EPA-R2-72-013
  .    ig72             Environmental Protection Technology Series
Winter Survival  of Fecal
Indicator Bacteria  in a
Subarctic Alaskan  River

                                 Office of Research and Monitoring
                                 U.S. Environmental Protection Agency
                                 Washington. D.C. 20460

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            RESEARCH REPORTING SERIES
Research reports of the  Office  of  Research  and
Monitoring,  Environmental Protection Agency, have
been grouped into five series.  These  five  broad
categories  were established to facilitate further
development  and  application   of   environmental
technology.   Elimination  of traditional grouping
was  consciously  planned  to  foster   technology
transfer   and  a  maximum  interface  in  related
fields.  The five series are;

   1,  Environmental Health Effects Research
   2.  Environmental Protection Technology
   3»  Ecological Research
   <*.  Environmental Monitoring
   5.  Socioeconomic Environmental Studies

This report has been assigned to the ENVIRONMENTAL
PROTECTION   TECHNOLOGY   series.    This   series
describes   research   performed  to  develop  and
demonstrate   instrumentation,    equipment    and
methodology  to  repair  or  prevent environmental
degradation from point and  non-point  sources  of
pollution*  This work provides the new or improved
technology  required for the control and treatment
of pollution sources to meet environmental quality
standards.

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                                                              EPA-R2-72-013
                                                              August 1972
           WINTER SURVIVAL OF FECAL INDICATOR BACTERIA

                               IN A

                     SUBARCTIC ALASKAN  RIVER
                                By

                        Ronald C. Gordon
                     Alaska Water Laboratory
                      College, Alaska  99701
                        Project 16100 FHB
                     Program Element
             NATIONAL ENVIRONMENTAL RESEARCH CENTER
                OFFICE OF  RESEARCH AND MONITORING
              U.S. ENVIRONMENTAL PROTECTION AGENCY
                     CORVALLIS,  OREGON 97330
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington. D.C. 20402 - Price SO cents

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                     EPA Review Notice
This report has been reviewed by the Environmental Protection
Agency and approved for publication.  Approval does not
signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
                              ii

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                               ABSTRACT
Survival of fecal indicator bacteria in a subarctic Alaskan river was
studied during the winter of 1969-70 when there was total ice cover and
the water temperature was 0°C.  Most of the domestic pollution entered
the river from one source.  Since no additional pollution entered down-
stream from this source, an uninterrupted study covering seven days of
flow time (210 river miles) was possible.  Nine sample stations were
established to obtain total coliform, fecal coliform, enterococcus and
water chemistry data.  Samples were collected four to eight times from
each station during the two week period of data collection* and a dis-
charge measurement was made at each station during the same period.
Bacteria survival was examined with and without consideration for the
effect of dilution.  After seven days flow time, total coliforms were
reduced to 3.2-6.5 percent of the initial count, fecal coliforms to
2.1-4.2 percent, and the enterococci to 18.1-37.3 percent depending on
dilution consideration.

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                         TABLE OF CONTENTS


SECTION                                                        PAGE


   I     SUMMARY AND CONCLUSIONS                                 1


  II     INTRODUCTION                                            3


 III     MATERIALS AND METHODS                                   5

              Selection of River to be Studied                   5
              Sample Station Selection and Sampling Schedule     5
              Sampling Techniques                                7
              Enumeration of Fecal Indicator Bacteria            7
              Chemical Analyses                                 11
              Hydrology                                         11


  IV     RESULTS                                                13


   V     DISCUSSION                                             29


  VI     ACKNOWLEDGEMENTS                                       33


 VII     REFERENCES                                             35


VIII     GLOSSARY OF TERMS                                      39

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                          LIST OF FIGURES
NUMBER                                                         PAGE

   1      Map of the Lower Tanana River Showing the Location
          of Sample Stations                                     6

   2      Rod Sampler with Attached BOD Bottle                   8

   3      Attaching Bacteriological Sample Bottle to Rod
          Sampler                                                9

   4      Obtaining Sample from Beneath Ice with Rod Sampler    10

   5      Discharge Measurements                                12

   6      Time of Flow Dye Study from Station T-600 to
          Station T-500                                         14

   7      Percent Survival of Total Coliform Bacteria With
          and Without Discharge Consideration                   22

   8      Percent Survival of Fecal Coliform Bacteria With
          and Without Discharge Consideration                   23

   9      Percent Survival of Enterococcus Bacteria With and
          Without Discharge Consideration                       24

  10      Comparison of Total Coliform, Fecal Coliform and
          Enterococcus Survival                                 25
                                 vi

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                          LIST OF TABLES
NUMBER                                                         PAGE

  1       Discharge at Each Sample Station and Velocity
          Estimates Between Stations                            16

  2       Total Coliform Bacteria at Each Sample Station
          Showing Actual Count Per 100 Ml and Count Adjusted
          for Dilution                                          17

  3       Fecal Coliform Bacteria at Each Sample Station
          Showing Actual Count Per 100 Ml and Count Adjusted
          for Dilution                                          18

  4       Enterococcus Bacteria at Each Sample Station Showing
          Actual Count Per 100 Ml and Count Adjusted for
          Dilution                                              19

  5       Arithmetic Mean of the Conductivity and pH, and
          Concentration of Dissolved Oxygen, Alkalinity and
          Nutrients at Each Sample Station                      27
                                  vn

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                               SECTION I

                        SUMMARY AND CONCLUSIONS
A winter survival study of fecal indicator bacteria (total and fecal
coliforms* and enterococci) was conducted on the 210-mile reach of
the Tanana River between its confluence with the Chena and Yukon
Rivers.  A large volume of domestic sewage effluent from the Fairbanks
area enters the Tanana at its confluence with the Chena.  Since the
Tanana receives no additional sewage effluent downstream from the
Chena, it was possible to conduct an uninterrupted survival study
for 7 days of flow time.  This study was conducted during February
and March of 1970 when there was total ice cover on the river and
the water temperature was 0°C.  Several conclusions can be drawn:

1.  The numbers of fecal indicator bacteria per 100 ml of sample
decreased progressively downstream.  However, they survived in
significant numbers throughout the 7 days of flow time with 380 total
coliforms, 88 fecal coliforms and 15 enterococci remaining per 100
ml of sample at the end of the time period.

2.  The greatest percentage reduction  in total and fecal coliform
numbers occurred during the first 1.2  days of flow time since they
decreased to 26.7-35.0 and 20.2-25.6 percent of the original numbers
respectively, depending on the method  of handling dilution.

3.  When the bacteria counts were adjusted for river flow in cubic
feet per second, the percent survival  decreased progressively down-
stream, and after 7 days of flow time, 3.26.5 percent of the total
coliforms, 2.1-4.2 percent of the fecal coliforms, and 18.1-37.3
percent of the enterococci remained viable, depending on the method
of handling dilution.

4.  Fecal coliform survival was 2.7-5.4 times greater than indicated
by winter survival data from more temperate climates, depending on
the method of handling dilution.

5.  Enterococci had a much higher survival rate than either total
or fecal coliforms after 7 days of flow time.

6.  Water was apparently lost from the river channel by infiltration
into groundwater reservoirs.

7.  The groundwater associated with the Tanana River may have been
somewhat contaminated if infiltration  from the river channel carried
bacteria with it.

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8.  No satisfactory correlation was established between the loss
of viability of bacteria and the observed changes in chemical parameters.

9.  Additional studies need to be conducted on several other aspects
of fecal microorganisms in arctic and subarctic rivers:

     A.  Summer data similar to the winter data is needed to ascertain
if a seasonal difference in indicator bacteria survival exists.

     B.  The survival of enteropathogenic bacteria must be studied
under the same conditions to obtain a correlation between pathogens
and indicators.

     C.  Other potential bacteriological water quality indicators
should be studied to determine their relationship to enteric pathogens
at low water temperatures.

     D.  The extent of fecal bacteria penetration into the groundwater
reservoirs should be determined.

     E.  An adequate disinfection method for sewage plant effluents
must be developed for use in the extreme environmental conditions
of Alaska.

     F.  Enteric virus survival at 0°C water temperature must be
exami ned.

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                             SECTION II

                            INTRODUCTION
Bacteria of the coliform and fecal streptococcus groups have been
used to indicate the sanitary bacteriological quality of water for
many years. The development and present usage of these bacteria to
indicate the potential presence of enteric pathogens have been reviewed
recently (7, 16).  Organic substrate concentration, pH, dissolved
oxygen concentration and water temperature are among the more obvious
factors affecting fecal bacteria survival and growth (2, 6).  In
view of these and other factors, the traditional concepts concerning
the quantitative relationship between fecal  indicator and enteric
pathogenic bacteria have come under scrutiny as Geldreich (17) pointed
out in his recent report.

The temperature of the suspending medium appears to have a significant
effect on the growth and survival of fecal bacteria.  Several low
temperature growth studies have been conducted with pure cultures
of Escherichia coli.  In 1934, Haines (20) found that a strain of
£. coli produced visable growth at 0°C after 29 days of incubation.
This early report of growth at 0°C has not been substantiated.  However,
Das and Goldstein (9) showed that during the first few hours of in-
cubation, £. coli had limited capacity to synthesize protein at 0°C.
Ingraham (24) found 8°C to be the minimum growth temperature and,
more recent work has shown the minimum growth temperature to be between
7.5 and 7.8°C (31).  Shaw  (30) found filaments produced at 6°C, and
stated that this was below the minimum growth temperature.  In their
review, Ballentine and Kittrell (5) stated that data from stream
studies did not show aftergrowth of coliforms at low temperatures.

Coliform and enterococcus survival studies have been conducted under
controlled laboratory conditions at temperatures between 0°C and
10°C in various suspending media  (19, 21, 22).  In all cases, the
survival rate at the low temperatures was higher than found in parallel
studies at 20° to 30°C.  Evidence from field studies is, perhaps,
not as clear cut as laboratory studies.  Nevertheless, there is evidence
that fecal bacteria survived longer in streams when the water tempera-
ture was low (winter conditions)  than when the temperature was warm
(summer conditions) (5, 14, 25).  In the reviews by Kittrell and
Furfari (25) and Ballentine and Kittrell (5), summer and winter water
temperatures were considered to be above and below 15°C respectively,
and their interpretation of temperature effect was based on this
arbitrary dividing line.

Water temperature of the arctic and subarctic rivers in Alaska is
0°C for about 6 months of the year, with total ice cover during most
of this period.  The temperatures are below  5°C for an additional

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4 months, and may exceed 15°C for about 2 weeks during the summer.
As a result of low year-around water temperatures, fecal bacteria
survival may be higher throughout the year than temperate climate
data indicate.  This may be more pronounced when the water temperature
is at or near 0°C.

This study was conducted to gain some insight into fecal indicator
bacteria survival during winter with total ice cover and 0°C water
temperature because:  [1] the probability of a higher survival rate
makes extrapolation from temperate climate data questionable; [2]
Ballentine and Kittrell (5) pointed out that few streams have suffi-
cient water travel time between sewage outfalls in which to obtain
survival data; and [3] Berg et_al_. (6) stated that there is a practical
need for survival time parameters.

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                             SECTION III

                        MATERIALS AND METHODS
Selection of River to be Studied
The Tanana River, one of the major rivers draining the interior of
Alaska, rises near the Canadian border and flows generally northwest
for several hundred miles to its confluence with the Yukon River.
The Tanana carries a heavy silt load during the summer, but clears
up in the fall and remains clear throughout the winter.  The Chena
River, which flows through Fairbanks, is a small tributary of the
Tanana River.  Raw domestic sewage and effluents from primary treat-
ment plants enter the Chena in the Fairbanks area causing a high
pollution level in the lower portion of the river.  The Chena enters
the Tanana a few miles below Fairbanks and is the major source of
pollution in the Tanana.  From the confluence of the Tanana and Chena
Rivers, the Tanana flows 210 miles to the Yukon (Figure 1).  Since
none of and villages on the Tanana below the Chena have sewage
collection systems, there are no significant additional sources of
fecal material.  The reach below the Chena confluence represents
a little more than 7 days flow time and provides an excellent op-
portunity to study fecal indicator bacteria survival.
Sample Station Selection and Sampling Schedule
Nine sample stations were selected and their locations are shown in
Figure 1.  Seven stations (T-100 to T-700) divide the Tanana River
into approximately equal reaches below its confluence with the Chena
River.  One station (T-800) above the Chena monitored the fecal bac-
teria from upstream.  The Chena River station (C-100) was downstream
from all domestic sewage sources in the Fairbanks area.

Field data was collected during the last week of February and the
first week of March 1970 because U.S. Geological Survey measurements
indicated that discharge is stable during this part of the winter.
Also, it was early enough in the year to ensure total ice cover,
and late enough to provide adequate daylight in which to sample all
stations each day.  Only two stations (C-100 and T-600) were acces-
sible by road so it was necessary to use an aircraft equipped with
skis for sampling.  Stations T-100 through T-800 were reached by
aircraft and C-100 by ground transportation.

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•«*ao'
            TANANA RIVER
              LOWIR SECTION
                    Figure 1.  Map of  the Lower Tanana River  Showing the Location  of Sample Stations

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Eight sampling trips were scheduled and accomplished, at least in
part, during the 2-week study period.  However, weather-related
problems encountered in the field prevented some sample stations
from being reached each day.  Samples were obtained as many as eight
times, but no fewer than four times from all stations.
Sampling Techniques
A gasoline powered ice auger was used to cut through the 3 to 6 feet
of ice encountered at each sample station.  There were only a few
occasions when it was not necessary to re-cut the hole each day.

A rod sampler (Figures 2, 3, and 4) was developed at the Alaska Water
Laboratory for obtaining bacteriological and dissolved oxygen samples
from below the ice.  Two models of this sampler were made from aluminum
tubing.  Both models were capable of extension to 12 feet, one tele-
scoping and the other in 3-foot breakdown sections.  The upper end
of each was equipped with a T-bar as a handle and the bottom with a
support and clamp to hold the sample bottle.  A rubber stopper
attached to a nylon line was placed in the top of the sample bottle.
When the bottle had been lowered to the desired depth, the stopper
was pulled and the bottle allowed to fill completely with water.

Samples for bacteriological analysis were taken in sterile, 1-liter
glass bottles attached to the rod sampler.  As soon as a sample was
brought to the surface, 50-100 ml of the water was poured from the
bottle and a glass stopper inserted.  Dissolved oyxgen samples were
taken in 300 ml BOD bottles.  The samples were fixed and the bottles
stoppered as soon as they were brought to the surface.  Samples for
chemical analysis were taken just under the water surface in hand-
held 500 ml polyethylene bottles.  All samples were transported to
the laboratory in ice chests for analysis.
Enumeration of Fecal Indicator Bacteria
Total coliforms (3), fecal coliforms  (16) and enterococci  (3) were
enumerated by the membrane filter method.  Total coliforms were grown
on m-Coliform Broth  (BBL) at 35°C for 24 hours, fecal coliforms on
m-FC Broth Base (Difco) for 24 hours  in a 44.5°C water bath, and
enterococci on m-Enterococcus Agar  (Difco) for 48 hours at 35°C.
Three volumes of sample were used for each analysis and plates were
made in triplicate at each volume.  The three plates from  the volume
giving the best count were evaluated  by the Q Test  (32) to reject
questionable results.  All results  from each sample station were
treated mathmetically (28) to determine the arithmetic mean, standard
deviation and 95 percent confidence limits.

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CO
                         Figure  2.   Rod  Sampler  with  Attached  BOD Bottle

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Figure 3.   Attaching Bacteriological  Sample Bottle to Rod Sampler

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Figure 4,   Obtaining Sample from Beneath Ice  with  Rod  Sampler

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Chemical Analyses
Dissolved oxygen was determined by the azide modification of the
idometric method as described in APHA Standard Methods (3).

Alkalinity, pH and conductivity were determined by the instrumental
methods described in FWPCA Methods for Chemical Analysis of Water
and Wastes (10).

A Technicon Auto Analyzer was used to determine ammonia nitrogen
by the sodium phenolate method (10), and nitrate nitrogen by hydrazine
reduction (10).

Total phosphorus was determined by the persulfate digestion method
(10).
Hydrology
Discharge was measured at each sample station by U.S. Geological
Survey personnel using the current-meter measurement procedure
described by Buchanan and Somers (8), and a typical measurement is
shown in Figure 5.

A dye study was conducted by U.S. Geological Survey personnel between
stations T-600 and T-500 to aid in determining the time required
for a water mass to move between stations.  Rhodamine B dye was
injected at T-600, and the passage of the dye was measured at T-500
using a Turner Model 111 Fluorometer with a high sensitivity door.
                                 11

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r-o
                                          Figure 5.   Discharge Measurements

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                             SECTION IV

                               RESULTS
The travel time between stations and the water volume at each station
was needed to interpret fecal indicator survival data.  The most
accurate time-of-travel information would have been obtained by a
dye study between each pair of stations, but such an extensive dye
study was impossible.  However, it was feasible to conduct a dye
study between one pair of stations.  The reach between the T-600
and T-500 stations was selected because the gradient was the approxi-
mate average of all reaches.

Before the dye study was conducted, the velocity estimated for this
reach was 1 mile per hour (mph) based on gradient.  Figure 6 shows
that the dye peak reached T-500 in 24 hours, which indicates the
water moved at 1.25 mph.  Since the Tanana is braided for most of
its length below Fairbanks, the smaller and slower channels considered
in the original estimate may have been frozen dry.  This would mean
that most of the water moved in the deeper and faster main channel.
The cubic feet per second (cfs) discharge at each sample station,
and  water velocity estimates between stations, are presented in
Table 1.  These velocity estimates were the basis for determining
the flow time between sample stations.

Since discharge usually increases as the water moves downstream,
there are some apparent anomalies in the data presented in Table
1.  The discharge increased 2490 cfs between T-600 and T-500 fol-
lowed by a decrease of 2780 cfs between T-500 and T-400.  The dis-
charge again increased 980 and 760 cfs respectively at T-300 and
T-200.  This was followed by a decrease of '1350 cfs at T-100.
These data indicate that far more water enters the Tanana than
shown by the overall increase of 100 cfs between T-600 and T-100.
The U.S. Geological Survey maintains a permanent gaging station at
the T-600 station.  Since discharge is measured regularly at this
station, it is probably the most reliable measurement obtained during
the study.  On the first day of the study, discharge at T-100 and
T-600 was nearly the same.  Because of this, discharge at T-100
was measured again on the last day of the study.  The two results
differed by only 2 percent which indicated an acceptable degree of
measurement reliability.  Thus, only a small portion of the discharge
anomaly can be attributed to measurement error.

The reason for the discharge anomalies evident in Table 1 has not
been established.  However, the most probable explanation is loss
of water from the channel to adjacent groundwater reservoirs.
                                13

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                          TABLE 1

             Discharge at Each Sample Station
                            and
            Velocity Estimates Between Stations
Station
C-100

Mouth of Chena

T-700

T-600

T-500

T-400

T-300

T-200

T-100
Discharge
Cu.Ft./Sec.
182



5630

7280

9770

6990

7970

8730

7380
Velocity
Estimate
Between
Stations
MPH*

0.93

1.50

1.50

1.25***

1.25

1.00

1.00

1.00

Reliability of
Measurement**
± 5%



± 5%

± 8%

± 8%

± 5%

± 8%

± 5%

± 5-8%
  * Based on the gradient between stations and the dye study
      results.
 ** Judgement of Hydrologist at time of discharge measurement.
*** Determined from dye study conducted between stations T-600
      and T-500.
                               15

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Anderson (4) reported that the Tanana River is in a large alluvial
flood plain consisting of well-stratified to lenticular silt, sand
and gravel with a low ice content.  He also stated that infiltration
and permeability are moderate to good, and that groundwater availa-
bility is good because of extensive saturated thickness.and abundant
recharge.  Abnormally dry conditions existed in the entire Tanana
valley for the 2 years prior to this study so the groundwater supply
may have been low.  In view of the area geology and the dry conditions,
the water lost from the Tanana River may have percolated into ground-
water reservoirs.

The arithmetic mean, standard deviation and 95 percent confidence
limits of the actual counts per 100 ml of river water at each sample
station are presented in Tables 2, 3 and 4 for total coliforms, fecal
coliforms and enterococci respectively.  The high counts at the C-100
and T-700 stations indicated that the Chena was the major fecal bacteria
source for the Tanana, while the numbers at T-800 represented background
counts in the Tanana from one or more sources above the Chena.  These
data also show that fecal bacteria numbers decrease progressively
between T-700 and T-100, and that a significant number of these bacteria
remained at the T-100 station.

Since fecal indicator bacteria came from both the Chena and upstream
on the Tanana, the survival study was started at T-700.  In order
to determine survival, it was necessary to adjust the bacteria counts
for dilution at each sample station downstream from T-700.  The discharge
anomolies (Table 1) created some problems so two methods of dilution
adjustment were employed. The resulting numbers of bacteria were
considered to represent survival at each station.  Sample calculations
for these two methods are as follows:

Method I - Count/100 ml Adjusted for Dilution With Water Loss

The dilution adjustment factors were calculated for each station
using the discharge measurements shown in Table 1.  The following
sample calculation is for station T-400:

     6990 cfs at T-400  _ -, «- j.--,„*.,,„, fs~+nv.
     5630 cfs at T-7QO  " 1>24 dllutlon factor

The actual counts per 100 ml obtained each day were multiplied by
the dilution factor to obtain the number of fecal bacteria present
at each station.  From these results, the data presented in the
appropriate portions of Tables 2, 3 and 4 were calculated.
                                 16

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                                    TABLE 2
Total Coliform Bacteria at Each Sample Station Showing Actual Count per 100 ml
                        and Count Adjusted for Dilution
G
o
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 — 1 — '
	
	
	
1,000
1,200
300
140
no
63
Count/100 ml Adjusted for
Dilution Without Water Loss
Arithmetic
Mean
—
—
—
—
—
1,900
1,100
940
780
Standard
Deviation
—
—
—
—
—
510
250
200
130
0)
o - —
C 1
Q>
-0 L-
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c +
O 10
0 +> C
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fc* E  -J-— -
	
	
	
	
	
420
190
150
98

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                                 TABLE 3
Fecal  Coliform Bacteria at Each Sample Station Showing Actual Count per 100 ml
                        and Count Adjusted for Dilution
Stati on
C-100
T-800
T-700
T-600
T-500
T-400
T-300
T-200
T-100
Actual Count/100 ml
Arithmetic
Mean
120,000
230
4,300
870
550
380
170
120
88
Standard
Deviation
80,000
120
1,100
230
170
190
64
55
37
Ol
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C 1

in T- s
en -i-*-*
66,000
120
880
-270
160
180
61
53
36
Count 100/ml Adjusted For
Dilution with Water Loss
Arithmetic
Mean
—
—
—
1,100
960
480
240
190
120
Standard
Deviation
—
—
—
290
300
230
91
86
49
0)
o .— «.
C 1
 C
•i- to
*« E OJ
in T- s
CT» _J^
	
	
	
340
280
220
87
82
47
Count 100/ml Adjusted For
Dilution Without Water Loss
Arithmetic
Mean
—
—
—
—
—
670
330
250
180
Standard
Deviation
—
—
—
—
—
320
120
110
76
0)
o — -
1 i
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                               TABLE 4
Enterococcus Bacteria at Each Sample Station Showing Actual  Count per 100 ml
                      and Count Adjusted for Dilution









c
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ID
•M
00
C-100
T-800
T-700
T-600
T-500
T-400
T-300
T-200
T-100

Actual Count/100 ml




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oo o
530
1.7
19
4.7
14
6.7
2.7
6.4
4.4

O -M C
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440
1.6
18
8.0
11
6.4
2.6
6.1
4.2
Count/100 ml Adjusted for
Dilution with Water Loss




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6.1
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8.3
3.9
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01 1]«—
—
—
—
10
19
7.9
3.7
9.5
5.5
Count/ 100 ml Adjusted for
Dilution Without Water Loss




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Method II - Count/100 ml  Adjusted for Dilution Without Water Loss

With this method, it was  assumed for dilution purposes that no water
was lost from the river channel.  Thus, the discharge either increased
(as indicated by actual measurement) or remained constant (where actual
discharge decreased) at each station proceeding downstream.  This
resulted in stations T-700 through T-100 having discharges of 5630,
7280, 9770, 9770, 10750,  11510 and 11510 cfs respectively.  The dis-
charges presented here for stations T-400 through T-100 differ from
the actual discharge measurements shown in Table 1.   Therefore, the
dilution adjustment factors for these stations differ from those
used in Method I and the  calculation for T-400 is as follows:


     563^ els rt ETOO -1-74 dilution factor

Again, as with Method I,  the actual counts obtained  each day were
adjusted with the dilution factor to obtain the results in the
appropriate sections of Tables 2, 3 and 4.
The total coliform, fecal  coliform and enterococci counts at T-700
were considered to be 100  percent survival  because this was the sur-
vival study starting point.   Proceeding downstream (stations T-600
through T-100), a percent  survive! range was established from the
arithmetic means in Tables 2, 3 and 4.  Sample calculations showing
the possible range of total  coliform survival  at station T-400 are
as follows:

     (1)  Based on actual  count/100 ml:

            1,100/100 ml at T-400
           12,000/100 ml at T-700  x lco =  9-2 Percent survival


     (2)  Based on count/100 ml adjusted for dilution with water
loss:

            1,300/100 ml at T-400
           12,000/100 ml at T-700  x 10° =  10-8 Percent survival


     (3)  Based on count/100 ml adjusted for dilution without water
loss:

            1.900/100 ml at T-400  v lnrt   lc o
           12,000/100 ml at T-700  x 10° =  15-8 percent survival
                                20

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The total coliform percent survival range at each station from T-700
to T-100 is shown in Figure 7.  These data indicate that the most
rapid reduction in numbers occurred during the first 1.2 days of
flow time (T-700 to T-600) with 26.7-35.0 percent remaining viable.
The percent survival continued to decrease at each of the remaining
stations with 18.3-32.5 percent remaining viable at T-500 (2.2 days
of flow time), 9.2-15.8 percent at T-400 (3.4 days), 4.9-9.2 percent
(4.7 days), 3.8-7.8 percent (5.8 days), and 3.2-6.5 percent (7.0
days) at T-300, T-200 and T-100 respectively.

The percent survival range for fecal coliforms at each station is
presented in Figure 8, with composite winter data from Ballentine
and Kittrell (5) shown for comparison.  The most rapid reduction in
numbers again occurred during the first 1.2 days of flow time with
20.2-25.6 percent remaining at T-600.  The percent survival continued
to decrease at a slower rate downstream from T-600 with 12.8-22.3
percent, 8.8-15.6 percent, 4.0-7.7 percent, 2.8-5.8 percent and
2.1-4.2 percent remaining viable respectively at T-500 through
T-100.  After the first 1.2 days of flow time, the fecal coliform
decrease in the Tanana River proceeded at a much slower rate than
suggested by the composite data from Ballentine and Kittrell (5).
They indicated that 0.78 percent of these bacteria remained viable
after 6 days of flow time, while the Tanana data showed that 2.1-4.2
percent remained viable after 7 days.

The enterococcus data presented in Figure 9 indicate that reduction
in numbers of these bacteria proceeds at a much slower rate than
observed with total or fecal coliforms (Figures 7 and 8).  The 47.0-
60.2 percent survival at T-600 after 1.2 days of flow time contrasts
to 26.7-35.0 and 20.2-25.6 percent for total and fecal coliforms.
Because the numbers of enterococci per 100 ml did not change be-
tween T-600 and T-500 or between T-300 and T-200 (Table 4), the
percent survival either remained essentially constant or snowed an
increase depending on the method of handling discharge.  In spite
of the possible discrepancies caused by the discharge measurements,
the enterococci decreased at a much slower rate than either coliform
group.  This is manifested by the much higher percent survival at
all stations, 47.0-60.2, 48.2-83.1, 34.9-61.5, 24.1-44.6, 22.9-45.8
and 18.1-37.3 percent survival at stations T-600 to T-100 respectively.

The data from figures 7, 8 and 9, in which survival with dilution
and water loss was considered, are compared in Figure 10 to show
the relative rates of total coliform, fecal coliform and entero-
coccus decrease throughout the 7 days of flow time.  In general,
fecal coliforms in the Tanana appear to decrease somewhat more
rapidly than total coliforms with the difference in rate becoming
more accentuated during the third through the seventh day of flow
time.  Winter data from Ballentine and Kittrell (5) is also shown.
                                21

-------
  100
   50
D
(XI
h- 10
z
ui
U
tt
111 - A
a. 5.0
   1.0
I
                © WITHOUT DILUTION
                ^ WITH DILUTION (HATER Loss)
                Q WITH DILUTION (No WATER Loss)
I
            123456

            DAYS OF  TRAVEL  TIME
   Figure 7.  Percent Survival  of Total  Coliform Bacteria
             With and Without  Discharge Consideration
                           22

-------
100
              o WITKDW DIULTTION
              0 WITH DILUTION  (WATER Loss)
              0 WITH DILJTION  (No WATER Loss)
              A COMPOSITE WINTER DATA FROM
                BALLENTINE AND KITTRELL (5)
 Figure 8.
1234567

DAYS  OF  TRAVEL   TIME

Percent Survival of Fecal Coliform Bacteria
With and Without Discharge Consideration

               23

-------
  100

   80


   60
>
   40
Z
ui
U
tt
ui
   20
               o WITHOUT DILUTION
               $ WITH DILUTION (WATER Loss)
               a WITH DILUTION (No WATER Loss)


            I   .  I   .  I  .  I  .  I   .  I  .   I
            1    234567
            DAYS  OF TRAVEL  TIME
Figure 9.  Percent Survival of Enterococcus Bacteria
          With and Without Discharge Consideration
                        24

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100
o TOTAL COLIFORM
A FECAL COLIFORM
Q ENTEROCOCCUS
$ WINTER FECAL COLIFORM
  BALLENTINE AND KITTRELL (5)
      )     1     2     3    4     5    6    7

          DAYS  OF  TRAVEL TIME

 Figure 10.  Comparison of Total Coliform, Fecal Coliform
            and  Enterococcus Survival
                              25

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Their composite data indicate that warmer temperatures (up to 15°C)
result in a much more rapid reduction of fecal coliform numbers than
observed in 0°C water under total ice cover.  It is obvious from
this data that enterococci have a much greater capability to survive
than either total or fecal coliforms.

Several chemical parameters were measured during this study.  The
results indicated that the concentration of these components in the
Chena River differed considerably from the Tanana River in nearly
all cases as shown in Table 5.  Perhaps the most significant dif-
ferences were the pH and the concentrations of dissolved oxygen and
ammonia nitrogen at C-100 and T-700.  As the water moved downstream
from T-700, a continuous change took place in all chemical parameters*
except for the total phosphorus concentration which remained constant.
The magnitude of these changes was small and no correlation with
bacteria survival was established.
                                26

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                                                  TABLE 5
                     Arithmetic Mean of the Conductivity and pH,and Concentration of
                     Dissolved Oxygen, Alkalinity and Nutrients at Each Sample Station



c
o
•r—

rtJ

C/1
C-100
T-700
T-600
T-500
T-400
T-300
T-200
T-100

•a
 E
.r- 0

o to
3 O
•a jc
c E
o
0 3.
277
304
314
323
333
333
338
345

4-5
•r—
C CO
•r- fO

 (O
< SO
123
122
126
131
133
138
139
143








n:
OL
6.72
7.32
7.19
7.18
7.08
7.07
7.04
6.96


c
0) OJ
4J O>
10 O
i. %-r-
•4^ 4^ "**v.
••- -I- 0)
JSZ E
0.02
0.05
0.06
0.05
0.07
0.05
0.08
0.07


c
(d  i —
••-> o ->.
O -c en
1— Q- E
0.05
0.01
0.01
0.01
0.01
0.01
0.01
0.01
IV3

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                             SECTION V

                            DISCUSSION
No doubt, the discharge measurements (Table 1) accurately reflect
the water in the river channel.  However, these measurements did
indicate that the river suffered a net flow reduction in two reaches.
In view of this information, it is suggested that some volume of
water may also have been gained in these two reaches and that water
may well have been lost as well as gained in others.  It was not
possible to determine the volume of water contributed by each tri-
butary and groundwater source or the volume of water lost to ground-
water reservoirs, so the actual exchange of water must remain in
the realm of speculation.

It was assumed that the fecal indicator bacteria were uniformly dis-
tributed throughout the water column.  Hence, water leaving the channel
would carry bacteria with it and the effect of water entering would
be to dilute the numbers of bacteria per unit volume.  Therefore,
any loss or gain in discharge would affect the apparent bacterial
survival.  Because of the discharge indeterminates, the real discharge
effect on bacterial numbers could not be assessed.  However, discharge
adjustment did permit a range of possible effects to be established.
Minimum survival was observed when the number of bacteria counted
per 100 ml of sample were examined without regard for discharge.
Thus, any dilution effect would tend to increase survival and the
actual percent survival probably lies somewhere between the minima
and maxima shown in Figures 7, 8 and 9.  A preliminary survey, con-
ducted on the Tanana under similar conditions in 1968 (18) suggested
that survival of fecal indicator bacteria was, closer to the maxima
shown here.

These data (Figures 7, 8 and 9) show that significant numbers of
fecal indicator bacteria survive for an extended period in 0°C river
water under total ice cover.  This is in agreement with previous
laboratory and field studies which indicated that survival rates
at temperatures below 15°C were greater than above 15°C (5, 9, 14,
19, 20, 21, 22, 24, 25, 30).  However, the extent of survival demon-
strated in this study (Figure 10) appeared to exceed that reported
in previous field work (5, 14, 25).  This higher survival rate may
have been caused by one or more factors such as lower water tempera-
ture, total ice cover, physical or chemical characteristics of the
water, or a longer time in which to conduct the study without inter-
ruption by sewage from additional sources.
                                 29

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The actual disposition of the bacteria in water leaving the river
channel is unknown, but there are at least two possibilities which
are dependent on the porosity of the river bed.  If the water passed
through silt, the bacteria would probably have been filtered out
in the silt.  If sand or gravel were exposed, the bacteria would
have been carried some unspecified distance as the water percolated
down.  The bacteria trapped in the silt constitute a potential source
of increased numbers in the river channel under flow conditions which
would resuspend the silt, and those carried through sand or gravel
represent a source of contamination for groundwater reservoirs.

The role of fecal indicator bacteria is to indicate the possible
presence of enteric pathogenic bacteria.  Traditional concepts of
the indicator-pathogen relationships are being questioned in the
more temperate portions of the United States where this water quality
parameter was developed (6, 15).  Thus, it is imperative that similar
questions be asked here in Alaska where lower water temperatures
occur for longer periods.  Most of the available data concerning
survival of enteric pathogenic bacteria in water deal with Salmonellae.
This group of bacteria are the only enteric pathogens which can be
isolated from water with any degree of reliability; even so, satis-
factory quantitative methods for their isolation have not been developed
(15).  Laboratory studies have shown that Salmonellae are capable
of growth at 5°C (23, 26).  These organisms were isolated from the
Red River of the North during the winter (14), and there are indications
that they persist in streams for at Least as long as the fecal coliforms
or possibly even longer (5).  Because of the much larger numbers
of coliforms than pathogens initially present, the longer survival
of coliforms relative to pathogens may be more apparent than real
(6).  The presence of enteric infections in Alaska has been well
documented (11, 12, 27, 29, 35), and Salmonellae have been isolated
from the Chena River (27, 33).  Since fecal indicator bacteria survive
in large numbers for extended periods in at least one subarctic river,
it is probable that pathogens can also be isolated at the same sample
stations.  However, when Gallagher and Spino (15) summarized the
data from several field studies, they found little correlation between
quantities of total or fecal coliforms and the probable isolation
of Salmonellae.  Thus, the question:  Is there any number of fecal
indicator bacteria below which raw water can be considered safe for
human consumption?  People in many villages along the rivers in Alaska
still use raw water for drinking purposes without benefit of any
form of treatment.  Water consumed in some villages probably contains
enteric pathogens, and may be a source of enteric infection.

The Water Quality Standards for Alaska (1) do not cover raw water
used for drinking purposes.  The minimum treatment specified is dis-
infection, and the raw water for this purpose must average less
                                30

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than 50 total coil-forms per TOO ml in any month.  Since total coliforms
survive for an extended period, numbers may be far in excess of 50
per 100 ml.  Therefore, it is necessary to increase awareness of
the problem, provide proper drinking water treatment facilities and
adequately disinfect effluents from sewage treatment plants.
                                 31

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                            SECTION VI

                         ACKNOWLEDGEMENTS
The discharge measurements and dye study results were provided by
James Meckel, Steven Swingle, and Vernon Norman of the Fairbanks
Office, U. S. Geological Survey.
                               33

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                            SECTION VII

                            REFERENCES
1.    Alaska Department of Health and Welfare, Water Quality Standards
     for Interstate Waters within the State of Alaska and a Plan for
     Implementation and Enforcement of the Criteria (1967).

2.    Allen, L. A., Pasley, S. M., and Pierce, M. A. F., "Some Factors
     Affecting the Viability of Faecal Bacteria in Water," Journal
     General Microbiology, 7, pp. 36-43 (1952).

3.    American Public Health Association, Standard Methods for the
     Examination of Water and Wastewater, 2nd Edition, American Public
     Health Association, Inc., New York (1965).

4.    Anderson, G. S., "Hydrologic Reconnaissance of the Tanana Basin,
     Central Alaska," U. S. Dept. of the Interior, U. S. Geological
     Survey, Hydro!ic Investigations Atlas, HA 319, Sheet 2 (1970).

5.    Ballentine, R. K., and Kittrell, F. W., "Observation of Fecal
     Coliforms in Several Recent Stream Pollution Studies," Proceedings
     of the Symposium on Fecal Coliform Bacteria in Water and Waste-
     water, Bureau of Sanitary Engineering, California State Department
     of Public Health (1968).

6.    Berg, G., Scarpino, P. V., and Bergman, D., "Survival of Bacteria
     and Viruses in Natural Waters," Dept. of the Interior, Federal
     Water Pollution Control Administration  (1965).

7.    Brezenski, F. T., "State of the Art, Microbiological Pollution
     Indicators," Dept. of the Interior, Federal Water Pollution
     Control Administration, pp. 1-72 (1968).

8.    Buchanan, T. J., and Somers, W. P., "Discharge Measurements
     at Gaging Stations," Techniques of Water-Resources Investigations
     of the United States Geological Survey, Book 3, Chapter A8, Dept.
     of the Interior, U. S. Geological Survey (1969).

9.    Das, H. D., and Goldstein, A., "Limited Capacity for Protein
     Synthesis at Zero Degrees Centigrade in Escherichia Coli," Journal
     of Molecular Biology, 31, pp. 209-226 (19687!

10.  Federal Water Quality Administration, FWPCA Methods for Chemical
     Analysis of Water and Wastes, Dept. of the Interior, (1969).
                                 35

-------
11.  Fournelle, J. H., Rader, V., and Allen, C., "A Survey of Enteric
     Infections Among Alaskan Indians," Public Health Reports, 81,
     pp. 797-803 (1966).

12.  Fournelle, J. H., Wallace, I. L., and Rader, V., "A Bacterio-
     logical and Parasitological  Survey of Enteric Infection in an
     Alaskan Eskimo Area," American Journal  of Public Health, 48,
     pp. 1489-1497 (1958).

13.  Freeman, H. M.  "Current Practices in Water Microbiology," U. S.
     Dept. of the Interior, Federal Water Pollution Control Admin-
     istration (1970).

14.  Gallagher, T. P., Hagan, J.  E., Thomas, N. A., and Spino, D. F.,
     "Report on Pollution of the Interstate Waters of the Red River
     of the North (Minnesota-North Dakota)," U. S. Dept. of Health,
     Education and Welfare, Public Health Service (1965).

15.  Gallagher, T. P., and Spino, D. F., "The Significance of Numbers
     of Coliform Bacteria as an Indicator of Enteric Pathogens,"
     Water Research. Pergamon Press, 2, pp.  169-175 (1968).

16.  Geldreich, E. E., "Sanitary Significance of Fecal Coliforms in
     the Environment," U. S. Dept. of the Interior, Federal Water
     Pollution Control Administration, WP-20-3 (1966).
17.  Geldreich, E. E., "Applying Bacteriological Parameters to Recrea-
     tional Water Quality," Journal American Water Works Association,
     62, No. 2, pp. 113-120 (1970).

18.  Gordon, R. C., Unpublished Data, Environmental Protection Agency,
     Alaska Water Laboratory (1968).

19.  Gyllenberg, H., Niemela, S., and Sormunen, T.,- "Survival of
     Bifid Bacteria in Water as Compared with that of Coliform Bac-
     teria and Enterococci," Applied Microbiology. 8, No. 1, pp.
     20-22 (1960).

20.  Haines, R. B., "The Minimum Temperatures of Growth of Some
     Bacteria," Journal Hygiene.  34, pp. 277-282 (1934).

21.  Halton, J. E., and Nehlsen,  W. R., "Survival of Escherichia
     Coli in Zero-Degree Centigrade Sea Water," Journal Water Pol-
     lution Control Federation, 40, No. 5, Part 1, pp. 865-868 (1968).

22.  Hanes, N. B. Rohlich, G. A., and Sarles, W. B., "Effect of
     Temperature on the Survival  of Indicator Bacteria in Water,"
     Eutrophication Symposium, University of Wisconsin (1966).
                                36

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23.  Hendricks, C. W., and Morrison, S. M., "Multiplication and
     Growth of Selected Enteric Bacteria in Clear Mountain Stream
     Water," Water Research, Pergamon Press, 1, pp. 567-576 (1967).

24.  Ingraham, J. L., "Growth of Psychrophillic Bacteria," Journal
     of Bacteriology. 76, No. 1, pp. 75-80 (1958).

25.  Kittrell, F. W., and Furfari, S. A., "Observations of Coliform
     Bacteria in Streams," Journal Water Pollution Control Federation.
     35, No. 11, pp. 1361-1385 (1963).

26.  Matches, J. R., and Listen, J., "Low Temperature Growth of
     Salmonella," Journal of Food Science, 33, pp. 641-645 (1968).

27.  Miller, L., Personal communication, U. S. Public Health Service,
     Arctic Health Research Center (1970).

28.  Natrella, M. G., Experimental Statistics, National Bureau of
     Standards Handbood 91, U. S. Dept. of Commerce, National Bureau
     of Standards, pp. 2-2 and 2-3 (1963).

29.  Pauls, F. P., "Enteric Disease in Alaska," Arctic, 6, pp. 205-212
     (1953).

30.  Shaw, M. K., "Formation of Filaments and Synthesis of Macro-
     molecules at Temperatures Below the Minimum for Growth of
     Escherichia coli," Journal of Bacteriology, 95, pp. 221-230
     (1968).

31.  Shaw, M. K., Marr, A. C., and Ingraham, J. L., "Determination
     of the Minimal  Temperature for Growth of Escherichia coli,"
     Journal of  Bacteriology, 105, pp. 683-684 (1971).

32.  Skoog, D. A., and West, D. M., Fundamentals of Analytical
     Chemistry,  Holt, Rinehart and Winston, New York, pp. 58-60
     (1963).

33.  Van Donsel, D., Personal communication, U. S. Public Health
     Service, Arctic Health Research Center, College, Alaska  (1970).

34.  Weiss, C. M., "Adsorption of E. coli in River and Estuarine
     Silts," Sewage  and Industrial Wastes, 23, No. 2, pp. 227-237
     (1951).

35.  Williams, R. B., and Dodson, M. W., "Salmonella in Alaska,"
     Public Health Reports, 75, pp. 913-916 (1960).
                                37

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                            SECTION VIII

                         GLOSSARY OF TERMS
aftergrowth - increase of coliform bacteria numbers in the receiving
water after effluent discharge from a waste treatment plant.

arctic - area north of the 10°C isotherm for the warmest month and
the -10°C isotherm for the coldest month of the year.

background count - the number of coliform and enterococcus bacteria
in the water upstream from the major source of these organisms.

BOD bottle - a bottle designed for biochemical oxygen demand (BOD)
determinations and to contain samples for dissolved oxygen determina-
tion by the Winkler method.

braided - a stream flowing in several dividing and reuniting channels
resembling the strains of a braid.

count per 100 ml - standard method for reporting the numbers of bac-
teria of sanitary significance in water.

discharge - volume of water passing a given point in a stream per unit
time.

dissolved oxygen (DO) - elemental oxygen in solution.

effluent - flowing out; i.e., a sewer outfall into a stream.

enteric - pertaining to the lower intestinal tract.

enterococcus - a bacteria commonly found in significant numbers in
feces of human or other warm-blooded animals.

enteropathogen - microorganism which causes diseases in the intestinal
tract of humans or other warm-blooded animals.

fecal coliform - a total coliform bacteria subgroup which is specifically
found in the feces of humans and other warm-blooded animals.

filament - a continuous protoplasm filled tube produced by some bacteria
when cross walls, which produce normal cells, are not formed during cell
division.

gaging station - location at which the discharge of a stream is measured.
                                39

-------
groundwater - all water found beneath the surface of the ground.

indicator - bacteria generally found in large numbers in feces of
humans and other warm-blooded animals and when found in water indicate
the probable presence of enteropathogenic bacteria.

infiltration - movement of surface water into the ground.

isolation - separation of a species or strain of bacteria from other
bacteria which may be present as contaminants.

membrane filter method - a standard method for obtaining bacterial
cells from large volumes of water for enumeration of the number
present.

parameter - any one chemical or biological determination which defines
the condition of the system relative to that determination.

pathogen - an organism which causes a disease.

percolate - movement of water through the ground.

permeability - measure of the capacity of a material to transmit
water through its interstices.

pollution - addition to any material which tends to degrade water
quality with respect to a particular use.

primary treatment - a waste treatment process designed to remove
floating and settleable solids, and removes 30-40 percent of the
oxidizable organic material in solution, before discharge.

 ure culture - a single strain or species of bacteria free from other
  cteria.

raw domestic sewage - the water carried wastes from households before
it has received any form of treatment.

raw water - fresh water which is potentially useful for drinking
purposes but has received no treatment to remove foreign substances
which may be present.

Salmonellae - pathogenic bacteria which belongs in the genus Salmonella.

sterile - free from any form of life.

subarctic - areas where the mean temperature is higher than 10°C for
less than four months of the year and the mean temperature for the
coldest month is less than 0°C.


                                 40

-------
suspending medium - any liquid in which particles are suspended; e.g.,
bacteria in water.

temperate climate - any area north of the Tropic of Cancer not pre-
viously defined as arctic or subarctic.

total coliform - heterogeneous group of bacteria which meet certain
morphological and biochemical criteria, and are found in feces of
human and other warm-blooded animals, as well as in other environ-
mental situations.

viable - bacterial cells capable of growth and reproduction if appro-
priate conditions are present.

water column - a volume of water extending from the surface to the
bottom of a water body.

water mass - a unit volume of water traveling more or less as a
discrete unit.
                                41

-------
 SELECTED WATER                       i. Report No.
 RESOURCES ABSTRACTS
 INPUT TRANSACTION FORM
                                                           2.         3. Accession No.
                                                                     w
   4. Title  WINTER SURVIVAL OF FECAL INDICATOR BACTERIA            5. ReportDate
           IN A SUBARCTIC ALASKAN RIVER                            ,.
                                                                     8. Performing Organization
   7. Author(s)                                                       ,   Report No.
           RONALD C. GORDON
                                                                  10. Project No.

                                                                    16100  FHB
                                                                    11. Contract/Grant No.
 9. Organization

 ENVIRONMENTAL PROTECTION AGENCY, OFFICE OF  RESEARCH AND
 MONITORING, ALASKA  WATER LABORATORY, COLLEGE,  ALASKA
                                                                  13. Type of Report and
             .                                                        Period Covered
12. Sponsoring Organization

IS. Supplementary Notes

            Environmental Protection Agency report
            number EPA-R2-72-013,  August 1972.
  i .  bstract   survival  of fecal indicator bacteria in a subarctic Alaskan  river was
  studied during the winter of 1969-70 when  there was total ice cover  and  the water
  temperature was 0°C.   Most of the domestic pollution entered downstream  from this
  source.  Since no additional pollution entered downstream from this  source, an uninter-
  rupted study covering 7 days of flow time  (210 river miles) was possible.   Nine sample
  stations were established to obtain total  coliform, fecal coliform,  enterococcus and
  water chemistry data.   Samples were collected four to eight times from each station
  during the 2-week period of data collection,  and a discharge measurement was made at
  each station during  the same period.  Bacteria survival was examined with  and without
  consideration for the effect of dilution.   After 7 days flow time, total coliforms
  were reduced to 3.2-6.5 percent of the initial  count, fecal coliforms to 2.1-4.2
  percent, and the enterococci to 18.1-37.3  percent depending on dilution  consideration.
  17a. Descriptors
  *Rivers,*Enteric  Bacteria, *Winter, *Coliforms, *Bioindicators, *Streptococcus,
  *Bacteria, Alaska,  Water Pollution, Water  Temperature, Discharge Measurement,
  Dissolved Oxygen, Conductivity, Alkalinity,  Nitrogen Compounds, Hydrogen Ion
  Concentration, Phosphorus Compounds

  17b. Identifiers
  *Subarctic, *Total  Coliforms, *Fecal Coliforms, *Enterococcus, *Survival
  17c. COWRR Field & Group
  18. Availability             19. Security Class.
                             (Report)

                          20. Security Class.
                             (Page)
  Abstractor                             I Institution
                                          21. No. of     Send To:
                                             Pages
                                          22  Price     WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                                      U.S. DEPARTMENT OFTHE INTERIOR
                                                      WASHINGTON. D. C. 20240
WRSIC 102 (REV. JUNE 1971)                                                                 GPO 913.261

*U. S. GOVERNMENT PRINTING OFFICE : 1 972 —514-11(6 (27)

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