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A method of bioassay for the residual contact toxicity of insecticides Harris, Charles Ronald 1956

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A METHOD OP BIOASSAT FOR THE RESIDUAL CONTACT TOXICITY OP INSECTICIDES by CHARLES RONALD HARRIS A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1956 i i ABSTRACT A method of bioassay distinguishing r e s i d u a l contact from fumigant t o x i c i t y i s described. B a s i c a l l y the apparatus consists of a series of Buchner funnels set up i n se r i e s . Woven fi b e r g l a s s c l o t h was selected as the substratum. By substitution of a dye i n place of Insecticide, i t was possible, using colorlmetric analysis, to calculate the t o t a l milligrams of dye adhering to the c l o t h . I t was assumed that proportionate amounts of dye and i n s e c t i c i d e would be picked up. Musea domestica L.was used as the test insect. Two strains of f l i e s , designated as the SES and Ottawa cultures, were used. Fumigant ef f e c t was eliminated by application of negative pressure. Since fumigant e f f e c t i s proportionate to vapour pressure, the rate of evacuation varied f o r each i n s e c t i c i d e . Elimination of fumigant effect brought about a corresponding decrease i n mortality. Dosage-mortality data are given f o r s i x i n s e c t i c i d e s i n comparison to a "standard" i n s e c t i c i d e (dieldrin) u t i l i z i n g the concept of t o x i c i t y index ( Sun, 195>0). S t a t i s t i c a l analysis of the data indicate s i g n i f i c a n t heterogeneity i n eleven out of twenty-four experiments. An analysis of the Chi-square function i s presented. Dosage-mortality data f o r DDT are given. The SES culture was determined to be lip.x as r e s i s t a n t f o r females and 67x as r e s i s t a n t f o r males as the Ottawa culture, by i i i t o p i c a l a p p l i c a t i o n . Residual contact a p p l i c a t i o n indicated that females of the SES culture were 2$2x as r e s i s t a n t com-pared to the Ottawa culture. V a r i a t i o n within the SES culture i s discussed as a factor i n demonstrating the s e n s i t i v i t y of the technique. i v ACKNOWLEDGEMENTS A project of thi s type i s one i n which the complete cooperation of a l l involved i s necessary to bring i t to a sat i s f a c t o r y conclusion. Acknowledgement i s made to the following chemical companies f o r supplying samples of the te s t i n s e c t i c i d e s : American Cyanamid Company: parathion, malathion. C a l i f o r n i a Spray Chemical Corporation: lindane. The Dupont Corporation: ETN. The Geigy Corporation: DDT. Sh e l l Chemical Corporation: a l d r i n , d i e l d r i n , endrin. V e l s i c o l Corporation: heptachlor. I should also l i k e to acknowledge with thanks, the help of the Defense Research Board i n placing the f a c i l i t i e s of the S u f f i e l d Experimental Station at my disposal during the summers of 1954 and 1955, and f o r the loan of equipment during the 195i+.-55 and 1955-56 Winter Sessions at the University of B r i t i s h Columbia. Acknowledgement i s also made to the B r i t i s h Columbia Academy of Sciences f o r the Goethe Award f o r research during the Winter Session 1954-55 and to the National Research Council f o r the Studentship awarded during the Winter Session of 1955-56. I am indebted to Dr. K. Graham f o r h i s suggestions regarding the development of the experimental technique and f o r h i s corrections and c r i t i c i s m s of the manuscript. V Acknowledgement Is also made to Dr. J . Sanjean f o r h i s c r i t i c i s m s of the manuscript, and to others i n the Departments of Zoology and Chemistry f o r t h e i r advice from time to time. Acknowledgement i s made to Dr. S. Nash f o r guidance In the s t a t i s t i c a l analysis. I am deeply indebted to Dr. H. Hurtig, Head/ Entomology Section, S u f f i e l d Experimental Station, who f i r s t suggested the development of t h i s project, and without whose help and guldanoe, completion would have been impossible. Acknowledgement i s made to Mr. G. Humphreys, of the S u f f i e l d Experimental Station, f o r doing such an excellent job of rearing the large numbers of insects used i n te s t i n g , and to Mr. C. Watson, f o r dr a f t i n g the graphs contained i n the body of the thes i s . I would e s p e c i a l l y l i k e to thank Professor G.J. Spencer, to whom I owe a great deal f o r h i s advice and encouragement. v i LIST OP FIGURES Figure 1. Figure 2. Figure 3(A). (B). Figure 2j.. Figure 5. Figure 6(A). (B). Figure 7(A). (B). Figure 8(A). (B). Standard curve f o r A n i l i n e Green dye dissolved i n a benzene-mineral o i l (95:5) solvent mixture. Extraction of A n i l i n e Green dye from 50 mgm. fi b e r g l a s s c l o t h . Retention of A n i l i n e Green dye on f i b e r g l a s s c l o t h (0 - 30$ range). Retention of A n i l i n e Green dye on f i b e r g l a s s c l o t h (0 - 0.12$ range). T o x i c i t y of d i e l d r i n to Musca domestlca L. (SES c u l t u r e ) . T o x i c i t y of d i e l d r i n to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of parathion to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of d i e l d r i n to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of EPN to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of d i e l d r i n to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of endrin to Musca domestica L. (SES c u l t u r e ) . T o x i c i t y of d i e l d r i n to Musca domestica L. (SES cul t u r e ) . v l l Figure 9(A). T o x i c i t y of a l d r i n to Musca domestica L. (SES cul t u r e ) . (B). T o x i c i t y of d i e l d r i n to Musca domestlea L. (SES c u l t u r e ) . Figure 10(A). To x i c i t y of lindane to Musca domestica L. (SES cul t u r e ) . (B). T o x i c i t y of d i e l d r i n to Musca domestlea L. (SES c u l t u r e ) . Figure 11(A). T o x i c i t y of heptachlor to Musca domestica L. (SES culture). (B). T o x i c i t y of d i e l d r i n to Musca domestlea L. (SES c u l t u r e ) . Figure 12. T o x i c i t y of DDT to Musca domestica L. (SES c u l t u r e ) . Residual contact a p p l i c a t i o n . Figure 13(A). DDT content of benzene-mineral o i l (95:5) solution (0 - 0.06$ range). (B). DDT content of benzene-mineral o i l (95:5) solution (0 - 30$ range). Figure li | . . T o x i c i t y of DDT to Musca domestica L. (SES c u l t u r e ) . Topical a p p l i c a t i o n . Figure 15. T o x i c i t y of DDT to Musca domestica L. (Ottawa c u l t u r e ) . Topical a p p l i c a t i o n . Figure 16. T o x i c i t y of DDT to Musca domestica L. (Ottawa c u l t u r e ) . Topical a p p l i c a t i o n . Figure 17. T o x i c i t y of DDT to Musca domestica L. (Ottawa cu l t u r e ) . Residual contact a p p l i c a t i o n . Figure 18. T o x i c i t y of lindane and heptachlor to Musca domestica L. (Ottawa c u l t u r e ) . v i i i LIST OF PLATES Plate I.(a). Technique employed i n rearing Musca domestica L. (b). View of rearing room at S u f f i e l d Experimental Station. Plate I I . A series of f r e s h l y cut f i b e r g l a s s cloths treated with "Cenco" l a b e l varnish to prevent fraying. Plate I I I . (a). Method employed i n removing houseflies from cages. (b). Buchner funnels containing $0 f l i e s each, demonstrating fumigant and residual contact t e s t s . Plate IV. (a).A t y p i c a l 0 r u n n consisting of s i x dosage l e v e l s (three r e p l i c a t e s each) and a control series, (b).Observation chambers set up i n s e r i e s . Mortal-i t y counts were made at 2% and 1+8 hour i n t e r v a l s . Plate V. (a). Components of equipment used i n t e s t i n g fumigant e f f e c t , (b). Assembled equipment used i n t e s t i n g fumigant e f f e c t . Plate VI.(a). Technique used f o r t o p i c a l a p p l i c a t i o n of DDT. (b). F l i e s contained In observation chambers a f t e r t o p i c a l a p p l i c a t i o n of DDT. TABLE OF CONTENTS Page Biography i Abstract i i Acknowledgements i v L i s t of Figures v i L i s t of Plates v i i i I. INTRODUCTION 1 I I . HISTORICAL BACKGROUND 1+ I I I . MATERIALS . 12 Test insect 12 Insecticides 12 IV. METHODS 17 Rearing technique 17 Bioassay technique 20 V. EXPERIMENTAL DATA 36 Elimination of fumigant e f f e c t 36 Dosage-mortality data . . lj.1 VI. DISCUSSION OF RESULTS . . . 63 Rearing methods 63 S t r a i n of insects £>l+ Bioassay technique 66 S u s c e p t i b i l i t y of males and females 70 P a r a l l e l i s m of probit-regression l i n e s . . . . 71 Resistance of SES culture to DDT 72 Homogeneity of data 7k S e n s i t i v i t y of technique 78 VII. CONCLUSIONS 80 VIII. BIBLIOGRAPHY 8 l IX. APPENDIX 86 1 I. INTRODUCTION The f i e l d of i n s e c t i c i d e chemistry has expanded considerably during the past f i f t e e n years. Previous to World War I I , the Insecticides c h i e f l y i n use were inorganic compounds such as the arsenicals and lime-sulphur, and organic compounds such as nicoti n e , pyrethrum, anabasine, and rotenone, which are of botanical o r i g i n , as well as synthetics such as phenothiazine and p-dichlorobenzene. In 1939, the Geigy Corporation synthesized DDT (dlchlorodiphenyltrlchloroethane), and t h i s compound was only the f i r s t of a series of chlorinated hydrocarbon Insecticides. In 19^6, the f i r s t organic phosphate i n s e c t i c i d e s , developed during the war by Schrader, were released upon the market. Examples of these compounds are TEPP (tetraethylpyrophosphate), parathion (dlethyl-p-nitrophenol thiophosphate), and sehradan (octamethylpyrophosphoramide). Since t h i s time, many other organic compounds have been found to exhibit i n s e c t i c i d a l a c t i v i t y . The tremendous increase i n the number of i n s e c t i c i d e s necessitated the development of more adequate methods of evaluating I n s e c t i c i d a l a c t i v i t y . The evaluation of a compound, eithe r chemically or b i o l o g i c a l l y , requires f i r s t l y , a q u a l i t a t i v e estimate. Qualitative chemical analysis determines either the ions or functional groups present i n the compound. Qualitative b i o l o g i c a l analysis or "screening" determines the r e l a t i v e t o x i c i t y or non-toxicity of a compound to insects. Complete 2 evaluation of a compound necessitates also a quantitative estimate. Quantitative chemical analysis consists of a determination of the proportions of the constituents present, while quantitative b i o l o g i c a l analysis or "bioassay" consists of the determination of the proportional t o x i c i t y of a compound to cert a i n insects, i n comparison with a "standard" i n s e c t i c i d e . A bioassay technique depends to a large extent upon the type of i n s e c t i c i d e to be tested. Insecticides may be c l a s s i f i e d i n t o three main categories according to t h e i r form and route of entry: fumigant poisons, stomach poisons, and contact poisons. Contact poisons may be further subdivided into d i r e c t contact poisons and r e s i d u a l contact poisons. I f we are to be successful i n testing an i n s e c t i c i d e q u a n t i t a t i v e l y , we must develop a method of bioassay f o r each type of poison. Furthermore, i t has been shown that i n s e c t i c i d e s are somewhat s p e c i f i c i n respect to t h e i r t o x i c i t y to a given insect species. For example, DDT i s very toxic to houseflies, but much les s so to cockroaches. Hence i t i s necessary that several insects be used before a compound can be considered as f u l l y tested. There are already numerous methods of bioassay f o r each type of poison, but the m u l t i p l i c i t y of methods renders i t impossible to compare data obtained by one investigator with those obtained by others. The consequent need f o r standardization i s therefore being recognized by many Investigators. 3 I t has been the object of th i s research project to develop a method of bioassay f o r the re s i d u a l contact t o x i c i t y of i n s e c t i c i d e s , with the view that i t w i l l be only the f i r s t step toward the standardization of a testing technique. 4 I I . HISTORICAL BACKGROUND The development of bioassay techniques f o r fumigant poisons have proven to be much les s involved than techniques f o r stomach and contact poisons. In most cases, the apparatus used involves a closed chamber into which the v o l a t i l i z e d i n s e c t i c i d e Is passed (Cotton, 1943). The best known bioassay technique f o r comparing the t o x i c i t y of stomach poisons i s the poisoned l e a f "sandwich" technique (Campbell and Filmer, 1929). Numerous modifications of t h i s technique have been devised (Hansberry, 1943). A related technique involves feeding to insects measured drops of l i q u i d or food containing known amounts of i n s e c t i c i d e (Pearson and Richardson, 1933; Sun, 1953). S t i l l another technique involves i n j e c t i o n s Into the alimentary canal (Hansberry et a l , 1940)• The development of sa t i s f a c t o r y bioassay techniques f o r d i r e c t contact t o x i c i t y has presented many problems. However, considerable work has been done i n t h i s f i e l d , and as a r e s u l t , numerous methods of bioassay are now ava i l a b l e . One of the f i r s t techniques developed was the Peet-Grady spray chamber method (Peet and Grady, 1928). During the suc-ceeding years, t h i s method has been modified, but i s s t i l l i n use i n many laboratories concerned with the evaluation of household spray i n s e c t i c i d e s . Using t h i s technique, i t i s possible to evaluate the t o x i c i t i e s of in s e c t i c i d e s by reference to a "standard" i n s e c t i c i d e compounded by the 5 Chemical S p e c i a l t i e s Manufacturer's Association (GSMA). The present standard consists of a mixture of pyrethrins I and I I , which has been c a r e f u l l y standardized by both chemical and b i o l o g i c a l analysis (Blue Book, 1953). I t i s necessary that the test Insects be reared under conditions set up by the CSMA. A l l tests must be ca r r i e d out using the standard Peet-Grady spray chamber. Numerous other spray chamber methods have since been developed (Campbell and Su l l i v a n , 1938J Potter, 19ip.). A second type of bioassay technique f o r estimating d i r e c t contact t o x i c i t y i s that of i n j e c t i o n . Campbell (1932) injected i n s e c t i c i d e solutions d i r e c t l y into the blood stream of the insect using as an i n j e c t i o n pipette, a c a p i l l a r y tube drawn to a very f i n e point. Since t h i s time there have been numerous modifications of i n j e c t i o n equipment, involving the use of hypodermic needles and micrometer heads graduated i n very accurate volumes (Yeager and Munson, 19l|.5; Heal and Menusan, 19l|.8) . At present many screening techniques involve i n j e c t i o n of the i n s e c t i c i d e into the insect as a primary test, since complicating factors such as s o l u b i l i t y , permeability, and other physico-chemical factors may be avoided. Other types of bioassay f o r d i r e c t contact t o x i c i t y are t o p i c a l a p p l i c a t i o n of the i n s e c t i c i d e (0»Kane et a l , 1933; March and Metcalf, 191*9')» and immersion of the insect into a solution or emulsion of the i n s e c t i c i d e (Mcintosh, 19ii-7, 1949). 6 Some methods of bioassay f o r the determination of resid u a l contact t o x i c i t y have also been developed (Stringer, 1949J Krijgsman, 1949; Proverbs and Morrison, 1947; Barnes, 1945; Hoskins et a l , 1952). However, most investigators have made no attempt to d i f f e r e n t i a t e between r e s i d u a l contact t o x i -c i t y and combined residual contact-fumigant t o x i c i t y . I n v e s t i -gators attempting to eliminate the fumigant e f f e c t have met with l i t t l e success. Pradhan (1949) attempted to eliminate the toxic vapours by evacuating fumes upward through a funnel placed over the impregnated substratum. He found that fumigant e f f e c t could not be eliminated i n t h i s way. One of the main factors i n the development of a technique of t h i s type i s the method of app l i c a t i o n of the in s e c t i c i d e to the substratum. One of the most common methods of a p p l i c a t i o n i s to treat the substratum i n a spray tower, such as f o r example, a Potter tower (Potter, 1941) • I E n e sub-stratum i s covered with a uniform, known concentration of spray, removed, and placed within the t e s t i n g apparatus. A second method of application of i n s e c t i c i d e consists of dis-* solving the i n s e c t i c i d e i n a v o l a t i l e solvent, placing I t within a container, and swirling u n t i l the solvent evaporates (Hamraan, 1949; Krijgsman, 1949; Hoskins et a l , 1952). A t h i r d method involves d i s s o l v i n g the i n s e c t i c i d e i n a v o l a t i l e s o l -vent, and either dipping the substratum Into the solution (Proverbs and Morrison, 1947), or pip e t t i n g the solution d i r e c t l y on to the substratum (Stringer, 1949). 7 Certain other factors must also he taken into consideration before an accurate method of bioassay can be developed. For example, some investigators have demonstrated a d e f i n i t e r e l a t i o n s h i p between temperature and humidity, and the rate of knockdown (Lindquist et a l , 1914-5; 19l|6; Dakshinamurity, 19lj-8; Pradhan, 1949; Teotia et a l , 1950). Few d e f i n i t e data have been obtained, and at present there i s considerable c o n f l i c t i n g evidence. In general, the temperature r e l a t i o n s h i p seems to be much more l i m i t i n g than the humidity r e l a t i o n s h i p . The temperature and humidity ranges selected depend e n t i r e l y upon the conditions accept-able to the test insects, but i t i s absolutely necessary that these conditions be maintained at a l l times throughout te s t i n g . Wot only the temperature-humidity r e l a t i o n s h i p , but also the physico-chemical c h a r a c t e r i s t i c s of an i n s e c t -i c i d e a f f e c t t o x i c i t y . There i s a d e f i n i t e r e l a t i o n s h i p between t o x i c i t y and p a r t i c l e size and shape (McGovran et a l , 191+0; Mcintosh, 19l|-7, 194,9, 195D • Stringer (191*9) makes the following statement: "The experiments with DDT deposits from alcohol and acetone on No. 1 and No. 50 Whatman f i l t e r papers indicate that the solvent and the nature of the substrate exert an e f f e c t upon the deposit which i s r e f l e c t e d i n the degree of mortality. I t i s obvious that the a v a i l a b i l i t y of the deposit depends upon the c r y s t a l size and shape, the d i s t r i b u t i o n on the f i l t e r paper, and the i n s e c t - i n s e c t i c i d e r e l a t i o n s h i p . " 8 P a r t i c l e s i z e and shape depends to a large extent upon the solvent used, and the method of r e c r y s t a l l i z a t i o n (Mcintosh, 1947, 1949). The solvent used may be v o l a t i l e , f o r example, acetone or alcohol, or i t may be a non-volatile o i l such as mineral o i l or o l i v e o i l . Stringer (1949) has noted that an i n s e c t i c i d e applied i n an o i l solution i s much more toxic than a s i m i l a r concentration i n c r y s t a l l i n e form. He attributed this increase i n t o x i c i t y to the " s t i c k i n g 1 1 c h a r a 6 t e r i s t i c of the o i l . More recently, i t has been postu-lated that the increased t o x i c i t y of i n s e c t i c i d e applied i n o i l f i l m i s due to the f a c t that the o i l i s nearer to the chemical composition of the outer, impenetrable surface of the c u t i c l e , and hence penetration and absorption of the i n s e c t i c i d e w i l l be much simpler. When an i n s e c t i c i d e i s applied i n a c r y s t a l l i n e f i l m , i t i s necessary that the c u t i -cular o i l s dissolve the compound before penetration i s possible. Stringer (1949) has determined that the slope of the dosage-regression l i n e i s proportional to the o i l f i l m thickness. To eliminate the d i f f e r e n t i a l e f f e c t of t h i s o i l f i l m thick-ness, he used one o i l dosage and varied the concentration of the i n s e c t i c i d e . Investigators have also found that not only are the physico-chemical c h a r a c t e r i s t i c s of the i n s e c t i c i d e of importance, but also that I t Is necessary to "standardize" the test insects. For example, the age of the insects used In t e s t i n g i s important (Sun, 1950). McLeod (1944) states: " I t w i l l be necessary . . . to devise some method of reporting 9 s u s c e p t i b i l i t i e s separately according to sex or of securing constant sex r a t i o s between variates i n an experiment. 1 1 Furthermore, a rel a t i o n s h i p has been demonstrated between n u t r i t i o n and t o x i c i t y . Both the rearing medium of the larvae and the food of the adult have been found to influence the s u s c e p t i b i l i t y of an insect to an i n s e c t i c i d e (Wilkes et a l , 1948). Numerous methods have been developed f o r rearing the d i f f e r e n t species of test insects (Peet and Grady, 1928; Wilkes et a l , 191+-8; Piquett and Fales, 1952; McLintock, 1952; Granett and Haynes, 19i|4) • The o f f i c i a l method of the CSMA, adopted o r i g i n a l l y i n 1932, and since modified, i s the Peet-Grady Method f o r rearing houseflies and cockroaches (Blue Book, 1952). A problem of increasing importance at present Is the selection of the s t r a i n of t e s t insect to be used. For example, the GSMA ffstandard" s t r a i n of housefly, known as the N.A.I.D.M. s t r a i n , Is used i n the O f f i c i a l Peet-Grady method. However, i n d i f f e r e n t laboratories throughout the country, many d i f f e r e n t strains of f l i e s , c o l l e c t e d and reared under d i f f e r e n t conditions, are used as the test Insect. These st r a i n s of f l i e s vary considerably i n t h e i r s u s c e p t i b i l i t y to the various i n s e c t i c i d e s . A re l a t e d f a c t o r i s that of Insect resistance to in s e c t i c i d e s . In many places throughout the world, insects, e s p e c i a l l y the housefly, have developed resistance to inse c t -i c i d e s . March and Metcalf (191+.9), a f t e r t e s t i n g and comparing s i x strains of houseflies against DDT, stated: "The r e s u l t s 1G show . . . that the resistance of the Bellflower s t r a i n i s 333x, the San Jose s t r a i n 22x, the Ontario s t r a i n llpc, the Riverside s t r a i n 13x, and the Hyman s t r a i n ipc that of the laboratory s t r a i n of f l i e s . " Roadhouse (1953), using the same technique, found resistance 2000x that of the laboratory s t r a i n . Insects exhibiting DDT resistance may show cross-resistance, usually to c l o s e l y related compounds. March and Metcalf (1949) found that the Bellflower s t r a i n of insects showed a lesser degree of resistance to compounds structur-a l l y s i m i l a r to DDT, but not to compounds such as gamma-benzene hexachloride, chlordane, parathlon, or pyrethrlns. Other investigators have found that resistance to insect-i c i d e s such as gamma-benzene hexachloride, chlordane, a l d r i n , and d i e l d r i n , Is d i s t i n c t from DDT resistance, but that "se l e c t i o n by exposure to one of them ra i s e s the resistance of the f l y s t r a i n to the others." (Busvine, 1954)* A considerable amount of inv e s t i g a t i o n has been made into methods of analysing the t o x i c i t y data obtained. The usual method consists of the construction of dosage-mortality curves obtained by p l o t t i n g the percent mortality against the concentration. The curve obtained i s sigmoid i n shape, when plotted upon an arithmetical scale. By adopting the method of probit analysis, i t Is possible to obtain a l i n e a r r e l a t i o n s h i p , making i t simpler to draw conclusions as to the equation of the slope, and LD5J0 11 values ( B l i s s , 1934a, b; 1935a, b; Gaddura, 1933). However, many authors f e e l that dosage-mortality data alone do not give as complete a picture as i s necessary and, consequently, other methods of tes t i n g have been developed. For example, the survival time-mortality curve makes i t possible to estimate the time at which any given percentage of the individ u a l s w i l l succumb to a toxicant, but only at one dosage l e v e l ( B l i s s , 1937). At present there i s no conven-ient method by which dosage, mortality, and time of response can be in t e r r e l a t e d , although there are several methods of showing these values topographically (Hansberry and Chiu, 1940J Richardson and Haas, 1932). Although these topographic methods serve as v i s u a l aids i n judging compar-ative t o x i c i t y , there i s no simple mathematical treatment from which one can draw conclusions as to comparative t o x i c i t y . 12 I I I . MATERIALS  Test insect The t e s t insect selected was the common housefly, Musca domestlea L. Two strai n s of housefly were used i n the te s t i n g procedure. The f i r s t s t r a i n was designated as the SES culture. This culture, o r i g i n a l l y obtained from Dr. L.E. Ghadwiek, Army Chemical Center, Maryland, had been reared f o r a period of four and one-half years at the Entomology Section, Suf-f i e l d Experimental Station. The second s t r a i n was obtained i n August, 1955 from the Pre s t i c i d e s Testing Laboratory, Ottawa, through the kindness of Mr. W.S. McLeod. This s t r a i n was designated as the Ottawa culture. This culture had been reared through from the N.A.I.D.M. "lj.8 s t r a i n , or the o f f i c i a l test insect f o r the Peet-Grady method. At no time since 1948 was t h i s culture exposed to DDT contamination, but there was a pos-s i b i l i t y of chlordane contamination during 1948-49 (McLeod, W.S., personal communication). Fisher (1952) and Roadhouse (1953) used t h i s culture f o r the "standard" susceptible s t r a i n . Insecticides A t o t a l of nine i n s e c t i c i d e s was used. The ins e c t i c i d e s selected were a l l organic compounds developed within the l a s t few years. 13 D i e l d r i n (1 ,2 ,3 ,4 ,10 ,10-hexachloro -6 ,7-epoxy -1 , 4, 4a, 5 , 6 , 7 » 8 , 8 a-octahydro-l,l4 . , 5 » 8-dinieth.anonaphthalene). E m p i r i c a l f o r m u l a : C ^ H S O I Q O . S t r u c t u r a l f o r m u l a : Cl R e c r y s t a l l i z e d : 100$ pure. A l d r i n (l , 2 , 3 , 4 , 1 0 , 1 0-hexachloro-l , 4 , 4 a , 5 , 8 , 8 a -h e x a h y d r o - 1 5 > : 8-dimethanonaphthalene). E m p i r i c a l formula: C i2 H 8 G 1 6* S t r u c t u r a l formula: CI R e c r y s t a l l i z e d : 99.6$ pure. E n d r i n (1 ,2 ,3 ,4 ,10 ,10-hexachloro -6 ,7-epoxy-l,4* 4a, 5 , 6 , 7 , 8 , 8 a-octahydro-l , 4 , 5 , 8-endo-endo-dimethanonaphthalene). E m p i r i c a l formula: C ^ H Q C L Q O . S t r u c t u r a l f o rmula: CI Cl 14 R e c r y s t a l l i z e d : 99.6% pure. Heptaehlor (1,4,5,6,7,8,8-heptaehlor-3a,4,7,Ta-te trahydro-4»7-endomethanoindene). Empirical formula: C^H^Cl^. Structural formula: CI Gl R e c r y s t a l l i z e d : 99.6% pure. Parathion (o,o-diethyl o-p-nitr©phenyl phosphorothioate) Empirical formula: C^H^NO^PS. Structural formula: OC{Hs Technical t ,Thiophos n: 95$ pure. Malathion (S-(1:2-diearbethoxyethyl)-o,o-dimethyl phosphorodithioate). Empirical formula: C^H^O^PS^ Structural formula: (OH^O)gPS.SOH.0OO02H5 CH 2.00O0 2H 5 P u r i f i e d : 99.6% pure. EPN (o-ethyl O-p-nitrophenyl phenylphosphorothioate) Empirical formula: C^H^NO^PS. Structural formula: / V . - ! >OG2H5 R e c r y s t a l l i z e d : 100$ pure. Lindane (garama-l:2:3:4 : 5i6-hexachlorocyclohexane). Empirical formula: C^H^Cl^. Structural formula: Gl R e c r y s t a l l i z e d : 100$ pure. DDT (l:l : l - t r i c h l o r o - 2 : 2 - d i ( p - c h l o r o p h e n y l ) ethane). Empirical formula: G^H^Cl^. Structural formula: R e c r y s t a l l i z e d : 100$ pure. Henceforth i n thi s text, these compounds w i l l be referred to by the common name. 17 IV. METHODS Rearing technique (Plate la) Adult f l i e s Adult f l i e s were reared i n cages 12" x 12n x 12". Three sides and the top of the cage were covered with wire screening. The f l o o r of the cage was wood. A c l o t h sleeve entrance was attached to the front of the cage by means of a two inch s t r i p of adhesive tape. The cage was washed and dried i n sunlight a f t e r the removal of each generation of f l i e s , and a fr e s h l y laundered sleeve was attached. Cages containing the SES culture were kept separate from those con-taining the Ottawa culture. At the S u f f i e l d Experimental Station, f l i e s were reared i n a rearing room, approximately 6' x 12'. Temper-ature was maintained at 78 - 2 degrees P. Relative humidity was 75 - 5$. V e n t i l a t i o n was provided i n order to reduce odours and gases from fermenting media (Plate l b ) . At the University of B r i t i s h Columbia, the temperature i n the rearing room was maintained at 78 - 2 degrees P. Since the humidity was extremely low and var-i a b l e , i t was necessary to cover the cages with p l a s t i c sheeting. Humidity was controlled at 72 - 2% with the aid of a saturated BaClg solution. V e n t i l a t i o n was provided i n the room by means of an exhaust fan. 18 Approximately 2000 f l i e s were placed i n each cage. Pood was supplied i n the form of lump sugar. Water jars consisted of a 2f>0 ml. beaker inverted i n t o a h a l f p e t r i dish. Absorbent cotton dental r o l l s were placed between the edge of the p e t r i dish and the beaker. The f l i e s were therefore supplied with a constant source of water, but could not drown i n i t . Fresh food and water were placed i n each cage with each new generation of f l i e s . Two stock cages were set aside each week. In t h i s way, eggs were not co l l e c t e d from f l i e s more than three weeks old. I t was found to be impossible to c o l l e c t eggs i n any great quantity u n t i l the f l i e s were from seven to ten days old . Eggs The stock cages were provided d a i l y with milk soaked paper towels f o r ov i p o s i t i o n canned milk: ^ water). Eggs were c o l l e c t e d a f t e r a period of not longer than eighteen hours (overnight). The eggs were washed gently i n d i s t i l l e d water several times u n t i l thoroughly separated. A measured 700 eggs were then placed on top of the media. This was done by allowing the eggs to s e t t l e i n a calibr a t e d pipette (0.1 ml. s e t t l e d eggs = 700). Five ml. d i s t i l l e d water were used to measure and scatter the eggs over the media. 19 Larval medium Canned dog food (horsemeat) was used, as a sub-s t i t u t e f o r the o r i g i n a l method used at the S u f f i e l d Exper-imental Station employing lean ground beef, as the l a r v a l medium. Two types of containers were used, both of which were found to be acceptable. At the S u f f i e l d Experimental Station, p l a s t i c bread containers, 9" x 2j.w were used, while at the University of B r i t i s h Columbia, battery ja r s were u t i l i z e d . A t h i n layer of autoclaved sawdust was placed at the bottom of the container, and 500 grams of horsemeat, flattened i n the form of a hamburger, were placed on top. The eggs were scattered over the meat, and covered with a further layer of autoclaved sawdust. Twenty ml. of d i s t i l l e d water were then placed over the media i n order to supply the necessary moisture. Six l a r v a l containers were set up per day. The larvae of the SES culture pupated within eight days. However, the Ottawa culture was found to be extremely va r i a b l e . At the S u f f i e l d Experimental Station, they reached maturity within f i v e days. At the University of B r i t i s h Columbia, t h i s period lengthened to eight days. Pupae I t was found that the mature pupae migrated to the bottom portion of the media. Any remaining meat was removed Plate I. (a) Technique employed i n rearing Musca domestica L. (b) View of rearing room at the S u f f i e l d Experimental Station 20 and discarded. The pupae-sawdust mixture was sieved through a number 10 sieve. The pupae remained on top, while the sawdust passed through. The pupae, contained i n large (6M) h a l f p e t r i dishes, were placed i n the cages. Approximately 2200 pupae were placed i n each cage. Two cages of f l i e s were set up per day. P l i e s hatched within ij.8 hours. A l l of the pupae maturing on one day were combined before s e l e c t i o n was made f o r te s t i n g . P l i e s reared according to this method were found to be both large and uniform i n s i z e . Bioassay technique Testing room At the S u f f i e l d Experimental Station the testing and observation room was approximately 8* x 12*. Temperature was maintained at 78 - 2 degrees P. Relative humidity was maintained at 7f> - 5$. V e n t i l a t i o n was provided. In order to eliminate any p o s s i b i l i t y of contamination, a l l i n s e c t i -cides were mixed and applied to the substratum i n the formu-l a t i o n laboratory. At the University of B r i t i s h Columbia, te s t i n g was ca r r i e d out i n a laboratory maintained at a temperature of 7 8 - 2 degrees P. The humidity was not controlled and varied from 10$ to $$% with an average l e v e l of 30$. However, since the exposure period was only four hours, i t was assumed that any change i n t o x i c i t y due to v a r i a t i o n In humidity would be 21 n e g l i g i b l e . Observation chambers were stored i n a temperature cabinet maintained at 78 - 2 degrees P. Humidity was main-tained at 72 - 2% with the aid of a saturated B a C l 2 solution. Testing apparatus One of the major objectives i n the development of t h i s technique involved the separation of residual contaet e f f e c t from fumigant e f f e c t . Pradhan (19I4.9), working with gamma-BHC attempted to eliminate fumigant e f f e c t i n several d i f f e r e n t ways: (1) By confining insects over i n s e c t i c i d e f i l m s with open truncated cones. (2) By confining insects over i n s e c t i c i d e f i l m s covered by wire gauze or perforated zinc covers. (3) By confining insects over i n s e c t i c i d e f i l m s with perforated f i l t e r cones. (]+) By confining insects over i n s e c t i c i d e f i l m s within f i l t e r paper cones f i t t e d with exhaust draughts. (5) By confining insects over i n s e c t i c i d e f i l m s with perforated f i l t e r paper cones f i t t e d with exhaust draughts. He concluded that "as there was considerable toxic action even ... when there was no contact e f f e c t and the insects are confined by open cones, the complete elimination of fumigant e f f e c t i n the case of gamma-benzene hexachloride appears impossible." 22 In a s i m i l a r series of experiments c a r r i e d out during the spring of 1955, i t was found impossible to eliminate fumigant e f f e c t by exhausting a stream of a i r through a glass cylinder. A s t r i p of f i l t e r paper, the width and length of the cylinder, was impregnated with para-thion and placed i n the cylinder. A s t r i p of wire screening was placed over the f i l t e r paper at a distance of 1/8". F i f t y t e s t insects were placed on the screen, and the cylinder was stoppered at both ends with one-hole No. 7 stoppers. Each stopper was f i t t e d with a piece of 6 mm. glass tubing, the ends of which were covered with muslin. A vacuum l i n e was attached to the tubing at one end of the cylinder. In a series of tests using d i f f e r e n t concentrations of parathion, the fumigant mortality was always extremely high, regardless of the rate at which the a i r was evacuated from the cylinder. I t was concluded that i t was impossible to eliminate fumigant e f f e c t i n t h i s manner. From the data obtained by Pradhan, and from those obtained i n the preceding experiments, i t i s obvious that fumigant e f f e c t cannot be eliminated as long as the fumes are drawn past the insects. Accordingly, a method was sought In which the exhaust would not pass the insects. I t was suggested that i n an apparatus such as a Buchner funnel, the insects could be confined to the upper surface of the substratum, and, by u t i l i z i n g negative pressure, a continual flow of clean a i r could be supplied to them, while contaminated a i r was drawn o f f below. In 23 order to t e s t t h i s hypothesis, experiments were c a r r i e d out using 9.0 cm. desk type Buchner funnels. No. 1 Whatman f i l t e r papers were Impregnated with parathion solutions of varying concentration. Comparative tests f o r fumigant (P), th e o r e t i c a l contact (C), and fumigant-contact (F+C) were car r i e d out at the same time. Direct fumigant e f f e c t was measured by placing a 9.1 cm. c i r c l e t of nylon screening in from the impregnated f i l t e r paper. Test insects were placed within the funnel, which was then covered with muslin. The following r e s u l t s were recorded: Table I. Elimination of fumigant e f f e c t of parathion. Percent No. Rate of E f f e c t 24 hour Percent solution f l i e s evacuation mortality mortality (l./m./funnel) 4 x 10" 1 25 2 P 21 84 25 - P+C 25 100 25 2 C 25 100 2 x 10" 2 19 4 P 5 26 25 - P+C 24 98 24 4 C 24 100 2 x 10" 3 10 4 P 0 0 25 - P+C 25 100 25 4 C 16 64 At the 2 x 10 concentration, with the rate of evacuation at 4 l./m./funnel, the fumigant e f f e c t was completely eliminated. There was a corresponding decrease i n the mortality of the d i r e c t contact series as compared to the furaigant-plus-contact s e r i e s . Hence i t would appear possible 2k to eliminate the fumigant e f f e c t of parathion by exhausting the fumes through the substratum. Therefore, the basic apparatus was made up of a series of Buchner funnels attached to a vacuum system. Substratum In the previous experiments, the substratum was 9 cm. No. 1 Whatman f i l t e r paper. However, two objections arose regarding the use of the f i l t e r paper as the substratum. F i r s t , i t was determined that i n order to eliminate the fumigant e f f e c t of parathion, at approximately the LD50 concentration (2 x 10~^%), an evacuation rate of k./m./funnel was necessary. I t was obvious that due to the consistency of the paper, a much higher rate of evacuation would be necessary than that required i f a more porous substratum were used. Secondly, some question arose as to the amount of i n s e c t i c i d e absorbed by the paper, e s p e c i a l l y i f a method of a p p l i c a t i o n such as dipping the substratum into a solution of known concentration were involved (Proverbs and Morrison, 19k7). I t was postulated that d i f f e r e n t amounts of insect-i c i d e would be picked up on the paper, depending upon the chemical composition of the i n s e c t i c i d e . For these reasons, experiments using d i f f e r e n t substrata were carried out. When wire screen was used, i t was determined that the fumigant e f f e c t could be eliminated with approximately one-half the rate of evacuation required 25 f o r f i l t e r paper. Furthermore, there would be no question of absorption but merely of adherence of the i n s e c t i c i d e to the substratum. Unfortunately, i t was d i f f i c u l t to cut to size and impossible to re-use. Nylon screen was eliminated f o r the same reason. Results using woven f i b e r g l a s s c l o t h were much more encouraging (8 oz. weave). The fumigant e f f e c t could be e a s i l y eliminated with approximately one-half the rate of evacuation required f o r f i l t e r paper. At the same time, I t was e a s i l y cut, inexpensive, and re-usable a f t e r decontamination i n chromic acid, i f c a r e f u l l y treated. Although i t tended to fr a y around the cut edges, i t was found that "Cenco11 l a b e l varnish, painted around the cut edge, prevented t h i s . Therefore, woven f i b e r g l a s s c l o t h was chosen as the substratum f o r the i n s e c t i c i d e (Plate I I ) . Mode of application of i n s e c t i c i d e I t has previously been noted that numerous methods of a p p l i c a t i o n of the i n s e c t i c i d e to the substratum have been developed. In the search f o r a simple, yet adequate, method of application, three of the previously mentioned techniques were considered: application of the i n s e c t i c i d e to the substratum i n a spray tower (Potter, 19lp.), p i p e t t i n g the i n s e c t i c i d e onto the substratum (Stringer, 191*9; Barnes, 191*5) * and dipping the substratum in t o a known concentration of the i n s e c t i c i d e (Proverbs and Morrison, 191*7)» A search was made for a method to circumvent the elaborate procedure involved i n the use of a spray tower. o Plate I I . A series of f r e s h l y cut f i b e r g l a s s cloths treated with "Cenco" l a b e l varnish to prevent fraying. 26 I t has been noted that t o x i c i t y varies with p a r t i c l e size (Mcintosh, 1947, 1949, 19£l; McGovran et a l , 1940). I t i s obvious that i f an i n s e c t i c i d e i s pipetted onto the substratum, the concentration w i l l not be the same throughout, although the o v e r a l l concentration would compare fo r r e p l i c a t e s treated i n the same way. The i n s e c t i c i d e would tend to layer up from the point(s) of application, and at the same time, c r y s t a l structure would d i f f e r . I f , however, the Insecticide was applied i n an o i l solvent, rather than a more v o l a t i l e one such as acetone or alcohol, t h i s layering tendency would be reduced considerably due to d i f f u s i o n of the o i l over the substratum. However, the application of i n s e c t i c i d e i n t h i s manner could conceivably introduce a variable which should be eliminated i f at a l l possible. Experiments with A n i l i n e Green dye dissolved i n benzene indicated that i f the f i b e r g l a s s substratum were dipped into the dye solution, an even concentration was obtained. Since there i s no question of absorption of the i n s e c t i c i d e or dye into the f i b e r s , as there may be with f i l t e r paper, dipping the substratum into solutions of known concentration appeared to provide an adequate method of appli c a t i o n . At the same time, t h i s method i s not completely quantitative as f a r as determination of the t o t a l amount of i n s e c t i c i d e a v a i l a b l e . In order to estimate retention of i n s e c t i c i d e residue, a dye could be substituted, and the t o t a l amount picked up by the substratum could be analysed c o l o r i m e t r i c a l l y . I t i s necessary to assume that the amount 27 of i n s e c t i c i d e adhering to the c l o t h i s d i r e c t l y proportion-ate to the amount of dye adhering to the c l o t h . A n i l i n e Green dye, soluble up to 2|$ was selected. Before construction of a standard curve was possible, i t was necessary to select the solvent or solvent mixture to be used throughout the work. Since i n s e c t i c i d e s are much more toxic when applied i n an o i l f i l m , a solvent mixture consisting of 9$% benzene (thiophene free) plus l i g h t weight mineral o i l was used. I t was determined experimentally that a higher concentration of mineral o i l caused considerable mortality among the controls, presum-ably due to blocking of the trachea. A standard curve f o r A n i l i n e Green dye i n a 9i>:5 benzene-mineral o i l solvent mixture was constructed by making a series of d i l u t i o n s ranging from 1 x 10 % to 5 x 10 % (Table I I ) . Galvanometer readings (G) were taken on an Evelyn Photoelectric Colorimeter. The readings were con-verted to o p t i c a l densities (L) by reference to a standard table. Figure 1 shows the standard curve obtained by p l o t t i n g the dye concentration {%) against the o p t i c a l density (L). Table I I I shows the r e s u l t s obtained by extract-ing the t o t a l dye picked up on £0 mgm. f i b e r g l a s s c l o t h . Pieces of glass c l o t h , 5>0 mgm., were dipped into dye solutions of known concentration, ranging from 0.1$ to 3.0$, f o r a period of ten seconds. They were then removed, placed on f i l t e r paper and allowed to dry. Each piece of 28 Table I I . Calculation of standard curve f o r A n i l i n e Green dye dissolved i n a 95:5 benzene-mineral o i l solvent mixture. percent mgm. dye/ solution ml. solvent 1 X i o - i 1.0000 1 X 10-2 0.1000 9 X 10-3 0.0900 8 X 10-3 0.0800 7 X 10-3 0.0700 6 X 10-3 0.0600 5 X 10-3 0.0500 4 X 10-3 0.0400 3 X 10-3 0.0300 2 X 10-3 0.0200 1 X 10-3 0.0100 5 X 10-4 0.0050 4 X 10-4 0.0040 3 X 10-4 0.0030 2 X io-4 0.0020 1 X 10-4 0.0010 5 X io-5 0.0005 galvanometer reading (G) 4.50 1.3470 5.25 1.2800 5.75 1.2400 7.00 1.1550 7.25 1.1L00 10.25 0.9890 13.00 0.8860 17.25 0.7630 24.00 0.6200 41.25 0.3850 60.00 0.2218 67.50 0.1707 74.50 0.1278 81.50 0.0888 87-25 0.0593 93.00 0.0315 impregnated c l o t h was extracted with 10 ml. of 95:5 benzene-mineral o i l mixture. Five r e p l i c a t i o n s were made fo r each concentration. Figure 2 shows the curve obtained by p l o t t i n g the dye concentration (%) against the o p t i c a l density (L). I t should be noted, that by placing the c l o t h on the f i l t e r paper, a large amount of dye was absorbed into the paper from the lower surface of the c l o t h . However, due to the rapid evaporation of the solvent, the dye con-centrated upon the upper surface remained. Hence, the figur e obtained f o r the t o t a l Insecticide on the c l o t h was cl o s e r to the actual i n s e c t i c i d e a vailable. I t was necessary, therefore, that the upper surface of the impregnated substrate be used as the surfaee upon which the insects were introduced. Table I I I . Extraction of Percent Mgm. dye/ G L solution ml. s o l . 0.10 1.0 8 l 2 0.0888 81 0.091$ 75 2 0.1221 74 1 0.1293 77 1 0.1121 0.25 2.5 59 3 0.2236 6 l l 0.2129 583 0.2310 64 1 0.1922 59 0.2291 0.50 5.0 43 3 0.3620 41 0.3870 38 3 o.lp.20 4 I O 0.3870 44 2 0.3520 0.75 7.5 30 2 0.5160 34 0.4690 3 d 0.5200 36 2 0.4380 28 0.5530 1.00 10.0 2 3 1 0.6340 26 o.585o 243 0.6060 22 2 0.6480 25 1 0.5980 Green dye from 50 mgm. f i b e r g l a s s c l o t h . Average Percent Mgm. dye/ G L Average L solution ml. s o l . L 0.1088 1.25 12.5 17 2 0.7570 0.7100 20l 0.6490 183 0.7270 2 l l 0.6730 20 0.6990 0.2178 1.50 15.0 17 2 0.7570 0.7580 17 0.7700 18 1 0.7390 171 0.7630 17 1 0.7630 0.3800 2.00 20.0 14 1 0.8460 0.9010 l l 3 0.9300 13 0.8860 l l 2 0.9300 12 0.9210 0.4590 2.50 25.0 10., 1.0000 1.0360 9} 1.0340 l l 1 0.9490 7 2 1.1260 8 2 1.0710 0.6140 3.00 30.0 8„ 1.0790 1.0790 83 1.0590 8 1.0970 9 1.0460 8 1.0970 F I G . 2 FIG. I 30 Prom Figures 1 and 2, i t was possible to calculate the amount of dye per 50 mgm. f i b e r g l a s s c l o t h , and hence the amount of dye per gram of c l o t h (Figure 3 a, b). Procedure The f i b e r g l a s s c l o t h was e a s i l y cut into c i r c l e t s , 9 centimeters i n diameter, with the aid of a sharp s c a l p e l . The cut edges were painted with nCenco n l a b e l varnish. In order to simp l i f y calculations, and avoid weighing each c l o t h i n d i v i d u a l l y , the weight of each c l o t h was assumed to be 2 grams (actual weight range, 1.95 - 2.05 grams). Therefore the t o t a l amount of i n s e c t i c i d e on the c l o t h could be c a l -culated by multiplying the figure obtained from figu r e 3, by 2. Insecticide solutions were made up on a weight/ volume basis. 2$ ml. of solution were placed i n the large h a l f of a 9 cm. p e t r i dish, and each c i r c l e t of cl o t h was dipped i n d i v i d u a l l y f o r a period of ten seconds. The c l o t h was removed and allowed to dry on 9 cm. No. 1 Whatman f i l t e r paper. Three r e p l i c a t i o n s were made at each dosage l e v e l . 9.0 cm. desk-type Buchner funnels were set up i n series of three. I f the i n s e c t i c i d e was fumigant i n nature, the funnels were attached to the vacuum system through a series of exhaust manifolds. Rate of evacuation was measured with a Fisher and Porter P r e c i s i o n Bore Rotameter, with a capacity of 32.5 L. of a i r per minute. R E T E N T I O N OF ANILINE G R E E N DYE O N F I B E R G L A S S C L O T H (O —3 0 % R A N G E ) MILLIGRAMS DYE/GRAM CLOTH FIG.3A R E T E N T I O N OF ANILINE G R E E N DYE ON F I B E R G L A S S C L O T H (0— .12 % RANGE) .2 .3 A MILLIGRAMS DYE/GRAM CLOTH FIG- % % 31 The impregnated cloths were dried f o r a period of one hour before they were placed i n the funnels. The t e s t Insects (Musca domestica L.) were c a r e f u l l y reared according to the procedure previously described. The pupae were previously mixed, before they were, placed i n the cages, i n order that the population tested be as homogeneous as possible. The f l i e s were removed from the rearing cages with the a i d of a vacuum system. A glass cylinder 8 W i n length was stoppered at both ends with No. 7 rubber stoppers. A 3" length of 12 mm. glass tubing was inserted into each stopper. A wire cage was placed over the stopper attached to the vacuum l i n e , i n order that the f l i e s were not drawn into the system. A 2' length of p l a s t i c tubing was attached to the glass tubing at the i n l e t end of the cylinder. A 2^ M glass funnel with a outlet was attached to the p l a s t i c tubing (Plate I l i a ) . P l i e s removed from the cages In t h i s way were seldom injured. Approximately 500 f l i e s could be removed at one time without crowding. The f l i e s were anaesthetized with CO2 and immediately placed i n a cold room (£• C.), where they were segregated according to t h e i r sex. I t was found that f l i e s subjected to cold alone were d i f f i c u l t to i d e n t i f y as to sex, since they tended to aggregate into a s o l i d mass, while those anaesthetized with G02 recovered much too quickly f o r sorting i n large numbers. At the University of B r i t i s h Columbia, a cold room was not accessible Plate I I I . (a) Method employed i n removing houseflies from cages. (b) Buchner funnels containing f?0 f l i e s each, demonstrating fumigant and residual contact tests. 32 and the f l i e s were anaesthetized with an ether-alcohol mixture (50:50). Recovery was slow, and mortality i n the controls was higher. Experiments were ca r r i e d out to determine a sa t i s f a c t o r y type of covering f o r the funnels. Muslin and screen proved unsatisfactory since the f l i e s would s e t t l e on such coverings i n preference to the impregnated substrate. However, f l i e s would not s e t t l e to any extent on a trans-parent surface. For t h i s reason, "Saran" f i l m was used as a covering f o r the funnels. The f i l m was perforated with approximately t h i r t y small holes through which the f l i e s could not escape (Plate I l l b ) . The f l i e s were introduced to the three r e p l i c a t e s of each dosage at the same time, and to each dosage at ten minute i n t e r v a l s . Each r e p l i c a t e contained $0 f l i e s (Plate IVa). The exposure period was a r b i t r a r i l y placed at four hours. Within the f i r s t f i f t e e n minutes of t h i s period, any mortality due to mechanical i n j u r y was noted. Upon completion of the exposure period, the insects were removed from the funnels to separate glass cylinders, 6" i n length. A vacuum system s i m i l a r to that used i n removal from the cages was u t i l i z e d . A small square of l " adhesive tape, containing a hole, was stuck to the saran f i l m . A tapered glass rod, attached to the p l a s t i c tubing i n place of the glass funnel, was inserted through t h i s small opening. The f l i e s were removed i n d i v i d u a l l y , using a Plate IV. (b) Observation chambers set up In series. Mortality counts were made at 2k and k8 hour periods. 33 l i g h t vacuum. The r e p l i c a t e s of each dosage l e v e l were removed at ten minute i n t e r v a l s , exactly four hours a f t e r they were introduced. Each r e p l i c a t e was numbered and dated, and placed i n series (Plate IVb). M o r t a l i t y counts were made at 2i* and 1*8 hour i n t e r v a l s . Only those insects showing no movement whatsoever were counted as dead. Before constructing a dosage-mortality curve f o r re s i d u a l contact t o x i c i t y , i t was f i r s t necessary to deter-mine the fumigant properties of the compound. Fumigant action was determined i n a manner si m i l a r to that described i n the preceding experiments (Plate V a, b). F i f t y f l i e s were placed on a wire screen at a distance of ^ t t from the impregnated substratum. The funnel was covered with a laye r of perforated saran f i l m . After four hours exposure, the f l i e s were removed and placed i n the observation chambers. Observations were taken at 21* and ij.8 hour i n t e r v a l s . I f a compound was found to be fumigant i n nature, further experiments were carr i e d out to determine the rate of evacuation necessary to eliminate the fumigant e f f e c t at the approximate I D ^ l e v e l . I t was also necessary to determine the approximate range of concentrations over which mortality occurred. This could quite often be determined i n a single series of experiments using eight to ten dosage l e v e l s ranging from the approximate LDg£ dosage l e v e l downward. (a) Components of equipment used i n testing fumigant e f f e c t . (b) Assembled equipment used i n testing fumigant ef f e c t . 34 As was previously pointed out, a large number of factors a f f e c t the t o x i c i t y of an i n s e c t i c i d e to an insect. In the development of the present technique, an attempt was made to standardize external factors such temperature and humidity control, physico-chemical r e l a t i o n s h i p s , and rear-ing techniques. However, a l l b i o l o g i c a l material shows inherent v a r i a t i o n . I t has been shown experimentally, that with a l l external factors controlled, L D C J Q values f o r groups of insects tested at d i f f e r e n t times w i l l vary considerably (Krijgsman and Berger, 1949). Since the usual method of analysis consists of comparison of L D C J Q values, I t i s obvious that a large error could occur. " T o x i c i t y index - an improved method of comparing the r e l a t i v e t o x i c i t y of i n s e c t i c i d e s " was introduced by Sun i n 1950. I t i s defined as the " r a t i o between the I<I>cjg of a standard i n s e c t i c i d e and that of a test sample, m u l t i p l i e d by 100." That i s : T o x i c i t y index = ^50 o f t h e x 1 0 0 L D C J Q of test sample The t o x i c i t y index of the standard i s always equal to 100. Sun has shown that the t o x i c i t y index remains f a i r l y constant i f the external variables are c a r e f u l l y controlled, and i f the test i n s e c t i c i d e i s tested on the same day as the standard. He concludes that "the change In the L D ^ Q of the standard i n s e c t i c i d e caused by changes i n the s u s c e p t i b i l i t y of the f l i e s and the environmental conditions i s accompanied by a proportional change i n the L D C J Q of the t e s t sample." 35 The concept of t o x i c i t y index was incorporated into the present technique. D i e l d r i n was selected as the "standard" i n s e c t -i c i d e , since i t was found to possess no fumigant e f f e c t , and was intermediate i n the range of t o x i c i t y of the compounds to be tested, according to the r e s u l t s obtained by Sun (1950). In the determination of the dosage-mortality curve, four to seven dosages were selected over the approximate range of the test i n s e c t i c i d e . A. dosage-mortality curve was constructed f o r the standard i n s e c t -i c i d e on the same day that the te s t sample was used. With the equipment available, i t was necessary to perform two "runs" a day. Each run consisted of three dosage l e v e l s of the standard, three or four dosage l e v e l s of the tes t sample, and a control se r i e s . Therefore, 2200 insects, segregated as to sex, were required per day. The r e s u l t s obtained were analysed according to the method of probit analysis developed mainly by B l i s s (1934 a, *>; 1935 a, b), using the s i m p l i f i e d method of Finney (1952). To x i c i t y indices were calculated from the L D J - Q values. 36 V. EXPERIMENTAL DATA Elimination of fumigant e f f e c t In the design of the test apparatus, i t was demonstrated that fumigant e f f e c t could be eliminated by-appl i c a t i o n of negative pressure through a Buchner funnel. Woven f i b e r g l a s s c l o t h was selected as the substratum due to the rough weave, which made i t possible to eliminate fumigant e f f e c t more e a s i l y . Some Insecticides, such as DDT, show no s i g n i f i -cant fumigant action, while others, such as parathion, possess a strong fumigant e f f e c t . Therefore i t was necessary to determine the fumigant aetion, i f any, of each of the i n s e c t i c i d e s to be used i n te s t i n g . Each i n s e c t i c i d e was tested i n the following manner: f>0 f l i e s were placed within a Buchner funnel on a screen 9.1 cm. In diameter. The screen was supported on a glass t r i a n g l e at a distance of in from the impregnated substratum. The funnel was covered with perforated saran f i l m . A concentration of k.O mgm. of each i n s e c t i c i d e was used. Table IV shows the r e s u l t s obtained. The data show that f i v e of the nine i n s e c t i c i d e s tested i n t h i s manner show fumigant action at a concentration of k.O mgm., while four do not. Fumigant action i s , of course, proportional to the vapour pressure of the compound, as the figures i n Table IV indi c a t e . 37 Table IV, Fumigant action of t y p i c a l i n s e c t i c i d e s upon Musca domestica L. (SES c u l t u r e ) . Insecticide Fumigant action Vapour pressure (mm.Hg) D i e l d r i n A l d r i n Endrin Parathion EPN Malathion DDT Lindane. Heptachlor The data obtained i n Table I also indicate that the rate of evacuation i s a c r i t i c a l f a c t o r . At the approximate L D C J Q concentration f o r parathion (on f i l t e r paper), there was no mortality due to fumigant e f f e c t , i f the rate of evacuation was set at it l i t r e s of alr/minute/funnel. However, i f the rate of evacuation was lowered to 3 l i t r e s / minute/funnel, mortality rose to 1+6%. Therefore, i t was deemed necessary to choose an evacuation rate f o r each i n s e c t i c i d e showing fumigant action. Table V shows the method used to determine the rate of evacuation necessary to eliminate the fumigant ef f e c t of a l d r i n . At a con-centration of 0 .053 mgm., i t was possible to eliminate fumigant e f f e c t completely I f the rate of evacuation was set at two or more l i t r e s of air/minute/funnel. However, at a rate of 1 .5 l i t r e s of air/minute/funnel, mortality due to fumigant e f f e c t rose to 23%. Similar r e s u l t s were obtained using male houseflies which require a lower con-centration of i n s e c t i c i d e . As a general r u l e , the lower l i m i t of concentration used f o r females could be taken as s i m i l a r to DDT • s i m i l a r to lindane • 3.78 x 10 -S @ 2 0 ' C. 3.00 x 10-2 @ l o o - C. + 1.50 x 10"? © 2 0 * C. • 9 .40 x l O " 6 @ 20- C. • 3 .00 x 10-4 @ 25- G. 38 Table V. Determination of the rate of evacuation necessary to eliminate the fumigant e f f e c t of a l d r i n on Musca domestica L. (SES c u l t u r e ) . Concentration Sex Rate of % mort. % mort. (Total mgm.) evacuation fumigant fumigant-(l./m./f.) contact 0.075 0 © 0 100 100 0.068 0 93 97 0.060 0 76 98 0.060 3 2 32 0.053 2 0 30 0.053 1.5 23 0.053 1 4 4 96 0.034 ( f t f 2 0 62 0.034 1.5 16 the upper l i m i t f o r males, thus simplifying the amount of approximate t e s t i n g necessary before construction of the f i n a l curve. The lower concentration at which I t was necessary to test the male Insects showed a proportional amount of fumigant e f f e c t , and as a r e s u l t , the same rate of evacuation could be used f o r both males and females. Preliminary experiments established the necessity of determining the rate of evacuation separately f o r each i n s e c t i c i d e . This l e v e l was determined f o r each compound at the approximate LD9CJ l e v e l . The r e s u l t s obtained are shown i n Table VI. Elimination of fumigant e f f e e t should bring about a corresponding decrease i n r e s i d u a l contact mortality. The data obtained i n Table V, with a l d r i n , show t h i s decrease to some extent. However, i n order to demonstrate more c l e a r l y the decrease brought about through a p p l i c a t i o n of negative 39 Table VI. Rate of evacuation necessary f o r elimination of fumigant e f f e c t of some i n s e c t i c i d e s on Musca domestica L. (SES c u l t u r e ) . Insecticide Rate of evacuation (l./m./funnel) D i e l d r i n -A l d r i n 2 Endrin -Parathion 2.5 EPN -Malathion 2 DDT -Lindane 2.5 Heptachlor 1 pressure, i t was decided to run a series of p a r a l l e l tests using a l d r i n , lindane, and heptachlor. The resu l t s obtained are shown i n Table VII. Table VII. Comparison of res i d u a l contact mortality and combined residual contact-fumigant mortality. Test insect: Musca domestica L. 90 . . (Average of three r e p l i c a t e s : 50 f l i e s / r e p l i c a t e ) . Insecticide Concentration Rate of % mort. Rate of % mort. ( t o t a l mgm.) evacuation contact evacuation fumigant. (l./m./f.) (l./m./f.) contact A l d r i n 0.053 2 27 0 91 (SES culture) 0.060 I16 93 0.06k 60 93 0.068 k7 97 Lindane 0.023 2.5 18 0 85 (Ottawa 0.030 56 86 culture) 0.038 50 81 0.0k5 63 90 Heptachlor 0.018 1 31 0 60 (Ottawa 0.023 32 79 culture) 0.026 If3 83 0.030 68 81 ko In a l l cases there Is a marked decrease In mortality with the elimination of fumigant e f f e c t . I t w i l l no doubt be argued that the decrease i n mortality brought about i n t h i s manner may be due more to v o l a t i l i z a t i o n of the i n s e c t i c i d e over the four hour exposure period than to the elimination of fumigant e f f e c t . In order to test t h i s p o s s i b i l i t y , a series of experiments were c a r r i e d out i n which the period of evacuation was raised from four to s i x hours. The insects were subjected to the usual four hour exposure period, but evacuation was begun two hours p r i o r to the introduction of the insects. P a r a l l e l tests were made using the standard four hour evacuation-exposure period. Lindane, requiring the highest evacuation rate of 2.5 l./m./funnel, and heptachlor, requiring the lowest evacuation rate of 1.0 ,/m./funnel were tested. The r e s u l t s obtained are shown i n Table VIII. I f v o l a t i l i z a t i o n of the i n s e c t i -cide was p a r t i a l l y responsible f o r the decrease i n mortality recorded i n Table VII, then a two hour evacuation period p r i o r to the introduction of the insects should reduce the i n i t i a l t o x i c i t y of the i n s e c t i c i d e , and the mortality f o r the s i x hour evacuation period as compared to the four hour evacuation period should be lower. Prom the r e s u l t s shown i n Table VIII, t h i s would not appear to be the ease. The r e s u l t s with heptaehlor f o r the six hour evacuation period indicate a s l i g h t l y higher mortality as compared to those obtained using the four hour evacuation period. The r e s u l t s with lindane, with a higher rate of evacuation, are s l i g h t l y 41 lower, but not s i g n i f i c a n t l y so. This difference could be attributed more to b i o l o g i c a l v a r i a t i o n within the culture, than to v o l a t i l i z a t i o n of the i n s e c t i c i d e . Table VIII. E f f e c t on mortality of increased period of evacuation. Test Insect: Musca domestica L., 9 $ , (Ottawa cu l t u r e ) . Insecticide Concentration % mort.6 hrs. % mort.4 hrs. Rate of ( t o t a l mgm.) evacuation evacuation evacuation (l./m./f.) Lindane 0.030 37 50 2.5 0.038 56 63 0.045 69 76 0.053 71 72 Heptachlor 0.030 72 69 1 0.038 76 69 0.045 66 79 0.053 83 86 Dosage-mortality data Since d i e l d r i n was to be used as the standard i n s e c t i c i d e , composite curves f o r both male and female house-f l i e s were f i r s t constructed. Approximate tests Indicated that the dosage range was 0.035 - 0.112 mgm. f o r females and 0.015 -0.035 mgm. f o r males. In the f i n a l t ests, three to s i x r e p l i c a t i o n s were made at each dosage l e v e l . Ten dosages were tested f o r females and seven f o r males. The probits of the percent m o r t a l i t i e s a f t e r 4$ hours were plotted against the logarithms of the dosages given In terras of t o t a l m i l l i -grams on the c l o t h . In order to eliminate negative values, the logarithms were m u l t i p l i e d by a factor of 10. Following the method of Finney (1952), the probit-regresslon l i n e s were 42 calculated from the data. T o x i c i t y of d i e l d r i n to Musca domestica L., , (SES cultu r e ) . The data are recorded i n Table 1 of the Appendix. Figure 4 shows the probit-regression l i n e . The equation of the l i n e was found to be Y = 5.6269 + 6.3826x. The slope was calculated as b = 6.3826 - 1.0874* Chi-square3= 115.451, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . Since there i s no evidence of anything other than a l i n e a r r e l a t i o n s h i p , the calculations f o r heterogeneity were made by Introducing the heterogeneity factor = 14-431, as suggested by Finney. The LDcjo was calculated as m = 0.046 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.064 mgm. and 0.033 mgm. T o x i c i t y of d i e l d r i n to Musca domestica L., <?c?, (SES cul t u r e ) . The data are recorded i n Table 2 of the Appendix. Figure 4 shows the calculated l i n e . The equation was calculated as Y = -5.7065 + 7.5594 s, while the slope was found to be b = 7,5594 + - 0.7484. Chi-square^ = 16.831, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The heterogeneity f a c t o r was calculated as 3.366. The UDCJQ. I S 0.026 mgm.,with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.028 and 0.024 mgm. T O X I C I T Y OF DIELDRIN TO MUSCA DOMESTICA L. (SES. CULTURE) J i i I L_ i . 2 1.4 i.e i.a 2.0 L O G 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 4 TOXICITY OF DIELDRIN TO M U S C A DOMESTICA L. (SES. C U L T U R E ) 6.0 h X M E A N O SINGLE R E P L I C A T E 1.0 1.2 1.4 1.6 1.6 LOG 1000 X DOSE ( T O T A L MILLIGRAMS) FIG. 5 43 Calculation of the problt-regression l i n e As has been stated previously, from four to seven dosage l e v e l s were used i n the construction of the dosage-mortality l i n e . Except i n the case of the composite graphs f o r d i e l d r i n (Figure 4) and DDT (Figures 12 and 14), three r e p l i c a t i o n s were made at each dosage l e v e l . I t was suggested that the mortality within the r e p l i c a t e s be plotted i n d i v i d u a l l y on the graph i n order to show the spread of mortality within a single dosage l e v e l , rather than p l o t t i n g the mean mortality of the three r e p l i c a t e s . In order to demonstrate both methods, a dosage-mortality curve was calculated i n both ways. Figure 5 shows the calculated l i n e . Replications are shown with an M o w , means with an nx". To x i c i t y of d i e l d r i n to Musca domestica L., , (SES cu l t u r e ) . Replications of each dose calculated i n d i v i d u a l l y . The data are recorded i n Table 3 of the Appendix. The equation of the l i n e was found to be Y = -0.6343 + 3.7683x. The slope of the l i n e was b = 3.7683 -1.15*20. Chi-square 1 0 = 24-472, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The heterogeneity f a c t o r was found to be 2.4472. The LD^Q was calculated as m *= 0.031 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.054 mgm. and 0.027 mgm. T o x i c i t y of d i e l d r i n to Musca domestica L., , (SES culture). Each point represents the mean of three kk r e p l i c a t i o n s . The data are recorded i n Table k of the Appendix. The equation of the l i n e was found to be Y = -0.6591 + 3.7922x. The slope was calculated as b = 3.7922 - 0.7k00. Chi-square 2 = l . k 8 , Indicating no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated as 0.031 mgm., with f i d u c i a l l i m i t s at the 95$ probab-i l i t y l e v e l of 0.037 mgm. and 0.028 mgm. Comparison of the data shows that the calculated slopes and equations of the two l i n e s are s i m i l a r . Therefore, the LDcjg value i s s i m i l a r . However, there i s a large difference i n the Chi-square values, and hence there w i l l be a difference i n the f i d u c i a l l i m i t s . The calculated l i m i t s f o r the i n d i v i d u a l r e p l i c a t e s are 0.05k and 0.027 mgm., while the l i m i t s f o r the mean m o r t a l i t i e s are 0.037 mgm. and 0.028 mgm. In l i n e s showing a high Chi-square value, i t would be impossible to calculate any l i m i t s whatsoever, using the i n d i v i d u a l r e p l i c a t e method. McLeod (19kk) has obtained s i m i l a r r e s u l t s . He concludes that "the method of using the mortality from each concentration i n each r e p l i c a t e f o r the c a l c u l a t i o n of the regression l i n e i s not to be advocated f o r comprehensive experiments. For one thing, the c a l c u l a t i o n i s considerably more laborious. Moreover, the increase of the Chi-square due to the use of many points about the regression l i n e was not i n any way compensated f o r by the increase i n the 45 number of degrees of freedom." He concluded that "when draw-ing the regression l i n e i n t o x i c i t y t e s t s , the method of p l o t t i n g the mortality secured from the t o t a l s of many re p l i c a t e s i s easier and gives a lower value of Chi-square than does the method of p l o t t i n g the i n d i v i d u a l values secured i n each r e p l i c a t e . This i s natural enough since the averaging of the r e s u l t s from a number of r e p l i c a t e s tends primarily to remove the e f f e c t s of extreme v a r i a t i o n i n some of them." The method of p l o t t i n g the mean m o r t a l i t i e s f o r c a l c u l a t i o n of the probit-regression l i n e has been adopted i n t h i s work. However, i n order to i l l u s t r a t e the range i n mortality between r e p l i c a t e s of one dosage, these figures have been included i n the p» column of the s t a t i s t i c a l tables. The mean mortality at each dosage l e v e l i s shown i n the column p. Corrections f o r natural mortality In the controls, using Abbott's formula (Abbott, 1925), have been made upon the i n d i v i d u a l r e p l i c a t e s before c a l c u l a t i o n of the mean. Hence each l i n e represents from four to seven dosage l e v e l s with three r e p l i c a t i o n s of 50 f l i e s at each dosage l e v e l . Bioassay of t e s t i n s e c t i c i d e s  Parathion T o x i c i t y of parathion to Musca domestica L., , (SES c u l t u r e ) . The s t a t i s t i c a l data are recorded i n Table 5 of the Appendix. Figure 6(a) shows the calculated l i n e . The equation of the l i n e was found to be Y = -l8.ij.035 + li*.3000x. 1*6 The slope was calculated as b = lii .3000 i 0.9110. Chl-square^ » 2.272, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated as m == O.Oij.3 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of O.Olj.5 and O.Olj.0 mgm. To x i c i t y of d i e l d r i n to Musca domestica L . , $ $ , (SES c u l t u r e ) . Table 6 of the Appendix shows the s t a t i s t i c a l c a l c u l a t i o n s . Figure 6(b) shows the probit-regression l i n e . The equation was calculated as Y = -2.lj.956 + li.6ij.10x. The slope of the l i n e i s b = ij..6ij.l0 - 0.5770. Chi-square2 • 7.637, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The LTJ^Q value was calculated as m = O . O I L I mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of O.Oljlf. mgm. and 0.038 mgm. To x i c i t y of parathion to Musca domestica L . , <f<f, (SES c u l t u r e ) . The data obtained are recorded i n Table 7 of the Appendix. Figure 6(a) shows the calculated l i n e . The equation of the l i n e was found to be Y = -6.2999 + 7.0638x. The slope was calculated as b = 7.0638 - 2.1j!j.00. Chl«-square2 = 9.707, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated as m = 0.0ij.0 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.077 and 0.028 mgm. To x i c i t y of d i e l d r i n to Musca domestiea L . , cfd1, (SES c u l t u r e ) . Table 8 of the Appendix shows the data recorded. The calculated l i n e i s shown i n Figure 6(b). The equation of the l i n e was found as Y = 3.1+J23 + 6.3039x. The slope was T O X I C I T Y O F P A R A T H I O N T O M U S C A D O M E S T I C A ( S E S . C U L T U R E ) 1 1 1 l l 12 1.4 1.6 1.8 2.0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 6A T O X I C I T Y O F DIELDRIN T O M U S C A D O M E S T I C A L (SE.S. C U L T U R E ) 1 1 I I i I 1.2 1.4 1.6 1.8 2.0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 6B 47 calculated as b = 6 . 3 0 3 9 - 0.4850. Chi-square^ = 1.742, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LDtjQ was calculated as m = 0.022 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0 . 0 2 6 mgm. and 0 . 0 1 9 mgm. EPN T o x i c i t y of EPN to Musca domestica L., , (SES c u l t u r e ) . The data obtained are recorded i n Table 9 of the Appendix. The calculated l i n e i s shown i n Figure 7(a). The equation of the l i n e was calculated as Y = -2.7345 * 7.6879x. Chi-square^ = 4-1.635, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The heterogeneity f a c t o r of 10.lj.09 was taken into consideration. The L D ^ Q w a s calculated as m = 1.014 mgm. F i d u c i a l l i m i t s could not be calculated since the g value exceeded 1 . T o x i c i t y of d i e l d r i n to Musca domestica L.,0$, (SES c u l t u r e ) . S t a t i s t i c a l analysis of the data obtained i s recorded i n Table 1 0 of the Appendix. The calculated p r o b i t -regression l i n e i s shown i n Figure 7(h). The equation was calculated as Y = -1.0722 + 3.5521+x. The slope of the l i n e i s recorded as b = 3.5524 - 1.4360. Chi-square^ = 33-775, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The LDcjo value was calculated to be m = 0.052 mgm. Taking the heterogeneity f a c t o r of 8.1+438 into consideration, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were calculated 48 as 0.057 and 0.01+2 mgm. T o x i c i t y of EPN to Musca domestica L . , OV, (SES c u l t u r e ) . The calculations f o r the data recorded are shown i n Table 11 of the Appendix. The probit-regression l i n e i s shown i n Figure 7(a). Y,= -8.9082 + 8.l599x was calculated as the equation of the l i n e . The slope was found to be b = 8.1599 - 0.9l+20x. Chi-square^ = 5.1+46, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q was calculated as m = 0.506 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.532 and 0.482 mgm. To x i c i t y of d i e l d r i n to Musca domestica L . , <f<f, (SES c u l t u r e ) . S t a t i s t i c a l analysis of the data recorded i s shown i n Table 12 of the Appendix. The probit-regression l i n e i s shown i n Figure 7(b). The equation was calculated as Y = 5.1983 + 7.0870x. The slope was calculated to be b = 7.0870 - 1.9970. Chi-square^ = 66.378, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q was calculated as 0.028 mgm. Taking the hetero-geneity f a c t o r of 16.595 into consideration, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were calculated to be 0.035 mgm. and 0.020 mgm. Endrin T o x i c i t y of endrin to Musca domestica L . , $ $ , (SES c u l t u r e ) . S t a t i s t i c a l analysis of the data i s recorded In Table 13 of the Appendix. The calculated l i n e i s shown i n Figure 8(a). The equation was calculated as Y = -6.5840 + TOXICITY OF EPN TO MUSCA DOMESTICA L. (S£.S CULTURE) 7.0 L LOC 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 7A TOXICITY OF DIELDRIN TO MUSCA DOMESTICA L (S.E.S. CULTURE) J I I I L. 1.2 1.4 1.6 1 8 2 . 0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 7B 49 8.l650x. The slope was found to be b = 8.1649 - 2.2838. Chi-square2 == 13.727, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ 0 was calculated as m = 0.262 mgra. Since both the hetero-geneity f a c t o r and the greatly increased t value (due to the small number of degrees of freedom) had to be taken into consideration, i t was impossible to calculate the f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l . T o x i c i t y of d i e l d r i n to Musca domestica L . , $ $ , (SES cu l t u r e ) . Table 11L of the Appendix shows the data obtained. The probit-regression l i n e i s shown i n Figure 8(b). The equation was calculated to be Y = 0.2383 + 3.0869x. The slope of the l i n e was found to be b = 3.0869 - 0.8650. Chi-squareg = 2.388, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q was calculated to be m = 0.035 mgm. F i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were calculated as 0.042 and 0.020 mgm. Tox i c i t y of endrln to Musca domestica L . , dVf, (SES cul t u r e ) . The data obtained are shown i n Table 15 of the Appendix. This calculated l i n e i s shown i n Figure 8(a). The equation was found to be Y = 1.5225 + 3.6l33x. The slope was calculated as b = 3.6133 - 0.6750. Chi-square 2 = O.78I, , Indicating no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q w a s found to have a maximum l i k e l i h o o d estimate of 0.092 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.118 mgm. and 0.070 mgm. 50 T o x i c i t y of d i e l d r i n to Musca domestica L . , < f e T , (SES c u l t u r e ) . The data obtained are recorded i n Table 16 of the Appendix. Figure 8(b) shows the probit-regression l i n e . The equation was calculated as Y = 0.9743 + 3.k66kx. The slope of the l i n e was found as b = 3.k66k - 0.7030. Chi-square 2 = 0.813, in d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q w a s calculated to be m = 0.015 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.020 mgm. and 0.010 mgm. A l d r i n T o x i c i t y of a l d r i n to Musca domestica L . , £ $ , (SES cu l t u r e ) . S t a t i s t i c a l analysis of the data recorded i s shown i n Table 17 of the Appendix. The calculated l i n e i s shown i n Figure 9(a). The equation was found to be Y = 3.5653 + k.7295x. The slope was calculated as b = 4.7295 - 1.5530. Chi-square^ = 13*395, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D C J Q was calculated to be m = 0.065 mgm. Since i t was necessary to take both the heterogeneity fa c t o r and the greatly increased t value into consideration, i t was impossible to calculate f i d u c i a l l i m i t s f o r the 95$ p r o b a b i l i t y l e v e l . T o x i c i t y of d i e l d r i n to Musca domestica L . , $ 9 , (SES c u l t u r e ) . The data obtained appear i n Table 18 of the Appendix. The calculated l i n e i s shown i n Figure 9(b). Y =. I.96OI + 3.928lx was found to be the equation of the l i n e . The slope was calculated to be b = 3.9281 - 1.6k20. Chi-square3 « 21.290, TOXICITY OF DIELDRIN T O MUSCA DOMESTICA L. C&E.S. C U L T U R E ) 1 1 1 l i 1.0 1 . 2 1.4 1 .6 1 . 8 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 6B 5 i i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated to be m = 0.059 mgm. Since g was greater than 1, f i d u c i a l l i m i t s could not be calculated. T o x i c i t y of a l d r i n to Musca domestica L.,cfcT, (SES cu l t u r e ) . The s t a t i s t i c a l analysis of the data recorded appears i n Table 19. Figure 9(a) shows the probit-regression l i n e . The equation was calculated as Y = 0.8kl7 + k.3132x. The slope of the l i n e was found to be b = k.3132 - 0.k500. Chi-square^ = 7-590, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The LD50 was calculated as m a 0.022 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.026 mgm. and 0.020 mgm. Tox i c i t y of d i e l d r i n to Musca domestica L . j C f d 1 , (SES c u l t u r e ) . The data obtained are recorded i n Table 20 of the Appendix. The calculated l i n e i s shown i n Figure 9(b). The equation was found to be Y = -0.6591 + 3-7922x. The slope of the l i n e was calculated as b = 3.7922 - 0.7k00. Chi-square2 = l.k8, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D C J Q was calculated as m = 0.031 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.037 mgm. and 0.028 mgm. Lindane T o x i c i t y of lindane to Musca domestica L . , £ $ , (SES c u l t u r e ) . Table 21 of the Appendix shows the data recorded. The probit-regression l i n e i s shown i n Figure 10(a). The T O X I C I T Y O F A L D R I N T O M U S C A D O M E S T I C A L. (&E.& C U L T U R E ) LOG IOOO X DOSE (TOTAL MILLIGRAMS) FIG. 9A T O X I C I T Y OF D I E LDR IN T O M U S C A D O M E S T I C A L. (S.E.S. C U L T U R E ) 1 1 1 i i 1 . 2 1.4 1 . 6 1 . 6 2 . 0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 9B 52 equation was calculated to be Y = 0.4902 • 2.81*26x. The slope of the l i n e was found to be b = 2.8426 - 0 .58l5. Chi-square^ = 8.099, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q w a s calculated to be 0.039 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of O.Oij.3 mgm. and 0.031 mgm. Tox i c i t y of d i e l d r i n to Musca domestica L.,$$, (SES cult u r e ) . The data obtained are recorded i n Table 22 of the Appendix. The calculated l i n e i s shown i n Figure 10(b). Y = -2.0355 + 3.7909x was calculated as the equation of the l i n e , while the slope was found to be b = 3.7909 * 1.1540. Chl-square^ = 19.388, Indicating s i g n i f i c a n t heterogeneity of the points about the l i n e . The LDcjg was calculated as m = 0.072 mgm., with f i d u c i a l l i m i t s of 0.632 mgm. and 0.040 mgm., at the 95$ p r o b a b i l i t y l e v e l . T o x i c i t y of lindane to Musca domestica L . , ( fcf , (SES cul t u r e ) . Table 23 shows the data recorded. The probit-regression l i n e i s shown i n Figure 10 (a). The equation was calculated to be Y = 2.701+2 + 2.1925x. The slope of the l i n e was found to be b = 2.1925 - 0.6550. Chi-square^ = 19.045, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The LDcjo was calculated as 0.011 mgm. Taking the heterogeneity f a c t o r of 4 « 7 6 l into consideration, the f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were c a l -culated to be 0.016 mgm. and 0.00073 mgm. 53 T o x i c i t y of d i e l d r i n to Musca domestica L.jCflcf, (SES c u l t u r e ) . The data are recorded i n Table 2k of the Appendix. The calculated l i n e i s shown i n Figure 10(b). The equation was calculated to be Y = -2.3710 + 5.05l2x. The slope of the l i n e was calculated as b = 5.0512 * 1.3350. Chi-square]^ = 35.933* i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D C J Q was calculated as m = 0.028 mgm. Taking the heterogeneity f a c t o r of 8.983 into consideration, the f i d u c i a l l i m i t s were calculated to be 0.035 mgm. and 0.021 mgm. Heptachlor T o x i c i t y of heptachlor to Musca domestica L . ( S E S c u l t u r e ) . Table 26 of the Appendix shows the data obtained. The probit-regression l i n e i s shown i n Figure 11(a). The equation was found to be Y = -1.7567 + k.6686x. The slope of the l i n e was calculated as b = 4.6686 + l .k890. Chi-square^ -30.187, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The LDcjo was found to be m = 0.030 mgm. Taking the heterogeneity f a c t o r of 7«5k7 into consideration, the f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were calculated as 0.035 mgm. and 0.001k mgm. Tox i c i t y of d i e l d r i n to Musca domestica L., (SES c u l t u r e ) . The data obtained are recorded In Table 27 of the Appendix. Figure 11 (b) shows the calculated l i n e . The equation was found to be Y = 3.6260 + 5.1l56x. The slope of the l i n e was calculated as b = 5.1156 - 0.56k0. Chi-square^ == T O X I C I T Y OF L I N D A N E TO MUSCA DOMESTICA L. (SE.S CULTURE ; 7 . 0 h LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. IOA TOXICITY OF DIELDRIN TO M U S C A DOMESTICA L (S.E.S. CULTURE) «5 k 1 . 2 1 . 4 1 . 6 1 . 8 2 . 0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 10 B 5k 5.079, Indicating no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated to be m = 0.0k8 mgm., with f i d u c i a l l i m i t s at the 95$ prob-a b i l i t y l e v e l of 0.050 mgm. and 0.0k6 mgm. Tox i c i t y of heptachlor to Musca domestica L . , o V , (SES c u l t u r e ) . The data obtained are recorded i n Table 27 of the Appendix. Figure 11(a) shows the calculated l i n e . The equation was found to be Y » 0.0531 + k.2703x. The slope of the l i n e was calculated as b = k.2703 * 0.7321. Chi-square^ • 10.993, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LD50 was calculated to be m = 0.01k mgm. Taking the heterogeneity f a c t o r of 3.66k into consideration, the f i d u c i a l l i m i t s were calculated as 0.017 mgm. and 0.010 mgm., at the 95$ p r o b a b i l i t y l e v e l . T o x i c i t y of d i e l d r i n to Musca domestica L . , O V , (SES cu l t u r e ) . Table 28 of the appendix shows the data obtained. The calculated l i n e i s shown i n Figure 11(b). The equation was found to be Y = 0.8812 + 3.1719x. The slope was calculated as b » 3.1719 * 0.k526. Chi-square^ = k.359, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the p r o b i t -regression l i n e . The LD50 was found to be 0.020 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.023 mgm. and 0.018 mgm. Calculation of t o x i c i t y index As has been previously noted, t o x i c i t y index may be represented i n the following manner: T O X I C I T Y O F H E P T A C H L O R T O M U S C A D Q M F S T I C A |_ (SES. C U L T U R E ) 7 . 0 L L O G 1000 X D O S E ( T O T A L M I L L I G R A M S ) F I G . II A T O X I C I T Y O F DIELDRIN T O M U S C A D O M F . S T I C A L (S.E.S C U L T U R E ) 7 . 0 L 1 . 2 1 .4 1 . 6 1 . 8 2 . 0 L O G 1000 X D O S E ( T O T A L M I L L I G R A M S ) F IG . 1  B 55 LDcfo of standard i n s e c t i c i d e T o x i c i t y index = — - x 100 L D ^ Q of test i n s e c t i c i d e The t o x i c i t y indices of the six i n s e c t i c i d e s tested above are shown i n Table IX. Table IX. Tox i c i t y indices of some i n s e c t i c i d e s . (Standard i n s e c t i c i d e : D i e l d r i n == 100). Insecticide Sex LD£ 0 test ( t o t a l mgm.) standard T o x i c i t y Index Parathion 99 ercr 0.043 O.Olj.0 0.041 0.022 95 55 EPN 99 orer l.OLk 0.506 0.052 0.028 5 6 Endrin 99 0.262 0.092 0.035 0.015 13 16 A l d r i n 99 era* 0.065 0.022 0.059 0.031 91 141 Lindane 99 eW 0.039 0.011 0.072 0.028 185 255 Heptachlor 99 0.030 0.014 0.048 0.020 160 143 Bioassay of DDT Toxicit y of DDT to Musca domestica L., gg , (SES cul t u r e ) . In preliminary experiments with DDT, r e s u l t s were i r r e g u l a r . Using pure p,p*-DDT, I t was impossible to obtain m o r t a l i t i e s over 50$, regardless of the concentration of DDT used. A composite dosage-mortality curve was constructed using f i f t e e n concentrations of DDT ranging from 7 mgm. to 200 mgm. The data obtained are recorded i n Table 29 of the Appendix. Figure 12 shows the probit-regression l i n e . The 56 equation was found to be Y = k.0882 + 0.3k68x. The slope of the l i n e was calculated as b = 0.3k68 - 0.1111. Chi-square^ =• kl.608, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The LD^ 0 was calculated as m = k26 mgm. Taking the heterogeneity fa c t o r into consideration (3.201), the f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were 1980 mgm. and 87 mgm. To x i c i t y of DDT to Musca domestica L., (fo", (SES c u l t u r e ) . A composite dosage-mortality curve was constructed using nine concentrations of DDT ranging from 15 mgm. to 200 mgm. Table 30 of the Appendix shows the data recorded. The calculated probit-regression l i n e i s shown i n Figure 12. The equation was calculated to be Y = k.1732 + 0.3630x. The slope of the l i n e was calculated as b = 0.3630 * 0.2122. Chi-squarey = 30.155, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LD50 was calculated as m = 190 mgm. Taking the heterogeneity factor of k.308 into consideration, i t was found to be impossible to calculate f i d u c i a l l i m i t s f o r the 95$ p r o b a b i l i t y l e v e l . The obvious assumption to the extraordinary data presented above would be that the f l i e s possessed a high degree of resistance to DDT. However, i n order to confirm t h i s assumption, and also to prove the technique, i t was necessary to bioassay the t o x i c i t y of DDT to these insects i n another manner. The method of t o p i c a l application was adopted. Application of the Insecticide was accomplished by means of ah "Agla" micrometer syringe consisting of a TOXIC ITY OF DDT TO MUSCA DOMESTICA L. RESIDUAL C O N T A C T APPLICATION (S.E.S. CULTURE) i.o 1.2 1.4 1.6 1.8 2.0 L O G D O S E ( T O T A L M I L L I G R A M S ) 2.2 F I G . 12 57 c a l i b r a t e d glass hypodermic syringe attached to a r i g i d holder by a micrometer screw head, which i n turn operated the plunger. The peripheral scale of the micrometer head was divided i n such a manner, that each graduation corresponded to a volume of 0.2 lambda. In order to f a c i l i t a t e application of a small amount of i n s e c t i c i d e , the standard hypodermic needle was replaced by a s p e c i a l l y made ground glass dropper, projecting at r i g h t angles to the syringe. The apparatus was clamped firm l y to a ri n g stand at a convenient height (Plate V i a ) . The test insects were reared i n exactly the same manner as described above. The f l i e s were anaesthetized using GO2 and cold. Ten f l i e s of each sex were used at each dosage l e v e l , with each test being r e p l i c a t e d at lea s t three times. In order to avoid injury, an eyedropper attached to a small vacuum pump, was used to hold the insects while applying the i n s e c t i c i d e . The DDT was dissolved i n a 95:5 benzene-mineral o i l solvent mixture. Pour-tenths of a lambda of DDT were applied to the pronotum of each f l y . Dosage was calculated In terms of t o t a l micrograms of DDT (Figure 13 a, b). Following treatment, the f l i e s were placed i n small "dixie* 1 cups covered with muslin. A 2" length of absorbent cotton dental r o l l saturated with a 10$ sugar solution was provided f o r food (Plate VIb). The f l i e s were placed i n an observation room maintained at 78 - 2 degrees P. Humidity was maintained at 75 * 5$. Mortality counts were Plate VI. (b) P l i e s contained i n observation chambers afte r t o p i c a l application of DDT. D O T C O N T E N T O F B E N Z E N E - M I N E R A L O I L ( 9 5 : 5 ) S O L U T I O N M I C R O G R A M S D D T / 0 . 4 L A M B D A S O L V E N T FIG. 13 A D D T C O N T E N T O F B E N Z E N E - M I N E R A L O I L ( 9 5 : 5 ) S O L U T I O N M I C R O G R A M S D D T / 0 . 4 L A M B D A S O L V E N T F I G . I3B 58 made a f t e r k8 hours. Only those insects showing no movement whatsoever were considered dead. DDT (Topical application) T o x i c i t y of DDT to Musca domestica L . , , (SES cul t u r e ) . A composite dosage-mortality curve was constructed using 19 concentrations of DDT ranging from 1 to 50 micrograms. The data obtained are recorded i n Table 31 of the Appendix. The probit-regression l i n e i s shown i n Figure l k . The equation of the l i n e was calculated as Y = k.k929 + 0.2505x. The slope was calculated as b = 0.2$0$ - 0.126k. Chi-squareTj = 21.083, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D ^ Q was found to be m = 10.5? micrograms. Since g was so large, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l could not be worked out. To x i c i t y of DDT to Musca domestica L.,cTcT, (SES cul t u r e ) . A composite dosage-mortality curve was constructed using eleven concentrations ranging from 1.0 to 50 micrograms. Table 32 of the Appendix shows the data obtained. The calculated l i n e i s shown i n Figure l k . The equation was calculated as Y = k . 2 2 3 5 + 0.5907x. The slope of the l i n e was calculated as b = 0.5907 * 0.2265. Chi-square^ = 2k.53k, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D ^ Q was calculated as m = 2.063 micrograms. F i d u c i a l l i m i t s could not be worked out. In order to es t a b l i s h the degree of resistance to DDT present In the S u f f i e l d culture, comparative tests were made on the Ottawa culture, which possessed a low tolerance T O X I C I T Y O F D D T T O M U S C A D O M E S T I C A L . T O P I C A L A P P L I C A T I O N ( S . E . S . C U L T U R E ) 1.2 1.4 1.6 1.8 2 .0 2 .2 2 .4 L O G 10 X D O S E ( T O T A L M I C R O G R A M S ) F I G . 14 2 . 6 59 l e v e l to DDT (Roadhouse, 1953; Fisher, 1952). A l l tests were ca r r i e d out as indicated above. T o x i c i t y of DDT to Musca domestica L., 0.$, (Ottawa c u l t u r e ) . The data obtained are recorded i n Table 33 of the Appendix. Figure 15 shows the calculated l i n e . The equation was found to be Y = -2.8951 + k . 2 l5 lx . The slope of the l i n e was calculated as b = k . 2 l 5 l * 0.7270. Chi-square^ = k .5kk, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LD^ 0 was calculated as m = 0.075 micrograms, with f i d u c i a l l i m i t s at the 95$ prob-a b i l i t y l e v e l of O.O83 micrograms and 0.06k micrograms. T o x i c i t y of DDT to Musca domestica L.,<jrV, (Ottawa c u l t u r e ) . Table 3k of the Appendix shows the data obtained. The probit-regression l i n e i s shown i n Figure 15. The equation was calculated to be Y = -0.k0k3 + 3.6l79x. The slope of the l i n e was calculated as b = 3.6179 * 0.877k. Chi-square^ = 12.132, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the l i n e . The LDtjQ was calculated as m = 0.031 micrograms. Taking the heterogeneity f a c t o r of 2.k26 into consideration, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were 0.037 micrograms and 0.026 micrograms. The r e s u l t s recorded above were obtained using the Ottawa culture of f l i e s reared through three generations at the S u f f i e l d Experimental Station. During t h i s time the l a r v a l period shortened from eight to f i v e days, and a f t e r transfer to the University of B r i t i s h Columbia, lengthened again to eight days. 60 The tests by t o p i c a l application with DDT were repeated with seventh generation f l i e s . The following r e s u l t s were recorded: T o x i c i t y of DDT to Musca domestica L . , 9 9 , (Ottawa c u l t u r e ) . The data obtained are recorded i n Table 35 of the Appendix. Figure 16 shows the calculated l i n e . The equation of the l i n e was calculated as Y = -5.ij.066 + 5.2907x. The slope was found as b = 5.2907 - 1.2360. Chi-square^ = 0.833, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The L D C J Q was calculated as m =5 0.099 micrograms. F i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were calculated as 0.103 micrograms and 0.074 micrograms. T o x i c i t y of DDT to Musca domestica L . , cTcT, (Ottawa c u l t u r e ) . Table 36 of the Appendix shows the data obtained. The calculated probit-regression l i n e i s shown i n Figure 16. The equation was calculated as Y = -ll.81j.69 + 8.7235x. The slope of the l i n e was found to be b = 8.7235 - l.ij.020. Chi-square^ = 2 . i j .H, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The L D C J Q was calculated as m = 0.085 micrograms, with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.091 micrograms and O.O78 micrograms. DDT (Residual contact application) Female f i f t h generation f l i e s were also bioassayed against DDT by residual contact a p p l i c a t i o n . T O X I C I T Y O F D D T T O M U S C A D O M E S T I C A L. T O P I C A L A P P L I C A T I O N ( O T T A W A C U L T U R E ) LOG IOOO X DOSE (TOTAL MILLIGRAMS) T O X I C I T Y OF D D T T O M U S C A D O M E S T I C A L. T O P I C A L A P P L I C A T I O N (OTTAWA CULTURE ) • I J 1 1.6 l.e 2 . 0 2 . 2 2 4 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 16 61 T o x i c i t y of DDT to Musca domestica L., g$, (Ottawa c u l t u r e ) . The data obtained are recorded i n Table 37 of the Appendix. The calculated probit-regression l i n e i s shown i n Figure 17. The equation was found to be Y = —7.82+14.6 + 10.4598x. The slope of the l i n e was calculated as b = 10.4598 * 2.8335. Chi-square^ = 16.500, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LD50 w a s calculated as m = 1.690 mgm. Taking the heterogeneity fa c t o r of 5.500 into consideration, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l were 2.393 mgm. and 0.274 mgm. The r e s u l t s of the tests with DDT are summarized i n Table X. Table X. Degree of resistance to DDT of the SES culture as compared to the Ottawa culture ( f i f t h generation) Culture Method Sex I»Dt;o Degree of . . resistance SES r e s i d u a l contact c « 426 mgm. 252x Ottawa re s i d u a l contact 1.690 mgm. SES t o p i c a l _ 10.570 micrograms I I L L X Ottawa t o p i c a l 99 0.075 micrograms SES t o p i c a l j y i 2.063 micrograms 66.5x Ottawa t o p i c a l 0.031 micrograms Bioassay of lindane and heptachlor Lindane and heptachlor were bioassayed with f i f t h generation f l i e s of the Ottawa culture, by res i d u a l contact ap p l i c a t i o n . The r e s u l t s are recorded below: T o x i c i t y of lindane to Musca domestica L..yy. (Ottawa c u l t u r e ) . The data obtained were recorded i n Table 38 of the 62 Appendix. The calculated l i n e i s shown i n Figure 18. The equation was found to be Y = -3.1118 + 6.5603x. The slope of the l i n e was calculated as b = 6.5603 - 1.0337. Chi-square2 = 5.68k, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The was calculated as m = 0.017 mgm., with f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l of 0.018 mgm. and 0.016 mgm. Toxi c i t y of heptachlor to Musca domestica L . , g $ , (Ottawa c u l t u r e ) . Table 39 of the Appendix shows the data obtained. The calculated l i n e i s shown i n Figure 18. The equation was determined to be Y = -1.5928 + k.6366x. The slope of the l i n e was calculated as b = k.6366 * 1.7236. Chi-square2 = 11.063, i n d i c a t i n g s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . The LDcjo w a s calculated to be m = 0.026 mgm. The heterogeneity f a c t o r was 5.532. Due to the large g value, f i d u c i a l l i m i t s at the 95$ p r o b a b i l i t y l e v e l could not be calculated. TOXICITY OF DDT T O M U S C A DOMESTICA L. R E S I D U A L C O N T A C T APPLICATION (OTTAWA CULTURE) 2 . 8 3 . 0 3 . 2 3 . 4 3 . 8 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 17 TOXICITY OF LINDANE AND HEPTACHLOR T O MUSCA DOMESTIC A L. (.OTTAWA CULTURE) • I I 1 1 1 1 . 0 1 . 2 1 4 1 . 6 1 . 8 LOG I000 X DOSE (TOTAL MILLIGRAMS) FIG. 18 63 VI. DISCUSSION OF RESULTS  Rearing methods The r e s u l t s obtained by t o p i c a l a p p l i c a t i o n of DDT with the Ottawa culture demonstrate the necessity of a standardized rearing method. Roadhouse i n 1949 and 195-1 determined the LD^Q values f o r female f l i e s of the Ottawa culture as 0.037 micrograms and O.Okl micrograms. March and Metcalf (1949), using the same culture, estimated the LD^ 0 value as 0.033 micrograms. During the period 1951 "bo 1955, t h i s culture was never exposed to DDT contamination (McLeod, W.S., personal communication), and although the usual short-ening of the l i f e cycle took place, the f l i e s were vigorous and uniform. Yet, a f t e r rearing through only three gener-ations at the S u f f i e l d Experimental Station, the LD^Q value was experimentally determined as 0.075 micrograms. The f l i e s used by both Roadhouse and March and Metcalf were reared according to the standardized Peet-Grady method, i n comparison to the SES culture, which was reared upon canned horsemeat. I t was noted that the f l i e s reared i n the l a t t e r manner were larger and much more vigourous. An increase of the LD^Q value might, therefore, be expected. Furthermore, n u t r i t i o n might have some e f f e c t upon the physio-l o g i c a l responses of the Insect, bringing about an increase i n the tolerance l e v e l not a l l i e d with the increase i n size and vigour. I t would appear that i n the standardization of test insects f o r bioassay, the r e l a t i o n s h i p of n u t r i t i o n to 61+-t o x i c i t y i s a matter of some importance, and worthy of further i n v e s t i g a t i o n . The data presented i n Figures 15 and 16 f o r the t o x i c i t y of DDT to f i f t h and seventh generation f l i e s of the Ottawa culture show an Increase In the I J D ^ Q value from 0.075 to 0.099 micrograms f o r females, and 0.031 to 0.085 micrograms f o r males. Although these insects were c a r e f u l l y reared under the same conditions of temperature and humidity, humidity was controlled i n a d i f f e r e n t manner. At the Suf-f i e l d Experimental Station, the cages were covered with screen and kept i n a rearing room under controlled conditions. At the University of B r i t i s h Columbia, i t was necessary to control humidity with a saturated BaGl2 solution placed within the cage covered with p l a s t i c sheeting. Sun (191+7) has pointed out that the oxygen-carbon dioxide r e l a t i o n s h i p i n close quarters would a f f e c t the development of the Insect culture. Experimentally he was able to determine that the simultaneous fumigation of Sitophilus granarlus L. popul-ations of increasing density resulted i n less mortality. He correlates t h i s r e s u l t with r e l a t i v e a c t i v i t y and r e s p i r a t i o n rates. I t Is f e l t that the s i m i l a r r e s u l t s shown i n Figures 15 and 16 may be due to the excess CX>2 within the cages covered with p l a s t i c sheeting. Stra i n of insects The comparative data shown f o r the t o x i c i t y of DDT upon the SES and Ottawa cultures serve to point out the 65 importance of the s t r a i n of f l y selected. Test with various i n s e c t i c i d e s (Table IX) yielded r e s u l t s i n l i n e with those found by other investigators. However, tests with DDT, which were l e f t u n t i l l a s t , indicated that the females of the SES st r a i n were llj.lx and the males 76x as r e s i s t a n t to DDT as the Ottawa s t r a i n , when compared by t o p i c a l application (Table X). I t Is suggested that i n the s e l e c t i o n of the s t r a i n of t e s t insect, comparative tests f o r tolerance l e v e l s to DDT, and perhaps lindane, or chlordane be made, since resistance seems to develop most e a s i l y to these i n s e c t -i c i d e s (March and Met c a l f , 19li9; Busvine, 1954)' Furthermore, cross-resistance between compounds within the chlordane group and also compounds within the DDT group may occur i n some strains of f l i e s , but not i n others (Busvine, 19$k» March and Metcalf, 19l|.9; Bruce and Decker, 1950). Hence I t i s necessary that at no time during i t s l i f e h i s t o r y must the culture be subjected to i n s e c t i c i d e contamination, and that t h i s l i f e h i s t o r y be available to the investigator. Insect strains also show differences i n tolerance l e v e l s not due to resistance. For example, the L D C J Q values f o r heptachlor were 0.030 mgm. f o r the SES culture and 0.026 mgm. f o r the Ottawa culture. S i m i l a r l y , the L D ^ Q v*Iues f o r lindane were 0.039 mgm. for the SES culture and 0.017 f o r the Ottawa culture. Approximate r e s u l t s with parathion indicated that the tolerance l e v e l was s i g n i f i c a n t l y higher f o r the Ottawa culture as compared to the SES culture (XIL) . 66 These r e s u l t s would be i n l i n e with those found by other investigators. Therefore, the s t r a i n of insect selected i s a f a c t o r of considerable importance I f comparable r e s u l t s are to be obtained. Bioassay technique Concentration of i n s e c t i c i d e The method of dipping the substratum into an i n s e c t i c i d e solution of known concentration developed by Proverbs and Morrison (1947) was simple and f a i r l y adequate. The one serious objection i s the possible v a r i a b i l i t y i n the amount of i n s e c t i c i d e picked up on the f i l t e r paper. By substituting woven f i b e r g l a s s c l o t h , with non-absorbent properties, as the substrate, t h i s method would be adequate. However, an estimation of the t o t a l amount of i n s e c t i c i d e upon the c l o t h would give the Investigator a clearer idea regarding the general t o x i c i t y , and f o r t h i s reason, dosages have been reported i n terms of t o t a l milligrams on the cloth. I t must be emphasized, however, that by no means i s a l l of the i n s e c t i c i d e available to the insects, and the data obtained are e n t i r e l y upon a comparative ba s i s . The solvent or solvent mixture selected has an Important e f f e c t upon the t o x i c i t y of an i n s e c t i c i d e f o r three reasons. F i r s t l y , the type of solvent determines the quantity of i n s e c t i c i d e which w i l l adhere to the c l o t h . For example, s l i g h t l y l e s s Insecticide i s deposited upon the substratum i f acetone i s used i n place of benzene. Secondly, the type of solvent and method of r e c r y s t a l l i z a t l o n determine 67 the c r y s t a l structure of the residue, causing changes i n t o x i c i t y (Mcintosh, 191+7, 191+9), Thirdly, i f an o i l solution Is used, t o x i c i t y increases, since the o i l , which i s chemi-c a l l y related to the outer, o i l y , protective layer of the c u t i c l e , dissolves the i n s e c t i c i d e and aids i n penetration. Furthermore, Stringer (191+9) has pointed out that the b i o -assay of DDT f o r residual contact t o x i c i t y i s d i f f i c u l t i f the compound i s used i n the c r y s t a l l i n e form. Therefore, a small amount of l i g h t weight mineral o i l was added to the benzene, which had f i r s t been selected as the solvent ( 5 > : 9 $ ) . Since the concentration curves shown i n Figures 1 and 2 are constructed by colorimetric analysis of known di l u t i o n s of A n i l i n e Green dye i n the solvent mixture, i t i s necessary to assume that the amounts of i n s e c t i c i d e and dye picked up on the c l o t h are proportionate. The s e l e c t i o n of t h i s dye was unfortunate i n that i t was only soluble up to 2\%, A f t e r the appearance of DDT resistance, concen-trat i o n s up to 20$ were necessary. Hence, i t was necessary to assume that the straight l i n e r e l a t i o n s h i p held at higher concentrations i n the same manner as at lower concentrations, and the graph shown i n Figure 3a i s drawn by i n t e r p o l a t i o n . Elimination of fumigant e f f e c t The data obtained i n Tables V and VI indicate that i t i s possible to eliminate fumigant e f f e c t i f the f l i e s are not subjected to the exhaust fumes. This can be brought about by l i m i t i n g the f l i e s to the upper surface of the substratum, while the fumes are drawn o f f below. I t i s 68 necessary that a cer t a i n rate of evacuation be set f o r each i n s e c t i c i d e showing fumigant properties. Elimination of the fumigant e f f e c t brings about a corresponding decrease i n mortality (Table VII). I t has been argued that t h i s decrease i n mortality i s due both to the elimination of the fumigant e f f e c t and v o l a t i l i z a t i o n of the i n s e c t i c i d e , during the four hour exposure period. However, the data shown i n Table VIII indicate that even with an additional two hour evacuation period p r i o r to the four hour exposure period, there i s no s i g n i f i c a n t decrease i n mortality. Therefore, i t may be concluded that the four hour period of evacuation i s not c r i t i c a l as regards v o l a t i l i z a t i o n of the i n s e c t i c i d e . T o x i c i t y index Many previous investigators have u t i l i z e d the LDej0 value as the basis of comparison f o r t o x i c i t y of the various i n s e c t i c i d e s . Reference to Table IX indicates that upon the basis of L D C J Q values, the order of t o x i c i t y (from highest to lowest) would be: Females: heptachlor, lindane, parathion, a l d r i n , endrin, and EPN. Males: lindane, heptachlor, a l d r i n , parathion, endrin, and EPN. However, a summary of the LDcjg values f o r concurrent runs with d i e l d r i n shows considerable range (Table XI). 69 Table XI. LD^ Q v a r i a t i o n of d i e l d r i n . L D C J Q (mgm.): females L D ^ Q (mgm.): males 0.0k6 0.026 O.Olj.1 0.022 0.052 0.028 0.035 0.015 0.059 0.031 0.072 0.028 0.0i|.8 0.020 Hence, the range varies from 0.035 to 0.072 mgm. f o r females, and 0.015 to 0.031 f o r males. Obviously, i t would be inaccurate to compare the LDtjg of one i n s e c t i c i d e with another i f such a va r i a t i o n i s possible. The basic assumption i n the use of t o x i c i t y index i s that "any change i n the LD50 of the test sample i s accom-panied by a proportional change i n the LD^ Q of the standard i n s e c t i c i d e . " (Sun, 1950). However, these tests must be carried out at the same time, with a homogeneous population of insects, i f the. tests are to be placed upon an accurate comparative basis. Prom the t o x i c i t y indices recorded i n Table IX, the order of t o x i c i t y from highest to lowest would be: lindane, heptachlor, parathion, a l d r i n , endrin, and EPN, f o r both males and females. I t i s of interes t to note that the t o x i c i t y index varies considerably f o r males and females i n a l l cases, other than EPN. Evidence i n favour of si m i l a r t o x i c i t y indices with d i f f e r e n t s t r a i n s of f l i e s i s meagre, although Sun has determined them to be sim i l a r i n at le a s t one case. In the experimental data presented here, there i s l i t t l e apparent 70 s i m i l a r i t y of the t o x i c i t y indices of the i n s e c t i c i d e s tested against the SES culture, as compared to the o r i g i n a l r e s u l t s obtained by Sun. Sun, of course, used a mixed population consisting of equal numbers of males and females. However, the average of the t o x i c i t y indices obtained f o r male and female against one i n s e c t i c i d e should be si m i l a r to those obtained by Sun. This, however, i s not the case. For example, Sun obtained a t o x i c i t y index of i+9 - 7.k f o r a l d r i n , while the "average" t o x i c i t y index obtained f o r a l d r i n on the SES culture was 116. The r e s u l t s with parathion also bear out th i s conclusion. S i m i l a r l y , the t o x i c i t y index may be expected to vary i f d i f f e r e n t species of insects are used. Hence, a standardized bioassay technique would involve the determin-ation of t o x i c i t y indices f o r several common i n s e c t i c i d e s against several inseet species, i n order to f i r s t e s t a b l i s h a basis of comparison. Afte r the standardization of these common i n s e c t i c i d e s , i t would be possible to bioassay test samples, and compare the t o x i c i t y index with those obtained f o r the common i n s e c t i c i d e s . The data given here are presented only to demonstrate the technique, with no intention that they be accepted as a series "standard" t o x i c i t y i ndices. Relative s u s c e p t i b i l i t y of males and females The experimental data show conclusively that males are generally more susceptible than females. As a general 71 r u l e , the highest concentration needed i n the construction of the dosage-mortality curve f o r male f l i e s was equivalent to the lowest concentration needed f o r female f l i e s . This s i m p l i f i e d the amount of approximate t e s t i n g necessary. Although the probit-regression l i n e s f o r male and female are included upon the same axis i n the figures, i t should be emphasized that comparative observations must not be made, other than i n a very general sense, since these graphs were constructed at d i f f e r e n t times, with d i f f e r e n t generations of insects. The only accurate comparison as regards t o x i c i t y towards males and females i s that of t o x i c i t y index. P a r a l l e l i s m of the probit-regression l i n e s I t i s generally accepted that the probit-regression l i n e s constructed f o r male and female, with a given I n s e c t i -cide are p a r a l l e l . However, from the data presented above, I t i s obvious that there i s considerable v a r i a t i o n i n the slope of the probit-regression l i n e of d i e l d r i n , ranging from 3.0869 to 6.3825 f o r females and 3-1719 to 7.5594 f o r males, with an average slope of 4*3431 f o r females, and 5.2239 f o r males. Here again, I t must be emphasized that accurate comparisons cannot be made unless the experiments are run at the same time, using a "standardized" technique, with homogeneous populations of insects. Sun has recommended that the dosage-mortality l i n e be constructed with no le s s than four dosage l e v e l s f o r the test i n s e c t i c i d e and three f o r the standard i n s e c t i c i d e . I t was f e l t that t h i s small number of dosages was inadequate, 72 and i n a l l cases, the l i n e s were constructed using from four to seven dosage l e v e l s . An attempt was made to set these concentrations such that mortality did not exceed 90$ or f a l l below 20$, as recommended by Sun. In the bioassay of parathion against male f l i e s (Figure k), however, the m o r t a l i t i e s obtained are extremely low. Since there i s an extreme prolongation of the sigmoid curve at such a low l e v e l , i t i s f e l t ,that a decrease i n slope may be brought about. This, of course, would a l t e r the t o x i c i t y index. A s i m i l a r s i t u a t i o n exists i n the case of d i e l d r i n against endrin (Figure 8 b ) , where two of the four concentrations used yielded m o r t a l i t i e s over 90$. I t i s suggested that a l l tests should be c a r r i e d out between the 20 - 90$ range, since i t i s f e l t that the true comparative basis i s not established i f the range of one i n s e c t i c i d e i s extremely high, while that of the comparative i n s e c t i c i d e i s extremely low. Resistance of the SES culture to DDT I t has been shown i n Table X that by t o p i c a l application, females of the SES culture were l k l x and males 6?x as r e s i s t a n t compared to f i f t h generation f l i e s of the Ottawa culture. By residual contact application, females of the SES culture were determined to be 2$2x as r e s i s t a n t as f i f t h generation f l i e s of the Ottawa culture. This d i s -crepancy serves to demonstrate the necessity of describing i n d e t a i l the bioassay technique employed. I t would appear 73 l o g i c a l that resistance figures would be higher by r e s i d u a l contact application since the amount of i n s e c t i c i d e which an insect can pick up must have a l i m i t beyond which t o x i c i t y w i l l not increase i n proportion to concentration. But, by t o p i c a l application, a known amount i s applied and hence t o x i c i t y w i l l increase i n proportion to the concentration. I t i s d i f f i c u l t to explain the resistance developed by the SES culture to DDT. At no time during the preceding four and one-half years that the culture was reared at SES were any symptoms of DDT resistance noted. The culture was extremely vigourous and healthy at a l l times and was reared under the best possible conditions. I t i s suggested that the resistance developed over a period of time due to exposure to l e t h a l amounts of DDT i n the a i r . I t would appear that the T3DT was v o l a t i l i z e d i n steam from the mixing room at the rear of the building, and passed through the a i r -conditioning system. This theory i s substantiated to some extent by the changes i n l i f e cycle undergone by the Ottawa culture during the few generations that i t was reared at SES. The adult f l i e s would l i v e no longer than 10 days. However, symptoms of DDT poisoning were not apparent. I t i s further postulated that the shortening of the l a r v a l period from seven to f i v e days was caused by exposure to DDT rather than to the n u t r i t i o n change, since upon removal to UBC, the length of the l a r v a l period increased to eight days over two generations. I Ik Homogeneity of data The qu a l i t y of a bioassay technique can be judged to a considerable extent by the homogeneity of the data obtained. A summary of the pertinent data obtained i s shown i n Table XII. A further summary of the data obtained f o r DDT i s shown In Table XIII. Throughout the s t a t i s t i c a l analysis, I t was noted that the size of the Chi-square value generally increased with the size of the sample tested. For example, i n Figure 11+ the v a r i a t i o n of the points about the pr o b i t -regression l i n e i s extreme. The Chi-square-jj value was 21.083, i n d i c a t i n g no s i g n i f i c a n t heterogeneity of the points about the l i n e . The corresponding Chi-squareq value f o r males was 2\+.534, i n d i c a t i n g some s i g n i f i c a n t heterogeneity of the points about the l i n e . In the above examples, each point on the l i n e represents the average mortality of three r e p l i c a t e s of 10 f l i e s each. However, i n the case of Figure 12, where each point represents 150 f l i e s , there would appear to be much les s v a r i a t i o n about the probit-regression l i n e and hence a lower Chi-square value would be expected. However, t h i s value Is found to be Chi-square-^ = ij.1.608 f o r females, and 30.155 f o r males. Hence, s i g n i f i c a n t heterogeneity i s shown i n both cases. Other investigators have reported si m i l a r r e s u l t s . Potter (I9I4.I) secured s a t i s f a c t o r y Chi-square values using from f i f t y to one hundred insects i n the determination of each point on the regression l i n e . However, Morrison (1943), 75 Table XII. Summary of data obtained Insecticide Sex Chi-square Degrees of freedom Parathion female 2.272 5 (nsh) male 9.707 2 (nsh) D i e l d r i n female 7.637 2 (nsh) male 1.742 4 (nsh) EPN female 41.635 4 (sh) male 5.446 4 (nsh) D i e l d r i n female 33.775 4 (sh) male 66.378 4 (sh) Endrin female 13.727 2 (shj male 0.781 2 (nsh) D i e l d r i n female 2.388 2 (nsh) male 0.813 2 (nsh) A l d r i n female 13.395 3 (sh) male 7.590 2 (nsh) D i e l d r i n female 1.480 2 (nsh) male 21.290 2 .(sh) Lindane female 8.099 4 (nsh) male 19.045 4 (sh). D i e l d r i n female 19.388 4 (shj male 35.933 4 (sh) Heptachlor female 30.187 4 (sh) male 10.993 4 (sh) D i e l d r i n female 5.079 4 (nsh) - male 4.359 4 (nsh) (* sh = s i g n i f i c a n t heterogeneity} nsh geneity.) i n Tables 1-28 (Appendix). LD^0(mgm) F i d u c i a l l i m i t s - (mgm) 0.043 0.045 & 0.040 0.040 0.077 & 0.028 0.041 0.044 & 0.038 0.022 0.026 & 0.019 1.014 0.506 0.532 & 0.482 0.052 0.057 & 0.042 0.028 0.035 & 0.020 0.262 • 0.092 0.118 & 0.070 0.035 0.042 & 0.020 0.015 0.020 & 0.010 0.065 0.022 0.026 & 0.020 0.031 0.037 & 0.028 0.059 0.039 0.043 & 0.031 0.011 0.016 & 0.00073 0.072 0.632 & 0.040 0.028 0.035 & 0.021 0.030 0.035 & 0.0014 0.014 0.017 & 0.010 0.048 0.050 & 0.046 0.020 0.023 & 0.018 • no s i g n i f i c a n t hetero-uslng eight to ten r e p l i c a t e s of 150 f l i e s each, found hetero-geneity i n a l l cases except one. McCleod (1944) obtained s i m i l a r r e s u l t s . Moore and B l i s s (1942) obtained high Chi-square values i n thirteen out of twenty-one experiments using large numbers of 76 insects. In explanation they stated: "So large a number reduced the sampling error In estimating the percentage of dead aphids ... to a r e l a t i v e l y small value and exposed the heterogeneity of the four points about t h e i r computed curve." Although t h i s explanation i s acceptable, i t would seem that further investigation into the Chi-square value i s necessary i f a s a t i s f a c t o r y measure of homogeneity i s to be obtained. Accepting B l i s s 1 c r i t e r i o n f o r homogeneity as the 5$ p r o b a b i l i t y l e v e l , eleven of the twenty-four tests tabulated i n Table XII show s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . Therefore, cor-r e c t i o n must be made by applying the heterogeneity f a c t o r (Finney, 1952). In the c a l c u l a t i o n of the f i d u c i a l l i m i t s , both the heterogeneity f a c t o r and the corresponding t value must be taken into consideration. Finney states: "When very few degrees of freedom are available f o r estimating the heterogeneity fa c t o r , the corresponding t value ... becomes large i n order to allow f o r the u n r e l i a b i l i t y of the hetero-geneity factor and consequently the f i d u c i a l l i m i t s are widely spaced." Hence, i t i s f e l t that l i t t l e emphasis should be l a i d upon the f i d u c i a l l i m i t s obtained i n an experiment showing s i g n i f i c a n t heterogeneity, i f the degrees of freedom are less than three. Figures 8a (females) and 9b (males) are examples i n which i t i s impossible to c a l -culate the f i d u c i a l l i m i t s f o r the above reasons. I t i s accepted, however, that a p p l i c a t i o n of the heterogeneity factor to data with three or more degrees of freedom, i s quite acceptable. The f i d u c i a l l i m i t s obtained 77 throughout the remainder of the experiments were narrow, with the exception of EPN, i n which the Chi-square^ value = 41.635 made i t impossible to calculate the f i d u c i a l l i m i t s . Table XIII. Summary of data obtained f o r DDT. Culture Sex Chi-square Degrees of freedom L D ^ Q (mgm) F i d u c i a l l i m i t s (mgm) SES (residual) female male ij.1.608 30.155 13 7 (sh) (sh) 426.000 190.000 1980 & 87 SES (topical) female male 21.083 24.534 17 9 (nsh) (sh) 10.570 2.063 Ottawa (5th gen.) (topical) female male 4.544 12.132 5 5 (nsh) (sh). 0.075 0.031 0.083 & 0.06, 0.037 & 0.02 Ottawa (7th gen.) (topical) female male 0.833 2.411 it (nsh) (nsh) 0.099 0.085 0.103 & 0.07. 0.091 & 0.07 Ottawa (5th gen.) (topical) female 11.063 2 (sh) 0.026 -------Although i t i s generally accepted that males are more variable than females (March and Metcalf, 1949), the data shown i n the preceding tables do not corroborate t h i s conclusion. The l i m i t s obtained f o r males and females are equally as good. I t has been suggested that the l i n e a r r e l a t i o n s h i p i s inadequate as a measure of the probit - l o g dosage mortality r e l a t i o n s h i p . This view has been put forward by several authors (Wadley and Su l l i v a n , 1943; Morrison, 1943). Other authors have defended the method (Moore and B l i s s , 1942). In the preceding experiments, inspection of the diagrams 78 indicated no s i g n i f i c a n t departure from the straight l i n e r e l a t i o n s h i p , i n either a convex or concave manner. In t h e i r discussion of heterogeneity, Moore and B l i s s state: "Conclusions depend primarily, therefore, upon the differences between curves and consistency of these differences rather than inferences drawn from t h e i r degree of i n t e r n a l homogeneity." The L D ^ Q , upon which the t o x i c i t y Index i s calculated i s not altered by the i n t e r n a l homo-geneity of the l i n e and, hence, comparative data may be drawn from i t with considerable certainty. S e n s i t i v i t y of the technique One prerequisite of an accurate bioassay technique i s a high degree of s e n s i t i v i t y . The experimental data f o r lindane (Table 27 of the Appendix) show that the technique i s accurate to 0.01 mgm. I t i s possible that even smaller quantities of i n s e c t i c i d e could be bioassayed, i f necessary. The s e n s i t i v i t y of the technique i s demonstrated i n another manner. Prom the r e s u l t s obtained with DDT (Figures 12 and lk) to f l i e s of the SES culture, a very s i g n i f i c a n t amount of heterogeneity of the points about the probit-regression l i n e i s indicated. This v a r i a t i o n i s e n t i r e l y c h a r a c t e r i s t i c of the s t r a i n of f l i e s (Hurtig, H., personal communication). In contrast, the r e s u l t s obtained with DDT on the Ottawa culture of f l i e s show no s i g n i f i c a n t heterogeneity of the points about the probit-regression l i n e . Therefore, with t h i s technique, i t i s possible to demonstrate heterogeneity or homogeneity of response of an insect s t r a i n 79 to a given i n s e c t i c i d e . A further i n d i c a t i o n of the s e n s i t i v i t y of the technique i s the f a c t that i t was possible to q u a l i t a t i v e l y determine that the SES culture was r e s i s t a n t to DDT, and to quantitatively e s t a b l i s h the order of resistance, as com-pared to the Ottawa culture. S t i l l another Indication of the s e n s i t i v i t y of the technique was the quantitative d i f f e r e n t i a t i o n of the t o x i c i t y of DDT to f i f t h and seventh generation f l i e s of the Ottawa culture. A technique which can define differences such as these i n a quantitative fashion may be extremely u s e f u l . 80 VII. CONCLUSIONS A simple, yet adequate, bioassay technique d i s -tinguishing residual contact from fumigant t o x i c i t y i s described. Insecticide concentration was calculated i n t o t a l milligrams upon the f i b e r g l a s s c l o t h substratum. Using a 9$i$ benzene-mineral o i l mixture as solvent f o r the i n s e c t i c i d e s , the observed lower l i m i t s of detection were 0.01 mgm. The technique i s , perhaps, s t i l l more sen s i t i v e , but at present i s e n t i r e l y adequate f o r even the most toxic i n s e c t i c i d e s . The usefulness of the method i s not r e s t r i c t e d to distinguishing between r e s i d u a l contact and fumigant e f f e c t , but can be quickly and accurately used to q u a l i t a t i v e l y demonstrate resistance to a given i n s e c t i c i d e i n a species or s t r a i n of species of insect, and to quan t i t a t i v e l y e s t a b l i s h the order of t h i s resistance. The technique i s also u s e f u l i n demonstrating heterogeneity of homogeneity of response to a given i n s e c t -i c i d e by an insect species or s t r a i n of insect species. I t would, therefore, be extremely u s e f u l f o r quickly and simply demonstrating, with a minimum of equipment, the presence of absence of heterogeneity of response to a given i n s e c t i c i d e , or formulation thereof, i n a sample of a wild population c o l l e c t e d i n the f i e l d . 81 VIII. BIBLIOGRAPHY Abbott, W.S. 1925. A method of computing the effectiveness of an i n s e c t i c i d e . Jour. Econ. Ent., 18 (2): 265-67-Barnes, Sarah. 1945. The res i d u a l t o x i c i t y of DDT to bed-bugs (Cimex l e c t u l a r i s ) . B u l l . Ent. Res., %6 (3): 273-82. B l i s s , C.I. 1934a. The method of probits. Science, 79: 38-39. . 1934b. The method of probits - a correction. Science, 22s 409-10. • 1935a. The c a l c u l a t i o n of the dosage-mortality curve. Ann. App. B i o l . , 22: 134-67. . 1935b. The comparison of dosage-mortality data. Ann. App. B i o l . , 22: 307-33-. 1937. The c a l c u l a t i o n of the time-mortality curve. Ann. App. B i o l . , 2k. (4): 815-52. Bruce, W.N. and G.C. Decker. 1950. House f l y tolerance f o r i n s e c t i c i d e s . Soap and Sanit. Chem., 26 (3): 122-25, 145-47- ~"~ Busvlne, J.'R. 1954- Houseflies r e s i s t a n t to a group of chlorinated hydrocarbon i n s e c t i c i d e s . Nature, 17k (4434): 783-85. Campbell, F.L. and R.S. Filmer. 1929- A quantitative method of estimating the t o x i c i t y of stomach poison Insecticides. Trans. IV Intern. Congr. Entomol. (1928): 523-33-. 1932. Preliminary experiments on the t o x i c i t y of ce r t a i n coal tar dyes f o r the silkworm. Jour. Econ. Ent., 2j>: 905-17. • and W.N. Su l l i v a n . 1938. A metal turntable method f o r comparative tests of l i q u i d spary contact Insecticides. Soap. lk_(6): 119-Cotton, R.T. 1943. Testing fumigants. Pub. Am. Assoc. Advance. S c i . , 20: 144-51. Dakshinamurity, S. 1948. The common housefly, Musca  domestica L.. and i t s behaviour to temperature and humidity. B u l l . Ent. Res., 9^_: 339-57. 82 Finney, D.J. 1952. Probit Analysis. A s t a t i s t i c a l treatment of the sigmoid response curve. Cambridge Univ. Press, Second Ed. Fisher, R.W. 1952. The importance of the locus of ap p l i c a t i o n on the effectiveness of DDT f o r the housefly, Musca domestica L. (Diptera:Muscidae). Canad. Jour. Zool., 10: 254-66. Gaddura, J.H. 1933. Reports on b i o l o g i c a l standards. I I I . Methods of b i o l o g i c a l assay depending on a quantal response. Spec. Rep. Ser. Med. Res. Council, London, 183. Granett, P. and H.L. Haynes. 1944' Improved methods of rear-ing Aedes aegypti mosquitoes f o r use i n repellent studies. Proc. New Jersey Mosq. Exterm. Assoc., ^1: 161-68. Hamman, R.E. 1948. Factors involved i n poisoning German roaches by exposing them to surfaces treated with c h l o r i n -ated hydrocarbons. Jour. Econ. Ent., i j l (3): 5l6-17« Hansberry, R. and S.F. Chiu. 1940. Presentation of time-dosage-mortality data by three dimensional graphs. Jour.' Econ. Ent., 21 ( D * 139-41 •. ., W.W. Middlekauff and L.B. Norton. 1940. T o x i c i t y of nicotine administered i n t e r n a l l y to several species of insects. Jour. Econ. Ent., 21 : 511-17. . 1943* Testing stomach i n s e c t i c i d e s . Publ. Am. Assoc. Advance. S c i . , 20: 85-94' Heal, R.E. and H. Menusan, J r . 1948. A technique f o r the bloodstream i n j e c t i o n of insects and i t s application i n tests of certa i n i n s e c t i c i d e s . Jour. Econ. Ent., 41: 535-43. Hoskins, W.M., J.M. Witt, and W.R. Erwin. 1952. Bioassay of 1,2,3,4,5,6-hexachlorocyclohexane (Lindane); some factors influencing the contact of chemical and test insect and methods f o r standardizing the process. Analyt. Chem., 2^: 555-60. Hurtig, H. 1955. Personal communication. Krijgsman, B.J. and Nelly E. Berger. 1949. A simple method f o r the estimation of contact i n s e c t i c i d e s . B u l l . Ent. Res., itO: 355-58. Lindquist, A.W., H.G. Wilson, H.Q. Schroederer, and A.H. Madden. 1945. E f f e c t of temperature on knockdown and k i l l of houseflies exposed to DDT. Jour. Econ. Ent., 28 (2): 261-64. 83 ., A.H. Madden, and H.Q. Schroederer. 19i|6. E f f e c t of temperature on knockdown and k i l l of bedbugs and mosquitoes exposed to DDT. Jour. Kansas Ent. S o c , 12 (1): 13-15. March, R.B. and R.L. Metcalf. 191+9. Laboratory and f i e l d studies of DDT-resistant houseflies i n southern C a l i -f o r n i a . Cal. Dept. Agric. B u l l . , 2: 1-8. McGovran, E.R., C C . C a s s i l , and E.L. Mayer. 1940. P a r t i c l e size of Paris green as related to t o x i c i t y and repellency to the Mexican bean beetle. Jour. Econ. Ent., 525. Mcintosh, A.H. 1947. Relation between p a r t i c l e size and shape of I n s e c t i c i d a l suspensions and t h e i r contact t o x i c i t y . I. DDT suspensions against Tribolium castaneum. Ann. App. B i o l . , 2k'- 586-610. . 1949. Relations between p a r t i c l e size and shape of i n s e c t i c i d a l suspensions and t h e i r contact t o x i c i t y . I I . DDT and rotenone suspensions against Oryzaephilus surinamensis with some time-mortality studies. Ann. App. B i o l . , 36: 535-50. . 195l. P a r t i c l e size of i n s e c t i c i d a l sus-pensions and t h e i r contact t o x i c i t y . IV. Mechanisms of action of d i f f e r e n t sized p a r t i c l e s . Ann. App. B i o l . , ^8: 881-97. McLeod, W.S. 1944* Further refinement of a technique f o r tes t i n g contact i n s e c t i c i d e s . Canad.Jour.Res., 22 (D): 87-lOk. McLeod, W.S. 1955* Personal communication. McLintock, J . 1952. Continuous laboratory rearing of Cullseta lnornata ( W i l l ) . Mosq. News, 12 (3): 195-201. Moore, W. and C.I. B l i s s . 194-2. A method f o r determining i n s e c t i c i d a l effectiveness using Aphis rumleis and cer-t a i n organic compounds. Jour. Econ. Ent., (4): 544-53. Morrison, F.O. 1943* The standardizing of a laboratory method f o r comparing the t o x i c i t y of contact i n s e c t i c i d e s . Canad; Jour. Res., Sect. D, Zool. S c i . , 21 (3): 33-75. O'Kane, W.C., G.L. Walker, H.G. Guy, and O.J. Smith. 1933-Studies of contact i n s e c t i c i d e s . X. Penetration of arsenic into insects. New Hamp. Exp. Sta. Tech. B u l l . 5 4 . Pearson, A.M. and C.H. Richardson. 1933. The r e l a t i v e tox-i c i t y of Trisodium arsenite and arsenious acid to the housefly. Musca domestica L. Jour. Econ. Ent., 26: 486-93. 81* Peet, C.H. and A.G. Grady. 1928. Studies i n i n s e c t i c i d a l a c t i v i t y . I. Testing i n s e c t i c i d e s against f l i e s . Jour. Econ. Ent., 21: 612-1?. Piquett, P.G. and J.H. Pales. 1952. Rearing cockroaches f o r experimental purposes. U.S. Dept. Agric. B u l l . ET-301. Potter, C. 19l*l. A laboratory spraying apparatus and tech-nique f o r investigating the action of contact i n s e c t i c i d e s with some notes on suitable test insects. Ann. App. B i o l . , 22 (2): 11*2-59. Pradhan, S. 191*9. Studies on the t o x i c i t y of i n s e c t i c i d e f i l m s . I. Preliminary investigations on the concen-tration-time-mortality r e l a t i o n . B u l l . Ent. Res., J4O ( l ) : 1-25. . 191*9. Studies on the t o x i c i t y of i n s e c t i c i d e f i l m s . I I . E f f e c t of temperature on the t o x i c i t y of DDT film s . B u l l . Ent. Res., 1*0 (2): 239-65. Proverbs, M.D. and P.O. Morrison. 1914-7- The r e l a t i v e tox-i c i t y of DDT and related organic molecules. Canad. Jour. Res., Sect. D, Zool. S c i . , 2j?: 12. Richardson, C.H. and L.E. Haas. 1932. The r e l a t i v e t o x i c i t y of pyridine and nic o t i n e i n the gaseous condition of Tribollum confusum Duval. Iowa State C o l l . Jour. S c i . , 6 (3): 287-98. Roadhouse, L.A.O. 1953.' Laboratory studies of DDT-resistant houseflies (Diptera) i n Canada. Can. Ent., 8£. (9): 3l|-0-l*6. Stringer, A. 191*9. A simple method fo r assaying contact t o x i c i t i e s of in s e c t i c i d e s with r e s u l t s of tests with some organic compounds against Calandrla granarla L. Ann. App. B i o l . , 2£: 213-21*. Sun, Y.P. 19l*7. An analysis of some important factors a f f e c t i n g the r e s u l t s of fumigation tests on insects. Minn. Agric. Exp. Sta. Tech. B u l l . 177. . 1950. T o x i c i t y index - an improved method of comparing the r e l a t i v e t o x i c i t y of i n s e c t i c i d e s . Jour. Econ. Ent., 1*2 (1 ) : 1*5-53. Teotia, T.P.S. and Paul A. Dahm. 1950. The eff e c t of temperature, humidity, and weathering on residual t o x i -c i t i e s to the housefly of f i v e organic i n s e c t i c i d e s . Jour. Econ. Ent.. 1*3 (6): 861*-76. Wadley, P.M. and W.N. Su l l i v a n . 191*3. A study of the dosage-mortality curve. Jour. Econ. Ent., 26 (3). 367-72. 85 Wilkes, A., J.W. Bucher, M.B. Cameron, and A.S. West. 191+.8, Studies on the housefly. Canad. Jour. Res., Sect. D., Zool. S c i . , 26 (1): 8-25. Yeager, J.P. and S.C. Munson. 1945• The r e l a t i o n between poison concentration and survival time of roaches i n -jected with sodium metarsenite. Ann. Ent. Soc. Am., 2S: 559-600. 86 IX. APPENDIX S t a t i s t i c a l analysis of dosage-mortality data Table 1. T o x i c i t y of D i e l d r i n to Musca domestica L., QQ, (SES culture) n r p p Empirical Y nw nwx 0.112 1.05 150 147 98 98 0.076 2.88 150 lii3 95 95 0.06k 2.81 150 135 90 90 0.060 2.78 150 119 79 79 0.056 2.75 300 2k0 80 80 o.o53 1.72 300 i5o 5o 5o O.Oij.5 2.65 300 87 29 29 O.Olil 2.61 150 135 45 45 0.038 2.58 300 126 42 42 0.03k 2.53 300 5 i 17 17 0.000 - 150 0 0 -Probit 7.05 6.45 6.28 5.81 5.8k 5.oo 4-45 k.87 4-80 k.05 nwy 13.8 7.03 28.276 97.01k 85.16k 299-433 45.3 6.61 65.8 6.2k 119.098 kl0.592 7.2 6.k 6.0 5.8 75.k 5.81 134-212 438.074 5.5 174.3 5.80 305.025l010.940 5.3 184.8 4.99 317.856 922.152 4.9 190.3 4-47 313.995 850.641 4.6 90.1 4.89 145.061 440.589 4.4 167.3 4-84 264.334 809.732 4.1 l k l - 4 4-05 216.342 572.670 1148.5 1929.363 3351.837 x = 1.6799 y = 5.0952 l/Snw = O.OOO8707 Snwx Snwxy Snwy^ 3253.335 9908.370 30428.815 ?24j.l3? 9930491 29816.278 S n = 1 2.202 Sxy= 77.881 Syy=632.537 b » 6.3826 Y = -5.6269 + 6.3826x r2 (8) = 115.451 h.f. = 14.431 V ( b ) « 1 . 1 8 2 7 ; S.E. ( bj = - 0 . 0 8 7 4 ; .*. b = 6 . 3 8 2 6 - 1 . 0 8 7 4 Log LD^Q = m = 1 . 6 6 5 0 ; A n t i l o g m = 0 . 0 4 6 mgm. v(m) = 0 . 0 0 0 3 1 8 4 ; S.E.( mj = t 0 . O I 7 8 ; m = 1 . 6 6 5 0 - O .OI78 g = 1.1549 P.L. = 1.6647 i 0.1412 « 1.8059 - 1.5235 Antilogs = 0.064 mgm. and 0.033 mgm. 87 Table 2. T o x i c i t y of D i e l d r i n to Musca Domestica L. tf(f, (SES culture) X n x r p' p Empirical Y nw y nwx nwy Probit 0.038 2.58 150 123 82 82 5.92 6.2 55.6 5.87 87.848 326.372 0.034 2.53 150 128 85 85 6.0ii 5.9 70.7 6.03 108.171 426.321 0.030 2.48 300 222 74 74 5.64 5.5 87.2 5.6k 129.056 491.808 0.026 2.k2 150 80 53 53 5.08 5.1 95.2 5.08 135.18k 483.616 0.023 2.36 150 51 34 34 4-59 4.6 90.1 4.59 122.536 413.559 0.019 2.28 150 Lk 9 9 3.66 4.0 65.8 3.72 84.224 244.776 0.015 2.18 150 8 5 5 3.36 3.3 31.2 3-36 36.816 104.832 0.000 - i5o 0 0 - , k95.8 703.885 2491.28k x = 1.4196 y = 5.0248 1/Snw s 0.0020169 Snwx2 Snwxy Snwy2 b = 7.5594 1005.165 3582.007 12878.125 999.160 3536.613 12518.144 Y = -5-7065 + 7-5594* Sxx - 6.005 =45-394 Syy =359-98l = 16.831 h.f. a 3.366 V( b) - 0.5605; s . E . ( b ) = t 0.7487; b = 7-5594 * 0.7487 Log LD£o = m = 1.4163; A n t i l o g m = 0.026 mgm. V(m) = 0 . 0 0 0 1 1 8 9 ; S.E.( m) = 0.0109; m = l . k l 6 3 - 0.0109 g = 0.0648 Since g i s small P.L. = (.0109)(2.57) on e i t h e r side of m; 1.4163 - 0.0280 = 1.4443 and 1.3883 Antilogs ss 0.028 mgm. and 0.024 mgm. 88 Table 3. T o x i c i t y of d i e l d r i n to Musca domestica L . , Q 9 » (SES cul t u r e ) . Three r e p l i c a t i o n s of each dosage plotted i n d i v i d u a l l y A x n r p f p Empirical Y nw y nwx nwy k.9 31.7 4.70 46.916 lk8.990 k.9 31.7 4-65 46.916 ik7.ij.05 4.9 31.7 5.30 46.916 168.010 4.7 30.8 5.01 43.736 154.308 4.7 30.8 4.59 43.736 141.372 4.7 30.8 4.90 43.736 150.920 4.5 29.1 4.02 39.576 116.982 4.5 29.1 4-76 39.576 138.516 4.5 29.1 4.53 39.576 131.823 4.2 25.1 4.16 32.128 104.416 4.2 25.1 4.44 32.128 111.444 4.2 25.1 3.95 32.128 99.145 350.1 k87.068 1613.331 Probit 0.030 2.48 50 19 38 38 4.70 50 18 36 36 4.64 _ So 31 62 62 5.31 0.026 2.k2 50 25 50 50 5.00 50 17 34 34 4.59 50 23 46 46 4-90 0.023 2.36 50 7 14 14 3.92 5o 20 40 40 4-75 5o 16 32 32 4.53 0.019 2.28 50 10 20 20 4.16 50 14 28 28 4.42 50 7 14 14 3.92 0.000 - 50 0 0 - -50 0 50 0 x = 1.3912 y = 4.6082 1/Snw = 0.0028563 Snwx2 Snwxy Snwy2 b = 3.7683 679.464 2251.452 7485.195 677-621 224k.g07 7434.^2 Y = 0.6343 + sxx =1.843 Sxy =6.945 Syy =50.643 3-7683x x 2 = 24.472 (10) ^ h.f. = 2.4472 v ( b ) - 1-328; S.E. ( b ) = 1.152; .'. b = 3.7683 - 1.152 Log LD^ 0 = ra = 1.4952; A n t i l o g m = 0.031 mgm. V(m) = o.ooi^o4; s - E - ( m ) = * 0.038; .*. m = 1.4952 * 0.038 g = 0.4650; P.L. = 1.5854 1 .1488 = 1.7342 and I.4366 Antilogs = 0.054 mgm and 0.027 mgm. 89 Table k. T o x i c i t y of d i e l d r i n to Musca domestica L.,Q<£, (SES cul t u r e ) . Each point represents three r e p l i c a t i o n s . A X n r p» p. p Empirical Y nw y nwx nwy - Probit 0.030 2.k8 50 19 38 38 k6 k.90 k .9 95-1 k.90 190.748 465-990 50 18 36 36 1.1+2 50 31 62 62 0.026 50 25 50 50 43 k.82 k .7 92.k k.83 131.208 446.292 50 17 3k 3k 2.36 50 23 k6 k6 4.5 87.1 4-45 118.456 387.595 0.023 50 7 l k l k 29 4-45 50 20 kO kO 2.28 50 16 32 32 k .2 75.k k.19 96.512 315.926 0.019 50 10 20 20 21 k.19 50 l k 28 28 50 7 l k l k 0.000 - 50 0 0 0 - - - - - -50 0 0 50 0 0 350.0 486.92k 1615.803 X = 1.3912 7 = 4-6166 1/Snw = 0.0028571 Snwx2 Snwxy Sxwy2 b = 3-7922 679.257 225k.913 7k87.k68 677.klk 22li.7.92k 74^483 Y = -0.6591 + 3.7922x S__ = 1.843 S = 6.989 S =27.985 P xx xy yy X 2 = 1.48 (2) V ( b ) = 0.5426; S.E. ( b ) = t 0.740; b = 3-7922 - 0.740 Log L D ^ Q = m = I .492O; A n t i l o g m = 0.031 mgm. v(m) = 0.00150; S.E.( m) = t 0.038; .'. m = 1.4920 i 0.038 g = 0.1448 P.L. =- 1.5091 - 0.0638 ; 1.5729 and 1-4453 Antilogs = 0.037 mgm and 0.028 mgm. 90 Table 5- T o x i c i t y of parathion to Musca domestica L., QQ (SES culture) X x n r P " P ' P Empirical Y nw y nwx nwy - Probit 0.060 2.78 5o 50 100 100 97 6.88 7.1 16.4 6.82 29.192 111.848 5o 45 90 90 So So 100 100 o.o55 2.7k 50 49 98 98 92 6.41 6.5 39.9 6.40 69.426 255.360 5o 47 94 94 o.o5o 5o k 2 84 84 2.70 50 45 90 90 87 6.13 6.0 65.0 6.12 110.500 397.800 5o 37 ? k 7 T - So 48 96 96 5.74 0.0k8 2.68 k9 38 77 77 77 5.7 78.2 5.74 131.376 448.868 5o 35 70 70 _ 5o 42 84 84 0.045 2.65 50 22 44 43 48 4.95 5.2 92.5 4.95 152.625 457.875 5o 22 43 5o 29 58 58 0.0k2 2.62 Ij.9 28 57 57 40 4.75 4.7 89.4 4.75 144.828 424.650 50 21 42 41 50 11 22 21 0.038 2.58 k9 7 13 24 4-29 4.1 66.6 4.31 105.228 287.046 50 13 26 25 50 17 34 13 0.000 - 50 0 0 . 1 - — - - - - -5o 0 0 S o 2 4 448.0 7k3.175 2383.kk7 x = 1.6588 y = 5.3202 1/Snw = 0.0022321 Snwx2 Snwxy Snwy2 b = 14-300 1234.036 3970.733 12928.872 1323.832 3953.516 12680.kOO Y = -18.4035 + l4-300x s x x = I.264 3 ^ = 17.217 248.472 V ( b ) = 0.8305; S.E. ( bj = - 0.911; .'. b = 14.300 - 0.911 Log LD£o = m = 1.6366; A n t i l o g m = 0.043 mgm. v(m) = 0.00025; S.E.( m) =0.0158; g = 0.0156; P;L. = I.6366 -0.0310 ; . f - L . = 1.6676 and 1.6056; Antilogs = 0.045 mgm. and 0.040 mgm. 91 Table 6. T o x i c i t y of d i e l d r i n to Musca domestica L., QQ (SES culture) A x n r p" p' p Empirical Y nw y nwx nwy Probit 0.065 2.81 50 38 76 76 78 5.77 5.8 7k.k 5.77 13k.66k 429.288 50 ko 80 80 _ 5o 39 78 78 0.060 2.78 50 kO 80 80 79 5.81 5.7 78.7 5.80 lk0.086 456.460 50 39 78 78 _ 5o kO 80 80 0.050 2.70 50 36 72 72 70 5.52 5.k 88.7 5.52 150.790 k89.62k 5o 32 6k 6k 50 37 7k 7k 5.3 90.9 5.23 152.712 0.0k8 2.68 50 30 60 60 59 5.23 475-407 50 29 58 58 _ 50 30 60 60 0.0k5 2.65 50 30 60 60 63 5.33 5.2 92.5 5.33 152.625 493.025 50 33 66 66 - 50 32 6k 6k 0.038 2.58 50 20 kO kO 38 k.70 k .9 93.1 k.70 147-098 437-570 50 19 38 38 50 18 36 36 0.000 - 50 0 0 1 - - _ _ _ -50 0 0 50 2 4 518.3 877.975 2781.37k x = 1.6939 y = 5.3663 l/Snw = 0.001929k Snwx2 Snwxy Snwy2 b = k . 6k l Ik90.2k0 ij.725.lfO7 14997.966 1487.246 4711.512 14925.797 Y = -2.4956 + 4 .64lx Sxx = 2.994 Syy =13.895 S = 72.169 0 3 ^ J Xf = 7.637 (4) v ( b ) = ° - 3 3 4 ; S . E . ( D ) = ± 0.577; b = 4-641 1 0.577 Log LD^Q = m = 1.6l5; A n t i l o g m = O.Okl mgm. V(m) = 0.0001845; S . E . ( M ) = t 0.0135; m = 1.615 - 0.0135 g = 0.2764; P.L. = 1.6120 - O.0350 = 1.6470 and 1.5770 Antilogs = 0.044 mgm. and O.O38 mgm. 92 Table 7• To x i c i t y of parathion to Musca domestica L . , 66, (SES culture) A x n r p M p* p Empirical Y nw y nwx nwy Probit 0.038 2.58 49 16 33 32 38 4-70 4-9 90.5 4-70 11+2.990 1+25.350 50 20 40 39 50 22 44 43 0.034 2.53 5o 26 52 51 41 4-77 4-5 81.2 4.79 124.236 388.948 50 19 38 37 49 17 35 34 0.030 2.48 48 16 33 32 19 4-12 4.1 62.4 4.12 92.352 257.088 50 5 10 8 49 9 18 16 0.027 2.43 50 7 14 12 9 3.66 3.8 47.2 3.67 67.496 173.224 50 5 10 8 50 4 8 6 0.000 - 50 1 2 2 - - - - -50 1 2 P 1 2 28I .3 427.074 1244.610 x = 1.5182 y = 4.4244 l/Snw = 0.0035549 Snwx 2 Snwxy Snwy2 b = 7.0638 649.205 1895.343 5557 . i4i 648.390 1889.586 5506.768 Y = -6.2999+7.0638x sxx = ° - 8 l S sxy = 5.757 Syy =50.373 xy JJ X 2 = (2) h.f. = 4.852 v ( b ) = 5-9534; s.E. ( b ) = t 2.440; ,\ b = 7.064 t 2.440 Log LD^Q = m = 1.5996; Antilog m = 0.040 mgm. V ( m ) = 0.010; S.E.( m ) = - 0.100; g = 0.4546 F.L. = 1.6675 - 0.2210 = 1.8885 and 1.4465 Antilogs = 0.077 mgm. and 0.028 mgm. 93 Table 8. T o x i c i t y of d i e l d r i n to Musca domestica L . , Q Q , (SES c u l t u r e ) . ¥ ¥ A x n r p" p» p Empirical Y nw y nwx nwy Probit 0.038 2.58 $0 45 90 90 91 6.34 6.4 49.3 6.34 77.894 312.562 50 47 9 k 9 k 50 44 88 88 0.03k 2.53 50 46 92 92 89 6.23 6.1 59.3 6.22 90.729 368.846 50 43 86 86 _ 50 44 88 88 0.030 2.k8 50 42 84 84 82 5.92 5.8 73.5 5.91 108.780 434.385 50 41 82 82 50 40 80 80 0.027 2\k3 50 38 76 76 69 5 . 5 0 / 5.5 84.7 5.50 121.121 465.850 50 31 62 61 50 36 72 71 5.1 91.7 5.18 124.712 475.006 0.023 2.36 50 25 50 49 57 5.18 50 37 74 73 - So 25 5o 49 4.7 90.0 4.56 115.200 0.019 2.28 50 20 40 39 33 4-56 410.400 50 18 36 35 50 13 26 24 0.000 - 50 1 2 2 50 1 2 5o 1 2 kk8.5 638.k36 2k67.0k9 x = l . k 2 3 4 y = 5.5007 l/Snw = 0.0022295 Snwx2 Snwxy Snwy2 b = 6.3039 913.049 3538.558 13737.210 908.808 3511.823 13570.klk Y = -3.4723+6.3039x Sxx = 4.241 S x y =26.735 Syy =166.796 A — 1 . Jl+d (4) V ( b ) = 0.2358; S.E. ( b) = ± 0.485; b = 6.3039 ± 0.485 Log L E > £ Q = m as 1.3440; Antilog m = 0.022 mgm. V(m) = 0 * 0 0 1 S 3 2 ; S.E. ( m) = t 0.039; m = 1.3440 - 0 . 0 3 9 . g = 0 .023; P . L . 1.96 x 0.039 on either side of m = 1.3440 - O.0764 = l . k 2 0 4 and 1.2676 , Antilogs = 0.026 mgm. arid 0.019 mgm. 94 Table 9. To x i c i t y of E P N to Mu3ca domestica L . , Q Q , ( S E S culture) X x n r p" p* p Empirical Y nw y nwx nwy - Probit 1.350 0.130 50 50 100 100 86 6.08 6.0 65.8 6.08 74-354 400.064 50 50 100 100 50 29 58 58 1.275 0.106 50 25 50 50 76 5.71 5.8 75.4 5.70 83-392 429.780 50 45 90 90 50 ii4 88 88 1.200 0.079 50 32 6k 6k 62 5.31 5.5 86.6 5.30 93-441 458.980 50 17 ^ 49 k3 88 88 505.248 1.125 0.051 50 39 78 78 71 5.55 5.3 91.2 5.54 95-760 50 2k i t 8 ft8 48 1+2 n87 87 521.696 1.050 0.021 50 32 6k 6k 69 5.5o 5.1 95.2 5-48 97-199 50 i|2 ®k 84 50 29 58 58 0.975 1.99 50 26 52 52 31 4.5o 4.9 95.2 4.52 94.248 430.304 50 lk 3k 34 50 7 lk 14 0.000 50 0 0 0 - - - - - - -5o 0 0 5o 0 0 509.4 538.394 2746.072 x = 1.0569 7 = 5.3908 l/Snw = 0.0019631 Snwx Snwxy Snwy2 b = 7-6879 570.265 2911.805 14917.671 569.038 2902.372 14803.516 Y = 2.7345 + 7-6879x = 1.227 S = 9.433 Syy =114.155 ? . , ^ ™ X 2 = 41-635 (4) h.f. = 10.409 V( b) = 8.4833J S.E.( b) = i 2.9126; ,\ b = 7.6879 - 2.9126 Log L D ^ Q = m = 1.0061; Antilog m = 1.014 mgm. V(m) = ° - 0 0 ° 7 l 6 l ; S - E « ( m ) = * 0.0267; .*. m = 1.0061 ± 0.0267 g = 1-109 Since g i s so large i t i s not possible to calculate P.L. 95 Table 10. T o x i c i t y of d i e l d r i n to Musca domestica L., QQ , (SES culture) A x n r p" p 1 p Empirical Y nw y nwx nwy Probit 0.070 2.85 49 31 63 63 66 5.1+1 5.5 86.6 5.ki 160.210 468.506 50 35 70 70 5o 33 66 66 0.060 2.78 50 29 58 58 56 5.15 5.2 94.1 5.15 167.488 484.615 50 26 52 52 5o 29 58 58 0.050 2.70 50 30 60 60 60 5.25 5.0 94.9 5.25 161.330 498.225 50 33 66 66 49 26 53 53 0.048 2.68 50 18 36 36 39 4-72 4-9 95.2 4.72 159.936 449.344 50 15 30 30 50 25 50 50 0.045 2.65 50 29 58 58 56 5.15 4.8 94.1 5.16 155.265 485.556 5o 29 58 58 5o 26 52 52 0.038 2.58 50 10 20 20 23 4-26 4.6 89.5 4-23 lkl.klO 378.585 50 15 30 30 49 10 20 20 0.000 - 5 0 0 0 0 - - - - -50 0 0 50 0 0 ; : 554.4 945.649 2764.831 x = 1.7057 y = 4.9871 l/Snw = 0.0018038 Snwx2 Snwxy Snwy2 b = 3.5524 1617.104 4730.563 13873.853 1613.009 4716.016 13788.402 Y = 1.0722 + 3-552kx S ^ = 4,095 =14.547 Sjy= 85.451 0 Xf = 33-775 (4) h.f. = 8.4438 V ( b ) = 2.0620} S.E. ( b ) = ± 1.436; .'. b = 3.5524 - 1.4360 Log LD. = n = 1.7093; Ant i l o g m. = 0.052 mgm. 50 V ( m ) = 0.2843; S.E. ( m ) = t 0.5332; m = 1.7093 ± 0.5332; g = 1.2628 P.L. a 1.6920 i 0.0646 = 1.7566 and 1.62?4 Antilogs = 0.057 mgm. and 0.0k2 mgm. 96 Table 11. T o x i c i t y of EPN to Musca domestica L . , d c f , (SES culture) A x n r p" p' p Empirical Y nw y nwx nwy probit 0.750 1.88 50 46 92 92 92 6.41 6.5 39.9 6.4,0 75.012 255-360 50 48 96 96 50 44 88 88 0.705 1-85 49 45 92 92 91 6.34 6.2 54*6 6.33 101.010 345.618 50.41 82 82 50 50100100 0.638 1.81 49 33 67 67 75 5-67 5.9 69.4 5.65 125.614 392.110 50 39 78 78 50 40 80 80 0.600 I.78 49 39 79 79 74 5-64 5-6 82.0 5-64 145-960 462.480 50 44 88 88 50 29 58 57 0.563 1.75 49 46 94 94 70 5.52 5-3 89-7 5-52 156.975 495-144 49 35 71 71 50 23 46 45 0.525 1-72 49 13 27 26 52 4.05 5-1 92.8 5.05 159.616 468.640 50 23 46 45 50 43 86 86 0.000 - 5 o 0 0 1 - - - -50 1 2 50 0 0  428.4 764.187 2419.352 x = 1.7838 y = 5.6474 l/Snw = 0.0023343 Snwx2 Snwxy Snwy2 b = 8.1599 1364.307 4324.966 13745-701 1363.169 4315,680 13664.482 Y = 8.9082 + 8.l599x S M = 1.138 S "= 9.286 S ™ = 81.219 0 ^ 5 7 x 2 6 ) = 5.446 V ( b ) = °* 8 7 8 7J s * E - ( b ) = ± 0-94 20; b = 8.1599 - 0.9420 Log LD^Q = m = 1.7044; A n t i l o g m = 0.506 mgm. V ( m ) ; 0.0001181; S.E.( m) = 0.011; m = 0.506 ± 0.011 g = 0.049; Since g i s small, P.L. = 1.96 x 0.011 on either side of m = 1.7044 - 0.0216 = 1.7260 and 1.6828 = 0.532 mgm. and 0.482 mgm. 97 Table 12. T o x i c i t y of D i e l d r i n to Musca Domestica L.,(£f, (SES culture) X x n r p" p' p Empirical Y nw y nwx nwy Probit 0.038 2.58 50 43 86 86 71 5-55 5.9 65-5 5.50 103.1*90 360.250 50 38 76 76 _ , 39 20 51 51 0.03k 2.53 50 ko 80 80 73 5.61 5.6 83.7 5.61 128.061 469.557 50 32 6k 6k 50 37 7k 7k ^ 0.030 2.48 Ij.9 43 88 88 83 5.95 5.3 91.2 5.86 134-976 534-432 49 42 86 86 50 37 74 74 0.027 2.43 50 26 52 52 56 5.15 4.9 95.2 5.15 136.136 490.280 50 37 74 74 50 21 42 k2 0.023 5.36 49 11 21 21 14 3.92 4.4 83.I 4-00 113.016 332.400 50 6 12 12 50 5 10 10 0.019 2.28 49 3 6 6 13 3-87 3.9 59.9 3.8? 76.672 231.813 50 11 22 22 50 6 12 2 0.000 - 50 0 0 0 - - -49 0 0 49 0 0 , k78.6 692.351 2418.732 x = 1.4466 y = $.0$3& 1/Snw = 0.0020894 Snwx2 Snwxy Snwy2 b = 7.0870 1005.727 3528.461 12499.020 1001.567 3498.979 12223.703 Y = -5.1983+7.0870x S ^ = k.160 Sn y =29.482 Syy = 275.318 x (4) = 0 0 •378 h.f. = 16.5945 V ( b ) = 3 * 9 8 9 ° 5 s - E - ( b ) 5 8 * 1-9970; /. b = 7.0870 ± 1.9970 Log LD^Q = m = 1.4390; Antilog m = 0.028 mgm. v(m) = 0.000695; S.E.( m) = 0.026; m = 1.4390 - 0.0260 g = 0.6138; P.L. = 1.4270 ± .1186 = 1.5456 and I.3083 Antilogs = 0.035 mgm. and 0.020 mgm. 98 Probit 5.88 5.9 70.2 5.87 IO6.704 412.074 5.61 5.5 87.1 5.6l 128.908 488.631 4.61 4-9 95.1 4.62 134.091 439.362 4.61 4.3 79.7 4-65 107.595 370.605 Table 13. To x i c i t y of endrin to Musca domestica L., QQ , (SES culture) X x n r p H p» p Empirical Y nw y nwx nwy 50 42 8k 84 49 42 86 86 0.300 T.48 50 37 74 74 73 50 36 72 72 50 36 72 72 0.285 1.41 50 16 32 32 35 50 16 32 32 50 20 40 4o 0.225 1.35 50 13 26 26 35 50 19 38 38 50 20 40 40 0.000 50 0 0 0 - - -50 0 0 50 0 0  ' 332.1 477.298 1710.672 x = 1.4372 y = 5.1511 l/Snw = 0.0030111 Snwx2 Snwxy Snwy2 b = 8.1649 687.294 2469.342 8913.258 685.978 2458.597 8811.799 Y = -6.5840 + 8.l649x s x x = 1-316 S ^ I o . 7 4 5 Syy ==101.459 . x (2) ~ 13.727 h.f. = 6.864 V( b) = 5.2158; S.E. ( b) = t 2.2838; .\ b = 8.1649 - 2.2838 Log LD^ 0 = m = I.4190; A n t i l o g m = 0.262 mgm. v(m) = 0.003348; S.E. ( m ) = 0.0183; g = 1.4466 P.L. cannot be calculated at the 95$ p r o b a b i l i t y l e v e l . 99 Table l k . T o x i c i t y of d i e l d r i n to Musca domestica L., QQ , (SES culture) A X n r P" P» p Empirical Y nw y nwx nwy Probit 0.068 2.83 49 4 i 84 83 84 5-99 5.9 64.3 5.99 117.669 385.157 5o 42 84 83 2.78 5o 43 86 85 0.060 5o 39 78 76 75 5.67 5.7 72.5 5-67 129.050 411.075 5o 38 76 74 2.72 50 39 78 76 5.5 78.6 5.44 135.192 0.053 5o 34 68 65 67 5.44 427.584 5o 36 72 70 2.65 5o 34 68 65 0.045 5o 32 64 61 66 5 . 4 i 5.3 82.4 5.41 135.960 445.784 50 27 54 51 5o 43 86 85 0.000 - 48 1 2 7 49 0 0 48 9 18 297.8 517.871 1669.600 x = = 1.7390 y = -. 5.6064 I/Snw = 0.003580 2 P Snwx Snwxy Snwy* b = 3-0869 901.907 2907.537 9375.633 900.572 290^.416 9360.52k Y = 0.2383+3.0869x S^=-1^3$ S ^ = k.121 S y y = 15.109 2 x^ 2 ) = 2 . 3 8 8 + + V, x = 0.7491; S.E. ^ = - 0.8650; .'. b = 3.0869 - 0.8650 (b) (b) Log LD^Q = m = 1.5425; Antilog m = 0.0349 mgm. V(m) = 0-00338; S.E. ( m ) = ± 0.058; g = 0.3018 P.L. = 1.4576 - 0.1608 = 1.6184 and 1.2968 Antilogs = 0.042 mgm. and 0.020 mgm. IQO Table 15- T o x i c i t y of endrin to Musca domestica L.cfcf, (SES culture) x n r P H P 1 p Empirical Y nw y nwx nwy - Probit 0.233 1.37 SO k8 96 96 93 6.k8 6.5 39.2 6.k8 53.70k 25k.016 50 k6 92 92 0.195 - t 9 kk 90 90 1.29 50 kl ^ ^ 87 6.13 6.2 53.9 6.12 69.531 329.868 k9 k3 88 50- ko 80 80 0.158 1.20 5o ko 80 8k 83 5.95 5.8 73.5 5.9k 88.200 k36.590 5o k2 8k 8k 5o k3 86 86 8k.7 5.50 9k'.017 0.128 l . n 5o 38 75 75 69 5.5o 5.5 k65.850 5o 33 66 65 5o 3k 68 67 0.000 - 5o 0 0 2 - «. 5o l ;.2 $o 2 k 251.3 305.k52 lk86.32k x = 1.2155 y = 5.91k5 l/Snw = 0.0039793 Snwx2 Sxwxy Snwy2 b = 3.6133 373.k66 l8lk.532 8820.335 371.273 1806.608 8790.923 Y = 1.5225 + 3.6l33x Sxx= 2 ' 1 9 3 Sxy = ?- 9 2k 3^=29.412 x u r 0 , 7 8 1 V ( b ) = 0.k559; S - E . ( D ) = - 0.6750; b = 3.6133 - 0.6750 Log LD^Q = m = 0.962k; An t i l o g m = 0.092 mgm. v(m) = 0'Q092; S.E.( m) = - 0.0958; /. m = 0.962k - 0.0958 g = 0.13kl F.L. = 0.9585 - 0.1132 « 1.0717 and 0.8k53 Antilogs = 0.118 mgm. and 0.070 mgm. 101 Table 16. T o x i c i t y of d i e l d r i n to Musca domestica L., db", (SES culture) n r P w p' p Empirical Y nw y nwx nwy Probit k9 1+5 92 92 93 6.48 6.5 39.4 6.48 63-434 255.312 So 1+6 92 92 1+9 1+7 96 96 75-735 50 1+5 90 90 91 6.34 6.3 49.5 6.34 313.830 1+9 45 92 92 50 46 92 98 5.9 69.9 5.95 99.258 415.905 50 39 78 78 83 5.95 5o 45 90 90 50 41 92 82 443.868 50 30 60 59 74 5.64 5.7 78.7 5.64 107.032 5o 40 80 80 5o 42 84 84 50 0 0 1 - - - - -5o 0 0 So 1 2 237.5 345.459 1428.915 x = 1,4546 y = 6.0165 l/Snw = 0.0042105 Snwx Snwxy Snwy b = 3.4664 504.514 2085-458 8622.153 502.492 2078.449 8597.044 Y = 0.9743+3.466kx S x x = 2.022 S x v = 7.009 S _ = 25-109 0 y 7 7 x 2 2 ) = 0.813 V( b)= 0.4945; s « E . ( b ) = t 0.7030; .*. b = 3.4664 - 0.7030 Log LD . = m = 1.1613; A n t i l o g m = 0.015 mgm. 50 V(m) = 0.0038905; S.E.( m) = i 0.0623; /. m = 1.1613 - 0.0623 g = 0.2282; P.L. = 1.1527 - 0.1568 = 1.3095 and 0.9959 Antilogs = 0.020 mgm. and 0.010 mgm. 102 Table 17. T o x i c i t y of a l d r i n to Musca domestica L.,QQ, (SES culture) * A x n r p" p' p Empirical Y nw y nwx nwy Probit 0.068 2.83 50 18 36 36 47 4-92 5.1 95-1 4-92 174.033 467.892 50 20 4o 40 50 32 64 64 0.064 2.81 5o 30 60 60 60 5.25 5.0 95.5 5.25 172.855 501.375 5o 3k 68 68 50 26 52 52 49 12 24 50 24 48 0.060 2.78  24 45 4.87 4.9 94.5 4-87 168.210 460.215 '  48 5o 32 64 64 0.053 2.72 50 8 16 16 27 4-39 4.6 90.1 4.38 154.972 394.638 50 20 40 40 50 12 24 24 0.045 2.65 50 16 32 32 25 4.33 4.3 79.7 4-33 131.505 345.101 50 12 24 24 50 10 20 20 0.000 50 0 0 0 - - - - -50 0 0 5o 0 0  454.9 801.575 2169.221 x = 1.7621 y = 4.7686 1/Snw = 0.0021983 Snwx2 Snwxy Snwy2 b = 4-7295 1414.297 3831.108 10398.829 1412.kkS 3822.363 10344.074 Y = -3.5653+4-7295x S ^ ^ m 3 ^ = - ^ S y y = ^-755 = i 3 ^ 9 5 h.f. = 4-465 V ( b ) = 2.4147; S.E. ( b ) = ± 1.5530; .*. b = 4-7295 - 1.5530 Log I*D 0^ = m = 1.8110; A n t i l o g m = 0.065 mgm. V(m) = 0-0°S72; s - E - ( m ) = - 0.0756; m = 1.8110 t 0.0756 g = 1.0917; •"• impossible to calculate f i d u c i a l l i m i t s . 103 Table 18. T o x i c i t y of d i e l d r i n to Musca domestica L . , Q Q (SES culture) n r Ptt p» p Empirical Y nw.. y nwx nwy Probit 5o 27 Sk Sk 6S 5.39 5.2 92.9 5.38 168.Ik9 k99.802 k8 26 5 k % So k3 86 86 k9 23 52 52 51 5.03 5.0 91.0 5.03 162.890 k57.730 SO 23 k6 k6 k9 27 kS SS 161.852 k22.509 SO 18 36 36 30 k.k8 k.8 9k. 1 k.k9 So 11 22 22 So 16 32 32 k9 9 18 18 27 k.39 k.5 85,k k.39 IkO.910 37k.906 k9 19 39 39 k9 12 2$ 2S 387.531 So 17 3k 3k 35 k . 6 l k . k 83.7 k.63 13k.757 So 8 16 16 So 28 $6 56 So 0 0 0 mm mm - - - mm mm So 0 0 50 0 0 kk7.1 768.558 21k2.k78 x = 1.7189 y = k.7919 l/Snw = 0.0022366 2 P Snwx Snwxy Snwyc b = 3.9281 1323.768 3693.213 10328.ii88 1321.139 3682.886 10266.633 Y = -1.9601+3.928lx S = 2.629 S = 10.327 S = 61.855 ? XX xy 33 X = 21.290 (3) h.f. = 7.096 = 2.6991; S.E. ( b) = t 1.6k20; /. b = 3.9281 ± 1.6k20, Log LD^o = m = 1.7718; A n t i l o g m = 0.0590 mgm. V ( m ) = 0.001518; S.E. ( m ) = t 0.039; g = 1.7689 Since g i s so large, i t i s impossible to calculate f i d u c i a l l i m i t s . 10k Table 19. T o x i c i t y of a l d r i n to Musca domestica L., 66, (SES culture) 50 kl 82 82 50 46 92 92 0.03k 2.53 50 k-0 80 80 81 50 43 86 86 49 38 76 76 0.030 2.48 50 37 74 74 76 50 37 74 74 50 40 80 80 0.023 2.36 49 30 61 61 48 49 13 27 27 50 28 56 56 0.019 2.28 50 31 62 62 37 49 3 6 6 50 21 42 k2 0.000 - 49 0 0 0 -50 0 0 50 0 0  Empirical Y Probit nw y nwx nwy 5.77 6.0 65.8 5.75 103.964 378.350 5.88 5.8 74.9 5.88 114.597 440.kl2 5.71 5.6 83.7 5.70 123.876 477.090 4.95 5.0 94.2 4.95 128.112 466.290 4-67 4.6 89.5 4-67 114.560 417.965 408.1 585.109 2180.107 x = 1.4337 y = 5.3421 l/Snw = 0.0024504 P p Snwx Snwxy Snwy b = 4•3132 843.800' 3146.865 11744.580 838.894 3125.704 11646.328 Y = -0.84l7+4*3132x S___. =4.906 S__ = 21.161 Syy = 95.252 0 7 1 X 2 3 ) =7.590 y ^ b ) = 0.2038; s.E. ( b ) = ± 0.4510; .'. b = 4.3132*0.4510 Log LD = m = 1.3544; A n t i l o g m = 0.022 50 V(m) = 0' o o 8 6S; s.E. ( m ) = t 0.0294; /. m = 1.3544 - 0.0294 g = 0.0k2 Since g i s small, f i d u c i a l l i m i t s = 0.0294 * 1.96 = 0.0568 = 1.3544 - 0.0568 = 1.4112 and 1.2976. Antilogs = 0.026 mgm. and 0.020 mgm. 105 Table 20. T o x i c i t y of d i e l d r i n to Musca domestica L., dc? * (SES culture) A x n r p n p» p Empirical Y nw y nwx nwy Probit 0.030 2.2+8 50 19 38 38 4 ° 2+.90 It.9 95-1 4-90 12+0.748 2+65.990 50 18 36 36 50 31 62 62 0.026 2\2|2 50 25 50 50 2+3 2+..82 2+.7 92.2+ 2+.83 131.208 2+46.292 50 17 34 34 50 23 36 36 0.023 2.36 50 7 14 14 29 4-45 4.5 87.1 4.45 118.456 387.595 50 20 40 40 50 16 32 32 0.019 2.28 50 10 20 20 21 4.19 4.2 75-4 4-19 96.512 315.926 50 14 28 28 50 7 14 14 0.000 50 0 0 0 - - - - -50 0 0 5o 0 0  350.0 486.924 1615.803 x = 1.3912 y = 4.6166 l/Snw = 0.0028571 Snwx2 Snwxy Snwy2 b = 3.7922 679.257 2254-913 8487.468 677.414 2247.924 7459.483 Y = -0.6591 + 3.7922x S x x =1.843 S x y = 6 . 9 8 9 Syy =27.985 2 X(2) = V ( b ) = 0.5426; s - E - ( b ) = ° - 7 4 0 ; .'. b = 3.7922 t 0.740 Log LD^ G = ra = 1.4920; A n t i l o g m = 0.031 mgm. V(m) = ° - 0 0 1 5 j s- E-(m) = " ° - ° 3 8 0 ; . m = 1.4920 - 0.0380 + g = 0.l2ji+8; P.L. = 1.5091 - ©.0638 = 1.5729 and 1.2+453 Antilogs = 0.037 mgm. and 0.028 mgm. 106 Table 21. T o x i c i t y of lindane to Musca domestica L.,QQ, (SES culture) A x n r p" p* p Empirical Y nw y nwx nwy Probit 0.068 2.83 50 30 60 60 75 5.68 5-7 79.7 5.67 145.851 ij.5l.899 50 38 76 76 50 44 88 88 0.060 2.78 50 29 58 58 73 5.61 5.6 83.7 5-61 148.986 469.557 50 kO 80 80 50 40 80 80 0.56 2.75 50 36 72 72 69 5-50 5-5 8?.2 5.50 152.600 479.600 50 ij.0 80 80 50 27 5k 5k 0.053 2.72 50 37 7k 74 66 5-kl 5-4 90.1 5.kl 154-972 k87.kkl 50 30 60 60 50 32 6k 6k 0.045 2.65 50 81 k2 k2 49 k.98 5.2 9k.1 k.97 155.265 467.677 50 25 50 50 50 28 56 56 O.Okl 2.61 50 36 72 72 57 5.18 5.1 95.2 5.18 153.272 k93.136 50 25 50 50 k.9 2k ij.8 ij.8 0.000 - 5 0 0 0 0 - - - -50 0 0 k9 0 0  530.0 910.9k6 28k9.310 x = 1.7188 y = 5.3761 1/Snw = 0.0018868 2 ? Snwx Snwxy Snwy b = 2.8k26 1568.659 k905.700 15350.038 1565.703 k897.297 15318.052 Y = 0.k902+2.8k26x S = 2.956 S = 8.k03 =31.986 P * ™ X 2 , = 8.099 (4) V ( b ) = °- 3 3 82? S * E * ( b ) = " ° . S 8 l 5 j •'. b = 2.8426 - 0.5815 Log LTJCJQ = m = 1.586; A n t l l o g m = 0.039 mgm. VCm) = 0.001002; S.E. ( m ) = t 0.0316; g = O.I63. P.L. = 1.5600 - 0.0720; 1.6320 and I.488O Antilogs = 0.043 mgm. and 0.031 mgm. 107 Table 22. T o x i c i t y of d i e l d r i n to Musca domestica L.,QQ (SES culture) * A X n r p' p Empirical Y nw y nwx nwy Probit 0.070 2.85 20 ^ k3 1+9 k.98 5.0 93.9 k.98 173.715 1+67.622 50 29 58 58 2.78 50 23 1+6 1+6 1+16.72k 0.060 5o ll+ 28 28 31 k.50 k .7 92.k k . 5 l I6k..k72 5o 11+ 28 28 2.70 5o 19 39 39 0.050 50 29 58 58 1+2 1+.80 k.k 83.7 k .8k 11+2.290 1+05.108 5o llj. 28 28 2.68 5o 22 10+ 1+1+ 0.01+8 5o 7 Ik l k 23 32 k.26 k .3 79.7 k.26 133.896 339.522 5o 16 32 2.65 5o 11 22 22 0.01+5 5o 15 30 30 20 k.16 1+.2 75.1+ k.16 12k.klO 313.661+ 5o 6 12 12 2.58 5o 9 18 18 0.038 5o 5 10 10 12 3.83 3.8 60.7 3.83 95.906 232.1+81 5o k 8 8 5o 9 18 18 0.000 • 5o 0 0 0 - _ — _ _ _ 5o 0 0 5o 0 0 k85.8 83k.689 2175.21 1 I = 1.7181 y = = l+.k77k 1/Snw = 0.002058k Snwx2 Snwxy Snwy2 b = 3.7909 11+37.779 3751.017 9810.513 lk3k.lkk 3737.237 9738.887 Y = -2.0355+3.7909x Sxx = 3 . 6 3 5 = 1 3 . 7 8 0 S =71.626 0 ^ 7 7 x 2 = 19.388 (1+) h.f. = k.8k7 V ( b ) = 1.3331+; S.E. ( b ) = I . l 5 k 0 ; .'. b = 3.7090 - 1.151+0 Log LD^ 0 = ra = 1.8550; A n t l l o g m = 0.072 mgm. V(m) = 0.0021+; S.E. ( m ) = ± 0.0k93; g = 0.717 P.L. = 2.2020 - 0.5990 = 2.8010 and 1.6030 Antilogs = 0.632 mgm. and O.Oi+O mgm. 108 Table 23. T o x i c i t y of lindane to Musca domestica L.. db", (SES culture) X x n r P" P' P Empirical Y nw y nwx nwy Probit 0.030 2.k8 5o 36 72 70 77 5.7k 6.0 61.9 5-71 91.612 353.kk9 5o k l 82 81 0.027 2.k3 5o k l 82 81 5o 43 86 85 88 6.18 5.9 66.k 6.1k 9k.952 k07.696 5o k6 92 92 0.023 2.36 5o 44 88 88 5o 52 8k 83 81 5.88 5.7 7k.6 5.87 101.456 k37-902 5o 37 7 k 73 0.019 2.28 5o kk 88 87 50 35 70 68 66 5.kl 5.5 81.0 5.kl 103.680 k38.210 5o 2k & 0.015 2.18 5o 43 66 85 5o 25 50 47 56 5.15 5.3 85.2 5.15 110.536 438.780 50 23 k6 43 0.011 2.0k 50 39 78 77 50 3k 68 66 50 5.00 k . 9 8k.2 5.00 87.568 421.000 5o 22 kk k l 48 23 k6 0.000 - 5o 3 6 5 - - - - -50 3 6 k8 2 4 JfeS3.fJt 579.804 2497.037 x = 1.2791 y = 5.5086 l/Snw = 0.0022060 Snwx2 Snwxy Snwy2 b = 2.1925 751.761 3218.166 12827.365 740.690 3193.893 13755.115 Y = 2.70k2 + 2.1925x =11.071 s x v = 24.273 72.250 o 7 ^ zr{k) = 19.045 H.f. = 4.761 Sxx V( b) = 0.4300; S.E.^j = t 0.6550; b = 2.1925 - 0.6550. Log LD^Q = m = 1.0470; A n t i l o g m = 0.0110 mgm. V(m) = ° - ° ° 6 9 9 ; s - E - ( m ) = 1 0.0836; .'. m = 1.0470 - O.O836 g = 0.6910; P.L. = 0.5300 - 0.6650 = 1.1950 and 1.8650 Antilogs = 0.016 mgm. and 0.00073 mgm. 109 Table 2k. T o x i c i t y of d i e l d r i n to Musca domestica L.td(jf (SES culture) r p" P' P Empirical Y nw y nwx nwy Probit 31 62 62 7k 5.6k 5.6 83.1 5.6k 131.298 k68.68k 36 73 73 k3 86 86 k l 82 82 70 5.52 5.k 89.5 5.52 136.935 k9k.0k0 37 Z k 7k 26 53 53 k58.l50 27 5k 5k k6 k.90 5.2 93.5 k.90 138.380 17 3k 3k 2k k9 k 9 93.2 5.k8 133.276 5lk.736 kO 82 82 69 5-50 k .9 23 k8 k8 37 76 76 89.5 k .3k 121.720 388.k30 21 k3 k3 25 k.33 k .6 12 1, M-13 0 27 o 27 22 k.23 k .3 76.6 k.23 98.0k8 32k.018 k 8 8 1k 30 30 0 0 0 - - - - - - -0 0 0 0 525.k 759.657 26kk.Q58 x = l .kk59 y = 5.0325 l/Snw = 0.0019033 Snwx2 Sxwxy Snwy2 b = 5.0512 1103.398 38k7.825 13k70.629 1098.360 3822.377 13306.133 Y =-2.2710+5.05l2x =5.038 S x v = 25.tk8 Syy = 16k.k96 P * = 35.933 h.f. = 8.983 V ( b ) = 1.7830; S.E. ( b ) = t 1.3350; /. b = 5.0512 - 1.3350 Log LD^ Q = m = l .k390; Antilog m = 0.028 mgm. v(m) = 0.0006733; S.E. ( m ) = 0.0259;.'.". m = l.k390 - 0.0259 g = 0.5k8; P.L. = l.k310 t 0.1070 = 1.5380 and 1.32k0 Antilogs = 0.035 mgm. and 0.021 mgm. 110 Table 25. T o x i c i t y of heptachlor to Musca domestica L., QQ (SES culture) A x n r p" p' p Empirical Y nw y nwx nwy Probiit 0.060 2.78 50 43 86 86 89 6.23 6.5 39.1 6.16 69.598 240.856 50 k8 96 96 0.053 2.72 50 43 86 86 50 46 92 92 94 6.55 6.3 48.7 6.52 83.764 317.524 50 47 94 94 0.045 2.65 50 48 96 96 50 46 92 92 76 5.71 5.9 68.1 5.69 112.365 387.489 50 37 74 73 O.Okl 2.61 50 32 6k 63 k8 42 88 88 85 6.04 5.8 71.6 6.01 115.276 430.316 50 42 84 83 0.038 2.58 50 42 84 83 50 47 94 94 83 5.95 5.7 76.6 5.93 121.028 454-238 50 36 72 71 0.030 2.k8 50 42 84 83 50 36 72 71 47 4-93 5.2 89.3 4.92 132.164 439.356 50 13 26 24 50 24 48 46 0.000 - 50 2 4 3 - - - - - . - -50 1 2 50 1 2 393.4 634.195 2269.779 x = 1.6121 y = S%n l/Snw = 0.0025419 Snwx2 Snwxy Snwy2 b = 4*6686 1025.781 3674.973 13200.203 1022.377 3659.081 13095.822 Y = -1.7567+4-6686x S = 3.404 S = 15-892 S _ =104.381 xy JJ x 2 30.187 (4) h.f. = 7.547 V ( b ) = 2.2171; S.E.( b) = t 1.4890; .*. b = 4.6689 - 1.4890 Log LD^ 0 = m = 1.4473; A n t i l o g m = 0.030 mgm. v(m) = 0-003636; s - E - ( m ) = - 0.0603; .'. m = 1.4473 - O.O603 g =0.786; P. L. = 0.8k20 - 0.7044 = 1.5464 and 0.1376 Antilogs = 0.035 mgm. and 0.0014 mgm. I l l Table 26. T o x i c i t y of d i e l d r i n to Musca domestica L., g o (SES culture) A x n r P" P' P Empirical Y nw y nwx nwy Probit 0.068 2.83 50 ko 80 79 79 5.6k 5.7 76.6 5.6k lk0.178 432.024 50 3k 68 67 50 38 76 75 0.060 2.78 50 k2 8k 83 72 5.58 5.5 83.k 5.58 148.452 465-372 50 32 6k 63 50 35 70 69 462.574 0.053 2.72 50 28 56 55 57 5.18 5.2 89.3 5.18 133.596 50 26 52 50 _ 50 3k 68 67 5.0 0.049 2.69 l\B 27 56 55 47 k.92 88.7 k .93 149.903 437.291 50 18 36 3k 50 27 5k 53 87.1 k.97 Ik3 .7 l5 0.045 2.65 50 32 6k 63 k8 k .9k k .8 432.887 49 22 kk k2 _ 50 20 kO 38 0.038 2.58 50 19 38 36 26 k.36 k.k 75.2 4.36 118.816 327.872 50 13 26 2 k 50 10 20 18 0.000 50 2 4 3 - mm - mm mm mm -5o 1 2 5o 1 2 500.3 854.660 2558.020 x = 1.7083 y = 5.1130 l/Snw = 0.0019988 2 2 Snwx Snwxy Snwy b = 5.1156 1463.151 4385.915 13166.336 1460.011 4369^852 13079.085 Y = -3.6260+5.Il56x S__ = 3.140 S x y =16.063 Syy = 87.251 0 xx ™ xf = 5.079 (4) V ( b ) = ° - 3 i 8 5 ; s.E. ( b ) = 0.5640; b = 5 . n 5 6 ± 0.5640 Log L D ^ Q = m = 1.6862; A n t i l o g m = 0.048 V( m) = 0.00008229; S.E.( m) = -.0.0091; g = 0.2391; P.L. = 1.6793 - 0.0206 = 1.6999 and 1.6587 Antilogs = 0.050 mgm. and 0.046 mgm. 112 Table 27. T o x i c i t y of heptachlor to Musca domestica L.,(£o*> (SES culture) A x n r p" p* p Empirical Y nw y nwx nwy Probit 0.026 2.kl $0 k l 82 92 79 5-8l 6.0 65.8 5.79 92.778 380.982 50 kO 80 80 f?o 38 76 76 0.023 2.36 50 kk 88 88 85 6.Ok 5.9 70.7 6.03 96.152 k26.321 50 1+2 8k 8k 50 k l 82 82 0.019 2.28 50 3k 68 68 72 5.58 5.5 86.6 5.58 110.8k8 k83.228 k9 37 75 75 50 36 72 72 0.015 2.18 50 38 76 76 59 5.23 5.1 93.9 5.23 110.802 k91.097 k9 19 39 39 k9 31 63 63 0.011 2.0k 50 18 36 36 26 k.36 k . 5 86.0 k.36 89.kk0 37k.960 k8 9 19 19 50 11 22 22 0.000 - 99 0 0 0 - - - - -98 0 0 100 0 0  x = 1.2k07 y = 5.3513 l/Snw = 0.002k8lk Snwx2 Snwxy Snwy2 b = k.2703 627.233 270k.966 11676.277 620.397 2675.77k Il5k0.62k Y = 0.0531+k.2703x = 6.836 S„__ = 29.192 =135.653 ? xx xy w = 1 0 < 9 9 3 h.f. 3.66k. v ( b ) = 0-5360; S.E. ( b ) * i 0.7321; ,\ b = k.2703 - 0.7321 Log LD^Q = m = l « l 5 8 k ; A n t i l o g m = 0.01k mgm. V(m) ; ° - 0 0 ° 6 9 7 5 ; s - E - ( m ) = " 0.026k; .*. m = l . l 5 8 k - 0.26k g = 0.297; P.L. = 1.1236 - 0.1070 = 1.2306 and 1.0166 Antilogs = 0.017 mgm. and 0.010 mgm. 113 Table 28. T o x i c i t y of d i e l d r i n to Musca domestica L.,c£f, (SES culture) A x n r p" p' p Empirical Y nw y nwx nwy Probit 0.038 2.58 50 kO 80 80 81 5.88 5.9 74.5 5.88 117.710 438.060 50 42 84 84 50 39 78 78 0.034 2.53 50 40 80 80 80 5.84 5.8 76.9 5.84 117.657 449.096 50 4o 80 80 50 40 80 80 0.030 2.48 50 36 72 72 73 5.61 5.6 82.6 5.61 122.248 463.386 50 40 80 80 49 33 67 67 0.026 2.42.49 40 82 82 61 5-28 5-4 85.3 5.23 121.126 446.119 50 26 52 52 5o 24 48 48 0.023 2.36 50 27 54 54 55 5.13 5.2 85.3 5.13 116.008 437-589 50 30 60 60 5o 25 50 50 0.019 2.28 49 21 43 43 50 5-05 5-0 81.5 5-05 104.320 411-575 50 32 64 6k 50 24 48 48 0.000 - 99 0 0 0 - - - -98 0 0 .100 0 0  486.1 699.069 2645-825 x = 1.4382 y =5-4430 l/Snw = 0.0020572 Snwx2 Snwxy Snwy2 b = 3-1719 1010.224 3820.489 1 4 4 5 4 - 5 9 7 1005.343 3805.007 14401.131 Y = 0.8812+3-1719x S = 4.631 S = 15-482 S = 53-466 xx xy yy x 2 = k.359 (4) + V^ b ) = 0.2049} s - E - ( b ) * •* 0.4526} .". b = 3-1719 - 0.4526 Log LD^ 0 = m = 1.2985; A n t i l o g m = 0.020 mgm. v ^ = 0.0006019; S.E., . = 0.0245; .". m= 1.2985 - 0.0245 g =0.0782. Since g i s small, F.L. = (1.96)(.0245)= -0.048 on e i t h e r side of m = 1.34&5 and 1.2505 Antilogs = 0.023 mgm. and 0.018 mgm. I l k Table 29. T o x i c i t y of p.p.-DDT to Musca domestica L., 9 9 , (SES c u l t u r e ) . Residual contact a p p l i c a t i o n n r p» p Empirical Y Prob i t nw 200 2.30 l£u 65 43 43 160 2.20 150 64 43 43 120 2.08 150 60 40 40 97 1.99 150 59 39 39 80 1.90 150 69 46 46 73 1.86 150 69 46 46 65 1.81 150 54 36 36 57 1.76 100 34 34 34 44 1.64 i5o 5 i 34 34 35 1.54 i5o 47 31 31 20 1.30 150 65 43 43 15 1.18 150 69 46 46 13 1.11 150 39 26 26 9 0.95 150 32 21 21 7 0.85 150 30 20 20 0 - i5o 0 0 -4.82 4.9 95.2 4.82 4.82 4.8 94.1 4.82 4,75 4.8 94.1 4.75 .4.72 4-8 94.1 4-72 4.90 4.7 92.4 4.90 4.90 4.7 92.4 4.90 4.64 4.7 92.4 4.64 4,59 4.7 61.6 4.59 4.59 4.6 90.1 4.59 4.50 4.6 90.1 4.51 4.82 4 .5 87.2 4.85 4.90 4-5 87.3 4-93 4.36 4.5 87.2 4.36 4.19 4-4 83.7 4.21 4.16 4 4 83.7 4.18 nwx 218.960 207.020 195.728 187.259 175.560 171.864 I67.244 108.416 147.764 138.754 113.360 102.896 96.792 79.515 71.145 nwy 458.864 453.562 446.975 444.152 452.760 452.760 428.736 282.744 413.559 406.351 422.920 429.896 380.192 352.377 349.866 2182.277 6175.714 x = 1.6464 2 Snwx 3853.816 3592.858 S = 260.958 . y = 4-6592 1/Snw = 0.007540 Snwxy Snwy2 b = 0.3468 10258.061 28846.612 10167.573 28773.627 Y = 4.0882+0.3468x .485 S = 72.985 s x y = 90 x ^ l 3 ) = 41.608 h.f. = 3.201 V ( b ) = 0 - 0 1 2 3 ; s « E ' ( b ) = " 0.111; .\ b = 0.3468 - 0.1110 Log LTJ^Q = m = 2 . 6 2 9 2 ; Antilog m = k26 mgm. V, » = 1.7747; S.E., . = - 1.3510; .\ m = 2.6292 - 1.3510 vm; (m; g = 0.1108 + P.L. = 2.6169 - 0.6798 = 3.2967 and 1.9371 Antilogs = 1980 mgm and 87 mgm. 115 Table 30. T o x i c i t y of p.p.-DDT to Musca domestica L., (SES cu l t u r e ) . Residual contact a p p l i c a t i o n A x n r p» p Empirical Y nw y nwx nwy Probit 200 2.30 150 95 63 63 5 . 33 5 . 0 95.5 5 -33 219.650 409.015 160 2.20 150 60 kO kO k.75 5 . 0 95.5 k.75 210.100 453.625 120 2.08 150 kO kO kO k.75 k .9 95.2 k . ? 5 198.016 452.200 97 1.99 100 37 37 37 k.67 k .9 95.2 k.67 189.448 4 4 4 - 5 8 4 73 1.86 150 69 k6 k6 4 . 90 k . 9 95.2 k.90 177.072 k66.k80 65 1.81 150 81 5k 5k 5 . 10 4 . 8 9k.1 5 . 1 1 170.321 480.851 57 1.76 150 65 43 43 k.82 k .8 9k.1 k.82 165.616 k53.562 27 l . k 3 150 56 37 37 4 . 6 7 4 - 7 92.4 4 . 6 7 132.132 431.508 15 1.18 150 51 34 34 4 . 5 9 4 . 6 90.1 4 . 5 9 106.318 413.559 0 - 150 0 0 - - -, 847.3 1568.673 4105.384 x = 1.8514 , y = 4-8453 l/Snw = 0.0011802 Snwx2 Snwxy Snwy2 b = O.363O 2999.813 7635.326 19934.374 2904.207 7600.619 19891.620 Y = 4.1732+0.3630x S ^ = 95.606 Sxy = 34.707 S = 42.754 p xx ^ yy x ^ = 30^55 h.f. = 4 .308 v(b> = 0 . 0 4 5 1 ; S.E.( b ) = - 2 1 2 2 ; .'. b = O.363O - 0 . 2 1 2 2 Log LD^ 0 = m = 2.2777; A n t l l o g m = 190 mgm. V( m) = 0 . 1 0 0 5 5 ; s.E. ( m ) = i 0.3170; .'. m = 2.2777 - 0.3170 g = 1.9010 Since g i s so large, i t i s impossible to calculate f i d u c i a l l i m i t s . 116 Table 31. T o x i c i t y of p.p.-DDT to Musca domestica L., op , (SES c u l t u r e ) . Topical application A X n r P P Empirical Y nw Probit 5o.o 1.70 30 15 50 k6 4.90 5.2 16. 7 40.0 1.60 30 21 70 68 5.47 5.2 16. 7 30.0 1.48 20 12 60 57 5.18 5.1 11. 1 20.0 1.30 30, 12 40 36 '4.64 5.1 16. 7 i5.o 1.18 30' 17 57 § k 5.io 5.1 16. 7 10.0 1.00 20 11 55 52 5.05 5.0 11. 1 7.0 0.85 30 18 60 57 5.18 5.0 16. 6 6.5 0.81 30 15 5o 46 4-90 5.0 16. 6 6.0 0.78 30 12 40 36 4-64 5.0 16. 6 5.5 0.74 30 14 47 44 4.85 4-9 16. 4 5.0 0.70 30 18 60 57 5.18 4.9 16 •4 4-5 0.65 30 17 57 54 5.io 4-9 16. >4 4.0 0.60 30 19 63 60 5.25 4.9 16. 3.5 o.54 25 9 30 25 4-33 4-9 13 6 3.0 0.48 30 18 60 57 5.18 4-9 16. 4 2.5 o.4o 30 11 55 52 5.05 4.8 16 .0 2.0 0.30 30 14 47 44 4-85 4.8 16. .0 1.5 0.18 30 9 30 25 4-33 4.8 16 .0 1.0 0.00 29 12 40 36 4.64 4.7 14 .9 0.0 on* 2 7 MB - - a m J 4-90 5.46 5.18 4-65 5.10 5.05 5.18 4-90 4-65 4.85 5.18 5.io 5.25 4-37 5.18 5.05 4.85 4-36 4.64 nwx 45.090 43-420 27.528 38.4lO 36.406 22.200 30.710 30.046 29.548 28.536 27.880 27.060 26.240 20.944 24.272 22.400 20.800 18.880 14.900 nwy 81.830 91.18,2 57498 77.655 85.170 56.055 '85.988 81.340 77.190 79.540 84.952 83.64o 86.100 59.432 84.952 80.800 77.600 69.760 69.136 297.3 535.270 1469.820 x = 1.8004 Snwx2 1026.285 963.720 S x x ^ f e y = 4.9439 xy Snwxy 2661.989 2646.319 = 15.670 Snwy2 7291.643 7266.635 s y y l/Snw = 0.0033636 b = 0.2505 Y = 4.4929+0.2505* (17) = 21.083 V ( b ) = ° - 0 1 5 9 ; s« E.(b) = " ° « i 2 6 4 ; .'.13 = 0.2505 ± 0 . 1 2 6 4 Log L D ^ Q = m = 2.0244* A n t i l o g m = 10.57 If V(m) = ° - ° 6 6 3 3 j s - E - ( m ) = * 0.2575} /. m = 2.0244 - 0.2575 g =0.9729 Since g i s so large, P.L. cannot be calculated. 117 Table 32. T o x i c i t y of p.p.-DDT to Musca domestica L.,<3&", (SES culture). Topical application X X n 5o.o 1.70 30 40.0 1.60 30 30.0 l . k 8 30 20.0 1.30 30 10.0 1.00 30 7.5 0.88 30 .5.0 0.70 30 2.5 O.I4.O 30 2.0 0.30 20 1.5 0.18 30 1.0 0.00 30 0.0 - 30 p' p 22 73 70 2ii 80 78 2,5 83 82 24 80 78 21 70 67 24 80 78 12 40 36 15 50 46 11 55 52 17 57 54 14 47 43 2 7 -Empirlcal Y Probit 5.52 5.77 5.92 5.77 5.44 5.77 4-64 4.90 5.05 5.10 4.82 5.8 5.7 5.7 5.6 5.4 5.3 5.2 5.0 5.0 4.9 4.8 nw 13.8 14.5 14.5 15.2 16.2 16.5 16.7 16.6 11.1 16.4 16.0 5.50 5.77 5.90 5.76 5.44 5.73 4.64 4.90 5.05 5.10 4.82 nwx 37.260 37.700 34-960 34-960 32.500 31.020 28.390 23.240 14.430 19.352 16.000 nxy 75.900 83.665 85.550 87.552 88.128 94.545 77.488 81.340 56.055 93.640 77.120 167.5 310.712 890.983 x = 1.8550 y = 5.3193 Snwx Snwxy Snwy^ 629.722 1684.287 4769.871 576.370 1652.771 4739.407 S = 53-352 S = 31.516 S w =30.1*64 77 1/Snw = 0.0059701 b = O.5907 Y = 4.2234+0.5907x X 2 = 24.534 (9) h.f. = 2.726 V ( b ) = ° ' 0 ^ ' S - E * ( b ) = - ° - 2 2 6 S ; .*. b = 0.5907 - 0.2265 Log LD^ 0 = m = 1.3145; A n t l l o g m = 2.063 V(m) = ° ' ° 8 9 4 ; s « E . ( m ) = * 0.2989; .'. m = 1.3145 - 0.2989 g = 0.7483 F i d u c i a l l i m i t s impossible to work out due to large difference between m and x and size of g. 118 Table 33. T o x i c i t y of p.p.-DDT to Musca domestica L.,QQ, (Ottawa cul t u r e ) . Topical application A x n r p" P ! p Empirical Y nw y nwx nwy Probit 0.138 1.1k 10 9 90 90 90 6.28 6.2 11.1 6.28 23.754 69.708 10 9 90 90 10 9 90 90 0.125 1.10 10 9 90 90 79 5.81 6.0 13.2 5.79 27.720 76.k28 10 8 80 79 10 7 70 69 0.113 1.05 10 9 90 90 86 6.08 5.8 15.1 6.05 30.955 91.355 10 9 90 90 10 8 80 79 0.100 1.00 10 8 80 79 72 5.58 5.6 16.7 5.58 33.400 93.186 10 6 60 59 0.088 2.95 10 8 80 79 10 6 60 59 55 5.13 5.3 18.5 5.12 36.075 94.720 10 4 kO 38 0.075 2.88 10 7 70 69 10 5 50 48 42 4.80 5.0 19.1 4.80 35.908 91.680 10 2 20 18 0.050 2.71 10 6 60 59 10 2 20 18 31 4.50 4.3 16.0 7.52 27.360 72.320 10 4 kO 38 10 4 I4.O 38 0.000 - 10 1 10 3 - - - - -10 0 0 10 0 0 109.7 215.172 589.397 x = 1.9615 y = 5.3728 1/Snw = 0.0091158 2 2 Snwx Snwxy Snwy b = 4 « 2 l 5 l 423.943 1164.054 3204.876 k22.05l 1156.078 3166.717 y =-2.8951+4.2l5lx S„_ = 1.892 = 7.975 S =35.159 P " ^ 7 7 x 2 5 ) = 4.544 v ( b ) = 0.5285; s . E . ( b ) = - 0.7270; .*. b = 4.2151 - 0.7270 Log LD^ 0 = m = 1.8731; Antllog m = 0.075 X V(m) = 0 * 0 0 ° 7 4 6 ; S - E « ( m ) - " 0.0273 g = 0.1143; P.L. = 1.8617 - 0.0580 =1.9197 and 1.8037 Antilogs = O.O83 * and O.O64V 119 Table 3k. T o x i c i t y of p.p.-DDT to Musca domestica L., (Ottawa c u l t u r e ) . Topical application X X n r p" p' P Empirical Y nw 7 nwx nwy Probit 0.063 2.79 10 8 80 79 76 5.71 6.1 11.7 5.62 20.943 65.754 10 8 80 79 2.70 10 7 70 69 0.050 10 8 80 79 83 5.95 5.7 15.3 5.93 26.010 90.729 10 9 90 90 2.58 10 8 80 79 0.038 10 8 80 79 66 5.1+1 5.3 17.6 5.kl 27.808 95.216 10 5 50 49 2.k0 10 7 70 69 0.025 10 7 70 69 55 5.13 k-1 17.1 5.14 23.940 87.894 10 3 30 28 2.38 10 7 70 69 0.02k 10 6 60 59 42 k.80 k . 6 16.5 k . 8 l 22.770 79.365 10 3 30 28 2.32 10 4 ko 38 59.100 0.021 10 1 10 7 12 3.83 k-k 15.0 3.9k 19.800 10 0 0 0 2.30 10 3 30 28 0.020 10 3 30 28 lk 3.92 4-3 l k . l 3.97 18.330 55.977 10 1 10 7 10 1 10 7 0.000 - 10 1 10 3 - - - - - - -10 0 0 10 0 0 107.3 159.601 534.035 x = 1.4874 Y = 4-9770 l/Snw = 0.0093197 2 2 Snwx Snwxy Snwy b = 3.6179 240.546 805.738 2711.283 237.395 794.338 2657.907 Y = -0.4043+3.6l79x S x x = 3.151 -11.400 Syy = 53.376 2 h.f. = 2.4264; V ( b ) = ° .7700; s . E . ( b ) * t 0.8774; b = 3.6179 - 0.5774 Log LD^ 0 ss m = 1.4938; A n t i l o g m = 0.031 * v ( m ) = 0.001730; s . E . ( m ) = ± 0.0415; /. m = 1.4938 * o . o 4 i 5 ; g = 1.601; F.L. = 1.4950 i 0.0749 = 1.5699 and 1.4201; Antilogs = 0.037 ^  and 0.026 If 120 Table 35. T o x i c i t y of p.p.-DDT to Musca domestica L. QQ (Ottawa cul t u r e ) . Topical application A X n r P R T P 1 P Empirical Y nw y nwx Probit 0.15I 1.20 10 9 90 90 90 6.28 6.2 11.1 6.28 2k.k20 10 9 90 90 10 9 90 90 0.150 1.18 10 9 90 90 87 6.13 6.1 12.1 6.13 26.378 10 9 90 90 1.09 10 8 80 80 0.125 5 3 30 30 70 5.52 5.6 ll+.O 5.52 29.260 10 6 60 60 1.05 10 9 90 90 0.113 10 5 50 50 67 5.1+k 5.1+ 18.0 5.1+1+ 36.900 10 8 80 80 1.00 10 7 70 70 0.100 10 5 5o 50 63 5.33 5.2 18.8 5.33 37.600 10 5 5o 50 2.97 10 9 90 90 0.094 10 5 5o 50 1+7 k.92 5.0 19.1 1+.93 37.627 10 2 20 20 10 7 70 70 0.000 - 10 0 0 0 - - - - - • -10 0 0 10 0 0 93.1 192.185 513.,'1+1+8 x = 2.06k3 y = 5.5l50 l/Snw = 0.01071+11 Snwx2 Snwxy Snwy b = 5.2907 397.351 1063.215 2850.029 396.725 1059.903 2831.67k Y = 1+066+5.2907x S = 0.626 S__ = 3.312 S — = 18.355 ? 7 7 x 2 k ) = 0.833 ¥ ( b ) = 1*S97k; S.E. ( bj --t 1.2630; .'. b = 5.2907 1.2630 Log LD = m = 1.9670; Antllog m = 0,099 * 50 V(ra) = 0.000923i+; S.E. ( m ) = t 0.0303; m = 1.9670 - 0.303 G = 0.2192; P.L. = 1.9397 - 0.0727 = 2.012k and I.867O Antilogs = 0.103 i and 0.071+ H 121 Table 36. T o x i c i t y of p.p«-DDT to Musca domestica I>.,d(f, (Ottawa c u l t u r e ) . Topical application X X n r p" p» P Empirical Y nw y nwx •nwy Probit 0.138 T.Ik 10 9 90 90 97 6.88 6.8 5-4 6.88 ii . 5 5 6 37.152 10 10 100 100 10 10 100 100 0.125 1.10 10 9 90 90 93 6.k8 6.5 8.1 6.k8 17.010 52.ij.88 10 10 100 100 1.05 10 9 90 90 0.113 10 8 80 80 80 5.8k 6.0 13.2 5.83 27.060 76.956 10 8 80 80 10 8 80 80 0.100 1.00 10 6 60 60 73 5.61 5.6 16.7 5.61 33-400 93.687 10 9 90 90 2.95 10 7 70 70 0.088 10 6 60 60 67 5 - 4 4 5.2 18.8 5-43 36.660 102.08k 10 8 80 80 2.88 10 6 60 60 0.063 10 3 30 30 27 k.39 4-5 17.k k.39 32.712 76.386 10 k kO kO 10 1 10 10 0.000 mm 10 0 0 0 10 0 0 10 0 0 79.6 158.398 I138.753 x = 1.9899 y = 5.5120 l/Snw = 0.0125628 Snwx2 Snwxy Snwy2 b = 8.7235 315.710 877.534 2459.616 315.200 87,3.085 2418.394 Y = -11.8469+8.7235x = 0.510 = 4.449 Syy =kl.222 g x (4) = 2 ' k 1 1 ^xx V ( b ) = 1.9608; S.E.(fc) = t i .k020; .*. b = 8.7235 1 1 - 4 0 2 0 Log LD^ 0 = m = 1.9312; A n t i l o g m = 0.085 V •V(m) = ° - 0 0 0 2 5 3 ; S - E ' „ ( m ) = ~ 0.0159; .". m = 1.9312 - 0 . 0 1 5 1 ; g = 0.0963; F.L. = 1.9249 - 0.0334 = 1-9583 and 1.8915; Antilogs = 0.091 V and 0.078V 122 Table 37. T o x i c i t y of p.p'-DDT to Musca domestica L . , Q Q , (Ottawa cul t u r e ) . Residual contact application A x n r p" p» P Empirical Y nw y nwx nwy Probit 2.06 0.31k 50 33 66 65 7k 5.6k 5.8 75.k 5.63 99.076 k2k.502 50 Ij.6 92 92 50 33 66 65 0.57 0.272 k7 35 75 75 70 5.52 5.k 88.3 5.52 112.318 k87.kl6 50 36 72 71 50 32 6k 63 95.2 k.75 117.667 1.72 0.236 50 17 31+ 33 kO . k.75 5.1 452.200 50 20 kO 39 50 2k k8 k? 1.50 0.176 50 23 k6 5.5 35 k.62 4.5 87.2 k.62 102.547 k02.86k 50 21 i[2 i j l 50 10 20 18 1.13 0.053 50 5 10 8 13 3.87 3.3 31.2 k.21 32.85k 131.352 50 10 20 18 50 7 l k 12 0.00 - ij.9 0 0 2 - - - - -50 3 6 50 0 0 377.3 k6k.k62 1898.33k x = 1.2310 y = 5.031k l/Snw = 0.002650k Snwx2 Snwxy Snwy2 b = 10.4598 572.kk5 23kk.0k3 96k2.656 571.760 2336.878 9551.211 Y = -7.8kk6+10.k598x S_„ =0.685" S = 7.165 S =91.kk5 p x^ 3 ) = 16.500 xx ^ xy 77 y h.f. = 5.500 V ( b ) = 8.0292; s « E . ( b ) - - 2.8335; .'. b = 10.J+598 - 2.8335 Log LD^ 0 = m = 1.2280; A n t l l o g m = 0.169 mgm. V(m) = 0.00013k0; S . E . ^ = - 0.0115; m = 1.2280 - 0.0115 g = 0.9907; P. L. = 0.908k * .k706 = 1.3790 and 0.k378 Antilogs = 2.393 mgm. and 0.27k mgm. 123 Table 38. T o x i c i t y of lindane to Musca domestica L.,QQ, (Ottawa cul t u r e ) . Residual contact application A x n r p" p* p Empirical Y nw y nwx nwy Probit 0.053 2.34 50 2k k8 k8 71 5-55 5.6 83.1 5-55 111.354 461.205 49 43 87 87 _ 50 39 78 78 0.045 2.28 50 33 66 66 69 5-50 5.2 94-1 5-48 120.448 515.668 50 31 62 62 50 4o 80 80 O.038 2.24 50 32 64 64 5 l 5.02 5-0 95.5 5.03 118.420 480.365 50 15 30 30 _ 50 30 60 60 0.030 2.20 50 9 18 18 37 4.69 4.7 92.4 4.67 110.880 431.508 50 16-32 32 50 30 60 60 0.000 50 0 0 0 - - - -50 0 0 50 0 0  - . 365.1 461.102 1888.746 x = 1.2629 y = 5.1732 l/Snw = 0.0027389 2 2 Snwx Snwxy Sxwy b = 6.5603 583.284 2391.533 9816.927 582.347 2385.386 9770.916 Y = -3XLl8+6.5603x S x x = ° ' 9 3 7 S x y = 6.1k 7 S y y =46.011 A = 5.68k (2) V ( b ) = 1.0672; S.E. ( b ) = ± 1.0337; .". b = 6.5603 - 1.0337 Log LD^ 0 = ra = 1.2365; Antilog m = 0.017 mgm. = 0.0000808; S.E., . = - 0.0089; m = 1.2365 - 0.0089 m^; (m) g = 0.0952; Since g i s small, P.L. = (.0089)(1.96) on it h e r side of m = 1.2365 - 0.0174 = 1.2539 and 1.2191 Antilogs = 0.018 mgm. and 0.016 mgm. e 12k Table 39. T o x i c i t y of heptachlor to Musca domestica L.,0^> (Ottawa cul t u r e ) . Residual contact a p p l i c a t i o n anplrical x nw y nwx nwy Probit 5.47 5.3 90.1 5.46 133.348 491.946 n r P M P* P 5o 30 60 60 68 46 29 63 63 5o 40 80 80 5o 25 50 50 43 5o 25 5o 5o 5o 15 30 30 5o 22 44 44 32 5o 18 3? 36 5o 8 16 16 5o 16 32 32 31 5o 26 52 52 5o 4 8 8 5o 0 0 0 50 0 0 5o 0 0 4.53 4.7 92.4 4.54 125.664 419.496 357.7 496.638 1732.957 x = 1.3884 y = 4.8447 l/Snw = 0.0027956 2 p Snwx Snwxy Snwy*- b = 4«°366 691.405 2414.703 8446.750 689.542 2406.065 8395.636 Y = -1.5928 + 7.6366X S =1.863 S = 8.636 S =51.114 2 xx y y X f = 11.063 (2) h.f. 5.532 V ( b ) = 2.9708; S.E. ( b ) = t 1.7236;.". b = 4.6366 - 1.7236 Log LD^ 0 = m = 1.4219; Antilog m = 0.026 mgm. V. , = 0.0008733; S.E., , = t 0.0295 g = 2.5535; F.L. cannot be calculated due to large g value. 

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