fl -S

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A POISONOUS PRINCIPLE IN CERTAIN COTTON-SEED

MEALS’

ALBERT C. CRAWFORD

From the Pathological Division of the Bureau of Animal Industry, United States
Department of Agriculture

Received for publication, February 21, 1910

Cotton-seed oil and cotton-seed meal are now of great economic

importance. These products are manufactured from cotton
seeds. The oil is obtained in a crude condition by expressing
the seeds. This process is facilitated and the yield of oil increased
by first heathig the seeds so as to split up their cells and thus free
the oil. This heating is also said to aid the separation of the oil
from a portion of the proteins. The compressed residues are
known as cotton-seed cakes, which on being ground constitute
the cotton-seed meal.
In this country most of our cotton seeds are derived from
Gossypium hirsutum (L), formerly named Gossypium herbaceum.
These are known in the United States as the “Upland” seeds.
Sea Island cotton seeds, or Gossypium barbadense (L), have been
introduced into the United States and now yield about 1 per
cent of the total cotton crop.
The seeds of Gossypium barbadense after ginning are practically
naked, so that they are not decorticated before being made into
meal and there is as yet no separation made of the hulls from the

1 As the full report on the work done by the Bureau of Animal Industry on cot-
ton-seed meal will not be ready for publication before several months, and as
several of the Experiment Stations are now engaged in studying this problem, it
was deemed wise to publish an abstract of the laboratory work done in this office,
so that to coordinate, if possible, the work. On this basis the Secretary of Agri-
culture readily agreed to the publication of the present paper. A more detailed
report of this work is to appear in the bulletin series of the Bureau of Animal
Industry. The success of this work is due to the enthusiasm of Dr. John R. Mohler,
Chief of the Pathological Division.
 1

520 ALBERT C. CRAWFORD

kernels in the meal of commerce. The seeds of Gossypium hirsu-

turn are covered with a short fuzz. so that to get rid of this it is
necessary to remove the hulls from the seeds before the meal is
made.
As far back as 1769 or 177O, the Moravians at Bethlehem, Pa.,
expressed the oil, and in 1799 a patent was obtained in this
country for its extraction from the seeds. In 1783 a prize was
offered at London’ to planters in the British West Indies who
would express the oil from a definite amount of cotton seeds and
make the residue into cakes for stock feed ; but though this prize
was offered for several years it was never awarded and very little
progress was made in introducing these industries. The seed not
used for planting was practically thrown away. In this country
the use of cotton-seed meal as a stock feed arose mainly from the
exigencies of the Civil War, the southern planter then being corn-
pelled by necessity to use this material for his stock, A small
percentage of the meal is now consumed in the manufacture of
bread.
The feeding experiments and Uinical reports of the Bureau of
Animal Industry, of Dinwiddie,5 and others, have shown that
small quantities of cotton-seed meal can be fed to hogs with
profit. This is especially true when it is mixed with other feed
as a balance ration,. and particularly if fed discontinuously and
for not over three weeks, but if fed in large amounts some of the
animals succumb.
Adult cattle are not so sensitive as hogs to the injiu’ious action
of this feed, and the meal may be fed to them with greaterimpunity.
All domestic animals, and especially young ones, are said to be
liable to be affected by the, meal when it is improperly used.
The injurious action seems to depend upon the amount of meal
given. Cases of poisoning by this feed have been reported in
England, France, Denmark, Germany, and the United States.
2 Bishop, J. L.: History of American Manuf act., vol. 2, p. 81, 1866
‘Trans. Soc. Instituted at London for Encouragement of Arts, vol. 1, p. 252,

1806. -

Annual Report Bureau of Animal Industry, Dept. of Agric ulture for 1906,
J). 161, 1907. . S

Dinwiddie, 11. H.: Arkansas Agric. Exper. Sta. Bull. 85, 1905; Bull. 76, 1903.
 S T 2 1’ ‘‘- ‘-‘.- -S.,-- 5, S ‘S :-

A POISONOUS PRINCIPLE IN CO11ON-SEED MEALS 521

There are, however, so many reports of animals fed without injury

that the variations in the reports can only be explained by pre-
supposing a difference in the various cotton-seed meals used.
On macroscopic examination some of the animals dying from
cotton-seed meal poisoning show edema of the lungs, hemorrhagic
gastro-enteritis with ulceration of the stomach and intestines,
nephritis, and hepatitis. The myocardium may appear cloudy
and hemorrhages have been seen on the endocardium and also
on the epicardium. There is usually some congestion of the
central nervous system and collection of bloody fluid in various
serous cavities. The changes in the liver have been compared to
those found in phosphorus poisoning. These dropsical effusions
and nephritis are especially noted in the more chronic cases. On
the’ other hand, in some cases no characteristic macroscopic lesions
were found.
The clinical symptoms are such as would be expected from
these pathological lesions, that is, emaciation, disturbance of
the respiration and in the animal’s gait, hematuria, at times
bloody diarrhea and icterus, muscular weakness, abortion, and
in cattle, ulcers of the cornea with blindness. A rise in tempera-
ture has been noted in certain animals, but this may be due to a
secondary infection. The first published notice dealing with
this injurious action was apparently that of Voelker. This
appeared in England in 1859.
Some of the work performed by previous investigators suggested
that the injurious action of cotton-seed meal was due to a tox-
albumen, or a toxic alkaloid, such as muscarin or neurin, while
the inference from other work, especially that of von Nathusius,
was that it related to parasitic organisms found in the meal.
An analogy was drawn between cotton-seed meal poisoning and
Texas. fever; but while very numerois species of organisms have
been found in cottonseed meal no pathogenic ones have been
proved to be constantly present. However, it must be remem-
bered that certain of the reported cases of poisoning by this meal
6 Von Nathusius, W.: Zeits. ti. landw. Central-vereins d. Provinz-Sachsen, vol.
42, p. 263, 1888; Zopf: W. Beitr. z. Physiol. ii. Morphol. niederer Organismen.,
vol. 1, p. 57, 1892.
 ‘- . - S ________

522 ALBERT c. CRAWFORD

may be due to organisms, such as the anthrax bacillus, which the

meal may accidentally contain ; but these are isolated cases and do
not necessarily present the pathological lesions found in cases of
true cotton-seed meal poisoning.
The poisonous property cannot be claimed to be due to decom-
position of the feed, as fresh undecomposed cotton-seed meal will
produce this action. Other investigators attribute the injurious
effects to the indigestibility of the contained fibers, which leads
to intestinal obstruction. The amount of these fibers varies
from 2 to 20 per cent. Against this view is the fact that at
autopsy no cotton fibers are found in the intestines of animals
which have died from experimental cotton-seed meal poisoning.
It has been claimed, also, that there was a resin present in cotton-
seed meal which injured young animals, but there are no pub-
lished data to corroborate this statement.
Cholin’ and betain8 have been obtained from cotton-seed meal,
but the peculiar toxic action cannot be traced entirely to them as
the clinical symptoms and pathological lesions induced by
cotton-seed meal do not correspond to poisoning by these bodies.
There is no experimental evidence to show that cholin fed by
mouth is poisonous, although the evidence shows that if injected
subcutaneously it acts toxic to animals. Betain is claimed to
be nonpoisonous. Cholin was believed to yield the toxic com-
pound neurin9 on decomposition, and experiments of Schmidt
would suggest that cholin chlorid under the action of hay infusion
could undergo a transformation into a body resembling neurmn.
If this transformation occurred a poisonous action of this feed
could be perhaps explained; but this conversion of cholin into
neurin has not been proved to take place in the meal. Neurin
has been found to be normally present in animals in the adrenal
glands. Modrakowski’#{176} has recently noted that old cholin is

Boehm, R.: Archiv f. exper. Pathol. vol. 19, p. 88, 188.5; Sitz. d. Gesells. z.
Beforderung d. gesam. Naturw. z. Marburg. 1883, p. 23, 1884.
S Ritthausen, H. and Weger, F.: Journ, f. prakt. Chem. n.s., vol. 30, p. 32,
1884; Maxwell: W. Amer. Chem. Journ., vol. 13, p. 469, 1891.
‘Brieger, L. Zeits. f. klin. Med. vol. 10, p. 268, 1886; Gram: C. Archiv f. exper.
Pathol. vol. 20, p. 116, 1886.
1o Modrakowski, G.: Archiv. f. gesam. Physiol. vol. 124, p. 601, 1908.
 S

.S. i

45

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 523

much more toxic than the freshly prepared base. B#{246}hm,by

oxidation with nitric acid of cholin derived from cotton-seed
meal, has obtained a muscarin-like body which, on subcutaneous
injection, was toxic to cats. Werenskiold found in the meal a
basic body which resembled cholin in certain of its chemical
properties, but nothing is known as to the pharmacological action
of this base.
Tietze believed the trouble resulting from feeding with cotton-
seed meal to be due to its high protein content. The meal as a
rule does not contain over 0. 1 per cent sand, and death cannot
be traced to the mechanical action of this small amount of material
However, there are cases of death reported from feeding on meal
in which pieces of iron were accidentally mixed; but these cases
of injury to the stomach must not be confused with the true cases
of poisoning. In high grade meal such pieces of iron are removed
by an electro-magnet. It has been shown that the feeding of
proteins” to rabbits induces a hypersensibility to such compounds,
but it is still a question if anaphylaxis plays any part in cotton-
seed meal poisoning.

PHARMACOLOGICAL EXPERIMENTS

The first point in the present investigations was to find some

animal, preferably one of the herbivora, which could be used in
the laboratory and, by feeding it extracts of cotton-seed meal,
reproduce the main clinical symptoms and pathological lesions
which have been produced by feeding this meal to stock. Rab-
bits were selected because they do not vomit and are of a con-
venient size to handle. The problem was not to find a principle
in the meal which would kill on subcutaneous injection only, but
to find one which does so after administration by mouth. Numer-
ous bodies kill on subcutaneous injection, but unless they are
given in large doses do not so act by mouth.
The published reports of the experiments with cotton-seed meal

1 BOrnstein, F.: Cent. f. Bakter., vol. 50, p. 374, 1909.

FrCS - . - -- S ,-.‘S- S sf

524 ALBERT C. CRAWFORD

on rabbits and guinea pigs have varied ; some, as those of O1ig12

and Konig were negative, while those reported by Klien and Din-
widdie were positive as to a poisonous action on these animals.
The meal used in this work was made from the decorticated seed.
It had a brown color and an oily fresh odor.
Extracts were first made by treating the meal with distilled
water for 48 hours in the thermostat at 37.5#{176}C. An extract
corresponding to 200 grams was fed to a rabbit in two doses on
successive days. During 13 days’ observation this animal
showed no symptoms. The meal was digested” at thebody tem-
perature (37.5#{176}C.), first with pepsin for twenty-four hours, and
then with pancreatin for the same length of time. This extract
was concentrated in vacuo, and 30 cc. of the fluid, representing 75
grams of the meal, were fed on July 6, 1908, to a rabbit weighing
1683 grams. On the following day it was fed the same amount
of extract. This animal died during the night of July 7. The
stomach was intensely inflamed.
In certain of the following experiments we must consider not
only the injurious action of one or more compounds, but also the
effects of concentrated solutions upon the stomach walls. When
the extract corresponding to 200 grams was fed in a period of
four days no injurious action resulted.
After being in the laboratory about nine months, 100 grams of
the same meal were digested and the extract fed April 3, 1909, to
a rabbit weighing 1265 grams. This animal died within one hour
after the feeding. The stomach and duodenum were reddened.
Control feedings with distilled water and also with pepsin and
pancreatin proved inactive. In most cases the gross pathological
lesions suggest primarily the action of some intense irritant;
in some the macroscopic changes are very slight, and death
can be best explained in such cases as due to absorption of
poisonous principles, especially through injured gastro-intestinal
mucosa!.

12 Olig, A.: Zersetzung pflanzlich. Futter-u. Nahrungsmittel durch Bakterien.

Dissert. Munster, 1903.
‘3 In this paper the term digest-ion refers to digestion with pepsin and pancreatin.
r------’ - ---- S

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 525

EXPERIMENTS WITH VARIOUS SPECIES OF COTTON SEEDS

Upland Cotton Seeds

An important question now arises as to whether the toxic

agent is present in all stages of the manufacture of the meal, and
as to whether it is common to all species of cotton seeds. If it
is not present in all seeds it is desirable to see if those in which
it is absent would meet the demands of commerce. Seeds of
Gossypium hirs’utum (Upland seeds) , the same speciesfrom which
the meal previously mentioned was made, were obtained from
Dr. D. N. Shoemaker, of the Bureau of Plant Industry, and were
apparently normal, showing no indication of disease. Two hun-
dred grams of these seeds, the variety which has received the name
of ‘ ‘ Pride of Georgia, “ were ground and digested and the digestion
products fed, March 20, to a rabbit weighing 2342 grams. It
was almost impossible to entirely free these seeds from lint.
An estimate has been made, that after final delinting, Upland
seeds contain about 10 per cent lint. On this basis only 180
grams of the seeds, free from lint, were really used. This rabbit
died at night. The autopsy showed some erosions of the gastric
mucosa with bright red hemorrhages in the stomach walls. One
hundred and fifty grams of the same seeds were digested and fed
on March 25 to a rabbit weighing 2060 grams. On March 30 the
rabbit weighed 2036 grams and showed no symptoms.
Two hundred and fifty grams of this “Pride of Georgia” were
extracted with ether in a Sohxlet extractor to remove oils, etc.,
as it was found that the digestion product of 200 grams of the
seeds not freed from the oil, etc., was very thick and difficult to
feed. Such an extraction would remove any possible admixed
rhus poison. After the ether extraction, the residue was then
digested and concentrated to 92 cc. On March 25, 46 cc. repre-
senting an extract of 100 grams, was fed to a rabbit weighing
1694 grams. On March 26 the same amount of solution was
fed and the animal weighed 1662 grams. On April 3 the rabbit
weighed 1302 grams.
Two hundred grams of Upland seeds, known as “Keenan,”
526 ALBERT C. CRAWFORD

were ground and digested and the extract fed to a rabbit weigh-
ing 1 175 grams. This animal died in one hour. The stomach
and duodenum were inflamed and there was slight tympanites
present, but the lungs appeared normal. One hundred and fifty
grams of the same seeds were similarly treated and fed, March
27, to a rabbit weighing 1657 grams. On April 1, it weighed 1641
grams and showed no symptoms.
Some of the variety of Upland seeds known as ‘ ‘ Hawkins”
were ground and 200 grams were digested. The solution was
fed March 27, at 3 :35 p.m. to a rabbit weighing 2075 grams.
This animal died at night. The post-mortem examination showed
the stomach intensely inflamed. Of thesameseeds l50grams were
similarly treated and the extract fed to a rabbit weighing 1132
grams. This rabbit died in one hour. The stomach and duode-
num were inflamed and there was marked tympanites present,
but the lungs were normal.
From the variet.y of Upland seeds known as “Columbia,” 200
grams were ground and digested and the extract fed to a rabbit
weighing 1447 grams. This animal died at night. The autopsy
showed that the stomach and duodenum were reddened, but the
lungs were normal. Of the same seeds 150 grams were similarly
digested and the extract fed on March 27, to a rabbit weighing
2140 grams. On March 31, the’ animal weighed 2188 grams.

Sea Island Cotton Seeds

Sea Island cotton seeds seemed especially suitable for laboratory

work as they could be almost entirely freed from lint by ginning.
The “Centerville” Sea Island cotton seeds were first used. These
had the black hull found in the fully ripe seeds. Two hundred
grams of these seeds were finely ground and digest&i for twenty-
four hours with pepsin and twenty-four hours with pancreatin.
These digestive products were then concentrated to 126 cc. and
one-half of this extract was fed on February 19 to a rabbit weigh-
ing 2182 grams. On February 20 the remainder of the extract
was fed to this rabbit which then weighed 2167 grams. On
March 1, its weight was 2140 grams.
 -‘ - 7 5 S - S s S #{182} S - . S 5 5 5 T

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 527

Two hundred grams of these seeds were ground, placed in a

Sohxlet extractor and extracted with ether until the ether returned
colorless. The ether extract weighed roughly 49 grams. After
this extraction the seeds were digested with pepsin and pan-
creatin and the extract concentrated to 65 cc. This was fed in
one dose on February 25 to a rabbit weighing 1506 grams. On
March 8, the animal weighed 1605 grams.
Two hundred grams of control meal made from Upland seeds,
after similar ether extraction, were digested and the extract fed
in one dose to a rabbit. This animal weighed 1215 grams and
died in about two hours after feeding.
Three hundred grams of these Sea Island seeds were finely
ground and digested as above. The fluid was concentrated to
140 cc. Seventy cubic centimeters representing 150 grams of
the ground seeds were fed March 1, 1909, to a rabbit weighing
2140 grams. On March 2, the feeding was repeated. The
animal now weighed 2109 grams. On March 8 it weighed 2152
grams, showing a gain in weight.
Two hundred and fifty grams of the same seeds were digested
and the extract fed in one dose on March 20 to a rabbit weighing
2030 grams. March 25, the weight was 2060 grams.
Seeds and meal made from Sea Island cotton seeds were
obtained from a factory in Madison County, Florida. In this
county where these seeds were grown the Sea Island and the
Upland cotton belts overlap.
Of these seeds 200 grams were ground and digested and the
extract fed April 15 to a rabbit weighing 1917 grams. The fluid
used in this case amounted to about 70 cc. The animal died one
hour and fifty-five minutes after the feeding. The stomach walls
were slightly reddened, while the upper portion of the duodenum
was markedly irritated. The mesenteric vessels were dilated;
the lungs seemed normal and the heart was relaxed; the kidneys
were congested.
Two hundred grams of the meal obtained from this factory were
digested and the extract fed April 14 to a rabbit weighing 1227
grams. This animal died at night. The gastric walls were
much inflamed. One hundred and fifty grams of the seeds from
 I

528 ALBERT C. CRAWFORD

which the meal was made were digested and the extract fed on
April 24 to a rabbit weighing 1669 grams. On April 29 the
animal weighed 1690grams, thus showing a gain inweight. Two
hundred grams of these seeds were extracted in the Sohxlet
extractor with ether until the ether returned colorless. After
the extraction the seeds weighed 161 grams. The seeds were
then digested and the extract fed April 22 to a rabbit weighing
1974 grams. After the feeding the animal became very sick and
death was expected. On Apri 23 it weighed 1850 grams; on
April 26, 1949 grams.

COTTON-SEED MEAL MADE FROM SEA ISLAND COTTON SEEDS

Some fresh meal, made from last season’s Sealslandcottonseeds,

was received from Blackshear, Georgia. This meal had a dark
color and gave a fresh but slightly oily odor. Mr. W. A. Orton,
of the Bureau of Plant Industry, through whom the seeds were
obtained, stated that seeds used in Blackshear consisted of about
95 per cent of the Seabrook Sea Island cotton seeds with about
5 per cent hybrids formed by the crossing of these seeds with the
Upland variety. These Seabrook Sea Island seeds showed only
slight botanical differences from the Centerville variety.
Of this meal 200 grams were extracted with ether (U. S. P.)
for two days and the residue after this extraction was digested.
These digestion products were fed on March 18 to a rabbit weigh-
ing 1622 grams. This animal died in one hour and fifty minutes.
The duodenum was much more noticeably reddened than the
stomach, which was merely reddened in areas. Two hundred
grams of this meal after the ether extraction weighed 173 grams.
One hundred and fifty grams of the same meal were digested
and fed on March 16 to a rabbit weighing 2377 grams. Its weight
on March 19 was 2349 grams. Of the same meal 180 grams
were digested and the extract fed April 3 to a rabbit weighing
1267 grams. This animal died in one hour and twenty-two
minutes after feeding. The stomach was found reddened and the
large intestines were markedly distended with gas. At present
there seems to be a prejudice among the dealers against market-
ing meal made from Sea Island seeds.
 ‘ .,

A’ POISONOUS PRINCIPLE IN COTTON-SEED MEALS 529

CONDITIONS INFLUENCING THE FORMATION OF A POISONOUS

PRINCIPLE

It can be seen that the digestion extracts of Centerville Sea

Island cotton seeds were apparently harmless to rabbits even
when fed in large amounts ; yet extracts of the meal made from
similar seeds induced death in the same manner as those from the
meal found on the market made from the Upland seeds. The
toxic principle must, then, have developed in the manufacture
of the meal. The process of manufacture consists in crushing
the seeds and cooking the grist in steam-jacketed metallic vessels
under from 40 to 100 pounds steam pressure. When m,aking meal
from cotton seeds the temperature inside the kettle in cooking is
said as a rule, not to exceed 220#{176}
F. (104.7#{176}C.). The length of this
heating varies from 15 to 45 minutes. The heated mass is then
subjected to a pressure of from 3500 to 4000 pounds to express
the oil.
Of the seeds obtained from Blackshear, Ga., 200 grams were
ground and digested and after concentration the extract was fed
April 1 to a rabbit weighing 2207 grams. On April 7 it weighed
2334 grams. The same firm in Blackshear, Ga., which sent the
seeds also sent meal made from such seeds under factory condi-
t-ions. In making this meal the seeds were heated for 40 minutes
under 80 pounds pressure.
Of this meal 200 grams were digested and the extract fed April
3 to a rabbit weighing 1372 grams. This animal died in less than
one hour. The stomach and duodenum were found markedly
reddened. The remainder of the intestines were not examined
and the autopsy otherwise proved negative to macroscopic
examination.
To simulate roughly the conditions of manufacture, 250 grams
of ground Centerville Sea Island seeds, which were proved to be
harmless, were placed in a wide enameled can, then heated for
one hour and twenty-two minutes in an oil bath, the oil of which
had a temperature of from 110 to 118#{176}
C. Before heating they
were of a light-yellow color, but afterwards became slightly
darker. The mass was then digested and the extract fed on
530 ALBERT C. CRAWFORD

March 12 to a rabbit weighing 1767 grams. On March 25, it

weighed 1699 grams
Two hundred and fifty grams of these seeds after grinding were
heated in the oil bath, the oil of which registered from 135 to
145#{176}
C. The heating was continued one hour. The seeds on
heating turned somewhat darker. This mass was digested and
the extract fed on March 12 to a rabbit weighing 2084 grams.
On March 19 the animal weighed 2040 grams. Two hundred
grams of the same seeds were heated intermittently for twenty-
three hours at periods of about eight hours in the oil bath, the
oil of which registered 1 10#{176}
C. The seeds were then digested
and the extract fed on April 1 to a rabbit weighing 2006 grams;
April 10 it weighed 1965 grams.
Two hundred grams of these seeds were heated in an oil bath
eleven and a half hours. The oil of this bath registered 120#{176}
C.
but the actual temperature to which the seeds were heated was
not noted. The mass was then digested and the extract fed
April 1 to a rabbit weighing 1641 grams. On April 2 the weight
of the animal was 1528 grams. The animal died at night. The
autopsy showed that the stomach and upper portion of the duo-
denum were slightly reddened.
Of the same seeds 200 grams were similarly heated for one hour
in a bath, the oil of which registered 160#{176}
C. The cooked mass
had the brown appearance seen in meal made from the Sea Island
seeds. It was digested and the extract fed to a rabbit weighing
2176 grams. This rabbit died at night. The autopsy showed
that the stomach and duodenum were reddened and that there
were petechia! in the gastric walls.
Two hundred grams of the same ground seeds were heated in
the bath so that the temperature of the mass ranged between 100-
105#{176}C. This is the temperature to which the Upland seeds are
usually subjected. The oil in the bath registered about 126#{176}
C. There was only slight darkening of the color, but the
meal did not appear as dark as that obtained from the factory.
This temperature was held for one hour and the mass was fre-
quently stirred. After the heating the seeds were digested and
the extract fed on April 8 to a rabbit weighing 2264 grams. This
r.wr!:._Sr T - ‘ -‘ - - - - -5 ..- - S : ‘ ‘ -- - “ -‘“-‘- S. -- “ ; ‘ -.S,

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 531

animal died at night. The autopsy showed that the stomach

was markedly inflamed and that the duodenum was somewhat
hemorrhagic. The lungs showed merely hypostatic congestion.
Cotton seeds when stored heat readily, and it remains a prob-
lem to see what part this autogenous heating or bacteria may
play in the production of a toxic principle. An important point
to consider in making meal from Sea Island seeds, which contain
only small quantities of the poisonous constituent, is to avoid
high heating.

EXPERIMENTS WITH COTTON-SEED OiL

Cotton-seed oil has long been used in human alimentation, and

this usage has been endorsed by the Comit#{233} consultive de 1’Hy-
giene de France and also by a committee appointed by the Spanish
government. As far back as 1667 Du Tertre’4 reported that an
oil made from flowers of the cotton plant was used as a dressing
for old ulcers by the natives of the Antilles.
After feeding a large dose of the crude cotton-seed oil (25 cc.)
to a rabbit its weight steadily fell and remained low, and when a
moderate dose (15 cc.) was fed and this was followed by repeated
small ones the animal died showing irritation of the gastro-intesti-
nal canal. Lendrich’5 noted that after the daily administration
of cotton-seed oil his rabbits emaciated, but readily assimilated
the same dose of oil when given intraperitoneally.
After feedings with purified cotton-seed oil, or with olive oil,
there was a loss in weight but the animals did not die. After feed-
ing pure cod liver oil the animal died. The loss in weight was
small in the case of feeding the purified cotton-seed oil. The fact
that the cotton-seed oil gave no red reaction to litmus paper would
suggest that the loss in weight, noted after feeding the crude oil,
was not due to free oleic acid. This acid has been recently shown
to play an important role in the production of certain forms of

‘4 Du Tertre, R. P.: Hist. g#{233}n. des Antilles, vol. 2, p. 150, 1667.

‘ Lendrich, K.: Zeits. f. Ijntersuch. d. Nahrungs-u. Genuesmittel. vol. 15,
p. 326, 1908.
532 ALBERT C. CRAWFORD

anemia. Oils” interfere with gastric digestion in man, and this

fact must be taken into consideration in experiments on such
animals as rabbits.

CHEMICAL EXPERIMENTS

Chemists, basing their opinion on the work of B#{246}hmand Max-

well, believed that the poisonous properties of this meal were due
to cholin. Maxwell, while his results are not strictly quantita-
tive, estimated the mixed chiorids of cholin and betain as 0.24
per cent. This is too small an amount to explain the toxicity of
the meal, for the recent experiments of von Hoesslin’7have shown
that even two grams of cholin hydrochiorate, the more toxic of
these two bodies, can be fed by mouth to a rabbit without any
serious result. The writer found that the toxic principle of
certain cotton-seed meals could be precipitated in some cases by
lead acetate from aqueous solutions. It is said that cholin is not
so precipitated.
Ritthausen has called attention to the presence of over 3 per
cent raffinose in cotton-seed meal. Fischer and Niebel have
found that this sugar is not hydrolyzed in the intestine of the
horse, and Pautz and Vogel showed that it is unchanged in the
dog’s intestine. The administration of over 6 grams of raffinose
failed to induce symptoms in rabbits.
The Bureau of Chemistry determined the ash content of this
meal to be 5.76 per cent and reported the absence of barium, lead,
or antimony, but that there were distinct traces of copper and
arsenic in 25 grams of the meal. The presence of copper may be
easily accounted for as the meal is made in copper vessels. Em-
merling has found meal which was poisonous to stock, yet gave
no reaction for metals or alkaloids.
Ether and ethyl alcohol (45 per cent) extracts of 200 grams of
the meal did not kill, and after treatment of the meal with 95 per
cent ethyl alcohol for three days the digestive products of the
residue caused emaciation, and death followed only after sixteen

“Coure, D. M. an(l Munson, J. F.: Archives Intern. Med., vol. 1, p. 61, 1908.
‘ von Hoesslin, W.: Beitr. z. ehem. Physiol. u. Pathol. vol. 8, p. 33, 1906.
!; _..;_,-,_ .. - - S S S

A POISONOUS PRINCIPLE IN CO’flON-SEED MEALS 533

days, showing that prolonged contact of strong alcohol interfered,

in some way, with the activity of the injuriou compound.
The digestion products of 150 grams of the meal, if fed in 75-
gram doses for two successive days, induced death on the last
day of the feeding. The concentrated digestion products of 200
grams of the meal was taken as a starting point for the chemical
work. This amount was greater than the toxic dose, so as to
allow for loss during manipulation and also for the power of the
stomach to render certain poisons less active. Such a solution
could be heated to a fairly high temperature without apparent
injury. Shakings from such a product by the Dragendorif
method with petroleum ether, benzol, ether, and chloroform proved
inactive ; acid amyl alcohol, however, was active.
Lead, mercury, and copper acetate precipitated a toxic agent
and the filtrate remained inactive, provided the precipitation was
carefully done. The lead precipitate on decomposing with H2S
and concentrating in vacuo yielded a red gummy material which,
when fed to rabbits, produced an intense gastro-enteritis and
induced death in about one hour. If not concentrated too far
a white gelatinous colloid separated on cooling. This seemed to
separate best if the decomposed lead precipitate was not filtered
carefully from the PbS, and failed to reappear on reprecipitation
with lead acetate. Apparently the same colloid appeared on
dialyzing off the salts from the original digestion products. This
colloid when fed in suspension produced no symptoms, while
the filtrate was active.
The concentrated solution above referred to, when placed in a
vacuum desiccator, yielded a gummy matrix and5 needle-shaped
crystals appeared in about ten days, but the number of crystals
was small. On neutralizing this decomposed lead precipitate
withNaOH the solution induced death four hours after feeding
it to a rabbit weighing 792 grams. The autopsy of this animal
was negative, suggesting that death was due to the absorption
of a poisonous body rather than to gastro-intestinal irritation.
However, the clinical symptoms were about the same as those
seen after feeding the acid solutions.
The concentrated digestion product and also the decomposed lead
534 ALBERT C. CRAWFORD

precipitate from this amount of meal were made alkaline with

NaOH, but though distilled, in some cases with a current of air,
in other cases in an atmosphere of hydrogen, in others in vacuo,
and in others with steam, the distillates when caught in acetie
acid water failed to produce death, showing the toxic action was
not due to a volatile poison. By this boiling in alkaline condi-
tion the solution lost much of its toxicity.
On decomposing the lead precipitate with H2S the resulting
solution became markedly acid although every precaution was
taken to prevent acids from reaching it. Thisreaction suggested
the presence in the meal, either of some acid, or acid salt, or per-
haps an acid body such as a saponin.
According to Tereg’8 the acidity of extracts of cotton-seed meal
is due largely to phosphoric acid; but a portion may be due to
lactic acid. Withers and Fraps’ claim there is only a small
amount of organic acids present in cotton-seed m’eal. The acid-
ity of the aqueous extract of 1 gram of this meal, examined
by Street2o, corresponded to .004 gram of NaOH, when either
phenophthalein or Toepfer’s test were used as an indicator, but
oniy to .0026 gram when litmus was used.
On pouring the concentrated solution of the decomposed lead
precipitate into 95 per cent ethyl alcohol a white precipitate
resulted. This precipitate quickly settled and could be filtered
off in a short time. It was pharmacologically inactive. The
filtrate, which was of a golden color, would on freeing from
alcohol, usually kill rabbits in a few hours.
After this precipitation the filtrate was concentrated in vacuo
and methyl alcohol added. On pouring ether into the solution
a precipitate gradually settled. This was gummy in character
and yielded a solution with water which foamed. Such a solu-
tion induced death in a rabbit. The animal showed erosions of
the gastric mucosa. The ether precipitate failed to yield crystals
from a methyl alcohol solution. The fact that the poisonous

18 Tereg, J.and Arnold, C.: Tier#{228}rztl. Arzneibuch, pt. 3, p. 438, 1892.

‘ Withers, W. A. and Fraps, 0. S.: North Carolina Exper. Sta. Bull., 179, 1901.
“ Street, J. P.: Proc. 25 Ann. Conven. Assoc. Offic. Agricultural Chemists,
Bureau Chemistry Bull., 122, p, 163, 1909, United States Department of Agricul-
ture.
‘:-: -‘- --S #{149} S ,

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 535

principle was precipitated by lead and copper acetate, and that

these precipitates, on decomposition with H2S, yielded solutions
markedly acid to litmus, suggested the presence of a saponin, as
the partial, if not the sole cause of the trouble. This seemed
especially probable since the pathological lesions resulting from
feeding cotton-seed meal corresponded in some respects to those
due to poisoning by saponins. However, none of the methods
yet proposed for isolating saponins proved satisfactory for yielding
an active compound.
It was noted that after precipitating the acid solution with 95
per cent alcohol the filtrate, on concentration, would give a pro-
cipitate on the addition of much water. The filtrate now had
less color and still induced death in rabbits. If washed charcoal
was added to this filtrate and the whole warmed for a few minutes
in the water bath, the solution became practically colorless, but
on exposure to air took on a slight color. It would kill rabbits in
a few hours.
This solution on standing in a desiccator yielded a colored gum
and after about eight weeks crystalline needles formed. These
crystals were separated from the gummy matrix by means of a
porous plate. A solution of them proved inactive, while the
gummy matrix, when similarly fed, killed rabbits in about one
hour. The animals showed the typical gastro-enteritis such as
resulted from feeding the original digestion products of the meal.
The fact that the crystals were physiologically inactive suggested
that the active principle in this extract was an amorphous body.
It has been found that cotton-seed meal contains considerable
phosphoric acid. Now certain phosphoric acids form amorphous
gummy masses and crystallize only under peculiar conditions
and it was suspected .that the toxicity of this meal was due to
one of these acids. If this surmise proved correct, the fact
could easily be explained that Sea Island cotton seeds, while
themselves harmless, could by simply heating be made to yield
a meal which proved poisonous, since it is known that orthophos-
phoric acid can be converted into the meta- or pyre-phosphoric
acid by heat. Thus orthophosphoric acid when heated to even
100#{176}C. in the presence of metaphosphoric acid, yields pyre-
 - . - -- ,.-S S #{149} . S / , --

536 ALBERT C. CRAWFORD

phosphoric acid, and metaphosphates are obtained from salts of

orthophosphoric acid by heating. Pyrophosphoric acid is a soft
glassy mass2’ which crystallizes only at -10#{176}C, and after three
months’ standing. -

The gummy mass containing the crystals was treated with

commercial absolute ethyl alcohol to remove inorganic salts and
the filtrate evaporated to about 5 cc., then treated with watei
and filtered. The filtrate was precipitated with lead acetate, and
the lead precipitate washed with distilled water and stirred for
several minutes with dilute acetic acid, then washed with distilled
wateruntilthe washings wereneutral in reaction. Theprecipitate,
after decomposing with H2S, yielded a solution which was color-
less and very acid in reaction. It induced death in rabbits in
about one hour. This solution after neutralization with NaOH
was fed to a rabbit weighing 922 grams. The animal died in
about twenty hours and on macroscopic examination showed
no characteristic lesions.
The solution gave no protein reaction with Millon’s reagent,
by the xantho-proteic, by the biuret reaction, or with tri-chlor-
acetic acid, so that the poisonous agent in it was not a tox-albu-
men. Fehling’s solution gave no reduction, either before or after
hydrolysis with a few drops of HC1, indicating the absence of
glucosides, while the failure of certain alkaloidal reagents to pro-
cipitate would suggest the absence of alkaloids in amounts suffi-
cient to explain the effects of the extract.
After neutralizing the solution it gave a white precipitate with
Ag NO,,22 which was insoluble in excess of sodium pyrophosphate,
but soluble in excess of the free acid. (The ‘white precipitate
which silver gives with a metaphosphate is soluble in an excess of
sodium metaphosphate.) This precipitate was soluble in NH4HO
and HNO,. It was not AgCl, as silver also produced a precipitate
in a decomposed copper precipitate solution where the chlorides
would be eliminated. Barium chloride yielded a white precipi-
tate, apparently insoluble in sodium pyrophosphate, and not
reprecipitable by NH4HO. Ammonium molybdate gave no pre-
21 Giran, H.: Comp. Rend. Acad. d. Sci., vol. 135, p. 961, 1902.
22 Arnold, C. and Werner, G.: Chem. Zeits. vol. 29, p. 1326, 1905.
 - ,--. ‘ ,5 5 S, S .5 5 -

.. - .

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 537

cipitate in
the cold, but on heating, a heavy yellow precipitate
formed. Magnesia mixture gave at once a heavy white precipi-
tate. The acetates of the metaLs, lead, mercury, iron and cad-
mium all gave precipitates, while the chlorides gave them only
in certain cases. After adding Zn and 112504 and hetting, the
vapors which resulted produced some slight yellow color in contact
with solid AgNO3, but no more than traces of phosphorous acid
could be suspected.
These heavy precipitates indicated the presence of a phosphoric
acid and that one of these acids, or their salts, played a part
in the poisonous action of the meal; but as has been shown by
Schulz and also by Gamgee, Priestley and Larmuth sodium
orthophosphate (Na,HPO4 + 12 H,O) is harmless; therefore the
poisonous action of the meal must be traced to the presence of
either meta- or pyre-phosphoric acid.
After neutralizing with NaOH to get rid of any free 112504,
formed by oxydation of the H2S, the solution was acidified with
acetic acid. On adding Cu SO4 solution a heavy bluish-white
precipitate formed, which was soluble in an excess of the mother
fluid. Zinc sulphate gave a white precipitate with the same
solution, likewise soluble in a large excess of the acid solu-
tion. These reactions according to Arnold and Werner would
indicate pyrophosphoric acid. Cobaltamine solution did not
yield distinctive results with control solutions of either meta-
or pyre-phosphoric acids or their sodium salts. The precipitate
with magnesia mixture was soluble in acetic acid and also in excess
of a sodium pyrophosphate (Na4P2O,) solution, and also soluble
in a large excess of magnesia mixture. According to Arnold and
Werner, the precipitate which metaphosphoric acid gives with
magnesia mixture is insoluble in acetic acid, magnesia mixture,
and also in excess of alkaline metaphosphate, while that obtained
from pyrophosphoric acid is soluble in acetic acid, in excess of an
alkaline pyrophosphate, and in excess of magnesia mixture. Egg
albumen was immediately precipitated by the acid concentrated
solution, even 11 the cold. This may be perhaps explained as due
to H2SO4, formed by the oxydation of the H2S, or to some unknown
factor, and not to a possible metaphosphoric acid, because on dis-
 . - - - - - . - S- 75

538 ALBERT C. CRAWFORD

solving the magnesia-mixture precipitate in acetic acid, egg albumen

gave only a cloud, and this was soluble in an excess of a solution
of pyrophosphoric acid, while a solution of pyrophosphoric acid
with a fair excess of acetic acid caused a gelatinization of egg
albumen, which was soluble in an excess of the acid, suggesting
merely quantitative differences in the behavior toward egg albu-
men. Arnold and Werner state that they have tested all the
published reactions for the three phosphoric acids, but have found
all unsatisfactory, and in their list o differential tests for the salts
of these acids omit that with egg albumen.23
An acetic-acid solution of the precipitate formed by magnesia
mixture was made alkaline with NaOH. This gave no precipi-
tate on adding Nylander’s solution, but a white precipitate
quickly appeared on adding a control solution of sodium meta-
phosphate. The precipitation by alkaline bismuth solution is
one of the few tests which Arnold and Werner consider distinc-
tive of a metaphosphate. Copper acetate gave a bluish-white
precipitate apparently insolubh in acetic acid, but soluble in
NH4HO. A control solution of di-sodium hydrogen phosphate
gave a heavy dark blue precipitate with this reagent, while sodium
pyrophosphate gave a bluish-white one. Ferric chloride gave
a white precipitate. Uranyl acetate gave a yellow-white precipi-
tate, soluble in a large excess of the acid solution, but the pre-
cipitate obtained by means of uranyl acetate from a solution of
the magnesia-mixture precipitate seemed almost white. The fact
that AgNO3 gave a white, and not a yellow precipitate, would
indicate that orthophosphoric acid was not present, or if so,
in such slight amounts that its yellow color was masked.
A similar solution to the one used for making these qualitative
tests was evaporated in a platinum bowl and then treated for
about forty-eight hours with concentrated H2S04, so as to convert
the pyro- into ortho-phosphoric acid. The solution was made
alkaline with NHS4HO and then acidified with acetic acid. This

23Rona says “Der Fallung durch Metaphosphorsaure liegt nach Biltz weniger

eine chemische als eine Zustandsaffinit#{228}t zugrunde, die mit der kolloidalen
Natur der Metaphosphorsaure zusammenhhngt.” (Oppenheimer, C. Handb. d.
Biochemie, vol. 1, p. 272, 1908.)
 -“S--

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 539

gave with AgNO, a canary-yellow precipitate, indicating the for-

mation of orthophosphoric acid.
A similar solution made from 200 grams of the meal was acidu-
lated with acetic acid and precipitated with copper acetate. This
precipitate was dissolved with NH4HO, and after acidulating
with acetic acid, the solution was precipitated with lead acetate.
This precipitate was washed with distilled water until the wash-
ings gave no acid reaction. After decomposing with 1125 it
induced death in rabbits. The filtrate from the copper precipi-
tation proved inactive.
A similar solution was treated with magnesia mixture. The
precipitate was dissolved in water with the aid of a small amount
of dilute HNOS. To get rid of the free acid the solution was
then rendered alkaline with NaOH, and the precipitate which
formed, was redissolved by the aid of acetic acid. The solution
was then precipitated by lead acetate and the precipitate decom-
posed with H2S; this, after concentration, was fed July 26, 1909,
to a rabbit weighing 1294 grams. The animal died that night.
The gastric mucosa was eroded and cauterized. In the upper
portion of the small intestines the mucosa seemed eroded. The
lungs appeared normal. The stiprarenals were not enlarged.
From these experiments one would argue that the poisonous
action of the meal is due to a pyrophosphoric acid compound,
but it is not yet proved that this acid exists as such in cotton-
seed meal. However, it is probably present in the digestion
products of the meal as a salt of this acid, and in the extract the
acid was freed by treatment with lead. It is to be presumed
that in the seeds and meal complex phosphorus compounds,
under the influence of the digestive ferments, break into com-
pounds of this acid. In extracts of our meal the reactions do
-not point to the presence of a metaphosphate, but as the ortho-
is readily transformed into the meta- or pyro-phosphoric acid,
it would be expected to find reactions for met.aphosphates in
some cotton-seed meals.
Dancy24 has called attention to the fact that the phosphoric

24 Dancy, F. B.: Proc. 7 Ann. Convent. Assoc. Offic. Agric. Chemists. Division
of Chemistry. Bull. 28, p. 161, 1890; United Stat.es Department of Agriculture.
 . ..‘.S 5 5 .. S . . 5 5 S

540 ALBERT C. CRAWFORD

acid of cotton-seed meal reacted differently to molybdic solution

than one would expect from orthophosphoric acid. Harding’
has already claimed that meta-, pyro-, and ortho-phosphoric
acids are present in cotton-seed meal and suggested “ that some
of the peculiar, and in certain cases injurious and even fatal
effects produced by the use of cotton seed and cotton-seed meal
as feeding stuffs may be due, in a measure at least, to the pres-
ence of these meta- and pyro- compounds. “ Hardin gives no
pharmacological tests to substantiate his statements.
Gamgee, Priestley and Larmuth have shown by subcutaneous
injections into rabbits that sodium orthophosphate (Na,HPO4)
is almost nontoxic, whereas sodium meta- and pyro- phosphates
are much more toxic. These data have been corroborated by
Schulz.27 The former noted that after the injection into animals
of sodium pyrophosphate the lesions induced in certain cases
were “identical with those observed in some cases of phosphorus
poisoning,” and add that “in its effects on the general nutrition
the poison has shewn great resemblance to the poisonous element
phosphorus * * for in cases where death has been delayed
there has been very marked fatty degeneration of the kidneys,
of the muscular substance of the heart, with slighter degeneration
of the liver.” These investigators were unable to induce death
by feeding these salts by mouth. A sodium salt of phosphorous
acid, after similar injection, also induced death in rabbits and on
post-mortem examination the lesions resembled those seen after
phosphorus poisoning. The corresponding salt of hypophosphor-
ous acid proved inactive. Some of the lesions found in cotton-seed
meal poisoning are said to resemble those seen in phosphorus
poisoning.
The biological experiments with meta-and pyro-phosphates need
to be repeated, as much doubt exists as to their real chemistry

2’. Hardin, M. B.: Proc. 9 Ann. Convent. Assoc. Offic. Agric. Chemists. Bureau
of (‘hemistr. Bull. 35, p. 50, 1892; also Report of Chemist. Fifth Ann. Report
South Carolina Exper. Station, 1892, p. 16, 1893.
Gamgee, A. Priestley, .1. and Larmuth, L.: Journ. of Anat, and Physiol., vol.
11, p. 255, 1876-7.
27Schulz, H.: Arch. f. exper. Pathol., vol. 18, p. 174, 1884. -
 , S S 7 ‘W5T55 ‘ ‘ :- - - -‘- . ..-; - ‘-:- ‘ - #{149}- - - #‘V7 ‘

A POISONOUS PRiNCIPLE IN CO1FON-SEED MEALS 541

and there are varieties of these salts with the same percentage
formulae. S

The total amount of phosphoric acid in Sea Island cotton seeds

has been estimated by Shiver to average from 1.2 to 1.6 per cent,
while the meal made from these seeds averaged 1.71 per cent.
Meal made from the decorticated seeds contains 1.26 to 4.62 per
cent phosphoric acid, with an average of 2.88 per cent, while
Uplands seeds gave on an average 1 .27 per cent. The Bureau of
Chemistry reported that the meal used in these experiments con-
tained phosphorus corresponding to 2.05 per cent P2O5. This
determination was made by the magnesium-nitrate-method.

PHARMACOLOGICAL EXPERIMENTS WITH SALTS OF VARIOUS PHOS--

PHORIC ACIDS

Sodium Melaphosphate

Sodium salts of these acids were selected on account of the

slight toxicity of the sodium ion.
On July 1, 1909, a rabbit weighing 1104 grams was fed with an
aqueous solution2s of 2 grams of sodium metaphosphate. On
July 2 the animal weighed 1058 grams and was given the same
amount of metaphosphate. On July 3 the weight was 974 grams.
The animal died at night. There were some hemorrhage in
the stomach walls. The gastric mucosa peeled off easily and
there was a decided increase of mucus on its walls. The duodenal
mucosa was hemorrhagic. The lungs seemed normal. The liver
was apparently not congested and the kindeys appeared a trifle
dark.

Sodium Pyrophosphate29 (Na4P2O7 + 10 H20)

On October 13, 1909, a rabbit weighing 1280 grams was fed with
0.5 gram of sodium phyrophosphate dissolved in 25 cc. of water.
This feeding was repeated every week day until it had received

28 About 50 cc. of water were used for solution of each salt unless otherwise
specified. The Sodium metaphosphate was made by Dr. B. Herstein;
“ Combinations of cholin with pyrophosphoric acid have not yet been made.
542 ALBERT C. CRAWFORD

3.5 grams of the salt. On October 19 this animal weighed 1285

grams ; October 21, 1210 grams; October 27, 1 150 grams.
On October 14, 1909, a rabbit weighing 929 grams was fed with
0.7 gram of sodium pyrophosphate in 25 cc. of water. On the
15th the animal weighed 862 grams and was similarly fed. The
animal died at night and on post-mortem examination there was
found an enteritis with some serum in the peritoneal cavity.
On July 12, 1909, a rabbit weighing 842 grams was fed with 1
gram of this salt in about 40 cc. of water. On July 13 the weight
of the animal was 879 grams ; July 20, 870 grams.
On June 30, 1 gram of the same was fed in a similar manner to
a rabbit weighing 975 grams. On the following day it weighed
922 grams; on July 2, 848 grams. The animal died on this day.
The gastric walls showed petechiae and the mucosa was softened.
The lungs appeared normal.
One gram of sodium pyrophosphate was fed in solution, June
28, to a rabbit weighing 1004 grams. On June 29 the weight of
the animal was 992 grams. The same amount of the salt was
again fed. On June 30, it weighed 975 grams and the dose was
repeated. On July 1, the animal weighed 922 grams; on July 2,
when the animal died the weight was 848 grams. The stomach
was found much contracted and showed petechiae in its walls,
while its mucous membrane was softened. The liver appeared
dark and its surface seemed irregular. The kidneys seemed
darker than normal. The lungs seemed normal.
On June 28, 2 grams of the same salt were similarly fed to a
rabbit weighing 968 grams. On June 29 its weight was 835 grams.
On June 30 the animal died. It then weighed 768 grams. The
autopsy showed a large ulcerated bleeding area in the cardiac
region of the stomach. The gastric mucosa appeared cauterized
and the upper portion of the duodenal mucosa seemed some-
what reddened. The lungs appeared normal. The left ventricle
of the heart was contracted. The liver appeared passively con-
gested. The kidneys were pale.
It will be interesting t.o see if the toxicity of this salt is in-
creased by administering it in colloidal suspensions which delay
its absorption in the gastro-intestinal tract.
 “V ,.- ‘ ,- . _‘4’ - _

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 543

Sodium Orthophosp/zate (NaHPO4 + 1f H20)

On September 4, four grams of di-sodium hydrogen phosphate

dissolved in 50 cc. of water, were fed to a rabbit weighing 1649
grams. On September 7, this animal weighed 1744 grams and
the feeding was repeated. On Septembet 8, the animal weighed
1689 grams, and again received four grams more of this salt in
a similar solution. On September 10 it weighed 1744 grams.
On September 7, 1909, a rabbit weighing 1915 grams was fed
with a similar solution of four grams of this salt. On September
8, the animal was similarly fed and on this date it weighed 1918
grams. The following day the weight had increased to 1930
grams and on September 10, it weighed 1942 grams. Of course if
larger doses were given there would follow the so-called “salt
action,” but this is not a true poisonous one and is commort to
concentrated solutions of all salts, even to our harmless sodium
chloride.

CHEMICAL DIFFERENCES IN VARIOUS COTTON-SEED MEALS

On examining other samples of cotton-seed meal, the results

observed were rather different from those obtained from the
original lot. A sample from a second bag of Upland meal was
digested and the solution precipitated with lead acetate; unlike
,the first meal, the lead filtrate, after freeing from lead with H2S,
killed rabbits, while the precipitate was inactive, unless small
rabbits were used. This lead precipitate gave a decided bluish-
white precipitate with copper acetate. Magnesia mixture also
produced a heavy precipitate. Silver nitrate gave a heavy precipi-.
tate which had at first an almost pure yellow color, suggesting
the presence of ortho- and some pyro- phosphoric acid, but
mainly the ortho-acid. The lead filtrate gave no precipitate
with copper acetate, while AgNO, gave a yellow-white precipitate
which quickly turned brown. Magnesia mixture gave no immedi-
ate precipitate. Uranyl acetate and ammonium molybdate.
produced merely a slight cloud. -

Two hundred grams of this meal were digested and the digestion:
544 ALBERT C. CRAWFORD

products were precipitated with magnesia mixture. This precipi-

tate was dissolved in water with the aid of a little HNO3 and free
acid was eliminated by making the fluid alkaline with NaOH.
After acidulating with acetic acid, lead acetate now gave a pro-
cipitate which, on decomposing with H2S, induced death in a
rabbit a few hours after feeding it. This animal showed the
characteristic hemorrhagic gastritis.
Two hundred grams of the same meal were similarly treated and
the lead precipitate, after decomposing, was fed to a smaller
rabbit, in two equal portions on successive days. At the time of
the first feeding, September 17, the animal weighed 930 grams;
On September 18, 835 grams. Death occurred on September 19.
The walls of the stomach and the upper portion of the small intes-
tines appeared cauterized, and a large hemorrhagic area was noted
in the gastric walls.
The precipitate from magnesia mixture was washed with water
to remove soluble chlorides, and then treated with a trace of
dilute HNO3. The ifitrate gave a white precipitate with AgNO3.
Some of this filtrate was precipitated with copper acetate so as to
leave behind any possible trace of chlorides. This copper precipi-
tate on decomposing with H2S now gave a slightly yellowish-
white precipitate with AgNO8. A portion of the magnesia-mixture
precipitate was treated for about 60 hours with concentrated HNO.
and dissolved in water. Silver nitrate then gave a canary-yellow
precipitate.
Evidently in this meal pyrophosphoric acid exists in a form not
precipitable by lead acetate, unless freed by some other reagent. -

This meal, according to the analysis of the Bureau of Chemistry,

contained phosphorus estimated as 2.57 per cent P2O5.
One hundred grams of the Upland seeds known as “Keenan”
were ground and the digestion products precipitated with lead ace-
tate. Both the lead precipitate and filtrate, after freeingfrom lead,
induced death in rabbits. These seeds contained phosphorus
corresponding to 1.58 per cent P2O5. After similar treatment
of 150 grams of the “Pride of Georgia” the lead precipitate
induced death in two days, while feeding the filtrate merely caused
some loss in weight.
 -T-% -; -‘-j , -:--, - -- ‘.- -: -‘r - - 1

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 545

Two hundred grams of the Centerville Sea Island seeds, after

similar digestion, were precipitated with lead acetate. The lead
precipitate, on freeing from lead, was fed to a rabbit weighing
1262 grams. This animal died in a few hours. The stomach
and duodenum appeared cauterized, while the stomach was much
eroded. The lungs seemed normal. This solution gave a bluish-
white precipitate with copper acetate. Magnesia mixture pro-
duced a heavy precipitate. Silver nitrate gave a precipitate
which appeared decidedly yellow, suggesting a mixture of the
ortho- and pyrophosphoric acid. The filtrate, after freeing from
lead with H2S, was fed to a rabbit weighing 1222 grams. This
animal died in about one hour and twenty minutes after the feed-
ing. The stomach was hemorrhagic. The lead filtrate gave no
precipitate with magnesia mixture, while copper acetate yielded
a moderate precipitate which gradually reduced. Silver nitrate
gave a heavy dirty brown precipitate which also reduced. It is
probable that this filtrate owed its toxicity to some principle other
than pyro-phosphoric acid. These seeds contained phosphorus
estimated as 1.45 per cent P2O5.
it is interesting to note that digestion products of the Sea
Island seeds, when not treated with lead, failed to kill. In some
cases by precipitation with lead, the phosphoric and other
acids would be freed, and thus the extract become more toxic
than one would expect from the simple digestion products
of the same amount of the meal. A certain amount of free
orthophosphoric acid will also kill. This freeing of phosphork
acid must be kept in mind in all pharmacological experiments wish
cotton-seed meal when heavy metats are used, and one cannot rely
entirely upon the biological test as a quantitative method in studying
the action of such cotton-seed meal extracts, but must depend upon a
combined chemical #{231}ind
biological test. Perhaps the fact, that the
pyrophosphoric acid in certain meals is precipitable by lead
acetate, may aid in determining what variety of seeds were used
in making the particular meal.
 . . S.- 5 : -.

546 ALBERT C. CRAWFORD

ESTIMATION OF PYROPHOSPHORIC ACID IN COTTON-SEED MEAL

EXThACTS

There are as yet no satisfactory methods for the direct quanti-

tative determination of meta-, and pyro-phosphoric acids. When
salts of either are present these are converted into orthophosphates
and then estimated as such. Weber considers the use of H2SO4
as preferable to all other acids for this conversion Berthelot .

and Andr#{233} precipitated a weighed amount of pyrophosphoric

acid with magnesia mixture and treated for some time the pro-
cipitate with warm concentrated HNO8. They found that by
a second precipitation with magnesia mixture practically all of
the original pyrophosphoric acid could be accounted for.
The direct quantitative estimation of pyrophosphoric acid in
cotton-seed meal seemed associated with especial difficulties, so
that it was decided to determine the amount of this acid in the
purified extract, such as the one used for making the qualitative
tests for pyrophosphates, and thus see if the amount of this acid
present, expressed in terms of its sodium salts, would explain
quantitatively death in rabbits.
Two hundred grams of the original meal were digested and the
digestion products treated in a similar way to the extract used in
making the pyrophosphoric acid tests, that is, by precipitation
with lead, alcohol and copper. This solution was precipitated
with magnesia mixture and the precipitate treated for about 48
hours with concentrated HNO3. Finally after driving off the
acid the residue was dissolved in water and reprecipitated with
magnesia mixture. One-tenth of this solution yielded 0.1 gram
of anhydrous magnesium pyrophosphate, or 1 gram in the total
extract. This corresponds to 2 grams Na4P,O7 + 10 H2O. It
has been shown that this amount of extrac.t, and also this quan-
tity of sodium pyrohosphate, will in rabbits produce a hemor-
rhagic gastritis followed by death.
Twenty grams of meal taken from the second lot, that is, the
one which gavean active filtrate with lead acetate, were digested

Berthelot
3#{176} and Andr#{233},G.: Compt. Rend., vol. 123, p. 773, 1896.
 ,. - -- - . .

- S

A POISONOUS PRINCIPLE IN COTTON-SEED MEALS 547

and precipitated with magnesia mixture. This precipitate was

treated with concentrated HNOS and reprecipitated with mag-
nesia mixture. This yielded 0.244 gram of anhydrous magnesium
pyrophosphate, which would correspond to 4.8 grams Na4P2O, +
10 1120 in 200 grams of the meal.
This determination of the amount of pyrophosphoric acid in the
precipitate with magnesia mixture from the crude digestion pro-
ducts of the meal does not represent the full phosphoric acid con
tent of the meal, as the filtrate and perhaps the unextracted resi-
due may contain phosphoric acid, but the determination was
made merely to explain chemically why such extracts would kill
animals.

CONCLUSIONS FROM THE LABORATORY WORK

1. The chief poisonous principle in certain cotton-seed meals

is a salt of pyrophosphoric acid. In some, this salt seems to be a
simple one, presumably inorganic, while in others it is more com-
plex, perhaps an organic one. Probably this difference in the com-
binations of pyrophosphoric acid may aid in explaining the varia-
tion in toxicity of different meals. In certain cotton-seed meals
one would expect to find salts of metaphosphoric acid entering
into this action.
2. To be harmful the pyrophosphates must be in such a form
that they can be absorbed, or the pyrophosphoric acid ionized
in the gastro-intestinal tract.
3.. The harmlessness of certain cotton seeds and meal is mainly
due to the fact that in them the phosphoric acid exists largely,
if not entirely, as a compound of ortho-, and not as one of the
other phosphoric acids. Small amounts of pyrophosphates can
apparently be borne without injury. The amount of the salt
which may be permitted in cotton-seed meal should be determined.
4. The laboratory experiments would indicate that it is not
wise to feed much oil to herbivora, so that the removal of the oil
before making the seeds into meal is logical.
5. Many of the varieties of Upland cotton seeds will yield to
digestion a product which is poisonous to animals, but certain
 S...

548 ALBERT C. CRAWFORD

Sea Island cotton seeds yield harmless extracts and are, there-
fore, practically harmless; but if, on making meal from them, the
temperature rises high a poisonous principle develops. However,
Sea Island seeds obtained from Florida proved poisonous, even
without heating, approaching in this respect certain varieties of
the Upland seeds.
6. It will be an important problem to determine the relation
of the character of the soil, etc., to the production of the poison-
ous body in cotton seeds. Also to ascertain if other than the
ortho- acid exists in soil suitable for cotton seeds and find if the
pyrophosphoric acid is derived from the soil or du to aphysio-
logical characteristic of these seeds. What part fertilizers play
in this process remains to be studied, and the question as tc
whether various organisms influence the transformation of these
acids remains to be investigated.
7. Perhaps by crossing Upland plants harmless seeds of this
variety can be obtained. The economic problem in the produc-
tion of cotton-seed meal is to find what variety of seeds, when
grown on a particular soil, and under definite climatic conditions,
produce seeds which yield no poisonous principle, and not to
plant any variety of seeds indiscriminately, irrespective of the
conditions.
8. At present it would seem to be the wisest course to test each
lot of meal by a combined biological and chemical method, and
if any excess of poisonous properties is present, to utilize such meal
for fertilizing purposes, or use for feed with the greatest caution,
and reserve for more indiscriminate feeding those which prove
harmless to experimental animals.
9. These investigations would logically lead to a study of the
action of various other pressed cakes used as feed, such as those
made from peanuts, soya beans, etc., about the poisonous proper-
ties of which there is still some doubt. The question as to whether
pyrophosphates will induce an hypersensibility and thus increase
the activity of pathogenic organisms should be investigated.
The phosphates in corn need examination. The study of the
relation of fagopyrism to buckwheat would be suggested.