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Comprehensive Analysis of the Great Lakes Top Predator Fish for Novel Halogenated Organic Contaminants by GCxGC-HR-ToF Mass Spectrometry Sujan Fernando, Aikebaier Renaguli, Michael Milligan, James J. Pagano, Philip K. Hopke, Thomas M. Holsen, and Bernard S. Crimmins Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05999 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Environmental Science & Technology

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Comprehensive Analysis of the Great Lakes Top Predator Fish for Novel Halogenated Organic Contaminants by GCxGC-HR-ToF Mass Spectrometry

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Sujan Fernando 1, Aikebaier Renaguli 2, Michael S. Milligan 3, James J. Pagano 4, Philip K. Hopke 5, Thomas M. Holsen 1,6, Bernard S. Crimmins 1,6,7*

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6 7 8 9 10 11 12 13 14 15 16 17 18

Center for Air Resources Engineering and Science, Clarkson University, 8 Clarkson Ave., Potsdam, NY, 13699, USA 2 Institute for a Sustainable Environment, Clarkson University, 8 Clarkson Ave., Potsdam, NY, 13699, USA 3 Department of Chemistry and Biochemistry, State University of New York at Fredonia, Houghton Hall, Fredonia, NY, 14063, USA 4 Environmental Research Center, State University of New York at Oswego, Oswego, NY, USA 5 Department of Chemical and Biomolecular Engineering, Clarkson University, 8 Clarkson Ave., Potsdam, NY, 13699, USA 6 Department of Civil & Environmental Engineering, Clarkson University, 8 Clarkson Ave., Potsdam, NY, 13699, USA 7 AEACS, LLC, Alliance, OH 44601

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*Corresponding author: [email protected], tel (202) 368-6926

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ABSTRACT

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The US Environmental Protection Agency’s Great Lakes Fish Monitoring and

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Surveillance Program (GLFMSP) has traced the fate and transport of anthropogenic

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chemicals in the Great Lakes region for decades. Isolating and identifying halogenated

24

species in fish is a major challenge due to the complexity of the biological matrix. A non-

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targeted screening methodology was developed and applied to lake trout using a 2

26

dimensional gas chromatograph coupled to a high resolution time of flight mass

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spectrometer (GCxGC-HR-ToF MS). Halogenated chemicals were identified using a

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combination of authentic standards and library spectral matching, with molecular formula

29

estimations provided by exact mass spectral interpretation. In addition to the halogenated

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chemicals currently being targeted by the GLFMSP, more than 60 non-targeted

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halogenated species were identified. Most appear to be metabolites or breakdown

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products of larger halogenated organics. The most abundant compound class was

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halomethoxyphenols accounting for more than 60% of the total concentration of

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halogenated compounds in top predator fish from all five Great Lakes illustrating the 1

ACS Paragon Plus Environment


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need and utility of non-targeted halogenated screening of aquatic systems using this

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platform.

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Keywords: Great Lakes Fish, Halogenated Contaminants, Comprehensive Two-Dimensional Gas Chromatography, High Resolution Mass Spectrometry

INTRODUCTION

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The Great Lakes region spans more than 750 miles and is home to nearly 50 million

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people in the United States and Canada. As the world’s largest fresh water system, the

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region contains 84% of North America’s surface fresh water.1 The region is also home to

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numerous manufacturing industries and significant amounts of agricultural land.2 As a

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result, safeguarding the well-being of the Great Lakes has been a vital mission of the

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United States Environmental Protection Agency (US EPA) since its establishment. As

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part of the US EPA’s Great Lakes Fish Monitoring and Surveillance Program

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(GLFMSP), top predator fish are routinely analyzed from each of the five Great Lakes for

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numerous contaminants including mercury and organic compounds including;

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polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated

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diphenyl ethers (PBDEs).3-6 Halogenated organic compounds represent one of the largest

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groups of chemicals found in the environment and have been studied extensively over the

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past four decades due to their persistent, bioaccumulative and toxic (PBT) properties.7

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Environmental research on the Great Lakes has a long history of discovering new threats

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to the environment. The vast amount of shoreline contains a variety of development

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categories including: industry, metropolitan, agriculture, suburban and remote areas

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where atmospheric deposition is the primary source of toxics exposure. The shoreline

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diversity allows the region serve as an indicator of North America’s recovery from legacy

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toxic chemicals (PCBs) and exposure to new chemicals of concern. In addition to the

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volumes of historical data on halogenated contaminants, there have been many

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discoveries of new organochlorine PBTs in the Great Lakes region including; Marbon8,

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dechlorane plus isomers9, polychlorinated carbozoles10, methoxylated polybrominated 2


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diphenoxybenzenes11, and hexabromocyclododecane12 to name a few. The detection of

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these novel compounds suggests that as regulations are passed to phase-out and/or restrict

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select halo-PBT compound production, alternatives will be produced with similar

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physicochemical properties that may result in similar environmental fate and toxicities.

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Recent in-silico work has also compiled a list of 610 chemicals, a subset of which are

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halogenated organics, with high production volumes that are expected to exhibit PBT

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properties.13 Also of concern are reagents and byproducts formed in the manufacturing

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of commercial products or metabolites/breakdown products of contaminants which may

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have similar or greater toxicity than the parent compound.

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To unravel the complex mixture of organohalogen compounds impacting the lakes,

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advanced separation techniques are necessary. The recent advancements and

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commercialization of 2-dimensional chromatography (GCxGC) has increased the peak

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capacity of gas chromatography systems by > 10-fold resulting in the isolation of over

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10,000 components in complex matrices. However, a major challenge of GCxGC

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analysis of complex samples is dealing with the large number peaks in the resulting 2D

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chromatograms. Banding of related chemical species in the 2D chromatographic plane

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can help organize the data structure and the use of mass spectral reference libraries

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further aids in the identification of compounds, although, authentic standards are crucial

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for confirmation. When identifying novel contaminants for which no such resources are

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available the task is much more challenging. A few recent studies have used the unique

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isotopic distribution of bromine and/or chlorine containing compounds to filter data.14

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Previous work using GCxGC has developed fingerprints for dolphin blubber15,16, fish

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oils17, sediments14, and benthic organisms.18 Several complex mixtures and novel classes

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of compounds have been resolved, including polyhalogenated polycyclic aromatic

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hydrocarbons18, alkyl bipyrroles16, chlorinated paraffins19, nonylphenols20 and legacy

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species (PCBs, PCDD/Fs).21,22

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Therefore, it is critical that advanced analytical techniques (e.g. GCxGC) are refined and

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applied to environmental matrices such as biota, sediment and water to identify and

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monitor new chemicals of concern before levels reach the point of biochemical effects. 3



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One such advance is the adaptation of a high resolution mass spectrometer to a

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commercial GCxGC platform. Traditionally, systems relied on a GCxGC equipped with a

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nominal mass MS23 or a 1D chromatography front end coupled to a high resolution mass

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spectrometer (HRMS) for analysis of complex samples.24 The coupling of GCxGC to

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HRMS provides the enhanced separation needed to resolve coelutions for more accurate

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peak identification and the enhanced MS resolution needed for component specificity and

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identification.

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A variety of plotting techniques such as Kendrick plots have been used to capitalize on

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mass defect differences between compounds.25 A combination of mass defect filtering

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and plotting has proven especially useful for isolating halogenated compound classes.26

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In addition to library matching, unknown identification can be performed using molecular

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formula deconvolution of the HRMS molecular ion and mass spectral fragment

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interpretation.

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The focus of the current study was to utilize a GCxGC coupled to a High Resolution

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Time of flight mass spectrometer (GCxGC-HRT) to develop semi-quantitative

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halogenated (chlorine and bromine) contaminant profiles in top predator fish (lake trout

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and walleye) from each of the Great Lakes. The profiles represent an integrated signal of

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volatile and semi-volatile halo-PBT burdens in top predator fish in each of the lakes.

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Applying this technique illustrates the expansion of GLFMSPs role beyond legacy

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chemicals to contemporary chemical monitoring to protect the Great Lakes region from

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emerging contaminants of concern.

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EXPERIMENTAL

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Chemicals

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List of chemicals purchased and the sources are listed in the Supporting Information (SI).

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Methods 4


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Lake trout were collected from each of the Great Lakes in 2014 with the exception of

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Lake Erie for which walleye were used.

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elsewhere.4 Briefly, approximately 50 fish were collected from each lake and combined

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into 10 composites of 5 whole fish each. For the current chemical screening effort, an

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equal amount of the 10 composites were combined for each lake resulting in a single

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homogenate containing 50 fish or a mega composite (MC). An aliquot of the MC (10g)

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from each lake was combined with 15g of drying agent (sodium polyacrylate) and

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extracted with DCM using an Accelerated Solvent Extractor (ASE350, Dionex). Prior to

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extraction 100 ng of phenanthrene-d10 was added as a recovery standard to each sample.

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Lipids were isolated via gel-permeation chromatography (GPC) followed by elution

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through a Primary Secondary Amine (PSA) bonded silica SPE cartridge for fatty acid

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removal. Purified extracts were concentrated to approximately 100 µL followed by the

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addition of 100 ng of pyrene-d10 to serve as an injection standard. A sample blank

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consisting of 15 g of drying agent spiked with 100 ng of phenanthrene-d10 (recovery

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standard) was extracted and treated (GPC and SPE) in the same manner as the samples.

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Extracts were analyzed using a Pegasus 4D GC×GC-HRT (LECO, St. Joseph, MI). A

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detailed description of the procedures is provided in the SI.

Detailed sampling procedures were presented

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Quality Assurance

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The phenanthrene-d10 recovery ranged from 78% to 94% for all samples and blanks

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analyzed. No halogenated compounds reported in the current work were detected in the

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blanks. The concentrations of 2-chloro-4-methoxyphenol and 2-bromo-4-methoxyphenol

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were determined using authentic standards. The response factor (RF) of 2-chloro-4-

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methoxyphenol was used to estimate the levels of all other chloromethoxyphenols

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detected in the samples. For the remaining compounds detected where authentic

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standards were not available, concentrations were based on a global RF generated from

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Cl4 to Cl7 PCBs in the standard mix. The total concentration of PCBs was also estimated

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using this global RF. In the case of PBDEs, a similar global RF was determined using an

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average RF for Br4 to Br6 PBDEs using an authentic standard mix. As a result, the

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concentrations presented in the current work are semi-quantitative. 5



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Standard Reference Materials (SRM) from the National Institutes of Standards and

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Technology (NIST, Gaithersburg, MD) were extracted and analyzed along with the five

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fish MCs. The SRMs were lake trout tissue collected from Lakes Superior (SRM 1946)

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and Michigan (SRM 1947) in 1997. The total PCB and PBDE concentrations for SRMs

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1946 and 1947 measured using the global RF described above were within 11% of the

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certified values. The Base Monitoring element of GLFMSP quantifies PCBs and PBDEs

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using targeted isotope dilution methods. Total PCB and PBDE (t-PCB, t-PBDE,

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respectively) concentrations measured using the GCxGC-HRT and global response

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factors were also within 17% of the values obtained from targeted methods3 (Table S1).

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Extracts were injected 3x each to assess reproducibility of profiles and concentration

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estimates with the latter typically varying less than 10%.

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Data Processing

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All data processing was completed using vendor software (Leco, ChromaTOF v1.90.60)

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and Microsoft Excel. To isolate the halogenated compounds, a bromo/chloro (Br/Cl)

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mass spectral filter was applied to take advantage of the unique isotopic distribution of

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compounds that contain one or more bromine and/or chlorine atoms.27 While the

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vendor’s software includes a Br/Cl filter function, we found many halogenated species

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with S/N1 per lake) are

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needed to infer similar sources or connection between these classes of compounds.

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The SRM tissue analyzed was collected from Lakes Superior and Michigan (SRM 1946

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and 1947, respectively) in 1997. Many of the HaloMeOPs detected in MC samples were

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also present in the SRMs, including the highly abundant 2-chloro-4-methoxyphenol and

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2-bromo-4-methoxyphenol compounds. The ClMeOP, BrMeOP, t-PBDE and t-PCB

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concentrations from the SRMs were compared to those observed in 2014 for Lakes

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Superior and Michigan using the GCxGC-HRT semi-quantitative method (Figure 4). The

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SRM (collected in 1997) exhibited higher levels of PCBs and PBDEs as well as

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ClMeOPs and BrMeOP. It should be noted that the SRMs consist of lake trout fillets and

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the 2014 mega composites are whole fish composites. Whole fish typically contain

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elevated PBT concentrations (and lipids) due to the inclusion of skin and fatty organelle

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tissue31, specifically the liver.32 As the detoxifying organ with elevated lipid levels,

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compared to muscle tissue, liver tissues may also contain elevated levels of the PBTs and

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possibly metabolites such as ClMeOP and BrMeOP. Therefore, it is possible that whole

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fish composites collected in 1997 would contain even higher levels of both PCBs and

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HaloMeOPs compared to the SRMs. The magnitude of this increase is difficult to

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quantify with the available data. However, the SRM results provide evidence that these

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species have been present in the Great Lake’s fish at significant concentrations (>PCBs)

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for over 20 years.

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The correlation between PCBs and HaloMeOPs in fish suggests similar sources. PCB

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metabolism in fish typically results in the formation of hydroxy and methylsulfonyl

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analogs.33,34 We found no evidence in the literature suggesting PCBs are transformed into

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ClMeOP analogs by fish or other biota. Debromination is the dominant transformation

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route for PBDEs.35 Again, no evidence has been presented to suggest a link between

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PBDEs metabolism and BrMeOP. 10


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The HaloMeOPs detected in fish may also belong to the large group of chemicals

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classified as halogenated natural products (HNPs) which consists of more than 5000

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compounds.36 These compounds are formed by living organisms (fungi, bacteria) and

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during natural abiogenic processes (volcanoes, forest fires). Although the HaloMeOPs

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detected in the current study have not been reported, compounds of a similar nature such

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as chlorinated phenols, chlorinated anisyl metabolites and chlorinated hydroquinone

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methyl esters are reportedly produced in large quantities by numerous fungi.30

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Thousands of new marine natural products have been reported in recent years which

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includes numerous HNPs.37 HNPs have been reported in marine mammals which

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include chlorinated and brominated bipyrroles as well as methoxypolybrominated phenyl

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ethers.30 A HNP similar in structure to those identified in the current work was reported

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in wild boar.38 The compound was confirmed as tetrachloro-p-methoxyphenol and is

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produced by a specific fungal species that are present in food consumed by wild boar.

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The octanol-water coefficient (Log Kow) of 2-chloro-4-methoxy-phenol and 2-bromo-4-

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methoxy phenol was compared to that of the most abundant PCBs in fish. The Log Kow

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for these two HaloMeOPs as predicted by the EPA’s EPI suite software is 2.24 and 2.48

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respectively. The Log Kow for the most abundant PCBs found in fish (Cl4-Cl7) range

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from approximately 6 to 9. Based on these facts it appears the HaloMeOPs may have

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low bioaccumulation potential compared to PCBs yet they are found in whole fish at

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higher levels. Further research is needed to identify sources and the toxicities of these

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species as they represent the largest burden of halogenated organic species detected in

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Great Lakes trout.

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Beyond the HaloMeOP, numerous other halogenated species were observed that are not

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currently on the targeted legacy list. The sum of these species accounted for 5% to 17%

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of the total halogenated species burden in fish. Several of these have been previously

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reported in fresh water fish; penta- and hexa- chlorobenzene39, pentachloroanisole40,

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polychlorinated vinylbenzenes41, polychlorinated naphthalenes42, polychlorinated

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terphenyls43, and polychlorinated diphenylethers.44 These compounds along with their

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approximate concentrations are displayed in Table S3 (SI). 11



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A number of compounds were also detected which appear to be novel or emerging

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contaminants (Table 3). One of the compounds detected in Lakes Michigan and Ontario

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and confirmed with an authentic standard (ID confidence level 1) was 3,6-dichloro-9H-

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carbazole. This compound represents one of many halogenated carbazole species

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previously detected in soil and sediment.10 The 3,6-dichloro-9H-carbazole is of

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significant interest due to the similarity in toxicity to dioxins.10 Also detected and

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confirmed with an authentic standard was triclosan methyl ether in Lake Erie. This

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compound is a byproduct of the antimicrobial agent triclosan which is present in many

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household and personal care products.45 The presence of 2,4,6-tribromoanisole was

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confirmed in all lakes. This compound is a fungal metabolite/conversion product of the

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fungicide 2,4,6-tribromophenol.46 These compounds were detected at approximately 1-2

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ng/g ww range.

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In cases where authentic standards were not available, tentative identities were

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determined for a number of compounds using the NIST 2014 mass spectral library

333

matching and accurate mass confirmation (ID confidence level 2). One of the

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compounds tentatively identified was a dichloro phenylpyrimidine. This compound has a

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similar mass spectrum and retention time to that of 4,6-dichloro-2-phenylpyrimidine

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(common name Fenclorim) and may represent an isomer. Fenclorim is used as a

337

herbicide safener for rice to limit phytotoxicity of certain herbicides.47 A monochloro

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phenylpyrimidine was also detected and may represent an impurity or dechlorinated

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breakdown product. Other transformation products tentatively identified included

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dichlorobenzophenone, a bacterial metabolite of DDT15, and dichlorostilbene, a DDD

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rearrangement product.48 Also identified was tetrachloro-dimethoxybenzene (or

342

tetrachloroveratrole) which is a biotransformation product of bleached pulp mill

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effluent.49 Dichloroanthracene, a halogenated polycyclic aromatic hydrocarbon (PAH),

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was also detected and is considered highly toxic. Dichloroanthracene isomers were

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detected at high concentrations in a plastic fire previously26 and have been shown to

346

bioaccumulate in fresh water organisms in an exposure study.18 These compounds were

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detected at approximately 1-2 ng/g ww range. 12


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In the case of halogenated features for which authentic standards and library matches

349

were not available, molecular formulas were derived based on accurate mass

350

measurements (< 5 ppm) and isotopic profile match. For some of these formulas

351

corresponding structures can be found in the ChemSpider database. These compounds

352

have been assigned an ID confidence level of 3. Although it is possible to select a

353

structure(s) that is consistent with the fragmentation data observed in the acquired EI

354

mass spectra, confirmation can only be achieved with an authentic standard. As a result

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these compounds are assigned a tentative ID of ‘Unknown’. Unknown 19 of this group

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was detected as three potential isomers. The most abundant isomer was observed in all

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lakes at an estimated concentration ranging 35 to 61 ng/g ww. The molecular formula for

358

Unknown 10 is consistent with that of a mixed halogenated (Br/Cl) methoxyphenol and

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was detected at relatively low abundance (0.1 ng/g ww) in Lake Ontario. This compound

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may represent a metabolite of a larger Br/Cl-organic contaminant. The mass spectra of

361

all compounds classified as ‘Unknown’ in Table 3 are provided in SI for reference.

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For molecular formulas where no corresponding structures were found, an ID confidence

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level of 4 was assigned. One of these features identified as ‘Unknown 4’ was assigned the

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molecular formula C9H10ClO2. This feature was detected in fish from all the Lakes at

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relatively high estimated concentrations ranging from 10 to 58 ng/g ww. Also, the

366

isotopologue pattern of some of these features appears to contain both bromine and

367

chlorine atoms suggesting the presence of mixed halogenated species. These compounds

368

are of special interest due to the potentially high toxicities50 and novelty of combining

369

both Br and Cl in an organic molecule. Previous work suggests this type of conformation

370

is derived from combustion of polymeric materials treated with flame retardants.51 Also

371

detected were features containing halogenated profiles for which no meaningful

372

molecular formulas were found (ID confidence level 5). Table 3 lists these species along

373

with an estimate of their concentrations. Ambiguity in assigning structures/molecular

374

formulae for the observed features may be the result of missing molecular ions. Without

375

the molecular ion, it is difficult assign a molecular formula without additional

376

information such as an authentic standard and/or library spectral reference. However, the 13



377

current methodology catalogs all of the identified features of halogenated compounds for

378

the GLFMSP. Future spatiotemporal surveillance is still possible without a priori

379

knowledge of the chemical identity.

380

The overreaching goal of this study was to explore the efficacy of a newly available

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GCxGC high resolution time of flight mass spectrometer to create halogenated

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contaminant profiles in trout collected in the each of the Great Lakes. This method will

383

not replace targeted monitoring of legacy chemicals as detection limits are > 10-fold

384

above targeted sector based HRMS methods. However, we propose that the current

385

methodology could be used as a screening tool for legacy halogenated contaminants or

386

where halogenated chemical fingerprinting is desired. The full scan, exact mass data,

387

combined with the resolving power of GCxGC enabled resolution of thousands of

388

compounds. Using embedded scripting capabilities, ~200 halogenated species were

389

isolated. The majority of compounds/features detected are not part of GLFMSP’s routine

390

monitoring schedule and likely have multiple sources including natural products and

391

transformation products of legacy halogenated contaminants.

392

Many of the detected halogenated compounds have not been identified and/or confirmed.

393

But the retention times and mass spectra for all of the halogenated features are cataloged

394

for future experiments. The current study does illustrates how the GCxGC-HRT can be

395

used to assess the spatial distribution of halogenated species in a system (Great Lakes

396

Region) through comprehensive screening and isolation halogenated features prior to

397

identifying each component. The significant intensity associated with a newly detected

398

halogenated feature resulted in the discovery of a new chemical class (HaloMeOPs) that

399

exhibits concentrations in lake trout greater than PCBs. These findings illustrate the

400

utility of this approach and add to the Great Lakes Fish Monitoring and Surveillance

401

Program’s directive to monitor and assess the health of the Great Lakes by identifying

402

new potential chemical threats.

403 404

Acknowledgments 14


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Funding for this work was provided by the Great Lakes National Program Office under

406

the United States Environmental Protection Agency, grant nos. GL96594201, GL

407

00E00454, GL 00E01505. We wish to thank the Program Manager Elizabeth Murphy and

408

many people who assisted in sample collection and processing. Although the research

409

described in this article has been funded wholly or in part by the United States

410

Environmental Protection Agency, it has not been subjected to the Agency's required peer

411

and policy review and therefore, does not necessarily reflect the views of the Agency and

412

no official endorsement should be inferred.

413 414 415

Supporting Information

416

at DOI:XXXX

The Supporting Information is available free of charge on the ACS Publications website

417

A brief description of the chemicals used and suppliers, the script used for isolating

418

halogenated features in the data files, A table summarizing the comparison between

419

PCB and PBDE concentrations determined by the current method and targeted

420

methods, A table detected in this study that are also monitored as part of GLFMSP,

421

and a table summarizing halogenated compounds detected that are not monitored by

422

GLFMSP, but reported in other studies.

423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

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667 668 669


Table 1. Confidence levels assigned for identification of the halogenated organics in the fish extracts. Confidence Identification Level

Confirmed with

Criteria

1

Confirmed structure

Authentic standard, Accurate mass measurement

The retention time and mass spectra of the acquired spectrum and standard are a match. Mass accuracy within 5 ppm.

2

Probable structure

Library spectrum match, Accurate mass measurement, Isotopic profile

Library match score 600 or higher, molecular formula is within 5 ppm mass error and isotopic profile is in agreement.

3

Tentative structure(s)

Accurate mass measurement, Isotopic profile

Molecular formula is within 5 ppm mass error and isotopic profile is in agreement. Corresponding structures available in ChemSpider database.

4

Molecular formula

Accurate mass measurement Isotopic Profile

In this case the selected molecular formula is within 5 ppm mass error and isotopic profile is in agreement.

5

Exact mass of interest

Isotopic Profile

In this case no meaningful molecular formula(s) are obtained for the observed m/z.

21



Page 22 of 29

Table 2. A list of the halogenated methoxyphenols detected in the fish extracts from the Great Lakes along with the approximate concentrations derived from the GCxGC-HRT semi-quantitative method. Tentative ID

1D RT (s)

2D RT (s)

ID Confidence Level

Superior

Huron

Michigan

Erie

Ontario

Cl1 dimethylpropylphenol

1160.5

2.9

2

0.9

ND

ND

ND

ND

Cl1 dimethylpropylphenol

1295.5

2.8

2

8.9

2.9

ND

1.7

3

2-Chloro-4-methoxyphenol

1610.7

2.0

1

971

971

1706

1212

857

Cl1 methoxyphenol

1695.7

1.9

2

26.3

44.2

10.3

44.2

11.6

Cl1 methoxyphenol

1715.8

1.9

2

9.9

15

29.7

29.9

ND

2-Bromo-4-methoxy-phenol

1740.8

2.0

1

99.7

137

83.5

173

89.5

Cl1 methoxyphenol

1745.8

1.9

2

0.1

11.9

19

11.4

ND

Cl1 methoxyphenol

1770.8

1.9

2

ND

ND

0.5

11

4.2

Cl1 methoxyphenol

1790.8

1.9

2

ND

8.8

ND

ND

ND

Cl2 methoxyphenol

1820.8

2.1

2

16.3

0.6

10.6

7.8

4.4

Cl1 methoxyphenol acetate

1875.8

2.2

2

3.9

ND

ND

11

4

Cl1 methoxyphenol

1910.9

1.8

2

ND

1.1

ND

ND

ND

Cl2 methoxyphenol

1940.9

2.0

2

3.7

3.4

3.4

1.6

2.3

Cl1 methoxyphenol acetate

1965.9

2.2

2

1

ND

2.3

1.5

ND

Cl1 methoxyphenol

2266.1

1.8

2

34.5

53.4

77.7

46.2

66.9

1176

1249

1943

1551

1043

Total Concentration (ng/g ww)

Concentration (ng/g ww)

ND denotes samples for which the compound of interest was not detected.

22


Page 23 of 29


Table 3. List of the novel contaminants identified in fish extracts from the Great Lakes along with their approximate concentrations derived from the GCxGC-HRT semi-quantitative method. ID Observed 1D RT (s) 2D RT (S) Confidence Monoisotopic Mass Level 1090.4 3.2 3 214.1122 1285.5 2.6 3 186.0443

Peak

Tentative ID

1 2

Unknown 1 Unknown 2

3

Unknown 3

1475.0

2.2

3

4

Unknown 4

5

Unknown 5

1490.6 1515.7

2.7 2.7

4 4

6

Unknown 6

1530.7

2.2

3

Concentration (ng/g ww) Formula

Superior

Huron

Michigan

Erie

Ontario

C12 H19 ClO C9H11ClO2

10.4 21.6

13.3 9.1

4.5 5.9

3.5 3.1

15.5 10.4

188.0598

C9H13ClO2

15.9

10.1

2.0

1.4

10.2

185.0365 131.0259

C9H10ClO2

57.7

35.6

15.9

10.4

28.9

C6 H8ClO

9.7

1.7

5.1

0.5

14

C8 H9 ClO 2

ND

1.9

ND

ND

ND

ND 0.1

ND

1.4

0.2

0.9

2.1

0.7 4.3

7

Unknown 7

1570.7

2.3

3

172.0288 186.0445

C9H11ClO2

ND

ND

8

2,4,6-Tribromoanisole

1650.7

2.7

1

341.7876

C7 H5 Br3 O

0.4

9

Unknown 8 Unknown 9

2.0 2.3

3 3

172.0286 216.0913

C8 H9 ClO 2

10

1665.7 1670.7

0.1 1.8

C11 H17ClO2

44.1

38.6

0.6

0.4 0.4

13.0

11

Unknown 10

1890.1

2.2

3

235.9230

C7H6BrClO2

ND

ND

ND

ND

0.1

12

1,2,4,5-tetrachloro-3,6-dimethoxybenzene

1890.9

2.6

2

273.9116

C8H6 Cl 4 O2

0.3

0.2

0.3

0.3

0.8

13

Chlorophenyl-pyrimidine

2025.9

2.2

2

190.0296

C10 H7ClN2

ND

ND

ND

ND

2.1

14

Unknown 11

2121.0

3.1

4

384.8060

C10 H4Cl 7O

2.0

0.7

1.7

0.5

0.7

15

Unknown 12

2201.0

2.9

4

370.8307

C8HBrCl 3F4N

ND

ND

ND

ND

1.8

16

Dichlorophenyl pyrimidine

2231.0

2.2

2

C10 H6 Cl 2 N2

ND

ND

ND

ND

1.8

17

Unknown 13

2271.1

2.8

4

223.9907 404.7912

C10H5 Cl 8

0.3

0.1

0.2

ND

0.6

18

Unknown 14

2276.1

3.2

4

276.8907

C8H6Cl 5

0.5

ND

0.3

ND

ND

19

9,10-Dichloroanthracene

2321.1

2.6

2

246.0000

C14 H8 Cl 2

ND

ND

0.6

ND

ND

20

Triclosan Methyl Ether

1 2 4

C13 H9 Cl 3 O2 C13 H8Cl 2O

ND

0.1 ND ND

2.2 ND ND

2,2'-Dichlorostilbene

4 2

2.4 4.5 ND

24

3.0 2.2

ND 1.2 ND ND

1.6

2551.0 2616.2

301.9668 249.9953 336.8674 338.8836

ND

2,4'-Dichlorobenzophenone Unknown 15 Unknown 16

2.4 2.3 2.4

ND

21 22 23

2426.1 2451.2 2496.2

248.0152

C14H10 Cl 2

ND

0.1

1.8

0.4

1.0

25

Unknown 17

5 4

N/A

0.1

0.1

0.1

ND

ND

Unknown 18

2.6 2.3

439.7630

26

2636.0 2736.3

370.8279

C11 HCl 6N2

2831.4

2.1

2856.4

2.1

2966.4

2.1

2936.0 3541.7

2.1 2.2

27 28

Unknown 19

29 30

Unknown 20

31

3,6-Dichloro-9H-carbazole

3 5 1

280.0500

C10 H7 Cl 6 C10 H9 Cl 6

C14 H13ClO4

0.7 ND 0.2

ND

1.3

ND

ND

ND

51.4

60.8

34.8

51.4

38.5

ND

8.8

ND

1.0

1.1

ND

ND

ND

0.4

0.5

265.2523

N/A

ND

ND

1.3

ND

ND

234.9953

C12 H7 Cl 2 N

ND

ND

2.1

ND

1.7

ND denotes samples for which the compound of interest was not detected.

23



Figure 1. (a) A selected region of the Total Ion Current (TIC) 2D-chromatogram of a fish extract from Lake Michigan which had undergone lipid removal by GPC. The black dots in the chromatogram are peak markers. Approximately 10,000 peaks were detected in this case. (b) Displays the subset of peaks classified as halogenated with the use of a Br/Cl mass spectral filter. In this case 117 peaks were present. (c) Chromatogram of the same fish extract shown in (a) and (b) which had undergone SPE cleanup for fatty acid removal following lipid removal by GPC. The chromatogram displays the subset of peaks classified as halogenated by the Br/Cl mass spectral filter. The removal of the fatty acids resulted in an increase in the number of halogenated compounds detected. In this case 169 peaks were detected.

24


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Page 25 of 29


Figure 2. The total concentration (ng/g ww) of halogenated organics detected in the 2014 fish mega composite extracts from the Great Lakes derived from the GCxGC-HRT semiquantitative method. The detected halogenated organics have been categorized into compounds currently being monitored by GLFMSP which include PCBs and a group of other contaminants which consists of PBDEs, DDTs, chlordane, oxychlordane, nonachlor, dieldrin and toxaphene. Those compounds identified that are not currently being monitored were categorized into halomethoxyphenols and other contaminants which are described in detail in Tables 2 and 3.

25



Figure 3. (a) The total concentration (ng/g ww) of PCBs and ClMeOP for each of the Lakes. The concentrations were derived from the GCxGC-HRT semi-quantitative method. In this case a relationship was observed (R2=0.75, p=0.00047) but the small number of samples prohibits concluding this source from the current data set. (b) The total concentration of PBDEs and BrMeOP for each of the Lakes. In this case a significant negative correlation was observed (R2=0.77, p=0.00035).

26


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Figure 4. Comparison of the t-ClMeOP, BrMeOP, t-PCB and t-PBDE concentrations (ng/g ww) in fish collected from Lake Superior and Michigan from 2014 and 1997. The concentrations for 1997 were derived from the analysis of SRM 1946 and 1947. All concentrations were derived from the GCxGC-HRT semi-quantitative method.

27



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Comprehensive Analysis of the Great Lakes Top Predator Fish for

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