'E'HE BACTERIAL TRANSLOCATION or
RADIOACTIVE PHOSPHORUS THROUGH
A Lona ecosvsraM

Thesis for the Beam of M. S.
MlCHIGAN STATE UNIVERSITY
AMchaeI E. Bender 7
I962

43.6. (3.434
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Michigan State
University

 

 

 

THE BACTERIAL TRANSLOCATION 0F RADIOACTIVE PHOSPHORUS
THROUGH A LOTIC ECOSYSTEM

By
MICHAEL E. BENDER

AN ABSTRACT

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1962

Approved

 

 

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ABSTRACT

[The recent availability of radioisotopes has enabled
ecologists to fo1low nutrient cycles in many habitats with
great precision.. Radioactive phosphorus (P32) has been
widely employed in the study of the phosphorus cycle in
lentic environments. The investigators of these lake systems
have found that phytoplankton and bacteria are the primary
fixers of phosphorus in the community.

In 1958, 1959, and 1960, inorganic P32 was applied to
a stream system, the West Branch of the Sturgeon River.

The transfer of P32 was followed throughout the biota of the
stream and was found to follow the trophic levels rather
closely.

In 1961, an experiment was designed to evaluate the
importance of bacteria in the distribution of phosphorus

within the stream system. ‘2, coli 0111 was selected as the

'carrier for the P32, and experiments on its growth and up-

take of P32 were performed. The organism was found to
incorporate nearly 100% of the P32 available when the normal
phosphorus concentration of the media was reduced to .3 mg/
100 ml.

A bacterial culture containing 25 me of P32 was added
to the stream system on July 13, 1961. It was discovered
that upon addition to the stream only a small proportion of
the P32 was released into the soluble state; approximately
90% of the activity was held by the bacterial cells.

In the three previous years of the experiment, nearly
all of the 23 mo of P32 added to the stream was fixed within
the study area, but in this experiment only half of the act-
ivity added was retained within the area.

. During the remainder of the study period; the various
consumer organisms within the system showed lower activity
densities than would have been expected if the bacteria
remaining within the system distributed the P32 as was en-
countered in previous years.

An analysis of P32 uptake in various stream sections
revealed heterogeneity of activity fixation. One section
showed low uptake dUring all four years of the experiment.
The most plausable hypothesis for this finding seems to be
that lower populations of energy fixers are found in this

section.

THE BACTERIAL TRANSLOCATION or RADIOACTIVE PHOSPHORUS
THROUGH A LOTIC ECOSYSTEM

By
MICHAEL E. BENDER

A THESIS

Submitted to
Michigan State university
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife
196 2

ACKNOWLEDGMENTS

During the preparation of this thesis, the author
was aided and encouraged by many people. The author is
particularly indebted to Dr. Robert C. Bell whose enthusiasm
for the proJect made its completion possible, and to
Dr. F. F. Hooper whose many ideas proved valuable through-
out the study period. In the field, the author was assisted
by Richard Bennett, Thomas Wthalik, and fellow graduate
student, James Bacon. For the assistance of these people,
the author is deeply grateful. Dr. W. Carl Latta and his
staff at the Pigeon River Trout Research Station also
contributed a great deal to the project. The author also
wishes to thank Dr. Philip J. Clark for his assistance with
the statistical analyses. The author was assisted by his
wife, Pat, throughout the study in typing, preparing of the
figures, and proofreading.

The study was made possible through a grant held
by Dr. R. C. Ball and.Dr. F. F. Hooper from the Atomic
Energy Commission. The author was supported by a Graduate
.Research Fellowship from the Institute for Fisheries Research

of the Michigan Department of Conservation.

11

TABLE OF CONTENTS

INTROD‘ICTION O O O O O O O O - O O O 0

Description of the Study Area .

Sampling Stations . . . .
Station 3 . . . . .
Station 5 . . . . .
Station 8 . . . . .

St8t10n12 e e e e e »

Station 14 . . . . .

Station 16 . . . . .

METHODS AND PROCEDURES . . . .
General Methods . . . .

The Isotope . . . . . .
Measurement of Activity .

Correction Factors . . .

The Bacterial Carrier . . . .

Test for Presence of Members of

Preliminary Bacterial Studies of the

Coliform
Group by Membrane Filter Technique

..

West Branch of the Sturgeon . . . . .

Normal Bacterial Flora . . . .

Rate of Coliform Mbvement in the

Stream System . . . . . . . . .

Growth of E. coli and Uptake of P32 . .

Sampling Methods . . . . .
Bacterial Methods .
Field . . . . .

Page_

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1h

15
15
16
19
27
27 ’
27

Laboratory . . . . . .

O

waterMEthOdseeeeeeeeeeee

Field . . . . . . . . .

Laboratory . . . . . .

Fish and Lamprey . . . . . .

Field . . . . . . . . .

Laboratory . . . . . ~.

Fish Biomass . . . . .

Addition of the Isotope . . . . .
RESULTS . . . . . . . . . . . . . . .

Bacteria as Phosphorus Carriers . .-. .
Mean Activity Levels for E. coli Cells

Effect of the Stream Environment on

the Bacterial Cells . . . . . . .

Activity Flow and Uptake in Various

Stream Sections . . . . . . . . .

Fish Activity Levels . . . . . .

Fish Biomass . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . .
APPENDIX I . . . . . . . . . . . . . .
APPENDIX 11 . . . . . . . . . . . . .
APPENDIX III . . . . . . . . . . . .
APPENDIX IV . . . . . . . . . . . . ;
APPENDIX V . . . . . . . . . . . . . .
APPENDIX VI . . . . . . . . . . . . .
APPENDIX VII . . . . . . . . . . . . .

32
35 '
35
35
36
36
36
37

39
he '
‘17

53

57

81
82
8h
87
88
89
9o
91
92
93

' Figure
1.

30'
n.

5.
6.’
7.

9.
10.

11.

12.

13.

1h.

15.

LIST OF FIGURES,

Map of the West Branch of the Sturgeon

River area, showing sampling stations

and_site of isotope entry

Flow of g. coli B through the stream
ayatemeeeeeeeeeeeeeeee

Phosphorus utilization bng, coli 0111 .

Uptake of P32 by E. coli 0111 . . . . .

Final uptake of P32 by E. coli 0111 . .

0

Growth of E. coli 0111 in the P32 medium .

Total water activity at collecting
stations during the passage of the
isotope O O O O O O 0 O O O O 0 O O O 0

Peak activity levels reached at the

Activity of acid and water wash filter

membranes at the collecting sites . .

Activity of acid and water wash filtrates

at the collecting sites .

O Q 0 O 0

Activity of the filter membranes and
number of E. coli 0111 organisms per
milliliter at tfie coIIecting sites . . . .

Total and soluble activity passage by
the collecting stations in microcuries . .

Total microcuries passing the.
points in the four years of the invest-

. collecting stations during isotOpe flow

sampling

igations on the stream system . . . . .

Micrbcuries uptake per square yard of the
stream sections in all four years of the

StUdyeeeeeeeeeeeeeeee

Proportion of the activity available to
the activity uptake per square yard in
the various stream sections in all four

years of the Study . . . .

Page

18
2h
26
29
30
Al

Ah

#6

52

56

59

62

65

Figure Page
16. Comparison of fish activity levels

in 1959 and 1961 o e e e e e e e e e e e e 71
17. .Comparison of fish activity levels

in 1959 and 1961 e e e e e e e e e e e e e 73
18. Activity density of fish at collecting

Bites 8 811d 12 e e e e e e e e e e e e .. e 76

19. Activity uptake rates for fish at-
Station 8 O 0 O O O O O O O O O 0 O O O O 78

20. Activity uptake rates for fish at
station 12 . . . . . . . . . . . . . . . . 80

vi

5.

LIST or TABLES '

Mean activity levels for E, coli Cells . .

variation in mean cell count between
stations.................

Uptake in microcuries in the various
stream sections in all four years of
the investigation . . . . . . . . . . . .

A Friedman analysis of variance on
the proportional uptake values . . . . . .

Uptake of P32 by aquatic plants during
1960 in the various stream sections. . . .

vii

Page
50

5h

66

67

LIST OF APPENDICES

Appendix ' _ I ' Page
‘1 Biochemical Characteristics of E, coli . . 87
II Growth Medium for E, coli 0111 . . . . . . 88

III Uptake Rates for Muddlers at Station 12
1n19598nd1961............. 89

IV Brown Trout Uptake Rates at Station 8
1n19593nd196leeeeeeeeeeeee 90

V Brown Trout Uptake Rates at Station 12
in 1959 and 1961 . . . . . . . . . . . . . 91 .

VI Activity values from Acid and Water
Wash Samples During the IsotOpe Passage . 92

VII Fish Biomass Estimations of the West
Branch for the Years 1958, 1959, 1960,
mdlg6leeeeeeeeeeeeeeeee 93

viii

INTRODUCTION

Phosphorus and nitrogen have long been recognized as
the major limiting elements in many habitats. The nitrogen
cycle has been called a near perfect cycle since the element
is circulated through and returns to various trOphic levels
by fairly definite routes. The phosphorus cycle, however,
is not a perfect one because the means of returning phospho-
rus to the cycle may be inadequate to compensate for the loss
(Odum, 1959). At the present time through the efforts of
Hutchinson and Bowen (1950), Rigler (1956), Hayes, et.al.
(1952) and others who have used P32 to study the cycle in
lakes, a fairly definite pattern of phosphorus exchange has
been established. Stream systems, however, have not been
investigated as well.

In l95h, Alexander and Grzenda studied the effects of
phosphorus fertilization on Hoffman Lake and its effluent
stream, the West Branch of the Sturgeon River. These authors
found an increase in periphyton growth downstream from any
detectable phosphorus increase in the water. They attribut-
ed these results to minute increases in phosphorus concen-
*trations below the sensitivity of normal chemical procedures.

In order to follow the pathways of phosphorus trans-
fer within the stream system, P32 was employed as a tracer
in four separate experiments during 1958, 1959, 1960, and
1961.

The trOphic levels sampled during the study were the

consumers, various species of aquatic insects, other inver-
tebrates and fish, and the primary producers, the periphyton.
The 1958 experiment was conducted by Borgeson, Clifford,
and Bryant. These authors found that upon addition of the
isotOpe, the periphyton populations showed immediate uptake

of P32

and rapidly lost this activity which was then bio-
logically incorporated into other organisms. The organisms
which showed initially high activity levels were the plant
feeding forms. Predators and omnivores had the next highest
activity and the scavengers the least. Brown trout, §glmg_
trutta, and muddlers, Cottus cognatus, showed similar act-
ivity levels.

In 1959, Knight followed the transfer of P32 through
the system and found much the same general pattern of phos-
phorus uptake. He noted, however, that much of the actitity
during the initial isotope passage was in the form of par-
ticulate matter. He then postulated that either diatoms
and/or bacteria were responsible for the initial fixation
of phosphorus. Using activity-free-periphyton substrates
cultured outside of the experimental area he was able to
detect, by placing these substrates in the stream immediate-
ly after the isotope passage, a recyling of activity within
an hour after the isotOpe passed.‘

In the 1960 experiment, Zettelmaier found much the
same uptake patterns throughout the stream biota.

Ball and Hooper (1962) have reviewed the results of

the above authors and have calculated uptake values for the

several stream sections.

In the three previous years, the uptake pattern of
'P32 has been shown to follow the various trophic levels
rather closely. The organisms responsible for the immediate
removal of inorganic P32 are unknown. The results of P32
studies on lakes have shown that phytoplankton and bacteria
are responsible for the initial removal. Knight (1961) has
hypothesized that bacteria may be the organisms responsible
for the initial removal in the West Branch of the Sturgeon
River.

In the 1961 experiment, the P32 was incorporated into
bacterial cells and then added to the stream system. The
bacterium, Escherichia 921;, was selected as a carrier organ-
ism because the absence of coliform organisms from the stream
system would permit its separation from the normal stream
flora. Before addition of the bacteria to the stream, the
organisms’growth and uptake of P32 were followed in labora-
tory experiments. With the trophic relationships in the
stream already well established, it was believed that initia-
ting the phosphorus cycle in the particulate form would
define the relationships and importance of bacteria in the

distribution of phosphorus in a stream ecosystem.

Description of the Study Area

The West Branch of the Sturgeon River originates at
Heffman Lake, a 128 acre hard-water lake located in Charle-
voix.County, Michigan. From its origin, the river flows in
a northeasterly direction for 1h miles before entering the
Sturgeon River near Wolverine, Michigan.

As the West Branch of the Sturgeon River continues
its northeasterly flow through Cheboygan, Otsego, and Charle-
voix Counties, the vegetation encountered along its course
consists chiefly of birch, aspen, cedar, tamarack, and _
balsam fir. Cedar, tamarack, aspen, alder, and ninebark are
located along the margin of the river.

The West Branch of the Sturgeon River is a coldAwAter
stream with the temperature of the water varying between
52° F. and 58° F. during the summer months (Clifford, 1959).
The cool temperature of the water can be attributed to the '
numerous springs and tributaries which enter the stream and
the shade produced by overhanging cover.

The study area of the river, located in (T.33 N, R.

3 W), consisted of a section approximately 5,280 yards in
length with the stream bottom varying from sand and gravel
to silt and detritus. The general conditions of the stream
within this area are as follows, I

The stream flow through the study area has a mean of

A3.75 cubic feet per second (Knight, 1961). During the

.summer, rains rarely affected the stable water level or

' h

brought turbidity to the normally clear water. The total
phosphorus concentration in the stream water of the study
area is approximately 6 parts per billion. A high dissolved
oxygen content exists in the stream due to churning and mix-
ing of the water with air. The-aquatic life supported by
the stream is rich in abundance and variety.

Among the main vegetational growths found in the
stream were beds of 92233 32,; water moss, Fontinalis anti:
pygetica; and water cress, Nasturtium officinale. Vegetation
was fairly abundant for the first 550 yards of the stream,
scarce for the next #80 yards, and again abundant plant beds
for the remainder of the study area.

The West Branch of the Sturgeon River was inhabited
by the following fishes: brown trout, Salmg.trutta; rainbow
trout, Salmo_gairdnerii; brook trout, Salvelinus fontinalis;
eastern slimy sculpin, Cottus cognatus; and northern mottled
sculpin, Cottus bairdii.

Aquatic insects present in the stream were those
species associated with swift currents and cold and clean
waters. The orders found in the stream were Odonata, Ephe-
meroptera, Plecoptera, Trichoptera, Diptera, Megaloptera,

Coleoptera, and Hemiptera.

Sampling Stations

 

In a stream system such as the West Branch, a variety
of habitats exist. In former years, 16 stations were estab-
lished which included such factors as shade, stream flow,
bottom type, and vegetation composition. or these establish-
ed sampling points, 6 were used as stations in the 1961
experiment and were numbered 3, 5, 8, 12, IA, and 16 (Figure
1). '

Collections of aquatic plants, periphyton, and aquatic
invertebrates were made at stations 3, 8, 12, and 1h; fish and
lampreys were collected only at stations 8 and 12. The
following is a description of the permanent collecting stat-

ions and also of those stations used to collect water samples.

Station 3 This site was located 300 yards below the addition
point. The immediate area is well shaded, but above the
station, the stream is fairly open to sunlight. Vegetation
in the area consists of th£g_§2,; sparse water cress, Nagy
turtium officinale; Potomogeton pectinatus; and some Ragga:
gglgg_gp, The water has an average depth of 12.8 inches

(Zettelmaier, 1962). The stream flow at this point was
' 38073 Cfs.

Station 5 This station was used only for the collection of
water samples. It is located 550 yards from the addition
point. The majority of the area between 3 and 5 is heaVily
shaded. The flow at this point was 38.73 cfs.

Station 8 This site was located in an open portion of the

 

stream 1,030 yards below the addition point. The vegetation
in this segment consists mainly of Ehggg_gp,; water moss,

thtinalis antipyretica; Potomogeton pectinatus; and 5222!?
gglgg_gp, The mean water depth at this site was 17.2 inches

(Zettelmaier, ibid.). The stream flow was u3.u8 cfs.

Station 12 This station was located in an open section of
the stream 2,580 yards below the isotope addition point. The
vegetation consisted mainly of Qh2£§_§p.; Ranunculus 52,;
Pptomogeton pectinatus; and water moss, Fontinalis antipyb
3.9.2.122. The mean water depth was 13.3 inches (Zettelmaier,

ibid.). The vrow '- at this site was u7.53 cfs.

Station 1h This station was located in a fairly open section
of the stream 3,280 yards below the point of isotope entry.
Vegetation consisted of Chagawgp,3 Potomogeton pectinatus;
Ranunculus 52,; water moss, Fontinalis antipyretiea; and

water cress, Nasturtium officinale. The mean depth in this
area was 12.2 inches (Zettelmaier, ibid.). The stream .

flow_ was h9.72 cfs.

Station 16 The partially shaded site chosen for this station
was located 5,280 yards below the addition point and was used
only for the collection of water samples. The water

flow at this site was 52.5 cfs.

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METHODS AND PROCEDURES

General Methods

The general methods of following the phosphorus cycle
through the stream system are as follows:
1. A study area approximately 5,280 yards in length was es-
tablished on the West Branch of the Sturgeon River. Taking
into consideration various ecological factors, sixteen samp-
ling stations were established in the study area.
2. A fluorescein dye marker, to alert the sampling crews,
was added to the stream each year preceding addition of the
isotope, and in 1961, preceding all bacteria additions.
3. The isotope was added to the stream as inorganic P32 in
the first three years of the experiment. Each year approx-
imately 23mc.were added to the stream at a constant rate
over a period of 30 minutes.
A. Six sampling stations (3, 5, 8, 12, 1A, and 16) were used
to follow the initial isotope passage over a period of l to
2 hours.
5. Four of the sampling stations (3, 8, l2, and 1A) were used
to sample the activity of the stream biota throughout the
remainder of the study period.
The Isotope

The selection of an isotOpe for the study of an aquatic
environment is dependent upon the element which is to be

studied and upon the physical characteristics of the isotope

10

11

which may make it acceptable as a study tool. The radioiso-
tope of phosphorus (P32) has been used in various investi-
gations of the phosphorus cycle in lakes and on the West
Branch of the Sturgeon River.

The radioisotope of phosphorus (P32) is a beta ray
emitter with a maximum energy of radiation of 1.712 New
(Chase, 1960) and has a half-life of 1h.3 days. The isotope
was supplied by the Oak Ridge National Laboratory, Oak Ridge,
Tennessee as phosphate (Pou) in weak HCl.

The procedures used to incorporate the isotOpe into
the bacterial cells will be described fully in a later
section.

The method by which the isotOpe was added to the _
stream has been described by Borgeson (1959), Clifford (1959),
and Knight (1961).

Measurement of Activigy

Activity was measured with a Nuclear Measurements
Corporation windowless,gas-flow,pr0portiona1 counter,mode1
FCC—10A; coupled to a decade scaler, model DS-lA; and a
single unit counter and scaler, model number PC-3A.

In order to represent activity values obtained in a
meaningful fashion, the following correction factors have
been given by Robeck, et.a1. (l95h).
§§1f Adsorption: .

Self adsorption is an interference which causes a
lower observed count due to the residue adsorption of some

of the particles, especially those of low energy.

12

Back Scatter:

 

Back scatter is the interference which causes an in-
crease in observed counts due to the deflection of radiation
by the sample support or shielding.

Decay Factor: I

Decay factor is due to the decrease with time of the
number of radioactive atoms in a sample. Counts must be
corrected to time zero for decay. The table given by Kins-
man (1957) was utilized to arrive at the correction factor.
Background: .

Background radiation is caused by natural cosmic
radiation, and/or radioactive substances in or near the
counter. The determination of background was carried out
daily by Operating the counters for 30 minutes with muffled
planchets. The background observed on that day was then
subtracted from the readings made that day.

VOlume Factor:

Due to the differences in sample size of the various
materials collected, all counts were corrected to coudts per
gram or to counts per milliliter.

During this.study period, counts were corrected as
indicated below:

corrected value = ( cpm - Bg ) x (Vf) x (Df)

where:

Bg = background
Vf 3 velume

Df = decay

13

Correction of counts per minute to millicuries and
microcuries can be derived from the following factors given

by Robeck, et.a1. (ibid.).

l curie (c) 3.7 x 1010 disintegrations '
per second (dps)

3.7 x 10“ dps

1 microcurie (uc)

2.22 x 106 dpm
l/2.22 x 106 = h.5 x 10"7 no

1 dpm

If results are desired in terms of microcuries, then

microcuries = ( cpm - Bg ) x (Vf) x (Df) x (1.001x10'6)

with a counter efficiency of usfi
The Bacterial Carrier

Escherichia gel; was selected as the carrier organism
for the radioactive phosphorus in the 1961 experiment on the
West Branch of the Sturgeon River. _§, 221;.belongs to the
coliform group of bacteria which are restricted in habitat
to the intestinal tract of man and other animals and can be
distinguished from the normal stream flora which is lacking
in coliform organisms.

The organism can be characterized by the properties
found in the Appendix I.

Many strains of the bacterium have been separated on
the basis of biochemical differences and phage specificity.
Strain B was originally selected as the carrier, but due to
difficulty resulting from low pH of the unbuffered mediumper
possible mutant phage, strain 0111 was substituted later in

the experiment.

err,

1h

Labaw, et.a1. (1959) stated that 2.72% of the dry
weight of an 8 hour culture of _E_. 3211 consisted of phospho-
Ims. The partitioning of phosphorus within the organism
was divided as follows: 66% in ribose compounds, 19% des-
oxyribose nucleic acid, 12% phospholipids, and 3% unknown.
Their experiments on uptake of P32 by E. 931; closely para-
lleled the findings of this investigation. The authors
found that bound phosphorus was not released into the medium
as cell death proceeded. Uptake of phosphorus corresponded
to an increase in optical density and to cell multiplication
as it did in this study.

The usual growth medium for coliform organisms con-
tains beef extract and other phosphorus carrying substances.
In order to eliminate the normal phosphorus content of the
media, the growth medium given in Appendix II was utilized.
Test For Presence of Members of Coliform Group berembrane
Filter Technique

The filter technique for determination of coliforms
is considered as a standard test for coliform enumeration
in the 11th edition of Standard Methods 1960. The method
consists of filtration of a water sample through a membrane
which retains the bacteria, transfer of the membrane to suit-
able culture media, and enumeration of the type colonies
after incubation.

In this study the sample was obtained by placing the
sample bottle completely below the surfaceof the water with

the neck pointed in the direction of the current. Samples

15

were filtered through an apprOpriate membrane (eg. RA Milli-
pore with a pore diameter of .h5 p) and the filter funnel
rinsed three times with 20 to 30 m1. of buffered water
solution. Membranes were removed and placed on a surface
of an agar plate.
The cultures were then incubated 20 t 2 hours at 35°C.
in an inverted position in an incubator with 100% humidity.
The only deviation in the methods outlined above
from those listed in Standard Methods was the use of agar
surface instead of an adsorbent pad upon which the membranes
were placed.
_ The colonies developing on cosine methylene blue
agar may be described as (l) typical- nucleated, with or
without metallic sheen; (2) atypical- OpaqUe, unnucleated
mucoid after 2h hours incubation, pink; (3) negative, all
others.
Preliminegy_Bacterial Studies on the West Branch_gf the
Sturgeon

Before utilization of the coliform organism, E. coli,

 

as a carrier of radioactive phosphorus in the stream system,
the following points were investigated.
Normal Bacterial Flora '

The normal bacterial flora of the stream was analysed.
to detect the presence of normal coliform organisms.

On June 27,1961, a 500 ml. water sample was collected
at station 8._ The sample was diluted and aliquots of it

filtered and plated on E.M.B. agar. The stream water seemed

16

fairly high in normal flora (1.27 x 103) organisms per m1.,
but no coliforms were-found.

Samples for normal flora were taken at stations 8,
12, 1A, and 16 on July 1 and July 10; no coliforms were
found. A

It seems safe to believe that coliforms are not nor-
mally present in the stream system, therefore, any detected
in the system originated from experimental addition.
Rate of Coliform Movement in the Stream System

In order to have an idea of sampling intervals needed
to detect the movement of radioactive g. 9211, the following
experiments were conducted.

Three temporary sampling stations were located at
200, ADD, and 500 yards below permanent station 1h. On
June 30, July 1, and July 10, _E_’. 32;; B was added to the
West Branch of the Sturgeon River below station 1h. The
cultures of bacteria were diluted in 55 gallons of stream
water and added to the stream at a constant rate over a
period of 30 minutes. Before addition of the bacteria, a
fluorescein dye marker was released and samples were taken
get 15 minute intervals after passage of the dye.

The first trial showed inconclusive results due to
sampling errors or faulty addition methods. .

The results of the second and third trials on rate of
movement of bacteria showed that sampling intervals of 15 min.
could detect movement with accuracy. Figure 2 shows the

passage of E. coli past the sampling stations during the

17

Figure II. Flow of E. coli g through the stream system.

 

1100 r

1000 "

900

800

T

700 "

600.

per/ml

(DI 500 r-

.0011

ml .
1:00 .

300 )-

200 ..

100 *-

 

18

Station 1

 

___ Station 2

— -— Station 3'-

l l l

15 30 1&5
Time in Minutes

19

third trial.

The total number of bacteria added to the stream on
the third trial was 1.3 x 1010; the results show that a
number of this magnitude could easily be detected downstream.
Growth of E. coli and Uptake of P32

E, 2211 is a facultative aerobe but will attain a
higher growth peak and Optical density with adequate aera-
tion. The organism can survive between 15°C. to h80C.,
indicating wide temperature tolerance, with Optimum growth
conditions from 350C to 37°C. g. g_o_1_i_ _g_1_1_1_ was found to
reach the peak of its growth curve between 12-15 hours at
3500.‘ '

During the final uptake experiment with _E. coli B,

 

an initial uptake of 8550f the P32 occurred within four
hOurs. The culture continued at this uptake level until 12
hours of growth had elapsed; upon filtration of this culture,
all Of the activity was found in the filtrate. These results
could be attributed to several factors: the media being used
contained no buffer and the pH of the culture at this time
was n.8, thus lysis of the cells due to low pH is a possi-
bility. _E_. 9211 organisms are, however, active fermentors
of lactose and can usually withstand fairly acid conditions.
Another possibility exists, that of a lysogenic bacteria
phage (i.e. a stage in which virulent phage coexists with
resistant bacteria) which by spontaneous mutation could

have caused near complete lysis of the culture. The real

nature of the difficulty could not be determined with the

20

available data, therefore a phage resistant strain of E,
ggli_glll_was substituted for strain B for the remainder of
the experiment.

Using §, 2211.911}; analytical studies of its growth
and uptake of P32 were conducted. The Object of these
studies was to determine how the greatest percent uptake of
P32 could be achieved and maintained.

Krumholz and Foster (1957) review some factors which
have been Observed to have an effect upon the uptake of
radioisotOpes; one of these factors is the concentration
of the material in the environment. Boroughs, et.al. (1957)
have shown by using the algae Nitzchia 52,, that as the
concentration of zinc in the environment decreased there
was a greater percent Of uptake by the cells.

In preliminary studies, E. 92;; B showed P32 uptakes
of 7A and 8A percent when 6 milligrams Of P31 per 100 m1.
of media were used as a growth supplement. It was believed
that by decreasing the normal phosphorus to a level Just
sufficient for good growth that a maximum uptake of P32
could be obtained.

The growth curve for a bacterial population consists
of three areas defined as follows:

(1) The lag phase is a period when only a slight increase in
the bacterial population is noted. The length of this phase
is directly related to the effective inoculum.

(2) The exponential growth phase is the phase during which

the cells are replicating at a constant and geometric rate.

21

The rate of bacterial growth in this phase is determined

by limiting factors. These may be intrinsic in the physio-
logical potentiality Of the cell, or they may be extrinsic
in that they may be environmental factors, such as concen-
tration of an essential element.

(3) The plateau phase is the phase in which cells are dying
and multiplying at equal rates. The decreasing rate of cell
division can be accounted for in many ways with the factors
differing under different environmental conditions. One

of the most important factors is the accumulation Of end
products of metabolism such as alcohols and organic acids
from carbohydrates, to toxic concentrations.

Under constant cultural conditions, one factor in the
environment, such as concentration of a nutrient substance,
can be varied so that its growth determining factors can be
evaluated. Thus, when an element is limiting in the medium,
a slower rate of growth might be expected: also the plateau
in the growth curve should be reached before the normally
produced toxic products have accumulated (Burrows,l959).

rhéfiflhdtht of phosphorus used by a bacterial culture
can therefore be determined by a slower growth rate and by
leveling of the normal growth curve at an earlier time than
would occur in unlimited phosphorus media.

Four cultures Of _E_. gall Q_1_1_1_ were inoculated, and
all conditions held constant except for varying phosphorus
concentrations. At one hour intervals, samples were removed

from each culture and optical densities were determined.

22

At the plateau in each growth curve, a sample of the culture
was analysed for phosphorus utilization by calculating the
ratio of total phosphorus present to organic phosphorus.
Figure 3 shows the growth curves of the cultures in varying
concentrations of phosphorus. ‘

The cultures containing the following concentration
of phosphorus showed corresponding percent uptakes of total
.phosphorus present.

.3 mg of phosphorus per/100 ml 97.6%

1.5 mg of phosphorus per/100 ml h3.%% '
3.0 mg of phosphorus per/100 m1 53.1%
6.0 mg of phosphorus per/100 ml 7.0%

The results of the above experiment show a high per
cent utilization of phosphorus when .3 mg of phosphorus
per 100 ml of media were used as a growth supplement.

A trial uptake experiment was run in which .3 mg of
phosphorus per 100 ml were used as a growth supplement to
.1 Of a millicurie of P32. The optical density of the
culture was brought to ho Klett units with an inoculum cul-
ture. The culture was incubated at 35°C. and aeriated with
a magnetic stirring bar. Within two hours, the bacteria
had accomplished a 99.9% incorportation of P32. Figure A
shows uptake percentage plotted along with Klett,units to
_indicate increasing growth.

On July 12,1961, 11.1 millicuries of P32 were added
to each Of four 1,000 ml flasks containing 500 ml of broth.

Each flask was inoculated with enough E. coli 0111 to bring

23

I.

Figure III. Phosphorus utilization by _E. coli 0111.

Klett Units

24

220
20 ' ,
O’o/o
o/
/
180 ./
9/ //-—
J /
160 °/ /

1&0 _ /. /

120_ ,l / ,x’“~~.
/

100L 0 / I,

80 - / / I,

60- J /’ II,

 

0/° /' [I Mg of P31/100 m1
I
ho 0/- -—l I’ —o—
o// ”I 6
7/,» -- ——
20 o/°//” 3
"’ / I —: :/ -—-_
,/,°/”"
az’5::1:” '''' '3
.v/J”
0 l I l 1 1 L l LII:1 l l i_4__

 

l l
1 2 3 H 5 6 7 8 9 10 ll l2 13 1A 15
Time in Hours

25

p
I .
Eur

110

100

90

80

70

50

no

Klett Units and Per Cent

 

 

26

 

 

30- .
20 _
' Klett units
.- , _—__ % Uptake P32
10 -/
I
l
()i L i L
0 2 3 A

Time in Hours

27

the Optical density to 20 Klett units‘. Carrier phosphorus
was added to bring the total phosphorus concentration to .3
mg per 100 ml. The cultures were-incubated at 35°C and
aeriated with magnetic stirring bars. Samples were removed
every hour, checked for percent uptake, and a portion of the
sample was used to give growth curve information. Figure 5
shows the percent uptake by the culture of E, 2212.912};

and Figure 6 shows the growth curve of the organism.

.Fifty ml.-aliquots of the cultures of bacteria were
filtered through a 10-gram'covering of Celite and a Milli-
pore membrane. The Celite ($102) and membrane were then
removed and placed in an ice bath to slow bacterial metabol-
ism. The procedure was repeated until 1,020 ml of the
culture had been filtered. Final assay of this material
showed that 25.0 millicuries were present in bacterial cells.
These cells were then stored in the ice bath until addition
to the stream system. -
Bacterial Methods
£1212

Stations 3, 5, 8, 12, and 1h were designated as
collecting sites for bacterial samples.- The samples were
collected in 1,000-m1 polyethylene bottles. After a sample
was collected, it was placed in a burlap bag suspended in
the stream to prevent warming and hence subsequent multipli-

cation Of the bacteria. One sample-was collected about

‘ Tfie TormuIa reIaEIng KIeff unIEs Io opIcaI HensIEy Is as
follows: 1000 x D g R where: D is density
-‘_37_-'

R is Klett reading

28

 

Figure v. Final uptake of P32 by~§_. coli 0111.

29

 

Klett Units '

 

fl-—

 

pcoo flow can nudes 930g

   

 

3

Time in Hours

30

Figure VI. Growth of E. coli 0111 in the P32 medium.

Cells Per/M1

10

107

 

31'

l l l

A 5 6
Time in Hours

 

32

15 - 20 minutes prior to the anticipated arrival of the
dye; and then six samples were collected, beginning 10
minutes after arrival of the dye, at 10 minute intervals.
Laboratory

The samples were brought to the laboratory and stored
on ice until processing was begun.

Analysis to determine the number of E. 293.1 organisms
per/ml was carried out first because undue holding of the
samples might have resulted in erratic counts. .Dilutions
of the samples, expressed in-terms of 1 ml of stream water,
were carried out as follows:

(1) Each liter bottle was rotated at least ten times to
insure complete suspension of the material.

(2) Two ml of the 1 liter sample were removed with a ster-
ile 2 ml pipette and added to 20 ml of distilled water in
order to yield a 10.1 dilution.

(3) One ml of 1 liter sample was removed and added to 100
ml of distilled water to give a 10"2 dilution.

(A) One ml of the 10"2 dilution was removed and added to
10 m1 of distilled water to yield a 10'3 dilution.

Beginning with station number 1h, dilutions of the
samples for each sampling period were filtered through
Millipore membranes. Filtration of the samples taken prior
to the addition of the isotope were carried out before
any other samples were filtered in order to determine if
any coliforms were present. The filtrations of all other

samples were begun with the highest dilutiOn of that sample

33

(i.e. 10'3), and filtration progressed to the lowest (10‘1)
dilution. The fiIters were rinsed with 10 m1 of distilled
water after each filtration was-completed. Between each
sample filtration, the filter apparatus was rinsed with

100 ml of distilled water. Filter membranes were removed
with forceps and placed in prelabeled petri dishes of E.M.B.
agar. The petri dishes were incubated at 350C in an invert-
ed position and were removed for counting at 20 and MO hours.

Following the processing of the water samples for
determination of bacterial numbers, samples to determine
activity levels were processed. 7

Beginning with station number 1h, a 200-mll aliquot
of each sample taken at that station was filtered and washed
with 100 ml of distilled water. The filter membranes were
removed and glued to planchets which were placed in-a drying
oven at 60°C. The filtrates were placed in prelabeled
beakers and processed as water samples.

Filters and flasks were washed with .01 N RC1 and
then with distilled H20 before the next sample was processed.
After completing the‘processing of this group of samples,
an identical procedure was followed using 100 m1 of .01 N
HCl acid as a wash in place Of the water wash..

The following flow sheet shows the procedure in

sequence.

200 m1
1. Filtered

2. Rinsed with 100
m1 HOH

Filter:
(a) Removed

(b) Glued to a
planchet

(c) Dried at 6000.
(d) Counted:

counts equal total

solids
Filtrate:

(a) Boiled
(b) Acidified

(c) Transfered to
a planchet

(d) Counted:

counts equal
water soluble

3“

1000 M1 SAMPLE

200 ml 3 m1

1. Serial dilutions
-made up to 10'3

1. Filtered

2. Rinsed with 100
ml of .01 N H01 2. Filtered
Filter: 3. Filtersremoved
and transfered to
E.M.B. agar for

bacterial enumerat-

(a) Removed

(b) Glued to a ion

planchet 0
0 h. Incubated at 37 C

(c) Dried at 60 C. for 20 and no hours

(d) Counted:

counts equal in-
corporated activity

Filtrate:
(a) Boiled
(b) Acidified

(c) Transfered to
a planchet

(d) Counted:

counts, in excess

of water soluble,
equal acid soluble
or adsorbed activity

35

Water Methods
Field

 

Water collection sites were designated at stations
3, 5, 8, 12, lh, and 16. Samples were collected in 1&0 ml
polyethylene bottles. Sampling was begun at the initial
sighting of the dye, and samples were collected at 5 minute
intervals for a period of 70 minutes and at 10 minute inter-
vals at stations la and 16 for a period of 1&0 minutes.
Laboratory

The water samples were prepared for activity deter-
minations as outlined by Robeck, et. al. ($2292) with the
exception of modification of volumes due to differences in
sizes.

Each sample bottle (1&0 ml ) was acidified by an
addition of three milliters of concentrated nitric acid.
The sample was mixed, and a 50 m1 aliquot was removed for
evaporation. The 50 ml aliquot was placed in a beaker and
and evaporated until the contents had been reduced to a
volume which could be placed in a planchet. The beaker was
then rinsed with 2 N nitric acid, and the washings were
then added to the planchet. The material in the-planchet
was evaporated to dryness and placed in a muffle furnace at
6000C until red hot. The planchet was then placed in an
aluminium tray and transfered to the counting laboratory
where the activity was determined.

Fish and Lamprgys

36

Field Methods

 

Fish and lampreyswere collected at two week inter-
vals at stations 8 and 12 for radiological analysis. Brown
trout, brook trout, rainbow trout, muddlers, and lamprey
were collected at station 8. The above species were collect-
ed at station 12, except for the brook trout which only
rarely occurs at station 12.

All species were collected by electro-fishing with a
230 volt, direct current generator as the power source. The
generator was placed 1. a small boat which had a 10 foot
copper strip on the bottom to serve as the negative pole.

The boat was pulled behind the shocking crew in which each
of two men carried small poles with a capper sheet wrapped
at one end to serve as positive poles.

Lampreys were collected from Chara or silt beds, and
muddlers were collected from under.logs and along rocks.

In order to obtain a representative sample of each
species, at least four individuals of a species were collect-
ed.

Laboratory Methods

Usually, the fish and lampreys from each station were.
placed in plastic bags and frozen until the following morn-
ing when processing was begun. On a few occasions, however,
the samples were processed on the same day in which they
were collected.

All fish were measured and weighed before processing.

Individuals weighing 30 or more grams were homogenized with

37

an equal volume of distilled water. If an individual weigh-
ed less than 30 gramsi twice its weight in water was usually
added. All samples were homogenized in a Waring blender

for 30 seconds or until complete homogenization had been
completed.

A sample of the homogenate was then removed with a
pipetting device, placed on a preweighed planchet, weighed,
-and covered with concentrated nitric acid. After the sample
was completely digested, the planchet was placed in a muffle
furnace at 6000C and removed for counting when it became
red hot. ,

Samples of muddlers and lampreys usually consisted
of 3 or u individuals.

Fish Biomass _

EstimatiOns of the trout and muddler populations of
the West Branch of the Sturgeon River were conducted at
stations 3, 8, 12, and in.

A segment of stream 100 yards in length was selected
at each station for estimation of the trout population and
50 feet in length for the muddler population. The area was
enclosed on the upstream end by a chicken wire fence, and
the shocking was conducted in an upstream direction.

The muddler population was estimated before the
trout shocking was begun. Muddlers were captured in the
section, placed in M.S. 222, measured, clipped on their
left pectoral fin, and then weighed as a group. The fish,

after undergoing the above procedures, were placed in fresh

38

water. After they had revived, they were redistributed in
the 50 foot section. A 15 - 20 minute waiting period was
allowed to elapse, and the procedure-was carried out again
until enough returns were obtained to yield a fair estimate.

All species and size groups of trout in a 100 yard
section were captured; lengths and weights were recorded;
and the caudal fins were clipped. The trout were then re-
distributed in the section, and 20 - 30 minutes were allowed
to elapse in order to facilitate redistribution. The sec-
tion was then reshocked. ‘

Addition of the Isotope ‘

On July 13, 1961, the bacteria and Celite filtering
powder were transported to the stream in an_ice chest. The
mixture was added to a 55 gallon barrel and the barrel was
then filled with stream water. A fluoescein dye marker
was released 10 minutes before the addition of the isotope
was begun. The bacteria were then siphoned into the stream

at a constant rate for a period of 33 minutes.

RESULTS

When radioactive—phosphorus is released into an
aquatic environment, it rapidly disappears from the inorgan-
ic form. In lakes, aquatic bacteria and phytoplankton seem
to be the organisms responsible for its removal. Bacteria
have shown turnover times of 5 minutes in their.relation-
ship to radioactive phosphorus in the water (Hayes and
Phillips, 1958), hence the removal of phosphorus by bacteria
is extremely rapid. Reid (1961) states that if bacteria
receive a large share of the available inorganic phospho-
rus, they may seriously limit the production of the animal
mass in the community. The extent to which bacteria are
utilized by consumer organisms in the food web is not known.

Earlier experiments on the phosphorus cycle in the
West Branch of the Sturgeon River have indicated rapid in-
corporation of phosphorus into particulate matter (Knight,

1961). The cycle of phosphorus in the stream has been
’ followed extensively through the biota in each of 3 separate
experiments, and food chain relationships have been well 8
established (Ball and Hooper, 1962). It was the object of I
this investigation to add the isotope in the form of bacter-
ial cells and to follow the phosphorus cycle beginning at a
different trophic level. If a similar phosphorus cycle
was observed, the bacteria might be considered the phosphorus
fixers in the stream system. I

The activity flow curves are shown in Figure 7.

39

#0

Figure VII. Total water activity at collecting stations
during passage of the isotope. Time zero is arrival at

station of water mass carrying P32.

41

omH

 

OMH ONH Ofifl OOH 0a

.:ruetF¢anhnlilqlqurellcurlellvrl.

S ..

I‘l-

:a
NH lloll

m IIII

 

couvmvm

nausea: ca made
00

cm

on

 

NH

 

 

tw/eanutw deg saunoo

#2

These curves were computed from the data obtained by filtra-
tion of 200 ml of stream water and addition of activity

'or the filters and filtrates. The values plotted for stat-
ion 16 were obtained from the 50-m1 water samples.

The general shape of the curves at stations 3, 5, 8,
and 12 compare to the curves found in other years (Knight,
3219,, Zettelmaier, ibiQ;). The curves at stations lu and
16, however, are drawn out much longer than was heretofore
encountered. This condition is due to the small amount of
uptake at the upper stations with a progressive lenthening
of the spike* due to mixing and dilution.

The time at which the peak was reached varied between-
30 and no minutes from arrival of the water mass bearing
P32 at stations 3 through I“, but was not reached until
60 minutes at station 16. Graphic representation of the
peak values is shown in Figure 8.

Bacteria as Phosphorus Carriers

In order to determine if all the activity observed
in the solid state was incorporated into the bacterial cells,
an acid wash of 100 ml of .01 N HCl was applied to the
filter membranes. After this treatment, the filter should
show lower activity values than a comparable sample withe
out the acid treatment if the activity was adsorbed to the
surface of the cell. Figure 9 shows the activity values of

acid and water washed membranes.

 

* The term spike is used to designate the P32 added to the

stream water.

1*3

I

Figure VIII. Peak activity levels reached at the collecting

stations during isotOpe flow.

uh

 

 

 

 

 

 

 

 

 

 

 

 

11F

10 -

8%

b

. p .
6 5 h.

ax\op5cH: mom epssoo

r
3

 

12 1“ 16

Stations

8

”5

Figure IX. Activity of acid and water wash filter membranes

at the collecting sites.

Counts Per Minute/Ml

M6

 

 

 

 

 

 

 

 

 

 

 

 
      

 

 

 

 

- Acid Wash Station 1h
_ -_-_ Water Wash ————— ‘~\‘\§K\
\
I l I l L

0 10 20 30 40 50 60

Time in Minutes

47

The small and inconsistent variations between the
activity levels for the two types of rinse indicate that
the activity was retained within the cells and not adsorbed
to the cell surface. The variations were probably due to
differences in the aliquots taken from the main sample
bottle.

The activity levels for the filtrates of these samp-
les, Figure 10, correspond in magnitude to the values ob-
tained from the filters.

Mean Activity_Ieve1s for E. coli Cells

The cell count in the final culture of E, coli which
was added to the stream was 9 x 108 per ml 3 1,020 ml of
this culture were filtered and the total activity calculated
to be 25 millicuries. Assuming each cell in the culture
had incorporated an equal amount of P32, a cell should
contain .0278 counts.

Figure 11 shows the cell count obtained at each
station plotted with corresponding activity values. Util-
izing the points for which a cell count was recorded and
the corresponding activity values for that time period,
the mean activity for each cell was calculated to be .0218
cpm with confidence limits at the 95% level of .Olh8<u<'
.0288 cpm (shown in Table 1). These data indicate that
each cell had incorporated an equal amount of P32.

The occurrenceof activity values, in Figure 11,
with no detectable cell count is thought to be due to the

greater sensitivity of the radioactive counting procedure.

Figure x. Activity of the acid and water wash filtrates at

the various collecting sites.

Counts Per Minute/Ml

ZS
Qfi
16
L0

.5

15

13

10

“9

 

 

 

Station 3

 

Station 5

 

 

 

 

 

 

  

 

 

  
 

 

 

 

 

/
/
/
/ .
K
‘ ___-
Station 1h Acid Wash
_ Water Wash
.——-"'l
0 10 20 30 ho 50 60

Time in Minutes

50

Tablerl

Mean Cell Counts

 

 

Time Station Counts Per Cell Cells
10 3 6.&0 .030& 210
20 3 8.10 .0337 2&0
30 3 8.50 .01&6 580
&0 3 5.10 .0182 280
50 3 5.50 0 0
60 3 .02 0 0
10 5 2.50 .010& 2&0
20 5 6.&0 .0200 320

0 5 8.00 .0235 3&0
0 5 9.30 .0232 &00
50 5 &.60 .0270 170
60. 5 .&0 0 0
10 8 2.30 .0135 170
20 8 5.80 .01&5 1&0
30 8 6.80 .0106 6&0
&0 8 8.50 .0121 700
50 8 &.10 .O&10 100
60 8 .60 0 0
10 12 .60 .0085 70
20 12 2.90 .0111 260
30 12 2.70 .0128 210
&0 12 5.90 .0256 230
50 12 3.00 .0115 260
60 12 2.70 .0900 30
10 l& .20 0 0
20 l& 1.50 .0150 100
30 l& 2.10 .0150 1&0
&0 l& &.80 .018& 260
50 l& &.90 .0233 210
60 l& 2.90 0 0
32 .00027853

3 I .016689

1 - .0218

Confidence Limits =

.Oߣ<u<.

0288

51

Figure XI. Activity of the filter membranes and number of
E, coli 0111 organisms per milliliter at the collecting

sites.

s. coli/M1 x 102 and --- Activity in cpm/M1

52

 

 

Station 3

 

 

 

 

 

 

 

 

 

 

 

0 10 2O 30
Time in Minutes

 

53

Effect of the Stream Environment on the Bacterial Cells

In order to detect the effect of the stream environ-
ment upon the retention of activity by the cells, an analy-
sis of variance was computed on the data. If the cells as
they passed through the stream system lost appreciable
activity by transfer of phosphorus}? with the normal phos-
phorus present, the analysis should detect heterogeneity in
cell activity between the stations. The results of this
analysis, Table 2, showed at the 95% level that there was
no significant difference in activity per cell between the
different stations. These data indicate that there was no
appreciable loss of P32 from the bacterial cells after the
initial loss upon addition to the stream.

Hayes and Phillips (122g,) and others have shown
that upon addition of P32 to a lake the bacteria equilibr-
ate with the inorganic state in 5 to 20 minutes. In this
experiment, the filtrable activity passing each station,
Figure 12, remained constant except for one period when a
near complete loss of filtrable activity occurred; Immedie
ately after this loss, the level of filtrable activity re;
turned to the concentration at which it was present before
the loss. These data indicate a constant exchange level
between the bacteria and the water, but it is not possible
to say that the filtrable activity was due to inorganic
P32. Whittaker (1961) states that loss from algae back to

the water may be in the form of some organic compound.

5&

Table-2
variation in Mean Cell Count Between Stations

 

 

Station 20 _géme 10" R2
3 337 1&6 182 665 R1 &&2,225
5 200 235 232 667 R2 &&&,889
8 1&5 106 121 372 R3 138,38h
12 111 128 256 &95 By 2&5,025
l& 150 150 18& &8& R5 23&,256
Total 9;; 733 9;: 2.633

T2 889.2u9 585.225 950.625

 

S S rows = €112 - T2
3 'ES

3 3 col. = ET? _ T2
5 15'

s 3 total = ax? - T2
13'

S 3 error ’ S S total - S S rows - S S col.

82 rows = S S rows
"_1T""

32 col.‘ = S S col.
.__§___.

 

S2 error = S S error
8*
F - 32 rows
error

F 33% 31.1129

55

Figure XII. Total and soluble activity passage by the

collecting stations in microcuries.

56

 

 

Acid Sample

Water
-'-'-' Sample

 

 

—

 

Acid SampIT-

Water

““SmMe

  

 

 

3 2 1
men x on assesses oaosfiom

8

.u.

sod x oa.apa>aso< doses

16

l&

12
Stations

57

Activity Flow and yptake in Various Stream Sections

Differences or similarities in the uptake of P32
by various stream sections may have significance in the
basic productivity and in the distribution of phosphorus
within the stream system. In order to determine the uptake
of the isot0pe in various stream sections, the total passage
of the isot0pe by each station was computed. The values
found in the computation are shown in Table 3. Figure 12
shows the activity flow values plotted logarithmically with
distance from the entry point. A comparison of the act-
ivity flow values for the years 1958, 1959, 1960, and 1961
is shown if Figure 13. A large difference can be noted bet-
ween the passage of the isotope in the-bacteria and inorganic
form. In the 1958 experiment, less than 1 mo passed out of
the area; in 1959, all of the P32 was removed within the
area: in 1960, 1.5 mc passed through the system, but in the
1961 experiment, 13 me passed through the study area. The
large amount of activity passing through is an indication
that the bacteria were not the organisms responsible for
the distribution of phosphorus in the stream system. Since
the experimental conditions were similar on all additions
of the isotope, the same general pattern of uptake might be
expected if the bacteria were the organisms responsible for
the removal of P32 from the inorganic state.

The area of each stream section was computed and the
uptake of P32 in the sections was determined (Table 3).

Figure 1& shows the uptake in microcuries per square yard

58

Figure XIII. Total microcuries passing the sampling points
in the indicated years.

Microcuries

 

l l

59

l

1961

_o_

——— 1959

—- — 1960
___. 1958

 

 

35

12
Stations

1&

60

Heaps w

weopOHdHoamu

 

 

 

annexe
mooapos mp. «swam annexe mom was ammo cmmoxo «one
m-m e.wam pm.umc w.<mw .qom poem
um c.6um mmm .pma .oou
-HN HMBOW‘N Hummm / 0 “OH epmm
Hmnpe m.mom m.mwe .eme .emm
“elem mo.<oo ”.mow .oms .sos
m-m r.w<m m.wmm H.rm4 .wpu Home
mum aroma =.oma H.0pu .mmo
mtum u=.omm m.omo .mom .moe
Hmnpe .wmm mom .Hmo .mom
u=-um .p .666
mum =.w<m Ho.oom m.mup .eqm Homo
m- e.owm ram .Hpm .owm
muum H=.omm m.om~ .row .emo
no-0: m.wmm cm» .umr .Hmo .
Heuum pm.moo w.omm .Hmw .mme
mum =.wqm m.mum H.rmo .mem Home
wum c.0wm mmq .mum .02:
muum H=.omm m.eqm .mom .Hme
Hmnue m.umm
tram Hm. moo 3. J3. nudl. m

61

Figure XIV. Microcuries uptake per square yard of the

stream sectionsin the indicated years.

62

mmmfl wmma
o

 

 

 

 

 

 

 

 

 

 

 

J

 

.Hanm
NNNHHH
' paex bs/eweadn satanooaotw

h- :-

m.m
©.m

m.m

 

 

 

63

of stream section in all four years of the investigation.
Upon examination of Figure 1&, it is evident that section A
shows the greatest amount of uptake in all years; unfortun-
ately, these figures can not be related to productivity
since the P32 available for uptake does not remain constant
in all sections. The uptake values were transformed into

32per square yard

proportions of the amount of available P
to the amount of uptake per square yard forming a more
logical relationship. The proportions found in the analysis
are shown in Table 3 and are graphically represented in
Figure 15. AA Friedman two-way analysis of variance was
computed on the data, Table &; the analysis showed heter-
ogeneity at the 99% level. Thus the analysis indicates

that the proportional uptake rates do not remain constant
throughout the study area. Two possible explanations for
these results are: (l) the phosphorus is more available

for uptake in some sections than in others; (2) some sections
of the stream are lower in productivity, populations of
energy fixers, and therefore can not remove similar pro-
portions of P32. The stream section between stations 5 and
8 (sectionB) shows 10w proportional uptake in all four
years. The habitat in section B is predominantly a riffle
and run area which should allow growth of energy fixing
13eriphyton and higher aquatic plants. In 1960, the only
:year for which separate plant biomass estimates are avail-

able, this section had the lowest biomass estimate, Hence,

6&

Figure XV. Pr0portion of the activity available to the act-
ivity uptake per square yard in the various stream sections

in the indicated years.

65

m

mea

Q o

m

m

Q

ooma
o

m

4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exeqda teuotqaodoad

 

 

1958
1959
1960
1961

66

VTable &

Friedman Analysis of Variance

A B C D E
5 l 2 3 u
3 2 & 5 1
3 1 & 2 5
.2. _E_. .1. _L .9.
l6 6 13 ll 1&

k
2 5. (RJ)2 - an (M)
X = 12 J = l

4.

X
ID
II

1 . 716 = 71.6
10’

where N number of rows

k =~number of columns

RJ sum of ranks in Jth column
and the section with the
lowest uptake was ranked

as 1

67

a possible explanation for the lower uptake of section B
could be that of a lower population of primary producers to
fix inorganic P32. Uptake values in microcuries per gram

of aquatic plants, the day following isotope entry, in the
various sections during 1960 are shown in Table 5. The
amount of uptake per gram of plants was analysed by an analy-
sis of variance which at the 95% level showed no heterogen-
eity between stream sections. Therefore, the low proportional
uptake in section B can not be attributed to a lower plant
biomass because the uptake per gram of plant remained fairly
constant throughout the stream.

Table 5. Uptake of P32 by aquatic plants during 1960 in

designated areas.

 

 

 

Plant Biomass 2 Uptake 2 Pr0portional Uptake

Grams Per Yard Per Yard Uptake Per Gram Per Gram
A 501 .7713, .03 .0003&19
B 359 .1261 .O& .OOO3&12
C 86& .1830 .21 .0002118
D 1,602 .3201 .30 .0001998

 

Several possible hypothesis for a stable uptake of
P32 by plants in the various sections are as follows:
(1) The recycling of P32 by periphyton from the upper
stations allowed uptake of P32 by plants in the lower sec-
tions, while plants in the upper sections could only lose

activity.

68

(2) The uptake of P32 per gram of plant represents only a

portion of the P32 present, but it is a constant proportion

to the amount of P32 in a usable state. This hypothesis

does not seem reasonable when one considers the proportional

uptake of P32 (i.e. the amount of P32 available to the amount

incorporated) by the plants in different sections, as is
shown in Table 5. The plants in the lower stream sections
removed a greater proportion of the activity present than
did the plants in the upper sections.

(3) Physiological differences in plant metabolism due to
sunlight, rate of stream flow, and other factors would in-
fluence P32 uptake, but the importance of these factors is
not known.

The low preportional uptake in section B can not be
attributed to low plant biomass, and since no periphyton
production figures are available, the reasons for the sec-
tionh low proportional uptake can only be hypothesized.
Section B is predominantly a riffle and run area supplied
with abundant sunshine; the possibility of photoinhibition

of the existing periphyton populations is a consideration.

The area may not have had comparable populations of periphy-
ton due to a type of substrate less productive in periphyton

or other limiting features not presently recognized.

Fish Activity Levels

The uptake of radioisotopes of biologically essential

elements by aquatic organisms may occur by absorption

through membranes exposed to the surrounding water, through

V

69

engulfment of food or inert particles which-contain the rad-
ioactive material, or by adsorption onto exposed surface

areas (Foster, 1959). Phillips, et.a1. (1957) have shown

that brook trout in aquaria were able to take up P32 from

the water, but the utilization from the diet was over 200
times that of dissolved P32. Krumholz and Foster (3)19.)

also stated that the major mode of accumulation of radioactive
materials by fish occurs through ingestion.

The activity levels of the fish food organisms during
this study were generally lO-fold lower than those of pre-
vious years. The relationship and importance of a high
level of activity in these food organisms can be seen in a
comparison of the activity values for fish from 1959 to 1961,
Figures 16 and 17. -

Due to the large differences in accumulation of P32
by fish in the 1959 and 1961 experiments, the patterns of
P32 uptake were obscured. To make possible a comparison of
uptake patterns in these experiments, regression equations
for uptake rates were computed; these are found in Appendices
III,IV and V.The slopes of the regressions (the rate of P32
uptake) are.dependent on several factors such as water temp-
erature, basal metabolism, species differences, and food
substances. The uptake rates for different years and species
,were compared by an analysis of variance and no significant
differences were found. If the P32 were reaching the fish
through radically different pathways due to incorporation
of the P32 into the bacterial cells, different rates of

7O

.
5

uc X 10'3/g
c>

uc X lO‘h/g

ID

H

0

7/10 15

71

 

Station 8 Muddlcrs

   

-———1959
___1961

 

 

Station 12 Muddlers

-’-~
dfl" -----~
---’

 

 

 

 

Station 12
Lamprey

 

 

 

1 L l l L l I l I _1

22 26 29 8/5 9 12 19 23 26 9/1 7
July August . September

 

72

18
‘
an

73

 

Station78Brown Trout

p
I

__——-~- .—
——-‘— _~
--——— --

 

uc X 10'3/g
(3

Station 12 Brown Trout

 

 

”
J"
’
’

-‘-—--~--’
"‘
-’

I I I I I I J I I I I

O

 

 

7/10 15 22 26 29 8/5 9 12 19 23 26 9/1 7
July August September

7&

uptake might be—expected. The finding of similar uptake
rates indicates that the fish received the P32 from a source
similar to that of other years. The magnitudes of activity
density, however, are so reduced that the densities could
have originated from trophic pathways followed by the small
amount of soluble P32 present at the time of addition of

the isot0pe.

Figure 18 shows the activity curves for all fish
collected during the 1961 study; the uptake rates for these
fish are shown in Figures 19 and 20.

The mean counts per gram of each species considering

the entire study period were as follows:

Station 8 Station 12
Lamprey 93 11&
Brown Trout 130 ’ 22& m
Brook Trout 138 ' ' 292 (2 fish)
Rainbow Trout 172. ~ -258

Muddler s 167 221

The mean activity levels for brown and rainbow trout
at stations 8 and 12 were compared by a two way analysis of
variance. The analysis showed a significantiiiffercnce in
activity between the stations at the 95% level. Comparison
between the species by a one-tailed test showed that the
rainbows had significantly greater mean activity levels than'
the browns at the 90% level. Differences in activity levels
.for fish at stations 8 and 12 have also been recognized by

75

Figure XVIII. Activity density of fish at collecting sites
8 and 12.

Microcuries/g X 10'”

OHIOU)

‘ U.) 0

76

 

 

 

 

 

 

 

 

 

 

 

 

 

Lamprey
__ /"’—-——————-—.
’ Rainbow Trout
L
/
’/
Brook Trout
' Station 8
F ".
—--- Station 12
I L
7/26 8/9 8/23
July August September

77

Figure XIX. Activity uptake rates for the fish at station 8.

H
0
NM

5‘ moot-

Counts Per Minute/g
:0

S
(DI-4H

l I

 

 

 

15

78

Rainbow
--_—Bmm

-- - — Brook
I I J l I L L L

20 25 30 35 to' us so 55
Time in Days

79

Figure XX. Activity uptake rates of the fish at station 12.

Counts Per Minute/g
a>

’2'.

(DH

 

80

 

— -. — Muddlers

 

 

- Rainbow
_______M_ Brown
I I l I J I I I I I L
0 5 10 15 20 25 30 35 &O &5 50 55

Time in Days

81

Knight (3:29) who attributes the findings to downstream
drift. Nelson (1958) noted that rainbow trout accumulated
more activity than brook trout in a lake system.
Fish Biomass

The fish population of the West Branch of the Stur-
geon River during the 1961 investigation had the following

 

composition.

No. in No. per Lb. per

Station Species 100 Yd. Acre Acre

3 Brook 22 125 3.&

. Brown 21 119 29.0

Rainbow 19 102 &.7

Muddler 115 3,928 27.6

8 .Brook 115 65& 13.5

Brown 19 108 .9

Rainbow 27 153 6.2

Muddler 11& 3,89& 27.&

12 Brook 3 16 1.9
Brown 22 121 18.9 '

Rainbow 17 93 2.8

Huddler 333 11,028 77.9

1& Brook 6 33 “-5

Brown 26 1&3 20.5

Rainbow 27 1&9 3.7
Muddler 199 3,931 27.7'

 

SUMMARY

7 Bacterial cells have been shown to play an important
role in the distribution of P32 in lakes. The results of
this study, however, seem to negate a similarity between
lake and stream environments in the transfer of phosphorus,
at least at the primary level. Fixation of P32 by the stream
biota in earlier years of the investigation of the West
Branch proved more efficient than in the present study. It
seems possible, in fact, that the bacteria lost to the.
stream system held most of their P32 or did not transfer it
in a form available to the stream biota.

The exchange of P32 with normal P31 in the stream
system was demonstrated to be of low magnitude, 10 - 15% of
the incorporated P32. Rice (1953) using algal cells showed
that cells grown in low phosphorus media incorp0rated more
P32 into the organic and unexchangeable state/than did cells
grown in media of high phosphorus content. The bacteria
utilized in this experiment were grown under minimal phos-
phorus concentrations which may account for the small portion
of exchangeable activity.

The activity levels reached at all tr0phic levels
investigated were 5-to-20 fold lower than in previous years.
Since an amount of P32 approximately one half of other years
was retained in the area, the activity levels reached
should theoretically have been higher if the same paths

were followed. The uptake rates of P32 for fish have been

82

83

shown to be similar to those observed when the P32 was added
in the inorganic form; this seems to indicate pathways of
phosphorus transfer not radically different from earlier
years. Uptake of P32 by periphyton and plants showed levels
of activity easily attributable to the soluble activity
present (Bacon,unpublished). The above considerations in-
dicate that the organisms responsible for fixation of P32
within the stream are most probably not bacteria.

Uptake of P32 per square yard by the uppermost
stream section was high in all years, while the uptake in
the lower sections was greatly reduced. This is probably
due to uptake of the isotope decreasing its availabilty per
unit area in the lower sections. Proportional uptake levels
for the stream sections revealed that all sections of the
stream were fairly uniform in proportional uptake, except
for section B. It was thought that the low uptake observed
in section B was due to low plant biomass, but updn analysis
it was discovered that the uptake of P32 per gram of plant

in all sections was fairly constant.

 

LITERATURE CITED

Alexander,Gaylord R. 1956. The fertilization of a marl lake.
Master' s Thesis, Michigan State University.

American Public Health Association. 1960. Standard methods
for the examination of water, sewage and industrial
wastes. 11th ed. Amer. Public Health Assoc., New York.

522 PP-

Ball, Robert C. and Frank F. Hooper. 1962. Translocation of
P32 in a trout stream ecosystem. (in press) Atomic
Energy Proceedings, Radioecology Conference.

Borgeson, David P. 1959. The movement of radioactive phos-
phorus through a stream ecosystem. Master's Thesis,
Michigan State University.'

Boroughs, Howard, Walter A. Chipman,'and Theodore R. Rice.
1957. Laboratory experiments on the uptake, accumula-
tion, and loss of radionuclides by marine organism.
get. Seed. Science -- Mat. Res. Council. Publ. No. 551:

0 - 7.

,Bryant, William C. 1960. M0vement of radiophosphorus through
the invertebrate community of a trout stream. Master' s
Thesis, Michigan State University.

Burrows, William. 1959. Textbook of microbiology. W.B.
Saunders Company, Philadelphia. 95& pp.

Chase, Grafton D. 1960. Principles of Radioisotope Methodo-
10 y. Burgess Publishing Company, Minneapolis, Minn.

28 pp.

Clifford, Hugh F. 1959. Response of periphyton to phosphorus
introduced into a Michigan trout stream. Master's
Thesis, Michigan State University.

Foster, R.F. 1959. Radioactive tracing of the movement of an
essential element through an aquatic community with
specific reference to radiophosphorus. Pub. della

stazione Zool. di napolic (Press), 31: 3& - 62.

Grzenda, Alfred R. 1955. The biological response of a trout
stream to headwater fertilization. Master's Thesis,
Michigan State University.

Hayes, F. R., J. A. McCarter, M. L. Cameron, and D. A. Living-

stone. 1952. On the kinetics of phosphorus exchange
in lakes. Jour. Ecology, &O: 202 - 216.

8&

 

85

Hayes, F.R., and J.E. Phillips. 1958. Lake water and sediment.
IV. Radiophosphorus equilibrium with mud,p1ants,and
bacteria under oxidized and reduced conditions. Limnol.
Oceanogr., 3: &59 - &75.

Hutchinson, G.E., and V. T. Bowen. 1950. Limnological stud-
ies in Connecticut. IX. A quantitative radiochemical
study of the phosphorus cycle in Linsley Pond. Ecology,
31: l9& - 203.

Kinsman, S. 1957. Radiological health handbook. U.S. Dept.
of Health, Education, and Welfare, Public Health Ser-
vice. Robert A. Taft Sanitary Engineering Center,
Cincinnati, Ohio. 355 DP.

Knight, Allen W. 1961. The translocation of radiophosphorus
through an aquatic ecosystem. Master's Thesis, Michigan
State University.v

Krumholz, Louis A., and Richard F. Foster. 1957. Accumulation
and retention of radioactivity from fission products
and other radiomaterials by fresh-water organisms.
Sgt. Acad. Science -- Nat. Res. Council. Publ. No. 551:
" 950

Labaw, L.W., V.M. Mosley, and R.W.G. Wyckoff. 1950. Radio-
active studies of the phosphorus metabolism of Escher-
ichia coli. Jour. Bacteriol. 59: 251 - 262.

Nelson, Wesley C. 1958. The uptake of radioactive phosphorus
' by the fish species of upper Camp Lake, Colorado. State
of Colorado, Dept. of Game and Fish, Progress Report.

Odum, Eugene P. 1959. Fundamentals of ecology. W. B. Saunders
Company, Philadelphia. 5&6 pp.

Phillips, A.M., Jr., H.A. Podoliak, D.R. Brockway, and
R.R. vaughn. 1957. The nutrition of trout. Cortland
Hatchery Rept. 26, New Yerk Cons. Dept., Fish. Res.
Bull. 21. 93 Pp. _ .

‘Rcid, George K. 1961. Ecology of inland waters and estuaries.
Reinhold Publishing Corporation, New York. 375 DP.

Rice, T. R. 1953. Phosphorus exchange in marine phytoplankton.
Fishery Bull., U. S. Fish and Wildl. Serv., 80: 77 - 89.

Rigler, F. H. 1956. A tracer study of phosphorus cycle in
lake water. Ecol., 37: 550 - 556.

Robeck, G.G., Croswell Henderson, and Ralph C. Palange. 195&.
Water quality studies on the Columbia River. U.S. Dept.
of Health, Education and Welfare, Public Health Service
Robert A. Taft Sanitary Engineering Center, Cincinnati,
Ohio.

86

Whittaker, R.H. 1961. Experiments with radiOphosphorus
tracer in aquarium microcosms. Ecol. Monogr., 31:
157 - 188 e

Zettelmaier, John L. 1961. The translocation of radiophos-
phorus through a lotic ecosystem. Master's Thesis,
Michigan State University.

I
I

87

APPENDIX I

Biochemical Characteristics of‘g, coli

Litmus Milk A.C.G.R. Xylose A.G.
Nitrates reduced X Inositol -
Bouillon X Arabinose A.G.
Potato X Mannitol A.G.
Dulcitol A.G. Indol x'
Maltose A.G. V0ges-Proskauer -
Dextrose . A.G. Methyl Red -
Lactose A.G. H23 -
Sucrose -

A 3 Acid

C = Coagulation

G = Gas

R 3 Reduction

X = Activity

No Activity

88

APPENDIX II

Growth Medium for E. coli Olll

L-Glutamic Acid 2.0 g Distilled Water 1,000 m1
Glycine 7.5 g Boron A .01
Glucose 2.0 g Copper .10
Glycerol 1.0 g Iron .20
NaCl 5.9 g Manganese .02
MgSOh .1 g Molybdenum , .02‘
Ca012 .1 g Zinc 2.00

mmononcnan

 

89

APPENDIX III

Uptake Rates for Muddlers at Station 12

a1»:

I

B»
m

10

Counts Per Minute/g

 

1961 1

-—-- 1959 2

lt—f—fis—fl?‘ W'it‘
. 3U 50
1 Time in Days
activity read on the right—hand scale
2
activity read on the left-hand scale.

 

 

 

 

Counts Per Minute/g

 

90

APPENDIX IV
& _ Brown Trout Uptake Rates at Station 8
/
/
3 - ,,’
/
/
1961 1
---- 1959 2
1 I I .1 I I I I I I I

 

I

 

 

O 5 10 15 2O 25 3O 35 &O &5 50
Time in Days

1
activity read on the right-hand scale

2
activity read on the left-hand scale

55

91

APPENDIX V

& _ Brown Trout Uptake Rates at Station 12

 

 

 

 

 

43
0
.p
5
S:
H
S
h
0
m
m
.p
G
:3
O
c) -& - _
lo2
2.. 1
1961 1
—--- 1959 2
1 I I I I I I I I I I I
0 5 10 15 20 25 3o 35 &0 &5 50 55

1 Time in Days
activity read on the right-hand scale
2

activity read on the left-hand scale

92

APPENDIX VI

Activity Values from Acid and Water
Wash Samples During the Isot0pe Passage

1 2
Total Total Mean Acid Water Mean
Activity Activity Total Soluble Soluble Soluble
Passing Passing Passing Passing Passing Passing

 

 

 

 

Station

3 25,790 22,599 25,6&& 2,551 2,&22 2,527
5 19,872 18,999 19,&25 1,106 9&3 ' 98A
8 16,972 20,178 18,568 1,73& 2,628. 2,33u
12 15.935 15,890 16,090 2,051 2,386 2,3&2
1& 16,517 15.999 16,251 2,02& 2,085 2,161
16 13,068 13,068 ._____ ‘

1

from water wash samples

2
from acid wash samples

93

APPENDIX VII

Fish Biomass Estimations of the West Branch for the Years
1958,1959. 1960, and 1961

 

Muddlers

 

Pound of
Year Trout per Acre Lb. per Acre Total
1958 86.0 16.0 101.8
(Bryant,.
1960)
1959 20.0 106.6 126.6
(Kni t,
1961
1960 55.0 18.5 -u 61.5
(Zettelmaier, -
1962)
1961 30.2 &0.0 70.2