Selective biodegradation of naphthenic acids and a probable link between mixture profiles and aquatic toxicity.

The toxicity of oil sands process-affected waters (OSPW) from the Athabasca Oil Sands (AOS) in northern Alberta, Canada, is related to a relatively persistent group of dissolved organic acids known as naphthenic acids (NAs). Naphthenic acids are a complex mixture of carboxylic acids, with a general formula C(n)H(2n+Z)O2, where n indicates the carbon number and Z specifies the number of rings in the molecule. The present study is the first to evaluate the potential for the selective biodegradation of NAs and the associated reduction in aquatic toxicity of 2 OSPWs, maintained under 2 different hydraulic retention times and increased nutrient availability (nitrate and phosphate), using flow-through laboratory wetland microcosms over a 52-wk test period. High-performance liquid chromatography/quadrupole time of flight-mass spectrometry analysis was used to track the changes in NA mixture profiles, or "fingerprints," in each treatment over time. Based on first-order degradation kinetics, more rapid degradation was observed for NAs that had lower carbon numbers and fewer degrees of cyclization (NA congeners with carbon numbers 11-16 and Z series -2 to -4; half-lives between 19 and 28 wk). Within the NA mixture fingerprints, the 2 most persistent groups of homologues were also identified (NAs with carbon numbers 17-20 and Z series -6 to -12; half-lives between 37 and 52 wk). The persistence of this group of NAs may aid in explaining the residual chronic toxicological response as measured by the Microtox bioassay (effective concentration for 20%), after the degradation of the more labile fractions of NA mixtures in OSPW.

Degradation and aquatic toxicity of naphthenic acids in oil sands process-affected waters using simulated wetlands.

Oil sands process-affected waters (OSPWs) produced during the extraction of bitumen at the Athabasca Oil Sands (AOS) located in northeastern Alberta, Canada, are toxic to many aquatic organisms. Much of this toxicity is related to a group of dissolved organic acids known as naphthenic acids (NAs). Naphthenic acids are a natural component of bitumen and are released into process water during the separation of bitumen from the oil sand ore by a caustic hot water extraction process. Using laboratory microcosms as an analogue of a proposed constructed wetland reclamation strategy for OSPW, we evaluated the effectiveness of these microcosms in degrading NAs and reducing the aquatic toxicity of OSPW over a 52-week test period. Experimental manipulations included two sources of OSPW (one from Syncrude Canada Ltd. and one from Suncor Energy Inc.), two different hydraulic retention times (HRTs; 40 and 400 d), and increased nutrient availability (added nitrate and phosphate). Microcosms with a longer HRT (for both OSPWs) showed higher reductions in total NAs concentrations (64-74% NAs reduction, p<0.05) over the test period, while nutrient enrichment appeared to have little effect. A 96 h static acute rainbow trout (Oncorhynchus mykiss) bioassay showed that the initial acute toxicity of Syncrude OSPW (LC50=67% v/v) was reduced (LC50>100% v/v) independent of HRT. However, EC20s from separate Microtox® bioassays were relatively unchanged when comparing the input and microcosm waters at both HRTs over the 52-week study period (p>0.05), indicating that some sub-lethal toxicity persisted under these experimental conditions. The present study demonstrated that given sufficiently long HRTs, simulated wetland microcosms containing OSPW significantly reduced total NAs concentrations and acute toxicity, but left behind a persistent component of the NAs mixture that appeared to be associated with residual chronic toxicity.

Petroleum coke adsorption as a water management option for oil sands process-affected water.

Water is integral to both operational and environmental aspects of the oil sands industry. A water treatment option based on the use of petroleum coke (PC), a by-product of bitumen upgrading, was examined as an opportunity to reduce site oil sands process-affected water (OSPW) inventories and net raw water demand. Changes in OSPW quality when treated with PC included increments in pH levels and concentrations of vanadium, molybdenum, and sulphate. Constituents that decreased in concentration after PC adsorption included total acid-extractable organics (TAO), bicarbonate, calcium, barium, magnesium, and strontium. Changes in naphthenic acids (NAs) speciation were observed after PC adsorption. A battery of bioassays was used to measure the OSPW toxicity. The results indicated that untreated OSPW was toxic towards Vibrio fischeri and rainbow trout. However, OSPW treated with PC at appropriate dosages was not acutely toxic towards these test organisms. Removal of TAO was found to be an adsorption process, fitting the Langmuir and Langmuir-Freundlich isotherm models. For TAO concentrations of 60 mg/L, adsorption capacities ranged between 0.1 and 0.46 mg/g. This study demonstrates that freshly produced PC from fluid cokers provides an effective treatment of OSPW in terms of key constituents' removal and toxicity reduction.

Petroleum coke and soft tailings sediment in constructed wetlands may contribute to the uptake of trace metals by algae and aquatic invertebrates.

The fate of trace metals in pore water collected from wetland sediments and organisms exposed to petroleum coke were evaluated within in situ aquatic microcosms. Oil sands operators of Fort McMurray, Alberta, Canada produced 60 million tonnes of petroleum coke by 2008, containing elevated concentrations of sulphur and several trace metals commonly seen in oil sands materials. This material may be included in the construction of reclaimed wetlands. Microcosms were filled with a surface layer of petroleum coke over mine-waste sediments and embedded in a constructed wetland for three years to determine how these materials would affect the metal concentrations in the sediment pore water, colonizing wetland plants and benthic invertebrates. Petroleum coke treatments produced significantly elevated levels of Ni. We also found unexpectedly higher concentrations of metals in "consolidated tailings" waste materials, potentially due to the use of oil sands-produced gypsum, and higher background concentration of elements in the sediment used in the controls. A trend of higher concentrations of V, Ni, La, and Y was present in the tissues of the colonizing macrophytic alga Chara spp. Aeshnid dragonflies may also be accumulating V. These results indicate that the trace metals present in some oil sands waste materials could be taken up by aquatic macro-algae and some wetland invertebrates if these materials are included in reclaimed wetlands.

Estimating the in situ biodegradation of naphthenic acids in oil sands process waters by HPLC/HRMS.

The oil sands industry in Northern Alberta produces large volumes of oil sands process water (OSPW) containing high concentrations of persistent naphthenic acids (NAs; C(n)H(2n+Z)O(2)). Due to the growing volumes of OSPW that need to be reclaimed, it is important to understand the fate of NAs in aquatic systems. A recent laboratory study revealed several potential markers of microbial biodegradation for NAs; thus here we examined for these signatures in field-aged OSPW on the site of Syncrude Canada Ltd. (Fort McMurray, AB). NA concentrations were lower in older OSPW; however parent NA signatures were remarkably similar among all OSPW samples examined, with no discernible enrichment of the highly cyclic fraction as was observed in the laboratory. Comparison of NA signatures in fresh oil sands ore extracts to OSPW in active settling basins, however, suggested that the least cyclic fraction (i.e. Z=0 and Z=-2 homologues) may undergo relatively rapid biodegradation in active settling basins. Further evidence for biodegradation of NAs came from a significantly higher proportion of oxidized NAs (i.e. C(n)H(2n+Z)O(3)+C(n)H(2n+Z)O(4)) in the oldest OSPW from experimental reclamation ponds. Taken together, there is indirect evidence for rapid biodegradation of relatively labile Z=0 and Z=-2 NAs in active settling basins, but the remaining steady-state fraction of NAs in OSPW appear to be very recalcitrant, with half-lives on the order of 12.8-13.6 years. Alternative fate mechanisms to explain the slow disappearance of parent NAs from OSPW are discussed, including adsorption and atmospheric partitioning.

Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride.

Black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 mM KF and 5 mM KF) in the presence and absence of 60 mM NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 mM NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 mM NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K(r) by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K(r) in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 mM KF + 60 mM NaCl were significantly reduced compared with the 5 mM KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.

A first approximation kinetic model to predict methane generation from an oil sands tailings settling basin.

A small fraction of the naphtha diluent used for oil sands processing escapes with tailings and supports methane (CH(4)) biogenesis in large anaerobic settling basins such as Mildred Lake Settling Basin (MLSB) in northern Alberta, Canada. Based on the rate of naphtha metabolism in tailings incubated in laboratory microcosms, a kinetic model comprising lag phase, rate of hydrocarbon metabolism and conversion to CH(4) was developed to predict CH(4) biogenesis and flux from MLSB. Zero- and first-order kinetic models, respectively predicted generation of 5.4 and 5.1 mmol CH(4) in naphtha-amended microcosms compared to 5.3 (+/-0.2) mmol CH(4) measured in microcosms during 46 weeks of incubation. These kinetic models also predicted well the CH(4) produced by tailings amended with either naphtha-range n-alkanes or BTEX compounds at concentrations similar to those expected in MLSB. Considering 25% of MLSB's 200 million m(3) tailings volume to be methanogenic, the zero- and first-order kinetic models applied over a wide range of naphtha concentrations (0.01-1.0 wt%) predicted production of 8.9-400 million l CH(4) day(-1) from MLSB, which exceeds the estimated production of 3-43 million l CH(4) day(-1). This discrepancy may result from heterogeneity and density of the tailings, presence of nutrients in the microcosms, and/or overestimation of the readily biodegradable fraction of the naphtha in MLSB tailings.

Ozonation of oil sands process water removes naphthenic acids and toxicity.

Naphthenic acids are naturally-occurring, aliphatic or alicyclic carboxylic acids found in petroleum. Water used to extract bitumen from the Athabasca oil sands becomes toxic to various organisms due to the presence of naphthenic acids released from the bitumen. Natural biodegradation was expected to be the most cost-effective method for reducing the toxicity of the oil sands process water (OSPW). However, naphthenic acids are poorly biodegraded in the holding ponds located on properties leased by the oil sands companies. In the present study, chemical oxidation using ozone was investigated as an option for mitigation of this toxicity. Ozonation of sediment-free OSPW was conducted using proprietary technology manufactured by Seair Diffusion Systems Inc. Ozonation for 50min generated a non-toxic effluent (based on the Microtox bioassay) and decreased the naphthenic acids concentration by approximately 70%. After 130min of ozonation, the residual naphthenic acids concentration was 2mgl(-1): <5% of the initial concentration in the filtered OSPW. Total organic carbon did not change with 130min of ozonation, whereas chemical oxygen demand decreased by approximately 50% and 5-d biochemical oxygen demand increased from an initial value of 2mgl(-1) to a final value of 15mgl(-1). GC-MS analysis showed that ozonation resulted in an overall decrease in the proportion of high molecular weight naphthenic acids (n> or = 22).

Metabolism of BTEX and naphtha compounds to methane in oil sands tailings.

Naphtha, comprising low molecular weight aliphatics and aromatics (C3-C14), is used as a diluent in processing of bitumen from oil sands. A small fraction (<1%) is lost to tailings waste and incorporated into mature fine tailings (MFT). BTEX (benzene, toluene, ethylbenzene, and xylenes) and whole naphtha were assessed for biodegradation under methanogenic conditions using MFT from an oil sands tailings settling basin. MFT spiked with 0.05-0.1% w/v of BTEX compounds produced up to 2.1 (+/-0.1) mmol of methane during 36 weeks of incubation. Metabolism of 0.5-1.0% w/v naphtha in MFT yielded up to 5.7 (+/-0.2) mmol of methane during 46 weeks of incubation. Gas chromatographic analyses showed that BTEX degraded in the sequence: toluene > o-xylene > m- plus p-xylene > ethylbenzene > benzene. Only 15-23% of whole naphtha, mainly n-alkanes (in the sequence: nonane > octane > heptane) and some BTEX compounds (toluene > o-xylene > m-xylene), was metabolized. Other naphtha constituents, such as iso-paraffins and naphthenes, remained unchanged during this period. These results suggest that the microbial communities in the MFT can readily utilize certain fractions of unrecovered naphtha in oil sands tailings and support methanogenesis in settling basins. Current study findings could influence extraction process, MFT management, and reclamation options.

Naphthenic acids in athabasca oil sands tailings waters are less biodegradable than commercial naphthenic acids.

Naphthenic acids (NAs) are natural constituents in many petroleum sources, including bitumen in the oil sands of Northern Alberta, Canada. Bitumen extraction processes produce tailings waters that cannot be discharged to the environment because NAs are acutely toxic to aquatic species. However, aerobic biodegradation reduces the toxic character of NAs. In this study, four commercial NAs and the NAs in two oil sands tailings waters were characterized by gas chromatography-mass spectrometry. These NAs were also incubated with microorganisms in the tailings waters under aerobic, laboratory conditions. The NAs in the commercial preparations had lower molecular masses than the NAs in the tailings waters. The commercial NAs were biodegraded within 14 days, but only about 25% of the NAs native to the tailings waters were removed after 40-49 days. These results show that low molecular mass NAs (C < or =17) are more readily biodegraded than high molecular mass NAs (C > or =18). Moreover, the results indicate that biodegradation studies using commercial NAs alone will not accurately reflect the potential biodegradability of NAs in the oil sands tailings waters.

Polyacrylamide added as a nitrogen source stimulates methanogenesis in consortia from various wastewaters.

Polyacrylamides are widely used as flocculants to enhance clarification of drinking waters and domestic wastewaters, for stabilization of agricultural soils, and to aid in managing mine tailings. The flocs produced with polyacrylamide may be deposited into retention areas that become anaerobic. Although it is unlikely that the carbon backbone of these polymers would be cleaved by microbial activity, the amide group could serve as a nitrogen source for microorganisms. Previous studies have shown that aerobic bacteria utilize the nitrogen from polyacrylamide. This study assessed whether methanogenesis was stimulated when an anionic polyacrylamide (Magnafloc LT27AG) was the sole fixed nitrogen source in serum-bottle microcosms. Microorganisms from two oil sands tailings sources, and a domestic anaerobic sewage sludge were used, with benzoate or acetate provided as carbon and energy sources. In each inoculum-substrate combination, the presence of polyacrylamide-enhanced methane production, indicating that polyacrylamide may stimulate microbial activities in anaerobic environments that are rich in fermentable carbon, but lack nitrogen sources.

Measuring naphthenic acids concentrations in aqueous environmental samples by liquid chromatography.

Naphthenic acids are found in wastewaters from petroleum refineries and oil sands extraction plants. Currently, the concentrations of these toxic carboxylic acids are determined by extracting them into methylene chloride and measuring the absorption of the carboxyl group by Fourier-transform infrared (FTIR) spectroscopy. An improved HPLC method, that is simpler and faster than the FTIR method, was used to detect the 2-nitrophenylhydrazides of the naphthenic acids at concentrations as low as 5 mg l(-1). Analyses of 58 oil sands water samples showed that the naphthenic acids concentrations determined by FTIR were on average 11% higher than those determined by HPLC.

Aerobic biodegradation of two commercial naphthenic acids preparations.

Naphthenic acids (NAs) have a variety of commercial uses including as emulsifiers and wood preservatives. They have been identified as being the main component responsible for the acute toxicity in produced waters in the oil sands operations in northeastern Alberta, Canada. NAs comprise a complex mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula CnH(2n+Z)O2, where n indicates the carbon number and Z specifies hydrogen deficiency from ring formation. In this study, commercial preparations of NAs were shown to be degraded in aerobic cultures from oil sands process-affected waters. High-performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) were used to monitor the concentrations and composition of the NA mixtures during biodegradation. Within 10 days of incubation, the NAs concentrations dropped from about 100 to <10 mg/L. This was accompanied by the release of about 60% of carbon from the NAs as CO2 and the reduction of toxicity of the culture supernatant, as measured by the Microtox assay. GC-MS results demonstrated that biodegradation changes the composition of the complex mixture of these NAs and that the lower molecular weight acids (with n = 5-13) were degraded more readily than the high molecular weight acids.

A statistical comparison of naphthenic acids characterized by gas chromatography-mass spectrometry.

Naphthenic acids are complex mixtures of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula C(n)H(2n+z)O(2), where n is the carbon number and Z specifies a homologous family. These acids have a variety of commercial uses, including being used as wood preservatives. They are found in conventional and heavy oils, and in the oil sands of northeastern Alberta, Canada. Naphthenic acids are major contributors to the toxicity of tailings waters that result from the oil sands extraction process. Eight naphthenic acids preparations (four from commercial sources and four from the oil sands operations) were derivatized and analyzed by gas chromatography-mass spectrometry. The composition of each mixture was summarized as a three-dimensional plot of the abundance of specific ions (corresponding to naphthenic acids) versus carbon number (ranging from 5 to 33) and Z family (ranging from 0 to -12). The data in these plots were divided into three groups according to carbon number (group 1 contained carbon numbers 5-14, group 2 contained carbon numbers 15-21, and group 3 contained carbon numbers 22-33). A t-test, using arcsine-transformed data, was applied to compare corresponding groups in samples from various sources. Results of the statistical analyses showed differences between various commercial naphthenic acids preparations, and between naphthenic acids from different oil sands ores and tailings ponds. This statistical approach can be applied to data collected by other mass spectrometry methods.

Isolation and characterization of naphthenic acids from Athabasca oil sands tailings pond water.

A laboratory bench procedure was developed to efficiently extract naphthenic acids from bulk volumes of Athabasca oil sands tailings pond water (TPW) for use in mammalian oral toxicity testing. This solvent-based procedure involved low solvent losses and a good extraction yield with low levels of impurities. Importantly, labour-intensive centrifugation of source water to remove solids was avoided, allowing processing of much larger volumes of water compared with previous protocols. Naphthenic acids, present at an estimated concentration of 81 mg/l, were procured from 515.5 l of TPW at an overall extraction efficiency of approximately 85%. By using distillation to recover and recycle solvent, a high solvent:water ratio was maintained while actual solvent consumption was limited to 70 ml per liter of water processed. Electrospray ionization mass spectrometry suggested a highly heterogeneous naphthenic acid mixture that exhibited nearly identical proportions of monocyclic, polycyclic, and acyclic acids with molecular weights primarily between 220 and 360. Biphenyls, naphthalenes, and phenanthrene/anthracene were the most prominent impurities detected, but their levels were low (< or = 13 microg/l) even in a concentrated solution of the naphthenic acids (8549 mg/l). Naphthenic acids stored at 4 degrees C at this concentration were stable, exhibiting no significant change in concentration over a 10-month period. This bulk isolation procedure should be useful to others needing to process large volumes of tailings or other source water for the purpose of procuring moderate amounts of naphthenic acids.

Characterization of naphthenic acids in oil sands wastewaters by gas chromatography-mass spectrometry.

The water produced during the extraction of bitumen from oil sands is toxic to aquatic organisms due largely to a group of naturally occurring organic acids, naphthenic acids (NAs), that are solubilized from the bitumen during processing. NAs are a complex mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula CnH(2n + Z)O2, where n is the carbon number and Z specifies a homologous family. Gas chromatography-electron impact mass spectrometry was used to characterize NAs in nine water samples derived from oil sands extraction processes. For each sample, the analysis provided the relative abundances for up to 156 base peaks, with each representing at least one NA structure. Plotting the relative abundances of NAs as three-dimensional bar graphs showed differences among samples. The relative abundance of NAs with carbon numbers < or = 21 to those in the "C22 + cluster" (sum of all NAs with carbon numbers > or = 22 in Z families 0 to -12) proved useful for comparing the water samples that had a range of toxicities. A decrease in toxicity of process-affected waters accompanied an increase in the proportion of NAs in the "C22 + cluster", likely caused by biodegradation of NAs with carbon numbers of < or = 21. In addition, an increase in the proportion of NAs in the "C22 + cluster" accompanied a decrease in the total NAs in the process-affected waters, again suggesting the selective removal of NAs with carbon numbers of < or = 21. This is the first investigation in which changes in the fingerprint of the NA fraction of process-affected waters from the oil sands operations has corresponded with measured toxicity in these waters.

Acute and subchronic mammalian toxicity of naphthenic acids from oil sands tailings.

Naphthenic acids are the most significant environmental contaminants resulting from petroleum extraction from oil sands deposits. In this study, a mixture of naphthenic acids isolated from Athabasca oil sands (AOS) tailings pond water was used in acute and subchronic toxicity tests with rodents, in order to assess potential risks posed to terrestrial wildlife. Dosages were chosen to bracket worst-case environmental exposure scenarios. In acute tests, adult female Wistar rats were given single po dosages of naphthenic acids at either 3, 30, or 300 mg per kg body weight (mg/kg), while adult male rats received 300 mg/kg. Food consumption was temporarily suppressed in the high-dose groups of both sexes. Following euthanasia 14 days later, histopathology revealed a significant incidence of pericholangitis in the high-dose group of both sexes, suggesting hepatotoxicity as an acute effect. Other histological lesions included brain hemorrhage in high-dose males, and cardiac periarteriolar necrosis and fibrosis in female rats. In subchronic tests, naphthenic acids were po administered to female Wistar rats at 0.6, 6, or 60 mg/kg, 5 days per week for 90 days. Results again suggested the liver as a potential target organ. The relative liver weight in the high-dose group was 35% higher than in controls. Biochemical analysis revealed elevated blood amylase (30% above controls) and hypocholesterolemia (43% below controls) in high-dose rats. Excessive hepatic glycogen accumulation was observed in 42% of animals in this group. These results indicate that, under worst-case exposure conditions, acute toxicity is unlikely in wild mammals exposed to naphthenic acids in AOS tailings pond water, but repeated exposure may have adverse health effects.