatially within a water body (e.g., cells m a y be transported by wind currents). Treated

sam p les should be taken at the treated water tap of the treatment plant or within the distribution system

.

Sa m p les, in am ounts required by the analytical laboratory, should be collected in glass containers, as studies indicate that the toxin, if present, can b e adsorbed to the plastic. Agencies may also wish to sam p le for algal identification. S p ecies identification, especially f r o m sites pos itiv e f o r toxin, can provide additiona l inf o rm ation regard ing the source, conditions and type of othe r toxins that might be present.

1 A field test kit could be used as a screening tool to determ ine the presence or absence of toxin in a water supply. If the presence of toxin is detected in a sam p le using the field test kit, th e sampling agen cy will need to subm it a sam p le to a recognized laboratory for confirm a tory analytical analysis (see Step 3).

Step 3: Send both rawand treated sa mples for microcystin-LR analysis Both raw and treated samples should be sent (i n coolers) to the laboratory for analysis as soon as possible (preferably within 24 hours) . Sampling agencies s hould contact their local or regional departm e nts of hea lth or environm ent for infor m ation on laboratories capable of conducting toxin anal yses. Upon receipt of sam p les from sa mpling agencies and in order to avoid unnecessary costs, the laboratory should store the treated sam p les until the r e s u lts of the m i crocys tin -L R analysis o f the raw sa mples are av ailab l e. A

m i crocystin-LR result of >1.0 μ g/L in the raw sam p le will cause further action, as outlin ed in S t eps 4–10.

Step 4: Microcystin

- LR >1.0 μg/L (raw ) Results will be reported to the sam p ling agenc y, as per Step 12 (raw). A result of >1.0

μ g/L should cause further action, as o u tlin ed in Steps 5–10.

Step 5: Perform toxin analys is of tr eated w a ter samples Testing will be perform e d on treate d sam p les when a level of >1.0 μ g m i crocystin-L R /L is found in a raw sam p le from the sam e site.

Step 6: Microcystin

- LR >1.5 μg/L (treated)

62A result of >1.5 μ g m i crocystin -LR/L f o r the tre a ted sam p le will cau se th e sam p ling agency to take appropriate action, as outlin ed in S t eps 7–10. Microcy s tin

- LR results of >1.5 μ g/L in a treated su pply will res u lt in a res ampling of the treated sup p ly as soon as possible (Step 9) and notifica tion of the comm unity and appr opriate agencies (S tep 7).

Step 7: Notify community and agencies Upon receiv i ng the results, the s a m p ling ag ency will follow s t andard p r otocol for notif ying co mmunities and appropria te agenc i es. Considera tio n will be g i v e n as to which agency should be taking the lead role in this regard. Additiona lly, th e inve stiga tion should tak e into accoun t the history o f the s ource/supply and the type of treatm e nt at the plant. Dialysis treatm e nt units in the community should also be notified, especially if it is a first-tim e occurrence for bloom s on this supply. A Health Canada fact sheet on

m i crocystin-LR ( It’s Your Health: B l ue-Green Algae (Cyanobacteria) and their Toxins ) is available to help convey inform ation to comm unities.

Step 8: Consultation and decision-making In the case of comm unit y (m unicipal) supplie s, risk assessm ent discussions should take place regard ing addition a l action to be taken. Health ag encies, m unicipal councils an d the lead regulatory agency responsible for m uni cipal system s should be included in these discussions. The risk assessm ent discussion process should include, for exam ple, such item s as the history of the site, the size and loc a tion of the blo o m , available tre a tm ent technology (if a treated site), us es of the source water (recrea tional versus dom e stic uses) and m onitoring of the environm ental conditions that m i ght affect the bloom (e.g., wind). Where possible, lysing of the bloom by the a ddition of copper sulphate or Blue Stone should be avoided, as this action will imm e di ately release toxin from the cells into the water supply. If the bloom is a common occurrence, nutrient m onitoring m a y be considered. Weekly m o nitoring should be continued. In the case of non-comm unity (non-m unicipal) supp lies

, the sam p ling agency will consult with he alth ag encies and agencies having treatm e nt expertise.

Note: See Step 10.

Step 9: Resample treated supply Following receip tof res u lts ind i cating th at the treated water contain s >1. 5 μ g

m i crocystin- L R/L, the tr eated supp lywill be resa mpled as so on as possib l e or as determ ined by the sam p ling agency.

Step 10: Alternative su pply or treatment adjustment During the S t ep 8 decision-m aking process, discussions regarding treatm e nt adjustm e nts or alternative supplies should be reviewed (Step 10). The lead agency should advise the owner of the supply of any treatm ent options , such as additional granular activated carbon filtration or o z on ation. It sho u ld be poi n t ed out that b o iling is not effective in reducing or rem oving these toxins, although som e point-of-use devices may be effective. Note: As bloom s m a y be of short duration (ranging from days to weeks), health and environm ent agencies may recomm e nd, after co nsultations with the community (see Step 8), that cons um ers seek alternative s upplies of safe drinking water until there is no longer a visible bloom and the toxi n level has dropped below 1.5 μ g/L.

63Step 11 (raw ): Microcystin-LR <1.0 μg/L A result of <1.0 μ g m i crocystin -LR/L in the r a w water will c ause the s a mpling agen c y to continue to visually m o nitor the r a w water f o r the recurrence of bloom s and resam p le if
necessary.

Step 11 (treated): Microcystin-LR <1.5 μg/L A result of <1.5 μ g m i crocystin -LR/L in the trea ted wate r will cau se the sam p ling agency to continue to m onitor the ra w water as per Step 11 (raw).

Step 12 (ra w ): Send results to ag encies Microcy s tin

- LR results f r om laboratories (and f i eld te st kit r e sults when available ) will b e reported to the sam p ling agency as so on as possib l e from ti m e of receipt o f the sam p le. Sa m p ling agencies are respons ible for reporting results to the water supply owner.

Step 12 (tre a ted): Send results to a g encies Treated wa ter m i crocystin-LR resu lts will be re p o rted to the sam p ling agency as soon as possible f r o mthe tim e of the positiv e raw water results f o r th e sam e sam p ling site. Sa m p ling agencies are responsib le for reporting results to th e water supply owner and/or responsible agencies.

64Blue-green algae and their tox i ns – Great Britain’s perspectiv e

HuwJames, WRc-NSF, Henley Road, Med menham, Marlow , Bucks. SL7 2HD. jam e s_h@wrcplc.co.uk And Anthony Ll oyd, Drinking Water Inspectorat e, 2/E5 Ashdown House, 123 Victoria Street, Lon d on SW1E 6DE Anthony.Lloyd@defra.gsi.gov.uk

ABST RACT

Details of research described in this paper can be found under Foundation for W a ter Research publications at www. fwr.or g

This paper reviews outputs from the Unite d Kingdom ’ s collaborative research program on algal toxins and their si gnificance in drinking water tr ea tment. Inf o rm ation on th e DW I policy towards control of algal toxins in water supplies and current water industry control m e a s ures is also given.

INTRODUCTION

Blue-green algae in some raw water sources had caused sporadic operational problem s , such as blockage of raw water filters or th e production of tastes and odors in finished waters, for water suppliers in the UK for decades. However, in 1989 following well publicized blue-green algal bloom s, som e of whi c h occurred in raw water storage reservoirs, it was considered possible that m a mm a lian toxins produced by som e of these algae cou l d pose a threat to the health of drinking water consum ers. The most commonly encountered species in the 1989 incidents were Microcystis aeruginosa

, Aphanizomenon
sp., Anabaena sp. and Oscillatoria sp. Ani m al toxicity tests on sam p les fr om 78 water bodies indicated that 68 per cent were toxin producing.

At this tim e , there was n o reliable inf o rm ati on regarding the presence of algal toxins in raw or treated waters in the UK, no toxicolo gical inform ation relating to the potential route of exposure existed, and it was not know n whether water treatm e nt processes then in use in the UK were ef fectiv e in r e moving toxins (if present) from raw waters. To address thes e deficiencies, a national collabo rative research program was i n itiated in 1990 by the then Departm e nt of the Environm en t and the National Rivers Authority (now the Environm ent Agenc y ). In 1991 and 1992, contributions to funding were m a de by UK Water Industry Research Ltd. The research program was m a na ged by the F oundation for Wat e r Resear ch.

R&D PRO G RAMME (1990-1996)

This research program had several objectives, as follows:

65• developm ent of sensitive analytical m e thods to allow toxins to be quantified in raw water sources and drinking waters;

• design of a toxicity testing program to es tablish levels of concern for the toxins;

• investigatio n of the ef f e ctiv eness of various water treatm ent processes used in the production of drinking waters for toxin rem oval.

At the tim e this research began there were several factors that influenced the approach that could be adopted. K nowledge concerning the toxins produced by blue-green algae was lim ited, although it was considered that the toxin of greatest relevance to the UK was the cyclic heptapeptide hepatotoxin m i crocys tin- L R, so resear ch ef f o rts were in itially focused on this com pound. The fact that it was one of the first hepatotoxins to be commercially available was also influential, as pure standards are required to develop quantitative analytical methods, to use for toxi city testing and for use in water treatm ent studies. S i milar work was also undertaken for the neurotoxin anatoxin-a, which was also considered to be of relevance w ith re spect to UK water supplies.

DEVELOP M ENT OF ANALYTI CAL METHODS

It was necessary to develop quantitative an alytical m e thods for toxins in w a ter to estab lish wh ether they w e re present in raw wa ters and drinking wate rs, to provide a check of the dosing levels used in the toxicity te sting program and to determ ine the efficiency of re m oval when various water treatm ent pr ocesses were applied to toxin-containing waters. It was also a requirem ent that th e m e thods developed should be usable on a routine basis by water company laboratory sta ff; this constraint ruled out the use of advanced techniques e.g. liquid chro m a tography-m a ss spectrom e try (LCMS).

Microcy stin -LR

Work on m e thod developm ent comm enced in ear ly 1991 and as noted earlier, this w a s one of the then known microc ystin congeners that was commercially available. A literature survey revealed that although there were several reports of its determ ination in algal cells, little inf o rm ation was availabl e on its an alysis in water s a m p les. The optim ized method developed involved solid-pha se extraction followed by analysis using high perform a nce liquid chrom a tography (HPL C) with ultra-violet (UV detection). Nodularin, a cyclic pentapetide toxi n produced by blue-green algae ( Nodularia sp. )

norm a lly found in brackish waters, was used as an internal standa rd. A lim ited evaluation of the m e thod for drinking waters and reservoi r w a ters in the autumnof 1992 showed that at the 1 μ g/l level, the relative standard devi ation was less than 10%. The m e thod was then perform a nce tested by five laboratories in 1993. This involved the analysis of over one hundred sam p les by each labo ratory, and dem onstrated that the lim it of detection was 0.5 μ g/l.

It was also shown that el ectrospray ionization L C MS could be used to provide confirm a tory data.

66In 1996 another analytical m e thod, which de term ined som e additional m i crocystin congeners (-RR, -YR) and nodul arin, was performance tested. It was show n to be satisfactory and could be applied to determ ine other m i crocystins, but the lack of commercially available p u re stand a rd s only allo wed it to be tested for the aforem entioned toxins.

Anatoxin-a

An analytical m e thod foranatoxin-a in wate r, which involved solid-phase extraction and analysis of the extracts us ing HPLC with UV detection, was developed in 1993. The lim it of detection of this m e thod was 1 μ g/l. In 1994, it was shown that a custom -synthesized inte rnal s t an dard (d 3 -homoanatoxin-a) im proved the perform a nce of the m e thod.

TOXICITY TESTI NG

Microcy stin -LR

The only published inform ation relating to the tox i city of m i crocystin -LR that was available p r ior to th ese s t udies was th at its LD 50 via intra-peritoneal (i.p.) injection in

m i ce was about 50 μ g/kg. The toxicity-testing program

, devised in consultation with the contract toxicology laboratorythat undertook the work, cons isted of an acute toxicity study to compare the toxicity vi a the oral and i.p. r outes, an investigation of the effe cts on the fetus in the m ouse, a nd a repeat dose st udy over thirteen week s by the oral route, again in the mouse. The m a in reason for using the m ouse rathe r then the ra t in the latter two studies was the ava i lability and cost of pure m i crocystin- L R. Even when using th e mouse, the cost of the pure toxin used was over $30,000.

The m ouse oral LD 50 in the acute study wa s found to be about 5000 μ g/k g, while the rat oral LD 50 w a s greater than 5000 μ g/kg. The m o use i.p. LD50 was between 50 and 158

μ g/kg. This m eant that the difference in acute toxicity between i.p. injection and oral dosing was between 30 and 100-fold, with the m ouse being slightly m o re sensitive than the ra t.

The No Observed Adverse Effect Level (NOAEL) f o r developm ental to xicity (da ily oral adm i nistration during organogenesis) was 600 μ g/kg bodyweight per day. In the 13 week repeat dose study, the clear NOA EL for pathological changes in the liver of the m ous e when adm i nistered orally by gavage once daily was 40 μ g/kg bodyweight per day. The effects observed at 200 μ g/kg bodyweight per day were relatively m i nor and few ani m als were affected.

In term s of risk assessm e n t the 40 μ g/kg bodyweight per day dose would be 400 tim es greater th an that received by a 10 kg child drinking 1 liter of water per day, and the

m a rgin of sa fety would be m u ch greater than this.

67Anatoxin-a

Toxicity d a ta relating to the acu te ef f ects of th is neurotoxin were alr eady available, but in order to undertake a better-inform e d risk asse ssm ent it was co nsidered n e cessary to carry out som e longer-term studies, so a 28-day oral in vivo toxicity study and a developm ental toxicity screening study were carried out in th e mouse. A series of in vitro and in vivo screening studies were also done in order to properly ch aracterize the pharm acological activ ity of an atoxin

- a re lative to nico tine (ana toxin-a was known to be a stereospecific nicotinic agonist).

In the pharmacological screening studies anatoxi n-a elicited s i m ilar respo n ses to tho s e of standard nicotine solutions, but it was a m o re potent agonist than nicotine. In a twentyeight day oral repeat dos e study the NOAE L fou nd was 0.098 m g /kg bodyweight per day. The NOAE L for teratogenicity over the period of organogenesis was 2.46 mg/kg bodyweight per day. The conclusion of this latter study (that anatoxin-a was not a specific developm ental toxicant in the m ouse) is in agreem ent with an earlier study carried out in the ham s ter.

Based on the m o st conservative NOAEL from these studies, a guideline value of 1 μ g/l for drinking water would provide a significant m a rgin of safety of about three orders of

m a gnitude for a 10 kg child consum ing 1 liter of water per day.

TREATMENT STUDI ES

Because work already p ublished h a d shown th at convention a l water treatm ent processes (chem i cal coagulation, s a nd filtratio n, chlorina tion) were not particularly effective in

rem oving microcystin-L R, advanced water treatm ent processes were inv e stig ated.

Microcy stin -LR

It was show n that activ ated carbon adsorpti on, o x idation and nanofiltr ation could all be used to rem ove this toxin from lowland rive r-de r ived rese rv oir wate r. Powdered activated carbon (PA C ) dosing was effective, but the de gree of rem o val was strongly dependent on the dose applied and wood-based P A C was m o r e effective than those derived from coal
or coconut source m a terials. Coal, peat a nd wood based granular activated carbon (GAC) were also effective in rem oving m i crocystin-LR although rapid breakthrough of the toxin occurred.

Ozonation and treatm ent with potassium perm anganate under practicab le water treatment dosing conditions were the m o st effective oxidative processes, with higher rem oval occurring when dosed into clarified or sand fi ltered water. Chlorination (applied chlorine dose 1.7 m g /l) was effective, but dependent on pH e.g. at pH5, rem oval was >90% within 30 m i nutes, but at pH7 90% rem oval was only obtained after 22 hours and at pH9

rem oval was even slower.

68W h en using a polym eric nanof iltr atio n m e m b rane with a nom inal m o lecular weight cu toff of 200, a nd a perm eate to retentate ratio of 0.25, com p lete rejection of m i crocystin occurred and no toxin accum u la ted on the m e mbrane itself.

Anatoxin-a

Oxidative processes also rem oved anatoxin-a, although as for m i crocystin-LR, they were more effective when applied to treated waters. For both ozone and potassium
perm anganate, a dose of 2 m g /l gave a reducti on of >90 percent in the dissolved toxin concentration. However, no significant rem oval was observed with chlorination.

PAC and GAC were effective in removing an atoxin-a, although rela tively high doses of PAC were required to achieve >85 percent t oxin rem oval. Results obtained with G A C suggested that for anatoxin-a biode gradation occurs on the G A C.

OTHER ST UDIES

Monitoring

Since the analytical m e thods described above were developed, severa l surveys of raw and treated waters have been carri ed out in England and W a les, and in Northern Ireland. T h e most com p r e hensive of these surveys was conducted in Northern Ireland to allay concerns regarding drinking water produced by a new drinking water treatm ent works taking raw water from a water body where th ere have been blue-green algal bloom s (usually Osc illa toria sp.) in the past. A total of tw o hundred and seventy three sam p les were analyzed during the period June 1998 – July 1999 for m i crocystin-LR and anatoxina. In one of these sam p les, anatoxin-a was detected at about 1 μ g/l and its presence confirm e d using GCMS following derivatisati on of the extract. This concentration found was not considered to be of significance to hum an health. No m i crocystin-LR was detected in any of the samples.

Toxin persistence

The analytical m e thods have also been app lied to study the persiste nce of m i crocystin-LR and anatoxin-a in raw water reservoirs. Fo r m i crocystin -LR the half-life in reservo i r water was found to be ab out six days, and with ad dition of bed sedim e nt this was reduced to about 3 days. The half-life of anatoxin-a wa s found to be 4-5 days in reservoir water at pH8 in the presence of bed sedim e nt. At pH4, anatoxin-a is persistent but as it is unlikely that natural waters at this pH would support populations of anatoxin-a producing bluegreen algae, this finding was not con s idered sign ificant.

GUIDELINES AND S T ANDARDS FOR ALG A L TOXINS

The water industry in England and Wales was privatized in 1989 and the Governm e nt ’s technical regulator for the industry is the Drinking W a ter Inspectorate (DW I). The Water Supply (W ater Quality) Regul ations (1989), which DW I enfor ces, cover som e 55 specific

69param e ters that do not include algal toxins. However, the regulations require that no substance m a y be present in drinking waters atco ncentration s that would cause a risk to health. In th is respec t wa ter u tilities would be required to m o nitor for algal toxins, if a risk s itua tio n existed. In the UK, water utilitie s currently base that risk assessm ent on the potential for algal loadings to overw helm tr eatment processes and contam inate supplies.

In respect of regulatory standards, there are insuf f i cient d a ta to determ ine health -based guidelines or standards for even a representati ve selec tion of the to xins. T h e best studied is m i crocystin LR, although uncertainties exist, particularly w ith regard to its tum o r prom oting capability. G u ideline values have only been set for m i crocystin LR/ m icrocystins. W HO proposed a provisional guideline value in 1998 for m i crocystin LR and Health Canada set a guideline value for microcystins at about the sam e ti m e

. Both of these were based on the data ge nerated by the UK nationa l research program.

Som e data have been generated for anatoxin-a that provides sufficient inform ation to give a reasonable level of reassurance regarding the concentrations encountered in UK waters. Data on cylindrosperm opsin have been recen tly produced in Australia bu t these data have

a num ber of problem s with regard to reporti ng and interpretation. It is understood that W H O will consider the data in the r o lling re vision of its Guideline s f o r Drinking W a ter Quality. There is pressure from some of the groups working on toxins to produce “standards” for toxins but reports from W HO indicate that th is is not s een as an appropriate approach.

At the Global Meeting on the WHO Gu idelines in Tokyo in May 2002, there was considerable discussion of blue-green algae and their toxins. The ge neral view was that toxic blooms need to be taken seriously. Ther e w a s particu l ar concer n in parts of Asia, where there can be significan t growths throughout the year. The m a in issue is clearly one for developing countries where there are m a ny sm all, under-resourced, supplies with only basic trea tment. I twas genera llyag reed that a preventative a pproach is preferred and that any attem p t to control th e ri sk by routine m onitoring for toxins was not practicable. Analytical m e thods are com p licated and not sufficiently robust fo r routine m onitoring purposes. Standards are very expensive and are available for only a few toxins. In addition, there are alm o st certainly a nu m b er of as yet unidentified toxins.

Currently, setting a standard based on the few toxins for which there were adequate data could be construed as potentially m i sleading because the absence of a particular toxin does not indicate the absence of a problem . In addition, the potential for changes in the
presence an d absence of toxins m eans that sam p ling to give an appropriate level of reassurance could be problem a tical. Preven tion of bloom s for m ing is the best way forward, although this m a y present som e diff iculties. Control of eutrophication is an im portant is sue f o r the E nvironm ent Agency in the UK and at the Europ e an leve l; it will be an im portant consideration in the I mplem entation of the W a ter Fr am ework Direc tiv e.

It is und ers t ood that the WHO approach will b e s upported by guidelines for som e toxins, developed w h en there are adequate data but the production of guideline values for an

70increasing list of toxins is seen as potentially counter-pr oductive. The WHO paragraph to be included in volum e 1 of the revise d guidelines currently reads as follows:

Cyanobacteria occur widely in lakes, reserv oirs, ponds and slow flowing rivers. Many species are known to produce toxins, a number of which are of concern for health. T h ere are many cyanotoxins, w h ich vary in structur e and may be found within cells or released into water. There is wide variation in the to xicity of recognized toxins (including am ongst different varieties of a single toxin, e.g., Microcystins) and it is likely that further toxins remain unrecognized.

The health h azard is pr imarily associated with o vergrowth (bloom) events. Such blo o ms may develop rap i dly and they may be of short duratio n. In most circumstances, but not all, they are seasonal.

Analysis of these substances is also d ifficult although rapid methods are becoming availab l e fo r a small nu mber, e.g, microcys tin s, in addition a nalytical s t a ndards are frequently not available. The preferred appr oach is, therefore, monitoring of source water for evidence of blooms, or bloom form ing potential, and increased vigilance where such even ts occur.

A variety of actions are available to decr ease the probability of bloom occurrence and some effective treatments ar e available for removal of cy anobacteria or cyanotoxins. For these reasons, monitoring of cyanotoxins is not the preferred focus of routine monitoring and is prima r ily used in response to bloom events. Whilst gu ideline values are derived where sufficient data exist, they are intended to inform the in terpre tation of data fr om the above monitoring and not to indicate that ther e is a requirem e nt for routine monitoring by chemical analysis.

OPERATI O NAL ISS U ES IN THE UK

Because of the com p rehensive treatm ent pro cesses that are alrea dy in place at water
treatm ent works, m onitoring for algal toxins is norm ally only undertaken when there is a risk that algal cell concentrations could a dvers ely affect treatm ent processes. Algal bloom s occur seasonally in m a ny i m pounding rese rvoirs where water is abstracted from lowland rivers. These rivers drain agricu ltural catchm ents and there are usually significant inputs of treated sewage effluent . Although phosphate rem ova l is practiced at

som e locations, this treatm ent rarely achieve s the overall reduction in phosphorus input needed to lim it developm ent of alga l bloom s.

Operation a l m easures focus on forced m i xing/aeration sys t em s. These are aim e d prim arily at avoiding stratification but also lim it algal growth by reducing light penetration. Multip le d r aw-off levels are used to facilitate s e lection of best quality water and where alternative sources are available, continuous fluorim etry is used to enable switching of sources to reduce algal load in raw water. Monitoring of reservoirs is

norm a lly carried out from boats, with sa m p les taken throughout the water column. Measurem ents include: a-chlorophyll, partic ulate organic carbon, algae identification and

71estim ation of abundance and inorganic algal nutri ents. These data are assessed in the light of

potentia l to cause blo c king of f

ilter s and no water utilitie s c a rry out routine ana l ysis f o r algal toxins.

Public ac ces s is perm itte d on m a ny lowland rese rvoirs and c ontact wa ter sports ar e commonly practiced. Livestock grazing to the wa ter’s edge is rarely allowed but dogs and horses m a y drink from r e servoirs, especially where footpaths and bridleways border the

reservo i r. W h ere access to reservo i rs is pe rm itted, warning sig n s alert th e public to the risk of contact with water, or allowin g anim als access to the water. These signs are usually p e r m anently installed, r a the r than be ing deployed only when an algal bloom i s underway.

CONCL US IONS

The com p re hensive R&D program t h at was funded by the UK governm e nt and the w a ter industry addressed the potential threat to dr inking water supplies from blue-green algal toxins. The research outputs have enabled a better understanding of the problem s posed by these toxins and the w a ys in which the poten tial risk s can b e effectivelyaddressed. The m a in pa rt of this program was conducted over the period from 1991-1996 and the cost to the funding bodies was in ex cess of 1.5 m illion dollars.

In the UK, there have been relatively few probl em s relating to blue-green algal toxins and drinking waters since 1989, and there wa s only a lim ited demand for m o nitoring for

m i crocystins and anatoxin-a during th e period 1992-1996. The only significant monitoring that has been undert aken since was for water samp les from Northern Ireland between June 1998-July 1999. A further indicatio n of the lack of de m a nd for m onitoring is illus t ra ted by the f act that with one exception (WRc-NSF), no c ontract laboratories

curren tly offer the analys is of blue-green algal toxins.

Most lowlan d waters in the UK receive m u ltip le barrier trea tm ent, often including oz one and/or carbon treatm ent. The national rese arch program provided reassurance that, as long as algal loadings are not allowed to overwhelm treatm e nt, exposure to algal toxins via drinking water would not cons titu te an id entif iable r i sk to health. Th is situa tion co uld of course change if water tem p eratures and nutrient inputs continue to increase.

The m a in potential p r oblem f

o r the UK water in dustry at present concerns disposal of f ilter b ackwash water f r o m those water trea tm ent works which experience signif i can t algal b l ooms in the i r raw water stor age res e rvoirs. The Environm ent Agency is actively seeking to place dis c harg e consents o nthe di spos al of such waters (particularly where there is little dilution by the receiving wa ters), although no consents have yet been applied.

72Summary o f: WHO Guidelines Presented b y : Ian Falconer, D.Sc.

Dr. Ian R. Falconer, from the Cooperative Research Center for W a ter Quality and Treatm e nt and the University of Adelai de Medical School, gave an additional presentation on the W o rld Health Orga nization (WHO) and their approach to cyanobacterial toxin guidelines. WHO dri nking water guidelines, based on potential hazards to hu m a n health , were first rel eased in 1 985, and have undergon e num erous revisions. Cyanobacterial toxins were cons idered as part of the W HO drinking water guidelines in April 1997, following the producti on of a WHO m onograph on the subject. The group that produced the m onograph recomm ended that a Guideline Value of 1

m i crogram per liter be m a de for m i crocysti n, based largely on anim al and hum a n data f r om Australia.

Dr. Falconer discussed research, m u ch of which was his own, on m i crocystin. The tole rable d a ily intake (T DI) was dete rm ined from 10-13 week oral dosing tests in m i ce and pigs. T h e NOAEL i nm i ce was 40 μ g/kg/day, and the L OAEL in pigs was 100

μ g/kg/day. The TDI was created by dividing the NOAE L in m i ce, 40 μ g/kg/day by three ten-fold safety factors, yielding 0.04 μ g/kg/day. The W H O then generated a guideline value by m u ltip lying the TDI by a 60 kg body m a ss and a 0.8 p r oportion of dose due to drinking water and dividing by 2.0 L/day water consum ption. This yields 0.96 μ g/L, or roughly 1.0 μ g/L.

As Dr. Falconer m e ntioned, the Chem ical Substances W o rking Group reviewed the

m a tter of cyanobacterial toxins in May of 2002. The Toxic Cyanobacteria in W a ter monograph, origin ally w r itten in 199 7, is to be revised and reissued. The WHO will reconsider cylindrospermopsin, for which an oral toxicity study was completed, and other cyanobacterial toxins in 2003/4. In light of actions by the WHO, Australia, Canada, New Zealand, Poland and Brazil have establishe d a guideline for m i crocystin, and have provisional standards for othe r cyanobacterial toxins. La stly, m onitoring and water
treatm ent techniques are being im proved, and at te ntivene ss to cyanobacterial tox i ns is rising.

73Laboratory Methods fo r the Study of Harmful Cyanobacteria Water blooms: “The Cyano-HABs”

Wayne W. Carmichael Professor-Aquatic Biology/Toxicology

Departmen t of Biological Sciences Wright State University Dayton, Ohio 45435 U.S.A.

Tel: 937-775-3173 Fax: 937-775-3320 E-mail: w a yne.carmichael@w right.edu

University w e bsite address: http://biology.w right.edu/faculty/carmichael/labhome.htm

Background

Increasingly, harmful algal bloom s (HABs) are bei ng reported worldwide due to several factors, prim arily - eutrophication, clim ate change and more scientific investigation (Hallegraeff et al., 1995). HAB organism s include those causing: PSP (paralytic shellfish poisoning), D SP (diarrhetic shellfish poisoning), NSP (neurotoxic shellfish poisoning ), ASP (amn esic shellfish poisoning) and CTP (cyanobacteria toxin poisoning). All but CTP organism s are m a inly a m a rine occurrence. CTPs occur in freshwater lakes, ponds, rivers and reservoirs throughout the world. Organi sm s responsible include an estim a ted 40 genera but the m a i n o n e s a r e A n ab a e n a , A p h a n i z o m e non , C y l i n dr o s p e rm opsis

, Lyngbya

, M i c r o c y s t i s , N o s t o c a n d O s cillato r ia ( P lanktothrix) (Carpenter and Carm ichael, 1995). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins with biot oxins being responsible for acute lethal, acute, chronic and sub-chronic po isonings of wild/dom estic anim als and hum a ns. The biotoxins include the neur otoxins; anatoxin-a, anatoxi n-a(s) and saxitoxins plus the hepatotoxins;

m i crocystins, nodularins and cylindrosperm ops in (Carm i chael, 1997; 2001) (Table 1).

Table 1. Nam e and Producer Organi sm for the Biotoxic Cyanotoxins

------------------ --------------- --------------- --------------- --------------- --------------- --------------- NAME PRODUCE D BY

------------------ --------------- --------------- --------------- --------------- --------------- --------------- Neurotoxins

Anatoxin-a Anabaena, Aphanizomenon, Oscilla toria Hom o -Anatoxin-a ( P lanktothrix)

Anatoxin-a(s) Anabaena, Oscilla toria (Planktothrix)

Paraly tic Sh ellf ish Poiso n s

74(Saxitox i ns) Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Planktothrix, Trichodesmium,

Liver Toxins

Cylindrosperm opsin Aphanizomenon, Cylindrospermopsis, Rhaphidiopsis, Umezakia

M i c r oc ys ti ns Anabaena, Aphanocapsa, Hapalosiphon, Microc ystis, Nostoc, Oscilla toria ( P lanktothr i x)

, Synechococcus

Nodularins Nodularia (brackish wa ter)

Contact Irr i tant-Der mal Toxins

Debrom oapl ysiatoxin, Lyngbyatoxin Lyngbya (marine ) Aplysiatoxin Schizothrix (m arine)

Concern for anim al and hum a n health im pair m e nts due to toxic cya nobacteria arises from several sources of inform ation. The m a in one concerns reports of anim al poisonings f r om ingestion of toxic water bloom s beginning withthe later part of the 1800's. It was not until the 195 0' s, with pioneering wor k

of scientists in Canada, the United States, Australia and South Africa, that we began to understand that cyanob acteria could indeed produce highly toxic compounds. The anim al and hum a n intoxication reports were first summarized in the 1960' s by Schwimmer and Schwimmer (1964, 1968). These reports have been updated by several works incl uding Sivonen (1990), Carm ichael (1992), Ressom

, et al. (1994) an d Yoo, et al. (1995). In addition, a major effort to com p ile all available inform ation on toxic cy ano b acteria in cluding issues of hum a n health, safe water practices, m a nagem e nt, prevention and rem e diation has been published by the W o rld Health Organization (Chorus and Bartram

, 1999). This book is designed as a m a nual for water and he alth au thor ities inaddres sing all is su es of toxic cyanobacteria that they may be confronted with.

Along with the W HO ma nual cam e a study on the risk assessm e nt of cya notoxins in drinking and bathing waters. Risk assessm ent of m i crocystins (as Microcystin-LR)
indicate that a level of 1 μ g L -1 should be considered a gu ideline value for m a xi m u m allowable concentration (MAC) based upon an adult consum ption of 2 L day -1 . Other MAC' s f o r the neurotox ins and cylin drosper m opsin will n o t be set un til more basic toxico logy and epidem iology are availab l e. In ad dition, the S t ate of Oreg on in the U. S.A. has set a regulatory level of 1 μ g g -1 for m i crocystins (as Micr ocystin-LR) in hum an food supplem ents m a de from the filam e ntous cyanobacterium A p h a n i z o m e non f l o s

- a qu a e (AFA). In th is case the A F A is not th e producer o f m i crocystin but because it is harv ested from a natural lake (Klam a th Lake), other cy ano b acteria p r es ent as a no rm al part of the

75h a rv e s t e d a l g a e ( i

. e

. , M i c r o c y s t i s a e r u g i n o s a a known producer of m i crocystins ) are the source of this hepatotoxins. Health authoritie s from several countries are also evaluating the risks of cyanotoxins in algae and m a y al so adopt guideline or re gulatory levels for

m i crocystins in drinking and recreati onal waters and algae food products.

Confir m a tion of hum a n deaths from cyanot oxins is lim ited to exposure through renal dialysis. Microcystins w e re im plicated as the m a jor contributing factor in live r f

a ilur e and death of at least 52 hum ans, in 1996, at a haem odialysis center in Caruaru, Brazil (Jochim s en, et al. 1998, Carm ichael, et al. 2001). This tragic but avoidable event points to the im portan ce of understanding cy anotoxins as health h azards in drink i n g waters and then comm unicating this inform ation, in cluding the correct identification of cyanobacteria responsible for poisonings, to wa ter authorities and public health officials. Since m o st of the worlds reservoir and lake bas e d water supp lies are sub j ect to incr ea sing nutrient leve ls, it is pr ob able th at ep isode s of cyanotoxin pois oning will c ontinue un le ss

m easures are taken to improve our understandi ng of their role in water-based diseases. For these reasons it is important to consid er the prevention, m a nage m e nt and m itigation of CyanoHABs.

Prevention, Management and Mitigation St rategies for Control of Cyanobacteria and Their Toxins

The first goal in the control of cyanobact eria water bloom s in public water supplies should be the im ple m entation of ways to prevent or at least minim i ze those conditions, which lead to their excessive growth. In a ddressing these grow th requirem ents the two

m a in problem s are nutrients and light (C horus and Bartram 1999-Ch. 8). Critical to nutrient and light control is proper managem e nt of the water supply watershed and the water reservoir. There are tw o m a in ways this can be done. They are term ed ‘top-dow n’ and ‘bottom-up control’. Top down control is also term ed bio-m a nipulation and is the

m a nage m e nt of higher trophic organism s to m a xi m i ze grazing of algae and thus reduce cyanobacteria abundance. The bottom

- up approachis to restrict the supply of essential nutrients and thus restrict growth. T h e key act ion for nutrient control of cyanobacteria is reduction of external phosphorus and nitrogen. In this ar gum e n t, bottom

- up control is extended to include the restrict ion to supply of all resources. Therefore, the m a nipulation of the light clim ate by artificial m i xing is also included in bottom

- up control.

The m a nage m e nt of cya nobacteria water bloom s is best approached by the developm ent of a risk-m anagem ent fr am e w ork. This fram ework should define the needed approaches for situation assessm ent, m a nage m e nt actions (often referred to as the Alert Levels Fram ework) , and appropriate planning and response (Chorus and Bartram 1999, Ch. 6-7).

The m itigation, or m i nim i zation, of the harm ful effects of a cyanobacteria water b l oo m can be approached in three m a in ways (Chorus and Bartram 1999-Ch. 9). The first involves selecting the best ab straction m e thod or location for bringing water into the
treatm ent plant. This can be done by choosing an optim um position for o ff take, or by abstracting s u rface water through ban k or ground filtration. T h e second major way to

m itigate cyanobacteria water bloom s is to choo s e the optim um water treatm ent

76technique(s) in the water treatm ent plant. Th ese include the proper choice of coagulation, precip itation

, sedim e ntation, filtration and adsorption techno lo gies for water bloom

m a nage m e nt . The third and final way to m itig ate cyanobacteria water b l oo m s is with chem icals.

Chem ical co ntrol of alg a e in water supply storages has b een a widespread water quality

m itigation p r actice for o v er 100 years. Copper su lphate h a s g e nerally been the alg aecide of choice and records of its use date f r om 1890 in Europe (Sawyer 1962), 1904 in the U.S. (Moore and Kellerm an 1905), and since at least the m i d-1940’s inAu stralia (Burch et al. 2001).

An extensive survey of water utilities in th e U.S. and Canada in the 1980' s indicated that copper sulp hate is by far the m o st widely us ed algaecide, alth ough other alternatives are used under som e circumstances (Casitas Muni cipal W a ter District 1987). A summ ary of the rang e of com pounds that hav e been us ed as algaecides is g i ven in Tab l e 2.

TABLE 2: Common algaecides an d their formulations (after Burch et al. , 1998)

COMPOUND FORMUL ATION Copper sulphate CuSO 4 .5H 2 O Copper II alkanolamine complex Cu Alkanolam ine .3H 2 O ++

Copper - ethylenediamine complex [Cu(H 2 NCH 2 CH 2 NH 2 ) 2 (H 2 O) 2 ] ++ SO 4

Copper - triethanolamine complex Cu N(CH 2 CH 2 OH) 3 .H 2 O

Copper citrate Cu 3 [(CO OC H 2 ) 2 C(OH)COO] 2 Potassium permanganate KMnO 4 Chlorine Cl 2 Lime Ca(OH) 2 Barley stra w

In summ ary, for m itigation of cyanobacteria l water b l oom s the first priority should b e

rem oval of intact cells, using selected abstraction points followed by separation techniqu es s u ch as coag ulation or mem b rane filtration. Chem ical treatment m e thods have and are being evaluated w ith chlorination and ozonati on being effective for the destruction of residual dissolved m i crocystins and cylindrosperm opsin at moderate w a ter tem p eratures. Possible tem p erature effects on these oxidation reactions are currently unknown. Anatoxin-a can be effectively rem oved using ozone, although chlorine is relatively ineffective. Oxidat ion techniques do not appear to be the best m e thod for t h e treatm ent of saxitoxins u nder norm a l treatm ent plant operating conditions. Powdered

77activated carbon can be effective for the rem oval of all toxins, except, perhaps, a few

m i crocystins, provided the appropriate carbon, and the correct dose is applied. Granular activ ated carbon filters s how a lim ited lifetim e for the adso rption of m o st

m i crocontam inants, including cyanotoxins. Th e biodegradation of cyanotoxins across GAC f ilters shows great potenti al a s a tre a tm ent process.

Laboratory methods fo r the Study of Harmful Cyanobacteria Water blooms

In order to effectively study CyanoHABs, it is necessary to consider several distinct areas including:

• Ecology and Physiology of Cyanobacteria W a ter bloom s

• Sa m p ling, Identification (Taxonomy) and Quantification of Cells

• Culture and Handling of Toxic Cells

• Preparation and Processing of Cells, Ti ssues and Other Matrices for Toxin Analyses

• Cyanotoxin Detection an d Analyses Methods

- Biological – Bioassay

- Biochem i cal - i.e. Immunologi cal (ELISA); Enzym e (PPIA)

- Analytical - HPLC, MS, NMR

- Genetic – PCR and others

Laboratory studies focus on the latter four ar eas and our laboratory work has em phasized the last topic area-detection and analyses of cyanotoxins.

Over the past 30 years thes e studies have involved:

- Biological - Bioassay

* Sm all anim al - m ouse , invertebrate LD and LC50

* Microbial

- Biochem i cal - i.e. Immunological (ELI SA); Enzym e (PPIA); Cell Receptor

- Analytical - HPLC, MS, NMR

78- Genetic – PCR

* 16S rRNA

* nifH (gene for dinitrogen reductase-N2 fixation)

* cpcBA, IGS (intergenic spacer regi on between two phycobilisom e subunits),

* MCY ( m icrocystin gene)

* Other toxin genes (CYN, Antx-a) are being studied

Recent Methods for Anal yses of Microcystins

A current m e thod used in our laboratory for ex traction and analyses of Microcystins is given in Fig. 1.

Fig. 1. Flow diagram for extraction and analyses of Microcystins.



### S a m p l e ( C e l l s , o r o t h e r s )



### S upe r n a t a n t E x t r a c tio n w ith 5 % H O A c a q

. or M e O H

P a ss t h ro u g h O D S (o r H L B )

c o lu m n ( c a r tr id g e )



### ODS ( o r HL B ) c o l u m n

W a s h w ith w a te r E l u t e w i t h M e O H



### T oxi n f r a c t i o n


### L C /M S a n a l y s is 

### S ELD I

- T O F M S D i r e ct a n al y s i s



### P u r e t o x i n H P L C s p a r a tio n 

### S a m p l e ( C e l l s , o r o t h e r s )



### S upe r n a t a n t E x t r a c tio n w ith 5 % H O A c a q

. or M e O H

P a ss t h ro u g h O D S (o r H L B )

c o lu m n ( c a r tr id g e )



### ODS ( o r HL B ) c o l u m n

W a s h w ith w a te r E l u t e w i t h M e O H



### T oxi n f r a c t i o n


### L C /M S a n a l y s is 

### S ELD I

- T O F M S D i r e ct a n al y s i s



### P u r e t o x i n H P L C s p a r a tio n

79LC/MS and MS/MS are done using an LCQ quadropole ion-trap m a ss spectrom eter (Therm oquest, USA). The m a ss spectra are obtaine d using direct infusion or flow injection with MeOH/H 2 O (1:1) mobile phase. The positive or negative ESI m ode is used. For

m i crocystins collision energy of 35-40% for MS/MS is used. Figure 2 shows the typical LC/MS conditions for m i crocystins.

Fig. 2. LC/MS Conditions for Analyses of Microcystins



### LC c o ndi ti o n s M o bile phas e: ACN/ 0

. 02 %HF B A H 2 

### O/0.02%HF BA
or CAN/0.05%TF A H 2 

### O/0.05%T F A
Col u mn: C18 (3 μ , 050x020, Meta Chem )

or S Y M M E RY C8 (3.5 μ , 100x021, W a te rs)
G r adient conditions : 0-1 mi n: 20% org a nic
2-7 mi n: 60% org a nic
7.1-15 mi n: 20% org a ni c Fl o w r a t e : 0.2 ml/min.

M S c o ndi ti ons P o s i tiv e or ne g a ti v e ESI
Sourc e v o l t a g e: 5.0kV Capillar y temperature: 2 5 0oC

80Using these conditions m i crocystin-LR can be de tected on column, in the positive mode, at less than 10 pg (Fig. 3).

Fig. 3. Chrom a togra m of 15.6 pg m i crocystin-L R on column (3 μ C18).

RT

: 0. 00 - 15. 00 SM : 7G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Ti m

e ( m

i n ) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100RelativeAbundance R T : 8. 24 M A : 6585 9 M H : 10271 B P : 995.43 NL : 1

. 0

3 E4
Ba

s e Pe

a k m/

z = 994. 5- 995. 5 F : +

c E

S I F u ll m

s 2 995. 50@ 20. 00 [ 995. 00- 996

. 0 0 ] M

S LR

cu

r v e

_ 8

81We have developed a rapid and efficient m e thod to analyze mi crocystins/nodularins using surface-enhanced laser desorption i onization-tim e-of flight m a ss spectrom e try (SELDI-TOFMS) with Ciphergen protein chip reader system. SELDI-TOFMS is a novel approach that was introduced by Hutchens (Hutchens 1993). This technique retains the target com p ounds on a solid-ph a se chrom a togr aphic surface (chip), s ubsequently ionized by laser and detected by TOFMS. The chip sys t e mwith SELDI-TOF MS allows f o r peptide and protein analysis with high sens itive f r om a variety of com p lex biological

m a terials such as cell, serum

, blood, plasm a , and tissue, et al., with lim ited sam p le preparation (Jssaq 2002 ).

SELDI-TOF MS analysis was carried out us ing the Ciphergen protein chip system (Ciphergen, Palo Alto, C A ). Ciphergen Protei n Chip Software 3.0 was used. The analysis procedure was adapted from the Ciphergen user s guide with the follo wing m odifications.

Two μ L of sam p le dilute d in distilled water we re applied to the spot on the chip and allowed to air dry. This wa s followed by washing with 3 μ L of distilled water and allowed to dry. To the dry spot, 0.7 μ L of Energ y Absorbing Molecule (EAM) was added. The s a m p le chip was transferred to the ch ip read er an d analyzed f o llowing desorption of the bound target by short intense probes from a nN2 320 nm

- UV laser. The

m a ss spectra were determ ined by tim e- of -f

light in a m a ss spectrom e ter.

An exa m ple of the results for SELDI

- TOF m a ss spectra of m i crocystins-L R, -YR, -RR and Nodularin are shown in Figure 4.

Figure 4. SELDI-TOF mass spectr a of m i crocystins-LR, -YR, -RR and Nodularin. Chip: H4. Sa m p le concentratio n : 1

μ g/mL each. Sa m p le volum e: 2 μ L. Laser intens ity : 19 0. EAM

c o n c e n t r a t i o n : 3 0 %
satura ted CHCA solution

82SELDI-TOF MS has also been used in our laboratory to analyze m i crocystin bound to the protein phosphatase enzym e co m p lex. This is useful in allowing the tim e course of

m i crocystin binding to be observed in vitro.

These and other m e thods being developed in our laboratory and othe rs around the world will prov ide new techniq u es f o r obse rving not on ly m i crocystins but a l so other cyanotoxin s in environmental m a trices. Resu lts o b tained fro mthese m e thods will help docum ent the growing incidence of cyanotoxins in water based dis ease and provide the basis for water m a nage ment techniques desi gned to im prove water quality in order to

m i ni m i ze the hum an and anim al health im pacts from CyanoHABs.

A c know ledgements

Past and present work on CyanoHABs has i nvolved m a ny students, staff and colleagues. The following agencies and persons supported the work summ arized in this paper:

• Florida (Funding sources)-FMR I and SJ W M D through FMRI

• W r ight State Univ.-Charles Friday, Jerom e Servaites, Moucun Yuan, John Blakelock, Jennifer Ott, Laurel Carm ichael

• UNC-IMS-Hans Paerl and crew

• Canada-E.A.D. Allen

• Univ. Illinois-Ken Rine h a rt-NIH Sub c ontra ct

• Australia-N RCET-Glen Shaw, Geoff Eaglesham

References

Burch, M.D., Chow, C.W.K., Hobson, P. 2001. Algicides for control of toxic cyanobacteria. Proceed ings of the 2001 W a ter Quality Technology Conference, Am erican Water W o rks Associatio n, Nashville Tennessee.

Burch, M.D., Velzeboer, R.M.A ., Chow, C.W.K., Stevens, H.C., Bee, C.M., House, J. 1998. Evalu a tion of cop p er alg aecid e s for the co ntrol of alg a e and cyano b acteria

. Urban Water Research Association of Australia, Research R e port, No. 130 , April 1998. UWRAA, Melbourne.

Carm ichael, W.W. 1997. The Cyanotoxins. in Advances in B o tanical Research (ed Callow, J.) 27, 211-256 Academ ic Press, London.

Carm ichael, W.W. 2001. Health Effects of Toxin Producing Cyanobacteria: "The CyanoHABS". Hum a n and Ecological Risk Assessm e n t

. 7 (5): 1393-1407.

83Carm ichael, W.W., Azevedo, M.F.O., An, J.S., Molica, R.J.R., Jochim sen, E.M., Lau, S., Rinehart, K.L., Shaw, G.R., Eagelsham, G.K. 2001. Hum a n Fatalities from Cyanobacteria: Chem ical and Biological Evidence for Cyanotoxins. Environm ental Health P e rspectives. 109 (7):663-668.

Carpenter, E.J., Carm ichael, W

. W. 1995. Ta xonomy of Cyanobacteria. in Hallegraeff, G.M. et al. (eds.) Manual on Harmful Marine Microalgae p. 373-80. IOC Manuals and Guides No. 33 UNESCO.

Casitas Municipal W a ter District .1987. Current Methodology for the C ontrol of Algae in Surface Reservoirs. American W a te r W o rks Ass o ciation, Denver, CO.

Chorus, I., Bartram

, J. (eds.) 1999. Toxic Cy anobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Managem e nt. World health Organization, E&FN Spon, Routledge, London.

Hallegraeff, G.M., Anderson, D.M., Ce m b ella , A.D. (eds.) 1995. Manual on Harmful Marine Microalgae 551 pp. IOC Manua ls and G u ides No. 33 UNESCO.

Hutchens, T. W

. ; and Yip, T. T. 1993. New desorption strategies for the m a ss spectrom e tric analysis of m acrom ole c ules. Rapid. Commun. Mass Spectrom . 7: 576-580.

Jssaq, H. J.; Veenstra, T. D.; Conrads, T. P.; and Felschow, D. 2002. The SELDI-TOF MS Approach to proteom c s: protein profiling and biom arker identification.

Biochem

. Bi ophys. Res. Commun. 292: 587-592.

Jochim sen, E.M., Carm i c hael, W

. W

. , An, J.S., Cardo, D.M., Cookson, S.T., Hol m es, C.E.M., Antines, M.b. de C., Filho, D.A. de Meb, Lyra, T.M., Burreto, V.T.S., Azevedo, S.M.F.O. and Jarvis, W

. R. 1998. Liver Failure and Death Following Exposure to Microcystin Toxins at a He m odialysis Center in Brazil. NewEng. J. Medicine. March 26, 1998, V. 338(13): 873-878.

Moore, G.T., Kellerm an, K.F. 1905. Copper as anAlgaecide and Disinfectant in W a ter Supplies. Bull. Bureau P l ant. Indus. U.S.D.A. 76: 19-55.

Ressom R., Soong, F.S., Fitzgerald, J., Turcz ynowicz, L., Saadi, O.E., Roder, D., Maynard, T., Falconer, I. (eds.) 1994. H ealth Effects of Toxic Cyanobacteria (blue-g r een algae) Natio nal Health and Medical Research Council, Australian Govt. Pub. Service, Canberra.

Sawyer, C.N. 1962. Ca uses, Effects and Control of Aquatic Growths. J. WPCF. 34: 279- 288.

84Sivonen,K. 1990. Toxic Cyanobacteria in Finnish Fr esh W a ters and the B a ltic Sea. Ph.D. Academ ic Dissertation in Microbiology, University of Helsinki, Finland.

Schwi m m e r, D., Schwi m m e r, M. 1964. Algae and Medicine. in Algae and Man. D.F. Jackson (ed.) Plenum Pr ess, New York, pp. 368-412.

Schwi m m e r, D., Schwi m m e r, M. 1967. Medica l Aspects of Phycology. in Algae, Man and the Environm ent. D.F. Jackson (ed.) Syracuse University Press, pp. 279-358.

Yoo, R.S., Carm ichael W.W., Hoehn, R.C., Hrudey, S.E. (eds.) 1995. Cyanobacterial (Blue-green Algal) Toxins: A Resource Guide 229 pp. AWWA Research Foundation and Am erican W a ter Works Association, Denver.

85CYA NOB A CTER IA T O X I NS: Florida Department of Health, Bureau of Laboratories Activiti es Jam es E. Evans, Ph.D. Marek Pawlowicz , Ph.D.

History of cyanobacteria toxin testing at the Departm e nt of Hea l th, Bureau of Laborator i es

Bureau of Laboratories involvem e nt in cy anobacteria testing began soon after the creation of the Florida Harmful Algal Bloom Tas k Force (HABTF) in 1998. In response to a HABTF Technical Advisory Group report identifying cyanobacteria as a research priority, the St. John’s R i ver W a ter Mana gem e nt District (SJ R W M D) initiated a collaborative study to characteri ze and identify algal toxins in Florida’s surface waters. Participan ts in this studyincluded th e SJ RW MD, W r ight State Un iversity , the Flor ida Departm e nt of Health, the Florida Marine Re s earch Institu te, and the Flo r ida Fish and W ildlife Conservation C o mm ission. The su rvey of Florida’s surface waters was conducted in 1999. This survey was followed up the following year with a m onitoring of surface waters iden tified in th e 1999 survey, selected water treatm e nt plants, and blo o m events.

The Florida Departm e nt of Health, Bureau of Laboratories Envir onm ental Microb iolog y Unit – Jacksonville, perf orm e d enzym e linked immunosorbent assays (ELISA) for

m i crocystin and m ouse bioassays for general toxi city in supp ort of the in itia l survey s as well as the follow-up studies. The findings of these studies are undoubtedly docum e n ted elsewhere in this series o f m onographs and will, therefore, no t be rep eated . However, the

cum u lative findings led the Departm e nt of Heal th to a decision that cy an obacteria to xins were an issue requiring f u rther investigati on from a public health perspective. To adequately support future public health inves tigations, it was determ ined that laboratory capabilities needed to be e xpanded to allow for more com p rehensive and quantitative analyses. T o this end, f unding for the enha ncem ent of laboratory capacity was sought and obtained via supplem ental funding from a CDC/Florida Cooperative Agreem ent (Pfiesteria-Related Illness Surveillan ce and Prevention Gr ant #U50/CCU415409-04).

Selection and im plementation of new analytical m e thodology

In the selection of analytical instrum e ntation to enhance and expand current laboratory capabilities, the Bureau of Laboratories had a number of needs in m i nd. The first was to expand capabilities to include other toxins of interest beyond the m i crocystins. There was also a desire for m o re sensitive, specific, and reproducible techniques with

m e thodologies that could identify and quantitat ive m u ltiple an alytes/analo gs in a sing le analytical run. Finally, with considerations for the future, it was desired that any newly acquired cap abilities con f or m to accepted pr actices and be ad aptable to p o tential monitoring and regulatory program s.

ELISA has distinct advantages in that proced ur es are relatively stra ightforward and that assays are available in comm er cial kit form . In addition, ELISA m e thods can prove to be
very sens itiv e, however, such claim s are ofte n not dem onstrated in routine use. ELISA

86m e thods are also often plagued by analog cros s reactivity. Such non- specificity m a y or

m a y not be well characterized, complicating th e interpretation of results. Reproducibility is also a pro b lem with ELISA assays as th ey are highly dependent on technique and the extent of sample preparati on required. W h ile ELISA has proven itself as a valuable screening assay, the la boratory sought to m ove toward a m o re quantitative analytical approach.

The m ouse bioassay has also proven to be a va luable screening tool for general toxicity of water containing cyanobacteria toxins. However, the assa y is inherently non-specific and m a y be com p licated by other unknown contam inants in a given water sam p le. It is also a non-quantitative assay, having proven to be difficult to correlate with the am ount or type of toxin present. There are also concerns with cost, ethics, and relevance to hum a n toxicity that lim it the m ouse bioassay’s utility.

Given the lim itation of the past screening assays used and in consider ation of the outlined needs, the laboratory cho s e an analytical instrum e nt system that provided a

com p rehensive solution. The system is com p rised of high perform a nce liquid chrom a tography with m u ltip le detection com pone nts. Both ph oto diode array and f l uorescenc e detec t ors a s well as a m a ss spec tro m eter will b e available to of f e r a wide range of capabilities for the detection of the various cya nobacteria toxins. Such a multiple d e tector s e t-up was designe d to allow f o r the an alys is of toxins b y the cur r ently accepted an alytical m e thodology as well as for the develop m ent of techniques to determ ine multip le tox i n s and the i r an alogs with in the sam e analytical run

. This syste m provides specific, sensitive, quantitative, and reproducible analytical capability that was lacking withthe screenin g assays. T h ere are li m i tations in that standard control m a terial must be available to properly quantitate a given toxin, however, such standards are becom i ng more readily availab l e as interes t and research in the field advances. An additional lim itation that bears m e ntion, as with any quantitative technique for the cyanobacterial tox i ns, is that the relations hip betw een concentration and hum a n health effects is unknown.

Future plans

While the immediate plans for the Department of Health, Bureau of Laboratories focuses around im plem enting the HPLC ana l ysis system previously described, other ongoing labora t ory a c tiv ities will allow f o r th e inve stigation of other assay and analytical techniques. Alternate anal ytical techniques such as gas chrom a tography - m a ss spectrom e try (GC-MS), and liquid chrom a tography – tandem m a ss spectrom e try (LCMS/MS) are available for exam ination of app licability to toxin analysis. In addition, bioassay an d m o lecular analyses currently in us e in othe r pub lic he alth initia tives with in the labo rato r y allow the possibility o f applying these techniques as well.

In addition to the investigation of other a ssay and analytical m e thodologies, the addition of

laborato r y capabilitie s allows f o r the assu m p tion of projects to consider the various public health im plications of cyanobacteria toxin exposure. On e such study supported by the CDC/Florida DOH Cooperative Agreem ent (P fiesteria-Related Illness Surveillance and Prevention Grant #U50/CCU415409-04) is a study designed to determine the

87effectivenes s of hom e filtration dev i ces in th e removal of cyanobacteria toxins from potable wa te r. This stud y will se ek to identi f y f ilter m a trice s that ar e ef f ective in the

rem oval of different cyanobacteria toxins. It is expected that the ability of a given filter type to effectively rem ove toxin will be de pendent upon such variab les as the type of toxin, filter com position, filter d e sig n, filter capacity, filter size, and water quality to

nam e just a few. The results of this st udy could provide a basis for advising consum e r s how to prevent or m i nim i ze exposure to cyanob acteria toxins in the absence of standards or scien tif ic evidence of actual healthrisks.

Conclusions

In conclus i o n, the Depart m e nt of Health Labo ratory is expan d ing capac ity and capability to perf orm analyses f o r c y anobacte r ia toxins. The labora t ory is comm itted to working with public health partners to address i ssues surrounding cyanobacteria toxin exposure and possible links to hum an health effects.

The laboratory wishes to acknowledge th e CDC/Florida Cooperative Agreem ent (#U50/CCU415409-04), the Florida Harmful Al gal Bloom Task Force, and the Departm e nt of Health, Division of E nvir onm ental Health, Bureau of Environm ental Epidem iology for their support.

The authors also wish to acknowledge wo rk and dedication of the staff of the Environm ental Microbiology Laboratory, Environm ental Chem istry Laboratory, and the entire Florida Departm e nt of Health, Bureau of Laboratories in their continued efforts to protect public health by providing diagnos tic screening, m o nitoring, reference,

em ergency, and research laboratory services.

88Current EPA Work on Algal Toxins in Drinking Water James L. Sinclair Office of Ground Water and Drinking Water U.S. EP A Cincinnati, OH

The Safe Drinking W a ter Act Am endm ents (SDWA) of 1996 (PL 104-182) authorizes regulation of previously unr egulated drinking water cont am inants in pub lic water system s. This act requires that a list of contam inants (Contam inant Candidate L i st, or CCL) to be considered for regulation be published 18 m onths after enactm ent of the SDWA (1998) and every 5 years thereafter. Contam inants placed on the list are not currently regulated, are known or thought to occur in public water system s, and m a y require regulation. At least five contam inants are required to be c onsidered for regulation every 5 years. Contam inants m a y be regulated if they have an adverse health effect on people, are known to occur or th ere is a subs tantial likelihoo d that they will occur in public water system s, and regulation presents a meaningful opportunity for health risk reduction in people served by public water system s.

The Federal Register notice announcing the Contam inant Candidate L i st (63FR10274) includes a list of research topics where suffi cient inf o rm ation is requ ired to m a ke a regulatory determ ination on contam inants. These topics include health effects of
contam inants, control of contam inants by dri nkin g water treatm ent, occurrence in water, and analytical m e thods that are used to get occurrence information. Specific
contam inants are lis t ed as needing researchin one or m o re of these areas before a regulatory determ ination can be m a de. For al gae and their toxins, research is needed on health effects, treatm ent, o ccurrence and analytical m e thods.

The research that is needed to m a ke regul atory decisions m a y be done through research organiz a tion s that includ e rese arch f a cilities of th e EPA. Addition a lly, in the cas e of
contam inant occurrence, occurrence inform ati on m a y be gotten from surveys carried out by these research organizations or it m a y also be gotten from a public water supply monitoring regulation, the Unregulated Contam i n ant Monito ring Rule (64FR50555, Scharfenaker, 2001). The UCMR requires that certain public water supplies m onitor for selected contam inants for one year or less, depending on the monitoring option used. The
contam inants to be m onitored under the UC MR a r e picked from the CCL. The UCMR monitoring list is revised every 5 years on a schedule where it is published one year later than the CC L. The first UCMR monitori ng list w a s published in 1999, and the second UCMR list will be due in 2004.

Research on algae and th eir toxins in the requ ired areas has been planned or initiated at several EPA research laboratories. H ealth effects projects being done at E P A’s National Health and Environm ental Effects Research Laboratory (NHEERL) in Research Triangle Park, NC, includes a project with W r ight State University to study biom arkers of exposure to m i crocystins. The sam e group also recently completed a project analyze

m i crocystins in sam p les from Brazilian patient s w ho received dialysis water containing

m i crocystins. A different laboratory of NH EERL is looking at the effects of anatoxin-a

89on the behavioral responses of rats. This work may be expanded to the affect of anatoxin-a on m ouse behavior, and future work may be done on behavioral responses of laboratory anim als to saxitoxins. Th e EPA NHEERL laboratory in Gulf Breeze, FL, is conducting a project with the University of Miam i to look at the affect of long term exposure of fish to m i crocystin and cylindr ospermopsin in two Florida lakes in which each of these cyanobacteria is a do m i nant species.

EPA projects on the affect of water treatm e nt on algal cells or toxins has been started or proposed in three areas. The EPA’s Nati onal Risk Management Research Laboratory (NRMRL) in Cincinnati is beginn in g a proje c t o nf iltra tion o f algal ce lls. This project will look at rem oval of c e lls during filter rip e ning , at the b e gi nning of the filter cycle, and during break t hrough, at the end of the filter cy cle. The filter will be a du al m e dia sand and anthracite filter. Th ree species of cyanobacteria will be used, including Microcystis which is unicellu lar, and Anaebena and Planktothrix, which are filam e ntous. The experim e nta l var i able s will b e the run tim e, and the loading rate. NRMRL in Cincinna ti also has a treatm e nt project with Univer sity of Wisconsin, the W i sconsin State Laborato ry of Hygiene, and Lake Superior S t at e University. This pr oject will look at the affect of chlorine on m i crocystin LR and an atoxin-a. and the infl uence of pH on toxin fate during exposure to chlorine. The experi m e nts m a y include the affect of chlorine on intracellular and extracellu lar toxin s . An investig ator of the O ffice of Ground W a ter and Drinking Water in W a shington, DC, has proposed studying the fa te of algal toxins during bank filtration. The prim ary m eans of reducing the toxins wo uld be biodegradation of toxins by m i croorganisms in riverbank sedim e nt s. This project has yet not been funded.

EPA has several analytical m e thod developm en t projects planned or under way, however, before m e thod developm ent can begin, analytic al chem istrystandard s are needed. T h e EPA’s Office of Ground Water and Dri nking W a ter (OGW DW ) Technical Support Center (TSC ) in Cincinnati is funding work at Wright State U n iversity to obtain cylindrospermopsin and anatoxin-a standards. T h e TSC has still not located a source of

m i crocystin LA. The Na tional Exposure Resear ch Laboratory (NERL) in Cincinnati is doing a project to develop a single micr o LC/MS m e thod for s e veral m i crocystin congeners, anatoxin-a and is planning to work on cylindrosperm opsin if a standard can be gotten. Separation will be done with m i crobore HPLC and e l ectrospray tim e of flight

m a ss spectrom e try will be used for detecti on an d identification. OGWDW, TSC, in Cincinnati, is starting a proj ect to develop an LC/MS m e thod that will be standardized and validated so that it can be run by commerc ial labs th at an alyze s a m p l e s for the E P A’s Unregulated Contam ina n t Monitoring survey fo r algal toxins. A c oncentration and cleanup step will be needed b e f o re analys is. The goa l will b e to m a ke a single LC/MS

m e thod for 5 or 6 m i crocystins, anatoxin-a and cylindrospermopsin. OGWDW

, TSC, in Cincinnati also is funding th e initial steps toward developm ent of immunoassay kits for cylindrospermopsin and anatoxin-a through W r i ght State University. Conjugates and haptens will be developed and given to a private com p any for developm ent of the immunoassay kits. The National Center fo r Environm ental Research (NCER) in Washington, DC, also offered a sm all busin ess grant under their SB IR program for the developm ent of these kits in 2002. NERL inCi ncinnati is w o rking with a com p any, Optec, to make an autom a ted instrum e nt for detection of algal toxins which uses

90aptam e rs. Aptam e rs are oligonucleotides that bind to specific m o lecules including algal toxins. They can be regenerated and reused. NERL in Cincinnati is planning to develop a taqm an PCR m e thod for identification of 6 diffe rent genera of toxic cyanobacteria. This m e thod would give results in 2 to 3 hours and would be quantitative. OGWDW, TSC in Cincinnati is dev e loping real-tim e P CR for a m i crocys tin gene. Th is m e thod will be used in a project to look at m i croc ystin production under differing environm ental conditions. OGWDW, TSC in Ci ncinnati is starting a projec t with Lake Superior State University. has been working on a standardi zed m i croscopic cell c ount m e thod that could be used for all of the tox i c genera of con cern. Th is m e thod will b e used w ith th e UCMR
survey.

EPA would like to conduct a risk assessm ent fo r algal toxins, if res ources are av ailab l e. The National Center for Environm ental Assess ment (NCEA) in Cincinnati would like to look at quantitative structure- activity relations for cylindros perm opsin and m i crocystins. NCEA is also interested in doing a risk assessm ent for the m i crocystins, cylindrospermopsin and anatoxin-a for the United States.

EPA has done or is doing 3 sm all occurrence surv eys for algal toxins in the last 2 years. NERL in Ci ncinnati has worked with USGS to survey 2 lakes and a river during the summer of 2001 for m i crocystin. The sam p les were archived and haven’t been analyzed yet. OGW D W, TSC in Cincinna ti with Lake S uperior S t ate University did a survey in the summ er of 2001 for m i crocystin. Cell c ounts were also done. The samples were archiv ed an d will be an alyzed this s u mm er. This summer OGWDW , TSC is conducting a survey with Lake Superior State U n ivers ity for cylindrosperm opsin. Source water and
trea ted wate r are b e ing s u rveyed a t6 utilitie s. These were picked based on their past history of having cylindrosperm opsin in the water. The sam p l e s from this survey are being archiv ed until there is an an aly tical capab ility for cylin drosperm opsin.

EPA has a separate regulation to get occurrence infor m ation on CCL contam inants in drinking water system s. This is the Unregul ated Contam ina n t Monitoring Rule (UCMR). Algal toxins weren’t m onitored under the first UCMR which goes from 1999 to 2004 because there were no validated m e thods that c ould be used for the survey. It is lik ely that they will be m onitored under the next UCMR m onitoring list, or UCMR2, which is due to be prom ulgated in 2004. Since it takes at least a year to write regulations that are published in the Federal Register, to m eet the 2004 deadline fo r publishing UCMR2, it will be necessary to h a v e valid ated m e thods for the UCMR by the end o f 2003 or early 2004. Monitoring will probably be done for the toxins that have standards available. This includes 5 or 6 m i crocystins, cylindr osperm opsin, and anatoxin-a. Based on the status of analytical m e thods, it is likely th at there will be an L C /MS m e thod and prob ably

a m i croscopic cell count m e thod in tim e fo r UCMR2 in 2004, but screening m e thods for the toxins, and genetic m e thods for toxin gene identification won’t be ready in tim e

. Based on the cost of the analyses, the UMCR survey for algal toxins will probably be

sm all, including 300 or fewer system s

.

91Therefore, these projects show that EPA is im pl em enting research to get the inform ation it needs to m a ke a regulatory determ ination fo r algal toxins although it still doesn’t have all the needed inform ation.

The com p ilation of the s econd CCL will be very im portant for future stud ies of algal toxins, because algal toxins c ould be kept on the CCL or th ey could be dropped off of it which would affect EPA research and regulatory activities. The second CCL is due to be published in February 2003, and it will likelybe very sim ilar to CCL1. Future CCLs could be different, though. For future CCLs, EPA m u st consider an approach

recomm ended by the National Research Counc il for picking CCL contam inants. The strategy that the NRC recommended was to start with a universe of about 80,000
contam inants and pare the list down to a pr e-CCL by using a neural network program in conjunction with expert judgm e nt. The N RC also recomm ended screening potential

m i crobial co ntam inants for the pre-C C L by chara c ter i zing the mwith quantitativ e structure activity relatio ns for chem i cals, or v i ru lence factor activ ity relations or VFARs for m i croorganism s. The VFAR appr oach is to de tect v i ru lenc e f actors tha t

m i croorganism s have. These virulence factors could be detected e ither by genotypic or phenotypic expressions. This appro ach could af fect the sele ction of algal toxins for future CCLs since the selection criteria could be different th an those used to pick CCL1
contam inants. For CCL2, though, it is likely, th ough not assured, that algal toxins will

rem a in on the CCL, and will con tinu e d to be con s idered f o r a regula t ory d e term ination

.

Reference

Scharfenaker, M.A. 2001. Making sense of th e Unregulated Contam ina n t Monitoring Rule. JAWWA 93(6):24-42

92Summary of Florida D e partment of Environmental Pro t ection’s Activities R e lated to Cyanobacterial Toxins

Presented b y : Richard Drew , M.S. Chief, Bureau of Water Faci lities Regulation, Division of Water Reso urce Management

The Departm e nt of Environm ental Protecti on [DEP] has se veral program s that either influence th e presen ce o r reoccurren ce of cyanob acteria and their as sociated toxins o r are im pacted by the pres enc e of the cyanobacte r ia. I will lim it this discu ssio nto two program s that influence the nutrient balance of F l orid a surface wa ters that in turn crea te a suitable environm ent for that group of organism s; and one program

, drinking water, that is directly affected by its presence.

The NPDES or Nationa l Pollutan tDischarge E l im ination program regulates the treatm e nt and disposal of industrial and dom estic wastew ater. Specific effluent lim its control the

am ounts of nutrients that can be d i scharged to surface waters, that in turn could prom ote eutrophication and the presence of cyanob acte r ia . The nutrie n t lim its con t ained in facility perm its are bas e d on com p l i ance with water quality standard s, and the stand a rds are related to the designated use or cl assification of the wa ter body receiving the discharge.

Direct was t ewater facility discharg es to shel lf ish areas [ C lass II] and to water bod ies that serve as a source of drinking wa ter [ C lass I] are prohibited in Florida. E v en indirect discharges must dem o nstrate th at w h at will be discharged will not affect the designated use of the downstream waters. W h ile there are prohibitions for direct discharges to Class I and II wa te rs, rec r ea tio nal wate rs [Class III] can rece ive dis c harges fro mwastewater f acilities. H o wever, thes e disc harges, like indirect discharges to Class I an d II wate rs, are only allowed if it is d e monstra t ed bythe f acil ity perm it applicant th at th e y will not af f ect the designated use of the water body. In re lation to cyanobacteria, the perm it applicant must dem o nstrate th at th e nutrients added to the receiv i ng water will not c ause alg a erelated pigm ents to exceed levels found in healthy wate rs. T h erefore, the NPDES
program has the ability to lim it nutrient addition s f o r any wastewate r f

aci lity in the state.

The rem a ining nutrient s ources reach ing surface waters and p r om oting algae bloom s are derived from non-point sources, such as storm water runoff from construction areas, urban storm water, poorly operated residentia l wastewater treatm ent system s [septic tank system s], irrigation from agriculture/rightof ways/golf courses, and atm o spheric deposition. The TMDL or Total Maxim u m Daily Load program exa m ines all water bodies in the state and for those determ ined to be im paired a process is set in m o tion. First the app r opria te leve ls of pollutants are defined that will protec t the designated use of the water body; then the sour ces of th ose pollu tan t s are identified; and finally, an im ple m entation plan is developed to reduce the lo ading to the appropriate level. It is in this program that the agency hopes to cont rol all the nutrient s ources supporting the occurrence of algae blo o m s . Here the concep t of watershed m a nage m e nt is applied to reduce nutrient loadings from all the sources in th e basin.

93A DEP program that can be directly affected by the presen ce of cy anobacteria and their toxins is the drinking water program . Fort unately the poten tia l f o r this to b ecom e a problem in Florida is greatly reduced by a va st system of aquifers that provide ground water to a majority of Flor ida’s population. Only 20 of about 6,400 public water system s (PW S s) use surface water. These 20 PW Ss supply roughly 10 % of Florida’s drink i ng water. Geographically, only one of the 20 system s is located in the northern part of the state. The rest are south of Orlando. Mo st are located within a 50-m ile wide band running fro mW e st Palm Beach on the eastern co ast to Charlotte Harbo r and Ft Myers to the west. M o st of these surface waters ar e slow moving and affected by water con t ro l structures. Fluctuating levels of organic color, iron, and acid ity affect the quality [taste and odor] of the water and dictate the leve l of treatm ent used at these sites.

Drinking water system s [WTPs ] are required to meet a num ber of federal regulations that have been adopted by Florida. The regulati ons are even m o re com p lex for system s that draw their s ource water from surface waters. Th e treatm ent processes us ed at surface WTPs is based on the quality [including taste, clarity, and odor] of the water supply. As a result th es e system s are typically v e ry respon sive to changes in the sour ce water character and take steps quickly to minim i ze citizen com p laints. In regard to public notification, the Department requires annually the producti on and public distribution of Consum er Confidence Reports that s u mm arize th e quality of the water p r o v ided to the public. Recently, the E P A and in turn the DEP have adopted m o re stringent rules focused specifically on system s that use surf ace waters as th eir sour ce w a ter. Surface Water Treatm ent, Filter Backwash Recycli ng, an d Stage 1 Disinfectan ts/Disinfection Byproducts Rules are exam ples of these regulations.

While there are no regulations th at d i rectly add r ess the treatm ent of cyano b acteria and its toxins or establish sp ecif i c wate r qua lity s t andard s f o r these toxins, the w a ter tre a tm ent plant m u st satisfy colo r and odor req u irem ents of DEP and EPA. To this end, the facility must adopt a set of m a na gem e nt strategies to handle the changing water quality of the surface water body serv ing as its sour ce. Many o f these m a na gem e nt strategies hav e also been shown to be effective at rem ovi ng or controlling cyanobacteria and its t toxins.

Managem e nt strategies are site specific and can b e catego r ized as contro lling the im pacts to the source water, pretreatm e nt practic es, and in-plant and post-plant treatm e nt techniques.

Watershed m a nage m e nt was previously discu ssed and attack s the s ource of the problem – nutrients. H o wever, depending on the size of the watershed feeding the so urce water, the im ple m entation of watershed m a nagem e nt tec hniques m a y be: im practical [technically or financially] in controlling nut rient loads to the basin; or, im practical in achieving long
term results; or, m a y not be as effective as originally hoped evenover the long term

. This leaves the W T P with the entire respons ibility of handling th e algae bloom problem

.

Pretreatm e nt practices include physical, chem ical or biologic al treatm ent of the source
water in s itu

, or prio r to enter i ng the f acility’s tre a tm ent process train. So m e in-lake

94m a nage m e nt techniques, such as herbicide applications, are mere ly tem porary fixes, attacking the problem of algae bloom s as theyhappen. Use of a herbicide or algalcides [e.g., copper sulfate] can be m o st effective whenused to m a intain low numbers of algae, instead of application on a bloom when it can re lease tox i ns from the dead cells. It also appears to be less effective with c ontinued use in the sam e water body.

Other types of lake m a nagem e nt practices ei ther try to rem o ve the nutrien ts f r om the water [chemical coagulants] or create an unsuitable environm ent for the developm ent of a bloom , [aeration, pH adjustm e nt]. Anothe r technique is the use of off-line wetland
trea tm ent system s that f ilte r [ b iologically o r phy sica lly] nutr i ents out of the water and into wetland vegetation and sedim e nts.

A drinking water treatment facil ity can also develop alternat ive sources of water to be used when bloom s appear in their prim ary drinking water supply, i.e., above ground

reservo i rs, aquifer storag e and recov e ry sy stem s, backup wells, use of adjacent water utility water.

Treatm e nt at the plan t, p a rticularly those facilities drawing s u rface waters, can invo lv e

severa l f iltra tion trea tm ent techn i que s that will ef f ectively re move the algal ce lls, including in-bank filtration, coagulation and filtration, fl occulation and filtration, and other form s of m e m b rane filtr ation, s u ch as rev e rse osm o sis and nanofiltration. Som e WTP practices applied d u ring bloom s, such as th e isolation o f sludge and filter b ackwash water to waste can prevent the reintroduction of cells and toxins into the treatm e nt process train. Disinfectants comm only used for pathogen removal, like chlorine and ozone can be partially effective at removal of the certain toxins. Powdered and Granular Activated Carbon [PAC & GAC] can be effec tive m easures at rem oving som e of the toxins, particularly PACs. Com b ining PAC or GAC with one of the dis i nfectants can im prove the rem oval effi ciency. Som e oxidant s, like potassium perm anganate, have also been shown to be especially effective at rem oving the toxins.

Surface waters in Flo r id a commonly used as sour ce waters are, unfortunately, often high in color and other organics, and have odors characteristic of stagnant wetlands. Fortunately, the techniques used to produ ce a good tasting, odor-f ree, clear drinking water, and com p liant with DEP/EPA dr inking water quality standards and new regulations, require em ploym e nt of a series of treatm ent techniques that also seem to be very effectiv e at rem oving cyanobact eria and theirs associated toxins.

The evaluation of treatm e nt te chniqu es to effectively rem ove cyanobacteria and their toxins is in the early stages of discovery, as evidenced in recent publications [Chorus and Bartram [ Ed.], 1999; Carm ichael, 2001; Chor us[Ed.], 2001]. W a ys to m e asure the presence of toxins, especially at the low c oncentrations observed inthe environm ent are still being developed. S t andardization of analytical m e thodologies and sam p ling protocols must be done to allo w the WTP to have tools for de tecting the toxins and have assurance th at the v a lues reported, for the increas i ng num ber of toxins, are accurate.

95Several system s in Florida ar e participating in current studies funded by the Am erican Water W o rks Association Research F oundation and the St. Johns River Water Managem e nt District to exam ine the occurrence, quantity, and treatabili ty of algal toxins at drinking water treatment syst em s. In addition, DEP is working with th e surface water WTPs in a workgroup form at to enhance the ex change of experiences each of the WTPs is having in dealing with this and other related problem s. EPA is also evaluating cyanobacteria-derived group of toxins to de term ine if they will establish a m a xim u m concentration level [MC L ] for one or m o re of the m o re common cyanobacters.

Where does this leave D E P, as regulators? On one hand, the inform ation to date for Florida W T Ps leads one to conclude only a sm all portion of the state’s drinking water system s are susceptible to potential introducti on of cyanobacteria and its toxins. A high level of treatm e nt is now being provided at th ese few system s caused by the nature of the water th ey n o w treat, an d this trea tment base d on the res earchto date appears to be effective at controlling the intr oduction of toxins into the di stribution system s. On the other hand, a fuller understanding of the problem and the eff ectiveness of treatm ent is still evolving. W e should m onitor this inform ation and di ssem i nate the results to the WTPs as soon as possible. W e should also continue to work with the TMDL program to
contro l the n u trient inpu ts to the watershe ds con t aining th e surface waters supplying th ese WTPs. Controlling and lowering the nutrien ts will reduce and hopefully elim inate the developm ent of these algae bloom s and the release of their toxins.

References:

Carm ichael, W.W., 2001. ASSESSM ENT OF BLUE-GREEN ALGAL TOXINS IN RAW AND FINISHED DRINKING W A TER. AWWARF Public. No. 90815. 179pgs.

Chorus, I., and J. Bartram, eds. 1999. Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, Monitoring and Management. London, UK: E&FN Spon. 416pgs.

Chorus, I., ed. 2001. Cyanotoxins: Occurrence, Causes, Consequences. Berlin : Springer. 357pgs.

96Aquatic Toxins Program Florida Department of Health Richard Clark, MS

On August 13-14, 2003, the Florida Departm e nt of Health hosted a m eeting in Sarasota, FL, entitled Health Effects of Exposure to Cyanobac teria Toxins: State of the Science

. Many cyano b acteria researchers, water treatm e nt plant personnel, and health care professionals gathered to exchange infor m a tion and further Florida’s knowledge on this new area of science. The Florida Departm e nt of Health es tab lished their Aquatic Toxins Program in 1997 to track illn esses from persons exposed to algal toxins. The program created the Aquatic Tox i n or Harm ful Alga l Blo o m Hotline (888-232-86 35) at that tim e and began tracking illnesses and exposures to harmful algae. This program also participa t es in m a ny research and edu cation a l ac tivities.

Aquatic Toxins Surveillance

The surveillance activities of the program began in response to pfies t er ia concerns in 1997. Pfies t eria pis c ic id a is an es tuar ine d i nof la gella te th at is believed to cause hea l th problem s in hum a ns, but the results of year s of research are c onflicting and it has not been dete rmine if pfiesteria actually produces a t oxin. Florida does have pfies t er ia in its estuarine water bod ies, b u t we have h a d no c onfirm e d illnesses related to exposure to this organism

. The Aquatic Toxins Program also works clos ely with the Food and W a terborne Illness Program to track illnesses of people who ha ve been exposed to an algal toxin through food sources. Florida has cases of Neurotoxic Shellfish Poisoning (NSP), ciguatera fish poisoning, p u ffer-fish poisoning a nd scom broid fish poisoning each year. Each of these illn esses is d u e to a tox i n that is of algal or ba cte r ial o r igin.

Persons can also becom e ill f r om dire ct contact with the a l gal toxins. Skin rash, nasa l and throat irritation, headache, and breathing pr oblem s can be associated with Red Tide ( Karenia brevis ) and cy anobacteria. The program receives hundreds of reports of illn esses fro mpersons exposed to red tide e ach y ear. So far, there h a ve b e en no confirm e d reports of illness relate d to cyanobacteria in Florida.

Research Activ i ties

The Aquatic Toxins Program is an active particip ant in m a ny research projects in F l orida. We collaborate on Red Tide studies with The National Center of Environm ental Health, Centers of Disease Control and Preventi on, University of Miam i Medical School, Lovelace Respiratory In s titu te, Flo r id a Departm e nt of Enviro nm ental Pro t ection, University of North Carolina W ilm ington, and Mote Marine Lab. The goal of these hum a n health studies is to es tab lish h o w red tide af f ects th e environm ent and people. The studies involve environm ental m onitoring, ai r m o nitoring, and hum a n health m onitoring including: nasal and throat swabs, urin e test, spirom etry and questionnaires.

97The Aquatic Toxins Program was also able to support the Depart m e nt of Health’s Laboratory in Jacksonville, FL, in building cap ac ity to tes twater f o r cy an obacte r ia an d their toxin s . The equipment is now in place and their first research p r oject is underway. This project is tes ting ho w ef f ective hom e f iltration devices ar e at rem oving cyanobacteria and their toxins.

The Aquatic Toxins Pro g ram will also be pa rticipating in a p u ffer-fish study with The Florida M a rine Research Institute. Nineteen people had become ill after eating pufferfish caught in the Titu sville, FL, area in 2002. The fish had bioaccum u lated saxitox i n in their tissue, which had not been previously id entif ied in the litera ture. Re search will determ ine the toxicity of the 16 different species of puffer-fish and how they are distr i buted in Florida. T h is project will a l so determ ine f r om what specie s of algae was the toxin’s origin and what other organism s m a y c ontain the toxin.

Education

One of the m a in goals of the Aquatic Toxi ns Program is education and it has created

m a terials for use by the county health depa rtm e nts, the public and the m e dia. A “Cyanobacteria Update” is released to the c ounty health departm e nts when there is any new infor m ation of interest. The program is very active in providing assistance to the county health departm e nts when they need to produce a press release related to pufferfish or red tide.

Prom otional item s have been created for the public to get the hotlin e number out. Cyanobacteria m a gnets and key chains have b een created alon g with red tide visors and key totes. Scientists and county health de partm e nt staff distribute these item s where people are recreating near wate r bodies. These item s have the hotline number printed on them and they encourage people to call in if they feel they have been exposed to

som e thing in the wate r that has m a de them ill.

The coordinator of the program has given m any educational presenta tions at national and local scientific m eetings, public m eetings, c ounty health departm e nts and wildlife parks.

Miscellaneous Aquatic Toxins Program Activities:

• Letters written fro m FDOH to EPA rega rding setting recrea tional and drinking water guidelines for cyanobacteria toxins.

• Investigations into reported cases.

• Creation of the physician workgroup

• FDOH is an activ e m e mber of the Florida Harm ful Bloom Task Force

• Cyanobacteria Literature Collection and review

98• Red Tide Literature Collection and review

• Participation in the production of profe ssional posters and presentations to be given at scientific m eetings

• Coordination of conference calls

• Response to m e dia calls, em ails , and questions from the public

• Response to reports of swi m m e r’s itch, cercarial derm atitis, naegleria (amoeba that c auses m e ningitis), jellyf ish stin gs, f i re cor a l, f i sh bites, e t c.

• Production of DOH education plans, business plans, m e dia contact reports, etc.

• Coordination of the State of the Scie nce Cyanobacteria m e eting: choosing speakers, setting the agenda, providing na m e tags, travel reimbursem e nt, travel coordination, notebooks, and contracts with Mote and supporting agencies.

Conclusion

The Florida Departm e nt of Health’s m i ssion is to prom ote and protect the health and safety of all people in F l orida throug h the de livery of quality public health services and the prom otion of health care standards. The Aquatic Toxins Program fol l ows this

m i ssion throughout its activ ities in education, re search and health surveillance. In Florida, harmful algal bloom s like red tide and cyanobacteria are comm on, therefore, there is a po tential f o r hu m a n illnes s

. The progr am will continue surve i llance f o r hea lth effects of harmful algael bloom s and to s upport F l orida’s research regarding related

em erging health con cern s

.

99Summary o f: Wate r Treatment Presented b y : Bruce MacLeod, M.S.

Mr. Bruce W. MacLeod, from the Manatee County Utility Operations Departm e nt, discussed the approach utilit ies take regarding cyanobact erial toxins. Mr. MacLeod began by discussing local news paper articles related to cyanob acterial tox i ns. These articles, in Mr. MacLeod’s opinion, are likely to fright en the public. The Manatee County facility is a large one, which often uses unique treatm e nt schem e s to address sitespecif i c p r o b lem s . General m a nagem e nt strateg i es f o r the u t ilitie s inc l ud e the pr even tion of bloom s and the rem oval of toxins. Bl oom s can be prevented using lake aeration, algaecides, inactivation of phosphorous, la ke shading, disruption and w a tershed

m a nage m e nt . Toxins can be rem oved from water by treatm ent with chlorine, ozone, potassium perm anganate, UV and m e mbranes. Each of these rem oval m e thods has its own problem s, such as taste changes, tim e dependence and added costs.

The Lake Manatee facility has a num ber of unique chara c te ris tics that im pact trea tm ent. It has a 125 square m ile watershed and an instream reservoir da ting back to 1967. The reservoir covers 1800 acres and has an aver age depth of 12.8 feet and a volum e of 12 billion gallons when full. The broad, sha llow sho r eline allows f o r algal g r owth on the periphery of the lake. The water is soft, aci dic, highly colored and nitrog en-lim ited, with a total organic carbon (T OC) range from 10- 30 mg/L. The temperature ranges from 15- 30 ° C, with tem p eratures above 25 ° C 65% of the tim e. Anabaena and Oscillatoria have been found recently. Also, a Micro c y s tis b l oom was observed several years ago. Bloom s

are m o re common in the winter, which is the dry season.

Bloom s have been prevented at Lake Manatee by spraying copper sulfat e from an airboat,

at tem p eratures between 21-30 ° C. Problem areas are spraye d at filam e nt counts of 2 filam e nts/mL, and other areas ar e sprayed at levels above 2 filam e nts/ m L . Mr. McLeod’s experiences have suggested that copper sulf ate has had little if any im pac ton algal densities, with fragm e ntation of filaments being the only change. Anabaena circinalis was viable, even at copper levels of 1 m g /L. Copper m a y stimulate the growth of actinomycetes, and other organism s th at produce taste and odor com pounds.

According to Mr. MacL eod, the watershed is being m a naged in a num ber of ways, which should reduce nutrient loading. First, effort s are m a de to encourage best m a nage m e nt practices (BMPs) for agricultural lan d uses . Hom e owners on the watershed are educated about the use of xeriscaping and other low fe rtilizer m e thodologies. A watershed overlay district was established to lim it housing density and prevent the use of reclaim e d water. Manatee County has been purchasing waters hed land since 1987, following a successful local ballot initiative. Thus far 43%, or 35,000 acres, of the waters hed is publicly owned.

A m a jor focus at Lake Manatee is odorant rem o val. Prior to 1988, a qualitative m e thod to determ ine the proper amount of active ca rbon was used. This m e thod wasted m o ney, due to chasing the effective carbon dose and still yielded complaints from water

consum ers. Between 1988 and 1989, an in-hous e quantitative m e thod wa s developed, a
treatm ent goal was set, optim ization studies were perform e d a nd odorant levels were

100monitored in raw and treated water. As a result of these efforts, the number of

com p laints decreased.

Current activities include HPLC/MS m e thod developm ent for anatoxin, LPS and cylind r osper m opsin, bench-sca l e m e mbrane stud ies with m i crocystin-sp iked f ilte r effluent at the University of South Flor ida, expanded toxin m onitoring and powdered activated carbon (PAC) treatm e nt studies. Mem b rane filters have been found to reduce

m i crocystin-LR in water sam p les by over 94%, as well as 60-96% of TOC, at the benchscale. P ilot-scale s tudies will be con ducted at Lake Manatee by the end o f 2002.

Mr. MacLeod closed by discussi ng water industry needs related to cyanobacterial toxins. These inclu d e acute and chronic acceptable concentrations; depe ndable standards for anatoxin, cylindrosperm opsin and endotoxins ; rapid, easy and rugged testing m e thods and site-specific treatm e nt studies.

101