Evaluation of ryania for the protection of stored wheat and shelled corn from insect attack

Historic, archived document

Do not assume content reflects current
scientific knowledge, policies, or practices,

Marketing Research Report No. 24 5

LIBRARY

'-"^■■'^ T f^DjAL RECORD

JUL 2 9 !958

Evaluation of Ryania
for the Protection of
Stored Wheat
and Shelled Corn
from Insect Attack

■'AKTMLNT Or «uM^,ULTUHE

Marketing Research Division
Agricultural Marketing Service
U.S. DEPARTMENT OF AGRICULTURE

WARNING

No tolerances have been established for the use of
.ryania as an insecticidal treatment for the prevention
of insect infestation in stored grain. The tests re-
ported herein were exploratory studies to develop in-
formation that could be used in considering the estab-
lishment of tolerances. Until such a tolerance is
announced ryania protective treatments should not
be used.

This report is the third of a group presenting re-
sults of tests with various insecticidal dusts and
sprays applied to stored grain for protection against
insect attack. These reports are a part of a broad
program of research to reduce the cost of marketing
farm products, including the cost of preventing in-
sect infestation in stored grain.

July 1958

For sale by the Superintendent of Documenta, U. S. Government Printing Office
Washington 25, D. C. - Price 15 cents

CONTENTS

Page

Summary

Backgrovmd and purpose of the work ^^

Materials . . . !

Techniques

Sampling methods

Statistical analyses

Residue determinations .

Bioassay tests .

Tests with wheat .

Group I - Exploratory tests with farm-stored seed wheat in Reno County," Kans.',
begun in 1952 ^

Group II - Tests with CCC-owned wheat at Beloit and La Crosse, Kans. , begun

in 1953 y

Tests with shelled corn n

Group III - Tests with CCC-owned shelled corn at Beattie, Kans., begun in

September 1952 jq

Group IV - Tests with CCC-owned shelled corn in Illinois begun in May 1953 ... 12
Group V - Tests with CCC-owned shelled corn in Saline County, Mo. , begun in

July 1953 15

Comparative abundance of the species of insects 17

Finding s 21

ACKNOWLEDGMENTS

This report is based on research conducted by a number of individuals and groups.
The performance tests were conducted by A. C. Apt, J. K. Quinlan, J. H. Schesser,
G. H. Spitler, G. D. White, W. K. Whitney, and H. E. Fearing, of the Stored-Grain
Insects Laboratory, Manhattan, Kans.

Determination of residues of ryania on treated wheat and in flour milled from treated
wheat were made by S. B. Penick and Company. This company also supplied the ryania
formulations.

The milling tests were made in cooperation with the Kelly Milling Company, Hutchin-
son, Kans. The commercial grade of grain used in the tests was determined by the Board
of Grain Supervisors, Chicago, 111.

Ill

SUMMARY

This report is the third of a series presenting results of tests with various insecti-
cidal dusts and sprays applied to stored grain for protection from insect attack. It
summarizes the tests with ryania begun in September 1952 and concluded in January 1956.

The studies were conducted under actual storage conditions both on the farm and at
CCC bin sites. At the latter tests were nnade with wheat and shelled corn stored in the
standard circular 3, 250-bushel metal bins of the Commodity Credit Corporation, U. S.
Department of Agriculture, and also on shelled corn in 3, 000-bushel rectangular wooden
bins .

After the dusts were applied, the grain was sampled monthly to determine the insect
population trends and the moisture content of the grain. These samples were used to es-
timate the levels of residue, to observe any change in the potency of the residues in bio-
assay tests, and to establish comnnercial grades. In tests with wheat, the level of residue I
was also traced through the milling process.

Application rates of ryania ranged from 20 to 100 pounds per 1, 000 bushels in wheat,
and from 20 to 60 pounds in corn. These rates practically eliminated any initial insect
populations, and kept the grain almost insect-free for as long as 33 months.

Milling tests showed that 80 to 90 percent of the ryania could be removed in the scour-
ing process preparatory to milling, and that relatively snnall amounts were carried into
the feed and flour fractions.

There was little change in the overall moisture content of the grain during the storage
period, and there was no apparent effect on the behavior of the protective dusts attribu-
table to variations in moisture content. There were no changes in the commercial grade
of the wheat during the period of test. A large portion of the corn was downgraded be-
cause of damage from moisture accumulation in the surface layer during the cooler
months of the year. One series treated with ryania -sulfoxide formulation was downgraded
because of odor.

In wheat, 9 species of stored-grain insects were found, the most abundant being the
dermestids, the lesser grain borer, the flat grain beetle, and the saw-toothed grain
beetle. In corn, 16 species were noted, the most abundant being the red flour beetle, the
flat grain beetle, the saw-toothed grain beetle, and the Angoumois grain moth.

IV

EVALUATION OF RYANIA FOR THE PROTECTION OF STORED
WHEAT AND SHELLED CORN FROM INSECT ATTACK

By H, H, Walkden and H. D. Nelson

Stored-Product Insects Laboratory

Manhattan, Kans.l

BACKGROUND AND PURPOSE OF THE WORK

Grain handling and grain storage practices have changed during the past few years,
and have resulted in the storage of grain for much longer periods of time, sometimes as
long as 5 years. Reserve stocks of grain, particularly corn and wheat, have accumulated
as a result of increased yields and the price support program. The safe storage of these
reserve stocks has necessitated new and improved methods of preventing damage by in-
sect attack. Emphasis is now being placed on preventive rather than curative measures
of insect control.

In line with this trend, intensive studies were begun in the fall of 1952 to explore the
possibility of applying insecticides directly to stored wheat and shelled corn to prevent
the development of insect infestations. The use of insecticides for this purpose poses
numerous problems. The fate of the resulting insecticidal residues must be ascertained
to determine whether they will be within safe limits for the ultimate consumers of grain
products--both human and livestock- -and whether the grade of the grain is affected by
the treatments.

This report is the third of a series presenting the results of the tests with various
protective treatments. The data presented herein pertain to the studies in which ryania
dusts were used. The data cover several phases of this complex problem; for presenta-
tion they are divided into five groups. The tests with both wheat and shelled corn are
tabulated below:

Group I. Exploratory tests with farm-stored seed wheat in Reno County, Kans, ,
begun in June 1952.

Group II. Tests with CCC-owned wheat at Beloit and LaCrosse, Kans., begun in
June 1953.

Group III. Tests with CCC-owned shelled corn at Beattie, Kans., begun in September
1952.

Group IV. Tests with CCC-owned shelled corn in Illinois, begun in May 1953.

Group V. Tests with CCC-owned shelled corn in Saline Co. , Mo. , begun in July 1953.

MATERIALS

Three formulations of ryania were used in these tests. All were in dust form but no
diluent was required since the ground ryania contains approximately 0.25 percent of
ryanodine, the active agent and has good dusting properties.

1 This laboratory is a field station of the Stored-Product Insects Section. Biological Sciences Branch. Marketing Research Division.
Agricultural Marketing Service. U. S. Department of Agriculture.

- 1 -

1. Grovmd ryania wood or stems (95 percent passes through a

200-mesh screen) 100. percent

2. Ground ryania 95. 8 percent

Sulfoxide 3.2 percent

Inert 1-0 percent

3. Ground ryania 95.8 percent

N-Propyl isome 3.2 percent

Inert !• percent

Most of the information in the following paragraphs was taken from an unnumbered
publication of S. B, Penick & Co., 50 Church Street, New York 8, N. Y. , dated 1953 and
entitled "Ryania, a botanical insecticide."

Ryania is a tropical American genus of shrubs and snnall trees belonging to the
family Flacourtiaceae, the principal source of the insecticide being the stem wood of
Ryania speciosa Vahl, a species native to Trinidad. Other species occur in different
areas of northern South America and in the Amazon basin.

Ryania was discovered in the course of a cooperative research program by the
Research Laboratories of Merck and Co. , Rahway, N. J. , and the Department of
Entomology of Rutgers University and the New Jersey Agricultural Experiment Sta-
tion, This program was directed toward the survey of materials of botanical origin
from all parts of the world in search of new materials of insecticidal value.

The first examination of Ryania under field conditions was made at the New Jersey
Agricultural Experiment Station in 1943 using the ground root of Ryania speciosa at
which time it was found to be outstandingly effective against the European corn borer.

An extensive investigation of the chemistry of Ryania principles has been con-
ducted in Research Laboratories of Merck and Company. The active principles can
be extracted with water and many organic solvents and quantitative extraction may
be secured by the use of water, methanol, or chloroform. The active principle of
Ryania is an alkaloid which has been designated as "Ryanodine".

Ryanodine is neutral to litmus and forms no salts. It is soluble in water, alcohol,
acetone, ether, and chloroform; difficulty soluble in benzene; and insoluble in
petroleum ether. The distribution coefficient of Ryanodine for ether/water is 1.3.
The ultra-violet absorption spectrum shows a maxinnum at 2685 A°(E:% cm. = 352)
in alcohol solution. A tentative empirical formula of C25H35NO9 or C26H37NO9 has
been suggested for Ryanodine. The structural formula has not been established.
Ryanodine has approximately 700 times the insecticidal potency of the stem wood of
Ryania speciosa , and 0.25% of Ryania is considered to be Ryanodine.

Work with the physiological effects of Ryania indicate that: Ryanodine exerts a
highly selective action on muscle tissue. It has been postulated that "Ryanodine acts
by interfering with the high energy phosphate system in striated muscle." The char-
acteristic action of Ryania is such as to produce a rapid cessation of normal activity,
often without producing death for several days. "Insects in the state of 'flaccid paraly-
sis' would be incapable of normal activity but could not be classified as dead. "

To establish the safety of use of powdered Ryania stems as an insecticide, an ex-
tensive investigation was undertaken by the Merck Institute for Therapeutic Research.

- 2 -

This work has been reported by Heal and Kuna (1948)2. They established the acute
oral toxicity of powdered stem of Ryania speciosa to be 1200 mg./kg. in rats, 150
mg./kg. in dogs, more than 400 mg./kg. in monkeys, 650 mg. /kg. in rabbit's, 650
mg. Ag. in mice, 2500 mg./kg. in guinea pigs, and more than 3,000 mg.Ag. in
chickens. Chronic oral toxicity studies with this same material demonstrated that
it wcvs possible to maintain rats, chickens or guinea pigs for at least 5 months on a
diet 'containing 1 percent of the Ryania powder without producing symptoms of cumu-
lative poisoning. Direct comparisons of this Ryania powder with DDT and cube
powders in chronic administration showed that Ryania powder is tolerated in larger
amounts and for a longer period of time than either DDT or cub^.

TECHNIQUES

The tests were conducted in three types of bins: (1) Farm bins of wood construction
with capacities ranging from 200 to 1,000 bushels; (2) USDA circular metal bins, 16 feet
in height and 18 feet in diameter with a rated capacity of 3,250 bushels; (3) CCC rec-
tangular wooden bins, 16 feet wide, 24 feet long, and 10 feet to the eaves with a rated
capacity of 3, 000 bushels.

The farm-stored wheat was dusted by estimating the nvunber of bushels per truck load
and sifting the required dosage of ryania uniformly over the surface of the load. The
truck was unloaded into an auger hopper and the grain elevated into the bin. This method
of handling was expected to produce a uniform distribution of the insecticide on the wheat.
These treatments were applied at harvest in late June 1952.

In the CCC metal bins the dust was applied by hand as the grain ran from the auger,
or by a mechanical applicator attached to the auger tube, or by aliquots as each truck
load was emptied into the hopper of the auger.

In the rectangular wooden bins the applications were made by a mechanical applicator
attached to the auger tube.

Sampling Methods

All of the sampling was done with an 11-celled grain trier equipped with extension
handles. Samples were drawn monthly for determination of insect populations and mois-
ture content of the grain.

In the farm bins, where wheat did not exceed 5 feet in depth, the samples were taken
vertically from the center and near the wall, and horizontally from the top 2 to 3 inches
in the center.

In the circular metal bins, the samples were taken vertically in the center of the bins
from the top, middle, and bottom 5 feet, from the top 5 feet in each quadrant at a point
about 3 feet from the wall, and horizontally from the top 2 to 3 inches at the center.

In the rectangular wooden bins, the samples were taken vertically from the top and
bottom 5 feet in the center and at a point about 3 feet from the gable ends of the bins.

Statistical Analyses

The treated lots of grain in the CCC bins were compared with the untreated controls
and with each other on the basis of the number of "bin months" of protection. The number
of bin months of protection was computed by dividing the total number of months of pro-

2 Heal. Ralph. E.. and Kuna. Samuel. Toxicological and pharmacological smdies on the powdered stem of Ryania speciosa .
a plant insecticide. Jour. Pharmacol, and Expt. Therapeutics 93(4): 407-413. 1948.

- 3 -

tection for a series of bins having the same treatment by the number of bins in that series.
The treatment was considered to give protection until the insect population reached the
level at which it would be designated as "weevily" under the provisions of the U.S. Grain
Standards current at the time of the tests. The grain was designated as "weevily" if a
1,000-gram sample contained living stored-grain insects as follows: Wheat : 2 or more
weevils; or 1 weevil and 3 or more bran beetles; or 5 or more bran beetles. Corn : 2 or
more weevils; or 1 weevil and 5 or more bran beetles; or 25 or more bran beetles. In
order to classify the insects for grading purposes, the rice weevil, granary weevil, lesser
grain borer, and the Angoumois grain moth were designated as weevils, and all other
stored-grain insects as bran beetles.

The data thus obtained were tested statistically for reliability by the analysis of vari-
ance method. This method of statistical analysis establishes the significance of nnean dif-
ferences between treated lots or between treated lots and the untreated controls.

Residue Determinations

As no chemical method for the determination of ryania residues in wheat are known,
the residues were estimated by exposing mosquito larvae to extractions from samples of
treated grain. This nnethod is not as satisfactory as a chemical one would be, but it does
indicate the degree of ryania residue present.

Bioassay Tests

Bioassay tests were conducted for the purpose of observing any changes in the po-
tency of the ryania during the storage period after treatment. In these tests 25 adults of
rice weevils or of confused flour beetles were confined in 3 ounces of corn or wheatfrom
both the treated and the untreated bins and the resulting mortalities were recorded after
1 and 3 weeks.

TESTS WITH WHEAT

Group I: Exploratory Tests with Farm-Stored Seed Wheat in
Reno County, Kans. , begun in 1952

AH of the wheat used in these tests was of good, dry storage quality, the moisture
content ranging from 11,0 to 12.0 percent. A total of 10 farm bins were treated with
ryania dust: 2 at a dosage rate of 60 pounds per 1,000 bushels of wheat, 5 at 75 pounds,
and 3 at 100 pounds.

Insect Population Trends

The monthly samples were taken to the laboratory and screened, and the number and
species of stored-grain insects were recorded. In all of the 10 bins in which wheat re-
mained in storage for from 6 to 20 months, only 2 insects--! flat grain beetle and 1 In-
dian-meal moth- -were found during the entire period. These two insects were in a bin
receiving the 60-pound dosage rate. The results of these tests demonstrate that ryania
gave alnnost complete protection against insects over a period of 20 nnonths, and could be
presumed to protect for an indefinite period. The reason for the variable lengths of stor-
age periods was that the wheat could be removed at the option of the cooperating farmer.

Insecticidal Residues

The first sannples for estimating the residue were drawn in July 1952, a month after
the ryania was applied. The results are given in table 1. The estinnated level ranged from
54 percent from the 75-po\md dosage rate to about 70 percent from the 60- and 100-pound

TABLE 1. -Residues of ryaMa in fam-stored wheat 1 month after treatment-

determined by the bioassay method '

Application rate and test number

60 lb. /I, 000 bu.

1

2

Mean

75 lb. A, 000 bu.

3

A

5

6

7

Mean

100 lb. /I, 000 bu

8

9

10

Mean

Ryanodine^

.p.m.

1.8
1.7
1.8

3.1

.5

.6

.3

4.0

1.7

2.3
2.5
3.9
2.9

Calculated on the basis of an estimated average of 0.25 percent of ryanodine in the
pulverized ryania.

In April 1953, two 1 -gallon samples were drawn from one bin in the farm-stored
series that had received the 60-pound dosage rate. One sample was cleaned and scoured
by the premilling process, and the other was left as it came from the bin. Table 2 shows
that 31 percent of the amount of ryania applied was recovered in the unsecured sample,
and of that amount 79 percent was removed by the cleaning and scouring process. These
samples were not nnilled,

TABLE 2. --Residues of ryania in wheat before and after cleaning and scouring
preparatory to milling; dosage rate 60 pounds per 1,000 bushels

Sample treatment

Ryania recovered

Ryanodine
recovered

Unscoured, as wheat came from bin:-""

First portion

Second portion

Third portion

Mean

Scoured, single sample

P. p.m.

0.75

.75

.80

.77

.16

""" The unscoured sample was divided into 3 portions for bioassay tests.

A third set of samples, taken in June 1953, showed that approximately 90 percent of
the ryania had been rennoved in the cleaning and scouring process. These samples were
not milled.

- 5

In January 1954 a fourth set of samples were taken from 2 bins of wheat that had re-
ceived the 75-pound dosage rate. The check sample was taken from a bin of vmtreated
wheat of the 1953 crop. These samples were milled with an AUis-Chalmers experimental
flour mill, and residues estimated for uncleaned wheat, scoured wheat, nnilled feed (bran,
shorts, and germ), and flour. Virtually all of the ryania was removed in the scouring
process; less than 1 percent of the original application was carried through the milling
process into the feed and flour fractions (table 3).

TABLE 3. — Residue in various milling products from wheat treated with ryania dust at the
rate of 75 poiinds per 1,000 bushels; treated June 1952 and milled January 1954

Milling fraction test number and
moisture

Proportion of
original weight of
wheat

Amount of ryania
fo\md

Wheat as drawn from bin:

11 (moisture 11. 5fo)

12 (moisture 11.3^o)

Mean

Wheat after cleaning and scouring:

11

12

Mean

Bran, shorts, and germ:

11

12

Mean

Flour :

11

12

Mean

Percent
100

C)

P. p.m.

^195

^2,625

1,410

^24
15

29.2
30.3

70.8
69.7

^12
^12

^9

Mean of 2 tests.

^ Mean of 4 tests. The great excess over the amount applied may have been due to
agglomeration of the ryania as the bin was filled.

^ Not determined.

^ These values are not significant since they merely indicate the lower limit of the
assay method for the size of samples used in the bioassays.

Changes in the Moisture Content During Storage

There was virtually no change in the moisture content of the wheat during the storage
period, nor was there evidence that the protective treatment had any effect on the trans-
fer of moisture or the amount of moisture.

Changes in the Commercial Grade

To obtain information on the possible downgrading of wheat treated with ryania dust,
three 1,'000-grann samples were treated at dosage rates of 60, 75, and 100 pounds per
1, 000 bushels. These samples were submitted to the Chicago Board of Grain Supervisors
for an opinion as to the effect of the treatments on the commercial grade of the wheat,
particularly on the feel and odor. The opinion of the Board is quoted below:

The appearance is very likely not affected because no change is noted in the ap-
pearance of the three samples submitted. We do detect something of an odor and a

feeling which we recognize as not entirely natural. This odor and feel is more notice-
able progressively with the samples containing the larger amounts of insecticide. Wc
believe that the presence of the insecticide is more noticeable in the feel of the wheat
than it is by odor, though immediately after smelling the samples some of us ex-
perienced a slight burning of the nose. Because of this it is possible that wheat
treated with larger amounts of the insecticide may in some instances be not accept-
able on a straight numerical grade.

Two months after treatment (August 1952) samples were drawn from each of the 10
treated bins and submitted to the Chicago Board of Grain Supervisors for an opinion as to
the feel and odor of the wheat. The opinion of the Board is quoted below:

The 10 samples treated with ryania dust at a dosage of 60, 75, and 100 pounds
per 1, 000 bushels of wheat, are very dry and in the present state do not have an ob-
jectionable feel although you can detect evidence of treatment from the gritty-like
feel of your hands. This gritty-like feel remains until your hands are washed. The
samples treated at the rates of 60 and 100 pounds feel better than the samples treated
with 75 pounds. [Note: Unknown to the inspector the 75-pound samples and the 100-
pound samples had been inadvertently reversed in numbering. This would account
for the difference noted by the inspector.] All samples have an odor not natural to
wheat. We will not say it is objectionable but again we find that it burns our nostrils,
and when our lips are moist we experience a burning sensation after smelling the
samples.

Subsequently a number of samples taken from the treated bins were submitted for
commercial grade and all were acceptable as to feel and odor.

Group II: Tests with CCC-Owned Wheat at Beloit and LaCrosse, Kans.,

Begun in 1953

The wheat used in theSe tests was of the 1952 crop, delivered from farm storage and
placed in CCC storage in the spring of 1953.

These studies were directed toward determining the degree of protection afforded by
the different dosage levels, and the length of time that the ryania retained its potency
under the conditions iinposed by metal bin storage. The degree of protection was deter-
mined by observation of the insect population trends, and the rate of degradation shown
by the bioassay tests.

The ryania dusts were applied in June 1953 to wheat at the Beloit and LaCrosse bin
sites. Six bins were treated at the rate of 20 pounds per 1, 000 bushels, 6 bins at 30
pounds, and 6 at 50 pounds. Six untreated bins served as checks. The ryania was applied
in aliquots to each truck load as the wheat was \inloaded from the trucks into the auger
hopper. Each bin was sampled monthly from June 1953 to January 1956 to determine in-
sect population trends and the moisture content of the wheat. Initial and terminal samples
were taken for commercial grades.

• Insect Population Trends

The insect populations in the treated and check bins are presented in table 4. All
three levels of treatment gave excellent protection from June 1953 to October 1954.

At the 20-pound dosage rate, the insect population in one bin (test 16) increased to
the weevily level in October 1954; another in March 1955 (test 14); another m October
1955 (test 18); the three remaining bins carried through until the tests were terminated
in January 1956.

At the 30-pound dosage rate, the insect populations were kept below the weevily level
throughout the entire storage period,

- 7 -

TABLE i.— Inflect

populations in wheat

following applications of ryania dust at e

pecified rates

, June

1953

Living insects found per

1,000 grajos of

wheat

in—

Application rate of ryanla
and test munber

1953

1954.

June

July

Aug.

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

Apr.

May

June

July

Aug.

Sept.

20 lb./l,000 bu.

Ho.

.5

.3

.3
1.0

.3

No.

1.0

.3

1.0

4.3

.5

No.

.3

.3

.3

.8
2.0
1.5

No.

.3

1.0

.3

.3

.5

.3

2.0
1.0
2.8

.3
1.3

.5

No.

.3

.8
C

3U.5

1.5

2.0

.5

.3

.5

No.
2.0
.5

.8

.3
.8

.8

1.0

.3

.3

.5

No.
0.8

1.0

35.8

No.

.3

.3
.3

.5

.8

.3

.5

36.3
2.3

No.

.3

.5

H.5

No.

.5

.3

No.

.3

No.

.3

.3

.3

No.

.8

No.

.8

4.5

No.

.3

.3

°.3

.3
.8

3.5

.3

No.

16

17

18

30 lb./l,000 bu.

19

20

.3

23

24

50 lb. A, 000 bu.

25

27

29

1.5

31

33

3.0

1.5

35

36

Application rate of ryanla
and test number

Living insects found per 1,000 grams of wheat In--

1954— Continued

Dec.

1955

Feb. Mar

Apr.

May June July Aug. Sept. Oct

20 lb. /1, 000 bu.

13

14

15

16

17

18

30 lb./l,000 bu.

19

20

21

22

23

24

50 lb. /1, 000 bu.

25

26

27

28

29

30

No treatment--controlB

31

32

33

34

35

36

No.

.8

^7.8

4.0

2.8

^8.8

yvo.

1.0

1.5

.3
.3
^9.0

3.5

No.

.5

2.5

No.

.3

1.0

No.

.3

.3
3.8

No.

^6.0

.5

2.3

No.

.3

.3

1.0

No.

.3

No.

.3

1.5

No.

No.
0.3

.5

.3

.5
^45. 8

^10. 3

No.

No.

0.3

2.5

.3

1.8
38.5

.8

.3

2.3

.3
.5

3.8
.5
.3

1.0

.3
327.5

.8

.5
2.0

No samples taJcen wherever dash appears.
^ Series terminated.
3 Test terminated; grain reached weevlly stage. After this count the bin was fumigated.

At the 50-pound dosage rate, the insect population reached the weevily level in one
bin in November 1954 (test 30); another in August 1955 (test 29); another in October 1955;
and the three remaining bins carried through until the series was terminated in January
1956.

All of the untreated controls became weevily before the end of the observation period.

- 8

Statistical Analyses of Performance

As stated previously the criterion for the elimination of a bin of wheat from a series
was whether the grain would be designated as weevily according to U. S. grain standards
The mean number of months of protection for a series of bins was designated as the num-
ber of bin-months of protection. The bin-months of protection for the treated series com-
pared with those of the controls are given below:

Treatment Bin-months of protection

No treatment--controls 12. 3

Ryania dust per 1, 000 bushels of wheat:

20 pounds 26. 8

30 pounds 32.

50 pounds 27. 8

The differences between the treated lots and the untreated controls, or between
treated lots, were tested for significance by the method of analysis of variance. This
analysis showed that there were significant differences. The least significant differences
between means were then computed. On the basis of this calculation there were found to
be no significant differences between the means of the treated lots, but the differences be-
tween the treated lots and the untreated controls were significant.

This analyses showed that the period of protection was significantly longer in the
treated series than that in the untreated controls. In a comparison of the treated lots,
the analysis showed that there were no significant differences between the three dosage
rates.

Bioassay Tests

Bioassay tests were conducted in order to observe any changes in the potency of the
ryania during the storage period subsequent to treatment. In these tests 25 adults of rice
weevils were confined in 3 ounces of wheat from both the treated and untreated bins, and
the resultant mortality was recorded after 1 and 3 weeks. Table 5 shows that at the
lowest dosage rate (20 pounds) there was a trend toward decreasing potency, but that at
the two higher dosages the killing power remained about the sanae throughout the 15-
month observation period.

Changes in the Moisture Content

The wheat used in these tests was dry, mostly below 11.0 percent moisture content.
The moisture determinations of the samples drawn at monthly intervals during the ob-
servation period showed that there was a continual transfer of moisture within the grain
mass. There was but little change in the overall moisture content, the usual moisture
accumulation being noted in the surface grain during the cooler nnonths of the year, fol-
lowed by a redistribution of it during the warmer months. No effect of moisture on the
protective treatment was observed in this series of tests.

Changes in the Comnnercial Grade

The commercial grades of the different bins of wheat, established at the beginning of
the study, at quarterly intervals during the observation period, and at the termination of
the observations in each bin, showed that there was no change in grade either because of
treatment or changes in grade factors.

TESTS WITH SHELLED CORN

These studies with shelled corn were directed toward the determination of the degree
of protection against insect infestation provided by the ryania formulations, changes m

- 9 -

TABLE 5. --Mortality of adult rice weevils in bioassay tests with samples taken at
intervals from wheat treated with ryanla protective diist

Mortality of weevils

in samples taken in-

--

Application rate and
length of confinement

1953

1954

July

Oct.

Jan.

Apr.

July

Oct.

20 Ib./l^OOO bu.

1 week »

Percent

100
100

100
100

100
100 .

1

5 •

Percent

40
100

63
100

72

100

1
10

Percent

70
79

76
100

83

100

2
3

Percent

15
74

17
96

16
95

2

Percent

4
59

39
99

25
96

2
2

Percent
22

3 weeks •

87

30 lb. A, 000 bu.

1 week. ••...•.•.•«••..•.•.

28

3 weeks

96

50 lb. /I, 000 bu.

1 week

24

3 weeks . » . «

No treatment — controls

1 week

98

7

3 weeks <

7

the moisture content, and changes in the connniercial grades during the period of observa-
tion. The results of three groups of tests are presented herewith.

Group ni: Tests With CCC-Owned Shelled Corn at Beattie, Kans. ,

Begun in September 1952

The corn used in these tests was of the 1949 crop, and had been placed in storage in
rectangular wooden bins in September 1950. The tests with ryania were begun in Septem-
ber 1952. Three formulations of ryania were used as stated in the section on materials.
The dusts were applied with a mechanical applicator attached to the auger tube, as the
corn was turned from one bin to another. Two bins were treated with the ryania dust at
45 poxxnds per 1, 000 bushels and Z at 60 pounds, 2 with ryania plus sulfoxide at 45 pounds
and 1 at 60 pounds, and 2 with ryania plus n-propyl isonne at 45 pounds and 1 at 60 pounds.
After the bins were filled with the treated corn, a capping of dust was applied to the sur-
face corn by running dust alone through the auger and depositing it on the surface corn.
The corn was then leveled and raked, so that the insecticide was distributed throughout
the top 6 inches of corn. Approximately 10 poiinds of dust were used in capping each bin.

A series of three untreated bins served as controls, and these were not turned.

Insect Population Trends

The bins were sampled prior to treatment to establish the initial insect population
density and distribution. After treatment the corn in each of the bins was sannpled at
monthly intervals and exannined for insects. The results are given in table 6. The insect
populations in the treated series were held at a low point throughout the observation
period, whereas those in the control series increased to the weevily level by September
1953.

Statistical Analyses of Performance

The data on the perfornnance of the ryania formulations were handled in the same
manner as for wheat. Bins were dropped fronn a series if they became weevily, and the
bin-months of protection were computed in the same manner as for wheat.

10 -

^^^^±Z^!-L'''^''.iri^i:;::^^^ -.u.aUo„. . a...... ...... 3„p.... „„

Formulation,

application rate,

and test number

Ryania

A5 lb. /I, 000 bu.

38

60 lb
39.

./1, 000 bu.

40

Ryania-

45 lb

41.

sulfoxide
./l.OOO bu.

42

60 lb
43.

./1, 000 bu.

45 lb. A, 000 bu.
44

45

60 lb
46.

A, 000 bu.

No treatment --controls
47

48

49

1952^

Sept,

^o.
16.7
53.3

15.3
9.7

Oct.

3.0

12.0

2.3

Ho.
1.0
3.7

2.0
1.3

74.0
2.2

53.5

2.3

15.3

^1.7

3.0

12.0

2.3

Nov.

No.
0.7
2.3

1.0
.4

2.7

Dec.

No.

Living Insects found per 1,000 grama of ahcUcd com in-

1953

Jan.

No.

3.0

Feb.

4.0

12.7

1.3

4.7
1.3
1.3

5.1
3.0

No.

.4

Mar,

iVo.

2.3

7.7

Apr.

1.4
2.3

.4 .7 1.4

;vo.

0.7

May

1.4
3.0

1.4
1.7
1.4

No.
0.4

July

2.7

.4

No.

Aug.

.4
9.0

.4

No.

Sept.

2.7

''63.4

3.0

No.
0.4
.2

20.0
^■30.0

A'o.

1.3

Nov.

jVo.

Dec

A'o.

•(*)

•('•)

-(')

Ao.

1.0

Formulation,

application rate,

and test number

Ryania

45 lb. A, 000 bu.

37

38

60 lb. A, 000 bu.

39

40

Ao.

Living insects found per 1,000 grams of shelled com in-continued

1954 — continued

June

No.

July

Ryania-sulfoxide
45 lb. A, 000 bu.

41

42

60 lb. A, 000 bu.
43

Ryanla-n-propyl isome
45 Ib.A.OOO bu.

44

45

60 lb. A, 000 bu.
46

No treatment--controls

47

48

49

No.

Aug,

No.

Sept.

yvo.

1.0

Oct.

No.

No.

.7

No.

1955

No.

1.0

2.0

Apr.

_L

No.

May

No.

July

Aug.

Sept.

A'o.

0.7

Oct.

Ao.
1.0

1.7

1.0

Nov. Dec

No.
'0.

First samples ta]£en prior to treatment.

No samples taken wherever dash appears.
^ Series terminated.

Series terminated. Downgraded to sample grade because of commercially objectionable foreign odor due to treatment.
^ Terminated - excess moisture.
'^ Insect population reached the "weevily" level. Bin dropped from series and fumigated. ■* '

The bin-months of protection for the Group III series of tests are tabulated below:

Treatment Bin-months of protection

No treatment- -controls 11

Ryania dust per 1, 000 bushels of corn:

45 pounds 40

60 pounds 40

Ryania- -n-propyl isome per 1,000 bushels of corn:

40 pounds 40

- U -

The bins treated with the ryania- sulfoxide formulation are not included in the tabula-
tion because the sulfoxide imparted an objectionable odor to the corn. The records for
the bin receiving the 60-pound dosage rate of the ryania-n-propyl isome formulation were
omitted because the corn had to be disposed of due to excessively high moisture before
the test was completed.

When tested for significance by the method of the analysis of variance, the mean dif-
ference between the treated series was not significant, but the difference between the
treated lots and the untreated controls was highly significant. The variation ratio "F"
was 1401.7, a much greater value than the 5.41 required for significance for this group.

The highly significant difference between the treated lots and the controls demon-
strated that the dosage rates were greater than needed for effective protection over the
40-nnonth observation period.

Bioassay tests

In the bioassay tests, the depletion of the ryania and the synergized formulations of
it proceeded very slowly during the period of observation (table 7).

Changes in the Moisture Content

The com used in these tests had been in storage for 2 years before the protective
dusts were applied, and was dry enough for storage. The usual seasonal transfer of
moisture within the grain mass was noted, but there was no overall change in the mois-
ture content. There was no apparent effect on the performance of the protective dusts
attributable to variations in moisture content.

Changes in the Commercial Grade

No change in commercial grade of the corn was noted except in that treated with the
ryania- sulfoxide formulation. In the opinion of the Chicago Board of Grain Supervisors,
this corn was not acceptable because of a rancid lard odor. Such corn was graded Sample
COFO (commercially objectionable foreign odor). Corn treated with the other formula-
tions was acceptable.

Group IV: Tests with CCC-Owned Shelled Corn in niinois

Begun in May 1953

The corn used in these tests was of the 1952 crop and in May 1953 was shelled on
the farm and delivered to CCC bin sites in Illinois and stored in USDA 3, 250-bushel
metal bins. The dusts were applied with a mechanical applicator attached to the auger
tube as the corn was unloaded from the delivery trucks.

One series of 10 bins was treated with ryania dust at a dosage rate of 20 poiinds per
1, 000 bushels, and another series of 1 1 bins at 30 pounds per 1, 000 bushels. A third series
of 19 bins receiving no treatment served as controls.

Insect Population Trends

After the bins were filled, samples were drawn at monthly intervals in the manner
described for circular metal bins (p. 3). The samples were taken to the laboratory and
the number of insects and the moisture content determined. The results are given in
table 8. Even though much of the corn was of high moisture content, and thus provided
a favorable environment for insect population increase, none of the treated series be-
canne weevily. Six of the 19 bins in the untreated control series became weevily within 4
months after they were filled, and 5 others developed insect populations exceeding 10 per
1,000 grams of corn. The entire series had to be terminated in November 1953 as much
of the corn stored on these sites became unmanageable because of excessively high moisture
content.

- 12 -

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13

TABLE 8. — Insect populations in shelled

dust at spe

com in Illinois following application of lyani J
cified rates, Afey 1953 1

Dosage rate and
test number

Living insects found per 1,000 grams of shelled com in — 1

June

July

Aug.

Sept.

Oct.

Nov.^

20 lb. A, 000 bu.

50

Number

1.3

.5

.3

.3

0.3
0.5

1.0

0.5

.3
1.0

.5

.1
.5

Number

.5

0.3

.3
.3

2.5

.3

.8

.3
7.3
3.3
2.3
2.0

11.5
8.3

^1.0
3.8

5.0
.8
5.5
3.0

.5
4.3
1.8
4.5

Number
0.3
.3
1.5

\8

^0

1.0

3.0

1.5

^0
lO
.5

1.0

3.5

234.5

239.0

2.0

4.8

7.5

20.0

S.3

^2.8
3.5

^0
2.5

^1.8

1.8

222.3

1.5

6.0

Number
0.8
.5
^0

\5
K3

.8
.3

^0

.3

^12.3
2.5

17.3

2.0
^5.5

249.3

^6.8
212.3

Number

4.0

.8

^0

1.3
^0
^0

^0
.3

14.0
15.0
17.5

231.5
9.5

Number

51

0.3

52

53

0.3

54

55

56

57

58....,

59

30 lb. A, 000 bu.

60

62 o

.8

64

65

67

69

70

No treatment — controls

71

0.5

73

75

20.3

12.3
16.5

11.3

77

79

80

81

83

85

87

89

- ■'■ The Illinois series of bins had to be tenninated in November 1953 as much of the com
stored on these sites became unmanageable because of high moisture content.

2 Test terminated; grain reached weevily stage. After this count the bin was ftmiigated.

14

statistical Analysis of Perfo rmahce

The data on the performance of ryania in stored corn in Illinois (Group IV) were
handled in the same manner as for Group III, The bin months of protection for the Group
IV series are given below:

Treatment Bin-months of protection

No treatment--controls 3.8

Ryania dust per 1, 000 bushels of corn:

20 pounds 4.7

30 pounds 4.8

When tested for significance by the method of the analysis of variance there were no
significant differences between the means of either the treated bins or between the
treated bins and the untreated controls. The variation ratio of 2. 56 was less than the
value of 3. 25 required for significance for this group. This was due to the short tinne
that the series were under observation.

Changes in Moisture Content

As noted previously, much of the corn put into storage on CCC bin sites in the spring
of 1953 contained too much moisture to store safely. The moisture content of the corn
ranged from 11.4 to 16. 1 percent, with some loads running as high as 19.0 percent. For
this reason much of it became unmanageable.

Changes in the Commercial Grade

The commercial grades of the bins of corn under observation taken at the beginning
and end of the storage period showed that there was no downgrading due to the treatment.
Of 18 bins in Group IV, 10 were graded sample on receipt at the bin site, and the other
8, received as number 1, 2, or 3 yellow, were downgraded to sample at the end of
storage because of the increase in the amount of total damage.

Group V: Tests with CCC-Owned Shelled Corn in Saline
County, Mo. , begun in July 1953

The corn used was of the 1952 crop and was delivered to CCC bin sites in Saline
County, Mo. , during July 1953. The dusts were applied with a mechanical applicator
attached to the tube of the loading auger as the corn was unloaded from the delivery
trucks.

Three series of bins were treated with ryania dust at rates of 20, 30, or 50 pounds
per 1,000 bushels of corn.

A fourth series of 7 bins receiving no treatment served as controls.

Insect Population Trends

After the treatments had been applied, samples were drawn at monthly intervals in
the manner previously described for circular metal bins. The samples were taken to the
laboratory and the number of insects and the moisture content determined. The results
are given in table 9. In the treated series excessive moisture in the corn caused more
trouble than insect infestation.

In the series treated with 20 pounds per 1, 000 bushels, one bin of corn became
weevily in March 1955, another in August 1955, one was dropped because of excessive
moisture content, and the fourth was carried through until the test was terminated m
February 1956,

- 15 -

TABLE 9." Insect populations In sheUed com follo»liig application of ryanla dust at specified rates in Saline County, Mo., in August 1953

Application rate of
ryanla and test number

20 lb./l,000 bu.

91

92

93

94

30 Ib.A.OOO bu.

95

96

97

98

50 lb./l,000 bu.

99

100

101

102

No treatment — controls

103

104

105

106

107

108

109

Living insects found per 1,000 grams of shelled com in

1953

Aug.

No.

.3
.3

1.3

1.8

2.0

.3
.3

Sept.

JVo.

1.3
.5
.3

2.3

.5
.5

1.5

2.5

1.3
1.0

10.8
K.3
8.0
12.0
8.3
5.0
3.5

Oct.

No.
4.5
4.3
1.0
4.5

1.0
5.5
2.8
1.8

.3

5.0

■^32.8
^92.8
^17.5

4.5
^37.5

8.0
^49.8

No.
23.3

8.0
2.3
1.0

4.5
'29.5
^13.0

3.5

.5
8.3

1.3
.8

2.8
1.8

Dec.

No.

1.0

5.3

Jan.

Feb.

2.0
2.3

1.0
4.3

No.

.3

No.

Apr.

No.

.3

May

No.

2.3

1.0

3.3

2.5

1.5
.3

No.

0.3
.5
.5

3.3

1.5
.3

July

No.

'13.3

Aug.

No.

1.0
2.0

.3

Sept.

No.
0.8

.5

6.8

Oct.

No.

.5

2.5

8.8

3.5

Nov.

No.
0.3

8.3

Dec.

No.

16.5

Application rate of
ryanla and test number

Living insects found per 1,000 grams of shelled com in—

1955

Jan.

20 lb./l,000 bu.

91

92

93

94

30 lb.A,000 bu.

95

96

97

98

50 lb./l,000 bu.

99

100

101

102

No treatment — controls

103

104

105

106

107

108

109

No.

2.0
22.3

No.
0.3
.5
"0

Mar.

No.
0.3
1.0

^200.0

Apr.

No.

1.3

May

No.

0.3

Jxme

No.

July

No.

1.8

Aug.

^2.3
1.8

No samples taien wherever dash appears.
Test terminated; grain reached weevily stage. After this count the bin was fumigated.
•" Series terminated.

I>ropped from series because of excessive moisture.

Sept.

No.
1.5

1.0

Oct.

No.
0.8

Nov.

1.0

.3

^2.3

Dec.

JVo.
2.3

1956

Jan.

^3

In the series treated with 30 pounds, two bins became weevily in November 1953,
one had to be dropped because of excessive moisture, and the fourth was carried through
until the test was terminated in February 1956.

In the series treated with 50 pounds, the first weevily bin did not appear until Octo-
ber 1955, two had to be dropped because of excessive moisture, and the fourth was car-
ried through until the test was terminated in February 1956.

In the untreated controls, the insect populations increased to the weevily level in 5
bins in October 1953, in 1 bin in July 1954, and in another in November 1954.

Statistical Analyses of Performance
The data on the performance of the formulations used in Group V were handled in the
same manner as for the preceding groups. Bins were droppedfrom a series if they became

- 16 -

weevily and the number of bin-months of protection were computed. The bin-months of
protection for the Group V series are tabulated below:

Treatment Bin-months of protection

No treatment- -controls (^ i

Ryania dust per 1, 000 bushels of corn:

50 pounds 35 q

30 pounds Y2.. 3

20 pounds 24. 7

The differences between the treated bins and the untreated controls, or between
treated bins, were tested for significance by the method of the analysis of variance. This
analysis showed that there were significant differences. The least significant differences
between means were then computed. On the basis of this calculation, there were found
to be significant differences between the untreated controls and the dosage rate of 50
pounds; the untreated controls and the dosage rate of 20 pounds; the 50 and the 30-pound
dosage rates; and the 30 and the 20-pound dosage rates.

This analysis showed that the period of protection was significantly longer in the
corn treated with 50 pounds and 20 pounds than in the untreated controls. The difference
between the 30 -pound rate and the controls was not significant due to the wide variability
and small number of bins in the treated series that were not adversely affected by exces-
sively high moisture content of the corn.

Changes in the Moisture Content

The corn used in the Group V tests was not sorted as to moisture content as it was
delivered to the storage site. The result was that there were wide differences in the
moisture contents of the different lots of corn put into the same bin. In average samples
taken in August 1953 soon after filling the bins the moisture content ranged from 11.6
to 13.5 percent. Samples drawn in January 1956 showed that there had been but little
change in moisture content during the storage period.

Changes in the Commercial Grade

No changes in the commercial grade were noted as being due to treatment. However,
of 19 bins in the series, 15 of them were downgraded one or more grades because of in-
creases in the grade factor "total damage". This was due largely to the accumulation of
moisture in the surface corn which caused molding and caking.

COMPARATIVE ABUNDANCE OF THE SPECIES OF INSECTS

The monthly samples drawn from the bins were taken to the laboratory and the num-
ber and species of stored-grain insects were recorded. The comparative abundance of
the different species in wheat and shelled corn is given in tables 10 and 11, expressed as
percentages of the total number of insects found during 3-month periods.

In the treated wheat (table 10), 7 species of stored-grain insects were found, the most
abiindant species being the dermestids (Trogoderma spp.) followed by the lesser grain
borer and the flat grain beetle. In the vintreated controls, 9 species were recorded, the
most abundant species being the lesser grain borer, followed by the dermestids and the
saw-toothed grain beetle.

In the treated corn (table 11), 15 species of stored-grain insects were noted, the
most abundant species being the red flour beetle, the Angoumois grain moth, and the
foreign grain beetle, in the order named. In the untreated corn I6 species were found,
the most abundant being the red flour beetle, the flat grain beetle, and the saw-toothed
grain beetle.

- 17 -

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- 20 -

FINDINGS

The following points were demonstrated in the tests reported herein:

1. Ryania formulations protected stored wheat and shelled corn through long periods of
storage.

2. Dosage rates of 20 pounds per 1,000 bushels or higher in wheat and in shelled corn
provided excellent protection from insect infestation through two full summer seasons
after the year in which they were applied.

3. Bioassay tests showed that degradation of the ryania in the treated grain was very
slow.

4. About 80 to 90 percent of the ryania could be removed from the treated grain by a
simple sifting.

5. The residues in the milling fractions of wheat were about equally distributed in the
feed and flour fractions and were reduced to relatively small amounts as related to
the dosage rate.

6. The moisture content of the grain had no observable effect on the treatments.

7. The commercial grade of the grain was not affected except that of the shelled corn
treated with the ryania -sulfoxide formulation, which imparted an objectionable odor.

8. Nine species of stored-grain insects were found in wheat and 16 species in shelled
corn. The dominant species in wheat were the dermestids, the lesser grain borer,
the flat grain beetle, and the saw-toothed grain beetle; in corn, the red flour beetle,
the flat grain beetle, the saw-toothed grain beetle, and the Angoumois grain moth.

21 - « u. S, GOVERNMENT PRINTING OFFICE 1958 — 468439