U.S. Environmental Projection Agency, Office of Research and Development Formation and Control of Cyanobacterial Toxins Thomas Speth, Nicholas Dugan, Joel Allen, Darren Lytle, Heath Mash, Toby Sanan, Jody Shoemaker, Jay Garland, and Michelle Latham

2 Harmful Algal Blooms (HABs)  Overgrowth of autotrophs close to the shore of a water body  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, and weather patterns Cyanobacterial HAB (CyanoHab) Bacteria that produce a wide variety of toxins and are considered HABs because they exhibit some similar characteristics with algae (e.g., photosynthesis). Often referred to as blue-green algae

20 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 Office of Water is currently drafting Health Advisories for microcystin-LR and cylindrospermopsin 3 Current Regulations/Guidance EPA’s informational webpage cyanohabs

4 Source Water Impacts on Drinking Water Crop Land 65% Urban 10% Septic 7% Pasture 14% Nitrogen Load Source Distribution to Harsha Lake in Ohio  Excessive nitrogen and phosphorous levels can cause harmful algal blooms  Agriculture is often the largest contributor of nitrogen load into waterways  Algal blooms put pressure on drinking water facilities, requiring operational changes that can be costly and not well understood  Algal/cyanobacteria strains bloom under different conditions, at different times  Different strains produce different toxins at varying amounts Problem:

Monitoring for HABs 5 (cyanobacteria)  Different algal/ cyanobacterial strains bloom under different conditions, at different times  Chlorophyll peak may be detected without cyanobacteria peak Chlorophyll peak but absence of BGA peak RFU

Cyanobacteria Strains & Associated Toxins 6 Cyanobacteria Taxa  Strains produce different toxins at different amounts  Toxins can have multiple variants Example: Over 80 known microcystin variants Toxins analyzed by USEPA

7 Extracellular Toxins

Dissolved (toxin released from cell)  Solids removal processes ineffective  Typical disinfectants may not be effective enough (e.g., permanganate, chlorine)  More effective treatments are expensive and plants typically do not have them in place (e.g., GAC) Treatment for Cyanobacteria Toxins 8 Particulates (toxin in cell)  Solids removal processes effective  Do not want to lyse cell or toxin will be released Microcystin ToxinMicrocystis (cells) Toxin within the cell and those that are dissolved require different treatment processes

Analytical Methods 9 LC-MS/MS  EPA uses three LC-MS/MS methods (2 triple quad and 1 high resolution MS)  EPA has tested for 7 microcystin congeners/variants (over 80 known)  Currently evaluating 13 congeners/variants  Lysis techniques can give total toxin concentration (internal and external to cell)  Requires significant expertise and analytical equipment ELISA  Broad-based method (measures 80+ toxin congeners/variants)  Example: Ohio’s ELISA-based guidance is 1 ug/L  EPA has tested ELISA under different sample handling procedures  Lysis techniques can give total toxin concentration (internal and external to cell)  Easy and relatively inexpensive

10 Development of Analytical Methods Drinking water: Multi-Lab Verification Complete Method 544: Microcystins and nodularin Ambient water: Method Development Method 544: Microcystins and nodularin Method 545: Cylindrospermopsin and anatoxin-a Comparison of ELISA and LC/MS/MS: Compare results of ELISA measurements and LC/MS/MS concentrations of microcystin variants Detection of cyanobacterial toxins

Harmful Algal Blooms in Ohio  Celina (population 10,400) Summer 2013: > 100 µg/L total microcystins and nodularin in treatment plant influent  Carroll Township (population 2,000) September 2013: > 3 µg/L total microcystins and nodularin in finished water Ohio EPA “Do not drink” advisory  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 10

12 Carrol Township 2013 First Do Not Drink Advisory (Ohio) Carroll Township finished water: – 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 On Advisory 48 Hours

Toledo (August 2014) Do Not Drink Advisory (Ohio) Could not bring in water from another utility Had to adjust treatment ORD Technical Assistance Performed multiple analyses (ELISA and LC-MS/MS) Provided technical assistance to the city and state on sample handling and analytical procedures Results USEPA’s initial results were in agreement with the elevated toxin levels previously reported by the Ohio EPA, which they used as the basis for recommending that the City issue a ban Joint sampling, analysis, and evaluation of results allowed for agreement on sample handling and analytical procedures Later results, in concert with the Toledo utility and Ohio EPA data, showed decreasing levels of toxins, confirming the plant was reaching its drinking water goal

Lake Erie Depth 14 WestEast Source: National Oceanic and Atmospheric Administration Red: Shallow Blue: Deep

Lake Erie Bloom (August 6, 2014) Sources:National Oceanic and Atmospheric Administration; National Aeronautics and Space Administration Cyanobacteria Index

16 Lake Erie Field Studies Monitoring cyanobacteria toxins through numerous treatment plants on Lake Erie Fate/transport of toxins through drinking water treatment systems Bench- and Pilot-Scale Studies Bench-scale permanganate, ozone, and activated carbon trials Pilot installation anticipated in time to be operational for the 2015 bloom Will support OW technical guidance to regions, states, and municipalities Lake Erie DW Treatment Plant Study

Typical Treatment Train Drinking water treatment system similar to Toledo plant 17 Flocculation Particulate (cell) removal Dissolved (toxin) removal: Not completely effective Dissolved (toxin) removal: Helpful but may not be effective enough To distribution system and consumer tap Source water with cyanoHAB Toledo plant intake 3 miles offshore Finished water water Flocculation

Western vs. Eastern Basin 18 Western basin intakes had higher nutrient loadings in early summer Western basin intakes had higher blooms levels later in summer

Through Treatment (Cells) 19 Most of the cells are taken out by the clarifier effluent

Cell Removal Treatment Facility Cyanobacteria cells removed during bloom peak sampling event 1 - plant influent to clarifier effluent (%) Toledo98 Oregon99 Ottawa94 Sandusky Cleveland99 1 Bloom peak sampling event was 9/9/13 for Toledo, Oregon, Ottawa, and Cleveland; 8/14/13 for Sandusky. 2 Sandusky operates two clarification stages. Other Lake Erie plants see similar results

Through Treatment (Toxin) 21 If toxin remains in the cell, most of it is removed before the filter

Effect of Permanganate 22 Inactivates cells Releases toxins into solution while at the same time destroying them

Through Treatment (Toxin) 23 Permangate reducing total and increasing extracellular toxin Powdered activated carbon reducing the extracellular toxin Particulate removal removes the intracellular toxin

24 Effect of Ozone Analyzed by LC-MS/MS

Expand the number of participants that have an incentive to purchase credits: WWTP to avoid expensive nitrogen removal upgrades DWTPs to protect source water Recreationists to protect beaches 25 Goal: reduce nutrient loadings at lowest cost Excessive nutrients have numerous deleterious effects such as increasing HAB potential Utilizing Lake Harsha, but program must be transferable to other watersheds East Fork Watershed and Lake Harsha as a case study Preliminary data from 2014 hyperspectral flyover: Drinking Water Intake Water Quality Trading Other impacts of nutrients Can result in increased GHG emissions GHG emissions from nutrient-stressed reservoirs can be significant Multiple reservoirs will be sampled in 2015

High Frequency Monitoring Tools 26 Physico-chemical –Temperature –pH –ORP –Specific Conductance –Turbidity –Dissolved Oxygen –Total Organic Carbon –Dissolved Organic Carbon –NO 3 -N –UV-Vis spectral profile Fluorescence BBE Algae Online Analyzer Green Algae Cyanobacteria Brown (diatoms) Cryptophyta Total Chlorophyll YSI Phycocyanin Total Chlorophyll Toxin Toxicity Online Toxicity Monitors Laboratory Assays Analytical Quantification ELISA LC-MS/MS

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 can serve as an effective barrier against exposure Preliminary evidence indicates that common doses of oxidants (permanganate and ozone) are sufficiently high to damage cells and release toxins, yet too low to completely degrade the released toxin Treatment facilities are currently adding oxidants and powdered activated carbon (PAC) at significant expense and uncertain effectiveness More must be known about the formation and control of cyanobacteria and their toxins to assure safe drinking water

28 Disclaimer 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.

Questions? 12