EPA-660/3-74-019
August 1974
Ecological Research Series
Research and Development of a
Selective Algaecide to Control
Nuisance Algal Growth
\
UJ
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
EPA REVIEW NOTICE
This report has been reviewed "by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views
and policies of the Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
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EPA-660/3-74-019
August 1974
RESEARCH AND DEVELOPMENT OF A SELECTIVE
AL6AECIDE TO CONTROL NUISANCE ALGAL GROWTH
BY
B. L. PROWS
W. F. McILHENNY
CONTRACT NO. ~68~-TJr-0782
PROGRAM ELEMENT 1BA031
ROAP/TASK 21 AIZ 06
PROJECT OFFICER
THOMAS E. MALONEY
PACIFIC NORTHWEST WATER LABORATORY
WATER QUALITY OFFICE
ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
PREPARED FOR
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
I !!! ! -
For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C. 20402 - Price $2.96
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ABSTRACT
The primary objective of this project was tL1 determine under
natural, open-field conditions, the efficsciy of two can-
didate algaecides, Compound No. 23 (2,5-Dichloro-3,4-dinitro-
thiophene) and No. 73 (p-Chlorophenyl-2-thienyl iodonium
chloride) from Phase II of the multiple phase developmental
program. Specific efforts were also directed toward further
delineation of the toxicological and environmental persistence
properties of the candidate compounds, as well as further
development of a possible biological-chemical control system.
Data from the field tests conducted under a wide variety of
conditions in four geographically diverse regions of the
United States revealed a distinctive pattern of selective
blue-green algal control for both experimental compounds.
Compound No. 23 was eliminated from the test series due to
unacceptable fish toxicity.
A whole-pond field study involving the use of a phagocytic
organism, Ochromonas ovalis, as a biological control system,
was inconclusive due to the apparent inability of the organism
to survive under the existing environmental conditions.
Continued laboratory screening tests of some 70 additional
compounds produced two additional candidate compounds,
No. 136 (2,2'-(l,2-Ethenediyl)bisbenzoxazole) and
No. 176 (l,2-Dichloro-4-(isothiocyanatomethoxy) benzene).
/
Continuation of testing of candidate compounds under field
conditions is recommended.
ii
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TABLE OP CONTENTS
Page
Abstract ii
List of Figures iv
List of Tables v
Acknowledgements ix
Sections
I Conclusions 1
II Recommendations 3
III Introduction **
IV Experimental Procedure 15
V Discussions 29
VI References 121
VII Appendices
ill
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LIST OF FIGURES
Figure
Number page
1 Open Field Test System 22
2 North Carolina Test Site 31
3 Chowan River - First Test, Test Compound No. 23
vs. M. Cyanea and M._ in cert a 35
4 Chowan River - Second Test, Test Compound No. 73
vs. M. cyanea and M._ incerta 38
5 Compound Depletion Patterns, Chowan River -
First Test 4l
6 Minnesota Test Site 49
7 Lake Sallie and Muskrat Lake - Linological
Sampling Stations and Algaecide Testing Stations. 50
8 Compound Depletion Patterns, Second Minnesota
o
10
n
12
13
14
Test - Muskrat Lake, Test Compound No. 73.
Northern Texas Test Site
Whole Pond Test - North Texas, Compound No. 73
Fish Toxlcity Tests - Blue Gills, Test Compound
No. 73
Influence of Three Test Compounds on the Phago-
60
66
67
Rl
87
f\ r*"
95
cytic Activity of Ochromonas danica at 0.2 ppm . 112
iv .
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LIST OP TABLES
Table
Number
1 Chowan River - First Test, Anabaena spp.
and Microcystis montana . 33
2 Chowan River - Second Test, Oscillatoria
tenuis and Agmenellum quadruplica'tum"!~. ... 34
3 Chowan River - First Test, Microcystis cyariea
and Microcystis incerta 36
4 Chowan River - Second Test, Anabaena spp. and
Microcystis cyanea 39
5 Chowan River - Second Test, Oscillatoria
planctonica and Microcystis incerta 40
6 Chowan.River - Second Test, Compound Depletion
Patterns 42
7 Chowan River - First Test, Compound Depletion
Patterns 43
8 Degradation-Absorption Check on Compound No. 73
Under Controlled Conditions 45
9 Chowan River - Second Test, Dissolved Oxygen
Levels 47
10 Lake Sallie - First Test, Compound Depletion
Patterns ,. . 52
11 Lake Sallie - First Test, Test Compound Action
on Blue-Green Algae Aphanizomenon and C.
naegellanum 53
12 Lake Sallie - First Test, Test Compound Action
on Blue-Green Algae Anabaena and' Microcystis. . 54
13 Lake Sallie - First Test, Total Blue-Green
Algae 56
14 Lake Sallie - First Test, Total Algal Cell
Counts x loVml 57
v
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LIST OP TABLES continued
Table
Number Page
15 Lake Sallie - First Test, Test Compound
Action on Oocystls and Fraglllaria ....... 58
16 Muskrat Lake - Second Minnesota Test,
Compound Depletion Patterns ........... 61
17 Muskrat Lake - Second Minnesota Test,
Anabaena and Aphani zomenon ........... 62
18 Muskrat Lake - Second Minnesota Test,
Coelosphaerium and Raphidiopsis ......... 63
19 Muskrat Lake - Second Minnesota Test,
Oscillatoria .................. 6H
20 Diamond Lake - First Test , Compound
Depletion Patterns . . . . » .......... 69
21 Diamond Lake - First Test, Blue-Green Alga
Anabaena .................... 70
22 Diamond Lake - First Test, Diatoms - Synedra
and Stephanodiscus ........... .... 71
23 Diamond Lake - First Test, Fluorometric
Relative Intensity Readings ........... 72
24 Diamond Lake - First Test, Chlorophyl "A" and
Carotenoid Levels ................ 74
25 Diamond Lake - Second Test, Compound Concen-
trations .......... k ......... 76
26 Diamond Lake - Second Test, Blue-Green Alga
Anabaena .... ................ 78
27 Diamond Lake - Second Test, Green Alga
Gleocystis ..... . . . . * ......... 79
28 Diamond Lake - Second Test, Staurastrum and
Stephanodiscus ......... ........ 80
29 North Test Test Site - Monitored Test Compound
Levels ..................... 84
vi
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LIST OF TABLES continued
Table
Number Page
30 North Texas - Whole Pond Test, Oscillatoria
and Agmenellum 85
31 North Texas - Whole Pond Test, Anabaena and
Microcystis 88
32 Phase III Field Test Results, Algal Control -
Compound No. 73 89
33 Phase III Field Test Results, Algal Control -
Compound No. 23 90
34 Fish Toxicity Tests - Blue Gills, Compound
No. 23 92
35 Fish Toxicity Tests - Blue Gills, Compound
No. 73 94
36 Fish Toxicity Tests - Rainbow Trout, Compound
No. 73 96
37 Laboratory Screening Tests, Test Compounds
versus Anabaena flos^-aquae 97
38 Laboratory Screening Tests, Test Compounds
versus Microcystis aeruginosa 104
39 Algaecidal Activity of CuSO»»5H20 Against
Two Species of Blue-Green Algae 106
40 Algaecidal Activity of Cutrine Against Two
Species of Blue-Green Algae. 1°7
41 pH Sensitivity Tests on Compounds No. 23 and
No- 73 Against Microcystis 1°8
42 Algaecidal Activity - Biomass Dependence Test
versus Anabaena flos-aquae 1°9
43 Influence of Test Compound No. 117 at 0.2 ppm
on the Phagocytic Activity of Four Species
of Ochromonas
vii
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LIST OP TABLES continued
Table
Number
114
44 Ochromonas Storage-Viability Study
viii
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ACKNOWLEDGEMENTS
The work done in connection with this project was
coordinated by Dr. B. L. Prows, principal investigator,
with the assistance of W. P. Mcllhenny, project director.
C. P. Ward assisted in the laboratory and field work.
We are indebted to each of the site investigators in
the four principal test areas, for their interest, cooper-
ation, and efforts in gathering background data and direct-
ing the field tests in their respective areas: Dr. B. J.
Copeland and Jim McKenzie of the North Carolina State
University; Dr. J. K. Neel and Dave Brakke of the Univer-
sity of North Dakota; personnel from the Lake Sallie,
Minnesota State Fish Hatchery; Dr. H. Horton and Jim Rybock
of Oregon State University; Dr. B. D. Vance and his graduate
students from North Texas State University.
Appreciation is also expressed to the various state officials
whose cooperation made the site utilization possible, and to
Drs. M. J. Wynne and G. T. Cole of the Department of Botany,
University of Texas at Austin, who served as consultants and
investigators on Biological Algal Control mechanisms.
The support of The National Environmental Research Center of
the United States Environmental Protection Agency, and of
Dr. T. E. Maloney of the National Eutrophication program,
who was the Federal Project Officer, is gratefully acknowledged.
ix
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SECTION I
CONCLUSIONS
Data collected under a wide variety of conditions in four
geographically diverse regions of the United States indi-
cate a rather distinctive pattern of blue-green algal
control by both of the two prime candidate algaecidal
compounds, No. 23, 2,5-Dichloro-3,4-dinitrothiophene; and
No. 73» p"-Chlorophenyl-2-thienyl iodonium chloride. Other
supporting data regarding compound selectivity, environ-
mental persistence and fish toxlcity was definitive.
The following specific conclusions are reached:
1. Compound No. 73» having met all of the original objec-
tives, has emerged as the best candidate. It is an
effective chemical for algal control; it is safe to
applicators, fish, and other higher aquatic plants and
animals; it has a fairly rapid degradation pattern
under open atmospheric conditions, with a half-life of
one to two days; and it also exhibits a fairly high
degree of specificity for the target algae, particularly
Anabaena, Microcystis, Aphani zomenon» and Oscillatoria.
It is relatively inactive against diatoms and most green
algae.
2. Compound No. 23 also proved to be an effective algae-
cid and met most of the criteria, but was found to be
toxic to fish life and thus had to be eliminated from
the test program.
3. Through the continuing laboratory screening of 70
additional compounds; No. 136, 2,2'-(l,2-Ethenediyl)
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bisbenzoxazole; and No. 176, l,2-Dichloro-4-(lsothio-
cyanatomethoxy) benzene; exhibited good algaecidal
properties, showing 91 to 97 percent control of Anabaena
at the 0.8 ppm level.
Continued efforts toward development of a biological-
chemical control system have resulted in further invest-
igations of the phagocytic algal organism, Ochromorias
ovalis, which has proven to be a voracious feeder on
the blue-green alga Microcystis aeruginosa, and have
shown that Ochromonas can retain some viability for more
than 70 days at room temperature when imbibed in acti-
vated charcoal or cotton and polyester fibers; whole
pond field tests with this organism for control of
Microcystis were unsuccessful as the Ochromonas was
apparently unable to cope with the new environmental
conditions present at the time.
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SECTION II
RECOMMENDATIONS
In view of the positive results obtained thus far in the
long-range development program, and the relatively good
prognosis for successful achievement of the original
objectives, it is recommended that the research program
be continued at least to Phase IV, of the general develop-
ment plan, under government funding. There are certain
inherant uncertainties and areas which should be investi-
gated in greater depth to determine whether the project
should be advanced, terminated, or modified at the
conclusion of Phase IV.
It is suggested that this next ph^se should Include:
1. Additional field tests under natural conditions,
especially in larger single ponds, small lakes, and
fish hatchery empoundments. to obtain more informa-
tion on the algal control efficacy of the candidate
compound.
2. Further tests to determine the compound's environmental
safety, Including expanded animal and plant toxicities
and compound degradation patterns under natural conditions.
3. Continued laboratory testing, as new compound structures
shown to be potential algaecides are synthesized in
proprietary programs, or otherwise become available for
preliminary screening.
4. Continued Investigation of the biological control
phenomena by flagellated phagocytes, especially with
regard to the development of practical application methods,
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SECTION III
INTRODUCTION
All surface waters contain dissolved and suspended materials
which serve as nutrients and help support the growth of
algae and many other forms of aquatic life, the numbers and
variety of which are determined by the amounts and kinds
of nutrients which are available (Palmer, 1962). A certain
amount of natural eutrophication in our fresh water systems
is tolerable, and even desirable, for the support of fish
life and the necessary accompanying biota. Excessive
eutrophication, however, upsets the natural aquatic ecologi-
cal balance and causes many troublesome aesthetic and economic
problems.
In comparatively recent times the availability of adequate
supplies of good quality fresh water has come to be regarded
as one of our most valuable natural resources, and the con-
trol of nuisance algae growth and aquatic weeds is one of
the major concerns of the Environmental Protection Agency
(Prescott, 1970). To this end a cooperative plan of action
has been formulated for the collection of a sufficient range
of comparable data on the degree and extent to which nutrient
loading in our fresh water lakes is correlatable with the
rate at which eutrophication is developing (EPA, 1973)
Algae are found as common and natural inhabitants of all sur-
face waters and are especially abundant where the water is
exposed to direct sunlight. The portion of the available
solar radiation spectrum utilized is in the visible, infrared,
and ultraviolet regions, in the approximate ratio of 60:45:1,
respectively (Brown, 1973).
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To date more than 18,000 species of algae have been identified
(Palmer, 1962) but only a relatively small number of these,
principally the blue-greens, are considered to be notable
nuisance species.
Unlike the other groups of small microscopic organisms all
species of algae contain chlorophyll. Algae are responsible
for an estimated 90 percent of all photosynthetic activity
on the earth (Meyer, 1971). One pound of algae growth will
produce about 15 pounds of oxygen (MacKenthun and Ingram, 1964).
The constant increase in urbanization, accompanied by changes
in ground cover and surface soil, together with such phenomena
as forest fires, over-grazing, deforestation, and agriculture,
having increased run-off and reduced soil seepage to the
extent that, as estimated by some authorities, the underground
water table in the Eastern half of the U.S. has been lowered
about 60 feet in the last 50 years (Palmer, 1962). This, in
addition to a large increase in the use of ground waters, has
created severe shortages in many areas. Thus, as population
and industrial demands increase, attention has, of necessity,
turned more and more to lakes, streams, and reservoirs in order
to meet these needs.
A need for an efficient and economically feasible method for
controlling certain algal species has arisen as a result of
'excessive eutrophication of natural waters due to the con-
tinued increase in the wastes produced by the human population
as well as from indescriminate use of available water supplies.
Although there is no universal agreement among scientists
and environmentalists as to the particular elements which
are responsible for excessive algal populations, the most
commonly suspended nutrients are phosphates and nitrates.
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It has been observed that algae blooms are often associated
with waters which receive sewage effluents or other waters
which are rich in these components. Thus, it is widely
believed that in many bodies of fresh water, growth of
phytoplankton tends to be limited by the supply of inorganic
phosphate (Tailing, 1962). MacKenthun and McNab (1961) made
studies of several Wisconsin stabilization ponds and con-
cluded that the annual per capital contribution of soluble
phosphorous and inorganic nitrogen was 1.1 and 4.1 pounds,
resspectively.
When the physical and chemical conditions of a body of water
become optimal for a particular algal species, proliferation
may take place in such abundance as to produce visible aggre-
gations of floating algal masses. To produce such "algal
blooms" the combined growth conditions usually become optimal
for only one species at a time. Thus an algal bloom usually
contains one heavily predominant species.
Many types of algae, particularly the blue-greens, tend to
impart obnoxious tastes and orders to the water, clog intake
screens and rapid sand filters of water treatment plants, and
produce unsightly collections of debris on shores, making the
water unsuitable for many desired uses.
The presence of algae in water supplies has been known to
cause digestive upsets or even death to certain warm-blooded
animals due to toxic substances released to the water. A
number of case histories are recorded in which -certain genera
of Cyanophyta (blue-green algae) such as Mlcrocystis.
Aphanizomenon and Anabaena are known to have caused animal
deaths, particularly in areas where the wind may have concen-
trated the algae into leeshore areas (MacKenthun and Ingram,
1964).
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A rather common occurrance resulting from heavy algal
blooms is a severe fish kill due to the depletion of
dissolved oxygen in the water. Even though algae do impart
oxygen to the water, through photosynthesis during daylight
hours, the metabolic and catabolic processes taking place
in living plant cells continuously utilize oxygen both day
and night. Should a heavy cloud cover reduce the oxygen-
producing phot©synthetic activity of the algae to an abnor-
mally low level over a several-day period, the dissolved
oxygen level during the night, when photosynthesis ceases
and oxygen-consuming digestive processes still continue,
may fall below the critical level required to sustain life
in many species of fish, and massive fish kills may result.
Game fish such as trout, which have high dissolved oxygen
requirements are usually the first to be affected in such
cases.
The pH of the water will tend to increase, as algae extract
carbon dioxide from the water for photosynthetic action, thus
reducing the amount of soluble carbonic acid in the water, as
well as the intermediately soluble bicarbonates and the nearly
insoluble monocarbonates, usually causing part of the latter
to precipitate. Water storage and transport problems may also
be caused by the depolarizing action of the oxygen produced
during photosynthesis (Palmer, 1962).
The literature documents the damage to our inland waters in
recent years as a result of excessive algal growth. For
example, Lake Washington in 1959* contained a maximum phyto-
plankton population of 1.5 x 106 uVrnl, of which only 15 per-
cent was made up of blue-green algae. By 1963, the phytoplank-
ton population had increased ten-fold and consisted of 95 per-
cent blue-greens (Bartsch, 1967).
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In contrast to most species of non-flagellated algae which
settle to the bottoms of lakes in calm weather, many plank-
tonic blue-greens exhibit an "upside-down" characteristic,
accumulating as dense scums on the surface, which may then
be blown by the wind into thick windrows and piled upon the
shorelines. Often an accompanying offensive "pig-pen" odor
results from the decaying material (MacKenthun and Ingram,
1964). Also, bad tastes as well as decomposition products
are imparted to the water which may become toxic to animals
and humans alike (Bartsch, 1967).
The problems associated with nuisance algal control are
being investigated through the National Eutrophication Research
Program of the Environmental Protection Agency. Basically
the investigative efforts fall into four broad categories:
mechanical, biological, ecological and chemical. Mechanical
approaches involve the engineering and design of machines and
equipment for underwater mowing, raking, and harvesting of
certain algae and higher aquatic weeds. Biological control
mechanisms being investigated include viruses, insects,
algae-eating fish, and phagocytic organisms. Ecological
approaches include diversion of nutrient-rich waters, flushing
of lakes and ponds with nutrient-poor water and nutrient-
removal by aquatic plant crops, or by flocculation and adsorp-
tion methods. Due to the great variety and complexity of
conditions in which algae problems may exist, it is unlikely
that any one single approach to the control of unwanted algae
would be suitable in all cases. Without question, one of the
basic solutions to the control of algae lies in the control
of nutrient levels in the water. The nutrients and the
required nutrient levels are not well understood, and the
difficulties involved in controlling excessive eutrophication
are so great that alternative methods of algae control will
likely persist in the future for a considerable length of time.
8
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For many situations, a chemical approach to the control of
nuisance algae growths will be suitable, particularly in
smaller lakes, streams and impoundments where algae "blooms"
are imminent and the need to restore ecological balance
exists.
Chemical approaches (the use of algaecides) have not been
as thoroughly investigated because of the difficulties
encountered in developing a compound which will selectively
kill or inhibit reproduction of the target algal species
without adversely affecting the other more desirable forms
of aquatic life.
For a number of years, the most widely used algaecidal
compound on the market has been copper sulfate. Despite
its extensive usage, copper sulfate has disadvantages such
as toxicity to desirable aquatic life at higher concentrations,
non-biodegradability, accumulations of copper salts in bottom
muds, and corrosive properties to paint and equipment
(Bartsch, 195*0. Hasler (19^7) and Kuentzel (1969) point out
the possible deleterious effects of lake-bottom accumulations
of copper sulfate on lake ecology. Investigations conducted
in Wisconsin and Minnesota by Moyle (19^9) indicate that
certain algae, particularly the blue-green alga Aphanizomenon
seem to have acquired an increased tolerance to copper sulfate
as a result of many years of successive treatments; in this
case up to five times as much copper sulfate was needed for
control of Aphanizomenon as was required 20 years earlier.
There is no federally registered chemical available for the
selective control of nuisance species of blue-green algae
in surface waters which are to be used for potable purposes.
A considerable amount of time and effort has been expended
in recent years in search of a compound which would replace
copper sulfate as an algaecide, and at the same time prove
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to be safe to non-target organisms, non-cumulative in
the environment, and be economically feasible to use for
such purposes (MacKenthun, et al, 1964).
It is important to understand that the development of a
compound for widespread use in the environment, particularly
where recreational and potable waters are concerned, is a
long-range and expensive process.
Phase I of this contractual effort to "Develop a Selective
Algaecide to Control Nuisance Algal Growth", was initiated
April 5, 1969. The primary objective was to develop a
compound which would control the growth of various species
of blue-green algae effectively, safely, and economically,
while exhibiting a minimum impact on other forms of life
in the aquatic environment.
The following criteria were specified as general guidelines
for compound selection and development. A satisfactory
algaecidal compound must have:
1. High activity against the target algae, specifically,
against selected species of blue-green algae.
2. Low toxicity levels for mammals, fish, and other
desirable aquatic organisms.
3. Low toxicity to terrestrial plants
4. No questionable elements such as arsenic, mercury
or other heavy metals, in its structure.
The approach to the problem involved initially making a
computer search of some 80,000 compounds in Dow's computer
listing of compounds in order to select those having the
10
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highest probability of meeting the established criteria
for an ideal algaecide. Most of these compounds had al-
ready been screened against at least one species of algae
and also for activity against higher aquatic plants, fish,
and some terrestrial plants and animals. A final hand
selection was then made from the computer printout, elim-
inating those compounds which contained heavy metals and
those which were likely to be costly, or which possessed
inherent or demonstrated undesirable properties, such as
high toxicities to fish, terrestrial plants, or mammals.
The candidate compounds, thus selected, were then divided
into several priority groups according to the established
selection criteria.
Within the 12-month contractual period, 33 compounds were
screened using the two target species of blue-green algae
suggested by EPA's Federal Water Quality Office, Corvallis,
Oregon. Each compound was tested against cultures of
Microcystis aeruginosa and Anabaena flos-aquae at a concen-
tration of 2.0 ppm in constant-temperature water bath shakers
(24°C) at 80 oscillations per minute under a cool-white
fluorescent light intensity of 100 foot-candles. Compound
activity was expressed as percent control, as compared to
control cultures which were run simultaneously under identical
conditions. Cell growth was monitored by visual inspections,
cell counts, and relative intensity readings using a fluoro-
microphotometer. Those compounds which passed the first
screening test with at least 80 percent control at 2.0 ppm
against both test species were selected for more detailed
testing at 1.0, 0.5» and 0.1 ppm. Of the 33 compounds tested,
Compound Nos. 23 (2,5-Dichloro-3,4-dinitrothiophene), 15
([5-Chloro-2-(p-nitrophenoxy)phenyl]phenyl iodonium chloride),
8 (4-Amino-2,5-dibromophenyl thiocyanate), and 24 (1,1-Di-
methyl tetradecyamine hydrochloride) were effective at these
11
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concentrations and were selected as the prime candidate
compounds for further research through Phase II of the
long-range algaecidal development program (Prows, 1971)-
A phagocytic organism, identified as Ochromonas oval is,
was discovered which showed promise as a means of control-
ing Microcystis. In addition, it was found that the growth
and activity of Ochromonas was enhanced by low concentra-
tions of some of the test compounds being studied.
As specified in the contractual work statement efforts were
to be directed toward the long-range goals of developing
an algaecidal compound which would:
1. be safe to non-target organisms
2. exhibit a high degree of specificity for the target algae
3. be economical
4. be safe to applicators
5. be non-persistent in aquatic systems
Phase II of the algaecide development program was begun
June 30, 1971- Initially a computerized structure search
of more than 100,000 compounds was made in order to select
the analogs of the following four Phase I prime candidates:
2,5-Dichloro-3,4-dinitrothiophene; [5-Chloro-2-(p-nitro-
phenoxy)phenyl] phenyl iodonium chloride; 4-Amino-2,5-di-
bromophenyl thiocyanate; and 1,1-Dimethyl tetradecylamine
hydrochloride. This endeavor resulted in the selection of
1309 analogs, which were each subjected to rapid agar-plate
screening tests. Forty-one compounds emerged from these
tests as candidates for final laboratory screenings. Although
six of these compounds showed high levels of activity against
12
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the target blue-green algal species, four proved to be
unacceptable because of poor economic feasibility, environ-
mentally unacceptable properties, or difficulty or danger
in compound production.
At the conclusion of Phase II, Compounds No. 23 (2,5-Di-
chloro-3,4-dinitrothiophene) and 73 ([p-Chlorophenyl]-2-
thienyl iodonium chloride) were selected as final candidates
based on superior algaecidal activity, environmental
acceptability, economic feasibility, and freedom from human
health and handling hazards (Prows and Mcllhenny, 1973).
i
Three additional species of Ochromonas were discovered
during Phase II which exhibited phagocytic activity against
Microcystis aeruginosa. However, the original species
discovered during Phase I exhibited the greatest activity
and showed some activity improvement when used with low
levels of certain test compounds.
The Phase III effort was begun on November 28, 1972. The
objectives of this third phase were to test the candidate
compounds under natural field conditions to obtain informa-
tion on the algal control efficiency, and of combined
biological-chemical systems in naturally occurring field
situations. The stability and environmental stability of
the compounds of concern were to be determined.
The initial approach was to synthesize large enough quantities
for use in field tests. Analytical test procedures were to
be developed in order that the persistence, adsorption, and
degradability rates could be followed during and after treat-
ment. The compounds were then to be field tested in small
lakes and ponds having blooms of nuisance algae. Careful
13
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monitoring as well as the analysis of water, bottom muds,
and higher aquatic plants. Toxicity studies on fish and
mammals were to be undertaken. The biological and chemical-
biological systems using Ochromonas were also to be further
studied.
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SECTION IV
EXPERIMENTAL PROCEDURES
The primary objective of this research project was to
determine under natural, open-field conditions, the efficacy
of two prime candidate algaecides, No. 23 (2,5-Dichloro-
3,^-dinitrothiophene) and No. 73 (p-Chloro-phenyl-2-thienyl-
iodonium chloride) which were final candidates at the con-
clusion of the Phase II effort. Specific efforts were to be
directed toward further delineation of the toxicological
properties and environmental persistence of these compounds,
with objectives focused on further defining an effective
chemical or biological-chemical system which would control
/
the growth of blue-green algae safely and economically while
exhibiting a high degree of specificity for the target algae.
In order to have sufficient quantities of the candidate com-
pounds to allow the toxicology tests to be run and to have
sufficient for each of the four field tests planned for various
sectors of the country, a re-synthesis of the two test com-
pounds was necessary. Twenty pounds of Compound No. 23 was
manufactured according to specifications by Pharm-Eco Corpora-
tion, Simi Valley, California. A like quantity of Compound
No. 73 was made in several successive batches at Dow's Organic
Synthesis Labs in Midland, Michigan.
ANALYTICAL PROCEDURES
During the developmental stages of a pesticide, particularly
if it is to be used in the aquatic environment, it is man-
datory that a fast and accurate means be developed for
detecting compound residuals as a function of elapsed time.
15
-------
In this case the procedure had to be capable of monitoring
traces of the compound in very dilute concentrations,
preferably in the parts per billion range, .since initial
treatment concentrations were planned at around 1.0 ppm,
and it was desirable to follow the compounds' persistence,
adsorption, and degradation rates for several weeks following
each treatment.
COMPOUND NO. 23
Both test compounds were examined by differential pulse
polarography but the lowest detection limit achievable
for Compound No. 73 was 3.0 ppm and the method was therefore
unacceptable for this compound. The instrument used was a
Princeton Applied Research Model 174 Polarographic Analyzer
equipped with Model 174/70 drop timer to record the differ-
ential pulse polarograms.
A standard solution of Compound No. 23, prepared in methanol,
was found to possess two polarographic waves in a sodium
acetate supporting electrolyte, with half-wave potentials of
-0.13 and 0.27v versus a saturated calomel reference electrode
(SCE). Sodium acetate itself gave no response in this poten-
tial region, but when high instrument sensitivities were
employed to achieve the desired response, interference due to
impurities appeared. After electrochemical purification for
a week (or longer), the sodium acetate was found to be free
of interferences at the desired level of sensitivity. When
laboratory distilled water and purified sodium acetate were
used, a useful response was obtained from 20 ppb of the
compound. Instrument response was linear with concentrations
from 20 ppb to 20 ppm.
16
-------
A sample of Tittabawassee River water was obtained upstream
from Dow in Midland, Michigan, to check for interferences
from natural waters. Purified sodium acetate was added to
the river water and a polarographic scan was recorded. No
interferences were observed in the potential region of
interest.
At this point, it was desired to establish the stability of
solutions of the compound so that proper shipping arrangements
could be made, since test sites were scheduled for various
parts of the United States. Initial dosage levels were
expected to be 1.6 and 0.8 ppm, and therefore, a 0.6 ppm
stock solution was selected for stability studies. Aliquots
of this solution were determined polarographically at various
time intervals. The compound remained stable at this concen-
tration for at least a week under ordinary laboratory condi-
tions .
Stability was also checked in Tittabawassee River water and
similar results were obtained, although the background signal
from untreated water increased with time, which would limit
detection of trace amounts. Stability was also checked on
refrigerated and frozen samples since it was planned to
ship samples packed in dry ice. No adverse effects were ob-
served.
From this procedure it was concluded that 2,5-Dichloro-3,4-
dinitrothiophene could be determined polarographically in
natural waters down to the 20 ppb level. The method proved
to be quite specific, due to the presence of two polarographic
waves.
17
-------
COMPOUND NO. 73
Liquid chromatography was finally selected as the analytical
method of choice for determination of Compound No. 73- The
test compound concentrations were monitored at the elution
end of the column by a Perkin Elmer "1250" ultraviolet
detector at the 254 nanometer range.
The original gradient elution system developed was later
modified by a more efficient and time-saving procedure.
In the gradient method, the water sample was injected into
a strong cation exchange resin (VYDAC-SCX). The water and
organics were first eluted with a 1:1 water/methanol solution;
the iodonium chloride, which had been retained on the column
was then gradient eluted with a dilute (0.02 M) sodium
perchlorate solution in 1:1 methanol/water. It was also
found that methanol was as effective a mobile phase solvent
as the water/methanol system. After the iodonium chloride
was eluted, the column was then washed for ten minutes with
the original solvent to remove all sodium ions. Due to the
column washings, associated with the gradient system, a sample
run lasted 20 to 30 minutes. Thus only about 15 samples, plus
the appropriate standards, could be run per day with this
method.
Recent work with chlorophenols has demonstrated that penta-
chlorophenol and tetrachlorophenol can be separated by ion
exchange chromatography using constant composition elution
with a weakly ionic mobile phase. Applying this principle
to the iodonium chloride/water system, it was found that by
using a'constant composition mobile phase consisting of 0.01 M
sodium perchlorate in methanol, the iodonium chloride was suf-
ficiently retarded in passing through the column to be separated
from the unretarded water and organics. With this method a
18
-------
30-sample shipment could be analyzed in three hours. There
were also no diffuse negative peaks associated with the
solvent change preceding the iodonium chloride peak. The
ionic strength needed to Just separate the bacteriostat from
the water/organics was constant for a given column but changed
when a new column was packed.
A series of standard solutions was prepared and chromato-
graphed with this technique. The chromatographic response
was found to be linear in the 0.3 to 5.0 ppm range. For a
given standard the reponse was also found to be reproducible
to within ±1.5 percent of the mean response.
Thus, the analysis of 4-Chlorophenyl-2-thienyl iodonium
chloride in natural water was simplified by replacing a two-
solution gradient elution system with a single-solution
procedure. The resulting 80 percent reduction in analysis
time permitted prompt analysis of large numbers of samples,
thereby circumventing problems of degradation in storage.
The linearity and reproducibility of this method was found
to be very good with standard solutions.
FIELD TESTS
Selection of Sites
It was proposed that test sites be selected in at least four
different sectors of the United States so as to provide a
good climatic cross-section with naturally occurring algal
blooms. The general areas were selected with the advise of
EPA personnel.
Specific sites were selected in connection with algologists
whose previous work and interest had been associated with the
19
-------
limnological aspects of lakes and streams in the local areas
where the particular Universities were located. The following
criteria were used as a basis for selection of the test
sites:
1. past history of algal populations and blooms
2. freedom from animal and human interactions
3. proximity of test sites to research personnel and
laboratory facilities
4. prognosis of appropriate agency approval for permission
to perform the tests
On a site-selection tour of the proposed test areas early in
May, 1973, final details and arrangements for carrying out
the field testing programs were made with the appropriate
university investigators. Suitable bodies of water with
reasonable proximities to the institutions concerned were
chosen.
The following is a list of the selected sites, together with
the university professors cooperating as area investigators:
1. Chowan River, North Carolina
Dr. B. J. Copeland, University of North Carolina
2. Lake Sallie, Minnesota
Dr. Joe K. Neel, University of North Dakota
3. Diamond Lake, Oregon
Dr. Howard Horton, Oregon State University
4. Slidell, Texas
Dr. Dwain Vance, North Texas State University
20
-------
During the inital site selection tour, contacts and liaison
was established by the cooperating investigators with the
appropriate government and state agencies at each site to
assure their cooperation and to obtain the necessary approvals
for the tests to be performed. No problems with state or
local approval were encountered.
Field Procedures
Due to the limited amount of test compound available, and
the need to contain the test compounds in small areas because
of their experimental nature, it was decided to use 60-gallon
plastic containment bags as the test vessels at each site,
except for the North Texas site, where a whole pond was to be
treated.
Six test vessels were designed for use at each site. These
were supported by polyurethane flotation collars, filled with
ambient lake waters, and held in position by means of auxiliary
flotation devices which were anchored to the lake bottom with
concrete anchors (Figure 1). A self-priming DC pump, powered
by a 12-volt battery was used as an aid in filling the test
vessels to a measured 60-gallon depth with ambient algae-
infested lake water.
The water in the vessels was then treated by Dow personnel
with selected, pre-determined concentrations of test compound,
with some vessels serving as controls, in addition to the sur-
rounding lake water itself.
Water samples were taken from the lake and from each vessel
immediately before and following treatment, for the purposes
of compound concentration monitoring, as well as the monitoring
of algal population numbers as a function of time.
21
-------
Figure 1.
OPEN FIELD TEST SYSTEM
Polyurethane auxiliary
flotation device
25" OD
Polyurethane
60-gallon
polyethylene
bag
30-lb. concrete anchor
.x^A ^ L& -_ -^ ^&~ ^
-& / ~^^w- ^_ ^-^fe
22
-------
Approximately 200 ml samples were taken and placed in plastic
biological "twirl pack" bags. Those collected for algal
monitoring were treated with formalin solution as preserva-
tive measure to assure counting validity. Samples which were
taken for compound monitoring were placed in a second pro-
tective bag before freezing and were then packed in dry ice
for shipment via air freight to Midland, Michigan, for
analysis.
Water chemistry data, which varied somewhat with individual
preferences of the site investigators, but always included
temperature, pH, dissolved oxygen, and alkalinity, were taken
before, during, and following treatment according to the
recommended schedule.
SAMPLE SHIPMENT
In order to eliminate inconsistencies in sample handling and
shipment and to help assure rapid and safe delivery via air
freight, the following suggestions were made in a letter sent
to each site investigator:
All samples which are collected for compound analysis and
sent to Midland, Michigan, should be:
1. Sealed securely in primary collection bags (200-300 ml)
and then placed in a second protective plastic bag.
I
2. Double labeled - (a) pencil writing on a cardboard
label, placed inside the second
bag.
(b) writing on outside or primary
collection bag with a permanent-
ink felt tipped pen.
23
-------
3. Uniformly labeled as follows:
I-C (this Is control no. 1, with no test
compound)
II-C1 (this is control no. 2, with no test
compound)
III-23-1.6 (test compound no. 23 at 1.6 ppm)'
IV-23-0.7 (test compound no. 23 at 0.8 ppm)
V-73-1.6 (test compound no. 73 at 1.6 ppm-)
VI-73-0.8 (test compound no. 73 at 0.8 ppm)
Vll-lake (ambient lake water outside of test
vessel)
4. Quick-frozen as soon as possible after sample
collection and retained in a frozen state until
packaged for shipment.
5. Packaged in a sturdy, well-insulated shipping carbon,
with at least four pounds of dry ice packed around,
but protected from, direct contact with the sample
bags (utilizing newspapers, or other good packing
materials).
6. Package should be labeled "fragile" and sent via air
freight by the most direct routing possible using
shipping labels which specify the presence of biological
samples, packed in dry ice, and a specific request to
handle with care, and to deliver on or before a specified
date. The only two major air lines which services the
Midland, Michigan, area directly are North Central and
United.
-------
FORMULATION AND DISPENSING
A rather extensive study was undertaken in order to deter-
mine the optimum procedures for formulation of the two
compounds. Several solvent systems, balanced to a specific
gravity of 1.00 were utilized in the study. Compound No. 23,
having a low water solubility, was found to be readily
soluble in both acetone and xylene. However, when dissolved
in acetone the compound was observed to crystallize and fall
out of solution when the concentrate was combined with water
in the normal temperature range one would expect to find in
lakes and streams. The utilization of xylene as the basic
solvent required the addition of about 19 percent perchloro-
ethylene, to yield the desired 1.00 specific gravity balance.
The use yof a surfactant as an emulsifying agent was also re-
required.
The principal governing factor for the optimum formation of
molecular compounds in such cases is the realization of the
best hydrophylic-lipophylic balance, permitting the formation
of lytotropic mesomorphous phases or dispersion into miscelles,
The optimum formulation for Compound No. 23 was determined as
a 25:75 ratio of anionic :nonionic surfactant in a xylene-
perchloroethylene solvent system.
Compound No. 73 was found to be 0.42 percent soluble in water
at room temperature and about 4 to 5 percent soluble in
methanol. The increased solubility in methanol was not con-
sidered sufficient justification in itself to prompt its
use as a solvent^ which would tend to escalate the economics
of the compound when considered for large-scale use. However,
the compound was solubilized in methanol for use in certain
portions of some of the field tests to determine whether that
particular solvent system would affect the Compound's algae-
cidal activity.
25
-------
A day or two prior to treatment, primary concentrates of the
test compounds were made up in pre-determined aliquots in the
laboratory and then taken to the treatment site. For later
treatments the procedure was modified by taking unmixed, pre-
weighed quantities of the original compound to the test sites
and combining with lake water in brown 500 ml bottles to
make a "use" concentrate just prior to treatment in order to
avoid the possibility of compound degradation. After the
"use" concentrates were prepared they were kept away from
direct sunlight as an additional safeguard against possible
photodegradation. Also, the tests were not begun until near
sundown, for the same reasons.
At the North Texas test site where a whole-pond treatment was
undertaken, a "use" concentrate of Compound No. 73 was made
by addition of a pre-determined portion of the compound to
500 gallons of water in a commercial sprayer, and continuously
agitated for one hour before application. The concentrate
was then sprayed onto the surface of the pond just before sun-
down.
LABORATORY SCREENING TESTS
According to plan, screening tests were to be continued on
candidate compounds as they were sent to this laboratory
from various sources within Dow and elsewhere. The basic
screening procedure was modified slightly from that which
was used in the Phase III work (Prows and Mcllhenny, 1973).
All primary screening tests were run for four days against
Anabaena only, at 1.6 ppm and 0.8 ppm. If secondary tests
were run they included tests down to O.U ppm, or lower if
deemed expedient, against both target algal species, Anabaena
flos-aquae and Microcystis aeruginosa.
26
-------
In preparation for the screening tests, stock cultures of
Anabaena and Microcystis were grown in 1000 ml flasks,
cultured in Gorham's medium under 100 foot-candles cool,
white light. Twenty-four hours prior to treatment standard
inoculum cultures were made up from the initial stock cul-
tures by placing sufficient algal cells in fresh Gorham's
medium to bring the standard inoculum to a relative inten-
sity of 0.20. After 24 hours of acclimation in the new
medium, the inoculum was ready for use. At "0-day" for a
particular test, 29 ml of standard inoculum was placed in
each of the 125 ml culture flasks and then inoculated with
1.0 ml formulated test compound of the appropriate concen-
tration such that the 1:30 dilution factor would reduce the
final concentration to the desired levels.
The test vessels and their controls were then kept under
a constant illumination of 100 foot-candles, cool white
fluorescent lighting, and agitated at 80 excursions per
minute in 24°C water bath shakers throughout the four-day
test period.
Culture growth in each test flask was monitored by cell counts
where appropriate, and by relative intensity readings, taken
with an AMINCO fluoromicrophotometer, equipped with a blue
mercury-vapor fluorescent lamp for the primary illumination
source. Light from the lamp was filtered through a No. 55^3
band pass filter before entering the sample. Subsequent light
being emitted from the fluorescing chlorophyll molecules thus
provided an extremely sensitive method of determining in vitro
the relative number of living algal cells remaining, without
having to go through a time-consuming chlorophyll extraction
procedure.
27
-------
After substracting the background Intensities read for each
test sample the final relative intensities compared to
those of the control cultures gave a basis for computation
of the percent control as exhibited by various concentrations
of the test compound in question.
28
-------
SECTION V
DISCUSSION
The primary research efforts of the Phase III program have
been to determine the algal control efficacy of two
selected algaecidal compounds. These two compounds, No. 23
(2-Dichloro-3,4-dinitrothiophene) and No. 73 ([p-Chloro-
phenyl]2-thienyl iodonium chloride) emerged from the Phase
II study as prime candidates for field testing because of
their effectiveness against two of the commonly troublesome
blue-green algal species, Anabaena flos-aquae and Microcystis
aeruginosa.
PIELD TEST RESULTS
The four test sites were diversely located to give a good
cross-section of climatic conditions. Because each test
was monitored by a different group, and because the tests
were conducted sequentially there are slight differences in
the methods of handling the field data.
The general plan was to include two tests at each site dur-
ing the 1973 summer and fall seasons. Two tests were made
at each site, except for North Texas.
In each case the polyurethane flotation bobs for the enclosure,
the flotation collars, and the 60 gallon plastic test bags
were prepared at Dow and sent to the area investigators pre-
vious to the tests. The complete sets of data collected at
each site during each test are included in this report in
Appendix B.
29
-------
Chowan River, North Carolina
The North Carolina test site was located on the west shore-
line of the Chowan River, about 20 miles southeast of
Ahoske, NC (Figure 2). The Chowan River is about four miles
wide at that point. Heavy blue-green algal blooms of
Anabaena, Aphanlzomenan, and Microcystis had been reported
as having occurred in the particular section of the River
in previous years, being especially common during the summer
of 1972. A fertilizer plant, located about 30 miles upstream
is reported to have been responsible for a considerable amount
of nutrient input to the river, which may have contributed to
optimal "bloom" conditions.
The specific location of the test site was located Just
to the North of the Perry-Wynn Pish Processing Plant, the
owners of which were cooperative and agreeable for the
tests to be conducted about 200 yards offshore from their
property.
The first test was initiated on June 14, 1973, and a second
test was conducted on August 7, 197 3» when a much heavier
bloom was present.
Specific clearance for conducting tests with experimental
compounds was requested and received from the North Carolina
State Department of Air and Water Resources by the area
investigator, Dr. B. J. Copeland, North Carolina State Univer-
sity.
Of the six 60-gallon test vessels prepared for the field
tests, two contained only ambient river water, with no test
compound added; to serve as controls, in addition to the
surrounding river water itself. The other four vessels
30
-------
Tigure 2. North Carolina Test Site
VIRGINIA
^
Bu
"NORTH \CAROLINA
A = Test Site
B = United Piece Dye Works
C C. P. Industries -
Fertilizer Plant
rffjeesboro
_-'
Potecasi
oGamville
iunda,ry line
-------
were treated with sufficient quantities of pre-formulated
test compound concentrations of Compound No. 23 and Mo. 73»
to bring the initial algaecide levels to 0.8 ppm and 1.6 ppm
concentrations in the respective chambers.
The pre-weighed test compound aliquots were dissolved in
brown 500 ml bottles at the site, and then wrapped in
aluminum foil to serve as a light barrier and minimize the
possibility of photodegradation. As an added precaution
the test compounds were not administered to the test vessels
until near sundown.
The algal data, as reported by the North Carolina State
University investigators, indicated 100 percent activity
against Microcystis montana by both Compounds No. 23 and
and No. 73 at concentrations down to 0.8 ppm (Table 1).
Compound No. 23 was similarly active against Anabaena. The
action of Compound No. 73 against Anabaena was inconclusive
from the data received. Of the two blue-green algae,
Oscillatoria and Agmenellum, the former was brought under
nearly 100 percent control by both compounds at all con-
centrations, while the latter was affected very little by
either compound (Table 2).
An unsuspected phenomena was revealed by the specific
activity of Compound No. 23 against Microcystis cyanea,
compared to its relatively low activity against Microcystis
incerta (Figure 3). Compound No. 73 showed 100 percent
activity against M. cyanea at the 1.6 ppm level but the
population returned somewhat at 0+4 days (similar to the
action of Compound No. 23). Little or no activity was noted
against IYL_ incerta (Table 3). Further verification of this
32
-------
TABLE 1
Chowan River - First Test
(cells/liter x 10")
Sample
Ambient River Water
Controls (Avg. )
Compound No. 23-0.8 ppm
Compound No. 23-1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
Anabaena spp.
0-days
_L
15
26
11
16
12
10
12
Ik
HT
10
13
0+1 day
28
11
25
19
0
0
1
0
0
0
0
0+U days
*
13
0
0
0
1
0
28
0
~0
0+11 days
_5
0
10.
0
_L
0
Microcystis montana
33
-------
uo
fr
TABLE 2
Chowan River - Second Test
Oscillatoria tenuis AND Agmenellum quadriduplicatum
Sample
Controls
Compound No. 73 - 1.0 ppm
Compound No. 73 - 2.0 ppm
Compound No. 73 - 2.0 ppm*
Compound No. 73 - 3.0 ppm
(cells /I
0-day
16
93
16
IbT
16
98
16
98
11
Ta
iter x 10")
0+1 day
?
?
0
9F
0
26"
0
W
16
\
0+2 days
18
9
U
38-
10
35
6
31
0
*9
0+3 days
?
?
0
W
lU
70
if
1
10
0+6 days
U6
13
_?_
102
12
7F
_5_
29
U
?0
"Compound solubilized in methyl alchol
. Oscillatoria
Agmenellum
-------
125r-
o
X
r
£
c/i
01
O
c
o
M
(O
O.
O
(O
O)
Figure 3.
Chowan River - First Test
Test Compound No. 23 % 0.8
ppm vs. M^ cyanea & M_._ incerta
O Microcystis cyanea
/\ Microcystis incerta
» Test disrupted by adverse weather
O
11
Time (days)
*test disrupted by adverse weather
35
-------
TABLE 3
Chowan River - First Test
Microcystis cyanea AND Microcystis incerta
(cells/liter x 10")
Sample
Ambient River Water
Controls (Avg.)
Compound No. 23 - 0.8 ppm
Compound No. 23 - 1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
0-day
0
105
79
*f
£
0+1 day
8
11
0
5lT
0
115
16
36"
days
125
6h_
73
a
37
8
10
S9
21
0+11 days
JL
53
21
75
ii
52
73
KEY:
.Microcystis cyanea
Microcystis incerta
36
-------
specific activity is seen by examination of the data from
the second Chowan test conducted in August. One hundred
percent activity was observed against M_._ cyanea at 2.0 ppm,
but little or no significant activity was registered against
5-L ihcerta (Figure 4 ).
The target blue-green alga Anabaena was brought under about
95 percent control by Compound No. 73 at 1.0 ppm during the
second Chowan River test (Table 4).
The blue-green algae Oscillatoria planctonica was nearly 100
percent controlled at 0+2 days, but the population returned
to normal levels at 0+6 days (Table 5 ). It is strongly
suspected that the population return was the result of a
considerable amount of intermixing with river water, which
may have occurred during turbulent weather, or from a par-
tially ruptured bag.- Oscillatoria tenuis was drastically
affected by all four concentrations of Compound No. 73; in
contrast, Compound No. 73 showed little or no control of
Agmenellum quadruplicatum at the 1.0 ppm level and only
partial control at the 2.0 and 3.0 ppm levels (Table 2).
The compound depletion patterns of both test compounds in
the Chowan River samples from the two tests were similar
(Figure 5). During the second test, after six days, com-
pound No. 73 had been depleted to near or below the 0.1 ppm
detection limit in every test vessel (Table 6).
The determined initial compound concentrations in the first
test were only about 50 percent (or less) of the calculated
values, even though the samples arrived at Dow's Midland,
Michigan location still frozen and in good condition (Table 7).
37
-------
«
o
O)
o
o
£»
10
Q.
O
o.
(O
<
Figure 4.
Chowan River - Second Test
Test Compound No. 73 at 2.0
ppm vs. ML cyanea and M. incerta
O Microcystis cyanea
/\ Microcystis incerta
0
38
-------
U)
TABLE ^
Chowan River - Second Test
Anabeana spp. AND Microcystis cyanea
(cells/liter x 10°)
Sample
Controls
Compound No.
Compound No.
Compound No.
Compound No.
73 - 1.0 ppm
73-2.0 ppm
73 - 2.0 ppm*
73 - 3.0 ppm
0-day
113
"98
1U8
"28
138
-62
138
63
109
60
0+1 day
139
32
27
32
8
25
0
0
0+2 days
138
32
JL
5
4
2
0
JL
0
0+3 days
|
_5
21
29
6
0
11
2
0+6 days
12
0
18
21
31
10
0
20
0
Compound soluMlized in methyl alcohol
Anabaena
KEY:
Microcystis
-------
4=-
O
TABLE 5
Chowan River - Second Test
Oscillatoria planctonica AND Microcystis incerta
(cells/liter x 10°)
Sample 0-day 0+1 day
Controls
Compound No.
Compound No.
Compound No.
Compound No.
73 - 1.0 ppm
73 - 2.0 ppm
73-2.0 ppm*
73 - 3.0 ppm
^^^^^^^M
101
To
108
98
62
62
62
30
118
-8T
-L-
~97
o
25
1
70
71
lUl
31
6§
o
0
51
8
25
21
o
28
o
73
62
73
81
50
112
.
73.
1U7
*Compound solubilized in methyl alcohol
Oscillatoria
: Microcystis
-------
Figure 5.
Compound Depletion Patterns
Chowan River - First Test
l.Or
Compound No. 23
/\ Compound No. 73
0.8
(Theoretical Treatment Level)
Q. '
O
(B
*->
C
0)
o
o
0.6
234
Time After Treatment (Days)
ill
-------
I\J
TABLE 6
Chovan River - Second Test
COMPOUND DEPLETION PATTERNS
values im ppm
Sample 0-day 0+1 day 0+2 days 0+3 days 0+6 days
Ambient Lake
Control
Compound
Compound
Compound
Compound
No.
No.
No.
No.
Water <0.1 <0.
73
73
73
73
- 3
- 2
- 2
- 1
.0
.0
.0
.6
ppm
ppm
ppm*
ppm
<0.1
2.85
2.07
1.99
0.92
<0.
1.
0.
0.
0.
1
1
76
71
90
37
<0.1
<0.l
0.20
0.31*
0.33
<0.1
<0.1
<0.1
<0.1
0.11
O.lU
<0.1
-------
TABLE 1
CO
Sample
Ambient River Water
Control Vessel No. 1
Control Vessel No. 2
Compound No. 23 - 0.8 ppm
Compound No. 23 - 1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
Chowan River - First Test
COMPOUND DEPLETION PATTERNS
values in ppm
0-day 0+2 days 0+3 days
0 0
<0.05 <0.05
<0.05 <0.05
0.39 0.15
0.39 0.06
o.Uo 0.15
0.30 0.10
0+U days
0
<0.05
<0.05
<0.05
<0.05
0+6 days 0+11 days
0
<0.05
<0.05
__ __
-------
The analysis of samples from the second Chowan treatment
showed Initial concentrations very close to the calculated
values (Table 6), except in the 1.6 ppm test vessel in
which the measured concentration was only 0.92 ppm.
The mechanism of degradation is not yet fully understood.
Photodegradation is believed to be one of the mechanisms,
since a laboratory test run on samples prepared at 1.6 ppm
of Compound No. 73 sent to Dow's Midland location for
analysis revealed a compound loss of 56 percent on a sample
which had been exposed to diffused sunlight for 30 minutes
before shipment. This compared to only a 25 percent loss
of an identical sample which was not exposed to sunlight
(Table 8). The use of plastic bags for sample containers
did not prove to be deleterious, as had been previously
suspected, but shipping samples in an unfrozen state did
cause considerable sample loss. Both glass and plastic-
contained, unfrozen samples degraded 19 to 25 percent in
nine days without light, which indicates that biodegradation
is also involved in the depletion of Compound No. 73.
Water chemistry samples were collected on a regularly scheduled
basis during both of the Chowan River tests and the deter-
minations were made either on the site or later at a field
test station.
The phosphates were reported as reactive P0i» = , total filter-
able, and total unfilterable phosphates. No particular
pattern was seen among the various stations, although some-
what higher levels of phosphates were often, but not always,
noted in samples taken from the various test vessels, as
compared to ambient river water samples. The NHi»+ and nitrite-
-------
TABLE 8
Sample No.
I-73-G
Degradation-Absorption Check on
Compound No. 73 Under Controlled Conditions
Conditions
in capped glass bottle,
unfrozen
Initial
Concentration
1.6 ppm
Final
Cone.
0+9 days
1.2 ppm
II-73-P
in plastic bag, unfrozen 1.6 ppm
1.3 ppm
III-73-GF in capped glass bottle,
quick frozen in dry ice
1.6 ppm
broken
IV-73-PF in plastic bag, quick 1.6 ppm
frozen in dry ice
V-73-GS in capped glass bottle, 1.6 ppm
previously exposed to open
atmosphere for one hour
under overcast sky
1.7 ppm
0.7 ppm
-------
nitrogen levels reported were fairly consistant with those
in the ambient river water, except in the chambers contain-
ing Compound No. 23 where considerably higher levels were
noted up to 0+3 days, ranging as high as 11.4 mg/1. The
nitrate-nitrogen levels remained higher in the 1.6 ppm
tests using Compound No. 23 through 0+4 days. Increased
nitrogen concentrations in these vessels were attributed
to the nitrogen components in the molecular structure of
Compound No. 23, as the most probable source.
The dissolved oxygen levels did not show any significant
pattern, and varied from a high of 9.02 ppm in the river
at 0-day, to a low of 2.20 ppm at 0+3 days in the test
chamber treated with 2.0 ppm of test Compound No. 73»
solubilized in methyl alcohol (Table 9). In this vessel,
which was added to the experimental design to determine
whether such a formulation would enhance algaecidal activity,
361 mg of test compound was dissolved in approximately one
i
pint of methanol before adding to the 60-gallon test chamber.
In this procedure the methanol concentration was about 0.2
percent, which is well below the 10,000 ppm methanol toxicity
limit for trout. However, the high BOD of methanol did show
up significantly in the tests where it was used as a solubil-
izing agent.
During the first Chowan test the temperature varied from a
high of 31.0°C to a low of 26.6°C. However, among the test
chambers during any given monitoring period there was little
or no difference in temperature and there seemed to be no
significant correlation between temperature and test results.
The same was generally true of salinity measurements which,
for all practical purposes, were below the detection limit
throughout both test periods.
46
-------
TABLE 9
Chowan River - Second Test
DISSOLVED OXYGEN LEVELS
Sample
0-day 0+1 day 0+2 days 0+3 days 0+6 days 0+9 days
Ambient River Water
Control
Compound
Compound
Compound
Compound
No.
No.
No.
No.
73
73
73
73
- 3.0
- 2.0
- 2.0
- 1.0
9.02
8.13
ppm 8.09
ppm 7 . 9l*
ppm* 7.58
ppm 8 . 36
8.
8.
6.
6.
6.
6.
03
15
65
51
1*1*
63
7.52 5
6.91* 5
6.21* 5
6.12 5
5.9!* 2
6.ll* 5
.06
.37
.25
.1*9
.20
.00
6.ll*
5
7
7
3
7
.97
.65
.86
.28
.71*
7.29
6.1*1
6.55
6.85
7.15
6.1*2
*Solubilized in methyl alcohol
-------
Lake Sallie - First Minnesota Test
This test site which was located about five miles southwest
of Detroit Lakes, Minnesota, (Figure 6) had been the subject
of extensive limnological studies for a number of years,
headed primarily by Dr. Joe K. Neel, University of North
Dakota who also served as area investigator for this project.
This moderately-sized lake had been in times past very
popular for sports fishing, but in recent years had developed
serious algal blooms due to heavy nutrient input from sewage
lagoons which emptied into Muskrat Lake and which then flowed
into Lake Sallie from the east. A state fish hatchery was
located at that point between Muskrat Lake (which was used
for the second test in that region) and Lake Sallie. Proper
liaison was made with the cognizant state agencies and formal
approval was received by the area investigators before the
test program was begun.
Lake Sallie is about six miles wide, east to west, and
about ten miles long. The tests were put into operation
at Station 15 across the Lake from the fish hatchery, about
500 yards from the west shore (Figure 7).
The first blue-green algal bloom of the season appeared
the first of July. The test compounds were administered
in calm and sunny weather to each test chamber, at the
calculated 1.6 and 0.8 ppm levels.
Water samples taken before and immediately following test
initiation arrived in rather poor condition the fourth day
after shipment. Most of the samples,were completely thawed,
and some of the plastic "twirl pack" bags were ruptured,
Fortunately, enough sample in each case remained to allow
for fairly accurate determinations to be made, although with
some difficulty.
48
-------
Figure 6. Minnesota Test Site
^s^ji^^^^^i^
\fa: C'4 \Alsen\J Milton- cLt.il M
\ HamJ>den\ F, .fNjkom* 1 [ 1 D,
iCknrfi,{<£ \ ra'fd«)- tdlnbura VM^,,*
_^. Loy*'*pi' \ i
^@JoUtoy * fV M«nit|«
te^ervoir I V ,^
v.y'Vr>y Ao:
IjVT^iDoW^J""1 '*
-------
Figure 7.
Lake Sallie and Muskrat Lake - Linologlcal Sampling Stations
and Algaeclde Testing Stations*
N
21
MUSKRAT
LAKE
22*
15*
20
'12
11
16
19
17
LAKE
SALLIE
10
'Pelican
River
Scale:
1 in.
1 mi.
50
-------
As may be seen from an examination of Table 10 only about
25 to 50 percent of the compound, with which the water was
initially treated, was still detectable in the samples at
the time they arrived for analysis. Compound No. 23 suffered
a greater loss than its companion. The depletion pattern
was fairly rapid for both compounds, verifying previous data
from the Chowan River tests. The concentration levels of
both compounds dropped below the 0.02 ppm and 0.05 ppm
detection limits by 0+4 days.
Although variations In available and total P0i» In the ambient
lake water and test vessels were found, no conclusive pattern
was discernable. Practically no N0z~-nitrogen was measured
in any of the samples and the N03--nitrogen levels remained
rather consistent In the control vessels, compared to the
lake water itself, as did the carbonate and bicarbonate
levels. The water temperatures were fairly stable, ranging
from 23 to 25.5°C, as were the pH readings which ranged from
8.4 to 9.0 during the six-day test period (appendix C).
The algal toxicity of both compounds was immediate and quite
effective on Aphanizomenon and three species of Anabaena but
it lasted for only two days. The 1.6 ppm concentration of
each compound was somewhat more effective than the 0.8 ppm
level, achieving 100 percent control of these two species
at 0+1 day (Table n and Table 12).
Both compounds also affected Coelosphaerium haegellanum
through 0+1 days. It can be noted that this blue-green
species was not as rapidly affected as were the two fila-
mentous groups and that it did not suffer complete "wipe
outs" as they did. The effects on Mlcrocystis were incon-
clusive from this experiment.
51
-------
TABLE 10
Lake Sallie - First Test
COMPOUND DEPLETION PATTERNS
values in ppn
Sample 0-days 0+2 days 0+U days 0+6 days
Ambient Lake Water
Control Vessel No. 1
Control Vessel No. 2 <0.02
Compound No. 23 - 0.8 ppm* 0.39 <0.02 <0.02 <0.02
Compound No. 23 - 1.6 ppm* 0.25 O.OU <0.02 <0.02
Compound No. 73 - 0.8 ppm O.liU <0.05 <0.05
Compound No. 73 - 1.6 ppm 1.2 0.05 <0.05
"Compound No. 23 vas determined by differential pulse polarography
Compound No. 73 was determined "by liquid chromatography
-------
UJ
TABLE 11
Lake Sallie - First Test
TEST COMPOUND ACTION ON THE BLUE-GREEN ALGAE
Aphanizomenon AND C. naegelianum
(cells/ml x 10*)
Sample
Ambient Lake Water
Control
Compound No. 23 - 0.8 ppm
Compound No. 23-1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73-1.6 ppm
0-day
U9.5
72.6
81.6
2U.2
iST"
0.0
SO
sP
0.0
0+1 day
166
120
7U.8
0.0
9T6-
p.p
2.2
US. 2
0.0
19.2
ti+2 days
1U"8~
ill
199
66
100
30.8
TO"
127
196
itJ
O+U days
iJF
Ip7
95.7-
170
93.5
129
66
91.2
67.1
9O"
0+6 days
168
100
161
103
126.3
100
iJfl
129
^
"BIT
Aphanizomenon
* c. naegelianum
-------
VJl
TABLE 12
Lake Sallie - First Test
TEST COMPOUND ACTION ON THE BLUE-GREEN
ALGAE Anabaena AND Microcystis
(cells/ml x 10*)
Sample
Ambient Lake Water
Control
Compound No. 23 - 0.8 ppm
Compound No. 23 - 1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73-1.6 ppm
._,, Anabaena
0-day
18.7
81.2
91.2
120
0.87
95.2
0.0
6.2
0.0
72.8
0+1 day
gi
.
o.g
0.0
6776"
28!o
0.0
53.2
0+2 days
120
131
117
58.7
7576
32.5
TOT
93.7
30.8
55.0
36. k
Q+h days
102
"iO"
Hi-
97.5
53.2
1O
90.0
71.2
56.0
0+6 days
186
177
131.6
206
72.8
198
~6lT
*3r
67.2
155
106
Microcystis
-------
Altogether 30 species of algae were identified and
enumerated in ambient lake water and in each test vessel
(see appendix). The total count of ten species of blue-
greens reached a maximum of 59,600 cells/ml at 0+1 day
1m ambient lake water and a minimum of 6,750 cells/ml with
Compounds No. 23 and 73 at the 0.8 ppm level during the
same period (Table 13). The total of all algal cells
dropped to a low of 29,800 cells/ml in the 1.6 ppm vessel
of Compound No.23 at 0+1 day, compared to 81,900 cells/ml .
in the surrounding lake water (Table 14).
It is also noteworthy that both algaecides are apparently
quite specific for blue-greens, with the green alga Oocystis
and the diatoms Staurastrum, Fragillaria, Synedra, and
Stephanodiscus being affected little or not at all (Table 15).
Muskrat Lake - Second Minnesota Test
The second test in the northern section of the United States
was begun on Muskrat Lake, a smaller lake adjacent to Lake.
Sallie on August 28. Muskrat Lake is smaller and better
protected from the wind. The severe wave action had upset
some of the test vessels in the previous test, and caused
difficult sampling problems. The algae bloom was denser than
in Lake Sallie.
The flotation equipment was assembled, anchored into place,
and filled with ambient water by mid-afternoon, but the test
compound was not administered until near sundown, according
to plan, to minimize photodegradation-' during the initial
action period. Only Compound No. 73 was used in this, and
all subsequent tests.
55
-------
VJ1
TABLE 13
Lake Sallie - First Test
TOTAL BLUE-GREEN ALGAE
(cells/ml x 102)
Sample 0-day 0+1 day 0+2 days 0+lt days 0+6 days
Ambient Lake
Control
Compound
Compound
Compound
Compound
No.
No.
No.
No.
Water
23
23
73
73
- 0.8
- 1.6
- 0.8
- 1.6
ppm
ppm
ppm
ppm
^^«IHWBM^
282
335
227
111
167
81*
597 520
381 573
67.5 310
78 222
67.5 U51*
79.5 195
393
U62
1*17
U23
285
288
596
596
555
613
^Tl
^I-T2
-------
TABLE Ik
Lake Sallie - First Test
TOTAL ALGAL CELL COUNTS X 102/ml
(includes 30 species of "blue-greens, greens,
diatoms, euglenophyta and dinophta)
Sample
Ambient Lake
Control
Compound
Compound
Compound
Compound
No.
No.
No.
No.
Water
23
23
73
73
- 0
- 1
- 0
- 1
.8
.6
.8
.6
ppm
ppm
ppm
ppm
0-day
1+29
526
510
339
352
338
0+1 day 0+2 days
819
693 910
3U8 516
298 Ull
327 651
360 iH7
Q+k days
75U
852
718
735
52U
6U5
0+6 days
753
873
792
88U
62k
610
-------
VJl
oo
TABLE 15
Lake Sallle - First Test
TEST COMPOUND ACTION ON
Oocystis and Fragillaria
(cells/ml x 10* )
Sample
Ambient Lake Water
Control
Compound No. 23 - 0.8 ppm
Compound No. 23 - 1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
0-day
22.1
WT
23.8
36.0
80.0
56
62.9
w%
28.9
58.8
UU.2
58.0
0+1 day
90.1
50. U
71. U
66.0
57.8
70.8
e'
30.6
67.2
U5.9
90.0
0+2 days
93.5
U9~72
lU2
"snr
23^5.
36.0
91.8
30.0
61.2
25.2
62.9
TO
0+U days
^
l89
1*9.2
171
~Ub~8
163
"387IT
113
28.8
1UU
TO
0+6 days
7.2
1^6
337^
13jt_
^TF
170
33.6
88. U
32~nr
39.1
27?^
KEY: °°Cystis
Fragilaria
-------
The flotation devices were anchored to bottom anchors and
to a small wooden platform at Station 22 (Figure 7), to
aid in servicing and collecting samples from the test cham-
bers, rather than from a boat as before. This design seemed
to be a considerable improvement and eliminated some of the
sampling problems encountered previously during windy weather.
The tests were conducted at the 0.4, 0.8, and 1.6 ppm levels,
using only Compound No. 73. Initial monitored compound levels
ranged from 63 to 82 percent of the calculated values. The
pattern of declining test compound concentrations as a function
of elapsed time after initiation of the tests is shown in
Figure 8. Compound levels generally dropped below the 0.05
ppm detection limit at 0+6 days, although 0.18 ppm still
remained in the 3.2 ppm (initial) vessel at 0+9 days. No
compound was detected in the 0.8 ppm vessel at 0+3 days
(Table 16).
Algae concentrations in the lake during the test were fairly
high, with Osclllatorla being the predominant species and
two other blue-greens, Aphamizomenon and Anabaena, ranking
second and third (Appendix B).
Anabaena and Aphamizomenon were both drastically affected by
the test compound, with no living cells remaining after the
first day. No population recovery of these two species was
noted within the nine-day monitoring period (Table 17).
Raphldiopsls curvata was similarly affected, but the activity
against Coelosphaerlumnaegelianum was variable and inconclu-
sive (Table 18). The test compound registered no appreciable
effect against Oscillatoria (Table 19).
59
-------
Figure 8.
Compound Depletion Patterns
Second Minnesota Test - Muskrat Lake
Test Compound No. 73
Initial Theoretical Level - 3.2 ppm
Initial Theoretical Level
Initial Theoretical Level
Initial Theoretical Level
1.6 ppm
0.8 ppm
0.4 ppm
Q.
Q.
O)
o
Time (Days)
60
-------
TABLE 16
Muskrat Lake - Second Minnesota Test
Compound Depletion Patterns
values in ppm
Sample 0-day 0+1 day 0+2 days 0+3 days 0+6 days 0+9 days
Control
Compound
Compound
Compound
Compound
Compound
No.
No.
No.
No.
No.
73
73
73
73
73
- 3.2
- 1.6
- 0.8
- 0.8
- O.U
ppm
ppm
PPm
ppm*
ppm
<0.05
2.62
1.20
0.50
0.56
0.27
<0.
1.
0
0
0
0
05
86
.86
.22
.31
.19
<0.05 <0.05
1.03 0.63
0.39
O.lU <0.05
0.18 O.ll
0.09
<0
0
0
<0
<0
<0
.05
.37
.12
.05
.05
.05
<0.05
0.18
<0.05
<0.05
<0.05
<0.05
*SoluMlized in methyl alcohol
-------
a\
ro
TABLE 17
Muskrat Lake - Second Minnesota Test
Anabaena AND Aphani zomenon
(cells/ml x 10")
Sample
Ambient Lake Water
Control
Compound No. 73 - O.U ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 0.8 ppm*
Compound No. 73 - 1.6 ppm
Compound No. 73 - 3.2 ppm
0-day
U9.8
T^IT~~
U6
5sT
J^l
50U
113
UOU
__2
252
93.2
262
7.62
218
0+1 day
1U.1
381
iii
39T
0
0
0
0
0
0
0
0
0
0
0+2 days
56.7
303
501
2U5
0
0
0
0
0
0
0
0
0
0
0+3 days
288
75.6
95.8
0
0
0
0
0
0
0
0
0+6 days
13.7
210
69.7
0
0
0
0
0
0
0
0
0
0
0
0+9 days
13.6
190
9U.1
0
0
0
0
0
0
0
0
0
0
0
*Solubilized in methyl alcohol
Anabaena
KEY:
Aphanizomenon
-------
TABLE 18
Muskrat Lake - Second Minnesota Test
Coelosphaerium AND Raphidiopsis
(cells /ml x
Sample
Ambient Lake Water
Control
Compound No. 73 - 0.4 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 0.8 ppm*
Compound No. 73 - 1.6 ppm
Compound No. 73 - 3.2 ppm
0-day
108
61.3
25.2
6.1
56.3
40.1
6.72
49.1
19.2
ifrrs
1^
19b
lit. 6
106
0+1 day
27.1
51.7
36.9
88.7
45.8
0
0
0
0
0
8.4
0
0
0
10*)
0+2 days
23.0
5374"
89.5
71.1
0
0
0
0
0
0
lii
0
7.68
0
0+3 days
29.9
72.0
46.3
3oT?
6.72
0
6.48
0
0
0
0
0
0
0
0+6 days
314
31.8
20.2
0
6.24
0
0
0
0
0
11.0
0
0.25
0
0+9 days
31.4
20.1
19.4
0
4.32
0
0
0
0
0
6.48
0
0
0
*Solubilized in methyl alcohol
KEY:
Coelosphaerium
Raphidiopsis
-------
TABLE 19
Muskrat Lake - Second Minnesota Test
Oscillatoria
(cells/ml x
Sample
Ambient Lake
Control
Compound No.
Compound No.
Compound No.
Compound No.
Compound No.
Water
73 - 0.1* ppm
73 - 0.8 ppm
73 - 0.8 ppm*
73 - 1.6 ppm
73 - 3.2 ppm
0-day
1*2.7
1*3.5
38.8
51.3
1*5.2
31.8
39.1
0+1 day
1*0
1*9
39
26
27
36
32
.3
.0
.2
.7
.8
.2
.8
10")
0+2 days
39
35
27
39
21*
28
37
.3
.1*
.6
.0
.7
.0
.1
0+3 days
1*1*
35
23
30
28
26
.2
.1*
.6
.1*
.5
.6
0+6 days
33
20
35
21
27
25
Uo
.6
.2
.1*
.1*
.1
.1
.7
0+9 days
39.7
2l*.0
26.7
26.0
26.2
27.8
32.2
*SoluMlized in methyl alcohol
-------
The water chemistry data, taken only at "0" and 0+9 days
did not reveal any significant patterns of deviation of
test vessels from the control values (Appendix B). The
temperature dropped 4°C during the nine-day test period.
The pH varied from a low of 8.4 in the lake to a high of
9.2 in the 1.6 ppm test chamber at 0+9 days.
Diamond Lake - First Test
After some rather extensive investigations, Diamond Lake,
a high altitude lake located inland about 60 miles south-
east of Eugene, Oregon, was selected as the west coast
test site (Figure 9). Dr. Howard Horton of the Department
of Fisheries and Wildlife, Oregon State University, served
as the area investigator for the project.
Diamond Lake is a resort area, famous for trout fishing
and both summer and winter sports. The Diamond Lake Lodge
campgrounds and numerous summer homes surrounding the Lake
contribute nutrients into the lake via ground seepage from
septic tank leach lines. In recent years, over-eutrophication
of the lake has resulted in troublesome algal blooms.
The specific location of the test site was on the northeast
end of the Lake, several hundred feet offshore from the
forest guard station at point "B" (Figure 10).
The first test was begun on August 26, 1973. About one
hour before sundown, samples were taken from the lake and
from each test vessel, before treatment, to furnish a base-
line for the water chemistry data and existing algal popula-
tions. The test compound primary concentrates were then
added to the appropriate test chambers at initial concentra-
tions ranging from 0.4 to 3-2 ppm. Samples were then taken
-------
Figure 9. Oregon Test Site
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«8«J7S?^ ««^C
^a^.^'
^MrfP=W4^^1>^R^
s^>*%^^^^rf^-^f
& ^ ^^FR Si:^
»SS&*SLi ^^'#f\^-^' Hol ^^r^^^^^&,)
kyinditttt.-~ . . /T"ff .-- \ I ^/. .,..,. -^oHo'«liiHMCi.~,-. In a r/\. ^--JNA . ' \A
-------
Figure 10.
DIAMOND LAKE
I MILE
CONTOURS IN FCC!'
67
-------
from each station for compound analysis, quick frozen, and
reserved for shipment to Midland.
The weather was calm and clement and the prognosis was
for continued good weather. However, a severe storm at
0+5 days upset the system sufficiently to rupture some of
the test bags and invalidate any further test data.
The water samples sent via air freight from the Oregon site
to Midland for analysis arrived in poor condition after being
in transit for four days. No reasonable pattern of compound
residual could be discerned (Table 20).
The algal data received from Diamond Lake indicated the
presence of Anabaena as the most abundant algal species.
Up to 98 percent control of Anabaena was achieved at 0+3
days down to the 0.4 ppm level. Nearly 100 percent control
was achieved at all other concentrations as early as the
first day after treatment. Control was still at the 98
percent level at 0+3 days before the test was disrupted by
inclement weather. No significant return of Anabaena was
noted during this time in any of the test chambers (Table 21).
The test compound seemed to be selective for Anabaena, the
dominant algal species, although an algaestatic effect was
evident against Stephanodiscus and Synedra at the 0+2 and
0+3 day periods (Table 22). No other algal species monitored
seemed to be adversely affected (Appendix B).
The fluorometric readings taken on samples from each station
corresponded fairly well with the decline in blue-green algal
population. Also, a good correlation was achieved between
the relative intensity readings of acetone-extracted samples
and the raw samples (Table 23).
68
-------
TABLE 20
Diamond Lake - First Test
COMPOUND DEPLETION PATTERNS
values in ppm
Sample 0-days 0+1 day 0+2 days 0+3 days
Ambient Lake Water <0.1 <0.1 <0.1 <0.1
Control Vessel No. 1 <0.1 <0.1 <0.1 <0.1
Compound No. 73 - O.U ppm
Compound No. 73 - 0.8 ppm 0.67 0.65 0.05 0.0
Compound No. 73 - 0.8 ppm* 0.30 0.57 0.0 0.0
Compound No. 73 - 1.6 ppm 1.07 1.13 0.2U 0,13
Compound No. 73 - 3.2 ppm 0.22 0.75 0.20 0.05
*Solubilized in methyl alcohol
69
-------
TABLE 21
Diamond Lake - First Test
BLUE-GREEN ALGA Anabaena
. Sample
Ambient Lake Water
Control Vessel No. 1
Compound No. 73 - 0.1* ppm
Compound No. 73 - 0.8 ppm
Compound No. 73-0.8 ppm*
Compound No. 73-1.6 ppm
Compound No. 73 - 3.2 ppm
(cells/ml)
0-days 0+1 day
1*277 1*^81*
1*11*1* 5981*
1*336 2189
5198 o
5287 21
1*396 36
3l*l6 1*2
0+2 days
3370
3207
238
89
0
71*
0
0+3 days
21*05
3312
76
1*2
31
21*
29
*Solubilized in methyl alcohol
70
-------
TABLE 22
Diamond Lake - First Test
DIATOMS - Synedra AND Stephanodiscus
(cells /ml)
Sample
Ambient Lake Water
Control Vessel No. 1
Compound No.
Compound No.
Compound No.
Compound No.
Compound No.
73 - O.U ppm
73 - 0.8 ppm
73-0.8 ppm*
73-1.6 ppm
73 - 3.2 ppm
0-days
803
576
968
37F
1013
5.9
1067
Tnr
n8o
7 = 9
1019
7-U
1213
-57T
0+1 day
1106
10.1
805
ITS"
8U3
TT73
676
5.2
109**
656
5.8
1019
~378~
0+2 days
13^7
13.1
2688
~O
1013
IT
g66
378"
1267
6.3
939
7.2
1115
0+3 day
1201
lUT?
536k
12. U
986
1.8
8UO
2.5
1213
TT5
900
3.8
977
~
*Solubilized in methyl alcohol
KEY:
Synedra
Stephanodiscus
71
-------
TABLE 23
Diamond Lake - First Test
FLUOROMETRIC RELATIVE INTENSITY READINGS
Sample
0-days 0+1 day
0+2 days 0+3 days
Ambient Lake Water
Control Vessel No. 1
Compound No. 73 - 0.1* ppm
Compound No. 73 - 0.8 ppm
Compound No. 73-0.8 ppm*
Compound No. 73-1.6 ppm
Compound No. 73 - 3.2 ppm
0.35
0.1*1*
0~T*3~
0.53
0.1*5
0.35
0.56
0.55
0.52
0.57
0.38
0.1*1
0.1*1
0.32
0.1*3
0.36
0.31
0.35
0.1*2
0.39
0.30
0.25
0.21
0.18
0.15
0.22
0.19
0.16
0.09
0.05
0.09
0.08
0.08
0.12
0.08
0.08
0.06
0.20
oTIF
0.28
0.17
0.15
o.oi*
0.06
o.oi*
0.05
0.09
0.06
0.06
o.oU
0.03
*Solubilized in methyl alcohol
KEY:
acetone extraction
raw samples
72
-------
The chlorophyll "a" and carotenold patterns showed a general
decline with increasing time, as compared to the controls,
but were not always consistently reduced with increased
algaecide concentration, as might have been expected (Table 24).
The temperature in the lake was 16°C at the start of the
test, gradually declining to 14°C at 0+3 days. The pH was
fairly stable in the 8.9 to 9.1 range. The conductivity
of the water did not vary significantly during the three-day
test period, but was fairly constant in the 44 to 46 micromhos/
cm range. Neither the dissolved oxygen nor the total alka-
linity departed significantly from daily control values.
The dissolved oxygen increased from a low of 8.1 ppm at the
beginning to a high of 9.0 ppm at 0+3 days, primarily because
of temperature reduction and increased wind action (see
Appendix C).
Diamond Lake - Second Test
The second test at this site was initiated September 17, 1973,
following the same basic research plan. A bloom of Anabaena
was reported to be persisting, although somewhat reduced.
The Weather was moderate and fairly calm, although the skies
were rather heavily clouded.
The test chambers were all anchored into place and filled with
ambient lake water by about 6 pm at which time the test com-
pound aliquots were administered to the respective 60 gallon
vessels to yield initial concentrations of 1.0, 0.2, 0.4,
0.8 and 1.6 ppm. One test bag contained only lake water to
serve as a comparative control. The lake itself served as a
second control.
73
-------
TABLE 2l*
Diamond Lake - First Test
CHLOROPHYL "A" AND CAROTENOID LEVELS
(mg/m3)
Sample
0-days
0+1 day
0+2 days
*Solubilized in methyl alcohol
KEY:
Chloro-phyl "A"
Carotenoids
0+3 days
Ambient Lake
Water
Control Vessel No. 1
Compound No.
Compound No.
Compound No.
Compound No.
Compound No.
73 - 0.1* ppm
73 - 0.8 ppm
73-0.8 ppm*
73-1.6 ppm
73 - 3.2 ppm
11.1*2
3.01*
8.81
2.56
9.33
2.56
1.76
10.91
2.67
9.81*
2.56
10.72
2.83
11.1*2
3.01*
11.1*6
"CToJ
8.25
2.21*
7.59
2.08
8.08
2.19
7.32
2.03
1*.78
2.51
11.1*2
3.09
ll*.06
3.73
_3^56
0.96
2.91*
0.69
2.78
0.80
3.21*
1.23
1.97
11.1*1
3.01*
10.1*3
3.31
2.91*
0.75
0.32
2.78
1.01
2.89
1.01
1.86
0.59
-------
The next morning it was evident that a storm front was moving
in which increased in severity as the day progressed. The
0+1 day samples were collected in the afternoon with some
difficulty due to rain, wind, and high wave action. The
storm persisted and increased in severity throughout the
night and part of the next day, showing signs of clearing
toward evening. However, an examination of the test devices
during the afternoon sampling trip revealed that all of the
test bags were torn due to pressures caused by violent wave
action.
The next day the barometer was up, the weather was calm and
intermittently sunny again, and it appeared that the storm
front had moved beyond the local area. After some delibera-
tion the decision was made to try to obtain new plastic
bags, double them on the flotation collars for added strength,
and put the test into operation again, with the hopes of
successfully completing the test and obtaining the desired
data. The collars had to be cut down to adapt them to the
smaller-sized bags which were available. The compound con-
centrations had to be adjusted accordingly.
The tests were put into operation and the base-line samples
were taken as before, completing the last test just as night-
fall was approaching. Unfortunately, another storm front
moved into the area. But, the storm was less severe and
the double bags seemed to hold better.
The monitored initial concentrations agreed fairly well with
the calculated values except for samples from Vessel No. IV,
in which it appears that the 0-day and the 0+1 day samples
may have been inadvertently interposed. The compound concen-
trations in all test vessels had fallen below the 0.05 ppm
detection level at 0+3 days (Table 25) in accordance with pre-
viously determined patterns for Compound No. 73.
75
-------
TABLE 25
Diamond Lake - Second Test
COMPOUND CONCENTRATIONS
ppm
Sample
0-day
0+1 day
0+2 days 0+3 days
Ambient Lake
Compound
Compound
Compound
Compound
Compound
No.
No.
No.
No.
No.
Water
73
73
73
73
73
- U.8
- 2.U
- 1.2
- 0.6
- 0.3
ppm
ppm
ppm
ppm
ppm
0.
3.
2.
1.
0.
0.
A^
05
68
2k
07
15(?)
25
*
3.55
1.53
*
0.3K?)
0.09
*
1.12
0.07
*
<0.05
<0.05
#
<0.05
<0.05
*
<0.05
<0.05
76
-------
The monitored algal populations, as reported by the Oregon
State University investigators, showed that Anabaena was
eliminated at all concentrations down to the 0.6 ppm level,
within one day after treatment. A fairly large population
returned at 0+2 days, however. It is believed that re-
inoculation may have occurred due to adverse weather con-
ditions and accompanying heavy wave action on the lake
(Table 26). Control Vessel No. 1 and one other vessel
were lost early in the test period, as a result of the tur-
bulent weather.
The green alga Gloeocystis was not significantly affected at
any test compound concentration used (Table 27). It appears
that the desmid, Staurastrum may have been affected initially
since the cell count dropped to zero at 0+1 day in the 0.3 ppm
and 0.6 ppm vessels and that the vessels were then re-inoculated,
The cell count in the 4.8 ppm vessel was only partly reduced
however. A similar uncertainty exists with the activity of
Compound No. 73 against the diatom, Stephanodiscus (Table 28).
Whole Pond Test - North Texas Site
Small, whole ponds, located near Denton, Texas, were proposed
as test sites, in order to duplicate natural circumstances
as much as possible.
Several 0.5 to 1.0 acre fishery ponds at the Lewisville State
Fish Hatchery had been selected as the test sites of choice,
but no suitable blue-green blooms appeared and stabilized
during the summer or fall seasons, as had occurred in previous
years. Consequently, another pond with an existing bloom in
the same general vicinity was chosen. The 0.272-acre farm-
pond selected was about 30 miles Northwest of Denton, Texas,
(Figure 11) and contained a heavy bloom of blue-green algae.
77
-------
TABLE 26
Diamond Lake - Second Test
BLUE-GREEN ALGA Anabaena
(cells/ml)
Sample
0-days 0+1 day
0+2 days 0+3 days
Ambient Lake
Water
Control Vessel Ho.
Compound
Compound
Compound
Compound
Compound
No.
No.
Wo.
No.
No.
73
73
73
73
73
- 0.
- 0.
- 1.
- 2.
- k.
1
3
6
2
k
8
ppm
ppm
ppm
ppm
ppm
12*4 112
108 68
188 kO
ikQ 0
76
6k
112 0
72
72
ho
6k
0
2k
68
6k
lou
6k
78
-------
TABLE 27
Diamond. Lake - Second Test
GREEN ALGA - Gloeocystis
Sample
Ambient Lake Water
Control Vessel No. 1
Compound No. 73 - 0.3 ppm
Compound No. 73-0.6 ppm
Compound No. 73 - 1.2 ppm
Compound No. 73 - 2.1* ppm
Compound No. 73 - 1*.8 ppm
(cells/ml)
0-days
1*0
1*8
1*8
58
16
1*1*
28
0+1 day OH
20
0
8
56
1*1*
0+2 days 0+3 days
1*0
1*0
36
28
36
52
32
1*8
32
80
79
-------
TABLE 28
Diamond Lake - Second Test
Staurastrum AND Stephanodisous
(cells/ml)
Samples
0-days 0+1 day
0+2 days 0+3 days
Ambient Lake Water
Control Vessel No. 1
Compound No. 73 - 0.3 ppm
Compound No. 73-0.6 ppm
Compound No. 73 - 1.2 ppm
Compound No. 73 - 2.U ppm
Compound No. 73 - ^.8 ppm
28 20
H88 W
U U
68 156"
U 0
160 228
12 0
216 tinr
0
IbT
2U
"52T
16 12
172* 176"*
12
56T
12
252
12
53T
8
272*
8
To
Uo
55T
12
58T
16
356-
12
328"*
*Some cells showing signs of degeneration
KEY:
Staurastrum
Stephanodiscus
80
-------
Figure 11. Northern Texas Test Site
- - ^^d^t: -Y»« Ai^n?^--
piiotfe
PO""TU^? *"aii [i2i
Cclini
J 1?*J. *r .1. IK*. Pftnc.ton 1 MentV \
3) u tiiiiMcKinneySP^^J^jj^Fi^
r siM.n
tur rJ».w«^ii
f.wij|.,ji|rco
'Atttrf^
M.illtyi'l
!5J1 Xv "V ' I X.
aJ^Lw^TS^feiboro l< -«,"^
u£ J&s^±\ Bly5°n(5"y ^TV^j. 1 & * ^LlT^l lluslin'P J^,v-°'l"\-i'*"'nTnT13>- JSl ^rrt
"~ -"grfml f ^J^sf^^J^V?^
^^^^^^S^nN^ - zT**""" *" ' """""*Md^'
Sf| TRmi" S _/>Bii)v^-y > ^tjy lown/fi J?
UnT P^«i "ra *§ .Mineral/I. ~\ {'"'"TT - *"*|
l?*"Mi"n J* pi ,-d vVells / - Ni" / I
r\" a!.^l >>-£frTJ«l1!@^,^-/W«at^e4.orl11
*tcSd' * V'n'
't.op«.»c/ ytic''<|y/
!i^6?
lF,ll>
Meridian
-------
The pond is somewhat lower than the rest of the terrain and
protected by surrounding trees. Dr. Dwain Vance of North
Texas State University served as area investigator, assisted
by several of his graduate students.
The primary concentrate for the test was made up on the site
by combining 332 grams of Compound No. 73 with 300 gallons of
water in a commercial sprayer and agitating continuously for
several hours while the 60-gallon control vessels and accom-
panying flotation devices were being prepared.
Two 60-gallon plastic bags were filled with ambient lake
water, one to be used as a control and the other for a
biological control test, utilizing the phagocytic organism,
Ochromonas ovalis. Before spraying the primary test compound
concentrate onto the pond's surface water samples were taken
for algal background data, and the control bags were covered
with plastic to prevent mixing of the algaecide with the
controls. The compound concentrate was sprayed onto the sur-
face of the water from a boat as well as from shore to give
an even spread and full coverage. The pond was treated with
a calculated 1.0 ppm concentration of test compound No. 73.
Application of the test compound was completed as the sun was
low in the horizon to minimize the possibility of photodecom-
position.
Several post-treatment water samples were taken at various
points on the pond and then combined to produce representative
samples. These were then quick-frozen and stored until ready
for shipment together with all other samples which were col-
lected up to 0+3 days; these were then sent air freight to
Dow's analytical lab at Midland for analysis as before.
82
-------
The analytical data obtained showed an initial concentration
of 0.42 ppm, less than one-half of the calculated value,
immediately following treatment. Rapid adsorption, due to
the high amount of biomass and possible compound biodegra-
dation may have been responsible for the low initial levels
detected.
A heavy storm moved into the area on the evening of the
first day after treatment, during which time three-inches of
rainfall was recorded. The water analysis data showed the
compound concentration in the pond at this time to have
dropped below the 0.05 ppm detection limit. None of the
control samples or subsequent pond samples registered any
detectable amounts of the test compound (Table 29).
The algal densities in the test pond, as determined by the
University investigators, were very high, ranging up to
250,000 cells/ml in the case of Agmenellum quadruplica'tum
the dominant species. Microcystis and Osclllatoria ranged from
10 to 60,0000 cells/ml; Anabaena was also present to a lesser
extent.
The results of the whole-pond test were not entirely con-
clusive. Although the test compound level at 0+1 day fell
below the 0.05 ppm detection limit, Oscillatoria in the pond
was reduced to "0" count from 0+1 day through the end of the
17-day test period. However, Oscillatoria cell counts were
noted, intermittently, in only one of the control vessels
during this same period. Agmenellum quadrupllcatum counts
showed a marked reduction from an initial count of 250,000
cells/ml to 17,000 cells/ml at 0+17 days, but no Agmenellum
cells were found in samples taken from the control chambers
after the first day (Table 30).
83
-------
TABLE 29
North Texas Test Site
MONITORED TEST COMPOUND LEVELS
Compound No. 73
(ppm)
Sample 0-day 0+1 day 0+2 days 0+3 days 0+6 days 0+17 days
Pond* O.U2 <0.005 <0.005 <0.005 <0.005 <0.005
Control Vessel No. 1 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005
Control Vessel No. 2 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005
"Initial calculated concentration =1.0 ppm
-------
TABLE 30
CD
VJ1
North Texas - Whole Pond Test
Oscillator!a AND Agmenellum
Sample
Ambient Pond Water*
Control Vessel No. 1
Control Vessel No. 2
0-day
60
250
60
250
60
250
(cells /ml x
0+1 day
0
72
U
0
0
0
103)
0+2 days
0
111
0
0
0
0
0+3 days
0
131
0
0
0
0
0+6 days
0
20
_i
0
0
0
0+17 days
0
17
~
*Treated at 1.0 ppm
KEY:
Oscillatoria
Agmenellum
-------
Microcystis cell counts in the pond varied greatly, from
10,000 cells/ml initially to a maximum 150,000 cells/ml
at 0+1 day, to "0" from 0+6 days on to the end of the 17-
day test period. At the same time, the controls dropped
from 10,000 cells/ml to "0" at 0+2 days, with no return
of this species (Figure 12). No Anabaena cells were
detected after the first day following treatment, until
the last samples collected at 0+17 days, in which a heavy
bloom of 200,000 cells/ml was found (Table 31).
This observed phenomena shows an apparent shift in biological
equilibrium dynamics due to competition reduction but it
causes difficulties in assessing the algaecidal activity
of Compound No. 73 in a whole pond.
Due to the inconclusive results from the whole-pond test it
was especially desirable to conduct a second test of the
same type. But the season was already well advanced at
this time, and further attempts to find a suitable pond with
a naturally occurring blue-green algal bloom was unsuccessful.
ALGAL CONTROL
As one of the primary objectives of the Phase III effort
the algal control efficacy of the prime candidate compounds
under natural "bloom" conditions was to be determined.
Data collected under widely varied conditions in geographically
diverse regions of the United States show a rather distinctive
pattern of selective blue-green algal control.
As may be seen by examination of Table 32 and Table 33,
good control was exerted over most of the blue-green algae
by both test compounds used at each site, with the exception
of Microcystis incerta, and under certain conditions
86
-------
Figure 12.
Whole-Pond Test - North Texas
Compound No. 73 at 1.0 ppm Vs. Microcystis
150
140
130
120
(0
£ 110
X
i 100
< 40
30
20
A
O Pond
Control
456
Time (Days)
16 1.8 20
87
-------
OD
OO
TABLE 31
North Texas - Whole Pond Test
Anataena AND Microcystis
Sample
Ambient Pond Water*
Control Vessel No. 1
Control Vessel No. 2
0-day
1
10
1
10
1
10
(cells /ml x
0+1 day
0
0
0
10
0
0
10s)
0+2 days
0
150
0
0
0
0
0+3 days
0
So
0
0
0
0
0+6 days
0
0
0
0
0
0
0+17 days
200
0
zzz.
zzz.
"Treated at 1.0 ppm
KEY:
Anabaena
Microcystis
-------
TABLE 32
PHASE III FIELD TEST RESULTS
Algal Control - Compound No. 73
Species
Chowan River Sallie Muskrat Diamond Lake Slidell Pond
oo
VD
BLUE-GREENS
Anabaena
Microcystis spp,
M. cyanea
M. incerta
Agmenellum
Oscillatoria
Coelosphaerium
Raphidiopsis
Aphanizomenon
GREENS
Gloeocystis
Oocystis
DIATOMS
Staurastrum
Fragillaria
Synedra
Stephanodiscus
1
3
3
1
1*
3
3
2
2
1
1
1
1*
1
2
2
1*
1
1
1
KEY: 1 = good control (80-1005?)
2 = moderate control (40-80$)
3 = little or no control (0-40$)
NOTE: double listings is indic-
ative of varied results
between the first and
second tests
^population return observed before end of test period
-------
TABLE 33
PHASE III FIELD TEST RESULTS
Algal Control - Compound No. 23
vo
o
Species
N. Carolina Minnesota Oregon N. Texas
Chowan River Sallie & Muskrat Diamond Lake Slidell Pond
BLUE-GREENS
Anabaena
Microcystis spp
M. cyanea
M. incerta
Agmenellum
Oscillatoria
Aphanizomenon
GREENS
Gloeocystis
Oocystis
DIATOMS
Staurastrum
Pragillaria
Synedra
Stephanodiscus
1*
1
1
2
3
1*
1
2
KEY:
1
2
3
good control (80-1005?)
moderate control (40-80$)
little or no control (0
*population return observed later in test period
-------
Oscillatoria, Coelosphaerlum, and Agmenellum quadrupllcatum.
Since variable results were obtained on the control of these
latter four species, it is suggested that this may have been
due to a difference in algal strains, which were not differ-
entiated by the university investigators in the reported data.
Those results which were somewhat inconclusive, from the
available experimental evidence, are indicated with a question
mark. Population returns of Anabaena were noted in a number
of cases, but it is possible that this may have been due to
re-contamination of the vest vessels during turbulent weather
conditions. Good control of Microcystls, as well as Anabaena
was achieved at all sites except in Minnesota. Raphidiopsis
and Aphanizomenpn were controlled well by both test compounds
at each site where these species were found'to be present at
the time of treatment.
In no case was either the green algae or diatoms affected
significantly by either test Compound No. 23 or No. 73, thus
verifying the algal control specificity of the algaecides in
question.
TOXICOLOGY
Candidate test compound No. 23 and No. 73 were submitted
to Dow's Waste Control Group for preliminary ranging, and
subsequent full-scale toxicity tests.
The preliminary ranging tests on Compound No. 23 indicated
a fish toxicity level which was too high to allow its continued
consideration for use as an algaecide where it would be
administered directly to natural water supplies. One hundred
percent kills were noted at concentrations down to 0.1 ppm,
with a partial kill at 0.065 ppm within the first 24 hours
(Table 3*1). Consequently, Compound No. 23 was withdrawn from
the list of candidate test compounds.
91
-------
TABLE 34
FISH TOXICITY TESTS - BLUE GILLS
Compound No. 23
2,5-Dichloro-3,4-dinitrothiophene
24-Hour
Concentration Toxlclty Effect
0.8 ppra 100$ kill
0.1 ppm 100? kill
0.065 ppm partial kill
0.037 ppm no kill
92
-------
Compound No. 73, as tested against both blue gills and
rainbow trout, proved to be safe to fish life when used in
the recommended ranges below 10 ppm. The highest toxicity
on blue gills was reported from the 48-hour test, where
the lethal concentration to 90 percent of the fish (LC90)
showed an average value of 177 ppm (Table 35 and Figure 13).
The minimum average lethal concentration to 90 percent of
the rainbow trout (LC»0) was 209 ppm at 72 hours (Table 36).
LABORATORY SCREENING
A continuing portion of the algaecide development program
included routine testing of additional compounds.
During Phase III 70 new compounds were tested for algaecidal
activity by the previously established primary screening
test procedures. Some of these were re-tests on a few of
the more promising Phase II compounds, with new formulations.
All were tested against Anabaena flos-aquae at 0.8 ppm.
Eight were tested at higher and lower concentrations, ranging
from 1.6 ppm down to 0.2 ppm (Table 37). Three were subjected
to tests against both Microcystis aeruglnosa and Anabaena flos-
aquae for greater sensitivity delineation (Table 38).
Five of these compounds tested exceeded the 90 percent control
level against Anabaena within the four-day test period. Only
two: No. 136, 2,2'-(l,2-Ethylenediyl)bisbenzoxazole and
No. 176, l,2-Dichloro-4-(isothiocyanatomethoxy)benzene were
considered to be of continuing interest when other known
factors such as compound instability, toxicity potential,
manufacturing difficulties, and economics were taken into
consideration.
Compound No. 136 showed 91 percent activity against Anabaena
at the 0.8 ppm level and exhibited 91* percent control of
93
-------
TABLE 35
PISH TOXICITY TESTS - BLUE GILLS
Compound No. 73
p-Chlorophenyl-2-thienyl iodonium chloride
Test
LC
s o
Test
LC10
LCSo
LiCg o
24 HOUR TLM*, ppm
Range
48 HOUR TLM*, ppm
Lower Limit
61
140
131
1£
U
'pper Limit
180
172
389
72 HOUR TLM*, ppm
Range
Lower Limit
35
107
89
Upper Limit
165
137
417
Average
Values
105
155
226
Average
Values
76
121
192
Test
LC10
LCs o
LC 9 o
Test
LC 10
LCg o
LCg o
Range
Lower Limit
72
124
126
96 HOUR
Upper Limit
141
145
248
TLM*, ppm
Range
Lower Limit
45
85
96
Upper Limit
137
155
295
Average
Values
101
134
177
Average
Values
78
115
169
*Tolerance Limit with 95$ confidence limits
-------
400
300
Figure 13.
Fish Toxicity Tests - Blue-Gills
Test Compound No. 73 - 48 hour TLM
g
(O
o
n-
(O
c
(I)
o
c
o
o
200
100
0 10 20 30 40 50 60 70
Sensitivity (% Kill)
80 90
100
95
-------
TABLE 36
FISH TOXICITY TESTS - RAINBOW TROUT
Compound No. 73
p-Chlorophenyl-2-thienyl iodonium chloride
24 HOUR TLM*, ppm
Test
LClo
LCBO
LC,0
Ran
Lower Limit
123
176
193
ge
Upper Limit
176
194
277
Average
Values
147
185
231
72 HOUR TLM*, ppm
Ranee
Test
LCxo
LCso
LC0o
Lower Limit
96
161
154
Upper Limit
178
180
284
Average
Values
131
170
209
48 HOUR TLM«, ppm
Test
LC10
LCSO
LC90
Test
LC10
LC9o
LC90
Range
Lower Limit Upper Limit
105 178
170 191
182 307
96 HOUR TLM«, ppm
Range
Lower Limit Upper Limit
39 245
140 171
98 615
Average
Values
137
180
236
Average
Values
98
155
246
Tolerance Limit with 95* confidence limits
-------
TABLE 37
LABORATORY SCREENING TESTS
Test Compound versus Anabaena flos-aqUae
Serial
Number
74
78
82
96
98
121
124
126
127
136
values in
Name of Compound
(p-Bromophenyl)-2-thlenyl
iodonium chloride
2-Thienyl-p-tolyliodonium
chloride
( o-Chloropheny 1 ) -2- thieny 1
iodonium chloride
2 , 5-Dibromo-3 , 4-dinitro-
thiophene
Tetrachlorothiophene
N'-(3-(l,l-Dimethylethyl)-4-
nitrophenyl)-N,N-dimethyl urea
N- ( 4-Buty 1-2-nitropheny 1 ) -
acetamide
7 , 8-Dihydro-6H-pyrrolo ( 1 , 2-e )
purin-4-01
2-Tert-butyl-4-nitrophenol
2 , 2 ' - ( 1 , 2-Ethenediy 1 ) bisbenzoxa-
ppm
Activity
1.6
100
94
95
100
23
100
at Various
0.8
95
65
94
100
23
9
0
0
12
91
Cone en
0.4
59
48
92
95
14
68
tration
0.2
41
zole
146 5-Butyl-2-methyl-lH-benzi-
midazole
-------
TABLE 37 continued
vo
CO
Serial
Number
14?
148
149
150
151
152
153
154
155
156
157
Name of Compound
Pentachlorophenol, compound
with 2-(2,4,5-Trichlorophen-
oxy)ethanamine (1:1)
N'-(4-((6-Chloro-4-tri-
fluoromethy1)-2-pyridiny1)
oxy)-N,N-dimethyl urea
5-Nitro-2-thiophene-
carboxaldehyde, oxime
N-(4-((6-Chloro-2-pyridiny1)
oxy)phenyl) acetamide
N-(4-((6-Chloro-2-pyridinyl)
oxy)phenyl)-Nf-methyl urea
N'-(4-((2,6-Dichloro-4-
pyridiny1)oxy)pheny1)-N,N-
dimethyl urea
2,5-Bis((4-methyIpheny1)
sulfonyl)-3,4-dinitro-
thiophene
N-(3-Chlorophenyl)-2-
isoxazolidinecarboxamide
N-(4-Chlorophenyl)-2-
isoxazolidinecarboxamide
3-(Acetyloxy)-4-bromo-
butanoic acid, methyl ester
N »-(4-Ac etyIpheny1)-N,N-
dimethyl urea
Activity at Various Concentrations
1.6 0.8 0.4 0.2
0
0
0
0
0
8
0
18
6
0 '
-------
TABLE 37 continued
Serial
vo
VD
Number
158
159
160
161
162
163
164
165
166
167
168
Name of Compound
N,N-Dimethy1-N'-(4-((6-
(methylthio)-2-pyridinyl)-
oxy)phenyl) urea
N'-(4-((6-Chloro-2-pyridiny1)
oxy)phenyl)-NsN-diethyl urea
2- (2,4-Dichlorophenoxy)-
3-nitropyridine
Tris(dimethylamino)(hydroxy-
phenyl=methyl) phosphonium,
hydroxide, inner salt
N-(4-((6-Chloro-2-pyraziny1)
oxy)phenyl)-N,N-dimethyl urea
N'-(4-((3-Chloro-2-pyridiny1)
oxy)phenyl)-N,N-dimethyl urea
N'-(4-EthyIphenyl)-N,N-
dimethyl urea
N»- (1|- ((3,5-Dichloro-2-
pyridiny1)oxy)pheny1)-N,N-
dimethyl urea
4-Amino-6-(l,l-dimethyl=ethyl)-
3-(methylthio)-l,2,4-triazin-
5(4H)-one
lH-Imidazol-2-ylphenyl-
diazene
2-Chloro-6-(4-methoxy-
phenoxy) pyridine
Activity at Various Concentrations
1.6 0.8 0.4 0.2
0
0
2
10
0
0
0
0
13
0
-------
TABLE 37 continued
Serial Activity at Various Concentrations
Number Name of Compound 1.6 0.8 0.4 0.2
169 N'-(4-(2,6
(trifluoromethy1)phenoxy)=
phenyl)-N,N-dimethyl urea
170 2-(4-Chlorophenyl)-2,3,5,6- -- 14
tetrahydroimidazo(2,1-b)-
thiazole, monohydrochloride
171 2,3,5,6-Tetrahydro-2-(2- 0
naphthalenyl)-imidazo(2,l-b),
monohydrochloride
172 2-Hydroxy-N-phenyl-3- ~ 4
pyridinecarboxamide
o 173 Nf-(3-Chlorophenyl-N-methoxy- 0
0 N-methyl urea
176 , l,2-Dichloro-4-(isothiocyanato- 97
methoxy) benzene
177 l-(2,4,5-Trichlorophenoxy)= 65
thiocyanic acid, ethyl ester
178 2-((2-(Dimethylamino)ethyl) 0
amino)3,4-dihydro-l(2H)-
isoquinolinone, dihydrochloride
179 2-Phenyl-5H-(l,2,4)triazolo- 0
(l,5-b)isoindole
180 2-(3-Methylphenyl)-5H-(l,2,4)- 0
triazolo(l,5-b)isoindole
-------
Serial Activity at Various Concentrations
Number Name of Compound 1.6 0.8 0.4 0.2
182 ((4,5-Dimethoxy-l,2-phenylene)- 0
bis=(imino(thioxomethylene)))bis-
carbamic acid, dimethyl ester
183 3-(4-(l,l-dimethyl=ethyl)phenyl)- 0
2,3,5,6-tetrahydroimidazo(2,1-b)
thiazole
184 Phenyl-2-thienyl methanone, o- 28
((methyl=amino)carbony1) oxime
185 Bis((l,l'-biphenyl)-4-yl)- 0
ethanedione
186 ((3,5-Dichloro-6-fluoro-2- 65
pyridinyl)oxy)methyl ester
thiocyanic acid
187 2-(4-(Ethoxyphenyl)2,3,5,6- 5
tetrahydroimidazo(2,1-b)thiazole,
monohydrochloride
188 N-(l-(4-Bromo-2,5-dichlorophenoxy)- 0
2,2,2-trichloroethy1)formamide
189 N'-(4-Ethyl-3-nitrophenyl)-N,N- 3
dimethyl urea
190 4-((3,5,6-Trichloro-2-pyridinyl)- 0
oxy)phenol)
191 N'-(3-Chloro-4-((6-chloro-2- 18
pyridiny1)oxy)=pheny1)-N,N-
dimethyl urea
192 N'-(4-((2-Chloro-6-methyl-4- 0
pyrimidinyl)=oxy)phenyl)-N,N-
dimethyl urea
-------
TABLE 37 continued
Serial Activity at Various Concentrations
Number Name of Compound 1.6 0.8 0.4 0.2
193 N'-(4-((6-Chloro-2-pyridinyl)oxy)- ~ 41
3-(trlfluoromethyl)phenyl)-N,N-
dimethyl urea
194 N,N-Dimethyl-N'-(4-((6-(trifluoro- 6l
methyl)-2-pyridinyl)thio)phenyl)
urea
195 N-(l-Methylethyl)-4-phenoxy- 0
benzenamine
196 N'-(4-((6-Bromo-2-pyridinyl)thio) ~ 0
phenyl)-N,N-dimethyl urea
197 l-(3,3-Dichloro-l-methylenebutyl)- 20
3,5-dimethyl benzene
198 p-(p-Nitrophenylthio) phenol 0
199 l-((4-Nitrophenyl)methyl)piperidine 0
200 (5-Chloro-2,4,dimethoxyphenyl)- 0
carbamic acid, 2,4,5-trichloro-
phenyl ester
201 2,4-Dibromo-3-methyl-6-nitro- 0
phenol
202 2>4-Dichloro-3-methyl-6~nitro- 0
phenol
203 N-Bromophenyl-2-chloro- 0
acetamide
204 4-((6-Fluoro-2-pyridinyl)=thio) 0
phenol
-------
TABLE 37 continued
Serial
Number
205
206
Name of Compound
l-Chloro-2-(methylsulfonyl)-
ethane
2,4,5-Trichloro-3-methy1-6-
nitrophenol
Activity at Various Concentrations
1.6 0.8 0.4 0.2
o
CO
-------
TABLE 38
LABORATORY SCREENING TESTS
Test Compounds versus Mlcrocystis aeruginosa
values in ppm
Serial Activity at Various Concentrations
Number Name of Compound 1.6 0.8 0.4 0.2
136 2,2'-(l,2-Ethenediyl)bisbenzoxazole 100 9^ 91* 8?
192 N'-(4-((2-Chloro-6-methyl-4-pyri- -- 53
midinyl)=oxy)phenyl)-N,N-dimethyl urea
197 l-(3,3-Dichloro-l-methylenebutyl)- 6M
3,5-dimethyl benzene
-------
Mlcrocystis down to 0.4 ppm. Compound No. 176 exhibited 97
percent control at the 0.8 ppm level when tested against
Anabaena.
Two commercial algaecides, copper sulphate and Cutrine
(copper sulphate with an ethanolamine complexing agent)
were tested at a wide range of concentrations against both
target species of algae to give a comparative base (Table 39
and Table 40).
Compound Activity as a Function of pH
A test series was run with compounds No. 23 and No. 73 at
0.8 ppm versus Microcystis aeruginosa at variable pH values
ranging from pH 6 to pH 9. Compound No. 23 showed a
decreasing activity at pH values below 9.0. The activity
of Compound No. 73 however, exhibited no pH-dependence In
the pH ranges and concentrations tested (Table 41).
Correlation of Compound Activity with Biomass
Because of the results of the whole-pond test in which the
biomass was very high, a laboratory test was designed to deter-
mine the relative effect of increasing algal population
densities on the algaecide activity of Compound No. 73. As
determined with doubling numbers of Anabaena cells, ranging
from approximately 0.5 x 106 cells/ml to 9.0 x 106 cells/ml
no significant reduction in algaecidal activity was noted,
except at the highest algal density used. At this density,
9 to 10 million cells/ml, only 84 percent control was found
compared to 100 percent control exhibited by each of the
others (Table 42 ). Up to a relatively dense culture, there
appears to be no relationship between culture density and
algaecidal effectiveness.
105
-------
TABLE 39
ALGAECIDAL ACTIVITY OF CuSO»-5H20 AGAINST TWO SPECIES OP BLUE-GREEN ALGAE
Three-Day Test
Anabaena flos-aquae
Microcystis aeruginosa
H
O
a\
Concentration
ppm
1.0
0.8
0.6
0.4
0.2
Control No. 1
Control No. 2
Initial
Relative
Intensity
0.18
0.18
0.18
0.18
0.18
0.18
0.18
Final
Relative
Intensity
0.07
0.08
0.11
0.49
0.60
0.56
0.55
Percent
Control
87
86
80
16
0
__
Initial
Relative
Intensity
0.21
0.21
0.21
0.21
0.21
0.21
0.21
Final
Relative
Intensity
0.02
0.04
0.05
0.15
0.41
0.44
0.43
Percent
Control
100
91
90
66
0
-------
TABLE 40
ALGAECIDAL ACTIVITY OP CUTRINE AGAINST TWO SPECIES OF BLUE-GREEN ALGAE
Three-Day Test
Anabaena flos-aquae
Microcystis aeruginosa
Concentrati on
ppm
4.0
3.0
2.0
1.0
0.5
Control No. 1
Control No. 2
Initial
Relative
Intensity
0.18
0.18
0.18
0.18
0.18
0.18
0.18
Final
Relative
Intensity
0.03
0.08
0.17
0.56
0.53
0.56
0.55
Percent
Control
95
85
70
0
0
__
Initial
Relative
Intensity
0.21
0.21
0.21
0.21
0.21
0.21
0.21
Final
Relative
Intensity
0.35
0.37
0.82
0.36
0.43
0.44
0.43
Percent
Control
92
91
81
17
0
__
-------
TABLE 41
pH SENSITIVITY TESTS ON COMPOUNDS NO. 23
(2,5-DICHLORO-3,4-DINITROTHIOPHENE) AND NO. 73 (P-
CHLOROPHENYL-2-THIENYL IODONIUM CHLORIDE) AGAINST Mlcrocystls
Test Compound Concentration - 0.8 ppm
Serial Number
and pH Value
23 - pH 6
pH 6 control
23 - pH 7
pH 7 control
23 - pH 8
pH 8 control
23 - pH 9
pH 9 control
73 - PH 6
pH 6 control
73 - pH 7
pH 7 control
73 - pH 8
pH 8 control
73 - pH 9
pH 9 control
Initial
Relative
Intensity
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
.21
.44
.20
.51
.046
.65
.036
.60
.015
.53
.016
.61
.022
.58
.019
.60
Percent
Control
57
64
95
100
100
100
100
100
108
-------
TABLE 42
ALGAECIDAL ACTIVITY - BIOMASS DEPENDENCE TEST
versus
Anabaena flos-aquae @ 1.6 ppm
Test Vessel
Time & Relative Intensities
Final R.I. Control
Initial R.I. 0+4 Days %
I-Control
II
II-Control
III
Ill-Control
IV
IV-Control
V-Control
VI
Vl-Control
2,
2,
1,
1,
0,
0,
0
0
0
0
5
5
0.25
0.25
0.12
0.12
0.06
0.06
0.56*
1.50
0.39**
0.82
0.20**
0.55
0.16**
0.48
0.11**
0.37
0.05**
0.20
84
100
100
100
100
100
* Residual R.I. - 0.32
**No living cells microscopically detectable
109
-------
BIOLOGICAL CONTROL SYSTEMS
Compound Activity Enhancement
During the course of Phase I investigations, a bi-flagellated,
yellowish-pigmented algal organism, identified as Ochromonas
ovalis, was discovered which was found to ingest cells of
the target blue-green algal Microcystis aeruglnosa. The
phagocytic activity of this organism was found to be enhanced
by the presence of certain test compounds at low levels
which alone exhibited only marginal algaecidal activity.
Through Phase II additional synergistic compounds were
identified, and environmental and growth parameters, which
exhibited a positive affect on the phagocytic activity of
various species of Ochromonas, were delineated.
An Investigation of this phenomena with respect to three
compounds (Nos. 114, 117 and 119) received for testing
during Phase III verified the previously obtained data.
Ochromonas danica in the presence of Compound No. 117
(N-((U-(6-cyano=2-pyridinyl)oxy)phenyl)-N,N-dimethyl urea)
and No. 119 (N'-(M(6-bromo-2-pyridinyl)oxy)phenyl)-N,N-
dimethyl urea) both produced 100 percent control of
Microcystis in six days, compared to 92 percent control in
the same period of time where no test compound was used.
Compound No. 114 showed no activity-enhancing effect at
the 0.2 ppm level, as did No. 117 and No. 119 (Figure 14,
Table 43, and Appendix E). In no case however, was the
system improvement considered to be of sufficient magnitude
to warrant further investigation with the compounds tested.
The phagocytic activity enhancement phenomenon may still be
valid, however, should a compound with sufficient optimizing
properties be discovered.
110
-------
TABLE 43
INFLUENCE OF TEST COMPOUND NO. 117 - N((4-(6-cyano-2-pyridinyl)oxy)phenyl-N,N-dimethylurea
AT 0.2 PPM ON THE PHAGOCYTIC ACTIVITY OF FOUR SPECIES .OF Ochromonas
CELL COUNTS
Species
0. 2>06trop+(M)+(T)
0. dontea+(M)+(T)
0. malhcanensis +
0, oualis+(M)+(T)
Controls
0. £>astrop+(M)
0. daniaa + (M)
0. malTzameneis+CM)
0. 0*,Ms+(M)
(M) only
Culture No. 1
(M) only
Culture No. 2
0 -
(M)xlOe
0.84
0.84
0.84
0.84
0.84
0.84
' 0.84
0.84
0.84
0.84
- days
(Och)xlO"
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
_
0 +
(M)xlO°
0.47
0.73
0.98
0.07
0.63
0.42
0.90
0.07
1.20
1.16
1 day
(Och)xlO"
1.80
0.65
0.30
3.0
0.35
0.40
0.50
3.5
^^
___i_
0 +
(M)xlO6
0.0
0.19
0.95
0.0
0.03
0.53
1.10
0.0
1.20
1.06
2 days
(OchJxlO*
7-3
1.9
0.70
9-1
4.5
2.60
0.45
8.6
_M^
^MB
0 +
(M)xlOe
0.0
0.0
0.57
0.0
0.0
0.34
0.75
0.0
4.35
4.25
6 days
(CchjxlO"
12.9
13.7
10.5
0.40
12.2
2.0
11.2
8.2
m^i-u-
^w
Percent
Control
100
100
86
100
100
92
82
100
Key:
(M) = Mioroeystis aerug-inosa
(Och) = Oohvomonas
(T) = Test chemical
-------
Figure lU.
[nfluence of Three Test Compounds
on the Phagocytic Activity of
Ochromonas danica at 0.2 ppm
With Compound
Without Compound
100
w
o
J-t
CJ
o
75
50
25
!»*+»**+
/********* *
»*«*******
/»*********»
/» ****** *
« t * *
******
******** *^*4
******
********* » <
*«*«»*
******
******
***
V******* »
******
No. 114
No. 117
Test Compound
No. 119
112
-------
Storage-Viability Study
A comprehensive study, involving 108 separate tests, was
undertaken to determine the various conditions under which
the Ochromonas ovalls and Ochromonas bastrop species would
be able to maintain viability and return to normal activity
upon re-culturing. Each species was subjected to seven
different types of substrate media, four different moisture
states, and three widely different temperature conditions.
After 15 days storage time under the specified conditions,
samples from each culture were re-cultured in Gorham's medium
for three days and then examined microscopically. Those
cultures which showed viable Ochromonas cells still present
for 44- and 70-day periods are indicated with an asterisk
in Table 44. Both species survived the full 70-day test
period in moist activated charcoal, in Jellied liquid
nutrient agar at room temperature, and when imbibed in cotton
or polyester fibers under air-dried conditions.
In no case of survival was the number of reviving cells very
great but the fact that cell viability was retained at all
furnishes a basis for the possibility of storage of substan-
tial culture volumes especially since the cells were able,
in certain substrates, to survive dry or partially dried
conditions for such a long period of time.
Field Tests
The planned approach toward further development of the phago-
cytic organisms, for the natural control of Mlcrocystls
blooms was to introduce a culture of Ochromonas ovalls into
a Mlcrocystis-infested body of water, or into an isolated
portion thereof. This chrysomonad algal species had proven
to be a voracious feeder on Mlcrocystis aeruglnosa during
113
-------
Conditions which Produced
_ Positive Results _
Gorham's medium - refrigerated
Gorham's medium - room temperature
Gorham's medium - refrigerated,
evaporated (near dryness)
Gorham's medium - room temperature,
evaporated (near dryness)
Topsoil - refrigerated, sterilized,
moist
Topsoil - room temperature,
sterilized, moist
Topsoil - refrigerated, sterilized,
dry
Topsoil - room temperature,
, dry
Topsoil - refrigerated, non-steril-
ized, moist
Activated charcoal - room tempera-
ture, moist
Nutrient agar (gel) - refrigerated
Nutrient agar (gel) - room
temperature
TABLE 44
Ochromonas STORAGE-VIABILITY STUDY
0+44 days
0+15 days
0. ovalis
**
*
**
0. bastrop
#*
««
**
0. ovalis 0. bastrop
0+70 days
0. ovalis 0. bastrop
-------
TABLE HH continued
Conditions which Produced 0+15 days 0+44 days 0+70 days
Positive Results Q. ovalis 0. bastrop 0. ovalis 0. bastrop 0. ovalis 0. bastrop
Liquid nutrient agar - refrigerated * ** * * *
Liquid nutrient agar - room
temperature ** ** ? ? * *
Polyester fibers - refrigerated,
dried **
Polyester fibers - room
temperature, dried * * ? ? * *
Polyester fibers - refrigerated
moist * *
._, Cotton fibers - refrigerated,
M moist * ** ? ? *
VJI
Cotton fibers - room temperature,
moist * * * ? *
Cotton fibers - room temperature,
dried * * * * *
* Viable organisms present after 15 days storage or longer
**Copious number of viable organisms present after 15 days or longer
-------
extensive laboratory and small-scale field test situations.
Verification of these results under natural conditions
was attempted as a sub-portion of the whole-pond test in
North Texas.
Sixty-gallon plastic bags, identical to those used for con-
trol chambers, were filled with ambient pond water and
covered with plastic previous to administration of test
compound to the pond. The dense cultures of Ochromonas
were added to each test chamber sufficient to yield an
Ochromonas density of about 200 cells/ml. The initial
Microcystis population was monitored at 10,000 cells/ml.
At 0+1 day water samples were taken from each vessel and
examined microscopically to determine algal population
densities. Data from this and subsequent monitorings indi-
cated that the physical, chemical, and/or biological inter-
ference conditions were apparently such that the Ochromonas
could not cope with them and this algal species did not
survive.
Ultramicrostructure and Mode-of-Action Study
An important aspect of the biological control study was
undertaken by Drs. Michael J. Wynne and Gary Cole of the
University of Texas Botany Department. An extensive invest-
igation on the sub-cellular organelle structure of Ochromonas
danica and their mode of ingestive action was made during
Phase III. The complete report is contained in Appendix A.
In summary, Wynne and Cole found that the chrysomonad alga
(Ochromonas) is a weak autotroph, primarily because of
insufficient chlorophyll, and therefore requires other
organic materials for sources of nitrogen, and for carbon
116
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energy sources. Consequently, the endocytic mode of
nutrition is common with this organism, of which the toxic
blue-green alga Microcystis aeruginosa is a common target
when present in the same culture.
A study of the efficiency and mechanism by which this
phagocytic organism engulfs and ingests cells of Microcystis
utilizing various light and electron-microscopic techniques,
was the primary focus,of this investigation. The action of
Ochromonas on Microcystis is suggestive of a mechanism for
this nuisance algal species.
Dense cultures of Ochromonas danica were prepared by the
University investigators and added to existing healthy
stock cultures of Microcystis. The decline in Microcystis
cell count as a function of time is shown graphically in
Table 1, Appendix A. The rate of endocystis is seen to
be very rapid during the initial 10 to 15 minute contact
period, after which the action declines markedly.
A series of light micrographs taken at successive time
intervals show various stages of engulfment of Microcystis
by CK_ danica and subsequent intercellular digestion of the
former (Figures 1-4, Appendix A). Ingestion has been
observed to consistently occur at the anterior end of the
phagocyte. When Microcystis is initially engulfed it
becomes invaginated in the primary food vacuole and then
immediately begins to migrate toward the posterior pole of
the cell. This food vacuole soon fuses with the larger
secondary food vacuole and releases its contents into the
digestive organelle. These events may occur several suc-
cessive times, resulting in the accumulation of as many as
six to eight Microcystis cells in the secondary phagosome,
which reaches its maximum volume when the vacuole becomes
117
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distended to full capacity, at which time CK_ danlca ceases
any further engulfment.
The precise extent of digestive activities In this stage
Is unknown, but it Is suggested that at least the outer,
mucilaginous sheath of the blue-green alga Is removed.
A unique cross-fracturing freeze-etch method with scanning
electron micrography techniques was employed by Drs. Wynne
and Cole in further investigations of the digestive processes
within the secondary phagosome. Almost complete breakdown
of the engulfed cells is indicated in the secondary phago-
some (SP) in Figure 4, Appendix A.
Figures 11 through 15, Appendix A, show Mlcrocystls in
successively later stages of digestion, in which the photo-
synthetic lamellae are seen to gradually depart from their
recognizable pattern to one of discontinuity. The ends of
these membranes apparently tend to fuse and give the appear-
ance of circular vessicles.
A freeze-etched, cross-fractured golgi complex, or dictyosome,
is shown just above the nucleus in Figure 21, Appendix A.
This organelle, which is responsible for the production of
digestive enzymes, was in an active state of proliferation
at the time of fixation, and treatment with acid phosphates
resulted in the accumulation of electron-rich deposits con-
centrated in the vessicles arising from the golgi cisternae,
as seen in Figure 22 .
A number of other kinds of microorganisms, including brewer's
yeast and several species of bacteria, have also been observed
to be Ingested within the food vacuoles of 0. danica. Figure
118
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25, Appendix A, shows seven partially digested bacteria
enclosed with a secondary phagosome.
Observations of endocytotic activity under experimental con-
ditions seem to indicate that initiation of the action
depends upon a chance contact between the two cells. Since
invagination seems to always occur at the anterior end of
the chrysomonad, its beating flagella tend to create micro-
currents over the surface of the cell, thus causing any
posterior contacts to be moved in that direction. Aaronson
postulated that the flagella may be involved in the accretion
of particles on the cell surface and thus provide the organ-
sim with a mechanism of sampling material from the environment.
Since Microcystis aeruginosa is a common bloom-producing
alga and one which is capable of releasing toxic substances,
identified as cyclic polypeptides, in sufficiently high
concentrations to kill a variety of animals the concept of
developing a natural control mechanism which could be intro-
duced at an appropriate time, is intriguing.
In this study, the rate of endocytosis was determined by
observing the rate of decrease in concentration of Microcystis
in a culture which contained a known concentration of 0.
danica. The uptake of Microcystis was most rapid during the
first ten minutes after the two cultures were combined, at a
calcualted 7.0 x 10* cells/ml/min. The rate of invagination
then decreased to 1.0 x 10* cells/ml/min during the next
20 minutes primarily due to two factors: reduction of blue-
green cell concentration, thus reducing the number of change-
encounters with Ochromonas cells, and many of the Ochromonas
cells becoming gorged with blue-green algae and thus render-
ing themselves Incapable of additional feeding until the
ingested cells had decomposed.
119
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It is suggested that the effectiveness of utilizing such
a phagocytic organism in the biological control of Microcystis
blooms would depend on fulfillment of at least the following
four conditions; a continuing rapid rate of intercellular
digestion of Microcystis* the maintenance of high concentra-
tions of Ochromonas in the environmental niche, the selective
action on Microcystis by Ochromonas as a preferential food
source and the detoxification of Microcystis cells during
the digestive process, all of which will require further
investigation.
120
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SECTION VI
REFERENCES
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Ochromonas . Arch. Mikrobiol. 29:39-44, 1973.
Bain, R. C., Jr. Algal Growth Assessments by Fluorescence
Techniques. Proceedings of the Eutrophication Biostimula-
tion Assessment Workshop, U. S. Department of the Interior,
Federal Water Pollution Control Administration, 1969.
Bartsch, A. F. Practical Methods for Control of Algae
and Water Weeds. Public Health Rpt. 69:749-757, 1954.
Bartsch, A. F. Proceedings of a Symposium Jointly
Sponsored by University of Washington and Federal Water
Pollution Control Administration. U. S. Department of
the Interior, 1967.
Battelle, Columbus Laboratories. Effects of Chemicals on
Aquatic Life. In: Water Quality Data Book, Vol. 3.
U. S. Environmental Protection Agency, 1971.
Brown, R. L. Effects of Light and Temperature on Algal
Growth. Calif. Dept. of Water Res, 1973.
Bueltman, C. G. Provisional Algal Assay Procedure. Joint
Industry-Government Task Force on Eutrophication, 1969.
Cortell, J. M. The Role of Herbicides in the Preservation
of our Urban and Industrial Water Resources. Weeds, Trees,
and Turf, 6:12-28, 1970.
Drews, G. Fine Structure and Chemical Composition of the
Cell Envelopes. In: The Biology of Blue-Green Algae,
Botanical Monographs, Vol. 9» Carr, N. G. and Whit ton,
B. A. (eds.). Berkeley, Univ. of Calif. Press, 1973-
pp. 99-116.
Environmental Protection Agency. Eutrophication Research
Highlights. Pacific Northwest Environmental Research
Laboratory, September 1973-
Faust, S. D. Fate of Organic Pesticides in the Aquatic
Environment Am. Chem. Soc., Washington, DC, 1972.
Fitzgerland, G. P. Algaecides. Univ. of Wis., Madison, 1971.
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Goddard, E. D. Molecular Association in Biological and
Related Systems. Am. Chem. Soc., Washington, DC, 1968.
Gorham, P. R. Toxic Algae as a Public Health Hazard.
JAWWA. 56:1481-8, 1964.
Hasler, A. D. Antibiotic Aspects of Copper Treatment of
Lakes. Wis. Acad. Sci., Arts and Lett. 39:97-103, 194?.
Holm-Hansen, 0., et al. Pluorometric Determination of
Chlorophyll. Inst. of Marine Resources, Scripps Institute
of Oceanography, 1966.
Highes, E. 0., P. R. Gorham and A. Zehnder. Toxicity of a
Unialgal Culture of Microcystis aeruginosa. Canadian J.
of Microbiol. 4:225-236, 1958.
Hutchinson, G. E. A Treatise on Limnology. Vols. I and II,
John Wiley & Sons, Inc., New York, NY, 196?.
Jackson, Daniel P. Algae, Man and the Environment. Syracuse
University Press, Syracuse, NY, 1967.
Katz, M. and A. R. Gaufin. The Effects of Pollution on the
Fish Population of a Midwestern Stream. Trans. Am. Pish Soc.
82:156-165, 1953-
Keup, L. E., W. M. Ingram, and K. M. MacKenthun. Biology of
Water Pollution. U. S. Department of the Inteiror, Federal
Water Pollution Control Administration, June 1968.
Kuentzel, L. E. Bacteria, Carbon Dioxide, and Algal Blooms.
JWPCF. 41:10,1737-1747, 1969.
Lewin, R. A. Physiology and Biochemistry of Algae. Academic
Press, New York, NY, 1962.
MacKenthun, K. M. and W. M. Ingram. Limnological Aspects
of Recreational Lakes. U. S. Dept. of Health, Ed. and
Welfare, 1964.
MacKenthun, K. M. and W. M. Ingram. Biological Associated
Problems in Fresh Water Environments - Their Identification,
Investigation, and Control. U. S. Dept. of the Interior,
Fed. Water Poll. Control Adm., 1967.
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MacKenthun, K. M. and C. D. McNabb. Stabilization Pond
Studies in Wisconsin. JWPCF 33(12):1234-1251, 1961.
Malueg, K. W., et al. Lake Restoration by Nutrient
Removal from Waste Water Effluent. National Environmental
Res. Center, Office of Res. and Monitoring, 1973.
/
Meyer, J. H. Aquatic Herbicides and Algaecides. Noyes
Data Corporation, 1971.
Middlebrooks, E. J., T. E. Maloney, E. P. Powers, and
L. M. Knack. Proceedings of the Eutrophication-Biostimula-
tion Assessment Workshop. Sanitary Eng. Res. Lab, Univ.
of Calif, and U.S. Dept. of the Interior, Fed. Water
Pol. Control Adm., 1969.
Moyle, J. B. The Use of Copper Sulphate for Algae Control
and its Biological Implications. Am. Assoc. for the
Advancement of Science, Washington, DC, 1949.
Nichols, M. S., T. Henkel, and D. McNall. Copper in Lake
Muds from Lakes of the Madison Area. Trans. Wis. Acad.
Sci., Arts and Lett. 38:333-350, 1946.
Okino, T. Studies on the Blooming of Microcystls aeruginosa
Jap. J. Bot. 20:381-402, 1973-
Otto, N. E. and T. R. Bartley. Aquatic Weed Control Studies.
U. S. Dept. of the Interior, Bureau of Rec., Res. Rpt. No. 2,
1966.
Palmer, C. M. Algae in Water Supplies. U. S. Dept. of
Health, Ed., and Welfare, Washington, DC, 1962.
Prescott, G. W. How to Know the Fresh Water Algae.
Wm. C. Brown Publishing Company, 1970.
Prows, B. L. Development of a Selective Algaecide to
Control Nuisance Algal Growth. U. S. Dept. of the
Interior, Washington, DC, 1971.
Prows, B. L. and W. F. Mcllhenny. Development of a Selective
Algaecide to Control Nuisance Algal Growth - Phase II.
U. S. Environmental Protection Agency, 1973.
Reazin, G. H., Jr. On the Dark Metabolism of Golden Brown
Algae, Ochromonas malhamensis. Am. J. Bot. 4l:9, 771-777>
1954.
123
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Reazin, G. H., Jr. The Metabolism of Glucose by the Alga
Ochromonas malhamensis. Plant Phys. 31:4, 229-303, 1956.
Smith, G. M. The Fresh Water Algae of the United States.
McGraw-Hill Book Company, New York, NY, 1950.
U. S. Environmental Protection Agency. Water Quality Data
Book, Vol. 3. Effects of Chemicals on Aquatic Life, 1971.
Vance, B. D. Sensitivity of Microcystis aeruginosa and
Other Blue-Green Algae and Associated Bacteria to Selected
Antibiotics. J. of Phycology. 2:125-128, 1966.
Weiss, C. M. The Relative Significance of Phosphorus and
Nitrogen as Algal Nutrients. Univ. of N. Carolina, Chapel
Hill, NC, 1970.
Zajic, J. E. Properties and Products of Algae. Proceedings
of the Symposium on the Culture of Algae sponsored by the
Am. Chem. Soc., Plenum Press, NY, 1970.
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SECTION VII
APPENDICES
125
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APPENDIX A
ENDOCYTOSIS OF MICROCYSTIS AERUGINOSA BY OCHROMONAS PANICA
by
Garry T. Cole
and
Michael J. Wynne
Department of Botany, University of Texas
Austin, Texas 78712
126
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ABSTRACT
a
Ochromonas danica, a chrysomonad alga which demonstrates
a high degree of nutritional versatility, is capable of
feeding on the toxic blue-green alga, Mlcrocystis
aeruginosa Kutz. In this paper, light-microscopic,
electron-microscopic, and cytochemical examinations of
endocytosis in 0_._ danlca are reported with particular emphasis
on the vicissitudes of phagosomal and lysosomal activities
during intracellular digestion. A diagrammatic interpretation
of the function of organelles associated with endocytosis
is presented.
127
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INTRODUCTION
Ochromonas danlca Prings. demonstrates an unusual versatility
of nutritional modes. This chrysomonad alga is a weak auto-
troph, primarily because of insufficient chlorophyll, and
therefore, requires some organic substances in the media to
serve both as a source of nitrogen compounds and as a carbon
and energy source. Pringsheim (43) suggested that limited
photosynthesis helps to keep Ochromonas alive during long
periods in axenic culture but, in the absence of an added
carbon source, photosynthesis is insufficient to provide all
the organic compounds necessary for continued multiplication.
However, Aaronson and Baker (3) have reported that 0. danica
can grow photoautotrophically in a substrate-free medium in
5 percent C02 on a shaker and illuminated with fluorescent
lamps. If particles of organic material are added to axenic
cultures of 0_. danica, or, if the cultures become contamin-
ated with other microorganisms such as bacteria, this chryso-
monad also demonstrates adeptness in endocytotic activity.
Pringsheim (43) considered the nutritional versatility of
Ochromonas to be a primitive character, perhaps reflecting
the mixed trophic levels of ancestral forms. It is the
endocytotic mode of nutrition which will be explored in this
paper. The efficiency by which the chrysomonad engulfs and
ingests Microcystis aeruginosa Kutz., a toxic blue-green alga,
is suggestive of a mechanism of biological control and was
examined using light-microscopic and electron-microscopic
techniques.
128
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MATERIALS AND METHODS
Pure cultures of Ochromonas danica and Mlcrocystis
aeruginosa were obtained from the Indiana Culture
Collection (L1298) and Dow Chemical Company, Freeport,
Texas, respectively. A culture of M._ aeruginosa was also
obtained from Dr. Pranklyn Ott (#0510). The culture tech-
niques as well as the thin-section and freeze-etch prepara-
tory procedures for these organisms were outlined in an
earlier paper (12). Light-microscopic studies of endocytosis
were performed using a Zeiss universal microscope equipped
with Nomarski objectives and a high-intensity light source.
Kodak Plus-X film was used.
For estimates of the rate of endocytosis, the following
procedure was designed. Axenic cultures of 0^ danica and
M^_ aeruginosa were first grown as outlined above, except
that equal volumes of inocula of the former were added to
200 ml of Ochromonas media in 500 ml Erlenmyer flasks and
subjected to continuous agitation (2.2 ops) in a Gyrotory
shaker (New Brunswick Sci. Co.). After four days, cell
counts of 0_._ danica were estimated using a Spencer (No. 1*192)
hemacytometer. Concentrations of 2.8 x 106 - 3.5 x 10s
cells/ml were recorded. Similarly, cell counts were esti-
mated for M_._ aeruginosa after three weeks' growth in station-
ary culture. Concentrations of about 1.9 x 106 cells per ml
were routinely recorded. Clumping of M._ aeruginosa cells
affected both our estimates of cell concentration in axenic
culture and our determination of the rates of endocytosis
in the mixed condition. The latter problem arose from the
fact that p_._ danica was unable to engulf large clumps of
cells, but the chrysomonads would cluster in large numbers
about the mass of blue-green algae and thus significantly
affect the validity of the hemacytometer values. However,
we found that vigorous shaking of the test tube which
129
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contained the M._ aeruglnosa culture for approximately five
minutes on a Vortex mixer before combining it with 0. danica
considerably reduced this problem. Aliquots of 0^ danica
and M»_ aeruginosa at known cellular concentration were then
mixed. One-mi samples were subsequently removed from the
mixed cultures of three-minute intervals, immediately fixed
with one drop of 1 percent formalin, and the number of re-
maining WL_ aeruglnosa cells was recorded using the hemacyto-
meter. These successive recordings of blue-green algal cell
counts were continued for a total period of 30 minutes for
each mixed culture examined.
The electron-microscopic, cytochemical procedures for
locating acid phosphatase activity (25) in 0_._ danica was
based on the technique reported by Stoltze et_ aJL. (5D» except
that our material was incubated at 37°C for 60 minutes. The
reaction was monitored under the light microscope by with-
drawing cell samples from the Gomori media at 15-minute
intervals, centrifuging, and then resuspending the cells in
ammonium sulfide. The cells were mounted on microscope slides
and examined for the degree of precipitation. Our control
experiment included incubation at pH 5.0 without the 6-
glycerophosphate substrate. Observations of sectioned
material before and after uranyl acetate and lead citrate
staining indicated that no artifacts were introduced by the
post-staining procedures.
OBSERVATIONS
A sequence of light micrographs illustrates different stages
of engulfment of MI. aeruginosa by 0_._ danica (Figures 1 and 2)
and subsequent intracellular digestion of the former (Figures
3 and 4). In Figure 1, contact between a blue-green alga
(arrow) and 0_._ danica has occurred at the anterior end of the
130
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chrysomonad, adjacent to its contractile vacuole (cv).
It is in this region of CL_ danica that engulfment consist-
ently occurred. In the same chrysomonad cell in Figure 1,
another blue-green alga (arrowhead) is enclosed by a vacuole
which has produced a distinct bulge on the lateral surface
of the phagocyte. A lateral protrusion of the cell membrane
encasing a Microcystis cell is even more evident in Figure 2.
At the posterior end of 0_._ danica in Figure 2, a Microcystis
cell is contained by another vacuole. It is suggested that
two major kinds of vacuoles form during endocytosis in
2i. danica. When M._ aeruginosa is initially engulfed by the
chrysomonad, the prokaryote is enclosed by a primary food
vacuole, or, primary phagosome (pp), which immediately
begins to migrate toward the posterior pole of the cell. It
is this organelle and its contents which cause the lateral
distention of the phagocyte. The primary phagosome soon
fuses with the larger secondary food vacuole, or secondary
phagosome (sp, Figures 2, 3, and 4), and releases its con-
tents into this digestive organelle. The preceeding succes-
sive events can occur several times resulting in the accum-
ulation of six to eight Microcystis cells in the secondary
phagosome (Figure 3). Soon after engulfment of the first
blue-green alga, 0,._ danica cells lose their characteristic
pyriform shape and become almost spherical. The cell reaches
its maximum volume when the secondary phagosome becomes dis-
tended to its full capacity as indicated In Figure 3. At
this stage, 0_._ danica ceases further engulfment of M_._
aeruginosa, and the blue-green algal cells contained by the
secondary phagosome appear to be digested simultaneously.
The precise extent of digestive activities within the primary
phagosome is unknown, but we suggest that at least the outer,
mucilaginous sheath of the blue-green alga (17) is removed.
Digestion within the secondary phagosome has been demonstrated
by the transmission electron-microscopic and freeze-etch
131
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micrographs which follow. Almost complete breakdown of
M_._ aeruginosa cells is indicated in the secondary phagosome
in Figure 4.
The rate of endocytosis was based on the rate of decrease
of M. aeruginosa when mixed with a culture of 0_._ danica.
As outlined above, known volumes and concentrations of
constantly agitated cultures of 0._ danica were combined with
an equal volume of known cell concentration of M._ aeruginosa.
The mean concentration of 0_._ danica was 3.1 x 106 cells/ml
and the average concentration of M._ aeruginosa in axenic
culture was 1.9 x 106 cells/ml. The results of the hemacyto-
meter counts of free M^ aeruginosa cells in the presence of
0. danica, after varying periods, were compiled and assimilated
in a graph which is presented in Table I. The greatest rate
of decrease of M._ aeruginosa (i.e., rate of endocytosis)
occurred within the first 10 minutes after mixing the two cul-
tures. The actual decrease in the concentration of blue-green
algal cells during this initial period, as indicated in Table
I, was 7-0 x 105 cells/ml. In contrast, the decrease in con-
centration of M^ aeruginosa during the subsequent 20 minute
period was only 1.0 x 105 cells/ml.
Figure 5 shows a freeze-etched, cross-fractured secondary
phagosome of 0^_ danica which contains the partially digested
remains of Microcystis and Ochromonas cells. The latter
demonstrates the cannibalistic nature of the chrysomonad
even in the presence of adequate food material. However, the
degree of cannibalization when cultures of 0. danica and M.
aeruginosa were mixed was minimal and was, therefore, not
taken into consideration when preparing Table I. Upon compar-
ison of the blue-green alga enclosed by the secondary phago-
some in Figure 5 with the free-living, freeze-etched cell in
Figure 6, it is evident that the outer wall of the ingested
M. aeruginosa has been digested, most of its vacuoles have
132
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disappeared, and its photosynthetic lamellae are hyper-
trophied and irregularly arranged. Crystalloids are
frequently present in the cytoplasm of M^ aeruginosa
(Figures 6 and 7). The periodicity of the lattice pattern
of the cross-fractured structure is demonstrated in Figure
6. These inclusions resemble the cytoplasmic structures
identified earlier as polyhedral bodies (37, 42). The
crystalloids are electron dense in blue-green algal cells
which are present in secondary phagosomes of 0._ danica
(Figure 11) and appear to decompose rather slowly (Figure 14).
The thin-sectioned phagocyte in Figure 8 shows M_._ aeruginosa
enclosed within a secondary phagosome. The posterior portion
of the chrysomonad has collapsed slightly as a result of the
fixation procedure, but the phagosomal membrane and plasma-
lemma can still be distinguished. This section demonstrates
many of the characteristic ultrastructural features of CK_
danica. The single, anteriorly located dictysome (D) lies
adjacent to the contractile vacuole. The large nucleus (N)
is located between the lobes of the chloroplast, and the
outer membrane of the nuclear envelope is continuous with a
sac encompassing the chloroplast (arrows), referred to as
the chloroplast ER (9, 23, 24, 50). A large number of mito-
chondria are distributed around the perimeter of the cell.
Also noteworthy is the sparsity of endoplasmic reticulum
which is characteristic of 0_._ danica.
In the posterior region of the cell in Figure 8, above the
secondary phagosome, a distinct clusture of vesicles is
present. Most of these organelles, which are shown at
higher magnification in Figure 9, contain membrane fragments,
diffuse material, and small vesicles. These contents seem to
be common to all vesicles of similar size which are located
133
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adjacent to the secondary phagosome (Figures 11 through
14, and 16 through 18). In addition, another kind of
vesicle is present in this region of the cell, which is
coated with pentagonal and hexagonal subunits (arrow in
Figure 9 and 10). These subunits may entirely encase the
membrane of the vesicle (Figure 10), or, only partially
coat its outer surface (Figures 13 and 18). Similar pro-
files were illustrated in an earlier ultrastructural
examination of p_._ danica in which the subunits formed the
coating of certain vesicles arising from the Golgi complex
(12). Suggestions of the function of these structures in
association with the process of endocytosis are presented
in the discussion.
The blue-green algal cell in Figure 8 lacks its outer,
mucilaginous sheath, but the wall is still intact which sug-
gests that the cell had moved from the primary to secondary
phagosome Just prior to fixation. Of course, it is quite
probable that the secondary phagosome in Figure 8 also con-
tained other M^ aeruginosa cells. This is also true for the
contents of the secondary phagosome shown in Figure 11. In
this case, early signs of cytoplasmic decomposition of the
blue-green alga are demonstrated. Myelin figures, which have
been attributed to pathological alterations of cells (28,46)
and cytoplasmic degeneration (13) in a number of different
organisms, are found associated with the breakdown products
of blue-green algae at early (Figure 11) and late stages of
digestion (Figure 14). Electron transparent regions in
the cytoplasm of the ingested cell in Figure 11 are also
visible. Outside the Microcystis cell, but within the lumen
of the secondary phagosome in Figure 11, are clusters of
ribosomes which are distributed about the perimeter of the
digestive organelle. Appressed to the outer surface of the
phagosomal membrane are numerous ER lamellae. Endoplasmie
-------
reticulum was frequently observed associated with the
outer circumference of secondary phagosomes at various
stages of the digestive process (Figures 14, 16 and 1?)
even though it was sparse in other parts of the cell.
The phagosomal membrane in Figure 11 is highly convoluted.
As Microcystls is further decomposed, the degree of con-
volution of this membrane decreases (c.f. Figures 11, 14,
16 and 17) .
Of particular interest in Figure 11 is the partially coated
vesicle (arrow) lying adjacent to the phagosomal membrane.
The contents of this vesicle are typical of those uncoated
vesicles of similar dimension found next to the secondary
phagosome (Figures 9 and 13). At higher magnification
(Figure 12) the coated surface is demonstrated to be com-
posed of subunits similar to those illustrated in Figure 10.
Uncoated vesicles in Figure 13 are Juxtaposed to tangentially
sectioned evaginations of the phagosomal membrane, which is
suggestive of an origin of these vesicles through a process
of blebblng of the secondary phagosome. In addition, numerous
smaller vesicles containing diffuse material are found
adjacent to both the surfaces of the secondary phagosome and
large vesicles and are distributed randomly through the
cytoplasm in this region of the cell (arrows in Figures 13
and 18).
The outer wall of M.. aeruginosa in Figure 13 has decomposed
and, even though the photosynthetic lamellae are still
oriented in a recognizable pattern, they are discontinuous.
Only remnants of photosynthetic lamellae can be found in
Figure 14. Fragments of membrane have accumulated within
the phagosome and appear to be emerging from the partially
decomposed blue-green algal cell. This phenomenon is more
apparent in Figure 15. Some of the membranous fragments
(arrows in Figure 15) are continuous with membranes of the
135
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almost completely digested M._ aeruginosa cell. A tendency
for these fragments to form loops within the secondary
phagosome is demonstrated by Figures 14 through 16.
Apparently the ends of these membranes may subsequently fuse
and, in transverse section, therefore, would give the
appearance of vesicles (arrowheads in Figure 15).
Figures 16 and 17 illustrate secondary phagosomes which
contain the remains of blue-green algal cells in progessively
later stages of digestion. Figure 16 demonstrates a prolif-
eration of the amount of membrane loops and fragments within
the digestive vacuole, while both phagosomes show a marked
decrease in the amount of electron dense material compared
to earlier stages of Microcystls decomposition (c.f. Figures
11 and 14). All that remains of the M._ aeruginosa cells in
Figure 17 are short, linear membrane fragments and some
diffuse material. In the cytoplasm of 0. danlca in Figure 17
electron-dense deposits have accumulated which are inter-
preted to be lipid droplets. Also in this region of the
same chrysomonad, large, distinctive vesicles (rb) are present
which do not appear to be structurally connected to the
secondary phagosome and their contents consist of only
electron-dense deposits and some diffuse material.
Posterior to the secondary phagosome in Figure 18, two
vesicles with partially coated surfaces (c.f. Figure 12)
are visible. Both vesicles lie adjacent to the cell mem-
brane, which at several locations in this section, is
associated with diffuse and rather fibrous material. At
one region (com) the inner surface of the plasmalemma is
coated with subunits similar to those present on the surface
of vesicles. The outer surface of the freeze-etched cell
membrane of 0^ danica in Figure 19 (arrows) demonstrates
evaginations which are suggestive of exocytotic activity on
the cell surface. The freeze-etch micrograph of 0. danica
136
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in Figure 20 reveals numerous irregularities on the surface
fracture of the plasmalemma and membrane surrounding the
large secondary phagosome (arrows). Around the cytoplasmic
boundary of the phagosome, many small vesicles appear close-
ly appressed to the membrane surface. As previously indi-
cated in thin sections, phagosomal membrane blebbing and
fusion are suspected to occur along this interface (Figures
11 and 13). It is suggested that a relationship may exist
between these activities and at least some of the circular
surface patterns on the phagosomal membrane demonstrated in
this freeze-etch micrograph. Similarly, some irregularities
of the surface of the cell membrane may be indicative of
exocytotic activity.
At the apex of 0_._ danlca in Figure 21, a freeze-etched,
cross-fractured dictyosome is shown Juxtaposed to the
anterior surface of the nucleus. The Golgi complex was
in an actively proliferating state at the time of fixation.
When endocytotic cells were treated with acid phosphatase
accumulations of electron-dense deposits were concentrated
in vesicles arising from Golgi cisternae (Figure 22). In
the secondary phagosome shown in Figure 23, similar electron-
dense deposits resulting from the acid phosphatase reaction
were dispersed over the surface and throughout the decomposing
M._ aeruginosa cell. Localized acid phosphatase positive
reactions were also shown in large vesicles surrounding the
secondary phagosome (Figures 23 and 24).
Ochromonas danica has a voracious appetite for many dif-
ferent kinds of microorganisms including several species of
bacteria and brewer's yeast (1). Figure 25 shows at least
seven partially digested bacteria enclosed by a secondary
phagosome. Current studies of endocytosis of conidia of
Oidiodendron truncatum (Robak) Barren, an imperfect fungus,
by (X_ danica will form the basis of a future communication.
137
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DISCUSSION
The initiation of endocytosis of Microcystis aeruginosa
by Ochromonas danica seems to depend on chance contact
between the two cells, at least under our experimental
conditions. If a Microcystis cell comes into contact with
the posterior end of the chrysomonad, the beating flagella
of the latter create a microcurrent over the surface of
the cell which causes the blue-green alga to be carried
toward its anterior end. It is in this region, Just post-
erior to the flagella, where engulfment occurs. Aaronson
(2) has also observed that flagellar movements caused
India ink particles added to the medium to flow over its
sides and toward the anterior end of the organism. He has
suggested that the flagella may be involved in accretion
of particles on the cell surface of 0_._ danica and thus pro-
vide the organism with a mechanism of sampling material
from its environment.
Microcystis aeruginosa is a well known bloom-producing blue-
green alga (4l) which is capable of releasing toxic com-
pounds (7, 26, 33), identified as cyclic polypeptides, into
its aquatic environment in sufficiently high concentration
to kill a variety of animals, except waterfowl (27). The
possibility occurred to us that the voracious appetite
which £._ danica demonstrates for this blue-green alga
could be utilized in developing a biological control system.
Examination of endocytosis with the light microscope
demonstrated that a large number of Microcystis cells could
be rapidly engulfed by a single Ochromonas cell. Under
defined experimental conditions it was possible to estimate
the rate of endocytosis. Jacques (30) suggested that the
rate of endocytosis of an organism could be defined in two
138
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different ways. In one case, it could be the rate of invag-
ination of the cell membrane during erigulfment estimated on
the basis of the number of phagosomes of an average diameter
formed by the phagocyte per unit time. In the second case,
the rate of endocytosis could be extrapolated from the rate
of change in concentration of a defined substance in the
medium which is being engulged by the phagocyte. Our
estimate is based on the rate of decrease in concentration
of M*. aeruginosa in a culture which contains a known con-
centration of O._ danica. Uptake of the blue-green alga was
most rapid during the -first 10 minutes after mixing the two
cultures. The actual rate of decrease of M._ aeruginosa
during this interval (Table I) was 7.0 x 104,cells/ml/min.
On the other hand, sampling mixed cultures during the last
20 minutes of the experimental period indicated that the
rate of decrease of M._ aeruginosa was only 1.0 x 10" cells/
ml/min. One explanation of this reduction in the rate of
uptake demonstrated in Table I is that the occurrence of
endocytosis, at least in our artificial culture conditions,
is dependent on chance contact between M_._ aeruginosa and its
predator. As the concentration of the former drops because ,
of endocytosis, there is a proportional decrease in the
number of chance encounters with 0^ danica. Light-microscopic
studies of living cells have indicated that cannibalism in
0_._ danica is relatively rare in these mixed cultures and is,
therefore,"Ignored in our statistical analyses. However, the
rate of decrease of Microcystis cells is not linear (Table I)
because many 0_._ danica cells soon become gorged with blue-
green algae (Figure 3) and are then incapable of additional
feeding until the ingested cells have decomposed. Therefore,
an initial rapid uptake of cells (i.e., first 10 minutes in
Table I) is followed by a digestive period. Fewer 0^ danica
cells function as phagocytes during the later part of the
experimental period, thus further reducing the number of effect-
139
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ive encounters with M»_ aeruginosa cells* These factors
would collectively contribute to a marked reduction in
the rate of endocytosis in the mixed cultures after an
initial period of very rapid cell uptake. The effective-
ness of 0^ danica in biological control of this blue-green
alga would depend on fulfillment of at least the following
conditionsi 1) rapid rate of intracellular digestion of
M. aeruginosa; 2) maintenance of high concentrations of
0. danica in the environmental niche; 3) selection of M.
aeruginosa by Oj_ danica as the preferential food source;
and 4) detoxication of M_._ aeruginosa during the digestive
process. All these factors require investigation.
The mechanism of engulfment of Microcystis cells involves
formation of a primary phagosome by 0^_ danica. This struc-
ture first appears at the anterior end of the chrysomonad
when the blue-green alga contacts the cell membrane of the
phagocyte in this region. Schuster et_ §.^.(49) suggested
that the anterior region of a feeding CK_ danica cell consists
of a "cytoplasmic tongue" differentiated by fibers arising
from the kinetosomes and pinocytotic activity on the cell
surface. A minimal amount of digestion likely occurs within
the primary phagosome, because it is only a transitory struc-
ture which soon after formation, about 30 to 60 seconds later,
fuses with the secondary phagosome located at the posterior
end of 0. danica. However, it is suggested that the mucilagin-
ous sheath of M._ aeruginosa is at least partially removed in
the primary phagosomes, which of course assumed the presence
of intraphagosomal hydrolase enzymes. Aaronson (1) has pre-
sented a lengthy list of hydrolases which have been found in
0_._ danica. There is also evidence that this chrysomonad is
capable of secreting a wide variety of hydrolases into its
environment (36). The effects of these secreted proteins on
the food material which is ingested is unknown.
-------
The large posterior vacuole of 0_._ danlca has been referred
to as the 'leucosin vacuole1 in earlier studies of both
axenic cultures (43) and cultures which are feeding on
other organisms (15). We prefer to associate the term
leucosin with the large, posterior vacuole and storage
product which occur in axenic cultures of the chrysomonad
grown under conditions of adequate light and carbon supple-
ment. In this case, the vacuole functions as a storage
site of leucosin, a polymer of glucose and mannitol composed
of 1:3 and 1:6 glucosidic linkages and a characteristic
reserve product of the Chrysophyta (45). In mixed cultures,
this same vacuole assumes an alternate function as a hetero-
phagosome and, at this time, is probably less involved in
storage of leucosin. As previously indicated, the posterior
food vacuole, or, secondary phagosome, receives ingested
organic material from the primary phagosome at the time of
fusion of these two inclusions.
The characteristic shape of 0_._ danica in axenic culture is
pyriform with a distinctive elongate tail. However, when
these cells feed on M. aeruginosa, and other microorganisms
which we have examined, the chrysomonads assume an almost
spherical shape. Retraction of the tail is witnessed at
the time 0_._ danica engulfs its first Microcystis at the
anterior end of the cell. As the secondary phagosome
expands with an increasing number of ingested blue-green
algae, the chrysomonad becomes even more spherical. Bouck
and Brown (10) have examined the biogenesis of microtubules
in correlation with cell shape in 0^_ danica and have sug-
gested (11) that "the beak and rhizoplast sites could exert
control over the position and timing of the appearance, the
orientation, and the pattern of microtubule distribution.,.".
When the authors exposed Ochromonas to colchicine or hydro-
static pressure (11), the microtubules disassembled, and the
141
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cells became spherical. When these artifical conditions
were removed, the characteristic pyriform cell shape
reappeared with concomitant assembly and orientation of
microtubules. We suggest that the onset of endocytosis
in 0_._ danica also has a disruptive effect on the anterior
microtubule nucleating sites, which causes initial dis-
assembly of the cytoplasmic, skeletal system and subsequent
retraction of the tail. The increased cellular turgor
pressure established by the enlarged secondary phagosome
perhaps causes additional disassembly of microtubules, and
also has an inhibitory effect on regeneration of these
structures. After digestion of the Microcystis cells, the
chrysomonads slowly regain their pyriform shape, provided
that they do not continue endocytotic activity. Resumption
of this shape by 0_._ danica is probably correlated with the
reassembly, orientation, and distribution of microtubules
controlled by the kinetobeak and rhizoplast nucleating
sites of the cell.
Within two to five minutes after a Microcystis cell is
encased by the secondary phagosome, a high degree of cyto-
plasmic activity, reminiscent of Brownian movement, adjacent
to the upper surface of the digestive vacuole, is visible
with the light microscope. Thin sections of this same region
of the cytoplasm of 0^ danica, fixed at a comparable stage
of intracellular digestion of M._ aeruginosa, indicate that
the particles observed with the light microscope are vesicles
clustered in high concentration about the upper surface of
the secondary phagosome (Figure 8). Both ultrastructural
and cytochemical evidence indicates a close structural-
functional interrelationship between these vesicles and the
secondary phagosome. The vesicles, which appear to arise by
a process of blebbing of the phagosomal membrane (Figure 13),
contain acid phosphatase (Figure 24) as does the secondary
142
-------
phagosome itself (Figure 23). Closer observation of the
organelles surrounding the secondary phagosomes revealed
two additional kinds of vesicles: smaller vesicles of
fairly uniform size which contain only diffuse electron-
translucent material (Figures 13 and 18), and coated
vesicles of variable diameter (Figures 10 through 12 and 18).
De Duve (16) suggested that primary phagosomes, which arise
as a result of endocytosis, are initially devoid of digestive
enzymes. He further postulated, basing his concepts primarily
on observations of polymorphonuclear leukocytes, that the
primary phagosome receives its digestive enzymes, such as
acid phosphatase, from primary lysosomes. The latter
membrane-bounded organelles serve as storage sites for
newly synthesized enzymes not yet involved in digestive
events and are capable of fusing with the primary phagosome,
releasing their contents into the vacuolar lumen, and thus,
initiating the intracellular digestive process. Daems,
Wisse, and Brederoo (1*0 identified another category of
hydrolase-containing organelles in endocytotic cells as
secondary lysosomes, which arise as blebs of the phagosomal
membrane. The authors stated that these organelles contain
undigested material, which has been translocated from the
heterophagosome, and some of the products of digestion within
the secondary lysosome eventually find their way out and
into the cytoplasm without the escape of lysosomal enzymes.
Primary lysosomes may fuse with secondary lysosomes, pre-
sumably to replenish the supply of digestive enzymes in
the latter. We suggest that the large vesicles shown in
Figures 8 and 9 are secondary lysosomes based on the facts
that they occur in high concentration in the vicinity of the
secondary phagosome (Figure 8), apparently arise from blebs
of the phagosomal membrane (Figure 13), and contain acid
phosphatase (Figure
143
-------
Daems ejt al. (I1!) also pointed out that lysosomes fuse
only with phagosomes, or with each other, and that they
are apparently incapable of fusing with membranes of
other organelles. They proposed that this latter phen-
omenon, which is not well understood, exemplifies "fusion
incompatibility". Of importance to this concept is the
origin of the membranes encompassing each of the digestive
organelles in the endocytotic cell. The membrane of the
primary phagosome is of plasmalemma origin, while the
ontogeny of the secondary phagosome is unknown. However,
fusion of the two kinds of phagosomes demonstrates their
membrane compatibility. Considerable evidence has
accumulated for the origin of primary lysosomes from Golgi
cisternae (14, 16, 18, 19, 32, 40, 51). Figure 22 demon-
strates positive reaction for the test of the presence of
acid phosphatase in the Golgi cisternae of 0_._ danica. The
dictyosome of the endocytotic chrysomonad appears to .
actively proliferate vesicles (Figure 21) and may account
for most of the synthesis and compartmentalization of
digestive enzymes in the cell. A sparsity of endoplasmic
reticulum in 0_._ danica, especially in the vicinity of the
Golgi complex, led us to speculate earlier (12) that another
cyto-membrane system, the nuclear envelope and vesicles
arising from proliferation of its outer membrane, is also
associated with this synthetic process (12). However, some
ER lamellae are observed juxtaposed to the secondary phago-
some (Figures 11, 14, 16 and 17). Trucket and Goulomb (52)
observed in regions of root nodules of Pisum satlvum L.
where bacterial parasitism occurs, that the process of lyso-
some formation from Golgi proliferation alone seemed insuf-
ficient considering the degree of lytic activity in the cells;
and the authors, therefore, speculated that "endoplasmic
reticulum then shunts the dictyosomes and directly buds
phytolysosomes". Such a function may also be associated with
-------
with ER in (K_ danica. Matile (35) concluded that the
relatively large amounts of RNA found in isolated food
vacuoles of plants are due to the presence of ribosomes.
In thin sections of secondary phagosomes of 0^_ danica
(Figure 11), an abundance of ribosomes can be found in the
matrix. It is proposed that phagosomes are capable of a
certain degree of autonomous synthesis of lysosome enzymes.
Considerable evidence is available that coated veiscles
play a role in endocytosis (14, 18, 20, 39). The origin
of coated vesicles, however, is still rather controversial.
Localized, invaginated regions of the plasmalemma of CK_
danica frequently demonstrated a coating of pentagonal
and hexagonal subunits on the inner, cytoplasmic surface of
the membrane (12, and Figure 18). It has been suggested
that coated vesicles may arise by micropinocytosis of these
regions of the cell membrane (14, 53) and function as endo-
cytotic vesicles which may later fuse to form large food
vacuoles (39). Franke and Herth (20) observed that the
outer surfaces of coated indentations of the cell membranes
of Dlnobryon sertularia Ehr., another chrysomonad alga,
were associated with diffuse, electron dense material which
they suggested was of "glycoproteinaceous character".
Similar observations are reported above for 0. danica
(Figure 18). Roth and Porter (44) had proposed earlier
that coated vesicles may be specialized for cellular uptake
of protein. Evidence is also available that coated vesicles
arise directly from Golgi cisternae (12, 18, 22, 34).
Holtzmann e_t al.(29) proposed that these vesicles function
in transport of acid phosphatase from the dictyosome to
heterophagosomes. We suggested earlier (12) that coated
vesicles, arising from the Golgi, may subsequently fuse
with other organelles such as the plasmalemma or secondary
lysosomes (Figure 18) and "by some unknown mechanism deposit
145
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their coated surface upon the inner protein layer of the
cell membrane". Kanaseki and Kadota (31) proposed that the
coated surface of vesicles is an apparatus to control the
"infolding and fissioning mechanism" of the plasmalemma.
Their suggestion was based primarily on evidence that
myosin-like material apparently comprises the subunits and
this provided the necessary contractile properties to the
membrane.(6, 8). Perhaps the coated surfaces of secondary
lysosomes is also associated with blebbing and fission of
its membrane. It would appear, on the basis of current
evidence, "that the group of coated vesicles comprises a
functionally heterogeneous population" (14).
As suggested previously secondary lysosomes arise from
blebbing of the membrane encompassing the secondary phago-
some (Figures 11 and 13). These lysosomes, which commonly
contain smaller vesicles as well as membrane fragments and
heterogeneous material, may also be referred to as multi-
vesicular bodies. However, caution is required in use of
this terminology since evidence exists for a considerable
variation in function and origin of structures generally
referred to as mutlivesicular bodies (14). Our cytochemical
studies of 0_._ danica indicated that the multivesicular organ-
elles surrounding the secondary phagosome contain acid
phosphatase. Friend (21) suggested that these lysosome
enzyme-containing structures may become multivesicular by
penetration of small vesicles (e.g., primary lysosomes)
through the outer membrane, or, by invagination and budding
of the membrane itself. In Q._ danica, it is assumed that
fusion between a number of functionally different organelles
in the region of digestive activities is possible, necessi-
tating a high degree of fusion compatibility between their
respective membranes. For example, secondary lysosomes may
146
-------
fuse with primary lysosomes and with each other. Secondary
lysosomes may arise from evaginations of the secondary
phagosome and may subsequently fuse with the latter. The
freeze-etch, surface fracture of the secondary phagosome
in Figure 20 shows small circular scars which are reminescent
of the circular pockets illustrated by the surface fractures
of cell membranes of Tetrahymena pyriformis W. These membrane
irregularities were attributed to the fusion of mucocysts
(47, 48). Similar scars were demonstrated on the cell mem-
brane of Chlorella Beij., which were considered to have
resulted from fusion with Golgi vesicles (38).
Secondary lysosomes gradually gather indigestible products
of endocytosis and finally become so filled with residues
of digestion that they no longer participate in the segre-
gation of material ingested by the cell (14). Such conver-
sion of secondary lysosomes to residual bodies is indicated
in Figure 17. These organelles and their contents may be
gradually transformed into lipoidal deposits with a con-
comitant decrease in hydrolase enzymes contained by the
lysosomal membrane (14). Accumulation of lipid deposits
in CL_ danica has also been suggested to be indicative of
aging (5, 49). The freeze-etch micrograph of 0_._ danica
in Figure 19, demonstrating evaginations of the cell mem-
brane, is suggestive of a mechanism of exocytosis. Secre-
tion of membranes extracellularly by £._ danica has also been
demonstrated in thin sections and has been proposed to be
associated with an excretory process (4).
Our concepts of the various activities of phagosomes,
lysosomes, and coated vesicles in 0_._ danica are presented in
a diagrammatic interpretation of intracellular digestion in
Figure 26. More experimental evidence is required in order
to validate or refute this interpretation of the sequence
147
-------
of events associated with endocytosis. One possible,
future research approach is to use autoradiographic tech-
niques involving labelled isoprenoids of the photosynthetic
lamellae of ]YL_ aeruglnosa, which appear to be rather slowly
decomposed (Figures 13 through 15)» and to trace the pro-
gressive dissipation of these radioactive products through-
out the cytoplasm of 0. danica.
148
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ACKNOWLEDGEMENTS
The authors are grateful to Dr. B. L. Prows of the Dow
Chemical Company for donating strains: of Microcystls
aeruginosa and for providing advice and encouragement
during the course of this project. Dr. H. C. Bold read
the manuscript and offered suggestions. The project was
supported by a Research Corporation Grant (BH-752) and a
Dow Chemical Company Grant-in-Aid of Research to the
senior author.
149
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51. Stoltze, H. J., Lui, N. S. T., Anderson, 0. R. &
Roels, 0. A. 1969. The influence of the mode of
nutrition on the digestive system of Ochromonas
malhamensis. J. Cell Biol. 43:396-409.
52. Trucket, G. & Coulomb, PH. 1973. Mise en evidence
et Evolution de systeTne Phytolysosomal dans les
cellules des differe"ntes zones de nodules radicularies
de Pols (Pisum sativum L.). Notion d'he'te'rophagie.
J. Ultrastruct. Res. 43:36-57.
53. Yamada, E. 1955. The fine structure of the gall
bladder epithelium of the mouse. J. Biophys. Biochem.
Cytol. 1:445-58.
155
-------
TA^LE I
Cole and Wynne, 1973
O
z
O
U
O
O
2.2 -
2.0 -
1.8 -
1.6 -
1.4 -
1.2 -
uu U
u_0 1.0
O 2
Z
O
P 0.6
<
i
Z 04
in w .*t
U
° no
U °'2
0.0
ib
CONCENTRATION OF
OCHROMONAS DANJCA
IS 3.1 x
106CELLS/ml
15 20 25
TIME ( min )
30
35
156
-------
Figures 1-7
157
-------
Figures 8-10
**
158
-------
Figures 11-15
159
-------
Figures 16-19
}
9*
«MT"
160
-------
Figures 20-25
161
-------
Figure 26
26
ev
162
-------
Figures 1-4
Figures 5-7
LEGENDS FOR FIGURES
Interference contrast (Nomarski) light
micrographs of 0_._ danica at various stages
of endocytosis of KL_ aeruginosa. Arrow
indicates free-living M»_ aeruginosa. cv,
contractile vacuole; F, flagellum; pp,
primary phagosome; sp, secondary phagosome.
Figures 1-4, X 2,000.
Freeze-etch micrographs of 0_._ danica (Figure
5) and M._ aeruginosa (Figures 6 and 7).
Figure 5 illustrates a secondary phagosome
containing partially digested Ochromonas (0)
and Microcystis (M) cells. The free-living
M. aeruginosa cell (Figure 6) contains a
crystalloid (C), which at higher magnification
(Figure 7), demonstrates a distinct lattice
(arrows), cm, cell membrane; L, lipid droplet;
pi, photosynthetic lamella; V, gas vacuole.
Figure 5, X 11,7*10. Figure 6, X 32,000.
Figure 7, 67,200.
Figures 8-10 Thin sections of 0. danica which contains a
M._ aeruginosa cell in its posterior, secondary
phagosome (Figure 8). Figure 9 is a higher
magnification of secondary lysosomes concen-
trated just above secondary phagosome. A
coated vesicle (arrow in Figure 9 and 10) is
also shown. Arrows in Figure 8 indicate con-
tinuities between nuclear envelope and chloro-
plast ER. D, dictyosome; N, nucleus. Figure
8, X 14,7^0. Figure 9, X 37,200. Figure 10,
X 111,600
163
-------
Figures 11-15
Figures 16-1?
Figure 18
Thin sections of secondary phagosomes and
surrounding organelles of 0_._ danlca at
progressive stages of digestion of M.
aeruginosa. A partially coated vesicle
(arrow in Figure 11) is shown at higher mag-
nification in Figure 12. Arrows in Figure
13 show small vesicles, suggested to be pri-
mary lysosomes, juxtaposed to the phagosomal
membrane and membranes of larger vesicles,
identified as secondary lysosomes. Figure 15
shows a late stage of the intracellular
digestion of M._ aeruginosa. Arrows indicate
fragments of membranes which are partially
detached from the decomposed cellular material.
Arrowheads indicate detached loops of membrane,
the ends of which have fused.
reticulum; mf, myeline-figure,
X 18,000. Figure 12, X 36,600,
X 50,000. Figure 14, X 24,600,
X 67,500.
er, endoplasmic
Figure 11,
Figure 13,
Figure 15,
Thin sections of secondary phagosomes and
surrounding organelles at progressive stages
of digestion of M._ aeruginosa. rb. residual
body. Figure 16, X 30,000. Figure 17,
X 21,600.
Thin section through cytoplasm of 0_._ danica
adjacent to secondary phagosome showing
partially coated vesicles, and coated region
of cell membrane (com). Arrows indicate
primary lysosomes. X 40,800.
164
-------
Figure 19 Freeze-etch, cross-fracture of 0_._ danlca
showing evaginations of the cell membrane
(arrows). Ch, chloroplast. X 23,500.
Figure 20 Freeze-etch, surface fracture of the cell
membrane and secondary phagosome of 0^_ danica.
Arrows Indicate circular scars on the
fractured, phagosomal membrane. X 11,390.
Figure 21 Freeze-etch cross-fracture of the dictyosome
of 0^ danica. X 23,200.
Figures 22-24 Thin sections of endocytotic 0_._ danica cells
subjected to the modified Gomori reaction
which is used to localized acid phosphatase
activity. The latter is indicated by the
electron-dense deposits in Golgi cisternae
(Figure 22), in the secondary phagosome
associated with partially digested M._
aeruginosa cell (Figure 23), and in secondary
lysosomes (Figure 24). Figure 22, X 26,400.
Figure 23, X 22,400. Figure 24, X 25,200.
Figure 25 Thin section of £._ danica showing partially
digested bacterial cells enclosed by secondary
phagosome and intact bacteria outside the
phagocyte. X 17,390.
Figure 26 Diagrammatic interpretation of cellular
processes associated with endocytosis of
M. aeruginosa by 0^ danica.
165
-------
Table I Graphical demonstration of the rate of
endocytosis which is interpreted as the
rate of decrease of free M^_ aeruginosa
cells in the presence of a known concen-
tration of 0. danica.
166
-------
APPENDIX B
DETAILED SCREENING DATA
167
-------
COMPOUND ACTIVITY OP SELECTED ALGAECIDAL COMPOUNDS AGAINST
Anabaena WHICH WAS CONTAMINATED WITH AN UNKNOWN
SPECIES OF FILAMENTOUS GREEN ALGAE AT VARIOUS CONCENTRATIONS
CO
Serial Compound Concentration
Number Name ppm
23
73
2,5-Dichloro-3,4-
dinitrothiophene
p-Chlorophenyl-2-
thienyliodonium-
chloride
3.2
1.6
0.8
3-2
1.6
0.8
Relative
Intensity
.20
.20
.20
.20
.20
.20
Relative
Intensity
.058
.058
.054
.030
.037
.052
Percent
Control
79
79
81
89
87
80
Control
.20
.28
-------
COMPOUND ACTIVITY - SUPPLEMENTARY SCREENING DATA
Anabaena flos-aquae
Serial
Number
74
Name of Compound
vo
78
82
96
(p-Bromophenyl)-2-thienyl
iodonium chloride
1.6 ppm
0.8 ppm
0.4 ppm
2-Thienyl-p-tolyliodonium
chloride
1.6 ppm
0.8 ppm
0.4 ppm
(o-Chlorophenyl)-2-thieny1
iodonium chloride
1.6 ppm
0.8 ppm
0.4 ppm
2,5-Dibromo-3,4-dinitro-
thiophene
1.6 ppm
0.8 ppm
0.4 ppm
Initial
Relative
Intensity
.23
.27
.18
.28
.19
.18
.36
.32
.22
.27
.26
.23
Final
Relative
Intensity
.04
.05
.37
.06
32
.47
.05
.06
.08
.04
.04
.05
Percent
Control
100
95
59
94
65
48
95
94
92
100
100
95
-------
Serial
Number
98
Name of Compound
Tetrachlorothlophene
1.6 ppm
0.8 ppm
0.4 ppm
Control
No. 1
No. 2
No. 3
Initial
Relative
Intensity
.23
.21
.19
.19
.18
.18
Final
Relative
Intensity
.70
.70
.78
.93
.97
.82
Percent
Control
23
23
14
i-1
-j
o
-------
COMPOUND ACTIVITY SCREENING OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Serial
Number
121
124
163
164
165
N'-(3-(l,l-Dimethylethy)-4-
nitrophenyl)-N,N-dimethyl urea
N- ( 4-Buty 1-2-nitrophenyl ) -
acetamide
N ' - ( 4- ( ( 3-Chloro-2-pyridiny 1 )
oxy)phenyl)-N,N-dimethyl urea
N'-(4-Ethylphenyl)-N,N-
dimethyl urea
N » - ( 4- ( ( 3 , 5-Dichloro-2-
pyr idiny 1 ) oxy ) pheny 1 ) -N ,N-
dlmethyl urea
Control
Initial
Relative
Intensity
(1) .29
(2) .31
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.19
.19
.30
.28
.47
.43
.48
.44
.21
.20
Final
Relative
Intensity
(1) .66
(2) .62
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.73
.69
.90
.90
.70
.71
1.10
.95
.70
.70
Percent
Control
9
0
0
0
0
-
-------
ro
COMPOUND ACTIVITY OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Serial
Number
126
127
136
4-day
Name of Compound
7 , 8-Dihydr o- 6H-pyrr olo ( 1 , 2-e )
purin-4-01
2-Tert-butyl-4-nitrophenol
2,2'-(l,2-Ethenediyl)bisben-
zoxazole
Control
peri
od^
Initial
Relative
Intensity
(1) .29
(2) .31
(1)
(2)
(1)
(2)
(1)
(2)
.28
.28
.29
.32
.30
.29
Final
Relative
Intensity
(1) 1.05
(2) 1.0
(1)
(2)
(1)
(2)
(1)
(2)
.75
.91
.08
.10
.98
.89
Percent
Control
0
12
91
-------
(JO
COMPOUND NUMBER 136 - 2,2'-(l,2-Ethenediyl)bisbenzoxazole
AGAINST Microcystis aeruginosa AND Anabaena flos-aquae AT VARIOUS CONDITIONS
Anabaena flos-aquae
Concentration
ppm
1.6
1.6'
0.8
0.8'
0.4
0.4'
0.2
0.2'
Control
Control'
Initial
Relative
Intensity
.35
.36
.27
.27
.26
.26
.25
.24
.26
.28
Final
Relative
Intensity
.04
.05
.069
.080
.27
.26
.92
.83
1.4
1.3
Percent
Control
100
94
80
35
«M*
Microcystis aeruginosa
Initial
Relative
Intensity
.28
.25
.22
.22
.21
.21
.21
.21
.21
.21
Final
Relative
Intensity
.066
.066
.070
.072
.066
.070
.11
.20
1.1
1.2
Percent
Control
100
94
94
87
-------
LABORAORY SCREENING TESTS
ALGAECIDAL ACTIVITY OF COMPOUND NO. 136
(2,2'-(l,2-Ethenedlyl)bis-benzoxazole)
AT VARIOUS CONCENTRATIONS OVER AN EXTENDED TEST PERIOD
Anabaena flos-aquae
Concentration
0 . 8 ppm
0.4 ppm
0 . 2 ppm
0 . 1 ppm
Control No. 1
Control No. 2
0,8 ppm
0 . 4 ppm
0 . 2 ppm
0 . 1 ppm
Control No. 1
Control No. 2
0-days
R.I.
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0+4 days
R.I.
0.11
0.16
0.19
0.27
0.39
0.40
Microcystis
0.51
0.13
0.37
0.48
0.54
0.54
%
Control
70
57
52
33
aeruginosa
90
76
31
10
0+21 days
R.I.
1.1
1.4
2.7
3.1
5.2
2,
1,
20
95
2.55
,05
.90
Control
76
68
28
21
30
23
2.70
174
-------
COMPOUND ACTIVITY SCREENING OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Serial
Number
146
147
148
149
150
Name of Compound
5-Butyl-2-methyl-lH-benzi-
midazole
Pentachlorophenol, compound
with 2-(2,4,5-Trichlorophen-
oxy)ethanamine (1:1)
N ! - ( 4- ( ( 6-Chloro-4-tri-
fluoromethyl)-2-pyridinyl)
oxy )-N,N-dimethyl urea
5-Nitro-2-thiophene-
carboxaldehyde, oxime
N-(4-( (6-Chloro-2-pyridinyl)
oxy)phenyl) acetamide
Control
Initial
Relative
Intensity
(1) .40
(2) .40
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.24
.24
.21
.21
.28
.28
.33
.34
.24
.25
Final
Relative
Intensity
(1) .40
(2) .40
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.79
.80
1.50
1.10
.76
.79
1.00
1.00
.73
.75
Percent
Control
0
0
0
0
0
-
-------
COMPOUND ACTIVITY SCREENING OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
a
ON
Serial
Number
151
152
153
154
155
Name of Compound
N-(H-((6-Chloro-2-pyridinyl)
oxy)phenyl)-N' -methyl urea
N '- ( 4- ( ( 2 , 6-Dichloro-4-
pyrldinyl)oxy)phenyl)-N,N-
dimethyl urea
2 , 5-Bis ( ( 4-methylpheny 1 )
sulfonyl)-3 ,4-dinitro-
thiophene
N-(3-Chlorophenyl)-2-
isoxazolldlnecarboxamide
N-(4-Chlorophenyl)-2-
isoxazolidinecarboxamide
Control
Initial
Relative
Intensity
(1) .23
(2) .23
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.66
.64
.28
.26
.34
.30
.2?
.26
.24
.25
Final
Relative
Intensity
(1) 1.20
(2) 1.00
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
1.60
1.60
.06
.75
.90
.90
70
.51
.73
.75
Percent
Control
0
0
8
0
18
-------
COMPOUND ACTIVITY SCREENING OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Initial
Serial
Number
156
157
158
159
160
Name of Compound
3-(Acetyloxy)-4-bromo-
butanoic acid, methyl ester
N'-(4-Acetylphenyl)-N,N-
dimethyl urea
N ,N-Dimethy 1-N ' - ( 4- ( ( 6-
(methy Ithio ) -2-pyridiny 1 ) -
oxyjphenyl) urea
N ' - ( 4- ( ( 6-Chloro-2-pyridinyl)
oxy)phenyl)-N,N-diethyl urea
2- ( 2 , 4-Dichlorophenoxy )-
3-nitropyridine
Control
Relative
Intensity
(1) .24
(2) .25
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.31
.30
.61
.61
.64
.65
.2?
.30
.24
.25
Final
Relative
Intensity
(1) .60
(2) .80
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.83
.80
1.20
1.00
.90
1.30
.72
.75
.73
.75
Percent
Control
6
0
0
0
2
-
-------
-a
CO
COMPOUND ACTIVITY SCREENING OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Initial
Serial
Number
161
162
Name of Compound
Tris(dimethylamino) (hydroxy-
phenyl=methyl) phosphonium,
hydroxide , inner salt
N ' - ( 4- ( ( 6-Chloro-2-pyraziny 1 )
oxy)phenyl)-N,N-dimethyl urea
Control
Relative
Intensity
(1) -28
(2) .26
(1)
(2)
(1)
(2)
.45
.43
.24
.25
Final
Relative
Intensity
(1) .68
(2) .67
(1)
(2)
(1)
(2)
.85
.88
.73
.75
Percent
Control
10
0
-
-------
COMPOUND ACTIVITY SCREENING OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Serial
Number
166
167
168
169
Initial
Relative
Name of Compound Intensity
4- Amino-6- ( 1 , 1-dimethy l=ethy 1 ) -
3- (methylthio)-l , 2 , 4-triazin-
5(4H)-one
lH-Imidazol-2-ylphenyl-
diazene
2-Chloro-6- ( 4-methoxy-?
phenoxy) pyridine
N ' - ( 4- ( 2 , 6-dinitro-4-
(trifluoromethyl)phenoxy)=
phenylJ-NjN-dimethyl urea
Control
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.55
.50
.42
.39
.45
.52
.52
.50
.21
.50
Final
Relative
Intensity
(1) .90
(2) .90
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
1.10
1.14
.94
1.07
1.05
1.05
.70
.70
Percent
Control
13
0
0
0
-
-------
oo
o
COMPOUND ACTIVITY SCREENING OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Serial
Number
170
171
172
173
Name of Compound
2-(4-Chlorophenyl)-2,3, 5,6-
t etrahydrolmidazo ( 2 , 1-b ) -
thlazole, monohydrochlorlde
2,3,5, 6-Tetrahydro-2- ( 2-
napht haleny 1 ) -imldazo ( 2 , 1-b ) ,
monohydrochlorlde
2-Hydroxy-N-phenyl-3-
pyrldlnecarboxamide
N'-( 3-Chlorophenyl-N-methoxy-
N-methyl urea
Control
Initial
Relative
Intensity
(1) .20
(2) .20
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.20
.19
.20
.23
.36
.28
.21
.20
Final
Relative
Intensity
(1) .63
(2) .56
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.62
.61
.64
.75
.93
.82
70
.70
Percent
Control
14
11
4
0
-
-------
COMPOUND ACTIVITY OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
CD
Serial
Number
177
178
179
180
Four-Day Test
Compound Name
1- ( 2 , 4 , 5-Tr ichlorophenoxy ) =thiocyanic
acid, ethyl ester
2- ( ( 2- ( Dimethy lamino )ethyl )amine) 3 , 4-
dihydro-l(2H) isoquinolinone, dihydro-
chloride
2-Phenyl-5H-( 1,2,4 )triazolo( 1,5-b )-
isoindole
2-(3-Methylphenyl)-5H-(l,2,4)tri-
azolo( 1,5-b) isoindole
CuSOu«5H20
Control
Initial
Relative
Intensity
(1) .20
(2) .20
(1)
(2)
(1)
(2)
CD
(2)
(1)
(2)
(1)
(2)
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
(1) .27
(2) .32
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
1.05
.96
1.0
1.30
.90
.79
.10
.15
.95
.75
Percent
Control
65
0
0
0
85
-------
COMPOUND ACTIVITY OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
oo
Serial
Number
183
184
185
186
Four-Day Test
Initial
Relative
Compound Name
3-(4-(l,l-Dimethyl=ethyl)phenyl)-
2,3,5, 6-t etrahydroimidazo ( 2 , 1-b )
thiazole
Phenyl-2-thienyl methanone, o-
( (methyl=amino)carbonyl)oxime
Bis< (l,l'-biphenyl)-4-yl)-
ethanedione
( (3,5-Dichloro-6-fluoro-2-
pyridinyl)oxy)methyl ester
thiocyanic acid
CuSO,,»2H20
Control
Intensity
(1) .20
(2) .20
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
(1) 1.10
(2) 1.10
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.55
.68
.95
.81
30
.31
.10
.15
.95
.75
Percent
Control
0
28
0
65
85
-------
COMPOUND ACTIVITY OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Pour-Day Test
CO
U)
Serial
Number
176
182
193
194
Compound Name
1,2-Dichloro-4-(isothiocyanato-
methoxy)benzene
((4,5-Dimethoxy-l-2-phenylene)-
bis=(imino(thloxomethylene)))bis-
carbamic acid, dimethyl ester
NT-(4-((6-Chloro-2-pyridinyl)oxy)-3-
(trifluoromethyl)phenyl)-N,N-
dimethyl urea
N,N-Dimethyl-N'-(4-((6-(trifluoro-
methyl)-2-pyridinyl)thio)phenyl)urea
Control
Initial
Relative
Intensity
(1) .20
(2) .20
(1)
(2)
.20
.20
Final
Relative
Intensity
(1) .10
(2) .09
(1)
(2)
.70
.88
Percent
Control
97
0
(1)
(2)
(1)
(2)
(1)
(2)
.20
.20
.20
.20
.20
.20
(1)
(2)
(1)
(2)
(1)
(2)
.45
.37
.40
.68
.75
41
61
-------
COMPOUND ACTIVITY OP SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos-aquae AT 0.8 PPM
Four-Day Test
Serial
Number
187
188
189
190
Compound Name
2- ( 4-Ethoxypheny 1 ) 2,3, 5, 6-t etra-
hydroimidazo( 2 ,l-b)thiazole
N- ( 1- ( 4-Bromo-2 , 5-dichlorophenoxy ) -
2,2, 2-tr ichloroethy 1 ) f ormamide
N _ ( 4-Et hy 1-3-nitropheny 1 ) N , N-
dimethyl urea
4- ( ( 3 ,5 , 6-Trichloro-2-pyridiny 1)-
oxy ) phenol
CuS04«5H20
Control
Initial
Relative
Intensity
(1) .20
(2) .20
(1)
(2)
(1)
(2)
(1)
(2)
CD
(2)
(1)
(2)
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
(1) .88
(2) .84
(1)
(2)
(1)
(2)
(1)
(2)
(D
(2)
(D
(2)
1.40
1.30
.as
.95
1.00
1.00
.08
.08
.es
.92
Percent
Control
5
0
3
0
91
-------
CD
COMPOUND ACTIVITY SCREENING TESTS OF ALGAECIDAL COMPOUND AGAINST
Anabaena flos-aquae AND Mlcrocystls aeruglnosa AT 0.8 PPM
Microcystis
Serial
Number
192
197
Compound
Name
Initial
Relative
Intensity
N'-(4-((2-Chloro-6- .20
methyl-4-pyrimidinyl)=
oxy) pheny 1)-N,N- .20
dimethyl urea
l-(3,3-Dichloro-l-
methylenebutyl )-
3,5-dimethyl benzene
Control
.20
.20
.20
Final
Relative Percent
Intensity Control
.53
41
.53
.66
.62
.74
Initial
Relative
Intensity
.20
.20
.20
.20
.20
Anabaena
Final
Relative
Intensity
.92
.91
.52
.52
.65
Percent
Control
0
20
__
-------
COMPOUND ACTIVITY OF SELECTED ALGAECIDAL COMPOUNDS
AGAINST Anabaena flos aquae AT 0.8 PPM
Four-Day Test
Serial
Number Compound Name
191 N ' - ( 3-Chloro-4- ( ( 6-chloro-2-pyri-
diny 1 ) oxy ) pheny 1 ) -N, N-dimethy 1 urea
195 N-(l-Methylethyl)-4-phenoxybenzen-
amine
oo
** 196 N'-(4-((6-Bromo-2-pyridinyl)thio)
pheny l)-N,N-dimethyl urea
CuSO««5H20
Control No. 1
Control No. 2
Initial
Relative
Intensity
-(1) .20
(2) .20
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
(1)
(2)
.20
.20
,20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
(1) .74
(2) .74
(1)
(2)
(1)
(2)
CD
(2)
(1)
(2)
(1)
(2)
1.50
1.40
.77
.96
.08
.08
.88
.92
.68
.75
Percent
Control
18
0
0
91
-------
LABORATORY SCREENING TESTS
ALGAECIDAL ACTIVITIES OF SELECTED TEST COMPOUNDS AGAINST Anabaena flos-aquae
FOR EXTENDED PERIODS
Test Concentration 0.8 ppm
Compound Number and Name
198 p-(p-Nitrophenylthio) phenol
199 l-((U-Nitrophenyl)methyl) piperidine
200 ( 5-Chloro-2 ,*-dimethoxyphenyl )-carbamic
acid, 2,^,5-trichlorophenyl ester
201 2 , U-Dihr omo-3-methyl-6-nitrophenol
202 2 ,*-Dichloro-3-methyl-6-nitrophenol
203 N-Bromophenyl-2-chloroacetamide
20* U-( (6-Fluoro-2-pyridinyl)=thio)phenol
205 l-Chloro-2-(methylsulfonyl) ethane
206 2 , U , 5-Tr ichloro-3-methyl-6-nitrophenol
Control No. 1
Control No. 2
0-day
R.I.*
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0,20
0.20
0+fy days
R.I.«
0.53
0.^9
0.56
0.52
0.53
0.53
0.58
0.52
0.5U
0.56
O.U5
Control
0
0
0
0
0
0
0
0
0
-
_
0+7 days
R.I.*
0.88
0.87
1.12
1.05
1.02
1.00
0.98
0.89
0.91*
0.85
0.80
0+10 days
0.87
l.lk
1.17
1.15
1.16
1.1*
1.15
l.lU
1.1*
1.13
1.12
Control
22
0
0
0
0
0
0
0
0
-
_
*averaged fluoromicrophotometric relative intensity readings
-------
APPENDIX C
WATER CHEMISTRY DATA
FIELD TESTS
188
-------
00
Chowan River - First Test
N03-N
nderogram-atoms per liter
Sample Q-&ay 0+2 days 0+3 days 0+U days 0+5 days 0+11 days
Ambient River Water
Control Vessel No.
Control Vessel No.
Compound No.
Compound No.
Compound No.
Compound No.
23 -
23 -
73 -
73 -
1
2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
0.80
0.36
0.81
0.60
0.28
0.16
0.
0.
0.
0.
0.
0.
0.
30
31
35
26
66
18
12
0.25
0.18
0.22
0.12
O.U5
0.26
0.
0.
0.
0.
0.
2.
0.
21
25
1*6
1U
85
OU
27
0
0
0
0
0
0
0
.09
.16
.29
.kk
.26
.11
.19
0.23
0.29
O.U2
0.28
O.U7
O.U5
O.U7
-------
Chowan River - First Test
NHt»-N
microgram-atoms per liter
SamPle 0-day 0+2 days 0+3 days 0+U days 0+5 days 0+11 days
Ambient River Water
Control Vessel No
Control Vessel No
Compound No. 23 -
Compound No. 23 -
Compound No. 73 -
Compound No. 73 -
. 1
. 2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
2.88
2.1*3
8.52
9.51
2.U
3.36
2.85
2.98
U.36
5-73
11.1*0
3.70
2.67
2.50
3.26
2.95
5.80
7-31
5.97
3.36
5.52
10.78
6.08
k.29
6.01
U.02
3.19
2.19
2.98
3.57
U.56
3.7k
2.78
3.67
3.12
U.46
9.58
5.15
10.1
5.15
5.60
-------
Cfcowan River - First Test
S02-N
microgram-atoms per liter
Sample 0^-day 0+2 days 0*3 days 0+4 days 0+5 days 0+11 days
VQ
Ambient Eiver Water
Control Vessel No.
Control Vessel No.
Compound No. 23 -
Compound No. 23 -
Compound No. 73 -
Compound No. 73 -
1
2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
0.08
0.08
0.32
O.N
0.08
0.07
0.07
0.08
0.09
0.18
0.78
0.1^
0.13
0.08
0.07
0.09
0.27
0.32
0.08
0.08
0.09
0.08
0.09
0.09
0.10
0.08
0.08
0.07
0.07
0.08
0.07
0.08
0.07
0.09
0.08
0.08
0.08
0.09
0.08
0.09
0.08
-------
Chovan River - First Test
REACTIVE P0i»
microgram-atoms per liter
ro
Sample
Ambient River Water
Control Vessel No. 1
Control Vessel No.
Compound No.
Compound No.
Compound No.
Compound No.
23 -
23 -
73 -
73 -
. 2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
0-day
0.68
0.22
0.58
0.6U
0.8U
0.16
0+2 days
0.2U
0.58
0.16
1.36
0.20
0.26
0+3
0.
0.
0.
0,
0.
0.
0.
days
26
16
26
Ik
10
30
26
o+u
0.
0.
0.
0.
0.
0.
0.
days
U6
1*8
35
66
38
3k
0+5
0.
0.
0.
0.
0.
0.
0.
days
26
26
1*8
Ik
36
32
36
0+11 days
o.Uo
0.72
O.U8
1.06
0.56
0.7U
-------
Chowan River - First Test
TOTAL FILTERABLE P0i»
microgram-atoms per liter
u>
Sample
Ambient River Water
Control Vessel No.
Control Vessel No.
Compound No. 23 -
Compound No. 23 -
Compound No. 73 -
Compound No. 73 -
1
2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
0-day
1.80
0.70
1.14
1.14
1.50
1.20
0+2
0.
0.
1.
0.
2.
0.
0.
days
80
84
46
74
84
94
86
0+3
0.
1.
0.
0.
0.
1.
0.
days
66
20
90
44
64
16
66
0+4
0.
1.
1.
1.
1.
1.
0.
days
94
24
38
38
50
20
94
0+5
0.
0.
1.
0.
1.
0.
1.
days
96
78
10
90
06
84
06
0+11 days
1.24
1.22
1.50
0.96
2.10
1.10
1.74
-------
Chowan River - First Test
TOTAL UNFILTERABLE POt,
microgram-atoms per liter
vo
-Cr
Sample
Ambient River Water
Control Vessel No
Control Vessel No
Compound No. 23 -
Compound No. 23 -
Compound No. 73 -
Compound No. 73 -
. 1
. 2
0.8 ppm
1.6 ppm
0.8 ppm
1.6 ppm
0-day
2.68
1.96
2.16
2.2U
,3.50
2.2U
0+2
2
3
3
2
U
2
2
days
.7U
.1U
.30
.76
.1*6
.20
.lU
0+3
2
2
2
2
2
2
2
days
.30
.2U
.16
.30
.36
.52
.02
0+U
2
2
3
2
3
3
2
days
.60
.58
.06
.80
.Uo
.00
.90
0+5
2
2
2
2
2
2
3
days
.5U
.72
.62
.36
.76
.56
.12
0+11 days
3.10
3.5U
U.OO
3.UO
5.10
3.51*
3.56
-------
Chowan River - First Test
TOTAL CHLOROPHYLL
microgram-atoms per liter
Sample
Ambient River Water
M
vo
VJl
Control Vessel No
Control Vessel No
Compound No. 23 -
Compound No. 23 -
Compound No. 73 -
Compound No. 73 -
. 1
. 2
0.8 ppm
1. 6 ppm
0.8 ppm
1.6 ppm
12.1*3
11.02
7.15
6.80
1.1*0
36.58
11.08
Q+2 days
33.81*
10.01
21.08
8.33
U.80
7.H
3.38
0+3 days
22.3U
25.57
22.31*
U.OO
3.UO
7.01
U.UU
0+U days
27.00
22. U9
26.16
32.66
22. U2
31.50
lU.50
0+5 days
2U.61
21.70
19. 2U
28.62
26.98
16.03
17.96
0+11 days
17.17
22.97
U0.08
28.78
31.03
30.83
28.63
-------
Chowan River - First Test
Agmenellum AND Oscillatoria
(cells/liter x 10")
Sample
0-days 0+1 day 0+U days 0+11 days
Ambient River Water
Controls (Avg. )
Compound No. 23 - 0.8 ppm
Compound No. 23-1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
u
3
11
10
88
10
^
36
10
6
IP.
n
12
0
53.
i
2Q.
0
U8
58-
106
11
58
0
31
2
13
4
T
120
T
*§
11
7
KEY:
Agmenellum
Oscillatoria
196
-------
Lake Sallie - First Test
TEMPERATURE AND pH VARIATIONS
Sample 0-days 0+2 days 0+1* days 0+6 days
AX.- 4. T 1 TT 4. 25.5 2U.5 23.0 2k. 0
Ambient Lake Water frV ~~5o A ft A:Q
O t T" O "7 O*O O t ^T
Control Vessel No. 1
25.5 2k,5 23.0 2U.O
Control Vessel No. 1 go '8 9 ""Of ' Q Q
A Q 25.5 2lt.O 22.5 2U.O
Compound No. 23 - 0.8 ppm "90 ""8.9 3.8 9.9
__. temp...-(°C)
KEY>
2U 0 22 5 2U.O
Compound No. 23 - 1.6 ppm -*** -
25 5 2U 5 23 0 23.
Compound No. 73 - 0.8 ppm -~ -gj|- - -
.. 25.5 2U.O- 23.0
Compound No. 73-1.6 ppm -*-* -- --
197
-------
Lake Sallie - First Test
AVAILABLE AND TOTAL POi, (ug/l)
Avail-
' Total POif
198
Sample 0-days 0*2 days 0+1* days 0+6 days
MMent U*e Water g$i g£ ftgL £^5.
Control Vessel No. 1
00^0! vesse, »o. 2 £gl g£L gg* ££L
Compound Ko. 33- 0.8 pp* S^i ^ 2^ ^
Ground NO. 23 - 1.6 pp. §^ ggi 2^§i 2^1
Compound Ho. 73 - 0.8 ppm £°15. 2025. £^|0 Oj||£
COMPOS »o. 73 - 1.6 PP. $&. §^ §^ ^
-------
Sample
Lake Sallie - First Test
N02-N and NQ3-N (yg/l)
0-days 0+2 days 0+U days 0+6 days
Ambient Lake Water
Control Vessel No. 1
Control Vessel No. 2
Compound No. 23 - 0.8 ppm
Compound No. 23-1.6 ppm
Compound No. 73 - 0.8 ppm
Compound No. 73 - 1.6 ppm
0
0.015
0
O.OlU
0
0.017
0
0.015
0
0.014
0
O.OlU
0
0.017
0
o.oiu
0
0.050
0.001
0.016
0
0 . Oil*
0
0.010
0
oToiU"
0
0.037
0
O.lU
0
0.015
0.002
o.oiu
0
0.014
0
O.OlU
0.001
0.012
0
0.015
0
0.014
0
O.OlU
0
O.OlU
KEY:
N02-N
N03-N
199
-------
Lake Sallie - First Test
CQ3 AND HC03 (yg/l)
Sample 0-days 0+2 days 0+U days 0+6 days
U8 52 UO
Compound No. 73-0.8 ppm ^ -*jj- jjsjj
Ambient Lake Water 20
155" 132 OT 132
Control Vessel No. 1 -- ~
U8 UU
Control Vessel No. 2 ^ ^ ^6 13U
52 U8
Compound No. 23-0.8 ppm £|£ j=jj-
Compound No. 23 - 1.6 ppm ||| -52* -^2. ^22,
150 132
Compound No. 73 - 1.6 ppm ^g ^|g- j££ 132
200
-------
Lake Sallie - First Test
NH3-Nitrogen
Sample 0-days 0*2 days 0+1* days 0+6 days
Anibient Lake Water 0,080 0.065 0.035 0.105
Control Vessel No. 1 <- -
Control Vessel No. 2 0.050 0.030 0.035 0.060
Compound No. 23 - 0.8 ppa 0.115 0.055 0.035 0.090
Compound No. 23 - 1.6 ppm O.lfcO 0,050 0.035 1.80
Compound No, 73 - 0.8 ppm 0.015 0.03Q 0.035 0.080
Compound No. 73 - 1.6 ppm 0.065 0.080 0.065 0.090
201
-------
Muskrat Lake - Second Minnesota Test
NH3- NITROGEN
Sample 0-days 0+9 days
Ambient Lake Water 0.065 0.100
Control 0.065 0.065
Compound No. 73 - 3.2 ppm 0.080 0.155
Compound No. 73 - 1.6 ppm 0.050 0.065
Compound No. 73 - 0.8 ppm 0.050 0.115
Compound No. 73 - O.lt ppm 0.050 0*065
302
-------
Muskrat Lake - Second Minnesota Test
TEMPERATURE AND pH VARIATIONS
Sample 0-days 0+9 days
0)1 n on ft
Ambient Lake Water
n 4- n 2U.O 20.0
Contro1 -O- To
Compound No. 73 - 3.2 ppm -Q ' O"'Q
, _ . , 2k. 0 20.0
Compound No. 73-1.6 ppm fa Q g g
Compound No. 73 - 0.8 ppm '
-,-,«) 2^.0 20.0
Compound No. 73 - O.U ppm
temp.. (°C)
pH
203
-------
Muskrat Lake - Second Minnesota Test
NOa-N AND N03*N (yg/1)
__^ _ Sample 0-day 0+9 days
Ambient Lake Water
KEY:
0 . 001 0*001
Compound No. 73 - 3.2 ppm
Compound No. 73 - 1.6 ppm n ^
Compound No. 73 - 0.8 ppm §719"
Compound No. 73 - 0.4 ppm 9 ^
204
-------
Muskrat Lake - Second Minnesota Test
CO, AND HCOS ALKALINITIES (yg/1)
Sample 0"-day 0+9 days
24 12
Ambient Lake Water j|^ pr|-
Control $ jff
Compound No. 73 - 3.2 ppm j|^ JJTJ-
72
Compound No. 73 - 1.6 ppm
Compound 'No, 73 - 0.8 ppm
Compound No. 73 - 0.4 ppm
KEY:
205
-------
Muskrat Lake - Second Minnesota Test
SOLUBLE AND TOTAL PO* (vg/D
_ Sample _ _ 0-day 0+9 days
Ambient Lake Water
Control |4
d , o
Compound No. 73 - 3.2 ppm fU-i-
o ^
Q Q **
Compound No. 73 - 1.6 ppm ~-|- u*
Compound No. 73 - 0.8 ppm §-
<- y -I-«i
Compound No. 73 - 0.4 ppm
. Soluble FOu
> Total P0tt
206
-------
Diamond Lake - First Test
GREEN A
Sample
Ambient Lake
Water
Control Vessel No. 1
Compound No.
Compound No.
Compound No.
Compound No.
Compound No.
73 - O.U ppm
73 ~ 0.8 ppm
73 * 0.8 ppm*
73 - 1*6 pp»
73 - 3.2 ppm
LGAE - Staurastrum & <
(cells/ml)
0-days 0+1 day
11.7 lit. 9
2575 377t
.,
P4.
00.5
17.1
30.2
uM
13.0
30.2
11^-
30.2
10.6
3673"
16.7
29.5
16J.
30.2
13.7
vfT?
1U.U
20.2
&4
27.4
Sloeocystis
0+2 days
12.1
13.0
rft?
lit. 6
EsT?
l»t. 6
T^n?
ILi
6075
16.2
23.0
16.6
EoTs
0+3 days
8.6
TIT
18.9
Ttt
11^1
33.5
16.0
79.2
17.1
U6.1
18. U
15T^
Solubilized In methyl alcohol
Staurastrum
* Oloeocystis
207
-------
Diamond Lake - First Test
Fragilaria AND Eudorina
(cells /ml)
Sample
Ambient Lake
Water
Control Vessel No. 1
Compound No.
Compound No.
Compound No.
Compound No.
Compound No.
73 - O.U ppm
73 - 0.8 ppm
73 - 0.8 ppm*
73-1.6 ppm
73 - 3.2 ppm
0-days
2.9
0
2.2
0
0
0
0
2VL
2.9
3.6
0
0+1 day
2^9
U.O
2.9
3.1
5 o
ij. T
2.9
27?
U/L
0
5.6
0+2 days
1.1
2i9
276
2i9
Sal
0
0
2.9
2T?
U.O
2.9
0+3 de
7.9
11.5
0
10.1
15.8
279"
6.1
0
6.8
2.2
*SoluMlized in methyl alcohol
. Fragilaria
Eudorina
208
-------
Diamond Lake - First Test
DISSOLVED OXYGEN AND TOTAL ALKALINITY LEVELS
ppm/(mg/l)
Sample
0-days 0+1 day
0+2 days 0+3 days
Ambient Lake
Water
Control Vessel No
Compound
Compound
Compound
Compound
Compound
No.
No.
No.
No.
No.
73 -
73
73 -
73 -
73 -
. 1
O.U
- 0.
0.8
1.6
3.2
ppm
8 ppm
ppm*
ppm
ppm
8
17
8
17
8
17
8
17
8
17
8
17
8
17
.2
.0
.1
.0
.1
.0
.2
.0
.2
.0
.2
.0
.2
.0
15.0
8.2
i6Tb~
8.2
17.0
8.2
16.5
o 3
itTo
8.2
8.3
j* J
1670
17.
8.
17.
8.
17.
8.
17.
8.
17,
8.
17.
8.
-------
APPENDIX D
COMPOUND pH-DEPENDENCE
AND PERSISTENCE TESTS
210
-------
pH SENSITIVITY TESTS ON COMPOUNDS NO. 23
(2,3-DlCHLORO-3,1t-DlNITROTHIOPHENE) AND NO. 73 (P-
CHLOROPHENYL-2-THlENYL IODONIUM CHLORIDE) AGAINST Anabaena
Test
Compound Concentration - 0.8 ppm
Initial
Serial Number Relative
and pH Value
23 - pH 6
pH 6 control
23 - pH 7
pH 7 control
23 - pH 8
pH 8 control
23 - pH 9
pH 9 control
73 - PH 6
pH 6 control
73 - PH 7
pH 7 control
73 - PH 8
pH 8 control
7.3 - PH 0
ntt Q control
Intensity
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
.20
Final
Relative
Intensity
.013
1.15
.018
1.18
.020
1.10
.025
1.09
.032
1.40
.021
1.10
.023
1.18
.026
1.10
Percent
Control
100
100
100
100
V100
100
100
100
211
-------
Depletion of Compound No. 73 Under
Natural Open Environmental Conditions
2.0 r
IV)
M
ro
Location:
Time :
Lake Sallie, Minnesota
July 10-16, 1973 - first treatment
0
Theoretical initial concentration
Monitored concentration in test vessel
2 3
Elapsed Time (days)
-------
2.0 r-
1.5
Depletion of Compound No. 23 Under
Natural Open Environmental Conditions
Location: Lake Sallie, Minnesota
Time : July 10-16, 1973 - first treatment
Theoretical initial concentration
Q,
rv>
M
uo
as
SH
-P
c
0)
o
c
o
o
1.0
Monitored concentration in test vessels
0.5
Elapsed Time (days)
-------
ru
2.0 r
D.
a
c
o
TO
0)
O
C
o
o
Depletion of Compound No. 23 Under
Natural Open Environmental Conditions
Location: Chowan River, North Carolina
Time : June 15-26, 1973 - first treatment
1.5
Theoretical Initial Concentration
Monitored concentration in test vessels
0.5
I
2 3
Time (days)
-------
Depletion of Compound No. 73 Under
Natural Open Environmental Conditions
2.0
Location: Chowan River, North Carolina
Time : June 15-26, 1973 - first treatment
1.5
6
O,
o.
c
o
1.0
Theoretical initial concentration
Monitored concentration in test vessel
0)
o
c
o
o
0.5
I
2 3
Time (days)
-------
APPENDIX E
Ochromonas STORAGE AND ACTIVITY
ENHANCEMENT STUDY
216
-------
Ochromonas STORAGE AND PERSISTENCE STUDY
Parameters:
I. Two species (0. ovalis and 0. bastrop)
,11. Seven types of media or imbibing substances
Gorham's medium
Liquid and solid nutrient agar
Topsoil
River silt
Charcoal
Polyester fibers
Cotton fibers
III. Pour states
Liquid
Dried or partially dried (wet)
Sterile
Non-sterile
IV. Three temperatures (all in total darkness)
Room temperature (23°C)
Refrigerator temperature (10°C)
Freezer temperature (-4°C)*
V. Storage period - 15 days; subsequent incubation period
3 days
"Samples subjected to below freezing temperatures are not
tabulated because in no case did any of the Ochromonas survive
217
-------
Ochromonas STORAGE AND PERSISTENCE STUDY
At 0+70 Days
Conditions Which Produced Positive Results
0. ovalisiQ. bastropi
Topsoil - refrigerated, sterilized,'* moist
Topsoil - room temperature, sterilized, moist
Activated Charcoal - room temperature, dry
Activated Charcoal - refrigerated, moist
Activated Charcoal,- room temperature, moist
Solid Nutrient Agar - room temperature
Liquid Nutrient Agar - refrigerated
Liquid Nutrient Agar - room temperature
Polyester Fibers - room temperature, dry
Polyester Fibers - room temperature,,,moist
Cotton Fibers - room temperature, dry
Cotton Fibers - refrigerated, moist
Cotton Fibers - room temperature, moist
218
-------
Ochromonas LONGEVITY STUDY
Conditions Which Produced
Positive Results at 0+44 Days
0. ovalis
0. bastrop
Activated charcoal - refrigerated - moist
Activated charcoal - room temperature,
moist
Solid nutrient agar- room temperature
Liquid nutrient agar - refrigerated
Polyester fibers - room temperature,
dried
Cotton fibers - room temperature, moist
Cotton fibers - room temperature, dried
219
-------
INFLUENCE OF TEST COMPOUND NO. 119 - N'-(4-((6-bromo-2-pyridinyl)oxy)phenyl)-N,N-dimethylurea
AT 0.2 PPM ON THE PHAGOCYTIC ACTIVITY OF FOUR SPECIES OF Oahromonas
CELL COUNTS.'
r\j
f\J
o
Species
0. donica+(M)+(T)
0. malhamen8is+
(
0.
Controls
0. bastrop+W)
0. danioa+(W)
0. malhamensis+CM.)
0. ovalis+(W
0 -
-days
(M)xlO° (Och)xlO"
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0 +
1 day
(M)xlO° (Och)xlO*
0.48
0.61
0.74
0.04
0.63
0.42
0.90
0.07
2.3
0.35
0.20
4.1
0.35
0.40
0.50
3.5
0 +
(M)xlO6
0.0
0.22
0.76
0.0
0.03
0.53
1.10
0.0
2 days
(OchjxlO*
7.0
0.80
0.80
6.0
4.5
2.60
0.45
8.6
0 +
(M)xlO°
0.0
0.0
0.09
0.0
0.0
0.34
0.75
0.0
6 days
(Och)xlO*
11.0
10.8
5.3
2.0
12.2
2.0
11.2
8.2
Percent
Control
100
100
98
100
100
92
82
100
(M) only
Culture No. 1
(M) only
Culture No. 2
0.84
0.84 -
1.20
1.16
1.20
1.06
4.35
4.25
Key:
(M) = Mierocystis
(Och) = Ocfocmonas
(T) * Test chemical
-------
ro
ro
INFLUENCE OF TEST COMPOUND NO. 114 - N, N-diethy1-2-2 (2,4,5-trichlorophenoxy) ethanamine
AT 0.2 PPM ON THE PHAGOCYTIC ACTIVITY OF FOUR SPECIES OF Ochromonas
CELL C 0 U'NT S
Species
0. fcastrop+(M)+(T)
0. dan£ea+(M)+(T)
0. nKiThemenais +
nvrt-ffT^
VI"/T\-I- /
0. 0vaZis+(M)+(T)
Controls
0. 2>astrop+(M)
0. donica + (M)
0. malhemensis+W
0. ovalis + (M)
0 -
(M)xlO6
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
days
(Och)xlO"
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0 + 1 day
(M)xlO6
1.08
1.07
0.88
0.91
0.63
0.42
0.90
0.07
Second Order
(M) only -
Control No. 1
(M) only -
Control No. 2
0.84
0.84
u
1.20
1.16
(Och)xlO*
0.25
0.20
0.20
0.25
0.35
0.40
0.50
3.15
Controls
__
^mfff
0 + 2
days
(M)xlO° (Och)xlO"
1.08
0.97
1.13
1.05
0.03
0.53
1.1
0.0
(Mieroaystis
1.20
1.06
<.01
<.01
<.01
.10
4.5
2.6
0.45
8.6
only)
^_^_
0 +
(M)xlO°
1.65
1.74
2.58
0.08
0.0
0.34
0.75
0.0
4.35
4.25
6 days
(Och)xlO*
9.5
15.1
0.15
6.3
12.2
2.0
11.2
8.2
_
_
Percent
Control
61
59
40
98
100
92
82
100
_
Key:
(M) = Miaroeystis
(Cch) = Oohfomonas
(T) = Test compound
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Report No.
,i **>
3, Accession No.
w
4. Title
RESEARCH AND DEVELOPMENT OF A SELECTIVE
ALGAECIDE TO CONTROL NUISANCE ALGAL GROWTH
7. Author(s)
Prows, B. L. and W. F. Mcllhenny
Dg OiganuatJon
The Dow Chemical Company
Texas Division
Freeport, Texas 77541
, OffilWResearHsh and Development
15. Supplementary Notes
Environmental Protection Agency report number
EPA 660/3-74-019, August 19?U
10, Project No.
11. Contract/GranJ/No.
, Contract Jto^, 68-01-0.7$
^ifcXifrttiuEtai :";
. AThftcprlmary objective of this project was to determine under natural, open-field
conditions, the efficacy of two candidate algaecides, Compound No. 23 (2,5-Dichloro-
3,4-d1n1trothiophene) and No. 73 (p-Chlorophenyl-2-thienyl iodonlum chloride) from Phase
II of the multiple phase developmental program. Specific efforts were also directed
toward further delineation of the toxicological and environmental persistence properties
of the candidate compounds, as well as further development of a possible biological-
chemical control system.
Data from the field tests conducted under a wide variety of conditions in four
geographically diverse regions of the United States revealed a distinctive pattern of
selective blue-green algal control for both experimental compounds. Compound No. 23 was
eliminated from the test series due to unacceptable fish toxicity.
A whole-pond field study involving the use of a phagocytic organism, Ochromonas
oval is, as a biological control system, was inconclusive due to the apparent Inability
dif the organism to survive under the existing environmental conditions.
Continued laboratory screening tests of some 70 additional compounds produced two
additional candidate compounds, No. 136 (2,2'-(l,2-Ethenediyl)b1sbenzoxazole) and No.
176 (l^-DlchlonM-Msothlocyanatomethoxy) benzene).
Continuation of testing of candthte compounds under field conditions 1s recommended.
17a. Descriptors
*alg1cides,
nuisance algae,, biocontrol, toxicity, environmental persistence.
17b. Identifiers
Chowan River, NC; Lake SalUe, MN; Diamond Lake, OR
.17c, COWRR Field & Group
05C
18. Availability
19. Security
20 focuntj- Class.
(Page)
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
UA DEPARTMENT OP THE INTERIOR
WASHINGTON. D.C. 2OI4O
Abstractor
Bernard L. Prows^
Institution
The Dow Chemical Company
WRSIC 102 (REV. JUNE 1971)
Q P O 488-935
------- |