The effect of oil sands process-affected water and naphthenic acids on the germination and development of Arabidopsis

Oil sands mining in the Athabasca region of northern Alberta results in the production of large volumes of oil sands process-affected water (OSPW). We have evaluated the effects of OSPW, the acid extractable organic (AEO) fraction of OSPW, and individual naphthenic acids (NAs) on the germination and development of the model plant, Arabidopsis thaliana (Arabidopsis). The surrogate NAs that were selected for this study were petroleum NAs that have been used in previous toxicology studies and may not represent OSPW NAs. A tricyclic diamondoid NA that was recently identified as a component of OSPW served as a model NA in this study. Germination of Arabidopsis seeds was not inhibited when grown on medium containing up to 75% OSPW or by 50 mg L⁻¹ AEO. However, simultaneous exposure to three simple, single-ringed surrogate NAs or a double-ringed surrogate NA had an inhibitory effect on germination at a concentration of 10 mg L⁻¹, whereas inhibition of germination by the diamondoid model NA was observed only at 50 mg L⁻¹. Seedling root growth was impaired by treatment with low concentrations of OSPW, and exposure to higher concentrations of OSPW resulted in increased growth inhibition of roots and primary leaves, and caused bleaching of cotyledons. Treatment with single- or double-ringed surrogate NAs at 10 mg L⁻¹ severely impaired seedling growth. AEO or diamondoid NA treatment was less toxic, but resulted in severely impaired growth at 50 mg L⁻¹. At low NA concentrations there was occasionally a stimulatory effect on root and shoot growth, possibly owing to the broad structural similarity of some NAs to known plant growth regulators such as auxins. This report provides a foundation for future studies aimed at using Arabidopsis as a biosensor for toxicity and to identify genes with possible roles in NA phytoremediation.     

Diethylaminoethyl-cellulose clean-up of a large volume naphthenic acid extract

The Athabasca oil sands of Alberta, Canada contain an estimated 174 billion barrels of bitumen. During oil sands refining processes, an extraction tailings mixture is produced that has been reported as toxic to aquatic organisms and is therefore collected in settling ponds on site. Investigation into the toxicity of these tailings pond waters has identified naphthenic acids (NAs) and their sodium salts as the major toxic components, and a multi-year study has been initiated to identify the principal toxic components within NA mixtures. Future toxicity studies require a large volume of a NA mixture, however, a well-defined bulk extraction technique is not available. This study investigated the use of a weak anion exchanger, diethylaminoethyl-cellulose (DEAE-cellulose), to remove humic-like material present after collecting the organic acid fraction of oil sands tailings pond water. The NA extraction and clean-up procedure proved to be a fast and efficient method to process large volumes of tailings pond water, providing an extraction efficiency of 41.2%. The resulting concentrated NA solution had a composition that differed somewhat from oil sands fresh tailings, with a reduction in the abundance of lower molecular weight NAs being the most significant difference. This reduction was mainly due to the initial acidification of tailings pond water. The DEAE-cellulose treatment had only a minor effect on the NA concentration, no noticeable effect on the NA fingerprint, and no significant effect on the mixture toxicity towards Vibrio fischeri.     

Electrochemical mineralization and detoxification of naphthenic acids on boron-doped diamond anodes

Electrochemical oxidation (ELOX) with boron-doped diamond (BDD) anodes was successfully applied to degrade a model aqueous solution of a mixture of commercial naphthenic acids (NAs). The model mixture was prepared resembling the NA and salt composition of oil sands process-affected water (OSPW) as described in the literature. The initial concentration of NAs between 70 and 120 mg/L did not influence the electrooxidation kinetics. However, increasing the applied current density from 20 to 100 A/m² and the initial chloride concentration from 15 to 70 and 150 mg/L accelerated the rate of NA degradation. At higher chloride concentration, the formation of indirect oxidative species could contribute to the faster oxidation of NAs. Complete chemical oxygen demand removal at an initial NA concentration of 120 mg/L, 70 mg/L of chloride and applied 50 A/m² of current density was achieved, and 85% mineralization, defined as the decrease of the total organic carbon (TOC) content, was attained. Moreover, after 6 h of treatment and independently on the experimental conditions, the formation of more toxic species, i.e. perchlorate and organochlorinated compounds, was not detected. Finally, the use of ELOX with BDD anodes produced a 7 to 11-fold reduction of toxicity (IC₅₀ towards Vibrio fischeri) after 2 h of treatment.     

Acute toxicity of aromatic and non-aromatic fractions of naphthenic acids extracted from oil sands process-affected water to larval zebrafish

The toxicity of oil sands process-affected water (OSPW) has regularly been attributed to naphthenic acids, which exist in complex mixtures. If on remediation treatment (e.g., ozonation) or on entering the environment, the mixtures of these acids all behave in the same way, then they can be studied as a whole. If, however, some acids are resistant to change, whilst others are not, or are less resistant, it is important to establish which sub-classes of acids are the most toxic.     

A detailed field-based evaluation of naphthenic acid mobility in groundwater

An anaerobic plume of process-affected groundwater was characterized in a shallow sand aquifer adjacent to an oil sands tailings impoundment. Based on biological oxygen demand measurements, the reductive capacity of the plume is considered minimal. Major dissolved components associated with the plume include HCO₃, Na, Cl, SO₄, and naphthenic acids (NAs). Quantitative and qualitative NA analyses were performed on groundwater samples to investigate NA fate and transport in the subsurface. Despite subsurface residence times exceeding 20 years, significant attenuation of NAs by biodegradation was not observed based on screening techniques developed at the time of the investigation. Relative to conservative tracers (i.e., Cl), overall NA attenuation in the subsurface is limited, which is consistent with batch sorption and microcosm studies performed by other authors. Insignificant biological oxygen demand and low concentrations of dissolved As (< 10 µg L^− 1) in the plume suggest that the potential for secondary trace metal release, specifically As, via reductive dissolution reactions driven by ingress of process-affected water is minimal. It is also possible that readily leachable As is not present in significant quantities within the sediments of the study area. Thus, for similar plumes of process-affected groundwater in shallow sand aquifers which may occur as oil sands mining expands, a reasonable expectation is for NA persistence, but minimal trace metal mobilization.     

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).     

Detection of naphthenic acids in fish exposed to commercial naphthenic acids and oil sands process-affected water

Naphthenic acids are a complex mixture of carboxylic acids that occur naturally in petroleum. During the extraction of bitumen from the oil sands in northeastern Alberta, Canada, naphthenic acids are released into the aqueous phase and these acids become the most toxic components in the process-affected water. Although previous studies have exposed fish to naphthenic acids or oil sands process-affected waters, there has been no analytical method to specifically detect naphthenic acids in fish. Here, we describe a qualitative method to specifically detect these acids. In 96-h static renewal tests, rainbow trout (Oncorhynchus mykiss) fingerlings were exposed to three different treatments: (1) fed pellets that contained commercial naphthenic acids (1.5mg g(-1) of food), (2) kept in tap water that contained commercial naphthenic acids (3mg l(-1)) and (3) kept in an oil sands process-affected water that contained 15mg naphthenic acids l(-1). Five-gram samples of fish were homogenized and extracted, then the mixture of free fatty acids and naphthenic acids was isolated from the extract using strong anion exchange chromatography. The mixture was derivatized and analyzed by gas chromatography-mass spectrometry. Reconstructed ion chromatograms (m/z=267) selectively detected naphthenic acids. These acids were present in each fish that was exposed to naphthenic acids, but absent in fish that were not exposed to naphthenic acids. The minimum detectable concentration was about 1microg naphthenic acids g(-1) of fish.     

Elemental and spectroscopic characterization of fractions of an acidic extract of oil sands process water

‘Naphthenic acids’ (NAs) in petroleum produced water and oil sands process water (OSPW), have been implicated in toxicological effects. However, many are not well characterized. A method for fractionation of NAs of an OSPW was used herein and a multi-method characterization of the fractions conducted.     

Effects of oil sands process-affected waters and naphthenic acids on yellow perch (Perca flavescens) and Japanese medaka (Orizias latipes) embryonic development

Syncrude Canada Ltd. is currently developing environmentally acceptable oil sands process-affected water management methods as part of their land reclamation strategy. Surface waters of the “wet landscape” reclamation option characteristically have elevated concentrations of sodium sulphate and naphthenic acids (NAs), with low levels of PAHs. The following experiment compared early-life stage responses of yellow perch (Perca flavescens) to those of Japanese medaka (Oryzias latipes) when exposed to Mildred Lake settling basin (MLSB) surface water and a commercial sodium naphthenate (Na-NA) standard. Perch eggs were fertilized and incubated in: 100%, 50%, 20%, 4%, 0.8%, and 0.16% dilutions of MLSB water, as well as 20, 10, 5, 2.5, and 1.25 mg/l solutions of the commercial standard. Medaka embryos were exposed to the same treatments, post-fertilization. Both species demonstrated an increase in the incidence of deformity, and a decrease in length at hatch as NA concentrations increased. MLSB surface water contained higher levels of NAs than the commercial standard, however, showed consistently higher NA threshold effect concentrations for both species. Significant differences between the MLSB water and the Na-NA standard suggest that they contain NA congeners with different toxicity, or other compounds such as PAHs. Species differences in thresholds could be explained by the difference in developmental stage in which the exposures were initiated.     

Comparison of GC-MS and FTIR methods for quantifying naphthenic acids in water samples

The extraction of bitumen from the oil sands in Canada releases toxic naphthenic acids into the process-affected waters. The development of an ideal analytical method for quantifying naphthenic acids (general formula C(n)H(2n+Z)O(2)) has been impeded by the complexity of these mixtures and the challenges of differentiating naphthenic acids from other naturally-occurring organic acids. The oil sands industry standard FTIR method was compared with a newly-developed GC-MS method. Naphthenic acids concentrations were measured in extracts of surface and ground waters from locations within the vicinity of and away from the oil sands deposits and in extracts of process-affected waters. In all but one case, FTIR measurements of naphthenic acids concentrations were greater than those determined by GC-MS. The detection limit of the GC-MS method was 0.01 mg L(-1) compared to 1 mg L(-1) for the FTIR method. The results indicated that the GC-MS method is more selective for naphthenic acids, and that the FTIR method overestimates their concentrations.     

Estimating naphthenic acids concentrations in laboratory-exposed fish and in fish from the wild

Naphthenic acids (NAs) are the most water-soluble organic components found in the Athabasca oil sands in Alberta, Canada, and these acids are released into aqueous tailing waters as a result of bitumen extraction. Although the toxicity of NAs to fish is well known, there has been no method available to estimate NAs concentrations in fish. This paper describes a newly developed analytical method using single ion monitoring gas chromatography-mass spectrometry (GC-MS) to measure NAs in fish, down to concentrations of approximately 0.1mgkg(-1) of fish flesh. This method was used to measure the uptake and depuration of commercial NAs in laboratory experiments. Exposure of rainbow trout (Oncorhynchus mykiss) to 3mg NAsl(-1) for 9d gave a bioconcentration factor of approximately 2 at pH 8.2. Within 1d after the fish were transferred to NAs-free water, about 95% of the NAs were depurated. In addition, the analytical method was used to determine if NAs were present in four species of wild fish - northern pike (Esox lucius), lake whitefish (Coregonus clupeaformis), white sucker (Catostomus commersoni), walleye (Sander vitreus) - collected from near the oil sands. Flesh samples from 23 wild fish were analyzed, and 18 of these had no detectable NAs. Four fish (one of each species) contained NAs at concentrations from 0.2 to 2.8mgkg(-1). The GC-MS results from one wild fish presented a unique problem. However, with additional work it was concluded that the NAs concentration in this fish was <0.1mgkg(-1).     

Naphthenic acid biodegradation by the unicellular alga Dunaliella tertiolecta

Naphthenic acids (NAs) are a major contributor to toxicity in tailings waste generated from bitumen production in the Athabasca Oil Sands region. While investigations have shown that bacteria can biodegrade NAs and reduce tailings toxicity, the potential of algae to biodegrade NAs and the biochemical mechanisms involved remain poorly understood. Here, we discovered that the marine alga Dunaliella tertiolecta is able to tolerate five model NAs (cyclohexanecarboxylic acid, cyclohexaneacetic acid, cyclohexanepropionic acid, cyclohexanebutyric acid and 1,2,3,4-tetrahydro-2-naphthoic acid) at 300 mg L⁻¹, a level which exceeds that of any single or combination of NAs typically found in tailings ponds. Moreover, we show that D. tertiolecta can metabolize four of the model NAs. Analysis of NA-amended cultures of D. tertiolecta via low resolution gas chromatography-mass spectrometry allowed us to quantify decreasing NA levels, identify metabolites, and formulate putative mechanisms of biodegradation. Degradation of cyclohexanebutyric acid and cyclohexanepropionic acid proceeded via β-oxidation and resulted in the transient accumulation of cyclohexaneacetic acid and cyclohexanecarboxylic acid, respectively. Cyclohexanecarboxylic acid was metabolized via 1-cyclohexenecarboxylic acid suggesting that further degradation may occur by step-wise β-oxidation. When D. tertiolecta was inoculated in the presence of oil sands tailings water from the Athabasca region, biodegradation of single-ring NAs was observed relative to controls. This result corroborates the trend we observed with the single-ring model NAs.     

Sequential UV-biological degradation of chlorophenols

The sequential UV-biological degradation of a mixture of 4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP) was first tested with each pollutant supplied at an initial concentration of 50 mg l(-1). Under these conditions, the chlorophenols were photodegraded in the following order of removal rate: PCP>TCP>DCP>CP with only CP and DCP remaining after 40 h of irradiation. The remaining CP (41 mg l(-1)) and DCP (13 mg l(-1)) were then completely removed by biological treatment with an activated sludge mixed culture. Biodegradation did not occur in similar tests conducted with a non-irradiated mixture due to the high microbial toxicity of the solution. UV treatment lead to a significant reduction of the phytotoxicity to Lipedium sativum but no further reduction of phytotoxicity was observed after biological treatment. Evidence was found that the pollutants were partially photodegraded into toxic and non-biodegradable products. When the pollutants were tested individually (initial concentration of 50 mg l(-1)), PCP, TCP, DCP, 4-CP were photodegraded according to first order kinetic model (r2>99) with half-lives of 2.2, 3.3, 5.7, and 54 h, respectively. The photoproducts were subsequently biodegraded. This study illustrates the potential of UV as pre-treatment for biological treatment in order to remove toxicity and enhance the biodegradability of organic contaminants. However, it also shows that UV treatment must be carefully optimized to avoid the formation of toxic and/or recalcitrant photoproducts and results from studies conducted on single contaminants cannot be extrapolated to mixtures.     

A review of the occurrence, analyses, toxicity, and biodegradation of naphthenic acids

Naphthenic acids occur naturally in crude oils and in oil sands bitumens. They are toxic components in refinery wastewaters and in oil sands extraction waters. In addition, there are many industrial uses for naphthenic acids, so there is a potential for their release to the environment from a variety of activities. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. This is a complex group of carboxylic acids with the general formula CnH(2n+Z)O2, where n indicates the carbon number and Z specifies the hydrogen deficiency resulting from ring formation. Measuring the concentrations of naphthenic acids in environmental samples and determining the chemical composition of a naphthenic acids mixture are huge analytical challenges. However, new analytical methods are being applied to these problems and progress is being made to better understand this mixture of chemically similar compounds. This paper reviews a variety of analytical methods and their application to assessing biodegradation of naphthenic acids.     

Mixture toxicity of the antifouling compound irgarol to the marine phytoplankton species Dunaliella tertiolecta

This study examined the toxicity of irgarol, individually and in binary mixtures with three other pesticides (the fungicide chlorothalonil, and the herbicides atrazine and 2,4-D), to the marine phytoplankton species Dunaliella tertiolecta. Standard 96-h static algal bioassays were used to determine pesticide effects on population growth rate. Irgarol significantly inhibited D. tertiolecta growth rate at concentrations > or = 0.27 micro g/L. Irgarol was significantly more toxic to D. tertiolecta than the other pesticides tested (irgarol 96 h EC50 = 0.7 micro g/L; chlorothalonil 96 h EC50 = 64 micro g/L; atrazine 96 h EC50 = 69 micro g/L; 2,4-D 96 h EC50 = 45,000 micro g/L). Irgarol in mixture with chlorothalonil exhibited synergistic toxicity to D. tertiolecta, with the mixture being approximately 1.5 times more toxic than the individual compounds. Irgarol and atrazine, both triazine herbicides, were additive in mixture. The toxicity threshold of 2,4-D was much greater than typical environmental levels and would not be expected to influence irgarol toxicity. Based on these interactions, overlap of certain pesticide applications in the coastal zone may increase the toxicological risk to resident phytoplankton populations.     

Toxicity of fenamiphos and its metabolites to the cladoceran Daphnia carinata: the influence of microbial degradation in natural waters

The acute toxicity of an organophosphorous pesticide, fenamiphos and its metabolites, fenamiphos sulfoxide, fenamiphos sulfone, fenamiphos phenol, fenamiphos sulfoxide phenol and fenamiphos sulfone phenol, to a cladoceran, Daphnia carinata was studied in both cladoceran culture medium and natural water collected from a local river. The toxicity followed the order: fenamiphos>fenamiphos sulfone>fenamiphos sulfoxide. The hydrolysis products of fenamiphos, F. sulfoxide (FSO) and F. sulfone (FSO(2)) (F. phenol, FSO phenol and FSO(2) phenol) were not toxic to D. carinata up to 500microgl(-1) water, suggesting hydrolysis reaction leads to detoxification. Also the toxicity was reduced in natural water compared to the cladoceran culture medium due to microbial mediated degradation of toxicants in the natural water. Fenamiphos and its metabolites were stable in both cladoceran water and filter-sterilised natural water while these compounds showed degradation in unfiltered natural water implicating the microbial role in degradation of these compounds. To our knowledge this is the first study on acute toxicity of fenamiphos metabolites to cladoceran and this study suggests that the organophosphate pesticides are highly toxic to fresh water invertebrates and therefore pollution with these compounds may adversely affect the natural ecosystems.     

Bioaccumulation and critical body residue of PAHs in the amphipod,Diporeia spp: additional evidence to support toxicity additivity for PAH mixtures

Polycyclic aromatic hydrocarbons (PAHs) are considered to act additively when exposed as congener mixtures. Additive internal concentrations at the site of toxic action is the basis for recent efforts to establish a sum PAH guideline for sediment-associated PAH toxicity. This study determined the toxicity of several PAH congeners on a body residue basis in Diporeia spp. These values were compared to the previously established LR(50) value for a PAH mixture based on the molar sum of PAH congeners and demonstrated similar LR(50) values for individual PAH. These results support the contention that the PAH act at the same molar concentration whether present as individual compounds or in mixture. Aqueous exposures were conducted for 28 d, and the water was exchanged daily to maintain the exposure concentration. The concentration in the exposures declined by an average of 22% between water exchanges across all compounds, and ranged from 11% to 32%. The toxicokinetics were determined using both time-weighted-average (TWA) and time-variable water concentrations and were not statistically different between the two source functions. Toxicity was determined for both mortality and immobility (failure to swim on prodding) and on both a TWA water concentration and a body residue basis. The LC(50) values ranged from 1757 microg l(-1) for naphthalene after 10 d exposure to 79.1 microg l(-1) for pyrene after 28 d exposure, and the EC(50) ranged from 1587 microg l(-1) for naphthalene after 10 d exposure to 38.2 microg l(-1) for pyrene after 28 d exposure. The LR(50) values for all congeners at all lengths of exposure were essentially constant and averaged 7.5+/-2.6 micromol g(-1), while the ER(50) for immobility averaged 2.6+/-0.6 micromol g(-1). The bioconcentration factor declined with increasing exposure concentration and was driven primarily by a lower uptake rate with increasing dose, while the elimination remained essentially constant for each compound.     

Combined UV-biological degradation of PAHs

The UV-photolysis of PAHs was tested in silicone oil and tetradecane. In most cases, the degradation of a pollutant provided within a mixture was lower than when provided alone due to competitive effects. With the exception of anthracene, the larger pollutants (4- and 5-rings) were always degraded first, proving that UV-treatment preferentially acts on large PAHs and thereby provides a good complement to microbial degradation. UV-photolysis was also found to be suitable for treatment of soil extract from contaminated soils. The feasibility of UV-biological treatment was demonstrated for the removal of a mixture of phenanthrene and pyrene in silicone oil. UV-irradiation of the silicone oil led to 83% pyrene removal but no phenanthrene photodegradation. Subsequent treatment of the oil in a two-phases partitioning bioreactor (TPPB) system inoculated with Pseudomonas sp. was followed by complete phenanthrene biodegradation but no further pyrene removal. Totally, the combined process allowed 92% removal of the PAH mixture. Further work should focus on characterizing the photoproducts formed and studying the influence of the solvent on the photodegradation process.     

Biological and chemical analysis of the toxic potency of pesticides in rainwater.

A newly developed method for measuring the integrated esterase inhibiting potency of rainwater samples was applied in practice, and the results are compared to the toxic potency calculated from concentrations of 31 organophosphate (OP) and carbamate pesticides, out of a total of 66 chemically analyzed pesticides. In addition, the general toxic potency of the rainwater samples was evaluated in a microtiter luminescence assay with Vibrio fischeri bacteria. Rainwater samples were collected over four consecutive 14-day periods in both open and wet-only samplers. The esterase inhibiting potency of the open rainwater samples (expressed as ng dichlorvos-equivalents/l) corresponded well with the chemical analyses of the rainwater samples collected by both types of samplers (r = 0.83-0.86). By far, the highest esterase inhibiting potency was found in a sample collected in an area with intense horticultural activities in June, and was attributed to high concentrations of dichlorvos, mevinphos, pirimiphos-methyl and methiocarb. The esterase inhibiting potency of this sample was equivalent to a dichlorvos concentration of 1380 ng/l in the rainwater, which is almost 2000 times higher than the maximum permissible concentration (MPC) of dichlorvos set for surface water in Netherlands. Maximum individual concentrations of dichlorvos and pirimiphos-methyl even exceeded the EC50 for Daphnia, suggesting that pesticides in rainwater pose a risk for aquatic organisms. Not all responses of the luminescence-assay for general toxicity could be explained by the analyzed pesticide concentrations. The bio-assays enable a direct assessment the toxic potency of all individual compounds present in the complex mixture of rainwater pollutants, even if they are unknown or present at concentrations below the detection limit. Therefore, they are valuable tools for prescreening and hazard characterization purposes.