U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2018–5092

Prepared in cooperation with the U.S. Army Corps of Engineers

Understanding the Effect of Salinity Tolerance on 

Cyanobacteria Associated with a Harmful Algal Bloom in 

Lake Okeechobee, Florida
Cover. Dolichospermum circinale stained with SYTOX® Green. Photograph by Barry H. Rosen, U.S. Geological Survey, July 17, 2015, using an 

epifluorescence microscope.
Understanding the Effect of Salinity 

Tolerance on Cyanobacteria Associated 

with a Harmful Algal Bloom in Lake 

Okeechobee, Florida

By Barry H. Rosen, Keith A. Loftin, Jennifer L. Graham, Katherine N. Stahlhut, 

James M. Riley, Brett D. Johnston, and Sarena Senegal

Prepared in cooperation with the U.S. Army Corps of Engineers

Scientific Investigations Report 2018–5092

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

RYAN K. ZINKE, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2018

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Suggested citation:

Rosen, B.H., Loftin, K.A., Graham, J.L., Stahlhut, K.N., Riley, J.M., Johnston, B.D., and Senegal, S., 2018, 

Understanding the effect of salinity tolerance on cyanobacteria associated with a harmful algal bloom 

in Lake Okeechobee, Florida: U.S. Geological Survey Scientific Investigations Report 2018–5092, 32 p., 

https://doi.org/10.3133/sir20185092.

ISSN 2328-031X (print)

ISSN 2328-0328 (online)

ISBN 978-1-4113-4247-7
iii

Acknowledgments

This work was supported by the U.S. Army Corps of Engineers, the U.S. Geological Survey 

(USGS) Priority Ecosystems Greater Everglades Program, and the USGS Environmental Health 

Program. Assistance with laboratory processing was provided by Stephanie Pena.
iv
v

Contents

Acknowledgments........................................................................................................................................iii

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Methods...........................................................................................................................................................3

Initial Bloom Material...........................................................................................................................3

Biomass Indicator—Chlorophyll-a ....................................................................................................3

Biomass Indicator—Cell Numbers....................................................................................................3

Live Organism Physiological Assessment........................................................................................5

Microcystin Analysis by Enzyme-Linked Immunosorbent Assay .................................................5

Additional Studies.................................................................................................................................5

Results .............................................................................................................................................................5

Initial Bloom Material...........................................................................................................................5

Biomass Indicators...............................................................................................................................5

Microcystin............................................................................................................................................6

Physiological Response to Salinity..................................................................................................12

Physiological Response as Determined by SYTOX® Green Visualization .................................12

Physio-Chemical Conditions .............................................................................................................21

Discussion.....................................................................................................................................................30

Summary and Conclusions.........................................................................................................................31

References....................................................................................................................................................31

Figures

1. Map showing the location of Lake Okeechobee, the tributary system in

and out of the lake, and Eagle Bay, where samples were taken for this study,

July 7, 2017.....................................................................................................................................2

2. Photograph showing area where water and bloom materials were collected

for bloom bioassay, 2017 Eagle Bay, Lake Okeechobee, Florida ..........................................4

3. Photograph showing a 1-liter graduated cylinder with initial bloom material...................4

4. Photographs showing the initial cyanobacterial community in the water

sample collected July 7, 2017, from Eagle Bay, Lake Okeechobee, Florida........................6

5. Graph showing chlorophyll-a concentrations during the bioassay.....................................7

6. Graph showing Microcystis aeruginosa abundance in response to salinity.....................8

7. Graph showing Dolichospermum circinale abundance in response to salinity ................9

8. Graphs showing microcystin response to salinity................................................................10

9. Photographs showing colony morphology of Microcystis aeruginosa in various

salinity treatments after 1 day of exposure............................................................................12

10. Photographs showing colony morphology of Microcystis aeruginosa in various

salinity treatments after 4 days of exposure..........................................................................13

11. Photographs showing disintegration of some of the Microcystis aeruginosa

colonies in salinity treatments greater than 10 psu after 1 or more days of

exposure.......................................................................................................................................13

12. Photographs showing response of Dolichospermum circinale filament

morphology to salinity after 1 day of exposure......................................................................14
vi

13. Photographs showing response of Dolichospermum circinale filament

morphology to salinity after 2 days of exposure....................................................................14

14. Photographs showing response of Dolichospermum circinale filament

morphology to salinity after 8 days of exposure....................................................................14

15. Photographs showing extracellular mucilage, at arrows, in

Dolichospermum circinale, day four at 25 psu and Microcystis aeruginosa,

day three at 15 psu .....................................................................................................................15

16. Photographs showing response of Microcystis aeruginosa after 1 day of

exposure to treatments with different salinities....................................................................16

17. Photographs showing response of Microcystis aeruginosa after 2 days of

exposure to treatments with different salinities....................................................................17

18. Photographs showing response of Microcystis aeruginosa after 3 days of

exposure to treatments with different salinities....................................................................18

19. Photographs showing response of Microcystis aeruginosa after 4 days of

exposure to treatments with different salinities....................................................................19

20. Photographs showing response of Microcystis aeruginosa after 8 days of

exposure to treatments with different salinities....................................................................20

21. Photographs showing response of Dolichospermum circinale after 1 day

of exposure to treatments with different salinities ...............................................................22

22. Photographs showing response of Dolichospermum circinale after 8 days

of exposure to treatments with different salinities ...............................................................23

23. Graph showing high frequency, optical sensor data for dissolved oxygen

concentrations in three treatments.........................................................................................24

24. Graph showing high frequency sensor data for temperature ............................................25

25. Graph showing high frequency sensor data for specific conductance............................26

26. Graph showing high frequency sensor data for pH..............................................................27

27. Graph showing high frequency, optical sensor data for chlorophyll-a

fluorescence................................................................................................................................28

28. Graph showing high frequency, optical sensor data for phycocyanin

fluorescence................................................................................................................................29
vii

Conversion Factors

International System of Units to U.S. customary units

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft) 

kilometer (km) 0.6214 mile (mi)

kilometer (km) 0.5400 mile, nautical (nmi) 

meter (m) 1.094 yard (yd) 

Area

square kilometer (km2) 247.1 acre

square kilometer (km2) 0.3861 square mile (mi2)

Volume

liter (L) 33.81402 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

liter (L) 61.02 cubic inch (in3) 

Flow rate

meter per second (m/s) 3.281 foot per second (ft/s) 

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

U.S. customary units to International System of Units

Multiply By To obtain

Flow rate

foot per second (ft/s) 0.3048 meter per second (m/s)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as 

°F = (1.8 × °C) + 32

Supplemental Information

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 

25 °C).

Concentrations of chemical constituents in water are given in either milligrams per liter (mg/L) 

or micrograms per liter (μg/L).
viii

Abbreviations

cells/mL cells per milliliter

FDEP Florida Department of Environmental Protection

LM light microscopy

μWm-2 microwatts per square meter

MCLR microcystin-LR

mL milliliters

μL microliter

μg microgram

μm micrometer

NaCl sodium chloride

PRSD percent relative standard deviation

psu practical salinity units

RFU relative fluorescent units

SFWMD South Florida Water Management District

USGS U.S. Geological Survey

UV ultraviolet

WB wide blue
Understanding the Effect of Salinity Tolerance on 

Cyanobacteria Associated with a Harmful Algal Bloom in 

Lake Okeechobee, Florida

By Barry H. Rosen,1 Keith A. Loftin,1 Jennifer L. Graham,1 Katherine N. Stahlhut,1 James M. Riley,2

Brett D. Johnston,1

 and Sarena Senegal1

Abstract

In an effort to simulate the survival of cyanobacteria as 

they are transported from Lake Okeechobee to the estuarine 

habitats that receive waters from the lake, a bioassay 

encompassing a range of salinities was performed. An overall 

decline in cyanobacteria health in salinity treatments greater 

than 18 practical salinity units (psu) was indicated by loss of 

cell membrane integrity based on SYTOX® Green staining, 

but this loss varied by the kind of cyanobacteria present. 

Microcystis aeruginosa was tolerant of salinities up to 18 psu; 

however, higher salinities caused leaking of microcystin from 

the cells. Dolichospermum circinale, another common bloomformer in this system, did not tolerate salinities greater than 

7.5 psu. Stimulation of mucilage production was observed and 

is likely a mechanism used by both species to protect organism 

viability. At 7.5 psu, microcystin increased relative to 

chlorophyll-a, providing some evidence of biosynthesis when 

M. aeruginosa is exposed to this salinity. This study indicates

that as freshwater cyanobacteria are transported to brackish

and marine waters, there will be a loss of membrane integrity

which will lead to the release of cellular microcystin into the

surrounding waterbody. Additional research would be needed

to determine the exact effect of salinity on this relationship.

Introduction

Lake Okeechobee (located at 27o north latitude and 

81o west longitude) is a large shallow lake (1,900 square 

kilometers [km2]) that receives inflow from the Kissimmee 

River and other smaller tributaries (fig. 1). The bloom 

material for this study came from Eagle Bay on the north 

side of the lake. An earthen dike that surrounds the lake was 

constructed to control adjacent flooding. When lake stage 

threatens the integrity of the dike, water is released to the 

Atlantic Ocean through the St. Lucie Canal and to the Gulf of 

Mexico through the Caloosahatchee River. Releases follow 

the Lake Okeechobee Regulation Schedule, developed and 

implemented by the U.S. Army Corps of Engineers and the 

South Florida Water Management District (SFWMD), to 

ensure the lake is at a low enough stage to accommodate 

summer season runoff and rainfall (U.S. Army Corps of 

Engineers, 2008). 

Lake Okeechobee is classified as a moderately eutrophic 

(Brezonik and Engstrom, 1998) shallow waterbody that has 

undergone ecological changes because of external nutrient 

loading from agriculture (Havens and others, 1996) and, more 

recently, by internal loading of phosphorus from lakebed 

sediments (Pollman and James, 2011). Ample phosphorus and 

other nutrients create the ideal conditions for cyanobacterial 

blooms and have been documented in the lake since 1970 by 

the SFWMD (1989). The first large-scale bloom occurred in 

1986, was dominated by Dolichospermum circinale (formerly 

Anabaena circinalis), and covered approximately 310 km2

. 

Some of the conditions that lead to cyanobacterial 

blooms are warm temperatures, sunlight, water-column 

stability, and sufficiently high concentrations of nitrogen 

and (or) phosphorus. While all cyanobacteria need ample 

phosphorus to thrive, inorganic nitrogen limitation allows a 

subset of cyanobacteria, those capable of using atmospheric 

nitrogen, to be more successful. Dolichospermum (formerly 

Anabaena) has the ability to fix nitrogen (Rosen and others, 

2017), compared to Microcystis, a nonnitrogen fixing genus, 

that needs to acquire nitrogen from inorganic sources. 

During the summer of 2016, regulatory discharges 

from Lake Okeechobee into the Caloosahatchee River 

and the St. Lucie Canal and Estuary occurred during an 

extensive cyanobacterial bloom (National Aeronautics and 

Space Administration, 2016). The Florida Department of 

Environmental Protection (FDEP), in conjunction with the 

Florida Fish and Wildlife Commission, reports cyanobacteria 

species composition and microcystin concentrations weekly in 

Lake Okeechobee and attending waterways (FDEP, 2018). In 

late June 2016, a large cyanobacterial accumulation occurred 

1

U.S. Geological Survey.

2

U.S. Army Corps of Engineers.
2 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

laf18-0875_fig 01

Eagle Bay

Caloosahatchee

River

75

95

LAKE OKEECHOBEE

PALMBEACH

HENDRY

GLADES

COLLIER

POLK

LEE

HIGHLANDS

MARTIN

BROWARD

OKEECHOBEE

STLUCIE

INDIAN RIVER

OSCEOLA

DESOTO

HARDEE

CHARLOTTE

BREVARD

Fisheating Creek

Taylor Creek

Arbuckle Creek

Caloosahatchee River

Blue Cypress Creek

Prairie

Creek

Fivemile Creek

Istokpoga

Creek

Kissimmee River

ATLANTIC OCEAN

St. Lucie Canal

Port Mayaca

Indian River Lagoon

EXPLANATION

Sample sites

81°30' 81° 80°30'

27°30'

27°

26°30'

Base from U.S. Census Bureau-TIGER, 1990 and U.S. Geological Survey

digital data, 1999, 1:100,000

0 5 10 15 20 MILES

0 5 10 15 20 KILOMETERS

Study

area

FLORIDA

Figure 1. The location of Lake Okeechobee, the tributary system in and out of the lake, and Eagle Bay, where samples were taken 

for this study, July 7, 2017.
Methods 3

in the St. Lucie Estuary, prompting the Governor of Florida 

to issue a state of emergency for three affected counties. The 

FDEP data indicated that Lake Okeechobee and (or) the canal 

system had existing populations of cyanobacteria (FDEP, 

2017) that were likely transported downstream when water 

was discharged from Lake Okeechobee. Local and coastal 

basin nutrient runoff, with cyanobacterial proliferation in the 

backwater areas, also contributed organisms to estuarine and 

marine habitats, mainly in the St. Lucie Estuary and Indian 

River Lagoon. In 2005, a similar event occurred with a large 

cyanobacteria population in the St. Lucie Estuary, which 

occurred during regulatory discharges from Lake Okeechobee 

(Phlips and others, 2012).

Salinity tolerance determines how long cyanobacteria 

originating from freshwater habitats will survive at the 

salinities typical of estuarine and marine environments 

(Batterton and Van Baalen, 1971; Orr and others, 2004; Tonk 

and others, 2007), and collectively, these studies indicate 

that many freshwater cyanobacteria experience mortality at 

salinities between 15 and 21 practical salinity units (psu). In 

the Dutch delta and its associated impoundments, such as Lake 

Volkerak in the Netherlands, water management strategies 

have been developed for Microcystis blooms (Verspagen and 

others, 2016) based on salinity tolerance in their systems.

Using cyanobacteria isolated from Lake Okeechobee, 

the U.S. Geological Survey (USGS), in cooperation with 

the U.S. Army Corps of Engineers, conducted a 4-day 

laboratory bioassay with four different salinity treatments 

and a control. Response variables included morphological 

changes to cyanobacterial filaments and colonies, changes 

in growth rate, physiological indicators, intracellular and 

extracellular (released) microcystin, and an approximation 

of cell membrane integrity. Collectively, these data provide 

converging lines of evidence about how Lake Okeechobee 

cyanobacteria might respond to increased salinity under 

natural conditions as water mixes in downstream estuaries. 

Additional data related to this study can be found in King 

and others (2018a, b).

Methods

The organisms used for the study were collected from 

Lake Okeechobee and brought into the laboratory in Orlando, 

Fla., for this study. 

Initial Bloom Material 

Water and floating bloom material were collected 

from the upper 10 cm of the water surface at noon on 

July 7, 2017, from Eagle Bay, Lake Okeechobee, Fla. (latitude 

27°11'42.19"N, longitude 80°49'46.24"W). Approximately 

35 liters (L) of water and bloom material (fig. 2) were 

collected in two large carboys and immediately transported 

to the laboratory, which was approximately 3 hours away, 

without being cooled or held in the dark. All water and 

bloom materials were blended and mixed thoroughly in the 

laboratory and distributed to eighteen 1-L glass graduated 

cylinders (fig. 3). The 1-L graduated cylinders were used to 

simulate a water column approximately 35 centimeters (cm) 

in depth to allow the diurnal migration typically exhibited by 

planktonic cyanobacteria. Cylinders were incubated in a westnorthwest window, and they were treated as batch reactors. 

Daily temperature and solar irradiance was recorded by using 

a Li-Cor LI-200/R pyranometer. Average temperature ranged 

from 23 to 26 degrees Celsius (oC), and light readings yielded 

8.9 microwatts per square meter (μWm-2; plus or minus one 

standard deviation). 

Approximately 20 hours after collection, triplicate 

cylinders were dosed with sodium chloride (NaCl) to increase 

salinity by 7.5, 10, 15, or 18 g L-1 to yield 7.5, 10, 15, or 

18 psu; NaCl was not added to three control cylinders. The 

time zero samples were taken just prior to dosing the cylinders 

with the NaCl. Time one began after 24 hours of exposure, 

at approximately 9:00 every morning. The water in the 

cylinders was thoroughly mixed by completely inverting the 

cylinders 5 times, and the volume needed for all the analytical 

procedures was quickly poured from the cylinder. During 

the time period of the experiment, no attached algae grew on 

the cylinder walls. Although not part of the original planned 

bioassay, some of the original sample was placed into water 

with higher salinities (20, 25, 30, and 35 psu) in graduated 

cylinders 2 days after the main bioassay was started. The 

assessment of these treatments was limited to microscopic 

observations and physiological assessment.

Biomass Indicator—Chlorophyll-a

Chlorophyll provided an overall indication of algal 

biomass. Ten-milliliter (mL) subsamples collected from 

each cylinder daily were filtered as described in Hambrook 

Berkman and Canova (2007), stored (frozen with desiccant), 

and quantified by using a modification of U.S. Environmental 

Protection Agency Method 445.0 (Arar and Collins, 1997). 

Instead of acetone extraction, samples were extracted in 

heated ethanol (Sartory and Grobbelar, 1986), and the 

fluorometer was modified with a flow-through cell (Knowlton, 

1984). Samples were analyzed in duplicate, and the results 

were reported as an average. 

Biomass Indicator—Cell Numbers 

A 10-milliliter (mL) sample from each of the 18 cylinders 

was collected by 10-mL graduated pipette dispensed into 

a 15-mL screw-cap plastic centrifuge tube, preserved with 

100 microliters (μL) of Lugol’s iodine, and refrigerated at 

4 oC until it was processed. For counting, the samples were 

homogenized by vigorous shaking to disperse the colonies, an 

aliquot was withdrawn, and the precise weight was determined 

(x 0.0001 gram [g]) with an Ohaus Explorer EX224 Analytical 

Electronic Balance. The weight was considered equivalent 

to the volume (1 mL = 1 g) and evenly distributed under a 
4 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Figure 2. Area where water and bloom 

materials were collected for bloom bioassay, 

2017 Eagle Bay, Lake Okeechobee, Florida. 

(Photograph by Barry H. Rosen, U.S. Geological 

Survey.)

Figure 3. A 1-liter graduated cylinder with 

initial bloom material. (Photograph by Barry H. 

Rosen, U.S. Geological Survey.)
Results 5

22-square-millimeter (mm2) glass coverslip. Counting was 

conducted microscopically at 400x by enumerating the 

cyanobacteria observed in a linear strip along the full length 

of the coverslip. The diameter of the strip was measured with 

a stage micrometer. Using the volume distributed under the 

coverslip per unit area, the number of organisms per unit 

volume was calculated. For Microcystis, its dense, colonial 

form does not allow an exact direct cell count. We were able 

to enumerate the number of colonies and optically image 

a subset of colonies to obtain an average number of cells 

per colony. This average number of cells per colony was 

multiplied by the number of colonies found in each sample to 

get estimated cell count. 

Live Organism Physiological Assessment 

Approximately 0.020 mL of live material taken from 

each salinity treatment was examined daily to assess the 

health of cells by following the protocol of Rosen and others 

(2010). Microscopic observations were made with a BX51 

Olympus microscope with differential interference contrast 

and epifluorescence using ultraviolet and wide blue excitation 

sources. SYTOX® Green (Invitrogen, Carlsbad, Calif.), a 

deoxyribonucleic acid (DNA) stain excluded from live cells, 

was added (Rosen and others, 2010) to assess if cells lost 

cellular membrane integrity.

Microcystin Analysis by Enzyme-Linked 

Immunosorbent Assay

For analysis of total microcystin, a 10-mL subsample 

was taken daily from each cylinder and frozen. For analysis 

of dissolved microcystin, the filtrate (Millipore Type TSTP 

3.0-micrometer [μm], 25-millimeter [mm] diameter) from 

a 10-mL subsample was collected daily from each cylinder 

and frozen. These samples were processed and analyzed as 

described in Loftin and others (2016), except that an Abraxis 

streptavidin amplified enhanced sensitivity (SAES) enzymelinked immunosorbent assay kit was used with a 5-psu sodium 

chloride microcystin-LR (MCLR) calibration curve custom 

made by Abraxis, Inc. (Warminster, Pa.). A four-parameter 

curve-fit was used for calibration. Measurements of kit diluent 

and laboratory reagent water were below the kit minimum 

reporting level (0.10 microgram per liter [μg/L]), 0.75-μg/L 

MCLR kit controls were analyzed every 10th sample, and 

28 percent of samples were laboratory replicates. Mean kit 

control recovery was 106 percent, mean percent relative 

standard deviation (PRSD) was 9.6 percent, and laboratory 

replicate PRSD was 20 percent. Particulate microcystin 

was calculated by subtracting dissolved concentration 

from total concentration.

Additional Studies

The influence of circulation was evaluated in unamended 

water and bloom material. Three 4-L glass beakers were filled 

with 2.5 L of water and bloom material, set atop magnetic 

stir plates, and separately circulated at velocities of 0.00 foot 

per second (ft/s; “nonstirred”), 0.27 ft/s, and 0.95 ft/s. The 

beakers were sealed with plastic to prevent evaporation. 

Velocity was measured with a USGS pygmy current meter 

by using the 0.6-depth method (Turnipseed and Sauer, 2010). 

High-frequency sensor data were collected at 5-minute 

intervals. Data were collected by using a multiparameter 

sonde (Xylem/YSI EXO2), calibrated in accordance with 

manufacturer protocols (YSI Incorporated, 2017) to measure 

water temperature, specific conductance, dissolved oxygen, 

pH, chlorophyll-a fluorescence, and phycocyanin fluorescence. 

The high-resolution visualization resulting from these data 

provides insight into the biogeochemical processes in relation 

to the results from less frequently collected discrete analyses 

(Downing and others, 2017).

Results

The majority of the results center on the salinity bioassay 

conducted; however, ancillary data were collected from the 

same water and bloom material to provide some understanding 

of how stirred conditions affect physio-chemical parameters. 

Initial Bloom Material 

The bloom material collected from Lake Okeechobee 

on July 7, 2017, contained three species of Microcystis: M. 

aeruginosa, M. flosaquae, and M. wesenbergii. Microcystis 

aeruginosa was the most frequently observed species of 

this genus. Also present were Dolichospermum circinale,

Planktolyngbya contorta, Planktolyngbya limnetica, and 

Cuspidothrix tropicalis (fig. 4). Microcystin concentration in 

the surface scum collected directly from Lake Okeechobee 

was 560 μg/L, indicating the presence of microcystinproducing species.

Although rare in the initial community of organisms, 

two additional cyanobacteria genera grew during the 

experimental treatments. Planktothrix was found on day 

eight of the experiment in water with salinity of 15 psu, and 

Cuspidothrix tropicalis was found on day four in water with 

salinity up to 18 psu.

Biomass Indicators 

Chlorophyll-a concentrations and cyanobacterial 

abundance were used to quantify changes in algal biomass 

during the bioassay (figs. 5–7). For chlorophyll-a, all 

concentrations declined from day zero to day four, with the 
6 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

A B C

D E F

G

Figure 4. The initial cyanobacterial community in the water sample collected July 7, 2017, from Eagle Bay, Lake Okeechobee, Florida. 

A, Microcystis aeruginosa (most abundant); B, M. flosaquae; C, M. wesenbergii; D, Dolichospermum circinale; E, Planktolyngbya 

contorta; F, Cuspidothrix tropicalis; G, Planktolyngbya limnetica.

greatest declines in the control (0 psu) and 7.5-psu treatments 

(fig. 5). The 10, 15, and 18 psu treatments showed greater 

amounts of chlorophyll-a than the lower salinity treatments.

Microcystis aeruginosa and Dolichospermum circinale

were the dominant cyanobacteria in the initial community 

used for the bioassay. For Microcystis, all of the treatments 

showed an initial increase in the number of cells during the 

first time interval from time zero through the first 24 hours, 

and then the number of cells decreased during the next 

24-hour time period (fig. 6), including the control treatment 

with no added salt. Because the bloom collected from Eagle 

Bay had been thriving in full sunlight, this adaptation to a 

laboratory setting was an anticipated response. 

Dolichospermum circinale abundance in the control and 

all of the treatments substantially decreased during the first 

24 hours of the bioassay (fig. 7).

Microcystin 

Microcystin was reported as a total concentration in 

whole water and as a dissolved phase in the water after the 

cells were removed by filtration. Total concentration (fig. 8A) 

decreased to less than 20 μg/L in the control. The 7.5- and 

10-psu salinity treatments initially declined but increased to 

more than 40 μg/L by the end of the 4-day bioassay. 

The dissolved phase (fig. 8B) is a combination of 

microcystin leaked from live or unhealthy cells and from 

those cells that died and released microcystin. In the control, 

the amount of dissolved-phase microcystin remained less 

than 2 μg/L, with the largest amounts in the 15- and 18-psu 

treatments (12–13 μg/L) and in the 7.5- and 10-psu treatments 

(8–10 μg/L). At all the salinities greater than the control, the 

amount of dissolved microcystin increased over time (fig. 8B), 

indicating that some of the cells in the treatments were 

leaking cellular microcystin and cell lysis was occurring. The 

partitioning of microcystin between the amount retained in 

cells (calculated particulate) and the dissolved phase (fig. 8C) 

has a similar pattern as the total microcystin (fig. 8A), with 

more retained or produced in the 7.5- and 10-psu treatments.

The calculated particulate microcystin to chlorophyll-a

ratio was examined (fig. 8D) to normalize the toxin 

concentration to a cell abundance surrogate (chlorophyll-a). 

Cell abundance was not used for this calculation because 

chlorophyll-a was a more precise measurement then cell 

abundance, given the difficulty and potential variability in 

estimating cell numbers. In addition, particulate microcystin 

and particulate chlorophyll-a are both intracellular 

constituents. Using this calculation, by day four, the 

7.5- and 10-psu treatments showed an increase in this 

ratio that is approximately double the ratios in the 15- and 

18-psu treatments. 
Results 7

100

150

200

250

300

350

400

450

0 1 2 3 4

Days

Chlorophyll-a, in micrograms per liter

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

Figure 5. Chlorophyll-a concentrations during the bioassay. 
8 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

0

200,000

400,000

600,000

800,000

1,000,000

1,200,000

0 1 2 3 4

Cells, per milliliter

Days

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

Figure 6. Microcystis aeruginosa abundance in response to salinity. The error bars are standard error of the mean.
Results 9

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

180,000

0 1 2 3 4

Cells, per milliliter

Days

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

Figure 7. Dolichospermum circinale abundance in response to salinity. The error bars are standard error of the mean.
10 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

0

5

10

15

20

25

30

35

40

45

50

Microcystin concentration, in micrograms per liter

Microcystin concentration, in micrograms per liter

0 1 2 3 4

Days

0 1 2 3 4

Days

A

B

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

0

2

4

6

8

10

12

14

16

Figure 8. Microcystin response to salinity: A, total microcystin; B, dissolved phase; C, calculated particulate; D, calculated 

particulate microcystin:chlorophyll-a ratio. The error bars are standard error of the mean.
Results 11

Microcystin concentration, in micrograms per liter Ratio of particulate microcystin to chlorophyll-a

0 1 2 3 4

Days

0 1 2 3 4

Days

C

D

0

5

10

15

20

25

30

35

40

45

50

0

0.05

0.1

0.15

0.2

0.25

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

EXPLANATION

0 Practical salinity

units (psu)

Standard error of 7.5 psu

the mean 10 psu

15 psu

18 psu

Figure 8. Microcystin response to salinity: A, total microcystin; B, dissolved phase; C, calculated particulate; D, 

calculated particulate microcystin:chlorophyll-a ratio. The error bars are standard error of the mean.—Continued
12 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Physiological Response to Salinity 

Overall, the Microcystis aeruginosa colonies maintained 

integrity during the first 24 hours of exposure at salinities 

up to 25 psu (fig. 9; 30 and 35 psu were not photographed at 

this time interval). After 4 days, complete disintegration of 

all Microcystis aeruginosa colonies did not occur, even up to 

35 psu (fig. 10). Although most colonies remained intact, a few 

disintegrated to small clusters and individual cells (fig. 11).

Dolichospermum circinale was much less abundant, 

leaving a less complete picture of salinity effects. Intact 

filaments were present after 1 day; however, the cells seem to 

be separating in the 20- and 25-psu treatments (fig. 12). After 

2 days of exposure, Dolichospermum has additional separation 

of cells at higher salinity (fig. 13). Dolichospermum circinale

was tolerant of 7.5 psu and was in good condition after 8 days 

(fig. 14), but was not found at any salinity greater than 7.5 psu 

by the end of the bioassay.

In addition to overall morphological changes in colonies 

and filaments of these two genera of cyanobacteria, evidence 

of adaptation was observed microscopically. The production 

of mucilage was observed enveloping the filaments and the 

colonies (fig. 15).

Physiological Response as Determined by 

SYTOX® Green Visualization 

Cell health was analyzed by using epifluorescence 

microscopy in conjunction with SYTOX® Green, a DNA stain 

that emits green light in the presence of DNA. If the cellular 

membrane of an organism is intact, SYTOX® Green does not 

penetrate, providing an indication of cell health. In figures 

16–22, the denotation is (1) LM—organisms illuminated 

with differential interference light microscopy for overall cell 

and colony structure; (2) UV—organisms illuminated with 

ultraviolet light, with variations in pigment color an indicator 

of cell health; (3) WB—organisms illuminated with “wide 

blue” as the baseline color before adding SYTOX® Green; (4) 

SYTOX® Green—organisms stained with this dye are bright 

green when DNA is present (Rosen and others, 2010). The 

photographs are representative of the overall condition of the 

colonies or filaments in the treatments. 

After 1 day of exposure, the SYTOX® Green is only seen 

outside of the Microcystis colony, with no penetration of the 

stain into the cells in all treatments (fig. 16). After 2 days of 

exposure, the SYTOX® Green penetrated the Microcystis cells 

in the 18-, 20-, and 25-psu treatments as seen in figure 17 

where the cells are green compared to the 15-psu treatment. 

After 3 days of exposure, the SYTOX® Green is seen only 

external to the Microcystis colonies, with no penetration of 

the stain into the cells in all treatments, with the exception 

of the 15-psu treatment (fig. 18). In the 15-psu treatment, the 

SYTOX® Green penetration and the pigment shift to yellow 

under UV indicate that this colony was no longer viable. After 

4 days of exposure, with one of two Microcystis colonies in 

the 15-psu treatment and all of the colonies in treatments of 

20 psu or greater salinity, the SYTOX® Green penetration 

and the pigment shift to yellow under UV indicate that these 

colonies were no longer viable (fig. 19). After 8 days of 

exposure, the 18-psu treatments had some Microcystis colonies 

that were still viable and some that were not, while all lower 

salinity treatments had viable colonies (fig. 20).

0 psu 7.5 psu 10 psu 15 psu 18 psu

 20 psu 25 psu

Figure 9. Colony morphology of Microcystis aeruginosa in various salinity treatments after 1 day of exposure.
Results 13

0 psu 7.5 psu 10 psu 15 psu 18 psu

 20 psu 25 psu 30 psu 35 psu

Figure 10. Colony morphology of Microcystis aeruginosa in various salinity treatments after 4 days of exposure.

10 psu, 72 hours 18 psu, 24 hours

20 psu, 48 hours 25 psu, 48 hours

Figure 11. Disintegration of some of the Microcystis aeruginosa colonies in salinity treatments greater than 10 psu after 1 or more 

days of exposure.
14 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

7.5 psu 10 psu 20 psu 25 psu

Figure 12. Response of Dolichospermum circinale filament morphology to salinity after 1 day of exposure. 

0 psu 20 psu 25 psu

Figure 13. Response of Dolichospermum circinale filament morphology to salinity after 2 days of exposure.

0 psu 7.5 psu

Figure 14. Response of Dolichospermum circinale filament morphology to salinity after 8 days of exposure.
Results 15

A

B

Figure 15. Extracellular mucilage, at arrows, in A, Dolichospermum circinale, day four at 25 psu; B, Microcystis 

aeruginosa, day three at 15 psu. 
16 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Figure 16. Response of 

Microcystis aeruginosa

after 1 day of exposure 

to treatments with 

different salinities 

(psu, practical salinity 

units; LM, differential 

interference microscopy; 

UV, epifluorescence 

microscopy; WB, wideblue epifluorescence 

microscopy; WB and 

SYTOX® Green, wideblue microscopy with 

the DNA stain SYTOX®

Green added. Blank boxes 

indicate that data were not 

available).

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu

10 psu

15 psu

18 psu

20 psu

25 psu
Results 17

Figure 17. Response of 

Microcystis aeruginosa

after 2 days of exposure 

to treatments with 

different salinities 

(psu, practical salinity 

units; LM, differential 

interference microscopy; 

UV, epifluorescence 

microscopy; WB, wideblue epifluorescence 

microscopy; WB and 

SYTOX® Green, wideblue microscopy with the 

DNA stain SYTOX® Green 

added).

25 psu

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu

10 psu

15 psu

18 psu

20 psu
18 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

 Figure 18. Response of 

Microcystis aeruginosa

after 3 days of exposure 

to treatments with 

different salinities 

(psu, practical salinity 

units; LM, differential 

interference microscopy; 

UV, epifluorescence 

microscopy; WB, wideblue epifluorescence 

microscopy; WB and 

SYTOX® Green, wideblue microscopy with 

the DNA stain SYTOX®

Green added).

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu

10 psu

15 psu

18 psu

20 psu

25 psu
Results 19

Figure 19. Response of 

Microcystis aeruginosa

after 4 days of exposure 

to treatments with 

different salinities 

(psu, practical salinity 

units; LM, differential 

interference microscopy; 

UV, epifluorescence 

microscopy; WB, wideblue epifluorescence 

microscopy; WB and 

SYTOX® Green, wide-blue 

microscopy with the DNA 

stain SYTOX® LM UV WB WB and SYTOX® Green added). Green

0 psu

7.5 psu

10 psu

15 psu

18 psu

20 psu

30 psu

25 psu

35 psu
20 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Figure 20. Response of Microcystis aeruginosa after 8 days of exposure to treatments with different salinities (psu, practical 

salinity units; LM, differential interference microscopy; UV, epifluorescence microscopy; WB, wide-blue epifluorescence 

microscopy; WB and SYTOX® Green, wide-blue microscopy with the DNA stain SYTOX® Green added. Blank boxes indicate that 

data were not available).

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu

10 psu

15 psu

18 psu
Results 21

After 1 day of exposure, Dolichospermum was much less 

abundant in the treatments, hence the sparse observations (fig. 

21). The Green was observed to have penetrated one of two 

filaments observed after 1 day of exposure at 20 psu. 

Dolichospermum filaments were seen in the control and 

in the 7.5-psu treatment, but were absent in salinity greater 

than 7.5 psu after day one. After 8 days, the only treatments 

with any Dolichospermum were the control and the 7.5-psu 

treatment, and Dolichospermum were rare in both samples 

(fig. 22). The few filaments observed in both of these 

treatments were viable and appeared healthy. 

Physio-Chemical Conditions

Ancillary to the bioassay itself, a continuously recording 

sonde was used to collect physical and water-chemistry data 

from water associated with the initial cyanobacteria sample 

collection and a simulation of water flow without any salinity 

treatments. Material was placed in 4-L beakers that were either 

stirred or not stirred. The nonstirred treatment is the most 

similar to the actual bioassay in the sense that the graduated 

cylinders used for the bioassay were not stirred. Dissolved 

oxygen initially showed a small increase to 4 milligrams per 

liter (mg/L), then decreased and maintained a fairly constant 

level of 3 mg/L in the nonstirred treatment. A slight diurnal 

pattern was present that dissipated after day two (fig. 23). 

Both stirred treatments had higher dissolved oxygen than the 

nonstirred treatment, with the highest concentration of oxygen 

at the fastest rate of stirring, 0.95 ft/s, reaching 8 mg/L.

Temperature varied diurnally from a low of 23 oC 

to a high slightly greater than 27 oC in the nonstirred 

treatment (fig. 24). Water in both stirred beakers (on a 

magnetic plate with a stir bar) had elevated temperatures 

that were 2 to 4 oC higher than temperatures in the 

nonstirred treatment.

In the nonstirred treatment, specific conductance was 

307 microsiemens per centimeter at 25 degrees Celsius (μS/

cm at 25 °C) on the first day of the test; specific conductance 

increased to 322 μS/cm by the end of the test (fig. 25). The 

stirred treatments had the same initial specific conductance as 

that of the nonstirred treatment; however, specific conductance 

was approximately 30 μS/cm greater in the stirred treatments 

than in the nonstirred treatment by the end of the test. 

The pH peaked at just over 7.3 in the nonstirred treatment 

(fig. 26). In the stirred treatments, pH values were higher on 

average. In the treatment that was stirred at a rate of 0.27 ft/s, 

the maximum pH was 7.6. In the treatment that was stirred at a 

rate of 0.95 ft/s, the maximum pH was 7.9. 

For all treatments, chlorophyll-a fluorescence exhibited 

the greatest decrease during the first day as the bloom was 

moved from Lake Okeechobee to the laboratory setting 

(fig. 27). The decrease of chlorophyll-a fluorescence in 

the nonstirred treatment leveled off for the duration of the 

experiment; however, fluorescence in both stirred treatments 

was higher than in the nonstirred treatment, and chlorophyll-a

fluorescence increased in the stirred treatments after day three. 

Phycocyanin fluorescence decreased substantially across 

all treatments over the course of the experiment, though 

fluorescence did remain higher in the treatment that was 

stirred at the fastest rate (0.95 ft/s; fig. 28).
22 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Figure 21. Response of Dolichospermum circinale after 1 day of exposure to treatments with different salinities (LM, differential 

interference microscopy; UV, epifluorescence microscopy; WB, wide-blue epifluorescence microscopy; WB and SYTOX® Green, 

wide-blue microscopy with the DNA stain SYTOX® Green added. Blank boxes indicate that data were not available).

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu

10 psu

20 psu

25 psu
Results 23

Figure 22. Response of Dolichospermum circinale after 8 days of exposure to treatments with different salinities (filament at 

blue arrow for image at upper right, among the Microcystis colonies; psu, practical salinity units; LM, differential interference 

microscopy; UV, epifluorescence microscopy; WB, wide-blue epifluorescence microscopy; WB and SYTOX® Green, wide-blue 

microscopy with the DNA stain SYTOX® Green added).

LM UV WB WB and SYTOX® Green

0 psu

7.5 psu
24 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

laf18-0875_fig 23

0

1

2

3

4

5

6

7

8

9

Dissolved oxygen, in milligrams per liter

Date and time

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

Figure 23. High frequency, optical sensor data for dissolved oxygen concentrations in three treatments: one not stirred, one 

stirred at 0.27 foot per second (ft/s), and one stirred at 0.95 ft/s.
Results 25

laf18-0875_fig 24

Date and time

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

15

17

19

21

23

25

27

29

31

33

35

Temperature, in degrees Celsius

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

Figure 24. High frequency sensor data for temperature.
26 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

laf18-0875_fig 25

Date and time

Specific conductance, in microsiemens per centimeter at 25 degrees Celsius

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

305

310

315

320

325

330

335

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

Figure 25. High frequency sensor data for specific conductance.
Results 27

laf18-0875_fig 26

Date and time

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

7.1

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8.1

pH

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

Figure 26. High frequency sensor data for pH.
28 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Date and time

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

1.5

2.5

3.5

4.5

5.5

6.5

Chlorophyll-a, in relative fluorescence units (RFU)

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

laf18-0875_fig 27

Figure 27. High frequency, optical sensor data for chlorophyll-a fluorescence.
Results 29

0.95 foot per second

0.27 foot per second

0.00 foot per second

EXPLANATION

Treatment, stirred at

laf18-0875_fig 28

Date and time

07/10/17

12:00

07/11/17

00:00

07/11/17

12:00

07/12/17

00:00

07/12/17

12:00

07/13/17

00:00

07/13/17

12:00

07/14/17

00:00

0

5

10

15

20

25

30

Phycocyanin, in relative fluorescence units (RFU)

Figure 28. High frequency, optical sensor data for phycocyanin fluorescence.
30 Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida

Discussion

It is frequently assumed that when cyanobacteria from 

freshwater lakes, reservoirs, and rivers reach saline estuaries, 

they will die off after some period of time. The hydrologic 

dynamics in these transition areas influence the salinity 

conditions that these organisms experience. In a study that 

included a period of augmented flow from Lake Okeechobee 

in 2005, Phlips and others (2012) found that Microcystis 

aeruginosa persisted in the St. Lucie Estuary. The Phlips 

study reported that the South Fork of the St. Lucie Estuary 

had dense surface accumulations of cyanobacteria, with 

chlorophyll-a reaching 2,863 μg/L–1 at the surface, and the 

cyanobacteria seem to have come from the St. Lucie Canal. 

Only a few studies have attempted to define the effects of 

salinity on cell growth, cell death, toxicity, pigmentation, and 

cell morphology (Batterton and Van Baalen, 1971; Orr and 

others, 2004). Tonk (Tonk and others, 2007) found the salinity 

tolerance of Microcystis did not exceed 20 psu.

The bloom used for this bioassay was collected 

from Eagle Bay, Lake Okeechobee, on July 7, 2017, and 

brought into a laboratory setting. The bloom was moved 

from an open-water habitat where it experienced wind and 

wave activity and nutrient and light variability associated 

with an open waterbody. Moving the community from 

its natural environment into the laboratory and into batch 

growth conditions (the graduated cylinders) altered many 

parameters that would equate to physiological stress on the 

organisms; however, the approach used in this study ensured 

physiological variables were similar across the treatments, 

leaving salinity changes as the only variable. The responses of 

dissolved oxygen and pH indicate the loss of a typical diurnal 

pattern that likely existed in the lake (figs. 23 and 26). The 

increase in specific conductance (fig. 25) may be explained 

by greater evaporation in the stirred treatments, which were 

also warmer than the nonstirred treatment (fig. 24) because 

of the heat produced by the stir plates. On a cellular level, if 

the cells were being disrupted and leaking cellular contents 

mineralized by bacteria, it could also explain the increase in 

specific conductance, as well as being a possible factor for the 

observed decline in fluorescence.

Overall, the control and treatments showed a downward 

trend in chlorophyll-a and phycocyanin fluorescence over 

time (figs. 27 and 28), and numerous possibilities exist 

for this phenomenon, including moving organisms from a 

high-light environment to a laboratory setting and changing 

nutrient dynamics that affect pigment fluorescence (Rosen and 

Lowe, 1984). 

The water collected contained the targeted organism, 

Microcystis aeruginosa, with a density of approximately 

400,000 cells per milliliter (cells/mL). This original sample 

contained ample microcystin (560 μg/L), which allowed us to 

monitor the presence of microcystin in cells and the amount 

released to the water as a result of salinity stress. Another 

major bloom-forming organism from the lake (Rosen and 

others, 2017), Dolichospermum circinale, was present at 

an initial density that was more than 140,000 cells/mL. The 

response of these two organisms provided information on the 

colonial and filamentous forms of cyanobacteria, Microcystis

and Dolichospermum, respectively. 

Generally, cyanobacteria health declined in salinity 

treatments greater than 18 psu as indicated by a loss of 

cell membrane integrity. In any given waterbody, the 

cyanobacteria colonies and filaments are not identical; rather, 

they are in various physiological states. Some colonies and 

filaments contain healthy cells that are likely better able to 

withstand stress, such as salinity changes, while others are 

unhealthy and more easily disrupted. Mucilage production, 

a mechanism commonly observed in response to desiccation 

in cyanobacteria (Pereira and others, 2009; Rosen and 

Mareš, 2016) and salinity (Ozturk and Aslim, 2010), was 

also an indicator of response to salinity in Microcystis and 

Dolichospermum (fig. 15). For mucilage production to 

occur, the filaments/colonies have to be alive and able to 

synthesize compounds that are excreted from the cells. The 

extracellular polymeric substance is mainly composed of 

complex polysaccharides that serve as a protective boundary 

between the cells and the water that surrounds them (Kehr 

and Dittmann, 2015). Nitrogen limitation has also been found 

to increase polysaccharide production in Microcystis cells, 

and light intensity seems to play an important role (Yang and 

others, 2012).

Initially, individual Microcystis colonies contained 

hundreds of cells (fig. 4A), and the effect of salinity on 

colony integrity did not show a single pattern. Some colonies 

maintain their overall shape with several hundreds of cells, 

even after 4 days of exposure in treatments up to 35 psu 

(fig. 10), while other colonies broke up into individual cells at 

10 psu or greater (fig. 11). Although some cells of Microcystis

lost integrity after 2 days of exposure to salinity greater than 

18 psu, as indicated by being stained green with SYTOX®

Green (fig. 17), other cells and colonies were left intact even 

after 4 days of exposure to salinity up to 20 psu. Cell mortality 

occurred after 4 days of exposure to salinity of 25 psu or 

greater (fig. 19), even if the colonies appeared to be intact. 

This range in colony breakdown in response to salinity is 

likely because of the natural variability in the health of each 

individual colony in a bloom, similar to the concept of the 

“paradox of the plankton” (Hutchinson, 1961). 

Dolichospermum circinale cells decreased substantially 

in all treatments during the first 24 hours, indicating this 

species is not tolerant of the salinities in the ranges tested 

(fig. 7). Filaments were initially coiled (fig. 4D), but became 

shortened fragments within 1 day of salinity exposure 

(fig. 12). Separation of the cells in a filament was observed 

and likely the cause of fragmentation. Dolichospermum 
References 31

tolerated the lowest salinity treatment, 7.5 psu (fig. 14B), 

for 8 days, but filaments were not observed in the higher 

salinities in this timeframe. From these observations, this other 

common Lake Okeechobee bloom-forming organism is much 

less tolerant of salinity compared to Microcystis. Although 

some of the fragments were stained with SYTOX® Green 

after 2 days of exposure to the 7.5-psu treatment, this salinity 

does not appear to cause complete mortality, as indicated 

by filamentous colonies that were still viable after 8 days at 

7.5 psu. Dolichospermum circinale did not tolerate conditions 

greater than 7.5 psu.

Total, dissolved, or calculated microcystin 

concentrations, as total, dissolved, or calculated particulate, 

increased over time and were greater in the salinity treatments 

compared to the control. The amount of microcystin was 

greatest in the dissolved phase at the two highest salinities 

(fig. 8B), indicating that either a greater amount leaked from 

the cells in general or that cellular contents were released 

by dead cells. The particulate microcystin and chlorophyll-a

give us information about the cellular content of organisms, 

hence we gain a more direct understanding of the cellular 

microcystin response as a function of salinity. The 7.5- and 

10-psu treatments contained a greater amount of microcystin

per unit of chlorophyll, indicating toxin stimulation at these

concentrations. In contrast, in the 15- and 18-psu salinity

treatments, the microcystin to chlorophyll ratios were similar

to or lower than in the control, suggesting the inhibition

of microcystin at these salinities. Additional experiments

are needed to determine the mechanisms by which salinity

stimulates or inhibits microcystin production.

Summary and Conclusions

This study documents an overall decline in cyanobacteria 

health in salinity treatments greater than 18 psu. A dominant 

bloom in this system, Microcystis aeruginosa, was tolerant 

of salinities up to 18 psu; however, higher salinities caused 

leaking of microcystin from the cells. Dolichospermum 

circinale, another common bloom-former in this system, did 

not tolerate salinities greater than 7.5 psu. Both organisms 

produced extracellular mucilage as a protective response 

to salinity, which is a common ecological strategy in 

cyanobacteria undergoing desiccation. At 7.5 psu, microcystin 

increased relative to chlorophyll-a, providing some evidence 

of biosynthesis when M. aeruginosa is stressed at these 

salinities. This study indicates that as freshwater cyanobacteria 

are transported to brackish and marine waters, there will be 

a loss of membrane integrity which will lead to the release 

of cellular microcystin into the surrounding waterbody. 

Additional research would be needed to determine the exact 

effect of salinity on this relation.

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Batterton, J.C., Jr., and Van Baalen, C., 1971, Growth 

responses of blue-green algae to sodium chloride 

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Downing, B.D., Bergamaschi, B.A., and Kraus, T.E.C., 

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https://water.usgs.gov/owq/FieldManual/Chapter7/7.4.pdf.

Havens, K.E., Aumen, N.G., James, R.T., and Smith, V.H., 

1996, Rapid ecological changes in a large subtropical 

lake undergoing cultural eutrophication: Ambio, v. 25, 

p. 150–155.

Hutchinson, G.E., 1961, The paradox of the plankton: The 

American Naturalist, v. 95, no. 882, p. 137–145.

Kehr, J.C., and Dittmann, E., 2015, Biosynthesis and function 

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U.S. Geological Survey

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Rosen and others—Effect of Salinity Tolerance on Cyanobacteria Associated with a Harmful Algal Bloom in Lake Okeechobee, Florida—SIR 2018–5092 

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