Toxic Algod 1 

Blue-Green Algae in St. Johns River, I 

Mark Au bell 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 oxygenicphotosynthetic bacteria containing 

chlorophyll-a and accounting for up to 40 

percent of planetary oxygen production. 

Amongst the oldest and most diverse 

lineages of bacteria, they have radiated 

into nearly every aquatic and terrestrial 

ecosystem. However, it is their prevalence 

in freshwater ecosystems that has caused 

the greatest amount of interest and 

concern in recent years. 

As anthropological eutrophication 

escalates, cyanobacteria are an 

increasingly common component 

of freshwater systems, especially in 

southern, sub-tropical systems. Excellent 

competitors at high-nutrient levels, 

they can rapidly form extensive bloom, 

often consisting of a nearly unialgal 

assemblage, presenting a number 

of problems, especially for potable 

waters. First, cyanobacteria tend 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 cyanobacteria produce secondary 

metabolites that can adversely impact 

aquatic ecosystems. 

One of the primary concerns with 

cyanobacteria stems from the production 

of cyanotoxins. Cyanotoxins are a 

class of chemical compounds typically 

released during lysis of mature colonies. 

While over 40 species have been 

documented to release toxic compounds, 

the most common genera are species or 

strains of Anabaena, Aphanizornenon, 

Cylindrospermopsis, and Microcystis, 

with several new genera recently 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, 

liver toxicity. Given the preponderance of 

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 

predetermined and consisted of Doctors 

Lake, Eagle Point (Shands Bridge), 

the Palatka Pier, Crescent Lake, Lake 

George, Little Lake Harris at Hickory 

Pt, Lake Monroe, Me Jesup, and Lake 

Washington. Geographical distribution 

of sample sites is shown in Figure 1. 

Sample sites were pre-determined based 

upon recreational use, 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 horizontal Van Do 

(2.2 L) water sampler or by directly 

immersing sample containers in ambia 

water. Water samples that were analya 

for toxigenic cyanobacteria were 

preserved with 10 percent lugols solufi 

and viewed microscopically using an 

inverted microscope equipped with ph 

contrast optics. 

Toxin Analyses 

Microcystins. Enzyme Linked 

Immunosorbent Assay (ELISA) was 

utilized for the determination of the 

concentration of total microcystins 

(MCs) present. Antibody-coated pW 

standards, and all reagents were su* 

by Abraxis LLC (Product No. 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 

quantification of cylindrospermopsk 

(CYN) and anatoxin-a (ANTX-A) for% 

water samples analyzed. 

Results and Observations 

Potentially Toxigenic Cyanobach~ 

Currently, no guideline levels exist for I 

management (monitoring, testing, po& 

andlor closure) of surface waters for 

potentially toxigenic cyanobacteria ad 

cyanotoxin content for water bodies tha 

support recreational activities in the U.! 

The World Health Organization, howev 

does suggest concentration levels for 

the increased monitoring and warning B 
Fort Mdanzas 

,Onnond-by-the-Sea 

lrmond Beach 

ELns Orange 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 

100,000 cells/ml (alert level 2), which is 

approximately equivalent to 2-4 pg/L and 

20 pgL of microcystin-LR, respectively 

Falconer et al. 1999). Australia has 

recently developed recreational guidelines 

for adults and children of 45 pg1L and 15 

pglL for total cyanotoxins, respectively 

(Burch 2005). The Florida Department of 

Bealth and the Public Health Technical 

Committee of the Florida Harmful Algal 

Bloom Task hope to address these 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 

throughout the late spring (May) and 

continuing into the early fall months 

(October/November). Data collected 

during the sampling period (MayOctober) of this project show that the 

concentration of 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 

concentration levels (> 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 sampling period (100 

percent) while Lakes Washington and 

Monroe were never observed to have 

population levels above 100,000 cells/ 

rnl. and were only observed to 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 environmental conditions 

varied during the year, which emphasizes 

the need for continuous monitoring 

with short intervals between monitoring 

events. It should be noted, however, that 

patterns of occurrence are impossible to 

determine with such a limited time frame 

of monitoring and usually need multiple 

years (five-ten) of frequency data before 

a true understanding can 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 

Cylindrospermopsis raciborskii 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 indicative of a significant 

level of associated cyanobacteria. One 

exception to this correlation is with the 

MC data found for Lake Jesup. Moderate 

levels (2-4 pa) of MC were determined 

for July-Sept. without significant levels (> 

50,000 cellslml) of either Microcystis or 

Anabaena (MC sources) present. 

In September of 2005 a major 

Microcystis (primarily M. aeruginosa) 

bloom occurred in the St. Johns River 

between Jacksonville and Crescent City, 

Florida, a span of over 100 river miles. 

Microcystin concentrations ranged 

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 of the 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 

citizens to be aware of bloom scum 

formations and describing potential health 

risks. Also during this bloom event, the St. 

Johns River Water Management District 

sampled several times per week and 
Table 1: Frequency of Water Samples Containing Potentially Toxin Producing Cyanobacteria Greater than Suggested World Health Organization 

Alert /Monitoring Levels. 

Sample Frequency (%) Frequency (%) Maximum Sampling 

Site Name >20,000 cellslml >100,000 cellslml 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. Total Microcystin Concentration Levels at Recreational Water Sites in the St. Johns 

River, Florida. 

issued weekly press releases to the media 

to alert the public as to where the bloom 

had concentrated and what stretches of the 

river may need to be avoided. Concurrent 

blooms of M. aeruginosa developed in 

the St. Lucie River, Me Okeeehobee, 

and the Caloosahatchee River. 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 accumulations at Governors' 

Creek and Doctors Lake in the St. Johns 

River system. 

MCs are a family of liver toxins 

and potentially can act as tumorpromoting agents (Falconer 1991). MCs 

can be produced by numerous species 

of Microcystis, as well as species of 

Planktothrix, and Anabaena. The most 

widely observed genus in Florida is, by 

far, Microcystis. MCs have been shown 

to cause animal mortalities during bloom 

events (as reviewed in Ressom et al. 

1994) and were the causative agent 

responsible for the death of three dogs 

last year in Minnesota (unpublished data, 

Steve Heiskary; Environmental Outcomes 

Division, Minnesota Pollution Control 

Agency). 

As a rule, CYN is not observed nearly 

as often as MC in the St. Johns River and 

its basin and any detectable level (> 0.05 

pg/L) is usually evidence for the presence! 

of Cylindrospermopsis. As seen with MC, 

CYN was found more frequently during r , 

July-October. Lake Jesup had the highest 1 ' 

(0.4 - 1.6 pa) levels of CYN (JulyOctober) of any site monitored during this ' 

study. These data correlated well with the i 

levels of Cylindrospermopsis observed 

during the same period. 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 ? 

significant levels (> 40,000 cells/ml) ' 

of Cylindrosperrrwpsis. What was of I 

particular interest 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 counts higher 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 

(Hawkins et al. 1985). CYN has also been 

shown to be genotoxic (Humpage et al. 

2000) and, potentially, can act as a tumor 

promoter similar to that of MCs (Falco 

and Humpage 2001). 

The predominant cyanobacteria 

species responsible for the production or 

ANTX-A in northeast Florida is Anabaem 

circinalis, but species of Aphanizomenon 

can also produce this compound. ANTXA has been the least-observed of 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 of Anabaena and 

of Florida complicated normal seasonal 

variation in 2004. The significance of 

those events to the severe Microcystis 

blooms of 2005 is uncertain, but given 

the occurrence of Microcystis blooms 

throughout Florida 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 cganobacteria do 

persist in the St. Johns River. The effect 

that these toxins have on human health 

and the overall health of the river is not 

well-understood. It is clear, based on the 

other sources of ANTX-A continue to be 

documented. Palatka Pier, Lake George, 

and Doctors Lake had Anabaena (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 

Multiple hurricane events in the state 

I e w \P l.' &?q9 4' Q 4 recent events, that the St. Johns River 

'" J and its basin maintain the potential to 

experience significant toxin producing 

Range of Concentrations blooms when the necessary environmental 

conditions arise. A continuation of 

4. Frequency of Microcystin Concentrations Analyzedfrom Water Samples Collected from this sampling and monitoring study or 

e Bloom of Microcystis mruginosa (September - October, 2005) in the St. Johns River. something similar is recommended. If 

feasible, both an inshore and offshore 

sampling program should be implemented 

to cover the areas of greatest risk for 

human exposure. One way this might 

be performed is by the use of a public 

information system hotline 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. Wind Driven Surjace toll-free number (1 -877-TOX-ALXiA) for 

Accumulation of M. aerugugisa such reports. 

Cells in Governors Creek, St. The identification and quantification Johm River. of dominant MC variants (i.e., LR, RR, 

etc.), particularly with Microcystis blooms 

of the size observed in 2005, would be 

Sum-NE a 
in transformed human white cells 

indicate clastogenic and aneugenic 

action of the cyanobacterial toxin, 

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. aeruginosa at the Shands 

Summer of 2005 (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 differences in 

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 

Cylindrospermopsis raciborskii are 

certainly included in this group as 

well as any other cyanobacteria found 

in the St. Johns River and its basin. 

Cylindrospermopsis raciborskii 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 Canadian Alzheimer's patients, but 

was not detected in the brain tissues of 

14 other 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 

determinations of BMAA levels in the St. 

Johns River should be of interest. 

References 

Burch, M. ISOC-HAB, 2005. Interagency 

International Symposium on 

Cyanobacterial Harmful Algal Blooms. 

September 6-10. Research Triangle 

Park, NC. 

Cox, P., S. Banack, S. Murch, U. 

Rasmussen, G. Tien, R. Bidigare, 

Bridge near Green Cove Springs, FL during the 

J. Metcalf, L. Morrison, G. Codd, 

and B. Bergman. 2005. Diverse 

taxa of cyanobacteria produce 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 Safe Practices (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 initiation by 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) Cylindrospermopsis raciborskii 

(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. 1994 Health 

Effects of Toxic Cyanobacteria (BlueGreen Algae). Australian National 

Health and Medical Research Council, 

Looking Glass Press. 108 pp. 

Dr. Mark Aubel 

currently performs 

cylindrospermopsin, 

anatoxi ncl (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 Harmful Alaal 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 phycologist for 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 

University of North 

Florida in Jacksonville. 

He has hen working 

with the St. Johns River 

Water Management 

District to examine 

--- orld's Largest Selrn+;-7 I 

he ecology 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 

program within 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. X 

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