Environ. Sci. Technol. 2008, 42, 1290–1295
Influence of Molecular Structure on the Biodegradability of Naphthenic Acids XIUMEI HAN,† ANGELA C. SCOTT,‡ PHILLIP M. FEDORAK,‡ MAHMOUD BATAINEH,† AND J O N A T H A N W . M A R T I N * ,† Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Toxicology, 10-102 Clinical Sciences Building, University of Alberta, Edmonton, AB, T6G 2G3, Canada, and Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E9, Canada
Received September 4, 2007. Revised manuscript received November 9, 2007. Accepted November 21, 2007.
Large volumes of toxic aqueous tailings containing a complex mixture of naphthenic acids (NAs; CnH2n+ZO2) are produced in northern Alberta by the oil sands industry. Because of their persistence and contribution to toxicity, there is an urgent need to understand the fate of NAs under a variety of remediation scenarios. In a previous study, we developed a highly specific HPLC-high resolution mass spectrometry method for the analysis of NAs. Here we apply this method to determine quantitative structure-persistence relationships and kinetics for commercial NAs and NAs in oil sands process water (OSPW) during aerobic microbial biodegradation. Biodegradation of commercial NAs revealed that the mixture contained a substantial labile fraction, which was rapidly biodegraded, and a recalcitrant fraction composed of highly branched compounds. Conversely, NAs in OSPW were predominantly recalcitrant, and degraded slowly by first-order kinetics. Carbon number (n) had little effect on the rate of biodegradation, whereas a general structure-persistence relationship was observed indicating that increased cyclization (Z) decreased the biodegradation rate for NAs in both mixtures. Time to 50% biodegradation ranged from 1 to 8 days among all NAs in the commercial mixture, whereas half-lives for OSPW NAs ranged from 44 to 240 days, likely a result of relatively high alkyl branching among OSPW NAs. It is anticipated that these data will facilitate development of strategic solutions for remediating billions of cubic meters of OSPW stored, or predicted to be generated, in Northern Alberta.
Introduction The oil sands industry in northern Alberta is currently undergoing rapid development. In surface mining operations, bitumen is extracted from the oil sands using a caustic hot water extraction method (1, 2). During this process, naphthenic acids (NAs) are solubilized and concentrated in oil sands process water (OSPW) which must subsequently be stored indeterminately in large tailings ponds due to a zero * Corresponding author phone: (780) 492-1190; fax: (780) 4927800; e-mail:
[email protected]. † Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Toxicology. ‡ Department of Biological Sciences. 1290
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discharge policy (1, 3). Oil sands companies continue to accumulate OSPW, and it was estimated that more than 0.6 billion m3 of OSPW were stored at Syncrude’s Mildred Lake site in 2005 (4), thus total OSPW stored by the industry is expected to exceed 1 billion m3 today. Fresh OSPW has both acute and chronic toxicity to aquatic organisms, and this has largely been attributed to NAs (5–8). NA concentrations in fresh OSPW may be as high as 120 mg L-1, and while some remediation occurs slowly through microbial processes, a persistent fraction of NAs remains after many decades, and aged OSPW has not been reported to decrease below 19 mg L-1 NAs (9–11). The nature of this persistent fraction remains poorly characterized, thus hampering strategic approaches to remediation of OSPW. NAs are a complex mixture of saturated cyclic and noncyclic carboxylic acids having the general formula of CnH2n+ZO2, where n is the carbon number, and Z is a negative even integer related to the number of rings in the molecule (e.g.: Z ) 0, no rings, Z ) -2, 1 ring, Z ) -4, 2 rings, etc.) (5, 12). Accurate characterization of complex NA mixtures, particularly in environmental samples, has been a substantial challenge and has limited the extent to which the environmental fate of NAs could be studied. Mass spectrometry is the most commonly used tool today, and the general approach is to classify NA mixtures by the relative response of each mass (i.e., m/z) corresponding to a particular combination of n and Z. All existing studies on the environmental distribution or fate of NAs in OSPW have relied on low resolution methods such as GC-MS (4, 13) or electrospray (14) and atmospheric pressure chemical ionization-MS (3). However, the accuracy of these low resolution methods is now recognized to be prone to substantial misclassification (i.e., for n ) 10-20) and falsepositive detection (i.e., for n < 10 and n > 20) of NAs in OSPW when compared to the same, or similar, samples analyzed by high-resolution mass spectrometry (HRMS) (15, 16). These analytical difficulties have made it difficult to study the structure-persistence relationship of NAs accurately or quantitatively, but general findings relating to NA biodegradability have been reported. Herman et al. (17, 18) found that microorganisms indigenous to OSPW were capable of degrading both commercial NA mixtures and NAs isolated from OSPW. This early finding led to more detailed studies with similar communities of mixed microbes. Clemente et al. (13) and Scott et al. (4) found that biodegradation of commercial NAs was rapid under aerobic conditions, but that NAs from OSPW degraded much more slowly (4), even when comparing NAs with the same n and Z classification. Following the development of a new analytical method for NAs using HPLC/HRMS (15)swhich permitted accurate classification of NAs by n and Z and which provided additional information on the relative extent of alkyl branching for each NA classswe presented evidence that the extent of alkyl branching may explain the persistence of OSPW NAs versus commercial mixtures. The present work has built on these early observations by further exploring the influence of NA molecular structure (n, Z, and alkyl branching) on microbial degradation kinetics through application of HPLC/HRMS. For the first time, quantitative structure-persistence relationships are provided for NAs. Understanding the reasons why NAs persist in OSPW may facilitate development of strategic solutions for remediation of the large volumes of water currently stored by the oil sands companies. 10.1021/es702220c CCC: $40.75
2008 American Chemical Society
Published on Web 01/09/2008
Experimental Section Chemicals and Reagents. Seven model NA compounds: 1-methyl-1-cyclohexane carboxylic acid (C8H14O2; Z ) -2), trans-4-pentylcyclohexane-1-carboxylic acid (C12H22O2; Z ) -2), 2,2-dicyclohexylacetic acid (C14H24O2; Z ) -4), 2-hexyldecanoic acid (C16H32O2; Z ) 0), octadecanoic acid (C18H36O2; Z ) 0), 5-β-cholanic acid (C24H40O2; Z ) -8), and the internal standard (also used as a model compound), tetradecanoic acid-1-13C (C14H28O2; Z ) 0), were purchased from SigmaAldrich Canada (Oakville, ON, Canada). 4-Butylcyclohexanecarboxylic acid (C11H20O2; Z ) -2) and 4-tert-butylcyclohexanecarboxylic acid (C11H20O2; Z ) -2) were purchased from TCI American Organic Chemicals (Portland, OR). Refined Merichem NAs were provided by Merichem Chemicals and Refinery Services LLC (Houston, TX). OSPW was provided by Syncrude Canada Ltd. Microbial Biodegradation. NA biodegradation experiments were conducted using previously established protocols (4). Briefly, triplicate incubations and sterile controls for each mixture (OSPW NAs and Refined Merichem NAs) were maintained at room temperature on a shaker at 200 rpm. Whereas OSPW already contained a viable microbial community, the commercial NA mixture had to be inoculated. Microorganisms indigenous to Syncrude OSPW were added to the incubations with Refined Merichem NAs such that the resulting microbial density was comparable to that of the OSPW incubations. Initial NA concentrations were approximately 50 and 100 mg L-1 in the incubations with OSPW NAs and Refined Merichem NAs, respectively. Incubations with Refined Merichem NAs were maintained for 28 days with intermittent sampling at day 0, 2, 4, 8, 11, 15, 21, and 28. Incubations with OSPW NAs were maintained for 98 days with intermittent sampling at day 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, and 98. At each sampling time, 3 mL was removed from each triplicate experiment (incubations and sterile controls) and combined in order to minimize the number of analyses while generating pseudoaverage data. The pH of samples was adjusted to 10.5 with 0.1 M NaOH and samples were centrifuged for 10 min at 12,000g. The supernatants (∼9 mL) were added to 5 mL of methanol, for preservation, and the resulting solutions were stored at 4 °C. Analysis of NAs by HPLC/HRMS. Samples were prepared by filtering through 0.45 µm syringe filters and were further diluted with methanol (0.1% formic acid) to get a final solution containing 60% methanol by volume. The internal standard, tetradecanoic acid-1-13C (C14H28O2), was added to each sample (500 ng) to correct for any instrumental sensitivity and retention time drift. Instrumental analysis was performed by capillary HPLC/ HRMS using the method as previously reported (15). A microHPLC system (Series 1100, Agilent Technologies, Waldbronn, Germany) and a reverse-phase capillary column (Aquasil, 150 × 0.5 mm, 3 µm, Thermo) were used for separations. Negative ion mass spectra were collected by HRMS using an API QSTAR Pulsar i mass spectrometer (Applied Biosystem/ MDS Sciex, Concord, ON) equipped with an ion spray source. Analyst QS 1.1 software (Applied Biosystem, Foster City, CA) was used for data acquisition and analysis. Quantitative Analysis. Absolute quantification was not possible since there are no appropriate standards for such complex mixtures, however the current method is internally quantitative such that the relative concentration changes can be determined based on relative response to the internal standard, for each NA isomer classes (i.e., for each Z and n combination). Relative response was plotted over time to determine the biodegradation kinetics for each NA isomer class. Depending on the shape of the kinetic plots, SigmaPlot 2004 (Version 9.0, Systat Software, San Jose, CA) was used to generate either (i) a biodegradation rate constant (and halflife) by linear regression when kinetics could be fit to a first
order model, or (ii) the time to 50% biodegradation (t50) when kinetics were best fit to a 5-parameter sigmoid model (eq 1); whereby C represents concentration at time t of biodegradation, C0 is the minimum analyte concentration after biodegradation, t0 corresponds to the time when C is equal to 50% of a, a is the difference between starting analyte concentration and minimum analyte concentration, b is the time at which C is 75% of a minus the time when C is 25% of a, and the c term controls the degree of asymmetry of the sigmoidal curve between the lag phase and C0. For Refined Merichem NAs, a first-order kinetic model was used to obtain biodegradation half-lives for Z ) 0 NAs (Figure S-1 in the Supporting Information), while the 5-parameter sigmoid model was used to obtain t50 for all other NAs (Figures S-2, S-3, and S-4). For OSPW, the firstorder kinetic model was used to obtain biodegradation halflife for all NAs (Figures S-5, S-6, S-7, and S-8). C ) Co +
[
a (t - to) 1 + exp b
(
)]
(1)
c
Results and Discussion Biodegradation of Refined Merichem NAs. The NA profiles for Refined Merichem at days 0 and 28 of incubation (Figure 1A) clearly show the readily biodegradable nature of this commercial mixture. Only 1.7% of the total initial NAs remained after this time period, and the residual profile was in stark contrast to what was initially present. For example, all of the Z ) 0 NAs were nondetectable after 28 days, and the Z ) -2 class, which was initially the most abundant Z series, was mostly nondetectable. In contrast, the NA isomer classes that remained were predominantly the polycyclic (i.e., more negative Z) fractions, which were relatively minor components of the original mixture. While it was qualitatively clear that the structure of NAs influenced their biodegradation rates, a quantitative assessment of the structure–reactivity relationships was performed by fitting the data to kinetic models. However, not all data could be fit to the same kinetic model. For example, the Z ) 0 NAs degraded very rapidly, with no discernible lag period, and were nondetectable at early stages in the experiment. Plots of natural log (ln) relative response versus time could be fit to a first-order kinetic model (Figure S-1) and rate constants (and half-life) estimated from their respective slopes. In contrast, the kinetics for all other NAs in Refined Merichem were best modeled by a sigmoid function (Figures S-2, S-3, and S-4). A typical plot of the biodegradation kinetics for cyclic Refined Merichem NAs could be divided into three periods corresponding to (i) a lag period, (ii) biodegradation of the labile fraction, and (iii) a remaining persistent fraction (Figure 2). To characterize the kinetics and enable comparison to Z ) 0 NA half-lives, t50 was interpolated from the equations of the sigmoid lines for each NA isomer class. Furthermore, to assess how NA structure affected the lag period and the remaining persistent fraction, the times to 10% (t10) and 80% (t80) biodegradation were also interpolated from the sigmoid lines. Biodegradation kinetics for Z ) -8, -10, and -12 NAs could not be calculated because the relative abundance of these NAs in the initial mixture was low and too few detectable data points were observed after incubation was initiated. The t50 ranged from 1 to 8 days for all NAs in Refined Merichem and tended to increase with increasing cyclicity, following the general trend: Z ) 0 < Z ) -2 < Z ) -4 < Z ) -6 (Figure 3A). The lag period also showed a similar structure–reactivity relationship, whereby t10 followed the same rank order (Figure 3B). As noted earlier, an actual lag period for Z ) 0 NAs was not discernible from the data collected, but t10 was included in the plots nonetheless. Since VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Three-dimensional plots showing relative NA response versus carbon number (n) and Z series before and after biodegradation for (A) Refined Merichem NAs and (B) NAs in OSPW. Percentage given indicates NAs remaining after biodegradation.
FIGURE 2. Typical plot of the biodegradation kinetics for cyclic Refined Merichem NAs (n ) 16, Z ) -6) showing three biodegradation periods, i.e. lag period, labile fraction, and persistent fraction. t10, t50, and t80 are also indicated. the lag period differed among NA classes, we suggest that the presence of the lag period was largely due to preferential biodegradation of NAs with less negative Z, rather than variations in the microbial consortium. The t80 (Figure 3C) was also calculated and ranged from 2 to 15 days among all NAs. The structure–reactivity trends were the same as t10 and t50, increasing with increased cyclicity. Because of the persistent fractions remaining, t80 was also a good reflection 1292
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of the relative proportion of NAs remaining after 28 days. For example, the Z ) -6 NAs had the longest t80 and were also the dominant Z-class remaining after 28 days (Figure 1A). Consistent with the results here for a complex mixture, biodegradation studies with model NA compounds (19, 20) also showed that a bicyclic NA surrogate was biodegraded slower than either monocyclic or linear NA surrogate compounds. The effect of carbon number (n) on the rate of biodegradation was examined by linear regression within each Z series (Figure 3). Although some statistically significant slopes were observed (p < 0.05), no consistent trends were evident. For example, t50 and t80 for Z ) -4 NAs was positively associated with n, however, t10 for Z ) -4 NAs showed no correlation with n. Although increasing n may play a small role in relative persistence for some NA classes, cyclicity had a much greater influence on persistence than n. As presented previously (15), the average retention time for each isomer class shifted toward earlier retention times at later stages of sampling throughout this 28-day biodegradation study with Refined Merichem. Retention time of NAs on the linear C18 stationary-phase column was assumed to be inversely proportional to the extent of alkyl branching among the multiple isomers because this column should have maximal selectivity for the least branched NAs due to the increased van der Walls forces. Based on this assumption, we hypothesized that the shift toward earlier retention times was due to preferential biodegradation of the least alkylsubstituted fraction, leaving behind the highly branched fraction within each NA isomer class.
FIGURE 4. Plot of half-life versus carbon number (n) and Z series for the most abundant NAs in OSPW. A first order kinetic model was used to calculate the half-life. *Indicates a statistically significant slope (p < 0.05).
FIGURE 3. Biodegradation time required to reduce the concentration of individual Refined Merichem NA isomer classes by (A) 50%, (B) 10%, and (C) 80%. For NAs with Z ) 0, a first order kinetic model was used for the calculation, while a 5-parameter sigmoid model was used for all other NA series. *Indicates a statistically significant slope (p < 0.05). To validate this hypothesis, two model NA compounds (4-butylcyclohexanecarboxylic acid and 4-tert-butylcyclohexanecarboxylic acid) were separated by the current method (Figure S-9). The tert-butyl isomer eluted 1.6 min earlier and was almost fully resolved from its more hydrophobic analogue, supporting that retention time (by this method) can indeed be used as a relative measure of average alkyl branching in complex mixtures. Furthermore, the linear internal standard (n-tetradecanoic acid; n ) 14, Z ) 0) eluted at the extreme right side (i.e., late eluting) of the broad chromatographic peak for this isomer class in Refined Merichem (Figure S-10). We are therefore confident that our previous hypothesis was correct, and thus that the sigmoidal shapes of the biodegradation kinetic plots indeed arise from the relative persistence of more highly branched isomers. Alkyl branching has previously been shown to increase the recalcitrance of model alkyl-substituted cycloalkane carboxylates to biodegradation by microbes indigenous to oil sand tailings (17, 18), and this is consistent with our findings here for a complex mixture. Overall, the results from biodegradation studies conducted with Refined Merichem NAs demonstrate that increasing cyclicity and increased alkyl branching lead to slower and less complete biodegradation, which may leave a persistent fraction behind. Biodegradation of NAs in OSPW. The NA profiles for OSPW at days 0 and 98 of biodegradation (Figure 1B) clearly showed that biodegradation of OSPW NAs proceeded much more slowly than for the commercial NAs (Figure 1A). On average, 52% of total NAs remained after 98 days of biodegradation when the study was finally halted due to insufficient volumes remaining for sampling. All kinetic data were best modeled by a first-order exponential decay model,
allowing half-lives to be calculated for all NA isomer classes. Whereas the half-lives (or t50) for Refined Merichem NAs varied from only 1 to 8 days, the half-lives for OSPW NAs ranged between 44 and 240 days (Figure 4). These results are similar to those of Scott et al. who, using HPLC and GC-MS as analytical tools, found that commercial NAs were biodegraded completely within 14 days, whereas only 25-30% of total NAs in OSPW were removed after 40-49 days (4). Using GC-MS, Del Rio et al. showed that >95% of commercial NAs were degraded after 4 weeks, while NAs from OSPW were more recalcitrant and were only partially degraded (20). In the present study using HPLC/HRMS, 98% of commercial NAs were biodegraded after 28 days, while 48% of OSPW NAs were biodegraded after 98 days; based on the summed response for all detected NAs. Despite limitations to the specificity of analytical techniques used in the past, it is reassuring that total NA analysis (i.e., disregarding n and Z classification) yielded similar results to the current method despite obvious differences in the observed NA profiles. OSPW NA biodegradation kinetics were studied here without purifying the NAs from other dissolved components, whereas the Refined Merichem NAs are obtained from petroleum. During the extraction of bitumen from the oil sands, other acidic fractions may be coextracted into OSPW including cresols, mercaptans, and thiophenols (7, 9). Therefore, it was a concern whether other dissolved organics may have influenced OSPW NA biodegradation kinetics here. However, Scott et al. (4) addressed this question by comparing the biodegradation of commercial NA mixtures in the presence or absence of OSPW, and the presence of OSPW did not slow the biodegradation of Refined Merichem NAs. For example, in the absence of OSPW, about 90% of Refined Merichem NAs were biodegraded after 10 days, while in the presence of OSPW, only 7 days were needed to biodegrade the Refined Merichem NAs (4). Therefore, the dissolved compounds in OSPW caused no measurable delay on Refined Merichem NA biodegradation rates, and thus we may assume that the minor components in OSPW also had no significant effect on the biodegradation rate of OSPW NAs in this study. Commercial NAs, such as Refined Merichem, are typically prepared from petroleum sources that have not undergone extensive biodegradation (21), whereas OSPW NAs come from bitumen which is considered to be extensively biodegraded petroleum. The difference in the recalcitrance of commercial NAs and OSPW NAs is hypothesized to be a function of relatively high alkyl branching of OSPW NAs. In much the same way that biodegradation of commercial NAs left behind a recalcitrant fraction (Figure 1A), many years of slow biodegradation in OSPW, or perhaps thousands of years of in situ biological processing in oil sands, has yielded this recalcitrant fraction of NAs. We previously showed direct evidence for relatively high alkyl branching of OSPW NAs by comparing the average retention times to Refined Merichem NAs (i.e., for the same n and Z) by HPLC/HRMS (15). Similarly, VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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GC analysis of the carboxylic acid fraction from samples of undegraded and highly degraded oil indicated that homologous series of saturated n-acids were abundant in the undegraded oil, whereas branched and cyclic carboxylic acids were predominant in the highly degraded oil, appearing as an unidentified and unresolved complex mixture in gas chromatograms (22). The weight of evidence currently suggests that a high degree of alkyl branching is the principal factor that differentiates easily biodegradable commercial NAs from persistent OSPW NAs. Structure-persistence relationships were also examined for OSPW NAs based on Z. Although the biodegradation halflives were orders of magnitude higher for NAs in OSPW (Figure 4), the same general relationship between Z and relative persistence was observed (i.e., Z ) -2 < Z ) -4 < Z ) -6 < Z ) -8). This confirms that, among highly branched NAs, cyclization remains a major factor contributing to persistence. The effect of n on the biodegradation of OSPW NAs was also examined within each Z series. Linear regression (Figure 4) indicated that the half-lives of Z ) -6 NAs were significantly (p < 0.05) associated with increasing n, but no statistically significant trends were evident for Z ) -2, -4, or -8 NAs. Therefore, as observed for the commercial NAs mixture, n played a minor role compared to cyclicity, but is not always insignificant. The effect of n on biodegradation has been reported in past studies using GC-MS (13, 20), but the accuracy of these findings is now suspect due to the low resolution MS methods that are prone to misclassification or false-positive detection (15). Mechanisms of NA Biodegradation and Influence of Alkyl Branching. Several mechanistic pathways have been proposed for the biodegradation of aliphatic and alicyclic carboxylic acids, including β-oxidation (23–26), combined R- and β-oxidation (27, 28), and aromatization pathways (29–31). β-oxidation is the preferred route by which most microorganisms degrade aliphatic and alicyclic carboxylic acids (26, 30, 31) and, thus, is the most likely mechanism by which biodegradation occurred in these laboratory studies. For example, in screening experiments, 32 out of 33 isolates from mud, water, and soil used β-oxidation (30). For noncyclic (e.g., Z ) 0) carboxylic acids, the presence of a quaternary carbon at the R or β position, or a tertiary carbon at the β position, will prevent β-oxidation (25). When a tertiary carbon is present at the R position, and no branching occurs at the β position, β-oxidation is not necessarily prevented but will be impeded due to steric hindrance. The effect of tertiary and quaternary carbons, β or R to the carboxyl group, can also explain why increased cyclization resulted in increased persistence for all NAs studied in this work. The tertiary carbons of a ring are less favorable for microbial oxidation than tertiary carbons of a noncyclic NA, because the rings create a further steric hindrance. β-oxidation of n-alkyl-substituted cyclohexanes may form either cyclohexylacetic acid or cyclohexyl carboxylic acid, depending on whether the alkyl chain contains an even or odd number of carbons, respectively. Cyclohexylacetic acid is resultantly more recalcitrant to biodegradation because the β-oxidation reactions are blocked by the tertiary carbon of the ring (26, 28). However, this compound was effectively degraded by a combined R- and β-oxidation pathway (28) that produced cyclohexylcarboxylic acid in the first step (Roxidation) followed by straightforward β-oxidation. Therefore, even carbon numbered n-alkyl-substituted cyclohexanes can be degraded with a higher degree of mineralization using this combined pathway than by β-oxidation alone, so long as R-oxidation is also not inhibited by a tertiary or quaternary carbon. However, microorganisms capable of utilizing cyclohexylacetic acid by R-oxidation are not widely distributed (28). 1294
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FIGURE 5. Chromatograms of three hydroxyl-NAs (C12H21O3-, n ) 12, Z ) -2, m/z ) 213.1496; C11H17O3-, n ) 11, Z ) -4, m/z ) 197.1183; C10H15O3-, n ) 10, Z ) -4, m/z ) 183.1027) identified at day 21 of incubation for Refined Merichem NAs. For comparison, the chromatograms of the three hydroxyl-NAs at day 0 are also shown. Cyclic NAs, such as cyclohexanecarboxylic acid, can also be degraded by aromatization. This pathway involves parahydroxylation and the subsequent formation of an aromatic intermediate, p-hydroxybenzoic acid (29, 30). Alkyl substitution or the presence of an adjacent ring at the para position will block the aromatization process. Based on the mechanism of the aromatization pathway (29), if a quaternary carbon is present at any position of the ring, the aromatization process will also be prevented. In the present study, analysis of incubation supernatants for the biodegradation of NAs did not reveal analogous aromatic intermediates. For the three pathways discussed above, the first step is always production of a hydroxylated intermediate (25, 28, 29). In the current study, analysis of incubation supernatants identified new chromatographic peaks with exact masses corresponding to these intermediates at day 15 and 21 of biodegradation in the commercial NA mixture (Figure 5). These peaks were not present at day 0 and had disappeared by day 28, suggesting that the intermediates were further oxidized. Oxidized NAs with one or two hydroxyl groups were identified in OSPW at day 0 of the incubations. However, their concentrations did not change throughout the 98-day biodegradation period, perhaps because the initial hydroxylation is rate limiting and any products are rapidly mineralized. Overall, commercial NAs and OSPW NAs are most likely mineralized by β-oxidation, while R-oxidation and aromatization may contribute to the degradation of any one compound. Based on the mechanisms of these pathways, and all relevant literature, increased cyclicity and alkyl branching decreases biodegradation by these pathways, consistent with what was observed in the current experiments.
Acknowledgments Warren Zubot (Syncrude Canada Ltd.) is thanked for providing samples of OSPW. J.W.M. and P.M.F. acknowledge financial support for this research through the Canadian Water Network (CWN, Strategic Research Grant). J.W.M. also acknowledges financial support from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant), an Alberta Ingenuity New Faculty Award, and Alberta Health and Wellness for support of laboratory operations. A.C.S. acknowledges support from an NSERC PGS-M award.
Supporting Information Available Figures showing determination of t10, t50, and t80 for commercial NAs, half-lives for OSPW NAs, and the retention times
of model NA isomers. This information is available free of charge via the Internet at http://pubs.acs.org.
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