Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms

Journal of Great Lakes Research xxx (xxxx) xxx

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Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms Dilrukshika S.W. Palagama a, David Baliu-Rodriguez a, Brenda K. Snyder b, Jennifer A. Thornburg a, Thomas B. Bridgeman b, Dragan Isailovic a,⇑ a b

Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606, United States Lake Erie Center and Department of Environmental Sciences, University of Toledo, Toledo, OH 43606, United States

a r t i c l e

i n f o

Article history: Received 7 May 2019 Accepted 2 January 2020 Available online xxxx Communicated by R.E. Hecky

Keywords: HABs Microcystins Quantification Liquid chromatography-mass spectrometry

a b s t r a c t Liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) were used to provide qualitative and quantitative information about microcystin (MC) congeners in western Lake Erie. Samples were collected at eight open-water locations on selected days during harmful algal blooms (HABs) in 2016 and 2017. Seven MCs were identified and 20 MCs were tentatively identified using highresolution mass accuracies and a unique fragment (Adda m/z 135). The most abundant MC was MC-LR, followed by MC-RR, MC-YR, and MC-LA, and these congeners were quantified. Total (extracellular and intracellular) MC concentrations ranged from 0.068 to 14.88 mg/L in 2016, and from 0.050 to 10.15 mg/L in 2017, with averages of 2.71 and 1.86 mg/L, respectively. Near-shore sites in Lake Erie had higher MC concentrations and Microcystis biovolumes than off-shore sites. This implies that nutrient loading from the Maumee River greatly influences Maumee Bay, and this influence decreases with distance from the river. Consequently, six MCs (MC-LR, MC-RR, MC-LA, MC-YR, MC-LW, and MC-LF) were quantified in water samples collected from the Maumee River and the Maumee Bay shore of Lake Erie in 2017, and MC-RR was the most abundant. The total MC concentrations in river samples ranged from 0.17 to 305.03 mg/L. Additionally, an MC degradation product (linear MC-LR) was detected at all open-water locations, and data indicated an increase in its concentration towards the end of the bloom. The trends for 2016 and 2017 HABs are comparable in terms of spatial distribution and MC congeners produced, though the intensity and peak dates change. Ó 2020 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction Lake Erie supports a robust ecosystem and economy due to its diverse biota and contributions to the industrial and tourism sectors, and it supplies water to a population of over 11 million (Watson et al., 2016). Increased cyanobacterial populations have threatened western Lake Erie biota and water use. An increase in nutrients in Lake Erie provides a eutrophic environment for the cyanobacteria that flourish during the late summer months. The resulting harmful algal blooms (HABs) are problematic because of their size and their toxic byproducts (Ho and Michalak, 2017). Western Lake Erie is particularly susceptible to cyanobacterial growth due to its shallow depth, which averages 7.3 m (MacIsaac et al., 1992). Its largest watershed drains agricultural and suburban landscapes via the Maumee River as well as sewage and fertilizer runoff (Wu et al., 2009) from other watersheds. Nutrients ⇑ Corresponding author. E-mail address: [email protected] (D. Isailovic).

originating from the watershed, particularly phosphorus and nitrogen, allow for excessive growth of phytoplankton when the ambient temperature increases above 25 °C and when radiation from sunlight is the most intense (Bigham et al., 2009). The increase in temperature and nutrient availability changes the composition of the phytoplankton from diatoms to green algae to cyanobacteria (Bigham et al., 2009). Cyanobacteria are responsible for several toxins present in western Lake Erie; the most common and among the most harmful are microcystins (MCs) (Chorus and Bartram, 1999). Cyanobacteria, such as Microcystis, Dolichospermum, Nostoc, and Planktothrix, produce MCs (Berry et al., 2017; Rinta-Kanto et al., 2009a,b). Cyanobacteria may also cause problems due to their sheer mass. Microcystis, the most common cyanobacterium found in western Lake Erie, congregates and forms potentially enormous algal blooms that choke narrow waterways, block sunlight, and deplete oxygen (Golnick et al., 2016). The water becomes hypoxic when dissolved oxygen levels fall below 2 mg/L, and affected biota may suffocate (Watson et al., 2016). The cyanobacterial genera

https://doi.org/10.1016/j.jglr.2020.01.002 0380-1330/Ó 2020 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

Dolichospermum, Nostoc, and Planktothrix are often present and abundant in rivers and bays, but contribute little to the algal biovolume of the open waters of western Lake Erie (Jankowiak et al., 2019; Rinta-Kanto et al., 2005, 2009b). Various methods have been used to investigate phosphorus and nitrogen loading, algal biovolume, dissolved oxygen content, and chlorophyll a concentration in Lake Erie in an effort to characterize trends in algal abundance (Bridgeman et al., 2012; Chaffin et al., 2014a; Conroy et al., 2005; Golnick et al., 2016; Ho et al., 2017; Kane et al., 2014; Kim et al., 2014; Maccoux et al., 2016; Matisoff et al., 2016; Millie et al., 2009). Microcystis and MC-LR concentrations have also been detected and quantified in different locations of western Lake Erie (Bridgeman et al., 2013; Foss and Aubel, 2015; Rinta-Kanto et al., 2009a, 2005; Wang et al., 2009). Several MC congeners were identified and quantified in Lake Erie using LC-MS and LC-MS/MS, including MC-LR, MC-RR, MC-YR, MC-LY, and MC-WR (Foss and Aubel, 2015). Real-time PCR and ELISA were applied to quantify Microcystis and MCs, respectively (Foss and Aubel, 2015; Rinta-Kanto et al., 2009a, 2005; Wang et al., 2009). Additionally, MC degradation products such as linear MC-LR and a tetrapeptide were detected in bench experiments with bacteria and water from Lake Erie (Thees et al., 2019). Many studies have found a relationship between nutrient discharge from the Maumee River and cyanobacterial blooms in western Lake Erie (Bridgeman et al., 2012; Chaffin et al., 2014a; Conroy et al., 2005; Maccoux et al., 2016; Matisoff et al., 2016). Phosphorus loading from the river influences the development of harmful algal blooms when combined with recycling of existing phosphorus sources in the lake’s bottom sediments (Bridgeman et al., 2012; Matisoff et al., 2016). Studies show that bloom size depends on the amount of phosphorus arriving from the western Lake Erie watershed (Conroy et al., 2005; Ho and Michalak, 2017; Stumpf et al., 2012). Environmental conditions, such as water pH, wind, and temperature, may delineate chlorophyll a and phytoplankton biovolume patterns, and competition for resources can also affect bloom size (Millie et al., 2009). Further research, including determination of different MCs during HABs, is necessary to understand these relationships and their impact on algal toxicity. In this study, LC-MS and LC-MS/MS were used to identify and quantify MC congeners present at eight locations in western Lake Erie during 2016 and 2017 algal blooms. Microcystis biovolume data was also collected from the same sites and correlated to MC concentration. Water samples from the Maumee River and the western Lake Erie shore were collected in 2017, and MC congeners were quantified in those samples. The spatial and seasonal differences in MC congeners and MC concentration in western Lake Erie were studied over two years, and the influence of the Maumee River on production of microcystins is investigated in different areas of western Lake Erie. Experimental Reagents HPLC-grade acetonitrile, methanol, and water were purchased from Fisher Scientific (Pittsburgh, PA, USA). HPLC-grade formic acid (FA) was obtained from Sigma (St. Louis, MO, USA). Standard solutions of 500 mg/L of MC-LR, 500 mg/L of MC-LA, and 100 mg/L of MC-RR were bought from Cayman Chemical Company (Ann Arbor, MI, USA). MC-LW, MC-LF, and MC-YR were purchased as solids from Enzo Life Sciences (Farmingdale, NY, USA) with purities > 95%. Materials and instrumentation Sep-pak C18 cartridges were obtained from Waters Corporation (Milford, MA, USA). Surfactant-free cellulose acetate membrane

filters (0.45 mm) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Three and 10 mL syringes were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Two mL clear glass vials and glass inserts were obtained from Sigma (St. Louis, MO, USA). The heated vacuum concentrator was from Eppendorf (Hamburg, Germany). An Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source was used in this study. The HPLC was a Shimadzu Prominence (Shimadzu Technologies, Addison, IL, USA) equipped with a DGU-20A3 degasser, two LC-20AD binary pumps, a SIL20A HT autosampler, and a CBM-20A system controller. HPLC separation was performed using an XBridge C8 column (100 mm
3. 0 mm ID, 3.5 lm particle size, Waters, Milford, MA). A C8 guard column (20 mm
3.0 mm ID, 3.5 mm, Waters, Milford, MA) was used. Lake Erie and Maumee River site description and water sample collection Lake samples were collected at eight open-water locations (MB20, 7M, 8M, 4P, GR1, MB18, Crib, and Buoy) in western Lake Erie (Fig. 1A, Electronic Supplementary Material (ESM) Table S1) during 2016 and 2017 algal blooms. MB20, MB18, and Buoy are near-shore sites with depths  2.5 m. The three intermediate sites, 8M, 7M, and Crib, have depths of 5–6 m. Two off-shore sites, GR1 and 4P, have depths of 8.5–10 m. Samples were collected between July 7th and August 10th, 2016, and July 31st and October 13th, 2017. Depth-integrated water samples were collected at each site every 5 to 21 days between 9 am and 4 pm using a flexible tube sampler that sampled the water column from the surface to within 1 m of the lake bottom. The tube sampler was emptied into a bucket and stirred thoroughly, producing a water sample that was integrated across all depths (Golnick et al., 2016). Amber glass vials for toxin analysis were filled from the bucket and stored on ice for transport to the laboratory. Upon return to the laboratory, a portion of the sample water was filtered through 0.45-mm membrane filters to remove cells. Both whole-water and filtered samples were processed within 5 h of initial collection and then placed in 20 °C storage in amber glass vials until analysis. Surface water samples were collected from five shore sites (L1-L5) and from eight locations in the Maumee River in 2017 (Fig. 1, ESM Table S1). All samples were stored at 20 °C until analysis. Lake and river water sample preparation Both extracellular and total MCs were analyzed in open-water samples. Extracellular MCs were analyzed from previously-filtered samples. Total MC concentration was determined from wholewater river and coastal samples by freezing and thawing samples three times to lyse cells before filtration. Solid-phase extraction (SPE) was performed to purify and preconcentrate the samples. Biovolume collection During collection of lake water samples, additional samples were taken for the determination of Microcystis biovolume. Microcystis and other plankton were collected at each site between 9 am and 4 pm in vertical tows from near the lake bottom to the surface using a 0.5-m diameter, 112-lm mesh net equipped with a flow meter to calculate net efficiency. All plankton tows were concentrated to a volume between 100 and 500 mL and immediately preserved in 4% buffered (pH = 7.5) sugar-formalin. Microcystis in the tows was separated from remaining plankton by a 48-hr settling procedure, and biovolumes of the settled colonies were

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

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Fig. 1. Sampling sites (Google Maps) in (A) Western Lake Erie, and (B) Maumee River. Water samples were collected from Western Lake Erie at eight open-water locations (MB20, MB18, Buoy, 8M, 7M, Crib, GR1, and 4P) in both 2016 and 2017, and five locations along the shore in 2017 (L1-L5). Maumee River samples were collected only in 2017.

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

determined in graduated 1 L Imhoff cones. To obtain an areal estimate of Microcystis (mL/m2) present at each sample location and date, the average biovolume (mL) from the replicate tows measured in Imhoff cones was divided by the area of the plankton net (0.196 m2). Extraction of microcystins A stock solution of MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) was prepared in methanol and stored at 20 °C. Matrix-matched external standard calibration solutions were made by diluting the stock solution in MC-free lake water. SPE was performed for calibration standards, lake samples, and river samples. Frozen lake and river samples were thawed to room temperature and vortexed. Five mL of water were transferred into a 50 mL centrifuge tube, and SPE filtration was performed. The SPE cartridges were conditioned with 2 mL of 90:10 methanol:water (v/v) containing 0.1% FA and equilibrated with 2 mL of 0.1% FA. The samples were loaded onto the cartridges and washed with 2 mL of 0.1% FA. MCs were eluted with ~1.75 mL of 90:10 acetonitrile:water (v/v) containing 0.1% FA. The solvent was evaporated using a vacuum concentrator and the residue was reconstituted in 500 lL of 90:10 methanol:water (v/v) containing 0.1% FA for LC-MS analyses. Liquid chromatography and mass spectrometry Initially, MCs were detected and identified using HPLC-orbitrapMS. Elution was achieved using a binary gradient of 0.05% FA in water (mobile phase A) and acetonitrile containing 0.05% FA (mobile phase B) with a flow rate of 0.3 mL/min. The column was equilibrated with 20% of mobile phase B for 30 min prior to initial sample injection. The gradient started with 20% B, was increased to 90% B in 12 min, was decreased to 20% B over 2 min, and was held for 6 min at 20% B until the next injection. ESI-full scan-MS in positive ion mode was used for MC detection. The ESI parameters were described in a previously developed method (Palagama et al., 2017). MS/MS was performed simultaneously to identify the Adda fragment (m/z 135). Higher-energy collisioninduced dissociation was used to fragment MC precursor ions. High-resolution MS (HRMS) was used to detect MC ions with mass accuracy < 3 ppm in 200–2000 m/z range using the orbitrap mass analyzer, and fragment ions were analyzed using the linear ion trap mass analyzer in 50–2000 m/z range. SIM-MS and MS/MS were performed for further confirmation of tentatively identified MCs. The most abundant MCs were separated and quantified using a previously developed HPLC-ESI-SIM-MS method (Devasurendra et al., 2018; Palagama et al., 2017). The mobile phases, flow rates, and injection volumes were the same as formerly mentioned. The gradient started with 20% B and was increased to 60% B from 0.1 to 2 min, 70% B from 2 to 7 min, 90% B from 7 to 12 min, and then was decreased to 20% B over 2 min. The column was re-equilibrated for 6 min at 20% B. ESI-SIM-MS in positive ion mode was used for MC quantification (Devasurendra et al., 2018; Palagama et al., 2017). SIM channels were monitored for singly-charged protonated ([M + H]+) ions of MC-LR (m/z 995.56), MC-YR (m/z 1045.54), MC-LA (m/z 910.49), MC-LF (m/z 986.52), MC-LW (m/z 1025.53), MC tetrapeptide (m/z 615.34), and linear MC-LR (m/z 1013.56), and for the doubly-charged protonated ion ([M + 2H]2+) of MCRR (m/z 519.79) using the orbitrap mass analyzer. All samples were analyzed in triplicate. MS and MS/MS data were acquired and processed using Xcalibur software (Thermo Scientific). The extractedion chromatogram (EIC) peak areas of monoisotopic MC ions were used for data calculation. The relative abundance of the linear MCLR ion (m/z 1013.56) was used to understand MC-LR degradation trends during HAB events.

Results and discussion Detection and identification of MCs in open water Open-water samples collected during 2016 and 2017 algal blooms were purified using SPE and subjected to LC-HRMS and MS/MS to identify MC congeners. A prior study indicated that 5 mL of water is sufficient to achieve very low limits of quantification (LOQs) of MCs in water (Palagama et al., 2017). Initially, full scan mode was used to search for monoisotopic masses of MC ions that were reported in the literature (Meriluoto et al., 2017). SIM channels were created for those ions, and MS/MS was performed simultaneously to check for characteristic MC fragmentation patterns. Two factors were used to tentatively confirm that the ion corresponded to an MC: HRMS spectra with good mass accuracy, and MS/MS spectra with Adda fragment signal at m/z 135. The MC ions were singly- or doubly-charged ([M + H]+ or [M + 2H]2+), and their isotopic distribution patterns are shown in ESM Fig. S1. Twenty-seven MC congeners were identified across both years after analyzing total MCs by LC-HRMS, and are listed in Table 1. Twenty-three out of 27 MCs were detected with mass accuracy < 3 ppm, and the other 4 MCs had mass accuracy  5.3 7 ppm. All MCs had the characteristic Adda fragment ion at m/z 135 ([C9H10O + H]+), and most MCs showed fragment ions at m/z 213 ([Glu + Mdha + H]+) and m/z 375 ([C11H14O + Glu + Mdha + H]+). Seven MC structures (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, MC-LW and linear MC-LR) were confirmed by comparing the MS/ MS pattern to an MC standard or to literature (Meriluoto et al., 2017). The structures of the 20 other MCs could not be definitively assigned because some MCs have the same exact masses, and signal intensity was too low for detailed MSn spectra or amino acid sequence determination. Furthermore, MC standards were not available for most congeners, and structures were tentatively assigned based on the most common MC with the best mass accuracy. Although their structures could not be confirmed, mass accuracies of those ions were excellent and correlate well with literature values (Meriluoto et al., 2017). Additional experiments should be performed to confirm the structures of those MCs. Twenty MC congeners were detected in both 2016 and 2017 (Table 2). Three congeners ([D-Asp3, ADMAdda5]MC-LR, [ADMAdda5]MC-LR, and [D-Asp3]MC-(H4)YR) were only found in 2016, and 4 congeners ([Asp3, Dha7]MC-LY, [Asp3, MeSer7]MC-LR, [D-Ser1, ADMAdda5]MC-LR, and MC-HphR) were only found in 2017. This may be due to fluctuations in the very low concentrations of these MCs, or due to differences in MC expression by cyanobacteria. Detection of both highly abundant and rare MCs is important in order to obtain information on their appearance in Lake Erie water and investigate their toxic effects. Rare MCs may potentially become more abundant in Lake Erie due to climate change and changes in land use patterns. Therefore, their detection and toxicity to humans and animals should be closely monitored. According to literature, the source of MCs in Lake Erie are Microcystis, Dolichospermum, Planktothrix, and Nostoc among others, and those genera of cyanobacteria can be present in western Lake Erie (Rinta-Kanto et al., 2005, 2009b). Microcystis is the most common and has been thought to be the primary MC producer (Chaffin et al., 2014b). Further studies should be conducted to confirm the diversity of toxin-producers in locations where high MC concentrations were observed.

MC distribution based on location in the lake Twenty-seven identified MCs (Table 1) were found in the 8 open-water locations (MB20, MB18, Buoy, 8M, 7M, Crib, GR1, and 4P) (Table 2). During both years, MC-LR, [Mdhb7]MC-LR, MC-RR,

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx Table 1 MCs identified in open-water samples with their chemical formulas, calculated and experimental m/z values, and mass accuracies. Microcystina 3

7

ǂ

[D-Asp , Dha ]MC-RR [Dha7]MC-RRǂ MC-RRǂ MC-LA MC-AR [D-Asp3, Dha7]MC-LR [Asp3, Dha7]MC-LYy [Dha7]MC-LR MC-LF MC-LR [Asp3, MeSer7]MC-LRy MC-LY [D-Asp3,ADMAdda5]MC-LR* [Mdhb7]MC-LR Linear MC-LR [ADMAdda5]MC-LR* MC-LW MC-M(O)R MC-FR [D-Asp3]MC-YR [D-Asp3]MC-(H4)YR* [D-Ser1, ADMAdda5]MC-LRy MC-HphRy MC-YR [Asp3, ADMAdda5, Dhb7]MC-RR [D-Glu(OMe)6]MC-YR MC-WR

Structureb

Chemical formula

Experimental m/z

Calculated m/z

Mass accuracy (ppm)

Tentative Tentative Confirmed Confirmed Tentative Tentative Tentative Tentative Confirmed Confirmed Tentative Tentative Tentative Tentative Confirmed Tentative Confirmed Tentative Tentative Tentative Tentative Tentative Tentative Confirmed Tentative Tentative Tentative

C47H72N13O12 C48H74N13O13 C49H76N13O12 C46H68N7O12 C46H69N10O12 C47H71N10O12 C50H68N7O13 C48H73N10O12 C52H72N7O12 C49H75N10O12 C48H75N10O13 C52H72N7O13 C49H73N10O13 C50H77N10O12 C49H77N10O13 C50H75N10O13 C54H73N8O12 C48H73N10O13S C52H73N10O12 C51H71N10O13 C51H75N10O13 C50H75N10O14 C53H75N10O12 C52H73N10O13 C49H74N13O13 C53H75N10O13 C54H74N11O12

505.7726 512.7800 519.7906 910.4925 953.5067 967.5239 974.4875 981.5410 986.5234 995.5577 999.5530 1002.5179 1009.5316 1009.5727 1013.5679 1023.5565 1025.5328 1029.5071 1029.5400 1031.5199 1035.5508 1039.5459 1043.5580 1045.5350 1052.5510 1059.5514 1068.5520

505.7716 512.7825 519.7899 910.4921 953.5091 967.5248 974.4870 981.5404 986.5234 995.5561 999.5510 1002.5182 1009.5353 1009.5717 1013.5666 1023.5510 1025.5343 1029.5074 1029.5404 1031.5197 1035.5510 1039.5499 1043.5560 1045.5353 1052.5524 1059.5510 1068.5513

1.98 4.88 1.35 0.44 2.52 0.93 0.51 0.61 0.00 1.61 2.00 0.30 3.67 0.99 1.28 5.37 1.46 0.29 0.39 0.19 0.19 3.85 1.92 0.29 1.33 0.38 0.66

Confirmed: mass accuracies  1.61 for MS peaks with appropriate MS/MS fragmentation pattern. a Abbreviations: ADMAdda, O-acetyl-O-demethylAdda; Asp, aspartic acid; Dha, dehydroalanine; Dhb, dehydrobutyrine; (H4)Y, 1,2,3,4-tetrahydroalanine; Hph, homophenylalanine; MeSer, N-methylserine; M(O), methionine S-oxide. b Tentative: mass accuracies  5.37 for MS peaks, and Adda fragment at m/z 135 was detected. y MCs detected only in 2017 samples. * MCs detected only in 2016 samples. ǂ Doubly-charged ions.

Table 2 Distribution of MCs identified in western Lake Erie during 2016 and 2017 algal blooms; D: Detected with S/N ratio > 3; –: Not Detected, S/N ratio < 3. Microcystin

2016

2017

MB20 [D-Asp3, Dha7]MC-RR [Dha7]MC-RR MC-RR MC-LA MC-AR [D-Asp3, Dha7]MC-LR [Asp3, Dha7]MC-LY [Dha7]MC-LR MC-LF MC-LR [Asp3, MeSer7]MC-LR MC-LY [D-Asp3,ADMAdda5]MC-LR [Mdhb7]MC-LR Linear MC-LR [ADMAdda5]MC-LR MC-LW MC-M(O)R MC-FR [D-Asp3]MC-YR [D-Asp3]MC-(H4)YR [D-Ser1, ADMAdda5]MC-LR MC-HphR MC-YR [Asp3, ADMAdda5, Dhb7]MC-RR [D-Glu(OMe)6]MC-YR MC-WR Total number of MC congeners

D D D D D – – D D D – D – D D D D D D D – – – D D D D 20

2016

2017

MB18 – D D D D – D D D D D D – D D – D D D D – D D D D D D 22

D D D D D – – D – D – D – D D – D – D D – – – D D D – 16

2016

2017

8M D D D D D – D D – D – D – D D – D – D D – D D D – D – 18

– D D D D D – D – D – D D D D D – D D D D – – D D D D 20

2016

2017

Buoy D D D D D D D D – D – D – D D – D D D D – D D D D D – 21

– – D D – – – D – D – – – D D – – D D D – – – D – D – 11

2016

2017

7M D – D D D – D – – D – D – D – – – D D D – – D D – D – 14

– – D D – – – – – D – – – D – – – D D D – – – D – D – 9

2016

2017

Crib – – D D D – – – – D – D – D – – – D D D – – D D – D – 12

– – D D – – – – – D – – – D – – – D D D – – – D – D – 9

2016

2017

GR1 – – D D D – – – – D – – – D – – – D D D – – D D – D – 11

– – D D – – – – – D – – – D – – – – – D – – – D – – – 6

2016

2017

4P – – D D D – – – – D – – – D – – – – D D – – – D – – – 8

– – D D – – – – – D – – – D D – – – – D – – – D – – – 7

– – D D – – – – – D – – – D D – – – D D – – – D – – – 8

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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MC-YR, [D-Asp3]MC-YR, and MC-LA were identified at every site. Five MCs were only present at a single location: [D-Asp3, Dha7] MC-LR, [D-Asp3, ADMAdda5]MC-LR, and [D-Asp3]MC-(H4)YR were only found in 8M, and the latter two only in 2016. [Asp3, MeSer7] MC-LR and MC-LF were only present in MB20, and the former only in 2017. The greatest diversity of MCs was in 8M, with 25 of the 27 congeners identified in either 2016 or 2017. 8M was followed by MB20, MB18, Buoy, 7M, Crib, GR1, and 4P, with 24, 19, 16, 12, 11, 8, and 8 different congeners across both years, respectively. The Buoy and 8M sample sites had the greatest year-to-year difference, with only 56% and 64% common congeners, respectively. All other sites had 75% common congeners. Nine MC congeners were detected at the Toledo water intake (Crib) in both 2016 and 2017. Differences in MC congeners may be affected by both time and location. Environmental factors can affect cyanobacterial presence and their MC production. MC genes may not always be expressed, and the concentration of some MCs may be below the method detection limit. Although Microcystis is the dominant genus in western Lake Erie HABs, genetic studies should be performed to confirm the presence of specific cyanobacteria. Overall, toxin populations were similar, with 74% of MC congeners found in both years. This indicates that the cyanobacterial bloom is repeating, in keeping with research showing that Microcystis blooms may be seeded by overwintering cells resting in the lake sediments from the previous year (Kitchens et al., 2018). More MC congeners can be found at near-shore locations than off-shore locations, likely because nutrient loading from the Maumee River leads to an increase in growth of cyanobacteria in the Maumee Bay area. Furthermore, different cyanobacterial species originating in the Maumee River may flow into the lake, which leads to a greater number of MC congeners. Quantification of extracellular MCs in western Lake Erie MCs produced by cyanobacteria are sequestered in bacterial cells and released upon cell lysis. MCs found freely in water are called extracellular MCs, and are quantified by filtering water without breaking bacterial cells. Extracellular MCs were quantified in samples collected at 8 locations in western Lake Erie during 2016 and 2017 algal blooms (ESM Fig. S2). MC-LR, MC-RR, MC-YR, and MC-LA were the most abundant in extracellular water samples, and other identified MCs were not quantified due to low abundance and/or lack of standards. In 2016, the highest extracellular concentration was generally observed for MC-LR, followed by MC-RR, MC-YR and MC-LA. MCLR, MC-RR, and MC-YR were detected at all locations from early July to mid-August, and the greatest abundances were observed in mid to late July. Contrary to this trend, MC-LA increased in abundance in mid-August for all locations despite very low or negligible concentrations in early July. At MB18, 7M, Crib, and GR1, the MCLA concentration was higher than the MC-YR concentration in midAugust, and at GR1 and Crib, MC-LA had a higher concentration than MC-RR, and nearly as high as MC-LR. In the beginning of the season, MC-YR had a higher concentration than MC-LR, MCRR and MC-LA at MB20, Crib, 8M, MB18, Buoy, and 4P. Similar observations were made for 2017 extracellular water samples. MC-LR, MC-RR, MC-YR and MC-LA were quantified while other MCs were below the quantification limit. MC-LR was the most abundant MC congener at every location, followed by MCRR. The highest concentration of all quantified MCs was on August 21. MC-YR and MC-LA were present in very low concentrations throughout the summer, and therefore trends were difficult to discern. MC-YR was present at every date and location during the season, while MC-LA was sometimes not detected. Average MC-LR, MC-RR, MC-YR, and MC-LA extracellular concentrations on sampling dates during 2016 were 0.111, 0.074,

0.050, and 0.017 mg/L, respectively (Fig. S3). The average concentration ratios of MC-LA to MC-LR, MC-YR to MC-LR, and MC-RR to MCLR were 0.15, 0.45, and 0.67. The concentration ratios of MC-LA and MC-YR to MC-LR had significant deviations from the average during the bloom. The ratio of MC-LA to MC-LR increased by about 4x from July to August (~0.10 to 0.41), and MC-YR/MC-LR was 1.15 in July but fell to 0.45 in mid-August. Possibly, MC-YR was released at a higher rate at the start of the season, and MC-LA was released at a higher rate at the end of the season, but this may also be due to different degradation rates of MCs in lake water. The ratio of MC-RR to MC-LR only had slight deviations, and the concentration of MC-RR was about two thirds that of MC-LR. Average extracellular MC-LR, MC-RR, MC-YR, and MC-LA concentrations in samples collected during 2017 were 0.092, 0.036, 0.0086, and 0.012 mg/L, respectively (Fig. S3). The average concentration ratios of MC-LA, MC-YR, and MC-RR to MC-LR were 0.13, 0.092, and 0.36, respectively. The values did not change significantly during the season, indicating that the congeners were released in the same proportions. However, the ratio is not constant year-to-year. MC-YR/MC-LR and MC-RR/MC-LR concentration ratios decreased in 2017, while MC-LA/MC-LR stayed about the same. Though all extracellular MCs had lower concentrations in 2017 than 2016, MC-RR and MC-YR saw a steeper decline (ESM Fig. S3). The difference in MC congener ratios may be due to higher production of MC-LR relative to other MCs in 2017. Table 3 summarizes the range and the average extracellular concentrations of four MCs at each location, as well as the peak bloom dates in 2016 and 2017. The extracellular MC concentration of 2016 samples ranges from 0.021 to 0.68 mg/L, with an average of 0.21 mg/L. Peak concentrations were observed on July 20th or July 25th in all locations except for GR1 and 4P, which peaked on August 10th. In 2017, the extracellular MC concentration range was from 0.024 to 0.40 mg/L, and the average was 0.14 mg/L. The concentrations for all locations peaked on August 21st. In both years, higher average extracellular MC concentrations were seen in near-shore sites than off-shore sites. Quantification of total MC concentration in western Lake Erie Samples were frozen and thawed three times to release cellbound MCs in order to determine total (intracellular and extracellular) MC concentration (Fig. 2). Although 27 MC congeners were identified in 2016 and 2017, only four (MC-LR, MC-RR, MC-YR, and MC-LA) were above the quantification limit. Because extracellular MC concentration offers a snapshot of the MCs being produced by cyanobacteria, trends should be similar for total and extracellular MCs. As expected, the most prevalent congener was MC-LR, followed by MC-RR, MC-YR, and MC-LA. These MCs were detected at every location in both years, with the exception of MC-YR in GR1 on September 8th, 2017. During summers of 2016 and 2017, the total concentration of the four quantified MCs in our samples did not exceed USEPA-recommended limit for recreational exposure to MCs of 8 mg/L (www.epa.gov, 2019) at all open water locations except MB20 (Fig. 2). The average concentrations of MC-LR, MC-RR, MC-YR, and MCLA during sampling dates of the summer of 2016 were 1.34, 0.80, 0.26, and 0.22 mg/L, respectively, while in 2017, the average concentrations were 0.97, 0.46, 0.13, and 0.10 mg/L, respectively (Fig. 3). The average total concentration of each MC was lower in 2017 than in 2016, which correlates well with extracellular concentration data. The total MC concentration was about ten times higher than the extracellular MC concentration in both years. The average ratios of MC-LA/MC-LR, MC-YR/MC-LR, and MC-RR/MCLR in 2016 were 0.17, 0.20, and 0.60, respectively, and 0.11, 0.13, and 0.47, respectively, in 2017. These ratios were significantly lower in 2017 than 2016, meaning that MC-LR concentration was

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx Table 3 The range and average extracellular MC concentrations of four MCs (MC-LR, MC-RR, MC-YR, and MC-LA) at eight locations on sample collection dates in 2016 and 2017. Location

MB20 MB18 Buoy 8M 7M Crib GR1 4P Overall a

2016 extracellular MC concentration (mg/L)

2016 peak bloom date

Range

Average

0.070–0.49 0.100–0.44 0.096–0.42 0.083–0.68 0.065–0.34 0.082–0.34 0.021–0.18 0.059–0.18 0.021–0.68

0.31 0.25 0.24 0.28 0.16 0.19 0.12 0.12 0.21

2017 extracellular MC concentration (mg/L)

7/20 7/20 7/25 7/20 7/20 7/20 8/10 8/10

2017 peak bloom date

Range

Average

0.046–0.26 0.056–0.38 0.058-0.40a 0.047–0.30 0.041–0.30 0.024–0.20 0.037–0.16 0.051–0.26 0.024–0.40

0.14 0.14 0.23a 0.14 0.11 0.12 0.10 0.13 0.14

8/21 8/21 8/21 8/21 8/21 8/21 8/21 8/21

Samples only collected on two dates.

MC-LA

12.0 8.0 4.0 0.0

6.0

3.0

7/31/2017

8.0

2.0 1.0 0.0 7/7/2016

7/20/2016 Date

8/10/2016

8/21/2017 9/8/2017 Date

9/22/2017

D) 2017: MB18

C) 2016: MB18

3.0

6.0

4.0

2.0

0.0 7/31/2017

8/21/2017 9/8/2017 Date

9/22/2017

F) 2017: Buoy

E) 2016: Buoy

6.0

5.0

Concentration (μg/L)

Concentration (μg/L)

B) 2017: MB20

9.0

8/10/2016

Concentration (μg/L)

Concentration (μg/L)

7/20/2016 Date

4.0

6.0

Total

MC-LR

0.0 7/7/2016

5.0

MC-RR

12.0

A) 2016: MB20

Concentration (μg/L)

Concentration (μg/L)

16.0

MC-YR

4.0 3.0 2.0

4.0

2.0

1.0 0.0

0.0 7/7/2016

7/20/2016 7/25/2016 Date

8/10/2016

7/31/2017

8/21/2017 Date

Fig. 2. Total (extracellular and intracellular) concentration of four MCs (MC-LR, MC-RR, MC-YR, and MC-LA) at 8 locations during sample collection dates in 2016 and 2017.

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

8.0

6.0 4.0 2.0 0.0

2.0

7/31/2017

3.0 I) 2016: 7M

4.0

2.0

0.0

8/21/2017 9/8/2017 Date

9/22/2017

J) 2017: 7M

2.0

1.0

0.0 7/7/2016

7/20/2016 Date

8/10/2016

7/31/2017

6.0

4.0 3.0 2.0

8/21/2017

9/8/2017 Date

9/22/2017

10/13/2017

L) 2017: Crib

K) 2016: Crib Concentration (μg/L)

Concentration (μg/L)

4.0

8/10/2016

Concentration (μg/L)

Concentration (μg/L)

7/20/2016 Date

6.0

5.0

6.0

0.0 7/7/2016

8.0

H) 2017: 8M

G) 2016: 8M Concentration (μg/L)

Concentration (μg/L)

8.0

4.0

2.0

1.0 0.0

0.0 7/7/2016

7/20/2016 8/1/2016 Date

8/10/2016

7/31/2017 8/21/2017 9/8/2017 9/22/2017 10/13/2017 Date

Fig. 2 (continued)

higher relative to other MCs in 2017. The ratios were similar to the extracellular MC ratios, with one exception. MC-YR/MC-LR in 2016 was 0.45 for extracellular MCs, but 0.20 for total MCs. The difference suggests that MC-LR and MC-YR were released and/or degraded at different rates in those two years. Total (extracellular and intracellular) concentrations of four MCs and the MC concentration ranges at each location on selected dates during two years are shown in ESM Table S2. The MC concentrations at all sites ranged from 0.068 to 14.88 mg/L in 2016, and from 0.05 to 10.15 mg/L in 2017. The average MC concentration was 2.71 mg/L during the 2016 collection period, and 1.86 mg/L during the 2017 collection period. The highest concentration was observed at MB20. Generally, the near-shore sites had the highest MC concentrations, and the off-shore sites (GR1 and 4P) had the lowest. In 2016, the peak MC concentrations were observed

between July 20th and August 10th, and in 2017, the peak was likely between August 21st and September 9th. MC concentrations measured for selected days of 2016 and 2017 were compared to understand the peak bloom seasons. The average total concentration of MCs during the beginning, middle, and end of the collection periods are shown in ESM Fig. S4. In 2016, the beginning, middle, and end dates are July 7th, July 20th, and August 10th. In 2017, those dates are July 31st, August 21st, and September 22nd. The MC concentrations at the beginning of the seasons are similar (0.45 mg/L in 2016 and 0.36 mg/L in 2017). The middle saw more deviation, as the 2017 bloom was 46% larger (3.24 vs 4.72 mg/L). By the end of the season, MC concentration in 2016 was ~10x greater than in 2017 (4.31 vs 0.41 mg/L). These deviations may be due to differences in collection periods. In 2016, sample collection ended on August 10th, but peak MC

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

4.0 M) 2016: GR1

0.8

Concentration (μg/L)

Concentration (μg/L)

1.0

0.6 0.4 0.2 0.0 7/20/2016 Date

2.0

1.0

8/10/2016

7/31/2017

2.0 O) 2016: 4P Concentration (μg/L)

Concentration (μg/L)

3.0

0.0 7/7/2016

2.0

N) 2017: GR1

1.5 1.0 0.5 0.0 7/7/2016 7/20/20168/10/2016 Date

8/21/2017

9/8/2017 9/15/2017 Date

9/22/2017 10/13/2017

8/21/2017

9/8/2017 9/15/17 Date

9/22/2017 10/13/2017

P) 2017: 4P

1.5

1.0

0.5

0.0 7/31/2017

Fig. 2 (continued)

Quantification of MCs in water samples collected on the Lake Erie shore and the Maumee River

3.00

Concentration (μg/L)

2.50 2016 2.00 2017 1.50 1.00 0.50 0.00

MC-LA MC-YR MC-RR MC-LR Total

Fig. 3. Averages of the total (extracellular and intracellular) concentrations of each MC congener at 8 sites during the sample collection dates in 2016 and 2017.

concentrations in 2017 were later during the summer, e.g., on August 21st. Seasonal coverage was limited in 2016, specifically in MB20, MB18, GR1, and 4P locations, potentially constraining the ability to capture peak blooms. Ideally, the collection period would show a decrease in MC concentration, as seen in late September and October of 2017. It is therefore possible that the 2016 sample collection period did not properly represent the 2016 algal bloom. The total concentration of four MCs at each site on a given day are given in Fig. S5. Dates were chosen for each year to represent the beginning, middle, and end of the sampling period. Sites are roughly arranged in location nearest to furthest offshore. The trend shows that MC concentration generally decreases as a function of distance from Maumee River. It should be noted that total MC concentration relates to the four most abundant MCs that were quantified.

Surface water samples were collected from five sites (L-1, L-2, L-3, L-4, and L-5; Fig. 1B) on the Lake Erie shore on August 25th and September 4th, 2017, and cells were lysed to measure total MC concentration. MC-LR, MC-RR, MC-YR, MC-LW, MC-LF and MC-LA were quantified using LC-MS (Palagama et al., 2019). The most abundant MC congener was MC-RR, followed by MC-LR. All six MCs were detected in water samples collected at every location in August, but MC-LA, MC-LF and MC-LW were not detected at sites L3, L4, and L5 on September 4th. The MC concentration ranged from 3.7 to 13,605.9 mg/L, and average concentrations of MCRR, MC-LR, MC-YR, MC-LW, MC-LF and MC-LA were 2105.5, 733.5, 84.5, 32.1, 11.0, and 5.7 mg/L, respectively (Palagama et al., 2019). MC concentrations were significantly higher than in openwater samples. The same six MCs were detected in open-water samples, but MC-RR was the most abundant on the shoreline, as opposed to MC-LR. MC-LF and MC-LW could not be quantified in open-water locations due to low concentrations, but are present in significant amounts along the lake shore. The increased concentration on the shore relative to open waters may be explained by lake currents or by wind. Water samples in shore locations were collected from the surface, where buoyant Microcystis cells may accumulate. Open water samples were collected at depths as great as 10 m, where Microcystis cells may not be as abundant. MCs were also quantified in Maumee River samples collected on September 25th or 26th, 2017 (Table 4) during an unusually large Microcystis bloom event in the river. The same six congeners were quantified as in Lake Erie shore samples. Similar to the lake shore samples, the most abundant MC congener was MC-RR, followed by MC-LR. Total MC concentration ranged from 0.17 to 305.03 mg/L, and average concentrations of MC-RR, MC-LR, MC-YR, MC-LA, MC-LF and MC-LW were 22.81, 11.57, 5.78, 8.75, 3.89, and 5.06 mg/L, respectively. Although the concentration of MCs

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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Table 4 Total concentrations of six MCs in 2017 Maumee River samples. Location

2017 Date

Concentration (mg/L) MC-LR

MC-RR

MC-YR

MC-LA

MC-LF

MC-LW

Total

Walbridge park Bayshore Boat Launch Maritime Plaza Middleground Metro Park Maritime Museum The Docks

9/25 9/25 9/25 9/25 9/25 9/25

0.09 2.69 20.65 15.47 54.68 16.98

0.19 4.70 22.07 34.25 128.49 20.99

0.02 0.68 3.44 5.03 41.89 5.25

D 0.26 9.42 9.98 34.21 4.77

D 1.23 0.52 5.41 20.21 5.24

D 1.31 0.61 8.54 25.54 6.51

0.30 10.87 56.71 78.67 305.03 59.73

The Docks Napoleon Middleground Metro Park Cullen Park

9/26 9/26 9/26 9/26

1.30 0.05 2.75 1.06

4.38 0.08 10.93 2.02

0.52 0.00 0.71 0.30

ND ND 2.01 0.58

0.25 0.01 1.24 0.89

0.31 0.03 1.68 1.01

6.76 0.17 19.33 5.87

detected in lake shore samples was much higher than in river samples, similar trends in the MC congeners were observed. The difference in concentration of MCs quantified along the shore compared to the river may be due to the river’s current. The river flows downstream and the water is constantly replenished. The bacteria and MCs are expelled into Maumee Bay, where they may be concentrated onto the shore by wind and wave action. High levels of MC-RR and the presence of MC-LF and MC-LW in both the river and the lake shore implies that those MCs may originate from cyanobacteria more commonly present in the river and find their way to the Lake Erie shore. Microcystis biovolume and microcystin content Microcystis biovolume and MC content (mg of four quantified MCs per mL of Microcystis) at collection sites in western Lake Erie in 2016 and 2017 are given in ESM Table S3 while Fig. 4 shows biovolume and MC content trends from selected sites to represent near-shore, intermediate, and off-shore locations. MC content is the toxin mass per volume of Microcystis. Significant spatial and seasonal heterogeneities were observed in both biovolume and MC content. The highest biovolumes in both years were observed in MB20, followed by MB18, Buoy, 8M, 7M, Crib, GR1, and 4P, with slight deviation in the intermediate locations. The greatest biovolume, 715.25 mL/m3, was observed in MB20 in 2016, with a decrease in 2017. However, all other sites had greater peaks in 2017 than 2016. The yearly peaks were observed in late July to mid-August in 2016, and shifted to late August and early September in 2017. MC content in 2016 was highest at 4P and MB20 on July 20th (0.10 and 0.095 mg/mL, respectively), and at 4P on August 21st and 8M on July 31st (0.035 and 0.036 mg/mL, respectively) in 2017. In both years, MC content decreased at most sites at the end of the sample collection period. The exceptions were Buoy, where MC content was approximately constant in both years, and Crib in 2017, where MC content increased. Overall, MC content was more than two times higher in 2016 than in 2017. MC-LR degradation product distribution Due to their cyclic structures, MCs are relatively stable under a wide pH and temperature range (Schmidt et al., 2014). However, the toxins degrade naturally through photodegradation and biodegradation (Schmidt et al., 2014). MC-LR degradation was investigated in Lake Erie during two seasons. Linear MCLR (ESM Fig. S6A) and MC tetrapeptide (ESM Fig. S6B) were detected with excellent mass accuracies (<3.0 ppm) as [M + H]+ ions at 1013.5679 and 615.3388 m/z, respectively. The tetrapeptide was tentatively identified because its standard and MS/MS spectrum were not available. Abundance of the

tetrapeptide was lower than the abundance of linear MC-LR, and this peptide was not detected in most of the locations during sample collection dates in both years. Monoisotopic-ion peak areas of linear MC-LR were compared at each location over two years to understand the spatial and seasonal distribution of degradation products (ESM Fig. S7). Linear MC-LR was detected in all locations, and peak areas showed an increase of linear MC-LR towards the end of the bloom. The ion was not detected on the first sampling date of both years. Generally, peak areas increased as the season progressed. The highest abundance of linear MC-LR was in MB20; and on average, nearshore sites had the greatest abundance, while off-shore sites had the least. No linear MC-LR was detected at Buoy in 2017, likely because only two samples were collected during that season. Generally, abundance was lower in 2017 than 2016. Linear MC-LR was the most prominent at the end of the summer, possibly due to accumulation of this degradation product.

General observations for cyanobacterial blooms in 2016 and 2017, and possible rationales Sampling locations were selected to represent diverse environmental conditions in western Lake Erie. Results indicate that the MC congeners and their concentrations, and Microcystis biovolumes varied among sites. This study suggests that MC congeners are widespread across western Lake Erie, with more variety and accumulation in Maumee Bay. The Maumee River and Lake Erie shore samples had similar trends in the presence of MC congeners. The greatest total MC concentrations and Microcystis biovolumes were observed mostly in Maumee Bay. As expected, areas that experience intense bloom events have higher MC concentrations. MC content decreased as the season progressed (Fig. 4). A possible cause is that cyanobacteria depleted the nutrients necessary for MC production by the end of the bloom season, and thus were not able to continue producing MCs, as has been observed in other studies (Gobler et al., 2016). The Maumee River is the major source of sediments and nutrients entering western Lake Erie, and the collection sites in western Lake Erie are affected by the river’s flow (Baker et al., 2014; Bridgeman et al., 2013; Matisoff et al., 2016). Near-shore sites experience greater influence of the river, and the Maumee River can be attributed to the growth of existing cyanobacteria and the introduction of different cyanobacterial species containing ratios of MCs that differ from the lake. The high proportion of MC-RR found at Maumee Bay shoreline sites suggests river influence at these sites and has relevance for nearby beach users, since MC-RR may be less toxic to mammals than MC-LR (Ito et al., 2002). The Maumee River influence is reduced as distance from the river and lake depth increase, as evidenced by the declining MC concentration in intermediate and off-shore sites.

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

D.S.W. Palagama et al. / Journal of Great Lakes Research xxx (xxxx) xxx

11

Fig. 4. Microcystis biovolume and MC content distribution representing near-shore, intermediate, and off-shore sites during 2016 and 2017 algal blooms.

Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002

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MCs in western Lake Erie were most abundant during late July to early September. This correlates with favorable weather conditions for cyanobacterial growth. Genetic and environmental factors such as temperature, pH, wind, and sunlight affect the variation in MC production in Lake Erie (Bigham et al., 2009; Marmen et al., 2016; Wood et al., 2008). In Florida lakes, Microcystis growth increases when the ambient temperature rise from 25 to 28 °C and when radiation from sunlight is most intense (Bigham et al., 2009). Also in Florida lakes, the phytoplankton community changes from diatoms to green algae to cyanobacteria with increasing temperature (Bigham et al., 2009). Annual climate variations may change the timing of bloom events in Lake Erie, which explains the temporal difference in peak MC concentration in 2016 and 2017. Otherwise, the western Lake Erie algal bloom is similar in both years.

Conclusion Results indicate that MC congeners and concentration, Microcystis biovolume, and mass of MCs per Microcystis biovolume (MC content) varied spatially and temporally between 8 sites in western Lake Erie. More MCs congeners were found at near-shore than off-shore locations. MC-LR, MC-RR, MC-YR, and MC-LA were quantified in open-water sites, and in addition to those MCs, MC-LW and MC-LF were quantified in the Maumee River and western Lake Erie shore. MC-LR was the most abundant in open-water samples, and MC-RR was the most abundant in river and shore samples. Peak MC concentrations were observed between late July and early September, but MC-LR degradation products generally increased after the season peaks. Most locations had elevated (1.0 mg/L) total MC concentrations during both years. The highest MC concentrations and Microcystis biovolumes were found in near-shore sites, and the lowest in off-shore sites. This implies that nutrient loading from the Maumee River stimulates cyanobacterial growth in Maumee Bay, and the river’s influence decreases with distance from the shore. Similar results in MC congeners and spatial distribution of the MCs in western Lake Erie suggest that the bloom is repeating year-to-year. Differences include intensity of the bloom and peak bloom dates. These results are useful for researchers to predict when elevated MC concentrations are likely to occur in Lake Erie based on Microcystis biovolume and other environmental factors. This study can lead to development of an MC database for Lake Erie, and could further advance understanding of the risk of algal toxicity. Although research was conducted in western Lake Erie, this study may be useful to understand potential MC trends in other lakes with cyanobacterial blooms. Overall, this research will assist in monitoring and understanding MC-associated problems in and around Lake Erie. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the Harmful Algal Bloom Research Initiative grants from the Ohio Department of Higher Education. Funding and support for the Orbitrap Fusion instrument was provided by the Air Force Office of Scientific Research (DURIP 14RT0605). The authors also gratefully acknowledge The University of Toledo and The University of Toledo Lake Erie Center.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jglr.2020.01.002.

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Please cite this article as: D. S. W. Palagama, D. Baliu-Rodriguez, B. K. Snyder et al., Identification and quantification of microcystins in western Lake Erie during 2016 and 2017 harmful algal blooms, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2020.01.002