STUDY
1 OF FECflL
U. S. ENVIRONMENTAL PROTECTION AGENCY
ARCTIC ENVIRONMENTAL RESEARCH LABORATORY
COLLEGE, ALASKA 99701
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PRELIMINARY STUDY
COMPARATIVE WINTER SURVIVAL OF FECAL BACTERIA
IN A SUBARCTIC RIVER
by
Dale J. Van Donsel
Ronald C. Gordon
Charlotte V. Davenport
WORKING PAPER NO. 28
U. S. Environmental Protection Agency
Arctic Environmental Research Laboratory
College, Alaska
Associate Laboratory of
National Environmental Research Center
Corvallis, Oregon
Office of Research and Development
May 1974
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A Working Paper presents results of investigations which are, to some
extent, limited or incomplete. Therefore, conclusions or recommendations,
expressed or implied, are tentative. Mention of commercial products or
services does not constitute endorsement.
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m
ABSTRACT
A preliminary investigation of Salmonella survival in a subarctic
Alaskan river was conducted during March 1973, when there was total ice
cover and 0°C water temperature. Since most of the domestic pollution
entered the river from one source and there were no additional sources
downstream, it was possible to examine survival without interruption for
seven days flow time. Six Salmonella serotypes were isolated, and
salmonellae were still recoverable both quantitatively and qualitatively
after seven days flow time in the presence of 330 total coliforms/100 ml
and 60 fecal coliforms/100 ml.
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TABLE OF CONTENTS
PAGE
INTRODUCTION
MATERIALS AND METHODS 4
RESULTS AND DISCUSSION 12
CONCLUSIONS 19
REFERENCES 20
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LIST OF FIGURES
NUMBER PAGE
Map of the lower Tanana River showing the location
of sample stations 5
Gasoline powered ice auger being used to cut through
ice at each sample station 6
Samples for quantitative analysis of salmonellae
and fecal indicator bacteria being dipped from ice
hole 7
Setting up tripod to support line with attached
qualitative Salmonella sampler and station marker
flag 9
Method of retrieving line with attached qualitative
Salmonella sampler 10
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VI
LIST OF TABLES
NUMBER PAGE
Salmonellae/Liter and Fecal Indicator Bacteria/
100 ml of Sample Obtained at Each Sample Station
During the March 21, 1973. Sample Run 13
Salmonellae/Liter and Fecal Indicator Bacteria/
100 ml of Sample Obtained at Each Sample Station
During the March 27, 1973 Sample Run 14
Salmonella Serotypes Isolated at Each Sample
Station from Samples Used for Quantitative and
Qualitative Analysis 16
Most Frequently Isolated Salmonella Serotypes
in the United States During 1972 (4) , 17
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INTRODUCTION
Decreasing the suspending medium temperature has shown to increase
survival time of enteric bacteria and viruses (1, 9, 11, 16, 17, 19, 20).
Survival time appears to increase continuously as the temperature decreases,
so the longest survival would be expected when the temperature is at or near
0°C. However, most of the survival studies described in these reports were
conducted in the laboratory using pure cultures, rather than directly in
rivers receiving the enteric microorganisms discharged in effluent from
waste treatment systems. Thus, information to fill the practical need for
survival time parameters, as stated by Berg et al. (3) in 1966* still re-
mains to be obtained. It has also been stated that coliforms must be quan-
titatively related to the presence of enteric pathogenic bacteria if coliform
enumeration is to continue as a useful criterion of health risk from polluted
waters (17).
Increased enteric microogranism survival at low suspending medium
temperatures becomes more significant when viewed in relation to current
treated wastewater disinfection practice. Recent studies (12, 13) have
demonstrated that chlorine disinfection at low effluent temperatures may
not effectively remove the fecal indicator bacteria before the effluent
is released into the receiving water. Therefore, disinfection under these
circumstances may not provide an adequate barrier to the spread of disease
caused by enteric pathogenic bacteria. There is also very little chance
that enteric viruses are being removed in the disinfection process.
In an effort to obtain low temperature survival time data, a survival
study was conducted in a subarctic Alaskan river (11). This study demon-
strated that a significant number of total coliforms, fecal coliforms
and enterococci remained viable after seven days flow time when the water
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temperature was essentially 0°C under total ice cover. The fecal coliform
results were compared with temperate climate winter data (2), and the percent
survival was found to be approximately five times greater at 0°C than at the
-warmer temperatures. That study did not include salmonellae, so quantita-
tive survival relationships are not available.
In 1911, Ruediger (20) demonstrated that salmonellae survived in much
greater numbers for a much longer period in an ice covered river than during
warm weather. He used pure culture suspensions of the bacteria in celloidin
sacs which he placed in the river and sampled at various time intervals.
More recently, Gallagher et al. (9) found salmonellae to be extremely per-
sistent in the Red River under ice cover. They isolated two Salmonella
serotypes 73 miles and four days travel time downstream from the outfall
source. In addition, salmonellae were consistently isolated from river
water when there were 1000 or more fecal col iforms/100 ml (9), and were
occasionally isolated when the fecal coliforms ranged from 4 to 20/100 ml
(5, 6, 8, 9, 21).
Approximately 36 percent of all surface waters examined throughout the
world have been found to contain enteric viruses (1). Even though enteric
viruses have been implicated as the etiological agent in numerous diseases,
infectious hepatitis virus is the only one for which definite epidemiological
evidence has been obtained showing water as the mode of transmission (17).
However, the extended survival of viruses in water makes this a potential
route for viral disease transmission (1).
The need for survival time data and quantitative relationships in
arctic and subarctic rivers, where the water temperature is 0°C for about
six months each year, cannot be overemphasized since low temperatures
enhance enteric microorganism survival causing an increased potential
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health hazard downstream. As a result, this preliminary investigation was
conducted to determine whether salmonellae could be isolated at the same
stations where fecal indicator bacteria had previously been isolated and
to establish, if possible, a quantitative relationship under low tempera-
ture conditions.
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MATERIALS AND METHODS
Selection of River to be Studied
The reach of the Tanana River studied is shown in Figure 1. The descrip-
tion of the river and reasons for selecting this particular river for study
have been described previously (11).
Sample Station Selection and Sampling Schedule
Nine sample stations were selected (Figure 1). T-100 through T-800
are essentially in the same locations as previously described (11). Station
T-900 was selected to obtain background data upstream from all known sources
of domestic pollution.
Two sampling trips were conducted during this preliminary study. All
stations were visited on March 21, 1973, for the purpose of collecting
samples for quantitative fecal indicator bacteria and Salmonella enumera-
tion, and placing long term qualitative Salmonella samplers (Moore swabs).
A second trip was made on March 27, 1973, to obtain anot&er set of samples
for quantitative analysis, and to retrieve the swabs.
Sampling Techniques
A gasoline powered ice auger was used to cut through the 1-1/2 to 4
feet of ice encountered at each sample station (Figure 2). Ice chips were
skimmed from the water surface, and the samples for quantitative analysis
collected by dipping from the water surface (Figure 3). Samples for fecal
indicator bacteria enumeration were collected in sterile, wide mouthed,
screw capped, 1-liter, polypropylene containers. These containers were
transported in ice chests to minimize freezing problems.
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'!"*
• 8*.
64*10
TANANA RIVER
LOWIR IECTIOH
tn
Figure 1. Map of the lower Tanana River showing the location of sample stations
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.-.
Figure 2. Gasoline powered ice auger being used to cut through ice at each sample station
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Figure 3. Samples for quantitative analysis of salmonellae and fecal indicator bacteria being dipped
from ice hole
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8
For Salmonella quantitation, samples were collected in 5-gallon Cubi-
tal ners. There appeared to be little or no ice formation in these large
volume samples. Qualitative Salmonella samples were obtained with the gauze
swabs as described by Spion (22). After the samples for quantitative analysis
were obtained, the line with the gauze swabs attached was lowered through the
hole in the ice, and attached to a tripod for support while the hole was
freezing closed (Figure 4). The tripod also supported a flag which marked
the sample station. In order to retrieve the gauze swabs after a week in the
water, it was necessary to cut another hole in the ice just downstream from
the first hole. A steel rod with a hook on the end was used to catch the
line and pull it up through the new hole (Figure 5). The guaze swabs were
placed in 18-ounce Whirl-Pak bags, and transported to the laboratory in ice
chests to minimize freezing.
Fecal Indicator Bacteria Enumeration
Total coliforms, fecal coliforms and fecal streptococci were enumera-
ted by the membrane filter method (13). All sample volumes were filtered
in triplicate, and volumes up to 200 ml were used in an effort to obtain
numbers of colonies on each filter in the statistically valid range.
Quantitative and Qualitative Salmonella Enumeration
The presence of salmonellae was quantitatively determined by the most
probable number (MPN) procedure. The diatomaceous earth method of Hammar-
strom and Ljutov (14) was used to concentrate the water samples. Sterile dia-
tomaceous earth (0.5 gm/1) was added to the water. For convenience the membrane
filters were removed from sterile, disposable, membrane filter holders (Fal-
con Plastics, number 7103) and replaced with filter pads of the type accom-
panying membrane filters. A slurry of 0.5 gm diatomaceous earth was
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,
Figure 4. Setting up tripod to support line with attached qualitative Salmonella sampler and station
marker flag
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Figure 5. Method of retrieving line with attached qualitative Salmonella sampler
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11
spread on the filter pad, and 12 liters of sample was vacuum filtered.
To remove substances that caused erratic salmonellae isolation, the diato-
maceous earth plug on the pad was washed five times with sterile buffered
distilled water and three times with Dulcitol Selenite Enrichment Medium
(DSE) (18). The concentrate plug was then removed from the filter pad and
homogenized in a small amount of DSE. Additional DSE was added to bring the
total concentrate volume to 120 ml (100:1 concentration of the original
sample). The concentrate was then thoroughly mixed, and one 50-ml, five
10-ml, five 1-rnl and five 0.1-ml portions were immediately planted into
the MPN series in DSE and incubated at 41.5°C. These portions were equi-
valent to 5,000, 1,000, 100 and 10 ml volumes of the original sample.
After 24 and 48 hours of incubation, material from the MPN tubes was
streaked on Hektoen Enteric Agar (HE) and Xylose Lysine Desoxycholate
Agar (XLD), and incubated for 24 hours at 35°C.
For the qualitative determination of salmonellae, each gauze swab
was divided into three equal portions. One portion was placed in a flask
of DSE and two in Tetrathionate Broth. Incubation was at 41.5°C and streaks
were made on HE and XLD at 24 and 48 hours with incubation at 35°C.
Suspect Salmonella colonies on HE and XLD were picked to Triple Sugar
Iron Agar (TSIA) slants. Colonies producing reactions indicative of sal-
monellae in TSIA tubes were further screened for urease, indoTe and lysine
decarboxylase production before being identified by the serological methods
of Edwards and Ewing (7). Additional biochemical tests were performed only
if warranted by questionable screening or serological results.
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12
RESULTS AND DISCUSSION
The objectives of this limited preliminary investigation were: [1] to
determine whether salmonellae could be isolated as many days flow time
downstream from any source of domestic pollution as previously isolated
fecal indicator bacteria (11), and; [2] to establish whether or not a quan-
titative relationship exists between salmonellae and fecal indicator bacteria.
Because this quantitative relationship was to be based on the number of
bacteria/unit volume of river water, discharge measurement were not necessary
at any station.
The results presented in Tables 1 and 2 show that the numbers of fecal
indicator bacteria decreased progressively downstream from the T-700 to the
T-100 station in a manner comparable with earlier results (11). The T-700
station (Figure 1) is located downstream from the last source of domestic
pollution while T-900 is upstream from any source. The fecal coliform
numbers were lower than expected from the previous study. However, the
m-Fecal Coliform Broth Base (BBL lot #204625) may have been a contributing
factor. This medium gave a final reaction pH of 7.2, which is within the
accepted 7.2-7.6 pH range. On further checking, it was found that the broth
base had a pH of about 6.4 before rosalic acid addition and heating. This
is 0.5 to 0.7 pH units lower than obtained with other broth base .lots from
the same manufacturer. Fecal coliform recovery from unchlorinated primary
sewage effluent was examined using the above mentioned broth base lot, and
recovery was compared directly with two other lots from the same manufacturer.
The results indicated that the broth base used during this study recovered
only 48-60 percent as many fecal coliforms as the other two lots. This
suggested that the fecal coliform numbers reported in Tables 1 and 2 may
be considerably lower than the actual number/unit volume present in the
river.
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13
TABLE 1
Salmonellae/Liter and Fecal Indicator Bacteria/100 ml of
Sample Obtained at Each Sample Station
During the March 21, 1973 Sample Run
Salmonellae
Station
No.
T-900
T-800
T-700
T-600
T-500
T-400
T-300
T-200
T-100
Total
Col i forms
(No./lOO ml)
2300
9600
1700
920
920
370
410
320
Fecal
Col i forms
(No./lOO ml)*
130t
1700
89t
160
110
64
36
34
Fecal
Streptococci
(No./lOO ml)
<0.5§
43
130
9t
15t
15t
9t
lit
13t
No. /liter
MPN
0.1#
0.1
<0.1#
0.1
0.1
0.1
0.8
1.7
<0.1#
95% Confidence
Limits
No. /liter
<0.05 0.4
<0.05 0.4
<0.05 0.4
<0.05 0.4
0.2 1.9
0.5 4.7
* These numbers are probably 48-60 percent of the actual number; see text for explanation.
§ No colonies on any filter when triplicate filters were examined,
# No salmonellae isolated from any water volume examined.
t Average of less than 20 colonies per filter when triplicate filters were examined.
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14
TABLE 2
Salmonellae/Liter and Fecal Indicator Bacteria/100 ml of
Sample Obtained at Each Sample Station
During the March 27, 1973 Sample Run
SaTmonellae
Station
No.
T-900
T-800
T-700
T-600
T-500
T-400
T-300
T-200
T-100
Total
Col i forms
(No./lOO ml)
**
3600t
6600
1600
2300
960
470
630
330
Fecal
Col i forms
(No./lOO ml)*
__**
480
170t
64t
260
120
43
63t
60t
Fecal
Streptococci
(No./lOO ml)
_**
73
50
26
26
12
10
21
6t
No. /liter
MPN
**
<0.1#
0.3
0.1
0.1
0.3
0.1
0.1
0.3
95% Confidence
Limits
No. /liter
<0.05 0.8
<0.05 0.4
<0.05 0.4
<0.05 0.8
<0.05 0.4
<0.05 0.4
<0.05 0.8
* These numbers are probably 48-60 percent of the actual number; see text for explanation.
** No samples obtained from this station because of open water.
# No salmonellae isolated from any water volume examined.
t Average of less than 20 colonies per filter when triplicate filters were examined.
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15
Salmonellae were quantitatively measurable in samples from the T-800
through T-100 stations during one or both sampling trips (Tables 1 and 2).
Since these were the same stations at which fecal indicator bacteria were
also found, it is apparent that low indicator bacteria numbers do not pre-
clude quantitative measurement of salmonellae. The most probable number
(MPN) of salmonellae present at the various sample stations suggests that
the numbers of these bacteria do not decrease with flow time. However, the
95 percent confidence limits of the MPN indicate that the salmonellae may
be decreasing in a manner similar to the indicator bacteria. A much more
detailed study must be conducted before relative survival rates and quan-
titative relationships between salmonellae and fecal indicator bacteria
can be established.
When Salmonella isolates from both quantitative and qualitative samples
were examined, six serotypes were found (Table 3). Four serotypes were
found in the quantitative samples and five were obtained from the swabs.
S. thompson was the only serotype isolated at every station from T-700 to
T-100 and was the most frequent isolate from quantitative samples. Thus.
SN, thompson was probably present in much larger numbers than the other
salmonellae. Insufficient data are available to ascertain the numerical
relationship of the other isolates, or to determine if survival character-
istics vary among the serotypes.
The 10 most frequently isolated Salmonella serotypes from human and
nonhuman sources during 1972 are shown in Table 4. Three of the serotypes
found in the Tanana River were among those most frequently isolated from
human sources and three from nonhuman sources. S. typhimurium and S. derby
appear under both headings, while S. thompson was ranked among those most
frequently isolated from human sources and S. senftenberg from nonhuman
sources.
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16
TABLE 3
Salmonella Serotypes Isolated at Each Sample Station From
Station
Number
T-Qnn
T-800
1 / UU
T-600
1 OUU
T-200
T-100
Samples Used for Quantitative and Qualitative
Quantitative Samples
March 21, 1973 March 27, -1973
derby None
thompson group .B
non-motile
LI lUIHJJo Ul 1 Li lUli i|p oU * 1
•fhnmnc nn "t" hnmncnn
thompson thompson
None thompson
senftenberg
Analysis
Qualitative
Samples
**
derby
*
thompson
senftenberg
typh.imurium
derby
*
*
*
thompson
senftenberg
thompson
paratyphi B
typhimurium
** No samples obtained from this station because of open water.
* Swab not recovered.
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17
TABLE 4
Rank
1
2
3
4
5
6
7
8
9
10
in the United States During
Human
typhimurium*
newport
enteritidis
infantis
hei del berg
saint-paul
thompson*
derby*
oranienburg
javiana
1972 C4)
Source
Nonhuman
typhimurium*
oranienburg
senftenberg*
saint-paul
newport
anatum
montevideo
eimsbuettel
derby*
hei del berg
*Isolated from Tanana River.
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18
The results presented in Table 3 indicate that the method used to re-
cover the gauze swabs was not entirely satisfactory since the swabs were
not recovered from four stations. The problem was apparently caused by the
insulating snow layer having been removed from the ice around the hole through
which the line and attached swabs were lowered. During the ensuing week, a
cone of ice developed below the lower edge of the ice cover and encased the
line on which the swabs were attached. The cone extended deeper than could
be reached with the hook so it was impossible to retrieve the swabs.
Efforts are currently in progress to develop a means of overcoming this
serious problem.
Circumstances did not permit the examination of enteric viruses during
this preliminary investigation. In view of the available information about
extended virus survival at low water temperatures (1), isolation and identi-
fication of enteric viruses should constitute a major effort during any
detailed enteric microorganism survival study. Field methods for concen-
tration of viruses from large volumes of water are available (15), and one
or more of these should be applicable under low temperature conditions,
making a virus survival study feasible.
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19
CONCLUSIONS
1. Salmonella serotypes survive for at least seven days flow time down-
stream from the last source of domestic pollution when the water temperature
is 0°C and there is total ice cover.
2. After seven days flow time, a variety of Salmonella serotypes are still
recoverable both quantitatively and qualitatively when total and fecal coli-
forms are present in low numbers.
3. The results from this preliminary study were too few to determine the
extent to which fecal indicator bacteria numbers would indicate Salmonella
density. The results do suggest that a survival relationship may exist.
However, a more detailed study is necessary to assess the quantitative
relationship.
4. Enteric virus survival should also be examined under the same low
temperature conditions.
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20
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s
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* U. S. GOVERNMENT PRINTING OFFICE: 1974-697-595 /54 REGION 10
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