I28
T H E J O U R N A L OF I N D L ' S T R I A L A N D ENGINEERI-VG C H E M I S T R Y
investigations on the chemistry of glycerophosphates, a n d resulting conclusions, yet I fully realize t h a t much remains t o be done in order t o define, without a doubt, t h e characteristics of t h e various isomeric glycerophosphoric esters, which naturally can be achieved only after t h e various compounds shall have been synthesized in such a way as t o exclude t h e possibility of other compounds being formed. 'C'ntil such time t h e figures given for t h e solubilities, for instance, must be regarded only as tentative. LABORATORIES O F THE MONSANTO CHEMICAL W O R K S ST. LOUIS, IvfISSOURI
TOXICITY OF VARIOUS W O O D PRESERVATIVES1 B y C. J. HUMPHREY AND RUTH M. FLEMING Received January 8, 1914
During t h e past three years t h e writers have been conducting toxicity tests a t t h e Forest Products Laboratory, MadisonJ Wisconsin, on various wood preservatives. These tests have been limited t o substances in actual use in t h e industry or t o those compounds submitted by cooperators as having a possible preservative value. The toxicity of a given substance is not necessarily a n ultimate criterion of its service value, b u t when correlated with other suitable properties, such a s cost, ease of injection, permanence, non-corrosive action o n metals a n d wood, fire resistance, a n d ease a n d safety in handling it assumes high importance in influencing our judgment as t o what may normally be expected from t h e substance. T h e writers have confined their attention t o toxicity only, a n d with t h e view of bringing t h e essential d a t a t o t h e attention of t h e industry t h e more important results of our investigations t o d a t e are here presented. While we are attacking t h e problem'from two angles, namely, by t h e quick method of mixing t h e preservative with agar culture media a n d inoculating with fungi, a n d b y t h e slower a n d probably more conclusjve method of testing small injected wood blocks, t h e present paper will consider only the former, a s t h e latter work is not yet far enough along t o report upon. Our method is essentially this: Agar culture media of t h e following formula is prepared: Extract of 1 Ib. lean beef in 1000 cc. distilled water. 25 grams Lofflund's malt extract. 20 grams agar-agar. (Carefully filtered but reaction not adjusted; slightly acid.)
Seventeen, cc. of this medium are measured out into jo cc. glass-stoppered bottles, using a specially graduated 17 cc. pipette, t h e bottles then being placed in clamp-frames a n d sterilized with steam a t 100' C. for 2 5 , 20 a n d 20 minutes, respectively, on three successive days. T h e handling of t h e preservatives involved slig4t modifications for individual cases, b u t in all instances the concentrations reported are based on the actual weight of preservative in 20 cc. agar-preservative mixture. The character of t h e substance determined the method of introducing it i n t o t h e agar medium. With inorganic salts soluble in 'water, from 3 t o I O per cent solutions (grams per roo cc. solution) were prepared a n d measured out into 50 cc. glass-
' Published by permission of the Secretary
of Agriculture
V O ~6. , NO. 2
stoppered bottles from either a I O or 2 5 cc. staridardized cc., respectively. burette graduated in I / ~ o or T O each bottle sufficient distilled water was added t o make 3 cc. All other preservatives were weighed out .into similar bottles on a n analytical balance, a n d distilled water likewise added t o make 3 cc. I n t h e case of certain thick viscous oils, namely, wood t a r , wood creosote, coal t a r creosote a n d its fifth fraction, which do not readily emulsify with water, j t o 331/8 per cent stock emulsions were prepared, using equal amounts of gum arabic and preservative a n d diluting with water t o t h e desired concentration; these emulsions were then used in place of t h e crude preservative. I n a few instances where t h e preservatives were very low in toxic properties, more t h a n t h e specified 3 cc. were necessary in order t o secure t h e higher concentrations, a n d in these cases i t became necessary t o t a k e into consideration t h e excess of preservative and t o reduce t h e amount of agar by just this a m o u n t . in order t h a t t h e combined volume might not exceed 2 0 cc. After t h e preservative had been introduced i n t o t h e bottles as indicated, t h e stoppers were sealed in with a rubber-glycerine burette cock grease a n d t h e bottles were then sterilized along with t h e agar containers. After sterilization both agar a n d preservative were heated on t h e water b a t h a n d t h e former was poured into t h e preservative bottle in a sterile culture box, thoroughly shaken, a n d then poured into sterile petri dishes 100 mm. in diameter a n d I O mm. deep. After cooling, t h e surface of t h e medium was inoculated a t t h e center with a weft of fungus mycelium 5 t o 6 mm. square cut from a petri dish culture 2 t o 3 weeks old. T h e test dishes t h u s prepared were then placed in a n incubator a n d held a t approximately 25' C. for from 4 t o 10 weeks, usually 4 t o 6. For each set of concentrations a check culture, using 1 7 cc. agar media plus 3 cc. of distilled water, was prepared. T h e method as above outlined is not, b y a n y means, intended t o furnish t h e last word o n t h e toxicity of a preservative, b u t it has t h e advantage of being quickly a n d easily applied a n d gives us a t least valuable indications. T h e principal objection is t h e possibility in some instances t h a t t h e preservative may combine with certain constituents of t h e culture media, thus rendering part of it inert. This is known t o be t h e case with such compounds as zinc chloride, copper sulfate a n d mercuric chloride. With t h e oils this objection prdbably does not enter so seriously, b u t even here i t would be difficult t o say what might occur in such heterogeneous compounds a s t h e various t a r distillates a n d similar substances of highly complex constitution. Another variable which only a large number of tests can correlate is t h e difference in susceptibility among different fungi. Since molds are, as a rule, much more resistant t o toxic agents t h a n t r u e wooddestroyers, t h e writers have avoided their use, as well as t h a t of bacteria a n d yeasts. E v e n among t h e t r u e wood-destroying fungi considerable variation exists,
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
Feb., I 9 1 4
c
129
T H E J O C R S d L OF I + I T D C S T R I dL d,VD E S G I S E E R I S G C H E J f I S T R Y as will be seen i n t h e tables following. b u t this is usually within narrower limits. Both t h e fungi used b y t h e m-iters grow well on t h e media employed, Fomes a i t i i o s z t s F r . covering t h e surface of t h e petri dish in a b o u t 2 0 d a y s a n d Fomes piizicola (Sw.) F r . in a b o u t I j. Table I shows a list of t h e preservatires tested, together with salient properties, a n d t h e concentration necessary t o prevent growth of t h e organisms. I n this table t h e preservatives have been grouped according t o their nature, such as coal t a r , water gas t a r , a n d wood t a r distillation products, petroleum oils a n d water-soluble inorganic salts, in order t o give a n easy direct comparison. I n general t h e table shows t h a t t h e tm-o fungi react, i n most cases, quite differently t o t h e same substance, F o m e s piizicola, as a rule, being a far less resistant organism t h a n Fonzes annosus. With F o m e s anzosais t h e first three fractions of coal t a r creosote are considerTABLE11-PRESERVATIVES
YO^. 6 , S O .2
properties a n d t h e o . g g j oil proving a t least, or more, toxic t h a n coal t a r creosote. Petroleum oils, according t o t h e three samples tested, have low antiseptic value. Cresol-calcium in these tests shows a high toxicity a n d t h e poor results reported against i t in practice are apparently due t o a change in chemical constitution which did not t a k e place under our method of testing. Zinc chloride, in t h e commercial form, is slightly more toxic t o Fonzes a i t i z o ~ i i a~ n, d far less toxic t o F o m e s piiiicolu, t h a n coal t a r creosote. Sodium fluoride is over tTTice as toxic t o Fomes a m o s u s , a n d a b o u t one a n d one-half times as toxic t o Foines p i n i c o l a , as coal t a r creosote. While t h e work has n o t progressed f a r enough with zinc sulfate t o give conclusive results, t h e indications are t h a t it compares very favorably with both zinc chloride a n d coal t a r creosote.
-1RRASGED I S ORDER OF TOXICITY ASD
SHO'iVISO
RATIOTO COAL TARCREOSOTE Killing point
Fomes annosus
Fomes pinicola 7
Ratio t o coal t a r creosote No. 1074 Preservative Coal t a r creosote, Fraction 11, No. 1106.. . . . . . . . . . . . . . . Sodium fluoride, No. 1929 Cresol-calcium, S o . 2098. Coal t a r creosote, Fraction I, No. 1094.. . . . . . . . . . . . . . . . Coal t a r creosote, Fraction 111, S o . 1107. . . . . . . . . . . . . . W a t e r gas t a r creosote, sp. gr. 0.995, hTo. 2235.. ........ Zinc chloride, KO. 2239.. Zinc sulfate, No. 171 1 . . .. Coal t a r creosote, S o . 10 Wood creosote, F o . 1099. ............................ Wood tar, No. 1561.. . C. A. Wood preserver, No. 1 9 3 1 . . .................... Spirittine wood preserver, No. 1932.. . . . . . . . . . . . . . . . . . S. P. F. Carbolineum, No. 1844 ............ Holzhelfer, S o . 2097, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coal t a r creosote, Fraction I\', No. 1108. . . . . . . . . . . W a t e r gas t a r creosote, sp. gr. 1.042, hTo. 2233 ....... Avenarius Carbolineum, S o . 1843, . . . . . . . . . . . . . . . . Fuel oil, No. 1103 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kerosene, No. 1847.. . . . . . . . . . . . . . . Coal tar creosote, Fraction V. No. 1109. . . . . . . . . . . . . . . Copperized oil, No. 1 0 9 5 , . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. S. Special, No. 2696 ........... W a t e r gas t a r creosote, sp. gr. 1.058, KO.1101.. . . . . . . . . Sapwood antiseptic, No. 161 1 . . . . . . . . . . . . . . . . . . . . . . . .
P e r cent
Lbs. cu. ft.
0.225
0.140 0.156 0,087-0.174 0.187 0.203
0.25 0.14-0.28 0.30 0.325* Around 0 . 4 5 0.50 Around 0.50 0.55 0 65* 1.25 1-1.5 1-2 2.25 Above 2 . 5 3 3* 3-4 5.25 Above 6 Above 34 33* 40 Above 45 Above 40 Above 7 5
ably more toxic, a n d t h e last t w o fractions much less toxic, t h a n t h e oil itself; with Fomes piizicola only t h e last fraction is less toxic. This indicates t h e advantage, from a toxicity standpoint, of removing a t least a p a r t of t h e heavier t a r oils. T h e carbolineums a n d similar products which consist, i n t h e main, of t h e higher boiling constituents of creosote in all cases proved much less toxic t h a n coal t a r creosote. Wood creosote from Douglas fir appears t o compare very favorably with coal t a r creosote, b u t t h e softwood creosote, under t h e t r a d e n a m e of Spirittine IYood Preserver, which has a much lower specific gravity, appears t o be much less effective. I n t h e case of water gas t a r creosotes t h e toxicity increased rapidly with decreasing specific gravity, t h e heavier 1.0j 8 oil possessing very low antiseptic
Lbs. cu. f t . Per c e n t 0.094 0.15 0.094 0.15
2.5 2.2 3.9-2 . O 1.8 1.7
1.5 1.5
0.140
...
.....
1. o 1.8
0.312
1.1
0.3
0.468
0.75
0.140 0.125 0.468
0.225 0.20 0.75
...
0.343 0.405 0.78 0.6-0.9 0.6-1.2 1,404
..
0,078
..
.....
, .
.....
..
..
..
0.84 0.44 0.55-0.37
1.12 0.3
0.55-0.2i
..
.. ..
..
..
0.24
..
..
..
.......
.....
2.059 1.9-2.5 3.27
0.16 0.18-0.14 0.104
...
..... .....
..
..
..
0.017 0.015 0.0140.0160 , 007-
0.029 0.0056 0.0045
4.867 25+ 31.2-k
0.0056
25C
...
20.59 24.96 28f
2546.8-
0.078
1,8
..
..
0.ij
0.187
..
..
..
0.225 0.125
...
...
*
... ...
...
...
0.125" , . .
0.30
...
...
7 . so*
Above 40 Above 50 Above 40
...
S o n e Such Special, which is claimed b y t h e manufacturers t o mater-proof a n d give a hard finish t o timber, as well as t o prevent or stop decay, proved very low in toxic properties, a n d even stimulated growth i n high concentrations ; h o w eve r t h e physic a! prop er t i es , wheninjected intowood, m a y b e s u c h a s t o exclude fungus growth a n d t h u s substantiate t h e claims made for it. I n Table I1 t h e preservatives are arranged in t h e order of their toxicity t o F o m e s u n n o s i i s , t h e concentrations are reduced t o pounds per cubic foot a n d t h e ratios of their efficiency as compared with coal t a r creosote are presented. H a d t h e preservatives been listed i n their order of toxicity t o F o m e s p i n i c o l a t h e arrangement would have been somewhat different a n d -1venarius Carbolineum a n d creosote Fraction IT would have appeared higher in t h e scale, a n d creosote Fraction I a n d zinc chloride considerably lower. ~
F e b . , 1914
T H E J O C R S A L 0 F I S D L-S T RI-4 L A S D E S G I S E E RI S G C €1E M I S T K F
It is seen t h a t t e n of t h e twenty-five preservatives tested fall belov I per cent, or more accurately 0 . 6 j per cent, for their killing point for Pomes aizmsrrs; P o m e s piiricola was used with only fourteen of t h e m b u t of these ten fell below I per cent! or more accurately, o.;j per cent. I t is interest.ing t o note t h a t sodium fluoride a n d Fraction I 1 of coal t a r creosote head t h e list with nearly equal ?oxicities. Just what constituents of t h e naphthalene fraction are t h e effective ones is not known a t present, b u t t h e work of other investigators indicates t h a t pure naphthalene has low toxic properties. T h e very favorable results froin sodium fluoride would place this in t h e first r a n k of mater-soluble preservatives. This. together n-ith several other fluorine compounds, has come into considerable use abroad, particularly in Austria, a n d i t is t h e opinion of t h e writers t h a t we have i n these substances very efficient wood preservatives which c a n be a d a p t e d t o use in m a n y situations. T h e i n d u s t r y particularly needs some substance which c a n safely be recommended for building timbers. I n comparing t h e two carbolineums with coal t a r creosote i t is seen t h a t approximately 3 0 per cent of t h e former distils below 3 2 0 ' C., while 74 per cent of t h e creosote comes over. This indicates t h e greater toxicity of t h e lower boiling constituents. Xs t o what causes t h e greater toxicity of S. P. F. Carbolineum over -4venarius Carbolineum we are n o t prepared t o s a y , b u t t h e fact t h a t t h e former is higher in t a r acids is very suggestive. T h e poor sholring made by copperized oil against both fungi in di c a t e s t h a t ad ding co p p er i n t his form t o 1o w -t o xi c petroleum or vegetable oils is of very doubtful value. I n conclusion, t h e writers are prone t o t a k e a T-ery conservative position when i t comes t o analysis of t h e d a t a presented a n d generalizations d r a v n therefrom. TVe wish t o strongly emphasize again t h e fact t h a t toxicity alone does not necessarily gi\-e a direct comparison of t h e service value of preservatives, a n d t h a t all t h e s t a t e m e n t s made comparing different. substances refer t o t h e toxic properties only. However, we do feel t h a t t h e results here set forth n-ill prove of considerable ].due in outlining further tests n-hich r i l l serve t o bring out actual sen-ice values. \Ye wish t o record our acknon-ledgments t o Dr. H a r m Iletcalf. Pathologist in Charge, Laboratory of Forest Pathology, Bureau of Plant I n d u s t r y , TTashington. D. C . ; t o l l r . Howard F. Tl'eiss, Director of the Forest Products Laboratory, C . S. Forest Service, N a d i s o n , TTisconsin. for facilities a n d material placed at our disposal; also t o l l r . Ernest Bateman, Chemist in Forest Products, Forest Products Laboratory, mho has supplied all t h e d a t a on t h e physical a n d chemic a1 prop e r t i e s of t h e preservatives e x a mi ne d . LABORATORY OF FOREST B L R E A UOF
PAIIIOLOGY
PI.AS1 ISDUSTRY
h I A D I S O X , xvISCOSSIS
A RAPID METHOD FOR DETERMINING THE PERCENTAGE O F CASEIN IN MILK B y '&'. 0 ,
\t''aLKER
Received February 14, 1913
During t h e past fern years dairymen have been
131
giving a good deal of attention t o t h e advisability o f taking into account t h e percentage of castyin n-hen paying patrons for milk t o be used in cheese-making. Some a d r o c a t e paying on a f a t basis only, others on t h e f a t a n d casein basis. It is not t h e object of this article t o discuss t h e merits of either of these methods, b u t t o outline a process which has been tried out i n t h e author's laboratory ' f o r quickly a n d simply a r riving at t h e a m o u n t of casein in milk. I t is well k n o n n t h a t several methods have been suggested of late, t h e most promising probably being t h e centrifugal method of H a r t . This has, apparently, yielded satisfactory results in m a n y cases, b u t has been found t o give varying results with preserved samples, a n d with fresh milk, under different conditions of temperature, etc. Research into t h e constitution of t h e protein molecule has revealed t h e probability of i t s consisting of a complex linkage of a large n u m b e r of amino acids. T o illustrate this linking u p of amino acids we may use t h e following equation, which shows how one molecule of a simple amino acid, such as amino-propionic acid, m a y become bound t o a molecule of another amino acid such as amino-acetic acid: H H H 0 , I \C--d-H / I H-C--C--C //O H-0 , '0-H H-- hT- H I HH-KN-H
+
H
H
H
0 =
H-C--C-C,
'C-C-H
+HOH
Both t h e molecules of amino-propionic acid a n d amino-acetic acid, as indicated above, possess a n alkaline (amino) group,
H-1-H, as well as a n acidic
0 , a n d when t h e y combine we m a y group. -C/ 'OH have t h e union taking place in t h e manner indicated, in which t h e alkaline (amino) group of one molecule interacts with t h e acidic group of t h e other molecule, t h u s leaving a n alkaline group a n d a n acidic group still existing in t h e product, which m a y be looked upon, for our purpose, as a r e r y simple representative of the proteins. It has further been observed t h a t when formaldehyde is added t o proteins, the neutral character of t h e molecule disappears, with t h e result t h a t t h e acidic property predominates strongly. I t is thought t h a t t h e formaldehyde reacts with t h e alkaline groups forming methylene derivatives, t h u s l e a r i n g t h e acidic groups free t o act. These acidic groups m a y now be neutralized with s t a n d a r d alkali, a n d if t h e value of t h e alkali in t e r m s of protein be known, t h e percentage of protein present m a y be estimated.
