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- ‘TECHNICAL Bee No. 134 NovEMBER, 1929

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RED-SQUILL POWDERS
AS RATICIDES

BY
J. C. MUNCH

Consulting Pharmacologist

JAMES SILVER

Associate Biologist

AND

E. E. HORN

Associate Biologist

Division of Predatory-Animal and Rodent Control
Bureau of Biological Survey

Unitep States DEPARTMENT OF AGRICULTURE, WaAsHINGTON, D. C.

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Novemser, 1929

TECHNICAL BULLETIN No. 134

UNITED STATES DEPARTMENT OF AGRICULTURE
WASHINGTON, D. C.

RED-SQUILL POWDERS AS RATICIDES

By J. C. Muncu,! Consulting Pharmacologist, JAMES SILVER, Associate Biologist,
and E. KE. Horn, Associate Biologist, Division of Predatory-Animal and Rodent
Control, Bureau of Biological Survey

CONTENTS
Page Page
Purpose of investigation___......___.-------- 1 | Results of investigation—Continued.
Source and structure of squill_______________- 2 Toxicity of white squill to rats_.______-___ 22
Results of previous investigations____________ 2 Toxicity of calcium oxalate to rats_______ 22
wo DETREI ae eg Se i 2 Preparation of red-squill powder on a
LenS Clerc, ae ee ee 3 semicommercial basis_______-__________ 22
it es See S28 00S) Oe BE ie DOES oat 3 Relative susceptibility of white rats and
Use of squill as rat poison______--.------- 4 of wild (brown) rats to squill powders_ 24
Experimental procedure____........--------- 5 Effect of red-squill powders on domestic
Preparation ofjpowders_____ +... -.-=_--- 5 and@voinermanimals. 2. 2 toe 8 5 oe 25
MePuCuiGLiecning oo 9 Yields of toxic squill powders (‘‘rat
Effects of squill on rats.._.__...-_-.____- 10 Wists se tas i Be ee oe aly 27
Results of investigation_______._____________- 10 Stability of squill powders______________- 31
Effect of temperature on toxicity___-_____ 19 A CEQDEATICRL OSES. 4 ee we WT en ee 31
Direct eet drying versus preliminary air Commercial squill rat poisons___________ 31
Gregan sabes tT) tev BON ET 19 Extraction of toxic principles by various
Effect al fermentation on toxicity and SOlVeHiasepl- Pa tagrriiiies 3} lee aries 32
TE ae 60) (6) ee a en | oS 20 | Suggested method of preparation of toxic
Effect of variability of squill bulbs on SOUUIGHOMNGOCL Se. ee 33
Sci ts ie 5 SEE) Sets 8 CR at ee DORI AOOMCHISIONS§_.-vommen 1S a3) 20 35> 3 en 34
Effect of intensity of color of squill bulbs Miteratwrew ited: ct 52 ts Le Lek 35
GrNCOMIGIGY sc2 S435 6 oe tae Sa es 21

PURPOSE OF INVESTIGATION

The principal requirements for an ideal rat poison are safety,
effectiveness, and economy. The rat poisons in most general use
to-day (arsenic, barium carbonate, phosphorus, and strychnine) have
toxic properties that seriously menace the safety not only of wild
and domestic animals, but also of human beings. Red squiil is rela-
tively safer and more nearly approaches the ideal rat poison. Animals
other than rats usually refuse to eat red squill in the concentrations
used for rat baits. The difficulty of obtaining a uniformly toxic
preparation of red squill has retarded its development for this pur-
pose. To contribute to the information needed for the production
of a potent red-squill preparation for the efficient destruction of rats,
the investigation here reported was undertaken.

Acknowledgment: The work here reported was a cooperative study by members of the Bureaus of Chem-
istry, Biological Survey, and Plant Industry. The writers wish to express their thanks to the following for
assistance in various ways Curing the four years of the relay yp Bureau of Chemistry—N. G. Bar-
bella, R. M. Hann, D. B. Jones, G. L. Keenan, B. A. Linden, E. W. Schwartze, K. A. Smith, and C. G.
Spencer; Bureau of ioe Survey—C. C. Carr, F. N. Jarvis, and M. A. Stewart; Bureau of Plant In-
dustry—O. F. Black, J. W. Kelley, A. F. Sievers, and W. W. Stockberger.

1 Formerly pharmacologist of the Bureau of Chemistry.
53580°—29—_1 1

2 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

SOURCE AND STRUCTURE OF SQUILL

Squill, Urginea maritima (L) Baker, also called U. scilla Steinheil,
is a perennial plant growing wild on the coast of southern Italy,
Sicily, and Sardinia, and elsewhere along the Mediterranean Sea.
The bulbs produced are pear shaped, usually from 3 to 6 inches in
diameter and from 300 to 2,000 grams in weight (pl. 1), and are
composed of closely overlapping fleshy scales. ‘The outer scales are
dry, brittle, and reddish brown; the inner scales vary from light
yellowish white to deep cherry or mahogany; and the central ones
(core) are usually white. _ :

There are two commercial varieties of squill—white squill, which
is marketed as dry, white scales and is the product official in various
pharmacopeeias, and red squill, which until recently was obtainable
only in the fresh state. Both the viscous, mucilaginous juice and
the dry scales of each variety are irritating to the skin. This sting-
ing effect upon the skin, as well as the acrid prickly taste, is commonly
attributed to calcium-oxalate raphides. White squill is used in human
medicine as a heart tonic, emetic, diuretic, and nauseant expectorant.
Various European investigators have recommended red squill as a
rat poison.

Microscopic examination of squill was made by George L. Keenan,
of the Food, Drug, and Insecticide Administration, who states:

A microscopic examination of red-squill powder shows that the anatomical
structure is similar to that of white squill. ‘The powdered bulb of both varieties
consists of fragments of parenchyma cells, spiral vessels, and long raphides of
calcium oxalate, some of which are surrounded by dried masses of transparent
refractive material, the mucilage sheath. The longest acicular crystals are ap-
proximately 1 millimeter long. Short raphides are present also. The greatest
diagnostic difference is the presence of pigment cells in the red-squill scales and
their absence in the white. The red pigment cells are numerous in the scales
of the red squill and are readily found in the powdered material.

To facilitate its identification, the red squill was reduced to a fine powder
and examined microscopically in a drop of menstruum consisting of an acidified
chloral-hydrate-glycerol solution (made by dissolving 45 grams of crystals of
chloral hydrate in 25 cubie centimeters of dilute hydrochloric acid (1 : 8) and
10 cubic centimeters of glycerol). The slide was not warmed. ‘The red coloring
matter gradually diffused out of the cells, giving the pigment masses a reddish
appearance. Long raphides of calcium oxalate were found in the dried mucilag-
inous material, many in close proximity to the red masses.

When a powdered dry bait containing 5 per cent of red squill was examined
according to the method just described, the pigment cells were readily found.

RESULTS OF PREVIOUS INVESTIGATIONS

CHEMICAL

An exhaustive review of the chemical studies on squill since 1741
is given by George (127). The active principle has not been definitely
isolated and identified. Many of the products obtained by extrac-
tion with various solvents have proved to be impure mixtures. In
many instances it is not plain whether red or white squill was studied.
George states that the active constituents of squill are (1) scillitin,
C,,H,;03, a yellow, very bitter, nitrogen-free glucoside, slightly solu-
ble in water (0.18 per cent) and soluble in alcohol, which was isolated

2 Reference is made by italic numbers in parentheses to ‘‘ Literature cited,” p. 35.

PEATE 1

Tech. Bul. 134, U. S. Dept. of Agriculture

Woelea

(WNILIMWW VSANISYN)

81nd T1INSS-G3ay

RED-SQUILL POWDERS AS RATICIDES 3

by Kopaczewski (18) as the toxic principle of squill; (2) scillidiuretin,
a strong diuretic, also isolated by Kopaczewski; (3) xanthoscillide,
a yellow crystalline glucoside, insoluble in chloroform, but soluble
in boiling alcohol, isolated by Buschmann (2); and (4) a water-soluble
toxic substance isolated, but not identified, by Ewins (8). George
obtained a red glucoside from African squill, Urginea_burker, which
corresponded in properties to Merck’s scillitoxin. (Scillitoxin has
not been chemically identified as a definite chemical entity.) Gala-
vielle and Cristol (11) found the same chemical principle in Scilla
autumnalis L. grown in the neighborhood of Montpellier, France, as
in S. maritima. <A patent has been granted to Stoll and Suter (27)
for a process of isolating a physiologically active glucoside, “Scillaren,”’
from white squill. The chemical composition and formula are not
given in the patent.
PHYSIOLOGICAL!

Lewin (21) reported that animals receiving squill showed a slower
pulse, dyspnea, and pyrexia, and died on account of respiratory fail-
ure. Danysz and Kopaczewski (6) stressed the paralysis of the hind
limbs, convulsions, and characteristic gyrations of rats following the
consumption of squill. They state that the respiration is greatly
depressed and that after several gasps the animal dies. Gunn and
Heathcote (14) found that the subcutaneous minimum lethal dose of
the glucoside prepared by Ewins’s process (8) was 5 milligrams per
kilo of body weight for the rabbit and 150 milligrams per kilo for
the rat; also, that much stronger concentrations were required to
stop the isolated rat heart than to stop the isolated rabbit heart in
the same space of time. They concluded:

It is, of course, still possible that the rat may be more susceptible than other
domestic animals to squill taken by the mouth; but this, if true, must be due, say,
to some constituent of squill other than the glucoside or to some difference in
action than the action on the heart. The possibility that this alleged hyper-
susceptibility of the rat may be due merely to the fact that the rat is the only
domestic animal that will eat squill has not been sufficiently excluded * * #*,
The rat is markedly less susceptible than the rabbit.

The present experiments tended to confirm the conclusions of Gunn

and Heathcote. Death following squill is apparently produced by
respiratory rather than cardiac action.

TOXICITY

_ The toxicity of squill (whether of the red or of the white variety
is not always stated) has been studied by a number of investigators
since the first experiments by Orfila in 1818 (22, v. 2, pp. 99-102).
The reports are summarized in Table 1.

The great apparent variation in toxicity may be attributed to
differences in products, in methods of preparation, and in adminis-
tration. Relatively enormous quantities of squill are apparently
required to injure animals other than rats.

’ While this bulletin was being prepared two articles by Winton (28, 29) were published. Winton
concluded that ‘‘ the cardiac glucoside and the rat-poisoning principle in red squills are distinct substances.
The former occurs in about equal amounts in red and white squills. The rat-poisoning substance, however,
is present in significant amounts in red squills only.”

4 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

Taste 1.—L fect of squill preparations upon animals (reported in literature)

Reference Animals Product Dose Result
“ Dried bulb_______- l'gramie. ee: Death
soul inte 5 cae Sats, -- sons re~eee lB ba pissce- tor d SAMs. art seereeeal Do
ee" ried bulb_______- Tey Ree ee Do
#opecsewakl (18) -~--|.—--+ G0. n+ senoon Welling £. -cacea! Img./kg..-----...- Do.
Red-squill powder | 260 to 1,800-+mg./kg_ Do.
W hite-squill | 9,000 mg./kg_________ Survived.
Claremont (6)...-.---|--<_- Py eceee SALT ee powder.
Urginea burkei | 2,000 mg./kg___-____- | Death.
powder.
i ee gape wees oy marion marta torrie SEL: GEE ee oe ah te SOE fee ok See | Do.
(7).
Howarth (1/6, pp. 16- |_____ DS SNS ee Soe Scillitin. ¢ .. _F 0.02 to.0.05 mg__...- Do.
67). Squil- e kt SP ie.) 2 *___ Se | Do.
Orin (Ati V.~2; *pp, |} Degser ivy wezrctn 5 > Fresh bulb_______- 246 ounces__________- Do.
Lewin (#2) - 22-1222 Guinea pigs_.__.-_.- Scillitoxin________- 05 amg.._-_ cL LLL Do.
Dogs and rabbits____}_____ eee ee 100 to 500 mg_______- Do.
| Dogs, cats, and hogs_| Fresh bulb_______- 0.25 to 2 grams_~_3__ Vomiting, diar-
: rhea, diuresis.
Hertwig (10, Dp. 196) - - = a (2) Be. Pos. ee ee et ae 45 prams. oo sees Death.
Horse and cow-____-_-_|_____ Mee eee SQ Prame-s SS ee Diarrhea
; 2 hersesa: a sahcee_ URE EE Peey eee a 6Digramseyoiss2s_2_ 3 Death.
Kopaczewski (18)__-_| Guinea pigs________- orci | = 2 0 aa T Mg /Re eee Do.
(TDidgs- fii ieee $00) 1s ig) JER Aes Go) 2212. BE Do.
Claremont (4)_------- Cats SEES ONS ESTE Bat VOLO Me SUORIS 2, [Ke ne Do.
Leaps. 7 eee Aker get Meo. 2. Soee on Bir Re ie oe ee Do.

USE OF SQUILL AS RAT POISON

The Danish antirat law of 1907 forbade the use of strychnine,
arsenic, and sulphurous preparations in rat control but permitted
various squill preparations. Aumann (J) lists nine commercial
squill preparations offered for sale in Germany in 1912; he also states”
that practice had confirmed the laboratory finding that squill prepa-
rations are twice as efficient as raticides as are the bacterial poisons.
Smith (26) found that only one of five chemical extracts on the
English market in 1921 killed rats. Aqueous, alcoholic, and acetic-
acid extracts of dried white squill failed to kill rats, although each of
three alcoholic extracts of dried red squill proved fatal.

Claremont (4, 5) reported an intensive study of squill as a rat
poison. The lethal doses of various red-squill powders ranged from
260 to more than 1,800 milligrams per kilo of body weight to rats
and did not seem to bear any relation to the chemical characteristics
of the preparations. The reducing sugars ranged from 3.32 to 10.45
per cent; and the total sugar after inversion from 6.94 to 73.73 per
cent. Hydrochloric acid intensified the red color of the extract.
Some powders and pastes on the British market were found to be
toxic to rats. -White-squill powders failed to kill in doses of 9,000
milligrams per kilo of body weight. South African squill, Urginea
burkei, gave more mucilage than the ordinary varieties, and only
one of three powders killed, the lethal dose of it being 2,000 mulli-
grams per kilo of body weight. A drying temperature of 60° C.
appeared to produce a satisfactory powder, although the lethal prin-
ciple was not destroyed in preparing squill biscuits at temperatures
around 100°. The powder preserved its toxicity when stored in
air-tight containers. Cold and hot water extracts were toxic, but
alcohol failed to extract the toxic principle. Wild rats were rather
more susceptible than tame rats. Microscopic examination showed
no specific characteristic except the large number of raphides. Clare-
mont concluded that the toxicity of squill preparations can not be
inferred from analytical results but must be determined by feeding
experiments on rats.

RED-SQUILL POWDERS AS RATICIDES 5

Kobert (17) quotes Kunkel’s findings that red squill is more potent
than white, that the outer scales act more intensely than the inner
scales, and that the bulbs gathered in summer are more potent than
those collected in the spring.

Lantz (19), Lereboullet (20), George (12), Howarth (/6), and
Schlupp (23) report the suitability of red squill as a rat poison under
various conditions. Howarth states further that “it 1s, moreover,
regarded as comparatively harmless to domestic animals.”” W. K.
Tonkin, rat officer of Middlesex County, England, reports * that
phosphorus pastes, barium carbonate, and one or two proprietary poisons have
on occasion been used, but freshly extracted squill, both as a lethal rat poison
and by reason of its being comparatively harmless to domestic animals, has
been found of greater use than any other poison.

Boulenger (3) found that squill solution was the most effective rat
poison, being three times as toxic as barium carbonate to rodents and
also less harmful to domestic animals. _ ;

Finally, the British Ministry of Agriculture and Fisheries (13)
recommended the use of squill:

Baits containing barium carbonate or red squill * * * are recommended
by the ministry, and if used as directed, and with proper precautions, are less
dangerous than baits containing such poisons as arsenic, strychnine, and phos-
phorus * * *, Red squill is the safer, and it is advisable to use it in pref-
erence to other toxic agents for application on farms and in places where special
care is necessary owing to the presence of poultry, livestock, domestic animals,
or stored food supplies * * *. Owing to the peculiar chemical nature of red
squill in its various forms, as sold for the destruction of rats and mice, users of
these preparations should satisfy themselves that they obtain guaranteed toxic
red-squill raticides.

Recapitulating, the literature indicates that red squill is toxic to
rats whether fed as the fresh bulb or as a powder, that it is essen-
tially nontoxic to animals other than rats, and that white squill is
nontoxic to rats (25). Apparently, the toxic principle has not been
consistently isolated by chemical methods.

EXPERIMENTAL PROCEDURE
PREPARATION OF POWDER

In the preliminary experiments, 24 powders were prepared from
samples taken from a 900-pound keg of squill bulbs imported from
Cagliari, Sardinia, in the fall of 1923 and kept in storage at 4° to 5° C.
This entire series of powders was prepared by Otis F. Black and
James W. Kelley, of the Office of Drug, Poisonous, and Oil Plants,
Bureau of Plant Industry, United States Department of Agriculture.

The outer, dry, reddish-brown husks, which were found to be less
toxic than the inner scales, were stripped from all bulbs. The bulbs
were sliced and, in most instances, air-dried for several hours and
placed in long glass tubes. A current of air was passed over the
surface. The temperature was increased over a period of several
days from room temperature to a given maximum, which was main-
tained until constancy in weight indicated that the products were
thoroughly dried. They were then removed and ground in a drug
mill until the powder passed through a 40-mesh sieve. These powders
contained 1 to 2 per cent of moisture. After being ground, all powders
were placed in tightly closed screw-top vials. Details of time and
temperature are given in Table 2.

4 Personal communication.

TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

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RED-SQUILL POWDERS AS RATICIDES 9

As the results of these preliminary experiments indicated that
squill is of value as a rat poison, the investigational work was con-
tinued, material from a second shipment of approximately a ton of
squill bulbs from Sardinia being used. Upon reaching the United
States in the fall of 1925, the 200-pound bags of squill were stored
at 4° to 5° C. Samples were drawn from time to time for the prepara-
tion of powders.

Essentially the same method of preparation was used as in the
preliminary experiments. As previous investigation had indicated
the desirability of using high temperatures in order to check fer-
mentation or enzyme action quickly, an electric oven that could be
set to within 1° was used for drying most of the powders in this
series. In some instances it was desirable to duplicate commercial
conditions. For such drying a commercial steam drier was used.
Dried material was ground to pass through a 40-mesh sieve and packed
in jars. The approximately ¥-inch-thick slices obtained in the com-
mercial runs by using a hand-operated slicing machine proved to be
too thin, as a large quantity of mucilaginous juice was exuded, facil-
itating fermentation and materially retarding drying. Slices half an
inch thick were found to be more satisfactory.

By using the method finally adopted for the preparation of squill
powders (p. 33), it has been possible to reproduce powders having

the same toxicity.
METHOD OF FEEDING

Most of the toxicity determinations were made on white rats.
Wherever feasible, results on wild (brown) rats were also obtained.
All rats were held in cages for about a week to make the experimental
animals essentially uniform. No rats weighing less than 100 grams
each, and when possible only those that weighed between 150 and
250 grams each, were used. None of the rats received food for 18
hours, but all were given water freely, to insure a somewhat constant
appetite and to favor the rapid consumption of the experimental diet.
As previous experiments had indicated that rats readily consume
1 per cent of their body weight, efforts were made to feed approxi-
mately this quantity of total food.

Animals were weighed and placed in separate cages. The ordinary
laboratory rat food—99 per cent of a mixture of whole-milk powder
(one-third) and whole wheat flour (two-thirds) and 1 per cent of salt—
was passed through a 40-mesh screen. ‘The sample of squill to be fed
was thoroughly mixed with this food, so that approximately 1 per cent
of the animal’s body weight would be fed in giving the desired dose of
squill. The weighed squill-rat-food mixtures were placed in individual
glass sponge cups. The time when these cups were placed in the
cages was noted, and frequent inspections were made to determine
the time at which all food had been consumed. The dish was then
removed and the rat left without food and water until the next
morning.

Frequent inspections were made to determine the onset of symptoms
of squill poisoning. Several times the fatal dose of fresh scales or of
food containing squill powder has been readily eaten. Concentra-
tions up to 40 per cent of squill powder in rat food have been rapidly
and completely devoured, indicating that the presence of calcium-

53580°—29——_2

10 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

oxalate raphides does not deter a starved rat from eating these
products. On the day following the feeding of an experimental diet,
animals that were not in an obviously depressed condition or that
were not gyrating viciously were offered the regular diet and water.
All rats that died within five days after having been fed squill, and
in which squill symptoms had been produced, were considered to
have been killed by squill.

The detailed Secialine obtained from about 3,000 white and 400 wild
(brown) rats are reported in Tables 3, 4, and 5. The preparations as
made were given ‘‘P. C.” (pharmacology card) numbers to identify
them. As these experiments were carried on over several years, the
date of each series of feeding tests is given to show that there was at
least a general agreement in susceptibility of test animals throughout
the entire investigation. The quantities of dried squill powder fed
are given in grams. The numerator of each fraction shows the
number of rats dying from the indicated dose within a period of five
days; the denominator shows the total number of rats fed the indi-
cated dose. The MLD (minimum lethal dose) was selected as the
smallest dose of a dried squill powder, in milligrams per kilogram body
weight, that killed all, or practically all, the rats within five days.

EFFECTS OF SQUILL ON RATS

Rats that have eaten a toxic dose of squill soon become somewhat
lethargic. From 4 to 14 hours later they usually exhibit character-
istic tremors and sensory depression in the hind legs. This is fol-
lowed by progressive paralysis of the trunk and forelegs. Respira-
tion increases in rate and becomes labored. The animal next starts
a peculiar, extremely characteristic rotation on its long axis. These
gyrations have continued, at intervals, for half an hour and some-
times for 24 hours. The animal rolls over and over for some time,
and then stops, apparently through fatigue. After resting, it re-
sumes this gyration. Stimuli, such as a current of air, cause
prompt resumption of gyrations. Few rats that showed this symp-
tom recovered. Post-mortem examinations of a number of animals
indicated acute cardiac dilatation, and hyperemia of the gastrointes-
tinal tract and omentum. Although no cause for death other than
acute cardiac dilatation was observed, in some instances the heart
beat was detected after the cessation of respiration, indicating that
respiratory paralysis played a part in producing death by squill.

RESULTS OF INVESTIGATION

During the progress of this investigation a number of individual
problems were studied. For clarity of presentation, the results ob-
tained in each separate study are reported under separate headings.
The data given in Tables 6 to 13 are drawn from the results in Tables
2, 3, 4, and 5,

11

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19

RED-SQUILL POWDERS AS RATICIDES

EFFECT OF TEMPERATURE ON TOXICITY

Powders made at 80° to 100° C. were more toxic than those made
at other temperatures. Apparently they were more toxic than the
fresh bulbs used in their manufacture, even after an 80 per cent
allowance had been made for the moisture content of the bulbs.
The powders made at room temperature (25° C.) and at 60° C. were
less toxic than the fresh bulbs (on a dry basis), and also less toxic
than powders made at 80° C. Temperatures above 100° C. seemed
to decompose the active principle. Somewhat similar findings have
been reported by Claremont (5). Although heating rarely increases
toxicity, such a result is not unknown, as in the conversion of quinine
into quinotoxin. Further investigation is necessary to determine the
factors underlying this apparent increase in toxicity.

It was concluded that the most toxic powders were produced at a
drying temperature of 80° to 100° C. (Table 6.)

_TasLe 6.—Effect of temperature on toxicity of red-squill powders

[Mg. per kgm. of body weight]

Equiv- Minimum lethal dose of squill dried at—
alent
mini-
mum
Series No; aa
ose 0 ° ° ° ° ° ° °
aaudtiod DUG, 602'SE SOC: "1002 CG.) L162 Cz | 120% C: 1140° C,
fresh
bulb
ECMO MILIRUON) ser sre eR eee PE es 750 AU Di Lea a aha gael otees] (OEE. MN (A
2 GhotediT OVER) hss ore 12h ees be 1,500 | 1,000 | 2,000 | 2, 500+ 3,000+)________
2 (Waedtnt OVON)E 2 er TS D5 Oe ESE ote eae 750 | 1,500+| 750 750 2, 000
A(CleCETIG OVEN) 3 70 250) 11250 500 7 5% | en |, Se iit a

DIRECT OVEN DRYING VERSUS PRELIMINARY AIR DRYING

,One portion of each of seven pairs of samples prepared from the same
composite lots of squill bulbs was weighed and placed directly in the
drying oven at the desired temperature. The other portions were
exposed on open watch glasses in the air for five days, after which they
were dried at the same temperatures as the first set. (Table 7.)

TABLE 7.—Effect of type of drying on toxicity

Minimum lethal dose
of squill powder—

a Sample No.

Minimum lethal dose
of squill powder—

Suee Sample No. eT
Direct ie tag rt Direct oS
: nary air : nary air
oven dried ated oven dried dried
Mg. per kg.| Mg. per kg. Mg. per kg.| Mg. per kg.
of body of body of body of body
weight weight weight weight
Lee a a 1, 000 iis || gE RO y Ps Meee: Las ee 1, 000 500
Oat ZUe sa eos en 250 AAS DY A CaN OF Wy ean a ae 500 750
LOE 2 eae 350 Ci) S12 Wy 6: ee cl 500 625
oo EG (+ TE SETS 500+ 350

_ No difference was found in one set, but the variations in toxicity
in the other sets suggested that other factors played more significant
réles in the development of toxicity. Direct oven drying is a simpler

20 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

and more convenient procedure. It was concluded that direct oven
drying gives as good results as preliminary air drying in the produc-
tion of toxic squill powders.

EFFECT OF FERMENTATION ON TOXICITY AND YIELD OF POWDER

The 1925 experiments showed that sliced composites of fresh squill
bulb fermented rapidly upon exposure to the air. Accordingly, an
experiment was conducted to determine the effect of fermentation on
the toxicity of squill powders, and incidentally to determine whether
the fermentation was conducted upon the glucose of the toxic gluco-
side (or glucosides).

Portions of chopped composite taken from a fermenting lot and
dried formed series 1. (Table 8.) One lot (P. C. 719A) was made
by directly drying the unfermented squill in an electric oven at 80° C.
Another (P. C. 719AF) was made from the same composite, which had
stood in a closed fruit jar for 11 days at room temperature (25° C.).
By this time the fermentaion had reached the alcoholic stage, that is,
the odor of alcohol was apparent when the jar was opened. From
500,000,000 to 800,000,000 bacteria, from 5,000,000 to 18,000,000
yeasts, and 10,000 molds per gram were found in different parts of -
the material.’ After fermentation had reached the acetic-acid stage,
sample P. C. 719AAF was taken.

TaBLEe 8.—Effect of fermentation on yield and toxicity of red-squill powders

Yield Toxicity
Series No.
Unfer- Alco- Acetic Unfer- Alco- Acetic |
mented holie acid mented holie acid
Mg. per | Mg. per | Mg. per
kgm. of | kgm. of | kgm. of
body body body
Per cent | Per cent | Percent | weight weight weight
Lien add ts beceisasa-salers 12 : 12. 1, 000 2 2, 000
pag ee Chee Bic ae a SEE ae, Pa CS Oe) EE Se ee es es ee SS 250-350) Wea ee 750
Sebi $s 75 Ee ee eo Ase 2S eee eee ae S00=1; 000. |e. sae eee 750

For series 2, a mixture of bulbs comprising the whitish-pink (P. C.
720) and the pink-red (P. C. 721), and for series 3 a mixture of the
deep-red (P. C. 722) and the nearly red (P. C. 723) color groups of
bulbs (p. 21) were allowed to rot to the slimy, oozy stage and were
then sliced and dried in a steam-heated commercial drying oven.
Samples P. C. 767 and P. C. 768 were produced.

In series 1 the yield and the toxicity decreased during fermentation.
The sample taken during the stage of acetic-acid fermentation gave
only half as much powder as the unfermented bulb, and it was only
half as toxic to rats. A similar loss in activity was noted in series 2.
Incomplete data were obtained in series 3, owing to lack of material.

It was concluded that chopped bulbs should be dried immediately
to avoid decreases in yield and toxicity following fermentation.

EFFECT OF VARIABILITY OF SQUILL BULBS ON TOXICITY

Variations in the toxicity of squill preparations have been reported
in the literature. Origin, climatic conditions during growth, and
season of harvesting are reported to be factors in the toxicity of squill.

5 These determinations were made by B. A. Linden, of the Food, Drug, and Insecticide Administration,

RED-SQUILL POWDERS AS RATICIDES 21

In the 1924 experiments, the minimum lethal dose to white rats of the
powders made simultaneously from two individual bulbs was found
to be 250 and 3,000 milligrams per kilo of body weight. In the 1925
experiments six powders were made at the same time, and dried in the
same oven at 80° C. simultaneously. Each powder was made from
a single bulb, three from large bulbs (weighing around 750 to 1,000
grams each), and three from small bulbs (weighing around 200 grams
each). The lethal doses to white rats were found to be 500, 500, and
2,500 milligrams per kilo of body weight for the powders from the
large bulbs, and 250, 250, and over 5,000 milligrams per kilo of body
weight for powders made from the small bulbs. No characteristic
differences in the appearance of the bulbs or powders were apparent.

A sample of red-squill bulbs from Sardinia was found somewhat
more toxic than one grown in Sicily during the same year.

Powders were prepared from composites of various portions of squill
bulbs to see if material differences in toxicity could be found. The
minimum lethal dose to white rats of the powder from the outer dry
husks and scales was over 3,000 milligrams per kilo of body weight,
that of the middle fleshy scales was 500 and 750 milligrams per kilo
of body weight, and that of the central core was 1,000 and over 3,000
milligrams per kilo of body weight.

The readily available outer dry husks were stripped from all bulbs
before the bulbs were sliced, in the preparation of squill powders.
Although the core was less toxic than the middle fleshy scales, it
constituted so small a portion of the bulb that it was not deemed
necessary to remove it.

It was concluded that powders should be made from composites of
a number of bulbs, and that the outer dry husks should be rejected.

EFFECT OF INTENSITY OF COLOR OF SQUILL BULBS ON TOXICITY

The color of the middle, fleshy scales of squill varies in intensity
from a light whitish pink to a deep mahogany. A sackful of bulbs
was arbitrarily divided into four color groups, and powders were
prepared from six bulb composites of each group by two methods:
(1) One portion of the chopped composite was directly oven-dried
at 80° C. for 45 hours to constant weight; (2) the remainer of the
chopped composite was exposed to the air for 46 hours at room tem-
perature (25° C.) and then dried in the electric oven at 80° C. for
93 hours to constant weight. The quantities of bulbs in each color
class, and the minimum lethal doses to white rats are given in Table 9.

TABLE 9.—Effect of intensity of color of squill bulbs on toxicity

Minimum lethal
dose of powder

Sample No. Color of bulbs Quantity of bulbs | ™ade by—

Method 1} Method 2

Mg. per | Mg. per

| kgm. of | kgm. of
y ody

bod b
Kgm. Per cent | weight weight
0 7 | ae oe ae se 8 Wahitish pink:> cr sax2 666 ee tc f 3.0 3.9 350 250
12 sie) Cin el Lae ed le gles JESUS (2) (em ee pete ne i ai et Si 9.0 Psy 350 400
aot): a ae Neary ned es Boles eS). eee PR 35. 0 45.6 1, 000+ 500
(PCr. eae DOYS EFE FG Rea oa aie 29. 8 38. 8 500+ 350
ge ee

22 TECHNICAL BULLETIN 134, U. 8. DEPT. OF AGRICULTURE

The powders made by one method were about as toxic as those by
the other in all but the nearly red group. The whitish-pink bulbs
gave the most toxic powder and the nearly red ones the least, although
still satisfactorily potent. As nearly red bulbs comprised almost
half the shipment, 1t was not deemed desirable to reject them, even
though they were less toxic than those of the other groups. To
facilitate the application of experimental results to commercial
conditions, all powders thereafter were made from run-of-shipment
bulbs, without regard to color. Properly selected light-colored
bulbs, however, might give more toxic powders.

TOXICITY OF WHITE SQUILL TO RATS

White squill is reported to be botanically identical with red squill
and to have essentially the same microscopic appearance, except for
the lack of pigment cells. The minimum lethal dose of three samples
of white-squill powder fed to rats could not be determined, but it
was more than 18,000 milligrams per kilo of body weight. White
squill is apparently nontoxic to rats.

TOXICITY OF CALCIUM OXALATE TO RATS

The presence of calcium oxalate in squill powders has been uni-
versally recognized. Fliickiger and Hanbury (9, p. 691) report the
presence of 3.07 per cent of CaC,O,.3 H,O in a sample of dried white
squill. A dose of 1,000 milligrams per kilo of body weight of calcium
oxalate (equivalent to 33,000 milligrams per kilo of squill, calculated
from Flickiger’s assay) was fed to rats without producing any
apparent effect. Since calcium oxalate is present in white as well as
in red squill, and since the squill powders that kill rats in doses
around 200 milligrams per kilo would contain only 6 milligrams per
kilo of calcium oxalate in the lethal dose, it was concluded that
calcium oxalate itself could not be the primary cause of death in rats
dying after eating red-squill powders.

PREPARATION OF RED-SQUILL POWDER ON A SEMICOMMERCIAL BASIS

From 10 to 30 kilograms of squill bulbs were dried in a 65 by 150
by 200 centimeter (25 by 60 by 80 inches, approximately) drier,
holding six trays, each 27.5 by 90 centimeters (12 by 36 inches), and
heated by closed steam coils. The temperature was kept constant
within 4° or 5° C. during a givenrun. The outer scales were stripped
off and the bulbs were sliced by hand or by slicing machines onto
tared trays, until the trays were covered to a depth of 2 to 3 inches.
The trays were placed in the oven, which had previously been heated
to the desired temperature. Weights were taken from time to time
until constancy of weight indicated that the bulbs had dried to
equilibrium. ‘The dried bulbs were then mixed and ground in an
electric mill until the product passed through a 40-mesh sieve. The
ground powder, after being again thoroughly mixed, was placed in
screw-top cardboard mailing tubes for protection from the air.
After six months to a year, the powder in these cardboard tubes
solidified into a hard cake, but no change in toxicity could be detected.
(Table 10.)

RED-SQUILL POWDERS AS RATICIDES 23

TaBLE 10.—Effect of semicommercial preparation on toxicity of red-squill powders

Run No. aneple Color of bulbs Fermentation tage eo
Mg. per
kgm. of

body
PG weight

W244 | Deep rediand nearly rede 21) 229-. le sarit eet Nioneti shi. te) 222
1 T24Br|pee- 3 ‘Jie Sue * eee SESE a=) 2 SNS IETS Sight * 330-3 ae 750
Sse a ase ap Aa Winigishpinkiand pink red. £22 32! "2 2" _'__ s)None_-....--.--._. 500
Gaza es ol SES Rl Ae PER POS EE OPS ae ee Siehiree 2s 2 FE 625
BUESIS ELS 7 lad of BY [SARS Le styl mi yo “aye "Neel a eee cig eee ae INONGtee-e hs 8 500
eae oe, VIO ee i ee eee ee ome eee re a, en see cal eae Of ae 750
Won «eee Oke. 2S Se eee ee ee ore 6 (0) See eee ae 625
As est geey 767 MW nitish pimkand pink'red= 2. _2_ 9. v= 222 eet Acetic acid_____ =. — 750
768 Deep ted and néanly.red 4-2-5. = Sess ea tte ee. COLE ss: [eeyet 2 750

For the first trial run, the outer scales were removed from about
150 kilograms of fresh bulbs, which were then arbitrarily divided
into two groups, deep red and nearly red (P. C. 724), and whitish
pink and pink red (P. C. 725). One portion of each group was sliced,
weighed, placed directly in the oven, and dried, at temperatures rang-
ing from 68° to 75° C., the average being 72°. The remainder was
sliced, weighed, and left exposed to the air for four days. Slight
fermentation had started when the material was loaded into the oven
and dried at the same temperature as the first group.

For the second run (P. C. 760) a composite of 250 kilograms of
bulbs, irrespective of color, was used. The outer husks were removed,
and the bulbs were sliced into large chunks. The weighed. material
was dried at once at a temperature ranging from 70° to 80° C.

For the third run (P. C. 770) a composite was made from 2 sack-
fuls of bulbs. A machine was employed to make uniform slices ap-
proximately a quarter of an inch thick. This brought out much of
the mucilaginous material from the bulbs and reduced them to a more
slimy consistency before drying. Conditions were therefore unin-
tentionally made more favorable for fermentation. The use of sharp
instruments in slicing the bulbs is advisable to avoid bringing out an
excess of the mucilaginous juices. During the first three days of
drying the oven temperature was maintained at 40° to 50° C. On
the fourth day it was brought up to 80°, where it was held during the
rest of the drying period.

One portion of the fourth run (P. C. 775) consisted of the remainder
of the bulbs used in the preparation of P. C. 770. They were also
finely sliced. In addition, bulbs that had been stored at 4° to 5° C.
for three months were used. Fermentation during storage had been
marked. The bulbs were soft and spongy, with the leaves separat-
ing, and were dripping mucilaginous slime. Two composites were
made. The whitish pink (P. C. 720) and pink red (P. C. 721) groups
were consolidated as P. C. 768; the deep red (P. C. 722) and nearly
red (P. C. 723) groups were consolidated as P. C. 767. The spongy
bulbs were sliced onto a tray and placed directly in the oven to dry.
During the fourth run the oven temperature was held at approxi-
mately 80° C.

Although the temperature was not held so constant as in the elec-
tric oven, the products obtained in the steam oven corresponded
reasonably well in toxicity with corresponding samples from the same
composite dried in the electric oven. Thus, the minimum lethal dose

24 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

of P. C. 727A, dried in an electric oven, was 500 milligrams per kilo
of body weight, and that of P. C. 724A and of P. C. 725A, dried ina
steam oven, was the same.

Sample P. C. 760 in the second run and P. C. 775 in the fourth run
were made at 80° C., and both were more toxic than P. C. 770 in the
third run, which was held at 40° to 50° for three days before being
heated to 80°. This suggests that a rapid rise to 80° for the fresh
bulb tends to produce the most toxic squill powder.

The rate of drying was followed only until uniform weight was
indicated. At 80° C.it seems that drying would be complete in about
three days.

Although food containing 40 per cent of squill powder was readily
eaten, 10 per cent concentrations were used in most instances. With
this concentration, a rat consuming 1 per cent of its body weight of
food would consume 0.1 per cent of its body weight of squill, or 1,000
milligrams of squill per kilo. Accordingly, squill preparations having
a lethal dose at or below 1,000 milligrams per kilo appear to be
sufficiently toxic for commercial purposes. Most of the experimental
powders killed in materially smaller doses.

The production under semicommercial conditions on a semicommer-
cial scale of squill powders having similar toxicities indicates the
possibility of successfully manufacturing squill powders of a satis-
factory degree of toxicity on a commercial basis.

RELATIVE SUSCEPTIBILITY OF WHITE RATS AND OF WILD (BROWN) RATS TO SQUILL
POWDERS
From the literature it would seem that wild (brown) rats are more
susceptible to squill than white rats. The lethal doses found in the
investigations here reported, when the same powder was fed to the
two forms, are given in Table 11.

TaBLeE 11.—Relative susceptibility of white rats and of wild (brown) rats to squill

powders
jit ameeaas leaienl | | | Minimum lethal
dose for— dose for—
Sample No. A, aye Eo Ratio || Sample No. 1 ae
“| White Wild White Wild
| Fats | rats rats rats
RUB S2ENO IGM SURG oe
Mg. per | Mg. per Mg. per | Mg. per
kgm. of | kgm. of kgm. of | kgm. of
boty: |, | body ! body body
Pe. weight weight PC weight weight
16. 2-5 hee es 750 250 3.0 Be = 4, SR oe oe 1, 000 500 (2. 0+)
Sf 2 Sere Se 500 500 1.0 RAGA. AS gee 500 .0
< SuSE bg FA es 250 | 200 1.25 VF 25 1s Rie Repti ie Site 7 500 L5
TOA ae ee | 2,000 | 500+) (4.0—) pi Seed OT ihe ee 500 300+} (1.7-—)
fe RL we 250 | 250 1.0 y ys il ee 625 500 1.3
ry): Soe Re | 250 150 1.7 THA 7 2 5) 500 500+; (1.0—)
yo te SEE TO 350 | 350 1.0
(y+, PEERS ED EES yk! 500 (i. 0+) A yerams 5 lo... ee eee 1.5

In some series no difference in susceptibility was noted; in others
the wild rats appeared to be about three times as sensitive. Lack of
material and the difficulty of obtaining a fairly large supply of wild
rats for feeding purposes at any definite time prevented more accurate
determinations of differences in susceptibility of the two forms.

RED-SQUILL POWDERS AS RATICIDES 25

Results obtained in these 14 series confirm Claremont’s findings (5)
that, on the average, less squill powder is required to kill wild rats
than is required to kill white rats.

EFFECT OF RED-SQUILL POWDERS ON DOMESTIC AND OTHER ANIMALS

Previous investigators have stated that squill, although toxic to
rats, is not toxic to cats, dogs, chickens, and other domestic animals.
As no data showing the minimum lethal dose of red squill to these
animals were found in the literature, a series of experiments was
undertaken to determine the toxicity of squill to animals other than
rats.

CATS

Squill powders were mixed with whole-milk powder, and various
proportions were added to 100 grams of lean hashed meat, which was
then fed to cats. In food containing 10 to 25 parts of squill powder
per million the unusual flavor was detected, and the food was eaten
slowly but completely. The quantities of food containing higher
concentrations of squill that cats consumed within 24 hours were then
determined. (Table 12.)

TaBLe 12.—Relative percentages of squill-meat baits eaten by cats in a 24-hour

f f

Percent- | Concentration of squill (parts per Percent-

Concentration of squill (parts per of :
million) age eaten million) age eaten
\
|
De Set ewes ee tee : na rea ee 8
ee IG FONSI iis | ager Ss ce bre sn | 2
0 3 MS ip a eee ee a ee eee 7
"WELL TEs tite CR ea 75

As rat baits are customarily exposed in concentrations of 10 per
_ cent, or 100,000 parts per million, cats probably will not eat such food.

DOGS

Dogs refused ground meat containing 1,000, 2,500, and 5,000
parts of squill powder per million of meat. When squill powder
suspended in water was injected into the stomach by stomach tube,
emesis and diarrhea followed in several instances after doses of 100
milligrams per kilo of body weight. As dogs refused food containing
a small concentration of squill powder, such as 1,000 parts per million,
it does not seem that they would be hurt by squill. Two separate
instances have been noted where dogs gulped down squill rat baits
containing 10 per cent of squill mixed with sausage. Emesis was the
only effect noted.

CHICKENS

No effect was observed following the injection into chickens’ crops
of quantities less than 2,000 milligrams of squill powder per kilo of
body weight. Diarrhea followed the injection of 2,000 and 3,000

illigrams per kilo. Squill powder was mixed with cracked corn and
with laying mash in 10 per cent concentrations. After one or two
picks, chickens refused to eat such food. Two chickens were placed
on a diet containing 10 per cent of squill powder in growing mash.

26 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

After refusing to eat for several days, they consumed the mash with-
out much hesitation. This food was continued for two weeks, during
which time the chickens apparently grew as well as controls fed on
untreated mash. Odom Stewart, formerly of the Bureau of Biological
Survey, in experiments with baby chicks at Raleigh, N. C., concluded
that chicks are not likely to eat enough feed containing 10 per cent
of powdered red squill in 1, 2, or 3 feedings to cause death, even when
fed it to the exclusion of other foods.

PIGEONS

After a few picks, pigeons refused to eat mash containing 10 per
cent of squill powder. Injection of 2,000 milligrams of squill powder
per kilo of body weight into the crop failed to produce any noticeable
effect. The injection of 3,000 and 4,000 milligrams per kilo resulted
in emesis, but no other untoward effect could be noted.

PIGS

A dose of 250 milligrams of squill powder per kilo was given in a
gelatin capsule at 10.45 a. m. to a 4-month-old pig weighing about
16 kilograms. No evidence of squill action was noticeable until
about 4 p. m., when signs of gastric distress developed. The pig
vomited several times during the night. At 9 o’clock the next morning
it was unable to stand without definite ataxia, and consistently
refused food, but drank water freely. Some food was eaten at noon
but it was vomited about 3 p.m. The pig’s tail was limp and straight,
although at the time of feeding it was tightly curled. On the second
day after the injection the pig had practically recovered.

After a 9-day intermission, to permit complete recovery from the
first dose, a mixture of cracked corn and middlings containing 10
per cent of the same squill powder was offered. The pig took only
one or two bites, then grunted, refused further food, and vigorously
rubbed its snout on the cage walls and floor. This reaction was
probably due to the sting of the calcium-oxalate raphides. Half an
hour later, and again the next morning, the same squill food was
refused, but untreated food was readily consumed.

Post-mortem examination showed nothing abnormal, except some
evidence of irritation in the stomach. When the body weight of a

ig is considered, it is readily apparent that the quantity of squill
bait required to cause toxic effects would need to be very large. For
this particular animal, the quantity of squill power given would be
sufficient to kill 16 kilograms (35 pounds) of rats. Under ordinary
conditions of rat baiting, it hardly seems possible that a pig would
consume enough poisoned bait to cause trouble.

WOODCHUCKS

Injected in aqueous suspension into the stomach of woodchucks,
the minimum lethal dose of sample P. C. 18 was found to be 500
milligrams per kilo of body weight (the same as for rats). Vomiting,
however, frequently followed even smaller doses. Baits containing
squill were refused. Squill does not seem to be suitable for use in
controlling woodchucks,

RED-SQUILL POWDERS AS RATICIDES a7

PRAIRIE DOGS AND POCKET GOPHERS

Prairie dogs and pocket gophers refused to eat freshly exposed
squill baits. After rains had washed off the squill, the remaining
baits were readily consumed.

From these feeding and stomach-tube experiments, it seems safe
to conclude that squill mixtures containing 5 to 10 per cent of squill
powder either will not be eaten by animals other than rats, or will
produce emesis with direct removal of the poison. Probably, there-
fore, squill powders deserve the reputation accorded them in the
literature of being generally harmless to farm animals. Of course,
failure to produce death may be due to the animal’s failure to eat
and retain enough squill powder, rather than to relatively high species
resistance or insusceptibility.

YIELDS OF TOXIC SQUILL POWDERS (‘‘RAT UNITS’’)

Generally speaking, squill bulbs lose 80 per cent of their weight
during drying. The yields of powder obtained when samples were
dried under stated conditions are given in Table 13. The weight of
powder obtained in following a given process is not significant of
the efficiency of the process unless the toxicity of the powder obtained
is also considered. Two kilograms of a highly toxic powder may
represent a better yield than 4 kilograms of a relatively nontoxic
preparation. It is necessary to know both the yield and the toxicity
in judging the efficiency of producing a squill powder under a given
set of conditions,

28 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

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tars Ale os 1] eae Be | Sai: HRN (AR aet i | a wa ey Ou geee Swe OCD oes oS ees Oe Fe ee ee ee a arr eee el
Vw ian eS ll! ll 0°82 892 HO i OP See Pees co” 1 Re Se CT BS ee, eT Oe

30 TECHNICAL BULLETIN 134, U. 8. DEPT. OF AGRICULTURE

In an attempt to express in a single figure the results of combining
the two variables, yield and toxicity, the so-called “rat units” of
powders have been calculated. A “rat unit” is the yield in grams
from 1 kilogram of fresh squill bulbs divided by the minimum lethal
dose in grams per kilo of body weight. It is the number of kilograms
of rats that should be killed by the powder obtained from a kilogram
of fresh squill bulbs treated by the method under consideration. For
example, if a 20 per cent yield was obtained by a certain method, and
the powder had a minimum lethal dose of 500 milligrams per kilo
rasan weight, the rat unit for the method would be calculated as
follows:

Yield =20 per centX1 kilogram of fresh bulbs=200 grams
Toxicity =500 milligrams per kilo=0. 5 grams

Another method for making a powder with 200 rat units would
obviously be less valuable, being but half as efficient. The rat units
determined for a number of series of samples are given in Table 14.

TaBLE 14.—Yields of toxic squill powders

Samp] Method of ee Sampl Method of nee
ample et fe) - mum ample ethod o . mum
No. drying Yield | jethal | Rat |! “No. drying Yield | jethal | units

dose dose

Mg. Mg.

per per

kgm. kgm.

Per | of body Per | of body
cent | weight PG. cent | weight

y (ee SO Eresh bulb. 4,000 | 250 yaks Rees Electric oven___| 22.3 500 450
rey: peewee Electric oven_-_- 23.2 | 1,000 | 230 Sate te eee e-S 23.9 500 480
71I9AF_ ONES) | Lee Et 17.8 | 2,000 90 f(y SS ite 2s do. S55 =! 19.5 750 260
TOA A 2 do.) ise 12.0 | 2,000 60 yi (aes as 6 (i = See 22. 5 1, 250 180
‘(Se Desiccator_____- 23.5 | 1,250 190 cf bs ih ss ln aes do. se-see 5 22.5 750 300
719A_____| Electric oven___| 23.2 1,000 | 230 NOE-f See ee de: 235 22.0} 2,000 110
cv) ae age. : ie a ee De 12.0 250 480 fi Cr Ake Reed PIER eos do. 622332 21.0 1, 250 170
GaAs ee peo ss 12.9 350 370 16. 22) SOs ee 23. 5 1, 250 190
Peas pe HO. 4 5-aee 21.5 430— || 724A_____ Steam oven_-_-_-_- 21.5 500 430
yr.) ees Gok = 7 seek 24.4 | 1,000+/) 240— }: Semen (ines (1s SD 20. 6 500 410
vi. 2 eee! Mee "en ela | 21.5 625 | 350 SESE Be ode S dos ts 20. 4 500 410
ee re ee A 10.1 250 400 (i (| eee Pee dos sees 22. 2 750 300
Va at es WS BO a eet ate 11.9 400 | 300 fi (et SN Ree dor 21:2 625 340
9.71 5 a ey (I Te des abet 20. 7 350 | 590

The rat unit for the fresh bulb (P. C. 761) was 250. Essentially
the same value was obtained for P. C. 719A in the unfermented stage.
Fermentation to the alcoholic stage reduced the rat unit to 90, and
further fermentation to the acetic-acid stage caused a drop to 60,
showing the loss in activity during fermentation to be progressive.
Freezing caused a decrease to about 200 rat units. The rat units of
powders prepared under laboratory conditions ranged from 110 to
590, while the values for powders made under semicommercial condi-
tions ranged from 300 to 430. Attention to manufacturing details
would be expected to raise the rat units for commercial powders
above 500. Indeterminate values for two of the P. C. No. 719
series prevent careful comparison, but the A series averaged less than
350, whereas the B series averaged 420 rat units. This would sug-
gest that preliminary air-drying treatment given the B series pre-
served the toxicity or aided in its development. The fact that

RED-SQUILL POWDERS AS RATICIDES ol

many powders have rat units greater than 250, the value for fresh
squill bulbs, also suggests that the toxic principle in squill bulbs may
be linked to some plant ingredient in a thermolabile condition, and
that the toxic principle is liberated by heating to 80° to 100° C.

STABILITY OF SQUILL POWDERS

Two red-squill powders stored in screw-top vials in the light for 15
months showed no change in lethal dose. A 10 per cent squill bis-
cuit that had been baked was as toxic a year later as the squill con-
tent would require. A number of powders tested over a period of a
year have shown no evidence of loss of activity.

ACCEPTANCE TESTS

To determine the palatability of 10 per cent squill biscuit as com-
pared with that of commercial samples of 5 per cent arsenious-oxide
biscuit and 30 per cent barium-carbonate biscuit, several dishes were
placed in a cage containing a starved white rat. Each dish contained
more poisoned food than would be necessary to cause death by itself.
After exposure from one to five hours, the dishes were removed, and
the quantities of food consumed were determined. A number of
tests were run simultaneously. With the weight of squill biscuit eaten
considered as unity in each case, the ratio of control biscuit to 10 per
cent squill biscuit was 2.6:1.0; that of 5 per cent arsenious-oxide
biscuit to 10 per cent squill was 0.17 :1.0; and that of 30 per cent
barium-carbonate biscuit to 10 per cent squill biscuit was 0.93 : 1.0.
The absolute quantities of poisons consumed would then be in the
ratio of 250 squill to 750 barium carbonate to 20 arsenious oxide.
These figures are roughly proportional to the minimum lethal doses
of the poisons under consideration. Barium carbonate is approxi-
mately one-third as toxic as squill powder; fed in three times the
concentration, about equal quantities of bait are consumed. As the
toxicity of arsenious oxide depends upon its degree of fineness, closer
comparisons are not available.

A field-acceptance test in comparison with phosphorus baits is re-
ported by Teall.6 At Vienna, Austria, an organized 2-day rat-kill-
ing campaign was held in January, 1927, phosphorus baits being used
exclusively. Of 1,460,000 baits exposed, about 20 per cent were taken.
In March, 1927, another 2-day campaign was undertaken, during
which only squill baits were used. Of 1,260,000 baits exposed, more
than 50 per cent were taken. It was noted that squill was taken in
many houses where phosphorus bait had not been touched.

The degree of acceptance of squill baits varies with the relative
palatability to rats of the foods with which the squill is mixed. Direc-
tions for preparing and distributing red-squill baits have recently
been published by the Department of Agriculture (24, p. 8, 9).

COMMERCIAL SQUILL RAT POISONS

Three liquid squill extracts that had been offered for sale as rat
poisons were tested. One was one-fourth and the other two were
less than one-tenth as toxic as their labels would indicate. Two
squill biscuits and three squill powders were found to correspond in
toxicity to the labeled claims for squill content. —-

6TEALL, G. ORGANIZATION AND RESULTS OF THE FIRST VIENNA RAT-KILLING CAMPAIGN. (Second sup-
plementary report to Report No. 6025 dated May 20, 1925, in reply to department’s unnumbered instruc-
tion of March 28, 1925, File No. 1027-1848.) [1927.] (Original. on file in Department of State, not seen-
Copy on file in U. S. Dept. Agr., Bur. Biol. Survey.]

32 TECHNICAL BULLETIN 134, U. 8. DEPT. OF AGRICULTURE

EXTRACTION OF TOXIC PRINCIPLES BY VARIOUS SOLVENTS

To obtain information regarding the chemical nature of the toxic
principle of red squill, a mixture of two powders (P. C. 724A and
P. C. 725A) was taken for intensive study. Seventy-five-gram por-
tions in Soxhlet thimbies were extracted with water, with 95 per
cent ethyl alcohol, with acetone, and with chloroform, until.there was
no further appearance of color. The solutions were concentrated in
a current of warm air, and the resulting solid extracts were dried in
an electric oven at 80° C. and finally in a vacuum desiccator over
calcium chloride. The residues were removed from the Soxhlet thim-
bles and dried in the electric oven at 80°. Feeding tests were made
with the original mixed powder and with some of the extracts and
residues.

The water extraction, started on January 20, 1926, was run from
8.30 a.m. until 5 p. m. daily for 19 days. The dark-red mucilaginous
extract, possessing an odor of molasses, was concentrated on a steam
bath, then dried in an-oven, and finally allowed to stand over calcium
chloride in a desiccator until constant in weight. It was ground to
pass through a 40-mesh sieve. Both the extract (P. C. 751) and the
exhausted residue (P. C. 750) were very low in toxicity, indicating
that the active principle is decomposed by this treatment.

The alcoholic extraction for 19 days removed a much smaller
quantity of mucilage, but apparently as much color as did the water
extraction. The residue caked and became very hard upon exposure
to the air. The solid extract (P. C. 749) had a rather oily feel,
and was the most toxic extract in this series. The lethal dose of
the residue (P. C. 748) exceeded 5,000 milligrams per kilo of body
weight. The residue therefore is inert (nontoxic).

Acetone extraction gave a small yield of lemon-yellow crystals
(P. C. 755) and no mucilage or color. Feeding tests showed that
the very fluffy residue (P. C. 754) was two-thirds as toxic as the
original powder, indicating that a portion of the toxic principle is
destroyed during the treatment with acetone. |

Chloroform gave the same result as acetone. The solid extract
(P. C. 757) was small and oily. The fluffy residue (P. C. 756) was
somewhat less toxic than the original powder. The acetone and the
chloroform extracts were too small to be used for feeding tests.

Distilled water was repercolated through another 75-gram charge.
Channels formed, producing incomplete extraction. So much muci-
lage dissolved that after seven days further repercolation was found to
be impossible. The thick, molasseslike percolate had a sweetish odor
and reduced Fehling’s solution. Evaporated to a very dark-brown,
tough, solid extract (P. C. 759), it was readily eaten by rats. The
lethal dose was 1,500 milligrams per kilo of body weight. The incom-
pletely extracted residue was not fed to rats. asa Ye

To study further the effect of water in extracting the toxic prin-
ciple, 75 grams of squill powder were placed in a beaker with about
800 cubic centimeters of water and warmed on a steam bath. Twice
a day the solvent was removed in a Buchner funnel, and a fresh
charge of water was added. After 22 days the extraction appeared
complete. The aqueous solution was concentrated to a solid extract
(P. C. 753). The residue (P. C. 752) was a dark-brown, tough sheet,
heavily laden with glittering calcium-oxalate crystals. Both the

RED-SQUILL POWDERS AS RATICIDES 33

extract and the residue were nontoxic, showing that the active prin-
ciple is totally destroyed by this treatment.

Table 15 shows the yield, the lethal doses, and the proportion of
toxic principle from the original powder that appeared in the various
extracts and in the residues.

TaBLE 15.—LEffect of method of extraction on yield and toxicity of squall

Yield eS ee ak: total toxicity
Method of extraction Solvent used SS eee eee

Solid | Resi- Solid Resi- Solid | Resi-

extract| due extract due extract | due

Mg. per | Mg. per
kg. of body|kg. of body
Per cent|Per cent) weight weight |Per cent| Per cent
Onipimalypowier. (255. As fase bese eee ite el ek LODE 4253 te 3 CODY) rans bs 1

Percviations 2%... 2221 LC) ae 2 eres 18.5 | 81.5 1 BOO, bd circent een Perea
PSS tIOM este.) Te eS OG age fh a ah a lp 82.6 | 17.4 5,000+| 5,000+} 0.8—| 1.7—
PUBIC Segoe Sg he (Gt OS 44.3 any 3, 500 4, 000+ 6.3 7.0—
POON Ey ee Le Fe Alcohol (95 per cent) ___ 14.0 | 86.0 200 5, 000+] 35.0 8.6—

Woe. Seto thas 3 ih APCOUONSE 22 eb beast 1.3 to Bt ld So ee iene Bs ae TOON Eee oe 3x 66. 0

D0 ee coer E Chloroform: 22. 2. -.4. ye a ay a eee ee 625y los. oe 80.0

Exhaustion with water on the steam bath, or in a Soxhlet thimble
at 100° C., caused almost total destruction of the toxic principle in
the extracts as well as in the residues. As dry heat at 100° C. did
not appear to cause any decrease in toxicity, it seems evident that
this loss in potency is related to the hydrolysis of the toxic glucoside.
The alcoholic residue was nontoxic, and the acetone and choroform
residues were somewhat less potent than the original powder. Even
though the boiling point of each of these three solvents is less than
80°, it seems that any method of extraction tried caused some destruc-
tion of the toxic principle. The alcoholic extract possessed one-third
the activity of the original powder.

Apparently the toxic principle of red squill is soluble in alcohol,
but not in water, acetone, or chloroform. Water and alcohol extracted
appreciable quantities of mucilage and reducing sugars. Extraction
of the toxic principle suggests itself in the study of the chemistry of
the active principle. It seems evident, however, that the cost of
undertaking this on a commercial scale would far outweigh any pos-
sible benefit of marketing a more toxic preparation. Therefore no
further experiments in this direction were conducted.

SUGGESTED METHOD OF PREPARATION OF TOXIC SQUILL POWDERS

Based upon the results obtained during a 3-year series of experi-
ments in the manufacture of red-squill powders, the following method
is offered as furnishing the most toxic product: ?

Remove the outer dry husks from fresh red-squill bulbs obtained as soon as
feasible after digging, and slice the bulbs transversely into sections one-fourth
to one-half inch thick. Place the sliced composite as soon as possible in a drying
oven, which has been previously heated to 80° C., and dry to constant weight
at that temperature. Grind the dried material so that it will pass through a
40-mesh sieve. Pack the powder in hermetically sealed containers.

7It is possible that other manufacturing conditions might yield products of equal toxicity.

34 TECHNICAL BULLETIN 134, U. S. DEPT, OF AGRICULTURE

Powders made by this process should kill white rats in doses approx-
imating 250 to 500 milligrams per kilo of body weight, and should
be stable for several years.

Because of the variations in toxicity of different lots of squill, the
minimum lethal dose of every lot of squill powder should be deter-
mined by feeding it to rats in the laboratory before it is marketed.
It should be fed in 10 per cent concentrations in ordinary rat food
to white rats that have been deprived of all food for 18 hours. The
minimum lethal dose is the smallest dose that kills all the rats within
fivedays. Atleast five rats should be fed with each dose tested. This
is necessary to insure essentially uniform toxicity in squill powders
distributed commercially. As a result of feeding tests, great varia-
tions in the potency of successive lots of powders may be reduced
by properly mixing powders of higher degrees of toxicity with less-
potent preparations, to produce the same standard potency. Such
procedure would put a premium on the production of the most toxic
squill powders, but would also permit the utilization of less toxic
preparations.

As a standard of toxicity it is suggested that commercial squill
powders having a minimum lethal dose of 1,000 milligrams per kilo
of body weight be marketed, and that commercial squill baits having
a minimum lethal dose of 10 grams per kilo be prepared. Such baits
would contain 10 per cent of the standard squill powder (that is, a
squill powder with a minimum lethal dose of 1,000 milligrams per
kilo). If this standard bait is used, it would be necessary that a
rat eat only 1 per cent of its body weight to obtain enough poison
to kill it. Many of the wild rats studied during this investigation
weighed between 250 and 400 grams (8 to 14 ounces). From 2.5 to
4 grams (one-twelfth to one-seventh of an ounce) of such a squill
bait would be necessary to kill them. These experiments indicated
that rats will readily eat much larger quantities of such baits.

CONCLUSIONS

Powdered red squill is toxic to rats; white squill is not.

Powders prepared by directly drying unfermented, sliced red squill
bulbs in an oven at 80° C. are usually more toxic than those pre-
pared under- other conditions. The lethal dose of squill powders
prepared by this method is usually about 250 milligrams per kilo
of body weight for white rats; wild (brown) rats are killed by some-
what smaller doses.

Cats, dogs, chickens, and pigeons were not seriously harmed by
squill powder. Food poisoned with squill either was not eaten or,
if eaten, was promptly vomited. Consequently it has been indicated
that red squill is nontoxic to these animals under normal conditions
and when exposed in the concentration recommended for rat poisons.

Because of variations in toxicity, squill powders should be tested
before being marketed and adjusted so that 10 grams of 10 per cent
squill bait will kill a minimum of 1 kilogram of rat. (One ounce
will kill 7 pounds of rats.)

(1)

(2)

(3)

(4)

(5)

(6)

(7)
(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

RED-SQUILL POWDERS AS RATICIDES 35

LITERATURE CITED

AUMANN. :
1912. VERGLEICHENDE UNTERSUCHUNGEN UBER DIE WIRKSAMKEIT BAK-
TERIELLER UND CHEMISCHER RATTENVERTILGUNGSMITTEL. Centbl.
Bakt. [ete.] 63: 212-221, illus.
BuUSCHMANN, E.
1919. UNTERSUCHUNGEN UBER DIE CHEMISCHEN BESTANDTEILE VON BUL-
BUS SCILLAE. Arch. Pharm. 257: 79-86.
BouLEeNGER, E. G
1920. REPORT ON METHODS OF RAT DESTRUCTION. Zool. Soc. London
Proc. 1919: [227]-244.
CuaREmont, C. L.
1921-22. NATIONAL RESEARCH ON RATDESTRUCTION. Journ. Roy. Sanit.
Inst. 42: 311-318.

1922. NoTES ON THE ANALYSIS AND USE OF RED SQUILL IN RAT POISONS.
Analyst 47: 60-67.
Danysz, J., and KopaczEewskI, W.
1914. SuR LES PROPRIETES TOXIQUES DU PRINCIPE DE LA SCILLE.
Compt. Rend. Soc. Biol. [Paris] 77: 59-61.
DUJARDIN-BEAUMETZ.
1923. LADERATISATION. Journ. Pharm. et Chim. 27: 461—467; 28: 17-25.
Ewins, A. J.
1911-12. A NEW WATER SOLUBLE ACTIVE CONSTITUENT OF SQUILLS.
Journ. Pharmacol. and Expt. Ther. 3: 155-160.
Fiicxicer, F. A., and Hansury, D.
1879. PHARMACOGRAPHIA. A HISTORY OF THE PRINCIPAL DRUGS OF
VEGETABLE ORIGIN, MET WITH IN GREAT BRITAIN AND BRITISH
INDIA. Ed. 2. 803 p. London.
F ROHNER, E.
1901. LenRBUCH DER TOXIKOLOGIE FUR THIERARTZE. Ed. 2, 356 p.
Stuttgart. (Ref. Hertwig, p. 196.)
GALAVIELLE, and CrisToL, P.
1922. LE SCILLA AUTUMNALIS L. ETUDE CHIMIQUE DE SES PRINCIPES
actirs. Bul. Sci. Pharmacol. 29: 29-31.
GeEorGgE, E.
1925. ‘THE ACTIVE PRINCIPLES OF THE AFRICAN SQUILL, URGINEA BURKEI
(SLANGKOP) AND ITS SUITABILITY OR OTHERWISE AS A SUBSTI-
TUTE FOR URGINEA MARITIMA IN COMMERCE. Journ. So. African
Chem. Inst. 8: 14-27.
[Great Britain]. MINistry oF AGRICULTURE AND FISHERIES.
1923. THE DESTRUCTION OF RATS. [Gt. Brit.] Min. Agr. and Fisheries
Leaflet 244, 8 p.
Gunn, J. A., and Hratucorts, R. Sr. A.
1920-21. THE ALLEGED HYPERSUSCEPTIBILITY OF THE RAT TO SQUILL.
Journ. Physiol. (Physiol. Soc. Proc. Oct. 16, 1920) 54: Ixxxv—
Ixxxvi.
Hartwicn, C.
1889. es DIE MEERZWIEBEL. Arch. Pharm. (3 R. 27) 227: [577]-588,
illus.
HowartuH, W. J.
1921. REPORT OF THE MEDICAL OFFICER OF HEALTH OF THE CITY OF
LONDON FOR THE YEAR 1920. Med. Off. Health [London] Ann.
Rpt. 1920, 76 p.
KosBeErt, R.
1902-06. LrEHRBUCH DERINTOXIKATIONEN. Ed. 2, 2v., illus. Stuttgart.
KopaczEwsk!, W.
1914. UBER DIE PHYSIOLOGISCHEN WIRKUNGEN DES SCILLITINS UND
SCILLIDIURETINS. Biochem. Ztschr. 66: [501]—508.
Lantz, D. E.
1917. ge at RATS AND MICE. U.S. Dept. Agr. Farmers’ Bul. 896, 24
p., illus.

36 TECHNICAL BULLETIN 134, U. S. DEPT. OF AGRICULTURE

(20) LEREBOULLET, P.
1916. LA LUTTE CONTRE LES RATS DES TRANCHEES. Paris Médical 6
Année: 165.
(21) Lewin, L.
1903. TraIT& DE ToxicoLocre. Translated and annotated by G.
Pouchet. 1120 p., illus. Paris.
(22) Orrina, M. P.
1818. TraiT& DES POISONS TIRES DES REGNES MINERAL, VEGETAL ET
ANIMAL ON TOXICOLOGIE GENERALE, CONSIDEREE SOUS LES
RAPPORTS DE LA PHYSIOLOGIE, DE LA PATHOLOGIE ET DE LA
MEDECINE LEGALE. Ed. 2, rev., corrected, andenl. 2 v. Paris.
(23) Scuuiupp, W. F.
1921. THE DESTRUCTION OF RODENTS BY THE USE OF POISONS. Union
So. Africa Dept. Agr. Bul. 4 (1921), 34 p.
(24) Sriver, J.
1927. Rat controu. U.S. Dept. Agr. Farmers’ Bul. 1533, 21 p., illus.
(25)

1929. RAT CONTROL AIDED BY DEVELOPMENT OF EFFECTIVE NEW POISONS.
U. 8S. Dept. Agr. Yearbook, 1928: 518—521, illus.
(26) Smitn, W. F.
1921. Liquip EXTRACT OF RED SQUILL (SCILLA MARITIMA) AS A RAT
poison. Analyst 46: 178-180.
(27) Srouu, A., and Suter, E.
1925. HEART-AFFECTING PURE GLUCOSIDE FROM BULBUS SCILLAE AND
PRODUCING THE SAME. (U. S. Patent No. 1516552). Commr.
Patents Ann. Rpt. 1924: 582. .
(28) Winton, F. R.
1927. THE RAT-POISONING SUBSTANCE IN RED SQUILLS. Journ. Pharma-
col. and Expt. Ther. 31: 123-136, illus.

(29)
1927. A CONTRAST BETWEEN THE ACTIONS OF RED AND WHITE SQUILLS.
Journ, Pharmacol. and Expt. Ther, 31; 137-144.

U.S. GOVERNMENT PRINTING OFFICE: 1929

ORGANIZATION OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE

October 26, 1929

Secretary of Agriculiure....-..--+1--+-----. ArtTHuR M. Hype.

Po masranh Weer Clary. 222 8 ISL 8 R. W. Dunuap.
Director of Scientific Work.__------------- A. F. Woops.
Director of Regulatory Work__--.---------- WALTER G. CAMPBELL.
Director of Extension Work___------------ C. W. WARBURTON.

Director of Personnel and Business Admin- W. W. StocKBERGER.
istration.

Pirecior of Information... 4-22 -._5--..----- M. S. E1sENHOWER.
pene each et ot ok R. W. WILLIAMS. .

WMeediner Bureau. 3-0 ni elk Cuartes F. Marvin, Chief.
Bureau of Animal Industry-_-------------- JoHN R. Mouuemr, Chief.
Bureau of Dairy Industry_....------------ O. E. Resp, Chief.

Bimeau of Plant Indusiry...2...-.=.---=--- Witi1am A. Taytor, Chief.
Mme earings eee ye ok R. Y. Sruart, Chief.
Bureau of Chemistry and Soils___---------- H. G. Kniaut, Chief.
haseau of Eniomology—--~--=-2---2.----- C. L. Maruatt, Chief.
Bureau of Biological Survey_.....--------- Paut G. Repineton, Chief.
Bureau of Public Roads__.---------- pie ie Tuomas H. MacDona tp, Chief.
Bureau of Agricultural Economics___.------ Nits A. Ousen, Chief.
Bureau of Home Economics. -------------- Louise STANLEY, Chief.
Plant Quarantine and Control Administration. C. L. Maruatt, Chief.
Grain Futures Administration.------------ J. W. T. Duve., Chief.

Food, Drug, and Insecticide Administration... WALTER G. CAMPBELL, Director of
Regulatory Work, in Charge.

; Office of Experiment Stations.__..---------- E. W. Auuen, Chief.
Office of Cooperative Extension Work____---- C. B. Smiru, Chief.
11 UCN OAS Ra ics pas a pa ag CLARIBEL R. Barnett, Librarian.

This bulletin is a contribution from

Bureau of Biological Survey_._..------.-- . Paut G. Repineton, Chief.
Division of Predatory-Animal and Ro- Stanueny P. Youna, Principal Biol-
dent Control. — ogist, in Charge.

Photomount
Pamphlet
Binder

Gaylord Bros.Inc.

‘Makers
Syracyse, N. Y.
PAT. JAN 21, 1908

Sam ng ne nes Se ae a ae a —