Comprehensive two-dimensional gas chromatography-mass spectrometry of complex mixtures of anaerobic bacterial metabolites of petroleum hydrocarbons.

Anaerobic biotransformation of petroleum hydrocarbons is an important alteration mechanism, both subsurface in geological reservoirs, in aquifers and in anoxic deep sea environments. Here we report the resolution and identification, by comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS), of complex mixtures of aromatic acid and diacid metabolites of the anaerobic biodegradation of many crude oil hydrocarbons. An extended range of metabolites, including alkylbenzyl, alkylindanyl, alkyltetralinyl, alkylnaphthyl succinic acids and alkyltetralin, alkylnaphthoic and phenanthrene carboxylic acids, is reported in samples from experiments conducted under sulfate-reducing conditions in a microcosm over two years. The range of metabolites identified shows that the fumarate addition mechanism applies to the alteration of hydrocarbons with up to C8 alkylation in monoaromatics and that functionalisation of up to three ring aromatic hydrocarbons with at least C1 alkylation occurs. The GC×GC-MS method might now be applied to the identification of complex mixtures of metabolites in samples from real environmental oil spills.

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. The unfractionated OSPW acidic extract was characterized by elemental analysis, electrospray ionization-Orbitrap-mass spectrometry (ESI-MS), and an esterified extract by Fourier Transform infrared (FTIR) and ultraviolet-visible (UV) absorption spectroscopy and by comprehensive multidimensional gas chromatography-MS (GCxGC-MS). Methyl esters were fractionated by argentation solid phase extraction (Ag(+) SPE) and fractions eluting with: hexane; diethyl ether: hexane and diethyl ether, examined. Each was weighed, examined by elemental analysis, FTIR, UV, GC-MS and GCxGC-MS (both nominal and high resolution MS). The ether fraction, containing sulfur, was also examined by GCxGC-sulfur chemiluminescence detection (GCxGC-SCD). The major ions detected by ESI-MS in the OSPW extract were assigned to alicyclic and aromatic 'O2' acids; sulfur was also present. Components recovered by Ag(+) SPE were also methyl esters of alicyclic and aromatic acids; these contained little sulfur or nitrogen. FTIR spectra showed that hydroxy acids and sulfoxides were absent or minor. UV spectra, along with the C/H ratio, further confirmed the aromaticity of the hexane:ether eluate. The more minor ether eluate contained further aromatics and 1.5% sulfur. FTIR spectra indicated free carboxylic acids, in addition to esters. Four major sulfur compounds were detected by GCxGC-SCD. GCxGC-high resolution MS indicated these were methyl esters of C18 S-containing, diaromatics with ≥C3 carboxylic acid side chains.

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. In the present study we therefore assayed the acute toxicity to larval fish, of a whole acidified OSPW extract and an esterifiable naphthenic acids fraction, de-esterified with alkali: both fractions were toxic (LC50 ∼5-8mgL(-1)). We then fractionated the acids by argentation solid phase extraction of the esters and examined the acute toxicity of two fractions: a de-esterified alicyclic acids fraction, which contained, for example, adamantane and diamantane carboxylic acids, and an aromatic acids fraction. The alicyclic acids were toxic (LC50 13mgL(-1)) but the higher molecular weight aromatic acids fraction was somewhat more toxic, at least on a weight per volume basis (LC50 8mgL(-1); P<0.05) (for comparison, the monoaromatic dehydroabietic acid had a LC50 of ∼1mgL(-1)). These results show how toxic naphthenic acids of OSPW are to these larval fish and that on a weight per volume basis, the aromatic acids are at least as toxic as the 'classical' alicyclic acids. The environmental fates and other toxic effects, if any, of the fractions remain to be established.

Synthesis and toxicity of some metabolites of the microbial degradation of synthetic naphthenic acids.

Some ill-defined carboxylic acids, termed 'naphthenic acids' (NA), are best known as important constituents of the >720billionlitres of process-affected water associated with the expanding oil sands industries. Other NA are components of some immature and biodegraded crude oils and these may enter the environment via produced water discharges from oil production platforms. Yet others are used as biocides and in the manufacture of steel radial tyres and these may also enter the environment through disposal and/or weathering. The environmental fate of NA, including the mechanisms of biodegradation, therefore needs to be better understood. In order to better elucidate such mechanisms, previously we studied the biodegradation in the laboratory of some alkylcyclohexylbutanoic synthetic NA. However, we could only tentatively identify the metabolites produced. In the present study we report the synthesis and characterisation of six alkylcyclohexylethanoic NA. Each was characterised by gas chromatography-mass spectrometry (GC-MS; trimethylsilyl esters) and we show by co-chromatography that these were indeed the metabolites. Also, a preferential degradation of the trans- isomers was revealed. Assessment of the toxicity of the synthetic NA (Microtox assay), revealed that the relative toxicity of the alkylcyclohexylbutanoic acids was reduced by biotransformation to the alkylcyclohexylethanoic acids, as observed recently for the corresponding aromatic acids. Very recent studies have shown that at least one commercial NA mixture contains cyclohexylbutanoic and alkylcyclohexylethanoic acids, suggesting that the biotransformation of the acids studied herein may be quite relevant to the environmental fate of such acids. A similar study of the acid extractables of one oil sands process-affected water sample suggests that the synthetic acids may be less good 'models' for oil sands NA. The consortia of microbes present in oil sands process-affected water may also be different to those used herein. However, the heterogeneity of oil sands process water is well-known and further detailed studies will need to be made in order to establish whether degradation of oil sands NA proceeds by beta oxidation as observed for the acids herein, or whether the oil sands acids are more resistant to bioremediation.

A derivatisation and liquid chromatography/electrospray ionisation multistage mass spectrometry method for the characterisation of naphthenic acids.

Naphthenic acids (NAs) are partially uncharacterised complex mixtures of carboxylic acids, resulting from the microbial oxidation of petroleum hydrocarbons. They are associated with the fouling of pipelines and process equipment in oil production and with corrosion in oil refineries. As by-products of the rapidly expanding oil (tar) sands industries, NAs are also pollutants and have proved to be toxic to a range of organisms. They also have important beneficial uses as fungicides, tyre additives and, paradoxically, also in the manufacture of corrosion inhibitors. These features make the characterisation of NAs an important goal for analytical chemists. Here we describe the synthesis of amide derivatives of NAs for characterisation by liquid chromatography/electrospray ionisation multistage mass spectrometry (LC/ESI-MS(n)). The method was applied to commercially available carboxylic acids, novel synthetic NAs, commercial NAs refined from crude oils, crude oil NAs and Athabasca oil sands NAs. In addition to confirming the number of alicyclic rings and length of alkyl side chain substituents (confirming information from existing methods), the MS(n) results provided further structural information. Most important of these was the finding that bi- to polycyclic acids containing ethanoate side chains, in addition to alkyl substituents, were widespread amongst the oil and oil sands NAs. The latter NAs are known end members of the beta-oxidation of NAs with even carbon number alkanoate chains. Since such NA mixtures are toxic, they should be targets for bioremediation. Bioremediation of NAs can also be monitored better by application of the methods described herein.

Method for assessing the chronic toxicity of marine and estuarine sediment-associated contaminants using the amphipod Corophium volutator.

Acute sediment toxicity tests do not test key life stage events such as moulting and reproduction and therefore do not reveal the longer-term effects of contaminant exposure. A laboratory method is described for determining the chronic toxicity of contaminants associated with whole sediments. The test is conducted using neonates of the estuarine amphipod Corophium volutator at 15 degrees C, salinity 25 psu and a 12 h light:12 h dark photoperiod. The endpoints are survival and growth after 28 days and survival, growth and reproduction of amphipods upon termination of test i.e. reproduction within all control vessels (ca 75 days). The sediment chronic toxicity test was used to investigate the effects of sediments spiked with environmentally relevant preparations of slightly weathered Alaskan North Slope crude oil, including a water-accommodated-fraction (WAF) and a chemically-dispersed (Corexit 9527) WAF. Sediment oil concentrations were quantified using ultra-violet fluorescence. The amphipods exposed to chemically dispersed oil had higher mortality and lower growth rates than control-, Corexit 9527- and WAF-exposed organisms, resulting in reduced reproduction. The described method supplements the standard acute sediment test and would be particularly useful when long-term ecological effects are suspected but acute tests reveal no significant mortality. The sediment chronic test reported herein has shown that sediment that was not evidently toxic during 10-day acute tests could have population-level effects on sediment-dwelling amphipods.

Analysis of oilfield produced waters and production chemicals by electrospray ionisation multi-stage mass spectrometry (ESI-MSn).

Large quantities of diverse polar organic chemicals are routinely discharged from oil production platforms in so-called produced waters. The environmental fate of many of these is unknown since few methods exist for their characterisation. Preliminary investigations into the use of multistage electrospray ionisation ion trap mass spectrometry (ESI-MSn) show its potential for the identification and quantification of compounds in specialty oilfield chemicals (corrosion inhibitors, scale inhibitors, biocides and demulsifiers) and produced waters. Multiple stage mass spectrometry (MSn) with both positive and negative ion detection allows high specificity detection and characterisation of a wide range of polar and charged molecules. For example, linear alkylbenzenesulfonates (LAS), alkyldimethylbenzylammonium compounds, 2-alkyl-1-ethylamine-2-imidazolines, 2-alkyl-1-[N-ethylalkylamide]-2-imidazolines and a di-[alkyldimethylammonium-ethyl]ether were all identified and characterised in commercial formulations and/or North Sea oilfield produced waters. The technique should allow the marine environmental effects and fates of some of these polar compounds to be studied.

Effects of temperature on polyunsaturation in cytostatic lipids of Haslea ostrearia.

Unusual chemicals produced by the-'blue oyster' diatom, Haslea ostrearia, include the water-soluble blue pigment marennine and numerous polyunsaturated sesterterpene oils or haslenes. Aqueous extracts of the alga exhibit in vitro and in vivo activities against human lung cancer cells and anti-HIV effects. Here we report that three haslenes also demonstrate in vitro cytostatic action against a human lung cancer cell line. The most active haslene is the most unsaturated and unsaturation in the haslenes increases with increasing algal growth temperature.