In Vitro Assessment of Endocrine Disrupting Potential of Naphthenic

Article pubs.acs.org/est

In Vitro Assessment of Endocrine Disrupting Potential of Naphthenic Acid Fractions Derived from Oil Sands-Influenced Water Liane A. Leclair,† Lani Pohler,‡ Steve B. Wiseman,‡ Yuhe He,‡ Collin J. Arens,† John P. Giesy,‡,§ Stephen Scully,⊥ Brian D. Wagner,⊥ Michael R. van den Heuvel,† and Natacha S. Hogan*,‡,∥ †

Canadian Rivers Institute, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada § Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada ∥ Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada ⊥ Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada ‡

ABSTRACT: Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, 1H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.

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INTRODUCTION

Exposure to oil sands-influenced waters has been reported to induce a range of toxicological effects in fishes including endocrine disruption. Studies have demonstrated decreased plasma sex steroid concentrations and reduced gonad development associated with elevated CYP1A activity in yellow perch (Perca f lavescens).2,3 Reduced sex steroids have also been

In 2010, the Athabasca oil sands industry in northern Alberta had accumulated just under 840 million m3 of tailings and process-affected water.1 Under a zero-discharge policy, tailings and affected water must be stored on site within tailings ponds or incorporated back into a reclaimed landscape. This waste material contains particulate matter (sand and silt) as well as inorganic and organic compounds such as metals, ions, naphthenic acids (NAs), and polycyclic aromatic hydrocarbons (PAHs). © 2015 American Chemical Society

Received: Revised: Accepted: Published: 5743

January 6, 2015 March 31, 2015 April 2, 2015 April 2, 2015 DOI: 10.1021/acs.est.5b00077 Environ. Sci. Technol. 2015, 49, 5743−5752

Article

Environmental Science & Technology observed in goldfish (Carassius auratus)4 and fathead minnows (Pimephales promelas)5 exposed to aged oil sands-affected process waters (OSPW), as well as decreased sex steroid production in gonadal tissue from slimy sculpin (Cottus cognatus) collected within tributaries of the Athabasca river in the Athabasca oil sands area relative to fish from a reference site.6 Explants of ovarian and testicular tissue from goldfish exposed to OSPW also had decreased synthesis of testosterone (T) and 17β-estradiol (E2).4 In vitro studies have shown that OSPW from an active settling basin (receiving raw fresh OSPW) can decrease production of T and increase E2 in a steroidogenesis assay using H295R cells7 as well as affect androgen and estrogen receptor signaling.8 The constituents or mechanisms of oil sands-influenced waters responsible for the endocrine disrupting effects are largely unknown. NAs have been suspected of being involved in reproductive responses largely because they are the dominant class of organic compounds present at mg/L concentrations,3,5 not because of any mechanistic evidence. NAs are composed of acyclic, monocyclic, and polycyclic carboxylic acids, with the general formula of CnH2n+ZO2, where n represents the carbon number and Z is zero or a negative, that specifies the hydrogen deficiency resulting from ring formation or double bonds.9 Fresh (subject to little environmental weathering or biodegradation) NA mixtures have been shown to be responsible for acute lethality in aquatic biota in oil sands-influenced water.9,10 Fathead minnows exposed to NAs extracted from oil sandsinfluenced waters (which we define as any water being influenced by either anthropogenic or natural chemical influence from oil sands bitumen) spawned fewer eggs, and males had fewer secondary sexual traits as well as lower levels of T and 11-ketotestosterone.11 Recently, the aromatic components of oil sands derived NAs have been shown to possess weak estrogen agonist activity.12 There is a need for further research on mechanisms of toxicity to determine whether NAs derived directly from oil sands sources are able to influence reproductive pathways directly. In vitro tools are best suited for this approach. The objective of this study was to assess the potential for NAs purified from OSPW to interact with receptor-mediated and steroidogenic reproductive pathways. Given the consistently observed CYP1A activity in fishes in vitro and the possible involvement of Ah receptor-active compounds in reproductive toxicity, the ability of NAs to induce CYP1A is also important. NAs were extracted using an acid precipitation method, separated from humic material, and further purified into four fractions. Four well-established in vitro assays were used to screen for compounds that (1) bind the arylhydrocarbon receptor (AhR) using H4IIE-luc cell line, (2) interfere with steroid biosynthesis using the H295R steroidogenesis cell line, and (3) exhibit estrogenicity, androgenicity, antiestrogenicity, and antiandrogenicity using the using the yeast androgen screen (YAS) and the yeast estrogen screen (YES). Fractions were chemically characterized by high resolution mass spectrometry (HRMS), 1H nuclear magnetic resonance (NMR), and attenuated total reflectance (ATR) infrared spectroscopy to relate their chemical nature to activity observed in the chosen bioassays.

Syncrude Canada, Fort McMurray, Canada. This water was chosen as it represented tailings water that had been aged approximately 17 years at the time of study in a storage pond open to the elements. Pond 10 water has also been observed to contain 40 mg/L of naphthenic acids, one of the higher levels at experimental ponds in the area, providing for the greatest possible yield of naphthenic acids for the effort. Furthermore, waters in the Syncrude experimental pond complex from the exact same tailings source have been found to show reproductive effects in fishes.3,5 Extraction methods were based on those developed by Frank et al.13 and modifications thereof detailed by MacDonald et al.14 (Figure 1). Further modifications described here were developed in order to capture all components of the mixture in our fraction, particularly that part of the mixture that did not reprecipitate in the last step and to characterize the neutral extract. The result of this was four fractions that captured the maximum possible amount and diversity of the NAs extracted. Briefly, 4000 L of water was acidified to pH 2 ± 0.2 with H2SO4 (Sigma, Oakville, Canada), and the precipitate was removed and redissolved in pH 10 ± 0.2 with 0.1 M NaOH (Sigma). Particulate matter was removed via centrifugation of the basic solution, and humic material was removed via DEAE cellulose filtration. Liquid−liquid extraction with dichloromethane (DCM) was performed to remove neutrals, and the DCM was removed by nitrogen evaporation (henceforth called the “DCM fraction”). The NAs were reprecipitated after adjustment to pH 2.0 and spun at 17 000g for 15 min. The pellet was washed with distilled water and freeze-dried to produce a solid material (henceforth called the “main fraction”). The supernatant was passed through a C18 cartridge and eluted with 100% MeOH (called “C18 MeOH” fraction) followed by 1:1 MeOH/0.1 M NaOH (“C18 NaOH” fraction). Those four fractions were (1) the DCM fraction, (2) the main fraction, (3) the C18 MeOH fraction, and (4) the C18 NaOH fraction were subsequently used for chemical analyses and in vitro bioassays. In Vitro Bioassay Justification. Hormone mimics or antagonists of estrogen or androgen receptor-mediated processes have been shown to play major roles in endocrine disruption. While receptor binding can be conducted for fishes, the inability of receptor binding approaches to differentiate agonism from antagonism led to the choice of a reporter gene assay that has the potential to determine both mechanisms. As there are currently no commonly available fish-based bioassays to suit this purpose, the well-established YES and YAS bioassays were chosen. While these are based on the mammalian (human) androgen and estrogen receptors, studies have shown that receptor binding affinity between mammals can vary as much as between mammals and fishes.15 Steroidogenesis is commonly disturbed in organisms exposed to endocrine disrupting substances, and there is only one established bioassay for steroidogenesis that is based on an immortal cell line, the H295R human cell line. Short of steroidogenesis in fish explants, which can have high variability and can usually only be conducted at particular times of year, this bioassay represents one of the only tools available for this end point. While there are multiple bioassays for Ah-receptor active substances, most depend on measurement of catalytic activity of induced CYP1A enzymes that can be inhibited by other compounds in complex mixtures such as those used here. The H4IIE-luc reporter gene assay was chosen as it measures Ah-receptor binding without the use of a catalytic end point

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METHODS Naphthenic Acid Extraction. NAs used in this study were extracted by acid precipitation from 17 year old tailings pond water from a small tailings storage pond (Pond 10) located at 5744

DOI: 10.1021/acs.est.5b00077 Environ. Sci. Technol. 2015, 49, 5743−5752

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Environmental Science & Technology

Figure 1. Procedure for bulk extraction and fractionation of NA extracted from oil sands-influenced waters.

incubated for 24 h. Media was removed and fractions dissolved (in 0.05% v/v, 1:1 NaOH/DMSO) were diluted in media and added to the plates in triplicate wells. Final NA concentrations ranged from 0.005 to 5 mg/L with time points at 24, 48, and 72 h conducted in two separate trials. Cells were harvested at each time point, and luciferase activity was measured by use of the SteadylitePlus Kit (PerkinElmer, MA, USA). Dose−response curves for the NA fractions were compared with that obtained using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Wellington Laboratory, Guelph, ON) to determine TCDD equivalent concentration. H295R Steroidogenesis Assay. The H295R steroidogenesis assay was performed according to established protocols.18,19 H295R adrenal cells were obtained from ATCC (CRL-2128) and propagated at 37 °C and 5% CO2 in a 1:1 mixture of DMEM/Hams F-12 medium supplemented with 2.5% Nu-Serum (BD Biosciences, San Jose, CA, USA), 1% ITS + Premix (BD Biosciences), and 1.2 g/L Na2CO3. Cells were plated in 24 well plates at a density of 3 × 105 cells/mL and incubated for 24 h. Media was removed, and media containing final concentrations for each fraction from 0.005 to 5 mg/L (four concentrations total) dissolved in 1:1 NaOH:DMSO (0.05% v/v final carrier volume) was added and incubated for 48 h. Forskolin (0.1 and 10 μM) and prochloraz (0.3 and 3 μM) were used as positive controls. Media was collected and measured for testosterone, progesterone, corticosterone, and androstenedione by high-performance liquid chromatography (HPLC)/mass spectrometry (MS) using an aqueous 0.1% formic acid in a nanopure and methanol gradient delivered at a flow rate of 250 μL/min. Samples were injected onto a Betasil C18 column (Thermo Electron Corporation, Waltham, MA) connected to a C18 guard column. A triple quadruple mass spectrometer, SpectraMax

CYP1A determination and has been shown to be more sensitive than other forms of the H4IIE bioassay.16 Cytotoxicity. Cytotoxicity was conducted only using the H4IIE-luc cells since, based upon experience in the laboratory, cytotoxicity between H295R and H4IIE cells varies by only 20%. The YES and the YAS bioassays incorporate an absorbance measure of cell density/proliferation during the bioassay, so cytotoxicity is incorporated into every bioassay. The WST-1 bioassay was performed with cells to determine cytotoxic concentrations of each fraction using the H4IIE-luc cells. The principle is based on cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells to form a yellow formazan dye. A lesser absorbance is therefore interpreted as a decrease in cell viability. H4IIE-luc rat hepatoma cells were used,17 and cells were propagated in DMEM/F-12 media containing 10% fetal bovine serum (FBS) at 37 °C, 5% CO2. Cytotoxicity of fractions were evaluated by exposing 8 × 104 H4IIE-luc cells to concentrations of fractions ranging from 0.05 to 50 mg/L (4 concentrations plus controls) for 24 h with 3 replicates. A 30 min treatment with the tetrazolium salt WST-1 (Roche Applied Science, Indianapolis, IN) was performed, and the absorbance was measured at 450 nm using a POLARStar OPTIMA microplate reader (BMG Labtech). Aryl Hydrocarbon Receptor Transactivation Assay. The assay to determine the potential for each fraction to activate a reporter gene through binding to the AhR was conducted as previously described17 with modifications. The amount of AhR-induced luciferase was quantified using the LucLite(R) Reporter Gene Assay System (PerkinElmer, Netherlands). H4IIE-luc cells were propagated in DMEM containing 10% FBS at 37 °C, 5% CO2. Cells were plated to a concentration of 8 × 104 cells/mL in 96-well plates and 5745


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Nuclear Magnetic Resonance. Proton (1H) nuclear magnetic resonance (NMR) characterization data were collected at 298 K on a Bruker AV-300 spectrometer operating at 300.1 MHz with chemical shifts reported in parts per million (ppm) downfield of SiMe4. Each fraction was dissolved in dDMSO (Cambridge Isotope, St Léonard, QC, Canada) and held in NMR tubes (5 mm, Sigma). Spectra data was analyzed for peak intensities and chemical shifts. Statistics. NA mixtures in the H4IIE-luc assay were quantified as their TCDD equivalent factor; YES were evaluated as 17β-estradiol (agonist) and 4-hydroxytamoxifen (antagonist) equivalent factors, and YAS cell assays were evaluated for DHT (agonist) and flutamide antagonist equivalent factors. Nonlinear logistic dose vs effect best fit curves, Y = Xmin + ((Xmax − Xmin)/(1 + ((X/EC50) ^Hillslope))), were used to determine the EC50 (n = 3) for each treatment on a mass basis since no molecular weight can be attributed to the NA mixture. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) was used for these analyses. H295R cell line assay data were analyzed by testing for normality and homogeneity of variance (Levene’s and BrownForsythe tests, respectively) with appropriate transformations where those assumptions were not met. A full factorial two-way ANOVA comparing both trials and dose effect was performed for the H295R cell line assay followed by a posthoc test of treatments against solvent controls by the use of Dunnett’s test. Statistical analyses were conducted using STATISTICA (v8.0, Statsoft Corporation, Tulsa, OK) with an experiment-wise alpha of 0.05.

190 (Molecular Devices Corp., Sunnyvale, CA, USA), operating in positive electrospray ionization multiple reaction monitoring (MRM) mode was used to measure hormone concentrations. Yeast-Based Screening Assays. The yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to quantify the estrogenic, antiestrogenic, androgenic, and antiandrogenic activity of the NA fractions. Genetically modified (recombinant) yeast (Saccharomyces cerevisiae) cells were cultured, and assays were performed as per Routledge and Sumpter20 with some modifications. Yeast cells were cultured in a 250 mL conical flask in media for 24 h at 28 °C. The fractions dissolved in 5% NaOH in DMSO were serially diluted in 2-fold steps, and 4 μL of each concentration was transferred to a 96well flat-bottom microtiter plate in triplicate to give final concentrations in media of 0.019−9.9 mg/L (10 concentrations). YES standard and fraction plates tested were incubated at 38 °C for 72 h, and YAS standard and fractions plates were incubated for 24 h at 38 °C and 48 h at room temperature. Plates were subsequently read on a microplate reader using a Bio-Tek ELx800UV plate reader (USA) at 550 and 630 nm. The 630 nm optical density reflects the yeast cell number; thus, cytotoxicity is incorporated into the bioassay. While the H4IIE cytotoxicity bioassay showed effects at greater concentrations used for YES and YES, no effects on cell density were observed at any of the concentrations used here. Absorbance derived from the exposure to fractions was compared with a standard curve for E2 and dihydroxytestosterone (DHT) for estrogenicity and androgenicity, respectively, and curves of 4-hydroxytamoxifen and flutamide for antiestrogenicity and antiandrogenicity, respectively, to determine equivalents of the respective standards. High Resolution Mass Spectrometry. Each fraction was examined by high resolution mass spectrometry (HRMS) using a Thermo Scientific Velos Orbitrap mass spectrometer equipped with an electrospray ionization interface. The sample was infused into the mass spectrometer at a rate of 2.5 μL min−1. The mass spectrometer was equipped with an electrospray ionization (ESI) source operated in negative mode. Analyses were recorded with the highest mass resolution mode (100 000), and the observed ions [M − H]− from m/z 90 to 400 were used to determine the relative concentration of the NAs that fit the formula CnH2n+ZO2, where Z is the hydrogen deficiency (an indication of the number of rings and/ or double bonds). Acid extractable structures not corresponding to the CnH2n+ZO2 formula are known to be present in weathered oil sands material, particularly O3 and O4 NAs, presumed to be alcohols of the primary NAs structures. In addition to the O2 structures described above, O3 and O4 NAs were evaluated using their predicted molecular mass. Ratios of O2, O3, and O4 NAs were derived by summing the total ion intensity of all ions within those three groups corresponding to m/z ratios of possible structures from to 5 to 30 carbons and from Z = 0 to Z = −30. Attenuated Total Resonance Infrared Spectroscopy. Fractions were characterized using attenuated total resonance (ATR) infrared spectroscopy to determine the functional groups found in each fraction. The data were collected at 293 K on a Bruker Alpha spectrometer with an optical resolution of 0.9 cm−1 equipped with an Alpha-P ATR accessory with a diamond crystal. Dry samples (C18 NaOH, C18 MeOH, main) were grounded to a fine powder before applying them directly to the plate. The spectra were then analyzed and compared for peak location and intensity.

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RESULTS Toxicity, Endocrine Disruption, and Ah-Receptor Binding of Fractions. The WST-1 assay showed cytotoxicity at 50 mg/L of each fraction tested. On the basis of these results, final concentrations in media used for the H4IIE-luc and H295R cell assays ranged from 0.005 to 5 mg/L. The DCM fraction exhibited AhR agonist activity at 5 mg/L after an incubation period of 24 h (Figure 2). This corresponded to a TCDD equivalent concentration of 1.99 × 10−6 mg/L after 24 h, and activity dissipated at 48 and 72 h. The other three fractions tested did not cause transactivation after 24, 48, or 72 h.

Figure 2. Dose−response curve of TCDD and NA fractions using H4IIE-luc cells after 24 h. Samples were assayed in triplicate, and two trials were conducted. Average, SEM, and nonlinear fit curve are shown (n = 2). 5746


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Figure 3. Concentration (ng/mL) of androstenedione (A), testosterone (B), progesterone (C), and corticosterone (D) in media of H295R cells exposed to NA fractions for 48 h. Incubation with the carrier NaOH/DMSO (0.05% v/v) was included as a control (C) as well as prochloraz (P) or forkoslin (F) as modulators of steroidogenesis. Samples were assayed in triplicate, and two trials were conducted. Average and SEM are shown (n = 2/treatment). Asterisk indicates a significant difference between the exposed and control group within each experiment by two-way ANOVA with Dunnett’s test.

also suggested by ATR infrared spectrometry by the broad shoulder peak around 3500−3200 cm−1 (Figure 5). Each fraction demonstrated a carboxylic acid dimer and a broad carboxylic acid OH stretch signature underlying the CH bend at 1700 and 3200−3000 cm−1, respectively. 1H NMR analysis of each fraction demonstrated that there was aromatic content in all fractions (Figure 6A). Unresolved peaks in the 6.8−7.4 ppm region suggested monocyclic aromatic moieties in all fractions (Figure 6B). A more complex pattern of conjugated aromatic rings is suggested by the peaks at 7.7 ppm in the DCM and main fraction only. However, on the basis of aromatic/aliphatic proton ratios from the proton integrations, the DCM fraction had a lesser proportion of aromatic protons than the other fractions (Table 2). The two C18 fractions contained the greatest relative quantity of aromatic protons. The DCM fraction was the only fraction that had the clear presence of olefinic protons in the 5−6 ppm region.

The H295R steroidogenesis assay was used to determine whether fractions would affect production of steroid hormones (androstenedione, testosterone, progesterone, and corticosterone) (Figure 3). Exposure to the main fraction at 5 mg/L resulted in a significant increase in corticosterone production compared to media collected from cells exposed to solvent control. Exposure to 0.05−0.5 mg/L of the C18 MeOH fraction resulted in greater production of progesterone when compared to the solvent control although no dose−response relationship was observed. The DCM and C18 NaOH fractions had no effect on hormone production at any concentration tested. Neither of the fractions of NAs had estrogenic effects in the YES assay. The DCM, main, and C18 MeOH fractions of NAs all showed antagonized effects of 17β-estradiol (Figure 4A). The 4-hydroxytamoxifen equivalent factor for those fractions was 3.7 × 10−3, 1.2 × 10−3, and 2.8 × 10−4 for the DCM, main, and C18 MeOH fractions, respectively (Table 1). There was no androgenicity present in any of the fractions at the concentrations tested. The DCM and main NA fractions showed an antagonistic effect in the YAS (Figure 4B). The flutamide equivalent factor for those fractions was 5.9 and 4.72 for DCM and main NA fractions, respectively (Table 1). Spectroscopic Analyses. HRMS demonstrated that all fractions contained a suite of compounds with m/z ratios consistent with NAs. The C18 MeOH and C18 NaOH fraction were generally enriched in NAs with 14 or less carbons and depleted in NAs with 15 or more carbons when compared to the main fraction (Table 2). In addition, the C18 MeOH fraction had a greater proportion of NAO3 and NAO4 presumably caused by one or two additional OH groups (Table 2) as HRMS analysis did not show −2 charge with any of the NAO3 or NAO4 ions. Additional hydroxyl groups were

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DISCUSSION There were stimulatory effects of NA fractions on steroid hormone production. There was no measurable estrogenic or androgenic activity in any of the fractions as assessed by YES and YAS. Antiestrogenic and antiandrogenic activity was detected in most of the fractions and was greatest in the DCM fraction. Only the DCM fraction was found to contain AhR agonists. The results for antiestrogenicity, antiandrogenicity, and AhR agonism were not consistent with increased aromaticity of extracted fractions according to 1H NMR. It was likely that neutral compounds present in the DCM fraction caused the AhR potency observed in H4IIE-luc cells after 24 h exposure. Planar compounds such as polycyclic aromatic hydrocarbons (PAHs), biphenyls (PCBs), and 2,3,7,8tetrachlorodibenzodioxin (TCDD) induce CYP1A activity.21,22 5747


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properties.23,24 However, the waters used in that study were relatively fresh as opposed to the aged waters used in the present study. Furthermore, the AhR agonist compounds found in the DCM fraction are concentrated from 4000 L of aged tailings water, and enriched in that fraction, extrapolating to the original tailings water, the concentration would have been approximately 0.2 ng/L as 2,3,7,8-TCDD. The acid precipitation technique was not designed for the extraction of neutrals, and no information is available as to the efficiency of the coextraction of neutrals; so, this is likely an underestimate of what might be present. The environmental relevance of the H4IIE-luc bioassay results were supported by the observations that tailings waters of the same origin consistently showed CYP1A induction in fishes exposed to tailings-influenced waters of the same origin.2,3,25,26 Also consistent with previous results was that the main NA fraction used here also failed to induce CYP1A in rainbow trout as measured by EROD activity in previous studies.14,27 Aromatic compounds found in the DCM fraction are suggestive of a structure containing at least two benzene rings. On the basis of the 1H NMR spectra, the two benzene rings are likely not conjugated, as such conjugated rings would show peaks in the spectrum at higher than 8 ppm. The peak present around 7.7 ppm is consistent with compounds such as dibenzothiophenes or other heterocycles. Dibenzothiophenes and heterocyclic aromatic compounds have been used in previous investigations as simple analogs of organic S found in petroleum materials.28 Dibenzothiophenes, heterocycles, and PAHs have previously been found in oil sands tailings pond in sediments29 as well as in insect larvae and adult insects.30 The presence of hydroxy acids (O3 compounds) has been reported in OSPW in northern Alberta.31 The present study also identified oxidized NAs and showed that they are removed from the main NA fraction by the extraction process. Despite rapid degradation, other NA compounds remain recalcitrant for decades and we would speculate that diamondoid-type structures could be resistant to bacterial degradation. The lesser molecular weight and more water-soluble C18 fractions showed little potency in the assays conducted here, with the exception of some weak antiestrogenicity. These fractions contained a larger proportion of NAO3 and NAO4 when compared to the DCM and main fraction. The NAO3 and NAO4 compounds would be less likely to precipitate with the addition of H2SO4 due to greater polarity imparted by one or two hydroxyl or ether groups. The smaller average molecular weight of the C18 fractions would also have contributed to

Figure 4. Dose−response curves for estrogen receptor antagonist (YES) activity (A) and androgen receptor antagonist (YAS) activity (B) of each NA fraction determined using the yeast screening assays. NaOH/DMSO (5% NaOH) was used as a solvent control. Average, SEM, and nonlinear best fit curve are shown (n = 3).

Furthermore, the CYP1A induction observed was attenuated after 48 and 72 h, which suggested that compounds found in this fraction are labile and biotransformed within a day, consistent with PAHs. However, the results of 1H NMR analyses suggested that the AhR-active fraction was not more enriched in aromatic moieties, and IR showed that this fraction was still dominated by NAs. Neutral chemicals, such as PAHs, may have comprised only a small portion of the mixture. In previous studies employing the same assay, OSPW from WestIn-Pit, an active settling basin, did not show AhR-active

Table 1. EC50 Concentrations and Toxicity Equivalency Factors (TEF) for Each Fraction and Standards for the YES, YAS, and H4IIE-luc Assaya EC50 (mg/L) YES

a

YAS

DCM C18 MeOH C18 NaOH main

0.45 5.88 9.57 1.43

1.49 11.34 16.37 1.87

4-hydroxyTamoxifen flutamide TCDD

0.0017 − −

− 8.81 −

TEF H4IIE-luc Fractions 9.82 − − − standards − − 1.96 × 10−5

YES

YAS

H4IIE-luc

0.0037 0.00028 0.00017 0.0012

5.90 0.78 0.52 4.72

1.99 × 10−6 − − −

− − −

− − −

− − −

Hyphens indicate either no significant activity in the respective bioassay or an inability to calculate an EC50 (less than 50% of maximal response). 5748


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Environmental Science & Technology Table 2. Average (SEM) and Most Abundant Carbon and Z Number for Each Fractiona average fractions C18 MeOH C18 NaOH DCM main

carbon 12.62 13.08 15.28 15.35

(0.23) (0.27) (0.27) (0.34)

most abundant Z number −3.75 −4.08 −4.38 −5.33

(0.20) (0.07) (0.06) (0.24)

NA (%)

carbon

Z number

O2

O3

O4

aromatic/aliphatic ratio

14 14 16 15

−4 −4 −4 −4

88.08 96.06 96.42 99.82

9.79 3.37 2.97 0.15

2.14 0.57 0.61 0.03

1:5.7 1:2.7 1:35.4 1:26.3

a

NA percentages demonstrate the relative concentration of each compound family, the NA O2, NA with one additional oxygen (O3), and NA with two additional oxygens (O4) (charge of −1) based on parent ion intensity.

Figure 5. ATR infrared spectroscopy of four NA fractions extracted from oil sands process-affected waters. (A) hydroxyl groups, (B) CH bend, and (C) carboxylic acid dimer.

their greater water solubility. The higher relative aromatic content of these fractions might also suggest that they were contaminated with some residual humic material not captured by the DEAE cellulose. The antiestrogenic and antiandrogenic activity demonstrated herein is in accordance with previously observed effects of NAs or oil sands-influenced waters. In the only other similar study, NAs present in North Sea offshore produced water were weak estrogen receptor (ER) agonists and androgen receptor (AR) antagonists.32 This differs from the results of the present study; however, the chemical nature of the material used in the respective studies could vary significantly. It should also be cautioned that antiandrogenicity and antiestrogenicity found in the present study does not demonstrate that this effect occurs due to competitive binding at the receptor level. As one possible example, the fractions may influence the potency or bioavailabilty of T or E2 by altering bioavailability as has been observed for other hydrophobic organic compounds.33 Reproductive-endocrine disruption has been reported in several fishes exposed to OSPW and extracted constituents with suppression of steroidogenesis being proposed as a predominant mechanism of action.7,34 The extracted fractions in the present study did not alter steroid hormone production in H295R cells. The difference in effects observed in this study, versus the results of previous in vitro studies, may be due to several factors. For example, in the in vitro study by He et al.34

where steroidogenesis was affected, whole fresh OSPW was applied to the cells. This would contain not only unweathered NAs but also potentially salts and ammonia not contained in our extracts that sought to isolate the effects of naphthenic acids. Aging of OSPW has also been shown to change the NA profile; therefore, the compounds responsible for the effects on steroidogenesis may have undergone degradation in aged OSPW used in this study. The most important conclusion of this study was that naphthenic acids derived from oil sands-affected waters can act through a mechanism of steroid antagonism at environmentally relevant concentrations. The novel aim of this study was to separate out NAs from other components of the mixture such as neutrals, salts, and ammonia so that conclusions could be definitely made regarding the endocrine disrupting effects of this family of compounds. Antiandrogenic/antiestrogenic potency of NAs demonstrated here was found at concentrations that would be found in oil sands-influenced waters. Reproductive impacts in yellow perch were not obvious at concentrations less than 7 mg/L of NAs35 but did become apparent at concentrations of 13 mg/L NA.3 A number of other studies using fathead minnow have found reproductive effects, in some cases in the same pond water, at NA concentrations within this same range,5,11,36 and thus steroid antagonism by NAs could be a potential mechanism for reproductive effects observed in vivo. The YES and YAS EC50s ranged from 5749


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Figure 6. 1H NMR of the main fraction (A) and a comparison of four NA fractions (B) extracted from oil sands process-affected waters. Spectra were centered on the aromatic region for comparison.

disrupters at concentrations found in tailings waters and in a number of reclamation scenarios. While this may have been suspected previously, this is one of the only studies to separate these components from the rest of the oil sands mixture. Second, neutral compounds, such as PAHs, may also be relevant as these have an alternative mechanisms of action in this mixture (that can also influence reproduction), though the precise identity of these compounds has yet to be determined.

approximately 0.5 to 16 mg/L NA, well within the range observed to cause effects on fishes. While the compounds responsible for the antiandrogenic and antiestrogenic effects seen here are not known, steroidal aromatic NAs37 as well as alkylphenols35,38 have been previously reported in the Athabasca OSPW. Recently, steroidal NAs have been shown to be weakly estrogenic to zebrafish.12 The research conducted has important implications for the management of tailings water in oil sands reclamation. As a zero discharge industry with nearly a billion cubic meters of tailings and tailings-contaminated waters stored, the need to eventually release these wastewaters is inevitable. This study and others have shown that oil sands-influenced waters can be aged nearly two decades and still disrupt endocrine function, which can result in adverse effects on reproduction of fish. When those waters are released, they must be treated in such a way that they pose little environmental impacts. However, this is very challenging when the environmental impacts of the mixture are uncertain and there are no guidelines for what constitutes safe release. This research contributes to that challenge in two ways. First, NAs are of concern as potential endocrine

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AUTHOR INFORMATION

Corresponding Author

*Phone: 306-966-6862; fax: 306-966-4151; e-mail: natacha. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Syncrude Canada, Suncor Energy, Shell Albian Sands, Total E&P Canada, and Canadian Natural Resources Limited under the auspices of Canadian Oil Sands 5750


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Network for Research and Development and by NSERC CRD grants and Canada Research Chairs held by Michael R. van den Heuvel and John Giesy.

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In Vitro Assessment of Endocrine Disrupting Potential of Naphthenic

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