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Molecular Markers and Environmental Organic Geochemistry: An Overview Robert P. Eganhouse U.S. Geological Survey, 12201 Sunrise Valley Drive, Reston, VA 20192

It is said that we live in an Information Age. Yet, throughout Man's tenure on Earth the ambient environment has been providing information on a continual basis. In the case of olfactory and taste senses, information is supplied and detected in the form of chemicals. So, in essence, we have been using chemical cues to discriminate among various substances for the entirety of our collective existence. [A good example is wine tasting.] In this book we will be interested in the detection of a particular category of chemicals, molecular markers, that can, under appropriate circumstances, provide information about sources of organic material. Some molecular markers are produced by humans either intentionally or inadvertently, whereas others are strictly biosynthetic. The characteristic shared by all molecular markers is the fact that their structures are linked to specific origins. Thus, the observation of one of these compounds in the environment necessarily signals the presence of a specific source material. In principle, when several markers are found, one can begin to unravel contributions made by multiple sources (source apportionment). This becomes particularly relevant when non-specific contaminants are also present because the markers can be used to indirectly estimate contributions of contaminants made by different sources. The chapters to follow examine a variety of molecular markers having diverse origins. However, for purposes of organization we have placed markers into three general categories. Contemporary Biogenic Markers (Chapters 2-7) are compounds synthesized by microorganisms and/or vascular plants that are used as cellular constituents. These can be found in living organisms themselves, or, if persistent, as residues that have undergone little or no alteration in the modern environment. The second category, Fossil Biomarkers (Chapters 8-11), includes compounds that are present in fossil fuels and ancient sediments as a result of the burial of biogenic organic matter and its subsequent alteration. As such, these markers have clear structural relations to contemporary biogenic markers. The last category we have called Anthropogenic Markers (Chapters 12-26). These compounds fall into two general classes based on their real or perceived toxicity and source specificity. The first is This chapter not subject to U.S. copyright. Published 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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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY

populated by synthetic (or sometimes natural) organic compounds which are not considered to be pollutants per se but whose presence in the modern environment is directly related to human activity. Examples include the fecal sterol, coprostanol, and synthetic surfactants used in commercial detergents. The second class of anthropogenic markers is composed of contaminant assemblages which are known to be toxic and whose pervasive presence in the environment is entirely or primarily attributable to industrial activity. These include such compounds as the PCBs (polychlorinated biphenyls) and PAHs (polycyclic aromatic hydrocarbons), toxaphene and chlorinated pesticides. In the chapters to follow the reader will see how these various types of markers can be used to gain a better understanding of our contemporary environment and to assess the impact of human activities on it. Many types of information can be derived from studies of these molecules, information that frequently cannot be obtained by conventional biogeochemical approaches or by direct examination of the contaminants themselves. In this sense they serve not only as source indicators but also as molecular 'process probes'. Historical Perspective Origin of the 'Molecular Marker Concept. Our current appreciation of the molecular marker concept comes from the discipline of organic geochemistry. Early work by Alfred Treibs on the organic constituents of shales, bitumens and crude oils led to the discovery in 1934 of a geochemical class of compounds having the tetrapyrrole structure, the petroporphyrins (V). Based on similarities between their core molecular structures, Treibs proposed that the petroporphyrins were actually derived from chlorophylls synthesized by ancient plants (Figure 1). He hypothesized that following death, cellular constituents of plants such as the tetrapyrrole pigments undergo a series of biogeochemical transformations (2). [The processes responsible for these transformations are collectively referred to as diagenesis by geochemists.] Whereas the vast majority (« 99.9%) of biologically produced organic matter is remineralized during diagenesis, the fundamental architecture of some of the more stable (or stabilized) molecules may be preserved (5). When this occurs over geologic time, a type of 'molecular' fossil (4) is formed, the presence of which offers strong evidence of a biological origin. The pathway proposed by Treibs between a recognizable biological precursor and a diagenetic product was highly significant at the time because it established the first unequivocal molecular evidence for the biological origin of petroleum and simultaneously ushered in the era of molecular organic geochemistry. In the context of the present book, his findings provide a compelling demonstration of the utility of the molecular marker approach in explaining complex natural phenomena. Environmental Organic Geochemistry: Formative Years (1950-70). Between the time of Treibs' original reports and the early 1950s progress in the field of organic geochemistry continued to be limited by the low resolving power and poor sensitivity of available analytical technologies. It was only with great difficulty that individual compounds could be isolated from complex matrices and identified at trace levels.



EGANHOUSE

An Overview

Q Phytyl—O-C.

o'\*°

CH3 Chlorophyll a

O

Chelation

Demetalation, Saponification

COOH

Decarboxylation Reduction

Aromatization

COOH

COOH

Figure 1. The Treibs Scheme for diagenetic conversion of chlorophyll a to petroporphyrins.



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Environmental chemistry as we know it today simply did not exist. However, at the outset of the second half of this century steady advances in analytical chemistry spurred growth in the earth sciences, notably in the disciplines of organic geochemistry, and later, oceanography. The rapid development and widespread implementation of gas chromatography (GC), high pressure liquid chromatography (HPLC) and mass spectrometry (MS) made possible, for the first time, the analysis of a wide variety of environmental materials. These materials were soon found to contain extraordinarily complex mixtures of natural and synthetic organic compounds {cf., references in 5-7). During this period organic geochemistry witnessed significant growth as these powerful analytical tools enabled a greatly improved understanding of the architecture of biological molecules, their processing in contemporary environments and eventual transformation to fossil organic matter {cf., references in 810). Meanwhile, the large-scale production of a diverse array of synthetic organic substances had begun (77), and global combustion of fossil fuels was increasing steadily (72). This resulted in the introduction of many persistent organic contaminants to the environment {e.g. DDT, PCBs, phthalates, dioxins, PAHs, etc.). By the mid-1960s, it was abundantly clear that human activities had culminated in widespread, if not global, pollution, and that the effects of certain persistent contaminants {e.g. DDT) on wildlife were a matter of grave concern. Rachel Carson's Silent Spring, in particular, identified some of the more insidious dangers of synthetic organic chemicals (75). This landmark book, along with several dramatic catastrophes [e.g. Torrey Canyon Oil spill-1967 {14), Yusho incident-1968 (75); Minimata disease1953 (76)], eventually drew attention to the problems of environmental degradation and served to catalyze the environmental movement. Ultimately, this led to the formation of the U.S. Environmental Protection Agency (in 1970) and similar agencies throughout developed and developing nations of the world. Modern Era of Environmental Organic Geochemistry (1970-present). Subsequent to 1970 the disciplines of environmental organic chemistry and organic geochemistry evolved largely along parallel, but separate, paths. This, despite the fact that many of the same analytical techniques are used by scientists belonging to these groups, and many of the same or related processes are of common interest. The molecular marker approach, a cornerstone of modern organic geochemistry, has only gradually been embraced by environmental chemists. To some extent, this is not unexpected. Research in environmental organic chemistry has traditionally focused on the identification and quantitative determination of contaminants in environmental matrices (water, air, soils, sediments, biota). The primary motivation has been the need to understand and predict the behavior, fate and effects of potentially toxic organic substances. Consequently, attention has usually been confined to the pollutants of interest, whereas the value of seemingly innocuous 'markers' may have been viewed as incidental to the task at hand. Organic geochemists, by contrast, have concentrated on establishing the dominant geochemical pathways and mechanisms by which biologically derived organic matter is transformed over geologic time into complex natural deposits such as coal, oil shales and petroleum. The approach is decidedly broad and holistic in character, incorporating as it does all available



1. EGANHOUSE

An Overview

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molecular and geologic information for purposes of providing a self-consistent and unified picture of the processes under study. A major product of this comprehensive effort over the last three decades has been the identification and structural elucidation of many classes of compounds that are commonly found in geological materials (e.g. steranes, triterpanes, acyclic isoprenoids, etc.; cf., Chapter 8) as well as the biological precursors from which they were presumably derived {cf, Chapters 2-7). These compounds have come to be known in the organic geochemistry community as 'biomarkers', a term that bespeaks their biosynthetic origin (17,18). There has also been considerable growth in the closely related field of biogeochemistry which is concerned with processes responsible for the transformation and cycling of biogenic organic matter in the contemporary environment. Biogeochemistry plays a central role in studies of global cycling of the elements and the impact of the biosphere on climate. The first applications of the molecular marker approach in environmental studies were reported in the 1960s. Early work with the fecal sterol, coprostanol, showed that this compound was produced by bacteria in the intestinal tracts of mammals via hydrogenation of cholesterol and could be detected in receiving water bodies and sediments impacted by human wastes (19). Thus, it was suggested that coprostanol might have potential as a molecular tracer of human fecal pollution (20,21). In subsequent years a large number of researchers conducted studies of the behavior and distribution of coprostanol in many aquatic environments. However, when considered individually, these investigations tended to befragmentedand limited in scope. For example, no systematic efforts have yet been undertaken to evaluate the stability of coprostanol under a broad spectrum of geochemical conditions or to determine its basic physical properties, and only limited information exists on the source specificity of this compound. In the late 1960s, the occurrence of several oil spills (Torrey Canyon-1967, Buzzard's Bay-1969, Santa Barbara Channel-1969) caught the attention of the public and led to cross-fertilization among the disciplines of organic geochemistry, oceanography and environmental chemistry. As a result, many field and laboratory studies of the fate and effects of oil in aquatic ecosystems were conducted during this period (22). Pioneering work by Max Blumer and co-workers demonstrated how the molecular marker approach could be used effectively to differentiate biogenic and petroleum hydrocarbons (23,24) as well as to track the fate of petroleum and other fossil hydrocarbons in the environment (25,26). To a large extent this early work established the basis for our current understanding of acute episodes such as the grounding of the Exxon Valdez (27). However, it also resulted in the recognition that chronic inputs of petroleum were far more important quantitatively than the highly publicized and readily observable spills (22). This, in turn, stimulated much research into the composition of organic matter in municipal wastes (28-30), urban runoff (31,32) and discrete industrial discharges (33). During the late 1970s and early to mid-1980s, a new class of molecular markers emerged (cf, Chapter 12). Several synthetic organic compounds, not ordinarily considered toxic and many of which are related to commercial detergents [e.g. vitamin E acetate (34), LCABs: long-chain alkylbenzenes (30,35), LAS: alkylbenzenesulfonates (36,37), alkylphenolpolyethoxylates (38), TAMs: trialkylamines (39)], were observed in municipal wastes and waste impacted environments. A flurry of research on the origins of these compounds ensued. As



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information concerning their properties and environmental occurrence accumulated, attention was increasingly directed at understanding their transport and ultimate fate. Research on these compounds continues today (cf., Chapters 12-21) and is complemented by ongoing efforts to search for new markers such that there now is a greatly expanded list of molecules at the environmental organic geochemist's disposal (40,41). During this same period (late 1970s-1980s), the unique lipid signatures of many classes of microorganisms were elucidated (42). In time this yielded valuable information that is being used today to characterize the structure and activity of microbial communities and to assess their nutritional status (43; cf. Chapters 3-6). During the last 10 years, the number of environmental applications in which molecular markers play a role has expanded, and there is growing evidence of an ongoing convergence among organic geochemistry, microbial ecology and environmental chemistry as disciplinary barriers show signs of breaking down. The talents and experience of classical organic geochemists have increasingly been brought to bear on environmental problems such as apportionment of natural and anthropogenic sources of organic matter in atmospheric aerosols (e.g. 44; cf, Chapters 7,11) and studies of ground water (45; cf, Chapter 26) and soil contamination (46; cf, Chapter 14). At the same time, there appears to be more recognition among environmental chemists of the utility of these powerful tools (47). In some cases, the development of new analytical capabilities is reinforcing this evolutionary trend (48-50). The result is that the science of environmental organic geochemistry is steadily moving in the direction of a more comprehensive approach in which anthropogenic markers, contemporary biogenic markers and fossil biomarkers are used together to answer complex environmental questions (51,52; e.g. Chapter 16). Atoms and Molecules as Information Carriers Molecular markers can carry information in three principal ways: 1) the isotopic composition of constituent atoms, 2) three dimensional molecular structure, and 3) the composition of assemblages of contemporaneously formed (i.e. co-genetic) compounds (homologs and isomers). We will have a brief look at these manifestations of molecular information and attempt to illustrate their utility for the three categories of molecular markers. Stable Isotopic Composition. With the exception of the PAHs (49) and more recently, chlorinated hydrocarbons (53), anthropogenic markers have not often been examined for their isotopic composition. Therefore, most of the discussion to follow will focus on the stable isotopic composition of contemporary and ancient residues of biogenic organic matter. Within the atomic nuclei of the light elements involved in life processes (C, N, H, O, S), differences in the number of neutrons give rise to stable and/or unstable isotopes. The isotopic composition of the atoms comprising a given biomolecule depend on the isotopic composition of the component building blocks, fractionations (mostly kinetic) that occur during uptake and at steps along the biosynthetic reaction pathway, and the structure of the molecule itself (e.g. chain length, functional groups). Within a single organism this gives rise to intermolecular



1. EGANHOUSE

An Overview

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and intramolecular variations in the isotopic composition of the constituent biochemicals. In addition, because of differences in the biosynthetic pathways used by various autotrophic organisms (e.g. C3, C4, CAM plants), the isotopic Composition of organic matter produced during photosynthesis varies (54). Together, these variations and the large fractionation associated with photosynthesis and dissimilatory pathways provide isotopic signatures that can be exploited for purposes of determining the source of organic matter and/or the effects of diagenesis. Until the last decade, the vast majority of measurements of stable isotopic composition of organic matter were made on bulk organic matter or somewhat less complex fractions isolated from it. Examples of such fractions include the humic acids, kerogen, extractable organic matter, chitin, hydrocarbons, fatty acids and so on. Although much has been learned about the major biogeochemical cycles and their variation over time from these analyses (55), the fractions nonetheless comprise complex mixtures of individual organic compounds and, thus, reflect multiple origins. In addition, the effects of diagenesis (and catagenesis- thermal breakdown of kerogen) serve to further obscure the isotopic signature of the originally deposited organic matter. Hence, the amount and/or quality of information that can be extracted from such analyses is necessarily limited and dependent upon the type of sample and the degree to which thefractionshave be refined. With the advent of compound-specific isotope analyses including stable isotope ratio monitoring-GC/MS (56) and compound-specific radiocarbon analysis (50) some of these limitations have now been overcome. These techniques permit the chromatographic separation and isotopic analysis of individual compounds. Although environmental applications are just beginning to emerge, this combined molecular-isotopic approach is likely to yield new insights into the origins of selected natural (e.g.tt-alkanes,perylene, unsaturated long chain alkenones, etc.; 57) and anthropogenic compounds (e.g. PAH; 58). Molecular Structure. The second locus of information in a molecule is its three dimensional structure. Here, isomerism is often the structural information-bearing feature of paramount importance. There are two types of isomerism. Structural isomerism is characterized by compounds having the same molecular formula but differing in the positions of substituent atoms. Examples of structural isomerism are found among many complex molecular assemblages including those: 1) synthesized industrially (LCABs, PCBs), 2) produced as a result of human activities (e.g. PAHs, dioxins), 3) formed by living organisms (e.g. fatty acids and aliphatic hydrocarbons) and 4) generated diagenetically or during thermal alteration of kerogen (e.g. alkylbenzenes; 59). Stereoisomerism, on the other hand, results from the presence of one or more chiral carbons in a molecule. Stereoisomers are compounds having the same molecular formula and substitution pattern but differing in the way the atoms are oriented in space. Stereoisomers are further classified as either enantiomers (mirror images of each other-one or more chiral carbon) or diastereomers (non-mirror imagesmore than one chiral carbon). A special subclass of diastereomers, geometric isomers, includes unsaturated compounds which have hindered rotation about the double bond(s). Biosynthesis leads to the production of organic structures which are uniquely suited to a specific biological function. Examples include the phospholipids,



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amphipathic molecules that are key structural constituents of the lipid bilayer of cell membranes (cf., Chapter 2), and bacteriohopanepolyols which are used as membrane rigidifiers by many prokaryotes (cf, Chapter 8). As the number of atoms in a molecule increases, the number of structures that are theoretically possible grows exponentially. Because of the highly directed nature of biosynthesis, however, biological molecules having more than about 20 carbon atoms represent a vanishingly small proportion of the total number of possible structures for a given molecular formula. In short, although biomolecules show great diversity, they are structurally limited by design. Upon death of the host organism the molecular constituents of cells suffer a variety of fates (Figure 2). Diagenesis leads to nearly complete destruction of labile biochemicals, but some of the smaller molecules are preserved intact. Meanwhile, various reactions (i.e. condensation, reduction, decarboxylation, dehydration, aromatization, rearrangement, etc..) bring about transformation of biomolecules to many other species. Resistant biomacromolecules such as the algaenans are preserved as a component of the insoluble organic matter in sediments by virtue of their inherent stability (60), whereas (otherwise labile) functionalized lipids may rapidly become sequestered in the insoluble organic matrix during early stages of diagenesis (61-63). All of these diagenetic products, though more diverse than the parent biomolecules, still represent only a small fraction of the total number of theoretically possible compounds. Subsequent burial and continued heating (i.e. catagenesis) causes breakdown of the insoluble macromolecular organic matter (kerogen) and further alteration of persistent low molecular weight species resulting in the production of the complex mixtures we know as fossil fuels. In addition to producing restricted 'types' of molecular structures, biosynthetic reactions are stereospecific. This results in molecules with unique stereochemistries that often do not have the thermodynamically favored configuration (e.g. sterols, acyclic isoprenoids; see references in 17, Chapter 8). In these cases, diagenesis can lead to isomerization. Thus, what may have started off as a simple mixture of stereochemically unique biomolecules ends up as a complex assemblage of isomers due to defunctionalization followed by rearrangement and isomerization reactions. [This type of process has been demonstrated in laboratory simulations as shown in Figure 3.] The extent of isomerization and the chiral carbons affected during these reactions are variable and depend upon the structure of the compound in question as well as a variety of diagenetic parameters related to the environment of deposition, mechanism of isomerization, and burial history (time, temperature, depth, etc..) of the sediments. Petroleum geochemists have exploited these relationships in efforts to assess the maturity of source rocks, migration processes, biodegradation, oil maturation and to establish oil-oil and oil-source rock correlations (17,18,65). In summary, whereas similarities in structure between contemporary biogenic markers and fossil biomarkers can be used to infer precursor-product relations, structural differences reflect the effects of post-depositional diagenetic processes. In either case, molecular structure and in particular, isomerism, is the key to understanding functional relationships. Compounds synthesized industrially may be thought of as an 'exogenous' manifestation of biosynthesis. Here, as in biosynthesis, the formation of specific molecular structures is directed by living organisms (i.e. humans) using controlled reactions with particular functions in mind. Nevertheless, there are important



EGANHOUSE

An Overview

MINERALISATION BIOTRANSFORMATION

MINERALISATION BIOTRANSFORMATION

EXTANT

BIOMACRO-

LMW BIO-

BIOMASS

MOLECULES

MOLECULES

"CLASSICAL PATHWAY"

W LU

z

SELECTIVE PRESERVATION

INCORPORATION

INCORPORATED LMW BIOMOLECULES KEROGEN + BITUMEN

RESISTANT BIOMACROMOLECULES

SELECTIVE PRESERVATION

•NATURAL VULCANISATION-

SULPHUR-RICH MACROMOLECULES

CO LU LU

1

THERMAL DISSOCIATION AND DISPROPORTIONATE

OIL

A U P H . & A R O M . HC

COAL

+ NSO COMPOUNDS

GAS

THERMAL DISSOCIATION AND DISPROPORTIONATION

Figure 2. Pathways by which biogenic organic matter is converted to fossil fuels. (Reproduced with permission from reference 60. Copyright 1989 Elsevier Science Ltd.)



Figure 3. Model studies of the diagenetic transformation of 4-methyl sterenes: a) partial gas chromatogram of the products of acid treatment of 4-methylcholest-4-ene, b)proposed pathways leading from 4-methyl stanols to steranes and chasteranes. (Reproduced with permission from reference 64. Copyright 1986 Elsevier Science Ltd.)


o o 8 g §


Figure 3. Continued.



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distinctions to be made between biogenic and synthetic molecules. First, in most cases synthetic chemicals are not also produced intracellularly by living organisms. Thus, the presence of a synthetic organic compound such as DDT in the environment is taken as unequivocal evidence of an anthropogenic source. Second, chemical synthetic processes involving optically inactive reactants that lead to molecules bearing one or more chiral carbons result in production of racemic (non-optically active) enantiomeric mixtures. Because of the presence of chiral carbon atoms in biomolecules and the stereoselectivity of enzyme-controlled reactions, the products of biosynthesis are usually optically active (i.e. composed of only one of two possible enantiomers). Therefore, even if biogenic and synthetic molecules were to have the same gross structures, they could, in principle, be distinguished on the basis of stereochemistry. [A more common application, however, is the use of enantiomeric composition to identify the actions of biological processes on synthetic compounds (66; cf., Chapter 23).] Similarly, diagenetic/catagenetic molecular markers (i.e. biomarkers) are not synthesized industrially on a large scale. Consequently, there is usually little difficulty in distinguishing between diagenetic/catagenetic molecules and synthetic ones in modern environments. Molecular markers having no chiral carbons or having the same stereochemistry in both living organisms and fossil deposits are few (9). The most notable example is the normal alkanes. As discussed below, homolog distributions are often used to distinguish between biogenic sources of w-alkanes and those derivedfromthermal alteration of organic matter (cf, Chapters 9-10,16). Molecular Assemblages. In addition to the specific molecular structures that typically result from biosynthesis, diagenesis/catagenesis and chemical synthesis, characteristic distributions of homologous series of compounds are often produced (e.g. «-alkanes, w-fatty acids, sterols, alkylcyclohexanes, LCABs, etc.). As noted above, the complexity of these distributions is sometimes increased by the presence of isomers (either structural or stereochemical). This gives rise to assemblages of homologs and isomers (e.g. long chain alkenones, triacylglycerols, alkylbenzenes alkylphenolpolyethoxylates, LCABs) which can be viewed as both source indicators and process probes. Depending upon the pathway of formation, these assemblages may provide a unique signature that can be used to differentiate among sources of contributing materials (cf, Chapters 7-11,16). Upon this basis, numerous compositional indices such as the CPI (carbon preference index; 67) and 5p/5

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