Estuanes and Coasts Vol. 30, No. 3, p.

551-561 June 2007

Improving Fluorometry as a Source Tracking Method to Detect

Human Fecal Contamination
PETER G. HARTEL 1'*, JENNIFER L. MCDONALD 2, LISA C. GENTIT 2, SARAH N . J . HEMMINGS 1,
KAREN RODGERS 1, KATY A. SMITH 2, CAROLYN N. BELCHER 2, ROBIN L. KUNTZ 1,
YAmTZA RaVEP.A--Tom~ES3, ERNESTO OTERO~, and EDUAtmO C. SCHRODE~4

1 Department of Crop and Soil Sciences, 3111 Plant Sciences, University of Georgia, Athens, Georgia
30602-7272
2 Marine Extension Service, 715 Bay Street, University of Georgia, Brunswick, Georgia 31520-4601
3 Department of Marine Sciences, University of Puerto Rico, Mayagiiez, Puerto Rico 00681
4 Department of Agronomy and Soils, College of Agricultural Sciences, University of Puerto Rico,
Mayagiiez, Puerto Rico 00681
ABSTRACT: In a continuing effort to develop inexpensive source tracking methods to detect human fecal contamination in
environmental waters, targeted sampling was combined with fluorometry. Targeted sampling works by identifying h o t s p o t s
of fecal contamination through multiple samplings over ever-decreasing distances. Fluorometry identifies human fecal
contamination by detecting optical brighteners, primarily from laundry detergents. Because organic matter fluoresces and
interferes with fluorometry, two locations were chosen for sampling: waters relatively low in organic matter at Mayagiiez Bay,
Puerto Rico, and waters relatively high in organic matter at St. Simons Island, Georgia. In Puerto Rico, targeted sampling and
fluorometry quickly and easily identified two hot spots of human fecal contamination in the Yagiiez River, which flows
through the city of Mayagiiez. Another source tracking method, detection of the esp gene, confirmed their human origin. O n
St. Simous Island, targeted sampling and fiuorometry identified two h o t s p o t s of potential human fecal contamination.
Detection of the esp gene confirmed the human origin of one site but n o t the other, most likely because background organic
matter fluorescence interfered with fhiorometry. A separate experiment showed that adding a 436-um emission filter to the
fluorometer reduced this background fluorescence by > 50%. With the 436-nm Filter in place, another sampling was
c o n d u c t e d o n St. Simons Island, and the second h o t s p o t was identified as fecal contamination from birds. As long as the
fluorometer was equipped with a 436-nm filter and organic matter c o n c e n t r a t i o n s w e r e c o n s i d e r e d , targeted sampling
combined with fluorometry was a relatively inexpensive method for identifying human fecal contamination in water.

Introduction samplings over ever-decreasing distances. Because

Source tracking identifies the host origin of fecal
contamination in water. T h e host origin may be
identified directly by various phenotypic and geno-
typic methods, or indirectly by various chemical
methods. Each of these methods has its advantages
and disadvantages, and selection of a particular
m e t h o d is usually based on the method's cost,
reproducibility, discriminatory power, ease of in-
terpretation, and ease of performance (Simpson et
al. 2002; USEPA 2005).
O u r effort has concentrated primarily on de-
veloping inexpensive methods because most of the
c o m m u n i t i e s in which we work c a n n o t afford
expensive methods. One way we have reduced the
cost was by developing targeted sampling (Hartel
2002; Kuntz et al. 2003). This m e t h o d works like the
children's game of hot and cold, where hot spots o f
fecal c o n t a m i n a t i o n are identified by multiple
*Corresponding author; tele: 706/542-0898; fax: 706/542-
0914; e-mail: pghartel@uga.edu
the m e t h o d considers flow conditions and requires
each sampling be conducted in just one day, the
m e t h o d minimizes bacterial changes with flow
(Hartel et al. 2004), time (Jenkins et al. 2003),
geography (Hartel et al. 2002), and animal diet
(Hartel et al. 2003). The m e t h o d also requires local
knowledge to help identify suspicious sites. Tar-
geted sampling has been successfully c o m b i n e d with
an expensive genotypic source tracking method,
ribotyping, in estuarine waters during calm (base-
flow) conditions (Kuntz et al. 2003), and combined
with several inexpensive source tracking methods in
marine and estuarine waters during both calm and
stormy (stormflow) conditions (McDonald et al.
2006). Although targeted sampling can identify
many sources of fecal contamination based on
c o m m o n sense (e.g., cattle standing in the water),
it cannot otherwise identify or confirm sources
specifically.
One relatively inexpensive source tracking meth-
od to identify h u m a n sources of fecal contamination
specifically is fluorometry. Fluorometry identifies

© 2007 Estuarine Research Federation 551

552 P . G . Hartel et al.
TABLE 1. Likely cause of fecal contamination w h e n certain
n u m b e r s of fecal bacteria a n d levels of optical b r i g h t e n e r
(fluorometric units) are observed. High n u m b e r s of fecal
bacteria are defined as exceeding the regulatory llmlt for the
particular location.
Fecal bacteria numbers Opucal brightener Likely cause
High High Failingonsite waste disposal
system or leaking sewer pipe
High Low Human (e.g., outhouse) or
other warm-blooded animals
Low High Gray water in storm water sys-
tem
Low Low No evidence of fecal contam-
ination
h u m a n fecal c o n t a m i n a t i o n by detecting optical
b r i g h t e n e r s (also called f l u o r e s c e n t w h i t e n i n g
agents) in water. Optical brighteners are com-
p o u n d s a d d e d primarily to laundry detergents,
and because these brighteners emit light in the
blue range (415-445 n m ) , they c o m p e n s a t e for
undesirable yellowing in clothes (Kaschig 2003).
In the United States, 97% of laundry detergents
contain the optical brighteners DSBP (4,4'-bis(2-
sulfostyry) biphenyl) or DAS1 (4,4'-bis[ (4-anilino-6-
morpholino-1,3,5-triazin-2-yl) a m i n o ] stilbene-2,2'-
disulfonate; H a g e d o r n et al. 2005). Because house-
hold p l u m b i n g systems mix effluent f r o m washing
machines and toilets together, optical brighteners
are associated with h u m a n sewage in septic systems
and wastewater t r e a t m e n t plants. In o r d e r to use
optical brighteners to properly detect h u m a n fecal
contamination, they must be c o m b i n e d with counts
of fecal bacteria (Table 1). Effluent f r o m a wastewa-
ter t r e a t m e n t plant contains optical brighteners
(Poiger et al. 1998), but unless the wastewater
treatment plant fails to disinfect its effluent prop-
erly, it will not contain viable fecal bacteria, and the
plant does not r e p r e s e n t a significant source of
h u m a n fecal contamination.
Results of studies that have c o m b i n e d fluorometry
with counts of fecal bacteria are contradictory.
Kerfoot and Skinner (1981) were able to detect
plumes of septic tank leachate in several U.S. lakes
by c o m p a r i n g fluorometric values with n u m b e r s of
fecal coliforms. H a g e d o r n et al. (2005) studied
a n u m b e r of rivers, bays, and estuaries in Virginia
and New York. T h e y were able to confirm the
h u m a n origin of their high fecal bacterial counts
a n d positive f l u o r o m e t r i c values with a n o t h e r
genotypic source tracking method, pulsed field gel
electrophoresis. McDonald et al. (2006) c o n d u c t e d
a limited study c o m b i n i n g targeted sampling and
fluorometry during calm conditions on Sea Island,
Georgia. O n e site containing a strong fluorescent
signal and high n u m b e r s of fecal enterococci was
confirmed positive for h u m a n c o n t a m i n a t i o n with
other relatively inexpensive source tracking meth-
ods.
T h e r e are instances where there was no correla-
tion b e t w e e n f l u o r o m e t r y a n d counts of fecal
bacteria. Close et al. (1989) tested 86 samples from
67 New Zealand wells and observed no correlation
(r = 0.11) between fluorometry and counts of fecal
coliforms. Wolfe (1995) also observed no significant
correlation between f l u o r o m e t r y and counts of
either fecal coliforms or fecal streptococci in South
Carolina (U.S.) waters.
T h e m o s t likely reason for these contradictory
results is organic matter. It has long been known
that o r g a n i c m a t t e r in w a t e r fluoresces w h e n
exposed to ultraviolet (UV) light (Kalle 1949).
Fluorescence can serve as a proxy for total organic
c a r b o n (Smart et al. 1976). T h e p r o b l e m o f
b a c k g r o u n d organic matter fluorescence has led
some investigators to conclude that fluorometry is
of limited value as a source tracking m e t h o d
(Boving et al. 2003). T h e p r o b l e m of background
organic matter fluorescence m u s t be resolved if
fluorometry is to be used as a source tracking
m e t h o d , especially for the southeastern United
States, w h e r e waters are c h a r a c t e r i z e d by low
t o p o g r a p h i c relief, extensive floodplain swamps,
and rich organic b o t t o m sediments.
O n e solution to reducing this b a c k g r o u n d organ-
ic m a t t e r fluorescence may be to change the
excitation:emission filters of the f l u o r o m e t e r to
e n h a n c e its resolution for certain wavelengths.
Because organic matter in environmental waters
has b r o a d b a n d , featureless emission spectra (Chen
and Bada 1992) and the emission spectra of optical
brighteners are maximal in the 415-nm to 445-nm
range, restricting the emission filter to within this
wavelength range may e n h a n c e the resolution of
the f l u o r o m e t e r to detect optical brighteners.
We c o n d u c t e d a study c o m b i n i n g targeted sam-
pling a n d fluorometry in two locations: Puerto Rico,
where organic matter concentrations in the water
were relatively low and were unlikely to affect
fluorometric results, and Georgia (U.S.), where
organic matter concentrations in the water were
relatively high and were likely to affect fluorometric
results. In an e x p e r i m e n t to reduce b a c k g r o u n d
organic matter fluorescence, we tested two fluorom-
eters, identical except one was e q u i p p e d with a 410-
n m to 600-nm emission filter and the other was
e q u i p p e d with a m o r e restrictive 436-nm emission
filter. We c o n f i r m e d the f l u o r o m e t r i c data by
isolating Enterococcus species f r o m the water, specif-
ically d e t e r m i n i n g the p e r c e n t a g e of Enterococcus
faecalis and detecting the esp (enterococcal surface
protein) gene in Enterococcus faecium isolates. High
percentages (--> 30%) of Ent. faecalis are usually
associated with h u m a n s a n d s o m e wild birds

(Wheeler et al. 2002; Kuntz et al. 2004), and this
m e t h o d has been tested successfully in environmen-
tal waters (e.g., McDonald et al. 2006). The esp gene
in Ent. faecium enrichments is detected in 97% o f
h u m a n sewage and septic samples and not in any
n o n h u m a n animal feces (Scott et al. 2005). This
m e t h o d has also been tested successfully in envi-
r o n m e n t a l waters (e.g., Francy et al. 2006).
Me~o~
TARGETED SAMPLING LOCATIONSAND DATES
T h e first sampling location was Mayagfiez Bay,
which is on the western e n d of Puerto Rico. The bay
is d o m i n a t e d by Mayagfiez, a modestly sized city
(population 98,000). The bay has a history of fecal
contamination problems (Otero 2002). Organic
matter concentrations in the water of the bay are
low ( < 2 mg 1-1; Otero unpublished data).
In addition to using local knowledge to identify
suspicious locations and considering flow condi-
tions, targeted sampling requires multiple sam-
plings over ever-decreasing distances. To satisfy
these requirements, three baseflow samplings were
c o n d u c t e d in Puerto Rico. The first sampling was
c o n d u c t e d in Mayagfiez Bay on February 2, 2005,
and 30 water samples were obtained. T h e r e was
insufficient time to resample all fecal hot spots (e.g.,
Majagual and Sabalos Creeks), and a second sam-
pling was d o n e only in one location, the Yagfiez
River, a 20.8-km river that originates in the Urayo~in
Mountains and flows t h r o u g h the city of Mayagfiez
before emptying into Mayagfiez Bay. The second
sampling was conducted from the m o u t h of the
Yagfiez River, through the city of Mayagfiez, to
a point 3.75 km upstream. To the greatest extent
possible, resampling included all tributaries, pipes,
and suspicious locations. U n d e r these conditions,
24 water samples were obtained, but no fluoromet-
ric data. This sampling identified two hot spots o f
fecal contamination, one near the m o u t h of the
river and the other from a pipe 3.3 km upstream.
T h e third sampling was c o n d u c t e d on February 4,
2005. On this sampling, duplicate water samples
were obtained at each of the two hot spots and were
analyzed for fluorometry, numbers of Escherichia coli,
and presence o f the esp gene.
T h e second sampling location was St. Simons
Village on the southern e n d of St. Simons Island,
Georgia. St. Simons, o n e o f Georgia's barrier
islands, is approximately 17 km long and 4 km wide
at its widest point. Since April 2004, when the
G e o r g i a E n v i r o n m e n t a l P r o t e c t i o n Division
switched from fecal coliforms to fecal enterococci
as an indicator of fecal contamination, the beach at
the southern end of the island has had chronic
beach advisories (Cheney personal communica-
Improving Fluorometry for Fecal Detechon 553
tion). Dissolved organic carbon (DOC) concentra-
tions average 5 to 28 m g C 1-1 in these waters
(Alberts and Takfics 1999).
Three baseflow samplings were conducted on St.
Simons. The first sampling was on July 3, 2005, and 66
water samples were obtained. Fluorometric values
were obtained with each water sample. In addition to
the beach and storm drains along the southern
portion of the island, a creek draining the interior
marsh 6f the island, Postell Creek, was also sampled.
The second sampling was conducted on upper
Postell Creek on April 27, 2006, and 23 water samples
were obtained. T h e sampling was conducted during
an ebbing spring tide because these sampling
conditions yield the highest numbers of fecal bacteria
(Boehm and Weisberg 2005). The water from one
storm drain had an unusual greenish cast to it and
local residents were queried about this color. It was
determined that the color originated from a local
business using Aquashade (Applied Biochemists,
Germantown, Wisconsin) to control aquatic plant
growth in a pond. The active ingredients of Aqua-
shade are two herbicidal dyes: Acid Blue 9 and Acid
Yellow 23. Because the concentration of Aquashade
in the water was unknown, this p r o d u c t was pur-
chased locally and serially diluted from 10 -1 to 10 -6
before testing it for its fluorescence.
The first sampling suggested h u m a n fecal con-
tamination in a storm drain in St. Simons Village
and county did maintenance work on the storm
drain. After the maintenance work was completed,
a third sampling, just off the storm drain, was
conducted on J u n e 12, 2006. A total o f 19 samples
was obtained.
SITE COORDINATESAND TOTAL ORGANIC
CARBON ANALYSES
At each sampling point, site coordinates were
taken with a global positioning system device
(Model GPSMAP 175, Garmin International Inc.,
Olathe, Kansas). Sampling site coordinates were
converted to ArcView 3.2 shapefiles and incorporat-
ed into a geographic information system database.
Total organic carbon (TOC) was d e t e r m i n e d by the
h i g h - t e m p e r a t u r e combustion m e t h o d (Method
5310B; Clesceri et al. 1998).
SAMPLE COLLECTION AND ESTIMATINGNUMBERS OF
FECAL BACTERIA
Duplicate water samples were collected aseptically
in stand-up, 120-ml (4 oz.) Whirl-Pak bags (Nasco,
Modesto, California) and were placed on ice in
a cooler. Samples for estimating numbers of fecal
bacteria were processed within six hours.
U n d e r normal circumstances, fecal enterococci
are the fecal indicator bacteria of choice for marine
554 P.G. Hartel et al.
and estuarine waters (Clesceri et al. 1998). In
Puerto Rico, the Colilert system (for estimating
n u m b e r s of E. coli; IDEXX Laboratories, Westbrook,
Maine) was used for all water samples instead of the
Enterolert system (for estimating n u m b e r s o f fecal
enterococci; I D E X X Laboratories) b e c a u s e the
Enterolert system h a d an unacceptably high num-
ber of false-positive wells (Hartel et al. 2005). Only
Puerto Rican samples processed with the Enterolert
system were used for the detection of the esp gene
(see next section) at the two hot spots on the
Yagfiez River. In Georgia, the Enterolert system was
used exclusively.
To estimate n u m b e r s of E. coli or fecal entero-
cocci, water s a m p l e s were diluted with sterile
distilled water to 10 -1 and 10 ~ in sterile manufac-
turer-supplied polystyrene bottles and were treated
as per the m a n u f a c t u r e r ' s instructions. T h e n u m b e r
of positive wells was converted to a most-probable-
n u m b e r (MPN) value based on the dilution factor
and manufacturer-supplied MPN tables.
ENTEROCOCCUS SPECIATION AND DETECTION OF THE
ESP GENE
To obtain Ent. faecalis and Ent. faecium isolates,
positive Enterolert Quanti-tray wells were obtained
from selected locations in Puerto Rico a n d Georgia.
The two locations in Puerto Rico were a site near
the m o u t h of the Yagiiez River, and a drainage pipe
3.3 km upstream f r o m the m o u t h o f the river.
During the first sampling in Georgia, three loca-
tions were sampled: u p p e r Postell Creek, a p o n d
f r e q u e n t e d by ducks n e a r Lord Avenue, a n d a storm
drain near Beachview and Mallory Streets identified
by local residents as one possible site of h u m a n fecal
contamination. During the second sampling, three
locations were sampled: u p p e r Postell Creek, a storm
drain n e a r a housing d e v e l o p m e n t (The Grands),
and a n o t h e r storm drain (near East Beach Cause-
way). During the third sampling, the same locations
as the first sampling were resampled except the
p o n d sample was replaced with a Virginia Street
storm drain sample. This storm drain empties the
p o n d into the ocean.
Fecal enterococci were obtained as described by
McDonald et al. (2006) and were speciated according
to Manero and Blanch (1999). Although testing for
the esp gene only requires an enterococcal enrich-
m e n t (Scott et al. 2005), between 50 a n d 100 isolates
of Ent. faecium were specifically identified f r o m each
site and were spotted on a 0.45-gm m e m b r a n e
contained on a 5-cm Petri plate with m E I agar
(Becton-Dickinson, Sparks, Maryland). Because only
3% of Ent. faecium isolates contain the esp gene,
obtaining this large n u m b e r of isolates increased the
possibility of obtaining an isolate with this gene,
assuming it was present. Plates were incubated at 41
-+ 0.5°C for 24 h and were sent by overnight mail to
Biological Consulting Service o f N o r t h Florida
(Gainesville, Florida) for detection of the esp gene.
FLUOROMETRY
Fluorometry was c o n d u c t e d with a field fluorom-
eter (Model 10-AU-005, T u r n e r Designs, Sunnyvale,
California) set to detect long wavelength optical
brighteners (excitation wavelength, 360 nm; emis-
sion wavelength, 410-600 n m ) as described by the
manufacturer. Water samples were refrigerated in
the d a r k at 4°C and were processed within 24 h.
Each water sample was analyzed in the discrete
m o d e at r o o m t e m p e r a t u r e (20-25°C) and was read
within 30 s to avoid heating effects by the UV lamp
in the fluorometer. The i n s t r u m e n t was checked
with known concentrations of a commercial liquid
laundry d e t e r g e n t (Tide, P r o c t e r and Gamble,
Cincinnati, Ohio) to ensure that the f l u o r o m e t e r ' s
low limit of detection was stable. This detergent was
selected because it is c o m m o n l y used and it contains
the optical brightener DAS1 (Hartel unpublished
data). At a sensitivity setting of 50%, a fluorometric
value o f 100 was equal to 100 m g of commercial
d e t e r g e n t 1-1 of distilled water. H a g e d o r n et al.
(2003) suggested that any site with an optical
density > 100 was positive for optical brighteners,
and this limit was the one selected to identify sites of
concern in Georgia waters. This limit was too high
for Puerto Rican waters because they contained
such low a m o u n t s of organic matter, and the
calibration setting was c h a n g e d such that 50 mg of
c o m m e r c i a l detergent 1-1 of local seawater was equal
to 100 fluorometric units, a n d after conducting
a preliminary sampling, an arbitrary limit of 30
fluorometric units was chosen to identify sites of
concern. T h e sensitivity setting was left unchanged.
In addition to arbitrary fluorometric limits to
identify sites of concern, an arbitrary limit for E. coli
was selected for Puerto Rico because the C o m m o n -
wealth relies on fecal coliforms, not E. coli, as
a measure of fecal contamination. In Puerto Rico,
the limit for a grab sample in waters involving indirect
h u m a n contact is 4,000 fecal coliforms p e r 100 ml
(Junta de Calidad Ambiental de Puerto Rico 1990).
An arbitrary limit of 1,000 E. coli per 100 ml was
chosen to identify sites of concern. In Georgia, the
federal limit for a single grab sample applies, and this
limit, 104 fecal enterococci p e r 100 ml (USEPA
2002), was the one chosen to identify sites of concern.
EFFECT OF 410-NM TO 600-NM VERSUS 436-NM
EMISSION FILTER
Four concentrations of Suwannee River Natural
Organic Matter (0, 20, 80, a n d 200 mg 1 1; Catalog
No. 1R101N, International H u m i c Substances Soci-

ety, St. Paul, Minnesota) were mixed with four
concentrations of commercial laundry detergent (0,
75, 150, and 300 m g 1 ~; Tide) in all possible
combinations in distilled water, and the fluoromet-
ric value was r e c o r d e d after 0 a n d 24 h. T h e
fluorometric values were r e c o r d e d with two identi-
cal Model 10-AU-005 fluorometers, except o n e was
e q u i p p e d with a 410-nm to 600-nm emission filter
a n d the o t h e r was e q u i p p e d with a 436-nm emission
filter obtained f r o m the manufacturer. Each com-
bination of organic matter and detergent was d o n e
in triplicate. The data were normalized with a square
r o o t transformation and analyzed with a regression
m o d e l (REG procedure, Version 8.02, SAS, Cary,
N o r t h Carolina) using the numerical values associ-
ated with the a m o u n t o f optical brighteners a n d
organic matter in each cuvette for each filter type.
Suwannee River Natural Organic Matter contains
52.5% carbon (International H u m i c Substances
Society 2006), so the concentrations of organic
m a t t e r u s e d in the e x p e r i m e n t , 20, 80, a n d
200 m g 1 l, are the equivalent of 10.5, 42, a n d
105 m g organic C 1-1, respectively.
Results
MAYAGI)EZBAY, PUERTO RICO
With one exception (site 14), all offshore samples
(sites 1-17) in Mayagfiez Bay contained < 1,000 E.
coli per 100 ml (Fig. 1). Site 14, just offshore o f
Majagual and Sabalos Creeks, contained 4,106 E. coli
p e r 100 ml. T h e fluorometric value was low (average
< 7 units). In contrast to the offshore samples, four
of the five urban rivers or creeks draining into
Mayagfiez Bay contained > 1,000 E. coli per 100 ml
a n d all five had fluorometric values of concern.
Because of insufficient time, only the worst location
for fecal counts, the Yag/iez River, with 12,033 to
> 24,192 E. coli per 100 ml, was selected for
a second sampling.
W h e n resampled, n u m b e r s of E. coli in the 3.75-
km section of the Yagfiez River ranged from 137 to
242,000 E. coli per 100 ml (Fig. 1, u p p e r inset). Two
sites on the river, 2 (242,000 E. coli p e r 100 ml) a n d
20 (120,000 E. coli per 100 nil), were selected for the
third sampling (relabeled A and B, respectively).
U p o n resampling, site A, a stream draining a p o n d
into the river, had an average fluorometric reading
o f 60 and 15,800 E. coli p e r 100 ml, and the esp g e n e
was detected. Site B, a pipe near a textile mill. h a d
an average fluorometric reading of 91 and 14,830 E.
coli p e r 100 ml, and the esp gene was detected.
ST. SIMONS ISLAND, GEORGIA (FIRST SAMPLING)
All surf zone samples f r o m St. Simons beach (sites
6-14) contained --- 52 fecal enterococci p e r 100 ml
(Fig. 2). All beach samples had fluorometric values
ImprovingFluorometry for Fecal Detection 555
< 100. Sites 1-5, 15-17, and 64A-66A were either
ditches, storm drains, or a pond. With the exception
of site 1, all were sites o f concern for fluorometry,
and, with t h e exception o f the storm drains at sites 2
a n d 64A, all were sites of c o n c e r n f o r fecal
enterococci as well. The storm drain at site 2 had
low n u m b e r s o f fecal enterococci (41 per 100 ml),
but had the highest fluorometric value (662 units)
of all the sites sampled. Site 3 was a ditch that
drained into site 4, a p o n d f r e q u e n t e d by ducks.
T h e p e r c e n t a g e o f Ent. faecalis f r o m this ditch was
33%, and the esp gene was not detected. Site 5 was
a storm drain located at Beachview and Mallory
Streets, and contained the highest n u m b e r of fecal
enterococci (19,863 per 100 ml) a m o n g all the sites
in the first sampling. T h e percentage of Ent. faecalis
f r o m this storm drain was only 20%, but the esp gene
was detected. Sites 15-16, 63A, a n d 64A-66A were
storm drains responsible for collecting r u n o f f from
local residential areas.
High n u m b e r s for fecal enterococci a n d fluoro-
metry were also observed in Postell Creek. In lower
Postell C r e e k (sites 18-32), fecal e n t e r o c o c c a l
n u m b e r s d e c l i n e d with distance f r o m site 32
(1,259 fecal enterococci per 100 ml), which was
furthest upstream, to site 18, the m o u t h o f Postell
Creek (31 fecal enterococci per 100 ml) with few
exceptions (sites 19-21 a n d 24). T h e m a j o r excep-
tion, site 21, drains the section o f Postell Creek
where high counts of fecal enterococci f r o m storm
drains at sites 15-17 are located. Like fecal
enterococci, fluorometric values also declined with
distance from site 32 (320 fluorometric units) to 18
(65 fluorometric units).
In u p p e r Postell Creek (Fig. 2), all sites were of
concern for fecal enterococci, which r a n g e d from
246 (site 56A) to 5,794 (site 60A) per 100 ml.
Excluding storm drains, fluorometric values were
also consistently high, averaging 323 units. When
fecal enterococci were obtained f r o m u p p e r Postell
Creek, the p e r c e n t a g e of Ent. faecalis was 56% and
the esp gene was not detected.
FLUOROMETRYEXPERIMENT
No significant differences were observed between
the values o b t a i n e d with a f l u o r o m e t e r e q u i p p e d
with a 410-nm to 600-rim emission filter and
a f l u o r o m e t e r e q u i p p e d with a 436-nm emission
filter for distilled water containing 0, 75, 150, and
300 m g of laundry d e t e r g e n t 1-1 a n d no organic
matter (Fig. 3). T h e emission filter had no signifi-
cant effect on fluorometric values w h e n d e t e r g e n t
was present a n d organic matter was not. When
o r g a n i c m a t t e r was a d d e d to w a t e r with n o
commercial d e t e r g e n t in the f l u o r o m e t e r contain-
ing the 410-nm to 600-nm emission filter, the
f u o r e s c e n c e increased f r o m 0 to 126 units at
556 P.G. Hartel et al.

I~
N "lr'==~.
I

- 18°14'N

Mayag0ez
Boca R ~ and Oro Creek
- 18"13'N

I___

,r Creek
- 18°12'N

MayagCiezBay

0 uan

Fig. 1. Location of three samplings during calm (baseflow) conditions in Puerto Rico (lower inset) around Mayagilez Bay and in the
Yagfiez River. The Yagfiez River (dashed box, enlarged in the upper inset) was the site of the second and third samplings. Each location
shows the site number (top number or letter), fluorometry (middle number; no dimension; not shown if not done), and Eschenchza coh
(bottom number; number per 100 ml). If the fluorometric value exceeds 30, or the number of E. coh exceeds an arbitrary limit (1,000 E. coh
per 100 ml), then the site is of concern and the number is shaded. Except for the data boxes, light gray areas define ocean and dark gray
areas define land.

20 m g o f o r g a n i c m a t t e r , to 233 u n it s at 80 m g o f o r g a n i c m a t t e r 1-1 was a d d e d to distilled water, t he

o r g a n i c m a t t e r , a n d d e c r e a s e d to 164 u n i t s at
200 m g o f o r g a n i c m a t t e r . W h e n o r g a n i c m a t t e r
was a d d e d to w a t e r with n o c o m m e r c i a l d e t e r g e n t in
t h e f l u o r o m e t e r c o n t a i n i n g th e 4 3 6 - n m e m i s s i o n
filter, t h e f l u o r e s c e n c e i n c r e a s e d f r o m 0 to 59 units
at 20 m g o f o r g a n i c m a t t e r , to 96 units at 80 m g o f
o r g a n i c m a t t e r , a n d d e c r e a s e d to 53 u n i t s at 200 m g
o f o r g a n i c m a t t e r . W h e n 20, 80, o r 200 m g o f
organic matter fluoresced, but the presence of the
4 3 6 - n m e m i s s i o n filter was a b l e to r e d u c e t h e
f l u o r e s c e n c e significantly ( > 5 0 % ) . T h i s r e d u c t i o n
was m a i n t a i n e d r e g a r d l e s s o f t h e l a u n d r y d e t e r g e n t
c o n c e n t r a t i o n . As o r g a n i c m a t t e r c o n c e n t r a t i o n
i n c r e a s e d , it also q u e n c h e d t h e overall rate o f
f l u o r e s c e n c e i n c r e a s e with i n c r e a s i n g d e t e r g e n t
c o n c e n t r a t i o n s . At 200 m g o f o r g a n i c m a t t e r 1-1,
 Improving Fluorometry for Fecal Detection 557

2 19
Postell\\~ SeaIsland
Creek \ U ~ ,
-'"~'~ ~ I-~..i09"30"N- ID~';~I~E'~"~7~E'~L'~JE~"~Il~rJ J~'~]

~ . _ 1 ~ 2 8 2 ~r/ ~ 12~ ~ ~ ml Eli! I ~

"~ 32
• 320 Ui
7 ' ~25c
~ / ~ ~u ~u

Atlantic Ocean A B
~]
II;tl I~:}:ll IFI E I [ ]
nnr~i II~lu
~lm]

i I ~ l Im~"~n
~ I IC~
l y I'11 ~ ~ Ln'Q1 4911
165 ,
H I L ~~I -,.-E~II~ 4o4 • 41o

C
Fig. 2. Location of sampling sites in and around St. Simons Island, Georgia, during cahn conditions. In A, the dashed box at the bottom
is shown in D and the dashed box to the right is shown in B and C. In A and B (first sampling), each location shows the site number (top
number), fluorometry (middle number; no dimension), and fecal enterococci (bottom number; number per 100 ml). If the site number
has an asterisk, then it is a storm drain. If the fluorometric ~lue is > 100, or the number of fecal enterococci exceeds the federal maximum
allowable for a grab sample (104 fecal enterococci per 100 ml), then the site is of concern and the number is shaded. In C (second
sampling) and D (third sampling), each location shows the site number (top number), fluorometry (top middle number; no dimension),
fecal enterococci (bottom middle number; number per 100 ml), and total organic carbon (TOC; bottom number, mg 1-1). Except for data
boxes, white areas define ocean, hght gray areas define marsh, and gray areas define land.

n o t o n l y was t h e r e n o i n c r e a s e in f l u o r o m e t r i c value t r a n s f o r m e d (to e n s u r e n o r m a l i t y ) , the g e o m e t r i c

with i n c r e a s i n g d e t e r g e n t c o n c e n t r a t i o n , b u t also
the f l u o r o m e t r i c values were lower t h a n those at
80 m g of o r g a n i c m a t t e r 1 1. W h e n the f l u o r o m e t r i c
values for b o t h filters were c o m p a r e d at 0 a n d 24 h,
the r e a d i n g s at 0 h were significantly h i g h e r t h a n
those at 24 h (data n o t shown, p < 0.0001).
ST. SIMONS ISLAND, GEORGIA (SEcoND SAMPLING)
T h e s e c o n d s a m p l i n g o f St. S i m o n s I s l a n d was
r e p e a t e d e x c e p t f l u o r o m e t r y was o n l y c o n d u c t e d
with a 4 3 6 - n m e m i s s i o n filter. W h e n u p p e r Postell
C r e e k was r e s a m p l e d o n April 27, 2006, 17 c r e e k
a n d six s t o r m d r a i n s a m p l e s were o b t a i n e d (Fig. 2).
All sites were o f c o n c e r n for fecal e n t e r o c o c c i , a n d
w h e n the n u m b e r s o f fecal e n t e r o c o c c i were log-
m e a n o f fecal e n t e r o c o c c i for all l o c a t i o n s i n u p p e r
Postell C r e e k ( e x c e p t for the storm d r a i n s ) f r o m the
first a n d s e c o n d s a m p l i n g s were 696 a n d 474 fecal
e n t e r o c o c c i p e r 100 ml, respectively. T h e average
n u m b e r o f fecal e n t e r o c o c c i was similar b e t w e e n the
two s a m p l i n g s , b u t with the 4 3 6 - n m e m i s s i o n filter
in place, o n l y the six s t o r m d r a i n s a m p l e s e x c e e d e d
100 f l u o r o m e t r i c u n i t s . E x c l u d i n g t h e s e s t o r m
drains, the average f l u o r o m e t r i c value for samples
collected f r o m u p p e r Postell Creek d e c r e a s e d f r o m
a n average o f 323 u n i t s to a n average of 49 units.
Two s t o r m d r a i n samples, sites 61B a n d 62B ( n e a r
A r n o l d R o a d ) , h a d a n average o f 4,674 fecal
e n t e r o c o c c i p e r 100 ml a n d a n average f l u o r o m e t r i c
value o f 191 units. T h e n e x t s t o r m d r a i n , site 63B
558 P . G . Hartel et al.
400 -
300 -
o3
200 •
u were variable, r a n g i n g f r o m 96 to 2,333 fecal
E 100
,7 v v
'0 ;~ ,'~0 2'2~
L a u n d r y d e t e r g e n t , m g 1-1
Fig. 3. Fluorometric values obtained from distilled water
a m e n d e d with 0 (circles), 20 (triangles), 80 (squares), a n d 200
(diamonds) m g organic matter 1-1 a n d 0, 75, 150, a n d 300 m g
commercial laundry d e t e r g e n t (Tide) 1-1. T h e a m o u n t s o f organic
matter, 20, 80, a n d 200 m g 1 1, are the equivalent of 10.5, 42, and
105 m g C 1 l, respectively. T h e optical brightener in t h e detergent
was m e a s u r e d with two identical fluorometers (Model 10-AU-005,
T u r n e r Designs, Sunnyvale, California), except o n e was e q u i p p e d
with a standard 410-nm to 600-nm emission filter (open symbols),
and the other was e q u i p p e d with a 436-nm emission filter (closed
symbols). T h e lines for 0 m g organic matter 1 1 are nearly
identical a n d are s u p e r i m p o s e d on each other. Each p o i n t is the
average of three replicates. Error bars are 1 standard deviation.
Where no error bars are shown, the symbol is larger t h a n the
error bars.
(The Grands), h a d > 24,192 fecal enterococci per
100 ml, but a slightly lower fluorometric reading
(130 units). T h r e e storm drain samples, sites 64B,
65B, and 66B (near East Beach Causeway), had
between 5475 a n d > 24,192 fecal enterococci per
100 ml and an average fluorometric value of 113
units. Percentages of Ent. faecalis exceeded 30% at
all three locations, but the esp g e n e was n o t
detected. T O C concentrations ranged f r o m 6 to
16 mg organic C 1 1. The fluorescence o f Aqua-
shade in water was negligible (0 to 2 fluorometric
units), regardless o f serial dilution (10 1 t o 1 0 - 6 ) .
ST. SIMONS ISLAND, GEORGIA (THIRD SAMPLING)
The third sampling was the same storm drain
system in the first sampling (Fig. 2). In between first
and third samplings, the storm drain was worked on
by the county D e p a r t m e n t of Engineering Services.
W h e n r e s a m p l e d o n J u n e 12, 2006 (baseflow
conditions), counts at the highest part of the storm
drain a r o u n d the m a i n t e n a n c e site (sites A, B, C,
and D) r a n g e d f r o m 20,630 to > 241,920 fecal
enterococci per 100 ml. T O C concentrations at sites
A, B, and D were also extremely high, a n d ranged
f r o m 165 to 911 m g organic C 1 1. F r o m the
m a i n t e n a n c e site, fecal enterococcal counts gener-
ally decreased with increasing distance until counts
were 51 fecal e n t e r o c o c c i p e r 100 ml in the
retention p o n d (site O; same as site 4 in Fig. 2).
T O C concentrations also generally decreased with
increasing distance from the m a i n t e n a n c e site to
16 m g organic C 1-1 in the retention pond. Beyond
the retention p o n d , where the water flowed in
a storm drain to the sea, counts of fecal bacteria
e n t e r o c o c c i p e r 100 ml, a n d T O C values were
slightly elevated (24 and 30 m g organic C 11).
Fluorometric values were of c o n c e r n at five sites
(sites A, B,J, R, and S). Percentages ofEnt, faecalis at
three locations, sites C, J, and R, were < 30% and
300 the esp g e n e was not detected at any of the three
locations.
Discussion
Previous studies have already shown that targeted
sampling, which consists of multiple samplings over
ever-decreasing distances and considers flow condi-
tions a n d local knowledge, is able to identify hot
spots o f fecal contamination quickly and easily in
marine a n d estuarine waters during calm conditions
(Kuntz et al. 2003) and during both calm and
stormy conditions (McDonald et al. 2006). Here,
targeted sampling was c o m b i n e d with fluorometry
to help differentiate h u m a n fecal contamination
from o t h e r sources. Because organic matter fluor-
esces a n d has the capacity to i n t e r f e r e with
fluorometry, the c o m b i n e d m e t h o d was tested in
two locations, Puerto Rico, which had waters with
a low organic matter content, and St. Simons Island,
which h a d waters with a higher organic matter
content. As expected, without any changes to the
fluorometer, the combination was m o r e successful
in Puerto Rico than in St. Simons Island.
In the case of Puerto Rico, the most likely source
of fecal contamination to the Yagfiez River at the
two hot spots was h u m a n s because the n u m b e r s of
E. coli were high, the fluorometric values were high,
and the esp gene was detected (Table 2). Detection
of the esp gene suggests that h u m a n s were the
source. T h e wavelength of the emission filter in the
f l u o r o m e t e r was u n i m p o r t a n t b e c a u s e organic
m a t t e r concentrations were low ( < 2.0 m g 1 l)
and b a c k g r o u n d organic m a t t e r fluorescence was
minimal (average fluorometric value in the bay was
< 7 units).
In the case of St. Simons Island, the organic
matter concentrations were at least threefold higher
(--> 6 m g 1-1) and, as expected, b a c k g r o u n d organic
matter fluorescence interfered with fiuorometry in
d e t e r m i n i n g the sources of fecal contamination.
Even so, despite contradictory fluorometric data for
m a n y sites during the first sampling, the enterococ-
cal estimates and fluorometric data agreed on the
presence or absence of sources o f fecal contamina-
 Improving Fluorometry for Fecal Detection 559

TABLE 2. Summary of suspected fecal sources that had either > 1,000Escherichiacoltper 100 ml (Puerto Rico) or > 104 fecal enterococci
per 100 ml (Georgia, U.S.) and high fluorometric values, which were confirmed by the percentage of Enterococcusfaecahsand detection of
the esp gene in Ent. faeaum isolates. Fluorometric values > 30 (Puerto Rico) or > 100 (Georgia) units are associated with optical
brighteners from laundry detergents in water (= human presence), high percentages (> 30%) of Ent. faecalis are associated with bird or
human feces, and detection of the esp gene is associated with the presence of human feces. Total organic carbon (TOC) is given because
organic matter affects fuorescence.
Fecalbacteria TOC Fluorometncunits No. of Ent espgene
Locauon F~gure,Site (no.per 100ml) (mg1 ~) (no dimension) faerahs(%) detected Suspectedsource
Mayagfiez Bay, Puerto Rico (no 436-nm emission filter added to fluorometer)
Yagtiez River 1, A 15,800 a <2.0 60b NAd Yes Human
Yagfiez River 1, B 14,830a <2.0 91b NA Yes Human
St. Simons Island, Georgia (first sampling; no 436-nm emission filter added to fluorometer)
Beachview and Mallory storm drain 2A, 5 19,863 NA 173b 78 (20) Yes Human
Duck pond 2A, 4 173 NA 222b 95 (33) No Unknown
Upper Postell Creek 2B, Average 696c NA 323b'c 176 (56) No Unknown
St. Simons Island, Georgia (second sampling; 436-nm emission filter added to fluorometer)
Upper Postell Creek 2C, Average 474¢ NA 49c 72 (50) No Bird
Upper Postell (East Beach Causeway 2C, 66B >24,192 14 101 64 (45) No Unknown
storm drain)
Upper Postell (The Grands storm 2C, 63B >24,192 10 130 97 (39) No Unknown
drain)
St. Simons Island, Georgia (third sampling; 436-nm emission filter added to fluorometer)
Beachview and Mallory storm drain 2D, B 61,310 512 207 189 (22) No Not bird or human
Lord Avenue storm drain 2D, J 2,950 17 153 90 (28) No Not bird or human
Virginia Street storm drain 2D, R 2,333 30 232 141 (10) No Not bird or human
aE. coli;all others are fecal enterococci.
b410-nm to 600-nm emission filter only (all others, 436-nm emission filter).
CAverage of sites in Fig. 2, excluding storm drains.
a NA, not analyzed.

t i o n for a few sites. I n t h e surf z o n e (sites 6 - 1 4 ) , Delaware wastewater t r e a t m e n t p l a n t s ( H a r t e l et al.

w h i c h h a d water s a m p l e s with low f l u o r o m e t r i c
values a n d low n u m b e r s o f fecal e n t e r o c o c c i , fecal
c o n t a m i n a t i o n was n o t a c o n c e r n . F o r site 1, a s t o r m
p i p e l o c a t e d o n a s e c t i o n o f b e a c h rarely visited by
b e a c h g o e r s , the relatively low n u m b e r s o f fecal
e n t e r o c o c c i (213 p e r 100 ml) a n d n o f l u o r e s c e n c e
(0 units) suggested that fecal c o n t a m i n a t i o n was n o t
a p r o b l e m . F o r site 2, the low n u m b e r s o f fecal
e n t e r o c o c c i b u t e x t r e m e l y h i g h f l u o r o m e t r i c value
(662 u n i t s ) s u g g e s t e d t h e p r e s e n c e o f o p t i c a l
b r i g h t e n e r s i n gray water. This s t o r m d r a i n c a r r i e d
r u n o f f f r o m local r e s i d e n c e s a n d businesses, in-
c l u d i n g a L a u n d r o m a t , a n d these r e s i d e n c e s a n d
b u s i n e s s e s were the likely source.
B e y o n d these few sites, the source t r a c k i n g results
were contradictory. For site 5, the storm d r a i n at
Beachview a n d Mallory Streets, the source o f fecal
c o n t a m i n a t i o n was likely to b e h u m a n s . F l u o r o m e t -
ric values were h i g h (173 u n i t s ) a n d the esp g e n e was
d e t e c t e d , b u t this d e t e c t i o n was n o t s u p p o r t e d by
h i g h p e r c e n t a g e s o f Ent. faecalis (20%). P e r c e n t a g e s
o f Ent. faecalis > 30% o f all fecal e n t e r o c o c c i
isolated are usually associated with h u m a n s a n d
s o m e wild birds ( W h e e l e r et al. 2002; K u n t z et al.
2004). If h u m a n s were a source, t h e n the p e r c e n t -
age o f Ent. faecalis s h o u l d have e x c e e d e d 30%. Low
p e r c e n t a g e s o f Ent. faecalis (15% a n d 23%) have
b e e n o b s e r v e d i n the p r i m a r y e f f l u e n t of two o f 32
I n press), so this result is possible b u t u n u s u a l . For
site 3, a s t o r m d r a i n e m p t y i n g a d u c k p o n d (site 4),
the p e r c e n t a g e o f Ent. faecalis was h i g h ( > 30%),
a n d the esp g e n e was n o t detected, which suggests
a b i r d source, b u t the f l u o r o m e t r i c value was high
(222 u n i t s ) , w h i c h suggests a h u m a n source. T h e
r e m a i n i n g sites, all in Postell Creek, c o u l d n o t be
resolved for t h e same reason: a h i g h p e r c e n t a g e o f
Ent. faecalis (56%) a n d n o d e t e c t i o n o f the esp g e n e
suggested a b i r d source, while c o n s i s t e n t l y h i g h
f l u o r o m e t r i c values (average 323 u n i t s ) i n t h e u p p e r
p o r t i o n o f the c r e e k suggested a h u m a n source.
T o r e d u c e the p r o b l e m of o r g a n i c m a t t e r in-
t e r f e r e n c e , a n e x p e r i m e n t was c o n d u c t e d with two
f l u o r o m e t e r s , i d e n t i c a l e x c e p t o n e was e q u i p p e d
with a 4 1 0 - n m to 6 0 0 - n m e m i s s i o n filter a n d the
o t h e r h a d a 4 3 6 - n m e m i s s i o n filter. W h e n n o
o r g a n i c m a t t e r was p r e s e n t , there was n o significant
d i f f e r e n c e b e t w e e n the filters. W h e n o r g a n i c m a t t e r
was p r e s e n t , the 4 3 6 - n m filter r e d u c e d f l u o r e s c e n c e
> 50% c o m p a r e d with the o t h e r filter. W h e n the
f l u o r o m e t e r e q u i p p e d with the 4 3 6 - n m filter was
u s e d d u r i n g t h e s e c o n d s a m p l i n g o f u p p e r Postell
Creek, the average f l u o r o m e t r i c value d e c r e a s e d
f r o m 323 to 49 units. This r e d u c t i o n m e a n t that
data for Postell C r e e k n o l o n g e r c o n f l i c t e d , a n d the
low f l u o r o m e t r i c value ( < 100 u n i t s ) , h i g h p e r c e n t -
age o f Ent. faecalis (56%), a n d a b s e n c e o f the esp
560 P.G. Hartel et al.
g e n e suggested t h a t t h e source of the h i g h n u m b e r s
o f fecal e n t e r o c o c c i was likely to be birds.
T h e r e was a clear limit to f l u o r o m e t r y : as the
a m o u n t o f o r g a n i c m a t t e r i n c r e a s e d , t h e rate of
i n c r e a s e i n f l u o r o m e t r i c values with i n c r e a s i n g
d e t e r g e n t c o n c e n t r a t i o n s b e g a n to d e c r e a s e , such
that by 80 a n d 200 m g of o r g a n i c m a t t e r 1-1, the
o r g a n i c m a t t e r q u e n c h e d optical b r i g h t e n e r fluo-
r e s c e n c e a l m o s t entirely, regardless of t h e e m i s s i o n
filter. F l u o r o m e t r y does n o t work above 80 m g of
o r g a n i c m a t t e r 1-~ ( e q u i v a l e n t T O C o f 42 m g 1-1)
a n d was of n o value i n d e t e c t i n g optical b r i g h t e n e r s
i n p o r t i o n s o f the s t o r m d r a i n system d u r i n g the
t h i r d s a m p l i n g (with T O C c o n c e n t r a t i o n s as h i g h as
911 m g 1-1). Even so, given that D O C c o n c e n t r a -
tions average f r o m 5 to 28 m g C 1-1 for s o u t h e a s t e r n
U.S. estuaries (Alberts a n d Tak~tcs 1999), a n d f r o m
5 to 40 m g C 1-1 for s o u t h e a s t e r n U.S. rivers (Alberts
et al. 1990), w i t h p a r t i c u l a t e o r g a n i c c a r b o n
c o n c e n t r a t i o n s a v e r a g i n g a b o u t 10% o f D O C values
(Alberts et al. 1990), f l u o r o m e t r y s h o u l d work for
m o s t G e o r g i a estuaries a n d rivers.
Even with the 4 3 6 - n m filter in place i n the
f l u o r o m e t e r , t h e r e were still sites with low T O C
values w h e r e the d a t a were c o n t r a d i c t o r y , with high
n u m b e r s of fecal e n t e r o c o c c i a n d h i g h f l u o r o m e t r i c
values s u g g e s t i n g a h u m a n source, a n d a h i g h
p e r c e n t a g e o f Ent. faecalis a n d a b s e n c e o f the esp
g e n e s u g g e s t i n g a b i r d source. T h e s e sites were all
s t o r m drains, i n c l u d i n g the s t o r m d r a i n s o n Postell
Creek ( s e c o n d s a m p l i n g ) , a n d m o s t o f t h e storm
d r a i n i n St. S i m o n s Village (third s a m p l i n g ) .
T h e r e are two r e a s o n a b l e e x p l a n a t i o n s for these
c o n t r a d i c t o r y results. S t o r m d r a i n s may b e a special
case. Regrowth o f fecal b a c t e r i a has b e e n o b s e r v e d
in storm d r a i n s ( M a r i n o a n d G a n n o n 1991), a n d
this r e g r o w t h m a y c o m p l i c a t e such tests as de-
t e r m i n i n g the p e r c e n t a g e o f Ent. faecalis o r detect-
i n g the p r e s e n c e o f the esp g e n e . T h e r e is s o m e
e v i d e n c e that Ent. f a e c i u m isolates c o n t a i n i n g the esp
g e n e survive differentially c o m p a r e d with o t h e r Ent.
faecium isolates w i t h o u t the g e n e (Scott et al. 2005).
O t h e r o r g a n i c c o m p o u n d s that fluoresce at the
same wavelengths as optical b r i g h t e n e r s may be
p r e s e n t . A l t h o u g h A q u a s h a d e d i d n o t f l u o r e s c e at
the same w a v e l e n g t h s as optical b r i g h t e n e r s , com-
p o u n d s like diesel fuel a n d m o t o r oil d o ( H a g e d o r n
p e r s o n a l c o m m u n i c a t i o n ) . O n e possible s o l u t i o n to
this p r o b l e m m a y b e to expose the water s a m p l e to
UV l i g h t ( H a g e d o r n p e r s o n a l c o m m u n i c a t i o n ) .
Because optical b r i g h t e n e r s d e g r a d e q u i c k l y i n the
p r e s e n c e o f s u n l i g h t ( K r a m e r et al. 1996), they may
b e m o r e sensitive to p h o t o d e g r a d a t i o n t h a n o r g a n i c
m a t t e r a n d o t h e r o r g a n i c c o m p o u n d s , a n d it m a y be
possible to d i s t i n g u i s h b e t w e e n the two b a s e d o n
t h e i r p h o t o d e c a y rates. S u c h e x p e r i m e n t s are
c u r r e n t l y in progress.
ACKNOWLEDGMENTS
We thank Paul Andrews, Doug Atkinson, Cristina I. Castro,
Jared A. Fisher, Keith W. Gates, Jennifer K. Knighton, Tom R.
Maddox, Karen A. Payne, Steven Smith, Linda V~lez, and Tristan
Wohlford for their technical assistance. This research was partially
funded by grants awarded to the University of Georgia by the
Puerto Rico Water Resources and Environmental Research
Institute and Environmental Protection Division, Georgia De-
partment of Natural Resources, through the U.S. Environmental
Protection Agency under the provisions of Section 319(h) of the
Federal Water Pollution Control Act. S. N. J. Hemmings was
supported by a fellowship through the Office of Ocean and
Coastal Resource Management, National Oceanic and Atmo-
spheric Administration.
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Recezved, October 9, 2006
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Accepted, March 3, 2007