j. ¿9

CHEMICALS EVALUATED AS

INSECTICIDES AND REPELLENTS

AT ORLANDO, FLA.

Compiled by W. V. King [• AUG 11 195?' *j

LZjPVtLLE BfigS

/

U. S. DEPARTMENT OF AGRICULTURE

Agricultural Research Service Agriculture Handbook No. 69
Acknowledgments

Most of the staff at the Orlando laboratory have taken

part in these studies. During the war years the work was

under the general direction of E. F. Knipling, director of

the laboratory, L. C. McAlister, Jr., assistant, and H. A,

Jones, adviser in chennistry. After the war to the end of

the period covered in this report, the laboratory was under

the direction of W. V. King, with B. V. Travis and W. C.

McDuffie as assistants and F. H. Babers and C. V. Bowen

as advisers in chemistry. The following persons were in

charge at one time or another of the different projects under

which the tests were conducted:

Louse control: G. W. Eddy, R. C. Bushland, C. N.

Smith.

Control of chiggers: F. M. Snyder, F. A. Morton,

C. N. Smith.

Mosquito larvicides: C. C. Deonier, J. C. Keller.

Insecticides for control of house flies, mosquitoes,

and cockroaches: A. W. Lindquist, A, H. Madden,

J. B. Gahan, I. H. Gilbert.

Repellents: B. V. Travis, F. A. Morton, C. N.

Smith.

J. A. Fluno, Helen Rodriguez-Torrent, and Anna C.

Maquire assisted in the preparation of this report.

- >i
CHEMICALS EVALUATED AS INSECTICIDES

AND REPELLENTS AT ORLANDO, FLA.

Compiled by W. V. King

Entomology Research Branch

Agricultural Research Service

U. S. DEPARTMENT OF AGRICULTURE

Agriculture Handbook No. 69

Issued May 1954

For sale by the Superintendent of Documents, U. S. Government Printing Office

Washington 25, D. C. - Price ,S2.25
Contents

Page

Source of compounds tested ••... 1

Classification of chemicals 2

Test insects 3

Evaluation of materials against the body louse 3

Adulticides 4

Ovicides 5

Knock-down 5

Mosquito larvicides 6

Flies and mosquitoes 7

Space sprays 7

Residual sprays 8

Cockroach toxicants 9

Flea toxicants 9

Tick toxicants 11

Chigger toxicants • H

Mosquito repellents 13

Tests on skin 13

Tests on cloth 14

Rinsing and washing tests 15

Wearing tests 15

Flea repellents 16

Tick repellents 17

Synergists 18

Pyrethrum • 18

Pyrethrum-DDT 18

Summary of results of screening tests 19

Explanation of abbreviations used in table 1 21

Table 1.--Relative effectiveness of chemicals tested as insecticides and repellents 22

Supplement to table 1 340

Table 2.--Larvicide tests against southern house and yellowfever mosquitoes 345

Table 3.--Tests of space and residual sprays against house flies

and yellow-fever mosquitoes 346

Table 4.--Tests against cockroaches 360

Table 5.--Additional tests of toxicants against fleas •.. •. 365

Table 6.--Additional tests of toxicants against ticks 373

Table 7.--Additional mosquito-repellent tests 375

Table 8.--Tests of repellents against fleas and ticks 386

Table 9.--Tests of pyrethrum synergists against body lice 395
CHEMICALS EVALUATED AS INSECTICIDES AND REPELLENTS

AT ORLANDO, FLA.

Compiled by W. V. Kingl/

INVESTIGATIONS to develop control measures for insects and arachnids of medical importance to the Armed Forces were started at the

Orlando, Fla., laboratory of the Bureau of Entomology and Plant Quarantine in April 1942. Many compounds were tested for insecticidal and

repellent action by simple laboratory procedures designed to eliminate

ineffective chemicals from further consideration and to provide leads

for more detailed studies of the promising ones. At first emphasis was

placed on the control of body lice, mosquitoes, and chiggers. Later

house flies, cockroaches, ticks, and fleas were included.

The results obtained with about 7,000 compounds against the first

group were summarized in 1947 in Bureau publication E-733. These

compounds were received before November 1945. Since then about

5,400 compounds have been received for testing, and more data have

been obtained on the original 7,000. Several thousand additional samples,

consisting chiefly of formulations of insecticides and repellents, were received for testing or prepared in the laboratory. The present report includes the results with about 11,000 compounds received prior to July 1952.

It does not include compounds of which the exact chemical composition

was unknown or held in confidence at the request of the submitting agencies.

The main table (table 1) gives the results of 14 tests against 7 species.

At least 2,000 compounds were evaluated by each test. Shorter tables

(tables 2-9) give the results with smaller numbers of compounds against

other species or in other types of tests. Altogether there were 45 different kinds of tests against 15 species representing 7 families.

Some of the compounds were not subjected to certain types of tests

because they were not soluble enough or were not available in sufficient

quantity, and some were not tested as repellents because they had an

obnoxious odor or were found to be toxic or irritating to the skin.

It is believed that this handbook will be of value to entomologists,

chemists, and others interested in the development of new insecticides

or in the relation of chemical structure to insecticidal activity.

Source of Compounds Tested

The compounds were obtained from three general sources--(1) the

Division of Insecticide Investigations of the Bureau of Entomology and

Plant Quarantine, (2) other Government agencies and university groups

operating under contracts with the Office of Scientific Research and

Development (OSRD), and (3) commercial sources.

^l Formerly entomologist. Bureau of Entomology and Plant Quarantine. Retired July 1953.

(1)
Most of the compounds from the Division of Insecticide Investigations

were prepared especially for tests as insecticides in connection with its

regular program, but a number of them were prepared for tests as

repellents under the OSRD program.

Beginning about June 1944, several universities, under the OSRD

program, prepared compounds especially for tests as repellents or

submitted materials available from their stock shelves. The Orlando

code numbers assigned to materials from the university groups are as

follows:

Columbia University 4900-4999

Harvard University 5500-5999 and 12000-12225

University of Illinois 11000-11320

University of Maryland 6000-6631

University of Minnesota 10000-10065

Ohio State University 7000-7499 and 13000-13242

Stanford University 10500-10594

University of Wisconsin 11500-11803

Classification of Chemicals

To present the results of the numerous tests in a condensed form

the chemicals have been divided into four classes according to their

relative effectiveness for the purpose indicated. The least effective

materials are placed in class 1 and the most effective ones in class 4,

The best of the class 4 compounds are designated as 4A. The definitions

of the classes are given in the discussion of the screening procedures

with the different kinds of insects.

The class 4 compounds and some in class 3 were subjected to further

tests designed to select materials showing promise for practical use and

to determine their limitations. Materials given low ratings should not

be entirely discounted, as they might be of use against other species or

in other preparations. One of the striking features of the results is the

variation in effectiveness shown by many of the compounds against

different species of insects.

In Bureau publication E-733 the compounds were designated by the

names under which they were submitted, provided they were correct

and as descriptive as possible. In this handbook the Chemical Abstracts

system of nomenclature is employed, and the compounds are listed in

alphabetical order under these names. A few officially approved common

names or trade names are listed with a cross reference to the chemical

names.
3

Test Insects

The common and scientific names of the species used in the screening

tests or mentioned in the text are as follows:

Body louse Pediculus humanus humanus L.

Head louse Pediculus humanus capitis Peg.

Crab louse Phthirus pubis (L,)

Common malaria mosquito -- Anopheles quadrimaculatus Say

Yellow-fever mosquito Aedes aegypti (L,)

Southern house mosquito Culex quinquefasciatus Say

Salt-marsh mosquitoes Chiefly Aedes taeniorhynchus (Wied>)

and sollicitans (Wlkr.)

House fly Musca domestica L.

American cockroach Periplaneta americana (L.)

Australian cockroach Periplaneta australasiae (F.)

German cockroach Blattella germánica (L.)

Cat flea Ctenocephalides felis (Bouché)

Oriental rat flea Xenopsylla cheopis (Rothsch.)

Lone star tick Amblyomma americanum (L.)

Chiggers . Trombicula spp., chiefly splendens

Ewing and alfreddugesi (Oud.)

Evaluation of Materials Against the Body Louse

(Table 1)

A colony of body lice has been maintained in the laboratory since

1942. The lice were obtained from naturally infested persons and at

first were fed solely on humans. Later a strain adapted to feeding on

domestic rabbits was developed, and the colony has been maintained

on these animals since 1946. No essential difference in susceptibility

to insecticides was found between the lice reared on the two kinds of

host.

DDT was among the first materials tested against this louse. Although

it has proved highly effective, it acts rather slowly and is not ovicidal.

Since a faster killing agent was desirable and an ovicide was required

for certain preparations, tests were continued with all the chemicals

received to determine their contact toxicity to adult lice, their speed of

action, and their ovicidal value. The information thus accumulated,

particularly with respect to preparations that could be substituted for

DDT, proved of inestimable value when DDT-resistant lice were encountered in Korea in 1951.

The body louse proved to be an excellent general test insect, as it

is easily handled and the rabbit strain is conveniently and economically

maintained in any numbers desired. This louse is also a good test
species for the study of pyrethrum synergists, and screening tests for

this purpose were conducted with selected compounds. The results are

discussed in another section of this report (see page 18).

Adulticides

To determine the initial kill and lasting effect of the different compounds against the body louse, circular woolen cloth pads 1 l/2 inches in

diameter were dipped in 1-percent acetone solutions of the test materials.

The pieces of cloth absorbed an amount of liquid almost equal to their

dry weight, giving a concentration of insecticide about the same as in

the solution. The compounds not soluble in acetone were dissolved in

chloroformj, benzene, ether, alcohol, water, or some other suitable

volatile solvent. The innpregnated pads were suspended on pins and

allowed to dry at room, temperature for 1 to 2 hours. Each pad with

10 young adult lice was then placed in a 50-ml. beaker and held at

86^ F. and a relative humidity of 75 to 80 percent for 24 hours. If a

material gave complete kill or knock-down within this period, new lice

were placed in the beakers daily until one or more of the lice survived.

Slightly affected lice, if still able to crawl, were included as survivors.

The testing usually was continued after one or two survivors were

recorded, in order that no promising materials might be passed over

inadvertently. For rating the screening results, however, the occurrence of one survivor was the criterion.

This method of testing, known as the beaker method, proved to be

an efficient and rapid technique for making preliminary evaluations of

previously untried compounds. Since materials that had been shown to

be toxic to lice by other procedures also proved toxic by this method,

it appeared to be a reliable screening-test method. The standard

adopted for the best group (class 4) was low. For example, DDT and

certain other m.aterials when tested by this technique remained effective for several months, whereas the minimum standard for class 4 was

10 days. This low standard was adopted so that an effective and possibly

practical insecticide would not be overlooked. Even so only 5.5 percent

of the test compounds rated class 4.\

The effectiveness against adults was classed as follows:

1. Ineffective (one or more lice survived initial exposure

to freshly treated pad).

2. Effective (no survivors) on the initial exposure but ineffective on the second exposure started 1 day after treatment.

3. Effective on the second exposure but failed on or before

the 10th day.

4. Effective for 10 days or more.

4A. Effective for 31 days or more.
Ovicides

At the beginning of the louse-control investigations there was no

preparation available that would remain completely effective throughout

the incubation period of the eggs. The use of a long-lasting material

such as DDT all but did away with the need of a body louse ovicide.

However, the research on ovicides was continued until January 1951

since an efficient ovicide was needed for the control of head and crab

lice.

For evaluating the compounds as ovicides, cloth pads approximately

1/2 inch square, bearing 100 to 200 eggs 4 to 6 days old, were dipped in

alcoholic solutions of the test compounds, allowed to dry, and then placed

in an incubator at 86 F. and a relative humidity of approximately 80 percent. The compounds not soluble in alcohol were dissolved in some other

nonovicidal solvent, such as acetone, chloroform, benzene, ether, or

water. The percent kill was determined after hatching was completed

in an untreated lot of eggs. Usually about 96 to 100 percent of the untreated eggs hatched.

Routijiely all the materials were tested at 5 percent, and those

giving complete kill of eggs at this concentration were also tested at

1 percent. When the compounds were not sufficiently soluble or only

a minute amount was available for testing, it was necessary to use

lower concentrations. A few of them, which gave kills of 90 percent

or more at 1 percent, were placed in class 3. About 50 others were

omitted as being unclassifiable in the system used. A few compounds

that gave 100 percent kill at 5-percent concentration were not tested

at 1 percent. They rated class 3-4.

The ovicide classes were as follows:

1. 0 to 50 percent kill of eggs treated with a 5-percent solution.

2. 51 to 99 percent kill with a 5-percent solution.

3. 100 percent kill with a 5-percent and up to 99 percent with

a 1-percent solution.

4. 100 percent kill at 1 percent or lower.

4A. 100 percent kill at 0.25 percent.

Knock-Down

Since it was important to find an insecticide that was fast-acting

against the body louse and other medically important insects, observations were usually made on the knock-down properties of the various

compounds by the beaker method (see page 4 ). Observations were

made 15 and 30 minutes and 1 and 3 hours after the lice had been

placed on the impregnated pads.
The knock-down classes are shown below.

1. Incomplete in 24 hours.

2. Complete in 24 hours but not in 3 hours.

3. Complete in 3 hours but not in 1 hour.

4. Complete within 1 hour.

4A. Complete within 15 minutes.

Mosquito Larvicides

(Tables 1 and 2)

A colony of common malaria mosquitoes has been maintained in the

laboratory since 1943. The original stock for this colony was obtained

from the Malaria Control Branch, Division of Health and Safety, Tennessee Valley Authority, Wilson Dam, Ala. The initial larvicide

screening tests were conducted on this species, but some of the compounds that reached class 4 and others that appeared to be of interest

were also tested against the yellow-fever m.osquito and the southern

house mosquito. Yellow-fever mosquito larvae were obtained from a

colony that has been maintained in the Orlando laboratory since about

1939, having been started from wild adults caught locally. Larvae of

the southern house mosquito were reared from egg rafts obtained by

exposing oviposition tubs containing polluted water on the laboratory

grounds. The relative susceptibility of the three species has shown

some variation, but as a rule the malaria mosquito larvae were the

most susceptible and those of the yellow-fever mosquito next.

For the tests reported here early fourth-instar larvae were transferred by means of a small bobbinet-cloth strainer from the rearing

pans to a porcelain pan or large glass crystallizing dish containing

distilled water. Larvae of the same age from several pans were mixed

together, and from this collection 20 or 25 larvae were taken at random

for each test.

A suspension of each compound was prepared from a stock solution

containing 1 part of the compound to 1,000 parts of acetone. Some of

the compounds insoluble in acetone were finely ground and mixed with

a small amount of wetting agent. In the later years if a compound was

not soluble in acetone or water, it usually was not tested as a larvicide.

For the initial test a suspension containing 10 p. p. m. of the compound was made by adding 2.5 ml. of the stock solution to 225 ml. of

water in a 400-ml. beaker. The larvae in 25 ml. of water were poured

into this suspension. The beakers were held in a constant-temperature

room at 70^-80^ F., and mortality readings were made at the end of 24

to 48 hours. Larvae unable to come to the surface after being forced

to submerge was classed as dead. An untreated lot included with each

set of tests usually showed no mortality in 48 hours. If mortality
exceeded 15 percent, the entire series was discarded and the tests were

repeated on a new lot of larvae. Compounds that caused less than 50

percent mortality at 10 p. p.m. were considered nontoxic and were dropped

from further consideration; those that gave a higher mortality were retested

at 1 and 0,1 p. p. m. and then at lower concentrations if indicated.

In suspensions of 10 p. p. m. difficulty was encountered in keeping a

few of the compounds dispersed. A note was made if a suspension flocculated and a precipitate was formed or, as was more frequently the case,

if the material floated to the surface. The materials that came to the

surface naturally gave higher mortalities than the dosage justified,

because of the high concentration to which the surface-inhabiting

anopheline larvae were subjected. Rarely did a suspension separate

visibly at the lower concentrations.

Larvae of the common malaria mosquito are excellent test insects

for evaluating the effectiveness of chemical compounds. They are susceptible to toxic materials, the criterion of death is easily established,

and they represent an important group of insects. The yellow-fever and

southern house mosquitoes can also be used satisfactorily for this purpose and are less expensive to rear, but they may show greater variation

in rate of development within a lot or in susceptibility from lot to lot.

Mosquito larvae also show effects from either stomach or contact

poisons.

Class 4 was set at 1 p. p. m. It contained many compounds that were

much less toxic than DDT, which has a lethal concentration of approximately 0.01 p. p.m. and was later selected as a standard of comparison.

Therefore class 4A indicates the compounds effective at 0.01 p. p.m.

The compounds were classified as follows according to larval

mortality:

1. Less than 50 percent at 10 p. p. m.

2. 50 to 94 percent at 10 p. p.m.

3. 95 to 100 percent at 10 p. p.m. but less than 50 percent

at 1 p. p. m.

4. 50 to 100 percent at 1 p. p. m.

4A, 95 to 100 percent at 0.01 p. p.m.

Flies and Mosquitoes

(Table 3)

Space Sprays

A colony of house flies that had been maintained in the laboratory

since 1943 and the laboratory colony of yellow-fever mosquitoes were

used in these tests.
8

The tests were conducted in 100-cubic-foot cabinets into which 2 ml.

of a 2-percent solution of the test compound in cyclohexanone was sprayed

with a DeVilbiss-type GS nozzle under a pressure of 12-14 pounds per

square inch. After a settling time of 1 minute, 50 flies were placed in

each of two screen cages 2 1/2 inches in diameter and 8 l/2 inches long.

These cages, each attached to the lower end of a pendulumlike arm,

were introduced through a hole in the top of the cabinet and swung back

and forth for 2 minutes. Immediately afterward two cages containing

the same number of mosquitoes were similarly exposed for 10 seconds.

After the exposure the insects were transferred to clean cages and held

in an air-conditioned room at 80^ F. and about 75-percent relative humidity. Knock-down was recorded after 30 minutes and mortality after

24 hours. The tests were replicated on another day. They were usually

rerun when mortality in control cages exceeded 10 to 15 percent, or

when a 1-percent DDT standard gave less than 85 percent kill. Compounds causing 50 percent or higher mortality at 2 percent were retested

at reduced concentration.

The compounds were classified as follows according to mortality:

1. 0 to 9 percent with a 2-percent solution.

2. 10 to 49 percent with a 2-percent solution.

3. 50 to 100 percent with a 2-percent but less than 50 percent

with a 1-percent solution.

4. 50 percent or more with a 1-percent solution.

Residual Sprays

In the early work plywood boxes having an inside surface area of

7/8 square foot were sprayed with an acetone solution of the tesl material

at the rate of 50 mg. of toxicant per square foot (4.4 ml. of a 1-percent

solution). From 50 to 100 house flies or yellow-fever mosquitoes of

both sexes were introduced 1 and 4 weeks after treatment and at intervals

of 4 weeks thereafter until the mortality dropped below 90 percent. Knockdown was recorded at 2 and 5 hours and mortality at the end of 24 hours

of continuous exposure.

Beginning in April 1947, cloth panels were used instead of plywood

boxes, and in January 1949 plywood panels were substituted for the cloth

panels. For the tests on cloth, pieces of cotton twill 6 inches square

were treated in duplicate with an acetone solution containing enough of

the compound to give a deposit of 100 mg. per square foot (2.5 ml. of a

1-percent solution made up to 8 ml. with acetone). About 50 insects

were exposed on each piece of cloth under a Petri dish for 24 hours.

For the tests on plywood panels, a piece of gum plywood 1 foot square

was sprayed with 10 ml. of a 1-percent acetone solution of the compound to give a deposit of 100 mg.,and the length of exposure was reduced to 2 hours. Only a few tests were run after this change was made.
The compounds were classified as follows according to mortality:

1. Less than 20 percent after 1 week,

2. 20 to 89 percent after 1 week.

3. 90 to 100 percent after 1 week but less than 100 percent after

4 weeks.

4. 100 percent after 4 weeks.

Cockroach Toxicants

(Table 4)

About 800 compounds were tested against adults of the American

cockroach. Most of the roaches were obtained from heavily infested

buildings, but a colony was also maintained in the laboratory. A few

tests were made with the Australian cockroach when the American

cockroach was not available. In a limited number of tests with dusts

and also in some with impregnated cloth panels German cockroaches

from a laboratory colony were used.

Two types of tests were made, dust and residue tests.

For the dust tests 0.25 gram of pyrophyllite containing 10 percent

of the compound was scattered over the bottom of a 6-inch battery jar,

and 5 or 10 adult roaches were introduced. Knock-down was recorded

after 3 and 5 hours and mortality after 24 and 48 hours. The results

obtained with compounds rated in class 3 or 4 were confirmed by additional tests.

For the residue tests three pieces of cotton cloth 6 inches square

were dipped in an acetone solution containing enough of the compound

to give a deposit of 200 mg. per square foot. The cloths were stapled

to plywood panels, and 5 male roaches, confined under Petri dishes,

were exposed on each for 2 hours and then transferred to clean jars!

Knock-down was recorded after 3 and 5 hours and mortality after 24

and 48 hours.

In both types of tests the compounds were classified as follows

according to the mortality after 48 hours:

1. Less than 10 percent.

2. 10 to 50 percent.

3. 51 to 90 percent.

4. 91 to 100 percent.

Flea Toxicants

(Tables 1 and 5)

A colony of cat fleas was maintained in the laboratory on dogs and

rabbits and a colony of oriental rat fleas on white rats for the testing of

compounds for possible use on clothing to prevent flea bites. These

tests were discontinued in January 1949, when it was decided that a

search for flea repellents offered greater promise.
10

Patches of cotton cloth 4 inches square were treated with 0.222 gram

of the test compound in acetone to give a deposit of about 2 grams per

square foot. The next day 6 cat fleas less than 2 days old were exposed

on the cloth for 60 minutes, and the time of knock-down of the third and

sixth fleas was recorded. If all 6 were down in 60 minutes, the tests

were repeated daily to the 14th day and every 2 weeks thereafter. Beginning in January 1948, the tests were made at weekly intervals for 4 weeks

and then at 2-week intervals.

Additional cloths were treated with class 3 and 4 compounds and

subjected to 15-minute rinsing tests in cold water. Those that were

still effective after two rinses were washed in hot, soapy water and then

rinsed for 5 minutes. The cloths were tested after each rinse or wash

until they failed to knock down the fleas.

Most of the class 3 and 4 compounds were also tested for toxicity to

rat fleas in comparison with cat fleas.

Known toxicants were also tested as dusts against cat and rat fleas

for possible use in flea control in buildings or in rat habitats. Quart

mason jars were prepared with a thin layer of dry earth on the bottona,

over which 200 mg. of dust (equivalent to 4 grams per square foot) was

sprinkled. The dusts used contained 0.5, 0.05, or 0.005 percent of the

toxicant on gypsum snow-white filler. Twenty fleas less than 24 hours

old were held in each jar for 24 hours, and the mortality was then recorded. The tests were replicated, usually on consecutive days, with

three lots of fleas, and effectiveness was compared with that of recrystallized technical DDT tested concurrently.

The classification of the compounds in the different kinds of tests

was as follows:

Insecticidal Effectiveness

1. Ineffective on initial test (fleas not all down in 60 minutes).

2. Effective for 1 to 5 days.

3. Effective for 6 to 10 days.

4. Effective for more than 10 days,

4A. Effective for more than 20 days.

Speed of Knock-Down

1. Not all down in 60 minutes,

2. All down in 41 to 60 minutes.

3. All down in 21 to 40 minutes.

4. All down in less than 20 minutes.

Rinsing and Washing Tests

1. Did not withstand 60 minutes of rinsing.

2. Withstood 60 minutes of rinsing or 15 minutes of washing,

3. Withstood 30 to 60 minutes of washing.

4. Withstood 90 minutes of washing.
11

Dust Tests (mortality)

1. 0 to 9 percent with 0.5-percent dust.

2. 10 to 49 percent with 0.5-percent dust.

3. 50 to 100 percent with ( .5-percent dust but less than 25

percent with 0.05-percent.

4. 25 percent or more with 0.05-percent dust.

Tick Toxicants

(Tables 1 and 6)

A colony of lone star ticks was maintained in the laboratory by

feeding the adults on dogs and the immature stages on rabbits. At

first this colony was used for testing both toxicants and repellents,

but the toxicant tests were discontinued in June 1949.

The 4-inch-square cloth patches as prepared for the flea tests

(2 grams of toxicant per square foot) were used also for the tests on

ticks. Six larvae from 30 to 90 days old were exposed on the patches

for 60 minutes, and the knock-down time of the third and sixth ticks

was recorded.

The classes were the same as for fleas. Aging and rinsing and

washing tests were also the same as for fleas, except that only class

4 compounds were included in washing tests.

Nymphal and adult ticks were tested on the class 4 compounds with

an exposure period of 180 minutes instead of 60. The classification on

speed of knock-down in these tests is shown below.

1. Not all down in 180 minutes.

2. All down in 121 to 180 minutes.

3. All down in 61 to 120 minutes.

4. All down in less than 60 minutes.

Chigger Toxicants

(Table 1)

In early studies at the Orlando laboratory it was found that dimethyl

phthalate applied to the outer clothing would prevent attachments of

chiggers for several weeks or until the garments were washed. To

protect troops in the field, however, it was desirable to find a material

that would resist exposure to water and laundering. A large number of

compounds were subjected to screening tests before this phase of the

work was discontinued in 1949.

At first the tests were conducted in the field in places having heavy

natural infestations of chiggers. Later a laboratory colony of Trombicula

splendens was established, and beginning in 1946 larvae from this colony

were used.
12

Small patches of cotton cloth were impregnated with an acetone

solution of the compound at the rate of 4 grams per square foot. When

dry each patch was fastened to a glass plate, and 5 chiggers were confined on it within a rubber ring such as is used on mason jars, and then

covered with another glass plate. A material was considered to be effective if the chiggers were immobilized within 15 minutes. The materials selected by these tests, therefore, were fast-acting toxicants

rather than true repellents.

If effective, the impregnated cloth was rinsed in cool water for 48

hours and tested again. For this purpose a mechanical rinsing apparatus

was used, which consisted of a wooden box with 12 quart-size glass jars

around its periphery rotated by an electric motor. The materials were

placed in the jars with 600 ml. of water, and rotated at 39 r.p. m. If

the cloths were still effective, fresh samples impregnated at 2 grams

per square foot were washed in an electric washer for 10 minutes at

120 F. in 20 gallons of water containing 0.7 percent of soap and then

rinsed twice for 5 minutes in equal volumes of cool water. Samples

were washed and tested until they failed to immobilize chiggers in

15 minutes.

Early in this work benzyl benzoate showed such promise that it was

tested on a larger scale, and clothing impregnated with it was found to

withstand at least two washings. Treatment of clothing with this material was adopted by the Army. In the screening tests, therefore, the

benzyl benzoate-impregnated cloth was also washed and tested as a

standard of comparison for the materials found to be effective after the

48-hour rinsing. An untreated cloth was also included in the tests as a

check.

In 54 tests benzyl benzoate always stopped chiggers after the first

washing. There were 6 failures after the second washing, 20 after the

third, 18 after the fourth, 9 after the fifth, and 1 after the sixth.

Beginning in 1946, cloth patches impregnated at the rate of 2 grams

per square foot were used in the first as well as subsequent tests. The

day after treatment 5 chiggers were exposed for 15 minutes and the individual knock-down, or stopping, times recorded and averaged. If all

were down in 15 minutes, the treated cloth was rinsed in cool water for

15 minutes and retested after drying. If still effective, the cloth was

subjected to repeated washings in an electric washer as described above.

The classification of compounds was as follows:

Insecticidal Effectiveness

1. Ineffective on initial test (chiggers not all down within 15

minutes).

2. Effective on initial test but ineffective after a 15-minute rinse.

3. Effective after the rinse but not after three washings.

4. Effective after three or more washings.

4A. Effective after five washings.
13

Speed of Knock-Down

1. Not all down in 15 minutes,

2. All down in 11 to 15 minutes.

3. AU down in 6 \to 10 minutes.

4. All down in 1 to 5 minutes.

Mosquito Repellents

(Tables 1 and 7)

Nearly all compounds received at the Orlando laboratory were

routinely tested for repellency to the yellow-fever mosquito. Most

of them were also tested in the laboratory against the common malaria

mosquito, and many of the better ones were tested in the field against

mixed populations of salt-marsh mosquitoes.

Samples of liquid chemicals were submitted to the U. S. Food and

Drug Administration for skin-irritation tests on rabbits. If no adverse

effects were observed, the compounds were then tested for repellency

on the skin of human subjects. Solid compounds were dissolved in

acetone and used to impregnate cotton cloth. Many of the liquids were

also tested in the same way.

A test was made by covering one forearm of the subject with a liquid

chemical or a treated cloth and then exposing it in a cage containing

2,000 to 4,000 unfed mosquitoes. The other arm was treated with another

material. The mosquitoes were of various ages, since new ones were

added each day. An arm of one of the persons in the testing group was

left untreated, and was introduced into the cage at intervals to check on

the biting rate, which was usually 50 to 75 per half minute. The mosquitoes in a cage were discarded or given a rest when the biting rate

dropped below 20. The testing rooms were maintained at 80^ to SS^F.

and a relative himiidity of 75 to 85 percent.

Both white and colored subjects were employed in the tests, and

more than 300 were used during the course of the work. Consequently,

there was a wide range in repellent times owing to individual differences.

Because of these differences, as well as variations in the activity or

avidity of mosquitoes from day to day, it was found advisable to replicate

the tests as many times as possible in the follow-up study of the more

effective compounds.

Tests on Skin

For the skin applications liquids and low-melting solids were tested

by rubbing 1 ml. of the chemical over one of the forearms of two to four

men. Another compound was applied to the other arm. Gloves were

worn to protect the hands, and the treated arms were exposed in the

cages for 3 minutes at intervals of about 30 minutes until the first bite

was received.
14

The time between treatment and the first bite was adopted as the

criterion of effectiveness and is referred to as the protection time.

The first bite, however, is sometimes accidental because of a rubbed

spot on the arm or an exceptionally refractory mosquito. Beginning in

January 1947, the exposures were continued until the second bite was

received. If this occurred in the same exposure period as the first

bite or in the following one, it was considered to confirm the first bite

as indicating the failure of the repellent. Otherwise the first bite was

disregarded, and the exposures were continued until two bites were

received in the same or consecutive exposure periods.

With the malaria mosquitoes the repellent times were almost always

much shorter, and the classes were accordingly adjusted to half the protection times selected for the yellow-fever and salt-marsh mosquitoes.

The repellent classes based on protection time (minutes) were as

follows:

Yellow-fever and Common malaria

Class salt-marsh mosquitoes mosquito

1 0-60 0-30

2 61-120 31-60

3 121-180 61-90

4 180 or more 90 or more

4A 300 or more 150 or more

Tests on Cloth

In the early tests on cloth it was found necessary to have the cloth

worn by a human subject to obtain satisfactory results. Women's

mercerized-cotton stockings were employed. A measured section a

short distance above the ankle was impregnated with the compound at

a rate equivalent to 3.3 grams per square foot. The stocking was

spread on a rack to dry and then hung indoors on a line. The usual

procedure was to treat one day and make the first test the following

day. The stocking was drawn over the arm, with the treated portion

about midway of the forearm. The hand was protected with a glove,

and the arm was exposed for 1 minute in a test cage.

If five bites were received, the treatment was considered noneffective. When applied to cloth, repellent materials remain effective for

several days or weeks, as compared with hours in skin applications.

If less than five bites occurred, the exposures were continued daily

until the 14th day and at weekly or biweekly intervals thereafter.

Beginning in January 19 48, the tests were run weekly until the fourth

week and at 2-week intervals thereafter.
15

Beginning in 1946 the stocking tests were used for screening liquid

as well as solid repellents, and usually only the compounds rated in class

3 or 4 by these tests were evaluated by skin applications. This change

made it possible to test a given number of compounds in a much shorter

time Moreover, since the materials in the stockings were in contact

with the skin in a dry state and for only a very short time, there was

thought to be so little danger from skin irritants that preliminary skinirritation tests on rabbits could be dispensed with. Compounds that

appeared from their chemical composition to be potentially dangerous

were omitted, however. Later, at the suggestion of the Army Environmental Health Laboratory, a piece of thin nylon netting was placed next

to the skm under the stocking to prevent direct contact of the test chemical. Compounds that were effective on cloth were submitted to that laboratory for skin-irritation tests on rabbits, and if no adverse effects

were observed they were subjected to skin tests.

The compounds were classed as follows:

1. Ineffective on the initial test (five or more bites in 1 minute)

2. Effective for 1 to 5 days.

3. Effective for 6 to 10 days.

4. Effective for more than 10 days.

4A. Effective for more than 21 days.

Rinsing and Washing Tests

A stocking freshly treated with each class 4 compound was subjected

to one or more 15-minute rinses in cool water and after each rinse was

tested against yellow-fever and common malaria mosquitoes. If still

effective after the second rinse, the stocking was washed for 15 minutes

m hot soapy water and then rinsed for 5 minutes. Only a small proportion of the repellents withstood exposure to water, and these tests were

not continued systematically in the later part of the work.

The classes for these tests were as follows:

1. Ineffective after first rinse.

2. Withstood 15-minute rinse.

3. Withstood 30 minutes of rinsing but no washing.

4. Withstood 15 or more minutes of washing.

Wearing Tests

It was found that the wearing of repellent-treated clothing reduced

considerably the duration of effectiveness below that of clothing hung on

a line at room temperature. Testing after 8 hours of wear was therefore used as another method of evaluation of the class 4 repellents.

Tests were run in duplicate in the laboratory against the yellow-fever

mosquito and in the field against salt-marsh mosquitoes. Some tests

were run also against the common malaria mosquito.
16

The classes were as follows, based on the hours of wear withstood

in the better of the two tests:

1. 0 to 8 hours.

2. 16 to 24 hours.

3. 32 to 40 hours.

4. 48 hours or more.

Flea Repellents

(Table 8)

The tests of flea repellents were conducted in a wooden pen. This

pen had an outer wall about 60 inches square by 16 inches high, and an

inner wall 46 inches square by 14 inches high, which left a moat about

6 inches wide between the walls. The bottom of the pen was covered

with plywood. The upper 3 inches of the inner wall was treated with a

flea repellent, and a band of tanglefoot was applied near the top of the

outer wall to prevent escape of the insects. A layer of dry hay was

spread over the bottom of the inner pen, and large numbers of reared

cat fleas were released into it.

In March 1950 tests with cat fleas were discontinued and only oriental

rat fleas were used, since they are easier to rear and the two species

had been found to give similar results with most materials. Rat fleas,

however, are less active than cat fleas and tend to stay in the straw;

so the straw was removed and the bottom of the pen covered with white

muslin.

Most of the materials selected for testing were compounds that had

been placed in class 3 or 4 in the mosquito-repellent tests. Two types

of tests, patch tests and pen tests, were conducted.

For the patch tests a piece of cotton cloth 4 inches square was

impregnated with 2 grams of the test material per square foot dissolved in acetone. After the acetone had evaporated, the patch was

attached to a small paddle and swung back and forth within jumping

distance of the fleas in the pen. A similar untreated patch was swung

at the same time for comparison. When a number of fleas were noted

on each patch, the patches were raised out of jumping range and after

1 minute the fleas remaining on each were counted. Four such counts

were totaled, and the percent repellency was calculated by the formula

100(a-b)4 a, in which a. was the number on the check and b the number

on the treated patch. Tests were run on the day after treatment, then

at weekly intervals for 4 weeks, and at 2-week intervals thereafter

until the repellency dropped below 80 percent on 2 consecutive days.

The pen tests consisted of a semipractical evaluation of the materials,

in which human subjects wore treated stockings in the pens for 15minutes,

Compounds placed in classes 3 and 4 in the patch tests were evaluated by
17

this method. Mercerized-cotton stockings were impregnated from

ankle to top with 3.4 grams of the test compound in 50 ml. of acetone

to give a dosage of about 2 grams per square foot of cloth. The stocking

feet were left untreated to avoid contamination of the pen, and short

socks were worn under the stockings to prevent the fleas from biting

the feet. Usually two men were in the pen at the same time, wearing

between them one untreated and three treated stockings The fleas

that crawled to the top of the stockings were removed and counted, and

the percent repellency was calculated by the formula given above

Testing intervals were the same as for the patch tests, except that

after the fourth week, if the treatment was still effective, the tests

were made at monthly instead of biweekly intervals. Effectiveness in

these tests was based on 90 percent instead of 80 percent repellency.

The classes for both kinds of tests were as follows:

1. Ineffective after 1 day.

2. Effective for 1 to 5 days.

3. Effective for 6 to 10 days.

4. Effective for more than 10 days.

Tick Repellents

(Table 8)

A pen similar to that used in the flea tests was used for testing tick

repellents, and the tests in general were similar. Large numbers

of nymphs of the lone star tick from the laboratory colony were released into the hay on the bottom of the pen.

For the patch tests the 4-inch square of cloth was impregnated with

the test chemical at the rate of 2 grams per square foot, as in the flea

tests. A black line was drawn across the middle of the patch, and after

it was attached to the paddle a folded strip of clean paper toweling 3/4

inch wide was fastened to its lower edge. The paddle was held in a

vertical position and the paper strip brought in contact with the infested

hay. The ticks crawled onto the paper strip. Those that crossed the

treated cloth to the marked line were counted, and a similar count was

made with an untreated cloth. Tests were made at the same intervals

as in the flea tests until the repellency dropped below 80 percent.

The pen tests were conducted in the same manner as those with

fleas, except that men's white cotton socks were used instead of long

stockings. They were treated from the ankle to the top, a distance of

about 10 inches, with 1.11 grams of repellent in 30 ml. of acetone to

give a dosage of approximately 2 grams per square foot.

The classes for both types of tests were the same as for the flearepellent tests.
18

Synergists

(Table 9)

Pyrethrum

Body lice were employed for the testing of materials as possible

synergists for pyrethrum. Most of the compounds so tested were related to compounds known to have some synergistic effect. They were

tested in the ratio of 10 parts to 1 part of pyrethrins (5 parts of a

pyrethrum extract containing 20 percent of pyrethrins in kerosene).

Circular pads of woolen cloth, similar to those used in the beaker

tests (see page 4 ), were prepared in duplicate by dipping in an acetone

solution containing 0.25 percent of the test compound and 0.025 percent

of pyrethrins. Twenty adult lice were placed on each pad and observed

for mortality at the end of 24 hours. If none of the lice survived, the

mixture was retested at reduced concentrations. If some mortality

occurred at the original concentration, a test was usually run on duplicate pads treated with a 0.25-percent or weaker solution of the compound

alone to distinguish between synergistic action and primary toxicity.

For some of the compounds there were not sufficient data to make

a distinction between classes 2 and 3; they rated class 2-3.

The classes were selected as follows on the basis of louse mortality:

1. Less than 80 percent at 0.25 percent of Synergist.

2. 80 percent or nnore at 0.25 percent of Synergist.

3. 80 percent or more at 0.125 percent of Synergist.

4. 80 percent or more at 0.05 percent of Synergist.

Pyrethrum-DDT

About 3,800 compounds were tested against house flies and common

malaria mosquitoes in a search for materials that would increase the

knock-down and lethal properties of a standard spray formula containing

1 percent of DDT and 0.1 percent of pyrethrins. Ten percent of the test

compound was added to this formula, and the technique employed was

similar to that described for the spray tests (see page 8).

The results of this work are given in Bureau publication E-775.

Some of these compounds have been retested as synergists for either

DDT or pyrethrum alone, but no further work has been done with

synergists for combinations of these two materials.
19

Summary of Results of Screening Tests

The number of compounds evaluated in each kind of test and the number

found to be in each class are given below.

Table T _ X _ 11« 1 /» I

Number of compounds

No.

inseci ana Kina oi test Total Class Class Class Class Class

X 1 \-/ #

tested 1 2 3 4 4A

1. Lice

Ins 10,748

8,889Í/

7,888 932 1,333 358 237

Ov 5,257 2,050 1,213 342 20

KD 10,514 7,880 2,067 336 203 28

Mosquito larvae

Mosq. larvicide 10,598 8,625 1,141 429 368 35

Fleas

Ins 2,302 1,987 235 43 16 21

KD 2,296 1,981 186 82 47 — «•

Ticks

Ins 2,261 1,520 479 102 65 95

KD 2,119 1,435 261 259 164 . M

Chiggers

Ins 7,397 3,769 2,371 1,065 127 65

KD 7,379 3,758 317 699 2,605

Mosquito repellents

Skin: YF 4,274 2,384 1,210 296 318 66

M 3,918 1,720 1,672 389 126 11

Clothe s: YF 6,283 4,097 '871 374 368 573

M 2,032 1,761 211 35 15 10

Mosquito larvicides

(additional tests)

Southern house 93 2 6 11 60 14

Yellow-fever 98 6 16 10 60 6

Fly and mosquito sPi-ays ,

Space: Flies 595^^ 524 29 18 21 - -

Mosq

1,335^

401 119 34 10 .^

Residual: Flies 1,223 63 14 29 - -

Mosq 1,302 1,061 181 39 21 --

Cockroaches

Dusts: Am 743 263 276 134 70 ...

Ger 83 36 19 10 18 ~. «

Residue: Ger 63 46 9 1 7 « ^
20

Summary of Results of Screening Tests.--Continued.

m I— 1

Insect and kind of test

Number of compounds

Table

No. Total Class Class Class Class Class

tested 1 2 3 4 4A

5. Fleas (additional tests)

Cat fleas: Washing

tests 54 34 11 6 3 --

Dusts 605 451 98 25 31 --

Rat fleas: Ins 65 41 8 7 5 4

KD 65 41 5 8 11 --

Dusts 60 21 8 19 12 --

6. Ticks (additional tests)

Nymphs: Ins 130 45 42 10 13 20

KD 133 45 29 27 32 -.

Adults: Ins 132 94 29 3 2 4

KD 132 95 16 14 7 -«

Larvae: Washing 136 72 28 25 11 --

tests

7. Mosquito repellents

(additional tests)

Salt-marsh: Skin 142 37 42 19 29 15

Cloth 316 54 62 46 61 93

Wearing tests

Salt-marsh 145 70 45 11 19 --

Yellow-fever 12 10 2 0 0 — M

Malaria 10 8 2 0 0 --

Washing tests

Yellow-fever 737 556 125 41 15 --

Malaria 19 15 2 1 1 --

8. Flea and tick repellents

Fleas: Patch 621 359 128 60 74 — M

Pen 151 55 22 13 61 --

Ticks: Patch 1,089 348 284 188 269 --

Pen 367 52 58 51 206 --

9. Pyre^hrum synêrgists

Lice 545^ 352 26 45 69 . mm

1/ 7 compounds rated class 3-4.

2/ 3 compounds rated class 3-4.

3/ 6 compounds rated class 3-4.

V 53 confipounds rated class 2-3.
21

Explanation of Abbreviations Used in Table 1

Lice

Ins = Insecticidal effectiveness based on 24-hour exposures

of adult body lice in beaker tests.

Ov = Ovicidal effectiveness,

KD = Speed of knock-down, or paralysis, of body lice when

exposed to freshly treated cloth.

Mosquito larvae

Mosq. larvicide = Effectiveness against fourth instars of

the common malaria mosquito.

Fleas

Ins = Insecticidal effectiveness based on 60-minute exposures

of cat fleas on impregnated cloth.

KD = Speed of knock-down of cat fleas when exposed on

freshly treated cloth.

Ticks

Ins = Insecticidal effectiveness based on 60-minute exposures

of larvae of the lone star tick on impregnated cloth.

KD = Speed of knock-down of larvae of the lone star tick on

freshly treated cloth.

Chiggers

Ins = Insecticidal effectiveness based on 15-minute exposures

on impregnated cloth before and after laundering.

KD = Speed of knock-down on freshly treated cloth.

Mosquito repellents (skin and clothes)

YF = Tests against yellow-fever mosquitoes.

M = Tests against common malaria mosquitoes.
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Ethane, 1 - ( 2 - chloroethoxy) - 2 - (trichlorophenoxy) -Ethane, l-(2-chloroethoxy)-2-(2, 4, 6-

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Ethane, 1-chloro-1-nitroEthane, 1 - (£- chlorophenoxy) - 2 - (2 -iodoethoxy) -Ethane, l-cyclohexyloxy-2-isoamoxyEthane, l-(o-cyclohexylphenoxy)-2-

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Ethane, 1 - (o-cyclohexylphenoxy) - 2- (2-phenoxyethoxy) -Ethane, 1, 2-dibenzyloxy

Ethane, 1, 2-dibromoEthane, 1, l-dibromo-2, 2-bis(£-bromophenyl)-Ethane, 1, l-dibromo-2, 2-bis(£-chlorophenyl)-Ethane, 1, 2-dibromo-l, 1-dichloroEthane, 1, 2-dichloroEthane, dichloro-2, 2-bis(chlorophenyl)-

Ethane, 1, 1-dichloro-2, 2-bis(£-chlorophenyl)-

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Ethane, 1, 1-dichloro-2, 2-bis(£-methoxyphenyl)-

Ethane, 1,1- dichloro- 2 - (o - chlorophenyl) - 2 -

(p- chlorophenyl) -

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