Bioconcentration of Dissolved Organic Compounds from Oil Sands

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Bioconcentration of Dissolved Organic Compounds from Oil Sands Process-Affected Water by Medaka (Oryzias latipes): Importance of Partitioning to Phospholipids Kun Zhang, Steve B. Wiseman, John P. Giesy, and Jonathan W. Martin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01354 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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Environmental Science & Technology

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Bioconcentration of Dissolved Organic Compounds from Oil

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Sands Process-Affected Water by Medaka (Oryzias latipes):

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Importance of Partitioning to Phospholipids

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Kun Zhang†, Steve Wiseman‡, John P Giesy‡

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±§∥⊥

, Jonathan W. Martin†*

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† Division of Analytical & Environmental Toxicology, Department of Laboratory Medicine &

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Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3

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‡ Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada

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§ Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK,

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Canada

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±Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East

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Lansing, MI, USA

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∥School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China

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⊥State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

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Nanjing University, Nanjing, People’s Republic of China

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*Corresponding Author

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Phone: 1-780-492-1190; fax: 1-780-492-7800; e-mail: [email protected]; mail: 10-102

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Clinical Sciences Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2G3.

ACS Paragon Plus Environment

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ABSTRACT: The complex mixture of dissolved organics in oil sands process-affected water

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(OSPW) is acutely lethal to fish at environmentally relevant concentrations, but few

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bioconcentration factors (BCFs) have been measured for its many chemical species. Japanese

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medaka (Oryzias latipes) were exposed to 10% OSPW, and measured BCFs were evaluated

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against predicted BCFs from octanol-water distribution ratios (DOW) and phospholipid

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membrane-water distribution ratios (DMW). Two heteroatomic chemical classes detected in

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positive ion mode (SO+, NO+) and one in negative mode (O2-; also known as naphthenic acids)

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had the greatest DMW values, as high as 10,000. Estimates of DMW were similar to and correlated

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with Dow for O+, O2+, SO+ and NO+ chemical species, but for O2- and SO2- species the DMW

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values were much greater than the corresponding Dow, suggesting the importance of electrostatic

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interactions for these ionizable organic acids. Only SO+, NO+ and O2- species were detectable in

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medaka exposed to OSPW, and BCFs for SO+ and NO+ species ranged from 0.6 to 28 L/kg,

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lower than predicted (i.e. 1.4 to 1.7×103 L/kg), possibly due to biotransformation of these

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hydrophobic substances. BCFs of O2- species ranged from 0.7 to 53 L/kg, similar to predicted

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values and indicating that phospholipid partitioning was an important bioconcentration

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mechanism.


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INTRODUCTION

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The oil sands of northern Alberta, Canada, are among the largest proven petroleum

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reserves in the world, and in 2014 production of crude bitumen reached 1.9 million barrels per

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day.1 For each barrel of bitumen produced, greater than one barrel of acutely toxic2 oil sands

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process-affected water (OSPW) is added to growing inventories in tailings ponds.3 The seepage

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of OSPW from tailing ponds to the Athabasca River and its tributaries is of concern, and a

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detailed water chemistry study suggested that OSPW was already reaching the Athabasca River

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through groundwater flow.4 Moreover, future end-pit lakes containing aged or treated OSPW will

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be hydraulically connected to the natural aquatic system.5 Commissioned in late 2012, the first

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full scale end-pit lake is located on the site of Syncrude Canada Ltd. and is called Base-Mine

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Lake. This water is not currently discharged, but it is now important to study the fate and

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behavior of chemicals dissolved in its OSPW.

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The toxicity of OSPW is attributable to a complex mixture of dissolved organic

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compounds derived from bitumen. Applications of ultrahigh-resolution mass spectrometry

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characterization show the presence of thousands of acidic and non-acidic heteroatom-containing

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chemical species in OSPW. Each species is identifiable by its unique empirical chemical formula

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(derived from accurate mass) containing carbon, hydrogen and various combinations of oxygen,

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sulfur or nitrogen heteroatoms (e.g. CxHxOxSxNx). Most of the chemical species are not pure

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compounds, but are themselves complex mixtures of structural isomers that are exceedingly

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difficult to separate and identify.6, 7 For qualitative purposes, the thousands of species can also be

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binned into classes based on their empirical formula and described by numbers of oxygen, sulfur

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or nitrogen, and whether they are detected in negative (-) or positive ionization (+) modes: i.e.

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OX+/-, NOX+/-, SOX+/-, S2OX+/-, NOXS+/-, N+/-, and S +/-8. For example, all simple carboxylic acids


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are detected in negative mode and are binned to the O2- empirical formula class (commonly

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termed naphthenic acids) whereas polar neutral compounds such as dihydroxyl, diketo, or keto-

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hydroxyl species are detected in positive mode and binned to the O2+ class.

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As a first estimate of the bioconcentration potential of organic compounds in OSPW, in

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previous work polydimethylsiloxane (PDMS) coated stir bars were used to estimate the octanol-

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water distribution ratio (DOW), or apparent KOW, for 2114 organic species at native OSPW pH

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(pH=8.4). Using a limited training set of neutral and ionizable compounds, results of this study

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demonstrated the unexpected hydrophobic nature of certain chemical species in OSPW, in

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particular for NO+ and SO+ species whose DOW were as great as 203,000.9 PDMS and octanol are

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used as surrogates for storage of neutral compounds in lipids of biota, thus partitioning from

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water to PDMS or octanol can be used to satisfactorily predict bioconcentration potential for

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neutral organic compounds, including ionizable compounds that are predominantly neutral in test

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solutions.10

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However, for ionizable organic compounds that are predominantly charged at

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physiological pH, the extent of partitioning into biota would likely be underestimated by use of

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partitioning to octanol or PDMS.11 This is because lipids in biota consist not only of neutral

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storage lipids, but also of membrane lipids with polar and ionizable head groups (e.g.

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phospholipids).12 DOW cannot adequately describe the interaction of ionizable compounds with

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such membranes because it neither mimics the ordered structure and anisotropy of biological

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membranes, nor electrostatic interactions between ionizable compounds and zwitterionic head

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groups in bilayer membranes.11-13 For example, it has been reported that at pH 7.4, affinities of

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18 drugs for membranes were greater than affinities predicted based on Dow.14 Moreover,

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phospholipids make up the functional compartments of cells and organelles, thus measurements


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of membrane-water partitioning for chemical species in OSPW would not only allow more

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accurate prediction of bioconcentration, but also of acute toxicity prediction for chemicals acting

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through mechanisms involving membrane disruption.

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The traditional method to assess partitioning of organic compounds from water to

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membranes is equilibrium dialysis with liposomal systems, which is accurate but time-

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consuming. Faster alternative methods have been developed, including retention factors on

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chromatographic stationary phases containing immobilized artificial membranes (IAM)

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consisting of phospholipids.15 Nevertheless, electrostatic interactions with incompletely shielded

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charged surfaces of IAM columns can lead to biased results.16 An emerging rapid method

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involves solid-supported lipid membranes, composed of silica beads coated with a realistic

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phospholipid bilayer. This system has been shown to be capable of high-throughput and accurate

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determination of phospholipid membrane-water distribution ratios. 14

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Most previous in vivo bioconcentration studies with OSPW have focused only on

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naphthenic acids, since these were previously assumed to be major contributors to toxicity of

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OSPW.17, 18 An effects-directed analysis study recently confirmed that naphthenic acids are a

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major cause of the acute toxicity, but that other polar neutral substances containing nitrogen and

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oxygen (NO+ empirical formula class) and sulfur and oxygen (SO+ empirical formula class) also

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contributed.19 Naphthenic acids have been detected in tissues of rainbow trout fingerlings

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exposed to OSPW, and also in tissues of wild fishes collected from rivers near oil sands

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deposits.17, 18 Based on muscle of rainbow trout (Oncorhynchus mykiss) that had been exposed to

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a commercial mixture of naphthenic acids, the BCF of one species of naphthenic acid with 13

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carbons and 3 double bond equivalents (DBE) (C13H22O2) was estimated to be approximately 2

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at pH 8.2, and approximately 4 at pH 7.6.18, 20 However, the DOW for naphthenic acids increases


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with number of carbons and decreases with increasing DBE.9 Thus, data for a single naphthenic

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acid species is insufficiently informative, and a more detailed in vivo study of bioconcentration is

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needed to assess the accumulation potential for the wide range of organic chemical species in

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authentic OSPW.

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In this study, phospholipid membrane-water partitioning constants were measured for all

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detectable OSPW chemical species using solid-supported lipid membranes (TRANSIL), and

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BCFs were predicted based on a QSAR model that combined partitioning to storage lipids and

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phospholipids of fish. These predictions were then compared to empirical BCFs measured in

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juvenile Japanese medaka (Oryzias latipes) exposed to 10% OSPW.

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MATERIALS AND METHODS

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Chemicals and Materials. Acetic acid, formic acid, dichloromethane (DCM), methanol

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acetonitrile (HPLC grade) and water (Optima grade) were purchased from Fisher Scientific (Fair

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Lawn, NJ). Lauric-d23 acid (C11D23CO2H) internal standard was purchased from Sigma-Aldrich

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(St. Louis, MO, USA) and progesterone (2,3,4-13C3C18H30O2, 99%) internal standard was from

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Cambridge Isotope Laboratories, Inc.( Tewksbury, MA, USA). TRANSILXL membrane affinity

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kit was obtained from Sovicell GmbH (Leipzig, Germany).

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OSPW Sample. The OSPW sample was collected July 15, 2014 on the site of Syncrude Canada,

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Ltd. (Fort McMurray, Alberta, Canada) from Base-Mine Lake. This body of water was originally

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an active tailings pond, but is now the first end-pit lake in the Athabasca oil sands region.

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Constructed from OSPW overlying fluid fine tailings, Base Mine Lake was commissioned in

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December 2012, at which time the supply of fresh tailings and OSPW was turned off. Thus,


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OSPW in Base Mine Lake now represents a relatively “aged” sample. The OSPW sample was

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collected by pumping into 20 L high-density polyethylene pails from a floating barge. After

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collection, the OSPW was kept at 4 °C in a walk-in refrigerator until use. The fish exposures

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began September 17th 2014, and membrane partitioning experiments were done with the same

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water in October 2014.

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Stock Preparation for Phospholipid Membrane Distribution Testing. A total of 1 L of OSPW

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was filtered with a 0.45 µm filter (Millipore, Billerica, MA) to remove suspended solids. The pH

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of the filtered

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water was reduced to 2 using 95% H2SO4 and then extracted with 2×200 mL of DCM. The

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extract was combined and evaporated to near dryness with a rotary evaporator (model R-210,

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Buchi, Toronto, Ontario, Canada). The remaining volume was transferred to a 20 mL glass vial

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and taken to full dryness under a gentle stream of nitrogen at room temperature (Turbovap LV,

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Biotage, Charlotte, NC). This was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain a

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1000× stock solution of dissolved organics in OSPW. This concentrated stock was then used to

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prepare 50× stock solutions for the phospholipid partitioning experiments.

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Determination of Phospholipd Membrane Distribution. The phospholipid membrane

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partitioning for each organic species in OSPW was evaluated using the TRANSILXL membrane

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affinity kit which is designed to measure the distribution coefficient of test compounds between

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phosphatidylcholine membranes and aqueous Dulbecco’s phosphate buffered saline (DPBS)

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buffer at pH 7.4. Membranes are supplied as surface-modified silica beads non-covalently coated

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with a phospholipid bilayer (phosphatidylcholine) in aqueous DPBS buffer at pH 7.4. Each kit


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was composed of eight vials with varying contents of total membrane, which was used in

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accordance with the user guide. Briefly, two vials containing only buffer served as controls to

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measure background and any non-specific adsorption. The other six vials contained silica beads

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with total membrane contents ranging from 0.14 to 4.40 µL lipid per mL of buffer. An aliquant of

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10 µL of 50-fold stock solution was added to every vial. After vortex mixing for 15 s, mixtures

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were incubated overnight. To separate membrane and adsorbed content from the medium, vials

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were centrifuged for 10 min at 750 g and supernatant was collected for identification and semi-

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quantification of species by HPLC-Orbitrap.

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Calculation of Phospholipid Membrane-Water Distribution Ratios (DMW). DMW is defined as

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the ratio between the total concentration of a chemical species in the membrane (cl) to the total

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concentration of the same species in buffer (cb) (Equation 1).

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‫ܦ‬ெௐ = ௖ ೗

௖

(1)

್

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The terms cl and cb can be calculated for each vial by use of the mass balance (Equation 2).

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݊௧ = ܿ௕ × ܸ௕ + ܿ௟ × ܸ௟

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where nt is total mass of the species, and Vb and Vl are the volumes of buffer and total lipid

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membrane in every vial, respectively. Equation 2 can be rearranged (Equation 3) so that DMW can

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be determined from the slope of a line by plotting the ratio of nt divided by cb as a function of Vl

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for each vial.

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௡೟ ௖್

(2)

௖

= ௖ ೗ × ܸ௟ + ܸ௕ = ‫ܦ‬ெௐ × ܸ௟ + ܸ௕

(3)

್

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The TRANSILXL membrane affinity kit is generally recommended for measuring log

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DMW values >2 because compounds with lower log DMW typically yield supernatant

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concentrations in the assay that can deviate only marginally from control signals. However, in


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the current study, statistically significant log DMW values could be determined down to 1.2 with

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“good” Transil quality index scores (i.e. TQI >7). Thus, in the current study, log DMW of 1.2 was

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an operationally defined limit of detection for significant membrane binding.

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As previously noted, most chemical species in OSPW are mixtures of structural isomers,

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and the current DMW measurements, as well as previous DOW measurements, are weighted

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averages for all components sharing the same chemical formula.9 Assuming that all isomers of a

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chemical species are not equivalent (i.e. have different true DMW or DOW), it must be

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acknowledged that there is no single distribution coefficient that can link aqueous concentration

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of a mixture to the corresponding concentration of the same mixture in a hydrophobic phase (e.g.

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storage lipid, or phospholipid).21 This is because it can depend on variable composition of the

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mixture, and to some extent on the ratio of water:hydrophobic phase. Nevertheless, for the

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current measurements of DMW the water:phospholipid ratio was varied and resulted in straight

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line relationships, thus the impact of the latter limitation is likely only minor here.

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Bioconcentration of Chemical Species by Fish. Juvenile medaka were selected from a culture

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tank and randomly placed into 30-L glass tanks at 25± 1 oC. The experimental setup consisted of

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the following treatment tanks, with each tank containing 9 fish: 2 tanks of control (i.e. laboratory

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dilution water, pH 8.1) female medaka, 2 tanks of control male medaka, 3 tanks of female

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medaka exposed to a 10-fold dilution of OSPW (pH 8.24±0.12) and 3 tanks of male medaka

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exposed to a 10-fold dilution of OSPW. To remove suspended solids, OSPW was filtered

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through a 0.45 µm glass filter paper (Millipore, Billerica, MA) before dilution. A semi-static

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experimental condition was used, with 50% of test water (15 L) renewed every 24 h in each tank.

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Exposure water pH (mean = 8.24±0.12), which is consistent with the pH in the Athabasca River


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system (pH 8.0-8.5),22 and dissolved oxygen concentration (>70%) were monitored daily in each

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tank. The photoperiod was 16:8 h light/dark, and fish were fed approximately 30 mg flake food

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per tank per day.

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A single fish from each exposure tank and each control tank was collected at each

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predetermined time during the uptake phase of the experiment: 6, 24, 72, 144, and 288 h.

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Samples of water (1 L) were collected below the surface of each tank into polypropylene bottles

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at the same time that fish were sampled. At 288 h, the remaining fish were transferred to tanks

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containing clean fresh water, in which they were allowed to depurate. Extra care was taken to

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reduce contamination of water during the depuration phase rinsing fish for 10 min in an

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intermediate 10 L tank containing clean water before being transferred to the tanks containing

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clean water where depuration would be followed. During the depuration phase, one fish from

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each tank and each control tank were sampled at each time: 6, 24, 72, and 144 h. Samples of

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water (1 L) were also collected at 6 and 72 h of the depuration phase. During the entire

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experiment, no fish died in any of the exposures.

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Studies were performed in the Aquatic Toxicology Research Facility (ATRF) at the

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University of Saskatchewan and medaka were from a culture maintained in the ATRF. All

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handling of fish and exposures were in accordance with protocols approved by the University of

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Saskatchewan Committee on Animal Care and Supply and Animal Research Ethics Board

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(UCACS-AREB; # 20090108)

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Identification and Semi-Quantification of Chemical Species in Fish and Water. After

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spiking with recovery standards, whole fish were homogenized in 1.5 mL plastic (polypropylene

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copolymer) centrifuge tubes using a TissueLyser II ball mill with stainless steel beads (Qiagen,


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Hilden, Germany). Homogenates were first extracted with 2×1 mL DCM and then extracted

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twice again with 1 mL DCM after adding 50 µL formic acid to adjust the pH. The extract was

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dried under gentle high-purity nitrogen gas, and taken up in 400 µL of 1:3 (v:v)

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water:acetonitrile. Solutions were centrifuged at 16089 g for 10 min to pellet the protein, and

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supernatants were transferred to hybrid SPE-precipitation cartridges (for phospholipid removal)

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with the zirconia coated silica as stationary phase (500 mg, 6 mL, Sigma-Aldrich, St. Louis, MO,

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USA) preconditioned with 6 mL 1% formic acid in acetonitrile. Cartridges were then eluted with

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6 mL formic acid in acetonitrile, then 6 mL 1% ammonium formate in methanol. Extracts were

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dried under gentle nitrogen, taken up in 200 µL of acetonitrile and analyzed by use of HPLC-

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Orbitrap after filtration with 0.22 µm syringe filters (Millipore, Billerica, MA, USA). Water

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samples from the fish tanks were spiked with internal standards and analyzed directly by HPLC-

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Orbitrap after filtration with 0.22 µm syringe filters. All fish sampled were extracted and

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analyzed individually, and all analytes detected in samples were blank subtracted by comparison

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to control fish.

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Instrumental Analysis. An Accela HPLC (Thermo Fisher Scientific, San Jose, CA, USA)

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coupled to an Orbitrap Elite, hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA,

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USA) was used for identification and quantification of species. Chromatographic separation was

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carried out on an XSELECT CSH C18 XP column (130 Å, 2.5 µm, 3 mm×150 mm) (Waters,

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Milford, MA, USA) at 25 °C with a flow rate of 0.5 mL/min. An injection volume of 20 µL was

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used for supernatant sampled from TRANSILXL membrane affinity kit, 10 µL for extracts of

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medaka and 50 µL was used for samples of water. Mobile phases consisted of (A) 0.1% acetic

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acid in water, and (B) 100% methanol, and the gradient flow was 5% B for 1 min, followed by a


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linear gradient ramp to 90% B at 9 min, to 99% B over 5 min, and returning to 5% B in 1 min

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followed by a 4 min hold prior to the next injection.

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The Orbitrap was operated with an electrospray (ESI) source operating in either positive

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or negative mode; separate injections of samples were made for each mode. Needle voltage was

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set at +/− 5 kV, while the sheath, aux, and sweep gas flows were set to 40 (arbitrary unit), 5

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(arbitrary unit) and 2 (arbitrary unit), respectively. Capillary temperature was 300 °C.

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Acquisition was performed in scan mode from m/z 100 to 500, with m/z resolving power set to a

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nominal value of 240,000. Mass calibration and tuning was done externally by direct infusion of

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Pierce ESI negative ion calibration solution, or Pierce LTQ Velos ESI positive ion calibration

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solution (Thermo Fisher Scientific, Rockford, USA). The negative ion mode calibration solution

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covered a mass range from m/z 265 to m/z 1880 and the positive ion mode calibration solution

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covered a mass range from m/z 74 to m/z 1822, and both confirmed good m/z accuracy (

Bioconcentration of Dissolved Organic Compounds from Oil Sands

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