Ecological Research Series
ALGAL METABOLITE INFLUENCE ON
BLOOM SEQUENCE IN
EUTROPHIED FRESHWATER PONDS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-081
July 1976
ALGAL METABOLITE INFLUENCE ON BLOOM
SEQUENCE IN EUTROPHIED FRESHWATER PONDS
By
Kathleen Irwin Keating
EPA Research Grant
RA 801387
Project Officer
Thomas E. Maloney
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
Prepared for
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
WASHINGTON, D. C. 20460
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products consti-
tute endorsement or recommendation for use.
ii
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ABSTRACT
I. Bloom sequence in Linsley Pond, Connecticut, was monitored for
three years. Bloom dominant algae were isolated in culture, heat-
labile, bio-active substances in cell-free filtrates of these cultures
were tested against each of the dominants. Enhancing, or neutral,
effects on successors; and inhibiting, or neutral, effects on predeces-
sors were consistently observed. Lake waters exhibited parallel
effects. Additionally, inhibition patterns suited differences in year-
to-year patterns of in situ blooms. This widespread correlation of in
situ events with in vitro phenomena indicates that extracellular pro-
ducts of bloom dominant algae are significant in bloom sequence deter-
mination in eutrophied fresh waters.
II. Spring diatom bloom density varied inversely with the preceding
winter's blue-green population density. Diatom blooms, when they
occurred, ended when available silica was depleted. Generalized in
situ and in vitro inhibition of diatoms by blue-greens was traced to
heat-labile, dialysable products of blue-greens. After separation and
concentration via ether extraction or ultrafiltration active substances
were returned to growth media. Preliminary evidence suggests that
inhibition involves interference with silica availability.
III. The feasibility of biological programming of bloom sequence in
eutrophied lakes is considered.
This report was submitted in fulfillment of EPA Research Grant
RA 801387 under the sponsorship of the Environmental Protection
Agency. Work reported was completed as of May 1, 1975.
iii
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CONTENTS
ABSTRACT iii
LIST OF FIGURES V
LIST OF TABLES vii
ACKNOWLEDGEMENTS viii
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III CORRELATIONS OF O[ SITU AND I£ VITRO EVENTS
AND THEIR IMPLICATIONS
III:I Correlation: Bloom Sequence and Filtrate
Activity 4
III:II Correlation: Bloom Sequence, Filtrate
Activity, and Activity of Linsley Pond Waters 6
IV WATER QUALITY MANAGEMENT PROSPECTS: PROGRAMMING
BLOOM SEQUENCE (Non-Mathematical)
IV:I In Situ Exclusion by Blue-Green Metabolites 16
IJt:II A Strategy for Sequence Control 17
V INTRODUCTION
V:I Eutrophication 18
V:II An Hypothesis 20
V:III Significant Related Research 24
VI FIELD STUDY
VI:I Site Choice and Sampling 30
VI:II Physical and Chemical: Methods 32
VI:III Physical and Chemical: Results, Discussion 34
VI:IV Bloom Sequence Monitoring: Methods 49
VI:V Bloom Sequence Monitoring: Results,
Discussion 51
VII LABORATORY STUDY
VII:I Bioassay 72
VII:II Biological Activity in Filtrates of Bloom
Dominant Organisms and of Natural Pond Waters 78
VII:III Preliminary Characterization of
Heat-Labile Diatom Inhibitor: Methods 88
VII:IV Preliminary Characterization of Heat-
Labile Diatom Inhibitor: Results and Discussion 93
VII:V Effects on Diatom Inhibition Produced by Si
Addition to Filtrates, to Linsley Pond Water 102
REFERENCES 110
APPENDICES 121
iv
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FIGURES
Mo. Page
1. Basic Model 22
2. Carbon Dioxide: Annual Cycle 35
3. pH: Annual Cycle 36
4. Total Alkalinity: Annual Cycle 38
5. Comparison of 1972 Alkalinity (by depth) with Lake
Morphology 40
6. Thermal Profile: Annual Cycle 41
7. Depth of Thermocline: Three Years 42
8. Dissolved Oxygen: Annual Cycle 43
9. Hydrogen Sulfide Horizon: Three Years 44
10. Secchi Depth: Three Years 46
11. B. 0. D. (5 day) 48
12. Phytoplankton Bloom Sequence, Linsley Pond. "Winter, 1971
thru Fall, 1974. 52
Key for Figure 12 on page 54
13. 1971-1972, Winter 55
14. 1972, Spring 58
15. Depth of Oscillatoria rubescens Bloom Populations
in the Lake—for Comparison 59
16. 1972, Late Spring and Summer 61
17. 1972-1973, Fall and Winter 63
18. 1973, Spring and Sunmer 65
19. 1973-1974, Fall and Winter 66
20. 1974, Late Winter and Spring. 1974, Summer and Fall 67
21. Oscillatoria rubescens. Depth of Population Compared
to H2S Boundary 69
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FIGURES (continued)
No.
22. Zooplankton Occurrence Compared to Blue-Green Algal
Occurrence. 1971-1973. 71
23. Comparison of Growth Response to Various Levels of Added
Silica in Pre- and Post Diatom-Bloom Pond Waters.
February 14, and June 13, 1974. Filter-LJterilized Only 106
24. Comparison of Growth Response to Various Levels of Added
Silica in Pre- and Post Diatom-Bloom Pond Waters.
February 14, and June 13, 1974. Autoclaved Only 107
25. Comparison of Growth Response to Various Levels of Added
Silica. Pre-Diatorn-Bloom Pond Waters.
Filter-Sterilized and Autoclaved 108
26. Comparison of Growth Response to Various Levels of Added
Silica. Post-Diatom-Bloom Pond Waters.
Filter-Sterilized and Autoclaved 109
VI
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TABLES
No. Page
1. Correlations between Bloom Sequence and Filtrate Effects 4
2. Seasonal Variations Which Correlate with Filtrate Effects 7
3. Comparison of In Situ and In Vitro Population Densities 9
4. Trifold Correlations: Bloom Sequence; Filtrate Effects,
and Biological Activity of Collected Linsley Pond
Water Samples 10
5. Summary: Heat-Labile Biological Effects of Blue-Green
Filtrates 79
6. Effects of Blue-Green Algal Filtrates on Blue-Green Algae 81
7. Suiaiaary: Heat-Labile Biological Effects of Freshly-
Collected, and Freezer-Stored Pond "Waters 84
8. Growth in F vs A Assays of Both Freshly-Collected and
Freezer-Stored Portions of the Linsley Pond Water
Sample Collected on Karch 18, 1973 67
9. Comparison of Dialyzable and Non-Dialyzable Portions of
a Producer Filtrate Before and After Storage:
Storage at Either 5°C or Frozen 96
10. Results of Dilution Series Tests of Activity of Filtrates
after Passage thru Ultrafilters 97
11. Comparison of Control and Test Culture Maxima in Ether
Extraction Procedures 99
12. Comparison of Inhibitory Effect of Bacterized and Axenic
Producer Cultures of Anabaena sp. (538) 101
13. Effects of Silica Addition on the Heat-Labile Diatom
Inhibition Exhibited by Filtrates of Blue-Green Algae 103
14. Diatom Growth Maxima in Pond Water Collected at the End of
the 1973 Diatom Bloom: With and Without Silica Addition 105
VII
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ACKNOWLEDGEMENTS
To my advisors, Luigi Provasoli, G. Evelyn Hutchinson, and Richard
Miller for their advice and criticism, and to Irma Pintner, Graham
Berlyn and Robert Trench for their guidance in laboratory procedures
and techniques, my thanks. I also wish to thank Francis Drouet,
Stjepko Golubic, and Ruth Patrick for their essential aid with the
taxonomy of many of the organisms involved in this study.
My thanks also to the Linsley Pond Association for their cooperation
and assistance during my study of their pond, to the Batkin family
for their encouragement and support, and to Ruth Allen, Edward
Bonneau, Richard Brugam, Douglas Conklin, Linda Cunningham, Dennis
Cunningham, Marta Estrada, Gail Ferris, Michael Hartman, Susan
Kossuth Murphy, Karen Glaus Porter, David Schoenberg, and Elizabeth
Schultz for their willing sharing of ideas and techniques, and
especially to Carol Dixon for her extraordinary typing, editing, and
proofreading skills.
I am also grateful to my parents, Alda and William Irwin, for that
special brand of baby sitting which only a family may provide.
I wish to acknowledge fellowship support from the Forestry and Environ-
mental Science Department at Yale; research support''from the U. S.
Environmental Protection Agency; the guidance of Mr. Allyn Richardson
and Dr. T. E. Maloney in the development of my grant proposal, the
assistance of Haskins Laboratories, Inc. in the administration of the
research grant and their hospitality in the laboratory, and the
encouragement of the Administration of the Department of Environmental
Science at Cook College, Rutgers University where the final work for
this project was completed.
viii
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SECTION I
CONCLUSIONS
GENERAL
1. While not the sole parameter of control, the extracellular metabo-
lites of planktonic bloom dominant algae play a most significant role
in the determination of bloom sequence in a eutrophied freshwater pond.
2. Certain extracellular metabolites of planktonic blue-green algae
substantially inhibit the growth of planktonic diatoms in culture and
in the natural sequence in Linsley Pond.
3. Preliminary tests indicate that the inhibition of diatom growth by
blue-green algal metabolites may be widespread in freshwater lakes.
4. When the elimination of excessive nutrient inflow is not practical,
biological management, or programming, of blooms in eutrophied lakes
should be attempted. An hypothetical plan is offered to modify the
unsatisfactory conditions in one lake which would cost approximately
$500 per annum while providing a more satisfactory lake from both
aesthetic and food chain points of view; Section IV.
SPECIFIC
1. Bloom sequence, cell-free filtrates of bloom dominant blue-green
algae, and freshly collected lake waters provided in vitro and in situ
examples of algal allelopathic effects on blue-greens, diatoms, flagel-
lates, and green algae.
2. Cell-free filtrates of eight planktonic blue-greens which dominated
in Linsley (three year sequence) all produced growth inhibiting effects
on the 30 plus varieties of planktonic Linsley diatoms tested, and on
the non-Linsley diatoms tested.
3. Two non-Linsley blue-greens produced inhibitory effects on most,
but not all, of the Linsley and non-Linsley diatoms tested.
4. The presence of bacteria in producer and, or, assay cultures dimin-
ished, but did not eliminate, the effects noted above.
5. The substance responsible for diatom inhibition in cell-free fil-
trates of the blue-green Anabaena sp. (538) can be separated and con-
centrated by ether extraction or by ultrafiltration without loss of
activity.
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6. This diatom inhibiting substance is heat-labile; dialysable; and
can be separated into the following three fractions:
1) Inhibitor #1
Color: Peach-pink (Winkler pink)
MW: Less than 12,000 and greater than 10,000
Size: 20-50 fi (as indicated by dialysis and Amicon
ultrafiltration)
Other: Oily to touch
2) Enhancer #1
Color: Vivid raid-yellow
MW: Less than 10,000 and greater than 1,000
Size: 10-20 ft
Other: Adheres to filter, therefore may be a steroid.
Is masked by Inhibitor #1 at full strength,
but not at dilution.
3) Inhibitor #2
Color: None
MW: Less than 500
Size: Less than 10 ft
Other: May be an artifact of bioassay procedures,
especially of autoclaving.
7.' The addition of moderately high quantities of silica overcomes in
part, or totally, the heat-labile inhibitory effects of blue-greens on
diatoms.
8. Densities of spring diatom blooms in Linsley Pond are inversely
proportional to* those of winter blue-green blooms.
9. Waters collected at the end of spring diatom blooms in Linsley Pond
support very low diatom growth; diatom growth can be restored by the
addition of silica.
10. Blue-green bloom populations develop in Linsley Pond after the
spring diatom bloom wanes.
11. Zooplankton populations in Linsley vary inversely with blue-green
populations.
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SECTION II
RECOMME NDA TIONS
1. The biological management of bloom sequence, as a means to amelio-
rate conditions of eutrophication when nutrient elimination is not
practical, should be thoroughly explored. Demonstration projects in
severely eutrophied small lakes should be attempted. While Linsley is
a likely target, and an hypothetical program for such control is out-
lined in this report, Section IV, the very special value of Linsley to
the scientific community may make any form of intervention undesirable.
The suggested program could be readily adapted to other lakes in similar
condition if suitable data concerning the bloom sequence of those lakes
were available. While the approach vould be similar, specific manage-
ment techniques must be tailored to the lake in question.
2. Further work should be undertaken to determine how general the blue-
green inhibition of diatom growth is. This would allow the application
of management techniques to incorporate additional controls for undesir-
able blooms.
3. Since preliminary study suggests that substances in the cell-free
filtrates of_ blue-green cultures interfere with in vitro growth of the
crustacean, Mpina macrocopa_, and since zooplankton populations in many
freshwater lakes 'have been found to vary inversely with blue-green
populations, the possibility that blue-green metabolites may effect the
next trophic level should be explored carefully. The rapid disappear-
ance of zooplankton during blue-green blooms has been tied to food
preference and digestability problems in prior studies. But no indica-
tion has heretofore been presented that there are allelopathic, extra-
cellular metabolite, effects.
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SECTION III
CORRELATIONS OF IN SITU —IN VITRO EVENTS
AND THEIR IMPLICATIONS
III; I CORRELATION OF BLOOM SEQUENCE WITH BIOLOGICAL ACTIVITY IN
FILTRATES OF DOMINANT BLUE-GREEN AIXSAE
The correlation between bloom sequence and heat-labile filtrate effects
presents a consistent picture of bloom dominant blue-greens producing
1) inhibiting or neutral (-, 0) effects on predecessors, and 2) enhanc-
ing or neutral (+, 0) effects on successors (Table 1). Therefore, the
extracellular metabolites in filtrates show a capacity to improve the
competitive position of the organism which will dominate in an impend-
ing bloom by accelerating the elimination of the predecessor and by
increasing the growth potential of the new dominant. In addition fil-
trate studies suggest an exclusion capacity; i-e_., the metabolic pro-
ducts of blue-green bloom dominants appear to exclude from the sequence
in eutrophied waters, seasonal blooms which commonly occur in oligo-
trophic and mesotrophic waters. Naturally, such biological activity
cannot be considered the only basis for a specific organism dominating
a bloom, but it does substantially limit the number of organisms likely
to achieve such dominance. This limit, when correlated with the many
physical and chemical parameters of growth control, would provide a very
few, if not one, possible dominant for any given place in the bloom
sequence.
Although the next organism studied may prove to be the first exception,
there are presently no indications that in vitro metabolite activity
works in opposition to the in situ bloom sequence. That is, none of
the filtrates of bloom dominants enhance the growth of a predecessor,
nor do any inhibit the growth of a successor. While this is reassur-
ing, it is not essential to the acceptance of the basic premise of
this study. There are many factors involved in the final determination
of sequence, and the competitive advantage of metabolite effects could
reasonably be overcome by a combination of other factors. It has not,
however, been necessary to resort to this rationale, and this absence
of incongruous effects for a period of three years suggests that these
metabolite effects are not frequently masked.
Table 1. Key
+ Positive; Enhancement
Negative; Inhibition
0 Neutral
? Uncertain; Untested
.. .. No Occurrence
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TABLE 1. Correlations Between Dloom Sequence and Filtrate Effects
Bloom Sequence 1971-1974
(Dominant Organisms in
Order of Appearance)
Months of Occurrence
Oscillatoria rubescens
(535) NDJFMA
Oscillatoria rubescens
(739) AMJJ
Anabaena sp.
(538) Jun,
Anabaona sp.
(762) JAS
Oscillatoria rubescens
(535) JAS(O)
Pseudanabaena galeata
(597) 0
Oscillatoria sp.
(776) MD(J)
Synechccoccus sp.
(91) AM
Anabaena sp.
(765) JJA(S)
Anabaena sp. (762)
A(S5
Predecessor and
Effect of Dominant
on Predecessor as
DcVerrninod by
Dioass.iy of
Filtrates
(535) 0
(739)
(739) -
(739) 0
(535) 0
(535) -
(597) -
(765) -
Successor and
Effect of Dominant
on Successor as
Determined by
Bioassay of
Filtrates
(739) 0
(538) +
(538) +
(762) 7
(535) 0
(762) ?
(535) +
(597) +
(776) +
(776) 0
(765) +
Contcm[xjraries and
Effect of Dominant
on Contemporariea as
Determined by
Bioassay of
Filtrates
(538) + start
(739) - end
{"5) - end
(762) 7
(597) + start
(776) 0
(597) - end-
'
(762) ?
(765) ~ «nd
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The events listed in Table 2 provide examples of another form of corre-
lation, that of year-to-year variations in seasonal patterns which
parallel filtrate effects. Negative filtrate effects correspond to the
absence of organisms which had dominated the lake during the same
season, but in a different year. This exclusion is of the same form
as the diatom exclusion discussed below.
The question of the population density of in situ blooms and that of
in vitro filtrate cultures is of great significance to the propriety of
the extrapolation from in vitro effects to in situ occurrences. Popu-
lation counts were taken for each of the filtrate producing cultures.
The maximum in situ population and the range of in vitro populations
are compared in Table 3. Since those effects which influence a compe-
titive situation in nature need only subtly influence a single consti-
tuent of that situation and since, in contrast, any in vitro effect
must be obvious to be accepted as possible, it is assumed that an in
vitro population density three to four times that of the in situ popu-
lation would not be unreasonably dense for purposes of extrapolation.
Actually, the inhibitory effect of blue-green filtrates against diatoms
was found to be still present at 33% of its original level (100% F);
therefore, an in situ population one half to one third of the in vitro
population could be expected to provide an effect strong enough to be
consistently detectable in the F vs A assays. As can be seen in Table 3,
most in vitrj) population densities were within reasonable limits. Only
the Pseudanabaena galeata (597) cultures (8-13x), and one of the
Synechecoccus sp. (91) cultures (160x) produced comparatively dense in
vitro populations. This low population density reflects measures taken
to avoid excessive growth in laboratory cultures. Lake water with a
very minimal nutrient addition (I1s% ESj) was employed as the basic cul-
ture medium. The use of artificial media, while essential to some
experimental design, was not desirable during this study.
III:II THE CORRELATION OF BOTH BLOOM SEQUENCE AND FILTRATE EFFECTS
WITH THE BIOLOGICAL ACTIVITY OF LINSLEY POND WATERS
This correlation, because it represents a tri-fold comparison, is more
difficult to document and to graphically communicate than is that between
filtrate effects and sequence only. As indicated in Table 4, much of
the information essential to a thorough exploration of these multi-
faceted correlations is lacking. However, for the pond water samples
which were tested many events which clearly show a correspondence between
sequence-filtrate data (Table 1, Section III:I) and pond water data were
observed. The following examples are of special interest.
September 5, and September 19, 1972
Pond water from September 19 inhibited the growth of Oscillatoria
rubescens (535) in vitro. In 1971 this organism had produced a bloom
population before turnover and this bloom population maintained dominance
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TABLE 2.
SEASONAL VARIATIONS WHICH CORBELATE WITH FILTRATE EFFECTS
ORGANISM
TIME OF ANTICIPATED BLOOM
CORRELATION BETWEEN FILTRATE EFFECT AND
IN SITU EVENT
Oscillatoria rubescens
(535)
On set i. August
Duration* August—April
1973 Anabaena sp. (765) bloomed just prior
the anticipated onset of the (535) bloom.
No (535) bloom occurred.
Filtrate: no filtrate was produced by
(765), but the unialgal bloom in the
pond was exceedingly dense and water
collected at the peak of the (765)
inhibited growth of (535)
Oscillatoria rubescens
(739)
Onsets April
Durationi April-July
1973 Synechecoccus sp. (91) bloomed at the
time of (739) onset.
No (739) bloom occurred.
Filtrates of (91) inhibit growth of
(739).
Anabaena sp. (538)
Onset» early June
Durationi June
1973 Synechecoccus sp. (91) bloomed just
prior to the anticipated onset of the
(538) bloom.
No (538) bloom occurred.
Filtrates of (91) inhibit growth of (538).
1973 Anabaena sp. (765) bloomed at the antici-
pated time of the (538) bloom.
No (538) bloom occurred.(765)water inhibited
(538), see (535) above for explanation.
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TABLE 2 (continued)
SEASONAL VARIATIONS WHICH CORRELATE WITH FILTRATE EFFECTS
ORGANISM
TIME OF ANTICIPATED BLOOM
CORRELATION BETWEEN FILTRATE EFFECT AND
IN SITU EVENT
Synechecoccus sp. (91)
Onset: April
Duration* April, May
1972 Oscillatoria rubescens (739) bloomed
at the anticipated time of the (91)
bloom.
No (91) bloom occurred.
Filtrates of (739) inhibit growth of
(91) in culture*.
Anabaena sp. (765)
Onset: June
Duration: June-August
1971 Anabaena sp. (538) bloomed at the
anticipated time of the (765) bloom.
No (765) bloom occurred.
Filtrates of (538) inhibit growth of
(765) in culture.
1971 Oscillatoria rubescens (739) bloomed
at the anticipated time of the (765)
bloom.
No (765) bloom occurred.
Filtrates of (739) inhibit growth of
(765) in culture.
* (91) and (739) may be mutually exclusive. Filtrates do not suggest that the preceding organisms
determine which will bloom. In 1971 the Predecessor was O. rubescens (535) which is neutral to both.
In 1972 there was no preceding bloom. _____^_____^__^^____^_^________^_^_-_.i
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TABLE 3.
COMPARISON OF IN SITU AND IN VITRO POPULATION DENSITIES
(Listed in thousands of organisms per ml)
ORGANISM
Oscillatoria rubescens (535)
Oscillatoria rubescens (739)
Anabaena sp. (538)
Pseudanabaena galeata (597)
Osciira~toria sp. (776)
V
Synechecoccus sp. (91)
Aphanizomenon flos-aquae (766)
Anabaena sp. (762)
IN SITU
POPULATION
MAXIMUM*
16
17
6
5
9
1000
13
IN VITRO
POPULATION
RANGE*
4-55
12-21
11-33
40-63
7-17
460-160,000
11-12
12
* Filamentous forms calculated at a length of 0.4 mm.
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TABLE 4.
TRIFOLD CORRELATIONS: BLOOM SEQUENCE; FILTRATE EFFECTS; AND
BIOLOGICAL ACTIVITY OF COLLECTED
LINSLEY POND WATER SAMPLES
SYMBOLS
. - = no occurrence
? = effect unknown
+ = positive effect-enhancement
= negative effect-inhibition
0 = no effect noted
( ) = parentheses enclosing effect sign
means effect determination unclear
++ = very strong positive effect-
enhancement
10
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TABLE 4. TRIFOLD CORRELATIONS: BLOOM SEQUENCE; FILTRATE EFFECTS, AND
BIOLOGICAL ACTIVITY OF COLLECTED LINSLEY POND WATER SAMPLES
DATE OF
POND H20
8/1-8/8/72
Fresh
9/5/72
Freah
9/19/72
Thaw
11/17/72
Thaw
DOMINANT
ORGANISM
Oscillatoria
rubescens(535)
max.
Anabacna sp.
(762)max.
Anabaena sp.
(762) end
Oscillatoria
rubescens (535)
Anabaena sp.
(762) end
Oscillatoria
rubescens (535)
Oscillatoria
sp. (776) nax.
ACCESSOR
• •
• •
I'seudanabaena
qaleata (597)
I'seudanabaena
galeata (597)
Pseudanabaena
galeata (597)
Pseudanabaena
galeata (597)
Oscillatoria
sp. (776)
EFFECTS 0
POND H 0
• •
* •
7
?
0
0
_
M SUCC.of
FILT.
OF
DOM.
• •
. .
_
interval
+
—
interval
+
(0)
PREDECESSOR
Oscillatoria
rubescens (739)
Anabaena sp.
(762)
0. rubescens
(739)
Oscillatoria
rubescens
(535)
Anabaena sp.
(762)
Oscillatoria
rubescens (535)
Oscillatoria
rubescens (535)
Anabaena sp.
(762)
Oscillatoria
rubescens (535)
Pseudanabaena
qaleata (597)
EFFECTS O
'OND H20
—
?
7
_
7
?
7
7
.
^
_
>J PRED.of
FILT.
OF
DOM.
(0)
7
_
_
?
(0)
-
7
(0)
-
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TABLE 4. (continued)
tvj
DATE OF
Pfun IT A
1/7/73
3/18/73
FjresH
Thaw
5/13/73
Fresh
6/2/73
Fresh
7/17/73
Thaw
8/13/73
Fresh
DOMINANT
UKljANISM
Oscillatoria
sp, (776) late
Flagellates
Astcrionolla
formosa(OOO)
Oscillatoria
sp. (776) end
Synechecoccus
sp. (91) max.
Asterionella
formosa(800)
end (second)
Anabaena sp.
(765)max.
Anabaena sp.
(765)end
SUCCESSOR
flagellates
Flagellates
Asterionella
formosa (800)
Oscillatoria
sp. (776)late
Synechecoccus
sp. (91)
Asterionella
formosa (800)
Anabaena sp.
(765)
Anabaena ep.
(765)
Anabaena sp.
(762) weak
bloom
Clear water
for over
3 mos.
EFFECTS C
POND 1^0
(+)
mixed
+
(0)
+
_
?
++
?
• •
>M SUCC. OK
FILT.
OF
DOM.
mixed
• •
* •
_
++
?
?
• •
PREDECESSOR
i
Oscillatoria sp.
(776)
Flagellates
Oscillatoria
SO f776J 1 at*A
Asterionella
formosa (800)
Synechecoccus
en (91)
Anabaena sp.
(765)
Anabaena sp.
(765)
KFFECTS (
1JOND H20
|
+
mixed
0
0
-H-
0
y> PItED. OF
FILT.
OF
DOM.
(0)
• •
* *
7
7
7
-------
TABLE 4 (continued)
DATE OF
POND H20
2/14/74
6/13/74
DOMINANT
ORGANISM
Oscillatoria
sp. (776) low
Pseudanabaena
galeata (597)
low
Flagellates
high
Diatoms, high
Oscillatoria
rubescens
(535)
SUCCESSOR
Diatoms
Flagellates
Oscillatoria
rubescens
(535)
i
EFFECTS C
POND HjO
_
mixed
+
v succ. or
FILT.
OF
DOM.
• •
• •
0
PREDECESSOR
Oscillatoria
sp. (776)
very low
Pseudanabaena
galeata (597)
very low
Diatoms
Flagellates)
(monad) (317)
Chlamydoroonas
(298)
Sphaerellopai
(598)
Synura uvella
(43)
FFECTS a
OND H20
—
-
_
•f
—
-
I PRED. OF
FILT.
OF
DOM.
_
•f
?
0
-------
in the pond throughout the J.97J.-J972 winter. When O. rubescens (535)
appeared in late summer 1972, it was expected that it would again pro-
duce a bloom population which would dominate through the winter (1972-
1973). The basis for the sudden demise of this bloom early in September
was explored. No sure explanation was found. Pond waters, however,
were consistently inhibitory to (535). Pond waters from September 5 and
September 19 were found to be inhibitory to Anabaena sp. (762), Aphani-
zomenon flos-aquae (766), Anabaena sp. (765), and Anabaena circinalis
(769), all of these were blue-greens which had been present in the water
at the end of August, 1972. Thus the pond water exhibited in vitro the
same generalized inhibition of blue-greens which sequential events in
the pond had displayed in situ. It is probable that inorganic nutrient
limitation played a part in the rapid drop in phytoplankton in early
September since both F and A assays produced less than expected popula-
tion densities (as compared to experimental cultures in waters collected
in other seasons). However, nutrient limitation alone cannot account
for F vs A differences since F and A inorganic nutrient conditions are
the same.
November 17, 1972
Waters from this date inhibited the growth of Pseudanabaena galeata
(597) in vitro. The dominant organism at this time was Oscillatoria
sp. (776). Osci1latoria sp. (776) filtrates also produced inhibition
of the in vitro growth of P_. galeata (597) . Since the (776) bloom
replaced a (597) bloom, the pond water and filtrates apparently exhi-
bited in vitro the same inhibition of this organism as did the sequence
in situ.
March 18, 1973
In contrast to its usual universally negative effect on diatom growth,
this sample of Linsley water enhanced the in vitro growth of Asterionella
formosa (800) only, and this sample was taken at the time of the first
diatom bloom of the study period—a bloom dominated by A_. formosa (800).
May 13, June 2, July 17, and August 13, 1973
In 1972 Oscillatoria rubescens (739) bloomed in early spring and Ana-
baena sp. (538) bloomed in late spring; but in 1973 Synechecoccus sp.
(91) bloomed in early spring and Anabaena sp. (765), which is quite
different morphologically from (538), bloomed in late spring.
Filtrates of Oscillatoria rubescens (739) enhanced growth of Anabaena
sp. (538) and inhibited growth of Anabaena sp. (765). Filtrates of
Synechecoccus sp. (91) enhanced growth of Anabaena sp. (765) and inhi-
bited growth of Anabaena sp. (538). Thus it appeaxs that both enhance-
ment and exclusion are involved in the determiantion of bloom sequence
in the springs of 1972 and 1973.
Further, filtrates of Anabaena sp. (538) inhibited the growth of Ana-
14
-------
baena sp, (765), Unfortunately, the only bloom dominant which never pro-
duced a filtrate for bioassay was Anabaena sp. (765), but the pond waters
from July 17, 1973, -during an immense, essentially unialgal, bloom of
Anabaena sp. (765) inhibited the growth of Anabaena, sp. (538). Thus a
mutual exclusion also appears to be involved in the determination of
bloom sequence in the springs of 1972 and 1973.
Pond waters from June 2, 1973 taken after the bloom of Synechecoccus sp.
(91), enhanced the in vitro growth of Anabaena (765). Pond water from
July 17, six weeks later, was still able to enhance in vitro growth of
Anabaena sp. (765). At that time the jji situ bloom of Anabaena (765)
was at its maximum. It remained at maximum for approximately one more
week.
By August 13, the (765) bloom had subsided, and the pond waters were
quite free of any form of blue-green bloom. Pond waters from that date
were found to be neutral to the growth of (765) in vitro; however, a
lag in the growth of both F and A tests was noted. Both F and A cul-
tures were less dense than those in pond water collected a month earlier,
suggesting depletion of nutrients in late summer 1973 similar to that
in late summer 1972.
Anabaena sp. (765), while it grows very slowly in maintenance culture,
readily produces dense cultures in the filtrates of Synechecoccus sp.
(91) and in pond waters taken during or immediately after blooms of
Synechecoceus sp. (91). The addition of a small amount of the cell-
free filtrate of Synechecoccus sp. (91) was found to provide a more
satisfactory maintenance culture for (765). The heat-stable factor
responsible for this phenomenon was not isolated.
In conclusion, although the correlation of filtrate effects, sequential
events, and pond water effects cannot assure that the same stimuli are
producing similar responses, the multiplicity of these correlations
strongly supports such an assumption. It should be noted that in most
respects frozen water samples gave results similar to those from
freshly collected samples, but the possibility that some changes may
occur during the freeze-store-thaw process cannot be ignored (Table 8;
Section VII:II). Therefore, it is suggested that future studies of
correspondence between in vitro and in situ events should be done using
freshly collected samples of pond water. This would provide more reli-
able bioassays.
15
-------
SECTION IV
WATER QUALITY MANAGEMENT PROSPECTS:
PROGRAMMING BLOOM SEQUENCE
The information developed during this study suggests a new approach to
water quality management in moderate-sized eutrophic lakes—sequence
control. This relatively inexpensive method, if successful, would offer
a practical alternative to sewage treatment and phosphate elimination.
The information in Section IV:I is the basis for the hypothetical
sequence control plan for Linsley Pond outlined in Section IV:II. This
information includes data specific to Linsley and data of a general
nature for fresh waters. The plan represents an attempt to replace
some of the blue-green blooms in Linsley by diatom blooms because the
diatoms are aesthetically (taste, smell, touch) and ecologically (strong
link in food chain) preferable to blue-greens. They also reduce the
available nutrients in lake water by carrying nutrients down into the
hypolimnion as they senesce and these nutrients are not recycled until
after the fall overturn. In contrast blue-greens lyse in the epilimnion,
releasing their nutrients for immediate recycling by subsequent blooms.
Improving conditions for the zooplankton community by increasing their
food supply and by eliminating some of the blue-greens should, in turn,
produce a healthy fish population. This chain of events occurred
naturally in Linsley in 1973, and is discussed in Section VI:IV.
IV: I IN SITU EXCLUSION BY BLUE-GREEN METABOLITES
Probable Exclusion of Diatoms by Blue-Greens^
1) Spring diatoms which commonly produce blooms in oligotrophic and
mesotrophic lakes require high N, P, and Si and they develop their blooms
at low temperatures during short days.
2) Diatom growth in Linsley has apparently been prevented in the spring
by the accumulation of metabolites produced by blue-greens growing dur-
ing the winter. When blue-green growth was low in winter, spring diatom
blooms developed (Figure 12, Section VI:V).
3) Preliminary evidence suggests that the addition of Na2SiO3'9H2O can
overcome this inhibition.
4) Spring diatom blooms which occurred in Linsley ended when available
silica was low. Available silica need not equal measured, dissolved
inorganic silica; see VII:V.
16
-------
Probable Exclusion of Zooplankton by Blue-Greens
1) Zooplankton populations do not develop in Linsley when the waters
are dominated by blue-green blooms.
2) Zooplankton populations which develop when blue-greens do not domi-
nate Linsley waters rapidly disappear with the onset of blue-green
bloom conditions.
3) Preliminary tests indicate that the cell-free filtrates of blue-
green algae may inhibit the growth of zooplankton. Also/ other investi-
gators have shown that blue-greens are in general a poor food choice for
zooplankton.
IV:II A STRATEGY FOR SEQUENCE CONTROL
1) Winter blue-green blooms should be eliminated by a commonly accepted
algicide. CuSO4 might be applied immediately after overturn. This
would remove the cause of spring diatom bloom exclusion. While a second
treatment might be considered if a late winter blue-green bloom develops/
it may be sufficient to eliminate some/ rather than all, blue-green win-
ter growth.
2) Spring diatom growth should be enhanced by the addition of. silica.
Silica would lessen the effect of remaining blue-green metabolite inhi-
bition and would extend the diatom bloom when natural silica was
depleted. Because it is a fertilizer only for diatoms and a few other
desirable forms/ it would not stimulate unwanted growth.
3) These efforts should increase the period of diatom domination and
decrease nutrient levels left in the epilimnion when the diatoms
senesce. Ideally, this would shorten any subsequent (late summer)
blue-green bloom and would decrease its density.
4) Zooplankton growth should be significantly improved by the decrease
in antagonistic organisms (blue-greens) and the increase in food organ-
isms (diatoms and other desirable forms).
17
-------
SECTION V
INTRODUCTION
V: I EUTROPHICATION
Widespread, excessive eutrophication (Hutchinson, 1969) of fresh waters,
which is a direct result of either inadequate waste water treatment or
of contamination by runoff waters (Biggar and Corey/ 1969; Cooper, 1969;
Weibel, 1969), is presently controlled either by measures aimed at the
elimination of bloom-stimulating waste -products by tertiary waste water
treatment and a concomitant modification in land use practices/ or by
measures intended to eliminate the bloom organisms themselves via the
application of chemical herbicides (Mulligan, 1969). Successful control
with the former approach is not only exceedingly expensive (U. S.
Department of the Interior, 1968)/ but is also dependent on the coopera-
tion and compliance of great numbers of individuals/ corporations, and
local, national and international governing bodies (U. S. Government
Printing Office, 1972); and success with the latter, chemical, approach
is fraught with unknown ecological repercussions (Crosby and Tucker/
1966).
Phosphate Control—Selected Successes
The success of Edmondson (1972) in the Lake Washington project settled
the question of the practical value of eliminating phosphate from inflow
waters, even if no parallel attempt to eliminate the standing load of
phosphate in lake waters and, or, sediments is made. This success also
indicates that a real prospect of rapidly reversing the conditions of
cultural eutrophication exists (Hasler, 1947; Tanner, et_ al^, 1972) if
wholesale phosphate removal (from inflow), diversion, precipitation,
etc., is undertaken.
In a series of whole lake experiments in Ontario Schindler (1974) con-
trolled the level of algal bloom by various regimes of nutrient addition
and concluded that in such natural lake waters phosphate addition alone
is capable of initiating algal bloom conditions. By dividing a single
lake in two with a plastic and nylon sea curtain he demonstrated that
while additions of nitrate, phosphate, and organic carbon do produce
extraordinary bloom development, additions of only nitrate and organic
carbon produce no changes in the usual flora of the lake; i-e_., a popu-
lation of mixed diatoms. Unfortunately/ these accomplishments do not
alter the fact that in many cases mechanical or chemical phosphate
elimination is unrealistic, representing untenable public expense.
Legislation banning high phosphate detergents in New York State (Murphy,
1973) has led to marked improvement in the degree of cultural eutrophica-
tion observed in Onondaga Lake and suggests a reasonably inexpensive
18
-------
method of control. In spite of such attendant drawbacks as; 1) the re-
quirement of. long term cooperation of the public in not purchasing "boot-
leg" high phosphate detergents due to the lack of a safe and effective
substitute; and, 2) the likelihood that non-detergent phosphate sources
alone may in some waters contribute sufficient phosphate to support
severe cultural eutrophication—such legislative action may provide sub-
stantial improvement in many small, urban-locked, bodies of water.
Biological Control
In contrast, biological control is highly desirable both as a more eco-
logically sound alternative to the use of chemical herbicides and as a
practical adjunct to waste water treatment and phosphate control.
Biological control, however, requires an intimate knowledge of the many
physical and biological factors which promote and sustain the natural
sequence of bloom populations/ and this information is presently insuf-
ficient in both quantity and scope.
Established Parameters of Bloom Sequence Control
Bloom sequence has been shown to depend in part on such factors as:
-total solids (Provasoli, ejt al^, 1954; Provasoli and Pintner,
I960);
-photoperiod (Dunshaw, 1973; Reynolds, 1973);
-light intensity (Ignatiades and Smayda, 1970a; Sorokin and
Krauss, 1958);
-accumulation of pesticides (Cope, 1962; Mensel, et^ al^., 1970;
Wurster, 1968);
-salinity (Ignatiades and Smayda, 1970a);
-temperature (Conover, 1956);
-carbondioxide-pH regime (Brock, 1973; Goldman, 1973; Shapiro,
1973);
-availability of nutrients (Chu, 1942, 1943); especially of
silica (Hamilton, 1969) and trace metals (Horne and Goldman,
1974; Ignatiades and Smayda, 1970b).
Certain biological factors are also considered significant in the deter-
mination of qualitative and, or, quantitative characteristics of bloom
populations. Among these are:
-buoyancy regulation (Fogg and Walsby, 1971);
-organic micronutrients (Carlucci, 1974; Carlucci and Cuhel,
1974; Carlucci and Shimp, 1974; Hagedorn, 1971a, 1971b;
Owada, et al., 1972; Provasoli, 1960; Schwartz, 1972);
-selective zooplankton grazing (Porter, 1973);
-pro- and antibiotic effects of extracellular metabolites of,
and the direct competition for nutrients of, vascular
plants (Fitzgerald, 1969; Hasler and Jones, 1949; Wetzel,
1969; Wetzel and Allen, 1971).
19
-------
In the last decade (Safferman, 1964; Shilof 1971f 1973; Stewart, 1969)
the possibility that viruses may be common in blue-green algae has
prompted studies into the possibility of selectively employing cyano-
phages to eliminate blooms. During the discussions following both his
1971 and 1973 papers, Shilo expressed doubt as to the feasibility of
the controlled use of cyanophage infection in this manner. His conclu-
sions were based on the specificity of the phages as compared to the
variety of blue-greens in most natural waters and to capacity of these
algae to produce additional phage resistant mutants. Still, M. Kraus
(personal communication, 1973) is of the opinion that cyanophage occur-
rence is so ubiquitous that they must be considered a possible factor
in the determination of the natural complement and density of blue-green
blooms.
To date, these many physical, chemical, and biological parameters,
although they have been shown to be significant in the specific cases
referenced above, have not provided sufficient explanation for bloom
sequence in general (Fogg, 1953, 1966, 1971; Hutchinson, 1941, 1944,
1971 lecture; Lucas, 1961); therefore, additional control mechanisms
must be sought. The effects of the metabolic products of bloom dominant
organisms on bloom sequence is explored in this study.
V:II AN HYPOTHESIS
Perspective
The involvement of biological effects in the determination of species
sequence was first suggested by Apstein in 1896 and reiterated by Putter
in 1908. Yet for decades the possibility that biological factors may
modulate bloom sequence in freshwater communities was neglected until
Lucas (1947) postulated that external metabolites might be ecologically
important.
That algae produce extracellular metabolites (Fogg and Boalch, 1958;
Forsberg and Taube, 1967; Schwimmer and Schwimmer, 1964; Steeraan-Nielsen,
1952; reviews by Fogg, 1962, 1966; Lucas, 1947; Saunders, 1957), and
that these metabolites can effect the growth of other algae (Carlucci
and Bowes, 1970a, 1970b, 1972; Droop, 1968; Lefevre, et^ al_., 1952; Pratt,
1966; Proctor, 1957; reviews by Hartmann, 1960; Pourriot, 1966) are
now established facts. However, among the many studies of interspecific
metabolite effects there are very few attempts to demonstrate a corre-
lation of in vitro activity with in situ occurrence. Rather, since the
bulk of this work deals directly with in vitro phenomena, the implied
probable in situ significance of these effects encounters the many pit-
falls inherent in such generalizations (Hutchinson, 1966).
20
-------
Basic Premise and Model
The basic premise of this study is that some, or all, of the organisms
which dominate freshwater blooms produce biologically active extracellu-
lar metabolites in sufficient quantity and of appropriate quality to
effect the sequence of blooms in situ. Figure 1 represents a simple
model based on this premise. That is, if organisms A, B, and C produce
blooms in the order a, b, c, then several distinct, but synergistic,
metabolite effects may be expected.
Inclusive;
I. It is likely that organism A produces an extracellular metabolite
which will enhance, the growth of organism B in such a manner as to
improve the competitive position of B, thus encouraging a bloom of
organism B (among the many possible candidates) to replace the ori-
ginal bloom. Subsequently, it is also likely that a similar chain
of events would encourage organism C to replace organism B.
II. It is likely that organism B produces an extracellular metabo-
lite which will JjflhiJoJUt the growth of organism A in such a manner as
to improve the competitive position of B, thus encouraging a bloom
of organism B (among the many possible candidates) to replace the
originaJL bloom. Subsequently, it is also likely that a similar
chain of events would encourage organism C to replace organism B.
Exclusive;
III, If organism A produces an extracellular metabolite which inhi-
bits a second organism, X, which might otherwise be expected to
bloom at the time (season) immediately following organism A, it is
likely that the competitive position of organism X would be suffi-
ciently damaged so as to eliminate, or exclude, a bloom of organism
X from the in situ sequence. Diatom exclusion is an example of
this phenomenon; Sections VII:II and, especially, VII:III and VII:IV.
It is not suggested that this combination of enhancement and inhibition
is solely responsible for bloom sequence, but rather that it is one of
the significant contributors to the determination of bloom sequence in
general.
Experimental Design
To give greater credence to the extrapolation of in vitro results to the
explanation of in situ phenomena three different approaches to the pro-
blem of metabolite involvement in bloom sequence determination were
employed.
I. The in situ physical and chemical conditions, and the natural
bloom sequence of the study pond (Linsley), were monitored for a
21
-------
Number of
- Cells
ro
IS)
H )
z o
G «
W i .
z .
n
w
(A W
Z H
o
0
tr« a
o o
o o
X W
tr<
W "
w
to s
G W
M ^
Z >
O 0)
W O
IT"
H
H
W
EXCLUSIVE
Inhibition
INCLUSIVE
^ Inhibition
Enhancement
JTIKE
-------
period of three years to determine if changes in the usual limnolog-
ical parameters were accompanied by specific changes in bloom
sequence, Section VI.
II. Those algae which were observed to be dominant in blooms dur-
ing this three year monitoring period were isolated/ established
in culture (axenic when possible), and employed to produce rela-
tively large batch cultures. Batch cultures were harvested by
passage through a 0.45p Millipore filter, and the resulting cell-
free filtrates were bioassayed for biological activity which might
parallel in situ events, Sections III and VII.
III. Large water samples were taken during periods of peak bloom
and these samples were bioassayed in the same manner as were the
cell-free filtrates for biological activity which might parallel
in situ events, Sections III and VII.
Special Problems Associated with Algal Metabolite Studies
Certain problems with the interpretation of algal metabolite studies are
especially frequently encountered. For example: the practice of employ-
ing algal species from culture collections. Because these organisms are
generally isolated from different environments, the extrapolation of
interspecies_effects from in vitro experiments to specific in situ occur-
rences is tenuous at best.
During this study most of the algae, filtrate producers and assay organ-
isms, were isolated directly from Linsley Pond. The only exceptions were
organisms intentionally chosen as "non-Linsley" algae for tests designed
to provide information concerning the possible extrapolation of the con-
clusions of this study to fresh waters other than Linsley. It is of
some interest that the very consistent pattern of diatom inhibition by
heat-labile metabolites of blue-greens was not as obvious when a set of
experimental organisms from several different sources was used.
Another problem, which develops when organisms are fresh isolates, is
that they are often established in bacterized culture—with the effect
that no distinction can be made between the effects of algal and bac-
terial metabolites. It is true that in some instances employing bacter-
ized algal cultures is unavoidable because it is not always possible to
separate an alga (especially a blue-green) from its accompanying
bacterial community. However, since the bacterial population, and thus
its influence on the system, may radically change when the natural com-
munity is transferred from a lake to a test tube, efforts must be made
to distinguish algal from bacterial effects.
During this study a variety of bacterized and axenic filtrate producers,
and bacterized and axenic assay organisms are employed. To aid in the
differentiation of algal from bacterial effects a mixed culture of Lins-
ley bacteria was added to an axenic producer culture and results for the
23
-------
subsequent bioassay were compared to results from bioassays of the
axenic culture's filtrate.
An additional common criticism of studies relating algal metabolite
effects to natural occurrences involves dilution. There is concern that
the effects observed in laboratory tests can be accurately interpreted
only for in vitro circumstances and cannot be extrapolated to the natural
situation. This is because the very dense growth in test tube cultures
could be expected to produce a far greater- concentration of metabolites
than could be produced in situ, where the relatively large volume of
water could dilute the population of producers, and where environmental
conditions, especially nutrient levels, are quite different. In turn
this would dilute the in situ concentration of metabolites to a point
where the biological activity demonstrated in the "artificially" concen-
trated in vitro tests could not be significant. Artificial media often
provide quite unnatural levels for certain nutrients, especially for the
trace elements whose absolute quantities may be critical to in situ
growth but very difficult to measure or to reproduce.
During this study population densities in producer cultures were similar
to those of the natural waters (Table 3, Section III:I). Since enriched
natural pond water was used as the basis for experimental media, in vitro
levels for most trace elements are assumed to be similar to in situ
levels. —
V:III SIGNIFICANT RELATED RESEARCH
Algal Metabolites In Situ
In two extensive studies relating either to salt marsh (Lee, et. al^ , 1970)
or ocean (Aubert, 1963-1971) communities, the involvement of algal extra-
cellular metabolites in a variety of ecologically significant phenomena
was investigated. Bloom sequence determination was not among the major
interests of either study; however, the conclusions in both that metabo-
lites are present in salt marsh and, or, ocean waters in sufficient
quantity to produce observable biological repercussions (especially
Aubert's bactericidal effects) weigh strongly against the dilution argu-
ment.
A significant proof of the importance of extracellular metabolite effects
in the determination of dominance in freshwater algal blooms was provided
by Williams (1971) when he presented conclusive evidence that an extra-
cellular inhibitor is produced by Anabaena flos-aquae, and that this sub-
stance is in part responsible for the periodic dominance of Lake Nelson,
New Jersey, by A. flos-aquae. Because his primary goal was to demonstrate
metabolite involvement in a single bloom situation in nature, field study
covered only that short period during which A. flos-aquae dominated the
lake waters. He offered a strong basis for his conclusions with parallel
results obtained from in situ and in vitro studies.
24
-------
Antibacterial Effects of Algal Extracellular Metabolites
Aubert, et_ al. (1963-1971), have developed a strong in vitro case for
the algal antibacterial effect they believe is demonstrated in situ by
rapid dissipation of sewage bacteria in the Mediterranean Sea. Though
they have included the algae among those organisms secreting antibac-
terial substances, they have not excluded the possibility that their
role may be minor in comparison to the role of marine bacteria, as Moebus
(1972a, 1972b, 1972c) has suggested, or to the more commonly proffered
explanations of anti-coliform activity which consider sedimentation,
predation, and lack of reproduction to be the critical factors.
Sieburth (1964) with less ill vitro evidence has tied the anti-coliform
activity of sea water samples from Narragansett Bay to the irregular
blooms of Skeletonema costatum and has presented a less strenuous in
vitro argument to substantiate his thesis; i_.e_., his cultures require
bacteria and additional phytoplankters to develop a strong antibiotic
effect (his axenic cultures of S_. costatum show no anti-coliform
activity).
The work of several others has also served to substantiate the in situ
antibacterial activity of algae. Steeman-Nielsen (1955), using light
and dark bottle techniques to measure oxygen consumption, noted that the
algae growing in "light" bottles inhibited bacterial respiration and
thus interfered with measurement techniques. J^Jrgensen (1962) isolated
chlorophyllides from filtrates of a variety of naturally growing algae
and found them to be inhibitors of bacterial growth. Jones (1959) found
the soluble organic extracts of sea water collected during a "red tide"
bloom of Gonyaulax polyhedra to be highly antibacterial. Interestingly,
he noticed a zone of stimulation surrounding the zone of inhibition on
agar plates which is reminiscent of certain effects of plant hormones
(Bentley-Mowat and Reid, 1969), indicating stimulation at low concentra-
tions, and inhibition at high concentrations. This is only one example
of the complicated array of activity to be expected from algal metabo-
lites. This might also have been the dilution-stimulation Pratt (1942)
thought he was witnessing in his Chlorella experiments; or it might have
been several extracellular metabolites as J^rgensen and Steeman-Nielsen
(1961) and JfJrgensen (1962) proved Pratt's "Chlorellin" to be; or it
might have been an indication of some totally unknown phenomenon peculiar
to this alga.
In 1966 Duff, Bruce and Antia surveyed the antibacterial range and
potency of Bacillariophyceae, Chrysophyceae, and Cryptophyceae and con-
cluded that these are more generally potent antibacterially than are
the Chlorophyceae or the blue-greens. Their algal samples, used after
drying as a source of active substance, were harvested from axenic mass
cultures derived from a variety of geographic origins. The assay bac-
teria were from type collections, or were fresh isolates from the sea.
Two ecologically significant theses were postulated: 1) the selective
activity of their algal stains against Gram-positive bacteria (especially
25
-------
Staphylococcus) may account for the prevalence of Gram-negative bacteria
in the seas (also suggested by the work of Saz, e_t al_., 1963}; and
2) the specific value" of the antibacterials, in terms of natural selec-
tion, may be to control epiphytic bacteria. This epiphytic association
would eliminate the aforementioned dilution arguments against in situ
antibiotic effects.
Other work with epiphytic associations includes that of Fitzgerald (1969)
who found that Cladophora in nitrogen-poor situations were free of
epiphytes (including other algae). An evolutionarily selective value
may be found in the coincident excretion of substances toxic to epiphytes
at that time when the Cladophora is experiencing nutritional stress.
Similarly, HcLachlan and Craigie (1964) found that Fucus vesiculosis
produced an inhibition of unicellular algae (likely epiphytes and compet-
itors) , and Jffrgensen (1956) demonstrated an inhibition by planktonic
diatoms and Chlorophyta on epiphytes.
In 1970 Davis and Gloyna determined that axenic cultures of freshwater
green and blue-green algae were mildly inhibitory to enteric bacteria
and decidely inhibitory to pathogenic forms. Their parallel experiments
in waste stabilization ponds produced similar results; however, the
endemic bacterial communities of these ponds provided cultures of
Flaveobacterium and Brevibacterium which, they noted, were more effective
than the algae tested in eliminating enteric forms.
Fogg (1962) expressed the opinion that "when vigorously growing cultures
of algae are exposed to contamination, it is often observed that rela-
tively few bacteria develop, an effect which could conceivably be due to
their suppression by antibacterial agents". He was, nevertheless, still
dissatisfied with the experimental proof of the importance of in situ
algal extracellular metabolites in 1966; and in 1971 he indicated (Fogg
and Walsby) additional dissatisfaction with all prior explanations of
blue-green dominance of blooms in eutrophied waters and offered,
instead, a partial explanation on buoyancy.
Concentration, Dilution, arid Identification of Algal Metabolites
When the effects of toxic "red tides" (Gilbert, 1974) and the effects of
the "fast death" factors produced by certain strains of Microcystis
aeruginosa and Anabaena flos-aquae are considered (Gorham, 1964; Fogg,
1962), it is reasonable to assume that algal extracellular metabolites
do occur in sufficient quantities in situ to cause metabolic reactions
in other aquatic organisms. Schwimmer and Schwimmer, in 1964 , offered
an intriguing discussion of the various documentations of algal toxi-
cosis in vertebrates, including humans. More recently Quick (1973)
found the "whirling death" fish kills in Biscayne Bay to be due to the
presence of Anacystis sp., and Aziz (1974) obtained a diarrhea toxin
from a new strain of M. aeruginosa. However, proofs relative to in situ
potency are few and, as such, they provide a basis neither for generali-
zation, nor for the assumption that biologically active algal metabolites
26
-------
do commonly produce effects on other algae.
This present study considers the possibility that algal metabolites may
be sufficient in quantity, and appropriate in quality, so to influence
the growth and reproductive rates of other algae that the presence of a
bloom population of a "producer" species or strain (for strains differ
biochemically, Gorham, 1964) will determine the presence or absence of
a bloom population of some other, "sensitive", species.
A warning concerning the peculiarities of nature and our inability to
reconstruct its complicated array of variables is inherent in Fogg's
(1966) caution (a) that stationary numbers in nature and in culture may
not be the overt manifestation of similar phenomena; and (b) that the
massive release of some metabolites "occurs only under particular circum-
stances which do not necessarily occur in laboratory cultures, but which
may occur regularly under natural conditions". This latter warning
follows his commentary on earlier experiments with glycolic acid excre-
tion, wherein natural marine and fresh waters showed the results of very
high excretions of fixed carbon while cultures showed insignificant
levels (below).
Duff, et al., (1966) concluded that the green algae are generally less
active antibiotically than other forms, thus substantiating Krogh's
(1930) early work indicating a frugality in green algae which he general-
ized to the other algal forms. This lack of evidence for the excretion
of large quantities of metabolite does not suggest a lack of activity.
One need only consider the microquantities of plant hormones necessary
to promote growth, flowering, abscission, etc., to appreciate this.
Berland, et al., (1972) found that in order to demonstrate an antibac-
terial effect on their most sensitive assay organism in filtrates of
Stichochrysis immobilis, they had to use a twenty-fold concentration of
the filtrate. Kroes (1971, 1972) found that a ten to twenty-five-fold
concentration was necessary. He concluded that the four types of extra-
cellular inhibitors he found were not ecologically significant because
of this concentration requirement and suggested pH effects as the
really significant factor in supposed inhibitory effects. To properly
consider the doubts raised by concentration requirements one must con-
sider Fogg's warnings (1966, above) and the more recent report of
Ignatiades and Fogg (1973) that, depending on culture conditions, any-
where from 2.1% to 87.4% of the total carbon fixed by a single organism
nay be excreted.
In order to present parallel, or proportional, arrays of data from in
situ algae and surrounding natural waters, Whittaker and Vallentyne (1957)
analyzed lake waters to determine the array of sugars they contained.
This grouping (maltose, sucrose, glucose, fructose, galactose, arabinose,
ribose, xylose, and two unknowns) of sugars they then compared with
sugars found in, or produced by, various modules of the lake ecosystem
(mud, free swimming bacteria, larvae, phytoplankton). They concluded
27
-------
that the open water phytoplankton were the only plausible source of these
sugars. The phytoplankton produced large quantities of these sugars
directly and also produced quantities of polysaccharides which could be
easily decomposed into these sugars.
Bentley (1960) found two plant auxins, which behave identically in chro-
ma tographic separation, in phytoplankton and in sea water samples. Since
this activity pattern significantly differed from that of the products
of other organisms from the same waters, she concluded that the auxins
in the water sample originated in the phytoplankton. Further proof was
provided by Steaman-Nielsen (1952). Using 1*C tracers he followed
carbon through fixation and out into organic matter filtered from lake
water. Many similar experiments by other investigators followed.
A few values, if noted with proper perspective, can be enlightening.
Attempts were made as early as 1930 to determine precisely what portion
of fixed carbon (or nitrogen) is excreted by various algae. Braarud
and F^yn (1930) estimated that a marine Chlamydomonas released approxi-
mately 30% of its organic production. This figure compares reasonably
with Lewin's 1956 estimate that Chlamydomonas excreted from 40-60% of
its total organic product into its mucilaginous capsule, and with the
35-40% excretion estimate of Antia, et al., (1963) . In contrast Fogg
reported (1966) a 95% excretion level in fresh waters under certain
circumstances, but found that the more usual levels ranged between 7%
and 50% (usually varying inversely with population density). He sug-
gested that similar levels could be expected in marine waters. He lie-
bust's (1965) in situ and in vitro estimates were somewhat lower for
"healthy" organisms than were those of Fogg, or of Antia. Hellebust
distinguished healthy (4-16%) and senescent (17-38%) colonies. In con-
trast Nalewajko (1966) reported in vitro excretion for healthy organisms
at less than 2%. However, Nalewajko, used young log cultures and
allotted one hour for labelling (early products); while Hellebust, used
young log cultures and allotted four days for labelling (full array of
products with possible recycling). Thus superficial comparison which
suggests great disparity in data can be quite misleading. As Fogg (1962)
commented "measurements of the amounts of extracellular material can
have no precise significance unless the physiological history of the
system and its environmental conditions are defined".
Vitamins as Algal Extracellular Metabolites
Provasoli (1963) suggested three possible areas of in situ metabolite
activity; 1) the removal, or deactivation, of inhibitions; 2) the pro-
duction of specific inhibition; and 3) the production of necessary
nutrients, or growth factors. Much study has been devoted to a portion
of his third category. Vitamins and other "growth factors" are required
by more than 50% of the algae (Provasoli, 1963; personal communication,
1975; Saunders, 1957). They are also well established as products of
algal metabolism (Bobbins, et al., 1951; Carlucci and Bowes, 197Oa, 1972;
Bentley, 1958). Excretion, lysis, and decomposition each play a part in
28
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releasing those vitamins produced by large masses of algae in fresh and
marine waters. Vitamins are, therefore, more an extracellular metabolite
in Lucasls sense than in Fogg*s. Fogg limits the term "extracellular
metabolite" to those metabolic products which are leaked into the sur-
rounding milieu by healthy, growing cells — an essential limitation in
many studies. Lucas's less stringent definition is, perhaps, more
suited to the present ecosystem study, wherein the presence and avail-
ability of metabolites, more than the mode of production, is significant.
As early as 1943 the cycles of thiamine and biotin in lake waters were
studied by Hutchinson (in 1946 he and Setlow added niacin) . Sufficient
quantities of these vitamins were present in the pond waters studied for
the needs of the vitamin requiring algae. In 1956 Cowey found that the
B12 in the sea is also present at adequate levels and Droop (1957) con-
firmed abundant B^2 in a variety of marine habitats; however, his later
work with the Bi2 binding factor does suggest that ambient levels may not
actually represent available levels (1968) .
The biotic origin and utilization of aquatic vitamin stores was confirmed
directly in vitro by studies of vitamin production by marine bacteria
(Burkholder and Burkholder, 1956; Menzel and Spoehr, 1962) and indirectly
in situ by the many observations that concentrations of vitamins in the
open sea are generally lower than they are in coastal waters where the
greater portion of primary production is localized. Until 1970, however,
the source of these marine and freshwater vitamin stores was believed to
be bacterial. It was then that Carlucci and Bowes (1970a, 1970b) , by
confirming in vitro production of vitamins by several species of marine
algae, provided a strong argument for the inclusion of algae among not
only the users of, but also the contributors to, the in situ vitamin pool.
The level of vitamins in natural waters is determined by a balance between
producers and consumers, and variations in vitamin levels in the sea have
been found to correlate with population increases of vitamin requiring
algae. In 1956 Cowey found that a drop in Bi2 concentration coincided
with May- June diatom blooms (many diatoms used exogenous 612) > and in
1959 Vishniac and Riley observed a drop in B12 levels which parallelled
a drop in N03 during blooms of Skeletonema costatum in Long Island Sound,
suggesting a direct correlation between NO3 utilization in cell growth
processes and the consumption of
The widespread occurrence of vitamins in measurable quantities in both
marine and freshwater environments (e_.£. , B12 in Linsley Pond throughout
1973 was approximately 4 mv%) , and the demonstrated capacity of bacteria
and algae both to use and to produce these vitamins are clear examples of
the extracellular metabolites of one group of organisms being of appro-
priate quality and sufficient quantity in situ to effect the metabolism
of a second group of organisms.
29
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SECTION VI
FIELD STUDY
VI:I SITE CHOICE AND SAMPLING
Site Choice
Since Linsley Pond is readily accessible, since it is presently evidenc-
ing the effects of cultural eutrophication, and since there is an exten-
sive literature relating to Linsley (see exhaustive bibliography in
Cowgill, 1970), it was chosen as the body of natural water about which
essential field data would be catalogued and from which experimental
organisms and materials would be collected.
Linsley is a medium sized (approximately 94,400 n2; mean depth 6.7 m;
maximum depth 14.8 m—Hutchinson, 1938) kettle lake approximately 25 m
above sea level. It drains a basin about 2Q times its own area (Riley,
1939). There are approximately 100 single family dwellings in the
drainage basin, 11 of which are located close to the water's edge.
The main inflow stream (approximately 2.5 x 10^ m3 wk~^—Hutchinson, 1941) ,
which enters the lake at its southwestern extremity is from Cedar Lake, a
slightly larger, though somewhat shallower, body of water which is
located in very similar environs less than 500 meters to the east of
Linsley. The stream connecting these two bodies of water passes along
the northern edge of a golf course. Surface water flows across this well-
fertilized lawn and into the stream. Twin Lakes Road runs north-south
between the two lakes and storm drainage is piped directly from the road
into the pond. There is a secondary inlet stream on the northern side of
the pond which provides a conduit for the waters which wash the lawns and
septic fields of houses on the northern slope of the drainage basin. The
outflow is at the northwestern "corner" of the lake. When precipitation
is heavy, the swamp on the west-northwest edge of the lake carries some
of the excess water.
A variety of organisms have been reported to have produced bloom popula-
tions in Linsley in the past. At present the annual cycle is dominated
by blue-green blooms which are irregularly broken by brief periods of
relatively bloom-free waters and on rare occasions by spring diatom blooms,
but the dinoflagellates no longer appear in any quantity. To offer back-
ground information and to provide a basis for comparison with blooms noted
during this study, a table of information relating to prior blooms in
Linsley Pond is included in Appendix A.
30
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Field Trips
Field study of the pond was carried on from fall, through summer, 1974.
During the first growing season, 1972, weekly field trips were made. The
decision to establish a weekly sampling schedule was based on the comments
of Lund and Tailing (1957) and on my own past observations, both suggest-
ing that significant, but unexpected, quantitative and qualitative changes
may pass unnoted if monthly observations are relied upon. During the
later growing seasons field trips were more widely spaced; i.e_., 1973,
every two weeks; 1974, once per month. The less precise monitoring of
field conditions during the last two years was dictated by time limita-
tions. Winter field work, 1971-1974, was on a less regular basis, 1-3
trips per month, depending on weather conditions and, especially, on the
thickness of ice cover.
Sampling Station
Since preliminary work suggested very little variation in phytoplankton
population levels when samples were confined to the open waters of the
pond, a single sampling station at the approximate center of the pond
was established. Landmarks on each of the four shores were used to pro-
perly locate the aluminum rowboat from which samples were taken. Depth
measure was also used as one of the criteria for determining the precise
location of the sampling station. Riley's bathymetric map of the pond
(1939) indicates a fairly narrow, 14 m deep, trough running east-west
along the center of the lake bottom. Depth at the station varied from
13-14 meters. This variance reflected both the changes in the actual
water level of the pond and a moderate variation in the precise location
of the station. No depth measure was accepted (i,-e_., location was assumed
to be inaccurate) when less than 13' m and, although Hutchinson reports a
14.8 m maximum depth in 1938, no depth greater than 14 m was recorded
during the 1971-1974 sampling period.
Sampling Regime
Samples were taken at 0, 2.5, 5, 9, and 13.5 (bottom) m. During the first
year of the sampling period Secchi, temperature, H2S boundary, pH,
alkalinity, hardness, D.O., and CO2 were checked regularly. Alkalinity
and CX>2 measures were done every other week; D.O. was checked on those
alternate dates when alkalinity and CO2 were omitted—a twice monthly
basis for each of these parameters. From fall, 1972, onward alkalinity,
CO2/ and D.O. measures were omitted from the regular sampling program.
All depth samples were taken with a Meyer sampler as described by Ruttner,
1953.
31
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VI: II PHYSICAL AND CHEMICAL: METHODS
Temperature and Dissolved Oxygen
Temperature was taken with a thermistor attached to the oxygen probe of
a Precision Scientific Galvanic Cell Oxygen Analyser (D.O. meter) .
After D.O. measure was dropped from the sampling schedule a -20 to 110°C
mercury-in-glass thermometer was employed.
Dissolved oxygen was measured every 0.5 m when the D.O. meter was em-
ployed/but was measured only at 0, 2.5, 5, 9, and 13.5 m when the Winkler
method (as described in Welch/ 1948) was used. The Winkler determination
was employed either as a check on the accuracy of, or as a substitute
for, the D.O. meter measurement. The meter was checked against a Winkler
determination once every six weeks, usually when the probe required
refurbishing.
D.O. samples for the Winkler determination were carefully siphoned out
of the Meyer bottle and into standard B.O.D. bottles. The siphoning
technique was sufficiently effective in avoiding 02 contamination of
samples to allow for zero mg/1 oxygen measures to be obtained from bottom
water samples.
Secchi
Although the Secchi measurement cannot be equated with light measurement
in a direct or simple manner, there is some indication that the point of
disappearance of a 10 cm, all white, disc approximates the depth of 5%
light penetration (Hutchinson, 1957; Yoshimura, 1938); therefore, a 10
cm, all white, disc was used and values were determined according to the
methods described in Welch (1948). To afford some consistency relative
to the possible effects of the angle of incidence of sunlight on Secchi
readings this test was done at approximately 1:00 p.m. on the day of any
given field trip.
H2S
H2S boundary was determined by odor. Samples for this measure were taken
at 0.5 m intervals beginning just above the thermocline and descending
until the characteristic H2S odor was detected.
The pH of each depth sample was measured immediately at collection with
Hydrion Lo-Ion, dye impregnated, pH "paper". This paper was designed
for use in unusually dilute solutions such as natural fresh waters and,
unlike Hydrion pH paper, produces readings which correspond closely to
pH meter readings for such solutions. A second reading was made on a pH
meter when samples were brought into the laboratory. Lo-Ion and meter
readings were always similar. High pH values obtained with Lo-Ion paper
32
-------
in situ during midsummer afternoons usually dropped by the time samples
were returned to the laboratory; however, a second Lo-Ion reading, taken
in the laboratory, and the meter reading remained similar.
Alkalinity and CO2
Alkalinity and CO2 were measured titraraetrically using the methods
described in Welch (1948).
Water Samples for Biological Activity Tests
In addition to the samples for phytoplankton counts a five to twenty-
five liter surface sample was taken for later laboratory assay of the
biological activity of natural waters. Upon return to the laboratory
this sample was first prefiltered thru fiberglass, then passed thru
sterile (H.A., 0.45 ;a) Millipore filtration apparatus and stored in
sterile, non-linear polyethylene containers at -20° (chest freezer).
According to the technical laboratory at Falcon Plastics, the prester-
ilized 2% liter containers employed for storage were made of a purified
plastic resin which Dupont claims carried no trace metal contaminants.
On occasion a portion of this large sample was not stored, but was in-
stead processed and assayed for biological activity immediately after
collection. The bioassay procedure is described in Section VII:I. This
immediate assay of biological activity was intended to serve as a com-
parison for those assays which were performed on the same water samples
after freezing, storage and thaw. After some experience with this com-
parison it was determined that freshly collected samples provided more
reliable information than did freezer-stored samples; therefore, assay
of freezer-stored water samples was discontinued.
Weather
Ambient temperature, cloud cover, and precipitation were noted for each
field trip.
Biological Oxygen Demand
The B.O.D. of samples from each of the five depths was measured twice
during 1972. Measure was based on the difference between saturated D.O.
levels at time zero and D.O. levels after five days incubation at 20°C
in total darkness. B.O.D. bottles were immersed in water for the dura-
tion of tests to assure that no oxygen could enter and contaminate test
samples. All oxygen measures were determined by the Winkler method as
described in Welch (1948).
33
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VI: III PHYSICAL AND CHEMICAL: RESULTS AND DISCUSSION
Introduction
Total alkalinity, CO2» pH, dissolved oxygen, Secchi, H2S horizon, thermo-
cline (including temper attire in depth) , and phytoplankton population
levels were monitored on a weekly basis thru the first year of this study.
Data was collected for 0, 2.5, 5, 9, and 13.5 m depths to permit consid-
eration of the significance of vertical changes. Since the first year's
data indicated no unexpected perturbations in the curves generated by
CC>2, alkalinity, and dissolved oxygen measures; since these tests required
considerable time during any given field trip; and since indirect moni-
toring of these parameters is sufficient for detection of extraordinary
changes; these three measures were omitted from the sampling program in
the second year and thereafter. Indirect monitoring of CC>2 and alkalin-
ity was accomplished by monitoring pH, and indirect monitoring of dis-
solved oxygen was possible via the monitoring of the H2S horizon.
Dissolved CO2
During the winter months CO2 remained low and constant thru the water
column (Figure 2) . This reflected a bacterial community of moderate
size and a low population of zooplankters. Thru the growing season,
however, CO2 varied with depth. The low (to zero) dissolved CC-2 in
surface waters during midsummer afternoons reflected a high rate of
carbon fixation and demonstrated the capacity of the blue-green bloom
organisms, which consistently dominated the open waters of Linsley, to
use available dissolved CO2 before resorting to the bicarbonate ion as a
carbon source.
2 was always measured at approximately 3:00 p.m. On two occasions,
July 18, and August 8, 1972, surface CO2 was measured at 11:00 a.m. and
again at 3:00 p.m. CO2 levels at 11:00 a.m. were found to be 3.5 and
4.0 ppm respectively, while in both cases, surface CO2 was 0 ppm at
3:00 p.m.
During the growing season the level of dissolved CO2 in bottom waters
reflected bacterial activity on, or near, mud surfaces. Bacterial
.activity increased in mid to late summer as the level of freshly
deposited bottom detritus increased. The increase in dissolved CC>2 at a
depth of approximately 5 m on March 28, and on October 23, 1972, probably
reflects an increase in zooplankton — Keratella in March and Cyclops in
October. No attempt was made at these early dates to determine numbers
of- zooplankton, but in both cases the zooplankters named were observed
in 5 m phytoplankton counting samples.
During the three years of this study the pH of Linsley Pond waters at
0, 2.5, 5, 9, and 13.5 m depths remained fairly constant (Figure 3 offers
-------
•
•'
DEPTH
(m)
•i
12-
FREE C02
0 ppm
18 ppm
29 . 4? ,
0 ppm
6 ppm
36 ppm
20 MO
30 ppm
6 ppm
20 ppm
, 2p . "p
6 ppm
5 ppm
3/28/72 5/22
7/18
8/8
9/12
10/3
10/31
7 ppm
J I
7 ppm
21)
11/17/72
TIGURE 2. Carbon dioxide: Annual Cycle,
-------
:•
pH
9-
8-
7-
8-
•'
PH
:
-
"V
N
1971
.
—••—"»" Timi
P. .
,
Surface
Bottom
i--
^••^M
5?^
— -*
"
F
L —
_^-^
^^*
1 /\
^^^
1
-
A
1
\S
N
\/
->
>
S
t
_\
/
/
/
V
A.
/ V
J
/
S
/
^
\x
-S
N
A
\ ><•
j |A
1
K/
\
S
S
/
W?
0
1972
FIGURE 3. pH: Annual Cycle.
-------
a comparison of the extremes; i-e_., surface and bottom measures). The
only abrupt changes noted corresponded to high productivity periods
(midsummer afternoons) when dissolved CO2 in surface waters was depleted
by the high rate of carbon fixation of phytoplankton. The single low
reading (7.0) in late June, 1972, reflected two unusual circumstances:
first, an exceptionally heavy rainfall two days before the field trip
(the radio reported a total of 12 inches) the runoff of which diluted
the phytoplankton population (the period between the rainfall and the
field trip was insufficient for recovery); and second, moderate cloud
cover during the morning hours which limited productivity.
Through the year readings from surface and 2.5 m depths were generally
similar, as were readings at 9 and 13.5 m. Five meter readings usually
fell midway between surface and bottom levels except when H2S dominated
the hypolimnion, in which case pH readings for the entire hypolimnion
were similar. No unexpected readings were taken during the three years
of this study. A graph of the first year is provided (Figure 3) to
demonstrate the annual cycle. Surface readings in general were slightly,
but consistently, higher than bottom readings, reflecting both the
elimination of dissolved CC>2 during periods of productivity in surface
waters, and the higher organic load and decomposition rate in bottom
waters and adjacent mud surfaces.
This stability reflects the natural bicarbonate buffering system of
Linsley. During laboratory work dealing with the F vs A assays, a con-
sistent return of pH values in A (autoclaved) tests to the original F
(filtered) levels, usually within a week of autoclaving, also suggests
a natural buffering capacity in Linsley Pond waters.
Alkalinity
No unexpected phenomena were indicated by unusual perturbations of alka-
linity curves (Figure 4); however, a most interesting correlation between
alkalinity measure and lake morphology in the 1970's and Hutchinson's
1938 commentary concerning this same correlation was noted.
In 1938 Hutchinson discussed the similarity between the variation of
alkalinity (HCO3~) with depth and the morphology of the lake bottom.
Using Linsley as a model, he demonstrated a correlation between the curve
resulting from the graphing of the ratio of mud surface to volume and the
curves resulting from the graphing of late summer alkalinity levels by
depth. He noted a similarity in both first and second derivative (similar
slope and points of inflection).
Thirty-five years later the same correlation can be seen between these
two parameters. Although his alkalinity measurement was much more pre-
cise (measurements were taken at one meter intervals) than are the
measurements of the present study (0, 2.5, 5, 9, and 13.5 m only), the
resulting alkalinity in depth curve is quite similar to earlier deter-
minations. The level of alkalinity has increased for all depths (i..e_.,
37
-------
•ico -
ppm
no-
•.:,
70-
50-
30-
-
N
1971
•
2
'13
i —
iii
.Bra
m
n
D
•— m
j
•WMMM
— -^—
F
M
TOTAL ALKALINITY
— — —
i
-i
1 IVI I
A
1
•<
X
y
/
,X—
n
V J •
k
\
\
V
"
>^*
'•f'
•*»
•IN
r
I
x^
y
**~^
s
^
^
****
J A
^^
<^~
\
\
1 — ^v
TV
-~~*\\^
\S
....>
i
^
o
1972
FIGURE 4. Total Alkalinity: Annual Cycle.
-------
the curve is displaced vertically), but the slope and inflection are
still similar to those of the mud surface to volume and projected mud
surface to volume curves (Figure 5).
The only significant exception to this correlation is shown in the alka-
linity curve for late June, 1972 when bottom alkalinity levels dropped
precipitously after a period of unusually heavy rainfall and concomitant
heavy runoff. This very dense runoff water, laden with the materials
accumulated during its rapid flow toward the lake, may reasonably be
interpreted to have entered the otherwise closed water system of the
hypolimnion directly as it flowed down the surrounding hillsides, along
the mud surfaces of the littoral zone, and finally, along the bottom mud
of the pond.
Thermal Profile and Thermocline
A thermal profile of Linsley was determined for every field trip. The
annual cycle of Linsley"s thermal regime is presented for 1972 (Figure 6).
Although the profile (in depth) provided more information concerning the
thermal stratification of the lake on any given date, the simple location
of the approximate depth of the center of the thermocline (Figure 7)
when plotted thru the entire study period offered a more informative pic-
ture of the water dynamics resulting from the thermal cycle. This repre-
sentation of the vertical movement of the thermocline permits comparison
not only of the timing and length of overturns, but also of the long
stable stratifications of summer waters, and of the relatively brief,
less stable, winter stratification periods—during which the entire body
of lake water was very close to a single temperature, extremes being no
more than 4°C apart.
Dissolved Oxygen and H2S Horizon
As with previously discussed parameters, dissolved oxygen profiles gene-
rated by the data of this study represented those to be expected for a
medium-sized, eutrophied, lake such as Linsley (Figure 8). In less
productive winter and spring waters the clinograde oxygen distribution
common to eutrophied lakes occurs. In the highly productive summer
months, however, the total elimination of dissolved oxygen from the
hypolimnion produced an orthograde curve with the appropriate absolutely
uniform vertical distribution of oxygen (albeit at zero).
Like a thermal profile (in depth), an oxygen profile can offer a clear
description of the condition of lake waters only on a single occasion.
But the simple location of the zero D.O. boundary, when plotted against
time, can provide additional meaningful information relating to the annual
cycle of the dynamics of the putrefication process which diminishes the
aesthetic acceptability of highly productive waters. In Linsley the zero
D.O. depth was always a close approximation of the H2S horizon (Figure
9).
39
-------
HC03"
140-
130-
120-
110-
100-
90-
80-
70-
60-1
Ratio
FIGURH 5. COMPARISON OF 1972 ALKALINITY (BY DEPTH)
WITH LAKK MORPHOLOGY
Ratio—Mud surf ace: Volume*
Ratio —Projection of mud
surface:Volume*
— — J-—'- Pond Alkalinity 10/3/72
— — - p°nd Alkalinity 8/1/72
*Adapted from Ilutchinson, 1938.
1 9
DEPTH
-------
I
DEPTH
(m)
o
4
•
T n
J.U
12
14-
THE
63 °C
a3°c
:RM.
2fl°C
40°C
*L 1
so°c
45°C
3ROFI1
2L4°C
!
/
/
/
^2UC
_E
29LO°C
z
/
'
/
/
&2UC
220°C
1
.
L^
z
nr
\
\
&5°C
9t3°C
ks°^
95°C
9L5°C
49 °C
49 °C
10 0 10 0 10 0 B 20 0 10 20 30 0 20 30 MO 0 10 0 10 0 10
1/3 3/20 5/22 7/18
9/12 11/7 11/17 12/2/72
FIGURE 6, Thermal Profile: Annual Cycle.
-------
THERMOCLINE
DEPTH 0.
2
r.
g
o
;ll
, ! .
V !^_
. . •
: ;:
1 : •
| ••••
i
N ID
i/
J-
i
j
-"
—
F
1
M
/
V
.
A
M
*— x
J
J
— ^
A
^"^
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A S
(m) 2-
1972
1973
1974
FIGURE 7, Depth of Thermocline. Three Years.
Spring and Fall Overturns indicated in Bottom
Margin by Solid Black Bar.
-------
'-..
DEPTH
(m)
DEPTH
•
rl
8
2-
DISSO
7 . 6 ppm
1
I
/
8. 4ppm
5 10
LVED
12. Ippm
r
--*-
QMG
9 . 8ppm
/
/
1
1
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0 . Oppm I 0 . Oppm
5 10 5 10
EN
7.7ppm
x*
1
0 . Oppm
5 10
8 . 9ppm
^
^
/
O.Op
5 1
pm
0
10.2 ppm
f""""
ilffJ
0 . Oppm
5 10
11. 2 ppm
^
}
0 . Oppm
5 10
11. 6 ppm
1 1. 5 ppm
5 10
12/26/71 3/28
5/22
7/18
9/12
10/17 11/7 11/17/72
FIGURE 8. Dissolved Oxygen: Annual Cycle.
-------
AND
DEPTH
(m)
N |D sj |F |M | A |M
1972
|A |s |o IN jo *j |F |M |A M |j [J|A js |o |N |D fej |F [M |A |M
1973 1974
A s
FIGURE 9. Hydrogen Sulfide Horizon: Three Years.
Spring and Fall Overturns Indicated in Bottom Margin
by Solid Black Bar.
D. 0. = Zero Line Included for Comparison: One Year.
-------
Some minor discrepancy between these two parameters appears in the graph
in Figure 9. Although this may be interpreted as the result of very
limited mixing at th'e interface of oxygenated and H2S dominated waters,
I believe it to be an artifact of measuring techniques which required
sampling for H2S and testing for D.O. to be temporally, and therefore,
almost certainly, spatially, separated. This latter explanation is
additionally favored if one considers the capacity of modules of lake
water to be in constant motion, not only in relation to the sampling
station (boat), but also in relation to each other. Also, the "mixing"
explanation could only be applied to those occasions which show overlap-
ping of the two regions. It could not be applied to those occasions
wherein the two parameters do not touch.
Evidence of the changing conditions in Linsley waters during this study
is found in the three year "H2S Boundary" graph (Figure 9). That the
14 m deep basin which holds Linsley's waters was filled with H2S laden
waters to within 2.5 m of its surface in the summer of 1972, but carried
at least 6 m of H2S-free water during midsummer, 1973 (doubling the depth
of acceptable water), and improved again during midsummer, 1974, is a
clear demonstration of the slow improvement in the condition of Linsley "s.
waters. This is, unfortunately, less improvement than would have been
assumed based on the visual and tactile judgements which accompany recre-
ational contact; however, this contrast in conclusions drawn from the
subjective recreational contact and the objective monitoring of H2S
boundary attests to the value of H2S boundary data as a rapid measure of
bloom productivity.
Secchi
Among the tools of science, the Secchi disc is unique. It is highly
valued for its consistent performance—yet its measurements of a physical
quantity must be expressed with a unit which describes length, and which
has only relative meaning when used for Secchi readings. Yoshimura (1938)
may have been accurate in his contention that a 10 cm, all-white, Secchi
(as used in this study) disappears at the depth at which light is approx-
imately 5% of the ambient light level; there are, however, a variety of
patterns and sizes of Secchi discs. With, or without, a tie-in to some
physically defined unit, the Secchi can provide a general measure of
conditions in any given body of water; and a few notes relative to the
color, opacity, dominant plankton, and detritus taken at time of the
Secchi reading provide an excellent, very rapid, assessment of such con-
ditions .
Any single Secchi reading offered an immediate basis for the description
of Linsley as highly eutrophied; however, there was little additional
input from a single measure if taken by itself. In this study the
greatest value of a Secchi measure (Figure 10) was its relative similar-
ity, or difference, to each of the other Secchi measures of the study.
During a bloom period a rise or fall of 10 to 20 cm in the Secchi depth
provided factual input for an immediate in situ decision as to whether a
45
-------
SECCHI
-
DEPTH ft
(m)
1
k
f
\i
\ n
12^
1 1,
1 .;
1 i
1
i !
1
;
1
t
| 1
•
N |D
i "
• *
i •
i i
i
i
' I1""
j
i
: i
1
j \r |M
1972
— •"^
A
.
•••*i
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P
•c<
p
/**».
1*
>v\
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19
I——,
F
73
4.
\.
«
M
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^
V.
\
|M
^. ,
\
U
^
f
i
I
1
j
.— ^
1^
\
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Is
'•>
|c
f
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1
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1
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— —
m
4
>-•
I
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19
F
74
*** V
1
M
».
U
*«s
M
1 . i. J
i
1 i
"^.L '
• • i '
; i
1 1 .
'Lii
i 1
1 I
_ - 1
i 1
1 ! 1
|J b IA Is
FIGURE 10. Secchi Depth: Three Years.
S pring and Fall Overturns indicated in Bottom Margin
by Solid Black Bar.
-------
specific week marked the beginning, or the end of a bloom; or whether
the phytoplankton community was similar to that of the previous sampling.
Without the input from the Secchi such judgements would have been based
on visual memories of the appearance of samples taken during a previous
field trip, and this would have been the only basis for such judgements
until several hours later when time-consuming population counts were
completed back in the laboratory.
Thus, the weekly progression of the Secchi depth (Figure 10} proved
especially valuable in the field. The obvious difference between 1972
and 1973 summer readings provided immediate assurance at the start of
each field trip that Linsley was markedly clearer in 1973 than it was in
1972. Later in the study similar comparisons indicated that conditions
in 1974 were similar to those of 1973. These facts would not have been
immediately obvious during field trips if only the general appearance of
surface samples was considered. For example, the plankton in surface
samples was not as dense in 1974 as it had been in 1973. This gave the
erroneous impression that waters were less dominated by algal blooms.
Actually, the 1973 bloom was a surface phenomenon, and the 1974 bloom
was a subsurface phenomenon, both populations had similar effects on the
overall quality of the water.
Biological Oxygen Demand
The biological oxygen demand ofO, 2.5, 5, and 13.5 m water samples was
measured on February 23, 1972 and on August 8, 1972 (Figure 11). Results
indicated that Linsley B.O.D. levels were within the range of those from
other moderately eutrophied lakes. When compared to the levels of as
high as 50 mg per liter in secondary sewage effluents, Linsley values
were low. When compared to the almost zero levels of oligotrophic moun-
tain waters, however, Linsley values were moderately high.
General Discussion of Physical and Chemical Results
The physical and chemical parameters monitored during the study were
found to be within the ranges expected for a lake of Linsley's size and
degree of eutrophication. The improvement in the condition of the lake
from the summer of 1972 to the summers of 1973 and 1974 is most readily
demonstrated by the increase in Secchi depth and by the decrease in the
volume of H2S dominated waters. It is probable that this improvement is
the result of the correction of a single faulty septic system. Raw
sewage was being diverted directly into the pond via an outlet stream on
the northeastern boundary of the lake. Corrective measures were apparently
made only in response to the threat of legal action against the offending
property owner. There is a possibility that at present sewage is again
being diverted into the pond because the endemic Oscillatoria rubescens
(535) population has produced its first bloom since the winter of 1971-
1972, and that bloom was supported by the high nutrient level during the
time period when raw sewage was entering the pond.
47
-------
B.O.D
DEPTH
(m)
Feb. 23,
1972
2.5
2.5
6.5
Aug. 8,
1972
6.5
FIGURE 11. Biological Oxygen Demand—5 Days.
Expressed as the Difference between
D.O. ppm (Saturated) on Day 0 and
D.O. ppm on Day 5.
48
-------
The original determination of sewage in the inflow was made by an engin-
eering firm. They reported very high (105/ml) Escherichia coli counts
in the stream. After correction, the E_, coli count was very low (102/ml)
Periodic check on this particular stream is now planned by members of
the Linsley Pond Association to assure the permanent compliance of the
offending party with the legal requirements regarding his septic system.
VisIV BLOOM SEQUENCE MONITORING: METHODS
Phytoplankton
Samples for phytoplankton counts were taken at 0, 2.5, 5, 9, and 13.5 m.
200 ml portions were stored in glass bottles which were, in turn, placed
into individual styrene thermal containers. Phytoplankton counts were
made immediately after return to the laboratory on the day of any given
field trip. Preserving specimens for later counting was not considered
satisfactory because many forms, especially the prokaryotes, were dis-
torted or destroyed by the chemical action of preservatives and this
distortion, or destruction, was considered to interfere with accurate
counting and species recognition.
The majority of counts were made at 400x and lOOOx (oil) on a standard
Zeiss microscope using a Modified Neubauer "Brightline" Hemacytometer.
Exceptionally small organisms, for example the Synechecoccus sp. (91)
which occurred in significant numbers in the spring of 1973, were
counted at 520x and lOOOx (oil) with the aid of the interference phase
contrast and dark field equipment of a Galileo L.G. 5 microscope. Counts
of the Synechecoccus sp. (91) were 400-500% higher when the more sophis-
ticated equipment was employed. This discrepancy reflects the generally
small size and low pigment density of most prokaryotic forms.
A Sedgewich-Rafter counting chamber was used for counting larger forms,
especially the Anabaena sp. (765) which dominated during the summer of
1973. Counts of larger organisms were also made by passing a 200 ml
sample thru a Millipore (H.A., 0.45p) filter with grid markings, and
counting organisms at lOOx and 200x with the aid of a Leitz inverted
scope. This filtration method worked well only for very large forms such
as the aforementioned Anabaena sp. (765), Coe1osphaerum, or Eudorina, and
for more rugged forms such as Trachelmonas and Ceratium. Volvox colonies,
in contrast, were frequently unrecognizable after filtration and were,
therefore, counted only in the Sedgewich-Rafter chamber.
Although the standard is somewhat arbitrary, 100 organisms were considered
to be the standard counting sample. This means that from 5 to 20 separate
sample drops were utilized in most of the counting procedures. This
standard was held only for major constituents of the population. Organ-
isms present at 1000/ml or less were represented by fewer individuals for
practical reasons. The 100 cell count is suggested by Lund and Tailing
(1957) as a reliable numerical sampling of individual organisms in popu-
49
-------
lation counts.
Brook, e£ al, (19711, used a measure of turbidity to approximate popula-
tion density for Oscillatoria agardhii and were satisfied that they were
measuring the correct parameter with the turbidimeter. On several occa-
sions, parallelling this method, an O.D. meter which accepts test tubes
(made for Haskins Laboratory by Dr. Fred Kavanaugh)* was used to obtain
population profiles in depth of Oscillatoria rubescens (a form of O.
agardhii which carries sufficient phycoerythrin to give a bloom a red-
dish-brown, or orange, tint). The correspondence of O.D. to counts was
fairly good; however, ma-giimim populations produced O.D. values in the
lowest part (least reliable) of the O.D. meter range and the possibility
of inaccuracy was considered too great to permit the substitution of
O.D. for direct counting.
Zooplankton
Monitoring of zooplankton populations was not one of the goals of the
original proposal for this study. Zooplankton were specifically omitted:
first, because preliminary. survey indicated very few zooplankters in the
open waters of Linsley Pond; and second, because the mechanics of col-
lecting even a moderately accurate numerical sampling of zooplankton are
cumbersome and, as with all collection techniques, become increasingly
more complex as the density of the population to be sampled drops.
Incidental data relating to zooplankton counts, however, proved to be of
such interest that it would be a greater error to ignore it than to in-
clude it, even while conscious of its weaknesses. The quantitative
aspects of zooplankton population monitoring in this study are only
meaningful in judgements concerning relative changes and cannot, there-
fore, be considered as measures of absolute numbers.
Zooplankton counts were made by passing approximately 200 ml of surface
water samples (collected by dipping and scooping water into jars) thru a
(H.A., 0.45u) Millipore filter with grid markings. This "counting" of
zooplankton was prompted by the dramatic appearance of zooplankton in
great numbers early in the 1973 growing season. In the Fall of 1971 and
thru the growing season of 1972 it was common to pass as much as 10
liters of surface pond water (collected for purposes of storage for later
bioassay) thru a large fiberglass filter (held in a Buchner funnel) and
to note no zooplankton in the "concentrate" above the filter. Some
zooplankters are known to actively avoid the suction current caused by
submerging a container; however, these large samples in 1972 usually pro-
*See Appendix D for additional information re this meter.
50
-------
duced no zooplankton whatsoever (curiosity, prodded by this total absence,
occasionally prompted careful search), In contrast in 1973 these same
large samples, taken with the same containers, in the same fashion, evi-
denced hundreds, at times thousands, of zooplankters.
In addition, because I have the Ceriodaphnia and Eubosmina from Linsley
in culture at the present; and because (although I have repeatedly failed
in attempts to initiate cultures of Cyclops, Keratella, and the rather
rarely noted Calinoid cyclopod from Linsley) I have had considerable
experience in attempting to catch, and ample opportunity observing, the
zooplankters of Linsley; I have noted that while Cyclops and the Calinoid
are very active in avoiding suction currents, the Eubosmina/ Ceriodaphnia
and Keratella are not.
Considering all the above facts, it is at least certain that the 1972
zooplankton population was extremely low in number, and that the 1973
population was dramatically higher.
VT:V BLOOM SEQUENCE MONITORING: RESULTS AND DISCUSSION
Introduction
The phytoplankton bloom sequence of Linsley Pond was monitored for three
years (winter, 1971 thru fall, 1974—Figure 12). The pond flora was
dominated by an almost continuous series of blue-green blooms. Brief
occurrences of diatom blooms in the springs of 1973 and 1974 were the
only occasions during this study when bloom dominants were not blue-green
algae. Figure 12 is offered to allow a general view of the three years
of the study; however, detail of bloom sequence events is more readily
gleaned from a study of the enlarged segments of this graph which are
included within the discussions of each seasonal segment (key on page 54).
1971-1972, Winter
Thru the first winter of this study (Figure 13) the open waters of Lins-
ley were dominated by a bloom of Oscillatoria rubeseems (535). The
population level which prevailed throughout the cold months was estab-
lished prior to turnover. A preliminary field trip to Linsley in the
fall of 1972 indicated that, if the total population of O. rubescens
(535) can be approximated from surface to 11 meter observations only
(counting samples were taken from irregular sampling stations—this was
prior to the establishment of a single, lake-center, station), then the
population level which prevailed throughout the cold months was estab-
lished prior to turnover. This point is important in the discussion of
a possible approach to control of Linsley's eutrophication problems by
the use of biological and chemical controls to program the bloom sequence
in situ, see Section IV.
51
-------
H A M
Volune expressed es mm-2
Figure 12. Key to Symbols on page 54.
52
-------
I I
BLOOM SEQUENCE
LINSLEY POND
1971-1974
FIGURE 12
Winter
1973-74
'1974
DIAL
' ^
53
-------
FIGURE 12. Phytoplankton Bloom Sequence of Linsley
Pond. Winter, 1971 to Fall, 1974.
Oscillatoria rubescens (535)
(winter strain)
.....*...... Oscillatoria rubescens (739)
(summer strain)
.._._._ Anabaena sp. (538)
Anabaena sp. (762)
(or Aphanizomenon
elenkinii (762)
Anabaena sp. (765)
(large form)
Pseudanabaena galeata (597)
Oscillatoria sp. (776)
Synechecoccus sp. (91)
Flagellated forms
Diatoms
54
-------
mn3xlO :
per al
130-
100-
80-
60-
40-
200-
100-
20-
FIGURE 1-3.
1971-1972, Winter.
Oscillatoria rubescens (S35)-
Inset indicates other occurrences of O. mbescens (535).
55
-------
Distinguishing Winter and Summer Forms of Oscillatoria rubescens
Oscillatoria rubescens (535) is a filamentous, non-heterocystous, blue-
green algae which exhibits a positive phototrophic response both ill situ
and in vitro. This phototrophisra, and several additional characteristics,
serve to distinguish the winter strain, (535), from the summer strain,
(739) , of O. rubescens. The cell height to cell diameter ratio in O.
rubescens (535) is less than or equal to, one—while that of O. rubescens
(739) is greater than, or equal to, one. Additionally, different growth
characteristics on agar—colony form, color, ease of establishment—dis-
tinguish the two strains. The colors of liquid cultures also provide
points of difference. A healthy, liquid culture of O. rubescens (535) is
a dark green-brown to khaki. A pink to light Winkler "peach" color
appears in liquid cultures of O. rubescens (535) in late senescence. In
contrast, healthy, liquid cultures of O. rubescens (739), the summer
dominant, appear green-brown to orange-brown and develop a white-orange
hue in late senescence. Cultures on agar maintain colors identical to
the colors of healthy liquid cultures of the same strain with only a
slight change toward the colors of senescence. Agar cultures dry out
before they reach senescence. They maintain vitality considerably longer
than do liquid cultures; some have survived thru as long as nine months.
Only the drying and separating of the agar ended these cultures.
The summer form, (739), produces growth on agar which resembles thinly
stacked filaments, patterned in long, thin, sweeping curves. Eventually
filaments are singly and evenly dispersed thru the entire volume of the
agar—suggesting an inherent capacity for motility with no concomitant
phototrophic response. The winter form, (535) however, which is diffi-
cult to establish in culture on agar (many attempts produced only one
successful culture), does not produce thin, sweeping curves, but is
rather made up of thick, slightly curving, colonies of parallel filaments.
These colonies, less definable as to shape, but a consistent dark green-
brown color, do not spread thru the volume of the agar. Instead they
remain on the surface, in apparently good condition, until the agar dries
(sole example: five months).
Neither of these Osci 1 latoria rubescens strains has reproduced in the ab-
sence of its accompanying bacterial community. In both cases, single,
axenic, trichomes have been isolated from agar and placed in test tubes.
Sterility tests have shown these "cultures" to be bacteria-free. Tri-
chomes appear intact and viable after several months, yet no apparent
growth, reproduction, or disintegration occurs. The result of this
apparent dependence on some bacterial metabolite, or process, is that
both strains are represented in the Linsley culture collection as bacter-
ized isolates only. The addition of filter-sterilized media, collected
from a bacterized, healthy culture of p_. rubescens might provide a suc-
cessful axenic culture of these organisms. This procedure could not be
included in this study, but offers promise for future work.
56
-------
Oscillatorla rubescens C535). , winter dominant, was successfully cultured
in liquid fronj an opaque, red, plastic container of stored pond water
(5°C in a darkened cold rooml.. The inoculum for this culture was taken
from the stored water after approximately four weeks in storage and
although growth, or reproduction, during this four-week period cannot be
documented, the organisms obviously survived and maintained (or improved)
vitality while in total darkness—an indication of heterotrophic tenden-
cies. This, too, offers opportunity for future study; a^.e^, the investi-
gation of the heterotrophic nature of these blue-green aerobic (possibly
also anaerobic - see Figure 21, this section) prokaryotes.
Oscillatoria rubescens as Opposed to O_. agardhii as the Proper Taxonomic
Placement for the 197T-1972 Winter-Summer Dominant Blue-Green Organism—
To avoid a misinterpretation of the appearance of Linsley Pond during
blooms this separate section has been included. The two dominant species
of Oscillatoria; i..e_., (535) and (739) , were designated as O. rubescens,
rather than as 0. agardhii, solely on the basis of the visible presence
of phycoerythrin; however, this pigment is not sufficiently concentrated
to cause Linsley to turn a blood red color, as do the European lakes
dominated by blooms of (X rubescens. Linsley takes on only an orange-
tan color during its extremely dense blooms. Thus, these two p_. rubescens
strains, while qualifying technically as members of the species, do not
exhibit the most infamous characteristic of the type.
1972, Spring
In the spring of 1972 the 0. rubescens (535) population was replaced by
O_. rubescens (739), summer form (Figure 14). The breaking and deteriora-
tion of (535) was accompanied by the appearance of new healthy filaments
of (739). The graph in Figure 12 indicates an additional reason for
separating these two similar organisms into distinct, overlapping popula-
tions; it clearly shows two distinct population peaks (February and June).
One of the previously listed bases for this separation of (535) and (739)
into two distinct strains, the phototrophic response peculiar to (535),
is displayed by a graph plotting the vertical location in the pond waters
of the bulk of the bloom population (Figure 15). In contrast to the
apparently erratic location in depth of the bulk of the (535) population
thru the winter, note the constant depth (at 2.5 m) of the bulk of the
(739) population thru the spring, and its relatively constant location
in, or near, surface waters in early summer.
The location in depth of the O. rubescens (535) winter bloom was dependent
on the phototrophic response of (535); i.£., on a very dark (heavy over-
cast) winter day the bulk of the bloom would remain near the lake bottom
(an early morning check in January, 1972, suggested that the bulk of this
population was near the bottom of the lake each morning) while on a very
bright day the bulk of the population would be near the surface. It was
57
-------
ram3xlO~'
Per i,l120_
100-
00-
100-
WINTER
1571-72
FIGURE 14. 1972. Spring.
Oscillatoria -rubescens (739) .
Inset indicates occurrence of
O. rubescens (739) .
-------
DRPTH
(a)
0-
2.5-
13.5-
(535) Oscillatoria rubescens.
(739) Oscillatoria rubescens•
H
M
FIGURE 15- Depth of Oscillatoria rubescens (535) and (739)
bloom populations in the lake—for comparison.
59
-------
essential to determine light conditions for the entire morning of a given
field trip, rather than simply light conditions wat collection", in order
to predict the depth at which the (5351 population would be found; how-
ever, with a little practice prediction became quite accurate. In large
filtrate cultures (535) was observed to migrate to the brighter side (still
relatively low illumination) of the flask. (739) did not exhibit any
form of photokinesis.
During the 1971-1972 winter-spring time period many other organisms were
noted from week to week. None appeared in quantity sufficient to be of
significance when compared to the volume of the ^. rubescens blooms. It
was, nevertheless, during this period when most of the organisms repre-
sented by cultures in the Linsley collection were obtained.
Cyclotella sp_. (211) appeared in the cold surface waters of the pond in
January and, as Gordon Riley (1940) suggested, was apparently not native
to Linsley but was rather washed down annually from Cedar Lake. Since
the Anabaena sp. (538), which produced a heavy bloom in late spring,
1972, produces a heat-labile substance which is toxic to Cyclotella sp.
(211), one may conclude that the inability of this organism to establish
a permanent (if small) population in Linsley is the result of its
inability to coexist with at least one of the bloom producing organisms
of the pond.
1972, Late Spring and Summer
In the late spring of 1972 Anabaena sp. (538) produced a bloom population
which became co-dominant with that of O. rubescens (739) (Figure 16) .
Both the Anabaena sp. (538) and the O. rubescens (739) blooms had sub-
sided by the end of July, 1972. At that time two significant forms,
morphologically similar to (538) and (739), replaced them as dominants.
The first was O. rubescens (535), the winter dominant. Its population
developed rapidly, reaching levels in August similar to those of the
previous fall, 1971. The second significant organism was actually a
combination of a few single (young colonies) filaments of Aphanizomenon
flos-aquae (766), and a large population of a second heterocystous blue-
green (762) which is tentatively identified as either Anabaena sp. (762) ,
or as Aphanizomenon elenkinii (762). It is of interest that the in_ situ
appearance of (762) was very similar to that of the spring bloom domi-
nant, Anabaena sp. (538). It can be distinguished from (538) by the more
conical shape of its terminal cell (a significant feature of the genus
Aphanizomenon) but its heterocysts appear very similar to those of (538).
It does not form flakes.
With, the O. rubescens (535) and Anabaena sp. (762) bloom populations came
three additional, non-dominant, heterocystous, filamentous, blue-green
forms: Anabaena sp. (765), sparsely distributed, of unusually large
size—ranging up to 2mm long; Anabaena circinalis (769), similar in size
to (762) and (538) but with a very different heterocyst pattern (even
interspacing with heterocysts often in pairs); and the aforementioned
60
-------
•am xlO
FIGURE 16. 1972. Late Spring
and Slimmer.
Inset for comparison.
739-C)- rubescens
762-Anabaena sp.
535-0^. rubescens
538-Anabaena sp.
61
-------
Aphanizomenon flps-aqua& (766) in its readily identifiable flake form.
None of these were represented by large populations in 1972, Signifi-
cantly in 1973 when the spring bloom of (538} was absent, and when
Synechecoccus sp_, C91) produced an early spring bloom Anabaena sp. (765)
bloomed. Filtrate studies indicate that (91) produces an enhancer for,
and (538) produces an inhibitor for, Anabaena sp. (765).
In September, 1972, the second appearance of 0. rubescens (535) (the
dominant of the previous winter) ended abruptly, as did those of (762) ,
(765), (766), and (769). This sudden ending, which also occurred in
September, 1973, does not seem to correlate with unexpected physical or
chemical changes in the pond; however, monitoring trace metals proved
impractical, and a sudden, if minor, change in the level of these or some
other critical nutrient might account for the sudden, generalized popu-
lation drop. Cowgill (1971), in her comprehensive study of the elements
in Linsley water (by depth and by date, weekly), found ample supplies of
nutrients in August. Unfortunately, her study ends at the beginning of
September and, therefore, can offer no assurance that the August levels
were carried over into the next month. Tests of pond water samples from
late August, 1973, when an extensive bloom of (765) ended, indicated no
F vs A difference in the growth of Anabaena sp. (765) and no appreciable
improvement in growth when waters were charcoal treated to remove organ-
ics. This suggests nutrient limitation rather than allelopathic effects.
1972-1973, Fall and Winter
In late September, 1972 the waters of Linsley were relatively free of
phytoplankton (Figure 17). In early September a blue-green, Pseudana-
baena galeata (597), which had appeared sporadically throughout the
prior year, appeared in quantity. By mid October it had produced a
bloom. In November this bloom was replaced by Oscillatoria sp. (776) .
This bloom, (776), waned slowly. It was not totally eliminated until the
spring of 1973. These organisms cannot easily be distinguished during
counting procedures? however, by filtering a large sample (one liter or
more) the color of the dominant organism can be determined. Pseudana-
baena galeata (597) is a vivid blue-green color, Oscillatoria sp. (776)
is green-brown to orange-brown. Neither of these latter blooms, (597)
or (776), represented the same, or even similar, volume as the bloom of
O_. rubescens (535) during the first (1971-1972) winter. The waters of
Linsley, as can be seen by comparing the maxima and duration of 1971-1972
and 1972-1973 winter blooms (Figure 12), were considerably free of blue-
green algae the second winter than they were the first. This is another
point of significance to the discussion of bloom sequence control,
Section IV.
After the blue-green population had dropped in February, 1973, the waters
of the second winter sustained a fairly high population of flagellates,
and this mixed flagellate population persisted thru the spring of 1973.
62
-------
rnnTxlO
per ml
120-
100-
80-
66H
40-
20-
XIJITCE 1572-73
55R76)
rS-l , ^ -
i i
O N D |J F
'1973
A M
JJ AS
MA
?>-M S»^u.«r«
I ^-v-
i^/. .L. [ .^rMr
1LLM jj
FIGURE 17. 1972-1973, Fall and Winter.
Insert for comparison.
597-Pseudanabaenj3 galeata
776-Oscillatoria^ sp.
63
-------
1973 , Spring and
The first diatom bloom to occur during this study was produced in the
spring of 1973 (Figure 181 , This bloom was dominated by Asterionella
formosa (800} f and persisted from early March to mid June, 1973, A
sudden drop in the graph in early May (Figures 12 and, or, 18) reflects
heavy rain and the washing out, by heavy runoff, of the A. forroosa (800)
population. The timing of the field trip at that point in the graph
was such that A. formosa (800) gave the appearance only of waning. The
occurrence of Synechecoccus sp. (91) at that time may have been partially
responsible for the slow recovery of A^ formosa (800) population since
Synechecoccus sp. (91) filtrates consistently produced a negative effect
on the growth of A. formosa (800) in filtrate studies.
The diatom bloom ended approximately three weeks later in mid June, 1973.
This was at the same time as a drop in available silica. Data supporting
the premise of silica limitation of the diatom populations in Linsley is
included in Section VII, and is also discussed in Section VIII.
The onset of an exceptionally dense bloom of the very large Anabaena sp.
(765) , which had appeared in late summer, 1972, marked the end of desir-
able conditions in the 1973 growing season. In mid September, 1973, the
waters of Linsley Pond were once again relatively free of phytoplankton.
1973-1974, Fall and Winter
Although £. rubescens (535) was present in low numbers from January thru
March, 1974, neither of the O_. rubescens strains (535, winter; 739,
summer) from the first annual cycle (1971-1972) appeared in quantity dur-
ing the 1973-1974 fall and winter; nor did either appear in quantity
during the preceding (1973) growing season (Figure 19) . P seudanabaena
galeata (597) and Oscillatoria sp. (776) of the second fall and winter
cycle (1972-1973) were also present but only in low numbers in this
third winter (1973-1974) . The combined populations of (597) and (776)
in the late winter and early spring are shown on the graph as a single
double-peaked bloom. They could not be securely distinguished during
counting procedures. Generally, however, when compared to the previous
two winters (Figure 12) , Linsley was relatively free of blue-green
blooms in the third winter.
1974, Late Winter and Spring
The population of flagellates in this third winter was similar to, but
less than, that of the second winter. The diatom occurrence, however,
was quite distinct from all previously monitored seasons. In mid January
diatoms began to appear in Linsley. By late . February a bloom population
was established (Figure 20) . This bloom included Asterionella formosa
(800) , the dominant form in the less extensive 1973 spring diatom bloom,
but was essentially a mixed diatom bloom. Most forms resembled Synedra
and, or, Fragilaria. This mixed population, which persisted until early
64
-------
mn3xlO~3
per ml
120 -
100 -
00 -
60-
40-
20-
200-
100-
1973-73
FLAGELA
(765)
,(800)
DIJF MAMJ JASO
1973
A
n
FIGURE 18. 1973, Spring and Svruner.
Insert for comparison.
765-Anabaena sp.
800-Asterionella formosa
65
-------
nnn3xlO~3
per ml
120-
100-
8O-
6O-
40-
20-
200-
100-
1975-74
(535)|
J J A S O
(597)
a
(776)
n
.
\
FIGURE 19. 1973-1974, Fall and Winter.
Insert for comparison.
535-O. rubescens
597-Pseudanabaena galeata
776-Oscillatoria sp.
66
-------
-3
per ml
120-
100-
80-
60-
40-
20-
200-
10O-
V/V
DlATOMS
L'.nll.^TW
ND|J'F/M AMJ JA
(597)8(776)
FIGURE 20. 1974, Late Winter and Spring.
1974, Summer and Fall.
Insert for- comparison.
535-O. rubescens
597-Pseudanabaena galeata
776-Oscillatoria sp.
67
-------
June, was consistently well distributed thru the water column.
As in the spring of 1973, the 1974 spring diatom bloom ended in early
June when the available silica level was insufficient to sustain growth
and reproduction in the large diatom population, see Section VII :V for
further consideration of this point.
1974, Summer and Fall
In early June the mixed populations of flagellates and diatoms waned,
and Oscillatoria rubescens (535) once again appeared in Linsley (Figure
20). This appearance of O_. rubescens (535) was quantitatively less than
the O_. rubescens (739) bloom two years before the summer of 1972. It
also differed in a qualitative way; i_.e_., it appeared only in the deeper
waters, whereas the (739) bloom in 1972 maintained a depth of 0-3m.
Whether, or not, the first (535) bloom (fall, 1971) was also distributed
thru the water column is uncertain because samples taken during the very
first field trip were only from shore points. The surface waters of
Linsley during the latter part of the summer of 1974 appeared to be free
of algal blooms. The deeper waters, however, maintained a fairly dense
population of O. rubescens (535).
By the end of the summer in 1974 a high population of O. rubescens (535)
had developed in the hypolimnion of Linsley. The presence of H2S did not
seem to hamper the development of these organisms in these deep and
relatively dark (well below the 5% of ambient light—Secchi depth) waters
of the pond. In the laboratory both of the O. rubescens strains, but
especially (535), establish high populations in moderate or subdued
light (10 to 20 foot candles).
Figure 21, below, locates the depth of the late summer-fall population of
O. rubescens (535) and compares this distribution to the H2S horizon.
The population is graphed both by cell count and by O.D. The similarity
of these curves above the H2S horizon attests to the validity of O.D.
measures as approximations of cell counts (especially since these values
are within the least accurate part of the O.D. range, accuracy and repeat-
ability improve at higher values) . The consistent O.D. reading of 0.015
below the H2S horizon reflects the even distribution of bacteria thru the
hypolimnion. Haters appeared cloudy when test tube samples were examined
visually. The O.D. readings above zero in the 0-3.5m depths reflect the
presence of phytoplankters other than O_. rubescens.
This winter, 1974-1975, the p_. rubescens (535) bloom survived thru turn-
over and thru the winter months, but did not equal the density of the
1971-1972 winter C535) bloom. A diatom bloom dominated by Asterionella
formosa (800) developed in the spring. Thus the moderate winter blue-
green growth in the waters of both 1972-1973 and 1974-1975 allowed an A.
formosa (800) dominated spring diatom bloom to develop suggesting a
greater tolerance for blue-green allelopathic effects may exist in A.
formosa them in the other diatoms of Linsley.
68
-------
Line Separating O and H S dominated waters.
Qpcillatoria rubescens (535) cell count.
Optical Density of Raw Water Sample.
DEPTH
(ml
2 —
4 —
6 —
8 —
10—
12-
14-
Cells
(1000)
O. D.
(x 1000)
T
8
"T
10
T
12
10
15
20
25
30
FIGURE 21. Oscillatoria rubosccns. Depth
of natural population compared to depth
of H S boundary. (Cell Count and O. D.)
69
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1971-1973, Zooplankton Populations
The zooplankton population of Linsley Pond is reported for the first two
years of this study. The contrast between these two years prompted the
focus of attention on ths zooplankton (Figure 22),
During the first year very few zooplankters were noted even in the large
surface samples which were collected for bioassay. In contrast high
populations were observed in all samples during the spring of the second
year. With the onset of the Anabaena sp. (765) bloom in mid June, 1973,
the zooplankton population once again dropped to such low levels that
none, or only a very few, were noted even in the large bioassay samples.
This unexpected, high population of zooplankters was accompanied by the
first (during the study period) signs of healthy fish in the pond. Along
the shores of the lake not only a variety of minnows and catfish, but
also a large population of blue-gills, who had carefully staked out
claims to all available territories along the Yale landing, appeared.
Although there were supposedly fish in the lake in the first summer, not
a single living specimen was observed either along the shore or in the
open water during any of the weekly field trips. There were, however,
quite a few dead fish noted in the first year.
In general the fish population appeared far more numerous, and healthier,
in the summer of 1973 than they had in the summer of 1972.
70
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C = Cyclops Observed in Sample
B = Eubosmina Observed in Sample
D = Ceriodaphnia Observed in Sampl*
Each Rectangle above a
Letter indicates
Approximately 200/Liter
LL
BD .
ccc D DC fr
,K$M.CK
crccc BC c
KD C
tJCURE 22. Zooplankton occurrence compared
to blue-green algal occurrence 1971-1973.
I
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SECTION VII
LABORATORY STUDIES
VII:I BIOASSAY
Culture Collection
Isolation—A variety of methods were employed to isolate phytoplankton
from the pond. Larger forms were picked from pond water samples/ washed,
and placed in separate test tubes containing 10 ml portions of enriched
pond water (enrichment was ESi, described under Maintenance, below).
Smaller forms were isolated by plating several drops of freshly collected
pond water on Oxoid, Specially Purified, lonagar #2, with various nutri-
ent additions; or by inoculating test tubes containing 10 ml aliquots of
various nutrient media with 0.1 ml aliquots of freshly collected water.
Inocula were taken from 0, 2.5, 5, 9, and 13.5 meter pond water samples.
The basic liquid to which agar and, or, nutrients were added was either
distilled water, or pond water from 0, 2.5, 5, 9, or 13.5 meters. Agar
was washed (Sands and Bennet, 1964) or unwashed. In some cases pH was
controlled and intentionally set at one unit intervals from 6 thru 11.
Nutrient additions included various combinations of vitamins and inor-
ganic salts. Many of the combinations employed have been published as
growth media, or as useful media additions. A listing of media employed
may be found in Appendix B. The most universally successful growth
medium is a 10 ml aliquot of charcoal treated pond water with 1.5% ESisi
enrichment (see details of preparation under Maintenance, below).
The great variety of nutrient enrichments produced an equal variety of
dominant organisms in these mixed cultures of pond organisms. Unialgal
cultures were obtained from these primary cultures, or in some cases from
secondary, or tertiary, cultures, by picking organisms (with a drawn
glass micropipette) from promising colonies either on the agar plates or
in the test tube cultures. Colonies were selected at 200x or 400x, but
physical removal of selected organisms with the pipette was done under
lOOx or 200x using an inverted Leitz microscope.
Any culture labelled "clone" is the result of a single cell (or single
organism in the case of multicellular forms) producing a new culture
after having been isolated in a test tube containing 10 ml of enriched
pond water.
Maintenance—Stock cultures are maintained at 18-23°C, under 10 200 foot
candles of light, in a natural pond water medium designated "B medium".
B medium is prepared with 50% pre-fall turnover pond water, preconditioned
by one year (or more) of storage in darkness at approximately 5°C, and
50%. post-fall turnover pond water similarly preconditioned. Pond water
portions are charcoal treated (4 grams powdered, activated charcoal per
72
-------
liter, stirred constantly for one hour( passed thru a Millipore, H.A-,
O.45ji filterlf jnjixedf distributed into 1Q ml test tube portions, and
autoclaved for 20 minutes, ESjgi at 1,5% (ES^f Provasoli, 1968; with
150 ml% silica, as Na2SiC»3 •-9H2O added to the basic ESj formula) is added
asceptically just prior to inoculation of cultures..
Axenization—The elimination of bacterial contaminants from unialgal
cultures was accomplished either by streaking on agar and picking clean
colonies, or by ultra-violet treatment. Washing, either by repeated
transfer of organisms of interest from sterile bath to sterile bath, or
by allowing organisms of interest to rest upon filters which are profusely
flushed with sterile medium, did not prove useful when dealing with blue-
green algae (it was not necessary with eukaryotic algae), probably because
the mucilaginous sheath permits a very close association between the alga
and its accompanying bacterial community.
Treatment of bacterized cultures with ultra violet light proved an effec-
tive method of axenization. A 10 ml culture of the organisms of interest
was poured into a 10 ml covered Petri dish, a 3 cm stirring bar was added,
and the covered Petri dish was placed on a magnetic stirring table. This
assemblage was then placed under a transfer hood equipped with a u.v.
tube which was approximately 20 cm above the dish. The cover of the
Petri dish was removed and the contents of the dish were exposed to the
u.v. light for one minute intervals. After each such exposure a small
sample of the culture was aseptically removed and inoculated into a test
tube containing 10 ml of sterile B medium.
Exposure times ranged from 1 to 20 minutes. In the most successful
attempts a minimum exposure of five minutes was necessary to eliminate
bacterial growth. In several attempts bacteria survived as much as 20
minutes of u.v. exposure. Actually, the u.v. treatment often proved more
lethal to the prokaryotic algae than to bacteria. The condition and age
of cultures, and a degree of chance, determined the outcome of such
efforts. The assumption of bacteria-free status was based on the methods
described under Sterility Testing, below.
Organisms—At minimum the following families are represented in the Lins-
ley Pond culture collection:
MYXOPHYTA (blue-greens)
Chroococcaceae, Oscillatoriaceae, Nostocaceae
EUGLENOPHYTA
Phacotaceae, Euglenaceae
CHRYSOPHYTA (diatoms)
Synuraceae, Coscinodiscaceae, Tabellariaceae, Diatomaceae,
Fragilariaceae, Naviculaceae, Surirellaceae
73
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CHLOROPHYTA
Chlamydomonadaceae, Volvocaceae, Tetrasporaceae, Oocystaceae,
Scenedesmaceae, Ulotricaceae, Trentepohliaceae, Cladophoraceae/
Zygnemataceae, De smidiaceae.
Due to the difficulties involved in, and the time consumed by, the proper
taxonomic treatment of an algal organism, there are many cultures which
have not been identified as to genus or species with certainty. Quite
naturally those organisms which have been employed in assays were given
priority and were more certainly placed taxonomically than were those
which have not yet been employed experimentally. Unialgal cultures
which represent unique organisms were routinely added to the culture col-
lection. The intent is the inclusion of as many members as possible of
the planktonic (a few of the cultured organisms are from littoral mats)
community of this single pond so that they are available in culture for
future experimentation.
For the same reason a Eubosmina and a CerLodaphnia from Linsley are main-
tained in culture. These two, along with a Keratella and a Cyclops, are
the major zooplankters of the pond. Although in vitro experiments can
never be assumed to provide information which related directly to an in
situ event, the possibility that valid information may be obtained is
increased when organisms from the same location are employed. This is
particularly significant when strains, or "variants", of algae are in-
volved since algal strains differ not only in nutritional and environ-
mental requirements but also in the array and quantity of metabolites
produced.
Dr. Stjepko Golubic and Dr. Francis Drouet provided guidance with the
taxonomic identification of blue-green algae of significance to this
study and Dr. Ruth Patrick provided similar aid with the diatoms of sig-
nificance. Additional taxonomic information relative to the blue-green
(Myxophyta) algae employed in this study may be found in Keating (1975,
Appendix C).
Sterility Testing—The determination of bacteria-free status was done
both visually via light microscopy (aided by dark field, phase contrast,
and, or, oil immersion techniques), and biologically via inoculation of
small aliquots of cultures to be tested into semi-solid test media con-
sisting of 0.25% Qxoid lonagar #2 in D.A. or S.T.P. media (see Appendix
B for nutritional complement of media).
Filtrate Production
A series of blue-green algae have been employed as producers of filtrates.
Those of Linsley origin have all produced bloom populations during the
last three years. The two non-Linsley blue-greens employed were: 1) Nos-
toc muscorum from the Indiana Collection via Dr. Luigi Provasoli's culture
collection; and 2) Nostic sp_. via Edward Bonneau, from the Connecticut
74
-------
Riyer cultures, established and maintained by Peter Bonanomi,
Filtrate Producing Organisms;
Oscillatoria rubescens de Candolle (winter strain) (O_. agardhii)
C535) *
filamentous, single trichomes, planktonic, non-heterocystous,
green-brown, blooms in fall thru winter, bacterized, very obvious
phototrophic response.
Oscilla-toria rubescens de Candolle (summer strain) ((X agardhii)
(739, 746)
filamentous, single trichomes, planktonic, non-heterocystous,
orange-green-brown, blooms in spring thru midsummer, bacterized,
no obvious phototrophic response.
Pseudanabaena galeata Bocher (597)
filamentous, single trichomes, planktonic, non-heterocystous,
vivid blue-green, blooms in early fall thru early winter, axenic
(this may be the organism originally designated as the type
organism for Oscillatoria rileyi)
Oscillatoria sp. (776)
filamentous, single trichomes, planktonic, non-heterocystous,
green-brown, blooms in mid winter, axenic.
Anabaena sp. (538)
filamentous, single trichomes, planktonic, with heterocysts,
green, blooms in late spring, axenic.
Anabaena sp. (762) or Aphanizomenon elenkinii Kisel (762)
filamentous, forms flakes, planktonic, with heterocysts, green,
blooms in late summer (minimal bloom in 1972) , bacterized.
Synechecoccus sp. (91)
coccoid, single cells, planktonic, green-blue-green, blooms in
spring, axeni c.
Nostoe muscorum
non-Linsley, filamentous, single trichomes, heterocystous, deep-
green, Indiana collection, axenic.
Nostoc sp.
non-Linsley, filamentous, colonial, probably non-planktonic,
heterocystous, green, collected from Connecticut River 1969,
from the collection of Peter Bonanomi, University of Connecticut,
forms globular colonies, axenic.
*Numbers—Linsley Pond culture collection designation.
75
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Thawing of Stored Pond Water
Large samples of pond water were filter-sterilized and stored at -20°C
immediately after collection. These samples were latex thawed and assayed
for biological activity. Individual 2.5 liter polyethylene containers
were placed in supportive dishes and allowed to thaw overnight at room
temperature (16 hours at 22°C), Prior to processing the sample into F
vs A assays, a sterility test was taken to assure that the sample had
remained sterile during storage.
Basis—preliminary work (involving isolation of various algal species)
indicated that population maytmg in unialgal cultures established in pond
water differ, and that this difference depends on the treatment of the
pond water prior to inoculation. Pond water was either 1) filter-steril-
ized; 2) filter-sterilized and autoclaved; 3) charcoal-treated and fil-
ter-sterilized, or 4) charcoal-treated, filter-sterilized, and auto-
claved. Further study indicated that charcoal treatment (described above
under Culture Collection; Maintenance, and originally employed because
it was found to remove most traces of certain organic compounds from pond
water) added materials to the pond water. However, there remained a con-
sistent, obvious, difference between population maxima in filter-steril-
ized, and filter-sterilized and autoclaved, water samples. This sug-
gested the presence in the pond water of heat-labile compounds worthy of
scrutiny, and this possibility initiated the pattern for assay followed
throughout this study.
The Assay— the assay is based on a comparison of the growth of selected
organisms in test tubes containing 10 ml portions of pond water. The
pond water to be tested is either filter-sterilized, or filter-sterilized
and autoclaved (these two treatments are usually simply referred to as
F and A) prior to inoculation with assay organisms. 1.5% ESi enrichment
(no silica addition) is added just prior to inoculation of cultures.
This assay is also employed when the biological activity of producer fil-
trates is studied. The term "producer filtrate" (or "filtrate") refers
to that water in which a culture of an alga has been grown and from which
the "producer" algae have been removed by passage thru a 0.45n (H.A.)
Millipore filter. These large producer cultures are established in B
medium, described above under Maintenance.
In order to allow the pH of autoclaved portions to return to the original
F level, a minimum of one week was allowed to pass between autoclaving
and inoculation. This return was consistent and no pH distinction between
F and A (filtered and autoclaved) tests greater than 0.1 pH units was
noted. During this equilibration period the tests were stored in test
tubes at 0-2°C in darkness.
76
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Assay Organisms—a taxonpmic variety of diatomsf blue^-green algae, green
algaer and flagellated forms were used as assay organisms, A. listing of
assay organisms is included in Appendix C, The selection of assay organ-
isms depended on the specific information sought in a given set of exper-
iments .
Growth maxima were determined by O,D. readings. A densitometer which
accommodates test tubes rather than cuvetts, and which was built by Dr.
Fred Kavanaugh for Haskins Laboratory, was employed for all readings of
growth maxima.* The capacity of this meter to accept test tubes permitted
O.D. readings to be taken without decanting (and contaminating) the grow-
ing cultures and, thereby, permitted constant surveillance of culture
growth over extended periods of time (usually several weeks). Immediately
before O.D. readings were taken the test tube containing the culture to
be measured was thoroughly shaken on a Vortex Genii.
Light and Temperature—cultures and experimental tests were grown under
24 hour light provided by cool white fluorescent bulbs. During tests
light intensity varied between 400 and 600 foot candles. This light
variation was dependent on both the age of an individual light tube and
on the placement of a specific test tube either directly over (illumina-
tion was from below during tests), or one to five cm to either side of,
a light tube. Since it was impractical to eliminate these light varia-
tions, each set of test tubes which was to be used as the basis of a
single comparison and interpretation of F vs A growth was carefully, and
consistently, placed in the incubator to minimize or eliminate this
variation; i.£., each such set of tubes was always placed parallel to, and
equidistant from, the light tube. If light variation was due to the aging
of a light tube, all tests in a set experienced a similar variation.
Glassware—for experiments 10 ml portions of media were employed in
20 x 125 Pyrex test tubes with linerless screw caps. All test tubes were
scrubbed clean in a solution of 7X, rinsed in tap water, and washed in
an automatic dishwasher (using recommended concentrations of 7X for auto-
matic washing) with a 30-minute wash, 30-minute rinse, and 30-second dis-
tilled water rinse. Tubes were then heated for a three-hour cleaning-
cycle in a General Electric oven equipped with P7 (electric oven clean-
ing) . This baking represents over 2.5 hours at a temperature between
900-10000F. The intent of such heat treatment was to assure disruption
of the carbon backbone of all organic compounds which might be present
in test tubes. After baking, tubes were rerinsed in the automatic dish-
washer in distilled water for 30 seconds (to remove any film of ash
remaining after baking, no visible film was noted) and were dried in an
oven.
*Additional information relative to this O.D. meter may be found in
Appendix D.
77
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Filtrate cultures were grown in 10QQ jojl portions of B medium in 2500 ml
Corning. #4422 Pyrex extra xi.de, conical, culture JElasks, Culture flasks
Cand a.11 other incidental glassware used for preparing and distributing
media.) were scrubbed in a. 7X solution, rinsed in tap water, rerinsed in
distilled water, and allowed to air dry» Culture flasks were purchased
new and were employed solely for filtrate production.
VII: II BIOLOGICAL ACTIVITY IN FILTRATES OF BLOOM DOMINANT ORGANISMS AND
IN NATURAL POND WATERS
Heat-Labile Biological Activity in Filtrates of Bloom Dominant Blue-
Green Algae
Filtrates of blue-green algae were tested against a variety of assay
organisms. The results are summarized in Table 5. The blue-green fil-
trates produced a complicated array of positive, negative, and neutral
effects on the assay organisms employed. A generalized negative effect
of blue-green filtrates on diatoms was noted quite early in the study,
and this phenomenon was singled out for study in depth. This study is
summarized below in Sections VII s III and VII: IV.
Because similar results obtained from separate filtrates are more valid
than similar results obtained from replicate tests of the same filtrate,
at least two separate filtrate cultures (from two to twenty-two) were
established and harvested for each of the bloom dominants. Also, repli-
cate tests were employed for each of the separate filtrate studies.
With the exception of one species, Anabaena sp. (765), which was diffi-
cult to maintain in culture, and which was not successfully grown in a
culture suitable for filtrate production, each of the blue-green organ-
isms which were observed to be bloom dominants during the study period
(1971-1974) was employed as a filtrate producer. In any study of a single
filtrate four separate tests (2F and 2A) of any organism employed for
assay were run in parallel. Due to time and space limitations only one
filtrate culture (1000 ml) was produced for Anabaena sp. (762) and for
each of the non-Linsley blue-greens. In these cases 4F and 4A tubes were
included for each organism tested.
The number of filtrates tested for each producer organism is listed in
Table 5 in the lower left hand corner of the PRODUCER box (first column
on left). The name, Linsley culture identification number, and condition
(axenic or bacterized) of each filtrate at harvest are also included in
this PRODUCER box. Because it was not always possible to assay every
sensitive organism when a given filtrate was being studied, most of the
assay categories (four columns on right—Table 5, under the heading ASSAY
ORGANISMS) involved less than the total number of filtrates produced by
the particular producer organism involved. This is especially true of
Anabaena sp. (538) which produced 22 filtrates. In order to convey the
number of filtrates actually used in any given assay category, the number
of filtrates used in the determination of effects for each of the ASSAY
78
-------
TABLE 5. SYMBOLS
Neg., Pos., Neu. = Filtrate had a negative, positive, neutra
effect on assay organisms.
(Neg.), (Pos.), (Neu.) = Filtrate had a very weak or
uncertain negative, positive, neutral effect on assay
organism.
Numbers
- Lower left hand corner of PRODUCER name box = number
of separate filtrates of the organism harvested.
-Upper right hand corner of effects box:
top number = number of filtrates used for
second number = number of different organisms
used for
Organisms are listed by name for each set of
assays,
- Immediately following symbol for effect on assay
organism (i.e., pos., neg., neu.) = the number
of assay organisms giving that result.
79
-------
TABLE 5. Summary: Heat-Labile Biological Effects of Blue-Green
Filtrates.
PRODUCER.
(Axenic)
(Dacterized)
Oscillatoria
rubescens
, (535> Bac.t
Oscillatoria
rube seen s
2 <739> Bad,
Anabaena sp.
(538)
22 Ax.
Pseud-
anabaena
galeata
2 <597> Ax.
Oscillatoria
sp.
3 <776> Ax.
Syneche-
coccus sp.
(91) A*
2 AX.
Aphani-
zomenon
flos-aquae
7~(T66) Bact.
Anabaena sp.
(762)
No s toe
muscorum
(Indiana).
1 Ax-
Nostoc sp.
(Bonanomi)
1 Ax.
ASSAY ORGANISMS
DIATOMS
Neg. 5 I
Neu. 3
Neg. 6 *
Neg. 29 "
2»
Neg. 7 e
(Neu.)l
(Neg.) 5 £
(Neu.)l
2
Neg. 9 ,
2
Neg. 4 7
Neu. 3
Neg. 2 *
(Pos.)l 3
Neg. 4 *
Neu. 4
Neg. 3 \
Neu. 5
BLUE-GREENS
>
Pos. 5
(Neu.) 3
Neg. 3 ^
Pos. 3
Neu. 2
Neg. .3 s
Pos. 3 *
Neu. 2
X
Neg. 4 ,
(Pos.) 1
Neu. 3
1
Neg. 6 ,
(Neu.)- 2
2
Neg. 4 ,
Pos. 4
1
Neg. 2 a
Pos. 3
Neu. 3
Neg. 5 l
Pos. 3 •
(Neg.) 1 1
Pos. 4
Neu. 3
omit
GREEN ALGAE
(Neg.) 1 J
Pos. 2
(Neg.) 1 1
(Pos.) 2
Neg. 2 2
Pos. 3 1S
Neu. 8
t
(NegJ 2 ,
Pos. 7
Neg. 5 s
(Neu.)l
2
Neg. 2 7
Pos. 3
Neu. 2
1
G
Pos. 3
(Neu.)3
Omit
(Neg.)l 3
(Neu.)2
omit
FLAGELLATES
(NegJi ,o
Pos. .6
Neu. 3
(Neg)l ,1
Pos.'S
Neu. 6
.omit
1
Neg.. 2 ,,
Neu. 2
l
Neg. 2 s
(Pos.)l
(Neu.) 3
1
Neg. 2 5
Pos. 3
1
s
(Pos.)l
Neu. 4
Omit
(Neg.)l *
(Neu.) 2
omit.
80
-------
ORGANI.SMS categories (diatoms, blue--greens f green algae, flagellates) is
included as the top number of the right in each effects box (under ASSAY
ORGANISMS) in Table 5. The second number on the right in thi.s same box
is the number of different assay organisms used for each category, (See
Appendix C for a listing of organisms tested, and specific effects on each
organism). The number following each of the symbols for negative, posi-
tive, or neutral (neg., pos.f neu.) is the number of organisms responding
in that manner to the specific filtrate.
Table 6/ Effects of Blue-Green Algal Filtrates on Blue-Green Algae, is
provided to permit a more detailed analysis of the interactions among the
bloom dominants. The data for one of the non-Linsley blue-greens is
included for comparison.
TABLE 6. SYMBOLS
- The effect of producer filtrate on assay organism
was negative.
+ The effect of producer filtrate on assay organism
was positive.
0 The effect of producer filtrate on assay organism
was neutral .
(+) The effect was weak or uncertain.
(0)
81
-------
PRODUCER
CD
to
ASSAY ORGANISM
Oscillatoria
rubescens
(535) winter
Oscillatoria
rubescens
(739) summer
Anabaena
sp.
(538)
Pseudanabaena
galeata
(597)
Oscillatoria
sp.
(776)
Synechecoccus
sp.
(91)
Aphanizomenon
f los -aquae
(766)
Anabaena
sp.
(765)
vj
ffl
to
(762
1 Anabaena sp. _J
-
-
*
-
-
*
+
-
I (non-Linsley) £
I muscorum £
!
s
n
-L)
*
0
+
+
-
-
0
(0)
^
a»
(
| f los-aquae J»|
+
1 Aphanisomenon^
*
+
0
+
0
-
0
H •
(PI
) Synechecoccus j
-
-
-
-
+
*
+
•^J *Q
*j •
a*
-
<-,
-
0
-
-
(0)
u?
-o
(S!
| galeata £;
0
1 Pseudanabaena _
0
-
M
(0)
+
-
-
w •
00
(538'
*
Anabaena 1
-
0
0
-
+
+
-
•si
u
U)
^^
C7.
I rubescens SJ
1 Oscillatoria
0
-
-
+
0
(-,
+
-
^^ M Irt
un 3 ifl
U) 3* p
(J\ (D r1'
X^ (/) >^
1 — '
ft» 01
3 ft
tn o
n
H-
0)
(535)
(0)
(0)
*
*
-
0
+
-
TABLE 6. Effects of Blue-Green Algal Filtrates on
Blue-Green Algae.
-------
Heat-Labile Biological Activity of Freshly-Collected and Freezer-Stored
Linsley Pond Waters
Freshly-collected and freezer-stored pond waters were assayed for biologi-
cal activity. Results of these assays are summarized in Table 7. The
number of assay organisms used in each of the assay categories (diatoms,
blue-greens, green algae, flagellates) is included in each of the effects
boxes (beneath the heading ASSAY ORGANISMS). The number following each
of the symbols for negative, positive, or neutral is the number of
organisms responding in that manner to the specific pond water sample.
(See Appendix C for a listing of assay organisms used for both filtrate
and pond water tests.)
Two assays were conducted using pond water collected on March 18, 1973.
One of these assays was done with freshly-collected pond water and one
with freezer-stored (and thawed) pond water. The intent of this duplica-
tion was to determine whether or not the freezing of pond water samples
would significantly alter the heat-labile biological activity of the pond
water (as demonstrated by diatom assays). In only one case, Asterionella
formosa (800), was a different assay result indicated (Table 8). When
freshly-collected pond water was assayed, A_. formosa (800) grew slightly
better in F than in A pond water samples; however, when freezer-stored
pond water was assayed, A. formosa (800) grew considerably better in A
than in F samples. This suggests a significant change in the heat-labile
inhibitory capacity of this March 18, 1973 pond water during the freeze-
store-thaw procedure. But closer examination of these results indicates
that a different interpretation may be necessary because A. formosa (800)
achieved similar growth maxima in the F assays of both portions (fresh
and frozen) of the March 18, 1973 Linsley Pond water. There was, however,
a significant difference in the growth maxima in the two A assays. Auto-
claved samples of the freshly-collected pond water supported growth maxima
similar to those of both types of filtered-only samples, however, auto-
claved samples of the freezer-stored pond water supported more than twice
that level.
Five other diatoms and one blue-green were also used in assays of these
two portions of the March 18 water. All five of these diatoms produced
a negative result in both freshly-collected and freezer-stored samples.
One other of these five diatoms exhibited a change in autoclaved samples
which was similar to the change in autoclaved samples of A_. formosa;
i.e. , in each case the autoclaved sample of freezer-stored pond water
supported higher growth than did the autoclaved sample of freshly-collected
water. This result suggests that during the freeze-store-thaw procedure
a significant change occurred in some unexplored heat-stable inhibitor.
That is, the heat-stable inhibitor expresses inhibition in freshly-
collected samples, but loses some, or all, of its inhibitory potential
during the freeze-store-thaw procedure. This interpretation requires the
assumption that the heat-stable inhibitor is masked by the presence of
the more potent heat-labile inhibitor in F assays of freshly-collected
water. Other interpretations are possible.
83
-------
TABLE 7. SYMBOLS
Neg., Pos., Neu. = Pond water had a negative, positive,
neutral effect on assay organisms.
(Neg.)» (Pos.), (Neu.) = Pond water had a very weak or
uncertain (or only one organism wa-s so effected) effect
on assay organism.
Numbers in upper right hand corner of effects (assay
organism) box = number of organisms tested.
Numbers immediately following symbol for effect on assay
organism (i.e., pos., neg., neu.) = the number of
assay organisms giving that result.
84
-------
TADLE 7. Summary: Hc-at-Lrtbi le Biological Effects of
Freshly-Collected, and Freezer-Stored Pond Waters.
DATE OF
COLLECTION
(fresh)
( thaw )
8/1/72
8/8/72
(fr)
9/5/72
(fr)
9/19/72
(th)
11/17/72
(th)
1/7/73
(fr)
3/18/73
(fr)
3/18/73
(th).
5/13/73
(fr)
6/2/73
(fr)
ASSAY ORGANISMS
DIATOMS
1
(Neg.) l
omit
7
Neg. 7
9
Neg. 8
(Neu.) 1
3
Neg. 3
18
Neg. 17
(Pos.) 1
9
Neg. 9
8
Neg. 8
9
Neg. 9
BLUE-GREENS
2
(Neg.) 1
(Pos.) 1
4
Neg. 4
8
Neg. 3
Pos. 2
Neu. 3
8
Neg. 4
( Pos .) 1
Neu. 3
9
Neg. 3
Pos. 6
1
(Pos.) 1
8
Neg . 7
(Pos.) 1
omit
7
Neg. 2
Pos. 3
Neu. 2
GREEN ALGAE
6
Neg. 4
Neu. 2
omit
9
Neg. 3
Pos. 3
Neu. 3
10
Neg. 4
(Pos.) 1
Neu. 5
6
(Neg.) 1
Pos. 3
Neu. 2
omit
9
Neg. 5
(Pos.) 1
Neu. 3
omit
2
(Pos.) !
(Neu.) !
FLAGELLATES
1
(Neg.) 1
omit
7
Neg. 2
Pos. 3
Neu. 2
8
Neg. 3
Neu. 5
1
(Pos.) 1
orait
8
Neg. 4
(Pos.) 1
Neu. 3
omit
omit
85
-------
TABLE 7- (continued)
DATE OF
COLLECTION
(fresh)
( thaw )
7/17/73
(th)
8/13/73
(fr)
2/14/74
(fr)
6/13/74
(fr)
ASSAY ORGANISMS
DIATOMS
7
Neg. 7
7
Neg. 7
8
Neg. 8
13
Neg. 13
BLUE-GREENS
8
Neg. 3 *
Pos. 4
(Neu.) 1
5
Neg. 4
Neu. 1
8
Neg. 6
(Pos.) 1
(Neu.) 1
8
Neg. 5
(Pos.) 2
(Neu.) 1
GREEN ALGAE
6
(Neg.) 1
Neu. 5
2
(Neg.) 1
(Neu.) !
8
Neg. 2
Neu. 6
9
Neg. 6
Pos. 2
(Neu.) 1
FLAGELLATES
9
Neg. 4
(Pos.)l
Neu. 4
omit
4
Neg. 2
Neu. 2
10
Neg. 8
(Pos.)l
(Neu.)l
86
-------
TABLE 8. Maximum Growth as Expressed by O. D. for
the Organisms Employed in F vs A Assays of Both
the Freshly-Collected and the Freezer-Stored
Portions of the Linsley Pond Water Sample
Collected on March 18, 1973.
ORGANISM
FRESHLY-COLLECTED
FREEZER-STORED
Asterionella formosa
(800)
Surirellc sp. (352)
Synedra sp. (299)
Fragilaria sp. (99)
Cyclotella sp. (211)
Tabellaria sp. (764)
Synechecoccus sp.
(91)
F A
65 55
120 240
60 110
60 95
80 110
45 155
380 220
Result
w
F A
75 145
100 200
65 140
55 75
135 270
50 100
360 230
Result
(+)
87
-------
This is only one of, the many indications of inhibitory and, ox, stimula-
tory metabolite effects which. a.re not heat-labilef and therefore, which
haye not been considered in this study. These numerous additional meta-
bolite effects offer many challenging problems for future study.
VII:III PRELIMINARY CHARACTERIZATION OF THE HEAT-LABILE SUBSTANCE
RESPONSIBLE FOR INHIBITION OF DIATOM GROWTH: METHODS
Methods described below were employed for individual experiments in con-
junction with the activity bioassay described above. These experiments
were designed to provide additional information relative to the inhibition
of diatom growth potential by a metabolite of blue-green algae. Anabaena
sp. (538) was employed as the producer organism due to its consistent
growth characteristics and to its reliable production of filtrates.
Although a variety of diatoms were used as assay organisms Nitschia
frustulum v. indica (Skvortzow) (224) was employed as the preliminary
assay organism because of its relatively small size (good dispersal for
optical density readings), consistently high population densities, and
freen single cell, growth habit (in contrast to cells in a gelatinous
matrix with, or without, attachment to glass vessel walls).
Heat Lability
In order to further examine the characteristics of the heat-labile diatom
inhibitory capacity apparent in both Linsley Pond waters and producer
filtrates a series of tests were pre-heated to 60°, 90°, and 121«>C (auto-
clave) . Tests were heated to designated temperatures and maintained at
such for 20 minutes. The autoclaved tests were also subjected to the
usual 15 Ibs. pressure of the autoclave. Controls were non-heated,
filter-sterilized water samples from the same source as the test cultures.
To provide additional insight into the effects of the temperature varia-
tions a dilution series was established for each of the target tempera-
tures. As with other dilution series, tested test samples were mixed to
designated dilution percentages with autoclaved portions of the same
water as that of the test samples. Dilutions were 100%F, 66%F/ 33%F, 0%F
(0%F representing 100%A).
Volatility
To determine whether the heat-labile inhibition of diatom growth by fil-
trates of blue-green algae is the result of a volatile substance, N2 at
20 psi was bubbled—via a Supreme "fine pore" aquarium air stone—thru
a sample of filtrate for a period of 20 minutes. This sample was then
prepared as a set of F vs A assays. For comparison and control a sample
of the same filtrate (with no N2 bubbling) was prepared as a set of the
usual F vs A tests.
-------
Dialysis
To determine whether -the heat-labile inhibition of diatom growth by the
filtrates of blue-green cultures is the result of a dialyzable molecule,
a. 1000 ml filtrate was dialyzed against sucrose; i_,e_, t the 1000 ml fil-
trate was confined within the dialysis tubing and the tubing was buried
under 5 Ibs. of ordinary granulated Jack Frost refined sugar.
Approximately 100 ml of liquid remained inside the tubing after 24 hours.
This was considered the "non-dialyzable" portion. The "dialyzable" por-
tion of this filtrate was considered to be contained in that deep yellow-
brown area forming a halo in the bed of wet sugar surround the tubing.
This grainy yellow-brown portion of sugar was scooped up and added to a
graduated cylinder. Its volume was then increased to approximately 100
ml by the addition of distilled water. Sugar granules were totally
dissolved. This 100 ml dialyzable portion and 100 ml non-dialyzable
portion were then separately filter sterilized by passage thru 0.45u
(H.A.) Millipore filters into sterile containers.
The amount of sugar in neither of these portions could be accurately
determined with the equipment available; therefore, a comparison control
was employed by the use of dilution series with the dialyzable and non-
dialyzable portions serving as additions. An identical set of dilution
series tubes was autoclaved and served as additional comparison and con-
trol. Dilutions were by additions of 0, 0.1, 0.5, 1.0, 2.5, and 5.0 ml
of the dialyzable (or non-dialyzable) material to 10 ml aliquots of B
medium. The final, most concentrated, test was set at 50% dilution by
combining 5 ml of test solution with 5 ml of B medium.
There were thus four dilution series established:
-non-dialyzable F -dialyzable F
-non-dialyzable A -dialyzable A
Ultrafiltration
To determine the size of molecules involved in the inhibition of diatom
growth by blue-green filtrates a series of ultra-filters were used to
separate by size the metabolites contained in such filtrates.
Amicon ultrafiltrates were employed in an Amicon Model 52 stirred cell
ultrafiltration unit. Filters and pore sizes (based on retention of pro-
tein molecules) were as follows:
Particles Excluded
PM10
UM2
UM05
Size
20 A°
10 AO
MW
10,000
1,000
500
89
-------
Dilution series were established tp determine the effects of each molecu-
lar size group* Dilutions wer.e of F samples with A; 1QQ%F, 66%Ff 33%F,
and Q%F (Q*F representing a. test with 10Q%A.l, The "A" used for dilution
series wa.s simply a portion of autoclayed filtrate; i_^e_,, it was not
autoclaved filtrate which ha.d been passed thru the ultrafilters. To
provide additional comparison and control a portion of the same filtrate
was processed thru the usual F vs A assay.
Ether Extraction of Lipids
To determine if the substance responsible for the heat-labile inhibition
of diatom growth by blue-green filtrates could be extracted by ether, a
300 ml portion of filtrate was extracted with 600 ml of Mallinckrodt
Ether for Fatty Acid Extraction by shaking in a 4 liter separatory funnel
for 45 minutes. The sample was then allowed to settle for 30 minutes
after which the ether fraction was placed in a graduated cylinder and
evaporated with- N2 to approximately 40 ml. The water fraction of this
primary extraction was extracted a second time with 300 ml of ether in a
2 liter separatory funnel. This extraction was allowed to settle for
16 hours, after which this ether fraction was combined with the first
ether fraction (40 ml from first extraction) and both were reduced via
N2 evaporation to 6 ml of concentrated extract. Presumably this was a
50x concentration of the ether soluble lipids from the original blue-
green filtrate.
To test the biological activity of the ether fraction; i_.e_., lipid frac-
tion, 200% and 500% concentrations of the original filtrate level (prior
to extraction) were established by adding 0.4 ml and 1.0 ml, respectively,
to 1O ml aliquots of B medium. Identifical F and A tests were established
in this manner.
Additional controls for the multiple variables in these tests were estab-
lished as follows:
(A) To verify the existence of the inhibitory activity in the
original filtrate, a portion of the filtrate that provided the
original 300 ml for the ether extraction was assayed via the
usual F vs A assay.
(B) 0.4 ml and 1.0 ml of ether extract were necessary, respect-
ively, for the 200% and 500% concentration tests; therefore,
0.4 ml and 1.0 ml of ether alone were added to B medium to ascer-
tain the effects, if any, of ether-only additions to B medium.
Identical F and A tests were established.
(C) 0,4 and 1.0 ml of ether were added to portions of the original
filtrate to determine the effects of ether on the original inhi-
bitory activity. This was to explore the possibility that the
simple addition of ether might destroy the inhibitor, thus negating
all results of the experiment. Identical F and A tests were estab-
lished.
90
-------
(D) To determine if any, mostf or all, of the inhibitory activity
was removed by the extractionf 1Q ml portions of the water frac-
tion were placed directly into test tubes. Identical F and A
tests were established.
(E) B medium with no additions (excepting the usual ESj addition)
was employed in an F vs A assay to determine the effects of reauto-
claving B medium,
Addition of ESj Metals and of EDTA
To determine if distinctions evidenced in F vs A assays were the result
of some unknown effect on the heirarchy of chelation in filtrates, the
trace metals included in ESi (Fe, Mn, Zn, Co) were added to both F and
A tests. Metals were added as chlorides at 1, 5, 10, 50, and 10 mg%
(by cation weight). The metals in ESj were chosen for two reasons:
first, because they represent metals commonly found to be of significance
in the culture of algae; and second, because the addition of ESj to tests
represented a possible causitive factor in the F vs A distinction; i_-e_.,
if the chelation capacity of the filtrate was altered during autoclaving,
the addition of equal amounts of ESj to F and A tests may not actually
represent the addition of equal amounts of available ESj metals to F as
to A tests.
Similarly, to determine the chelation effects of EDTA, Na2EDTA was added
to 100%, 200%, 300%, and 400% of the ESi level (calculated at an ESj addi-
tion rate of 1.5%); i_-e^. , Na2EDTA was added (by weight of molecule) at
9.3, 18.6, 27.9, and 37.2 %, to both F and A tests.
Addition of Vitamins and of Selecj:ej3 Organic Nutrient Sources
To determine whether the capacity of blue-green filtrates to inhibit
diatom growth involves the inactivation (binding) of vitamins, a multi-
vitamin (Vitamins, 8A; see Appendix B for complete listing) mix was added
to both F and A tests of one filtrate. The vitamin mix was added at 0.2,
0.4, 0.8, 1.6, and 2.0 ml%. This represents vitamin levels in excess of
known requirements, or in excess of commonly employed concentrations
(0.1 ml%)—when requirements are unknown.
Because an exhaustive survey of the possible organic nutrient sources
which might somehow effect the capacity of blue-green filtrates to inhibit
diatom growth was impractical, only liver oxoid, yeast extract, and dex-
trin were considered. The addition levels tested for these three sub-
stances were 0.1, 0.5, 1.0, 5.0, and 10.0 rog%. Sterile additions were
made to both F and A tests. Comparison and control was established by
the usual F vs A assay of the same filtrate.
Although these three compounds are generally considered to contain a
variety of organic molecules which might reasonably be used as nutrient
sources by auxotrophic, or heterotrophic, algae, these tests in no way
91
-------
exhaust the possible nutrient sources which might be involved in the
inhibition. The testing of these complex organics might, howeyer., sug-
gest a limit to. the probability that such nutrients are involved.
Non-Linsley Organisms
In order to determine whether the inhibition of diatom growth by heat-
labile metabolites of blue-green algae is a phenomenon peculiar to
Linsley Pond, or whether it is of more general occurrence in fresh water,
non-Linsley algae were selectively employed.
Filtrate Production—two non-Linsley blue-green algae were employed as
filtrate producers: Nostoc muscoruin, from the Indiana collection and
Nostoc sp,, from E. Bonanomi's Connecticut River collection. These were
grown in the same incubator with the same light, temperature, and media
as were Linsley producers; and filtrates of these non-Linsley producers
were employed in standard P vs A assays of heat-labile inhibition.
Assay Organisms-—non-Linsley diatoms were employed to determine if the
inhibition of diatom growth by natural pond waters, and by filtrates of
blue-green cultures, was restricted to diatoms from Linsley Pond.
Difficulty with Flagellates—because, as a group, the flagellated forms
collected from Linsley are bacterized, axenic non-Linsley flagellates
were employed in tests of the sensitivity of flagellates. These tests
did not replace tests of Linsley flagellates, but rather, both sets of
flagellates were employed in parallel tests.
Effects of Addition of Bacteria to Axenic Producer Cultures
To more accurately determine the effects of bacteria on the heat-labile
inhibition of.diatom growth by the filtrates of blue-green algae, a
1000 ml producer culture of axenic Anabaena sp. (538) was infected with
a 10 ml portion of the bacterial community of the Oscillatoria rubescens
(535) culture. O. rubescens (535) was separated from its bacterial com-
munity by passing 10 ml of the culture thru a l.Op Millipore filter. The
strength of the inhibitory capacity of the resulting filtrate was com-
pared with that of a filtrate of the same Anabaena sp. (538) grown
axenically.
Results of this comparison permit consideration of the effects of bacteria
in four distinct situations:
-bacteria in producer culture, but not in assay culture;
-bacteria in both producer and assay cultures;
-bacteria neither in producer culture, nor in assay culture;
-bacteria not in producer culture, but in assay culture.
92
-------
This array of comparisons is useful in the interpretation of bacterial
effects on inhibitory capacity of filtrates, in general; and in the inter-
pretation of assays of filtrate activity for those producer organisms
which, were never successfully separated from bacteria, in particular,
Effects of Age of Producer Culture on Inhibitor Strength.
To determine if the heat-labile inhibitory substance is released through-
out the life of a culture or released mainly at lysis, one half of a fil-
trate was harvested after one week's growth, the second half was har-
vested after 15 days growth (lysis). These two halves were then subjected
to the F vs A assay and results were compared for differences in inhibi-
tory capacity which would correlate with culture age at harvest.
Effect of Lower Mn Levels in Producer Cultures
To determine if the Mn level in producer cultures was in excess of the op-
timum level for blue-greens, ESj was formulated with all Mn omitted from
the nutrient complement. Thus only the Mn which occurred naturally in
the Linsley water (which was the basic liquid for the B medium in which
producer cultures were grown) was present in these producer cultures.
The inhibitory capacity of this filtrate (as demonstrated by the usual
F vs A assays) was then compared to that of a producer culture (grown in
parallel) which had contained the usual ESj nutrient addition.
VII:IV PRELIMINARY CHARACTER!ZATION OF HEAT-LABILE SUBSTANCE RESPONSIBLE
FOR INHIBITION OF DIATOM GROWTH: RESULTS AND DISCUSSION
Introduction
The consistent, heat-labile, inhibition of diatom growth by filtrates of
blue-green algae and by Linsley Pond waters was considered of sufficient
significance to warrant a detailed study. Chemical, and, or, physical
characteristics of the heat-labile molecule postulated to be the basis
for tlftls negative activity were sought.
All experiments aimed at the preliminary characterization of the heat-
labile inhibitor were done with filtrates of Anabaena sp. (538) . If only
a single assay organism was to be used, Nitschia frustulum (224) was
chosen. The limit to only one, or a few, assay organisms was essential
in these more complex experiments. The multiple controls; e_.c£., in the
ether extraction experiment, would have required prohibitive numbers of
replicate tests if a large number of assay organisms had been employed.
93
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Heat-Lability
The heat-labile characteristic of the inhibitor was the first to be
studied. It was originally found that the inhibitor was eliminated by
autoclaving (15 psi, 120°C) for 20 -minutes. Further study indicated that
the inhibitor was partially eliminated by 20 minutes at 90°C (atmospheric
pressure!, but was not measurably diminished by 60°C for 20 minutes. If
D.C, medium (Provasoli, McLaughlin, Droop, 1957) was added prior to auto-
claving, the inhibitory substance was spared. In preliminary tests of
the effects of the inhibitor on marine diatoms the inhibition occurred
in both F and A tests if the D.C. was added to the filtrate prior to
autoclavingj however, if D.C. was added to the filtrate after autoclaving,
then the inhibition in A tests was eliminated. Although studies have not
been conducted to determine which of the constituents of the D.C. medium
was responsible for the sparing of the inhibitor during autoclaving,
other work Cconceming the vitamin 83.2 found in Linsley waters) has indi-
cated that Tris buffer alone is sufficient to produce the sparing effect
of D.C. for other organic molecules. This suggests that the rise in pH,
as dissolved CO2 is driven out of the water, rather than the high tempera-
ture, destroys the inhibitor.
Volatility
The inhibitor is non-volatile. -Loosely capped filtrates can be stored
for over one month (providing they are kept in darkness at 0-5°C and are
axenic) without substantial loss of activity. To further explore the
possibility that the inhibitor was a volatile substance N2 was vigorously
bubbled (5 psi) thru an uncapped, freshly-harvested filtrate (150 ml) via
a Supreme "very fine" aquarium air stone for 20 minutes. No measurable
loss of activity occurred.
Dialysis
The inhibitor is a dialyzable molecule. Dialysis against sucrose was suf-
ficient to isolate the heat-labile inhibitor from the filtrate. This
inhibitor can be returned to test tube cultures and will produce F vs A
assays similar to those of untreated filtrate. Pore size in the dialysis
tubing employed for this work is 50ft, the tubing retains molecules with
a mw of 12,000 or more. These results place an upper limit on the size
of the inhibitory molecule at 50R and an upper limit for its mw at 12,000.
The high concentration of sugar in these experiments somewhat limited the
growth in all tests, both in the dialyzable and non-dialyzable F and A
tests.
Separate portions of the dialyzable and non-dialyzable segments of the
filtrate were stored for a second assay. One half of each portion was
frozen, the other half was stored at 0-5°C. An F vs A assay was run on
these separately stored portions approximately six months later. It was
found that freezing destroyed the inhibitor while cold storage preserved
it. Williasm (personal communication, 1972) has suggested that freezing
94
-------
might initially cause a minor disruption of the structure of the organic
molecule (or molecules) responsible for biologica.1 activity, but that
this form of disruption could be corrected by allowing the frozen sample
to rest undisturbed for an unspecified (but relatively long) period. He
further speculated that the high concentration of sugar in such samples
might mechanically interfere with the recovery of structural integrity
of organic molecules in frozen samples.
The growth maxima (expressed as O.D.) of each of the conditions discussed
above are provided in Table 9 for illustration and comparison.
Ultrafiltration
Ultrafiltration provided more detailed information concerning the size
and character of the inhibitor. Ultrafiltration separated the inhibitor
into three distinct molecules:
(1) An heat-labile inhibitor which is retained by the Amicon PM10
Ultrafilter, with a pore size of 20R and a nominal retention weight
based on 90% retention of protein molecules of 10,000.
(2) An heat-labile enhancer which passes thru the 20fi Amicon Ultra-
filter (PM10) and which is retained by the lOfi Amicon Ultrafilter
(UM2). The nominal retention weight of this filter (based on pro-
teins) is a mw of approximately 1,000. The influence of this
enhancer is effectively masked by the inhibitor described above (1).
(3) An heat-labile inhibitor which passes thru both of the previously
mentioned ultrafilters and which also passes thru the UM05 ultra-
filter with a mw limit of 500 and a pore size of 10ft.
Summary—(1) Heat-labile inhibitor; mw between 1,000 and 10,000; size:
20-50fl. Peach-pink color, oily to touch.
(2) Heat-labile enhancer; mw between 1,000 and 10,000; size:
10-20ffl. Vivid mid-yellow color, adheres to filter (suggesting
a possible steroid since the UM2 membrane filter is known to
bind steroids).
(3) Heat-labile inhibitor; mw between 500 and 0; size: 0-108.
The determination of an inhibiting or enhancing quality of these molecules
was accomplished by sets of dilution series tests. These indicated the
presence of an enhancer by diminished growth with greater dilution, and
the presence of an inhibitor by increased growth with greater dilution
(Table 10). It should be mentioned that the larger inhibitor (1) is
similar in color (peach-pink) and tactile effect (oily) to the inhibitory
substance extracted by ether, below. The smaller of the inhibitors (3)
has not been characterized to my satisfaction, and may yet prove to be an
artifact of the assay system; i..£., since assays were done with diatoms,
95
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TABLE 9 Comparison of Dialyzable and Non-Dialyzablc
Portions of a Producer Filtrate Before and After
Storage. (Growth Maxima expressed as O.D.)
Description of Sample
Dialyzable r no storage
Non-Dialyrable, no storage
Dialyzable, stored at 5°C
(6 BOS.)
Non-Dialyrable, stored at 5°C
(6 BOS.)
Dialyzable* stored frozen
(6 mO«.)
Non-Dialyrable, stored frozen
f
155
48O
155
375
410
450
A
450
390
400
325
395
435
1.0 ml sample (as described in text) added to
10 ml test tube portions of B
96
-------
TABLE 10. Results of Dilution Series Tests of Activity
of Filtrates after Passage thru Ultrafliters
Percentage Dilution
0%
33*
66%
100\
(No filtration)
180
170
190
380
(Thru PM10 u.f.)
275
410
385
380
(Thru UM2 and UM05)
200
215
245
380
Heat-Labile
Molecular
Species Present
-1 +2 -3
+2 -3
-3
Interpretation
The combined strength of
-1 and -3 even when diluted.
cannot be overcome by +2
Now that -1 is removed, -3
can be diluted sufficiently
so that +2 (33%) can express
itself. The 66% dilution is
to great for +2 to withstand.
Diluting -3 allows increased
growth. -3 is sufficiently
strong to still be evident
at the 33% level.
97
-------
a small, but: additional portion of silica (leached from test tubes during
autoclaving, nay be present in A tests.
Ether Extraction
The inhibitor can be extracted from the filtrate with ether. This sub-
stance is peach-pink color and is oily to the touch. An ether extract,
concentrated and added to B medium at approximately 200% of the original
level (in freshly harvested 16-day-old filtrates), though not immediately
lethal, prevents cell growth and division. A 500% concentration similarly
employed causes bleaching and disintegration of the assay inoculum (Table
11). There is an indication that some of the heat-labile inhibition
remains in the water fraction after extraction.
Other Nutrient Additions
No pattern of effects was observed as a result of the additions of FeCl,
MnCl, ZnCl, CoCl, Vitamins 8A (see Appendix B for listing), liver oxoid,
yeast extract:, or dextrin. Results with Na2EDTA additions require addi-
tional study for interpretation. Higher levels appear to improve growth
in both F and A tests, but there is no pattern presently clear as to an
effect which would eliminate inhibition.
Possible Generalization of Inhibition of Diatoms by Heat-Labile Metabo-
lites of Blue-Green Algae
As can be seen from results summarized in Table 5, filtrates of the two
non-Linsley blue-greens tested evidenced inhibitory, or neutral, effects
on the growth of Linsley diatoms. While the limit of testing to only
two non-Linsley blue-greens makes generalization tenuous at best, there
are two points which must be considered: first, these tests suggest a
likely generalization of diatom inhibition by blue-greens (tests of non-
Linsley diatoms also suggest this); and second, though the distribution
of negative and neutral results in Table 5 may not make it self-evident,
close observations of these tests indicate a much more obvious inhibition
when producer and assay organisms are from the same body of water. This
obvious, and common, form of inhibition might not have been recognized if
original test organisms had been from various collections (therefore,
from various bodies of water). This point gives great significance to
the choice of experimental organisms. When ecologically significant
principles are sought, it is most desirable that experimental materials
including, especially, organisms be from the same ecosystem. Morphologi-
cally (taxonomically) identical organisms may be very dissimilar in their
physiological requirements.
Non-Linsley assay diatoms included LP and Hillsdale, pinnate diatoms,
from central New York State (L. Provasoli isolates) ; a Fragilaria croten-
ensis from Fuller Pond in northwestern Connecticut (K. Glaus-Porter iso-
late) ; a mixed Fragilariaceae culture and a Tabularia sp. from the North
Brunswick Reservoir, Connecticut (J. Lehman isolates); Navicula pellicu-
98
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TABLE 11 Comparison of Control and Test Culture
Maxima in Ether Extraction Procedures
Description of Test
Untreated filtrate
Untreated filtrate with
0.4 ml ether added
Filtrate after ether
extraction
B Medium*
B Medium with
1.0 ml ether added
B Medium with
0.4 ml ether added
B Medium with
l.O ml ether and
500% concentration of
Inhibitor
B Medium with
0.4 ml ether and
200V concentration of
Inhibitor
F
190
220
275
455
410
420
0
O
A
300
285
350
470
520
470
5,40
510
•Comparisons of growth in test cultures must be made with growth in
the B Medium control. Tests were run in B Medium with extract, or
other, additions. B Mediusi has a higher concentration of nutrients
than does the filtrate. Filtrates were tested to insure that this
filtrate would exhibit the basic inhibition. To date all filtrates
have exhibited the inhibition; however, its presence cannot be
assumed.
99
-------
losa (from the Indiana collection) ,- and Skeletonema costatum and Thaias-
siosira from L. Provasoli's collection. All evidenced an inhibition of
growth by filtrates of Linsley blue-greens. A minimal testing of the
marine diatoms suggests that this inhibition also effects marine forms;
however, the ecological significance of the inhibition in freshwater
systems cannot be readily generalized to marine systems because of 1) the
obvious dilution possibilities in ocean waters; 2) the minimum occurrence
of blue-green blooms in marine environments, and 3) the myriad of differ-
ences in the chemical effects of fresh and marine waters. Nevertheless,
a study of Tricodesmi \rm blooms might prove interesting at some future
date.
Effects of Bacteria on Diatom Inhibition
Some of the producer cultures and some of the assay cultures used in this
study were bacterized. This bacterization did not totally eliminate the
inhibition of diatom growth produced by filtrates of blue-green algae,
but it did appear that the presence of bacteria in either the producer or
the assay culture lessened the potency of the inhibitor and, further, it
appeared thai: the presence of bacteria in both the producer and the assay
culture had an even greater effect on the inhibition. To determine if the
presence of bacteria (presumably some of the bacteria would be able to
metabolize the inhibitory molecules) did actually lessen inhibition an
axenic culture of Anabaena sp. (538) was infected with a 10 ml aliquot of
bacterized (algae-free) filtrate from one of the Oscillatoria rubescens
(535) cultures. This newly bacterized culture was then used as the inocu-
lum for a 1000 ml producer culture. The comparison of the inhibitory
capacity of this bacterized producer of Anabaena sp. (538) with the inhi-
bitory capacity of an axenic producer culture of Anabaena sp. (538) indi-
cated that the original premise was correct. That is, a comparison of
"bacterized" F tests with axenic F tests indicates greater growth (less
inhibition) in the "bacterized" group. In general the "bacterized" A
tests while they did support greater growth than "bacterized" F tests,
did not achieve the maxima of axenic A tests. This may reflect the com-
petitive use of some essential nutrients, such as PO4, by the bacteria in
the bacterized producer cultures. Table 12 provides a comparison of
results obtained from this work.
Mn Effect
Although the subject requires considerable additional study, it is worthy
of note that the higher Mn level in producer cultures enriched by l>s% ESj,
as compared to producer cultures enriched by ESj which was formulated
without Mn, apparently lessened the level of inhibition exhibited by fil-
trates of these cultures. This suggests a stronger competitive position
for blue-greens in waters with low natural stores of Mn—reminiscent of
the findings (under very different circumstances) of R. Patrick (1969).
100
-------
TABLE 12. Comparison of Inhibitory Effect of Bacterized and
Axenic Producer Cultures of Anabaena sp. (538)
Assay
Oraanism
Nitschia
frustulum
(224)
Synedra
famnilica
(202)
Synedra sp.
(299)
Fragilaria sp.
(99)
Cyclotella sp.
(211)
Nitschia sp.
(352)
Tabellaria sp.
(764)
Asterionella
formosa (800)
Bacterized
F
240
180
105
70
190
130
80
115
A
370
120
115
75
215
370
100
125
Axenic
F
180
90
80
65
65
130
110
110
A
380
120
120
80
170
245
140
140
101
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VII:V EFFECTS ON DIATOM INHIBITION OF SILICA ADDITION TO FILTRATES OF
BLUE-GREEN ALGAE AND TO FREEZER-STORED AND FRESHLY-COLLECTED
LINSLEY POND WATERS
Methods
To determine the effects of silica addition on the diatom inhibition evi-
denced in F vs A assays, silica—as Na2SiO3—was added to assays of both
blue-green filtrates and natural pond waters. Initially, additions were
established at 20 mg% (by weight of hydrated molecule). Later 1, 5, 10,
and 20 mg% additions were used.
The second set of addition levels was employed in tests designed not only
to determine effects of Si addition on assays, but also to determine the
availability of silica in the natural waters of the pond at the times of
collection.
Results and Discussion
A suggestion by L. Margulis (personal communication, 1973) that the blue-
green algae might effect an inhibition of diatom growth by binding silica,
making it less available to diatoms even at times when silica levels were
apparently sufficient, prompted the study of silicate addition as a means
of overcoming the heat-labile inhibitor. Na2SiO3«9H2O was added to fil-
trates and to pond waters, and F vs A assays were established.
Silica addition did improve growth in most cases, but this improvement
often occurred in both F and A assays with the result that there was
still considerably less diatom growth in F than in A cultures (Table 13).
These results were promising, however, since the addition of other sub-
stances in a variety of attempts to overcome inhibition produced no
effects. Additionally, in some cases the added silica did appear to elim-
inate the inhibition.
Some organisms responded negatively to high Si addition levels and this
response occurred at lower levels of silica addition in A tests than it
did in F tests. This disparity suggests an initially greater available
Si.pool in A tests than in * tests. Since the absolute silica level in
both F and A tests must be very similar (autoclaving of A tests could not
account for the leaching of as much as 20 mg% of silica from test tubes),
the possibility of a heat-labile silica binder, or inactivator, must be
considered. Further work on this point is needed to clarify its signifi-
cance and to determine whether this silica effect is a part, or the whole,
of the inhibitory effect which can be studied via .ether extraction,
ultrafiltration, or dialysis (see discussions above).
The annual cycle of silica levels in Linsley, as reported by Cowgill
(1970), do not suggest that simple silica limitation can be the basis for
the absence of diatom blooms in the pond. Laboratory tests, however,
indicate that silica is a determining factor in the ending of those spring
102
-------
TABLE 13 .The'Effects of Silica Addition on the Heat-Labile Diatom
Inhibition Exhibited by Filtrates of Blue-Green Algae. (20mg» added)
PRODUCER
Anaoaena sp. (538)
With Silica Addition
Without Silica Addition
Assay F
Organism
Interpre-
tation
Nitschia
frustulum 420
(224)
270
220
420
420 is the max.
growth for this
culture. Too
little, or too
much silica limits.
Svnedra sp.
(299) 180
180
75
105
180 is the max.
growth for this
culture. A levels
are inadequate Si
addition does not
exceed tolerance of
assay organism.
Fragilaria sp.
(99) 125
125
75
90
125 is the max.
growth for this
culture. A levels
are inadequate Si
addition does not
exceed tolerance of
assay organism.
Cvclotella sp.
(211) O
330
140
There is a differ-
ent kind of inhib-
ition here. (It is
heat-labile toxicity.
PRODUCER
Synechecoccus sp. (91)
With Silica Addition
Without Silica Addition
Assay
Organism
Interpre-
tation
Svnedra
famnilica
(202)
20
50
A non-heat-
labile inhibi-
tion is present
Silica addition
has no effect.
Growth is poss.
ible if fil-
trate is char-
coal treated.
(Max. - 170)
Fragilaria sp.
(99) 130
170
Nitschia
frustulun 300
(224)
155
80
115
Silica addition
has no effect
on inhibition.
Generally
increases
growth.
245
330
300-330 is the
max. growth
for this cult.
Too little or
too much Si
limits growth.
103
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diatom blooms which do occur. Water collected immediately after the end
of the spring diatom bloom in 1973 was tested for its ability to support
diatom growth. The addition of silica (as Na2SiO3*9H2O at 20 mg%) tripled
the growth of most of the diatoms tested (Table 14). The addition of
increasing amounts of silica to Linsley water collected prior to the 1974
diatom bloom did produce increasingly positive effects on the growth of
assay diatoms. The same additions to water collected at the end of the
sane* 1974, bloom also produced significant improvements in growth;
however/ changes were notably greater in post bloom waters. Figures 23
thru 26 illustrate this distinction between pre- and post-diatom bloom
waters for the spring of 1974.
As demonstrated in Figure 23 when filter' sterilized water was tested,
nmyiimmi growth (approximately O.D. 350) for pre-bloom water was achieved
by the addition of 5 mg% sodium silicate. This same growth mnxima was
achieved for post-bloom filtered water by the addition of more than 20
mg%. When autoclaved water samples are compared (Figure 24) maximum
growth in pre-bloom waters is achieved with an addition of only 1 mg%
sodium silicate while more than 10 mg% is required for post-bloom maxima.
Both situations indicate a much greater silica deficit in post-, then in
pre-bloom waters; however, if autoclaved and filtered samples from the
same date are considered, an additional informative contrast can be ob-
served. In pre-bloom waters (Figure 25) A samples required only 1 mg% to
achieve growth may JIM between 300 and 400, but F samples required 5 mg%.
Similarly in post-bloom waters (Figure 26) F samples required 20 mg%
addition to achieve growth ma-rima of O.D. 300, while autoclaved samples
required only 10 ng%. Suggesting that some of the silica in the water
samples for either date was not available to the diatoms until after the
water samples were autoclaved. Circumstantial evidence only that some
of the heat-labile diatom inhibition exhibited during this study is
related to silica availability.
104
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TABLE 14 Diatom Growth Maxima in Pond Water Collected at the
End of the 1973 Diatom Bloom (May, 13, 1973) With
and Without Silica Addition
(20 mgt added)
Organism
Nitschia
frustulura
(224)
Synedra
famnilica
(202)
Synedra sp.
(299)
Fragilaria sp.
(99)
Cyclotella sp.
(211)
Nitschia sp.
(352)
Tabellaria sp.
(764)
Asterionella
fornosa (BOO)
With Silica Addition
T
315
120
140
125
230
210
160
185
A
315
170
190
155
28O
230
205
205
Without Silica Addition
F
145
70
45
30
60
70
55
60
A
335
120
85
95
160
245
65
115
105
-------
O.D.
FIGURE 23. COMPARISON OF GROWTH RESPONSE TO'VARIOUS LEVELS 07 ADDED SILICA
IN PRE- AND POST DIATOM-BLOOM POND WATERS.
FEBRUARY 14, AND JUNE 13, 1974
FILTER-STERILIZED ONLY.
300-
Nitschia frustulum (224)
200-
100-
10
20
-------
O.D
300-
200-
100-'
FIGURE 24 COMPARISON OF GROWTH RESPONSE TO VARIOUS LEVELS OF ADDED SILICA
IN PRE- AND POST DIATOM-BLOOM POND WATERS._
FEBRUARY 14, AND JUNE 13, 1974
AUTOCLAVED.
Nitschia frustulum (224)
3
mg%
10
20
-------
O
00
FIGURE 25 COMPARISON OP GROWTH RESPONSE TO VARIOUS LEVELS OP ADDED SILICA
PRE-DIATOM-BLOOM POND WATERS.
O.D
(2/14/74) A
300-
200-
100-
Nitschia frustulum (224)
3
mg%
10 20
-------
O.D
FIGURE 26 COMPARISON OF GROWTH RESPONSE TO VARIOUS LEVELS OF ADDED SILICA
POST-DIATOM-BLOOM POND WATERS.
•JUNE 13, 1974
o
vo
300-
200-
100-
Nitschia frustulum (224)
35
mg%
10 20
-------
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120
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APPENDIX A
SOME PREVIOUSLY REPORTED BLOOMS IN LINSLEY POND
Linsley Pond has been considered moderately eutrophic, or mesotrophic,
for at least four decades. Many occurrences of "blooms" have been
reported by other investigators. Often such reports are simply descrip-
tive additions which were incidental to the main interest of a given
paper, but which were included for background, or interpretative, value.
A more thorough understanding of the historic development of conditions
of eutrophication in Linsley Pond will be available in the near future
when R. Brugam completes his study of cores taken from Linsley in con-
junction with his dissertation research at Yale.
Listed below are some of the reports of blooms in Linsley.
Organism Descriptive Terms Date of Bloom Source of
Employed Information
Synedra 5140/liter Approx. 1938 Riley, 1938
growing season
Tabellaria 2170/liter
Fragilaria 640/liter
Anabaena, Micro- extensive blooms August & Sept. Riley, 1939
cystis f 1936
Coelosphaerium
Dinophyceae and occasionally great September 1937 Riley, 1939
Chrysophyceae blooms to June 1938
Fragilaria dominant form in September 1937
blooms
Anabaena and most important in August 1937
Coelosphaerium blooms
Oscillatoria dominant genus most of winter
and early spring
1937-1938
peridinians, great phytoplank- January 1938
Cyclotella, ton burst-seeded
Mallomonas from inflow water
Eudorina
121
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APPENDIX A
SOME PREVIOUSLY REPORTED BLOOMS IN LJNSLEY
Descriptive Terms
Employed
spring phytoplank-
ton burst
Organism
Oscillatoria,
Synedra, Melo-
siraj
ScenedesmuSr
peridinians
Dinobryon, Frag- sparser bloom than
ilaria, Asterion- above
ella
Small Oscillatoria, Bloom
larger peridinians
(Clorophyceae)
Anabaena
(never dominant)
large amount
POND (cont'd.)
Date of Bloom
April 1938 sur-
face, and thru
June in deeper
waters
May-June 1938
surface
Mid-winter
1937-1938
August 22,
1937
Source of
Information
Riley, 1939
Hutchinson,
1941b
circi—
nalis
Oscillatoria
water-bloom
Bloom
large population
June 25, 1938
July 25-
August 5, 1938
September 1941-
1942
Hutchinson,
1943
Fragilaria croto-
nensis,
Synedra acus,
Aaterionella
fo:
Diatoms
abundant
dominated
general state-
ment
April 1937
Hutchinson,
1944
Oscillatoria and
other Myxophyceae
Dinobryon
vernal maxima
dominant
other years
than 1938-1941
May-June 1938-
1941
122
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APPENDIX A
SOME PREVIOUSLY REPORTED BLOOMS IN LINSLEY
Organism
Fragilaria
crotonensis and
Anabaena circi-
nalis
Oscillatoria
blue-green
Scenedesmus sp.
and Asterionella
formosa
Oscillatoria
Synedra acus
Fragilaria croto-
nensis
Anabaena circi-
nalis
Oscillatoria pro-
lific^ and O_.
rileyi
Dinobryon
Aphani zome non
Dinobryon
Oscillatoria and
Aphani zomen on
Oscillatoria
prolifica
Oscillatoria
prolifica
Anabaena and
Oscillatoria
Descriptive Terms
Employed
POND (cont'd.)
Date of Bloom
enormous development July 1937-1941
relatively large
considerable popu-
lation
transitory develop-
ments
rather monotonous
immense population
enormous multipli-
cation
enormous multipli-
cation
bulk of plankton
bloom
(graph maxima)
spring
maxima after blue-
greens or diatoms
immense population
in deeper water (6 m)
Bloom
main autumnal
dominant but poor
growth
late summer
1938-1941
autumnal 1938-
1941
winter 1938-
1941
winter 1938-
1941
1942
April 17, 1937
end July 1937
August 3
August 10,
1937
Spring 1938
(March)
May-June 1938
Spring 1939
March, April,
and June 1939
July 1944
April 1945
November 1945
October 14,
1952
Source of
Information
Hutchinson,
1944
Hutchinson,
1946
Benoit, 1957
123
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APPENDIX B
MEDIA USED DURING THIS STUDY
The following media were used with some frequency during this study.
Other media were tested but those listed were found to be of greatest
value.
ESj - enrichment
pH 7.8
(Provasoli, 1968)
350 mg%
50 mg%
2.5 mg%
25 ml
10
0.5 mg%
25^
500 mg%
to 100 ml
(Provasoli, unpublished)
150 mg% added to ESj
Use 2 ml of stock per 100 ml natural water
FeEDTA (Provasoli, 1968)
351 mg Fe(NH4)2(SO4)2'6H2O
330 mg Ha2EDTA
H2O to 500 ml
Na2 glycerophosphate
FeEDTA
P II metals
Vitamin Bi2
Thiamine
Biotin
Cu (as Cl)
Tris
H2O
~ enrichment
124
-------
P II Metals (Provasoli, 1968)
Na2EDTA 100 mg%
Fe(EDTA) 1 mg%
Boron 20 rog%
Mn (as Cl) 4 rog%
Zn (as Cl) 0.5 mg%
Co (as Cl) 0.1 mg%
H20 to 100 na
FW (Provasoli, unpublished)
pH 7.0-7.5
*K2 glycerophosphate 0.63 mg*
Ca(N03)2-4H20 4.2 mg%
Ca3 citrate-7H2O 4.3 mg*
MgS04-7H2O 0.7 mg%
MgCl2-6H2O 1.7 mg%
FeEDTA 0.1 mg%
Mn (as Cl) 0.001 mg%
Zn (as Cl) 0.03 mg%
ethyl silicate 3.3 mg%
l.O mg%
H2O to 100 ml
*substituted Na for K (sonetimes)
125
-------
DC
pH 7.8-8.0
NaCl
KC1
NaNO3 _
Ca (as Cl)
Tris
K2HP04
Na2Si03-9H20
P II metals
FeEDTA
fliiamine
Biotin
B12
NaH glutamate
Glycine
Na lactate
Sucrose
Na acetate
H2O
(Proyasoli, McLaughlin, and Droop, 1957)
1.8%
60 mg%
50 mg%
0.5%
10 mg%
0.1%
3 rog%
20 rog%
3 ml/100
0.01 mg%
10 f %
0.1^ %
O.Sj' %
50 mg%
50 mg%
50 mg%
50 mg%
50 mg%
to 100 ml
126
-------
cy II
(Provasoli, unpublished)
pH 7.6
NH4 acetate (CH3COONH4)
P II metals
FeEDTA
Ca (as Cl)
KC1
glycerophosphoric acid
B12
83 vitamins
Tris (Sigma 121)
83 Vitamins
Thiamine
Biotin
PABA
Folic acid
Nicotinic acid
Thymine
Inositol
Ca panthotenate
H2O
20 mg%
1 ml/100
0.04 mg%
1 mg%
3 mg%
10 mg%
5 mg%
0.5 ml/100
33.3 mg%
to 100 ml
(Provasoli, McLaughlin, and Droop, 1957)
5 mg%
10 f %
100 ^%
20 Y %
1 mg%
30 mg%
50 mg%
1 mg%
to 100 ml
127
-------
DA
(D'Agostino and Provasoli, 1970)
CV7
pH 6.5
K2HPO4
Va-3 citrate
MgS04-7H2O
Fe (EDTA)
Thiotone
Trypticase
Yeast extract
H2O
Agar
2 rag%
2 mg%
2 mg%
0.2 mg%
60 mg% (Baltimore Biological
Labs)
16 mg%
5 mg%
to 100 ml
25% for semi-solid
pH 7.6
Ca {as Cl)
NaNO3
MgSO4-7H2O
Na2 glycerppbosphate
KC1
B12
Biotin
Glycylglycine
P IV metals
{Provasoli, unpublished)
2 mg%
10 mg%
4 mg%
5 mg%
5 mg%
0.01|- %
o.oir %
50 mg%
1 ml/100
H2O
to 100 ml
128
-------
(thiamine)
CV-i + thiamine
p IV Metals
Na2EDTA
FeEDTA
Mn (as Cl)
Zn (as Cl)
Co (as Cl)
Mo
H2O
Ger 8/10
Ca (as NO3)
MgSO4-7H2O
Na2CO3
FeEDTA
Citric acid
H2O
(Provasoli, unpublished)
10 j %
(Provasoli and Pintner, I960)
100 mg%
4 mg%
1 mg%
0.5 mg%
0.1 jng%
O,5 ntg%
to 100 ml
(Provasoli, unpublished)
0.96 mg%
1.0 mg%
2.5 mg%
2.0 mg%
4.4- mg%
0.056 mg%
O.39 mg%
to 100 ml
129
-------
TA
pH 7.2
Trypticase
Thiotone
KOI
Na acetate
KH2PO4
•fliiamine
H-,0
FD II
pH 6.5
Ca3 citrate
NH4NO3
glycerophosphate
FeEDTA
Vitamins 8A
VT metals
H20
(Provasoli, unpublished)
0.1%
0.1%
0.01%
0.01%
0.05%
0.005%
0.05 mg%
to 100 ml
(Provasoli, unpublished)
5 mg%
2 mg%
2.5 mg%
1.0 mg%
0.07 mg%
0.2 ml/100
O.I ml/100
to 100 ml
130
-------
VT Metals (Proyasoli, unpublished)
Zn 10
Mn 4
Co °-3
Cu 0.03 rng%
V 0.1 rog%
Ti 0.3 mg%
Cr 0.01 mg%
Ni 0.01 mg%
Sr 30
Mo °-
131
-------
'fhe following broad range vitamin addition was sometimes added
with ESj when new isolates were sought.
Vitamins 8A (Tatewaki and Provasoli, 1963)
tfiiamine HCl 20 mg%
Biotin 50 y%
B12 5 y*
Folic acid 0.25 mg%
PABA 1 mg%
Nicotine acid 10 mg%
tfhymine 80 mg%
Chlorine H2 citrate 50 mg%
Inositol 100 mg%
Putrescine 2HC1 4 mg%
Riboflavin 0.5 »g%
Pyridoxamine 2HC1 2 mg%
Orotic acid 26 ing %
Folinic acid 20Jf%
Ca panthotenate 10 mg%
Pyridoxine 2H 4 mg*
H2O to 100 ml
132
-------
The following trace metal additions were at times added with
when new isolates were sought.
Guillard Metals (Guillard, 1974)
1.0 ml% of this stock = Guillard's recommendations.
Na2 EDTA 43.6 mg% (of Na2 EDTA)
(amt. of element) (amt. of compound)
FeCl2*6H2O Fe 6.5 mg% 31.5 mg%
CuSO4-5H2O Cu 25v % 0.1 mg%
ZnSO4-7H2O Zn 50jr% 0.22 mg%
CoCl4-6H2O Co 25 y% 0.1 mg%
MnCl2-4H2O Mn 0.5^% 1.8 mg%
Na2MoO4-2H2O Mo 25^% 0.06 mg%
H3BO3 B 1.7 mg% 10.0 mg%
H2O to 100 ml
133
-------
U II Metals (Aldrich and VJilson, 1960)
0.3 ml of this stock = Provasoli's recommendation.
Na2EDTA 15 mg%
FeCl2-4H2O 0.1 mg%
CuCl2 0.1 mg%
MnCl2-4H20 1.0 mg%
ZnCl2 0.5 mg%
NiCl2*6H2O O.I mg%
A1C13-6H2O 0.5 mg%
CoCl2*6H2O 0.5 mg%
RbCl 1.0 mg%
BaCl2 0.1 mg%
0.5
0.5
0.5 mg%
SrCl2-6H2O 0.5 rog%
Na2MO4*2H2O 0.5 mg%
113603 0.5 mg%
CsCl 0.5 mg%
(NH4)2Ce(NO3) 0.1 mg%
CdCl2-2*jH2O 0.1 mg%
SnCl2-2H2O 0.1 mg%
RuCl3 0.1 rag%
RhCl 0.1 mg%
134
-------
APPENDIX C
LISTING OF SPECIFIC EFFECTS OF FILTRATES ON INDIVIDUAL ORGANISMS
PRODUCER: Oscillatoria rubescens (535) - bacterized
EFFECT ASSAY ORGANISM
0 Oscillatoria rubescens
(535)b*
0 O. rubescens (739)b
+ Oscillatoria sp. (776)a
+ Pseudanabaena galeata
(597)a
+ Anabaena sp. (538)a
-I- Anabaena sp. (765) b
+ Aphanizomenon flos-aquae
(766)b
0 Synechecoccus sp. (91)a
EFFECT ASSAY ORGANISM
0 Nitschia frustulum (224)a
Nitschia sp. (352)a
Asterionella formosa (800)b
Synedra sp. (299)a
0 Synedra famnilica (202)a
0 Fragilaria sp. (99)b
Tabellaria sp. (764)b
Cyclotella (211)b
Ankistrodesmus convolutus
(105)a
Scenedesmus quadricauda
(314)a
Staurastrum sp. (207)a
+ Trachelmonas sp. (778)a
0 Synura uvella (43)b
Chlamydomonas sp. (298)b
+ C. reinhardtii (+)a
non-Linsley
+ C. reinhardtii (-)a
non-Linsley
0 C. reinhardtii (+ & -)a
non-Linsley
+ Euglena gracilis-a
non-Linsley
0 monad (317)b
+ Wolnscynskia limnetica-a
non-Linsley
+ Dinobryon cylindricum-b
non-Linsley
*An a or b following culture identification means assay culture was
either axenic or bacterized.
135
-------
PRODUCER: Osciliatoria rubescens (739) - bacterized
EFFECT ASSAY ORGANISM
0 Oscillatoria rubescens
(53S)b
- O. rubescens (739)b
0 Oscillatoria sp. (776)a
+ Pseudanabaena galeata
(597)a
+ Anabaena sp. (538)a
- Anabaena sp. (765)b
-I- Aphanizomenon flos-aquae
(766)b
— Synechecoccus sp. (91)a
EFFECT ASSAY ORGANISM
Nitschia frustulum (224)a
Nitschia sp. (352)a
- Asterionella formosa (80O>
- Synedra sp. (299)a
Fragilaria sp. (99) b
Tabellaria sp. (764)b
Ankistrodesmus convolutus
(105)a
Scenede smus quadricauda
(314)a
Staurastrum sp. (207)a
0 Trachelmonas sp. (778)a
0 Synura uvella (43)b
0 Chlamydomonas sp. (298}b
0 C. reinhardtii (+)a
non-Linsiey
— C. reinhardtii (-)a
non-Linsley
+ C. reinhardtii (+ fi -)a
non-Linsley
0 Euglena gracilis-a
non-Linsley
0 ronad (317)b
+ Wolnscynskia limnetica-a
non-Linsley
+• Dinobryon cylindricum-b
non-Linsley
136
-------
PRODUCER: Oscillatoria sp. (776) - axenic
EFFECT ASSAY ORGANISM
- Oscillatoria rubescens
(535}b
- O. rubescens (739)b
0 Oscillatoria sp. (776)a
- Pseudanabaena galeata
(597)a
- Anabaena sp. (538)a
0 Anabaena sp. (765)b
- Aphanizomenon flos-aquae
(766)b
- Synechecoccus sp. (91) a
EFFECT ASSAY ORGANISM
Nitschia frustulura (224)a
Nitschia sp. (352)a
- Asterionella formosa (800)b
0 Synedra sp. (299)a
Synedra famnilica (202)a
- Fragilaria crotenensis
non-Linsley
Kirchneriella obesa (104)a
Ankistrodesmus convolutus
(105)a
Scenedesmus quadricauda
(314)a
Staurastrum sp. (207)a
Closterium sp. (499)a
chlorococcal green algae
(307)a
Eudorina sp. (767) b
+ Sphaerellopsis sp. (598)b
- Chlamydomonas sp. (298)b
0 C. reinhardtii (+ & -)a
non-Linsley
0 Euglena gracilis-a
non-Linsley
0 monad (317)b
137
-------
PRODUCER: Pseudanabaena galeata (597) - axenic
EFFECT ASSAY pRGANISM
O Oscillatoria rubescens
(535)b
0 O. rubescens (739)b
0 Oscillatoria sp. (776)a
- Pseudanabaena galeata
(597)a
- Anabaena sp. (538)a
- Anabaena sp. (765)b
- Aphanizomenon flos-aquae
(766)b
+ Synechecoccus sp. (91)a
EFFECT ASSAY ORGANISM
Nitschia frustulum (224)a
Nitschia sp. (352)a
0 Asterionella fornosa
Synedra sp. (299)a
Synedra famnilica (202)a
Fragilaria sp. (99)b
Cyclotella (211)a
Fragilaria crotenensis
non-Linsley
+
+
Kirchneriella obesa (104) a
Ankistrodesmus convolutus
(105) a
Scenede sinus quadricauda
(314)a
Staurastrum sp. (207) a
Closterium sp. (499) a
chlorococcal green algae
(307) a
0 Sphaerellopsis sp. (598)b
0 Chlamydononas sp. (298)b
C. reinhardtii (+ & -)a
non-Linsley
monad (317)b
138
-------
PRODUCER: Anabaena sp. (538) - axenic
EFFECT ASSAY ORGANISM EFFECT ASSAY ORGANISM
Oscillatoria rubescens
(535)b
O. rubescens (739)b
Oscillatoria sp. (776)a
Pseudanabaena galeata
(597)a
Anabaena sp. (538)a
Anabaena sp. (765)b
Aphanizomenon flos-aquae
(766)b
Synechecoccus sp. (91)a
Nitschia frustulum (224)a
Nitschia sp. (352)a
Asterionella forirosa (800) b
Synedra sp. (299)a
Synedra famnilica (202)a
Fragilaria sp. (99)b
Tabellaria sp. (764)b
Cyclotella (211)a
Fragilaria crotenensis
non-Linsley
•f
0
Kirchneriella obesa (104)a
Ankistrcx3esmus convolutus
(105)a
Scenedesmus guadricauda
(314)a
Staurastrum sp. (188)a
Staurastrum sp. (200)a
139
-------
PRODUCER: Anabaena sp. (762) -bacterized
EFFECT ASSAY ORGANISM EFFECT ASSAY ORGANISM
- Oscillatoria rubescens - Nitschia frustulum (224)a
(535)b - Asterionella formosa (800)b
O. rubescens (739)b + Synedra sp. (299)a
- Oscillatoria sp. (776)a
— Pseudanabaena galeata
(597)a
•f Anabaena sp. (538)a
- Anabaena sp. (765)b
+ Aphanizomenon flos-aquae
(766)b
+ Synechecoccus sp. (91)a
140
-------
PRODUCER: Aphanizoroenon flos-aquae (766) - bacterized
EFFECT ASSAY ORGANISM
+ Oscillatoria rubescens
(535)b
0 O. rubescens (739)b
+ Oscillatoria sp. (776)a
0 Pseudanabaena galeata
(597)a
+ Anabaena sp. (538)a
0 Anabaena sp. (765)b
— Aphanizomenon flos-aquae
(766)b
0 Synechecoccus sp. (91) a
EFFECT ASSAY ORGANISM
Nitschia frustulum (224) a
Nitschia sp. (352)a
0 Asterionella fornosa (800)b
0 Synedra sp. (299)a
0 Fragilaria sp. (99)b
Tabellaria sp. (764)b
Cyclotella (211)a
0 Kirchneriella obesa (104)a
+ Ankistrodesmus convolutxis
(105)a
O Scenedesmus quadricauda
(314)a
0 Staurastrum sp. (185)a
+ Closterium sp. (499)a
+ chlorococcal green algae
(307)a
+ Synura uvella (43)b
0 Chlamydomonas sp. (298)b
0 C. reinhardtii (+ & -)a
non-Linsley
0 Euglena gracilis-a
non-Linsley
0 nonad (317)b
141
-------
PRODUCER: Synechecoccus sp. (91) - axenic
EFFECT ASSAY ORGANISM EFFECT ASSAY ORGANISM
Oscillatoria rubescens
(535)b
O. rubescens (739)b
Oscillatoria sp. (776)a
Pseudanabaena galeata
(597)a
Anabaena sp. (538)a
Anabaena sp. (765)b
Aphanizomenon flos-aquae
(766)b
Synechecoccus sp. (91) a
Nitschia frustulum (224)a
Nitschia sp. (352)a
Asterionella fornosa (800)b
Synedra sp. (299)a
Synedra famnilica (202)a
Fragilaria sp. (99)b
Tabellaria sp. (764)b
Cyclotella (211)a
Fragilaria crotonensis
non-Linsley
0
0
Xirdhneriella obesa (104)a
Ankistrodesmus convolutus
(105)a
Scenedesmus quadricauda
(314)a
Staurastrum sp. (188)a
Staurastrum sp. (200)a
Sphaerellopsis sp. (598)b
- Chlantydomonas sp. (298) b
+ C. reinhardtii (+ & -)a
non-Linsley
•f Euglena gracilis-a
non-Linsley
+ monad (317)b
142
-------
APPENDIX D
THE O.D. METER EMPLOYED DURING THIS STUDY
The optical density meter built by Dr. Fred Kavanaugh for Dr. Provasoli
functions in a manner similar to that of the Spec-20 with a test tube
insert module; i.e.., it accepts test tubes and does not require decant-
ing of cultures into cuvettes. As mentioned in the text, this permitted
close surveillance of culture growth throughout the lifespan of a culture.
Density units were read from 0.000 thru 0.999 and expressed as reading
x 1000 to eliminate the necessity for a decimal. Repeatability was
excellent, consistent readings were obtained to the nearest thousandth
on all occasions. It was necessary to mark each test tube to insure
that it was not rotated for different reading occasions. This was the
result of test tubes being considerably less consistent in terms of thick-
ness and pregrowth transparency than would be cuvettes which were made
especially for spectrometer reading. A mark was placed on each test tube
prior to inoculation at the position in which a zero reading could be
obtained. Actually, even if this precaution were not to be taken, the
ordinary test tubes were remarkably consistent, varying at maximum from
0.000 to about 0.025 when rotated in the light beam.
To provide assurance as to the consistent performance of this meter
(which is unfamiliar apparatus to all except those who have spent time in
Dr. Provasoli's laboratory), its readings are compared on the following
page to those of a Beckraan Spectrometer set at 425.
To indicate the consistent relationship of O.D. readings to cell counts
the following pages contain translation graphs based on organisms com-
monly employed for experimental purposes.
D-l. HASKINS O.D. READINGS COMPARED TO BECKMAN SPECTROMETER
READINGS WITH SPECTROMETER SET AT 425.
D-2. CELL COUNT VS O.D. FOR SKELETONEMA COSTATUM
D-3. CELL COUNT VS O.D. FOR COCCOLITHUS HUXLEYI
D-4. CELL COUNT VS O.D. FOR NITSCHIA FRUSTULUM V. INDICA
SKVORTZOW
143
-------
OS
D-l.
08-
HASKIH'S O. D. READINGS COMPARED TO
BECKMAN SPECTROMETER READINGS
WITH SPECTROMETER SET AT 425
(Data assembled by
Marta Estrada-for
this study)
07-
06*
01
00-
0*2 0*.3 0'.
RASKIN'S O. D. METER
o!s
0.'6
0.7
144
-------
Millionr.
of
Cells
10-
6-
in
D-2. CELL COUNT VS. 0. D. FOR
Skeletoncma costatun
LOG
SENESCENCE
0.1
i
0.2
i
0.3
0.4
0. D.
0.4
t
0.3
0.2
o'.i
0.0
-------
0-3. CELL COUNT VS. 0. D. FOR
Coecolithut huxleyi
STATIONARY
.SENESCENCE
<
0.1
0.2
0.3
i
0.4
0.4
0.3
0.2
0.1
0.0
0. D.
-------
CELL COUNT VS. 0. D. FOR
Nitechia frustuluro v. indica
(224)
0.1
0. D.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing
1. REPORT NO.
EPA-600/3-76-081
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Algal Metabolite Influence on Bloom Sequence In
Eutrophied Freshwater Ponds
5. REPORT DATE
July 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kathleen Irwin Keating
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Haskins Laboratories, Inc.
Osborn Memorial Laboratory
165 Prospect Street
New Haven, Connecticut 06520
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
RA 801387
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Corvallis Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97330
13. TYPE OF REPORT AND PERIOD COVERED
final report
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
I. Bloom sequence in Linsley Pond, Connecticut, was monitored for three years. Bloom
dominant algae were isolated in culture, heat-labile, bio-active substances in cell-
free filtrates of these cultures were tested against each of the dominants. Enhancing,
or neutral, effects on successors; and inhibiting, or neutral, effects on predecessors
were consistently observed. Lake waters exhibited parallel effects. Additionally,
inhibition patterns suited differences in year-to-year patterns of in situ blooms.
This widespread correlation of in situ events with iji vitro phenomena Indicates that
extracellular products of bloom~3bminant algae are sTgnificant in bloom sequence deter-
mination in eutrophied fresh waters.
II. Spring diatom bloom density varied inversely with the preceding winter's blue-
green population density. Diatom blooms, when they occurred, ended when available
silica was depleted. Generalized in situ and in. vitro inhibition of diatoms by blue-
greens. After separation and concentration via ether extraction or ultrafiltration
active substances were returned to growth media. Preliminary evidence suggests that
inhibition involves interference with silica availability.
III. The feasibility of biological programming of bloom sequence in eutrophied lakes
is considered.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
phytoplankton blooms, bloom sequence, algae
diatoms, blue-green algae, algal metabolite;
eutrophication
08/H
8. DISTRIBUTION STATEMENT
release to public
19. SECURITY CLASS (ThisReportj
unclassified
21. NO. OF PAGES
156
20. SECURITY CLASS (Thispage)
unclassified
22. PRICE
EPA Form 222O-1 (»-73|
148
U. S. GOVERNMENT PRINTING OFFICE: 1976—697-7151110 REGION 10
------- |