A Thesis Entitled Diel Vertical Distribution of Microcystis And

A Thesis

entitled

Diel Vertical Distribution of Microcystis and Associated Environmental Factors in the

Western Basin of Lake Erie

Eva L. Kramer

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Biology

______Dr. Thomas Bridgeman, Committee Chair

______Dr. Timothy Davis, Committee Member

______Dr. Daryl Moorhead, Committee Member

______Dr. Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo

December 2018

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Diel Vertical Distribution of Microcystis and Associated Environmental Factors in the Western Basin of Lake Erie by

Eva L. Kramer

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology

The University of Toledo December 2018

Harmful algal blooms comprised of the cyanobacteria Microcystis have recently caused multiple “do not drink” advisories in Ohio communities that draw their drinking water from Lake Erie, including the city of Toledo. Microcystis colonies are able to regulate their buoyancy and have a tendency to aggregate in thick scums at the water’s surface on a diel cycle under certain conditions. The city of Toledo’s drinking water intake draws water from near the bottom of the water column, thus a concentration of the bloom near the surface would present an opportunity to minimize Microcystis biomass and microcystin toxin entering the drinking water system. To better understand the vertical distribution of Microcystis over diel cycles, five temporally intensive sampling events were conducted from 2016-2017 under calm weather conditions near the drinking water intake in the western basin of Lake Erie. In-situ vertical profiles of algal concentrations and environmental parameters were recorded, and water samples were collected from discrete depths for algal identification and laboratory analysis of chlorophyll a, phycocyanin, microcystin, and nutrient concentrations. A scum was observed on only one of the five sampling events; unexpectedly, Microcystis decreased near the surface in the middle of the day during three of the five events. Total water

iii column Microcystis decreased throughout each sampling event, possibly due to settling.

Toxin distributions did not reflect cyanobacterial distributions. Therefore, real-time monitoring of cyanobacteria and toxin remain important tools for the management of

Toledo’s drinking water.

This thesis is dedicated to those who have made it possible through their constant support and love: my husband, Greg Lytmer; my parents, David and Peggy Kramer; and my

Forest Family, you know who you are.

Acknowledgements

I would like to thank my advisor, Dr. Thomas Bridgeman, for all of his help and guidance with this research. I would also like to thank my committee, Dr. Timothy Davis for his help and expertise in algal ecology, and Dr. Daryl Moorhead for his guidance on data analysis. This work would not have been possible without the additional lab and field support of my colleagues at the Lake Erie Center: Brenda Snyder, Zach Swan, Alex

Lytten, Ken Gibbons, Joe Turner, Lucas Arend, Jenna Houdashelt, Rachel Lohner, and

Pam Struffolino. Special thanks to Christian Moldaenke of bbe Moldaenke for field and instrumentation support and Dr. Douglas Kane for field assistance. Further assistance was provided by the National Oceanic and Atmospheric Administration’s Great Lakes

Environmental Research Laboratory; I would like to thank Dr. Mark Rowe for guidance on data analysis and David Fanslow for providing FlowCam data. I would also like to thank Dr. Dragan Isailovic and Dilrukshika Palagama for providing liquid chromatography-mass spectrometry data. Finally, I would like to thank the Lake Erie

Center for use of their boat and laboratory space, and the Ohio Department of Higher

Education for funding this study.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... ix

List of Figures ...... x

List of Abbreviations ...... xiii

1 Diel Vertical Distribution of Microcystis in the Western Basin of Lake Erie ...... 1

1.1 Introduction ...... 1

1.2 Methods...... 6

1.2.1 Field Sampling ...... 6

1.2.2 Vertical Profiles ...... 7

1.2.3 Full Sample Collection ...... 8

1.2.4 Laboratory Analysis ...... 9

1.3 Results ...... 13

1.3.1 Overview of Study Results ...... 13

1.3.2 Sampling Events ...... 16

1.3.2.1 August 3-4, 2016 ...... 16

1.3.2.2 August 18, 2016 ...... 23

1.3.2.3 August 9-10, 2017 ...... 28

vii 1.3.2.4 August 15, 2017 ...... 33

1.3.2.5 September 25, 2017 ...... 38

1.3.3 Depth-Integrated Microcystis Biomass Estimates ...... 43

1.3.4 Analytical Methods Comparisons ...... 45

1.4 Discussion ...... 51

1.4.1 Vertical Distributions of Microcystis ...... 51

1.4.2 Capturing the Full Water Column...... 52

1.4.3 Implications for Drinking Water Management ...... 54

1.4.4 Recommendations for Future Studies ...... 55

References ...... 57

A Supplementary Figures ...... 62

viii

List of Tables

1.1. Sampling event dates and time ranges ...... 7

1.2. Summary of all measured parameters and sources of final data ...... 12

List of Figures

1-1. Location of Toledo drinking water intake and sampling site ...... 6

1-2. MODIS satellite imagery of WBLE bloom conditions on sampling dates ...... 13

1-3. Comparison of total microcystin concentrations across all sampling events...... 16

1-4. Chlorophyll a concentrations measured using the YSI. August 3-4, 2016 ...... 17

1-5. Chlorophyll a concentrations measured using the FP. August 3-4, 2016...... 18

1-6. Temperature profiles measured using the YSI. August 3-4, 2016 ...... 19

1-7. ODO concentrations measured using the YSI. August 3-4, 2016 ...... 20

1-8. pH measured using the YSI. August 3-4, 2016 ...... 20

1-9. Vertical profiles of algal concentrations measured with the FP on August 3, 2016. 21

1-10. Cyanobacteria and microcystin concentrations at discrete depths. August 3-4, 2016

...... 22

1-11. Chlorophyll a concentrations measured using the YSI. August 18, 2016 ...... 23

1-12. Chlorophyll a concentrations measured using the FP. August 18, 2016 ...... 24

1-13. Temperature profiles measured using the YSI. August 18, 2016 ...... 24

1-14. ODO concentrations measured using the YSI. August 18, 2016 ...... 25

1-15. pH measured using the YSI. August 18, 2016 ...... 25

1-16. Vertical profiles of algal concentrations measured with the FP on August 18, 2016

...... 26

x 1-17. Cyanobacteria and microcystin concentrations at discrete depths. August 18, 2016

...... 27

1-18. Chlorophyll a concentrations measured using the YSI. August 9-10, 2017...... 29

1-19. Total chlorophyll concentrations measured using the FP. August 9-10, 2017 ...... 29

1-20. Temperature depth profiles measured using the YSI. August 9-10, 2017 ...... 30

1-21. ODO concentrations measured using the YSI. August 9-10, 2017 ...... 30

1-22. pH measured using the YSI. August 9-10, 2017 ...... 31

1-23. Vertical profiles of algal concentrations measured with the FP on August 9, 2017.

...... 31

1-24. Cyanobacteria and microcystin concentrations at discrete depths on August 9-10,

2017...... 32

1-25. Chlorophyll a concentrations measured using the YSI. August 15, 2017 ...... 33

1-26. Total chlorophyll concentrations measured using the FP. August 15, 2017 ...... 34

1-27. Temperature depth profiles measured using the YSI. August 15, 2017 ...... 34

1-28. ODO concentrations measured using the YSI. August 15, 2017...... 35

1-29. pH measured using the YSI. August 15, 2017 ...... 35

1-30. Vertical profiles of algal concentrations measured with the FP on August 15, 2017.

...... 36

1-31. Cyanobacteria and microcystin concentrations at discrete depths on August 15,

2017...... 37

1-32. Chlorophyll a concentrations measured using the YSI. September 25, 2017 ...... 39

1-33. Total chlorophyll concentrations measured using the FP. September 25, 2017...... 39

1-34. Water temperature measured using the YSI. September 25, 2017 ...... 40

xi 1-35. ODO concentrations measured using the YSI. September 25, 2017 ...... 40

1-36. pH measured using the YSI. September 25, 2017 ...... 41

1-37. Vertical profiles of algal concentrations measured with the FP on September 25,

2017...... 41

1-38. Cyanobacteria and microcystin concentrations at discrete depths on September 25,

2017...... 42

1-39. Estimates of water column cyanobacterial biomass over each sampling event...... 44

1-40. Comparison of chlorophyll a measurements ...... 46

1-41. Comparison of Microcystis and cryptophyte concentrations as measured from

Lugol’s-preserved samples and in-situ with the FP ...... 48

1-42. Comparison of microcystin concentrations measured with ELISA and LC-MS .... 50

A-1. Example FlowCam image ...... 62

xii

List of Abbreviations

DMF ...... DiMethylFormamide DNA ...... DeoxyriboNucleic Acid

ELISA ...... Enzyme-Linked ImmunoSorbent Assay EPA ...... Environmental Protection Agency

FP ...... bbe FluoroProbe Submersible Spectrofluorometer

HAB ...... Harmful Algal Bloom

LC-MS ...... Liquid Chromatography-Mass Spectrometry

MODIS ...... MODerate resolution Imaging Spectroradiometer

NOAA ...... National Oceanic (and) Atmospheric Administration NOAA GLERL ...... National Oceanic (and) Atmospheric Administration’s Great Lakes Environmental Research Laboratory

ODO ...... Optical Dissolved Oxygen

PAR ...... Photosynthetically Active Radiation

WHO ...... World Health Organization WBLE ...... Western Basin (of) Lake Erie

YSI EXO2 ...... YSI EXO2 Water Quality Sonde

xiii Chapter 1

Diel Vertical Distribution of Microcystis and Associated Environmental Factors in the Western Basin of Lake Erie

1.1 Introduction

Drinking water supplies around the globe are facing increasing threats from Harmful

Algal Blooms (HABs) due to anthropogenic pressures and climate change (Michalak et al. 2013, Visser et al. 2016). While there has been much debate on the precise definition of a HAB, the term is generally applied when growth of phytoplankton results in densities high enough to cause negative effects to the environment, economy, or public health (Smayda 1997). Often, the negative health effects come from toxins produced by the phytoplankton. There are many species of algae that create these toxins, and the toxins themselves are diverse in potency and mechanism (Carmichael 1997). In the fresh waters of the Laurentian Great Lakes, the most problematic HAB species are the cyanobacteria Microcystis, Planktothrix, Aphanizomenon, and Dolichospermum which produce cyanotoxins including cytotoxins, neurotoxins, and hepatotoxins (Carmichael

and Boyer 2016). Many of these organisms are widespread; toxic blooms of Microcystis have been documented on every continent except Antarctica. In the shallow, nutrient-rich waters of the western basin of Lake Erie (WBLE), blooms of Microcystis have become an annual problem for communities along the lake shore, impacting beach use and municipal drinking water intakes (Steffen et al. 2014, Watson et al. 2016).

Microcystis is a genus of cyanobacteria which often form globular colonies of spherical cells aggregated in a polysaccharide matrix. The formation of colonies may help Microcystis avoid predation and harbor symbiotic bacteria (Rohrlack et al. 1999,

Yang et al. 2008, Shen et al. 2011). Some strains of Microcystis are capable of producing the toxin microcystin (Ouellette et al. 2006, Dyble et al. 2008, Davis et al. 2009), a hepatotoxin which has been linked to both acute and long-term health effects in humans and other animals (Falconer 2005, Zhang et al. 2015, Carmichael and Boyer 2016). There is also growing evidence that microcystins can accumulate in plant and fish tissues, leading to concerns about human and animal consumption of these foods (Corbel et al.

2014, Wituszynski et al. 2017).

In addition to the health risks associated with cyanotoxins, there are also short- and long-term socioeconomic impacts of HABs. Many freshwater lakes are important resources for nearby communities, and HAB impacts on these communities can be significant and diverse. The WBLE provides drinking water as well as recreation, tourism, fisheries, real estate, and transportation for the region. In 2011, a significant

Microcystis bloom in the WBLE was estimated to have caused nearly $71 million in lost economic benefits to the region (Bingham et al. 2015). Additionally, two “do not drink” advisories have been issued in the past five years due to microcystin concentrations in the 2

WBLE overwhelming water treatment plants. In September 2013, the community of

Carroll Township was under a brief advisory before temporarily connecting to a neighboring, unaffected water supply. In August 2014, the city of Toledo and its suburbs were under a “do not drink” advisory, leaving 400,000 people without a reliable source of drinking water for over two days (Jetoo et al. 2015). While these treatment plants both had systems in place to help remove microcystin, the concentrations in the source water were too high to be completely removed and toxin concentrations in the finished water exceeded the 1 ppb drinking water advisory threshold. The Toledo drinking water advisory, in particular, has greatly increased public awareness of the dangers posed by

HABs and the treatment plant now has better monitoring and treatment resources than in

2014. However, removing the toxin is very costly and concerns of increasing bloom severity mean that new mitigation strategies should be considered.

One possible strategy is HAB avoidance utilizing the diel vertical migration cycle of

Microcystis (Westrick et al. 2010). Microcystis cells contain gas vacuoles which can be synthesized or crushed to regulate their buoyancy and therefore their position in the water column (Deacon and Walsby 1990). Buoyant velocity is also driven by the amount of carbohydrate ballast in the cell (Ibelings et al. 1991), the strain of Microcystis (Xiao et al.

2012), and the size of the colonies (Yang et al. 2009, Zhu et al. 2014, Rowe et al. 2016).

Buoyant velocity has been shown to increase with water temperature, possibly due to increased consumption of carbohydrates for lipid and protein synthesis (You et al. 2018).

Studies have shown there may be a diel cycle to buoyancy such that Microcystis tends to rise toward the surface in the morning and early afternoon during calm conditions, and sink in the evening (Ganf 1974, Takamura and Yasuno 1984, Hunter et al. 2008, Cui et 3

al. 2016). This pattern has often been informally observed in western Lake Erie. One explanation for this cycle is the production and consumption of carbohydrate ballast

(Ibelings et al. 1991). Cells accumulate carbohydrates as they photosynthesize during the day, which increases the colony density and leads to sinking. They then consume the ballast as they respire overnight, which decreases colony density and causes the colony to float upwards. This explanation is supported by experiments with Microcystis which relate rates of density change to photo irradiance (Visser et al. 1997). This diel cycle can be interrupted by wind-induced mixing, even at relatively low wind speeds of 3-4 m/s

(Ha et al. 2000, Cao et al. 2006, Ma et al. 2015), though larger colonies are better able to resist mixing by stronger winds than small colonies (Wu and Kong 2009, Aparicio

Medrano et al. 2013). Recent modeling efforts have suggested that Microcystis tend to concentrate in the surface mixed layer, the depth of which is determined by wind and water temperature conditions (Rowe et al. 2016), and that daily cycles in wind-driven turbulence could explain observed Microcystis migrations (Zhu et al. 2018). The effect of wind is important for Microcystis in the WBLE, where wind is a significant driver of vertical mixing and wind speeds of >7 m/s are able to break down weak thermal stratification and fully mix the water column (Loewen et al. 2007, Boegman et al. 2008).

Microcystis grows very well and typically outcompetes other phytoplankton in warm, nutrient-rich water (Paerl and Huisman 2008), leading to late summer HABs in the

WBLE which typically peak between the months of July and September (Bridgeman et al. 2013). It is during these late summer months that drinking water supplies are under the greatest threat, as many near-shore municipalities draw their drinking water from the

WBLE. The city of Toledo’s drinking water intake is located about 5 km offshore in the 4

WBLE in 6-7 m of water. The opening of the intake is located about 1 m off the bottom; therefore, during dense bloom conditions, pumping rates could be increased when the

Microcystis is concentrated at the surface, and decreased when it sinks low in the water column. This would result in lower concentrations of HABs and, possibly, microcystin entering the treatment plant, which would lower treatment costs and better protect public health.

To investigate the feasibility of this strategy and better understand Microcystis vertical migration, five temporally intensive sampling events were conducted from 2016-

2017 under varying weather conditions near the Toledo drinking water intake. In-situ vertical profiles of algal concentrations and environmental parameters were recorded.

Water samples were also collected from discrete depths for algal identification and laboratory analysis of chlorophyll a, phycocyanin, microcystin, and nutrient concentrations. Relationships among algal distributions and environmental conditions were analyzed for trends and significant correlations to provide a better understanding of the nature and drivers of Microcystis diel vertical migration in the WBLE.

Based on the current understanding of Microcystis and previous studies, we expected the following distributions:

 During calm conditions, Microcystis would concentrate near the surface of the water at midday

 During windy conditions, Microcystis would be evenly distributed through the water column

 At night, Microcystis would be evenly distributed through the water column

 Intracellular microcystin distributions would reflect Microcystis distributions

1.2 Methods

1.2.1 Field Sampling

Samples were collected at one site near the City of Toledo’s drinking water intake

(41.701752 N, 83.260765 W, Figure 1-1). Water depth at the site was between 6 and

6.5m during the study. Temporally intensive sampling was conducted five times over two

HABs seasons. Sampling dates were chosen based on bloom density and predicted weather conditions; dense blooms and calm days were targeted to best capture vertical migration undisturbed by mixing. In-situ vertical profiles were conducted every 1-2.5 hrs, complemented by full sample collection at every other time point (2-5 hr intervals)

(Table 1.1). Sampling interval was adjusted for each sampling event to collect as much data as possible given the available crew. Data types are summarized in Table 1.2. All sampling was conducted from the University of Toledo’s research vessel.

Figure 1-1. Location of Toledo drinking water intake and sampling site () in Lake Erie (Google 2017).

Table 1.1. Sampling event dates and time ranges. Sampling interval is the time between vertical profiles, full sampling was conducted with every other vertical profile.

Sampling Dates Start Time End Time Total Duration Sampling Interval

Aug 3-4, 2016 9:00AM 1:00PM 28.0 hrs 2.0 hrs

Aug 18, 2016 6:00AM 9:00PM 15.0 hrs 2.5 hrs

Aug 9-10, 2017 5:00AM 1:00AM 20.0 hrs 2.5 hrs

Aug 15, 2017 5:00AM 8:00PM 15.0 hrs 1.0 hr

Sep 25, 2017 7:30AM 1:30PM 6.0 hrs 1.0 hr

1.2.2 Vertical Profiles

To determine the vertical distribution of HAB species and other algal groups, in- situ vertical profiles were conducted using a YSI EXO2 sonde (YSI EXO2, Xylem, Inc.) and bbe FluoroProbe (FP, bbe Moldaenke, GmbH). The YSI EXO2 was equipped with sensors to measure depth, temperature, conductivity, turbidity, pH, optical dissolved oxygen (ODO), chlorophyll a, and phycocyanin. The FP is a submersible spectroflourometer which uses fluorescence to detect concentrations of algal pigments and then classifies algal groups using pigment fingerprints to determine concentrations of cyanobacteria, green algae, diatoms, and cryptophytes. The FP also records total chlorophyll concentration and is equipped with a depth sensor. Both instruments were slowly lowered over the side of the boat at a rate of 5 cm/sec (20 sec/m) in continuous data logging mode, pausing at each meter below the surface to capture high density observations from the water’s surface to the bottom. The YSI EXO2 recorded data approximately every 1.5 seconds in continuous data logging mode. The FP takes several

readings per second, then averages them over user-defined time intervals and records only the averaged readings. FP averaging intervals were varied between 3 and 10 seconds to maximize data quality. The depth sensor on the FP failed during the 2017 sampling season, and thereafter depths were determined by marking 1 meter increments on the FP cable, pausing at each meter, and manually recording the depths and times of each pause.

Environmental conditions were recorded at each vertical profile time point including wind speed and direction, Secchi depth, cloud cover, wave height, and photosynthetically active radiation (PAR) profile. Wind speed, direction, and wave height are directly related to vertical mixing, which can overcome Microcystis’ buoyancy.

Secchi depth, cloud cover, and PAR profile provide information about light availability through the water column and ultraviolet radiation exposure. Additionally, a buoy operated and maintained by LimnoTech (Ann Arbor, MI) was moored a few hundred meters from our site which logged weather and wave conditions every 10 minutes. Data from this buoy was downloaded and used to calculate average wind speeds, predominant wind directions, and average wave heights for each sampling event.

1.2.3 Full Sample Collection

To complement the in-situ measurements and determine the vertical distribution of microcystin toxin, grab samples of water were collected at 3 discrete depths (1 m, 3 m, and 5 m below surface) using a Van Dorn sampler. These samples were collected with every other vertical profile due to the time required to collect and preserve the samples.

Each sample was filtered and preserved for various laboratory analyses. Unfiltered water was frozen in 250 mL polypropylene bottles for total nutrient analysis and in 50 mL 8

amber glass vials for total microcystin concentrations. Water filtered through 0.45 µm cellulose filters was frozen in 250 mL polypropylene bottles for dissolved nutrient analysis and in 50 mL amber glass vials for extracellular microcystin concentrations.

Water was also filtered in duplicate through GF/F glass microfiber filters; these filters were retained and frozen for extracted chlorophyll a and phycocyanin quantification.

Unfiltered water (100 mL) was preserved using a final concentration of 1% formalin for algal identification using the FlowCam (Fluid Imaging Technologies, Inc.). Additional unfiltered water (300 mL) was preserved in a 400 mL glass jar using Lugol’s solution for algal identification and quantification using microscopy. Finally, 200 mL of water was filtered through a sterile 5 µm filter. The filtrate was then pushed through a Sterivex filter

(MilliporeSigma) using a 60 mL syringe. Both the 5 µm and Sterivex filters were frozen for genetic analysis.

1.2.4 Laboratory Analysis

Microcystin samples (both filtered and unfiltered) were subjected to three freeze/thaw cycles to ensure release of intracellular toxins. They were then analyzed using the Abraxis enzyme-linked immunosorbent assay kit for detection of microcystins

(ELISA, Abraxis, Inc.) according to U.S. EPA Method 546. Samples which initially exceeded the upper limit of the ELISA method (5 µg/L), were diluted 10x and reanalyzed. Prior to ELISA analysis, 10 mL of each of these samples was reserved and stored at -80 ºC; 41 of these samples were analyzed in the Chemistry Department of the

University of Toledo for the presence and concentration of microcystin congeners using liquid chromatography-mass spectrometry (LC-MS) (Palagama et al. 2017). Data from 9

the LC-MS analysis were provided for use in this research (Personal communication, D.

Palagama).

Chlorophyll was extracted from GF/F filters using 10 mL of dimethylformamide

(DMF) and quantified using a Turner 10AU Fluorometer (Turner Designs, Inc.) (Speziale et al. 1984). Phycocyanin samples were analyzed by the National Oceanic and

Atmospheric Administration’s Great Lakes Environmental Research Laboratory (NOAA

GLERL).

Samples preserved with Lugol’s solution were concentrated 12x by overnight settling and preserved for long term storage using a final concentration of 1% formalin.

To confirm FP measurements, a subset of these samples were examined using microscopy. Microcystis colonies and cryptophyte cells were identified and quantified by viewing 1 mL of concentrated, preserved sample on a gridded Sedgewick-Rafter counting cell (Wildco Wildlife Supply Company) with an inverted compound microscope at 400x

(Leica Microsystems GmbH).

Formalin-preserved samples were transported to NOAA GLERL for imaging flow cytometry with a FlowCam. This instrument passes sample through a very thin flow cell, where each particle is spectrally analyzed and photographed, then identified and counted.

This analysis was intended to validate FP measurements of cyanobacteria and cryptophytes, however, the resulting image quality proved insufficient to identify cryptophytes and smaller Microcystis colonies (Figure A-1).

Filters for genetic analysis were transported to Drs. George Bullerjahn and Mike

McKay at Bowling Green State University. DNA extraction was performed using a

PowerWater Sterivex DNA Isolation Kit (MO BIO Laboratories, Inc.). Illumina MiSeq 10

sequencing of the short 16s rRNA gene was used to determine bacterial species composition. Data from this analysis were not included in this thesis.

Nutrient samples (total and dissolved) were stored at -80 °C. A SEAL

AutoAnalyzer 3 HR (SEAL Analytical, Inc.), which colorimetrically quantifies nutrient concentrations, was used to determine concentrations of total and soluble reactive phosphorus.

Table 1.2. Summary of all measured parameters and sources of final data Parameter Data Source Water Depth Vessel sonar Cloud Cover Field observer Wind Speed Anemometer Wind Direction Compass Wave Height Field observer PAR Profile PAR meter Secchi Depth Secchi disk Water Temperature YSI Conductivity YSI ODO YSI pH YSI Turbidity YSI Chlorophyll a (in-situ) YSI Phycocyanin (in-situ) YSI Total Chlorophyll FP Cyanobacteria FP Green Algae FP Diatoms FP Cryptophytes FP Algal Identification (Lugol’s) Microscope Algal Identification (1% formalin) FlowCam Total Nutrients SEAL AutoAnalyzer Dissolved Nutrients SEAL AutoAnalyzer Total Microcystin ELISA kit Extracellular Microcystin ELISA kit Chlorophyll a (extracted) Fluorometer Phycocyanin Fluorometer

1.3 Results

1.3.1 Overview of Study Results

Figure 1-2. MODIS satellite imagery of WBLE bloom conditions on sampling dates (or closest date not obscured by cloud cover). Boxes contain date, average wind speed, and average wave height. Yellow arrows indicate prevailing wind direction and average wind speed. The sampling site is marked by the diamond ().

A range of bloom and mixing conditions were observed over the five sampling events (Figure 1-2). Higher wind and wave conditions were observed in 2016, which was considered a “mild” bloom year according to NOAA’s Lake Erie Harmful Algal Bloom

Seasonal Forecast. Calmer weather was targeted for sampling in 2017, and though the bloom was “significant” according to NOAA, much of the biomass was concentrated away from the drinking water intake. Observed wave heights ranged from 0.14-0.61 m and most often were seen to decrease from morning until late afternoon, then increase

from evening into the night. Observed water temperatures ranged from 19.2-27.9 °C, with warmer temperatures in 2016 than 2017. Typically, temperature, pH, and dissolved oxygen distributions showed a well-mixed water column in the morning, evening, and night, with the exception of September 25, 2017, when an ephemeral thermocline was observed (Figure 1-34). This thermocline was evidenced by a 2 °C drop in temperature from 3-4 m below the surface to the bottom which persisted throughout the sampling event. Additionally, dissolved oxygen concentration and pH both declined precipitously near the bottom of the water column during this sampling event (Figure 1-35, Figure 1-

36), with dissolved oxygen dropping from 9 mg/L in the top half of the water column to

4.5 mg/L at the bottom and pH dropping from 9.2 in the top half of the water column to

8.7 at the bottom. Temperature, dissolved oxygen, and pH all increased near the surface during midday on each sampling date, indicating surface warming and active photosynthesis due to sunlight.

In-situ chlorophyll a concentrations measured with the YSI EXO2 showed similar distributions of algae as the FP throughout the study, though FP readings tended to be higher than YSI EXO2 readings (Figure 1-40). FP readings of chlorophyll a concentrations were highly correlated with extracted chlorophyll a analyzed in the lab (r2

= 0.86), although measurements of extracted chlorophyll a were about double those of the FP. This suggests that both the FP and YSI EXO2 are underreporting true chlorophyll a concentrations in the field. FP algal classification revealed that total chlorophyll was often driven by algal groups other than cyanobacteria during the sampling events.

Diatoms were often the most prevalent algal group, though cryptophytes occasionally made up a significant portion of total chlorophyll and cyanobacteria made up the majority 14

of chlorophyll measured on August 3, 2016. Microscope identification of Lugol’s- preserved samples revealed that Microcystis was by far the most prevalent cyanobacteria present throughout the study, with other genera such as Dolichospermum and

Planktothrix being only occasionally observed.

Extracellular microcystin concentrations were consistently low throughout the study, ranging from 0.03-0.42 µg/L. 81% of these samples were below the Ohio EPA reporting limit of 0.3 µg/L, and 15% were below the Abraxis ELISA detection limit of

0.1 µg/L. Total microcystin concentrations varied throughout the study, ranging from

0.19-3.49 µg/L, with one outlier measured at 8.22 µg/L. This outlier, sampled 1 m below the surface on August 9, 2017 at 3:00 pm, initially exceeded the upper limit of the ELISA method (5 µg/L), therefore was diluted 10x and reanalyzed. However, LC-MS analysis of the undiluted sample determined a concentration of only 3.43 µg/L. In both years, the highest toxin concentrations were observed during the first sampling event of the season in early August and toxin concentrations decreased later in the season (Figure 1-3).

Figure 1-3. Comparison of total microcystin concentrations across all sampling events, as measured with the ELISA method. Toxin concentrations were highest in early August, and decreased as each sampling season progressed.

1.3.2 Sampling Events

1.3.2.1 August 3-4, 2016

Wave heights did not vary much during the day (0.33-0.38 m), but nearly doubled during the night (0.61 m) before subsiding the following day (0.28 m) (Figure 1-4). No samples were collected between 11:00 pm and 5:00 am due to rough lake conditions.

Chlorophyll a concentrations were well mixed through the water column in the morning, evening, and night, but increased near the surface during midday on both days (Figure 1-

4, Figure 1-5). Cyanobacterial concentrations, however, were well mixed throughout the sampling event; the increased chlorophyll a concentrations near the surface were instead due to green algae and cryptophytes (Figure 1-9). No scums were observed during this sampling event. Total microcystin concentrations varied throughout the day and throughout the water column (Figure 1-10). While toxin was evenly vertically distributed

in the morning and the evening, increases were observed at 3 m below the surface (the middle of the water column) on each day at 1:00 pm. These increases were not present in the cyanobacterial distributions. During the night, microcystin concentrations near the surface were substantially higher than those near the bottom, despite corresponding in- situ cyanobacterial concentrations being evening distributed. Microcystis colony concentrations measured using microscope counts did show a slight increase in colonies present at the surface.

Figure 1-4. Chlorophyll a concentrations measured using the YSI EXO2 on August 3-4, 2016. Wave heights are included in each panel. Increased chlorophyll concentrations near the surface were observed on both days at 1:00pm.

Figure 1-5. Chlorophyll a concentrations measured using the FP on August 3-4, 2016. Wave heights are included in each panel. FP chlorophyll distributions agreed with YSI EXO2 measurements, including the concentration of chlorophyll near the surface at 1:00pm on both days.

Figure 1-6. Temperature profiles measured using the YSI EXO2 on August 3-4, 2016. Wave heights are included in each panel. Surface warming was observed beginning in late morning, continuing into late afternoon before mixing completely at night.

Figure 1-7. ODO concentrations measured using the YSI EXO2 on August 3-4, 2016. ODO concentrations increased near the surface during midday, consistent with active photosynthesis. Low concentrations near the bottom on August 3 suggest ephemeral stratification may have been present from the previous day and broke down by the morning of August 4.

Figure 1-8. pH measured using the YSI EXO2 on August 3-4, 2016. pH distribution closely resembled ODO distribution, consistent with diel photosynthesis near the surface and oxygen depletion near the bottom.

Figure 1-9. Vertical profiles of algal concentrations measured with the FP on August 3, 2016. Total chlorophyll distribution was driven by distributions of green algae and cryptophytes throughout the day, while cyanobacteria remained well-mixed and diatom concentrations were very low.

Figure 1-10. Cyanobacteria and microcystin concentrations at discrete depths on August 3-4, 2016. Top row: Cyanobacteria were well mixed from top to bottom during this event with little evidence of rising or sinking. Dashed lines indicate discrete sample depths. Middle row: Total (●) and extracellular () microcystin concentrations. Total toxin was well-mixed in the morning and concentrated in the middle of the water column in the middle of the day. Unexpectedly, during the night much higher concentrations of toxin were present near the surface than at the bottom. Bottom row: Microcystis colony concentrations counted using microscopy. A slight increase near the surface is present at night which was not recorded by the FP cyanobacteria profile.

1.3.2.2 August 18, 2016

Wave heights declined throughout the day, from 0.44 m at 6:00 am to 0.17 m at 4:00 pm (Figure 1-11). An equipment malfunction during the 11:00 am YSI EXO2 profile resulted in the loss of the bottom half of the profile data. Chlorophyll a increasingly concentrated towards the bottom throughout the day. While cyanobacteria did appear to sink throughout the day, the chlorophyll a on this date was mostly composed of diatoms, of which a significant portion sank in the evening (Figure 1-16). No scums were observed on this day. Microcystin concentrations were very low throughout this sampling event (< 1 µg/L). There was an increase in toxin concentrations near the bottom at 11:00 am, however this did not correspond with an increase in cyanobacteria near the bottom

(Figure 1-17).

Figure 1-11. Chlorophyll a concentrations measured using the YSI EXO2 on August 18, 2016. Wave heights are included in each panel. Chlorophyll appeared to sink throughout the day, becoming more concentrated toward the bottom of the lake.

Figure 1-12. Chlorophyll a concentrations measured using the FP on August 18, 2016. Wave heights are included in each panel. FP chlorophyll distributions agreed with YSI EXO2 measurements.

Figure 1-13. Temperature profiles measured using the YSI EXO2 on August 18, 2016. Wave heights are included in each panel. Surface warming peaked in the afternoon with a 2 degree difference from top to bottom, and vertical mixing was observed in the evening.

Figure 1-14. ODO concentrations measured using the YSI EXO2 on August 18, 2016. Wave heights are included in each panel. ODO concentrations were initially well-mixed but increased in the top half of the water column and decreased near the bottom over the course of the day.

Figure 1-15. pH measured using the YSI EXO2 on August 18, 2016. Wave heights are included in each panel. pH distribution closely resembled ODO distribution, consistent with diel photosynthesis near the surface and oxygen depletion near the bottom.

Figure 1-16. Vertical profiles of algal concentrations measured with the FP on August 18, 2016. Diatoms sank throughout the day. Cyanobacterial concentrations were low but also appeared to sink throughout the day. Green algae were concentrated near the surface in midday but evened out by nightfall. Cryptophytes were not present.

Figure 1-17. Cyanobacteria and microcystin concentrations at discrete depths on August 18, 2016. Top row: Cyanobacteria were well mixed from top to bottom in the morning, with evidence of sinking throughout the afternoon and evening. Dashed lines indicate discrete sample depths. Bottom row: Total (●) and extracellular () microcystin concentrations. Microcystin concentrations were very low and mostly evenly distributed. There was an increase in total toxin concentration near the bottom at 11:00 am which was not reflected in the chlorophyll or cyanobacteria measurements.

1.3.2.3 August 9-10, 2017

Wave heights decreased from 0.24 m to 0.14 m throughout the day, and increased to

0.26 m during the night (Figure 1-18). A thin but visible scum of Microcystis was present on the surface of the water in the middle of the day. Extra effort was made at

12:30 pm to read the scum with the FP by jiggling the sensor window at the very surface of the water. The results of this effort can be seen in the dramatic increase in surface concentrations of chlorophyll a and cyanobacteria recorded by the FP, however, this technique was not used elsewhere in the study (Figure 1-19). It is therefore impossible to compare these surface concentrations to others in the study. Total microcystin concentrations were higher near the surface than the bottom, with the exception of the initial 5:00 am sample (Figure 1-24). A particularly high toxin concentration was reported by the ELISA analysis 1 m below the surface at 3:00 pm. However, LC-MS analysis of the undiluted sample determined a concentration of only 3.43 µg/L. Given the distribution of toxin data analyzed in this study and the relationship between the ELISA and LC-MS toxin measurements (Figure 1-42), it is likely that this lower concentration is more accurate.

Figure 1-18. Chlorophyll a concentrations measured on August 9-10, 2017 using the YSI. Wave heights are included in each panel. A slight increase near the surface is seen at 3:00pm. The YSI EXO2 did not read the surface scum we observed. Both YSI EXO2 and FP use fluorescence to detect chlorophyll and may be photo-inhibited at the surface on sunny days.

Figure 1-19. Total chlorophyll concentrations measured on August 9-10, 2017 using the FP. Wave heights are included in each panel. Chlorophyll was concentrated at the surface from late morning until late afternoon, after which it appeared to sink. With the exception of the surface at 12:30pm, the YSI EXO2 and FP reveal similar distributions of chlorophyll.

Figure 1-20. Temperature depth profiles measured on August 9-10, 2017 using the YSI. Wave heights are included in each panel. Surface warming was observed in the afternoon, but mixed completely overnight.

Figure 1-21. ODO concentrations measured on August 9-10, 2017 using the YSI. Wave heights are included in each panel. A large increase in surface ODO occurred throughout the afternoon, then mixed deeper into the water column during the night.

Figure 1-22. pH measured on August 9-10, 2017 using the YSI. Wave heights are included in each panel. Vertical profiles of pH closely resemble those of ODO, especially during midday. In the evening, ODO decreases with depth, but pH increases in the middle of the water column before decreasing again towards the bottom.

Figure 1-23. Vertical profiles of algal concentrations measured with the FP on August 9, 2017. Diatoms dominated the phytoplankton community in the morning and night, but a large concentration of cyanobacteria and cryptophytes was measured at the surface in midday. These surface concentrations are gone by nightfall, but no corresponding increase in concentration is observed lower in the water column.

Figure 1-24. Cyanobacteria and microcystin concentrations at discrete depths on Aug 9- 10, 2017. Top row: Cyanobacteria were well mixed from top to bottom in the morning, concentrated near the surface at midday, and mixed again at night. Dashed lines indicate discrete sample depths. Middle row: Total (●) and extracellular () microcystin concentrations. * = 8.2 µg/L. Microcystin was initially much higher at the bottom than the surface, but the distribution flipped later in the morning, and surface concentrations remained higher than bottom concentrations throughout the rest of the sampling event. Bottom row: Microcystis colony concentrations counted using microscopy.

1.3.2.4 August 15, 2017

Winds were stronger than anticipated and no scum was visible. Wave heights were consistently 0.23 m through the sampling event, except for a drop to 0.17 m around 4:00 pm (Figure 1-25). Cyanobacteria were well mixed throughout the day, and diatoms made up the majority of the algae present (Figure 1-30). Diatom and, therefore, chlorophyll a distributions showed evidence of sinking throughout the day, with concentrations near the bottom increasing and concentrations higher in the water column decreasing. Total microcystin concentrations were low throughout the sampling event and little difference was observed from the surface to the bottom.

Figure 1-25. Chlorophyll a concentrations measured on August 15, 2017 using the YSI. Wave heights are included in each panel. Chlorophyll was well mixed from top to bottom throughout the sampling event.

Figure 1-26. Total chlorophyll concentrations measured on August 15, 2017 using the FP. Wave heights are included in each panel. Chlorophyll profiles were similar between the FP and YSI, though the FP showed greater evidence that algae near the bottom were sinking in the second half of the day.

Figure 1-27. Temperature depth profiles measured on August 15, 2017 using the YSI. Wave heights are included in each panel. Surface warming occurred in the afternoon and into the evening but was more evenly distributed than observed during other sampling events.

Figure 1-28. ODO concentrations measured on August 15, 2017 using the YSI. Wave heights are included in each panel. ODO near the bottom remained fairly constant but increased in the upper half of the water column throughout the afternoon and evening.

Figure 1-29. pH measured on August 15, 2017 using the YSI. Wave heights are included in each panel. Vertical profiles of pH closely resemble those of ODO.

Figure 1-30. Vertical profiles of algal concentrations measured with the FP on August 15, 2017. Diatoms dominated the phytoplankton community and sank towards the bottom throughout the day, while other groups were present in very low concentrations that were evenly distributed through the water column.

Figure 1-31. Cyanobacteria and microcystin concentrations at discrete depths on August 15, 2017. Top row: Cyanobacteria were well mixed from top to bottom in the morning, concentrated near the surface at midday, and mixed again at night. Dashed lines indicate discrete sample depths. Bottom row: Total (●) and dissolved/extracellular () microcystin concentrations. There was a slight increase in surface toxin at 10:00am, but levels were otherwise constant at around 1 µg/L.

1.3.2.5 September 25, 2017

Colonies were visually observed rising towards the surface in the morning, but a scum never formed. Overall concentrations of algae and toxin were much lower than earlier in the season. Water temperature had also decreased and an ephemeral thermocline, persisting from the previous day, was observed (Figure 1-34). ODO concentrations and pH also showed evidence of a thermocline and declined sharply near the bottom of the water column (Figure 1-35, Figure 1-36). Wave heights were low, decreasing from 0.23 m in the morning to 0.15 m by midday (Figure 1-32). FP data for this date was very noisy, and rolling averages were used to reduce the noise for data visualization. This was likely due to the very low algal concentrations present. The FP also experienced a malfunction during the 12:30 pm profile and the top half of the profile data was lost (Figure 1-33). Algal distributions were noticeably different in the top and bottom halves of the water column (Figure 1-37). The bottom half of the water column was primarily cyanobacteria whose distribution stayed constant throughout the sampling event. All algal groups were present in the top half of the water column, and cyanobacteria were observed concentrating 0.5 m below the surface at 8:30 am and 11:30 am. However, this increased subsurface concentration decreased at 9:30 am and disappeared at 10:30 am. Microcystin concentrations were very low throughout this sampling event, and tended to be higher near the bottom (Figure 1-38). Given the low oxygen and low temperature, it is possible the stressed cyanobacteria present near the bottom were producing more toxin than those closer to the surface.

Figure 1-32. Chlorophyll a concentrations measured on September 25, 2017 using the YSI. Wave heights are included in each panel. Slightly elevated concentrations were observed near the surface, especially at 9:30 am. The bottom half of the water column remained constant throughout the sampling event.

Figure 1-33. Total chlorophyll concentrations measured on September 25, 2017 using the FP. Wave heights are included in each panel. A large concentration of chlorophyll was measured just below the surface at 8:30 am, and concentrations were generally higher close to the surface. The bottom half of the water column did not change during sampling. A computer issue resulted in the loss of some data during the 12:30 pm profile. FP readings were very noisy on this date; original data are presented here. For all other analyses and plots, rolling averages were calculated to reduce noise.

Figure 1-34. Water temperature measured on September 25, 2017 using the YSI. While temperatures were mostly constant in the top half of the water column, a dramatic decline in temperature beginning around 3-4 m below the surface suggests an ephemeral thermocline persisted from the previous day.

Figure 1-35. ODO concentrations measured on September 25, 2017 using the YSI. Wave heights are included in each panel. The thermocline led to oxygen depletion at the bottom of the water column, resulting in concentrations as low as 4 mg/L. Very little change in ODO was seen in the top half of the water column.

Figure 1-36. pH measured on September 25, 2017 using the YSI. Similar declines as ODO were observed in the lower half of the water column, resulting in a pH range of approximately 8.6-9.3 through the water column.

Figure 1-37. Vertical profiles of algal concentrations measured with the FP on September 25, 2017. All algal concentrations were very low, with total chlorophyll not exceeding 5 µg/L. In the morning, cyanobacteria and cryptophytes were most concentrated near the surface, then sank towards midday. Cyanobacterial concentrations were steady at about 1 µg/L in the bottom 2 m of the water column. Diatoms were concentrated in the middle and at the bottom of the water column. Diatoms sank through the sampling but did not seem to cross the thermocline, suggesting the thermocline may have prevented those diatoms closer to the surface from sinking to the bottom. Green algae in the top half of the water column increased through the morning but were almost completely absent below the thermocline.

Figure 1-38. Cyanobacteria and microcystin concentrations at discrete depths on September 25, 2017. Top row: Cyanobacterial distribution remained constant in the bottom half of the water column, but varied near the surface. Dashed lines indicate discrete sample depths. Bottom row: Total (●) and dissolved/extracellular () microcystin concentrations. All toxin concentrations were very low, < 0.8 µg/L. Concentrations were higher at the bottom than the surface.

1.3.3 Depth-Integrated Microcystis Biomass Estimates

FP cyanobacteria profiles were used to estimate total Microcystis present in the water column at each sampling point. This was done to investigate how much total

Microcystis biomass was changing, i.e. whether changes in concentration profiles were due to redistribution through the water column or if biomass was being lost or gained through horizontal movement, settling, or scum formation. All profile data was trimmed to only include depths from 0.1-5.5 m to ensure comparable estimates. Cyanobacteria profile data was then integrated over those depths to estimate the amount of Microcystis in the water column. Unexpectedly, decreases in biomass were observed through each sampling event (Figure 1-39). In both 2016 sampling events, biomass estimates at the end of the sampling event were half of their initial amount. The estimates for the 2017 sampling events showed more fluctuation but still appeared to decline throughout the event. The reason for this is not clear, it is possible that Microcystis was floating up above 0.1 m or sinking below 5.5 m during the calm conditions targeted for sampling.

Figure 1-39. Estimates of water column cyanobacterial biomass over each sampling event. Decreases in total biomass were observed during each sampling event, especially during the afternoon and evening.

1.3.4 Analytical Methods Comparisons

Several parameters in this study were measured using more than one analytical method. This was done to corroborate the results and investigate potential biases in the methods. Chlorophyll a was profiled in-situ with both the YSI EXO2 and FP, and also analyzed in the lab by extracting pigment from discrete-depth samples and quantifying with a fluorometer. Cyanobacteria and cryptophyte concentrations were profiled in-situ with the FP and also quantified with microscopy using Lugol’s-preserved, discrete-depth samples. Microcystin toxin samples were analyzed using the Abraxis ELISA method and separately analyzed by LC-MS.

Chlorophyll a measured in the lab correlated well with in-situ FP measurements, with r2 = 0.86 (Figure 1-40). The relationship was not 1:1, however, as chlorophyll a concentrations were always higher when pigments were extracted rather than measured in-situ. There was one exceptionally high extracted chlorophyll a reading of 98.9 µg/L, which was likely an outlier caused by an error in the lab analysis. FP and YSI EXO2 readings of chlorophyll a did not correlate as well as expected, with r2 = 0.43. The YSI

EXO2 readings tended to be noisier than the FP, possibly due to the differences in sampling interval and data processing between the instruments. The FP takes several readings and averages them over user-defined intervals (3 and 10 second intervals were used in this study), while the YSI EXO2 reports individual readings every 1.5 seconds.

The relationship was also not 1:1, the FP tended to read higher concentrations of chlorophyll than the YSI. This could be due to differences in the calibrations of the instruments.

(a)

(b)

Figure 1-40. Comparison of chlorophyll a measurements. (a) Extracted chlorophyll concentrations measured in the lab compared to in-situ FP measurements. Though not 1:1, the FP measurements correlated highly with extracted chlorophyll measurements. (b) Comparison of YSI EXO2 and FP in-situ measurements. Most measurements correlated well, but more variability was present in the YSI EXO2 measurements. 46

FP measurements of cyanobacteria and cryptophyte concentrations were verified using microscope counts of Lugol’s-preserved samples. This was not an ideal comparison as it was not possible to count individual Microcystis cells under the microscope, and an attached camera and software would have been required to measure colonies and estimate cell densities. Instead, microscope counts are reported as colonies/mL, though cells/colony varied considerably. Despite that, a correlation of r2 = 0.54 was found between the FP and Lugol’s Microystis concentrations (Figure 1-41). There was some concern that the FP may be misidentifying some Microcystis as cryptophytes due to higher-than-expected cryptophyte concentrations in some profiles. Therefore, cryptophyte cells were also identified and counted under the microscope, and

Microcystis:cryptophyte ratios were calculated and compared between the Lugol’s samples and FP profiles. This was the best comparison that could be made, however, it was not expected that ratios of Microcystis colonies to cryptophyte cells would match in- situ pigment concentrations. Nevertheless, a weak positive correlation was observed, with r2 = 0.30. Cryptophyte cell concentrations from the Lugol’s samples correlated well with

FP measurements of cryptophytes, with r2 = 0.85, and similar patterns were observed in the depth distributions between the two methods. This verifies the high concentrations of cryptophytes sometimes measured by the FP.

(a)

(b) (c)

Figure 1-41. Comparison of Microcystis and cryptophyte concentrations as measured from Lugol’s-preserved samples and in-situ with the FP. (a) Ratios of Microcystis:cryptophyte concentrations. In the Lugol’s method, Microcystis is measured as colonies/mL and cryptophytes as cells/mL. The FP measures each group as a concentration in µg/L. (b) Microcystis and (c) cryptophyte concentrations compared between the two methods.

ELISA-quantified microcystin concentrations were verified using LC-MS. LC-

MS, while more costly per sample than ELISA, has a much lower detection limit, higher precision and accuracy, and can quantify individual microcystin congeners as well as total microcystins (Palagama et al. 2017). There was a positive, linear relationship between the two methods (r2 = 0.47), though the relationship worsened at higher toxin concentrations (Figure 1-42). Notably, the sample from 1 m below the surface on August

9, 2017 at 3:00 pm measured 8.2 ug/L microcystin with ELISA but only 3.43 µg/L with

LC-MS. This still represents a substantial concentration of toxin, more than three times the World Health Organization’s drinking water guideline of 1 µg/L (WHO 2011) and more than three times the concentration near the bottom of the water column at that time.

The ELISA method tended to report lower toxin concentrations than the LC-MS.

(a)

(b)

Figure 1-42. Comparison of microcystin concentrations measured with ELISA and LC- MS. (a) Extracellular microcystin and (b) all (extracellular and total) microcystin. The two instruments generally agreed, with the exception of one total toxin sample from Aug 9, 2017. The ELISA method (after diluting the original sample) indicated a concentration three times higher than that measured by the LC-MS. 50

1.4 Discussion

1.4.1 Vertical Distributions of Microcystis

Based on previous studies and anecdotal observations, we expected to see

Microcystis rise to the surface in the late morning, form a scum, and then sink back down in the afternoon during calm, sunny weather. While calm weather was targeted for each sampling date, different patterns in vertical distribution were observed during each sampling event. During three of the five sampling events (August 3-4, 2016; August 18,

2016; and August 15, 2017), there was no evidence of Microcystis accumulating near the surface of the water at any time of day. While cyanobacteria were evenly distributed throughout the day on August 3, 2016, on August 4 and August 18, 2016 cyanobacterial concentrations near the surface actually decreased in the early afternoon (Figure 1-10,

Figure 1-17). While cyanobacterial concentrations did not change much in the lower half of the water column, it is possible much of the cyanobacteria settled to the bottom, beyond the range of the FP sensors.

On both August 9 and September 25, 2017, there was evidence of Microcystis accumulating near the water’s surface, though the timing varied (Figure 1-24, Figure 1-

38). On August 9, increased concentrations near the surface coincided with a slight decrease in concentrations near the bottom. This, combined with the observation of scum formation, suggests Microcystis was floating up to the surface during midday. On

September 25, 2017, a thermocline was present at 3-4 m below the surface; physical conditions and algal distributions differed substantially above and below the thermocline.

Below the thermocline, Microcystis was well-distributed and did not change much

throughout the morning. Above the thermocline, however, higher concentrations of

Microcystis were observed near the surface at 8:30 and 11:30 am, but these higher concentrations were not measured at 9:30 am, 10:30 am, or 1:30 pm. These alternating high and low surface concentrations most likely were caused by horizontal patchiness, with varying densities of bloom moving through the sampling site.

Distributions of microcystin toxin were often surprisingly different from

Microcystis distributions, and no significant correlation was observed between cyanobacterial and total toxin concentrations. Extracellular toxins remained consistently low throughout the study and were evenly distributed from top to bottom. There did not appear to be any significant releases of toxin from the cells, which indicates cell lysis and would have caused a noticeable increase in extracellular toxin concentrations. Instead, nearly all variation in toxin levels was observed in the total microcystin concentration, meaning the toxin was contained within intact cyanobacterial cells or bound to particulates. It is therefore puzzling that total toxin concentrations did not correlate well with Microcystis concentrations. There may have been substantial variation in toxin concentration within each cell, and the variation in toxin distribution could be evidence that the more toxic cells were not evenly distributed through the bloom.

1.4.2 Capturing the Full Water Column

One of the biggest challenges of this study was accurately collecting data on the full water column, from the surface to the bottom. Though data were collected with a variety of methods, algal concentrations at and near the water’s interfaces with air and sediment proved difficult to quantify. In-situ instruments measure algal concentrations 52

using fluorescence, which can be inhibited near the surface by sunlight. Additionally, scums which float on the surface may be highly concentrated but only a few millimeters thick. Even though the instruments are “continuously” logging, these scums are often too thin to be measured accurately, and the presence of the instrument at the water’s surface disrupts and mixes the scum. Care must also be taken not to hit the bottom with the instrument, which disrupts the sediment, drastically altering water conditions and clogging sensors with mud. Similar considerations must be taken when collecting water at discrete depths with a Van Dorn or similar sampler. While there are no sensors to be fouled or inhibited, disrupted sediments can contaminate water samples near the bottom.

Methods are being developed to accurately and precisely collect surface water with these samplers and quantify associated scums, but at the time of sampling these methods had not yet resolved issues of surface disruption or appropriate collection volume.

The result of these challenges is that data from the water’s surface and bottom is missing or subject to high uncertainty throughout this study. At one sampling point (Aug

9, 2017 at 12:30 pm, Figure 1-19), extra effort was made to measure the water’s surface with the FP by holding the sensor at the surface of the water, and the data appear to represent the thin scum that was observed. However, this method has not been verified or repeated, so these data cannot be compared to any other data. The incompleteness of water column data means that the full extent of vertical movement cannot be described.

When measured algal distribution changed, we were unable to determine with certainty whether the changes were due to vertical or horizontal movement. The estimates of water column biomass described in section 1.3.3 were intended to elucidate whether total water column biomass was staying constant while vertical distributions changed, if horizontal 53

currents were changing the total water column biomass. However, large decreases in total biomass were observed through each sampling event. Without measurements of the complete water column, it is impossible to determine whether the bloom was actually becoming less dense at our sampling site during these times, or if algae was moving above or below our sampling window (rising to form a scum or settling to the bottom).

1.4.3 Implications for Drinking Water Management

No strong diel trends were observed in the Microcystis or microcystin distributions, except that Microcystis tended to mix evenly through the water column overnight. Due to the shallowness of the lake at the drinking water intake, we observed that even moderate wave conditions can cause enough mixing to distribute cyanobacteria through the water column. Toxin concentrations sometimes varied substantially from top to bottom, even in conditions which evenly mixed Microcystis, as seen on August 3-4,

2016. During very calm events, there was evidence that Microcystis concentrations near the surface increased. It is not clear whether the increase in surface concentrations caused a decrease in concentrations lower in the water column, however, as concentrations near the intake depth (approximately 5m below the surface) tended to stay constant throughout each sampling event. It may also be possible for a small percentage of Microcystis to float and form a scum, while the majority of colonies remain lower in the water column.

If these results are indicative of “normal” conditions at the drinking water intake, then

HAB avoidance based on diel migration would not be very effective at this site.

Therefore, managers of Toledo’s drinking water treatment plant should continue the strategies they are already utilizing: real-time monitoring of cyanobacteria in the intake 54

and surrounding waters, frequent testing of toxin concentrations, and pre-emptive treatment to remove cyanobacteria and toxins. Hopefully, results from this study can inform future studies and lead to a greater understanding of cyanobacterial dynamics in the WBLE.

1.4.4 Recommendations for Future Studies

This ambitious study sought to describe changes in cyanobacterial distribution over diel cycles and relate those changes to environmental factors which may be driving them. However, over the course of the two years of sampling, many challenges were faced, and many lessons learned. Algal distributions measured using different methods all correlated well with one another, showing similar patterns in vertical distribution. These different methods did not share 1:1 relationships, though, so care should be taken not to compare concentration magnitudes between methods (for instance, comparing extracted chlorophyll near the surface to FP-measured chlorophyll concentrations near the bottom).

Many different kinds of data were sampled at each sampling point, which could be streamlined in the future to allow denser algal profile data to be collected with fewer crew. The FP provided the best correlation to established in-lab methods while collecting much denser data than possible with discrete samples, and is very useful for determining algal distributions through much of the water column. The top and bottom of the water column remain challenging to monitor, and future studies should explore how best to capture a complete in-situ water column distribution. It would also be helpful to collect in-situ vertical algal profiles hourly over the course of several consecutive days. This would allow better understanding of how consistent vertical distributions are from day to 55

day. HAB conditions can change rapidly during the bloom season, and spacing events out over two seasons meant that different bloom conditions and compositions were observed on each sampling event. Microcystin remains expensive and labor-intensive to quantify, and this study would have greatly benefited from an accurate in-situ toxin sensor to provide denser vertical data on toxin distribution, as the difference between toxin and cyanobacterial distributions was one of the most unexpected results of this study. To differentiate vertical and horizontal movement, continuous measurements of vertical and horizontal currents at the study site should be included in future studies of vertical migration, or other sources of these data (such as hydrodynamic model outputs) should be included. This would help determine how much vertical mixing Microcystis can overcome, and how much distributions are being affected by the horizontal movement of the bloom through the basin. Experiments to isolate vertical from horizontal movement could be conducted by constructing a 6-7 m long, 1 m wide, transparent tube which would be deployed in the lake and filled with water from the sampling site. Profiling the water within the tube in addition to the open-lake water column would provide valuable data on vertical movement independent of horizontal currents.

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Appendix A

Supplementary Figures

Figure A-1. Example of FlowCam output from August 3, 2016 at 1:00 pm, 1 m depth. Image quality is sufficient to identify large colonies of Microcystis, but insufficient to identify smaller colonies or cryptophytes.