ALTERATION OF THE PRODUCTIVITY OF A
TROUT STREAM BY THE ADDITION

OF PHOSPHATE

The“: I05- I'Im Dogma o? M. S.
MICHIGAN STATE UNIVERSITY

David Lee Corr-ell

1958

 

 

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IIIIIIIIII IIIIIIIIIIIIIIII

31293 00992 8304

 

 

ALTMIW OP '1'!!! PRODUCTIVITY OF A “001' 8'!!!“

BY m ADDITIW OF PHOSPHATE

3!
DAVID m cmu.

A THESIS

Sub-itted to The College of Agriculture of Hichigsn State
University of Agriculture and Applied Science
in pertisl fulfillment of. the requiruents
for the degree of
MASTER OF SCIENCE

Department of Fisheries end wildlife

1958

 

.— 5’ ' if?

'3: 7'3. :0

David Lee Correll

ABSTRACT

Dis-onitn phosphate was added to the weet branch of the Sturgeon
River at a point approxinately four strea- miles above where it crosses
U. S. Highway 27 near Wolverine. At tines the preaence of excessive
phosphorus was detected as far downstrean as Highway 27.

In the period in which phosphate was added, increased periphyton
growth at a point about one and a half niles downstre‘ was show. the
ratio of phosphorus to organic nitrogen in the periphyton population at
all tines, both upstre- and downstream, was found to be one to ten by
weight.

No change in volme of benthos one and a half niles downstrean
fro. the point of phosphate addition could be correlated with this
addition.

A study of the co-position of the pignent ccqlex in ninety-five
percent ethanol extracts of periphyton fro. the west branch of the
Sturgeon liver and a ninety percent acetone extract of fresh periphyton

fro- the Red Cedar liver was carried out.

@oI'JI‘C e; “M

ii

WW8

The author wishes to express his appreciation and gratitude to
Dr. Robert 6. Ball and Dr. Frank P. Hooper for their guidance and
advice during this project. The author is also indebted to 141'.
Lowell Keup, who helped carry out the field work; and Dr. Philip J.
61*, for his assistance in the statistical analysis of the data.

this study was carried out under a graduate research fellowship

fro. the Institute for Fisheries Research, Ann Arbor, Michigan.

iii

TABLE OF CONTENTS

INTRODUCTION.............
GENERAL DESCRIPTION OF THE STUDY AREA.
DESCRIPTION OF SAMPLING STATIONS . . .
METHODS.
Fertilization...........
Physical..............
BottonFaune............
Chemical..............
RESULTS
Physical..............
BottomFauna............
Chemical..............
OONGLUSION..............
APPENDIX
Introduction............
Experimental
RedCedarRiver.........

West Bramh of Sturgeon River. .

iv

10

15
22

25

91
105

TABLEl
TAKE 2

TAKEB

TAKELI

TABLE5

TAKE 6

TAKE?

TAKES

TABLE 9

TABLE 10

TAKEll

TABLEJZ

TAKE 13

TAKE Ill»

LIST OF TABLES

Temperature Date

Total Volumes of Bottom Fauna-Station 3A
in Cubic Centimeters per Square Foot During
Six Periods of the Sumner

Total Volumes 01' Bottom Fauna-Station 7
in Cubic Centimeters per Square Foot
During Six Periods of the Sumner

Total Numbers of Bottom Fauna-Station 3A
per Square Foot During Six Periods of
the Summer

Total Numbers of Botton Fauna.Stat ion 7
per Square Foot During Six Periods of the
Summer

Water Hardness in Parts per Million

Hydrogen-ion Concentration of Water in
pH Units

Total Vater Borne Phosphorus in Parts
per Billion

Soluble Ortho Phosphate in Water kpressed
in Parts per Billion

Harvey Units of Pigment per Subshingle-
Station 3A During Six Periods of the Sumner

Harvey Units of Pigment per Subshingle-
Station 7 During Six Periods of the Summer

Milligrams of Organic Nitrogen per Sub-
shingle-Station 3A During Six Periods of
the Summer

Hilligrans of Organic Nitrogen per Sub-
shingle-Station 7 During Six Periods of
the Summer

Micrograms of Phosphorus per Subshingle
for Station 3A During Six Periods of the
Sumner

Page
19-21

28

29
31

35

’42

50-51

55-56

57-53

62.63

TAKEH

TABLE 16

TABLE 17

TAKE 18

TABLE 19

TLBLEZO

TABLE 2.1

TABLE 22

TABLE 23

TABLE 2“

TABLE 25

LIST 01“ news (Cont.)
Page

Micrograms of Phosphorus per Subshingle
for Station 7 During Six Periods of the
Summer 64-65

Ratio of Phosp o to Organic Nitrogen
in Periphyton _: Station 3A (’1 g. p/mg.N)
During Six Periods of the Summer 66

Ratio of Phosphorus to Organic Nitrogen in
Periptwton-Station 7 (’13. p/ng.N) During
Six Periods of the Sumner 67-68

Ratio of Pigment to Organic Nitrogen in
Periplwton-Station 3A (Harvey units pigment/mg.
N) During Six Periods of the answer 75

Ratio of Pigment to Organic Nitrogen in
Periphyton—Station 7 (Harvey units pigment]
mg.N) During Six Periods of the Sumner 76.77

Ratio of Phosphorus to Pigment in Periphyton
Station 3A ( g. p/Harvey units pigment)
During Six eriods of the Sumnr 81

Ratio of Phosphorus to Pigment in Peri-
phyton-Station 7 (yg. p/Harvey units

pigment) During Six Periods of tin Summer 82-83
Total Phosphorus in Bottom Organisms 81+
Red Cedar River Periplvton Separations 108

Absorbeney Data Obtained with a Beckman
Model B Spectrophotometer on Pigments
from Red Cedar River Periphyton 109.110

Absorbency Data Obtained With a Beclman

Model B SpectrOphotoneter on Pigments of
Periplvton from the West Branch of Sturgeon

River 120

vi

LIST OF FIGURES

Page
Figure 1 Map of Study Area Showing Sampling Stations 3

Figure 2 Schematic Diagram of Apparatus Used in
Fertilization of the West Branch of the
Sturgeon River ' 7

Figure 3 Temperature Curve. Station 7. July 17.
g 1957. Degrees F. 16

Figure 1; Water Temperatures. Stations 3A and 6.
in Degrees P. (All readings taken
between 8 A.M. and 12 noon) .1?

Figure 5 Water temperatures. Stations 7 and 8.
in Degrees I". (All readings taken
beWeen 1 P.M. and 5 RM.) 18

Figure 6 Relative Guage Height at Station 7 23
Figure 7 Total Volumes of Bottom Fauna in Hillil-

iters per Surber Sample at Six Times

During the Summer. (Mean 3 2 standard

deviations of the mean). 2“
Figure 8 Total Numbers of Bottom Fauna per

Surber Sample at Six Time During the

Sumner. (Mean g 2 standard deviations ‘

of the mean). 30
Figure 9 Total Haziness of Water. Stations 3 and 7 33
Figure 10 Total Phosphorus in Water. Stations 3A and 6 37
Figure 11 Total Phosphorus in Water. Stations 7 and 8 38

Figure 12 Total Phosphorus in Water During Period of
July 24 to August 29. 39

Figure 13 Total Phosphorus in Water Durim Period
August 1 to August 18 (Idealized by
grouping and averag ing) 1+0
Figure 11+ Soluble "0rtho" Phosphate in Water Durirg
Period August 8 to August 18 1+3

vii

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

15

16

17

18

19

20

21

22

23

21+

25

26

LIST or FIGURES (Cont.)

Means of Pigment per Unit Area in Harvey
Units During Six Periods of the Sumner

Harvey Units per Unit Area During Six
Periods of the Summer (Mean per sub-shingle
g 2 standard deviations of the mean)

Mean Milligrams of Organic Nitrogen per
Unit Area During Six Periods of the Summer

Organic Nitrogen per Unit Area in Milligrams
Nitrogen During Six Periods of the Summer.
(Means g 2 standard deviations of the mean)

Mean Total Phosphorus per Unit Area During
Six Periods of the Sumner

Total Phosphorus per Unit Area During Six
Periods of the Sinnner (Means g 2 standard
deviations of the means)

Mean Ratio of Total Phosphorus (in I’ g.)
to Organic Nitrogen (in mg.) During Six
Periods of the Stunner

Mean Ratio of Total Phosphorus (in g.)

to Organic Nitrogen (in mg.) D Six
Periods of the Summer. (Means 3 2 standard
deviations of the means)

Mean Ratio of Pigment (in Harveys) to
Organic Nitrogen (in mgs.) During Six
Periods of the Summer

Ratio of Pigment (in Harveys) to Organic
Nitrogen (in mg.) During Six Periods of
the Summer (Mean 3 2 standard deviations
of the mean)

Mean Ratio of PhOSphorus (in g.) to
Pigment (in Harveys) During ix Periods
of the Summer)

Ratio of Total Phosphorus (in g.) to
Pigment (in Harveys) During Sgt Periods of
the Sumner (Means g 2 standard deviations
of the means)

viii

Page

52

53

59

69

70

73

71+

79

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 3”

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure I+0

LIST (1’ FIGURES (Cont.)

Absorption Spectra of Red Cedar Periphyton
Pigment (Fract. A)

Absorbency Spectra of Red Cedar Per iphyton
Pigment . Fraction C

Absorbency Spectra of Red Cedar River
Periptvton Pigment. Initial Mixture

Absorbency Spectra of Red Cedar River
Periplwton Pigment . Fraction 1' .

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction 3'.

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction 6'

Absorbency Spectra of Red Cedar River
Periphyton Pigment . Fraction 2"

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction 2" (after
transfer to ettwl alcohol)

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction it"

Absorbency Spectra of Red Cedar River
Periplwton Pigment . Fraction 8'

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction 9'

Absorbency Spectra of Red Cedar River
Periplvton Pigment Fraction 9' (trans-
ferred to ettwl alcohol and at a known
concentration of 70 rig/1.)

Absorbency Spectra of Red Cedar River
Periphyton Pigment. Fraction 11'

Absorbency Spectra of West Branch of
Sturgeon River Periplvton Pigment.
Initial Mixture

Page

92

93

95

96

98

101

102

103

101+

106

107

Figure 41

Figure 1&2

Figure 43

Figure 141+

Figure 45

Figure 46

LIST or FIGURES (Cont.)

Absorbency Spectra of West Branch of the
Sturgeon River Periphyton Pigment.
Fraction 1

Absorbency Spectra of West Branch of
Sturgeon River Periphyton Pigment.
Fraction 7

Absorbency Spectra of West Branch of
Sturgeon River Periplvton Pigment.
Fraction 8

Absorbency Spectra of Ethyl Alcohol
Extract of Cedar Wood (from shingles)

Absorbency Spectra of West Branch of
Sturgeon River Periphyton Pigment (sample
10. period E. station 3A)

Absorbency Spectra of West Branch of the
Stm-geon River Periphyton Pigment (sample
7. period D. station 3A) in Ethyl
Alcohol

Page

113

111+

115

117

118

119

mooucnm

Civilization with its expanding populations and rapidly developing
industrialization has created an ever increasing problem of pollution.
One important phase of this problem is the municipal pollution of
stre-s. In order to handle this problem in such a way as to serve
the best interests of men we must gain a much more thorough under-
standing of the normal biology of relatively unpolluted stre-s. We
list also study the effects of the addition of know amounts of
extraneous materials.

The present study is the fourth in a series of experiments on
the effects, both biological and chemical. of the addition of inorganic
nitrogen and phosphorus to the west branch of the Sturgeon River. In
all three of the previous studies (1954-56). these elements were
applied to Hoffman Lake, the source of the stream concerned (erenda.
1956; Colby, 1957; Carr, ll.S.). During the present study dimmeonim
phosphate was added directly to the are. in a continuous flow for
a short period in August, 1957. The effects of this addition were
studied in cooperation with Keup 01.3.).

Although there have been many publications on the subject of the
fertilization of ponds and lakes, very few studies of this type have
been made on streams. Hunt-an (1948) observed an increase in pro"

duction as a result of inorganic fertilization of a “rec in Nova

Scotie.

CEREAL DBSCRIPTIW OF STUDY AREA

The west branch of the Sturgeon liver is a cold, clear trout
stream which originates in noffman Lake, Charlevoix County. liiehigan;
and joins the main branch of the Sturgeon River at Wolverine in
Cheboygen County. Hoffman Lake is a marl lake of about 120 acres
and the outflow from it is about one cubic foot per second. The
are. flows through the northwest corner of Otsego County and on
into Cheboygen County.

We to the large umber of moraines in the area, the watershed
is restricted to a small area and the surface runoff is only rarely
a major contribution to the volt-e of flow. The stream picks up the
bulk of its water from springs and the outflow of several nail
lakes and beaver ponds. A.large part of the watershed is within
the Pigeon River State Forest. however there are a number of summer
cottages on both noffman Lake and the stren. There are also some
scattered farms on the watershed. Figure 1 shows the study area

and adjacent roads in detail.

Figure 1

Map of Study Area
Showing Sampling
Stations

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WEST BRANCH STURGEON RIVER
AREA

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DESCRIPTIG 0P ammo STATICBS

All sampling stations are marked on the map (fig. 1). The
principal control station was located on the Charlevoix-Otsego
County line and was designated as 3A. The stre. is .all (approx.
ten cu. ft. per sec.)* at this point and is often broken up into many
channels through a heavy arbor vitae m. The flow is relatively
slow as compared to downstream and the water temperature fluctuates
more widely. About half a mile upstre- there is a beaver d-, which
also has an effect on the stren.

The next station used extensively was located in Cheboygen
County about two miles north of the Otsego-Cheboygen County line.
This station was designated as 6 and is not far downstre- from a
point where the stre- leaves an arbor vitae m which extends
almost continuously from the source of the stream to this point.

At this station the base flow is about 30 cubic feet per sscond“
and the bottom of the stre- is gravel in most places.

Station 7 is about one and a half stream miles downstre- from
station 6 and about three miles north of the Otsego-Cheboygen County
line. The base flow at this station is about 45 cubic feet per
-sscond* and the bottom is marked by the presence of numerous large
9.9.! beds, Nithin this stretch a brook, which originates in

“vet-a1 inactive beaver ponds, joins the stren.

 

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Station 8 is located about one stremn mile downstream from
station 7 and is at the point where Fun-er Creek joins the stream.
Fulmer Creek is a small brook which drains Fulmer Lake to the north
of station 8. There is also a small Spring fed brook which joins
the stre- from the south and sometimes brings in nutrients from
a cow pasture.

Station 27 is the place at which the stream crosses U. S.

Highway 27. This station was only used on a temporary basis.

METHODS
Fertilisation

Four hundred and ten pounds of diemsonium phosphate, (“021004,

which was rated 21-53-0 (NH3-P205-K) was divided into 35 eleven and

a half pound portions and each portion was put in a plastic bag.
These bags were then transported as needed to the fertilisation
site and each was mixed with ten gallons of stream water in a
galvanised tub and poured into the fertilisation apparatus (fig. 2) .

The fertilisation apparatus was installed about one hundred
yards upstream frum the bottom sapling area at station 6. The
barrels were placed on the bank and the sediment trap was located
on a log in such a manner as to direct the jet of dim-onium
phosphate into the main core of the current. Fertilisation pro-
cesded from 2:45 p.m. August 8, 1957 until sometime in the early
morning of August 17 for a total of about eight and a half days or
lbout 204 hours .

The calculation of flow at the point of fertilisation based
on the figure of 30 cubic feet per second was 6.7 x 106 pounds of
“tar per hour. The total .ount of fertiliser added contained
Shout 95 pounds of phosphorus and 71 pounds of nitrogen. The rate
°f Oddition was as even as conditions permitted but fluctuated
Mat due to variations in height of the head of solution in

"'9 barrels, which mounted to about 18 inches. During the ninth

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and tenth of August there were also some periods when the rate was
greatly reduced due to particles clogging the jet tip. However,
the assumption is made here that the rete was uniform. This would
give additions of 0.466 pound of phosphorus per hour and 0.348
pound of nitrogen per hour. In ten-s of parts per billion this
would be 70 parts per billion (p.p.b.) phosphorus and 52 p.p.b.
nitrogen. These figures give an approxneetion of the average rate
of addition of nutrients at station 6 and may be multiplied by
two-thirds for corresponding values to be expected at station 7

if dilution were the only factor to be considered. This would

give values of 47 p.p.b. phosphorus and 35 p.p.b. nitrogen.

Physical

T-perature
Air and water temperatures were taken with a Taylor pocket

ther-ometer at each station on all sampling trips along with the
tine of day. Air temperatures were taken in the shade, but over

the stren. Notes were also taken on weather conditions.

Gauge Height
A depth gauge calibrated in hundredths of feet was fastened

in a permanent position at station 7 where the stre. is confined
between two vertical concrete bridge abutments. The gauge was

installed on July 12 and readings were recorded for the remainder

of the study .

Water-mass )iovement Data

The time required for a patch of water dyed green with

fluorescein dye to move from station 6 to various points downstream

was measured .

Bottom Fauna

Six sets of bottom samples were taken at both station 3A and

station 7 at two week intervals from July 5 to September 13. The
"Plcs were taken with a Surber s-pler in gravel riffles. Bach

“‘1 ¢0nsisted of a ten smaple transect imsediately upstream from the
l“"Vltius saqle transect. The samples were transferred to pint

bottles containing enough formalin to make a final concentration of

“M": five percent formaldehyde.

10

The benthic fauna in the eagles were separated by floatation
using a saturated sugar solution. This work was done by prisoners
at the state prison camp at Heterloo, Michigan. Total volumes were
determined with ten ml. calibrated centrifuge tubes and ten ml.
burettes. Counts of total nuders of organisms were also made.
These data were then analysed statistically by the use of an F

test for homogeneity.

Chemical

Rater Chemistry

hardness
Hardness was determined in parts per million (p.p.m.) using the

versonate method? Titre Ver and Mono Var were used in the determina-
tion.

mlkalinitz
Total alkalinity was determined in p.p.n. using the titration
method described in Ellis, westfall, and Ellis (1948). Methyl orange

and phenolphthalein were used as indicators.

hydrogen-ion concentration

Model H
A heckmannpn Iieter was used to determine pl! on fresh water

Iqles .

5&2}. ghosmrus
Total phosphorus was determined by a modification of the method

in lllis, flestfall, and Ellis (1948). A Klett-SI-erson Photoelectric
cGlorimeter was used with a red (660 millimicron) filter. Results

“e expressed as parts per billion (p.p.b.).
'2‘ a a 0g No. 14., page 5. Hach Chemical Co. Amos, Iowa.

ll
soluble, ortho phosphate
A series of phosphorus tests were run during fertilization in
which digestion of the sample was omitted. Otherwise the samples
were treated as in total phosphorus determinations. Since this
method will detect all soluble ortho phosphates and the di-oniu
phosphate added comes under this heading, this test proved to be a

valuable aid in tracing the progress of the fertilization.

Periphyton
Samples of periphyton were collected on cedar shingles which

were sawed to a uniform twelve by four inch sise. These shingles
were attached at the butt and to logs and extended downstream
parallel to the current. Bach shingle had two slots cut in it in
such a way that it could easily be split into three pieces, each
twelve by one and one-fourth inches. Average calculated surface
area of actual subshingles was 38.76 square inches, of which 16,125
square inches was the area of the upper surface. These shingles
Vere fastened along the stream in sets of ten at both station 3A
and station 7 and left for two week intervals. The sets were re-
leced five times, each individual shingle being replaced in the
‘lme spot by the new one. The six consecutive sets were labeled
Pcriods A through I and are referred to by this convention for the
I'-'e-ainder of the text. As each shingle was raoved it was split
1“to three subshingles, each of which was sealed in a plastic bag.
1"'0 parts were frozen until the periphyton could be analysed for

°r8anic nitrogen and total phosphorus. The remaining one was

12

scraped into a white enameled pan and washed with distilled water.
NILstmn

The scrapings and wash water were filtered throng Ano. 1 filter

paper in Buchner funnels. The filtered material was extracted with

95 percent ethanol in one ounce glass bottles. The bottles were

stored in complete darkness until they could be analysed for pigments.

In no case was any effort made to remove invertebrates from the

periphyton complex before analysis.

piesnts

The 95 percent alcohol extracts were filtered through/$31.“?
filter paper and the residue washed with enough 95 percent alcohol
to bring the volume of extract up to 50 ml. The color of this
solution was then read in a Klett-Sunerson Photoelectric Calorimeter
using a 660 millimicron filter and the reading obtained was converted
to Harvey units (Harvey, 1934) by comparison with a standardisation
curve. ,The Harvey units can be converted to absorbency units by
mltiplying by the factor 12 x 10’3.
organic nitrogen

One frosen subshingle from each shingle was thawed and then
'craped and washed as in the pigment determination, then transferred
to a 300 ml. Erletneyer flask. It was then acidified with sulfuric
‘cid, concentrated by boiling, and analysed by a semi-micro Ijeldahl
firocedure as described by Belcher (1945) . Results are expressed in
“illigr-s organic nitrogen per unit area (subshingle).
$2.1. Eggnog-us

One frosen subshingle from each shingle was thawed and then

‘craped and washed as in the pigment determination, then transferred

13
to a 300 ml. Erlmeyer flask and analysed in the sue manner as the
water eagles for total phosphorus. Results are expressed in micro-
gr-s phosphorus per unit area (subshingle) .
fiiphzton gags;

Three ratios were calculated for each shingle; phosphorus to
pigment, phosphorus to nitrogen, and pigment to nitrogen.
statistical analysis

All six types of periphyton chemical data were analysed by the

application of r tests and multiple range tests (Duncan, 1957).

Bottom Organisms

On July 22 and again on August 18 samples of M, mayfly naiads
(flaggenia), stonefly naiads (Receptors), and dragonfly naiada
(Odonata) were collected and frosen until they could be analyzed.
Rough volumes were run on the samples before freezing using 25 m1.
centrifuge tubes and a 25 ml. burette. These samples were later
digested and analysed for total phosphorus in the same nanner as the

water s-ples. [lore acid was used in digesting the samples, however.

Isolations and Identifications of Pigments

A large volume of the ethanolic’extracts of pigments from the
“It branch of the Sturgeon River and a large volume of an acetone
eattract of pigments from periphyton growing in the Red Cedar River
“POtre- from the, Michigan State University campus were separately
e"‘Pfirated to dryness in a vacuum desiccator under reduced lighting.
These samples were then repeatedly fractionated by column chromatog-

r‘Phy using powdered sucrose and anhydrous alumina as adsorbents and

14

a solvent system of petroleus ether-benzene (9:1) . Various developers
were used which incorporated petroleum ether, benzene, and isopropyl
alcohol.

A Bechan Model B Spectrophotometer was used to follow the
progress of the fractionations and curves were plotted for various
separated components. The results of this work are reported in the

appendix.

RESULTS

Physical
Tnperature

A rather large diurnal fluctuation in water temperature occurs
in the stream, especially on clear, warm days. This fluctuation is
well illustrated by figure 3, which was recorded during a period of
this type of weather. Figures 4 and 5 show the seasonal temperature
fluctuations for stations 3A, 6, 7, and 8. It is easily seen that
the highest water temperatures occurred during the period from the
middle of July into early August at stations 7 and 8. Furthermore,
the lowest overall tqeratures and the least fluctuation in temper-
atures were recorded at station 6.

Apparently station 3A hasnhigher temperatures and larger
fluctuations due to the fact that the strema is sluggish and divided
into many .all channels at this point. By the time it reaches
Station 6 it has gained a large volume of cold spring water and
this tends to stabilise the temperature at a fairly low value. The
Breatest fluctuation observed during the course of the study at
station 6 was eight degrees Fahrenheit.

Stations 7 and 8 are subjected to more intense solar radiation
‘34 therefore have a greater range of fluctuation. It is interesting
‘0 hate the close parallel in temperatures at these two stations.

All available temperature and weather data are recorded in table 1.

FigureB

Temperature Curve.
Station 7. July 17.
195?, Degrees F.

16

Temperature

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Time of Day

Air

Water

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22

Gauge height

In the period from July 12 to September 17 the stre- level
underwent only minor fluctuations even though this period included
several rains in early Septuber. The data is plotted in figure 6.
, The maxi-am fluctuation during this period was less than three inches.
lsrly in the study, however, the stress: was considerably higher and
roily. This was particularly true on June 29, at which time the

stream was at least twelve inches above base flow.

Hater-mass Movement Date

aim-must l, at which time the stre- was near base flow, a
pateh'of water was dyed green at station 6. It required two hours
and twenty minutes for the dye to reach station 7 and an additional

hour and fifteen minutes to reach station 8.

Button Fauna

Total Values of Bottom Fauna

The means of the total volumes of bottom fauna for stations 3A
and 7 are plotted in figure 7 along with twice the standard deviation
of the mean. It can be seen by this rough nethod that no two
“Incentive periods show a difference that is valid at a 95 percent
confidence limit at either station. There are, however, significant
differences between some pairs of values at both stations.

The application of an F test to station 3A data showed that the
m. Vere not significantly different at the five percent level.

Th” 1': may be said with considerable assurance that the volumes

23

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25
of bottom fauna at station 3A, the control for this study, had little
if any seasonal fluctuations.

An F test on the data from station 7 also showed that these
means were not significantly different at the five percent level.

The data and statistical analysis are su-arised in tables 2 and 3.

Total Hmbers

The data and a limited mount of statistics concerning them are
mind in tables 4 and 5. A rough plot of the means and twice
the standard deviations of the means is shown in figure 8 for both
station 3A and station 7. Nothing further was done with total
numbers statistically since it is easily seen that it would be
difficult and certainly meaningless to attempt to correlate the
erratic fluctuations shown without breaking the samples down into

taxonomic units.

Chemical

Hater Chaistry
hardness

In general hardness was a function of surface runoff. Heavy
rains resulted in lower hardness and when the stream was at base
flow the hardness was highest, since the spring water was saturated
“Ch calcixn bicarbonate. Table 6 records the data obtained for
Itationa 3n, 6, 7, 8, and 27. On August 22 a sample was taken at
th‘ Paint where the stream originates in the lake. At this point

the hag-an... was 150 p.p.m., while on the same day the water at

TABLE 2 26

Total Values of Bottom Fauna-Station 3A in Cubic Centimeters per Square
Foot During Six Periods of the Suer

s-ple
no. A. n c 0 s r
1 0.25 0.32 0.17 0.66 0.89 0.74
2 1.10 0.31 1.28 0.70 0.40 0.85
3 0.18 0.26 1.01 1.33 0.58 0.82
4 0.36 0.40 0.76 0.58 0.88 0.43
5 0.28 0.23 1.41 0.48 1.68 0.37
6 1.53 0.22 0.37 0.42 0.67 0.15
7 0.15 0.12 0.23 1.37 2.97 0.64
8 0.14 0.14 1.45 0.33 0.55 0.36
9 0.21 0.33 0.57 1.28 0.62 0.27
10 0.94 1 18 0.99 1.59 0.96 0.78
sum 5.14 3.51 8.24 8.74 10.20 5.41
E 0.51 0.35 0.82 0.87 1.02 0.54
.sr? 4.82 2.06 8.85 9.59 15.76 3.51
flxxfln 2.64 1.23 6.79 7.64 10.40 2.93
h? 2.18 0.83 2.07 1.95 5.36 0.58
var. 0.24 0.09 0.23 0.22 0.60 0.06
mu. dev. 0.49 0.30 0.48 0.46 0.77 0.25
(m2 26.42 12.32 67.90 76.39 104.04 29.27
381 - 15.78, at - 59
ss, - 2.81, df - 5
85., - 12.97, df - 54
fi-oon
s
s2 - 0.240
‘0
F - 2.34
9% _.
X - mean (3)2 II at- squared
\ 322 - at? of squares SS1- - sum of squares total
‘“ n2 - In ' SSg - mm of squares between groups
vu~ " ”“1““. 33H - 81- of uares withi o s
sta. dmw. - standard deviation cg - mean squar': between, Ermuegn
square

within

TABLE 3 27

Total Values of Bottom Fauna-Station 7 in Cubic Centimeters per Square
Foot During Six Periods of the Suer

eagle

no. A B c n r r
1 0.11 0.08 0.33 0.28 0.25 0.47
2 0.15 0.05 0.22 0.18 0.14 0.21
3 0.08 0.04 0.39 0.17 0.17 0.15
4 0.05 0.06 0.24 0.07 0.20 0.04
5 0.11 0.02 0.36 0.08 0.26 0.14
6 0.13 0.20 0.13 0.10 0.11 0.20
7 0.10 0.19 0.19 0.15 0.22 0.12
8 0.11 0.14 0.17 0.15 0.07 0.22
9 0.15 0.37 0.22 0.23 0.60 0.21
10 0.14 0.22 0.23 0.29 0.17 0.35
sum 1.13 1 37 2.48 1.70 2.19 2.11
x 0.11 0.14 0.25 0.17 0.22 0.21
:22 0.14 0.30 0.68 0.34 0.67 0.58
(ml/n 0.13 0.19 0.62 0.29 0.48 0.45
8:2 0.01 0.11 0.06 0.05 0.20 0.13
veer. 0.00 0.01 0.01 0.01 0.02 0.01
m. dev. 0.03 0.11 0.08 0.07 0.15 0.12
(m2 1.28 1.88 6.15 2.89 4.80 4.45

831- - 0.67, df - 59
sa-oamdt-s
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6% - 0.020
.3 - 0.011
r-Lw

28

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TABLE 6

Hater Hardness in Parts per lullion

sta. 3A

192
175
194
187
194
194
196
193

194

194
199
192
195

ate. 6

188
168
196
187
195
195
191
188
196
196
197
194
193
195
195
193
196
191
196
194
196

Std.

190
170
194
186
193
193
190
193
196
196
196
196
192
194
193
193
192
193
196
193
195

7

sta. 8

188
173
193
196
197
196
193
201
202
199
196
194
197
195
193
195
195
197
195
196

sta. 27

197
197
192
200
196
200
196
195
197
196
195
197

31

32
station 3A had a hardness of 194 p.p.m. The highest value recorded
in this study is 202 p.p.m. at station 8 on August 11. The lowest
value, 168 p.p.m., was found at station 6 on July 4 during the
period in which the water level was very high due to heavy rains
over a prolonged period. The data from stations 3A and 7 are
plotted in figure 9.
alkalinity

(k1 June 27 alkalinity was run on stations 3A, 6, 7, and 8.
All detectable alkalinity was methyl orange alkalinity and checked
within two parts per million with total hardness run on the same
auples. It appears from this data that all or almost all of the
alkalinity and hardness was due to calcium bicarbonate in solution.
udrogen-igg concentration

The pH of the water samples was restricted to the range between
7.8 and 8.4 with only one exception and the majority were between
8.0 and 8.3. These values are recorded in table 7. It is inter-
esting to note that the lowest pH values were recorded from August 8
to August 12, but it is not very likely that this fact had anything
to do with the addition of fertiliser. This series of low values
was also found at station 3A and may have been due to considerable
haunts of organic acids being produced in the upper swampy section
of the stream and in the beaver ponds since it was warm weather and
the water was law.
353 zhosphorus

“IO data compiled for the results of total phosphorus determina-

ti‘m' ere shown in table 8. Hormel levels are about ten parts per

33

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34

TIBLB 7

Hydrogen-ion Concentration of water

in pH Units

sta. 8 sta. 27

sta. 7

6

sta.

sta. 3A

date

0 e e339 s092322222 . e s
a e a e e e e e e e e e e e e s s s e e
. O . . O

.887 .878888888 .

3232331.. .99232112231395.

8.8.8.88.8.8 .7788.8.8.8.8.8.8.8.8.8.

323333109912212223222
888888887788888888888

323333089923323214332
888888877788888888888

1110.12.16. . . . . .1 . . .434?”
O O O O O s O I
8.8.8.8.8.88.7. . . . . .8 . . .8.8.8.8.
7 185 01234567829 2
4444444o4444444444444
677778888888888888899

TABLE 8

totsl Hster Borne Phosphorus
in Psrts per Billion

dsts* sts. 3A** sts. 6 sts. 7 sts. 8 sts. 27
6-27 11 11 20 9 ..
7-4 11 17 13 17 ..
7-11 1 6 5 5 ..
7-18 6 4 4 4 ..
7-25 8 7 5 5 7
8-1 4 3 2 5 0
8-8 (sub 14 13 17 17 8
8-8 (8pc) . . 38 22 12 13
8-9 (ls-D .. 16 25 10 9
8-9 (Gs-0 .. 21 18 15 28
8-9 (9slb .. 23. 19 13 7
8-10 .. l9 l9 l4 ..
8-11 . 31 24 51 27
8-12 .. 71 51 32 37
8-13 . 71 55 47 32
8-14 .. 69 39 51 41
8-15 10 49 .. 29 25
8-16 .. 65 38 33 28
8-17 .. 8 18 25 26
8-18 .. 5 4 4 10
8-22 11 14 11 13 ..
8-29 20 12 14 29 . .
9-5 9 10 ll 9 ..
9-12 11 13 10 13 ..

’ . fertiliser spplisd August 8-17, 1957.
** Control Ststion.

36
billion or less at all stations. There are a number of factors
which can raise this level, however.

Heavy rain tends to bring organic debris and nutrients into
the stress: in the surface runoff. During long warn periods the
stream has a higher phosphorus level due to aore rapid decay of
organic materials in such places as beaver ponds. Life cycles of
various aquatic plants way also play a role in this pheno-enon.
Higher values than nor-a1 were found on June 27, July 4, and the
warning of August 8. The results of the entire study period for
stations 3A and 6 are plotted in figure 10 and for stations 7
and 8 in figure ll.

During the period in which the dimniua phosphate was added
there can be no doubt that the addition was soaawhat erratic and
the water s-ples taken are only an attupt to obtain a rough idea
of the count of phosphorus loving downstream This period for
stations 6, 7, 8, and 27 is shown in figures 12 and 13. It is
interesting to note that when fertilisation was stopped early in
the morning on August 17, there was a sharp drop in phosphorus
values back to the noraal range. its rise in values for August 29
at both stations 3A and 8 could be due to rain which is recorded
in the gauge height data as a rainy period. It could also be due
t° "saturation of phosphorus fro. upstreal in the case of station 8.

Although it is quite possible that there was a tine lapse in
u“ build-up of phosphorus in stations which were progressively
firth“ downstrea, this cannot be shown in a clear-cut Ianner due

to a part“ fro. the only mrning of August 9 to 10:00 a... on

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on

41
August 10 during which the fertilizer was added at an erratic rate
due to the filter becosdng plugged several tunes.

About August 18 it*was noted that there was a fairly heavy
growth of filamentous green algae in the part of the stream above
the point of fertilizer input (station 6). On August 22 water
samples were taken along the upper section of the stream and all
had total phosphorus values around 22 to 24 p.p.b. Samples of the
filamentous algae were found to be made up of Spirogzga and llougeotia
in a proportion of roughly two to one (Keup, 3.8.). 111s affects of
these slightly higher nutrient levels seems, however, not to have
been very great in terms of increased periphyton growth on shingles.
The proportion of total phosphate available to the algae nay have
been much lower. lhere is no way of knowing from this data, how
much soluble phosphorus was present at this time.
soluble orthofiphosphata

Determinations of soluble ortho-phosphate were only made during
the period from the evening of August 8 to the morning of August 18.
The data is recorded in table 9. It is interesting to note that the
highest values recorded correspond very closely with the values

theoretically calculated as average values assuming no biological
uPtaka. The calculated values were 70 p.p.b. and 47 p.p.b. for
stations 6 and 7 respectively and the highest values are 70 p.p.b.

”1d ’05 p.p.b. The data for stations 6 and 7 are plotted in figure 14.

date
8-8 (8pm)
8-9 (lamb
8-9 (6am)
8-9 (9am)
8-10
8-11
8-12
8-13
8-14
8-15
8-l6

8-l7

8-18

Soluble Ortho Phosphate in Water
Expressed in Parts per Billion

sta. 6
0
8

12

20
SS
64
65
70
59
69

IABLB 9

sta. 7

ll

13

38
36

36
39
38

ate. 8

l

11

30
39
28
33
33
19

sta. 27
6
0

17
20
3O
21
23
23

19

42

“3

.3 .563 o...
m can: ooanom mean—5 93.3
5 Sansone .038. 9338

i 853m

 

b 8.33.5

m 831%

OH

ON

on

 

on

nouns: Jed curd

Periphyton

zines-.22

The lean pigment values in Harvey units per unit area (sub-
shingle) for each period at stations 3A and 7 are shown as a
histogras in figure 15. The three fold change in absorbency as
represented here would be even larger if the fact that the pigment
couple: doesn't follow the Lambert-Beer Law at values above 16
Harvey units were taken into account. This fact has become evident
as a result of work being carried out by Drehner 01.8.). The
corrected value would be much higher for station 7, period D.

In order to show how significant the change is and to compare
with station 3A, the means and twice the standard deviation of the
lean are plotted in figure 16. It is easily seen that all the nuns
for both stations are in a fairly compact group with the exception
of station 7, period D.

P tests showed both sets of means to be significantly different
even at the one percent level. Using the nethod of Duncan (1957) it
was found that for station 3A, periods D, c, D. and 3 could not be

shown to be significantly different at the five percent level and
siailarly periods A and P are not significantly different at this
level. However, A and P are significantly different frou B, c, D,
and I at the five percent level.

For station 7 it was found that periods A and D are each
“Snificantly different frot- all other periods at the one percent

level. It is obvious that this would also be true at the five

“5

Figure 15

Keane of Pigment per Unit
Area in Harvey Units During
51: Periods of the Summer

 

 

Harvey Units

50

30

WT

 

 

 

 

 

 

 

 

Closed Bars = Station 3A
Open Bars 8 Station 7

 

 

 

 

 

 

 

 

1+6

one: 3%
a
83.325 235%
no n48 tom 505 ggméa
SEE Soto.“ fin no
u.. ms 5 as a...
d

0H 0.53.—

 

 

 

 

_l

 

 

 

 

1
[I]

J
30

70

J
20

filo

 

 

J l L
c: o on
V5 “01 4'18

Z. “014313

Harvey Units

47

percent level. Periods D, E, and P can not be shown to be significant-
ly different at the one percent level. The data and statistical
analysis for stations 3A and 7 are shown in tables 10 and ll
respectively.

It is easily seen from this date that the addition of diasnoniun

phosphate did increase the amount of pipent per shingle to a large

extent, but the amount of pigsent was also influenced by other factors.

0 genie nitrogen

The mean values of organic nitrogen per unit area (subshingle)
in.nilligrans nitrogen are shown as a histogram in figure 17. The
change at station 7 fro. period 0 to D is alsost three fold while
the values for station 3A decrease throughout the study period.
Figure 18, which shows the means and twice the standard deviations
of the neans, points out the large variance found in organic nitrogen
values. The only seen which is different from those adjacent to it
at the 95 percent confidence level is station 7, period D.

P'tests showed both sets of means to be heterogenous at the one
percent level and further tests showed that for station 3A periods
C. D, E, and P are not significantly different at the five percent
level and similarly periods A and B are not significantly different
at this level. Periods A and B are significantly different from
periods c, D, B, and I at this level.

It was found for station 7 that in terns of organic nitrogen
demlolihent periods 3, C, E, and l" are not significantly different
it“ “Ch other at the five percent level and sinilarly periods

A’ 3’ “Id 3 are not significantly different fro- each other.

TABLE 10

Harvey Units of Pignent per Subshing1e* Station 3A

salple

no. A B C
1 19.4 20.2 12.1
2 be“ 14.0 1206
3 10.4 22.8 20.9
4 5.1 20.8 18.2
5 6.4 10.4 13.0
6 3.0 21.6 11.6
7 6.6 14.9 13.9
8 7.0 20.1 18.5
9 5.3 18.0 11.9
10 ... 16.6 13.2
s1- 67 .6 179 .4 145.9
E 7.5 17.9 14.6
313 700.5 3,356.8 2,228.1
(KHz/u 507.7 3,218.4 2,128.7
8:2 192.7 138.4 99.4
var. 24.1 15.4 11.1
sta. dev. 4.9 3.9 3.3
(m2 4,569.8 32,184.4 21.286.8

88., - 2,864, df - 58
88, - 1,547, df - s
85H - 1.317, df - 53

8% - 309

.5- 24.8

r- 12.4

*surfac. area equals 38.76 square inches

During Six Periods of the Sui-er

U

N

~13~h38~hDL’O\hD~IC>

HHHNHU
"O‘DHONQQ
0

wN
«#0

191.8

19.2

4,103.7
3,678.7

425.0

47.2

6.9

36,787.2

28.7
13.1
27.8
19.2
17.3
27.4
20.8
15.2
15.0
13.3

197.8

19.8

3,912.5

339.9

37.8

6.1

39,124.8

48

H

fl
UUIUIONmeyO 'I
e s e 0 e O
O OO~HN~DU~OJ>QU

..s

122.0
13.6
3.7

5,625.0

(a)

(b)

(c)

(d)

(«2)

TABLE 10 (CONT.)

Multiple Range Test*-Station 3A

Source df
Between treat-ants 5
Error 53
l'p - s rp
p: 2 3 4
51 ‘p3 2.84 2.99 3.09
57. 3'9: 14.20 14.95 15.45
Code; a b c
X: 7.50 7.51 14.59
oz 10 9 10
Test Sequences: at 51 level

24.84

3.15
15.65

d
17.94
10

49

5.0
6
3.21
16.05
e f
19.18 19.78

10 10

(f—b)‘ greater than 1'5; (f-c)’ not greater than 334

(e-b)‘ greater than R'g; (e-c)‘ not greater than 3'3

(d-b)‘ greater than 1'3; (d-c)‘ not greater than l'z

(c-b)‘ greater than 3'2

(b-a)’ not greater than R'z

Conclusions: at 52 level

(c, d, e, f) can not be shown to be different
(a, b) can not be shown to be different

Man (1957)

 

 

TABLE 11

Harvey Units of Pig-ant per Subshingle—Station 7

sue-ple
no. A
1 11.0
2 11.2
3 12.8
4 8.7
5 7.7
6 14.2
7 10.4
8 9.1
9 9.5
10 10.5
81. 105.1
x 10.5

1::2 1,138.4

(mzln 1,104.6

Ibrz' 33.8
‘vaur. 3.7
sta. dev. 1.9

(m2 11,046.0

n c
18.3 10.9
23.7 11.9
21.6 11.9
18.5 13.0
17.4 12.1
23.2 16.6
20.6 15.0
20.6 15.7
27.6 17.8
25.8 20.6

217.3 145.5
21.7 14.6
4,823.0 2,206.0
4,722.0 2,117.0
101.0 89.0
11.2 9.9

3.3 3.1
47,219.0 21,170.0

ssr - 14,281, df - 58
ssB - 12,906, df - 5
ss" - 1,375, df - 53

oi - 2,581.0

oi - 25.94

7 - 99

During Six Periods of the Su-aer

e e
GOQQNHH¢

33338338 e

30,571.0
30,327.1

244.0

27.1

5.2

303,270.5

50

B F
11.9 9.1
. . 11.2
12.1 14.2
17.4 12.8
34.2 22.4
18.2 30.7
33.4 18.2
23.0 14.2
22.9 18.7
27.8 19.7
200.9 171.2
22.3 17.1
5,033.5 3,288e6
4,484.5 2,930.9

549.0 357.7
0.1 39.7
0.4 6.3

40,360.8 29,309.4

 

(e)

(a)

(b)

(c)

(d)

(e)

51

TABLE 11 (CONT.)

‘Hultiple Range Test-Station 7

Source df
Between Treat-ants 5
Error 53
R p - s ap
p: 2 3 4
12. : 3.79 3.95 4.06
11 R'p: 19.33 20.14 20.71
Code; a b c
x: 10.51 14.6 17.1
n: 10 10 10
Test Sequences: at 12 level

(f-i)’ greater than 3'2

n.s. e

25.94 5.1

5 6

4.14 4.20
21.14 21.42

d e f
21.7 22.3 55.1
10 9 10

(e-b)‘ greater than 3'4; (e-c)‘ not greater than l'3

(d-b)’ greater than 3'3

(c-a)’ greater than 3'3: (c-b)‘ not greater than ['2
(b-a)‘ not greater than 1'2

Conclusions:

at 11 level

(c, d, e) can not be shown to be different
(b, c) can not be shown to be different
(a, b) can not be shown to be different

Figure 17

Kean Milligrams of Organic
Nitrogen per Unit Aroa Durim
Six Periods of the Summer

52

52

 

 

 

Milligrams

3.2...

3.0...

2.8..

2.6

2.4

2.2

2.0

1.8

1.6

1.14

1.2

1.0

0.8

006*

 

 

Closed Bars :1: Station 3A
Open Bars :- Station 7

 

 

 

 

 

 

Periods

 

 

 

 

 

 

 

 

 

53

.348. on... «6 838.898 unseen...» N
.4: 9333 33555 on» no neoanom
Ham means: unwound: engagin—

5 no.3 flap pom unwound: 3:498

ma charm

Quads:

m...“

 

 

— J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L J_ L l

J

 

1. “0111913

vs uomzs

54

Period D is significantly different from all others at this
level.

Although the organic nitrogen data was not as distinctly
indicative as the pigment data, it does show a statistically valid
increase in organic nitrogen during the period in which fertiliser
was added.

The data and statistical analysis for stations 3A and 7 are
shown in tables 12 and 13 respectively.

total phosphorus

The mean values of phosphorus in micrograms of phosphorus
per unit area (subshingle) are plotted as a histogram in figure 19.
There is almost a four fold increase in phosphorus at station 7
between periods c and D.

Pigure 20 shows the mean phosphorus values and twice the
standard deviation of the means. The only set which is not within
the grouping is station 7, period D.

P tests showed the periods of both stations to be heterogenous
at the one percent level. Multiple range tests on station 3A indicate
that periods c, D, E, and P can not be shown to be significantly
different from each other at the five percent level and similarly
periods A and B are not significantly different at this level.
However, these two groups of means are significantly different
from each other at this level.

‘Hultiple range tests for station 7 show that all periods ex-
cept period D are not significantly different at the five percent

level. Period D is significantly different from all others at this
level.

 

TABLE 12 55

Hilligrams of Organic Nitrogen per Subshingle-Station 3A
During Six Periods of the Suuer

enple

no. A n c n n r
1 5.59 2.19 1.91 0.60 0.95 0.75
2 0.98 1.61 0.96 1.64 0.78 0.52
3 2.03 4.43 0.75 0.71 0.33 0.80
4 1.03 2.02 0.89 1.10 1.82 1.13
5 1.60 1.38 1.61 0.80 1.23 0.60
6 3.32 1.61 1.40 0.95 0.79 1.00
7 1.32 1.79 1.52 0.88 1.28 0.88
8 0.88 1.44 0.77 0.55 1.25 0.63
9 2.84 2.92 1.03 1.27 0.63 0.66
10 ... 1.06 1.78 1.13 0.63 0.58
sum 19.59 20.45 12.62 9.63 9.69 7.55
'i 2.18 2.04 1.26 0.96 0.97 0.76
xx? 61.56 50.52 17.61 10.27 11.06 6.05
(Ex)2/n 42.64 41.82 15.93 9.27 9.39 5.70
8:2 18.92 8.70 1.68 1.00 1.67 0.35
var. 2.36 0.97 0.19 0.11 0.19 0.04
sta. dev. 1.5 0.99 0.43 0.33 0.43 0.20
(2132 383.78 428.20 159.26 92.74 93.90 57.00

83, - 48.02, at - 58
33B - 15.70, df - 5
ssu - 32.32, at - 53

a; - 3.1
4-051

F-5.0

(a)

(b)

(c)

(d)

(e)

56

TABLE 12 (can .)

lultiple Range Test-Station 3A

Source df m. s. s
Between freshnents 5
Error 53 0 .6098 0 .78
' I
R P s sp
p: 2 3 4 5 6
5! 8?: 2.84 2.99 3.09 3.15 3.21
52 R'p: 2.22 2.33 2.01 2.66 2.50
dads: a h c d e f
x. 0.76 0.96 0.97 1.26 2.04 2.18
n 10 10 10 10 10 9
rest Sequences: at 51 level

(f-d)‘ greater than 1'3; (f-e)‘ not greater than 3'2

(e-d)‘ greater than 8'2

(d-a)‘ not greater than 8'4

Conclusions: at 51 level

(a, b, c, d) can not be shown to be different
(a, f) can not be shown to be different

Milligrams of Organic Nitrogen per Subshingle-Station 7
During Six Periods of the Suner

TABLE 13

57

s-ple
no. A D c D I! P
1 1.16 0.99 0.80 3.37 1.81 0.75
2 2.35 1.28 0.88 3.29 ... 1.08
3 1.86 2.70 ... 2.20 1.45 0.92
4 1.98 0.99 1.15 4.41 1.53 1.01
5 2.29 1.26 0.79 3.44 1.56 0.73
6 1.14 1.47 0.91 1.97 0.74 1.31
7 2.58 1.05 3.08 2.56 2.14 1.40
8 2.57 1.73 0.58 3.53 1.70 0.92
9 1.68 3.73 1.04 3.49 1.59 0.86
10 2.89 1.40 0.96 3.07 1.28 1.37
a:- 20.50 16.60 10.19 31.33 13.80 10.35
i’ 2.05 1.66 1.13 3.13 1.53 1.04
212 45.23 34.61 16.02 102.80 22.34 11.27
(IXJZIn 42.02 27.56 . 11.54 98.16 21.16 10.71
ex? 3.21 7.05 4.48 4.64 1.18 0.56
‘var. 0.36 0.78 0.56 0.52 0.15 0.06
sta. dev. 0.60 0.89 0.75 0.72 0.38 0.25
(2232* 420.25 275.56 103.84 981.57 190.44 107.12

831 - 46.98, df - 57
88B - 25.86, df - 5
ssw - 21.12, df - 52
a; - 5.17
ea - 0.4062

I II 12.73

(a)

(b)

(c)

(d)

(e)

58

TABLE 13 (CWTJ

Multiple Range Test-Station 7

Source df 111.8. s
Between Treatments 5
Error 52 0.4062 0.64
R' -
p ' a‘1)
: 2 3 4 5 6
51 8p: 2.86 2.99 3.09 3.15 3.21
51 3'9: 1.82 1.91 1.98 2.02 2.05
Code: a b c d e f
X: 1.04 1.13 1.53 1.66 2.05 3.13
n: 10 9 9 10 10 10

Test Sequences: at 51 level
(f-e)’ greater than ['2
(e-b)’ greater than 3'4; (e-c)’ not greater than 3'3

(d-a)‘ not greater than 3'4

Conclusions: at 51 level

(a, b, c, d) can not be shown to be different
(c, d, e) can not be shown to be different

Figure 19

Mean Total Phosphorus per Unit
Area During Six Periods of the
Summer

59

Hicrograms

520

080

360

320

280

160

120

 

Closed Bar = Station 3A
Open Bar = Station 7

 

 

 

 

 

 

 

 

Periods

 

 

 

 

 

 

 

60

e Ag;

8: .3 86335: 2483....
N fl 2.83.5 .8556 on»

no Scion 5m wfihfi «83.
fine .84 3353f in;

ON chamdh

60

55.883:

CON

ow

 

 

17

can 0mm
q

_

94m
4

4'

8H 8H
«1 4

EU

 

 

 

 

q

o:
n

 

Z. 11014349

TC “01' 4318

61

The data on phosphorus show the most striking effects of
fertilization of the three analytical methods partly due to the accu-
racy of the analytical method itself. The data and statistical
analyses are shown for stations 3A and 7 in tables 14 and 15
respectively.
periphyton ratios

phogphorus 32 organic nitrggen: The ratios and statistical
data for stations 3A and 7 are shown in tables 16 and 17 respectively.
A histogram of the means of the ratios for stations 3A and 7 is shown
in figure 21. Figure 22 shows a plot of the means and two standard
deviations of the means. This method shows, roughly, that no period
at either station is significantly different from the adjacent
periods at the 95 percent confidence level.

An 1" test on station 3A shows that the means are not significantly
different even at the 25 percent level. An F test on all periods at
both stations gives a value that shows that the means are not
significantly different at the one percent level, but are signifi-
cantly different at the five percent level.

A multiple range test of this set of twelve means indicates
that all but station 7, period D can not be shown to be significantly
different at the five percent level. Station 7, period D can not
be shown to be significantly different from station 7, period c or
station 3A, periods A and D.

These data show that with the exception of the period of
fertilization at the downstream station, the phosphorus to nitrogen

ratio is statistically valid and could be representative of a fairly

62
TABLE 14

liicrogrmas of Phosphorus per Subshingle for Station 3A
During Six Periods of the Summer

sample
no. A B C D E P
l 182 218 87 230 59 137
2 148 295 118 70 48 54
3 435 185 98 109 50 93
4 132 112 94 86 48 92
5 265 98 69 68 70 78
6 213 133 54 103 67 85
7 162 418 78 112 50 80
8 138 90 92 88 91 80
9 151 132 88 95 191 90
10 ... 81 95 73 47 29
sum. 1,826 1,762 873 1,034 721 818
X 203 176 87 103 72 82

313 445,360 415,420 78,927 126,952 69,449 73,848
(EXJZIn 370,475 310,464 76,927 106,916 51,984 66,912

xx? 74,885 104,956 2,714 20,036 17,465 6,936
var. 9,361 11,361 302 2,226 1,941 771
ltd. dev. 97 108 17 47 44 28

(I232 3,334,276 3,104,644 762,129 1,069,156 519,841 669,124
as, - 371,360; df - 58
as, - 144,368; df - 5
33w - 226,992; df - 53
a; - 28,874
.3 - 4,283
r-mn

(a)

(b)

(C)

(d)

(e)

63

TABLE 14 (CONT.)

Multiple Range Test-Station 3A

Source df m.s. s
Between Treatments 5 .
Error 53 4,283 65.4
R p - s 2p
p: 2 3 4 5 6
51 s - 2.84 2.99 3.09 3.15 3.21

51 R': 185.74 195.55 202.09 206.01 209.93

Code: a b c d e f
X: 72.1 81.8 87.3 103.4 176.2 203.0
n: 10 10 10 10 10 9

Test Sequences: at 51 level

(f-d)‘ greater than R'3; (f-e)’ not greater than R'z
(e-d)‘ greater than R'z

(d-a)’ not greater than R'4

Conclusions: at 52 level

(a, b, c, d) can not be shown to be different
(e, f) can not be shown to be different

64
TABLE 15

iHicrograms of Phosphorus per Subahingle for Station 7
During Six Periods of the Summer

sample
no. A. B c D B P
l 124 58 205 340 127 41
2 132 115 62 375 ... 78
3 151 202 68 592 104 56
4 151 133 97 542 90 66
5 119 149 112 370 113 64
6 140 116 87 502 120 137
7 149 214 160 635 187 184
8 81 79 144 552 348 98
9 125 198 130 462 129 108
10 131 131 106 692 142 75
sum. 1,303 1,395 1,171 5,062 1,360 907
X 130 140 117 506 151 91

11; 173,711 219,000 154,487 2,689,594 242,323 98,871

(£1)2/n 169,781 194,600 137,124 2,562,384 205,511 82,265

3:2 3,930 24,500 17,363 127,210 36,812 14,606
var. 437 2,722 1,929 14,134 4,602 1,845
sta. dev. 21 52 44 119 68 43

(3‘92 1,697,809 1,946,000 1,371,241 25,623,844 1,849,600 822,649
33, - 1,452,743; df - 58
3s, - 1,226,322; df - 5
33,, - 226,421; a: - 53
o; - 245.264

.6 - 4,272
' r - 57.41

(a)

(b)

(C)

(d)

(e)

65

TABLE 15 (CONT.)

Multiple Range Test-Station 7

Source df m.s. a
Between Treatments 5

Error 53 4,272 65.4
R' It I 8

p: 2 3 4 5 6
51 SD: 2.84 2.99 3.09 3.15 3.21
52 R'p: 185.74 195.55 202.09 206.01 209.93

Code: a b c d e f
x: 90.7 117.0 130.3 140.0 151.1 506.2
n: 10 10 10 10 9 10
Test Sequences: at 52 level

(f-e)’ greater than R'z

(e-a)’ not greater than R's

Conclusions: at 51 level

(a, h, c, d, e) can not be shown to be different

TABLE 16

66

Ratio of Phosphorus to Organic Nitrogen in Periphyton-Station 3A
0P8- Pins. 3) During Six Periods of the Summer

sample
no. A B
1 32.56 99.54
2 151.02 183.23
3 214.29 41.76
4 128.16 55.45
5 165.62 71.01
6 64.16 82.61
7 122.73 233.52
8 156.82 62.50
9 53.17 45.21
10 ... 76.42
sum 1,088.53 951.25
3 120.95 95.12
xx? 160,241 126,489
(2132/6 131,655 90,488
8:? 28,586 36,001
(3‘92 1,184,898 904,877
var. 3,573 4,000
sta. dev. 60 63
331 - 240,455;
883 I 16,538;
SS" - 233,917;
of - 3,308
.6 - 4,414

P O 0.749

C
45.55
122.92
130.67
105.62
42.86
38.57
51.32
119.48
85.44
53.37
795.80

79.58
75,797

63,330
12,467

633,298
1,385
37

df - 58
df - 5
df I 53

383.33
42.68
153.52
78.18
85.00
108.42
127.27
160.00
74.80
64.60

1,277.80

127.78
248,990

163,277
85,712

1,632,773

9,524

98

62.11
61.54
151.52
26.37
56.91
84.81
39.06
72.80
303.17
74.60

932.89
93.29
146,033
87,028
59,004

P

182.67
103.85
116.25
81.42
130.00
85.00
90.91
126.98
136.36
50.00

1,103.44

110.34
133,904

121,758
12,146

870,284 1,217,580

6,556
81

1,350
37

TABLE 17

67

Ratio of Phosphorus to Organic Nitrogen in Periphyton-Station 7
Gig. P/Is. N) During Six Periods of the Sun-er

sample

no. .A B C D
1 106.90 58.59 256.25 100.89
2 56.17 89.84 70.45 113.98
3 81.18 74.81 ... 269.09
4 76.26 134.34 84.35 122.90
5 51.96 118.25 141.77 107.56
6 122.81 78.91 95.60 254.82
7 57.75 203.81 51.95 248.05
8 31.52 45.66 248.28 156.37
9 74.40 53.08 125.00 132.38
10 45.33 93.57 110.42 225.41
sum 704.28 950.86 1,184.07 1,731.45
1' 70.43 95.09 131.56 173.14
It? 56,689 110,554 199,140 341,501
(I!)2/n 49,601 90,413 155,780 299,792
Ix? 7,088 20,140 43,359 41,709
(1!!)2 496,010 904,135 1,402,022 2,997,919
var. 788 2,238 5,420 4,634
sta. dev. 28 47 54 68

P Test on Data of Station 3A;+ Station 7
SS: - 458,918; df - 116

833 -
ssu - 373,385; df - 105

85,533; df - ll

- 7,776

I 3,556

as a?» of»

- 2.187

E
70.17

71.72
58.82
72.44
162.16
87.38
204.71
81.13
110.94
919.47

102.16
113,502
93,936
19,566

845,425
2,446
49

P
54.67
72.22
60.87
65.35
87.67
104.58
131.43
106.52
125.58

54.74
863.63

86.36
82,190
74,586

7,605

745,857
845

29

(a)

(b)

(c)

(d)

(e)

(f)

68

TABLE 17 (CONT.)

HMltiple Range Test-Station 3A«+ Station 7

Source df m.s. s
Between Treatments 11
Error 105 3,556 59.7
R. m g g
P p
p: 2 3 4 5 6 7
52 sp: 2.80 2.95 3.05 3.12 3.18 3.22
5% R'p: 167.2 176.1 182.1 186.3 189.8 192.2
p: 8 9 10 11 12
5% sp: 3.26 3.29 3.32 3.34 3.36
52 R'p: 194.6 196.4 198.2 199.4 200.6
Code. a b c d e f
X: 70.43 79.58 86.36 93.29 95.09 95.12
n: 10 10 10 10 10 10
Gods: g h i j k 1
X: 102.16 110.34 120.95 127.78 131.56 173.14

n: 9 10 9 10 9 10

Test Sequences: at 52 level
(l-h)‘ greater than R's; (l-i)‘ not greater than R'a

(k-a)’ not greater than 3'11

Conclusions: at 52 level

(a, b, c, d, e, f, g, h, i, j, k) can not be shown to be
different
(i, j, k, 1) can not he shown to be different

Nicrograms of Phosphorus per Nilligram of Organic Nitrogen:

EX
12 - 101.15

Figure 21

Mean Ratio of Total Phosphorus
(in fig.) to Organic Nitrogen
(in mg.) During 81:: Periods of
the Summer.

69

Ratio

160 1.. F”

150 _

Closed Bars 2 Station 3A
140 Open Bars = Station 7

 

130

10

909-

 

30

20..

10..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B C D

Periods

70

Aeneas one no 333809 Eugen
m u 885 .4658 8» B 8346.—
fim 9:445 Ads :3 286.5; 358.6 a...
Eu.— :.3 9.8535 H33. no oapwm nae:

mm 0.563

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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a. as a o m as c: o m 4

16 “014948

A “014399

220

180

160

140

100

20

Ratio

 

71

homogeneous population. If all the data are combined and the units
are equalized this ratio becomes 1.01 g. phosphorus per 10.0 grams
organic nitrogen. Ketchum (1949), and Ryther (1956) obtained ratios
within the general range of one 3. phosphorus to five g. organic
nitrogen in cultures of freshwater algae and marine algae,
respectively.

The effects of phosphorus storage by algae undoubtedly have
an influence upon the phosphorus to nitrogen ratio. It is possible
that the periphyton quickly became established on the shingles and
stored phosphorus in the cells until fertilization ceased, after
which there was a four day period in which the periphyton could
have grow enough to reduce the ratio of phosphorus to nitrogen
almost back to normal.

Einsele (1941) found that the planktonic algae of Schleinsee
were capable of storing up to ten times the necessary amount of
phosphorus per cell. The fact that planktonic algae are capable
of storing phosphorus is also shown by Lund (1950). This work was
done on Asterionella formosa in various English lakes. Lund also
illustrated that algae can take up phosphorus from lake water when
the concentration is as low as one part per billion. Work done on
the Red Cedar River by Crsenda (14.8.) tends to show the same type
of phenomenon in periphyton. In his studies he has found a periphyton
phosphorus to nitrogen ratio of about one to one during some periods.
However, it may be true, in some situations, that species composition
of the periphyton community is what changes rather than the phosphorus

to nitrogen ratio in a given species.

72

The data on the phosphorus to organic nitrogen ratio in the west
branch of the Sturgeon River are especially interesting since it
effectively proves that phosphorus is the limiting nutrient for
periphyton growth in the stream rather than nitrogen. This is true
since the amount of nitrogen added was considerably lower than
phosphorus added, but the maount of nitrogen per subshingle increased
several fold. It is possible that nitrogen would have become the
limiting factor if the addition was continued over a period of some
length.

M _t_¢_1_ organic nitrogen: Figure 23 is a histogram of the
means of the ratios for stations 3A and 7 at various times during
the «user. A combination of progressively decreasing organic
nitrogen values for station 3A and depressed pigment values early
in the study at both stations tends to give a skewed graph. The
reasons for the low pigment values are probably related to higher
water turbidity and lower temperatures. Figure 24 shows these means
and two standard deviations of the means.

Further analysis shows that periods B, c, D, R, and P from
station 7 and periods B, c, and P from station 3A cannot be shown
to be significantly different at the five percent level.

Although there is a certain nount of stability in the ratio
of pigment to organic nitrogen, it is by no means as constant as the
phosphorus to nitrogen ratio. The data and statistical analyses are

recorded for stations 3A and 7 in tables 18 and 19 respectively.

Figure 23

HOOD Ratio of Pigmnt (in

B W) to Organic Nitrogen
(in us.) During 31: Peri:
of the Summer.

73

20__

19 _

18 P

 

 

 

 

 

 

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Periods

74

 

.23: 65 no

neonate 2481.6 m a. 565
pagan 8... «6 83.84 5m 9:58
n.9— :.3 :omonfiz 35.30 on
Shotgun :3 page no Sea

a 995

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.3
—T
L.
L_1
J 1
to an
V: “011348

A norms

38

34

30

26

22

18

14

10

Ratio

TABLE 18

Ratio of Pigment to Organic Nitrogen
(Harvey units pigmenthmg. N) During

sample

no. A
1 3.47
2 4.49
3 5.12
4 4.95
5 4.00
6 0.90
7 5.00
8 7.95
9 1.87
lo . . .
sum 37.75
'x' 4.19
It? 191.43
(mzln 158.34
3:2 33.09
(£102 1,425.06
var. 4.14

sta. dev. 2.0

B
9.22
8.70
5.15

10.30
7.54
13.42
8.32
13.96
6.16
15.66
98.43

9.84
1,077.54
968.85
108.69

9.688.46
12.08

3.5

C

6.34
13.12
27.87
20.45
8.07
8.29
9.14
24.03
11.55
7.42
136.28

13.63
2,390.56
1,857.22

533.34

18,572.2
59.26
7.7

75

in Periphyton-Station 3A
Six Periods of the Summer

D

46.67
5.30
45.49
15.09
26.62
20.21
18.64
25.82
16.06
13.01
232.91

23.29
7,061.60
5,424.71
1,636.89

54,247.1
181.88

13.5

E
30.21
16.79
84.24
10.55
14.06
34.68
16.25
12.16
23.81
21.11

263.86

26.39
11,227.1
6,962.2
4,264.9

69,622.1
473.88

21.8

P
13.73
7.31
10.50
13.19
8.83
7.90
12.16
8.10
8.48
5.17
95.37

9.54
978.67
909.54

69.13
9,095.44
7.68

2.8

76

TABLE 19

Ratio of Pigment to Organic Nitrogen in Periphyton-Station, 7
(Harvey units pigment/Ins. N) During Six Periods of the Sufier

sample
no. A B c D E P
1 9.48 18.48 13.62 17.92 6.57 12.13
2 4.77 18.52 13.52 16.14 ... 10.37
3 6.88 8.00 ... 24.14 8.34 15.43
4 4.39 18.69 11.30 13.65 11.37 12.67
5 3.36 13.81 15.32 17.09 21.92 30.68
6 12.46 15.78 18.24 29.34 24.59 23.44
7 4.03 19.62 4.87 22.66 15.61 13.00
8 3.54 11.91 27.07 14.90 13.53 15.43
9 5.65 7.40 17.12 15.36 14.40 21.74
10 3.63 18.43 21.46 14.04 21.72 14.38
sum 58.19 150.64 142.52 185.24 138.05 169.27
E 5.82 15.06 15.84 18.52 15.34 16.93
£13 419.64‘ 2,458.76 2,573.51 3,672.13 2,433.00 3,230.48
(EX)2/n 338.61 2,269.24 2,256.88 3,431.39 2,117.53 2,865.23
3x2 81.03 189.52 316.63 240.74 315.47 365.25
(xx)2 3,386.08 22,692.4 20,311.9 34,313.9 19,057.8 28,652.3
Vlr. 9.00 21.06 39.58 26.75 39.43 40.58

sta. dev. 3.0 4.6 6.3 5.2 6.3 6.4

(s)

(b)

(c)

(d)

(9)

TABLE 19 (CWTJ

‘Nultiple Range Test-Station 3A;+ Station 7

Source df
Between Treatments 11
Error 105
’ u
R p s 2p
p: 2 3 4
5! sp: 2.80 2.95 3.05
52 R'p: 24.64 25.96 26.84
8 9 10
5! sp. 3.26 3.29 3.32
51 1'9: 28.69 28.95 29.22
Code: a b c
X: 4.19 5.82 9.54
n: 9 10 10
Codg: g h i
x: 15.34 15.84 16.93
n: 9 9 10
Test Sequences: at’SZ level

(1-1)'
(k-e)'
(J-b)'
(1-b)'
(h-b)'
(e-s)'
(d-a)'

Conclusions:

(5. k.
(:9 39
(c. d.
(b. c.
(a. b.

greater
greater
greater

greater

than R'4; (l-j)’
than R'7; (k-f)‘
than R'9; (j-c)’
than R'g
greater than R'y
greater than R's; (e-b)‘
not greater than R'g

at 51 level

m.s. a
77.66 8.8

5 6 7
3.12 3.18 3.22
27.46 27.98 28.34
11 12

3.34 3.36
29.39 29.57

d e f
9.84 13.63 15.06
10 10 10

j k 1
18.52 23.29 26.39
10 10 10

not greater than R'
not greater than R'5
not greater than R's

not greater than R'g

can not be shown to be different

i, j, k) can not be shown to be different
f, g, h, i, j) can not be shown to be different
e) can not be shown to be different
d) can not be shown to be different

78

phosphorus _t_o piggents: Pigure 25 shows in histogram form the
means of the ratios for stations 3A and 7. The reason for the ex-
cessively high value during period A is low pigment values which seem
to be associated with high water, as explained previously. Pigure 26
is a plot of the means and two standard deviations of the means.

An P test on station 7 showed the means to he heterogeneous even
at the one percent level. Purther analysis showed that periods B,

C, D, E, and P for station 7 and periods C, D, and E for station 3A
can not be shown to be significantly different at the five percent
level. The data and statistical analysis for stations 3A and 7 are
recorded in tables 20 and 21 respectively.

The phosphorus to pigment ratio is similar to the pigment to
nitrogen ratio in that it is usually fairly close to a mean value,
but there are many exceptions and this ratio is by no means as useful
as the phosphorus to nitrogen ratio.
bottom organius

The results of total phosphorus analysis of various benthic
organisms are reported in terms of micrograms phosphorus per milliliter
of organi-s in table 22. It was felt by the author that there would
be a value in knowing at least the approximate amount of phosphorus
occurring in these organisms. However, the values are not very
accurate, since volumes were measured crudely and not enough samples
were taken to carry out a statistical analysis.

whether a .all increase in the percentage of phosphorus in

these organisms took place after fertilization is not known, but

79

1'18an 25

Mean Ratio of Phoephom
(3.11 #80) to Pigmnt (3.11

Ham”) Dm‘ing 31:: Period;
of the Sumnr.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Periods

 

 

 

 

80

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N a. 23: Sean on... no 83.84
one union “Photon :3 283.— o...
Adm as 88885 H369 no canoe

wN 0.93m

 

 

 

 

 

 

 

 

 

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4.
30

4.
22

 

 

E} 4a;
H F .o
H
r* .
B a —’ b ‘Ko
[] A
‘02
l I 1 1 1 ii .41 4L7 1,
m. u: c: :3 a: «e (3 a: ‘4

V8 “014949

6 “014929

Ratio

81

TABLE 20

Ratio of Phosphorus to Pigment in Periphyton-Station 3A
(us. P/llsrvey units pigment) During Six
Periods of the Summer

sample
no. A B C D E P
1 9.38 10.79 7.19 8.21 2.06 13.30
2 33.64 21.07 9.36 8.04 3.66 14.21
3 41.83 8.11 4.69 3.37 1.80 11.07
4 25.88 5.38 5.16 5.18 2.50 6.17
5 41.41 9.42 5.31 3.19 4.05 14.72
6 71.00 6.16 4.66 5.36 2.44 10.76
7 24.54 28.05 5.61 6.83 2.40 7.48
8 19.71 4.48 4.97 6.20 5.99 15.69
9 28.49 7.33 7.39 4.66 12.73 16.07
10 ... 4.88 7.20 4.96 3.53 9.67
sum. 295.88 105.67 61.54 56.00 41.16 119.14
‘2 32.88 10.57 6.15 5.60 4.12 11.91
It? 12,197.32 1,666.18 400.46' 340.55 265.64 1,525.76
(IXJz/u 9,727.22 1,116.62 378.72 313.60 169.42 1,419.43
Ix; 2,470.10 549.56 21.74 26.95 96.22 106.33
(I!)2 87,544.97 11,166.2 3,787.17 3,136.00 1,694.15 14,194.3
‘ver. 308.76 61.06 2.44 2.99 10.67 11.81

sta. dev. 17.6 7.8 1.6 1.7 3.3 3.4

TABLE 21

Ratio of Phosphorus to Pigment in Periphyton-Station 7
(gig. P/Rarvey units pigment) During Six
Periods of the Summer

sample
no. A B C D E
1 11.27 3.17 18.81 5.63 10.67
2 11.78 4.85 5.21 7.06 ...
3 11.80 9.35 5.71 11.15 8.60
4 17.36 7.19 7.46 9.00 5.17
5 15.45 8.56 9.26 6.29 3.30
6 9.86 5.00 5.24 8.68 6.59
7 14.33 10.39 10.67 10.95 5.60
8 8.90 3.83 9.17 10.49 15.13
9 13.16 7.17 7.30 8.62 5.63
10 12.48 5.08 5.14 16.06 5.11
sum 126.39 64.59 83.97 93.93 65.80
2 12.64 6.46 8.40 9.39 7.31
812 1,655.81 470.80 860.07 963.94 586.94
(BX)2/o 1,597.44 417.19 705.10 882.28 481.07
3:2 58.37 53.61 154.97 81.66 105.87
(BIJZ 15,974.43 4,171.87 7,050.96 8,822.84 4,329.64
‘var. 6.49 5.96 17.22 9.07 13.23
sta. dev. 2.5 2.4 4.1 3.0 3.6

as, - 818.67, or - 58

53a - 324.34, at - 5

ss" - 494.33, df - 53
8% - 64.87

82

P

4.50
6.96
3.94
5.16
2.86
4.46
10.11
6.90
5.78
3.81

54.48

5.45

336.66

296.81

39.85

2,968.07

4.43

2.1

(a)

(b)

(e)

(d)

(8)

TABLE 21 (CONT.)

'Nultiple Range Test-Station 3Au+ Station 7

Source df
Between Treatments 11
Error 106
' C
R P s zp
p: 2 3 4
51 8p: 2.80 2.95 3.05
52 R'p: 16.80 17.70 18.30
p: 8 9 10
5% sp: 3.26 3.29 3.32
51 R'p: 19.56 19.74 19.92
Codg, a b c
X 4.12 5.45 5.60
n 10 10 10
Codg: g h i
X: 8.40 9.39 10.57
n: 10 10 10
Test Sequences: at 52 level
(l-k)‘ greater than R'z

(k-e) ' greater
(j-c)’ greater
(i-a)‘ greater

Conclusions:

(f. 8.
(d. e.
(b. e.
(a. b.

h: 1, 39
f. s. h.
‘19 e, f9
c9 d9 es

m.s.
35.52

5 6
3.12 3.18
18.72 19.08
11 12
3.34 3.36
20.04 20.16
d e
6.15 6.46
10 10

j R
11.91 12.64
10 10

83

3.22
19.32

than R'7; (k-f)‘ not greater than R'

then R'g; (j-d)‘ not greater than R'7
than R'9; (i-h)‘ not greater than R'g
(h-a)’ not greater than R'g

at 52 level

k) can not be shown to be different

i, 1) can not be shown to be different

g, h, i) can not be shown to be different
f, g, h) can not be shown to be different

TABLE 22

Total Phosphorus in Bottom Organisms

84

 

 

 

 

 

 

 

 

 

date organism sample volume ,1 g. P/ml. mean
7-22 Chara 3.0 m1. 200
7-22 " 5.6 ml. 172
7-22 " 5.0 m1 245
206
8-18 Chara 6.6 m1. 250
8-18 " 9.5 m1. 266
258
7-22 Stoneflies 1.6 .1. 1,875
8-18 Stoneflies 3.7 m1. 1,311
7-22 Neyflies 4.0 m1. 812
7-22 " 1.8 m1. 472
642
8-18 11.375116. 7.3 ml. 911
8-18 " 4.9 ml. 1,163
8-18 " 5.2 m1. 1,082
1,052
7-22 Dragonflies 3.4 m1 1,235
7-22 " 0.5 ml 1,999
7-22 " 3.3 m1 985
1,406
8-18 Dragonflies 1.0 m1. 1,425

 

 

85
there was no large increase. Even if a small change could be shown,
other factors such as life cycles could also influence the percentage.

It is evident that 92323 has only about one-fourth as high a
percentage of phosphorus as the insects studied. It is also
interesting to note that none of the analyses indicated more than

0.2 percent phosphorus (wet weight).

CNCLUS 1:11

It was concluded from.the results of this study that the
addition of inorganic phosphate to the west branch of the Sturgeon
River resulted in a large increase in the prflmary production in a
section extending at least several miles down the stream. Coloniza-
tion and growth upon new substrates was no more rapid after the
cessation of fertilization than before. The phosphorus to nitrogen
ratio in the periphyton complex.was one to ten by weight during the
entire study with the possible exception of the period of fertilisa-
tion. There is good reason to believe that phosphorus is the
limiting nutrient in primary production.

No significant increase in total volumes of bottom fauna at
station 7, the downstream station, could be correlated with

fertilization or its after effects.

APPENDIX
Introduction

There has been a concerted effort for many years to develop a
method of measuring primary production. The greatest problem has
been the difficulty of getting an exact, quantitative method. This
is especially difficult in lotic situations since current is a
factor. Any method which is developed must he of a type which can
be applied in an efficient manner to have any practical value.

One channel of effort has been based upon the fact that certain
types of pigments are essential to the photosynthetic process.

These methods are based upon the extraction and measurement of
pigments found in the plants which are carrying out primary production
in a given case.

Harvey (1934) formulated a method for the estimation of the
quantity of chlorOphyll present in an extract based upon a visual
comparison with a set of inorganic standards. This method was the
basis for much work in the fields of Limology and Oceanography. In
a modification of this method in which absorbency of the extract
is measured in the region from 640 to 700 millimicrons with a
photoelectic calorimeter and a correction is made at higher absorb-
encies for the deviation from the Lambert-Beer Law, Grzenda (11.8.)
has correlated the amount of pigment with dry organic weight of

periphyton in the Red Cedar River.

88

One of the more important recent attempts to improve on this
type of measurement is based upon the light absorbed by a 90 percent
acetone extract using a spectrophotometer at certain specific wave-
lengths (Richards, 1952) and the use of nomographs to simplify
calculations of the components causing this absorbency (Duxbury,
1956). In this method certain assmsptions as to the composition of
the pigment complex must be made, unless supplmnentary studies are
made. If all the pigments which absorb light in this area are
known and their specific absorbency can be determined, this method
should give accurate results.

It is the belief of the author that before any method of this
type can completely succeed, a more thorough understanding of the
pigment complex in algae is necessary. The physiological importance
of the various pigments needs further study and the relative stability

of the quantities of these pigments within the cell should be known.

Spectroscopy and Chromatography
(historical)

The pigment complex found in algae is complicated and has been
studied a great deal. A good review of the subject is found in
11:35 £939; _o_§ thcologz (Smith, 1951) in the section on pigments,
which is written by R. R. Strain. The xanthophylls are discussed
more thoroughly in £e_a_f_ gnthophylls (Strain, 1938) .

Pigments maybe separated efficiently by chromatography. Lind
(1953) made some rough separations by means of two-dimensional

ascending paper chromatography. However, column chromatography

89

has proven more useful in most applications. The subject of
chromatography is discussed on an introductory level by Brimley
(1953) and many applications and references to specific methods

are given by Zechmeister (1950). A thorough review of the literature
on the physical properties of piglents in general is given by

Zacheile (1941).

The structures of chlorophyll _a_ and b have been known for a
umber of years. Chlorophyll g has been found to be a magnesium
complex lacking phytol, and it is probably a modified magnesium
pheoporphyrin (Granick, 1949) . Chlorophylls _a and _l_>_ are found in
the Chlorophyta, ’- and g in the Bacillariaceae, and only a in the
Cyanophyta, (Smith, 1951). Although the chlorophylls are the only
pigments which are known to take a direct part in photosynthesis,
the Cyanophyta also possess phycobilin type pigments which are
protein containing pigments that absorb in the green range of the
spectrum and fluoresce in green light. These pigments are believed
to act to aid in energy transfer to the red-absorbing chlorophylls
by absorbing green light and fluorescing in a lower frequency
(French, 1952).

It is interesting to note that although the red peak of
chlorophyll _a_ is generally quoted as about 665 millimicrons, upon
direct measurement in leaves the peaks were found to be shifted about
twelve millimicrons toward the red end of the spectrum (Shpolskii,

1947) . This may be due to the association of chlorophyll with

proteins in the chloroplast.

9O

Chlorophyll, when acted upon by the enzyme chlorophyllase in
the presence of ethanol, forms ethyl chlorophyllide in which the
phytol is replaced by ethanol. Weak acids remove the magnesium
from chlorophyll to form pheophytins and the action of a strong
acid upon either the chlorophyll, the ethyl chlorophyllide, or the
pheophytin yields a pheophorbide in which both the phytol group and
the magnesium are missing (Bonner, 1950).

In order to detect which pigment is being studied after
separations and to follow the progress of the separations, spectro-
grams of the visible range are usually used. The absorbency peaks
for the algal pigments are given by Smith (1951). Bacteriochlorophyll
_a_ solutions have maxima at 360, 390, 570, and 770 millimicrons
(Holt, 1954). Protochlorophyll has peaks at 435, 530, 575, and 625
millimicrons (Smith, 1948). Holt (1952) also reported the
absorption spectra of ethyl chlorophyllides .a_ and _l; as being the
same as the original chlorophylls. He reports a shift of the peaks
to 408, 500, 532, 605, and 645 millimicrons for the pheophorbide
of chlorophyll _a_ and peaks of 432, 525, 600, and 645 millimicrons
for the pheophorbide of chlorophyll b. He reports the same spectra
for the pheophytins as for the pheophorbides. This would mean that
the removal of the alcohol or a change of the alcohol would have no
effect on the spectra.

Using ethanol Bvstigneev (1954) reports a reversible shift of
chlorophyll _a_ to a smaiquinonoid form with peaks at 415, 518, 585,

and 665-670 millimicrons. In aerobic conditions this form shifts

to the pheophyt in .

91

Experimental

Red Cedar River

Fresh periphyton material was obtained from the Red Cedar River
in Ingham County in the water upstream from the sewage treatment
plant at Hilliaston. In a preliminary experiment a small amount
of 90 percent acetone extract was fractionated in a column packed
with powdered sucrose. The calms were always packed with a
slurry of adsorbent in the solvent. Petroleum ether and benzene
(9:1) was used as a solvent. Four fractions were obtained. Fractions
A and C are shown in figures 27 and 28. Fraction A contains a large
amount of chlorophyll a and probably a relatively small amount of
chlorophyll 5. Similarly fraction C probably contains a considerable
amount of chlorophyll b, which is obscured by some persistent
chlorophyll g. It will be noticed in all of the experimental
results that there tends to be a shift of absorbency peaks several
millimicrons toward the blue and, probably due to instrumental error.

In order to get a better idea of the pigments present in this
complex another sample was obtained and a large initial volume of
90 percent acetone extract was transferred to a petroleum ether,
benzene solvent. This was run through a powdered sucrose column 16
millimeters in diameter and about 30 centimeters in length. By
visually controlled fractionation 19 fractions of eluent were
obtained and the pigment remaining in the sugar was extracted to

form fraction 20 .

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A graph of the spectra of the total mixture is shown in figure 29.
Fraction 1 was run on a ten millimeter column of alumina dried at
60’ C. and broken into three more fractions. Fraction 1' is shown
in figure 30 and has a single peak at about 448 millimicrons. In
all probability this reddish pigment was fucoxanthin, which is the
most abundant xanthophyll found in diatoms. Fraction 3' is shown
in figure 31 and is identical with fraction 9 ' and will be dis-
cussed later. It is shown here only to point out that two different
fractionation methods arrived at precisely the same peaks for this
important constituent of the pigment complex.

Fractions 7 through 11 were combined and rechromatagrsphed in
precisely the sme manner as the original mixture, except that the
pigments were separated on the basis of color regions within the
column rather than elution. The column was cut into six color
regions to form fractions 4' through 9' and the color that came
through made up fraction 10'. Fraction 6' is shown in figure 32.

It was an olive green color. This fraction was shown to be composed
of at least two pigments by combining it with fraction 5', a similar
fraction, and then rechromatagrsphing on a ten millimeter column of
sucrose and separating into fractions 1" through 4" on the basis of
colored bands.

‘ Fraction 2" is shown in figure 33. It is believed to represent
almost entirely chlorophyll _a_ in an umnodified form. This fraction
was dried under reduced light in a vacuum desiccator and weighed.
The weight of the sample was 2.3 milligrams. This was redissolved

in ten milliliters of ethanol and the spectra determined i-ediately.

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The spectrogram is shown in figure 34. The peak which was

originally at about 428 millimicrons shifted to about 415 millimicrons
while the red peak rusined unchanged. This corresponds to the

A results of Evstigneev (1954) as described earlier.

Fraction 4" is shown in figure 35. This was an orange pigment
and the peaks are at about 442 and 465 millimicrons. It was probably
violaxanthin, which is found in the green algae.

Fraction 8' is shown in figure 36 and is quite apparently a
combination of several pigments. Further fractionations were of
no value due to lack of sufficient yields. The author would pro-
pose that the peaks are a result of a combination of the chlorophyll
intermediate described by Evstigneev (1954) and a pigment with peaks
at about 442 and 473 millimicrons. This might be lutein which
absorbs at 446 and 476 millimicrons and is a major pigment in the
green algae, or it might be diadinoxanthin which is found in‘diatoms
and absorbs at 448 and 478 millimicrons.

Fraction 9' was a definitely blue-green pigment with a very
high specific absorbency. It is shown in figure 37 and has definite
peaks at 408, 502, 532, 605, and 662 millimicrons. This pigment
occurs in fresh periphyton and since it has a strong peak at 662
millimicrons and occurs in large enough quantities to be easily
separable, the method used by Richards (1952) would be very mis-
leading if applied to this material. The peaks found for this
pigment correspond to the peaks reported by Holt (1954) for
pheophorbide 3 except for the red peak at 662 millimicrons. This

pig-ant is only adsorbed weakly by sucrose. Fraction 9' was dried

101

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in a vacuum desiccator under reduced lighting and found to weigh 0.7
milligrams. This amount was dissolved in ten milliliters of 95 per-
cent ethanol and the spectrogram determined imediately. The result
is shown in figure 38. It is interesting to note that the use of
alcohol as a solvent doesn't shift these peaks.

Fractions 14 through 17 were combined and rechromatagrsphed
using a ten millimeter column of alumina. The column was separated
into four fractions an the basis of color bands and the fractions were
labeled 11' to 14'. Fraction 11' is shown in figure 39. It is an
orange pigment with a single peak at about 442 millimicrons. It is
probably neofucoxanthin A (447 my) , neofucoxanthin B (446 mg), or a
combination of the two. Both pigments are found in fair amounts in
diatoms.

An outline of these fractionations and the colors of the
fractions is shown in table 23. The spectrophotometer data, which
was obtained using a Beckman Hodel B Spectrophotometer, is shown

in table 24.

West Branch of the Sturgeon River

In order to determine what was actually measured when the
absorbencies of 95 percent ethanol extracts of periphyton were
determined in the west branch of the Sturgeon River study, a
chromatographic separation was carried out on a large volume of
the extract. The extract was dried in a vacuum desiccator under
reduced lighting and chromatographed in a 16 millimeter column

packed with sucrose. The eluent was separated into seven fractions

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TIBLB 23

108

Red Cedar River Periphyton Separations

 

 

 

MIXTURE
sucrose
alumina Fraction
Fract. l - yellow-greenf“ "
n 2 _ grew 11
" 3 - green
" 4 - green
" 5 - green ’
" 6 - dark green” ‘Fract. 4' -
Fract. 7 - " 5' -
fl 8 - H 6| -
" 9 - dark sucrose ’ " 7' -
" 10 - green " 8' -
n 11 _ J u 9! _
Fract.12 - dark green L " 10' -
" l3 -_ggeen-yell
Fract l4 - Fract.ll' -
ll 15 - II 12' _
n 16 - yellow 1 n 13! _
I! 17 _ ll 14' _

 

Fract.18 :dark green
19 - light green
20 - light green

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* Fract. 5' - green
" 6' - olive green

Fract.

sucrose

1" - light green
2" - dark green
3" -

4" - 2nd orange

1' - red
2' - red-green
3' - green

light green
green *
olive green*
green
green-yellow
blue-green
yellow

orange
green-yellow
green-yellow
green

1st orange and green

109
TABLE 24

Absorbency Data Obtained with a Beckmmn Model B Spectrophotommter on
Pigments from Red Cedar River Periphyton

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Have- Absorbency
length fract. fract. mixture fract. fract. fract.
in.lw1 A c 1- 3: 6.
400 0.229 0.272 0.432 0.241 0.810 0.560
405 0.250 0.320 0.490 0.264: 0.835 0.625
410 0.271 0.348 0.543 0.285 0.845 0.698
415 0.279 0.372» 0.572 0.313 0.750 0.740
420 0.292 0.391 0.610 0.348 0.510 0.800
425 0.329 0.420 0.672 0.370 0.322 0.845
430 0.360 0.460 0.750 0.390 0.187 0.880
435 0.368 0.460 0.738 0.404 0.118 0.855
440 0.336 0.418 0.658 0.422 0.094 0.795
445 0.392 0.375 0.578 0.440 0.083 0.720
450 0.279 0.350 0.523 0.444 0.081 0.695
455 0.2417 0.324 0.481 0.431 0.080 0.655
460 0.191 0.302 0.430 0.420 0.080 0.622
465 0.150 0.295 0.392 0.411 0.072; 0.620
470 0.103 0.298 0.369 0.400 0.077 0.602
475 0.083 0.284, 0.352 0.378 0.075 0.555
480 0.065 0.260 0.318 0.342 0.070 0.469
485 0.056 02221 0.273 0.302 0.069 0.390
490 0.048 0.170 0.211 0.257 0.081 0.292
495 0.045 0.134 02124 0.2127 0.097 0.228
500 0.036 0.104 0.126 0.173 0.108 0.170
-505 0.032 0.085 0.105 0.136 0.104 0.136
510 0.029 0.070 0.080 0.110 0.084 0.101
_230 0.023 0.042 0.056 0.064 0.049 0.060
530 0.024 0.034 0.043 0.048 0.083 0.042
540 0.025 0.025 0.035 0.040 0.049 0.030
550 0.024 0.023 0.034 0.035 0.028 0.026
560 0.028 0.023 0.039 0.032__ 0.029 0.028
570 0.040 0.028 0.051 0.030 0.022 0.031
580 0.044 0.030 0.059 0.929 0.023 0.033
590 0.040 0.027 0.052 0.028 0.035 0.032
600 0.049, 0.033 0.060 0.024 0.060 0.040
610 0.047 0.041 0.071 0.020 0.057 0.046
_620 0.052 0.043 0.075 0,019 0.036 0.042
625 0.053 0.038 0.071 0.018 0.035 0.039
630 0.053 0.033 0.061 0.017 0.031 0.034
635 0.045 0.034 0.049 0.016 0.037 0.036
640 0.048 0.040 0.063 0.016 0.044 0.054
645 0.058 0.059 0.092 0.017 0.078 0.083
650 0.094 0.093 0.155 0.011 0.137 0.140
655 0.137 0.133 0.233 0.010 0.223 0.197
660 0.161 0.159 0.298 0.015 0.318 0.210
'9665 0.143 0.138 0.272 0.016 0.338 0.167
670 0.095 0.101 0 185 0.013 0 252 0.106
::§80 0.027 0.027 0.038 0.013 0.058 0.023

 

 

 

 

TABLE 24 (CM.)

110

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Have- Absorbency
length fract. fract. fract. fract. fract. fract. fract.
in m“ 8' 9'-AC 9 ' -3T 11 ' 2"-AC 2"-ET 4"
400 0.530 0.870 0.462 0.425 0.558 0.400 0.710
405 0.574 0.920 0.482: 0.463 0.615 0.412; 0.785
410 0.620 0.940 0.483 0.502 0.658 0.462 0.900
415 0.628 0.770 0.433 0.540 0.680 0.490 0.985
420 0.578 0.522 0.328 0.563 0.700 0.482 1.08
_425 0.515 0.307 0.210 0.582 0.725 0.468 1.12
430 0.485 0.150 0.114 0.612 0.685 0.450 1.19
435 o.4gg_ 0.080 0.058 0.628 0.490 0.383 1.23
440 0.490 0.058 0.039 0.642 0.282 0.308 1.26
445 0.485 0.043 0.024 0.641 0.158 0;2Q6 1.26
450 0.455 0.040 0.022 0.620 0.100 0.138 1.23
455 0.401 0.039 v0.018 0.600 0.078 0.105 1.21
460 0.373 0.039 0.018 0.565 0.061 0.085 1.21
465 0.373 0.048 0.025 0.5522 0.055 0.072 1.21
470 0.384 0.049 0.024 0.520 0.048 0.064 1.20
475 0.382, 0.048 0.025 0.480 0.046 0.057 1.08
480 0.330 0.041 0.018 0.420 0.044 0.057 0.930
485 0.238 0.049 0.022 0.366 0.042 ..... 0.7692
490 0.161 0.062 0.032 0.298 0.037 0.044 0.590
495 0.110 0.085 0.043 0.242 0.034 ..... 0.444
500 0.080 0.105 0.050 0.191 0.030 0.039 0.350
505 0.063 0.107 0.055 0.161 0.027 ..... 0.258
510 0.046 0.084 0.045 0.131 0.028 0.036 0.189
_220 0.031 0.046 0.031 0.092 0.033 0.037 0.112_
530 0.035 0.084 0.038 0.070 0.035 0.040 0.067
540 0.022 0.050 0.043 0.060 0.033 0.036 0.043
550 0.020 0.031 0.025 0.057 0.032 0.040 0.031
560 0.021 0.029 0.022 0.052 0.049 0.051 0.021__
570 0.020 0.021 0.017 0.051 0.059 0.056 0.023
580 0.020 0.022 0.017 0.056 0.051 0.061 0.023
590 0.024 0.036 0.025 0.063 0.055 0.059 0.024
600 0.030 0.065 0.042: 0.080 0.080 0.07] 0.019
610 0.031 0.064 0.038 0.094 0.095 0.087 0.023
620 0.024» 0.042 0.028 0.105 0.083 0.086 0.017
625 0.017 0.037 0.028 0.101 0.077 0.089 0.022
630 0.009 0.035 0.020 0.095 0.068 0.078 0.020
635 0.027 0.038 0.026 0.078 0.087 0.077 0.020
640 0.040 0.054 0.032, 0.088 02224 0.106 0.025
'—645 0.055 0.084 0.048 0.090 0.208 0.146 0.027
650 0.082 0.154 0.081 0.093 0.343 0.207 0.034
'—655 0.120 0.267 0.134 0.097 0.450 0.266 0.042
39290 0.147 0.358 0.188 0.096 0.470 0.322_20.042
'565 0.142 0.373 0.205 0.095 0.357 0.301 0.042
670 0.108 0.277 0.167 0.080 0.238 0.222 0.036
780 0.031 0.062 0.041 0.072 0.049 0.072 0.021

111
by visual control. The upper, middle, and lower parts of the column
were extracted to give fractions 8, 9, and 10 respectively. A
spectrogram of the orginal mixture is shown in figure 40.

Fractions 1 and 2 were combined and rechromatagrsphed on a ten
millimeter column of alumina. A red layer (fract. 1') was separated
and is shown in figure 41. It is the belief of the author that this
material represented the chromatophore grouping of a phycobilin
type pigment which was no longer connected with the protein portion
of the molecule. The phycobilins are known to be unstable in
solvents at room temperature.

Fraction 7 is shown in figure 42. It is a golden-colored
pigment with a single peak at about 445 millimicrons. It may be
one of the neofucoxanthins.

Fraction 8 is the component of greatest interest, since it
represents the bulk of the pigment measured in this study. It is
shown in figure 43. The major peaks are at 415 and 645 millimicrons.
:The red peak corresponds to the value reported by Holt (1954) for
pheophorbide _a_, and the blue peak corresponds to the value reported
by Bvstigneev for the semiquinonoid form of chlorOphyll _a_.

Since the periphyton was obtained by scraping periphyton from
wood shingles, some of the cedar wood was soaked for several months
in 95 percent ethanol and the resulting solution's spectra determined.
The spectrogram is shown in figure 44. The amounts of wood fibers
in the samples would have been very anall and the peak is at 452
millimicrons which doesn't correspond with any of the separated

components. The absorbency in the 660 millimicron region is also
negligible.

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116

To illustrate the amounts of the red pigment (fract. 1') found
in some samples, the spectra of an unusually reddish sample was
determined and is shown in figure 45. The sample was station 3A,
period 8, sample 10. Its graph is essentially a line with a negative
slope. There is only a slight peak at 415 millimicrons due to the
presence of a little pheophorbide g, or whatever the pigment in
fraction 8 is.

An unusually green sample from station 3A, period D, sample 7
is shown in figure 46. It has peaks at 417 and about 650 mdllimicrons.
The spectrophotometric data for the figures are shown in table 25.
It is evident from this analysis that the pigments measured in the
study were not chlorophylls, but rather the decomposition products
of chlorophyll. If the method is understood to yield only a
relative index to periphyton abundance, this in no way invalidates
the method. If measurements of actual quantities of various
pigments are to be.made, it is evident that ethanol should not be

used as a solvent.

117

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TIBLE 25

120

Absorbency Data Obtained with e Becknan nodel B Spectmphotoneter on
Pigments of Periphyton fronxthe'weat branch of the Sturgeon River

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hive-

length mixture frect. frect. fract. cedar 3Ar10-B 3A—7-D
in up. 1 ' 7 8 extract in non
400 0.501 0.865 0.369 0.204 0.196 0.496 0.183
405 0.521 0.870 0.3822 0.210 9:290 0.480 0.193
410 0.543 0.875 0.410 0.226 0.214 0.500 0.225
415 0.559 0.875 0.420 0.222» 0.224 0.521 0.255
420 0.545 0.865 0.422 0.233 0.238 0.508 0.244
425 0.504 0.850 0.419 0.227 0.248 0.463 0.200
430 0.468 0.825 0.414 0.222 0.252 0.433 0.180
435 0.451 0.815 0.421 0.220 0.259 0.412 0.164
440 0.438 0.800 0.427 0.221 0.272 0.402 0.159
445 0.420 0.780 0.429 9;§§9 0.282 0.395 0.151
450 0.405 0.767 0.418 0.214 0.281 0.389 0.143
455 0.380 0.750 0.398 0.210 0.273 0.377 0.136
460 0.359 0.730 0.374 0.199 0.259 0.367 0.122
465 0.333 0.720 0.352 0.196 0.251 0.353 0.115
470 0.310 0.685 0.330 0.187 0.243 0.339 0.108
475 0.290 0.665 0.310 0.177 0.241 0.331 0.099
480 0.260 0.645 0.279 0.164 0.230 0.317 0.088
485 0:237 ..... 0.244 ..... 0.299 0.300 0.074
490 0.210 0.600 0.210 0.127 0.179 0.276 0.061
495 0.189 ..... 0.184 ..... 0.154 0.260 0.052
500 0.165 0.560 0.150 0.093 0.132 0.248 0.045
505 0.149 ..... 0.125 ..... 0.119 0.239 0.039
510 0.136 0.520 0.100 0.064 0.108 0.230 0.034
520 0.110 0.488 0.064 0.042 0.099 0.213 0.027
530 0.094 0.462 0.045 0.037 0.093 0.207 0.021
540 0.085 0.440 0.032 0.029 0.091 0.202 0.019
550 0.080 0.418 0.027 0.029 0.088 0.200 0.018
560 0.074 0.390 0.020 0.027 0.079 0.195 0.019
570 0.068 0.357 0.019 0.024 0.071 0.186 0.017
580 0.066 0.327 0.018 0.025 0.062 0.174 0.017
590 0.068 0.304 0.019 0.026 0.056 0.166 0.021
600 0.064 0.281 0.013 0.022 0.046 0.154 0.022
610 0.057 0.260 0.014 0.020 0.039 0.139 0.021
620 0.053 0.2§Z_ 0.012__A 0.017 0.033 0.122 0.923
625 0.056 ..... ..... ..... 0.031 0.118 .....
630 0.056 0.229_ 0.013 0.018 0.024 0.108 0.026
635 0.059 0.218 ..... 0.020 0.021 0.106 .....
640 0.076 02218 0.025 0.023 0.017 0.104 0.038
645 0.085 0.218 0.028 0.023 ..... ..... 0.044
650 0.082 0.212 0.023 0.022 0.012 0.101 0.049
655 0.069 0.209 0.0204 0.018 ..... ..... 0.045
660 0.060 0.297 0.020 0.017 0.008 0.078 0.039
665 0.051 ..... 0.017 0.016 ..... ..... 0.032
670 0.042 0.169 0.012 0.025 0.0022 04044 0.028
680 0.024 0.143 0.005 0.010 0.003 0.032 0.021

 

 

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