TYPICAL R.4T \\'ORK-THE OF ENORMOUS Loss
RAT LOSSESARE PREVENTABLE BY THE USE OF RED SQUILLBAITS
CAUSE
IN THE
FOOD IhDUSTRIES
0
0 Csurtesy. C . S. Bureau of Biolooical Survey
Red Squill Investigations Effectiveness of Red Squill Extracts as Raticides ROBERT E. BUCK AND C. R. FELLERS Rlassachusetts State College, Amherst, Mass.
Toxic extracts can be prepared from red squill powder and are efficient raticides. Methyl and ethyl alcohols are the most efficient solvents tried for the extraction of the toxic principle from red squill. Extracts prepared with the Soxhlet extraction apparatus are more toxic than those prepared by shaking or stirring. Wheat bran is a suitable and inexpensive carrier for the dried extract. Short extraction periods remove inore toxic material from coarselj ground powder than from finely ground. Rats, which do not receive a lethal dose of red squill preparations in the initial feeding, will repeatedly- ccInsume more baits containing extracts. This is not true of powder baits. Toxic extracts can be prepared on a large scale b y a percolation method. Field tests using baits containing extracts from red squill demonstrate their efficacy as raticides.
HE previous paper in this series (3) showed that red squill can be used successfully as a raticide and that its effectiveness and the safety of its use make it a poison of unique action. The atlvantages of red squill extracts over red squill powder are that extract baits are more palatable and can be readily st.andartlieed as t o toxicity, and rats will eat the baits repeatedly. ComequeIitly, such extract baits are exceedingly valuable xn followup campaigns in cases where the rat9 are not completely exterminated by the first baiting. Clareinont (1) reported that the toxic principle of red squill could be extracted with cold and hot' water, but not with alcohol. Munch, Silver, and Horn (2) found that the toxic principle ivas soluble in alcohol hut not in water, acetone, or chloroform. Kumerous extracts, both in the laboratory and on a commercial scale, were prepared in the present study.
T
Preparation of Extracts for Toxicity -4ssays The preliminary work on the extraction of the toxic principle from red squill made use of a Soxhlet ext,raction apparatus in an effort to determine the most effective and practical solvent. After preparing the liquid extracts, the solutions containing the active principle were concentrated and dried on various substances in an attempt to make them easier to handle and use and t o render them more palatable to the rats. Lactose and wheat bran were finally used as the dry1497
INDUSTRIAL AND ENGINEERING CHEMISTRY
1498
ing agents. The amounts added to the liquid extracts were dependent on the desired strength of the finished product. If the extract was weak, only a small amount of the carrier was added, so that the rat would not have to eat an excessive amount of the material in order to get a toxic dose. After evaporating the solvent, a dry, easily-handled substance, free from undesirable characteristics due to the solvent, was
VOL. 27, NO. 12
maximum amount of the toxic constituent from red squill powder by this method. Cellosolve (ethylene glycol monoethyl ether) also gave good results as an extracting agent but not quite as good as the methyl and ethyl alcohols. Such solvents as water, diethyl ether, acetone, and ethylene dichloride were of no use and a t best extracted only traces of the toxic principle. The results of feeding tests on the above extracts are given in Table I. I n all cases 15 grams of red squill powder and 100 cc. of solvent were used. During these extractions the completion of the extraction of the red color from the red squill could be used as a fair indication of the completion of the extraction of the toxic principle. Effect of Particle Size on Extractability A portion of the red squill powder was screened and ex-
+Thermostat
FIGURE 1.
PERCOL.4TOR S E T - U P
obtained. This dry product was weighed and the relation of its weight to the weight of the original red squill powder from which the extract was made, was determined. I n this manner a direct comparison of the toxicity of the prepared extract and the squill powder from which the extract was prepared could be made, and the effectiveness of the solvent determined. The figures for doses of the various extracts are in terms of the original dried squill powder, rather than of the material as it was fed to the rats. The method of conducting the toxicity tests on the rats was described in the first paper of this series (S), and in this case the procedure was the same except that the extract dried on a suitable base was mixed with the regular diet of the rat colony instead of the powder. It was noted in the first feeding tests that the rats did not show any preference between the baits containing the extracts dried on wheat bran or on lactose. As a result, in all subsequent tests the extracts were dried on bran. Soxhlet Extracts By the use of the Soxhlet extraction apparatus with methyl and ethyl alcohols, practically 100 per cent of the toxic principle was extracted from the squill. Methyl alcohol was probably slightly more efficient than ethyl. The limited results also indicated that the 4-hour extraction period with methyl alcohol is the shortest extraction which will remove the TABLE
Extn. Period Hours 1" 2 4 8, dried on
CHaOH CHIOH Synthetic C H I O H CpHsOH CiHaOH CiHsOH CpHsOH
0
TABLE 11. TOXICITY DETERVISLTIOU OF METHYL~ L C O H O L EXTRACTS PREPARED BY SHAKING AND STIRRING" M e t h o d of Preparation
8 16
8-
lactose
41
2
S 8, dried on Blactose
4 8 4 4
2
4
............ 2/4 414
Stirring
2/2 2/2
2/2 2/2
......... 2/2 2/2
2/2
2/2
......... ......... .........
iji
2j2
2j2
1/1 2 / 2 2/2 6/6
2/2 4/4
Oii ...
ai2
0/2
074 0/4
All extracts dried on bran except where indicated.
2/2
Q
0/4 214 4/4
...
4/4 2/2
... ... ...
2/2
...
:$:::: 2/2 4/4 4/4
...
4/4
4/4 4/4 4/4
2/2 3)i 4/4 4/4 3/4
515
5/5
15
R a t i o of squill powder t o alcohol by weight, 1 to 6.
as that prepared from the coarse powder. 1Iechanical packing and tunneling of the fine powders in the extractor or percolator prevents complete extraction. I n coarse powders these factors are largely obviated and more of the toxic principle is removed.
5/5
2/2
5/5
15
60 90 120
4ji
...
...
30 45
5/5b 5/5
.. .. ..
.
Feeding Level, Jlg / K g 700 600 500
30
Feeding Level, bIg. per Kg. of Body Weight: 2000 1000 900 SO0 700 600 600 400
......... .........
Time
Mzn. Red aquill powder Shaking
DETERMINATIONS O F SOXHLET EXTRACTS O F RED I. TOXICITY SWILLPOWDER BY VARIOUSSOLVENTS
Solvent
Cellosolve Cellosolve Water E t h y l ether C~HICIQ Acetone
tracts were prepared from the coarse and fine powders. I n a short time a more complete extraction of the toxic principle could be made from a coarse (50-mesh) powder than from a h e (300-mesh) powder. The 30-minute methyl alcohol Soxhlet extract which was prepared from the coarse powder had approximately the same toxicity as the 2-hour extract which was prepared from the unscreened powder. The extract prepared from the fine powder was not nearly as toxic
4/4
718
4/4 4/4
0/4 0/4 4/4
3/5
... ... ... ... iji ... ...
4/4 3/4
. . . . . . 2/4 ::: ............ ............... ............... ...............
b T h e numerator of each fraction indicates t h e number of rats dying within 3 d a y s ; the denominator indicates t h e total number of r a t s f e d
Extraction by Shaking Another series of extracts was prepared using a shaking method of extraction. Methyl alcohol was used as the solvent in all instances. The ratio of solvent to powder was 6 to 1 by weight. The red squill powder and m e n s t r u u m were placed in a closed container and shaken in a shaking machine a t approximately two hundred and fifty agitations per minute. The results showed that the method of extraction was not nearly as efficient as the Soxhlet extraction method; only about 30 per cent of the toxic principle was extracted in 6 hours. The results of this series of experiments are given in Table 11. Soaking the red squill powder before the extraction process was started caused the particles of the powder to swell, thereby facilitating the penetration of the solvent into the powder and increasing the accessibility of the toxic principle.
INDUSTRIAI, AND ENGINEERING CHEMISTRY
DECEMBER, 1935
1499 ~~
TABLE 111.
Extract NO. LS-100 LS-101
Ratio. Solvent t o PowCharge Vol. der by oi oi Powder Solvent W e i s h t Grams Cc. 6:1 250 1875
. . ...
LS-102 LS-103 LS-104
250 188 8
1875 1420
6:l
LS-105
136 6
1030
...
LS-106 LS-107
250
1875
SUhIMliRY O F REDSQUILL EXTRACTS PREPARED BY
Solyent
O
Alcohol r u n through charge once Extract from LS-100 r u n through s a m e charge again E x t r a c t from LS:lOl r u n through same charge t h i r d time Alcohol r u n through charge once E x t r a c t from LS-103 run through second charge E x t r a c t from LS-104 r u n through third chnrge Alcohol r u n through charge once E x t r a c t from LS-106 r u n through same charge eecond time E x t r a c t from LS-107 r u n through s a m e charge third time Soaked i n hot solvent 4 t o u r s Soaked i n hot solvent 4 hours Soaked in hot solvent 4 hours Soaked i n hot solvent 4 hours
CHIOH
CHaOH
6:1
LS-109 LS-110 LS-111 LS-112
250 250 250 250
18i5 1875 1875 1875
6:1 6:1 6: 1 6:l
LS-113 LS-114 LS-115 LS-116 LS-11; LS-118 LS-123 LS-124.1
250 250 250 250 250 250 250
940 940 625 940 1875 940 1875 1875
3:1
...
CHIOH 50% CHaOH 3:1 {SOY CzHsOH &“OH 2:l CHIOH 3:l CHIOH 6:1 CHIOH 3:l CHlOH 6:1
. ....
.... .
LS-124B
KO.
Liters 95 3:1
Toxicity oi Temp. Marc. of E x - P o n d e r ,--Toxicity of E x t r a c t 3000 500 300 Ex1000 750 tractracted m g . / k g . mg./kg , m g . / k g . nig./kg. b l g . / k g tion
-
Description of Extraction
..
LS-108
PERCOLATIOX
CHaOH
LS-1000
22.68
LS-1001
22,68
95
3:1
CHIOH
1,s-1002
34.02
125
3:l
CHaOH
1
Soaked in hot solvent 4 hours Soaked i n h o t solvent 4 hours Soaked i n hot solvent 4 hours Soaked i n solvent 3 d a y s Soaked i n hot solvent 3 days Soaked in alcohol 6 days Soaked i n hot solvent 3 d a y s Fresh alcohol poured ol-er marc f r o m LS-123’eoaked for 3 d a y s E x t r a c t irom LS-124.1 poured over new charge of powder: soaked for 3 da!, 3
c.
%
25
14.6
4/4
3/4
3/4
0/4
25
14 6
2/4
4/4
3/4
0/4
...
25 25 25
14 6 6 6 5.3
4/4 4/4 4/4
114 2/4 4/4
1/4
0/4
1/4
2/4
0/4 0/4
313 4/4 4/4
26
6.2
114
014
0/4
0/4
4 /4
23
3.2
0/4
0/4
0/4
0/4
...
25
2.4
0/4
2/4
0/4
0/4
...
26 60 i0 60 60
2 0 11 5 6 3
1/4 0/4 2/4 4/4 4/4
1/4 2/4
23:
0/4 1/4 0/4 414 4/4
0/4 0/4 0/4
3/3 2/3
4/4 4/4 4/4 4/4
4/4 4/4 4/4 4/4
4/4
4/4
4/4 4/4 4/4 3/4 4/4 3/6 6/8
. .
..
014
4/4
4/4
..
0/4
60 60 60 25 60 25 60
Extraction by Stirring Methyl alcohol extracts, prepared by stirring in an electric Mixonette for 2 hours, extracted only about 10 per cent of the toxic principle. The ratio of powder to methyl alcohol was 6 to 1 by weight. The results of these experiments are given in Table 11. This ~ v a qthe leaqt effective method of extraction tried. Extraction by Percolation Later, red squill extracts were prepared using larger amounts of powder. These extracts were prepared in a waterjacketed percolator, fitted with a thermostatic heat control. A diagram of the percolator is given in Figure 1. The red squill powder was tied loosely in a cheesecloth bag and placed in the percolator, and the solvent was poured over it. The ertraction methods and their effectiveness in removing the toxic principle of red squill are given in Table 111. The results from extracts prepared from 50- to 75-pound lots of povder are also given in Table 111. The extracts prrpared from the large lots of pox-der were as toxic as those prepared on a small laboratory scale, practically 100 per cent of the toxic ratkilling principle being extracted. Canned baits containing red squill extracts were distributed for field tests over a period of several months and the results
6.6
9.4 4.2 7.9 0 9 10 9 3 9 4 5
4/4
3: :%
60
.,..
60
14.3
414
Soaked i n solvent 4 hours; extract drawn off 55 a n d poured back over,powder twice Soaked in solvent overnight. placed i n percol a t o r , ~ o a k e di n 112 total &ount of alcohol 58 Soaked’in solvent overnight: placed i n percol a t o r ; soaked in I / > t o t a l a m o u n t of alcohol 55
However, a 6-hour period of soaking before extraction did not produce a more toxic extract than a 2-hour period.
14 6
... ... ,. . ..
.. .
...
1/4 114 2/4
?/4 2/4 4/4 4/4 1/4 4/4
...
4/4
4/4
. ,.
., .
2/4
,...
. ,.
12/12
12/12
10/12
0/4
...
. .
818
718
X/6
0/4
of these tests (compiled by E. 31. Mills of the U.S. Bureau of the Biological Survey) are as follows : Number Number Number Number
using baits obtaining good results obtaining fair results obtaining poor results
260 141 29 90
In these field tests the extract baits were used to follow up red squill powder baits distributed from 1 t o 3 months previously. The powder baits consisted of meat, fish, and cereals put up in separate cans; the ext,ract bait consisted of cereal only. Thus the rats, which had ftlready been once baited, were offered no choice of baits, and the bait offered mas known to be inferior in palatability to either meat or fish. When these factors are considered, the results must be classed as very satisfact’ory. The continued use of extract? in follow-up baits is warranted. Further study is being given to this entire problem of red squill extracts, particularly in the production of concentrated preparations. Literature Cited (1) Claremont, C. L., Analyst, 47, 60-7 (1922). (2) Munch, J. C., Silver, J., and Horn, E. E., C . 3. Dept. Agr., Tech. Bull. 134 (19291. (3) O’Connor, BI. G., Buck, R. E.; and Fellers, (3. R.,IND. E ~ G . CHEM.,27, 1377-80 (1935).
RECEIVED M a y 2 6 , 1935. Presented ( b y title) before t h e Division of Apricultural and Food Chemistry a t t h e 8 9 t h Meeting of t h e American Chemical Society, R‘ew Y o r k , N. Y . , April 22 t o 26, 1935. Contribution N o . 228 of t h e Massachusetts Agricultural Experiment Station.
