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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 t h e s i s as conforming t o the standard r e q u i r e d  from c a n d i d a t e s f o r the  degree o f MASTER OF ARTS  Members o f the Department o f  THE UNIVERSITY OF BRITISH COLUMBIA April,  1956  ii  ABSTRACT A method o f b i o a s s a y d i s t i n g u i s h i n g r e s i d u a l c o n t a c t from fumigant t o x i c i t y i s d e s c r i b e d .  B a s i c a l l y the apparatus  c o n s i s t s o f a s e r i e s o f Buchner f u n n e l s s e t up i n s e r i e s . Woven f i b e r g l a s s c l o t h was s e l e c t e d as the substratum. By s u b s t i t u t i o n of a dye i n p l a c e o f I n s e c t i c i d e , i t was p o s s i b l e ,  using  c o l o r l m e t r i c a n a l y s i s , t o c a l c u l a t e the t o t a l m i l l i g r a m s o f dye  adhering t o the c l o t h .  I t was assumed t h a t  proportionate  amounts o f dye and i n s e c t i c i d e would be p i c k e d up. Musea domestica L.was used as the t e s t i n s e c t .  Two  s t r a i n s o f f l i e s , designated as the SES and Ottawa c u l t u r e s , were used. Fumigant e f f e c t was e l i m i n a t e d by a p p l i c a t i o n o f negative pressure.  Since fumigant e f f e c t i s p r o p o r t i o n a t e  to vapour p r e s s u r e , the r a t e o f e v a c u a t i o n v a r i e d f o r each insecticide.  Elimination  a corresponding  decrease  o f fumigant e f f e c t brought about i n mortality.  Dosage-mortality  data a r e g i v e n f o r s i x i n s e c t i c i d e s  i n comparison t o a "standard" i n s e c t i c i d e ( d i e l d r i n ) u t i l i z i n g the concept  o f t o x i c i t y index ( Sun, 195>0).  Statistical  a n a l y s i s o f the data i n d i c a t e s i g n i f i c a n t h e t e r o g e n e i t y i n eleven out o f twenty-four Chi-square  experiments.  An a n a l y s i s o f the  f u n c t i o n i s presented. Dosage-mortality  data f o r DDT a r e g i v e n . The SES  c u l t u r e was determined t o 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 c u l t u r e , by  iii topical application.  Residual contact a p p l i c a t i o n  t h a t females of the SES  indicated  c u l t u r e were 2$2x as r e s i s t a n t com-  pared to the Ottawa c u l t u r e . Variation within  the SES  c u l t u r e i s d i s c u s s e d as  f a c t o r i n demonstrating the s e n s i t i v i t y o f the  technique.  a  iv ACKNOWLEDGEMENTS A p r o j e c t o f t h i s type i s one i n which the complete c o o p e r a t i o n o f a l l i n v o l v e d i s necessary  to bring i t to a  satisfactory conclusion. Acknowledgement i s made t o the f o l l o w i n g chemical companies f o r s u p p l y i n g samples o f the t e s t American Cyanamid Company: p a r a t h i o n ,  insecticides: malathion.  C a l i f o r n i a Spray Chemical C o r p o r a t i o n : l i n d a n e . The Dupont C o r p o r a t i o n : ETN. The Geigy C o r p o r a t i o n : DDT. S h e l l Chemical C o r p o r a t i o n : a l d r i n , d i e l d r i n , e n d r i n . V e l s i c o l Corporation: heptachlor. I should a l s o l i k e to acknowledge w i t h thanks, t h e h e l p o f t h e Defense Research Board i n p l a c i n g the f a c i l i t i e s of t h e S u f f i e l d Experimental  S t a t i o n a t my d i s p o s a l d u r i n g  the summers o f 1954 and 1955, and f o r t h e l o a n o f equipment d u r i n g t h e 195i+.-55 and 1955-56 Winter Sessions a t the U n i v e r s i t y o f B r i t i s h Columbia. Acknowledgement i s a l s o made to the B r i t i s h Columbia Academy o f Sciences f o r the Goethe Award f o r r e s e a r c h d u r i n g the Winter S e s s i o n 1954-55 and t o the N a t i o n a l Research C o u n c i l f o r the Studentship  awarded d u r i n g the Winter S e s s i o n  of 1955-56. I am indebted t o Dr. K. Graham f o r h i s r e g a r d i n g t h e development o f the experimental  suggestions  technique and  f o r h i s c o r r e c t i o n s and c r i t i c i s m s o f the manuscript.  V  Acknowledgement I s a l s o made to Dr. J . Sanjean f o r h i s c r i t i c i s m s o f the manuscript, and t o o t h e r s i n the Departments of Zoology and Chemistry f o r t h e i r a d v i c e from time t o time. Acknowledgement i s made t o D r . S. Nash f o r guidance In the s t a t i s t i c a l  analysis.  I am deeply i n d e b t e d t o Dr. H. H u r t i g , Head/ Entomology S e c t i o n , S u f f i e l d E x p e r i m e n t a l S t a t i o n , who f i r s t suggested the development o f t h i s p r o j e c t , and without whose h e l p and guldanoe,  completion would have been i m p o s s i b l e .  Acknowledgement i s made t o Mr. G. Humphreys, o f the S u f f i e l d E x p e r i m e n t a l S t a t i o n , f o r doing such an e x c e l l e n t j o b of r e a r i n g the l a r g e numbers o f i n s e c t s used i n t e s t i n g , and to Mr. C. Watson, f o r d r a f t i n g the graphs c o n t a i n e d i n the body o f t h e t h e s i s . I would e s p e c i a l l y l i k e to thank P r o f e s s o r G.J. Spencer,  t o whom I owe a g r e a t d e a l f o r h i s a d v i c e and  encouragement.  vi LIST OP FIGURES F i g u r e 1.  Standard curve f o r A n i l i n e Green dye d i s s o l v e d i n a benzene-mineral o i l (95:5) s o l v e n t mixture.  Figure  2.  E x t r a c t i o n o f A n i l i n e Green dye from 50 fiberglass  F i g u r e 3(A).  - 30$  range).  R e t e n t i o n o f 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  F i g u r e 2j..  cloth.  R e t e n t i o n o f 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  (B).  mgm.  - 0.12$  range).  T o x i c i t y o f d i e l d r i n t o Musca domestlca L. (SES c u l t u r e ) .  Figure  5.  T o x i c i t y o f d i e l d r i n t o Musca domestica L. (SES c u l t u r e ) .  F i g u r e 6(A).  T o x i c i t y o f p a r a t h i o n t o Musca domestica L. (SES c u l t u r e ) .  (B).  T o x i c i t y o f d i e l d r i n t o Musca domestica L. (SES c u l t u r e ) .  Figure 7(A).  T o x i c i t y o f EPN t o Musca domestica L. (SES c u l t u r e ) .  (B).  T o x i c i t y o f d i e l d r i n t o Musca domestica L. (SES c u l t u r e ) .  Figure 8(A).  T o x i c i t y o f e n d r i n t o Musca domestica L. (SES  (B).  culture).  T o x i c i t y o f d i e l d r i n t o Musca domestica L. (SES c u l t u r e ) .  vll F i g u r e 9(A).  T o x i c i t y o f a l d r i n t o Musca domestica L . (SES  (B).  T o x i c i t y o f d i e l d r i n t o Musca domestlea L. (SES  F i g u r e 10(A).  culture).  T o x i c i t y o f l i n d a n e t o Musca domestica L. (SES  (B).  culture).  culture).  T o x i c i t y o f d i e l d r i n t o Musca domestlea L . (SES c u l t u r e ) .  F i g u r e 11(A).  T o x i c i t y o f h e p t a c h l o r t o Musca domestica L. (SES  (B).  culture).  T o x i c i t y o f d i e l d r i n t o Musca domestlea L. (SES c u l t u r e ) .  F i g u r e 12.  T o x i c i t y o f DDT t o Musca domestica L. (SES c u l t u r e ) . R e s i d u a l c o n t a c t  F i g u r e 13(A).  DDT content o f benzene-mineral o i l (95:5) solution  (B).  (0 - 0.06$ r a n g e ) .  DDT content o f benzene-mineral o i l (95:5) solution  F i g u r e li|..  (0 - 30$ r a n g e ) .  T o x i c i t y o f DDT to Musca domestica L . (SES c u l t u r e ) . T o p i c a l  F i g u r e 15.  Topical  Topical  application.  T o x i c i t y o f DDT to Musca domestica L. (Ottawa c u l t u r e ) . Residual contact  F i g u r e 18.  application.  T o x i c i t y o f DDT to Musca domestica L. (Ottawa c u l t u r e ) .  F i g u r e 17.  application.  T o x i c i t y o f DDT t o Musca domestica L . (Ottawa c u l t u r e ) .  F i g u r e 16.  application.  application.  T o x i c i t y o f l i n d a n e and h e p t a c h l o r to Musca domestica L. (Ottawa  culture).  viii LIST OF PLATES Plate  I . ( a ) . Technique employed i n r e a r i n g Musca domestica L. ( b ) . View o f r e a r i n g room a t S u f f i e l d E x p e r i m e n t a l Station.  Plate  II.  A s e r i e s o f f r e s h l y cut f i b e r g l a s s c l o t h s t r e a t e d w i t h "Cenco" l a b e l v a r n i s h to prevent fraying.  P l a t e I I I . ( a ) . Method employed i n removing h o u s e f l i e s from cages. ( b ) . Buchner f u n n e l s  containing  $0 f l i e s  each,  demonstrating fumigant and  residual  contact  tests. Plate  IV.  (a).A t y p i c a l r u n 0  n  c o n s i s t i n g o f s i x dosage l e v e l s  (three r e p l i c a t e s each) and (b).Observation  chambers s e t up  a control series, i n series. Mortal-  i t y counts were made a t 2% and 1+8 hour i n t e r v a l s . Plate  V.  ( a ) . Components o f 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 effect.  Plate  V I . ( a ) . Technique used f o r t o p i c a l a p p l i c a t i o n o f ( b ) . F l i e s contained  In o b s e r v a t i o n  t o p i c a l a p p l i c a t i o n of  DDT.  DDT.  chambers a f t e r  TABLE OF CONTENTS Page Biography Abstract Acknowledgements L i s t of Figures L i s t of Plates I. II. III.  IV.  V.  i i i iv vi viii 1  INTRODUCTION HISTORICAL BACKGROUND  1+  MATERIALS .  12  Test i n s e c t  12  Insecticides  12 17  METHODS R e a r i n g technique  17  B i o a s s a y technique  20 36  EXPERIMENTAL DATA  36  E l i m i n a t i o n o f fumigant e f f e c t Dosage-mortality VI.  data  . .  DISCUSSION OF RESULTS  lj.1 . . .  63  R e a r i n g methods Strain of insects  VIII. IX.  £>l+  B i o a s s a y technique  66  S u s c e p t i b i l i t y o f males and females  70  Parallelism of probit-regression l i n e s  VII.  63  . . . .  71  R e s i s t a n c e o f SES c u l t u r e t o DDT  72  Homogeneity o f data  7k  S e n s i t i v i t y o f technique  78  CONCLUSIONS  80  BIBLIOGRAPHY  8l  APPENDIX  86  1 I.  INTRODUCTION The  field  o f i n s e c t i c i d e chemistry has expanded  c o n s i d e r a b l y d u r i n g the past f i f t e e n y e a r s .  P r e v i o u s to  World War I I , the I n s e c t i c i d e s c h i e f l y i n use were i n o r g a n i c compounds such as the a r s e n i c a l s and l i m e - s u l p h u r , and o r g a n i c compounds such as n i c o t i n e , pyrethrum, anabasine,  and  rotenone,  which are o f b o t a n i c a l o r i g i n , as w e l l as s y n t h e t i c s such as phenothiazine  and p-dichlorobenzene.  I n 1939, the Geigy  C o r p o r a t i o n s y n t h e s i z e d DDT ( d l c h l o r o d i p h e n y l t r l c h l o r o e t h a n e ) , and t h i s compound was o n l y the f i r s t o f a s e r i e s o f c h l o r i n a t e d hydrocarbon I n s e c t i c i d e s . I n 19^6, the f i r s t o r g a n i c phosphate i n s e c t i c i d e s , developed  d u r i n g the war by Schrader,  r e l e a s e d upon the market.  were  Examples o f these compounds are  TEPP ( t e t r a e t h y l p y r o p h o s p h a t e ) , p a r a t h i o n ( d l e t h y l - p - n i t r o p h e n o l thiophosphate),  and sehradan (octamethylpyrophosphoramide).  S i n c e t h i s time, many other o r g a n i c compounds have been found to  exhibit i n s e c t i c i d a l The  activity.  tremendous i n c r e a s e i n the number o f i n s e c t i c i d e s  n e c e s s i t a t e d the development o f more adequate methods o f evaluating I n s e c t i c i d a l The  activity.  e v a l u a t i o n o f a compound, e i t h e r c h e m i c a l l y o r  b i o l o g i c a l l y , requires f i r s t l y ,  a qualitative  estimate.  Q u a l i t a t i v e chemical a n a l y s i s determines e i t h e r the i o n s o r f u n c t i o n a l groups p r e s e n t i n the compound.  Qualitative  b i o l o g i c a l a n a l y s i s o r " s c r e e n i n g " determines the r e l a t i v e t o x i c i t y o r n o n - t o x i c i t y o f a compound t o i n s e c t s .  Complete  2 e v a l u a t i o n o f a compound n e c e s s i t a t e s a l s o a q u a n t i t a t i v e estimate.  Q u a n t i t a t i v e chemical a n a l y s i s c o n s i s t s o f a  d e t e r m i n a t i o n o f the p r o p o r t i o n s o f the c o n s t i t u e n t s p r e s e n t , while q u a n t i t a t i v e b i o l o g i c a l a n a l y s i s or "bioassay" c o n s i s t s of the d e t e r m i n a t i o n o f the p r o p o r t i o n a l t o x i c i t y o f a compound to c e r t a i n i n s e c t s , i n comparison w i t h a "standard"  insecticide.  A b i o a s s a y technique depends t o a l a r g e extent upon the type o f i n s e c t i c i d e t o be t e s t e d .  I n s e c t i c i d e s may be  c l a s s i f i e d i n t o three main c a t e g o r i e s a c c o r d i n g to t h e i r form and r o u t e o f e n t r y : fumigant p o i s o n s , stomach p o i s o n s , and contact poisons.  Contact poisons may be f u r t h e r s u b d i v i d e d  i n t o d i r e c t c o n t a c t p o i s o n s and r e s i d u a l c o n t a c t p o i s o n s .  If  we a r e to be s u c c e s s f u l i n t e s t i n g 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 o f b i o a s s a y f o r each type o f p o i s o n . Furthermore, i t has been shown t h a t i n s e c t i c i d e s are somewhat s p e c i f i c i n r e s p e c t t o t h e i r t o x i c i t y to a given insect species.  F o r example, DDT i s v e r y t o x i c t o  h o u s e f l i e s , but much l e s s so t o cockroaches.  Hence i t i s  n e c e s s a r y t h a t s e v e r a l i n s e c t s be used b e f o r e a compound can be c o n s i d e r e d as f u l l y t e s t e d . There are a l r e a d y numerous methods o f b i o a s s a y f o r each type o f p o i s o n , b u t the m u l t i p l i c i t y o f methods renders it  i m p o s s i b l e t o compare data o b t a i n e d by one i n v e s t i g a t o r  w i t h those o b t a i n e d by o t h e r s .  The consequent need f o r  s t a n d a r d i z a t i o n i s t h e r e f o r e b e i n g r e c o g n i z e d by many Investigators.  3 I t has been the o b j e c t o f t h i s r e s e a r c h p r o j e c t to develop a method o f b i o a s s a y f o r the r e s i d u a l c o n t a c t  toxicity  of i n s e c t i c i d e s , w i t h the view t h a t i t w i l l be o n l y the f i r s t step toward the s t a n d a r d i z a t i o n o f a t e s t i n g  technique.  4 II.  HISTORICAL BACKGROUND The development o f b i o a s s a y techniques f o r fumigant  poisons have proven to be much l e s s i n v o l v e d than techniques f o r stomach and c o n t a c t p o i s o n s .  I n most cases, the  apparatus  used i n v o l v e s a c l o s e d chamber i n t o which the v o l a t i l i z e d i n s e c t i c i d e I s passed  (Cotton, 1943).  The b e s t known b i o a s s a y technique f o r comparing the t o x i c i t y o f stomach poisons i s the poisoned l e a f "sandwich" technique (Campbell and F i l m e r , 1929).  Numerous m o d i f i c a t i o n s  o f t h i s technique have been d e v i s e d (Hansberry, 1943). A r e l a t e d technique i n v o l v e s f e e d i n g to i n s e c t s measured drops of l i q u i d o r food c o n t a i n i n g known amounts o f i n s e c t i c i d e (Pearson and Richardson, 1933;  Sun, 1953).  Still  another  technique i n v o l v e s i n j e c t i o n s Into the a l i m e n t a r y c a n a l (Hansberry et a l , 1940)• The development o f s a t i s f a c t o r y b i o a s s a y techniques f o r d i r e c t c o n t a c t t o x i c i t y has presented many  problems.  However, c o n s i d e r a b l e 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 o f b i o a s s a y are now a v a i l a b l e . One  o f the f i r s t techniques developed was the  spray chamber method (Peet and Grady, 1928).  Peet-Grady During the suc-  ceeding y e a r s , t h i s method has been m o d i f i e d , but i s s t i l l i n use i n many l a b o r a t o r i e s concerned w i t h the e v a l u a t i o n o f household  spray i n s e c t i c i d e s .  U s i n g t h i s technique, i t i s  p o s s i b l e to e v a l u a t e the t o x i c i t i e s o f i n s e c t i c i d e s by r e f e r e n c e 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  A s s o c i a t i o n (GSMA).  The present standard c o n s i s t s o f a mixture o f p y r e t h r i n s I and I I , which has been c a r e f u l l y s t a n d a r d i z e d by both and b i o l o g i c a l a n a l y s i s (Blue Book, 1953).  chemical  I t i s necessary  that the t e s t I n s e c t s be r e a r e d under c o n d i t i o n s s e t up by the CSMA.  A l l t e s t s must be c a r r i e d out u s i n g t h e standard  Grady spray chamber.  Peet-  Numerous other spray chamber methods  have s i n c e been developed  (Campbell  and S u l l i v a n , 1938J  P o t t e r , 19ip.). A second type o f b i o a s s a y technique f o r e s t i m a t i n g d i r e c t contact t o x i c i t y i s that o f i n j e c t i o n .  Campbell (1932)  i n j e c t e d i n s e c t i c i d e s o l u t i o n s d i r e c t l y into the blood  stream  o f the i n s e c t u s i n g as an i n j e c t i o n p i p e t t e , a c a p i l l a r y drawn t o a very f i n e p o i n t .  tube  Since t h i s time t h e r e have been  numerous m o d i f i c a t i o n s o f i n j e c t i o n equipment, i n v o l v i n g t h e use o f hypodermic needles and micrometer heads graduated i n very a c c u r a t e volumes (Yeager and Munson, 19l|.5; H e a l and Menusan, 19l|.8) .  A t p r e s e n t many s c r e e n i n g techniques i n v o l v e  i n j e c t i o n o f t h e i n s e c t i c i d e i n t o the i n s e c t as a primary  test,  s i n c e c o m p l i c a t i n g f a c t o r s such as s o l u b i l i t y , p e r m e a b i l i t y , and o t h e r p h y s i c o - c h e m i c a l f a c t o r s may be avoided. Other types o f b i o a s s a y f o r d i r e c t c o n t a c t 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 o f the i n s e c t i c i d e (0»Kane e t a l , 1933; March and M e t c a l f , 191*9')» and immersion o f the i n s e c t i n t o a s o l u t i o n o r emulsion o f t h e i n s e c t i c i d e  19ii-7, 1949).  (Mcintosh,  6 Some methods o f b i o a s s a y f o r the d e t e r m i n a t i o n o f r e s i d u a l c o n t a c t t o x i c i t y have a l s o been developed  (Stringer,  1949J Krijgsman, 1949; Proverbs and M o r r i s o n , 1947; Barnes, 1945;  Hoskins e t a l , 1952).  However, most i n v e s t i g a t o r s have  made no attempt t o d i f f e r e n t i a t e between r e s i d u a l c o n t a c t t o x i c i t y and combined r e s i d u a l contact-fumigant t o x i c i t y .  Investi-  gators attempting t o e l i m i n a t e the fumigant e f f e c t have met w i t h little  success.  Pradhan  (1949) attempted to e l i m i n a t e the  t o x i c vapours b y e v a c u a t i n g fumes upward through a f u n n e l p l a c e d over the impregnated  substratum.  He found t h a t fumigant  e f f e c t c o u l d n o t be e l i m i n a t e d i n t h i s way. One o f t h e main f a c t o r s i n the development o f a technique o f t h i s type i s the method o f a p p l i c a t i o n o f t h e i n s e c t i c i d e t o the substratum.  One o f the most common methods  o f a p p l i c a t i o n i s t o t r e a t t h e substratum i n a spray tower, such as f o r example, a P o t t e r tower ( P o t t e r , 1941) •  sub-  I E n e  stratum i s covered w i t h a u n i f o r m , known c o n c e n t r a t i o n o f spray, removed, and p l a c e d w i t h i n the t e s t i n g apparatus.  A  second method o f a p p l i c a t i o n o f i n s e c t i c i d e c o n s i s t s o f dis-* 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 v e n t , p l a c i n g I t w i t h i n a c o n t a i n e r , and s w i r l i n g u n t i l t h e s o l v e n t evaporates (Hamraan, 1949; Krijgsman, 1949; Hoskins e t a l , 1952).  A third  method i n v o l v e s 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 vent, and e i t h e r d i p p i n g the substratum I n t o the s o l u t i o n (Proverbs and M o r r i s o n , 1947), o r p i p e t t i n g the s o l u t i o n d i r e c t l y on t o the substratum ( S t r i n g e r , 1949).  sol-  7 C e r t a i n other f a c t o r s must a l s o he taken i n t o c o n s i d e r a t i o n b e f o r e an a c c u r a t e method of b i o a s s a y can be developed.  F o r example, some i n v e s t i g a t o r s have  a d e f i n i t e r e l a t i o n s h i p between temperature  demonstrated  and humidity,  and the r a t e o f knockdown ( L i n d q u i s t e t a l , 1914-5; 19l|6; Dakshinamurity, Few  19lj-8; Pradhan, 1949;  T e o t i a e t a l , 1950).  d e f i n i t e data have been obtained, and a t p r e s e n t t h e r e  i s c o n s i d e r a b l e c o n f l i c t i n g evidence.  In g e n e r a l , 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 s e l e c t e d depend e n t i r e l y upon the c o n d i t i o n s a c c e p t able t o the t e s t i n s e c t s , but i t i s a b s o l u t e l y n e c e s s a r y t h a t these c o n d i t i o n s be maintained a t a l l times  throughout  testing. Wot  o n l y the temperature-humidity  relationship,  but a l s o the p h y s i c o - c h e m i c a l c h a r a c t e r i s t i c s o f an i n s e c t icide affect toxicity.  There i s a d e f i n i t e  relationship  between t o x i c i t y and p a r t i c l e s i z e and shape (McGovran e t al,  191+0; Mcintosh, 19l|-7, 194,9, 195D  makes the f o l l o w i n g statement:  "The  •  S t r i n g e r (191*9)  experiments w i t h  d e p o s i t s from a l c o h o l and acetone on No.  1 and No.  DDT  50  Whatman f i l t e r papers i n d i c a t e t h a t the s o l v e n t and the n a t u r e o f the s u b s t r a t e e x e r t an e f f e c t upon the d e p o s i t which i s r e f l e c t e d i n the degree o f m o r t a l i t y .  I t i s obvious  t h a t the a v a i l a b i l i t y o f the d e p o s i t depends upon the c r y s t a l s i z e and shape, the d i s t r i b u t i o n on the f i l t e r paper, 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 . "  and  8 P a r t i c l e s i z e and shape depends t o a l a r g e  extent  upon the s o l v e n t used, and the method o f r e c r y s t a l l i z a t i o n (Mcintosh,  1947, 1949).  The s o l v e n t used may be v o l a t i l e ,  f o r example, acetone o r a l c o h o l , o r i t may be a n o n - v o l a t i l e o i l such as m i n e r a l  o i l or o l i v e o i l .  S t r i n g e r (1949) has  noted t h a t an i n s e c t i c i d e a p p l i e d i n an o i l s o l u t i o n i s much more t o x i c than a s i m i l a r c o n c e n t r a t i o n  i n c r y s t a l l i n e form.  He a t t r i b u t e d t h i s i n c r e a s e i n t o x i c i t y t o the " s t i c k i n g c h a r a 6 t e r i s t i c o f the o i l . More r e c e n t l y , i t has been  1 1  postu-  l a t e d t h a t the i n c r e a s e d t o x i c i t y o f i n s e c t i c i d e a p p l i e d i n o i l f i l m i s due t o the f a c t t h a t the o i l i s n e a r e r t o the chemical composition o f the o u t e r , impenetrable s u r f a c e o f the c u t i c l e , and hence p e n e t r a t i o n and a b s o r p t i o n i n s e c t i c i d e w i l l be much s i m p l e r .  o f the  When an i n s e c t i c i d e i s  a p p l i e d i n a c r y s t a l l i n e f i l m , i t i s n e c e s s a r y t h a t the c u t i c u l a r o i l s d i s s o l v e the compound before p e n e t r a t i o n i s p o s s i b l e . S t r i n g e r (1949) has determined t h a t the slope o f the dosager e g r e s s i o n l i n e i s p r o p o r t i o n a l t o the o i l f i l m  thickness.  To e l i m i n a t e the d i f f e r e n t i a l e f f e c t o f t h i s o i l f i l m t h i c k ness, he used one o i l dosage and v a r i e d the c o n c e n t r a t i o n o f the  insecticide. I n v e s t i g a t o r s have a l s o found t h a t n o t o n l y are the  physico-chemical  c h a r a c t e r i s t i c s o f the i n s e c t i c i d e o f  importance, b u t a l s o t h a t I t I s n e c e s s a r y t o " s t a n d a r d i z e " the t e s t i n s e c t s .  F o r example, the age o f the i n s e c t s used  In t e s t i n g i s important (Sun, 1950). McLeod (1944) s t a t e s : " I t w i l l be n e c e s s a r y . . . to d e v i s e some method o f r e p o r t i n g  9 s u s c e p t i b i l i t i e s s e p a r a t e l y a c c o r d i n g to sex or o f s e c u r i n g constant sex r a t i o s between v a r i a t e s  i n an experiment.  11  Furthermore, a r e l 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 . the  Both the r e a r i n g  medium o f  l a r v a e and the f o o d o f the a d u l t have been found t o  influence  the s u s c e p t i b i l i t y o f an i n s e c t  (Wilkes e t a l , 1948). for rearing  to an i n s e c t i c i d e  Numerous methods have been developed  the d i f f e r e n t s p e c i e s o f t e s t i n s e c t s  (Peet and  Grady, 1928; Wilkes e t a l , 191+-8; P i q u e t t and F a l e s , McLintock, 1952; G r a n e t t and Haynes, 19i|4) •  The  1952;  official  method of the CSMA, adopted o r i g i n a l l y i n 1932, and m o d i f i e d , i s the Peet-Grady Method f o r r e a r i n g  since  houseflies  and cockroaches (Blue Book, 1952). A problem o f i n c r e a s i n g the  importance a t p r e s e n t I s  s e l e c t i o n o f the s t r a i n o f t e s t i n s e c t  example, the GSMA  standard"  ff  to be used. F o r  s t r a i n o f h o u s e f l y , known as  the N.A.I.D.M. s t r a i n , I s used i n the O f f i c i a l method.  However, i n d i f f e r e n t l a b o r a t o r i e s  Peet-Grady  throughout the  country, many d i f f e r e n t s t r a i n s o f f l i e s , c o l l e c t e d and r e a r e d under d i f f e r e n t c o n d i t i o n s , are used as the t e s t I n s e c t . These s t r a i n s o f 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 t o the v a r i o u s  insecticides.  A r e l a t e d f a c t o r i s that o f Insect resistance to insecticides. especially icides.  In many p l a c e s throughout the world,  the h o u s e f l y , have developed r e s i s t a n c e  insects, to insect-  March and M e t c a l f (191+.9), a f t e r t e s t i n g and  s i x strains of houseflies  a g a i n s t DDT,  s t a t e d : "The  comparing results  1G show . . . that the r e s i s t a n c e o f the B e l l f l o w e r s t r a i n i s 333x, the San Jose s t r a i n 22x, the O n t a r i o  s t r a i n llpc, the  R i v e r s i d e s t r a i n 13x, and the Hyman s t r a i n ipc t h a t o f the laboratory strain of f l i e s . "  Roadhouse (1953), u s i n g the  same technique, found r e s i s t a n c e 2000x t h a t o f the l a b o r a t o r y strain. I n s e c t s e x h i b i t i n g DDT r e s i s t a n c e may show c r o s s r e s i s t a n c e , u s u a l l y t o c l o s e l y r e l a t e d compounds. Metcalf  March and  (1949) found t h a t the B e l l f l o w e r s t r a i n o f i n s e c t s  showed a l e s s e r degree o f r e s i s t a n c e t o compounds s t r u c t u r a l l y s i m i l a r t o DDT, but n o t t o compounds such as gammabenzene h e x a c h l o r i d e ,  chlordane, p a r a t h l o n ,  or pyrethrlns.  Other i n v e s t i g a t o r s have found t h a t r e s i s t a n c e t o i n s e c t i c i d e s such as gamma-benzene h e x a c h l o r i d e ,  chlordane, a l d r i n ,  and d i e l d r i n , I s d i s t i n c t from DDT r e s i s t a n c e , b u t t h a t " s e l e c t i o n by exposure t o one o f them r a i s e s the r e s i s t a n c e o f the f l y s t r a i n t o the o t h e r s . " A considerable  (Busvine,  1954)*  amount o f i n v e s t i g a t i o n has been  made i n t o methods o f a n a l y s i n g the t o x i c i t y data  obtained.  The u s u a l method c o n s i s t s o f the c o n s t r u c t i o n o f dosagem o r t a l i t y curves obtained a g a i n s t the c o n c e n t r a t i o n .  by p l o t t i n g the percent The curve obtained  mortality  i s sigmoid  i n shape, when p l o t t e d upon an a r i t h m e t i c a l s c a l e . By adopting the method o f p r o b i t a n a l y s i s , i t I s p o s s i b l e t o o b t a i n a l i n e a r r e l a t i o n s h i p , making i t s i m p l e r t o draw conclusions  as t o the equation o f the s l o p e , and LD5J0  11 v a l u e s ( B l i s s , 1934a, b; 1935a, b; Gaddura, 1933).  However,  many authors f e e l t h a t d o s a g e - m o r t a l i t y data alone do not g i v e as complete a p i c t u r e as i s n e c e s s a r y and,  consequently,  o t h e r methods o f t e s t i n g have been developed. F o r example, the s u r v i v a l t i m e - m o r t a l i t y curve makes i t p o s s i b l e t o estimate the time a t which any g i v e n percentage o f the i n d i v i d u a l s w i l l succumb t o a t o x i c a n t , but o n l y a t one dosage l e v e l ( B l i s s , 1937).  A t present there i s no conven-  i e n t method by which dosage, m o r t a l i t y , and time o f response can be i n t e r r e l a t e d , although there are s e v e r a l methods of showing these v a l u e s t o p o g r a p h i c a l l y (Hansberry Chiu, 1940J Richardson and Haas, 1932).  and  Although these  topographic methods serve as v i s u a l a i d s i n j u d g i n g compara t i v e t o x i c i t y , t h e r e i s no simple mathematical  treatment  from which one can draw c o n c l u s i o n s as t o comparative toxicity.  12 III.  MATERIALS Test insect The  t e s t i n s e c t s e l e c t e d was the common h o u s e f l y ,  Musca domestlea the t e s t i n g  L.  Two s t r a i n s o f h o u s e f l y were used i n  procedure.  The f i r s t s t r a i n was designated as the SES c u l t u r e . T h i s c u l t u r e , o r i g i n a l l y o b t a i n e d from Dr. L . E . Ghadwiek, Army Chemical of  Center, Maryland,  had been r e a r e d f o r a p e r i o d  f o u r and o n e - h a l f y e a r s a t the Entomology S e c t i o n , Suf-  f i e l d Experimental The  Station.  second s t r a i n was o b t a i n e d i n August, 1955  from the P r e s t i c i d e s T e s t i n g Laboratory, Ottawa, the kindness o f Mr. W.S. McLeod. as the Ottawa c u l t u r e .  through  T h i s s t r a i n was d e s i g n a t e d  T h i s c u l t u r e had been r e a r e d  from the N.A.I.D.M. "lj.8 s t r a i n , o r the o f f i c i a l t e s t for  the Peet-Grady method.  through insect  A t no time s i n c e 1948 was t h i s  c u l t u r e exposed t o DDT contamination, but t h e r e was a poss i b i l i t y o f chlordane contamination d u r i n g 1948-49 (McLeod, W.S., p e r s o n a l communication).  F i s h e r (1952) and Roadhouse  (1953) used t h i s c u l t u r e f o r the "standard" s u s c e p t i b l e strain. Insecticides A t o t a l o f n i n e i n s e c t i c i d e s was used. i n s e c t i c i d e s s e l e c t e d were a l l o r g a n i c compounds w i t h i n the l a s t few y e a r s .  The developed  13 Dieldrin  ( 1 , 2 , 3 , 4 , 1 0 , 1 0 - h e x a c h l o r o - 6 , 7 - e p o x y - 1 , 4, 4a,  5,6,7»8,8a-octahydro-l,l4.,5»8-dinieth.anonaphthalene). C^HSOIQO.  Empirical formula: Structural  formula:  Cl  Recrystallized: Aldrin  100$  pure.  (l,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-  h e x a h y d r o - 1 5 > : 8-dimethanonaphthalene). Empirical Structural  C  formula:  i2 8 6* H  G1  formula:  CI  Recrystallized: Endrin  99.6$ p u r e .  ( 1 , 2 , 3 , 4 , 1 0 , 1 0 - h e x a c h l o r o - 6 , 7 - e p o x y - l , 4 * 4a,  5,6,7,8,8a-octahydro-l,4,5,8-endo-endodimethanonaphthalene). Empirical formula: C ^ H Q C L Q O . Structural  formula: 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 - h e p t a e h l o r - 3 a , 4 , 7 , T a te trahydro-4»7-endomethanoindene). Empirical formula: C ^ H ^ C l ^ . S t r u c t u r a l formula: CI Gl  R e c r y s t a l l i z e d : 99.6% pure. P a r a t h i o n ( o , o - d i e t h y l o-p-nitr©phenyl phosphorothioate) Empirical formula:  C^H^NO^PS.  S t r u c t u r a l formula:  OC H {  Technical Malathion  t,  s  95$ pure.  Thiophos : n  (S-(1:2-diearbethoxyethyl)-o,o-dimethyl  phosphorodithioate). E m p i r i c a l formula:  C^H^O^PS^  S t r u c t u r a l formula: (OH^O)gPS.SOH.0OO0 H 2  CH .00O0 H 2  P u r i f i e d : 99.6% pure.  2  5  5  EPN  (o-ethyl O-p-nitrophenyl Empirical formula:  phenylphosphorothioate)  C^H^NO^PS.  S t r u c t u r a l formula:  V . - !  /  R e c r y s t a l l i z e d : 100$ Lindane  pure.  >OG H 2  5  (garama-l:2:3:4 5i6-hexachlorocyclohexane). :  E m p i r i c a l formula:  C^H^Cl^.  S t r u c t u r a l 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).  E m p i r i c a l formula: G^H^Cl^. S t r u c t u r a l formula:  R e c r y s t a l l i z e d : 100$  pure.  H e n c e f o r t h i n t h i s t e x t , these compounds w i l l be r e f e r r e d to by the common name.  17 IV.  METHODS Rearing technique ( P l a t e l a )  Adult  flies A d u l t f l i e s were r e a r e d i n cages 12" x 12  n  x  12".  Three s i d e s and the top o f the cage were covered w i t h wire screening.  The f l o o r o f the cage was wood.  A c l o t h sleeve  entrance was a t t a c h e d to the f r o n t o f the cage by means o f a two i n c h s t r i p o f adhesive tape.  The cage was washed and  d r i e d i n s u n l i g h t a f t e r the removal o f each g e n e r a t i o n o f f l i e s , and a f r e s h l y laundered sleeve was a t t a c h e d .  Cages  c o n t a i n i n g the SES c u l t u r e were kept separate from those  con-  t a i n i n g the Ottawa c u l t u r e . A t the S u f f i e l d Experimental S t a t i o n , f l i e s were r e a r e d i n a r e a r i n g room, approximately 6'  x 12'.  a t u r e was maintained a t 78 - 2 degrees P.  R e l a t i v e humidity  was 75 - 5$.  Temper-  V e n t i l a t i o n was p r o v i d e d i n order t o reduce  odours and gases from fermenting media ( P l a t e l b ) . A t the U n i v e r s i t y o f B r i t i s h Columbia, the temperature  i n the r e a r i n g room was maintained a t 78 - 2  degrees P.  Since the h u m i d i t y was extremely low and v a r -  i a b l e , i t was n e c e s s a r y to c o v e r the cages w i t h p l a s t i c sheeting. of  Humidity was c o n t r o l l e d a t 72 - 2% w i t h the a i d  a s a t u r a t e d BaClg s o l u t i o n .  V e n t i l a t i o n was p r o v i d e d i n  the room by means o f an exhaust f a n .  18 Approximately 2000 f l i e s were p l a c e d i n each cage. Pood was s u p p l i e d i n the form o f lump sugar.  Water j a r s  c o n s i s t e d o f a 2f>0 ml. beaker i n v e r t e d i n t o a h a l f dish.  petri  Absorbent c o t t o n d e n t a l r o l l s were p l a c e d between the  edge o f the p e t r i d i s h and the beaker.  The f l i e s were  t h e r e f o r e s u p p l i e d w i t h a c o n s t a n t source o f water, but c o u l d n o t drown i n i t .  F r e s h f o o d and water were p l a c e d i n  each cage w i t h each new g e n e r a t i o n o f f l i e s . Two stock cages were s e t a s i d e each week.  In t h i s  way, eggs were not c o l l e c t e d from f l i e s more than three weeks old.  I t was found t o be i m p o s s i b l e t o c o l l e c t eggs i n any  g r e a t q u a n t i t y u n t i l the f l i e s were from seven t o t e n days old. Eggs The stock cages were p r o v i d e d d a i l y w i t h m i l k soaked paper towels f o r o v i p o s i t i o n  canned m i l k : ^ w a t e r ) .  Eggs were c o l l e c t e d a f t e r a p e r i o d o f n o t l o n g e r than eighteen hours ( o v e r n i g h t ) .  The eggs were washed g e n t l y i n  d i s t i l l e d water s e v e r a l times u n t i l t h o r o u g h l y s e p a r a t e d . A measured 700 eggs were then p l a c e d on top o f the media. T h i s was done by a l l o w i n g the eggs t o s e t t l e i n a c a l i b r a t e d p i p e t t e (0.1 ml. s e t t l e d eggs = 700).  F i v e ml. d i s t i l l e d  water were used to measure and s c a t t e r the eggs o v e r the media.  19 L a r v a l medium Canned dog f o o d (horsemeat) was used, as a subs t i t u t e f o r the o r i g i n a l method used a t the S u f f i e l d  Exper-  i m e n t a l S t a t i o n employing l e a n ground beef, as the l a r v a l medium.  Two  types o f c o n t a i n e r s were used, both of which  were found t o be a c c e p t a b l e .  At the S u f f i e l d Experimental  S t a t i o n , p l a s t i c bread c o n t a i n e r s , 9" x 2j. were used, w h i l e w  a t the U n i v e r s i t y o f B r i t i s h Columbia, b a t t e r y j a r s were utilized.  A t h i n l a y e r o f a u t o c l a v e d sawdust was p l a c e d  a t the bottom o f the c o n t a i n e r , and 500  grams of horsemeat,  f l a t t e n e d i n the form o f a hamburger, were p l a c e d on t o p . The eggs were s c a t t e r e d over the meat, and covered w i t h a f u r t h e r l a y e r o f a u t o c l a v e d sawdust.  Twenty ml. o f d i s t i l l e d  water were then p l a c e d over the media i n o r d e r to s u p p l y the necessary moisture.  S i x l a r v a l c o n t a i n e r s were s e t up per  day. The l a r v a e o f the SES c u l t u r e pupated w i t h i n e i g h t days.  However, the Ottawa c u l t u r e was  variable.  extremely  A t the S u f f i e l d E x p e r i m e n t a l S t a t i o n , they reached  m a t u r i t y w i t h i n f i v e days. Columbia,  found to be  At the U n i v e r s i t y of B r i t i s h  t h i s p e r i o d lengthened t o e i g h t  days.  Pupae I t was  found t h a t the mature pupae migrated to the  bottom p o r t i o n o f the media.  Any remaining meat was  removed  Plate I.  (a) Technique  employed i n r e a r i n g Musca domestica L.  (b) View o f r e a r i n g room a t the S u f f i e l d Experimental Station  20 and d i s c a r d e d . a number 10  The pupae-sawdust mixture was  sieve.  The pupae remained on top, w h i l e the  sawdust passed through.  The pupae, c o n t a i n e d i n l a r g e  h a l f p e t r i d i s h e s , were p l a c e d i n the cages. 2200 pupae were p l a c e d i n each cage. s e t up per day.  s i e v e d through  Two  (6 ) M  Approximately  cages o f f l i e s were  P l i e s hatched w i t h i n ij.8 hours.  A l l o f the  pupae maturing on one day were combined b e f o r e s e l e c t i o n  was  made f o r t e s t i n g . P l i e s r e a r e d a c c o r d i n g to t h i s method were found to be both l a r g e and u n i f o r m i n s i z e . B i o a s s a y technique T e s t i n g room A t the S u f f i e l d E x p e r i m e n t a l S t a t i o n the t e s t i n g and o b s e r v a t i o n room was was maintained a t 78 maintained a t 7f>  approximately 8*  - 2 degrees P.  - 5$.  x 12*.  Temperature  R e l a t i v e h u m i d i t y was  V e n t i l a t i o n was  provided.  In order  to e l i m i n a t e any p o s s i b i l i t y o f contamination, a l l i n s e c t i c i d e s were mixed and a p p l i e d t o the substratum i n the formulation  laboratory. At the U n i v e r s i t y o f B r i t i s h Columbia,  testing  was  c a r r i e d out i n a l a b o r a t o r y maintained a t a temperature o f 78-2  degrees P.  from 10$  The h u m i d i t y was not c o n t r o l l e d and v a r i e d  to $$% w i t h an average l e v e l o f 30$.  the exposure p e r i o d was  o n l y f o u r hours, i t was  However, s i n c e assumed t h a t  any change i n t o x i c i t y due to v a r i a t i o n In h u m i d i t y would be  21 negligible.  O b s e r v a t i o n chambers were s t o r e d i n a  c a b i n e t maintained a t 78 t a i n e d a t 72 Testing One  - 2 degrees P.  temperature  Humidity was  - 2% w i t h the a i d o f a s a t u r a t e d B a C l  2  main-  solution.  apparatus o f the major o b j e c t i v e s i n the development of  t h i s technique i n v o l v e d the s e p a r a t i o n o f r e s i d u a l contaet e f f e c t from fumigant e f f e c t . gamma-BHC attempted  Pradhan (19I4.9), working w i t h  t o e l i m i n a t e fumigant e f f e c t i n s e v e r a l  d i f f e r e n t ways: (1)  By c o n f i n i n g i n s e c t s over i n s e c t i c i d e f i l m s w i t h open t r u n c a t e d cones.  (2)  By c o n f i n i n g i n s e c t s over i n s e c t i c i d e f i l m s covered by wire gauze o r p e r f o r a t e d z i n c c o v e r s .  (3) By c o n f i n i n g i n s e c t s over i n s e c t i c i d e f i l m s w i t h perforated f i l t e r  cones.  (]+) By c o n f i n i n g i n s e c t s over i n s e c t i c i d e f i l m s w i t h i n f i l t e r paper cones f i t t e d w i t h exhaust (5)  draughts.  By c o n f i n i n g i n s e c t s over i n s e c t i c i d e f i l m s w i t h p e r f o r a t e d f i l t e r paper cones f i t t e d w i t h exhaust draughts.  He concluded t h a t "as there was  considerable toxic  action  even ... when there was no c o n t a c t e f f e c t and the i n s e c t s are  c o n f i n e d by open cones, the complete  e l i m i n a t i o n of  fumigant e f f e c t i n the case o f gamma-benzene h e x a c h l o r i d e appears i m p o s s i b l e . "  22 I n a s i m i l a r s e r i e s o f experiments c a r r i e d out d u r i n g the s p r i n g of 1955,  i t was found  impossible to  e l i m i n a t e fumigant e f f e c t by exhausting a stream o f a i r through a g l a s s c y l i n d e r .  A s t r i p o f f i l t e r paper, t h e  width and l e n g t h o f the c y l i n d e r , was impregnated w i t h t h i o n and p l a c e d i n the c y l i n d e r .  A s t r i p of wire  was p l a c e d over the f i l t e r paper a t a d i s t a n c e o f  para-  screening 1/8".  F i f t y t e s t i n s e c t s were p l a c e d on the screen, and the c y l i n d e r was stoppered stoppers.  a t both ends w i t h one-hole No. 7  Each stopper was f i t t e d w i t h a p i e c e o f 6  g l a s s tubing, the ends o f which were covered w i t h  mm.  muslin.  A vacuum l i n e was a t t a c h e d t o the t u b i n g a t one end o f the cylinder. of  In a series of tests using d i f f e r e n t  p a r a t h i o n , the fumigant m o r t a l i t y was always  concentrations extremely  h i g h , r e g a r d l e s s o f the r a t e a t which the a i r was from the c y l i n d e r . to  I t was concluded  t h a t i t was  evacuated impossible  e l i m i n a t e fumigant e f f e c t i n t h i s manner. From the data obtained by Pradhan, and from  obtained i n the p r e c e d i n g experiments, i t i s obvious  those that  fumigant e f f e c t cannot be e l i m i n a t e d as l o n g as the fumes are drawn past the i n s e c t s .  A c c o r d i n g l y , a method was  sought In which the exhaust would n o t pass the i n s e c t s . I t was suggested  t h a t i n an apparatus such as a  Buchner f u n n e l , the i n s e c t s c o u l d be c o n f i n e d t o the upper s u r f a c e o f the substratum, and, by u t i l i z i n g  negative  p r e s s u r e , a c o n t i n u a l f l o w o f c l e a n a i r c o u l d be s u p p l i e d to  them, w h i l e contaminated a i r was drawn o f f below.  In  23 order t o t e s t t h i s h y p o t h e s i s , experiments u s i n g 9.0 cm. desk type Buchner f u n n e l s .  were c a r r i e d out No. 1 Whatman  f i l t e r papers were Impregnated w i t h p a r a t h i o n s o l u t i o n s o f varying concentration.  Comparative t e s t s f o r fumigant ( P ) ,  t h e o r e t i c a l c o n t a c t ( C ) , and fumigant-contact c a r r i e d out a t the same time.  D i r e c t fumigant  (F+C) were e f f e c t was  measured by p l a c i n g a 9.1 cm. c i r c l e t o f n y l o n s c r e e n i n g i  from the impregnated  n  f i l t e r paper.  T e s t i n s e c t s were  p l a c e d w i t h i n the f u n n e l , which was then covered w i t h m u s l i n . The f o l l o w i n g r e s u l t s were r e c o r d e d : Table I . Percent solution  4 2 2  x  x  x  10" 10" 10"  1  2  3  2  25 25 25  2  19  4  -  25 24 10 25 25  -  4 4 -  4  P P+C C  21 25 25  84 100 100  P P+C C  5 24 24  26 98  P+C C  P  25 16  100 0  0  100 64  c o n c e n t r a t i o n , w i t h the r a t e o f e v a c u a t i o n  4 l./m./funnel,  eliminated.  e f f e c t of parathion.  Rate o f Effect 24 hour Percent evacuation mortality mortality (l./m./funnel)  No. flies  A t the 2 x 10 at  E l i m i n a t i o n o f fumigant  the fumigant  e f f e c t was completely  There was a corresponding decrease i n the  m o r t a l i t y o f the d i r e c t c o n t a c t s e r i e s as compared t o the furaigant-plus-contact s e r i e s .  Hence i t would appear p o s s i b l e  2k to the  e l i m i n a t e the fumigant e f f e c t o f p a r a t h i o n by exhausting fumes through the substratum.  T h e r e f o r e , the b a s i c  apparatus was made up o f a s e r i e s o f Buchner f u n n e l s a t t a c h e d to a vacuum  system.  Substratum In  the p r e v i o u s experiments, the substratum was  9 cm. No. 1 Whatman f i l t e r paper.  However, two o b j e c t i o n s  arose r e g a r d i n g the use o f the f i l t e r paper as the substratum. F i r s t , i t was determined t h a t i n o r d e r t o e l i m i n a t e the fumigant e f f e c t o f p a r a t h i o n , a t approximately the LD50 c o n c e n t r a t i o n (2 x 10~^%), was n e c e s s a r y . of  an e v a c u a t i o n r a t e o f k./m./funnel  I t was obvious t h a t due t o the c o n s i s t e n c y  the paper, a much h i g h e r r a t e o f e v a c u a t i o n would be  n e c e s s a r y than t h a t r e q u i r e d i f a more porous were used.  substratum  Secondly, some q u e s t i o n arose as t o 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 d i p p i n g the substratum i n t o a s o l u t i o n  of  known c o n c e n t r a t i o n were i n v o l v e d (Proverbs and M o r r i s o n ,  19k7).  I t was p o s t u l a t e d t h a t d i f f e r e n t amounts o f i n s e c t -  i c i d e would be p i c k e d up on the paper, depending upon the chemical composition o f the i n s e c t i c i d e . For  these reasons, experiments u s i n g d i f f e r e n t  s u b s t r a t a were c a r r i e d o u t . When w i r e s c r e e n was used, i t was determined that the fumigant e f f e c t c o u l d be e l i m i n a t e d w i t h approximately o n e - h a l f the r a t e of e v a c u a t i o n r e q u i r e d  25 for  f i l t e r paper.  Furthermore,  t h e r e would be no q u e s t i o n  of a b s o r p t i o n but merely o f adherence o f the i n s e c t i c i d e to the substratum.  U n f o r t u n a t e l y , i t was  s i z e and i m p o s s i b l e to r e - u s e . for  the same reason.  d i f f i c u l t t o cut to  Nylon screen was  R e s u l t s u s i n g woven f i b e r g l a s s  were much more encouraging  (8 oz. weave).  cloth  The fumigant  c o u l d be e a s i l y e l i m i n a t e d w i t h approximately r a t e o f e v a c u a t i o n r e q u i r e d f o r f i l t e r paper. time, I t was  eliminated  one-half  effect the  A t the same  e a s i l y c u t , i n e x p e n s i v e , and r e - u s a b l e a f t e r  decontamination  i n chromic  acid, i f carefully treated.  Although i t tended to f r a y around the cut edges, i t was found t h a t "Cenco edge, prevented was  l a b e l v a r n i s h , p a i n t e d around the cut  11  this.  T h e r e f o r e , woven f i b e r g l a s s  cloth  chosen as the substratum f o r the i n s e c t i c i d e ( P l a t e I I ) . Mode of a p p l i c a t i o n  of i n s e c t i c i d e  I t has p r e v i o u s l y been noted t h a t numerous methods of a p p l i c a t i o n o f the i n s e c t i c i d e t o the substratum have been developed.  I n the s e a r c h f o r a simple, y e t adequate,  method of a p p l i c a t i o n ,  t h r e e of the p r e v i o u s l y mentioned  techniques were c o n s i d e r e d : 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  i n a spray tower ( P o t t e r , 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 191*5) * and d i p p i n g the substratum  (Stringer,  191*9; Barnes,  i n t o a known c o n c e n t r a t i o n  o f the i n s e c t i c i d e (Proverbs and M o r r i s o n , 191*7)» A s e a r c h was e l a b o r a t e procedure  made f o r a method to circumvent  the  i n v o l v e d i n the use o f a spray tower.  o  Plate I I .  A s e r i e s of f r e s h l y c u t f i b e r g l a s s c l o t h s t r e a t e d w i t h "Cenco" l a b e l v a r n i s h to prevent f r a y i n g .  26 I t has been n o t e d t h a t t o x i c i t y v a r i e s w i t h p a r t i c l e s i z e (Mcintosh, 1947,  1949,  McGovran et a l , 1940). I t  19£l;  i s obvious t h a t i f an i n s e c t i c i d e i s p i p e t t e d onto substratum, the c o n c e n t r a t i o n  w i l l not be the same throughout,  although the o v e r a l l c o n c e n t r a t i o n t r e a t e d i n the same way.  The  the  would compare f o r r e p l i c a t e s  i n s e c t i c i d e would tend to l a y e r  up from the p o i n t ( s ) of a p p l i c a t i o n , and c r y s t a l s t r u c t u r e would d i f f e r .  a t the same time,  I f , however, the  Insecticide  was  a p p l i e d i n an o i l s o l v e n t , r a t h e r than a more v o l a t i l e  one  such as acetone or a l c o h o l , t h i s l a y e r i n g tendency would  be reduced c o n s i d e r a b l y substratum.  due  the  However, the a p p l i c a t i o n of i n s e c t i c i d e i n t h i s  manner could c o n c e i v a b l y eliminated  to d i f f u s i o n o f the o i l over  introduce  a v a r i a b l e which s h o u l d  be  i f at a l l possible. Experiments w i t h A n i l i n e Green dye  dissolved i n  benzene i n d i c a t e d t h a t i f the f i b e r g l a s s substratum were dipped i n t o the dye obtained.  s o l u t i o n , an even c o n c e n t r a t i o n  Since there i s no q u e s t i o n  i n s e c t i c i d e or dye  of absorption  i n t o the f i b e r s , as there may  f i l t e r paper, d i p p i n g  the  was of  be  the  with  substratum i n t o s o l u t i o n s of known  concentration  appeared to p r o v i d e an adequate method of  application.  At the same time, t h i s method i s not  completely  q u a n t i t a t i v e as f a r as d e t e r m i n a t i o n of the t o t a l amount of insecticide available. i n s e c t i c i d e residue,  I n o r d e r to estimate r e t e n t i o n  a dye  c o u l d be  s u b s t i t u t e d , and  t o t a l amount p i c k e d up by the substratum c o u l d be colorimetrically.  of  the  analysed  I t i s n e c e s s a r y to assume t h a t the amount  27 o f 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 p r o p o r t i o n -  ate to the amount of dye adhering Green dye,  s o l u b l e up to 2|$  was  to the c l o t h . A n i l i n e selected.  Before c o n s t r u c t i o n o f a standard p o s s i b l e , i t was  necessary  curve  was  to s e l e c t the s o l v e n t o r s o l v e n t  mixture to be used throughout the work. Since  insecticides  are much more t o x i c when a p p l i e d i n an o i l f i l m , a s o l v e n t mixture c o n s i s t i n g o f 9$% benzene (thiophene l i g h t weight m i n e r a l o i l was experimentally  used.  I t was  free) plus  determined  that a higher concentration of mineral o i l  caused c o n s i d e r a b l e m o r t a l i t y among the c o n t r o l s , presuma b l y due  to b l o c k i n g o f the A standard  trachea.  curve f o r A n i l i n e Green dye  benzene-mineral o i l s o l v e n t m i x t u r e was  c o n s t r u c t e d by making  a s e r i e s o f d i l u t i o n s r a n g i n g from 1 x 10 (Table I I ) .  Galvanometer readings  Evelyn P h o t o e l e c t r i c Colorimeter.  i n a 9i>:5  % to 5 x 10  (G) were taken on  F i g u r e 1 shows the standard  p l o t t i n g the dye c o n c e n t r a t i o n  {%)  an  The r e a d i n g s were con-  v e r t e d to o p t i c a l d e n s i t i e s (L) by r e f e r e n c e to a table.  %  curve o b t a i n e d a g a i n s t the  standard by  optical  density (L). Table I I I shows the r e s u l t s o b t a i n e d by e x t r a c t i n g the t o t a l dye p i c k e d up on £0 mgm. P i e c e s o f g l a s s c l o t h , 5>0 mgm.,  fiberglass  were dipped  i n t o dye  s o l u t i o n s o f known c o n c e n t r a t i o n , r a n g i n g from 0.1$ 3.0$,  f o r a p e r i o d o f ten seconds.  p l a c e d on f i l t e r paper and allowed  cloth.  to  They were then removed, to dry.  Each p i e c e  of  28 Table I I .  percent solution 1 1 9 8 7 6 5 4 3 2 1 5 4 3 2 1 5  X X X X X X X X X X X X X X X X X  io-i  10-2 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-4 10-4 10-4  io-4  10-4  io-5  C a l c u l a t i o n o f standard curve f o r A n i l i n e Green dye d i s s o l v e d i n a 95:5 benzenem i n e r a l o i l s o l v e n t mixture. mgm. d y e / ml. s o l v e n t  galvanometer r e a d i n g (G)  1.0000 0.1000 0.0900 0.0800 0.0700 0.0600 0.0500 0.0400 0.0300 0.0200 0.0100 0.0050 0.0040 0.0030 0.0020 0.0010 0.0005  1.3470 1.2800 1.2400 1.1550 1.1L00 0.9890 0.8860 0.7630 0.6200 0.3850 0.2218 0.1707 0.1278 0.0888 0.0593 0.0315  4.50 5.25 5.75 7.00 7.25 10.25 13.00 17.25 24.00 41.25 60.00 67.50 74.50 81.50 87-25 93.00  impregnated c l o t h was e x t r a c t e d w i t h 10 ml. o f 95:5 benzene-mineral o i l m i x t u r e . f o r each c o n c e n t r a t i o n .  F i v e r e p l i c a t i o n s were made  F i g u r e 2 shows the curve  by p l o t t i n g the dye c o n c e n t r a t i o n density (L).  obtained  (%) a g a i n s t the o p t i c a l  I t should be noted, t h a t by p l a c i n g the c l o t h  on the f i l t e r paper, a l a r g e amount o f dye was absorbed i n t o the paper from the lower s u r f a c e o f the c l o t h . However, due t o the r a p i d e v a p o r a t i o n  o f the s o l v e n t , t h e dye con-  c e n t r a t e d upon the upper s u r f a c e remained.  Hence, the f i g u r e  obtained f o r the t o t a l I n s e c t i c i d e on the c l o t h was c l o s e r to the a c t u a l i n s e c t i c i d e a v a i l a b l e .  I t was necessary,  t h e r e f o r e , t h a t the upper s u r f a c e o f the impregnated be used as the surfaee upon which the i n s e c t s were  substrate  introduced.  Table I I I . Percent solution  0.10  Extraction of  Mgm. dye/ G ml. s o l .  1.0  L  8 l 0.0888 2  81  0.091$  Green dye from 50 mgm. Average L  Percent solution  0.1088  1.25  Mgm. dye/ ml. s o l .  12.5  75 0.1221 74 0.1293 77 0.1121 1  1  2.5  59 0.2236 3  6 l l 0.2129  0.2178  1.50  15.0  583 0.2310 64 0.1922 59 0.2291 5.0  43 41 38 4I 44  3  3  O 2  0.75  7.5  30 34 3d 36 28  2  2  1.00  10.0  23 26 243 22 25  1  2  1  L  0.7570 0.7100 0.6490  2ll 20  0.6730 0.6990  17  0.7570 0.7700  0.7580  2  2  17  1  1  0.3620 0.3870 o.lp.20 0.3870 0.3520  0.3800  0.5160 0.4690 0.5200 0.4380 0.5530  0.4590  0.6340 o.585o 0.6060 0.6480 0.5980  0.6140  2.00  20.0  14  25.0  0.7270  0.7390 0.7630 0.7630  ll3 13 l l 12  0.9300 0.8860 0.9300 0.9210  0.8460  0.9010  10.,  1.0000  1.0360  1  2  2.50  Average L  17 20l  18 171 17  1  0.50  G  183  2  0.25  fiberglass cloth.  9} 1.0340 ll 0.9490 1  7 8  2  3.00  30.0  2  1.1260 1.0710  8„ 1.0790 83 1.0590  8  9  8  1.0970  1.0460 1.0970  1.0790  FIG.  2  FIG. I  30 Prom F i g u r e s 1 and 2,  i t was p o s s i b l e  to calculate  the  amount o f dye p e r 50 mgm. f i b e r g l a s s c l o t h , and hence  the  amount o f dye p e r gram o f 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 c u t i n t o  circlets,  9 c e n t i m e t e r s i n diameter, w i t h the a i d o f a sharp s c a l p e l . The  c u t edges were p a i n t e d w i t h C e n c o n  n  l a b e l varnish.  In  o r d e r t o s i m p l i f y c a l c u l a t i o n s , and a v o i d weighing each c l o t h i n d i v i d u a l l y , the weight o f each c l o t h was assumed t o be 2 grams ( a c t u a l weight range, 1.95 the  grams). T h e r e f o r e  t o t a l amount o f i n s e c t i c i d e on t h e c l o t h c o u l d be c a l -  culated by  - 2.05  by m u l t i p l y i n g  the f i g u r e obtained from f i g u r e  3,  2. Insecticide  s o l u t i o n s were made up on a w e i g h t /  volume b a s i s . 2$ ml. o f s o l u t i o n were p l a c e d i n the l a r g e h a l f o f a 9 cm. p e t r i d i s h , and each c i r c l e t o f c l o t h was dipped i n d i v i d u a l l y f o r a period  o f t e n seconds. The c l o t h  was removed and allowed t o d r y 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 a t each dosage  level. 9.0  cm. desk-type Buchner f u n n e l s were s e t up i n  series of three. the  I f the i n s e c t i c i d e was fumigant i n nature,  f u n n e l s were a t t a c h e d to the vacuum system through a  s e r i e s o f exhaust m a n i f o l d s . measured w i t h a F i s h e r with a capacity  o f 32.5  Rate o f e v a c u a t i o n was  and P o r t e r P r e c i s i o n Bore Rotameter, L. o f a i r p e r minute.  RETENTION  O F ANILINE  GREEN  DYE O N F I B E R G L A S S  CLOTH  (O — 3 0 % R A N G E )  MILLIGRAMS DYE/GRAM CLOTH FIG.3A  RETENTION  O F ANILINE  GREEN  DYE ON FIBERGLASS  ( 0 — .12 % R A N G E )  .2  .3  MILLIGRAMS DYE/GRAM CLOTH FIG- % %  A  CLOTH  31 The impregnated of  c l o t h s were d r i e d f o r a p e r i o d  one hour b e f o r e they were p l a c e d i n the f u n n e l s . The t e s t I n s e c t s (Musca domestica L.) were  c a r e f u l l y r e a r e d a c c o r d i n g t o the procedure p r e v i o u s l y described.  The pupae were p r e v i o u s l y mixed, b e f o r e they  were, p l a c e d i n the cages, i n o r d e r t h a t the p o p u l a t i o n t e s t e d be as homogeneous as p o s s i b l e .  The f l i e s were  removed from the r e a r i n g cages w i t h the a i d o f a vacuum system.  A glass cylinder 8  W  i n l e n g t h was stoppered a t  b o t h ends w i t h No. 7 rubber s t o p p e r s . mm.  A 3" l e n g t h o f 12  g l a s s t u b i n g was i n s e r t e d i n t o each s t o p p e r .  A wire  cage was p l a c e d over t h e stopper a t t a c h e d t o the vacuum l i n e , i n o r d e r t h a t the f l i e s were not drawn i n t o the system.  A 2' l e n g t h o f p l a s t i c t u b i n g was a t t a c h e d t o the  g l a s s t u b i n g a t the i n l e t end o f the c y l i n d e r . funnel with a  A 2^  M  glass  o u t l e t was a t t a c h e d to the p l a s t i c t u b i n g  (Plate I l i a ) . P l i e s removed from the cages I n t h i s way were seldom i n j u r e d . at  Approximately  one time without crowding.  w i t h CO2 and immediately  500  f l i e s c o u l d be removed  The f l i e s were a n a e s t h e t i z e d  p l a c e d i n a c o l d room (£• C . ) ,  where they were segregated a c c o r d i n g t o t h e i r sex. I t was found t h a t f l i e s s u b j e c t e d t o c o l d alone were d i f f i c u l t t o i d e n t i f y as t o sex, s i n c e they tended t o aggregate  into a  s o l i d mass, w h i l e those a n a e s t h e t i z e d w i t h G02 r e c o v e r e d much too q u i c k l y f o r s o r t i n g i n l a r g e numbers.  A t the  U n i v e r s i t y o f B r i t i s h Columbia, a c o l d room was n o t a c c e s s i b l e  Plate I I I .  (a) Method employed cages.  i n removing h o u s e f l i e s from  (b) Buchner f u n n e l s c o n t a i n i n g f?0 f l i e s each, demonstrating fumigant and r e s i d u a l contact tests.  32 and  the f l i e s were a n a e s t h e t i z e d w i t h an e t h e r - a l c o h o l  mixture ( 5 0 : 5 0 ) .  Recovery was slow, and m o r t a l i t y i n the  c o n t r o l s was h i g h e r . Experiments were c a r r i e d out t o determine a s a t i s f a c t o r y type o f c o v e r i n g f o r the f u n n e l s .  Muslin  and  s c r e e n proved u n s a t i s f a c t o r y s i n c e the f l i e s would  settle  on such c o v e r i n g s i n p r e f e r e n c e  substrate.  t o the impregnated  However, f l i e s would n o t s e t t l e t o any extent on a t r a n s parent  surface.  F o r t h i s reason,  a c o v e r i n g f o r the f u n n e l s . approximately  "Saran" f i l m was used as  The f i l m was p e r f o r a t e d w i t h  t h i r t y s m a l l h o l e s through which the f l i e s  c o u l d n o t escape ( P l a t e I l l b ) . The  f l i e s were i n t r o d u c e d t o the three r e p l i c a t e s  o f each dosage a t the same time, and t o each dosage a t t e n 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 f o u r hours.  exposure p e r i o d was a r b i t r a r i l y p l a c e d a t W i t h i n the f i r s t f i f t e e n minutes o f t h i s  p e r i o d , any m o r t a l i t y due t o mechanical i n j u r y was n o t e d . Upon completion  o f the exposure p e r i o d , the i n s e c t s were  removed from the f u n n e l s t o separate g l a s s c y l i n d e r s , 6" i n length.  A vacuum system s i m i l a r t o t h a t used i n removal  from the cages was u t i l i z e d . tape, c o n t a i n i n g a  A s m a l l square o f l " adhesive  h o l e , was stuck to the saran  film.  A tapered g l a s s r o d , a t t a c h e d t o the p l a s t i c t u b i n g i n p l a c e o f the g l a s s f u n n e l , was i n s e r t e d through t h i s opening.  small  The f l i e s were removed i n d i v i d u a l l y , u s i n g a  P l a t e IV.  (b) Observation chambers s e t up In s e r i e s . M o r t a l i t y counts were made a t 2k and k8 hour p e r i o d s .  33 l i g h t vacuum.  The r e p l i c a t e s o f each dosage l e v e l were  removed a t t e n minute  i n t e r v a l s , e x a c t l y f o u r hours  they were i n t r o d u c e d .  after  Each r e p l i c a t e was numbered and  dated, and p l a c e d i n s e r i e s ( P l a t e IVb). M o r t a l i t y counts were made a t 2i* and 1*8 hour i n t e r v a l s .  Only those i n s e c t s  showing no movement whatsoever were counted as dead. Before c o n s t r u c t i n g a d o s a g e - m o r t a l i t y curve f o r r e s i d u a l c o n t a c t t o x i c i t y , i t was f i r s t n e c e s s a r y t o d e t e r mine the fumigant p r o p e r t i e s o f the compound.  Fumigant  a c t i o n was determined i n a manner s i m i l a r t o t h a t d e s c r i b e d i n the p r e c e d i n g experiments ( P l a t e V a, b ) .  Fifty  were p l a c e d on a wire s c r e e n a t a d i s t a n c e o f ^ impregnated substratum.  from the  The f u n n e l was covered w i t h a  l a y e r of perforated saran f i l m . the  tt  flies  A f t e r f o u r hours exposure,  f l i e s were removed and p l a c e d i n the o b s e r v a t i o n  chambers.  O b s e r v a t i o n s were taken a t 21* and ij.8 hour  intervals. If  a compound was found t o be fumigant i n n a t u r e ,  f u r t h e r experiments were c a r r i e d out t o determine the r a t e of the  e v a c u a t i o n n e c e s s a r y t o e l i m i n a t e the fumigant e f f e c t a t approximate I D ^ l e v e l . I t was a l s o n e c e s s a r y t o determine the approximate  range o f c o n c e n t r a t i o n s over which m o r t a l i t y o c c u r r e d . T h i s c o u l d q u i t e o f t e n be determined i n a s i n g l e s e r i e s o f experiments u s i n g e i g h t t o t e n dosage l e v e l s r a n g i n g from the  approximate LDg£ dosage l e v e l  downward.  (a) Components of equipment used i n t e s t i n g effect.  (b) Assembled effect.  equipment used i n t e s t i n g  fumigant  fumigant  34 As was  previously pointed  out, a l a r g e number of  f a c t o r s a f f e c t the t o x i c i t y o f an i n s e c t i c i d e t o an i n s e c t . I n the development of the p r e s e n t made to s t a n d a r d i z e  technique, an attempt  e x t e r n a l f a c t o r s such temperature  and  h u m i d i t y c o n t r o l , p h y s i c o - c h e m i c a l r e l a t i o n s h i p s , and ing  techniques.  inherent  I t has been shown e x p e r i m e n t a l l y ,  with a l l external factors controlled,  LDCJQ  values  (Krijgsman and Berger, 1949).  t o x i c i t y of i n s e c t i c i d e s " was  introduced  the  usual  values,  LDCJQ  I t i s obvious t h a t a l a r g e e r r o r c o u l d occur. index - an improved method o f comparing the  vary  Since  method o f a n a l y s i s c o n s i s t s of comparison of  that  for  groups o f i n s e c t s t e s t e d a t d i f f e r e n t times w i l l  "Toxicity  relative  by Sun  1950.  in  I t i s d e f i n e d as the " r a t i o between the I<I>cjg o f a i n s e c t i c i d e and  rear-  However, a l l b i o l o g i c a l m a t e r i a l shows  variation.  considerably  was  standard 100."  t h a t o f a t e s t sample, m u l t i p l i e d by  That i s : T o x i c i t y index = ^50 LDCJQ  The  t o x i c i t y index o f the  Sun has if  o  f  t  h  e x  1  0  0  o f t e s t sample  standard  100.  i s always equal t o  shown t h a t the t o x i c i t y index remains f a i r l y  constant  the e x t e r n a l v a r i a b l e s are c a r e f u l l y c o n t r o l l e d , and i f  the t e s t i n s e c t i c i d e i s t e s t e d on the same day standard. He standard  as  the  concludes t h a t "the change In the L D ^ Q of  i n s e c t i c i d e caused by changes i n the  o f the f l i e s and  the  susceptibility  the environmental c o n d i t i o n s i s accompanied  by a p r o p o r t i o n a l change i n the  LDCJQ  of the t e s t sample."  35 The  concept o f t o x i c i t y index was i n c o r p o r a t e d i n t o the  present  technique. D i e l d r i n was s e l e c t e d as the "standard"  i c i d e , s i n c e i t was found t o possess no fumigant  insecteffect,  and was i n t e r m e d i a t e i n the range o f t o x i c i t y o f the compounds t o be t e s t e d , a c c o r d i n g t o the r e s u l t s o b t a i n e d  by Sun (1950). I n the d e t e r m i n a t i o n o f the d o s a g e - m o r t a l i t y curve, f o u r t o seven dosages were s e l e c t e d over the approximate range o f the t e s t i n s e c t i c i d e . A. dosagem o r t a l i t y curve was c o n s t r u c t e d f o r the standard  insect-  i c i d e on the same day t h a t the t e s t sample was used. With the equipment a v a i l a b l e , i t was n e c e s s a r y t o perform two "runs" a day.  Each r u n c o n s i s t e d o f three dosage l e v e l s o f  the standard, three o r f o u r dosage l e v e l s o f the t e s t and a c o n t r o l s e r i e s .  T h e r e f o r e , 2200 i n s e c t s ,  sample,  segregated  as t o sex, were r e q u i r e d per day. The r e s u l t s o b t a i n e d were analysed a c c o r d i n g t o the method o f p r o b i t a n a l y s i s developed m a i n l y by B l i s s (1934 a, *>; 1935 a, b ) , u s i n g the s i m p l i f i e d method o f F i n n e y (1952). LDJ-Q  values.  T o x i c i t y i n d i c e s were c a l c u l a t e d from the  36 V.  EXPERIMENTAL DATA E l i m i n a t i o n o f fumigant e f f e c t In  the d e s i g n o f the t e s t apparatus, i t was  demonstrated t h a t fumigant e f f e c t c o u l d be e l i m i n a t e d bya p p l i c a t i o n o f n e g a t i v e p r e s s u r e through a Buchner f u n n e l . Woven f i b e r g l a s s c l o t h was s e l e c t e d as the substratum due to  the rough weave, which made i t p o s s i b l e t o e l i m i n a t e  fumigant e f f e c t more e a s i l y . Some I n s e c t i c i d e s , such as DDT, show no  signifi-  cant fumigant a c t i o n , w h i l e o t h e r s , such as p a r a t h i o n , possess a s t r o n g fumigant e f f e c t . T h e r e f o r e i t was n e c e s s a r y to  determine the fumigant a e t i o n , i f any, o f each o f the  i n s e c t i c i d e s t o be used i n t e s t i n g .  Each i n s e c t i c i d e was  t e s t e d i n the f o l l o w i n g manner: f>0 f l i e s were p l a c e d w i t h i n a Buchner f u n n e l on a s c r e e n 9.1 cm. In diameter.  The  s c r e e n was supported on a g l a s s t r i a n g l e a t a d i s t a n c e o f i  n  from the impregnated substratum.  The f u n n e l was covered  w i t h p e r f o r a t e d saran f i l m .  A c o n c e n t r a t i o n o f k.O mgm. o f  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 t h a t f i v e o f the n i n e i n s e c t i c i d e s  t e s t e d i n t h i s manner show fumigant a c t i o n a t a c o n c e n t r a t i o n of  k.O mgm.,  w h i l e f o u r do n o t .  Fumigant  action i s , of  course, p r o p o r t i o n a l t o the vapour p r e s s u r e o f the compound, as the f i g u r e s i n Table IV i n d i c a t e .  37  Table IV,  Fumigant a c t i o n 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 a c t i o n  Dieldrin Aldrin Endrin Parathion EPN Malathion DDT Lindane. Heptachlor The  Vapour p r e s s u r e  s i m i l a r to DDT s i m i l a r to l i n d a n e  • •  + • •  3.78 3.00  x 10-S x 10-2  1.50 9.40 3.00  x  LDCJQ  p a p e r ) , there was  concentration f o r parathion no m o r t a l i t y due  r a t e o f e v a c u a t i o n was  At  C.  the  (on f i l t e r  lowered to 3  litres/  to choose an e v a c u a t i o n r a t e f o r each Table V shows the  method used to determine the r a t e of e v a c u a t i o n e l i m i n a t e the fumigant e f f e c t o f a l d r i n .  c e n t r a t i o n o f 0 . 0 5 3 mgm., fumigant e f f e c t completely a t two  C. G.  s e t at it l i t r e s of a l r / m i n u t e / f u n n e l .  i n s e c t i c i d e showing fumigant a c t i o n .  set  ©20* @ 20@ 25-  m o r t a l i t y r o s e to 1+6%. T h e r e f o r e , i t was  deemed n e c e s s a r y  to  6  C. C.  to fumigant e f f e c t , i f the  However, i f the r a t e of e v a c u a t i o n was minute/funnel,  10"?  x lO" x 10-4  @ 20' @ loo-  data obtained i n Table I a l s o i n d i c a t e t h a t  the r a t e o f e v a c u a t i o n i s a c r i t i c a l f a c t o r . approximate  (mm.Hg)  i t was  necessary  A t a con-  p o s s i b l e to e l i m i n a t e  I f the r a t e o f e v a c u a t i o n  o r more l i t r e s of a i r / m i n u t e / f u n n e l .  at  a r a t e o f 1.5  to  fumigant e f f e c t r o s e to 23%.  was  However,  l i t r e s o f a i r / m i n u t e / f u n n e l , m o r t a l i t y due S i m i l a r r e s u l t s were  o b t a i n e d u s i n g male h o u s e f l i e s which r e q u i r e a lower conc e n t r a t i o n of i n s e c t i c i d e .  As a g e n e r a l r u l e , the lower  l i m i t o f c o n c e n t r a t i o n used f o r females c o u l d be taken  as  38 Table V.  D e t e r m i n a t i o n o f the r a t e o f e v a c u a t i o n n e c e s s a r y t o e l i m i n a t e the fumigant e f f e c t o f a l d r i n on Musca domestica L. (SES c u l t u r e ) .  Concentration ( T o t a l mgm.)  Sex  0.075 0.068 0.060 0.060 0.053 0.053 0.053 0.034 0.034  Rate o f evacuation (l./m./f.)  % mort. fumigant  0 0 0 3 2 1.5 1 ( f t f 2 1.5  100 93 76 2 0 23 4 4 0 16  0 ©  % mort. fumigantcontact  100 97 98 32 30 96 62  the upper l i m i t f o r males, thus s i m p l i f y i n g the amount o f approximate t e s t i n g n e c e s s a r y b e f o r e c o n s t r u c t i o n o f the f i n a l curve.  The lower c o n c e n t r a t i o n a t which I t was  n e c e s s a r y t o t e s t the male I n s e c t s showed a p r o p o r t i o n a l amount o f fumigant  e f f e c t , and as a r e s u l t , the same r a t e  of e v a c u a t i o n c o u l d be used f o r both males and females. P r e l i m i n a r y experiments  e s t a b l i s h e d the n e c e s s i t y  o f determining the r a t e o f e v a c u a t i o n s e p a r a t e l y f o r each insecticide.  T h i s l e v e l was determined  a t the approximate LD9CJ l e v e l .  f o r each compound  The r e s u l t s o b t a i n e d are  shown i n Table V I . E l i m i n a t i o n o f fumigant  e f f e e t should b r i n g  about a corresponding decrease i n r e s i d u a l c o n t a c t m o r t a l i t y . The data o b t a i n e d i n Table V, w i t h a l d r i n , show t h i s to some e x t e n t .  decrease  However, i n order t o 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 o f n e g a t i v e  39 T a b l e V I . Rate o f e v a c u a t i o n n e c e s s a r y f o r e l i m i n a t i o n of fumigant e f f e c t o f 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 o f evacuation (l./m./funnel)  2.5 2 -  Dieldrin Aldrin Endrin Parathion EPN Malathion DDT Lindane Heptachlor  2  2.5 1  p r e s s u r e , i t was d e c i d e d to r u n a s e r i e s o f p a r a l l e l u s i n g a l d r i n , l i n d a n e , and h e p t a c h l o r .  tests  The r e s u l t s o b t a i n e d  a r e shown i n Table V I I . Table V I I . Comparison o f r e s i d u a l c o n t a c t m o r t a l i t y and combined r e s i d u a l contact-fumigant m o r t a l i t y . Test i n s e c t : Musca domestica L. 90 . . (Average o f t h r e e r e p l i c a t e s : 50 f l i e s / replicate). Insecticide  C o n c e n t r a t i o n Rate o f % mort. Rate o f % mort. ( t o t a l mgm.) e v a c u a t i o n c o n t a c t e v a c u a t i o n fumigant. (l./m./f.) (l./m./f.) c o n t a c t  Aldrin (SES c u l t u r e )  0.053 0.060 0.06k 0.068  2  27 I16 60 k7  0  91 93 93 97  Lindane (Ottawa culture)  0.023 0.030 0.038  2.5  18 56 50  0  85 86 81  Heptachlor (Ottawa culture)  0.018 0.023 0.026  1  31 32 If3  0  0.0k5  0.030  63  68  90  60 79 83  81  ko I n a l l cases there I s a marked decrease In m o r t a l i t y w i t h the e l i m i n a t i o n of fumigant  effect.  I t w i l l no doubt be argued t h a t the decrease i n m o r t a l i t y 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 o f the i n s e c t i c i d e over the f o u r hour exposure p e r i o d than to the e l i m i n a t i o n o f fumigant  effect.  to t e s t t h i s p o s s i b i l i t y , a s e r i e s o f experiments out i n which the p e r i o d o f e v a c u a t i o n was s i x hours.  I n order were c a r r i e d  r a i s e d from f o u r to  The i n s e c t s were s u b j e c t e d t o the u s u a l f o u r hour  exposure p e r i o d , but e v a c u a t i o n was the i n t r o d u c t i o n o f the i n s e c t s .  begun two hours p r i o r to  P a r a l l e l t e s t s were made  u s i n g the standard f o u r hour evacuation-exposure  period.  Lindane, r e q u i r i n g the h i g h e s t e v a c u a t i o n r a t e o f  2.5  l./m./funnel, and h e p t a c h l o r , r e q u i r i n g the lowest  evacuation  r a t e o f 1.0  ,/m./funnel were t e s t e d .  are shown i n Table V I I I . c i d e was  The r e s u l t s o b t a i n e d  I f v o l a t i l i z a t i o n of the  insecti-  p a r t i a l l y r e s p o n s i b l e f o r the decrease i n m o r t a l i t y  r e c o r d e d i n Table V I I , then a two hour e v a c u a t i o n p e r i o d p r i o r to the i n t r o d u c t i o n of the i n s e c t s 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 m o r t a l i t y f o r the s i x hour e v a c u a t i o n p e r i o d as compared to the f o u r hour e v a c u a t i o n p e r i o d should be lower.  Prom the r e s u l t s shown  i n Table V I I I , t h i s would not appear t o be the ease.  The  r e s u l t s w i t h h e p t a e h l o r f o r the s i x hour e v a c u a t i o n p e r i o d i n d i c a t e a s l i g h t l y h i g h e r m o r t a l i t y as compared to those o b t a i n e d u s i n g the f o u r hour e v a c u a t i o n p e r i o d .  The  w i t h l i n d a n e , w i t h a h i g h e r r a t e o f e v a c u a t i o n , are  results slightly  41 lower, but n o t s i g n i f i c a n t l y so.  T h i s d i f f e r e n c e c o u l d be  a t t r i b u t e d more t o b i o l o g i c a l v a r i a t i o n w i t h i n the c u l t u r e , than t o v o l a t i l i z a t i o n o f the i n s e c t i c i d e . Table V I I I . E f f e c t on m o r t a l i t y o f i n c r e a s e d p e r i o d o f evacuation. Test I n s e c t : Musca domestica L., 9 $ , (Ottawa culture). I n s e c t i c i d e C o n c e n t r a t i o n % mort.6 h r s . % mort.4 h r s . Rate o f ( t o t a l mgm.) evacuation evacuation evacuation (l./m./f.) Lindane  Heptachlor  0.030 0.038 0.045 0.053  37 56 69 71  50 63 76 72  2.5  0.030 0.038 0.045 0.053  72 76 66 83  69 69 79 86  1  Dosage-mortality  data  Since d i e l d r i n was t o 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 housef l i e s were f i r s t c o n s t r u c t e d .  Approximate t e s t s I n d i c a t e d t h a t  the dosage range was 0.035 - 0.112 mgm. f o r females 0.035 mgm. f o r males.  and 0.015 -  I n the f i n a l t e s t s , three t o s i x  r e p l i c a t i o n s were made a t each dosage l e v e l .  Ten dosages  were t e s t e d f o r females  The p r o b i t s o f  and seven f o r males.  the percent m o r t a l i t i e s a f t e r 4$ hours were p l o t t e d a g a i n s t the l o g a r i t h m s o f the dosages g i v e n I n terras o f t o t a l grams on the c l o t h .  I n order t o e l i m i n a t e n e g a t i v e v a l u e s ,  the l o g a r i t h m s were m u l t i p l i e d by a f a c t o r o f 10. the method o f Finney  milli-  (1952),  Following  the p r o b i t - r e g r e s s l o n l i n e s were  42 calculated  from the data.  T o x i c i t y o f d i e l d r i n t o Musca domestica  L.,  , (SES  culture). The data a r e r e c o r d e d i n T a b l e 1 o f t h e Appendix. F i g u r e 4 shows the p r o b i t - r e g r e s s i o n  line.  The e q u a t i o n  of the l i n e was found t o be Y = 5.6269 + 6.3826x. The s l o p e was c a l c u l a t e d  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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n  l i n e . Since t h e r e i s  no evidence o f a n y t h i n g o t h e r than a l i n e a r  relationship,  the c a l c u l a t i o n s f o r h e t e r o g e n e i t y were made by I n t r o d u c i n g the h e t e r o g e n e i t y f a c t o r = 14-431, as suggested by Finney. as m = 0.046 mgm., w i t h f i d u c i a l  The LDcjo was c a l c u l a t e d  l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.064 mgm. and  0.033 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica L., <?c?, (SES culture). The data a r e r e c o r d e d i n T a b l e 2 o f t h e Appendix. F i g u r e 4 shows the c a l c u l a t e d calculated  line.  The equation was  as Y = -5.7065 + 7.5594 , w h i l e the slope was s  found t o 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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n was c a l c u l a t e d  line.  as 3.366.  The h e t e r o g e n e i t y f a c t o r  The  UDCJQ. I S 0.026  mgm.,with  f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.028  and 0.024 mgm.  TOXICITY  O F DIELDRIN T O MUSCA DOMESTICA L . (SES. CULTURE)  TOXICITY  OF DIELDRIN (SES.  6.0  TO MUSCA  h  X  O  J i.2  LOG  i  1.4  i  i.e  1000 X DOSE (TOTAL FIG. 4  I L _  i.a  2.0  MILLIGRAMS)  D O M E S T I C A L.  CULTURE)  1.0  1.2  M E A N  SINGLE R E P L I C A T E  1.4  1.6  1.6  LOG 1000 X DOSE ( T O T A L MILLIGRAMS)  FIG.  5  43 C a l c u l a t i o n o f the p r o b l t - r e g r e s s i o n  line  As has been s t a t e d p r e v i o u s l y , from f o u r t o seven dosage l e v e l s were used i n the c o n s t r u c t i o n o f the dosagemortality line.  Except i n the case o f the composite graphs  f o r d i e l d r i n (Figure 4) and DDT ( F i g u r e s 12 and 14), three r e p l i c a t i o n s were made a t each dosage l e v e l . I t was suggested t h a t the m o r t a l i t y w i t h i n the r e p l i c a t e s be p l o t t e d i n d i v i d u a l l y on the graph i n order  to show the spread o f  m o r t a l i t y w i t h i n a s i n g l e dosage l e v e l , r a t h e r than p l o t t i n g the mean m o r t a l i t y o f the three r e p l i c a t e s .  In order to  demonstrate b o t h methods, a d o s a g e - m o r t a l i t y curve was F i g u r e 5 shows the c a l c u l a t e d l i n e .  c a l c u l a t e d i n both ways.  R e p l i c a t i o n s are shown w i t h an o , means w i t h an x " . M  w  n  T o x i c i t y o f d i e l d r i n t o Musca domestica L . ,  , (SES  c u l t u r e ) . R e p l i c a t i o n s o f each dose c a l c u l a t e d i n d i v i d u a l l y . The The  data are r e c o r d e d i n Table 3 o f the Appendix.  equation o f the l i n e was found t o be Y = -0.6343  + 3.7683x. 1.15*20.  The slope o f the l i n e was b = 3.7683 -  Chi-square  heterogeneity  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  o f the p o i n t s about the p r o b i t - r e g r e s s i o n  l i n e . The h e t e r o g e n e i t y  f a c t o r was found t o be 2.4472.  The LD^Q was c a l c u l a t e d as m *= 0.031 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.054 mgm. and  0.027 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica L . , culture).  Each p o i n t r e p r e s e n t s  , (SES  the mean o f three  kk replications. The  data are recorded i n Table k o f the  Appendix.  The  equation o f the l i n e was  Y = -0.6591 + 3.7922x. b = 3.7922 - 0.7k00. no  The  slope was  Chi-square  2  found to be  c a l c u l a t e d as  = l.k8,  Indicating  s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the  probit-regression l i n e .  The L D ^ Q was  c a l c u l a t e d as  0.031 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ probabi l i t y l e v e l of 0.037 mgm. and 0.028 mgm. Comparison of the data shows t h a t the c a l c u l a t e d s l o p e s and equations  o f the two  l i n e s are  T h e r e f o r e , the LDcjg value i s s i m i l a r . a l a r g e d i f f e r e n c e i n the Chi-square  similar.  However, there i s v a l u e s , and hence  there w i l l be a d i f f e r e n c e i n the f i d u c i a l l i m i t s .  The  c a l c u l a t e d 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., w h i l e 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. Chi-square  I n l i n e s showing a h i g h  v a l u e , i t would be impossible to c a l c u l a t e any  l i m i t s whatsoever, u s i n g 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  t h a t "the method of u s i n g the m o r t a l i t y from each c o n c e n t r a t i o n 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 o f the r e g r e s s i o n l i n e i s not to be advocated f o r comprehensive experiments.  F o r one  more l a b o r i o u s .  t h i n g , the c a l c u l a t i o n i s c o n s i d e r a b l y  Moreover, the i n c r e a s e o f the  Chi-square  due  to the use  of many p o i n t s about the r e g r e s s i o n l i n e  was  not i n any way compensated f o r by the i n c r e a s e i n the  45 number of degrees of freedom."  He  concluded t h a t  "when draw-  i n g the r e g r e s s i o n l i n e i n t o x i c i t y t e s t s , the method o f p l o t t i n g the m o r t a l i t y  secured from the t o t a l s of many  r e p l i c a t e s i s e a s i e r and  g i v e s a lower value of  than does the method of p l o t t i n g the secured i n each r e p l i c a t e .  Chi-square  i n d i v i d u a l values  This i s natural  enough s i n c e  averaging of the r e s u l t s from a number of r e p l i c a t e s primarily  to remove the  the  tends  e f f e c t s of extreme v a r i a t i o n i n some  o f them." The  method o f p l o t t i n g the mean m o r t a l i t i e s  c a l c u l a t i o n of the p r o b i t - r e g r e s s i o n t h i s work. mortality  been adopted i n  However, i n order to i l l u s t r a t e the range i n  between r e p l i c a t e s o f one  have been i n c l u d e d i n the p» The mean m o r t a l i t y column p.  l i n e has  for  dosage, these  column of the  figures  statistical  a t each dosage l e v e l i s shown i n  Corrections for natural mortality  In the  tables.  the controls,  u s i n g 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 b e f o r e c a l c u l a t i o n of the mean.  Hence each l i n e r e p r e s e n t s from f o u r to seven dosage l e v e l s w i t h three r e p l i c a t i o n s of 50 B i o a s s a y of t e s t  f l i e s at each dosage l e v e l .  insecticides  Parathion T o x i c i t y o f p a r a t h i o n to Musca domestica L., The  , (SES  s t a t i s t i c a l data are r e c o r d e d i n Table 5  the Appendix. e q u a t i o n of the  F i g u r e 6(a) l i n e was  shows the c a l c u l a t e d  line.  culture). of The  found to be Y = -l8.ij.035 + li*.3000x.  1*6 The  s l o p e was c a l c u l a t e d  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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n calculated  The L D ^ Q was  line.  as m == O.Oij.3 mgm., w i t h f i d u c i a l l i m i t s a t the  95$ p r o b a b i l i t y l e v e l o f O.Olj.5 and O.Olj.0 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica L . , $ $ ,  (SES c u l t u r e ) .  Table 6 o f the Appendix shows the s t a t i s t i c a l calculations.  F i g u r e 6(b) shows the p r o b i t - r e g r e s s i o n  The equation was c a l c u l a t e d  line.  as Y = -2.lj.956 + li.6ij.10x. The  slope o f 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 h e t e r o g e n e i t y of the p o i n t s about the l i n e . The LTJ^Q value was c a l c u l a t e d  as m = O . O I L I mgm.,  w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f O.Oljlf. mgm.  and 0.038 mgm.  T o x i c i t y o f p a r a t h i o n to Musca domestica  L . , <f<f, (SES c u l t u r e ) .  The d a t a obtained are r e c o r d e d i n T a b l e 7 o f the Appendix.  F i g u r e 6(a) shows the c a l c u l a t e d  line.  o f the l i n e was found t o be Y = -6.2999 + 7.0638x. was c a l c u l a t e d  as b = 7.0638 - 2.1j!j.00.  The e q u a t i o n The s l o p e  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 h e t e r o g e n e i t y of the p o i n t s about the p r o b i t - r e g r e s s i o n  line.  The L D ^ Q was c a l c u l a t e d as  m = 0.0ij.0 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.077 and 0.028 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestiea  L . , cfd , (SES c u l t u r e ) . 1  Table 8 o f the Appendix shows the d a t a r e c o r d e d . The c a l c u l a t e d  l i n e i s shown i n F i g u r e 6 ( b ) . The equation o f  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 (SES.  T O  M U S C A  D O M E S T I C A  T O X I C I T Y  O F  DIELDRIN (SE.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  M U S C A  D O M E S T I C A  L  C U L T U R E )  I I i I 1.8 2.0 LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 6B 1  1  1.2  1.4  1.6  47 c a l c u l a t e d as b =  6.3039  -  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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n  The LDtjQ was c a l c u l a t e d as  line.  m = 0.022 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0 . 0 2 6 mgm. and  0.019  mgm.  EPN T o x i c i t y o f EPN t o Musca domestica  L.,  , (SES c u l t u r e ) .  The data obtained a r e recorded i n Table 9 o f the The c a l c u l a t e d l i n e i s shown i n F i g u r e 7 ( a ) .  Appendix. The  equation o f the l i n e was c a l c u l a t e d 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  h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n line.  The h e t e r o g e n e i t y f a c t o r o f 10.lj.09 was taken  consideration.  The L D ^ Q  w  a  s  into  c a l c u l a t e d as m = 1.014 mgm.  F i d u c i a l l i m i t s c o u l d n o t be c a l c u l a t e d s i n c e the g v a l u e exceeded 1 . T o x i c i t y o f d i e l d r i n t o Musca domestica  L . , 0 $ , (SES c u l t u r e ) .  S t a t i s t i c a l a n a l y s i s o f the d a t a o b t a i n e d i s r e c o r d e d i n Table 1 0 o f the Appendix. The c a l c u l a t e d regression  probit-  l i n e i s shown i n F i g u r e 7 ( h ) . The e q u a t i o n was  c a l c u l a t e d as Y = -1.0722 + 3.5521+x. i s r e c o r d e d as b = 3.5524 - 1.4360.  The slope o f the l i n e 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 h e t e r o g e n e i t y o f the p o i n t s about the line.  The LDcjo v a l u e was c a l c u l a t e d t o be m = 0.052 mgm.  Taking the h e t e r o g e n e i t y f a c t o r o f 8.1+438 i n t o  consideration,  f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l were c a l c u l a t e d  48 as 0.057 and 0.01+2 mgm. T o x i c i t y o f EPN to Musca domestica L . , OV,  (SES c u l t u r e ) .  The c a l c u l a t i o n s f o r the data r e c o r d e d are shown i n T a b l e 11 o f the Appendix.  The p r o b i t - r e g r e s s i o n l i n e i s  shown i n F i g u r e 7 ( a ) . Y,= -8.9082 + 8.l599x was c a l c u l a t e d as the equation o f the l i n e . b = 8.1599 - 0.9l+20x.  The slope was found t o be  C h i - s q u a r e ^ = 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 h e t e r o g e n e i t y o f the p o i n t s about the l i n e . The L D ^ Q was c a l c u l a t e d as m = 0.506 mgm., w i t h f i d u c i a l  limits  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. T o x i c i t y o f d i e l d r i n t o Musca domestica L . ,  <f<f,  (SES c u l t u r e ) .  S t a t i s t i c a l a n a l y s i s o f the data r e c o r d e d i s shown i n Table 12 o f the Appendix.  The p r o b i t - r e g r e s s i o n l i n e i s  shown i n F i g u r e 7 ( b ) .  The equation was c a l c u l a t e d a s  Y = 5.1983 + 7.0870x.  The slope was c a l c u l a t e d to be  b = 7.0870 - 1.9970.  C h i - s q u a r e ^ = 66.378,  indicating  s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the l i n e . The L D ^ Q was c a l c u l a t e d as 0.028 mgm.  Taking the h e t e r o -  g e n e i t y f a c t o r o f 16.595 i n t o c o n s i d e r a t i o n , f i d u c i a l  limits  a t the 95$ p r o b a b i l i t y l e v e l were c a l c u l a t e d t o be 0.035 mgm.  and 0.020 mgm. Endrin T o x i c i t y o f e n d r i n t o Musca domestica  L . , $ $ ,  (SES c u l t u r e ) .  S t a t i s t i c a l a n a l y s i s o f the data i s recorded I n T a b l e 13 o f the Appendix.  The c a l c u l a t e d l i n e i s shown i n  F i g u r e 8 ( a ) . The equation was c a l c u l a t e d as Y = -6.5840 +  TOXICITY OF EPN TO MUSCA DOMESTICA L. (S£.S CULTURE)  TOXICITY OF DIELDRIN TO MUSCA DOMESTICA L (S.E.S. CULTURE)  7.0 L  J 1.2  LOC 1000 X DOSE (TOTAL FIG. 7A  MILLIGRAMS)  I  I  1.4  1.6  LOG 1000 X DOSE (TOTAL FIG. 7B  I 18  L. 2.0  MILLIGRAMS)  49 8.l650x.  The slope was found t o 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 h e t e r o g e n e i t y o f t h e p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . was c a l c u l a t e d as m = 0.262 mgra.  The L D ^  0  S i n c e both the h e t e r o -  g e n e i t y f a c t o r and the g r e a t l y i n c r e a s e d t v a l u e (due t o the s m a l l number o f degrees o f freedom) had t o be taken  into  c o n s i d e r a t i o n , i t was i m p o s s i b l e t o c a l c u l a t e the f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y  level.  T o x i c i t y o f d i e l d r i n t o Musca domestica  L . , $ $ ,  (SES c u l t u r e ) .  Table 11L o f the Appendix shows the data o b t a i n e d . The p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 8 ( b ) . The equation was c a l c u l a t e d t o be Y = 0.2383 + 3.0869x. The slope o f the l i n e was found t o 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 h e t e r o g e n e i t y o f the p o i n t s about the l i n e . be m = 0.035 mgm.  The L D ^ Q was c a l c u l a t e d t o  F i d u c i a l l i m i t s a t t h e 95$ p r o b a b i l i t y  l e v e l were c a l c u l a t e d as 0.042 and 0.020 mgm. T o x i c i t y o f e n d r l n t o Musca domestica  L . , dVf, (SES c u l t u r e ) .  The data obtained a r e shown i n Table 15 o f the Appendix.  T h i s c a l c u l a t e d l i n e i s shown i n F i g u r e 8 ( a ) .  The equation was found t o be Y = 1.5225 + 3.6l33x. was c a l c u l a t e d as b = 3.6133 - 0.6750.  Chi-square  The slope 2  = O.78I, ,  I n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y o f t h e p o i n t s about the l i n e .  The L D ^ Q  w  a  s  found t o have a maximum l i k e l i h o o d  estimate o f 0.092 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.118 mgm. and 0.070 mgm.  50 L.,<feT,  T o x i c i t y o f d i e l d r i n t o Musca domestica  (SES c u l t u r e ) .  The data obtained a r e recorded i n Table 16 o f the F i g u r e 8(b) shows the p r o b i t - r e g r e s s i o n  Appendix.  equation was c a l c u l a t e d  as Y = 0.9743 + 3.k66kx.  the l i n e was found as b = 3.k66k - 0.7030.  l i n e . The The s l o p e of  Chi-square  2  = 0.813,  i n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n  line.  The L D ^ Q  w  a  s  calculated  t o be  m = 0.015 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.020 mgm. and 0.010 mgm. Aldrin T o x i c i t y o f a l d r i n t o Musca domestica S t a t i s t i c a l analysis i n Table 17 o f the Appendix.  L . , £ $ ,  (SES c u l t u r e ) .  o f the d a t a r e c o r d e d i s shown The c a l c u l a t e d  l i n e i s shown i n  F i g u r e 9 ( a ) . The equation was found t o be Y = 3.5653 + k.7295x. The  slope was c a l c u l a t e d  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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n be m = 0.065 mgm.  line.  The  was c a l c u l a t e d t o  Since i t was n e c e s s a r y t o take both the  h e t e r o g e n e i t y f a c t o r and the g r e a t l y consideration,  LDCJQ  increased t value  i t was i m p o s s i b l e t o c a l c u l a t e  into  fiducial limits  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 o f d i e l d r i n t o Musca domestica L . , $ 9 , (SES c u l t u r e ) . The data o b t a i n e d appear i n Table 18 o f the Appendix. The c a l c u l a t e d  l i n e i s shown i n F i g u r e 9 ( b ) . Y =. I.96OI  3.928lx was found t o be the equation o f the l i n e . was c a l c u l a t e d  t o be b = 3.9281 - 1.6k20.  +  The slope  Chi-square3 « 21.290,  TOXICITY OF DIELDRIN T O MUSCA DOMESTICA L. C&E.S. C U L T U R E )  1 1.0  1 1.2  1 1.4  l  i  1.6  1.8  LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 6B  5i i n d i c a t i n g s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . m = 0.059 mgm.  The L D ^ Q was c a l c u l a t e d t o be  S i n c e g was g r e a t e r than 1, f i d u c i a l  limits  c o u l d n o t be c a l c u l a t e d . T o x i c i t y o f a l d r i n to Musca domestica The  s t a t i s t i c a l a n a l y s i s o f the data r e c o r d e d  appears i n Table 19. line.  L.,cfcT, (SES c u l t u r e ) .  F i g u r e 9(a) shows the p r o b i t - r e g r e s s i o n  The equation was c a l c u l a t e d as Y = 0.8kl7 + k.3132x.  The slope o f the l i n e was found t o 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 h e t e r o g e n e i t y o f the p o i n t s about the l i n e .  The LD50 was c a l c u l a t e d as  m a 0.022 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.026 mgm. and 0.020 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica  L.jCfd , 1  (SES c u l t u r e ) .  The data obtained a r e r e c o r d e d i n Table 20 o f the Appendix.  The c a l c u l a t e d l i n e i s shown i n F i g u r e 9 ( b ) . The  equation was found t o be Y = -0.6591 + 3-7922x.  The slope  o f the l i n e was c a l c u l a t e d as b = 3.7922 - 0.7k00. C h i square2 = l . k 8 , i n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . The L D C J Q was c a l c u l a t e d as m = 0.031 mgm., with f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.037 mgm. and 0.028 mgm. Lindane T o x i c i t y o f l i n d a n e t o Musca domestica  L . , £ $ , (SES c u l t u r e ) .  Table 21 o f the Appendix shows the data r e c o r d e d . The p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 1 0 ( a ) . The  TOXICITY  OF  ALDRIN (&E.&  TO M U S C A DOMESTICA  CULTURE)  L.  TOXICITY  OF  1.2  FIG.  9A  MILLIGRAMS)  TO MUSCA  DOMESTICA  (S.E.S. C U L T U R E )  1 LOG IOOO X DOSE (TOTAL  DIELDRIN  LOG 1000  1  1.4  1 1.6  i 1.6  i 2.0  X DOSE (TOTAL MILLIGRAMS) FIG.  9B  L.  52 equation was c a l c u l a t e d  t o be Y = 0.4902 • 2.81*26x. The  slope o f the l i n e was found to be b = 2.8426 - 0 . 5 8 l 5 . C h i - s q u a r e ^ = 8.099,  i n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y  o f the p o i n t s about the l i n e .  The L D ^ Q  w  a  s  calculated  t o be  0.039 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f O.Oij.3 mgm. and 0.031 mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica L . , $ $ , (SES c u l t u r e ) . The data o b t a i n e d a r e recorded i n Table 22 o f the Appendix.  The c a l c u l a t e d  l i n e i s shown i n F i g u r e 10(b).  Y = -2.0355 + 3.7909x was c a l c u l a t e d  as the equation o f the  l i n e , w h i l e the slope was found t o be b = 3.7909 * 1.1540. C h l - s q u a r e ^ = 19.388,  Indicating  of the p o i n t s about the l i n e .  s i g n i f i c a n t heterogeneity  The LDcjg was c a l c u l a t e d as  m = 0.072 mgm., w i t h f i d u c i a l l i m i t s o f 0.632 mgm. and 0.040 mgm., a t 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 o f l i n d a n e t o Musca domestica  L.,(fcf,  Table 23 shows the data r e c o r d e d . r e g r e s s i o n l i n e i s shown i n F i g u r e 10 ( a ) . calculated  t o be Y = 2.701+2 + 2.1925x.  (SES c u l t u r e ) .  The p r o b i t The equation was  The s l o p e o f the  l i n e was found t o 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 h e t e r o g e n e i t y o f the p o i n t s about the l i n e .  The LDcjo was c a l c u l a t e d  as 0.011 mgm.  Taking the h e t e r o g e n e i t y f a c t o r o f 4 « 7 6 l i n t o c o n s i d e r a t i o n , the f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l were c a l c u l a t e d t o be 0.016 mgm. and 0.00073 mgm.  53 T o x i c i t y o f d i e l d r i n to Musca domestica  L.jCflcf, (SES c u l t u r e ) .  The data a r e r e c o r d e d i n Table 2k o f the Appendix. The c a l c u l a t e d l i n e i s shown i n F i g u r e 10(b). The equation was c a l c u l a t e d t o be Y = -2.3710 + 5.05l2x.  The s l o p e o f  the l i n e was c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . as m = 0.028 mgm.  The  LDCJQ  was c a l c u l a t e d  Taking the h e t e r o g e n e i t y f a c t o r o f 8.983  i n t o c o n s i d e r a t i o n , the f i d u c i a l l i m i t s were c a l c u l a t e d t o be 0.035 mgm.  and 0.021 mgm.  Heptachlor T o x i c i t y o f h e p t a c h l o r to Musca domestica  L . ( S E S  Table 26 o f the Appendix shows the data  culture).  obtained.  The p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 11(a). The equation was found t o be Y = -1.7567 + k.6686x.  The slope o f  the l i n e was c a l c u l a t e d as b = 4.6686 + l . k 8 9 0 .  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 h e t e r o g e n e i t y o f t h e p o i n t s about the l i n e .  The LDcjo was found  to be m = 0.030 mgm.  Taking the h e t e r o g e n e i t y f a c t o r o f 7«5k7 i n t o c o n s i d e r a t i o n , the f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l were c a l c u l a t e d as 0.035 mgm. and 0.001k mgm. T o x i c i t y o f d i e l d r i n t o Musca domestica  L.,  (SES c u l t u r e ) .  The data obtained a r e r e c o r d e d I n Table 27 o f the Appendix.  F i g u r e 11 (b) shows the c a l c u l a t e d l i n e . The  equation was found t o be Y = 3.6260 + 5.1l56x. the l i n e was c a l c u l a t e d as b = 5.1156 - 0.56k0.  The slope o f C h i - s q u a r e ^ ==  TOXICITY  7.0  OF L I N D A N E T O M U S C A D O M E S T I C A (SE.S C U L T U R E ;  L.  TOXICITY  OF DIELDRIN TO M U S C A (S.E.S. C U L T U R E )  «5 k  h  1.2  LOG  DOMESTICA  1000  X DOSE ( T O T A L FIG. IOA  MILLIGRAMS)  LOG  1000  1.4  1.6  1.8  X DOSE ( T O T A L FIG.  10 B  2.0  MILLIGRAMS)  L  5k 5.079,  I n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s  about the p r o b i t - r e g r e s s i o n l i n e .  The L D ^ Q was c a l c u l a t e d  t o be m = 0.0k8 mgm., with f i d u c i a l l i m i t s a t the 95$ proba b i l i t y l e v e l o f 0.050 mgm. and 0.0k6 mgm. T o x i c i t y o f h e p t a c h l o r t o Musca domestica  L . , o V , (SES c u l t u r e ) .  The data obtained a r e r e c o r d e d i n Table 27 o f the Appendix.  F i g u r e 11(a) shows the c a l c u l a t e d l i n e . The equation  was found t o be Y » 0.0531 + k.2703x.  The slope o f the l i n e  was c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about the probit-regression l i n e . m = 0.01k mgm.  The  LD50  was c a l c u l a t e d to be  Taking the h e t e r o g e n e i t y f a c t o r o f 3.66k  i n t o c o n s i d e r a t i o n , the f i d u c i a l l i m i t s were c a l c u l a t e d as 0.017 mgm. and 0.010 mgm., a t the 95$ p r o b a b i l i t y T o x i c i t y o f d i e l d r i n t o Musca domestica  L . , O V ,  level. (SES c u l t u r e ) .  Table 28 o f the appendix shows the data o b t a i n e d . The  c a l c u l a t e d l i n e i s shown i n F i g u r e 11(b).  was found t o be Y = 0.8812 + 3.1719x. as b » 3.1719 * 0.k526.  The equation  The slope was c a l c u l a t e d  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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t regression l i n e .  The  LD50  was found t o be 0.020 mgm., w i t h  f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.023 mgm. and 0.018 mgm. Calculation of toxicity  index  As has been p r e v i o u s l y noted, t o x i c i t y index may be r e p r e s e n t e d i n the f o l l o w i n g manner:  TOXICITY  OF HEPTACHLOR (SES.  7.0  TO MUSCA  D Q M F S T I C A |_  CULTURE)  L  TOXICITY  OF  DIELDRIN T O M U S C A (S.E.S  7.0  1000  X  DOSE  (TOTAL FIG.  II A  MILLIGRAMS)  CULTURE)  L  1.4  1.2  L O G  DOMF.STICA  L O G  1000  X  DOSE  1.6  1.8  (TOTAL FIG.  11  B  2.0  MILLIGRAMS)  L  55 LDcfo o f 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 insecticide  The t o x i c i t y i n d i c e s o f the s i x i n s e c t i c i d e s  t e s t e d above  are shown i n Table IX. Table IX.  T o x i c i t y i n d i c e s o f 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 Parathion  Sex  99 ercr  99  LD£ test 0  0.043  O.Olj.0  ( t o t a l mgm.) standard  Toxicity Index  0.041 0.022  95 55  0.052  orer  0.506  0.028  5 6  Endrin  99  0.262  0.035 0.015  13 16  Aldrin  99  era* 0.022  0.065  0.059 0.031  91  141  99  0.039 0.011  0.072 0.028  185 255  0.030 0.014  0.048 0.020  160 143  EPN  Lindane  eW Heptachlor  99  l.OLk  0.092  Bioassay o f DDT Toxicity  o f DDT t o Musca domestica L., gg , (SES c u l t u r e ) . In p r e l i m i n a r y experiments  w i t h DDT, r e s u l t s were  irregular.  Using pure p,p*-DDT, I t was i m p o s s i b l e t o o b t a i n  mortalities  over 50$, r e g a r d l e s s o f t h e c o n c e n t r a t i o n o f DDT  used.  A composite  d o s a g e - m o r t a l i t y curve was c o n s t r u c t e d  u s i n g f i f t e e n c o n c e n t r a t i o n s o f DDT r a n g i n g from 7 mgm. t o 200 mgm.  The data o b t a i n e d a r e recorded i n T a b l e 29 o f the  Appendix. F i g u r e 12 shows the p r o b i t - r e g r e s s i o n l i n e .  The  56 e q u a t i o n was found t o be Y = k.0882 + 0.3k68x.  The s l o p e o f  the l i n e was c a l c u l a t e d as b = 0.3k68 - 0.1111.  C h i - s q u a r e ^ =•  kl.608, i n d i c a t i n g s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the l i n e .  The L D ^ was c a l c u l a t e d as m = k26 mgm. 0  T a k i n g the h e t e r o g e n e i t y f a c t o r i n t o c o n s i d e r a t i o n (3.201), the f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l were 1980 mgm. and 87 mgm. T o x i c i t y o f DDT t o Musca domestica L., (fo", (SES c u l t u r e ) . A composite  d o s a g e - m o r t a l i t y curve was c o n s t r u c t e d  u s i n g n i n e c o n c e n t r a t i o n s o f DDT r a n g i n g from 15 mgm. t o 200 mgm.  Table 30 o f the Appendix shows the data r e c o r d e d . The  c a l c u l a t e d p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 12. The equation was c a l c u l a t e d t o be Y = k.1732 + 0.3630x. The slope of the l i n e was c a l c u l a t e d as b = 0.3630 * 0.2122. C h i squarey = 30.155, i n d i c a t i n g s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . c a l c u l a t e d as m = 190 mgm.  The LD50 was  Taking the h e t e r o g e n e i t y f a c t o r  o f k.308 i n t o c o n s i d e r a t i o n , i t was found t o be i m p o s s i b l e to c a l c u l a t e 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  level.  The obvious assumption t o the e x t r a o r d i n a r y data presented above would be t h a t the f l i e s possessed a h i g h degree o f r e s i s t a n c e t o DDT. t h i s assumption,  However, i n order t o c o n f i r m  and a l s o t o prove the technique, i t was  n e c e s s a r y t o b i o a s s a y the t o x i c i t y o f DDT t o these  insects  i n another manner. The method o f t o p i c a l a p p l i c a t i o n was adopted. A p p l i c a t i o n o f the I n s e c t i c i d e was accomplished b y means o f ah " A g l a " micrometer s y r i n g e c o n s i s t i n g o f a  T O X I C I T Y OF DDT T O M U S C A D O M E S T I C A L. R E S I D U A L C O N T A C T APPLICATION (S.E.S. C U L T U R E )  i.o  1.2  LOG  1.4  1.6  DOSE (TOTAL F I G . 12  1.8  2.0  MILLIGRAMS)  2.2  57 c a l i b r a t e d g l a s s hypodermic syringe attached to a r i g i d by a micrometer screw head, which i n t u r n operated The  holder  the plunger.  p e r i p h e r a l s c a l e o f the micrometer head was d i v i d e d i n  such a manner, t h a t each g r a d u a t i o n volume o f 0.2  lambda.  corresponded t o a  I n o r d e r to f a c i l i t a t e  application  o f a s m a l l amount o f i n s e c t i c i d e , the standard hypodermic needle was r e p l a c e d by a s p e c i a l l y made ground g l a s s dropper, p r o j e c t i n g a t r i g h t angles t o the s y r i n g e .  The apparatus  was clamped f i r m l y t o a r i n g stand a t a convenient  height  (Plate V i a ) . The  t e s t i n s e c t s were r e a r e d i n e x a c t l y the same  manner as d e s c r i b e d above. The f l i e s were u s i n g GO2 and c o l d .  Ten f l i e s  anaesthetized  o f each sex were used a t each  dosage l e v e l , w i t h each t e s t b e i n g r e p l i c a t e d a t l e a s t times.  three  I n order t o a v o i d i n j u r y , an eyedropper attached t o  a small vacuum pump, was used t o h o l d the i n s e c t s w h i l e a p p l y i n g the i n s e c t i c i d e .  The DDT was d i s s o l v e d i n a  benzene-mineral o i l s o l v e n t mixture.  95:5  Pour-tenths o f a lambda  o f DDT were a p p l i e d t o the pronotum o f each f l y .  Dosage  was c a l c u l a t e d I n terms o f t o t a l micrograms o f DDT (Figure 13 a, b ) .  F o l l o w i n g treatment, the f l i e s were p l a c e d i n  small "dixie*  1  cups covered w i t h m u s l i n .  A 2" l e n g t h o f  absorbent c o t t o n d e n t a l r o l l s a t u r a t e d w i t h a 10$ s o l u t i o n was p r o v i d e d f o r food  (Plate VIb).  p l a c e d i n an o b s e r v a t i o n room maintained Humidity was maintained  a t 75 * 5$.  sugar  The f l i e s were  a t 78 - 2 degrees P.  M o r t a l i t y counts were  P l a t e VI.  (b) P l i e s contained i n o b s e r v a t i o n 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.  DOT  CONTENT  OF BENZENE - MINERAL  (95:5)  MICROGRAMS  OIL  SOLUTION  DDT/0.4  LAMBDA  SOLVENT  FIG. 13 A DDT  CONTENT  OF  BENZENE - MINER A L  (95:5)  MICROGRAMS  OIL  SOLUTION  DDT/0.4 FIG. I3B  LAMBDA  SOLVENT  58 made a f t e r k8 hours.  Only those i n s e c t s showing no movement  whatsoever were c o n s i d e r e d dead. DDT ( T o p i c a l a p p l i c a t i o n ) T o x i c i t y o f DDT t o Musca domestica L . , , A composite  (SES c u l t u r e ) .  d o s a g e - m o r t a l i t y curve was c o n s t r u c t e d  u s i n g 19 c o n c e n t r a t i o n s o f DDT r a n g i n g from 1 t o 50 micrograms. The data obtained a r e r e c o r d e d i n Table 31 o f the Appendix. The p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e l k . The e q u a t i o n o f the l i n e was c a l c u l a t e d as Y = k.k929 + 0.2505x. The s l o p e was  c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about The L D ^ Q was found t o be m = 10.5? micrograms.  the l i n e .  Since g was so l a r g e , f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l c o u l d n o t be worked o u t . T o x i c i t y o f DDT t o Musca domestica L.,cTcT, (SES c u l t u r e ) . A composite  d o s a g e - m o r t a l i t y curve was c o n s t r u c t e d  u s i n g e l e v e n c o n c e n t r a t i o n s r a n g i n g from 1.0 t o 50 micrograms. Table 32 o f the Appendix shows the data o b t a i n e d . The c a l c u l a t e d l i n e i s shown i n F i g u r e l k . The equation was c a l c u l a t e d as Y = was  k.2235  + 0.5907x.  The slope o f the l i n e  c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about t h e probit-regression l i n e . micrograms.  The L D ^ Q was c a l c u l a t e d as m = 2.063  F i d u c i a l l i m i t s c o u l d n o t be worked out.  In o r d e r t o e s t a b l i s h the degree o f r e s i s t a n c e t o DDT p r e s e n t I n the S u f f i e l d c u l t u r e , comparative  t e s t s were  made on the Ottawa c u l t u r e , which possessed a low t o l e r a n c e  TOXICITY TOPICAL  1.2  1.4  LOG  OF  DDT  MUSCA  APPLICATION  1.6  10  TO  X  1.8  DOSE  (S.E.S.  2.0  (TOTAL  FIG.  DOMESTICA  14  2.2  L.  CULTURE)  2.4  MICROGRAMS)  2.6  59 l e v e l t o DDT (Roadhouse, 1953; F i s h e r , 1952). A l l t e s t s were c a r r i e d out as i n d i c a t e d above. T o x i c i t y o f DDT to Musca domestica  L., 0.$,  (Ottawa c u l t u r e ) .  The data o b t a i n e d a r e recorded i n Table 33 o f the Appendix. F i g u r e 15 shows the c a l c u l a t e d l i n e .  The equation  was  found t o be Y = -2.8951 + k . 2 l 5 l x .  was  c a l c u l a t e d as b = k . 2 l 5 l * 0.7270. Chi-square^ = k.5kk,  The slope o f the l i n e  i n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e .  The L D ^ was c a l c u l a t e d as 0  m = 0.075 micrograms, w i t h f i d u c i a l l i m i t s a t the 95$ proba b i l i t y l e v e l o f O.O83 micrograms and 0.06k micrograms. T o x i c i t y o f DDT t o Musca domestica L.,<jrV, (Ottawa c u l t u r e ) . Table 3k o f the Appendix shows the data o b t a i n e d . The p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 15. The equation was  c a l c u l a t e d t o be Y = -0.k0k3 + 3.6l79x.  The s l o p e o f the  l i n e was c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about the l i n e . micrograms.  The LDtjQ was c a l c u l a t e d as m = 0.031  Taking the h e t e r o g e n e i t y f a c t o r o f 2.k26 i n t o  c o n s i d e r a t i o n , f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y  level  were 0.037 micrograms and 0.026 micrograms. The r e s u l t s recorded above were o b t a i n e d u s i n g the Ottawa c u l t u r e o f f l i e s r e a r e d through t h r e e g e n e r a t i o n s a t the S u f f i e l d Experimental S t a t i o n .  During t h i s time the  l a r v a l p e r i o d shortened from e i g h t t o f i v e days, and a f t e r t r a n s f e r t o the U n i v e r s i t y o f B r i t i s h Columbia, a g a i n t o e i g h t days.  lengthened  60 The t e s t s by t o p i c a l a p p l i c a t i o n w i t h DDT were r e p e a t e d w i t h seventh g e n e r a t i o n f l i e s .  The f o l l o w i n g r e s u l t s  were r e c o r d e d : T o x i c i t y o f DDT t o Musca domestica  L.,99,  (Ottawa c u l t u r e ) .  The data o b t a i n e d are r e c o r d e d i n Table 35 o f the Appendix. of  F i g u r e 16 shows the c a l c u l a t e d l i n e . The equation  the l i n e was c a l c u l a t e d as Y = -5.ij.066 + 5.2907x. The  s l o p e 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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . m =5 0.099 micrograms.  The  LDCJQ  was c a l c u l a t e d a s  F i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y  l e v e l were c a l c u l a t e d as 0.103 micrograms and 0.074 micrograms. T o x i c i t y o f DDT t o Musca domestica  L . , cTcT, (Ottawa c u l t u r e ) .  Table 36 o f the Appendix shows the data o b t a i n e d . The c a l c u l a t e d p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 16. The  equation was c a l c u l a t e d as Y = -ll.81j.69 + 8.7235x. The  slope o f the l i n e was found t o be b = 8.7235 - l.ij.020. Chi-square^ = 2 . i j . H , of  i n d i c a t i n g no s i g n i f i c a n t h e t e r o g e n e i t y  the p o i n t s about the l i n e .  The  LDCJQ  was c a l c u l a t e d as  m = 0.085 micrograms, w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.091 micrograms and O.O78 micrograms. DDT ( R e s i d u a l c o n t a c t a p p l i c a t i o n ) Female f i f t h g e n e r a t i o n f l i e s were a l s o b i o a s s a y e d a g a i n s t DDT by r e s i d u a l c o n t a c t a p p l i c a t i o n .  TOXICITY  OF  DDT TO  TOPICAL  MUSCA  DOMESTICA  L.  APPLICATION  (OTTAWA  TOXICITY  OF DDT T O M U S C A DOMESTICA TOPICAL APPLICATION (OTTAWA CULTURE)  CULTURE)  LOG IOOO X DOSE (TOTAL MILLIGRAMS)  L.  1.6  •  l.e  I 2.0  J 2.2  1 2 4  LOG 1000 X DOSE (TOTAL MILLIGRAMS) FIG. 16  61 T o x i c i t y o f DDT to Musca domestica L., g $ ,  (Ottawa c u l t u r e ) .  The data o b t a i n e d are r e c o r d e d i n Table 37 o f the Appendix. The c a l c u l a t e d p r o b i t - r e g r e s s i o n l i n e i s shown i n F i g u r e 17.  The equation was found t o be Y = —7.82+14.6 + 10.4598x.  The slope o f the l i n e was c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . c a l c u l a t e d as m = 1.690 mgm. of  The LD50  w  a  s  Taking the h e t e r o g e n e i t y f a c t o r  5.500 i n t o c o n s i d e r a t i o n , f i d u c i a l l i m i t s a t 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 o f the t e s t s w i t h DDT are summarized i n Table X. Table X. Degree o f r e s i s t a n c e to DDT o f the SES c u l t u r e as compared to the Ottawa c u l t u r e ( f i f t h generation) Culture SES Ottawa  Method r e s i d u a l contact r e s i d u a l contact  Sex  c  «  I»Dt;o . .  Degree o f resistance  426 mgm. 1.690 mgm.  252x  10.570 micrograms 0.075 micrograms  SES Ottawa  topical topical  _ 99  SES Ottawa  topical topical  j y i 2.063 micrograms 0.031 micrograms  IILLX  66.5x  B i o a s s a y o f l i n d a n e and h e p t a c h l o r Lindane and h e p t a c h l o r were b i o a s s a y e d w i t h generation f l i e s application.  fifth  o f the Ottawa c u l t u r e , by r e s i d u a l c o n t a c t  The r e s u l t s are recorded below:  T o x i c i t y o f l i n d a n e to Musca domestica L..yy.  (Ottawa c u l t u r e ) .  The data obtained were r e c o r d e d i n T a b l e 38 o f the  62 The c a l c u l a t e d l i n e i s shown i n F i g u r e 18. The  Appendix.  equation was found t o be Y = -3.1118 + 6.5603x. The slope o f the l i n e was c a l c u l a t e d 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 h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n  line.  The  was c a l c u l a t e d as  m = 0.017 mgm., w i t h f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y l e v e l o f 0.018 mgm. and 0.016 mgm.  L.,g$,  T o x i c i t y o f h e p t a c h l o r to Musca domestica  (Ottawa c u l t u r e ) .  Table 39 o f the Appendix shows the data obtained. The c a l c u l a t e d l i n e i s shown i n F i g u r e 18. determined t o be Y = -1.5928 + k.6366x. was c a l c u l a t e d as b = k.6366 * 1.7236.  The equation was  The slope o f the l i n e Chi-square2 = 11.063,  i n d i c a t i n g s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the probit-regression 0.026 mgm.  line.  The LDcjo  w  a  s  c a l c u l a t e d t o be m =  The h e t e r o g e n e i t y f a c t o r was 5.532. Due t o the  l a r g e g v a l u e , f i d u c i a l l i m i t s a t the 95$ p r o b a b i l i t y c o u l d n o t be c a l c u l a t e d .  level  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. RESIDUAL 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  T O X I C I T Y OF L I N D A N E AND H E P T A C H L O R T O M U S C A DOMESTIC A L. (.OTTAWA CULTURE)  • 1.0  I 1.2  I 14  1 1.6  1 1.8  1  LOG I000 X DOSE (TOTAL MILLIGRAMS) FIG. 18  63 VI.  DISCUSSION OF RESULTS Rearing methods The  DDT  r e s u l t s o b t a i n e d by t o p i c a l a p p l i c a t i o n o f  w i t h the Ottawa c u l t u r e demonstrate the n e c e s s i t y o f a Roadhouse i n 1949 and  s t a n d a r d i z e d r e a r i n g method.  195-1  determined the LD^Q v a l u e s f o r female f l i e s o f the Ottawa c u l t u r e as 0.037 micrograms and O.Okl micrograms. Metcalf  (1949), u s i n g the same c u l t u r e , estimated  v a l u e as 0.033 micrograms. t h i s c u l t u r e was W.S.,  During  March and the  0  the p e r i o d 1951 "bo 1955,  never exposed to DDT contamination  p e r s o n a l communication), and  LD^  although  (McLeod,  the u s u a l  short-  ening of the l i f e c y c l e took p l a c e , the f l i e s were v i g o r o u s and uniform.  Y e t , a f t e r r e a r i n g through o n l y three gener-  a t i o n s at the S u f f i e l d Experimental was  S t a t i o n , the LD^Q value  e x p e r i m e n t a l l y determined as 0.075 micrograms. The  f l i e s used by both Roadhouse and March  and  M e t c a l f were r e a r e d a c c o r d i n g to the s t a n d a r d i z e d Peet-Grady method, i n comparison to the SES upon canned horsemeat.  I t was  c u l t u r e , which was  reared  n o t e d t h a t the f l i e s  reared  i n the l a t t e r manner were l a r g e r and much more An  vigourous.  i n c r e a s e o f the LD^Q value might, t h e r e f o r e , 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 I n s e c t , b r i n g i n g about an i n c r e a s e in  the t o l e r a n c e l e v e l not a l l i e d w i t h the i n c r e a s e i n s i z e  and v i g o u r .  I t would appear t h a t i n the s t a n d a r d i z a t i o n o f  t e s t i n s e c t s f o r bioassay,  the r e l a t i o n s h i p o f n u t r i t i o n to  61+t o x i c i t y i s a matter o f some importance,  and worthy o f  further investigation. The data presented i n F i g u r e s 15 and 16 f o r the t o x i c i t y o f DDT t o f i f t h and seventh g e n e r a t i o n f l i e s o f the Ottawa c u l t u r e show an Increase I n the  IJD^Q  value from 0.075  to 0.099 micrograms f o r females, and 0.031 t o 0.085 micrograms for  males.  Although these i n s e c t s were c a r e f u l l y r e a r e d  under the same c o n d i t i o n s o f temperature  and h u m i d i t y ,  humidity was c o n t r o l l e d i n a d i f f e r e n t manner.  A t the Suf-  f i e l d E x p e r i m e n t a l S t a t i o n , the cages were covered w i t h s c r e e n and kept i n a r e a r i n g room under c o n t r o l l e d c o n d i t i o n s . At the U n i v e r s i t y o f B r i t i s h Columbia,  i t was n e c e s s a r y t o  c o n t r o l humidity w i t h a s a t u r a t e d BaGl2 s o l u t i o n p l a c e d w i t h i n the cage covered w i t h p l a s t i c s h e e t i n g .  Sun (191+7)  has p o i n t e d out t h a t the oxygen-carbon d i o x i d e r e l a t i o n s h i p i n c l o s e q u a r t e r s would a f f e c t the development o f the I n s e c t culture.  E x p e r i m e n t a l l y he was a b l e t o determine  simultaneous  t h a t the  f u m i g a t i o n o f S i t o p h i l u s g r a n a r l u s L. p o p u l -  a t i o n s o f i n c r e a s i n g d e n s i t y r e s u l t e d i n l e s s m o r t a l i t y . He c o r r e l a t e s t h i s r e s u l t w i t h r e l a t i v e a c t i v i t y and rates.  respiration  I t I s f e l t t h a t the s i m i l a r r e s u l t s shown i n F i g u r e s  15 and 16 may be due t o the excess CX>2 w i t h i n the  cages  covered w i t h p l a s t i c s h e e t i n g . Strain of insects The comparative  data shown f o r the t o x i c i t y o f DDT  upon the SES and Ottawa c u l t u r e s serve t o p o i n t out the  65 importance of the insecticides  s t r a i n of f l y s e l e c t e d . T e s t w i t h  (Table  IX) y i e l d e d r e s u l t s i n l i n e w i t h those  found by o t h e r i n v e s t i g a t o r s . were l e f t  various  However, t e s t s w i t h DDT,  which  u n t i l l a s t , i n d i c a t e d t h a t the females o f the SES  s t 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 a p p l i c a t i o n (Table X ) .  I t I s suggested t h a t 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 i n s e c t , comparative t e s t s f o r t o l e r a n c e t o DDT,  and  resistance  perhaps l i n d a n e ,  levels  o r chlordane be made, s i n c e  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 and  between compounds w i t h i n  a l s o compounds w i t h i n  s t r a i n s of f l i e s , Metcalf,  but not  the chlordane group  the DDT group may occur i n some i n o t h e r s (Busvine, 19$k» March  19l|.9; Bruce and Decker, 1950). Hence I t i s n e c e s s a r y  t h a t a t no time d u r i n g  i t s l i f e h i s t o r y must the c u l t u r e be  s u b j e c t e d to i n s e c t i c i d e contamination, and h i s t o r y be a v a i l a b l e to the Insect l e v e l s not for mgm.  due  that t h i s  life  investigator.  s t r a i n s a l s o show d i f f e r e n c e s i n  t o r e s i s t a n c e . F o r example, the  h e p t a c h l o r were 0.030 mgm. f o r the SES  LDCJQ  tolerance values  c u l t u r e and 0.026  f o r the Ottawa c u l t u r e . S i m i l a r l y , the L D ^ Q * I u e s f o r v  l i n d a n e were 0.039 mgm. f o r the the Ottawa c u l t u r e .  SES  c u l t u r e and  0.017 f o r  Approximate r e s u l t s w i t h p a r a t h i o n  i n d i c a t e d t h a t the t o l e r a n c e for  and  l e v e l was  s i g n i f i c a n t l y higher  the Ottawa c u l t u r e as compared to the SES  culture  (XIL) .  66 These r e s u l t s would be investigators.  i n l i n e w i t h those found by  T h e r e f o r e , the  s t r a i n of insect  selected  a f a c t o r of c o n s i d e r a b l e importance I f comparable are  to be  other is  results  obtained.  B i o a s s a y technique Concentration of The  insecticide  method of dipping  the  substratum i n t o  an  i n s e c t i c i d e s o l u t i o n of known c o n c e n t r a t i o n developed M o r r i s o n (1947) was  Proverbs and The  one  serious objection  i s the  amount of i n s e c t i c i d e p i c k e d up  simple and possible on  by  f a i r l y adequate.  variability in  the f i l t e r paper.  the  By  s u b s t i t u t i n g woven f i b e r g l a s s c l o t h , w i t h non-absorbent properties,  as  the  s u b s t r a t e , t h i s method would be  adequate.  However, an e s t i m a t i o n o f the  t o t a l amount of  upon the c l o t h would g i v e the  Investigator a clearer  r e g a r d i n g the  g e n e r a l t o x i c i t y , and  insecticide idea  f o r t h i s reason, dosages  have been r e p o r t e d i n terms of t o t a l m i l l i g r a m s on the I t must be the  cloth.  emphasized, however, t h a t by no means i s a l l of  insecticide available  o b t a i n e d are  to the  insects,  e n t i r e l y upon a comparative  The  the  Firstly,  t o x i c i t y of an the  data  basis.  s o l v e n t or s o l v e n t mixture s e l e c t e d  Important e f f e c t upon the t h r e e reasons.  and  has  insecticide  an for  type of s o l v e n t determines  q u a n t i t y of i n s e c t i c i d e which w i l l adhere to the example, s l i g h t l y l e s s I n s e c t i c i d e  cloth.  i s d e p o s i t e d upon  the For  the  substratum i f acetone i s used i n p l a c e of benzene. Secondly, the  type of s o l v e n t 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 s t r u c t u r e o f the r e s i d u e , c a u s i n g changes i n toxicity  (Mcintosh,  191+7,  T h i r d l y , i f an o i l s o l u t i o n  191+9),  Is used, t o x i c i t y i n c r e a s e s , s i n c e the o i l , which i s chemic a l l y r e l a t e d to the outer, o i l y , p r o t e c t i v e l a y e r of  the  c u t i c l e , d i s s o l v e s the i n s e c t i c i d e and a i d s i n p e n e t r a t i o n . Furthermore, S t r i n g e r assay of DDT  (191+9)  has  p o i n t e d out that the b i o -  f o r r e s i d u a l 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. T h e r e f o r e , s m a l l amount o f l i g h t weight m i n e r a l o i l was  added to the  benzene, which had f i r s t been s e l e c t e d as the s o l v e n t S i n c e the c o n c e n t r a t i o n curves  a  (5>:9$).  shown i n F i g u r e s 1  and 2 are c o n s t r u c t e d by c o l o r i m e t r i c a n a l y s i s of known d i l u t i o n s of A n i l i n e Green dye i s necessary  i n the s o l v e n t mixture, i t  to assume t h a t the amounts o f i n s e c t i c i d e  dye p i c k e d up on the c l o t h are p r o p o r t i o n a t e . o f t h i s dye was to  2\%,  to 20$ were n e c e s s a r y .  The s e l e c t i o n  o n l y s o l u b l e up  r e s i s t a n c e , concenHence, i t was  necessary  assume t h a t the s t r a i g h t l i n e r e l a t i o n s h i p h e l d a t h i g h e r  concentrations and  i n t h a t i t was  A f t e r the appearance o f DDT  t r a t i o n s up to  unfortunate  and  i n the same manner as a t lower c o n c e n t r a t i o n s ,  the graph shown i n F i g u r e 3a i s drawn by i n t e r p o l a t i o n . E l i m i n a t i o n of fumigant The  effect  data o b t a i n e d i n T a b l e s V and VI i n d i c a t e  t h a t i t i s p o s s i b l e to e l i m i n a t e fumigant e f f e c t i f the f l i e s are not  subjected to the exhaust fumes.  brought about by l i m i t i n g the f l i e s  T h i s can  be  to the upper s u r f a c e of  the substratum, w h i l e the fumes are drawn o f f below.  It i s  68 necessary  t h a t a c e r t a i n r a t e of evacuation be  insecticide  showing fumigant p r o p e r t i e s .  E l i m i n a t i o n o f the  fumigant e f f e c t b r i n g s about a corresponding m o r t a l i t y (Table V I I ) .  set f o r each  decrease  I t has been argued t h a t t h i s  in decrease  i n m o r t a l i t y i s due both to the e l i m i n a t i o n o f the fumigant e f f e c t and v o l a t i l i z a t i o n o f the i n s e c t i c i d e , d u r i n g f o u r hour exposure p e r i o d .  the  However, the data shown i n Table  V I I I i n d i c a t e t h a t even w i t h an a d d i t i o n a l two hour  evacuation  p e r i o d p r i o r to the f o u r hour exposure p e r i o d , there i s no s i g n i f i c a n t decrease concluded  i n m o r t a l i t y . T h e r e f o r e , i t may  be  t h a t the f o u r hour p e r i o d o f e v a c u a t i o n i s n o t  c r i t i c a l as regards v o l a t i l i z a t i o n o f the Toxicity  insecticide.  index  Many p r e v i o u s i n v e s t i g a t o r s have u t i l i z e d the  LDej  0  v a l u e as the b a s i s of comparison f o r t o x i c i t y o f the v a r i o u s insecticides. b a s i s of  LDCJQ  Reference to Table IX i n d i c a t e s t h a t upon the v a l u e s , the order o f t o x i c i t y (from h i g h e s t  to lowest) would  be:  Females: h e p t a c h l o r , l i n d a n e , p a r a t h i o n , a l d r i n , e n d r i n , and  EPN.  Males: l i n d a n e , h e p t a c h l o r , a l d r i n , p a r a t h i o n , e n d r i n , and  EPN.  However, a summary o f the LDcjg v a l u e s f o r concurrent w i t h d i e l d r i n shows c o n s i d e r a b l e range (Table X I ) .  runs  69 Table XI. L D ^ v a r i a t i o n o f d i e l d r i n . Q  LDCJQ  (mgm.): females  LD^Q  (mgm.): males  0.0k6 O.Olj.1 0.052  0.026 0.022 0.028  0.035 0.059 0.072 0.0i|.8  0.015 0.031 0.028 0.020  Hence, the range v a r i e s from 0.035 t o 0.072 mgm. f o r females, and 0.015 t o 0.031 f o r males.  Obviously, i t would be  i n a c c u r a t e t o compare the LDtjg o f one i n s e c t i c i d e w i t h another i f such a v a r i a t i o n i s p o s s i b l e . The b a s i c assumption i n the use o f t o x i c i t y i s t h a t "any  change i n the  LD50  o f the t e s t sample i s accom-  panied by a p r o p o r t i o n a l change i n the L D ^ o f the  standard  Q  i n s e c t i c i d e . " (Sun, 1950).  index  However, these t e s t s must be  c a r r i e d out a t the same time, w i t h a homogeneous p o p u l a t i o n o f i n s e c t s , i f the. t e s t s are t o be p l a c e d upon an accurate comparative  basis.  Prom the t o x i c i t y i n d i c e s r e c o r d e d i n  Table IX, the o r d e r o f t o x i c i t y from h i g h e s t t o lowest would be: l i n d a n e , h e p t a c h l o r , p a r a t h i o n , a l d r i n , e n d r i n , and EPN,  f o r both males and females. I t i s o f i n t e r e s t t o note t h a t the t o x i c i t y  index  v a r i e s c o n s i d e r a b l y f o r males and females i n a l l cases, o t h e r than  EPN. Evidence  i n favour o f s i m i l a r t o x i c i t y  indices  w i t h d i f f e r e n t s t r a i n s o f f l i e s i s meagre, although Sun determined  them t o be s i m i l a r i n a t l e a s t one case.  experimental data presented here, t h e r e i s l i t t l e  has  I n the  apparent  70 s i m i l a r i t y o f the t o x i c i t y i n d i c e s o f the i n s e c t i c i d e s a g a i n s t the SES c u l t u r e , as compared to the o r i g i n a l  tested  results  o b t a i n e d by Sun. Sun, o f course, used a mixed p o p u l a t i o n c o n s i s t i n g o f equal numbers o f males and females. However, the average  o f the t o x i c i t y i n d i c e s o b t a i n e d f o r male and  female a g a i n s t one i n s e c t i c i d e should be s i m i l a r t o those obtained by Sun.  T h i s , however, i s n o t the case. F o r example,  Sun obtained a t o x i c i t y index o f 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 o b t a i n e d f o r a l d r i n on the SES c u l t u r e was 116.  The r e s u l t s w i t h p a r a t h i o n a l s o bear out  this conclusion. S i m i l a r l y , the t o x i c i t y index may be expected to v a r y i f d i f f e r e n t s p e c i e s o f i n s e c t s are used. Hence, a s t a n d a r d i z e d b i o a s s a y technique would i n v o l v e the determina t i o n o f t o x i c i t y i n d i c e s f o r s e v e r a l 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 a b a s i s o f comparison.  establish  A f t e r the s t a n d a r d i z a t i o n o f these  common i n s e c t i c i d e s , i t would be p o s s i b l e t o b i o a s s a y t e s t samples, and compare the t o x i c i t y index w i t h those o b t a i n e d f o r the common i n s e c t i c i d e s . The data g i v e n here a r e p r e s e n t e d o n l y t o demonstrate the technique, w i t h no i n t e n t i o n t h a t they be accepted as a s e r i e s "standard" t o x i c i t y  indices.  R e l a t i v e s u s c e p t i b i l i t y o f males and females The experimental data show c o n c l u s i v e l y t h a t males are g e n e r a l l y more s u s c e p t i b l e than females.  As a g e n e r a l  71 r u l e , the h i g h e s t c o n c e n t r a t i o n needed i n the c o n s t r u c t i o n o f the d o s a g e - m o r t a l i t y curve f o r male f l i e s was  equivalent to  the lowest c o n c e n t r a t i o n 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 n e c e s s a r y . Although the p r o b i t - r e g r e s s i o n l i n e s f o r male and female  are  i n c l u d e d upon the same a x i s i n the f i g u r e s , i t should be emphasized t h a t comparative  o b s e r v a t i o n s must n o t be made,  o t h e r than i n a v e r y g e n e r a l sense, s i n c e these graphs were c o n s t r u c t e d a t d i f f e r e n t times, w i t h d i f f e r e n t of  insects.  generations  The o n l y a c c u r a t e comparison as regards  towards males and females  i s t h a t of t o x i c i t y  toxicity  index.  P a r a l l e l i s m o f the p r o b i t - r e g r e s s i o n l i n e s I t i s g e n e r a l l y accepted t h a t the p r o b i t - r e g r e s s i o n l i n e s c o n s t r u c t e d f o r male and female, w i t h a g i v e n I n s e c t i c i d e are p a r a l l e l .  However, from the data presented above,  I t i s obvious t h a t there i s c o n s i d e r a b l e v a r i a t i o n i n the slope of the p r o b i t - r e g r e s s i o n l i n e o f 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, w i t h an average slope of 4*3431 f o r females, 5.2239 f o r males.  and  Here a g a i n , I t must be emphasized t h a t  a c c u r a t e comparisons cannot be made u n l e s s the  experiments  are run a t the same time, u s i n g a " s t a n d a r d i z e d " technique, w i t h homogeneous p o p u l a t i o n s of i n s e c t s . Sun has recommended t h a t the d o s a g e - m o r t a l i t y  line  be c o n s t r u c t e d w i t h no l e s s than f o u r dosage l e v e l s f o r the t e s t i n s e c t i c i d e and three f o r the standard I t was  insecticide.  f e l t t h a t t h i s s m a l l number o f dosages was inadequate,  72 and i n a l l cases, the l i n e s were c o n s t r u c t e d u s i n g from f o u r to  seven dosage l e v e l s .  concentrations f a l l below 20$,  An attempt was  made to s e t these  such t h a t m o r t a l i t y d i d not exceed 90$ as recommended by Sun.  p a r a t h i o n a g a i n s t male f l i e s  In the bioassay  extreme p r o l o n g a t i o n o f the sigmoid  Since there i s an  curve at such a  l e v e l , i t i s f e l t ,that a decrease i n slope may  A s i m i l a r s i t u a t i o n e x i s t s i n the case o f d i e l d r i n where two  o f the f o u r  used y i e l d e d m o r t a l i t i e s over 90$. all  low  be brought  T h i s , of course, would a l t e r the t o x i c i t y  endrin (Figure 8 b ) ,  of  ( F i g u r e k ) , however, the  m o r t a l i t i e s obtained are extremely low.  about.  or  index. against  concentrations  I t i s suggested t h a t  t e s t s should be c a r r i e d out between the 20 - 90$  range,  s i n c e i t i s f e l t t h a t the true comparative b a s i s i s not e s t a b l i s h e d i f the range of one  i n s e c t i c i d e i s extremely  h i g h , w h i l e t h a t o f the comparative i n s e c t i c i d e i s extremely low. Resistance  of the SES  c u l t u r e to  DDT  I t has been shown i n Table X t h a t by a p p l i c a t i o n , females of the SES 6?x  topical  c u l t u r e were l k l x and males  as r e s i s t a n t compared to f i f t h g e n e r a t i o n f l i e s of the  Ottawa c u l t u r e . the SES  By r e s i d u a l c o n t a c t a p p l i c a t i o n , females of  c u l t u r e were determined to be 2$2x as r e s i s t a n t  f i f t h generation f l i e s  of the Ottawa c u l t u r e . T h i s  as  dis-  crepancy serves to demonstrate the n e c e s s i t y of d e s c r i b i n g i n d e t a i l the b i o a s s a y  technique  employed.  I t would appear  73 l o g i c a l that r e s i s t a n c e f i g u r e s would be h i g h e r by r e s i d u a l c o n t a c t a p p l i c a t i o n s i n c e the amount of i n s e c t i c i d e which an i n s e c t can p i c k up must have a l i m i t beyond which  toxicity  w i l l not i n c r e a s e i n p r o p o r t i o n  But,  topical  to c o n c e n t r a t i o n .  by  a p p l i c a t i o n , a known amount i s a p p l i e d 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 e x p l a i n the r e s i s t a n c e developed by  the SES  c u l t u r e to DDT.  f o u r and  one-half  were any  symptoms of DDT  was  At no  time d u r i n g  y e a r s t h a t the c u l t u r e was r e s i s t a n c e noted.  extremely vigourous and h e a l t h y  the  preceding  reared at The  culture  a t a l l times and  r e a r e d under the b e s t p o s s i b l e c o n d i t i o n s .  SES  was  I t i s suggested  t h a t the r e s i s t a n c e developed over a p e r i o d of time due exposure to l e t h a l amounts of DDT appear t h a t the T3DT was  i n the a i r .  to  I t would  v o l a t i l i z e d i n steam from the mixing  room at the r e a r o f the b u i l d i n g , and passed through the a i r c o n d i t i o n i n g system.  T h i s theory  i s substantiated  to some  extent by the changes i n l i f e c y c l e undergone by the Ottawa c u l t u r e during SES.  The  the few  generations  a d u l t f l i e s would l i v e no  However, symptoms of DDT further postulated  that i t was  reared  at  l o n g e r than 10 days.  p o i s o n i n g were not apparent.  t h a t the s h o r t e n i n g  from seven to f i v e days was  of the l a r v a l  caused by exposure to DDT  It i s period rather  than to the n u t r i t i o n change, s i n c e upon removal to UBC,  the  l e n g t h of the l a r v a l p e r i o d i n c r e a s e d  two  generations.  to e i g h t days over  I  Ik  Homogeneity o f data The q u a l i t y o f a b i o a s s a y technique can be judged to  a c o n s i d e r a b l e extent by the homogeneity o f the d a t a  obtained.  A summary o f the p e r t i n e n t data obtained i s shown  i n Table X I I .  A f u r t h e r summary o f the data obtained f o r DDT  i s shown In Table X I I I . Throughout the s t a t i s t i c a l a n a l y s i s , I t was noted t h a t the s i z e o f the Chi-square v a l u e g e n e r a l l y i n c r e a s e d w i t h the s i z e o f the sample t e s t e d .  F o r example, i n  F i g u r e 11+ the v a r i a t i o n o f the p o i n t s about the p r o b i t r e g r e s s i o n 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 h e t e r o g e n e i t y o f the p o i n t s 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 h e t e r o g e n e i t y of  the p o i n t s about the l i n e . I n the above examples, each  p o i n t on the l i n e r e p r e s e n t s the average m o r t a l i t y o f three r e p l i c a t e s o f 10 f l i e s each.  However, i n the case o f  F i g u r e 12, where each p o i n t r e p r e s e n t s 150 f l i e s ,  t h e r e would  appear to be much l e s s v a r i a t i o n about the p r o b i t - r e g r e s s i o n l i n e and hence a lower Chi-square value would be expected. However, t h i s value I s found t o be Chi-square-^ = ij.1.608 for  females, and 30.155 f o r males.  Hence, s i g n i f i c a n t  h e t e r o g e n e i t y i s shown i n both cases. Other i n v e s t i g a t o r s have r e p o r t e d s i m i l a r r e s u l t s . P o t t e r (I9I4.I) secured s a t i s f a c t o r y Chi-square values u s i n g from f i f t y to one hundred i n s e c t s i n the d e t e r m i n a t i o n o f each p o i n t on the r e g r e s s i o n l i n e .  However, M o r r i s o n  (1943),  75 T a b l e X I I . Summary o f d a t a o b t a i n e d i n T a b l e s 1-28 (Appendix). Insecticide  Sex Chi-square Degrees o f LD^ (mgm) F i d u c i a l l i m i t s freedom (mgm) 0  Parathion  female male  2.272 9.707  5 (nsh) 2 (nsh)  0.043 0.040  0.045 & 0.040 0.077 & 0.028  Dieldrin  female male  7.637 1.742  2 (nsh) 4 (nsh)  0.041 0.022  0.044 & 0.038 0.026 & 0.019  EPN  female male  41.635 5.446  4 (sh) 4 (nsh)  1.014 0.506  0.532 & 0.482  Dieldrin  female male  33.775 66.378  4 (sh) 4 (sh)  0.052 0.028  0.057 & 0.042 0.035 & 0.020  Endrin  female male  13.727 0.781  2 (shj 2 (nsh)  0.262 0.092  • 0.118 & 0.070  Dieldrin  female male  2.388 0.813  2 (nsh) 2 (nsh)  0.035 0.015  0.042 & 0.020 0.020 & 0.010  Aldrin  female male  13.395 7.590  3 (sh) 2 (nsh)  0.065 0.022  0.026 & 0.020  Dieldrin  female male  1.480 21.290  2 (nsh) 2 .(sh)  0.031 0.059  0.037 & 0.028  Lindane  female male  8.099 19.045  4 (nsh) 4 (sh).  0.039 0.011  0.043 & 0.031 0.016 & 0.00073  Dieldrin  female male  19.388 35.933  4 (shj 4 (sh)  0.072 0.028  0.632 & 0.040 0.035 & 0.021  H e p t a c h l o r female male  30.187 10.993  4 (sh) 4 (sh)  0.030 0.014  0.035 & 0.0014 0.017 & 0.010  female male  5.079 4.359  4 (nsh) 4 (nsh)  0.048 0.020  0.050 & 0.046 0.023 & 0.018  Dieldrin -  (* sh = s i g n i f i c a n t h e t e r o g e n e i t y } n s h • no s i g n i f i c a n t h e t e r o geneity.) u s l n g e i g h t to t e n r e p l i c a t e s  o f 150 f l i e s each, found h e t e r o -  g e n e i t y i n a l l cases except one. McCleod (1944) o b t a i n e d s i m i l a r results.  Moore and B l i s s (1942) o b t a i n e d h i g h Chi-square v a l u e s  in thirteen  out o f twenty-one experiments u s i n g l a r g e numbers o f  76 insects. reduced  I n e x p l a n a t i o n they s t a t e d : the sampling  dead aphids  "So l a r g e a number  e r r o r In e s t i m a t i n g the percentage o f  ... t o a r e l a t i v e l y s m a l l value and exposed the  h e t e r o g e n e i t y o f the f o u r p o i n t s about t h e i r computed  curve."  Although t h i s e x p l a n a t i o n i s a c c e p t a b l e , i t would seem t h a t f u r t h e r i n v e s t i g a t i o n i n t o the Chi-square  value i s n e c e s s a r y  i f a s a t i s f a c t o r y measure o f homogeneity i s to be o b t a i n e d . 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 o f t h e twenty-four  tests  t a b u l a t e d i n Table X I I show s i g n i f i c a n t h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e .  Therefore, cor-  r e c t i o n must be made by a p p l y i n g t h e h e t e r o g e n e i t y (Finney, 1952).  factor  I n the c a l c u l a t i o n o f the f i d u c i a l  limits,  both t h e h e t e r o g e n e i t y f a c t o r and the corresponding  t value  must be taken i n t o c o n s i d e r a t i o n .  Finney s t a t e s :  "When  v e r y few degrees o f freedom are a v a i l a b l e f o r e s t i m a t i n g the h e t e r o g e n e i t y f a c t o r , the corresponding  t value  ... becomes  l a r g e i n order t o a l l o w f o r the u n r e l i a b i l i t y o f the h e t e r o g e n e i t y f a c t o r and consequently w i d e l y spaced."  the f i d u c i a l l i m i t s a r e  Hence, i t i s f e l t t h a t 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 o b t a i n e d i n an experiment showing s i g n i f i c a n t h e t e r o g e n e i t y , i f the degrees o f freedom a r e l e s s than t h r e e .  F i g u r e s 8a (females) and  9b (males) a r e examples i n which i t i s i m p o s s i b l e t o c a l c u l a t e 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, t h a t a p p l i c a t i o n o f the h e t e r o g e n e i t y f a c t o r t o data w i t h three o r more degrees o f freedom, i s q u i t e a c c e p t a b l e .  The f i d u c i a l l i m i t s  obtained  77 throughout the remainder o f the experiments were narrow, w i t h the e x c e p t i o n o f EPN, i n which the C h i - s q u a r e ^ v a l u e = 41.635 made i t i m p o s s i b l e t o c a l c u l a t e the f i d u c i a l Table X I I I . Culture  Summary o f data o b t a i n e d f o r DDT. Chi-square Degrees o f freedom  Sex  limits.  LD^Q  (mgm) F i d u c i a l limits (mgm)  426.000 190.000  1980 & 87  female SES ( r e s i d u a l ) male  ij.1.608 30.155  13 (sh) 7 (sh)  female male  21.083 24.534  17 (nsh) 9 (sh)  10.570 2.063  female Ottawa (5th gen.) male (topical)  4.544 12.132  5 (nsh) 5 (sh).  0.075 0.031  0.083 & 0.06, 0.037 & 0.02  female Ottawa (7th gen.) male (topical)  0.833 2.411  (nsh) (nsh) it  0.099 0.085  0.103 & 0.07. 0.091 & 0.07  female Ottawa (5th gen.) (topical)  11.063  2 (sh)  0.026  SES (topical)  -------  Although i t i s g e n e r a l l y accepted t h a t males a r e more v a r i a b l e than females (March and M e t c a l f , 1949), the data shown i n the p r e c e d i n g t a b l e s do n o t c o r r o b o r a t e t h i s conclusion.  The l i m i t s o b t a i n e d f o r males and females a r e  e q u a l l y 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 o f t h e p r o b i t - l o g dosage relationship.  mortality  T h i s view has been put forward by s e v e r a l  authors (Wadley and S u l l i v a n , 1943; M o r r i s o n , 1943). authors have defended the method (Moore and B l i s s ,  Other  1942).  In the p r e c e d i n g experiments, i n s p e c t i o n o f the diagrams  78 i n d i c a t e d no s i g n i f i c a n t departure from the s t r a i g h t  line  r e l a t i o n s h i p , i n e i t h e r a convex or concave manner. In  t h e i r d i s c u s s i o n of h e t e r o g e n e i t y , Moore and  B l i s s s t a t e : "Conclusions depend p r i m a r i l y , t h e r e f o r e , upon the d i f f e r e n c e s between curves and c o n s i s t e n c y of these d i f f e r e n c e s r a t h e r than i n f e r e n c e s 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  toxicity  Index i s c a l c u l a t e d i s not a l t e r e d by the i n t e r n a l homog e n e i t y of the l i n e and, hence, comparative  data may  be  drawn from i t w i t h c o n s i d e r a b l e c e r t a i n t y . S e n s i t i v i t y of the One  technique  p r e r e q u i s i t e o f an a c c u r a t e b i o a s s a y  i s a h i g h degree o f s e n s i t i v i t y . l i n d a n e (Table 27 i s a c c u r a t e to 0.01  The experimental d a t a f o r  of the Appendix) show t h a t the mgm.  technique  technique  I t i s p o s s i b l e t h a t even s m a l l e r  q u a n t i t i e s of i n s e c t i c i d e c o u l d be b i o a s s a y e d , i f n e c e s s a r y . The  s e n s i t i v i t y o f the technique i s demonstrated  i n another manner. ( F i g u r e s 12  Prom the r e s u l t s o b t a i n e d w i t h  DDT  and l k ) to f l i e s of the SES c u l t u r e , a v e r y  s i g n i f i c a n t amount o f h e t e r o g e n e i t y of the p o i n t s about the probit-regression line 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 o f f l i e s p e r s o n a l communication). w i t h DDT  (Hurtig,  H.,  I n c o n t r a s t , the r e s u l t s obtained  on the Ottawa c u l t u r e o f f l i e s  show no  significant  h e t e r o g e n e i t y o f the p o i n t s about the p r o b i t - r e g r e s s i o n l i n e . T h e r e f o r e , w i t h t h i s technique, i t i s p o s s i b l e to demonstrate h e t e r o g e n e i t y or homogeneity o f response  of an i n s e c t  strain  79 to a g i v e n i n s e c t i c i d e . A f u r t h e r i n d i c a t i o n of the s e n s i t i v i t y o f the technique i s the f a c t t h a t i t was p o s s i b l e to q u a l i t a t i v e l y determine  t h a t the SES c u l t u r e was r e s i s t a n t to DDT,  and to  q u a n t i t a t i v e l y e s t a b l i s h the o r d e r of r e s i s t a n c e , as compared to the Ottawa c u l t u r e . S t i l l another I n d i c a t i o n of the s e n s i t i v i t y o f the technique was the q u a n t i t a t i v e d i f f e r e n t i a t i o n o f the t o x i c i t y o f DDT to f i f t h and seventh g e n e r a t i o n f l i e s o f the Ottawa c u l t u r e . A technique which can d e f i n e d i f f e r e n c e s such as these i n a q u a n t i t a t i v e f a s h i o n may be extremely u s e f u l .  80 VII.  CONCLUSIONS A simple, y e t adequate, b i o a s s a y technique  dis-  t i n g u i s h i n g r e s i d u a l c o n t a c t from fumigant t o x i c i t y i s described. I n s e c t i c i d e c o n c e n t r a t i o n was  calculated i n  t o t a l m i l l i g r a m s 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  the i n s e c t i c i d e s , the observed were 0.01  mgm.  The technique  as s o l v e n t f o r  lower l i m i t s o f d e t e c t i o n i s , perhaps, s t i l l more  s e n s i t i v e , but a t present i s e n t i r e l y adequate f o r even the most t o x i c  insecticides.  The u s e f u l n e s s o f the method i s not r e s t r i c t e d to d i s t i n g u i s h i n g between r e s i d u a l c o n t a c t and fumigant e f f e c t , but can be q u i c k l y and a c c u r a t e l y used to q u a l i t a t i v e l y demonstrate r e s i s t a n c e to a given i n s e c t i c i d e i n a s p e c i e s or s t r a i n o f s p e c i e s o f i n s e c t , and  to q u a n t i t a t i v e l y  e s t a b l i s h the o r d e r of t h i s r e s i s t a n c e . The  technique  i s also useful i n  demonstrating  h e t e r o g e n e i t y o f homogeneity of response to a g i v e n  insect-  i c i d e by an i n s e c t s p e c i e s o r s t r a i n o f i n s e c t s p e c i e s . I t would, t h e r e f o r e , be extremely u s e f u l f o r q u i c k l y and simply demonstrating,  w i t h a minimum of equipment, the  presence o f absence o f h e t e r o g e n e i t y o f response to a g i v e n i n s e c t i c i d e , o r f o r m u l a t i o n t h e r e o f , i n a sample o f a w i l d p o p u l a t i o n c o l l e c t e d i n the  field.  81 VIII.  BIBLIOGRAPHY  Abbott, W.S. 1925. A method o f computing the e f f e c t i v e n e s s o f an i n s e c t i c i d e . J o u r . Econ. Ent., 18 ( 2 ) : 265-67Barnes, Sarah. 1945. The r e s i d u a l t o x i c i t y o f DDT t o bedbugs (Cimex l e c t u l a r i s ) . B u l l . E n t . Res., %6 (3): 273-82. B l i s s , C.I.  38-39.  1934a.  The method o f p r o b i t s .  S c i e n c e , 79:  . 1934b. The method o f p r o b i t s - a c o r r e c t i o n . S c i e n c e , 22 409-10. s  • curve.  1935a. The c a l c u l a t i o n o f the d o s a g e - m o r t a l i t y Ann. App. B i o l . , 22: 134-67.  . 1935b. The comparison o f d o s a g e - m o r t a l i t y d a t a . Ann. App. B i o l . , 22: 307-33. curve.  1937. The c a l c u l a t i o n o f the t i m e - m o r t a l i t y Ann. App. B i o l . , 2k. (4): 815-52.  Bruce, W.N. and G.C. Decker. 1950. House f l y t o l e r a n c e f o r i n s e c t i c i d e s . Soap and S a n i t . Chem., 26 (3): 122-25,  145-47-  ~"~  Busvlne, J.'R. 1954- H o u s e f l i e s r e s i s t a n t t o a group o f c h l o r i n a t e d hydrocarbon i n s e c t i c i d e s . Nature, 17k (4434):  783-85.  Campbell, F.L. and R.S. F i l m e r . 1929- A q u a n t i t a t i v e method o f e s t i m a t i n g the t o x i c i t y o f stomach p o i s o n I n s e c t i c i d e s . Trans. IV I n t e r n . Congr. Entomol. (1928): 523-33. 1932. P r e l i m i n a r y experiments on the t o x i c i t y of c e r t a i n c o a l t a r dyes f o r the silkworm. J o u r . Econ.  Ent., 2j>: 905-17.  • and W.N. S u l l i v a n . 1938. A metal t u r n t a b l e method f o r comparative t e s t s o f l i q u i d spary c o n t a c t Insecticides. Soap. lk_(6): 119Cotton, R.T. 1943. T e s t i n g fumigants. Advance. S c i . , 20: 144-51.  Pub. Am. Assoc.  Dakshinamurity, S. 1948. The common h o u s e f l y , Musca domestica L.. and i t s behaviour t o temperature and h u m i d i t y . B u l l . E n t . Res., ^9_: 339-57.  82 F i n n e y , D.J. 1952. P r o b i t A n a l y s i s . A s t a t i s t i c a l treatment of the sigmoid response curve. Cambridge Univ. P r e s s , Second Ed. F i s h e r , R.W. 1952. The importance o f the l o c u s o f a p p l i c a t i o n on the e f f e c t i v e n e s s o f DDT f o r the h o u s e f l y , Musca domestica L. (Diptera:Muscidae). Canad. J o u r . Z o o l . ,  1 0 : 254-66.  Gaddura, J.H. 1933. 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Ent., 2 1 511-17. :  . 1943* T e s t i n g stomach i n s e c t i c i d e s . Assoc. Advance. S c i . , 20: 85-94'  P u b l . Am.  H e a l , R.E. and H. Menusan, J r . 1948. A technique f o r the bloodstream i n j e c t i o n o f i n s e c t s and i t s a p p l i c a t i o n i n tests of certain insecticides. J o u r . Econ. E n t . , 41:  535-43.  Hoskins, W.M., J.M. W i t t , and W.R. Erwin. 1952. Bioassay of 1 , 2 , 3 , 4 , 5 , 6 - h e x a c h l o r o c y c l o h e x a n e ( L i n d a n e ) ; some f a c t o r s i n f l u e n c i n g the c o n t a c t o f chemical and t e s t i n s e c t and methods f o r s t a n d a r d i z i n g the p r o c e s s . A n a l y t . Chem.,  2^: 555-60.  H u r t i g , H.  1955.  P e r s o n a l communication.  Krijgsman, B.J. and N e l l y E . Berger. 1949. A simple method f o r the e s t i m a t i o n o f c o n t a c t i n s e c t i c i d e s . B u l l . E n t .  Res., itO: 355-58.  L i n d q u i s t , A.W., H.G. W i l s o n , H.Q. Schroederer, and A.H. Madden. 1945. E f f e c t o f temperature on knockdown and k i l l o f h o u s e f l i e s exposed t o DDT. Jour. Econ. E n t . ,  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 o f bedbugs and mosquitoes exposed to DDT. J o u r . Kansas E n t . S o c , 12 (1): 13-15. March, R.B. and R.L. M e t c a l f . 191+9. Laboratory and f i e l d s t u d i e s o f D D T - r e s i s t a n t h o u s e f l i e s i n southern C a l i fornia. C a l . Dept. A g r i c . B u l l . , 2: 1-8. McGovran, E.R., C C . C a s s i l , and E.L. Mayer. 1940. Particle s i z e of P a r i s green as r e l a t e d t o t o x i c i t y and r e p e l l e n c y to the Mexican bean b e e t l e . J o u r . Econ. Ent., 525. Mcintosh, A.H. 1947. R e l a t i o n between p a r t i c l e s i z e and shape of I n s e c t i c i d a l suspensions and t h e i r c o n t a c t t o x i c i t y . I . DDT suspensions a g a i n s t T r i b o l i u m castaneum. Ann. App. B i o l . , 2k'- 586-610. . 1949. R e l a t i o n s between p a r t i c l e s i z e and shape o f 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 a g a i n s t O r y z a e p h i l u s surinamensis w i t h some t i m e - m o r t a l i t y s t u d i e s . Ann. App. B i o l . , 36: 535-50. . 195l. P a r t i c l e s i z e o f i n s e c t i c i d a l suspensions and t h e i r c o n t a c t t o x i c i t y . IV. Mechanisms o f a c t i o n o f d i f f e r e n t s i z e d p a r t i c l e s . Ann. App. B i o l . , ^8: 881-97. McLeod, W.S. 1944* F u r t h e r refinement o f a technique f o r t e s 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*  P e r s o n a l communication.  McLintock, J . 1952. Continuous l a b o r a t o r y r e a r i n g o f C u l l s e t a l n o r n a t a ( 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 e f f e c t i v e n e s s u s i n g Aphis r u m l e i s and c e r t a i n o r g a n i c compounds. J o u r . Econ. Ent., (4): 544-53. M o r r i s o n , F.O. 1943* The s t a n d a r d i z i n g of a l a b o r a t o r y method f o r comparing the t o x i c i t y of c o n t a c t i n s e c t i c i d e s . Canad; J o u r . Res., S e c t . D, Z o o l . S c i . , 21 (3): 33-75. O'Kane, W.C., G.L. Walker, H.G. Guy, and O.J. Smith. 1933S t u d i e s o f c o n t a c t i n s e c t i c i d e s . X. P e n e t r a t i o n o f a r s e n i c into insects. New Hamp. Exp. S t a . Tech. B u l l . 5 4 . Pearson, A.M. and C.H. Richardson. 1933. The r e l a t i v e t o x i c i t y o f Trisodium a r s e n i t e and a r s e n i o u s a c i d t o the h o u s e f l y . Musca domestica L. J o u r . Econ. E n t . , 26: 486-93.  81* Peet, C.H. and A.G. Grady. 1928. S t u d i e s i n i n s e c t i c i d a l activity. I. Testing i n s e c t i c i d e s against f l i e s . J o u r . Econ. Ent., 21: 612-1?. P i q u e t t , P.G. and J.H. P a l e s . 1952. Rearing cockroaches f o r experimental purposes. U.S. Dept. A g r i c . B u l l . ET-301. P o t t e r , C. 19l*l. A l a b o r a t o r y s p r a y i n g apparatus and t e c h nique f o r i n v e s t i g a t i n g the a c t i o n of c o n t a c t i n s e c t i c i d e s w i t h some notes on s u i t a b l e t e s t i n s e c t s . Ann. App. B i o l . , 22 (2): 11*2-59. Pradhan, S. 191*9. S t u d i e s on the t o x i c i t y o f i n s e c t i c i d e films. I . P r e l i m i n a r y i n v e s t i g a t i o n s on the concent r a t i o n - t i m e - m o r t a l i t y r e l a t i o n . B u l l . Ent. Res., J4O ( l ) :  1-25.  .  191*9. S t u d i e s on the t o x i c i t y o f i n s e c t i c i d e f i l m s . I I . E f f e c t o f temperature on the t o x i c i t y of DDT f i l m s . B u l l . E n t . Res., 1*0 (2): 239-65. Proverbs, M.D. and P.O. M o r r i s o n . 1914-7- The r e l a t i v e toxi c i t y of DDT and r e l a t e d organic molecules. Canad. J o u r . Res., Sect. D, Z o o l . 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 p y r i d i n e and n i c o t i n e i n the gaseous c o n d i t i o n o f T r i b o l l u m confusum Duval. Iowa State C o l l . J o u r . S c i . , 6 (3): 287-98. Roadhouse, L.A.O. 1953.' Laboratory s t u d i e s of D D T - r e s i s t a n t h o u s e f l i e s (Diptera) i n Canada. Can. Ent., 8£. (9): 3l|-0-l*6. S t r i n g e r , A. 191*9. A simple method f o r a s s a y i n g c o n t a c t t o x i c i t i e s o f i n s e c t i c i d e s with r e s u l t s of tests with some o r g a n i c compounds a g a i n s t C a l a n d r l a g r a n a r l a L. Ann. App. B i o l . , 2£: 213-21*. Sun, Y.P. 19l*7. An a n a l y s i s o f some important f a c t o r s a f f e c t i n g the r e s u l t s o f f u m i g a t i o n t e s t s on i n s e c t s . Minn. A g r i c . Exp. S t a . Tech. B u l l . 177. . 1950. T o x i c i t y index - an improved method o f 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. T e o t i a , T.P.S. and P a u l A. Dahm. 1950. The e f f e c t o f temperature, humidity, and weathering on r e s i d u a l t o x i c i t i e s t o the h o u s e f l y of f i v e o r g a n i c i n s e c t i c i d e s . J o u r . Econ. Ent.. 1*3 (6): 861*-76. Wadley, P.M. and W.N. S u l l i v a n . 191*3. A study of the dosagem o r t a l i t y curve. J o u r . Econ. Ent., 26 (3). 367-72.  85 Wilkes, A., J.W. Bucher, M.B. Cameron, and A.S. West. 191+.8, S t u d i e s on the h o u s e f l y . Canad. J o u r . Res., S e c t . D., Z o o l . 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 p o i s o n c o n c e n t r a t i o n and s u r v i v a l time o f roaches i n j e c t e d w i t h sodium m e t a r s e n i t e . Ann. E n t . Soc. Am., 2S: 559-600.  86 IX.  APPENDIX  S t a t i s t i c a l a n a l y s i s o f d o s a g e - m o r t a l i t y data Table 1.  T o x i c i t y o f D i e l d r i n to Musca domestica L., QQ, (SES c u l t u r e ) r  Empirical Probit 1.05 150 147 98 98 7.05 2.88 150 lii3 95 95 6.45 6.28 2.81 150 135 90 90 2.78 150 119 79 79 5.81 2.75 300 2k0 80 80 5.8k 1.72 300 i5o 5o 5o 5.oo 2.65 300 87 29 29 4-45 2.61 150 135 45 45 k.87 2.58 300 126 42 42 4-80 k.05 2.53 300 5 i 17 17 150 0 0 n  0.112 0.076 0.06k 0.060 0.056 o.o53 O.Oij.5 O.Olil 0.038 0.03k 0.000  p  p  Y  nw  nwx  7.2 13.8 7.03 28.276 97.01k 6.k 45.3 6.61 85.16k 299-433 6.0 65.8 6.2k 119.098 kl0.592 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  90.1 4.89 145.061 440.589  4.6  4.4 167.3 4-84 264.334 809.732 4.1 l k l - 4 4-05 216.342 572.670  1148.5 y = 5.0952  x = 1.6799  S = n  1929.363 3351.837  l/Snw = O.OOO8707  Snwx Snwxy Snwy^ 3253.335 9908.370 30428.815 ?24j.l3? 9930491 29816.278 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 ( ) « 1.1827; S.E. j = - 0.0874; b  ( b  Log LD^Q = m = 1 . 6 6 5 0 ; v  .*. b = 6 . 3 8 2 6  - 1.0874  A n t i l o g m = 0 . 0 4 6 mgm.  (m) = 0 . 0 0 0 3 1 8 4 ; S . E . ( j = t 0 . O I 7 8 ; m = 1 . 6 6 5 0 - O.OI78 m  g = 1.1549 P.L. = 1.6647 i 0.1412 « 1.8059 - 1.5235 A n t i l o g s = 0.064 mgm.  nwy  and 0.033 mgm.  87 T o x i c i t y o f D i e l d r i n to Musca Domestica L. tf(f, (SES c u l t u r e )  Table 2.  X  n  0.038 0.034 0.030 0.026 0.023 0.019 0.015 0.000  x  2.58 2.53 2.48 2.k2 2.36 2.28 2.18 -  r  p' p  Empirical Probit  150 123 82 82 150 128 85 85 300 222 74 74 150 80 53 53 150 51 34 34 150 Lk 9 9 150 8 5 5 i5o 0 0  5.92 6.0ii 5.64 5.08 4-59 3.66 3.36  Y  nw  y  nwx  6.2 5.9 5.5 5.1 4.6 4.0 3.3 -  55.6 70.7 87.2 95.2 90.1 65.8 31.2  5.87 6.03 5.6k 5.08 4.59 3.72 3-36  87.848 108.171 129.056 135.18k 122.536 84.224 36.816  k95.8 x = 1.4196  y = 5.0248  Snwx 1005.165 999.160 Sxx - 6.005  Snwxy 3582.007 3536.613 =45-394  2  326.372 426.321 491.808 483.616 413.559 244.776 104.832 ,  703.885 2491.28k s 0.0020169  1/Snw  Snwy 12878.125 12518.144 Syy =359-98l  nwy  b = 7.5594  2  Y = -5-7065 + 7-5594* = 16.831  h . f . a 3.366 V( )  - 0.5605; s . E .  b  Log V  (m)  ( b )  = t 0.7487; b = 7-5594 * 0.7487  LD£o = m = 1.4163; A n t i l o g m = 0.026 =  0  .  0 0 0 1 1  89;  mgm.  S . E . ( ) = 0.0109; m = l . k l 6 3 m  - 0.0109  g = 0.0648 S i n c e g i s s m a l l P.L. = (.0109)(2.57) on e i t h e r s i d e o f 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 o f d i e l d r i n to Musca domestica L . , Q 9 » (SES c u l t u r e ) . Three r e p l i c a t i o n s o f each dosage plotted individually A  x  n  0.030 2.48 50 50 _ So 0.026 2.k2 50 50 50 0.023 2.36 50 5o 5o 0.019 2.28 50 50 50 0.000 50 50 50  r  p  p Empirical Probit  f  38 38 36 36 62 62 50 50 34 34 46 46 14 14 40 40 32 32 20 20 28 28 14 14 0 -  19 18 31 25 17 23 7 20 16 10 14 7 0 0 0  5.31 5.00 4.59 4-90 3.92 4-75 4.53 4.16 4.42 3.92  -  y  Snwx 679.464 677-621 =1.843  Snwxy 2251.452 224k.g07 Sxy =6.945  nwx  31.7 4.70 46.916 31.7 4-65 46.916 31.7 5.30 46.916 30.8 5.01 43.736 30.8 4.59 43.736 30.8 4.90 43.736 29.1 4.02 39.576 29.1 4-76 39.576 29.1 4.53 39.576 25.1 4.16 32.128 25.1 4.44 32.128 25.1 3.95 32.128  nwy  lk8.990 ik7.ij.05 168.010 154.308 141.372 150.920 116.982 138.516 131.823 104.416 111.444 99.145  k87.068 1613.331  y = 4.6082  2  xx  4.64  k.9 k.9 4.9 4.7 4.7 4.7 4.5 4.5 4.5 4.2 4.2 4.2  nw  350.1  x = 1.3912  s  4.70  Y  1/Snw = 0.0028563  Snwy 7485.195 7434.^2 Syy =50.643 2  b = 3.7683  Y = 0.6343 + 3-7683x x  2  (10)  = 24.472 ^  h . f . = 2.4472 v  - -328; S . E .  (b)  = 1.152;  1  ( b )  .'. b = 3.7683 - 1.152  Log L D ^ = ra = 1.4952; A n t i l o g m = 0.031 mgm. 0  V  (m)  =  o.ooi^o4; - - ( ) = * 0.038; .*. m = 1.4952 * 0.038 s  E  m  g = 0.4650; P.L. = 1.5854  1  .1488 = 1.7342 and I.4366  Antilogs  = 0.054 mgm and 0.027 mgm.  89 T a b l e k.  A  T o x i c i t y o f d i e l d r i n t o Musca domestica L.,Q<£, (SES c u l t u r e ) . Each p o i n t r e p r e s e n t s t h r e e replications. X  n  p» p.  r  -  2.k8  0.030 0.026 0.023 0.019 0.000  50 50 50 1.1+2 50 50 50 2.36 50 50 50 2.28 50 50 50 50 50 50  38 38 36 36 62 62 50 50 3k 3k k6 k6 lk l k kO kO 32 32 20 20 28 28 lk l k 0 0 0 0  19 18 31 25 17 23 7 20 16 10 lk 7 0 0 0  p Empirical Probit  Y  nw  y  nwy  k6  k.90  k . 9 95-1 k.90 190.748 465-990  43  k.82  k.7  29  4-45  4.5 87.1 4-45 118.456 387.595  21  k.19  k . 2 7 5 . k k.19  -  92.k k.83 131.208 446.292  -  -  96.512 315.926  350.0  7 = 4-6166  X = 1.3912  nwx  -  -  486.92k 1615.803  1/Snw = 0.0028571  Snwx Snwxy Sxwy b = 3-7922 679.257 225k.913 7k87.k68 677.klk 22li.7.92k 7 4 ^ 4 8 3 Y = -0.6591 + 3.7922x S__ = 1.843 S = 6.989 S =27.985 xx xy yy X = 1.48 (2) 2  2  P  2  V  ( b  )  Log v  (m)  = 0.5426; S . E .  ( b )  = t 0.740;  b = 3 - 7 9 2 2 - 0.740  L D ^ Q = m = I . 4 9 2 O ; A n t i l o g m = 0.031 mgm. = 0.00150; S.E.( ) = t 0.038; m  .'. 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-  x  X  T o x i c i t y o f p a r a t h i o n to Musca domestica L., QQ (SES c u l t u r e ) n  r  P"  P  P'  -  0.060 2.78 5o 50 5o 45 So So o.o55 2.7k 50 49 5o 47 5o o.o5o 2.70 50 45 5o 37 - S o 48 0.0k8 2.68 k9 38 5o 35 _ 5o 42 0.045 2.65 50 22 5o 22 5o 29 0.0k2 2.62 Ij.928 50 21 50 11 0.038 2.58 k9 7 50 13 50 17 0.000 - 50 0 5o 0 So 2 k  2  Empirical Probit  100 10097 90 90 100 100 98 98 92 94 94 84 84 90 90 87 ? 7 96 96 77 77 77 70 70 84 84 44 43 48 43 58 58 57 57 40 42 41 22 21 13 24 26 25 34 13 0 .1 0 k  nw  Y  y  7.1 16.4 6.82  29.192  111.848  6.41  6.5 39.9 6.40 69.426  255.360  6.13  6.0 65.0 6.12 110.500  397.800  5.74  5.7 78.2 5.74 131.376  448.868  4.95  5.2 92.5 4.95 152.625  457.875  4.75  4.7 89.4 4.75 144.828  424.650  4-29  4.1 66.6 4.31 105.228  287.046  T  -  —  -  -  y = 5.3202  Snwx Snwxy 1234.036 3970.733 1323.832 3953.516 x x = I.264 3 ^ = 17.217 2  Snwy 12928.872 12680.kOO 248.472 2  = 0.8305; S . E . j = - 0.911;  ( b  -  -  4  x = 1.6588  V )  nwy  6.88  448.0  s  nwx  ( b  7k3.175 2383.kk7  1/Snw = 0.0022321 b = 14-300 Y = -18.4035 + l4-300x  .'. 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.( ) =0.0158; g = 0.0156; P;L. = I.6366 -0.0310 m  ;.f-L. Antilogs  = 1.6676 and 1.6056; = 0.045 mgm. and 0.040 mgm.  91  Table 6. T o x i c i t y o f d i e l d r i n t o Musca domestica L., Q Q (SES c u l t u r e )  A  x  n  nw  r p" p' p E m p i r i c a l Y Probit  0.065 2.81 5038 50 ko _ 5o 39 0.060 2.78 50kO 50 39 _ 5o kO 0.050 2.70 50 36 5o 32 50 37 0.0k8 2.68 50 30 50 29 _ 50 30 0.0k5 2.65 50 30 50 33 50 32 0.038 2.58 50 20 50 19 50 18 0.000 50 0 50 0 50 2  76 76 78 5.77 80 80 78 78 80 80 79 5.81 78 78 80 80 72 72 70 5.52 6k 6k 7k 7k 60 60 59 5.23 58 58 60 60 60 60 63 5.33 66 66 6k 6k kO kO 38 k.70 38 38 36 36 0 1 0 4  y  nwx  5.8 7k.k 5.77 13k.66k  429.288  5.7 78.7 5.80 l k 0 . 0 8 6  456.460  5.k 88.7 5.52 150.790  k89.62k  5.3 90.9 5.23 152.712  475-407  5.2 92.5 5.33  152.625  493.025  k . 9 93.1 k.70 147-098  437-570  -  _  _  _  518.3 x = 1.6939  Snwxy ij.725.lfO7 1487.246 4711.512 Sxx = 2.994 Syy =13.895 S ^ 3  v  (b) = °-334;  S.E.  ( D )  -  877.975 2781.37k l/Snw = 0.001929k  y = 5.3663  Snwx2 Ik90.2k0  nwy  Snwy 14997.966 14925.797 = 72.169 2  J  b = k.6kl Y = -2.4956 + 4 . 6 4 l x 0  Xf = 7.637 (4)  b = 4-641  = ± 0.577;  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  g = 0.2764;  = t 0.0135;  m = 1.615 - 0.0135  P.L. = 1.6120 - O.0350 = 1.6470 and 1.5770 Antilogs  = 0.044 mgm. and O.O38 mgm.  92 Table 7•  A  T o x i c i t y of parathion (SES c u l t u r e )  x  n  rp  t o Musca domestica L . , 66,  p* p E m p i r i c a l Y Probit  M  nw  y  nwx  nwy  0.038 2.58 49 16 33 32 38 50 20 40 39 50 22 44 43 0.034 2.53 5o 26 52 51 41 50 19 38 37  4-70 4-9 90.5 4-70 11+2.990  1+25.350  4-77  4-5 81.2 4.79 124.236  388.948  0.030 2.48 48 16 33 32 19 50 5 10 8 49 9 18 16  4-12  4.1 62.4 4.12  92.352  257.088  3.66 3.8 47.2 3.67 67.496  173.224  49 17 35 34  0.027 2.43 50 7 14 12 9 50 5 10 8 50 4 8 6 0.000 50 1 2 2 50 1 2 P 1 2  -  -  - -  427.074 1244.610  28I.3 x = 1.5182 y = 4.4244 Snwx 2 Snwxy Snwy 649.205 1895.343 5557.i4i 2  s  xx  648.390 =°- S 8 l  s  l/Snw = 0.0035549 b = 7.0638  1889.586 5506.768 x y = 5.757 Syy =50.373 xy JJ  Y = -6.2999+7.0638x X  =  2  (2) h . f . = 4.852 v  ( b ) = 5-9534; s . E .  ( b )  = t 2.440;  Log L D ^ Q = m = 1.5996; V  ( m )  = 0.010; S . E . (  m )  , \ b = 7.064 t 2.440  A n t i l o g m = 0.040 mgm. =  - 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 o f d i e l d r i n to Musca domestica (SES c u l t u r e ) .  A x  n  r p " p»  0.038 2.58 $0 45 50 47 50 44 0.03k 2.53 50 46 50 43 _ 50 44 0.030 2.k8 50 42 50 41 50 40 0.027 2\k3 50 38 50 31 50 36 0.023 2.36 50 25 50 37 So 25 0.019 2.28 50 20 50 18 50 13 0.000 50 1 1 50 5o 1  k  88 92 86 88 84 82 80 76 62 72 50 74 5o 40 36 26 2 2 2  9  6.34  6.4  49.3 6.34  77.894  312.562  88 92 86 88 84 82 80 76 61 71 49 73 49 39 35 24 2  89  6.23  6.1 59.3 6.22  90.729  368.846  82  5.92  5.8  73.5 5.91 108.780  434.385  69  5.50/  5.5 84.7 5.50 121.121  465.850  57  5.18  5.1  91.7 5.18 124.712  475.006  33  4-56  4.7  90.0 4.56 115.200  410.400  V  Log V  = 0.2358;  ( b )  (m)  LE>£Q =  0*  y = 5.5007  x y  Snwxy Snwy 3538.558 13737.210 3511.823 13570.klk =26.735 Syy =166.796  S . E . ) = ± 0.485; (b  g = 0.023;  b = 6.3039 Y = -3.4723+6.3039x A  (4)  — 1 . Jl+d  b = 6.3039 ± 0.485  = m as 1.3440; A n t i l o g m = 0.022 0 0 1  638.k36 2k67.0k9 l/Snw = 0.0022295  2  S  mgm.  S 3 2 ; S . E . ) = t 0.039; m = 1.3440 - 0 . 0 3 9 . (m  P . L . 1.96 x 0.039 on e i t h e r s i d e o f m =  1.3440 - O.0764 = l . k 2 0 4 and 1.2676 , Antilogs  nwy  nwx  y  k  x = l.k234 2  ¥  Y  kk8.5  Snwx 913.049 908.808 Sxx = 4.241  ¥  Empirical Probit  p  90 90 91 9  nw  L . , Q Q ,  = 0.026 mgm.  arid 0.019  mgm.  94 Table 9.  X  x  T o x i c i t y o f E P N t o Mu3ca domestica ( S E S culture) n  r  p*  p" -  p Empirical Probit  1.350 0.130 50 50 100 100 86 50 50 100 100 50 29 58 58 1.275 0.106 50 25 50 50 76 50 45 90 90 50 ii4 88 88 1.200 0.079 50 32 6k 6k 62 50 17 ^ 49 k3 88 88 1.125 0.051 50 39 78 78 71 50 2k i t ft 48 1+2 n87 87 1.050 0.021 50 32 6k 6k 69 50 i|2 ®k 84 50 29 58 58 0.975 1.99 50 26 52 52 31 50 lk 3k 34 50 7 lk 14 0 0.000 50 0 0 5o 0 0 5o 0 0 8  Y  L . , Q Q ,  nw  y  6.0 65.8 6.08 74-354  400.064  5.71  5.8 75.4 5.70 83-392  429.780  5.31  5.5 86.6 5.30  93-441  458.980  5.55  5.3 91.2 5.54  95-760  505.248  5.5o  5.1 95.2 5-48  97-199  521.696  4.5o  4.9 95.2 4.52  94.248  430.304  8  -  -  -  7 = 5.3908  x = 1.0569  h . f . = 10.409 V ( ) = 8.4833J b  S ^  -  Snwxy 2911.805 2902.372 = 9.433  b  (m)  =  °-  0 0  °7l6l;  S  - «( ) E  m  = * 0.0267;  -  538.394 2746.072  l/Snw = 0.0019631  Snwy 14917.671 14803.516 Syy =114.155 ™  S . E . ( ) = i 2.9126;  -  2  b = 7-6879  Y = 2.7345 + 7-6879x . , X = 41-635 (4) ?  2  ,\ b = 7.6879 - 2.9126  Log L D ^ Q = m = 1.0061; A n t i l o g m = 1.014 mgm. V  nwy  6.08  509.4  Snwx 570.265 569.038 = 1.227  nwx  .*. m = 1.0061 ± 0.0267  g = 1-109 S i n c e g i s so l a r g e i t i s not p o s s i b l e t o c a l c u l a t e P.L.  95 T a b l e 10.  A  x  Toxicity of dieldrin (SES c u l t u r e ) n  0.070 2.85 49 50 5o 0.060 2.78 50 50 5o 0.050 2.70 50 50 49 0.048 2.68 50 50 50 0.045 2.65 50 5o 5o 0.038 2.58 50 50 49 0.000 - 5 0 50 50  r p" p  t o Musca domestica L . , QQ ,  p Empirical Probit  1  31 63 63 66 35 70 70 33 66 66 29 58 58 56 26 52 52 29 58 58 30 60 60 60 33 66 66 26 53 53 18 36 36 39 15 30 30 25 50 50 29 58 58 56 29 58 58 26 52 52 10 20 20 23 15 30 30 10 20 20 0 0 0 0 0 0 0  Y  nw  468.506  5.15  5.2 94.1 5.15 167.488  484.615  5.25  5.0  94.9 5.25 161.330  498.225  4-72  4-9 95.2 4.72 159.936  449.344  5.15  4.8 94.1 5.16 155.265  485.556  4-26  4.6  378.585  -  89.5 4-23 l k l . k l O  -  -  1617.104 1613.009 S ^ = 4,095 h . f . = 8.4438 V = 2.0620} S . E . ( b )  -  ;  Snwxy  4730.563 4716.016 =14.547  ( b )  Snwy  2  13873.853 13788.402 Sjy= 85.451  = ± 1.436;  945.649 2764.831  l/Snw = 0.0018038  y = 4.9871  2  nwy  5.5 86.6 5.ki 160.210  554.4  x = 1.7057  nwx  5.1+1  :  Snwx  y  b = 3.5524  Y = 1.0722 + 3-552kx 0  Xf = 33-775 (4)  .'. b = 3.5524 - 1.4360  Log LD. = n = 1.7093; A n t i l o g m. = 0.052 mgm. 50 V ( ) = 0.2843; S . E . m  ( 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 T o x i c i t y o f EPN t o Musca domestica L . , d c f , (SES c u l t u r e )  Table 11.  A  x  n  r p" p ' p E m p i r i c a l probit  0.750 1.88 50 46 92 92 50 48 96 96 50 44 88 88 0.705 1-85 49 45 92 92 50.41 82 82 50 50100100 0.638 1.81 49 33 67 67 50 39 78 78 50 40 80 80 0.600 I.78 49 39 79 79 50 44 88 88 50 29 58 57 0.563 1.75 49 46 94 94 49 35 71 71 50 23 46 45 0.525 1-72 49 13 27 26 50 23 46 45 50 43 86 86 0.000 - 5 o 0 0 1 50 1 2 50 0 0  Y  nw  y  6.41  6.5 39.9 6.4,0  75.012  255-360  91  6.34  6.2 54*6 6.33 101.010  345.618  75  5-67  5.9 69.4 5.65 125.614  392.110  74  5-64  5-6 82.0 5-64 145-960  462.480  70  5.52  5-3 89-7 5-52 156.975  495-144  52  4.05  5-1 92.8 5.05 159.616  468.640  -  x = 1.7838  -  -  -  y = 5.6474 Snwxy 4324.966 4315,680 S "= 9.286 ^  2  S  V  M  (b)  Log  =  °*  8 7 8 7  J  s  * -(b) E  =  nwy  92  428.4  Snwx 1364.307 1363.169 = 1.138  nwx  2  0-94 0; 2  ±  LD^Q = m = 1.7044;  l/Snw = 0.0023343  Snwy 13745-701 13664.482 S ™ = 81.219 5  7  764.187 2419.352  b = 8.1599 Y = 8.9082 + 8.l599x 0  x  2 6 )  = 5.446  b = 8.1599 - 0.9420  A n t i l o g m = 0.506 mgm.  V ( ) ; 0.0001181; S.E.( ) = 0.011; m = 0.506 ± 0.011 m  g = 0.049;  m  Since g i s small, P.L. = 1.96 x 0.011 on e i t h e r  s i d e o f m = 1.7044 - 0.0216 = 1.7260 and 1.6828 = 0.532 mgm. and 0.482 mgm.  97 Table 12.  X  T o x i c i t y o f D i e l d r i n t o Musca Domestica L.,(£f, (SES c u l t u r e )  x  n  r  0.038 2.58 50 50 _ , 39 0.03k 2.53 50 50 50 0.030 2.48 Ij.9 49 50 0.027 2.43 50 50 50 0.023 5.36 49 50 50 0.019 2.28 49 50 50 0.000 50  p" p' p  43 38 20 ko 32 37 43 42 37 26 37 21 11 6 5 3 11 6 0 49 0 49 0  86 76 51 80 6k 7k 88 86 74 52 74 42 21 12 10 6 22 12 0 0 0  86 76 51 80 6k 7k 88 86 74 52 74 k2 21 12 10 6 22 2 0  Empirical Probit  Y  71  5-55  5.9  65-5 5.50 103.1*90  360.250  73  5.61  5.6  83.7 5.61 128.061  469.557  83  5.95  5.3  91.2 5.86 134-976  ^ 534-432  56  5.15  4.9  95.2 5.15 136.136  490.280  14  3.92  4.4  83.I 4-00 113.016  332.400  13  3-87  3.9  59.9 3.8?  231.813  -  nw  -  y  ,  y = $.0$3&  x = 1.4466  Snwxy 3528.461 3498.979 S n =29.482  2  76.672  nwy  -  k78.6  Snwx 1005.727 1001.567 S ^ = k.160  nwx  y  692.351 2418.732  1/Snw = 0.0020894  Snwy 12499.020 12223.703 Syy = 275.318 2  b = 7.0870 Y = -5.1983+7.0870x x  (4)  =  00  •378  h . f . = 16.5945 V  (b)  =  3  *  9 8 9  ° 5 - -(b) s  E  58  * 1-9970;  Log LD^Q = m = 1.4390; v  / . b = 7.0870 ± 1.9970  A n t i l o g m = 0.028 mgm.  (m) = 0.000695; S.E.( ) = 0.026; m = 1.4390 - 0.0260 m  g = 0.6138;  P.L. = 1.4270 ± .1186 = 1.5456 and I.3083 Antilogs  =  0.035 mgm. and 0.020 mgm.  98 Table 13. X  x  T o x i c i t y o f endrin to Musca domestica L., QQ , (SES c u l t u r e ) n  50 49 0.300 T.48 50 50 50 0.285 1.41 50 50 50 0.225 1.35 50 50 50 0.000 50 50 50  r  p  H  p» p E m p i r i c a l Probit  42 8k 84 42 86 86 37 74 74 73 36 72 72 36 72 72 16 32 32 35 16 32 32 20 40 4o 13 26 26 35 19 38 38 20 40 40 0 0 0 0 0 0 0  s  y  nwx  70.2 5.87 IO6.704  5.61  5.5  87.1 5 . 6 l  4.61  4-9  95.1 4.62 134.091  439.362  4.61  4.3  79.7  370.605  -  4-65 107.595  -  y = 5.1511 Snwxy 2469.342 2458.597 S^Io.745  477.298 1710.672  l/Snw = 0.0030111  Snwy 8913.258 8811.799 Syy ==101.459  b = 8.1649  2  Y = -6.5840 + 8.l649x . ( 2 ) ~ 13.727  h . f . = 6.864 V ( ) = 5.2158; S . E . ) = t 2.2838; Log LD^ v  (b  0  = m = I.4190;  (m) = 0.003348;  S.E.  .\ b = 8.1649 - 2.2838  A n t i l o g m = 0.262 mgm. ( m )  = 0.0183;  412.074  128.908 488.631  x  b  nwy  5.9  332.1  x = 1.4372 2  nw  5.88  '  Snwx 687.294 685.978 xx= 1-316  Y  g = 1.4466  P.L. cannot be c a l c u l a t e d a t the 95$ p r o b a b i l i t y l e v e l .  99 Table l k .  X  A  T o x i c i t y o f d i e l d r i n to Musca domestica L., QQ , (SES c u l t u r e ) n  0.068 2.83  49 5o 5o 0.060 2.78 5o 5o 50 0.053 2.72 5o 5o 5o 0.045 2.65 5o 50 5o 0.000 48 49 48  -  r P" P» 4i 42 43 39 38 39 34 36 34 32 27 43 1 0 9  83 83 85 76 74 76 65 70 65 61 51 85 7  84 84 86 78 76 78 68 72 68 64 54 86 2 0 18  p  Empirical Probit  Y  nw  y  nwx  84  5-99  5.9  64.3  5.99 117.669  385.157  75  5.67  5.7  72.5 5-67 129.050  411.075  67  5.44  5.5  78.6  5.44 135.192  427.584  66  5.4i  5.3  82.4  5.41 135.960  445.784  517.871 1669.600  297.8 x == 1.7390  y =-. 5.6064  2 Snwx 901.907 900.572 S^=-1^3$  Snwxy 2907.537 290^.416 = k.121  V, = 0.7491; (b) x  Log V  (m)  S^  S.E.  =  0-00338;  P.L. = 1.4576  I/Snw = 0.003580 P  S y y  Snwy* 9375.633 9360.52k = 15.109  +  ^ = - 0.8650; (b)  LD^Q = m = 1.5425; S.E.  ( m )  nwy  Y = 0.2383+3.0869x 2  x^  2)  =2.388  +  .'. b = 3.0869 - 0.8650  A n t i l o g m = 0.0349 = ± 0.058;  b = 3-0869  mgm.  g = 0.3018  - 0.1608 = 1.6184 and 1.2968 A n t i l o g s = 0.042 mgm.  and 0.020  mgm.  IQO Table 15- T o x i c i t y o f e n d r i n to Musca domestica (SES c u l t u r e ) x  n  r  P  P  H  p Empirical Probit  1  -  0.233 1.37 SO k8 96 50 k6 92 -1.29 50 t 9 kk 90 0.195 kl ^ k9 k3 50- ko 80 0.158 1.20 5o ko 80 5o k2 8k 5o k3 86 0.128 l . n 5o 38 75 5o 33 66 5o 3k 68 0.000 5o 0 0 5o l ;.2 $o 2 k  96 93 92 90 87 ^ 88 80 8k 83 8k 86 75 69 65 67 2  nw  Y  y  25k.016  6.13  6.2 53.9 6.12 69.531  329.868  5.95  5.8 73.5 5.9k 88.200  k36.590  5.5o  5.5 8k.7 5.50 9k'.017  k65.850  «.  2  y = 5.91k5 Sxwxy l8lk.532 1806.608 Sxy = ? - k  2  1 9  9 2  305.k52 lk86.32k  l/Snw = 0.0039793  Snwy 8820.335 8790.923 3^=29.412 2  b = 3.6133 Y = 1.5225 + 3.6l33x x  V  = 0.k559; - . ( ) S  E  D  ( b )  Log LD^Q = m = 0.962k; v  (m)  =  = - 0.6750;  u r  ,  7  8  1  b = 3.6133 - 0.6750  / . m = 0.962k - 0.0958  g = 0.13kl F.L. = 0.9585 - 0.1132 « 1.0717 and 0.8k53 Antilogs  0  A n t i l o g m = 0.092 mgm.  0'Q092; S.E.( ) = - 0.0958; m  nwy  6.5 39.2 6.k8 53.70k  251.3  Snwx 373.k66 371.273 Sxx= ' 3  nwx  6.k8  -  x = 1.2155  L.cfcf,  = 0.118 mgm. and 0.070 mgm.  101 Table 16.  T o x i c i t y o f d i e l d r i n to Musca domestica L . , db", (SES c u l t u r e ) n  r P  k9 1+5 So 1+6 1+9 1+7 50 1+5 1+9 45 50 46 50 39 5o 45 50 41 50 30 5o 40 5o 42 50 0 5o 0 1 So  w  92 92 96 90 92 92 78 90 92 60 80 84 0 0 2  nw  nwx  y  92 92 96 90 92 98 78 90 82 59 80 84 1  93  6.48  6.5 39.4 6.48  63-434  255.312  91  6.34  6.3 49.5  6.34  75-735  313.830  83  5.95  5.9 69.9  5.95  99.258  415.905  74  5.64  5.7  78.7 5.64  107.032  443.868  -  -  -  -  237.5 x = 1,4546  S  y = 6.0165  Snwx 504.514 502.492 = 2.022  x x  S  x v  Snwxy 2085-458 2078.449 = 7.009  y  0.4945;  V( )= b  s  « .( ) E  b  S_ 7  =  t  0.7030;  (m)  =  345.459 1428.915  l/Snw = 0.0042105 Snwy 8622.153 8597.044 = 25-109  b = 3.4664 Y =  0.9743+3.466kx  0  x  7  2  2 )  = 0.813  .*. b = 3.4664 - 0.7030  Log LD . = m = 1.1613; A n t i l o g m = 0.015 50 V  nwy  p  -  Empirical Probit  Y  p'  mgm.  0.0038905; S.E.( ) = i 0.0623; /. m = 1.1613 - 0.0623  g = 0.2282;  m  P.L. = 1.1527 - 0.1568 = 1.3095 and 0.9959 Antilogs  = 0.020 mgm.  and 0.010  mgm.  102 Table 17.  A  x  T o x i c i t y of a l d r i n (SES c u l t u r e ) n  0.068 2.83 50 50 50 0.064 2.81 5o 5o 50 0.060 2.78 49 49 50 5o 0.053 2.72 50 50 50 0.045 2.65 50 50 50 0.000 50 50 5o  to Musca domestica L.,QQ, *  r p" p' p E m p i r i c a l Probit 18 20 32 30 3k 26 12 24 ' 32 8 20 12 16 12 10 0 0 0  Y  nw  4-92  5.1  95-1 4-92 174.033 467.892  60  5.25  5.0  95.5 5.25 172.855 501.375  45  4.87  4.9  94.5 4-87 168.210 460.215  27  4-39  4.6  90.1 4.38 154.972 394.638  25  4.33  4.3  79.7 4-33 131.505  -  -  -  36 36 47  4o 40  64 60 68 52 24 48 48 64 16 40 24 32 24 20 0 0 0  64 60 68 52 24 48 64 16 40 24 32 24 20 0  y  -  nwx  y = 4.7686  Snwx 1414.297 1412.kkS S ^ ^ m  Snwxy 3831.108 3822.363 3 ^ = - ^  2  1/Snw Snwy 10398.829 10344.074 = ^-755 2  S  y  y  801.575 2169.221 = 0.0021983  b = 4-7295 Y = -3.5653+4-7295x ^ =  i  3  9  5  h . f . = 4-465 = 2.4147;  V( ) b  S.E.  ( b )  Log I*D^ = m = 1.8110; 0  V  (m)  =  0-0°S72;  g = 1.0917;  s  = ± 1.5530;  - - ( ) = - 0.0756; E  m  .*. b = 4-7295 -  A n t i l o g m = 0.065  345.101  -  454.9 x = 1.7621  nwy  1.5530  mgm.  m = 1.8110 t 0.0756  •"• i m p o s s i b l e to c a l c u l a t e f i d u c i a l  limits.  103 Table 18.  T o x i c i t y of d i e l d r i n t o Musca domestica (SES c u l t u r e ) n 5o k8 So k9 SO k9 SO So So k9 k9 k9 So So So So So  50  r P 27 26 k3 23 23 27 18 11 16 9 19 12 17 8 28 0 0 0  tt  Sk 5 86 52 k6 kS 36 22 32 18 39 2$ k  3k  16 $6 0 0 0  p» p  Empirical Probit  Y  Sk 6S  5.39  5.2  92.9 5.38 168.Ik9  k99.802  52 51 k6 SS 36 30 22 32 18 27 39 2S 3k 35 16 56 0 mm  5.03  5.0  91.0  5.03 162.890  k57.730  k.k8  k.8  9k. 1 k.k9  161.852  k22.509  k.39  k.5  85,k k.39  IkO.910  37k.906  k.6l  k.k  83.7 k.63  13k.757  387.531  % 86  S XX  -  mm  nw..  y  -  -  kk7.1  x = 1.7189 2 Snwx 1323.768 1321.139 = 2.629  L.,QQ  y = k.7919  S xy  Snwxy 3693.213 3682.886 = 10.327  nwx  nwy  mm  mm  768.558 21k2.k78  l/Snw = 0.0022366 Pc  Snwy 10328.ii88 10266.633 S = 61.855 33  b = 3.9281 Y = -1.9601+3.928lx ? X = 21.290 (3)  h . f . = 7.096 = 2.6991; S . E . ) = t 1.6k20; (b  Log LD^o = m = 1.7718; V  ( m )  = 0.001518; S . E .  / . b = 3.9281 ± 1.6k20,  A n t i l o g m = 0.0590 mgm. ( m )  = t 0.039;  g = 1.7689  Since g i s so l a r g e , i t i s impossible t o c a l c u l a t e f i d u c i a l  limits.  10k T o x i c i t y of a l d r i n t o Musca domestica L., 66, (SES c u l t u r e )  Table 19.  Empirical Y Probit 50 50 0.03k 2.53 50 50  378.350  k-0 80 80 81 43 86 86  5.88  5.8  74.9  5.88 114.597  440.kl2  37 74 74 76  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  37 74 74  49 50 50  0  0  0 0 0 0  0  408.1  x = 1.4337  y = 5.3421  P  Snwx 843.800' 838.894  =4.906  S___.  3125.704 S__ = 21.161  = 0.2038; s . E .  b)  ( b )  11646.328  Syy = 95.252  Log LD = m = 1.3544; 50 V  (m)  =  0'  o o 8 6  S; s.E.  ( m )  1  = ± 0.4510;  585.109 2180.107 l/Snw = 0.0024504  p Snwy 11744.580  Snwxy 3146.865 7  y^  nwy  65.8 5.75 103.964  49 3 6 6 50 21 42 k2  -  nwx  6.0  40 80 80 30 61 61 48 49 13 27 27 50 28 56 56 0.019 2.28 50 31 62 62 37 0.000  y  5.77  kl 82 82 46 92 92  49 38 76 76  0.030 2.48 50 50 50 0.023 2.36 49  nw  b = 4•3132 Y = -0.84l7+4*3132x 0  X  2 3 )  =7.590  .'. b = 4.3132*0.4510  A n t i l o g m = 0.022 = 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. A n t i l o g s = 0.026 mgm. and 0.020 mgm.  105 T o x i c i t y o f d i e l d r i n t o Musca domestica L., dc? * (SES c u l t u r e )  Table 20.  A  x  n  r  p  n  p» p  Empirical Probit  0.030 2.2+8 50 19 38 38 4 ° 50 18 36 36 50 31 62 62 0.026 2\2|2 50 25 50 50 2+3 50 17 34 34 50 23 36 36 0.023 2.36 50 7 14 14 29 50 20 40 40 50 16 32 32 0.019 2.28 50 10 20 20 21 50 14 28 28 50 7 14 14 0.000 50 0 0 0 50 0 0 5o 0 0  Y  nw  y  nwx  2+.90  It.9 95-1 4-90 12+0.748  2+65.990  2+..82  2+.7  92.2+ 2+.83 131.208  2+46.292  4-45  4.5  87.1 4.45 118.456  387.595  4.19  4.2  -  -  75-4 4-19 -  S  x x  Snwx 679.257 677.414 =1.843  y = 4.6166  2  S  x y  Snwxy 2254-913 2247.924 =6.989  s  - -( ) E  b  b  Log LD^ = ra = 1.4920; G  V  (m)  =  °-  0 0 1  5 j - -(m)  g = 0.l2ji+8;  s  E  =  =  l/Snw = 0.0028571  Snwy 8487.468 7459.483 Syy =27.985 2  °-740;  315.926  486.924 1615.803  b = 3.7922 Y = -0.6591 + 3.7922x 2 X  V ( ) = 0.5426;  96.512  -  350.0 x = 1.3912  nwy  (2)  =  .'. b = 3.7922 t 0.740  A n t i l o g m = 0.031 mgm. " °-°380;  . m = 1.4920 - 0.0380  + P.L. = 1.5091 - ©.0638 = 1.5729 and 1.2+453 A n t i l o g s = 0.037 mgm. and 0.028 mgm.  106 Table 21.  A  x  T o x i c i t y of l i n d a n e t o Musca domestica L.,QQ, (SES c u l t u r e ) n  r  p" p* p  Empirical Probit  0.068 2.83 50 30 60 60 75 50 38 76 76 50 44 88 88 0.060 2.78 50 29 58 58 73 50 kO 80 80 50 40 80 80 0.56 2.75 50 36 72 72 69 50 ij.0 80 80 50 27 5k 5k 0.053 2.72 50 37 7k 74 66 50 30 60 60 50 32 6k 6k 0.045 2.65 50 81 k2 k2 49 50 25 50 50 50 28 56 56 O.Okl 2.61 50 36 72 72 57 50 25 50 50 k.9 2k ij.8 ij.8 0.000 - 5 0 0 0 0 50 0 0 k9 0 0  Y  nw  y  nwx  5.68  5-7 79.7 5.67 145.851  ij.5l.899  5.61  5.6 83.7 5-61 148.986  469.557  5-50  5 - 5 8?.2 5.50 152.600  479.600  5 - k l 5 - 4 90.1 5 . k l 154-972  k87.kkl  k.98  5.2 9k.1 k.97 155.265  467.677  5.18  5.1 95.2 5.18 153.272  -  -  y = 5.3761  2  S  V  (b)  Snwx 1568.659 1565.703 = 2.956  =  °-  3 3 8  2?  S  S  *  Snwxy k905.700 k897.297 = 8.k03  * *( ) E  Log LTJCJQ = m = 1.586; V  Cm)  =  0.001002;  S.E.  910.9k6 28k9.310  1/Snw = 0.0018868 ?  Snwy 15350.038 15318.052 =31.986 ™  " °.S8l5j  =  b  b = 2.8k26 Y = 0.k902+2.8k26x P  X , (4) 2  = 8.099  •'. b = 2.8426 - 0.5815  A n t l l o g m = 0.039 mgm. ( m )  P.L. = 1.5600 - 0.0720;  k93.136  -  530.0 x = 1.7188  nwy  = t 0.0316;  g =  O.I63.  1.6320 and I.488O  A n t i l o g s = 0.043 mgm. and 0.031 mgm.  107  Table 22.  A  T o x i c i t y o f d i e l d r i n t o Musca domestica L.,QQ (SES c u l t u r e ) *  n  X  r  0.070 2.85  50 50 0.060 2.78 5o 5o 5o 0.050 2.70 50 5o 5o 0.01+8 2.68 5o 5o 5o 0.01+5 2.65 5o 5o 5o 0.038 2.58 5o 5o 5o 0.000 • 5o 5o 5o  Empirical Y Probit k.98 k3 1 + 9 5.0 ^ 58 58 1+6 1+6 28 28 31 k.50 k.7 28 28 39 39 58 58 1+2 1+.80 k.k 28 28 10+ 1+1+ k.26 k.3 Ik l k 23 32 32 22 22 30 30 20 k.16 1+.2 12 12 18 18 10 10 12 3.8 3.83 8 8 18 18 0 0 0 0  nw  p' p  20 29 23 ll+ 11+ 19 29 llj. 22 7 16 11 15 6 9 5 k  9 0 0 0  _  y  nwx  93.9 k.98 173.715  1+67.622  92.k k . 5 l I6k..k72  1+16.72k  83.7 k . 8 k 11+2.290  1+05.108  79.7 k.26 133.896  339.522  75.1+ k.16 12k.klO  313.661+  60.7 3.83  232.1+81  — _  95.906  _  _ 83k.689 2175.21  k85.8 1I = 1.7181  Snwx 11+37.779 lk3k.lkk Sxx = 3 . 6 3 5  y == l+.k77k  2  ^  Snwxy 3751.017 3737.237 =13.780 S 7  1/Snw = 0.002058k  Snwy 9810.513 9738.887 =71.626 2  7  b = 3.7909 Y = -2.0355+3.7909x x  = 1.3331+; S . E .  ( b )  Log V  (m)  LD^ =  0  ( b )  = I.l5k0;  = ra = 1.8550;  0.0021+; S . E .  0 2  = 19.388  (1+)  h . f . = k.8k7 V  ( m )  .'. b = 3.7090 - 1.151+0  A n t l l o g m = 0.072 mgm.  = ± 0.0k93;  g = 0.717  P.L. = 2.2020 - 0.5990 = 2.8010 and 1.6030 Antilogs  nwy  = 0.632 mgm. and O.Oi+O mgm.  108 Table 23.  X  x  T o x i c i t y of l i n d a n e (SES c u l t u r e ) n  0.030 2.k8 5o 5o 5o 0.027 2.k3 5o 5o 5o 0.023 2.36 5o 5o 5o 0.019 2.28 50 5o 5o 0.015 2.18 5o 50 50 0.011 2.0k 50 5o 48 0.000 5o 50 k8  r  P" P' P  36 kl kl 43 k6 44 52 37 kk 35 2k 43 25 23 39 3k 22 23 3 3 2  72 82 82 86 92 88 8k 7 88 70  70 81 81 85 92 88 83 73 87 68  k  & 85  66 50 k6 78 68 kk k6 6 6 4  Empirical Probit  Y  nw  y  6.0  61.9 5-71  91.612  353.kk9  88  6.18  5.9  66.k 6.1k  9k.952  k07.696  81  5.88  5.7  7k.6 5.87  101.456  k37-902  66  5.kl  5.5  81.0 5 . k l 103.680  k38.210  5.15  5.3  85.2 5.15 110.536  438.780  5.00  k.9  8k.2 5.00  421.000  -  -  -  x = 1.2791  S  Snwxy 3218.166 3193.893 = 24.273  s  x v  579.804  2497.037  l/Snw = 0.0022060  Snwy 12827.365 13755.115 72.250 2  ^  7  87.568  -  y = 5.5086  2  nwy  5.7k  JfeS3.fJt  Snwx 751.761 740.690 x x =11.071  nwx  77  47 56 43 77 66 50 kl 5  to Musca domestica L.. db",  b = 2.1925 Y = 2.70k2 + 2.1925x o zr = 19.045 {k)  H.f. = 4.761 V ( ) = 0.4300; S . E . ^ j = t 0.6550; b  b = 2.1925 - 0.6550.  Log LD^Q = m = 1.0470; A n t i l o g m = 0.0110 mgm. V  (m)  =  °-°°699;  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 A n t i l o g s = 0.016 mgm. and 0.00073 mgm.  109 Table 2k.  T o x i c i t y o f d i e l d r i n to Musca domestica (SES c u l t u r e ) r  p" P' P  31 62 36 73 k3 86 k l 82 37 k 26 53 27 5k 17 3k 2k k9 kO 82 23 k8 37 76 21 k3 12 1, M- 0 13 27 k 8 1k 30 0 0 0 0 0 0  Z  62 7k 73 86 82 70 7k 53 5k k6 3k k  o 27 22 8 30 0 -  y  Snwx 1103.398 1098.360 =5.038  nwx  S  x v  *  nwy  5.6k  5.6  83.1 5.6k 131.298  k68.68k  5.52  5.k  89.5 5.52 136.935  k9k.0k0  k.90  5.2  93.5 k.90 138.380  k58.l50  5-50  k.9  93.2 5.k8 133.276  5lk.736  k.33  k.6  89.5 k . 3 k 121.720  388.k30  k.23  k.3  76.6 k.23  32k.018  -  -  y = 5.0325  2  f  Y  -  -  525.k x = l.kk59  t  Empirical Probit  9  82 69 k8 76 k3 25  nw  L. d(j  Sxwxy 38k7.825 3822.377 = 25.tk8  98.0k8  -  -  759.657 26kk.Q58  l/Snw = 0.0019033  Snwy 13k70.629 13306.133 Syy = 16k.k96 2  b = 5.0512 Y =-2.2710+5.05l2x P  = 35.933  h . f . = 8.983 V  ( b )  = 1.7830;  S.E.  ( b )  Log LD^ = m = l.k390; Q  v  (m) = 0.0006733; S . E .  g = 0.5k8;  = t 1.3350;  /. b = 5.0512 - 1.3350  A n t i l o g m = 0.028 mgm. ( m )  = 0.0259;.'.". m = l.k390 - 0.0259  P.L. = l.k310 t 0.1070 = 1.5380 and 1.32k0 Antilogs  = 0.035 mgm. and 0.021 mgm.  110 Table 25.  A  T o x i c i t y o f h e p t a c h l o r to Musca domestica L., QQ (SES c u l t u r e ) n  x  0.060 2.78 50 50 50 0.053 2.72 50 50 50 0.045 2.65 50 50 50 O.Okl 2.61 k8 50 50 0.038 2.58 50 50 50 0.030 2.k8 50 50 50 0.000 50 50 50  r  p" p' p  43 k8 43 46 47 48 46 37 32 42 42 42 47 36 42 36 13 24 2 1 1  86 86 89 96 96 86 86 92 92 94 94 94 96 96 92 92 76 74 73 6k 63 88 88 85 84 83 84 83 94 94 83 72 71 84 83 72 71 47 26 24 48 46 4 3 2 2  Empirical Probiit  Y  nw  y  6.23  6.5  39.1 6.16  69.598  240.856  6.55  6.3  48.7 6.52  83.764  317.524  5.71  5.9  68.1 5.69 112.365  387.489  6.04  5.8  71.6 6.01 115.276  430.316  5.95  5.7  76.6 5.93 121.028  454-238  4-93  5.2  89.3 4.92 132.164  439.356  -  -  -  -  393.4 x = 1.6121 Snwx 1025.781 1022.377 = 3.404  y = S%n Snwxy 3674.973 3659.081 = 15-892  2  S  S xy  nwy  nwx  . -  -  634.195 2269.779  l/Snw = 0.0025419 Snwy 13200.203 13095.822 S _ =104.381 JJ  b = 4*6686  2  Y = -1.7567+4-6686x x  2 (4)  30.187  h . f . = 7.547 = 2.2171; S.E.( ) = t 1.4890;  V( ) b  b  Log L D ^ = m = 1.4473;  A n t i l o g m = 0.030 mgm.  0  v  (m)  =  0-003636;  g =0.786;  s  - E  ( m  .*. b = 4.6689 - 1.4890  )  = - 0.0603;  .'. m = 1.4473 - O.O603  P. L. = 0.8k20 - 0.7044 = 1.5464 and 0.1376 A n t i l o g s = 0.035 mgm. and 0.0014 mgm.  Ill Table 26. T o x i c i t y o f d i e l d r i n to Musca domestica L., g o (SES c u l t u r e )  A  x  n  0.068 2.83 50 50 50 0.060 2.78 50 50 50 0.053 2.72 50 50 _ 50 0.049 2.69 l\B 50 50 0.045 2.65 50 49 _ 50 0.038 2.58 50 50 50 0.000 50 5o  r  Empirical Probit  P" P' P  68 76 8k 6k 70 56 52 68 56 36 5k 6k kk kO 38 26 20 4 2 2  67 75 83 63 69 55 50 67 55 3k 53 63 k2 38 36 2  nw  y  nwx  5.7  76.6 5.6k l k 0 . 1 7 8  432.024  72  5.58  5.5  83.k  5.58  148.452  465-372  57  5.18  5.2  89.3  5.18  133.596  462.574  47  k.92  5.0  88.7  k . 9 3 149.903  437.291  k8  k.9k  k.8  87.1 k.97 I k 3 . 7 l 5  432.887  26  k.36  k.k  75.2 4.36 118.816  327.872  k  18 3 -  -  mm  mm  mm  y = 5.1130  x = 1.7083  V  (b)  S  x y  Snwxy 4385.915 4369^852 =16.063  = °-3i85; s.E.  ( b )  Log L D ^ Q = m = 1.6862; V( ) m  = 0.00008229;  -  mm  500.3  2 Snwx 1463.151 1460.011 S__ = 3.140 xx  nwy  5.6k  ko 80 79 79  3k 38 k2 32 35 28 26 3k 27 18 27 32 22 20 19 13 10 2 1 5o 1  Y  854.660 2558.020  l/Snw = 0.0019988  2 Snwy 13166.336 13079.085 Syy = 87.251 ™  b = 5.1156 Y = - 3 . 6260+ 5 . Il56x 0  xf = 5.079 (4) b = 5 . n 5 6 ± 0.5640  = 0.5640;  A n t i l o g m = 0.048  S . E . ( ) = -.0.0091; m  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.  A  x  n  r  p" p* p  0.026 2 . k l $0 k l 50 kO f?o 38 0.023 2.36 50 kk 50 1+2 50 k l 0.019 2.28 50 3k k9 37 50 36 0.015 2.18 50 38 k9 19 k9 31 0.011 2.0k 50 18 k8 9 50 11 0.000 99 0 98 0 100 0  Snwx 627.233 620.397 = 6.836  (b)  =  0-5360;  Log LD^Q V  (m)  nw  y  nwx  nwy  5-8l  6.0  65.8 5.79  92.778  380.982  6.Ok  5.9  70.7 6.03 96.152  k26.321  5.58  5.5  86.6 5.58 110.8k8  k83.228  5.23  5.1  93.9 5.23 110.802  k91.097  k.36  k.5  86.0 k.36  37k.960  -  -  -  y = 5.3513  Snwxy 270k.966 2675.77k S„__ = 29.192 xy  2  Y  -  l/Snw = 0.002k8lk  Snwy 11676.277 Il5k0.62k =135.653 2  w  89.kk0  b = k.2703 Y = 0.0531+k.2703x ? =  1  0  <  9  9  3  3.66k.  h.f. v  Empirical Probit  82 92 79 80 80 76 76 88 88 85 8k 8k 82 82 68 68 72 75 75 72 72 76 76 59 39 39 63 63 36 36 26 19 19 22 22 0 0 0 0  x = 1.2k07  xx  L.,(£o*>  T o x i c i t y o f h e p t a c h l o r to Musca domestica (SES c u l t u r e )  ;  °-  l«l58k;  =  m  0 0  °6975;  g = 0.297;  =  S.E.  s  * i 0.7321;  ( b )  ,\ b = k.2703 - 0.7321  A n t i l o g m = 0.01k mgm.  - - ( ) = " 0.026k; .*. m = l . l 5 8 k - 0.26k E  m  P.L. = 1.1236 - 0.1070 = 1.2306 and 1.0166 Antilogs  = 0.017 mgm. and 0.010 mgm.  113 Table 28.  A  x  Toxicity of dieldrin (SES c u l t u r e ) n  0.038 2.58 50 50 50 0.034 2.53 50 50 50 0.030 2.48 50 50 49  r  p" p' p  kO 42 39 40  80 84 78 80 80 80 72 80 67  4o  40 36 40 33  80 81 84 78 80 80 80 80 72 73 80 67  0.026 2.42.49 40 82 82 61  50 26 5o 24 0.023 2.36 50 27 50 30 5o 25 0.019 2.28 49 21 50 32 50 24 0.000 99 0 98 0 .100 0  52 52 48 48 54 54 55 60 60 50 50 43 43 50 64 6k 48 48 0 0 0 0  to Musca domestica L.,c£f,  Empirical Probit  Y  nw  y  Snwx  5.9  74.5 5.88 117.710  438.060  5.84  5.8  76.9 5.84 117.657  449.096  5.61  5.6  82.6 5.61 122.248  463.386  5-28  5-4  85.3 5.23 121.126  446.119  5.13  5.2  85.3 5.13 116.008  437-589  5-05  5-0  81.5 5-05 104.320  411-575  -  -  -  S xx V^  b )  y =5-4430 Snwxy  2  1010.224 1005.343 = 4.631  = 0.2049}  b  Log L D ^ = m = 1.2985; 0  v  ^  = 0.0006019;  g =0.0782.  Snwy  - - ( ) * •* 0.4526} E  699.069 2645-825  l/Snw = 0.0020572 2  3820.489 14454-597 3805.007 14401.131 S = 15-482 S = 53-466 xy yy s  nwy  5.88  486.1 x = 1.4382  nwx  b = 3-1719  Y = 0.8812+3-1719x x  = k.359 (4) +  2  .". b = 3-1719 - 0.4526  A n t i l o g m = 0.020 mgm.  S.E., . = 0.0245;  .". m = 1.2985 - 0.0245  Since g i s small, F.L. = (1.96)(.0245)= -0.048  on e i t h e r side o f m = 1.34&5 and 1.2505 A n t i l o g s = 0.023 mgm. and 0.018 mgm.  Ilk T o x i c i t y o f p.p.-DDT t o Musca domestica L., 9 9 , (SES c u l t u r e ) . R e s i d u a l c o n t a c t a p p l i c a t i o n  Table 29.  n 200 160 120 97 80 73 65 57 44 35 20 15 13 9 7 0  r  p» p  Empirical Y Probit  2.30 l£u 65 43 43 4.82 2.20 150 64 43 43 4.82 2.08 150 60 40 40 4,75 1.99 150 59 39 39 .4.72 1.90 150 69 46 46 1.86 150 69 46 46 4.90 1.81 150 54 36 36 4.90 1.76 100 34 34 34 4.64 1.64 i 5 o 5 i 34 34 4,59 1.54 i 5 o 47 31 31 4.59 1.30 150 65 43 43 4.50 1.18 150 69 46 46 4.82 1.11 150 39 26 26 4.90 0.95 150 32 21 21 4.36 0.85 150 30 20 20 4.19 4.16 i5o 0 0 -  nw  nwx  nwy  4 . 9 95.2 4 . 8 2 218.960 4 . 8 9 4 . 1 4 . 8 2 207.020 4 . 8 94.1 4 . 7 5 195.728 4-8 9 4 . 1 4-72 187.259 175.560 4 . 7 9 2 . 4 4.90 171.864 4 . 7 92.4 4.90 I67.244 4.7 92.4 4 . 6 4 108.416 4 . 7 61.6 4.59 147.764 4 . 6 90.1 4.59 4 . 6 9 0 . 1 4.51 138.754 113.360 4 . 5 8 7 . 2 4.85 102.896 4 - 5 87.3 4-93 96.792 4 . 5 8 7 . 2 4.36 79.515 4-4 83.7 4.21 4 4 83.7 4.18 71.145  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  S  y = 4-6592  2 Snwx 3853.816 3592.858 = 260.958 .  Snwxy  10258.061 s  xy  1/Snw = 0.007540 Snwy  2  28846.612  10167.573 28773.627 = 90 .485 S = 72.985  b = 0.3468 Y = 4.0882+0.3468x x^  l 3 )  = 41.608  h . f . = 3.201 V  (b)  Log  =  0-  LTJ^Q  0 1  23;  s  = m =  E  b  2.6292;  V, » = 1.7747; vm; g = 0.1108  « '( )  = " 0.111;  .\ b = 0.3468 - 0.1110  A n t i l o g m = k26 mgm.  S.E., . = - 1.3510; (m;  .\ m = 2.6292 - 1.3510  +  P.L. = 2.6169 - 0.6798 = 3.2967 and 1.9371 A n t i l o g s = 1980 mgm and 87 mgm.  115 Table 30.  T o x i c i t y of p.p.-DDT t o Musca domestica L., (SES c u l t u r e ) . R e s i d u a l contact a p p l i c a t i o n  A  x  n  200 160 120 97 73 65 57 27 15 0  2.30 2.20 2.08 1.99 1.86 1.81 1.76 l.k3 1.18 -  150 150 150 100 150 150 150 150 150 150  r  p» p  95 63 60 kO kO kO 37 37 69 k6 81 5k 65 43 56 37 51 34 0 0  Empirical Y Probit  63 kO kO 37 k6 5k 43 37 34 -  5.33 k.75 k.75 k.67 4.90 5.10 k.82 4.67 4.59  nw  5.0 5.0 k.9 k.9 k.9 4.8 k.8 4-7 4.6  ,  2  nwy  219.650 210.100 198.016 189.448 177.072 170.321 165.616 132.132 106.318 -  409.015 453.625 452.200 444-584  k66.k80 480.851 k53.562 431.508 413.559 -  1568.673 4105.384 l/Snw = 0.0011802  , y = 4-8453 Snwxy 7635.326 7600.619 Sxy = ^ 34.707  nwx  95.5 5 - 3 3 95.5 k.75 95.2 k . ? 5 95.2 k.67 95.2 k.90 9k.1 5 . 1 1 9k.1 k.82 92.4 4 . 6 7 90.1 4 . 5 9 847.3  x = 1.8514 Snwx 2999.813 2904.207 Sxx ^ = 95.606  y  Snwy b = O.363O 19934.374 19891.620 Y = 4.1732+0.3630x Syy= 42.754 p ^ 30^55 2  x  =  h . f . = 4.308 v  (b>  =  0 . 0 4 5 1 ;  Log LD^ V( ) = m  0  S.E.(  b )  = m = 2.2777;  0 . 1 0 0 5 5 ;  s.E.  =  -  2 1 2 2 ;  .'. b = O.363O -  0 . 2 1 2 2  A n t l l o g m = 190 mgm. ( m )  = i 0.3170;  .'. m = 2.2777 - 0.3170  g = 1.9010 Since g i s so l a r g e , i t i s impossible t o c a l c u l a t e f i d u c i a l limits.  116 T a b l e 31. T o x i c i t y o f p.p.-DDT t o Musca domestica L., o p , (SES c u l t u r e ) . T o p i c a l a p p l i c a t i o n  A  n  r  P  P  30 30 20 30, 30' 20 30 30 30 30 30 30 30 25 30 30 30 30 29  15 21 12 12 17 11 18 15 12 14 18 17 19 9 18 11 14 9 12 2  50 70 60 40 57 55 60 5o 40 47 60 57 63 30 60 55 47 30 40 7  k6 68 57 36  X  5o.o 40.0 30.0 20.0 i5.o 10.0 7.0 6.5 6.0 5.5 5.0 4-5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.0  1.70 1.60 1.48 1.30 1.18 1.00 0.85 0.81 0.78 0.74 0.70 0.65 0.60 o.54 0.48 o.4o 0.30 0.18 0.00 on*  §  Empirical Y Probit  4.90 5.47 5.18 '4.64 5.io 5.05 5.18 4-90 4-64 4.85 5.18 5.io 5.25 4-33 5.18 5.05 4-85 4-33 4.64  k  52 57 46 36 44 57 54 60 25 57 52 44 25 36  -  MB  5.2 5.2 5.1 5.1 5.1 5.0 5.0 5.0 5.0 4-9 4.9 4-9 4.9 4-9 4-9 4.8 4.8 4.8 4.7  -  nw  J  nwx  16. 7 16. 7 11. 1 16. 7 16. 7 11. 1 16. 6 16. 6 16. 6 16. 4 16 •4 16. >4 16. 13 6 16. 4 16 .0 16. .0 16 .0 14 .9  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  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  a  Snwx  535.270 1469.820  V  2661.989 2646.319 = 15.670 xy  x x ^ f e  (b)  =  °-  0 1  59;  s  « .(b) E  =  Log L D ^ Q = m = 2.0244* V  (m)  =  °-°6633j  Snwy  Snwxy  1026.285 963.720 S  l/Snw = 0.0033636  y = 4.9439  2  s  2  7291.643 7266.635 s  " °«i264;  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  m  297.3 x = 1.8004  nwy  b = 0.2505 Y = 4.4929+0.2505*  yy  = 21.083 (17) .'.13 = 0.2505 ± 0 . 1 2 6 4  A n t i l o g m = 10.57 If  - - ( ) = * 0.2575} /. m = 2.0244 - 0.2575 E  m  g =0.9729 Since g i s so l a r g e , P.L. cannot be c a l c u l a t e d .  117 Table 32. T o x i c i t y o f p.p.-DDT t o Musca domestica L.,<3&", (SES c u l t u r e ) . T o p i c a l a p p l i c a t i o n  X  X  5o.o  n  1.70 1.60 l.k8 1.30 1.00 0.88 0.70 O.I4.O 0.30 0.18 0.00  40.0 30.0 20.0 10.0  7.5 .5.0 2.5 2.0  1.5  1.0 0.0  -  30 30 30 30 30 30 30 30 20 30 30 30  p' p 22 2ii 2,5 24 21 24 12 15 11 17 14 2  73 70 80 78 83 82 80 78 70 67 80 78 40 36 50 46 55 52 57 54 47 43 7 -  Empirlcal Y Probit 5.8 5.7 5.7 5.6 5.4 5.3 5.2 5.0 5.0 4.9 4.8  5.52 5.77 5.92 5.77 5.44 5.77 4-64 4.90 5.05 5.10 4.82  nw 13.8 14.5 14.5 15.2 16.2 16.5 16.7 16.6 11.1 16.4 16.0  nwx  x = 1.8550  S  Snwxy 1684.287 1652.771 = 31.516  S  310.712 890.983  Snwy^ 4769.871 4739.407 S =30.1*64 77  (b)  =  °' ^' 0  S  - *(b) E  =  Log L D ^ = m = 1.3145; 0  V  (m)  =  °'°894;  s  « .( ) E  m  - °-  2 2 6  b = O.5907 Y = 4.2234+0.5907x  w  h . f . = 2.726 V  S;  75.900 83.665 85.550 87.552 88.128 94.545 77.488 81.340 56.055 93.640 77.120  1/Snw = 0.0059701  y = 5.3193  Snwx 629.722 576.370 = 53-352  37.260 37.700 34-960 34-960 32.500 31.020 28.390 23.240 14.430 19.352 16.000  5.50 5.77 5.90 5.76 5.44 5.73 4.64 4.90 5.05 5.10 4.82  167.5  nxy  X  2  (9)  = 24.534  .*. b = 0.5907 - 0.2265  A n t l l o g m = 2.063 = * 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 t o work out due t o l a r g e between m and x and s i z e o f g.  difference  118 Table 33.  A  T o x i c i t y o f p.p.-DDT to Musca domestica L.,QQ, (Ottawa c u l t u r e ) . T o p i c a l a p p l i c a t i o n  x  n  0.138 1.1k 10 10 10 0.125 1.10 10 10 10 0.113 1.05 10 10 10 0.100 1.00 10 10 10 0.088 2.95 10 10 10 0.075 2.88 10 10 10 0.050 2.71 10 10 10 10 0.000 10 10  r p" P 9 9 9 9 8 7 9 9 8 8 6 8 6 4 7 5 2 6 2 4 4 1 0 0  90 90 90 90 80 70 90 90 80 80 60 80 60 kO 70 50 20 60 20 kO I4.O 10 0 0  p  !  90 90 90 90 79 69 90 90 79 79 59 79 59 38 69 48 18 59 18 38 38 3  Empirical Probit  Y  nw  6.28  6.2  11.1 6.28  23.754  69.708  79  5.81  6.0  13.2 5.79 27.720  76.k28  86  6.08  5.8  15.1 6.05  30.955  91.355  72  5.58  5.6  16.7 5.58  33.400  93.186  5.3  18.5 5.12  36.075  94.720  55  5.13  42  4.80  5.0  19.1 4.80 35.908  91.680  31  4.50  4.3  16.0 7.52  27.360  72.320  -  -  -  -  y = 5.3728  2 Snwx 423.943 k22.05l S„_ = 1.892  Snwxy  1164.054  1156.078 = 7.975  " v  ^  -  215.172 589.397  ( b ) = 0.5285; s . E . ( ) = - 0.7270;  Log L D ^ = m = 1.8731; 0  1/Snw = 0.0091158  2 Snwy 3204.876 3166.717 S =35.159 7  b  nwy  90  109.7 x = 1.9615  nwx  y  b = 4«2l5l y =-2.8951+4.2l5lx P  x  7  2 5 )  = 4.544  .*. b = 4.2151 - 0.7270  A n t l l o g m = 0.075 X  (m) * °746; - « ( ) - " 0.0273 g = 0.1143; P.L. = 1.8617 - 0.0580 =1.9197 and 1.8037  V  =  0  0 0  S  E  m  A n t i l o g s = O.O83 *  and O.O64V  119 T a b l e 3k.  X  X  T o x i c i t y o f p.p.-DDT t o Musca domestica L., (Ottawa c u l t u r e ) . T o p i c a l a p p l i c a t i o n n  r p" p' P  0.063 2.79 10 10 10 0.050 2.70 10 10 10 0.038 2.58 10 10 10 0.025 2.k0 10 10 10 0.02k 2.38 10 10 10 0.021 2.32 10 10 10 0.020 2.30 10 10 10 0.000 10 10 10  8 8 7 8 9 8 8 5 7 7 3 7 6 3 4 1 0 3 3 1 1 1 0 0  -  80 80 70 80 90 80 80 50 70 70 30 70 60 30 ko 10 0 30 30 10 10 10 0 0  79 79 69 79 90 79 79 49 69 69 28 69 59 28 38 7 0 28 28 7 7 3  Empirical Probit  Y  nw  nwx  7  76  5.71  6.1  11.7 5.62  20.943  65.754  83  5.95  5.7  15.3 5.93  26.010  90.729  66  5.1+1  5.3  17.6 5 . k l  27.808  95.216  55  5.13  k-1  17.1 5.14  23.940  87.894  42  k.80  k.6  16.5 k . 8 l  22.770  79.365  12  3.83  k-k 15.0 3.9k  19.800  59.100  lk  3.92  4-3  lk.l  3.97  18.330  55.977  -  -  -  -  -  -  S  x  x  Y = 4-9770  2 Snwx 240.546 237.395 = 3.151  -  159.601 534.035  107.3 x = 1.4874  nwy  l/Snw = 0.0093197  2 Snwxy Snwy 805.738 2711.283 794.338 2657.907 -11.400 Syy = 53.376  b = 3.6179 Y = -0.4043+3.6l79x 2  h . f . = 2.4264; V  (b)  Log v  ( m )  = °.7700;  s.E.  ( b )  L D ^ ss m = 1.4938; 0  = 0.001730;  g = 1.601;  s.E.  * t 0.8774;  b = 3.6179 - 0.5774  A n t i l o g m = 0.031 * ( m )  = ± 0.0415; /. m = 1.4938  *  o.o4i5;  F.L. = 1.4950 i 0.0749 = 1.5699 and 1.4201; Antilogs  = 0.037 ^ and 0.026 If  120 Table 35.  A  T o x i c i t y o f p.p.-DDT t o Musca domestica L. QQ (Ottawa c u l t u r e ) . T o p i c a l a p p l i c a t i o n n  X  0.15I 1.20 10 10 10 0.150 1.18 10 10 10 0.125 1.09 5 10 10 0.113 1.05 10 10 10 0.100 1.00 10 10 10 0.094 2.97 10 10 10 10 0.000 10 10  r  P  9 9 9 9 9 8 3 6 9 5 8 7 5 5 9 5 2 7 0 0 0  90 90 90 90 90 80 30 60 90 50 80 70 5o 5o 90 5o 20 70 0 0 0  R T  P  Empirical Probit  P  1  90 90 90 90 90 80 30 60 90 50 80 70 50 50 90 50 20 70 0  Y  nw  y  90  6.28  6.2  11.1  6.28  2k.k20  87  6.13  6.1  12.1  6.13  26.378  70  5.52  5.6  ll+.O  5.52  29.260  67  5.1+k  5.1+  18.0  5.1+1+  36.900  63  5.33  5.2  18.8  5.33  37.600  1+7  k.92  5.0  19.1  1+.93  37.627  -  -  -  -  -  Snwx 397.351 396.725 = 0.626  Snwxy 1063.215 1059.903 S__ = 3.312  2  S  y = 5.5l50  l/Snw = 0.01071+11 b = 5.2907  Snwy 2850.029 2831.67k S — = 18.355 7  Y = ? x  7  2  k )  ¥  (b)  1  =  *S97k; S . E . j --t ( b  Log LD = m = 50 V  (ra)  G =  =  1.9670;  0.000923i+; S . E .  0.2192;  1.2630;  • -  192.185 513.,'1+1+8  93.1 x = 2.06k3  nwx  .'. b = 5.2907  1+066+5.2907x = 0.833 1.2630  A n t l l o g m = 0,099 * ( m )  = t 0.0303;  P.L. = 1.9397 - 0.0727  m =  1.9670 - 0.303  = 2.012k and I.867O  A n t i l o g s = 0.103 i  and 0.071+ H  121  Table 36.  X  I>.,d(f,  T o x i c i t y o f p.p«-DDT to Musca domestica (Ottawa c u l t u r e ) . T o p i c a l a p p l i c a t i o n n  X  r p" p» P  Empirical Probit  0.138 T.Ik 10 9 90 90 97 10 10 100 100 10 10 100 100 0.125 1.10 10 9 90 9093 10 10 100 100 10 9 90 90 0.113 1.05 10 8 80 80 80 10 8 80 80 10 8 80 80 0.100 1.00 10 6 60 60 73 10 9 90 90 10 7 70 70 0.088 2.95 10 6 60 60 67 10 8 80 80 10 6 60 60 0.063 2.88 10 3 30 30 27 10 k kO kO 10 1 10 10 0.000 mm 10 0 0 0 10 0 0 10 0 0  Y  nw  y  nwx  •nwy  6.88  6.8  5-4 6.88  ii.556  37.152  6.k8  6.5  8.1 6.k8  17.010  52.ij.88  5.8k  6.0  13.2 5.83  27.060  76.956  5.61  5.6  16.7  5.61  33-400  93.687  5-44  5.2  18.8 5-43  36.660 102.08k  k.39  4-5  17.k k.39  32.712  79.6 x = 1.9899  y = 5.5120  Snwx 315.710 315.200 ^xx = 0.510  Snwxy 877.534 87,3.085 = 4.449  2  158.398 I138.753  l/Snw = 0.0125628  Snwy 2459.616 2418.394 Syy =kl.222 2  b = 8.7235 Y = -11.8469+8.7235x g  (4) .*. b = 8.7235 x  V  = 1.9608;  ( b )  S.E. ) = t i.k020; (fc  Log LD^ = m = 1.9312; V  =  °-  0 0 0 2  g = 0.0963;  53;  S  =  1  2  '  k  1  1  1-4020  A n t i l o g m = 0.085 V  0  • (m)  76.386  - ' „ ( ) = ~ 0.0159; E  m  .". m = 1.9312 -  0.0151;  F.L. = 1.9249 - 0.0334 = 1-9583 and 1.8915; Antilogs  = 0.091 V  and 0.078V  122 Table 37.  A  T o x i c i t y o f p.p'-DDT t o Musca domestica L . , Q Q , (Ottawa c u l t u r e ) . R e s i d u a l contact a p p l i c a t i o n  x  n  r p" p»P  Empirical Probit  5.6k 2.06 0.31k 50 33 66 65 7k 50 Ij.6 92 92 50 33 66 65 5.52 0.57 0.272 k7 35 75 75 70 50 36 72 71 50 32 6k 63 1.72 0.236 50 17 31+ 33 kO . k.75 50 20 kO 39 50 2k k8 k? k.62 1.50 0.176 50 23 k6 5.535 50 21 i[2 i j l 50 10 20 18 1.13 0.053 50 5 10 8 13 3.87 50 10 20 18 50 7 l k 12 0.00 ij.9 0 0 2 50 3 6 50 0 0  -  Y  nw  y  Snwx  75.k 5.63  99.076  k2k.502  5.k  88.3 5.52 112.318  k87.kl6  5.1  95.2 k.75 117.667  452.200  4.5  87.2 k.62 102.547  k02.86k  3.3  31.2 k.21 32.85k  131.352  -  -  y = 5.031k Snwxy  2  572.kk5 571.760 S_„ =0.685" xx ^  23kk.0k3 2336.878 Sxy = 7 . 1 6 5  nwy  5.8  -  377.3  x = 1.2310  nwx  -  k6k.k62 1898.33k  l/Snw = 0.002650k Snwy  2  96k2.656 9551.211 S77 =91.kk5  b = 10.4598  Y = -7.8kk6+10.k598x p x^ = 16.500 y  3 )  h . f . = 5.500 V  ( b )  = 8.0292;  s  « . E  (  b  )  Log LD^ = m = 1.2280; 0  V(m) = 0.00013k0; g = 0.9907;  - - 2.8335;  .'. b = 10.J+598 - 2.8335  A n t l l o g m = 0.169 mgm.  S . E . ^ = - 0.0115;  m = 1.2280 - 0.0115  P. L. = 0.908k * .k706 = 1.3790 and 0.k378 Antilogs  = 2.393 mgm. and 0.27k mgm.  123 Table 38.  A  T o x i c i t y o f l i n d a n e to Musca domestica L.,QQ, (Ottawa c u l t u r e ) . R e s i d u a l contact a p p l i c a t i o n  x  n  0.053 2.34 50 49 _ 50 0.045 2.28 50 50 50 O.038 2.24 50 50 _ 50 0.030 2.20 50 50 50 0.000 50 50 50  r p" p* p 2k k8 k8 43 87 87 39 78 78 33 66 66 31 62 62 4o 80 80 32 64 64 15 30 30 30 60 60 9 18 18 16-32 32 30 60 60 0 0 0 0 0 0 0  Empirical Probit  Y  71  5-55  5.6  83.1 5-55 111.354  461.205  69  5-50  5.2  94-1 5-48 120.448  515.668  5l  5.02  5-0  95.5 5.03 118.420  480.365  37  4.69  4.7  92.4 4.67 110.880  431.508  -  -  -  -  . x = 1.2629  S  V  2 Snwx 583.284 582.347 xx= °'  ( b )  9 3 7  = 1.0672;  S  7  S.E.  ( b )  Log L D ^ = ra = 1.2365; 0  ^m; = 0.0000808; g = 0.0952;  S  y  y  nwx  nwy  -  365.1  y = 5.1732 Snwxy 2391.533 2385.386 x y = 6.1k  nw  461.102 1888.746  l/Snw = 0.0027389  y  2 Sxwy 9816.927 9770.916 =46.011  = ± 1.0337;  b = 6.5603 Y = -3XLl8+6.5603x A  = 5.68k (2) .". b = 6.5603 - 1.0337  A n t i l o g m = 0.017 mgm.  S.E., . = - 0.0089; (m)  m = 1.2365 - 0.0089  Since g i s s m a l l , P.L. = (.0089)(1.96) on  ei t h e r s i d e o f m = 1.2365 - 0.0174 = 1.2539 and 1.2191 Antilogs  = 0.018 mgm. and 0.016 mgm.  12k Table 39.  T o x i c i t y o f h e p t a c h l o r t o Musca domestica L.,0^> (Ottawa c u l t u r e ) . R e s i d u a l c o n t a c t a p p l i c a t i o n n  r P 30 29 40 25 25 15 22 18 8 16 26 4 0 0 0  5o 46 5o 5o 5o 5o 5o 5o 5o 5o 5o 5o 5o 50 5o  P* P  M  60 63 80 50 5o 30 44 3? 16 32 52 8 0 0 0  60 63 80 50 5o 30 44 36 16 32 52 8 0  anplrical Probit  x  5.47  5.3  90.1 5.46 133.348  491.946  4.53  4.7  92.4 4 . 5 4 125.664  419.496  68  nw  y  S xx  2 Snwx 691.405 689.542 =1.863  32 31  y = 4.8447  2414.703  2406.065 = 8.636  S y  496.638 1732.957  l/Snw = 0.0027956  p Snwy*8446.750 8395.636 =51.114  Snwxy  S  b = 4«°366 Y = -1.5928 + 7.6366X 2  y X  f  h . f . 5.532 V  ( b )  = 2.9708;  Log LD^  0  S.E.  ( b )  = m = 1.4219;  V. , = 0.0008733; g = 2.5535;  nwy  43  357.7 x = 1.3884  nwx  =  (2)  = 11.063  t 1.7236;.". b = 4.6366 - 1.7236  A n t i l o g m = 0.026  mgm.  S.E., , = t 0.0295  F.L. cannot be c a l c u l a t e d due to l a r g e g v a l u e .  

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