e20495a

Determination of Faecal Pollutants in

Torrens and Patawalonga Catchment
Waters in South Australia Using Faecal
Sterols
Iryanti Suprihatin, Howard Fallowfield, Richard Bentham & Nancy Cromar
Department of Environmental Health, Flinders University
GPO Box 2100, Adelaide SA 5001

ABSTRACT

KEYWORDS
Faecal sterols, pollution, water management

INTRODUCTION

Since the first South Australian Catchment Water Management Boards (CWMB) were
founded in 1995, they have had to deal with on going water quality problems arising from
the frequent presence of high numbers of thermotolerant coliform bacteria indicative of
faecal contamination. The concentrations of thermotolerant coliforms in catchment waters
often exceed the guidelines set by the National Health and Medical Research Council
(NHMRC) (Ralph, et.al.,1998).
In response to these water quality problems, the Catchment Boards have been conducting
monitoring of two main metropolitan water catchments: the Torrens and Patawalonga.
Torrens catchment is constituted of several creeks and reservoirs covering three sub-
catchments: urban, lower rural and upper rural (Ralph, et.al., 1998). The Patawalonga
2
catchment supports about 208km urban area and comprises six sub-catchments, which
are collected in the Patawalonga basin and then drained to the Gulf of St. Vincent.

To be able to effectively manage the pollution, sources of the contamination have to be

identified. The microbiological method, by enumeration of faecal indicator bacteria is not
capable of doing so; thus other methods which are able to distinguish the sources of faecal
contamination are being evaluated (Tabak et.al., 1972; Walker et.al., 1982; Venkatesan
et.al., 1986; Leeming et.al., 1996).
Faecal sterols including coprostanol (5-cholestan-3-ol), epicoprostanol (5-cholestan- 3-
ol) and cholestanol (5-cholestan-3-ol) have been successfully used as chemical
biomarkers of faecal pollution in water (Yde et.al., 1982; Pierce and Brown, 1984;
Venkatesan et.al., 1986; Writer et.al., 1995; Jeng et.al., 1996; Leeming et.al., 1996). The
composition of steroids in animal faeces (including humans) is determined by diet,
biosynthesis of sterols from other precursors and intestinal flora (Leeming et.al., 1996).
Consequently, the profile of sterols is unique for each animal and therefore can be used as
an aid in indicating pollution sources. Coprostanol is considered to be the major human
faecal steroid indicator. However, if coprostanol coexists with ethyl coprostanol it indicates
herbivores faeces. High levels of cholesterol and sitosterol with minimal coprostanol may
indicate dog or birds faecal contamination (Ralph et.al., 1998). The objective of this
research was to determine both abundance and sources of faecal pollutants in Torrens
and Patawalonga catchment waters.

METHODS

Sampling locations
Five sites were selected for steroid profiling from the Torrens catchment namely Waterfall
Gully at First Creek, Castambul at Sixth creek, Merchant, Gorge and Gumeracha.
Waterfall Gully and Castambul represented sites at the junctions of the First creek and
Sixth creek with the Torrens, while Merchant is an upstream site on the Sixth creek. The
Gorge and Gumeracha sites are reservoirs on the Torrens itself. All these sampling sites
fall in the upper and lower rural catchment sections, which cover pastoral and agricultural
area. Septic tanks are commonly used in this section. The Patawalonga catchment was
represented by Brownhill creek with two collection sites: Mitcham, located upstream and
Airport by the end of the creek near the collection basin.

Sampling
Approximately 20 litre water samples were collected monthly from selected sites along
both catchments in May-July 2000 (Winter) and January-February 2001 (Summer). In
January and February, only one site from the Patawalonga catchment was sampled as the
other site (Mitcham) was dry.

Sample Preparation
Particulates from five litre water samples were filtered in triplicate using glass fibre filter
0
paper (GF/C, Whatman) and oven dried at 105 C for 24 hours.

Extraction
Extraction was conducted using a modification of the lipid extraction method of Bligh and
Dyer (1959). Samples (the dried filter paper containing particulates) were percolated in a
miscible chloroform: methanol: water mixture for 24 hours. The mixture was extracted
using water and chloroform, and chloroform layers collected and saponified using
methanolic potassium hydroxide. The products were then extracted using a hexane-
chloroform mixture. The sterols were collected from hexane layers and concentrated by
drying under nitrogen.

Silylation
The isolated steroids were silylated using a modification of the method described by Ralph
et.al., (1998). Samples were resuspended in chloroform (1 mL) and dimethyl formamide
(20 µL) followed by the addition of bis(trimethylsilyl) trifluoro acetamide (80 µL). The
0
solutions were then heated at 60 C for 60 minutes.
Quantitation of steroids
The steroid content of the silylated samples was determined using a gas chromatograph
(Varian's Star 3400CX) with mass spectrometer (Varian's Saturn 2000). The column was
0 0
programmed with an initial temperature of 50 C (1 minute), increased to 220 C at
0 0 0
30 C/minute held for 1 minute, and finally increased to 300 C at 5 C/minute and held for
0
10 minutes. The injector temperature was set at 250 C and carrier gas was He at 15 psi.
Sterols were quantified by reference to standard solutions (Sigma). The analyses were
performed in triplicate for each site where possible. The results show the mean of three
replicate analyses with error bars representing standard deviations. Results without error
bars represent the means of duplicates.

RESULTS AND DISCUSSION

Winter Samples
In the May sampling period, coprostanol and cholesterol were the major steroids detected
in Torrens catchment (Figure 1). No cholestanol was identified in this catchment,
suggesting that the sterols were derived from faecal contamination (Nishimura, 1982;
Leeming et.al., 1998). The coprostanol concentration was similar to that of cholesterol in
the Waterfall Gully sampling site, suggesting that the pollution may include human faeces.
This was inferred as human faeces comprise 60% of its steroid profile as coprostanol
(Leeming, 1996). In the other sites, the concentration of coprostanol was insignificant
compared to cholesterol and or betasitosterol suggesting non-human sources. Dogs or
birds (magpies and rosellas) may have some contribution to this steroid profile (Ralph
et.al., 1998).

In June and July (Figures 2 and 3 respectively), cholesterol was found to be the largest
contributor of the steroid pollutants in all sites with the exception of Gorge (July), where
coprostanol was higher than cholesterol. This suggests that the faecal contaminants were
possibly from human origin (Gorge, July) and dog or birds at other sites. The levels of the
faecal pollution indicated by sterol concentrations, decreased by a factor of 10 in July,
probably because of lower rainfall.

The Patawalonga samples (Figure 4) show that the steroid pollution was dominated by
cholesterol and betasitosterol at all times during the three months. Again, these profiles
suggest dog or birds as sources of the faecal pollution.
0.0 5 0
Wa te r fa ll
0.0 3[s5 Go r g e
te C
0.0 2ro
0
ls
0.0 010
], a
m
g/ s

t
a
m

b
u

e
 r cha a
nt
Gu
mer
ach

0.0 005

0.0 000
Coprostanol Cholesterol
Betasitosterol
C o m p ou nd N am e

Figure 1 The steroid profile of sampling sites in the Torrens catchment in May 2000

0.03
W
a
[s
0.02te t
ro e
l], r
m
g/ f
a
ll
G
o
r
g
e

C
a
s
t
a
m
b
u
l
M
e
r
c
h
a
n
t
G
u
m
 e c
r h
a a

0.01

0.00
Coprostanol Cholesterol
Betasitosterol
Compound Name

Figure 2 The steroid profile of the Torrens catchment in June 2000

0.0075
W
a
[s t
0.0050
te
ro e
ls r
], f
m
a
ll
G
o
r
g
e

C
a
s
t
a
m
b
u
l
M
e
r
c
h
a
n
t
G
u
m
e
r
 a h
c a

0.0025

0.0000
Coprostanol Cholesterol
Betasitosterol
Compound Name

Figure 3 The steroid profile of sampling sites of the Torrens catchment in July 2000
A
0.006 i
[s
r
te
ro
0.004 P
ls
],
o
m
0.002
g/ r
t

a
y

i
r
P

o
r
t

J
u
n
e

A
i
r
P

o
r
t

J
u
l
y

M
 it u
c n
h e
a
m M
i
M t
a c
y
M h
it a
c m
h
a J
m u
l
J y

0.000
Coprostanol Cholesterol
Betasitosterol
Compound Name

Figure 4 The steroid profile of the sampling sites of the Patawalonga catchment in May-July 2000

Summer Samples
In the summer months (January and February 2001), the steroid
concentrations were generally lower compared to the winter results in the
Torrens catchment. The GC-MS performance was optimised and the results
became more quantitatively significant. In January (Figure 5), cholesterol
was found at the highest concentrations at all sites along the Torrens
catchment. The steroid profiles suggest dog and birds may contribute
significantly to the faecal contamination observed.

In February (Figure 6), the steroid pollutants in the Torrens catchment were
dominated by cholestanol. The detection of relatively high concentrations
of cholestanol suggests that the steroid contamination was non-faecal in
origin or it was not ‘freshly’ introduced. The steroid content usually
indicates faecal contamination if the coprostanol to cholestanol ratio is
greater than 0.4 (Leeming, 1996). The presence of relatively high cholestanol
might indicate that the steroids were resuspended from the river bed on
sample collection due to low water level, because as the most
thermodynamically stable stanol, cholestanol is always present in pristine
sediment (Nishimura, 1982).

Waterf all Gumeracha Merchant Castambul Gorge

stigmastanol

sitosterol

stigmasterol

cholestanol
 cholesterol

epicoprostanol

coprostanol

0 0.001 0.002 0.003

[steroid], mg/L
 Figure 5 The steroid profiles of the sampling sites in the Torrens catchment in January

w aterfall Gumeracha Merchant Sixth

stigmastanol Gorge

sitosterol

stigmasterol

cholestanol

cholesterol

epicoprostanol

coprostanol

0 0.001 0.002 0.003 0.004 0.005

[steroid], mg/L

Figure 6 The steroid profiles of sampling sites in the Torrens catchment in February.

.
The Mitcham site at the Patawalonga catchment was dry over the summer sampling
periods. The results shown in Figure 7 are from the Airport site only. Compared to the
Torrens catchment, the concentrations of sterols in this site are higher. The high
concentration of cholestanol detected suggests the steroid contamination may not be of
faecal origin (Nishimura, 1982).

stigmastanol February
January
sitosterol

stigmasterol

cholestanol

cholesterol

epicoprostanol

coprostanol

0 0.02 0.04
[steroid], mg/L

Figure 7. The summer steroid content of Patawalonga catchment (Airport site only).
The results shown in this paper are comparable with other environmental sampling
conducted previously in Australia (Ralph et.al., 1998; Leeming, 1996). The sterol
concentrations detected in this research ranged from 5 ng/L (coprostanol at the Airport in
July) to 34 g/L (stigmasterol at the Airport in January). Previous work on the Torrens
found 0.5 g/L (stigmasterol) to 13 g/L (brassicasterol) (Ralph et.al., 1998). Leeming
(1996) reported his finding on sterol content of lakes and creeks in the Wyong region of
New South Wales, to range from 2 ng/L (24-ethylcoprostanol) to 3.8 g/L (cholesterol)
(Leeming, 1996).

The large degree of variation shown in the winter results was due to reduced instrumental
sensitivity which has subsequently been addressed. These results present a qualitative
rather than quantitative indicator of pollution.

CONCLUSION

This research demonstrates an initial comparison between a number of sites on two

Adelaide metropolitan catchments in winter and summer. Three main sterols were
detected during winter sampling periods: coprostanol, cholesterol, and betasitosterol.
Cholesterol was found at high levels at all sites, coprostanol was identified at low
concentrations with two occasions when its level was higher than cholesterol, although not
significant (i.e. at the Waterfall Gully in May collection and at Gorge in July). Betasitosterol
was found to be inconsistent in terms of abundance and sampling sites.

These results may suggest that human, dogs, birds and cows may be responsible for the
faecal contamination. Since no ethyl coprostanol was detected, the pollution is probably
not herbivorous in origin but this remains to be established.

In the summer sampling periods, the steroid contaminants detected were coprostanol,
cholesterol, cholestanol, stigmasterol, betasitosterol, and stigmastanol. Amongst other
steroid compounds, cholesterol was the most frequently found at relatively high levels. The
steroid profiles found in the Torrens may indicate faecal contamination originating from
dog or birds, while in the Patawalonga the profiles suggest they may be non faecal steroid.

ACKNOWLEDGEMENTS

This research was partly funded by the South Australian Catchment Water Management
Boards. AusAID provided the scholarship for Iryanti Suprihatin.

REFERENCES

Bligh, E.G. and Dyer, W.M. (1959). A Rapid Method of Total Lipid Extraction and
Purification. Can.J.Biochem. and Physiol. 35, 911-917.

Düreth, S., Herrmann, R. and Pecher, K. (1986) Tracing Faecal Pollution by Coprostanol
and Intestinal Bacteria in an Ice-Covered Finnish Lake Loaded With Both Industrial and
Domestic Sewage. Water, Air, and Soil Pollution. 28: 131-149.

Ferezou,J., Gouffier, E., Coste, T., and Chevalier, F. (1978). Daily elimination of faecal
sterols by humans. Digestion, 18, 201-212.
Grimalt, J. O., Fernández, P., Bayona, J. M. and Albaigés, J. (1990) Assessment of Fecal
Sterols and Ketones as Indicators of Urban Sewage Inputs to Coastal Waters. Environ.
Sci. Technol. 24(3): 357-363.

Hatcher, P.G., and McGillivary, P.A. (1979). Sewage contamination in the New York Bight,
coprostanol as an indicator. Envir.Sci.Technol., 13, 1225-1229.

Jeng, W. L., Wang, J. and Han, B. C. (1996) Coprostanol distribution in Marine Sediments
Off Southwestern Taiwan. Environ. Pollut. 94(1): 47-52.

Leeming, R., Nichols, P. D., Ball, A. and Ashbolt, N. (1996) Using Faecal Sterols from
Humans and Animals to Distinguish Faecal Pollution in Receiving Waters. Wat. Res. 30
(12), 2893-2900.

Leeming, R. (1996). Coprostanol and related sterols as tracers for faecal contamination in
Australian aquatic environment. PhD thesis at Australia National University, Canberra.

Leeming, R., Nichols, P. D. and Ashbolt, N. (1998). Distinguishing sources of faecal

pollution in Australian inland and coastal waters using sterol biomarkers and microbial
faecal indicators. Research Report No. 204, Urban Water Research Association of
Australia, Melbourne.

McCalley, D. V., Cooke, M. and Nickless, G. (1980) Coprostanol in Severn Estuary

Sediments. Bull. Environ. Contam. Toxicol. 25: 374-481.

Murtaugh, J.J., and Bunch, R.L (1967). Sterols as a measure of faecal pollution. J. Wat.
Pollut. Control Fed., 39, 404-409.

Nichols, P. D., Leeming, R., Rayner, M. S. and Latham, V. (1993) Comparison of the
Abundance of Faecal Sterol Coprostanol and Faecal Bacterial Groups in Inner-shelf
Waters and Sediments Near Sydney, Australia. J. Chromatography 643 189-195.

Nishimura, M. (1982) 5isomers of Stanols and Stanones as Potential Markers of

Sedimentary Organic Quality and Depositional Paleoenvironments. Geochim. Cosmochim.
Acta 46 423-432.

Pierce, R. H. and Brown, R. C. (1984) Coprostanol Distribution from Sewage Discharge

into Sarasota Bay, Florida. Bull. Environ. Contam. Toxicol. 32: 75-79.

Ralph, J.P., Fallowfield,H., and Cromar, N. (1998). Sourcing Faecal Pollution in Torrens
and Patawalonga Catchment Waters and Sediments by Analysis of Faecal Steroids. A
technical report to the Torrens and Patawalonga Catchment Water Management Boards.
Unpublished.

Tabak, H.H., Bloomhuff, R.N., and Bunch, R.L (1972). Coprostanol : A positive tracer of
fecal pollution. Developments in Industrial Microbiology, 13, 296-307.

Venkatesan, M. I., Ruth, E. and Kaplan, I. R. (1986) Coprostanols in Antarctic Marine

Sediments : A Biomarker for Marine Mammals and non-Human Pollution. Marine Pollut.
Bull. 17(12): 554-557.
Walker, R. W., Wun, C. K. and Litsky, W. (1982) Coprostanol as an Indicator of Faecal
Pollution. CRC Crit. Rev. J. 12 (2), 91-112.

Writer, J. H., Leenheer, J. A., Barber, L. B., Amy, G. L. and Charpa, S. C. (1995) Sewage
Contamination in the Upper Mississipi River as Measured by the Faecal Sterol,
Coprostanol. Wat. Res. 29(6): 1427-1436.

Yde, M., De Wulf, E., De Maeyer-Cleempoel, S. and Quaghebeur, D. (1982) Coprostanol

and Bacterial Indicators of Faecal pollution in the Scheldt Estuary. Bull. Environ. Contam.
Toxicol. 28 129-134.

CONTACT
Dr. Nancy Cromar at Nancy.Cromar@flinders.edu.au