Hexabromocyclododecane (HBCDD) Flame Retardant in Top

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Characterization of Natural and Affected Environments

Hexabromocyclododecane (HBCDD) Flame Retardant in Top Predator Fish across Canada and its 36-Year Temporal Trends for Lake Ontario Guanyong Su, Daryl McGoldrick, Mandi G. Clark, marlene evans, Melissa Gledhill, Christine Garron, Alain Armellin, Sean Backus, and Robert J. Letcher Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01052 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Environmental Science & Technology

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Hexabromocyclododecane (HBCDD) Flame Retardant in Top Predator Fish across

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Canada and its 36-Year Temporal Trends for Lake Ontario

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Guanyong Su †,‡, Daryl J. McGoldrick†,*, Mandi G. Clark†, Marlene S. Evans†, Melissa

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Gledhill†, Christine Garron†, Alain Armelin†, Sean M. Backus†, Robert J. Letcher§,*

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†

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Water Science & Technology Directorate, Science and Technology Branch, Environment and Climate Change Canada, Burlington, ON L7S 1A1, Canada

‡

Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of

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Environmental and Biological Engineering, Nanjing University of Science and Technology,

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Nanjing 210094, P. R. China

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§

Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada

14 15 16 17

*Corresponding authors:

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Daryl J. McGoldrick; Tel: 1-905-220-1173; E-mail: [email protected]

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Robert J. Letcher; Tel.: 1-613-998-6696; E-mail: [email protected]

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1

ACS Paragon Plus Environment


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Abstract

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Hexabromocyclododecane (HBCDD) is a high concern environmental pollutant due to its

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persistent, bioaccumulative and toxic properties. The spatial distribution of HBCDD was

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investigated in top predator fish (Lake Trout, Walleye or Brook Trout) collected in 2013

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(n=165) from nineteen sampling sites and in 2015 (n=145) from twenty sites across Canada.

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HBCDD was measurable in at least one sample at each sampling site regardless of sampling

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year with the exception of Walleye from the south basin of Lake Winnipeg (2013). Sampling

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sites in or near the Laurentian Great Lakes had greater ΣHBCDD concentrations compared to

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locations to the west or east. The greatest mean ΣHBCDD concentration was 72.6 ng/g lw in

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fish from Lake Huron-Goderich (2015). Regardless of the sampling sites, α-HBCDD was the

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dominant congener followed by γ-HBCDD, whereas β-HBCDD was barely detectable. In fish

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from the same waterbody there were comparable α/γ isomer concentration ratios. The greatest

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ratio was 20.8 in fish from Lake Ontario, whereas the lowest ratio was 6.3 for fish from Lac

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Memphrémagog (Québec) likely related to more recent emissions of technical HBCDD

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mixture. Temporal trends of HBCDD in Lake Trout from Lake Ontario showed a significant

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decreasing trend for γ-HBCDD with a half-life estimate of 10 years over a 36-year period

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(1979-2015), and for α-HBCDD with a half-life of 11 years over the years of 2008 to 2015.

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The proportion of α-HBCDD to ΣHBCDD increased significantly during 1979 to 2015. The

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present study provided novel information on the isomer-specific HBCDDs in Canada

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freshwater fish.

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Keywords: Hexabromocyclododecane; Isomers; Flame Retardants; Temporal Trend; Spatial

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Distribution; Canada 2


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Introduction

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1,2,5,6,9,10-Hexabromocyclododecane (HBCDD) has a cyclic ring structure of twelve

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carbon atoms of which six carbons are bonded to bromine atoms. HBCDD is used as an

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additive flame retardant (FR) in extruded (XPS) and expanded (EPS) polystyrene foams,

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textiles, electronics and plastic materials.1-3 HBCDD is of very high environmental concern

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due to its persistent, bioaccumulative and toxic properties and susceptibility to long-range

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transport.4-10

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HBCDD is composed of 16 theoretical diastereomer structures, but the commercial

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HBCDD mixture is primarily composed of three stereo-isomers, alpha-(α-; 12 %), beta-(β-;

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6 %) and gamma-(γ-; 82 %) HBCDD.11-13 HBCDD has been commercially manufactured and

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used since the 1960s, and thus has been used as a FR for half a century.11, 14, 15 The global

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annual production of HBCDD was estimated to be 16000 t, 16700 t and 21951 t in the year of

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1999, 2001 and 2006, respectively.14, 15 Given its persistence, bioaccumulation, potential for

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long-range transport and toxicity, HBCDD was listed to Annex A of the Stockholm

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Convention of Persistent Organic Pollutants in 2013, but with specific exemptions for

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production and use in XPS and EPS in building materials.10, 16, 17 In Canada, HBCDD was

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added to the list of Toxic Substances (Schedule 1) of the Canadian Environmental Protection

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Act (CEPA, 1999) and is a Chemical of Mutual Concern under Annex 3 of the 2012 Protocol

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amending the Great Lakes Water Quality Agreement between Canada and the United States

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of America (GLWQA, 2012). Existing data has demonstrated that HBCDD (mainly α- and

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γ-isomers) is globally ubiquitous in aquatic environments with high detection frequencies in

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biota. Meng et al. determined HBCDD in twelve fish species from South China, and found 3



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that HBCDD was detectable in more than 70% of 60 analyzed samples with concentrations

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ranging from not detectable (ND) to 2.9 ng/g lipid weight (lw).18 In a recent study, Guo et al.

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reported HBCDD in 11 of 15 fish samples collected in the Great Lakes basin with a

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concentration range of 4-122 ng/g lw.19 Very recently, Su and coworkers investigated a broad

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suite of flame retardants in various predator and prey fish species collected from Lake

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Ontario and Lake Erie, and found that ∑HBCDD was quantifiable in most samples with the

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exception of Freshwater Drum (Aplodinotus grunniens) and Round Goby (Neobious

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melanotomus) from Lake Erie, and with concentrations ranging from ND to 17.3 ng/g ww.5

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Overall, HBCDD monitoring studies in Canadian freshwaters aquatic environments including

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sediment and biota are generally scarce and limited to a few regions such as the Great Lakes

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and Lake Winnipeg.20-23

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It takes a sufficient number of years of monitoring to generate a dataset with an annual

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range of coverage to permit statistical tests that are robust enough to detect temporal

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changes.24-26 Due to the recent interest in HBCDD by regulatory agencies there are only a few

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environmental studies reporting temporal trends of HBCDD.27, 28 The studies that have been

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published are limited to trends of HBCDD in herring gull eggs from the German North and

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Baltic Sea coasts (1998-2008) and Laurentian Great Lakes (1982-2006)29,

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organisms (2009-2012) from Chinese Taihu Lake31, human serum samples (2002-2015) from

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Australian South East Queensland32, seabird eggs (2003-2014) from Canadian Arctic, or

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adipose tissues of polar bears (1983-2010) from East Greenland6. The trends for HBCDD

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reported in these studies show both increases and decreases over time depending on location

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and/or species.27

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, aquatic

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The present study had two primary objectives. Firstly, to determine the spatial distribution

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of total and isomer-specific HBCDD concentrations in Lake Trout (Salvelinus namaycush),

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Walleye (Sander vitreus) and Brook Trout (Salvelinus fontinalis) collected from freshwater

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lakes across Canada. This information will be useful as a temporal baseline with which to

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compare future measurements of HBCDD in the monitoring of Lake Trout from across

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Canada to evaluate the effectiveness of the risk management actions taken to reduce the

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levels of HBCDD in the Canadian environment. Secondly, over a 36 year time period to

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examine the temporal trends (1979-2015) of isomer-specific HBCDD in Lake Trout from

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Lake Ontario by using a combination of published data, retrospective analysis of preserved

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samples, and contemporary monitoring.

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Materials and Methods

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Sampling Information

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Fish were collected from water bodies across Canada consisting of lakes, rivers, and

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reservoirs ranging from remote regions in northern Canada. Most fish were captured using

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bottom set gill nets between the months of June and October. The exceptions were fish from

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Lake Athabasca, Great Bear Lake, Reindeer Lake, and Cold Lake which were collected

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through the ice in December and fish from Kejimkujik National Park for which collection

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with rod and reel was more effective. Lake trout were the most common species collected

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across the monitoring network; however, at locations where Lake Trout are not present,

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Walleye, or Brook Trout were collected instead. These three species occupy the uppermost 5



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trophic levels of the water bodies where they were captured.

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Upon collection, the fish were frozen as soon as possible, transported to the Canada

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Centre for Inland Waters in Burlington, Ontario, Canada, and maintained at -20 °C until

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laboratory processing. Prior to processing, fish were partially thawed at 5 °C, measured for

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total length and weight, sexed, and aging structures are removed (coded wire tags, scales

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and/or otoliths). Each individual whole fish was then cut into pieces and homogenized

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through a HOBART model 4622 commercial meat grinder five times. Sub-samples of whole

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fish homogenate were then placed in solvent rinsed jars and either held at -20 °C until

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submitted for laboratory analysis (contemporary monitoring) or were added to the (Canadian)

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National Aquatic Biological Specimen Bank (NABSB) at -80 °C until chemical analysis

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(retrospective monitoring).33

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Three subsets of fish homogenate samples were analyzed for total HBCDD or individual

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α-, β- and γ-HBCDD isomers. The first subset was for a contemporary investigation of the

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spatial distribution of total HBCDD in top predator fish across Canada collected in 2013

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(Table S1). Nineteen sampling sites and 165 individual adult fish were studied and 5-10

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individual whole fish homogenates were analyzed for each of the sampling sites. The detailed

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biological information (i.e. length, weight, sex, age) for all 165 fish, and exact longitude and

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latitude of the sampling sites are provided in Table S1. Lake trout were captured at all lakes

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except for the western basin of Lake Erie, two locations in Lake Winnipeg, St. Lawrence

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River, and Lake Diefenbaker where Walleye were caught. Brook Trout were captured from

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Kejimikujik Lake-Rogers site.

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The second subset was comprised of 145 individual Lake Trout or Walleye collected in 6


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2015 (Table S2) for a recent investigation of the recent spatial distribution of HBCDD

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isomers in Canadian lakes, and in combination with the results from 2013, establish a

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baseline of the levels of HBCDD in Canadian fish. Twenty sampling sites were studied and

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the detailed biological (i.e. length, weight, sex, age) and location information is listed in

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Table S2. For most of the sampling sites Lake Trout were collected with four exceptions

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where Walleye were collected: St. Lawrence River-Cap-Santé, Columbia River, Lake

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Winnipeg-South Basin and Lake Diefenbaker-Saskatchewan Landing.

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The third subset of data was generated for a retrospective study of temporal changes of

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isomer-specific HBCDD in Lake Ontario (Table S3). For each sampling year from 2004 to

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2012, nine to ten samples were removed from the NABSB and submitted for analysis. This

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filled the gap between ECCC’s contemporary monitoring and a previous study of archived

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samples by Ismail et al. who reported on halogenated FRs including HBCDD in archived

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Lake Trout samples collected from Lake Ontario from 1979-2004.22 As a result, this study

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examined a 36-year trend (1979-2015) for HBCDD isomers in Lake Trout from Lake Ontario.

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All Lake Trout specimens selected from Lake Ontario were in the age range of 4 to 6 years to

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minimize age and growth dilution effects on the HBCDD concentrations.

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Measurement of HBCDD Isomers in Whole Fish Homogenates

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Sample analysis of all three subsets of fish were carried out at two laboratories; the

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National Lab for Environmental Testing (NLET) (ECCC; Burlington, ON, Canada) and

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SGS-AXYS Analytical Services Ltd (Sidney, BC, Canada). Specifically, the first subset of

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frozen specimens was analyzed for total HBCDD (rather than for individual HBCDD isomers) 7



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using a gas chromatography-mass spectrometry (GC-MS) based method using routinely by

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the participating NLET laboratory (Table 1). Briefly, fish homogenate samples were thawed,

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10g of material removed and blended with NaSO4. Surrogate and method spikes were then

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added to the sample prior to being subjected to soxhlet extraction with dichloromethane

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(DCM). The extracts were evaporated to 2mL and lipids removed by gel permeation

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chromatography (GPC) and additional clean-up by silica gel column. Samples were brought

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to a final volume of 1mL prior to quantitation by gas chromatography-mass spectrometry

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(GC-MS). The lone peak was chromatographically resolved and resulting from the response

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of the isotopic bromide anions m/z 79 and 81 that were monitored.

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The second and third subsets of frozen specimen samples were analyzed for α-, β- and

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γ-HBCDD isomers following method MLA-070 (SGS-AXYS Analytical Services Ltd).

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Briefly, approximately 10 g of thawed fish homogenate was spiked with

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surrogates of each isomer, subjected to soxhlet extraction with DCM, lipids were removed

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from the extracts by GPC and the extract were further cleaned with a Florisil column prior to

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quantification using liquid chromatography-mass spectrometry (LC-MS-MS). Two MRM

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transitions were monitored for the HBCDD isomers (m/z 640.5 and 79/81) and 13C12-labeled

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HBCDD (m/z 650.6 and 79/81), respectively. each isomer.Mean recoveries of

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13

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respectively. Detection limits were 0.15, 0.10, and 0.14 ng/g ww for the α-, β- and γ-HBCDD

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isomers, respectively.

13

C-labeled

C12-α-HBCDD, 13C12-β-HBCDD and 13C12- ɣ -HBCDD were 73±14, 52±19 and 64±20 %,

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To validate the results from two GC-MS and LC-MS based methods, for a small batch

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(n=30) of fish homogenate samples, duplicate samples were analyzed by both NLET and 8


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SGS-AXYS Analytical Services Ltd. The results of analyses found that mean ∑HBCDD in

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extracts generated by NLET was 1.4 ng/g, that was significantly 32% greater (paired t-test, p

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< 0.0001) than those generated by SGS-AXYS. The differences between the laboratories are

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likely the result of instrumentation and extraction procedures. The bias (±32%) between

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NLET and AXYS quantified samples is comparable to the average within site coefficients of

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variation for these sample sets of 33 and 39%, respectively.

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Data Analysis

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Method detection limit (MDL) was defined as a minimum amount of analyte producing

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a peak with three times signal-to-noise ratio (S/N) for GC-MS, and the MDL for the

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∑HBCDD concentrations generated from GC-MS (i.e. the first subset of fish, Table 1) was

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0.005 ng/g ww. MDLs for the isomer-specific HBCDD concentrations generated by

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LC-MS-MS were determined as lower method calibration limits (LMCLs). LMCLs are

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determined by prorating the concentration of the lowest calibration limit for sample size and

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extract volume. The following equation is used: ((lowest level cal conc.) x (extract

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volume))/sample size. The typical extract volume is 1 mL. The LMCL was 0.1 ng/g ww for

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all three HBCDD isomers. Arithmetic mean HBCDD concentrations were determined using

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the Kaplan-Meier method in the NADA package of the free statistical software package R to

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accommodate censored values34, 35. Comparisons of HBCDD concentrations among n ≥ 3

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sampling groups were conducted by one-way analysis of variance (ANOVA) and temporal

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trends were assessed using the slope of linear regression models. Statistical analyses and

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generation of figures were performed using GraphPad Prism software. All statistical 9



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hypotheses were assessed at α = 0.05.

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Characterization of Temporal Trend for HBCDD

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A statistically significant negative linear relationship was observed between γ-HBCDD

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concentration and sampling years over the period of 1979 to 2015, and the linear

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relationships for ∑HBCDD or other HBCDD isomers were not significant. To this end, fitted

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linear curves were generated for γ-HBCDD between Ln(C/C0) and the sampling year. The

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half-life of γ-HBCDD was calculated according to the following two equations:

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Ln (C/C0) = S × [Year]

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t1/2 = -ln(2) ÷ S

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where S is the slope of fitted curve. C0 is the chemical concentration of initial year (1979),

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and C is the chemical concentrations of [Year]. [Year] is the time period (unit: year) from the

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initial year of 1979. t1/2 is the half-life.

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Non-linear modelling of 2-segment piecewise regression was also applied to

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investigate non-monotonic trends in HBCDD over the period of 2004 to 2015. The

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two-segment model that best fit the time series and the change point were determined using

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the non-linear modelling package in JMP (SAS Institute) with α = 0.05 and a convergence

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criterion of 0.00001 and the following equation:

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If [Year] ≤ change point

Ln (C) = S1 × [Year] + I1

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If [Year] ≥ change point

Ln (C) = S2 × [Year] + I2

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C and [Year] are the HBCDD concentrations and sampling year, respectively. S1 and I1 are

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the slope and intercept of the first segment, and S2 and I2 are the slope and intercept of the 10


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second segment.

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Results and Discussion

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Spatial Distribution of HBCDD

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Regardless of the 2013 or 2015 sampling year or laboratory differences, HBCDD

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showed very similar spatial distribution patterns in the fish from freshwater lakes across

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Canada. As shown in Figures 1 and 2, fish from sampling sites in or near the Laurentian

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Great Lakes had much greater concentrations of ∑HBCDD compared to the sampling sites in

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the western or eastern regions of Canada. For fish samples collected in 2013, ∑HBCDD was

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quantifiable in 148 of 165 analyzed samples with concentrations ranging from 90%) with small amounts of α- and β-HBCDD.23, 36,

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compositions, and primarily contain high proportions of α-HBCDD relative to minor

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contributions from β- and γ-HBCDD.36,

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bioaccumulation or biotransformation of α-HBCDD as compared to β- and γ-HBCDD, which

and 0.8-5.8% for β-HBCDD

Biotic samples from same sampling locations normally exhibit different HBCDD isomer

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Thus, our monitoring data suggest greater

17



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is in good agreement with previous studies.

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To further explore the HBCDD isomer pattern change as a function of sampling year,

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we plotted the α-HBCDD/∑HBCDD concentration ratios versus sampling year, and observed

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a significant (p < 0.0001) increasing linear trend for α-HBCDD/∑HBCDD concentration

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ratios over 36 years (Figure 5). On the contrary, a significant decreasing linear trend (p

Hexabromocyclododecane (HBCDD) Flame Retardant in Top

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