New Theory on Wood -
Preservation R. W. FINHOLT', MAURICE WEEKS2, AND CLAYTON HATHAWAY3 Union College, Schenectady, N. Y .
I
NDUSTRIAL users of wood who have realized the seriousness of our dwindling forest resources have searched for u-oodpreserving chemicals. There are chemicals effective against all three of the great destroyers-fire, insects, and fungi. I t is the chemicals which are toxic t o wood-destroying fungi that concern this paper. In considering fungitoxicity, Bateman and Henningson (3) regarded the solubility problem as crucial since the toxicity of a homologous series first increased with molecular weight and then fell off, The low toxicity of higher homologs was attributed to insufficient water solubility. Their theory was based on tests on aqueous'media and not on wood itself, but it has since been found that comparative tests on wood and on aqueous media do not always agree even qualitatively (6). This paper presents toxicity evaluations of some normal alcohols and amines using both types of media. These are of particular interest because the alcohols behaved as predicted by the Bateman-Henningson theory, while the amines did not. The same qualitative results were obtained with alcohols on both wood and malt-agar media while the amines gave far different results on the two test media. It will be shown that these apparently discordant facts can be explained by a relatively simple theory. COMPARATIVE TESTS OF NORMAL ALCOHOLS A N D AMINES
One test was a variation (6) of the agar-dish method which employs a gel containing the test substance, agar, and malt extract; toxicity is determined by the concentration of toxic needed to prevent fungal growth on the surface of the gel. Baechler ( 1 , B ) had tested the series of normal alcohols and a few amines by this method, but since the fungus that he used (Madison 517) doas not destroy wood readily, it was also necessary t o determine fungitoxicities for an actual wood destroyer to compare results on both wood and agar. Although i t would have been desirable to use several fungi, the comparative work was limited t o only one-Madison 534 (Lentinus Zepideus). The other test was the soil-block method of Flerov and Popov (6), as systematized by Leutritz (7). Wood blocks are placed on a fungus culture growing on untreated feeder blocks in direct contact with moist soil in a bottle. The soil acts as a water reservoir and also supplies nutrilik. After 10 to 20 weeks, decay is estimated from loss in weight and change in appearace of the exposed test blocks. This soil-block method is much closer to natural conditions than the agar-dish method, but it lacks precision and is more time consuming. The results of the two tests on a series of alcohols are shown in Figures 1 and 2. Figure 1 shows the relationship on maltagar between the apparent inhibition point (AIP) and chain length of 6- to 18-carbon alcohols using two fungi-Madison 517 and 534. The top curve of Figure 2 plots the alcohol chain length against per cent decay (loss in weight) of wooden test blocks. This loss in weight represents the approximate protection that the alcohols give at 6 pounds per cubic foot. Other
concentrations could have been plotted, but this concentration shows nicely the difference in fungitoxicities of the alcohols. The 10- and 11-carbon alcohols mark the peak toxicity in the agar-dish test while the 12-carbon alcohol is ineffective. Figure 2 shows that, when alcohols are tested on wood, the peak effectiveness is a t 12 carbons. The 3% weight loss level of Figure 2 represents a level of no decay, while the 30% level represents decay of control blocks. The line C-D is somewhat lower than would be expected from the agar-dish test results; it seems likely, however, that the higher alcohols have mechanical effects such as waterproofing of wood that would be absent in an agar medium. The alcohol series give similar qualitative toxicity curves for the two media and one curve could be roughly predicted from the other. The differences come in the shifting of peak toxicity from an 11-carbon length in agar t o a 12-carbon length in wood, and with the evidence of some toxicity for 14- t o l&carbon alcohols in wood. The amines present a different picture. Figure 3 shows t h a t the agar-dish test curve for the amines looks much like that of the alcohols. The amines in this range are not aa toxic as the alcohols a t peak toxicity, but are more toxic than the corresponding alcohols a t the ends of the curve. I n other words, the rate of change of toxicity of amines with chain length is not so rapid ws with the alcohols. The amines in wood, however, give an entirely different picture. The soil-block tests of the lower curve of Figure 2 demonstrate that all of the amines prevent decay (a 3% weight loss is a level of no decay) even at a concentration of 0.6 pound per cubic foot, which w w one tenth that of the alcohols. This means that the relative toxicities of alcohols and amines are reversed when tested on the two mediums. The amines are all toxic in wood but are much less toxic in agar. The alcohols are more toxic than the amines when tested in agar, but are far less toxic than the amines when tested in wood. NEW THEORY ON WOOD PRESERVATION
A new theory is putlined here that offers a good explanation for the alcohol-amine turnabout and that also offers an explanation for other toxicity reports. This theory is that there must be at least two broad types of fungal poisons; one type interferes with the metabolism system in some way inside of the fungus membrane while the other type interferes with some essential system completely outside of the fungus membrane. The first type of fungitoxic could interfere in any or all of the essential enzyme systems that operate inside of the fungus membrane, such as oxidation-reduction enzymes involved in sugar oxidation. The second type of fungitoxic could affect the fungus by preventing it from obtaining nutrient in its normal manner. This could be done by substances that would denature the hydrolytic enzymes necessary t o break down wood into usable nutrient, such as cellobiose or glucose. If the hydrolytic enzymes were so deactivated there would, of course, be no penetration of the wood by exploring fungus hyphae and the wood could not be rotted. The nutrient supply could also be disrupted by substances that would form stable chelates with essential minerals, such as trace
1 Present address, General Eleotric Co.,Locomotive and C. E. Laboratory, Erie, Pa. a Present address, General Electric Co., Hanford. Wssh. a Present address, Chemistry Department, Purdue University, West Lafayette, Ind.
101.
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
102
amounts of iron, soluble nitrogen compounds, phosphates, or other essential trace materials. Other ways in which materials might be toxic to fungus either inside or outside of the membrane wall can be readily visualized. It is important t o examine carefully the postulate that these two types of fungitoxics actually exist and that this existence explains the known facts concerning fungitoxicity.
.3.2
-
.I
-
a4
I-:
s
z
-
0
-
3 -03-
.oe
-
.01
-
U .oo2
J 8
10 II 12 14 No. OF CARBONS IN CHAIN
16
Figure 1. Relationship between AgarDish Toxicity and Chain Length of Aliphatic Alcohols
EXTERIOR FUNGITOXICS. Considering exterior fungitoxics first, it seems reasonable from both a practical and theoretical standpoint that some fungitoxic substances can be fully operative entirely outside of the cell membrane of the fungus. The woodpreserving industry has bent every effort to make their preservatives as water insoluble as possible sa as to achieve permanence. One has only t o read a few of the patents that have been issued on production of all types of insoluble fungitoxics to appreciate the fact that preservatives can be both effective and extremely water insoluble. Creosote, chromated zinc chloride, mercury oleate, and pentachlorophenol are only four examples of the very insoluble materials that are being used commercially to good advantage. It has been tacitly assumed that these materials dissolve to the small level needed to prevent fungus growth; but such an assumption need not be made at all if one assumes instead that totally water-insoluble materials can deactivate some of the vital exterior enzymes. This is not t o say that many of the "insoluble" fungitoxics may not actually dissolve but t o affirm simply that such solution may not be necessary to explain their action. Besides this practical evidence there is laboratory proof that is more direct and convincing. The laboratory evidence is found in the comparative toxicity tests run on creosote fractions by Flerov and Popov (6) and in the similar comparative tests on amines reported in this paper. Both of these studies found that some materials were nontoxic when tested in agar but were toxic
Vol. 44, No. 1
when tested on wood. With creosote it was the higher boiling fractions that so behaved, and with the normal amines (see Figure 2), it was the 16- and 18-carbon amines that had especially striking differences. The work of Popov and Flerov was confirmed in this laboratoiy by using a 330' to 340' C. creosote fraction which was inert in agar but active in wood. With both the creosote and the amines in agar the suspensions were submicroscopic in size (particles under 1 micron) so there was no question about the dispersion, but even with this good emulsion the fungus hyphae grew down through the agar and the rate of growth was the same as in the controls. I n wood, however, there was no hyphae penetration and no loss in weight of the wood even though Bome of the test blocks were completely covered by mycelium. If there was any possibility of the toxics functioning by passing into the fungus proper this striking difference would be impossible. Clearly these materials are acting outside of the fungus membrane. The most likely mode of action of these exterior toxicants is open to discussion, but one likely activity is the denaturing of the hydrolases excreted by the hyphae. This means that the hyphae could not penetrate the wood and could not convert the wood into food. Since the wood is not penetrated, it ie not rotted, even though the mycelium may cover the surface. Actually these exterior toxicants are toxic only indirectly and do not kill the fungus directly at all. The exterior toxicante are, of course, ineffective in agar-malt since there is a food source-maltoseready for use and no hydrolytic activity is needed outside of the membrane. Other types of exterior toxicants may exist, but hydrolase denaturation seems to be a good explanation for the action of these long-chain amines and high boiling creosote fractions. These toxicants also do not have to dissolve in water to be effective since the water-soluble enzymes come to the toxicants and are then denatured, much like ion exchangers. INTERIOR FUNGITOXICS. It seems self-evident that some types of toxic materials operate inside of the membrane in view of the general mode of action of toxic materials on both plants and animals. This class of fungitoxics must be water soluble in most cases (although fat solubles may be possible), but molecular size is also a limiting factor with this class. Examples of this class
'"t 35
I ALCOHOLS AT 6.0 LBS./CU.FT.
10
5 ,
6
4
,
#
1
1
8
W l l 1 2 ' 1 4 I6 18 NUMBER OF CARBONS IN CHAIN
1
I
I
.
I
.
I
,
I
Figure 2. Wood Block Decay as a Function of Chain Length of Normal Alcohols and Amines
are the 10- and 11-carbon alcohols which are toxic on both wood and in agar. Since the higher alcohols are nontoxic in either wood or agar it can be reasoned that none of the alcohols have exterior fungitoxic function because exterior fungitoxics are independent of molecular weight. The amines of 10- and 12carbon length have both exterior and interior fungitoxicities. This is shown in Figures 2 and 3 ; these amines have interior functions because they are toxic in agar. They probably also have exterior functions since the higher amines have this function solely. -Highly insoluble materials cannot be transported in an aqueous
INDUSTRIAL A N D ENGINEERING CHEMISTRY
January 1952
system and water solubility is one limiting factor for this type of fungitoxic. This was the essential viewpoint of Bateman and Henningson in their theory. Besides this factor i t can be shown that molecular s h e is a limiting factof and that, in the straightchain organic compounds, this molecular sixe is reached at 10- to 12-carbon chains. The pase presented here for molecular s h e limits rests on the toxic effects of three series of compoundsamines, acids, and their sodium salts as tested in agar. Baechler (1,P) reported that the sodium salts and free carboxylic acids both reached maximum toxicity in agar at an 11-carbon chain length. As seen in Figure 2 the amines reached maximum toxicity in agar at 10 carbons when tested with the same funmsMadison 517. The fact that these three classes of compounds reach toxic limits a t about the same chain length proves that molecular size is a limiting factor because one of these series must be water soluble regardless of the pH of the water environment. If the solution is basic the sodium salts would all be soluble and there would be no great difference between solubility of a l h a r b o n salt and a 14carbon salt. If the solution were basic enough the acids would also dissolve and behave like the sodium salts. The amines would be unaffected and some peak beyond which the amines were too insoluble to be effective might be expected. Since all three series behave alike, 'however, a baaic pH is inconsistent with a simple solubility picture of toxicity. The reverse case can be made for an acidic pH in which the amines would be soluble and the acids and salts would not be soluble. In a neutral medium the salts would dissolve, but the amines and the free acids would be insoluble. Since one of the series must be water soluble at all chaii lengths, within limits,but none of the series has toxic higher members, it must follow that water solubility per se is not the only limiting factor for toxicity. The solutions are actually strongly acidic, which is due in some cases, at least, to oxalic acid production (IO).
TABLE I. SUMMARY Aloohol in Toluene, % Hexanol
Retention Lba. per Cubio'Foot
% Weight Lose
16
1.24 2.20 5.15 9.20
30.2 a0.8 28.1 27.5
4 8 15
1.37 2.57 4.83 9-43
28.8 27.8 17.8 17.0
1.30 2.64 4.Bo 9.49
8.8 6.4 6.6 7.8
1.42 2.74 4.95 10.01
8.8 4.4 6.a 6.8
1.18 2.50 5.00 10.22
8.8 3.a 2.8 2.0
1.28 2.64 4.82 10.10
17.4 4.1" 10.2 11.2
1.21 a.49 8.96
io.63
19.9 11.8' 20.9 11.2
1.18 2.a9 4.48 7.99
20.4 28.3 12.8 18.4
4
8 30 Ootanol
ao
Decanol 4 8 16
ao
Hendecanol 4 8 16
ao
Dodecanol 4 8 16 30 Tetradecsno 4 8 15 30 Hexadecanol 4 8 16
30 Ootadecanol 4 8
15
ao 8
SOIL-BLOCK TESTSON NORMAL ALCOHOLS
OF
Contaminated by molds.
103
By assumkg that a maximum molecular size exists beyond which no materials are interior fungitoxicants, .all difticultiea vanish. It then becomes perfectly reasonable that alcohols, amines, sodium salts, F d acids should all reach maximum toxicities at approximately the same chain length. There may be molecular s h e openings in the cell walls that simply cannot accommodate molecules any larger than a certain critical she. This accounts nicely for the very abrupt change in toxicities found when one goes from a l k a r b o n chain t o a l2-car bon chain
-01
8
0 12 14 * 16 No. OF CARBONS IN CHAIN
18
Figure 3. Relationship between Agar-Dish Toxicity and Chain Length of Aliphatic Amines USESFOR THE NEWTHEORY.This theory explains or a t least offers a possible explanation for several peculiar phenomena. The turnabout behavior of amines and alcohols is now explainable. The alcohols are more effective interior fungitoxicants than the amines and hence are more effective in agar, while the amines are more effective exterior toxicants and hence are more effective in wood. It is now possible to understand why some materials that test poorly in agar may be good wood preservatives nevertheless. Others have found that many materials seem to require a much higher concentration to be effective in wood than in agar; this theory would lead to the prediction that anything effective on agar ought also t o be effective on wood in the same or a lower concentration range, allowing for the adsorptive effect of wood. It has been found in this laboratory that the differences in toxic concentration, in grams per ml., in agar and wood are slight when testing alcohols, provided that the same amount of mycelium is present at the start of the test. All of the testing on wood has used large amounts of mycelium while the agar tests have always used small amounts of mycelium; this difference in quantity produces differences in the quantitative toxic concentration, although qualitatively the results are the same. This theory helps to explain the ditrerences in rot resistance of various woods although there are many other factors to be considered. It also offers a possible explanation for the combined fungus and insect protection offered by some preservatives. If a fungicide is acting by denaturing wood hydrolases it will not only prevent fungus attack, but insects that eat the protected wood cannot digest the wood since the hydrolases produced in their inteatines would also be denatured. Thii theory predicts that no molecules over a critical size can funption as interior fungitoxics, and it predicts that a variety of exterior fungitoxics can be found which need not be water soluble.
INDUSTRZAL AND ENGINEERING CHEMISTRY
104
TABLE 11.
SUMMARY OF
Amines in Toluene, % Octylamine 2 4
8 15 30
Decylamine 2 4
8
15 30
Dodecylamine 2
4 8
15
Tetradecylamine 2 4 8
15 Hexadecylamine 2 4 8 15
Octadecyladno 2 4
6)
3.6
SOIL-BLOCK TESTS O N NORMiaL AMINES Retention, Lbs. per Cubic Foot
% Weight Loss
0.65 1.27 2.61 4.67 9.97
3.2 4.7 3.0 4.2 6.5
0.62 1.27 2 61 4.84 9.51
3.1 2.4 3.9 2.7 5.5
0.61 1.05 2.60 4.67
3.8
0 67 1,20 2.51 4 58
3.6 3.2 3.0
0.65 1.38 2.53 4.95
3.7 3.7 3.2 7.2
8.67 1.33
4.0 3.p 3. a 4.9
2.52
4.81
3.3 3.5 6.0
Vol. 44, No. 1
were put into test over about a month's time, and during this time coincident runs with alcohols and creosotes gave normal decays. Furthermore, the controls run a t the same time and with the same cultures gave a uniform 30 to 32y0 decay. The soil-block tests were like those of Leutritz ( 7 ) as modified by Duncan and Richards ( 4 ) . The test blocks were carefully machined 3/4-in~hsquare blocks of kiln-dried Southern Pine (Pinus palzistris-Miller) and their decay mas tested against Lerzlznus Zppidezts (Madison 5 3 3 ) . The blocks were put under a 15-mrn. vacuum for 15 minutes priw to impregnation with toluene solutions of the test chemical and then were allowed to soak under vacuum for 15 minutes more. The tests were usually run with two blocks of the same concentration per &ounce bottle, and incuhation was for 10 weeks a t 26.7" C. Ordinary garden soil of low organic content wm used and was wet to 40% of its dry w-eight for test use.
6.2
The use of both types of testing combined with this theory should enable one t o identify pure exterior toxicants at once and, b y indirect evidence, Lo identify pure interior toxicants as well. As a pure research tool this differential testing technique may lead to a much broader understanding of the nature of fungitoxicity and enzyme deactivation. This type of test may also find application in study of other types of fungus besides the wood dostroycrs, since the same line of reasoning can be applied to other fields.
T.4BLE
111.
Number of Carbon Atoms
SLXIMARY O F A G B R - D I S H
ALCOHOLS Apparent Inhibition Point. Madison 317
n nw
ti 8 10
0,014 0.009
Over Over Over Over Over
11 12 14 16 18
TABLE Iv.
5 5 5
5
XORMAL
Apparent Inhibition Point, Madison 534 n IR 0,023 0.004 0.0045 0.13 Over 5 Over 5 Over 5
SUMMARY O F A4GAR-DISH TESTS ON N O R M A L
Nimiber of Carbon Atoms 8 IO 12
14 18 18 Q
5
TESTSO N
AMINES
Apparent Inhibition Point, Madison 517
Apparent Inhibition Point. AMadison534
0.08 O.O
