# 1 Toxic 

## Algod



# Blue-Green Algae in 

# St. Johns River, 

# I

Mark Aubell Peter D'Aiuto, Andrew Chapman, Dale Casamatta, Andrew Reich, Sharon Ketchen, and Chris Williams

Introduction 

# C yanobacteria, or blue-green algae, are a group of oxygenicphotosyntheticbacteria containing chlorophyll-a and accountingfor up to 40 percent of planetary oxygen production. Amongst the oldest and most diverse lineages ofbacteria, they have radiated into nearly every aquatic and terrestrial ecosystem.However, it is their prevalence in freshwaterecosystemsthat has caused the greatest amount of interest and

concern in recent years. As anthropologicaleutrophication escalates, cyanobacteria are an increasingly common component of freshwater systems, especially in southern, sub-tropical systems. Excellent competitors at high-nutrientlevels, they can rapidly form extensive bloom, often consisting of a nearly unialgal assemblage,presenting a number of problems, especially for potable
waters. First, cyanobacteriatend to be inedible or noxious to other aquatic organisms. Second, as blooms senesce, they contribute to large-scale anoxia

as cells lyse and are degraded-often leading to extensive fish kills, Finally, many cyanobacteriaproduce secondary metabolitesthat can adversely impact aquatic ecosystems. One of the primary concerns with cyanobacteria stemsfrom the production of cyanotoxins. Cyanotoxins are a class of chemical compounds typically released during lysis of mature colonies. While over40 specieshave been documented to release toxic compounds, the most common genera are species or strains of Anabaena, Aphanizornenon, Cylindrospermopsis, and Microcystis, with several new generarecently being confirmed as toxin-producing.These toxins are typically differentiated into five major classes based on tissue or organ specificity:hepatotoxins, neurotoxins, cytotoxins, dermatotoxins, and irritant toxins. Of these, the most common are 

### hepatotoxic microcystins (MC), which tend to dominate most freshwaters. Produced primarily by Microcystis aeruginosa,several other genera, including Anabaena, Anabaenopsis, Planktothrix, and Nostoc, have recently also been documented to produce these toxins, which can lead to skin irritation, kidney problems, and, most significantly, livertoxicity. Given the preponderanceof eutrophic lentic and lotic water systems in Florida,coupled with the extensive recreation value of these systems,it is essential to monitor the cyanotoxin levels. However, at present there is no federal

### drinking water standard for these compounds. The purpose of this study

was to monitor cyanotoxin levels in the St. Johns River.

Methods 

### Sample sites. Sample sites were predeterminedand consisted of Doctors Lake, Eagle Point (Shands Bridge), the Palatka Pier, CrescentLake, Lake George, Little Lake Harris at Hickory 

### Pt,Lake Monroe, Me Jesup, and Lake Washington. Geographicaldistribution of sample sites is shown in Figure 1. Sample sites were pre-determined based upon recreationaluse, previous historical

occurrence of toxigenic blooms, and potential for being used as a resource for drinking water production. 

### Algal analyses.Water samples were collected by using a horizontalVan Do 

### (2.2

### L) water sampleror by directly 

### immersing samplecontainers in 

### ambia
water. Water samples that were analya for toxigenic cyanobacteriawere preserved with 10percent lugols solufi and viewed microscopically using 

### an inverted microscope equipped with 

### ph
contrastoptics.

Toxin Analyses 

### Microcystins. Enzyme Linked ImmunosorbentAssay (ELISA) was utilized for the determinationof the 

### concentration of total microcystins (MCs) present. Antibody-coatedpW standards, and all

### reagents were su* by Abraxis LLC (ProductNo. 52011). The level of sensitivity for 

### microcystin using this method yas 

### approximately 

### O P&*

Anatoxin-a and Cylindrospermop, A 

### liquid chromatographic/mass spectrometric (LCIMSIMS) system

was utilized for the identification and quantificationof cylindrospermopsk (CYN) and anatoxin-a (ANTX-A) 

### for% water samples analyzed.

Results and Observations 

### Potentially Toxigenic Cyanobach~ Currently,no guideline levels existfor I 

### management (monitoring, testing, 

### po& andlor closure) of surface waters for potentially toxigenic cyanobacteria

### ad cyanotoxin content for water bodies 

### tha supportrecreational activities in the 

### U.! The World Health Organization, howev does suggest concentrationlevels for the increased monitoring and warning BFort Mdanzas

,Onnond-by-the-Sea

lrmond Beach

Orange 

# ELns

Beach

coma Beach

Figure I.Map of the St. Johns River sampling sites.

recreational users that bloom conditions exist due to an increase in the probability of human health risks. These levels

are 20,000 cellslml (alert level 

### 1) and 100,000cells/ml (alert level 2), which is approximately equivalent to 2-4pg/L and 20

### pgLof microcystin-LR, respectively Falconer et al. 1999).Australiahas recently developed recreationalguidelines 

### for adults and children of 45pg1L and 15 

### pglL fortotal cyanotoxins,respectively (Burch2005). The Florida Department of 

### Bealth and the Public Health Technical Committee of the Florida Harmful Algal BloomTask hope to addressthese issues relevant to the state of Florida in the very


### near future. In general, the St. Johns River maintains a strong population of potentially toxigenic cyanobacteria throughoutthe late spring (May) and continuinginto the early fall months (October/November).Data collected during the samplingperiod (MayOctober) of this project show that the concentrationof potentially toxigenic cyanobacteria,on several occasions, increased to greater than 500,000 cellslml (Table 1).Population densities
were, however, site-specific and not all sites were shown to maintain high concentrationlevels (> 20,000 cellslml.). For example, the Hickory Pt. site in Little Lake Harris, a permitted bathing
water location, exhibited concentration levels of greater than 100,000 cellslml. throughout the samplingperiod (100 percent) while Lakes Washington and Monroe were never observed to have population levels above 100,000 cells/ 

### rnl. and were only observedto have concentration levels above 20,000 cellslml twice (Lake Monroe in mid/ late May). Most water bodies showed rpical "up-and -downw fluctuations in levels as weather, nutrient input, and environmentalconditions varied during the year, which emphasizes the need for continuousmonitoring with short intervalsbetween monitoring
events. It should be noted, however, that patterns of occurrence are impossibleto determine with such a limited time frame of monitoring and usually need multiple
years (five-ten) of frequency data before

a true understandingcan be extracted. Inter-annual variation in algal population dynamics and subsequently toxin production can be significant. During the term of this project, species of Microcystis and Cylindrospermopsisraciborskii were the dominant toxin producers observed.



### Cyanotoxins. Microcystins and cylindrosperrnopsin, but not anatoxin-a,

were positively identified at samples sites in the St. Johns River, Figures 2 and 

### 3 provide monthly levels of MC and CYN, respectively. In most cases, the presence of toxin was indicativeof a significant level of associated cyanobacteria. One exceptionto this correlation is with the MC data found for Lake Jesup. Moderate levels (2-4pa) of MC were determined for July-Sept. without significantlevels (> 50,000 cellslml) of either Microcystisor Anabaena (MC sources)present. In Septemberof 2005 a major Microcystis (primarily M. aeruginosa) bloom occurred in the St. Johns River between Jacksonville and Crescent City, Florida, a span of over 100river miles. Microcystin concentrationsranged from non-detectable to over 1,400 pg/L (70x greater than the World Health Organization alert level 2), indicating the patchiness of bloom events. Figure 4 shows the frequency of toxin levels reported for this event.This bloom event persisted from late August through the end of October and was concentrated on the eastern shore ofthe St. Johns River due to westerly winds. For the first time in the state of Florida,the St. Johns County Health Department issued a health advisory for the St. Johns River informing citizensto be aware of bloom scum formationsand describingpotentialhealth risks. Also during this bloom event, the St. Johns River Water Management District sampled several times per week andTable 1: Frequency of WaterSamples Containing Potentially Toxin Producing Cyanobacteria Greaterthan Suggested WorldHealth Organization

Alert /MonitoringLevels.

Sample Frequency (%) Frequency (%) Maximum Sampling Site Name >20,000 cellslml >100,000cellslml Concentration Events

Doctors Lake Eagle Pt. (Shands Bridge) Palatka Pier Crescent Lake Lake George Little Lake Hanis (Hickory Pt. 

### - Beach) Little Lake Harris (Hickory Pt. 

### - Docks) Lake Monroe Lake Jesup Lake Washington

May '05 June '05 July '05 Aug '05 

### Sept '05 

### Oct '05 

### Date of Collection

Figure 2. TotalMicrocystin ConcentrationLevels at Recreational WaterSites in the St.Johns River, Florida.



### issued weekly press releases

### to the media
to alertthe public as to where the bloom had concentrated and what stretchesof the river may need to be 

### avoided. Concurrent blooms of 

### M. aeruginosa 

### developed in the St. Lucie River, Me Okeeehobee, and the CaloosahatcheeRiver. It should also be 

### noted that cylindrospermopsin

was positively identified at sample sites located within this bloom area and shows the possibility of multiple toxins being produced simultaneously. Figures 5 and 

### 6 show bloom accumulationsat Governors' Creek and DoctorsLake in the St. Johns River system. MCs are a family of liver toxins and potentiallycan act 

### as tumorpromoting agents (Falconer 1991).MCs

can be 

### produced by numerous species ofMicrocystis, 

### as well as species of Planktothrix, 

### andAnabaena. 

### The most widely observed genus in Floridais, by far, Microcystis. 

### MCs have been shown
to 

### cause animal mortalities during bloom
events (as reviewed inRessom et 

### al. 1994)and were the causative agent responsiblefor the death of three dogs last year in Minnesota (unpublished data, Steve Heiskary;EnvironmentalOutcomes Division,MinnesotaPollution Control Agency). As a rule, CYN is not observednearly

as often as MC 

### in the St. Johns River and its basin and any detectablelevel (>

### 0.05 pg/L) is 

### usually evidence for thepresence! of Cylindrospermopsis.

### As seen with 

### MC, CYN was found more frequently during r , 

### July-October. Lake Jesup had the highest 

# 1 ' 

### (0.4 

### - 1.6pa) levels of CYN (JulyOctober) of any site monitored during this ' 

### study.These data correlated well with the i 

### levels of Cylindrospermopsis 

### observed during the sameperiod. Palatka Pier, 

## 1 Lake Monroe,Lake George, and Eagle i 

### Pt. were all found to have detectable 

### '
levels (> 

### 0.05 pa) of CYN present and 

# ? significantlevels (>

### 40,000 

### cells/ml) 

# '

of Cylindrosperrrwpsis.

### What was of I 

### particularinterest was that Lake Jesup, with thbhghest CYN levels, did not have

# I

the highest Cylindrospermopsis 

### cell count. Palatka Pier, Lake George, and Eagle Pt. 

### 1 all had cell countshigher than Lake Jesup, A


### and Doctors Lake not only had the highest Cylindrospermopsis 

### cell count (> 

### 375,000 cells/mL),but no detectable level of 

### CYN. Cylindrospermopsis raciborskii. 

### CYN is classified as a hepatotoxin but 

### can affect other tissues such as the kidneys, adrenal glands, lungs, and intestines (Hawkinset al. 

### 1985).CYN has also been shown to be genotoxic (Humpage et al. 2000) 

### and, potentially, can act 

### as a tumor promoter similarto that of MCs (Falco and Humpage 2001). The predominant cyanobacteria speciesresponsible for the productionor 

### ANTX-A in northeast FloridaisAnabaem circinalis,

### but speciesofAphanizomenon



### can also produce this compound.

### ANTXA has been the least-observedof the three toxins but, as a neurotoxin, can

### be of greatest concern and has caused the only reported human death in the U.S. attributed to cyanotoxins.

### ANTX-A was not found at any detectable level (>

### 0.05

### pa) 

### for any of the sample sites included in this study,but species ofAnabaena 

### and

of Florida complicated normal seasonal variation in 2004. The significance of those events to the severeMicrocystis 

### blooms of 2005 is uncertain,but given the occurrenceofMicrocystis 

### blooms throughoutFlorida in 2005 and the precipitation levels associated with the hurricanes; the increased 

### xun-off may have significantly increased nutrient loads and coupled with the heat of the summer primed the middle and lower St 

### Johns River Basin for the large blooms and toxin levels observed. As previously documented, populations of toxigenic cganobacteriado persist in the St. Johns River. The effect that these toxins have on human health and the overallhealth of the river is not well-understood. 

### It is clear, based on the other sources of ANTX-A continueto be documented. Palatka Pier, Lake George, and Doctors Lake hadAnabaena 

### (primary 

### ANTX-A source)cell counts 

### >50,000 cells/mL.This information, in addition
to some of 

### the Cylindrospermopsis 

### data, reinforces the need for toxin monitoring

as the only way of accurately determining the presence and level of cyanotoxins.

Conclusions and Recommendations Multiplehurricane events in the state

I 

# e w 

# \P l.' &?q9 

# 4' Q 

# 4 recent events, that 

### the St. Johns River 

# '" J and its basinmaintain the potential 

### to experience significanttoxin producing Range of Concentrations 

### blooms when the necessary environmental conditions arise.A continuation of

4. Frequency ofMicrocystin Concentrations Analyzedfrom WaterSamples Collected from 

### this sampling and monitoring study or

eBloom ofMicrocystismruginosa (September

### - October,2005)in the St.Johns River. 

### something similaris recommended.

### If feasible,both an inshore and offshore sampling program should be 

### implemented
to coverthe areas of greatestrisk for human exposure. One way this might be 

### performed is by the use of a public information systemhotline that can accept phone calls from people who observe algal cell accumulations and instruct field personnel, for sampling purposes,

as 

### to where bloom events are occurring. Greenwater Laboratories does maintain a Figure 5. WindDriven Surjace 

### toll-free number (1 

### -877-TOX-ALXiA)for AccumulationofM.aerugugisa 

### such reports. Cells in Governors Creek,St. 

### The identification and quantification JohmRiver. 

### of dominantMC variants (i.e., 

### LR,RR, etc.),particularly with Microcystis 

### blooms of the size observed in 2005, would be

Sum-NE 

# ain transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterialtoxin, cylindrospermopsin.Mutation Res., 472: 155:161. - Ressom, R.. F.S. Soong,J. Fitzgerald,

### L.

Turczynowicz, 0.El Saadi,D. Roder,

### T.

Figure 6. Bloom of

### M. aeruginosaat the Shands Summer of2005 (News4Jax.com).
highly recommended. 

### In addition to providing further data it would also help in evaluating the level of toxicity and potential risk levels due to differencesin toxicity among the different MC isomers. Production of P-N-methylaminoL-alanine (BMAA), a neurotoxic amino acid, has recently been associated with the majority of cyanobacteria
genera,Microcystis,Anabaena, and Cylindrospermopsisraciborskii are certainly included in this group as well as any other cyanobacteria found in the St. Johns River and its basin. Cylindrospermopsisraciborskii actually produces some of the highest levels of BMAA. BMAA has been suggested

as a possible cause of the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALSIPDC). BMAA was recently discovered in the brain tissues of nine CanadianAlzheimer's patients, but

was not detected in the brain tissues of 14other Canadians who died of causes unrelated to neurodegeneration. (Cox
et al. 2005). With the increased interest in BMAA due to its potential global human exposure, some preliminary determinationsof BMAA levels in the St. Johns River should be of interest.

References Burch, M. ISOC-HAB, 2005. Interagency

International Symposiumon CyanobacterialHarmful Algal Blooms. September6-10. Research Triangle Park, NC. Cox, P., S. Banack, S. Murch, U.

Rasmussen, G. Tien,R. Bidigare, Bridge near Green Cove Springs, FLduring the

J. Metcalf, 

### L. Morrison, G. Codd, and B. Bergman. 2005. Diverse
taxa of cyanobacteriaproduce P-Nmethylamino-L-alanine,a neurotoxin amino acid. Proc. Natl. Acad. Sci. USA 102:5074-5078. Falconer, I.R. 1991.Tumor promotion and

liver injury caused by oral consumption of cyanobacteria.Environ. Toxicol. Water Qual.,6(2), 177-184 Falconer,

### I., J. Bartram, I. Chorus,T.

Kuiper-Goodman,H. Utkilen,M. Burch, and G. Codd. 1999.Safe levels and SafePractices (Chapter 5). In I. Chorus and J. Bartram (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public health Consequences, Monitoring, and Management.London1 New York: E & 

### FN Spon Publishers. Falconer, I.R. and A.R. Hurnpage. 2001 Preliminary evidence for in vivo tumour initiationby oral administration of extracts of the blue-green alga Cylindrospermopsis raciborskii containing the toxin cylindrospermopsin.Environ. Toxicol. 16(2): 192-195. Hawkins, P.R., M.T.C. Runnegar, A.R.B.

Jackson, and I.R. Falconer. 1985. Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsisraciborskii (Woloszynska) Seenaya and Subba Raju isolated from domestic water supply reservoir.Appl. Environ.

### Microbial. 50, 1292-1295. Humpage, A.R., M. Fenech, P. Thomas,

and I.R. Falconer. 2000 Micronucleus induction and chromosome loss Maynard, and I. Falconer. 1994Health Effects of Toxic Cyanobacteria (BlueGreen Algae).Australian National Health and Medical Research Council, Looking Glass Press. 108pp.

Dr. Mark Aubel currently performs cylindrospermopsin, anatoxincl (LC/MS/MS), and microcystin analyses (LC/MS/MS and ELISA) I GreenWater Laboratories CyanoLab. GreenWater Laboratories/CyanoLab, established in 2001. is the only private analytical laboratory in the U.S. dedicated to the identification and quantification of toxigenic cyanobacteria and cvanotoxim in source waters and is currently initiating protocols to provide cyanotoxin standards and certified reference materials on a commercial level Dr. Aubel is currently developinn and implementing methdds for the iientification and quantification of BMAA, toxins associated with Lyngbya species, and the saxitoxins.

Dr. Christopher Williams is an aquatic toxicologist who currently



### . acts as the laboratory manager for GWL/CL. Dr. Williams was the project coordinator for the Cyanobacteria Survey Proiect that performed the first statewide survey for cyanotoxins. This project, developed 

### by the Florida HarmfulAlaal Bloom Task Force. " identified microcystins, cylindrospermopsin and anatoxin-a as common components ir Florida surface waters.

Andrew Chapman is

a nationally recognized expert in the field of cyanobacteria identification and taxonomy, and was the lead phycologistfor the Cyanobacteria Survey Project.Peter D'Aiuto is a phycologist and water quality specialist at 

### GWLI 

# a. Mr. D'Aiuto previousl) worked at the USGS in Ahamonte Springs, FL, where he assisted with monitoring water quality irr Lake Okeechobee.

Dr. Dale Casamatta is an assistant professor of biology at the Universityof North Florida in Jacksonville. He has henworking with the St. Johns River Water Management District to examine --- 

# orld's Largest Selrn+;-7 

# I

heecology and management of cyanobacterial blooms and asscciated algal assemblages. His research interests are cyanobacterial systematics, phycology, and aquatic microbial ecology.
Andy Reich has been he program coordinator for the aquatics toxin programwithin the Division of Environmental Health at the FDOH in Tallahassee, Florida, since 2004. His duties include the coordination of epidemiologic and research activities of the Aquatic Toxins Program.

Sharon Ketchen has worked in the environmental field in both the public and private sectors for 

### 15 years. She first ioined the staff of the Horida Department of Mealth in 2000 as an environmentul manager in the Drinking Water Toxins Program, and has been with the Aquatic Toxins Program since 2003. 

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