I'HE JOURNAI, OF I N D U S T R I A L A N D ENGINEERlNG C H E M I S T R Y
July, 1922
617
Studies in Wood Decay' 111-The
Toxicity of Western Yellow Pine Crude Oil to Lenxites Saepiaria Fries By Henry Schmitz LABORATORY OF FOREST PRODUCTS, SCHOOL OF FORESTRY, UNIVERSITY OF IDAHO, M o s c o w , IDAHO
The low toxicity and high rate of evaporation of the crude oil from the distillation of Western yellow pine make it of little value as a general wood preservative. I t may, however, be adapted to special uses, as in the manufacture of shingle stains, preservative paints, etc. The toxicity has been determined with sawdust from three species of wood as culture media: lowland white fir. Douglas fir, and sugar maple. The toxic point varies slightly with the species of wood used. I t is suggested that an ideal way to determine the toxicity of wood preservatives to wood-destroying fungi would be to set up such a culture series as was used in this work, and to determine the actual decomposition of the wood as indicated by the loss in weight of the flasks. The disadvantage would be that at least three months would be necessary, during which a volatile preservative would evaporate considerably. The petri dish method was also used in this investigation.
ROM time to time, this laboratory has received inquiries concerning the possibility of employing the oil obtained by the destructive distillation of Western yellow pine as a wood preservative. Since, also, it is shown in a recent publication cooperated in by this Laboratory2 that the yield of this oil is approximately 53.0 gal. per cord, and since definite information concerning the toxicity of such oils to wood-destroying fungi does not seem to be generally available, the present piece of work was undertaken. Any substance which is to be used as a wood preservative must have other important properties besides being toxic to the growth of wood-destroying fungi, but even though a substance may fulfil all these other requirements, its use will be limited if it is not toxic to wood-destroying fungi. PRODUCTION OF OIL The oil was produced by heating the wood directly in a small retort approximately 12 in. in diameter and 36 in. long. The vapors were led through a worm condenser, and the distillate was collected in a separatory funnel. After settling, the water was drawn off and the oil transferred to a storage container. The oil was used as collected, with no attempt a t redistillation. PHYSICAL PROPERTIES Crude Western yellow pine oil is a heavy sirupy liquid, acid in reaction, and of sharp pungent odor. It much resembles crude petroleum in general appearance. This sample of oil gave the following analytical results:
F
Sp. gr. a t 38O Per cent water
1.0141 0.70
When it was fractionally distilled in a ~OO-CC.,round-bottom, distillation flask with the side tube a t the center of the neck, the following results were obtained: Distillation Temperature 150; 200 250 300 Residue above 300'
;
Per cent 10.43 15.55 13.20 11.07 49.57
Received November 21, 1921. I. W. Cook, Henry Schmitz and Louis Grant, "The Availability of Western Wood Oils for Flotation Concentration," University of Idaho, Bull. 16, No. 13 (1921),1. 1
Whenever the rate of distillation fell below four drops per minute, the temperature was raised to the next highest distillation temperature. This is a t variance with the standard system advocated by the American Society for Testing Materials for the fractional distillation of coal-tar creosote. However, it seems to the writer that there is little in favor of employing the same system of fractional distillation for two substances SO entirely different as coal-tar creosote and crude mood oil. The above distillation temperatures are not suggested as a standard method of analysis of a wood oil, but they seem rational for the present purpose. It is assumed that the 150" C. fraction will include the wood alcohol, water, acetic acid, etc., that the 150" to 200" C. will contain the terpenes, and that the higher fractions and the residue will contain the rosins and tars.
TOXICITY OF WESTERN YELLOW PINEOIL ADDEDTO VARIOUS KINDSOF SAWDUST In a recent paper by Schmitz and Zeller,3 the large discrepancies between a method employing nutrient agar and one employing sawdust as a culture medium are shown. In the present work, the exact method outlined in the previous paper was employed. Ethyl alcohol, in which the oil was entirely soluble, was used as the solvent. After impregnation, the sawdust was exposed to the air for 24 hrs. in order to allow the alcohol to evaporate. Five-gram samples were then placed in 8-011. bottles. Before sterilization, the bottles were tightly plugged with steamed corks and the corks securely tied to prevent them from blowing out during the steaming process. Sterilization was effected by steaming for 1 hr. in an Arnold sterilizer. Sfter sterilization, the corks were replaced by sterile cotton plugs and the flasks inoculated with Lentites sacpiaria in the usual manner. The cotton plugs were then covered with two thicknesses of paraffined tissue paper to avoid excessive evaporation from the flasks, the paper being secured by means of a rubber band. Three series were prepared: one with lowland white fir (Abies grandis) sawdust, one with Douglas fir (Pseudotsuga tnzifolia) sawdust, and the last with sugar maple (Acer saccharum) sawdust. Crude yellow pine oil was added in amounts to make the following percentage concentrations, based upon the air-dry weight of the sawdust: 0.5, 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. The culture flasks were kept under observation for 21 days and examined a t frequent intervals in order to determine in which flasks and to what extent growth had taken place. DETERMINATION OF TOXIC CONCENTRATION-In order that there may be no confusion as to how the toxic concentration, which is defined as the minimum percentage of the oil which will completely inhibit the growth of the fungus, is determined, this phase of the work is discussed in some detail. In a culture series, it is found that the growth of the fungus in the control flasks and in flasks containing small amounts of preservative is relatively copious. With the aid of a hand lens, the mycelium can easily be seen on the inner surface of the flask. Placing the inoculum near the wall of the flask simplifies this observation. With an increase in the amount
* THISJOURNAL,
13 (1921),621.
618
THE JOURhTAL OF INDUSTRIAL A N D EhTGINEERING CHEMISTRY
of preservative, growth of the fungus hecomes less extensive, but can still be found permeating the sawdust. With a further increase in concentration of the preservative, the growth of the fungus is confined to the inoculum. This type of growth can usually be easily observed on the cut surfaces of the inoculum. Finally, a concentration of the preservative is reached where there is no growth, even €rom the inoculum. The lowest concentration where all gro%th is inhibited is considered to be the toxic concentration. If there is any doubt as to whether limited growth has taken place, the inoculum can be removed from the culture flask and the cut surfaces examined by means of a hand lens. With very little experience, it is quite easy to determine whether or not growth has taken place. It is undoubtedly true that actual decay may be inhibited at a lower concentration than that determined in the above manner. However, when growth is very limited, it is practically impossible to determine whether this growth is entirely from the inoculum or whether the mycelium is obtaining a part of its food material by actually decomposing the sawdust. The point a t which no growth whatsoever takes place is sharp and definite and the personal factor in its determination is reduced to a minimum. The toxic concentration (as above defined), based on the dry weight of sawdust, was found to be between 9 and 10 per cent when white fir sawdust was employed as the culture medium, between 10 and 11 per cent when Douglas fir SRWdust was used, and between 11 and 12 per cent when hard maple was used. The determinations were checked and rechecked, and the same results were obtained in each case. EVAPORATION OF oIL-The legitimate objection might be raised that by exposing the impregnated sawdust to the air for a period of 24 hrs. the more volatile fractions of the oil would evaporate. I n order to determine the rate of evaporation of the oil from the sawdust when exposed to the air, 5-g. samples of the sawdust were impregnated with crude oil and the various fractions of the oil in the manner previously described. These samples were exposed to the air and weighed after a 24-hr. exposure. The average results of a duplicate series, after correcting for the moisture changes indicated by the control, are given in Table I. TABLEI-RATE OF EVAPORATION O F CRUDE WESTERN YELLOW PINE OIL AND VARIOUS FRACTIONS OR THE OIL FROM IMPREGNATED DOUGLAS FIR SAWDUST Loss in Weight Per cent OIL after 24 Hrs. Exposure Fraction below 150’ C . . 68 Between 15O0-2OO0 C.. 42 6 Between 20O0-25O0 C.. Between 25O0-3OO0 C.. 0 Residue above 300’ C.. 0 Crude oil.. ................................... 9 Control (sawdust without addition of oil). 2
....................... ....................... ....................... ....................... .......................
......
Since 9 per cent of the oil was lost by evaporation by exposing the impregnated sawdust to the air for 24 hrs., it is necessary to make a corresponding correction of the toxic concentration previously determined. The corrected toxic concentration would be between 8.2 and 9.1 per cent, when lowland white fir sawdust was used as the culture medium, between 9.1 and 10.0 per cent when Douglas fir sawdust was used, and between 10.0 and 11.0 per cent when sugar maple sawdust was used. It is of course recognized that these figures would not represent the actual toxicity of the original oil since the more volatile portions would be lost by the sawdust being exposed to the air. This difficulty is not encountered in determining the toxicity of coal-tar creosote because little or no loss in weight occurs during this stage of the operation. If the toxic concentrations were based upon the net weight of the sawdust (5 g. sawdust plus 25 g. distilled water) they would of course be much lower, namely, between 1.4 and 1.5 per cent for lowland white fir sawdust, between 1.5 and
Vol. 14, No. 7
1.7 pet cent for Douglas fir sawdust, and between 1.7 and 1.8 per cent for sugar maple sawdust. It would appear to the writer, howeTer, that it is far more rational to compute the toxic concentration on the dry weight of the sawdust. The ideal way in which to determine the toxicity of wood preservatives to wood-destroying fungi would be to set up a culture series as above described and to determine the actual decomposition of the wood as indicated by a loss in weight of the individual culture flasks. There are, however, several decided disadvantages to such a method, the most important of which would be (1) that at least a 3-mo. incubation period would be necessary before reliable results could be obtained, and (2) with such substances as coal-tar creosote, wood tar or any other volatile substance, a loss in weight would occur during the final drying to constant weight, as the result of the evaporation of the substance. With nonvolatile substances such as zinc chloride, sodium fluoride, etc., this method can be used and is now being used in the laboratory, in connection with an investigation of the effects of “soil alkali’’ on the rate of decay of wood.
TOXICITY OF
THE
OIL WHEN ADDEDTO NUTRIENT AGAR
In a recent paper, Humphrey and his associates4 point out what they consider to be the advantages of the petri dish method for determining the toxicity of a wood preservative to wood-destroying fungi. While this method has certain advantages, it also has disadvantages, the most important of which, in the opinion of the writer, is that there is not of necessity any relation between the toxic concentration determined by this method and the toxic concentration when sawdust is used as the culture medium. Coal-tar creosote may be ten times as toxic when mixed with nutrient agar as it is when used in connection with sawdust, while wood tar may be only five times as toxic in the former case as in the latter case. These figures are not to be taken as absolute, but merely illustrative. In order to show what relationship, if any, exists between the results obtained by the two methods the following experiment was made: 100 g. of yellow pine crude oil were exposed to the air in a beaker until a 9 per cent loss in weight occurred. This corresponds to the loss of the oil taking place during the exposure of the impregnated sawdust to the air. A hard potato agar was prepared according to the formula: 0
Extract from Glucose
200 g. potato 20 g.
Agar Distilled water
40 g. 1000 cc.
Fifty-cc. samples of this medium were pipetted while hot into 125-cc. Erlenmeyer flasks, and the flasks and their contents were sterilized. After sterilization, the yellow pine crude oil was carefully added to the series to make the following percentage concentrations of the culture medium: 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, and 1.4. The flasks were then vigorously shaken until the agar was about to jell. The stoppers were removed and the contents of each flask approximately equally divided into two sterile petri dishes. After the medium in the dishes had cooled, the plates were inoculated from plate cultures of Lenzites saepiaria in the usual manner. Duplicate series were prepared in each case. The plates were placed in large empty desiccating dishes to prevent excessive drying and evaporation of the oil and incubated for 4 wks. a t 24’. Under the conditions above described the toxic concentration was found to be between 0.5 and 0.6 per cent. From this, it is evident that Western yellow pine oil has only very limited toxic properties. 4 C. J. Humphrey, Ruth M. Fleming and E. Bateman, THISJOURNAL, 13 (1921),618.
