Analysis of Microcystin-LR in the San Francisco Delta Following Drought Year

Western Canada Trace Organics Workshop

Tuesday April 28th, 2015

Max Mizel

Department of Civil and Environmental Engineering

University of California - Davis

Background – Harmful Algal Blooms (HABs)

• Toxins released by cyanobacteria (or blue-green algae)

• Anabaena, Aphanizomenon, Microcystis, Cylindrospermopsis, Planktothrix

• Toxins include hepatotoxins (microcystins, cylindrospermopsin), and neurotoxins

(anatoxin, saxitoxin)

• Conditions must promote growth AND production of toxins

• Poses considerable public health and environmental risks

• Drinking water, recreational use, local fish and wildlife (bioaccumulation)

Background – Harmful Algal Blooms (HABs)

• Favorable conditions include:

• Warm Temperatures

• High Light Intensity

• Low Flushing Time

• Little Turbulence

• High Nutrient Concentrations (e.g. Nitrogen, Phosphorus)

Background – HABs in San Francisco Delta

http://www.bayareacouncil.org/issues-initiatives/water-policy-overview/

http://ichthyologistbright.blogspot.com/2008/08/delta -in-distress.html

Google Earth

Background – HABs in San Francisco Delta

• Conditions in the SF Delta are ideal for HABs

• Microcystis transported via San Joaquin River, high ammonium provided by

Sacramento River (Lehman, 2014)

• First observed in the Summer of 1999

• Primarily composed of microcystis aeruginosa

• Microcystin (MC) congener LR has been identified since 2004

• Historical maximum total MC concentrations ~50 µg/L

• Detected during drought year

Background – Microcystins

Generic MC Structure MC-LR

Ohio EPA Threshold (2014) for Total MC Congeners:

Do Not Drink – 1 ppb

Recreation – 6 ppb

Do Not Use – 20 ppb

Drought Study – Questions

• Is a change in LR concentration observed with changes in

• Nutrients – Total Nitrogen, Nitrate, Ammonium

• Water Temperature

• Solar Irradiance/ Turbidity

Drought Study - Overview

• Samples collected twice/month from

July-December

• Ten Stations

• Water quality parameters measured

by DWR

• Past drought years have shown an

increase in microcystis cells, total

microcystin, increased nutrient

concentrations (Lehman, 2013)

• Correlation with Low

Streamflow, High Temperature

• Nutrients less important

Microcystin Sample Collection

• MC samples collected via boat tow and grab

• LR quantified based on previous results and

historical research

• Cellbound (dry weight)

• Cellbound in water column

• Dissolved

• Provided by DWR

• Algal mass on filter paper (filtered volume

provided) from Tow

• Duplicates and Triplicates randomly

chosen

• 30 mL filtered, frozen grab sample

75 µm Mesh Tow

Grab Sample

……………………………… ……………

MC-LR Analysis - Instrumentation

Chromatography

Degasser Agilent Infinity 1260

Bin Pump

HiP ALS

TCC

Column Agilent Poroshell 120 EC-C18, 2.1 x 100 mm, 2.7 µm

Solvent 0.1mM NH4F/ Methanol

Flow Rate 0.600 mL/min

Gradient

Time %B

0 20

0.9 20

4.5 100

10 100

Post time 2 min

Kindly assisted by Ralph Hindle

MC-LR Analysis - Instrumentation

Mass Spectrometry

Instrument Agilent 6530 QTOF

Source Dual Jetstream ESI

Acquisition

Gas Temp (◦C) 325

Gas Flow (L/min) 10

Nebulizer (psi) 40

Sheath Gas Temp 350

Sheath Gas Flow 11

VCap 4000

Nozzle Voltage 1500

Fragmentor 150

Skimmer 65 Kindly assisted by Ralph Hindle

MC-LR Analysis – Cellbound Extraction

Filter concentrated sample aliquot from tow( Whatman glass microfiber filters)

Weigh N2 evaporator vial, scrape cells from filter into vial. Add 10 mL MeOH. Sonicate 30 min

N2 evaporate to dryness (40ᴼC). Weigh. Record mass of cells

Add 5 mL 10% MeOH. Sonicate for 20 minutes

Filter through 0.22 µm Millex PVDF syringe filter. Freeze (-20ᴼC) until analysis

MC-LR Analysis – Cellbound LR (dry weight). . .

And my path to becoming an analytical chemist

• Total cell mass extracted was unknown

• Alternative approach for determining cellbound MC-LR (mg/kg)

• Total volume filtered

• cells/mL (qPCR)

• Average microcystin aeruginosa cell mass (dry weight) = 18 pg/cell (Li, 2014)

MC-LR Analysis – Cellbound LR (dry weight)

Excitement was short lived

• Results varied considerably

• Most concentrations orders of magnitude greater than reported elsewhere (ppms)

MC-LR Analysis – Cellbound LR in Water Column

• Quantitation did not rely on cell mass

• Samples n = 208

• Quantitation based on accurate mass of [M+H]+

• Confirmed by [M+2H]2+

• Calibration curve and MS/MSD every 50 samples

• 4 Data points (0.1, 1, 10, 100 ppb), R2 > 0.996

• %Recovery = 83-107% (n=5)

• LOD – 0.03 ppb

• LOQ – 0.1 ppb

• LR not detected in method blanks

MC-LR Analysis – 0.1, 1, 10, 100 ppb Standards

MC-LR Analysis – Site CV July 28th, 2014

MC-LR Analysis – Site CV July 28th, 2014

Mass Accuracy - 0.9 ppm

Cellbound MC-LR Results (ng/L)

0

5

10

15

20

26-May 15-Jul 03-Sep 23-Oct 12-Dec 31-Jan

SJCVORVCJP

0

5

10

15

20

25

ng

/L

RRATMIBIFT

AT BI

CV FT

SJ VC

Comments and Next Steps

• LR trends agree with past observations

• High temperature/ solar irradiance/ turbidity main contributor

• Light >> Nutrients

• Dissolved LR Analysis

• Statistical analysis of LR and WQ parameters

• Incorporation of all MC congeners

Special Thanks To - Thomas Young -PI, Civil and Environmental Engineering, UC-Davis Peggy Lehman

-Project Manager, CA Department of Water Resources

Swee Teh, Tomo Kurobe, and Chelsea Lam -Lead MC Researchers, Department of Veterinary and Medical Science, UC-Davis

Joe Weitzel -Global Marketing Manager, Agilent

Ralph Hindle -Method Development/ Analytical Consultation, Vogon Laboratory Services Ltd.