Occurrence of Fecal Coliforms in Surface Waters in the Tiger Point Golf Course
Drainage Area, Santa Rosa County, FL.
Fred J. Genthner, Roman S. Stanley and Michael A. Lewis
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Stanley, and Michael A. Lewio. 1997. Occurrence
o£ Fecal Coliforros in Surface Waters in the Tiger Point Golf Course Drainage
Area, Santa Rosa County, FL. U.S. Environmental Protection Agency, National I IDDADV
Health and Environmental Effects Research Laboratory, Gulf Ecology Division, LIDIl/lIll
Gulf Breeze, FL. 12 p. (ERL.GB 1008).
Internal report for Agency use only.
Occurrence of Fecal Coliforms in Surface Waters in the Tiger Point Golf Course
Drainage Area, Santa Rosa County, FL.
Fred J. Genthner, Roman S. Stanley and Michael A. Lewis
Summary
From 7/16/96 to 12/17/96 fecal coliforms were enumerated in water and oyster
tissue samples obtained from the Tiger Point Golf Course area. Unsanitary conditions
(fecal coliform counts exceeding a standard of 200 per 100 ml) were found in water
samples obtained from Boones Bayou and a ditch which drained into Boones Bayou.
Low fecal coliform numbers (sanitary conditions) were observed in water samples
collected from storage ponds receiving chlorinated effluent from the South Santa Rosa
Utility Company's wastewater treatment facility and from surface waters on the golf
course. Oysters (possessing < 20 fecal coliforms per 100 ml) placed 90 meters from
the mouth of Boones Bayou, accumulated and concentrated Escherichia coli and other
fecal coliforms.
Introduction
A municipal waste water treatment facility (WWTF) located on Tiger Point Golf
Course in Santa Rosa County, FL. utilizes its effluent in spray irrigation. The Tiger
Point WWTF stores treated, chlorinated effluent in holding ponds. From these ponds
the effluent is sprayed onto the golf course for irrigation. The ecological impact of spray
PROPERTY OF
EKV {ONMENTAL PROTECTION AGENCY
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irrigation on Santa Rosa Sound was assessed during 1996. One indicator of impact
was contamination by fecal coliforms which is the subject of this report.
Fecal coliforms were monitored in this study because they are the recommended
bacterial indicators for measuring the sanitary quality of recreational waters and
shellfish in the United States. Fecal coliforms are thermotolerant members of the total
coliform group. They are defined as gram-negative nonspore-forming rods that ferment
lactose in 24 ± 2 hr at 44.5 ± 0.2° C with the production of gas in a multiple-tube
procedure or produce acidity with blue colonies in a membrane filter procedure. In
1968 a National Technical Advisory Committee set the current water quality safety
standard of 200 fecal coliforms per 100 ml of water (1). For commercial oysters the
retail limit for fecal coliforms is 230 per 100 g. To determine if the above criteria were
exceeded, surface water samples and oysters, which accumulate and concentrate
bacteria, were analyzed for fecal coliforms to assess sanitary quality.
Materials and Methods
Duplicate water samples, collected from 7/16/96 to 10/11/96, were taken at a
depth of 0.1 meter, stored on ice, and analyzed in the laboratory within 6 hr of
collection. Location, time, and hydrological parameters were recorded for each sample.
Dissolved oxygen (mg/L), temperature (°C), pH and salinity (parts per thousand, %0)
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were measured with a Hydrolab Scout II1.
During September and December oysters, free of fecal coliforms, were caged at
selected locations for 12 and 21 days, respectively. Exposed oysters, 5 per location,
were analyzed for fecal coliforms. The direct membrane filter method (MF) and the
most probable number (MPN) method (2) were used to enumerate fecal coliform
bacteria in surface water samples. In reporting fecal coliform counts using the direct
membrane filter method, a percent verification correction factor was used. For
enumeration of fecal coliforms in oyster tissue, chlorinated effluent and turbid water
samples, a multiple tube fermentation, 5-tube MPN assay (3) was used. Escherichia
co//, a member of the fecal coliform group, was also enumerated using the MPN method
by transferring inocula from lactose broth tubes that showed gas or growth to EC broth
with 4-methylumbelliferyl-|3-D-glucuronide (MUG) and incubating at 44.5° C for 24 hr.
All EC tubes showing gas were scored positive for fecal coliforms and all EC tubes
showing fluorescence under long wave UV-illumination were scored as positive for £.
co//.
Results and Discussion
Sampling stations are briefly described in Table 1. Sampling dates, temperature,
salinity, dissolved oxygen, and pH of the water are also shown. Water temperatures
1Mention of trade names of commercial products does not imply
endorsement by the U.S. Environmental Protection Agency
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ranged from 26 to 32° C. A maximum pH of 9.7 was measured at the WWTF
emergency holding pond, Station 2. A minimum pH of 6.1 was measured at Boones
Bayou, Station 3. Salinities ranged from freshwater in the wastewater holding pond,
golf course ponds and drainage ditches of Stations 1, 2, 14, 16 and 19 to brackish
water (20 - 25 %o) in water samples taken from Santa Rosa Sound. Low dissolved
oxygen concentrations (< 1.0 mg/L) were measured in the drainage ditches (Stations 0
and 19) and in Boones Bayou.
Fecal coliform enumerations using both MF and MPN procedures are reported in
Table 2. To assure accuracy with our MF enumeration procedure, replicate samples
were analyzed by personnel at the Florida Department of Environmental Protection
(DEP). Replicate samples from Stations 3 and 19 were taken and analyzed on 8/12/96.
DEP reported a fecal coliform count of 3,500 and 3,380 per 100 ml from Stations 3 and
19, respectively. We obtained lower counts of 1,130 and 1,700 fecal coliforms per 100
ml from Stations 3 and 19, respectively. A possible explanation for this difference is
that our verification procedure differed from the method used by the DEP. We
calculated a percent verification and applied this figure as a correction factor to our
direct test results (2). This correction factor was not used by DEP. Instead, random
and atypical looking colonies were verified. Thus, counts we obtained (before
correction) of 2,260 and 3,050 fecal coliforms per 100 ml for Stations 3 and 19,
respectively, were in agreement with the results reported by the DEP.
To further validate our data of 8/12/96, we enumerated fecal coliforms from Station
3 using the MPN procedure. The MPN procedure is recommended as a quality control
4
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measure to be run in parallel with the MF method whenever a highly turbid sample is
encountered or when the coliforms may be injured (eg., chlorinated wastewater or high
salinities). The MPN technique yielded a fecal coliform count (95% CL) of 7,000 (2,300-
17,000) per 100 ml water (Table 2). This count (approximately 6X higher than the
count obtained by the MF method) suggests that the coliforms were injured in this water
sample, possibly due to the high salinity (21 %0 )(Table 1).
Water samples taken from the storage ponds receiving the chlorinated effluent
(Stations 1 and 2) yielded fecal coliform levels below the State standard of 200 fecal
coliforms per 100 ml. These results were confirmed with the MPN procedure to assure
that any chlorine-injured coliforms were being enumerated (Table 2). These data show
that the WWTF was discharging a final chlorinated effluent which meets the fecal
coliform standards for surface waters. This water is used to irrigate the golf course and,
samples taken from surface waters on the golf course also contained low numbers of
fecal coliforms, verifying the effectiveness of the chlorination.
This study revealed fecal coliform contamination in a residential section near the
western boundary of the golf course. Water samples from Stations 3 and 19 taken on
8/12/96 contained fecal coliforms in numbers that greatly exceeded the water quality
safety standard. Station 19 is a drainage ditch that flows between homes south of the
WWTF This ditch empties into Boones Bayou, Station 3, a heavily urbanized area.
The extent of fecal coliform contamination in Santa Rosa Sound adjacent to
Boones Bayou was also studied. Because fecal coliforms are inhibited in saltwater, we
chose oysters as biomonitors of fecal coliform contamination. Oysters are filter feeders
5
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which readily accumulate coliforms and other bacteria from their environment.
Therefore, these bivalves were used at different locations during September and
December in Santa Rosa Sound to determine the extent of the fecal contamination.
Results of these experiments are presented in Table 3.
Oysters possessed (< 20) fecal coliforms at the start of both the September and
December experiments (Table 3). In the September experiment, oysters caged at the
mouth of Boones Bayou (Station 3, 15 meters from the drainage ditch) became heavily
contaminated with fecal coliforms (13,000 per 100 g) after 12 days of exposure.
Importantly, these fecal coliforms were shown to be Escherichia coll, which strongly
suggests that their source was from feces of warm-blooded animals. Heavy
contamination of these oysters was not surprising since fecal coliform counts of water
taken from this site (499 fecal coliforms per 100 ml of water) were higher than the
standard set by the State of Florida (Table 3). Fecal coliform numbers were below
detection limits in water samples at Stations 4 and 5. However, after 12 days the
transplanted oysters had accumulated 230 fecal coliforms per 100 g (Station 4) and 170
fecal coliforms per 100 g (Station 5). Also of importance was the fact that 74% and
41% of the fecal coliforms present in Station 4 and 5 oysters, respectively, were E. coli.
In approximately 3 days oysters can purge themselves of fecal coliforms when placed
into uncontaminated water. Thus, these oysters were probably exposed to a
continuous or repetitive input of fecal coliforms during the 12- day period in the
September experiment.
Results from the December experiment support the conclusions from the
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September experiment. Fecal coliforms were below detection limits in water sampled
from Stations 4, 9 and 12. However, oysters exposed at Stations 4 and 9 accumulated
and concentrated fecal coliforms from the water (Table 3). Station 4 is approximately
90 meters from the mouth of Boones Bayou. Oysters exposed at Station 12, a
relatively pristine site approximately 4 kilometers east of Boones Bayou, were not
contaminated with fecal coliforms. Oysters exposed at Station 9 contained 790 fecal
coliforms per 100g (Table 3). This result was unexpected. Station 9 is, however, at the
mouth of a small bayou. We have not done extensive sampling in this area and further
testing may be warranted. E. coll was not found in oysters from the December
experiment. Perhaps this coliform is not as cold-tolerant as other members of this
group and died before entering Santa Rosa Sound.
In conclusion, the data indicate that during this study the practice of spraying
chlorinated effluent onto the golf course and the overflow drainage did not result in a
fecal coliform contamination problem in the area. A beneficial supplemental study
would be to corroborate these findings after a heavy rain event. In contrast to the
above, high coliform counts in surface water samples were detected just southwest of
the golf course, and caged oysters at the mouth of a small bayou in this area became
heavily contaminated with fecal coliforms after 12 days of exposure.
Acknowledgments. We thank Cheryl Bunch, Bill Chandler, and Delores Spears of the
Florida Department of Environmental Protection and Robert Quarles of the U.S. EPA for
assistance in this project.
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References
1. National Technical Advisory Committee. Water Quality Criteria . Federal Water Poll.
Control Admin., Dept. of the Interior, Washington, DC. 1968.
2. Microbiological Methods for Monitoring the Environment Water and Wastes. R.
Bordner and J. Winter (eds.) U.S. Environmental Protection Agency EPA-600/8-78-017,
1978.
3. American Public Health Association (APHA). 1985. "Standard Methods for the
Examination of Water and Wastewater." 16th Edition. Amer. Publ. Health Assoc.,
Washington, DC.
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Table 1. Sampling stations and properties of the water samples. T : temperature, °C; S : salinity, parts per thousand; DO :
dissolved oxygen, mg/L
Station
0
1
2
3
4
5
6
Description
Drainage inlet
(Bay St.)
WWTF (Pond
for treated
effluent)
WWTF
(Emergency
holding pond)
Boones
Bayou
Sound (near
channel to
Boones
Bayou)
Sound
(Residential
dock)
Residential
Finger Canal
(SEof GC)
Sampling Dates
7/16/96
T
27
31
29
S
20
0.3
18
DO
0.5
6.4
2.9
PH
6.7
7.2
6.9
7/22/96
T
32
30
S
0.1
8.3
DO
6.9
1.1
PH
9.2
6.8
8/12/96
T
30
30
S
0.3
21
DO
5.8
0.8
pH
7.0
6.8
9/18/96
T
28
26
28
28
29
S
0.1
9
23
22
21
DO
11
1.5
5.7
5.5
4.8
PH
9.7
6.1
7.4
7.7
7.4
10/11/96
T
19
18
S
0.1
22
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Table 1. Cont.
Station
7
13
14
16
18
19
Description
Golf course
pond
(Waterfront
condos)
Culverts from
golf course
Freshwater
pond (E. side
ofGC)
Freshwater
pond (E. side
ofGC)
Sound -
undeveloped
area (E. side
ofGC)
drainage
ditch -
elephant ear
Sampling Dates
7/16/96
T
30
30
S
16
0.1
DO
4.1
8.2
PH
7.1
7.8
7/22/96
T
28
S
0.1
DO
0.9
PH
7 1
8/12/96
T
27
S
0.2
DO
2.6
PH
6.9
9/18/96
T
29
29
28
S
7.0
0.3
22
DO
3.6
14
6.3
pH
6.9
9.0
7.7
10/11/96
T
S
10
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Table 2. Fecal coliforms per 100 mis water. MPN : most probable number. Numbers in parentheses are 95%
confidence limits. NR : no result, too many colonies on membrane filter at highest dilution (1 ml filtered).
Sampling
Station
0
1
2
3
4
5
6
7
13
14
16
18
19
DATES
7/16/96
<10
<10
186
< 1
< 1
7/22/96
< 10
270
NR
8/12/96
< 20 MPN
1,130
7000 (2,300-1 7,000) MPN
< 10
1,700
9/18/96
117
499
<10
<10
22 estimated
count
37
45
<10
9/24/96
<10
300 estimated
count
10/11/96
40 (10-170) MPN
468
790 (250-1 900) MPN
103
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Table 3. Accumulation of fecal conforms by oysters placed in areas of Santa Rosa Sound receiving drainage from the Tiger Point
area. MF: membrane filter; MPN: most probable number (95% CL)
September Experiment
Measurements
fecal coliforms/
100 ml water
fecal coliforms/
100 g oysters
Escherichia
CO//7100 g
oysters
Beginning of exposure - 9/18/96
Station 3
499 MF
<20 MPN
<20 MPN
Station 4
<10MF
<20 MPN
<20 MPN
Station 5
<10MF
<20 MPN
<20 MPN
End of exposure - 9/30/96
Station 3
Not done
13,000
(3,500 - 30,000)
13,000
(3,500 - 30,000)
Station 4
Not done
230
(70 - 700)
230
(70 - 700)
Station 5
Not done
170(50-460)
70(10-170)
December Experiment
Measurements
fecal coliforms/
100 ml water
fecal coliforms/
1 00 g oysters
Escherichia
CO//7100 g
oysters
Beginning of exposure -11/26/96
Station 4
Not done
<20 MPN
<20 MPN
Station 9
Not done
<20 MPN
<20 MPN
Station 12
Not done
<20 MPN
<20 MPN
End of exposure - 12/17/96
Station 4
< 20 MPN
330(110-930)
< 20 MPN
Station 9
< 20 MPN
790(250-1900)
< 20 MPN
Station 12
< 20 MPN
20 (<5-70)
< 20 MPN
12
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