Samples · Web viewNitrate-Nitrite (dissolved inorganic nitrogen) was measured by cadmium reduction (EPA 353.2 and APHA 2012) using a flow injection analyzer (FIA) with autosampler

Supplement for

Proliferation of Microalgae and Enterococci in the Lake Okeechobee, St. Lucie, and

Loxahatchee Watersheds

E Kelly1,2,3, M Gidley3,5,6, C Sinigalliano3,5, N Kumar7, L Brand3,4, RJ Harris8, HM Solo-Gabriele1,2,3

1University of Miami Leonard and Jayne Abess Center for Ecosystem Science and Policy, Coral Gables, FL, USA

2University of Miami Department of Civil, Architectural and Environmental Engineering, Coral Gables, FL, USA

3NSF NIEHS Oceans and Human Health Center, Rosenstiel School of Marine and Atmospheric Science, University of Miami,

Miami, FL, USA

4University of Miami Department of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science

(RSMAS), Miami, FL, USA,

5National Oceanic and Atmospheric Administration (NOAA) Atlantic Oceanographic and Meteorological Laboratory (AOML)

Environmental Microbiology, Miami, USA

6University of Miami Cooperative Institute for Marine and Atmospheric Studies (CIMAS), Miami, USA

7University of Miami Department of Public Health Sciences, Division of Environment & Public Health, Miami, FL, USA

8Loxahatchee River District, Jupiter, FL, USA

For consideration for potential publication in: Water Research

Version Dated: December 20, 2019

Corresponding author: Helena Solo-Gabriele, Ph.D.e-mail address: [email protected]

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mailto:[email protected]

Supplementary Table S1. GPS coordinates for sites and water control structures

Site Latitude (decimal degrees) Longitude (decimal degrees)Canal Point 26.866056 -80.631639S308 26.984892 -80.621428S80 27.111633 -80.284981Roosevelt Bridge 27.202444 -80.258SE03 27.202778 -80.259167Dubois Park 26.943389 -80.074556

3536

3738

Site Visit Sample Location Latitude Longitude Microcystin(µ/L) FDEP Algal ID8/27/2018 Leighton 27.17 -80.26 2.9 Dominant taxon: Microcystis aeruginosa8/27/2018 A1A bridge 27.20 -80.21 6.7 Dominant taxon: Microcystis aeruginosa8/27/2018 Roosevelt 27.20 -80.26 27.0 Dominant taxon: Microcystis aeruginosa8/27/2018 North Fork 27.21 -80.28 4.8 Dominant taxon: Microcystis aeruginosa8/23/2018 SLE South Fork 27.20 -80.26 14.0 Dominant taxon: Microcystis aeruginosa8/23/2018 SLE N Fork 27.22 -80.28 2.9 Dominant taxon: Microcystis aeruginosa8/23/2018 C44S80 St. Lucie lock 27.11 -80.28 495.1 Dominant taxon: Microcystis aeruginosa8/22/2018 Bathtub Beach 27.19 -80.16 1.2 Dominant taxon: Microcystis sp.8/20/2018 SF near Rustic 27.13 -80.26 16.0 Dominant taxon: Microcystis aeruginosa8/16/2018 SF near Leighton 27.17 -80.26 2.3 Dominant taxon: Microcystis aeruginosa8/14/2018 Lake Okeechobee L004 26.98 -80.71 11.0 Dominant taxon: Microcystis aeruginosa8/14/2018 Lake Okeechobee 27.05 -80.75 2.6 Dominant taxon: Microcystis aeruginosa8/13/2018 S308C Port Mayaca 26.98 -80.62 1.1 Dominant taxon: Microcystis aeruginosa8/9/2018 C44S80 St. Lucie lock 27.11 -80.29 1.0 Dominant taxon: Microcystis aeruginosa8/6/2018 SF near Meridian 27.18 -80.27 4.5 Dominant taxon: Microcystis aeruginosa8/6/2018 NF near Seagate 27.20 -80.27 10.0 Dominant taxon: Microcystis aeruginosa8/2/2018 St. Lucie Canal @ Martin Highway 27.16 -80.25 30.0 Dominant taxon: Microcystis aeruginosa8/2/2018 SF Canal 27.18 -80.26 5.9 mixed algae; no dominant species in sample8/2/2018 SE near Central Marine 27.22 -80.26 110.0 Dominant taxon: Microcystis aeruginosa8/2/2018 SE Warner Creek 27.22 -80.23 3.1 Dominant taxon: Microcystis sp.7/30/2018 S Fork 27.18 -80.27 1.3 mixed algae; no dominant species in sample7/30/2018 St. Lucie Canal 27.14 -80.26 8.6 mixed algae; no dominant species in sample7/26/2018 Outside Frazier 27.20 -80.26 3.2 Dominant taxon: Microcystis aeruginosa7/26/2018 Warner Creek 27.22 -80.23 2.1 Dominant taxon: Microcystis aeruginosa7/26/2018 Roosevelt 27.20 -80.26 1.2 Dominant taxon: Microcystis aeruginosa7/26/2018 C44S80 St. Lucie Locks 27.11 -80.28 3.1 mixed algae; no dominant species in sample7/23/2018 SLR Canal @ de la Bahia 27.17 -80.25 2.5 mixed algae; no dominant species in sample7/19/2018 SLE near Central Marine 27.22 -80.25 8.3 Dominant taxon: Microcystis aeruginosa7/19/2018 St. Lucie Locks, C44S80 27.11 -80.29 4.7 Dominant taxon: Microcystis aeruginosa7/19/2018 Mouth of Warner Creek 27.22 -80.23 6.9 Dominant taxon: Microcystis aeruginosa7/18/2018 LZ30 - Lake Okeechobee 26.80 -80.86 8.8 Dominant taxon: Microcystis aeruginosa7/17/2018 CLV10A inside Lake Okeechobee 26.92 -80.63 2.4 Dominant taxon: Microcystis aeruginosa7/17/2018 L004 26.98 -80.71 3.1 Dominant taxon: Microcystis aeruginosa7/16/2018 Circle Bay Condos 27.18 -80.26 2.8 Dominant taxon: Microcystis aeruginosa7/16/2018 Middle Estuary North Shore 27.21 -80.25 6.1 Dominant taxon: Microcystis aeruginosa7/16/2018 South Fork @ Rustic Circle 27.13 -80.26 3.0 Dominant taxon: Microcystis aeruginosa7/16/2018 SLE near Leighton 27.17 -80.26 5.0 Dominant taxon: Microcystis aeruginosa7/16/2018 S308C 26.98 -80.62 2.8 Dominant taxon: Microcystis aeruginosa7/12/2018 C44S80 27.11 -80.28 15.0 Dominant taxon: Microcystis aeruginosa7/5/2018 Lake Okeechobee NW of Port Mayaca 26.99 -80.67 4.0 mixed algae; no dominant species in sample7/5/2018 Lake Okeechobee SW of Port Mayaca 26.94 -80.65 6.0 mixed algae; no dominant species in sample7/5/2018 C44S80 St. Lucie Lock 27.11 -80.28 154.4 Dominant taxon: Microcystis aeruginosa7/2/2018 SLE near Central Marine 27.22 -80.25 1.9 Dominant taxon: Microcystis aeruginosa7/2/2018 St. Lucie Estuary Poppleton Creek 27.19 -80.26 1.1 Dominant taxon: Microcystis aeruginosa7/2/2018 Lake Okeechobee S308C lakeside 26.98 -80.62 1.9 Dominant taxon: Microcystis aeruginosa6/27/2018 St. Lucie Locks, C44S80 27.11 -80.29 1.3 Dominant taxon: Microcystis aeruginosa6/25/2018 Port Mayaca 26.98 -80.62 2.2 Dominant taxon: Microcystis aeruginosa6/18/2018 Lake_Okeechobee_S352 26.86 -80.63 5.9 Dominant taxon: Microcystis aeruginosa

Supplementary Table S2. Results of surface water samples collected from Palm Beach and Martin Counties during the 2018 cyanobacteria bloom. Table displays all results over the established laboratory detection limits. Samples were collected by FDEP or SFWMD staff and samples analyzed at FDEP laboratory. Data access https://floridadep.gov/dear/algal-bloom/content/algal-bloom-sampling-results

39404142434445

https://floridadep.gov/dear/algal-bloom/content/algal-bloom-sampling-results

SUMMARY OF ANALYTICAL METHODS

1. Chlorophyll a and pheophytin

In this study, both spectrophotometry (e.g., monthly field sampling) and the fluorometric

method (e.g., microcosm experiments) were used to measure chlorophyll a and pheophytin. The

measurement of chlorophyll a is widely used to provide a measure of microalgae biomass during

blooms; confirmatory tests were performed by the Florida Department of Environmental

Protection (FDEP) to determine the presence of cyanobacteria. Chlorophyll a values are

generally reported as their corrected value, which is the value after acidification and are reported

as “chlorophyll a” in μg/L herein. Pheophytin is also reported in μg/L.

The spectrophotometry method was based on the Standard Methods 22nd Edition (APHA

2012), as modified by the Loxahatchee River District (LRD). Samples were filtered on Whatman

0.47 μm glass microfiber filters and preserved with 1 mL of 1% saturated magnesium carbonate

solution within 24 hours of collection and stored frozen. Within 28 days of collection samples

were ground (1 minute tissue grinder) and set in 90% aqueous acetone for approximately 24

hours; the mixture was centrifuged at 4000 rpm for 20 min to separate the ground filter from the

supernatant. The supernatant was analyzed spectrophotometrically (Perkin-Elmer Lambda 365

UV/Visible spectrophotometer), in which the sample was exposed to six different wavelengths of

light, within a range of 480-750 nm. The absorption was recorded for each wavelength. The

samples were acidified and re-exposed to the same six wavelengths of light; the absorbance was

recorded. The lowest practical range of determination with the spectrophotometric method used

was 1.0 µg/L. All samples were processed under dark conditions.

In the fluorometric method, 100 mL (or until the filter clogged) of sample water was

filtered on Whatman 47 mm glass microfiber filters. The filters were placed in vials and frozen,

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to be analyzed within 28 days. For analysis, the filters were transferred to a glass tube and 10 mL

dimethyl sulfoxide (DMSO) was added. The filter and DMSO set for 30 minutes under dark

conditions. Fifteen mL of acetone was then added, agitated, and the mixture was stored in the

freezer for 24 hours. Fluorometry (Turner Designs 10-AU Fluorometer with infrared sensitive

photomultiplier tube, calibrated with pure chlorophyll a) was then used to analyze the mixture

before acidification and after (to produce the degradation product pheophytin). The range can be

adjusted through dilution. All chlorophyll samples were processed under dark room conditions.

2. Enterococci

Enterococci was typically measured (n=11) using the membrane filtration (MF) method

(EPA method 1600; EPA 2009). One exception was made where the sample was analyzed using

chromogenic substrate (APHA 2012). For membrane filtration, 10 mL of sampled water was

filtered through a 0.45 μm sterile glass fiber membrane. The membrane was placed on selective

media (mEI) and incubated at 41ºC for 24 hours. The number of colony forming units (CFU)

present on the membrane were counted and multiplied by a dilution factor of 10 to provide units

of CFU/100 mL (EPA 2009). For chromogenic substrate, one pack of Enterolert chromogenic

media (IDEXX) was added to 100 mL of sample (saline sites were diluted 1:10 as stated in the

IDEXX protocol) and shaken to dissolve all chromogenic media. The mixture was then added to

a 51-Well Quanti-Tray, sealed, and incubated at 41ºC for 24 hours. Results were read under an

ultraviolet light and the number of wells that fluoresced were counted as positive. The number of

fluorescent wells were used to determine the Most Probable Number (MPN) from the MPN table

provided by IDEXX based on the 95% confidence interval; this MPN was multiplied by the

dilution factor to provide units of MPN/100 mL.

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3. Nitrate-Nitrite

Nitrate-Nitrite (dissolved inorganic nitrogen) was measured by cadmium reduction (EPA

353.2 and APHA 2012) using a flow injection analyzer (FIA) with autosampler (Lachat

QuikChem 8500 series) on 0.45 μm field-filtered samples. Samples were processed within 48

hours of collection. An ammonium chloride buffer pH adjusted to 8.5 with EDTA added to

reduce metal interference and sulfanilamide color reagent (10% concentrated phosphoric acid,

sulfanilamide and N-(1-naphthyl) ethylenediamine dihydrochloride) were injected as sample

flowed through a copper-treated cadmium column. Nitrate-nitrite was determined through the

intensity (absorbance) of the pink color produced by the sample according to the concentration;

calculated by a known standard calibration curve (R2 ≥ 0.999; n=6). Samples were read via

spectrophotometer flow cell with an absorbance wavelength of 520nm. Nitrate-nitrite was

measured in units of mg/L. The practical range of the determination was from 0.005 mg/L to 0.3

mg/L.

4. Total Kjeldahl Nitrogen (TKN)

The determination of total Kjeldahl nitrogen (TKN) is based upon the conversion of

organic sources of nitrogen (free ammonia, ammonia nitrogen, amino acids, proteins, and

peptides) to ammonium sulfate (EPA Method 351.2 1993 and APHA 2012). Samples were

preserved to pH < 2 with sulfuric acid and processed within 28 days. Samples were boiled at 200

°C for 2 hours to reduce organic nitrogenous components. Samples were then digested at 380 °C

for one hour in a solution of potassium sulfate, sulfuric acid, and cupric sulfate (copper) as the

catalyst. Measurements were then made using FIA with autosampler (Lachat QuikChem 8500

series) spectrophotometer (absorbance of 660nm), and standard calibration curve methods

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mentioned above. TKN is measured in units of mg/L. The practical range for the determination

was from 0.2 mg/L to 5 mg/L.

5. Total Nitrogen

Total nitrogen in this paper was reported as “nitrogen,” in mg/L. It was not measured

directly but was calculated by adding nitrate-nitrite and TKN.

6. Phosphorus and orthophosphate

Samples were analyzed for total phosphorus by persulfate oxidation digestion (121°C for

30 minutes). This process oxidizes all available forms of phosphate to orthophosphorus, thus

providing a measure of total phosphorus (TP). TP was then determined colorimetrically by

molybdate/ascorbic acid colorizing reagent (APHA 2012 4500-P A and standard method 4500-P

E) and analyzed spectrophotometrically (Perkin-Elmer Lambda 365 UV/Visible

spectrophotometer), with a practical range of 0.005 mg/L to 0.50 mg/L. Samples analyzed for

orthophosphate (dissolved reactive phosphorous) were field-filtered through a 0.45 μm

membrane filter and processed within 48 hours by colorimetric analysis without persulfate

oxidation digestion (SM4500-P F). Ammonium molybdate and antimony potassium tartrate

were combined as a color reagent and a 0.33N ascorbic acid buffer was added to create a blue

color change proportionate to orthophosphate concentration. Measurements were made using

FIA with autosampler (Lachat QuikChem 8500 series), spectrophotometer (absorbance of

880nm), and standard calibration curve methods mentioned above. The practical range of

orthophosphate was from 0.005 mg/l to 0.50 mg/l.

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7. Turbidity

Turbidity was measured in the laboratory within 48 hours using a Hach 2100Q turbidity

meter calibrated using 3 formalin standards (APHA 2012 method D1889-88A and EPA 180.1).

Results were reported in nephelometric turbidity units (NTU). The range of analysis was 0.1-

4000 NTU.

ADDITIONAL DETAILS ON LABORATORY QUALITY CONTROL ANALYSES

1. Quality Control for Nitrogen, Phosphorus, Chlorophyll, turbidity, conductivity, turbidity,

dissolved oxygen, and enterococcus by chromogenic substrate (EnterolertTM).

These parameters were analyzed through the Loxahatchee River District (LRD), a

National Environmental Laboratory Accreditation Program (NELAP) certified

laboratory. This laboratory uses EPA’s standard methods and also Standard Methods for

the Examination of Water and Wastewater, 22nd edition. The specific method of each

analyte and quality control criteria are given in Table S2.

The EPA methods and standard methods both state that the determined concentrations

from each method must be within +/-10% of the control sample (i.e., secondary standard

or the quality control sample). The analytical runs used for all of our samples fell within

this criterion.

The exception is chlorophyll a. As there is no published QC criteria for chlorophyll a,

historical data was used to determine QC acceptance criteria (+/-60%, based on the ability

to detect the true value of the control samples).

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Table S3: Table of Quality Control Acceptance Criteria for Loxahatchee River District Laboratory Activities

Parameter MethodBlank(mg/L)

LOQ (mg/L)

# Cal or Initial Stds.

CalibrationR2 or

%recovery2nd Std.

% recovery

Continuing Cal. Std. %

recovery LCS

Dup. Precision

RPD

Spike Accuracy

% recovery Hold TimeMethod of

Preservation

Enterococcus

Enterolert MPN

1 per lot of Quanti-Tray

10 MPN/100

ml

3 control organisms per lot of Enterolert

Within mfg accept criteria

N/A N/A(4) <51 &

Rlog < 0.361N/A 8 hrs

On ice,Refrigerate

< 6°C

TKNBlock dig.

FIAEPA 351.2

1 pre, (4)

1 post.< LOQ

0.2 6 to bracket sample range ≤ 0.995 90 – 110(1) 90 – 110(1) < 20(4) 90 – 110(3,4) 28 d H2SO4

pH < 2

NO2 + NO2Cd reductionFIA (low)

EPA 353.21 pre, (4)

1 post.< LOQ

0.005 6 to bracket sample range ≤ 0.995 90 – 110(1) 90 – 110(1) < 20(4) 90 – 110(3,4) 48 hrs No pres.

Field filtered

NO2 + NO2Cd reductionFIA (high)

EPA 353.21 pre, (4)

1 post.< LOQ

0.02 6 to bracket sample range ≤ 0.995 90 – 110(1) 90 – 110(1) < 20(4) 90 – 110(3,4) 28 d H2SO4

pH < 2

Ortho-Phoscolorimetric

SM4500-P E1 pre, (4)

1 post.< LOQ

0.005 6 to bracket sample range ≤ 0.995 90 – 110(1) 80 – 120(1) < 20(4) 90 – 110(3,4) 48 hrs No pres.

Field filtered

Total Phosphoruscolorimetric

SM4500-P E1 pre, (4)

1 post.< LOQ

0.005 6 to bracket sample range ≤ 0.995 90 – 110(1) 80 – 120(1) < 20(4) 85 – 115

(3,4) 28 d H2SO4pH < 2

Chlorophyll aSpectrophotometer SM10200H

1 pre, (4)

1 post.< LOQ

1 µg/L 1 in duplicate 40 - 160 N/A 40 - 160 < 30 N/A 28 dGlass filter, freeze

Conductivity,Electronic Meter EPA 120.1 1 pre, (4)

< LOQ2

µmhos/cmMinimum 2,Bracket range 95 – 105 95 – 105(1) 95 - 105(2,4)

+ at end ≤ 5 (4) N/A 48 hrsOn ice,Refrigerate

< 6°C

Turbidityturbidimeter EPA 180.1 1 DI H20(9)

< LOQ0.1 NTU 3 formazin,

quarterly 90 – 110

90 - 110(1)

1 10 NTU Formazin

std.

90 – 1101 10 NTU Formizin every

10 samp + end

≤ 10 (4) N/A 48 hrsOn ice,Refrigerate< 6°C

pHelectronic meter EPA 150.1 N/A N/A 2 – 3,

bracket range

95 – 105% eff.

of probe

±0.2 pH units ±0.2 pH units ≤ 5 N/A Within 15

min None

Dissolved Oxygenelectronic EPA 360.1 N/A N/A

1 initial, water saturated air

±0.5 mg/LConsult chart N/A

95 – 1051 at end,Saturated air

N/A N/A Immediately None

Footnotes for Table of Acceptance Criteria1) Secondary standards verify validity of method or calibration performance; Run after calibration or initial standards but before samples. 2) Laboratory Control Standards are fortified blanks that verify continuing method performance and are prepared in duplicate of the same analyte concentration as the spike. Blank used to prepare LCS must be treated same as sample; i.e. acid preserved, pH adjusted, filtered, etc.3) Matrix spike is typically, but not always the same sample as matrix duplicate. Spike concentration should be less than half of calibration or analytical range. 4) Run every 10 samples or matrix batch.5) Must meet 2.0 mg/L minimum DO depletion (initial – final) and 1.0 mg/L residual (final) DO for each test bottle.6) Dried to a constant weight @ 180C; i.e. weight change < 0.0005g.7) Choose sample volume to yield < 200 mg residue.8) Choose sample volume to yield between 2.5 – 200 mg residue and complete filtration within 10 minutes.9) Run every 20 samples or matrix batch.

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2. Quality Control for Enterococci by Membrane Filtration

The reproducibility of enterococci analysis by membrane filtration (EPA method 1600) was

determined by collecting 6 samples and analyzing them in quadruplicate. For each sample,

the mean, standard deviation, and coefficient of variation (COV) (standard deviation divided

by the mean) was determined. The average value of the COV was determined as 81.2%.

This value was then used to determine the expected standard deviation of a particular sample

by multiplying the enterococci value for the sample by the COV of 81.2%.

3. Quality Control for Chlorophyll a and Pheophytin by Fluorometry.

The reproducibility of chlorophyll a and the pheophytin measurements by fluorometry (used

for microcosm experiments only) was determined through the analysis of 6 samples analyzed

in quintuplicate as documented by Brand (2002). Computations were completed in a similar

fashion as for enterococci by membrane filtration. The average value of the COV was

determined to be 83.7%. This value was then used to determine the expected standard

devation of a particular sample by multiplying the chlorophyll a value for the sample by the

COV of 83.7%.

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Sample D_10 D_30 D_60

Uniformity Coefficient

Coefficient of Gradation

Canal Point (incubator) 2.90 4.83 7.35 2.53 1.09Canal Point (algae room) 2.98 5.17 8.19 2.75 1.10Canal Point (microcosm 4) 4.90 6.78 9.77 1.99 0.96Dubois Park (microcosm 4) 0.18 0.28 0.41 2.24 1.00

Supplementary Table S4. Grain size analysis. All of the samples were poorly/uniformly graded, which means that they were distributed within a narrow size range. The three samples from Canal Point can be classified as gravels and the Dubois Park sample can be classified as sand, based on the Unified Soil Classification System (USCS).

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Variable Roosevelt Bridge R2

Dubois Park R2

Rainfall 24 Hrs Before

0.001 0.002

Rainfall 3 Days Before

0.04 0.002

Rainfall Week Before

0.04 0.001

Air Temperature

0.001 NS

Water Temp 0.03 NSSupplementary Table S5. Result of linear regression for temperature and rainfall (quantitative variables) versus enterococci levels at Roosevelt Bridge and Dubois Park, 2000-2018. NS corresponds to correlations that were not significant.

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Parameter Canal Point Roosevelt Bridge Dubois ParkChlorophyll (μg/L) 33.27 9.44 3.06

Pheophytin (μg/L) 28.23 11.85 2.62

ENT (CFU/100 mL) 526 232 84

Nitrate-Nitrite mg/L 0.41 0.19 0.02

TKN mg/L 2.15 1.05 0.36

TN mg/L 2.55 1.21 0.37

Orthophosphorus mg/L 0.10 0.11 0.01

Total Phosphorus mg/L 0.22 0.19 0.03

Sampling Depth (m) 0.24 0.51 1.00

Water Temperature (ºC) 26.29 24.76 25.03

pH 8.54 7.79 7.88

Salinity ppt 0.18 10.24 33.77

DO (mg/L) 7.00 6.46 4.84

Turbidity (ntu) 171.4 31.5 6.4

Air Temperature (F) 78.58 78.17 73.92

Wind (mph) 6.5 7.6 8.3

Humidity (%) 61.6 68.8 76.2

Supplementary Table S6. Monthly sampling means for chlorophyll, enterococci, nutrients, and basic physical-chemical and meteorological data.

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Site Month

Enterococci per 100 mL (CFU) or MPN*

Nitrite-

Nitrate

(mg/L)

TKN (mg-N/L)

TN (mg/L)

Orthophosphorus (mg/L)

Phosphorus (mg/L)

Chlorophyll a ug/L

(corrected)

Pheophytin (ug/L)

Canal Point

September 18 0.39 1.70 2.09 not analyzed 0.209 121.7 5.8

October 60 0.46 1.29 1.75 not analyzed 0.168 8.0 5.6November 336 0.47 2.82 3.29 not analyzed 0.260 17.4 22.5

December 104* 0.70 unavailab

le unavailab

le 0.072 0.254 11.2 11.2

January 30/<10* 0.69 2.60 3.29 not analyzed 0.170 9.6 21.5February 400 0.60 1.70 2.30 0.091 0.277 7.6 11.2March 1000 0.58 4.80 5.38 0.423 0.423 17.3 26.3April 10 0.44 2.30 2.74 0.239 0.239 46.9 51.9May 1650 0.19 1.40 1.59 0.121 0.121 6.3 10.7

June 1390 0.28 2.20 2.48 0.103 0.103 99.7 105.2July 1110 0.06 2.00 2.06 0.100 0.367 35.1 45.5August 200 0.03 3.00 3.65 0.075 0.08 18.4 21.4

Roosevelt Bridge


October 100 0.35 1.56 1.91 not analyzed 0.249 6.4 5.6November 112 0.39 1.68 2.07 not analyzed 0.192 4.5 4.5


le unavailab

le 0.101 0.168 4.4 2.8

January 20 /10* ≤0.02 0.70 0.70 not analyzed 0.118 21.4 31.4February ≤10 ≤0.02 0.50 0.50 0.064 0.089 4.8 6.7March 30 0.01 0.70 0.71 0.095 0.130 6.9 8.3April 10 0.02 0.40 0.42 0.103 0.149 4.1 5.7May 100 0.03 0.40 0.43 0.128 0.138 3.9 5.0June 1020 0.32 1.50 1.82 0.173 0.295 15.2 18.8July 860 0.08 1.50 1.58 0.127 0.253 21 24.0August 390 0.08 1.20 1.28 0.122 0.220 17.1 23.0

Dubois Park


October 10 0.04 0.55 0.59 not analyzed 0.047 10.1 3.0November 23 0.05 0.71 0.76 not analyzed 0.044 3 1.8


le unavailab

le 0.023 0.033 2.7 1.4

January 10 /≤10* 0.03 0.40 0.43 not analyzed 0.038 3 5.3February 40 0.02 ≤0.2 ≤0.2 0.009 0.015 2.2 2.8March 200 ≤0.005 0.50 0.50 ≤0.005 0.014 1.1 1.4April 30 0.01 ≤0.2 ≤0.2 ≤0.005 0.021 2 2.7May 330 0.01 ≤0.2 ≤0.2 ≤0.005 0.007 2.6 3.4June 110 0.01 0.30 0.31 0.013 0.015 1.7 2.1July 170 0.01 0.50 0.51 0.018 0.023 2.5 3.2August 30 0.03 ≤ 0.2 ≤ 0.234 ≤ 0.005 0.010 1.3 1.9

*=Most Probable Number (MPN).

Supplementary Table S7. Monthly sampling data for cyanobacteria, enterococci, and nutrients.

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230231

232233

Site monthTime of

collectionSample

Depth (m)Temperature

(˚C) pHTide or wave

height

Salinity (ppt) Dissolved Oxygen

(mg/L)

Turbidity from lab analysis

(NTU)

Canal Point

September 4:58 PM 0.60 34.6 9.2 >1 foot 0.22 unavailable 49.8 October 5:39 PM 0.32 31.1 8.0 >1 foot 0.19 5.29 62.8November 2:20 PM 0.30 24.5 8.4 2-3 feet 0.18 6.50 304December 12:55 PM 0.26 18.8 8.2 1-2 feet 0.18 8.61 516January 12:42 PM 0.15 13.2 8.1 1 foot 0.13 9.02 254February 12:48 PM 0.21 19.6 8.8 >1 foot 0.17 8.10 149March 1:14 PM 0.17 20.8 8.6 1-2 feet 0.18 8.73 384April 1:06 PM 0.06 30.0 8.9 >1 foot 0.18 8.93 70.5May 12:00 noon 0.09 27.6 8.3 >1 foot 0.19 8.02 70.7June 12:20 PM 0.26 31.7 8.6 1 foot 0.17 7.60 74.4July 12:39 PM 0.20 30.5 8.6 1-2 feet 0.18 6.78 81.4August 11:51 AM 0.21 33.1 8.8 1-2 feet 0.16 6.41 40.7

Roosevelt Bridge

September 2:04 PM 0.51 28.8 7.6 high 2.28 unavailable 47.2 October 4:01 PM 0.58 28.2 7.8 Flood tide 0.20 5.44 91.3November 12:26 PM 0.82 24.2 7.9 Flood tide 0.18 6.00 71.9December 11:02 AM 0.57 18.6 7.7 Low 2.20 7.46 28.7January 10:58 AM 0.36 15.5 8.2 Flood tide 12.40 10.54 15February 11:04 AM 0.34 21.6 8.4 Flood tide 17.55 8.23 8.52March 11:31 AM 0.57 19.7 7.8 Ebb tide 26.46 7.10 9.2April 11:14 AM 0.38 26.8 7.7 Ebb tide 27.29 5.33 7.2May 10:13 AM 0.59 25.4 7.3 Ebb tide 26.26 5.07 7.2June 10:25 AM 0.56 28.1 7.5 Ebb tide 1.89 5.30 50.7July 10:50 AM 0.57 30.4 7.8 Ebb tide 3.26 5.42 18.4August 10:09 AM 0.30 29.8 7.8 Low 2.87 5.22 22.1

Dubois Park

September 12:15 PM 0.51 29.4 8.0 high 35.10 unavailable 14.5 October 2:31 PM 0.97 29.6 7.8 Flood tide 24.30 4.90 4.59November 10:49 AM 1.10 25.0 7.8 Ebb tide 27.60 4.80 8.84December 9:31 AM 0.91 19.8 7.6 Flood tide 32.90 5.49 5.9January 9:22 AM 0.93 16.9 7.8 Flood tide 26.90 7.50 22.4February 9:29 AM 1.03 22.4 8.4 Low 36.86 6.45 4.83March 9:45 AM 1.27 21.6 7.9 Ebb tide 38.32 5.72 4.2April 9:35 AM 0.79 26.1 7.9 Ebb tide 37.65 4.86 3.9May 8:50 AM 1.19 25.7 7.5 high 37.90 5.16 1.8June 8:50 AM 1.38 26.9 7.9 Ebb tide 35.54 4.50 1.7July 9:08 AM 1.14 29.5 7.9 Ebb tide 34.47 4.01 3.6August 8:49 AM 0.8 27.4 8.0 Flood tide 37.67 4.73 0.6

Supplementary Table S8. Monthly sampling means for basic physical-chemical data

234235236237238239240241242243244

245246247248

Supplementary Figure S1. Microcosm experiment setups. CP=Canal Point, RB=Roosevelt Bridge, DP=Dubois Park.

unfiltered

filteredDPRBB

CP

Water only Algae room conditions 19ºC with

alternating light and dark cycles

DPRBB

CP

Microcosm Experiment 1

CP/DI2:1

DP/DI2:1

CP/DP2:1

CP/DI2:1

Water only Incubator conditions 41ºC dark

DP/DI2:1

CP/DP2:1


Water and sediment Algae room conditions 19ºC with


CP

DPCP

CP


CPCP

Water and sediment Incubator conditions 41ºC dark

Water and sediment Algae room conditions 19ºC with


CPCP


Water only Algae room conditions 19ºC with


249250251252253254255

257258259260261262263264265266267268269270271272273274275276277278279

Supplementary Figure S2. Microcosm experiment 1: Water from all sites, chlorophyll and enterococci measured every 12 hours.

0 12 24 360

5

10

15

20

25

30

35

0

0.5

1

1.5

2

2.5

3

3.5

4

Hours

Chlo

roph

yll µ

g/L

Ente

roco

cci (

norm

alize

d)

Unfiltered FilteredSymbol Water Symbol Water

Chlorophyll a Lake Okeechobee Lake Okeechobee

Roosevelt Bridge Roosevelt BridgeDubois Park Dubois Park

Enterococci Lake Okeechobee Lake OkeechobeeRoosevelt Bridge Roosevelt Bridge

Dubois Park Dubois Park

280281282283284285

287288289

290291292293294295

0 12 24 360

2

4

6

8

10

12

14

0

0.2

0.4

0.6

0.8

1

1.2

Hours

Chlo

roph

yll a

µg/

L

Ente

roco

cci (

norm

alize

d)

Incubator Algae Culture RoomSymbol Dilution 1 Dilution 2 Symbol Dilution 1 Dilution 2

Chlorophyll a Lake Okeechobee

Deionized Water (DI)

Lake Okeechobee

DI

Dubois Park DI Dubois Park DI

Lake Okeechobee

Dubois Park Lake Okeechobee

Dubois Park

Enterococci Lake Okeechobee

DI Lake Okeechobee

DI

Not included in analysis

Dubois Park DI Dubois Park DI

Lake Okeechobee

Dubois Park Lake Okeechobee

Dubois Park

Supplementary Figure S3. Microcosm experiment 2: Water from Dubois Park and Lake Okeechobee, chlorophyll a and enterococci measured every 12 hours. Microcosms are 50:50 dilutions of Dilution 1 and Dilution 2 per table legend. Sample size n=1 per treatment.

296

297298299300301

References

American Public Health Association (APHA), American Water Works Association, Water Environment Federation. 2012. Standard Methods for the Examination of Water and Wastewater, 22nd Edition. E.W. Rice, R.B. Baird, A.D. Eaton, L.S. Clesceri, editors. da.

EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI) December 2009 https://nepis.epa.gov Viewed 1/22/18

EPA Methods for the Determination of Inorganic Substances in Environmental Samples, EPA/600/R-09/100, August 1993 https://nepis.epa.gov Viewed 1/22/18

EPA Methods for the Determination of Inorganic Substances in Environmental Samples, EPA/600/R-09/100, Method 353.2: “Nitrogen, Nitrate-Nitrite” 1993 https://nepis.epa.gov Viewed 1/22/18

Florida Department of Environmental Protection Algal Bloom Sampling Results 2018 https://floridadep.gov/dear/algal-bloom/content/algal-bloom-sampling-results Viewed 3/16/19

IDEXX Laboratories, Inc., 2015, Enterolert and Quanti-Tray Technical Documents https://www.idexx.com/en/water/water-products-services/enterolert/ Viewed 11/13/18

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