Applications of Biomarkers for Identifying Sources of Natural and

Chapter 8

Downloaded by UNIV MASSACHUSETTS AMHERST on October 4, 2012 | http://pubs.acs.org Publication Date: July 1, 1997 | doi: 10.1021/bk-1997-0671.ch008

Applications of Biomarkers for Identifying Sources of Natural and Pollutant Hydrocarbons in Aquatic Environments 1

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John K. Volkman , Andrew T. Revill , and Andrew P. Murray 1

Division of Marine Research, CSIRO, GPO Box 1538, Hobart, Tasmania 7001, Australia Australian Geological Survey Organisation, GPO Box 378, Canberra, Australian Capital Territory 2601, Australia

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Over the past decade, many thousands of compounds have been identified in crude oils using high resolution capillary gas chromatography-mass spectrometry (GC-MS) and other techniques. Some of these compounds have distinctive chemical structures which are closely related to the organic compounds produced by plants, bacteria and algae. These 'biomarkers' are used in petroleum exploration to identify the likely source rocks from which different oils were derived. The same compounds can also be valuable for identifying sources of petroleum contamination in the marine environment, for recognising natural constituents and as tracers for physical and biological processes that affect hydrocarbon distributions such as dissolution, evaporation, biodegradation, bioturbation, and uptake by biota. Some applications of petroleum biomarkers to environmental studies of hydrocarbon pollution are reviewed in this paper. Hydrocarbons are ubiquitous in modern marine environments. Those derived from large oil spills due to shipping accidents have tended to capture the attention of the media, public and regulators. However, there are many other sources, and in order to differentiate between them it is necessary to examine the types and distribution of hydrocarbons present. Petroleum geochemists have developed a wide variety of biomarkers for use in petroleum exploration studies (7,2) which can also be useful for identifying contamination of modern environments by crude oil and petroleum-derived products (e.g. 3,4,5). Biomarkers are defined here as those compounds, or groups of compounds, which have distinctive structures which can be related, through reasonable transformation pathways, to compounds produced by living organisms. The term "biomarker" is also used in a different context in the field of ecotoxicology, and so alternatives such as "signature lipid" (especially in studies of Recent environments), "chemical fossil" or "molecular fossil" (in ancient sediments), "biogeochemical marker", molecular marker and "chemicalfingerprint"are also used by geochemists. 110

© 1997 American Chemical Society

In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Identifying Sources of Hydrocarbons

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This paper provides an introduction to the wide range of biomarkers now being used by petroleum geochemists together with some examples of the use of these compounds for identifying the sources, transport and fates of natural and pollutant hydrocarbons in aquatic environments. For more information on the composition of oils and the geochemical applications of biomarkers the reader is referred to textbooks by Peters and Moldowan (7), Tissot and Welte (6) and Engel and Macko (7). Analytical Methods Crude oils are composed primarily of hydrocarbons with small amounts of polar functionalized compounds such as porphyrins, and compounds containing sulfur, oxygen or nitrogen (collectively referred to as the NSO fraction or "resins plus asphaltenes"). Aliphatic hydrocarbons contain either no double bonds (alkanes or saturates) or one or more double bonds (alkenes). Alkanes and alkenes may be straight-chain (e.g. w-heptadecane, n-Cn- structure I, Figure 1), branched (e.g. pristane and phytane; structures VI,V, respectively) or cyclic (e.g. steranes and hopanes; structures XVI-XXI and XXTV-XXXIV, respectively). Alkenes are minor constituents of most oils. Aromatic hydrocarbons commonly contain from 1 to 5 carbocyclic fused rings; examples include naphthalene, phenanthrene, chrysene and perylene (structures XLIII-XLV, XLVIII, respectively). Many methods have been proposed to measure the amount of petroleum in the marine environment (8). The concentration of total hydrocarbons can be measured by direct weighing of the hydrocarbon fraction of the total solvent extract. However, accurate data can be difficult to obtain when the sample size is small. Also, elemental sulfur must be removed from the extract, usually by treatment with activated copper. Thin layer chromatography-flame ionisation detection (TLC-FID) provides an alternative with a reproducibility of about ± 8% (9,10). The TLC-FID technique is particularly well suited to the analysis of samples contaminated with heavier oils, biodegraded oils or those which have already lost light-ends due to evaporation or water washing. However TLC-FID is not suited to the analysis of highly volatile oils and condensates. Spectroscopic methods can provide a rapid measure of oil in seawater, but they provide little indication of the source of the oil, are difficult to calibrate and, hence, can be inaccurate. Total polycyclic aromatic hydrocarbons can be estimated using ultraviolet fluorescence spectroscopy (UVF) which involves the excitation of the sample with UV light and then measuring the light emitted at longer wavelength by the excited multi-ring aromatic compounds in the sample (77). UVF is a very sensitive technique, but it provides little information about oil composition. Some limited fingerprinting information can be obtained using synchronous scanning of the excitation and emission wavelengths. High resolution capillary gas chromatography with its high sensitivity and resolving power (particularly when 50 or 60 m long narrow-bore fused silica capillary columns are used) coupled with mass spectrometry can provide a great variety of detailed information on the structures of compounds present. The mass spectrometer can be a simple bench-top quadrupole or ion trap instrument or a sophisticated MSMS or triple quadrupole system. Source-specific biomarkers (steranes, diasteranes, hopanes, methylhopanes etc.; see Figure 1 for structures) can be measured by established GC-MS techniques such as selected ion monitoring (12-14), as described below, or by using metastable reaction monitoring with MS-MS systems. These In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Figure 1. Chemical structures of some biomarkers commonly encountered in crude oils and contaminated sediments as well as some of their biological precursor molecules such as phytol (IV), sterols (XV) and bacteriohopanetetrol (XXIII). Note that "R" refers either to a hydrogen or alkyl group substituent of general formula CnH2n+i or to the stereochemistry at a chiral centre in the molecule (contrast XVII with XVIII which show S and R stereochemistries at position 20 in the sterane side-chain).


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XVII 14P, 17p(20S)

X X 4a-Methyl-24-ethylsterane

Identifying Sources of Hydrocarbons

XVHI

14p, 17p(20R)

XLX 14a, 17XVI). These processes tend to leave the sterol-derived side-chain intact, and so different proportions of C 7, C 8 and C 9 constituents can be used to characterize the oil (21,22). Sterane mass spectra have major fragment ions at m/z 217 and 218 which are commonly used to characterize the sterane distribution. An example is shown in Figure 2. Steranes occur in a number of isomeric forms having different stereochemistries at positions 5, 14, 17, 20 and 24 (cf, XVI-XIX). All but the latter isomers are separable on non-polar capillary columns so that sterane distributions are quite complex. The ratio of 5a(H),14a(H),17a(H) to 5a(H),14P(H),17P(H) steranes (ocaa/ccpp) is often used as an index of thermal maturity, but most crude oils have similar proportions of these isomers which limits the use of this ratio in environmental studies. A ratio of 20S and 20R isomers of about 1.0 is typical of mature crude oils. If the 20R isomer greatly predominates in a contemporary sediment sample, this could indicate the presence of hydrocarbons from eroded sediments which are thermally immature. A high abundance of C i and C22 steranes relative to C27-C29 steranes is typical of oils of high thermal maturity. A predominance of C29 steranes, such as in Australia's Bass Strait oils (peaks 912; Figure 2), is usually associated with source rocks containing primarily higher plant organic matter although some exceptions are known. The predominance of C 7 steranes (peaks 3,4,6,7) and only slightly lower abundances of C28 steranes and C 9 steranes is typical of oils derivedfrommarine algal source rocks such as thosefromthe Middle East (e.g. Kuwait Crude; Figure 2). The distributions of 4-methylsteranes, as shown by mass chromatograms of the m/z 231 ion, can provide additional evidence for algal-derived organic matter since the presence of the four major isomers of dinosterane (XXI) as well as 4a-methyl-24-ethyl-C o steranes (XX) is usually associated with marine depositional environments (23). Lacustrine sediments commonly, but not always, contain a much greater abundance of the latter 4amethylsterane. Diasteranes (XXII) are found in most oils and are usually fingerprinted using the m/z 259 mass chromatogram although they are also readily discernible in m/z 217 mass chromatograms (peaks 1,2,5,8; Figure 2). A high diasterane abundance is 2

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Figure 2. Mass chromatograms for m/z 217 showing the steranes and diasteranes in oils from Kuwait and Bass Strait, a 1:1 mixture (wt./wt.) of these two oils and a contaminated sediment from Port Phillip Bay, Victoria, Australia. The Kuwait and Bass Strait crude oils contain similar amounts of the C 5a,14a,17a-20S-24-ethylcholestane (peak 9; 44 and 40 ppm respectively), but the Kuwait crude oil contains much less of the C 9 20S-diacholestane (peak 5; 11 and 40 ppm respectively), so the C 7:C 9 ratio of the steranes and diasteranes in the polluted sediment is very different from either oil, but clearly similar to a 1:1 mixture of the two oils. Peak identifications: 1 - C 7 20S-diacholestane (XXII); 2 - C 20R-diacholestane (XXII);. 3 - 20S-5a,14a,17a-cholestane (XIX); 4 20R- 5a,14p,17P-cholestane (XVIII); 5 - C 20S-24-ethyldiacholestane (XXII); 6 -20S-5a,14p,17P-cholestane (XVII); 7 - 20R-5a,14a,17a-cholestane (XVI); 8 - C 20R-24-ethyldiacholestane (XXII); 9 - 20S-5a,14a,17a-24-ethylcholestane (XIX); 10 - 20R-5a,14p,17p-24-ethylcholestane (XVIII); 11 - 20S5a,14p,17P-24-ethylcholestane (XVII); 12 20R-5ot,14a,17a-24ethylcholestane (XVI).



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often associated with oils from clastic source rocks containing clays which catalyse the steroid backbone rearrangement. The Bass Strait oils provide a good example of this. Figure 2 illustrates the use of steranefingerprintingto ascertain the sources of hydrocarbon pollution in polluted sediments from Port Phillip Bay (Australia). These profiles are consistent with an approximately 1:1 mixture of the locally produced Bass Strait Crude oil from the offshore Gippsland Basin and an oil of Middle Eastern, carbonate origin such as those from Kuwait. The latter are used in Australia as a feedstock for lubricating oils because the Bass Strait Crudes are too waxy for this purpose. Of particular note is the presence in the sediment of a high proportion of C27 steranes (peaks 3, 4, 6 and 7) mainlyfromthe Middle East oil in conjunction with high amounts of the C g diasteranes (peaks 5 and 8) mainlyfromthe Bass Strait crude oils. This example highlights the wide variation in absolute concentrations of particular biomarkers in different oil types which must be considered when determining the source of oil polution based on biomarker ratios. 2

Hopanes and Methylhopanes. Hopanes are found in almost all ancient sediments and crude oils (20,24). They are derived from oxygenated analogues such as the bacteriohopanetetrols (e.g. XXIII) found in most bacteria and cyanobacteria (24). Their distribution is usually recorded using a m/z 191 mass chromatogram (Figure 3). In mature samples, the 17a(H),2ip(H)-isomers (XXVI, XXVII) greatly predominate over the 17P(H),21a(H) isomers (moretanes; XXV). However, the isomer commonly found in living organisms is 17P(H),2ip(H) (e.g. XXTV), so that in polluted sediments all three isomers can be found as well as hopenes (containing double bonds at various positions in the ring system) derivedfromindigenous bacteria. An unusually high proportion of the C29 hopane (XXXI, where R=CH ) is often associated with oils derivedfromcarbonate source rocks oils, which includes most of those from the Middle East (peak 3, Figure 3). These oils also show a slightly enhanced abundance of the C extended hopanes (peaks 17,18; Figure 3) compared with the C34 homohopanes (peaks 15, 16; Figure 3). The ratio of the two C27 hopanes, Ts (18a(H)-22,29,30-trisnorneohopane; XXXII) and Tm (17

Applications of Biomarkers for Identifying Sources of Natural and

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