Winter Survival of Fecal Indicator Bacteria in a Subarctic Alaskan River


 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
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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

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

5630