Ohio s Use of qpcr as a Cyanobacteria Screening Tool

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1 Ohio s Use of qpcr as a Cyanobacteria Screening Tool Ohio AWWA Technology Conference September 26, 2017 Heather Raymond Ohio EPA HAB Coordinator

2 Outline Evaluation of qpcr Assay as a Cyanobacteria Screening Tool Microcystins Saxitoxins Comparison of qpcr Assay to Phytoplankton Enumeration How to Interpret qpcr data Next Steps Microcystins in Water Treatment Plant Residuals

3 Cyanobacteria Screening qpcr Assay Overview Commercial multiplex quantitative polymerase chain reaction (qpcr) assay. Identifies and quantifies the presence of genes unique to: Cyanobacteria: 16S rdna Microcystins production: mcye gene Cylindrospermopsin production: cyra gene Saxitoxins production: sxta gene Fast: 2-3 hours Scalable Utilizes certified reference material Specific Ohio EPA conducts all compliance sample analysis from June 2016 December 2017 Ohio EPA draft method: Ohio EPA Lab certification in (current assay in use)

Cyanobacteria Screening (qpcr) 2016 Sampling Summary Public Water Systems (119): Cyanobacteria Screening (qpcr)

aspx Biweekly raw water sampling (paired with microcystins) Gene detections triggers follow up sampling by Ohio

Lakes (24): Cyanobacteria Screening (qpcr) Monthly (May-October) Paired with: microcystins, saxitoxins,

4 Cyanobacteria Screening (qpcr) 2016 Sampling Summary Public Water Systems (119): Cyanobacteria Screening (qpcr) OAC Chapter (Effective June 1, 2016): epa.ohio.gov/ddagw/rules.aspx Biweekly raw water sampling (paired with microcystins) Gene detections triggers follow up sampling by Ohio EPA for saxitoxins and cylindrospermopsin PAIRED SAMPLES 2016: 1850 Microcystins, 228 Saxitoxins Targeted Inland Lakes (24): Cyanobacteria Screening (qpcr) Monthly (May-October) Paired with: microcystins, saxitoxins, cylindrospermopsin, and other water quality parameters (including phytoplankton enumeration 3 times per year) PAIRED SAMPLES IN 2016: 168 (128 with phytoplankton)

Source Waters Microcystins Detections June Dec.

5 Source Water Microcystins Detections at Ohio PWSs Data Available for 119 Public Water System Source Waters Microcystins Detections June Dec Microcystins Detections PWS Intakes

6 Frequency of Source Water Microcystins Detections > 1.6 ug/l at Ohio Public Water Systems Number of Microcystins Detections >1.6 ug/l Response Sampling ( ): samples >1.6 ug/l microcystins, out of 3583 total samples (21%). - 44% of samples were > 0.30 ug/l microcystins. Routine Monitoring (2016): samples >1.6 ug/l microcystins, out of 3512 total samples (5%) - 11% of samples were > 0.30 ug/l microcystins.

7 qpcr as Screening Tool for Microcystins Microcystins detected in raw water at 45 PWSs (38%) and mcye detected at 57 PWSs (48%) mcye detections at all 25 Lake Erie PWSs Microcystins detections at 22 Lake Erie PWSs Out of 1850 paired PWS samples: 100% of microcystins >1.6 µg/l had paired mcye gene detections. 100% of microcystins >5 µg/l had mcye detections > 5gc/µL 90% of microcystins detections >1.6 ug/l had mcye detections >5 gc/µl Less than 2% of samples (22 sites, 32 samples) had microcystins detections without mcye detections: 19 of the 22 sites had gene detections in either prior or post sampling events The remaining three PWSs had only one low level ( µg/l) microcystins detection in 2016; all had trace mcye gene copies

8 qpcr Screen for Microcystins Public Water System (PWS) Data Microcystins (µg/l) # samples, (# PWS) < (120) (40) > (9) mcye %, range (Gene Copies/µL) 5% 29 66% % K 16S, Total Cyanobacteria range (Gene Copies/µL) 67K 8 59K 1.4K 300K

001 Type Inland Lake PWS Intake qpcr Microcystin (Gene Copies/µL) Positive relationship with

9 qpcr mcye Screen for Microcystins Microcystins (µg/l) 1 Lake- Prior mcye, Possible inhibition or extracellular cyanotoxins Correlations Spearman Kendall rho= 0.61 tau= 0.59 p << p << Type Inland Lake PWS Intake qpcr Microcystin (Gene Copies/µL) Positive relationship with microcystins and mcye Limited microcystins occurrence without mcye Extracellular toxin Inhibition for qpcr due to high biomass

10 Inland Lake Outlier Composition and Microcystins Dynamics Phyto ID, biovolume (µm 3 /L) Planktothrix Microcystis MC Microcystins (µg/l) Pseudanabaena Non-MC Cyanobacteria mcye (GC/µL) Microcystins concentration associated with Planktothrix 170 May June July Aug Sept Oct May June July Aug Sept Oct May June July Aug Sept Oct Site A Site B Site C Microcystins without corresponding mcye Relatively low concentration, possibly persistent extracellular High abundance of non-mc producing cyanobacteria may inhibit detection of low levels of Planktothrix and mcye Microcysitins & mcye

11 Example: Source Water With Consistently High Microcystins & mcye Concentrations Microcystins, mcye /1/16 7/11/16 8/20/16 9/29/16 11/8/16 12/18/16 mcye gene (gc/µl) Microcystins (µg/l)

12 Example: mcye Detections Precede Microcystins Detections in Central Lake Erie Basin 3.5 Microcystins, mcye /30/16 7/9/16 8/18/16 9/27/16 11/6/16 PWS1 mcye (GC/µL) PWS2 mcye (GC/µL) PWS1 Microcystins (µg/l) PWS2 Microcystins (µg/l)

13 Example: 16s and mcye Trends Microcystins, mcye Total Cyanobacteria, 16S 0 Jun-16 Jul-16 Jul-16 Aug-16 Sep-16 Oct-16 Nov-16 Dec-16 0 Microcystins (µg/l) mcye (GC/µL) 16S (GC/µL)

14 Example: 16s and mcye trends Microcystins, mcye Jun-16 Jul-16 Jul-16 Aug-16 Sep-16 Oct-16 Nov-16 Dec Total Cyanobacteria, 16S Microcystins (µg/l) mcye (GC/µL) 16S (GC/µL) Microcystins trend with mcye genes Non-toxic cyanobacteria bloom (16s) in August

2017 Lake Erie qpcr mcye Data Summary mcye detections at 22 of 25 PWSs and Microcystins detections at 18 PWSs - West Basin: mcye preceded microcystins at all four water systems by 1-4 weeks.

15 2017 Lake Erie qpcr mcye Data Summary mcye detections at 22 of 25 PWSs and Microcystins detections at 18 PWSs - West Basin: mcye preceded microcystins at all four water systems by 1-4 weeks. - Central Basin (East of Cleveland): mcye preceded microcystins detections at 6 of 7 PWSs by 1-2 weeks. Trace detection (0.31 ug/l at Painesville not preceded by mcye). - Central Basin (Vermillion Cleveland): no microcystins - Sandusky Subbasin: mcye preceded microcystins at half (3) PWSs. Trace detections at three PWSs not preceded by mcye detections (impacted by Sandusky Bay bloom). Photo Credit: Toledo Aerial Media

16 Impact of Sandusky Bay Bloom on Lake Erie From NOAA Lake Erie Bulletin Public Water Systems

17 Source Water Saxitoxins Detections at Ohio PWSs Finished Water Saxitoxins Detections (below threshold) Saxitoxins Detections 2016 Saxitoxins PWS Intakes

18 qpcr as Screening Tool for Saxitoxins and Cylindrospermopsin sxta detections triggered response sampling at 33 PWSs (22%), including many with no historic saxitoxins occurrence and three Lake Erie intakes. 15 of those PWSs detected saxitoxins in raw water (12%) 6 PWSs had finished water detections (none above thresholds) Less than 1% percent of samples had saxitoxins detections without corresponding gene detections (includes 168 paired inland lakes samples) Only one cyra detection, no cylindrospermopsin detections in 2016 cyra & cylindrospermopsin detections in 2017

qpcr sxta Screen For Saxitoxins Correlations Spearman rho= 0.58 p << 0.001 Kendall tau= 0.49 p << 0.

Water Threshold sxta (Gene Copies/µL) Positive relationship with saxitoxins and sxta Saxitoxins

19 qpcr sxta Screen For Saxitoxins Correlations Spearman rho= 0.58 p << Kendall tau= 0.49 p << Ohio EPA Recreation Threshold Type Inland Lake Saxitoxins (µg/l) PWS Intake Ohio EPA Drinking Water Threshold sxta (Gene Copies/µL) Positive relationship with saxitoxins and sxta Saxitoxins occurrence without gene copies Only 1 Inland Lake: Lake Hope STX= µg/l Extracellular or benthic source

20 14 Example: sxta Triggers Saxitoxins Sampling sxta Saxitoxins /30/16 6/19/16 7/9/16 7/29/16 8/18/16 9/7/16 9/27/16 10/17/16 11/6/16 11/26/16 12/16/16 0 sxta (GC/µL) Raw Water Saxitoxins (µg/l) Finished Water Saxitoxins (µg/l) Microcystin (mcye) genes and microcystins also detected; Cylindrospermopsin (cyra) genes also detected (cylindrospermopsin was not detected)

21 Example: Simultaneous Saxitoxins and Microcystins Microcystins, mcye Saxitoxins, sxta, 16S 0 5/30/16 7/19/16 9/7/16 10/27/16 12/16/16 Microcystins (µg/l) mcye (GC/µL) 16S (GC/µL) 0.01 Saxitoxins (µg/l) sxta (GC/µL)

22 2017 Lake Erie qpcr sxta Data Summary sxta detections at four Lake Erie PWSs, including two with no prior sxta detections Saxitoxins detections in Central Basin (offshore) No saxitoxin detections at Lake Erie intakes Photo Credit: Brenda Snyder

10 different potential saxitoxin-producing genera found in multiple habitat zones (pelagic, benthic, periphyton, etc.

23 Example: Use of qpcr to Help Identify Source of Saxitoxins and Target Reservoir Management Low concentrations of saxitoxins detected in drinking water (below Ohio EPA threshold) from late July September, Extracellular saxitoxins predominated all samples. 10 different potential saxitoxin-producing genera found in multiple habitat zones (pelagic, benthic, periphyton, etc.) in multiple (all sampling) locations. Follow-up qpcr analysis revealed highest sxta gene copies in benthic samples. qpcr data used to target algaecide application.

24 Comparison Between Phytoplankton Identification/Enumeration & qpcr One PWS Lake Example 26 Inland lakes, 76 paired samples 5 lakes (19 samples) with microcystins detections 7 lakes (10 samples) with saxitoxins detections

25 Example: Phytoplankton Enumeration versus qpcr Microcystins, mcye /30/16 7/19/16 9/7/16 10/27/16 12/16/ Total Cyanobacteria, 16S mcye (GC/µL) Microcystins (µg/l) Total Cyanobacteria qpcr (GC/µL) Total Cyanobacteria count (cells/µl)

26 Microcystin-producing Cyanobacteria, mcye, & Microcystins mcye (GC/µL) Microcystins (µg/l) Non-detect Low, Med, High, > Microcystin-producing Cyanobacteria (cells/µl) High cell counts generally associated with elevated microcystins (May-October). Non-detects were mcye positive in 2% of samples, but had cell counts of concern in 42% of samples.

27 Saxitoxin-producing Cyanobacteria, sxta, and Saxitoxins sxta (GC/µL) Saxitoxins Non-detect Detected Saxitoxin-producing Cyanobacteria (cells/µl) Saxitoxin producing cyanobacteria are ubiquitous (96% samples) Saxitoxins detected at relatively low cyanobacteria abundance Two instances with no saxitoxin-producing cyanobacteria present with sxta and saxitoxins detected

28 Inland Lake Examples: Saxitoxin-producing Cyanobacteria, sxta, and Saxitoxins Cyanobacteria (cells/µl) Saxitoxins (µg/l) & sxta (GC/µL) 0 Site A B C D 1 D 2 D 3 D 4 E 1 E 2 F 0.01 Cylindrospermopsis Aphanizomenon Saxitoxins Planktothrix Dolichospermum (Anabaena) sxta

29 Cyanobacteria Enumeration and 16S Phtyo ID, Total Cyanobacteria (cells/µl) qpcr 16S, Total Cyanobacteria (GC/µL) In most cases, cyanobacteria have more than one gene copy per cell Relationship is variable between samples possibly due to cyanobacteria composition Large difference in cell size among cyanobacteria taxa Difference in number of gene copies per cell among cyanobacteria taxa

30 qpcr Data Interpretation mcye Any mcye detection may indicate onset of a bloom, and could provide early warning. More severe blooms, indicated by microcystins concentrations >5µg/L, were always associated with mcye >5GC/µL. Based on literature, 1.4 gene copies per cell for Microcystis aeruginosa. sxta sxta detections were associated with saxitoxins, but in some cases low sxta was associated with higher saxotoxins concentrations. Based on literature, 2.5 gene copies per cell for Anabaena circinalis. 16S 16s detections indicate presence of cyanobacteria, not cyanotoxins. Based on literature, gene copies per cell.

31 Off-season qpcr monitoring Ohio EPA will maintain shipping hubs and provide qpcr analysis through December 31, Ohio EPA will provide revised off-season monitoring schedules to PWSs by October 6.

32 Summary sxta is an effective, specific, screen for saxitoxins mcye is an effective, specific, screen for microcystins Multi-plex functionality of assay works 16S is not an effective screen for cyanotoxins, but potentially useful for assessing susceptibility Phytoplankton enumeration not effective screen for saxitoxins, may be useful for microcystins at recreational concentrations. Ohio EPA will continue to use qpcr assay as screen for saxitoxins and cylindrospermopsin and started using as additional screen for microcystins in 2017.

Molecular Methods: Next Steps Ohio EPA: Provide qpcr analysis to PWSs at no cost through December 31, 2017 Refine sampling and analysis method to better address high biomass samples (scums, benthic

33 Molecular Methods: Next Steps Ohio EPA: Provide qpcr analysis to PWSs at no cost through December 31, 2017 Refine sampling and analysis method to better address high biomass samples (scums, benthic samples) Lab certification (ongoing) USEPA: Assist with method validation- interlab comparison Sequence samples for 16s NOAA/BGSU: Metagenomics: Identify cyanobacteria sources of saxitoxins and cylindrospermopsin RNA analysis: Evaluate gene expression ALL: Publish results, help inform national policy Agency Priority in 2017 OBHE HAB Grant RPF

Microcystins Accumulation in Water Treatment Residuals Study Goals Determine MCs occurrence in a variety of residual types. Investigate persistence of MCs in WTR over time.

1 - >100 ug/l 1 to 9 MGD treatment plants with and without lime softening and with and without powdered activated carbon.

34 Microcystins Accumulation in Water Treatment Residuals Study Goals Determine MCs occurrence in a variety of residual types. Investigate persistence of MCs in WTR over time. Evaluate microcystins (MCs) analytical methods for water treatment residual (WTR) matrices. Study Design Four sites: Raw water microcystins concentrations ranged from >100 ug/l 1 to 9 MGD treatment plants with and without lime softening and with and without powdered activated carbon. Collected multiple discrete and composite samples at each site representing varying sludge ages. Three extraction methods: Freeze-Thaw (3 cycles) 75% Acidified Methanol Extraction EDTA Enhanced Methanol Extraction (Chen, 2016) Three analysis methods: MC-ADDA ELISA LC-MS/MS MMPB LC-MS/MS Individual Microcystins Variant Analysis

Microcystins Accumulation in Water Treatment Residuals Initial Findings Complex WTR matrices require an advanced extraction step. Extraction method performance varied by residual composition.

35 Microcystins Accumulation in Water Treatment Residuals Initial Findings Complex WTR matrices require an advanced extraction step. Extraction method performance varied by residual composition. Microcystins were detected in all WTR samples, regardless of WTR age. LC-MS analysis confirmed presence of microcystins variants. In general, microcystins concentrations in WTR were greater than concentrations in raw water. Additional Sampling and Analysis Two additional sites with low historic microcystins concentrations. Representative composite sampling. Evaluate additional established soil analysis methods: Synthetic Particulate Leaching Test (SPLT) ASTM Shake Method

Identify potential for biodegradation of microcystins in WTR. Evaluate potential for plant uptake.

36 MCs in Residuals: Next Steps Ohio Board of Higher Education HAB Grant: Further evaluate extraction and analytical methods for determining concentrations of microcystins in water treatment residuals (WTR). Determine fate of microcystins in WTR. Identify potential for biodegradation of microcystins in WTR. Evaluate potential for plant uptake. Ohio EPA Division of Materials Waste Management: Address interested party comments and release revised water treatment plant residuals beneficial reuse general permits.

37 Questions? (614)

Drinking Water from the Ohio EPA Perspective. Susan K. Schell Environmental Manager Ohio EPA Division of Drinking and Ground Waters

Drinking Water from the Ohio EPA Perspective Susan K. Schell Environmental Manager Ohio EPA Division of Drinking and Ground Waters Overview Legislation update Asset Management Lead Contingency Plans Operator