Degradation of naphthenic acids by sediment micro-organisms.

AIMS: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. METHODS AND RESULTS: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a 14C-labelled monocyclic NA surrogate [14C-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [14C-decahydronaphthoic acid (DHNA)] was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. CONCLUSIONS: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

The effects of salinity on naphthenic acid toxicity to yellow perch: gill and liver histopathology.

Naphthenic acids (NAs) are naturally occurring saturated linear and cyclic carboxylic acids found in petroleum, including the bitumen contained in the Athabasca Oil Sands deposit in Alberta, Canada. The processing of these oil sands leads to elevated concentrations of NAs, as well as increased salinity from produced waters as a result of ions leaching from the ores, the process aids, and the water associated with the deeper aquifers. These changes can result in waters that challenge reclamation of impacted waters associated with oil sands development. Laboratory tests examined the effects of salinity on NA toxicity using local young-of-the-year yellow perch exposed to a commercially available mixture of NAs (CNA) and an NA mixture that was extracted from oil sands process-affected water (ENA), with and without the addition of sodium sulfate (Na(2)SO(4)). Gill and liver histopathological changes were evaluated in the surviving fish after 3 weeks of exposure. At 6.8 mg/L ENA and 3.6 mg/L CNA, 100% mortality was observed, both with and without the addition of salt. Exposure of yellow perch to 25% of the NA required to give an LC100 (0.9 mg/L CNA; 1. 7 mg/L ENA) resulted in high levels of gill proliferative (epithelial, mucous, and chloride cell) changes, a response that was increased with the addition of 1g/L salt (Na2SO4) for the ENA. The significance of these changes was a reduced gill surface area, which likely caused a reduction in both the transport of NAs within the fish and the exchange of vital respiratory gases. While the gills were affected, no liver alterations were identified following NA or NA+salt exposures. Differences in the chemical composition of the NAs tested may explain the differences in the lethality and histopathology of yellow perch.

Responses of red-osier dogwood to oil sands tailings treated with gypsum or alum.

The application of composite or consolidated tailings (CT) technology provides Alberta's oil sands industry with a means of reducing the volume of the fines fraction in extraction tailings and allows for faster reclamation and revegetation of mining sites. This study examined the effects of coagulant aids (gypsum and alum), used in the production of CT, on the ion content, growth, and survival of greenhouse-grown red-osier dogwood (Cornus sericea L. subsp. sericea). Seedlings were planted in gypsum-CT and alum-CT substrates, and compared with those planted in reclamation material (salvaged peat and till). The seedlings were bottom-watered with one of the following: (i) Hoagland mineral solution prepared in deionized water (Epstein, 1972); (ii) Hoagland solution in gypsum-based CT release water; or (iii) Hoagland solution in alum-based CT release water. Pore water of CT substrates and CT release waters had similar chemical characteristics, including salinity levels. However, plants in CT substrates had higher concentrations of ions (particularly Na and B), reduced growth, and higher mortality than plants in reclamation material and treated with CT waters. The presence of H2S indicated low-oxygen conditions in the CT substrates, while in the reclamation materials with CT release water treatments, no evidence of sulfides was observed. Low-oxygen conditions in the CT substrate treatments may have interfered with plant exclusion mechanisms for Na and B. Therefore, substrate properties may modify responses of reclamation plants to pore water chemistry due to the effects on oxygen availability to roots.

Aquatic reclamation in the Athabasca, Canada, oil sands: naphthenate and salt effects on phytoplankton communities.

Microcosm experiments with natural indigenous phytoplankton communities were conducted to assess the effects of waters from oil sands extraction processes, emphasizing the naphthenate and salt constituents. Process waters of varying ages (zero to eight years) remediation histories, and chemical composition were obtained from outdoor mesocosms and inoculated with phytoplankton assemblages from a reference lake in the study area. Community composition measures, including percentage model affinity (PMA) and canonical correspondence analysis (CCA), revealed significant community effects of water from systems less than five years old, with naphthenate concentrations greater than 20 mg/L, compared to water from the reference lake. Canonical correspondence analysis, PMA, and regression analyses further showed that naphthenate concentration was significantly correlated with community structure. Using CCA, groups of taxa characteristic of waters with > 20 mg/L naphthenates (including Botryococcus braunii, Gloeococcus schroeteri, Cosmarium depressum, Chrysococcus rufescens, Chromulina spp., Ochromonas spp., and Keratococcus spp.) were identified. Salinity, as reflected in conductivity, was positively correlated with naphthenate concentration and itself appeared to influence the community structure. The results confirmed an important role for naphthenates in ecological effects of process waters from oil sands mining, but the influence of covarying factors such as salinity requires further investigation.

Naphthenic acids and surrogate naphthenic acids in methanogenic microcosms.

Naphthenic acids (NAs) are a complex mixture of naturally occurring acyclic and cyclic aliphatic carboxylic acids in petroleum. In the Athabasca oil sands. NAs have been identified as the largest component of dissolved organic matter in the tailings waters from oils sands extraction processes. They are the major contributor to the acute toxicity of the fine tailings wastewaters at the oil sands extraction plants in northeastern Alberta, Canada. In this study, three sources of NAs were studied, including commercially available NAs, those extracted from oil sands process-affected waters, and individual naphthenic-like surrogate compounds. Analysis by gas chromatography-mass spectrometry demonstrated differences between the commercial and extracted NAs. The NAs derived from the process-affected waters showed a short-term inhibition of methanogenesis from H2 or acetate, but with time the populations resumed methane production. It has been postulated that microbial metabolism of the carboxylated side chains of NAs would lead to methane production. The two NA mixtures failed to stimulate methanogenesis in microcosms that contained either oil sands fine tailings or domestic sewage sludge. However, in microcosms with sewage sludge, methanogenesis was stimulated by some surrogate NAs including 3-cyclohexylpropanoic acid at 400-800 mg/L, 5-cyclohexylpentanoic acid at 200 mg/L or 6-phenylhexanoic acid at 200 and 400 mg/L. When added at 200 mg/L to methanogenic microcosms containing fine tailings, 3-cyclohexylpropanoic and 4-cyclohexylbutanoic acids produced methane yields that suggested mineralization of the side chain and the ring.

Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste.

In the past decade, the large tailings pond (Mildred Lake Settling Basin) on the Syncrude Canada Ltd. lease near Fort McMurray, Alta., has gone methanogenic. Currently, about 60%-80% of the flux of gas across the surface of the tailings pond is methane. As well as adding to greenhouse gas emissions, the production of methane in the fine tailings zone of this and other settling basins may affect the performance of these settling basins and impact reclamation options. Enumeration studies found methanogens (10(5)-10(6) MPN/g) within the fine tailings zone of various oil sands waste settling basins. SRB were also present (10(4)-10(5) MPN/g) with elevated numbers when sulfate was available. The methanogenic population was robust, and sample storage up to 9 months at 4 degrees C did not cause the MPN values to change. Nor was the ability of the consortium to produce methane delayed or less efficient after storage. Under laboratory conditions, fine tailings samples released 0.10-0.25 mL CH4 (at STP)/mL fine tailings. The addition of sulfate inhibited methanogenesis by stimulating bacterial competition.

Biodegradation of naphthenic acids by microbial populations indigenous to oil sands tailings.

Organic acids, similar in structure to naphthenic acids, have been associated with the acute toxicity of tailings produced by the oil sands industry in northeastern Alberta, Canada. Bacterial cultures enriched from oil sands tailings were found to utilize as their sole carbon source both a commercial mixture of naphthenic acids and a mixture of organic acids extracted from oil sands tailings. Gas chromatographic analysis of both the commercial naphthenic acids and the extracted organic acids revealed an unresolved "hump" formed by the presence of many overlapping peaks. Microbial activity directed against the commercial mixture of naphthenic acids converted approximately 50% of organic carbon into CO2 and resulted in a reduction in many of the gas chromatographic peaks associated with this mixture. Acute toxicity testing utilizing the Microtox test revealed a complete absence of detectable toxicity following the biodegradation of the naphthenic acids. Microbial activity mineralized approximately 20% of the organic carbon present in the extracted organic acids mixture, although there was no indication of a reduction in any gas chromatographic peaks with biodegradation. Microbial attack on the organic acids mixture reduced acute toxicity to approximately one half of the original level. Respirometric measurements of microbial activity within microcosms containing oil sands tailings were used to provide further evidence that the indigenous microbial community could biodegrade naphthenic acids and components within the extracted organic acids mixture.

Paired-ion reversed-phase high-performance liquid chromatography of phenol sulfates in synthetic mixtures, algal extracts and urine.

Phenol sulfate esters have been analyzed by paired-ion reversed-phase high-performance liquid chromatography. The method provided direct, rapid chromatography of phenol sulfates in crude extracts of the red alga (Polysiphonia lanosa (2,3-dibromo-4,5-dihydroxybenzyl alcohol 1',4-disulfate), of the brown alga Ascophyllum nodosum (1,2,3,5-tetrahydroxybenzene 2,5-disulfate), and in rat urine (resorcinol mono- and disulfates). Detector response (254 nm) was linear within the approximate range from 30--125 ng to 5--10 microgram. Semipreparative scale chromatography provided sufficient amounts of purified phenol sulfates for further analysis by paper electrophoresis.