U.S. Environmental Projection Agency, Office of Research and Development

Analysis and Monitoring of Algal Toxins in Fresh and Coastal/Estuarine Environments

US EPA Cincinnati OH Heath Mash

14th Annual EPA Drinking Water Workshop August 22, 2017

U.S. Environmental Protection Agency, through its Office of Research and Development, funded and managed, or partially funded and collaborated in, the research described herein. It has been subjected to the Agency’s peer and administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the author(s) and do not necessarily reflect the views of the Agency, therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use.

&EPA Harmful Algal Blooms (HABs)

HABs result in approximately $2.2B of associated costs annually in the US, from restricted use of recreational waters, declining waterfront real estate value, spending on recovery of biodiversity, and drinking water treatment.

Wide variety of taxa can produce blooms

Typically detrimental to the aquatic system and can be harmful to humans and land animals (contact and consumption)

Blooms are dependent on numerous factors, including nutrient loading, temperature, water flow and weather patterns

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Microcystis

Oscillatoria

Nostoc

Anabaenopsis

Aphanocapsa

Haplosiphon

Pseudanabaena

Synechocystis

Synechococcus

Microcystin and other Cyanotoxin Toxin Producers

Largest population of cyanotoxin producers in the Great Lakes: Microcystis aeruginosa Anabaena circinalis Anabaena flos-aquae Aphanizomenon flos-aquae Cylindrospermopsis raciborskii

Source: NOAA

Speciation of toxin producers in Lake Erie nearest Toledo in: 2013: Microcysis sp. 80 -99% Other microcystin producers 1 - 20%

2014: Microcysis sp. 85 -95% Other microcystin Producers 2 - 15% Non -microcystis producers 0 - 2% Dolichospermum 0 – 5%

Source: USGS, 2015

Source Water Impacts on Drinking Water

Problems facing drinking water treatment:

Excessive nitrogen and phosphorous levels can cause harmful algal blooms Agriculture (non-point source) is often the largest contributor of nitrogen load into

waterways Forecasting is difficult because algal/cyanobacteria strains bloom under

different conditions at different times Additionally, an algal bloom may not necessarily produce toxins

Treatment is still impacted due to biofouling, taste and odor concerns, increasing disinfection by-product potential, etc.

Algal blooms put pressure on drinking water facilities, requiring: - Immediate operational changes (i.e. PAC addition) can be

costly, with varying effectiveness - Possible shut-off of services, public relations challenges - Costly facility upgrades

4

Current Regulations/Guidance

5

21 states have recreational water guidelines for harmful algae blooms

Three states (MN, OH, and OR) have implemented standards or guidelines that apply to cyanotoxins in drinking water

EPA’s informational webpage http://www2.epa.gov/nutrient -policy -data/cyanobacterial -harmful -algal -blooms -cyanohabs

EPA’s Office of Water has released its Health Advisory Level for microcystin-LR and cylindrospermopsin

• The Harmful Algal Bloom and Hypoxia Research and Control Amendments Act of 2014 – Delegates primary responsibility to NOAA in advancing the scientific understanding and ability to

detect, monitor, assess, and predict HAB and hypoxia events in marine and freshwater

--

--

- -

-

--

Current Regulations/Guidance

– OH • ”Do not Drink” Advisories

– 0.3 μg/L Tot MYC Child < 6 yr & Sensitive Pop – 1.6 ug/L Tot MYC Child > 6 yr & Adults

• “Do Not Use” Advisory – 20 ug/L

• USEPA Health Advisory Level for microcystin-LR and Cylindrospermopsin “Drinking Water Health Advisory for the Cyanobacterial Microcystin Toxins” “Drinking Water Health Advisory for the Cyanobacterial Toxin Cylindrospermopsin “

– 0.3 μg/L MYC (0.7 μg/L CYL) 10 Day Infants/Young Children – 1.6 μg/L MYC (3.0 μg/L CYL) 10 Day Adults

• Safe Drinking Water Act – Contaminant Candidate List 4 (draft) includes cyanotoxins

• Microcystin LR, Anatoxin a, Cylindrospermopsin

– Unregulated Contaminant Monitoring Rule (2018 2020) • US EPA Method 544

– MN 0.04 μg/L as MYC LR – OR 1.0 μg/L as MYC LR

• State Health Advisory Levels (MYCs)

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Lake Erie and the Ohio River are Major Drinking Water Sources

29 Ohio and 2 Michigan communities intake water directly from Lake Erie (Western and Central Basins)

Subsequent communities purchase either treated or untreated water from these primary DWTPs and may subject the water to further treatment

The Ohio River is a major source of drinking water

along its entire reach

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Ohio In 2013 and 2014

Celina (population 10,400) Summer 2013: > 100 µg/L total microcystins and nodularin in treatment plant influent

Carroll Township (population 2,000)

September 4, 2013 = 1.4 ug/L

September 5, 2013 = 3.6 ug/L

Switched to emergency connection with Ottawa County

Began flushing distribution system

Ohio EPA’s first “Do Not Drink” advisory issued due to microcystin

On Advisory 48 Hours

Toledo (population ~500,000) September 2013: Detectable, but < 1 µg/L toxin in finished water August 2014: >1 µg/L total microcystins and nodularin in finished water, Ohio EPA “Do Not Drink” advisory

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OEPA 2015 Ohio River Bloom

Microcystins Concentration (ug/L) RM = River Mile N = 62 as of 9-11-15 Some data may be prel iminary

0 Below Detection (<0.30)

0 0.30- 1.6

0 1.6 - 6

0 6 - 20

• >20 .. Lock and Dams

-- Ohio River

Majot Tribs

LJ LAKES

D Ohio River Basin

Markland RM 531 .5

2015 Ohio River HAB

Wheellllg

Belleville RM 20 3 .9

W illow Island RM 161 .7

• Approximate Dates:August 19th through

October 29th, 2015

• Main Contributor:Microcystisaerugenosa

• 3x107 cells/ml1x105 typicallyreferenced as level for water impairment

• Numerousrecreational wateradvisories

Source: Ohio River Sanitation Commission

Microcystin Toxin Variants

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MYCs are cyclic heptapeptides Varying strains produce different toxins

at different rates and quantities Exist in multiple variants

113+ known microcystin variants

Significant differences in hydrophobicity

and pKa

Species can be multi-charged

Variants differ in potencyEstimated cytotoxic IC50 values

Shimizu, Kumiko, et al. Toxins 6.1 (2013): 168-179.

All MYCs include the ADDA (3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyl-4(E), 6(E)-decadienoic acid, red) and methyldehydroalanine (MDHA, purple)modified amino acids. Leucine (green) and arginine (blue) residues are sitesof structural diversity, referred to as positions X and Z, respectively.

Microcystin-LR OMe

NH

O

N

COOH

O

N

CH2

O

NH

ONH

O

NH

O

NH

O

COOH

NH

NH2

NH

ADDA

R, Arginine

L, Leucine

Dissolved (toxin released from cell)

Solids removal processes ineffective

Typical disinfectants or dosages may not beeffective (e.g., permanganate, chlorine)

More effective treatments are expensive andplants typically do not have them in place(e.g., GAC)

Particulates (toxin in cell)

Solids removal processes effective

Do not want to lyse cell or toxin willbe released

Microcystin ToxinMicrocystis (cells)

Dissolved or Particulate?

11

Toxin within the cell and those that are dissolved require different sample processing and treatment

OEPA Lake Harsha Cyanobacteria Bloom Study

Lake Harsha Cyanobacteria Bloom Study

12

Study Site Lake Harsha (East Fork State Park)

EMB

EFLS/LD

BOUY

ENN

– 5 Sites• Inlet (ENN)• Beach (EMB)• Buoy near drinking water intake

(BOUY)• Drinking water intake – Surface (EFLS)• Drinking water intake – Intake at depth

(EFLS)

– Weekly sampling March-November– 3x/week during observed bloom– Bi-hourly sampling for 4 hours on

representative days

13

Sample Handling and Analytical Methods

Sample Container

Preservative and dechlorination agents added adapted from USEPA Method 544

Sample

Placed at 4oC until Processed

Sample Filtered through 0.8 um Filter

Filtrate Extracellular

Retentate-Solvent Extracted

Combined Total

Other Analysis – Chlorophyll-a (USEPA 445.0)

– Phycocyanin– Water Quality Parameters

(i.e. Nutrients, Metals)

Enzyme Linked Immunosorbent Assay (ELISA) Broad-based method

Total toxin concentration congeners/variants

Abraxis Microcystins/Nodularins (ADDA),ELISA Kit, Microtiter Plate

LC/MS – Two LC/MS/MS methods

Triple quad and IT/HRMS (Orbitrap)

– Analytical m ethods includes 13 commercially-available MC congeners/variants

– Anatoxin A and Cylindrospermopsin (USEPA545)

– MMPB Oxidation (Total M C methodology)(2-methyl-3-methoxy-4-phenylbutyric acid)

14

y Parameters

Microcystin Analytical Methods

Mass Spectroscopy

Algaltoxin Formula Mmi of Measured

Species [M+H]+ (Da)

Microcystin LF C52H71N7O12 986.523 Microcystin LR C49H74N10O12 995.556 Microcystin LW C54H72N8O12 1025.532 Microcystin LY C52H71N7O13 1002.518 Microcystin RR C49H75N13O12 519.792c

Microcystin WR C54H73N11O12 1068.543 Microcystin YR C52H72N10O13 1045.535 Nodularin C41H60N8O10 825.450 [D-Asp3-(E)-Hhb7] Microcystin-HphR C52H72N10O12 1029.540 [D-Asp3-(E)-Dhb7] Microcystin-RR C48H73N13O12 512.786c

Microcystin-N-Methyl-LR C50H76N10O12 1009.572 Microcystin-HilR C50H76N10O12 1009.572 Microcystin-HtyR C53H74N10O13 1059.551 [D-Asp3] Microcystin-RR C48H73N13O12 1024.557 [D-Asp3] Microcystin-LR C48H72N10O12 981.540

o LC-IT/HRMS (Orbitrap)o Dionex Ultimate 3000 UPLC

o 3400RS Analytical Pump o 3400SD Loading Pump o Equan Autosampler

o Thermo Discovery Orbitrapo MS/MS product scan o Primary and confirmatory ions observedo Internal standards added to samples and standardso 8 point standard curve used (0.0 to 1000 ppt)

o Internal Standardso 13C,15N2 17-Amino Geldanamycin (225 ng/L)o Clarithromycin (56.25 ng/L)o 13C6-Paclitaxel (562.5 ng/L)o Tacrolimus (56.25 ng/L)o Virustomycin A (1125 ng/L)o d5-Atrazine (45 ng/L)

15

2016 Study Season – Total Targeted MC Concentration

Total Concentration of Known MYC Variants

– Similar targeted MCs concentrationpattern seen between EMB and ENNsites

– Similar pattern appeared betweenEFLS and BOUY sites

16

2016 Study Season – Total Targeted MC Concentration

Total Concentration of Known MYC Variants

– Delayed response in peak MC concentrations between the surface (EFLS) and depth sample (EFLD)

17

2017 Lake Harsha Monitoring Study –Comparing Methodologies

BOUY Site Fixed Camera Station

Fixed Camera Prediction of Cyanobacteria

July 18th at 10:30 am

Satellite Imaging

Estimated Cell Counts

July 18th at 10:49 am

EFLD/EFLS = 1,023,293 cells/ml • Camera Prediction: BUOY = 676,083 cells/ml • 10:30 am - 98.3% Probability of Bluegreens EMB = 323,594 cells/ml • 11:30 am 100% Probability of Bluegreens ENN = 1,258,925 cells/m

Courtesy of Jim Lazorchak/Blake Schaeffer 18

2017 Lake Harsha Monitoring Study

Targeted LC/MS/MS MC Concentration

Total Targeted MC Concentration

EFLD/EFLS =1244.3 ppt EFLD/EFLS = 1,023,293 cells/ml

Satellite Imaging

Estimated Cell Counts

July 18th at 10:49 am

BUOY = 1179.7 ppt BUOY = 676,083 cells/ml EMB = 1725.3 ppt EMB = 323,594 cells/ml ENN = 1843.8 ppt ENN = 1,258,925 cells/m

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OEPA Lake Erie Drinking Water Treatment PlantStudy

Lake Erie Drinking Water Treatment Plant Study

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Lake Erie Blooms 2014 vs. 2015 -Toxic vs. Non-Toxic Producing Blooms

August 2014

Chl-a Index

Chl-A Index

Chl-a Index

August 2015

Source: NOAA/NASA 21

Chlorophyll-a Concentration Observed2014

Month Sampled

April May June July August September October

Chlor

ophyl

l-A (µ

g/L)

0

20

40

60

80

100

120

DWTP 1 DWTP 2 DWTP 3 DWTP 4 DWTP 5 DWTP 6 DWTP 7

Chlorophyll-a, a proxy indicator, concentrations peak in August-September, consistent with the observed HAB occurrence

Large fluctuations show need for an increased sampling frequency 22

Toledo

Oregon

Ottawa

Sandusky

ClevelandAshtabula

Toledo

Oregon

Ottawa

Sandusky

ClevelandAshtabula

Nutrient and Bloom Event Flux 2014

Inor

gani

c ni

troge

n (m

g/L

as N

)

4

3

2

1

0

Nitrogen

Plant 3 Plant 1 Plant 5 Plant 2 Plant 7 Plant 8

t rlyne s er e eru b bJu Ju gu m ctob

emA te O voepS N

110

100

90

80

70

60

50

40

30

20

10

0

Chlo

roph

yll A

(ppb

)

Plant 3 Plant 1 Plant 5 Plant 2 Plant 7 Plant 8

tlyne suJu Ju guA

Chlorophyll-a

er erb ber bm ote ct emO voepS N

Western basin intakes had Western basin intakes had higher nutrient loadings in higher blooms levels later

early summer in summer

23

110

Seasonal Bloom Dynamics

Apr May Jun Jul Aug Sep Oct Nov

Chl

orop

hyll

(µg/

L)

0

10

20

30

40

50

60

70

80

90

100

110

0

10

20

30

40

50

60

70

80

90

100 Plant 2: 2013 bloom season Plant 2: 2014 bloom season

Similar concentration was observed at the peak of the bloom for each year

Plant 2 2013 vs. 2014

Sampling Date

4/1/2014 5/1/2014 6/1/2014 7/1/2014 8/1/2014 9/1/2014 10/1/2014 11/1/2014

Chl

orop

hyll-

A C

once

ntra

tion

(µg/

L)0

20

40

60

80

100

120

DWTP 1 DWTP 2 DWTP 3 DWTP 4 DWTP 5 DWTP 6 DWTP 7

2014 Season

Peak of a bloom season does not necessarily occur at the same time in a given year

There may be more than one major bloom event 24

w uent

hed 4 PAC

uent nORa sM l lff ff nit s E E it Po Fos er erP i tf li iFarlC

DWTP 1 DWTP 2 DWTP 3 DWTP 4 DWTP 5 DWTP 6 DWTP 7

Peak Event

Chlor

ophy

ll a (µ

g/L)

100

10

1

0.1

0.01

0.001

Chlorophyll-a Conc. Indicate Biomass is Effectively Removed Post-Clarification

Raw

Post MnO4

PostPAC

Clarifie

r eff

Filter e

ff

Finished

Chl

orop

hyll

a (u

g/L)

0.001

0.01

0.1

1

10

100

May 19 July 1 July 21

Most of the cell removal work is accomplished prior to filtration

Plant 4 (2014)

As an proxy measure of intact cells, chlorophyll-a concentrations decreases across the treatment train

Most cell removal was accomplished prior to filtration 25

Toxin

Con

cent

ratio

n by

LC/

MS/

MS

(µg/

L)

0

2

4

6

8

10

Extracellular Toxin Total Toxin

Release

LC-MS/MS

Raw Post-MnO4 Post-PAC Filter Influent Filter Effluent Plant Tap

Treatment Stage

Treatment Stage

Raw Post-MnO4 Post-PAC Filter Influent Filter Effluent Plant Tap

Toxi

n C

once

ntra

tion

by E

LISA

(µg/

L)

0

20

40

60

80

100

120

Extracellular Toxin Total Toxin

Release

Credit: Nick Dugan USEPA

ELISA

Toxin Propagation through DWTP - ELISA vs LC-MS/MS

Intracellular toxin release following MnO4 addition Added at crib intake, some residency/ contact time occurs before entering at the head of the treatment train

- Zebra mussel and taste & odor control

Unlike ELISA, total MYC measured by LCMS/MS decreases after MnO4 addition

- However, only 8 MYC (and NOD) variants were measured

­

26

[MCs

] by

LC/M

S/M

S an

d M

MPB

, ug/

L

8000

7000

6000

5000

4000

3000

2000

1000

0

y = 0.3704x + 166.51 R² = 0.7282

LC/MS/MS (Congener)

> 32 MC Congeners

0 1000 2000 3000 4000 5000 6000 7000 [MCs] by ELISA, ug/L

Courtesy of Toby Sanan

Comparison of Total MCs by ELISA and LC/MS/MS (Lake Harsha)

Comparison of Total MCs by ELISA and LC/MS/MS (Lake Harsha)

Lake Harsha Sample SRM Chromatograph Why the difference?

• Non-targeted MC variants • Need to ensure similarly

massed variants are not misidentified

chromatography and confirmation ions are important

ND ND ND

Total Toxin Measurements Through Treatment by LC/MS/MS

Extracellular Toxin Measurements Through Treatment by LC/MS/MS

Toxin Speciation and Propagation - LC-MS/MS

Treatment Stage

Raw Post MnO4 Post PAC Filter Effluent Finished To

tal T

oxin

by

LC/M

S/M

S (µ

g/L)

0

2

4

6

8

10

MYC-LR MYC-RR MYC-YR MYC-LA MYC-WR MYC-LY

Total MYC Measured through Treatment

Treatment Stage

Raw Post MnO4 Post PAC Filter Effluent Finished

Extra

cellu

lar T

oxin

by

LC/M

S/M

S (µ

g/L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

MYC-LR MYC-RR MYC-YR MYC-LA MYC-WR MYC-LY

Extracellular MYC Measured through Treatment

NDND

Significant diversity in toxin variants Both extracellular and total MYC concentrations decrease

through the treatment train; no MYC observed in finished water 29

Ozonation and IntracellularToxin Release

Ozone effectively lysed cyanobacteria However, the applied ozone dose was not sufficient to further remove

toxins from post-ozonation water 30

OEPA Conclusions

Lake Erie water quality was significantly degraded in the western basin as compared to the eastern basin

The bulk of toxin in treatment facility influents was intracellular Therefore, if this holds true, a facility originally designed for particulate

control (conventional particulate removal strategies - coagulation, flocculation, sedimentation, filtration) are effective in removing biomass, hence intracellular toxins trapped within intact cells, and can serve as an effective barrier against exposure

Powdered activated carbon reduces the extracellular toxin - Treatment facilities are currently adding oxidants and powdered

activated carbon (PAC) at significant expense and uncertain effectiveness

Preliminary evidence indicates that common doses of oxidants (i.e. permanganate) are sufficiently high to damage cells and release toxins, yet may be too low to completely degrade the released toxin

More must be known about the formation and control of cyanobacteria and their toxins to assure safe drinking water 31

Special Thanks to: • The various Lake Erie DTWPs • Ohio EPA • Christy Muhlen, Maily Pham, Joel

Allen, Kit Daniels, Dana Macke, Toby Sanan

Questions?

EPA’s informational webpage http://www2.epa.gov/nutrient-policy-data/cyanobacterial-harmful-algal-blooms-cyanohabs

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