Oil Sands Derived Naphthenic Acids Are Oxidative Uncouplers and

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Ecotoxicology and Human Environmental Health

Oil Sands-Derived Naphthenic Acids are Oxidative Uncouplers and Impair Electron Transport in Isolated Mitochondria Kate Rundle, Mahmoud Sharaf, Don Stevens, Collins Kamunde, and Michael R. Van Den Heuvel Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02638 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Oil Sands-Derived Naphthenic Acids are Oxidative Uncouplers and Impair Electron Transport in Isolated Mitochondria

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Kate I. Rundle1, Mahmoud S. Sharaf2, Don Stevens2, Collins Kamunde2, and Michael R. van den Heuvel1,3* 1

Canadian Rivers Institute, Department of Biomedical Science, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada, C1A 4P3 2

Department of Biomedical Science, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada, C1A 4P3 3

Canadian Rivers Institute, Department of Biology, University of Prince Edward Island, Charlottetown, Canada, C1A 4P3

* Author to whom correspondence should be addressed Department of Biology University of Prince Edward Island 550 University Avenue Charlottetown, Canada C1A 4P3 ph: 1-902-388-0895 fax: 1-902-566-0740 [email protected]


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ABSTRACT

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cause numerous acute and chronic effects in fishes. However, the mechanism of toxicity underlying these

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effects has not been fully elucidated. Due to their carboxylic acid moiety and the reported disruption of

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cellular bioenergetics by similar structures, we hypothesized that NAs would uncouple mitochondrial

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respiration with the resultant production of reactive oxygen species (ROS). Naphthenic acids were

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extracted and purified from 17-year-old oil sands tailings waters yielding an extract of 99% carboxylic

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acids with 90% fitting the classical O2-NA definition. Mitochondria were isolated from rainbow trout

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liver and exposed to different concentrations of NAs. Mitochondrial respiration, membrane potential, and

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ROS emission were measured using the Oroboros fluorespirometry system. Additionally, mitochondrial

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ROS emission and membrane potential were evaluated with real-time flow cytometry. Results showed

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NAs uncoupled oxidative phosphorylation, inhibited respiration, and increased ROS emission. The

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effective concentration (EC50) and inhibition concentration (IC50) values for the endpoints measured

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ranged from 21.0 to 157.8 mg/L, concentrations similar to tailings waters. For the same endpoints,

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EC10/IC10 values ranged from 11.8 to 66.7 mg/L, approaching concentrations found in the environment.

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These data unveil mechanisms underlying effects of NAs that may contribute to adverse effects on

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organisms in the environment.

Naphthenic acids (NAs) are predominant compounds in oil sands-influenced waters. These acids

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Keywords: oil sands, naphthenic acid, mitochondria, ROS, membrane potential

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INTRODUCTION The Athabasca oil sands in Alberta, Canada are among the largest oil deposits in the world. In

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2016, approximately 2.5 million barrels of crude oil were being produced every day and an estimated 165

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billion barrels remained in oil reserves.1 Most of Alberta's crude oil is produced using the Clarke hot

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water extraction method, where large volumes of hot water and a caustic soda (NaOH) are added to

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promote the separation of bitumen that is further processed into synthetic crude oil.2,3 Large volumes of

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waste water, commonly referred to as oil sands process-affected water (OSPW) are produced during this

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process and are stored in tailings ponds. At the end of 2013, these tailings covered an area of

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approximately 220 km2 and housed over 975 million liters of OSPW.1,2 OSPW contains residual bitumen,

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dissolved salts, minerals, and a complex mixture of inorganic and organic compounds.2,4

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Naphthenic acids (NAs) make up a large proportion of the organic compounds present in OSPW

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and have been implicated for much of the observed OSPW toxicity of various species.2,4 These acids are a

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mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids that are natural components of

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petroleum.4,5 Classical NAs refer to compounds that follow the general formula CnH2n+ZO24, where n is the

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carbon number and z is the hydrogen deficiency, though this definition has been broadened to include

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carboxylic acids with additional oxygens and compounds containing N or S, the definition we used

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herein. Organisms exposed to NAs extracted from OSPW exhibit inhibition of growth;6,7 disruption of the

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endocrine system and reproductive physiology;8 increased developmental abnormalities;9,10 and exposure

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is correlated with opportunistic disease in fishes.11 NAs are considered bioavailable and have been

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suggested to act via narcosis or physical disruption of the cell membrane.12,13 However, because of the

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complexity of NAs, a model of multiple mechanisms of toxic action also has been suggested.7,14

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While no specific mechanistic studies have been conducted on NAs, their carboxylic acid moiety

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suggests that NAs could have the potential to disrupt mitochondrial membrane permeability and

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consequently uncouple oxidative phosphorylation (OXPHOS). Supporting this theme, another group of

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carboxylic acids prevalent in pulp and paper effluents, resin acids, were found to alter mitochondrial

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membrane integrity, uncouple OXPHOS, and decrease ATP production.15,16 Uncoupling of OXPHOS is


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the disconnection of two reactions. The first reaction is the movement of electrons through a series of

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protein complexes in the inner mitochondrial membrane, referred to as the electron transport system

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(ETS), causing the creation of a hydrogen ion gradient known as the proton motive force. The second is a

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phosphorylation event by ATP synthase powered by the proton motive force that is responsible for

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converting ADP to ATP.17 When OXPHOS is uncoupled, the proton motive force is dissipated without

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being used to produce ATP.17 While mild uncoupling has been associated with a decrease in production of

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reactive oxygen species (ROS), uncoupling that occurs in mitochondria where the ETS has been inhibited

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increases production of ROS.18 Reactive oxygen species play a role in cell signaling,19 but in excess, can

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impose oxidative stress, leading to the release of cytochrome c from mitochondria resulting in apoptosis.20

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We hypothesized that NAs can uncouple OXPHOS and alter the overall mitochondrial function.

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This hypothesis was tested by examining the effects of NAs on the respiration of mitochondria isolated

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from rainbow trout liver. Based on this, we predicted that NAs would uncouple OXPHOS and inhibit the

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ETS, and that these effects would lead to oxidative stress. These predictions were tested using NAs

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extracted from aged OSPW using acid precipitation and by measuring oxygen consumption, ROS

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emission, and membrane potential of mitochondria isolated from rainbow trout liver using respirometry

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and real-time flow cytometry.

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MATERIALS AND METHODS Chemicals and Reagents Sucrose, potassium phosphate, EGTA, bovine serum albumin (BSA), aprotinin, malate,

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glutamate, adenosine diphosphate (ADP), and hydrogen peroxide were supplied by Sigma Aldrich

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(Oakville, ON). Tris-hydrogen chloride was from EMD Chemicals Inc. (Gibbstown, NJ, USA). The

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fluorescent dyes, Amplex red and H2DCFDA, as well as horseradish peroxidase (HRP) were from

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Invitrogen (Oregon, USA). The membrane potential indicator lipophilic cation tetraphenylphosphonium

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chloride (TPP+), was from Alfa Aesar (Heysham, England) and the fluorescent dye JC-1 was from

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Biotium Inc. (California, USA). Sulfuric acid and diethylaminoethyl (DEAE) cellulose used in the NA



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extraction were from Sigma Aldrich (Oakville, ON) and hydrochloric acid, dichloromethane (DCM),

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ammonium hydroxide, and acetonitrile were supplied by Caledon Laboratories Chemicals (Georgetown,

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ON). Oligomycin-A, carbonyl cayanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin-A

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were from Sigma Aldrich (Oakville, ON).

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Naphthenic Acid Extraction

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Naphthenic acids were extracted from 17-year-old tailings water from Syncrude Canada Ltd.'s

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experimental pond 10 based on previously described methods.21,22 In the present study, the water used

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was from the same source sample used in previous studies22,23,33 Briefly, 4,000 L of tailings water were

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acidified using sulfuric acid and the precipitate was removed, concentrated by centrifugation (17,000 × g)

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and dissolved in 0.1 M NaOH. The sample was then centrifuged again at 17,000 × g to remove any clay

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or insoluble material and the supernatant was vacuum filtered through cleaned DEAE cellulose to remove

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any humic material. The filtered supernatant was extracted using DCM in glass liquid-liquid extractors

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for 72 h to remove neutral compounds. The sample was again acidified with sulfuric acid and centrifuged

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to obtain NAs. The NA extract was washed several times with distilled water to remove residual sulfuric

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acid and was then freeze dried to obtain a solid material. All concentrations in this study are based on

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weighing out the dried solid material. As there are no analytical standards for NAs, this represents the

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most accurate concentration available.

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Naphthenic Acid Characterization

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Mass spectrometry to quantify the mixture was performed by direct injection of a solution of the

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extracted naphthenic acids into a Thermo Velos Orbitrap high-resolution mass spectrometer equipped

128

with an electrospray ionization interface in negative mode. Naphthenic acids dissolved in a 10 mM 1:1

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NH4OH:acetonitrile solution were introduced into the mass spectrometer by direct infusion at 3 µL/min.

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Ion intensity data at 100,000 mass resolution were evaluated against the predicted molecular weights of

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naphthenic acids according to the formula CnH2n+ZO2. Further chemical characterization using NMR and

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IR spectrometry was previously conducted on an earlier sample of NAs extracted by identical methods


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and is consistent with a pure mixture of carboxylic acids.23 The characteristics reflected in those analyses

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would be expected to be similar to the sample used.

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A sample of naphthenic acid extract was sent to Galbraith Laboratories (Knoxville, TN) for

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analysis of percent carbon, hydrogen, nitrogen, sulfur, and oxygen. Briefly, carbon, nitrogen, hydrogen,

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and sulfur were determined by combustion of the sample in pure oxygen at high temperature (920 – 1350

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°C) and the combustion products CO2, H2O, N2, and SO2 were automatically analyzed in a self-

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integrating, steady state thermal conductivity analyzer (PerkinElmer 2400 Series II CHNS/O

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Analyzer).24,25 Oxygen percentage was determined by pyrolysis of the sample in a helium atmosphere.

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After pyrolysis, N, H, and CO are produced and passed through an absorption filter where halogenated

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compounds were retained and N, H, and CO were separated via a chromatographic column. The amount

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of CO was automatically analyzed the PerkinElmer 2400 Series II CHNS/O Analyzer.26 Detection limits

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for C, H, N, and O were 0.5% and 0.1% for sulfur.

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Mitochondria Isolation

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Fish were obtained from Ocean Farms (Brookvale, PE) and were approximately 1 year old. They

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were maintained in a 250 L tank in the Atlantic Veterinary College Aquatics Facility and fed 1% of their

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weight daily. Mitochondria were isolated from rainbow trout (Oncorhynchus mykiss; mean fish weight

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135±11 g) livers using differential centrifugation.27 Mitochondria were re-suspended in mitochondrial

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respiration buffer (MRB: 10 mM Tris HCl, 25 mM KH2PO4, 100 mM KCl, 1 mg/mL BSA, 2 mg/mL

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aprotinin, pH 7.3) and immediately used for experiments. Protein concentrations of the mitochondrial

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suspensions were measured by the method of Bradford protein assay28 using BSA as a standard.

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Measurement of Mitochondrial Respiration, H2O2 Emission, and Mitochondrial Membrane

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Potential (∆Ψmt)

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Oxygen consumption, H2O2 emission (representing a portion of ROS emission), and

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mitochondrial membrane potential (∆Ψmt) were measured simultaneously using the Oxygraph-2k

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fluorespirometer (Oroboros Instruments, Innsbruck, Austria) as described previously.29 Briefly, oxygen

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concentration and changes in concentration per mg of mitochondrial protein were recorded in real-time



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(continuous measurement). The ∆Ψmt was obtained by measuring TPP+ uptake by mitochondria using the

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TPP+ ion selective electrode with respect to a reference electrode. Calibration with a known TPP+

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concentration was done at the beginning of each experiment. Lastly, the H2O2 concentration and rate of

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emission were measured using the Amplex red - HRP assay. Amplex red reacts with H2O2 in a 1:1

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stoichiometric reaction giving rise to the fluorescent product resorufin, which is measured by the

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Oroboros. A calibration curve was obtained using known concentrations (0-0.45 µM) of H2O2 at the

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beginning of each experiment.

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Isolated mitochondria (0.75 mg protein/mL) were first added to the Oroboros chambers, followed

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by the substrates for ETS complex I, malate and glutamate. State 3 respiration, where ATP is actively

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being produced, was initiated by the addition of ADP (6.25 µM). After all ADP was depleted,

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mitochondria entered state 4 respiration, when no ATP production takes place. To investigate the effects

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of NAs on different mitochondrial energetic states, dose response curves consisting of concentrations

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ranging from 20 to 320 mg/L NA were obtained during state 3 and then again during state 4 respiration.

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Here, NAs were added sequentially in 20 or 40 mg/L doses during each run to achieve predetermined

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cumulative target concentrations. Sequential addition was required in order to complete a full dose

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response will within the 4 h viability of mitochondria and to limit the number of live fish required. At

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least three biological replicates for each state were performed.

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Real-time Flow Cytometry Measurement of ∆Ψmt and ROS Emission

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Real time ∆Ψmt and mitochondrial oxidation state (representing a portion of ROS emission)

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measurements were done using fluorescent dyes JC-1 and H2DCFDA, respectively, as previously

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described.27 Isolated mitochondria were added to MRB containing 5 mM of malate and 5 mM of

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glutamate (state 2 respiration, high membrane potential, low respiration most comparable to state 4).

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During ∆Ψmt measurement, mitochondria were incubated with JC-1 for 10 min prior to beginning of the

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experiment. During ROS emission measurements, H2DCFDA was added approximately 200 s after

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beginning the experiment. A dose-response assessment was conducted using the same NA concentrations

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as in the Oroboros experiments. Doses of NA (20 or 40 mg/L) were added approximately 160 s apart and


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a sample size of 3 was obtained for each parameter. Data were gated and presented using Flowjo 7.6.5

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(TreeStar Inc., Oregon, USA). Three biological replicates for each parameter were performed.

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Statistical Analysis

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In order to obtain more consistent data for curve fitting, numbers were converted to a percentage

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of the control, which was represented by state 3, state 4, or state 2 depending on the measurement. The

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control measurement was used as 100% and subsequent measurements were converted to a percent by

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dividing the measured value by the control and multiplying by 100. Dose-response curves were prepared

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using Statistica v.13 (Dell, Oklahoma, USA) using a four-parameter logistic equation (three parameters

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where the effect variable started at zero) using a Quasi-Newton estimation method in order to determine

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the IC50 (inhibition concentration) or EC50 (effective concentration) for all endpoints. Curve fitting

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employed a standard loss function, (observed-predicted)2. Estimates of IC10 or EC10 were calculated using

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the equation of the curve. Curves were estimated for every replicate and the mean and 95% confidence

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intervals calculated from the three replicates of each endpoint.

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RESULTS AND DISCUSSION

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Characterization of Naphthenic Acid Extract

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Examination of NAs by negative ion mode direct injection HRMS revealed that our sample was

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99% carboxylic acids with 90.4% of the ion intensity conforming to the NA structural formula,

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CnH2n+ZO2. The most abundant ions had either 15 (30%) or 16 carbons (28%) and Z-values of -4 (40%) or

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-6 (34%) (Figure 1). Other studies23,30 have observed similar carbon numbers and the Z values as found in

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the sample examined herein. In the present study and previous studies, over 90% of compounds were in

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the O2 category and fit the classical NA formula.23,30 This was expected as the extraction method used in

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this study is specific to extracting amphipathic NAs that fit the classical NA formula.21 It is worth noting

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that when comparing purified NAs with those directly extracted from the same tailings in a previous

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study, the authors found that low and high molecular weight NAs tend to be depleted in the present



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extraction process due to the multiple acid precipitation steps and the dichloromethane solvent extraction

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steps, respectively.21,22

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While the majority of negative ions in the extract fit within the classical NA formula, NA

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compounds outside of the classical formula were also found. Of the ions identified, 2.0 % were O3

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structures that are potentially carboxylic acids with an alcohol (or ether) functional group. A total of 7.0

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% of the mixture was accounted for by O4 compounds that are likely dominated by compounds containing

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two carboxylic acid groups. Based on this, the mixture is estimated to be 99.4% carboxylic acids at a

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minimum, but may be higher due to traces of O5 compounds also identified in the mixture. Oxidized O3-,

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O4-, and even O5-NAs have been reported in various OSPW mixtures.31,32 However, these compounds are

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more water soluble than O2-NAs and therefore are less likely to precipitate out during extraction.22,23 A

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study examining the same tailings water that was directly extracted using C18 methods found that O3 and

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O4 compound accounted for 40% of the mixture.33

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Elemental analysis of our extract showed that 70.4%, 18.4%, and 10.6% of the sample by mass

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was C, O, and H, respectively. Nitrogen was not detectable in the mixture (< 0.5%), and sulfur was

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present at 0.25% indicating a relatively pure hydrocarbon mixture. Accounting for the O2, O3, and O4

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compounds, the elemental analysis suggested that the average carbon number was 11.14, which is lower

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than the weighted average carbon number from the mass spectrometry analysis of 15.5. This may have

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occurred if the sample was not completely dry at the time of analysis. An elemental analysis of seven NA

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extracts from a previous study showed a range of 59-71% carbon, 15-20% oxygen, 6-9% hydrogen, 0-

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0.7% nitrogen, and 1-6% sulfur.34 This analysis is considered consistent with our sample because previous

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studies have documented that OSPW and NA extract samples from different areas have varying chemical

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compositions and individual acid proportions.14,35 Overall, the results from all of the chemical analyses

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suggest that the compound extracted in the present study is a NA mixture with > 99% purity this mixture

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could be reliably used to estimate effects of OSPW-derived NAs on energy metabolism.

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NAs Uncouple Oxidative Phosphorylation


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An example of the effects of cumulative additions of NAs on mitochondrial oxygen consumption,

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∆Ψmt, and ROS emission are shown in Figure 2, where tracings of each of the three parameters during a

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representative experiment of state 4 respiration are overlaid. There is a period of instability for

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approximately 1 min, particularly for the ROS endpoint as seen by large spikes, and all readings were

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taken after the system returned to stability (2 min). The time of addition of mitochondria, necessary

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substrates for respiration, and sequential doses of NAs are indicated. After the addition of substrates, the

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mitochondria enter state 3 respiration, where ADP is being converted to ATP. The resultant movement of

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protons by the ETS to drive ATP synthase results in an increase of mitochondrial respiration (red line) as

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complex IV of the electron transport chain consumes oxygen to create proton motive force (blue line).

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When all added ADP is converted to ATP, the mitochondria enters into state 4, or a resting state, which is

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indicated by the reduction in respiration, and increase in ∆Ψm to a steady state. In state 4, the sequential

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doses of NA reduce ∆Ψm and this reduction in ∆Ψm triggers an increase in respiration to compensate.

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However, this was not consistently observed in the present study at higher doses of NA, there is an

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ultimate decrease in respiration as the electron transport chain presumably ceases to function at a level

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sufficient to sustain an increase respiration. Similarly, there is an increase in ROS emission (green lines,

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large spikes are only a transient effect after NA addition), but at higher doses, ROS emissions also

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decrease.

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To demonstrate the functionality of the system to predict mitochondrial dysfunction and provide

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examples of how effect at different levels of the ETS can be detected, a number of reference toxicants

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were used, including oligomycin, an inhibitor of ATP synthase, carbonyl cayanide-4-

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(trifluoromethoxy)phenylhydrazone (FCCP), a model uncoupler of OXPHOS, and antimycin-A, which

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binds to cytochrome C and inhibits electron transport (Figure S1). Oligomycin completely eliminates

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respiration (as ATP synthase is the only step that uses oxygen) with an increase in ∆Ψmt as ATP synthase

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is not utilizing the proton motive force, and causes no change in the ROS emission. FCCP decreases ∆Ψmt

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as it uncouples the proton gradient with an increase in respiration to compensate, and a slight increase in

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ROS. Antimycin-A almost completely stops respiration as it impairs the ETS with only a slight drop in



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∆Ψmt as the upstream complexes are still active. The subsequent back up of electrons causes substantive

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ROS generation.

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Dose-effect curves for all experiments combined show that successive additions of NAs resulted

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in consistent decreases in respiration during state 3 with almost complete cessation of respiration at the

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highest dose (ATP being produced; Figure 3a). When respiration is already low during state 4 there was a

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trend towards a 25% increase in respiration, however the effect was not consistent in all replicates as

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indicated by the high variability in the effect, the IC50 values were not calculated (ATP not being

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produced; Figure 3b). NAs showed a trend to dissipate ∆Ψmt by less than 10% during state 3 when

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membrane potential is already relatively low as compared to state 4 (ATP being produced; Figure 4a).

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However, this effect was not consistent and IC50 values could not be calculated for all replicates. During

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state 4, there was a consistent 20% drop in ∆Ψmt (ATP not being produced; Figure 4b). Real-time flow

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cytometry measurement was used to validate these results and a stronger (50%) dose-dependent

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dissipation of ∆Ψmt was observed (Figure 4c, d), though the effective concentrations using the Oroboros

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and flow cytometry methods were similar (Table 1). The uncoupling of mitochondrial respiration and

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dissipation of ∆Ψmt that were observed using two independent methods show that NAs can act as

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mitochondrial uncouplers. The implications of sequential additions of NAs were evaluated by comparison

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to the sequential addition of solvent controls (Figures S2 and S3) demonstrating the over time the system

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remained relatively stable (< 5% drift) with sequential solvent additions. Furthermore, the effects of only

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a single dose, equivalent to two sequential additions, of NA was investigated, and were found to have

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similar effects as sequential doses (Figure S1).

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The respective result for ∆Ψmt and respiration in state 3 and state 4 would be expected in the case

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of a mitochondrial uncoupler that effects both the proton gradient, and the ETS. In state 3 respiration,

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oxygen is being consumed at its maximum rate to maintain membrane potential to drive ATP synthesis,

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the dissipation of ∆Ψmt alone should not result in a decrease in respiration. Thus, these results indicate that

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the ETS is being inhibited independently of the reduction in ∆Ψmt. As there was almost complete

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reduction of respiration, and only partial reduction of ∆Ψmt, this also suggests that the inhibition of the


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ETS may be a more important mechanism. In contrast, in state 4, membrane potential is high, and oxygen

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consumption is low, thus dissipation of the proton gradient by NAs causes an increased consumption of

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oxygen that is indicative of the ETS attempting to restore the gradient. While disruption of the ETS may

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be occurring in state 4, it does not become apparent until higher doses of NAs where respiration comes to

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a plateau, and then appears to drop off (Figure 3b).

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The observed uncoupling effect of NAs are likely due to the ability of NAs to act as

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protonophores in which they are protonated in the intermembrane space and transported across the inner

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mitochondrial membrane into the mitochondrial matrix where they release the proton in the alkaline

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environment, thus dissipating the proton gradient (i.e., uncoupling OXPHOS).36,37 While and impact on

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the ETS is evident from the decrease in state 3 respiration, the precise cause of this parallel effect is not

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clear. Since there are multiple steps in the ETS, which are dependent on previous reactions, NAs could be

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having inhibitory effects at any of these potential steps that drive the ETS, including direct interference

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with ETS complexes.

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NAs Stimulate ROS Emission

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During both state 3 and 4 respiration dose responses using the Oroboros system, H2O2

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concentration increased steadily for the first half of the dose range, followed by a general plateau or

303

decrease (Figure 5a, b). Real-time flow cytometry measurements were again used to validate the results

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and a similar trend was observed (Figure 5c, d). The calculated EC50 and EC10 values were similar

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between both energetic states and measurement techniques, ranging from 11.8 to 36.7 mg/L (Table 1).

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The finding of stimulation of ROS emission by NAs measured by both Oroboros (H2O2 emissions) and

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flow cytometry (mitochondrial oxidation state) is consistent with earlier reports that OSPW induced the

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expression of genes related to oxidative stress in multiple species.38,39 The increased ROS emission

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measured lends mechanistic support to an earlier report that correlated oxidative stress due to OSPW

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exposure with developmental malformations observed in fathead minnow (Pimephales promelas)

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embryos.40 Thus, mitochondria, a major source of cellular ROS, may be an important target for NAs.7,39



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The underlying cause of increased ROS production by NAs is unknown. Generally, toxicants

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have been shown to increase ROS production by interacting with the ETS via two different mechanisms.41

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The first is by blocking electron transport thus increasing the reduction level of the ETS complexes

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upstream of the inhibition.41 This results in increased leakage of electrons from the ETS complexes with

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partial reduction of oxygen to form ROS.41,42 The second is that a toxicant may accept an electron from

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the ETS complexes and transfer it to molecular oxygen. This mechanism stimulates ROS production

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without inhibiting the ETS.41 We propose the first explanation is the most plausible mechanism of

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increased ROS emission by NAs due to the observed inhibition of state 3 respiration (Figure 3a, 4a). The

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inhibition of state 3 is indicated by the impairment of the ETS43, which is associated with an immediate

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increase in ROS and dissipation of ∆Ψmt after addition of NAs. ROS emissions were the most sensitive

322

endpoints with EC50/IC50 values 3 to 6-fold lower than the other endpoints. In addition 95% confidence

323

intervals for the median effective concentration for state 3 respiration and ROS generation using the

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Oroboros did not overlap, suggesting that ROS production can be detected well before effects on

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OXPHOS are apparent. The ROS emission increased up to a NA concentration of ~ 160 mg/L on the

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Oroboros system and ~ 120 mg/L during real-time flow cytometry, after which there was a drop in the

327

rate of ROS emission. The drop of ROS emission is likely due to impaired mitochondria function at

328

higher doses of NAs shown by almost complete inhibition of state 3 respiration by the 240 mg NA/L dose

329

(Figure 3a, 5a).

330

Environmental Relevance

331

Environmental concentrations of NAs are typically much lower than the EC50/IC50 values we

332

calculated, but EC10/IC10 estimates, particularly for ROS emission approach the high end of the range of

333

concentrations found in the receiving environment. Any comparison to environmental concentrations is

334

confounded by the many methods to determine such concentrations, including what is used as a standard,

335

as well as what the investigators consider to be NAs. In the present study, we were able to weigh

336

quantities of the NA mixture, which represented accurate concentrations given the mixture's high purity

337

as carboxylic acids. While our mixture consists of mostly O2-NAs, we believe toxicity of the source


Page 15 of 32


Rundle et al. 14

338

material would be relatively unchanged based on another study from our laboratory that exposed isolated

339

mitochondria to that source material (unpublished data). Recent estimates of surface water NA

340

concentrations using Fourier-transform infrared (FTIR) spectroscopy methods in local lakes and

341

experimental ponds, including Beaver Creek, Demonstration Pond, Bison Pond, Mildred Lake, and

342

Horizon Lake, showed NA levels ranging from < 1 to 14 mg/L.35 The FTIR method overestimates

343

measured total carboxylic acids as compared to HMRS by a factor of two.33 A study conducted on

344

different sites, Muskeg River and Jackpine Mines, found surface water concentrations > 10 mg/L for O2

345

NAs and groundwater levels in the order of 5 mg/L using ultra performance liquid chromatography-time

346

of flight-mass spectrometry (UPLC-TOF-MS) methods. Although given different standard mixtures, it is

347

difficult to compare to HRMS methods compared by our laboratory.44 Levels of NAs in the Athabasca

348

River using liquid chromatography-high resolution mass spectrometry (LC-HRMS) techniques showed

349

concentration of total NA (O2-species only, thus an under estimate of all NAs) ranging from 0.7 to 52

350

µg/L.45 In general, concentrations in most affected waters are well within the EC10/IC10 values determined

351

in this study suggesting they may cause at least 10% changes in mitochondrial function.

352

The effective concentration of NAs experienced by aquatic organism can also be expected to be

353

dictated by the potential of NAs to bioconcentrate. Partitioning of oil sands organic compounds in

354

polydimethylsiloxane showed that O2-species have apparent KOW (pH 8.4) that ranging from negligible to

355

as high as 100 46; with KOW increasing with carbon number and decreasing with double bond equivalents.

356

However, as most NAs are present in their naphthenate form at environmental pH (> 7.5),

357

bioaccumulation is likely to be low. In a study that exposed rainbow trout to 3 mg/L at pH 8.2,

358

bioaccumulation factor (BAF) was reported as ~2.47 However, that study was done with commercially

359

available NAs, known to be rich in free fatty acids, unlike the aged samples utilized herein that may have

360

higher BAFs. However, yellow perch (Perca flavescens) exposed to 13 mg/L NA (by FTIR) aged tailings

361

water in an experimental pond were found to contain non-detectable levels of NAs (< 1 mg/kg) in muscle

362

tissue.48 Those fish accumulated and excreted NAs as indicated by biliary NA levels as high as 200

363

mg/L.48 There is support for bioaccumulation of NAs in fish from the Athabasca River (where levels of



Page 16 of 32

Rundle et al. 15

364

NAs are much lower) that had tissue concentrations ranging from non-detectable to as high as 2.8 mg/kg.

365

NAs tend to concentrate at the highest levels in gills and liver49, but data on compound-specific BAF in

366

fish are sparse and inconsistent. There has not been any work regarding the subcellular partitioning of

367

NAs and further work is needed to estimate and characterize the concentration of NAs that could be

368

present in the mitochondria of living organisms.

369

The question of how much reduction in ATP production would result in cellular or organismal

370

impacts is also critical to the environmental relevance of the present results. Cells must produce ATP at

371

the same rate they consume it. Therefore, a cell that is producing 10% less ATP than the cell demands can

372

deplete its ATP within two minutes.50 Typically, full or partial uncoupling of OXPHOS and the

373

dissipation of ∆Ψmt decrease or prevent the production of ATP. Cytotoxicity studies using mouse and

374

human cells have reported rapid (< 48 h) partial cell death occurs with 30% reduction in ATP levels51 and

375

greater than 50% cell death occurs when ATP levels decline by half.51,52 At the organismal level, this may

376

limit available energy for the organisms to carry out normal physiological processes, possibly resulting in,

377

or at least contributing to, the toxic effects reported in various studies. The NA mixture in the present

378

study was observed to cause complete mortality in threespine stickleback (Gasterosteus aculeatus) at 15

379

mg/L, comparable to EC10/IC10 values reported here (authors unpublished data), suggesting that energetic

380

mechanisms may play a factor in mortality. While caution must be taken when interpreting in vitro results

381

in vivo, the EC10/IC10 values determined herein may represent concentrations that are relevant to sub-

382

lethal physiological effects of NAs in trout and perhaps in aquatic organisms in general. As such, we

383

conclude that oxidative uncoupling is a likely mechanism of sub-lethal toxicity that occurs at

384

concentrations of NAs present in the oil sands region of Canada.

385 386

ACKNOWLEDGEMENTS

387

This research was funded through NSERC Discovery grants to MRV and CK. The work was made

388

possible through NSCER RTI and CFI grants to MRV and CK. The authors wish to thank Gillian

389

MacDonald, Dr. Rabin Bissesseur, and Dr. Hebelin Correa for their assistance in completing this work.


Page 17 of 32


Rundle et al. 16

390 391

SUPPORTING INFORMATION

392

Raw Oroboros data traces for respiration, ROS, and membrane potential for positive controls oligomycin,

393

FCCP, and antimycin-A, solvent controls in state 3 respiration, solvent controls in state 4 respiration (S1-

394

S3).

395 396

REFERENCES

397 398

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399 400

(2) Allen, E.W. Process water treatment in Canada's oil sands industry: I. Target pollutants and treatment

401

objectives. J. Environ. Eng. Sci. 2008, 7, 123-138.

402 403

(3) Headley, J.V.; McMartin, D.W. A review of the occurence and fate of naphthenic acids in aquatic

404

environments. J. Environ. Sci. Health, Part A. 2004, 39 (8), 1989-2010.

405 406

(4) Clemente, J.S.; Fedorak, P.M. A review of the occurrence, analysis, toxicity, and biodegradation of

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naphthenic acids. Chemosphere. 2005, 60, 585-600.

408 409

(5) Madill, R.E.A.; Orzechowski, M.T.; Chen, G.; Brownlee, B.G.; Bunce, N.J. Preliminary risk

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assessment of the wet landscape option for reclamation of oil sands mine tailings: bioassays with mature

411

fine tailings pore water. Environ. Toxicol. 2001, 16 (3), 197-208

412 413

(6) Melvin, S.D.; Lanctôt, C.M.; Craig, P.M.; Moon, T.W.; Peru, K.M.; Headley, J.V.; Trudeau, V.L.

414

2013. Effects of naphthenic acid exposure on development and liver metabolic processes in anuran

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tadpoles. Environ. Pollut. 2013, 177, 22-27.



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(7) Wiseman, S.B.; Anderson, J.C.; Liber, K.; Giesy, J.P. Endocrine disruption and oxidative stress in

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larvae of Chironomus dilutus following short-term exposure to fresh and aged oil sands process-affected

419

water. Aquat. Toxicol. 2013a, 142-143, 414-421.

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(8) Kavanagh, R.J.; Frank, R.A.; Solomon, K.R.; Van Der Kraak, G. Reproductive and health assessment

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of fathead minnows (Pimphales promelas) inhabiting a pond containing oil sands process-affected water.

423

Aquat. Toxicol. 2013, 130-131, 201-209.

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(9) Marentette, J.R.; Frank, R.A.; Bartlett, A.J.; Gillis, P.L.; Hewitt, L.M.; Peru, K.M.; Headley, J.V.;

426

Brunswick, P.; Shang, D.; Parrorr, J.L. Toxicity of naphthenic acid fraction components extracted from

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fresh and aged oil sands process-affected waters, and commercial naphthenic acid mixtures, to fathead

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minnow (Pimephales promelas) embryos. Aquat. Toxicol. 2015, 164, 108-117.

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(10) Mohseni, P.; Hahn, N.A.; Frank, R.A.; Hewitt, L.M.; Hajibabaei, M.; Van Der Kraak, G. Naphthenic

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acid mixtures from oil sands process-affected water enhance differentiation of mouse embryonic stem

432

cells and affect development of the heart. Environ. Sci. Technol. 2015, 49, 10165-10172.

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(11) Hogan, N.S., K.L. Thorpe, and M.R. van den Heuvel. Opportunistic disease in yellow perch in

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response to decadal changes in the chemistry of oil sands-affected waters. Environ. Poll. 2018, 234, 769-

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778.

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(12) Frank, R.A.; Kavanagh, R.; Burnison, B.K.; Arsenault, G.; Headley, J.V.; Peru, K.M.; Van Der

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Kraak, G.; Solomon, K.R. Toxicity assessment of collected fractions from an extracted naphthenic acid

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mixture. Chemosphere. 2008, 72, 1309-1314.

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(13) He, Y.; Wiseman, S.B.; Zhang, X.; Hecker, M.; Jones, P.D.; Gamal El-Din, M.; Martin, J.W.; Giesy,

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J.P. Ozonation attenuates the steroidogenic disruptive effects of sediment free oil sands process water in

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the H295R cell line. Chemosphere. 2010, 80, 578-584

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(14) Reinardy, H.C.; Scarlett, A.G.; Henry, T.B.; West, C.E.; Hewitt, L.M.; Frank, R.A.; Rowland, S.J.

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Aromatic naphthenic acids in oil sands process-affected water, resolved by GCxGC-MS, only weakly

446

induce the gene for vitellogenin production in zebrafish (Danio rerio) larvae. Environ. Sci. Technol. 2013,

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47, 6614-6620.

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(15) Rabergh, C.M.I.; Isomaa, B.; Eriksson, J.E. The resin acids dehydroabietic acid and isopimaric acid

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inhibit bile acid uptake and perturb potassium transport in isolated hepatocytes from rainbow trout

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(Oncorhynchus mykiss). Aquat. Toxicol. 1992, 23, 169-180.

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effect of hyperbaric oxygen. Biochem. J. 1973, 134, 707-716.

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Mitochondrial redox signalling at a glance. J. Cell Sci. 2012, 125, 801-806.

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(20) Kowaltowski, A.J.; Vercesi, A.E. Mitochondrial damage induced by conditions of oxidative stress.

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(21) Frank, R.A.; Kavanagh, R.; Burnison, B.K.; Headley, J.V.; Peru, K.M.; Ven Der Kraak, G.;

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Solomon, K.R. Diethylaminoethyl-cellulose clean-up of a large volume naphthenic acid extract.

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Chemosphere. 2006, 64, 1346-1352.

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M.R. 2013. Immunotoxic effects of oil sands-derived naphthenic acids to rainbow trout. Aquat. Toxicol.

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2013, 126, 95-103.

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(23) Leclair, L.A.; Pohler, L.; Wiseman, S.B.; He, Y.; Arens, C.J.; Giesy, J.P.; Scully, S.; Wagner, B.D.;

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van den Heuvel, M.R., Hogan, N.S. In vitro assessment of endocrine disrupting potential of naphthenic

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acid fractions derived from oil sands-influenced waters. Environ. Sci. Technol. 2015, 49, 5743-5752.

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(24) ASTM D4239-83, Sulfur in the Analysis Sample of Coal and Coke Using High Temperature Tube

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Furnace Combustion Methods, Method C. In: Annual Book of ASTM Methods, Vol. 05.05; ASTM 1992.

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(25) Bance, S. Handbook of Practical Organic Microanalysis; Ellis Horwood, Ltd. 1980.

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(26) Thermo Fischer Scientific, FlashEA™ 1112 Elemental Analyzer Operating Manual.

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2016

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mitochondrial redox state and morphofunctional integrity. Free Radical Biol. Med. 2015, 84, 142-153.

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utilizing the principle of protein–dye binding. Anal. Biochem. 1976, 72, 248–254.

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coordinated activities of complex I and nicotinamide nucleotide trans- hydrogenase. BBA - Bioenergetics.

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2017, 1858, 955-965.

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(30) Headley, J.V.; Peru, K.M.; Fahlman, B.; Colodey, A.; McMartin, D.W. Selective solvent extraction

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and characterization of the acid extractable fraction of Athabasca oils sands process waters by Orbitrap

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mass spectrometry. Int. J. Mass Spectrom. 2013, 345-347, 104-108.

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(31) Huang, R.; McPhedran, K.N.; Gamal El-Din, M. Ultra performance liquid chromatography ion

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mobility time-of-flight mass spectrometry characterization of naphthenic acids species from oil sands

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process-affected water. Environ. Sci. Technol. 2015, 49, 11737-11745.

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(32) Wang, C.; Huang, R.; Klamerth, N.; Chelme-Ayala, P.; Gamal El-Din, M. Positive and negative

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electrospray ionization analyses of the organic fractions in raw and oxidized oil sands process-affected

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water. Chemosphere. 2016, 165, 239-247.

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(33) Leclair, L.A.; MacDonald, G.Z.; Phalen, L.J.; Köllner, B.; Hogan, N.S.; van den Heuvel, M.R. The

510

immunological effects of oil sands surface waters and naphthenic acids on rainbow trout (Oncorhynchus

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mykiss). Aquat. Toxicol. 2013, 142-143, 185-194.

512 513

(34) Grewer, D.M.; Young, R.F.; Whittal, R.M.; Fedorak, P.M. Naphthenic acids and other acid-

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extractables in water samples from Alberta: What is being measured? Sci. Total Environ. 2010, 408,

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(35) Arens, C.J.; Hogan, N.S.; Kavanagh, R.J.; Mercer, A.G.; Van Der Kraak, G.J.; van den Heuvel, M.R.

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Sublethal effects of aged oil sands-affect water on white sucker (Catostomus commersonii). Environ.

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Toxicol. Chem. 2015, 34(3), 589-599.

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(36) Kadenbach, B. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim. Biophys.

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Acta. 2003, 1604, 77-94.

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mitochondria. Biochemistry. 1986, 25, 1747-1755.

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McMaster, M.E.; Parrott, J.; Bickerton, G. Differential changes in gene expression in rainbow trout

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hepatocytes exposed to extracts of oil sands process-affected water and the Athabasca River. Comp.

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Biochem. Physiol., Part C. 2012, 155, 551-559.

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(39) Wiseman, S.B.; He, Y.; Gamal-El Din, M.; Martin, J.W.; Jones, P.D.; Hecker, M.; Giesy, J.P.

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Transcriptional responses of male fathead minnows exposed to oil sands process-affected water. Comp.

534

Biochem. Physiol. C: Pharmacol. Toxicol. 2013b, 157, 227-235.

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(40) He, Y.; Patterson, S.; Wang, N.; Hecker, M.; Martin, J.W.; Gamal El-Din, M.; Giesy, J.P.; Wiseman,

537

S.B. Toxicity of untreated and ozone-treated oil sands process-affected water (OSPW) to early life stages

538

of the fathead minnow (Pimephales promelas). Water Res. 2012, 46, 6359-6368.

539 540

(41) Turrens, J.F. Mitochondrial formation of reactive oxygen species. J. Physiol. 2003, 552 (2), 335-344.

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(42) Barrientos, A.; Moraes, C.T. Titrating the effects of mitochondrial complex I impairment in the cell

543

physiology. J. Biol. Chem. 1999, 23 (4), 16188-16197.

544 545

(43) Kalbácová, M.; Vrbacky, M.; Drahota, Z.; Melkova, Z. Comparison of the effect of mitochondrial

546

inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and

547

spectrofluorometry. Cytometry Part A. 2003, 52A, 110-116.

548

(44) Huang, R.; Chen, Y.; Meshref, M.N.A.; Chelme-Ayala, P.; Dong, S.; Ibrahim, M.D.; Wang, C.;

549

Klamerth, N.; Hughes, S.A.; Headley, J.V.; Peru, K.M.; Brown, C.; Mahaffey, A.; Gamal El-Din, M.

550

Characterization and determination of naphthenic acids species in oil sands process-affected water and

551

groundwater from oil sands development area of Alberta, Canada. Water Res. 2018, 128, 29-137.

552 553

(45) Arens, C.J.; Arens, J.C.; Hogan, N.A.; Kavanagh, R.J.; Berrue, F.; Van Der Kraak, G.J.; van den

554

Heuvel, M.R. Population impacts in white sucker (Catostomus commersonii) exposed to oil sands-derived

555

contaminants in the Athabasca River. Environ. Tox. Chem. 2017, 36 (8), 2058-2067.

556 557

(46) Zhang, K.; Pereira, A.S.; Martin, J.W. Partitioning for thousands of dissolved organic species in oil

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sands process-affected water. Environ. Sci. Technol. 2015, 49, 8907-8913.

559 560

(47) Young, R.F; Wismer, W.V.; Fedorak, P.M. Estimating naphthenic acids concentrations in

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laboratory-exposed fish and in fish from the wild. Chemosphere. 2008, 73, 498–505.

562 563

(48) van den Heuvel, M.R.; Hogan, N.S.; MacDonald, G.Z.; Berrue, F.; Young, R.F.; Arens, C.J.; Kerr,

564

R.G.; Fedorak, P.M. Assessing accumulation and biliary excretion of naphthenic acids in yellow perch

565

exposed to oil sands-affected waters. Chemosphere. 2014, 95, 691-627.

566



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567

(49) Young, R.F.; Martínez Michael, L.; Fedorak, P.M. Distribution of naphthenic acids in tissues of

568

laboratory-exposed fish and in wild fishes from near the Athabasca oil sands in Alberta, Canada.

569

Ecotoxicol. Environ. Saf. 2011, 74, 889–896.

570 571

(50) Moyes, C.D.; Schulte, P.M. Principles of Animal Physiology, 2nd ed.; Pearson Education Inc.: San

572

Fransico, U.S., 2008

573 574

(51) Lieberthal, W.; Menza, S.A.; Levine, J.S. Graded ATP depletion can cause necrosis or apoptosis of

575

cultured mouse proximal tubular cells. Am. J. Renal Physiol. 1998, 274 (2), F315-F327.

576

(52) Rogers, C.; Davis, B.; Neufer, P.D.; Murphy, M.P.; Anderson, E.J.; Robidoux, J. A transient increase

577

in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization

578

and ATP depletion during prolonged exposure to fatty acids. Free Radic. Biol. Med. 2014, 67, 330-341.

579 580 581 582 583 584 585


Page 25 of 32


Rundle et al. 24

586

Table 1. IC50 or EC50 and IC10 or EC10 values mg/L (95% Confidence Interval) calculated for each

587

parameter based on Oroboros and flow cytometry measurements. Curve fits were conducted on individual

588

replicates and parameters averaged. The mean variance explained by the curve fits for each parameter are

589

indicated. Parameters for which dose-effect curves could not be fitted for all replicates are indicated as no

590

consistent effect.

591 Parameter

Mean EC50/IC50

Mean EC10/IC10

Mean % Variance

(mg/L)

(mg/L)

Explained

Oroboros State 3

104.7 (34.2-175.1)

35.1 (13.9-56.2)

99.0

Oroboros State 4

no consistent effect


Oroboros State 3



Oroboros State 4

157.8 (58.4-257.2)

66.7 (25.5-107.9)

99.2

Flow Cytometry State 2

88.7 (61.5-115.8)

29.4 (0-85.5)

96.7

Oroboros State 3

21.0 (15.0-27.0

15.0 (1.1-29.0)

88.5

Oroboros State 4

36.7 (1-109.8)

24.1 (1.2-47.1)

87.0

25.6 (12.4-38.7)

11.8 (0.4-23.2)

66.8

Oxygen Consumption

Mitochondrial Membrane Potential

Reactive Oxygen Species

Flow Cytometry State 2

592



Page 26 of 32

Rundle et al. 25

593

Figure 1. Relative abundance of molecular ions present in naphthenic acid extract obtained using negative

594

ion direct injection HRMS. The Z-value represents the hydrogen deficiency of the compound. The

595

absolute value of Z divided by 2 gives the number of double bonds or rings in the compounds. Each Z-

596

value reported in the extract is indicated with a different colour.

597 598

Figure 2. Representative Oroboros tracing of a state 4 respiration dose response. This tracing includes all

599

three parameters, oxygen consumption (red line), mitochondrial membrane potential (∆Ψmt; blue line),

600

and ROS represented by H2O2 production (green line) overlaid. The units for H2O2 production are

601

pmol/mg protein/min and are represented in the right axis, while the ∆Ψmt (mV) are arbitrary for the

602

purposes of this figure. The transient spikes of H2O2 production (green line) are an artifact of addition of

603

NAs or substrates to the experiment chamber. At the beginning of the experiment, isolated mitochondria

604

and the necessary substrates malate and glutamate (MG) and ADP were added as shown with the first

605

three arrows. During the appropriate respiration state, NAs were added sequentially 3 minutes apart to

606

achieve a cumulative concentration of NAs, which is indicated by arrows (eg., NA20 indicates 20 mg/L

607

NAs). All data for analysis were collected once steady states were reached.

608 609

Figure 3. (a) Dose-effect curve for oxygen consumption during state 3 respiration using the Oroboros

610

system with the calculated IC50. (b) Dose-effect curve for oxygen consumption during state 4 respiration

611

using the Oroboros system (EC50 could not be consistently calculated for state 4). Error bars are standard

612

error of the mean. N=3 for each point.

613 614

Figure 4. (a) Dose-effect curve for mitochondrial membrane potential (∆Ψmt) during state 3 respiration

615

measured by the Oroboros system (IC50 could not be consistently calculated for state 3). (b) Dose-effect

616

curve for ∆Ψmt during state 4 respiration measured by the Oroboros system during state 4 respiration with

617

calculated IC50. (c) Dose response curve for ∆Ψmt during state 2 respiration measured by real-time flow

618

cytometry with calculated IC50. (d) Representative tracing for the effects of different doses of NA on ∆Ψmt


Page 27 of 32


Rundle et al. 26

619

during state 2 respiration measured by real-time flow cytometry where the x-axis is time and the y-axis is

620

the amount of fluorescence. The colour represents the density of the distribution of fluorescence. The

621

additions of the NA doses are indicated on the figure with arrows and are labeled to each corresponding

622

dose. Error bars are standard error of the mean. N=3 for each point.

623 624

Figure 5. (a) Overall dose-effect curve for H2O2 production during state 3 respiration measured by the

625

Oroboros system with calculated EC50. (b) Overall dose-effect curve for H2O2 production during state 4

626

respiration measured by the Oroboros system with calculated EC50. (c) Dose-effect curve for

627

mitochondria ROS during state 2 respiration measured real-time flow cytometry with calculated EC50.

628

EC50 valcues were calculated from individual replicates and then averaged. (d) Representative tracing for

629

the effects of different doses of NA on ROS emission during state 2 respiration measured by real-time

630

flow cytometry where the x-axis is time and the y-axis is the amount of fluorescence. The colour

631

represents the density of the distribution of fluorescence. The additions of the NA doses are indicated on

632

the figure with arrows and are labeled to each corresponding dose. Error bars are standard error of the

633

mean. N=3 for each point.

634



Figure 1 692x563mm (96 x 96 DPI)


Page 28 of 32

Page 29 of 32


Figure 2 1058x595mm (96 x 96 DPI)



Figure 3 1044x404mm (96 x 96 DPI)


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Oil Sands Derived Naphthenic Acids Are Oxidative Uncouplers and

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