Book of Legends 1.0.0.8 serial key or number

EPA/ August Ecological Research Series Research and Development of a Selective Algaecide to Control Nuisance Algal Growth \ UJ Office of Research and Development U.S. Environmental Protection Agency Washington, D.C.

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 RESEARCH REPORTING SERIES Research reports of the Office of Research and Monitoring, Environmental Protection Agency, have been grouped into five series. These five broad categories were established to facilitate further development and application of environmental technology. Elimination of traditional grouping was consciously planned to foster technology transfer and a maximum interface in related fields. The five series are: 1. Environmental Health Effects Research 2. Environmental Protection Technology 3. Ecological Research 4. Environmental Monitoring 5. Socioeconomic Environmental Studies This report has been assigned to the ECOLOGICAL RESEARCH series. This series describes research on the effects of pollution on humans, plant and animal species, and materials. Problems are assessed for their long- and short-term influences. Investigations include formation, transport, and pathway studies to determine the fate of pollutants and their effects. This work provides the technical basis for setting standards to minimize undesirable changes in living organisms in the aquatic, terrestrial and atmospheric environments. EPA REVIEW NOTICE This report has been reviewed "by the Office of Research and Development, EPA, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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 EPA/ August RESEARCH AND DEVELOPMENT OF A SELECTIVE AL6AECIDE TO CONTROL NUISANCE ALGAL GROWTH BY B. L. PROWS W. F. McILHENNY CONTRACT NO. ~68~-TJr PROGRAM ELEMENT 1BA ROAP/TASK 21 AIZ 06 PROJECT OFFICER THOMAS E. MALONEY PACIFIC NORTHWEST WATER LABORATORY WATER QUALITY OFFICE ENVIRONMENTAL PROTECTION AGENCY CORVALLIS, OREGON PREPARED FOR OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC I •!••!! ••••! - For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. - Price $

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 ABSTRACT The primary objective of this project was tL1 determine under natural, open-field conditions, the efficsciy of two can- didate algaecides, Compound No. 23 (2,5-Dichloro-3,4-dinitro- thiophene) and No. 73 (p-Chlorophenylthienyl iodonium chloride) from Phase II of the multiple phase developmental program. Specific efforts were also directed toward further delineation of the toxicological and environmental persistence properties of the candidate compounds, as well as further development of a possible biological-chemical control system. Data from the field tests conducted under a wide variety of conditions in four geographically diverse regions of the United States revealed a distinctive pattern of selective blue-green algal control for both experimental compounds. Compound No. 23 was eliminated from the test series due to unacceptable fish toxicity. A whole-pond field study involving the use of a phagocytic organism, Ochromonas ovalis, as a biological control system, was inconclusive due to the apparent inability of the organism to survive under the existing environmental conditions. Continued laboratory screening tests of some 70 additional compounds produced two additional candidate compounds, No. (2,2'-(l,2-Ethenediyl)bisbenzoxazole) and No. (l,2-Dichloro(isothiocyanatomethoxy) benzene). / Continuation of testing of candidate compounds under field conditions is recommended. ii

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 TABLE OP CONTENTS Page Abstract ii List of Figures iv List of Tables v Acknowledgements ix Sections I Conclusions 1 II Recommendations 3 III Introduction ** IV Experimental Procedure 15 V Discussions 29 VI References VII Appendices ill

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 LIST OF FIGURES Figure Number page 1 Open Field Test System 22 2 North Carolina Test Site 31 3 Chowan River - First Test, Test Compound No. 23 vs. M. Cyanea and M._ in cert a 35 4 Chowan River - Second Test, Test Compound No. 73 vs. M. cyanea and M._ incerta 38 5 Compound Depletion Patterns, Chowan River - First Test 4l 6 Minnesota Test Site 49 7 Lake Sallie and Muskrat Lake - Linological Sampling Stations and Algaecide Testing Stations. 50 8 Compound Depletion Patterns, Second Minnesota o 10 n 12 13 14 Test - Muskrat Lake, Test Compound No. • • • Northern Texas Test Site Whole Pond Test - North Texas, Compound No. 73 Fish Toxlcity Tests - Blue Gills, Test Compound No. 73 Influence of Three Test Compounds on the Phago- 60 66 67 Rl 87 f\ r*" 95 cytic Activity of Ochromonas danica at ppm . iv .

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 LIST OP TABLES Table Number 1 Chowan River - First Test, Anabaena spp. and Microcystis montana . 33 2 Chowan River - Second Test, Oscillatoria tenuis and Agmenellum quadruplica'tum"!~. 34 3 Chowan River - First Test, Microcystis cyariea and Microcystis incerta 36 4 Chowan River - Second Test, Anabaena spp. and Microcystis cyanea 39 5 Chowan River - Second Test, Oscillatoria planctonica and Microcystis incerta 40 6 steel-group.eu - Second Test, Compound Depletion Patterns 42 7 Chowan River - First Test, Compound Depletion Patterns 43 8 Degradation-Absorption Check on Compound No. 73 Under Controlled Conditions 45 9 Chowan River - Second Test, Dissolved Oxygen Levels 47 10 Lake Sallie - First Test, Compound Depletion Patterns ,. . 52 11 Lake Sallie - First Test, Test Compound Action on Blue-Green Algae Aphanizomenon and C. naegellanum 53 12 Lake Sallie - First Test, Test Compound Action on Blue-Green Algae Anabaena and' Microcystis. . 54 13 Lake Sallie - First Test, Total Blue-Green Algae 56 14 Lake Sallie - First Test, Total Algal Cell Counts x loVml 57 v

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 LIST OP TABLES continued Table Number Page 15 Lake Sallie - First Test, Test Compound Action on Oocystls and Fraglllaria 58 16 Muskrat Lake - Second Minnesota Test, Compound Depletion Patterns 61 17 Muskrat Lake - Second Minnesota Test, Anabaena and Aphani zomenon 62 18 Muskrat Lake - Second Minnesota Test, Coelosphaerium and Raphidiopsis 63 19 Muskrat Lake - Second Minnesota Test, Oscillatoria 6H 20 Diamond Lake - First Test , Compound Depletion Patterns . . . . » 69 21 Diamond Lake - First Test, Blue-Green Alga Anabaena 70 22 Diamond Lake - First Test, Diatoms - Synedra and Stephanodiscus 71 23 Diamond Lake - First Test, Fluorometric Relative Intensity Readings 72 24 Diamond Lake - First Test, Chlorophyl "A" and Carotenoid Levels 74 25 Diamond Lake - Second Test, Compound Concen- trations k 76 26 Diamond Lake - Second Test, Blue-Green Alga Anabaena 78 27 Diamond Lake - Second Test, Green Alga Gleocystis . . . . * 79 28 Diamond Lake - Second Test, Staurastrum and Stephanodiscus 80 29 North Test Test Site - Monitored Test Compound Levels 84 vi

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 LIST OF TABLES continued Table Number Page 30 North Texas - Whole Pond Test, Oscillatoria and Agmenellum 85 31 North Texas - Whole Pond Test, Anabaena and Microcystis 88 32 Phase III Field Test Results, Algal Control - Compound No. 73 89 33 Phase III Field Test Results, Algal Control - Compound No. 23 90 34 Fish Toxicity Tests - Blue Gills, Compound No. 23 92 35 Fish Toxicity Tests - Blue Gills, Compound No. 73 94 36 Fish Toxicity Tests - Rainbow Trout, Compound No. 73 96 37 Laboratory Screening Tests, Test Compounds versus Anabaena flos^-aquae 97 38 Laboratory Screening Tests, Test Compounds versus Microcystis aeruginosa 39 Algaecidal Activity of CuSO»»5H20 Against Two Species of Blue-Green Algae 40 Algaecidal Activity of Cutrine Against Two Species of Blue-Green Algae. 1°7 41 pH Sensitivity Tests on Compounds No. 23 and No- 73 Against Microcystis 1°8 42 Algaecidal Activity - Biomass Dependence Test versus Anabaena flos-aquae 1°9 43 Influence of Test Compound No. at ppm on the Phagocytic Activity of Four Species of Ochromonas vii

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 LIST OP TABLES continued Table Number 44 Ochromonas Storage-Viability Study viii

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 ACKNOWLEDGEMENTS The work done in connection with this project was coordinated by Dr. B. L. Prows, principal investigator, with the assistance of W. P. Mcllhenny, project director. C. P. Ward assisted in the laboratory and field work. We are indebted to each of the site investigators in the four principal test areas, for their interest, cooper- ation, and efforts in gathering background data and direct- ing the field tests in their respective areas: Dr. B. J. Copeland and Jim McKenzie of the North Carolina State University; Dr. J. K. Neel and Dave Brakke of the Univer- sity of North Dakota; personnel from the Lake Sallie, Minnesota State Fish Hatchery; Dr. H. Horton and Jim Rybock of Oregon State University; Dr. B. D. Vance and his graduate students from North Texas State University. Appreciation is also expressed to the various state officials whose cooperation made the site utilization possible, and to Drs. M. J. Wynne and G. T. Cole of the Department of Botany, University of Texas at Austin, who served as consultants and investigators on Biological Algal Control mechanisms. The support of The National Environmental Research Center of the United States Environmental Protection Agency, and of Dr. T. E. Maloney of the National Eutrophication program, who was the Federal Project Officer, is gratefully acknowledged. ix

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 SECTION I CONCLUSIONS Data collected under a wide variety of conditions in four geographically diverse regions of the United States indi- cate a rather distinctive pattern of blue-green algal control by both of the two prime candidate algaecidal compounds, No. 23, 2,5-Dichloro-3,4-dinitrothiophene; and No. 73» p"-Chlorophenylthienyl iodonium chloride. Other supporting data regarding compound selectivity, environ- mental persistence and fish toxlcity was definitive. The following specific conclusions are reached: 1. Compound No. 73» having met all of the original objec- tives, has emerged as the best candidate. It is an effective chemical for algal control; it is safe to applicators, fish, and other higher aquatic plants and animals; it has a fairly rapid degradation pattern under open atmospheric conditions, with a half-life of one to two days; and it also exhibits a fairly high degree of specificity for the target algae, particularly Anabaena, Microcystis, Aphani zomenon» and Oscillatoria. It is relatively inactive against diatoms and most green algae. 2. Compound No. 23 also proved to be an effective algae- cid and met most of the criteria, but was found to be toxic to fish life and thus had to be eliminated from the test program. 3. Through the continuing laboratory screening of 70 additional compounds; No. , 2,2'-(l,2-Ethenediyl)

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 bisbenzoxazole; and No. , l,2-Dichloro(lsothio- cyanatomethoxy) benzene; exhibited good algaecidal properties, showing 91 to 97 percent control of Anabaena at the ppm level. Continued efforts toward development of a biological- chemical control system have resulted in further invest- igations of the phagocytic algal organism, Ochromorias ovalis, which has proven to be a voracious feeder on the blue-green alga Microcystis aeruginosa, and have shown that Ochromonas can retain some viability for more than 70 days at room temperature when imbibed in acti- vated charcoal or cotton and polyester fibers; whole pond field tests with this organism for control of Microcystis were unsuccessful as the Ochromonas was apparently unable to cope with the new environmental conditions present at the time.

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 SECTION II RECOMMENDATIONS In view of the positive results obtained thus far in the long-range development program, and the relatively good prognosis for successful achievement of the original objectives, it is recommended that the research program be continued at least to Phase IV, of the general develop- ment plan, under government funding. There are certain inherant uncertainties and areas which should be investi- gated in greater depth to determine whether the project should be advanced, terminated, or modified at the conclusion of Phase IV. It is suggested that this next ph^se should Include: 1. Additional field tests under natural conditions, especially in larger single ponds, small lakes, and fish hatchery empoundments. to obtain more informa- tion on the algal control efficacy of the candidate compound. 2. Further tests to determine the compound's environmental safety, Including expanded animal and plant toxicities and compound degradation patterns under natural conditions. 3. Continued laboratory testing, as new compound structures shown to be potential algaecides are synthesized in proprietary programs, or otherwise become available for preliminary screening. 4. Continued Investigation of the biological control phenomena by flagellated phagocytes, especially with regard to the development of practical application methods,

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 SECTION III INTRODUCTION All surface waters contain dissolved and suspended materials which serve as nutrients and help support the growth of algae and many other forms of aquatic life, the numbers and variety of which are determined by the amounts and kinds of nutrients which are available (Palmer, ). A certain amount of natural eutrophication in our fresh water systems is tolerable, and even desirable, for the support of fish life and the necessary accompanying biota. Excessive eutrophication, however, upsets the natural aquatic ecologi- cal balance and causes many troublesome aesthetic and economic problems. In comparatively recent times the availability of adequate supplies of good quality fresh water has come to be regarded as one of our most valuable natural resources, and the con- trol of nuisance algae growth and aquatic weeds is one of the major concerns of the Environmental Protection Agency (Prescott, ). To this end a cooperative plan of action has been formulated for the collection of a sufficient range of comparable data on the degree and extent to which nutrient loading in our fresh water lakes is correlatable with the rate at which eutrophication is developing (EPA, )• Algae are found as common and natural inhabitants of all sur- face waters and are especially abundant where the water is exposed to direct sunlight. The portion of the available solar radiation spectrum utilized is in the visible, infrared, and ultraviolet regions, in the approximate ratio of , respectively (Brown, ).

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 To date more than 18, species of algae have been identified (Palmer, ) but only a relatively small number of these, principally the blue-greens, are considered to be notable nuisance species. Unlike the other groups of small microscopic organisms all species of algae contain chlorophyll. Algae are responsible for an estimated 90 percent of all photosynthetic activity on the earth (Meyer, ). One pound of algae growth will produce about 15 pounds of oxygen (MacKenthun and Ingram, ). The constant increase in urbanization, accompanied by changes in ground cover and surface soil, together with such phenomena as forest fires, over-grazing, deforestation, and agriculture, having increased run-off and reduced soil seepage to the extent that, as estimated by some authorities, the underground water table in the Eastern half of the U.S. has been lowered about 60 feet in the last 50 years (Palmer, ). This, in addition to a large increase in the use of ground waters, has created severe shortages in many areas. Thus, as population and industrial demands increase, attention has, of necessity, turned more and more to lakes, streams, and reservoirs in order to meet these needs. A need for an efficient and economically feasible method for controlling certain algal species has arisen as a result of 'excessive eutrophication of natural waters due to the con- tinued increase in the wastes produced by the human population as well as from indescriminate use of available water supplies. Although there is no universal agreement among scientists and environmentalists as to the particular elements which are responsible for excessive algal populations, the most commonly suspended nutrients are phosphates and nitrates.

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 It has been observed that algae blooms are often associated with waters which receive sewage effluents or other waters which are rich in these components. Thus, it is widely believed that in many bodies of fresh water, growth of phytoplankton tends to be limited by the supply of inorganic phosphate (Tailing, ). MacKenthun and McNab () made studies of several Wisconsin stabilization ponds and con- cluded that the annual per capital contribution of soluble phosphorous and inorganic nitrogen was and pounds, resspectively. When the physical and chemical conditions of a body of water become optimal for a particular algal species, proliferation may take place in such abundance as to produce visible aggre- gations of floating algal masses. To produce such "algal blooms" the combined growth conditions usually become optimal for only one species at a time. Thus an algal bloom usually contains one heavily predominant species. Many types of algae, particularly the blue-greens, tend to impart obnoxious tastes and orders to the water, clog intake screens and rapid sand filters of water treatment plants, and produce unsightly collections of debris on shores, making the water unsuitable for many desired uses. The presence of algae in water supplies has been known to cause digestive upsets or even death to certain warm-blooded animals due to toxic substances released to the water. A number of case histories are recorded in which -certain genera of Cyanophyta (blue-green algae) such as Mlcrocystis. Aphanizomenon and Anabaena are known to have caused animal deaths, particularly in areas where the wind may have concen- trated the algae into leeshore areas (MacKenthun and Ingram, ).

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 A rather common occurrance resulting from heavy algal blooms is a severe fish kill due to the depletion of dissolved oxygen in the water. Even though algae do impart oxygen to the water, through photosynthesis during daylight hours, the metabolic and catabolic processes taking place in living plant cells continuously utilize oxygen both day and night. Should a heavy cloud cover reduce the oxygen- producing phot©synthetic activity of the algae to an abnor- mally low level over a several-day period, the dissolved oxygen level during the night, when photosynthesis ceases and oxygen-consuming digestive processes still continue, may fall below the critical level required to sustain life in many species of fish, and massive fish kills may result. Game fish such as trout, which have high dissolved oxygen requirements are usually the first to be affected in such cases. The pH of the water will tend to increase, as algae extract carbon dioxide from the water for photosynthetic action, thus reducing the amount of soluble carbonic acid in the water, as well as the intermediately soluble bicarbonates and the nearly insoluble monocarbonates, usually causing part of the latter to precipitate. Water storage and transport problems may also be caused by the depolarizing action of the oxygen produced during photosynthesis (Palmer, ). The literature documents the damage to our inland waters in recent years as a result of excessive algal growth. For example, Lake Washington in * contained a maximum phyto- plankton population of x uVrnl, of which only 15 per- cent was made up of blue-green algae. By , the phytoplank- ton population had increased ten-fold and consisted of 95 per- cent blue-greens (Bartsch, ).

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 In contrast to most species of non-flagellated algae which settle to the bottoms of lakes in calm weather, many plank- tonic blue-greens exhibit an "upside-down" characteristic, accumulating as dense scums on the surface, which may then be blown by the wind into thick windrows and piled upon the shorelines. Often an accompanying offensive "pig-pen" odor results from the decaying material (MacKenthun and Ingram, ). Also, bad tastes as well as decomposition products are imparted to the water which may become toxic to animals and humans alike (Bartsch, ). The problems associated with nuisance algal control are being investigated through the National Eutrophication Research Program of the Environmental Protection Agency. Basically the investigative efforts fall into four broad categories: mechanical, biological, ecological and chemical. Mechanical approaches involve the engineering and design of machines and equipment for underwater mowing, raking, and harvesting of certain algae and higher aquatic weeds. Biological control mechanisms being investigated include viruses, insects, algae-eating fish, and phagocytic organisms. Ecological approaches include diversion of nutrient-rich waters, flushing of lakes and ponds with nutrient-poor water and nutrient- removal by aquatic plant crops, or by flocculation and adsorp- tion methods. Due to the great variety and complexity of conditions in which algae problems may exist, it is unlikely that any one single approach to the control of unwanted algae would be suitable in all cases. Without question, one of the basic solutions to the control of algae lies in the control of nutrient levels in the water. The nutrients and the required nutrient levels are not well understood, and the difficulties involved in controlling excessive eutrophication are so great that alternative methods of algae control will likely persist in the future for a considerable length of time. 8

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 For many situations, a chemical approach to the control of nuisance algae growths will be suitable, particularly in smaller lakes, streams and impoundments where algae "blooms" are imminent and the need to restore ecological balance exists. Chemical approaches (the use of algaecides) have not been as thoroughly investigated because of the difficulties encountered in developing a compound which will selectively kill or inhibit reproduction of the target algal species without adversely affecting the other more desirable forms of aquatic life. For a number of years, the most widely used algaecidal compound on the market has been copper sulfate. Despite its extensive usage, copper sulfate has disadvantages such as toxicity to desirable aquatic life at higher concentrations, non-biodegradability, accumulations of copper salts in bottom muds, and corrosive properties to paint and equipment (Bartsch, *0. Hasler (19^7) and Kuentzel () point out the possible deleterious effects of lake-bottom accumulations of copper sulfate on lake ecology. Investigations conducted in Wisconsin and Minnesota by Moyle (19^9) indicate that certain algae, particularly the blue-green alga Aphanizomenon seem to have acquired an increased tolerance to copper sulfate as a result of many years of successive treatments; in this case up to five times as much copper sulfate was needed for control of Aphanizomenon as was required 20 years earlier. There is no federally registered chemical available for the selective control of nuisance species of blue-green algae in surface waters which are to be used for potable purposes. A considerable amount of time and effort has been expended in recent years in search of a compound which would replace copper sulfate as an algaecide, and at the same time prove

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 to be safe to non-target organisms, non-cumulative in the environment, and be economically feasible to use for such purposes (MacKenthun, et al, ). It is important to understand that the development of a compound for widespread use in the environment, particularly where recreational and potable waters are concerned, is a long-range and expensive process. Phase I of this contractual effort to "Develop a Selective Algaecide to Control Nuisance Algal Growth", was initiated April 5, The primary objective was to develop a compound which would control the growth of various species of blue-green algae effectively, safely, and economically, while exhibiting a minimum impact on other forms of life in the aquatic environment. The following criteria were specified as general guidelines for compound selection and development. A satisfactory algaecidal compound must have: 1. High activity against the target algae, specifically, against selected species of blue-green algae. 2. Low toxicity levels for mammals, fish, and other desirable aquatic organisms. 3. Low toxicity to terrestrial plants 4. No questionable elements such as arsenic, mercury or other heavy metals, in its structure. The approach to the problem involved initially making a computer search of some 80, compounds in Dow's computer listing of compounds in order to select those having the 10

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 highest probability of meeting the established criteria for an ideal algaecide. Most of these compounds had al- ready been screened against at least one species of algae and also for activity against higher aquatic plants, fish, and some terrestrial plants and animals. A final hand selection was then made from the computer printout, elim- inating those compounds which contained heavy metals and those which were likely to be costly, or which possessed inherent or demonstrated undesirable properties, such as high toxicities to fish, terrestrial plants, or mammals. The candidate compounds, thus selected, were then divided into several priority groups according to the established selection criteria. Within the month contractual period, 33 compounds were screened using the two target species of blue-green algae suggested by EPA's Federal Water Quality Office, Corvallis, Oregon. Each compound was tested against cultures of Microcystis aeruginosa and Anabaena flos-aquae at a concen- tration of ppm in constant-temperature water bath shakers (24°C) at 80 oscillations per minute under a cool-white fluorescent light intensity of foot-candles. Compound activity was expressed as percent control, as compared to control cultures which were run simultaneously under identical conditions. Cell growth was monitored by visual inspections, cell counts, and relative intensity readings using a fluoro- microphotometer. Those compounds which passed the first screening test with at least 80 percent control at ppm against both test species were selected for more detailed testing at , » and ppm. Of the 33 compounds tested, Compound Nos. 23 (2,5-Dichloro-3,4-dinitrothiophene), 15 ([5-Chloro(p-nitrophenoxy)phenyl]phenyl iodonium chloride), 8 (4-Amino-2,5-dibromophenyl thiocyanate), and 24 (1,1-Di- methyl tetradecyamine hydrochloride) were effective at these 11

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 concentrations and were selected as the prime candidate compounds for further research through Phase II of the long-range algaecidal development program (Prows, )- A phagocytic organism, identified as Ochromonas oval is, was discovered which showed promise as a means of control- ing Microcystis. In addition, it was found that the growth and activity of Ochromonas was enhanced by low concentra- tions of some of the test compounds being studied. As specified in the contractual work statement efforts were to be directed toward the long-range goals of developing an algaecidal compound which would: 1. be safe to non-target organisms 2. exhibit a high degree of specificity for the target algae 3. be economical 4. be safe to applicators 5. be non-persistent in aquatic systems Phase II of the algaecide development program was begun June 30, Initially a computerized structure search of more than , compounds was made in order to select the analogs of the following four Phase I prime candidates: 2,5-Dichloro-3,4-dinitrothiophene; [5-Chloro(p-nitro- phenoxy)phenyl] phenyl iodonium chloride; 4-Amino-2,5-di- bromophenyl thiocyanate; and 1,1-Dimethyl tetradecylamine hydrochloride. This endeavor resulted in the selection of analogs, which were each subjected to rapid agar-plate screening tests. Forty-one compounds emerged from these tests as candidates for final laboratory screenings. Although six of these compounds showed high levels of activity against 12

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 the target blue-green algal species, four proved to be unacceptable because of poor economic feasibility, environ- mentally unacceptable properties, or difficulty or danger in compound production. At the conclusion of Phase II, Compounds No. 23 (2,5-Di- chloro-3,4-dinitrothiophene) and 73 ([p-Chlorophenyl] thienyl iodonium chloride) were selected as final candidates based on superior algaecidal activity, environmental acceptability, economic feasibility, and freedom from human health and handling hazards (Prows and Mcllhenny, ). i Three additional species of Ochromonas were discovered during Phase II which exhibited phagocytic activity against Microcystis aeruginosa. However, the original species discovered during Phase I exhibited the greatest activity and showed some activity improvement when used with low levels of certain test compounds. The Phase III effort was begun on November 28, The objectives of this third phase were to test the candidate compounds under natural field conditions to obtain informa- tion on the algal control efficiency, and of combined biological-chemical systems in naturally occurring field situations. The stability and environmental stability of the compounds of concern were to be determined. The initial approach was to synthesize large enough quantities for use in field tests. Analytical test procedures were to be developed in order that the persistence, adsorption, and degradability rates could be followed during and after treat- ment. The compounds were then to be field tested in small lakes and ponds having blooms of nuisance algae. Careful 13

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 monitoring as well as the analysis of water, bottom muds, and higher aquatic plants. Toxicity studies on fish and mammals were to be undertaken. The biological and chemical- biological systems using Ochromonas were also to be further studied.

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 SECTION IV EXPERIMENTAL PROCEDURES The primary objective of this research project was to determine under natural, open-field conditions, the efficacy of two prime candidate algaecides, No. 23 (2,5-Dichloro- 3,^-dinitrothiophene) and No. 73 (p-Chloro-phenylthienyl- iodonium chloride) which were final candidates at the con- clusion of the Phase II effort. Specific efforts were to be directed toward further delineation of the toxicological properties and environmental persistence of these compounds, with objectives focused on further defining an effective chemical or biological-chemical system which would control / the growth of blue-green algae safely and economically while exhibiting a high degree of specificity for the target algae. In order to have sufficient quantities of the candidate com- pounds to allow the toxicology tests to be run and to have sufficient for each of the four field tests planned for various sectors of the country, a re-synthesis of the two test com- pounds was necessary. Twenty pounds of Compound No. 23 was manufactured according to specifications by Pharm-Eco Corpora- tion, Simi Valley, California. A like quantity of Compound No. 73 was made in several successive batches at Dow's Organic Synthesis Labs in Midland, Michigan. ANALYTICAL PROCEDURES During the developmental stages of a pesticide, particularly if it is to be used in the aquatic environment, it is man- datory that a fast and accurate means be developed for detecting compound residuals as a function of elapsed time. 15

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 In this case the procedure had to be capable of monitoring traces of the compound in very dilute concentrations, preferably in the parts per billion range, .since initial treatment concentrations were planned at around ppm, and it was desirable to follow the compounds' persistence, adsorption, and degradation rates for several weeks following each treatment. COMPOUND NO. 23 Both test compounds were examined by differential pulse polarography but the lowest detection limit achievable for Compound No. 73 was ppm and the method was therefore unacceptable for this compound. The instrument used was a Princeton Applied Research Model Polarographic Analyzer equipped with Model /70 drop timer to record the differ- ential pulse polarograms. A standard solution of Compound No. 23, prepared in methanol, was found to possess two polarographic waves in a sodium acetate supporting electrolyte, with half-wave potentials of and v versus a saturated calomel reference electrode (SCE). Sodium acetate itself gave no response in this poten- tial region, but when high instrument sensitivities were employed to achieve the desired response, interference due to impurities appeared. After electrochemical purification for a week (or longer), the sodium acetate was found to be free of interferences at the desired level of sensitivity. When laboratory distilled water and purified sodium acetate were used, a useful response was obtained from 20 ppb of the compound. Instrument response was linear with concentrations from 20 ppb to 20 ppm. 16

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 A sample of Tittabawassee River water was obtained upstream from Dow in Midland, Michigan, to check for interferences from natural waters. Purified sodium acetate was added to the river water and a polarographic scan was recorded. No interferences were observed in the potential region of interest. At this point, it was desired to establish the stability of solutions of the compound so that proper shipping arrangements could be made, since test sites were scheduled for various parts of the United States. Initial dosage levels were expected to be and ppm, and therefore, a ppm stock solution was selected for stability studies. Aliquots of this solution were determined polarographically at various time intervals. The compound remained stable at this concen- tration for at least a week under ordinary laboratory condi- tions . Stability was also checked in Tittabawassee River water and similar results were obtained, although the background signal from untreated water increased with time, which would limit detection of trace amounts. Stability was also checked on refrigerated and frozen samples since it was planned to ship samples packed in dry ice. No adverse effects were ob- served. From this procedure it was concluded that 2,5-Dichloro-3,4- dinitrothiophene could be determined polarographically in natural waters down to the 20 ppb level. The method proved to be quite specific, due to the presence of two polarographic waves. 17

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 COMPOUND NO. 73 Liquid chromatography was finally selected as the analytical method of choice for determination of Compound No. The test compound concentrations were monitored at the elution end of the column by a Perkin Elmer "" ultraviolet detector at the nanometer range. The original gradient elution system developed was later modified by a more efficient and time-saving procedure. In the gradient method, the water sample was injected into a strong cation exchange resin (VYDAC-SCX). The water and organics were first eluted with a water/methanol solution; the iodonium chloride, which had been retained on the column was then gradient eluted with a dilute ( M) sodium perchlorate solution in methanol/water. It was also found that methanol was as effective a mobile phase solvent as the water/methanol system. After the iodonium chloride was eluted, the column was then washed for ten minutes with the original solvent to remove all sodium ions. Due to the column washings, associated with the gradient system, a sample run lasted 20 to 30 minutes. Thus only about 15 samples, plus the appropriate standards, could be run per day with this method. Recent work with chlorophenols has demonstrated that penta- chlorophenol and tetrachlorophenol can be separated by ion exchange chromatography using constant composition elution with a weakly ionic mobile phase. Applying this principle to the iodonium chloride/water system, it was found that by using a'constant composition mobile phase consisting of M sodium perchlorate in methanol, the iodonium chloride was suf- ficiently retarded in passing through the column to be separated from the unretarded water and organics. With this method a 18

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 sample shipment could be analyzed in three hours. There were also no diffuse negative peaks associated with the solvent change preceding the iodonium chloride peak. The ionic strength needed to Just separate the bacteriostat from the water/organics was constant for a given column but changed when a new column was packed. A series of standard solutions was prepared and chromato- graphed with this technique. The chromatographic response was found to be linear in the to ppm range. For a given standard the reponse was also found to be reproducible to within ± percent of the mean response. Thus, the analysis of 4-Chlorophenylthienyl iodonium chloride in natural water was simplified by replacing a two- solution gradient elution system with a single-solution procedure. The resulting 80 percent reduction in analysis time permitted prompt analysis of large numbers of samples, thereby circumventing problems of degradation in storage. The linearity and reproducibility of this method was found to be very good with standard solutions. FIELD TESTS Selection of Sites It was proposed that test sites be selected in at least four different sectors of the United States so as to provide a good climatic cross-section with naturally occurring algal blooms. The general areas were selected with the advise of EPA personnel. Specific sites were selected in connection with algologists whose previous work and interest had been associated with the 19

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 limnological aspects of lakes and streams in the local areas where the particular Universities were located. The following criteria were used as a basis for selection of the test sites: 1. past history of algal populations and blooms 2. freedom from animal and human interactions 3. proximity of test sites to research personnel and laboratory facilities 4. prognosis of appropriate agency approval for permission to perform the tests On a site-selection tour of the proposed test areas early in May, , final details and arrangements for carrying out the field testing programs were made with the appropriate university investigators. Suitable bodies of water with reasonable proximities to the institutions concerned were chosen. The following is a list of the selected sites, together with the university professors cooperating as area investigators: 1. Chowan River, North Carolina Dr. B. J. Copeland, University of North Carolina 2. Lake Sallie, Minnesota Dr. Joe K. Neel, University of North Dakota 3. Diamond Lake, Oregon Dr. Howard Horton, Oregon State University 4. Slidell, Texas Dr. Dwain Vance, North Texas State University 20

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 During the inital site selection tour, contacts and liaison was established by the cooperating investigators with the appropriate government and state agencies at each site to assure their cooperation and to obtain the necessary approvals for the tests to be performed. No problems with state or local approval were encountered. Field Procedures Due to the limited amount of test compound available, and the need to contain the test compounds in small areas because of their experimental nature, it was decided to use gallon plastic containment bags as the test vessels at each site, except for the North Texas site, where a whole pond was to be treated. Six test vessels were designed for use at each site. These were supported by polyurethane flotation collars, filled with ambient lake waters, and held in position by means of auxiliary flotation devices which were anchored to the lake bottom with concrete anchors (Figure 1). A self-priming DC pump, powered by a volt battery was used as an aid in filling the test vessels to a measured gallon depth with ambient algae- infested lake water. The water in the vessels was then treated by Dow personnel with selected, pre-determined concentrations of test compound, with some vessels serving as controls, in addition to the sur- rounding lake water itself. Water samples were taken from the lake and from each vessel immediately before and following treatment, for the purposes of compound concentration monitoring, as well as the monitoring of algal population numbers as a function of time. 21

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 Figure 1. OPEN FIELD TEST SYSTEM Polyurethane auxiliary flotation device 25" OD Polyurethane gallon polyethylene bag lb. concrete anchor .x^A ^ L& -_ -^ ^&~ ^ -& / ~^^w- ^_ ^-^fe 22

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 Approximately ml samples were taken and placed in plastic biological "twirl pack" bags. Those collected for algal monitoring were treated with formalin solution as preserva- tive measure to assure counting validity. Samples which were taken for compound monitoring were placed in a second pro- tective bag before freezing and were then packed in dry ice for shipment via air freight to Midland, Michigan, for analysis. Water chemistry data, which varied somewhat with individual preferences of the site investigators, but always included temperature, pH, dissolved oxygen, and alkalinity, were taken before, during, and following treatment according to the recommended schedule. SAMPLE SHIPMENT In order to eliminate inconsistencies in sample handling and shipment and to help assure rapid and safe delivery via air freight, the following suggestions were made in a letter sent to each site investigator: All samples which are collected for compound analysis and sent to Midland, Michigan, should be: 1. Sealed securely in primary collection bags ( ml) and then placed in a second protective plastic bag. I 2. Double labeled - (a) pencil writing on a cardboard label, placed inside the second bag. (b) writing on outside or primary collection bag with a permanent- ink felt tipped pen. 23

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 3. Uniformly labeled as follows: I-C (this Is control no. 1, with no test compound) II-C1 (this is control no. 2, with no test compound) III (test compound no. 23 at ppm)' IV (test compound no. 23 at ppm) V (test compound no. 73 at ppm-) VI (test compound no. 73 at ppm) Vll-lake (ambient lake water outside of test vessel) 4. Quick-frozen as soon as possible after sample collection and retained in a frozen state until packaged for shipment. 5. Packaged in a sturdy, well-insulated shipping carbon, with at least four pounds of dry ice packed around, but protected from, direct contact with the sample bags (utilizing newspapers, or other good packing materials). 6. Package should be labeled "fragile" and sent via air freight by the most direct routing possible using shipping labels which specify the presence of biological samples, packed in dry ice, and a specific request to handle with care, and to deliver on or before a specified date. The only two major air lines which services the Midland, Michigan, area directly are North Central and United.

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 FORMULATION AND DISPENSING A rather extensive study was undertaken in order to deter- mine the optimum procedures for formulation of the two compounds. Several solvent systems, balanced to a specific gravity of were utilized in the study. Compound No. 23, having a low water solubility, was found to be readily soluble in both acetone and xylene. However, when dissolved in acetone the compound was observed to crystallize and fall out of solution when the concentrate was combined with water in the normal temperature range one would expect to find in lakes and streams. The utilization of xylene as the basic solvent required the addition of about 19 percent perchloro- ethylene, to yield the desired specific gravity balance. The use yof a surfactant as an emulsifying agent was also re- required. The principal governing factor for the optimum formation of molecular compounds in such cases is the realization of the best hydrophylic-lipophylic balance, permitting the formation of lytotropic mesomorphous phases or dispersion into miscelles, The optimum formulation for Compound No. 23 was determined as a ratio of anionic :nonionic surfactant in a xylene- perchloroethylene solvent system. Compound No. 73 was found to be percent soluble in water at room temperature and about 4 to 5 percent soluble in methanol. The increased solubility in methanol was not con- sidered sufficient justification in itself to prompt its use as a solvent^ which would tend to escalate the economics of the compound when considered for large-scale use. However, the compound was solubilized in methanol for use in certain portions of some of the field tests to determine whether that particular solvent system would affect the Compound's algae- cidal activity. 25

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 A day or two prior to treatment, primary concentrates of the test compounds were made up in pre-determined aliquots in the laboratory and then taken to the treatment site. For later treatments the procedure was modified by taking unmixed, pre- weighed quantities of the original compound to the test sites and combining with lake water in brown ml bottles to make a "use" concentrate just prior to treatment in order to avoid the possibility of compound degradation. After the "use" concentrates were prepared they were kept away from direct sunlight as an additional safeguard against possible photodegradation. Also, the tests were not begun until near sundown, for the same reasons. At the North Texas test site where a whole-pond treatment was undertaken, a "use" concentrate of Compound No. 73 was made by addition of a pre-determined portion of the compound to gallons of water in a commercial sprayer, and continuously agitated for one hour before application. The concentrate was then sprayed onto the surface of the pond just before sun- down. LABORATORY SCREENING TESTS According to plan, screening tests were to be continued on candidate compounds as they were sent to this laboratory from various sources within Dow and elsewhere. The basic screening procedure was modified slightly from that which was used in the Phase III work (Prows and Mcllhenny, ). All primary screening tests were run for four days against Anabaena only, at ppm and ppm. If secondary tests were run they included tests down to O.U ppm, or lower if deemed expedient, against both target algal species, Anabaena flos-aquae and Microcystis aeruginosa. 26

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 In preparation for the screening tests, stock cultures of Anabaena and Microcystis were grown in ml flasks, cultured in Gorham's medium under foot-candles cool, white light. Twenty-four hours prior to treatment standard inoculum cultures were made up from the initial stock cul- tures by placing sufficient algal cells in fresh Gorham's medium to bring the standard inoculum to a relative inten- sity of After 24 hours of acclimation in the new medium, the inoculum was ready for use. At "0-day" for a particular test, 29 ml of standard inoculum was placed in each of the ml culture flasks and then inoculated with ml formulated test compound of the appropriate concen- tration such that the dilution factor would reduce the final concentration to the desired levels. The test vessels and their controls were then kept under a constant illumination of foot-candles, cool white fluorescent lighting, and agitated at 80 excursions per minute in 24°C water bath shakers throughout the four-day test period. Culture growth in each test flask was monitored by cell counts where appropriate, and by relative intensity readings, taken with an AMINCO fluoromicrophotometer, equipped with a blue mercury-vapor fluorescent lamp for the primary illumination source. Light from the lamp was filtered through a No. 55^3 band pass filter before entering the sample. Subsequent light being emitted from the fluorescing chlorophyll molecules thus provided an extremely sensitive method of determining in vitro the relative number of living algal cells remaining, without having to go through a time-consuming chlorophyll extraction procedure. 27

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 After substracting the background Intensities read for each test sample the final relative intensities compared to those of the control cultures gave a basis for computation of the percent control as exhibited by various concentrations of the test compound in question. 28

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 SECTION V DISCUSSION The primary research efforts of the Phase III program have been to determine the algal control efficacy of two selected algaecidal compounds. These two compounds, No. 23 (2-Dichloro-3,4-dinitrothiophene) and No. 73 ([p-Chloro- phenyl]2-thienyl iodonium chloride) emerged from the Phase II study as prime candidates for field testing because of their effectiveness against two of the commonly troublesome blue-green algal species, Anabaena flos-aquae and Microcystis aeruginosa. PIELD TEST RESULTS The four test sites were diversely located to give a good cross-section of climatic conditions. Because each test was monitored by a different group, and because the tests were conducted sequentially there are slight differences in the methods of handling the field data. The general plan was to include two tests at each site dur- ing the summer and fall seasons. Two tests were made at each site, except for North Texas. In each case the polyurethane flotation bobs for the enclosure, the flotation collars, and the 60 gallon plastic test bags were prepared at Dow and sent to the area investigators pre- vious to the tests. The complete sets of data collected at each site during each test are included in this report in Appendix B. 29

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 Chowan River, North Carolina The North Carolina test site was located on the west shore- line of the Chowan River, about 20 miles southeast of Ahoske, NC (Figure 2). The Chowan River is about four miles wide at that point. Heavy blue-green algal blooms of Anabaena, Aphanlzomenan, and Microcystis had been reported as having occurred in the particular section of the River in previous years, being especially common during the summer of A fertilizer plant, located about 30 miles upstream is reported to have been responsible for a considerable amount of nutrient input to the river, which may have contributed to optimal "bloom" conditions. The specific location of the test site was located Just to the North of the Perry-Wynn Pish Processing Plant, the owners of which were cooperative and agreeable for the tests to be conducted about yards offshore from their property. The first test was initiated on June 14, , and a second test was conducted on August 7, 3» when a much heavier bloom was present. Specific clearance for conducting tests with experimental compounds was requested and received from the North Carolina State Department of Air and Water Resources by the area investigator, Dr. B. J. Copeland, North Carolina State Univer- sity. Of the six gallon test vessels prepared for the field tests, two contained only ambient river water, with no test compound added; to serve as controls, in addition to the surrounding river water itself. The other four vessels 30

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 Tigure 2. North Carolina Test Site VIRGINIA • ^ Bu "NORTH \CAROLINA A = Test Site B = United Piece Dye Works C • C. P. Industries - Fertilizer Plant rffjeesboro _———-' Potecasi oGamville iunda,ry line

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 were treated with sufficient quantities of pre-formulated test compound concentrations of Compound No. 23 and Mo. 73» to bring the initial algaecide levels to ppm and ppm concentrations in the respective chambers. The pre-weighed test compound aliquots were dissolved in brown ml bottles at the site, and then wrapped in aluminum foil to serve as a light barrier and minimize the possibility of photodegradation. As an added precaution the test compounds were not administered to the test vessels until near sundown. The algal data, as reported by the North Carolina State University investigators, indicated percent activity against Microcystis montana by both Compounds No. 23 and and No. 73 at concentrations down to ppm (Table 1). Compound No. 23 was similarly active against Anabaena. The action of Compound No. 73 against Anabaena was inconclusive from the data received. Of the two blue-green algae, Oscillatoria and Agmenellum, the former was brought under nearly percent control by both compounds at all con- centrations, while the latter was affected very little by either compound (Table 2). An unsuspected phenomena was revealed by the specific activity of Compound No. 23 against Microcystis cyanea, compared to its relatively low activity against Microcystis incerta (Figure 3). Compound No. 73 showed percent activity against M. cyanea at the ppm level but the population returned somewhat at 0+4 days (similar to the action of Compound No. 23). Little or no activity was noted against IYL_ incerta (Table 3). Further verification of this 32

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 TABLE 1 Chowan River - First Test (cells/liter x 10") Sample Ambient River Water Controls (Avg. ) Compound No. ppm Compound No. ppm Compound No. 73 - ppm Compound No. 73 - ppm Anabaena spp. 0-days _L 15 26 11 16 12 10 12 Ik HT 10 13 0+1 day 28 11 25 19 0 0 1 0 0 0 0 0+U days * 13 0 0 0 1 0 28 0 ~0 0+11 days _5 0 0 _L 0 Microcystis montana 33

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 uo fr TABLE 2 Chowan River - Second Test Oscillatoria tenuis AND Agmenellum quadriduplicatum Sample Controls Compound No. 73 - ppm Compound No. 73 - ppm Compound No. 73 - ppm* Compound No. 73 - ppm (cells /I 0-day 16 93 16 IbT 16 98 16 98 11 Ta iter x 10") 0+1 day ? ? 0 9F 0 26" 0 W 16 \ 0+2 days 18 9 U 10 35 6 31 0 *9 0+3 days ? ? 0 W lU 70 if 1 10 0+6 days U6 13 _?_ 12 7F _5_ 29 U ?0 "Compound solubilized in methyl alchol . Oscillatoria Agmenellum

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 r- o X r— £ c/i 01 O c o •M (O O. O (O O) Figure 3. Chowan River - First Test Test Compound No. 23 % ppm vs. M^ cyanea & M_._ incerta O Microcystis cyanea /\ Microcystis incerta ••••••» Test disrupted by adverse weather O 11 Time (days) *test disrupted by adverse weather 35

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 TABLE 3 Chowan River - First Test Microcystis cyanea AND Microcystis incerta (cells/liter x 10") Sample Ambient River Water Controls (Avg.) Compound No. 23 - ppm Compound No. 23 - ppm Compound No. 73 - ppm Compound No. 73 - ppm 0-day 0 79 *f £ 0+1 day 8 11 0 5lT 0 16 36" days 6h_ 73 a 37 8 10 S9 21 0+11 days JL 53 21 75 ii 52 73 KEY: .Microcystis cyanea Microcystis incerta 36

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 specific activity is seen by examination of the data from the second Chowan test conducted in August. One hundred percent activity was observed against M_._ cyanea at ppm, but little or no significant activity was registered against 5-L ihcerta (Figure 4 ). The target blue-green alga Anabaena was brought under about 95 percent control by Compound No. 73 at ppm during the second Chowan River test (Table 4). • The blue-green algae Oscillatoria planctonica was nearly percent controlled at 0+2 days, but the population returned to normal levels at 0+6 days (Table 5 ). It is strongly suspected that the population return was the result of a considerable amount of intermixing with river water, which may have occurred during turbulent weather, or from a par- tially ruptured bag.- Oscillatoria tenuis was drastically affected by all four concentrations of Compound No. 73; in contrast, Compound No. 73 showed little or no control of Agmenellum quadruplicatum at the ppm level and only partial control at the and ppm levels (Table 2). The compound depletion patterns of both test compounds in the Chowan River samples from the two tests were similar (Figure 5). During the second test, after six days, com- pound No. 73 had been depleted to near or below the ppm detection limit in every test vessel (Table 6). The determined initial compound concentrations in the first test were only about 50 percent (or less) of the calculated values, even though the samples arrived at Dow's Midland, Michigan location still frozen and in good condition (Table 7). 37

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 « o O) o o •£» 10 Q. O o. (O < Figure 4. Chowan River - Second Test Test Compound No. 73 at ppm vs. ML cyanea and M. incerta O Microcystis cyanea /\ Microcystis incerta 0 38

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 U) TABLE ^ Chowan River - Second Test Anabeana spp. AND Microcystis cyanea (cells/liter x 10°) Sample Controls Compound No. Compound No. Compound No. Compound No. 73 - ppm ppm 73 - ppm* 73 - ppm 0-day "98 1U8 "28 63 60 0+1 day 32 27 32 8 25 0 0 0+2 days 32 JL 5 4 2 0 JL 0 0+3 days — |— _5 21 29 6 0 11 2 0+6 days 12 0 18 21 31 10 0 20 0 •Compound soluMlized in methyl alcohol Anabaena KEY: Microcystis

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 4=- O TABLE 5 Chowan River - Second Test Oscillatoria planctonica AND Microcystis incerta (cells/liter x 10°) Sample 0-day 0+1 day Controls Compound No. Compound No. Compound No. Compound No. 73 - ppm 73 - ppm ppm* 73 - ppm ••^^^^^^^M To 98 62 62 62 30 -8T -L- ~97 o 25 1 70 71 lUl 31 6§ o 0 51 8 —•— 25 21 o 28 o 73 62 73 81 50 . 1U7 *Compound solubilized in methyl alcohol Oscillatoria : Microcystis

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 Figure 5. Compound Depletion Patterns Chowan River - First Test steel-group.eu Compound No. 23 /\ Compound No. 73 (Theoretical Treatment Level) Q. ' O (B •*-> C 0) o o Time After Treatment (Days) ill

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 I\J TABLE 6 Chovan River - Second Test COMPOUND DEPLETION PATTERNS values im ppm Sample 0-day 0+1 day 0+2 days 0+3 days 0+6 days Ambient Lake Control Compound Compound Compound Compound No. No. No. No. Water