Microbiological findings of pollution studies on the East Gallatin River and its tributaries
by Theodore Allen Ehlke
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Theodore Allen Ehlke (1968)
Abstract:
A microbiological study was made on the Hagt Gallatin River near Bozeman, Montana, to determine if
pollution existed and if so, its extent and ways of measurement. Total numbers, coliform and
entero-coccic bacteria, anaerobic heterotrophs, sporeformers, ammonia and nitrite oxidizers,
denitrifiers, urea utilizers, aerobic and anaerobic cellulose decomposers and nitrogen fixers were
quantitatively determined at various stations.
The major sources of pollution were found to be a sewage outfall of the city of Bozeman and
agricultural areas bordering the river.
MICROBIOLOGICAL FINDINGS OF POLLUTION STUDIES
ON THE EAST GALLATIN RIVER AND ITS TRIBUTARIES
by
THEODORE ALLEN EIILKE
A thesis submitted to the Graduate Faculty in partial
fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Microbiology
Approved:
Head, Major Department
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1968
ill
ACKNOWLEDGMENT
The author would like t o ■take this opportunity to express his
,
gratitude to those who have.especially been o f help during the course
of this study&
; '
Sincere thanks are due Dr. John C, Wright for his guidance in •
this study and assistance in the preparation of this manuscript.
Sincere thanks are also due Drs. Kenneth L. Temple, William G. Walter
and Richard H.- McBee for their support and suggestions throughout the
duration of this study.
Thanks are also due Ray Soltero for his assistance in collection
of field data a n d .for making flow and chemical data available.
This project was supported by a National Institute of Health
Training Grant No. 5T01 A100131-08 and by a Federal Water Pollution
■
■
/
Control Administration Training Grant No. WP-5T1-WP-180~01. ■
iv
TABLE OF CONTENTS
Page
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lNTRODUCTION
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ABS Tl^-ZVCT
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DESCRIPTION OF THE STUDY AREA
METHODS
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S GimpIG Co XIQ c l.ion
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« © © (° ©•©
Collection H o m s
©
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Bacteriological Studies
©
©
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.
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,
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©
©
©
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©
10
^
o
o
o
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o
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o
o
o
Snoreformers
o
o
o
e
b
e
o
o
o
o
o
o
o
o
o
o
Nitrite Oxidizers
«
.
0
0
«
Cellulose Decomposers
=
.
O
.
.
.
0
0
13
.. . . .
o
«
10
© © © © © ©© © © ©
o
,
10
®
o
Ammonia Oxidizers
7
'
© © ©© © ©.- ©
Total Numbers (Thornton's Medium)
Anaerobes
.
0
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o » » < .
13.
00
'15
0
0
15
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.
«
.
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.
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0
0
16
.
=
=
=
=
.
.
.
.
.
16 .
Z
Nitrogen Fixation
0. '• ' 19.'
Denxcrrfxers i= = = = = = = = = = = = = = = =
-o
..
21
LJrea Utxlxzers
.
23
Flow Studies
RESULTS
O
O
O
1 O
0
X*Xo v7 S tudy
0
0
0
0
0
0
0
0
0
0
Summer 1967 Work
0
0
0
0
0
0
0
0
0
=
=
.
0
0
=
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
o
O
O
1O
O
O
O
O
.
«
»
«
.
0
0
.
o
o
.
.
.
.
0
0
.
0
0
0
. .
o
o
e
0
Coliform Organisms . . = . . . o .
Enterococcal Organisms
0
o
O
0
0
o
0
.
0
0
0
0
0
.
24
25
25
27
« . = «.
27
.
27
.
.
.
.
V
Page
Total Counts . . . . . .
28
24 Hour Studies
28
L ... .
DISCUSSION . . . . . . . . . .
44
SUMMARY
. . . . . . . . . . .
49
APPENDIX . . . . . . . . . . .
51
LITERATURE CITED . . . . . . . .
67
LIST OF TABLES
TABLE
,
Page
1
.
II
;
Aerobic cellulose medium © .
17
© . . . © ©
.
18
20
VIII
Anaerobic nitrogen fixation medium = = = . « =
21
Denitrifier medium „ = . © = » « = « © = „ = ©
22
Urea soil extract medium « » . « . = . © « © =
23
Results of a tracer study^ Elapsed time
in iAinutas © © © © © © © © © © © © © © © © © ©
23
Influent sewage flow at the treatment
plant (M,G.P=D„) at various times during
the day over the summer of 1967 © © © © © © ©
26
Flow times (minutes) between stations at the
stations sampled during 24 hour sampling
periods throughout the summer of 1967
© . . .
26
xi
XII
' ;'
16
Aerobic nitrogen fixation medium „ © © © . © •
- X
_
= = © . . © =
14
VII
IX
.
14'
Anaerobic cellulose medium
VI
' A
Anaerobic heterotroph medium (Based on
Thornton's medium) . . . . . o . . . , . . . . .
Stephenson's basal salts medium
iv
V
'
13
Anaerobic heterotroph medium (nutrient ■
bro th base)
@ o o <> o <* o o o « o * « * « » ©
■ III
;r.
Thornton's medium
XIII
XIV
XV
•
Number of organisms-per 100 ml in water
sample's taken from the sampling stations during
the summer of 1967 on 7/11, 7/19, 7/25, 8/1,
8/8 and 8/28 © © © © © © © © © © © © © © © © © .29
Total number of organisms per ml in water
samples taken at the sampling stations on
7/11, 7/19, 7/25 and 8/8 1967
30
vii
Page
XVI
XVII
XVIII
XIX
■ XX
XXI
XXII
Percent change in numbers between
scanons 0 0 0 0 0 0 0 0 0 0 0 0
Number of anaerobic heterotrophs per ml .in
water samples collected at the sampling • ' ’ •, ■
stations 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
35
Total number of aerobic heterotrophs per
ml in water samples taken at the sampling
stations 0 0 0 0 0 0 0 0 0 0 0 0 0
0 o o o o
36
MPN of anaerobic nitrogen fixing organisms
per ml in water samples taken at the
sampling s cations 0 0 0 0 0 0 0 0 0 0 0 0 0
37
MPN of denitrifying organisms p e r .ml in
water samples taken at the sampling stations
38
Number of nitrite oxidizing organisms per
ml in water samples taken at the sampling
stations O O O O O O O O O O O O O O O O O
O
39
O
40
Number of ammonia oxidizing organisms per
ml in water samples taken at the sampling
s t a t i o n s
XXIII
XXIV
XXVI
XXVII
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Number of -sporeforming organisms per ml in
water samples taken at the sampling stations
41
Number of urea hydrolyzing organisms per
ml in water samples taken at the sampling'
S cations
XXV
31
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Number of aerobic cellulose decomposing
organisms per ml in water samples taken
at the sampling stations = = 0 0 0 0 0 0 0 0
42-
43
Number of organisms per 100 ml in water
samples taken at the sampling stations
8/l"**2/b7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Number of organisms per 100 ml in watersamples taken at the sampling stations
8/15-16/67 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
59
vxii
Page
XXVIII
xxix
XXX
XXXI
XXXII
Number of organisms per 100 ml in water
,samples taken at the sampling stations
6
& u p p p p p p p p p p p p
p
u
p
■
6Q
Nuitiber of col!form organisms per 100 ml
in water samples taken at the sampling
stations during the summer of 1967
61
Number of enterococcal organisms per 100 ml
in water samples taken at the sampling
stations during the summer of-1967.• . . . . .
62
Total count of organisms per ml of water
samples' taken at the" sampling stations
during the summer of 1967, . . . . . . . . . .
63
Temperature in °C of water samples taken
at the sampling stations during 1967r 1968
64'
„ .
ix
LIST OF FIGURES
Page
Figures
1
2
3
Map of the upper East Gallatin River
system showing location of study area
and Stations o o e o o e o e o o o o o
.■ 5
6'
7
' : "s
9
10
o
Results of a 24 hour sample taken Aug„'
I & 2, 1967= o o o o o <a o o o w e o a o
e
9
e
v
o
o
0
0
0
2
Results of a 24 hour sample taken Aug,
I
4
e
&
2
^
1 9 6 7 o
0
c
o
0
o
0
o
o
o
0
0
0 * 0
0
Rate of net change between stations over
a 24 hour period Aug= I & 2, 1967, » » « = Results of a 24 hour sample taken Aug«
15 & 1 5 ^ 1967 0 0 0 0 O 0 0 0 0 0 0 0 0 0
0
0
0
0
- 3 3
34
52
Results of a 24 hour sample taken A u g0
15 St 16, 19670 Coliform organisms per
-L00 ml 0 0 0 0 0 0 0 0 0 ( 0 0 0 0 0 0 0 0 0 0 0
53
Rate of net change between stations■over
a 24 hour period Aug0 15 & 16, 1967 . 0 . . .
54
Results of a 24 hour sample taken Aug0
29 Ss 30, 1967. Enterococcal organisms
per 100 ml O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . 55
Results of a 24 hour sample taken A u g 0
29 Sc 30, 1967o Coliform organisms per
100 m I 0 0 0 0 O 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0
Rate of net change between stations over
a 24 hour period Aug= 29 & 30, 1967. O 0 0 0 0
56
57
ABSTRACT
A microbiological study v/as made on the Hagt Gallatin River near
Bozeman, Montana, to determine if pollution existed and if so, its
extent and ways of measurement. Total numbers, coliform and enterococcic bacteria, anaerobic heterotrophs, sporeformers, ammonia and
nitrite oxidizers, denitrifiers, urea utilizers, aerobic and anaerobic
cellulose decomposers and nitrogen fixers were quantitatively deter­
mined: at various stations.
The major sources of pollution were found to be a sewage outfall
of the city of Bozeman and agricultural areas bordering the river.
INTRODUCTION
At one time much of the microbiological work involving water
quality dealt with total numbers of bacteria as well as g o Iiform and
cnterococcic populations*
.
.
.
.
Recent work tends to include other
eco-
logical groups 'as well'so as to achieve a clearer picture of the
microbiological community*
'
Ryabov (1965) in a study of the Dnepr River and reservoirs of
the UcS.SoR. found that numbers of denitrifying organisms were a more
sensitive criterion of organic pollution than were other saprophytic
bacteria and suggested the need for their inclusion in water quality
studies.
.
Deufel (1965) found a rapid rise in numbers of Azotobacter in
Lake Constance (Switzerland) over an eight year period correlated with
a rise in organic content of the lake, and suggested the use of
Azotobacter numbers as a criterion of pollution.
Harrison, Keller and Lombard (1963), in a study of the Vaal River
(Union of South Africa) determined total numbers at 20°C and 37°C;
lactose fermenters, denitrifers, H^S producers, and citrate producers
as well as doing a'chemical analysis of the river mud bottom.
Only
slight changes in population were noted between wet and dry seasons,
and these were believed to be due to soil forms being washed .into the
river from runoff resulting from rainfall.
It appeared that the
populations consisted partly of forms indigenous to water, partly of
soil origin, and saprophytic bacteria which entered with the runoff
and settled out in the'
mud.
2
In a Polish study, Luchterowa (1962) determined numbers of glucose,
lactose, mannitol^ phenol, cellulose, urea and kerosene decomposing
bacteria as well as the denitrifying, ammonifying, and proteolytic
bacteria at two stations differing in the degree of pollution.
She
■found"hydrocarbon and urea decomposing organisms only at the station
of lesser pollution, and the others at both stations but in greater
numbers in the polluted zone.
Her results followed the second bio-
coenotial rule of Thienemann (1939) which states, that if the
conditions of life change from the normal situation, the number of
species decreases, while the number of individuals increases.
Felton, Cooney and Moore (1967) studied sulfur, ammonia, nitrite
and iron-oxidizing autotrophs, aerobic nitrogen-fixers, urea utilizers
cellulose decomposers and sporeformers, as well as aerobic and anaer­
obic heterotrophs i n •a. temporary pond near.Florenville, Louisiana.
The
bacteria were found to play a role in the nitrogen, carbon, and energy
cycles as decomposers and transformers, as a source of nutrilites and
as members of the food chain.
;
Lueschow and Mackenthun (1962) described a microscopic method of.
concentrating iron bacteria on a membrane filter and counting them
in situ on the filter.
XsFhen water was .sampled from municipal wells
highest- counts were obtained from infrequently used outlets and indi­
cated disuse rather than pollution.
Pintus (1961) studied the usefulness of coliform, enterococcal^
and Clostridium welchii (C. perfringens) populations as indicators of
3
pollution of surface waters and found that in treated waters the test
for GfIostridium was the most sensitive while in untreated waters the
teat for coliform organisms was most sensitive and Clostridium the
least sensitive.
.
He concluded that coliform and enterococcal
populations were indicative of recent pollution and Clostridium
indicated pollution of more remote•origin=
Bonde (1962) in a study of marine waters, concluded that.
Clostridium perfringens was a useful indicator of intermittent sewage
pollution,, that its detection was more precise than tests for coliform
bacteria and therefore it might be used to support Escherichia coll
counts.
In a later paper, Bonde (1966) restated his view that the
total coliform.group of bacteria was a poorly defined group which did
not meet all the requirements that a pollution indicator.system should
have and proposed that C„ perfringens would be a more useful.indicator.
He also proposed that under certain conditions green fluorescent
pseudomonads.would be more useful indicators.
. Rodina (1964) in Russia, found Clostridium pasteurianum to be
widely distributed in lakes, rivers, fishery ponds and soil.
The
development of this organism seemed to be related to the content andnature of organic substances in the water=
Much of the modern European work is-based on the saprobity
system originally devised by Kolkwitz and Marsson (1908, 1909) and by
Kolkwitz (1935, 1950).
The saprobity system is based on the observation
that a slow and evenly flowing river which has received a.',heavy load
4
of sewage shows distinct zones of decreasing pollution*
These zones
are termed polysaprobic (gross pollution), alpha-mesosaprobic, betamesosaprobic and oligosaprobic*
Pantle and Buck (1955a, 1955b)
define the zones in the following formula;
■
'
s = r. sh
"z:h
where S is the sap.robity index, s is the degree of saprobity, and h
is the frequency with which the single species occur.
The degree of
saprobity s is the indicator value of each species obtained from
Liebmann1s 'list of indicator organisms (Liebmann, 1951, 1962)»
The
frequency term h is obtained by ecological investigation of the water.
The frequency of each organism is noted and tabulated as follows:
oligosaprobic indicator organism
s = I
beta-mesosaprobic indicator organism
s = 2
alpha-mesosaprobic indicator organism
> s = 3
polysaprobic indicator organism
s = 4
species found only by chance
h = I
species occurring frequently
h = 3
species occurring in abundance,
h = 5
By using the above-mentioned formula and degree of pollution may be
estimated in the following manner:
5
Saprobity index
Degree of pollution
I.0-1 .5
Very slight (oligosaprobio)
I.5-2.5
Moderate (beta-mesosaprobic)
2 .5-3.5
Heavy (alpha-mesosaprobic)
3.5-4.0
Very heavy (polysaprobic)
Each zone offers optimal conditions for certain species and communi­
ties of organisms, that could be used as indicator organisms.
Since
the bacteria are mainly engaged in the decomposition of organic matter,
their numbers constitute very important criteria for determining.the
different zones of pollution.
Thus, in the saprobity system, both
stenoecic (limited to a narrow range of environmental conditions) and
euryoecic (ubiquitous) organisms are used.
Considerable work has been
done by Sladecek (1963.) in Czechoslovakia to subdivide the zones of
/
greater pollution and to characterize their ecosystems.
.
In a study of Czechoslovakian waters, Daubner (1963) determined
total numbers, heterotrophs, psychrophiles, mesophiles, sporeforming
psychrophiles and mesophiles, enterobacters, enterococci, and anaerobic
sporeformers.
An attempt was made to correlate numbers to flow volume,
temperature, and organic content of the water.
An inverse relationship
was found between bacterial numbers and flow volume and between non-'
sporeforming heterotrophs and sporeformers.
Discharge from sugar beet
refineries in Austria and Moravia caused considerable increase in
numbers o f ;heterotrophs 'during October, November, and December.
6
Sladecek and Katzova (1964) determined horizontal distribution
of Coliform9 mesophilic and psychrophilic bacteria in a fishpond near
Jankov, Czechoslovakia in an attempt to study their correlation■with
the presence of aquatic weeds.
The fecal population was very low.
There was no correlation between numbers of heterotrophs in surface
water layers and the presence of aquatic weeds.
The- purpose of this investigation was to determine numbers of
bacteria-of both fecal and non fecal origin at different seasons in
the East Gallatin River.
Various stations were selected to demonstrate
different zones in the saprobity system.
The results were examined to
determine if correlations existed between members of the groups
studied and temperature, time of day, season, agricultural practices,
organic.content of the water, and location within the study,area.
•DESCRIPTION OF THE STUDY AREA
.■
The East Gallatin River is formed by the confluence of Bozeman
and Rocky Creeks at n point on" half mils (0.8 km) north of
Bozeman, Montana.
2
The river drains a 148 square mile (383 km ) area
3
and has an average discharge of 86.3 c.f.s. (2.44 m /sec) (p.S„G.Se,
1963).
Bridger Creek joins the East Gallatin River at a point 1.3 miles
(2.1 Itin) below the origin of the East Gallatin.
Bozeman, Bridget, and
Rocky Creeks originate in forested areas and all of them drain
agricultural areas in the vicinity of Bozeman.
The stations were located as follows:
Station I was located, above Bozeman on Bozeman Creek a distance
of 3.7 miles (5.9 km) above the site, of the drainage of the sewage
effluent into the East Gallatin River.
Station 2 .was above a' slaughterhouse and stockyard on the East
Gallatin River about 0.8 mile (1.3 km) above the sewage effluent.
Station 3 was situated above the fish hatchery on Bridger Creek
about 3.4 miles (5.5 km) upstream from its junction with the East
Gallatin River.
Station 4 was' a sampling point below the fish hatchery on
Bridget Creek about 0.6 mile (0.96 km) above its confluence with the
East Gallatin River.
Station 5 was located below the slaughterhouse and stockyard on
the East Gallatin River about 0.5 mile (0.8 km) above the sewage
effluent and about 0.1 mile (0.16 km) above the Bozeman Creek effluent.
8
Station 6 was situated below Bozeman on Bozeman Creek about 0«,4
mile CO,,64 km) above the Sewage effluento
Station 7 was on the East Gallatin M v e r about 50 feet (0.015 km)
upstream from the sewage effluent.
Station 8 was the sewage effluent, which originates about 0.4
mile (0.64 km) from the treatment plant.
Station 9 was a sampling point- on the East Gallatin River about
0.2 mile „(0.32 km) below the sewage effluent.
This station was located
in the vicinity of the city sanitary land fill.
Station 10 was on the East Gallatin River about 1.5 miles (2.4 km)
below the sewage effluent.
This station is located in an agricultural
area and receives the flow from Bridget Creek into the East Gallatin
River at a point approximately one mile (1.6 km) below the sewage
effluent.
Station 11 was situated in an agricultural area on the East
Gallatin River about 5.0 miles (8 km) below the sewage effluent.
■■ Station 12 was on the East Gallatin River about '11.3 miles (18.2
km) below the sewage effluent.
'
-
X-
9
O Bozsman City S e w a g s Treatment Plant
0 Stockyards
L Slaughter House
IIi0O O 1
45" 45'
c
S
/ EAST
jGALLATIN
( RIVER
ETIDGER
CREEK
B CZ E1
1MA N
I Mile
.BOZEMAN
vTCREEK
J
Figure I.
Map of the upper East Gallatin River system showing
location of study area and stations
METHODS
Sample Collection.
Samples were collected from the sampling
stations- during the summer of 1967 and the spring and summer of 1968.
Collection was made by lowering a plastic bucket into the water and
rinsing the bucket in the water before obtaining a sample.
Samples
were placed in sterile 2 liter wide mouth .polyethylene bottles which
had been rinsed with a small amount of sample.
Temperature was
determined by a mercury- thermometer or by the thermistor probe of a
Precision Galvanic Cell Oxygen Analyzer.
Upon return to the laboratory, samples were stored at 4°C, if
analysis could not be done immediately.
However, all samples were
processed within 5 hours of collection.
Collection Hours.
During the summer of 1967 samples were collect­
ed between 0600 and 0730 hours except for the 24 hour runs which were
collected at 0600, 0900, 1200, 1500, 1800, 2100, 2400 and 0600 hours.
During the spring and summer of 1968 samples were collected between
1200 and 1300 hours..
•
Bacteriological Studies.
- ""
Water samples were taken at the various
'i
stations at weekly intervals, when possible, throughout the summer of
1967.
Total number, coliform and enterococcal populations were deter­
mined for each sample after return to the. laboratory.
Total counts were done using either membrane filter or conventional
plate method.
Materials used in the membrane filter count included
plastic Sterifil (a registered■trademark of the Millipore Corn.) filter
11
holders and b a s e , membrane filters of 47 mm diameter and 0.45 p. pore
size obtained from the Millipore Coirp,, ^ B- $1ford, Massachusetts.
The
procedure described in Standard Methods for the Examination of Uater
and Wastewater, 12th edition , s used for the membrane filter count in
the following manner.
The apparatus was sterilized at 121°C for 15 min
and was then aeseptically assembled when cool.
Dilutions of M O
I
2
, 10",
3
and 10
were made from each sample and filtered.
were filtered before the lower.
The higher dilutions
The apparatus was not sterilized
between samples but was rinsed with 100-200 ml sterile phosphate buffer
ed water.
The filters were then transferred to sterile 12 x 60 mm
Falcon (a registered trademark of Falcon Plastics) plastic disposable
petri dishes.
The filter was placed directly on the Tryptose Glucose
Extract-agar.
Petri dishes were then incubated inverted at 35°C for 22
hours in a closed vessel containing a small amount of water to prevent
desiccation.
A small quantity of 17. triphenyltetrazolium chloride was
added after incubation to facilitate counting.
-Some difficulty was -
encountered with spreaders, therefore a conventional technique was
adopted using Tryptose Glucose Extract agar (Difco) incubated at 35°C
for 48 hours.
v
.•
' For the coliform test M-endo broth (Difco) was used.
The medium,
in 2 ml amounts was added to a sterile pad in a 50 mm tight fitting
plastic petri dish (Miliipdre or Falcon).
After filtration the filter
was. placed on the pad and the inverted dish was incubated at 35°G for
22 hours In a closed container.
12
MPN coliform tests were also run, and lactose broth served as
the medium-
Tubes were read after 48 hours incubation at 35 G=
A
5 tube series was used for each dilution=
For the enterococci test, M-enterococcus agar (BBL) was used.
The medium in 10 ml amounts was poured into sterile 60 mm Falcon
plastic dishes.
The filter was placed directly on the agar- surface
and the inverted, petri dish was incubated at 35°C for 30 hours in a
closed container.
IujN enterococci determinations were also made.
In this case, azide dextrose broth.was used.
A 5 tube series was.
used for each dilution.
Counting.
At,the end of incubation all plates were counted
using a binocular dissecting scope and hand tally.
counts all colonies were counted.
For the total
In the enterococcic all pink or
red colonies'were counted as enterococcic organisms.
All colonies
with a metallic sheen were counted as coIiforms=
For coliform counts the total number was used as a basis for
determining the count as both types (dark red, with or without sheen)
were found to ferment brilliant green lactose bile broth.
In the spring and summer of 1968 the"program was expanded to
include: total numbers at 20 C and 35, C , anaerobes, sporeformers,
ammonia oxidizers, nitrite oxidizers, aerobic and anaerobic cellulose
decomposers, aerobic'and anaerobic nitrogen fixers, denitrifiers, and
urea utilizers.
)
13
Total !lumbers.
hetorotrophoa
Thornton's medium was used to cultivate aerobic
This medium wea selected on the rationale that the
greatest number of organisms would be of soil origin-
One set of
plates was incubated at 20°C and another at 35°C„
.
O
Thornton's Medium
S
MgSO^-YHgO.
0.2
S
CaCl-ZHgO
0.1
g
NaCl
0.1 ■ g
Fed3
6.002 S
KNO3
0-5
S
Aspargine
0.5
S
Mannitol
1.0
S
FO
TABLE 1«
g
iyipo^
Agar
iooo
Distilled water
, 7.2-7. 4
Anaerobes-
Two media were devised to allow growth of anaerobic
and faculative heterotrophs =
One was based on Thornton's medium and
the other utilized nutrient brothtechnique of McBee (1950) was used-
In both cases the roll tube
14
TABLE H o
Anaerobic heterotroph medium (Based on Thornton’s
medium)=
Oob
g
MgSO-ZKgC
0.1
S
CaCl2
0.05
S
NaCl
0.05
g
Eed3
OoOOl• S
NHaCI
0.25
S
Aspargine
0.25
g
Mannitol ■
0.5
S
NaIICO3
2.5
g
10.0
ml
Na thiogIyco11ate
0.1
S
Resazurin (0.1% solution)
0.5
ml
490.0
ml
Cysteine (2 =5% solution)
Distilled water
.
TABLE H I .
'
Anaerobic heterotroph medium (nutrient broth base)
Nutrient broth (Difco)
Cysteine (2*5% solution)
Na2CO3
Na sulfide
Resazurin (0*1% solution)
Distilled water
pH
7.0-7.4
4.0
S
10.0
ml
2.5 ' g
. 0.1
S
0.5
ml
490.0
ml
0
1
pH
*2
7
/
15
The medium was made up in a one liter balloon flask and oxygen
free carbon dioxide vraa bubbled, in until the indicator (r^daKurin)
was-reduced, indicating anaorobioois,
The medium, in ,10 ml amounts
was put into .18 x 150 mm test tubes containing 0 =2 g agar=
The tubes
were sparged with CO^ for 20 seconds, securely stoppered and stcril-.
ized for 15 minutes at 121°C=
Inoculation was done while the tubes
O
1
w e r e ■in a water bath at 47 C= After inoculation, the tubes were again
sparged with,CO^ for 20 seconds and then rolled until cool on a tube
rolling machine to produce a thin layer of medium around the tube
surface=
Tubes were then incubated at 23°C for two to three weeks=
Sporeformers=
Sporeforming organisms were selected by heating
an aliquot of the sample -to 85°C for 10 minutes and appropriate di­
lutions were plated on Thornton's agar=
Incubation was at 20°C for
48 - 72 hours or until growth was adequate for counting=
Ammonia oxidizers=
and nitrate=
These organisms oxidize ammonia to nitrite
The basal salts medium of Stephenson (1949), as modi­
fied by Mayeaux (1961) was used.
16
TAHLE IV.
Stephenson's basal salts medium
KIIgPO^
0< 75
C
0.25
S
■ 0.01
MgSO4-YHpO
0.03
S
0.01
S
1.00
8
Saturated phenol red solution
0.20
ml
Oxoid ionaga.r No. 2
O
O
Os
-s
EeSO4-THpO
S
MnSO4-Hp0
■
(NH4 )pSO4
■
1000
Distilled water
ml
Plates w e r e 'incubated at 28°C for 2 to 3 weeks
At the end of
incubation, 2 ml of a 1:20,000 solution of Rose Bengal was added to
each plate to facilitate count "/■-.g
Nitrite Oxidizers.
The basal salts medium of Stephenson (1949),
as modified by Mayeaux (1961) was also used to cultivate nitrite
oxidizers with the substitution of I,.0 g NaNO^ for (NH^^SO^ °
Plates
were handled in the same manner as for ammonia oxidizers=
Cellulose Decomposers=
Anaerobic and aerobic cellulose
decomposers were counted using the roll tube method of McBee (1950)=
Two different media were used:
17
TABLE V.
Anaerobic cellulose medium.
Ainer.al solution Ifo. I
36.5 ml
Mineral solution Mo. 2
'38.5 ml
0.5 ml
Resazurin (0.17. solution)
50.0 ml
Cellulose (57.)
0.5
Tea-St extract
Cysteine HCl (2.57.)
Ha thioglycollate
Na2CO3
Distilled water
S
' 10.0 ml
■ .
0.1
S
2.5
S
388
ml
Distilled water '
O
K2HPO^
L-J
Mineral solution No. I
500
S
ml
Mineral solution No. 2
.
KH2PO^
3.0
S
(rn^gSO^. . .
6.0
g
NaCl ■
6.0
S
MgSO^
0 =6
S
CaCl2
0.6
S
Distilled water
500
ml
18
TABLE VI.
Aerobic cellulose medium,.
K 2KPC4
■0.5
g
0.1
g
CaClg-ZKgO
0.05 s
NaCl
0.05 g
FeCl3
0.001 s
KNO3
0.25 g
Cellulose (57. floe)
Yeast extract•
Distilled water'
pH
50
0.5
45 0
ml
s
ml
7.2
The cellulose suspension vzas prepared by grinding a 57« solution
of Solka.Flop in a pebble mill for 72 hours.
T h e 'aerobic cellulose medium was made up and 10 ml aliquots were
distributed into 18 x 150 mm test tubes containing 0.2 g agar.
Tubes
were tightly stoppered and_sterilized in the autoclave for 15 minutes
at 121°C.
Inoculation was done in a water bath at 47°C , after which
the tubes were placed on a tube rolling machine and rolled until the
agar had solidified.
The stoppers were then replaced by sterile'metal
caps and the tubes were incubated at 28°C for 2 to 4 weeks.
On the second run it was found necessary to add 100 m g /I Captan
to inhibit fungi.
19
The anaerobic medium was made up in a one liter erlenmeyer flask
and gassed with CO^ until the resazurin was decolorized.
Ten ml
aliquots were distributed in 18 x 150 min test tubes containing 0.2 g
agar and gassed with CO^ for 20 seconds.
Tubes were tightly stoppered
and sterilized in the same manner as for the aerobic medium.
Inocul­
ation was .handled in the same manner as for. the anaerobic heterotrophs.
Tubes' were incubated at 28°C for 4 to 6 weeks.
Nitrogen Fixation.
Aerobic and anaerobic nitrogen fixation
was studied by the method of Po chon.: and Tardieux (1962).
The media
-X
used are described in Tables VII and- VIII.
:i
I
The soil extract was made by adding one liter of distilled
water to 1000 g soil and autoclaving the mixture for one hour at 121°C.
After cooling the supernatant was decanted and filtered to obtain
an amber liquid which was sterilized in 100 ml volumes in the autooclave at 121 C for 15 minutes.
7, or slightly alkaline.
:
The pH of the extract should be near
Soil was obtained near the study area, in
this case, from■the East Gallatin River and yielded a soil extract
with pH 7.4.
The aerobic nitrogen fixation medium]is designed to.enumerate
essentially Azotobacter.
The medium, in 5 ml amounts, was distributed
in 16 x 150 mm test'tubes and sterilized at 121°C for 15 minutes.
Inoculation was done by transferring a I ml aliquot of t h e ■appropri­
ate dilution to each of 5 tubes.
Five different dilutions were used,
(10^, 10^, 10^, 10^ and 10').' The tubes were incubated at 28°C for one
'
.!
::
-i
!(
: -'
20
week.
Those with a pellicle of Azotobacter were counted as positive
It was also necessary to confirm positives microscopically,
TABLE VII,
Aerobic nitrogen fixation medium.
Standard saline solution
50,0
Mannitol
10,0 \ g
Soil extract
10,0
ml
Solution of trace elements
1,0
ml
CaCO3
0.5
s
940,0
ml
5.0
. g
2.5
g
2.5
g
0.05
g
0.05
g
Distilled water
ml
Saline solution
' K 2HPO4
MSS°4^
NaCl
.
.
.
MnSOz,
Distilled water
1000.0
mV
Trace element solution
.K2MoO4 /
0.05
g
Na2B4 O,
0.05
g
" I crystal
Fed,
0.05
g
0.05
g
CuSOzi
0.05
g
ZnSO
0.05
g
MnSO.
4
Distilled water
0.05
g
CoNO3
' CdSO4
.
1000,0
■
ml
,1
21
TABLE VIII.
Anaerobic nitrogen fixation medium.
Standard saline solution
K2m 0 4
50.0
0.75
.
ml
S
0.IOH NaOH . .
33.0
ml
Glucose
10.0
g
.10.0
ml
1.0
ml
1000.0
ml
Soil ■extract
Trace elements
Distilled water Q t» S »
Anaerobic nitrogen fixation was studied by a technique also
described by Pochon and Tardieux (1962).
Essentially Clostridium
pasteurianum was the species cultivated.
The medium' was distributed in 17 x 150 mm test tubes, each
containing a durham tube.
Sterilization, inoculation and incubation
were done as described for the aerobic nitrogen fixers.
At the' end
of 7 to 15 days tubes showing the presence of gas were recorded as
positive.
Denitrifiers.
The denitrifying bacteria typically cause an evo­
lution of gaseous nitrogen from nitrate and nitrite.. The gaseous
products■of the reaction are
, N^O and sometimes NO.
Denitrifi­
cation is affected mainly by certain species of the genera Pseudomonas,
Achromobacter, Bacillus, and Micrococcus.
The bacteria responsible are
facultative anaerobes which adapt to the utilization of NO^
environments of low O0 tension.
or NO" in
The process is then analogous to
22
respiration as the nitrate or nitrite replaces 0? as a terminal
electron acceptor^
Denitrifiers were enumerated using a MPN technique
described in Methods o£ £ioil Analysis (1965), part 2.
TABLE IXo
Denitrifier medium=.
Solution A
KNO^
1.0
Aspargine
O
H
to
g
5.0
ml
17. Alcoholic solution
°
of brom thymol blue
Distilled water
' 500
ml
Solution B
Na Citrate
8.5 ' g
KH^ro^
1.0
g
MgSO4 -7H20 ' ■
1.0
g
CaCl2 -OH2O
0.2
g
FeCl3 -GH2O
0.05
g
Distilled water
500
ml
The solutions were mixed and the pH was adjusted to 7=0-7=2.
The medium in 10 ml amounts was placed in 15 x 125 mm test tubes each
containing a durham tube.
at 121°C for 15' minutes.'
The tubes were then.plugged and sterilized,
Sterile tubes were stored in the.dark, as
the color changes in the light.
Inoculation was done using a series of
23
5 dilutions (1C)\ IO^, IO^, 1 0 ’ and 10^) with 5 tubes per dilution;
IoO or Ool ml of .the appropriate'dilution being introduced=
were incubated at 28 G for 3 to 7 days=
Tubes
As the bacteria grew* the
color, initially greenish blue, changed to a deep,' intense blue as the
pH increased=
At the same time large amounts of
and captured in the durham tubes=
were evolved
Any tube showing both vigorous
gassing and deep blue coloration was recorded as positive=
By
consulting the appropriate probability table in Methods of Soil Analysis
(1965), part 2, readings can be converted to a MPN value per ml=
Urea Utilizers=
Bacteria hydrolyzing urea were cultivated on a
modified urea soil extract agar medium ,of Allen (1957).
TABLE X=
Urea soil extract medium.
Urea
Soil extract
Tap H2O
Agar
. pH
5.0
g
0 =5
g
. 100
ml
900
ml
15
g
7 =4-7 =6
An alternative medium was developed which was identical except
for. the addition of 10 drops of a 17. tincture of phenolphalein per
liter which, it was hoped, would make the colonies stand out.
After
O
.appropriate dilutions were plated, the cultures were incubated at 28 C
24
for approximately 4 to 7 days.
smooth white appearance.
Urea hydrolyzers normally- have a
Such colonies were counted using a New
Brunswick Scientific electronic colony counter.
'
'
■
' Flow Studies.
It was necessary to correct data of the 24 hour
studies for flow in order to determine net change between stations.
The' procedure of Odum (1956) and Wright (1967) was used.
■
Flow time
.
between stations was determined by detection of fluorescent rhodamine
B dye introduced upstream.
A Turner Fluorimeter model H O with
rustralc recorder and, continuous flow cell was used to detect the dye
downstream.
:
■
When flow time and bacterial populations have been determined
■■
data can be corrected for flow as follows: An arbitrary point in the
study area was designated zero and flow time between it and downstream
stations was calculated. , In this case station 7 was selected as zero.
Data were plotted a s ■received for the null point.(station 7) with time
as abscissa and number of organisms as ordinate.
Data for downstream
were displaced to the left in an amount equal to the flow time between
the station and .station 7.
For example, at 1200 hours on August 1st,
1967, the coliform count at station 9 was 33,000 per ml.
Since the
flow time in this case was 9 minutes, the point was plotted at 11:51 on
the time scale.
In this manner it was possible to study net changes of
the populations in the same body of water as it flowed downstream.
;
,
a
-
!
<
i1
h
i ,
:
RESULTS
Flow Study-
On July 22, 1968 an experiment was undertaken to
determine flow times within the Bozeman sewerage system;
At.1330
hours two liters of a 40% solution of rhodamine B in gla&iul- acetic
acid were dumped'into a sink in the laboratory. . The results are shown
in Table XI.
TABLE XI.
Results of a tracer study.
Elapsed time in minutes.
From laboratory to plant
80 ^ —
Retention in plant
30
Plant to river
10
Total time (Maximum flow time to ,river)
,
Minimum time (Firstdetected in river)
Plant flow (Cap. 4 M.G.P.D.)
120
;
105
4.1
The Bozeman sewage is treated as follows: The influent is
screened to remove large debris and ground to reduce the particle•
size.
The sewage is then passed into a clarification pond where the
large., particles settle out.
The supernatant is then chlorinated and
passes to the river via an.outfall located at station 8, about 0.4
mile (0,64 km) north of the treatment plant. - Sludge from the clarification pond is pumped to a series of two digestion tanks, then to
drying beds.
The sewage plant capacity is frequently exceeded and
needs secondary treatment facilities as well.
The volume of influent -sewage received by the treatment plant on
dates when 24 hour sampling was done is shown in Table XII.
TABLE X U .
Influent sewage flow at the treatment plant
(MoGcPiD0) at various times.during the day
over the summer of 1967.
Aug- I & 2
Aug, 29 & 30
0600
2.8
3.4
3.2
0900
3.5 '
3.9
3.9
1200
3.6
3.9
3.9
3.8 .
4.0
3.8
3.9
3.9
.
1500
•
Aug. 15 & 16
1800
3.8
..
2100
3.8
3.8
3.6
2400
3.6
3.6
3.5
0300
2.8
3.1
3.1
0600
2.8
3.1 '
3.0. .
The flow time increased during the summer as river flow decreased
(Table XIll)„
for this reason water took much longer to pass downstream
in late summer than in spring or early summer.
■ 'TABLE XIII.
Flow times -(minutes) between stations at the
•stations sampled during 24 hour sampling periods
.throughout the summer of 1967»
Stations
Date
7-9
August.1-2 -
.9
August 15-16
10
9-10
■ 70
V
\
August 29-30
11
10-11
116
103
133 .
104
165
27
Summer 1967 Vfork-
The results are recorded in Tables XIV through
■XVI and in Figures 2 through 10.
The microbial population tended to be
most numerous when the water warmed during the late summer.
Coliform Organisms.
shown in Table XIV.
Values for the coliform populations are
Counts above the outfall, with the exception of
one value, showed a slow increase in numbers as the water flowed through
Bozeman.
Bozeman Creek south of the city had a very low coliform
population but numbers increased sharply as the flow passed through
Bozeman and in fact it was the major contributor above station 7.
It is
possible that coliform organisms entered the stream by septic tank
drainage from nearby residential areas.
In contrast, the counts in
Bridger and Rocky Creeks did not show such rapid increases =
Between
stations 7 and 9 numbers increased 286%, a result of outfall pollution
at station 8.
Between stations 8 and 9,numbers decreased 53% due to
dilution by the East Gallatin River.
Despite the low numbers of
coliform organisms entering between stations 9 and 10 with the flow of
Bridget Creek, the counts increased 47% between these two stations.
satisfactory explanation has been-found.
No
Between stations 10 and 11
numbers decreased 55%, possibly as the result of an increased death
rate.
The population increased betweep. stations 11 and 12 possibly
because of an intermittent source of pollution.
Enterococcal Organisms.
Numerical values for the enterococcal
populations have been placed in Table XIV.
Generally the enterococcal
28
counts closely paralled the coIiforms, although they were much lower.
Counts in Rocky and Bridget Creeks showed a slow, increase in numbers
When flowing towards Bozeman but counts in Bozeman Creek increased' ■
sharply in it's passage through town.
:
Counts reached a maximum at
station 7 and decreased slightly downstream, tending to approach a
constant value.
Total Counts.
XV.
Results of the total count are presented in Table
The populations fluctuated greatly but tended to increase as the
water passed through Bozeman.
was from Bozeman Creek.
288%.
Above station 7 the major contribution
Between stations 7 and 9 numbers increased
. Numbers decreased 39% between 9 and 10 and increased 62% between
10 and 11.
24 Hour Studies.
On days when a 24 hour study was made, samples
were collected at 0600, 0900, 1200, 1500, 1800, 2100, 2400 and 0600
hours at stations 7 through 11.
Coliform and cnterococcal populations
were quantitatively determined by MPN as previously discussed.
times were used to correct results for flow.
Flow
Data for each 24 hour
study were plotted in the following manner: Coliform and enterococcal
numbers were corrected for flow time and plotted■against time.
Such
plots showed gross differences in the Same body of water at different
stations.
Figures 2 and 3 show plots for the data of August 1-2, 1967.
Mien the net differences between the stations are computed from plots
corrected for flow time the result is a net rate of change such as.is
29
TABLE XIV.
Number of organisms per 100 ml in water samples taken
from the sampling stations during the summer of 1967
on 7/11, 7/19, 7/25, 8/1, 8/8 and 8/28.
Coliform organisms
Station
min.
I
150
19,000
76,000
2
4,600
7,400
10,000
'3
3,500
4,500
6,000
. 4
.3,000
•5
3,000
6
7,300
7
mean
max.
8,100 '
16,000
9,400
16,000
23,000
42,000
. 4,900
13,000
45,000
3,000
100,000
540,000
9
16,000
49,000
160,000
10
6,100
72,000
160,000
11
2,000
40,000
120,000
■12
28,000
79,000
130,000
8
'
;
' -
Entcrococcal organisms
I
9
170
470
2
370
450
750
3
30
430
840
4
190 .
380 '
630
5
380
610
1,100'
6
770
1,300
2,100
7 .
460
3,400
11,000
8
30
2,400
19,000
9
300
2,400
11,000
’ 10
460
3,100
390.
2,600
7,900
2,900
6,300
11
12
1 ,100
'
11,000
30
TABLE XV.
Station
Total number of organisms per ml in water samples
taken at the sampling stations on 7/11, 7/19, 7/25,
and 8/8. 1967.
min.
mean
THaX o
.1
3,000
850
19,000
2
3,000
17,000
31,000
3
3,000
5,300
10,000
• 4
. . - 3,000
30,000
54,000
5
3,000
22,000
34,000
6 •
4,200
69,000
96,000
7
3,000
50,000
92,000
8
5.000
3,300,000
6,520,000
.9
8.000
190.000
320.000
10
4,200
120.000
320.000
11
2,800
191.000
470,000,
12
2,800
110.000
210.000
31
presented in Figure 4.
Data from the three 24 hour studies are summar­
ized in Table XVI„
TABLE XVlo
Percent change in numbers between stations.
Coliform population
I
-19.0
Co
• +738.0
% change
10+11
9-10
.7-9
Stations
Ln .
..
Enterococcal population
Stations
7-9
% change
-8.9
10-11.
9-10 '
-19.7
-46.4
..
Table XVI shows that, for coliform organisms a large increase in
numbers occurred between stations 7 and 9; a result of outfallu
pollutiono
"
'
’
'
A moderate decrease in numbers occurred between stations
9 and 10 and a large decrease between 10 and 11 was evident.
For the,.
enterococcal population a slight decrease in numbers occurred between
7 and 9, probably as a result of effluent chlorination.
A moderate
decrease was observed between 9 and 10 and great decrease occurred
between 10 and 11.
Two periods of peak demand on the river were observed, one
between 0900 and 1200, the other near 2400 hours.
It can. be seen, that
during periods of light pollution (morning), recovery (from the standpoint of coliform populations) is nearly complete at station 11.
:
'■ n
:•
1
r
r:
.
n o
ENTEROCOCCAL ORGANISMS PER IOO ml. x IOOO
Figure I.
...
-------
0600
STATION
STATION
STATION
STATION
STATION
1200
7
8
S
10
11
1800
2400
OSOO
HOUR
Results of a 24 hour sample taken August I & 2, 1967. MPN
enterococcal counts were determined on samples collected
at stations 7, 8, 9, 10 and 11 at 0600, 0900, 1200, 1500,
1800, 2100, 2400 and 0600 hours.
Data were corrected for
river flow using the procedure of Odum (1956) and Wright
(1967) described on page 24.
33
1500 -
STATION
STATION
STATION
STATION
STATION
7
8
9
IO
11
CU
2 0 0 i- /
IOO
/ •
0600
Figure 3.
1200
1800
HOUR
2400
0300
Results of a 24 hour sample taken August I & 2, 1967. MPN
coliform counts were determined on samples collected at
stations 7, 8, 9, 10 and 11 at 0600, 0900, 1200, 1500,
1800, 2100, 2400 and 0600 hours.
Data were corrected for
river flow as described on page 24.
34
P"
500 I-
OOI X Knoi I / '|W 001 K3c! SriolNVOKO NI 30NVH0
o
/
200
I
-
100 ^
•-0XV
/
?«"
O K
-100
:or
(
\
•
\
! COLSFORM
..-o
- £ 0 0 1- \
\
-- STATIONS 7-9
—
STATIONS 9-10
-- STATIONS IO-11
■500 1X
4
IOl_____L
OSOO
Figure 4.
I
1200
i
i
IS00
HOUR
'
I
2400
!
I
OSCO
Rate of net change between stations over a 24 hour period
August I & 2, 1967. The rate of net change was obtained
from Figures 2 and 3 by calculating the net difference
between stations 7-9, 9-10 and 10-11.
35"
TABLE XVTL.
Station
Number of anaerobic heterotrophs per ml in water
samples collected at the sampling stations.
7/3/68
5/15/68 ''
2
570
1,200
4 '
580
570
850
' 3,400
1 ,000
1,500
.1S
6
■
,
2,000
7
640
8
15,000
9
1,300
10
.1,300
2,500
11
940
1,800
15,000
%
Table XVII shows the anaerobic heterotrophic population.
2,600
On the
earlier date (5/15/68) little appreciable increase occurred above
station 7«
The largest increase was between stations 7 and 9, after
.
which numbers decreased and tended to approach populations at stations
2 through 7.
On the later date (7/13/68) numbers tended to be higher.
Only slight increases occurred between stations 7 and 9, with slight
decreases between 9 and 10 and rapid decreases between 10 and 11 that
approached figures recorded at stations 2 through 7.
36
;xviii.
Total number of aerobic heterotrophs per ml in
water samples taken at the sampling stations.
-
8/5/68
20*C
35°C
20°C
35*C
2
2,900
570
10,000
1,300
4
3,500
230
5,600
520
5
5,000
530
7,900
2,000
6
1,900
.830'
21,000
4,800
7
4,000
400
12,000
3,600
8
27,000
31,000
1,200,000
• 470,000
9
5,400
■ 550
78,000
35,000
10
2,800
310
79,000
30,000
11
3,700
370
45,000
13,000
Station
'
5/8/68
Population at 20°C
5/8/68
= 7.7
Population at 35°C
Population at 20°C
8/5/68
= 2.9
Population at 35°C
Table XVIII presents the aerobic heterotrophic population.
On the
earlier date (5/8/68) only slight changes occurred between stations.
On the-later date (8/5/68) significant increases occurred between
stations 7 and 9, with a tendency for a decrease downstream.
Above
station 7 and major contributor of these forms was Bozeman Greek, follow­
ed by Rocky and'Bridger Creeks =
If the count at station 8 is excluded,
it, can be ,shown that the population at 35 C was present in a lesser
proportion on the earlier date (5/8/68).
This suggests that organisms
■of soil origin were present to a greater degree during spring runoff.
37
TABLE XIXo
MPN of anaerobic nitrogen fixing organisms per ml
in water samples taken at the sampling stations=
5/1/68
Station
- 8/5/68
2
7 =9 ,
0=45
4
3 =3
0.2
'5
3.3
0.2
6
2.3
0.78
7
4.9
0.45
8
270=0
24.0
9
22.0
4.9
13.0 .
1.3
10
•
11
7 =9
0.2
Table XIX shows the population "of anaerobic nitrogen fixing
organisms=
Above station 7 very low numbers were evident but" a" Large
increase occurred between I and 9=
There was a decrease downstream
until the low figures recorded at stations 2 through 6 were similar •
to those at station 11=
Data for the later date indicated lower
numbers but the pattern was similar.
38
TABLE XX„
IfPM of denitrifying organisms per ml in water
samples taken at the sampling stations.
5/1/68
7/9/68
2
28
33
4
11
27
Station
5
6
7.9
'
350
12
■ .
6.4
7
- 48
40
8
470
35,000
■9'
330
1,300
10
140
240
' 11
21
1,300
Table XX presents the number of denitrifying organisms per ml.
Upstream from station 7 data indicated low populations except for the
value observed at station 6 on 7/9/68=
Greatest increases occurred
between stations 7 and 9 after which numbers tended to approach the
low value recorded fcpr stations 2 through 5.
major contributor was Bozeman Greek.
Above station. 7 the
■
39
TABLE XXI.
Number of nitrite oxidizing organisms per ml in
water samples taken at the sampling stations.
Station
1,000
4
560
240
5
1,200
2,000
■ 3,500
5,600
7
1,700
4,100
8
32,000
58,000
9
4,000
12,000
10
7,200
4,400
7,600
4,300
11
•
;
950
2
6 .
,
7/22/68
'5/20/68
Table XXI shows the population of nitrite oxidizing organisms.
Upstream of station 7 greatest numbers occurred at station 6 on both
dates.
Greatest increases occurred between stations 7 and 9.
On the
■later sampling date numbers below station 8 tended to decrease to
numbers recorded- for stations upstream, on the other hand, on the
■earlier date'they tended to increase.
40
TABLE XXII.
Number of ammonia oxidizing organisms per ml in
water samples taken at the sampling stations
5/20/68
7/22/68
2
1,800
1,850
4
5,700
900
5
7,400 -
3,200
6.
8,400
9,800
7
' 5,300
5,800
8
100,000
170,000
9
11,000
10,000
10
'16,000
10,000
14,000
■ 7,400
;ation
11:
Table XXII indicates the populations of ammonia oxidizing
organisms.
Only slight increases in numbers may be seen between the
dates sampled.
As previously noted station 6 had the highest numbers
of the stations upstream from station 7, and numbers approximately
doubled between stations 7 and 9; but between. 9 and 10 numbers in­
creased slightly or remained constant and then decreased slightly
downstream to station 11.
41
TABLE XXIlI.
Number of sporeforming organisms per ml in
water samples taken at the sampling stations.
Station
2 ■
' 5/8/68
8/5/68
■ 120
37
4
44
5
74
6■
35
7
63
'
21
31
.
91
_
48
7
51
9
58
80
10
90
55
130
93
. 8
'
11 .
‘ '
Table XXIII shows the sporeformer population.
Numbers on the
earlier date tended to be higher than those at the later sampling
date.
Great fluctuation was' apparent and appeared to be due to
agricultural practices in the vicinity of the sampling stations.
both cases maximum numbers occurred at station 11«' Station 4
(Bridger Creek) tended to contribute low numbers of sporeformers.
In
42
TABLE XXIV.■
Number of urea hydrolyzing organisms per ml in
water samples taken at the sampling stations.
7/22/68
Station
2
660
4
- 100
5
2,100
6
3,000
"..7
8 .
’
3,100
13,800
9
4,400
10
3,700
11
4,000
Table -XXIV presents the urea hydrolyzing population.
Only
one sample is available since modification to the formula was first
necessary.
Station 4 (Bridger Creek) had'the lowest numbers.
In
general moderate increases occurred below station 5 with only slight
increases between stations 7 and 9, below which numbers tended to
remain constant.
'i
43
’ TABLE XXVo
Number of aerobic cellulose decomposing organisms
per ml in water samples taken at the sampling
stations=
7/3/68
6/23/68
Station '
100
4
■ 210
25
170
60
.6
250
30
7
400
30
8
230
20
30
35
160
90
230
50
5.
9
'
10
■n
• ■
■
■i
50
2
Table XXV shows the numbers of aerobic cellulose decomposing
organisms=
Numbers tended to be highest on the earlier sampling
date, probably d u e .to runoff-
Considerable fluctuation was apparent
and seemed dependent on terrain and agricultural practices- .
The
population of anaerobic cellulose decomposing.organisms was low.
counts were between 2 and 5 organisms per ml.
ficient to warrant any inferences.
\
All
The data were insufr-
DISCUSSION
A downstream increase in numbers of coliform and enterococcic
organisms and total numbers was shown in the Bridger and Bozeman Creeks
as well as the East Gallatin River
itself.
Of the three streams
forming the East Gallatin River the major contributor to the microbial
groups was Bozeman.Creek, followed by Rocky and Bridger Creeks.
The;
great increase found in Bozeman Creek is no doubt due to its passage
through Bozeman.
The study revealed no real indication of pollution due to the
stockyard and slaughterhouse located on Rocky Creek but should not
preclude the.possibility of intermittent pollution from these sources.
It is possible that a significant portion of the .pollution of Bozeman
Creek comes from homes bordering the creek since most use a septic
tank.disposal system.
The traditional indicators of fecal pollution are coliform and
enterococcic populations.
A review of the data does show a four fold
increase in numbers of coliforms between stations 7 and 9 due to the
sewage outfall.
However, on dates of single samples numbers increased
497. between stations 9 and 10 and 99% between 11 and 12.
If it were
not for the influx of Bridger Creek between stations 9 and 10 the
increase would have been even greater.
The observed increases could.•
have been due to growth of organisms in the river itself or to pollution,
from adjacent agricultural areas.
Hanes, Rohlich and Sarles (1966) and
Scarce, Rubenstein-and ’Megtegian'(1964) in laboratory studies using
samples' of raw wastewater diluted with B.O.D. water found that coliform
45
populations could increase in numbers at IO0C 3 20°C, and 30°C but
that enterococcal populations could not.
In the absence of data to
confirm pollution of agricultural origin it -would seem that increases
in numbers could be attributed to growth.
Results of the flow time
data indicate that sufficient time existed particularly in late
summer.
The decrease in numbers of coliform organisms between stations
9 and 10 could have been due to an increase in the death rate, an-.
increase in predation, or to a settling out of the particulate matter
to which the bacteria are attached.
However, the most likely cause
is a dilution effect due to the flow of Bridger Creek entering between
stations 9 and 10.
In the case of. the enterococcal organisms the picture changes.
The mean values indicated a high at station 7 decreasing to a nearly
constant value .at the lower stations.
The low value recorded at the
sewage outfall may have resulted from the practice of chlorinating the
effluent.
The enterococcal .group is noticeably less resistant to
S' . . .
extremes of environment than is the coliform group. The results of
counts at stations 9, 10 and 11 would tend to agree with the findings
of Hanes, Rohlich and Series (1966) since,numbers, did not appear to
increase appreciably because of growth in the stream.
This could
indicate that fecal pollution below the outfall is minimal.
The 24 hour studies were set up to show periods of demand on
the river and recovery from pollution originating from the sewage
outfall.
It would seem that numbers of;enterococcal organisms do not
-
1j ■
46
serve as a reliable index of fecal pollution because of the low. values
observed at and below the outfall*
It is possible that in the absence
of chlorination results would have been completely different.
studies showed a more regular pattern.
Goliform
During periods of lesser
pollution (1400 to 1900 hours), recovery of the river although never
complete, was very, nearly so at station 11.
That is, numbers at station
11 tended to approach numerical values, at station 7.
During periods of
peak pollution (0900 to 11400, 2000 to 2400 hours) recovery was far
from complete even at station 11.due to the higher microbial load carried
by the river during these periods.
Aerobic heterotrophic counts were much higher at 20°C than,at 35°Co
This was also observed by Boyd and Boyd (1967) in a study of arctic
waters and by Harrison, Keller and Lombard,(1963) in a study of the Vaal
River (South Africa)•
If numbers, at 20 C ,are compared with numbers at
•35°C it is seen-that the proportion of,organisms at 35°C is much lower
for the.earlier sample (see Table XVIII).
This seems to indicate that
a greater proportion of soil organisms' and,.organisms attached to organic
matter entered with the runoff during the spring.
Anaerobic heterotrophs showed about a two-fold increase in numbers
between sampling dates except at the outfall.
This group could indicate
the level of organic pollution in the water.'
-It has been suggested by some sources, ,(Rodina, 1964) that anaerobic
nitrogen fixers (principally Clostridium pasteurianum) could serve as a
very sensitive criterion of organic pollution.,
i
My results indicate that
47
this could well be the case and further suggest using C. pasteurianum
as an indicator of pollution from soil sources.
I was unable to
verify the suggestion made by Deufel (1965) regarding use of
Azotobacter as an indicator of organic pollution.
The medium also
allowed growth of other organisms which made counts confusing. '
Ryabov (1965) and Luchterowa (1962) suggested the.use of denitrifiers as pollution criteria.
suggestion.
The results of this study supported this
The rise in numbers at .stations 6 and 8 probably indicate
organic but not necessarily fecal contamination at these points. (At
8 fecal contamination obviously occurs).
The rise in numbers between-
stations 10 and 11 has already been noted and probably indicates
pollution of an intermittent source.
:
The group of nitrite and ammonia oxidizing organisms is auto­
trophic and seems to be a sensitive index to the level of the partic­
ular substrate. • In general, numbers follow closely the nitrite and
ammonia concentrations found at each station (Soltero, •1968).
Chemical data indicated a low level of nitrite, below the outfall
which increased very slowly as the water passed downstream.
Numbers
of nitrite oxidizers follow this chemical change very closely.
Ammonia
concentration was found to be much higher than nitrite and to increase
slightly below the outfall to station 11, Soltero, (1968).
v
Numbers of
ammonia oxidizers were found proportionate, ;to ammonia levels, hence
they would be expected to be much more numerous than nitrite oxidizers.
48
Sporeformeirs do not seem to indicate fecal pollution but seem
indicative of pollution from soil origin*
This can be seen by the
high counts during runoff at irregular points in the study area.
High counts just below station 9 are easily seen by the dirt, concrete
blocks, etc. pushed into the river at this point.
It would seem that
pollution introduced between stations 10 and 11 is of soil, rather
than of fecal origin.
No chemical data were available for comparison with numbers of
urea hydrolyzing organisms.
The observed decrease 'between stations 9
and 10 seems to be a result of substrate dilution.
An increase in
numbers of urea utilizers between stations 10 and 11 would be expected
if organisms were to utilize the urea remaining at these points.
Changes in nupbefs tend to suggest that the origin of cellulose
decomposing organisms was from adjacent fields rather than fecal and .
organic sources and probably represents a good index of pollution from
soil origin.
This;Is supported by the higher numbers found at various
stations during spring runoff and low numbers found in the sewage
effluent.
SUMMARY
During the summer of 1967 coliform, enterococcal and total
microbial populations were determined at stations located on the
East Gallatin River, and on Bozeman, Bridger, and Rocky Creeks near
Bozeman, Montana,
On dates when single samples were collected (7/11,
7/19, 7/25 and 8/8) all three groups were quantitatively determined
at 12 stations.
On dates when samples were collected every 3 hours
during a 24 hour period (8/1 and 2, 8/15 and 16, and 8/29 and 30)
coliform, and enterococcal populations were quantitatively determined
at 5 stations, one.situated above, one out of, and 3 below the sewage
outfall.
During the spring and summer of 1968, total numbers of bacteria
and organisms classed as anaerobes, sporeformers, ammonia and nitrite,
oxidizers, aerobic and anaerobic cellulose'decomposers, aerobic and
anaerobic nitrogen'fixers, denitrifiers,■and urea utilizers were
quantitatively determined at 9 stations.
Except for urea utilizers,
data for each group were based on samples taken on two different dates
Above the sewage outfall the major pollution was from Bozeman
Creek and could,have been of fecal origin.
Below station 7 the sewage
outfall was a major contributor but pollution enters from nearby
agricultural areas as well.
There was some indication of partial
recovery from outfall pollution 5 miles downstream'of the sewage
outfall.
50
Results from determinations.of aerobic hcterotroph, anaerobic
nitrogen fixer, sporeformer, and cellulose decomposer populations
suggested the possibility of serious pollution from agricultural
sources during spring runoff.
i
Results from counts of the anaerobic nitrogen fixer, dcnitrifier,
ammonia .and nitrite oxidizer populations seem indicative of 'organic
and hence fecal pollution from the sewage outfall.
If so, their
numbers appear to indicate that under certain conditions partial
or nearly full recovery from the sewage effluent may occur within
5 miles below the outfall.
APPENDIX
ENTEROCOCCAL ORGANISMS PER IOO ml. x IOOO
52
Figure 5.
-- STATION
• * • STATION
--- STATION
---STATION
---STATION
"-(j -j-y.'
0600
I
•1’— ...
1800
HOUR
2400
0600
Results of a 24 hour sample taken August 15 & 16, 1967.
MPN enterococcal counts were determined on samples
collected at stations 7-11 at 0600, 0900, 1200, 1500,
1800, 2100, 2400 and 0600 hours. Data were corrected
for river flow using the procedure described on page 24.
53
COLIFORM ORGANISMS PER IOO ml. x IOOO
1500
• STATION
STATION
-- STATION
- - STATION
-- SiAi ION
IOOO
7
S
9
IO
11
400 -
200
:■
IOO -
0600
1200
1800
2400
0600
HOUR
Figure 6.
Results of a 24 hour sample taken August 15 & 16, 1967.
MPN coliform counts were determined on samples collected
at stations 7-11 at 0600, 0900, 1200, 1500, 1800, 2100,
2400 and 0600 hours.
Data were corrected for flow using
the procedure described on page 24.
54
-IOO
COLiFORM
-200
— STATIONS 7-9
--STATIONS 9-10
— STATIONS IO-Il
—
y
ENTEROCOCCAL
0600
1200
ISOO
2400
0600
HOUR
Figure 7.
Rate of net change between
August 15 & 16, 1967. The
ed from Figures 5 and 6 by
between stations 7-9, 9-10
stations over a 24 hour period
rate of net change was obtain­
calculating the net difference
and 10-11.
ENTEROCOCCAL ORGANISMS PER IOO ml. x IOOO
55
--...
-------
OSOO
STATION
STATION
STATION
STATION
S .ATiON
1200
7
S
9
IO
11
1300
2400
0300
HOUR
Figure 8.
Results of a 24 hour sample taken August 29 & 30, 1967.
MPN enterococcal counts were determined on samples
collected at stations 7-11 at 0600, 0900, 1200, 1500,
1800, 2100, 2400 and 0600 hours.
Data were corrected
for flow using the procedure described on page 24.
56
COLIFORM ORGANISMS
P E R IOO ml. x I O O O
!500 -
--IOOO -------
STATION
STATION
STATION
STATION
S iA.ION
7
8
9
10
II
o
500
400
Figure 9.
Results of a 24 hour sample taken August 29 & 30, 1967.
KPN coliform counts were determined on samples collected
at stations 7-11 at 0600, 0900, 1200, 1500, 1800, 2100,
2400 and 0600 hours.
Data were corrected for flow using
the procedure described on page 24.
57
C H A N G E IN O R G A N I S M S P E R IOO m l . / H O U R x I O O O
300 -
COLIFO RM
200
__ STATION'S 7 - 9
— STATIONS 9 - 1 0
— STA TIO N S 10-11
ENTEROCOCCAL
-IO -
2400
0600
OSOO
HOUR
Figure 10.
Rate of net change between stations over a 24 hour
period August 29 & 30, 1967. The rate of net change
was obtained from Figures 8 and 9 by calculating the
net difference between stations 7-9, 9-10 and 10-11.
58
TABLE
'
' Number of organisms per 100 ml in water samples taken
at the sampling stations 8/1-2/67.
X X V I o
PdIifairm
Time»
Itims
Stations
8 ’
9
10
. 11
11,000
28,000
161,000
35,000
35,000
7,000
54,000
35,000
54,000
14,000
2,000
350,000
■ 33,000
920,000
1500
4,000
49,000
49,000
12,000
13,000
1800
2,000
9,000
14,000
17,000
17,000
2100
7,900
160,000 ■
35,000
54,000
54,000
2400
2,000
1,600,000
240,000
220,000
33,000
0600
11,000
540,000
17,000
130,000
N>
O
O
O
7
0600
.
0900
1200
.
-
Enterococcal organisms
0600
3,300.
200
11,000
11,000
7,900
0900
3,300
17,000
7,000
1,300
1,700
1200
4,000
13,000
2,000
5,000
-
1500
5,000
2,000
2,000
2,000
2,000
1800
2,000
2,000
2,000
2,000
2,000
, 2,300
.4,900
3,300
4,900'
2400
2,100
54,000
4,900
4,900
4,900
0600
2,200,
200
3,300 .
1,700
1,400
2100 '
not determined
' . 2,300
59
TABLE XXVII„
Number of organisms per 100 ml in water samples taken
at the sampling stations 8/15 -16/67.
do 11 leriti organisms
i'
Stations
Time
8
7
.
11
92,000
24,000
160,000
17,000
92,000
79,000
160,000
24,000
17,000
1200 .
7,900
79,000
79,000
49,000
14,000
1500"
7,900
79,000
79,000
23,000
7,000
1800
4,900
240,000
27,000
23,000
14,000
2100
3,100
540,000
110,000
49,000
17,000
2400.
7,900
1,600,000
240,000
130,000
17,000
0600
7,000
2,000
5,000
31,000
92,000
•
0900
.
10
7,900
0600
'
9
Enterococcal organisms
200
1,700
3,300
4,900
' 35,000
13,000
7,900
200
200
' 500
3,300
2,300
2,300
200
200
1,700
. . 3,300
200
500
•1800
400
200
1,300
7,900
'soo
2100
2,200
11,000
4,600
3,300
200
2400
2,100
' 13,000
2,300
13,000
3,300
11,000
200
4,900
3,300
0600
3,300
0900
1200
'1500 .
■
;
'
0600
.
'
500 '
60
TABLE XXVIII.
Nurnbe::: of organisms per 100 ml in water samples
taken- at the sampling stations 8/29- 30/67.
Gollform organisms
Time
Stations
7
8
9
10
11
0600
7,900
7,900
17,000
17,000
35,000
0900
- .1,700 '
79,000
160,000
92,000
13,000
1200
13,000
540,000
79,000
79,000
' 11,000
1500
7,900
110,000-
49,000
33,000
70,000
1800
7,900
130,000
79,000
23,000
17,000
2100
.3,300
350,000
.130,000
23,000
14,000
2400
7,900
I,600,000
350,000
33,000
17,000
0600
4,900
17,000
54,000
160,000
35,000
Enterococcal organisms
0600
9,400
200
1,700'
4,900
3,100
0900
. 3,300
7,900
3,300
■ 1,100
2,200
1200
2,300
13,000
7,900
2,300
200
1500
2,200
13,000
4,900'
1,300
800
1800
7,900
2,300
2,200
2,300
1,100
2100
2,300
7,900
2,200
800
2400
13,000
28,000
13,000
4,900
800
0600
2,100
200
1,300
' 2,100
3,300
. 17,000 '
61 '
Number of coliform organisms per 100 ml in water
samples taken at the' sampling stations during the
summer of 1967.
TABLE XXIX.
Date
8/8 -
8/15
8/28
7/11
7/19
7/25
I
150
350
440
-
76,000
-
-
2
. 9,000
5,900
4,600
-
10,000
-
-
3
3,500
3,900
-
-
6,000
-
-
4
16,000
3,000
-
-
5,300
-
-
5
11,000
3,000
7,600
-
16,000
-
-
6
7,300
11,000
34,000
-
42,000
-
-
7
11,000
5,400
16,000
Il'000
11,000
45,000
7,900
7,000
7.900
4.900
8
30,000
3,000
270,000
28,000
540,000
52,000
92,000
2,000
7,900
17,000
9
31,000
16,000
25,000
161,000
17,000
140,000
24,000
5,000
54.000
35,000
130,000
160,000
160,000
31,000
160,000
35,000
2,000
120,000
17.000
92.000
35.000
35.000
Station
10
11
. .12'
11,000
38,000
80,000
6,100
17,000
-
9,400
13,000
28,000
8/1
130,000
-
- .not determined
i
-
17.000
17,000
-
TABLE XXX
Number of enterococcal organisms per 100 ml in water
samples taken at the sampling stations during the
summer of 1967»
Date
Station . 7/11
7/19
7/25
I
9
23
170
2
270
750
390
3
’ . 30 ' ' 840
8/8
8/15
8/28
470
-
-
-
370
-
-
-
-
420
-
-
-
-
630
-
-
"
540
-
-
-
1,000
-
—
8/1 .
4
190
330
■ 5"
400
i,ioo
. 380
6
770
2,100
1,300
7
460
■ 1,400
630
3,300
2,200
670
3,300
11,000
9,400
2,100
■8
19,000
920
3,000
200
200
30
200
200
200
200
9
' 1,500
940
2,400
11,000
3,300
300
1,700
500
1,700
1,300
460
11,000
1,700
480
3,300
.4,900
4,900
2,100
390
7,900
1,400
480
10
.720
11
.430
12
1,350
. 1,200
■ 1,000 ■
- not determined
6,300'
’
_
»■
1,100
4,900 .
3,300
—
3,100
3,300
'
-
63
TABLE XXXI.
Total count of organisms per ml of water samples
taken at the sampling stations during the summer
©£ 1967a
Date
Station
7/11-
7/19
7/25 .
8/8
I
8,600
3,000
19,000
3,300
2
3,000
31,000
30,000
5,600
3,000
-
3,000
54,000
34,000
-
3,000
5
34,000
30,000
-
3,000
6
96,000
88^000
89,000
4,200
7
69,000
92,000
35,000
3,000
8
6,520,000
-
5,000
■9
270,000
300,000
-
8,000
10
320,000
30,000
“
4,200
470,000
-
2,800
-
2,800
3
10,000 -
4
11
'
12
not determined
100,000 '
210,000
-
-
64
TABLE XXXII.-
O
Temperature in C of water samples taken at the
sampling ■stations during 1967 and 1968.
July and August 1967
Stations
7/11
7/19
,7/25
8/8
I
. 10.8
-
11.5
9.6
2
12.5
-
13.7
11.8
3
11.4
-
-
11.9
4
12.2
5
12.3
6
10.9
I
11.9
14.0
11.6
-
13.0
10.4
11.7
11.6
13.6
11.3
8
12.7
12.8
13.5 .
13.5
9
11.9
11.5
13.6
11.4
10
12.3
11.8
13.9
11.7
11
14.0
12.3
14.5
12.2
12
14.0
15.6
13.4
not determined
65
TABLE XXXII.
CONTINUED.
Aliquot I & 2, 1967
Time
Stations
8
7
9
10
11
0600.
13.0
13.7
13.1
13.3
13.6
0900
12.8
14.0
13.2
13.2
14.8
1200 '
15.1
15.3
15.3
15.1
1500
18 „6 ■
15.4
18.9
18.5
18.9
1800
20.1
15.6
19.5
19.5
20.0
2100
18.6
15.3
18.1
18.2
18.7
2400
16.5
15.0
16.2
16.3
16.6
0600
12.7
13.5
12.6
13.0
13.5
-
•
15.7
August 15 & 16, 1967
13.3
13.7
0900
. 12.4
14.5
12.7
12.6
13.4
1200
15.0
15.7
. 15.3
15.5
1500 ■
19.6
18.5
19.0
18.6
19.3
1800
20.5
16.3
20.1
19.7
20.4
2100
18.7.
18.2
18.2
2400
17.0
0600 •
14.0
16 =6
15.4
16.5
13.4
14.0
13.3
13.5
H
. 15.5
O
14.1
UO
14.0
H
CO
13:0
0\
0600
.
,
•
' 16.0
66
TABLE XXXII0
CONTINUED,
August 29 & 30, 1967
Time
Stations
7
8
9
11 ‘
10
0600
13.0
14.3
13.1
13.3
13.5
0900
12.6
14.7
13.0
13.0
13.5
1200
14.0
15.6
14.3
14.4
15.0
15.00
17.6
16.0
17.6
17.2
. 18.0
18.0
15.8
17.6
18.0
18.3
2100
16.6
15.4
16.4
16.6
17.1
2400
15.0
15.4
1&.7
15.0
15.3
0600
12.5
14.2
12.6
12.9
13.2
1800
-
Temperature in °C, 1968
Date
Stations
5/1
5/8
5/15
5/20
5/28
7/3
7/19
7/22
8/5
2.
5.0
4.5
4.0
5.5
8.0
13.0
16.2
15.3
15.0
4
6.0
5=0
4.6
5.8
.8.0
11.4
14.0
14.5
14.8
5.5
4.6
4.0
6.0
8.0
13.5
16.7
15.8
’ 16.0
7.0.
5.2
4.0
' 5.4
7.4
10.2
13.8
14.0
13.4
'5
.
6
:
C
•' 7
6.0
4.8
4.0
5.8
8.0
12.3
16.0
15.5
15.0
'8
12.0
12.0
12.5
13.0
13.1
14.8
15.5
16.0
16.2
"9"
6.0
5.3
4.5
6.0
8.0 '
12.5
16.2
15.8
15.4
10
6.0
5.0
4.6
6.0
8.4
12.5
15.5
15.4
15.3
11
6.3
5/0
4.8
6.2
8.5
13.0
16.0
16.0
15.6
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