Steroidal Aromatic 'Naphthenic Acids' - American Chemical Society

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Steroidal Aromatic ‘Naphthenic Acids’ in Oil Sands Process-Affected Water: Structural Comparisons with Environmental Estrogens Steven J. Rowland,*,† Charles E. West,† David Jones,† Alan G. Scarlett,† Richard A. Frank,‡ and L. Mark Hewitt‡ †

Petroleum and Environmental Geochemistry Group, Biogeochemistry Research Centre, University of Plymouth, Drake Circus, Plymouth PL4 8AA, U.K. ‡ Aquatic Ecosystems Protection Research Division/Water Science & Technology Directorate, Environment Canada, 867 Lakeshore Road, Burlington, ON, Canada L7R 4A6

bS Supporting Information ABSTRACT: The large volumes, acute toxicity, estrogenicity, and antiandrogenicity of process-affected waters accruing in tailings ponds from the operations of the Alberta oil sands industries pose a significant task for environmental reclamation. Synchronous fluorescence spectra (SFS) suggest that oil sands process-affected water (OSPW) may contain aromatic carboxylic acids, which are among the potentially environmentally important toxicants, but no such acids have yet been identified, limiting interpretations of the results of estrogenicity and other assays. Here we show that multidimensional comprehensive gas chromatography
mass spectrometry (GCxGC-MS) of methyl esters of acids in an OSPW sample produces mass spectra consistent with their assignment as C19 and C20 C-ring monoaromatic hydroxy steroid acids, D-ring opened hydroxy and nonhydroxy polyhydrophenanthroic acids with one aromatic and two alicyclic rings and A-ring opened steroidal keto acids. High resolution MS data support the assignment of several of the so-called ‘O3’ species. When fractions of distilled, esterified, OSPW acid-extractable organics were examined, the putative aromatics were mainly present in a high boiling fraction; when examined by argentation thin layer chromatography, some were present in a fraction with a retardation factor between that of the methyl esters of synthetic monoalicyclic and monoaromatic acids. Ultraviolet absorption spectra of these fractions indicated the presence of benzenoid moieties. SFS of model octahydro- and tetrahydrophenanthroic acids produced emissions at the characteristic excitation wavelengths observed in some OSPW extracts, consistent with the postulations from ultraviolet spectroscopy and mass spectrometry data. We suggest the acids originate from extensive biodegradation of C-ring monoaromatic steroid hydrocarbons and offer a means of differentiating residues at different biodegradation stages in tailings ponds. Structural similarities with estrone and estradiol imply that such compounds may account for some of the environmental estrogenic activity reported in OSPW acidextractable organics and naphthenic acids.

’ INTRODUCTION Widespread interest in reclaiming the large volumes of process-affected water (OSPW) resulting from the surface mining of oil sands, particularly those contained within the industrial leases in northeastern Alberta, Canada, has catalyzed a spate of studies attempting to identify the toxic constituents1
3 and numerous others attempting to delimit the toxic effects.4
6 Recently, we described the chromatographic resolution and mass spectral identification of the methyl esters of some of the first individual alicyclic acids in an acid-extractable OSPW mixture by multidimensional comprehensive gas chromatography
mass spectrometry3,7 (GCxGC-MS). The toxicity of these individual trito pentacyclic diamondoid acids to the bacterium Vibrio fischeri, measured in a screening assay and the modeled toxic effects on the water flea Daphnia magna, were those expected to result from nonspecific narcosis8 and exhibited a water solubility-controlled toxicity ‘cutoff’ at higher carbon numbers. The toxicity of these r 2011 American Chemical Society

acids was sufficient to explain the narcotic toxicity of some OSPW fractions.9 Whether the same acids are responsible for more specific toxicological actions remains to be examined. Components other than the alicyclic acids so far identified in OSPW may be more toxicologically important.2,10,11 Numerous examinations of OSPW acid-extractable organic matter by electrospray ionization high and low resolution mass spectrometry, sometimes coupled with liquid chromatography,12,13 have shown that the acid-extractables of OSPW contain compounds with four or more double bond equivalents (DBE). These are designated by a z number of g
8 in the formula CnH2n+zO2 which is often used to describe the acids. While such acids undoubtedly include Received: July 27, 2011 Accepted: October 7, 2011 Revised: October 5, 2011 Published: October 20, 2011 9806

dx.doi.org/10.1021/es202606d | Environ. Sci. Technol. 2011, 45, 9806–9815

Environmental Science & Technology tetracyclic (z =
8) and pentacyclic (z =
10) compounds,7 no hexacyclic (z =
12) species have been identified to date, and an alternative explanation for some of the z g
8 (four or more double bond equivalents) species is that they include monoaromatic species (gfour DBE) with zero, one, or more alicyclic rings. Evidence for this also includes distinctive synchronous fluorescence spectra (SFS) associated with OSPW and groundwater near a tailings pond.14 Characterization of these acids would begin to address important knowledge gaps about the toxicity and mobility of OSPW components and help to direct potential methods for remediating the toxicants. We now describe the chromatographic resolution and mass spectral identification of some individual steroidal aromatic acids in the acid-extractables from an OSPW by GCxGC-MS. We provide supporting evidence for the identifications from argentation thin layer chromatography, ultraviolet (UV) and SFS, including for authentic monoaromatic acids and postulate a reasoned argument for the origins of the acids. The presence of such acids may help to explain some of the environmental estrogenic activity of OSPW and other naphthenic acid mixtures.

’ MATERIALS AND METHODS The OSPW acidic extract was obtained from a previous study where the organic acids were extracted from 3000 L of fresh OSPW collected from the discharge into West In-Pit settling basin at Syncrude Canada Ltd.15 Distilled fractions from the same organic acid extract were also generated from a previous study.9 In the present study, acids were derivatized by refluxing with BF3-methanol or reaction with diazomethane (distilled fractions9). Authentic aromatic acids used were dehydroabietic acid (DHA) from Helix Biotech (Vancouver, BC, Canada) and 1,2,3, 4-tetrahydrophenanthrene-1,2-dicarboxylic acid dimethyl ester (TPDADE) and phenanthrene-4-carboxylic acid (PCA; both from Sigma-Aldrich (Oakville, ON, Canada). Methyl esters of cyclohexyl-3-propanoic and phenyl-3-propanoic acid used as TLC references were from previous studies.16 Argentation thin layer chromatography was conducted on the methylated OSPW extract with silica gel stationary phase containing 5% by weight silver nitrate to generate three TLC fractions. Synchronous fluorescence spectroscopy was performed as described previously14 using a Perkin-Elmer Luminescence spectrometer LS50B paired with FL Winlab 3 software (PerkinElmer, Norwalk, CT) for data collection. In brief, samples were scanned in a 1 cm quartz cuvette with a PTFE stopper (Hellman, Concord, ON, Canada) at 20 °C. SFS were collected at a Δλ of 18 nm in the 250
400 nm excitation wavelength range. The scan speed was 50 nm min
1 at a resolution of 0.5 nm, and the excitation and emission monchromator slit widths were set at 5 nm. The spectrum of each sample was blank-corrected with HPLC water (Caledon Laboratory Chemicals, Georgetown, ON, Canada) and then smoothed with a 5-point averaging adjacent method using OriginPro software ver. 8.01 (OriginLab Corp., Northampton, MA). Two-dimensional comprehensive gas chromatography
mass spectrometry (GCxGC-MS) analyses were conducted as described previously.3 Briefly, an Agilent 7890A gas chromatograph (Agilent Technologies, Wilmington, DE) fitted with a Zoex ZX2 GCxGC cryogenic modulator (Houston, TX, USA) interfaced with an Almsco BenchTOFdx time-of-flight mass spectrometer

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(Almsco International, Llantrisant, Wales, UK) was operated in positive ion electron ionization mode and calibrated with perfluorotributylamine. For GC-MS, extracts were examined on an Agilent GC-MSD (Agilent Technologies, Wilmington, DE, USA). This comprised a 7890A gas chromatograph fitted with a 7683B Series autosampler and a 5975A quadrupole mass selective detector. The column was a HP-5MS fused silica capillary column (30 m  0.25 mm internal diameter  0.25 μm film thickness). The carrier gas was helium at a constant flow of 1.0 mL min
1. A 1.0 μL sample was injected into a 300 °C splitless injector. The oven temperature was programmed from 40 to 300 at 10 °C min
1 and held for 10 min. High resolution MS accurate mass measurements were made using a Thermofisher LTQ Orbitrap XL high resolution mass spectrometer. The mass range was m/z 120
2000; mass accuracy ∼100 min GC1) contained several very well resolved components with clear mass spectra and molecular weights of around 300 Da and apparently six (z =
12) to eight (Z =
16) DBE. One group of well-resolved compounds was highlighted by selected ion mass chromatography of the m/z 145 base peak ion (Figure 1B), others by m/z 237, 223 and by high mass (>300 Da) ions. When a distilled fraction9 of the methyl esters of the same OSPW extract boiling at 220 °C was examined by GC-MS, several resolved components were observed by selected ion mass chromatography (e.g., m/z 145; Figure 1C), and when examined by GCxGC-MS the same compounds were once again very well resolved. Previously, low resolution electrospray ionization mass spectrometry indicated that the major acids in this fraction possessed six DBE (z =
12) with a median mass of 288 (M+) and carbon numbers of ca. 15
20.9 9807

dx.doi.org/10.1021/es202606d |Environ. Sci. Technol. 2011, 45, 9806–9815

Environmental Science & Technology

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Figure 1. A. Gas chromatography
mass spectrometry total ion current chromatogram of OSPW acid extractable fraction (methyl esters). Bimodal nodes in the unresolved profile occur at ca. 16 and 19 min retention time. One reasonably well resolved peak at 22 min. B. Comprehensive multidimensional gas chromatography
mass spectrometry (GCxGC-MS) selected ion mass chromatogram (m/z 145) of OSPW acid extractable fraction (methyl esters) illustrating high chromatographic resolution of putative aromatic acids. C. Gas chromatography
mass spectrometry selected ion mass chromatogram (m/z 145) of distilled OSPW acid extractable fraction (methyl esters) boiling at 220 °C, illustrating high chromatographic resolution of putative aromatic acids. D. Gas chromatography
mass spectrometry total ion current chromatogram of OSPW acid extractable argentation thin layer chromatography fraction (Rf 0.74
0.82) fraction (methyl esters). Unimodal node in the unresolved profile occurs at ca. 16 min retention time. Several reasonably well resolved peak at 22
23 min. E. Gas chromatography
mass spectrometry total ion current chromatogram of OSPW acid extractable argentation thin layer chromatography fraction (Rf 0.72
0.74) fraction (methyl esters). A unimodal node in the unresolved profile occurs at ca. 19 min retention time. Several reasonably well resolved peaks occur at 22
23 min. F. Gas chromatography
mass spectrometry total ion current chromatogram of OSPW acid extractable argentation thin layer chromatography fraction (Rf 0.66
0.72) fraction (methyl esters). A unimodal node in the unresolved profile occurs at ca. 22 min retention time. The well resolved peak at 22
23 min is due to bis-ethylhexylphthalate, possibly arising from contamination during sampling or analysis.

When we examined the OSPW methyl esters by argentation thin layer chromatography, we isolated three fractions with retardation factors (Rf) of 0.74
0.82 (Fraction 1), 0.72
0.74 (Fraction 2), and 0.66
0.72 (Fraction 3). Under these conditions, methyl cyclohexyl-3-propanoate had a Rf of 0.76
0.83 and methyl phenyl-3-propanoate an Rf of 0.64
0.72. When examined by GC-MS, these TLC fractions comprised three separate unresolved ‘humps’; fraction 1 maximizing at ca. 16 min, fraction 2 at ca. 19 min, and fraction 3 at ca. 22 min retention time (Figure 1 D-F). GC-MS selected ion mass chromatography (e.g., m/z 145) of the three TLC fractions revealed that the components with apparent molecular weights of around 300 Da in the OSPW were in fractions 1 and 2 only, each with slightly different distributions. The observations that fraction 2 and 3 components were more retained and that fractionation of the ‘hump’ of esters of OSPW into three separate nodes occurred by argentation TLC (Figure 1 D-F) suggest there was some aromatic (or double bond) character in the more retained acids. Examination of the unfractionated and TLC fractions of OSPW methyl esters by ultraviolet (UV) absorption spectroscopy revealed spectra with absorption maxima typical of benzenoid moieties at ∼265 nm and ∼295 nm (unfractionated, fractions 1 and 2) and ∼290 nm only (fraction 3). Steroidal Hydroxy Acids. Figure 2A and B show positive ion electron ionization mass spectra obtained by GCxGC-MS of the methyl esters of two apparently isomeric acids (I and II) in the unfractionated OSPW methyl esters, eluting about 16 s apart in

GC1 (see also, Table of Contents artwork). The apparent molecular ion m/z 310 is consistent with the methyl ester of either a monocyclic C19 acid or a C20 acid with eight DBE (z =
16). Previous high resolution mass spectrometry studies have shown that monocyclic acids are not abundant in OSPW,12,17 so we suggest the compounds are not monocyclic but are polycyclic C20 acids. Hexa- to octacyclic acids are unlikely since such hydrocarbons have rarely been reported in the oil sands bitumen or petroleum from which the oil sands acids are thought to originate.18 Rather, we favor the suggestion that the C20 acids comprise both aromatic (4 DBE) and alicyclic (1 DBE per ring) moieties. Other features of the spectra (Figure 2A and B) are the base peak ion at m/z 237, and abundant ions at m/z 221 and m/z 286. The latter ion suggests loss of 24 Da from the apparent molecular ion, which would be very unusual. These mass spectra (Figure 2A and B) show considerable similarities to the mass spectrum of 20 -[3β-hydroxy-5,7,9-estratrien-17-yl]propanoic acid (III; Figure 2C: reproduced with permission from NIST). The latter is also characterized by an apparent molecular ion at m/z 310, whereas the molecular weight is actually 328 Da. The m/z 310 ion is probably due to loss of water from the molecular ion. The intensity of the ion is approximately the same as that observed in the spectra shown in Figure 2A and B. Second, the spectrum of III is also characterized by a base peak ion at m/z 237 (Figure 2C) as shown by the spectra of the unknowns. The mass:charge ratios of the ions