Research Effect of Carboxylic Acid Content on the Acute Toxicity of Oil Sands Naphthenic Acids R I C H A R D A . F R A N K , * ,† KATHARINA FISCHER,‡ RICHARD KAVANAGH,§ B. KENT BURNISON,| GILLES ARSENAULT,⊥ JOHN V. HEADLEY,# KERRY M. PERU,# GLEN VAN DER KRAAK,§ AND KEITH R. SOLOMON‡ Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1, Centre for Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1, Department of Integrative Biology, University of Guelph, Guelph, ON, Canada N1G 2W1, National Water Research Institute, Environment Canada, Burlington, ON, Canada L7R 4A6, Wellington Laboratories, Guelph, ON, Canada N1G 3M5, and National Water Research Institute, Environment Canada, Saskatoon, SK, Canada S7N 3H5
Received July 28, 2008. Revised manuscript received October 24, 2008. Accepted October 27, 2008.
Fractions of methylated naphthenic acids (NAs) isolated from oil sands process-affected water were collected utilizing Kugelrohr distillation and analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. 1H NMR analysis revealed that the ratio of methyl ester hydrogen atoms to remaining aliphatic hydrogen atoms increased from 0.130 to 0.214, from the lowest to the greatest molecular weight (MW) fractions, respectively, indicating that the carboxylic acid content increased with greater MW. Acute toxicity assays with exposure to monocarboxyl NA-like surrogates demonstrated that toxicity increased with increasing MW (D. magna LC50 values of 10 ( 1.3 mM and 0.59 ( 0.20 mM for the respective lowest and highest MW NAlike surrogates); however, with the addition of a second carboxylic acid moiety, the toxicity was significantly reduced (D. magna LC50 values of 10 ( 1.3 mM and 27 ( 2.2 mM for the respective monocarboxyl and dicarboxyl NA-like surrogates of similar MW). Increased carboxylic acid content within NA structures of higher MW decreases hydrophobicity and, consequently, offers a plausible explanation as to why lower MW NAs in oil sands process-affected water are more toxic than the greater MW NAs.
Introduction The oil sands industry of the Athabasca Basin, located in northeastern Alberta, Canada, continues to grow both structurally and financially. Investment in oil sands exceeds * Corresponding author phone: (519) 888-4567 x36416; Fax: (519) 746-0614; e-mail:
[email protected]. † University of Waterloo. ‡ Centre for Toxicology, University of Guelph. § Department of Integrative Biology, University of Guelph. | Environment Canada, Burlington. ⊥ Wellington Laboratories. # Environment Canada, Saskatoon. 266
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all other industrial investment, and, since 2003, project applications have steadily increased and continued increases are proposed (1). Since the year 2000, nonconventional methods of oil production, including those involving oil sands, have grown in comparison to conventional methods. In 2000, production from nonconventional methods was approximately 604 700 barrels (bbl) day-1 (1 barrel ) 159 L), and conventional methods approximately 591 000 bbl day-1 (2). In 2007, production increased for nonconventional methods to an average of 1 130 000 bbl day-1; however, conventional production methods declined to an average of 543 000 bbl day-1 (1). Current estimates indicate that 173.2 billion bbl of recoverable bitumen are present within the Athabasca Basin and, at the current production rate of 1.25 million bbl of bitumen day-1, production will continue well into the future (1). As the costs associated with oil sands refining decrease because of technological advances, and conventional oil supplies decline globally, the oil sands projects of the Athabasca Basin are anticipated to expand (3). The extraction of bitumen from oil sands utilizes the Clark hot water extraction process (4). Resulting wastewater, commonly referred to as oil sands process-affected water (OSPW), contains sand, clay, unrecoverable bitumen and hydrocarbons, and is stored in ponds on site as part of a zero discharge policy (5). Toxicity studies on unaged OSPW have reported that aquatic organisms, including Salmo gairdneri and Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), are adversely affected at environmentally relevant concentrations (6, 7). The sodium salts of naphthenic acids (NAs), ranging in concentration from 40-120 mg L-1 in OSPW, have been identified as the primary toxic components (6, 8). NAs (Figure 1) comprise a diverse group of acyclic, monocyclic, and polycyclic carboxylic acids with the general formula of CnH2n+zO2, where n represents the carbon number and z is the homologous group series number related to the number of five- or six-carbon rings within the structure. NA mixtures are concentrated in OSPW through the extraction of bitumen from the Athabasca oil sands deposit (5). Field studies have demonstrated that the acute toxicity of OSPW decreases within 1 to 24 months (6), and that OSPW that had been allowed to degrade in tailings ponds for 4 weeks exhibited decreased toxicity to fathead minnows compared to fresh tailings (9). Similarly, following several weeks of biodegradation in a laboratory, commercial NA mixtures were no longer toxic in Microtox assays with Vibrio fischeri (10). Marked changes in the composition of NA mixtures occur following the biodegradation of fresh tailings. Indigenous microbial populations preferentially degrade NAs that contain 220 °C residue
0.130 0.132 0.138 0.167 0.214
Version 1.5 (34), which generated LC50 values with 95% CIs. Significant differences between LC50 values were identified by a lack of overlap between 95% CIs.
Results and Discussion Analysis by electrospray ionization mass spectrometry (ESIMS) of the collected fractions confirmed that the Kugelrohr fractionation allowed for the separation of methylated NAs from lower to larger MW compounds, as the distillation collection temperature increased (23). NAs have been considered to be monocarboxylic (22); therefore, the ratio of methyl ester hydrogen atoms to the remaining aliphatic hydrogen atoms should have steadily decreased with increased MW. However, this was not observed. In this study, methylation of NAs permitted easier measurement of the percentage of carboxylic acid groups present within NA structures by 1H NMR spectroscopy. The 1H NMR spectra for the methylated NA fractions (Figure S1, Supporting Information) demonstrated that the ratio of methyl ester hydrogen atoms (3.4-4.0 ppm) to other aliphatic hydrogen atoms (0.4-3.4 ppm) steadily increased with each fraction of greater MW (Table 2). To calculate this ratio, the integrated peak values from the 1H NMR analysis were compared between the methyl ester hydrogen atoms and the remaining aliphatic hydrogen atoms (i.e., 3:23.1 ) 0.130 for the 130 °C fraction). This increasing ratio suggests that there is a greater carboxylic acid content within NAs of higher MW. Further 1H NMR evidence supports the presence of multiple carboxylic groups within higher MW NAs. Hydrogen atoms within alkyl and cycloalkane moieties have a chemical shift between 0.4 and 1.5 ppm, and those attached to a carbon adjacent to a carbonyl carbon have a shift ranging between 2.0 and 3.0 ppm (28, 29). The increase in hydrogen atoms with a chemical shift between 1.6 and 3.2 ppm relative to the aliphatic hydrogen atoms at 0.4 to 1.5 ppm suggests an increasing number of carbon atoms adjacent to carbonyl carbon atoms. This result is also supportive of increased carboxylic acid content within the structures of greater MW NAs. Because of the compositional complexity of the methylated NA fractions, it is not possible to clearly identify all of the structures present. However, the combined data from the 1H NMR and ESI-MS analyses (23) indicate that there are greater numbers of carboxylic groups within more cyclic, higher MW structures. These observations were unexpected, considering the formerly accepted NA general formula of CnH2n+zO2. The observations also suggest that structural differences, other than just increased carbon content, may exist between lower and higher MW NAs and that these differences could influence toxicity and potentially explain why lower MW NAs elicit a greater toxic response. The 1H NMR chemical shift for protons to the R carbon atoms of alcohols appears in the region of 3.2-3.9 (28, 29); therefore, the possibility existed for an artificially enhanced methyl ester hydrogen signal (typical shift 3.4-3.7). Analysis by FTIR (Figure S2, Supporting Information) confirmed that
there was negligible alcohol content within the methylated fractions because of the lack of a detectable O-H stretching adsorption band in the region of 3200-3400 cm-1. In addition, the ratio between the methyl ester (carbonyl adsorption at 1735 cm-1) and hydrocarbon (alkyl C-H stretching adsorption in the 2850-2960 cm-1 region) signal intensities increased with increasing fraction collection temperature. This result further suggests increased methyl ester content within fractions of greater MW, indicating the presence of additional carboxylic acid groups within NA structures of higher MW. Within the 1H NMR spectra, there is also a noticeable increase in a peak appearing between 6.8 and 7.4 ppm in all of the fractions except the 130 °C fraction. These signals are characteristic of aromatic compounds (28, 29) and support the results of a recent study (35) which detected aromatic NA-like compounds in petroleum products using ESI-MS with a chip-based nanoelectrospray system. Previous analysis by gas chromatography with a mass spectrometer and 1H NMR of carboxylic acids extracted from California crude oil suggested the presence of aromatic rings fused to saturated rings and even indicated the location of the aromatic ring at the terminal end (36). Furthermore, analysis by 1H NMR of the acidic fraction of petroleum revealed a low concentration of aromatic hydrogen atoms relative to the remaining hydrogen atoms within the mixture (26). The extraction and purification procedure used in the present study to concentrate NAs from OSPW (27) required three washes with DCM to remove neutral organics such as polycyclic aromatic hydrocarbons. The DCM washes, in addition to the distillation process utilized to fractionate the NA mixture, make it unlikely that neutral organics were present. Accordingly, it is possible that OSPW contain larger NA-like compounds with unsaturated rings within their structures, an interesting possibility considering that the general NA formula (CnH2n+zO2) does not take into account unsaturated rings. As mentioned previously, 1H NMR analyses of the fractions collected from OSPW indicate that as NAs increase in size, additional carboxylic groups are present within the structures. Although compounds containing multiple carboxylic acid groups may not be considered “classical” NAs, they are present within the organic acid component of OSPW, contain structures and MW ranges similar to those of NAs, and therefore should be considered when assessing the toxicity of OSPW NA mixtures. The presence of more than one carboxylic acid moiety within a NA structure offers a plausible explanation as to why lower MW NAs exhibit a greater acute toxicity. For typical NA structures, the larger MW compounds should have a greater hydrophobicity, be more susceptible to bioaccumulation, and when assuming a narcotic mode of action, should elicit a greater toxic response. However, increased toxicity with larger MW NAs has not been observed in OSPW biodegradation assessments (6, 9, 10, 13), nor in fractions of NAs with differing MW from OSPW (23). A possible explanation for this result is the presence of larger MW NAs containing more than one carboxylic acid moiety. These multiple carboxylated structures would be less hydrophobic than lower MW monocarboxyl NAs and consequently be less toxic. Because of the limited availability of larger compounds, the NA-like surrogates investigated in this study had relatively low MWs (116.16-200.23 Da) in comparison to the NAs observed in OSPW (142-500 Da 13, 14). Also, the nonbranched acyclic surrogate structures were likely simpler than OSPW NAs, which are highly branched (14, 22). However, the goal of this study was to determine the effect that size, structure, and carboxylic acid content had on NA toxicity; therefore, the relationships observed from the studied surrogates in this study could exist for larger compounds more commonly found in OSPW NA mixtures. VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Calculated Effect Concentrations for the Vibrio fischeri Microtox and the Daphnia magna Acute Lethality Assays with Exposure to Eight NA-Like Surrogates (units are expressed in mg L-1 as well as mM)a Vibrio fischeri Microtox assay: calculated EC50 ( 95% CI
Daphnia magna acute lethality assay: calculated LC50 ( 95% CI
hexanoic acid (HA)
2222 ( 303 mg L-1 19.1 ( 2.6 mM
1166 ( 153 mgL-1 10.0 ( 1.3 mM
cyclohexanecarboxylic acid (CHCA)
1233 ( 238 mg L-1 9.6 ( 1.9 mM
855 ( 97 mg L-1 6.7 ( 0.8 mM
decanoic acid (DA)
57.0 ( 6.7 mgL-1 0.33 ( 0.04 mM
219 ( 64 mg L-1 1.3 ( 0.4 mM
cyclohexanepentanoic acid (CHPA)
13.0 ( 1.6 mg L-1 0.07 ( 0.01 mM
109 ( 38 mg L-1 0.59 ( 0.20 mM
succinic acid (SA)
74079 ( 3463 mg L-1 627 ( 29 mM (a)
3223 ( 261 mg L-1 27.3 ( 2.2 mM
adipic acid (AA)
68402 ( 31215 mg L-1 2996 ( 164 mg L-1 468 ( 214 mM (a,b) 20.5 ( 1.1 mM
NA surrogate (abbreviation)
1,4-cyclohexanedicar- 80185 ( 3695 mg L-1 boxylic acid (CHDCA) 466 ( 21 mM (b) cyclohexylsuccinic acid (CHSA)
5224 ( 376 mg L-1 26.1 ( 1.9 mM
2632 ( 128 mg L-1 15.3 ( 0.7 mM 1344 ( 246 mg L-1 6.7 ( 1.2 mM
a All concentrations (mM) not followed by a letter are significantly different, as defined by a lack of overlap between 95% confidence intervals (CIs). Values followed by the same letter are not significantly different.
The toxicity data for the NA-like surrogates (Table 3) confirmed that a reduction in toxicity was associated with the addition of a carboxylic group within the molecule. The Microtox Basic Test yielded EC50 values which decreased with greater MW in the monocarboxyl and dicarboxyl NA-like surrogates (Figure 2). Significant differences existed between all of the EC50 values derived for the monocarboxyl NA-like surrogates. When comparing the dicarboxyl NA-like surrogates, CHDCA was significantly more toxic than SA, and CHSA was significantly more toxic than the other three compounds. In addition, the response to the dicarboxyl NAlike surrogate eliciting the greatest effect, CHSA, was significantly less in the Microtox assay than the response to the monocarboxyl NA-like surrogate that elicited the weakest effect, HA. These results support the hypothesis that the acute toxicity of NA-like surrogates increases with increasing MW; however, the addition of a carboxylic group would cause a significant decrease in toxicity. The 48-h acute toxicity test using D. magna yielded similar results to the V. fischeri Microtox assay. The acute toxicity of the NA-like surrogates increased with increasing MW for both the monocarboxyl and dicarboxyl compounds, and the monocarboxyl NA-like surrogates elicited significantly greater toxic responses than dicarboxyl NA-like surrogates of similar MWs (Table 3; Figure 2). However, D. magna were more sensitive to dicarboxyl NA-like surrogates than V. fischeri in the Microtox assay. If aged OSPW contained a high concentration of multiple-carboxylated compounds and toxicity was only assessed using the V. fischeri Microtox assay, the calculated effect concentrations could be overestimated. Consequently, the toxicity of this aged OSPW could be underestimated toward potentially more relevant test organisms, in consideration of the fact that V. fischeri is a marine bacterium and oil sands tailings ponds are located freshwater environments. 270
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FIGURE 2. EC50 values ( the 95% confidence interval for Vibrio fischeri bioluminescence following 15 min exposure, and LC50 values ( the 95% confidence interval for Daphnia magna following 48-h exposure, to eight NA-like surrogates. The bioassays with the NA-like surrogates indicate that toxicity is likely a function of hydrophobicity. As the MW of the surrogates increased, so too did the acute toxicity, while if an additional carboxylic group was present in the compound, the toxicity was significantly decreased. The 1H NMR and FTIR results observed in this study confirmed that higher MW NA-like compounds have more than one carboxylic group within their structure and therefore would likely be less hydrophobic than the lower MW monocarboxylic NAs. The evidence within the 1H NMR analyses, in combination with the toxicity results using the NA-like surrogate compounds, supports narcosis as the probable mode of action for NA acute toxicity. Regardless of whether or not these multiple-carboxylated structures should be considered NAs, they are organic acids with structures and MW ranges similar to those of “classical” NAs and are present within OSPW. They are also a probable contributor to the observed decrease in toxicity for aged OSPW that has undergone microbial degradation of the lower MW component in the NA mixture.
Acknowledgments The authors would like to thank Wellington Laboratories for offering their facilities for the fractionation method development, the NMR Centre in the Physics Department at the University of Guelph for performing the 1H NMR analyses, and Dr. Robert Reed in the Department of Chemistry at the University of Guelph for dedicating time and resources for the FTIR analyses. We thank Lorna Deeth, Benjamin de Jourdan, Kelly Shannon, Derek Hillis, and Richard Brain for technical support. Financial support was provided in part by the Program for Energy Research and Development, Syncrude Canada Ltd., and the Canadian Water Network.
Supporting Information Available Table S1 provides the nominal test concentrations used for the Vibrio fischeri and Daphnia magna acute lethality assays with exposure to eight NA-like surrogates. 1H NMR spectra and FTIR spectra for the five collected NA fractions are depicted in Figures S1 and S2, respectively. This information is available free of charge via the Internet at http:// pubs.acs.org.
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