ate aeors Ces eee ee ae A ye P+ ine eee reee ar? Ae tel $0 8 On oe Oe ae - ~ "5 eA ey Seen’ Piteisatae ee Seo ee ee a -e- 5-8 (ies ny mise” = " > GE = oe > Fe 2 “i z Naas = ; S OSHLINS Sa luvugi7_LIBRARI ES SMITHSONIAN _ INSTITUTION NOILNLILSNI NVINOSHLINS S31yvusI7 > At, 7) | Wwe a w +3 a 6 @s & 5 A. < c < = < = BRAN OS 3 NN a = oc af cx Cc o c a) = a - m ea » a - - i) - re) =a fe) Ve ool 4 = Zz a a a 5 I HSONIAN INSTITUTION NOILALILSNI | NWINOSHLINS _S3 1YVYdit LIBRARIES SMITHSONIAN INSTITUTION > ae za i za -_ o = ro) — B a OF; o = o = o 2 o a Ly > : = Ne 2 5 £42 5 p lf > = > \ SS = > = i D> = Cre a 2 SNA a = = ee i ra no = SW 7) = Paw ~ w = a ise cs o z o z OSHLINS SSlYVEAII_ LIBRARIES SMITHSONIAN INSTITUTION NOILMLILSNI NVINOSHLINS S31uVuaIT_ = Vi See = =a z < z Vy = by J 5 =: 5 NS = 2 & > YM w th fi". 2 <% n an ~ We o AD a ae 9 G tei x ie} rc TAN OO fe) hp z Z Ee 2 E ENS le = = ; Sy > = > - Ser iy =a 2 wo Zz = P = 2 > 4SONIAN INSTITUTION NOILMLILSNI_NVINOSHLINS S3IYVYAI7 LIBRARIES SMITHSONIAN INSTITUTION z < e 4 re) ” Es =a = a = Zp) = w” = = Ta Zz z ” ; ep) tify ar) me n cs wn lJ Yr 2 us Yidy, ~ 2 a fe a = Yfy, % : Dy fo 4 < = _< = < A < Vt; = Be = ya 9: a Cc oc —¢ oe re) a 2) “Ss _ fe) = ro) = z ey) = 4 2 = Zz ai ISHLINS S3JIYWVYSIT LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLINS S31yuYvHSsIT oe z 2 Ze es z = Fa ce we. BE D 5 a zs re = x BS > r= > 2 > =) > NS = b = b - b a q a oD Bee tae Z : . 3 B 4ISONIAN NOILNLILSNI NVINOSHLINS S31IYVYSIT LIBRARIES SMITHSONIAN wn Fok 22) = a n = e2) = ‘S = e peek Qs = = = ’ s = 8 2 SU Ye 2 ® (te 2 8 S = 2 Yip f= IN Z = 2 > = > = ae ae = > Fe ” = ” Pree w Sie OSHLINS S3IYVYGIT LIBRARIES SMITHSONIAN INSTITUTION NOILALILSNI NVINOSHLIWS salyvugiy S NOILNLILSNI LIBRARIES NOILNLILSNI N \ ISONIAN INSTITUTION NOILALILSNI NVINOSHLINS S31a¥vud!1 LIBRARIES SMITHSONIAN INSTITUTION | a11 LIBRARIES SMITHSONIAN INSTITUTION NOILALILSNI INSTITUTION NOILALILSNI S3!1YVYEIT LIBRARIES INSTITUTION Ssalyvaudl INSTITUTION Sa1uV ISHLINS SAIYVYEIT LIBRARIES ‘’ INSTITUTION NOILALILSNI NVINOSHLINS S3I1YVudtT NVINOSHLINS S31YVYSITLIBRARIE NVINOSHLINS NVINOSHLIWS SMITHSONIAN NVINOSHLIWS SMITHSONIAN f ISONIAN INSTITUTION NOILALILSNI_ NVINOSHLIWS saluvugly LIBRARIES SMITHSONIAN INSTITUTION » LIBRARIES SMITHSONIAN aOR. NOILNLILSNI NOILNLILSNI 17_LIBRARIES SMITHSONIAN NOILNLILSNI NOILNLILSNI NVINOSHLIWS 7 a ad SHLINS SAIYVYdalt erate INSTITUTION NOILNLILSNI ae 17 LIBRARIES SMITHSONIAN 17 LIBRARIES I7_ LIBRARIES SalyVvudlt ee )N ON N )N ee ee ARES Saat a iN hel ese een Le es > SCS a - 1ES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLIWS S31uvugI7_-IBRARIES SMITHSONIAN _ INST uw = tw z * Zz Ma z e LN . =" x pa’ — a oe wat ip 4 = ax. % = = o = Te — = {> F > \AQAE J2 VVeE 2 = ‘2 = 7 ARV = Zin WS m & : g a SW 8 4 ; = 2) = wo = (an) = [ES (SMITHSONIAN INSTITUTION NOILNLILSNI_NVINOSHLINS Saluvugi _-IBRAR! ES SMI ASONIAN (iy i'e er = < = , oe = ae - x = =a 3 o = x t S WY : ae = 5 YQ Fs 5 a z WAY S ye O - Zr WSN O ian z ee \S Z, = Z E NS Zz Eee oe 2 = a >" = > = i = = eS 7) a Fn 7) Zz 7) < 2 7) NI_NVINOSHLINS S3INVYGIT LIBRARIES SMITHSONIAN INSTITUTION |, NOILMLILSNI_NVINOSHLINS | SI a w = yy, 2 uw ee : a a = a res o = Ly fed } my eo: a < SoA < C Ww WS < To) or 5 = 5 ES 6. Sar 1ES SMITHSONIAN INSTITUTION NOILNLILSNI NVINOSHLINS S31uvugIT LIBRARIES SMITHSONIAN _INS1 EAU aad BK os a - Ey > E Da “ea > = > gee 2 cs = ne = = 2 on J n os ” m 2 m Zz om ze m z w Zz pa diem = a) . = = (a7) LSNI_NYINOSHLINS | Sa lYVYGIT_LIBRARIES SMITHSONIAN ip NOLLNLILSNI_ NVINOSHLIWS | Sail ee = r= &s = < = < =< Zz = rR NS = = =| Fa = o =e re) ANS ayes ro) Be re) + JZ , - Z ENG 2 = 2 = z @ = > . = > Ss > Z = > a = 7) ame ~ 2 a = JES “SMITHSONIAN _ INSTITUTION NOILNLILSNI_NVINOSHLIWS Sa1uvuG17_LIBRARIES SMITHSONIAN _ INS i 8 z a & © & a eee = 2 Je A « = NG o ea = tel fy Ss EYL 3 < SQ = 5 i YUp 3 = Wp * : s& = : i ae Ey Z Zz =a 2 ee z Si ‘ z ISNI_NVINOSHLINS S3IYVYSIT LIBRARIES SMITHSONIAN INSTITUTION NOILNLILSNI_NVINOSHLIWS S31 = a r ei |S z ae 2 rs) NA : = Pe) = a 5 S\N ‘ a a wt ae =. z= = XS i SMITHSONIAN TES SMITHSONIAN INSTITUTION INS ‘= NOILNLILSNI NVINOSHLINS S3JIYVUYSIT LIBRARIES NS SMITHSONIAN INSTITUTION NOILNLILSNI_ NWINOSHLINS $3 NVINOSHLINS S31YVual NVINOSHLIWS SMITHSONIAN f NVINOSHLIWS SMITHSONIAN i NVINOSHLIWS SMITHSONIAN LSNI_ NVINOSHLIWS V¥dil LIBRARIES NOILNLILSNI NOILNLILSNI NOILNLILSNI NOILNLILSNI LIBRARIES SMITHSONIAN 1ES SMITHSONIAN INS ~~ INSTITUTION NOILNLILSNI NVINOSHLINS S31uVvudiy ak. I17_LIBRARIES SMITHSONIAN 11 LIBRARIES 117” LIBRARIES 317° LIBRARIES ZariSON™ ON JON aAnSON;™ ty ON ON = Wi f e i ¥ DH ORNS Ow, ss owen BELLE ALLE Np ee aon ey ee st “i “ ie, PP TA. WED. ie L Mr ; A Nee lis ~~ 6 1966 er ee Ft She INVESTIGATIONS IN FISH CONTROL onion oo pyres I. Laboratories and methods for screening fish-control chemicals 2. Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals United States Department of the Interior Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife UNITED STATES DEPARTMENT OF THE INTERIOR Stewart L. Udall, Secretary Frank P. Briggs, Assistant Secretary for Fish and Wildlife FISH AND WILDLIFE SERVICE Clarence F. Pautzke, Commissioner BUREAU OF SPORT FISHERIES AND WILDLIFE Daniel H. Janzen, Director The United States Department of the Interior, created in 1849, is concerned with rianagement, conservation, and de- velopment of the Nation's water, wildlife, fish, mineral, forest, and park and recreational resources. It has major responsibilities also for Indian and Territorial affairs. As America's principal conservation agency, the Department works to assure that nonrenewable resources are developed and used wisely, that park and recreational resources are conserved for the future, and that renewable resources make their full contribution to the progress, prosperity, and security of the United States, now and in the future. INVESTIGATIONS IN FISH CONTROL I. Laboratories and methods for screening fish-control chemicals Bureau Circular 185 2. Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals Bureau Circular 186 Washington, D.C. - June 1964 Investigations in Fish Control are reports on the results of work at the Bureau's Fish Control Laboratories at La Crosse, Wis., and Warm Springs, Ga. The two reports presented here are the first of several reports that are planned on work now under way. CONTENTS Laboratories and methods for screening fish-control chemicals, by Robert, E. Wennon and Charles! Ro Walken os 5. ..0 « ss weeneteee i-io Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals, by Charles R, Walker, Robert E. Lennon; and) Bernard We, Beree riper 7 trsicns loses sie) eae eaens a5 1-18 Bureau Circular 185 Page Column Line 8 1 39 1 3 3 13 ! 47 Bureau Circular 186 ] ] 16 2 | 46 2 1 h§ Be Fig. 1 5 2 17 6 2 2 8 l 2 10 2 23 10 2 28 17 1 -- ERRATA ltem properties instead of porperties olla fiberglass are empirical Methylethyl- acetic acid were occurred 122 OF Un | if on '' fibergalss int is tL emperical '' Methylethyl- '' (Table 2) a was 't occured tt 12 ia 7. Literature citation No. 5: add Biochim. Biophys. Acta., Vol. 53 INVESTIGATIONS IN FISH CONTROL 1. Laboratories and methods for screening fish-control chemicals By Robert E. Lennon, Fishery Research Biologist Charles R. Walker, Chemist Bureau of Sport Fisheries and Wildlife La Crosse, Wis. Se eee DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE Bureau of Sport Fisheries and Wildlife Circular 185 Washington, D.C. + June 1964 CONTENTS Other laboratory facilites . co) siete eos hom ds oie, Se Pish=holdirig faciitiesy .suc./aa s: «2 dene ees A ee li Laboratories and methods for screening fish-control chemicals By Robert E. Lennon, Fishery Research Biologist and Charles R, Walker, Chemist Bureau of Sport Fisheries and Wildlife La Crosse, Wis. Abstract.--This report describes the physical and technical facilities and the procedures of the Fish Control Laboratories at La Crosse, Wis., and Warm Springs, Ga. The laboratories emphasize screening of chemi- cals to find a variety of fishery management tools. Preliminary Screening ascertains whether a chemical in three concentrations has a desirable biological activity on eight species of fish in reconstituted water at 12° and 170 C, Delineative Screening ascertains effective concentrations (EC100) on eight species in reconstituted water (the method for which is described) at 120, 17°, 22°, and 27°. Intensive Screening of promising fish-control agents ascertains effects on 24 species of fish and on other aquatic organisms, at different temperatures and in waters of various qualities, in the laboratory and in the field. Fish Control Laboratories were established by the Bureau of Sport Fisheries and Wildlife at La Crosse, Wis., in 1959 and at Warm Springs, Ga., in 1963. The mission of the Lab- oratories is the development of means for efficient manipulation of fresh-water fish. In particular, safe and economical controls-- chemical, biological, electrical, or mechani- cal--are sought for undesirable populations in standing and flowing waters. The objectives are sufficiently broad to encompass investi- gation and development of any new tools that may be useful in fishery management, fish culture, or fishery research, At La Crosse, the buildings of the National Fish Hatchery were remodeled and expanded in 1960-62 to provide a large research facility (figs. 1 and 2), The subsidiary Laboratory at Warm Springs is new construction on the grounds of the National Fish Hatchery (fig. 3). Their locations offer contrasting advantages to be exploited through close coordination in the research on fish control; La Crosse Warm Springs Southern fishes. Warm climate. Warm water, Soft water. Northern fishes. Cold climate. Cold water. Hard water, In equipping and staffing the laboratories, early recognition was given to the potentials of chemical control agents. The bioassay (wet), chemistry, and physiology laboratories are concerned with general and selective toxicants, attractants, repellants, anesthetics, sterilants, spawning inducers, osmoregulators, marking dyes, medications for diseases, and sedatives and decontaminants for fish distribution. Em- phasis is on finding selective toxicants for longnose and shortnose gars, gizzard shad, goldfish, carp, squawfishes, white sucker, black bullhead, rock bass, green sunfish, pumpkin- seed, yellow perch, and freshwater drum. Ample justification for research on selective piscicides is contained in fishery literature, aa 133 Figure 1,--The Fish Control Laboratory at La Crosse, Wis, Figure 2,--The fish holding house at La Crosse, Wis, Figure 3,--The Fish Control Laboratory (foreground) and wet laboratory holding house at Warm Springs, Ga, Construction and grading were incomplete when the photograph was taken, LesVeaux (1959) listed 30 States that need con- trol of certain troublesome fishes. Among the qualities desired in selective toxicants are specificity to certain life stages or to certain fish, low cost, ease and safety of application, rapid degradation to nontoxic residues, harm- lessness to warm-blooded animals, and effec- tiveness at low temperatures, Applegate et al. (1961) reported on an effective and selective sea lamprey larvicide, Loosanoff, MacKenzie, and Shearer (1960) showed the possibilities of controlling certain shellfish predators with chemicals in marine environments. These and other studies stimulated interest in investiga- tions to find selective toxicants for various fresh-water fish. The American Fisheries So- ciety, for example, resolved at its 88th annual meeting in 1958 to recommend, to the Secre- tary of the Interior, an expansion of research in fish control. Congress in the same year made the first appropriation for establishment of the Fish Control Laboratory at La Crosse. FACILITIES Bioassay laboratories At La Crosse and Warm Springs there are wet laboratories for large-scale screening of chemicals against fish. Fiberglass or alumi- num troughs serve as water baths for bioassay vessels, and fiberglass or concrete tanks hold selected fish for experiments (figs. 4 and 5), Ground water is used for the water baths and fish holding, and temperatures are adjusted by means of thermostatically controlled im- mersion heaters or refrigeration units. Deionized water of at least 1 million ohms resistivity is reconstituted according to a formula developed at the Bureau's Fish- Pesticide Research Laboratory and is em- ployed as a test medium in the bioassay vessels. The following chemicals are added per liter of deionized water: 30 mg. of calcium sulfate, 30 mg. of magnesium sulfate, 48 mg. of sodium bicarbonate, and 3 mg. of potassium chloride. : Glass vessels are preferred for bioassays in the laboratory. Most of the screening is done in economical 1-gallon pickle jars which are used once and discarded. Five-gallon water buckets of glass are employed in advanced screening. They are reused after thorough de- contamination and washing which include sev- eral steps: a. Rinse jar with tap water. =< ae Figure 4,--View of wet laboratory at La Crosse showing batteries of bioassay vessels in concrete, aluminum, and fiberglass tanks, b, Add 6.3 grams of activated charcoal (1 gram/3 liters), fill jar with deion- ized water, and let stand over night. c, Empty and rinse; wash with strong detergent in hot tap water and rinse thoroughly. d. Sponge entire jar with 10- to 14-per- cent hydrochloric acid and rinse twice with deionized water, Whenever residual contamination is detected or suspected, the jar is discarded, All dis- carded bioassay glassware is smashed to pre- vent further use, All test solutions are discarded into a floor drain which continually carries at least 300 g.p.m. of waste water from the fish holding tanks. The dilution has been found to be more than sufficient to eliminate hazards. All fish used in the bioassays are disposed of in gas-fired incinerators of complete-com- bustion type. Other laboratory facilities Each Fish Control Laboratory has chem- istry, biochemistry, and physiology labora- tories as adjuncts to the bioassay facilities. Chemicals for testing are received in the chemistry laboratories, stored in fire- and explosion-resistant vaults, and prepared in proper solutions and dilutions for bioassay. Compounds showing promise as fish-control agents are investigated in the biochemistry laboratories to evolve methods for applica- tion, effective and economical formulations, possibilities for potentiation, means for mini- mizing side effects and hazards, and techniques for detoxification. In the final stages of de- velopment, a control agent is studied in the Figure 5,--View of a wet laboratory at Warm Springs showing a battery of bioassay vessels in a fiberglass tank, physiology laboratories to define its mode of action on fishes and other organisms, any chronic effects, the fate of residues in live animals, and the risks, if any, to consumers of treated fish. Fish-holding facilities Large quantities of fish are required for the chemical screening programs, At La Crosse, for example, 498,000 fish of 34 species were used in 1963. Most are obtained from Federal, State, or private hatcheries and rearing stations; it is more satisfactory to arrange small, frequent deliveries than attempt to maintain large quantities on hand for long periods in usable, disease-free con- dition, A holding house and outside pools are provided at each Laboratory for the main- tenance, feeding, sorting, and grading of the experimental fish (fig. 3 and 6). Outdoor bioassay pools An intermediate step between laboratory testing and field trials of promising fish- control agents is essential to detect and evaluate some of the physical or chemical factors that influence the performance of a candidate agent in natural waters, Race- ways and portable plastic pools are located at each Laboratory for this purpose. Figure 6,--Interior view of fish holding house at La Crosse, Inexpensive vinyl wading pools, 9 and 10 feet in diameter, 2.5 feet deep, and about 1,000 gallons in capacity, are set up as de- scribed by Lawrence and Blackburn (1962) in outdoor testing areas (figs. 7 and 8), Bottom soils of various types, pond or ground waters, aquatic plants, invertebrates, fish, and am- phibians are used in them as needed during chemical trials. Contaminated vinyl liners are economically replaced. The pools at La Crosse are employed only during the warm season, but those at Warm Springs are in operation all year, Ten- and 20-foot concrete raceways are used for extraordinary tests in running or standing water, Disposable vinyl liners are used when necessary to avoid harmful con- tamination of the raceways. METHODS The static bioassay is the first approach in screening chemicals for control agents. Constant-flow bioassays are reserved for advanced stages of testing. Bliss (1957) defined a bioassay as a deter- mination of the potency of a physical, chemi- cal, or biological agent by means of a bio- logical indicator, Noting its development during the past 20 or 30 years by scientists from many and diverse fields, he listed principles which characterize the modern bioassay: (1) Potency is a property of the drug, not of the response; (2) potency is relative, not absolute; (3) the assayed potency of an unknown is only an estimate of its true value; and (4) both the reliability Figure 8,--Vinyl bioassay pools at Warm Springs, 7 and efficiency of an assay are linked insep- arably with its design. Observance of these principles overcomes some major disad- vantages of the bioassay as a research tool. Fish have long been employed as biological indicators in bioassays of water pollutants, insecticides, herbicides, detergents, and other substances, The standards recom- mended by Doudoroff et al. (1951), Henderson and Tarzwell (1957), and Henderson (1960) for such tests have been widely accepted and applied, although difficulties in comparing studies have arisen because of the many kinds of fish involved. Douglas and Irwin (1962) pointed out that the results of inde- pendent bioassays often cannot be related because the comparative resistance of the many test fishes has not been established. They noted that certain species have been more useful in toxicity bioassays than others, and they emphasized the need for knowledge about the reactions of different species of fish when exposed to a particular toxicant. The large body of literature on methods and results of bioassays with fish has been helpful in defining the screening programs of the Fish Control Laboratories, In general, the practical methods proposed by the inves- tigators cited above are followed but with some modifications since we seek more in biological activities than acute toxicity only. Test chemicals The test chemicals are selected by staff chemists and biologists and are contributed by industry. Preference is given to compounds that have demonstrated biological activity or are suspected of possessing a useful activity against fish, It is also desirable to have as much information as possible before screen- ing on the nature and porperties of each chemical, its shelf life and stability, its solubility, and its potential hazards to inves- tigators, Security is respected, and precau- tions are taken with compounds and test data to protect the rights of contributors. The chemicals obtained for screening are arbitrarily classified as follows in order to facilitate scheduling of tests, observation of responses, and reporting of results; 1, Natural organic products; a. Animal extracts (steroids, proteins, etc.): b. Plant extracts (rotenoids, alkaloids, phenols, etc.), c. Fermentation products (antibiotics, eter 2. Synthetic organic products; a. Halogenated hydrocarbons. b. Nitrogen-bearing hydrocarbons (salicylanilides, carbamates, triazines, etc.), c. Phosphorus-bearing hydrocarbons. d, Sulfur-bearing hydrocarbons (mercapto, thioates, thiozines, etc.): e. Miscellaneous compounds and com- binations, 3. Inorganic products, The progress of a chemical through testing and development is depicted in figure 9, A compound is introduced into Preliminary Screening to detect whether it has activity against fish. If it has, it is advanced into Delineative Screening, where the effective concentrations are defined and the possible usefulness of the substance is declared, If results are favorable, the candidate is re- ferred to Intensive Screening, where it is fully evaluated in the laboratory and in the field as a fishery tool. Compounds which fail to meet requirements at any stage of screening are shelved or discarded immediately. Test fishes It is of utmost importance that the validity and comparability of the bioassays be assured by using only fish of selected species, of certain sizes, and in good condition as bio- logical indicators. The criteria are defined and strictly observed for each stage of screen- ing. Therefore, the rate of progress of a chemical through screening and development depends largely on the availability of the pre- scribed fishes. Time and Chemical PRELIMINARY SCREENING 6 to 12 mo. | 10 grams rejection DELINEATIVE SCREENING 6 to |2 mo. | 5O grams RENEE TION ECig9 evaluation rejection Consultation with supplier rejection INTENSIVE SCREENING 12 to 24mo. | kilogram Laboratory Physiology Outdoor pools Field testing rejection rejection rejection FINAL EVALUATION rejection acceptance Figure 9,-~Schematic on the progress of chemicals through screening, 9 TABLE 1.--Time required by certain fish held at 12° C. to empty the digestive tracts after food is withheld ) ; Number of Seo Voiding Species in Roan pound time (hours) Rainbow trout..ceccscssesecs 90 2,000 36 Wee eeeeescseesses River shiner...+cccess White sucker...+.s++- ouee Green sunfish....+-e++eseesees 45 355 60 Pumpkinseed...seseeesseeeere 45 177 84, Bluegill. ...sesscssccccevaes 60 368 84 Longear sunfish...-++.seesss 45 465 72 Lots of hatchery fish are requested for delivery at least 2 weeks prior to use in bio- assays. They are placed in the care of fish culturists, and everything possible is done to minimize stresses, During the first 10 days they are fed, prophylactically or therapeu- tically treated as necessary, and observed to evaluate them as test animals. Lots in which the mortalities exceed 10 percent within the period are not moved into the bioassay program, The fish for experiments are carefully graded to desired and uniform size and trans- ferred into the wet laboratories or outside pools 3 or 4 days before use. Food is withheld for as long as 96 hours before screening, depending on the life stage and species of fish. Generally, young fish and certain spe- cies require less time to empty the intestinal tract than others (table 1), The fish spend at least 24 hours in water similar to the test medium before being introduced into the bioassay vessels, A help in maintaining comparable results in bioassays is use of a recognized reference toxicant against a sample of fish from each test lot. We employ para-, para '-DDT (1,1, 1-trichloro-2, 2-bis(p-chlorophenyl) ethane), and the test is made coincidentally with the introduction of a lot of fish into the screening program; the test is repeated biweekly if the lot remains on hand. The sensitivity (EC50) of the sample is deter- mined for comparison with regression curves established for the species from experience or literature. Although costly, the test pro- vides the only measure of relative sensitivity of fish used in the bioassays. The value of reference tests was demon- strated during shakedown trials of facilities 10 and methods at La Crosse, For example, gold- fish from Missouri and Wisconsin had such widely divergent sensitivities that they bracketed those of other species. Also, the sensitivities within a lot of fish differ signifi- cantly if some specimens are exposed to pre- sumably harmless electric shocks like those experienced in electrofishing. Moreover, the stress of disease, malnutrition, temperature change, or altered water quality may influence sensitivity to a toxicant. Holding time is another factor, and we observed that the sensitivities changed greatly in a lot of gold- fish which was retained for 2 months. Saila (1953) emphasized the significance of this and showed that the resistance of his mosquito- fish to rotenone decreased in rough proportion to the length of time they were held. Responses of test fish We recognize that the observer may be the greatest source of error in bioassays with fish, The criteria of response are arbitrary and subject to individual interpretation. Moreover, they are complicated at the Fish Control Lab- oratories because effects in addition to and more subtle than acute toxicity are sought. To achieve individual and mutual consistency, the observers are trained to use definite criteria of response, Chemicals may have either short-term or long-term effects on fishes. Those of short term are typically identified with the following: 1, Acute toxicity: a. Selective toxicant. b. General toxicant. 2. Movement: a, Attractant. b.. Repellant. 3. Facilitating capture, handling, or transport; a. Anesthetic. b. Sedative. c. Osmoregulator. 4. Marking live fish: a. Immersion stain. b. Internal stain. 5. Therapy or prevention of disease; a. Bactericide. b. Parasiticide. c. Fungicide. d. Prophylactic. e. Antiseptic. The long-term activities of potential con- trol chemicals may be difficult to detect and evaluate. They are associated with-- 1. Control of reproduction: a. Hormonal spawning inducer, b. Hormonal spawning inhibitor, c. Sterilant. 2. Control of growth and development; a, Growth stimulant. b. Growth inhibitor. The criteria employed to evaluate the re- sponses of fish are rather refined, and are applicable to short-term responses, They are used as necessary at each observation period to identify fully the reactions of the fish. They are-- General behaviour: 1, No observable difference from control. 2. Quiescent. 3. Excitable, 4, Irritated, 5. Surfacing. 6, Sounding. 7. Twitching. 8. Motionless; a, Tetany. b. Flaccidity. 9. Swimming: a. Erratic; convulsive. b. Gyrating; skittering. 11 c. Inverted. d. On side. e. Against tank sides on bottom. Integument: 1, Pigmentation: a. No observable change, b. Light discoloration, c. Dark discoloration, d. Varidiscoloration, 2, External mucosa: a. No observable change. b. Shedding; patchy. c. Copious exudate. d. Coagulation. 3. Hemorrhagic. Respiration: 1, Respiratory rate: a. No observable change. b. Rapid. c. Slow. d. Irregular. e, Ceased, 2. Gulping air. 3. Structures, organs: No observable change. Mouth gaping. Hemorrhage in gills. Irritative response. Copious mucus in gills. e Ss ie PAP Alimentary responses: 1. No observable change. 2. Egurgitating mucus or other material. 3. Defecating mucus or other material. Nervous responses; 1. No observable change. 2. Sensitivity to stimuli: Positive Negative a. Exterior movement.. De eel Gay eaceeennsecereaan en C. (SOUNG : ciscasseeceeteuds dy ROUGH icc acesecesweatmeese e)) Electric probersd.e.c Moribundity: 1, No motion. 2. No respiration. 3. Distended operculum. 4, Opaque eyes. 5. Death. Recovery: 1. Complete, 2. Incomplete. Additional and more definitive observations are made of fish at the in-test, recovery, or postmortem stages in the biochemistry and physiology laboratories. Their objectives may be, for example, the modes of action and side effects of control agents. Screening Preliminary Screening.--Preliminary Screening is designed to detect whether a selected chemical at 0.1, 1, and 10 p.p.m, has a biological activity (see list of criteria above) against eight species of fish within 48 hours, It is a static bioassay in 1-gallon jars containing 2,5 liters of pre-aerated, reconsti- tuted water. No artificial aerating is done during the test. The temperatures of water 12 baths are 12° C, at La Crosse and 17° C. Warm Springs. at The fish include the following species; La Crosse Warm Springs Rainbow trout, Goldfish, White sucker, Yellow perch, Carp, Black bullhead. Green sunfish. Bluegill, Goldfish from the same gene pool and from the same source are employed for occasional toxicity checks between the two laboratories, At least 10 fish of each species are used with each concentration of chemical and as controls, They weigh between half a gram and 2 grams each, but the weight range of the fish in each test does not exceed 15 percent. They are distributed at one species per jar about 16 hours before the bioassay at a loading of 1 gram or less of fish per liter of test medium Thus, there may be but one or two fish per jar, and up to 10 jars may be needed for each concentration of chemical, A stock solution of a test compound is pre- pared within an hour of the bioassay. The solvents preferred are water, acetone, and ethanol, in that order. Because of their toxi- city to fish, precautions are taken that the concentrations of acetone and ethanol do not exceed 4 parts per thousand in the bioassay media, Furthermore, whenever these solvents are used, equal volumes of them are applied in control vessels. Thus the volume in con- trols is equal to the highest volume used in a dosage series. Small aliquots of a stock solu- tion are thoroughly mixed into bioassay media to avoid improper dilution or stratification, The responses of the fish in bioassays and controls are observed routinely at 0.75, 1.5, 3, 6, 24, and 48 hours, but so far as possible they are recorded on the first day and thereafter as often as the nature of a candidate compound warrants, If a compound has a desirable activity against the fish, it is held for further screenings, which are discussed briefly below. Delineative Screening.--The effective con- centrations (EC100) of a chemical against fish are determined in Delineative Screening. The vessels may be jars or troughs for static or flowing water trials, The water, aeration, species and size of fish, duration of tests, and controls are the same as in Preliminary Screening. A compound is first bioassayed at 12° C, to define concentrations which evoke all-or-none responses from the fish, The approach to these concentrations may be direct by bracketing and interpolation, or it may be indirect by probit analysis, whichever is the shorter. The EC100, if seemingly practical, is confirmed by seven replicate trials. A chemical which yields favorable results at 12° C, is retested at 17°, 22°, and 27° to evaluate the effects of temperature on the effective concentrations, At 22° and 279, rain- bow trout are omitted from the bioassays, and the loading rates for other species are half a gram or less per liter of test medium. If a compound succeeds in these trials, its potential as a fish-control agent is estimated, Information is sought on its possible applica- tion; on the source and manufacturing costs; on possible hazards, conflicts, or limitations in use; and on the size of the market. Only if it continues to appear promising is the chemi- cal promoted into the more elaborate and expensive Intensive Screening. Intensive Screening.--Intensive Screening is directed toward development of a promising chemical as a fish-control agent and the defi- nition of its advantages and limitations. These are broad objectives, and some subdivision of approaches is in order. A. Stages: The advanced tests of a com- pound are accomplished in the wet labora- tories, in outside pools, and finally in the field. They may include static or flowing water bioassays with durations of 24, 48, and 96 hours or longer, The vessels may include 1-gallon jars with 2.5 liters of test medium, S-gallon jars with 15 liters of medium, and fibergalss troughs of 4-, 8-, and 16-foot lengths, The outdoor pools include 1,000- gallon vinyl units and 10- and 20-foot concrete 13 raceways, Proportioning pumps are used in the flowing bioassays to achieve consistent concentrations of chemical, The initial trials of a compound in the field are conducted in waters closed to public use, These waters are found, for example, on the grounds of Federal and State fish hatcher- ies, on wildlife refuges, or on military reser- vations, Trials are manned or immediately supervised by the Laboratory staff. Subsequent experiments in the field may be accomplished by selected cooperators in State and Federal agencies, B, Varieties of fish: Sixteen species in addition to those included in Preliminary and Delineative Screening are employed, They are; 1, Brook trout. 2. Northern pike. 3. Fathead minnow. 4, Brook stickleback. 5. Pumpkinseed. 6. Longear sunfish. 7. Smallmouth bass. 8. Walleye. 9, Gizzard shad. 10. Golden shiner. 11. Bigmouth buffalo. 12, Brown bullhead. 13. Channel catfish. 14, Redear sunfish. 15, Largemouth bass. 16. White crappie. Other species may be used on occasion in advanced tests. C, Life stages; Various life stages of fish, from egg to mature adult, may be involved in the more advanced bioassays to detect the presence, absence, or variation of response to a promising chemical, It may be a great ad- vantage or disadvantage if a potential control agent is selective for one life stage and not for another. D. Other organisms; The effects of a potential chemical tool on other aquatic life must be assessed at least minimally in the laboratory before it is tested in the field. Some forms such as water fleas, snails, and plants are cultured for this purpose. Others, such as freshwater scuds, damselfly and mayfly nymphs, and tadpoles, are collected in the field. Static bioassays in glass jars are con- ducted with chemical concentrations and ex- perimental conditions similar to those of the fish assays. E. Temperature; The influences of water temperature on the biological activity of a candidate chemical are defined for all species and different life stages from bio- assays under ice and at 29, 79, 1290, 170, 220, and 27° C, It is especially desirable to de- velop compounds that may be applied effec- tively during cold seasons when recreational use of public waters is low. F, Water quality: It is recognized that water quality may exert tremendous influence on the activity of an introduced chemical. Ac- cordingly, the effectiveness of a candidate control is observed in hard and soft waters, in alkaline and acid waters, and in waters of low and high organic content, in the labora- tory and field. Initially, the formula for re- constituting deionized water is altered to de- tect trends. G, Formulations: Formulation is often the key to an effective biological activity or efficient application of a compound, Density and solubility, for example, may be extremely important factors in a fish-control agent. Vari- ous formulations with carriers, wetting agents, dispersing agents, potentiators, or inhibitors may be prepared or acquired for testing. Combinations of biologically active chemicals are tested to ascertain their potentials as multiple controls for fish and aquatic weeds, fish and parasites, or fish and amphibians, Moreover, the practicality of a control agent might depend on or be enhanced by an applicator's ability to modify or arrest its activity efficiently at a given point. Hence, the Intensive Screening includes experiments with possible deactivators, detoxifiers, or decontaminants for potential controls. Unsatisfactory results or hazardous side effects, and new information on manu- facturing, marketing, or competitive control 14 chemicals may contribute at any point in In- tensive Screening to the abandonment of further trials and development. Recording and reporting results The results obtained with each chemical are analyzed soon after completion of the Preliminary, Delineative, and Intensive Screenings, They are furnished to the con- tributor of the chemical immediately. After screening, we evaluate the compound as a potential fishery management tool, to deter- mine patent positions, to refer it for studies of residues and chronic effects, and to inves- tigate requirements for clearance and labeling. For compounds with negative results the screening data are segregated according to the classes of chemicals involved and will be published as soon as the accumulation of data warrants. The results with chemicals which succeed as fish-control agents will be reported individually and addressed primarily to fish managers or fish culturists. Thus, we plan to define the range of effective concentrations for certain target fishes in waters of different temperatures, different types, and of various qualities. SUMMARY The Fish Control Laboratories at La Crosse, Wis., and Warm Springs, Ga., are equipped to find and develop chemicals which may be used as fishery management tools, Objectives of the research are general and selective toxi- cants, attractants, repellents, anesthetics, sterilants, spawning inducers, marking dyes, medications for diseases, and sedatives for fish distribution. The facilities include chem- istry, physiology, and wet laboratories; fish holding structures; and outdoor pools and race- ways. The program involves three stages of chemical screening. Preliminary Screening detects whether a selected chemical at 0.1, 1, and 10 p.p.m. has an activity in static bioassays on eight species of fish in reconstituted water at 12° and ERAGE Delineative Screening defines the effective concentrations (EC100) of a chemical in static or flowing bioassays on eight species of fish in reconstituted water at 129, 179, 22°, and 27° «6C, Intensive Screening is reserved for chemi- cals which show great promise as fish-control agents. Effective concentrations are deter- mined in the laboratory, in outdoor pools, and in the field against 24 or more species of fish in various life stages from egg to adult, against selected aquatic organisms, and in waters of different temperatures and various qualities. The modes of action, side effects, chronic effects, and deactivators also are studied, as necessary. The results of screening are reported direct to contributors, They are also pub- lished by the Bureau. The effective concen- trations of fishery tools on target fishes under certain conditions are given. LITERATURE CITED Applegate, Vernon C,, John H, Howell, James W. Moffett, B. G. H. Johnson, and Manning A. Smith, 1961, Use of 3-Trifluormethyl-4-nitrophenol as a selective sea lamprey larvicide, Great Lakes Fishery Commission, Technical Report 1, 35 p, Bliss, C. L. 1957, Some principles of bioassay, American Scientists, vol, 45, No. 5, Dp. 449-466, Doudoroff, P., B. G. Anderson, G, E. Burdick, P. S. Galtsoff, W. B. Hart, R. Patrick, E. R. Strong, E. W. Surber, and W, M, Van Horn, 1951, Bio-assay methods for the evaluation of acute toxicity of industrial wastes to fish, 15 Sewage and Industrial Wastes, vol, 23, No, 11, Pp. 1380-1397, Douglas, Neil H., and W, H, Irwin, 1962, Evaluation and relative resistance of six- teen species of fish as test animals in toxicity bioassays of petroleum refinery effluents. Department of Zoology, Oklahoma State Univer- sity, Contribution No, 351, Mimeo, 40 p, Henderson, Croswell, 1960, Bioassay procedures, aims, and equipment, Biological Problems in Water Pollution, Tech- nical Report W60-3, The Robert A, Taft Sani- tary Engineering Center, U.S, Public Health Service, p, 246-248, Henderson, Croswell, and Clarence M, Tarzwell. 1957, Bio-assays for control of industrial efflu- ents, Sewage and Industrial Wastes, vol, 29, No, 9, P. 1002-1017, Lawrence, J. M., and R. D. Blackburn, 1962, Evaluating herbicidal activity of chemicals to aquatic plants and their toxicity to fish in the laboratory and in plastic pools, Auburn Univer - sity. Mimeo, 23 p, (In press, Proceedings of 16th Annual Conference of Southeastern Association of Game and Fish Commissioners, Columbus, SHEA LesVeaux, John F. 1959, Summary report of survey to evaluate the need for specific fish toxicants in sport fishing waters, Progressive Fish-Culturist, vol, 21, No, 3, Pp, 99-110. Loosanoff, V. L., C. L. MacKenzie, Jr., and L. W. Shearer, 1960, Use of chemicals to control shellfish pre- dators, Science, vol, 131, No, 3412, p, 1522- 1523, Saila, Saul B. 1953, Bio-assay procedures for the evaluation of fish toxicants with particular reference to rotenone, Transactions of the American Fisheries Society, vol, 83, p, 104-114, aa avneht’ & 3 rl F ae sone i or. ” Nomi Renee as “ah ares tgs { rhe - ett ay si 7 bite ‘ <—¢ oF ha | P a ' , pithy « ze qu : ~drts nity wt borreds ae Te NTO italy’ un patie a ee se pb Sh # otis thr baa ae erat’, clingy a deiae HE’ 4 = RD i "1s eror a 2 Ee saga OH Herel! eID. 3B hpi: bay mine wbrutsity aft aS Sa Heh i 5" 4h bist aia” tidiiae "a “Rbbetl bry ee a es | » oles 107 Gg ‘yise £ af She ity Nik memenin TOF Chagas Ai VSTIOGy 1 SLE snitesia i esti a: f ‘ a wig eis vx ‘iy ioipahey : 1 45 ‘aye is ; «eats dy hss Saf ‘Ueatur 1S % : 178 i nt “atu st ‘ iy Mey ¢ | actly etal ioe sna. am . lereern i, svete gt fat ne a . fps ey (Gene .A goienal be oes HOLE " yt Oh Idererye tj ia-b-! yelp Hicuf UT +f wa at Sal teen) olilo} via aszqmiat ‘ioe ovine S6.f sicgsh leohs tn espero i 4) I > nian a \ svakewel ad) steed ny nee " i ‘) (OB bi ne * GO Oe, iow etna ire he a al ¢ 6 oNeee be aySioh ra A lubes ati sya 9A had wih 3, 9 ott mystead aege wh ot nae a OU » i ae folsret A ant A. to” * ah AR EE 4 Ra F Oe, eT ES Seb - 16} BBodianeetem a phtaty 4 wien Of Bese bw am seit a ets ry Rin 4: ne = a n ea . ‘ i aT cal api : ‘| cD | Ny ah af eee Ad i ¥3 3 Vs ery mom el chs vical > fet ei, W dcsbviey ap‘annete dings INVESTIGATIONS IN FISH CONTROL 2. Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals By Charles R. Walker, Chemist Robert E. Lennon, Fishery Research Biologist Bernard L. Berger, Chemist Bureau of Sport Fisheries and Wildlife US DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE Bureau of Sport Fisheries and Wildlife Circular 186 Washington, D.C. - June 1964 ADSEGAGCE: 55.5: fo cerca ies ole ome ANEIMYCIN:, Sie ams + sa EE [¥ Sources and uses..... CONTENTS Composition and structure. . Physical and chemical properties Biological: activity .: cue cles « che SoG bloc Methods and materials ..... Laboratory festsry ares. au Fieldstests).. 22.02. are Aertel is. fel foiasace, “atte Sisl aciets Results of laboratory studies........ awe iuae ic EL OUES!: 2 5c< e055: olen ewee aren teuter el. FACTS OS emeciie aire NOL CSO Ee oud OCD REECHES)%. 4 sitemeter eae RHIRES be’. s sie ctor sere ee nee ateate a SUCKEMRS | .c:c cies ie) icrloleutemeiter te SUMEUSHES Sis fe omatewaertes Sticklebacks (2.2055... sears Minnows and carps..... Fresh-water catfishes. .. Other animals......... Results of field studies..... Tests in wading pools... Tests in hatchery ponds. . Discussion of field studies. Gonclusions. ss. Ges. seis lene leiternatunre cited.) aloes li ee ee coeeeeee coeeeeee eoeeeeee e eecceee oeeeeee eoeeveevee ee oeeee eee . . ©, ee) (01/6 ‘06. 0 ame) 0 io) @) « eee ee ° ooo ew @ e eee ° ° . ec eee eee ee oeese eoee ee oee e eosceeeee eoeeeseee ee © @ 6 0 « ee ° e eee e e e eoeee eeee . eoeee eeeee eeee eee coeeee e coeoee eee eeee e oeeeeee coeeeeeee eee eoeoeeee eoeceeeeee coeceoee eee e ee ee LSP REYO DS Be ee ee RFK OOVnonnonunwm@at NN Se SS ake —" ON — “I Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals By Charles R, Walker, Chemist Robert E, Lennon, Fishery Research Biologist and Bernard L, Berger, Chemist Bureau of Sport Fisheries and Wildlife Abstract,--Antimycin A, an antifungal antibiotic, has been suggested for use as a fish toxicant, Preliminary tests were made to evaluate its effects at concentrations of 0,01 to 120 p.p.b, on 24 species of fresh- water fish in the laboratory and 25 species in outdoor pools, Responses of a select group of other animals and aquatic plants are discussed, The antibiotic is a powerful fish toxicant, Carp and other rough fish were killed by small concentrations in short exposures at cool and warm tem- peratures, Longnose gar, bowfin, black bullheads, and yellow bullheads were relatively resistant to the quantities tested, Plankton, aquatic plants, bottom fauna, salamanders, tadpoles, and turtles were not harmed by pisicicidal concentrations, Antimycin A degrades rapidly in water, especially in the presence of free hydroxide, Detoxification occurred within 24 to 96 hours, Further studies are planned on the performance of antimycin A against various life stages of fish, on other aquatic ani- mals, and in waters of differing qualities and temperatures, The process of detoxification and the fate of residues deserve further attention, An objective of the Fish Control Labora- tories is the development of new fish toxicants that can be used safely and economically in the management of fish populations, Antimycin A exhibits properties desired in a candidate fish toxicant, It is lethal to certain target fishes in low concentration and on short ex- posure; it works in cool and warm water and in the presence of aquatic plants; it degrades rapidly in water and appears to leave no harm- ful residue. This report summarizes data obtained on antimycin A in the laboratory and small out- door pools and larger hatchery ponds. Devel- opment and efficacy of the compound as a fishery tool is to be further investigated. ANTIMYCIN Sources and uses Antimycin is an antifungal antibiotic isolated from the bacteria Streptomyces sp. and identi- fied by Dunshee, Leben, Keitt, and Strong (1949) at the University of Wisconsin, Follow- ing this discovery, at least seven species of Streptomyces were found to be producers of antimycin. Burger, Teitel, and Grunberg crystalized the antibiotic from two species of Streptomyces (Strong, 1956), Later at the University of Wisconsin, another culture pro- duced an antimycin-like product which showed promise as an antibiotic for plant pathogens (Lockwood et al., 1954), Harada and associates (Nakayama et al., 1956) in Japan discovered an antimycin- producing culture of Streptomyces kitazawaensis which differed from the first culture at the University of Wisconsin, but both produce an antitumor substance (carzinomyceticus). Re- search at the University of Tokyo by Watanebe et al. (1957) on S. blastmyceticus yielded an antibiotic called blastmycin which consists largely of antimycin A3. Harada et al. (1959) devoted special attention to the antifungal property of blastmycin as a control for rice blast disease (Piricularia oryzae) in Japan. Derse and Strong (1963) related that anti- mycin is an antibiotic of unusual chemical structure which is toxic to yeasts, other fungi, insects, and mammals, but not to bacteria. They also reported that it is extremely toxic to goldfish at 1 p.p.b. On the basis of this observation, on the rapid degradation of the chemical, and its much lower toxicity to higher animals, they suggested that antimycin may be useful in fish management. Composition and structure The complex structure of antimycin was elucidated by Dunshee et al. (1949), Tener et al. (1953), Strong (1956) and Strong et al. (1960), van Tamelen et al, (1959 and 1961), and Dickie et al. (1963), It is illustrated in founre is Lockwood et al. (1954) described antimycin as a complex made up of several active frac- tions which they identified from paper chromatograms as Aj, Ag, Ag, and Ay accord- ing to increasing Rp values, Liu and Strong (1959) determined that one or more of these Rp values were represented in antimycin A-35, antimycin A-102, blastmycin, and virosin, and they investigated them, Further study by Dickie and his associates (1963) established that the fractions differ only in the alkyl side chain (R) in figure 1. The anti- mycin A; and A, fractions are probably iso- meric with R = n-hexyl, and calculations of the elemental composition indicate that the emperical formula is CogH4gN9Og. The Ag and Az isomers bear the n-butyl side chain, and the emperical formula is perhaps C96H36N209. The percentage composition of fractions or isomers is very important to the biological activity of the antimycin complex. Physical and chemical properties The fermentation extracts of antimycin are dark, tarry substances which upon further purification yield a fine crystalline material. This nitrogenous, phenolic complex is charac- terized by solubility in polar organic solvents including ethanol, acetone, and chloroform; slight solubility in nonpolar solvents including petroleum ether, benzene, and carbon tetra- chloride; and relative insolubility in water and 5S-percent solutions of hydrochloric acid, sodium bicarbonate, and sodium carbonate (Keitt, Leben, and Strong, 1953), The infrared absorption spectrum of anti- mycin has been identified in isolates from several cultures, although the crystalline products appear to have different properties. These differences are attributed to the intri- cate composition of the antibiotic and the presence of impurities associated with sam- ples (Strong, 1956), For example, blastmycin has almost the duplicate IR spectrum of anti- mycin A-35 isolate, but the melting points are 1669-1679 and 140.59-141.5° C, respectively. Blastmycin is composed primarily of the anti- mycin Ag fraction with a trace of Ay in con- trast to antimycin A-35, antimycin A-102, and virosin, which contain additional subcompounds A; and Ap» (Strong, 1956; Liu and Strong, 1959), Antimycin is susceptible to alkaline degra- dation as indicated in figure 1. Hydrolytic cleavage occurs at the lactone carbonyl sites on the cyclic diester and leads to the forma- tion of antimycic acid or blastmycic and the neutral fragment (van Tamelen et al., 1961; Liu et al., 1960; and Tener et al., 1953). The degradation is rapid in water, and detoxifica- tion of 10 p.p.b. is accomplished within 7 days according to Derse and Strong (1963); it is accelerated in the presence of light, high alkalinity, and warm temperatures, Biological activity Antimycin is a powerful and highly selective inhibitor of the electron transport in oxidative CH o f mi coat CONH = ioe aG CHACH(CH), i “ re) CH, \, R NHCHO Antimycin A, ; R= n-hexyl : CogHggn30, Antimycin A, , R= n-butyl : CogHsgNo0g COOH CONH —CH fe) te) CHs HCOH OH | CH NHCHO 3 R’ R Blastmycic acid Antimycin lactone CoH 4NoO5 CHO: 14°"24~4 ° R =O0CCH,CH(C | COOH | i eee se R=N-CyHyg CONH—CH HCOOH : CreH Formic acid i624 H COH R=00CCH,CH(CH,)> OH ! 1 CH Be Cons Antimycic acid == peneD ponte CH Ag. [5 ee R=OH | / R=n-Celis «x —n-hexyl - ae. a Glycine + levulinic acid salicylic aci Cy, Hoo C, H.NO 11 2043 2S 2 C,H,NO, Rane actetic acid Ce5Hi9%% Figure 1.--Structure of antimycin and the assumed process of breakdown under alkaline conditions in the laboratory. phosphorylation systems (Strong, 1956), It retards the respiration of cells, and the selec- tive action.in the electron transport chain at the cytochrome b-(Coenzyme Q)-cytochrome c has made antimycin an indispensable reagent for enzyme studies, Its effects on the suc- cinic-oxidase system have been described as the "antimycin-A-blocked factor,'' Gottlieb and Ramachandran (1961) illustrated the site of action of antimycin and ascosin as follows; Substrate --> Pyridine nucleotide--> Flavoprotein--> _cytochrome b_ === < = SSCA? ili 85 “coenzyme Q~ cytochrome c--> cytochrome a--> oxygen Because of its extreme potency as an inhibitor of electron transport, Derse and Strong (1963) surmised that antimycin is absorbed into the gills and interferes with respiration in fishes, METHODS AND MATERIALS Crystalline antimycin A was supplied by the Wisconsin Alumni Research Foundatior from Kyowa Fermentation Company, Ltd., in Tokyo, Japan, This material was isolated from the culture of Streptomyces kitazawaensis and had the following fractions by weight: Aj, 40 per- cent; Ay, 20 percent; Az, 20 percent; and Ay, 10 percent. Although the fraction Az amounts to only 20 percent, it accounts for about 60 percent of the biological activity. Stock solutions were prepared with 100 mili- grams of crystalline antimycin A dissolved in 1 liter of acetone. They were renewed with each series of bioassays, although tests in- dicated that solutions in acetone are relatively stable up to 24 days. Crystalline material stored at room temperature for 2 years also remained stable, Laboratory tests The methods and facilities employed for evaluation of potential fish-control agents were described by Lennon and Walker (1964), The bioassays of antimycin A were conducted in slightly alkaline and medium hard, reconsti- tuted water at 129, 179, and 22° C, Twenty- four species of fish, representing nine fami- lies, were included (table 1). They were supplied by national fish hatcheries, the Wis- consin Conservation Department, and Ozark Fisheries, Inc., and each lot was graded to a desired size before use. Aliquots of the stock solution of antimycin A were diluted and stirred into the 1- or 5- gallon bioassay vessels in the presence of fish. The responses of the fish to the toxicant were observed at 24, 48, 72, and 96 hours, Other animals included in bioassays were water fleas (Daphnia magna), crayfish (Cambarus sp.), damselfly nymphs (/schnura sp.), tiger salamander (Ambystoma tigrinum), and bullfrog tadpoles (Rana catesbiana). They were stocked in bioassay vessels as follow: 10 water fleas or 2 damselfly nymphs in each 16-ounce jar, 1 crayfish or 2 bullfrog tadpoles in each 1- gallon jar, and 1 adult tiger salamander in each 5-gallon jar. Field tests Vinyl wading pools.--Only a few outdoor bioassays were made in 1962 and 1963 because only small quantities of toxicant were available. TABLE 1.--The 24 fishes used in laboratory tests of antimycin A = Size range Common name Technical name (grams) Gizzard shad...-e+e+eseeee Dorosoma cepedianum 12.0-15.0 Rainbow trout.......-..+- Salmo gairdneri 1.0- 1.6 Brown trout...-+seeeeeeee Salmo trutta 1.2- 1.4 Northern pike......-..++-. Esox lucius 0.5- 0.6 StonerOller....+ssesesees Campostoma anomalum 3.0- 4.0 Goldfish... --ceccccvcscece Carassius auratus 1.5- 2.4 CALPecccccenscccccvcccces Cyprinus carpio 0.6- 2.3 Golden shiner...'...-..+-- Notemigonus crysoleucas 1.0- 2.2 Fathead minnow..«........-. Pimephales promelas 0.9- 1.8 White sucker...-ceesecsee Catostomus commersoni 1.3- 2.8 Bigmouth buffalo......... Ictiobus cyprinellus 1.6- 2.5 Black bullhead........... Ictalurus melas 0.7- 2.3 Yellow bullhead........-. Ictalurus natalis 1.2- 2.2 Channel catfish.......... Ictalurus punctatus 1.5-1.8 Brook stickleback........ Eucalia inconstans 0.6- 1.0 Green Sunfish...ecsssseee Lepomis cyanellus 0.8- 2.5 Pumpkinseed...-+eseeeeeee Lepomis gibbosus 1.0- 2.3 BIUe LIT... ccc eccceereces Lepomis macrochirus 1.2- 2.4 Llongear sunfish.........- lepomis megalotis 1.0- 2.5 Largemouth basS...++-+-«- Micropterus salmoides 1.8- 2.9 White crappie..-.cesecees Pomoxis annularis 1.5- 3.0 Towa darter...cecceceseee Etheostoma exile 0.6- 1.2 Yellow perch....scccescee Perca flavescens 0.6- 3.0 Walleye...cecescsccccuces Stizostedion vitreum 0.4- 0.8 The test vessels were 1,000-gallon wading the course of tests according to standard pools similar to those described by Lawrence methods (American Public Health Association and Blackburn (1962), Some physical, chemi- et al., 1960), cal, and biological conditions characteristic of ponds were simulated or intrinsic, The Hatchery ponds.--The Wisconsin Conserva- physical aspects included bottom soils of sand tion Department provided two ponds for tests and loam, naturally varying temperatures, at the Delafield Warmwater Fisheries Re- turbidity, and natural light. The chemistry of search Station in September 1963, The sur- the well water in the pools was modified by face areas of ponds No, 2 and No. 5 are 0.47 physical and biological factors, and 0.78 acre respectively (fig. 2), Of the 18 pools, 9 had 3 inches of sand on Pond No. 2 was stocked with 18 species of the bottom, and 9 had 3 inches of silt loam, fish at the rate of 240 pounds per acre, and After the pools were filled, the following were pond No. 5 with 19 species at 225 pounds per introduced: Sagittaria latifolia, Elodea canadensis, acre, 1 week before antimycin was applied. Myriophyllum heterophyllum, Potamogeton nodosus, Samples of water, plankton, and bottom fauna P. pectinatus, Spirogyra spp., and phytoplankton. were taken from each pond soon after the They were established, and the water chemis- fish were stocked and again just before the try was stabilized, during the 4- to 8-week ponds were drained (table 2), periods before fish were added. Fingerling and adult fish were stocked 1 to 2 weeks be- ™ ease agiaivon tant end emt trout at eclected yotex temperatures 40, fore applications of the toxicant. 24 and 96 hours F Concentrations (p-p-b.) and survival Number | Temper- Species At 96 hours The rate of detoxification of the antimycin was observed, and some of the killed fish were shipped to the Wisconsin Alumni Research Foundation for mammalian toxicity tests. Bottom fauna were sampled and quantitated. Tay a Data were obtained on water chemistry during POND NO. 2 YY Chara * + Spirogyra °o Potamogeton CONTOUR ACRE-FEET CONTOUR ACRE - FEET O - I ft. 0.44 O -| ft. 0.68 | — 2ft. 0.33 1-2 ft. 0.63 VAP LE 0.01 2-3 ft. 0.42 3+ ft. 0.17 TOTAL 0.78 ——_— TOTAL 14.90 Figure 2,--Sketch of ponds No, 2 and No, 5 at the Delafield Warmwater Fisheries Research Station, 5 Two formulations of antimycin A were pre- pared for application at 10 p.p.b. Pond No. 2 received 9.72 grams of technical material in a carrier formulated by the S. B. Penick Com- pany to make up a total volume of 300 ml. Pond No. 5 received 23.37 grams of technical material dissolved in 300 ml. of acetone as a carrier, Each aliquot was mixed with 2 gal- lons of water and applied to a pond surface with a hand-powered garden sprayer. The applications were made from a rowboat in late afternoon, and frequent observations were made during the next 8 hours, Observa- tions and recovery of dead fish were made daily in the following 4 days. RESULTS OF LABORATORY STUDIES We found that antimycin A is toxic to the 24 species of fish tested, The toxicity varies among species and is correlated with water temperature and time. Trends in sensitivity reflect taxonomic relationships of the fishes, and variations in susceptibility among indi- viduals was more pronounced in some species than others. The following remarks pertain principally to the concentrations which delin- eate the all-or-none survival ECp to ECyo9 ranges, of fish at 24 or 96 hours in bioassays at 129, 17°, or 22° C, Data are shown graph- ically in figures 3 and 4, Among the 24 species, the group of fish most sensitive to antimycin A includes giz- zard shad, rainbow trout, brown trout, white sucker, Iowa darter, yellow perch, and wall- eye. All survived exposure to 0.08 p.p.b. for ‘24 hours at 12° C; all perished at 0.8 p.p.b. The group intermediate in sensitivity in- cluded northern pike, stoneroller, carp, golden shiner, fathead minnow, bigmouth buffalo, brook stickleback, green sunfish, pumpkinseed, bluegill, longear sunfish, largemouth bass, and white crappie (fig. 5). Concentrations of 0,1 and 1.6 p.p.b. defined their all-or-none survival in 24 hours at 12° C, GIZZARD| SHAD -—— — — 7 ee RAINBOW TROUT — — -—~———___, BROWN TROUT — — - -~>——4 rad NORTHERN PIKE — — — — — — — — ——— STONEROLLER — — — — — - —— — i eT GOLDFISH) ck eae ge Deas ee ey ye ae gt ee ——— GOLDEN SHINER — — — — —--—— -- SSS FATHEAD MINNOW-— — — — — — —— SSS WHITE SUCKER — — — — —— —_ BIGMOUTH BUFFALO — — — — — — — — TS BLACK “BULLHEAD PP he ota ae ai ASIEN (UWL Sa SS CHANNEL CATFISH — — — BROOK ‘STICKLEEBACK = = = = = FEE ATEN GREENUSUNFISH 2? oe a oy PUMPKINSEED - — — ee ee BRUEGI es a= mm Co TEMPERATURE mmm 12°C LONGEARGSUNEISHs—) — —s—h—o Soe 9 17° wa 22°C LARGEMOUTH BASS — — — — — — — — a as Winule! CRAPS = = SSS SSS [Esa 2 IOWA DARTER — — — -—- --- - —-————E YELLOW PERCH--— — ee WALCEYES OS =] = = SSeS Ss Sarees Sa nite Ea TT TRE =e eRe GLC ESS oan ke 0! 0.1 1.0 10 100 Figure 3,--The 24-hour responses of 24 fishes in the laboratory to antimycin A in p.p.b, The solid, plain, and cross hatched bars span the ranges between the EC, and ECy9q at 12°, 17°, and 22° C, GIZZARD SHAD— —- — — ee en RAINBOW TROUT — — — -————— OE pa ee a a a J) anos ay CS a et oe et — FATHEAD MINNOW — — — — — — —— io WHITE SUCKER — ~~ —— ———e BURCH IBULLREAD — — —- 4 — > eo — YELLOW BULLHEAD—- — - — —- —~--—-— - - CHANNEL CATFISH —- —- — -- -- ----- -— Sheen SUNFISH Sccp- - - eerie PUMPKINSEED — — — PERO eee BLUEGILL - — - - ~ - [Hee LONGEAR SUNFISH ~ ~ ~~~ LARGEMOUTH BASS — — — — — — — — REE IOWA DARTER -- - - -- - -- co. ae Se SSS TEMPERATURE mma (2°C ——! I7% GZ 22C Of 0.1 1.0 10 fue Figure 4,--The 96-hour responses of 16 fishes in the laboratory to antimycin A in p,p,b, The solid, plain, and crosshatched bars span the ranges between the ECp and ECyq9 at 12°, 17°, and 22° C, The more resistant group of fish was represented by goldfish, black bullhead, yellow bullhead, and channel catfish. The concentrations required for kills in 24 hours at 12° C were 20 p.p.b. for channel catfish, 80 p.p.b. for yellow bullhead, 100 p.p.b. for goldfish, and 120 p.p.b. for black bullhead. Increases in water temperature or duration of exposure made significant differences in the toxicity of antimycin A to fish in the three groups. For example, the toxicity to goldfish was increased tenfold at the higher tempera- ture of 17° C, Among catfishes, the toxicity was enhanced about twofold at 179°, At the maximum temperature of 22°, the black and yellow bullheads were about 10 times as tolerant to antimycin A as goldfish, but channel catfish were only slightly more resistant. For more detailed discussion on the toxicity of antimycin A, the species are grouped according to their respective families, The families, in turn, are presented in order of their sensitivity to the toxicant. Trouts Rainbow trout and brown trout were ex- tremely sensitive to antimycin A (table 2), At 12°, the rainbow trout succumbed to 0.6 p.p.b. in 24 hours and to 0.08 p.p.b. in 96 hours. At the same temperature, brown trout were killed by 0.4 p.p.b. in 24 hours and by 0.08 p.p.b. in 96 hours, Both species tolerated concentra~ tions of 0,1 p.p.b. for 24 hours, In 96-hour tests, the rainbow trout survived 0.02 p.p.b. whereas brown trout withstood 0,06 p.p.b. Herrings At 12° C,, all gizzard shad died within 24 hours upon exposure to 0.8 p.p.b. and within 96 hours at 0.1 p.p.b. (table 3), They were especially sensitive to the toxicant at 22°: MOST SENSITIVE INTERMEDIATE LEAST SENSITIVE TROUTS PIKES FRESHWATER CATFISHES >) Py @ 7, PERCHES SUNFISHES SOT oe | EE, < te) HERRINGS SUCKERS Gxt ax STICKLEBACKS a MINNOWS AND CARPS % < ; GOLDFISH <=. (ee ye Figure 5.--The order of sensitivity of 11 families of fish to antimycin A in the laboratory and field. concentrations of 0.04 p.p.b. caused complete died at 0.08 p.p.b. within 24 hours and at kills within 24 hours, and partial kills occured 0.06 p.p.b. within 96 hours. at 0.02 p.p.b. or more, It was noted that a narrow range of concentrations yielded all-or- | none survival, particularly at the higher tem- Pikes 2 perature and longer exposure. The fry and fingerlings of northern pike were difficult to use in bioassays because of Perches cannibalism and rapid growth. Nevertheless, f they exhibited great susceptibility to anti- The Iowa darter, yellow perch, and walleye mycin A, Complete kills were obtained in 24 were also very sensitive to antimycin A (table 4), All’specimens in 0.08) p/p:b> AGIOS cg, acres acc ey ee for 24 hours survived, but those in 0.66 p.p.b. died. The narrow range in concentrations which caused all-or-none survival was more apparent at 229 and 96-hour exposures. Yellow perch, for example, survived 0.02 p.p.b. for 24 hours and 0.01 p.p.b. for 96 hours; they Concentrations (p.p.b.) and survival hours by 0.8 p.p.b. at 12°, 0.2 p.p.b. at 17°, and 0.1 p.p.b, at 22°, In contrast, all speci- mens survived 0.4 p.p.b. at 12°, 0.08 p.p.b. at 17°, and 0.06 p.p.b. at 22°, Greater toxicity was detected in 48-hour exposures, but the concentrations related to all-or-none survival were not defined. Suckers The white sucker and bigmouth buffalo differed in their sensitivities to the toxicant, and the former was among the most sus- ceptible fishes tested (table 5), Concentra- tions greater than 0.06 p.p.b. produced par- tial kills of white suckers at 12° in 96 hours, and 0,22 p.p.b. caused complete kills, Even greater sensitivity was observed at 22°, The bigmouth buffalo, on the other hand, required concentrations of antimycin A in excess of 0.4 p.p.b. for complete kills in 96 hours at 12°, Sunfishes Green sunfish, pumpkinseed, bluegill, long- ear sunfish, largemouth bass, and white crappie were moderately sensitive to antimycin A TABLE 4.--Concentrations of antimycin A which caused all-or-none survival among Iowa daters, yellow perch, and walleye at selected water temper- atures in 24 and 96 hours Concentrations (p.p.b.) and survival Number | Temper- Iowa darters.... Yellow perch.... TABLE 5.--Concentrations of antimycin A which caused all-or-none survival among white sucker and bigmouth buffalo at selected temperatures in 24 and 96 hours Concentrations (p.p.b.) and survival Species Number | Temper- of ature fish | (“C.) 12 810 36 17 22 White sucker.... Bigmouth buffalo 12 (table 6), The concentrations required to cause complete kills of them at 12° ranged from 1 to 6 p.p.b. in 24 hours and from 0,2 to 0.8 p.p.b. in 96 hours, At 22°, killing concentrations ranged from 0,2 to 0.8 p.p.b. in 24 hours and from 0,08 to 0,4 p.p.b, in 96 hours, The pumpkinseed and bluegill were the more sensitive of the six species, and they were followed in order of decreasing sensitivity by longear sunfish, largemouth bass, white crappie, and green sunfish. Sticklebacks Brook sticklebacks were moderately sensi- tive to antimycin A at 12°, Concentrations of 5 p.p.b. killed all specimens within 24 hours, and partial kills occurred at concentrations greater than 0.5 p.p.b. The exposures beyond 24 hours failed to give consistent results. The condition of the fish was suspect because of difficulty in maintaining them without feeding during the longer test periods. Minnows and carps Stoneroller, goldfish, carp, golden shiner, and fathead minnow responded over a wide range of concentrations in an interesting pattern of susceptibility. In contrast to other families, the minnows exhibited greater vari- ation in response between species as well as between individual speciments (table 7). TABLE 6.--Concentrations of antimycin A which caused all-or-none survival among green sunfish, pumpkinseed, bluegill, longear sunfish, largemouth bass, and white crappie at selected water temperatures in 24 and 96 hours Concentrations (p.p.b.) and survival Number | Temper- Species of ature fish | (°°¢) None Green sunfish.... 396 12 6.00 0.80 See goa on 216 17 2-00 0.60 DO cjncencciecee 30 22 0.80 0.40 Pumpkinseed...... 480 12 2.00 0.20 aiieie atetats tava 120 tify 1.00 0.10 DeREeGecpogcn 180 22 0.20 0.08 Bluegill......... 1,053 12 1.00 0.40 DOwreteivin ele nic'v'e 360 v7. 0.60 0.10 TY sisteidinia wcities 200 22 0.20 0.08 Longear sunfish 240 12 0.20 2.00 0.40 eb scdacosec 48 17 0.08 0.40 0.20 DG sis.cisielvin visisin 48 22 0.08 0.40 0.20 Largemouth bass.. 800 12 0.20 <6.00 0.80 White crappie.... 180 12 0.60 >2.00 -- TABLE 7.--Concentrations of antimycin A which caused all-or-none survival among stoneroller, goldfish, carp, golden shiner, and fathead minnow at selected water temperatures in 24 and 96 hours Species boo lokoke) oo An outstanding highlight of the screening program was the discovery that carp are vulnerable to small concentrations of anti- mycin A, This prolific exotic is widely con- sidered a most undesirable species in game- fish waters and is difficult to control with existing means. At 12° all test carp were killed by 2 p.p.b. of antimycin in 24 hours and by 0.6 p.p.b. in 96 hours; at 17° all were killed by 1 p.p.b. in 24 hours and by 0.4 p.p.b. in 96 hours, Tem- peratures had less effect on toxicity to carpthan to most species. There were only slight dif- ferences due to temperature in 24-hour ex- posures and even less at 96 hours, All carp survived 0,08 p.p.b. The results on goldfish contrasted sharply with those on carp. In fact, the goldfish was the most tolerant of the minows tested against antimycin A, It required 100 p.p.b. for com- plete kills within 24 hours at 12°, but only 2 p.p.b. were needed for kills within 96 hours. Higher temperatures contributed to greater toxicities, and all goldfish perished within 96 hours when exposed to 1 p.p.b. at 17° and 0.6 p.p.b. at 22°, Stonerollers were among the more sensi- tive minnows. Concentrations of toxicant as low as 1 p.p.b. killed all specimens within 24 hours at 12°, but variations in suscepti- bility were observed; a concentration which killed on one occasion failed on the next. The golden shiner and fathead minnow were somewhat similar to the stoneroller in sen- sitivity, but all-or-none effects were delin- eated within a narrow range of concentrations. The golden shiners succumbed to 0.6 p.p.b. within 96 hours at 129, and survival was noted at 0.05 p.p.b. Fathead minnows died at 0.4 p.p.b, and survived at 0.08 p.p.b. Fresh-water catfishes The catfishes were significantly less sen- sitive to antimycin A than other families (table 8), Channel catfish were more suscep- tible than bullheads, They survived 24-hour exposures at 12° to 2 p.p.b. but perished at 20 p.p.b, All specimens died at 6 p.p.b. in 96-hour tests at 22°, The black bullhead was the more tolerant to the toxicant, and the yellow bullhead was only slightly less so. Concentrations of 120 and 100 p.p.b. respectively were required for complete kills in 24 hours at 12 . These concentrations are more than 100 times greater than those needed to kill fish of the most sensitive families. The bullheads were affected by somewhat smaller quantities of chemical at 17 . Never- theless, black bullheads tolerated 4 p.p.b. for 24 hours at 22°, and all died at 40.p.p.b. TABLE 8.--Concentrations of antimycin A which caused all-or-none survival among black bullhead, yellow bullhead, and channel catfish at selected water temperatures in 24 hours and 96 hours Namber |) Temper= Concentrations (p.p.b.) and survival Species. Black bullhead... Do 10 Other animals Four hundred water fleas were used in trials with antimycin A, At 12° C., specimens survived 1 and 0.5 p.p.b., but died in 100 p.p.b. in 24 hours and in 10 p.p.b. in 48 hours, Their susceptibility increased with temperature, At 22°, they survived 0.1 p.p.b., but died in 10 p.p.b. in 24 hours and in 0.5 p.p.b. in 48 hours. There were no mortalities among 20 cray- fish exposed to 10 p.p.b. of toxicant at 12° for 96 hours, Tests with 120 damselfly nymphs disclosed that the insects were relatively tolerant to antimycin A, At 12°, specimens survived 100 and 50 p.p.b. for 24 and 48 hours respectively, and 1,000 and 500 p.p.b. were required to kill them in the same time periods. At 229, they survived 50 and 10 p.p.b., but died at 500 and 100 p.p.b. in 24 and 48 hours, The observations were not continued to 96 hours because high mortalities began to occur among controls. Ninety-six tiger salamanders were exposed to antimycin A at 12°, Specimens survived 80 p.p.b. for 96 hours, but were killed by 600 p.p. b, Among the 40 bullfrog tadpoles tested for 24 hours at 12°, the individuals exposed to 20 p.p.b. of toxicant survived whereas those subjected to 40 p.p.b. perished, RESULTS OF FIELD STUDIES Tests in wading pools Results in 1962,--Some preliminary bio- assays were conducted in 18 pools in July and October, to determine the utility of the pools as bioassay vessels and to yield information on the performance of antimycin A outdoors, A shortage of toxicant limited the scope of the trials, and a scarcity of fish of desirable species, sizes, and condition affected their validity. A number of the species were wild fish which later proved to be unsatisfactory test animals because of variable sizes, heavy parasitism, and poor condition. The wading pools worked well as bioassay vessels, Fish, invertebrates, and plants did well in the test units and controls, There were some differences in the quantity of plankton and aquatic vegetation in the sand- and loam- bottom units because the latter were more fer- tile. The abundance of plants, we believe, con- tributed to increases in pH and alterations of alkalinity, and these in turn influenced the efficacy of antimycin A, Goldfish, golden shiner, black bullhead, blue- gill, largemouth bass, and yellow perch were exposed to 5 and 10 p.p.b, of toxicant in July. Most of them survived in the sand pools. The mortality was greater in the loam pools, es- pecially at 10 p.p.b., but in no instance did it reach 100 percent. The black bullheads ex- hibited high tolerance to the toxicant in all pools. Another series of tests was made in October with higher concentrations against rainbow trout, goldfish, golden shiner, bluntnose min- now, yellow bullhead, green sunfish, and yellow perch (table 9), The pH values in the pools at the time ranged from 7.5 to 9.9. Ninety to 100 percent of the trout, golden shiner, bluntnose minnow, green sunfish, and perch, and 60 per- cent of the goldfish were killed by 20 p.p.b. over sand and loam bottoms, At 40 p.p.b., there was very low survival among the trout, gold- fish, and sunfish, but nearly complete survival of bullheads. There appeared to be rapid degradation and detoxification of antimycin in the pools within 24 to 96 hours, depending on the initial con- centration and the pH. Small numbers of gold- fish, golden shiner, bluntnose minnow, blue- gill, and largemouth bass were stocked later in pools in which antimycin A had been present for 24 to 72 hours. No more than half of the golden shiners and bluegills perished within the following 2 days, Results in 1963.--The plants, plankton, and bottom fauna were permitted to develop in the pools for 2 months before toxicity trials. In July, acetone solutions of antimycin A were tested at 10, 20, 40, and 80 p.p.b. against eight species of fish of various sizes (tables 10, 11, 12, and 13), Golden shiners, bluegills, large- mouth bass, and yellow perch were the more TABLE 9.--Toxicity of antimycin A at 20 and 40 p.p.b. on adult and fingerling fish in sand- and loam-bottom pools [Mortalities are cumulative by observation periods] Antimycin A at 20 p.p.b. +p.b. Species Type myc p-p Antimycin A at 40 p.p.b of of 2 fish Botih, wunitbar’ Number dead in (hours) Number dead in (hours) -- of fish Adults: Rainbow trout...... Ceres cvcscsce sand DOceeccerccccccccccsesscsvssses loam Yellow bullhead....cccecceceseees sand DOecercccccccccvccvescceseveses loam Green sunfish.....sesesccesee eeee sand DOwccevccces on ccceeccecre wewace loam Fingerlings: GOTAEUSH <6 < wie ease cinnccnvevers cio sand DOwsevcccvcccscvccsvecrccssceves loam Golden shiner....csececcscsececce sand DOvccccccscsccccccvsccsecessscce loam Bluntnose MinNOW...+eseseeeeeeeee sand DOeececcccccccccscccnccscescscs loam Yellow perch....... vieeccccanavene sand Doweceees cece ccc ecerceccceeces loam sensitive, and they were killed by 10 p.p.b. within 48 hours over sand and loam bottoms, BN SraEe Carp and green sunfish perished at 10 p.p.b. TABLE 10.--Numbers and sizes of fish exposed to antimycin A in wading pools in July 1963 Susciee weight (grams) within 24 hours over loam bottoms and at 20 p.p.b. in the same time over sand soil. Some lots of goldfish died at 20 p.p.b., but all suc- cumbed at 40 p.p.b. A concentration of 80 Golden shiner.....scscecsceccacses ae ot ee 180 18.0 p.p.b. killed all fingerling and adult black Gréan-auutlenee ss ne dddtoviveea Bee 144 2.7 bullheads within 48 hours over sand bottom Bluegill... 270 0.8 but only one-half of them over loam. Dosecvaceccncccvecccrscvceeere 180 22.0 Largemouth bass...+.+++- acta 4 1.5 The green sunfish and yellow perch ap- Yellow perch.....+++.+4+ setee enone 2.5 peared at the surface of pools within 2 hours TABLE 11.--Toxicity of antimycin A at 10 and 20 p.p.b. on adult and fingerling fish in sand- and loam-bottom wading pools [Mortalities are cumulative by observation period] Antimycin A at 10 p.p.b. Antimycin A at 20 p.p.b. rs) ies i == i se specie Nombess Number dead in (hours) Number Number dead in (hours) of fish of fish Adults: Black DULTHeAd SS csc ca cisiac vassal sand 20 fe) LO FOAC AID TOOODCUOOOUAROOOTOOAI A loam 20 3 Bluegill..... ae ccieserecevceceore Sand 20 -- DO. ceccccccvcescccccccccsvcces loam 20 20 -- Fingerlings: Goldfish... ccccccceccnccsccecscnce Sand 20 6 -- DOs ia'e\cie.ciciciciviccieje cleisicieivie ele'eviaieis loam 20 rs 10 Carpe etecccccccccccvcccccccccces sand 20 14 == DO ve cleisie(eecnisjncespieauesbisssicins loam 20 20 14 GoldenySHineri cin /sislsls|sieinfeie vie elsiatets sand 14 14 -- DOs ewecccncccceevcvcssveccece loam 14 14 a= Black DULIDE Ades tslelelslsisielsielelalsieleieis sand ie} DO steiejajelelavelalsrelaie(e ofeyw/aiejele/stelslais oie loam te) Green sunfish...csecsscconccnucs sand -- DO. vcccccnccccvcccccccccccsccs loam a BL We PAA atsyoin\s elminie (o/s /n\u(arsiofatuinia’ninfe! sand ed DOnfemiviels ecccee ee eceesceereccce loam -- Largemouth baSS.......eseeesenes -- Yellow perch........ ee ececeecees DOs ccscccccccce eocceee eeeesces «Xa —— ae TABLE 12.--Toxicity of antimycin A at 40 and 80 p.p.b. on adult and fingerling fish in sand- and loam-bottom wading pools (Mortalities are cumlative by observation period] Species Terr Terre eee eee eee eee eee Yellow perch. . TABLE 13.--Average values of analyses made on water from sand- and loam- bottom wading pools before and after applications of antimycin A in July 1963 Unit of iam Pattee ters % 25 27 Resistivity........... at 20°c | 2803 2864 3037 3052 Dissolved oxygen...... p-p-m.O5 8.7 9.1 9.7 9.7 Carbon dioxide........ p-p-m.CO, 0.0 0.0 0.0 0.0 Hydrogen ion.......... pH 8.8 9.1 8.8 9.2 Total alkalinity...... p-p-m.CaC0, | 204.4 181.2 198.2 183.7 (as phenolphthalein) | ........... (30-7)! | (Gates ( 14.6) | ( 14.3) (as methyl orange)..| ........... (193.7) | (169.7) | (183.6) | (169.4) Total hardness........ 211.8 176.0 210.6 182.0 Calcium hardness...... 53.6 47.9 60.0 53.6 Btruestectrtoris scsiclcles'e a sss 0.0 0.0 0.0 Sulfate ion........... 25.8 18.3 abipy Total phosphorus...... 0.059 0.082 0.106 Ammonia nitrogen...... 0.399 0.710 1.100 Nitrite nitrogen...... 0.006 0.005 0.018 secon -p-m. . - 0.154 after exposure to the toxicant, and they ex- hibited a narcosislike condition. They showed little response to motion stimulus or handling with a dip net. Some of the larger bullheads behaved as if in distress and were subject to development of an unidentified funguslike condition on the body prior to death. Antimycin A at 80 p.p.b. Number Number dead in (hours) -- on eee A at 40 | Atinysin A at 40 ppb p-b. ae Number Number dead in | Number dead in (hours)-- | of fish of fish oa ee Se ee oe 0 2 ss 0 1 3 0 0 — 10) 19) 11 13 The trials in October included two formula- tions of antimycin. One was a solution in ace- tone, and the other an emulsifiable concen- trate, applied to pools at 1, 5, 10, and 100 p.p.b, against 10 species of fish. The pH values at the time in all pools were about 10, and the antimycin A degraded so rapidly that most fish escaped toxic effects (table 14), The TABLE 14.--Average values of analyses made on water from sand- and loam- bottom wading pools before and after applications of antimycin A in October 1963 2 loam aes en Arter Temperature...ssseeeee 6 15 Resistivity.....-...-- at 20°C 3396 Dissolved oxygen....-+. p-p.m.02 9.5 Carbon dioxide........ p-p.m.COz 0.0 Hydrogen ion..-....++. PH 9.8 Total alkalinity...... p.p.m.Cacd; 121.0 (as phenolphthalein) | ....-++.++. (34.0) (as methyl orange).. |] wscccceeesee (88.0) Total hardness.......- p.p.m.Caco. 154.0 Calcium hardness...... p-p-m.CaC03 = . = 35.0 Total iron....s++sses p-p.m.Fe~ 0.028 Sulfate ion......000.- P-p-m-SO, 14.0 Total phosphorus...... p-p-m.PO, 0.035 Ammonia nitrogen...... p-p-m.NH3 0.550 Nitrite nitrogen...... p-p-m.NO2 0.0 Nitrate nitrogen...... 0.0 Chloride ion.......... 13.6 exceptions were those individuals exposed to 100 p.p.b. It appeared that the acetone solu- tion of toxicant deteriorated sooner than the other preparation. Of the 10 species of fish, 7 species suc- cumbed totally to 100 p.p.b. of acetone- antimycin A, and 9 species to the emulsifiable formulation, within 24 hours over sand bot- toms; only carp, fathead minnow, bluegill, longear sunfish, and yellow perch died over loam bottoms, The black bullhead was the sole survivor of 100 p.p.b. over both bottom types. Neither preparation of toxicant caused 100-percent kills of any species within 96 hours at 5 or 10 p.p.b. In general, most of the susceptible fish showed signs of distress within a short time after exposure, and many came to the surface of the pools. The length of time which elapsed before death varied with the species and water temperature, and ranged from a few hours to several days. It is significant that all specimens which displayed symptoms of dis- tress eventually died. This suggests that the action of the toxicant on fish is irreversible, There were no grossly toxic effects by anti- mycin A on the plankton, bottom fauna, or aquatic plants during the course of the July and October trials, For example, in the four pools which received 20 p.p.b. of antimycin A in July, the average quantity of plankton was ‘0.0036 cc./1. (range: 0.0020 to 0.0044) before treatment and 0.0040 cc./1. (range: 0.0033 to 0.0061) at 20 days after treatment. The quan- tities in two control pools were 0.0047 and 0.0089 cc./1. during pretreatment sampling and 0.0022 and 0.0044 cc, /1. during post- treatment sampling. Tests in hatchery ponds There appeared to be a more rapid re- sponse of fish to the antimycin A which was formulated with an emulsifiable concentrate than with acetone, With the former prepara- tion in pond No, 2, fish surfaced within 4 to 6 hours after application, whereas in pond No. 5 there were no comparable effects for another 10 hours. By the end of the first full 14 day, we saw no Significant differences in the effects produced by the two formulations. Table 15 gives before and after water analyses for the two ponds, Northern pike were the first fish to exhibit distress, They surfaced and appeared to be in a state of narcosis which was followed by com- plete locomotor ataxia. The rainbow trout, white suckers, carp, walleye, and sunfishes followed in order with similar symptoms. The great majority of specimens were dead within 48 hours (tables 16 and 17). It is noteworthy that goldfish--a species which was relatively TABLE 15.--Analyses of water from ponds No. 2 and No. 5 at Delafield Warmwater Fisheries Research Station before and after applications of antimycin A in September 1963 mas ot, of [ Pond No. 2 Pond No. 5 Before | After Temperature..........- ay 21 15 Resistivity........++- at 20°C 2550 2600 Dissolved oxygen...... p-p-m.0, 6.7 6.9 Carbon dioxide........ p-p-m.CO, 3.4 0.0 Hydrogen ion.........- pH 8.0 8.4 Total alkalinity...... p-p-m.CaCO,| 210.0 202.0 (as phenolphthalein) | ..........- ( 0.0) ( 0.0) (as methyl orange)..| .....-..+-- (210.0) (202.0) Total hardness........ p-p-m.CaC0,| 213.0 220.0 Calcium hardness...... p-p-m.Cac0, 77.0 82.0 Manganese......--+-++- p-p-m.Mn° 0.0 0.0 Mayall TMs caccoachoda p-p-m.Fe® 0.00 0.05 Sulfate ion..........- p-p-m.SO, 44.3 38.0 Total phosphorus...... p-p-m. PO, 1.40 0.10 Ammonia nitrogen...... p-p-m.NH, 0.20 0.19 Nitrite nitrogen...... p-p-m.NO, 0.0 0.0 Nitrate nitrogen...... p-p-m.NO, 0.07 0.50 Chioride ion.......... p-p-m.CL 14.5 TABLE 16.--Effects of 10 p.p.b. of antimycin A in emulsifiable concentrate on 18 species of fish in pond No. 2 Number of fish dead at Total (Gomme )e= fish stocked Average length (inches) Average weight (grams) Species we oa ae 3 25.6 658 ie) eect sent ene 1 16.8 545 ie) 6) Rainbow trout..... 312 4.0 82 312 -- -- Northern pike..... uF 17.8 713 5 5 7 GolGRSH se elelnlese/e) 740 2.4 -- -- Gen eteteteletetatstetaleratete 18 1553) 17 18 | -- -- White sucker...... 4 els 3 3) 3 4 Black bullhead.... 600 3.8 lo) (0) lo) lo) Yellow bullhead... 4 8.3 0 te) ie} ie} Brown bullhead.... 1 4.2 lo) 0 to) ie} Rock bass......... 1 8.0 1 -- | -- == Green sunfish..... 3 3.8 (6) ie} 1 3 Pumpkinseed....... 13 4.6 BB -- pkiey-siblS Soe saeaeo 27 6.1 22 27 Black crappie..... "7 8.3 Das 7 Largemouth bass... 4 15.4 =e == Hybrid sunfish.... 1,400 -- Op un Wee Ssccooond. TABLE 17.--Effects of 10 p.p.b. of antimycin A in acetone solution on 19 Species of fish in pond No. 5 Number of fish dead at Average | Average Species length (inches) Longnose gar...... 6 Bowfin. ..esseeeees 8 Rainbow trout..... 1 Northern pike..... of Goldfish........+. 7 Carp. nccccescccee 3 White sucker...... 7 Black bullhead.... 7 Yellow bullhead... 7 Brown bullhead 4 Rock bas8......... 2 Green sunfish..... 2 Pumpkinseed....... 4.6 6.4 8.8 12.8 Black crappie..... Largemouth bass... Hybrid sunfish.... Walleye....ceccsee Drum eee eee weneee Pop aon tolerant to antimycin A in the laboratory--died in both ponds within 24 hours. The longnose gar, bowfin, black bullhead, yellow bullhead, and brown bullhead were the only species which were not affected greatly by the toxicant at 10 p.p.b. Seventy percent of them were recaptured alive when the ponds were drained after 20 days. The detoxification of antimycin A was moni- tored throughout the first 96 hours, It occurred within 72 hours after application, and fish placed in live cages after this time survived until the ponds were drained. Plankton was sampled in both ponds during the experimental period. In pond No, 2, the pretreatment quantity was 0.018 cc,/1 and the posttreatment quantity was 0.047 cc./1, Pond No. 5 had pretreatment and posttreatment quantities of 0.0035 and 0,039 cc. /1. None of the relatively minor changes was attributed to the toxicant. Also, there were no observable changes in the aquatic plants in the ponds. Pretreatment and posttreatment samples of bottom fauna were taken. We concluded that antimycin A was nontoxic to the 15 taxonomic groups which were represented in both ponds because there were no significant changes in species composition or numerical abundance (table 18), The midges were the more numer- ous in all samples, and they increased by 55 to 65 percent during the experimental period. The nymphs of mayflies, dragonflies, and 15 TABLE 18.--Abundance of bottom fauna in ponds No. 2 and No. 5 before and after treatment with 10 p.p.b. of antimycin A [Each collection consisted of 16 one-square foot samples Average number per square foot Horsehair worm (Nematomorpha).... Aquatic earth- worm Leech 5 (Oligocheata) (Hirundinea)...... coc noo NFO Owo Scuds (Amphipoda)....... 4 Mayflies (Ephemeroptera.... 9.0 145.3 6.5 Damselflies (Zygoptera)....... a ey 2.0 7.8 Dragonflies (Anisoptera)...... 0.0 0.7 0.5 Waterbugs (Hemiptera)....... 0.0 Rial? 1 Caddisflies (Trichoptera)..... 0.7 2.0 0.0 Water beetles (Coleoptera)...... ey 18.0 355 Mosquitoes (Culicidae)....... 0.0 0.0 0.0 Midges (Tendipedidae).... 209.7 388.0 422.0 Biting midges (Ceratopogonidae).. ats 0.0 1.0 Soldierflies (Stratiomyiidae)... 0.3 0.0 0.0 Snails (Gastropoda)....... 4.0 30.7 30.5 TOt@l ocscececvecuas 8 damselflies were also more abundant in the posttreatment samples. Care was taken to note any gross effects of the toxicant on frogs, salamanders, and tur- tles, but there were none, Discussion of field studies There was a lack of consistency in the per- formance of antimycin A in sand- and loam- bottom pools in July and October, 1962 and 1963, and in the hatchery ponds. The cause, we believe, was the chemistry of the waters and particularly the presence of the hydroxyl ion. An alkaline shift occurred in the wading pools as the plant biomass increased. The relatively hard, well water which was used to fill the pools was gradually softened be- cause of the decrease in calcium, There was a shift from bicarbonate (methyl orange alka- linity) to free hydroxide (phenolphthalein alkalinity), The measure of the acid-base shift was pH which rose from 7.5 upward to 10 or more, Diurnal fluctuations of several PH units are not uncommon in ponds, and the PH in wading pools ranged accordingly between morning and afternoon. The highest pH values were observed in late afternoon in the presence of abundant plants and sunshine, In this situation, the hydroxyl ions appear, and often they are not checked by buffering salts. Magnesium pre- vails as calcium ions are removed from solution, and the result is the sort of alka- line shift observed in softer waters, We assume that the relative success of the toxicity trials in hatchery ponds was due in large part to the fact that the water had high buffer capacity and little reserve alkalinity in the form of hydroxide. Thus, the antimycin A was not subject to immediate detoxification by action of free hydroxide, and the 10 p.p.b. were effective in killing fish. In contrast, the poorer results obtained in the wading pools reflected the greater con- centrations of free hydroxide present, In July 1963, the pools had approximately the same pH and total alkalinity as the hatchery ponds, but there was more free hydroxide present, Therefore, the degradation of the toxicant was more rapid, and 20 to 40 p.p.b. were needed to kill fish. The contrast was heightened by results in October 1963, The water was much softer and lower in buffer capacity, and there was even more free hydroxide present, The pH ranged up to 10, Under these conditions, there was almost immediate detoxification of the anti- mycin, and only partial fish kills were ob- tained at 100 p.p.b. CONCLUSIONS Antimycin A is a powerful toxicant to fresh-water fish. We observed the responses of many specimens to concentrations which ranged from 0.01 to 120 p.p.b. Among them, the carp--a most undesirable fish in many waters-=proved vulnerable to small concen- trations and short exposures at cool and warm temperatures, Other fishes which at times may be undesirable, such as goldfish, white suckers, green sunfish, and pumpkinseeds, were also killed. The sensitivities to antimycin A varied among species, and they were correlated with temperature and duration of exposure. The tests in the laboratory at 12°, 17°, and 22° Cc, > indicated that smaller quantities of toxicant or shorter exposures produced kills of fish in warmer waters, but the results at 12° were nonetheless satisfactory. There were three general degrees of sensi- tivity detected among the 24 species of fish in thé laboratory and a similar order among the 25 species used in outdoor trials. Indicative of the extremes in response, gizzard shad perished at 0.04 p.p.b. of toxicant whereas black bullheads survived 100 p.p.b. There also appeared to be a tendency for sensitivities to follow family lines, but species in the nine families tested exhibited great variations in susceptibility. For example, fingerling carp in the laboratory died within 24 hours upon exposure to 0.6 p.p.b. at 12°, but up to 100 p.p.b. were required for complete kills of goldfish, Observations in the laboratory and field demonstrated that antimycin A was less toxic to other animals, Water fleas were killed by 100 p.p.b. in 24 hours at 12°, but their sus- ceptibility increased at warmer temperatures or longer exposures, Crayfish were not harmed by 10 p.p.b. over 96 hours, and damselfly nymphs survived 50 p.p.b. for 48 hours, Tiger salamanders survived 80 p.p.b. for 96 hours at 12°, and bullfrog tadpoles were unharmed by 20 p.p.b. for 24 hours, The plankton in wading pools and hatchery ponds was not significantly affected during experiments, and there was no gross evidence of toxicity to filamentous algae, and submersed and emergent plants. No deleterious effects were detected on the composition, numbers, and growth of bottom fauna in hatchery ponds, Antimycin A degraded rapidly in water, and detoxification was complete within 24 to 96 hours under field conditions, The rate of molecular breakdown was accelerated sharply in the presence of free hydroxide, and this suggests a possibility for artificial detoxifi- cation. Bioassays with fish following the de- gradation of the toxicant revealed an absence of harmful residues in water. Further investigation on antimycin A as a fish toxicant is warranted in the laboratory and field, Studies are contemplated or in progress at the Fish Control Laboratories on its performance against various life stages of fish from egg to adult; against additional species; on minimum killing con- centrations and exposures; in waters of vari- ous chemistries; and at cold and warm tem- peratures, Appropriate formulations for standing and flowing waters are desirable. Further attention must also be given to the effects of the toxicant on other aquatic or- ganisms, The factors in water which con- tribute to degradation of the toxicant deserve study, and the nature and fate of residues require definition, Also--and depending on adequate supplies of toxicant--many, and more comprehensive, trials in the field are needed for full and fair evaluation of this material which has potential value in fishery manage- ment and research. LITERATURE CITED American Public Health Association, American Water Works Association, and Water Pollution Control Federation, 1960, Standard methods for the examination of water and waste-water, 11th, ed, American Public Health Association, New York, 626 p, Derse, P. H., and F, M, Strong, 1963, Toxicity of antimycin to fish, Nature, vol, 200, No, 4906, p, 600-601, Dickie, J. P., M. E. Loomans, T, M, Farley, and F, M, Strong, 1963, The chemistry of antimycin A, XI, N- substituted 3-formamidosalicylic amides, Journal of Medicinal Chemistry, vol. 6, p, 424- 427. Dunshee, B, R., C. Leben, G. W. Keitt, and F. M. Strong, 1949, Isolation and properties of antimycin A. Journal of the American Chemical Society, vol, T1, P. 2436-2437, Gottlieb, D,, and S, Ramachandran, 1961, Mode of action of antibiotics, 1, Site of action of ascosin, p, 391-396, Harada, Y., K. Nakayama, and F, Okamoto, 1959, Antimycin A, an antibiotic substance useful in prevention and treatment of imochibyo, a disease of rice, Japan, 2200, (Chemical Ab- stracts, vol. 53 (1959), p. 19286i). Cy Keitt, George W., Curt Leben, and Frank M, Strong, 1953, Antimycin and process for production, U.S. Patent Office. Patent No, 2,657,170, 10 p, Lawrence, J. M., and R, D, Blackburn, 1962, Evaluating herbicidal activity of chemicals to aquatic plants and their toxicity to fish in the laboratory and in plastic pools, Auburn Uni- versity, mimeo; Pp, 1-23, (/n press, Proceedings of 16th Annual Conference of Southeastern Association of Game and Fish Commissioners, Columbia, S.C.). Lennon, Robert E,, and Charles R, Walker, 1964, Investigations in Fish Control; 1 Laboratories and methods for screening fish-control chem- icals, Bureau of Sport Fisheries and Wildlife, Circular 185, Liu, Wen-chih, and F, M, Strong, 1959, The chemistry of antimycin A, VI. Separa- tion and properties of antimycin A subcompo- nents, Journal of the American Chemical So- ciety, vol, 81, p. 4387-4390, Liu, Wen-chih, E, E. van Tamelen, and F, M, Strong, 1960, The chemistry of antimycin A. VIII. De- gradation of antimycin A. Journal of the Ameri- can Chemical Society, vol. 82, p, 1652-1654, Lockwood, J. L., C. Leben, and G, W. Keitt, 1954, Production and properties of antimycin A from a new Streptomyces isolate, Phytopath- ology, vol, 44, p, 438-446, Nakayama, K., F. Okamoto, and Y. Harada, 1956, Antimycin A: Isolation from Streptomyces kitazawaensis and activity against rice plant blast fungi. Journal of Antibiotics (Japan) Ser, A 9, Pp. 63-66 (Chemical Abstracts, vol. 53 (1959), P. 19030h). Strong, F. M. 1956, Topics in microbial chemistry, John Wiley and Sons, Inc., New York, 166 p, Strong, F. M., J. P. Dickie, M. E. Loomans, E, E, van Tamelen, and R. S. Dewey. 1960, The chemistry of antimycin A. IX, Struc- ture of the antimycins, Journal of the American Chemical Society, vol, 82, p, 1513, Tener, G. M., F. Merlin Bumpus, Bryant R. Dunshee, and F, M. Strong, 1953, The chemistry of antimycin A. II, Degra- dation studies, Journal of the American Chemi- cal Society, vol. 75, p, 1100-1104, Tener, G. M., E. E. van Tamelen, and F, M. Strong, 1953, The chemistry of antimycin A, III, The structure of antimycic acid, Journal of the American Chemical Society, vol, 75, p, 3623- 3625, van Tamelen, E, E., F, M, Strong, and U, Carol Quarck, 1959, The chemistry of antimycin A, IV. Studies on the structure of antimycin lactone, Journal of the American Chemical Society, vol, 81, p, 750-751, van Tamelen, E, E,, J. P. Dickie, M, E, Loomans, R S. Dewey, and F, M, Strong, 1961, The chemistry of antimycin A. X. Struc- 18 ture of the antimycins, Journal of the A merican Chemical Society, vol, 83, P. 1639-1646, Watanabe, K,, T, Tanaka, K, Fukuhara, N, Miyairi, H, Yonehara, and H, Umezawa, 1957, Blastmycin, a new antibiotic from Streptomyces. Journal Antibiotics (Japan), vol, A10, p, 39-45, (Chemical Abstracts, vol, 53 (1959), p, 22221¢), a juswuidojaaep 4pbes {UBIIND [DYIOT spunodwiod jDuyje7 NOILVDIGVYF FUNIdVD FLVLINIVI juawidojaaesp ines Aviip Buipin6 1p314439)3 A isa i ge! ed {UDJPIDIYO 2uU0S UOHP[NdiuDW [BAZ] 19jDM uolKNpodyul 10jyedWo> UOHINpodyul JOjDpeig jusuidojaaep ipas S@uSDIDd BAI29/E¢ SINE IxO eal e|25 quesnrspesitd NOILING3Ie FAL9D3139S UOHDINdiuDW [8Ad| 18jD MA BSDESIP SNOjI@sUI BAIj29/95 uipays AsojyouBiul-uou 404 42985 sjupjjeday SjuD; DANY sjuDjjaday uoypindiupw s1jnDIpAy uoypindiupw a1jnDIpAy Apiip 1214499}3 J914IDg D211)99;9 LNIWIAOW LOldls3ad 4eiDg AUYLN] LN3AIad MIOHS DILOWSO INJAIad LYOdSNVUL JINVISISIY ISVISIA "ALIGNND43d ‘YOOIA ‘HLMOYD JAOUdWI Aiijiqoidopp 410) j2e]95 spunodwo) AiojpjnBeiowso UOHDZNO WII YONDINdIuUDW 481g sjucuIWIDju0I8G S@AIDp|S s2eUusouYy Jo4ju0> ainjosedwey ]O4jU0> r1]NDIPAY $1SO2IDU-01499}9 uolDiey @2UDISIS@1 BSDASIP 104 429/05 4yimoiB pidos 104 439/85 SSOUPIIM 10} 429/85 UO1j29]OS 420js poolg Josjuo> 44617 jupsseidep punos 4481] pejjosjuo> uoNDIpDYy yPOYs 314j30)3 souoWwIOLY sjuDji1945 jDAowes 40 UOKINpoYU! yud]g SNINMVdS LNIAId sUDINWs punos 1481] pejjouuoy yPoys 214)20]3 TVDINVH33W GNNOS ‘ALIDIY19373 ‘13H GNV 1VDISAHd ‘IHOIT - AOWINA LNVIGVY asodauynad TOULNOD HSI4 So | Buiumods jo uoypunp PUD UOSD_eS 104 428/85 SUOHJINpOwyUI JUD] g ]94jU0> r1NOIPAY SNINMVdS JDNANI TVOIWAHDOI8 GNV 1V5IW3HD SQOHLAW SINIOV IVIIDOO1OI# s, ~~ ——— ge i —- e UNITED STATES POSTAGE AND FEES PAID a DEPARTMENT OF THE INTERIOR U.S. DEPARTMENT OF THE INTE FISH AND WILDLIFE SERVICE BUREAU OF SPORT FISHERIES AND WILDLIFE WASHINGTON, D.C. 20240 sep «6 8 , ty DIVISION OF Fivcucs cist U. Se NATIONAL MUSEUM fs 4 J Ss INVESTIGATIONS IN FISH CONTROL 3. 4. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes Effects of Toxaphene on Plankton and Aquatic Invertebrates . in North Dakota Lakes Growth Rates of Yellow Perch -in Two North Dakota Lakes After Population Reduction with Toxaphene Mortality of Some Species of Fish to Toxaphene at Three Temperatures Treatment of East Bay, Alger County, Michigan with Toxaphene for Control of Sea Lampreys Effects of Toxaphene on Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island, Alaska United States Department of the Interior Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife INVESTIGATIONS IN FISH CONTROL Investigations in Fish Control, published by the Bureau of Sport Fisheries and Wildlife, in- clude reports on the results of work at the Bureau's Fish Control Laboratories at La Crosse, Wis., and Warm Springs, Ga., and reports of other studies related to that work. Though each report is regarded as a separate publication, several may be issued under a single cover, for economy. Current reports in this series are-- (Reports 1 and 2 are in one cover,) 1. Laboratories and Methods for Screening Fish-Control Chemicals, by Robert E, Lennon and Charles R. Walker. (Bureau Circular 185.) 1964. 15 p. 2. Preliminary Observations on the Toxicity of Antimycin A to Fish and Other Aquatic Ani- mals, by Charles R, Walker, Robert E, Lennon, and Bernard L. Berger. (Bureau Circular 186.) 1964. 18 p. (Reports 3 through 8 are in one cover,) 3. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes, by Dale L, Henegar. (Resource Publication 7.) 1966. 4, Effects of Toxaphene on Plankton and Aquatic Invertebrates in North Dakota Lakes, by Robert G. Needham. (Resource Publication 8.) 1966. 5. Growth Rates of Yellow Perch in Two North Dakota Lakes After Population Reduction with Toxaphene, by Donald C. Warnick. (Resource Publication 9.) 1966. 6. Mortality of Some Species of Fish to Toxaphene at Three Temperatures, by Mahmoud Ahmed Mahdi. (Resource Publication 10.) 1966. 7. Treatment of East Bay, Alger County, Michigan, with Toxaphene for Control of Sea Lam- preys, by William E, Gaylord and Bernard R. Smith. (Resource Publication 11.) 1966. 8. Effects of Toxaphene on Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island, Alaska, by William R. Meehan and William L. Sheridan. (Resource Publication 12.) 1966, 9. Relation of Chemical Structure to Fish Toxicity in Nitrosalicylanilides and Related Com- pounds, by Charles R. Walker, Roland J, Starkey, and Leif L. Marking. (Resource Publi- cation 13.) In press. 10. Evaluation of p,p'-DDT as a Reference Toxicant in Bioassays, by Leif L. Marking. (Re- source Publication 14.) In press. 11. Evaluation of an Electronic Method of Measuring Hematocrits of Fish, by Richard A, Schoettger and Arnold M, Julin. (Resource Publication 15.) In press. Fish Control Laboratories Bureau of Sport Fisheries and Wildlife U. S. Department of the Interior P. O. Box 862 La Crosse, Wisconsin 54602 INVESTIGATIONS IN FISH CONTROL 3. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes By Dale L. Henegar (Resource Publication 7, p. 1-16) 4. Effects of Toxaphene on Plankton and Aquatic Invertebrates in North Dakota Lakes By Robert G. Needham (Resource Publication 8, p. 1-16) 5. Growth Rates of Yellow Perch in Two North Dakota Lakes After Population Reduction with Toxaphene By Donald C, Warnick (Resource Publication 9, p. 1-9) 6. Mortality of Some Species of Fish to Toxaphene at Three Temperatures By Mahmoud Ahmed Mahdi (Resource Publication 10, p. 1-10) 7. Treatment of East Bay, Alger County, Michigan with Toxaphene for Control of Sea Lampreys By William E. Gaylord and Bernard R, Smith (Resource Publication 11, p. 1-7) 8. Effects of Toxaphene on Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island, Alaska By William R. Meehan and William L. Sheridan (Resource Publication 12, p. 1-9) United States Department of the Interior, Stewart L. Udall, Secretary Stanley A. Cain, Assistant Secretary for Fish and Wildlife and Parks Fish and Wildlife Service, Clarence F. Pautzke, Commissioner Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director NOTE Toxaphene has never been registered for use asa piscicide. The Bureau of Sport Fisheries and Wildlife has discontinued management use of toxa- phene at least until completion of definitive studies on detoxification in relation to water quality, residual toxicities, and short-term effects of its use on aquatic organisms. These six papers on toxaphene and its effects are published as contributions to an understanding of the chemical and its use in fish management. li INVESTIGATIONS IN FISH CONTROL 3. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes By Dale L, Henegar Chief, Fish Management Division North Dakota Game and Fish Department U.S. DEPARTMENT OF THE INTERIOR Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife Resource Publication 7 Washington ., January 1966 ADS tGhaAC tem wensreiouerienens ceoeeee Characteristics of toxaphene . Method of application eoeeesee Characteristics of the lakes... MeSE Mette enenencuenel RESUS ois eSWeureseremeles Odland Lake .... Brush lakes. sc.0 6 gas comenere ILC? IMEI Soo Kons Gumms Lake.... North Lake Metigoshe... South Lake Metigoshe... Red Willow Lake Frettum Lake ... North Lake Tobiason.... Bowbells Mine Lake..... Glen Ullin Reservoir..... South Lake Tobiason Nieuwsma Lake.. CONTENTS Cat Coulee Wakes eo chee ei tee Wolfe Butte Lake..... Aiebael LEIS 5 Goo oc Discussion and conclusions .. SUIMIMAI YA tenet on ce eee eneienenoue References ....ccec6 ° ° eee coeeeeeee . e eoeeeee ee @ ° ee ° eee eeeoeeveeeeeeee ee e eee eeeeevee eecee ee e eeeeeseve e eoee5uxee eeceevcee e e e eoeoeeneveeeee ee oeeeee ee e e eeeee e e eeeeceeeee ee ° eeoeeeeeeeeeee eee eooeoeeeeeeeweeeeee @ e e ceoeoeeeeeeeeeeeee ee e eoeoeoeoereeee @ eeeeveeeee eee @ eee ee eeeee eeeveee eeec3seeeee ° ee ° e eeoeec30ec3¢ee e e e ee ° e e e eeeee0nee30eee e ee eeeee eeoec5vsoeee e e e eeee eeecse eeee eeecee eeec5ee¢ee ee coeeeeee eoeeneeee eoeee eoeveeveeeeee ee Page MINIMUM LETHAL LEVELS OF TOXAPHENE AS A PISCICIDE IN NORTH DAKOTA LAKES By Dale L, Henegar, Chief, Fish Management Division North Dakota Game and Fish Department Abstract,--To determine minimum levels of toxaphene lethal to fishes in prairie lakes and reservoirs, 16 North Dakota lakes, ranging from 6,3 to 915 acres, were treated in 1959 and 1960 with concentrations of toxaphene ranging from 0,005 to 0.035 p.p.m, Physical and chemical studies were made of each area, hydrological maps were prepared, and test netting was carried out before and after treatment; application methods and equipment were according to North Dakota Game and Fish Department regulations, Mortality after treatment varied from incomplete, involving only young- of-the-year fish, to complete, A marked selective mortality pattern was noted; smaller fish succumbed at lower dosages; as the concentrations were increased, larger fish were killed, Duration of toxicity did not appear excessive--five of the Seven lakes in which mortality was complete were successfully restocked within 7 months after treatment, Toxaphene (chlorated camphene) has not been widely used as a piscicide for local field application because minimum lethal concen- trations in the field have not been determined, Considerable information has been gained from laboratory bioassay studies (Surber, 1948; Doudoroff et al,, 1953; Hooper and Grzenda, 1957; Henderson et al,, 1959), but the concen- trations indicated by such studies are not necessarily correct for field use, Prevost (1960) pointed out that results of controlled laboratory experiments do not always yield dosages giving similar results in the field, where there are a number of variables, both known and unknown, over which the field worker has little or no control, Gebhards (1960) in his review lists 14 West- ern States and 6 Canadian Provinces that have used toxaphene in fish-control programs in various formulations at concentrations ranging This publication is based on a thesis submitted to the Graduate Faculty, Department of Agriculture, South Dakota State College of Agriculture and Mechanic Arts, in partial fulfillment of the requirements for the degree of Master of Science, June 1961, from a low of 0,003 parts per million (p.p.m.) to a high of 0,61 p.p.m, Inconsistencies were emphasized in the review by the side varia- tions in results, Fifteen of the reports said an average concentration of 0.185 p.p.m, failed to kill all fishes in treated areas, while 23 reports mention that an average concentration of 0,139 p.p.m, caused complete mortality, Stringer and McMynn (1958) reported complete kills at from 0.010 p.p.m. to 0.036 p.p.m. In North Dakota, mortality of fish was com- plete when toxaphene was applied at a concen- tration of 0.070 p.p.m., which was not consid- ered the minimum lethal level (Henegar, 1958), It was to determine the desirable minimum concentration for management use in North Dakota that this study was initiated. Sixteen lakes were chosen for treatment during 1959 and 1960, All were test netted to determine existing populations of fish both before and after treatment, Physical and chem- ical characteristics were studied to establish criteria, Application of the toxaphene followed procedures commonly used by the North Dakota Game and Fish Department, For lakes with physical and chemical char- acteristics of those in the Great Plains, rec- ommended concentrations range from 0,025 to 0,030 p.p.m, as indicated in table 1, Concen- trations used on the project areas ranged from 0,005 to 0,035 p.p.m, TABLE 1.--Recommended concentration of toxaphene for various types of lakes. (William Cooper & Nephews, Chicago, Ill., 1958] Concentration of toxaphene HENS ne 25-30 DeDeDenseieee Unstratified, shallow, hard-water lakes of low transparency and high productivity. 15-20 p.p.Deesanee Unstratified, soft-water lakes of moderate or high transparency. 10-20 p.p.b...eeee Stratified lakes of moderate depth (mean depth less than 20 feet) and moderate to low transparency. 7.5-10 p.p.b...... | Stratified lakes of great depth (mean depth greater than 20 feet), high transparency, and low produc- tivity. + Qne report indicates that a concentration of 50-100 p-p-b. should be used in highly turbid waters containing suspended clay (Secchi disk reading less than 1 foot). I wish to express my appreciation to Marvin O, Allum, Assistant Professor of Zoology, for his suggestions, interest, and constructive criticism during this study, I also wish to thank Dr. Donald Progulske, Assistant Professor of Zoology, for his advice and suggestions on preparation of the manuscript, I am obliged to all the members of the Fishery Division of the North Dakota Game and Fish Department who generously contributed time and suggestions on the field work, CHARACTERISTICS OF TOXAPHENE Toxaphene is one of the group of toxicants known as cyclodione insecticides, which also includes aldrin, dieldrin, chlordane, heptachlor, endrin, and isodrin, Toxaphene is not as well characterized chemically as the others, and the precise nature of the compounds in the mixture of isomers is not known (Negherbon, 1959), Most frequently encountered formula- tions are 10- to 20-percent dusts, 40-percent wettable powders, and emulsifiable concen- trates of 4, 6, and 8 pounds per gallon, The commercial product used in the study was Agricultural Cooper-Tox No, 6 (toxaphene emulsifiable concentrate) manufactured by 4 William Cooper & Nephews, Chicago, Ill, The formulation contained 6 pounds of technical ' toxaphene per gallon, Toxaphene remains toxic for extended pe- riods of time following application as a pisci- cide, Ten Michigan lakes treated in 1949 and 1950 detoxified in from 8 to 33 months, Other Michigan lakes detoxified in from 2 to 10 months after being treated at a concentration of 0,010 p.p.m, (Hooper and Grzenda, 1957), Stringer and McMynn (1960) reported that several British Columbia lakes remained toxic for over 2 years after treatment, Application at 1,00 p.p.m, (2,7 lbs. per acre-foot) gave concentrations of from 0,40 to 0.50 p.p.m, toxaphene, Application rates do not appear to be im- portant to the duration of toxicity when applied within the median tolerance limits of fishes, Mayhew (1959) stated that the period of toxicity is more related to chemical and physical char- acteristics of treated lakes than to the concen- tration of toxaphene, The factors which influence detoxification are dilution, water temperature, water circu- lation, oxygen levels, turbidity, alkalinity, types of substrate, microorganisms, and ratio of water to bottom interface (Hemphill, 1954; Hooper and Grzenda, 1957; Rose, 1958; Hene- gar, 1958; Prevost, 1960), Detoxification takes place very slowly when ice cover is present and more rapidly when water temperatures are high and conditions are favorable for the growth of plankton and other microorganisms, METHOD OF APPLICATION ‘Toxaphene has been applied in various ways, the method usually depending on the formula- tion, The wettable powder has been applied by aircraft and by placing it in burlap bags and then towing in the wake of an outboard motor (Henegar, 1953), The emulsifiable liquid has been applied by aircraft, but most commonly by distributing premixed solutions with powder sprayers mounted in boats, One method is the metering of desired amounts into the wake of a moving outboard-powered boat (Stringer and McMynn, 1960), but unless this is carefully controlled the toxaphene solution may settle to the bottom in a comparatively undiluted state, In this study, a pumping system used by the North Dakota Game and Fish Department was employed (fig, 1), It consists of a 55-gallon drum connected to a motor-driven centrifugal pump with two outlet pipes terminating in common garden-type nozzles extending behind the boat transom on each side, During use, the nozzles are in contact with the surface of the lake to minimize airborne spray from coming in contact with the boat operator, The toxaphene was premixed in the barrel at a maximum ratio of 1 gallon to 5 gallons of water so that a minimum of 55 gallons of liquid was available for spraying on each lake, During application the spray boat, powered by an 18- horsepower outboard motor, was operated at full throttle, This allowed for a maximum coyv- erage of 200 acres an hour, On smaller lakes adjustment of the nozzles reduced application time to as little as 30 minutes, The spray unit was left intact in the boat after each project, when the boat was loaded onto a boat trailer, This reduced the time nec- essary to ready equipment for each treatment, Figure 1,--Spray boat used in application of toxaphene on project lakes, Areas for treatment were determined from prepared hydrographic maps of each lake, Lakes over 100 acres in size were subsec- tioned, and each subsection was treated as a separate unit, CHARACTERISTICS OF THE LAKES For study I selected 16 lakes, varying in size from 6,3 to 915 acres, in widely scattered areas of North Dakota, Seven are impound- ments, and nine are natural, Maximum depths ranged from 8 to 26 feet, and volume from 79 to 8,254 acre-feet (table 2), At the time of treatment, none of the lakes were chemically or thermally stratified, Water samples were taken from all lakes, and field analyses were made to determine chemical characteristics of the water (table 2), Concurrently, l1-gallon samples of water were forwarded to the North Dakota Health Depart- ment to check the accuracy of the field analy- ses, The variation in results between field and laboratory analyses was not Significant, With one exception, all the lakes had exten- sive growths of aquatic vegetation extending from shore outward to a depth of 8 feet, Potamogeton spp. and Myriophyllum spp, were particularly abundant, with Polygonum spp, and Sagittaria spp, prevailing less frequently, In all the lakes, primary bottom composition was silt, mud, clay, and organic material, Areas of sand, gravel, and rubble were of minor importance and were restricted to the natural lakes, TABLE 2.--Physical and chemical characteristics of project lakes Alkalinity yess Volume pH lies Hardness ieee depth Phenol- Methyl ia phthalein’ orange Oe Feet Acre-feet P.p.m. P.p.m. P.p.m. P.p.m. Odland Lake.....cscceeccesccse 712.9 8.2 120 180 171 414 Brush Lake.... eae 160.2 23 1,526.7 8.5 40 460 476 290 Long Lake.. 290.8 23 2,390.6 8.3 40 220 308 307 Gums Lake..seceeceeeees 173.6 8 853.7 9.0 84 442 821 816 North Lake Metigoshe........-- 670.8 22 7,588.5 8.8 20 210 205 317 South Lake Metigoshe.........- 915.0 22 8,254.4 8.7 30 240 238 326 Red Willow Lake...cssereeevece 129.9 26 1,272.8 8.0 24 178 239 364 Frettum Lake... escecceescscree 95.2 22 511.7 8.3 54 496 444 281 North Lake Tobiason........... 50.3 14 472.8 8.6 44 282 359 779 Bowbells Mine Lake............ 6.3 21 78.9 9.9 to) 800 2,385 4,100 Glen Ullin Reservoir.......... 12.3 18 144.4 8.0 60 420 136 418 South Lake Tobiason..........+ 40.1 10 286.9 8.5 106 460 547 1,452 Nieuwsma Lake 80.8 23 651.2 7-9 0 140 136 415 Cat Coulee Lake. 8.0 17 87.4 8.8 30 130 170 328 Wolf Butte Lake. 17.3 14 137.5 9.4 100 260 114 940 18.5 14 162.9 8.0 20 80 51 306 TEST NETTING Qualitative sampling of fish populations in all lakes was carried out before and after treat- ment (table 3), Three types of gear were used, gill nets, small-mesh seines, and small-mesh frame nets, The gill nets were experimental nylon nets, 250 by 6 feet, composed of 50-foot sections of increasing mesh size: 3/4, 1, 1-1/4, 1-1/2, and 2 inches, Total netting effort for each lake varied according to size: a greater number of sets were made in larger lakes, The nets were fished in diurnal and nocturnal periods to lend validity to the results, Although this type of gear is subject to considerable error, esti- mates of fish populations may be made from 6 the data, During the project period all lakes were gill-netted a total of 2,304 hours, repre- senting 96 individual sets, Shoreline seining with a 100- by 6-foot, . 1/4-inch-mesh, nylon bag seine was done where vegetation was not too dense, Data so gathered were of limited value owing to TABLE 3.--Fishes in project lakes. Rainbow trout, Salmo gairdneri Richardson. Northern pike, Esox lucius Linnaeus. Carp, Cyprinus carpio Linnaeus. Golden shiner, Notemigonus crysoleucas (Mitchill). Bluntnose minnow, Pimephales notatus (Rafinesque ). White sucker, Catostomus commersoni (Lacepede). Black bullhead, Ictalurus melas (Rafinesque). Brown bullhead, Ictalurus nebulosus (LeSueur). Orangespotted sunfish, Lepomis humilis (Girard). Bluegill, Lepomis macrochirus Rafinesque. White crappie, Pomoxis annularis Rafinesque. Black crappie, Pomoxis nigromaculatus (leSueur). Yellow perch, Perca flavescens (Mitchill). Walleye, Stizostedion vitreum vitreum (Mitchill). inconsistent results, Altogether, 71 drags were RESULTS made in project lakes, representing a coverage of 35,500 square feet. ODLAND LAKE Odland Lake was treated on August 12, 1960, with toxaphene at a concentration of 0,005 p,p.m, No effects of the toxaphene were observed for 10 hours following application; after this pe- riod, distressed young-of-the-year yellow perch and black bullheads were noted in the shallow bays and backwaters, Within 36 hours many of these small fishes were either lying dead on the bottom or washed ashore, Evident mortality ceased 48 hours after application, In the observation period only seven larger fish were noticeably affected, The small fish de- composed rapidly, and after 7 days evidence of their mortality disappeared, Small-mesh frame nets produced valid data On populations of fishes inhabiting the littoral zone, These nets were originally designed to sample young-of-the-year northern pike in heavily vegetated areas, The front frame of each net was 3 feet high and 4 feet wide (fig. 2); the net was 15 feet long and had round wooden hoops behind the front rectangular frame, The webbing was 1, 4-inch nylon dyed a dark brown, The single tunnel used the string type of con- struction rather than open orifice, and the net retained the trapped fishes very satisfactorily, The single, 50-foot lead from the front frame was of the same material as the body of the net, In use, the frame nets were placed at right angles to the shoreline, A net of this type can be placed in position by one man, and sets are made without using a boat, Representative : : 6 P the toxaphene was used, In this lake the net catches were made in all types of habitat, The : : : : frequencies did not show the true population frame nets were fished a total of 3,704 hours TABLE 4.--Concentrations of toxaphene applied and fish mortality in project . The lake was test-netted 48 days after treat- ment, Net frequencies (table 5) were not sig- nificantly changed from those arrived at before during the project period, ore Lake Patent Mortality Odland Lakes. esccsceccceccccnvcenss 0.005 Incomplete BES EAKE fe \cletuie)taletaielslate\staeinrujelaletn/ aie 0.010 Incomplete Tong Lake. .sccccccccccsievcscvecescs 0.010 Incomplete GUMMTIS HEA estat ote rele intaveinmievelslaieysiclataietaha's 0.010 Incomplete North Lake Metigoshe.....sccccorece 0.015 Incomplete South Lake Metigoshe......esceceone 0.015 Incomplete Red Willow Lake...... iocoopaecosaoge 0.015 Incomplete Frettum Lake....ssceccecceccscceces 0.020 Incomplete North Lake Tobiason................ 0.020 Incomplete Bowbells Mine Lake.........ssseeeee 0.625 Complete Glenn Ullin Reservoir.........-..-- 0.025 Complete South Lake Tobiason......... 0.025 Complete Nieuwsma Lake 0.025 Complete Cat Coulee Lake. 0.030 Complete Wolf Butte Lake. 0.030 Complete Jund Lake. sc ccasveccccevce 0.035 Complete TABLE 5.--Changes in test-netting frequencies following application of toxaphene in project lakes [Frequencies are computed as fish per hour per net both for frame and gill nets. Seining data not used] Frequency Concentration Reduction (p.p.m. ) Before After (Percent) treatment | treatment Odland Lake......sseeceeeee 0.005 6.91 6.94 None Brush Lake... 0.010 6.88 1.34 80.5 Long Lake 0.010 10.38 1.59 84.6 Gumms Lake. ...ccsessscccaes 0.010 34.46 7.96 76.9 North Lake Metigoshe....... 0.015 8.46 1.07 87.3 South Lake Metigoshe....... 0.015 6.46 1.11 83.2 Red Willow Lake......-..-+. 0.015 10.20 2-50 75.4 Frettum Lake.......eeeseees 0.020 6.36 -32 94.9 North Tobiason Lake........ 0.020 12.91 233 97.4 Bowbells Mine Lake......... 0.025 95 0.00 100.0 Glen Ullin Reservoir....... 0.025 6.91 0.00 100.0 South Lake Tobiason........ 0.025 7.29 0.00 100.0 Nieuwsma Lake....sseeeeeeee 0.025 5.89 0.00 100.0 Cat Coulee Lake.......++0-. 0.030 4.06 0.00 100.0 Wolf Butte Lake....sseeeeee 0.030 15.08 0.00 100.0 = 9 Jund Lake. cccccrcserevcrece 0.035 3.62 0.00 100.0 Figure 2,--Small-mesh frame net used to sample fishes in the littoral zone, structure, Young-of-the-year fishes taken during pretreatment test netting (table 6) were absent from the data, Average sizes of larger fishes remained relatively stable, reflecting insignificant mortality, BRUSH LAKE Brush Lake was treated on October 5, 1959, with toxaphene at a concentration of 0.010 p,p.m. Application preceded lake freeze-up by only 2 days. During the 2 days that observations could be made, no affected fish were found, The fol- lowing spring (157 days after treatment) thou- sands of partly decomposed yellow perch (2,1 to 6,1 inches) were washed on shore, Seventeen walleyes (9,8 to 12.3 inches) and five northern pike (10.1 to 13.1 inches) were recorded, Fur- ther observations for 5 days after breakup did not reveal additional current mortality. Ninety-six hours of posttreatment test net- ting starting on April 22, 1960, disclosed only a partial mortality among the fishes, It was evident from the results (table 7) that although mortality was heavy among the young-of-the- year and older yellow perch (2,1 to 5.9 inches), little difference in the abundance of the larger fishes could be found, Total test netting fre- quency was reduced by 80.5 percent (table 5), On May 27, 1960, Brush Lake was stocked with 29,000 northern pike fingerlings (2,000/1b.), On June 26, 1960, it was stocked with 48,000 walleye fingerlings (1,600/1b,) and on August 1, 1960, with 200,000 bluntnose minnows (1,000/1b.), TABLE 6.--Qdland Lake test netting data before and after treatment with 0.005 p.p.m. toxaphene [Combined data from all types of netting gear | Length (inches) Species Number Range Average Before treatment (134 hours): Bilaeks DU he Aceismielslatels\atel=|aieisislels|sialspe 418 4.0 - 11.6 5.8 DO statsl aintetelvinistotetelelels[=tetefate(ofalsleiatersicints 405 young-of-year -- VETVOW MDeUCHletalstalelelolsieiel=(eisl=i=/elsieleisinlare 44 3.4 - 6.5 5.5 GoldengShinesatelsislelstetele/slelsteletsicleia/eiale 27 5. = 625 6.1 Nomithe mnt ke@isiajats(aletulalsfels)stalstaie(s slaldiein ily 16.0 - 28.3 21.7 WHE MSM CKE T+ atelstmjateralelwlet={eteyel aie mnibnluate 11 EEX PETIM|Nts! s/s wet dyeyo «! lei shor als lales )e Pele aise BPemetietme ene > hee) see 13 DISCUSSION <2 uc, coc everemel tener oust) = ciel sto: Ael vou otal rene) PoMeu onaelseko tose ye acme 15 SUIMMMALY. Cre sk, success o tene: er wlle @ be) eiletten s soutetien ou ontoninns = owtamtas fonteiteme 16 REfEFENCES. a 2) sescn ciel heme eme cisco? ole) ole sproulemteMtee Na Mnueln = tok Be ai-moMalke 16 — EFFECTS OF TOXAPHENE ON PLANKTON AND AQUATIC INVERTEBRATES IN NORTH DAKOTA LAKES By Robert G, Needham, Fishery Biologist Montana Department of Fish and Game Abstract.--Effects of low concentrations of toxaphene on plankton and larger invertebrates were studied in four North Dakota lakes (a fifth lake, untreated, was a control). Brachionus, Keratella, Trichocerca, Asplanchna, Polyarthra, Conochiloides, Daphnia, Ceriodaphnia, Bosmina, and Cyclops were dominant zooplankters; none exhibited marked reduction after treat- ment at 5 to 34 p.p.b. Most phytoplankter populations showed no obvious changes after treatment, except Aphanizomenon, which increased in all lakes. The posttreatment increase in South Lake Metigoshe was especially noticeable, since there was no increase in untreated North Lake Metigoshe. Several of the plant-inhabiting and bottom fauna decreased slightly after treatment, but this did not appeartobethe result of toxaphene treatment. Tolerance levels for several zooplankters and other aquatic invertebrates were observed in controlled experiments. Rotifera was the most tolerant group, followed in order by Cladocera and Copepoda. Among larger in- vertebrates, Hirudinea, Hydracarina, and Gastropoda were the most tolerant, followed in order by Trichoptera, Odonata, Hemiptera, Ephemeroptera, Amphipoda, and Coleoptera. Use of toxicants in fishery management has provided considerable information concerning the effects of various poisons on fish, Muchless is known of the effects on the fish-food orga- nisms--several workers have reported some such effects: Hooper and Grzenda, 1957, in Michigan; Hoffman and Olive, 1961, and Cush- ing and Olive, 1957, in Colorado; and Stringer and McMynn, 1958, in British Columbia. The object of this study was to determine the effects of toxaphene at low concentrations on the plankton and certain other aquatic orga- nisms under natural and controlled conditions, This was made possible by the rough-fish re- moval program in North Dakota, which various concentrations of toxaphene were used, Investi- This publication is based on a thesis submitted to the Graduate Faculty, Montana State College, in partial ful- fillment of the requirements for the degree of Master of Science in Fish and Wildlife Management, March 1962, gations were carried out in four lakes--a natural lake in the north-central part of the State and three impoundments in the southwest. A fifth lake, untreated, was a control. The study extended from June through September of both 1960 and 1961. Dr. C, J. D. Brown directed the study; Dale L., Henegar, Chief of Fisheries, North Dakota Game and Fish Department, suggested the problem; I am indebted also to Dr. John C, Wright and Dr. G. W. Prescott for help in identifying plankton, to Dr. George F. Edmunds, Jr., for help in identifying aquatic insects, to Donald C. Warnick for help in field work, and to my wife Avis for help in analysing samples, Chemical analyses were made by the State Laboratories, The fish studies were by the North Dakota Fish and Game Department, which also provided financial aid under Dingell-John- son Projects F-2-R 7 and 9, The National Wild- life Federation granted a fellowship for the last year of the study. METHODS Surface water temperatures were obtained with a pocket thermometer, and depth tempera- tures with a reversing thermometer. Secchi disk readings were taken at all stations in con- junction with each collection series. The toxaphene used was an emulsified con- centrate containing 6 pounds of technical toxa- phene per gallon. Before application the toxa- . phene was diluted to 10 to 15 times with water to facilitate uniform distribution. It was applied to the water surface by spraying from a boat. Water samples were collected before and after toxaphene treatment in 1960, and once in 1961. A summary of the physical and chemical data is presented in table 1. Plankton samples were secured with a pump at 1.5 and 7.5 feet at all stations. All samples were taken while the boat was moving in order to avoid resampling the same water. Each sample contained 40 gallons of water, and two samples constituted a collection. Each sample was concentrated to 200 cc. with a No. 20 silk plankton net. Plankton counts consisted in total enumeration of all organisms in 1 cc., with the exception of a few abundant phytoplankters, which were counted by the differential method, employing 20 to 80 fields within a l-cc. sample. Plant-inhabiting organisms were collected with a metal device that I designed. This had an opening of 1 square foot and a height of 30 inches. Openings (4 by 6 inches) were cut on two sides to allow for drainage; these were covered by screen having 30 meshes per inch, A sliding plate was installed at the bottom to sever the plants near their roots. Samples were limited to water depths of 2 feet or less, since this device had to be operated manually, Ap- proximately 4,5 pounds (drained weight, 2-3 minutes) of plants were taken per sample in 1960, In 1961 this was reduced to approximately 12 ounces, since analyses showed this to be ade- quate, The number of square feet of bottom covered in each sample varied from 8 to 16 in 1960 and from 3 to 5 in 1961. Bottom organisms were taken with an Ekman dredge at depths ranging from 4 to 10 feet. Either 3 or 4 square feet were sampled at each station, Organisms from both plant and bottom samples were concentrated with a screen hav- ing 30 meshes per inch, Plant-inhabiting organisms and bottom fauna were sampled at the same stations, which were approximately 50 feet in diameter. These sta- tions had both abundant vegetation and open water. TABLE 1.--Physical and chemical data before and after toxaphene treatment for two lakes and three reservoirs in North Dakota. ’ [All chemical data except pH are expressed as parts per million; bottom temperatures were taken at depths of 9-12 feet] ss Temperature (°F) Secchi |p otar Total Tota Sampling dates eee aes (Gan solids Henanee pH alkalinity Chlorides Sulfates Iron Wolfe Butte Reservoir (treated Aug. 8, 1960): ANGe 5, L96D- cccecccccccvccccccccccvccessessseses 71.4 68.2 4.9 904 114 9.4 408 none 280 1.5 AUG~s 16, 1960... cccrecncccccccccccccccncscnsncees 68.6 68.2 4.2 1,025 114 9.1 463 none 348 1.1 Sept s: '7) 21960 sseecaemutisics seis aaleinee oacisem cmisieletint 64.0 63.7 7.9 == os == = == == == AUG 9, L96Leweceenncsccccccsenccncseccsasesecece 71.0 70.0 4.3 678 84 9.2 336 trace 164 1.0 atl : — Raleigh Reservoir (treated Aug. 4, 1960): AUgs 4, 1960... cecesccenseccssvecesss 721 70.5 3.2 266 184 8.4 153 none 86 1.0 Aug 15, 1960.... 71.7 68.8 4.7 336 196 8.7 143 none 96 0.5 Sept. 6, 1960... Ae 68.0 67.6 8.7 ae = = =e == 2s = BUGs 8, L961. cccccnccvccscveccccvccncassesesvese 71.0 68.2 11.0 380 180 9.8 200 28 143 0.5 South Lake Metigoshe (treated July 17, 1960): July 14, 1960. .ccecccecececvcvevcvesescsevevevere 71.8 68.5 7.9 270 228 8.8 224 none 27 0.2 JULY 21, 1960. ccccccccscccccccesvcvecaccccssesccs 72.0 68.4 8.4 279 222 8.5 214 none 53 0.3 Ree Cheval CBee AA sR Speers: Me aac tangas Han tn 64.6 62.3 8.3 = ze oe as == = = Septagl 5 LOGON some amae aareleiaieriarnates times aieelnete 60.2 59.1 6-4 = ss = == or == == July 19, 1961. .cenccccccvvcccccccccccccccscccecce 68.5 66.8 10.0 299 208 9.4 216 28 44 0.8 a 4 North Lake Metigoshe (untreated control): JULY 14, 1960. ccccccsccecccccccaccscccscevcescnce 71.8 66.8 8.5 281 232 8.6 224 none 33 0.9 July 21, 1960. .ccceencececccnccncesccnscncesccece 72.3 68.0 8.8 282 226 8.2 214 none 53 0.2 INGA Fag Wah gonaengdo soonoande saonaadeeaanoosue 64.9 61.9 6.4 == 5 == -- -- -- -- Odland Reservoir (treated Aug. 11, 1960): 66.1 2.2 414 184 8.2 187 none 165 0. 65.8 1.8 510 202 8.2 195 none 186 0.8 64.0 3.1 -- -- -- -- == aS =2 68.0 21 574 208 8.5 196 trace 259 0.5 WOLF BUTTE RESERVOIR DESCRIPTION Wolf Butte Reservoir, in southwestern North Dakota, has a surface area of 24 acres and a maximum depth of 9 feet. It has no permanent inlet or outlet, and water is supplied mainly by runoff. The bottom is muck, No marked thermal stratification was present, The area surround- ing the reservoir is primarily rangeland. Aquatic vegetation was very abundant at all depths less than 4 feet. Potamogeton pectinatus, P. richardsoni, and Myriophyllum exalbescens, were the dominant plants. A heavy mat of fila- mentous algae (Rhizoclonium) accompanied these plants at the water surface. TREATMENT Fish.--Toxaphene was applied at 35 p.p.b. on August 8, 1960, in an attempt to eradicate the fish population, This impoundment was heavily populated with green sunfish (Lepomis cya- nellus) black bullheads (Ictalurus melas), and a few large rainbow trout (Salmo gairdneri). Many green sunfish and black bullheads were found dead and dying after treatment. The reservoir was test-netted 1 week after eradication and again the following spring. Two 125-foot ex- perimental gill nets were set for 24 hours, and no fish of any species were taken. The reser- voir was test-netted again in August of 1961, when one 125-foot gill net and one frame net were set for 24 hours, The nets contained ap- proximately 475 black bullheads and 83 trout. Many young-of-the-year green sunfish were also observed, A trapping program later in the fall revealed several adult green sunfish. Plankton.--Four collections of plankton were made at one station near the center of the reservoir. Collections were made 3 days before treatment, and after treatment at 8 days, 30 days, and 366 days. The kinds and numbers of plankton are given for each collection in table 2, These are arranged in a phylogenetic order with the zooplankters first. Comparison in numbers per liter was made between pretreatment and posttreatment col- lections, Rotifers were represented by nine genera, Keratella and Asplanchna being the TABLE 2.--Number of plankters per liter in Wolf Butte Reservoir before and after toxaphene treatment at 35 parts per billion. [Treated Aug. 8, 1960] a a T eee After Before cos Aug. 5, 1960 i Ee E [ , Aug. 16, 1960 | Sept. 7, 1960 | Aug. 9, 1961 + See © —_ = —— Brachionus.... 1 “ -- 3 Keratella..... 91 15 2 1 LECONE.eseecee -- -- -- 1 Trichocerca... 1 -- -- -- Chromogaster.. ul 2 1 | -- Asplanchna.... 73 106 -- -- Polyarthra.... 7 3 1 2 Filinia....... 1 1 -- 1 Hexarthra..... 3 21 3 -- Daphnia.....++ 244 18 129 28 Simocephalus.. -- ee 1 ee Ceriodaphnia.. 4 9 18 -- Bosmina...+.++ 98 130 18 25 Chydorus.....«. 1 —- = aad Diaptomis..... 6 -- -- 2 CyclopS...-+..+. Ae 3 1 Lb Neuplii}...... 106 26 10 73 Pandorina..... 3 ab) -- -- Oedogonium.... 3 4 -- -- Cladophora.... -- -- -- 2 tr Rhizocloniun. . 4 1 8 1 Pediastrun.... 12 5 3 61 Coelastrum.... ad -- -- 3 Oocystis...... 1 3 -- -- Closteriopsis. 1 4 -- tr Tetraedon..... -- 1 -- -- Scenedesmus... 7 4 7 -- Mougeotia..... -- -- 1 -- Spirogyra..... 1 nh 52 -- Closterium... 1 al tr Cosmarium..... 4 1 2 -- Staurastrun.. 3 3 -- -- Desmidium..... 65 84 7 tr Botryococcus. -- 3 2 7 Diatoma......- 2 1 1 2 Navicula...... 2 6 -- -- Pinmlaria.... 1 — = == Pleurosigma... -- -- ab -- Cymbella...... ae tr -- -- Nitzschia..... aut 9 4 tr Camplyodiscus al -- -- -- Ceratium...... 5 2 -- 11 Synechocystis. 90, 067 589,405 149, 306 82,563 Polycystis.... 242 131 2 110 Merismopedia.. -- -- al -- Coelosphariun. 50 28 1 -- Lyngbya.....e. 8 2 9 -= Anabaena...... 61 58 1 “= Aphanizomenon. Gs 277 33,157 54,716 138 Nodularia..... -- 12 1 == + Includes nauplii of both Diaptomus and Cyclops. 2 Less than 1 per liter. most numerous, Keratella changed from 91 be- fore treatment to 15 at 1 week, 2 at 1 month, and only 1 at 1 year after treatment. Asplanchna increased from 73 before treatment to 106 at 1 week after treatment, but none were present in collections at 1 month or 1 year after treat- ment, Other rotifers were too scarce for com- parisons, Cladocerans were the most abundant zoo- plankters, with Daphnia and Bosmina appearing in large numbers, Daphnia decreased from 244 before treatment to 18 at 1 week, then increased to 129 at 1 month after treatment. Bosmina ex- hibited the reverse effect, and both were less abundant at 1 year after treatment. Copepoda were represented by Diaptomus, Cyclops, and undetermined nauplii. Six Diaptomus were taken before treatment, but none were found at 1 week or 1 month after treatment and only 2 at 1 year. 5 Cyclops decreased from 46 in the pretreatment collection to 3 at 1 week after treatment, but increased to 11 at 1 month, Nauplii decreased from 106 before treatment to 26 and 10 at 1 week and 1 month after treatment. Cyclops and nauplii were relatively abundant the following year, There were 16 genera of Chlorophyta, 8 of Chrysophyta, and 1 of Pyrrophyta in the col- lections. None exhibited numerical changes - which could be attributed to toxaphene treat- ment, Eight genera of Cyanophyta were present, and these were the most numerous algae. Synechocystis and Aphanizomenon were the most abundant genera. Synechocystis increased from 90,067 before treatment to 589,405 at 1 week after treatment, then decreased to 149,306 at 1 month. Aphanizomenon increased from 7,377 before treatment to 54,716 at 1 month after treatment. Polycystis, Coelospharium, and Anabaena decreased after treatment. Poly- cystis was abundant at 1 year, but Coelosphar- ium and Anabaena did not reappear 1 year after treatment, Most of the changes before and after treat- ment were small and could well be the result of normal fluctuations in the population or the result of sampling techniques. A few of these changes may have resulted from the toxaphene, but none were obvious. Plant-inhabiting organisms. --Aquatic-plant- inhabiting organisms were collected at two sta- tions on the same dates plankton was sampled. The numbers of organisms per pound of vege- tation for the four collections is presented in table 3. Nineteen genera were represented, but only seven were numerous. Gammarus varied throughout the study, but remained abundant. ‘Callibaetis, Caenis, and Ischnura decreased at 1 week and 1 month after treatment, but were more abundant at 1 year, Tendipes decreased from 44 before treatment to 9 at 1 week after treatment, while 48 were taken at 1 month and 25 at 1 year after treatment. Gastropoda (Physa and Gyraulus) increased from 771 before treat- ment to 1,107 at 1 week, 1,366 at 1 month, and 1,558 at 1 year after treatment. TABLE 3.--Number of plant-inhabiting organisms and bottom fauna in Wolf Butte Reservoir before and after toxaphene treatment at 35 parte per billion. [Plant-inhabiting organisms are expressed as the mumber per pound of plants and bottom fauna as the number per square foot of bottom. Treated Aug. 8, 1960. tr = less than 1 per pound or per square foot] T Before aes After Organiem | AUS+ 5» 1969 | aye. a6, 1960 | Sept. 7, 1960| Aug. 9, 1962 Plant | Bottom | Plant | Bottom | Plant | Bottom | Plant | Bottom Oligochseta... -- tr -- tr -- 3) Hirudinea....-. -- -- tr -- -- Amphipoda Gammarus...«. 63 6 172 tr 73 Hydracarina: Hydachnidae.. 5 -- tr -- 2 Ephemeroptera; Callibaetis.. 5 1 tr -- -- Caenis....... 6 3 1 tr -- Odonata: Sympetrum... tr -- tr tr -- Aeschna...... -- -- tr -- -- Ischnura....- 40 5 10 1 -- Hemiptera: PleGeccccccce -- -- tr -- -- Notonecta. ab -- -- == 3 SIgara..++.-. 3 -- -- -- tr Coleoptera: Haliplus..... 2 -- tr -- -- Copelatus.... tr -- -- -- tr Hydroporus... -- -- -- -- if Trichoptera: Hydroptila... -- al -- tr -- Diptera: Tendipes..... 44 28 9 2 48 Probezzia. tr 3 -- 2 -- ChrySopS...«+.- -- 1 -- al -- Gastropoda: PhYSA.+++eeee 123 2 325 af 156 Gyraulus....- 648 tr 782 1 1,210 Pelecypoda: Pisidium..... -- 3 -- 1 -- Numerical comparisons of the seven domi- nant genera revealed no marked changes before and after treatment. Reductions of Ephemerop- tera and Odonata in the first two posttreatment collections may be significant but could have resulted from an emergence. Bottom fauna,--These organisms were col- lected at the same stations as those used for plant-inhabiting organisms, Each collection consisted of 3 square feet of bottom. The num- ber of organisms per square foot of bottom is given for each collection (table 3), Thirteen genera were taken, but only Gammarus and Tendipes were abundant. Gammarus fluctuated — from 6 before treatment to less than 1 at 1 week, 44 at 1 month, and less than 1 at 1 year after treatment. The large number at 1 month after treatment resulted from a collection that contained considerable vegetation. Tendipes de- creased from 28 before treatment to 12 and 9 at 1 week and 1 month after treatment but in- creased to 25 at 1 year. A comparison of the number of bottom organisms before and after treatment revealed no marked changes. RALEIGH RESERVOIR DESCRIPTION Raleigh Reservoir, in southwestern North Dakota, has a surface area of 15 acres and a maximum depth of 18 feet. There are no perma- nent inlets or outlets, and the water is supplied mainly by runoff. The bottom is muck, covered by silt in some areas. No marked thermal stratification was present, The surrounding area is almost entirely rangeland, Aquatic vegetation was very abundant at all depths less than 3feet. Potamogeton pectinatus, P, richard- soni, Myriophyllum exalbescens, and Cerato- phyllum demersum were the dominant plants. Large amounts of filamentous algae (Rhizo- clonium) accompanied these plants in most areas, TREATMENT Toxaphene was applied at 25 p.p.b. on August 4, 1960, in an attempt to remove the entire fish population. A complete kill was not achieved and a second treatment was made at 90 p.p.b. on September 26, 1960. Fish.--Before treatment, two 125-foot ex- perimental gill nets and four frame nets were set for 24 hours. The frame nets contained sev- eral thousand golden shiners (Notemigonus crysoleucas), approximately 5,000 green sun- fish, and 1,200 white crappies (Pomoxis annu- laris) and black crappies (Pomoxis nigromacu- latus), The two experimental gill nets captured 13 white suckers (Catostomus commersoni), 11 black bullheads, and a few golden shiners, green sunfish, and crappies. Large numbers of the four most numerous species were found dead and dying after treatment. The reservoir was netted again 1 week after the first treat- ment, but with only two experimental gill nets set for 24 hours, These contained 10 white suckers and 5 black bullheads. Test-netting was discontinued since drought had lowered water levels to a point where restocking was imprac- ticable. Plankton.--Four collections were made at one station near the center of the reservoir. Collections were made 1 day before the first treatment and at 11, 33, and 371 days after the first treatment; a second treatment was made 53 days after the first, and the fourth collection was 318 days after this treatment. Quantities of plankters in pretreatment and posttreatment collections are shown in table 4, Rotifers were represented by 15 genera, but only Brachionus and Asplanchna were abundant. Brachionus de- creased from 114 before treatment to 108 at 11 days and 15 at 33 days after treatment. As- planchna varied from 24 before treatment to 194 at 11 days and 16 at 33 days after treat- ment, Only 3 Brachionus and 1 Asplanchna were TABLE 4.--Number of plankton per liter in Raleigh Reservoir before and after tregtment at 25 parts per billion. [Treated Aug. 4, and Sept. 26, 1960] After Before Gagan | ug. 12) 860 soe) cc 15, 1960 iL Sept. 6, 1960 | Aug. 10, 19612 Brachioms... 114 108 15 3 Keratella.... al) i 9 Af Platyias..... 2 tr 17 1 -- Lecane.-coeee tr 2 -- = Monostyla.... eT: 1 -- 5 Trichocerca.. 5 -- -- tr Chromogaster. al 3 -- at Asplanchna... 24 194 16 1 Polyarthra... 3 ab) 7 1 Synchaeta.... 1 1 13 -- Filinia...... tr 1 -- tr Testudinella. 1 -- tr -- Trochosphaera tr -- 1 -- Hexarthra.... 2 -- -- = Conochiloides 3 aah == 45 Daphnia.....-. 65 173 57 9 Ceriodaphnia. 44 156 100 al Bosmina....++ 314 283 50 -- Chydorus..... 4 17 1 -- Diaptomus 10 1 1 -- Cyclops...... 120 9 41 8 Nauplii?..... 190 85 19 7 Elakothrix... =5 =2 a} 73 Microspora... -- 1 3 =o Oedogonium... 9 3 ab -- Rhizocloniun. 7 5 3 i Golenkinia... -- 3 -- -- Pediastrum... 151 462 3 3 Coelastrum... ee 594 -- -- Oocystis..... 69 75 2 -- Chodatella... 4 1 -- -- Closteriopsis 18 462 1 =e Tetraedon.... any 89 -- -- Scenedesmus. . 727 3,038 133 1 Crucigenia... 17 264 4 -- Tetrastrum... -- 3 -- -- Mougeotia.... -- 1 al = Zygnema.....- 1 -- -- -- Spirogyra. . 2 4 19 107 Closterium... -- -- 2 tr Cosmarium.... 20 4 5 1 Staurastrum.. 4 9 -- -- Desmidium.... 925 1,189 4 -- Botryococcus. 5 8 7 -- Melosira..... 4 9 -- 2 Diatoma...... 8 -- 3 1 Synedra..-.-- 4 3 -- 3 Navicula..... 6 al ab -- Pinnularia... 1 -- 3h -- Frustulia.... -- tr 1 -- Gyrosigma.... ae tr -- -- Pleurosigma.. tr -- -- aed Gomphonema... tr 1 == =e Cymbella..... 4 4 1 == Nitzschia.... 8 14 7 8 Cymatopleura. 2 1 oe -- Camplyodiscus 2 -- -- -- Ceratium....- 24 rf 4 5 Synechocystis 54,161 6,275 2,312 601,057 Polycystis... 2,906 859 38 99 Merismopedia. 9 7 1 -- Coelospharium 2 25 94 4 Lyngbya.....- 10 1 8 1 Anabaena..... 5 4 16 2 Aphanizomenon 6 190 81,902 tr- Nodularia.... Pale “77 3,633 | 21 P| 3 + After the second treatment at 90 p.p.b. 2 Less than 1 per liter. 3 Ineludes nauplii of both Diaptomus and Cyclops. taken 371 days after treatment. All rotifers were very scarce at this time, and 6 of the original genera were not found, Cladocera was the most abundant zooplankter. Daphnia, Ceriodaphnia, and Bosmina were present in large numbers, Daphnia varied from 65 before treatment to 173 at 11 days, 57 at 33 days, and only 9 at 371 days after treatment. Ceriodaphnia increased from 44 before treat- ment to 156 at 11 days, then decreased to 100 at 33 days, and only 1 was taken at 371 days after treatment, Bosmina decreased from 314 before treatment to 283 at 11 days and 50 at 33 days after treatment and disappeared by 371 days. A few Chydorus were found in the pre- treatment and early posttreatment collections, but did not occur in the collection 371 days after treatment. Copepoda were represented by the young and adults of Diaptomus and Cyclops. Diaptomus changed from 10 before treatment to 1 at 11 days and 1 at 33 days, but none at 371 days after treatment, There were 120 Cyclops before treatment while collections after treat- ment showed 9 at 11 days, 41 at 33 days, and 8 at 371 days. Nauplii decreased from 190 before treatment to 85 at 11 days, 19 at 33 days, and 7 at 371 days after treatment. The Chlorophyta were represented by 21 genera. Pediastrum, Coelastrum, Closteriopsis, Tetraedon, Scenedesmus, Crucigenia, and Des- midium were the most abundant. All of these increased in the collection 11 days after treat- ment but were greatly reduced at 33 days and 371 days. Spirogyra was the most abundant of the Chlorophyta in the collection 371 days after treatment, but was scarce in the pretreatment and early posttreatment collections. The Chrysophyta contained 9 genera and the Pyrro- phyta 1, These were infrequently encountered, and no comparisons were made. The Cyanophyta were represented by 8 genera. Synechocystis, ~ Polycystis, Coelospharium, Aphanizomenon, and Nodularia were the dominant organisms, Synechocystis and Polycystis decreased in the first two posttreatment collections but were abundant at 371 days after treatment. Coelo- spharium and Aphanizomenon increased after treatment but were scarce at 371 days after treatment. Nodularia varied from 77 before treatment to 3,663 at 11 days, 21 at 33 days, and 3 at 371 days after treatment. 8 Changes after the first treatment (25 p.p.b.) are probably the result of normal population fluctuations, At 371 days after treatment water levels had dropped approximately 6 feet, the water was clear, and aquatic vegetation had in- creased, The severe reduction in nearly all plankters at this time may have been due to the drop in water levels or the possible consequent increased toxaphene concentration. Plant-inhabiting organisms,--Collections were made at two Stations on the same dates plankton was collected. The number of orga- nisms per pound of vegetation is presented for each collection (table 5), Nineteen genera were | taken, but only eight were abundant. Gammarus increased from 31 before treatment to 313 at 11 days, 569 at 33 days, and 334 at 371 days after treatment. Hydrachnidae decreased from 45 be- fore treatment to 27 at 11 days, 22 at 33 days, and 14 at 371 days after treatment, Callibaetis, Caenis, Ischnura, and Tendipes were markedly reduced in the first two posttreatment collec- tions, but all except Caenis were abundant at 371 days after treatment. Sigara decreased . from 39 before treatment to less than 1 at 11 days after treatment, and none were taken after TABLE 5.--Numbers of plant inhabiting organisms and bottom fauna in Raleigh Reservoir before and after toxaphene treatment at 25 parts per billion. [Plant inhabiting organisms are expressed as the mumber per pound of plants and bottom fauna as the number per square foot of bottom. Treated Aug. 4, and Sept. 26, 1960.] a Before pd Organism | AU8- 3» 1960 Sept. 6, 1960 | Aug. 10, 19612 q Plant | Bottom | Plant | Bottom ~ Oligochaeta... -- 4 =: 8 ss 28 Hirudinea..... 2 tr es wi ie poe = =a Amphipoda Gammarus..... 31 4 313 tr 569 zt Hydracarina: “ Hydrachnidae. 45 -- 27 -- 22 4 Ephemeroptera: Callibaetis.. 66 1 9 tr tr -- Caenis....-- 185 6 10 tr 6 -- Odonata: Sympetrum. .. -- -- af -- tr as. ANAXs se eeeeee -- -- -- -- -- == Aeschna....-- tr -- tr -- -- os Ischnura..... 109 2 21 1 8 = & Hemiptera: Notonecta. 2 -- tr -- tr -- : Sigara....... 39 2 tr -- -- =o Coleoptera: Copelatus.... | --| -- |) ee 2s at Hydroporus... 2 -- 7 -- 6 a Diptera: Chaoborus.... -- 1 -- -- -- a Tendipes..... 12 27 tr 2 1 71 : Probezzia.... tr -- tr -- -- == Chrysops....-. = = tr =< =e pes | Gastropoda PHYSA. cesses 5 -- als) -- 6 -- . Gyraulus..... 1,163 tr 1,695 4 398 = . Pelecypoda Pisidium..... -- tr -- 4 -- 4 1 after the second treatment at 90 p.p.b. 2 Less than one per pound or square foot. this time. There were 1,163 Gyraulus before treatment, 1,695 at 11 days, 398 at 33 days, and 38 at 371 days after treatment, Several changes were noted following treat- ment, some of which may be the result of the toxaphene, Reductions of Callibaetis, Caenis, Ischnura, and Tendipes may be significant; all but Caenis, however, were abundant 371 days after treatment. Stringer and McMynn (1958) reported that Ephemeroptera were killed at 30 p.p.b, toxaphene, The disappearance of Sigara after treatment appears to be the result of the toxaphene since they exhibited low tolerance levels in the controlled experiments (table 11). The reduction of Gyraulus at 371 days after treatment may be related to lowered water levels, since other workers (Hooper and Grzenda, 1957; and Stringer and McMynn, 1958) found Gastropoda to be unaffected by toxaphene at 100 p.p.b. Bottom fauna.--Four collections were made at two stations on the same dates plant-in- habiting organisms were collected, The number per square foot of bottom is given for each col- lection (table 5), Eleven genera were taken, but most of these were too scarce for comparisons, Oligochaeta increased throughout the study from 4 before treatment to 28 at 371 days after treat- ment. Cushing and Olive (1957) found an in- crease in Oligochaeta after treatment with 100 p.p.b. toxaphene. Ephemeroptera decreased from 7 before treatment to less than 1 at 11 days, and none were taken in succeeding col- lections. Tendipes decreased from 27 before treatment to 2 at 11 days and 6 at 33 days after treatment, then increased to 71 at 371 days after treatment. The reductions of Ephemeroptera and Tendipes may be significant. Stringer and McMynn (1958) reported that Ephemeroptera were killed at 30 p.p.b. of toxaphene, and Cush- ing and Olive found that a concentration of 100 p.p.b. eliminated Tendipedidae. SOUTH LAKE METIGOSHE DESCRIPTION South Lake Metigoshe is a glacial lake in the Turtle Mountains in north central North Dakota. It has an area of 915 surface acres and an average depth of 9 feet. Water is supplied mainly by runoff. Water levels fluctuate slightly owing to releases from an upstream reservoir. The major bottom materials are peat and muck,. No marked thermal stratification was present. Trees border most of the shoreline, Aquatic vegetation was common and was exceptionally abundant in the bays, Scirpus sp, occupied sev- eral large areas near shore, Myriophyllum exalbescens and Ceratophyllum demersum were present at most depths less than 15 feet. Other dominant plants were Potamogeton natans, P, pectinatus, P. richardsoni, P. zosteriformis, Najas flexilis, Sagittaria latifolia, Eleocharis palustris, and Polygonum amphibium., TREATMENT Toxaphene was applied at 10 p.p.b. on July 17, 1960, in an attempt to reduce the number of yellow perch (Perca flavescens) and black bull- heads, This was supplemented by 5 p.p.b. on July 19. Fish,--Several 250-foot experimental gill nets and frame nets (0,5-inch and 0, 25-inch mesh) were set at selected stations 1 week be- fore, 1 week after, and again 11 months after treatment; the netting efforts were 333, 290, and 120 hours, respectively. The fish taken are expressed as the number per 100 net-hours. Adult yellow perch were reduced from 900 be- fore treatment to 36 at 1 week and none at 11 months after treatment. Young-of-the-year were reduced from 610 before treatment to 6 at 1 week and none at 1 year after treatment. Young-of-the-year black bullheads decreased from 240 before treatment to 9 at 1 week and none at 11 months after treatment. Young-of- the-year northern pike (Exos lucius) decreased from 40 before treatment to 10 at 1 week and none at 11 months after treatment, Netting at 1 week after treatment did not show a reduction in adult black bullhead, northern pike, and wall- eye (Stizostedion vitreum), but several were found dead along shore at this time. No walleye were taken at 11 months after treatment, and bullheads and northern pike were greatly re- duced, The paucity of all species taken at 11 months after treatment may have been due to the residual effects of the toxaphene. Plankton,--Five collections were made at four stations on South Lake Metigoshe. These were made 2 days before treatment and at 4, 40, 60, and 367 days after treatment. The pre- treatment and the first two posttreatment col- lections at South Lake Metigoshe are compared with those made at four stations on North Lake Metigoshe, which was sampled on the same dates. North Lake Metigoshe is adjacent to South Lake Metigoshe and is connected by a channel approximately 30 feet wide; it was not treated until later in the fall and could there- fore be used as a control. The number of plankton per liter for all collections in both lakes is given in table 6. TABLE 6.--Number of plankton per liter in South and North Lake Metigoshe before and after toxaphene treatment at 15 parts per billion [South Lake Metigoshe treated July 17, 1960] Before, BANE = : July 15, 1960 Organism July 21, 1960 Aug. 26, 1960 sept. 15, 1960, July 19, 1961, South North South North South North South South T BrachionusS..+++eseeee Ghendcdnagsacundocoanccosone4 -- 2 1 1 © ip =o ab -- Gia ETHIE Sopcacencenaondbanoceamadccuse Eas 35 28 48 22 a 25 1 12 TRS CTI etotate fata ny oi ets Te Sete acto nle te eted settee ete fe fafetet +r -- -- -- il tr -- tr Monostyla......+sseee gonondgomoscdodsogenc a9 3 -- 2 -- 9 2 al, 2 Mrtehoe emse aetebtetetelstetaletefelatelaledalctelstleietaleterstelelatelelteletetet=ts 234 ala 83 86 Shi 5 3 3 Ascomorpha..... 1 3 5: 4 4 tr tr 1 Chromogaster... 180 2 tr 2 3 -- tr al, Asplanchna..... 28 3) 24 6 aL 12 -- 20 Polyarthra..... 4 27 35 34 42 32 28 16 Synchaeta...... 17 il 19 3) 10 2 i tr Filinia........ 3} 3 25 2 7 2 2 3 Testudinella... -- -- -- tr -- -- == tr Hexarthra...... ail 1 15 2 -- 1 == tr Conochiloides.. 182 14 160 34 al 10 -- tr Stephanoceros.. -- -- -- -- -- tr -- = Daphnia........ aL 9 1 2 9 5 16 41 SimOcephalUsy tr 2 =e -- tr -- -- tr -- -- Diptera: CHAODOTUS see eereecececerscsvccscsesccssssassssecs tr == iL -- -- -- -- a TENdiPeS..ceeeeseecessccsccccsccccccrscessscsesecs 9 5 3 6 pie 3 2 a8 PLODEZZIAs cccccccevccccccccsccccssccecesssessese tr a tr tr tr -- -- -- Gastropoda: 24 28 21 27 31 19 29 66 -- tr -- tr -- -- tr -- 47 56 24 44 51 105 86 10 -- tr tr -- tr -- tr -- Soe | rere + Represents less than 1 per pound. TREATMENT Fish.--Toxaphene was applied at 5 p.p.b. on August 11, 1960, to reduce young-of-the-year black bullheads and yellow perch. Large num- bers of these fish and several young-of-the- year northern pike, white crappie, and orange- spotted sunfish (Lepomis humilis) were found dead along shore the day after treatment. Adults of these species were not significantly reduced by treatment, since only a few were found dead and large numbers were taken in posttreatment test nettings. Plankton.--Four collections were made at each of two stations. These were made 1 day before treatment, and at 7, 28, and 362 days after treatment. The kinds and number of plankters per liter are given for each collec- tion (table 8), There were 10 genera of Rotifera taken; Brachionus, Keratella, Polyarthra, and Conochiloides were the most abundant and re- mained nearly constant before and after treat- ment, Cladocera were the most abundant zoo- plankters with Daphnia, Ceriodaphnia, and 12 Bosmina being most common. There were 22 Daphnia before treatment, 68 at 7 days, 40 at 28 days and 3 at 362 days after treatment. Ceriodaphnia varied from 31 before treatment to 66 at 7 days, 37 at 28 days, and only 1 at 362 days after treatment. Bosmina decreased from 459 before treatment to 24 at 28 days and 66 at 362 days after treatment. Copepoda were represented by adults and nauplii of Diaptomus and Cyclops. Cyclops decreased from 71 before treatment to 16 at 28 days after treatment, and nauplii decreased from 129 before treatment to 36 at 28 days after treatment, but both were again abundant at 362 days after treatment, Fourteen genera of Chlorophyta, 12 of Chryso- phyta, 1 of Pyrrophyta, and 5 of Cyanophyta were found. Melosira, Ceratium, Polycystis, and Aphanizomenon were the dominant orga- nisms of these groups. These increased at 7 days and 28 days after treatment. Approxi- mately the same numbers were found in post- treatment collections at 362 days as were found before treatment. Numerical comparisons of pretreatment and posttreatment collections showed no marked changes. TABLE 8.--Number of plankton per liter in Odland Reservoir before and after toxaphene treatment at 5 parts per billion. [Treated Aug. 11, 1960] ct BOGRPHPRPIRPREYD rb Poreobp + Represents less than one per liter. 2 Includes nauplii of both Diaptomus and Cyclops. Plant-inhabiting organisms.--Four collec- ions were made at two Stations on the same _ dates plankton were collected. The number of _ organisms per pound of plants is given for each collection (table 9), Nineteen genera were taken, with Gammarus, Hydrachnidae, Caenis, Isch- hura, Tendipes, Physa, Gyraulus, and Valvata _ being the most abundant. Caenis and Tendipes ecreased slightly after treatment, probably a ormal population fluctuation rather than a re- TABLE 9.--Number of plant-inhabiting organiems and bottom fauna in Odland Reservoir before and after toxaphene treatment at 5 parts per billion. [Plant-inhabiting organisms expressed as number per pound of plants and bottom fauna as number per square foot of bottom. Treated August 11, 1960] Before, After ae = teste eee Organism | 4¥8- 10, 1960 | 4.5. 18, 1960 | sept. 8, 1960 | Aug- 8, 1961 Plant ] Bottom | Plant | Bottom | Plant | Bottom | Plant | Bottom Oligochaeta... -- oo 1 tr ae Hirudinea..... tr -- -- 1 == Amphipoda Gammarus..... 195 2 303 6 76 tr Hydracarina: Hydrachnidae. 53 -- -- 4 ins 17 nee Ephemeroptera: CaeniS...ses. 21 2 tr 5 -- lu tr Odonata; Sympetrum... n -- -- -- == =e = Aeschna...... tr tr -- -- «= 2 med Ischmura..... 5 1 tr 13 1 11 os Hemiptera: Notonecta..-. tr -- -- 2 -- = ated Sigara....... tr -- -- iit -- «= mes Trichoptera: Hydroptila... -- Ab -- -- we == == Psychomyia... tr -- -- AS 2S == - Phryganea... tr -- -- == == = - Coleoptera: Haliplus..... 6 -- -- 1 = tr = Hydroporus. +. tr -- -- 2 me <= == Diptera: Tendipes..... 37 18 8 2 14 8 Probezzia.... -- -- -- = tr = tr Chrysops..... -= Gastropoda; + Less than 1 per pound or square foot. Gyraulus, Valvata, and Pisidium were abundant, None of these exhibited marked numerical changes that could be attributed to toxaphene treatment. EXPERIMENTS Six Rotifera, two Cladocera, and two Copepoda were tested at six toxaphene concentrations ranging from 50 to 1,000 p.p.b., to determine their tolerance levels. All tests were conducted in battery jars, each containing 8 liters of fil- tered lake water taken at the site where the organisms were collected. The water had an average temperature of 68° F,, a dissolved oxygen content of 9.8 p.p.m., total alkalinity of 341 p.p.m., and pH of 8.4. Before each experi- ment the jars were washed with steel-wool soap pads and rinsed, All organisms were collected by pumping lake water through a No, 20 plankton net and were then placed in the jars. The toxa- phene was diluted with water and applied to the water surface with moderate mixing, and after 24 hours the plankters were removed by siphon- ing into a No. 20 plankton net and concentrated to 25 cc, All organisms in 2 cc. of this sample were counted. To avoid collecting the dead and 13 affected plankters the jars were tilted and 200 day intervals. This was done to determine cc. were left in the bottom after drainage. Three whether large amounts of toxaphene were trials were conducted at each concentration, and accumulating because of inadequate washing, the number of organisms counted was compared since these minnows were found to have low with untreated controls, which were maintained tolerance levels (Hooper and Grzenda, 1957), for all experiments (table 10). The lowest concentration used in the experi- ments was 10 p.p.b., which produced 100 per- Larger invertebrates were tested to deter- cent mortality among the test fish while all mine the concentration at which 100 percent experimental organisms survived, No fathead survived for 24 hours and 100 percent were minnows died in the washed tanks, and it was killed (table 11), These tests were carried out assumed that the procedure was adequate. in galvanized tanks, each containing 20 gallons of lake water at 71° F, The water used, toxa- RESULTS phene application, and cleaning method were the same as for zooplankters. In most cases 10 to Marked reductions of rotifers were first ob- 20 organisms were used to calculate percent served at 500 p.p.b., cladocerans (Daphnia pulex survival and mortality. Controls were main- and Boxmina) at 250 p.p.b., and copepods at 100 tained for 2 weeks, then discontinued, and sur- p.p.b. (table 10), All genera in each group ex- vival was assumed to be 100 percent, with the hibited similar tolerance levels. Four trials exception of Gammarus which showed 91 per- employing 10 organisms each were conducted cent survival. with Daphnia magna at six concentrations, No effects were obvious at 50 to 400 p.p.b., how- Fathead minnows (Pimephales promelas) ap- ever retarded movements were observed at proximately 1 inch in length were placed in all 1,000 p.p.b., and movements had nearly ceased containers after washing, for 48 hours at 10- at 1,500 p.p.b. Prevost (1960) reported a median TABLE 10.--Comparison in mumber of zooplankters per cc. in 25-cc. concentrates from treated and control jars. 50 p-p.-b. 100 p-p-b. 250 p-p-b. 500 p.p-b. 750 p-p-b. 1,000 p.p.b. Organism and trial _— Control Treated Control Treated Control Treated Control Treated Control Treated Control Ese: 1 i 5 4 5 6 ie} 2 1 0 2 1 4 6 {0} ab 0 2 69 23 14 al 0 ie) 7 4 7 2 ay 4 10 ie) 10 0) 2 ak 4 5 5 5 5 2 le) (0) 4 1 0 0 111 133 14 6 10 10 8 ie) 41 2 4 ae 3 4 3 2 4 1 7 1 7 te) 20 1 19 26 14 35 14 14 22 11 19 1 3 fe) 6 5 4 3 22 8 17 9 6 il 4 ie} 7 6 7 2 3) 10 12 3 12 1 2 ie} Keratella: Trial 1. (0) al 9 9 9 4 2 ie) 0 fe) 16 1 Trial 2. ab it 35 at, 2 at 23 14 1 (6) 35 at Trial 3. 105 51 105 4l 1 al 12 3 12 a 18 2 8 10 18 14 18 22 8 3 8 ie} 7 ie} 12 7 14 13 8 9 98 48 12 10} 1 ie} abl 9 10 12 13 21 al fe) a 2 7 2 122 136 296 252 296 278 144 102 122 10 80 6 58 68 72 85 144 148 351 191 58 1 79 24 27 24 27 18 281 363 94 88 88 23 84 aE 24 21 10 8 10 3 11 7 24 1 6 0 11 16 1 2 11 10 8 1 11 ie} Z 6) 5 5 5 Ws 36 8 7 0 7 10} 28 ie) 34 43 75 79 79 Bb 29 4 34 0) 37 8 76 93 6 % 29 5 68 2 76 4 16 4 19 27 19 17 69 33 88 4 88 4 202 8 140 116 25 16 15 2 183 8 140 1 166 ie) 23 27 23 9 183 5 21 (o) 33 e) 23 1 52 36 52 3 50 2 122 ie) 122 ie) 32 ce) 20 12 14 6 5 2 12 ie) 20 1 28 0 4 6 5 6 12 1 58 3 4 ie} 14 0 10 14 10 8 10 le) 27 1 27 le) 85 ie} 53 49 63 40 63 15 46 1 53 2 48 ie} 80 71 48 ent 46 26 89 12 80 2 80 ab 50 58 50 46 59 29 73 19 73 7 152 9 > Represents both Diaptomus and Cyclops. 14 TABLE 11.--Percent survival of several aquatic invertebrates after 24 hours' exposure to toxaphene concentrations eS and toxaphene concentration “if Percent alive Hirudinea (2 trials): PGi eter sen cue ener Sava caiwcliccuatessiececuyy 100 Amphipoda: Gammarus (14 trials): Pp-p Hydracarina ra trials): 1,000 pep.Deceeccccccccceccereensecrecteeerereseee 100 emeroptera: Callibaetis (14 trials): Lb 0 100 84 40 lo) 100 81 33 le) 100 721 39 0 100 60 25 (0) Trichoptera: Limephilus (12 trials) 500 PePeDeveneccevcccvecccccssscscsessscsesssvecss 100 550 PePeDeceeeccevecccrenccccsrccsssesssceessccnes 49 600 PePeDeseeeecccesnccccccccssenscsssessseesssens 20 650 Ee Da wnccscccccccccccccccccccsceccscccscence 10) es laa tolerance limit (TL,,) of 0.037 p.p.m. for cladocerans, and Hooper and Grzenda (1957) found Daphnia magna to have a TL, of 1.5 p.p.m. at 55° F. Tolerance levels (100-percent survival) for the larger invertebrates are listed in decreas- ing order as follows; Hirudinea, Hydracarina, Gastropoda, Trichoptera, Odonata, Hemiptera, Ephemeroptera, Amphipoda, Coleoptera (table 11), Survival at concentrations between 100-percent survival and 100-percent mor- tality showed an approximate straight-line relation (table 11), Genera within each group did not exhibit similar tolerance levels. This was evidenced among members of Odonata, Hemiptera, and Coleoptera, Lowered tempera- tures produced marked increases in tolerance levels. In Lestes tolerance increased approxi- mately 35 percent by lowering the temperature 10 degrees. Hooper and Grzenda (1957) found mortality in fathead minnows increased approxi- mately threefold by raising the temperature from 50° F, to 75° F, Many of the findings are similar to those of Prevost (1960), however comparisons are difficult since he provided no temperature data. DISCUSSION Populations of plankton show many large variations throughout the year (Pennak, 1949; and Rawson, 1956), In the present study, the populations of organisms which could best illustrate posttreatment changes were not severely reduced, therefore no obvious effects could definitely be attributed to toxaphene treat- ment. Extensive fish removal can evidently be accomplished without seriously affecting the plankton, but large reductions in these orga- nisms occur at 100 p.p.b. (Wollitz, 1958; and Hoffman and Olive, 1961). However, they re- appear while the water is still toxic to fish (Tanner and Hayes, 1955), and begin repopu- lating before detoxification will permit fish survival, No marked reductions were observed among most of the larger invertegrates. Hooper and Fukano (1960) reported bottom fauna to be nearly as abundant in two Michigan lakes after treat- ment (10 p.p.b.) as before, but Stringer and McMynn (1958) found that Amphipoda was eliminated at 10 p.p.b. and Ephemeroptera at 30 p.p.b. Severe reductions in many of these organisms may be expected at higher concen- trations. Odonata, Ephemeroptera, Tendipedidae, and Chaoborus, were eliminated with 100 p.p.b. toxaphene (Hooper and Grzenda, 1957; and Cushing and Olive, 1957), Unionidae, Sphaeridae, Gastropoda, Oligochaeta, and Hirudinea appear to be more resistant (Hooper and Grzenda, 1957); and Stringer and McMynn, 1958), Field observations were supplemented by controlled experiments, since most organisms 15 tested were not reduced at concentrations used for fish removal, It should be recognized that lower tolerance levels probably exist under field conditions which involve longer exposure periods. SUMMARY Effects of different toxaphene concentrations on plankton and other aquatic invertebrates were studied under natural and controlled conditions, Five North Dakota lakes were in- cluded in the study, which extended from June through September of 1960 and 1961. Physical and chemical data are presented for each lake. Polyarthra, Keratella, Asplanchna, Cono- chiloides, Brachionus, Trichocera, Daphnia, Bosmina, Ceriodaphnia, and Cyclops were the dominant zooplankters. No marked reductions were observed after treatment with 5 to 35 p.p.b., but a marked reduction of many plankters followed the second treatment (90 p.p.b.) in Raleigh Reservoir. The Cyanophyta were the most abundant phytoplankters in all lakes. Aphanizomenon increased in all lakes after treatment, but other phytoplankters exhibited no consistent changes. Chlorophyta, Chryso- phyta, and Pyrrophyta contributed little to phytoplankton abundance. The most abundant plant-inhabiting orga- nisms and bottom fauna exhibited no marked changes after treatment. Gammarus, Physa, Gyraulus remained almost constant, while Callibaetis, Caenis, Ischnura, and Tendipes decreased slightly but were again numerous 1 year after treatment. Tests on several species of zooplankters showed Rotifera to be the most tolerant, fol- lowed by Cladocera and Copepoda. Reductions were in Rotifera at 500 p.p.b., in Cladocera at 250 p.p.b., and in Copepoda at 100 p.p.b. Ex- periments with the larger invertegrates showed Hirudinea, Hydracarina, and Gastropoda to be the most resistant to toxaphene, followed in order by Trichoptera, Odonata, Hemiptera, Ephemeroptera, Amphipoda, and Coleoptera. Survival among the larger invertegrates at intermediate concentrations between 100-per- cent survival and 100-percent mortality re- 16 vealed an approximate straight-line relation. Genera within each group exhibited dissimilar tolerance levels. REFERENCES Cushing, Colbert E,, Jr., and John R, Olive, 1957, Effects of toxaphene and rotenone upon the macroscopic bottom fauna of two northern Colorado reservoirs, Transactions of the American Fish- eries Society, vol, 86 (1956), p, 294-301, Hoffman, Dale A,, and John R, Olive, 1961, The effects of rotenone and toxaphene upon plankton of two Colorado reservoirs, Limnology and Oceanography, vol, 6, No, 2, p, 219-222, Hooper, Frank F,, and Kiyashi G, Fukano, 1960, Summary of experimental lake treatments with toxaphene 1954-58, Institute for Fisheries Research Michigan Department of Conservation, 17 p, (Manu- script), Hooper, Frank F,, and Alfred R, Grzenda, 1957, The use of toxaphene as a fish poison, Trans- actions of the American Fisheries Society, vol, 85 (1955), p. 180-190, Pennak, Robert W, 1949, Annual limnological cycles in some Colorado reservoir lakes, Ecological Monographs, 19:233=267, 1953, Fresh-water invertebrates of the United States, New York, Ronald Press Co, 769 pp. Prevost, G, 1960, Use of fish toxicants in the Province of Quebec, Canadian Fish Culturist, vol, 28, p, 13-35, Rawson, D, S, 1956, The net plankton of Great Slave Lake, Journal of the Fisheries Research Board of Canada, vol, 13, p. 56-127, Stringer, George E., and Robert G, McMynn, 1958, Experiments with toxaphene as a fish poison, Canadian Fish Culturist, vol, 23, p, 39-47, Tanner, H, A,, and M, L, Hayes, 1955, Evaluation of toxaphene as a fish poison, Colo- rado Cooperative Fishery Research Unit, Quarterly Report 1, p, 31-39, Ward, Henry B., and G, C, Whipple, 1959, Fresh-water biology, John Wiley & Sons, 1248 p, © Wollitz, R, E, 1958, The effects of certain commercial toxicants on the limnology of three cold water ponds near Three Forks, Montana, M,S, thesis, Montana State College, 63 p. INVESTIGATIONS IN FISH CONTROL 5. Growth Rates of Yellow Perch in Two North Dakota Lakes After Population Reduction with Toxaphene By Donald C, Warnick, Fishery Biologist U.S. DEPARTMENT OF THE INTERIOR Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife Resource Publication 9 Washington . January 1966 ADStract, << © << The study lakes . Age and growth . Discussion .... Summary and conclusions References .... CONTENTS Page GROWTH RATES OF YELLOW PERCH IN TWO NORTH DAKOTA LAKES AFTER POPULATION REDUCTION WITH TOXAPHENE By Donald C, Warnick, Fishery Biologist Abstract,--Growth rates of yellow perch that survived a toxaphene treat- ment in Brush and Long Lakes in North Dakota were calculated by the scale method for the 1960 and 1961 growing seasons, Brush Lake fish ex- hibited greatly increased growth rates for both growing seasons following the treatment, Increased growth rates were not evident for Long Lake fish until the 1961 growing season, At the end of the first full growing season after treatment the surviving yellow perch exceeded what may be consid- ered to be the minimum harvestable size of 7 inches, The approximate concentration of toxaphene for reducing the density of fish populations is believed to be 25 percent of the rate determined for fish eradication in most North Dakota waters, Waters overpopulated with desirable species generally produce few harvestable fish, be- cause of slow growth. Bennett (1962) stated that no fish of harvestable size were found in some waters thus affected; Eschmeyer (1936) made a similar observation concerning over- crowded populations of yellow perch (Perca flavescens), For lack of more efficient re- medial measures the use of piscicides has been recommended to reduce the numbers of the problem species, Relatively low concentrations of toxaphene (chlorinated camphene) in two North Dakota lakes substantially reduced the density of the yellow perch populations; the effect on other fish species was less obvious, The results re- ported (Henegar, 1965) were incidental to the determination of the minimum toxaphene con- centration necessary for fish control in that State, My study was started in 1960 to deter- mine the growth rates of the yellow perch sur- This publication is based on a thesis submitted to the Graduate Faculty, Department of Entomology-Zoology, South Dakota State College of Agriculture and Mechanic Arts, in partial fulfillment of the requirements for the degree of Master of Science, June 1963, viving in Brush and Long Lakes, and thus gain information concerning the suitability of toxa- phene for reducing the numbers of fish in overpopulated waters, The scale method was employed to calculate growth rates of Brush Lake fish for the 1960 and 1961 growing sea- sons, Posttreatment growth rates of Long Lake fish were determined for part of the 1960 growing season and for all of the 1961 season, Several authors reporting on the use of rotenone to thin overcrowded populations or to restore balance among fish species consid- ered the results favorable. Beckman (1941) noted that the growth rates of fish surviving the treatment of half of Booth Lake, Mich,, were too great to be accounted for by normal vari- ation, Substantially increased harvests, appar- ently the results of accelerated growth rates of remaining fishes, were reported by Swingle, Prather, and Lawrence (1953) following treat- ment of some Alabama ponds, Hooper and Crance (1960) stated that the use of rotenone was an effective and economical way to restore balance in certain fish populations, The use of toxaphene was recommended by several authors including Hemphill (1954) who first used the chemical for fish eradication, Cost of fish eradication with toxaphene is approximately 15 percent of the cost with rotenone, With recommended concentrations and methods for thinning overcrowded fish populations with these chemicals, toxaphene is even more economical, Definite information about this use of the poison and the subsequent results is conspicuously absent, Unfavorable results from the early use of toxaphene for fish eradication were not uncom- mon and tended to delay the acceptance of the piscicide for use in fishery management (Pre- vost, 1960), Consequences of a serious nature were the failure of the poison to kill all fish; the extended toxicity of some treated waters; and the reduction or elimination of many aquatic organisms, Hooper and Grzenda (1955) first suggested that such results were due to confusion concerning lethal concentrations and the belief was substantiated by the accumula- tion of additional evidence (Stringer and McMynn, 1960), Increased proficiency in using the chemical for fish eradication led to its acceptance for that purpose as indicated by Gebhards' 1960 review of past and proposed use in western states, Toxaphene concentrations used for fish erad- ication reportedly reduce or eliminate many fish-food and food-chain species, some of which do not reappear in quantity for extended periods (Stringer and McMynn, 1958), Relatively little is known concerning the effects of the lesser toxaphene application rates recommended for reducing the density of overcrowded fish popu- lations, A paucity of fish-food organisms in the North Dakota lakes--even for a compara- tively short period after toxaphene applica- tion--would affect growth rates of the surviving fish as indicated by scale analysis, _ Iam obliged to Dale L, Henegar, Chief of Fisheries, North Dakota Game and Fish De- partment, who brought to my attention the opportunity for this investigation, and to the fishery personnel who assisted with the field work, I wish to thank Marvin O, Allum, Asso- ciate Professor of Zoology, for his counsel during the study, and the other faculty mem- bers and fellow graduate students of South Dakota State College for their interest and assistance, 4 THE STUDY LAKES The minimum concentration of toxaphene required for fish eradication is determined on the basis of the physical and chemical charac- teristics of the water for which treatment is proposed in addition to certain biological con- ditions, and this is assumed to be true with regard to application rates for reducing the density of fish in overpopulated waters, Some physical and chemical characteristics of Brush Lake and Long Lake are presented in table 1, The following history is pertinent to the study of the posttreatment growth rates and is based on material presented by Henegar in 1961 (Henegar, 1966), Both lakes were treated with toxaphene to produce concentrations of approximately 0,010 parts per million (p.p.m,) using an emulsifiable concentrate containing 6 pounds of the active ingredient per gallon, Brush Lake was treated on October 5, 1959, and Long Lake on July 17, 1960, The method of application was that com- monly used by the North Dakota Game and Fish Department, and similar to that described by Stringer and McMynn (1958), Dilution of the waters by rainfall or by run- off was inconsequential after toxaphene treat- ment, because of unusual drought, Water levels receded somewhat during the course of the study, Rooted aquatic vegetation was along por- tions of the shoreline and in several small shallow areas of Long Lake at the time of treatment, but was nearly absent from Brush Lake because of the late-season treatment date. Apparently, all young-of-the-year yellow perch were eliminated from both lakes, TABLE 1.--Physical and chemical characteristics of the lakes Item Brush Lake Long Lake McLean County Bottineau County Central Location (North Dakota).....ceeceeees General area of State....eeseceececee Origin of lakes... scsceeeeee isfelainis) inte Bottom type. .ceeccesececscrcecscceces GUrface ACTES. occecccccnccccnccsasces 160 291 North Central Glacial ACTE LOCC. cee weeeenseuscnsercessccees Maximum depth (feet)....seeeeeeseeeee Average depth (feet).....seeeeeeeeeee 9.5 8.2 2 Phenolphthalein alkalinity (p.p.m.)?. 40 40 Methyl orange alkalinity (p.p.m. )?... 460 220 Hardness (p.p.m. )* Total dissolved solids (p.p.m.)*..... 290 307 + Condition on date of application. Observations established deaths of some young- of-the-year northern pike (Esox lucius) in Long Lake, but posttreatment netting disclosed they were not eliminated, The effect of the poison on adult fish of several species in both lakes was less evident than the effect on yellow perch, the dominant species, Excluding the young-of-the-year, yellow perch density was reduced approximately 91 percent in Brush Lake and 79 percent in Long Lake, The figures are derived from the results of test-netting just before and several months after poisoning, Netting results also indicated that a greater percentage of the smaller yellow perch (less than 140 millimeters) was elimi- nated than of the larger fish, Observation of Long Lake for several days after toxaphene application tended to substantiate the netting results, Populations of fathead minnows (Pimephales promelas) were established in the lakes after treatment, Brush Lake was stocked on May 27, 1960, and Long Lake on August 18, 1960, Intro- duction of minnows after toxaphene treatment is a general practice of the North Dakota Game and Fish Department, AGE AND GROWTH Varied evidence has been presented in sup- port of the validity of the scale method for the determination of the age and growth of fishes (Lee, 1920; Van Oosten, 1929), Similar evidence indicates that the method is valid for determin- ing the age and growth rates of yellow perch, Joeris (1957) indicated that additional evidence on the validity of the annulus world accumulate from the further study of Green Bay (Lake Michigan) perch, Jobes, as early as 1934, as- sumed the validity of the method for yellow perch, The North Dakota study was based on the assumption that the method is valid, Scale samples of yellow perch from the study lakes were obtained from specimens netted be- fore and after the poisoning, from poisoned fish, and from winterkilled specimens, It was apparent during analysis of the scale samples from Long Lake, July 12-17, 1960, that distinction of age classes would be difficult, Determination of the age composition for this group was dependent on the identification of all annuli for each scale sample, For many samples it could not be established whether the 1960 annulus had been formed, The samples from smaller fish (80-120 millimeters) generally evidenced annulus formation and some subse- quent growth, but the 1960 annulus was appar- ently unformed on some of the scales of larger fish, Because of relatively little scale growth the previous season, it could not be determined whether the annulus was recently formed and the later scale growth was of the 1960 growing season, or the annulus was unformed and the scale growth of the previous year was repre- sented, An error of 1 year would be introduced by the wrong choice, A similar difficulty was noted by Joeris (1957) during analysis of yellow perch scales, Beckman (1943) reported that the time of annu- lus formation may vary notably among species and within age groups of the same species, Annulus formation probably would have oc-~ curred before the July collection date with more favorable growth conditions, Even without this difficulty, determination of age classes would have been somewhat subjec- tive. Annuli were not distinct, and markings assumed to be false annuli were common, Con- sequently the age classes and specific growth rates of fish before treatment are not included, Posttreatment scale samples from both lakes were obtained after the interruption of growth for the 1961 season and before 1962 growth was begun, An annulus was assumed at the scale margin although none was evident, All discernible increase in scale growth of the Long Lake fish was included between the mar- gin and the annulus of the previous year, The scale growth of Long Lake fish during the 1960 season after poisoning was not distinguishable from previous scale growth, Growth incre- ments for the 1961 growing season are pre- sented in table 2, Errors other than mechanical are unlikely because of the distinctive scale growth and the absence of false annuli during that period, TABLE 2.--Calculated growth increments of yellow perch from Long Lake for the 1961 growing season {In millimeters] Total length at capture | Calculated growth increment Number of 3 tel [ Range Average Range Average 103-132 115 -- -- -- 178 -- 82 201-210 205 58-101 75 -- 219 -- 59 221-230 227 59-120 74 231-240 235 45-116 75 241-250 247 56-106 79 251-260 253 75-116 83 261-270 265 54-99 78 1 young-of-the-year in 1961. Scale samples were not obtained from Brush Lake fish until 2 years after treatment, Accel- erated scale growth was obvious between the scale margin and annuli of the 2 previous years, The calculated growth rates for the corresponding periods are contained in table 3, As in the scales of Long Lake fish, growth before poisoning was obscured by the presence of numerous false annuli, When an annulus of the year previous to those located for the preparation of tables 2 and 3 was obvious, as it was on some Scales, a direct comparison of scale growth before and after poisoning was made, On this basis the post- treatment growth during the first year was ap- proximately six times greater than for the previous year, Table 4 shows the relation of fish length to subsequent growth--both calculated, The fact that greater length increments were recorded for smaller fish lends validity to the scale method as applied here, A change in the size composition of Long Lake fish is evident in table 5, The numbers of fish in the last column represent a subsample of winterkilled specimens in addition to sev- _ eral obtained by qualitative test-netting, The numbers of fish in the other columns represent test-netting results at the times indicated, Ex- cluding the 17 young-of-the-year of 1961, the lengths of fish listed in the last column are approximately 75 to 100 millimeters greater than the lengths of fish listed in the previous column, Despite the time interval, only one growing season, 1961, is represented, The cal- culated average growth increment for the pe- riod was 82 millimeters, 6 TABLE 3.--Calculated growth increments of yellow perch from Brush Lake for the 1960 and 1961 growing seasons if In millimeters] Total length fish at capture Average 1960 increment Average 1961 increment Gusictdaleds ae biawisethisie’s 175-200 Cotstera(stetsietstepte cieiele aie 201-225 226-250 251-275 276-300 1 Young-of-the-year in 1960. TABLE 4.--Relation of fish lengths to subsequent growth increments (both calculated) for yellow perch from Long Lake [In millimeters] — Number of Calculated length, Growth increment 1961 fish ‘ip May 1961 Range 103-132 103-132 115 91-100 82-121 101 101-110 101-119 110 111-120 a = 121-130 ws 55 131-140 64-116 92 15 141-150 61-104 85 27 151-160 58-96 76 PERS coat tea eE nee 161-170 59-101 76 io eee soe. eee cae 171-180 67-89 75 B.. 181-190 45-82 67 1 191-200 = 66 201-210 = 54 + Young-of-the-year in 1961; lengths are measured total lengths. TABLE 5.--Toxaphene-effected change in the yellow perch population of Long Lake based on measured total lengths of fish taken during the study Number of fish length range Betorct trenton After treatment (July 1960) October 1960 1,010 66-100 MM. ccccencccevcccccccs 527 101-125 mm 312 126-150 mm 198 151-175 mm 196 176-200 mm 7 226-250 mm... 251-275 mm ie) ie) 17 ie) ie) af 12 75 19 124 x Young-of-the-year in 1961. DISCUSSION The yellow perch is a popular species in recreational fisheries, especially for winter fishing, but fish shorter than a total length of 7 inches or approximately 175 millimeters are not often sought and removed by fishermen, If — this length is considered the minimum harvest- able size, the growth rates recorded for the yellow perch from the North Dakota lakes are significant with respect to the short time re- quired for the improvement of recreational fisheries, All yellow perch surviving similar | treatment rates could be expected to exceed the minimum desirable size during the subsequent ~ growing season, and young-of-the-year after only two growing seasons, Young-of-the-year yellow perch commonly reach harvestable size in three or more grow- ing seasons except in overpopulated waters where growth is restricted, Growth rates of surviving fish were exceptionally rapid during the growing season following treatment--1960 for Brush Lake and 1961 for Long Lake--when compared with growth rates of yellow perch in other areas, Similar growth increments have not been recorded even for more southern lati- tudes with longer growing seasons (Carlander, 1953), On the basis of this study, better recrea- tional fishing cah be provided at low cost in some lakes and small impoundments overpopu- lated with yellow perch, Improved angling was assumed in the North Dakota lakes since more fish of harvestable size were produced, but the determination of increased harvests would be conclusive, Comparable results might also be expected following the thinning of other com- monly overpopulated species inasmuch as my study was opportunistic, not a deliberate selec- tion of species, Low toxaphene concentrations in North Dakota lakes have been observed to eliminate small fish of many species including the bull- head (Ictalurus melas), reported by Kallman, Cope, and Navarre (1962) to be somewhat re- sistant to the toxicant, The policy in North Dakota of introducing minnows in waters after treatment with toxaphene is based on this ob- servation, An assumed absence of prey fish may ex- plain the continued slow growth of yellow perch in Long Lake during the 1960 growing season, after the mid-July application of toxaphene, The May 27, 1960, introduction of 180,000 fathead minnows in Brush Lake evidently as- sured the presence of significant numbers for the same growing season and no unusual period of slow growth was apparent from scale analy- sis, The reduction or elimination of prey fish by the mid-July poisoning and the August 18, 1960, stocking of 200,000 fathead minnows, leaves doubt whether significant numbers were present in Long Lake until the 1961 growing season when growth rates of yellow perch were greatly accelerated, A need for more conclu- Sive information concerning the relation of prey Species to growth rates is indicated, Assuming need for the introduction of prey species, a definite advantage is apparent for the fall treatment of waters since stocking can be accomplished early in the subsequent grow- ing season, Treatment in April or May is prob- ably more advantageous than during the growing season, but conditions then are not favorable for rapid detoxification, and stocking of prey species might have to be delayed, The toxaphene treatment rate which allowed the survival of yellow perch in the North Dakota lakes was approximately one-third of the de- termined rate for fish eradication in most waters of that State, A belief that the reductions were excessive can be temporized since the possibility that an optimum number of fish survived in either lake is unlikely, and greater growth rates could hardly be expected, Test- netting of both lakes and observation after a partial winterkill of Long Lake in 1962 sub- stantiates the belief, Concentrations approxi- mating 0,008 p.p.m, (one-fourth of the minimum lethal rate) probably would have allowed the survival of more fish without significantly re- ducing growth rate, On the basis of a recent report by Kallman, Cope, and Navarre (1962), the presence of relatively large quantities of vegetation at the time of treatment could affect the outcome, especially in consideration of the low toxaphene concentrations required for the thinning of fish populations, It was indicated that high concen- trations of toxaphene are accumulated by cer- tain vegetative species in a relatively short time, thus essentially removing the chemical from the water--at least temporarily--and the further disposition of the chemical was un- known, Therefore, more consistent results might be obtained, with regard to the degree of reduction, by treatment during the absence of most aquatic vegetation, The appropriate reduction for any population necessarily depends on a variety of conditions, some unknown, The difficulty of determining the magnitude of fish populations, particularly after treatment, seriously affects an evalua- tion of the results, Additional information con- cerning the use of toxaphene for reducing the density of fish populations is needed for its greatest usefulness, SUMMARY AND CONCLUSIONS Yellow perch populations in two North Dakota lakes were substantially reduced by low toxa- phene concentrations, Growth rates of surviv- ing fish were determined by the scale method for 2 years after treatment, Greatly increased growth rates were evident for both growing seasons following the fall treatment of Brush Lake, Increased growth rates were not evident for Long Lake fish after the July 17, 1960, treatment until the 1961 growing season, All yellow perch surviving the poisoning exceeded what may be considered to be the minimum harvestable size during the first full growing season after treatment, Comparable results can be expected from similar and perhaps lesser reductions with toxaphene, Further use of toxaphene is recommended for reducing the density of yellow perch popu- lations and thus improving certain recreational fisheries, Other species might be similarly | managed, Reduction or elimination of prey species was believed to explain the continued Slow growth of Long Lake fish for approxi- mately 2 months after toxaphene application, Fall treatment is apparently the most timely, especially with regard to assuring the presence of significant numbers of prey species during the growing season, Numerous conditions affect the concentration of toxaphene needed for fish eradication, and the approximate concentration for reducing the density of fish populations is believed to be 25 percent of that rate, REFERENCES Beckman, William C, 1941, Increased growth rate of rock bass (Amblopites rupestris Rafinesque), following reduction in the density of the population, Transactions of the Amer- ican Fisheries Society, vol, 70, p, 143-148, 1943, Annulus formation on the scales of certain game fishes, Papers, Michigan Academy of Science, Arts, and Letters, vol, 28, p, 281-312, Bennett, George W, 1962, Management of artificial lakes and ponds, Reinhold Publishing Co,, New York, 283 p, Carlander, Kenneth D, 1953, Handbook of freshwater fishery biology with the first supplement, Wm, C, Brown Co,, Dubuque, Iowa, 281 p, Eschmeyer, R, William, 1936, Some characteristics of a population of stunted perch, Papers, Michigan Academy of Science, Arts, and Letters, vol, 22, p, 613-628, Gebhards, Stacy V, 1960, A review of toxaphene for use in fish eradica- tion, Idaho Department of Fish and Game, 14 p, Henegar, Dale L, 1966, Minimum lethal levels of toxaphene as a pisci- cide in North Dakota lakes, U,S, Bureau of Sport Fisheries and Wildlife, Investigations in Fish Con- trol No, 3, Hemphill, Jack E, 1954, Toxaphene as a fish toxin, Progressive Fish- Culturist, vol, 16, No, 1, p, 41-42, Hooper, Archie D,, and Johnie H, Crance, 1960, Use of rotenone in restoring balance to over= crowded fish populations in Alabama lakes, Trans- actions of the American Fisheries Society, vol, 89, No, 4, p, 351-357, Hooper, Frank F,, and Alfred R, Grzenda, 1957, The use of toxaphene as a fish poison, Trans- actions of the American Fisheries Society, vol, 85 (1955), p. 180-190, Jobes, Frank W. 1932, Preliminary report on the age and growth of the yellow perch from Lake Erie, as determined from a study of its scales, Papers, Michigan Academy of Science, Arts, and Letters, vol, 17, p, 643-652, Joeris, Leonard S, 1957, Structure and growth of scales of yellow perch of Green Bay, Transactions of the American Fish- eries Society, vol, 86, p. 169-194, Kallman, Burton J,, Oliver B, Cope, and Richard J, Navarre, : 1962, Distribution and detoxification of toxaphene in Clayton Lake, New Mexico, Transactions of the American Fisheries Society, vol, 91, No, 1, p. 14-22, Lee, Rosa M, (Mrs, T, L. Williams), 1920, A review of the methods of age and growth de- termination in fishes by means of scales, Ministry Agriculture and Fisheries, Fishery Investigations, ser, 2, 1-32, (Fredericton, New Brunswick), Prevost, G, 1960, Use of toxicants in the Province of Quebec, Canadian Fish Culturist, vol, 28, p, 13-35, ger, George E,, and Robert G, McMynn, 1958, Experiments with toxaphene as a fish poison, Canadian Fish Culturist, vol, 23, p, 1960, Three years’ use of toxaphene as a fishtoxicant in British Columbia, Canadian Fish Culturist, vol, 28, p. 37-44, Swingle, H, S,, E, E, Prather, and J, M, Lawrence, 1953, Partial poisoning of overcrowded fish popula- tions, Alabama Polytechnic Institute, Auburn, Ala, Van Oosten, John, 1929, Life history of the lake herring (Leucichythys artedi LeSeuer) of Lake Huron as revealed by its scales, with a critique on the scale analysis, U.S, Bureau of Fisheries, Document 1053, 250 p, nye 2 ¢ sd . 4 v jo ike Hel Pay ty ye Lage Deve Tt A Aa ie ive 7 bP ive ar oy “2 y Lshipiet HO ye: ED MAD: i rR) . aie 2 re «h Ath é i x7 ‘ , i Vike , \ \, aw ee inky * an es is i, hs ea s i uf Ls 4 it ips aah eae aN gia , Xe ihe ' ie <6 18 1 =) ; ; ‘ ts cet as re voor nae yuierwed, a Wi eget if ie, Pir ; ne \ jah aacaloet on Fg F a. uchiprane Ha 5 mh te , Phage lis ae eft cpaplai.. Ph fs ae he os ve ‘7 n on epee Te ab ei ; 1 ee A obs Gediny ‘ ~~ e- 7 , ts Aongh Meet. ee om de ort Over oem, lel Ge Lowe aed MI, Glo hie ; ror +) ail Santas Me A ty ws y °2 ora in Coe owed “oo Oi hardest Al y ite ©) in dao See 7 ere Fes, PA sg ONG. a, he ih praia A That ik St: Cea ale Sa ne ian ot Saloon Tari: OMA esr en 4M \, Nak i +h a ' t kta heyeliktey api ley Ga aie a vith Stee (ol Laie BU, ae i. nie i 2calgey Pogues oii « ' ~s > ¥ } tortie Th); bd uaa: ‘vt. ees %, Recta a? ged orailur 4 Olev- Dee, “hoa at ie ty Ka tity By i aN or Cee CT: lpia wr al jay thps, Sian kiny olor lien | ‘Syer pet roy ne payee hee say P hondd, et ing i i (nity ale ay Review ok Ate Re INVESTIGATIONS IN FISH CONTROL 6. Mortality of Some Species of Fish to Toxaphene at Three Temperatures By Mahmoud Ahmed Mahdi, Fishery Biologist U.S. DEPARTMENT OF THE INTERIOR Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife Resource Publication 10 Washington ., January 1966 CONTENTS ADSERACE arene 6) (6) (8: 10! <6) 6 a) ja oeoeeoeeeee ee @ Methods eeeneveeeeee eeeeeveesveeeeeeee Transter Or iishewercicrsielc ola isiel lak eiate Introduction of toxaphene specie snenenene ODSCEVALTONS accrcmemen cnenielcl ol steneienementente AMALY SUS iersje wu) enreneuel 017 ja’ see) e) ieite folfaticl folrelre RESULESHycactc srerclisulcdememalcincner + telonene olctteuleiemte Change with Elmers cys anes ot «