Effect of Ozonation on the Estrogenicity and Androgenicity of … · Published: June 15, 2011 r...

Published: June 15, 2011

r 2011 American Chemical Society 6268 dx.doi.org/10.1021/es2008215 | Environ. Sci. Technol. 2011, 45, 6268–6274

ARTICLE

pubs.acs.org/est

Effect of Ozonation on the Estrogenicity andAndrogenicity of Oil Sands Process-Affected WaterYuhe He,† Steve B. Wiseman,*,† Markus Hecker,†,‡ Xiaowei Zhang,§ Nan Wang,|| Leonidas A. Perez,^

Paul D. Jones,†,‡ Mohamed Gamal El-Din,|| Jonathan W. Martin,^ and John P. Giesy†,§,#,r,[,O,z

†Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada‡School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C8, Canada§State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China

)Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta, Canada^Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry,University of Alberta, Edmonton, Alberta, Canada

#Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, CanadarDepartment of Zoology, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan, United States[Department of Zoology, College of Science, King Saud University, Riyadh, Saudi ArabiaODepartment of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, ChinazSchool of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China

bS Supporting Information

’ INTRODUCTION

The oil sands in northeastern Alberta, Canada, are the secondlargest proven oil deposit on Earth.1,2 In current surface miningoperations, extraction of bitumen from oil sands involves theClarke hot water extraction method. This results in productionof oil sands process-affected water (OSPW). In accordancewith current zero-discharge policies, OSPW is stored on-site inactive settling basins, otherwise known as tailings ponds. It isestimated that greater than 1 billion m3 of OSPW are currentlystored on-site in the various settling basins.3 The volume ofOSPW stored in tailing ponds will continue to increase as mining

activities expand. Consequently, there is a need to pursueeffective means of remediating or reclaiming OSPW.

OSPW is acutely and chronically toxic to aquatic organisms,including fish.4 The toxicity of OSPW has been attributed in partto the persistent class of organic acids know as naphthenic acids(NAs).5,6 NAs are a group of acyclic, monocyclic, and polycyclic

Received: March 11, 2011Accepted: June 15, 2011Revised: June 14, 2011

ABSTRACT: There is increasing environmental concern about the volume ofoil sands process-affected water (OSPW) produced by the oil sands industry inAlberta, Canada. There is limited knowledge of the toxic effects of OSPW andone of the primary organic constituents, naphthenic acids (NAs), which arethought to be one of the toxic constituents of OSPW. OSPW and NAs can haveendocrine disrupting potential. The NAs in OSPW are persistent, but ozonationcan significantly reduce concentrations of NA, while increasing their biodegrad-ability, and consequently reduce OSPW toxicity. However, it is of concern thatOSPW ozonation might generate hydroxylated cycloaliphatics with endocrinedisrupting potential. In this study, the estrogen receptor- (ER) and androgenreceptor- (AR) mediated effects of OSPW and ozone-treated OSPW wereinvestigated in vitro by use of T47D-kbluc (estrogen responsive) and MDA-kb2(androgen responsive) cells. Ozonation neither attenuated nor intensified theestrogenicity of OSPW. The estrogenic responses to untreated OSPW and ozone treated OSPW were 2.58((0.22)-fold and2.48((0.13)-fold greater than those of controls, respectively. Exposure to untreated OSPW produced significant antiandrogenicityin the presence of 0.01, 0.05, or 0.1 nM testosterone (T), while ozone-treated OSPW produced significant antiandrogenicity in thepresence of 0.01 or 0.05 nM T. Exposure to untreated and ozone-treated OSPW also caused potentiation of androgen receptor-mediated effects of T. OSPW could cause estrogenic and antiandrogenic effects through receptor mediated pathways, and ozonationcan partially mitigate the OSPW antiandrogenicity as well as androgen potentiating effect, without increasing estrogen potency.

6269 dx.doi.org/10.1021/es2008215 |Environ. Sci. Technol. 2011, 45, 6268–6274

Environmental Science & Technology ARTICLE

carboxylic acids with the general formula of CnH2n+ZO2, where nrepresents the number of carbon atoms and Z is zero or anegative even integer related to the number of rings (or doublebond equivalents) in the molecule.7 However, NAs account for<50% of all compounds in the organic fraction of OSPW.8,9

Because toxicity of OSPW has been attributed primarily to NAs,these compounds have often been the target of remediationefforts. In-situ remediation relying on natural microbial degrada-tion can reduce a portion of NAs in OSPW, but chronic toxicitystill remains.10�12 Natural half-lives for NAs in OSPW are12.8�13.6 years.12 Consequently, more rapid methods of OSPWremediation are being tested. Treatment with ozone is capable ofdecreasing concentrations of NAs, thereby decreasing the toxi-city of OSPW.13 Moreover, treatment with ozone acceleratesmicrobial degradation of residual NAs.14 While little is knownabout the biological properties of ozone-treated NAs, studieshave warned that ozonation of wastewater could result in theproduction of endocrine disrupting compounds.15�18

The specific mechanism(s) of toxicity of OSPW and thecomponent NAs are unknown, although acute toxicity via physi-cal disruption of membranes, or narcosis, has been suggested.19

Little is known about the potential effects of OSPW, and NAs inparticular, on the endocrine axis. Significant alterations in con-centrations of testosterone (T) and 17β-estradiol (E2) have beenreported in goldfish (Carassius auratus) and yellow perch (Percaflavescens) that had been exposed to OSPW.20,21 Previous re-search has also demonstrated altered T and E2 steroidogenesis inH295R cells exposed to OSPW.22

Endocrine disruption by environmental contaminants hasbeen identified as a potential threat to humans and wildlife.The endocrine disrupting capabilities of some chemicals resultfrom their interaction with nuclear receptors, including theestrogen receptor (ER) and androgen receptor (AR). Disruptionof ER and AR signaling pathways can result in alterations ofphysiological processes, including early embryonic development,growth, and reproductive success or fecundity.23,24 Several invitro assays have been developed to screen for potential ER andAR agonists or antagonists. Among those are assays using theT47D-kbluc and MDA-kb2 cell lines that screen for direct-actingagonists and antagonists of the ER and AR, respectively.25,26

Here results of a study of the endocrine disrupting potency ofOSPW and ozone-treated OSPW by use of these reporter assaysare reported. The objectives of this study were to determine thefollowing: 1) in vitro androgenic effect of OSPW via the receptor-mediated signaling pathway by use of the MDA-kb2 cell line; 2)in vitro estrogenic effect of OSPW via the receptor mediatedsignaling pathway by use of the T47D-kbluc cell line; and 3)whether these baseline effects are modulated by ozonationof OSPW.

’MATERIALS AND METHODS

OSPW and Commercial NAs. OSPW was collected in Feb-ruary 2010, from the West-In-Pit (WIP), an active settling basinon the Syncrude Canada, Ltd. site (Fort McMurray, AB,Canada). The OSPW in the WIP is received from two othersettling ponds that receive input directly from the main bitumenextraction plant. A schematic illustration is provided by Hanet al.12 Concentration of NAs in the WIP-OSPW sample,determined by ultra pressure liquid chromatography high resolu-tion mass spectrometry (UPLC-HRMS), was 19.7 mg/L.27 Sinceprevious studies have reported activation of in vitro AR and ER

signaling by NAs, a commercial mixture of high purity NAsprovided by Merichem Chemicals and Refinery Services LLC(Houston, TX, USA) was used as a reference in this study so thatcomparisons of the results of this study could bemade to previousstudies.28�30 There are similarities and differences between theNAs in OSPW and the Merichem NAs mixture.14,31 The NAdistribution of both mixtures is similar by carbon number, withthe major clusters in the range of C12�C15.31 However, inOSPW the most intense ions by high pressure liquid chromatog-raphy/quantitative time-of-flight mass spectrometry (HPLC/QTOF-MS) are for Z = �4 (36%) and Z = �6 (36%), followedby Z =�12 and�10 (7.8%) > Z =�2 (6.7%) > Z =�8 (5.6%) >Z = 0 (0.4%). In theMerichemNAmixture the most intense ionswere for the Z = �2 (41%), followed by Z = �4 (32%) > Z = 0(20%) > Z =�6 (5.8%) > Z =�8 (1.7%) > Z =�10 (0.3%).31 Inaddition, OSPW contains a significant oxidizedNA fraction that isbarely detectable in the Merichem NAs mixture.14 Moreover,although NAs are considered one of the major toxic constituents,other organic compounds in OSPW may contribute to toxiceffects.Ozonation of OSPW. Ozonation of WIP-OSPW was con-

ducted at the University of Alberta (Edmonton, AB, Canada)following standard procedures.32,33 Ozone treatment was con-tinued until the total degradation of parent NAs reachedapproximately 90%, as determined by the remaining sum re-sponse of all UPLC�HRMS peak area corresponding to NAs.Consequently, the total concentration of NAs was decreasedfrom 19.7 mg/L in the untreated WIP-OSPW to 1.9 mg/L in theozone-treated WIP-OSPW.27 A detailed description of theozonation protocol is provided in the Supporting Information.T47D-kbluc and MDA-kb2 Cell Culture. The estrogenicity

and androgenicity ofOSPWwas tested in T47D-kbluc andMDA-kb2 cells, respectively, based on methods described by Wilsonet al.25,26 T47D-kbluc cells (ATCC CRL-2865) and MDA-kb2cells (ATCCCRL-2713) were obtained from the American TypeCulture Collection (ATCC, Manassas, VA, USA). T47D-kbluccells were maintained in RPMI culture media (Sigma) supple-mented with 2.5 g/L glucose, 10 mM HEPES, 1 mM sodiumpyruvate, 1.5 g/L NaHCO3, 0.2 U/mL insulin, and 10% normalFBS, at 37 �C in a 5% CO2 atm. MDA-kb2 cells were cultured inL15 media (Sigma, Mississauga, ON, Canada) supplementedwith 10% FBS (Sigma) at 37 �C, without CO2. For T47D-kblucand MDA-kb2 assays, additional withdrawal and assay/exposuremedia were also prepared by supplementation with 10% and 5%dextran-charcol treated FBS (Sigma), respectively, rather than10% normal FBS. All the media were filtered through a 0.22 μMbottle top filter (Corning) to avoid microbial contamination.T47D-kbluc and MDA-kb2 Cell Assays. To minimize the

baseline reporter gene activity, prior to experimental exposures,both cell types were subcultured in their respective withdrawalmedium for 24 h, and then experimental exposures were con-ducted by use of the corresponding assay/exposure medium. Forexposure experiments, T47D-kbluc cells were plated at 4 � 104

cells/mL, and MDA-kb2 cells were plated at 8� 104 cells/mL ina 96-well luminometer plate (Perkin-Elmer, Woodbridge, ON,Canada) with 250 μL of assay medium per well. Next, the assaymedium was replaced with either fresh assay medium as controlor exposure medium containing full-strength and differentdilutions of untreated OSPW, ozone-treated OSPW, or theMerichem NAs mixture. Exposure media were prepared thesame as standard assay medium, except that the nanopure waterwas replaced with untreatedOSPW, ozone-treatedOSPW, or the



Merichem NAs mixture. Dilutions of exposure medium wereprepared with assay medium. All exposure media containinguntreated OSPW, ozone-treated OSPW, or the Merichem NAsmixture were prepared fresh on the day of exposure, and the pHwas adjusted to 7.6. E2 or T standard curves were also includedon each plate. Stock standard solutions of E2 and T wereprepared in 100% ethanol, and 10-fold serial dilutions wereprepared in assay medium, with final concentrations rangingfrom 0.0001 to 10 nM and 0.001 to 10000 nM for E2 and T,respectively. The final concentration of ethanol in all exposureswas 0.1%. Control experiments demonstrated that this concen-tration did not affect cell viability or the reporter gene assays(data not shown). The estrogen receptor antagonist ICI 182,780(Faslodex) (Sigma, Mississauga, ON, Canada) was used toconfirm activation of ER signaling in T47D-kbluc cells. TheMDA-kb2 cell assay was performed with the glucocorticoidreceptor (GR) agonist dexamethasone (DEX) to determineany GR activity of untreated WIP-OSPW, ozone-treated WIP-OSPW, or the Merichem NAs mixture. No significant cytotoxi-city was observed in any of the treatments.After dosing with standards or exposure medium the cells were

incubated for 24 h, and luciferase activity was determined as ameasure of estrogenicity or androgenicity by use of the Steady-Gloluciferase assay system (Promega, Sunnyvale, CA, USA) accordingto the manufactures recommendations. Cells were washed oncewith PBS at room temperature. Twenty-five μL of lysis bufferwas added per well, and plates were incubated for 15 min atroom temperature. After cell lysis, 25 μL of the luciferase reagentwas added to each well, and luminescence was read by useof a POLARStar OPTIMA microplate reader (BMG Labtech,Offenburg, Germany).Data Analyses. All experiments were performed on two

separate occasions, with triplicate exposures performed for eachindividual experiment (n = 6). There was no significant differ-ence between the results obtained from the two experimentaldates. Statistical analyses were conducted by use of SPSS 16(SPSS Inc., Chicago, IL, USA). The normality of each data setwas assessed by the Kolomogrov�Smirnov one-sample test, andhomogeneity of variance was determined by the Levene’s test.

Significant differences were evaluated with a one way ANOVAwith Tukey’s HSD posthoc test. Differences with p < 0.05 wereconsidered significant. All data are presented as mean((standard deviations) fold change from controls.

’RESULTS AND DISCUSSION

Estrogenicity. Estrogenic responses were proportional toconcentrations of untreated WIP-OSPW, ozone-treated WIP-OSPW, and the Merichem NAs mixture (Figure 1). At full-strength (100%) concentrations, the estrogenic responses tountreatedWIP-OSPW and ozone-treatedWIP-OSPWwere 2.58((0.22)-fold and 2.48 ((0.13)-fold compared to control, re-spectively. Thus, ozonation neither attenuated nor intensified theestrogenicity of WIP-OSPW. Exposure to the Merichem NAsmixture stimulated a nonsignificant induction (2.18 ( 0.40-foldat 100%) in the estrogenic response compared to control levels.In coexposure experiments, the estrogenic response in the

cells coexposed to untreated WIP-OSPW or ozone-treatedOSPW, and E2, was significantly greater than the response toE2 alone at concentrations between 0 and 0.01 nM in bothcoexposure scenarios (Figure 2a and 2b). These results suggestthat neither untreatedWIP-OSPWnor ozone-treatedOSPWhassignificant ER antagonist properties, but rather, there is anadditive estrogenic effect of WIP-OSPW or ozonated OSPWwith E2. The estrogenic response in cells coexposed to theMerichem NAs mixture and E2 (Figure 2c) resembled theresponse of cells coexposed to E2 with untreated WIP-OSPWor ozone-treatedWIP-OSPW. At lesser concentrations of E2 (foruntreated and ozone-treatedWIP-OSPW: E2e 0.01 nM; for theNAs mixture: E2e 0.001 nM), significant additive estrogenicitywas observed. While the results suggest that ER agonists arepresent, given that the concentration of NAs in the Merrichemmixture was 60 mg/L while the concentration of NAs in theuntreated WIP-OSPW was 19.7 mg/L, the coexposure experi-ment indicated that the estrogenicity of the Merichem NAsmixture is weak compared to WIP-OSPW (no significantlygreater response at 0.01 nM E2). However, at greater concentra-tions of E2 the additive estrogenic effect of untreated and ozone-treated WIP-OSPW and the Merichem NAs mixture was notstatistically significant, probably due to saturation of the ER.To confirmwhether the estrogenic effects ofOSPWand ozone-

treated OSPW were due to binding to the ER, T47D-kbluc cellswere coexposed, with the potent ER antagonist ICI 182,780, tountreated OSPW, ozone-treated OSPW, or the Merichem NAsmixture, along (Figure 3). Co-exposure to ICI 182,780 attenuatedthe estrogenicity observed in cells exposed to untreated OSPW orozone-treated OSPW, such that levels were not significantlydifferent from control levels (Figure 3a and 3b). These resultsare consistent with estrogenicity of both untreated OSPW andozone-treatedOSPWbeing due to binding of some component ofthe OSPW mixture to the ER. Also, the estrogenic responseobserved for the Merichem NAs mixture was abolished bycoexposure with ICI 182,180 (Figure 3).There are several reports related to binding and activation of

hormone receptors by offshore effluent from oil productionplatforms, including crude oil and refined products.28�30,32,33

NAs present in North Sea offshore produced water were ERagonists and AR antagonists.35 OSPW is a mixture of inorganicand organic compounds, with NAs being the predominantorganic compound.34 Due to the complex chemistry ofWIP-OSPW it is difficult to identify specific ER agonist(s).

Figure 1. Concentration�response relationship for the estrogenic re-sponse in T47D-kbluc cells exposed to untreated OSPW, ozone-treatedOSPW, or the NAsmixture. Fold changes are relative to themedia control.An * indicates a significant change in response from control cells (p< 0.05).



However, the results from the exposures to the Merichem NAsmixture suggest that NAs present in WIP-OSPW are probably

responsible for the observed effects. The possibility that otherunidentified compounds present in OSPW, and that are notaffected by ozonation, are ER agonists cannot be eliminated.Because the T47D-kbluc cells contain both ERR and ERβ, it isunknown whether the agonist(s) preferentially bind to one of thesereceptors or have equal affinity for both.26

If the NAs present in WIP-OSPW are responsible for theestrogenic effects and ozonation removes NAs, it is inconsistentthat ozonation did not attenuate estrogenicity. Several studieshave demonstrated that ozonation of OSPW decreases NA levels,thereby attenuating the acute toxicity.13 In a previous study,ozonation of WIP-OSPW attenuated effects on steroidogenesisin the H295R cell line.22 Ozonation primarily targeted the WIP-OSPW NAs with more rings and was hypothesized to preferen-tially degrade NAs with greater extents of alkyl branching.14 Oneexplanation of the observed results is that less cyclic and lessbranched NAs present in WIP-OSPW that are unaffected byozonation act as the ER agonists. Alternatively, oxidized NAs (i.e.,CnH2n+ZO3) might be responsible for ER agonism of WIP-OSPW. Oxidized NAs are naturally present in untreated OSPWand are both degraded and formed during the ozonation processsuch that there is little net change in their total concentration.14,37

Therefore, if the oxidizedNAs are estrogenic, then ozonationmaynot attenuate observed effects.Androgenicity. Significant antiandrogenic response was ob-

served in cells coexposed to different concentrations of T withfull-strength untreated WIP-OSPW, ozone-treated WIP-OSPW,and the Merichem NAs mixture, compared to the same concen-trations of T alone (Figure 4). Themagnitude (M) of the anti-ARresponse in coexposed cells versus cells exposed to the corre-sponding concentrations of T alone is shown in Figure 5 and wasdetermined (eq 1)

M ¼ Fold change in AR response in coexposed cells-1Fold change in AR response in cells exposed to T alone-1

ð1Þ

Co-exposure with WIP-OSPW resulted in a significantantiandrogenic effect at T concentrations of 0.01, 0.05, and

Figure 3. Effect of the ER antagonist ICI 182,780 on the estrogenicityof 100% a) untreated OSPW, b) ozone-treated OSPW, or c) or the NAsmixture, alone or as a coexposure with 0.1 nM E2 and/or 1 μM ICI 182,780 in T47D kbluc cells. Fold changes are relative to the media control.Different letters represent significant differences between treatmentgroups (p < 0.05).

Figure 2. Concentration�response relationship for estrogenic re-sponse in T47D-kbluc cells coexposed to different concentrations ofE2 and 100% a) untreated OSPW, b) ozone-treated OSPW, or c) or theNAs mixture. An * indicates a significant change between treatmentgroups at the same concentration of E2 (p < 0.05).



0.1 nM (Figure 4a), in which the magnitude of the anti-ARresponse was 16( 6.5%, 47( 7.6%, and 75( 9.7%, respectively(Figure 5). Co-exposure with ozone-treated WIP-OSPW re-sulted in a significantly antiandrogenic effect at T concentrations

of 0.01 and 0.05 nM (Figure 4b), in which the magnitude of anti-AR response was 64 ( 12% and 70 ( 11%, respectively(Figure 5). Compared with untreated WIP-OSPW, significantlyless antiandrogenic capability was observed in cells coexposed toozone-treated OSPW and 0.01, 0.05, and 0.1 nM of T (Figure 5).Ozone-treated WIP-OSPW was less potent as an antiandrogenthan untreated WIP-OSPW. Similar to the WIP-OSPW andozone-treated WIP-OSPW, coexposure with NAs resulted in asignificant antiandrogenic effect at T concentrations of 0.01 (49(13%) and 0.05 nM (64( 13%) (Figure 4c). The antiandrogenicresponse in cells exposed to the Merichem NAs mixture isweaker than that of untreated WIP-OSPW but is greater thanthat of the ozone-treated OSPW.MDA-kb2 cells also contain glucocorticoid receptor (GR).29

To confirm the antiandrogenic response rather than an anti-glucocorticoid response, a coexposure experiment with the GRagonist, dexamathasone (DEX), was conducted (Figure S1,Supporting Information). Co-exposure to different concentra-tions of DEX with untreated WIP-OSPW, ozone-treatedWIP-OSPW, or the Merichem NAs mixture did not result insignificantly less response compared to the corresponding con-centrations of DEX alone, which suggests that neither theuntreated WIP-OSPW, ozone-treated WIP-OSPW, or theMerichem NAs mixture possess an anti-GR effect.Crude oil has been reported to have antiandrogenic effects.

There are several reports of steroid hormone receptor bindingand activation by offshore effluent fromNorth Sea oil productionplatforms, crude oil, and refined products in recombinant yeastassays.28�30,32,33 Significant AR antagonist potency has beendemonstrated in at least one of these effluents fractions contain-ing mixtures of NAs. In those reports the observed antiandro-genic effects were attributed to an enriched NA fraction and nothydroxylated polycyclic aromatic hydrocarbons (PAHs) as wasshown in a mammalian and human cell line study.38 Since ourprevious analyses of sediment-free WIP-OSPW detected noPAHs (unpublished data), it is unlikely that the effects observedin the present study are due to PAHs.Given the apparent antiandrogenic properties of untreated

WIP-OSPW and ozone-treatedWIP-OSPW, it was hypothesized

Figure 5. Magnitude of androgenic response in MDA-kb2 cells coex-posed to untreated OSPW, ozone-treated OSPW, or the NAs mixtureand T. Fold changes are relative to the same concentrations of T alone.An * indicates a significant change in response from the AR response ofcoexposure group of untreated OSPW and T (p < 0.05).

Figure 4. Concentration�response relationship for androgenic responsein MDA-kb2 cells coexposed to different concentrations of T and 100% a)untreatedOSPW, b) ozone-treatedOSPW, or c) or the NAsmixture. Foldchanges are relative to themedia control. An * indicates a significant changein between treatment groups at the same concentration of T (p < 0.05).



that coexposure with T and WIP-OSPW would have resulted inattenuation of the androgenic response. However, there was nosignificant difference in the response to T alone and in combina-tion with 100% untreated WIP-OSPW at the 0.05 nM T(Figure 3a). In fact, coexposure with WIP-OSPW significantlyincreased the androgenic response compared to that for 1, 10,and 100 nM of T alone (Figure 3a). The magnitude of thisresponse was 121 ( 8.5%, 140 ( 10.7%, and 118 ( 5.3%,respectively (Figure 4). At concentrations of T greater than100 nM there was no statistical difference in the androgenicresponse between cells exposed to T alone or in combinationwith 100% WIP-OSPW (Figure 3a). Similar responses wereobserved in cells coexposed to ozone-treated OSPW, resulting ina greater AR response compared to that for 1 or 10 nM of T alone(Figure 3b), and the Merichem NAs mixture, which resulted in asignificantly greater AR response compared to that for 1 nM of Talone (Figure 3c). Compared with untreated WIP-OSPW, themagnitude of the androgenic effect caused by ozone-treatedWIP-OSPW was significantly weaker in coexposure with100 nMT,while for theMerichemNAsmixture it was significantlyweaker in coexposure with 10 and 100 nM T (Figure 4).The mechanism of the increased androgenic response in cells

coexposed to moderate concentrations of T and untreated WIP-OSPW, ozone-treated WIP-OSPW, or Merrichem NAs is un-known. One explanation is that untreated WIP-OSPW, ozone-treated WIP-OSPW, and the NAs mixture might have altered thephysiological properties of the cell membrane, which resulted ingreater bioavailability of T and a greater AR response comparedto cells exposed to the same concentration of T alone. This typeof effect has been reported for other hydrophobic organiccompounds.39�41 Under such a scenario the antiandrogeniceffects of untreated WIP-OSPW, ozone-treated WIP-OSPW,and the Merichem NAs mixture would be considered weak sinceT is apparently able to compete with the antagonist for the ligandbinding domain of the AR. Further work is needed to fullyelucidate the mechanism of antiandrogenicity observed here.Regardless of themechanism(s) of action, ozonation attenuated

the antiandrogenic effects of WIP-OSPW on the AR. Thecompounds in WIP-OSPW and ozonated WIP-OSPW responsi-ble for the antiandrogenic effect are unknown, but to some extentNAs likely play a role, as evidenced by the experiments with theNAs. Considering the antiandrogenicity was still evident in cellsexposed to full-strength ozone-treated WIP-OSPW compared tothat for 0.01 and 0.05 nMT alone, in which 90% of NAs had beendegraded by ozone, ozonation was not able to completely abolishthe effects. This suggests that other unidentified compoundspresent in WIP-OSPW might possess antiandrogenic activity.Given that the same antiandrogenic effect was observed in cellsexposed to the NAs, which is composed entirely of lessermolecular weight NAs, these results suggest that the lessermolecular weight NAs might be responsible for some of theantiandrogenicity of WIP-OSPW. Whereas attenuation of antian-drogenicity was observed in cells exposed to ozone-treated WIP-OSPW alone, under a coexposure scenario both untreated OSPWand ozone-treated WIP-OSPW potentiated the effect of T, andozonation only attenuated the potentiating effect compared to thatfor 100 nM T alone. This result suggests that the componentof untreated WIP-OSPW responsible for this potentiatingeffect might be partially affected by ozonation. Because the sameeffect was also found in the NAs mixture, then that component ofWIP-OSPW may share properties with the Merichem NAsmixture.

In summary, exposure toWIP-OSPW as well as NAs can resultin both ER agonist and AR antagonist properties, and ozonationcan partially mitigate the potential antiandrogenic and androgenpotentiating effects of WIP-OSPW. However, the results of thecurrent study do not support the idea that ozonation reduces theestrogenic effect. To some extent, ozonation can be an effectivetreatment to reduce the effects of OSPW due to endocrinedisruption. Further study is needed to clarify the mechanism(s)of action of OSPW dissolved organics as ER/AR agonist/antagonists, including more detailed determination of the ERand ARmediated effects of OSPW and the effect of ozonation onthe in vivo responses of organisms.

’ASSOCIATED CONTENT

bS Supporting Information. A description of the ozonationof OSPW and the anti-GR effects ofWIP-OSPW, ozonatedWIP-OSPW, and the Merichem NAs mixture. This material is avail-able free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Fax: 306-970-4796. E-mail: [email protected].

’ACKNOWLEDGMENT

This research was supported by a research grant from theAlberta Water Research Institute to J.P.G, J.W.M., and M.G.E.-D.(Project # C4288). The research was supported, in part, by aDiscovery Grant from the National Science and EngineeringResearch Council of Canada (Project # 326415-07) and a grantfrom the Western Economic Diversification Canada (Project #6578 and 6807). The research was also supported by researchgrants from the Helmoltz-Alberta (HAI) Initiative to M.G.E.-D.and J.W.M. The authors wish to acknowledge the support of aninstrumentation grant from the Canada Foundation for Innova-tion. J.P.G. was supported by the Canada ResearchChair program,an at large Chair Professorship at the Department of Biology andChemistry and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong, The Einstein Professor Program of theChinese Academy of Sciences, and the Visiting Professor Programof King Saud University. The authors wish to thankWarren Zubotof Syncrude Canada Inc. for supplying the WIP-OSPW.

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SUPPORTING INFORMATION

Effect of Ozonation on the Estrogenicity and Androgenicity of Oil Sands Process-Affected

Water

Yuhe He, Steve B. Wiseman*, Markus Hecker, Xiaowei Zhang, Nan Wang, Leonidas A. Perez-

Estrada, Paul Jones, Mohamed Gamal El-Din, Jonathan W. Martin, John P Giesy

* Corresponding author

This supporting information includes:

� Ozonation of OSPW

� Exposure with GR agonist, dexamethasone

Summary of the number of pages, figures, and tables

� Number of pages: 4 (including cover page)

� Number of figures: 1

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Ozonation of OSPW. Ozonation of WIP-OSPW was conducted at the University of Alberta

(Edmonton, AB, Canada). An AGSO 30 Effizon ozone generator (WEDECO AG Water

Technology, Herford, Germany) was used to produce ozone gas from extra dry, high purity

oxygen. To obtain a stable ozone concentration in the feed-gas, the ozone generator was allowed

to stabilize for 10 min. The feed gas was sparged into the OSPW through a ceramic fine bubble

gas diffuser located at the bottom of a PVC plastic reactor. During the ozonation process, ozone

concentrations in the feed and off-gas lines were continuously monitored by two identical ozone

monitors (model HC-500, PCI-WEDECO). The ozone monitors were calibrated by use of the

potassium iodide (KI) method periodically according to Standard Methods for the Examination

of Water and Wastewater.26

Ozone treatment was continued until the total degradation of parent

NAs reached approximately 90%, as determined by the remaining sum response of all UPLC–

HRMS peak area corresponding to NAs. After treatment with ozone, the WIP-OSPW was

purged for 10 minutes with a purified nitrogen gas to strip residual ozone and oxygen from the

reactor. Ozonation of the OSPW reduced the NA concentration from 19.7 mg/L to 1.9 mg/L 25

.

The ozone residual in the reactor was measured using the Indigo method.26

The ozone dose for

this system can be calculated according to equation 1.27

∆O3 = L

t

L

outGoutGinGinGCdt

V

CQCQ−

−∫0

,,,, )(

(1)

where ∆O3 is the amount of utilized ozone (mg/L), CG,in is the ozone concentration in the feed

gas, which was calculated from reading the first ozone monitor (mg/L), CG,out is the ozone

concentration in the off gas, which was calculated from reading the first ozone monitor (mg/L),

CL is residue ozone concentration in the liquid phase (mg/L), VL is effective reactor volume (L),

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QG,in is feed-gas flow rate (L/min), QG,out is off-gas flow rate (L/min), and t is ozone contact time

(min).

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Figure S1. Dose-response relationship for glucocorticoid response in MDA-kb2 cells co-

exposed to different concentrations of DEX and 100% of untreated OSPW, ozone-treated

OSPW, and Merichem NAs. An * indicates a significant change between treatment groups at the

same concentration of DEX (p < 0.05).

Figure S1.

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