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Sterol paper


									Sterol Composition in Surficial Sediments in The Coastal mid-Atlantic Potomac River Basin


Cassi L. Walls and Gregory D. Foster1

Department of Chemistry and Biochemistry, George Mason University, MSN 3E2, Fairfax, VA


    Corresponding author: email to; fax, 703/993-1055


       The molecular composition of sterols in sediments collected along a 320 km longitudinal

transect of the Potomac River (mid-Atlantic United States) was investigated to identify the

primary sources of lipid organic matter along the physiographically diverse hydrologic

continuum of the Potomac River basin. Total sterol concentrations in sediments ranged from ~ 3

to 230 g g-1 dry weight. In most samples, cholesterol dominated although there were also

significant amounts of -sitosterol and brassicasterol sterols, which are terrestrially and algal

derived moieties, respectively. Ratios based on the sterol structure were used to determine the

relative contribution of terrestrial, sewage, or algal inputs. Factor analysis identified three suites

of sterol compounds that account for the majority of variability. These sterol suites represent

allochthonous terrestrial plants, allochthonous sewage, and autochthonous marine algae derived

organic matter. Samples collected from the upland freshwater sites had prominent terrestrial

sterol signatures while samples collected from the tidal region showed much greater algal

contributions. Sewage signatures were identified downstream of where the South Branch

Potomac River meets the Potomac located in the upland Ridge and Valley Province and around

the urbanized areas of Washington D.C. The sterol profiles revealed changes in the molecular

composition of sterol along a downstream gradient ranging from the upland Allegheny Plateau to

the Coastal Plain of the Potomac River basin.

Keywords: Sterols; Potomac River Watershed; Lipid biomarker compounds; Sediments; Factor


1. Introduction

       Carbon cycling and deposition in surface waters is affected by many ecological and

biogeochemical processes. The organic matter composition in surficial sediments of rivers

reflects recent depositional origin, internal production, and early diagenetic alterations of carbon

at the watershed scale. Assemblages of low molecular weight organic chemicals in sediments

can function as molecular markers that aid in detecting and tracking ecological perturbations and

water quality impacts derived from landscape changes, such as urbanization, the release of

sewage treatment effluent into natural water bodies, and the mode of transport and distribution of

organic contaminants in river runoff. Coastal watersheds near large population centers undergo

substantial modifications in surface geochemistry during destruction of natural forests for the

development urban structures such as roads and buildings. Sedimentary composition of biogenic

organic substances in coastal rivers may reflect these landscape changes over time. Thus,

organic geochemical studies that utilize molecular markers can provide a great deal of

information on the sources of organic material that is produced within the confines of the river or

is introduced from the terrestrial or anthropogenic environment as well as track landscape

changes and alterations.

       Sterols represent a group of geolipid markers that can differentiate between

allochthonous, autochthonous and anthropogenic lipid carbon sources in aquatic environments

across spatial (Bodineau et al., 1998; Colombo et al., 1996; Leeming & Nichols, 1998; Mudge &

Norris, 1997; Sicre et al., 1993; Venkatesan et al., 1987) and temporal scales (Canuel &

Martnes, 1993; Gonzalez-Oreja & Saiz-Salinas, 1998; Rohjans et al., 1998; Skerratt et al., 1995;

Yunker et al., 1995; Zimmerman & Canuel, 2000; Zimmerman & Canuel, 2001). Sterols were

identified in sediments from distinct hydrologic zones along the Potomac River to characterize

and compare sources and sinks of lipid organic matter in the watershed. The objective of the

sampling scheme was to collect samples in a manner that would clearly reveal the spatial

resolution from the upstream to downstream profiles in a transitional freshwater-to-marine

coastal river system. The central study hypothesis was that sedimentary sterol composition

varies along the hydrologic continuum of the Potomac River basin in a manner that depends on

riverine biogeochemistry, landscape, land use, and anthropogenic impacts of urban development

and agricultural activities.

1.1. Sterols as molecular markers

        Although lipids usually represent a small fraction of the TOC, they are robust molecular

markers/biomarkers of organic matter production because of the specificity of their biosynthesis

and their adaptation of biosynthetic pathways to environmental parameters (Colombo et al.,

1996; Saliot et al., 1991). Sterols are significant components of the lipid mixtures and are

among the most specific and diverse lipid biomarkers that can trace the contribution from algae,

higher animals, vascular plants, and sewage contamination (Hatcher & McGillivary, 1979;

Huang & Meinschein, 1979; Volkman, 1986). This, coupled with the relatively high resistance

of the sterol skeleton to extensive degradation after release into the environment, makes them

valuable as biomarkers (Leeming et al., 1996; Philp et al., 1976).

        Phytosterols and animal sterols have three main functions; they act as membrane

components, as hormones, and as steroid precursors (Bean, 1973). Lipid sterols are essential

cell membrane components of all eukaryotic cells that assist in the formation of the lipid bilayer,

which is the most fundamental structure of cell membranes. Most biologically produced sterols

are planar 3-hydroxy tetracyclic structures commonly containing a methyl- or ethyl- substituted

C7-C11 hydrocarbon side chain, and exhibiting a range of methyl-substitution (C4, C14) patterns

on the polycyclic nucleus with varying degrees and positions of unsaturation (C5, C7, C8),

(Jones et al., 1994; Smith et al., 1982; Yeagle, 1993). The rigid structure of the sterols, caused by

the fused ring system, provides the cell membrane integrity and stability thus, holds the

membrane together.

       The use of sterol biomarkers is well documented. Fecal sterols have been routinely used

to monitor and track the degree pollution in various watersheds by sewage waste effluent and

disposal sites (Brown & Wade, 1984; Chan et al., 1998; Churchland et al., 1982; Fattore et al.,

1996; Gonzalez-Oreja & Saiz-Salinas, 1998; Goodfellow et al., 1977; Grimalt et al., 1990;

Hatcher et al., 1977; Hatcher & McGillivary, 1979; Huang & W.G., 1976; Jeng & Han, 1994;

Laureillard & Saliot, 1993; Leeming et al., 1996; Leeming & Nichols, 1998; McCalley et al.,

1980; Mudge & Gwyn Lintern, 1999; Nichols & Leeming, 1991; Nichols et al., 1996b; O'Leary

et al., 1999; Pierce & Brown, 1984; Poon et al., 2000; Quemeneur & Marty, 1992; Quemeneur &

Marty, 1994; Sherwin et al., 1993; Takada et al., 1994; Venkatesan & Kaplan, 1990; Vivian,

1986; Writer et al., 1995). Sterol molecular biomarkers also have been used to characterize the

source and fate of organic matter in marine and coastal sedimentary environments (Bouloubassi

et al., 1997; Canuel & Martnes, 1993; Duan, 2000; Farrington et al., 1988; Harvey, 1994; Lee et

al., 1979; Lee et al., 1980; Nishimura, 1977; Rohjans et al., 1998; Smith et al., 1982; Smith et

al., 1983; Sun & Wakeham, 1999; Venkatesan et al., 1986; Venkatesan et al., 1987; Volkman et

al., 1987; Volkman et al., 1981). Furthermore, several estuaries have been evaluated for their

organic matter sources through the use of sterols as biomarkers, for example, the Mackenzie

River estuary in Canada (Yunker et al., 1995), the Changjiang mesotidal estuary in China (Lajat

& Saliot, 1990; Sicre et al., 1993), the macrotidal estuaries of the Loire River, Brittany, France

(Bodineau et al., 1998), the macrotidal Seine estuary of France (Thoumelin et al., 1997), the

Conwy Estuary of North Wales (Mudge & Norris, 1997), and the main stem Chesapeake Bay

(Zimmerman & Canuel, 2000; Zimmerman & Canuel, 2001).

1.2. Sterol sources

       In any sedimentary environment there are several potential sources of sterol lipid organic

matter such as, phytoplankton, macroalgae, vascular plants, yeasts, fungi, protozoa, lower plants,

zooplankton, and benthic fauna. The organic matter can be produced in situ or carried from

other areas by sedimentation processes, currents, or tides. Furthermore, the composition of the

organic matter can be affected by chemical and biological alterations, to the extent that the

distribution of sterol lipids may bear little resemblance to that produced in the overlying water

column (Volkman, 1986).

       Sterols display considerable structural diversity, particularly in the pattern of substitution

and unsaturation in their side-chains, making them good candidates as chemotaxonomic markers

(Jones et al., 1994). These chemotaxonomic features can be used to define input sources of

organic matter in various environments. Although few biogeolipids can be linked unequivocally

to one biological source, some useful correlations have been developed for differentiating

terrestrial, algal, and sewage contamination sources by identification and the analysis of sterol

distribution patterns. It should be noted that the potential chemical/biological modifications were

also examined. The sterol biomarkers and their corresponding sources are summarized in Table


1.2.1. Terrestrial Sterols

       Campesterol (C28; 24-methylcholest-5-en-3-ol), -sitosterol (C29; 24-ethylcholest-5-

en-3-ol) and stigmasterol (C29; 24-ethylcholesta-5,22-diene-3-ol) are common sterols in

epicuticular waxes of vascular plants {Scheuer, 1973 #273; Goad, 1972 #227; Heftmann, 1971

#265; Volkman, 1986 #102; Bayona, 1989 #209; Laureillard, 1993 #51; Knight, 1967 #283;

{Nishimura, 1977 #198}. Sterols other than these are relatively rare in higher plants (Patterson,

1970). However, Nishimura (Nishimura, 1977) has characterized stigmastanol as a vascular

plant biomarker and identified low concentrations of cholesterol and brassicasterol in some

higher plants. Although campesterol, stigmasterol and -sitosterol are the three most common

sterols in vascular plants, their use as biomarkers of terrigenous organic matter has been of

concern since they have also been reported in several other organisms (Volkman, 1986).

       Although C27 sterols are often dominant in plankton and C29 sterols are dominant in

higher plants (Nishimura, 1977), some investigators reported both cholestanol (C27) and -

sitosterol (C29) as the main sterols in some marine organisms such as phytoplankton,

macroalgae, and sponges {Laureillard, 1993 #51; Nishimura, 1976 #266; Matsumoto, 1982

#193; Robinson, 1984 #84; {Aiello, 1993 #298}. Furthermore, -sitosterol and stigmasterol also

have been found in several species of phytoplankton such as, diatoms, Prymnesiophycea,

Chlorophyceae, and cyanobacteria (Boon et al., 1983; Gagosian et al., 1983b; Goad & Goodwin,

1972; Matsumoto et al., 1982; Paoletti et al., 1976; Volkman, 1986; Volkman et al., 1981;

Volkman et al., 1990). Campesterol also has been found in some dinoflagellates and diatoms

(Volkman, 1986) as well as, in various marine and freshwater Chlorophyceae (Goad & Goodwin,

1972), and as the predominant sterol in rotifers (Nishimura, 1977).

       The relative abundance of the higher plant sterols, sitosterol/stigmasterol/campesterol,

has been found to be (11.5-31)/(0.5-1.3)/1 (Nishimura, 1977; Volkman, 1986). Thus, the ratios

of theses three sterols has been proposed as an indicator of terrestrial source (Volkman, 1986).

Despite the potential ambiguities, campesterol, -sitosterol, and stigmasterol have been

successfully used to trace terrestrial matter in estuarine and marine environments by the use of

absolute concentrations or terrestrial ratios (Harvey, 1994; Huang & W.G., 1976; Laureillard &

Saliot, 1993; Mudge & Norris, 1997; Saliot et al., 1982; Saliot et al., 1991).

1.2.2. Algal Sterols

       Many of the sedimentary sterols are known to originate from diatom sources, particularly

cholesterol, brassicasterol (24-methyl-cholesta-5,22-dien-3-ol), 24-methylenecholesterol (24-

methylene-cholesta-5,24(28)-dien-3-ol) campesterol, fuco/isofucosterol (24-ethylene-cholesta-

5,24(28)-dien-3-ol) and -sitosterol (Ballantine et al., 1979b; Barrett et al., 1995; Colombo et

al., 1996; Gillan et al., 1981; Kates et al., 1978; Nichols et al., 1990; Orcutt & Patterson, 1975;

Patterson, 1991; Volkman et al., 1980; Volkman et al., 1986; Volkman et al., 1981). 22-

Dehydrocholesterol (cholesta-5,22-dien-3-ol) is the major sterol of the diatom Biddulphia

sinensis (Smith et al., 1983; Volkman et al., 1980) which also contains, as minor components,

two unusual sterols – lanthosterol (cholest-7-en-3-ol) and 23,24-dimethylcholesta-5,22-dien-

3-ol (Smith et al., 1982). However, non-diatomaceous sources may be probable for 23,24-

dimethylcholesta-5,22-dien-3b-ol since it is only present in very low concentrations (Volkman et

al., 1980). Other minor sterols such as 23,24-dimethylcholset-5-en-3-ol, 24-norcholesta-5,22-

dien-3-ol and cholesta-5,22-dien-3-ol also have been attributed to diatoms (Ballantine et al.,

1979b; Smith et al., 1982; Volkman, 1986; Volkman et al., 1993; Volkman et al., 1981). In

addition, the C26 sterol, 24-nor-cholesta-5,22-dien-3-ol, has been identified in a phytoplankton

sample composed principally of diatoms (Boutry et al., 1971) and dinosterol has been observed

in the diatom Navicula sp. (Harvey, 1994). Since brassicasterol and 24-methylenecholesterol are

major constituents of many diatoms, they have been used in numerous cases as diatomaceous

biomarkers (Lee et al., 1980).

       In some cases, it is possible to assign an alga to a taxonomic group based on the presence

of only one sterol. Specifically, dinoflagellates contain the unusual 4-methylsterol dinosterol

(4,23,24-trimethyl-5a-cholest-22-en-3-ol) which has predominately been found in this algal

group (Alam et al., 1979; Boon et al., 1979; de Leeuw et al., 1983; Robinson et al., 1984;

Shimizu et al., 1976; Withers et al., 1979). Dinoflagellates contain unusual sterols having 7, 8

double bonds and unusual patterns of side-chain alkylation such as 23,24-dimethyl substitution

(Volkman, 1986) such as, 23,24-dimethylcholesta-5,22-dien-3-ol, 23,24-dimethylcholset-5-en-

3-ol (de Leeuw et al., 1983). Other unusual side-chains such as 24-nor, 27-nor, propylidine, or

cyclopropyl are mainly found in marine environments where they are derived from

dinoflagellates; and, brassicasterol has significant amounts present in other microalgae including

dinoflagellates (Volkman, 1986). Other minor sterol constituents of dinoflagellates include

cholesta-5,22-dien-3-ol, isofucosterol, 4,24-dimethyl-cholestan-3-ol, 4a-methylcholest-

8(14),22-dien-3-ol, 4,24 dimethylcholset-8(14),22-dien-3-ol, 24-methylcholest-7-en-3-ol,

24-methylcholestan-3-ol and cholesterol (Harvey et al., 1987; Volkman, 1986; Volkman et al.,

1981). In addition, the reduced form of dinosterol, dinostanol, also has been shown to occur in

some phytoplankton including dinoflagellates (Robinson et al., 1984). Dinosterol has been used

in numerous chemotaxonomic (Jones et al., 1983) and geochemical studies (Mackenzie et al.,

1982) as a dinoflagellate biomarker, since it is widely accepted as a specific product of

dinoflagellates (Boon et al., 1979).

       Green algae (Chlorophyta) produce a range of sterols including chondrillasterol,

poriferasterol, 28-isofucosterol, ergosterol, cholesterol, sitosterol, zymosterol, 24-

methylenecholesterol, clionasterol (Gibbon et al., 1968; Knight, 1967; Patterson, 1970;

Patterson, 1982; Volkman, 1986). Typically, 7, 5,7,7,22 unsaturation patterns are found in

many species of green algae (Holden & Patterson, 1982).

       Red algae (Rhodophyta) are known to produce cholesterol, desmosterol, 22-

dehydrocholesterol, fucosterol (Patterson, 1970). Additionally, cholesta-5,22-dien-3-ol is the

major sterol in red algae of the genus Porphyridium, but these are not abundant in seawater

(Volkman, 1986).

       Brown algae (Phaeophyta) are known to produce fucosterol, saringosterol, 24-

methylenecholesterol (Patterson, 1970) where fucosterol is the major sterol of nearly all

macroscopic brown algae (Volkman, 1986).

       In general, there is not a specific sterol that can be uniquely linked to one algal source.

Many of the sterols previously discussed are also found in other groups of algae. For example,

Volkman (Volkman et al., 1990) introduced the possibility that poorly studied groups of

microalgae may also contain 4-methyl sterols such as dinosterol. 24-ethylcholesta-5,24(28)-

dien-3-ol has been reported from various sources; brown algae are often regarded as sources for

the 24(28)E isomer, whereas green algae are regarded as producers for the 24(28)Z isomer

(Patterson, 1972; Patterson, 1982). Although brassicasterol and 24-methylenecholesterol are

often used as diatom biomarkers, they are found in many other algal groups (Volkman, 1986).

They are produced by many microalgae, coccolithophores (de Leeuw et al., 1983; Smith et al.,

1982; Volkman et al., 1981) dinoflagellates (Goad & Withers, 1982; Huang & Meinschein,

1979), and a number of Prymnesiophytes (Marlowe et al., 1984). 23,24-dimethylcholesta-5,22-

dien-3-ol and 23,24-dimethylcholset-5-en-3-ol have diatom (Volkman et al., 1993; Volkman

et al., 1981) and dinoflagellate (de Leeuw et al., 1983) sources. Furthermore, many species of

green algae, haptophyceae algae, diatoms, and dinoflagellate contain sterols such as 24-

norcholesta-5,22-dien-3-ol, 27-nor-24-methylcholesta-5,22-dien-3-ol, cholesta-5,22-dien-3-

ol, 23,24-dimethyl-5a-cholest-22-3-ol, 23,24-dimethylcholesta-5,22-dien-3-ol, cholesterol,

24-methylcholesta-5,22-dien-3-ol, 24-methylcholesta-5,24(28)-dien-3-ol, and dinosterol

(Alam et al., 1979; Bayona et al., 1989; Colombo et al., 1996; Goad & Withers, 1982;

Laureillard & Saliot, 1993; Volkman et al., 1986).

        It is apparent that many sterols are widely distributed and few can be considered as

characteristic of a particular algal class, thus making it difficult to elucidate a specific source to

these compounds. Because the sterol profiles for specific classes of algae overlap, and multiple

classes of algae are found in the Potomac River, the algal source of lipids were not separated into

individual algae classes. Therefore, the sterols of all algal classes were combined for use as a

source marker for overall algal input.

1.2.3. Fecal/Sewage Contamination Sterols

        Fecal sterols have been widely used to monitor the degree of pollution and trace sewage

contamination in a variety of environments. Coprostanol (5-cholestan-3-ol), epicoprostanol

(5-cholestan-3-ol), and cholesterol are abundantly found in human feces and are therefore

present in sewage-effluent and sewage-contaminated waters (Brown & Wade, 1984; Rosenfeld

& Hellman, 1971). Some higher animal’s feces (i.e., whales) contain cholesterol, coprostanol

and epicoprostanol. Although epicoprostanol is found at significant levels in marine mammalian

feces (Venkatesan & Santiago, 1989), it is only found at trace levels or not detected in other

animals (i.e., pigs, sheep, cows, horses, hens, seagulls, ducks, dogs, and cats) and human feces

(Leeming et al., 1996).

       Coprostanol is formed at significant amounts in the digestive tract of higher animals.

This occurs by anaerobic microbial degradation of the sterospecific reduction of the 5,6-double

bond of cholesterol (Eneroth et al., 1964; Eyssen et al., 1973; Martin et al., 1973; Rosenfeld et

al., 1954; Rosenfeld & Gallahger, 1964). Both the 5 and 5-stanols are produced by the

reduction of the 5 bond of cholesterol and its C28 and C29 analogues by mammalian intestinal

microorganisms. This reduction of the 5 sterols to the corresponding stanols appears to proceed

equally, regardless of structural differences in the side-chain (Rosenfeld & Hellman, 1971). Two

different pathways have been proposed for the reduction of cholesterol to coprostanol by

intestinal microorganisms. The first pathway involves the intermediate formation of 4-

cholesten-3-one, which is subsequently converted to coprostanone and coprostanol. However, in

vivo studies have shown that the favored conversion is the direct reduction of the 5 double bond

(Venkatesan & Santiago, 1989).

       Coprostanol constitutes about 60% of the total sterol pool in human feces (Ferezou et al.,

1978). Other animals such as pigs, sheep, cows, horses, hens, seagulls, ducks, and cats have

coprostanol in their feces, but total concentrations and amounts relative to other sterols are much

less (Ferezou et al., 1978; Leeming et al., 1996). Furthermore, coprostanol constitutes about

40% of the total sterols identified in raw sewage (Quemeneur & Marty, 1994). Although

epicoprostanol has only been found at trace levels in human feces, large quantities of it have

been identified in the digested sludge of sewage treatment plants. Thus, epicoprostanol levels

may provide a means of distinguishing between pollution of treated and untreated waste

(McCalley et al., 1981).

        Coprostanol concentration is unaffected by various treatments such as chlorination or

aeration of overlying water (Bartlett, 1987). However, coprostanol and cholestanol degrade

under the conditions found in aerobic wastewater treatment plants, in treatment effluent and in

seawater but are known to be refractory in anoxic sediments (Bartlett, 1987; Hatcher &

McGillivary, 1979; Venkatesan et al., 1986). Studies of sediments from freshwater systems have

shown that once coprostanol and cholestanol are buried in anaerobic sediments, they are

persistent (Nishimura & Koyama, 1977). Coprostanol is typically associated with particulate

matter and becomes quickly incorporated into the sediments due to its lipophilic nature (Marty et

al., 1996).

        A direct relationship between coprostanol levels and the degree of water pollution has

been observed (Dutka et al., 1974; Murtaugh & Bunch, 1967; Tabak et al., 1972). The amount of

coprostanol correlates well to faecal coliform counts (Goodfellow et al., 1977). Therefore,

coprostanol has been considered as an ideal indicator of anthropogenic pollution and has been

successfully used to trace sewage pollution in many diverse environments {Goodfellow, 1977

#35; Walker, 1982 #184; Grimalt, 1990 #36; Quemeneur, 1992 #79; Takada, 1994 #295;

Laureillard, 1993 #51; LeBlanc, 1992 #203; Nichols, 1993 #264; Sherwin, 1993 #294;

Venkatesan, 1990 #99; Chan, 1998 #15; Wun, 1976 #232; Hatcher, 1977 #233; Hatcher, 1979

#40; McCalley, 1980 #234; McCalley, 1981 #55; Brown, 1984 #200; Pierce, 1984 #236;

Eganhouse, 1988 #238; Nichols, 1991 #241; Green, 1992 #24; Chalaux, 1995 #240; Grimalt,

1990 #309; Escalona, 1980 #292; Vivian, 1986 #293; Jeng, 1994 #201; Murtaugh, 1967 #188;

Smith, 1968 #189; Dutka, 1974 #190; Dureth, 1986 #244}.

1.2.4. Cholesterol

       Cholesterol is commonly abundant in natural coastal and marine sediments and seawater.

In most cases, it is usually the major sterol encountered in environments with high productivity

and, thus, high organic matter supply (Gagosian et al., 1983b). In marine sediments and in the

overlying water column, cholesterol is generally attributed to zooplankton or other marine fauna,

since it constitutes the major sterol of most marine organisms {Gagosian, 1979 #211; {Huang,

1976 #44; Huang, 1979 #194; {Volkman, 1986 #102; Chan, 1998 #15}. Historically, the

presence of cholesterol has provided evidence of zooplankton and resident invertebrates input

into sediments either directly from their carcasses and feces or from their grazing activities

(Harvey, 1994). However, cholesterol is found in several alga taxa as well (Gagosian et al.,

1983a; Volkman et al., 1981). Cholesterol is abundant in most dinoflagellates and small

flagellate species (Volkman, 1986). It is also present in many diatoms and some species of

Prymnesiophycaean algae contain cholesterol as the major sterol (Volkman, 1986). Furthermore,

cholesterol is one of the primary sterols in raw sewage (Sicre et al., 1993). Therefore, cholesterol

is rather unspecific due to its presence in a wide range of organisms (zoo-, phytoplankton, fish or

mammals) and because of its ubiquity it is of limited utility as a more precise source indicator

(Morris & Culkin, 1977; Sicre et al., 1993).

1.2.5. Biotransformation of Sterols

       Significant concentrations of stanols are often found in sedimentary environments.

Stanols can originate from direct biogenic input or from bacterial hydrogenation of sterols.

Evidence from labeling studies (Gaskell & Eglinton, 1975) indicate that biohydrogenation occurs

in some sediments however, others report of substantial levels of stanols in some marine animals

and phytoplankton (Ballantine et al., 1979a; Ballantine et al., 1978; Ballantine et al., 1977;

Ballantine et al., 1976; Nishimura & Koyama, 1976; Nishimura & Koyama, 1977).

       Series of oxidation, dehydration and reduction reactions resulting in the conversion of 5

stenols into steroid ketones and stanols can occur by in situ transformations (Gagosian &

Heinzer, 1979). It has been shown that stenols can be transformed into stanols by microbial

activity under anaerobic conditions (Bjorkhem & Gustaffson, 1971; Eyssen et al., 1973; Gaskell

& Eglinton, 1976; Huang & Meinschein, 1978; Mackenzie et al., 1982; Nishimura, 1977;

Nishimura, 1982) although the rate of transformation is small (Gaskell & Eglinton, 1975). For

example, Nishimura and Koyama (Nishimura & Koyama, 1977) showed that cholesterol was

biologically hydrogenated into coprostanol and cholestanol in anoxic sediments over a period of

1200 days; however, the conversion of cholesterol to coprostanol was only 2-3%. Furthermore,

several studies have shown that in the absence of sewage inputs, coprostanol and cholestanol can

be produced via anaerobic diagentic transformation of cholesterol in sediments which receive

high labile organic matter and has existing reducing conditions (Gaskell & Eglinton, 1975;

Nishimura, 1982; Nishimura & Koyama, 1977; Taylor et al., 1981). Since only anaerobic

bacteria appear capable of biohydrogenating cholesterol to the coprostanol and such bacteria are

largely absent from aerobic waters, it appears that coprostanol does not occur naturally in fresh

or marine waters or in aerobic sediments. However, trace amounts of coprostanol have been

detected in non-polluted, aerobic sediments, which suggests that in situ hydrogenation of

cholesterol occurred in anaerobic conditions of micro-environments within the sediment

(Nishimura, 1977; Writer et al., 1995). Moreover, it has also been demonstrated that other

sterols such as dinostanol can be formed by the microbially mediated reduction of sterols that

often occurs at oxic-anoxic boundaries (Wakeham, 1989).

       Stanols can also be derived from biogenic sources such as diatoms (Nishimura &

Koyama, 1976; Nishimura & Koyama, 1977) and in some marine animals such as sponges,

echinoderms, jellyfish, tunicates and annelids (Ballantine et al., 1979a; Ballantine et al., 1978;

Ballantine et al., 1977; Ballantine et al., 1976). Therefore, organisms must be considered as a

source of stanols, particularly in oxic environments. It has been shown that cholestanol can be

biosynthesized by diverse aerobic organisms, including some phytoplankton, zooplankton, and

macrophyte species (Nishimura & Koyama, 1977; Robinson et al., 1984) and they are also found

in common algae such as diatoms (Gagosian et al., 1983b; Volkman, 1986; Volkman et al.,

1981). Some investigators reported cholestanol as the main sterol in some marine organisms

such as phytoplankton, macroalgae and sponges (Aiello et al., 1993; Laureillard & Saliot, 1993;

Matsumoto et al., 1982; Nishimura & Koyama, 1976). Furthermore, dinostanol has also been

shown to occur in some phytoplankton including dinoflagellates (Robinson et al., 1984) while

stigmastanol has been characterized as a vascular plant marker (Leeming & Nichols, 1998;

Nishimura & Koyama, 1977). Since stanols may occur via reduction processes or from natural

organisms, some authors have suggested to evaluate the stenol/stanol ratio to determine if the

primary source of the stanol is from specific source-organisms or by a bacterial conversion

(Gagosian & Heinzer, 1979). Although, the extent of such a contribution is often difficult to

determine (Volkman et al., 1981).

2. Materials and methods

2.1. Sampling sites

A description of the Potomac River watershed has been provided elsewhere (Walls and Foster,

unpublished article). Briefly, the Potomac River originates near Fairfax Stone, WV and

stretches ~650 km southeast from its headwaters to convergence with Chesapeake Bay at Point

Lookout, MD. It is the second largest tributary of Chesapeake Bay, with a watershed of 37,600

km2 and a historical annual flow of 330 m3/s at the river fall line at Chain Bridge, Washington,

D.C. (Mason & Flynn, 1975). Acting as the principal boundary between the states of Maryland

and Virginia, the river flows through six physiographic provinces from its headwaters to the

Chesapeake Bay (Fig. 1). Since the coastal Potomac River flows through such diverse

physiographic regions and has distinct hydrodynamic zones, it has multitude of sources of

geolipids and is ideal for evaluating the origin, distribution and application of sterols as

molecular markers at the watershed scale.

       In June 2000, Potomac River sediments were collected from upper Potomac River in the

Appalachian Province (A, incorporating the Allegheny Plateau, Ridge and Valley and Blue

Ridge Provinces together), Piedmont Province (P), freshwater tidal (FT), transition tidal (TT),

and saline tidal (ST) river. aboard a Boston Whaler using a petite Ponar grab (Wildco, Saginaw,

MI). Ponar grab samples were obtained from a Boston Whaler for the Piedmont and tidal

regions. Sediment grabs were initially placed in stainless steel (SS) trays, where surficial

sediments (top 2 cm) were transferred to amber glass jars using a SS spatula. The jars were

sealed with Teflon lined caps, wrapped with aluminum foil, labeled, placed in a zip lock plastic

bag, and stored in an ice chest until return to the laboratory. Sediments from the Appalachian

Province were collected using the Ponar while wading in the deeper pools of the river, and the

sediments were stored as described above. Upon arrival at the laboratory, sediment samples

were stored at –40 oC in a freezer prior to chemical analyses.

       Eight to ten sediment samples were collected throughout each region, except for the

Appalachian Province where fewer samples were collected. In order to assess variability, one

station in each distinct hydrologic region was sampled at least in triplicate. Additionally, four to

five samples each were collected throughout two small sub-basins. These samples were obtained

from an area that is heavily impacted by urban development (the urban Anacostia River) and the

other that is forested and relatively undeveloped (Chopowamsic Creek). A total of 33 sites were

sampled in the Potomac River basin, 25 in the mainstem Potomac River and 8 in the two

tributaries (Table 2).

2.2. Sediment bulk property analysis

    Moisture content, texture (as sand and silt/clay percentage), and total organic carbon and

nitrogen were analyzed in each sediment sample as previously described (Walls and Foster,

unpublished data).

2.3 Sterol analysis

    Sterols were extracted from sediments using procedures described by Harvey (Harvey,

1994). All solvents used were high purity (Burdick and Jackson brand, Fischer Scientific,

Pittsburgh, PA). Prior to analysis, all glassware was fired to 450 oC for 4 hours and solvent

rinsed (acetone and hexane) to remove impurities. A thawed 10 g (wet weight) sediment sample

was mixed with 10 ml of dichloromethane (DCM):methanol (MeOH) (1:1) and sonicated for 10

min three times sequentially with fresh solvent to extract the lipids. The extracts were combined

and evaporated to dryness under a stream of nitrogen gas. The extracted residue was subjected

to mild alkaline hydrolysis using 0.5 M KOH/MeOH and gentle heating (70 oC for 30 min).

After the sample was cooled, the neutral lipids were partitioned from the alkaline solution into 2

ml of hexane.

   The neutral lipid fraction containing the sterols was evaporated to dryness under a stream of

nitrogen gas and treated with bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Sigma Aldrich,

Saint Louis, MO) reagent that was amended with 25% pyridine (Fisher Scientfic, Somerville,

NJ) while heating at 50 oC for 15 minutes to convert the free hydroxyl groups of the sterols to

their trimethylsilyl-ether (TMS) derivatives. The excess reagent was evaporated to dryness, and

the extract was then re-dissolved into DCM for chromatographic analysis.

       Analysis of the final extracts was performed by gas chromatography/mass spectrometry

(GC/MS) using a Hewlett-Packard (Wilmington, DE) 5890 GC coupled to an HP 5970 mass

selective detector. The inlet was operated in the splitless mode with total and purge flows

adjusted to 30 and 3 mL/min, respectively. The GC/MS was fitted with an HP-5 60 m x 0.25

mm (id) fused-silica capillary column (0.25 m film), with the He carrier gas flow rate through

the column adjusted to 1 mL/min at 100 oC. A two-stage temperature program consisting of 50-

120 oC at the rate of 10 oC min-1, followed by a 3 oC min-1 rate to 300 oC was used in all

separations. All gas chromatographic data were evaluated using MSD Enhanced ChemStation

(Version B.01.00). Sterol quantitation was performed using cholestane (Sigma Aldrich) as the

internal injection standard. Structural identification of the sterols was determined by comparison

of retention times with both internal and external standards (Sigma Aldrich) and mass spectral

interpretation of the ion fragmentation (Jones et al., 1994; Smith et al., 1982). Depending on the

structure of the sterol, the detection limits ranged from 0.1 ppb to 1 ppb.

2.4 Statistical Analysis

    The composite sterol data were statistically evaluated using MINITAB (MINITAB Inc.;

release 12, University Park, PA). Parametric analyses, including ANOVA, Tukey’s pairwise

comparison, and the Student’s t tests, were used to identify differences in sterol compositions

among the hydrographic regions. The results of these tests were used to evaluate relative

differences among sample sites and were further correlated to ecological or hydrogeochemical

processes. Pearson’s Product Moment Correlation coefficient was selected for measuring

association between samples and the association between fatty acid and sterol distributions to

ensure that selected biomarkers were indicative of the biogenic sources. The data were also

evaluated through Factor Analysis using the principle component extraction method and the

Varimax rotation solution to identify the underlying factors associated with the variability in the

sterol distributions within the Potomac River basin. Only those variables with mean values and

standard deviations of similar magnitude were selected for factor analysis.

3. Results

       3.1. Sterol composition

       The results and analyses of the bulk sediment composition were described in a previous

report (Walls and Foster, unpublished article). It should be noted, based on these results, that

sites A1 and A4 were excluded from further analyses due to the extremely high sand content

which provided very little organic matter for further sterol characterization.

       The total-sterol concentrations in sediments ranged from 3 to 235 g g-1 dry wt in the

mainstem Potomac River, with the highest regional mean concentration observed in the saline

tidal region (134  68 g g-1) and the lowest regional mean concentration in the FT region (48 

11 g g-1). The high standard deviations among the sediment sterol concentrations are not

surprising given the substantial variability in sediment properties (e.g., grain size and TOC) in

the Potomac River. Furthermore, there was a significant correlation (Pearson’s correlation

coefficient r = - 0.39, p<0.01) between the silt content and total-sterol concentrations, indicating

a link with grain size distribution of the sediment that has been previously reported by several

investigators {Nichols, 1996 #66; Venkatesan, 1990 #99; {Poon, 2000 #77; O'Leary, 1999 #67;

Hatcher, 1979 #40; Chan, 1998 #15; Writer, 1995 #109}. When expressed as a percent of

sediment TOC, the results of the ANOVA and Tukey’s pairwise tests showed significant

differences (p<0.05) among the total-sterol concentrations where the ST region of the Potomac

River had higher concentrations than in the upland P, FT, and the TT regions (Fig. 2).

       The total-sterol concentrations found in the two tributaries ranged from 66 to 164 g g-1

dry wt, with the highest mean concentration detected in Chopowamsic River sediments (101 

43 g g-1) and the lowest in Anacostia River sediments (84  21 g g-1). There was no

significant difference in the total-sterol sediment concentrations between sediments collected in

the two tributaries (Student's t, p>0.05).

       A total of 51 individual sterols were identified in the mainstem Potomac River,

Chopowamsic Creek, and Anacostia River sediments, representing a broad range of lipid organic

matter sources. The structures of the identified sterols ranged from C26-C30 with various levels

of unsaturation, including fully saturated structures and sterols with double bonds at C-5, C-22,

C-8, C-8(14), C-24, and C-24(28). In addition to compounds with no alkylation at C-4, a

number of 4-methyl-sterols were also present. A complete list of all the identified sterols is

presented in Table 3. The most abundant sterols were cholesterol, cholestanol, brassicasterol,

24-methylenecholesterol, and campesterol throughout all the regions of the mainstem Potomac

River (Fig. 3). Dinosterol, 23,24-dimethylcholesta-5,22-dien-3-ol, and 4,24-

dimethylcholsestan-3-ol showed enrichment in the saline tidal sediments, while -sitosterol, -

sitostanol, stigmasterol, and cholesterol were distinctly more abundant in the freshwater (upland

and tidal) sediments. The sterols detected in Chopowamsic Creek and Anacostia River

sediments were similar to the mainstem Potomac River freshwater regions in composition. The

sterol selected as biomarkers shown in Fig. 5 represented >95% by mass of the total-sterols


       For spatial comparisons of carbon sources (Fig. 4), the sterols were arranged into

categories that directly related to sources (Table 1). It should be noted that site T7 (Gunston

Cove) was not included in evaluation of spatial sterols distributions because high concentrations

of polycyclic aromatic hydrocarbons (PAHs) in the sediments interfered with the sterol

identifications (P. McEachern, personal communication). In addition, sediments at sites A1 and

A4 were predominantly sand, which can be correlated to the very low concentrations of sterols

detected, therefore, these sites were also discarded. The sterols grouped according to source

specificity represented an average of 78  7% (mean  SD) of the total-sterol composition

measured in each sample for the mainstem Potomac River sediments, and 78  3% (mean  SD)

of the total-sterol for the Anacostia and Chopowamsic River sediments.

       Although considerable spatial variation within the distribution of sterols in the mainstem

Potomac River sediments existed throughout the freshwater regions of the Potomac River, a few

trends emerged (Fig. 4). The sterols grouped according to source and normalized to TOC had

higher proportions of terrestrial derived sources than algal or sewage sources in the upstream

Appalachian and Piedmont locations. Further downstream, a progression from a slight

predominance of terrestrial sources occurred in the freshwater tidal region to a more pronounced

enhancement of algal sources in the transition tidal region. In the saline tidal region, algal

sterols predominated, progressively increasing in relative abundance further downstream of site

T17 to a maximum at site T23. The percentage of sterols in the TOC pool increased sharply in

the saline tidal region. The relative abundance of terrestrial sterols remained relatively stable

throughout the entire river, although the upland regions had slightly higher concentrations than

the downstream tidal regions of the river. Throughout the entire river, the sewage sterols were

consistently lower in sediments relative to the terrestrial and algal sterols. However, there was a

slight enhancement of sewage sterols in the upper Appalachian regions and at sites T1, T2, and


       Terrestrial biomarker sterols (i.e., stigmasterol, -sitosterol, campesterol, and total of the

three) were compared to terrestrial LSCA fatty acids (>C22 long chain saturated fatty acids)

found in the Potomac River sediments (Fig. 5). Although the terrestrial sterols and fatty acids

were not significantly correlated and their relative abundances are different, they did follow the

same general trend. The terrestrial sterol concentrations tracked the LCSA concentrations up to

site T17. At site T17, there was a marked increase in the LCSA concentrations while the sterol

concentrations remained the same or decreased slightly. Downstream of site T17, the LCSA

concentrations returned a level that was observed upstream and then continued to show a slight

enhancement in the saline tidal region, while the terrestrial sterols showed an even greater

increase in concentrations.

       The sterol biomarker ratio C29/C27 and the fatty acid biomarker ratio C24/C16, used to

identify the relative importance of allochthonous versus autochthonous lipid sources in

sediments, were also compared along the Potomac River (Fig. 6). Larger values indicated an

increased importance in allochthonous inputs whereas, smaller values are indicative of

autochthonous inputs. Although the absolute values between these two ratios were very different

due to differences in the relative amounts of sterols and fatty acids in various organisms, there

were some similar trends among these two ratios. These ratios generally followed the same trend

in the upper Appalachian and Piedmont regions, which showed that the Appalachian region has

higher allochthonous inputs relative to the Piedmont region. These ratios also followed the same

trend in the transitional tidal region where the ratios remained fairly constant except for site T17

where both ratios sharply increased. However, these ratios were divergent in the freshwater tidal

sites (T1, T2, and T3) and in the saline tidal sites (T20-T25). The sterol biomarker ratio

decreased at sites T1, T2, and T3 whereas the fatty acid biomarker ratios increased. Similarly,

the sterol biomarker ratio increased in the saline tidal regions whereas, the fatty acid biomarker

ratios slightly decreased and remained relatively constant.

       The biomarker ratios coprostanol/(cholestanol + cholesterol) and

coprostanol/(cholestanol + coprostanol) were used to track the input of sewage sources in the

Potomac River sediments (Fig. 7). The higher ratios show that there was sewage input in the

upper Appalachian region, which is the home of numerous poultry farms, as well as in the

urbanized region of Washington D.C., which has numerous combined sewer outfalls (CSOs) and

wastewater treatment plant discharges. At sites T1, T2, and T3, the relatively high sewage

biomarker ratios (Fig. 7) correlate well with the low sterol biomarker C29/C27 ratios (Fig. 6).

The sewage biomarker ratios decreased downstream of T3 and increased slightly at sites T14 and


       Although there were no significant differences (Student's t, p>0.05) among most of the

individual terrestrial and algal sterol concentrations normalized to TOC between Chopowamsic

Creek and Anacostia River sediments, there was a significant difference in the individual sewage

sterol biomarkers as well as a shift in the relative abundances of the sedimentary sterol profiles.

The Chopowamsic Creek sediments showed the same relative abundance of

terrestrial>algal>sewage sterols in the sediments at all four sites (Fig. 8). Conversely, the

Anacostia River sedimentary sterol profiles showed a change in relative distribution. The

upstream-most site in the Anacostia River showed a sterol pattern most similar to the

Chopowamsic Creek sediments, although the sewage contribution was much greater in the

Anacostia River sediments. The downstream Anacostia sites showed a decrease in the relative

abundance of terrestrial sterols and an increase in sewage sterols. At site AR5, the prominence of

sewage sterols increased, which corresponded with a greater degree of shoreline development

and urban runoff through CSO discharges in the river as opposed to the relatively undeveloped

areas around the Chopowamsic Creek.

       Similarly, even there were no significant differences among most individual algal

biomarkers, the Chopowamsic Creek sediments also had significantly higher (Student's t,

p<0.05) terrestrial/algal biomarker ratios (stigmasterol/brassicasterol, sitosterol/ brassicasterol,

and campesterol/ brassicasterol) than those found in the Anacostia River. This indicated that

there was an enhanced deposition of terrestrial sources relative to algal sterols in the

Chopowamsic Creek as well as, an enhanced deposition of algal sterols relative to terrestrial

sources in the more urbanized Anacostia River. Furthermore, this sterol terrestrial/algal ratio

(sitosterol/brassicasterol) ratio was significantly correlated (Pearson’s correlation coefficient r =

0.81; p<0.05) to the fatty acid terrestrial/algal ratio (LCSA/PUFA) (Fig. 9).

       Three sewage biomarker ratios were used to track the input of sewage sources in the

Chopowamsic Creek and Anacostia River sediments (Fig. 10). The three ratios were:

coprostanol/(cholestanol + cholesterol), coprostanol/(cholestanol + coprostanol), and

coprostanol/(cholestanol + cholesterol). As with the individual sewage sterol biomarkers, the

Anacostia River sediments also had significantly higher (Student's t, p<0.05) sewage ratios than

those found in the Chopowamsic Creek where, sites A4 and AR5 had the highest ratios.

Although the algal fatty acid (PUFAs) biomarkers and algal sterol biomarkers were not

significantly correlated (p>0.05) to the sewage sterol ratios, the PUFAs and

coprostanol/cholestanol ratio followed the same trend in the Anacostia River (Fig. 11). This

suggestes that an increase in sewage input enhances algal production. The increase in sewage

sterol ratio and consequently the PUFA concentration at site AR4 is associated with the high

degree of shoreline development and urban runoff through combined sewer outfall discharges.

3.3. Factor analysis

       Factor analysis using the principal component extraction method and Varimax rotation

solution was used to identify meaningful geochemical trends in sterol distributions among

sediments in the Potomac River basin. Factor analysis was conducted using individual sterols

normalized to sediment TOC. Because the method requires fewer variables than observations,

only the sterols that had dominant representative sources were used. Therefore, the complete

data set was not used in order to meet the assumption of independence. Following the approach

of Zimmerman and Canuel (Zimmerman & Canuel, 2001), only those variables with mean

values and standard deviations of similar magnitude were selected for the factor analysis.

       The first three factors identified by the factor analysis accounted for 52%, 15%, and 13%

and of the total variance in the sterol data. A large portion of the total variance remained

unaccounted for, suggesting there are many additional minor factors that contribute to the high

variability in this complex system. However, the first three factors combined accounted for 80%

of the variability and appeared to represent interpretable, geochemical factors. All of the algal

sterols in addition to cholesterol were heavily loaded on Factor 1, indicating that Factor 1

represents autochthonous organic matter. Factor 2 was most heavily loaded with terrestrial

sterols, which suggests that Factor 2 represented natural allochthanous inputs. Factor 3 was most

heavily loaded with sewage sterols, indicating that Factor 3 represented anthropogenic inputs.

The factor score plot (Fig. 12) shows the relative influence of each Factors 1 and 2 on the sample

structure. Most of the samples were located around the origin, suggesting that they were nearly

equally dominated by allochthonous terrestrial and autochthonous inputs. Sites A2 and A3,

however, had a very high positive score for Factor 2 indicating that they were dominated by

allochthonous terrestrial inputs. Samples T22, T23, T24, and T25 were all located in the

southern most saline tidal region and had relatively high scores for Factor 1, which is indicative

of autochthonous inputs. The sample scores validated the interpretation of the factors as

indicators of lipid organic matter sources and their ability to represent spatial variations in

surficial sediments. These results were almost identical to the fatty acid factor analysis results

(Walls and Foster, unpublished data).

       The results of the cluster analysis (K-means), based on sterol concentrations normalized

to TOC, showed that only two predominant clusters of sterol profiles existed along the

downstream continuum of the Potomac River. The first cluster consisted of all A, P, and T

samples except for the downstream most sites T22 – T25; the second cluster consisted of T22 -

T25. The sterol profiles were very similar through the entire freshwater region of the Potomac

River up to the transition zone. Below the transition zone, the sterol profiles changed to a

predominantly marine autochthonous/algal source. Although the terrestrial sterol biomarkers

were fairly consistent between the two clusters, there was a clear difference in algal and sewage

biomarker concentrations between the two regions. The saline tidal cluster had much higher algal

and lower sewage biomarker concentrations. Again, these results are very similar to the fatty

acid cluster analysis results (Walls and Foster, unpublished data).

4. Discussion

4.1. Sterols in the mainstem Potomac River

       It has been widely acknowledged that the correlation among physicochemical factors,

such as organic carbon content and particle size, can interfere with interpretations of

geochemistry studies when based on comparing individual sterol concentrations (Brown &

Wade, 1984; Hatcher & McGillivary, 1979; Jeng & Han, 1994; Leenheer et al., 1995; Writer et

al., 1995). The partitioning interaction between non-ionic, non-polar molecules such as sterols

with organic coatings on particulate matter is the cause of this correlation (Leenheer, 1991).

Organic matter binding mechanisms are highly dependent on surface interactions. Due to the

large ratio of surface area to volume, smaller particles have a higher percent organic matter and

therefore tend to have higher levels of non-ionic, non-polar molecules associated with them. In

order to minimize interference caused by these correlations, use of appropriate sterol ratios is

considered a better parameter for making relevant comparisons and identifying trend among

samples (Chan et al., 1998; Poon et al., 2000; Writer et al., 1995). Therefore, biomarker ratios

have been widely used in identifying primary source inputs into sedimentary organic matter

(Bouloubassi et al., 1997; Dachs et al., 1997; Fattore et al., 1996; Grimalt & Albaiges, 1990;

Laureillard & Saliot, 1993; Mudge & Norris, 1997; Nishimura, 1977; Volkman, 1986).

       Compositional ratios assisted in identifying the relative importance of various organic

matter source inputs. Since campesterol, -sitosterol and stigmasterol are typical vascular plant

biomarkers and cholesterol is indicative of marine plankton input, ratios such as

campesterol/cholesterol, -sitosterol/cholesterol, and stigmasterol/cholesterol have been

suggested to be used to distinguish the allochthonous from the autochthonous organic material

(Bouloubassi et al., 1997; Grimalt & Albaiges, 1990; Mudge & Norris, 1997). Furthermore,

Because C27 sterols are often dominant in plankton and C29 sterols are dominant in higher

plants the C29/C27 ratio has also been suggested as useful indicator of the terrestrial input of

organic material (Laureillard & Saliot, 1993; Nishimura, 1977). The use of compositional ratios

revealed additional patterns that are not clearly seen in sterol relative abundance profiles as was

illustrated by the differences in Figures 5 and 6. Although all of these suggested ratios were

evaluated for this study, only the results of the C29/C27 ratios are presented because all four

ratios followed very similar trends. As previously discussed, the allochthonous (terrestrial) to

autochthonous ratios tended to decrease moving from upstream to downstream. The patterns of

C29/C27 sterol and C24/C16 fatty acid biomarker ratios showed that autochthonous inputs to

Potomac River sediments varied spatially in a seemingly rhythmic fashion of peaks and valleys

along the river transect in response to primary production, particle settling, and

resuspension/dispersion processes acting on sediments in fluvial and bed load transport. As

identified by the fatty acid biomarkers and supported by the sterol biomarker ratios, zones of

enhanced autochthonous particle settling (i.e., low ratio values) in the river were clearly evident

at site T6 and to a lesser extent T16 (Fig. 6).

       Furthermore, as illustrated from the C29/C27 sterol ratio and C24/C16 fatty acid ratio,

allochthonous sources contributed more to sediment lipid pools in certain regions of the river,

particularly at site T17 in the mixing zone (Fig. 6). This high terrestrial input was most likely due

to a localized source of organic matter from either terrestrial plants or submerged aquatic

vegetation (SAV), which are abundant in the tidal portion of the Potomac River. The significant

abundance of SAV in the tidal Potomac River may influence carbon dynamics and profiles of

sterols in sediments in the transition tidal region. As previously discussed, sterols such as, -

sitosterol and stigmasterol, which are indicative of continental plants are also found in seagrasses

(Attaway et al., 1971; Nichols et al., 1982; Volkman et al., 1981).

       However, it should be reiterated that inferences drawn from sterols regarding terrigenous

and marine sources must be made with caution (Volkman, 1986). As previously discussed,

although they are dominant sterols, neither cholesterol nor the C29 sterols (sitosterol or

stigmasterol) are absolutely unique to marine or terrestrial sources, respectively. Cholesterol is

also a dominant sterol in sewage treatment effluent. Figure 6 shows that the C29/C27 sterol

ratios are diminished at sites T1-T3, which happens to be the locations of several CSOs and the

wastewater treatment plants. At all of these sites, there is an increase in sewage effluent and,

therefore, higher cholesterol inputs into the river. Thus, the C29/C27 sterol ratios are

incorporating additional cholesterol that is not a source of autochthonous material. Furthermore

some C29 sterols (-sitosterol and stigmasterol) that are typically used as terrestrial biomarkers

have also been found in some algae. Since there is an enormous amount of algal production in

the saline region, as identified by the individual sterol and fatty acid biomarkers, there is likely

some additional C29 sterol contribution coming from algal sources, which consequently affected

this sterol biomarker ratio. Therefore, it is extremely important to consider the entire suite of

biomarkers and all of their potential sources when interpreting these results. It is not adequate to

simply check for the presence or absence of a particular sterol; rather the most reasonable source

of the sterol should be identified and determined whether this is consistent with other

information known about the sample and other lipid data (Volkman, 1986).

       Volkman (1986) suggested that an evaluation of campesterol/stigmasterol/-sitosterol

ratios is necessary in order to determine if these sterols are appropriate to use as terrestrial

biomarkers. For various higher plants, the relative abundance of these plant sterols has been

found to be 1/(0.5-1.3)/(11.5 –31) (Nishimura, 1977). Furthermore, in surface sediments of Loch

Clair, where organic matter inputs were attributed to higher plant origin, the ratios obtained were

1/1.6/6.6 (Cranwell & Volkman, 1981).      Dachs (1998)(Dachs et al., 1997) also suggested that

values greater than one for -sitosterol/campesterol and stigmasterol/campesterol ratios are

indicative of terrestrial input rather than phytoplankton. Harvey (1994) also suggests that a

substantial amount of -sitosterol is indicative of widespread input from higher plants. Figure 3

illustrates that -sitosterol concentrations are dominant in both the freshwater and marine regions

of the Potomac River. The campesterol/stigmasterol/-sitosterol ratios for the Potomac River

sediments were 1/0.2 –2.0/1.0 - 9.3. Although most sites had ratios indicative of terrestrial

input, the saline tidal region ratios appeared to have a more phytoplanktonic characteristic rather

than terrestrial. Additionally, all of the -sitosterol/campesterol and stigmasterol/campesterol

ratios were greater than one except for sites in the saline tidal region. These observations further

support that an additional contribution of the C29 sterols were coming from algal sources and

therefore affecting the C27/C29 biomarker ratio in the saline tidal region.

       In addition to ensuring that campesterol, stigmasterol, and -sitosterol are appropriate to

use as terrestrial biomarkers, the stanol molecules must also be addressed. Stanols in recent

sediments can be formed by bacterial reduction of stenols during sedimentation in the water

column and at the water/sediment interface (Gaskell & Eglinton, 1975; Smith et al., 1983).

Some researchers have suggested using the stanol/stenol ratio as an indicator of this

transformation process {Tian, 1992 #140; Rohjans, 1998 #86; Venkatesan, 1990 #99; Wakeham,

1989 #305; {Wakeham, 1995 #275; Bouloubassi, 1998 #248; Sicre, 1993 #90}. Canuel (Canuel

& Martnes, 1993) observed that the stanol/stenol ratio correlated with high rates of sulfate

reduction and production of bacterial fatty acids. Half of the Potomac River sediment

stanol/stenol ratios were significantly correlated with the bacterial fatty acids while the other half

were not correlated (Table 4). This indicates that the stanols found in the sediment were from

biogenic sources as well as biohydrogenation processes, but the extent of each a contribution is

difficult to determine. Therefore, it was assumed that the source of each stanol was the same as

its corresponding stenol.

           Multivariate analysis (factor analysis) was employed to gain further insight into the

relationships between samples (scores) and lipid sterols (variables) and to assist in confirming

that the source assignment was appropriate. Several authors have used multivariate analyses to

help identify organic matter sources (Colombo et al., 1996; Dachs et al., 1999; Dachs et al.,

1997; Mudge & Gwyn Lintern, 1999; Mudge & Norris, 1997; Yunker et al., 1995; Zimmerman

& Canuel, 2000). The results of the Potomac River sediment factor analysis showed that there

were three main factors that contributed to most of the variability. These factors consisted of an

autochthonous source (i.e., plankton), an allochthonous source (i.e., terrestrial plants), and an

anthropogenic source (i.e., sewage effluent). This factor analysis showed that a major portion of

the total variance of the data was related to the terrestrial-marine or vascular plant-algae gradient

in the estuary. Furthermore, the results provided confirmation that the sterol biomarkers were

adequately assigned to their appropriate source (i.e., all of the terrestrial sterols were grouped

together into one factor while all of the algal sterols were grouped together into a separate


           In addition to the Factor analysis, Pearson’s correlation of fatty acids and sterols also

assisted in confirming that the sterol biomarkers were reflective of their organic matter sources.

As shown in Table 5, all of the Anacostia River and Chopowamsic Creek fatty acids and sterols

ratios corresponding to the same source were significantly correlated. Although, the individual

sterols were not correlated to the fatty acids, the sterol ratios were correlated to the fatty acids.

This further supports the fact, as previously discussed, that ratios rather than individual sterols

should be evaluated in geochemical studies. These correlations also provide additional

confirmation that the sterol biomarkers were adequately assigned to their associated organic

matter sources. It can also be seen that the fatty acids and sterols associated to the same source

were not significantly correlated in the Potomac River sediments.

        The lack of fatty acid and sterol correlation in the Potomac River suggests that the

Potomac River contains older organic matter than the Anacostia River and Chopowamsic Creek.

Because the organic matter in the Potomac River is aged it is mixed together over time and is not

as reflective as the organic matter in the water column. Therefore, the biomarker profile

signatures become smudged and the sterol biomarkers can not be precisely correlated with the

fatty acid biomarker of the same source. Since the tributaries are more routinely scoured and

have more recent organic matter inputs, their sediments are less aged especially in the upstream

reaches. Therefore, the organic matter in the tributaries has not had sufficient time to be

degraded or altered. Thus, the sterol biomarkers can be more effectively correlated with the fatty

acid biomarkers of the same source in the smaller tributaries.

        Another observation that supports this conclusion is the fact that the PUFAs were

depleted relative to the terrestrial and bacterial fatty acids at sites T11 through T17 while the

algal sterols were enhanced. Consequently, the PUFAs and algal sterols were not significantly

correlated. This discrepancy can likely be explained by two observations. The first observation

pertains to the difference in the relative stability of the two different types of compounds.

PUFAs are typically an indication of fresh algal input because they are very labile and are

preferentially degraded by heterotrophic bacteria (Chuecas & Riley, 1969; Pohl, 1982; Scribe et

al., 1991). However, sterols are refractory molecules and are well preserved in a sedimentary

environment. Therefore, due to their refractory nature the algal sterol concentrations can appear

to be enhanced while concomitantly the labile PUFA concentrations appear reduced.

Furthermore, it has been shown that in the Seine estuary there was ultimately a seaward transport

of organic matter which was subsequently trapped in the estuarine turbidity maximum

(Thoumelin et al., 1997). Thus, more refractory compounds such the sterols can be trapped in the

transition zone and consequently enhanced in concentration levels while at the same time the

available PUFAs are being consumed and depleted.

       Although many fatty acid and sterol biomarkers and ratios were not significantly

correlated, it can still be concluded that the maximum phytoplankton productivity occurs in the

saline tidal waters. Most of the algal sterol biomarkers, especially those specific to

dinoflagellates (i.e., dinosterol), were at their maximum concentrations in the tidal saline regions

(Figs. 3 and 4). The results of several studies examining phytoplankton production in estuarine

environments support the present observations made on sterols in Potomac River sediments

(Fisher et al., 1988; Harding et al., 1986; McPherson et al., 1990). McPherson (McPherson et

al., 1990) observed that maximum productivity and biomass in transitional rivers occurs where

color associated with the freshwater inflow was diluted by seawater so that light and nutrients are

both more bioavailable. Furthermore, both Harding (Harding et al., 1986) and Fisher (Fisher et

al., 1988) observed that the chlorophyll and productivity maximum in the Chesapeake Bay

occurred seaward of the turbidity maximum where light penetration increased and sufficient

nutrients were present to support active phytoplankton growth. Marine phytoplankton

abundance was clearly evident in by the increased relative abundance of phytoplankton sterols in

the sediment organic carbon pool downstream of the transition zone.

       Individual sewage sterol concentrations and sewage ratios were used to identify sources

of sewage pollution in the Potomac River. Since coprostanol constitutes most of the total sterols

in human feces, its dominance tends to isolate humans from other animals and is, therefore, used

to track sewage input (Ferezou et al., 1978; Leeming et al., 1996). Coprostanol concentrations

have been used by many authors to track and monitor fecal contamination in aquatic systems

(Brown & Wade, 1984; Chan et al., 1998; Gonzalez-Oreja & Saiz-Salinas, 1998; Goodfellow et

al., 1977; Grimalt et al., 1990; Hatcher et al., 1977; Hatcher & McGillivary, 1979; Jeng & Han,

1994; Mudge & Gwyn Lintern, 1999; Nichols et al., 1996a; O'Leary et al., 1999; Pierce &

Brown, 1984; Poon et al., 2000; Venkatesan & Kaplan, 1990; Writer et al., 1995). The

sedimentary coprostanol concentrations found in these studies range from 0.004 to 390 g/g dry

sediment. Various authors have suggested various levels of coprostanol that could be indicative

of sewage contamination. Hatcher (Hatcher & McGillivary, 1979) suggested a coprostanol

threshold of 0.01 g/g that is indicative of sewage; Brown and Pierce (Brown & Wade, 1984;

Pierce & Brown, 1984) both stated that a level of 0.1 g/g is reflective of sewage input; O’Leary

(O'Leary et al., 1999) suggested that a concentration of 0.25 g/g is indicative of sewage input

while, Nichols (Nichols et al., 1996a) suggested that 0.5 g/g is indicative of significant

contamination. However, coprostanol concentrations as high as 3.5 g/g have been observed in

pristine locations (Grimalt et al., 1990). Therefore, there is no consensus of what coprostanol

concentration is indicative of polluted waterways. The coprostanol levels identified in the

Potomac River sediments ranged from 0.06 to 2.01 g/g and fall in the range of not being

impacted by sewage input to being significantly impacted by sewage discharge.

       As previously discussed, a more telling indication of sewage sterol input is the use of

sewage sterol ratios. Several ratios have been proposed and used to evaluate domestic sewage

input. The coprostanol/cholesterol ratio provides a relative measure of sewage contamination

and has been used by several authors (Fattore et al., 1996; Grimalt et al., 1990; Mudge & Gwyn

Lintern, 1999; Mudge & Norris, 1997; Nichols & Leeming, 1991; Nichols et al., 1996a;

Quemeneur & Marty, 1994; Takada et al., 1994). A ratio less than 1 indicates a strong input of

cholesterol from autochthonous biogenic sources and levels of 0.1 or less have been observed in

uncontaminated sites (Nichols, 1996). It has been proposed that gross sewage contamination is

associated by a ratio values of 1 to 2 since a ratio of >1 was found in particulate of raw sewage

from Toulon, Marlaix and Brest treatment plants (Quemeneur & Marty, 1994), and 4 for sewage

sludge from New York City (Takada et al., 1994).

       Another useful measure for assessing whether coprostanol found in sediments is of fecal

origin is gained by evaluating the coprostanol/cholestanol ratio. Cholestanol can be abundantly

found in uncontaminated sediments. Typically, coprostanol does not occur naturally in fresh or

marine waters or in aerobic sediments. Coprostanol can be formed from reduction in situ of

cholesterol in anaerobic sediments. However, some background concentration of coprostanol

might be expected even in pristine sediments and comparatively oxygenated sediments not

contaminated by fecal pollution due to the existence of anaerobic conditions in micro-

environments within the sediment (Nishimura, 1982). In uncontaminated sites the

coprostanol/cholestanol ratio is generally well below 0.3 whereas at sites known to be

contaminated by sewage the ratio is greater than 1 (Leeming & Nichols, 1998). However, it has

been suggested that ratios greater than 0.4 or 0.5 are indicative sites impacted by sewage input

(Leeming et al., 1996; O'Leary et al., 1999).

       The coprostanol/(cholestanol + coprostanol) ratio has also been used to examine sewage

input to sediments (Fattore et al., 1996; Grimalt et al., 1990; Jeng & Han, 1994; Mudge & Gwyn

Lintern, 1999; Mudge & Norris, 1997; Poon et al., 2000; Writer et al., 1995). In an environment

without fecal contamination, the hydrogenation of cholesterol to cholestanol seems to be

preferred in comparison to hydrogenation of cholesterol to coprostanol (McCalley et al., 1980;

Nishimura, 1982). Studies where microbial assemblages, obtained from marine sediments, were

allowed to grow in anaerobic media containing radiolabelled cholesterol have shown that nearly

equal concentrations of coprostanol and cholestanol are produced (Taylor et al., 1981).

Furthermore, in a sewage sludge containing radiolabelled cholesterol, coprostanol was observed

to be preferentially produced (Gaskell & Eglinton, 1975). Therefore the coprostanol / cholestanol

+ coprostanol ratio can be used to determine if sewage contributions or natural reduction

processes of cholesterol in the sedimentary environment are dominant. Low ratios correspond to

lower coprostanol content. Ratios between 0.7 and 1 are characteristic of urban pollution

whereas ratios in the order of 0.1-0.3 correspond to remote areas (Grimalt et al., 1990).

       The coprostanol/(cholestanol + cholesterol) ratio has also been used to examine sewage

contamination in sediments (Chan et al., 1998; Leenheer et al., 1995; Poon et al., 2000). Since

there appears to be no consensus on which ratio method is better in predicting sewage

contamination all of these ratios were evaluated for the Potomac River sediments. In general the

highest ratios were found in areas around Washington D.C. and in the upper Appalachian region.

The coprostanol/cholesterol values ranged from 0.05 to 0.33 where, Nichols (Nichols et al.,

1996a) suggested that values greater than 1 were indicative of sewage contamination and less

than 1 shows a strong input of biogenic autochthonous sources. All of the Potomac River sites,

including the Blue Plains wastewater treatment plant site did not have levels typically

characteristic of sewage contamination. The coprostanol/(cholestanol + cholesterol) ratios

ranged from 0.05 to 0.29 where (Grimalt et al., 1990) proposed the range of 0.7-1.0 as

characteristic of urban polluted sediment; none of the ratios here exceeded 0.7; similar behavior

has also been observed in Taiwan (Jeng & Han, 1994). The range of coprostanol/(cholestanol +

cholesterol) values were 0.02 – 0.17, while the range of coprostanol/cholestanol values were 0.05

to 0.41. Although all of the ratios followed the same trend along the Potomac River, the ranges

of values for most of the sewage ratios evaluated were not very wide. There seemed to be a

clearer separation between samples having low and high coprostanol/cholestanol values and,

therefore, it was considered the most suitable parameter for spatial comparison purposes.

       When comparing the various ratios to values from studies conducted in sewage impacted

systems, it appears that the Potomac River is not greatly impacted by sewage inputs. In fact, it

would be considered uncontaminated by the threshold values proposed above. However, the

highest ratio and coprostanol values along the Potomac River can be associated to specific

sewage input locations. Sites T1, T2, and T3 had elevated ratios compared to the rest of the sites

along the River (Fig. 7). Sites T1 and T2 are associated with the untreated sewage being

released by CSOs in the Washington D.C. area. During large storm events when runoff

overflows the sewage treatment system, raw sewage is discharged directly into various streams

(Lugbill & Berger, 1993). This runoff contains untreated sewage mixed with stormwater. There

are presently 60 CSO outfalls listed in the National Pollutant Discharge Elimination System

(NPDES) Permit issued by EPA to District of Columbia Water and Sewage Authority

(DCWASA, 2004). Site T3 is associated with the Blue Plains Advanced Wastewater Treatment

Plant, which is the largest advanced wastewater treatment facility of its type in the United States

with an average daily capacity of 370 million gallons of water per day (DCWASA, 2004).

(Quemeneur & Marty, 1994) Quemeneuer showed that in a physical-chemical treatment plant,

the removal efficiencies for dissolved sterols was 22% and for particulate sterols was 78% where

as, in a biological treatment plant, the removal efficiencies reached 93% for particulate and 95%

for dissolved sterols. It seems reasonable that the sewage ratios at site T3 are similar to those

found at T1 and T2. The amount of wastewater released at T3 is orders of magnitude higher

than the amount released at T1 and T2, however T1 and T2 release untreated sewage while the

effluent from T3 is biologically treated and depleted in the sterol concentration by up to 95%.

       Although not as high as the T1-T3 sites, all of the upland Appalachian and Piedmont sites

had elevated sewage ratios with A1 being the highest. A1, Pawpaw West Virginia, is

approximately 5 km from the mouth of South Branch Potomac River. The South Branch has

experienced environmental challenges due to the growth of the chicken industry. Over 95

million chickens are raised in the Potomac River headwater regions, particularly along the South

Branch River (AmericanRivers, 1998). Waste from the feedlots and farms and excess manure

run off into the Potomac. Moreover, in 1998 the USDA claimed that the number of poultry

houses and feedlots along the river directly correlates with widespread presence of bacteria

throughout the watershed (AmericanRivers, 1998). The sewage markers evaluated are used to

track human not chicken feces. Human feces contains about 300 times more coprostanol than

hen feces (Leeming et al., 1996). However, given the fact that there is a huge poultry farm

presence, which was correlated to high fecal bacteria levels in the river in this region, it appears

reasonable that the elevated sewage ratio levels are in part due to the poultry industry.

       Moreover, in 1990, there were 17 major wastewater treatment plants and several minor

plants located above the fall line that directly discharged their effluent into the Potomac River or

its tributaries (Lugbill & Berger, 1993). Although not as large as the Blue Plains treatment plant,

most of these plants utilize far less advanced treatment practices and therefore have the potential

to release higher levels of sterols. The presence of these facilities may also contribute to the

elevated sewage ratios in the upland regions of the Potomac River basin.

       It can be seen that the sewage ratio levels decline downstream of the Blue Plains

treatment plant (Fig 7). This is consistent with other studies that have shown the progressive

seaward decline of fecal sterols relative to total sterols, which represents dilution of sewage by

biogenic sterols (Goodfellow et al., 1977; Venkatesan & Kaplan, 1990). However, two sites

(T15 and T24) downstream of the Blue Plains facility had slight enhancements of the sewage

ratios. Both of these sites can be associated with some type of sewage input. T15 is associated

with the Aquia sewage treatment facility while T24 can be associated with the Ragged Point

Marina, which contains a sewage pump out facility for boats.

       The source of lipid organic matter in Potomac River sediments undergoes distinct

changes in the tidal region at the marine and freshwater boundary (Fig. 13), with a switch to

almost exclusively autochthonous sources in the marine region. A mass balance of lipid

biomarkers is illustrated in Fig. 13 to highlight the changing dynamics of organic matter

transport through a coastal river. Such changes in the origin of organic substances in the river

can affect the sources and fate to anthropogenic contaminants during land to sea transport.

4.2. Sterols in the Potomac River tributaries

       The results of the t-tests showed that the Anacostia and Chopowamsic regions were

significantly different in sewage input, with the Anacostia River having much higher input

levels. This is due to the fact the much of the CSOs flow directly into the Anacostia River

(Lugbill & Berger, 1993). Many studies have correlated the elevated levels of algal growth to

sewage release (Leeming & Nichols, 1998; Mudge & Gwyn Lintern, 1999; Nichols et al.,

1996b). Although the algal and sewage sterol concentrations were not significantly correlated,

there was a definite association between the increase sewage input and algal sterols and fatty

acids (PUFAs) (Fig. 11).

       The compositional ratios revealed indistinguishable patterns in the Anacostia versus

Chopowamsic analysis. The results of the t-tests showed that the two regions were not

significantly different in any of the individual terrestrial or algal sterol concentrations. However,

the tests did show significant differences in the autochthonous versus allochthonous ratios. This

suggests that terrestrial source is a more important input into the Chopowamsic lipid organic

matter than in the Anacostia organic matter. Conversely, there was a change in the

predominance of lipid biomarker sources in the Anacostia River through the downstream transect

(Fig. 14). The upstream tidal Anacostia River site showed a lipid profile very similar to

Chopowamsic Creek, where the terrestrial lipids predominated, but further downstream algal and

sewage sources became the most abundant. The downstream Anacostia River is the most urban-

development-impacted region of the Potomac River basin, and here the river is completely

channelized and surrounded by development. Several combined sewer outfalls, which discharge

raw sewage and impervious surface runoff in response to atmospheric precipitation, provide

heavy loads of pollutants to the river.

5. Conclusions

       Graphical analyses, compositional ratios, factor analysis, and cluster analysis of sterol

biomarkers were used to reveal spatial variability in the origin of lipid organic matter sources

along the hydrographic continuum of the Potomac River. The use of all of these tools allowed a

more complete geochemical interpretation of lipid organic matter source distributions.

       The use of graphical analyses and compositional ratios identified that the biomarker

profiles gradually change in each hydrographic region that reflect landscape, land use, and

anthropogenic impacts of urban development along the Potomac River and its tributaries. The

factor analysis identified three factors that account for 80% of the variability. Factor 1

represented autochthonous (plankton) input, Factor 2 represented allochthonous (terrestrial)

input, and Factor 3 represented anthropogenic allochthonous (sewage effluent). Furthermore, as

with the fatty acid study, the factor analysis and cluster analysis revealed that there are only two

distinct regions that have unique fatty acid biomarker signatures. One grouping consists of all A,

P and T (except T22-T25) samples while the other grouping consists of the southernmost

samples T22, T23, T24, and T25 located in the saline tidal region of the Potomac River. The

predominate difference between these two groupings is that the phytoplankton productivity

maximum occurs seaward of the turbidity maximum zone (T22, T23, T24, and T25) where there

is a strong algal fatty acid and sterol signature as compared to the up-river locations.

       The use of compositional ratios also revealed information about the relative importance

of the of source specific sterols into the organic matter. The algal inputs are more important

contributors to the organic matter in the seaward regions of the Potomac River. Additionally,

terrestrial sources are more important contributors in the forested Chopowamsic Creek compared

to the urban Anacostia River. Furthermore, there were a few definite sewage signature profiles

identified in the Potomac River basin. One of the sewage inputs occurs in the upland reaches of

the river which is associated to the non-point release of poultry farm fecal matter, another occurs

around the urbanized regions of the Washington D.C. area which is associated with CSOs and

the Blue Plains sewage treatment effluent, and another occurs along the Anacostia River which is

associated with CSOs.

       Based on the results of the fatty acid and sterol biomarker studies, it can be seen that a

combination of both lipid biomarkers are necessary to use when identifying sources of organic

matter and relating them to land use and geochemical processes. It is extremely important to

consider the entire suite of lipid biomarkers, all of their potential sources, and any additional

information pertaining to the sample and sample site when interpreting results from geochemical



       Sincere appreciation is given to Dr. Rodger Harvey and his research group of the

University of Maryland for technical assistance in the lipid extraction method. Mr. Tom Huff

and Mr. Phil McEachern of George Mason University (GMU) provided critical laboratory

coordination and support. The coordination of sediment collection by Dr. Paige Doelling Brown

(GMU) and the statistical assistance of Dr. Leila Barraj (Exponent, Inc.) were invaluable.

Financial support for this study was provided by the Jeffress Memorial Trust (Grant No. J-559).


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