__k___.§- .4. .‘é:_._.'_..a.._:. .8. This is to certify that the thesis entitled TIE EA’l'flthl'JP-l AND ILA" RALEITT OF A LABCPATOIK TECIE'IQTE FOR EVALUATING CCNTACT INSECTICIDES presented by William Erle Collins has been accepted towards fulfillment of the requirements for Masters degree “I'M ajor professor Date May 27, 1952 i ; i l I % I 4-. .V-O-‘n-u-ifl 1.- f I ‘I l . ‘.: I l i s THE EXTENSION AND REFINEMENT OF A LABORATORY TECHNIQUE FOR EVALUATING CONTACT INSECTICIDES By William Erle Collins AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1952 @ng—M / ABSTRACT The purpose of this work was to evaluate the use of filter papers as a test surface for contact killing with insecticides and to measure the effect of varying environmental conditions and experimental techniques. The problem required the~production of quantities of the confused flour beetle, Tribolium confusum, Duval, which was the test insect. These beetles were raised in whole wheat flour under relatively constant temper- ature and relative humidity. The amount of the insecticide on the filter papers was calculated on the basis of milligrams per square foot. The insecticide used was DDT, and the deposit was Obtained by pouring known amounts of a DDT suspension onto filter papers in a Buchner funnel. After the papers had dried, they were put in vials. Ten beetles were put in each test vial, and the vials were put in a rack which was at an angle that kept the beetles in constant contact with the test surface. The tests were conducted under varying conditions to determine the effect of concentration, the length of exposure time, the temperature, the age of the beetles, and the residual prOperty of the papers. The tests indicated that the length of exposure time, the deposit of the insecticide and the temperature had a decided effect on the results. It was clearly evident that there was greater mortality at the lower temper- ature of 59° Fahrenheit than at the higher temperature of 80° Fahrenheit. There was also evidence from the six-hour exposure with the adult beetles of different ages that the age of the beetle had a significant effect on the results.‘ It was found that there was a higher percent,mortality with beetles fifty-four to fifty-eight days old than with beetles seven days old. The age of the deposit of DDT on the filter papers did not effect the results. Storage conditions for the filter papers was at room temper- ature and relative humidity, and an air current was maintained to prevent the accumulation of dust on the papers. THE EXTENSION AND REFINEMENT OF A LABORATORY TECHNIQUE FOR EVALUATING CONTACT INSECTICIDES BY William Erle Collins A THESIS submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of .MASTER OF SCIENCE Department of Entomology 1952 \. \- ACKNOWLEDGMENTS Particular acknowledgment is made to Dr. Herman King of Michigan State College under whose guidance and inspiration this work has carried out. Appreciation is expressed to Professor Ray Hutson of Michigan State College for his help in selecting a thesis topic. Grateful acknowledgment is made to Gordon GUyer for his help in taking the pictures. The author is also grateful to other members of the Department of Entomology of Michigan State College for their helpful suggestions and assistance. ll. ll ll ll TABLE OF CONTENTS Page I INTRODUCTION---- ........ ----- ..... ------ .................... 1 Review of Literature------------------ ...... ------------2 II APPARATUS AND PROCEDURES--- ................................. h Rearing and Handling the Test Insects---------- --------- h Preparing the Filter Papers-----------------------------6 Preparation of the Test Vials----------—----------------7 Putting Beetles in Test Vials---------------------------8 The TeStS‘ ------C-C-------------------------------------8 Mortality counts--CD------------ o -nnon--------------- u-lz Phtes-IIOOn--------cum-unuocucnuo-u--------n---------n-13 III MENTATION OF DATA'----------"---------------------------16 Table8--------u-¢---------nn-----------------;--—----—-17 Gmph8------—--------- can---n--------------¢----u-coco-27 Iv DISCUSSION or RESULTS--------------------------------------3h MOrtality Readings-------------------------------------3h Results of Test Number One-----------------------------3h Results of Test Number Two-----------------------------36 Results of Test Number Three---------------------------37 Results of Test Number Fear----------------------------37 Results of Test Number Five------------------- ......... 37 V conchIONS-------------------- ------- ---------------------39 3LITERATURE CITED------------------------- ---------- - ....... hi I INTROHJCTION The testing of insecticides in the laboratory is a necessary pre- liminary to the more extensive experimentation in the field. Deny tests of contact insecticides involve the direct application of the insecticide to the insect. Because of the advent of many contact insecticides with long residual action, tests have been designed which involve the use of insecticide films on various surfaces. The use of filter papers as a surface upon which a film of insecticide is placed is a relatively new practice. The purpose of this experiment was to evaluate the use of filter papers as a test surface for contact killing and to measure the effect of varying environmental conditions and experimental techniques. The insect used in these tests was the confused flour beetle, Tribolium confusun, mm. The effects of the concentration of the insecticide, the length of exposure time, the temperature, the relative humidity, the age of the beetles, and the residual property of the filter papers were determined to some extent in order to ascertain which conditions should be con- trolled to reduce experimental error. The first test was to determine the effect of concentration and time on the mortality of the beetles, although all of the tests included these factors to some extent. The second test We designed to determine the effect of temperature. Test mmber three was to determine the effect of relative humidity. The effect of age of the beetles on the results was determined in test number four. The fifth test was designed to determine the residual effect of the filter papers treated with DDT. The confused flour beetle has long been recognized as a good labo- ratory test animal. It is both easy to raise and easy to handle. Early insecticidal tests involving this insect were primarily with fumigants since this insect is a serious pest in flour mills where this method of control is used. Review of Literature Tattersfield and Potter (1918) used films of insecticides on filter paper in contact tests against the confused flour beetle. Their pro- cedure called for the use of a Potter tower (Potter, 191d) to spray the insecticidal solution onto the filter papers. The beetles were then en- closed on the papers with inverted filter funnels. Since these beetles do not readily climb glass, they were kept in constant contact with the film on the filter paper. This method of applying the material to the filter papers was also used by Pradhan (19195.). The procedure was sanewhat simplified by Stringer (1911»9) who used a pipette to drop a known amount of an acetone Solution onto the papers. This gave a known amount of deposit on the filter papers. A much simpler though less accurate method was used by Morrison (1916) who merely dipped the papers into solutions of known concentration. The exact amount of deposit on the filter papers was not known, but the results from papers dipped in different solutions were com— Pared. Langford, Squires and Dozier (1951) tested insecticides against the Japanese beetle by using blotters which were dipped in the insecti- cide and allowed to dry thoroughly. Bartlett (1951) impregnated filter Papers with l milliliter of a standard acetone solution of the insecti- cide and allowed the papers to dry. Cotton, Frankenfeld and Dennis (19h8) used Tribolium confusum in tests with contact insecticides, but they used glass and.metal plates as test surfaces and enclosed the beetles on the film by the use of glass rings and inverted glass vials. Pradhan (l9h9a) in his tests used the insect Tribolium castaneum, Hbst., which is very similar to Tribolium confusum. He used filter papers and bolting cloth as the surfaces. The beetles were kept on the papers by the use of filter funnels, truncated wglass cones, glass rings, filter paper cones with both papers treated, and cones 5f perforated papers to allow for ventilation. Stringer (l9h9) 'used grain weevils, Calendra granaria L., as test insects. He used glass rings to enclose the beetles on the filter papers. Bartlett (1951) used adult Drosgphila on filter papers and made counts at various times to determine the median lethal concentration. Morrison (1916) lined the inside of a vial with the treated filter paper. The then enclosed a number of adult Drosophila in the vials for a twenty- 1iour hour period. II. APPARATUS AND PROCEDURES Rearing and Handling the Test Insects The establishment and maintenance of a large colony of the adult confused flour beetles to be used in the tests was the first undertaking. A large cabinet in which a relatively constant temperature could be main- tained, was available. Since numerous investigations by other workers had revealed that a higher temperature shortened the length of time for a life cycle to be completed, the temperature in the cabinet was maintained at its highest level, which fluctuated between 78° and 83° Fahrenheit. Temperature records were compiled for a period of fifteen weeks beginning with October 22, 1951. It was noted that Brindley (1930) maintained his colony of Tribolium confusum at a relative humidity of between sixty and seventy percent by the use of a saturated sodium chloride solution. Diamond Crystal Salt cunposed of 99.5 percent sodium chloride and 0.5 percent tri-calcium Phosphate was readily available and was used to make up the salt solutions . The solutions were mixed in two large enameled ware pans, and these were Placed at the bottom of the cabinet. A fan in the cabinet circulated the air and apparently kept the relative humidity constant throughout. Numer- ous readings of the relative humidity were made by the use of a Lloyd's Hygrodeik which was kept in the cabinet. These readings indicated that the relative humidity was around 69 percent. The solutions in the pans lost water quite readily and every two or three days, water had to be added to each pen. One hundred adult confused flour beetles were obtained as a breeding colony. Whole wheat flour of medium grind was obtained from a local mill as food for the beetles. The flour was sifted twice through a twenty-mesh screen before being used, in order to remove all of the larger particles. The flour was put in pint Jars to a depth of between one and one-half and . two and one-half inches. The one hundred adults were placed on the top of the flour of the first Jar on October 22, 1951, and the Jar was numbered and also marked with the date. According to Brindley (1930), the incubation period of the eggs was approximately five and one-half days. The adults were sifted fran the flour four days after they were put in the Jar. A twenty-mesh sieve was used to separate the beetles from the flour, and a rubber bulb aspirator was used to pick up the adults frm the sieve. Often, however, the beetles could be Jarred directly from the sieve into the next pint Jar. The flour containing the eggs was put back in the cabinet along with the next Jar which contained the adult beetles. On December 5, 1951, 363 adults were removed fran Jar number one and were used as more breeding stock. Jar number two yielded 1,177 adults and Jar number three yielded 995 adults. By using the adults from the first Bevan Jars as breeding stock, a large colony was soon built up. The cabinet which was used for rearing can be seen in Plate 1. The hagrodeik and temperature recorder are shown on the top shelf. Jars con- taining beetles can be seen on the top, second and third shelves. The pens with the salt solutions are at the bottmn of the cabinet. The one pan on the shelf was moved up in order to get a better picture of it. The thermo- stat for controlling the temperature is visible Just above the temperature recorder . Preparing the Filter Papers The amount of the insecticidal material on the filter papers was calculated on the basis of milligrams per square foot of surface. Deposits of 11!», 33, 61, 122 and 229 milligrams per square foot were used in the first test. Only two concentrations were used in tests two and three. In tests four and five, three concentrations were used. Checks of distilled water were prepared for all the tests. Fifty percent DDl‘ wettable powder* was used for all of the tests. The powder was weighed and then mixed in beakers with #00 milliliters of dis- ' tilled water. The powder was kept in suspension by the use of an electric mixer. When the suspension was thoroughly mixed, it was poured into a vial of known volume until the vial was overflowing. The suspension was then poured onto a number two Whatman filter paper in a Buchner funnel. A vacuum forced the liquid quickly through the paper, leaving the deposit of DDT. The vacuum was then turned off and a break made in the connection so that the paper could be lifted from the funnel. A dissecting needle was Placed under the edge of the filter paper, and the paper was then lifted until the fingers could be put under the paper. The filter paper was lifted out of the funnel and put on muslin to dry. For the control papers, a. vial filled with distilled water was poured over the papers. One vial Was used for each concentration of the material. Each vial was used for preparing ten papers and then was thoroughly washed before being used again. the muslin was used only once and then it was washed thoroughly. The equipent used to prepare the filter papers is shown in Plate II. The ¥ * Dow Chemical Cmpany mixer was kept high on the stand so that the beaker could be held in place and removed quickly. The suspension was poured into the vial before the particles had time to settle. In test number five the papers were stored for a period of thirty days before being used. After the papers had been dried on the muslin, they were transferred to wooden trays and put in a large wooden cabinet. None of the papers was touching any other paper on the tray. The cabinet was at roan temperature and relative humidity. A tube was attached to the cabinet and air was constantly blown through it. This kept the air circu- lating and helped to prevent dust from settling on the papers. Preparation of the Test'Vials The filter papers were put in 25 X 95 millimeter vials. Preliminary observations shorted that the adults would not crawl over the glass surface if the angle of the glass was steep and the glass was clean. This indi- cated that there was no need for a cover of any sort over the vial mouth. 'Jhe lack of a cover also aided in ventilation. The beetles, however, would crawl between the filter paper and the glass wall and escape exposure. has outer edge of the filter papers was coated with vaseline which held the Papers close to the glass and confined the beetles to the treated surface. The paper was picked up with a pair of tweezers and the vaseline was ap- Plied with a cotton swab. Another pair of tweezers was then used with the first pair to roll the filter paper and slide it into the vial. A soft cloth was used to wipe off the excess vaseline on the vial mouth, and a Swab was used to remove the excess vaseline around the edge of the filter Paper. The equipment used to prepare the test vials is shown in Plate III. The dissecting needle was used to adjust the position of the filter paper after it was in the vial. Putting Beetles in Test Vials The beetles were taken from the rearing cabinet and some flour was sifted from them so that only about one-quarter to one-half inch of flour was left with the beetles in the pint Jar. A small piece of cardboard was placed in the Jar, and the beetles readily climbed up on it. The beetles were then tapped slightly with a dissecting needle, and they dropped into the test vial. Ten adult beetles were put in each vial. The beetles were counted several times after they were in the test vials to prevent error, but several miscounts did occur. The time that the beetles were put in the vials was recorded to the half minute. The vials were then put in the rack which held them while the tests were being run. The equipment used to put the beetles in the vials is shown in Plate IV. The rack in which the vials were placed is shown in Plate V. The whole rack was adjustable so that the angle of the vials would be constant. The angle was set where the wall of the vials was too steep for the beetles to climb, and the batter: of the vial was also too steep. The Vials were turned so that the largest area of the filter paper was down. This kept the beetles in contact with the filter paper during the entire test. The Tests Test number one. The first test was conducted with beetles approxi- The papers were prepared with DDT deposits of 11+, t mately .four weeks old. 33, 61, 122 and 229 milligrams per square foot of surface. Check papers were prepared with distilled water. Each concentration was tested for time periods of one hour, three hours, six hours, and twenty- four hours. Each concentration was replicated five times in one day, and the whole test was repeated on three different days. This involved the testing of 3,600 beetles in 360 different vials. The vials, after the beetles had been put in them, were kept at room temperatures and humidities. After exposure, the papers were removed from the vials, and the beetles were tapped into recovery vials. The recovery vials were twenty-five millimeters in diameter with about three-quarters of an inch of whole wheat flour. The recovery vials were then put in the large cabi- net at a temperature of from 78° and 83° Fahrenheit.and a relative humidity of about 69 percent. The room temperature varied from 68° to 78° Fahren- heit, and the relative humidity varied.from.28 to 3h percent. The relative humidity in the room was read several times during one test. The recovery vials were placed in the cabinet one day before being used in order to bring their temperature constant with the chamber. Test number two. The second test was designed to determine the effect of temperature on the results. The papers were prepared with DDT deposits of 33 and Gl.milligrams per square foot. The check papers were prepared at the same time. The filter papers were used one day after the DDT deposit had been put on them. Two small constant temperature cabinets were used to test the effect of the two temperatures, 59° and 80° Fahrenheit, on the results. The relative humidity was held constant in the two cabinets with a saturated salt solution of Mg012.6320 which kept the relative humidity at approximately 33 percent for both temperatures (International Critical Tables). One of the small constant temperature cabinets is shown in Plate VI. These cabinets were also used for recovery purposes. The tests were replicated five times on two different days. This involved the use of 600 beetles in 60 vials. The beetles were approximately four weeks old. These beetles were put in the temperature cabinets one day before they were used. The exposure time was three hours and the rack was used to hold the vials in the constant temperature and humidity cabinets. After the three hours had elapsed, the vials were removed and the beetles transferred to recovery vials. After the transfer, the recovery vials were put back in the constant temperature cabinets in which the tests were run. The transfer of beetles was at room temperature for the 80° Fahrenheit test, and in a cold room.at 50° Fahrenheit for the lower temperature test. Eggt_gu!bgg three. This test was designed to determine the effect of the relative humidity upon the results. The papers were prepared with DDT deposits of 33 and 61 milligrams per square foot. Check vials were also prepared. The filter papers were used one day after the deposit had been placed on them. The temperature was held constant at 80° Fahrenheit. The relative humidities were maintained at approximately 33 percent and 69 per- cent. The 33 Percent relative humidity was maintained with a solution of magnesium chloride. The 69 percent relative humidity was maintained with a solution of Diamond Crystal Salt. The exposure time was three hours and the tests were replicated five times on two days. This involved the use of 600 beetles in 60 vials with beetles approximately four weeks old. The test vials containing the beetles were put in the cabinets for the 10 exposure period. The beetles were then changed to recovery vials and these put back into the test cabinets. The transfer to the recovery vials was at rocn temperature and humidity. Test M 2911;. This test was designed to detemine the effect of the age of the beetles on the results. The two ages of beetles used were seven days and fifty-four to fifty-eight days. The papers were pre- pared with DDT deposits of 33, 61, and 122 milligrams per square foot. Check papers were also prepared. The tests were run at a temperature of 80° Fahrenheit and a relative humidity of approximately 69 percent. The exposure times were three and six hours with five replicates on each of two days. This involved the use of 1,600 beetles in 160 vials. The vials containing the beetles were put in the cabinet for the ex- posure period. The beetles were transferred to recovery vials at roan temperature and humidity and the vials were put back into the test cabinet. The recovery vials as well as the beetles were put in the temperature cabi- net one day before being used. Test number five. This test was designed to determine the residual effect of the DDI' on the filter papers. The papers were prepared one day and thirty days before being used. The filter papers were prepared with 133'! deposits of 33, 61, and 122 milligrams per square foot. Check papers “are also prepared. The tests were run at a temperature of 80° Fahrenheit and a relative humidity of approximately 69 percent. The exposure times Vere three and six hours, with five replications on each of two days. This involved the use of 1,600 beetles in 160 vials. The vials containing the beetles were put in the test cabinet for the exposure periods. The beetles were transferred to the recovery vials at roan temperature and humidity. The recovery vials were then put back into the test cabinet to recover. The recovery cabinet is shown in Plate VI. Mortality Counts The beetles, after given time to recover, were removed fran the cabinets. The flour was then sifted fran the beetles with a twenty-mesh screen. The criterion for death was the inability of the beetle to walk on a flat surface. The very active beetles were picked up with a bulb aspirator directly from the sieve. The remaining beetles were put on a flat piece of paper and given some time to walk. A little practice showed that a beetle with a darker thorax was probably dead. The beetles were sometimes blown upon since, if they were alive, they would begin to walk quicker. After counting, the beetles were put back into the recovery vial and returned to the test cabinets. Two mortality counts were made for each vial. In the first test, the number of dead or moribund beetles in each vial was counted at the end of four days and eight days. In the other tests, the counts were made at the end of four days and seven days. Plate I - Chamber for rearing the test animals. Plate II - Apparatus for preparing the filter papers. 13 Plate III - Apparatus for putting filter papers in vials. 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