October 15, 19.110
I N D U S T R I A L dA7D EiVGISEERISG CHEMISTRY
mg. up to 5.5 nig. Cf the thirty comparisons twenty-four differ by less than 0.1 p. p. m. and only one differs by as much as 0.22 p. p. m. T a b l e V-Results of D u p l i c a t e D e t e r m i n a t i o n s of Boron i n Leaf S a m p l e s b y Modified C h a p i n M e t h o d i n R o u t i n e L a b o r a t o r y Work a t t h e . Limoneira L a b o r a t o r y (Results on basis dry weight of material) EXPT. SUBSAMPLE A SUBSAMPLE B DIFFERENCE P. p . m. P. P . m. P. P . m . 164 34 3fi 2 .~ ._ 162 90 69 1 1730 117 107 10 1720 142 149 0 169 186 180 6 1700 210 307 3 107 285 280 171a 352 300 52 140 452 465 13 138 509 a?? 13 14' 720 732 12 166 848 860 12 a Analyses made by Francis Scofield, scientific aide. All other analyses by author.
A similar series of comparisons has been made with leaf samples, the results of which are shown in Table V. This series represents approximately the range of boron content found in leaf material of citrus and walnuts in California.
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Of t1:e twelve comparisons six differ by less than 10 p. p. ni. and only one by as much as 52 p. p. m. These examples will indicate the range of difference found by this method as due to analytical error. Acknowledgment These modifications were worked out by the author while employed in the Chemical Laboratory of the Limoneira Company a t Santa Paula, Calif., under the direction of Charles A. Jensen. The author is indebted to the Limoneirs Company and to Mr. Jensen for the use of these hitherto unpublished contributions to the methods of boron determination. Literature Cited (1) Allen and Zies, J . Am. Ceram. Sac., 1, 739 (1918). ( 2 ) Chapin, J . A m . Chem. Soc., 30, 1691 (1908). (3) Dodd, Analysl, 52, 459 (1927). (4) Gooch, A m . Chem. J . , 9, 23 (1887). ( 5 ) Rosenbladt, Z . anal. Chem., 26, 21 (1887). ( 6 ) Ross and Deemer, A m . FeriiZieer, 52, 62 (1920). ( 7 ) Wherry, J . A m . Chem. Soc., 30, 1687 (1908).
A Suggested Toximetric Method for Wood Preservatives' Henry Schmitz and Others* DEPARTMEST OF AGRICELTERE, USIVERSITYOF ~ I I N N E S O TUSWERSITY A, FARM, ST. PAUL, MmX.
During t h e past fifteen or twenty years t h e toxicity (2) Test fungus or fungi to OXICITY studies deal be used. of numerous wood preservatives has been determined. only with the poisonous (3) Criteria to be used ,to Since almost every investigator has used a somewhat properties of a wood determine toxicity-i. e., indifferent method of making these determinations, preservative, and therefore do hibition of growth, killing concentration, effect on rate of m u c h confusion has resulted i n the interpretation of not give a complete picture growth, loss of weight, etc. t h e results obtained. It has been apparent for some of the value of any substance (4) Interpretation and sigthat as t h e number of workers increases the time as a wood preservative. I n nificance of toximetric values. situation will become worse instead of better. The addition to being toxic, a Cultural Methods need at this time for the formulation and general wood preservative must (a) adoption of a satisfactory toximetric method for wood Broadly speaking, the toxnot attack the wood, ( b ) not preservatives is urgent. icity of wood preservatives react with the wood or its conIn order to determine if a n agreement might be may be determined by either tents so as to become nonreached o n a method of testing of the toxic properties of two general methods. The toxic or less toxic, (c) not atof wood preservatives and on t h e interpretation a n d first involves determining the tack metal, ( d ) be easy to insignificance of t h e results obtained from such a test, resistance to decay of wood ject into wood, and (e) possess a group of workers m e t a t the Missouri Botanical Garimpregnated with the presome degree of permanence. den on December 12 and 13, 1929, to consider t h e quess e r v a t i v e material. The S o one requirement is more tion. A brief statement of t h e conclusions reached by wood may be either en masse important than any o t h e r , that group follows. or in the form of a fine sawsince a marked deficiency in dust or wood flour. I n so any may render an othervise good preservative of little value. Nevertheless a wood pre- far as toxicity tests are concerned, wood en masse and servative to be effective must be able to kill or inhibit the wood sawdust, even of the same kind of wood, are two growth of the organism against which protection is required. very different things. A limited amount of work only I n analyzing the problem of suitable methods of testing has been done in this country using treated wood en the toxicity of a wood preseryative, it is evident that at masse a s a culture medium to test the toxicity of wood preservatives. Impregnated sawdust has, however, been used least four distinct questions are involved, namely: by several investigators, but it appears that all the merits (1) Cultural methods-i. e., the kind of medium used, method and limitations of the method are not yet definitely known. of preparation, age, and size of inoculum, etc. Further work may show that the method has advantages Received June 7 , 1930. not yet recognized. * This statement m-as prepared jointly by the following individuals: The second method involves adding the preservative to Ernest E. Bateman, R . H . Collej-, S. R . Church, Carl Hartley, Robert E. nutrient agar, emulsifying the mixture when necessary by Waterman, Alfred I,. Kammerer, Hermann von Schrenk, E. B. Fulks, shaking, and determining the effect of the resulting niixture Ernest E. Hubert, C. S. Reeve, Kalter H. Snell, C. Audrey Richards, David H. Under, J . D. Burnes, and Henry Schmitz. The last named on tlie growth of the test fungus. Both of these methods member of the group acted as Secretary and endeavored to correlate as have certain advantages and disadvantages and neither gives far a s possible the divergent opinions and ideas of the various members of all the information desired. t h e group. Where coordination was impossible, alternative procedures are As a general rule German investigators have been inclined proposed.
T
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A N A L Y T I C A L EDI TI0.V
to use wood as the basic culture medium, while most American investigators have used nutrient agar. The desirability of using wood blocks impregnated with the Preservative is rather universally accepted, but until more confidence can be placed in the even distribution of the preservative in the test block their use will be greatly limited. Although the use of nutrient agar preservative mixtures to determine the toxic properties of the preservative may have limitations, it more nearly meets the requirements of a simple, rapid, controllable test than any of the others now available. Test Fungus Different species of wood-destroying fungi vary greatly in their resistance to toxic agents. Even within a given species of wood-destroying fungus, different physiological strains or races exist which also show considerable variation in their resistance to the same toxic agent. It is imperative, therefore, if the results of toxicity tests obtained by different investigators are to be comparable, that cultures of the same test fungus be used. A strain of Fomes annosus isolated from mine timber and distributed by the Office of Forest Pathology, Forest Products Laboratory, Madison, Wis,, has been used in making most of the toxicity tests in this country. It is, therefore, recommended that when only one test fungus is used it be the strain of Fomes annosus recommended by that office. Under certain conditions it will often be desirable to use test fungi other than Fomes annosus. I n such cases the following fungi3 are recommended: Lenzites trabea, Lentinus lepideus, Lenzites sepiaria, Poria incrassata, Coniophora cerebella, Polyporus vaporarius, Polystictus versicolor, Polystictus hirsutus, and Trametes serialis. Criteria to Be Used t o Determine Toxicity The influence of a toxic material on the test fungus may be evaluated by ( a ) its influence a t a given concentration on the rate of growth of the fungus, (b) the concentration of preservative in the nutrient medium necessary completely to inhibit growth of the fungus, or (c) the concentration necessary to kill the inoculum in a given length of time. It is recommended that both the concentration necessary totally to inhibit the growth of the test fungus and that necessary to kill the inoculum of the test fungus in 14 days be determined in the evaluation of the toxicity of wood preservatives. Note-“Total inhibition” shall mean no signs of growth either on the nutrient agar or on the inoculum. When a small amount of growth, confined to the inoculum, occurs i t shall be known as “trace.” T h e condition of the inoculum a t the end of the incubation period is determined by transferring i t from the test plates to nutrient agar slants. If no growth occurs on the slants after 14 days, the inoculum shall be con sidered “killed.”
Interpretation and Significance of Toximetric Values of Wood Preservatives The interpretation and significance of toximetric values have been the source of considerable misunderstanding and error. The toximetric value of wood preservatives, as determined by laboratory methods, shows the amount of wood preservative necessary to stop the growth and kill the test fungus under more or less controlled conditions. In commercial practice, however, it is of interest to know the amount of preservative that must be injected into the wood in order t o maintain a certain minimum amount of preservative for a given length of time. Laboratory studies of the toxicity of wood preservative do not give this information. Attempts to calculate the amount of preservatives which must be 8 I n order that authentic cultures of test fungi may he generally available to all investigators, some central agency must keep such cultures in stock. The Office of Forest Pathology, Forest Products Laboratory, Madison, Wis., is the logical agency to render this important service.
Vol. 2, No. 4
injected into the wood from laboratory studies of toxicity are therefore based upon an erroneous conception of the value and purposes of such studies. I n the interpretation of toximetric values of wood preservatives, the following facts must receive due consideration: (1) The higher the percentage concentration required completely to inhibit the growth and kill the test fungus the lower the toxicity of the preservative. Note-In complex mixtures there seems to be little or n o relation between the concentration causing complete inhibition of growth of the test fungus and the killing concentration.
(2) Toximetric values are not in themselves an index of the wood-preserving value of the substance tested. (3) Other factors, such as leaching, volatility, chemical stability, penetrability, cost, cleanliness, etc., must all be considered in the final evaluation of a wood preservative.
Adopted Toximetric Method for Wood Preservatives I n consideration of the facts mentioned in the previous discussion, it was agreed to follow in a general way the method developed and now used by the Office of Forest Pathology, Forest Products Laboratory, Madison, Wis. As new facts are discovered it may be desirable to modify the adopted method. This method involves the use of a preservative-nutrient agar mixture or emulsion in standard Petri dishes. A’ote-When testing the toxicity of very volatile substances, it may be found desirable, if not necessary, to substitute glass-stoppered Erlenmeyer Basks for Petri dishes as suggested by Bateman and Henningsen. The essential features of this method are described io the Proceedings of the American Wood Preservers‘ Association for 1923, pages 136 to 145,
The details of the method follow: (1) I n the preparation of the nutrient agar-preservative emulsions or mixtures, all weighings and measurements shall be made with reasonable accuracy. Note-The use of small glass ampoules is suggested in the preparation of nutrient agar-creosote emulsions. A known quantity of creosote is placed in the ampoule, the neck of which is then sealed. Further manipulation is described in paragraph (3). The preservative may also be weighed directly into sterile ground-glass-stoppered bottles and the desired amount of hot sterile agar added, When the latter procedure is followed the details of the method as described in paragraphs (2) a n d (3) must be slightly modified.
I t is suggested that variations in concentration of preservative shall be in accordance with the following system: Per cent
Per cent
Per cent
Per cent
0.9 0.8 0.7 0 6 0.5
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01
0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001
0.4 0.3 0.2 0.1
(2) The nutrient agar shall consist of Difco bacto-agar., . , , . . . . . . . . . , , , , , , , , . . . , , , . . Trommers plain diastasic extract of m a l t , . , . , , , , . Distilled water. . . . . . . . . . . . . . . , , , , , , . . . . . . . . . .
15 grams 25 grams 1000 cc.
steamed a t atmospheric pressure until the agar is dissolved. Immediately after steaming (in no case should re-steamed nutrient agar be used), the required amount of nutrient agar shall be weighed or measured out in a suitable container such as a glass-stoppered flask. The stopper is attached to the flask by means of a string or rubber band and a cotton plug inserted into the neck of the flask. The flask and contents shall be sterilized by steaming a t 10 pounds (0.7 kg. per sq. in.) pressure for 20 minutes. (3) After the flask has been taken from the sterilizer, the cotton plug is removed and the preservative added either directly or in a sealed glass ampoule. (The exterior of the ampoule may be sterilized by immersing in absolute alcohol for a few minutes and then flaming to remove the alcohol.) When ampoules are used they are broken, by means of sterile
INDUSTRIAL AND ENMNEERING CHEiMlSTRY
October 15, 1930
glass tongs, under the surface of the agar medium. The glass stopper is immediately inserted in the neck of the flask and the flask intermittently shaken until cool. The contents are then poured into 90 by 15 mm. Petri dishes. (4) Approximately 25 grams of nutrient agar-preservative mixture shall be placed in each Petri dish. Immediately after cooling the culture plates shall be inoculated with the test fungus. The inoculum should be approximately 1 em. square and should be taken from 14-day-old plates of fungus. The inoculum is placed near the center of the dish and the mycelium side is turned upwards. ( 5 ) The test plates shall be incubated for 14 days at 28' C. The amount of radial growth shall be measured daily for 6 days and every other day from 7 to 14 days. If photographs are taken of the test plates, this should be done on the thirteenth or fourteenth day. When the toxicity
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of non-volatile substances is determined, the plates may be placed directly in the incubator. When volatile substances are tested, some method, using either glass-stoppered flasks or bell jars covering the dishes, must be used to reduce the loss of the substance through volatilization. (6) If no growth occurs on the plates or inoculum after 14 days, the inoculum shall be transferred to standard maltagar slants to determine if it is dead or alive. I n making this transfer, the fungus growth on the inoculum shall be placed in direct contact with the nutrient agar slant. If no growth occurs in 14 days, the inoculum shall be considered to have been killed. (7) The results of the tests by the above toximetric method shall be stated for both total inhibition and killing point in terms of the percentage of preservative used in the medium.
Quantitative Analysis b y Monochromatic Transmission' Monroe Barnard and Paul McMichael AMERICAN PHOTOELECTRIC
CORPORATION,
18TH ST.
A
USEFUL derivation from Lambert's law has been developed which enables one to calculate the decimal proportions of the components in a mixture from precise measurements of light-transmission factors. Derivation of Formula
I n its simplest form, where there are only two components in the mixture, the law is expressed as follows: in which
T , = TInl. Tznl
T,,,= transmission factor of mixture at some particular
wave length TI = transmission factor of one ingredient of mixture at same wave length TZ = transmission factor of other ingredient of mixture a t same wave length n1 = decimal proportion of f i s t ingredient in mixture n2 = decimal proportion of second ingredient in mixture 721 n2 = 1
+
To facilitate computation, the formula may be expressed as follows:
Figure 1 shows the spectral-transmission curves of three liquids: A , yellow potassium chromate solution; B , blue ammoniacal solution of copper; and C, green liquid composed of equal parts by volume of solutions A and B. Application of Formula
Applying the foregoing formula to the transmission factors at any particular wave length, we secure the results given in Table I, in which the transmission factors of the yellow potassium chromate solution are designated T I , those of the blue ammoniacal copper solution Tz, and those of the 50-50 mixture as T,. Coasideration of the curves and of the proportions derived from application of the formula indicate that accuracy is best secured when the calculations are based upon determinations a t a wave length in a region where the variations in 1
Received July 23, 1930.
AND
THIRDAvE.,
N E W YORK,
N. Y.
the transmission factors are relatively small with large variations in wave length, and a t which there are considerable differences between the transmission factors of the components entering into the mixture. Table' I WAVELENGTH
mu 680 660 640 620 600 580 560 540 520 500 480
TRANSMISSION FAC 'ORs ~
TI 0.882 0.878 0.872 0.866 0.860
0.848 0.830 0.802 0.762 0.628 0.125
Ti
Tm
0.230 0.196 0.175 0.161 0.158 0.170 0.215 0.300 0.412 0.540 0.662
0.448 0.410 0.386 0.374 0.372 0.382 0.422 0.495 0.554 0.581 0.290
PROPORTIONS TI Ta
%
%
49.6 49.2 49.3 50.1 50.5 50.4 49.9 50.9 48.2 48.5 49.5
50.4 50.8 50.7 49.9 49.5 49.6 50.1 49.1 51.8 51.5 50.5
Where there are wide differences in the transmission factors of one of the components at wave lengths not widely separated -as, for example, those of the potassium chromate solution between 480 and 540 mp, or those of the ammoniacal copper solution between 480 and 560 mp-a very slight error in the adjustment of the spectrometer will introduce an error which the subsequent calculation will greatly magnify. Similarly, error may be introduced when the calculations are based upon determinations at a wave length where the transmission factors of the components are not appreciably separated. For example, in Table I, if the transmission factor found for the mixture at 500 mp had been 58.2 per cent instead of 58.1 per cent as recorded, then the proportions found by calculation would have been 49.6 and 50.4 per cent instead of 48.5 and 51.5 per cent as shown. I n other words, an error of 0.1 per cent in the measurement of the transmission factor a t this wave length resulted in a final error of 1.1 per cent, whereas a similar variation at 560 mp would have resulted in a change in the final result of only 0.2 per cent. Accuracy Obtainable
It is essential, of course, for the practical application of this method of quantitative analysis that all measurements of transmission factors be made with a high degree of accuracy, such, for example, as is obtainable with an A. P. C. photo-
