Advances in Distinguishing Groundwater Influenced by Oil Sands Process-Affected Water (OSPW) from Natural Bitumen-Influenced Groundwaters.

The objective of this study was to advance analytical methods for detecting oil sands process-affected water (OSPW) seepage from mining containments and discriminating any such seepage from the natural bitumen background in groundwaters influenced by the Alberta McMurray formation. Improved sampling methods and quantitative analyses of two groups of monoaromatic acids were employed to analyze OSPW and bitumen-affected natural background groundwaters for source discrimination. Both groups of monoaromatic acids showed significant enrichment in OSPW, while ratios of O2/O4 containing heteroatomic ion classes of acid extractable organics (AEOs) did not exhibit diagnostic differences. Evaluating the monoaromatic acids to track a known plume of OSPW-affected groundwater confirmed their diagnostic abilities. A secondary objective was to assess anthropogenically derived artificial sweeteners and per- and polyfluoroalkyl substances (PFAS) as potential tracers for OSPW. Despite the discovery of acesulfame and PFAS in most OSPW samples, trace levels in groundwaters influenced by general anthropogenic activities preclude them as individual robust tracers. However, their inclusion with the other metrics employed in this study served to augment the tiered, weight of evidence methodology developed. This methodology was then used to confirm earlier findings of OSPW migrations into groundwater reaching the Athabasca River system adjacent to the reclaimed pond at Tar Island Dyke.

Assessing spatial and temporal variability of acid-extractable organics in oil sands process-affected waters.

The acid-extractable organic compounds (AEOs), including naphthenic acids (NAs), present within oil sands process-affected water (OSPW) receive great attention due to their known toxicity. While recent progress in advanced separation and analytical methodologies for AEOs has improved our understanding of the composition of these mixtures, little is known regarding any variability (i.e., spatial, temporal) inherent within, or between, tailings ponds. In this study, 5 samples were collected from the same location of one tailings pond over a 2-week period. In addition, 5 samples were collected simultaneously from different locations within a tailings pond from a different mine site, as well as its associated recycling pond. In both cases, the AEOs were analyzed using SFS, ESI-MS, HRMS, GC×GC-ToF/MS, and GC- & LC-QToF/MS (GC analyses following conversion to methyl esters). Principal component analysis of HRMS data was able to distinguish the ponds from each other, while data from GC×GC-ToF/MS, and LC- and GC-QToF/MS were used to differentiate samples from within the temporal and spatial sample sets, with the greater variability associated with the latter. Spatial differences could be attributed to pond dynamics, including differences in inputs of tailings and surface run-off. Application of novel chemometric data analyses of unknown compounds detected by LC- and GC-QToF/MS allowed further differentiation of samples both within and between data sets, providing an innovative approach for future fingerprinting studies.

Use of the distributions of adamantane acids to profile short-term temporal and pond-scale spatial variations in the composition of oil sands process-affected waters.

Oil industry produced waters, such as the oils sands process-affected waters (OSPW) of Alberta, Canada, represent a challenge in terms of risk assessment and reclamation due to their extreme complexity, particularly of the organic chemical constituents, including the naphthenic acids (NA). The identification of numerous NA in single samples has raised promise for the use of NA distributions for profiling OSPW. However, monitoring of the success of containment is still difficult, due to the lack of knowledge of the homogeneity (or otherwise) of OSPW composition within, and between, different industry containments. Here we used GC×GC-MS to compare the NA of five OSPW samples from each of two different industries. Short-term temporal and pond-scale spatial variations in the distributions of known adamantane acids and diacids and other unknown tricyclic acids were examined and a statistical appraisal of the replicate data made. The presence/absence of individual acids easily distinguished the OSPW NA of one industry from those of the other. The proportions of tricyclic acids with different carbon numbers also varied significantly between the OSPW of the two industries. The pond-scale spatial variation in NA in OSPW samples was higher than the short-term (2 weeks) temporal variations. An OSPW sample from an aged pond was exceptionally high in the proportion of C15,16,17 compounds, possibly due to increased biotransformation. Such techniques could possibly also help to distinguish different sources of NA in the environment.

Bicyclic naphthenic acids in oil sands process water: identification by comprehensive multidimensional gas chromatography-mass spectrometry.

Although bicyclic acids have been reported to be the major naphthenic acids in oil sands process-affected water (OSPW) and a well-accepted screening assay indicated that some bicyclics were the most acutely toxic acids tested, none have yet been identified. Here we show by comprehensive multidimensional gas chromatography-mass spectrometry (GC×GC-MS), that >100 C8-15 bicyclic acids are typically present in OSPW. Synthesis or purchase allowed us to establish the GC×GC retention times of methyl esters of numerous of these and the mass spectra and published spectra of some additional types, allowed us to identify bicyclo[2.2.1]heptane, bicyclo[3.2.1]octane, bicyclo[4.3.0]nonane, bicyclo[3.3.1]nonane and bicyclo[4.4.0]decane acids in OSPW and a bicyclo[2.2.2]octane acid in a commercial acid mixture. The retention positions of authentic bicyclo[3.3.0]octane and bicyclo[4.2.0]octane carboxylic acid methyl esters and published retention indices, showed these were also possibilities, as were bicyclo[3.1.1]heptane acids. Bicyclo[5.3.0]decane and cyclopentylcyclopentane carboxylic acids were ruled out in the samples analysed, on the basis that the corresponding alkanes eluted well after bicyclo[4.4.0]decane (latest eluting acids). Bicyclo[4.2.1]nonane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane, bicyclo[4.2.2]decane and spiro[4.5]decane carboxylic acids could not be ruled out or in, as no authentic compounds or literature data were available. Mass spectra of the methyl esters of the higher bicyclic C12-15 acids suggested that many were simply analogues of the acids identified above, with longer alkanoate chains and/or alkyl substituents. Our hypothesis is that these acids represent the biotransformation products of the initially somewhat more bio-resistant bicyclanes of petroleum. Although remediation studies suggest that many bicyclic acids can be relatively quickly removed from suitably treated OSPW, examination by GC×GC-MS may show which isomers are affected most. Knowledge of the structures will allow the toxicity of any residual isomers to be calculated and measured.

Aquatic hazard assessment of a commercial sample of naphthenic acids.

This paper presents chemical composition and aquatic toxicity characteristics of a commercial sample of naphthenic acids (NAs). Naphthenic acids are derived from the refining of petroleum middle distillates and can contribute to refinery effluent toxicity. NAs are also present in oil sands process-affected water (OSPW), but differences in the NAs compositions from these sources precludes using a common aquatic toxicity dataset to represent the aquatic hazards of NAs from both origins. Our chemical characterization of a commercial sample of NAs showed it to contain in order of abundance, 1-ring>2-ring>acyclic>3-ring acids (∼84%). Also present were monoaromatic acids (7%) and non-acids (9%, polyaromatic hydrocarbons and sulfur heterocyclic compounds). While the acyclic acids were only the third most abundant group, the five most abundant individual compounds were identified as C(10-14) n-acids (n-decanoic acid to n-tetradecanoic acid). Aquatic toxicity testing of fish (Pimephales promelas), invertebrate (Daphnia magna), algae (Pseudokirchneriella subcapitata), and bacteria (Vibrio fischeri) showed P. promelas to be the most sensitive species with 96-h LL50=9.0 mg L(-1) (LC50=5.6 mg L(-1)). Acute EL50 values for the other species ranged 24-46 mg L(-1) (EC50 values ranged 20-30 mg L(-1)). Biomimetic extraction via solid-phase-microextraction (BE-SPME) suggested a nonpolar narcosis mode of toxic action for D. magna, P. subcapitata, and V. fischeri. The BE analysis under-predicted fish toxicity, which indicates that a specific mode of action, besides narcosis, may be a factor for fishes.

Can two-dimensional gas chromatography/mass spectrometric identification of bicyclic aromatic acids in petroleum fractions help to reveal further details of aromatic hydrocarbon biotransformation pathways?

RATIONALE: The identification of key acid metabolites ('signature' metabolites) has allowed significant improvements to be made in our understanding of the biodegradation of petroleum hydrocarbons, in reservoir and in contaminated natural systems, such as aquifers and seawater. On this basis, anaerobic oxidation is now more widely accepted as one viable mechanism, for instance. However, identification of metabolites in the complex acid mixtures from petroleum degradation is challenging and would benefit from use of more highly resolving analytical methods. METHODS: Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS) with both nominal mass and accurate mass measurement was used to study the complex mixtures of aromatic acids (as methyl esters) in petroleum fractions. RESULTS: Numerous mono- and di-aromatic acid isomers were identified in a commercial naphthenic acids fraction from petroleum and in an acids fraction from a biodegraded petroleum. In many instances, compounds were identified by comparison of mass spectral and retention time data with those of authentic compounds. CONCLUSIONS: The identification of a variety of alkyl naphthalene carboxylic and alkanoic and alkyl tetralin carboxylic and alkanoic acids, plus identifications of a range of alkyl indane acids, provides further evidence for 'signature' metabolites of biodegradation of aromatic petroleum hydrocarbons. Identifications such as these now offer the prospect of better differentiation of metabolites of bacterial processes (e.g. aerobic, methanogenic, sulphate-reducing) in polar petroleum fractions.

Profiling oil sands mixtures from industrial developments and natural groundwaters for source identification.

The objective of this study was to identify chemical components that could distinguish chemical mixtures in oil sands process-affected water (OSPW) that had potentially migrated to groundwater in the oil sands development area of northern Alberta, Canada. In the first part of the study, OSPW samples from two different tailings ponds and a broad range of natural groundwater samples were assessed with historically employed techniques as Level-1 analyses, including geochemistry, total concentrations of naphthenic acids (NAs) and synchronous fluorescence spectroscopy (SFS). While these analyses did not allow for reliable source differentiation, they did identify samples containing significant concentrations of oil sands acid-extractable organics (AEOs). In applying Level-2 profiling analyses using electrospray ionization high resolution mass spectrometry (ESI-HRMS) and comprehensive multidimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF/MS) to samples containing appreciable AEO concentrations, differentiation of natural from OSPW sources was apparent through measurements of O2:O4 ion class ratios (ESI-HRMS) and diagnostic ions for two families of suspected monoaromatic acids (GC × GC-TOF/MS). The resemblance between the AEO profiles from OSPW and from 6 groundwater samples adjacent to two tailings ponds implies a common source, supporting the use of these complimentary analyses for source identification. These samples included two of upward flowing groundwater collected <1 m beneath the Athabasca River, suggesting OSPW-affected groundwater is reaching the river system.

Diaromatic sulphur-containing 'naphthenic' acids in process waters.

Polar organic compounds found in industrial process waters, particularly those originating from biodegraded petroleum residues, include 'naphthenic acids' (NA). Some NA have been shown to have acute toxicity to fish and also to produce sub-lethal effects. Whilst some of these toxic effects are produced by identifiable carboxylic acids, acids such as sulphur-containing acids, which have been detected, but not yet identified, may produce others. Therefore, in the present study, the sulphur-containing acids in oil sands process water were studied. A fraction (ca 12% by weight of the total NA containing ca 1.5% weight sulphur) was obtained by elution of methylated NA through an argentation solid phase extraction column with diethyl ether. This was examined by multidimensional comprehensive gas chromatography-mass spectrometry (GCxGC-MS) in both nominal and high resolution mass accuracy modes and by GCxGC-sulphur chemiluminescence detection (GCxGC-SCD). Interpretation of the mass spectra and retention behaviour of methyl esters of several synthesised sulphur acids and the unknowns allowed delimitation of the structures, but not complete identification. Diaromatic sulphur-containing alkanoic acids were suggested. Computer modelling of the toxicities of some of the possible acids suggested they would have similar toxicities to one another and to dehydroabietic acid. However, the sulphur-rich fraction was not toxic or estrogenic to trout hepatocytes, suggesting the concentrations of sulphur acids in this sample were too low to produce any such effects in vitro. Further samples should probably be examined for these compounds.

Aromatic naphthenic acids in oil sands process-affected water, resolved by GCxGC-MS, only weakly induce the gene for vitellogenin production in zebrafish (Danio rerio) larvae.

Process waters from oil sands industries (OSPW) have been reported to exhibit estrogenic effects. Although the compounds responsible are unknown, some aromatic naphthenic acids (NA) have been implicated. The present study was designed to investigate whether aromatic NA might cause such effects. Here we demonstrate induction of vitellogenin genes (vtg) in fish, which is a common bioassay used to indicate effects consistent with exposure to exogenous estrogens. Solutions in water of 20-2000 µg L(-1) of an extract of a total OSPW NA concentrate did not induce expression of vtg in larval zebrafish, consistent with earlier studies which showed that much higher NA concentrations of undiluted OSPW were needed. Although 20-2000 µg L(-1) of an esterifiable NA subfraction of the OSPW NA concentrate did induce expression, this was of much lower magnitude to that induced by much lower concentrations of 17α-ethynyl estradiol, indicating that the effect of the total NAs was only weak. However, given the high NA concentrations and large volumes of OSPW extant in Canada, it is important to ascertain which of these esterifiable NA in the OSPW produce the effect. Up to 1000 µg L(-1) of an OSPW subfraction containing only alicyclic NA, and considered by most authors to be NA sensu stricto, did not produce induction; but, as predicted, 10-1000 µg L(-1) of an aromatic NA fraction did. Such effects by the aromatic acids are again consistent with those of only a weak estrogenic substance. These findings may help to focus studies of the most environmentally significant OSPW-related pollutants, if reproduced in a greater range of OSPW.

Diamondoid diacids ('O4' species) in oil sands process-affected water.

RATIONALE: As a by-product of oil sands extraction, large volumes of oil sands process water (OSPW) are generated, which are contaminated with a large range of water-soluble organic compounds. The acids are thought to be derived from hydrocarbons via natural biodegradation pathways such as α- and ß-oxidation of alkyl substituents, which could produce mono- and diacids, for example. However, while several monoacids ('O2' species) have been identified, the presence of diacids (i.e. 'O4' species) has only been deduced from results obtained via Fourier transform infrared (FTIR) spectroscopy, Fourier transform ion cyclotron resonance high-resolution mass spectrometry (FTICR-HRMS) and nuclear magnetic resonance ((1)H-NMR) spectroscopy and the structures have never been confirmed. METHODS: An extract of an OSPW from a Canadian tailings pond was analysed and the retention times and the electron ionization mass spectra of some analytes were compared with those of bis-methyl esters of authentic diacids by gas chromatography × gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS) in nominal and accurate mass configurations. RESULTS: Two diamondoid diacids (3-carboxymethyladamantane-1-carboxylic acid and adamantane-1,3-dicarboxylic acid) were firmly identified as their bis-methyl esters by retention time and mass spectral matching and several other structural isomers were more tentatively assigned. Diacids have substantially increased polarity over the hydrocarbon and monoacid species from which they probably derive: as late members of biodegradation processes they may be useful indicators of weathering and ageing, not only of OSPW, but potentially of crude oil residues more generally. CONCLUSIONS: Structures of O4 species in OSPW have been identified. This confirms pathways of microbial biodegradation, which were only postulated previously, and may be a further indication that remediation of OSPW toxicity can occur by natural microbial action. The presence and abundance of these diacids might therefore be useful as a measure of biodegradation and weathering.

Abundant naphthenic acids in oil sands process-affected water: studies by synthesis, derivatisation and two-dimensional gas chromatography/high-resolution mass spectrometry.

RATIONALE: The large volumes of 'supercomplex' mixtures of reputedly toxic organic compounds in acidic extracts of oil sands process-affected waters (OSPW) represent a challenging goal for complete characterisation. To date, comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS) has allowed the acquisition and interpretation of numerous electron ionisation mass spectra including many of those confirmed to be tricyclic and pentacyclic carboxylic acids by reference to the spectra and retention positions of authentic or synthetic compounds. This has allowed the toxicities of some of the identified acids to be determined and their environmental significance to be better assessed. METHODS: Synthesis, derivatisation (methyl, trideuteriomethyl and trimethylsilyl esters) and GCxGC/TOFMS with nominal mass and higher mass accuracy (ca. 5 ppm) were used to study three abundant unknown acids present in OSPW samples from two different industrial suppliers. RESULTS: GCxGC/TOFMS with nominal mass accuracy, of methyl, trideuteriomethyl and trimethylsilyl esters of three abundant acids in two OSPW samples, produced mass spectra consistent with their assignment as either C(16) tetracyclic acids or as isobaric pentacyclic C(15) hydroxy acids ('O(3)') or sulfur-containing ('SO(2)') species. The synthesis of several isomeric pentacyclic C(15) hydroxy acids and examination of the GCxGC retention times and mass spectra (nominal mass) of their derivatives suggested that the unknown OSPW acids were not hydroxy acids, and GCxGC/TOFMS with higher mass accuracy ruled out the possibility. The possibility that they were isobaric 'SO(2)' species could also be dismissed as this was inconsistent with accurate masses, the derivatisation reactions observed, the fragmentation patterns and the isotope distributions, which excluded the presence of sulphur. CONCLUSIONS: The data support the contention that the three abundant unknowns were indeed C(16) tetracyclic acids. An equally rigorous approach will be necessary to characterise further acids in such mixtures. This is important so that chemistry can be used to guide the search for toxic modes of action.

Isolation and estimation of the 'aromatic' naphthenic acid content of an oil sands process-affected water extract.

The naphthenic acids of oil sands process-affected water (OSPW) are said to be important toxicants. The major acids are stated to have alicyclic structures and recently, numerous of these have been identified, but some evidence suggests 'aromatic' acids are also present. The proportions of such acids have not been reported because they exist in so-called supercomplex mixtures with the alicyclic species. Their contribution to the toxicity of OSPW, if any, is therefore unknown. Here we report the use of multidimensional comprehensive gas chromatography-mass spectrometry (GC×GC-MS) with polar first dimension and non-polar second dimension GC columns and argentation solid phase extraction, to separate methyl esters of the acids of an OSPW supercomplex, into distinct fractions. A major fraction (ca 70%) was shown to contain acids (methyl esters) previously identified as alicyclic species. Authentic adamantane acid methyl esters were shown to chromatograph in this fraction. This fraction was isolated by argentation solid phase extraction (SPE) by elution with hexane. GC-MS and GC×GC-MS confirmed this to be the major fraction in the original supercomplex containing alicyclic acids (methyl esters). A second fraction shown to contain monoaromatic acids (methyl esters) by GC×GC-MS was unexpectedly abundant (ca 30% relative to the acyclic acids). The naphtheno-aromatic dehydroabietic acid was confirmed by co-injection with an authentic compound and several acids previously tentatively identified as naphtheno-monoaromatics were present. This fraction was isolated by argentation SPE by elution with more polar 5% diethyl ether in hexane. GC-MS and GC×GC-MS confirmed that the fraction represented a significant proportion of the original supercomplex. A further fraction, eluting from the argentation SPE column with more 5% diethyl ether in hexane in the same retention volume as authentic methyl naphthoate, contained, in addition to some of the second fraction, a third, much more minor complex. This had somewhat similar GC×GC retention characteristics to that of methyl naphthoate and the methyl ester of authentic fluorene-9-carboxylic acid. Spectra are reported. Non-alicyclic, mono- and possibly diaromatic acids are a much more quantitatively significant proportion of OSPW than previously realised. The individual acids need to be better identified and the toxicity of these and other SPE fractions studied in order to assess any possible environmental effects of OSPW.

Aqueous phototransformation of diazepam and related human metabolites under simulated sunlight.

Phototransformation of the widely used benzodiazepine pharmaceuticals diazepam and human metabolites nordiazepam, temazepam and oxazepam under simulated sunlight in water was investigated. Photolysis experiments were conducted in the presence and absence of humic acids. Half-lives for each of the benzodiazepine pharmaceuticals were <200 h (under all conditions) suggesting that phototransformation is an important process for such chemicals in the photic zone of receiving waters. Due to the observed phototransformation of the benzodiazepines, significant emphasis was placed on identification of the photoproducts. A total of fourteen photoproducts, including benzophenones, acridinones and quinazolinones or quinazolines was identified and measured by liquid chromatography-multistage mass spectrometry (LC-MS(n)). Phototransformation studies were also undertaken on authentic samples of two of the identified photoproducts, 5-chloro-methylaminobenzophenone and 2-amino-5-chlorobenzophenone, in order to establish the phototransformation pathways. Interestingly, these two photoproducts showed relatively higher persistence than some of the benzodiazepines, suggesting that the fate and effects of photoproducts should also be incorporated into future risk assessments and environmental models of the fate of benzodiazepines.

Predicted toxicity of naphthenic acids present in oil sands process-affected waters to a range of environmental and human endpoints.

Naphthenic acids (NAs) are considered to be a major toxic component of oil sands process-affected waters (OSPW) and are also widely used for industrial processes. The effects of previously identified NAs (54 in total), together with six alkylphenols, were modelled for a range of environmental and human toxicity related endpoints using ADMET predictor™ software. In addition to the models, experimental CALUX® assays were performed on seven tricyclic diamondoid acids. Most of the NAs modelled were predicted to have lethal median concentrations (LC(50)) >100 µM for the three aquatic species modelled. Polycyclic acids containing a single aromatic ring were predicted to be the most toxic to fathead minnows with LC(50)s typically ca 1 µM. Some of these compounds were also predicted to be the most carcinogenic (based on rat and mouse models), possess human estrogenic and androgenic activity and potentially disrupt reproductive processes. Some aliphatic pentacyclic acids also were predicted to exhibit androgenic activity and, uniquely amongst the compounds tested, act as substrates for the cytochrome P450 enzyme CYP3A4. Consistent with the models' predictions for the tricyclic acids, no estrogenic or androgenic activity was detected by ER/AR CALUX®. Further experimental validation of the predictions should now be performed for the compounds highlighted by the models (e.g. priority should perhaps be focused on the polycyclic monoaromatic acids and the aliphatic pentacyclic acids). If shown to be accurate, these compounds can then be targeted for toxicity reduction remediation efforts.

Differentiation of two industrial oil sands process-affected waters by two-dimensional gas chromatography/mass spectrometry of diamondoid acid profiles.

RATIONALE: Processing of the oil sands of Canada has produced large amounts of process-affected water (OSPW). Concerns have been raised over the possible environmental impacts of any leakage of OSPW from storage lagoons which contain toxicants, including organic acids. Natural weathering of oil sands deposits may also produce the toxicants, including the acids. Therefore, there is a need for differentiation of the possible natural and industrial sources of such toxicants and also for methods suitable for monitoring changes in the composition of OSPW during long-term storage. METHODS: Here we show in a simple preliminary study of the two samples currently available to us, by use of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/ToF-MS), the distributions of methyl esters of individual isomeric diamondoid acids in OSPW from lagoons with different histories and from different industrial operators. RESULTS: We show that the distributions of methyl esters of individual isomeric diamondoid acids, including methyladamantane carboxylic and ethanoic acids, identified by comparison with data for reference compounds, can be differentiated readily. The use of acids with known structures, each verified by authentic acids, known toxicities and known and/or predictable physicochemical properties, to distinguish the different sources is advantageous, since factors likely to control the fate and dispersion of the acids can then more easily be predicted. It is postulated that the differences observed in the relative amounts of some of the acids result from variable extents of bacterial transformation of the organic matter in OSPW. CONCLUSIONS: The differences in distributions of diamondoid acids clearly vary between the two samples of OSPW and may prove very useful for monitoring the fate of different sources of OSPW both in storage and in the wider environment, once a wider collection of representative samples is available for study.

Aerobic biotransformation of alkyl branched aromatic alkanoic naphthenic acids via two different pathways by a new isolate of Mycobacterium.

Naphthenic acids (NAs) are complex mixtures of carboxylic acids found in weathered crude oils and oil sands, and are toxic, corrosive and persistent. However, little is known about the microorganisms and mechanisms involved in NA degradation. We isolated a sediment bacterium (designated strain IS2.3), with 100% 16S rRNA gene sequence identity to Mycobacterium aurum, which degraded synthetic NAs (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and (4'-t-butylphenyl)-4-butanoic acid (t-BPBA). n-BPBA was readily oxidized with almost complete degradation (96.8% ± 0.3) compared with t-BPBA (77.8% ± 3.7 degraded) by day 49. Cell counts increased fourfold by day 14 but decreased after day 14 for both n- and t-BPBA. At day 14, (4'-butylphenyl)ethanoic acid (BPEA) metabolites were detected. Additional metabolites produced during t-BPBA degradation were identified by mass spectrometry of derivatives as (4'-carboxy-t-butylphenyl)-4-butanoic acid and (4'-carboxy-t-butylphenyl)ethanoic acid; suggesting that strain IS2.3 used omega oxidation of t-BPEA to oxidize the tert-butyl side-chain to produce (4'-carboxy-t-butylphenyl)ethanoic acid, as the primary route for biodegradation. However, strain IS2.3 also produced this metabolite through initial omega oxidation of the tert-butyl side-chain of t-BPBA, followed by beta-oxidation of the alkanoic acid side-chain. In conclusion, an isolate belonging to the genus Mycobacterium degraded highly branched aromatic NAs via two different pathways.

Steroidal aromatic 'naphthenic acids' in oil sands process-affected water: structural comparisons with environmental estrogens.

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 C(19) and C(20) 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 acid-extractable organics and naphthenic acids.

Toxicity of individual naphthenic acids to Vibrio fischeri.

Numerous studies have suggested that the toxicity of organic compounds containing at least one carboxylic acid group and broadly classified as "naphthenic acids", is of environmental concern. For example, the acute toxicity of the more than 1 billion m(3) of oil sands process-affected water and the hormonal activity of some offshore produced waters has been attributed to the acids. However, experimental evidence for the toxicity of the individual acids causing these effects has not been very forthcoming. Instead, most data have been gathered from assays of incompletely characterized extracts of the water, which may contain other toxic constituents. An alternative approach is to assay the individual identified toxicants. Since numerous petroleum-derived naphthenic acids and some in oil sands process water, have recently been identified, we were able to measure the toxicity of some individual acids to the bioluminescent bacterium, Vibrio fischeri. Thirty-five pure individual acids were either synthesized or purchased for this purpose. We also used the US EPA ECOSAR computer model to predict the toxicity of each acid to the water flea, Daphnia magna. Both are well-accepted toxicological screening end points. The results show how toxic some of the naphthenic acids really are (e.g., V. fischeri Effective Concentrations for 50% response (EC(50)) 0.004 to 0.7 mM) and reveal the influence of hydrophobicity and aqueous solubility on the toxicities. Comparison with measured toxicities of other known, but more minor, constituents of oil sands process water, such as polycyclic aromatic hydrocarbons and alkylphenols, helps place these toxicities into a wider context. Given the reported toxicological effects of naphthenic acids to other organisms (e.g., fish, plants), the toxicities of the acids to further end points should now be determined.