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

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1

Sterol

Composition

in Surficial Sediments
in
The Coastal mid
-
Atlantic Potomac River
Basin
(
USA)




Cassi L. Walls and Gregory D. Foster
1

Department of Chemistry

and Biochemistry
, George Mason University, MSN 3E2, Fairfax, VA
22030







1

Corresponding author: email to
gfoster@gmu.edu
; fax, 703/993
-
1055


2

Abstract

The molecul
ar 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 a
ccount 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 terrestri
al
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 Provin
ce 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.


Keyw
ords:

Sterols; Potomac River Watershed; Lipid biomarker compounds; Sediments; Factor
Analysis

3

1. Introduction

Carbon cycling and deposition in surface waters is affected by many ecological and
biogeochemical processes.
The organic matter composition in s
urficial sediments
of rivers
reflects
recent
d
epositional 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
or
ganic 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. Sedimen
tary 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 th
at 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.

S
terols represent a group of geolipid

markers
that can differentiate
between
allochthonou
s
,
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 i
n sediments from distinct hydrologic zones along the Potomac River to characterize

4

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 th
e 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 basi
n
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, th
ey 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, cou
pled 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 biolo
gically produced sterols
are planar 3

-
hydroxy tetracyclic structures commonly containing a methyl
-

or ethyl
-

substituted

5

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. Fe
cal 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 a
l.
, 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, 1
994; 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 environ
ments
(Bouloubassi

et al.
, 1997; Canuel & Martnes, 1993; Duan, 2000; Farrington

et al.
, 1988; Harvey, 1994; Lee

et
al.
, 1979; Lee

et al.
, 1980; Nishimura, 19
77; 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 mat
ter 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

6

(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
alteratio
ns
, 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 v
arious 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
.


1.2.1. Terrestrial Sterols


7

Campesterol (C28; 24
-
m
ethylcholest
-
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; Volkma
n, 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 o
rganisms 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 phyt
oplankton 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, 1
986)

as well as, in various marine and freshwater Chlorophyceae
(Goad & Goodwin,
1972)
, and as the predominant sterol in rotif
ers
(Nishimura, 1977)
.

The relative abundance of the high
er 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

8

of theses three sterols has been propose
d as an indicator of terrestrial source
(Volkman, 1986)
.
Despite the potential ambiguities, camp
esterol,

-
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; Mud
ge & 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; Colomb
o

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 (c
holesta
-
5,22
-
dien
-
3

-
ol
)
is the major ster
ol 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
-
diatomac
eous 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
-
die
n
-
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 b
een observed

9

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 pre
sence
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, pr
opylidine, 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
(Harv
ey

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)
.


10

Green algae (Chlorophyta) produce a range of sterols including chondrillasterol,
poriferasterol, 28
-
isofucosterol, ergosterol, cholesterol, sitosterol, zymost
erol, 24
-
methylenecholesterol, clionasterol
(Gibbon

et al.
, 1968; Knight, 1967; Patterson, 1970;
Patterson, 1982; Vo
lkman, 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, the
y are found in many other algal groups
(Volkman, 1986)
.
They are produced by many microalgae, c
occolithophores
(de Leeuw

et al.
, 1983; Smith

et al.
,
1982; Volkman

et al.
, 198
1)

dinoflagellates
(Goad & Withers, 1982; Huang & Meinschein,
1979)
, and a number of Prymnesiophy
tes
(Marlowe

et al.
, 1984)
. 23,24
-
dimethylcholesta
-
5,22
-

11

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. Furth
ermore, 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
-
dime
thylcholesta
-
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 wid
ely 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 S
terols

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 de
tected in other

12

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 anae
robic 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
redu
ction 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 t
he 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 concentrat
ions 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)
.


13

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 {Goodfe
llow, 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; Hatch
er, 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


14

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 pr
oductivity
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 constitu
tes 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
in
to 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 cholestero
l as the major sterol
(Volkman, 1986)
.
Furthermore,
cholesterol is
one of the primary
sterol
s

i
n 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

15

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, dehydra
tion 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 t
ransformation is small
(Gaskell & Eglinton, 1975)
. For
example, Nishimura and Koyama
(Nishimura & Koyama, 1977)

showed that cho
lesterol 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 se
wage 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 anaerob
ic
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 a
mounts 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
st
erols such as dinostanol can be formed by the microbially mediated reduction of sterols that
often occurs at oxic
-
anoxic boundaries
(Wakeham, 1989)
.


16

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.
, 197
6)
.
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 specie
s
(Nishimura & Koyama, 1977; Robinson

et al.
, 1984)

and they are also found
in common algae su
ch as diatoms
(Gagosian

et al.
, 1983b; Volkman, 1986; Volkman

et al.
,
1981)
. Some investigators reported cholestanol as the main sterol in some marine org
anisms
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
stigmast
anol 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 fro
m specific source
-
organisms or by a bacterial conversion
(Gagosian & Heinzer, 19
79)
. 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).
B
riefly, t
he Potomac River originates near Fairfax Stone, WV and

17

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
km
2

a
nd a historical annual flow of 330 m
3
/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 c
oastal 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

s
amples were obtained from a Boston Whaler for the Piedmont and tidal
regions. Sediment grabs were initially p
laced 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 stor
ed 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, sed
iment samples
were stored at

40
o
C 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, on
e

18

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 developmen
t (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
.
Sedim
ent 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

Ster
ols 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

o
C 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 co
mbined
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

o
C for 30 min).
After the sample was cooled, the neutral lipids were partitioned
from the alkaline solution into 2
ml of hexane.


19

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) reage
nt that was amended with 25% pyridine (Fisher Scientfic, Somerville,
NJ) while heating at 50

o
C 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
selectiv
e 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 flo
w rate through
the column adjusted to 1 mL/min at 100
o
C. A two
-
stage temperature program consisting of 50
-
120

o
C at the rate of 10

o
C min
-
1
, followed by a 3

o
C min
-
1

rate to 300

o
C 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 i
nternal and external standards (Sigma Aldrich) and mass spectral
interpretation of the ion fragmentation
(Jones

e
t 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, Unive
rsity Park, PA). Parametric analyses, including ANOVA, Tukey’s pairwise

20

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 relat
ive
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 s
terol distributions to
ensure that selected biomarker
s

were
indicative of the biogenic source
s
. The data were also
evaluated through Factor Analysis using the principle component extraction method and the
Varimax rotation solution to identify the underlyin
g 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 composi
tion

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 extr
emely high sand content
which provide
d

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 concentrati
on 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 variabili
ty in sediment properties (e.g., grain size and TOC) in
the Potomac River. Furthermore, there was a significant correlation (Pearson’s correlation

21

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 sedimen
ts (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 stero
ls 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
T
able 3. The most abund
ant 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 s
howed enrichment in the saline tidal sediments, while

-
sitosterol,

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

22

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

For spatial comparisons of carbon sources (Fig. 4), the sterol
s 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 (PAH
s) 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, therefor
e
,

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 f
ew
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 progres
sion 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 increas
ing 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

23

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 st
erols. However, there was a
slight enhancement of sewage sterols in the upper Appalachian regions and at sites T1, T2, and
T3.

Terrestrial biomarker sterols (i.e., stigmasterol,

-
sitosterol, campesterol, and total of the
three) were compared to terrestr
ial 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 d
id
follow the

same general trend. The terrestrial sterol concentrations
tracked
the LCSA concentration
s
up to
site T17. At site T17, there
was
a marked increase in the LCSA concentration
s

while the sterol
concentrations remain
ed

the same or
decreased
slightly. Downs
tream of site T17, the LCSA
concentration
s

return
ed

a level that was observed upstream and then continue
d

to show a slight
enhancement in the saline tidal region, while the terrestrial sterols show
ed

an even greater
increase in concentrations.

The sterol b
iomarker ratio C
29
/C
27
and the fatty acid biomarker ratio C
24
/C
16
, 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 indicate
d

an
i
ncreased 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

24

in the upper Appalachian and Piedmont regions, which showed that the Appalachian region has
higher allochthonous inputs
relative
to th
e 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
s
ites (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 whe
reas, 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
Potoma
c 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 decrease
d

downstream of T3
a
nd increased
slightly at sites T14 and
T24.

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 sedi
ments, 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

25

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
Ch
opowamsic Creek sediments, although the sewage contribution was much greater in the
Anacostia
River
sediment
s
. 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 correspond
ed

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, ev
en 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 t
errestrial/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. 1
0
). The three ratios were:
coprostanol/
(
cholestanol + cholesterol
)
, cop
rostanol/
(
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 Chop
owamsic Creek where, sites A4 and AR5 had the highest ratios.

26

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. 1
1
). This
suggeste
s

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
degr
ee 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 repre
sentative 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 de
viations 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
un
accounted 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, geoch
emical 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

27

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. 1
2
) 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 tha
t 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 score
s 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 z
one. 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 da
ta).


28


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 studi
es 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 co
atings 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 inpu
ts into sedimentary organic matter
(Boulou
bassi

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 orga
nic
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 dis
tinguish the allochthonous from the autochthonous organic material

29

(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
(Laurei
llard & 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 suggeste
d 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 f
rom upstream to downstream. The patterns of
C
29
/C
27

sterol and C
24
/C
16

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 aut
ochthonous 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 contribut
ed 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 aquat
ic
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 previousl
y discussed, sterols such as,

-

30

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 mad
e with caution
(Volkman, 1986)
.


As previously discussed,
although they are dominant sterols, ne
ither 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 a
n 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 ste
rol 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 appropri
ate to use as terrestrial
biomarkers. For various higher plants, the relative abundance of these plant sterols has been

31

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 w
ere
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 a
mount 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 rati
os 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, a
ll 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. Stanol
s 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 us
ing

the stanol/stenol ratio as an indicator of this
transformation process {Tian, 1992 #140; Rohjans, 1998 #86; Venkatesan, 1990 #99; Wakeham,
19
89 #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

32

stanol/s
tenol 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 e
xtent 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 use
d
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 t
hat 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). Th
is 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
adequate
ly 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
factor).

In addition to the Factor analysis, Pearson’s correlation of f
atty 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 s
ource were significantly correlated. Although, the individual

33

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 individua
l 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 ste
rols 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 a
nd 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 bio
markers 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.
There
fore, 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 observa
tion 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 significan
tly
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 becau
se they are very labile and are
preferentially degraded by heterotrophic bacteria
(Chuecas & Riley, 1969; Pohl, 1982; Scribe

et

34

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 subsequ
ently 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 biomark
ers, especially those specific to
dinoflagellates (i.e., dinosterol
)
, were at their maximum concentrations in the tidal saline regions
(Fig
s
. 3 and 4). The results of several studies examining phytoplankton production in estuarine
environments support th
e 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 transition
al 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 a
l.
, 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 w
ere 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.


35

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 sterol
s

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

there
fore
,

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.
, 199
9; 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 co
prostanol 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, 19
84)

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 us
ed to evaluate domestic sewage

36

input. The coprostanol/cholesterol ratio provides a relative measure of sewage contamination
and has been used by several authors
(Fattore

et al.
, 1996; Grima
lt

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 sourc
es 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 fro
m 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. Cholesta
nol 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, so
me 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
co
prostanol/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; G
rimalt

et al.
, 1990; Jeng & Han, 1994; Mudge & Gwyn

37

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 compa
rison 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 ch
aracteristic 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
cont
amination 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 fo
r 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
lev
el
s

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

38

characteristic of urba
n 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 Pot
omac 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 par
ameter 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 uncontaminat
ed 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 ratio
s

compared to the rest of the sites
along th
e 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 str
eams
(Lug
bill & 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 Au
thority
(DCWASA, 2004)
. Site T3 is associated with the B
lue 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 wa