Cyanobacterial Blooms: Toxins, Tastes, and Odors
USGS Kansas Water Science Center Algal Toxin Team Jennifer L. Graham, Keith A. Loftin,
Michael T. Meyer, and Andrew C. Ziegler
USDA-CSREES National Water ConferenceFebruary 4, 2008
Overview
• Cyanobacterial (Blue-Green Algal) Toxins and Taste-and-Odor Compounds
• Microcystin in the Midwest
• Research Needs
• USGS Studies
Cyanobacterial toxins are “…anthropogenically amplified, but basically a natural phenomenon…”
I. Chorus, 1993
Binder Lake, IA Aug 2006
Thomas Lake, NE May 2006
• Ecologic Concerns– Zooplankton avoidance or death– Accumulation by mussels– Fish kills– Losses to bird and mammal populations
• Economic Concerns– Added drinking water treatment costs– Loss of recreational revenue– Death of livestock and domestic animals– Medical expenses
• Health Concerns– Tastes-and-Odors
• Olfactory sensitivity at low concentrations (< 0.01 µg/L) • Chronic effects?
– Toxins• Human and animal illness and death• EPA contaminant candidate list• Drinking water - microcystin
– WHO guideline – 1.0 µg/L– Drinking-water treatment processes effectively remove most toxins
• Recreational water – WHO guidelines for microcystin– Low Risk - < 10 µg/L– Moderate Risk - 10-20 µg/L– High Risk - > 20 µg/L
• Known chronic effects
Toxins and Taste-and-Odor Compounds Produced by Cyanobacteria
Dermatoxins Hepatotoxins Neurotoxins Taste/OdorCYL MC ANA BMAA GEOS MIB
Colonial/Filamentous
Aphanizomenon X X X X X X
Anabaena X X X X X X ?
Cylindrospermopsis X X X
Microcystis X X X
Oscillatoria/Planktothrix X X X X X X
Unicellular
Synechococcus X X X X X
Synechocystis X X X
Cyanobacterial Toxins and Taste-and-Odor Compounds Are Not Produced By The Same Biochemical Pathway But Patterns in Distribution Are Similar
• Extreme spatiotemporal variability
• Lack of relation with cyanobacterial community composition or chlorophyll concentration
• Coupling with lake/river processes as influenced by physiochemical, biological, hydrological, and meteorological factors
Upper Gar, IA Aug 2006
Upper Pine, IA August 2006
Algal Toxins
SAX
ATX-A(s)
MC-LR, L
A, -YRATX-A
MC-RR
CYL (24
h)
CYL (5d
)
LD50
(g/
kg)
0100200300400500600
20002100
Cyanotoxins Exhibit a Wide Range of Toxicities and Toxic Effects and Are Currently Listed on the U.S. EPA Contaminant Candidate List
• Acute Toxicity– Neurotoxic– Hepatotoxic– Dermatoxic
• Chronic Toxicity– Carcinogen– Tumor Promotion– Mutagen– Teratogen– Embryolethality
Toxi
city
After Chorus and Bartram, 1999
Cyanobacteria Made the News in at Least 21 U.S. States During 2006
= news report
After Graham, 2006 USGS FS-2006-3147
At Least 35 U.S. States have Anecdotal Reports of Human or Animal Poisonings Associated with Cyanotoxins
= reported incident
During 1999-2006 Microcystin was Detected in INTEGRATED PHOTIC ZONE Samples from 78% of Lakes (n=359) and TOTAL Concentrations Ranged from <0.1 to 52 µg/L
After Graham and others 2004 and 2006
Trophic Gradient
Mean and Maximum TOTAL Microcystin Concentrations Significantly Increased Along the Natural Trophic Gradient in the Study Region
OH OP DT WL
Mic
rocy
stin
(g/
L)
0
1
2
3
4
530405060
a
a, b
b
cn=2546p<0.01
After Graham and others 2004 and 2006
maxima
a, b, and c indicate significant differencesin mean concentration
80% of All Lakes Sampled During 1999-2006 Had Maximum TOTAL Microcystin Concentrations ≤ 1 µg/L in Open Water Samples
After Graham and others 2004 and 2006
Microcystin Concentration (g/L)ND 0-1 1-5 5-10 10-20 20-60
% O
ccur
renc
e
0
20
40
60
80
100 Total n=355
61% of Lakes Sampled During 3-6 Years Always Had Detectable Microcystin During Summer, and Microcystin Maxima Were Greatest in These Lakes
Max
mim
um M
icro
cyst
in
Con
cent
ratio
n (
g/L)
Always Detected
OccassionallyDetected
n=101
n=65
100
1
0.1
0.01
0.001
10
After Graham and others 2004 and 2006
Seasonal Patterns in Microcystin Concentration are Unique to Individual Lakes and Peaks May Occur Anytime Throughout the Year
Marceline 1, MO
Mic
rocy
stin
(g/
L)
510152025
Bilby Ranch, MO
1
2
Harrison, MO
1
2
Mozingo, MO
1
2
Forest, MO
1
2
Sterling Price, MO
2004J F M A M J J A S O N D
1
2
Forest Lake, MO
Jan
Feb Mar
Apr
May
Jun
Jul
Aug Sep
Oct
Nov
Dec
Jan
Mic
rocy
stin
(g/
L)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Peak Microcystin Values May Occur in the Winter
Oscillatoria
2004
Seasonal Patterns Were Relatively Consistent Between Years in Some Lakes
Mozingo Lake, MO
Jan
Feb
Mar
Apr
May
Jun Ju
l Au
g Se
p Oct Nov
Dec
Jan
Mic
rocy
stin
(g/
L)
0.0
0.5
1.0
1.5
2.0
2.5
3.0 20042001
After Graham and Jones, 2006
Regionally, Microcystin Was Significantly Correlated With Factors That Affect Cyanobacterial Growth
Variable rs p-value n
Latitude 0.66 <0.01 800
Total Nitrogen (TN) 0.58 <0.01 795
Total Phosphorus (TP) 0.46 <0.01 795
Secchi -0.27 <0.01 796
pH 0.17 <0.01 507
Alkalinity 0.15 <0.01 432
TN:TP -0.15 <0.01 791
After Graham and others 2004
Regional Associations Between Microcystin and Environmental Variables Were Complex
TN:TP
010
020
030
040
050
0
TP (g/ L)
020
040
060
080
010
00
TN (m/ L)
020
0040
0060
0080
0010
00012
00014
00016
00018
000
Mic
rocy
stin
(g/
L)
pH2 4 6 8 10 12
Secchi (m)0 2 4 6 8 10
Alkalinity (mg/ L)0 50 10
015
020
025
030
035
0
r2=0.84
r2=0.70
r2=0.38 r2=0.99
r2=0.72 r2=0.94
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
After Graham and others, 2004
Microcystin Was Not Strongly Correlated With Measures of the Cyanobacterial Community
After Graham and others, 2004
Biovolume of Potential Microcystin Producers (m3/ L)
1e+5
1e+6
1e+7
1e+8
1e+9
1e+10
1e+11
1e+12
1e+13
Mic
rocy
stin
(g/
L)
0
1
2
3
4
5
6 r=0.31p<0.01
r2=0.96 r2=0.90 r2=0.83
Mozingo Lake, MO - Summer 2001
Log10 Dissolved Nitrogen2.7
02.7
52.8
02.8
52.9
02.9
53.0
0
Log 1
0 Mic
rocy
stin
1.0
1.5
2.0
2.5
3.0
3.5
Log10 Total Cations1.6
01.6
51.7
01.7
5
Log 1
0 Mic
rocy
stin
1.0
1.5
2.0
2.5
3.0
3.5
Log10 Chlorophyll>35 m0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Log 1
0 Mic
rocy
stin
1.0
1.5
2.0
2.5
3.0
3.5
Individual Lake Correlations Between Microcystin and Environmental Variables Were Linear
After Graham and others, 2006
Seasonal Patterns in Individual Lakes are Coupled with Seasonal Lake Processes, Including Stratification and Nutrient Loss from the Epilimnion
Epilimnion of Mozingo Lake, MO - Summer 2001
May June July Aug Sept
Diss
olve
d Ni
trog
en (
g/L)
500
550
600
650
700
750
800
850
900
Mic
rocy
stin
(ng/
L)
0100200300400500600700800900
1000
Net C
hlor
ophy
ll ( g
/L)
0
5
10
15
20
25
30
35
Tota
l Cat
ions
(mg/
L)
3840424446485052545658
NetChlorophyll
Microcystin
Nitrogen
Cations
After Graham and others, 2006
Factors Most Strongly Correlated With Microcystin Vary Among Lakes and Years
Mozingo Lake, MO
Chlorophyll > 35 m (g/ L)0 10 20 30 40 50
Mic
rocy
stin
(g/
L)
0.00.20.40.60.81.01.21.41.61.8 r2=0.30
p<0.01
Forest Lake, MO
Chlorophyll > 35 m (g/ L)0 2 4 6 8 10 12
Mic
rocy
stin
(g/
L)
0.0
0.1
0.2
0.3
0.4
0.5
0.6 r2=0.90p<0.01
Marceline 1, MO
Chlorophyll > 35 m (g/ L)0 10 20 30 40 50 60
Mic
rocy
stin
(g/
L)
0
5
10
15
20
25 r2=0.17p<0.01
Microcystin in Midwestern Lakes - Conclusions
• Microcystin is common in the Midwest and may reach levels that can cause health concerns
• Seasonal patterns in microcystin are unique to individual lakes and maxima may occur in any season
• Regional relations between microcystin and environmental variables are complex
• Microcystin and environmental variables may be tightly coupled in individual lakes, but relations vary among lakes and years
Binder Lake, IA Aug 2006
Elysian Lake, MN Aug 2006
Research Needs
• Certified Standards
• Consistent Sampling Protocols
• Robust and Quantitative Analytical Methods for a Variety of Toxins
• Distribution of Microcystin Variants and Other Cyanobacterial Toxins
• Long Term Studies to Identify the Key Environmental Factors Leading to Toxic/Taste-and-Odor Producing Blooms
• Methods for Early Detection
• Predictive Models
Cheney Reservoir, KS June 2003Photo Courtesy of KDHE
Thomas Lake, NE May 2006Photo from Omaha NBC News
Consistent Sampling Protocols – Sample Location is Important
After Chorus and Bartram, 1999
Concentrations of Toxins and Taste-and-Odor Compounds May Vary by Orders of Magnitude at Different Sample Locations Within a Lake
Cheney Reservoir, KS September 2006
Microcystin: 13 µg/LGeosmin: 0.25 µg/L
Microcystin: 4 µg/LGeosmin: Not Detected
Actinomycetes Bacteria Also Produce Geosmin and MIB and May Contribute to Taste-and-Odor Problems in Drinking Water Supplies
Consistent Sampling Protocols – Collection Technique is Important
ToxinToxin
IntracellularToxin
DissolvedToxin
SorbedToxin
IntracellularToxin
DissolvedToxin
SorbedToxin
Total Toxin Dissolved Phase Toxin
= +
Particulate Toxin
Plankton Net Sampling Whole Water Sampling Filter/Filtrate Sampling
Microcystin Concentrations Decreased with Decreases in Cyanobacterial Size Class
Size Class (m)> 100 53-100 35-53 10-35 1-10
Mic
rocy
stin
(g/
L)
0.0
0.2
0.4
0.6
0.8
1.0a
a, b
bbb
n=24
Graham and Jones, 2007
Letters indicate significant differencesin mean concentration
Net Size (m)
% U
nder
estim
ate
of
Tota
l Mic
rocy
stin
0
20
40
60
80
100
100 53 35 10
n=24
Use of Plankton Nets Consistently Underestimated Microcystin Concentrations Relative to Whole Water Samples
Graham and Jones, 2007
There are Currently Over 80 Known Microcystin Variants
From McKinnon, 2003
Analytical Methods for Cyanotoxins - Bioassays
BioassaysEnzyme-linkedimmunosorbent assays(ELISA) - Microcystins/Nodularin - Cylindrospermopsins - SaxitoxinsInhibition Assays - Protein Phosphatase Inhibition (Microcystins/Nodularin)Radioassays - Neurotoxicity (Anatoxins/
Saxitoxins)
AdvantagesEasy to UseRapidInexpensiveUseful screening toolsMay indicate toxicity
DisadvantagesCross-reactivityMatrix effectsSemi-quantitativeRadioassays use radio-
labeled isotopes
Analytical Methods for Cyanotoxins – Gas Chromatography
AdvantagesSpecificityIntermediate costQuantitative
DisadvantagesAvailability of analyticalstandards
Derivitization likely required
Not all compounds areamenable to derivitization
GC-FID requires furtherconfirmation
Sample concentrating maybe necessary
Gas Chromotography (GC)Flame ionization detector (FID)Mass spectrometry (MS)
Analytical Methods for Cyanotoxins – Liquid Chromatography
AdvantagesSpecificity
Derivitization not typicallynecessary
Many toxins amenable toLC techniques
Multi-analyte methodsare cost-effective
TOFMS good fordetermining unknowns(not quantitative)
DisadvantagesAvailability of analyticalstandards
Matrix effects
Expensive
Sample concentrating maybe necessary
Spectroscopic techniquesmay require furtherconfirmation
Liquid Chromotography (LC)UV-Visible (UV-Vis)FluorescenceMass spectrometry (MS)Tandem MS (MS/MS)Ion trap MS (ITMS)Time of flight MS(TOFMS)
Robust and Quantitative Analytical Methods - Capabilities of the USGS Organic Geochemistry Research Laboratory
http://ks.water.usgs.gov/Kansas/researchlab.html
Geosmin and MIB MRL: 5 ppt
Algal Toxin AnalysisLC/MS/MS Chromatogram
Pea
k In
tens
ity
Elution Time - Minutes
CYL
ATX
MC-RR
MC-LRMC-YR MC-LW
MC-LF
MC-LA
Toxin MRL’s: ~25 ppt
Robust and Quantitative Analytical Methods - Capabilities of the USGS Organic Geochemistry Research Laboratory
http://ks.water.usgs.gov/Kansas/researchlab.html
Geosmin and MIB MRL: 5 ppt
Algal Toxin AnalysisLC/MS/MS Chromatogram
Pea
k In
tens
ity
Elution Time - Minutes
CYL
ATX
MC-RR
MC-LRMC-YR MC-LW
MC-LF
MC-LA
Toxin MRL’s: ~25 ppt
MC-LYDeoxycylindrospermopsinLyngbyatoxin-aNodularin-R
Total Microcystin Comparison – ADDA Specific ELISA vs LC/MS/MS for –LR, -RR, -LY, -YR, -LA, -LW, and –LF variants
Total Microcystin by ELISA (g/L)
0 10 20 30 40 50
Tota
l Mic
rocy
stin
by
LC
/MS/
MS
(g/
L)
0
50
100
150
200
250r2=0.36n=22
ELISA (ADDA) can be a useful tool in conjunction with LC/MS/MS
Total Microcystin by ELISA (g/L)
0 10 20 30 40 50
Tota
l Mic
rocy
stin
by
LC
/MS/
MS
(g/
L)
0
50
100
150
200
250r2=0.94n=18
Implies microcystin or microcystin-like congeners were measured by ELISA, but not LC/MS/MS
Distribution of Microcystin Variants and Other Cyanobacterial Toxins – August 2006 Midwestern Cyanotoxin Lake and Reservoir Reconnaissance
• Objectives:– Characterize occurrence and co-occurrence of taste and odor compounds and
cyanotoxins– Determine the specific toxins by LC/MS/MS
• Design:– States: IA, KS, MN, MO (23 Lakes and Reservoirs)– Targeted Sampling: Blooms and Scums– Analyses:
• Taste and Odor – SPME GC/MS• Toxins – TOTAL and Dissolved Concentrations
– ELISA – Microcystins (ADDA), Microcystin LR, Cylindrospermopsins, Saxitoxins– LC/MS/MS – 7 microcystins (LR, RR, YR, LW, LA, LF, LY), Nodularin, Anatoxin-
a, Cylindrospermopsin, Deoxycylindrospermopsin, Lyngbyatoxin a • Water Chemistry• Chlorophyll• Phytoplankton
During August 2006 100% of BLOOMS Sampled (n=23) Had Detectable Microcystin, 83% Had Detectable Geosmin, and 26% Had Detectable Anatoxin
TOTAL Microcystin Maxima (12,500 – 18,030 µg/L) in BLOOM Samples Were Orders of Magnitude Greater Than Maxima for Other Compounds
(Anatoxin Maxima = 13 µg/L, All Other Maxmima < 1 µg/L)
Microcy
stin (E
LISA)
Microcy
stin (L
C/MS/M
S)
Geosm
in MIB
Anatoxin
Saxito
xin
Cylindro
sperm
opsin
Nodularin
Con
cent
ratio
n (
g/L)
0255075
100125150175200
12000
15000
18000
During August 2006 Toxins and Taste-and-Odor Compounds Co-Occurred in 87% of BLOOMS Sampled (n=23) and Anatoxin-a Always Co-Occurred with Geosmin
During August 2006 Toxins and Taste-and-Odor Compounds Co-Occurred in 87% of BLOOMS Sampled (n=23) and Anatoxin-a Always Co-Occurred with Geosmin
“Algae may make for stinky water, but it poses no health risks” -Concord Monitor, Concord, NH July 7, 2006
Although Toxins and Taste-and-Odor Compounds Frequently Co-Occurred Concentrations Were Not Linearly Related
Microcystin by ELISA (g/L)0 10 20 30 40 50
Microcystin by LC/MS/MS (g/L)0 50 100 150 200
Geo
smin
(g/
L)
0.0
0.2
0.4
0.6
0.8
1.0r2=0.04p=0.34n=22
r2<0.01p=0.67n=22
Cyanobacterial BLOOM with TOTAL Microcystin = 0.6 μg/L, Anatoxin = 0.1 μg/L , and Geosmin = 0.02 μg/L
Cyanobacterial BLOOM with TOTAL Microcystin = 12.3 μg/L, Nodularin = 0.1 µg/L, Geosmin = 0.02 μg/L, and MIB = 0.06 μg/L
Cyanobacterial BLOOM with TOTAL Microcystin = 18,000 μg/L, Cylindrospermopsin = 0.12 μg/L Saxitoxin = 0.04 μg/L, and Geosmin = 0.69 μg/L
Microcystin-LR and –RR Were the Most Common Microcystin Variants, and 41% of Lakes Had All 7 Measured Variants Present
Microcystin-LR and –RR Comprised the Majority of TOTAL Microcystin Concentrations
IA1
IA2
IA3
IA4
IA5
IA6
IA7
IA8
IA9KS1
KS2KS3
KS4KS5
MN1Mn2
MN3MN4
MN5MN6
MO1MO2
Tota
l Mic
rocy
stin
( g
/L)
02000400060008000
100001200014000160001800020000
LR RR LY YR LA LW LF
IA2
IA3
IA4
IA5
IA6
IA7
IA8
IA9KS1
KS2KS3
KS4KS5
MN1Mn2
MN3MN4
MN5MN6
MO1MO2
0
50
100
150
200
250
IA3
IA4
IA5
IA6
IA7
IA8
IA9KS1
KS2KS3
KS4KS5
MN1Mn2
MN3MN4
MN5MN6
MO1MO2
0
10
20
30
40
50
2006 Texas Reservoir Survey for DISSOLVED Microcystin in Surface Samples at OPEN WATER Locations
Results:
28% of reservoirs (n=36) haddetectable microcystin by ELISA
Maximum DISSOLVEDMicrocystin concentrations: < 1 µg/L
69% of reservoirs had detectable MIB
30% of reservoirs had detectableGeosmin
After Kiesling and others, in prep
Microcystins and Taste-and-Odor Compounds Frequently Co-Occurred in Texas Reservoirs
After Kiesling and others, in prep
2007 US EPA National Lake Assessment: ~1200 Lakes and ReservoirsTOTAL Microcystin in Integrated Photic Zone Samples
Preliminary Results:
33% of samples (n=711) haddetectable microcystin by ELISA
Mean TOTAL microcystin concentration: 0.97 µg/L
Maximum TOTAL microcystinconcentration: 74 µg/L
Sample Location and Type are Important
Study Sample LocationSample
Type n% Samples
with MCMaximum MC
(µg/L)
Graham and others 1999-2006
Open Water, Integrated Photic
Total 2546 39 52
Midwest Recon2006
Targeted Blooms, Bloom Grab
Total 23 96 12,500
Texas Recon2006
Open Water, Surface Grab
Dissolved 67 22 0.2
EPA NLA2007
Open Water, Integrated Photic
Total 711 33 74
Microcystin was measured by ELISA in all studies
Long Term Studies – Assessment of Water Quality in the North Fork Ninnescah River and Cheney Reservoir, 1997-Present
• Concerns– Taste-and-odor occurrences related to algal blooms
– Relation between watershed inputs and taste-and-odor causing algae
• Approach– Describe current and historical loading inflow
• Sediment Cores• Continuous Water-Quality Monitoring
– Describe physical, chemical, and biological processes associated with cyanobacteria and cyanobacterial by-products
• Discrete Samples• Real-Time Monitors
Cheney Reservoir, KS June 2003Photo Courtesy of KDHE
North Fork Ninnescah RiverMarch 2006
http://ks.water.usgs.gov/Kansas/studies/qw/cheney/
Early Detection and Predictive Models – Continuous Real-Time Water-Quality Monitors
• Real-Time Variables– Specific conductance, pH,
temperature, turbidity, dissolved oxygen
– Chlorophyll– Light– Blue-green algae– Nitrate
The J. W. Powell USGS Monitoring Station on Lake Houston, Texas
Station Developed by Michael J. Turco, Timothy D. Oden, William H. Asquith, Jeffery W. East, and Michael R. Burnich
http://waterdata.usgs.gov/tx/nwis/
Continuous Monitoring Allows the Identification and Description of Events that Occur Within Relatively Short Periods of Time
http://waterdata.usgs.gov/tx/nwis/
http://ks.water.usgs.gov/Kansas/rtqw/index.shtmlEstimated Geosmin Concentration 2003
Geosmin
ElevationEs
timat
ed G
eosm
in
Con
cent
ratio
n (μ
g/L)
Early Detection - Geosmin Concentrations in Cheney Reservoir Frequently Exceed the Human Detection Limit of 10 ng/L
log10(Geo) = 7.2310 - 1.0664 log10(Turb) - 0.0097 SCr2=0.71
After Christensen and others, 2006 USGS SIR 2006-5095 http://ks.water.usgs.gov/Kansas/rtqw/index.shtml
Jennifer Graham [email protected] (785) 832-3511
Additional Information Available on the Web:
Cyanobacteria - http://ks.water.usgs.gov/Kansas/studies/qw/cyanobacteriaCheney - http://ks.water.usgs.gov/Kansas/studies/qw/cheneyOlathe - http://ks.water.usgs.gov/Kansas/studies/qw/olatheRTQW - http://ks.water.usgs.gov/Kansas/rtqw/index.shtml
Keith [email protected](785) 832-3543