TECHNICAL REPORT NO. 93 TECHNICAL REPORT NO. 77

Survey of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

May 2018

Final Report

Prepared by: Mark Swinton Sandra Nierzwicki-Bauer Darrin Fresh Water Institute

For: The Lake Champlain Basin Program and New England Interstate Water Pollution Control Commission This report was funded and prepared under the authority of the Lake Champlain Special Designation Act of 1990, P.L. 101-596 and subsequent reauthorization in 2002 as the Daniel Patrick Moynihan Lake Champlain Basin Program Act, H. R. 1070, through the US EPA and the Great Lakes Fishery Commission. Publication of this report does not signify that the contents necessarily reflect the views of the states of New York and Vermont, the Lake Champlain Basin Program, the Great Lakes Fishery Commission, or the US EPA.

The Lake Champlain Basin Program has funded more than 90 technical reports and research studies since 1991. For complete list of LCBP Reports please visit: http://www.lcbp.org/media-center/publications-library/publication-database/

NEIWPCC Job Code: 0100-310-029 Final Report

NEIWPCC Job Code: 0100-310-029

Project Code: L-2016-058

Prepared by: Mark Swinton Sandra Nierzwicki-Bauer Darrin Fresh Water Institute

Date Submitted: January 2018

Date Approved: May 2018

Survey of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Contact Information ────────────────────────────────────────────────────────────────── Sandra Nierzwicki-Bauer, Associate Director

Darrin Fresh Water Institute 5060 Lake Shore Drive Bolton Landing, NY 12814

phone (518) 644-3541 FAX (518) 644-3640 [email protected] This is a Lake Champlain Basin Program funded project 54 West Shore Road Grand Isle, VT 05482 802.372.3213 www.lcbp.org Final Report Form v.1.2016 (Revised: 11/3/2016) Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

This project was selected for funding by the Lake Champlain Basin Program (LCBP) Steering Committee and it has been supported directly by an agreement or sub-award issued by the New England Interstate Water Pollution Control Commission (NEIWPCC). NEIWPCC manages LCBP’s personnel, contracts, grants, and budget tasks through a partnership with the LCBP Steering Committee.

Although the information in this document may have been funded wholly or in part by the United States Environmental Protection Agency (under agreement CE982720010), the National Park Service, or by the International Great Lakes Fishery Commission, through their respective contracts to NEIWPCC, it has not undergone review by the Agency, Service, or Commission, and no official endorsement of the content of the document should be inferred. The viewpoints expressed here do not necessarily represent those of NEIWPCC, the LCBP, the USEPA, the NPS, or the GLFC, nor does mention of trade names, commercial products, or causes constitute endorsement or recommendation for use.

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Executive Summary

Mercury and cyanotoxins in Lake Champlain pose health concerns to humans and the ecosystem. Mercury poisoning through the consumption of contaminated fish has been well documented for more than half a decade, typically with top predators posing the greatest threat. And while the most common route of cyanotoxin intoxication is exposure through drinking water and recreational contact, research has shown cyanotoxin levels in fish can reach concentrations that pose health risks, if consumed. The aim of this study was two-fold, 1) to reassess fish mercury throughout the lake to determine which species at what size pose a health concern, identify areas that are disproportionally impacted by mercury and assess long-term changes, along with 2) determining if cyanotoxins are present in fish, and if so, do concentrations in fish correlate with presence in water samples.

More than 600 fish of five species (smallmouth bass, walleye, lake trout, yellow perch and white perch) from the seven segments of Lake Champlain (South Lake, South Main Lake, Main Lake, North Main Lake, Malletts Bay, Northeast Arm and Missisquoi Bay) were analyzed for total mercury. While all fish species had specimens exceed the US EPA mercury advisory limit of 300 ppb, walleye and smallmouth bass had 38% (28/74) and 17% (27/157) of their specimens, respectively, exceed the USFDA action limit of 1000 ppb. Fish length and location were significant factors explaining mercury variability for the five species tested, however, no consistent trend was observed for location among species. Because these species include cold, cool and warm-water fish feeding from benthic and pelagic food webs along with different growth rates and efficiencies, utilizing fish mercury concentrations to determine lake segments that are disproportionately affected by mercury was inconclusive.

Assessing long-term mercury trends in fish shows a significant decrease in lake trout, walleye and yellow perch from their initial mercury surveys (1987-1990). Smallmouth bass and white perch did not show a significant decrease from their initial surveys in the mid-1990s. An unexpected finding was the increase in smallmouth bass and yellow perch mercury concentrations since the 2011 study. Similar findings have been documented in the Great Lakes region and Ontario with proposed explanations including enhanced deposition from Asia, invasive species and climate change. These along with impacts of Hurricane Irene in 2011 are plausible explanations for the increase in Lake Champlain fish mercury but require additional research.

Cyanotoxins (microcystins, anatoxin-a and cylindrospermopsin) were measured in water samples collected throughout the summer and fish samples during low and high bloom periods from the Main Lake and Missisquoi Bay. Analysis utilized HPLC coupled with tandem mass spectrometry able to detect microcystin metabolites, a technical advancement over ELISA that can react with non-microcystin metabolites leading to spuriously high values. However, all water and fish measurements were below the detection level agreeing with VT DEC data showing no microcystin or anatoxin in water samples during this time period. Although correlations of cyanotoxin concentrations between water and fish could not be compared due to non-detectable levels, the study validated the method used for microcystin detection and demonstrated microcystin, anatoxin and cylindrospermopsin did not bioaccumulate in fish as cyanotoxins were present in 2015.

Page 3 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Table of Contents

Page

Executive Summary ...... 3 1 Project Introduction ...... 5 2 Tasks Completed ...... 8 3 Methodology ...... 11 4 Quality Assurance Tasks Completed ...... 155 5 Deliverables Completed ...... 16 6 Conclusions ...... 54 7 References ...... 62 8 Appendices ...... 65

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Project Introduction This project focused on health risks associated with the consumption of fish contaminated by mercury and cyanotoxins. Mercury biomagnifies through the foodweb and top predatory fish can reach levels to warrant fish consumption advisories. The mercury portion of this study is part of a recurring study that began three decades ago that reassesses mercury in fish to help better inform the public of health risks. More specifically, this study was designed to answer how mercury burden in fish varies among species and location with special attention to the influence of body condition, measured as relative weight.

Cyanotoxins have become more prevalent in the last decade, likely a result of warmer waters and eutrophication. Typical exposure to cyanotoxins occurs through ingestion or direct contact but research has shown fish can accumulate cyanotoxins (Paerl and Paul 2012, O'Neil et al. 2012). Therefore, this study was designed to determine the extent to which Lake Champlain fish accumulate cyanotoxins, and how do the concentrations in water samples correlate to those in fish tissue.

MERCURY

Mercury is a ubiquitous pollutant that despite knowing the neurological, developmental and sometime fatal effects since the Minamata Bay, Japan poisoning during the 1950s (Selin 2009), is the most frequent cause for fish consumption advisories worldwide with >35% of the US freshwaters having some fish consumption advisories due to elevated methylmercury (Ward et al. 2010). Mercury is incorporated into the atmosphere from anthropogenic and natural sources, primarily in elemental form which can stay in the atmosphere for more than a year allowing it to travel around the world (UNEP 2013). When it enters the aquatic environment through atmospheric deposition and runoff, it can become methylated by sulfur and iron-reducing bacteria in anoxic environments (UNEP 2013). The preferential binding of methylmercury (MeHg) to thiol groups (Gabriel & Williamson 2004) enables methylmercury to associate with muscle which bioaccumulates within an organism and biomagnifies throughout the foodweb (Marvin- DiPasquale et al. 2009, Ward et al. 2010). It is the biomagnification in higher trophic levels that creates health concerns when predatory fish are consumed by humans, birds and other predatory animals. By systematically testing higher trophic level fish, advisories can be implemented to minimize the health risk to humans and potentially influence regulatory entities.

Mercury loading to Lake Champlain occurs primarily through tributary discharge (26 kg/yr) and atmospheric deposition (18 kg/yr) which account for 94% of all inputs (Gao et al. 2006). The South Lake experiences the greatest loading from tributaries with point source loading (2.7 kg/yr) from a wastewater treatment facility which results in this segment of the lake having the highest water mercury concentrations (Gao et al. 2006). However, mercury loading to the lake is not the primary factor controlling mercury bioaccumulation in lower trophic levels. If it was, zooplankton in the South Lake would

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain exhibit significantly higher mercury concentrations than any other segment of Lake Champlain, which is not the case.

Chen et al. (2012) determined that productivity and algal biomass had the greatest influence on mercury concentration in zooplankton. A brief assessment of zooplankton in 2009 (8/5-9/10) across 12 sites lake-wide determined the highest mercury concentrations in zooplankton were found in the least productive area of Lake Champlain (Malletts Bay) and the lowest mercury concentrations were found in the most productive area (Missisquoi Bay). Data collected from 2005-2008 on these two bays showed that zooplankton in Malletts Bay consistently had higher mercury concentrations than those collected from Missisquoi Bay (Chen et al. 2012). The effect of productivity on mercury burden is a well-established theory known as “bloom dilution” which states that if there is a greater biomass to distribute a finite amount of a conserved substance then the concentration of that substance will be lower on average than if fewer organisms were available to endure the burden. A study by Driscoll et al. (2007) showed that fish from waterbodies with total phosphorus (TP) concentrations < 30 µg/L were more likely to exceed the 300 ppb fish consumption advisory set forth by the US EPA than fish from lakes with TP > 30 µg/L.

To assess mercury concentrations in fish throughout Lake Champlain, the DFWI enlisted the expertise of Dr. Richard Bopp at RPI for mercury analyses and Lake Champlain International (LCI) to carry out the fish collection. Incorporating historic fish mercury data allowed for a long-term assessment of fish mercury in Lake Champlain, which appeared to be declining based on the last Lake Champlain fish mercury study (Johnson 2012). These data can be used to inform government agencies responsible for issuing fish consumption advisories in New York, Vermont and Canada.

CYANOTOXINS

Cyanobacterial blooms have become a frequent occurrence in both marine and freshwater systems around the world due to anthropogenic eutrophication and climate change (Paerl et al. 2011). Under ideal conditions, including ample light, warm water temperature (>20 C), excessive nutrients, and quiescent water, cyanobacteria blooms can become prolific and have the potential to produce cyanotoxins that raise health concerns for humans and the environment (Hudnell 2010). These toxins have been detected at multiple trophic levels and include: algae, zooplankton, macrophytes, macroinvertebrates, bivalves, crayfish, amphibians and fish (Papadimitriou et al. 2012, Wood et al. 2014, Wiegand & Pflugmacher 2005). Cyanotoxins became a health concern in Lake Champlain in 1999 when several dogs died as a result of cyanobacteria exposure (Shambaugh et al. 2015). Beginning in 2002, efforts by the LCBP working in partnership with NOAA’s MERHAB-LGL program created an annual cyanobacteria monitoring program that continues today under the state of Vermont. Microcystis blooms have been documented in most areas of Lake Champlain with the most frequently impacted segments being Missisquoi Bay and the Northeast Arm (Boyer et al. 2004,

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Mihuc et al. 2005; 2006). Over the 2000-2004 seasons, almost 600 water samples were tested for the full suite of cyanobacterial toxins including microcystins, anatoxin-a, PSP toxins and cylindrospermopsins (Boyer 2008). Only anatoxin-a and microcystins were found in these samples, with microcystin at detectable levels in nearly 50% of the samples and exceeding the WHO drinking water guidelines of 1 µg/L in 12% of the samples. Peak levels of microcystins have exceeded 200 µg/L or 10x the current WHO recreational contact limit in samples collected in the northern portions of the lake with the microcystin congeners LR, RR, LF, YR, FR and several dimethyl variations of these microcystins present (Boyer et al. 2004).

The most common method of microcystin intoxication is exposure through drinking water or recreational contact (Carmichael & Boyer 2016; Backer et al. 2010). However, microcystins can also accumulate in fish tissues, sometime at extremely high levels (see Schmidt et al. 2013 and references cited within). This raises the possibility of intoxication through foodweb transfer by consumption of fish exposed to microcystins. Detection of microcystins in fish tissue is complicated by the fact that fish rapidly metabolize the toxin through the GSH pathway, and also forms a series of demethylated derivatives (Schmidt 2014; H Raymond, Ohio EPA personal communication). Early studies often used enzyme-linked immunosorbent assays (ELISA) to detect the toxins due to their low concentration in fish tissues, however this assay cross-reacts with other non-microcystin metabolites in fish tissues which can lead to spuriously high values. For this reason, most researchers have shifted to using HPLC coupled with tandem mass spectrometry (LC-MS/MS) to measure microcystins in fish (Schmidt et al. 2013; 2014; Cadel-Six et al. 2014). This technique also has the advantage that it can identify the different metabolites in fish, providing some information on the rate of detoxification (Schmidt et al. 2014).

To assess cyanotoxins in water and fish samples, the DFWI enlisted the expertise of Dr. Greg Boyer at SUNY-ESF for cyanotoxin analyses and LCI to carry out the fish collection. LCI was responsible for collecting fish (smallmouth bass, yellow perch and brown bullhead) in the Main Lake and Missisquoi Bay during low and high periods of cyanobacterial blooms.

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Tasks Completed Task 1-QAPP was completed as described with quality control protocols for mercury and cyanotoxins as outlined by respective laboratories.

Task 2-PI meeting was completed as described with investigators from RPI, SUNY ESF and LCI meeting at LCI on May 24th to discuss study details and responsibilities of each entity.

Task 3-Water Collections were completed as described with VT DEC (Angela Shambaugh and Pete Stangel) coordinating and collecting water samples during their routine long-term sampling project, however, originally a total of 10 collections dates were anticipated but only 6 were collected because blooms were infrequent during 2016. Water samples were filtered in the field and sent to Dr. Boyer’s lab at SUNY ESF for analysis following protocols (collection, transport and analysis) outlined by Dr. Boyer. Samples were analyzed for microcystins, anatoxin and cylindrospermopsin as outlined in Section B of the QAPP.

Task 4-Tissue Plug Instruction was completed as described; all LCI employees and volunteers responsible for collecting tissue plugs were trained at LCI on June 8, 2016 as part of the LCI Fishing Derby Captains meeting.

Task 5-Fish Collection for Mercury was completed with slight modification. While more than 600 fish tissue samples were analyzed for total mercury (only 350 proposed), we were not successful in collecting a minimum of 10 smallmouth bass in the South Lake or walleye in the South Lake or the Main Lake (Table 1). There were four smallmouth bass specimens collected at the end of the project from South Lake but were not analyzed due to an irreparable malfunction of the mercury analyzer. Attempts to purchase a new instrument are underway and upon purchase these samples will be analyzed and the data shared with LCBP. Table 1. Total number of specimens collected and analyzed for each fish species in each lake segment. Red boxes indicate the species did not reside in that lake segment (lake trout). Yellow boxes indicate the species did reside in the lake segment but a minimum of 10 specimens were not collected despite multiple attempts. *Four samples were collected but could not be analyzed because the mercury analyzer had an irreparable malfunction.

Lake Walleye Smallmouth Yellow White Total Lake Segment Trout Bass Perch Perch South Lake 0 0* 22 14 36 South Main Lake 16 17 13 32 14 92 Main Lake 43 5 46 36 23 153 N. Main Lake 10 15 16 20 11 72 Mallets Bay 12 14 12 16 54 Northeast Arm 11 29 14 21 75 Missisquoi Bay 14 39 54 18 125 Total 69 74 157 190 117 607

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Task 6-Fish Collection for Cyanotoxin was completed with slight modifications. Originally a minimum of ten specimens for each species (smallmouth bass, yellow perch and brown bullhead) in each lake segment (Main Lake and Missisquoi Bay) were to be collected but bullhead were more difficult to sample than anticipated. Initially, sheepshead were substituted for bullhead because their dentition allows them to feed on filter feeding bivalves, like clams. Filter-feeders present a possible pathway for cyanotoxins to be passed to fish and humans. However, we were reminded that our collection permit did not include sheepshead, and therefore collections ceased. Analysis of 25-30 specimens was outlined in the proposal, 45 paired samples were analyzed (Table 2).

Table 2. Number of liver and muscle tissue samples analyzed for cyanotoxins in the Main Lake and Missisquoi Bay during the low bloom period.

Smallmouth Yellow Main Lake Bass Perch Sheepshead Number of fish 15 12 8 Number of liver samples 15 12 6 Number of muscle samples 15 12 4 Number of paired samples 15 12 2

Smallmouth Yellow Missisquoi Bay Bass Perch Sheepshead Number of fish 8 6 2 Number of liver samples 8 6 2 Number of muscle samples 8 6 2 Number of paired samples 8 6 2

Task 7-LCI Fishing Derby-A total of 203 samples were collected during the fishing derby allowing samples from trophy specimens to be acquired without having to sacrifice the animals using a non-lethal tissue plug technique.

Task 8-Quarterly Report-The June quarterly report was completed and sent to Eric Howe on June 30, 2016.

Task 9-Fish Sampling with VT F&W-Logistics made sampling difficult and we were not able to accompany Shawn Good on the electrofishing trip but did accompany VT F&W on a sampling trip in the Northeast Arm area.

Task 10-Fish Collection for Cyanotoxins-Sampling during high cyanobacteria density proved to be a challenge because typical large blooms with mats did not occur in 2016. Using the VDH Tracking Map, fish collection efforts were concentrated in Missisquoi Bay

Page 9 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain because of the more frequent occurrence of cyanobacteria (Category 2) and blooms in progress (Category 3) than were recorded in the Main Lake. Within the Main Lake only 5% of samples were classified as Category 2 and 3% as Category 3 compared to Missisquoi Bay with 9% of samples categorized as Category 2 and 23% as category 3. Additionally, the highest cell densities recorded in Missisquoi Bay were nearly 100-fold greater than that measured in the Main Lake (Shambaugh et al. 2017). Because this was a pilot study and the objective was to determine if we could detect cyanotoxins in fish tissue, we focused collection efforts in Missisquoi Bay to best position the study for success (Table 3). A total of 28 specimens were analyzed, 25-30 specimens were proposed.

Table 3. Number of liver and muscle tissue samples analyzed for cyanotoxins in Missisquoi Bay during high bloom periods.

Smallmouth Yellow Sheepshead Brown Largemouth Missisquoi Bay Bass Perch Bullhead Bass Number of fish 17 10 1 1 1 Number of liver samples 17 8 1 1 1 Number of muscle samples 17 10 1 1 1 Number of paired samples 17 8 1 1 1

Task 11-LCI Fishing Derby-Additional smallmouth bass samples were collected during Missisquoi Bay fishing derby for mercury but not cyanotoxins because blooms did not coincide with derby dates.

Task 12-Quarterly Report for September was completed but final collection numbers were not included as collections continued into 2017. The report was submitted to Eric Howe on September 30, 2016.

Task 13-Historic Mercury data was obtained from Neil Kamman, VT DEC, and was analyzed to assess long-term trends which are included in this report.

Task 14-Cyanotoxin Statistical Analysis was not able to be completed because all water and fish measurements were below the detection limit.

Task 15-Quarterly Report for December was completed but excluded any final numbers because specimen collections for mercury were ongoing. Report was sent to Eric Howe, Meg Modley and Jane Ceraso on December 29, 2016.

Task 16-Quarterly report submitted but extension requested due to ongoing specimen collection for mercury analyses and delays encountered with cyanotoxin analyses (delayed subcontract). Report was submitted to Meg Modley, Jane Ceraso and Heather Radcliffe on April 7, 2017.

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Task 17-Final Report (draft) submitted for review on January 30, 2018 to Meg Modley.

Methodology Task 3-Water Collection- VT DEC (Pete Stangel) collected water samples during their routine long-term monitoring trips. Water Samples (1-2 L) were filtered onto 0.7 µm GF filters in the field, kept on ice and sent to Dr. Boyer for cyanotoxin analysis. Samples were stored at -40°C until extraction and analysis. All extractions were completed on ice. Filters were extracted in 10 mL of 50% methanol containing 1% acetic acid with sonication to lyse cells (three 20 sec pulses at 320 watts). Samples were then centrifuged at 3500 rpm for ten minutes, after which the supernatant was filtered through a 0.45 µm nylon syringe filter into an HPLC vial. The methanolic extracts were analyzed for anatoxin-a and homoanatoxin-a by LC-MS/MS using a modification of the EPA method 545 for determination of anatoxin-a and cylindrospermopsin in drinking water (US EPA 2015a). Microcystins in the water samples were measured by LC-MS using a modification of the method of Harada (1996). Briefly, samples were separated on a 3.0 x 150 mm Ace 3 C18 (Advanced Chromatography Techniques) column using a 20 min 2- step gradient of 30-70% acetonitrile to water, both acidified to 0.1% with trifluoroacetic acid, at a flow rate of 0.3 mL min-1. Detection was at 239 nm (PDA) and by MS with electrospray ionization. For LC-MS, the total ion current was collected between m/z 750 and 1250 amu. Molecular ions of known microcystins were extracted from the total ion current and suspected positive peaks matched up with positive uv traces in the PDA spectrum indicative of microcystins. For both detection methods, unknown peaks that fell between the retention times of our most polar (MC-RR) and non-polar (MC-LF) standards were considered putative MC variants, and compared to the published list of MC variants by Lawton and Edwards (2001). For this work, we routinely screened for a subset of seventeen of these microcystin including LR (m/z 995.5), RR (m/z 1038.5), YR (m/z 1045.5), LA (m/z 932.5) and LF (m/z 986.5 + Na Adduct), as well as mRR (m/z 1052.4), mLR (m/z 1009.5), dLR(m/z 981.5) mRR (m/z 1052.4), homoYR (m/z 1059.5), AR (m/z 953.5), FR (m/z 1029.5), LW (m/z 1025.5 + Na adduct), WR (m/z 1068.5), mLA (m/z 926.6 + Na Adduct), LL (952.5 + Na adduct), LY +Na adduct) and Nodularin-R (m/z 825.5). Selective positive samples containing the first five congeners are then confirmed using LC-MS/MS (US EPA 2015b). These waters samples were extracted directly from filters and not subjected to solid phase extraction. Previous research (Boyer 2008) has shown that the extraction efficiency for these filters is between 90-100% for the three toxins in question. Thus, the raw results were not corrected for extraction efficiency.

Task 5-Fish Tissue Collection for Mercury-LCI coordinated with anglers to collect smallmouth bass, walleye, lake trout, yellow perch and white perch from each of the 7 lake segments, if the species resides in the lake segment. Trained LCI employee (Eric LaMontagne) collected tissue samples from the fillet which was stored in scintillation vials and frozen until sent overnight on ice to Dr. Bopp at RPI. Specimens were kept frozen until analyzed on a Milestone Direct Mercury Analyzer model-80 (DMA-80) with remaining samples kept frozen for re-analysis, if necessary. Clean techniques were

Page 11 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain utilized throughout collection and analysis to minimize any contamination issues. There is no sample preparation required for analyzing samples using the DMA-80.

Task 6 & 10-Fish Collection for Cyanotoxin-LCI coordinated with anglers to collect fish specimens for cyanotoxin analyses. Specimens were collected from the Main Lake and Missisquoi Bay by LCI and their volunteers during low cyanobacteria periods and during bloom periods. Specimens were brought to LCI where fish fillet and liver samples were taken, stored in scintillation vials and frozen. At the completion of the low-density cyanobacterial sampling, all samples were sent to Dr. Boyer overnight, on ice, for cyanotoxin analysis. Samples were received and inspected for defects. Samples that were damaged in shipping, had inconsistent identification numbers, or had incomplete identification information (i.e. missing fish biometrics) were retained, but not analyzed. All samples were stored at -40°C until lyophilization and extraction. In preparation for extraction, samples were lyophilized to dryness and then homogenized with a mortar and pestle. In large biomass samples, a subsample of tissue 0.2 g or 0.4 g was weighed in a centrifuge vial for extraction. In smaller samples, the entire tissue amount was extracted. Samples were extracted in 5 mL of 90% methanol acidified with formic acid. A nominal amount of the internal standard, nodularin-R, was added to assess microcystin recovery. To disrupt the tissue cells, samples were placed on ice and then macerated for 30 seconds with a Tissue Tearor  followed by sonication with three 20 sec pulses at 320 watts. Samples were then centrifuged at 3500 rpm for ten minutes (6°C), after which the supernatant was transferred to a glass vial and reduced to dryness under vacuum. In preparation for solid phase extraction (SPE), samples were reconstituted in 10% methanol acidified with formic acid.

Waters Oasis Sep Pak SPE cartridges were used to purify the samples and remove compounds that could interfere with analysis. Sep Pak cartridges were prepared by washing with 5 mL of 100% methanol followed by 5 mL of deionized water (DIW) acidified with 0.1% formic acid. Samples were individually transferred to SPE cartridges and allowed to equilibrate with the SPE cartridges for 10 minutes. After equilibration, samples were drawn through the cartridges under vacuum at an approximate rate of 5 mL per minute. To reduce sample loss, empty sample vials were rinsed with 2 mL of 10% methanol acidified with formic acid, which was then transferred to the SPE cartridges. The cartridges were again drained at 5 mL per minute. Cartridges were then rinsed with 5 mL of DIW acidified with 0.1% formic acid and eluted with 5 mL 90% methanol acidified with formic acid. Samples were then brought to dryness under vacuum and then reconstituted in 1 mL of acidified 90% methanol. Finally, samples were filtered through a 0.45 µm nylon syringe filter into HPLC vials. Samples were stored at -40°C until LC-MS/MS analysis. Lab blanks were prepared with the same 90% acidified methanol used to extract fish samples.

For microcystins and microcystin conjugates, analyte separation was achieved on a 4.6 x 150 mm Ace 5 C18 column using a 23 minute gradient of nanopure water and acetonitrile acidified with 0.02 % trifluoroacetic acid. The flow rate was variable and

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain ranged between 0.3 and 0.5 mL min-1. Samples were evaluated for the presence of microcystin LR, dLR, RR, dRR, the glutathione [Dha7]-microcystin conjugate (GSH-LR) and the cysteine [Dha7]-microcystin conjugate (Cys-LR). An electrospray LC-MS/MS method was used with 4 mass transitions to identify and confirm the microcystins LR, dLR (aka dimethyl LR or [D-Asp3] Microcystin-LR) and RR. Microcystin dRR (aka dimethyl RR or [D-Asp3] Microcystin-RR) used three transitions while GSH-LR and CYS- LR utilized two. While one mass transition was used for quantitation, the remaining transitions were used to confirm the presence of microcystin. The specific mass transitions, quantitation ions, and confirmation ions used for each microcystin are indicated in Table 4. Wherever possible the UV max absorbance spectra of microcystin was utilized as further confirmation for positive samples. However, interpretation of the UV spectra was often complicated from a high level of background noise due to the complex nature of the fish tissue matrix. Greater than 10% of samples were analyzed in duplicate as a QA/QC check.

Table 4. Quantitation and confirmation ions used to identify samples for the microcystin variants by LC-MS/MS. Quantitation ions were used to determine the concentration of the microcystin in the sample. Confirmation ions confirm the presence of the microcystin.

Mass Transitions (m/z) Microcystin Variant Quantitation Ion Confirmation Ions LR 995 135 995 107, 995 112, 995  155 dLR 981 135 981 93, 98 112, 981 155 RR 519 135 519 112, 519 127, 1035 135 dRR 512 135 512 124, 512 140 GSH-LR 1302 135 1302 213 Cys-LR 1116 135 1116 213

The 4.6 x 150 mm Ace 5 C18 column and sample extracts used for the microcystin analysis was also used for the anatoxin-a and cylindrospermopsins analysis. A 23 min gradient was used consisting of nanopure water and acetonitrile acidified with 0.1% formic acid at a flow rate of 0.5 mL min-1. Samples were evaluated for the presence of anatoxin-a, homo-anatoxin-a, cylindrospermopsin, epi-cylindrospermopsin and deoxycylindrospermopsin using electrospray LC-MS/MS. The method used 3 independent mass transitions to quantify and confirm the presence of the toxins. The mass transitions used are indicated in Table 5. Phenylalanine has the same mass transitions as anatoxin-a and can confound the identification of positive toxin samples. As a result, a phenylalanine standard was run as part of the method to ensure that we could differentiate between these two compounds by their retention time. At least 10% of samples were analyzed in duplicate as a quality control check.

Table 5. Quantitation and confirmation ions used to identify samples for anatoxin-a, homo-anatoxin-a, cylindrospermopsin, deoxycylindrospermopsin and phenylalanine by LC-MS/MS. Quantitation ions were used to

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain determine the concentration of the analyte in the sample. Confirmation ions were used to confirm the identity of the quantitation ion.

Mass Transition (m/z) Toxin Quantitation Ion Confirmation Ions Anatoxin 166 131 166 91, 166 149 Homo Anatoxin-a 180 131 166 105, 166 163 (epi)-Cylindrospermopsin 416 194 416 274, 416 336 Deoxycylindrospermopsin 400 194 400 274, 400 320 Phenylalanine 166 131 166 91, 166 149

For microcystins and their conjugates, anatoxin-a, homo-anatoxin-a and the cylindrospermopsins, the sample was considered positive only if they met three criteria including (1) the presence of a quantitation ion at the retention time of the standard, (2) the presence of at least one confirmation ion at the same retention time (3) the ratio of the quantitation to confirmation ion(s) intensities fell within accepted lab variation as determined using laboratory standards. In all cases, the calculated quantitation ion concentration was required to be above the individually calculated sample method detection limit to be reported. Samples meeting these criteria but with only one of two possible confirmation ions were considered “possible” positives. Those samples meeting the criteria and with two confirmation ions were considered “confirmed” positives.

Task 5 & 13- Statistical analysis of mercury data was conducted in SigmaPlot and R. All fish mercury concentrations were log transformed for ANalysis of COVAriance (ANCOVA) which relies on linear trends. Pairwise comparisons (Holm-Sidak method) were conducted on data that did not exhibit significant interactions between factor being tested and covariates.

Two random forest models were built, one for the current study and one utilizing data from all years using R 3.4.2 with the package Rattle (5.1.0). The models were built using fish length, weight, species and location for the current study; location was removed and year added when predicting log transformed mercury concentrations using the data for all years. The random forest models were made using 70% of the data for training with the remaining 30% used for testing. A randomization of the model was built using a seed of 42. For each model five hundred trees were built with two variables per split, missing data was imputed.

Page 14 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Quality Assurance Tasks Completed Mercury

Mercury measurements were all above the method detection limit of 0.20 ng (5 times average blank values). Standard Reference Materials (SRMs) and duplicate samples were normally within 10% of known (SRMs) or measured (duplicates) values; those exceeding 10% variance were re-analyzed. If re-analysis of SRMs that exceeded 10% variance did not reconcile the original measurement, all samples within that run were re- analyzed. Duplicate samples that exceeded the 10% variance were re-analyzed until reconciled. Samples that exceeded the 95% prediction intervals based on linear regressions of log transformed mercury data, were re-analyzed if possible. Due to an irreparable malfunction of the DMA-80, some outliers were not able to be re-analyzed and were not included in the statistical analyses but are included in the appendix. To ensure accuracy of measurements, multiple NIST SRMs were used including aqueous (dilution of Spex Certi-Prep PLHHG2-1Y, 100 mg/l mercury), sediment (NIST SRM 2702, 2704 and 2709) and fish muscle tissue (NIST SRM 1946). Variance of SRMs and duplicate measurements are shown in Figure 1.

Figure 1. Percent difference between known mercury concentrations and measured concentrations for Standard Reference Materials (fish, N=49; sediment, N=78) and duplicate sample measurements (N=61). Box represents 25th and 75th percentiles with median value represented by black line. Whiskers represent the 10th and 90th percentiles; circles indicate measurements were outliers for the measurement type. Values represent only initial measurements and not reanalyzed measurements. Red dashed lines represent 10% variance; any values beyond the 10% variance were reanalyzed to resolve discrepancy or data was not included in statistical analyses.

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Cyanotoxins

As there are no SRMs for cyanotoxins in tissues, the QA/QC protocols rely on the standard addition of nodularin to each sample. The instruments were cleaned at the start of each group of samples. Laboratory blanks were run every twenty samples and any positive value in the blank resulted in the sample set being discarded. Concentrations of the laboratory standards were verified by UV-VIS at the start of the sample season and the individual response factors monitored over the course of the study. Cleanliness of the mass spectrometer on any individual day was monitored by looking at the ratio of the response factor in the standard as determined by UV-VIS to the response factors as determined by mass spectrometer. Any ratio less than 2 (LC-MS) or 0.5 (LC-MS/MS) resulted in the sample set being discarded, the source cleaned and the analysis repeated.

The method for microcystin extraction, purification, and analysis was tested and validated by spiking store bought cod with the internal standard, nodularin-R (Nod), microcystin congeners (LR, RR. dRR and dLR), anatoxin-a and cylindrospermopsin to determine recoveries (Table 6). Both high (1 µg/L) and low (0.2 µg/L) concentrations of Nod and MC-LR were tested. The spiked cod with a low amount of MC-LR and Nod (0.2 µg/mL final concentration) had percent recoveries of 109% ± 8.6% and 102% ± 2%, respectively. The recoveries in high spikes (1 µg/mL final concentration) were 81% and 127%, respectively. The coefficient of variation for seven replicate spike injections was 8%. All spikes were done in duplicate. The final method was optimized to maximize recoveries.

Table 6. Percent recoveries of Nodularin, the microcystin congeners MC-LR, MC-RR, MC-dRR, MC-dLR, anatoxin-a, and cylindrospermopsin. Percent recoveries of Nodularin and MC-LR were tested at a low and high concentration (0.2 μg/mL and 1 μg/mL). The percent recoveries for low concentration spikes are presented first followed by the high concentration spikes in brackets.

Toxin Percent Recovery Standard Deviation CV Nodularin 102 (127) 2 2 MC-LR 109 (81) 9 8 MC-RR 72 5 8 MC-dRR 69 3 4 MC-dLR 66 9 14 Anatoxin-a 9 0.00 0.04 Cylindrospermopsin 2 0 3

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Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Deliverables Completed

Figure 2. Seven segments of Lake Champlain defined for fish mercury collections with stars indicating location of water sample collections for cyanotoxin analysis.

Page 17 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Mercury

This mercury study is the most extensive conducted to date, analyzing more than 600 fish, including five species from the seven segments of Lake Champlain. Species were statistically analyzed individually to determine if fish mercury varied spatially throughout the lake, and if so, was it consistent among species or groups (i.e. cold-water species). Data throughout this section will present the individual data points for each lake segment with the linear regression (log transformed Hg) along with the 95% confidence intervals and the 95% prediction interval. The significance of each regression is reported in the following table with the y-intercept, slope (bioaccumulation rate) and mercury estimates at two lengths, a typically standard length and a larger length representing specimens sampled through fishing tournaments. Only significant regressions were included in subsequent analyses to limit the error (i.e. ”noise”) allowing for a more rigorous analysis. When lake segment was a significant factor explaining fish mercury variance pair-wise comparisons were made among lake segments, an additional table is included to detail each pairwise comparison.

Smallmouth Bass

Adequate smallmouth bass specimens were collected from all segments of Lake Champlain with the exception of South Lake. LCI attempted to collect smallmouth bass from professional bass tournaments held at South Lake boat launches along with chartering boats targeting smallmouth bass. These efforts culminated in a total of four specimens that were received at the end of the project but were not analyzed due to an irreparable malfunction of the mercury analyzer utilized for this project. Current efforts are attempting to secure a new mercury analyzer, at which time, these four samples will be analyzed and data shared with LCBP.

Mercury concentrations in the 157 smallmouth bass specimens analyzed showed 136 (87%) had concentrations greater than the US EPA advisory limit of 300 ppb and 27 (17%) exceeded the US FDA action limit of 1000 ppb (Figure 3). South Main Lake, Main Lake, North Main Lake and Missisquoi Bay exhibited similar bioaccumulation rates while Malletts Bay and Northeast Arm were generally lower in mercury; the difference diminished in the Northeast Arm with size (Figure 4). At 14” (356 mm) smallmouth bass in Malletts Bay and Northeast Arm were still below the 300 ppb while all other sites were closer to 400 ppb (Table 7). At 18” (457 mm) mercury concentrations in South Main Lake smallmouth bass were approaching 1000 ppb and by 19” exceeded 1000 ppb (Figures 3 & 4, Table 7). Location and length were both significant factors explaining the variability in mercury concentrations (ANCOVA, factor: location (p<0.001), covariate: length (p<0.001), covariate: relative weight (p=0.917)). Pairwise comparisons between lake segments found Malletts Bay and the Northeast Arm to have significantly less mercury than other lake segments (Figure 4, Table 8). It is worth noting that smallmouth bass are a high pressure tournament species and can be moved to/from various lake segments by anglers. Therefore, observed differences among lake segments must be taken with caution, especially when the sample size is low.

Page 18 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 3. Length and mercury concentration (log ppb) for individual smallmouth bass in each lake segment and combined. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

Page 19 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Table 7. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in smallmouth bass from each lake segment with sample size (N), p-value and estimated mercury concentration at standard lengths of 14" (356 mm) and 18" (457 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported.

-3 Lake Segment N yo a (10 ) p-value [Hg] @ 14" [Hg] @ 18" Missisquoi Bay 39 1.48 3.07 <0.001 374 764 Northeast Arm 29 0.90 4.10 <0.001 229 594 Malletts Bay 14 1.48 2.66 0.018 267 496 North Main Lake 16 1.76 2.49 0.022 791 Main Lake 45 1.69 2.53 <0.001 390 702 South Main Lake 13 1.39 3.39 0.002 395 869

Figure 4. Linear regressions in individual lake segments. Location and length were significant factors explaining variance in fish mercury content (ANCOVA, factor: location (p<0.001), covariate: length (p<0.001), covariate: relative weight (p=0.917)). Significant pairwise differences exist between Malletts Bay and South Main Lake (p<0.001), Missisquoi Bay (p<0.001), North Main Lake (p=0.003) and Main Lake (p=0.026) along with significant differences between Northeast Arm and South Main Lake (p<0.001) and Missisquoi Bay (p=0.003). Significant differences are indicated by different letters in the legend. Dashed red lines represent the USEPA mercury advisory limit of 300 ppb and the USFDA action limit of 1000 ppb.

Page 20 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Table 8. Pair-wise comparisons (Holm-Sidak method) of smallmouth bass mercury concentrations among lake segments. Values represent p-values between lake segments. Those with significant differences are highlighted in yellow.

SMB SL SML ML NML MLB NEA MSB SL X NA NA NA NA NA NA SML X 0.356 0.812 <0.001 <0.001 0.073 ML X 0.859 0.026 0.607 0.751 NML X 0.003 0.155 0.633 MLB X 0.212 <0.001 NEA X 0.003 MSB X

Walleye

Walleye specimens were collected in adequate quantity in most areas of the lake with the exception of the Main Lake (only 5 specimens) and South Lake (no samples). Seventy-four specimens were analyzed in total with 63 (85%) specimens exceeding the US EPA advisory limit of 300 ppb and 28 (38%) exceeding the US FDA action limit of 1000 ppb (Figure 5). Walleye in the North Main Lake segment did not exhibit a significant mercury trend and were not included in the subsequent analyses. The remaining five segments tended to fit into two groups. Malletts Bay and Northeast Arm exhibited lower bioaccumulation rates but a much higher y-intercept resulting in higher mercury concentrations at 20” compared to other segments (Table 9). This higher mercury estimate could partially be due to the larger specimens collected (>18”), similarly the steep bioaccumulation rate in Main Lake could be influenced by the lack of smaller specimens to level out the regression (Figure 5). South Main Lake and Missisquoi Bay specimens included smaller sized walleye and exhibited bioaccumulation rates between Main Lake and Malletts Bay/Northeast Arm. Mercury estimates for 20” (508 mm) walleye show almost a three-fold difference between South Main Lake and Malletts Bay which diminishes to less than half for 24” (610 mm) specimens (Table 9). By the time walleye reach 24” in Malletts Bay, they are expected to exceed 1000 ppb with all sites expected to exceed 1000 ppb by 26.5” (673 mm, Figure 6, Table 9). Location and length were both significant factors explaining the variability in mercury concentrations (ANCOVA, factor: location (p=0.026), covariate: length (p<0.001), covariate: relative weight (p=0.120)). No significant interactions existed between location and length (p=0.134) or relative weight (p=0.496). Pairwise comparisons (Holm-Sidak Method) showed significant differences between South Main Lake and Malletts Bay (p<0.001), Missisquoi Bay (p<0.001), Main Lake (0.003) and Northeast Arm (p=0.005) (Figure 6, Table 10).

Page 21 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 5. Length and mercury concentration (log ppb) for individual walleye in each lake segment and combined. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

Page 22 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Table 9. Linear accumulation curves (y = yo + a*x) for mercury (log ppb) in walleye from each lake segment with sample size (N), p-value and estimated mercury concentration (ppb) at standard lengths of 20" (508 mm) and 24" (610 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported. Trends that were not significant are highlighted in gray.

-3 Lake Segment N yo a (10 ) p-value [Hg] @ 20” [Hg] @ 24" Missisquoi Bay 14 1.24 2.76 <0.001 439 839 Northeast Arm 11 1.91 1.67 0.015 573 849 Malletts Bay 12 1.94 1.78 <0.001 699 1061 North Main Lake 15 2.83 0.33 0.225 995 1075 Main Lake 5 0.22 4.43 0.003 827 South Main Lake 17 0.55 3.64 <0.001 251 589

Figure 6. Comparing linear regressions from individual lake segments shows a significant effect of location and length on mercury content (ANCOVA, factor: location (p=0.026), covariate: length (p<0.001), covariate: relative weight (p=0.120). With no significant interactions between location and length (p=0.134) or relative weight (p=0.496) pairwise comparisons showed South Main Lake had significantly less mercury from all other segments included in the analysis. Significant differences are indicated by different letters in the legend. Asterisk indicates data was not included in analysis because the regression was not significant. Dashed red lines represent the USEPA mercury advisory limit of 300 ppb and the USFDA action limit of 1000 ppb.

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Table 10. Pair-wise multiple comparisons (Holm-Sidak method) of walleye mercury concentrations between each lake segment. Values represent p-values between lake segments with significant differences highlighted in yellow.

Walleye SL SML ML NML MLB NEA MSB SL X NA NA NA NA NA NA SML X 0.003 NA <0.001 0.005 <0.001 ML X NA 0.793 0.756 0.967 NML X NA NA NA MLB X 0.380 0.858 NEA X 0.798 MSB X

Lake Trout

Lake trout were collected in adequate numbers from the three segments in which they are known to reside (South Main Lake, Main Lake and North Main Lake). Seventy-five specimens were analyzed with 73 (97%) exceeding the US EPA advisory limit of 300 ppb while only 1 specimen exceeded the USFDA action limit of 1000 ppb (Figure 7). South Main Lake did not exhibit a significant trend and was not included in subsequent analyses. Mercury trends in Main Lake and North Main Lake were similar with North Main Lake specimens being slightly under 300 ppb and Main Lake slightly over 300 ppb at 20” (508 mm), at 28” (711 mm) they had similar mercury concentrations and mercury in specimens from North Main Lake are expected to exceed Main Lake in specimens larger than 28” (Table 11, Figure 8). Location and length were significant factors explaining the variability in lake trout mercury (ANCOVA, factor: location p=0.017, covariate: length (p<0.001), covariate; relative weight (p=0.795)) as was the interaction between location and relative weight (p=0.010) limiting pairwise comparisons. Removing relative weight from the analysis because it was not a significant main covariate resulted in only length being significant (ANCOVA, factor: location p=0.722, covariate: length (p<0.001).

Page 24 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 7. Length and mercury concentration (log ppb) for individual lake trout in each lake segment and combined. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

Table 11. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in lake trout from each lake segment with sample size (N), p-value and estimated mercury concentration (ppb) at standard lengths of 20" (508 mm) and 28" (711 mm) based on linear regressions. Trends that were not significant are highlighted in gray.

-3 Lake Segment N yo a (10 ) p-value [Hg] @ 20" [Hg] @ 28" North Main Lake 10 1.77 1.34 0.028 282 528 Main Lake 43 2.01 1.00 <0.001 330 526 South Main Lake 16 2.45 0.4 0.158 450 543

Page 25 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 8. Comparing regressions from individual lake segments shows a significant effect of location and length on mercury content (ANCOVA, factor: location (p=0.017), covariate: length (p<0.001), covariate: relative weight (p=0.795)). A significant interaction between location and relative weight limits pairwise comparisons.

Page 26 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch

Yellow perch were collected in adequate numbers from all seven lake segments. In total 189 specimens were analyzed with 25 (13%) specimens exceeding the US EPA advisory limit of 300 ppb with no samples exceeding 1000 ppb. However, larger specimens (>12” or 305 mm) in South Main Lake and Northeast Arm do approach the US FDA action limit of 1000 ppb (Figure 9). Most of the yellow perch specimens collected were relatively small, only exceeding 10” (229 mm) in Northeast Arm, Main Lake and South Main Lake (Figure 9). Trends in Missisquoi Bay and Main Lake were not significant and were excluded from subsequent analyses. The remaining five lake segments showed mercury content in 9” (229 mm) yellow perch was similar in Northeast Arm, Malletts Bay and North Main Lake (155-177 ppb) with South Main Lake and South Lake estimates being slightly higher (198-232 ppb, Table 12). Comparisons above 9” is not possible due to the size range collected. Location and length were both significant factors explaining the variability in yellow perch mercury (ANCOVA, factor: location (p=0.040), covariate: length (p<0.001), covariate: relative weight (p=0.212)). A significant interaction between location and length (p=0.005) limits pairwise comparisons.

Page 27 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 9. Length and mercury concentration (log ppb) for individual yellow perch in each lake segment and combined. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

Page 28 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Table 12. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in yellow perch from each lake segment with sample size (N), p-value and estimated mercury concentration (ppb) at standard lengths of 9" (229 mm) and 12" (305 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported. Trends that were not significant are highlighted in gray.

-3 Lake Segment N yo a (10 ) p-value [Hg] @ 9" [Hg] @ 12" Missisquoi Bay 54 1.85 1.20 0.087 133 Northeast Arm 114 0.13 9.00 <0.001 155 750 Malletts Bay 12 1.33 3.83 0.004 161 North Main Lake 20 1.04 5.28 <0.001 177 Main Lake 36 2.05 1.50 0.078 247 322 South Main Lake 32 1.38 4.30 <0.001 232 491 South Lake 22 0.99 5.71 <0.001 198

Figure 10. Comparing regressions of yellow perch mercury content from individual lake segments shows a significant effect of location and length (ANCOVA, factor: location (p=0.040), covariate: length (p<0.001), covariate: relative weight (p=0.212)). A significant interaction between location and length (p=0.005) limits pairwise comparisons. Dashed red lines represent the USEPA mercury advisory limit of 300 ppb.

Page 29 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain White Perch

White perch were collected in adequate numbers from all seven lake segments. In total 117 specimens were analyzed with 29 (25%) exceeding the US EPA advisory limit of 300 ppb and a single sample exceeding the USFDA action limit of 1000 ppb. The single sample exceeding 1000 ppb was a 13+” white perch from the Northeast Arm, several other larger specimens (>12” of 305 mm) from the Northeast Arm and Main Lake approached 1000 ppb. Mercury trends in North Main Lake and South Main Lake were not significant, likely due to a limited size range of specimens (Figure 11). Specimens from North Main Lake were between 220 mm and 290 mm while South Main Lake specimens were between 200 mm and 240 mm with a single specimen at 330 mm. The specimens collected from Northeast Arm, Malletts Bay and Main Lake exhibited very similar bioaccumulation rates with Northeast Arm having a slightly higher y-intercept resulting in estimated mercury for 9” (229 mm) yellow perch being 253 ppb while Malletts Bay and Main Lake had similar estimates of 202 ppb and 197 ppb, respectively (Figure 12, Table 13). Mercury estimates for Missisquoi Bay were the lowest for 9” yellow perch at 102 ppb and South Lake was the highest at 302 ppb (Table 13). While the estimated mercury in 12” yellow perch only exceeded 500 ppb in the Northeast Arm, it is clear from individual data points that fish around this size can be much higher, approaching 1000 ppb (Figure 11, Table 13). Location and length were both significant factors explaining the variability in yellow perch mercury (ANCOVA, factor: location (p=0.005), covariate: length (p<0.001), covariate: relative weight (p=0.394)). A significant interaction between location and relative weight (p=0.002) limited pairwise comparisons. Removing relative weight as a covariate because it was not a significant main covariate resulted in a significant interaction between location and length which could not be reconciled and thus hinders pairwise comparisons.

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Figure 11. Length and mercury concentration (log ppb) for individual white perch in each lake segment and combined. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

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Table 9. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in white perch from each lake segment with sample size (N), p-value, mercury concentration (ppb) at standard length of 9" (229 mm) and 12” (305 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported. Trends that were not significant are highlighted in gray.

-3 Lake Segment N yo a (10 ) p-value [Hg] @ 9" [Hg] @ 12" Missisquoi Bay 18 0.13 8.21 <0.001 102 431 Northeast Arm 21 1.43 4.25 0.002 253 532 Malletts Bay 16 1.34 4.22 <0.001 202 North Main Lake 11 1.79 2.07 0.287 184 Main Lake 23 1.25 4.56 <0.001 197 437 South Main Lake 14 2.16 0.66 0.535 205 230 South Lake 14 0.77 7.47 <0.001 302

Figure 12. Comparing regressions of white perch mercury content from individual lake segments shows a significant effect of location and length (ANCOVA, factor: location (p=0.005), covariate: length (p<0.001), covariate: relative weight (p=0.394)). A significant interaction between location and relative weight (p=0.002) limits pairwise comparisons. Dashed red lines represent the USEPA mercury advisory limit of 300 ppb.

Page 32 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Random Forest Model

The random forest model agreed with the ANCOVA analyses identifying length as the factor explaining the greatest amount of variance in the mercury concentrations of Lake Champlain fish. A total of 607 samples from five species and seven lake segments were used to develop and test the random forest model to predict fish mercury concentrations. The random forest model was built using 424 of the 607 samples (70%) with the remaining 183 samples (30%) used to test the model’s ability to accurately predict fish mercury concentrations. The model explained 80.9% of the variance, the mean square of the residuals was 0.028. The models accuracy is determined by its ability to predict values not used to build the model. This model has an r2=0.76 when comparing the predicted and observed values (Figure 13). Through successive iterations of the model building with leaving one of the variables out a relative importance of each variable is calculated by determining the percent increase in the mean square error for each variable when it is eliminated from the model. This model identified length as the most important variable with an increase in MSE of 74% when eliminated from the model. Species, location and relative weight were less important with an increase of MSE of 46%, 41% and 12%, respectively.

Figure 13. Random forest model showing predicted vs observed mercury values for the 183 specimens tested using length, species, location and relative weight as factors. The model explained 80.9% of the variability with length being the most important factor.

Page 33 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Historic Trends To assess the changes in fish mercury over time in Lake Champlain, two approaches were taken. The first was to use all data from each study, regardless of where the sampling occurred and ask if there has been a significant change over time. This approach ignores the fact that location can significantly impact fish mercury, but the larger sample size provides greater confidence in the regression. Additionally, low sample size in some studies only allows this level of resolution; some study periods were combined to obtain an acceptable sample size (Table 14). The second approach was more rigorous, comparing mercury concentrations in individual lake segments between 2011 and 2016-2017 for a single species. The larger sample sizes required for this analysis mainly limits the comparisons to these two studies. This approach could only be carried out for smallmouth bass, yellow perch and white perch. Walleye were not collected in sufficient numbers and lake trout mercury trends were not significant in 2011. Additional study periods were included in some lake segments when sample size allowed but the results were either similar to whole lake comparisons or inconclusive because there was a lack of size overlap among studies.

Table 10. Sample size for each fish species (only species collected in current study) during each study period.

2016- 2011 2003- 1999- 1996- 1993 1990- 1987 2017 2004 2000 1998 1991 SMB 157 68 25 10

Walleye 74 6 31 26 28

Lake Trout 69 24 22 15 21 7

Yellow Perch 190 103 13 32

White Perch 117 79 22

Smallmouth Bass

Mercury data for smallmouth bass in Lake Champlain began in 1996 and was available during four studies (Figure 14). Linear regressions on fish mercury concentrations (log ppb) were significant in all studies and were included in the analysis (Table 15). Both year and length were significant factors explaining the variability in smallmouth bass mercury (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001)). Pairwise comparisons showed significant differences between 2011 and 2016 but no other years (Holm-Sidak, p<0.001). Estimated mercury concentrations for 14" (356 mm) smallmouth bass decreased from 352 ppb in 2011 to 266 ppb in 2016-2017 (Figure 15, Table 15). Comparisons among larger specimens (18” or 457 mm) were limited to the 2011 and 2016-2017 study due to smaller size collections in other studies. Again estimated mercury increased from 534 in 2011 to 690 ppb in 2016-2017 (Table 15).

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Figure 13. Length and mercury concentration (log ppb) for individual smallmouth bass for each study. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit (300 ppb) and the US FDA action limit (1000 ppb).

Table 11. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in smallmouth bass for each study year with sample size (N), p-value and estimated mercury concentration (ppb) at standard lengths of 14" (356 mm) and 18” (457 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported.

-3 Year N yo a (10 ) p-value [Hg] @14" [Hg] @ 18” 2016-2017 157 1.442 3.057 <0.001 339 690 2011 68 1.36 2.993 <0.001 266 534 2003-2004 25 1.993 1.371 0.029 303 1996-1998 10 1.711 2.347 0.007 352

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Figure 145. Comparing regressions of smallmouth bass mercury content (log ppb) from each study year shows significant differences for year and length (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001)). Mercury concentration were significantly greater in 2016 than 2011 (p<0.001), no other significant differences existed among study years. Red dashed lines represent the US EPA mercury advisory limit of 300 ppb and US FDA action limit of 1000 ppb. Comparing mercury concentrations within individual lake segments required a reanalysis of the 2011 data to determine which segments exhibited significant regressions. Summarized are regressions for individual lake segments along with significance and estimated mercury concentration (ppb) for 14" (356 mm) and 18” (457 mm) smallmouth bass (Table 16). While comparing smallmouth bass mercury concentrations lake-wide showed a significant increase in mercury between the 2011 and 2016-2017 studies, not all lake segments responded in a similar manner (Figure 16). Three lake segments were not included in the analyses; Missisquoi Bay and South Lake were not sampled in 2011 and the mercury trend in South Main Lake was not significant in 2011. Of the four remaining segments, only the Main Lake showed a significant change with mercury increasing 62% and 44% in 14” and 18” smallmouth bass, respectively (Figure 16, Table 17). The increase observed in North Main Lake was not significant; specimens collected were dominantly large specimens from a narrow size range. Mercury concentrations in the Northeast Arm were similar between study periods while concentrations in Malletts Bay continued to decrease by 12% and 22% for 14” and 18” bass, respectively. This change was not significant and while noted earlier, it is worth restating that smallmouth bass are a high pressure tournament species that can be transported throughout the lake by anglers potentially impacting results, particularly if the sample size is small.

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Table 12. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in smallmouth bass for each lake segment in 2011 with sample size (N), p-value and estimated mercury concentration (ppb) at standard lengths of 14" (356 mm) and 18" (457 mm). Gray highlight indicates the trend is not significant.

2011 SMB N yo a(10^-3) p [Hg] @ 14" [Hg] @ 18" Northeast Arm 16 1.221 3.4 >0.001 270 595 Malletts Bay 13 1.352 3.173 0.0001 303 634 North Main Lake 12 0.7565 4.394 0.0007 209 582 Main Lake 15 1.2973 3.044 0.0114 240 488 South Main Lake 11 -0.1539 5.947 0.0972 92 366

Figure 15. Length and mercury concentration (log ppb) for individual smallmouth bass in each lake segment and combined for the 2011 and 2016-2017 studies. Bioaccumulation rates based on linear regression are shown for 2011 (red) and 2016-

Page 37 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain 2017 (black). South Lake and Missisquoi Bay are not included because samples were not collected from those lake segments in 2011.

Table 17. Estimated mercury concentration (ppb) for 14” (356 mm) and 18” (457 mm) smallmouth bass in each lake segment in 2011 and 2016 based on linear regressions with the percent change in mercury calculated. Empty cells indicate the collections did not overlap the standard size in that lake segment. Significance (p-value) is listed for the comparisons (ANCOVA, factor: year, covariate: length) between the two studies for each lake segment, significant differences only observed in the Main Lake. Gray highlight indicates the regression was not significant.

2011 2016 2011 2016 p-value Lake Segment 14" 14" % ∆ 18" 18" % ∆ Missisquoi Bay 374 764 Northeast Arm 270 229 -15% 595 594 0% 0.83 Malletts Bay 303 267 -12% 634 496 -22% 0.56 North Main Lake 209 582 791 36% 0.40 Main Lake 240 390 62% 488 702 44% <0.01 South Main Lake 395 366 869

Walleye

Walleye sampling for mercury began in 1990 and has been revisited four additional times with all studies except 2011 exhibiting significant trends (Figure 17, Table 18). Estimated mercury in 20” walleye has decreased by half since 1990, however the slightly larger 24” walleye may have increased mercury since the 1999-2000 study (Table 18, Figure 18). The higher bioaccumulation rate in 2016-2017 is responsible for the differences observed with size. Year and length were significant factors along with the interaction (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001), year x length (p=0.001)). The interaction limits pairwise comparisons but comparing just the 1990-1991 and 2016- 2017 studies, a significant decrease is detected (ANCOVA factor: year (p<0.001), covariate: length (p<0.001)) in walleye mercury concentrations for specimens <700 mm (Figure 18).

Page 38 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 167. . Length and mercury concentration (log ppb) for individual walleye for each study. Bioaccumulation rate (black line) based on linear regression is shown along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines indicate the US EPA fish consumption advisory limit of 300 ppb (bottom) and the US FDA action limit of 1000 ppb (top).

Table 18. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in walleye for each study year with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 20" (508 mm) and 24” (610 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported. Trends that were not significant are highlighted in gray.

-3 Year N yo a (10 ) p-value [Hg] @ 20” [Hg] @ 24” 2016-2017 76 1.285 2.618 <0.001 412 762 2011 6 2.426 0.601 0.719 620 2004 31 0.754 3.827 <0.001 499 1999-2000 26 2.174 1.061 0.03 516 663 1990-1991 28 2.292 1.244 0.022 839 1124

Page 39 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 178. Comparing regressions of walleye mercury content (log ppb) from each study year shows significant differences for year and length (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001)) along with the interaction (p=0.001) limiting pairwise comparisons. Asterisks indicate a study without significant trends and were not included in the ANCOVA. Dashed red lines represent the US EPA advisory mercury limit of 300 ppb and the USFDA action limit of 1000 ppb.

Lake Trout

Lake trout samples collected for mercury analysis began 1987 with 5 additional samplings over the next 30 years. Unfortunately, lake trout length does not always relate directly to fish age meaning there may be young and old fish of the same length. This discrepancy resulted in only the 1987 and 2016-2017 studies having significant trends (Figure 19, Table 19). Comparisons of these two studies, completed 30 years apart, show mercury concentrations have significantly decreased (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001)) with no significant interaction (p=0.219)). The higher bioaccumulation rate in 1987 resulted in lake trout mercury decreasing in 24” and 28” specimens by 23% and 34%, respectively (Table 19, Figure 20). It should be mentioned that the 1987 study had a sample size of 7. Therefore, results should be interpreted cautiously. Individual lake segments were not compared between the 2016- 2017 and 2011 study periods because the 2011 regressions were not significant.

Page 40 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 18. Individual lake trout length and mercury content (log ppb) by study year showing regression (black line) along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Dashed red lines represent the US EPA advisory mercury limit of 300 ppb (bottom) and the USFDA action limit of 1000 ppb (top).

Table 139. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in lake trout for each study year with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 24” (610 mm) and 28” (711 mm) based on linear regressions. Trends that were not significant are highlighted in gray.

-3 Year N yo a (10 ) p-value [Hg] @ 24” [Hg] @ 28” 2016-2017 72 2.062 0.935 <0.001 429 533 2011 24 2.454 0.142 0.639 347 359 2004 22 2.338 0.6 0.223 506 582 2000 15 3.053 -0.381 0.349 662 605 1993 21 2.352 0.663 0.069 571 666 1987 7 1.756 1.62 0.033 555 809

Page 41 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 20. Comparing regressions of lake trout mercury content (log ppb) from each study year shows year and length were significant (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001), interaction: (p=0.219)). Different letters indicate significant differences between years; asterisks indicate years did not exhibit significant trends and were not included in ANCOVA. Red dashed lines represent the US EPA mercury advisory limit of 300 ppb and the USFDA action limit of 1000 ppb.

Page 42 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch

Yellow perch were collected and tested for mercury four times beginning in 1988 with all studies, except 2003-2004, exhibiting significant trends (Figure 21, Table 20). The remaining three study periods (1988-1989, 2011 and 2016-2017) exhibited similar bioaccumulation rates with year and length explaining a significant amount of variability in yellow perch mercury concentrations (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001), interaction (p=0.811)). Pairwise comparisons found all three periods to be significantly different from one another (Holm-Sidak, p<0.001) with the initial 1988- 1989 study year exhibiting the highest mercury content, the 2011 study year exhibiting the lowest mercury content and the current study an intermediate over the entire size range (Figure 22).

Figure 21. Individual yellow perch length and mercury content (log ppb) by study year showing regression (black line) along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Red dashed lines represent the US EPA mercury advisory limit of 300 ppb (bottom) and the USFDA action limit of 1000 ppb (top).

Page 43 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Table 20. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in yellow perch for each study year with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 7” (178 mm) and 10” (254 mm) based on linear regressions. Trends that were not significant are highlighted in gray.

-3 Year N yo a (10 ) p-value [Hg] @ 7” [Hg] @ 10” 2016 191 1.272 4.342 <0.001 111 237 2011 103 1.231 3.881 <0.001 84 165 2003-2004 13 1.8 2.207 0.132 156 229 1988-1989 32 1.406 4.307 <0.001 149 316

Figure 192. Comparing regressions of yellow perch mercury content (log ppb) from each study year shows significant differences for year and length (ANCOVA, factor: year (p<0.001), covariate: length (p<0.001) but not their interaction (p=0.811)). Pairwise comparisons indicate all years were significantly different from one another (p<0.001). Different letters indicate a significant difference; asterisk indicates the trend was not significant and not included in the analysis. Dashed red line represents the US EPA advisory mercury limit of 300 ppb.

Page 44 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Comparing mercury concentrations within individual lake segments required a reanalysis of the 2011 data to determine which segments exhibited significant regressions, summarized with significance and estimated mercury concentration (ppb) for 7" and 10” yellow perch (Table 21). Main Lake and South Main Lake did not exhibit significant trends in 2011 nor did Missisquoi Bay in 2016-2017 and were not included in subsequent analyses (Figure 23, Table 22). Additionally, there was an interaction between year and length in the Northeast Arm when comparing the two study periods limiting the pairwise comparison. Of the three remaining lake segments, specimens in the South Lake were relatively small and did not exhibit a significant difference between studies. However, mercury concentrations in the North Main Lake significantly increased between 2011 and 2016-2017 while mercury in yellow perch from Malletts Bay exhibited a significant decrease (Figure 23, Table 22).

Table 21. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in yellow perch for each lake segment during 2011 with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 7” (178 mm) and 10” (254 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported. Trends that were not significant are highlighted in gray.

2011 Yellow Perch N yo a(10^-3) p [Hg] @ 7” [Hg] @ 10” Missisquoi Bay 10 0.99 5.59 <0.01 97 Northeast Arm 17 0.85 5.36 <0.01 64 163 Malletts Bay 15 1.29 4.43 <0.01 120 260 North Main Lake 14 0.68 5.70 <0.01 49 134 Main Lake 17 2.09 -1.33 0.79 71 South Main Lake 5 0.87 5.63 0.12 200 South Lake 25 1.24 4.18 <0.01 96

Page 45 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 20. Length and mercury concentration (log ppb) for individual yellow perch in each lake segment and combined for the 2011 and 2016-2017 studies. Bioaccumulation rates based on linear regression are shown for 2011 (red) and 2016-2017 (black).

Page 46 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Table 2214. Estimated mercury concentration for 7” (178 mm) and 10” (254 mm) yellow perch in each lake segment in 2011 and 2016-2017 based on linear regressions with the percent change in mercury calculated. Empty cells indicate the specimens collected did not overlap the standard length. Significance (p-value) is listed for the comparisons (ANCOVA, factor: year, covariate: length) between the two studies for each lake segment, significant differences were observed in Malletts Bay and North Main Lake. Gray highlighted cells indicate the regression was not significant in that study and was not included in the analysis. Asterisk indicated a significant interaction between the factor and covariate in the ANCOVA which limits the pairwise comparison.

2011 2016 Lake Segment p-value 2011 7" 2016 7" % ∆ 10" 10" % ∆ Missisquoi Bay 97 116 143 Northeast Arm 64 54 -16% 163 261 60% * Malletts Bay 120 103 -14% 260 201 -23% 0.03 North Main Lake 49 95 95% 134 241 79% <0.01 Main Lake 71 207 270 South Main Lake 140 200 297 South Lake 96 101 6% 276 0.43

White Perch

White perch were collected for mercury analysis beginning in 1993 but few samples were collected sporadically over the next 11 years so they were combined into a single group to compare against the 2011 and 2016 mercury data (Figure 24). Analysis of the data showed that length was significant, year was not (ANCOVA, factor: year (p=0.392), covariate: length (p<0.001)). All three periods exhibited similar mercury concentrations over the entire size range collected (Figure 25, Table 23).

Comparing mercury concentrations within individual lake segments required a reanalysis of the 2011 data to determine which segments exhibited significant regressions, summarized with significance and estimated mercury concentration (ppb) for 9" (229 mm) and 12” (305 mm) white perch (Table 24, Figure 26). All lake segments exhibited significant trends in 2011 but the trends in North Main Lake and South Main Lake were not significant in the 2016-2017 study preventing their use in subsequent analyses. South Lake could not be compared between studies due to a significant interaction between study period and length. The remaining four lake segments exhibited similar mercury concentrations over the size ranges collected (Figure 26, Table 25). Mercury concentrations in Missisquoi Bay show a decrease in smaller specimens (9%) but an increase in larger sizes (12”), likely an effect of sampling mostly smaller fish in 2011, most samples <10”. Mercury concentrations in the Northeast Arm showed an increase of ~25% across all size ranges collected (~8”-12”) while Malletts Bay shows a continued decrease in mercury at 9” and 12”. White perch collected in Main Lake and South Lake were limited to mostly smaller specimens in one of the studies; all but one specimen from the Main Lake was <8” in 2011 and the 2016-2017 study had one sample >9”. The resulting regressions show a decrease of mercury in smaller (9”) white perch from the Main Lake and a slight increase in the South Lake, however the disproportional importance placed on a single large specimen when most specimens are small creates low confidence in the regression and should be viewed cautiously.

Page 47 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 214. Individual yellow perch length and mercury content (log ppb) by study year showing regression (black line) along with 95% confidence intervals (blue lines) and 95% prediction intervals (red lines). Red dashed lines represent the US EPA mercury advisory limit of 300 ppb (bottom) and the USFDA action limit of 1000 ppb (top).

Table 23 Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in white perch for each study year with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 9” (229 mm) and 12” ( 305 mm) based on linear regressions.

-3 Year N yo a (10 ) p-value [Hg] @ 9” [Hg] @ 12” 2016 117 1.276 4.437 <0.001 196 426 2011 79 1.116 5.049 <0.001 187 453 1993-2004 22 1.291 4.548 <0.001 215 477

Page 48 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 25. Comparing regressions of white perch mercury content (log ppb) from each study year shows significant differences for length only (ANCOVA, factor: year (p=0.392), covariate: length (p<0.001)). Dashed red line represents the US EPA advisory mercury limit of 300 ppb.

Table 2415. Linear accumulation curves (y=yo + a*x) for mercury (log ppb) in white perch for each lake segment during 2011 with sample size (N), p-value and estimated mercury concentrations (ppb) at standard lengths of 9” (229 mm) and 12” (305 mm) based on linear regressions, if samples collected did not overlap the standard length no value is reported.

2011 White Perch N yo a(10^-3) p [Hg] @ 9” [Hg] @ 12” Missisquoi Bay 15 0.81 5.65 <0.01 127 341 Northeast Arm 8 1.25 4.45 <0.01 187 407 Malletts Bay 15 1.08 5.54 <0.01 225 594 North Main Lake 12 0.49 7.01 <0.01 125 Main Lake 6 0.91 6.62 <0.01 269 South Main Lake 8 0.98 5.81 <0.01 204 564 South Lake 15 1.70 3.20 <0.01 270

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Figure 226. Length and mercury concentration (log ppb) for individual white perch in each lake segment and combined for the 2011 and 2016-2017 studies. Bioaccumulation rates based on linear regression are shown for 2011 (red) and 2016-2017 (black).

Page 50 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Table 25. Estimated mercury concentration for 9” (229 mm) and 12” (305 mm) white perch in each lake segment in 2011 and 2016-2017 based on linear regressions with the percent change in mercury calculated. Empty cells indicate the specimens collected did not overlap the standard length. Significance (p-value) is listed for the comparisons (ANCOVA, factor: year, covariate: length) between the two studies for each lake segment. Gray highlighted cells indicate the regression was not significant in that year and was not included in the analysis. Asterisk indicated a significant interaction between the factor and covariate in the ANCOVA which limits the pairwise comparison.

2011 2016 Lake Segment p-value 2011 9" 2016 9" % ∆ 12" 12" % ∆ Missisquoi Bay 127 102 -25% 341 431 21% 0.13 Northeast Arm 187 253 26% 407 532 23% 0.17 Malletts Bay 225 202 -11% 594 424 -40% 0.09 North Main Lake 125 184 Main Lake 269 197 -37% 437 0.75 South Main Lake 204 205 564 230 South Lake 270 302 11% *

Page 51 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Random Forest Model A random forest model was built using all the data (1170 samples) for the five species (smallmouth bass, walleye, lake trout, yellow perch and white perch) to develop the model examining mercury concentrations in Lake Champlain fish. Seventy percent of the samples (818) were used to build the model with the remaining 30% (358) used to test the model’s ability to accurately predict the mercury concentrations in Lake Champlain fish throughout all study periods, location was not included. The random forest model explained 74% of the variance, the mean square of the residuals was 0.038. The measure of the model’s accuracy was calculated from the predicted values vs observed values. This model has an r2= 0.73 when comparing the 358 predicted and observed values used for testing (Figure 27). Through successive iterations of the model building with leaving one of the variables out, a relative importance of each variable is calculated by determining the percent increase in the mean square error for each variable when it is eliminated from the model. This model identified fish length as the most important variable with an increase in MSE of 55% when eliminated from the model. Species and study period were less important with an increase of MSE of 35% and 30%, respectively.

Figure 237. Random forest model for all study years to predict mercury concentrations in Lake Champlain fish.

Page 52 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Cyanotoxins A method of extracting, purifying, and analyzing cyanotoxins in fish tissue using LC- MS/MS was developed and validated. Eighty-one Lake Champlain fish collected during high and low-bloom periods were analyzed for common congeners and metabolites of microcystin including LR, dLR, RR, dRR, CYS-LR and GSH-LR.

Microcystin Congeners Fish were analyzed for microcystin LR, dLR, RR, dRR, and the glutathione (GSH-LR) and cysteine conjugate (CYS-LR) of microcystin LR. All 153 fish samples (muscle and liver) tested negative for all microcystin congeners and conjugates assessed with this method. The average detection limit for the LR microcystin congener was 5.8 μg/kg ± 6.2 μg/kg with values ranging between 0.9 μg/kg and 37.9 μg/kg. Detection limits for the other microcystin congeners and a complete list of samples analyzed are included in Appendix D. Of the 153 samples analyzed, 39% were analyzed in duplicate as a QA/QC measure.

Anatoxin and Cylindrospermopsin All 153 fish samples tested negative for cylindrospermopsin and anatoxin-a. The median detection limit for anatoxin-a was 1.5 µg/kg while the mean was 309.9 μg/kg ± 433.5 μg/kg. Values ranged between a minimum of 0.1 μg/kg and a maximum of 2527.3 μg/kg. Of the 153 samples analyzed, 12% were analyzed in duplicate as a QAQC measure. The average detection limit for cylindrospermopsin was 26.2 μg/kg ± 28.6 μg/kg with values ranging between 2.4 μg/kg and 173.4 μg/kg. The limit of detection varies as a function of the tissue mass extracted and the final extraction volume. A complete list of samples tested for anatoxin-a and cylindrospermopsin in addition to sample specific detection limits is included in Appendix D.

Page 53 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Conclusions Mercury

All fish species tested had mercury concentrations that exceeded the US EPA advisory limit of 300 ppb and with the exception of yellow perch, had at least one specimen that exceeded the USFDA action limit of 1000 ppb. Smallmouth bass had 27/157 (17%) and walleye had 28/74 (38%) specimens exceed the USFDA limit, while lake trout and white perch had only a single specimen exceed 1000 ppb. ANCOVAs and the random forest model showed fish mercury concentrations were strongly influenced by length, species and location but not relative weight (Table 26). The link between mercury concentration and fish length has been well established and while age or trophic position may provide a better metric to explain mercury variability in fish, length is a universal metric that is easily conveyed to the public to address fish consumption advisories. The attempt to explain additional variability in fish mercury using body condition (relative weight) was unfruitful. Previous research has shown that body condition can help explain mercury variability in yellow perch, however expanding to other species resulted in contrasting trends. Greenfield et al. (2001) determined that yellow perch with lower relative weight (skinny) exhibited higher mercury levels than fish with higher relative weight, presumably from less efficient growth. The relationship held true for most of the yellow perch but a positive trend was observed in smallmouth bass throughout the lake and most of the walleye and lake trout (Table 27). This inconsistency suggests that body condition at time of capture is not adequate to generalize growth rates throughout a long-lived species’ entire life.

Table 26. P-values for ANCOVA analyses for each fish species using location as the factor, log ppb as dependent variable and length and relative weight as covariates. Only significant trends were included in these analyses. Significant trends are highlighted in yellow.

Smallmouth Walleye Lake Trout Yellow Perch White Perch Bass Location <0.001 0.002 0.017 0.040 0.005 Length <0.001 <0.001 <0.001 <0.001 <0.001 Relative weight (Wr) 0.917 0.053 0.795 0.212 0.394 Location x length 0.175 0.074 0.715 0.005 0.085 Location x Wr 0.203 0.035 0.010 0.713 0.002

Page 54 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Table 27. Correlations (positive or negative) between relative weight and mercury content for each species in each lake segment. Previous research has shown a negative relationship with body condition and mercury content due to abundant prey items and thus less exposure, but the trend only appears to be prominent in yellow perch.

Smallmouth Walleye Lake Trout Yellow Perch White Perch Bass Missisquoi Bay + - NA - - Northeast Arm + + NA - - Malletts Bay + + NA - - North Main Lake + + + - + Main Lake + + - - + South Main Lake + + + + - South Lake NA NA NA - +

While fish mercury was significantly influenced by location, no consistent trends existed for all five species among the lake segments. Mercury concentrations in fish are influenced by numerous variables, initially the amount of mercury being deposited to the aquatic environment through atmospheric deposition and runoff. Then it has to be methylated by bacteria in anaerobic environments (i.e. wetlands, sediments, hypoxic hypolimnion) and introduced into the food web (bacteria and algae). Fish will prey on a variety of items over their lifetime depending on their size. The prey items a fish will feed on begins with the habitat and feeding strategy of the fish. In this study we have cold- water (lake trout and walleye), cool water (smallmouth bass) and warm water (yellow and white perch) fish. The prey items can vary in each layer of the lake either by presence/absence, the nutritional quality or the mercury concentration. While walleye, lake trout and white perch feed mainly on fish as adults, smallmouth bass and yellow perch feed on a combination of insects, crayfish and fish which have direct links to both pelagic and benthic food webs. Even if fish are consuming the same type of prey item, if one is of higher quality the result will be more efficient growth leading to lower mercury exposure and thus lower mercury burden. All of these factors influence fish mercury concentrations making generalizations among species and lake segments extremely difficult. What can be said from the data collected is walleye and smallmouth bass pose the greatest health risk exhibiting the highest mercury concentrations (Figure 28).

Page 55 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Figure 248. Linear regressions for species in each lake segment, only significant trends were included. Red dashed lines represent the US EPA mercury advisory limit of 300 ppb (bottom) and the USFDA action limit of 1000 ppb (top).

Page 56 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Long-term Mercury Trends

Analyzing long-term fish mercury trends shows a general decrease in fish mercury concentrations since the initial collections for lake trout (1987), walleye (1990) and yellow perch (1988) but not for smallmouth bass or white perch. This may be explained by the later initial collections and small sample sizes of smallmouth bass and white perch. Smallmouth bass were initially collected in 1996 but only 10 specimens were analyzed and white perch collections were so few during initial samplings that years 1993 to 2004 were combined to obtain an adequate sample size. An unexpected observation was the significant increase in mercury concentrations in smallmouth bass and yellow perch since 2011. While analyzing lake-wide fish mercury concentrations between study periods ignores the differences that exist among lake segments, it provides greater confidence in the regression analysis due to larger sample sizes and a larger size range allowing generalizations to be made with confidence.

Assessing changes in fish mercury since 2011 within individual lake segments allowed spatial differences to be accounted for but sometimes at the expense of confidence in the regression due to smaller sample sizes and size ranges. Additionally, individual specimens outside a lake segment’s narrow size range can disproportionately influence the trend relative to using whole lake data. Comparing the whole lake trends for smallmouth bass, yellow perch and white perch between the 2011 and the 2016-2017 studies show similar bioaccumulation rates while individual segments can vary substantially (Figures 16, 23 & 26). Therefore, any contradicting trends between studies should have sample size and size range considered.

Assessing the change in fish mercury between the 2011 and the 2016-2017 studies identified significant increases in smallmouth bass and yellow perch mercury when using whole lake data but were heavily influenced by significant increases in the Main Lake and North Main Lake, respectively (Table 28). Many lake segments exhibited either little change or contradicting directional changes between small and large specimens and should be viewed with caution. Interestingly, the most consistent trend observed in any lake segment occurred in Malletts Bay with a consistent decrease in fish mercury for all three species at both small and large standard lengths. The findings from whole lake and individual lake segments raises two main questions:

1) Why is fish mercury increasing in some species since 2011? 2) What is unique about Malletts Bay that shows a continued mercury decrease in all species analyzed?

An reversal in fish mercury from a dominantly decreasing trend since peak fish mercury in the 1970s and 1980s has been documented in the Providence of Ontario and the Great Lakes Region for walleye and northern pike but with no definitive explanation (Gandhi et al. 2014, Monson et al. 2011).Possible explanations put forth for the increased fish mercury include increased mercury deposition from Asia, the introduction

Page 57 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain of invasive species altering foodweb dynamics and enhanced methylation due to warmer temperatures. These explanations are also feasible in Lake Champlain. Lake Champlain would likely receive similar mercury loading from Asia through atmospheric deposition as the Great Lakes and Ontario. The invasion of the alewife in the early 2000s could have altered the foodweb by increasing predation pressure on native smelt and/or large zooplankton species resulting in a less efficient foodweb. Water temperature in Lake Champlain has been increasing in recent years making increased methylation rates a likely possibility. Bacteria responsible for the methylation of mercury will increase activity with warmer temperatures. Benthic environments are ideal for mercury methylation which could lead to benthic fauna having elevated mercury levels. Benthic fauna are consumed by opportunistic feeders like smallmouth bass and yellow perch, the two species shown to significantly increase in mercury since 2011. Another possibility is the effect of Hurricane Irene in 2011 which likely delivered a substantial amount of mercury to the lake during high discharge periods. Also, the resulting high lake level could have methylated mercury from peripheral areas that typically remain dry. All of these theories are possible and will require additional research before any definitive statements can be made.

Malletts Bay exhibited consistent decreasing mercury trends for all three species assessed (smallmouth bass, yellow perch and white perch) with the yellow perch decrease being significant. No other lake segment showed as consistent of a trend as Malletts Bay with other lake segments often increasing in fish mercury, showing no change or trends contradicting each other depending on standard length estimates. The exact explanation for the continued decrease likely revolves around the constricted hydrology in Malletts Bay. Malletts Bay is unique in that it has relatively low productivity compared to other lake segments but is the only segment to experience regular hypoxia prior to fall turnover. This annual occurrence has resulted in the focusing of heavy metals in deeper regions of the Bay because heavy metals are liberated from sediments under low oxygen conditions and when turnover redistributes oxygen to the hypolimnion, the metals are re-precipitated to deeper sediments (McIntosh et al. 1997). Because mercury can be methylated under anoxic conditions and become incorporated into the foodweb, it does not respond in a similar fashion as other heavy metals. Mercury methylation could have depleted the more reactive (younger) sediment mercury resulting in lower concentrations than other areas of the lake. If warmer water temperatures are increasing methylation in sediments, this could explain why fish mercury in Malletts Bay are not responding the same as most of the lake. This is another hypothesis that could be tested by collecting sediment cores to analyze for mercury along with radionuclide dating. It is clear that our understanding of mercury in the Lake Champlain foodweb is far from complete and more research is required before we can definitively state why fish mercury in Malletts Bay continues to decrease while other areas remain stable or have begun to increase.

Table 28. Percent change in estimated fish mercury for small and large standard lengths between the 2011 and 2016-2017 studies in each lake segment. “No Fish” indicates fish were not collected from that lake segment in at least one of the

Page 58 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain studies, NA indicates collected sample size did not overlap standard length and NS indicates trend was not significant in the year indicated. Yellow highlight indicates a significant difference between study periods based on ANCOVAs.

Smallmouth Bass Yellow Perch White Perch 14" 18" 7" 10" 9" 12" Missisquoi Bay No Fish 2016 NS -25% 21% Northeast Arm -15% 0% -16% 60% 26% 23% Malletts Bay -12% -22% -14% -23% -11% -40% North Main Lake NA 36% 95% 79% 2016 NS Main Lake 62% 44% 2011 & 2016 NS -37% NA South Main Lake 2011 NS 2011 NS 2016 NS South Lake No Fish 6% NA 11% NA

Cyanotoxins

Water samples were analyzed for microcystins, anatoxin-a, and cylindrospermopsin with negative results for the three cyanotoxins measured. We unfortunately did not have algal biomass results to determine if these results were due to a lack of a bloom or was due to the presence of a non-toxic bloom. Separate analysis by VT DEC were also negative for cyanotoxins during this time period. Hence, it is likely that the blooms that occurred in Lake Champlain were non-toxic. In the present study we used a highly sensitive LC-MS method that monitored 17 common microcystins found in the Northeast as our screening method. Vermont DEC uses the ADDA specific ELISA assay for microcystins. While these two techniques use different approaches for measuring microcystins, the two methods agreed with each other, that all water samples were negative for cyanotoxins.

To protect human health, the World Health Organization (WHO) recommended the tolerable daily intake (TDI) for microcystins over the lifetime of an individual to be 0.04 µg microcystin-LR equivalents per kg of body weight per day (WHO, 2008). Ibelings and Chorus (2007) used the seasonal WHO guideline value of 0.4 µg/kg body weight/day to estimate a seasonal (daily exposure for several weeks during the cyanobacterial season) tolerable intake for microcystin-LR in food. Converting this laboratory value to the amount of fish that could be safely consumed is dependent upon several factors, including body weight of the affected individual, amount of contaminated fish consumed per day, the duration and mode of exposure and several uncertainty factors. Using a safety factor of 100, a body weight of 10 kg for a child and 75 kg for an adult and 100 g of contaminated fish being consumed per day, Ibelings and Chorus (2007) derived a seasonal value of 300 µg/kg of food for adults and 40 µg/kg of food for children. Levels for lifetime exposure to microcystin-LR using similar assumptions and safety factors would be tenfold lower. Using these numbers, none of the fish tissues tested in this study would have exceeded the seasonal value for adults. However due to the limited sample weight extracted, 12% of the 153 samples extracted had a detection limit >300 μg/kg (Appendix C). For those12%, the detection limit was not sufficient to meet this threshold criterion. This limitation becomes more critical for the child threshold value of 40 µg/kg. Approximately half of the samples analyzed (55%) did not have a detection limit

Page 59 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain sufficient to determine if the levels in fish exceeded 40 µg/kg. Measures such as increasing the amount of tissue extracted from 0.2 to 0.4 g and decreasing the final volume to 1 ml rather than 5 ml were taken later in the project to lower this detection limit. For these samples (n=52), >99% of the samples would have detected any microcystin LR in these fish tissues greater than 40 µg/kg. Lower detection limits were observed during the reanalysis of the samples for the microcystin congeners. During this analysis period all samples analyzed had sufficient detection limits for determining if the adult and children guidelines were being exceeded.

The same 81 fish evaluated for microcystins were also tested for anatoxin-a and cylindrospermopsin. All samples were found to be negative for anatoxin-a, homo- anatoxin-a, cylindrospermopsin (including the epi derivative) and the analog deoxycylindrospermopsin. No safe consumption guidelines have been established for anatoxin-a and cylindrospermopsin in fish. However, consumption guidelines can be derived following the calculations of Ibelings and Chorus (2007) and utilizing anatoxin-a and cylindrospermopsin references from the US EPA. For both the anatoxin-a and cylindrospermopsin, the amount of contaminated fish consumed per day was assumed to be 100 g per day and the average adult and child body weights were assumed to be 75kg and 10 kg, respectively. No TDI has been established for anatoxin-a due to insufficient data. However, one was estimated using the US EPA No Observable Adverse Effect Level (NOAEL) for anatoxin-a (50 µg/kg of body weight per day; D’Anglada et al. 2015) and dividing by a safety factor of 1000 to give a TDI of 0.05 µg/kg of body weight per day. Using this estimated TDI, a seasonal guideline for anatoxin-a in fish was derived to be 37.5 µg/kg for adults and 5 µg/kg for children. Using these values, 54% of the samples had detection limits sufficient for determining if both the adult and children guidelines were being exceeded.

For cylindrospermopsin, the USEPA’s references dose (Rfd) of 0.1 µg/kg of bodyweight per day (D’Anglada et al. 2015) was used to derive a safe toxin concentration in fish of 75 µg/kg for adults and 10 µg/kg for children. Ninety-seven percent of the samples had a detection limit sufficient to detect if the adult guideline was exceeded and 40% of the samples had a sufficient detection limit to determine if the children’s guideline values were exceeded.

The SPE step used in this method to purify the fish samples was optimized for microcystins and not anatoxin-a and cylindrospermopsin. Due to the limited availability of sample and cost, a separate clean-up method was not optimized for anatoxin-a and cylindrospermopsin. All of the water samples were negative for cylindrospermopsin (Appendix D) and cylindrospermopsin has not been detected in New York or Vermont waters and hence these limitations likely have no impact on human health. It is highly unlikely that the fish in this study contained cylindrospermopsins. Anatoxin-a has been reported in Lake Champlain at relatively low concentrations. However, as the water samples were negative for anatoxin-a, it is also unlikely that the fish contained the toxin.

Page 60 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Further studies should focus on refining the analytical methods for anatoxin-a in fish tissues.

While all water samples and fish samples (muscle and liver) were negative for cyanotoxins and thus limiting correlation analyses, two facts should be highlighted. First, the method used is appropriate and sensitive to the most common cyanotoxin, microcystin. And second, blooms with detectable microcystin were present in 2015 but no microcystin, anatoxin or cylindrospermopsin were detectable in fish during 2016 indicating no long-term bioaccumulation.

Page 61 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain References

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Page 62 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Harada K. 1996. Chemistry and detection of microcystins. In: “Toxic Microcystis” MF Watanabe, K Harada, WW Carmichael, H Fujiki, Eds., pp. 103-148. Hudnell HK. 2010. The state of U.S. freshwater harmful algal blooms assessments, policy and legislation. Toxicon 55: 1024-1034. Ibelings, B.W. & Chorus, I. 2007. Accumulation of cyanobacterial toxins in freshwater “seafood” and its consequence for public health: A review. Environmental Pollution. 150: 177-192. Johnson I. 2012. A synoptic assessment of mercury and re-evaluation of PCB’s in Lake Champlain Fishes. Lake Champlain Basin Program Technical Report No. 66. pp. 63. Lawton LA & Edwards C. 2001. Purification of microcystins. Journal of Chromatography A. 912: 191-209. Marvin-DiPasquale M, Lutz MA, Brigham ME, Krabbenhoft DP, Aiken GR, Orem WH & Hall BD. 2009. Mercury cycling in stream ecosystems. 2. Benthic methylmercury production and bed sediment-pore water partitioning. Environmental Science & Technology 43: 2726-2732. McIntosh A, Watzin M & Brown E. 1997. Lake Champlain sediment toxics assessment program. An assessment of sediment-associated contaminants in Lake Champlain Phase II. Lake Champlain Basin Program Technical Report No. 23B. pp. 593. Mihuc TB, Boyer GL, Satchwell MF, Pellam M, Jones J, Vasile J, Bouchard A & Bonham R. 2005. 2002 Phytoplankton community composition and cyanobacterial toxins in Lake Champlain, U.S.A. Verhandlungen des Internationalen Verein Limnologie 39: 328-333. Mihuc TB, Boyer GL, Jones J, Satchwell MF & Watzin MC. 2006. Lake Champlain phytoplankton and algal toxins: past and present. GLRC Great Lakes Research Review 7:18-21. Monson BA, Staples DF, Bhavsar SP, Holsen TM, Schrank CS, Moses SK, McGoldrick DJ, Backus SM & Williams KA. 2011. Spatiotemporal trends of mercury in walleye and largemouth bass from the Laurentian Great Lakes Region. Ecotoxicology 20: 1555-1567. Ohio EPA. 2015. Total (Extracellular and Intracellular) Microcystins - ADDA by ELISA Analytical Methodology, Version 2.0. Available online at http://www.epa.ohio.gov/Portals/28/documents/habs/HAB_Analytical_Methodolog y.pdf O'Neil JM, Davis TW, Burford MA & Gobler CJ. 2012. The rise of harmful cyanobacteria blooms: The potential roles of eutrophication and climate change. Harmful Algae 14: 313-334. Paerl HW, Hall NS & Calandrino ES. 2011. Controlling harmful cyanobacterial blooms in a world experiencing anthropogenic and climatic-induced change. Science of the Total Environment 409: 1739-1745. Paerl HW & Paul VJ. 2012. Climate change: Links to global expansion of harmful cyanobacteria. Water Research 46: 1349-1363.

Page 63 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Papadimitriou T, Kagalou I, Stalikas C, Pilidis G & Leonardos ID. 2012. Assessment of microcystin distribution and biomagnification in tissues of aquatic food web compartments from a shallow lake and evaluation of potential risks to public health. Ecotoxicology 21: 1155-1166. Schmidt JR. 2014. Fate and Detection of Microcystin-LR and its Metabolites in the Environment. PhD Thesis, Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York. Schmidt JR, Shaskus M, Estenik JF, Oesch C, Khidekel R & Boyer GL. 2013. Variations in the microcystin content of different fish species collected from a eutrophic lake. Toxins 5: 992–1009. Schmidt JR, Wilhelm SW & Boyer GL. 2014. The fate of microcystins in the environment and challenges for monitoring. Toxins 6: 3354–3387. Schneider JC, Laarman PW & Gowing H. 2000. Length-weight relationships. Chapter 17 in Schneider, JC (ed.) 2000. Manual of fisheries survey methods II: with periodic updates. Michigan Department of Natural Resources, Fisheries Special Report 25, Ann Arbor. Selin, NE. 2009. Global biogeochemical cycling of mercury: a review. Annual Review of Environment and Resources 34: 43-63. Shambaugh A, Chevrefils A & Winslow M. 2015. Cyanobacteria monitoring on Lake Champlain Summer 2014. Report prepared for the Lake Champlain Basin Program. pp. 106. Shambaugh A, Vose S, O’Brien B, Fisher L & Campbell H. 2017. Cyanobacteria monitoring on Lake Champlain Summer 2016 (Final Report for the LCBP April 2017). [UNEP] United Nations Environment Programme. 2013. Global Mercury Assessment. Available online at http://www.unep.org US EPA. 2015a. Method 545: Determination of cylindrospermopsin and anatoxin-a in drinking water by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS). Available online at https://www.epa.gov/dwanalyticalmethods/method-545-determination- cylindrospermopsin-and-anatoxin-drinking-water-liquid US EPA. 2015b. METHOD 544. Determination of microcystins and nodularin in drinking water by solid phase extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS). Available online at https://cfpub.epa.gov/si/si_public_record_report.cfm?dirEntryId=306953 Ward DM, Nislow KH & Folt CL. 2010. Bioaccumulation syndrome: Identifying factors that make some stream food webs prone to elevated mercury bioaccumulation. Annals of the New York Academy of Sciences 1196: 62-83. Wiegand C & Pflugmacher S. 2005. Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review. Toxicology and Applied Pharmacology 203: 201-218. Wood JD, Franklin RB, Garman G, McIninch S, Porter AJ & Bukaveckas. 2014. Exposure to cyanotoxin microcystin arising from interspecific differences in feeding habits among fish and shellfish in the James River Estuary, Virginia. Environmental Science & Technology 48: 5194-5202.

Page 64 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Appendices Appendix A………………………………………………………………………………….66 Mercury concentration of five fish species (smallmouth bass, walleye, lake trout, yellow perch and white perch) caught in the seven lake segments (South Lake, South Main Lake, Main Lake, North Main Lake, Malletts Bay, Northeast Arm and Missisquoi Bay). Data includes weight and length measurements, along with relative weight (Wr) which is a body condition metric that is calculated based on the weight and length of the fish. Each fish species has an ideal growth curve that predicts weight at each length. A value of 100 indicates the fish is in agreement with the ideal growth while fish with Wr above 100 indicate the fish weighs more than expected (i.e. plump) while values less than 100 indicate the fish weighs less than expected (i.e. skinny). Equations used to determine the relative weight of each fish were obtained from Schneider et al. (2000).

Near the end of the study, the DMA-80 experienced an irreparable malfunction which resulted in 30 samples not being included in the statistical analyses. Four samples were never analyzed because they were received after the malfunction, these are smallmouth bass from South Lake while the additional 26 samples were outside the 95% prediction intervals based on the regressions (highlighted) but could not be re-run due to the DMA- 80 irreparable malfunction.

Appendix B………………………………………………………………………………….89 Lake Champlain water sample results for microcystin, anatoxin-a, and cylindrospermopsin. All water samples test negative for all three cyanotoxins. Method detections limits are reported for each sample by analysis type. Average detection limits are also reported for each analysis. “F” indicates the sample was a filter.

Appendix C………………………………………………………………………………….90 Fish specimens analyzed for MC-LR including tissue type (M, Muscle; L, Liver), weights, recoveries and detection limit.

Appendix D………………………………………………………………………………….97 Detection limits for microcystin congeners, anatoxin-a and cylindrospermopsin for all fish samples tested. Method detection limits for CYS-LR, homo-anatoxin-a and deoxycylindrospermopsin are not reported but are similar (±20%) to their parent compound. Since the same fish samples were analyzed for all of the analyses (MC-LR, microcystin congeners, anatoxin-a and cylindrospermopsins) sample weights, tissue types and percent recoveries are not presented here, this information is in Appendix C.

Page 65 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Appendix A. Fish collected for mercury analysis with species, location, weight, length, relative weight (Wr) and mercury concentration for each sample tested. Highlighted cells indicate the mercury concentration was an outlier that could not be validated while no values indicate sample was collected but not run due to an irreparable malfunction of the mercury analyzer.

Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB Main Lake 0.29 8.75 222 85 0.127 2.104 SMB Main Lake 0.49 8.88 225 138 0.264 2.422 SMB Main Lake 0.44 9.00 229 119 0.129 2.111 SMB Main Lake 0.71 11.25 286 98 0.222 2.346 SMB Main Lake 0.90 12.25 311 96 0.304 2.483 SMB Main Lake 0.92 12.50 318 92 0.274 2.438 SMB Main Lake 0.96 13.00 330 85 0.317 2.501 SMB Main Lake 1.08 13.63 346 83 0.485 2.686 SMB Main Lake 1.24 13.75 349 93 0.565 2.752 SMB Main Lake 1.78 15.00 381 102 0.328 2.516 SMB Main Lake 2.10 15.50 394 109 0.378 2.577 SMB Main Lake 2.01 15.75 400 100 0.516 2.713 SMB Main Lake 3.39 16.25 413 153 0.446 2.649 SMB Main Lake 2.48 16.25 413 112 0.070 SMB Main Lake 2.14 16.50 419 92 0.721 2.858 SMB Main Lake 1.88 16.50 419 81 0.880 2.944 SMB Main Lake 1.87 16.99 432 74 0.651 2.814 SMB Main Lake 2.18 17.00 432 86 0.394 2.595 SMB Main Lake 2.98 17.00 432 117 0.838 2.923 SMB Main Lake 2.77 17.50 445 100 1.109 3.045 SMB Main Lake 2.82 17.88 454 95 0.476 2.678 SMB Main Lake 3.10 17.88 454 105 0.776 2.890 SMB Main Lake 3.22 18.00 457 107 0.425 2.628 SMB Main Lake 2.99 18.00 457 99 0.487 2.688

Page 66 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB Main Lake 3.39 18.00 457 112 0.781 2.893 SMB Main Lake 2.73 18.00 457 90 1.283 3.108 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB Main Lake 3.40 18.25 464 108 0.528 2.723 SMB Main Lake 2.78 18.25 464 88 0.569 2.755 SMB Main Lake 2.78 18.25 464 88 1.041 3.017 SMB Main Lake 2.86 18.50 470 87 0.654 2.816 SMB Main Lake 3.15 18.50 470 96 0.674 2.829 SMB Main Lake 3.13 18.50 470 95 0.836 2.922 SMB Main Lake 3.61 18.75 476 106 0.715 2.854 SMB Main Lake 2.74 18.75 476 80 1.247 3.096 SMB Main Lake 3.58 18.75 476 105 1.487 3.172 SMB Main Lake 3.60 19.00 483 101 0.357 2.553 SMB Main Lake 3.49 19.00 483 98 0.406 2.609 SMB Main Lake 3.30 19.00 483 93 0.776 2.890 SMB Main Lake 3.67 19.00 483 103 1.024 3.010 SMB Main Lake 3.85 19.00 483 108 1.093 3.039 SMB Main Lake 3.75 19.00 483 106 1.104 3.043 SMB Main Lake 4.08 19.00 483 115 1.474 3.168 SMB Main Lake 3.63 19.25 489 98 0.923 2.965 SMB Main Lake 3.75 19.50 495 98 0.685 2.836 SMB Main Lake 4.04 19.75 502 101 0.593 2.773 SMB Main Lake 3.55 20.00 508 86 0.720 2.857 SMB Main Lake 4.32 20.03 509 104 1.716 3.235 SMB Main Lake 3.95 21.00 533 82 0.516 SMB Mallets Bay 1.30 13.00 330 115 0.544 2.736 SMB Mallets Bay 1.25 13.00 330 111 0.679 SMB Mallets Bay 1.40 13.50 343 111 0.143 2.155 SMB Mallets Bay 1.45 13.50 343 115 0.187 2.272

Page 67 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB Mallets Bay 1.60 14.00 356 113 0.267 2.427 SMB Mallets Bay 1.50 14.00 356 106 0.281 2.449 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB Mallets Bay 1.55 14.50 368 99 0.220 2.342 SMB Mallets Bay 1.75 14.75 375 106 0.184 2.265 SMB Mallets Bay 1.65 15.00 381 95 0.142 2.152 SMB Mallets Bay 1.78 15.00 381 102 0.279 2.446 SMB Mallets Bay 2.75 17.75 451 95 0.433 2.636 SMB Mallets Bay 3.08 17.75 451 107 0.685 2.836 SMB Mallets Bay 3.15 19.00 483 89 0.324 2.511 SMB Mallets Bay 3.57 20.00 508 86 0.985 2.993 SMB Mallets Bay 4.02 20.50 521 90 1.097 3.040 SMB Missisquoi Bay 0.51 10.25 260 93 0.336 2.526 SMB Missisquoi Bay 0.69 11.75 298 83 0.306 2.486 SMB Missisquoi Bay 0.60 12.00 305 68 0.265 2.423 SMB Missisquoi Bay 0.93 12.75 324 88 0.179 2.253 SMB Missisquoi Bay 1.09 13.00 330 97 0.336 2.526 SMB Missisquoi Bay 1.21 13.25 337 101 0.154 2.188 SMB Missisquoi Bay 1.26 14.00 356 89 0.201 2.303 SMB Missisquoi Bay 1.36 14.25 362 91 0.352 2.547 SMB Missisquoi Bay 1.57 14.50 368 100 0.271 2.433 SMB Missisquoi Bay 1.65 14.50 368 105 0.305 2.484 SMB Missisquoi Bay 2.02 15.00 381 116 0.660 2.820 SMB Missisquoi Bay 1.91 15.50 394 100 0.381 2.581 SMB Missisquoi Bay 2.08 15.50 394 108 0.491 2.691 SMB Missisquoi Bay 2.39 15.50 394 125 0.547 2.738 SMB Missisquoi Bay 2.09 15.75 400 104 0.410 2.613 SMB Missisquoi Bay 2.55 16.00 406 121 0.626 2.797 SMB Missisquoi Bay 2.50 16.25 413 113 0.495 2.695

Page 68 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB Missisquoi Bay 2.26 16.50 419 97 0.650 2.813 SMB Missisquoi Bay 2.24 16.50 419 97 0.713 2.853 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB Missisquoi Bay 1.98 16.50 419 85 1.061 SMB Missisquoi Bay 2.50 16.75 425 103 0.723 2.859 SMB Missisquoi Bay 2.36 16.75 425 97 0.772 2.888 SMB Missisquoi Bay 2.51 17.00 432 99 0.611 2.786 SMB Missisquoi Bay 2.22 17.00 432 87 0.647 2.811 SMB Missisquoi Bay 3.17 17.10 434 123 0.788 2.897 SMB Missisquoi Bay 3.00 17.75 451 104 0.491 2.691 SMB Missisquoi Bay 2.78 17.75 451 96 0.667 2.824 SMB Missisquoi Bay 2.89 17.75 451 100 0.755 2.878 SMB Missisquoi Bay 2.66 18.00 457 88 0.788 2.897 SMB Missisquoi Bay 2.31 18.00 457 77 0.813 2.910 SMB Missisquoi Bay 2.06 18.00 457 68 0.925 2.966 SMB Missisquoi Bay 2.91 18.25 464 93 0.746 2.873 SMB Missisquoi Bay 3.69 18.25 464 117 0.949 2.977 SMB Missisquoi Bay 3.10 18.25 464 99 0.428 SMB Missisquoi Bay 3.68 18.50 470 112 0.710 2.851 SMB Missisquoi Bay 3.32 18.75 476 97 0.832 2.920 SMB Missisquoi Bay 3.46 18.75 476 101 0.847 2.928 SMB Missisquoi Bay 4.14 19.00 483 117 0.961 2.983 SMB Missisquoi Bay 3.03 19.38 492 80 1.313 3.118 SMB Missisquoi Bay 3.00 19.50 495 78 1.472 3.168 SMB Missisquoi Bay 3.81 19.75 502 95 0.875 2.942 SMB N. Main Lake 2.15 17.00 432 85 0.612 2.787 SMB N. Main Lake 2.37 17.50 445 86 0.731 2.864 SMB N. Main Lake 2.90 18.00 457 96 0.210 2.322 SMB N. Main Lake 3.21 18.25 464 102 0.878 2.943

Page 69 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB N. Main Lake 3.32 18.50 470 101 0.935 2.971 SMB N. Main Lake 2.76 19.00 483 78 0.882 2.945 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB N. Main Lake 2.83 19.00 483 80 1.323 3.122 SMB N. Main Lake 3.99 19.25 489 108 0.818 2.913 SMB N. Main Lake 3.74 19.25 489 101 0.923 2.965 SMB N. Main Lake 3.45 19.50 495 90 0.735 2.866 SMB N. Main Lake 3.65 19.50 495 95 1.203 3.080 SMB N. Main Lake 3.68 19.50 495 96 0.111 SMB N. Main Lake 3.92 19.75 502 98 1.104 3.043 SMB N. Main Lake 3.49 20.00 508 84 0.812 2.910 SMB N. Main Lake 4.08 20.00 508 98 0.871 2.940 SMB N. Main Lake 3.44 20.00 508 83 1.213 3.084 SMB N. Main Lake 3.73 20.00 508 90 1.330 3.124 SMB Northeast Arm 1.34 14.25 362 90 0.362 2.559 SMB Northeast Arm 2.37 16.50 419 102 0.297 2.473 SMB Northeast Arm 2.11 16.50 419 91 0.417 2.620 SMB Northeast Arm 2.61 17.00 432 103 0.331 2.520 SMB Northeast Arm 2.32 17.00 432 91 0.527 2.722 SMB Northeast Arm 2.88 17.25 438 109 0.527 2.722 SMB Northeast Arm 2.58 17.50 445 93 0.505 2.703 SMB Northeast Arm 3.64 17.50 445 131 0.562 2.750 SMB Northeast Arm 2.43 17.63 448 86 0.582 2.765 SMB Northeast Arm 2.77 17.75 451 96 0.538 2.731 SMB Northeast Arm 2.49 17.75 451 86 0.696 2.843 SMB Northeast Arm 3.09 18.00 457 102 0.412 2.615 SMB Northeast Arm 2.72 18.00 457 90 0.451 2.654 SMB Northeast Arm 2.69 18.00 457 89 0.581 2.764 SMB Northeast Arm 2.76 18.00 457 91 0.644 2.809

Page 70 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB Northeast Arm 2.64 18.00 457 88 0.661 2.820 SMB Northeast Arm 3.46 18.00 457 115 1.420 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB Northeast Arm 3.02 18.25 464 96 0.573 2.758 SMB Northeast Arm 3.14 18.50 470 96 0.522 2.718 SMB Northeast Arm 3.15 18.50 470 96 1.074 SMB Northeast Arm 3.41 19.00 483 96 0.532 2.726 SMB Northeast Arm 3.49 19.00 483 98 0.586 2.768 SMB Northeast Arm 3.20 19.25 489 87 0.930 2.968 SMB Northeast Arm 3.56 19.50 495 93 1.012 3.005 SMB Northeast Arm 3.41 19.50 495 89 1.123 3.050 SMB Northeast Arm 3.59 19.75 502 90 0.989 2.995 SMB Northeast Arm 4.25 19.75 502 106 1.151 3.061 SMB Northeast Arm 4.04 20.00 508 97 0.943 2.975 SMB Northeast Arm 3.28 20.00 508 79 1.078 3.033 SMB Northeast Arm 3.88 20.00 508 93 1.203 3.080 SMB Northeast Arm 3.83 20.25 514 89 0.779 2.892 SMB South Lake 0.90 13.00 330 80 SMB South Lake 3.41 18.00 457 113 SMB South Lake 3.17 18.75 476 93 SMB South Lake 3.90 19.00 483 110 SMB South Main Lake 1.00 12.00 305 113 0.191 2.281 SMB South Main Lake 1.11 13.00 330 99 0.513 2.710 SMB South Main Lake 2.74 17.00 432 108 0.658 2.818 SMB South Main Lake 3.00 17.50 445 108 0.757 2.879 SMB South Main Lake 3.34 17.50 445 121 1.404 3.147 SMB South Main Lake 2.80 18.00 457 93 0.538 2.731 SMB South Main Lake 3.22 18.25 464 102 0.558 2.747 SMB South Main Lake 3.61 18.50 470 110 0.823 2.915

Page 71 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain SMB South Main Lake 3.48 19.00 483 98 0.871 2.940 SMB South Main Lake 3.53 19.00 483 99 0.921 2.964 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) SMB South Main Lake 3.02 19.00 483 85 1.309 3.117 SMB South Main Lake 3.32 19.00 483 93 1.958 3.292 SMB South Main Lake 3.79 19.50 495 99 1.338 3.126 Walleye Main Lake 2.80 21.00 533 92 0.381 2.581 Walleye Main Lake 5.19 24.00 610 114 0.815 2.911 Walleye Main Lake 5.98 26.25 667 100 1.193 3.077 Walleye Main Lake 6.90 26.50 673 112 1.708 3.232 Walleye Main Lake 8.26 26.50 673 134 1.748 3.243 Walleye Mallets Bay 2.70 19.00 483 121 0.470 2.672 Walleye Mallets Bay 1.50 19.00 483 67 0.677 2.831 Walleye Mallets Bay 3.20 20.00 508 122 0.518 2.714 Walleye Mallets Bay 3.60 20.50 521 128 0.630 2.800 Walleye Mallets Bay 3.15 20.50 521 112 0.938 2.972 Walleye Mallets Bay 5.16 21.00 533 170 1.308 3.117 Walleye Mallets Bay 3.63 21.25 540 116 0.881 2.945 Walleye Mallets Bay 4.66 23.00 584 117 2.036 Walleye Mallets Bay 4.10 23.50 597 96 0.871 2.940 Walleye Mallets Bay 5.30 23.50 597 124 1.137 3.056 Walleye Mallets Bay 6.42 24.50 622 133 1.185 3.074 Walleye Mallets Bay 8.60 29.00 737 107 1.551 3.191 Walleye Mallets Bay 6.51 29.00 737 81 1.900 3.279 Walleye Missisquoi Bay 3.05 16.00 406 230 0.146 2.164 Walleye Missisquoi Bay 3.30 20.75 527 113 0.396 2.598 Walleye Missisquoi Bay 3.45 22.00 559 99 0.536 2.729 Walleye Missisquoi Bay 3.80 22.00 559 109 0.901 2.955 Walleye Missisquoi Bay 3.70 23.00 584 93 0.882 2.946

Page 72 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Walleye Missisquoi Bay 3.90 23.00 584 98 1.015 3.006 Walleye Missisquoi Bay 5.30 24.00 610 117 0.806 2.906 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Walleye Missisquoi Bay 4.50 24.00 610 99 0.935 2.971 Walleye Missisquoi Bay 4.30 24.25 616 92 1.015 3.006 Walleye Missisquoi Bay 4.80 24.25 616 102 1.358 3.133 Walleye Missisquoi Bay 5.80 24.50 622 120 0.901 2.955 Walleye Missisquoi Bay 6.20 25.50 648 113 1.128 3.052 Walleye Missisquoi Bay 6.40 26.50 673 104 0.816 2.912 Walleye Missisquoi Bay 7.40 28.00 711 102 0.892 2.950 Walleye N. Main Lake 8.39 19.00 483 375 1.193 Walleye N. Main Lake 3.70 22.00 559 106 0.872 2.941 Walleye N. Main Lake 5.07 23.25 591 123 0.270 2.431 Walleye N. Main Lake 4.81 23.25 591 117 1.095 3.039 Walleye N. Main Lake 5.87 25.00 635 114 0.783 2.894 Walleye N. Main Lake 6.06 25.00 635 118 1.179 3.072 Walleye N. Main Lake 6.90 25.50 648 126 1.213 3.084 Walleye N. Main Lake 6.33 26.00 660 109 1.218 3.086 Walleye N. Main Lake 7.25 26.50 673 118 0.937 2.972 Walleye N. Main Lake 6.72 27.00 686 103 1.128 3.052 Walleye N. Main Lake 7.30 28.00 711 101 0.695 2.842 Walleye N. Main Lake 9.11 28.00 711 126 1.736 3.240 Walleye N. Main Lake 9.40 28.50 724 123 1.672 3.223 Walleye N. Main Lake 8.70 28.75 730 111 1.175 3.070 Walleye N. Main Lake 7.98 29.00 737 99 0.889 2.949 Walleye N. Main Lake 8.35 29.50 749 98 1.535 3.186 Walleye Northeast Arm 2.34 19.50 495 97 0.454 2.657 Walleye Northeast Arm 4.89 22.00 559 140 1.034 3.015 Walleye Northeast Arm 4.34 23.00 584 109 0.816 2.912

Page 73 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Walleye Northeast Arm 5.04 25.00 635 98 0.630 2.799 Walleye Northeast Arm 6.09 25.00 635 118 0.669 2.825 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Walleye Northeast Arm 5.17 25.00 635 100 0.961 2.983 Walleye Northeast Arm 5.06 25.00 635 98 1.048 3.020 Walleye Northeast Arm 6.09 25.00 635 118 1.217 3.085 Walleye Northeast Arm 7.01 26.00 660 121 0.834 2.921 Walleye Northeast Arm 6.72 27.50 699 98 1.238 3.093 Walleye Northeast Arm 9.65 28.75 730 123 1.515 3.180 Walleye South Main Lake 2.88 18.00 457 151 0.180 2.255 Walleye South Main Lake 2.00 19.50 495 83 0.277 2.442 Walleye South Main Lake 2.25 19.75 502 89 0.218 2.338 Walleye South Main Lake 2.68 20.00 508 103 0.256 2.408 Walleye South Main Lake 2.90 20.00 508 111 0.523 2.719 Walleye South Main Lake 2.85 20.75 527 98 0.232 2.365 Walleye South Main Lake 3.03 21.00 533 100 0.191 2.280 Walleye South Main Lake 3.40 21.00 533 112 0.200 2.301 Walleye South Main Lake 3.00 21.00 533 99 0.373 2.572 Walleye South Main Lake 3.15 21.00 533 104 0.567 2.753 Walleye South Main Lake 3.25 22.25 565 90 0.220 2.342 Walleye South Main Lake 3.35 22.25 565 93 0.410 2.613 Walleye South Main Lake 3.85 22.75 578 100 0.192 2.282 Walleye South Main Lake 4.25 23.50 597 100 0.634 2.802 Walleye South Main Lake 6.18 24.00 610 136 0.742 2.870 Walleye South Main Lake 6.83 26.00 660 118 1.300 3.114 Walleye South Main Lake 6.86 28.00 711 95 1.464 3.166 Lake Trout Main Lake 2.32 18.75 476 107 0.374 2.573 Lake Trout Main Lake 2.12 19.00 483 94 0.327 2.515 Lake Trout Main Lake 2.85 20.00 508 107 0.356 2.551

Page 74 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Lake Trout Main Lake 3.11 21.00 533 100 0.369 2.567 Lake Trout Main Lake 5.95 26.25 667 94 0.413 2.616 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Lake Trout Main Lake 7.95 26.50 673 122 0.343 2.535 Lake Trout Main Lake 8.05 27.00 686 116 0.380 2.580 Lake Trout Main Lake 7.04 27.00 686 102 0.455 2.658 Lake Trout Main Lake 8.67 27.00 686 125 0.625 2.796 Lake Trout Main Lake 6.72 27.00 686 97 0.870 Lake Trout Main Lake 7.46 27.00 686 108 0.894 Lake Trout Main Lake 7.30 27.50 699 100 0.512 2.709 Lake Trout Main Lake 6.73 27.50 699 92 0.546 2.737 Lake Trout Main Lake 7.16 27.50 699 98 0.702 2.846 Lake Trout Main Lake 9.61 28.00 711 124 0.528 2.723 Lake Trout Main Lake 6.10 28.00 711 79 0.558 2.747 Lake Trout Main Lake 9.77 28.00 711 126 0.562 2.750 Lake Trout Main Lake 7.97 28.00 711 103 0.180 Lake Trout Main Lake 9.36 28.00 711 121 1.070 Lake Trout Main Lake 8.40 28.25 718 105 0.503 2.702 Lake Trout Main Lake 8.40 28.25 718 105 1.250 Lake Trout Main Lake 9.02 28.75 730 107 0.386 2.587 Lake Trout Main Lake 8.76 28.75 730 104 0.399 2.601 Lake Trout Main Lake 8.33 29.00 737 96 0.509 2.707 Lake Trout Main Lake 8.12 29.00 737 94 0.598 2.777 Lake Trout Main Lake 8.75 29.00 737 101 0.756 2.879 Lake Trout Main Lake 9.12 29.25 743 102 0.494 2.694 Lake Trout Main Lake 8.66 29.50 749 95 0.369 2.567 Lake Trout Main Lake 2.95 29.50 749 32 0.305 Lake Trout Main Lake 10.34 29.75 756 110 0.389 2.590 Lake Trout Main Lake 9.82 30.00 762 102 0.380 2.580

Page 75 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Lake Trout Main Lake 10.26 30.00 762 106 0.404 2.606 Lake Trout Main Lake 10.40 30.00 762 108 0.621 2.793 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Lake Trout Main Lake 8.97 30.00 762 93 0.662 2.821 Lake Trout Main Lake 8.37 30.00 762 87 0.898 2.953 Lake Trout Main Lake 9.75 30.50 775 96 0.745 2.872 Lake Trout Main Lake 8.91 30.75 781 85 0.682 2.834 Lake Trout Main Lake 10.15 31.00 787 95 0.537 2.730 Lake Trout Main Lake 10.38 31.00 787 97 0.612 2.787 Lake Trout Main Lake 9.67 31.00 787 90 0.674 2.829 Lake Trout Main Lake 10.26 31.00 787 96 0.805 2.906 Lake Trout Main Lake 9.50 31.25 794 86 0.753 2.877 Lake Trout Main Lake 10.43 31.50 800 92 0.661 2.820 Lake Trout Main Lake 12.40 32.00 813 105 0.548 2.739 Lake Trout Main Lake 11.36 32.00 813 96 0.687 2.837 Lake Trout Main Lake 9.64 32.00 813 81 0.832 2.920 Lake Trout Main Lake 9.25 32.25 819 76 1.029 3.012 Lake Trout Main Lake 12.80 32.50 826 103 0.584 2.766 Lake Trout Main Lake 10.78 32.50 826 86 0.972 2.988 Lake Trout N. Main Lake 2.10 20.00 508 79 0.283 2.452 Lake Trout N. Main Lake 1.75 21.00 533 56 0.324 2.511 Lake Trout N. Main Lake 4.50 23.00 584 108 0.344 2.537 Lake Trout N. Main Lake 4.10 23.00 584 99 0.391 2.592 Lake Trout N. Main Lake 4.20 24.50 622 83 0.419 2.622 Lake Trout N. Main Lake 5.25 26.00 660 86 0.216 2.334 Lake Trout N. Main Lake 9.19 27.50 699 125 0.836 2.922 Lake Trout N. Main Lake 8.49 29.00 737 98 0.744 2.872 Lake Trout N. Main Lake 11.00 30.50 775 108 0.497 2.696 Lake Trout N. Main Lake 10.61 31.25 794 96 0.701 2.846

Page 76 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Lake Trout South Main Lake 3.02 19.50 495 123 0.534 2.728 Lake Trout South Main Lake 4.00 23.00 584 96 0.420 2.623 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Lake Trout South Main Lake 4.30 24.00 610 90 0.327 2.515 Lake Trout South Main Lake 4.96 24.50 622 98 0.473 2.675 Lake Trout South Main Lake 6.12 25.00 635 113 0.481 2.682 Lake Trout South Main Lake 6.00 26.00 660 98 0.540 2.732 Lake Trout South Main Lake 5.56 26.00 660 91 0.560 2.748 Lake Trout South Main Lake 6.82 26.25 667 108 0.569 2.755 Lake Trout South Main Lake 7.50 27.00 686 108 0.632 2.801 Lake Trout South Main Lake 8.00 27.00 686 116 0.812 2.910 Lake Trout South Main Lake 7.75 27.50 699 106 0.637 2.804 Lake Trout South Main Lake 7.92 28.50 724 96 0.637 2.804 Lake Trout South Main Lake 8.51 28.75 730 101 0.484 2.685 Lake Trout South Main Lake 9.31 29.00 737 107 0.485 2.686 Lake Trout South Main Lake 10.46 30.00 762 108 0.620 2.792 Lake Trout South Main Lake 12.93 33.00 838 99 0.557 2.746 Yellow Perch Main Lake 0.06 5.75 146 80 0.183 2.262 Yellow Perch Main Lake 0.13 7.00 178 93 0.481 2.682 Yellow Perch Main Lake 0.15 7.50 191 86 0.279 2.446 Yellow Perch Main Lake 0.15 7.50 191 86 0.483 2.684 Yellow Perch Main Lake 0.18 7.75 197 93 0.510 2.708 Yellow Perch Main Lake 0.19 8.00 203 89 0.196 2.292 Yellow Perch Main Lake 0.25 8.25 210 106 0.109 2.037 Yellow Perch Main Lake 0.24 8.25 210 101 0.177 2.248 Yellow Perch Main Lake 0.22 8.50 216 85 0.161 2.207 Yellow Perch Main Lake 0.27 8.50 216 104 0.164 2.215 Yellow Perch Main Lake 0.25 8.50 216 96 0.314 2.497 Yellow Perch Main Lake 0.22 8.50 216 85 0.326 2.513

Page 77 of 100

Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch Main Lake 0.32 8.75 222 112 0.112 2.049 Yellow Perch Main Lake 0.28 8.88 225 94 0.214 2.330 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch Main Lake 0.28 9.00 229 90 0.284 2.453 Yellow Perch Main Lake 0.37 9.25 235 109 0.162 2.210 Yellow Perch Main Lake 0.34 9.25 235 100 0.243 2.386 Yellow Perch Main Lake 0.32 9.25 235 94 0.302 2.480 Yellow Perch Main Lake 0.35 9.50 241 95 0.112 2.049 Yellow Perch Main Lake 0.40 9.50 241 108 0.174 2.241 Yellow Perch Main Lake 0.41 9.50 241 111 0.201 2.303 Yellow Perch Main Lake 0.37 9.50 241 100 0.269 2.430 Yellow Perch Main Lake 0.40 9.63 244 104 0.141 2.149 Yellow Perch Main Lake 0.36 9.63 244 93 0.203 2.307 Yellow Perch Main Lake 0.43 9.75 248 107 0.338 2.529 Yellow Perch Main Lake 0.36 10.00 254 83 0.353 2.548 Yellow Perch Main Lake 0.49 10.25 260 104 0.214 2.330 Yellow Perch Main Lake 0.42 11.00 279 71 0.129 2.111 Yellow Perch Main Lake 0.63 11.00 279 107 0.535 2.728 Yellow Perch Main Lake 0.63 11.25 286 99 0.440 2.643 Yellow Perch Main Lake 0.71 11.75 298 98 0.497 2.696 Yellow Perch Main Lake 0.82 12.00 305 106 0.425 2.628 Yellow Perch Main Lake 0.98 12.25 311 118 0.195 2.290 Yellow Perch Main Lake 0.96 12.50 318 109 0.474 2.676 Yellow Perch Main Lake 0.81 12.50 318 92 0.527 2.722 Yellow Perch Main Lake 0.91 12.75 324 97 0.387 2.588 Yellow Perch Mallets Bay 0.14 7.00 178 100 0.084 1.924 Yellow Perch Mallets Bay 0.14 7.25 184 89 0.124 2.093 Yellow Perch Mallets Bay 0.16 7.75 197 82 0.119 2.076 Yellow Perch Mallets Bay 0.21 8.13 206 93 0.191 2.281

Page 78 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch Mallets Bay 0.25 8.25 210 106 0.084 1.924 Yellow Perch Mallets Bay 0.29 8.50 216 111 0.136 2.134 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch Mallets Bay 0.29 8.88 225 97 0.184 2.265 Yellow Perch Mallets Bay 0.31 9.25 235 91 0.202 2.305 Yellow Perch Mallets Bay 0.39 9.50 241 105 0.169 2.228 Yellow Perch Mallets Bay 0.51 10.13 257 113 0.173 2.238 Yellow Perch Mallets Bay 0.48 10.38 264 98 0.189 2.276 Yellow Perch Mallets Bay 0.51 10.50 267 100 0.254 2.405 Yellow Perch Missisquoi Bay 0.11 5.63 143 0.129 2.111 Yellow Perch Missisquoi Bay 0.09 6.25 159 92 0.170 2.230 Yellow Perch Missisquoi Bay 0.11 6.50 165 99 0.088 1.944 Yellow Perch Missisquoi Bay 0.10 6.50 165 90 0.095 1.978 Yellow Perch Missisquoi Bay 0.12 6.50 165 108 0.098 1.991 Yellow Perch Missisquoi Bay 0.12 6.50 165 108 0.112 2.049 Yellow Perch Missisquoi Bay 0.10 6.50 165 90 0.114 2.057 Yellow Perch Missisquoi Bay 6.75 171 0.081 1.908 Yellow Perch Missisquoi Bay 0.14 6.75 171 112 0.049 Yellow Perch Missisquoi Bay 0.12 6.88 175 90 0.176 2.246 Yellow Perch Missisquoi Bay 0.14 7.00 178 100 0.077 1.886 Yellow Perch Missisquoi Bay 0.15 7.00 178 107 0.134 2.127 Yellow Perch Missisquoi Bay 0.12 7.00 178 85 0.196 2.292 Yellow Perch Missisquoi Bay 0.14 7.13 181 94 0.086 1.934 Yellow Perch Missisquoi Bay 0.13 7.25 184 83 0.095 1.978 Yellow Perch Missisquoi Bay 0.16 7.25 184 102 0.097 1.987 Yellow Perch Missisquoi Bay 0.16 7.25 184 102 0.105 2.021 Yellow Perch Missisquoi Bay 0.14 7.25 184 89 0.124 2.093 Yellow Perch Missisquoi Bay 0.14 7.25 184 89 0.156 2.193 Yellow Perch Missisquoi Bay 0.13 7.25 184 83 0.194 2.288

Page 79 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch Missisquoi Bay 0.13 7.25 184 83 0.283 Yellow Perch Missisquoi Bay 0.13 7.38 187 78 0.359 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch Missisquoi Bay 0.17 7.50 191 97 0.088 1.944 Yellow Perch Missisquoi Bay 0.18 7.50 191 103 0.101 2.004 Yellow Perch Missisquoi Bay 0.17 7.50 191 97 0.101 2.004 Yellow Perch Missisquoi Bay 0.18 7.50 191 103 0.126 2.100 Yellow Perch Missisquoi Bay 0.16 7.50 191 91 0.126 2.100 Yellow Perch Missisquoi Bay 0.15 7.50 191 86 0.135 2.130 Yellow Perch Missisquoi Bay 0.17 7.50 191 97 0.141 2.149 Yellow Perch Missisquoi Bay 0.17 7.50 191 97 0.145 2.161 Yellow Perch Missisquoi Bay 0.18 7.50 191 103 0.147 2.167 Yellow Perch Missisquoi Bay 0.19 7.50 191 109 0.156 2.193 Yellow Perch Missisquoi Bay 0.23 7.63 194 125 0.125 2.097 Yellow Perch Missisquoi Bay 0.17 7.75 197 88 0.093 1.968 Yellow Perch Missisquoi Bay 0.15 7.75 197 77 0.290 Yellow Perch Missisquoi Bay 0.19 7.78 198 97 0.124 2.093 Yellow Perch Missisquoi Bay 0.23 7.88 200 113 0.088 1.944 Yellow Perch Missisquoi Bay 0.19 7.88 200 93 0.110 2.041 Yellow Perch Missisquoi Bay 0.25 8.00 203 116 0.088 1.944 Yellow Perch Missisquoi Bay 0.19 8.00 203 89 0.170 2.230 Yellow Perch Missisquoi Bay 0.20 8.00 203 93 0.173 2.238 Yellow Perch Missisquoi Bay 0.22 8.00 203 102 0.251 Yellow Perch Missisquoi Bay 0.20 8.00 203 93 0.264 Yellow Perch Missisquoi Bay 0.24 8.25 210 101 0.101 2.004 Yellow Perch Missisquoi Bay 0.19 8.25 210 80 0.111 2.045 Yellow Perch Missisquoi Bay 0.20 8.25 210 84 0.112 2.049 Yellow Perch Missisquoi Bay 0.24 8.25 210 101 0.112 2.049 Yellow Perch Missisquoi Bay 0.21 8.25 210 89 0.137 2.137

Page 80 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch Missisquoi Bay 0.25 8.50 216 96 0.088 1.944 Yellow Perch Missisquoi Bay 0.26 8.50 216 100 0.093 1.968 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch Missisquoi Bay 0.24 8.50 216 92 0.134 2.127 Yellow Perch Missisquoi Bay 0.29 9.00 229 93 0.095 1.978 Yellow Perch Missisquoi Bay 0.29 9.00 229 93 0.216 2.334 Yellow Perch Missisquoi Bay 0.29 9.13 232 89 0.103 2.013 Yellow Perch Missisquoi Bay 0.29 9.25 235 85 0.081 1.908 Yellow Perch Missisquoi Bay 0.32 9.25 235 94 0.358 Yellow Perch Missisquoi Bay 0.34 9.50 241 92 0.152 2.182 Yellow Perch Missisquoi Bay 0.45 9.75 248 112 0.159 2.201 Yellow Perch Missisquoi Bay 0.42 10.00 254 96 0.214 2.330 Yellow Perch Missisquoi Bay 0.46 10.00 254 106 0.234 2.369 Yellow Perch N. Main Lake 0.09 6.00 152 104 0.048 1.681 Yellow Perch N. Main Lake 0.08 6.00 152 93 0.058 1.763 Yellow Perch N. Main Lake 0.11 6.25 159 112 0.083 1.919 Yellow Perch N. Main Lake 0.09 6.25 159 92 0.093 1.968 Yellow Perch N. Main Lake 0.11 6.50 165 99 0.065 1.813 Yellow Perch N. Main Lake 0.11 6.50 165 99 0.068 1.833 Yellow Perch N. Main Lake 0.12 6.50 165 108 0.083 1.919 Yellow Perch N. Main Lake 0.10 6.50 165 90 0.087 1.940 Yellow Perch N. Main Lake 0.09 6.50 165 81 0.109 2.037 Yellow Perch N. Main Lake 0.10 6.75 171 80 0.071 1.851 Yellow Perch N. Main Lake 0.11 6.75 171 88 0.113 2.055 Yellow Perch N. Main Lake 0.13 7.00 178 93 0.079 1.898 Yellow Perch N. Main Lake 0.14 7.25 184 89 0.092 1.964 Yellow Perch N. Main Lake 0.18 7.75 197 93 0.142 2.152 Yellow Perch N. Main Lake 0.16 7.75 197 82 0.208 2.318 Yellow Perch N. Main Lake 0.21 7.75 197 108 0.214 2.331

Page 81 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch N. Main Lake 0.18 8.00 203 84 0.109 2.037 Yellow Perch N. Main Lake 0.21 8.25 210 89 0.091 1.959 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch N. Main Lake 0.43 10.00 254 99 0.201 2.304 Yellow Perch N. Main Lake 0.51 10.00 254 117 0.235 2.371 Yellow Perch Northeast Arm 0.13 6.50 165 117 0.054 1.732 Yellow Perch Northeast Arm 0.12 7.00 178 85 0.039 1.591 Yellow Perch Northeast Arm 0.13 7.00 178 93 0.052 1.716 Yellow Perch Northeast Arm 0.12 7.25 184 76 0.068 1.833 Yellow Perch Northeast Arm 0.18 7.50 191 103 0.051 1.708 Yellow Perch Northeast Arm 0.15 7.75 197 77 0.078 1.892 Yellow Perch Northeast Arm 0.18 8.00 203 84 0.085 1.929 Yellow Perch Northeast Arm 0.21 8.00 203 98 0.112 2.049 Yellow Perch Northeast Arm 0.15 8.00 203 70 0.141 2.149 Yellow Perch Northeast Arm 0.19 8.50 216 73 0.092 1.964 Yellow Perch Northeast Arm 0.43 9.75 248 107 0.189 2.276 Yellow Perch Northeast Arm 0.39 10.00 254 89 0.271 2.433 Yellow Perch Northeast Arm 0.56 11.00 279 95 0.387 2.588 Yellow Perch Northeast Arm 0.63 11.50 292 93 0.129 Yellow Perch Northeast Arm 0.68 12.00 305 88 0.887 2.948 Yellow Perch South Lake 0.12 6.75 171 96 0.056 1.748 Yellow Perch South Lake 0.13 6.75 171 104 0.103 2.013 Yellow Perch South Lake 0.14 6.75 171 112 0.124 2.093 Yellow Perch South Lake 0.13 7.00 178 93 0.059 1.771 Yellow Perch South Lake 0.14 7.00 178 100 0.132 2.121 Yellow Perch South Lake 0.12 7.00 178 85 0.158 2.199 Yellow Perch South Lake 0.20 7.25 184 127 0.070 1.845 Yellow Perch South Lake 0.13 7.25 184 83 0.136 2.134 Yellow Perch South Lake 0.16 7.50 191 91 0.148 2.170

Page 82 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch South Lake 0.18 7.50 191 103 0.149 2.173 Yellow Perch South Lake 0.15 7.50 191 86 0.151 2.179 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch South Lake 0.16 7.75 197 82 0.106 2.025 Yellow Perch South Lake 0.13 7.75 197 67 0.259 2.413 Yellow Perch South Lake 0.20 8.00 203 93 0.125 2.097 Yellow Perch South Lake 0.18 8.00 203 84 0.130 2.114 Yellow Perch South Lake 0.17 8.25 210 72 0.143 2.155 Yellow Perch South Lake 0.26 8.50 216 100 0.106 2.025 Yellow Perch South Lake 0.22 8.50 216 85 0.112 2.049 Yellow Perch South Lake 0.23 8.75 222 81 0.118 2.072 Yellow Perch South Lake 0.20 9.50 241 54 0.314 2.497 Yellow Perch South Lake 0.43 9.50 241 116 0.377 2.576 Yellow Perch South Lake 0.38 10.00 254 87 0.259 2.413 Yellow Perch South Main Lake 0.12 6.00 152 139 0.107 2.029 Yellow Perch South Main Lake 0.49 6.00 152 0.137 2.137 Yellow Perch South Main Lake 0.12 6.50 165 108 0.219 2.340 Yellow Perch South Main Lake 0.15 6.50 165 135 0.220 2.342 Yellow Perch South Main Lake 0.10 6.75 171 80 0.139 2.143 Yellow Perch South Main Lake 0.12 7.00 178 85 0.098 1.991 Yellow Perch South Main Lake 0.18 7.00 178 128 0.101 2.004 Yellow Perch South Main Lake 0.12 7.00 178 85 0.129 2.111 Yellow Perch South Main Lake 0.16 7.00 178 114 0.196 2.292 Yellow Perch South Main Lake 0.22 7.50 191 126 0.150 2.176 Yellow Perch South Main Lake 0.15 7.50 191 86 0.183 2.262 Yellow Perch South Main Lake 0.19 7.75 197 98 0.122 2.086 Yellow Perch South Main Lake 0.21 7.75 197 108 0.122 2.086 Yellow Perch South Main Lake 0.23 7.75 197 118 0.157 2.196 Yellow Perch South Main Lake 0.20 8.00 203 93 0.107 2.029

Page 83 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Yellow Perch South Main Lake 0.21 8.00 203 98 0.208 2.318 Yellow Perch South Main Lake 0.16 8.00 203 75 0.243 2.386 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) Yellow Perch South Main Lake 0.16 8.00 203 75 0.311 2.493 Yellow Perch South Main Lake 0.16 8.00 203 75 0.444 Yellow Perch South Main Lake 8.25 210 0.137 2.137 Yellow Perch South Main Lake 8.50 216 0.165 2.217 Yellow Perch South Main Lake 0.27 8.50 216 104 0.251 2.400 Yellow Perch South Main Lake 0.25 9.00 229 80 0.106 2.025 Yellow Perch South Main Lake 0.34 9.00 229 109 0.160 2.204 Yellow Perch South Main Lake 0.35 9.00 229 112 0.172 2.236 Yellow Perch South Main Lake 0.32 9.00 229 103 0.207 2.316 Yellow Perch South Main Lake 0.25 9.00 229 80 0.304 2.483 Yellow Perch South Main Lake 0.65 9.00 229 0.307 2.487 Yellow Perch South Main Lake 1.32 9.25 235 0.143 2.155 Yellow Perch South Main Lake 0.36 9.75 248 90 0.292 2.465 Yellow Perch South Main Lake 0.45 10.00 254 103 0.329 2.517 Yellow Perch South Main Lake 0.82 12.50 318 93 0.772 2.888 Yellow Perch South Main Lake 1.15 13.00 330 115 0.873 2.941 White Perch Main Lake 0.08 5.00 127 148 0.059 1.771 White Perch Main Lake 0.08 5.00 127 148 0.127 2.104 White Perch Main Lake 0.10 6.00 152 103 0.088 1.944 White Perch Main Lake 0.12 6.00 152 124 0.179 2.253 White Perch Main Lake 0.23 6.75 171 162 0.203 2.307 White Perch Main Lake 0.23 7.00 178 144 0.089 1.949 White Perch Main Lake 0.17 7.00 178 107 0.119 2.076 White Perch Main Lake 0.23 7.75 197 104 0.156 2.193 White Perch Main Lake 0.24 8.00 203 98 0.073 1.863 White Perch Main Lake 0.25 8.00 203 102 0.106 2.025

Page 84 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain White Perch Main Lake 0.25 8.00 203 102 0.215 2.332 White Perch Main Lake 0.28 8.25 210 104 0.071 1.851 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) White Perch Main Lake 0.29 8.25 210 107 0.071 1.851 White Perch Main Lake 0.25 8.25 210 93 0.216 2.334 White Perch Main Lake 0.29 8.50 216 98 0.095 1.978 White Perch Main Lake 0.35 8.50 216 118 0.142 2.152 White Perch Main Lake 0.37 8.50 216 124 0.225 2.352 White Perch Main Lake 0.33 8.50 216 111 0.245 2.389 White Perch Main Lake 0.34 8.75 222 104 0.107 2.029 White Perch Main Lake 0.38 9.25 235 97 0.217 2.336 White Perch Main Lake 0.54 10.38 264 96 0.354 2.549 White Perch Main Lake 1.17 12.50 318 114 0.750 2.875 White Perch Main Lake 1.33 13.50 343 101 0.920 2.964 White Perch Mallets Bay 0.23 7.88 200 99 0.153 2.185 White Perch Mallets Bay 0.26 8.13 206 101 0.228 2.358 White Perch Mallets Bay 0.27 8.25 210 100 0.142 2.152 White Perch Mallets Bay 0.28 8.25 210 104 0.155 2.190 White Perch Mallets Bay 0.31 8.50 216 104 0.143 2.155 White Perch Mallets Bay 0.28 8.50 216 94 0.152 2.182 White Perch Mallets Bay 0.28 8.50 216 94 0.185 2.267 White Perch Mallets Bay 0.32 8.75 222 98 0.183 2.262 White Perch Mallets Bay 0.31 8.75 222 95 0.194 2.288 White Perch Mallets Bay 0.33 8.75 222 101 0.202 2.305 White Perch Mallets Bay 0.31 8.88 225 91 0.221 2.344 White Perch Mallets Bay 0.34 9.00 229 95 0.163 2.212 White Perch Mallets Bay 0.36 9.00 229 101 0.205 2.312 White Perch Mallets Bay 0.57 10.00 254 114 0.236 2.373 White Perch Mallets Bay 0.56 10.25 260 103 0.374 2.573

Page 85 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain White Perch Mallets Bay 0.75 11.25 286 102 0.306 2.486 White Perch Missisquoi Bay 0.21 7.00 178 132 0.027 1.431 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) White Perch Missisquoi Bay 0.19 7.00 178 119 0.037 1.568 White Perch Missisquoi Bay 0.39 9.00 229 109 0.096 1.982 White Perch Missisquoi Bay 0.54 10.00 254 108 0.101 2.004 White Perch Missisquoi Bay 0.47 10.00 254 94 0.179 2.253 White Perch Missisquoi Bay 0.50 10.13 257 96 0.337 2.528 White Perch Missisquoi Bay 0.57 10.25 260 105 0.212 2.326 White Perch Missisquoi Bay 0.67 10.25 260 123 0.239 2.378 White Perch Missisquoi Bay 0.52 10.25 260 96 0.278 2.444 White Perch Missisquoi Bay 0.52 10.50 267 89 0.235 2.371 White Perch Missisquoi Bay 0.61 10.63 270 100 0.221 2.344 White Perch Missisquoi Bay 0.82 11.50 292 104 0.276 2.441 White Perch Missisquoi Bay 0.85 11.50 292 108 0.311 2.493 White Perch Missisquoi Bay 0.82 11.50 292 104 0.580 2.763 White Perch Missisquoi Bay 0.95 11.88 302 109 0.311 2.493 White Perch Missisquoi Bay 0.90 12.00 305 100 0.224 2.350 White Perch Missisquoi Bay 0.79 12.00 305 88 0.448 2.651 White Perch Missisquoi Bay 0.91 12.25 311 94 0.411 2.614 White Perch N. Main Lake 0.35 8.75 222 107 0.251 2.400 White Perch N. Main Lake 0.44 9.00 229 123 0.207 2.316 White Perch N. Main Lake 0.46 9.50 241 108 0.214 2.330 White Perch N. Main Lake 0.43 9.75 248 93 0.105 2.021 White Perch N. Main Lake 0.49 10.00 254 98 0.152 2.182 White Perch N. Main Lake 0.57 10.25 260 105 0.188 2.274 White Perch N. Main Lake 0.64 10.63 270 105 0.253 2.403 White Perch N. Main Lake 0.67 10.75 273 106 0.229 2.360 White Perch N. Main Lake 0.65 10.75 273 103 0.232 2.365

Page 86 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain White Perch N. Main Lake 0.67 11.25 286 91 0.323 2.509 White Perch N. Main Lake 0.77 11.38 289 101 0.241 2.382 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) White Perch Northeast Arm 0.36 8.50 216 121 0.230 2.362 White Perch Northeast Arm 0.27 8.75 222 83 0.364 2.561 White Perch Northeast Arm 0.43 9.13 232 115 0.224 2.350 White Perch Northeast Arm 0.45 9.50 241 106 0.434 2.637 White Perch Northeast Arm 0.57 9.63 244 129 0.101 2.004 White Perch Northeast Arm 0.47 9.75 248 102 0.286 2.456 White Perch Northeast Arm 0.57 10.50 267 97 0.637 2.804 White Perch Northeast Arm 0.91 11.25 286 124 0.538 2.731 White Perch Northeast Arm 0.96 11.50 292 122 0.204 2.310 White Perch Northeast Arm 0.58 11.50 292 74 0.697 2.843 White Perch Northeast Arm 0.96 12.00 305 106 0.280 2.447 White Perch Northeast Arm 0.79 12.00 305 88 0.291 2.464 White Perch Northeast Arm 0.90 12.00 305 100 0.750 2.875 White Perch Northeast Arm 0.97 12.00 305 108 0.775 2.889 White Perch Northeast Arm 1.15 12.25 311 119 0.440 2.643 White Perch Northeast Arm 0.93 12.50 318 90 0.874 2.942 White Perch Northeast Arm 1.02 13.00 330 87 0.852 2.930 White Perch Northeast Arm 1.06 13.00 330 91 0.864 2.937 White Perch Northeast Arm 1.06 13.00 330 91 0.892 2.950 White Perch Northeast Arm 1.32 13.25 337 106 0.345 2.538 White Perch Northeast Arm 1.26 13.50 343 96 1.022 3.009 White Perch South Lake 0.19 7.00 178 119 0.096 1.982 White Perch South Lake 0.19 7.00 178 119 0.130 2.114 White Perch South Lake 0.20 7.00 178 126 0.151 2.179 White Perch South Lake 0.20 7.25 184 112 0.145 2.161 White Perch South Lake 0.19 7.25 184 107 0.174 2.241

Page 87 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain White Perch South Lake 0.24 7.50 191 121 0.166 2.220 White Perch South Lake 0.28 8.00 203 114 0.152 2.182 Species Location weight (lbs) length (in) length (mm) Wr Hg (ppm) Hg (log ppb) White Perch South Lake 0.33 8.00 203 135 0.180 2.255 White Perch South Lake 0.24 8.00 203 98 0.228 2.358 White Perch South Lake 0.35 8.00 203 143 0.239 2.378 White Perch South Lake 0.33 8.50 216 111 0.186 2.270 White Perch South Lake 0.32 8.50 216 108 0.193 2.286 White Perch South Lake 0.30 8.50 216 101 0.231 2.364 White Perch South Lake 0.48 9.50 241 113 0.498 2.697 White Perch South Main Lake 0.29 8.00 203 119 0.167 2.223 White Perch South Main Lake 0.30 8.00 203 123 0.204 2.310 White Perch South Main Lake 0.27 8.00 203 110 0.217 2.336 White Perch South Main Lake 0.31 8.50 216 104 0.173 2.238 White Perch South Main Lake 0.35 8.75 222 107 0.157 2.196 White Perch South Main Lake 0.33 8.75 222 101 0.161 2.206 White Perch South Main Lake 0.30 8.75 222 92 0.357 2.553 White Perch South Main Lake 0.39 9.00 229 109 0.184 2.265 White Perch South Main Lake 0.37 9.00 229 104 0.218 2.338 White Perch South Main Lake 0.37 9.00 229 104 0.322 2.508 White Perch South Main Lake 0.35 9.13 232 94 0.206 2.314 White Perch South Main Lake 0.46 9.25 235 118 0.143 2.155 White Perch South Main Lake 0.38 9.25 235 97 0.193 2.285 White Perch South Main Lake 1.16 13.00 330 99 0.240 2.380

Page 88 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain

Appendix B. Lake Champlain water sample results for microcystin, anatoxin-a, and cylindrospermopsin. All water samples test negative for all three cyanotoxins. Method detections limits are reported for each sample by analysis type. Average detection limits are also reported for each analysis. “F” indicates the sample was a filter.

Page 89 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Appendix C. Fish specimens analyzed for MC-LR including tissue type (M, Muscle; L, Liver), weights, recoveries and detection limit.

Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2109 C103 L Yellow Perch No 0.4 0.2 0.1 1.0 132 62.2 16-2117 C133 M Yellow Perch No 5.5 4.3 0.2 4.0 123 201.3 16-2118 C135 M Yellow Perch No 4.7 3.7 0.2 6.2 71 313.8 16-2133 C178 L Yellow Perch No 1.1 0.9 0.1 5.4 74 657.5 16-2132 C170 M Yellow Perch No 4.3 3.4 0.2 5.5 77 266.5 16-2135 C180 L Yellow Perch No 1.5 1.1 0.2 4.9 52 316.7 16-2131 C169 L Yellow Perch No 0.9 0.7 0.2 4.0 96 266.5 16-2134 C179 M Yellow Perch No 5.1 4.1 0.2 5.3 89 281.2 16-2136 C181 M Yellow Perch No 5.6 4.4 0.2 4.8 74 233.7 16-2142 C191 L Yellow Perch No 1.3 1.0 0.3 1.0 87 12.9 16-2139 C187 M Yellow Perch No 3.5 2.7 0.2 4 .9 80 313.8 16-2144 C197 L Yellow Perch No 1.2 1.0 0.2 1.0 101 20.7 16-2190 C203 M Yellow Perch No 3.4 2.7 0.4 1.0 39 15.3 16-2191 C204 L Yellow Perch No 2.6 2.0 0.4 1.0 116 16.9 16-2192 C205 M Yellow Perch No 7.2 5.3 0.4 1.0 92 8.9 16-2193 C206 L Yellow Perch No 8.8 8.1 0.4 1.0 148 12.3 16-2199 C301 L Yellow Perch No 0.4 0.4 0.1 1.0 127 50.6 16-2200 C302 M Yellow Perch No 4.4 3.3 0.4 1.0 132 8.9 16-2212 C116 M Yellow Perch No 0.1 0.1 0.0 1.0 133 216.6 16-2213 C117 L Yellow Perch No 2.4 1.9 0.4 1.0 65 16.9 16-2216 C115 L Yellow Perch No 0.4 0.4 0.1 1.0 148 68.0 16-2217 C124 M Yellow Perch No 2.4 1.9 0.4 1.0 72 14.3

Page 90 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2233 C193 M Yellow Perch No 2.0 1.6 0.2 4 .9 87 162.5 16-2234 C183 L Yellow Perch No 0.2 0.2 0.0 1.0 84 147.2 16-2235 C194 M Yellow Perch No 1.8 1.5 0.3 1.0 74 15.1 16-2236 C184 L Yellow Perch No 0.3 0.3 0.1 1.0 84 81.2 16-2389 C953 M Yellow Perch No 2.6 2.1 0.4 1.0 64 14.3 16-2390 C954 L Yellow Perch No 0.6 0.5 0.1 1.0 104 39.4 16-2401 C966 M Yellow Perch No 19.4 1.8 0.4 1.0 49 35.6 16-2402 C965 L Yellow Perch No 0.7 0.6 0.1 1.0 103 40.1 16-2409 C975 M Yellow Perch No 0.7 0.6 0.1 1.0 57 40.1 16-2410 C974 L Yellow Perch No 0.7 0.6 0.2 1.0 117 31.4 16-2411 C977 M Yellow Perch No 2.6 2.1 0.4 1.0 62 12.7 16-2412 C976 L Yellow Perch No 2.0 1.7 0.4 1.0 90 13.5 16-2415 C168 M Yellow Perch No 2.9 2.3 0.2 1.0 85 316.4 16-2416 C113 L Yellow Perch No 0.8 0.6 0.2 1.0 134 37.5 16-2111 C112 L SMB No 13.6 10.3 0.2 4 .9 58 313.5 16-2126 C154 M SMB No 5.0 3.9 0.2 4.5 87 266.3 16-2178 C356 L SMB No 10.9 8.3 0.2 4 .9 83 161.2 16-2179 C357 M SMB No NA 6.9 0.2 5.7 55 218.9 16-2181 C359 M SMB No 7.8 6.0 0.2 5.0 65 317.6 16-2182 C360 L SMB No 8.1 6.1 0.2 4.7 67 179.1 16-2194 C207 M SMB No 8.5 6.7 0.2 4 .9 84 161.5 16-2195 C208 L SMB No 15.7 13.0 0.2 1.0 103 26.2 16-2196 C209 M SMB No 12.2 9.6 0.2 1.0 75 31.5 16-2201 C303 L SMB No 7.1 5.3 0.2 1.0 74 31.5

Page 91 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2249 C251 M SMB No 6.5 5.1 0.4 1.0 43 15.3 16-2250 C271 L SMB No 12.9 10.1 0.2 4.8 59 316.1 16-2253 C261 M SMB No 6.5 5.2 0.4 1.0 75 14.3 16-2254 C281 L SMB No 6.1 4.7 0.4 1.0 90 12.3 16-2256 C262 M SMB No 8.8 6.8 0.2 4 .9 56 162.5 16-2257 C272 L SMB No 15.9 11.8 0.4 1.0 104 14.3 16-2260 C274 M SMB No 6.3 4.9 0.4 1.0 49 14.3 16-2261 C273 L SMB No 8.7 6.8 0.4 1.0 65 11.9 16-2264 C282 M SMB No 5.1 3.9 0.2 1.0 78 26.2 16-2265 C292 L SMB No 13.8 10.5 0.2 4.7 78 155.6 16-2266 C285 M SMB No NA NA 0.2 1.0 92 26.2 16-2267 C284 L SMB No NA NA 0.2 5.0 69 165.5 16-2270 C294 M SMB No 8.0 6.2 0.2 5.0 55 319.6 16-2271 C295 L SMB No 6.0 4.6 0.2 5.0 50 325.5 16-2319 C821 M SMB No 0.7 0.6 0.2 1.0 73 32.0 16-2320 C820 L SMB No 3.6 2.8 0.4 1.0 99 14.3 16-2323 C824 M SMB No 0.8 0.6 0.2 1.0 71 40.6 16-2324 C823 L SMB No 5.3 4.2 0.4 1.0 73 16.1 16-2327 C826 M SMB No 0.5 0.4 0.1 1.0 73 67.7 16-2328 C833 L SMB No 5.4 4.3 0.4 1.0 116 16.9 16-2329 C832 M SMB No 0.3 10.3 0.1 1.0 77 77.5 16-2330 C831 L SMB No 7.8 6.1 0.4 1.0 92 12.7 16-2331 C834 M SMB No 2.7 2.1 0.4 1.0 50 16.1 16-2332 C829 L SMB No 5.8 4.6 0.4 1.0 83 16.0

Page 92 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2333 C836 M SMB No 3.2 2.7 0.4 1.0 51 17.0 16-2334 C835 L SMB No 6.1 4.8 0.4 1.0 43 16.0 16-2393 C957 M SMB No 4.0 3.1 0.4 1.0 65 15.4 16-2394 C967 L SMB No 3.2 2.5 0.4 1.0 76 12.3 16-2395 C959 M SMB No 5.6 4.4 0.4 1.0 106 12.3 16-2396 C958 L SMB No 2.7 2.1 0.4 1.0 115 8.9 16-2403 C969 M SMB No 5.3 4.0 0.4 1.0 108 8.9 16-2404 C968 L SMB No 6.2 4.5 0.4 1.0 76 8.9 16-2413 C979 M SMB No 6.6 5.2 0.4 1.0 58 11.9 16-2414 C978 L SMB No 3.7 2.8 0.4 1.0 55 316.4 16-2269 C293 M SMB No 9.3 7.3 0.2 1.0 80 31.5 16-2418 C283 L SMB No 7.9 6.1 0.2 5.0 68 192.2 16-2110 C110 M Sheephead No 6.7 5.2 0.2 4 .2 56 222.9 16-2119 C138 L Sheephead No 0.4 0.3 0.1 1.0 161 71.3 16-2120 C139 L Sheephead No 1.2 1.0 0.2 4 .7 31 355.0 16-2115 C130 M Sheephead No 9.1 6.9 0.2 6.7 44 325.4 16-2124 C148 M Sheephead No 5.7 4.5 0.2 6.0 69 306.8 16-2127 C158 L Sheephead No 0.9 0.7 0.2 6.8 82 398.7 16-2128 C159 L Sheephead No 5.2 4.0 0.2 4 .9 133 161.9 16-2141 C189 M Sheephead No 9.4 7.4 0.2 4.9 116 160.7 16-2145 C199 L Sheephead No 5.1 3.8 0.2 4 .7 50 181.2 16-2140 C188 L Sheephead No 4.9 3.6 0.2 5.0 102 166.9 16-2148 C218 M Sheephead No 6.0 4.6 0.2 4.8 58 185.5 16-2149 C220 L Sheephead No NA 1.8 0.2 4.9 124 162.9 16-2321 C822 M Yellow Perch Yes 0.2 0.2 0.1 1.0 104 114.6

Page 93 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2322 C830 L Yellow Perch Yes 0.3 0.3 0.1 1.0 117 72.9 16-2313 C807 M Yellow Perch Yes 0.4 0.3 0.1 1.0 126 22.7 16-2314 C817 L Yellow Perch Yes 0.2 0.2 0.1 1.0 98 113.5 16-2405 C971 M Yellow Perch Yes 0.5 0.4 0.1 1.0 71 53.8 16-2406 C970 L Yellow Perch Yes 0.2 0.0 NA 1.0 127 105.6 16-2387 C951 M Yellow Perch Yes 0.5 0.4 0.1 1.0 65 45.9 16-2388 C961 L Yellow Perch Yes 0.3 0.2 0.0 1.0 105 116.9 16-2397 C962 M Yellow Perch Yes 0.3 0.3 0.0 1.0 74 102.6 16-2398 C972 L Yellow Perch Yes 0.2 0.2 0.0 1.0 81 103.1 16-2407 C973 M Yellow Perch Yes 1.2 1.0 0.2 1.0 59 24.5 16-2408 C952 L Yellow Perch Yes 0.2 0.1 0.0 1.0 89 141.4 16-2210 C107 M Yellow Perch Yes NA 1.9 0.4 1.0 74 8.9 16-2211 C111 L Yellow Perch Yes 0.2 0.1 0.1 1.0 109 96.8 16-2263 C265 L Yellow Perch Yes 0.3 0.3 0.1 1.0 114 95.2 16-2419 C275 M Yellow Perch Yes 1.9 1.5 0.4 1.0 83 8.9 16-2245 C244 M Yellow Perch Yes 13.6 14.0 0.4 1.0 69 8.8 16-2246 C243 L Yellow Perch Yes 0.5 0.4 0.1 1.0 63 45.5 16-2251 C253 M Yellow Perch Yes 3.6 2.8 0.4 1.0 68 17.0 16-2252 C263 L Yellow Perch Yes 1.6 1.3 0.3 1.0 82 12.3 16-2262 C175 L Yellow Perch Yes 0.6 0.5 0.1 1.0 135 38.1 16-2284 C386 M Yellow Perch Yes 2.2 1.7 0.4 1.0 68 15.3 16-2297 C400 M Yellow Perch Yes 3.1 NA 0.4 1.0 67 15.3 16-2298 C399 L Yellow Perch Yes 0.4 NA 0.1 1.0 99 37.9 16-2221 C134 M Yellow Perch Yes 2.9 2.3 0.4 1.0 73 12.3 16-2222 C153 L Yellow Perch Yes 0.2 0.2 0.0 1.0 123 149.3 16-2225 C137 L Yellow Perch Yes 0.4 0.3 0.1 1.0 85 53.0

Page 94 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2226 C147 M Yellow Perch Yes 3.3 2.6 0.2 4.8 67 158.6 16-2285 C388 M Yellow Perch Yes NA NA 0.2 1.0 90 26.2 16-2286 C387 L Yellow Perch Yes 0.3 0.3 0.1 1.0 122 81.5 16-2214 C122 M Yellow Perch Yes 2.2 1.8 0.4 1.0 89 14.3 16-2215 C173 L Yellow Perch Yes 0.6 0.6 0.1 1.0 151 45.2 16-2227 C155 M Yellow Perch Yes 2.0 1.6 0.2 5.0 93 165.3 16-2228 C145 L Yellow Perch Yes 0.3 0.2 0.1 1.0 110 51.8 16-2385 C950 M SMB Yes 0.7 0.6 0.1 1.0 74 56.6 16-2291 C393 M SMB Yes 10.1 7.9 0.2 1.2 63 32.3 16-2292 C392 L SMB Yes 4.7 3.6 0.2 4.8 85 159.3 16-2289 C391 M SMB Yes 9.5 7.4 0.4 1.0 52 15.4 16-2290 C394 L SMB Yes 8.5 6.6 0.4 1.0 63 4.4 16-2247 C245 M SMB Yes 4.0 2.9 0.4 1.0 66 15.3 16-2248 C255 L SMB Yes 3.0 2.4 0.4 1.0 123 11.9 16-2275 C372 M SMB Yes 5.1 4.2 0.2 4.8 81 313.7 16-2276 C123 L SMB Yes 8.5 6.7 0.2 2.9 120 110.1 16-2277 C374 M SMB Yes 8.4 6.6 0.2 4.8 74 157.7 16-2278 C373 L SMB Yes 12.3 NA 0.2 5.0 80 163.6 16-2283 C381 L SMB Yes 19.6 13.7 0.2 4.9 89 161.8 16-2417 C382 M SMB Yes 7.8 2.2 0.2 4 .6 69 179.0 16-2229 C156 M SMB Yes 10.5 8.3 0.4 1.0 69 15.3 16-2230 C166 L SMB Yes 13.5 10.4 0.4 1.0 69 14.3 16-2231 C144 L SMB Yes 9.4 7.2 0.2 4.5 84 174.7 16-2232 C176 M SMB Yes 11.3 8.5 0.4 1.0 66 4.4 16-2272 C264 M SMB Yes 5.4 4.1 0.2 4.9 51 318.0 16-2223 C136 L Sheephead Yes 2.4 1.9 0.4 1.0 101 14.3

Page 95 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Starting Sample SUNY ESF Darrin FWI caught wet Dry Weight Extraction Nodularin Detection Identification Identification tissue in weight Weight extracted volume % limit number number type Species bloom (g) (g) (g) (ml) Recovery (µg/kg) 16-2224 C146 M Sheephead Yes 5.5 4.4 0.4 1.0 58 15.3 16-2273 C371 M Largemouth Yes 10.1 8.0 0.4 1.0 47 15.4 Bass 16-2274 C361 L Largemouth Yes 4.2 3.3 0.2 4.7 66 308.6 Bass 16-2399 C964 M Bullhead Yes 4.3 3.5 0.4 1.0 57 12.3 Catfish 16-2400 C963 L Bullhead Yes 4.5 3.6 0.4 1.0 60 12.3 Catfish

Page 96 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain Appendix D. Detection limits for microcystin congeners, anatoxin-a and cylindrospermopsin for all fish samples tested. Method detection limits for CYS-LR, homo-anatoxin-a and deoxycylindrospermopsin are not reported but are similar (±20%) to their parent compound. Since the same fish samples were analyzed for all of the analyses (MC-LR, microcystin congeners, anatoxin-a and cylindrospermopsins) sample weights, tissue types and percent recoveries are not presented here, this information is in Appendix C.

ATX CYL SUNY- Microcystin Limit of Detection (µg/kg) DFWI ID Detection Detection ESF ID Number Limit Limit Number LR dLR RR dRR GSH-LR (µg/kg) (µg/kg) 16-2109 C103 382.1 26.2 3.3 4.6 15.7 2.6 17.9 16-2117 C133 773.8 53.1 7.6 13.0 12.7 6.7 102.8 16-2118 C135 966.7 66.3 9.5 16.3 15.8 8.4 128.4 16-2133 C178 2527.3 173.4 24.7 42.6 41.4 22.0 335.7 16-2132 C170 1024.1 70.3 10.0 17.3 16.8 8.9 136.0 16-2135 C180 1006.9 69.1 9.8 17.0 16.5 8.8 133.7 16-2131 C169 1024.5 70.3 10.0 17.3 16.8 8.9 136.1 16-2134 C179 1081.0 74.2 10.6 18.2 17.7 9.4 143.6 16-2136 C181 898.3 61.6 8.8 15.1 14.7 7.8 119.3 16-2142 C191 151.6 10.4 1.3 1.8 6.2 1.0 7.1 16-2139 C187 997.6 68.4 9.8 16.8 16.3 8.7 132.5 16-2144 C197 0.3 15.3 1.6 2.2 7.4 1.2 8.4 16-2190 C203 0.1 2.4 0.9 1.2 4.2 0.6 4.8 16-2191 C204 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2192 C205 0.1 2.4 0.9 1.2 4.3 0.6 4.9 16-2193 C206 0.1 2.4 0.9 1.2 4.3 0.6 4.9 16-2199 C301 1081.0 28.5 3.6 5.0 17.0 2.8 19.4 16-2200 C302 415.7 7.1 0.9 1.2 4.3 0.6 4.9 16-2212 C116 3.0 133.4 15.3 26.4 25.7 13.7 208.4 16-2213 C117 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2216 C115 491.9 33.7 4.8 8.3 8.1 4.3 65.3 16-2217 C124 0.1 2.4 1.0 1.8 1.7 0.9 13.8 16-2233 C193 1020.8 70.0 8.9 12.3 41.8 6.9 47.7 16-2234 C183 953.4 65.4 8.3 11.5 39.1 6.4 44.6 16-2235 C194 130.9 9.0 1.3 2.2 2.1 1.1 17.4 16-2236 C184 526.2 36.1 4.6 6.3 21.6 3.6 24.6 16-2389 C953 0.1 2.4 4.7 3.8 2326.3 864.4 187.0 16-2390 C954 285.1 19.6 2.8 4.8 4.7 2.5 37.9 16-2401 C966 0.1 2.4 0.9 1.2 4.3 0.6 4.9 16-2402 C965 300.8 20.6 2.6 3.6 12.3 2.0 14.1 16-2409 C975 329.6 22.6 2.9 4.0 13.5 2.2 15.4 16-2410 C974 257.7 17.7 2.2 3.1 10.6 1.7 12.1 16-2411 C977 0.1 2.4 4.7 3.7 2325.1 863.9 186.9 16-2412 C976 117.3 8.0 1.0 1.4 4.8 0.8 5.5 16-2415 C168 1216.1 83.4 37.9 24.2 202.0 7.1 736.2

Page 97 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain ATX CYL SUNY- DFWI ID Detection Detection ESF ID LR dLR RR dRR GSH-LR Number Limit Limit Number (µg/kg) (µg/kg) 2.0 2.8 9.4 1.6 10.8 16-2111 C112 996.7 68.4 9.7 16.8 16.3 8.7 132.4 16-2126 C154 846.4 58.1 8.3 14.3 13.9 7.4 112.4 16-2178 C356 1012.5 69.5 8.8 12.2 41.5 6.8 47.4 16-2179 C357 1035.6 71.0 10.1 17.5 17.0 9.0 137.6 16-2181 C359 1.9 2.4 9.9 17.0 16.5 8.8 134.1 16-2182 C360 962.6 85.9 9.4 16.2 15.8 8.4 127.9 16-2194 C207 1014.7 69.6 8.8 0.0 41.6 6.9 47.5 16-2195 C208 207.8 14.3 1.8 2.5 8.5 1.4 9.7 16-2196 C209 0.3 11.2 1.8 3.2 3.5 7.8 113.2 16-2201 C303 103.8 11.2 1.8 3.2 3.5 7.8 113.2 16-2249 C251 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2250 C271 1005.1 69.0 8.7 12.1 41.2 6.8 47.0 16-2253 C261 0.1 2.4 4.7 3.7 2325.1 863.9 186.9 16-2254 C281 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2256 C262 1020.7 70.0 8.9 0.0 41.8 6.9 47.7 16-2257 C272 0.1 2.4 4.7 3.7 2325.1 863.9 186.9 16-2260 C274 0.1 2.4 4.7 3.7 2325.7 864.2 186.9 16-2261 C273 0.1 2.4 4.7 3.7 2325.7 864.2 186.9 16-2264 C282 207.8 14.3 1.8 0.0 8.5 1.4 9.7 16-2265 C292 977.8 67.1 8.5 0.0 40.1 6.6 45.7 16-2266 C285 207.8 14.3 1.8 0.0 8.5 1.4 9.7 16-2267 C284 1039.6 71.3 9.0 0.0 42.6 7.0 48.6 16-2270 C294 1016.0 69.7 9.9 17.1 16.6 8.8 135.0 16-2271 C295 1034.8 71.0 10.1 17.4 16.9 9.0 137.5 16-2319 C821 262.9 18.0 2.3 3.2 10.8 1.8 12.3 16-2320 C820 0.1 2.4 4.7 3.7 2323.9 863.5 186.8 16-2323 C824 249.4 17.1 2.4 4.2 4.1 2.2 33.1 16-2324 C823 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2327 C826 416.1 28.5 3.6 5.0 17.0 2.8 19.5 16-2328 C833 0.1 2.4 0.9 3.7 4.3 0.7 4.9 16-2329 C832 502.0 34.4 4.4 6.0 20.6 3.4 23.5 16-2330 C831 0.1 2.4 4.7 3.7 2323.9 863.5 186.8 16-2331 C834 0.1 2.4 4.7 3.7 2325.1 863.9 186.9 16-2332 C829 0.1 2.4 4.7 3.7 2321.6 862.6 186.6 16-2333 C836 104.0 7.1 0.9 1.2 4.3 0.7 4.9 16-2334 C835 0.1 2.4 4.7 3.7 2321.6 862.6 186.6 16-2393 C957 104.2 7.1 0.9 0.0 4.3 0.7 4.9 16-2394 C967 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2395 C959 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2396 C958 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2403 C969 0.1 2.4 0.9 2.1 4.3 0.6 4.9

Page 98 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain ATX CYL SUNY- DFWI ID Detection Detection ESF ID LR dLR RR dRR GSH-LR Number Limit Limit Number (µg/kg) (µg/kg) 16-2404 C968 0.1 2.4 0.9 2.1 4.3 0.6 4.9 16-2413 C979 0.1 2.4 4.7 3.7 2323.9 863.5 186.8 16-2414 C978 0.1 2.4 4.7 3.7 2325.1 863.9 186.9 16-2269 C293 0.3 11.2 1.8 3.2 3.5 7.8 113.2 16-2418 C283 1.8 52.9 7.4 13.1 17.5 91.0 625.3 16-2110 C110 0.1 19.5 18.0 17.1 142.4 5.0 518.7 16-2119 C138 0.6 10.0 20.0 15.8 9802.7 3642.4 787.9 16-2120 C139 1.6 25.7 23.7 22.5 187.5 6.6 683.1 16-2115 C130 1.8 28.5 26.3 24.9 207.8 7.3 757.1 16-2121 C140 0.1 67.3 0.9 1.2 4.3 0.6 4.9 16-2124 C148 980.4 2.4 9.6 16.5 16.1 8.5 130.2 16-2127 C158 2.2 34.9 32.2 30.5 254.6 8.9 927.8 16-2128 C159 1.5 23.1 21.4 20.3 169.0 5.9 615.9 16-2141 C189 1.4 23.0 8.8 12.1 41.4 5.9 47.2 16-2145 C199 1.4 22.1 20.5 19.4 161.8 5.7 589.4 16-2140 C188 1.5 23.9 9.1 12.6 43.0 6.1 49.0 16-2148 C218 1.4 22.7 20.9 19.9 165.6 5.8 603.5 16-2149 C220 1.4 23.3 8.9 12.3 41.9 6.0 47.9 16-2321 C822 704.6 48.3 6.1 8.5 28.9 4.8 32.9 16-2322 C830 599.0 41.1 5.2 7.2 24.5 4.1 28.0 16-2313 C807 532.2 36.5 5.2 9.0 8.7 4.6 70.7 16-2314 C817 821.5 56.4 8.0 13.8 13.5 7.2 109.1 16-2405 C971 441.7 30.3 3.8 5.3 18.1 3.0 20.7 16-2406 C970 867.8 59.5 7.6 10.4 35.6 5.9 40.6 16-2387 C951 387.4 26.6 3.4 4.7 15.9 2.6 18.1 16-2388 C961 846.6 58.1 8.3 14.3 13.9 7.4 112.5 16-2397 C962 843.2 57.8 7.3 10.1 34.5 5.7 39.4 16-2398 C972 846.6 58.1 7.4 10.2 34.7 5.7 39.6 16-2407 C973 0.3 4.0 2.8 3.5 29.5 1.0 107.3 16-2408 C952 1023.9 70.2 10.0 17.3 16.8 8.9 136.0 16-2210 C107 0.1 2.4 0.9 1.3 4.3 0.7 4.9 16-2211 C111 627.0 43.0 5.5 7.5 25.7 4.2 29.3 16-2263 C265 689.4 47.3 6.7 11.6 11.3 6.0 91.6 16-2419 C275 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2245 C244 0.1 2.4 1.6 2.1 17.2 0.6 62.6 16-2246 C243 373.1 25.6 3.2 4.5 15.3 2.5 17.5 16-2251 C253 0.1 2.4 1.6 2.1 17.3 0.6 63.1 16-2252 C263 0.2 3.3 1.3 1.7 5.9 1.0 6.7 16-2262 C175 331.5 22.7 2.9 4.0 13.6 2.2 15.5 16-2284 C386 1.4 2.4 0.9 1.2 4.2 0.7 4.8 16-2297 C400 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2298 C399 444.6 30.5 3.9 5.3 18.2 3.0 20.8 16-2221 C134 0.1 2.4 0.9 1.2 4.3 0.7 4.9

Page 99 of 100 Survery of mercury and cyanotoxin concentrations in fish tissues in Lake Champlain ATX CYL SUNY- DFWI ID Detection Detection ESF ID LR dLR RR dRR GSH-LR Number Limit Limit Number (µg/kg) (µg/kg) 16-2222 C153 917.6 63.0 8.0 11.0 37.6 6.2 42.9 16-2225 C137 447.5 30.7 3.9 5.4 18.3 3.0 20.9 16-2226 C147 1.4 22.7 3.0 19.9 165.5 5.8 603.2 16-2285 C388 0.1 4.7 1.8 2.5 8.5 1.4 9.7 16-2286 C387 0.3 36.1 4.6 6.3 21.6 3.6 24.6 16-2214 C122 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2215 C173 327.1 22.4 3.2 5.5 5.4 2.8 43.4 16-2227 C155 1.5 23.6 3.1 20.7 172.6 6.1 628.8 16-2228 C145 437.1 30.0 3.8 5.3 17.9 3.0 20.4 16-2385 C950 0.7 11.2 7.7 9.8 81.9 2.9 298.5 16-2291 C393 0.4 5.8 2.2 3.1 10.5 1.7 12.0 16-2292 C392 1.4 22.8 8.7 12.0 41.0 6.8 46.8 16-2289 C391 526.2 2.4 0.9 1.2 4.3 0.7 4.9 16-2290 C394 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2247 C245 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2248 C255 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2275 C372 1.4 22.7 3.0 19.9 165.7 5.8 603.7 16-2276 C123 0.8 13.5 5.8 10.0 9.7 5.2 78.6 16-2277 C374 1.4 22.5 15.5 19.8 164.6 5.8 599.9 16-2278 C373 1.5 23.4 8.9 12.3 42.1 7.0 48.1 16-2283 C381 867.8 23.1 8.8 12.2 41.7 6.9 47.5 16-2417 C382 1.4 21.9 2.9 19.2 159.8 5.6 582.4 16-2229 C156 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2230 C166 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2231 C144 1.3 21.4 9.2 15.8 15.4 8.2 124.7 16-2232 C176 0.1 2.4 1.6 2.1 17.3 0.6 62.9 16-2272 C264 1.4 23.0 3.0 20.1 167.9 5.9 611.9 16-2223 C136 0.1 2.4 1.6 2.1 17.2 0.6 62.8 16-2224 C146 0.1 2.4 0.9 1.2 4.3 0.7 4.9 16-2273 C371 0.1 2.4 0.9 1.3 4.3 0.7 4.9 16-2274 C361 1.4 22.3 2.9 19.6 163.0 5.7 593.8 16-2399 C964 0.1 2.4 0.9 1.2 4.2 0.7 4.8 16-2400 C963 0.1 2.4 0.9 1.2 4.3 0.7 4.9

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