ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 3716– 3728

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Monitoring source water for microbial contamination:

Evaluation of water quality measures

Jeanine D. Plummera,, Sharon C. Longb

a
Department of Civil and Environmental Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA
b
Department of Soil Science and Wisconsin State Laboratory of Hygiene, 263 Soils Building, 1525 Observatory Drive,
Madison, WI 53706, USA

ar t ic l e i n f o abs tra ct

Article history: Watershed management programs often rely on monitoring for a large number of water
Received 13 January 2007 quality parameters to define contaminant issues. While coliforms have traditionally been
Received in revised form used to identify microbial contamination, these indicators cannot discriminate among
30 April 2007 potential contaminant sources. Microbial source tracking (MST) can provide the missing
Accepted 6 May 2007 link that implicates the sources of contamination. The objective of this study was to use a
Available online 22 May 2007 weight-of-evidence approach (land use analysis using GIS, sanitary surveys, traditional
Keywords: water quality monitoring, and MST targets) to identify sources of pollution within a
Watershed watershed that contains a raw drinking water source. For the study watersheds, statistical
Water quality analyses demonstrated that one measure each of particulate matter (turbidity, particle
Rhodococcus coprophilus counts), organic matter (total organic carbon, dissolved organic carbon, UV254 absorbance),
Bifidobacteria and indicator organisms (fecal coliforms, enterococci) were adequate for characterizing
Coliphages water quality. While these traditional parameters were useful for assessing overall water
Microbial source tracking quality, they were not intended to differentiate between microbial sources at different
locations. In contrast, the MST targets utilized (Rhodococcus coprophilus, sorbitol-fermenting
Bifidobacteria, and male-specific coliphages) pinpointed specific sources of microbial
pollution. However, these targets could not be used for routine monitoring due to a high
percentage of non-detects.
& 2007 Elsevier Ltd. All rights reserved.

1. Introduction monitoring plans are needed to identify pollutant sources

The multiple barrier approach to providing safe drinking
water includes source water protection, treatment, and
maintenance of distribution system integrity. Development
of watershed management programs relies on an under-
standing of the impact of watershed activities and land uses
on receiving water quality. Human impacts pose a threat to
maintenance of water quality due to contaminant introduc-
tion. While land acquisition can permanently avoid these
issues, increasing population pressures make this a non-
viable option in many watersheds. Therefore, watershed
and develop appropriate remediation strategies.
Monitoring programs often include a large number of water
quality parameters in order to fully capture contaminant
issues in a watershed. Physical and chemical parameters of
interest include pH, temperature, dissolved oxygen (DO),
organic matter, particulate matter, and specific conductance.
Indicator organisms, such as total and fecal coliforms, are the
most commonly used measures of microbiological quality.
However, the use of coliforms as an indicator organism has
been questioned because pathogens have been isolated when
there have been low concentrations of fecal coliforms, and

Corresponding author. Tel.: +1 508 831 5142; fax: +1 508 831 5808.
E-mail address: jplummer@wpi.edu (J.D. Plummer).
0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2007.05.004
 ARTICLE IN PRESS
WAT E R R E S E A R C H
many viruses and protozoan cysts are more resistant to
disinfection than coliforms (AWWA, 1999). Coliform enu-
meration does not have the ability to discriminate among
potential sources of microbial contamination (Sinton et al.,
1998). Microbial source tracking (MST) can provide the
missing link that implicates the sources of contamination.
MST can be library independent or library based, and
involves the measurement of targets that are highly asso-
ciated with fecal contamination from a certain source
(Simpson et al., 2002). Understanding the origin of fecal
pollution allows for appropriate, targeted remedial measures
(Gilpin et al., 2003). Based on target, four major approaches to
MST have emerged: chemical methods, phenotypic methods,
host-specific methods, and molecular-based methods.
This study focused on host-specific indicators, defined as
library-independent MST targets that are shed/associated
with a specific host species such as humans but no others.
Many host-specific MST methods can be conducted using
standard bacteriological methods, which tend to be reprodu-
cible across investigations and utilize equipment and ex-
pertise typically available at water utility laboratories. The
cost for performing these methods is relatively low compared
to other methods (Sinton et al., 1998). In addition, if a host-
specific method is culture based, it provides a measure of
viable indicators. If environmental stresses have not inacti-
vated the MST indicator, there is a potential that associated
pathogens may not have been inactivated either. However,
further studies are necessary to determine specific infection
risk associations between indicators and pathogens.
Successful implementation of source tracking has been
demonstrated in several US watersheds, including Holmans
Creek, Virginia; Kensico Reservoir, New York; and Homosassa
Springs, Florida (Dart and Wiggins, 2003; Alderisio et al., 1996;
Griffin et al., 2000). Worldwide, MST has been applied with
some success in locations such as Australia (Ashbolt, 2005;
Ahmed et al., 2006), Spain, and the United Kingdom (Payan
et al., 2005).
Among the host-specific or library-independent methods,
the fecal coliform/fecal streptococcus (FC/FS) ratio, which is
based on the hypothesis that different animals excrete fecal
coliforms and fecal streptococci in different quantities, has
been strongly criticized due to inconsistent relationships
between the ratio and pollution sources (Pourcher et al., 1991;
Howell et al., 1995; Anderson et al., 1997). Other organisms
reported to be host specific include Clostridium perfringens,
Bacteroides spp., and phages of Bacteroides fragilis (Savichtcheva
and Okabe, 2006). With the exception of Bacteroides spp.,
host-specific MST targets can be analyzed using culture-
based or molecular-based methods. This study used sorbitol-
fermenting Bifidobacteria, Rhodococcus coprophilus, and F+RNA
coliphage groups.
Sorbitol-fermenting Bifidobacteria can be used to identify the
presence of human fecal or domestic wastewater pollution
because of its higher specificity to human fecal sources
(Resnick and Levin, 1981; Mara and Oragui, 1983). As an
obligate anaerobe, it is unlikely to multiply in environmental
waters and has exhibited short survival periods in the extra-
intestinal environment (Mara and Oragui, 1983). Prior
research has demonstrated that sorbitol-fermenting Bifidoba-
cteria were consistently isolated only from human and
4 1 (200 7) 371 6 – 372 8 3717
human-impacted samples (Mara and Oragui, 1983). In Africa,
Jagals et al. (1995) detected sorbitol-fermenting Bifidobacteria
at densities of hundreds to thousands per 100 mL downstream
from human settlements with limited sanitation, with no
detection upstream. In Massachusetts, USA, sorbitol-fermenting
Bifidobacteria were detected in tributary samples from neighbor-
hoods served by aging septic systems and were absent from
areas distant from potential sewage inputs (Long et al., 2003).
Research has consistently shown that R. coprophilus is
present in the feces of herbivores such as cows, donkeys,
goats, horses, and sheep, but is absent from the feces of
humans (Mara and Oragui, 1981; Jagals et al., 1995; Long et al.,
2002; Gilpin et al., 2003). It has also been isolated from the
feces of poultry reared in close proximity to farm animals,
and from fresh waters and wastewaters contaminated by
animal fecal material (Mara and Oragui, 1981, 1983). Based on
these findings, R. coprophilus has been suggested as an
indicator of grazing animal contamination. The long environ-
mental persistence of R. coprophilus, up to 120 days, could be a
problem because its presence would not necessarily indicate
recent fecal pollution (Oragui and Mara, 1983). Thus, locating
sample sites upstream and downstream of potential animal
sources could overcome this limitation.
Male-specific (F-specific) coliphages have rarely been de-
tected in individual human feces samples (Dhillon et al., 1976;
Osawa et al., 1981; Havelaar et al., 1986, 1990). Havelaar et al.
(1986) found F+RNA phages in the feces of pigs, broiler
chickens, sheep, and calves, but not in the feces of dogs,
cows, or horses. The proportion of human and animal
populations that carry F-specific coliphages are the order of
a few percent; however, sewage, which is a composite of
materials from populations of typically 100 s of connections
or more, always tests positive for F-specific coliphages (Long
et al., 2005a; Blanch et al., 2006).
Pollution sources can be identified as human or non-human
by F+RNA subgroup classification via either serotyping or
genotyping. Both of these methods have demonstrated
promising results in the differentiation of F+RNA environ-
mental isolate subgroups (Havelaar et al., 1990; Hsu et al.,
1995; Sobsey et al., 1995). F+RNA coliphages may be classified
into four subgroups (Furuse, 1987): subgroups II and III tend to
be isolated from human feces and wastewater; subgroup I is
usually isolated from the feces of non-human mammals and
birds; and subgroup IV phages are of mixed origin but mostly
animal. These groupings can be applied as a general rule, but
are not absolute. Recent research demonstrated that the
associations of F+RNA coliphage groups applied to 103 human
wastewaters tested, but failed for 35% of the 82 animal
samples tested (Blanch et al., 2006). However, the samples
tested by Blanch et al. (2006) were composites and not
individual fecal samples. All samples classified as human
were in fact wastewater and samples classified as animal
included slaughterhouse effluent or farm slurries.
While MST targets may identify the origin of fecal inputs,
many of the targets are not suitable for routine monitoring
because of a high percentage of non-detects or because of the
relatively long analysis times required, up to 3 weeks for
some targets. For example, the culture-based method for
R. coprophilus requires a 10–14-day incubation of MM3 plates,
followed by 4 days of exposure to light for proper colony color
 ARTICLE IN PRESS
3718 WAT E R R E S E A R C H
development before plate counts can be completed (Long
et al., 2002). Therefore, the objective of this study was to use a
weight-of-evidence approach (land use analysis using GIS,
sanitary surveys, traditional water quality monitoring, and
MST targets) to identify sources of pollution within a
watershed from which raw drinking water is obtained. The
suite of tools was evaluated for its ability to assess land use
impacts in general and for defining specific microbial sources.
Both of these objectives were aimed at providing manage-
ment agencies with the information necessary to focus
remediation efforts on critical microbial sources in a
watershed.
2. Materials and methods
This study was aimed at identifying non-point source
pollution inputs in a watershed, with an emphasis on
microbial sources that may constitute a public health risk.
Sampling locations within a watershed were determined to
isolate surrounding land uses. For example, sample sites were
located both upstream and downstream of a horse pasture to
ascertain the pasture’s influence on water quality. Grab
samples were collected from a total of nine sampling sites,
discussed further below, and analyzed for physical, chemical,
and microbial water quality as well as MST targets. The
impacts of season and precipitation were determined by
developing a year-round sampling program that included
both wet and dry weather flows. The data were analyzed to
determine relationships amongst water quality measures,
seasonal and precipitation influences, and the utility of MST
targets to identify pollution sources.
2.1. Watershed sites
Field and laboratory data were gathered from two sub-basins
(Malagasco Brook and Beaman Pond Brook) in the Wachusett
Reservoir watershed located in central Massachusetts, USA,
which serves as part of the water supply for Boston and the
Table 1 – Percent land use characteristics of subwatersheds
Land use
Malagasco Brook
subwatershed
Forested and open land 74
Agriculture o1
Residential
Low density 16
Medium density 3 17
High density 6 10
Commercial and industrial 0 6
Open water and wetlands o1
Total area 227 ha (562 acres)
% Impervious 12.2
41 (2007) 3716– 3728
surrounding communities. Table 1 shows the distribution of
land uses within each subwatershed. Both subwatersheds
consist predominantly of forested and open land, followed by
residential development. Twenty-five percent of the Malagas-
co Brook sub-basin is under residential development, includ-
ing 6% identified as high-density residential (highest absolute
area in the Reservoir District). A large multi-unit housing
complex, comprised of more than 100 units in 24 buildings, is
the dominant development in the sub-basin. Beaman Pond
Brook sub-basin has the third highest percentage of residen-
tial development in the Reservoir District (predominantly
medium density) and as a result has the third highest
percentage of impervious surfaces as well. This is significant
as impervious surfaces reduce infiltration rates and increase
runoff of pollutants. As little as 20% impervious surfaces can
highly degrade receiving water quality (Schueler, 1994; Mallin
et al., 2000). Historical records from the Department of
Conservation and Recreation (DCR) showed periodic elevated
fecal coliform measurements in the tributaries of these sub-
basins. Numerous land use types could have contributed to
the elevated bacterial levels and thus a sampling plan was
initiated to identify microbial inputs to the subwatersheds.
Six sample sites were selected in the Malagasco Brook
subwatershed as described in Table 2. Land uses that were
isolated included the swampy headwaters, a plant nursery,
and a condominium complex. In the Beaman Pond Brook sub-
basin, the sampling locations were designed to isolate
impacts from domestic farm animals and from septic
systems (see Table 2). In addition to the GIS data on land
uses within each subwatershed, characteristics of each
sampling site were determined through a field reconnais-
sance and personal interviews with the DCR wildlife biologist
to determine the presence/absence of wildlife activities.
Because of the compact nature of the landscape in New
England, it is possible that sampling sites may be influenced
by multiple upstream land uses in these subwatersheds.
However, the sites selected for this study provide a realistic
dataset, and thus are appropriate for thorough analysis of the
utility of an MST toolbox for identifying microbial inputs.
Percent of land use
Beaman Pond Brook Wachusett watershed
subwatershed
55 70
5 6
4 7
4
1
1
3 9
151 ha (373 acres) 30,307 ha (74,889 acres)
19.6 7.6
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Table 2 – Sample sites included in field study
Site Site location
ID
Malagasco Brook sub-basin
MB6 Headwaters of Malagasco Brook
MB5 Intersection of Malagasco Brook and School
Street
MB4 Edgebrook Drive within condominium complex
MB3 Edgebrook Drive within condominium complex
MB2 East Temple Street, downstream of
condominium complex
MB1 Confluence with Wachusett Reservoir on East
Temple Street
Beaman Pond sub-basin
BP3 Route 110 on DCR property; upstream of private
horse farm
BP2 Route 110, downstream of private horse farm
BP1 Route 110, downstream of residence
2.2. Water sampling and testing program
Samples were collected from Malagasco Brook and Beaman
Pond Brook subwatersheds from October 2003 to October
2004, with a total of 11 sampling events over 13 months. All
sites were sampled at least once during every season and at
least once during wet weather conditions (at least 1.3 mm
(0.05 inches) of rain in the 48 h preceding sampling). In these
watersheds, this is the minimum amount of rain needed
before runoff might occur, depending on antecedent condi-
tions (Rees, 2004). The samples were collected aseptically,
starting from the most downstream location, in sterilized
high-density polypropylene, wide-mouth, screw-capped bot-
tles (Nalge Nunc International, Rochester, NY). The bottles
were immersed in the tributary and filled to capacity while
standing and holding the bottle on the downstream side. Care
was taken to avoid disturbing the sediments as much as
possible. Samples were collected, transported, and handled in
accordance with US EPA quality assurance and quality control
(QA/QC) standards (US Environmental Protection Agency (US
EPA), 2001a).
At each sampling site, water temperature, specific con-
ductance, and DO were measured in the field using YSI field
meters (YSI, Yellow Springs, OH) in accordance with Standard
Methods (APHA, AWWA and WEF, 1998). Samples were then
transported to the laboratory for additional analysis. Labora-
tory analyses included measurements of physical and che-
mical water quality, indicator organism concentrations, and
MST targets. All physical and chemical water quality para-
meters were measured in duplicate and average results are
reported. pH was determined with a Fisher Scientific AB15 pH
Meter (Fisher Scientific, Pittsburgh, PA) calibrated with three
standard buffer solutions on the day of use. Turbidity was
measured using a Hach Model 2100N Ratio Turbidimeter
(Hach Company, Loveland, CO) calibrated to sealed standards
the day of use. UV254 absorbance, a surrogate measure of
dissolved organic matter, was analyzed on samples filtered
4 1 (200 7) 371 6 – 372 8 3719
Distance from Site description
prior site
Heavily wooded; swampy
0.23 km (0.14 miles) Downstream of nursery; three single family homes
adjacent to site
0.50 km (0.31 miles) Dense residential development
0.48 km (0.30 miles) Dense residential development
0.35 km (0.22 miles) Moderate residential development
0.42 km (0.26 miles) Protected access; heavily wooded
Swampy; low flow
0.10 km (0.06 miles) Overgrown; refuse leaves and manure near
embankment
0.42 km (0.26 miles) Residential property with cesspool; lightly wooded
through a glass fiber filter in a UV–Visible Spectrophotometer
Cary 50 (Varian, Mulgrave, Victoria, Australia). Particle counts
in the 2–400-mm range were determined using a light blockage
particle counter (PC 2400 PS, Chemtrac Systems Inc., Nor-
cross, GA). Total and dissolved organic carbon (TOC and DOC)
samples were preserved with acid on the day of collection,
stored at 4 1C, and analyzed within 2 weeks on a Shimadzu
TOC 5000-A analyzer (Shimadzu Corporation, Kyoto, Japan)
calibrated with potassium hydrogen phthalate standards.
Fecal coliforms and enterococci were enumerated using
membrane filtration techniques, Standard Methods 9222D
and 9230C, respectively (APHA, AWWA and WEF, 1998). The
fecal coliform method used m-FC agar (Difco, Detroit, MI)
plates with rosalic acid addition and incubation at 44.5 1C for
2472 h in a Neslab Exacal waterbath (Neslab Instruments,
Inc., Newington, NH). Enterococci enumeration used ME agar
incubated for 48 h at 4170.5 1C, followed by transfer to Esculin
Iron agar plates. The Esculin Iron agar plates were retained at
room temperature for 20–30 min, incubated at 4170.5 1C for
20 min, and then enumerated by counting pinkish red
colonies that had developed a black precipitate on the
underside of the filter. For both microbial indicators, samples
were processed in triplicate, and QA/QC was maintained
through use of appropriate positive and negative controls.
Three MST targets were also enumerated for each sample:
R. coprophilus above 50 cfu/100 mL, sorbitol-fermenting Bifido-
bacteria, and F-specific RNA coliphage serotypes. Among these
MST targets, a standardized method has only been estab-
lished for enumeration and isolation of F-specific coliphages.
Coliphages were enumerated using EPA Method 1602, the
single agar layer method (US EPA, 2001b). Single plaque-
forming units were isolated and serotyped using an antisera
neutralization assay (Watanabe et al., 1967; Hsu et al., 1995;
Long et al., 2005a). R. coprophilus was quantified via membrane
concentration, resuspension, and spread-plating as detailed
in Arango (2000) and Long et al. (2002). Sorbitol-fermenting
Bifidobacteria were enumerated using membrane filtration and
 ARTICLE IN PRESS
3720 WAT E R R E S E A R C H
anaerobic incubation on human bifid sorbitol agar at 35–37 1C
for 4872 h, followed by biochemical confirmation (Arango,
2000; Long et al., 2002, 2005a, b).
2.3. Statistical analysis
Statistical analyses were conducted to determine the value of
each parameter in relating to microbial water quality. So as
not to assume normality of any of the parameters, correlation
analyses were performed using the non-parametric Spear-
man’s rank correlation. The observations for each water
quality parameter were ranked and correlation analysis was
performed using Analysis Tools with Microsoft Excel Profes-
sional Edition 2003. The individual water quality parameters
were compared to microbial measures to identify relation-
ships. Analysis was conducted for each subwatershed dataset
separately, and for the entire dataset to determine if relation-
ships were watershed-specific. Critical values of r were
determined for the appropriate degrees of freedom for each
dataset comparison. The r values larger than the critical
value of r at the 95% confidence level were considered
significant. These analyses were also used to determine
which water quality measures were of most value in a
watershed monitoring program focused on minimizing
microbial risk and which could be eliminated to minimize
labor and costs.
Two-way analysis of variance (ANOVA) was used to
determine differences between seasons and precipitation
conditions for each watershed and the entire dataset. In this
way, the significance of season, occurrence of precipitation,
and the interactions between season and precipitation on
sampling design could be assessed. First, monitoring data
were reviewed for normality. Fecal coliform and enterococci
data were log transformed to achieve normality. Owing to the
very few samples in which R. coprophilus, sorbitol-fermenting
Bifidobacteria, and coliphages were detected, these parameters
were excluded from ANOVA. Seasons were defined by the
solar calendar where spring begins at the end of March,
summer begins at the end of June, fall begins at the end of
September, and winter begins at the end of December.
Precipitation data at the Worcester Regional Airport in
Worcester, MA, for the day of and the days preceding
sampling were obtained from the National Climatic Data
Center. Rain measuring greater than 1.3 mm or 0.05 inches
was recorded for four of the 11 sampling dates. Statistical
analyses were performed using the Mixed Procedure in SAS
version 9.0 (SAS Institute, Cary, NC). For this analysis,
p-values less than 0.05 were considered statistically significant.
3. Results and discussion
3.1. Land use and water quality relationships
A summary of physical and chemical water quality including
means and ranges for each sampling site is provided in
Table 3. In the Malagasco Brook subwatershed, specific
conductance ranged from 30 to 554 mS with means for the
sample sites ranging from 42 to 311 mS. In comparison, the
values in the Beaman Pond Brook subwatershed samples
41 (2007) 3716– 3728
ranged from 387 to 805 mS, with means for the sample sites
ranging from 572 to 658 mS. In general, specific conductance
values averaged less than 200 mS in Malagasco Brook (with the
exception of MB1, the most downstream site), while the
Beaman Pond Brook samples averaged greater than 500 mS. In
both subwatersheds, specific conductance increased with
downstream distance. For example, the average specific
conductance increased from 42 at MB6 (swampy headwaters)
to 311 mS at MB1 (confluence with the reservoir). While there
is no US water quality standard for specific conductance, it is
used as a general indicator of dissolved solids. Thus,
increasing concentrations downstream were anticipated.
Also, higher levels in the Beaman Pond Brook subwatershed
are consistent with the presence of agricultural lands and
the greater percentage of impervious surfaces in this
subwatershed.
Three measures of organic matter were included in this
study: TOC, DOC, and UV254 absorbance. In natural waters,
excess organic matter can impart an oxygen demand that
lowers DO concentrations and affects fish and other aquatic
species. In drinking waters, organic matter serves as a
precursor for disinfection byproducts and may enhance the
survival of microorganisms (Scherer et al., 1992; Ferguson
et al., 1996). Readily degradable organic matter can also lead
to indicator regrowth (LeChevallier, 1990; Bouteleux et al.,
2005). In Malagasco Brook, TOC and DOC levels were highly
variable depending on site and season. A pattern of decreas-
ing TOC and DOC from MB5 (nursery) to MB1 (confluence with
the reservoir) was apparent. The highest TOC recorded was
51.9 mg/L at MB5 in October 2003, compared to a low of
1.41 mg/L at MB1 in August 2004. In general, concentrations
were approximately twice as high at MB6 (swampy head-
waters) and MB5 (nursery) in comparison to MB1. High con-
centrations at the upstream sites in the fall can be attributed
to deciduous leaf litter and vegetation from the woodlands,
while site MB5 was elevated (greater than 20 mg/L) year-round
due to the influence of the plant nursery that stockpiled
compost and mulch products in uncovered piles. Lower
downstream concentrations indicate degradation without
significant forest influence to further increase concentra-
tions. In Beaman Pond Brook, organic carbon concentrations
remained below 7 mg/L year-round. The highest level was
6.11 mg/L TOC at site BP3 (upstream of horse farm) in
November, and the lowest level was 0.94 mg/L at BP1 (down-
stream of residence) in January. In general, TOC tended to be
highest at BP3, while DOC was highest at BP1.
Turbidity is an aggregate measure of particulate matter in
water while particle counts provide exact numbers of
particles per milliliter in specific size ranges. In the Malagasco
Brook subwatershed, there was no apparent trend of parti-
culate matter with regard to site. All turbidities were below
5 ntu with three exceptions: MB5 (nursery), MB3 (town-
houses), and MB2 (downstream of townhouses) in August
2004. Particle counts were consistent with turbidity readings.
In the Beaman Pond Brook subwatershed, there was a clear
pattern of elevated particulate readings at BP3 (upstream of
horse farm) in comparison to the downstream sites. The
average turbidity level at BP3 was 8.22 ntu, compared to 2.37
and 1.06 ntu at BP2 and BP1, respectively. Site BP3 had a
shallow flow that was difficult to sample from without
Table 3 – Physical and chemical water quality

Site Temperature (1C) Dissolved oxygen (mg/L) Specific conductance (mS) pH

Mean Low High Mean Low High Mean Low High Mean Low High

Malagasco Brook subwatershed

MB6 9.4 1.4 11.2 2.89 2.30 4.08 42.0 30.0 58.0 5.9 5.6 6.1
MB5 9.9 0.2 18.6 5.03 2.90 8.20 88.3 56.0 110 6.0 5.3 6.4
MB4 7.8 0.0 18.1 10.5 7.64 12.8 170 71.0 341 6.7 6.1 7.3
MB3 7.6 0.1 18.2 10.2 7.23 12.6 152 79.0 217 6.7 6.2 7.0

WAT E R R E S E A R C H
MB2 7.6 0.1 18.9 11.3 7.82 14.2 150 79.0 215 6.9 6.5 7.3

ARTICLE IN PRESS
MB1 6.9 0.8 13.9 11.8 9.20 15.2 311 172 554 6.9 6.6 7.0

Beaman Pond Brook subwatershed

BP3 7.2 1.0 10.5 9.16 6.83 13.7 572 387 678 6.7 6.3 7.0
BP2 9.4 4.0 14.7 10.5 8.37 15.3 614 461 762 7.0 6.3 7.5

4 1 (200 7) 371 6 – 372 8

BP1 8.3 2.0 15.6 9.58 7.81 14.3 658 446 805 7.1 6.3 7.8

Site Total organic carbon (mg/L) Dissolved organic carbon (mg/L) UV254 (cm1) Turbidity (ntu) Particle counts (#/mL)

Mean Low High Mean Low High Mean Low High Mean Low High Mean Low High

Malagasco Brook subwatershed

MB6 35.4 19.4 50.8 33.7 18.4 46.9 1.6 0.80 2.3 1.96 1.00 3.67 4190 3010 5630
MB5 38.9 23.1 51.9 37.5 20.9 50.1 2.1 1.1 2.8 2.78 0.83 5.10 5050 2230 10,920
MB4 25.0 9.26 44.8 24.4 8.89 44.1 1.3 0.43 2.4 1.98 1.14 2.59 4140 2370 6050
MB3 22.7 8.00 42.1 21.8 6.16 41.3 1.1 0.30 2.2 3.21 1.01 8.27 5580 2520 11,620
MB2 21.8 9.67 39.5 21.4 8.31 39.5 1.1 0.41 2.1 2.63 1.23 5.99 4440 1750 9740
MB1 14.4 1.41 26.4 14.3 1.49 25.7 0.73 0.046 1.3 1.18 0.26 1.96 2610 1320 4450

Beaman Pond Brook subwatershed

BP3 3.21 2.11 6.11 1.13 0.81 1.36 0.029 0.017 0.051 8.22 4.60 17.8 5600 3920 6880
BP2 1.98 1.00 3.61 1.60 0.89 2.24 0.046 0.023 0.083 2.37 0.93 6.84 3800 2460 4760
BP1 1.94 0.94 2.83 1.90 0.98 2.89 0.044 0.021 0.080 1.06 0.52 2.83 2580 1370 3770

3721
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3722 WAT E R R E S E A R C H 41 (2007) 3716– 3728

disturbing the underlying sediments. Thus, the high particu-
late numbers are a result of the site characteristics rather
than reflecting true sediment loadings from runoff.
Traditional microbial indicators included fecal coliforms
and enterococci. These indicators can potentially identify
areas of concern within a watershed with regard to fecal
inputs. Table 4 summarizes fecal coliform and enterococci
densities for the nine sampling sites. In the Malagasco
Brook subwatershed, fecal coliform densities ranged
from 2 to 2459 cfu/100 mL. Enterococci ranged from 1 to
3233 cfu/100 mL. Average enterococci densities were highest
at the sites within (MB4 and MB3) and just downstream of
(MB2) dense residential development. This is consistent with
the fact that enterococci are more numerous in human-
related fecal sources than in animal fecal sources (Wheeler
et al., 2002).
The highest indicator densities all occurred during spring
and summer, and the lowest densities during the fall and
winter. While these data appear to implicate the townhouse
development as a major contributor to fecal pollution, the
individual data points cannot conclusively support this
assessment. Considering the six sampling events in Mala-
gasco Brook, the highest density of fecal coliforms was found
at MB6, MB4, MB3, and MB2 on different sampling dates. In
the Beaman Pond Brook subwatershed, while the average
densities of fecal coliforms and enterococci were highest at
BP2 (downstream of the horse farm), there was no consistent
pattern in densities. The highest densities of indicator
organisms were found at each of the three different sites
during different sampling events. Although the indicator
organism data provide preliminary evidence of fecal inputs,
more specific information was needed to identify predomi-
nant microbial sources.
Table 4 also contains the MST data for the Malagasco Brook
and Beaman Pond Brook subwatersheds. Average values are
not provided as numerous samples were below detection
limits. R. coprophilus is an MST target indicative of grazing
animal activity. R. coprophilus may be found in low numbers in
soils or on vegetation; therefore, a threshold level of 50 cfu/mL
has been suggested to be indicative of animal fecal pollution
(Long et al., 2002). In the Malagasco Brook subwatershed,
R. coprophilus was detected at sites MB5 (nursery), MB4
(townhouse development), and MB3 (townhouse develop-
ment). Overall, there were four detections among 28 possible
samples, however never above the threshold limit of 50 cfu/mL.
Thus, grazing animal influences are not significant in this
subwatershed and the low densities are likely due to runoff
from vegetated areas. In the Beaman Pond Brook subwatershed,
R. coprophilus was detected at every site at least once, with the
highest densities at BP2 (horse farm) in five of six sampling
events. Densities were above the threshold for five out of 16
samples: three times at BP2 (horse farm) and twice at BP1
(residential development). In all but one sampling event,
densities were higher at BP2 than BP1, indicating the major
contributing source of pollution at BP2 (horse farm), with some
survival downstream of this source.
In the Malagasco Brook subwatershed, sorbitol-fermenting
Bifidobacteria were found at site MB4 (townhouse develop-
ment), and in decreasing densities at each site downstream
from MB4 with densities of 46, 16, 11, and 4 cfu/100 mL,
respectively. The values shown in Table 4 were all from the
October 2003 sampling event: sorbitol-fermenting Bifidobacter-
ia were detected at only one other time (4 cfu/100 mL at
site MB4 in August 2004). These data indicate a human fecal
source upstream of MB4, with decreasing densities
downstream indicative of transport and partial survival in
agreement with Arango (2000) and Long et al. (2002). As
sorbitol-fermenting Bifidobacteria were never detected upstream
of the townhouses, this is clear evidence of human inputs from
the development. The development was constructed in the
early 1970s, with 24 buildings and 17 septic systems (including
leach pits, leach fields, and distributed leach fields). It is also
significant to note that sorbitol-fermenting Bifidobacteria were
not detected in the winter sampling events in Malagasco Brook,

Table 4 – Microbial water quality

Site Fecal coliforms (cfu/ Enterococci (cfu/ R. coprophilus Sorbitol-fermenting Coliphages

100 mL) 100 mL) (cfu/100 mL) Bifidobacteria (cfu/ (pfu/100 mL)
100 mL)

Mean Low High Mean Low High Low High Low High Low High

Malagasco Brook subwatershed

MB6 252 2 734 16 1 31 BDLa BDL BDL BDL BDL BDL
MB5 199 3 568 232 1 1061 BDL 13 BDL BDL BDL 0.5
MB4 400 26 1664 611 12 2758 BDL 13 BDL 46 BDL 0.5
MB3 427 4 1832 698 13 3233 BDL 13 BDL 16 BDL 8.5
MB2 542 4 2459 292 10 1240 BDL BDL BDL 11 BDL 1.5
MB1 71 2 175 38 7 85 BDL BDL BDL 4 BDL 1.0

Beaman Pond Brook subwatershed

BP3 70 42 115 305 33 1000 BDL 13 BDL 8 BDL BDL
BP2 269 13 1364 323 38 1636 BDL 320 BDL 6 BDL 1
BP1 84 6 283 46 25 103 BDL 53 BDL 5 BDL BDL

a
Below detection limits.
 ARTICLE IN PRESS
WAT E R R E S E A R C H
when DO levels were high (8.2–15.2 mg/L). As a strict anaerobe
that is inactivated in the presence of DO, this MST target may
have limited applicability in cold climates when DO saturation
values approach 15 mg/L in 0 1C freshwater (US Geological
Survey, 2007).
In Beaman Pond Brook, sorbitol-fermenting Bifidobacteria
were detected upstream of the horse stable (BP3) in the winter
of 2004 at 8 cfu/100 mL. In combination with the enterococci
data with densities up to 1000 cfu/100 mL at this site, an
upstream residential area may be contributing to fecal
contamination. In the summer, sorbitol-fermenting Bifidobac-
teria were found downstream of the horse stable (BP2).
Unfortunately, the upstream (BP3) location could not be
sampled during this event due to lack of flow; therefore, no
conclusion can be drawn regarding the exact source. In the
spring, sorbitol-fermenting Bifidobacteria were detected at
5 cfu/100 mL downstream of the residential area (BP1) and
not at any other site, indicating an input from septic systems.
Considering both subwatersheds, R. coprophilus was de-
tected in 15 of 44 samples (34%) and sorbitol-fermenting
Bifidobacteria were detected in eight of 44 samples (18%).
Therefore, these MST targets have limited utility for routine
monitoring and derive their value as MST indicators only. As
with many of the MST methods, a positive result can provide
definitive proof of the presence of a specific source; however,
a negative result may be inconclusive because of the nature of
the survival and presence of inhibitors in environmental
samples. The methods used in this study were based on
culturing R. coprophilus or sorbitol-fermenting Bifidobacteria.
Culture-based methods have some value in measuring viable
indicators and potential for infectious pathogen risk over
quantification of nucleic acid targets alone. It has been
demonstrated that MST targets (i.e. Bacteroides spp.) quanti-
fied using real-time, quantitative PCR have been detected
throughout an industrial process train even after peroxide
oxidation treatment, whereas culturable sorbitol-fermenting
Bifidobacteria were only detected before oxidation (Olstadt,
2006). Thus, this study demonstrates the strength of a toolbox
or weight-of-evidence approach when applying MST so that
multiple measures may be used to provide evidence of
microbial contaminant sources. This approach is also pro-
moted by the MST scientific community (Rochelle and
DeLeon, 2006).
F-specific coliphages were detected in the Malagasco Brook
subwatershed in seven out of 28 samples, at every site except
MB6. Densities were below 10 pfu/100 mL in all cases. The
high percentage of negative samples and the low levels of
coliphages in these subwatersheds reflect the relatively small
animal and human populations within these areas. Unlike
fecal coliforms which are universally shed in the feces of
warm-blooded animals, male-specific coliphages have only
been demonstrated to be shed by a small percentage of the
human and animal population. In one study out of Hong Kong
that analyzed 13 cow, 21 pig, and 22 human fecal samples, no
male-specific coliphages were detected (Dhillon et al., 1976).
In another study conducted in the United States, less than 2%
of human fecal samples tested were positive for male-specific
coliphages while up to 60% of cow feces contained male-
specific coliphages (Long et al., 2005a). In contrast, composite
samples (wastewater and animal waste slurries) are typically
4 1 (200 7) 371 6 – 372 8 3723
close to 100% positive for male-specific coliphages (Long et al.,
2005a; Blanch et al., 2006). Varying prevalence limits the use of
male-specific coliphages to an MST target, as opposed to a
general fecal indicator, and also supports its use as one of a
number of measurements in a toolbox approach.
Typing results demonstrated a predominance of F+DNA
phages (93%) compared to F+RNA phages (7%). Research has
shown that F+DNA coliphages tend to dominate F+RNA
phages in waters with contamination of human origin (Long
et al., 2005a). This was true of samples in and downstream of
the townhouse development in Malagasco Brook. In the
Beaman Pond Brook subwatershed, there was only one
positive sample, at HF2 (horse farm). In both subwatersheds,
serotyping of the F+RNA coliphages isolates was inconclusive.
The value in F-specific coliphage typing is that the associa-
tions of the different groups with human (groups II and III)
and non-human (groups I and IV) fecal contamination have
been demonstrated to apply globally and have remained
stable for over three decades as reported by Japanese
researchers and others (Watanabe et al., 1967; Osawa et al.,
1981; Furuse, 1987; Long et al., 2005a; Blanch et al., 2006).
However, in watersheds where microbial sources are diffuse,
and therefore detection and densities may be low, coliphage
typing should be part of an MST toolbox.
Overall, each group of water quality measures alone
(physical, chemical, microbial, and MST) was not adequate
to characterize the non-point impacts within the watersheds
studied. A total of 14 water quality measures were collected
over all four seasons under both baseflow and stormflow
conditions in combination with land use data. Studies of this
magnitude can cost local governments or utilities tens of
thousands of dollars. Statistical analyses were conducted to
determine which water quality measures provide the most
useful information and which ones could be eliminated from
a watershed management sampling scheme to save costs.
3.2. Correlation analysis
As described above, non-parametric correlation analysis was
conducted to determine whether significant relationships
exist among microbial measures and water quality. In
addition, analyses by the subwatershed dataset were con-
ducted to determine whether relationships among para-
meters are watershed specific. Table 5 summarizes the
correlations between water quality parameters that are
statistically significant at the 95% confidence level for
Malagasco Brook, Beaman Pond Brook, and the complete
dataset. Significance was determined by calculating critical
Spearman’s r values. If the calculated r value was greater
than the critical value, the correlation was judged to be
significant. Few relationships among parameters were noted
for the Beaman Pond Brook dataset. This reflects the smaller
number of data observations and lower statistical power.
When considering the microbial parameters, the more
traditional microbial indicators (fecal coliforms, enterococci,
and coliphages) were related when all data were considered
and when only the Malagasco Brook data were considered.
Previous studies have reported moderate to strong correla-
tions between coliphage and fecal coliform densities in
sewage and treated waters (r ¼ 0.69; Wentsel et al., 1982)
 ARTICLE IN PRESS
3724 WAT E R R E S E A R C H 41 (2007) 3716– 3728

Table 5 – Statistically significant Spearman correlations* between microbial indicators and other water quality parameters

Watershed Malagasco Brook Beaman Pond Brook Entire dataset

Critical r (95% level) 0.37 0.50 0.30

Parameter Parameter r- Parameter r- Parameter r-

Value Value Value

Fecal coliforms Temperature 0.84 Temperature 0.74 Temperature 0.83

pH 0.42 DO 0.62 Turbidity 0.42
TOC 0.44 TOC 0.55 Particle counts 0.42
DOC 0.45 Enterococci 0.77
UV254 0.44 Coliphages 0.51
Turbidity 0.58
Particle counts 0.53
Enterococci 0.80
R. coprophilus 0.44
Coliphages 0.44

Enterococci Temperature 0.77 No significant correlations Temperature 0.66

pH 0.61 pH 0.41
TOC 0.44 TOC 0.31
DOC 0.45 DOC 0.34
UV254 0.44 UV254 0.31
Turbidity 0.40 Turbidity 0.31
Fecal coliforms 0.80 Fecal coliforms 0.77
R. coprophilus 0.44 Coliphages 0.48
Coliphages 0.59

R. coprophilus Temperature 0.47 No significant correlations Specific conductance 0.50

Turbidity 0.42 TOC 0.54
Particle counts 0.42 DOC 0.46
Fecal coliform 0.44 UV254 0.46
Enterococci 0.44

Sorbitol-fermenting pH 0.51 No significant correlations pH 0.42

Bifidobacteria

Coliphages Temperature 0.54 No significant correlations Temperature 0.45

Specific cond. 0.39 Particle counts 0.37
Turbidity 0.45 Fecal coliforms 0.51
TOC 0.50 Enterococci 0.48
DOC 0.48
UV254 0.48
Particle counts 0.44
Fecal coliforms 0.58
Enterococci 0.59
R. coprophilus 0.41

Water quality Temp. and DO 0.58 Temp. and DO 0.72 Temp. and DO 0.58
TOC, DOC, UV 40.99 Temp. and DOC 0.51 TOC, DOC, UV 40.95
Turbidity and particle 0.88 DO and TOC 0.68 Turbidity and particle 0.83
counts counts
pH and carbon 40.45 TOC, DOC, UV 40.53 pH and carbon 40.48
pH and specific 0.80 Turbidity and particle 0.80 pH and specific 0.58
conductance counts conductance

Malagasco Brook (28 data points per parameter), Beaman Pond Brook (16 data points per parameter), and the entire dataset (44 data points per
parameter).

and surface waters (r ¼ 0.387; Castillo et al., 1988), and
consistently high levels of coliphages in domestic wastewater
(Calci et al., 1998). Traditional microbial indicators were also
related to particulate matter and inversely related to organic
matter. Therefore, once experience with monitoring within a
watershed has been established, rapid measures such as
turbidity and UV254 absorption could be used to trigger
intensive microbial analyses. The lack of relationships
between sorbitol-fermenting Bifidobacteria and traditional
microbial indicators, and between R. coprophilus and tradi-
tional microbial indicators (except in Malagasco Brook)
supports the conclusion that these two MST targets are not
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 371 6 – 372 8 3725

Table 6 – Two-way ANOVA results (p-values) for water quality differences by site, season, and precipitation

Difference Season Precipitation Season by precipitation

by interaction

Dataset Malagasco Beaman All Malagasco Beaman All Malagasco Beaman All
Brook Pond data Brook Pond data Brook Pond data
Brook Brook Brook
Parameter

Temperature 0.002 0.610 0.031 o0.001 0.033 0.001 * 0.307 0.899

Dissolved 0.128 0.153 0.067 0.012 0.038 o0.001 * 0.399 0.986
oxygen
Specific 0.546 0.270 0.346 0.007 0.008 o0.001 * 0.352 0.584
conductance
pH 0.414 0.045 0.056 0.001 0.012 0.640 * 0.086 0.398
TOC o0.001 0.204 0.005 0.001 0.581 0.901 * 0.556 0.242
DOC o0.001 0.085 0.006 0.002 0.388 0.726 * 0.779 0.260
UV254 o0.001 0.077 0.008 0.002 0.109 0.719 * 0.086 0.247
Turbidity 0.592 0.358 0.457 0.067 0.662 0.234 * 0.701 0.799
Particle 0.056 0.973 0.084 0.102 0.582 0.059 * 0.902 0.990
counts
Log (fecal 0.004 0.520 0.036 0.026 0.410 0.014 * 0.670 0.070
coliforms)
Log 0.116 0.722 0.059 0.588 0.735 0.594 * 0.586 0.618
(enterococci)
R. * * * * * * * * *
cophrophilus
Sorbitol- * * * * * * * * *
fermenting
Bifidobacteria
Coliphages * * * * * * * * *

*Could not be calculated because of lack of adequate data pairs for each variable case.

suitable for routine microbial monitoring. While fecal coli-
forms and enterococci can be anticipated to occur in
relatively high concentrations due to any type of fecal
contamination, sorbitol-fermenting Bifidobacteria would only
be present if the fecal matter is of human origin and absent
for fecal matter arising from grazing animals. A non-detect on
the MST target would provide limited information on
potential public health risk. Thus, both a traditional indicator
and an MST target are needed to fully assess the pollution
level and source.
When considering an overall monitoring plan, a number of
correlations were significant for the Malagasco Brook subwa-
tershed and combined dataset. Microbial indicator densities
(fecal coliforms, enterococci, and coliphages) were all related,
while the measures of organic matter (UV254 absorbance, TOC,
and DOC) were highly related, and measures of particulates
(turbidity and particle counts) were also highly related. These
correlations between water quality measures are not likely
watershed specific. Therefore, to optimize information and to
minimize labor and expenditure of resources, a water manage-
ment agency might choose to measure only fecal coliforms,
UV254 absorbance, and turbidity.
3.3. Analysis of variance
A two-way repeated-measures ANOVA, which accounted for
correlation over time within the same site, was used to
determine the significance of season and precipitation. The
analysis was completed for every water quality parameter
that was measured. The data were analyzed separately for
each subwatershed and for the combined dataset. p-Values
for the ANOVA results are summarized in Table 6, with
statistically significant differences at the 95% confidence level
indicated in italics. Once again, the statistical power within
the Beaman Pond Brook dataset was relatively low; therefore,
few statistically significant relationships were observed. The
numbers of replicate analyses for each sampling site within
each watershed were relatively small (6 and 5, respectively)
and thus conferred low statistical power. Sample sites were
selected to hydrologically isolate specific land uses. Thus,
water quality at each sampling site was assumed a priori to be
different. Owing to these factors, further statistical analyses
for differences in water quality by site were not conducted.
For seasonal analysis, five, one, and five water quality
parameters were statistically different by season for Mala-
gasco Brook, Beaman Pond Brook, and the combined dataset,
respectively. Temperature was demonstrated to be affected by
season, which is as expected, and therefore is a benchmark as
to whether analyses were conducted properly. All three
measures of organic carbon were statistically affected by
season. These parameters are affected by snow cover and
deciduous tree life cycles. Once again, these parameters
indicate that analyses were conducted properly. Considering
the indicator organisms, fecal coliforms were different by
 ARTICLE IN PRESS
3726 WAT E R R E S E A R C H
season for the Malagasco Brook dataset and the combined
dataset. In the climate of New England, seasonal variations in
these microorganisms would be expected. In particular,
increased fecal indicator densities in summer months are
impacted by both increased inputs to surface waters and
increased survival times. These factors are influenced by
temperature, incident solar radiation, livestock management
practices, manure handling practices, and/or land store
recovery (Howell et al., 1995; Hunter et al., 1999; Edwards et al.,
2000). This supports the need to conduct monitoring in each of
the four seasons for watershed management purposes.
ANOVA was also conducted to determine differences in
water quality parameters during dry versus wet weather
flows. For this analysis, a rain event was defined as 1.3 mm
(0.05 inches) or more of precipitation in the 48 h preceding
sampling based on the advice of a hydrologist (Rees, 2004).
Eight, four, and four water quality parameters were statisti-
cally different by precipitation for Malagasco Brook, Beaman
Pond Brook, and the combined dataset, respectively. Tem-
perature, DO, and specific conductance were significantly
related to precipitation for all three datasets analyzed. pH was
significant when each subwatershed was considered indivi-
dually, but not in the combined dataset. This indicates that
constituents that affect pH behave in different manners
within watersheds. Organic carbon varied significantly with
precipitation in Malagasco Brook. This watershed is predo-
minantly forested land (74%) as compared to Beaman Pond
Brook (55%; Table 1). Therefore, greater amounts of organic
matter can be mobilized with runoff. Finally, fecal coliforms
were statistically affected by precipitation when considering
Malagasco Brook and the combined dataset. Increased
streamflows in other studies have demonstrated increases
in bacterial content (Kay and McDonald, 1980; Ferguson et al.,
1996). These results indicate that in order to capture true
microbial indicator loading within a watershed, monitoring
should include capturing both baseflow and stormflow
conditions.
Finally, the interaction between season and precipitation
was evaluated using ANOVA analysis—however, this was only
done for observations taken in spring and fall, since there
were no dates with precipitation in winter or summer.
Malagasco Brook was excluded as there were no dry observa-
tions in spring. There was no interaction observed in any
model for any response. The implications for the design of
watershed management programs are that monitoring
schemes need to include collection of samples over all
seasons and a variety of precipitation conditions.
4. Conclusions
In this study, traditional water quality parameters were useful
for assessing overall water quality, but were not designed to
differentiate between microbial sources. Statistical analyses
demonstrated that one measure each of particulate matter
(turbidity, particle counts), organic matter (TOC, DOC, UV254
absorbance), and indicator organisms (fecal coliforms, enter-
ococci) were adequate for characterizing water quality.
Measuring multiple parameters in each category did not
provide any added information. Therefore, drinking water
41 (2007) 3716– 3728
utilities would only need to include one of each of these
measures in their monitoring plan to keep budgets as
low as possible without compromising the value of infor-
mation gained. However, in order to fully understand
variability in microbial loadings within a watershed, monitor-
ing programs should include schemes to collect and analyze
samples each season and under different precipitation
conditions.
The three culture-based, library-independent MST targets
(R. coprophilus, sorbitol-fermenting Bifidobacteria, and coli-
phages) were selected for inclusion in this study based on
the non-point sources indicated by land use analyses
(residential development served by aging septic systems, a
plant nursery, and a horse pasture). Collection and analysis of
MST targets from samples over a number of hydrologic and
seasonal conditions is suggested for source water protection.
Diffuse and transient microbial sources in the study wa-
tershed often resulted in non-detects for the MST targets.
Negative results for a single sampling time should be viewed
as inconclusive; however, repeated negative results over time
under different hydrologic conditions can provide proof that a
source is being adequately controlled. On the other hand, a
positive result provides proof of the contribution of a specific
source. Therefore, a toolbox approach incorporating a num-
ber of sampling campaigns is necessary since a single non-
detection of viable indicators may result in erroneous
interpretations. The MST toolbox employed in this study
was useful for microbial source identification, but could not
be used as the only tool in a watershed management
program. Therefore, this study supports the integration of
land use analysis, traditional monitoring, and a suite of MST
targets to fully characterize non-point source and microbial
pollution in a drinking water subwatershed.
Acknowledgments
This work was funded in part by the Massachusetts Depart-
ment of Conservation and Recreation (DCR). The conclusions
and recommendations are those of the authors and not
necessarily those of DCR. We would like to thank the students
who assisted in sample collection and analysis: Malia Aull,
Tiffany Tauscher, and Craig Hebert. Special thanks to
Nicholas S. Keuler of the College of Agriculture and Life
Sciences Statistical Help Center, University of Wisconsin, for
his statistical advice.
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