AN ABSTRACT OF THE THESIS OF
Barry Scott Goodell for the degree of Doctor of Philosophy in
Forest Products presented on April 8, 1983
Title: Microdistribution and Retention of Chloropicrinin Douglas-fir
(Pseudotsuga menziesii (Mirb.) Franco]Heartwood
Abstract approved: Redacted for Privacy Le-r-44 Robert L. Krahmer
Quantitative and qualitative informationon the retention of chlorinated residues in chloropicrintreated wood is lacking.
Retention of chloropicrin and itschemical breakdown products in wood, and the effects of chloropicrintreatment of wood on invading decay fungi are explored in this thesis.
Chlorinated residues in amountsup to 1.5% (w/w, as determined by neutron activation analysis) were present in Douglas-firwood exposed to chloropicrin vapors for 240 days. These residues could not be totally removed by aeration,acetoneextraction, heating, or high vacuum treatment. Lesser amounts of chlorinated residueswere detected in wood exposed to chloropicrin forshorter periods of time or treated with smaller quantities of the chemical. Analysis of vaporsdesorbed from chlorpicrin-treated woodsuggests that chemically unaltered chloropicrin can be retained bywood up to 4 years after treatment. Decreased retention of chlorinated residues intreated wood with brown rot decay suggests thathydrogen bonding and van der Waals forces are responsible, at least in part, for this retention.
Wood does not swell when treated with chloropicrin.Although a monomolecular layer of chloropicrin may hydrogen bond to wood at some sites, lack of swelling indicates that chloropicrin molecules are retained by forces weaker than the hydrogen bonds which hold the lignocellulose matrix of wood together.
Covalent linkages may also form between chloropicrin, or chlori- nated breakdown products of chloropicrin, and wood.Energy dispersive
X-ray analysis showed that chloropicrin residues were present in greater concentrations in areas of the wood rich in lignin or extrac- tives. Covalent linkages between chloropicrin breakdown products and vanillin were detected in mass spectrographic analyses of chloropicrin/vanillin mixtures. Thin layer chromatography of extracts from chloropicrin treated wood indicated that chloropicrin may be bonding to dihydroquercetin (a phenolic wood extractive) and to other extractives in wood.
Microscopic observation of Douglas-fir wood exposed under severe laboratory decay conditions to Poria carbonica (a brown rot fungus), and to Ganoderma applanatum (a white rot fungus), showed that these fungi will attack chloropicrin treated wood, producing bore holes and erosion of cell lumen surfaces similar to that observed in untreated wood. Lysis and vacuolation of the fungal hyphae, particularly of G. applanatum, indicates that chloropicrin has an inhibitory effect on fungi in wood. Microdistribution and Retention of Chloropicrin
in Wood
by
Barry Scott Goodell
A THESIS
submitted to
Oregon State University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Completed April 8, 1983
Commencement June 1983 APPROVED:
Redacted for Privacy
Professor of Forest Products in charge of major
Redacted for Privacy
Head of Department of Forest Products
Redacted for Privacy
Dean of Graduate c ool
Date thesis is presented April 8, 1983
Typed by Julie Cone for Barry Scott Goodell DEDICATED TO
JODY ACKNOWLEDGEMENTS
My special thanks to Bob Krahmer for his guidance over the past three and one-half years; not only for his assistance with my thesis research but for his help in developing my teaching skills, and for his friendship. Thanks are also due to Mal Corden, Bob Graham and
Murray Laver for their insights into pathology, preservation and che- mistry, respectively. Without their research insights and their help in developing my skills as a researcher this work would not have been possible. I would like to acknowledge and thank Bob Graham, Bob
Krahmer and Helmuth Resch for the opportunities and funding they pro- vided me, allowing me to carry out not only my thesis research but also other projects which helped to broaden my background in wood technology and wood preservation/pathology.Ted Scheffer and Don
Miller also deserve my heartfelt thank you for their friendship and for the opportunities and insights they have given me in research out- side my thesis area.
Many people gave me technical assistance in work necessary for the development of the research presented in this thesis. Their excellent assistance is acknowledged below:
Electron Microscopy:
Alfred Soeldner- Instructor, Botany and Plant Pathology, OSU Christine Weiss Research Assistant, Botany and Plant Pathology, OSU Arnold Schmitt Research Assistant, Mining, Metalurgical and Ceramic Engineering, University of Washington
Mass Spectroscopy:
Donald Griffin - Chemist, Agricultural Chemistry, OSU Neutron Activation Analysis:
Michael Conrady - Chemist, Radiation Center, OSU Roman Schmitt - Professor of Chemistry, Radiation Center, OSU
Photography:
Grant Mitman - Teaching Assistant, Botany and Plant Pathology, OSU
Supplying Fungal Cultures:
Robert Zabel - Professor, College of Environmental Science and Forestry, State University of New York Jean Clark - Technologist, Forintek Canada Corporation, Western Laboratory Bradley Kropp - Mycologist, Botany and Plant Pathology, OSU
Thin Layer Chromatography:
Steven Danko - Research Associate, Agricultural Chemistry, University of Nebraska
Finally, I would like to thank my wife, Jody, not only for her patience and understanding during my graduate career, but also for the technical support she offered me while she worked toward her own degree.
Thanks to you all! TABLE OF CONTENTS
Page
INTRODUCTION 1
BACKGROUND 3
Retention of Chloropicrin 3 Bonding of Chloropicrin to Wood 4 Chemical Breakdown of Chloropicrin 6 Other Chemical Reactions of Chloropicrin 8 Fungal Attack of Chloropicrin Treated Wood 8
PROCEDURES 12
I. Sorbtion 12 Dimensional Change Occurring in Wood Treated 12 With Chloropicrin Non-Steady-State Desorbtion and Residue 13 Analysis
II. Neutron Activation Analysis 14 Preliminary Analysis of Background Chlorine 16 in Wood Analysis of Treated Samples 16 Sorbtion Samples 16 Non-decayed Wood 17 Decayed Samples 18
III. Energy Dispersive X-ray (EDX) Analysis to 19 Determine the Microdistribution of Chlorinated Residues
IV. Infrared and Mass Spectrographic Analysis 20 Analysis of Chloropicrin and Derivitized 20 Wood Components Analysis of Desorbed Gases from Chloropicrin 21 Treated Wood
V. Microscopy of Treated Wood Samples Infected With 22 Decay Fungi
RESULTS AND DISCUSSION 23
I. Sorbtion 23 Dimensional Change Occurring in Wood Treated 23 With Chloropicrin Non-Steady-State Desorbtion and Residue 23 Analysis Page
II. Neutron Activation Studies to Determine the 27 Amount of Chloropicrin Retained by Wood After Treatment Preliminary Analysis for Background Chlorine 27 in Wood Analysis of Treated Wood Samples 27 Sorbtion Samples 27 Non-decayed Wood 29 Decayed Samples 32
III. Energy Dispersive X-ray (EDX) Analysis to 36 Determine the Microdistribution of Chlorinated Residues
IV. Infrared and Mass Spectrographic Analysis 50 Analysis of Chloropicrin and Derivitized 50 Wood Components Analysis of Desorbed Gases from Chloropicrin 56 Treated Wood
V. Microscopy 58
CONCLUSIONS 68
SUGGESTIONS FOR FUTURE RESEARCH 70
BIBLIOGRAPHY 71
APPENDIX 1 79
APPENDIX 2 80
APPENDIX 3 81 LIST OF FIGURES
Figure Page
Sorbtion (-11-) and desorbtion (-II-) of chloropicrin 24 from Douglas-fir wafers (A) over time.
Sorbtion (--) and desorbtion (-II-) of chloropicrin 25 from Douglas-fir wafers (B) over time.
Chlorine retention in samples decayed to a range of 34 weight losses by Poria carbonica prior to treatment with chloropicrin.
Chlorine retention in samples decayed by Poria 35 carbonica prior to treatment with chloropicrin. Amount of decay in the samples determined by the percentage of alkalli soluble material.
Same regression as shown in Figure 4 minus three 37 data points (> 100 pg chlorine).
Scanning electron micrographs of Douglas-fir heartwood 38 treated for two weeks with liquid chloropicrin. Above, split radial surface, 500X; below, razored cross section, 500X.
Typical energy dispersive X-ray scan across chloro- 39 picrin-treated wood showing the absence of chloronated material in the sample. Peaks for gold and palladium from the coating material are present in the scan.
Representative energy dispersive X-ray spectral scan 41 across acetone extracted or heated control wafers. No detectable chlorine peak is present.
Energy dispersive X-ray analysis line scan across one 42 cell wall of an untreated, control wafer. No detect- able amounts of chlorine are present.
Representative energy dispersive X-ray spectral scan 43 across extracted or heated, chloropicrin-treated wafers.
Energy dispersive X-ray analysis line scan across a 44 cell wall of a heated, chloropicrin-treated sample that was sectioned dry.
Energy dispersive X-ray analysis line scan across a 45 cell wall of a heated, chloropicrin-treated sample that was sectioned wet. Figure Page
Representative energy dispersive X-ray line scan across 46 an acetone extracted or heated, chloropicrin-treated wood.
Energy dispersive X-ray line scan across ray cellwalls, 47 tracheid cell walls, and resin canal parenchyma cells with included deposits in extracted, chloropicrin- treated wood.
Energy dispersive X-ray line scan across cellwall 48 material and extractive materials deposited in the lumens of rays in extracted, chloropicrin-treated wood.
Energy dispersive X-ray line scan across fusiform 49 ray in heated, chloropicrin-treated wood.
Gas chromotograph of a reacted mixture of chloropicrin 52 (retention time = 0.52 m) and vanillin (retention time = 5.60 m). Reaction products present at retention times of 11.07 in and 17.99 m.
Mass spectrograph of chloropicrin/vanillin reaction 54 product at 5.60 m (Figure 17). Parent ion mass = 197.
Mass spectrograph of chloropicrin/vanillin reaction 55 product at 11.07 in (Figure 17). Parent ion mass = 270.
Mass spectrograph of chloropicrin vapor from treated 57 wood showing only the mass fragment for C-C13 (ion mass = 117, 118 and 119), and smaller mass fragments.
Perforation type bore holes produced by the fungus 59 Ganoderma applanatum in the cellwallsof Douglas-fir.
Erosion produced by the action of Ganoderma applanatum 60 on the lumen surface of Douglas-fir cellwalls.
Perforation type and typical holes produced by the 61 fungus Poria carbonica in the cellwallsof Douglas- fir.
Vacuolated and lysed hyphae of Ganoderma applanatum 63 in chloropicrin-treated Douglas-fir wood. 4000X.
Vacuolated hyphae of Ganoderma applanatum with clamp 64 connection in chloropicrin-treated Douglas-fir wood. 7500X.
Normal appearing hyphae of Ganoderma applanatum in 65 untreated Douglas-fir. 10,600X. Figure Page
Lysed hyphae of Poria carbonica in chloropicrin-treated 66 Douglas-fir. 7200X.
Normal appearing hyphae of Poria carbonica in untreated 67 Douglas-fir. 9400X.
Infrared spectral scan of chloropicrin in potassium 81 bromide.
Infrared spectral scan of vanillin (in potassium 82 bromide).
Infrared spectral scan of acetyl ferullic acid (in 83 potassium bromide).
Infrared spectral scan of d-catechin (in potassium 84 bromide).
Infrared spectral scan of tannic acid (in potassium 85 bromide).
Infrared spectral scan of cellobiose (in potassium 86 bromide).
Infrpred spectral scan of solka flok (in potassium 87 bromIde).
Infrared spectral scan of vanillin reacted with 88 chloropicrin and aerated for five minutes (in potassium bromide).
Infrared spectral scan of acetyl ferullic acid 89 reacted with chloropicrin and aerated for five minutes (in potassium bromide).
Infrared spectral scan of d-catechin reacted with 90 chloropicrin and aerated for five minutes (in potassium bromide).
Infrared spectral scan of tannic acid reacted with 91 chloropicrin and aerated for five minutes (in potassium bromide).
Infrared spectral scan of cellobiose reacted with 92 chloropicrin and aerated for five minutes (in potassium bromide).
Infrared spectral scan of solka flok reacted with 93 chloropicrin and aerated for five minutes (in potassium bromide). LIST OF TABLES
Table Page
Chlorine content of control and chloropicrin treated 28 wafers which were aerated and heated to 115°C for 5 hours or extracted with acetone.
Chlorine content of wood samples initially injected 30 with 50 pl of chloropicrin.
Chlorine content of ground wood treated with 31 chloropicrin, then aerated for various periods of time.
Comparison of the chloropicrin content of treated 33 ground wood after aeration with closed-tube assay data of the same wood. MICRODISTRIBUTION AND RETENTION OF CHLOROPICRIN IN WOOD
INTRODUCTION
Fumigants have been used commercially to control wood decay fungi
in transmission poles since the early 1970's (20). Since that time considerble research has been done to evaluate their field effec-
tiveness and determine their gross distribution in treated wood pro- ducts. Studies at Oregon State University have shown that chloro- picrin has remained effective against decay fungi in transmission poles for 12 years (21) and bioassays show that fungitoxic chloro- picrin vapors are present in wood samples removed from timbers up to 5 years following treatment (21).
Research on the nature of this retention of chloropicrin by wood, the breakdown of the chemical over time, and reinvasion of treated wood by fungi is lacking. If the amounts and location of chloropicrin remaining in wood following fumigation can be determined, this may provide insight into better methods for fumigant treatment and should suggest, depending on the nature of retention, if chloropicrin can be considered for use as a long-term wood preservative.
I investigated these problems and the potential for the use of chloropicrin as a long-term preservative through experiments designed
to determine:
The means of retention of chloropicrin and its degradation
products in wood.
The location and concentration of chloropicrin and its
degradation products within the wood structure. 2
The amounts of chlorine retained in chloropicrin-treated, decayed and sound wood, and the effects of chloripicrin treat- ment on the penetration and breakdown of wood by fungi. 3
BACKGROUND
Retention of Chloropicrin
One of the main advantages of using fumigants to treat wood is that gas molecules can move through the pits without surface tension interference (4), allowing the gas to rapidly diffuse through the wood structure. If the fumigant is not bound to the wood, however, and forces such as surface tension--which serve to retain conventional liquid preservatives in wood--are not active, then the fumigant may also readily diffuse out of the wood. It is known that fumigants will bond to organic matter and clays in the soil (15, 19) and low levels of residue of many fumigants have been recorded in stored grains after aeration (2). Smith (37) determined that residues of propylene oxide can remain in wood after aeration, and these residues are fungitoxic to mature mycelium of Lentinus lepidius.Low levels of chloropicrin residues remaining in stored grain products after fumigation have been reported but no quantitative data is available, and the fungitoxic nature of these residues was not studied (13).
Evidence that chloropicrin residues may be binding to wood was suggested in studies where significantly smaller quantities of chloro- picrin vapor diffused longitudinally in severely decayed wood than in less decayed wood (16). This suggested that more chemical was retained in the severely decayed wood because the greater surface area or micropores in the cell wall created by fungal degradation provided more sites for chemical bonding or physical entrapment of the chloro- picrin molecules. 4
Bonding of Chloropicrin to Wood
Cowling (11) determined that at weight losses above 40%, wood
decayed by the white rot fungus, Polyporous versicolor, absorbed
significantly more moisture than non-decayed wood (compared to oven
dry weights) when samples were exposed to various relative humidity
conditions. The reverse situation occurred in samples decayed by the
brown rot fungus, Poria monticola, where a general reduction in
hygroscopicity occurred with increasing decay at all stages. Cowling
suggested that in white rot decay, the increase in hygroscopicity may
have been due to the reduction in the amount of crystalline material
in the wood which he considered to be less hygroscopic; presumably
this material was also converted to more hygroscopic amorphous cellu-
lose. With brown rot decay he suggested that the amorphous cellulose may have been attacked preferentially causing a relative reduction in
hygroscopicity.
Schulze-Dewitz et al. (36) also found that brown rotted woods gen-
erally had greater relative sorbtion when the wood had been incubated
with fungi for 4 months. However, when sorbtion capacity was measured
in woods decayed to a range of weight losses between zero and 25% it
was found initially to decrease, then increase, then decrease again.
Even if no significant bonding of a fumigant to wood by hydrogen-
or covalent-type bonds occurs, if greater microvoid space is created
by the degradation of the wood material, this may allow greater amounts
of the chemical to be retained in decayed wood than in sound wood.
Molecules of polar liquids will move into the amorphous regions
of a ligno-carbohydrate matrix expanding this matrix in proportion to 5 the size and quantity of molecules introduced (32). Polar molecules unite with dry cell wall material by means of hydrogen bonding (32).
The dipole moment of chloropicrin is approximately the same as that for water (43) and hydrogen bonding should be possible between the oxygens of the chloropicrin molecule and the hydrogen molecules in wood. However, because chloropicrin contains no hydrogen, dipole- dipole interaction of its molecules will be much weaker than the hydro- gen bonding attraction (28) which occurs between water molecules and allows the buildup of multimolecular layers attached to the wood ma- trix (the cause of dimensional change/moisture relationships in wood).
The energy of the bonds between chloropicrin molecules, or between chloropicrin and wood molecules may be too low to overcome the attrac- tive forces holding the amorphous regions in wood together to limit swelling of wood in the presence of chloropicrin. Fumigants such as chloropicrin can be sorbed to surfaces by physical adsorbtion and held by van der Waals forces (8) or depending on environmental conditions can be chemically (covalently) bound to some extent (5). The physi- cally bound residues can be removed far more easily than residues bound covalently by means such as prolonged aeration, or by more drastic treatments such as the use of heat, solvents, or reduced pressure.
Cooper (personal communication, P. A. Cooper, 2/1/83) has ana- lyzed chloropicrin retention by wood, comparing its retention time on ground wood particles (used as the absorbtive phase in a gas chroma- tography column) to that of other solvents.He found that chloro- picrin had a retention time similar to that of non-polar solvents such as benzene. Analysis of the ground wood after treatment was not made to determine if covalently bound residues were present. 6
The persistence of chloropicrin residues in treated grain was
found to be greater when the grain was subjected to treatment for greater periods of time (48). Grain subjected to 112 hours of chloro- picrin treatment retained 29.62% residue after gas sweeping but only
8.56% residue was retained after gas sweeping if the treatment was for
0.5 hours. Chloropicrin (or breakdown residue) was not completely removed after heating of the grain samples to 1200-1300. This evi- dence suggests that chloropicrin strongly hydrogen bonds or possibly even covalently bonds to grain; however, no information concerning this, or the fungitoxic nature of this residue was given.
The preceding evidence suggests that chloropicrin could possibly bond at least in small quantities to other organic, cellulosic materials such as wood. Since chloropicrin is known to be very susceptible to nucleophillic attack (9, 14), the likelihood of a cova- lent attachment of chloropicrin in wood increases if weak bases occur naturally in the extractive materials.
Chemical Breakdown of Chloropicrin
Chloropicrin in the gas-phase can be photolytically decomposed to phosgene and nitrosyl chloride (27, 3). Moilanen et al. (27) found that 20 days were required to decompose half of the chloropicrin at an initial concentration of 14 ug/ml, when exposed to sunlight wave- lengths (> 290 nm). Chloropicrin was not observed to decompose in the dark over a 7 day sampling period.Hydrolysis of chloropicrin in aqueous solution at pH 7, when irradiated by a monochromatic light source at 254 nm was very rapid (9). In 10 hours greater than 95% of the chloropicrin in a 10-2 M solution had decomposed. Decomposition 7 was slower when the system was exposed to sunlight wavelengths, and
"almost no hydrolysis" was detected under conditions of ambient laboratory light, or in darkness.
Since the environment for chloropicrin in a treated timber would be largely protected from light, no photolysis, and little photohydro- lysis would be expected to occur except perhaps in the outer 2.5 mm of wood exposed to sunlight (7). Over a period of years however, small amounts of phosgene (C12C0) and nitrosyl chloride (Nod), and their photolysis and hydrolysis products NO, NO2, N204, C12, and H+ would possibly accumulate if sufficient moisture and ambient light were pre- sent. It is possible that the rate of the hydrolysis reaction may be increased if the acidity is lower than pH 7, as occurs in Douglas-fir heartwood.
One of the principle products of the photolysis and photohydroly- sis reactions, phosgene, is a highly toxic gas. Phosgene will undergo a strong nucleophyllic substitution reaction with alcohols producing transition chlorocarbonates and yielding alkyl carbonates (28). It is possible that phosgene could react with the free hydroxyl sites of wood to form covalent bridges between various wood components, which may confer some resistance to fungal attack. The predominant location for this reaction (if it occurs) would be in the outer shell of wood exposed to sunlight; however, even if limited to this site it may pro- vide a thin protective barrier to protect against biological invasion and degradation.
Hydrolysis of chloropicrin has also been investigated by researchers (33) working with wine products where chloropicrin was used to sterilize wine barrels. The degradation products C1CH2NO2 and 8
C12CHNO2 were isolated from wine, both of which had low toxic and
sterilizing potential. Neither of these products had been reported in previous literature concerning hydrolysis of chloropicrin.
Biological breakdown of chloropicrin by microorganisms has recently been studied by Castro (personal communication, C. Castro,
2/10/83). His preliminary findings are that a bacterium, Pseudomonas sp. is capable of degrading chloropicrin. This may have implications
in wood treatments with chloropicrin since species of Pseudomonas have been isolated from wood products (30, 24) some of which may have the ability to chemically degrade creosote.
Other Chemical Reactions of Chloropicrin
Chloropicrin is very susceptible to nucleophyllic attack by weak bases (9) and produces ortho esters (14), carbonate esters, Cl, NO2 and other products when reacted with alkoxides. Although heartwood is generally acidic, localized components may act as weak bases to par- ticipate in the reaction, and water within wood may also act as a weak base to produce small amounts of the breakdown products.
Fungal Attack of Chloropicrin-Treated Wood
The attack of wood by brown rot and white rot fungi has been well documented by microscopic studies (45, 25). In general, these studies show that white rot fungi produce a progressive thinning of the secon- dary cell wall, produce radial cracks or checks in this wall in early stages of decay, and may cause separation to occur within the wall structure or in the adjacent middle lamellae. In brown rot degraded wood, no progressive thinning has been observed and the wall retains 9 its original sound dimensions during early decay stages; collapse of the wall caused by uniform degradation throughout the wall will occur in later decay stages. The attack of the cell wall in brown rots is likely to be irregular even within single tracheids. Cracking or checking of the secondary cell wall also is apparent in later decay stages. Bore holes are produced by both types of fungi; however, bore hole production has been observed to occur more slowly and with smaller holes in white rot decay than in brown rot decay (42).
There is less information concerning the attack of preservative treated wood by these Basidiomycetes. Bravery et al. (6) studied the inhibition of attack of Polyporous versicolor in tri-n-butyl tin oxide treated wood. The hyphae of this fungus were able to colonize wood treated with low retentions of the chemical and to start enzymatic dissolution of the cell walls. Inhibition of the fungal action and lysis of the hyphae (denatured, collapsed cytoplasm) would occur after degradation had freed enough of the bound chemical from the wood cell walls. With increasing exposure to the decay fungus the preservative deposit in the walls was stripped away and fungal attack proceeded as in untreated wood.
Wilcox et al. (46) studied the action of brown rot fungi in wood treated with low levels of pentachlorophenol (PCP). The fungi were able to degrade PCP treated cell walls similarly to those of untreated wood, up to retention levels of 3%; however, the authors indicate that less shrinkage may have occurred in the decayed PCP treated wood.
Their data suggest that enzymatic degradation, to release the cell wall bound PCP, is necessary before fungal action is inhibited; but, they conclude that extracellular enzyme inhibition may also occur. 10
Schmidt et al. (35) have studied the germination of brown and white rot basidiospores on woods treated with low concentrations of chromated copper arsenate (CCA) and PCP. They found that germination was inhibited at lower concentrations of the chemicals than that which inhibits mycelial growth. Ungerminated spores on treated wood had degenerated or had become highly vacuolated.Where spores had ger- minated (at the lowest treatment concentrations used in the study--0.0001% W/W) the germ tubes were much shorter in the treated woods than on control samples.
In nucleophilic substitution reactions, chloropicrin can be attacked by any group with an unshared pair of electrons. Such groups might include the SH, OH, NH2, NH, COOH, aromatic OH and NH2 contained in all living organisms (8). Chloropicrin reacts with free thiol groups, oxidizing them to disulfide groups, and thus should react with proteins containing these groups and inhibit enzyme systems whose activity depends on the presence of free-SH groups (13). It can be concluded from this information that chloropicrin would act as a multisite inhibitor of cellular fungal activity, and would also inter- fere with the reactions of some extracellular enzymes. If a suf- ficient amount of free chloropicrin is retained by the wood it is likely that fungal growth will be inhibited before extensive coloniza- tion and enzymatic degradation can occur. Breakdown of chloropicrin may also occur in wood, and if so, some of the chloronated breakdown products, notably phosgene, if present in sufficient quantity should also inhibit fungal growth.
If appreciable amounts of chloropicrin do break down in wood and these products covalently link to the wood structure, then fungal 11 degradation of the wood cell wall may have to proceed to release the fungicidal moiety before inhibition of fungal growth occurs.However, at present it is not known if these types of reactions occur in wood, and if they do, if the products of these reactions are fungitoxic. 12
PROCEDURES
Technology is not yet available to perform a detailed chemical analysis of the individual chemical components in intact wood. In addition, many of the techniques and analytical instruments that I used in the research were designed only for elemental analysis, and I was frequently unable to distinguish between intact chloropicrin and chlorinated breakdown residues from chloropicrin. Despite these limi- tations, this work does explore the problem of chloropicrin residue retention and distribution in wood, in greater detail than previously reported and should provide a base for further research in this area.
I. Sorbtion
A. Dimensional Change Occurring in Wood Treated With Chloropicrin
Twelve Douglas-fir [Pseudotsuga menziesii (4irb.) Franco] blocks,
10 x 10 x 150 mm long were exposed to Poria placenta for 2 to 18 weeks in modified soil block chambers (Appendix I; personal communication,
T. C. Scheffer, 1982). After equilibrating the decayed blocks to about 10% moisture content, weight loss values were calculated. The blocks were grouped into three weight loss classes designated as: low, 1-8% weight loss; moderate, 8-16% weight loss; and high 16-30% weight loss. Three blocks from each group were placed in a dessicator containing a saturated atmosphere of chloropicrin; the remaining blocks were left untreated as controls. Periodically, over a 28 day period, the blocks were removed from the dessicator and measured across their tangential face to determine dimensional changes. The 13 dimensional change of the control blocks was also measured and the percentage change of the controls was subtracted from that of the treated blocks. An analysis of variance test was used to determine if significant dimensional change had occurred due to the adsorbtion of chloropicrin.
Be Non-Steady-State Desorbtion and Residue Analysis
Cooper (10) reported on the non-steady state diffusion of chloro- picrin from wood. The purpose of my work in this study was to extend the test period for desorbtion beyond that indicated in Cooper's work in an attempt to determine if total desorbtion could be achieved under ambient laboratory conditions in wafers that were about one tracheid length in thickness.
Douglas-fir wafers, 45 x 90 x 5 mm thick were cut to expose the end grain along the broadest surface. Four sets of ten wafers were equilibrated in an Amincol controlled environment chamber set at 22°C and 50% R.R. The air flow rate in the chamber was 4.2 m3/minute.
Following equilibration, two sets of wafers (designated set At and Bt) were placed above a liquid reservoir of chloropicrin and sealed in a dessicator within the Amine° chamber. The remaining two sets of wafers (control sets designated Ac and Bc) were sealed in a dessicator containing no chloropicrin. Weights of the wafer sets were taken periodically by briefly removing them from the dessicators and weighing on a balance sealed within the Aminco chamber. After 5,767 hours the treated wafers were removed from the dessicators and allowed
lAminco-Aire. American Instrument Company. Silver Spring, MD. 14
to desorb chloropicrin. The control wafers were removed from their
dessicator 267 hours after the treated wafers when the amount of
chloropicrin being desorbed was low enough that accumulation within
the chamber atmosphere would not appreciably affect the weight of the
control wafers. Desorbtion was continued for 3,191 hours with
periodic weighings of each wafer set. The percent weight change of
the control wafers, due to moisture fluctuations, was subtracted from
the treated wafers and the percent weight change of these wafers
calculated based on their original untreated weight.
Following the desorbtion weighings, one control and one treated wafer set (sets A) were placed in a 115°C oven for 5 hours. The other
two wafer sets (sets B) were extracted with acetone (3-1 hour changes, under 150 torr vacuum). All wafers were then reequilibrated to 22°C
and 50% relative humidity and their weights taken for determination of residue retention in the treated wafers.
Samples of the acetone extracts were analyzed by thin layer chro- matography on silica gel plates sequentially developed in 96:4,
chloroform/methanol, and 50:40:10, chloroform/ethyl acetate/formic
acid solvent systems. Chlorinated residues on the TLC plates were
detected with a silver nitrate bromophenol-blue spray reagent (40).
II. Neutron Activation Analysis
Methodology - All analyses were carried out in a TRIGA2 atomic research reactor located on the Oregon State University campus. Eight
2TRIGA Reactor Division, General Atomic Company, P.O. Box 81608, San Diego, CA 92138. 15
samples could be analyzed along with a standard solution of ammonium
chloride in each reactor run. All samples were sealed in high density
polyethylene vials and were irradiated in the reactor's rotating rack.
Initial experiments were undertaken to determine the appropriate flux,
activation time, and decay time needed for analysis of chlorine in 1 g wood samples. The following conditions were found to be suitable for
the analysis and were used throughout the NAA experiments:
n ) Reactor power - 100 KW (neutron flux approximately 3 x 1011 cm2 - sec Activation time - 15 minutes
Decay time - 45 to 160 minutes
Samples were counted on a multichannel pulse height analyzer with
a lithium-drifted germanium detector. A low level discriminator was
normally used to suppress the 0.845 and 1.81 MeV peaks of 56Mn and
other lower energy peaks which may have been present in the spectra,
allowing the 2.167 MeV peak of chlorine to be easily analyzed.
Live-count time for all samples was 200 seconds. Standard statistical
analysis of the data was done on a WANG 500 computer using program
4165 provided by the Radiation Chemistry Laboratory, OSU.3
In recent years activation analysis has been used in the field of
Forest Products to perform basic elemental analyses of wood (47, 30,
26) and charcoals (12), and for determination of chlorine in spent
bleaching pulp liquors (22). A good description of activation analy-
sis and gamma ray spectroscopy may be found in Chapters 7 and 17 in
the text of Wang et al. (41).
3R. Schmitt, project leader. 16
Preliminary Analysis of Background Chlorine in Wood
At the time this analysis was initiated (July 1980) accurate analyses for chlorine levels present in Douglas-fir wood were not available. Since that time, however, Cutter et al. (12) have reported finding of 67 ppm Cl in Douglas-fir wood. This amount was an average from an analysis of two replicate samples split from a 1-cm cube of
Douglas-fir (personal communication, Bruce E. Cutter, 8/10/80).
The purpose of my analysis was to determine if the amount of naturally occurring chlorine in Douglas-fir wood would interfere with my analysis of chloropicrin contributedresidues.
Seven 9 x 9 x 25 mm wood blocks were cut from random locations along a Douglas-fir board which was large enough to provide adequate test specimens for future NAA experiments. The blade of the saw, table and fence were wiped down with water, 95% ethanol, and acetone before cutting, and all samples were handled with clean polyethylene gloves to prevent chlorine contamination of the samples. The samples were sealed in clean polyethylene vials and theanalysis was carried out as described in the previous section.
Analysis of Treated Samples
1. Sorbtion Samples
Drill shavings from the heat treated or acetone extracted wafers
(from part I.B.) were analyzed by NAA as previously described. 17
2. Non-decayed Wood
Wood blocks - Thirty 9 x 9 x 150 mm long blocks of Douglas-fir, cut with the long axis oriented with the wood grain, were equilibrated at 22°C and 50% R.H. until a moisture content of approximately 11% was attained. One end of each block was sealed with paraffin film and paraffin wax, and a 5 mm diameter, 7 mm deep injection hole was bored into one side, 15 mm from the sealed end of each block. Each hole was then sealed with paraffin film and paraffin wax. Fifty pl of chloro- picrin was injected through the film into each hole and the hole then resealed. The samples were placed in polyethylene bags and incubated at ambient laboratory temperatures (21-23°C) within an exhaust hood for 3 weeks. A 25 mm segment was then cut 55 mm from the coated block end, immediately heat sealed in a high density polyethylene vial, and analyzed 4 as previously described.
Following analysis the samples were stored to allow the radioac- tivity to decay to background levels. All of the samples were then removed from their vials. Ten of the samples were allowed to aerate under ambient conditions within an exhaust hood for 24 hours. The remaining samples were ground in a Wiley mill (washed with water, alcohol, and acetone prior to grinding each sample) through #20 mesh screening and also aerated for I day.
The unground samples were sequentially reanalyzed after addi- tional aeration periods of 72 hours and 360 hours. Four of these
4Due to difficulty in scheduling reactor time the samples would remain sealed within the vials from one day to two weeks before analysis could be performed. 18 samples were then evacuated under a 10 torr (29.5 inches of mercury) vacuum for 24 hours and reanalyzed. Two of the samples were minced with a razor blade and aerated for 3 hours, before NAA.
Ground wood - Because of the chlorine contamination of the ground wood reported in the previous section, procedures had to be altered so that contaminant chlorine could be accounted for in the analysis.
Approximately 300 g of Douglas-fir was ground in a Wiley mill
(washed as previously described) through a #20-mesh screen.This wood was then thoroughly mixed to distributecontaminantmaterial evenly throughout, and four 0.5 g samples were taken for NAA. The average ppm chlorine in these samples was 36 with a standard deviation of 5.
The remainder of the ground wood was treated with chloropicrin at a rate of 50ulchloropicrin/g wood and then mixed in a sealed con- tainer for one hour. Four 0.5 g samples were then sealed in polyethy- lene vials for later NAA. The treated ground wood was aerated in an exhaust hood and occasionally mixed. Four replicate samples were then taken at 24 and 1,176 hours, and additional samples were taken after
480 and 816 hours after treatment.
Similarly treated ground wood was sampled immediately after mixing and after 24, 96, 480, and 1176 hours of aeration. Four dupli- cate samples were analyzed either by neutron activation or by a modi- fication of a closed-tube bioassay (34) using approximately 0.5 g of the ground wood rather than an increment core.
3. Decayed Samples
Weight loss vs. chlorine retention. - Forty eight 9 x 9 x 150 mm long blocks of Douglas-fir were decayed to a range of weight loss from 19
0-33% in decay chambers--1 liter canning jars containing malt agar-- inoculated with Poria carbonica. The blocks were supported above the agar by plastic screening and glass rods in an attempt to limit transfer of soluble material from the agar to the wood.After incuba- tion, blocks were equilibrated to approximately 11% moisture content.
Treatment, incubation, and preparation of the blocks for NAA were as described previously in this section. The average chlorine content of four control samples (decayed but not treated) was subtracted from the treated samples to correct for chlorine contributed to the samples by the medium or the fungus. Percent weight loss in each sample was regressed against the total ug of chlorine in each after treatment.
Alkali solubility vs. chlorine retention. - After the radioac- tivity had decayed to background levels, samples were ground through
#20 mesh screening and the percentage of alkali soluble material in each (1)5 determined. The percentage of alkali soluble material in the four control samples was regressed against their total chlorine content and this regression factor was used to correct for the amount of chlorine present in the treated samples. Percent alkali soluble material in each treated sample was regressed against the corrected ug of chlorine retained.
III. Energy Dispersive X-ray (EDX) Analysis to Determine the Microdistribution of Chlorinated Residues
Three 9 x 9 x 150 mm long Douglas-fir blocks were treated by sub- merging one end of the blocks in liquid chloropicrin for a 2 week
5Procedures suggest using duplicate 2 g samples but because of limited sample quantities, I assayed smaller amounts without replication. 20 period. The blocks were then razor sectioned to expose transverse surfaces and split to expose the radial surfaces. Immediately after sectioning, the blocks were placed in a high vacuum and coated with gold and palladium for analysis of chlorine by EDX analysis under the scanning electron microscope.
Radially split and razor cross sectioned samples of treated and control wafers from section I.B., and other thin wafers of Douglas-fir heartwood that had been treated in a saturated atmosphere of chloro- picrin vapor for 24 months were also prepared and analyzed by EDX ana- lysis. Surfacing of these blocks was done both on dry blocks and on blocks which had been wet with distilled water. Coating was with car- bon rather than gold/palladium. Analysis was carried out on a
Cambridge Stereo Scan - Mark IIA scanning electron microscope6 with an
EDAX7 X-ray analyzer.
IV. Infrared and Mass Spectrographic Analysis
A. Analysis of Chloropicrin and Derivitized Wood Components
Derivitives or synthesized components representative of the monomer or polymer units of the cellulosic, lignin, and extractive fractions of wood were mixed with chloropicrin and analyzed to deter- mine if chemical bonding could occur during treatment.
Infrared analysis. - Individual samples (- 0.25 g) of vanillin, acetyl ferullic acid, (+)-catechin, tannic acid, cellobiose, and solka
6Kent Cambridge Scientific, Inc., Morton Grove, IL.
7EDAX International, Inc., Prairieview, IL. 21 flok were saturated with 250 pl of liquid chloropicrin and then aerated for 5 minutes. Samples of the mixtures (- 5 mg) were then mixed with potassium bromide and pelletized for infrared analysis.
Each sample was again analyzed after approximately 45 and 90 minutes of aeration. Infrared scans of chloropicrin and untreated wood deri- vatives were also made for comparison.
Mass spectroscopy. - Approximately 5 mg of each of the components listed in the previous section were individually mixed with 2 ml of chloropicrin. Gas chromatographic (GC) analysis of these mixtures in- dicated that only the vanillin/chloropicrin mixture contained volatile components other than chloropicrin that would elute through the chroma- tograph column. Therefore, this was the only sample analyzed by mass spectroscopy (MS). Vanillin is a derivative of the lignin molecule in wood, and differs only in that the propyl side chain of the lignin mon- omer has been replaced by an aldehyde group. Because of chemical simi- larity between vanillin and the lignin monomer, many chemical reactions that lignin would undergo should be adequately represented by the van- illin molecule. Chromatographic separation of the vanillin/chloropic- rin components for the MS analysis was performed with a 10 foot by 2 mm I.D. pyrex column with a 7% OV 101 liquid phase coated on a 100/120 supelcoport support phase. The instrument used for this analysis was a Finnigan model 4023 GC-MS quadrupole mass spectrometer system.8
B. Analysis of Desorbed Gases From Chloropicrin Treated Wood
Matched 25 mm long increment core segments from a southern pine laminated timber that had been treated 4 years earlier (18) were
8Finnigan Co., San Jose, CA. 22 sealed in separate vials for a closed-tube assay (34) and for analysis of desorbed vapors by GC/MS. Douglas-fir wafers, 1 mm thick x 25 m long, that had been exposed in a saturated atmosphere of chloropicrin for 7 months and then aerated for 1 to 5 hours were also analyzed similarly. Mass spectroscope conditions were the same as those cited in the previous section.
V. Microscopy of Treated Wood Samples Infected With Decay Fungi
Douglas-fir wafers 1 x 10 x 130 mm long were exposed to a saturated atmosphere of chloropicrin for 35 weeks. Five wafers were then aerated for 1 hour and were immersed for 15 minutes in sterile water. The treated wafers and 5 untreated, sterilized, control wafers were then individually implanted in 2 week old malt agar cultures of
Ganoderma applanatum (Pers. ex S.F. Gray) Pat., Madison isolate
FP-57035-5 contained in 25 x 185 mm culture tubes. Tubes were loosely sealed with cotton plugs and incubated at 26°C. All cultures con- taining treated wafers were killed, and after 50 days these wafers were reimplanted in fresh cultures. Wafers were observed periodically under the light and electron microscopes to compare differences in fungal attack of treated and control wafers (see Appendix 1 for sample preparation for microscopy). The presence and type of bore holes pro- duced by the fungi and erosion of the lumen surface were the primary characteristics used to compare differences in attack. Observations of the hyphae were also made to determine if treatment may have effected morphological changes.
Similar experiments were also performed with the fungus Poria carbonica Overh., Madison isolate FP-105585. 23
RESULTS AND DISCUSSION
I. Sorbtion
Dimensional Change Occurring in Wood Treated with Chloropicrin
No significant dimensional change (F statistic, a = .05) in the blocks treated with chloropicrin was indicated by the analysis of variance. The lack of swelling suggests that if chloropicrin does hydrogen bond to wood, or to water molecules sorbed to the wood, the bonding sites are not located where the added bulk of the chloropicrin molecule would force the wood polymer chains to separate to any signi- ficant extent. Dipole attraction between chloropicrin molecules may provide a means for retention of the chemical in void spaces, but this attraction is not strong enough to act as a bulking agent and cause swelling by overcoming the attractive forces of wood.
Non-Steady-State Desorbtion and Residue Analysis
The percent weight change of the chloropicrin treated wafers
(sets A and B) during the adsorption and desorbtion of the wafers is plotted in Figures 1 and 2, respectively.With both wafer sets a hysteresis-type curve was produced; however, the desorption curves--if extrapolated-- would appear to stabilize at weights approximately 1 to
1.5 percent greater than the original weights of the samples. This suggests that residual chloropicrin, in some form, may be retained in wood for relatively long periods of time before total desorbtion (if this occurs) is achieved. HOURS DESORBT ION 0 0 0 0 08 in tr) §
12
9
0 0 0 :01 8 § in HOURS ADSOR BT ION
Figure 1. Sorbtion (-4,-) and desorbtion (-II-) of chloropicrin from Douglas-fir wafers (A)over time. HOURS DESORBTION 0 0 0 0 0 0 0 0 0 0 § 8 CO in ? 2 c,, 8
12 _
9 _
6 -
_ - -Nr-a--
1 1 I I I 0 0 8 0 0o 8 , o § o 0 0 - CJ ?,)K) IA 1 HOURS ADSORBTION
Figure 2. Sorbtion (--) and desorbtion (- -) of chloropicrin from Douglas-fir wafers (B)over time. 26
Treated and control wafers that were heated to 115°C and then reequilibrated to 22°C and 50% relative humidity lost 2.68% and 4.387. of their original untreated weights, respectively, indicating that some residue resulting from the treatment still remained in the wood.
Treated and control wafers that were acetone extracted and then
reequilibrated lost 0.87% and 0.34% of their original weight, respec-
tively, showing that somewhat more material was removed from the
treated wafers than the controls. This finding was also supported by
the darker color of the acetone extracts from the treated wood and suggests that chloropicrin treatment could possibly be solubilizing some fraction of the wood, allowing it to be more easily extracted.
TLC of the acetone extracts from the wafers in the chloroform/methanol solvent showed that two substances (Rf values of
0.10 and 0.44) eluted from the control extract whereas only one substance (Rf value of 0.10) eluted from the treated extract. The eluted materials were not identified but both could be distinguished
from dihydroquercetin (an extractive commonly isolated from Douglas-
fir wood), which did not move in this solvent system.When the TLC plates were developed in the second solvent system (chloroform/ethyl acetate/formic acid) a substance having the same Rf value as dihydro- quercetin was eluted from the control but only a faint trace was
eluted from the treated extract. The chlorine spray detector showed
that none of the eluted materials were chlorinated; however, chlorine was indicated at the baseline of the treated extract chromatogram and
to a lesser extent at the baseline of the control chromatogram. The
data suggest that chloropicrin may be binding to one of the uniden-
tified extractives eluted from the acetone extract of the control 27 sample (Rf value of 0.44 when developed in chloroform/methanol) and probably to a large portion of the dihydroquercetin in the wood, inhibiting these extractives' mobilities in the solvent systems tried on the TLC plates.
II. Neutron Activation Studies to Determine the Amount of Chlorinated Residues Retained by Wood After Treatment
Preliminary Analysis for Background Chlorine in Wood
The average background ppm chlorine in my untreated Douglas-fir samples was 2.14 with a standard deviation of 0.8. This was low enough so that it would not interfere with later analyses.
Analysis of Treated Wood Samples
1. Sorbtion samples
The amount of chlorinated residue remaining in the chloropicrin treated and control wafers after extraction with acetone or heating to 115°C for 5 hours is shown in Table 1.The amount of chlorinated material in the control samples was slightly higher than that obtained in the untreated samples from section II.A., possibly because of chlorine contamination on the drill bit used for sampling. However, this amount of chlorine is much smaller than the approximate 1% w/w
(9355 ppm) of chlorine residue in the aerated/heat treated samples or the 0.5% w/w (4950 ppm) chlorine residue in the aerated/extracted treated samples (Table 1). The data from the heat treated samples confirms that of the weight measurements of the samples (section I.B.) in showing that a small residue is present, but further indicates that 28
Table 1. Chlorine content of control and chloropicrin treated wafers which were aerated and heated to 115°C for 5 hours or extracted with acetone.
PPM chlorine Treated Control
Heated to 115°C 9355 ± 106 22 ± 3
Extracted with acetone 4950 ± 126 21 ± 2 29 this residue is directly contributed to the wood by chloropicrinor chlorinated breakdown residues of chloropicrin. Analysis of the ace- tone extracted samples indicates that a significant amount of these residues also re-mains in the wood which should have produced a net gain in weight of the treated samples. As a net loss in weight was observed in the treated samples this offers further evidence that chloropicrin may be altering some component in the wood, making it more soluble in acetone.
2. Non-decayed wood
Wood blocks and ground wood.--The data in Tables 2 and 3 show that amounts of chlorine in excess of 50 ppm can remain in chloro- picrin treated wood after aeration and vacuum treatment. The smaller amounts retained in these samples compared to the sorbtion samples
(Table 1) reflects the different method of treatment;a single small treatment and short incubation as opposed to constant exposure to the chemical over a relatively long period. Zakharenko et al. (48) deter- mined that chloropicrin residues were retained in greater amounts after aeration of treated grain if the grain were exposed to longer fumigation periods. This suggests that the differences in residue retentions in my samples may be caused by the length of exposure to chloropicrin as well as its vapor concentration. The data do suggest, however, that even when small dosages are applied, a persistent chloronated residue can remain in the wood long after treatment.
The slow desorbtion of chloropicrin from treated wood is also demonstrated in the closed-tube assay experiment conductedover a 1176 hour desorbtion period. If it is assumed that all the chlorinated TABLE 2. Chlorine content of wood samples initially injectedwith 50aof chloropicrin.
No aeration 24 Hour aeration 72 Hour aeration 360 Hour aerationEvacuated samples Sample IF ppm Cl ppm Cl ppm Cl ppm Cl ppm Cl
1 116 ± 4 98 ± 9 110 ± 4 105 ± 4 89 ± 8 2 146 ± 5 121 ± 8 118 ± 5 119 ± 6 129 ± 10 3 147 ± 5 99 ± 8 128 ± 5 105 ± 6 121 ± 10 4 160 ± 5 125 ± 9 145 ± 6 120 ± 5 127 ± 10 5 250 ± 8 174 ± 6 240 ± 9 203 ± 7 6 603 ± 16 498 ± 14 532 ± 16 569 ± 17 7 154 ± 7 130 ± 6 134 ± 7 143 ± 6 Minced samples 8 211 ± 8 196 ± 7 211 ± 10 160 ± 6 9 289 ± 8 295 ± 10 287 ± 8 250 ± 7 270 + 16 10 95 ± 4 79 ± 5 91 ± 4 85 ± 4 93T-6
11 86 ± 3 364 ± 26 12 346 ± 9 458 ±29 13 75 ± 4 158 ±16 14 122 ± 5 218 ±21 15 142 ± 6 229 ± 11 16 569 ±16 280 ±15 17 119 ± 4 18 98 ± 4 Due to excess chlorine 19 195 ± 6 introduced by grinding 20 103 ± 5 these samples could 21 174 ± 6 not be used and were 22 545 ± 15 not analysed 23 242 ± 9 24 224 ± 9 25 117 ± 4 26 52 ± 3 27 137 ± 5 28 171 ± 6 29 85 ± 4 30 216 ± 9 31
TABLE 3. Chlorine content of ground wood treated with chloropicrin, then aerated for various periods of time.
Chlorine content of treated and untreated wood (ppm Cl) Untreated Treated wood control No 24 Hrs. 72 Hrs. 480 Hrs. 816 Hrs. 1176 Hrs. aeration aeration aeration aeration aerationaeration
36± 3 14,800± 79± 3 77± 4 64± 2 73± 3 57± 3 1,900
27± 3 7,800 ± 85± 4 88± 5 76± 2 74± 3 67± 4 1,000
37± 3 7,450± 84± 4 84± 5 72± 3 70± 4 1,000
44± 4 7,350 ± 83± 4 75± 5 __ __ 72± 3 1,000 32 material volatilized from the wood is in the form of chloropicrin, in the last sampling period of 480 to 1176 hours (Table 4) an average of approximately 1 ppm of chloropicrin was desorbed from the 0.5 g samples into the 6 ml void space of the closed-tubes. If the desorb- tion rate over this period was constant, a concentration of 0.003 pg/ml of chloropicrin in the first 24 hours of incubation would have been present in the closed tubes. This concentration is apparently capable of inhibiting the growth of P. carbonica but it is very low compared to the 0.06 pg/ml minimum lethal dosages of chloropicrin determined previously for other decay fungi over a 24 hour period
(17). This assay was not continued long enough to determine if a chlorinated residue would be left in the wood when desorbtion was low enough to allow growth of the fungus in the closed-tube assay.
3. Decayed samples
The amount of chlorine (3 moles per mole of chloropicrin) retained in decayed and nondecayed blocks treated with chloropicrin is regressed against weight loss of the samples in Figure 3, and against the percentage of alkali soluble material in Figure 4. A weak negative correlation (R2 = -0.56) exists in the regression against weight loss indicating that with increasing decay, less chlorine is retained by the samples. A similar and somewhat stronger correlation (R2 =
-0.62) is apparent in the regression against percentage of alkali soluble material.
The three data points in these plots that appear to skew the regressions (> 100 pg chlorine) are from non-decayed samples that were analyzed in separate reactor runs from the decayed samples. 33
TABLE 4. Comparison of the chloropicrin content of treated ground wood after aeration with closed-tube assay data of the same wood.
% Growthl ppm Chloropicrin Not activated Activated2 in samples
No aeration 0 0 79-528
24 hour aeration 0 0 18-21
96 hour aeration 0 2 13-19
480 hour aeration 0 10-13
1176 hour aeration 0 8-13
1% growth calculated by dividing the totalmm of growth in the 4 treated tubes by that in 4 control tubes containing either activated or unactivated, untreated ground wood.
2Activated samples analyzed byneutron activation analysis prior to closed-tube analysis. :510. Ul Ul
LLI
4-
Li Li Y 0,1 0.10X
Figure 3. Chlorine retention in samples decayed to arange of weight losses by Poria carbonica prior to treatment with chloropicrin. # 44' 4 4.
4. t 0 . 4.
tS1 Fs, IS1 1St LA -0.0MX
LI L FR I NI E..:
Figure 4. Chlorine retention in samples decayed by Poria carbonica priorto treatment with chloropic- rin. Amount of decay in the samples determined by the percentage of alkalisoluble material. 36
Regressing the amount of chlorine against the percent alkali soluble matter in the samples without these data points (Figure 5) yields a significant regression (F-statistic = 14.7, F-significant a.05 = 4.2) with an R2 value of -0.57.
These data show a trend that generally conforms to the findings of researchers studying water sorbtion in brown rotted wood (11, 36), and suggest that hydrogen bonds may be important in retaining chemi- cally unaltered chloropicrin in wood. However, the data could also be interpreted to support the hypothesis that brown rot fungi alter, or selectively remove some wood component to which chloropicrin or its breakdown products may chemically link.
Both weight loss and alkali solubility were used as measures of the amount of fungal degradation present in the samples. Less chlori- nated residues were retained in samples with greater amounts of decay; however, a stronger correlation between decay and chlorine retention was apparent when the alkali solubility test was used to estimate de- cay. The alkali solubility data were generated from the same samples analyzed for chlorine content; these samples were cut from the larger blocks used for determining weight loss. Since the amount of decay can vary considerably within a sample of decayed wood, alkali solubility data should give a more accurate measure of decay in the samples ana- lyzed for chlorine content in this study than the weight loss data.
III. Energy Dispersive X-ray (EDX) Analysis to Determine the Micro- distribution of Chlorinated Residues
Representative micrographs and EDX spectra of the wood blocks treated with liquid chloropicrin for 2 weeks (Figures 6 and 7) show szs. ra
4.
4. 4. 4. 1-a-ra. sa 4.
4.
4. 4.
tsa 141 -0. MX
UE5C L. El IR I
Figure 5. Same regression as shown in Figure 4 minus three data points (> 100pg chlorine). 38
Scanning electron micrographsof Douglas-fir heartwood Figure 6. Above, treated for two weekswith liquid chloropicrin. section, split radial surface,500X; below, razored cross 500X. 39
27SEC 1577.2INf VS:1000,HS: 50EV/CH
05 -4 / 481 111 ED AX
Figure 7. Typical energy dispersive X-ray analysis scan across chloropicrin-treated wood showing the absence of chlorona- ted material in the sample. Peaks for gold and palladium from the coating material are present in the scan. 40 thatany chlorine present in the samples was below the detection limit of te instrumentation. EDX spectral scans included analyses of both lume surfaces and interior cell wall material.
Spectral scans for chlorine in wood samples taken from untreated
(conrol wafers) that had been acetone extracted or heated (section
I.B. showed that no detectable chlorine was present (Figure 8). This was donfirmed by a line scan across one cell wall in a heated, control wafer (Figure 9) which showed only the presence of background chlorine. Spectral scans for chlorine in eXttacted or heated, chlo opicrin7treated wafers (Figure 10) Showed that a significant amou t of chlorine was present in the samples confirming NAA data
(Tab e 1). Line scans along the cross sectional surface of treated samp es that were sectioned dry (Figure 11) and after wetting (Figure
12) how the presence of chlorine in the cell walls. Since wet sec- tion d wood produced a better surface for microscopy and this method did ot appear to affect retention of chlorine residues in the wood, it w s used for preparing the remainder of the treated samples. Scans acro s cell walls of treated, extracted or heated samples (Figure 13) show d that the entire cell wall contained detectable amounts of chlo inated materials but that concentration of chlorine tended to be grea er in the middle lamella region. Since the middle lamella region is c mposed mostly of lignin, the data suggest that chioropicrin resi- dues may be binding preferentially to lignin materials.
Line scans across ray tissue in chloropicrin treated, extracted
(Fig res 14 and 15) and heated (Figure 16) samples were also made.
All cans indicate that greater amounts of chlorinated materials may be p esent in the ray tissues. A spectral scan through a section of 41
I_ CL. C
14%; 5;9cpIs4 T a.:1 0 2 4 I-1 :I. 0 I< E: V :I. t C-1 :I. 0 1.sz: t.) CI
..""1494N444..tese."11;ef4er.A41..yr./.44,
o Ok 1E7: V X 1E: Ei :I. 0 4 N.; El: keP
Figure 8. Representative energy dispersive X-ray spectral scan across acetone extracted or heated control wafers. No detectable chlorine peak is present. 42
X16.08 764,
Figure 9. Energy dispersive X-ray analysis line scan across one cell wall of an untreated, control wafer. No detectable amounts of chlorine are present. 43
(L. .L.
PR 2006 2 0 0 f3 1E: (2: 0:I:Nur
c'=i.024 I--I 1 0 Is:V:I. t 3cI CA := C> FE V :I.C:st
4L
eix1
41,
I , 11 Ili . 'OAI. .
1 .-44,464tivitaiv )I )) ,) '4****1444`AI*44,49,,,,,...0.4..ms,
X 1E: :I.
Figure 10. Representative energy dispersive X-ray spectral scan across extracted or heated, chloropicrin-treated wafers. 44
Figure 11. Energy dispersive X-ray analysis line scan across a cell wall of a heated, chloropicrin-treated sample that was sectioned dry. 45
%Os 140 '1111 IDA%
woe- griptiftwa ineriassirimeAir,,,P11.11, *Mi 4111e0 11114, 't Att..
-X430 -766 03153
Figure 12. Energy dispersive X-ray analysis line scan across a cell wall of a heated, chloropicrin-treated sample that was sectioned wet. 46
47' F
I
LEL I _ Item,' 414 11I.1 'Veapt ..,Ef.,.1171"14 11
4
20KU---X1700 10U 767 03153RHIN
Figure 13. Representative energy dispersive Xray linescan across acetone extracted or heated, chloropicrintreated wood. 47
X 769 83153 RHS
Figure 14. Energy dispersive X-ray line scan across ray cell walls, tracheid cell walls, and a resin canal epithelial cell with included deposits, in extracted, chloropicrin-treated wood. 48
I
Figure 15. Energy dispersive X-ray line scan across cell wall mater- ial and extractive materials deposited in the lumens of rays in extracted, chloropicrin-treated wood. 49
yr
I
-10U768 03153 RHS
X-ray line scan acrossfusiform ray in Figure 16. Energy dispersive heated, chloropicrin-treatedwood. 50 ray parenchyma cell walls (Figure 14) indicates that large amounts of chlorinated materials are present in these walls and also apparently in the extractive layer which coats the interior of these walls
(Figure 15). Chlorinated materials within the cell walls of tracheids and deposits in the epithelial cells of resin ducts were not generally observed to be as highly chlorinated as ray parenchyma cell walls and extractive deposits (Figure 16).
These data indicate that chloropicrin or chlorinated breakdown products may selectively bind to lignin and to extractive material.
Since lignin is a complex phenolic material and many extractives are known to be phenolic in nature, chloropicrin residues may be binding to phenolic compounds.
The fact that no detectable chlorine residues were found in liquid chloropicrin-treated wood is puzzling. There are two possible explanations for the difference in residues retained in the liquid and vapor treated samples. (i) Vapor phase oxidation of chloropicrin may be required to form reactive degradation products which can then bind to the wood; liquid phase treatment would exclude most oxygen to limit this reaction. (ii) Since the vapor phase treatment continued for a much longer period of time, binding of chloropicrin to wood may be a time dependent phenomenon.
IV. Infrared and Mass Spectrographic Analysis
A. Analysis of Chloropicrin and Derivitized Wood Components
Infrared analysis.--Representative infrared scans of chloropicrin
(Figure 29, Appendix III) and the derived or synthesized compounds 51 from wood (Figures 30-35, Appendix III) were compared to scans of these same derivatives saturated with chloropicrin and allowed to aerate for 5 minutes (Figures 36-41, Appendix III). The major peaks for chloropicrin appeared to be superimposed over some of the treated derivative spectra and no new peaks were apparent. After 90 minutes no trace of chloropicrin could be detected in any of the spectra.
Absorbtion band shifts in the spectra in the range of 3200-3600 cm-1 could indicate that hydrogen bonding between chloropicrin and the hydroxyl groups on the derivative compounds was occurring, but no dif- ferences were noted in any sample when comparing untreated and treated spectra. Since a small percentage of each compound could be reacting with chloropicrin and the spectra of the reaction compound masked by that of the parent materials, this analysis indicates only that most of the derivative compounds tested remain unreacted with chloropicrin.
Mass spectroscopy.--Two small peaks in addition to those of chloropicrin and vanillin were present in the GC analysis of the reac- tion mixture of these two materials (Figure 17). These peaks were approximately 0.1% of the area of the chloropicrin peak. When unreacted chloropicrin and vanillin were injected simultaneously into the GC/MS only the peaks for the parent compounds were present, indi- cating that the reaction products were not artifacts produced in the injection port of the GC.
Mass spectral analysis of the first unidentified peak in Figure 17
(retention time = 11.07 m) yielded masses which corresponded to the isotopic fragments of the vanillin ion (average mie = 152), and also of a parent ion with a major isotopic peak mass of 197. Subtracting these two ion masses yields a mass of 152. Figure 17. Gas chromatograph of a reacted mixture of chloropicrin (retentiontime = 0.52 m) and vanillin (retention time = 5.60 m). Reaction products present at retention times of 11.07m and 17.99 m. 53
Taking into account the different isotopic masses present in the spectrum for both vanillin ion and the parent ion, and also the com- position of the parent chemicals injected into the GC/MS, the probable identity for a fragment with a mass of 45 would be an NO2 ion.Analy- sis of the second unidentified peak in Figure 17 (retention time =
17.99 m) yielded fragment masses corresponding to the masses of CC13+
(average m/e = 119), and to the vanillin molecule which has probably lost a proton reducing its major isotopic peak mass to 151 (Figure
19). A parent ion mass (m/e = 270) which corresponds to the sum of the masses of vanillin (minus a proton) and CC13+ is also present in the spectrum shown in Figure 19. In addition the isotopic fragment masses present about the 119 m/e peak and the 270 m/e peak in this spectrum are characteristic for the isotopes present in chlorinated materials. Breakdown of chloropicrin into NO2 and CC13+ transition states has been proposed to explain the mechanism for chloropicrin breakdown to phosgene and nitrosyl chloride (27), but experimental evidence of the existence of these products is lacking.My data indi- cate that a small portion of chloropicrin was broken down in the reac- tion mixture, and the breakdown products, NO2 and CC131- were cova- lently linked to the vanillin molecule at some point which was not determined in this study. Further, this analysis suggests a possible mechanism for the covalent attachment of chloropicrin breakdown resi- dues to wood.
As the IR analysis did not detect the reaction products of vanillin and chloropicrin, it is possible that some reaction with the other wood derivatives and chloropicrin may have occurred but the IR sensitivity was not great enough to detect the reaction products. HASS SPECTBUH 01/14/83 14:58:00 + DATA: BEACTIOHVAH 166 BASE 11/E:197 2:12 CALI: FC431' 05 RIC: 9680. SAHPLE: CHLOBOPICBIN OV101 185CISOT 165 TO 167 - N63 TO 064 - 874 TO 1/76 X1.00 100.0 197
50.0
79
65 109 93 122
H/E 59 100 150
Figure 18. Mass spectrograph of chloropicrin/vanillin reactionproduct at 5.60 m (Figure 17). Parent ion mass = 197. BASS SPECTBOH 01/14/83 13:18:00 + 12:32 DATA: VANPIC 0376 BASE (l/E:151 CALI: FC43P 05 RIC: SAINTE: COLODOPICRIN OV101 185C ISOT 251648. ENHANCED IS 15B 211 OTI 100.0 - 151 ,-- 37440.
50.0-
77 9 270 63 ss 233 105
---111 t1/E 50
Figure 19. Mass spectrograph of chloropicrin/vanillinreaction product at 11.07 m (Figure 17). Parent ion mass = 270. 56
B. Analysis of Desorbed Gases from Chloropicrin Treated Wood
Only one peak was present in the vapor samples analyzed in the
GC/MS. MS analysis indicated that this component was CC13+ (Figure
20). This ion does not exist in the natural state, chloroform CC13H being its most probable stable chemical relative. Chloroform has a different retention time from chloropicrin on the GC suggesting that chloropicrin was being broken down in the MS to produce the two fragments CC131- and NO2. Having no charge, NO2 would not be detected in this spectrograph and CC13+ would be the only ion apparent. When pure chloropicrin was injected directly into the MS without chroma- tographic separation, only the CC13+ ion was apparent, offering further confirmation that chloropicrin (and not a breakdown product) was the chemical present in the vapor samples.
The volatile components which would be produced in the oxidative degradation of chloropicrin [phosgene and nitrosyl chloride (27, 3)], if formed in wood, should rapidly react with hydroxyl sites or pheno- lic materials to produce alkyl carbonates or nitrosation products, respectively (28), releasing free chlorine. Chlorine would also react with wood components or cations in solution. If these reactions do occur, no significant amount of volatile material other than chloro- picrin would be detected in treated wood.
To determine if these breakdown products could attack wood derivi- tives, a preliminary experiment was performed where chloropicrin in vapor-phase was decomposed by heat, using procedures similar to that of Steacie et al. (39), and reacted with vanillin. The products iso- lated could not be positively identified from GC/MS data; however, no HASS SPECTRUH DATA: VANPIC 1307 BASE 11/E: 63 01/14/83 13:18:00 + 10:14 CALI: FC43P 05 RUC: 1214460. SAHPLE: CHLOROPICRIN OV101 I85C ISOT EHHANCED IS 15B 2N OT) 100.0- 63 - 146432.
17
58 50.0-
82
70 1 98
ti j 9'21' . 15? , , 011. ?51 21 .2fq , .340. , WE 50 100 150 250
Figure 20. Mass spectrograph of chloropicrin vapor from treated wood showing only themass fragment for CC134- (ion mass = 117, 118 and 119), and smallermass fragments. 58 product was chlorinated and the simple alkyl carbonates or ring nitro- sation products that would be expected from the reaction of phosgene or nitrosyl chloride with vanillin were not present. The large number of non-chlorinated compounds isolated suggests that heating of vanillin may have produced many of these products, and does not explain how chlorinated residues from chloropicrin may bind to wood.
Since many of the breakdown residues were probably not volatile enough to be analyzed by GC/MS, additional research should be done on this problem to determine if significant amounts of the oxidative degrada- tion products of chloropicrin are produced in wood and if these pro- ducts react with wood derivitives.
V. Microscopy
Periodic light microscopic observations of chloropicrin treated and control wafers showed that fungal attack did not occur until after
3 months incubation. This may have coincided with a reduction in moisture content of the wafers to a range favoring fungal attack.
In wafers attacked by G. applanatum, typical white rot bore holes were produced in the earlywood of both control and treated wafers at locations from 10 mm above, to depths up to 25 mm below the agar sur- face. Perforation type bore holes (Figure 21), similar to those previously reported (25, 29), were also present in this same region of the earlywood and in the latewood of both control and treated wafers.
Some erosion of the interior cell walls was also apparent, especially in the latewood (Figure 22).
Both perforation type and typical bore holes were observed in the treated and control wafers attacked by Poria carbonica (Figure 23). 59
Figure 21. Perforation type bore holes produced by the fungus Ganoderma applanatum in the cell walls of Douglas-fir. 60
I
Figure 22. Erosion produced by the action of Ganoderma applanatum on the lumen surface of Douglas-fir cell walls. 61
Figure 23. Perforation type and typical bore holes produced by the fungus Poria carbonica in the cell walls of Douglasfir. 62
Bore holes were present in some samples from 5 mm above, to 20 mm below the agar surface. The predominant attack, as evidenced by numbers of bore holes, occurred in the range of 1-5 mm below the agar surface.
For both fungi no differences in the amount or severity of attack--as evidenced by bore hole formation and erosion--were observed between control and treated wafers.
Observations of the cytoplasm in the cells of G. applanatum showed that vacuolation or lysis of the fungal cells, similar to that reported by Bravery et al. (6) in preservative-treated wood, was much more prominent in general in the treated samples (Figures 24 and 25) than in control samples (Figure 26). Extensive vacuolation of P. carbonica hyphae in treated wood was not observed although some lysis of cell walls was apparent (Figure 27) and contrasted with the unaf- fected hyphae present in control samples (Figure 28).
The attack of chloropicrin treated wood by the two decay fungi indicates that the treatment cannot be depended on to protect wood under extreme decay hazard conditions. The apparent disruption of the fungal protoplasm in the treated wood of at least the white rot spe- cies suggests that treatment residues in wood do have an inhibitory effect on the fungi and may possibly limit fungal attack of the wood under less severe decay exposure conditions. The differences observed in the disruption of the cellular material of the two fungi suggests that P. carbonica may be more resistant to the treatment than G. applanatum, possibly reflecting differences in the wood components utilized by the fungi and those to which chloropicrin or its breakdown residues may be bonding, or it may simply indicate the variability of residue retention in the samples. 63
1.45.
, :r 4/...Arn .1r' V1.V. °lc 1/4 ), ',":7r-.. st" :,.
, 1 : - A ,mi.,.....,t,2.7.,, ' e ' A 414 ,
...... 4 e,.....- .) . It, -i, . ...- ,. ,.... ,-... ..; O.' -. ..,:- ;.. . .0 i ., ..d.r - . ...,.. , , t4'14, ' e .
).41*
Figure 24. Vacuolated and lysed hyphae of Ganoderma applanatum in chloropicrin-treated Douglas-fir wood. 4000X. 64
Figure 25. Vacuolated hyphae of Ganoderma applanatum withclamp con- nection in chloropicrin treated Douglas-firwood. 7500X. 65
.11
II I
Figure 26. Normal appearing hyphae of Ganoderma applanatum inun- treated Douglas-fir. 10,600X.
1
1 66
fr
SI
Figure 27. Lysed hyphae of Poria carbonica in chloropicrin treated Douglas-fir. 7200X. 67
Figure 28. Normal appearinghyphae of Poria carbonica in Douglas-fir. 9400X. untreated 68
CONCLUSIONS
Wood does not swell when treated with chloropicrin; however,
the lower retention of chlorinated residues in wood with greater amounts of decay suggests that chloropicrin may hydrogen bond towood.
Hydrogen bonding between chloropicrin and wood (or water molecules sorbed to wood) may occur only at exterior microsites in the wood structure where the added bulk of the chloropicrin moleculewould not cause the wood lignocellulosic matrix to separate.Hydrogen bonds and van der Waals forces may play a significant role in theretention and slow release of chloropicrin as shown by the mass spectrographic analysis of volatiles from wood treated 4 years prior.
A 1-1.5% residue of chlorinated material is present in aerated treated wood. The residue cannot be removed by heating, repeated acetone extraction, or high vacuum treatment suggestingthat covalent bonds between chloropicrin breakdown products and wood maybe formed. Thin layer chromatography of acetone-extracted, treated wood also indicates that a chlorinated residue is covalently bound towood extractives.
Mass spectrograph data confirm that chloropicrin degradation products can covalently bond to phenolic wood derivitives suggesting that they could bond to phenolic wood extractives and to lignin.
Energy dispersive X-ray analysis shows that greater amountsof chlori- nated materials are found in phenolic rich sites of treated wood, offering further support that chloropicrin residues chemicallybond to lignin and phenolic extractives in wood. 69
Chloropicrin desorption studies and energy dispersive X-ray
analysis show that the amount of chlorinated residues retained by
treated samples is affected by the amount of chemical applied or the
length of contact time with the wood, or both. A time dependent
effect could be explained by the slow breakdown of chloropicrin to more reactive species which would react with wood; however, prelimi- nary studies to test the reactivity of chloropicrin degradation pro- ducts with wood derivitives were inconclusive.
Light microscopic analysis shows that chloropicrin treatment cannot be expected to protect wood from fungal attack under severe decay hazard conditions. Preliminary micromorphological analysis of
the fungi invading treated wood shows that the treatment does have an
inhibitory effect, especially on Ganoderma applanatum, as evidenced by
lysis and vacuolation of the fungal hyphae. It was not determined whether the fungi played an active role in releasing the fungitoxic moiety from the wood, or if this material was passively released into
the fungal environment. 70
SUGGESTIONS FOR FUTURE RESEARCH
This thesis uncovered many new problems which need to be examined in greater detail. Research in the following areas would help further our understanding of the microdistribution and retention of chloro- picrin in wood.
- Chemical and biological assays should be performed to
determine: (1) the time/concentration effects of chloropicrin
retention in wood, (2) the extent that extractive content of
wood affects chloropicrin retention, and (3) the fungitoxicity
of chloropicrin treated wood and its potential for use as a
long-term wood preservative after simulated leaching and
weathering treatments.
- Transmission electron microscopy studies should be done using
an electron dense stain to locate chlorine residues in the
wood ultrastructure. Fungal invasion of treated wood should
be studied to determine if enzymatic attack is necessary to
release chlorinated moieties from the wood.
- Specific mechanisms for the degradation of chloropicrin in
wood should be determined. Chlorinated residues in treated
wood, and the wood components these residues bond to should be
identified. Reactions of chloropicrin and wood derivitives in
the dark should be compared to light catalyzed reactions. 71
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Chem. Abstr. 1958. 52(4045h). APPENDICES 79
APPENDIX 1
Preparation of Samples for Light and Electron Microscopy
Light microscopy.--Wafers were soaked in 25% ethanol until soft
enough for sectioning on the sliding microtome.Ten to 20 pm thick,
50 mm long sections were made of the region to both sides of the point
of contact with the agar surface. Sections were stained with safrarin
0 and picroanaline blue according to procedures outlined by Wilcox
(44), and mounted in diluted Karo syrup.
Scanning electron microscopy.--Small sections above and below the agar were split, mounted, and air dryed for later coating under vacuum with gold/palladium.
Transmission electron microscopy.--Small 1 mm square samples were
removed above and below the agar, and were fixed and stained with 2.5% potassium permangenate for 30 minutes. Samples were imbedded in
Spurr's standard medium (38) and sectioned on the ultra microtome.
The sections were collected on copper wire grids for microscopic observation. 80
APPENDIX II
Modified Soil-Block Test
Fill incubation bottle about half full of screened soil (screening
to eliminate lumps). Use a measuring cup.
Place a circle or square (depending on shape of bottle) of filter
paper on top of the soil.
Pour onto the filter paper, evenly, sufficient water to give the
soil a moisture content of 60%, based on oven-dry weight of the
soil.9
Place the weighed test block on the filter paper (the block ordi-
narily is dry), close the bottle with the lid loosely turned down,
and autoclave about 15 minutes at 15 or 20 pounds of steam.
Allow the bottles to thoroughly cool, then inoculate the filter
paper.
Allow up to 3 weeks for the fungus to grow on the filter paper.
Shorter period is OK if the paper is covered earlier.
Incubate at 80°F for 12 weeks (or for a shorter period with small
blocks). The 12-week period is standard for 3/4 inch cubes.
9The water at step 3 should contain in solution enough malt extract to provide 5% extract by weight of soil (extract and soil propor- tions on dry-weight basis).
The oven-dry weight of soil in a bottle (on which the weight of water and malt extract to added is calculated) is the average deter- mination for 3 or 4 bottles which are extra for the purpose. (If the soil is measured into the bottles it can be assumed that the amounts in the test bottles are essentially the same as those in these extra bottles). APPENDIX III
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Figure 30. Infrared spectral scan of vanillin (in potassium bromide). 03 2.5 WAVE11114G111 114 111101014 3 4.3 S 5.6 6 6 6 7 5 II 10 II 12 7.5 4 16 IS 20 25 30 40 III I III Ilk I 11111111111111111 11111111111111111111111111 iii 111111111illiiililii Ilillii11111111111111i111, 1111111111111N III IliIIIIIIii11111111111111111111 Pa 111111111d' Jill JillI Ii11111111IIIIII IIII 1 [...ill 100 ,o ill'll 1 11 fir III 11111111111111'iliiiiIM.1111111111iiiiiiliiiiiiii10111111111'illill 111011111111111111111111111111111111111111 111111111111111101111,111111111!11101H1111111101111111,1111 /0 II i1 111ll IIllIl111[1 illumitilii,iiiiiiiiiitioniilluittilliiiiilitillii ictillill 111111,111,11,11111ilimilil'ill1111111,1115:1111,vimmilillmirillIllifiiiiiiillillium,or 60 11 Aricao1111;iiiiilinilitilimililiiilloilimiumirtiliiiim [111114 JIIIIII !,,illiiiiiii, iicitiiiii 11 1 I 1111111111111111111111111111111111111111111111111111B111111111111111111111111 '11111111 111 11111 lililli 1 III11111:1!!11111111111111111R1111111111111111111111111H11111111111111111111111111111 1111 5:
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Figure 31.Infrared spectral scan of acetyl ferullic acid (in potassium bromide). WAVElINGIN0021111010114 3.5 3 5 5.5 4 45. .$ . 1 14 16 II 20 25 30 40 I II 100 IF III 1 -- 1$Illill 171 iiiIIIIIIIIIIIIIIii6 121 1., Lit i 1W
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Figure 32.Infrared spectral scan of d-catechin (in potassium bromide). CO WAV8l1240121 II MICRONS 2.5 3 3.3 4 4.3 3 3.5 .
11311111II II71111 1111111111'i 100 100
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Figure 33.Infrared spectral scan of tannic acid (in potassium bromide). 03 ..vntwAnwoo 2.5 3.3 1 4.5 3 3.3 _.AS 74 . , 17 14 16 III20 25 30 40 741 100 I 1 1 111, 1111111111111111111111111 11 1 l' IR 1E1E1111 1 Trli'''''1:1Thrl:f'p; , 90 1 i1-111-1111111 INEr11111111111-111110M111'II -1°° 80 .. 111114r/iliplilli 1Jlitlifirill 1
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Figure 34. Infrared spectral scan of cellobiose (in potassium bromide). 100
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Figure 35. Infrared spectral scan of solka flok (in potassium bromide). CO 33 30 40
431 I i3 . 4 Wtr 'NG:"MCI7 100 90 31111111111111i Ii 1111436"t Iill
110 11111111M ri 11 11 IIII:111 ill 70 .. 1E1 I II Ill IIII 1111 INIIIIIIM1111111 I 1 [IllIIIII111111 INIIIII 1111M i. 4011111111 11111 ill 11111111111111 I =ME , 30 orni,...,,,... , 1.
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.ill IIIIIIII Illr IH 1111111111 I IIII 11111 . III 11111111 III III11113111 MI III II 4000 2000 11100 1600 1400 1200 1000 100 600 .0 ...... visrn e.s 1 500 400 300
Figure 36. Infrared spectral scan of vanillin reacted with chloropicrin and aerated forfive minutes (in potassium bromide). WAV1t11401/1 06 11114310140 2.5 3 15 4 to 3 .5.3 6.2 7 7 10 11 12 14 16 II 20 25 30 40 100 I 11111 11111111 i111111111111111111111,1111111 - Ur - 1 100 90 II 90 0111111 IUiIll 70 Tuairdlii
a600 '1 11111111111111111111 11=1111111111111:1
40111111a11111111111111111 Og4 30 30 11 20 11111111 1111 20
I I 10 I! 10'
4000 3000 0I 2000 11100 1600 1400 1200 1000 000 600 500 400 300
Figure 37. Infrared spectral scan of acetyl ferullic acid reacted with chloropicrinand aerated for five minutes (in potassium bromide). CX) 2.3 WA0801154o116SIMUILIN1 3.5 43 .5 45 7 /5 II 10 11 12 14 10 IS 20 23 20 40 100 Ill , 111111illoulmi III a i' li100 111111111111111111111111111111921 7,11111111111111M IISO 011111;1111111111111=1111111 II I IR70 111111111111 1IN11111111111111 1 IPIPI So 111111111111111111111111111111 a ff 14°1111E111111111111111111111 lamonnan 111--. -77-- I. iii 3d amonivi ii immumm .11 i 111111111111111111111ialli 111 ill 111 I 0 alcomilioniama , 1,i 6000 3000 ii III 1800 1 1600 1400 1200 1000 SOO 600 500 400 200
Figure 38. Infrared spectral scan of d-catechin reacted with chloropicrinand aerated for five minutes (in potassium bromide). vD 30 WAVR5146141 14 MOONS 3.3 4 4.5 5 6 66 1.3 9 10 11 0 4 0 000 0 0 100 ti chi ILI II11111 II '111III 11111 ill 111111111III 111 1 112111 I 1 1 100
90 i li 1111 II till IIIIIIII 11111 III 11111111 III 11111111111111 1111111111111111 rillail 111 11190
1 1 11 11111,1111 II HEISE 111 111 111II 11111 IM 1111111111111111111111111Ni Ill I I II 70 1 III II MN'1 111111 111 11mil iim cm1
1 nog III 111 it rim 11E1 1111E111 IIIimili1111mili111.11 11 l III 1ii I so II II 11111111 111111II 1E1 . t '1 'I EIN Illi" III
I III 111 II I 1111 III II .11 . i14° I II 1 1 ,.I 10. Illi iii111 30 INI IIIII III II II I II II 111 IIIII .' N II '11111 III I I I 11111 IIII III I . MIL II I II 1 11 1 1 Hi iiii ti ii 1 1 II 1 I1111 1ElsHI ' I111.111 ill HI 1 1111 1 III Ilirmi ii um iiiii mum" IIIII11111 ME II i,11! 4000 3000 1111111111111111111111111111110 IIIIIII111111 I 2000 1000 MO 1400 1200 000 BOO 00 500 400 300
Figure 39.Infrared spectral scan of tannic acid reacted with chloropicrin andaerated for five minutes (in potassium bromide). w600tultAn6469cimmu 3 3.3 4 43 3 3.3 6.5 7 7.3 II 10 11 9 12 14 16 11120 23 30 40 rill '.1 JIl HI III 131111 III111H1111 1.11/11i111111111111111/111li IIIIIIII[111111111111111:111111111111171111i11,111111iilliilligi'ii: HI hl II AI II IBA 400 ' k 1111 1 111't II ',114RI 90 t lb; 11211111111 11111111itill111111111111116',:ilign '111111111131111111111111.huh ' l'i -,' 90 11.1: 1. 1_011111 1111 II 111' 1111 IT111 Hill 111 j i MI 1 j 00 TI IIIl' it" 'WT. 1,11 I 11{,] I 11111111 IIIfikiIllhIiI I ' dii 1, I II I it! III I t II I' 6 I I II t i 41til I, I.111i 11 I 11 111 i I a w 111.1..11111LI.111311111iii T III hi II 3 ILI I ALL, ! 111 III g- 111111 vIji NI1111111111311111111111111 1111i41ii p IF; h,
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Figure 40.Infrared spectral scan of cellobiose reacted with chloropicrinand aerated for five minutes (in potassium bromide). k.o 2.3 W4913131010 44 IMC00113 3.3 4.3 9.5 100 H it it tillIll Hill I 100 90 ow- 90 RO Linomunassmitap
70
A 70 111111111111nallit A f" 60I 150 EIRIFINIENI MEM !01E1 1111111111111 L _ 30 1111111 1 301 20 11 IMO
.0 I111111111
11111111111 III; 9000 5000 2000 1 11 1600 1600 100 1200 SOO 00 SOO 400 300 WAVM111M11FR CM' INUM.1410 11.11.6.75 MUIV101.. CNNOODait.U MMUS MU USA.
Figure 41. Infrared spectral scan of solka flok reacted with chloropicrinand aerated for five minutes (in potassium bromide).