COMPARATIVE EFFECTIVENESS AGAINST THE GRANARY WEEVIL OF CONTACT, FUMIGATION AND REPELLENCY OF PYRENONE, PYRETHRINS, PIPERONYL BUTOXIDE, LINDANE AND DDT

DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of the Ohio State University

By YUNG CHANG CHAO, B.A., M.S.

The Ohio State University 1952

Approved bys CONTENTS Pago ACKNOWLEDGMENTS ...... Ill INTRODUCTION ...... 1 LITERATURE R E V I E W ...... 4 CULTURE METHODS ...... 11 EXPERIMENTATION ...... 15 Experimental Design ...... 15 Equipment ...... 16 Toxicants and. Adjuvants ...... 19 Formulations ...... 26 Application Technique ...... 31 Collecting D a t a ...... 40 RESULTS AND DISCUSSION ...... 44 STATISTIC ANALYSIS ...... 54 CONCLUSION ...... 57 SUMMARY ...... 59 ILLUSTRATIONS ...... 61 LIST OF ABBREBIAT IONS ...... 81 LITERATURE CITED ...... 82 AUTOBIOGRAPHY...... 86

I S00441 LIST OP TABLES Table Page 1 Formulation of Impregnated Dusts for 50-gram Stocks...... 29 2 Emulsion Concentrates for Experiments 4 and 5.. 31 3 Formulation of Emulsion Concentrate for the Study of Contact Toxicity...... 31 4 The Differences In Adult, Pupal and Larval Stages of Rice Weevil and Granary Weevil...... 32 5 Dosages of Active Ingredients in PPM of Wheat.. 35 6 Dosages of the Five Toxicants Applied to Wheat in PPM...... 37 7 Average Weight of Feces from Treated Wheat and Difference in Weight of Feces between Treated Wheat and Checks...... 47 8 Contact Effect of Toxicants Impregnated on Bags after 2§ Months...... 50 9 Comparative Effect of Lindane Impregnated Bags in Closed Jars and in Open Jars Showing by the Number of Adults Developed after 100 Weevils Being Exposed Six Days and Collected 50 Days after the Removal of 100 Beetles ...... 51 10 Showing the Results of Four-Week Fumigation by Lindane Impregnated on Bags against Immature Granary Weevils of 1-4 Weeks ...... 53

ii ACKNOWLEDGMENTS The author wishes to express his most sincere gratitude to Dr. Dwight M. DeLong under whose direction this work was

carried on. Dr. A. Peterson under whose direction this work was

inaugurated suggested the problem. Dr. Harold E. Gray, in charge of the Stored Product Insect Investigations, Division of Entomology, Department of Agriculture, Ottawa, Canada, suggested the study of pyrenone. Dr. W. E. Dove, Director of Entomology Division of Re­

search and Development Laboratory, U.S. Industrial Chemicals,

Inc., furnished a great deal of information on the use of pyrenone. Dr. Samuel S. Ristich offered much valuable information on the formulation of insecticides and helped in taking the photographs.

Dr. D. P. Miller kindly approved all the orders for equipment and materials employed In this work. Dr. Prank W. Pisk, in charge of the Matheison Chemical

Corporation Project at the Ohio State University, permitted the use of the ball mill and the taking of Its picture.

Matheison Chemical Corporation Project at the Ohio State University gave the facility of using a number of materials for insecticidal studies.

H i Ohio Seed Improvement Laboratory at the Ohio State

University helped to determine the water content of the samples of wheat with the Tag-Happenstall Moisture Meter and supplied some of the wheat used in tests* U.S. Industrial Chemicals, Inc. supplied the samples of pyrenone, pyrethrins, piperonyl butoxide and several diluent dusts•

California Spray Chemical Corporation furnished the sample of lindane* Attapulgus Clay Company supplied the sample of Atta- clay SP* To these men and companies that contributed much toward the work, the author is very grateful*

iv INTRODUCTION

Along with, the unprecedented, development of insecticides in the past decade a new approach to the control of the stored grain insects has been laid. Among the newer in­ secticides synthetic chlorinated compounds received immedi­ ate attention. However, toward the close of the decade at­ tention was turned to the combination of pyrethrins and their synergists. Among the chlorinated compounds lindane is es­ pecially promising for the control of stored grain insects because it has several unsurpassed properties. The so- called pyrenone, the combination of pyrethrins and piperonyl butoxide Is considered the most promising insecticide for controlling stored grain Insects because it gives not only rapid "knock-down," greater mortality and longer residual action but also offers safe use for human beings. In addi­ tion to the well-known properties of lindane and pyrenone, lindane is assumed to act as a fumigant and pyrenone and/or its ingredients as a repellent. These new properties are perhaps more favorable to the control of stored grain in­ sects. Their study may afford the bases of their new uses. However, a detailed survey of literature Indicates that little work has been done in connection with the new proper- 2

ties of lindane and pyrenone. The problem of Stored Grain Insect Control which was

chosen as the author's doctorate study was first specified

as Stored Grain Insect Control by Use of Newer Insecticides. A little later the problem was narrowed down to Fumigation

Effect of Lindane. Still later Repellence Effect of Pyre­ none and Its Ingredients, and Lindane; and Contact Effect

of the Same Toxicants were added to the study. Thus, the problem was finally shaped as Comparative Effectiveness,

against Stored Grain Insects,of Contact, Fumigation, and

Repellence of Lindane, Pyrenone, Pyrethrins and Piperonyl

Butoxide. Judging from the excellent properties and new uses of the selected insecticides the present form of the problem seems logical and advisable. During the study six experiments were carried out: experiments 1 and 6 for contact effect; experiments 2, 4 and 5, for fumigation effect; and experiment 3 for re­ pellence effect. These experiments were conducted by two methods: experiments 1, 2 and 3 were conducted by use of i impregnated dusts and experiments 4, 5 and 6, by use of im­ pregnated cloth bags.

The work is the result of four quarters investigations in the laboratory at the Ohio State University. One of the Important findings of the study Is the relative toxicity and stability of pyrenone and lindane. Another finding is the 3

effectiveness of lindane vapor against the adult and im­ mature stages of the granary weevil. LITERATURE REVIEW

To obtain the best, first-hand and up-to-date references

to the present work a detailed survey of literature was made,

'This survey indicated that the work on the evaluation of the

effect of DDT, BHC and pyrenone against stored grain Insects

was so crowded that only outstanding papers could be briefly

summarized in this review. The papers which are to be re­

viewed were essentially obtained from Biological Abstracts

and Bibliography of Agriculture, from 1941 to 1951, and some

other sources. In addition, some information was obtained

by personal communications. The applications of the three

insecticides to stored grain insects include three categories, namely: (1) control by spraying warehouses and boxcars, (2)

control by mixing chemicals with grain and (3) control by

impregnation of sacking. The category (1) will be omitted herein since it is not incorporated in the present work. The remaining categories are separately discussed as follows: I. Control by Mixing Chemicals with Grain. This cate­ gory will be dealt with under the following two topics. A. Protection of grain for seed purpose. It is most probable that far more work on this subject has been done than on any other subject dealt with later. Among many 4 5 workers along this line Cotton, Hammer, Farrar and Kulash 1 p deserve mention especially. Cotton * noted that an effec­ tive treatment consists of 3$ DDT in magnesium oxide applied at the rate of 0.05$, by weight, of the seed mixture or ig- oz of the mixture per bushel of seed. He also suggested BHC at 3 ^ 1 PPM for seed protection. Hammer found that 3% DDT dust at the rate of 1.5 ozs per bushel affords protection of seed corn against the rice weevil, Anguomois moth and Indian meal 4 moth for 223 days without affecting germination. Farrar demonstrated that satisfactory protection of fifteen kinds of seed against eight species of grain insects for a whole year results from the application of 2 .5 -20% DDT dust at the rate of 1.5 ozs per bushel. It is apparent that the results obtained by the three workers are very similar. Kulash ob­ tained the satisfactory results by spray or slurry method.

He treated husked, unhusked and shelled corn by spraying DDT solution. He also treated shelled corn by applying DDT emul­ sion or slurry.

B. Protection of grain for human consumption. 6 1. DDT. ZInkernagel found that wheat may be pro­ tected by 10$ DDT dust at the rate of 0.1$. It is shown that the residual left is only 9 PPM after the grain is cleaned and 4-6 PPM after milling which is below the United States official tolerance of 7 PPM. Ho ill effects were observed in laboratory animals that fed for seventy days on the treated 6 grain that had not undergone any of the normal cleaning

7 processes or on bread baked from it. Gay showed that DDT at the concentration of 4 PPM produces a 50$ kill of the rice weevil in ten days. For some years DDT has been practically applied to 8 stored grains for food to some extent in Switzerland, Bel- Q glum and some other parts of the world. However, from a recent personal communication it became known that the use of DDT in protecting grain for food purpose was much re- 8 duced. This is most probably due to the fact that the ac­ cumulation of DDT was shown in body tissues of mammals. 2, BHC. Slade'1'0 first observed that BHC is ex­ ceptionally toxic to the rice weevil when applied to grain at the concentration of 1 PPM. He also showed that no ef­ fect can be detected on flour subsequently made from wheat or in loaves of bread made from the flour when BHC is used at 1 PPM or at considerably higher concentrations. When it is used at 1 PPM the residual is practically nil in comparing with the residual of DDT, which is 4-6 PPM when applied at

100 PPM. Gay*7 found that BHC at 0.2 PPM gave a 50$ kill of the rice weevil in less than three days and that at 0.1 PPM it gave a 50$ kill in just under six days. Depending upon his results, Gay suggested the use of BHC at 0.4 PPM on the top six inches of wheat in storages. On this basis, one pound of BHC would be sufficient to treat 132,000 tons of 7 wheat. In 1948 the British Agricultural Research Council 1 T_ considered that the addition of 0.5 PPM of BHC to stored food could be safely recommended. In 1950 the British Toxi- oology Committee of Medical Research 11 recommended a maximum permissible concentratIon of 2.5 PPM of BHC in foodstuff. In

East Africa the use of BHC in controlling grain Insects is being extended. 12 A technique has been worked out whereby a dilute BHC dust is injected by air pressure into the bags 7 of grain. Since BHC is over forty times as effective as 1 I*5! DDT against the rice weevil, and since BHC is about ^ as toxic as DDT to mammals in chronic toxicity, and further, since BHC does not accumulate In body tissues of mammals as does DDT, it is probable that BHC is undoubtedly more likely to be used as the protectant of grain than Is DDT. In addi­ tion, BHC in the form of lindane is much less odorous than its crude form so that it Is doubtful that it will taint grain. However, the formation of dust clouds during handling induces the objection to its use in protecting grain in Britain,14

3. Pyrethrins and pyrenones. Since the admixture of DDT and BHC with grain is subject to deleterious sus­ picion a considerable Interest In grain protection is turned toward the pyrethrins. In addition to their safety, the dis­ covery of piperonyl butoxide, the best synergist of pyrethrins, gives the increasing hope of applying them as grain protec­ tants. 8

Beckley 1 5 found that pyrethrum powders at the concen­ tration of one pound per 2G0 pounds gives complete protec­

tion to bagged maize or wheat for 8.5 months. Watts16 found

that a combination of 0.4$ piperonyl butoxide and 0.08$ pyrethrins gives 100$ kill in one week. Extensive tests1^

in both the laboratory and the field conducted by U.S. In­ dustrial Chemicals, Inc. under different conditions during

1948-50 demonstrated that application of 100 pounds of py­ renone per 1000 bushels gives control to various grains for over nine months• II. Control by Impregnation of Sacking. As the pro­

tection of grain for human consumption by direct mixing with chemicals is subject to objection it would be more practical

to control grain insects by treating bags with chemicals. In addition, this method itself has its sound basis in fact. -IQ 1 Q Both Johns and Nasir observed that a great number of adult weevils migrate to the surface of bags from Inside the con­ tents. For this reason, the treated bags not only keep the insects outside the bags from entering, but also kill those present within the bags.

The investigations on the preventive measures against food product insects by treated packages were made by Parker20 In 1913. Later, in 1938 Stracener21 found that multiwall, metal-strip-sealed bags afford adequate protection of milled rice against Insects. Still later, the investigations22* 23»:24 #£5 9 along this line directed toward (1) the studies of biology and food habits of insects, (2) the tests of package materi­ als in relation to insect penetration, contamination and infestation, and (3) findings of repellents of insects for package treatments. A. DDT and BHC. As soon as DDT and BHC were found to act as marvelous residual poisons, they attracted immedi­ ate attention to their use in impregnation of sacking. Cot­ ton2® found that the paper or cotton bags which are filled with wheat flour receive protection against insects for sev­ eral months when they were treated with 5$ and 10$ DDT solu- 97 tion. Parkin showed that various kinds of sacks treated with 1% DDT solution insure proper protection to the con­ tents of bags. Nasir^® found that the application of 1.7 gm of DDT or BHC to half-size standard gunny bags offered satisfactory protection of wheat after six months. He strongly recommended the practical application of treated gunny bags to the protection of grain. He also suggested the use of treated gunny cloth as a covering for stacks of bags in case the bag treatment is impractical. However, the use of DDT is very discouraging owing to the fact that as many as 100 PPM28 are absorbed by the fatty and finely divided foodstuffs•

B. Pyrenone. In addition to safety and excellent contact toxicity with long residual effect, pyrenone was con- 10 sidered to act as a repellent. These characteristics make it very desirable for bag treatment. Smallman^® found that pyrenone Impregnated on cotton sacks will kill or repel in­ sects and will give a high degree of protection to the en- closed flour for at least ten months. Cotton30 demonstrated that pyrenone at the rate of 5 mg pyrethrins and 50 mg pipe­ ronyl butoxide per square foot of cotton cloth provided pro­ tection of flour against the confused flour beetle, the ca- delle and the Mediterranean flour moth for seven months. CULTURE METHODS

In furnishing insect cultures for insecticidal tests it is of great importance to start with a large and con­ stant supply of cultures as soon as possible.31 It Is equal­ ly important to supply a uniform diet to insect cultures and to maintain them under the condition of constant temperature and humidity so as to obtain the homogeneous populations of 31 test Insects. These essentials will Insure the speed of work as well as consistent data. During the course of the study special effort was made to achieve these essentials. The granary weevil was used in this study. Its cul­ ture was made up in 120 cc or 5 cm by 11 cm wide-mouth bot­ tles covered with cotton cloth called white rainbow bunting.

These cultures were established by the following procedure.

First, the water content of a sample of the wheat in use for a culture was determined by means of a Tag-Heppenstall

Moisture Meter. Before determining, the sample of wheat was kept under the condition of ordinary room temperature for at least 24 hours in case the wheat stock Is stored In a cold room. If otherwise, the error in water content read­ ings would occur. Secondly, an estimated amount of water was added to the wheat to raise its waiter content to 14$,3^

11 12 the optimum moisture level for weevil infestation. The amount of water to be added is the difference between the weight of the sample of wheat and its weight when its water content is raised to 14$>. The weight of the sample of wanted water content may be obtained by the following formula.

------122------X dried material of sample. 100 X wanted water content The dried material of the sample of wheat is derived by the following formula.

100 X Jm o w n g a ter., content x welght of aampl9.

This lot of wheat was allowed to condition in a closed earth­ enware crock for one week. During that period, daily turn­ over was made with the aid of a hand. Thirdly, a batch of

50 gm of the well-conditioned wheat was introduced, together with 200-300 weevils, into each bottle. Twenty to forty such bottles were prepared for a series of cultures. Finally, the bottles of the series of cultures were numbered and placed in an enameled pan. Around the margin of the pan a ring of a mixture of vaseline and mineral oil was smeared to prevent the enemies of cultures, such as mites and ants from entering.

After one week the weevils were removed from the bottles and transferred to the bottles of another series which were pre­ pared beforehand in the same manner as mentioned above. In doing this, a 10-mesh sifter,0 superimposed above a 20-mesh sifter with a pan below the latter and a lid above the former, 13

was employed. By means of this set of sifters, wheat was retained In the 10-mesh sifter; weevils, In the 20-mesh sifter; and feces, borings, dust and debris, in the pan. Four series of cultures were regularly kept going during the

study. The cultures were maintained at the constant temperature

of 25°C. and the constant relative humidity of 60^J5$3^ in one

of the temperature-controlling cabinets in a subbasement room.

This cabinet was a 36-lnch cube. Its back pressure valve re­

mained fully open at all times. Its solenoid valve was set

at 20. The required constant temperature was produced by

means of the electric thermoregulator manufactured by Eastern

Engineering Company. This regulator consists of a thermostat

and a relay operated by the former. It is shown in Fig. 1. The adjustment of the thermostat can be quickly and easily made with the accuracy of l/50°C.*^ Figure 2 shows the ther­ mostat in diagrammatic form. This thermostat is operated by the pressure of gas. When the temperature of the sensitive

liquid In the bulb of the element reaches its boiling point

It begins to vaporize and pushes back the mercury causing it to rise In the tube. Any rise of the mercury in the tube, however, Increases the pressure on the vapor in the bulb so that a higher temperature is required for it to boil. The surrounding temperature must increase slightly in order to rz f* cause further rise of the mercury. A 60-watt bulb was em- 14

ployed to furnish the heat, Furan, with the B.P. of 31°C.,

was chosen as the sensitive liquid and amyl acetate, as the

protective liquid in the bulb of the thermostat. Difficulty

was encountered in producing a small air bubble above the

sensitive liquid. The difficulty was finally overcome by

cooling the bulb, mercury and sensitive liquid on the flush of a water-cooler fountain before filling. The required rela­

tive humidity in the cabinet was regulated by the supersatu­ rated solution of sodium nitrite in a pan. Circulation of

air was induced by use of a pre-oiled 9-inch fan. It is

shown in Fig. 3. This fan is very satisfactory. It runs at all times without producing excessive heat. To reduce

the speed of air movement the fan was covered by a carton with a hole of six square inches.

The cultures manipulated in this way produced normal populations. However, on one occasion the population of a hymenopterous parasite.upon the granary weevil became so dense

that a much smaller number of weevils emerged from two of the

series of cultures. The population of weevils In most series of cultures is very high. In one case 10,360 granary weevils were removed from-ten bottles of a series of cultures, or an average of 1036 weevils from each bottle. Fifty gm of wheat

contains about 1500 grains, the amount of wheat used in each bottle. It Is thus apparent that more than two thirds of the grains of wheat in each bottle were infested. EXPERIMENTATION

This portion will cover the following topics: Experi­ mental Design, Equipment, Toxicants and Adjuvants, Formula­ tions, Application Technique, and Collecting Data. Among these topics, equipment, formulations and application tech­ nique will he emphasized. They are thus treated because they contain some original information, which is developed largely during the study. I. Experimental Design. The purpose of this work is to develop the.control measures of stored grain Insects by the best method of application of some newer insecticides, including pyrenone, pyrethrins, piperonyl butoxide, lindane and DDT. In achieving this purpose, six experiments were conducted by means of two methods. These experiments are given as follows:

Method 1. Tests made in glass bottles with grains of wheat coated with the selected insecticides in the form of impregnated dusts.

Experiment 1. Contact effects of pyrenone, lindane and DDT,,

Experiment 2. Fumigation effect of lindane. Experiment 3. Repellence effect of pyrenone, pyre— thrins, piperonyl butoxide, lindane and DDT.

15 16

Method 2. Tests made by Impregnating the selected insecticides on cloth bags in the form of water emulsions*

Experiment 4. Fumigation effect of lindane upon the adult of the granary weevil.

Experiment 5. Fumigation effect of lindane upon the immature stages of the granary weevil. Experiment 6. Contact effect of pyrenone, pyrethrins, piperonyl butoxide, lindane and DDT. II. Equipment. The important equipment used in ex­ perimentation includes a working set, an aspirator, a ball mill and a Tag-Heppenstall moisture meter. They are des­ cribed separately below.

A. Working Set. The working set is 3liown in Fig. 4. The set consists of an enaBieled pan, 18/12 in., a homemade funnel and two sieves. The funnel was made of the top por­ tion of a five pound jar. The size of the funnel stem Is very suitable for transferring wheat from one container to the other. A brass 10-mesh sieve, and a brass 20—mesh sieve provided with a bottom pan and a cover are made into a de­ pendable apparatus for separating weevils from grains of wheat or for separating dockage from wheat. B. A New Tyj© of Aspirator. A new aspirator which was modified by the.author was employed In counting the beetles. This aspirator was found so satisfactory that a great number of lots of weevils were obtained at a much shorter time without loss or injury. Its construction is 17

shown In Pig# 5# It is essentially made up of a T-shaped glass tube# Around the latter part of the stem of the tube an adaptor, which is made of a piece of celluloid sheet, is

attached to perform the connection between a 40 cc bakelite screw-cap vial used to contain weevils and the stem of the

glass tube. The connection was strengthened by fixing be­ neath the edge of the stem a bakelite screw-cap of the 40 cc

vial with the top removed. When picking up the weevils each time, a vial is screwed in the cap. At one end of the tube is a rubber stopper with a piece of glass tubing ^ inch in diameter passing through the center. A short arm at about 90 degrees is prepared by bending the latter portion of the

tubing extending into the tube. This tubing is so placed

that the short arm is directed toward the stem of the glass tube. Through this arm weevils are conducted into the stem

of the tube and then into the vial. A gentle curvature was made in the portion of the tubing extending out of the rubber stopper. At the other end of the tube is a rubber stopper mounted with the same type of brass blower-suction -unit as 37 devised by Woodbury and Barnhardt. This unit Is provided 38 with a brass venturi through which insects are blown up and also a brass tubing through which Insects are sucked up. C. Portable Ball Mill. The ball mill Is shown in Fig. 6. It is a very useful piece of equipment for the study. It Is used in mixing Impregnated dusts and In treating 18 wheat samples. It is essentially made of* a ^ HP Emerson motor, which, travels about 1725 rpm, and four rolling units mounted on a wood structure. The motor is mounted on a re­ movable and adjustable platform so that the slack can be taken up when the "A" belt Is shifted from one pulley to the next. A 12” shank on one of the drive rollers and a pulley cone with a 4 X 3 X 2 Inch shank arrangement on the pack shaft was used to get proper gear reduction. Each rolling unit consists of two rollers made of 5/8” or 3/4” cold rolled steel. These rollers are covered by soft rubber hose ‘They are set in self-adjustable pillow blocks with a brass bearing. The pillow blocks are set In a grooved ”2 X 4” or in an angle iron. They can be adjusted to take bottles of various sizes. The wood structure is made of ”2 X 4” pieces ripped lengthwise. Its dimensions are 30 X 20 X 36 Inches. It consists of two frames, one held above the other by a vertical ”2 X 4” in each of their corners. At the top of each frame are mounted two rolling \inits. D. Tag-Heppenstall Moisture Meter. A Tag-Heppenstall moisture meter was used in measuring the water content of samples of wheat for the study. This meter provides the data on water content in approximately one minute. It mea­ sures moisture by measuring the resistance to electrical cur rent flowing through the grain being tested between two re­ volving electrodes. Through calibration, the resistance in­ dicated bn the meter provides a direct index to the moisture 19 content. The meter is operated as follows: After standard­ izing the instrument by adjusting the rehostat, the motor is

started under no load, i.e. no material in hopper or between rolls. A sample of the grain to be tested is then placed in the hopper. Dial switch is turned from tap to tap until meter needle reads between 15 - 20. After average needle reading and tap setting are noted, the reading is converted

to moisture content. Adjustment of this preliminary reading

is then made for temperature to give the final actual mois­ ture content.

III. Toxicants and Adjuvants. Under this topic some

of the general and specific features of importance will be presented. A. Toxicants. The toxicants employed in the pre­ sent study include DDT, lindane, pyrenone, pyrethrins and piperonyl butoxide. They are separately discussed below.

1. DDT. DDT had unprecedented development as a synthetic Insecticide because of its several unusual pro­ perties. Its full chemical name is 2,2-bis (p-chlorophenyl)-1, 1,1-trichioroethane. Its structural formula is known ass

ci- Cl C1 Cl 20

It is soluble in most of the organic solvents. Acetone and xylene are most often used in laboratory studies. Acetone dissolves 58 gm of DDT, and xylene dissolves 57 gm of DDT 39 per 100 cc. The form of DDT used for this work is the aerosol grade, containing 97$ P, DDT and 3$ 0, P DDT, It is the product of Hercules Powder Co., Washington 99, Delaware, 2. Lindane. Lindane (Lnd.) is the purified prepara­ tion of BHC. It contains not less than 99$ of the gamma isomer of 1, 2, 3, 4, 5, 6-hexachlorocyclohexane, -which is

erroneously called benzene hexachloride (BHC). The struc­ tural formula of gamma isomer of BHC is as follows:40

Cl

Cl

Cl

Cl

This compound was selected because it has several unusual properties, which are surprising contact toxicity to grain

weevils, less stable but more fumigative effect, absence of accumulative toxicity in body tissues of mammals, and less flavoring effect upon foodstuffs. The sample in use is 21 claimed to be 100$. It is a white granule, manufactured by

California Spray Chemical Corporation. It costs about ten dollars per pound. 3. Pyrenone. Pyrenone (Pyrn.) has been a registered •17 trademark of U.S. Industrial Chemicals, Inc. since 1946. Recently It was also manufactured in Britain under the name

,,pybuthrin.ny:‘t Pyrenone or pybuthrin contains a combination

of pyrethrins and piperonyl butoxide. It is highly toxic

and surprisingly lasting. In addition to its contact tox­ icity, it is also assumed to act as a repellent. For these reasons, it is considered very promising for stored grain

insect control. The sample used in the present work Is

Pyrenone O.T. 50-5, which Is combined with 50 gm of techni­ cal piperonyl butoxide and 5 gm of pyrethrins per 100 cc.

Its specific gravity is 0.94^0.025 at 20° C. It Is a dark

amber liquid and is soluble in deobase in all proportions. It costs a little over five dollars per pound. 4. Pyrethrins. "Pyrethrins" is the term used to desig­ nate the four active constituents of pyrethrum, Chrysanthemum cineridefolium which are pyrethrin I and II and cinerin I and II. The striictural formulae of these four compounds are as follows: CINERIN I

CH, H t, H2 H 0— * C‘ ■ C — C I h 2c-

CINERIN II

CH,

H H2 H C -C -™-C

h 2c

PYRETHRIN I

CH,

H2 H 0__C -C — C

HgC, 0

PYRETHRIN II 23

The product of pyrethrins used in the present study is a

20% extract in a hydrocarbon solvent. The color of the ex­ tract varies from yellow to dark amber. This extract costs fifteen dollars per pound*

5. Piperonyl butoxide. Piperonyl butoxide is chemical­ ly known as 3, 4-methylene-dioxy-6-prophylbenzyl (butyl)

40 dielhylene glycol ether* c Its structural formula is given as follows:

CH,

ch2-ch2-ch3 -CHg-O-CHg-CHg-O-CHg-CHg- o-c4h9

The sample used in this work was obtained from U.S.

Industrial Chemicals, Inc. under the name technical piperonyl s butoxide, which is considered as 80^. It cost five dollars per pound. The product is a clear, pale-yellow, oily liquid.

It Is practically odorless. It has a specific gravity, at

c\ ^3-IS 25 C., of about 1.06. It is soluble In all dilutions in mineral oils, which are the accepted solvents for this pro- duc t,

Piperonyl butoxide is known as the best synergist of pyrethrins. It Increases the toxicity of pyrethrins tremen­ dously when it is admixed with pyrethrins, usually In the 2 4 proportion of 10:1. In addition, it stabilizes the toxicity of pyrethrins. However, when used alone piperonyl butoxide is slightly toxic to insects at higher concentrations. B. Adjuvants. The adjuvants used with the toxicants will be discussed under impregnated dusts and emulsion con­ centrates • 1. Impregnated dusts. The adjuvants used in impreg­ nated dusts consists of pyrophyllite, attaclay SP and ace­ tone . a. Pyrophyllite. Pyrophyllite Is chemically a 4 4 hydrous aluminum silicate, HgOAlgO^SIOg. ■ There are sev­ eral varieties of the mineral. The variety used In this work is in the form of flat or plate-like particles. It was found by several workers that this dust is non-abrasive against grain weevils. For this reason, it was selected as the di­ luent of the impregnated dusts. b. Attaclay SF. In preparing the satisfactory impregnated dusts of pyrenone, pyrethrins and piperonyl butoxide, an anticaking agent was found quite necessary.

The reason for this will be mentioned under the subject,

"Formulation.” Attaclay was first found to be excellent for anticaking. However, it is strongly advised that its use with pyrethrins and related compounds be not used because its strong absorptive power conceals their toxicity to some ex- 43:' tent. Finally, attaclay SF was selected. This product 25 was found as satisfactory as attaclay with, regard to anti­ caking efficiency but not as absorptive as attaclay* It is described chemically as a complex hydrated aluminum magne- 45 sium silicate. Its approximate chemical analysis Is as

follows: i

SiOg 67.0 # A1203 12.5#

MgO 11.0#

Feg03 ...... 4.0# GaO 2.5# Other ...... 3.0# Total 100.0# The product is developed by Attapulgus Olay Company, Phil­

adelphia 5, Pennsylvania. C. Acetone, CH^COCHg. In preparing DDT-impregnated dust and lindane-Impregnated dust, the active Ingredients

are necessarily dissolved in a solvent. Acetone was selected as a solvent. This compound is particularly suitable for

the purpose because of its tremendous solubility and its rapid vaporization.

2. Emulsion Concentrates. The adjuvants applied in these preparations include the two solvents, xylene and deo- base and the one emulsifier, Triton X-100. a. Xylene, CgH-^Q. Xylene was used as the solvent of DDT and lindane• It Is a good solvent for the two com- 26

pounds. It also vaporizes slowly. This property makes It

suitable for keeping in storage for a long time. However,

it was found very toxic to the granary weevil at the con­ centration of 0 .1$.

b. Deobase. Deobase or refined or deodorized kero­ sene was used as the solvent of pyrethrins products. Kero­ senes are believed to be composed of hydrocarbons having

10-16 carbon atoms per molecule. They are deodorized by 49 sulfonation process. c. Triton X-100. Triton X-100 was employed as the

emulsifier of the emulsions. It is one of the anionic soaps.

Its chemical name is alkyl aryl polyether alcohol. It is 46 manufactured toy Rohm and Haas Company. III. Formulation. Formulation very often presents a problem in the application of Insecticides. The potency of an insecticide will not be shown unless it is applied by

the appropriate formulation. A formulation is concerned with

the appropriate kinds of adjuvants, the suitable amount of

each ingredient, and the method of preparation. During the

study, Impregnated dusts and emulsions were used. The de­ velopment of the formulation and method of preparation of each of them will be discussed below. A. Impregnated Dusts.

1. Development of Formulation. Impregnated dusts were chosen for experiments 1, 2 and 3'. Stock supplies of 27 these were first prepared. Acetone, pyrophyllite and atta­ clay SF were employed in making stocks. Acetone was used only in the dusts of DDT and lindane in an amount sufficient to dissolve the required amount of the two toxicants. Since it was allowed to evaporate completely, its weight was not counted in the formulation. It was omitted from the dusts of pyrenone and of its ingredients because it is unnecessary for them. In addition, Its omission improved the dust na­ ture of the three products immensely. Pyrophyllite was selected as the diluent on account of its powerful absorp­ tion and its very little abrasive value for the control of grain weevils. The latter property makes It useful for the present study because It would not obscure any effects pro­ duced by toxicants. Twenty per cent of attaclay SF was added to each of* the five impregnated dusts. This dust was used in making DDT - and lindane - impregnated merely for the purpose of keeping the inert part of the two dusts the same as that of the rest. Otherwise, an undesirable .varied factor would be introduced into the study. However, this anticaking dust is quite necessary to prevent caking in the case of pyrenone and its Ingredients. This caking property arises from their adhesive nature and also from the necessary use of their large quantities due to the low concentration of pyrethrins and low toxicity of piperonyl butoxide. The de­ termination of the percentage of the toxicants contained in 28 their respective Impregnated dusts depended upon (1 ) the 26 official tolerance, (2) the formulation used by Cotton, G a y ^ and Watts and Berlin**"® and (3) the toxicity and con­ centration of their original forms. As the problem is con­ cerned with human food, an attempt was made to test the toxi­ cants at concentrations below the official tolerance only. One per cent each of DDT and lindane was prepared because of their high toxicity to insects and low tolerance for humans. One per cent of pyrethrins was selected because of the combination of the low concentration and limited amount capable of being added to the carrier. Five per cent of piperonyl butoxide was employed because of its lower toxicity and the limitation of the carrier. Two and a half per cent piperonyl butoxide and 0.25$ of pyrethrins were used in making pyrenone or 2.5$ of pyrenone O.T. 50-5 was used. This formulation was modified from the one de­ veloped by U.S. Industrial Chemicals, Inc. The formulation of the impregnated dust stocks of the five toxicants devel­ oped during the study is given in Table 1. 29

Table 1 Formulation of Impregnated Dusts for 50-gram Stocks

i DDT Lnd Pyrn Pyr PB 1 0 0 $ 1 0 0 $ 0 .T.5-50 1 0 0 $ 80$ Per­ Gm. Per­ Gm. Per­ Gm. Per- Gm. Per- Gm. cent cent cent cent cent Active Ingre­ 1 . 0 0.5 1.0 0.5 5 2.5 1.0 2.5 5.0 2.5 dient Pyro­ phyl­ 79.0 39.5 79.0 39.5 75 37.5 2 0 . 0 10 . 0 20. 0 1 0 . 0 lite Atta­ clay SF 2 0 . 0 1 0 . 0 2 0 . 0 10 . 0 20 10 79.0 37.5 75.0 37.5

Acetone 5.0 5.0

Each of the five stocks of impregnated dust were diluted by use of pyrophyllite according to the following plan. Concentration 1 = l/5 stock / 4/5 pyrophyllite Concentration 2 = l/5 concentration 1 / 4/5 pyrophyllite Concentration 3 = l/5 concentration 2 / 4/5 pyrophyllite Concentration 4 = l/5 concentration 3 / 4/5 pyrbphyllite Concentration 5 - l/5 concentration 4 / 4/5 pyrophyllite

2. Method of Preparation. The toxicants were weighed with an analytical balance with the accuracy of 0 . 0 0 1 gm while the adjuvants with a tortion balance with the accuracy of 0.1 gm. Wax weighing paper was used when solid substances were weighed. A small beaker was used when the liquid sub­ stances were weighed. Screw-lid jars 7 X 9 cm were employed 30 as the containers in which the dusts were prepared. The procedure of preparing the dusts of the organic chlorinated compounds differs from those of pyrethrins products. In preparing the former group of dusts, first, active ingredi­

ents, then acetone and finally pyrophyllite and attaclay SF were introduced into the container. In preparing the lat­

ter group of dusts, pyrophyllite and attaclay SF, then ac­ tive ingredients were put into the container. As soon as all the ingredients were admitted into containers the lumps were thoroughly broken up with a glass rod. Subsequently, 30 glass marbles, 15 mm In diameter were put into the con­ tainer. The mixing process was finished on a ball mill by rolling for two hours. Four times during the period the

jars were shaken In all directions with a hand..

B. Emulsion Concentrates. In experiments 4-6, which were conducted by use of the impregnated bags, emulsions were used. The ingredients of the emulsion concentrates were measured on the basis of weight. In fumigation tests against the adult weevils and immature weevils two differ­

ent lindane emulsion concentrates were prepared In order to

apply higher concentrations to bags for immature stages and lower concentrations to bags for adults without varying the

amount of concentrates. Otherwise, different stages would receive different quantities of xylene and Triton X-100, and the effects of lindane would be obscured because they are very toxic to insects at higher concentrations. The formu- 31 lations for the two experiments are given in Table 2.

Table 2 Emulsion Concentrates For Experiment 4 For Experiment 5 Lindane 0.2$ Lindane 1.0$ Triton X-100 10.0$ Triton X-100 10.0$ Xylene 98.8$ Xylene 89.0$

The ^formulation of emulsion concentrate for the study of con­ tact toxicity^was given in Table 3.

Table 3 Pyrn Pyr PB Lnd DDT Active Ingre­ dient 20$ 5$ 20$ 1$ 2$ Triton H o o X-100 10$ H 10$ 10$

Xylene 89$ 8 8 $ Deobase 70$ ■ 85$ 70$

V. Application Technique. This topic will cover the test insect, test grain and methods. They are discussed separately below. Test. Insect. The granary weevil, Sitophilus granarius L.

was used in this work. It was chosen because it is the most common of the serious insect pests of major stored grains. This beetle is easily confused with the rice weevil, Sito- 32 philus oryza L. but they are readily distinguished by the 417 4 a characteristics f given in the following table: Table 4 The Differences in Adult, Pupal and Larval Stages of Rice weevil and Granary Weevil

Stage Characteristics Rice Weevil Granary Weevil Punctures on Dense, round Sparse, oval pronotum Wings Present Absent Adult Two pairs of spots on elytra Present Absent Color Reddish brown Chestnut brown or blackish Size 2 *1 -2.8 mm 3-4 mm Well-developed, Rudimentary, al­ extending well most completely Pupal Wings beyond tips of concealed by elytra elytra Number of ab­ dominal segments, First three First four Larval above, divided segments segments into 3 areas Seta on middle lobe of hypo- None One pleurum

The populations of the two weevils which were used in o the study were derived from the cultures maintained at 25 C. and the relative humidity of 60^5^. The standardization of

the two beetles involves the following procedure. As soon as weevils started to emerge, they were removed by means of

the set of sifters mentioned above. The removal, once a week, continued until the weevils of the next generation were 33 expected to appear. The weevils of known age were reared in lots of 1 0 0 0 per 2 -liter utility jar half full of wheat. These beetles are then used in tests one week later. By this method beetles of 1-2 weeks of age were obtained. Be­ fore being used, they were starved overnight in an empty jar provided with strips of paper towels upon which the weevils remain. Before exposure to insecticidal surfaces they were counted directly into bakelite screw-cap vials of 40 cc in lots of definite numbers. Test Grain. In all of the experiments wheat was used. Several lots of high quality wheat with very little dust and debris was used throughout this work. The stocks of wheat were stored In a room at about 2° C. Before used each esti­ mated batch of wheat was conditioned under room temperature for 24 hours. Water content of a 3 ample-of the wheat was determined by means of a Tag-Heppenstall Moisture Meter. The water content of wheat employed during the work ranges

from 9.3 to 13.2$. The experiments were conducted In a basement room where temperature and relative humidity were rather constant during the study.

Methods. By means of three series of preliminary tests

in 1 2 0 cc glass bottles with impregnated dusts of the insec­ ticides used in this work it was Indicated that fumigation effect was shown only by lindane, contact toxicity by pyrenone, lindane and DDT and repellency effect -by all the five toxi­ cants. These results suggested the toxicants needed to evalu­ 34 ate the fumigation, contact and repellency effects. These

toxicants will he indicated in the following listed experi­ ments. All experiments which were conducted for this work included untreated checks and checks treated with the con- juvants which were the same in both quality and quantity as

those of treatments. As two different sorts of powerful

insecticides were corporated In the study, cautious steps have been taken to avoid contamination. First, any con­ tainer or apparatus which was once contaminated was carefully cleaned by detergent water, mixture of ysodiuto . dichromate

and sulfuric acid, and acetone. Secondly, these toxicants were always handled In the following order; piperonyl butoxide, pyrethrins, pyrenone, DDT and lindane and also from the low­

est concentration of each toxicant upwards provided that two

or more concentrations of two or more toxicants were applied. This work order not only eliminates the possible contamina­ tion but also saves the labor spent In washing. The detail of each experiment is given as follows:

Experiment 1. Comparative Effectiveness of Contact Toxicity of DDT, Lindane, Pyrenone, Pyrethrins and Piperonyl

Butoxide Determined by Use of Bottles*

In this experiment the experimental unit was a 120 cc wide mouth bottle. Into each bottle was placed 30 gms of treated wheat and 30 granary weevils. The bottle was cov­ ered with cotton cloth, called white rainbow bunting held in position by a rubber band. The labels of the bottles were hi made of a piece of 1-inch wide masking tape. It was marked 35 with, a red wax pen. Thirty such bottles were prepared for one test. Each test supplied the data of the eight expo­ sures, such as 1, 2, 3, 5, 7, 10, 12 and 14 days, to one dosage of a toxicant. One series of tests for the three dosages of the five toxicants would consist of fifteen tests or 450 experimental units. Samples of wheat for the tests were treated in five pound wide-mouth jars. Each of the impregnated dusts given in Table 2 was applied at the rate, by weight, of l/500 of wheat. The concentrations of active ingredients of these dusts in PPM of wheat are given in Table 5.

Table 5 Dosages of Active Ingredients in PPM of Wheat P y r n DDT End Pyr PB Pyr PB Dosage 1 0.16 0.16 0.04 0.4 0.16 0 . 8 Dosage 2 0.80 0.80 0 . 2 0 2.0 0 . 8 4.0 Dosage 3 ' 4.00 4.00 1.00 1 0 . 0 4.0 20 . 0

The mixture of wheat and a dust was rolled In a five pound jar on the ball mill as shown in Pig. 5, for about two hours. Three times during this period the bottles were hand-shaken in all directions to aid the mixing. In addi­ tion, a strip of glass plate about 5 cm wide and as long as the height of the five pound jar was placed in the jar to insure thorough mixing. By this method of treatment it was found that the dusts were evenly distributed over the wheat 36 and that little dust was left in the jars. The 30-gram samples of wheat were weighed in a 30-gram glass vial,

3 cm X 6.5 cm by a tortion balance with the degree of ac­ curacy of 0.1 gm. The treatments were completed by intro­ ducing into each bottle thirty granary weevils which were admitted into a vial previously. Experiment 2. Fumigation Effect of Lindane. This experiment was set up in a similar manner as ex­ periment 1. The differences between the two experiments are as follows. The experimental unit was two 120 cc wide-mouth bottles. One was filled with 30 gm of untreated wheat to­ gether with 30 granary weevils. The other was filled with 30 gm of treated wheat. The two bottles were held mouth to mouth by masking tape. Between mouths is a partition which was made of organdy cloth and filter paper with a round opening^1.5 cm in diameter near the center. This partition allows lindane vapor to pass through the opening from one bottle to the other and simultaneously to prevent the treated and untreated wheat from contact with each other. During testing, the experimental units were laid horizontally. The dosages were 0.8, 4, and 20 PPM instead of 0.16, 0.8 and 4 PPM.

Experiment 3. Repellence Effect of Pyrenone, Pyrethrins and Piperonyl Butoxide, Lindane and DDT Determined by Weight of Feces and Oviposition Capacity. 37 This experiment was also similar to experiment 1 . The differences "between them are as follows:

1 . The dosages for this experiment were so designed that their effects will be expected to lie below the sig­ nificant mortality and above the significant repellency.

The dosages of the five toxicants applied are given in Table 6 *

Table 6 Dosages of the Five Toxicants Applied to Wheat in PPM P y r n Pyi* PB Pyr PB Lnd DDT Dosage 1 0.0064 0.064 0.16 0 . 8 0.0064 0.16

Dosage 2 0.0032 0.032 . 0.80 0.032 0.80 Dosage 3 4.00

2. In order to avoid the errors of the weight of feces, wheat was sifted before it was treated, 3. One hundred weevils were exposed to the treated wheat for ten days. Experiment 4, Fumigation Effect of Lindane upon the Adult of the Granary Weevil.

A 15 X 20 cm cotton cloth mailing bag provided with a drawstring was utilized In this experiment. The fabric of the bag is known as 371*-4 unbleached sheeting, which means 37 inches in width and 4 yards per pound. The count is 48/48, which implies that both the warp and the filling is 38

48 threads per square inch. This hag was obtained from the

Ames Bag Company, Cleveland, Ohio. Bags were thoroughly, washed and dried in the laboratory before being treated. They were treated with the emulsion of lindane of such strengths that the finished cloth con­ tained 0.5 mg, 1.5 mg or 4.5 mg of lindane per square foot.

To deliver this strength to a 15 X 20 cm bag 0.14 gm, 0.42

gm or 1.26 gm of 0*2% lindane emulsion concentrate was used.

To supply the three replications of each concentration three bags were treated each time in a 1-liter beaker. The emulsion for each of the three bags was prepared with the required

amount of 0*2% lindane emulsion concentrate and 50 cc of wa­ ter. This amount of water was the exact amount needed to wet the bags thoroughly. The -uniform penetration was aided

by pressing out and sponging in the emulsion alternately with the hand for a few minutes. The treated bags were dried by hanging them over a string in the laboratory.

As soon as the bags became dried, each was filled with 200 gm of wheat, together with 100 granary weevils. The mouths of the bags were sealed with separate pieces of string. All three bags, treated at one time, were introduced into a 5-pound wide-mouth jar. Two identical series of tests were prepared at one time. In one series the mouths of the jars remained open, while in the other series the mouths were sealed. 39

Experiment 5. Fumigation Effect of Lindane upon the Immature Stages of the Granary Weevil• . The impregnation of hags for this experiment was com­ pleted in the same manner as in experiment 4 except that such strengths of lindane were furnished that the finished cloth contained 0,9 mg, 4.5 mg or 22.5 mg per square foot. The infestation of wheat required for this experiment was artificially introduced hy keeping 200 weevils in a 2 0 0 gm sample of wheat in a pint mason jar covered with a piece of cloth. One week later, these weevils were removed. Then, all the 2 0 0 -gram samples of wheat were dumped in a large earthenware crock and well mixed by turning it over by hand for a few minutes. Finally, 200 gm of wheat were returned to each jar. These samples were placed on racks of a case in the laboratory. Fifteen of them were chosen at intervals of one week; nine were introduced into treated bags at three concentrations, three in check bags and three in untreated bags. The rest of the procedure is the same as experiment 4.

Experiment 6 . Contact Effect of Pyrenone, Pyrethrins and Piperonyl Butoxide, Lindane and DD£. The cloth bags were treated by the same method as that used in experiments 4 and 5. The dosages of the emulsion concentrates given on page 3l were 320, 1600 or 3200 mg/sq ft in pyrenone and piperonyl butoxide-, 80, 400 or 800 mg/sq ft in pyrethrins, 16, 80 or 160 mg/sq ft in lindane, and 32, 40

160 or 320 mg/sq ft In DDT. Every three replications of a treatment were introduced in a five pound jar with 2 0 0 gm wheat. In each jar fifty weevils were admitted. VI. Collecting Data. Data-collecting in this work varied with different ex­ periments. In experiments 1, 2, 4 and 6 mortality was re­ corded, while in experiment 5 the number of emerged weevils and mortalities of them, and in experiments 3 the weight of feces was recorded. The procedure for each method will be described below. To obtain accurate mortality an attempt was made to interpret two conditions. One was the determination of the differentiation between the death feigning and the true death. The other was the determination of the border line whereby the moribund can be divided into the dead and the living with certainty. The granary weevil has the habit of feigning death. Evidently, errors would be introduced into

the data If the two kinds of death were to be confused with each other. As a matter of fact, It Is not difficult to dif­ ferentiate them merely by a fairly careful examination under a binocular microscope. In most cases, the truly dead were indicated by the stretched out legs and antennae and side- wise lying posture and also the dull appearance of body sur­ face. On the contrary, the beetles which were feigning death were usually characterized by folded legs and antennae, an up-down resting posture and a glossy appearance of body sur­ 4 1

face. Besides, they were determined by disturbing them with a sharp needle in case of doubtfulness. Likewise, the moribundlty presents a problem to data-collecting. In this study, a definite criterion was established to divide the moribund into the dead and the living. Those which walked about freely, although slowly, were considered as the liv­

ing, while those which moved about staggeringly or activated their legs or antennae or only joints of them were considered as the dead. The procedure in making mortality observation is as fol­ lows s Weevils were removed from the wheat in an experimen­ tal unit with the set of sifters. In experiments 1 and 2

beetles were removed by gently shaking the wheat over the

2 0 -mesh sifter in order to prevent the dust on the grains of wheat from being rubbed off. Among the beetles removed from a sample of wheat, active beetles were picked up, with the aid of the naked eye, by a pair of forceps. Then the inactive were examined under a binocular microscope and were divided into the living and the dead with care. A mortality data

table (see page 42 ) was prepared to record the mortality. The table is especially useful for experiment 1. With exper­ iments 1 and 2 , the mortality observations were made accord­ ing to the fixed exposure periods as 1, 2, 3, 5, 7, 10, 12

and 14 days. With experiments 4 and 6 mortality readings were taken one week and two and a half months afterwards re- 42

Mortality Dfeta Table

Expt, Number ____ Test Number ____ Testing Effect___ Testing Method___ Exposure From To

Toxleant s Concentration Replication 1 Replication 2 Replication 3 in PPM Mor i- Mori*- klor i- of wheat Alive bund Dead Alive bund Dead Alive i bupd Dead I

• r , ' i i ! I .... ! 1 1 i tF

I1 i * 1

. - .. j i !j ■ ; i !i Check *t ! 1♦ i i! Untreated : . ; • . ^ J 1 t i --- _ — i .. 1... . . i 43

spectively. The residual tests of all the experiments 1, 2 and 4 were made at the intervals of about three months.

With experiment 5 the data on number of emerged beetles and

their mortalities were read 50 days after the infestation of wheat samples. The results of experiment 4 were shown by the relative weight of feces accumulated during the exposure period. The feces were removed with a 20-mesh sieve. The feces taken

from each sample of wheat were stored in a vial. As the feces of all the samples were collected they were weighed with an analytical balance. The accuracy of weighings is within'the degree of 0 . 0 0 1 gm. Then the feces were trans­ ferred from one container to another, a camelhair brush being used to brush off the feces attached in the container. Before they were weighed, larger particles of debris and dust mixed with feces were removed under a binocular microscope. The number of dead beetles was recorded to check the weight of feces. RESULTS AND DISCUSSION

Results of a part of the study are summarized in Fig­ ures 7-10 in which average percentage mortalities for the three replications have been plotted against time or dosages. Results of another part of the tests are given in Tables 7-10. In addition, thepart of results in which the exact mortalities are not necessarily recorded will be stated in words. Checks and untreated controls have been omitted if their mortalities are negligible; but the data are included if their mortalities are significant.

Impregnated Dust Method Contact Effects of Impregnated Dusts. Figure 7 gives the results of comparative contact toxicity of pyrenone, lindane and DDT. From this Figure it will be seen that LDgQ Is obtained in two days from only pyrenone combined with 4 PPM pyrethrins and 40 PPM piperonyl butoxide and from lin­ dane at 0.8 PPM and 4 PPM, and practically no kill results from pyrenone combined with 0.16 PPM pyrethrins and 16 PPM piperonyl butoxide and from DDT at 0.16 PPM and 0.8 PPM. After three months only the three treatments which give LD&0 in two days at the beginning of the experiment remained un­ infested. The other treatments became slightly or heavily infested, depending upon exposure. Figure 8 shows the com­

44 45 parative contact toxicity between the initial tests and re­ sidual test3 with these three treatments. The residual testa indicate that all the three treatments retain a great part of their initial toxicity after three months. These tests also show that pyrenone Is more stable than lindane* This fact is very significant because stability plays an im­ portant role in controlling stored grain insects. After six months these three treatments are still free from In­ testation. This indicates strongly that pyrenone mixed with 4 PPM pyrethrins and 40 PPM piperonyl butoxide and lindane at 0.8 PPM and 4 PPM afford satisfactory protection of wheat against the granary weevil for at least six months* The percentage of kill obtained by the use of DDT Is somevfaat better than that reported by Gay. Gay found that DDT at 4 PPM gave a 50$ kill in ten days whereas in the pre­ sent tests DDT at the same dosage gives 78.8$ kill in the same exposure. But the results with regard to pyrenone and lindane are Inferior to those reported by Gay and Watts re­ spectively. Gay found that BHC at 0.2 PPM gave a 50$ kill in slightly less than three days, and Watts found that pyrenone mixed with 0 . 6 6 pyrethrins and 6 * 6 piperonyl butoxide gave 86$ kill in seven days. In the present tests lindane at 0.16 PPM gives a little over 50$ kill in two weeks and pyrenone mixed with 0.8 PPM pyrethrins and 8 PPM piperonyl butoxide af­ fords a 17.6$ kill in seven days. However, both Gay and Watts 46 used the rice weevil as the test insect while the author used the granary weevil* The rice weevil is known, to he more sensitive to insecticides than the granary weevil* Furthermore, Gay and Watts did not conduct the residual tests* Fumigation Effect of Lindane-Impregnated Dusts* Fig­ ure 9 shows the results of fumigation toxicity of lindane- impregnated dusts against the granary weevil. From this Figure the fumigation effect of lindane is obviously much inferior to its contact effect. At the beginning of the experiment the contact toxicity of lindane at 4 PPM produces 100$ kill in one day while its fumigation toxicity at a dosage of 20 PPM gives a 100$ kill in seven days* After three months, lindane at 0.8 and 4 PPM loses all of its fumigation power. This is evidenced by the occurrence of a large number of weevils in the bottle in which the test bee­ tles were exposed at the beginning of the experiment. On the contrary, its contact toxicity at 4 PPM after three months is still so effective as to give a 95.3$ kill In seven days* Repellence Effect of Impregnated Dusts. The total and average weight of feces after 1 0 0 weevils being exposed ten, days derived from five replications of each treatment with five insecticides at two or three concentrations and the differences between the average weight of feces from 47 each treatment and the average weight or feces from five replications of checks are given in Table 7. The results show that none of the Insecticides tested is strong enough, to starve the weevil when their dosages are not high enough to produce & significant kill by their contact toxicity. In addition, from the fact that all but three treatments which show high toxicity become infested after three months, it is evident that none of the Insecticides tested is good enough to repel oviposition by the weevil.

Table 7 Average Weight of Feces from Treated Wheat and Difference in Weight of Feces between Treated Wheat and Checks

BSCS Toxi­ Cone, in PPM Av. Feces Wt. of (Av. Feces Wt. of cants of Wheat Treated in gm. Treated) -(Av. Feces Wt. of ok)

Pyrn 0.0064 Pyr / 0.073 0 . 0 0 2 0.064PB 0.0032 Pyr / 0.072 0 . 0 0 1 0.032 PB 0.16 PPM 0.070 -0 . 0 0 1 Pyr '"O.r'FFM ... " ' " 0.067 -0.0o4 4 *>PM 0.062 -0.009 0.8 PPM 0.076' -o.ool PB 4 PPM 0.071 0 . 0 0 0 0.0684 PPM 0.074 0 .003 Lnd 0.632 PPM 0.078 0.(507 6.18 pM T ” “ 0.676 ^ 6 . 6 0 6 DDT 0.80 PPM 0.072 0 .001 CK 80# Pyrophyllite / 20% Alio clay SF 0,071 48 Impregnated Bag Method Contact Effect of the Impregnated Bags• Table 9 gives the number of the dead and the living outside of the bags which were treated with the five toxicants at three concen­ trations when fifty weevils were exposed in each jar con­ taining the three bags for two and a half months# This Table indicates that only lindane and DDT at practical con­ centrations afford satisfactory protection by their contact toxicity# In the bags treated with these two toxicants on­ ly 50 dead weevils are recorded. This implies that all the 50 test weevils are killed and that the immature stages of weevils are killed which are developed from eggs laid by the 50 weevils before they are killed or they are repelled to lay eggs before they die. Although 50 dead weevils are recorded from first level of concentration of pyrethrins and piperonyl butoxide* their concentrations seem too high to use practically. As to the remaining treatments, their toxicity Is not strong enough to kill all the test weevils and yet does not prevent them from reproducing normally. The number of beetles inside of the bags were also de­ termined# But in only a few cases a beetle was found in some bags, including checks and untreated controls# This perhaps indicates that the fabric for the bag is very re- sis tent to penetration by the weevil. The untreated bags made of the fabric, called 37rt - 4 - 48/48 unbleached sheet­ ing, therefore, afford the protection of grain against the 49

* 6 6 7 1 1 for at least two and a half months. Fumigation |3ffect of Lindane-Impregnated Bags upon the Adult Granary Weevil. Figure 7 shows the six days compara­ tive fumigation effect of the lindane-impregnated hags in closed fumigation jars and the open fumigation jars and also shows the six days residual fumigation test of the same two series of bags in comparison with the initial test. In either test the closed series gives higher kill. Like­ wise, In either series the fumigation effect drops consider­ ably after three months. At the beginning 50# kill is ob­ tained from the lowest concentration, namely; 0.5 mg/sq ft In either series. After three months 50# kill results from the highest concentration or 4.5 mg/sq ft in either series. The number of the dead and the living beetles produced dur­ ing the period of the initial and the residual exposure are given in Table 9. From the table the following facts are shown. First, It is revealed by the presence of dead bee­ tles in the bags treated with the highest dosage that none of the dosages applied Is high enough to prevent the weevil from laying eggs and to kill the Immature weevils. Secondly, the number of the dead and the living in closed fumigation jars is smaller than that in open fumigation jars in all cases. The number of the living beetles In checks of open jars Is over 65 times as large as that in the checks of closed jars. Associated with the occurrence of overwhelming­ ly large number of beetles in the open jar, wheat becomes Table 8 Contact Effect of Toxicants Impregnated on Bags after 2% Months

Toxicant Pyrn Pyr PB In d DDT Ckl Ck2 Unt

Cone, in mg per sq ft 320 1600 3200 80 400 800 320 1600 3200 16 80 160 32 160 320 Deo- Triton base X-100 Alive 535 23 6 1109 294 0 796 12 0 0 0 0 2 0 0 1179 580 1668 Dead 480 480 343 127 262 50 29 43 50 50 50 50 182 50 50 6 27 9

seriously infested, a great amount of humidity is produced, bags are badly destroyed where damp wheat occurs and bettles penetrate out of bags from sound places* On the contrary, none of the conditions mentioned above is observed in the closed jar. The difference in results of the two series of tests is undoubtedly significant. As the only known dif­ ference betteehsthem id aiy0on oxygenssupply it is most probable the differences could be due to oxygen deficiency. In a different experiment, the result of which will be

Of o 51 Table 9 Comparative Effect of Lindane Impregnated Bags In Closed jars (G) and in Open Jars (0) Showing by the Number of Adults Developed after 100 Weevils Being Exposed Six Days and Collected 50 Days after the Removal of 100 Beetles

Total Average Cone* Alive Dead Alive Dead mg/sq ft 0 C 0 C 0 C 0 C 0.9 2 1 6 378 145 7 2 126 .0 48.3 4.5 0 0 183 85 0 0 61.0 28.3 22.5 0 0 74 54 0 0 24.8 18.0 CK 12971 297 189 291 4327 99 63.0 97.0

presented later, it is found that the imnature weevils of three to four weeks of age in the grains of wheat do not complete their development in both checks and the bags im­ pregnated with lindane at three concentrations after these bags were enclosed in closed jars for four weeks. To con­ firm the fact, infestation of samples of wheat from the sealed jars has been determined by dissecting grains of the sample beeiring the oviposition plugs which was detected by staining with acid fuchain solution. It was found that about 1 0 % of grains of the samples was Infested with larvae* In addition, judging from the size these larvae were later instars. In general, oxygen assumption follows an u-shaped curve in developmental stages of insects. Hence, it is most probable that those larvae do not complete their development in closed jars die of oxygen deficiency. This experiment shows that later lnstars of the weevil are probably subject 52 to suffocation. This phenomenon deserves attention because it is concerned with the control of stored grain by air­ tight chambers, which has been one of the good primitive methods for stored grain insect control for many years in various countries* If the fact that late instars b£ the - weevil are subject to oxygen deficiency is true, it might be the evidence that air-tight containers affords an effec­ tive method for stored' grain control* Nasir found that satisfactory protection of wheat for six months was obtained by applying 1*7 gm BHC to half­ size standard gunny bags* If the standard bag is supposed to be 41 in* X 23 in* as adopted in the United States and if the BHC is supposed to be 12$, the dosage used by Nasir would be near 19 mg/sq ft. This concentration Is apparently much higher than the highest concentration applied in the present tests* Fumigation Effect of Lindane-Impregnated Bags upon the Immature Stages of the Granary Weevil at the Age of 1-4 Weeks* The results of this experiment ere given in Table 10. In this table the following facts are shown* First, lindane- impregnated bags at the concentration of 0.9 PPM and 4.5 PPM do not give satisfactory protection of grain for 50 days* This is indicated by the presence of a large number of adults in the bags treated by lindane during this period* Secondly, the significant number of adults emerged from one-week im— 53

mature weevils Indicates that one-week immature weevils are very resistant to lindane vapor at all its concentrations. Thirdly, a significant number of adults emerged from two- week checks but not from all of the treated samples. This fact suggests that two-week immature weevils start to be­ come susceptible to lindane vapor at the concentration ap­ plied. Fourthly, a negligible number of adults emerged from some of the samples of wheat treated in third or fourth week. This probably indicates that untreated bags in air­ tight jars alone can cause the immature weevils* death. The reason for this has been mentioned above.

Table 10 Showing the Results of Four-Week Fumigation by Lindane Im­ pregnated on Bags against Immature Granary Weevils of 1-4 Weeks

Total Average Cone• 1 WK 2 WK 3WK 4WK 1 WK 2 WK 3 WK 4 WK mg/sq ft A DAD A DAD A D A D A D A D 0.9 1124 0 2 1 0 0 12 6 374.6 0 0.6 0.3 0 0 4 2 4.5 489 0 0 0 0 0 0 0 163.0 0 0.0 0.0 0 0 0 0 22.5 2 114 3 3 3 12 0 0 0.6 38 1.0 1.0 1 4 0 0 Ck. 675 0 315 1 0 0 0 0 225.0 0 105 0.3 0 0 0 0 STATISTIC ANALYSIS

An attempt was made to interpret the data by statisti­ cal analysis for experiments 3 and 4 the result of which will not be clearly shown by such analysis. The results of statistical study concerning these two experiments are given as follows: The experiment 3 is concerned with the comparative effectiveness of lindane vapor between the open fumigation jar and closed fumigation jar in both initial and residual tests. The statistical study was made to ascertain whether the numerical value shown in Fig. 8 is significant. The U statistic developed by H. B. Mann and D. R. Whit­ ney is used to test the hypothesis that the percentage mor­ tality obtained an open fumigation jar Is equivalent to that by using a closed fumigation jar. The alternative , is that the closed jar will give a higher percentage mortality than the open jar. The following tables give the calculated values of the U obtained in the two tests and under each of the three concentrations•

The;:author wishes to express his indebtedness to Sta­ tistics laboratory, Ohio State University for this part of work.

54 55

Initial Test Concentration TJ p(u L U ) 0*5 0* .050

1*5 2 .200 4.5 0* .050

Residual Test Concentration TJ p(U^U) 0.5 1.5 .150 1.5 1 .100 4.5 1 .100

Where denotes significance at the 5% level. Thus we can reject the hypothesis and conclude that the percen­ tage mortality obtained by using closed jars is higher than the percentage mortality by using open jars only in the Initial test at concentration 0.5 and 4.5 mg/sq ft.

Experiment 3 was made to determine whether odor of in­ secticides coated on wheat is strong enough to prevent the insect from feeding or not by measuring the weight of feces accumulated during the exposre period. The results given in Table 7 show that pyrethrins act probably as a repellent. In this experiment one way analysis of variance is used to test the hypothesis that no difference exists in repellency between pyrethrins and checks. 56

The following table gives the calculated F value.

Analysis of Variance Source Sums of Squares df Mean square P P .05 F .01 Between 0.01830 3 0.0031 6.138 3.24 5.29 Means Within 0.008081 16 0.00505 Groups

The P value indicates that the hypothesis is accepted Thus it can be concluded that no difference exists in re- pellence between pyrethrins and checks. CONCLUSION

The present study contributes the following conclusive results. When these insecticides are used in the form of impregnated dusts the following results are obtained. Used as a contact toxicant, pyrenone at a dosage of 4 PPM pyre­ thrins and 40 PPM piperonyl butoxide and lindane at 0.8 PPM afford complete protection of wheat against the granary weevil for at least six months. As a fumigant, lindane at 20 PPM gives satisfactory protection of wheat for at least three months. None of the insecticides at the concentrations which give no significant mortalities show either distinct feeding repellency or oviposition repellency. Pyrethrins, however, seem to show a little feeding repellency. When these insecticides are applied to cloth bags their effects are shown as follows? DDT at 32 mg/sq ft and lin­ dane at 16 mg/sq ft and pyrethrins at 3.2 gm/sq ft give satisfactory protection of grain against the adult granary weevil for two and a half months. Lindane vapor at 0.5 mg/sq ft give 50# kill in six days at the beginning of the experiment, but 4.5 mg/sq ft Is required to give a 50# kill of adult weevils after three months. In addition, lindane vapor at 4.5 mg/sq ft affords satisfactory protection of grain against the adult stage of the weevil for three months.

57 58

However, it does not completely control Immature stages of the weevil when applied at 22,5 mg/sq ft to Infested grain for four weeks although the later instars of the beetle are susceptible to it* In addition, It was found that cloth bags made of 37” - 4 - 48/48 unbleached sheeting alone prevent penetration by the weevil for two and a half months. Furthermore, it was found that the late-Instar larvae of the weevil are suscep­ tible to oxygen deficiency. This might be the answer why air-tight containers serve as an effective method of stored grain insect control* SUMMARY

The present study started with the detailed survey of* literature. This survey supplied extensive up-to-date suggestive information on this study. Following the sur­ vey, the work on standard supply of cultures of the test in­ sect was developed. This requirement proved important in supplying consistent data especially when comparative tox­ icity between initial tests and residual tests is to be made. As soon as the cultures were adequate suitable formu­ lations of the toxicants tested were obtained by extensive tests. The chief part of the study included tests of ef­ ficient uses of the five insecticides in the form of im­ pregnated dusts mixed with wheat and in the form of emul­ sion impregnated on cloth bags. First of all, the results of the tests show that the most efficient use of these toxi­ cants is their application in the form of impregnated dusts. Pyrenone at a dosage of 4 PPM pyrethrins and 40 PPM pipe- ronyl butoxlde and lindane at 0.8 PPM were found to give complete protection of wheat against the granary weevil for at least six months. When applied as a fumigant, lindane at 22.5 mg/sq ft is not good enough to kill the early instars of the weevils, but it affords satisfactory protection against the adults for at least three months when applied to bags at

5$ 60 a dosage of 4,5 mg/sq ft. None of the insecticides under testing shows distinct feeding repellency or oviposition repellency for the granary weevil. The main findings for this study include the compara­ tive toxicity and stability of pyrenone and lindane for a period of six months, the evaluation of fumigation effect of lindane against adult and immature stages of the granary weevil and the evaluation of the feeding repellence and oviposition repellence of the selected toxicents. Pig* 1* Thermoregulator The thermostat mounted, in a wire cylinder*

✓ 65

Fig. 2 A Diagrammatic Review of Thermostat 64

ADJUSTABLE CON­ TACT ROD KNURLED CLAMPING TO RELAY NUT BINDING POSTS

FIXED CONTACT ROD

INSULATING BEAD

AIR BUBBLE .PROTECTIVE SENSITIVE LIQUID LIQUID MERCURY

G'T V Pig* 3 Pro-oiled 9-inch Fein

157

Pig. 4. A Working Set for Separating Weevils from Wheat

Pig. 5 A Portable Ball Mill, One or Two Jars on Each Rolling Unit 70

P i g S

\ \

RUBBER STOPPER RUBBER STOPPER

BLOWER PICK-UP TUBE SUCTION VENTURI TrSHAPED GLASS TUBE

RUBBER STOPPER

CHEESE CLOTH ADAPTOR

VERTICAL DIAGRAMMATIC SEC­ TION THROUGH SLOWER SUCTION UNIT (AFTER A. PETERSON)

£■'3- b -.73

Fig* 7* A Graph Showing the Comparative Toxicity of Pyrenone, Lindane and DDT ■ ■ ■;..■■■

74 PERCENTAGE MORTALITY

!■! ■#'

h3 *V h). *rJ W W

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~ M H CJ1 M » 0

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* r L Fig* 8* A Graph Showing the Comparative Toxicity and Stability of Pyrenone and Lindane 100 ■O—O Q

90

80

70

U 40 o’A

20

10

0

TIMS IN DAYS ^ r * 77

Pig. 9. A Graph Showing the Fumigation Effect of Lindane, When Testing in Glass Bottles by the TJse of Impregnated Dust 78

100

90

Q0

70

M& 60 £ O 5-3 M CO 00 CH< a C ‘5 !-i a,

30 Closed, Initial Open, Initial 20 Closed, Residual

10 Open, Residual

0 0.5 1.6 4 .5

003AGE II MG/sq FT

■ . . ■ ■ • ■ . . ■ ■ ; ■ " ' - y-i-.V -.ivc- I^.h. ■ . . , -i” Pig, 10* A Graph Showing the Fumigation Effect of Lindane upon the Adult of the Granary Weevil when Lindane Impregnated on Cloth Bags PERCENTAGE MORTALITY 100 70 50 60 30 40 90 20 80 10 0 Pis- Pis- o i INDAYS 10 4 PPM 4 20 PPM20 12 80 14 LIST OF ABBREVIATIONS

A - Alive BHC = Benzene Hexachlorocylohexane D s Dead DDT = DichlorodiphenyltrIchlorethane Lnd - Lindane Pyr - Pyre thr ins Pyrn - Pyrenone Ok s Check TJnt * Untreated LD5o s Lethal Dose PPM = Part per million LITERATURE CITED

1. Cotton, R. T., and J. C. Frankenfeld. 1945. Dust treatment for protecting stored seed. Seed World. Nov. 2, 1945. 2 . Cotton, R. T. 1946. Insect pests of stored seeds and their control. Seed Trade Buyer Guide. 1946. 3. Hammer, A. L. 1947. Dust for protecting seed corn from Insects. Jour. Econ. Ent. 404 574-75. 4. Farrar, M. D., and J. M. Wright. 1946. Admixture of DDT with crop seeds. Jour. Econ. Ent. 39:520-522. 5. Kulash, W. M. 1948. Control of the rice weevil and Angumois grain moth. Jour. Econ. Ent. 41:715-18. 6 . Zinkernagel, et* al. R. 1946. DDT to protect wheat from insects. Mittel Schweiz. Ent. Gesell 19:653-691. 7. Gay, F. J. 1947. The use of DDT and 666-impregnated dusts for the control of grain pests. Aust. Coun. Soc. and Ind. Res. Bull. 225:33-38. 8 . Geigy Company, Inc. 1951. Personal communication. 9. Mayne, R. 1948. The specific conditions found in farm storage of grain in Belgium and means used in reduc­ ing storage losses from insects and rodents. EAO Agricultural Studies No. 2. Preservation of grains in Storages, 110-114. 1 0 . Slade, R. 1945. Gamma-Isomer of Hexachlorocychlohexane. Soc. Chem. Ind. Jour. Ghem. and Ind. 40:314-319. 11. Imperial Chemical Industries Ltd. Technical Service De­ partment Report No. GCS/PC/32. 12. Pirie, H. 1951. Insecticides in industry and public health. Feb. 10, 1951. pp. 100—104. 13. HensIII, G. S. 1951. Lindane. Soap. Sept. 1951. p. 135. 14. Parkin, E. A. 1950. Control of stored product insects with contact insecticides. 8th Int. Cong. Ent. pp. 834-837. 82 83

15. Beckley, V. S. 1948. Protection of grain against weevils. Jour. East Africa Agric. 14(2)*71-76. Biol. Abst. 1951. Bo. 5134. p. 485. 16. Watts, 0. H. and P. B. Berlin. 1950. Piperonyl butox- ide and pyrethrins to control rice weevils. Jour. Econ. Ent. 43(3)*371-373. 17. U.S. Industrial Chemicals, Inc. Proven method for pre­ venting insect infestation of grain in storage. 18. Johns, B. M. 1947. An experiment with DDT against pests of stored products. Bull. Ent. Res. 38(2)8 347-352. 19. Nasir, M. M. 1947. Eradication of insect pests of stored grains. Curr. Sci. 15(4):98-99. 2 0 .Parker, W. B. 1913. A sealed paper carton to protect cereals from insect attack. TJ.S.D.A. Bull. 15. 2 1 . Stracener, C. B. 1938. Control of insect pests of milled rice by improved storage bags. Jour. Econ. Ent. 3(6)*687-8. 22.Essig, E. 0. et al. 1943. Impregnation for packaging materials. Jour. Econ. Ent. 36:822-29. 23. Linslay, E. G. 1944. DNOC as package Impregnant. Jour• Econ. Ent. 37*377-79. 24. Sweetman, H. B. and A. B. Baurne. 1944. The protec­ tive value of asphalt laminated paper against cer­ tain insects. Jour. Econ. Ent. 37(5)*605-9. 25. Frings, H. 1948. Ammonium salt as package impregnant. Jour. Econ. Ent. 41*413-16. 26. Cotton, R. T., et al. 1945. Tests of DDT against the insect pests of stored seed, grain and milled cereal products. TJ.S.D.A., B.E.P.Q, E-641. 27. Parkin, E. A. 1948. DDT impregnation of sacks for the protection of stored cereals against insect infes­ tation. Ann. Appl. Biol. 35(2)*233-242. 28. Butterfield, D. E., Parkin, E. A. and M. M. Gale. 1949. Transfer of DDT to food from sacking. Jour. Soc. Chem. Inc. 68*310-313. J 8 4 29. Smallman, B. N. 1949. Chemical control of insects. Chemistry in Canada. November, 1949. 30. Cotton, R. T. and J. C. Frankenfeld. 1947. Insect- proofing cotton bags. TJ.S.D.A., B.E.P.Q. E-783. 31. Pyenson, L. and Mennson, H. Jr. 1937. In culture meth­ ods for invertebrate animals by Needham et al. pp• 478-480• 32. Sheppard, H. H. 1943. Rearing insects that attack stored products. In Publications of the Anerican Association for the Advancement of Science. No. 20. pp. 36— 39. 33. Needham, J., et al. 1937. Live culture of grain wee­ vils. In their MCulture methods for invertebrate animals.M pp. 481-3. Comstock Publishing Co., Inc. Ithaca, New York. 34. Gay, H. E. 1943. Stored food insect. In A.A.A.S. No. 20. pp* 54—56. 35. Sun, Y. P. 1947. Methods of rearing insects for fumi­ gation experiments. In his "An analysis of some important factors affecting the results of fumi­ gation tests of insects.n Tech. Bull. 177. Univ. Minn. Agric. Eaqpt. Sta. pp. 6-9. 36. Description sheet of the thermoregulator, manufactured by Eastern Engineering Company. 37. Woodbury, E. N. and Barnhardt, C. S. Soap. September, 1939.

38. Peterson, Ja . A manual of entomological equipment and methods. PI. 160. Edwards Brothers, Inc., Ann Arbor, Mich. 39. Frear, D.E.H. 1948. Chemistry of insecticides, fungi­ cides and herbicides, p. 62. D. Van Nostrand Co., Inc., New York. 40. Brown, A. W. A. 1951. Insect Control by Chemicals. p. 101, 40. John Wiley and Sons, Inc., New York.

\ 41. Dove, W. E. 1951. Personal communication. 42. Wacks, H. 1947. Synergistic insecticides. Science. Vol. 105. No. 2733. pp. 530-531. -V85 43. Dove, W. E. 1947. Piperonyl butoxide, a new and safe insecticide for the household and field* Amer. Jour. Trop • Med. 27(3)s339• 44. Walkins, T. C., and L. B. Norton. 1947. A classifica­ tion of insecticides, dust diluents and carriers* Jour. Econ. Ent. 40(2)8 211-214. 45. Attapulgus Olay Co. Description sheets of Attaclay SP. 46. Chisholm, R. D. 1948. A review of DDT formulatiojn TJ.S.D.A., B.E.P.Q. ET-712. p. 10. 47. Cotton, R. T. 1921. Four Rhynchophara attacking corn in storage. Jour. Agr. Res. 20:605-614. 48. Metcalf, C. L. and W. P. Flint. 1928. Destructive and useful insects. McGraw Hill Book Co., Inc. New York. AUTOBIOGRAPHY

The author was born In Pingyoasien, Shansi Province,

China, June 10, 1911. He moved to Tatungsien, Shansi with his parents in 1914 and attended schools there up to the junior high school. He had senior high school education for one year in Taiyuan, the capital of Shansi, and com­ pleted it in Peking. In 1935 he received B.S. Degree in biology at National Mornial University, Peking. During 1935-

41 he was assistant in zoology and entomology and was a teach­ er of biological sciences In colleges and high schools. Dur­ ing 1941-46 he worked as a research assistant on the biology and control of several insects in Entomology Division at

Tsing Hua University, Chenkungsien, Yunnan Province. During

1946-48 he was an instructor at the same university. In 1948 he was awarded a scholarship by Tsing Hua University for the advanced study in the United States. In the same year he came to the United States and made his graduate study at the Ohio

State University, Columbus, Ohio. In 1949 he received M.S. Degree at the same university and was granted the university i scholarship for higher degree study. From the summer of 1949 to the winter quarter of 1952 he enrolled at the Ohio State University as a candidate for a Ph. D. Degree in entomology.

86