660-3-73-006
Ecological Research
DEVELOPMENT OF A SELECTIVE
ALGAECIDE TO CONTROL
NUISANCE ALGAL GROWTH
Office of Research and Deveiopmen:
U.S. Environmental Protection Agan.'v
Washington, O.C. 20450
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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.
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EPA-660/3-73-006
August 1973
DEVELOPMENT OF A SELECTIVE ALGAECIDE
TO CONTROL NUISANCE ALGAL GROWTH
By
Bernard L. Prows
William F. Mcllhenny
Project No. 16010 EDJ
Contract No. 68-01-0076
Program Element 1B1031
Project Officer
Thomas E. Maloney
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent ol Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.50
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EPA Review Notice
This report has been reviewed by the Water
Quality Office, 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.
ii
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ABSTRACT
The objective of this project was to develop a compound
which would effectively and economically control the growth
of nuisance species of blue-green algae with a minimum
impact on desirable forms of life in the aquatic environ-
ment .
A computerized structure search of more than 100,000 com-
pounds was made to select the analogs of the following four
Phase I prime candidates: 2,5-Dichloro-3,4-dinitrothiophene;
[5-Chloro-2-(p-nitrophenoxy)phenyl]phenyliodoniumchloride;
4-Amino-2,5-dibromophenylthiocyanate; 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
screening. At the conclusion of Phase II, compounds No. 23
(2,5-Dichloro-3,i*-dinitrothiophene) and No. 73 [(p-Chloro-
phenyl)-2-thienyliodoniumchlorideJ were selected as final
candidates, based on superior algaecidal activity, en-
vironmental acceptability, economic feasibility and freedom
from human health and handling hazards.
A non-pigmented flagellate, Oohromonas oval-is , which exhibited
phagocytic activity against the blue-green alga, Miaroeystis
aeruginosa, was discovered during Phase I.
Three additional species of Oohromonas were discovered during
Phase II which also exhibited phagocytic activity against
Miaroaystis aeruginosa. The original species discovered
during Phase I, exhibited the greatest activity and showed
some improvement in the presence of certain test compounds.
iii
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TABLE OP CONTENTS
Section
I. Conclusions
II. Recommendations
III. Introduction
IV. Experimental Procedures
V. Discussion
VI. Acknowledgements
VII. References
VIII. Appendices
1
3
i|
13
33
94
95
101
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LIST OF FIGURES
No. Page
1 Formulation Efficacy Tests 20
2 Open Environment Test Design (Shading
Roof and Support) 23
3 Test Vessles with Polyurethane Flotation
Collars 25
4 Phase I - Prime Candiate Algaecides 3^
5 Activity Comparison of Selected Compounds
at 0.8 ppm Concentration - 3 Day Tests 55
6 Compounds No. 23 and No. 73 versus Micro-
oystis and Anabaena 5&
7 Compound Activities versus Anabaena flos
aquae - 3 Day Tests 57
8 Phase III Test Compounds - Structure vs.
Activity 67
9 Degradation of Compound No. 23 in Well
Water and PW-M Medium 72
10 Phagocytic Activity of Ochromonas versus
Microcystis 78
11 Phagocytic Activity of Ochromonas versus
Microcystis 79
12 Influence of Compound No. 6 at 1.0 ppm on
the Phagocytic Activity of Ochromonas oval-is
on Microcystis aeruginosa 84
13 Influence of Compound No. 11 at 0.1 ppm on
the Phagocytic Activity of Ochromonas ovalis
on M-iarocyst-Ls aeruginosa 85
14 Ochromonas dan-Lea - Organelle Arrangement 89
15 Striated Cylinders Associated with Con-
tractile Vacuoles in 0. dan-ioa 91
16 Ochromonas danica - Showing Mitochondria,
with Dialated Cristae 92
vi
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List of Figures continued
17 Transmission Electron Micrograph of
Oohromonas danioa Showing Ingested
Miorooystis aeruginosa 93
18 Transmission Electron Micrograph of
0. danioa Showing Large Vacuole and Organ-
elle Arrangement 114
19 Oohromonas danioa - Mitochondria, with
Dialated Cristae and Bundles of Micro-
tubules 115
20 Miorooystis aeruginosa Nearing Dividing
Stage, Showing Mucopeptide Envelope 117
21 Miorooystis aeruginosa - Depicting Photo-
synthetic Lamellae 118
22 Oohromonas danioa - Transmission Electron
Micrograph Showing Striated Cylinders and
Size Relationship to M. aeruginosa 119
23 Oohromonas danioa - Transmission Electron
Micrograph Showing Contractile Vacuole at
Anterior End 121
24 Oohromonas danioa with Engulfed Mioro-
oystis 122
25 Oohromonas danioa - Details of Cytoplasm 123
26 0. danioa Containing Engulfed Microoystis
with Outer Envelope Pre-digested 125
27 Oohromonas danioa with Ingested Bacteria 126
vii
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LIST OF TABLES
N°JL Page
1 Analysis of Water from Well No. 4 28
2 PW-4 Culture Medium ("lX"-Solution) 2y
3 Results of Computerized Analog Structure
Search 36
4 Laboratory Screening Data - Test Compounds
versus Mioroaystis aeruginosa 38
5 Laboratory Screening Data - Test Compounds
versus Anabaena flos-aquae 44
6 Compound Formulation Optima 52
7 Prime Candidate Compounds Selected for
Economic Evaluation 54
8 Cost Comparison of Algaecidal Compounds
versus Mioroaystis aevuginosa 59
9 Field Tests - Test Compounds versus
Anabaena flos-aquae 63
10 Corrleation Analysis of Phase II Test
Compounds-Activity as a Function of
Compound Structure 65
11 Activity as a Function of Structure -
Class I 6:6
12 Persistence Study - Compound No. 23
(2,5-Dichloro-3,4-dinitrothiophene) at 1.0
ppm vs. Anabaena flos-aquae 71
13 Persistence Study - Compound No. 73 at 1.0
ppm using Anabaena as the Test Species 73
14 Field Study to Determine the Susceptibility
of M-iarocystis to Ochromonal Infestations in
Which Various Protective Coverings are
Utilized as Barriers 75
15 Phagocytic Activity of Four Species of
Oohromonas on Miovooystis aeruginosa 77
16 A Four Day Study of the Rate of Phagocytic
Activity of Various Species of Oohromonas
Against Micvoaystis aeruyinosa in Light and
Dark Conditions °°
viii
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List of Tables continued
17 Test Compound No. 14 Effect on Ochromonas
Activity 81
18 Enhancement of Ochromonas Activity by
Test Compound No. 6 83
19 Influence of Test Compound No. 11? at 0.2
ppm on the Phagocytic Activity of Four
Species of Ochromonas 86
20 Influence of Test Compound No. 119 at 0.2
ppm on the Phagocytic Activity of Pour
Species of Ochromonas 87
21 Influence of Test Compound No. 114 at 0.2
ppm on the Phagocytic Activity of Four
Species of Ochromonas 88
22 Laboratory Screening Tests - Compound No.
11 and 72 102
23 Laborotory Screening Tests - Compound No.
70 and 73 1Q3
24 Comparative Algaecidal Properties of
Various Formulations of Two Batches of Syn-
thesized .Compound No. 23 Against Anabaena
flos-aquae (3-day Test) 104
25 A Four Day Study of the Rate of Phagocytic
Activity of Various Species of Ochromonas
on Microcystis aeruginosa
26 Effect of Test Compound on Ochromonas
Activity 10?
27 Ochromonas Activity Enhancement Test at
0.1 ppm - Compound No. 11 108
28 Effect of Test Compound No. 21 on
Ochromonas Activity
ix
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SECTION I
CONCLUSIONS
A computerized structure search of over 100,000 compounds
revealed 1309 analogs of the four Phase I candidate com-
pounds. As a result of rapid agar-plate screening tests,
41 of these compounds were selected for more detailed test-
ing in the laboratory.
Although six of these compounds showed high levels of
activity against the target blue-green algal species,
Anabaena flos-aquae, four of them proved unacceptable be-
cause of poor economic feasibility, environmentally unac-
ceptable properties or difficulty or danger in compound
production.
The following conclusions are reached:
1. Compound No. 23 (2,5-Dichloro-3,4-dinitrothiophene)
is 100 percent active against Mierocystis aeruginosa
at concentrations to 0.2 ppm and 100 percent active
against Anabaena flos-aquae at concentrations as low
as 0.8 ppm. This compound is safe to applicators, if
ordinary safe handling procedures are observed, and ex-
hibits no known environmentally hazardous properties.
The estimated cost, for the control of Micvocystis
aeruginosa, is only 0.51 that of copper sulfate.
2. Compound No. 73 (p-Chlorophenyl-2-thienyliodoniumchloride)
is 94 percent active against Microcystis at 0.2 ppm "and
100 percent against Anabaena at 0.8 ppm with low environ-
mental and human handling hazards.
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3. Ochromonas ovalis was the most voracious, in its attack
on Micfoaystis aeruginosa, of the several species of
Oahromonas which were determined to have phagocytic
properties.
4. Compound No. 6 (l,l-Dimethyl-2-(a ,<=,«-trif luror-p-
tolyl)-urea) produced about 27 percent increase in
Ochromonal phagocytic activity.
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SECTION II
RECOMMENDATIONS
Since the research efforts in Phases I and II have indicated
that the two selected candidate compounds are relatively spe-
cific, technically effective and economically competitive,
it is recommended that investigations of their algaecidal
activity under natural field conditions and determination of
of persistance, degradability and toxicity, be undertaken.
Phase III of this effort should include:
1. Re-synthesis of multi-pound lots of the two candidate
compounds.
2. Testing of the compounds under natural field conditions
in various sectors of the country. These tests should
take place in small lakes or ponds having naturally
occuring blooms of nuisance algae.
3. Toxicity studies on fish and mammals, as well as gross
pathology, acute oral toxicity and human primary skin
irritation tests.
4. Development of analytical test procedures, In order that
persistence, adsorption and degradability rates can be
followed during and after treatment to determine the
environmental stability of the compounds.
5. Further development of biological-chemical control
systems in the laboratory, and testing for efficacy in
naturally occuring field situations.
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SECTION III
INTRODUCTION
The control of algae and aquatic weeds has become of
major concern to the Environmental Protection Agency in a
national effort to improve "the quality of life". Due to
the great diversity of species and the ubiquitous nature
of many species, there is hardly a body of water or moist
spot on the face of the earth which is devoid of algae
(Prescott, 1970) .
Algae are common and normal inhabitants of all surface
waters and are encountered in virtually all waters which
are exposed to sunlight. To date more than 18,000 species
have been identified (Palmer, 1962). Unlike the other
groups of small or microscopic organisms, all species of
algae possess chlorophyll and are responsible for an esti-
mated 90 percent of all photosynthetic activity on the
earth (Meyer, 1971). One pound of algae growth produces
about 15 pounds of oxygen (Mackenthun and Ingram, 1964) .
The need for control of certain nuisance algal species
has arisen in relatively recent times as a result of
excessive nutrification of surface waters due to an in-
creasing human population and to indiscriminate use of
available supplies of water.
Eutrophication of inland waters is caused by concentra-
tions of human population in urban areas and by inadequate
treatment of the resulting sewage runoff from urban areas.
Additional eutrophication occurs as a result of runoff
from fertilized agricultural lands from the natural proc-
esses of precipitation from the atmosphere, and interchange
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of bottom sediments which occurs periodically in lakes
and reservoirs (Mackenthun and Ingram, 1964).
Although there is no universal agreement among scientists
and environmentalists as to the particular elements which
are responsible for excessive algal populations, most com-
monly suspected are phosphorus and nitrogen. Algal blooms
are usually associated with waters which receive sewage
effluents which are rich in phosphorus and nitrogen. For
example, Mackenthun and McNab (1961) made studies of
Wisconsin waste stabilization ponds and concluded that the
annual per capita contributions were 4.1 pounds of inorgan-
ic nitrogen and 1.1 pound of soluble phosphorus.
Increasing urbanization and changes in ground cover and
surface soil due to such processes as fires, overgrazing,
deforestation and agriculture have increased surface runoff
and reduced soil seepage to the extent that, as estimated
by some authorities, the water table in the Eastern half
of this country 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
water shortages in many areas. Thus, as population and
industrial demands increase, attention has, of necessity,
turned more and more to lakes, streams and reservoirs to
meet these needs.
In view of the increasingly heavy demands on our existing
water supplies, it is of vital importance that the quality
and availability of these waters be preserved. Waters con-
taining dissolved and suspended materials not only support
the growth of algae, but many other kinds of aquatic life,
the numbers and abundance of which are governed by the
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amounts and kinds of nutrients available. Since the utili-
ty of natural waters is often determined by the abundance
and type of biota, adequate control of noxious species
becomes a major concern.
When the nutrients, temperature and light condition becomes
optimal for the growth of a particular species of algae,
proliferation may take place in such abundance as to pro-
duce a visible agregation of algal masses. To produce
such "algal blooms" the water and environmental conditions
usually become optimal for only one species at a time.
Thus, a bloom usually contains a heavily predominant species
of algae. Many algae, particularly blue-greens, may impart
obnoxious tastes and odors to the waters, clog intake
screens and rapid-sand filters of water treatment plants.
In addition, they may produce unsightly floating masses
or collections of debris on shores making the waters un-
suitable for many purposes.
Such nuisance algae may shorten filter runs in water treat-
ment plants, or otherwise hamper industrial and municipal
water treatment processes, impair areas of picturesque
beauty, lower waterfront property values, and, in some
cases, be toxic to certain warm-blooded animals which ingest
the water (Mackenthun and Ingram, 1964). Certain species
of Cyanophyta (blue-green algae) such as Miorocystis*
Aphanizomenon, and Anabaena are known to have caused animal
deaths by the production of toxic substances, particularly
in areas where the wind has concentrated the algae in lee-
shore areas (Mackenthun and Ingram, 1964).
Fish kills have resulted from the depletion of dissolved
oxygen in waters where the algal population has been very
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dense. Even though algae do impart oxygen to the water
through photosynthesis during daylight hours, the metabolic
and catabolic processes within plant cells utilize oxygen
continuously, day and night. If a heavy cloud cover should
reduce oxygen-producing photosynthetic activity to a low
level over a several-day period, the amount of dissolved
oxygen during subsequent nights may fall below the critical
level for many species of fish and fish kills may result.
The pH of the water will also tend to increase as the algae
increase their requirements for carbon dioxide during day-
light hours. This process of continuous removal of carbon
dioxide from the water results in an alteration in the rela-
tive amounts of soluble carbonic acid, intermediately soluble
bicarbonates and nearly insoluble monocarbonates, often
causing some of the latter to precipitate. The corrosive
activity of the water is also often increased as a result
of algal growth, causing water storage and transport prob-
lems due to the depolarizing action of the oxygen which is
produced (Palmer, 1962).
Many inland waters have been seriously impaired in recent
years as a result of algal growths. For example, Lake
Washington in 1959 contained a maximum of 1.5 x 106 yVml
of total phytoplankton volume, of which only 15 percent
was blue-green algae. By 1963 the phytoplankton population
had shown a ten-fold increase, of which 95 percent consisted
of various species of blue-green algae (Bartsch, 1967).
The blue-green algae are of particular concern for a number
of reasons. In contrast to most species of non-flagellated
algae which settle to the bottoms of lakes in calm weather,
many planktonic blue-greens exhibit an "upside-down" charac-
teristic and accumulate as dense scums on the surface which
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can then be blown into thick windrows and piled upon the
shore. Decomposed materials often produce an offensive
"pig-pen" odor (Mackenthun and Ingram, 1964). These pro-
ducts as well as the substances produced by several living
species may impart tastes to the water and be toxic to ani-
mals and humans alike (Bartsch, 1967).
Several approaches to the problem of nuisance algal control
are being investigated under the auspices of the National
Eutrophication Program of the Environmental Protection Agency,
Basically the approaches fall into four broad categories:
(1) mechanical, (2) biological, (3) ecological and (4) chem-
ical. Mechanical approaches involve the design and engin-
eering of machines for underwater mowing, raking and harvest-
ing of aquatic weeds and algae. Biological control mechan-
isms being investigated include viruses, insects, algae-
eating fish and phagocytic organisms. Ecological approaches
include diversion of nutrient-rich waters, flushing lakes
and ponds with nutrient-poor water, removal of nutrients by
sewage treatment processes and nutrient removal from bodies
of water by aquatic plant crops or other means.
Chemical approaches to the control of nuisance algae have
not been investigated thoroughly, even though much effort
has been expended in other aspects of pest control. The
problem is complicated by difficulties encountered in devel-
oping an algaecide which will selectively kill or stop re-
production of the target species without adversely affecting
the desirable forms of aquatic life.
Copper sulfate has, for a number of years, been the chemical
of choice for the control of algae. Despite its extensive
usage, such shortcomings as its toxicity at higher concen-
8
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trations to desirable aquatic life, non-biodegradability,
accumulation as copper salts in bottom muds and its corrosive
properties to paint and equipment (Bartsch, 1954), leave room
for the development of a more ideal algaecide. Hasler (1947)
and Kunkel (1969) point out the possible deleterious effects
of lake bottom accumulations of copper sulfate on lake ecol-
ogy, and investigations in Wisconsin and Minnesota conducted
by Moyle (1949) indicate that certain algae, particularly
the blue-green algae Aphanizomenon, seem to have acquired
an increased tolerance to copper sulfate as a result of many
years of treatment; two to five times as much copper sulfate
was required for control as was required 20 years earlier.
There is, at present, no federally registered chemical
available which is effective against nuisance species of
blue-green algae in surface waters from which drinking water
may be prepared. In recent years a considerable amount of
effort has been expended in search of a selective compound,
to replace copper sulfate, which would be safe to non-target
organisms, non-cumulative and economically competitive
(Mackenthun, et al, 1964).
Although it is likely that no single approach will be found
which will serve in all cases, it is possible that a chemical
approach to the control of|nuisance algal growths will be
suitable for many situations, particularly where algal
"blooms" are evident and an ecological balance needs to be
restored.
It should be emphasized that the development of a chemical
for widespread use in the environment, particularly where
recreational and culinary waters are concerned, is not a
short-term project. After a compound has been selected for
long-range development, as a result of proven activity,
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economics and initial environmental safety projections,
larger-scale field tests and complete toxicology studies,
together with environmental, persistence and handling
hazards tests, must be conducted. To avoid misleading
generalizations, experiments must be designed to give spe-
cific answers related to the compound in question. The
chances for success for a candidate compound must be very
good at this point to justify the time and expense neces-
sary to meet federal clearance requirements.
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 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
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 where 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.
10
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Within the 12-month contractual period, thirty-three
selected compounds were screened using the two target spe-
cies of blue-green algae suggested by EPA's Federal Water
Qaulity Office, Corvallis, Oregon. Each compound was
tested against cultures of Microcystis aeroginosa and
Anabaena flos-aquae at a concentration of 2.0 ppm in con-
stant-temperature water bath shakers (24°C) at 80 oscil-
lations per minute under a cool - white fluorescent light
intensity of 100 foot-candles. Compound activity was ex-
pressed 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-microphoto-
meter- 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-dibromophenylthiocyanate) and
24 (1,1-Dimethyltetradecyamine, hydrochloride) were effective
at these concentrations and were selected as the prime can-
didate compounds for further research through Phase II
of the long-range algaecidal development program.
i
A phagocytic organism, identified as Oehromonas ovalis,
was discovered which showed promise as a means of control-
ing Micvocystis. In addition, it was found that the growth
and activity of Oehromonas was enhanced by low concentra-
tions of some of the test compounds being studied.
In the work statement, as included in the contract agree-
ment, efforts were to be directed toward the long-range
11
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goal 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 econom-
ical, (4) be safe to applicators and (5) be non-persistent
in aquatic systems.
Included in the Phase II effort was to be the screening
of analogs of compounds that had exhibited good algaecidal
activity in the initial screening. This was to be done
because of the possibility that analogs of these compounds
might possess potential algaecidal activities equal to or
greater than the initial compounds. Also, the physical
and chemical properties of the selected compounds were to
be analyzed more completely in an effort to develop the
optimum formulations in terms of use in the aquatic envir-
onment .
The research was to continue in the identification of other
biological chemical systems and to continue the effort to-
ward optimizing the Oehromonas system discovered as speci-
fied under Contract No. 14-12-814. Phagocytic algae, nemo-
todes and protozoa were to be obtained and tested against
Miorooystis and Anabaena. Those showing promising algaecidal
activity were to be tested with algaecidal chemicals in an
effort to develop a feasible integrated control technique.
The three compounds found to be algaecidal in the initial
screening were to be tested on a pilot-scale, using plastic
wading pools. Analytical procedures for suitable and rapid
monitoring of the test compounds were to be developed./ Pre-
liminary mammalian and fish toxicities were also to be con-
ducted on the selected test compounds.
12
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SECTION IV
EXPERIMENTAL PROCEDURES
The primary objective of the presently reported effort
was to include all of the available analogs of the four
candidate compounds chosen through the initial screening
program and then to make a final selection of the prime
candidates for large-scale field testing. Specific efforts
were to be directed toward further defining an effective
chemical or biological-chemical system which would control
the growth of blue-green algae safely and economically,
and yet would be non-persistant in the environment and
exhibit a high degree of specificity for the target algae.
Analog Search
A computerized search was conducted to select from Dow's
library of over 100,000 compounds those which were analogs
of the four candidate compounds and were selected as a
result of the Phase I effort. It was felt that there would
be a good probability of finding other active algaecides
among those compounds with structures closely related to
the Phase I prime candidates.
The analog structure search was undertaken by personnel at
Dow's Computation Research Laboratory in Midland, Michigan.
The computer search revealed 1309 analogs which were
screened in a rapid test for algaecidal activity at Dow's
Western Division Agricultural Research Center, Walnut Creek,
California.
13
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Laboratory Tests
(
Rapid Screening
Stock cultures of Anabaena .flos-aquae were also sent to the
Western Division for use in an agar plate screening test
which was designed for rapid screening of the selected
analogs. Gorham's "Ix" nutrient medium was prepared with
agar, poured into culture plates, and then spotted with
test compounds at 1.0 and 0.2 ppm. Those which proved
positive within seven days at these concentrations were
tested at lower concentrations to the "break point" where
the compounds were only 50 percent active. It was not
necessary to test any compound at concentrations lower than
0.04 ppm. With the facilities and space available, about
200 compounds were tested per week until screening data had
been completed on all of the compound analogs.
Fine Screening
Those compounds which exhibited complete activity at 1.0
ppm or less were tested according to the previously estab-
lished screening test procedures, using Gorham's medium.
Early in the inception of Phase II the decision was made,
with the approval of the Project Officer, Dr. T. E. Maloney,
to use only Anabaena as the primary algal test species.
The decision was based on several factors, but principally
upon the extreme difficulty encountered in trying to grow
cultures of Miaroayetis in the open atmosphere at the
selected field test site, without contamination by the
phagocytic organism Ochromonas ovalis - which was found,to
be omnipresent in the area. Previous experience had con-
sistently revealed Anabaena to be the most resistant species
to algaecidal action, and any compounds which exhibited
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acceptable toxic properties against Anabaena proved to be
even more toxic to UiovoQ-ystis. Cultures of Microaystis
were maintained, however, for research on biological-chem-
ical control systems and also for use in compound persis-
tance studies where greater sensitivity at lower chemical
concentrations was desirable.
Gorham's medium was used for culturing all algae utilized
in the laboratory screening tests. The composition of this
medium (in milligrams per liter) was:
NaN03 496
MgSOn'7 H20 49
CaCl2«H20 36
Na2Co3 20
NaSiOa 58
ferric g
citrate
EDTA 1
(Hughes et al, 1958) The pH was adjusted to 7.8-8.2
(Kuentzel, 1969).
Anabaena stock cultures showed a better growth pattern
under 200 foot-candles of cool-white fluorescent light than
at lower intensities. Miopocystis cultures were cultured
at about 100 foot-candles.
The laboratory screening tests were conducted in much the
same fashion as those in Phase I (Dow, 1971) utilizing 30
ml algal culture in 125 ml test flasks. These were contin-
uously agitated at 80 excursions per minute in 24°C water
bath shakers under a constant cool-white fluorescent illum-
ination of 100 foot-candles. The light was furnished by
15
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four 48-inch, 25-watt cool-white fluorescent bulbs, diffused
and attenuated by layers of cheese cloth interposed between
the bulbs and the shaker assembly.
Culture growths were monitored by cell counts and by relative
intensity readings taken with an AMINCO fluoromicrophotometer
equipped with a blue mercury-vapor fluorescent lamp. Light
from the lamp was filtered through a 5230 band-pass
filter before entering the sample. Due to the extreme
sensitivity of this instrument to fluorescing chlorophyll
molecules it was possible to take these readings
without going through chlorophyll extraction procedures.
Six new compounds were tested for algaecidal activity using
the normal three-day laboratory screening procedures. These
compounds were selected separately from the analog structure
search on the basis of their compositions and structures,
as compared to other known algaecides.
Accelerated Screening
After the initial rapid screening tests were completed at
Dow's Western Division and the compounds to be put through
the secondary screening tests were selected, a stepped-up
testing schedule was adopted. In the new schedule the re-
maining test compounds were formulated by attempting a
water solution at first and then trying successively, ace-
tone, alcohol, toluene, xylene and finally methylene di-
chloride. In most cases where water or acetone proved to
be inadequate, xylene was used and balanced to a specific
gravity of 1.00 with the addition of about 19 percent per-
chloroethylene. The test compounds were then laboratory
tested at 0.5 ppm for three days against both target algal
species, Microcystis aeruginosa and Anabaena flos-aquae<
16
-------
All other testing conditions were the same as for the fine
screening tests. Cell growth was monitored daily by taking
relative intensity readings with the fluoro-microphotometer.
Initial and final cell counts were taken also, in the case
of M-iGVoaystis .
The tests were terminated if less than 70 percent or greater
than 95 percent activity was achieved against Anabaena. If
an activity between 70 and 95 percent was obtained, the
test was continued for another three days.
Culture Clean-up Procedures
It was discovered that field cultures of Anabaena which had
become contaminated with Ochromonas could be freed from
these organisms by a quick-freezing process. Due to the
more fragile nature of Ochromonas it was found that by sub-
jecting the mixed culture in a sealed container to a dry
ice-acetone bath for five minutes and then allowing to
warm to room temperature, the Ochromonas cells were ruptured
by the freezing process whereas most of the Anabaena cells
survived. After centrifuging and washing the "cleaned" cul-
tures several times a healthy culture of Anabaena, free of
Ochromonal contaminants, could be obtained.
Synergism
Several compounds were tested in combination to determine
whether the combined algaecidal activity would be greater
than either one alone. Equal quantities of each compound
were formulated and added to established culture of Mioro-
oystis and Anabaena for total test chemical concentrations
ranging from 0.8 to 0.05 ppm. After seven days the tests
were terminated and cell growth monitored by cell counts
and relative intensity readings.
17
-------
Compound Persistence
A test series was planned to determine the toxicity per-
sistence of the prime candidate compounds as functions of
time and environmental conditions, using bioassay tests
to determine residual toxicities. The experimental design
included using "World Health Organization" (WHO) standard
hard water, well water with PW-4 medium and deionized water
with Gorham's medium. One ppm of the test compound was
added to each type of water in 4-liter flotation bags in
the field and similarly in 500 ml flasks in the laboratory
to serve as controls. Monitoring of the compound degra-
dation was accomplished by bioassays utlizing Anabaena flos-
aquae cultured in the various waters initially, and subse-
quently at three-day progressive intervals. After a three-
day growth period for each progressive monitoring test,
final readings were taken on each culture and the results
were compared to controls which were grown under the same
conditions with no test compound added.
Formulation
During Phase I an acetone emulsifier, containing two sur-
factants, had been used successfully in formulating nearly
all of the compounds tested. Although this system and the
formulation procedure used had proven fairly successful,
it was deemed important that the best formulation possible
be developed in order that the maximum algaecidal activity
efficiency could be achieved.
Standard formulation optimization procedures were utilized
in determining whether a non-ionic, anionic
emulsifier should be used in establishing the hydrophilic-
lipophilic balance (HLB). Generally speaking, the better
"HLB" which can be attained, the smaller the emulsified
18
-------
particles, thus causing more surface contact area and a
higher rate of algaecidal activity
An 18-hole formulation board was made and the necessary
graduated centrifuge tubes and automatic syringes obtained
for a full scale formulation optimization study using the
three most probable solvents: toluene, xylene and perchloro-
ethylene, together with matched anionic and nonionic pairs
of Atlox, Tumulz and Sponto emulsifiers.
The Atlox emulsifier pair was tested with a 9-hole, 100:
100 expansion (Figure 1 ) in which all of the emulsifier
in the solvent contained only anionic emulsifier on one
end of the board and only nonionic emulsifier on the other
end. By progressive blending of equal portions, seven
other emulsifier ratio combinations were obtained. When
1.0 ml of each of these was added to 100 ml water in grad-
uated centrifuge tubes, shaken well and left standing for
three days, the most stable emulsion could be easily de-
tected by noting the tube with the least amount of settling.
For an even more precise detection another series was run
using a 50:100 expansion starting with a 1:1 emulsifier
ratio on the left end of the board and all nonionic emul-
sifier in the solvent on the right end. The solvent blend
was 81 percent xylene and 19 percent perchloroethylene to
give a specific density as near that of water as possible.
For the Atlox pair tested, the optimum blend was determined
to be 10 percent emulsifier at the ratio of 18.75 anionic
to 81.25 nonionic in the primary concentrate. In practice
this ratio was often changed when the test chemical was
dissolved in the primary solvent. In those cases additional
formulation tests were necessary to determine the optimum
"HLB" under various test compound loadings.
19
-------
Figure 1
FORMULATION EFFICACY TESTS
Dote 9-22"7.1
Solvent V-*—' ^^° Xylene; 19$ Perchloroethylene
Emolsifiers: I-fr Atlox 3404F Rioht Atlox 3^2 5F
Test Compound_Jione
100:100 Expansion
Use Concentrates
ooooooooo
Primary Concentrates
O O O O O
100:0 \ 75:25 50:50 f J 25:75 ( J 0:100
87.5-125 62.5:375 37.5:625 12.5:87.5
50:100 Expansion
Use Concentrates
ooooooooo
Primary Concentrates
o o o o o
50:50 S~\ 375:62.5 S~\ 25:75 (\ 125:875 £ ^ 0:100-
43.75:56.25 31.25:68.75 18.75:81.25 6.25:93.75
-------
Once a theoretical optimum had been determined for a par-
ticular compound, it was then necessary to verify its
algaecidal activity when reduced to actual practice. This
was done by bench-scale testing in the laboratory following
a procedure similar to the secondary screening test. This
task had to be completed for each compound recommended for
open field testing before actual testing of the compound
could begin.
Toxicology Tests
Sixty grams of Compound No. 23 in the crystalline form,
99 percent pure, was sent to Dow's Biomedical Research lab at
Midland, Michigan, where the compound was tested for pri-
mary skin irritation on rabbits, acute oral lethality
using rats and eye irritation on rabbits. A 10 percent
w/v primary concentrate formulation of this same compound
was also sent to Midland for similar toxicological evalu-
ation. This formulation was made up with a solvent-emul-
sifier system composed of 8l percent xylene and 19 percent
perchloroethylene, to which was added 10 percent Atlox
emulsifier with an anionic:nonionic ratio of 25:75.
Because of the N02 in the molecular composition of Compound
No. 23, the possibility of handling and storing hazards
existed and the compound was thus submitted for independent
drop weight and differential thermal analysis tests to
determine whether any mechanical shock or ordinary reactive
chemical handling hazards existed.
Field Tests
The following criteria were used in choosing a site where
the small-scale open-environment tests were to be conducted.
21
-------
It was felt that the site should be:
1. Remote from any chemical plants or areas where agri-
cultural chemicals were to be used.
2. Free from cattle and other domestic animal disturbances.
3. Remote from open waters and trees.
4. Easily accessible by an all-weather road.
5. Located where 110-volt power and an acceptable supply
of water could be made available at a reasonable cost.
With the above criteria in mind a site was selected near
Dow's Ag-Organic Department research facilities at Lake
Jackson, Texas.
Test Equipment
A shading roof structure of 4' x 8' sheets of corrugated
transluscent plastic was built (Figure 2 ). Six plastic
wading pools, six feet in diameter and 14 inches deep, with
enameled, corrugated metal sides were set up under the
shading roof. They were then lined with 6 mil guage poly-
ethylene film and filled with 200 gallons of well water
piped 300 feet from well number 4 to the test site. A
110-volt supply of electricity was also made available at
the test site.
Aside from the decision to use only Anabaena for the outdoor
algal test species, a few other modifications in experimental
design were necessary. The sides of the shading roof struc-
ture were covered with a layer of saran mesh screening to
help attenuate the wind in an attempt to modify conditions
22
-------
Figure 2
OPEN ENVIRONMENT TEST DESIGN
(Shading Roof and Support)
ro
4"x4" coiner supports
4'x8' sheets of corrugated translucent plastic roofing
710 pal. wadine pools. 14" deep, with corrugated metal sides
-------
sufficiently to allow for continued operation during the
winter months. Also, 300-watt heat lamps were suspended
above the pools to keep the water temperature from dropping
too low. None of these modifications, however, changed
the ambient conditions sufficiently to yield significant
results from the field tests conducted during cold weather.
Test Procedures
Sixteen-liter polyethylene bags, supported by polyurethane
flotation collars were used as test chambers and control
vessels in the earlier field tests (Figure 3). Later,
field test data on the two final candidate compounds were
obtained using 100 gallons of PW-4 media in each of three
test pools. After Anabaena cultures had become acclimated
in the pools and had grown to a concentration giving a rela-
tive intensity reading of 0.10, the pools were then inoculated
with 1.0, 0.3 and 0.1 ppm of the test compound respectively.
Controls were contained in floating 16-liter polyethylene
bags in each large pool. The cell growth, or decline, was
monitored daily by taking fluoro-microphotometer readings.
It was found that more consistent and uniform readings were
obtainable if a 200 ml sample, taken from a well-agitated
pool, was blended in a Waring blender for 15 seconds and
was then added immediately to the cuvette before settling
could take place.
Originally, laboratory screening tests were conducted on
both Microcystis aeruginosa and Anabaena fios-aquae. But,
early attempts to culture Microcystis in the field always !
resulted in contamination by a phagocytic organism identified
as Oehromonas oval-is. A series of comprehensive tests were
conducted at the selected open-environment test site to de-
termine the source and a possible means of controlling
-------
Figure 3
Test Vessles with Polyurethane Flotation Collars
25
-------
contamination by the organism. Extreme precautionary sani-
tation and handling procedures were used. Open-topped,
sterile, 5-gallon polyethylene carboys were placed under
the shading canopy at the test site and cultures of Micro-
aystis were allowed to incubate under ambient conditions.
The cultures were prepared with sterile deionized water and
Gorham's medium. At five days elapsed time, the Microcystis
cultures progressed to their log phase of growth, but Oahra-
monas infestation was observed and soon thereafter had taken
a heavy toll.
Other Microcystis cultures were started in a similar manner,
using PW-4 media and well water piped to the test site. All
of these cultures, including outdoor and laboratory controls
showed the presence of Oo'hyomonas within two days. It was
concluded, and later confirmed, that the well water itself
contained Oahvomonas- Culture medium which contained water
pumped directly from the well, rather than from the storage
tank was found to contain Oehromonas also, although it took
four or five days to detect their presence, indicating many
fewer organisms. Additional precautionary measures in-
cluded disinfecting the shed, grounds and water line from
the well to the test area, using 5-0 ppm Clorox solution.
It was found that Oahromonas was highly susceptible to
chlorine at this concentration. Subsequent tests included
culturing Microaystis in sterilized well water with PW-4
medium and also with sterile Gorham's medium in sterilized
wading pools and open battery jars at the test, site, with
capped controls incubated in the same vicinity as well as
under laboratory conditions. Sterilized mosquito netting'
was placed over one of the wading pool tests to avoid the
possibility of insect contact as being the vectors of
Ochromonas contamination. In spite of all these precautions,
all open vessels showed the presence of Oohromonas within
26
-------
five days or less, whereas laboratory and outdoor capped
controls remained uncontaminated.
Prom this study it was concluded that the phagocytic organ-
ism Oohromonas oval-is., was ubiquitous in this area, and
due to its destructive action against Miorocystis, it was
not possible to use Miaroaystis as an algal test species
for this portion of the study. Consequently, permission
was sought, and granted, by the Federal Project Officer to
use Anabaena as the sole test species. In view of the fact
that past experience had proven Anabaena to be consistently
the most resistant to previously tested compounds it was
felt that no vital information would be lost.
New Culture Medium
With the relatively large volumes of water required in the
field tests it was impractical to continue using deionized
water and Gorham's medium, as in the laboratory screening
tests. An analysis of water from well number 4 at the test
site was obtained (Table 1 ) and a new medium entitled "PW-4
medium" was formulated (Table 2 ). The new medium was tested
for efficacy with the well water, and it was found that low-
ering the pH of the "10x" concentrate to about 2.8 and then
readjusting with sodium hydroxide to a pH of 7.8-8.5, proved
to be an acceptable procedure for facilitating the solution
of the various nutrient medium additives.
Large Volume Algal Inoculum Cultures
In order to facilitate outdoor testing, test chambers
consisting of floating polyethylene bags were used. This
resulted in each test being scaled up by a factor in excess
of 500. For the final testing of the prime candidate com-
pounds the wading pools contained 15,000 times as much water
27
-------
TABLE 1
ANALYSIS OF WATER FROM WELL NO. 4
Component
HC03
C03
OH
Total Cl
Ca
Mg
Na
Fe
Si02
S04
TDS
TSS
Turbidity
Total nitrogen
pH = 8.4
Parts
Per
Million
430
0
0
580
29
16
440
16.7
5
1,5^5
7
14
0.03
28
-------
•TABLE 2
PW-4 CULTURE MEDIUM
("lX"-Solution)
Component
NaN03
K2HP04
MgS04
CaC03
Ferric Citrate
Citric Acid
EDTA
Mg/1
(Well Water)
240
40
32
15
6
6
1
29
-------
as in the laboratory screening test flasks. It was apparent
that a much larger quantity of algal innoculum would have
to be grown to accomodate such tests. A new bank of cool-
white fluorescent lights was- prepared to give a light inten-
sity of 200 ft-c. Large batches of Andbaena were grown in
4-liter flasks and in 5-gallon polyethylene carboys with
the tops cut off and then covered with Saran Wrap to protect
them from dust and foreign matter. The cultures were agi-
by hand daily and their growth patterns monitored by fluor-
ometric readings.
Resynthesis of Compound No. 23
Compound No. 23 (2,5-Dichloro-3,4-dinitrothiophene),
selected as the prime candidate compound was resynthesized
to obtain larger quantities than those available from the
compound library. Of the 100 grams obtained, 60 grams were
formulated and sent to Dow's toxicology lab at Midland,
Michigan for Class I toxicology tests. The remainder was
retained for further formulation studies and field confirm-
ation tests.
Cell Growth Monitoring Procedures
It was found that even Anabaena cultures grown freely in
the outdoors soon picked up •Qa'hvomonae, although the presence
of the Oohromonas did not seem to affect the growth of the
Anabaena. Since fluorometry had been the principal means of
monitoring cell growth of this species, the presence of
fluorescing Oohvomonas cells in the test chambers introduced
a considerable amount of error in the readings. Utilizing
previously obtained knowledge of the fragile nature of
Oohromonas, a reliable analytical method was developed. Six
milliliters of algal culture were measured in a graduated
centrifuge tube, capped tightly and then quick-frozen in a
30
-------
dry ice-acetone bath. The freezing process ruptured Ochro-
monaa cells but left Anabaena relatively unharmed. After
washing, centrifuging and resuspending to the same volume,
reliable relative intensity readings were then obtained.
Biological-Chemical Systems
Further investigations concerning the phagotrophy of the
algal species, Oohromonae ovalis, and other phagocytic or-
ganisms, were conducted in an attempt to improve on a bio-
logical algal control system. In cooperation with Dr. M.
J. Wynne, consultant from the University of Texas, cultures
of several other species of Oohpomonas were obtained and
tested with various compound additives, under varying con-
ditions, to determine optimum activity parameters. Tests
were conducted both in the laboratory and in the open at-
mosphere.
Algal cultures, after becoming well established in the lab-
oratory, were purposely infected with Oohromonae in various
stages of growth and in combination with other chemicals
and nutrient additives. Thirty milliliter quantities of
Mioroayatie culture were started in 125 ml Erlenmeyer flasks
in "lOx" Gorham's medium. The initial cell count was about
1.0 x 106 cells/ml. Various concentrations of test compounds
and other micronutrients were added to duplicate sets of
test flasks, with one additional flask serving as a control.
The flasks were agitated in water bath shakers at 80 excur-
sions per minute under cool - white fluorescent lights of
about 120 foot-candles intensity. When the cell counts had
reached 3.0 x 10s cells/ml, the test cultures were infected
with an Oohromonas inoculum of sufficient concentration to give
an initial cell count of 1.0 x 10* Ochromonas cells per ml.
31
-------
Cultures were monitored daily by microscopic examination.
Those combinations which exhibited the greatest phagocytic
activity enhancement were taken to the open environment and
tested in a fresh water canal with both open and closure-
capped flasks. The flasks were supported in a fresh water
canal by a polyurethane flotation device.
The viability of cultures of Ochromonas was also studied as
a function of time and storage conditions. This included
investigations of various types, of dispersing media, such as
powdered polyurethane, or other types of imbibing materials,
and their effects on longevity and viability -
Other types of phagocytic organisms were obtained from the
University of Texas Botany Department collection and were
cultured and tested in a similar manner. The modes of in-,
gestion and the ultramicro-structure of Oehromonas and activ-
ity rate comparisons between species were studied by Dr. M.
J. Wynne and Dr. Gary Cole of the University of Te.xasj at
Austin.
32
-------
SECTION V
DISCUSSION
By means of a computer search of some 80,000 compounds
during Phase I of this effort, thirty-three compounds were
selected and tested for algaecidal activity by primary and
secondary laboratory screening techniques. Four of these
compounds were found to be sufficiently active against the
two target blue-green algal species, Anabaena flos-aquae
and Mieroeyatis aevuginosa to be selected as candidates for
Phase II testing.
The objective of Phase II was to obtain information on the
algal control efficacy of the selected candidate compounds
and their active structural analogs, and on combined bio-
logical-chemical systems, in a controlled open environment.
Specific efforts were directed toward further defining an
effective chemical or biological-chemical system which would
control the growth of blue-green algae economically and yet
be safe to applicators, be non-persistent in the environment
and exhibit, a high degree of specificity.
Structure Search
The structures of the four successful Phase I compounds are
given in Figure 4 . Since closely related compounds often
exhibit similar biochemical properties it was felt that the
analogs of these compounds should be investigated to give
the broadest possible coverage in a search for compounds
having superior algaecidal properties.
A structure search of Dow's library of compounds was under-
taken at Dow's computation Research Laboratory in Midland,
33
-------
Figure 4
PHASE I - Prime Candidate Algaecides
4-Amino-2,5-dibromophenyl
-thiocyanic acid ester
1,1-Dimethyltetradecylamine
' -Hydrochioride
C,6H35N HCI
CH3
CtaH:
•NHjHCI
SCN
2j5-Dichloro-3,4 dinitrothiophene
Q-Chloro-2-(p-nitrophenoxy)phenyn
-phenyl lodonium chloride
02N
-------
Michigan. Prom this effort 1309 analogs were identified.
A breakdown of the number of analogs of each of the parent
compounds is given in Table 3 . One hundred ten of the
analogs were common to both compound No. 23 and No. 15.
Rapid Screening Tests
The printout containing the selected analogs was sent to
Dow's Western Division Agricultural Research Center at
Walnut Creek, California, to be put through a rapid coarse
screening test at the rate of about 200 compounds per week.
Initially the screening was at a concentration of 5.0 ppm
of the test compound on nutrient agar plates. Because of
the large number of compounds which were showing up as
"positives" at the 5-0 ppm level the test concentration was
reduced to 1.0 ppm, with active compounds being also tested
at 0.20 ppm and 0.04 ppm. Run downs to the point where
the compounds were less than 50 percent active were only
undertaken on those compounds which proved to be 100 percent
active at the 1.0 ppm level.
Of the two test algae, Anabaena consistently proved to be
more resistant than Miorocystis to the analogs of the Phase
I compounds tested. The decision was therefore made to use
only Anabaena in the rapid screening. This proved satisfac-
tory for giving the necessary initial screening information
on compound efficacy.
As a result of this testing program 41 compounds were chosen
to be tested through the laboratory fine screening program
used previously in Phase I.
The majority of the analogs were laboratory tested against
both test species of algae at 0.5 ppm for 3 days. Some of
35
-------
TABLE 3
RESULTS OP COMPUTERIZED ANALOG STRUCTURE SEARCH
Compound Serial Structure Search
Number Criteria No. of Analogs
8 aromatic thiocyanates 434
15 iodoniums 332
23 thiophenes 497
24 alkylamines 46
TOTAL 1309
36
-------
the compounds, before incorporation of an accelerated test
schedule, were tested at three levels (1.0 ppm, 0.3 ppm and
0.1 ppm) for seven-day periods. The modified three-day
testing program proved to be suitable for identifying com-
pounds worthy of further investigation.
Nearly all of the compounds tested proved to be more toxic
to Microcystis than to Anabaena. Of the 74 compounds tested
15 were at least 90 percent active against Microcystis at
0.5 ppm (Table 4). Four of these compounds (No. 50, 54, 55
and 72) proved to have activities of 90 percent or greater
against Microcystis at concentrations down to 0.1 ppm. Only
one compound, No. 6l, showed a comparable activity against
Anabaena within the same test period (Table 4 and 5). This
compound, however, was eliminated from the prime candidate
list early in the program due to its heavy metal component.
Only five compounds (No. 23, 50, 70, 72 and 73) showed
activites of 90 percent or better at 0.8 ppm against Anabaena
(Table 5). Compounds No. 23 and 73 both exhibited 100 per-
cent activity at 0.8 ppm but showed definate "break points"
in the 0.8-0.4 ppm range.
A number of the more promising Phase I compounds were
retested, using new formulation procedures, but no better
performance was found.
In addition to the Phase I compounds which were retested
and the 42 analogs selected for laboratory screening, six
other compounds (Nos. 98-103) were tested because, on the
basis of the structure and composition, good algaecidal pro-
perties could be expected. There were, however, no good
algaecides among this group. A series of sixteen additional
compounds were tested because of indicated algaecidal activi-
37
-------
TABLE 4
LABORATORY SCREENING DATA
TEST COMPOUNDS VERSUS Microcystis aeruginosa
Serial Activity (#) at Various Cone, (ppm)
Number Name of Compound 1.6 1.0 0.8 0.5 0.4 OTJ 0.2 0.1
1 3,3',4',5-Tetrachlorosalicylan- 0
Hide
2 4-Hydroxy-l-natphythiocyanic acid 0
3 5,5'-Dichloro-2,2'-dihydroxy- <10
benzophenone
6 l,l-Dimethyl-2-(cc,«,ec_trlfluoro- 0
^ p-toly)urea
CO
7 2-Benzyl-5,6-dimethylbenziraedi- 78
zole
15 (5-Chloro-2-(p-nitrophenoxy)- 100
pheny1)phenyliodoniumohloride
21 2,2'-Thiobis(4J6-dichloro- 44
phenol)
23 2,5-Dichloro-3,4-dinitrothio- 100 100 100 100 100 85
phene
24 1,1-Dimethyltetradecylamine, 100 100 100* 100 100* 100* 53*
hydrochloride
25 l,l-Dimethyl-9-octadecenylamine, 86
hydrochloride
28 Thio-m-(trifluoromethyl)carbonilic
acid, s-p-nitrophenylester
-------
TABLE 4 continued
LO
Serial
Number
31
33
34
50
51
52
53
5^
55
56
57
58
Name of Compound
n-Dodecyl-n,n-dimethy 1-1,3-
propanediamine, hydrobromide
Phenyltrichloromethylsulfide
1 , 2-Di-o-tolyhydrazine
Di-2-thienyliodoniumchloride
Phenyl-2-thienyliodonium-
bromide
Phenyl-2-thienyliodonium-
chloride
(p-Chlorophenyl) ( 2-thienyl)-
iodoniumbromide
(p-Chlorophenyl) (2-thienyl)-
iodoniumtrifluoroacetate
(p-Fluorophenyl)~2-thionyl-
iodoniumtrifluoroacetate
3-Pyridyl-2-thienyliodonium-
chloride, hydrochloride
Diphenyliodoniumtrifluoro-
acetate
Di-2-thienyliodonium-
Activity (%) at Various Cone, (ppm)
1.6 1.0 0.8 0.5 0.4 0.3 0.2
54
74
26
100* 99 100* 100*
95* 95*
99* 97*
93* 75*
100* 100 98*
100* 96*
30* 23*
100* 79*
64
0.1
98*
49*
71*
0*
92*
97*
<10*
46*
fluoborate
-------
TABLE 4 continued
Serial
Number
59
60
61
62
70
71
72
73
75
76
Name of Compound
3-Pyridyl-2-thienyliodonium-
trifluoroaceate
Phenyl-2-thienyliodonium-
trifluoroacetate
Tributylisothiocyantotin
3-Pyridyl-2-thienyliodonium-
bromide
2-Iodo-3,5-dinitrothiophene
2-Thienyl-m-tolyliodonium-
chloride
(m-Chlorophenyl)-2-thienyl-
iodoniumchloride
(p-Chlorophenyl)-2-thienyl-
iodoniumchloride
(p-Bromophenyl)-2-thieny1-
ioconiumchloride
2-Thieny1-(«,=,a-trifluoro
m-toly)icdoniumchloride
2-Thieny1-2,4-xylyliodonium-
chloride
Activity (%) at Various Cone, (ppm) _
ITS' TTo oTg" o~^5 oTT oTI o7 oTT
64
57
83
52
100* 100* 100* 100* 100* 65*
86
100* 100* 100*
100* 93*
100 100* 100 100* 60 94* 60*
100
34
25
60
33
-------
TABLE 4 continued
Serial
Number
77
78
79
80
81
82
83
84
85
86
87
Name of Compound
2-Thienyl-(«j«c,a:-trif luoro-m-
tolyl)iodoniumtetrafluoroborate
2-Thienyl-p-tolyliodonium-
chloride
2-Biphenlyl-2-thienyl-
iodoniumchloride
Isothiocyanatotriphenyltiri
Diphenyliodoniumbromide
(o-Chlorophenyl)-2-thienyl-
iodoniumchloride
2-Iodo-5-nitrothiophene
(p-Bromophenyl)-2-thienyltri-
fluoroacetate
(p-Fluoropheny 1 ) -2-thieny 1-
iodoniumbromide
Pyrazol-J4yl-2-thienyliodonium-
bromide
(2-Thienyl)(p-tolyl)iodonium-
Activity (%} at Various Cone, (ppm)
1.6 1.0 0.8 0.5 0.4 0.3 0.2 0.1
81
54
]
100
76
24
100* 100 100* 71* 58*
75
76
48
54
50
nitrate
-------
TABLE 4 continued
Serial Activity (%) at Various Cone, (ppm)
Number Name of Compound 1.6 1.0 0.8 0.5 0.4"OT3 072 OTT
88 (p-Chlorophenyl)-2-thienyl-p-. 100
toluenesulfonate
89 2,4-Dinitrothiophene 87
91 2-(Chloromethyl)-5-nitrothio- 82
phene
92 n-Allyl-5-bromo-3,^-dinitro- 48
2-thiophene-
w 93 2-Bromo-5-nitrothiophene 75
94 3-thiophenalanine 97* 100 97* 92* <20*
95 5-Nitro-2-thiophenecarbonitrile 76
96 2,5-Dibromo-3,4-dinitrothio- 88
phene
97 5-Nitro-2-thiophenecarboxamide 0
98 Tetrachlorothiophene 000
99 2,5-Dibromothiophene 000
100 l-Chloro-3-iodobenzene 000
101 l-Chloro-4-lodobenzene 000
-------
TABLE 4 continued
-tr
(JO
Serial
Number
102
114
115
116
117
118
119
CuSO,.5H2(
Cutrine
xylene-pe]
Activity (%) at Various Cone, (ppm)
Name of Compound 1.6 1.0 0.8 0.5 0.4 0.3 0.2 0.1
l-Chloro-4-iodosobenzene, di- 000
acetate
n,n-Diethyl-2-(2,4,5-trichloro- ^* 50* 57* 24*
phenoxy ) ethanamine
2-(4-Chlorophenoxy)-n-ethyl-
ethanamine
4-Butyl-2-nitrobenzeamine
n-((4-6-Cyanao-2-pyridinyl)oxy) 56* 52* 36* 0*
phenyl)-n,n-dimethylurea
n-(4-( (6-Bromo-2-pyridinyl)
oxy)phenyl)acet amide
n'-(4-((6-Bromo-2-pyridinyl) 82* 75* 64* 52*
oxy ) phenol ) -n, n-dimethy lurea
D 100 100 91 66 0
81 17 0
pchlor-atlox system at a 0
43.75t56.25 anionic to nonionic ratio
*
-day tests, all others are 3-day tests
-------
TABLE 5
LABORATORY SCREENING DATA
TEST COMPOUNDS VERSUS Anabaena flos aquae
Serial
Number
1
2
3
4=r
r~f
•4—
6
11
15
21
23
24
25
Name of Compound 1.6
3 , 3 ' j 4 ' , 5-Tetrachlorosalicyl-
anilide
4-Hydroxyl-l-napthylthiocyanic acid
5 > 5 ' -Dichloro-2 , 2 ' -dihydroxy-
benzophenone
l,l-Dimethyl-2-(«,°S°c-trifluror-
p-tolyl)urea
3,4,4' ,5' ,6-Pentachloro-2,2'-
methylenediphenol
( 5-Chlor o-2- (p-nitrophenoxy )
phenyl)phenyliodcniumchlorlde
2,2', -Thiobis- ( 4 , 6-dichlorophenol )
2,5-Dichloro-3,4-dinitrothio- 100
phene
1, 1-Dimethylt etradecylamine ,
hydrochloride
1 , 1-Dimethy 1-9-octadeceny 1-
Activity (#) at Various Cone, (ppm)
1.0 0.8 0.5 0.4 0.3 0.2 0.1
<10
17
19
22
61
0
0
100 57 44 42 44
60* 81 <20* <20* <20'
50
amine, hydrochloride
-------
TABLE 5 continued
Ul
Serial
Number
28
31
33
34
50
51
52
53
54
55
56
57
Name of Compound
Thio-m- (Trif luoro-methyl ) carbonic
acid, s-p-nitrophenyl ester
h-Dodecyl-n,n-diethy 1-1 , 3-pro-
panediamine, hydrobromide
Phenyltrichloromethylsulfide
1,2-Di-o-tolyhy.drazine
Di-2-thienyliodoniumchloride
Phenyl-2-thienyliodoniumbromide
Phenyl-2-thienyliodoniumchloride
(p-Chlorophenyl) (2-thienyl)io-
doniumbromide
(p-chlorophenyl) (2-thienyl)io-
doniumtrif luoroacetate
(p-fluorophenyl)-2-thionylio-
doniumtr if luoroacetate
3-Pyridyl-2-thienyliodonium
chloride, hydrochloride
Diphenyliodoniumtr if luoroacetate
Activity (%)
1.6 1.0 0.8
90*
50*
71*
<20*
94*
74*
0*
74*
at Various Cone, (ppm)
0.5 0.4 0.3 0.2 0.1
0
18
0
0
80* 45* 35*
48 53* 61*
64* 61*
<20* <20*
33 79* 0*
60* 0*
0* 0*
40* 0*
-------
TABLE 5 continued
cr.
Serial
Number
58
59
60
61
62
70
71
72
73
74
75
Name of Compound 1 . 6
Di-2-thienyliodoniumfluoborate
3-Pyridyl-2-thienyliodonium-
trifluoroacetate
Phenyl-2-thienyliodonium-
trifluoroacetate
Tributylisothiocyantotin
3-Pyridyl-2-thienyliodonium-
bromide
2-Iodo-3, 5-dinitrothiophene
2-thienyl-m-tolyliodoni urn-
chloride
(m-Chlorophenyl)-2-thienyliodonium-
chloride
(p-Chlorophenyl)-2-thienyliodonium-
chloride
(p-Bromopheynl)-2-thienyliodonium-
chloride
2-Thienyl-(oc4
-------
TABLE 5 continued
Serial
Number
76
77
78
79
80
81
82
83
Name of Compound
86
2-Thienyl-2,4-xylyliodonium-
chloride
2-Thi enyl-(<*,«, oc_trif luoro-m-tolyl)-
iodoniumtetrafluoroborate
2-Thienyl-p-tolyliodonium-
chloride
2-Biphenylyl-2-thienyliodonium-
chloride
IsothiocyanatotriphenyIt in
Diphenyliodoniumbromide
(o-Chlorophenyl)-2-thienyliodon-
iumchloride
2-Iodo-5-nitrothiophene
(p-Bromophenyl)-2-thienyltri-
fluoroacetate
(p_Fluorophenyl)-2-thienyliodon-
iumbromide
Pyrazol-4-yl-2-thienyliodonium-
bromide
Activity (%) at Various Cone, (ppm)
ITS' IToo78" o75 oTT oTT 072 oTI
31
20
57
60*
40
0
62
25
0
22*
37
0
0* 0*
-------
TABLE 5 continued
Serial
Number
87-
Name of Compound
Activity (%} at Various Cone, (ppm)
1.6
1.0
O.b
J=
CO
89
91
92
93
94
95
96
97
98
99
100
101
(2-Thienyl)(p-tolyl)iodonium-
nitrate
(p-Chloropheny1)-2-thieny1-p-
toluenesulfonate
2,4-Dinitrothiophene
2-(Chloromethyl)-5-nitrothio-
phene
n-Allyl-5-bromo-3,4-dinitro-2-
thiophene
2-Bromo-5-nitrothiophene
3-Thiophenalanine
5-Nitro-2-thiophenecarbonitrile
2,5,-Dibromo-3,4-dinitrothiophene
5-Nitro-2-thiophenecarboxamide
Tetrachlorothiophene
2,5-Dibromothiophene
l-Chloro-3-iodobenzene
l-Chloro-4-iodobenzene
0.2
0.1
0
13
12
41* 56 <20*
11
81 <10
0 0
0 0
0 0
0 0
0 0
0 0
<20* <20*
0'
0
0
0
0
0
-------
TABLE 5 continued
Serial
Number
102
104
105
106
107
108
109
110
111
112
113
114
Name of Compound 1.6
l-Chloro-4-iodosobenzenediacetate
3 , 4-Dichloro phenol
3 s 5-Dichlorophenol
3 , 5-Dichloro-4-hydroxybenzoic
acid
p , p ' -Methylenediphenol
Quinizarin
4 !-Fluoro-4-biphenylamine
4- ( 2-Amino-2-oxoethyoxy ) benzole
acid, methyl ester
n,n-Dimethyl-n»-(3-methyl-4-nitro-
phenyl)urea
n,n'-Dimethyl-6(methylthio)-l,3,5-
triazine-2 , 4-diamine
o-(l,2-Dimethylpropyl)-s-carbonodithoic
acid, ethyl ester
n,n-Diethyl-2-(2,4,5-trichlorophen-
Activity (%}
1.0 0.8
0
52
48
36
27
33
60
42
0
18
21
at Various Cone.
0.5 0.4 0.3
0
41
39
33
33
27
55
42
0
0
12
(ppm)
0.2 0.1
0
30
33
27
24
21
44
27
0
0
21
oxy)ethanamine
-------
TABLE 5 continued
VJl
o
Serial
Numb er
115
116
117
118
119
Name of Compound
2-(4-Chlorophenoxy)-n-ethylethan-
amine
4-Butyl-2-nitrobenzeamine
n-((4-6-Cyanao-2-pyridinyl)oxy)
phenyl)-n,n-dimethylurea
n-(4-((6-Bromo-2-pyridinyl)oxy)
phenyl)acetamide
n' - (I*- ((6-Bromo- 2-pyridiny 1) oxy)
phenyl)-n,n-dimethylurea
CuSO«»5H20
Cutrine
xyl:ene-perchlor-atlox system at a
43.75:56.25 anionic to nonionic ratio
Activity
at Various Cone. (ppm)
1.6 1.0 0.8 0.5 0.4 0.3 0.2 0.1
70
100
0
<10*
<10*
<10*
92
<10*
<10*
<10*
16
<10*
<10*
<10*
*7-day tests, all others are 3-day tests
-------
ty in an unrelated proprietary screening program. None of
those tested (Nos. 103-119) (Table 5) showed sufficient
algaecidal activity to warrant further testing.
Formulation Study
Each of the test compounds screened through the normal
testing procedure was initially prepared by first deter-
mining the compound's solubility in water. If the water
solubility was adequate no additional additives were used
in the preparation. Most compounds however, required prior
solution in acetone, xylene or toluene, sometimes with mild
heating. Emulsifiers were necessary with xylene or toluene
to bring about an emulsion for stabilization. With the aid
of a 9-hole formulation board, and equipment assembled for
this purpose the optimum formulation of each of the prime
candidate compounds was determined.
The optimum ratios of anionic to nonionic emulsifiers for
those compounds formulated are given in Table 6. A majority
of those compounds tested required no anionic emulsifier
(0:100 ratio). Also, it may be noted that Compound No. 23
required a 25:75 emulsifier ratio for optimum conditions
when tested with a 10 percent w/v test compound solution,
rather than the usual 4800 ppm (or 0.48 percent) laboratory
i
primary concentrate. Compound No. 73 was found to be suf-
ficiently soluble in acetone and water so that once a solution
was accomplished in acetone, dilution to testing concentra-
tions was possible without the use of emulsifiers.
Economic Evaluation of Prime Candidate Compounds
On the basis of activity, as determined by the laboratory
screening tests, and by elimination of those compounds which
contained environmentally unacceptable components, the list
51
-------
Compound
Number
ui
ru
6
8
10
21
23
54
55
57
70
80
23*
73
TABLE 6
COMPOUND FORMULATION OPTIMA1
Name of Compound
5,5'-Dichloro-2,2'-dihydroxy-
benzophene
1,1'-Dimethyl-2-(«,<*,«-tri-
fluoro-p-tolyl)urea
4-Amino-2,5-dibromophenyl-
thiocyanate
3,4,5-Trichloro-o-creosol
2,2' ,-Thiobis-(436-dichloro-
phenol)
2,5-Dichloro-3,4-dinitrothio-
phene
(p-Chlorophenyl)(2-thleny1)-
iodoniumtrlfluoroacetate
(p-Fluorophenyl)-2-thionyl-
iodoniumtrIfluoroacetate
Diphenyliodoniumtrlfluoro-
acetate
2-Iodo-3,5-dinitrothiophene
Isothiocyanatctriphenyltin
2,5-Dichloro-3,4-dinitrothio-
phene
(p-Chlorophenyl)-2-thienylidon-
iumchloride
Solvent System
8l/? xylene
195? perchloro-
ethylene
it
Tt
If
II
M
tt
M
II
M II
Acetone
Emulsifiers
Anionic Atlox
3404 +
Nonionic Atlox
3495F
Opt imum
Emulsifier
Ratio
0:100
IT
tt
n it
It M
II tt
0:100
0:100
0:100
25:75
" 31.25:68.75
0:100
0:100
0:100
25:75
0:100
25i75
1See Appendix C for detailed data
*For a 10$ w/v primary concentrate for large-scale field testing
-------
of compounds under active consideration was narrowed to six
prime candidates (Table 7). At this point an economic eval-
uation of each of these compounds was made in which not only
the costs of the basic building materials, but also the re-
action hazards and difficultures, were considered. Compound
No. 50 was found to be very difficult to synthesize and
involved a number of hazardous reaction steps. Compound
No. 72, though similar in structure to No. 73, was not only
high in component costs but involved more reaction steps
and was more difficult to make. Compound No. 24 was elimin-
ated from the prime candidate list on the basis of poor
activity against Anabaena as revealed in subsequent con-
firmatory tests. Also, the reactive steps for the production
of Compound No. 70 were found to be very difficult; es-
pecially the step involving the iodination of thiophene which
included the use of mercuric oxide or some other heavy metal.
Compound No. 23 was selected as the best candidate compound
remaining after the screening program for intensive invest-
with Compound No. 73, even though considerably
higher in cost, as the alternative choice.
On the basis of compound activity against Microcystis aerugin-
osa compounds No. 23 and 73 compare favorably with CuSOu«
i
5H20 and are much more active than equal concentrations of
Cutrine,a commercial chelated copper compound (Figures 5,6>&7)
The cost of treatment for control of Microaystis with Compound
No. 23 would be approximately 0.512 that of CuSOu«5H20, taking
into consideration the difference in activity and the cost
of solvents and emulsifiers necessary in the formulation.
The estimated cost of Compound No. 23 alone is 2.02 times
that of hydrated copper sulfate. On the same basis, Compound
No. 73 and Cutrine would be 3-01 and 15.1 times greater,
53
-------
TABLE 7
PRIME CANDIDATE COMPOUNDS SELECTED
FOR ECONOMIC EVALUATION
Compound
Number Name of Compound Descriminating Factors
23 2,5-Dichloro-3,4-dinitrothiophene use of emulsifiers
necessary in formulation
24 1,1-Dimethyltetradecylamine, insufficient activity
hydrochloride against Anabaena
50 Di-2-thienyliodoniumchloride very hazardous reaction
steps involved
70 2-Iodo-3,5-dinitrothiophene iodonation of thiophene
is difficult, requires
use of mercuric oxide
72 (m-Chlorophenyl)-2-thienylio- high cost of components
doniumchloride and more difficult than
No. 73 to make
73 (p-Chlorophenyl)-2-thienyl- high cost
iodoniumchloride
-------
Figure 5
ACTIVITY COMPARISON OF SELECTED COMPOUNDS
AT 0.8 PPM CONCENTRATION - 3 DAY TESTS
ui
ui
o
-------
COMPOUNDS NO. 23 & NO. 73 VERSUS MICROCYSTIS & ANABAENA
ui
100 r-
90 -
80
70
60
~ 50
40
30
20
10
(m)
(A)
• Comp. No. 23
• Comp. No. 73
j I
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 l.l 1.2 1.3
Concentration
1.4
1.5
1.6
-------
Figure 7
COMPOUND ACTIVITIES VERSUS ANABAENA FLOS AQUAE - 3 DAY TESTS
•Ar Compound No. 23
• Compound No. 73
CuSO*.5H20
OCutrine
1.5 2.0
Concentration (ppm)
3.0
4.0
-------
respectively, than copper sulfate for the same control
efficacy (see Table 8 ).
Compound Re-synthesis and Toxicology Tests
One hundred grains of Compound No. 23 were synthesized since
the existing supply was exhausted and larger quantities
were needed for the proposed field tests and toxicological
studies. The re-synthesis was facilitated by purchasing
2,5-dichlorothiophene and completing the synthesis by react-
ing with nitric acid in the presence of 30 percent fuming
sulfuric acid.
Sixty grams of the newly made compound were sent directly
to Dow's Chemical Biology Research Laboratories in Midland,
Michigan were Class I toxicology tests were conducted.
This included tests for primary skin irritation on rabbits,
acute oral lethality using rats and eye irritation tests
on rabbits.
The remaining 40 grams of Compound No. 23 were sent to this
laboratory where 30 grams were formulated into a 10 percent
w/v primary concentrate, using the pre-determined solvent
system and optimum emulsifier ratio. This was the concen-
trate which was diluted 1:100 with water to make a "use"
concentrate for the control of blue-green algae, the final
concentration at the control level being in the 0.4-0.8 ppm
range. The formulated concentrate was returned to Dow's
toxicology laboratory in Midland for similar tests on the
compound in its formulated state. The crystalline sample
submitted for the Class I toxicology tests was 99 percent
pure.
-------
TABLE 8
COST COMPARISON OP ALGAECIDAL COMPOUNDS VERSUS Microcystis aeruginosa
Concentration Ibs/acre ft. Comparative
Algaecide req. for control req. for control cost/acre ft. cost factor*
I - Test Compound No. 23 0.2 ppm 0.546 $ 0.415 0.512
(2,5-Dichloro-3,4-dintro-
thiophene), including form-
ulation agents
II - Test Compound No. 73 0.2 ppm 0.5^6 $ 2.19 3-01
(p-Chlorophenyl)-2-thienyl-
iodoniumchloride), including
formulation agents
III - Copper Sulfate 1.0 ppm 2.72 $ 0.81 1.00
(CuSOu»5HaO
IV - Cutrine 3-0 ppm 8.16 $12.25 15.1
*Compared to CuSOi, arbitrarily set at 1-00
-------
The toxicology report indicated that the acute oral lethal-
ity of the material was moderate and that there was little
liklihood of internal injuries resulting from acute ingestion
of amounts of the material one might encounter incidental to
industrial handling. It was emphasized, however, that
serious internal injuries could result from accidental or
deliberate ingestion of larger amounts.
Eye contact with the concentrated test material would likely
r^ult in moderate pain, severe conjunctival inflammation,
moderate iritus and severe corneal injury, with possible
impairment of vision if the eyes were not promptly and
thoroughly decontaminated. Prolonged skin contact with the
test material would likely result in slight redness and mod-
erate swelling. However, if skin contact was repeated or
contaminated clothing was worn for several days, slight
redness, severe swelling and a slight chemical burn could
result. The material may be absorbed through the skin in
acutely toxic amounts if contact is prolonged or repeated.
The toxicological report on the formulated sample was very
similar to that which resulted from studies of the concen-
trated compound with no significant differences in recommended
precautionary or safe handling procedures.
Shock Sensitivity and DTA Tests
One of the tasks to be completed during Phase II was the
determination of production or handling hazards, if any, of
the compounds selected as final candidates. Differential
thermal analyses (DTA) and shock sensitivity tests conducted
by two independent laboratories, both associated with Dow's
Reactive Chemicals Team, indicated the existence of no
hazards due to ordinary mechanical shock or small-scale pro-
60
-------
duction of test Compound No. 23. Out of ten drop-weight
tests for shock sensitivity no positives were reported. This
gave an E50>300 kg-cm, i.e., the energy required to yield
positive results from 50 percent of the tests when a 6 kg
mass is dropped from a height of 50 cm onto a small quan-
tity of encapsulated sample.
One of the differential thermal analyses (DTA) tests on
Compound No. 23 revealed endotherms at 88°C and 327°C but
the existance of no exotherms. However, a second test re-
vealed a fairly large exotherm starting at 275-290°C, serving
as a warning that a large quantity of the material might pro-
duce a fire or explosion hazard. Thus, it is recommended that
before multi-pound lots of the material are synthesized, larger
samples should be tested for heavy shock sensitivity using
blasting caps and tetryl boosters.
Overall, considering the results of the Class I toxicology
and Reactive Chemicals tests, it appears that the small-
scale production and handling of Compound No. 23 is safe,
if the prescribed precautions are properly observed-
After the selection of Compound No. 73 as a prime candidate
for further study, it was discovered that previous interest
in this compound for another use had resulted in the toxic-
ology and hazardous properties tests having already been
conducted. As indicated from the reports on file, this com-
pound also is considered to be relatively free of production
and personnel handling hazards.
Field Tests .
As the screening tests, economic evaluations and hazardous
properties tests progressed, and the list of potential candi-
date compounds became narrowed to a smaller number, field
61
-------
tests were undertaken on the prime candidates to determine
their behavior as algaecides under conditions more closely
simulating those which might be encountered in nature.
Of the four compounds which were tested under field con-
ditions only No. 23 (2,5-Dichloro-3,4-dinitrothiophene) and
No. 73 ((p-Chlorophenyl)-2-thienyliodoniumchloride) were
sufficiently active to justify their selection as final
candidates (Table 9 ). Compound No. 11 (3,^,4',5,6-Penta-
chloro-2,3f-methylenediphenol) had held much interest
earlier in the screening program due to its rapid action
on the algal cell walls, causing an almost immediate initia-
tion in loss of chlorophyll. This compound was later elim-
inated due to environmental questions regarding its phenyl
components and its poor activity at concentrations below
1.0 ppm. Compound No. 70 (2-iodo-3,5-dinitrothiophene)
exhibited 96 percent activity against Anabaena when screened
under laboratory conditions, but in the open atmosphere the
activity dropped to 82 percent, indicating probable photo-
degradation of the compound under those conditions.
The confirmed outdoor activity of Compound No. 23 showed
greater algaecidal properties than were indicated during
laboratory tests (Tables 4 and 9 )• The same was true
with Compound No. 73» except that due to the onset of cold
weather, attempted confirmatory tests were unsuccessful.
The relatively high activity at lower concentrations with
Compound No. 73 must be accepted with some question due to
the fact that control cultures under those conditions did
not show a healthy growth, although the growth pattern did
return to normal when samples were placed in warmer temper-
atures under laboratory conditions.
62
-------
TABLE 9
FIELD TESTS
TEST COMPOUNDS VERSUS Anabaena flos aquae
Compound
Number
cr>
U)
11
23
70
73
Name of Compound 1
3 , 4 , *» ' , 5 , 6-Pentachloro-2 , 2 ' -
methylenediphenol
2 , 5-Dichloro-3 , 4-dinitrothio-
phene
2-Iodo-3 , 5-dinitrothiophene
(p-Chlorophenyl)-2-thienyl-
Activity (%} at Various Cone, (ppm)
.6 1.0 0.8 0.6 0.4 0.3 0.2 0.1
82 87 54 31
100 100 100 38
100* 100* 100'
lodoniumchloride
*Colder weather test. Stock cultures before treatment were somewhat below par.
-------
Correlation Analysis - Activity as a Function of Structure
The basic philosophy underlying the initial effort of the
Phase II program to "Develop a Selective Algaecide" involved
the probability of there being a direct correlation between
known active algaecidal compounds and those having related
structures. With this in mind a computerized structure
search was conducted in which over 100,000 compounds were
screened to select analogs of the four surviving compounds
from Phase I.
Of the 1309 analogs selected (sec Table 3, page 36), 69
came through the rapid agar-plate screening tests as "posi-
tives". By comparing the results of the fine-screening
tests on these compounds with the related compound structures
it was observed that most of the compounds showing good
algaecidal properties were either substituted thiophenes
or combined iodonium-thiophenes. Of the 69 compounds
tested, 40 fitted into these two categories (Table 10).
Outside of the two mercurial compounds (Nos. 40 and
and one tin compound (No. 61) there were no compounds with
high algaecidal activity among any of the other groups. A
classification of the structures of the eleven best compounds,
together with the substituent components present in each case,
is given in Table 11 . In addition to the thiophene com-
ponent the most common elements associated with high algae-
cidal activity was iodine and chlorine, usually, but not
always in combination.
A comparison of the compound structures of the single and
double thiophenes, together with the combined iodohium
thiophenes, listed according to algaecidal ratings is shown
in Figure 8. Within this general group of compounds there
64
-------
TABLE 10
CORRELATION ANALYSIS OF PHASE II TEST COMPOUNDS
ACTIVITY AS A FUNCTION OF COMPOUND STRUCTURE
VJl
5-Membered Ring
Comb: 5^6-Membered Ring
Benzene Ring Types
Single
Com-
pound
No.
Class
41 (Hg) I
100
101
102
103
105
106
110
111
114
115
116
8
9
10
112
Key:
Class
Class
Class
III
III
III
II
II
II
II
III
III
III
III
II
III
III
III
Double Triple
Com-
pound
Com-
pound
No. Class No. Class
11
57
81
107
109
118
119
^tlvity against
- - 35
T f"'
III •
-100% at 1
C-95# at 1
-------
GT\
TABLE 11
ACTIVITY AS A FUNCTION OP STRUCTURE - CLASS I
STRUCTURES ATTACHMENTS ( ELEMENTAL COMPONENTS)
Compound No . kx' y
OP 9 O^ Lc-nc-acr s
41 x
70 *
23 *
94 x
96 ^
50
73
78
40
61
82
X
X.
X
x.
X. X
X X
X X
X
X X
X X
lodonium Br Cl N Hg Sn R-Group I
X. X
X. X.
X. X.
X
X. X.
X X
X. X
XX X
X X
X X
X X
-------
Hgure 8
PHASE I! TEST COMPOUNDS - STRUCTURE VS ACTIVITY
Single Thiophenes
Double Thiophenes
No. 23
OsN
\
Class I
N02
02N
Cl
No. 70 No. 94
CH2-CHNH2-COOH
\
No. 96
('' I \ / ^°2
s
02N
x.
NOa
\
•/\ /%,
s
No. 50
cr
Class II
None
None
No, 83
No. 89
No. 91
N02
02N
No. 92
1 'I 1 '
A. A A /
1 \ / I 0?.N \ /
H 02N \ / CH2CI
S' S
No. 93
No. 95
Class
N02
Br
H2-CH2CH=CH2
No. 97 No. 98
Br/\v /N02 02N\ / CN
o o
No. 99
Cl Cl
02N \
CONHz Cl
Cl Br
Br
No. 58
No. 86
-------
Figure 8 continued
PHASE II TEST COMPOUNDS - STRUCTURE VS ACTIVITY
Combination: Thiophene - Benzene ting - types
No. 78
Class I
r V
cr
oo
Class II
No. 52
cr
No. 54
QJU O,
I I
Cl
No. 72
cr
No. 55
I I
CFaCOz"
No. 74
Br
c
CH3
No. 76
CH3
c
No. 71
CHa
I I
cr
No. 85
I I
See Table 10 for key to toxicity classes.
-------
Figure 8 continued
Combination: Thiophene - benzene ring - types
Class
No. 51
Br'
No. 59
CFCOa"
No. 75
Jl jj
r^-/
cr
No. 87
CH3
No. 53
Cl
No. 60
CF3C02"
No. 77
BF7
" l
.x^
N03
See Table 10 for key to toxicity classes.
No. 56
0,
Br'
No. 62
Br
No. 84
Br
-------
does not seem to be any predominant activity-structure
relationship. However, it may be noted that chlorine was
always associated with Class I iodonium thiophenes, as well
as with the highly active single-thiophene, No. 23. Com-
paring Class I and III compounds Nos . 23 and 98, and also
No. 96 and 99, it would appear that the double N02 attach-
ments in the 3 and 4 positions have a definite positive
affect on algaecidal action. Also, the increased toxicity
of Class I Compound No. 70 over the Class III Compound No.
83 indicates an enhancing effect due to an additional N02
group.
The attachment of chlorine in the "meta" position of No. 72
shows a reduction in toxicity as compared to chlorine in
the "para" positions of Compound No. 73.
Compound Persistence Tests
A series of tests, planned to determine the toxicity per-
sistence of the two compounds selected as prime candidates,
revealed a clear pattern of compound decomposition in the
field within three to seven days, but very little breakdown
of the compound while stored under laboratory conditions
(Table 12). The breakdown under field conditions versus
laboratory conditions, as determined by toxicity reduction,
is depicted graphically in Figure 9 . It appears probable
that the decomposition is due to light action rather than
from atmospheric oxidation.
A similar breakdown pattern is shown with respect to Com-
pound No. 73 (Table 13).
70
-------
TABLE 12
PERSISTENCE STUDY - COMPOUND NO. 23 (2,5-Dichloro-3,4-dinitrothiophene)
at 1.0 PPM VS. Anabaena flos aquae
Values in Relative Intensity
Progressive Monitoring Time
Percent
0 - days
Test No.
I
I1
I" (control)
II
II'
II" (control)
III
III1
III" (control)
Components
1 H
1 H
1 H
2 H
2 H
2 H
3 H
3 -
3 -
- 4 + T
• 4 + T
h 4
h 4 + T""
I- 4 + T
I- 4
h 5 + T
i- 5 + T
H 5
Conditions
field
lab.
lab.
field
lab.
lab.
field
lab.
lab.
Initial
0
0
0
0
0
0
0
0
0
.07
.07
.07
.07
.07
.07
.07
.07
.07
Final*
0.02
0.02
0.15
0.02
0.02
0.39
0.02
0.02
0.33
3 days
Initial
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.16
Final*
0.57
0.46
0.54
1.34
0.03
1.50
0.70
0.02
0.36
7 days
Initial
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
Final*
0.35
0.01
0.32
0.34
0.01
0.30
0.21
0.00
0.76
Decomp-
osition
100
3
100
3
27
0
Key to Components: 1
2
3
4
5
T
World Health Organization (WHO) water
Well water
Deionized water
PW-4 media
Gorham's media
Test Compound at 1.0 ppm
*Final RI's were taken 3 days after initiation of each monitoring culture,
-------
Figure 9
DEGRADATION OF COMPOUND NO. 23
IN WELL WATER AND PW-4 MEDIUM
ro
co
CO
>
>>
X
o
U)
ea
-------
U)
II
- TABLE 13
PERSISTENCE STUDY - COMPOUND NO. 73 AT 1.0 ppm
USING Anabaena AS THE TEST SPECIES
(Values in Relative Cell Counts)
Progressive Monitoring Time
Test No.
I
I'
I"
Components
"WHO" water and
Compound No. 73
with PW4 media
Tl
control
u
-
Conditions
field
lab.
lab.
0 days
Initial Final*
1.09
1.09
0.0
1.13
3 days
Initial Final*
0.35
i
0.35
0.35
0
0
0
.0
.0
.40
7 days
Initial Final*
1.13
1.13
1.13
1.0
0.0
2-3
Percent
Decomp-
osition
45
0
-
well water with
PW4 media and
Compound No. 73
field
0.35 0.0
1.13 0.83
36
II'
II"
III
III1
III"
tl IT
control
Ion water and
Compound No. 73
w/Gorham's media
control
lab.
lab.
field
lab.
lab.
1
1
-
1
1
.09
.09
—
.09
.09
0.0
1.33
0.0
1.10
0.35
0.35
0.35
0.35
0.35
0
0
0
0
0
.0
.43
.0
.0
.51
1.13
1.13
1.13
1.13
1.13
0.0
2.3
3.11
0.0
3.5
0
-
89
0
-
*Final cell counts were taken 3 days after initiation of each monitoring culture .
Numbers indicate cell counts x 106, made after blending in a blender for 15 seconds.
-------
Biological Control Systems
Early in the Phase I study an organism, subsequently iden-
tified as Oahromonas ovalis, was discovered which was phago-
cytic to M-icrocyst-is aeruginosa. Since that time the effort
to develop an effective chemical algaecide has been paralleled
by an effort to determine the optimum activity parameters of
phagocytic organisms.
Attempted field studies on test compounds for the control of
Miorooystis were unsuccessful because of repeated infestation
by Oahromonas, which soon destroyed the algal test cultures.
A seven-day study was conducted to determine the mode of
Oahromonas infestation. Open-topped and muslin-covered
vessels containing Miarooyst-is cultures were set out at the
test site. All became infected within a few days. The only
ones in which Oahromonas did not appear were those covered
with Saran Wrap (Table 14). From this and other field exper-
iences it was concluded that Oohromonas was naturally ubi-
quitous in this locality and that because of its presence
it would not be possible to conduct successful field tests
using Miarooystis as the test species.
An attempt was made to find other organisms which might
also serve as biological agents to control the growth of
various species of blue-green algae.
Through the cooperation of Dr. M. J, Wynne, of the Department
of Botany, University of Texas at Austin, cultures of four
other species of Oahromonas were obtained. Three of these
(0. danica, 0. malhamensis and 0. minuta) were obtained from
the University of Indiana Algal Culture Collection. The
fourth strain (0. bastrop) was isolated from a pond at, Bastrop,
Texas and is being maintained as part of a teaching collection
at the University of Texas.
74
-------
TABLE 14
FIELD STUDY TO DETERMINE THE SUSCEPTIBILITY OP Microcystis TO OCHRMONAL
INFESTATIONS IN WHICH VARIOUS PROTECTIVE COVERINGS ARE UTILIZED AS BARRIERS
(A SEVEN DAY STUDY)
Initial
Microcystis
Initial
Ochromonas
Final
Microcystis
Final
Ochromonas
Cell Count X 10s Cell Count X 10" Cell Count X 106 Cell Count X 10"
Barrier Type
Muslin Cloth
Saran Wrap
Control (open top)
0.2
0.2
0.2
0
0
0
0.3
0.9
0.08
1.0
0
0.5
Effective
Ochromonal
Barrier
No
Yes
No
-------
Early tests conducted by Dr. Wynne at the University of Texas
indicated that three of the four Ochromonal species did
phagocytize Microcystis under laboratory conditions. 0.
minuta, a relatively small-sized species did not exhibit
this property. Under the laboratory conditions utilized in
that study, using a yeast-liver extract medium, 0. bastrop
was reported to be the most voracious feeder, with 0. mal-
hamensis showing the least activity of the three (see appen-
dix C). Studies conducted at this laboratory using Gorham's
medium produced basically the same results (Table 15).
Figures 10 and 11 show graphical comparisons of the various
activity rates as functions of time. 0. ovalis showed the
highest activity rate of any of the species tested, with
nearly complete depletion of Microcystis within the first
day after inoculation.
A test utilizing the four active species of Ochromonas in
light and dark conditions produced only slight differences
in phagocytic activity as a result of the absence or presence
of light (Table 16).
Phagocytic Activity Enhancement Systems
A number of experiments were conducted to determine possible
conditions which might positively effect the rate of growth
of Ochromonas or in some way cause an increase in their
ability to phagocytize Microcystis. A number of the test
compounds which were found to have low activities against
blue-green algae, proved to be stimulators to the growth
and phagocytic activity of Ochromonas. For example, in the
presence of Compound No. 14 the Ochromonas count was 20 x
10" cells/ml after two days incubation as compared to only 14
x 10" cells/ml in the flasks where no test compound was pre-
sent (Table 17 ). Also, in the same tests, at 0 + 3 days
76
-------
TABLE 15
PHAGOCYTIC ACTIVITY OP POUR SPECIES OP
Ochromonas ON Mlcrocystis aeruginosa
CELL COUNTS
Species
0. bastrop
0. danica
0. malhamensis
0. ovalis
Controls (avg)
0 +
(M)xlO6
0.84
0.84
0.84
0.84
days
(OCH)xlQ1*
1.0
1.0
1.0
.. 1.0
0 +
(M)xlO6
0.63
0.42
0.90
0.07
1 day
(OCH)xlO4
0.35
0.40
0.50
3.5
0 +
(M)xlO6
0.03
0.53
1.10
0.0
2 days
(OCTDxlO*
4.5
22.60
0.45
8.6
0 +
(M)xlO6
0.0
0.34
0.75
0.0
6 days
(OCHOxlO1*
12.2
2.0
11.2
8.2
Percent
Control
100
92
82
100
(M) only
0.84
1.18
1.13
4.30
-------
CO
o
u>
>^
o
Figure 10
PHAGOCYTIC ACTIVITY OF OCHROMONAS VERSUS MICROCYSTIS
Control
o-malhamensis
o-ovalis
Time (days)
-------
Figure 11
PHAGOCYTIC ACTIVITY OF OCHROMONAS VERSUS MICROCYSTIS
VD
o
o
S
o
Control
o-danica
if o-bastrop
Time (days)
-------
TABLE 16
A POUR DAY STUDY OF THE RATE OP PHAGOCYTIC ACTIVITY OF VARIOUS SPECIES
OF Ochromonas AGAINST Microcystis aeruginosa IN LIGHT AND DARK CONDITIONS
Initial
Microcystis
Cell Count X 106
. Ochromonas
Cell Count X 10"
Ochroraonas Species and
Lighting Conditions
Ochrononas malhemensis
a, in light
0 in dark
Oehromonas danlca
in light
in dark
Ochronpnas ovalis
In light
in dark
Ochpomonas bastrop
in light
in dark
Control - in light"
Control - in dark
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Microcystis
Cell Count X
0.03
0
0.58
0.56
0
0
0.01
0
1.65
1.02
Final
Ochrononas
10s Cell Count X 10"
3.0
3.0
2.0
1.6
3.2
2.6
3.0
2.6
—
Activity
%^
Control
100
100
65
U6
100
100
100
100
-------
TABLE 1?
TEST COMPOUND NO. 14 EFFECT ON Ochromonas ACTIVITY
Conditions
Components Added
Average Cell Counts
0 - Days
~~7M5 (Och)
X 106 X 104
0+1 Day 0+2 Days 0+3 Days
(M) (Och)
X 10s X 104 X 10s X 104 X 106 X 104
Test Chem. + (Och) 5.5 0.1 0.8 3.6 0.6 20 0.0 10
Test Chem. only
4.5 o.o 0.7 o.o 1.9 o.o 3.3 o.o
(Och) only
4.0 0.1 0.8 1.2 1.2 14.0 0.8 9-5
No Test Chem. or
(Och)
4.5 o.o 4.o o.o 4.6 o.o 5.2 o.o
(M) = Microcystis aeruginosa
(Och) = Ochromonas ovalis
-------
the Miorooystis culture was completely depleted when the
test compound was present, but 0.8 x 106 cells/ml were left
where no test chemical was used. A similar pattern is seen
with respect to Compound No. 6 (Table 18), except that the
growth of Oehromonas in the cultures with no test chemical
surpassed the others after the first 24 hours. This rela-
tionship is more easily seen in the graphical representation
shown in Figure 12. Also, test Compound No. 11 at 0.1 ppm
indicated some improvement to the system for control of
Miarooystis (Figure 13).
A comparative study of the phagocytic activity of the four
most active species of Ochromonas, with respect to three
selected test compounds, was made. The rate of Mierocystis
depletion resulting from the activity of 0. bastrop^ 0. danica
and 0, malhamensis- was greater in the presence of both com-
pounds, No. 11? and 119 at 0.2 ppm (Tables 19 and 20)•
These compounds, however, did not show any significant effect
on the growth or phagocytic activity of 0. oval-is. Compound
No. 114 produced no improvement in activity of any of the
four Ochromonal species tested (Table 21 ).
Ochromonal Microstructure and Mode-of-Action Study
Healthy dense cultures of 0, danica were prepared by Dr.
Wynne and his co-worker Dr. Gary Cole, University of Texas,
and examined by means of transmission electron microscopy
to determine the organelle arrangement within this type of
cell. In normal cells of Ochromonas danica a single,
large vacuole occurs in the posterior end of the cell
(Figure 14). The arrangement of the organelles is evident,
with the nucleus situated at the anterior end of the cell
and usually two fairly large chloroplasts on opposite sides.
The density of the vesicles at the anterior end are note-
82
-------
TABLE 18
Components Added
ENHANCEMENT OF OCHROMONAS ACTTIVTY
BY TEST COMPOUND NO. 6
Cell Density, cells/ml.
0 - Days
(M)(OchT
X IO6 X 104
0+1 Day
x io6
(Och)
X IO4
0+2 Days
(MT
X 10'
(OchT
X IO4
Test Chem. + (Och)
2.4
1.4
0.75
15
0.0
18
oo
(JO
Test Chem. only
(Och) only
2.4
2.4
0.0
1.4
1.4
0.0
12.7
2.8
0.0
0.0
No Test Chem. or
(Och)
2.4
0.0
0.0
4.1
0.0
(M) = Microcystls aeruginosa
(Och) = Ochromonas ovalis
-------
Figure 12
INFLUENCE OF COMPOUND NO. 6 AT 1.0 PPM ON THE
PHOGOCYTIC ACTIVITY AT OCHROMONAS OVALIS ON MICROCYSTIS AERUGINOSA
40
• "• (m) with (och) + test them.
O (m) + (och) - control No. 1
if (m) only - control No. 2
oo
X
J=
o
o
(och) only control No. 3
Time (days)
-------
Figure 13
INFLUENCE OF COMPOUND NO. 11 AT 0.1 PPM ON THE
PHAGOCYTIC ACTIVITY OF OCHROMONAS OVALIS ONMICROCYTIS AERUGINOSA
22 -
20 -
18 -
(m)with (och) + test compound
-—. (m) + (och) - control No. 1
—- (m) only - control No. 2
16t-
oo
VJl
14
12
s, 10
Elapsed Time (days)
-------
TABLE 19
INFLUENCE OF TEST COMPOUND NO. 11? AT 0.2 PPM ON THE
PHAGOCYTIC ACTIVITY OF FOUR SPECIES OF Ochromonas
Species
0. bastrop+(M)+(T)
Control*
0. danica+(M)+(T)
Control*
2? 0. malhamensis-*-
(MH(T)
Control*
0. ovalis+(M)+(T)
Control*
(M) only -
Control No. 1
(M) only -
Control No. 2
0 -
(M)xlO6 (
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
C E
days
OCH)xlO"
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
LL COUNTS
0 +
(M)xlO6
0.47
0.63
0.73
0.42
0.98
0.90
0.07
0.07
1.20
1.16
1 day
(OCH)xlO"
1.80
0.35
0.65
0.40
0.30
0.50
3.0
3.5
0 +
(M)xlO5
0.0
0.03
0.19
0.53
0.95
1.10
0.0
0.0
1.20
1.06
2 days
(OCH)xlO*
7.3
4.5
1.9
2.60
0.70
0.45
9.1
8.6
...
Percent
Control
100
98
84
54
16
<10
100
100
...
Key: (M) = Microcystis aeruginosa
(OCH) = ^Ochromonas sp .
(T) = Test compound
*Same algal components, but with no test compound
-------
TABLE 20
INFLUENCE OF TEST COMPOUND NO. 119 AT 0.2 PPM ON THE
PHAGOCYTIC ACTIVITY OF FOUR SPECIES OF Ochromonas
Species
0. bastrop+(M)+(T)
Control*
0. danica+(M)+(T)
Control*
0. malhamensis+
(M)+(T)
Control*
0. ovalis+(M)+(T)
Control*
(M) only -
Control No. 1
(M) only -
Control No. 2
0
(M)xlO
0.84
0.84
0.84
0.84^
0.84
0.84
0.84
0.84
0.84
0.84
C E
- days
0 (OCH)xlOH
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
LL COUNTS
0 +
(M)xlO6
0.48
0.63
0.61
0.42
0.74
0.90
0.04
0.07
1.20
1.16
1 day
(OCHjxlO"
2.3
0.35
0.35
0.40
0.20
0.50
4.1
3.5
0 +
(M)xlOB
0.0
0.03
0.22
0.53
0.76
1.10
0.0
0.0
1.20
1.06
2 days
(OCH)xlO"
7.0
4.5
0.80
2.60
0.80
0.45
6.0
8.6
...
Percent
Control
100
98
82
54
33
<10
100
100
___
Key: (M) = Microcystis aeruginosa
(OCH) = Ochromonas sp.
(T) = Test compound
*Same algal components, but with no test compound
-------
TABLE 21
INFLUENCE OF TEST COMPOUND NO. 114 AT 0.2 PPM ON THE
PHAGOCYTIC ACTIVITY OF FOUR SPECIES OF Ochromonas
Species
0. bastrop+(M)+(T)
Control*
O. danica+(M)+(T)
Control*
0. malhamensis+
(M)+(T)
Control*
0. ovalis+(M)+(T)
Control*
(M) only -
Control No.
(M) only -
Control No.
Key: (M) =
(OCH) =
1
2
0
(M)xlO6
0.84
0.84
0.84
0.84
0.84'
0.84
0.84
0.84
0.84
0.84
C E
- days
(OCH)xlO*
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
L L
(M)
1.
0.
1.
0.
0.
0.
0.
0.
1.
1.
C 0 U N
0 +
xlO6
08
63
07
42
88
90
91
07
20
16
1 day
(OCH)
0.
0.
0.
0.
0.
0.
0.
3.
—
T S
xlO*
25
35
20
40
20
50
25
5
-
CM)
1.
0.
0.
0.
1.
1.
1.
0.
1.
1.
0 +
xlO6
08
30
97
53
13
10
05
0
20
06
2 days
(OCH)xlO"
<0. 1
4.5
<0 . 1
2.6
<0 . 1
0.45
0.10
8.6
Percent
Control
74
54
<10
<10
<10
100
Mlcrocystis aeruglnosa
Ochromonas sp .
(T) = Test compound
*Same algal components, but with no test compound
-------
Figure l^l
Ochromonas danica - Organelle Arrangement
^Sgmmf& • ffSfmst
.
89
-------
worthy, as are the coiled or circular striated cylinders
located between the Golgi bodies and the nucleus, also near
the anterior end. These striated cylinders are found to
often be associated with the contractile vacuole (Figure
15)- The mitochondria surrounding the chloroplasts have
distinctly dialated cristae (Figure 16). In this same
figure the parallel-arranged bundles of microtubules around
the outer periphery of the cell are evident. These cyto-
skeletal structures are thought to serve by affording
rigidity and shape to the "naked" cells. A Miarocystis
cell is seen in close proximity at the lower right-hand
corner of. Figure 16.
In Figure 17 is shown an engulfed Microoystis cell within
0. danioa. The food vacuole seems to be distinct, at least
initially from the normally present vacuole. We could ex-
pect it to be probable for these two vacuoles to be able
to fuse. If this should be the case the prey may be cap-
tured in individual food vacuoles which secondarily merge
into a common reservoir. It appears, from this photomicro-
graph, that the outer envelope of the ingested Miorooystis
seems to have been enzymatically disintegrated and that the
discrete cell wall layer appears to be in the early stages
of disintegration at localized regions.
From optical microscopic examination of combined Ochromonas-
Miorocyst-is cultures it appears that Micvooystis cells are
phagocytized by Oahromonas only when fortuitous contact is
usually made, rather than by purposeful seeking. When con-
tact is established, a definite adhesion to the gelatinous
envelope seems to occur and engulfment is accomplished at,
that point. It is not uncommon to observe several Microoystis
cells engulfed within a single Ochromonas cell at a time.
90
-------
Figure 15
Striated Cylinders Associated with Contractile
Vacuoles in 0. danica
^"^•iV^*^?'"/^ f* '£•'
j* ^m
PP^w
91
-------
Figure 16
Ochromonas danica - Showing Mitochondria,
with Dialated Cristae
92
-------
Figure 17
Transmission Electron Micrograph of Oohfomonas do.nica
Showing Ingested Miorooystis aeruginosa
, f-j^. .<».• -;- .. ..>
•
fp
m
~^£v::,:
^^1*^. '!• '^^^¥$»
i.\
$SV
:'/--*>
£k?&
•M4
^rl
-• -'»;?
-:V
':
- '-' * f
-..,!
-••'.;•-";"- ^'
--••.•<
••^v/'-" .,
93
-------
SECTION VI
ACKNOWLEDGEMENTS
The work done in connection with this project was performed
by Dr. B. L. Prows, principal investigator, and C. P. Ward.
W. F. Mcllhenny was the project director.
Drs. Michael J. Wynne and Gary Cole of The Department of
Botany- University of Texas at Austin, served as consultants
and aiding investigators on the biological control aspect
of the project.
We are indebted to Paul Ludwig and personnel at Dow's
Ag-Organics Department for use of the field site and con-
struction of the physical facilities utilized for the small-
scale field tests.
The support of this project by the Water Quality Office,
Environmental Protection Agency, together with the interest,
advice and review of the final manuscription by T. E. Maloney,
the Project Officer, are acknolwedge with sincere appreciation.
-------
SECTION VII
REFERENCES
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Techniques", Proceedings of the Eutrophication Bio-
stimulation Assessment1workshop, U. S. Department of
the Interior, Federal Water Pollution Control
Administration. (1969)
Bartsch, A. P.: "Proceedings of a Symposium Jointly
Sponsored by University of Washington and Federal
Water Pollution Control Adminstration, U. S. Depart-
i
ment of the Interior. (1967)
Bartsch, A. P.: "Biological Aspects of Stream Pollution",
Sewage Works Journal, 20:292-302. (1948)
Bartsch, A. F. and W. M. Ingram: "Stream Life and the
Pollution Environment", Public Works, 90:104-114.
(1959)
Bueltman, C. G. (Chairman): "Provisional Algal Assay
Procedure", Joint Industry-Government Task Force on
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Cortell, J. M.: "The Role of Herbicides in the Preserva-
tion of our Urban and Industrial Water Resources",
Weeds, Trees and Turf, June: 12-28. (1970)
Davis, V. E.: "Managing Farm Fish Ponds", Farmers Bulletin
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95
-------
Faust3 S. D.: "Pate of Organic Pesticides in the Aquatic
Environment", American Chemical Society, Washington,
DC. (1972)
Fogg, G. E.: "Algal Cultures and Phytoplankton Ecology",
The University of Wisconsin Press, Milwaukee, Wiscon-
sin. (1965)
Fitzgerald, G. P-: "Algaecides", University of Wisconsin,
Madison, Wisconsin. (1971)
Gorham, R. P.: "Toxic Algae as a Public Health Hazard",
Journal of American Water Works Associations, 56 (11):
1481-1488. (1964)
Hasler, A. D.: "Antibiotic Aspects of Copper Treatment of
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(1947)
Holm-Hansen, 0. et_ al: "Fluorometric Determination of
Chlorophyll", Institute of Marine Resources, Scripps
Institute of Oceanography. (1966)
Hughes, E. 0., P. R. Gorham and A. Zehnder: "Toxicity of a
Unialgal Culture of M-icvoeyst-Ls aerguinosa", Canadian
Journal of Microbiology, 4:225-236. (1958)
Hutchinson, G. E.: "A Treatise on Limnology", Vols. I & II,
John Wiley & Sons, Inc., New York, NY. (1967)
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. Fish
Soc., 82:156-165. (1953)
96
-------
Keeney, D. R.: "The Pate of Nitrogen in the Aquatic
Ecosystems", University of Wisconsin, Madison, Wis-
consin. (1972)
Keup, L. E., Ingram, W. M. and Mackenthun, K. M.: "Biology
of Water Pollution", U. S. Department of the Interior
Federal Water Pollution Control Administration, June.
(1968)
Kuentzel, L. E.: "Bacteria, Carbon Dioxide and Algal Blooms",
J. W. P. C. P., 41:10, 1737-1747. (1969)
Kunkel, D. H.: "Algae Control in Ponds", Farm Pond Harvest,
Summer. (1969)
Lewin, R. A.: "Physiology and Biochemistry of Algae",
Academic Press, New York, NY (1962)
MacKenthun, K. M.: "The Practice of Water Pollution Bio-
logy "> U. S. Department of the Interior, Federal
Water Pollution Control Administration. (1969)
MacKenthun, K. M. and W. M. Ingrain: "Limnological Aspects
of Recreational Lakes", U. S. Department of Health,
Education and Welfare. (1964)
MacKenthun, K. M. and W. M. Ingram: "Biological Associated
Problems in Freshwater Environments—Their Identifi-
cation, Investigation and Control", U. S. Department
of the Interior, Federal Water Pollution Control
Administration. (1967)
MacKenthun, K. M. and C. D. McNabb: "Stabilization Pond
Studies in Wisconsin", J.W.P.C.F., 33 (12):123^-1251.
(1961)
97
-------
Meyer, J. H.: "Aquatic Herbicides and Algaecides", Noyes
.Data Corporation. (1971)
Middlebrooks, E. J., T. E. Maloney, E. F. Powers and L. M.
Knack: "Proceedings of the Eutrophication-Biostimu-
lation Assessment Workshop", Sanitary Engineering Re-
search Lab., University of California and U. S. Dept.
of the Interior, Federal Water Pollution Control
Administration. (1969)
Moyle, J. B.: "The Use of Copper Sulphate for Algae Control
and its Biological Implications", American Assoc. for
the Advancement of Science, Washington, DC. (19^9)
Nichols, M. S., T. Henkel and D. McNall: "Copper in Lake
Muds from Lakes of the Madison Area", Trans. Wis.
Acad. Sci., Arts & Lett., 38:333-350. (1946)
Otto, N. E. and T. R. Bartley: "Aquatic Weed Control Studies",
U. S. Department of the Interior, Bureau of Reclamation,
Research Report No. 2. (1966)
Otto, N. E. and T. R. Bartley: "Aquatic Pests on Irrigation
• Systems", U. S. Department of the Interior, Washington,
DC. (1965)
Outlook: "Urban Runoff adds to Water Pollution", Vol. 3,
No. 6. (1969)
Palmer, C. M. : "Algae in Water Supplies", U. S. Department.
'of Health, Education and Welfare, Washington, DC.' (1962)
Prescott, G. W.: "How to Know the Fresh Water Algae,
Wm. C. Brown Publishing Company. (1970)
98
-------
Prows, B. L. : "Development of a Selective Algaecide to
Control Nuisance Algal Growth", U. S. Department of
the Interior, Washington, DC. (1971)
Reazin, G. H., Jr.: "On the Dark Metabolism of Golden Brown
Alga, Oahromonas malhamensis, Am. Jour. Bot,, 41:9,
771-777- (195*0
Reazin, G. H., Jr.: "The Metabolism of Glucose by the Alga
Oohromonas 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)
Technomic Research Associates: "Agricultural Chemicals
Planning Program: Aquatic Herbicides", Technomic
Research Associates, Chicago, IL. (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)
Vance, B. D. and Smith, D. L.: "Effects of Five Herbicides
on Three Green Algae", Texas Journal Science, 20:4,
330-335- (1969). '
Weiss, C. M.: "The Relative Significance of Phosphorus and
Nitrogen as Algal Nutrients", University of North
Carolina, Chapel Hill, NC. (1970)
Yentsch, C. S. and D. W. Menzel: "A Method for Determina-
tion of Phytoplankton Chlorophyll and Phaeophytin by
Fluoroescence", Deep Sea Research, 10:221-231. (1963)
99
-------
Zajic, J. E. : "Properties and Products of Algae", Proceedings
of the Symposium on the Culture of Algae sponsored by
the American Chemical Society, Plenum Press, NY. (1970)
100
-------
SECTION VIII.
APPENDIX A.
Synergistic Activity Tests
101
-------
o
ro
TABLE 22 _ LABORATORY SCREENING TESTS
Compounds No. 11 % 72 (equal quantities of f.-arh)
.3,4,4',5',6-Pentachloro-2,2' -methylenediphenol and (m-Chlorophenyl)-2-thienyliodoniumchloride
Microcystis aeruginosa Anabaena flos-aquae
Initial
Cell Count
(Cells/mlxlO6)
0.8 ppm
Flask 1
Flask 2
0 . 4 ppm
Flask 1
Flask 2
0 . 2 ppm
Flask 1
Flask 2
0.1 ppm
Flask 1
Flask 2
0.05 ppm
Flask 1
Flask 2
Control
Control 1
Control 2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Final
Cell Count
(Cells/mlxlO6)
0.0
0.0
0.0
0.0
0.26
0.40
0.91*
0.86
li39
Compound
Activity
(% Control) Initial R.I. Final R.I.
inn °-27 °-12
100 0.27 0.12
100 °'27 °'?5
0.27 0.40
YR 0.27 0.47
IJ 0.27 0.47
^ 0.27 0.32
JJ 0.27 0.32
0.27 0.38
0.27 0.35
Compound
Activity
(% Control)
68
0
0
0
NOTE: R.I. = Relative Intensity
-------
TABLE 23 - LABORATORY SCREENING TESTS
Compounds- No. 70 & 73 (equal quantities of each)
2-Iodo-3,5- Dinitrothiophene and (p- Chlorophenyl) -2-thienyliodoniumchloride
Microcystis aeruginosa
Anabaena flos-aquae
o
U)
Initial Final Compound Compound
Cell Count Cell Count Activity Activity
(Cells/mlxlO6) (Cells/mlxlO6) (% Control) Initial R.I. Final R.I. (% Control)
0.8 ppm
Flask 1
Flask 2
0.4 ppm
Flask 1
Flask 2
0.2 ppm
Flask 1
Flask 2
0.1 ppm
Flask 1
Flask 2
0.05 ppm
Flask 1
Flask 2
Control
Control 1
Control 2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2?
0.36
1.41
1.39
100
100
100
loo
100
An
.80
°'27
°'27
0. 27
0.27
0.28
0.38
0.40
0.39
0.42
0.41
0.35
0.36
0.38
0.35
10
0
0
0
NOTE: R.I. = Relative Intensity
-------
TABLE 24
COMPARATIVE ALGAECIDAL PROPERTIES OP VARIOUS FORMULATIONS
OP TWO BATCHES OF SYNTHESIZED COMPOUND NO. 23
AGAINST Anabaena flos aquae (THREE-DAY TEST)
Relative Intensity Relative Intensity %
Initial Reading Final Reading Control
Components and ppm
23-A-1.6 ppm1 0.24 0.12 100
23-A-0.8 ppm1 0.24 0.12 100
23-A-0.4 ppm1 0.24 0.25 60
23-X(old)-1.6 ppm2 0.24 0.10 100
23-X(old)-0.8 ppm2 0.24 0.15 89
23-X(old)-0.4 ppm2 0.24 0.21 71
23-X(10%-old)-1.6 ppm3 0.24 0.10 100
23-X(10#-old)-0.8 ppm3 0.24 0.16 86
23-X(10$-old)-0.4 ppm3 0.24 0.22 69
23-X(105£-new)-1.6 ppm" 0.24 0.11 100
23-X(10#-new)-0.8 ppm" 0.24 0.17 83
23-X(10%-new)-0.4 ppm" 0.24 0.23 66
CuSO,,*- 1.6 ppm 0.24 0.033 100
CuSO,,*- 0.8 ppm 0.24 0.058 100
CuSOu*- 0.4 ppm 0.24 0.22 69
Control (no test chemical) 0.24 0.35
formulated with acetone and newly synthesized compound
2Formulated with an old Atlox-xylene-perchlor " use" concentrate
3Formulated with a previously synthesized compound (10$ by weight
in primary concentrate)
"Formulated with a newly synthesized compound (10% by weight in
primary concentrate)
*CuSO«,-5H20
104
-------
SECTION VIII.
APPENDIX B.
Phagocytic Activity of Oohromonas Against Miorocystis aeruqinosa
105
-------
TABLE 25
A FOUR DAY STUDY OP THE RATE OF PHAGOCYTIC ACTIVITY OF VARIOUS
SPECIES OF Ochromonas ON Microcystis aeruginosa
Initial
Microcystis
Cell CountXIO6
Ochromonas
Ochromonas
Ochromonas
Ochromonas
Ochromonas
Species
danica
malhamensis
mi nut a
sp.
1.
1.
1.
1.
5
5
5
5
Final
Activity
Ochromonas Microcystis Ochromonas %
Cell CountXKT Cell CountXIO6 Cell CountXlO"
1
8
7
8
.30 o
.0 0 6.1)
.0 6.1 3.7
.0 0 3-1
Control
100
100
0
100
(Freeport)
Control
1.5
5.9
*An unknown species found in the Freeport area
-------
TABLE 26
EFFECT OF TEST COMPOUND ON OGHROMONAS ACTIVITY
0+5 Days* 0+6 Days 0+7 Days 0+8 Days Q + 9 Days 0+12 Days
(M) (Och) iMl(Och) (M) fO~ch)~(M} (Och) (M) (Och) iMl fOch")
Components Added x 10s x 104 x 10s x 104 x 10s x 104x 106 x 104x 10s x 104 x 106 x 104
Test chemical + 1.4 0 0.25 0.2 0.09 1.0 0.1 1=0 0.05 5.0 0 2.2
Ochromonas
Test chemical only 1.7 0 1.37 0 1.6 0 1.51 0 1.0 0 0.32 0
Ochromonas only 1.6 0 1.23 1.0 0.12 3.5 0.07 5-1 0.02 10.0 0 9.0
Control (Nothing 0.7 0 0.5 0 0.32 0 0.13 0 0.18 0 0.5 0
added)
*Cultures of Microcystis were established and left to adapt under ambient conditions for
first 5 days and then appropriate test flasks were inoculated with Ochromonas and others
with test compound at 0.1 ppm.
-------
TABLE 27 - OCHROMONAS ACTIVITY ENHANCEMENT TEST AT 0.1 PPM
Components Present
Test Chemical + (OCH)
Test Chemical
H (M) + (OCH)
o
oo
(M) Only
11 - 5,4,4' , 5,6-Pentachloro-2,2 ' -methylenediphenol
0 - Days
(M) (OCH)
X106 X104
1.05 0.55
1.05
1.05 0.55
1.05
0+1 Day
(M) (OCH)
X106 X104
0.08 5.5
1.4?
-17 5-3
1.45
0+3 Days
(MJ (OCH)
X10S X104
o.o 5.8
2.02
o.o 6.15
2.06
SIF1>3 = 1.2
-------
Components Added
TABLE 28
EFFECT OF TEST COMPOUND No. 21 ON Ochromonas ACTIVITY
0 - Days 0+1 Day 0+2 Days 0+3 Days 0+4 Days 0+7 Days
lM) (Och) lM) (Och) lM) (Och) (M) (Och) ~W)(Och) (M) (Och)
x 106 x 10" x 106 x 10* x 106 x 10* x 106 x 10* x 106 x 10 * x 10* x 10*
Test chemical +
Ochromonas
Test chemical only
1.4 0 0.25 0.2 0.09 1.0 0.1 1.0 0.05 5-0 0 2.2
1.7 0 1.37 0 1.6 0 1.51 0 1.0 0 0.32 0
Ochromonas only
1.6 0 1.23 1.0 0.12 3-5 0.07 5-1 0.02 10.0 0 9-0
Control (nothing added) 0.7 0 0.5 0 0.32 0 0.13 0 0.18 0 0-5 0
-------
SECTION VIII.
APPENDIX C.
Studies of Microcystis-Ochromonas Interactions
by
Dr. M. J. Wynne
Dr. G. Cole
University of Texas
Department of Botany
Austin, Texas
110
-------
Studies of Miorooystis-Oahromonas Interactions
One phase of this project was to survey available cultures
of OahTomonas for phagocytic capabilities of other species
besides the original Dow isolate, 0. ovalis. Three species
were obtained from the Indiana Culture Collection, namely,
0. daniaa., 0. malhamensis^ and 0. minuta. Also, a strain
isolated from a pond at Bastrop, Texas, (0. bastrop} was
being maintained in the teaching collection of The Univer-
sity of Texas. This last mentioned Ochromonas comes closest
to 0. fragilisj using Huber-Pestalozzi's Key (1941), but
differs from that species in having stigmata (eyespots).
The three Indiana University species have been cultured in
Oohromonas medium: 0. daniaa yields excellent growth rapidly,
0. malhamensis produces moderate growth, 0. minuta grows
very slowly and does not yield large quantities under the
conditions utilized. The Oahromonas from Bastrop has been
grown in 1/3 BBM-soil water, which is not a highly enriched
medium in contrasts to the above Oohromonas medium.
Of these strains that have been examined besides the 0.
ovalis, only 0. minuta (a relatively small-sized species) does
not seem to phagocytize the Miovocystis; no cases of ingested
blue-green algal cells have been observed. 0. bastrop proved
to be the most voracious "feeder", often with several Mioro-
systie cells ingested per Ochromonas cell. The cells are
larger than those of 0. ovalis. 0. daniaa is also an
efficient feeder, but typically only one blue-green algal
cell will be ingested at a time. 0. malhamensis also ingests
blue-green algae but seems to be the least effective of the
three.
Ill
-------
Some difficulties have been encountered in obtaining good
growth of the Mioroaystis. The initial results were poor
to negligible growth using CG11 and BG10, two media used
successively for other blue-green algae. The reason for
this lack of good growth of M. aeTugino.sa is uncertain.
But lately we have used Gorham (IX) medium, as has been done
by Dow, and we are finally achieving better growth. Bub-
bling in a 3 percent C02-air mixture did not enhance growth.
'j
We have hopes of carrying out ultrastructural studies in-
volving the electron microscope, and preliminary results
were encouraging in regard to the fixation-images of both
the Miovooystis and the Oohromonas (in this case, 0. danioa).
We have delayed further fixations until we have achieved
denser growth of Miaroaystis, since low concentrations of the
blue-green alga would mean that few Oahromonas cells will
have engulfed blue-greens.
A survey of the literature on the mechanism of phagocytosis
(or endocytosis) is underway, this involving ultrastructural
investigations. There has been extensive work recently but
largely confined to the ciliates (Tetrahymena and Parameaium)
and amoebae. We have come across nothing specifically on
phagocytosis in Ochromonas , although some workers (as Bouck
and Gibbs) have examined the cellular details of Oahromonas .
So this area seems to be untouched and thus promising. Many
ciliates have cytostomes in which a series of food vacuoles
will be continually formed. The method in Ochromonas is
different. Phase-contrast light microscopy demonstrates a
very rapid engulfment, i.e., almost immediate engulfment when
/
contact is fortuitously made. These species of Oahvomonas do
have large vacuoles and the blue-green algae seem to be de-
posited in these large, already present vacuoles rather than.
in special food vacuoles.
112
-------
0. bastrop would at present seem to offer the best organism
of the group studies on further analyses of interactions with
Miaroaystis.
Some additional insight into the relationship between the
phagocytic Ochromonas (utilizing 0. daniea in this study)
and the engulfed Microcystis aeruginosa has been gained with
transmission electron microscopy (Hitachi HU-ll-E). A
better understanding of the organelle arrangements within
these two types of cells has also been achieved, and it seems
worth-while to discuss certain of these aspects in this adden-
dum to the original report submitted June 1, 1972. It might
also be pointed out that attempts have also been made at
uncovering three-dimensional details of these two cells by
means of the "freeze-etch" device accompanied by transmission
electron microscopy, but preliminary results are not yet
considered to be of sufficient quality to include in this
report. Yet that technique should provide us with a different
perspective of the two cells, once the problems of fixation
and preparation have been solved.
The following is a discussion of observations made on the
accompanying electron micrographs (Figures 18-27).
Figures 18 and 19
In normal cells of Oahromonas daniea, i.e., grown in axenic
culture, a single large vacuole occurs in the posterior
region of the cell. The arrangement of organelles is evident,
with the nucleus situated at the anterior-end of the cell
and with usually two chloroplasts located on opposite sides
of the cell. Note the density of vesicles in the anterior
region of the cell, with their probable origin from a Golgi
body .occupying a conspicuous position at this pole. We are
particularly interested in the coiled or circular, striated -
113
-------
Figure 18
Transmission Electron Micrograph of 0. danica
Showing Large Vacuole and Organelle Arrangement
-------
Figure 19
Ochromonas danica - Mitochondria, v.'ith
Dialated Cristae and Bundles of I'icrotubules
= -
115
-------
cylinders (Figure 18), which are at times associated with
contractile vacuoles (Figure 22). The mitochondria (Figure
19) have distinctively dilated cristae, which at first were
regarded as artifacts of fixation; however, Gibbs1 study
of mitosis in the same species (J. Phycol. 8(3), 1972) also
portrayed these swollen cristae. This would suggest they are
normal. In Figure 19 the parallel-arranged bundles of micro-
tubules are evident. These cytoskeletal elements function
to afford a rigidity to the shape of these naked cells. We
have consistently observed these microtubules, which are
particularly noticeable when they become concentrated at
the two poles of the cell. We have not encountered any men-
tion in the literature of their occurrence in these naked
chrysomonads.
Figures 20 and 21
Normal cells of Miorooyst-is aeruginosa grown in axenic cul-
ture. The fibrous nature of the nucopeptide envelope sur-
rounding the thin multi-layered wall proper is faint though
detectable. We often note osmiophilic (dark-staining) re-
gions in these cells, which is an aid in their quick recog-
nition, especially when they are later engulfed by the
Oohromonas. The photosynthetic lamellae are loosely arranged
which is characteristic of prokaryotic cells. A region of
different density in the central portion may be chromatin.
Figure 22
The size relationship is seen in this figure. It is critical
to catch the very earliest stages of contact and food vabuole
formation, and this seems at the moment to be a fortuitous
event of fixation. The process is rapid but should be possible
to visualize also at the EM-level. The two evagination
"blebs" on the opposite side of the cell are of uncertain
116
-------
Figure 20
Microeystis aeruginosa Near Dividing Stage
Showing Mucopeptide Envelope
117
-------
Figure 21
Microcystis aeruginosa - Depicting Photosynthetic Lamellae
118
-------
Figure 22
Ochromonas daniaa - Transmission Electron Micrograph
Showing Striated Cylinders and
Size Relationship to M. aeruginosa
119
-------
function. The narrow striated cylinders seem to be feeding
into the contractile vacuole near the nucleus of the Ochromon-
as.
Figure 23
Detailed view of a contractile vacuole of 0. danioa. In
addition to numerous fusing cylinders, a large number of cir-
cular vesicles are in the immediate vicinity of the contrac-
tile vacuole.
Figure 24
Engulfed Mioroeystis within 0. danioa. Our observations in-
dicate that the food vacuole is distinct, at least initially,
from the normal present vacuole. This duality is also seen
in Figure 27, in which the food vacuole contains bacteria
from a contaminated culture. We would expect it probable for
these vacuoles to be able to fuse. Perhaps the prey is cap-
tured in individual food vacuoles, which secondarily merge
into a common reservoir. It is uncertain whether the mater-
ial in the second vacuole of Figure 24 represents digested
products of Miarooystis or is a part of the Oahromonas cyto-
plasm. It might be mentioned that the envelope of the ingest-
ed Microeystis is no longer noticeable, and the discrete wall
layer appears to be in the early stages of disintegration at
localized regions,
Figure 25
Detail of Ochromonas cytoplasm. The proliferating Golgi
bodies as well as the outer nuclear envelope, which extends
out to surround the chloroplast(s), are both portrayed in
this electron micrograph.
120
-------
Figure 23
Ochromonas dan-ioa - Transmission Electron Micrograph
Showing Contractile Vacuole at Anterior End
* ;
*i* ..: . -. .' M;..%*. :f
•;-* vv-j -'•>•*?*?•-:.-.-;•*, - /. -;. *^>-
rV ^^--^-'K-v^-v^^.^^ ^
^'^kDl- ^'.^."/^..dw^r'tv.K "*.'•* 'i'l •*-*-•
191
-------
Figure 24
Ochromonas danica with Engulfed Microcystis
122
-------
F i gur e 25
Ochromonas danica - Details of Cytoplasm
-**., *>
?v»
3r'4&'*
• •« .*
123
-------
Figure 26
Micvocystis contained with Ochromonas. The blue-green cell
does not show obvious signs of digestion at this point, other
than the absence of the fibrous outer envelope. We would
expect there to be a production of lysosomes, which function
in the digestion process. Some vesicles are present, but
it may be too early for their abundance.
Figure 27
0. danioa from a bacterized culture, with a food vacuole of
bacteria. Two vacuoles are present, one with captured
bacteria and the other without. In this particular tube we
noted that the Oahromonas seemed to prefer the bacteria over
the Microcystis, which was also added. Note the less dense
appearance of the ingested bacteria than that of those on the
outside of the cell.
The problem of obtaining dense cultures of Microcystis for
inoculating into dense cultures of 0. danioa has been solved
by simply growing the blue-green under less intense illumin-
ation. We can now achieve rapid and dense growth of Miaro-
cystis.
124
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Figure 26
0. danica Containing Engulfed Microcystis
with Outer Envelope Pre-digested
125
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Figure 27
Oohromonas danioa with Ingested Bacteria
126
OU.S. OOVIRNMENT PRINTING OFFICE: 1973 546-310/69 1-3
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
/. Report .Vo.
w
Development of a Selective Algaecide to Control Nuisance
Algal Growth
07/73
Bernard L. Prows
William F. Mcllhennv
The Dow Chemical Company
Texas Division
Freeport, Texas
S. 1 .'forrtji .- Ores
No.
16010 EDJ
Contract No. 68-01-0076
I. Type : Repe, 2nd
Pi'iiod Cover erf
Qf
rifi' Oration
Environmental Protection Agency report number,
EPA-660/3-73-006, August 1973.
Development
The objective of this project was to develop a compound which would effectively and
economically control the growth of nuisance species of blue-green algae with a minimum
impact on desirable forms of life in the aquatic environment.
A computerized structure search of more than 100,000 compounds was made to select
the analogs of the following four Phase I prime candidates: 2,5-Dichloro-3,4-
dinitrothiophene; [5-Chloro-2-(p-nitrophenoxy)phenyljphenyliodoniumchloride; 4-Amino-
2,5-dibromophenylthiocyanate; and 1,1-Dimethyltetradecylamine, hydro-chloride. Through
this endeavor 1309 compounds were selected, 41 of which emerged from a rapid, agar-plate
screening as candidates for final laboratory screening tests.
A golden-brown flagellate, Ochromonas oval is, which exhibited phagocytic activity
against the blue-green alga, Microcystis aeruginosa, was discovered during Phase I.
Further research and development of biological-chemical control system included
studies involving several species of Ochromonas and conditions which would enhance
their phagocytic activity against Microcystis aeruginosa, with Ochromonas oval is
proving to be the most voracious feeder.
17a. Descriptors
*algicides, *selective chemical control, *algal control, *nuisance algal control,
Cyanophyta, laboratory assay, biocontrol.
17b. Identifiers
*selactive algiclde, *blue-green algae, *Anabaena, *Microcystis, *analogs,
test compound, phagocytic, selective screening. Qchromonas.
t~c. COWRR Field £ Group Q5G
s. Availability
19. Security Class.
f Re par.)
">0. Se< -rity Cl M,
21. No. of
Pages
Send To;
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
Bernard L. Prow
The Dow Chemical Company
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