United States

Department of

Agriculture

Forest Service

Southern Forest

Experiment Station

New Orleans,

Louisiana

General Technical Report

so-1 01

March 1994

Symposium on Current Research

in the Chemical Sciences

Third Annual Southern Station Chemical

Sciences Meeting

Third Annual Southern Station

Chemical Sciences Meeting

SYMPOSIUM ON CURRENT RESEARCH

IN THE

CHEMICAL SCIENCES

Alexandria, Louisiana

February 7-8, 1990

Edited by Timothy G. Rials

Each contributor submitted camera-ready dopy and is responsible for the

accuracy and style of his or her paper. The statements of the contributors do

not necessarily reflect the policy of the U.S. Department of Agriculture,

Forest Service or the Southern Forest Experiment Station.

i
Preface

The original charter for this annual meeting of chemical sciences personnel called for an

informal atmosphere for the discussion of common concerns and needs. The years have seen

the definition of our “common concern” evolve into a sharing of our efforts in applying the

science of chemistry to the resolution of problems faced by our forest resource. I believe this

expanded scope is significant because, as we pursue our special interests (physiology, ecology,

utilization), it is easy to forget that the objective - to enhance the security of the concept of

multiple-use forestry - remains constant. This affords several advantages, including an

enhanced awareness of the breadth of chemical sciences research that is being conducted, as well

as an increased familiarity with project resources and individual skills that are available across

the Southern Station’s laboratories. An additional benefit to be gained through this approach can

be found in the diverse backgrounds, philosophies, and perceptions that are collectively available

to address research problems. This meeting provides an opportunity to generate new approaches

and new perspectives far removed from those applied previously.

This report contains proceedings of the Third Annual Southern Station Chemical Sciences

Meeting. The collection of papers is divided into three sections addressing forest biology, forest

protection, and forest utilization. It effectively covers the life cycle of the forest with topics

ranging from seed coat physiology to the development of new adhesives for wood composites.

This report also illustrates the far-reaching role of chemistry through the discussion of subjects

as fundamental as polyflavanoid stereochemistry and as applied as nitrogen fixation in

agroforestry systems. In short, this proceeding defines the wide range of research problems

whose solution lies somewhere in the realm of chemistry.

I would like to express my gratitude to all of the participants in this symposium, and

especially to the authors who have contributed to this effort. I hope that this has proved to be

as beneficial an exercise to all of you as it has to me. Also, there are a number of individuals

whose contributions made the meeting a success, and they deserve a special note of thanks.

They include Mr. Raymond Paul and Mr. Mike Roessler who handled dinner accommodations,

Miss Debbie Wolfe who compiled this document, and Mrs. Becky Rials who did whatever else

was required. Finally, let me express my appreciation to R.W. Hemingway whose assistance

in every aspect of organizing this meeting was invaluable. Thank you all!

Timothy G. Rials

Southern Forest Experiment Station

Pineville, Louisiana

ii
CONTENTS

Forest Biology

Integration of Ion-Selective Electrodes into a Laboratory Analysis System,

Roger L. Baker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Comparing Elemental Compositions from Seed Leachates and Seed Coat Surfaces

ofPinustaedu,J.A.Vozzo.. . . . . . . . . . . .~. . . . . . . . . . . . . . . . . . . . 4

Application of the Incubation, Drying, and Separation Method to Pinus roxburghii

Seeds, R. V. Singhand J. A. Vozzo.. . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Comparative Seed Coat Anatomy of Dormant, Stratified, and Germinated pinus

koruiensis Seeds, Zhuahua Ning, J. A. Vozzo, and M. V. Bilan . . . . . . . . . . . . . . . . . . 1 1

Subterranean Clover: Nutrients For Pines, Henry A. Pearson, Michael L. Elliott-Smith,

Ahon Martin, Jr., and Virgil C. Baldwin . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

A Method for the Detection of Trace Quantities of Ethylene in the Atmosphere,

Lewis R. Brown, Gerard S. Pabst, Jr., James L. Spencer, Jr., and Ted H. Filer, Jr. . . . . . 21

Forest Protection

Optimization of Graphite Furnace Atomic Absorption Technique for

Determination of Rubidium in Southern Pine Beetle, Dendroctonus front&,

for Mark-Recapture Studies, W.T. Thoeny, A.E. Tiarks, and M.L. Elliott-Smith . . . . . . . . 25

Chemical Stimulation of Host Tree Antihiosis for Southern Pine Beetle,

Dendroctonus frontalis Zimm., Suppression, M. C. Miller, T. G. Rials, and

D.N.Kinn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Development of Analytical Methods for Escort Herbicide in Forest Environment

Samples, Joseph B. Fischer and Jerry L. Michael . . . . . . . . . . . . . . . . . . . . . . . . . 33

Evaluation of Worker Respiratory Exposure to Herbicide Residues in Prescribed

Fire Smoke: A Preliminary Report, C. K. McMahon and P. B. Bush . . . . . . . . . . . . . . . 37

Forest Utilization

NMR Experiments for Elucidation of the Structure of Flavanoid Oligomers:

Some Examples, *Richard W. Hemingway, Jan P. Steynberg, and Seiji Ohara . . . . . . . . 42

Principles and Applications of Relaxation Spectroscopy, Timothy G. Rials . . . . . . . . . . . 49

Use of Gel Permeation Chromatography in the Development of Resin Adhesives,

Chung-Y. Hse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Condensed Tannins: Must Enzymes Control the Stereochemistry in Proiisetinidin

Biosynthesis?, Jan P. Steynberg and Richard W. Hemingway . . . . . . . . . . . . . . , . . . 58

Can Wood Extractives be Used as Wood Protectants?, C. A. McDaniel and

Billies. Dunn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Integration of Ion-Selective Electrodes

into a Laboratory Analysis System

Roger L. Baker

INTRODUCTION

Ion-selective electrodes (ISE’s) allow the specific and

quantitative determination of substances, ranging from simple

inorganic ions through amino acids to complex organic

molecules. About 30 anions can be specifically and directly

detected with these electrodes, but in addition, other ions can

be determined indirectly through chemical complexation,

precipitation, or biochemical reactions that change the

activity of the indicated ion in the solution equilibrium

(Cammann 1979).

ISE’s appear to be an alternative to expensive equipment

often used for ion analysis. The wide range of applications,

low material requirements, and simplicity of the analytical

procedures have brought ISE’s into active use in analytical

chemistry for many disciplines. When compared to the

initial and per sample costs of other methods, the ISE’s

appear to be an answer for the small, budget conscious lab.

Time-consuming sample preparation including filtrations,

distillations, and extractions can often be avoided through the

use of ISE’s. Methods of analysis by ISE’s are usually

nondestructive and adaptable to very small sample volumes.

Analysis can be made of highly colored or viscous samples

without difficulty.

The [SE’s are not without problems though. Cammann

(1979) estimated that most problems relate back to a lack of

practical know-how on the part of the user and to difficulties

with the reference electrode. In addition, direct,

interference-free measurements of desired ‘ions can be

rendered difficult due to interferences from other ions.

Ion-selective electrodes sense the activity rather than the

concentration of ions in solution. The ISE’s are

electrochemical half-cells in which a potential difference that

is dependent on the concentration or activity of a particular

ion in solution arises across the electrode/electrolyte interface

(Cammann 1979). These interfaces are regions of space that

separate phases in such a way that material transport between

the outer, contacting phase is modified or inhibited compared

to transport that would occur when phases are in direct

contact (Cammann 1979, Lakshminarayanaiah 1976).

Membranes are generally liquids or solids and are usually

thick enough that they possess an inside region and two

outer-boundary-defining surfaces (or interfaces) that separate

the membrane from the exterior phase. Each interface is a

hypothetical surface that separates the physical-chemical

properties of the membrane from the outer phases where

another set of chemical and physical properties exists.

Roger Baker is research ecologist, formerly with U.S. Department

of Agriculture, Forest Service, Southern Forest Experiment

Station, Nacogdoches, TX 7.5961.

Location of the interfaces is not absolute because important

properties such as charge density and potential distribution

vary continually from one phase to another. Membranes for

ISE’s are immiscible or at least partly so with respect to the

bathing solutions or solid contacts. Hydrophobic organic

liquids and low water-solubility organic solids constitute the

main materials of membrane construction.

Membrane ISE’s are commonly classified according to the

type of membrane. The first ISE was actually a glass pH

electrode that measured the activity of the H+ ion. A

complete description of the different types of membrane

electrodes that have been developed is available in Bailey

(1976), Cammann (1979), and Ma and Hassan (1982). The

types of ion selective electrodes that are readily available

commercially are classified as: (1) liquid membrane

electrodes, (2) sealed or solid-state electrodes, and (3)

combination electrodes.

Liquid membrane electrodes have a screw-on sensing

module that contains an inert membrane through which a

layer of liquid ion exchanger establishes contact with the

sample. The exchanger is a water-soluble salt of an

organophosphoric acid and has high specificity. A gelled salt

bridge solution connects the liquid membrane with an internal

Ag-AgCl element. This type is represented by the calcium

and divalent ion electrodes.

The solid-state or sealed electrode is the most basic type.

A sensing membrane is permanently mounted in the tip, and

an ion potential develops across that membrane. Solid-state

electrodes require a reference electrode for use.

Combination electrodes incorporate both a solid ionsensing element and a reference electrode in a single stem.

These electrodes can be used with very small samples. Gassensing combination electrodes have been developed that use

a hydrophobic gas-permeable membrane to separate sample

solution from the electrode internal solution. Dissolved gas

in the sample solution diffuses through the membrane until

the partial pressure of the gas is the same on both sides of

the membrane.

CALIBRATION

Regardless of the evaluation method being employed, the

electrode should periodically be tested by means of a

calibration curve. Normal procedures start with a 0.1 M

solution of the indicated ion and progressively dilute until no

further dilution effect can be detected by the detecting

instrument. The calibration curve is obtained by plotting the

measured electro-motive force (EMF) values on a linear

ordinate scale against concentrations on a logarithmic

abscissa scale. The lower detection limit is defined as the

measured ion concentration at which the measured signal is

exactly twice as large as the background noise (Bailey 1976,

Cammann 1979).

Standards should bracket the concentration ranges as

closely as possible. The analytical measurement can never

be obtained more accurately than the calibration standards are

known or defined. Care is required, especially when

1
7

working with standard solutions less than 10e5 M, since

highly diluted, unbuffered solutions are unstable due to

irreversible absorption processes on the container walls.

Therefore, such solutions should always be freshly prepared.

MEASUREMENT

The most important and widely used techniques for

measuring ion activity are as follows: (1) direct

indication/concentration, (2) standard addition/subtraction,

(3) Gran’s plot, (4) potentiometric titration. These are noted

in increasing order of accuracy and precision and time

reqtlired per analysis.

1. Direct indication/concentration. This is the most

straightforward technique and is the one of choice whenever

possible. It is a rapid technique enabling measurements to be

completed in just 2 to 3 minutes and is the most familiar and

simple technique for workers. Typical accuracy of

measurement is approximately f2 percent (Bailey 1976,

Cammann 1979).

2. Standard addition/subtraction. In the standard

addition method, the ISE is immersed in the sample and the

equilibrium cell potential is recorded. A known volume of

a standard solution is then added, and the new equilibrium

cell potential, the change in potential, is measured. The

initial concentration of the sample may then be calculated

from the change in potential (Bailey 1976, Orion 1986). The

known addition is useful for measuring dilute samples,

checking results of direct calibration, or measuring total

concentration of an ion in the presence of an excess

complexing agent.

3. Gran’s plot. This method locates the equivalence

points in titrations based on linear titration curves. The

volume of titrant consumed is plotted on Gran’s plot paper

against the concentration of the ion to be determined, and a

straight line is obtained. As the volume of titrant added

increases, the concentration of the ion decreases linearly to

approach zero at the equivalence of the end point (Bailey

1976, Ma and Hassan 1982, Orion 1982).

4. Potentiometrlc titration. In potentiometric titration,

the sample is titrated with suitable titrant, and the decrease

or increase in titrant activity is followed with an ISE. The

end point of the titration is assumed to be at the inflection

point of the curve. Titrimetry is seldom the preferred

manual technique. Usually, when accuracy and precision are

important, the analysis may be done more rapidly and the

end point located more precisely by Gran’s plot method, if

available. The titration does enable a large number of ions

not directly sensed by ISE to be measured (Bailey 1976, Ma

and Hassan 1982).

EXPERIENCE

The author’s approach to the use of ISE’s was one of

optimism and of naivete. As Durst (1978) states ‘I... it is

usually not until one is actually using these sensors that the

problems and pitfalls become apparent, and may result in

serious errors and considerable aggravation to the unwary.

In many cases, the problem results because the analyst has

not familiarized himself with the particular idiosyncrasies of

his electrode system...often, the users approach the

application of ion-selective electrodes with great anticipation,

assuming that since they have used the pH electrode without

difficulties, the analogous ion-selective electrodes will be

equally well behaved. This naive approach often leads to

considerable disappointment because the potential sources of

trouble are almost as varied as the sensors and the

samples...common causes of difficulty can be identified:

electrode interferences and/or “fouling” of the sensors,

sample matrix effects, reference electrode instability, and

improper calibration of the measurement system.”

The study with ISE’s began with a need for determining

sulfate, orthophosphate, nitrate, and ammonia concentrations

from samples of rainfall, throughfall, and litter leachate.

Several companies have ISE systems available (Cammann

1979). A high-quality pH/ISE meter, a printer, a reference

electrode, and the fluoride, lead, and ammonia ISE’s were

purchased. Total investment (suggested retail price) would

be about $3,700. Additional investments were made in

pipettes, tips, and disposable polypropylene beakers.

Through the use of Gran’s plot titrations, sulfate ion

concentration can be obtained using the lead electrode (titrate

with lead perchlorate), and phosphate ion concentration can

be obtained using the fluoride electrode (uses lanthanum

nitrate and sodium fluoride). Satisfactory results were not

obtained for either ion. In retrospect, the results fit our

overall knowledge concerning sample analysis with ISE’s.

Most samples contained less than 0.1 ppm total phosphorous

and 1.0 ppm total sulfur, which approach the lower detection

limits of phosphate and sulfate. At times, it was difficult to

even plot the blank and standard. Currently, the

phosphorous and sulfate analyses are run by a local soiltesting lab, but the ISE phosphate and sulfate analyses will be

attempted using soil samples with higher levels of each ion

in the samples. These analyses will be approached with a

better understanding of ISE’s and the perils of their analysis.

No major problems have slowed ammonia and nitrate

measurement. The ammonia electrode with the nitrate test

kit is used for nitrate measurement, and is less subject to

interference from other ions than the nitrate electrode. The

nitrate test kit requires a different filling solution for the

ammonia electrode. There are two ammonia electrodes, one

electrode specifically for ammonia determination and one for

nitrate determination.

An oxygen electrode is currently used to measure

dissolved oxygen in surface and ground water samples. This

electrode has been trouble-free. and it can be used in the
determination of biochemical oxygen demand from future

samples.

Many minor problems have been faced during the 2 years

of sample measurement (or attempted measurement) but have

been overcome. Good lab procedures and recordkeeping

must accompany the sample measurement. All chemicals

should be dated when received and again when opened. The

shelf life of unopened standard solutions is about 3 years, but

once opened, their shelf life would be reduced to 1 year.

One standard solution could not be calibrated correctly after

repeated tries because of the age of the solution.

Equipment is available to automate the ISE sample

measurement, although the investment for that type of system

runs from $15,000 to $20,000. This cost is approaching the

investment required for other methods of sample

measurement. However, that would be a top-quality ISE

system, and measurement of additional ions could be added

to the system for a relatively small investment.

Future plans call for ISE measurement of chloride and

aluminum (fluoride electrode). These measurements will be

approached with a better understanding of the operations of

ISE’s and an awareness of possible troubles.

LITERATURE CITED

Bailey, P. L. 1976. Analysis with ion-selective electrodes.

New York: Heyden and Son Ltd. 228 p.

Cammann, K. 1979. Working with ion-selective

electrodes. New York: Springer-Verlag. 225 p.

Durst, R.A. 1978. Sources of error in ion-selective

electrode potentiometry. In: Freiser, Henry, ed. Ionselective electrodes in analytical chemistry. New York:

Plenum Press: 311-318. Vol. 1.

Lakshminarayanaiah, N. 1976. Membrane electrodes. New

York: Academic Press. 368 p.

Ma, T.S.; Hassan, S.S.M. 1982. Organic analysis using

ion-selective electrodes. New York: Academic Press: 184

p. Vol. 1, methods.

Orion Research Incorporated. 1982. Instruction manual,

lead electrode, model 94-82. Cambridge, MA. 31 p.

Orion Research Incorporated. 1986. Instruction manual,

ammonia electrode, model 95-12. Cambridge, MA. 36 p.
Comparing Elemental Compositions

from Seed Leachates and Seed Coat

Surfaces of Pinus taeda

J. A. Vozzo

INTRODUCTION

Leachate analyses of seeds imbibed in deionized water

have shown a relationship between seed health and electrical

conductance (Bonner 1988, Bonner and Vozzo 1987). The

poorer the seed quality of southern pines, the more electrical

conductance their leachate allows in deionized water.

Although the quantitative conductance may be measured in

pmhos by any of several techniques, there is no clear

qualitative analyses of the leachate. Additionally, Vozzo and

Song (in press) have proposed that a structural failure or

trauma of cell membranes in stressed seeds allows more than

usual active and passive translocation, thereby contributing,

to additional leachates and a higher conductance from poor

seeds in water. Data here present a view of associating

electrical conductance from leachate with leachate analyses

by atomic absorption and seed coat elemental analyses using

energy dispersive spectroscopy (EDS).

MATERIALS AND METHODS

Seed samples of loblolly pine were collected and selected

for a representative of a poor seed source and a good seed

source. Individual seeds were randomly selected from each

source to provide replications of each analytical technique.

Using these sources as examples of the relationship

between conductance and germination, seeds of both sources

were analyzed for a possible source of conductance by

qualitative and quantitative analyses of elements present in

their leachate and seed coat surfaces.

Conductivity readings were collected using the YS132

conductivity meter. Seeds were placed in 100 ml of

deionized water for 24 hours of imbibition, then strained

from their leachate. Electrical conductivity for leachates and

water controls was recorded in units of pmhos/gram of seed.

The YS132 conductivity meter has a built-in temperature

compensation. Bonner (1988) describes the use of both

multiprobe and single-probe conductivity meters for seed

leachate analyses.

Qualitative and quantitative leachate analyses were

modified from atomic absorption and spectrophotometric

methods developed for liquid samples by Switzer and others

(1988) and Worsham (1989, personal communications).

Nine grams of each seed sample were shaken with 25 ml of

double deionized water overnight at room temperature.

Extractions were filtered using a vacuum flask and 0.22

J.A. Vozzo is research plant physiologist at Forest Sciences

Laboratory, U.S. Department of Agriculture, Forest Service,

Southern Forest Experiment Station, Starkville, MS 39759.

pfilters. A Perkin Elmer 5000 was used for atomic

absorption observations and the Milton-Roy 1001 + provided

spectrophotometer data. Data reflect the average from three

replications of g-gram samples of the poor seed source and

the good seed source.

For EDS, 10 seeds of each source were observed at 3

random spots on the seed coat of each seed sampled. Seeds

were attached to opaque carbon planchets with small amounts

of epoxy and placed in an AMR 1OOOA scanning electron

microscope (SEM) equipped with a Kevex 5100 energy

dispersive x-ray spectrometer. Seeds were coated with

carbon using a Polaron Sputter Coater equipped with a

carbon evaporation unit to minimize specimen charging.

Data were collected with the following conditions being held

constant: magnification, duration of probe (200 set),

accelerating potential (30 KV), tilt (45’), consider lens

setting (spot size 3), and working distance (12 mm).

Spectral data were transmitted to a computerized smoothing

routine followed by background subtraction. X-ray counts

were recorded for each integrated elemental peak and total

counts for the spectrum, after background subtraction. The

elements detected were expressed as a percentage of total

counts after background subtraction.

RESULTS

The good seed source (LOB 55) germinated 80 percent

and averaged 10.5 pmhos/gram seed weight, whereas the

poor seed source (LOB 14) germinated 1 percent and

averaged 60.2 pmhos/gram. Germination was tested for 28

days under standard germination conditions.

Leachate from each seed source in deionized water was

analyzed by atomic absorption/spectrophotometry. Nine

elements, were detected in each of both seed sources. In

ascending atomic number, sodium (Na), magnesium (Mg),

phosphorous (P), potassium (K), calcium (Ca), manganese

(Mn), iron (Fe), copper (Cu), and zinc (Zn) were found in

significantly different amounts for each source’s leachate

(table 1).

Seed coat surface elemental composition was determined

by EDS. In this analysis, eight elements were detected on

the seed surface as follows in order of ascending atomic

number: aluminum (Al),silicon (Si), P, sulfur (S), chlorine

(Cl), K, Ca, and Fe. Of these eight seed coat surface

elements detected, only Ca is in significantly different

amounts between both seed sources, with the low

conductance source having twice as much Ca present as the

high conductance source (table 2).

Among the total 13 elements found, only P, K, Ca, and

Fe were commonly detected in both the leachate and on the

seed coat surface with only Ca different in both analyses (fig.

1). As seen in tables 1 and 2 and figure 1, the primary

source of ion contamination in the deionized water is from

the leachate coming out of the seed. This discounts the seed

coat contamination as the probable cause for increased

conductance in bad seeds.

i
Table 1 .--Elemental analysis of seed leachate from good seed source and poor seed source Pinus taeda seeds, expressed

in ppm’

N a W P K C a M n F e cu

Good seed source 6.32 A 1.04 A 5.33 A 55.29 A 0.30 A 0.20 A 0.61 A 5.33 A

Poor seed source 9.38 B 24.06 B 26.03 B 439.33 B 4.61 B 3.00 B 2.41 B 0.14 B

*Means not sharing letter in common differ significantly at 0.01 probability level by Duncan’s Multiple Range Test.

Zn

0.09 A

0.55 B

Table 2.--Elemental analysis of seed su@ace from good seed source and poor seed source Pinus taeda seeds,

expressed in share of total counts of spectral energies generated after background subtraction’

Al Si P s Cl K C a F e

Good seed source 1040 A 16449 A 109A 658 A 263 A 1777 A 2719 A 266A

Poor seed source 1121 A 15036 A 217 A 552 A 281 A 1855 A 13OOB 631 A

*Means not sharing letter in common differ significantly at 0.01 probability level by Duncan’s Multiple Range Test.

r

P K c a Fe

Elements Detected

Leachate (G,B) m Seed Coat Surface (G,B)

1Q’OOod *cm, B-bad aed nacmti~ly,

Figure 1 .--Percent of each element detected in both leachate

and seed coat surjace analysis.

DISCUSSION ACKNOWLEDGMENTS

As the conductance of electrical current is dependent upon The author wishes to acknowledge the following

ionic presence of contaminants in deionized water, the higher individuals for expertise in their respective interests: F. T.

conductance of LOB 14 than LOB 55 reflects a greater Bonner, single-probe electrical conductance; H. M.

amount of ionic loss from poorly germinating seeds than Worsham, atomic absorption and spectra-photometry; Ray

from better ones. Scheetz, EDS instrumentation.

Ca ions alone do not explain the increased conductance by

poorly germinating seeds as the better seed source shows

twice as much Ca loss as the poorer seed source. The

increased Ca coming from the seed coat surface alone does

not affect germination (table 2). However, the

disproportionately greater amounts of leached ions from the

poor seed source found by atomic absorption do readily

indicate the source of conductance as coming from inside the

seeds. The one exception is Cu (table 1).

There is a high level of Al from seed coat surfaces

detected by EDS. Al x rays may be impinging from the

stage assembly of the instrument itself. Even though the

seeds were mounted on carbon planchets, there is a

possibility of beam interaction with the stage. This allows a

possible source of Al x-ray excitation directly from the stage

or scattering from the curved seed coat surface.

The greater amounts of ions detected in the leachate than

on the seed coat surface, however, indicated that seed coat

contamination is not as major a contributor to conductance by

poor seeds as is the inherent potential of the seed itself to

leak ions when damaged.

Two important studies are now underway to supplement

this hypothesis. First, additional seed rinsing prior to

conductivity tests are in progress by Banner (1989, personal

communications). Secondly, high-voltage electron

microscopy studies by the author indicate cell wall trauma

associated with high conductance seeds. A cell wall trauma

would allow more ions to leach from the poorer seeds than

an intact cell wall allows from the low conductance seeds.
LITERATURE CITED

Bonner, F.T. 1988. Using leachate conductivity of bulked

samples to estimate seed quality. In: Proceedings,

Southern Forest Nursery Association; 1988 July 2.5-28;

Charleston, SC. Columbia, SC: Southern Forest Nursery

Association: 164-172.

Bonner, F.T. 1989. Personal communications.

Bonner, F.T.; Vozzo, J.A. 1987. Evaluation of tree seeds by

electrical conductivity of their leachate. Journal of Seed

Technology. 10: 142-150.

Switzer, G.L.; Nelson, L.E.; Shelton, M.G. 1988. Influence

of the canopy of loblolly pine plantations on the’

disposition and chemistry of precipitation. Mississippi

Agricultural Forestry Experiment Station. Bull. 154: l30.

Vozzo, J.A.; Song, M.J. [In press]. High-voltage electron

microscopy of cell walls in Finus cuedu L. seeds. In:

IUFRO symposium proceedings, seed problems of

multipurpose trees and other tropical and subtropical

species; 1989 August 19 - September 7; Canberra,

Australia.

Worsham, H.M. 1989. Personal communications.

6
Application of the Incubation, Drying, and

Separation Method to Pinus roxburghii Seeds

R. V. Singh and J. A. Vozzo

INTRODUCTION

The cultivation of coniferous plants in containers is

increasing in temperate zones of the world. Economically,

it’ is desirable to make use of every seed as well as each

container; i.e., to sow only one seed per container. This

depends on high seed quality if the percentage of empty

containers is to be kept low. Each container requires the

same time for seedling production and management, whether

a plant develops in it or not. Improvement of the seed

germination percentage can decrease the number of empty

containers. Therefore, it is highly desirable to remove the

dead seeds from a lot before precision sowing can be done.

Simancik (1965) studied the differences in the rates of

water uptake of empty, dead, and viable seeds for grading

the seed quality of several coniferous species. Seeds placed

in water sank to the bottom at different treatments depending

on their viability. However, a separation of viable and

nonviable seeds cannot be achieved by this method.

Simak (1970) has shown that Pinus silvestris seeds, after

being soaked in water for a few hours and subsequently

dried, will lose their water at different rates depending on

their viability. The water is lost faster from dead seeds than

from viable seeds. This led to a new technique developed

for separating filled-live from filled-dead lodgepole pine

seeds that relies on incubation-drying-separation (IDS)

regimes (Simak 1983, 1984). The IDS-method is based on

the previously described principle that viable seeds after

incubation (I) and subsequent drying (D) lose absorbed water

at a much slower rate than dead seeds. This causes

differences in weight and densities between live and dead

seed and makes it possible to use separation methods (S)

based on physical principles such as flotation and gravity

separation.

The objective of this study was to determine the

incubation, drying time, and separation gradients (solvent,

solute, and their specific gravities) for separation of filledlive from filled-dead Pinus roxburghii seeds.

MATERIALS AND METHODS

The seeds used in this experiment were purchased from

Mistletoe Sales - S&S Seeds, 5690 Casifas Pass Road,

Carpinteria, CA.

R.V. Singh is visiting scientist from G.B. Pant University,

Ranichauri, India; J.A. Vozzo is research plant physiologist at

Forest Sciences Laboratory, U.S. Department of Agriculture,

Forest Service, Southern Forest Experiment Station, Starkville, MS

39759.

Sucrose was dissolved in distilled water to make solutions

with specific gravities of 1.02, 1.04, 1.06, 1.08, 1.10, 1.12,

and 1.14. Distilled water was used as a control. Specific

gravity was determined by using standard hydrometers of

laboratory grade.

Separation of the seeds was “carried out by the IDS

method as follows:

I (Incubation): Four hundred seeds were initially

radiographed to determine empty and full seeds. Then, all

seeds were placed in an incubator between two moistened

blotter paper sheets and kept in this condition in a germinator

for 72 hours at 15 “C, 100 percent RH, and 1,000 lux

illumination.

D (Drying): After incubation, the seeds were surface

dried and spread out in a layer on blotter paper for 2-, 4-, 8- ,

and 16-hour drying.

S (Separation): After drying, the seeds were placed in

eight specific gravity solutions of sucrose and distilled water.

After 5 minutes, the buoyant seeds on the surface were

removed and counted. They represented the dead seed

fraction. The sunken .seeds on the bottom represented the

viable seed fraction. They were separated and counted. All

the sinking and floating seeds were placed in the germinator.

Germination was carried out at 20 “C/30 “C and observed

for germination after 7, 14, 21, and 28 days. Seedlings

having root lengths three times the length of seed were

considered as germinated. At the end of the germination

test, all ungerminated seeds were inspected by a cutting test.

In this IDS study, the seeds were designated empty or

full. The following categories were established:

Good ungerminated seeds = full, live

Abnormal germinated seeds = full, live

Rotten seeds = full, dead

Empty seeds = empty, dead.

In addition to the initial radiography and IDS-method to

determine full and empty seeds as well as live and dead

seeds, other seed sample history was analyzed: weight per

1,000 seeds, percentage of moisture loss in fresh seeds at 2-

hour intervals for 16 hours, and percentage of moisture loss

in germination twice in a week up to 28 days.

RESULTS

Average weight of Pinus roxburghii seeds was 110.4

g/1000 seeds. The range of moisture content loss in dry

seeds was 7.6 percent to 9.3 percent at 2-hour intervals over

a 16-hour period (fig. 1). In dry seeds and 16-hour imbibed

seeds, moisture content loss was 8.5 percent and 32.7 percent

respectively. During the germination trials, moisture content

losses varied from 37.9 percent to 73.7 percent while

germination increased 8 percent to 43 percent (fig. 2).
8.26 -

8.00 -

0.76 -

8.26 -

7.76 -

0 2 4 e 8 10 12 14 16

Drying Time (Hours)

Figure 1 .--Percentage moisture loss in dry st!&ifi;om 2 hours

to 16 hours drying. :,’ ,l?:hb*..$ar

,I,0

Days

Figure 2.--Percentage moisture loss in dry, 16 hours imbibed

and germinated seeds,and germination percentage

of seeds.

In radiography tests, an average 11 percent empty seeds

were observed in the 4-hour drying trial followed by 8

percent, 5 percent, and 4 percent in 2-hour, 8-hour, and 16-

-hour drying trials. No more than 5 percent damaged seeds

The object of these preliminary trials was to determine the

combination of drying time and sucrose gradient that gave

were recorded in ldhour drying trial (fig. 3).

separation of filled-live seeds comparable to those from

germination,trials. Pinus roxburghii had 73 percent normal

germination, so the goal was to find the combination of

drying time and gradient to separate 27 percent of the seeds.

According to the IDS-principle, the drying created a

difference in the water content and consequently in the

density between dead and viable seeds.

Two combinations (i.e. 4-hour drying, separated by 1.04

specific gravity sucrose solution, and 8-hour drying,

separated by 1.06 specific gravity sucrose solution) separated

filled-live from filled-dead Pinus roxburghii seeds (table 1,

figs. 4, 5).

2 Hrs 4 Hra 8 Hrs

Figure 3.--characteristics of seed lot x-radiography.

There are two reasons for a certain mixture of viable and

dead seeds in the floating and sinking fractions, respectively.

First, some of the germinable seeds have a low viability, and

their ability to retain water is low when they are dry.

Consequently, some of the seeds are removed together with

the dead seeds during the separation step. The number of

viable seeds in the floating fraction can be adjusted to some

extent by changing the length of the drying time after

incubation. Second, mechanically and insect-damaged seeds

easily absorb water and sink to the bottom together with the

viable seeds during the separation step.

The results of IDS-preliminary trials indicate that there is

a need for further research to determine the optimum

combinations of drying times and separation gradients for

separation of filled-live from filled-dead Pinus roxburghii

seeds.

The radiographs clearly show that the full and empty

seeds can be separated by the IDS-method with good

accuracy. The ungerminated seeds in sink- and floatfractions at the end of the germinated test were analyzed by

a cutting test showing a few ungerminated seeds in floatfraction were viable but failed to germinate.

Table l.--Genninarion @ercent) of IDS-treated Pinus

rQxb&r&&i seeds

Drying

time

Specific gravity Percent in Germination

of solution Fraction seed fraction pe-t

_ _ 1.0, D.W. control 100 7 3

4-hours 1.04, sucrose sink 72 94

float 28 50

&hours 1.06, SucroBe sink 74 86

float 26 1 5
Two hours drying

Specific gravity (sucrose)

0’ ” ” ” :, 1.00 I.02 1.01 Loo Loo I.4 112 1.14

Specific gravity (sucrose)

- Floaters ... + ... Sinkers - Floaters ... + ... Sinkers

Four hours drying

Eight hours drying Sixteen hours drying

100

a... D _ , , . . . . I “.. .,I1.00 !a2 m1 Loo I.00 LI) 1.12 LII

Specific gravity (sucrose)

- Floaters ... + ... Sinkers - Floaters ... + ... Sinkers

Figure 4.--Floater and sinker seed (percent) in d@erent specljic gravity solutions.

1.00 1.02 1.04 1.08 1.08 1.10 1.12 I.,4

Specific gravity (sucrose)

100

80

60

40

20

0

1.00 1.02 1.04 1.06 I.08 110 1.12 1.14

Specific gravity (sucrose)

_ 2 Hrs - + - 4 Hrs ...*... 8 Hrs -II- 18 Hrs

Figure 5.--Germination (percent) offloater and sinker seed in d@erent spec@c gravity solutions

at 2-16 hours drying.

9
CONCLUSION

Separation of Pinus roxburghii seeds by the IDS-method

gave positive results. Seeds with 73 percent normal

germination were clearly enhanced after IDS-treatment.

Removing 28 percent of mostly dead seeds after a 4-hour

drying period at room temperature by 1.04 specific gravity

sucrose solution increased the germination rate to 94 percent.

LITERATURE CITED

Simak, M. 1970. New uses of x-ray method for the analysis

of forest seed. IUFRO Section 22. In: Proceedings,

Working Group on Sexual Reproduction of Forest Trees;

Vaparanta, Finland. Vaparanta, Finland: IUFRO Working

Group 22: Vol. 2. 7 p.

Simak, M. 1983. A new method for improvement of the

quality of Pinus contorta seeds. In: Lodgepole pine:

regeneration and management. Gen. Tech. Rep. PNW157. Portland, OR: U.S. Department of Agriculture,

Forest Service, Pacific Northwest Forest and Range

Experiment Station: 39-41.

Simak, M. 1984. A method for removal of filled-dead seeds

from a sample of Pinus contorta. Seed Science and

Technology 12: 767-775.

Simancik, F. 1965. Seed germination capacity of some

conifers and its relation to the rate of seed sinking in

water. Praha: Lesnicky Casopsis, 1965: 61-70.

10
MATERIALS AND METHODS Comparative Seed Coat Anatomy of

Dormant, Stratified, and

Germinated Pinus koraiensis Seeds

Zhuahua Ning, J. A. Vozzo, and M. V. Bilan

INTRODUCTION

Korean pine (Pinus koraiensis Sieb. et Zucc.) is a fiveneedle, mountain tree species with natural distribution in

northeastern China, southeastern Siberia, South Korea, and

central Honshu in Japan. This pine begins to produce cones

at the age of 80, but good seed production can rarely be

expected until the tree is 120 years old. Large crops usually

occur at 2- to 3-year intervals.

The long period of seed dormancy and the low percentage

of seed germination are the major problems in establishing

Korean pine. Some researchers reported that seed dormancy

depends on the abscissic acid balance in the seed (Chen and

Wang 1987). Others investigated the relations between seed

coat and dormancy. Wang and others (1986) suggested the

Korean pine seed dormancy has little relation with

mechanical resistance or gas impermeability of hard seed coat

and perhaps it depends on hormonal balance in the seeds.

But Zhang and others (1981) reported that its hard testa

constituted the main barrier to respiratory gas exchange,

especially the penetration of oxygen into the seed. They

propose that this might be a factor in dormancy control,

whereas the inner coat appeared to affect seed germination

ability in a manner unrelated to gas exchange.

Stratification remains the best pretreatment for

overcoming dormancy and hastening seed germination.

Asakawa (1955, 1956 a,b, 1961, and 1964) reported that

rudimentary and dormant embryos enlarged during

stratification. He also found that during pretreatments,

glucose and stachyose appeared, whereas sucrose and

raffinose decreased. He further mentioned that the embryo

enlargement may also be explained by an increase in the

ability to absorb water after pretreatment. Chen and Wang

(1987) stated that during stratification the abscissic acid level

decreased in the outer seed coat and endosperm but increased

in the inner seed coat and embryo.

In this paper, scanning electron microscopy (SEM)

photomicrographs show comparative seed coat anatomy of

dormant, stratified, and germinated Korean pine seeds.

Zhuahua Ning is graduate student and M.V. Bilan is professor, at

School of Forestry, Stephen F. Austin State University,

Nacogdoches, TX 75962; J.A. Vozzo is research plant

physiologist, at Forest Sciences Laboratory, U.S. Department of

Agriculture, Forest Service, Southern Forest Experiment Station,

Starkville, MS 39759.

1 1

Seeds were collected from a natural Korean pine forest on

Lesser Xinggan Mountain, Heiliongjiang, China, in 1983 and

stored at 4 “C. Before stratification,~seeds were soaked in

running water at 15 “C for 1.5 days, then stratified at 4 “C.

After stratification for 4 months, seeds were placed in a

germinator at alternating 20 “C to 30 “C! with 12 hours of

photoperiod until the radicle emerged 7 mm in length. Seed

coat samples were collected separately from dormant,

stratified, and germinated seeds. Three-mm squares were cut

from the seed coats. Squares were fixed in 4 percent

glutaraldehyde buffered with 0.07M cacodylate at pH 7.2 for

1 hour. After being rinsed three times in the buffer, squares

were dehydrated in a graded ethanol series and dried in a

DCP-1 Critical Point Dryer, then coated in a Hummer

Sputter Coater. All observations were made with a Hitachi

S-405A scanning electron microscope.

RESULTS

The mature Korean pine seed is 16-17mm long, obovoid

in shape, and has a yellowish brown color. The seed coat

has three distinct layers (fig. 1): a thin scleritied outer layer

formed by 3-4 layers of macrosclereids, a thick sclerified

middle layer formed by lo-15 layers of osteosclereids, and

a thin, papery membranous layer (fig. 2).

The seed surface (outer layer of the seed coat) appears to

be layered also, with a wax and lipid coating layer on top

(fig. 3), a large, narrow macrosclereid layer on the bottom,

and a netlike matrix in between (fig. 4). Surface texture of

dormant seeds was different from that of stratified and

germinated seeds by having obvious wax and lipid deposition

(fig. 3), whereas the amount of wax and lipid on the seed

surface decreased during stratification and germination (fig.

5, 6). The wax and lipid top layer and the netlike matrix

almost disappeared during seed germination (fig. 6). There

are definite pores on the surface of the stratified and

germinated seeds (fig. 5, 6). The pores vertically run

through outer and middle layers of seed coat. The outline of

each cell on the surface of stratified and germinated seeds

can be seen more clearly than on dormant seeds (fig. 4, 5,

6). The intercellular spaces are larger on the surface of

stratified and germinated seeds than on dormant seeds (fig.

4, 5, 6), perhaps due to decreased wax on their seed

surfaces.

The middle layer of seed coat consists of lo-15 layers of

osteosclereids (fig. 1, 7), which are typically bone shaped,

with secondary walls and very small cell lumen. The

secondary wall shows prominent simple pits (fig. 7, 8). The

middle layer of the seed coat of stratified and germinated

seeds is less dense than that of dormant seeds.

The inner layers of the seed coat of dormant, stratified,

and germinated seeds are similar. This layer is derived from

the inner, fleshy layer of the integument. During seed

maturation, the fleshy layer of the integument is crushed and
Figure I.--Seed coat of Pinus koraiensis, x 50; o = outer layer of seed coat; m = middle layer of seed coat; i =

inner layer of seed coat; mr = micropylar region.

Figure 2.4nner layer of seed coat, x 250.

12
Figure 3.--Su@ace of dormant seed, x 150; wl = wax and lipid deposits.

Figure 4.--Sueace of dormant seed, x 250; n = net layer on the seed coat su#ace; MS = macrosclereid layer on

the seed coat suvace.

1 3
Figure 5.--Sut$ace of stratljied seed, x XXI; p = pore; is = intercellular space.

Figure k--B&ace of germinated seed, x 300.

14
Figure 7.--Middle layer of seed coat of stra@ed seed, x 500; OS = osteosclereid; lu = cell lumen.

Figure 8.-Middle layer of seed coat of germinated seed, x 1500; pt = pit.

1 5
pressed into a papery membrane that lines the seed coat and

wraps around the gametophyte and remnants of the nucellus.

DISCUSSION

The stratification treatment influenced both rate and

germination. Seeds stratified for 90 days germinated 18.51

percent after 30 days, whereas seeds stratified for 120 days

germinated 38.95 percent after 30 days (fig. 9). Unstratified

seeds barely germinated. Germination tests verified that the

longer the stratification period, the higher the germination

percentage. Some researchers suggested that Korean pine

seeds should be stratified for at least 6 months (Xu 1978).

Entry of water into the seed is greatly influenced by the

nature of the seed coat. Heavy deposits of wax and lipid on

the surface of dormant Korean pine seeds may repel moisture

because of their hydrophobic characteristic. These deposits

also plug the pores and intercellular spaces on the seed coat.

After stratification, the amount of deposits decreased. The

decrease may be partially due to soaking the seeds in running

water for 1.5 days before stratification and partially due to

the stratification process itself. During soaking, the running

water forced seeds to rub against each other, affecting the

integrity of the wax and lipid layer on the surface of the seed

coat. During stratification, the deposits were further

decomposed.

Like Pinus elliottii and P. taeda, Korean pine seed

surface has a netlike matrix. Vozzo (in press) reported that

this may suggest a hyaline layer of fungal presence,

indicating a symbiotic relation between seeds and fungi.

Stratification may provide a moist environment for fungal

growth on the seed coat surface, and fungi may serve as a

scarification process that would help water and gas exchange

to satisfy prerequisite germination conditions. Fungi may

also decompose the deposits of wax and lipid on the seed

coat surface during stratification.

50

I I

Days

During stratification, the seed coat density decreased.

The cleft of the seed coat in the micropylar region loosened

to allow increased imbibition and also lessened the

mechanical resistance of the seed coat to later radicle

emergence during germination.

Pores running through the seed coat may offer channels

for water and gas exchange. The pits on the surface may

also function as translocation channels, since the thick

secondary wall layers in the pit region were completely

interrupted.

Further studies will emphasize elemental composition of

the seed coat; ultrastructural changes; and translocation

patterns of carbohydrates, lipids, and proteins of dormant,

stratified, and germinated Korean pine seeds.

LITERATURE CITED

Asakawa, S. 1955. Hastening the germination of Pinus

koraiensis seeds. Preliminary study on chemical changes

during pretreatment. Journal of the Japanese Forest

Society 37(4): 127-132.

Asakawa, S. 1956a. Further investigation on hastening the

germination of Pinw koraiensis seeds. Journal of the

Japanese Forest Society 38(l): l-4.

Asakawa, S. 1956b. On the water absorption by Pinus

koraiensis seeds. Journal of the Japanese Forest Society

38(4): 125-129.

Asakawa, S. 1961. A preliminary study concerning the

relationship between an embryo and endosperm during

stratification. Journal of the Japanese Forest Society

43(6): 195-196.

Asakawa, S. 1964. A brief report on the respiration of Pinus

koraiemis seeds. Journal of the Japanese Forest Society

46(100): 336-338.

Chen, J.; Wang, W: Z. 1987. Determination of ABA content

of Pinus koraiensis seeds during their dormancy,

stratification, and germination by means of radioimmunity. Journal of the Northeast Forest University

15(l): 7-12.

Vozzo, J. A. [In press.] Comparative morphology of seed

coat surfaces for seven species of Pinus.

Wang, W. Z.; Chen, J.; Liu, E. J. 1986. Study on relations

between seed coat and dormancy of Pinus koraiensis

seeds. Journal of the Northeast Forest University 14(2):

84-86.

Xu, Z. B. 1978. Korean pine forest. Beijing, China: Pub.

House Chinese For.

Zhang, L. C.; Guo, W. M.; Cheng, Y. S. 1981.

Experimental investigation on the physiology of

afterripening of Korean pine seed. Journal of the

Northeast Forest University 4(4): 43-50.

Figure 9.--Germination of Korean pine seeds ajier treatment.

16
c

Subterranean Clover: Nutrients For Pines

Henry A. Pearson, Michael L. Elliott-Smith,

Alton Martin, Jr., and Virgil C. Baldwin

INTRODUCTION

Forest soils in the South are generally low in available

nitrogen. Therefore, legumes and other nitrogen-fixing

plants could benefit growth by nitrogen cycling among the

host legume plant, the soil, and associated plants growing on

a particular site. Annual nitrogen-fixation rates of up to 200

kg/ha were reported from subterranean clover (TrijXium

subterruneum L.) production under longleaf pine (Pinus

palustris Mill.) stands in southern Mississippi (Hagedom and

others 1980). Supposedly, about 80 percent of the fixed

nitrogen in legume pastures is transferred to the soil as plant

and animal residues (Date 1970). Southern pines regenerated

in subterranean clover pastures are potential beneficiaries of

nitrogen-fixation by the clover. Nitrogen can be removed

from the site through timber harvesting, forage grazing, and

soil leaching (Haines and DeBell 1979).

In the sandhills of South Carolina near the Savannah

River Project, common sericea lespedeza (Lespedezu cuneatu

[Dum.-Cours.] G. Don) apparently fixed nitrogen in soils to

the point of increasing foliar nitrogen contents of sand pine

(P. clausa [Chapm. ex Engelm.] Vasey ex Sarg.) by the third

growing season (Jorgensen 1985). Similar results by the

fourth growing season were obtained in loblolly pine (P.

tueda L.) using common sericea lespedeza, “Caricea”

lespedeza (an improved variety of common sericea), and

Thunberg lespedeza (L. thunbergii [DC.] Nakai) in North

Carolina where stemwood volumes were greater on the

lespedeza treatments (Schoeneberger and Jorgensen 1989).

The purpose of this study was to determine whether

subterranean clover, a cool season legume, fixes nitrogen in

tame pastures sufficiently for it to be incorporated into the

soil and subsequently translocated into associated loblolly and

slash (P. elliottii Engelm.) pines. Specifically, this

agroforestry study focused on comparing nitrogen content of

the pine foliage from trees growing in tame pastures with and

without subterranean clover. These preliminary results may

reflect the value of subterranean clover in nutrient cycling

and subsequent pine growth when used in agroforestry.

STUDY METHODS

The study area was located in a 4-ha tame pasture within

the southeast range unit, Palustris Experimental Forest,

Henry Pearson is project leader, Michael L. Elliott-Smith is

biological laboratory technician, Alton Martin, Jr. is range

technician, and Virgil C. Baldwin is research forester, at Southern

Forest Experiment Station, U.S. Department of Agriculture, Forest

Service, Pineville, LA 71360.

McNary, Louisiana. The Unit was cleared in 1977; ink-the

fall of 1980, the pasture was burned, disked, fertilized, and

planted to subterranean clover (Pearson and others 1990).

Clover seed were inoculated with Rhizobium trifolii at the

time of planting (Hagedom and others 1983). In the late

summer of 1983 and subsequent years, randomly selected 0.4

ha paddocks were disked and treated with herbicide

(ROUNDUPR: isopropylamine salt of N-(phosphonomethyl

gylcine)) to remove the summer, warm season growing

grasses and to encourage fall and winter production of

subterranean clover. During the ensuing winter, a third

treatment paddock (the control) was established (subterranean

clover removed from the paddock by applying herbicide

during early March). Each year during late August or early

September, 53 kg/ha each of P,O, [23 kg/ha elemental

phosphorus (P)] and K,O [44 kg/ha elemental potassium (K)]

‘were top dressed over all paddocks.

At the time of control treatment, loblolly and slash pine

seedlings (O-l) were planted in all three treatment paddocks.

Three rows of seedlings of each pine species were planted

within each 0.4-ha paddock; each row served as replication

for the chemical analyses. Pines were planted 1.3 m within

rows, which were 6.7 m apart; pine density was about 1,165

seedlings/ha. Each replication contained 45 seedlings for

each pine species.

Pine seedling heights were measured after the second and

third years, and both heights and diameters were measured

at tree age 5. However, the fifth-year tree measurements are

not reported in this paper, since analyses are not complete.

By the end of the third growing season, no differences in tree

heights were noted (unpublished data).

Following the 1987 (samples collected May 1988) and

1988 (samples collected January 1989) growing seasons,

composite pine foliar samples from 10 trees within each 45-

tree replication row were collected. Foliage was analyzed

for tree age (4- and 5-year-old trees), foliar location (upper

and lower one-third of the tree crown vertical height), stage

of growth (mature vs. immature needles), cultural treatments

(none, disking, and chemical application), and species

(loblolly and slash pine). Nutrient analyses, including

nitrogen, phosphorus, and potassium, were conducted at the

Alexandria Forestry Center, Pineville, Louisiana. The pine

foliar samples were digested in sulfuric acid with copper

sulfate added as a catalyst. Nitrogen was determined using

an ammonia-specific electrode (Powers and others 1981),

phosphorus was determined calorimetrically (John 1970), and

potassium was determined by atomic absorption

spectrophotometry. Soils on the paddocks were analyzed

during August 1985, by the Feed and Fertilizer Laboratory,

Louisiana State University, Baton Rouge. Although not used

in these evaluations, averages for the soil analyses on the

different cultural treatments are provided for background

information (table 1).

Percentage of ground cover of subterranean clover was

estimated during the spring each year from 20 plots (0.22

m2) systematically located every 5 m apart on 2 diagonal

1 7
Table 1 .--August 1985 soil analyses on d@erent cultural

treaxmerus

Cultural Treatments

Attribute Control Disk Chemical

Organic matter (percent) 2.2 2.7 2.0

l l

P (w/kg)

5

Na (mgkz)

11 13 10

K (mdkg) 47 30 2 2

Ca bwkd 1323 974 1216

Mg (w/kg) 46 60 4 3

PH 6.8 6.0 6.7

*Missing data

transects across each quarter of a 0.4-ha paddock for a total

of 80 estimates/paddock. A yield-ground cover equation,

developed by double-sampling (Pearson and others 1990),

was used to estimate subterranean clover yields:

Y = 49.1 c

where Y is kg/ha ovendry yield and C is percentage of

ground cover; the standard error of estimate was 1,505 kg/ha

and the coefficient of determination (9) was 0.91.

RESULTS AND DISCUSSION

Subterranean clover yields during spring averaged 4,040

and 3,457 kg/ha on the disk and chemical cultural treatments,

respectively (table 2). The control paddock had an average

residual clover yield of 380 kg/ha; however, a high

proportion of this yield during the latter years was volunteer

white clover (T. repens L.), possibly transported by cattle

grazing other areas and the study paddocks. Crude protein

content of the subterranean clover during winter and spring

varied from 18 to 23 percent (Pearson 1983); phosphorus

content varied from 0.20 to 0.27 percent during the same

period (Pearson and Barnett 1984).

Immature growing pine needles collected during spring

had higher nitrogen content than mature needles on 4-year

old pines regardless of species (table 3). Loblolly pine

needles had higher nitrogen content than slash pine needles

for both immature and mature stages of growth. Apparently,

loblolly pines utilize nitrogen from the soil more efficiently

than slash pines or at least translocated more nitrogen in the

plant to Lhe needles. Phosphorus and potassium content of

the needles responded similarly to maturity and species--

immature needles had higher analyses than mature needles,

and loblolly pine needle analyses were higher than slash pine.

Needles in the upper one-third of the crown had more

nitrogen than needles in the lower third for the 1987 (fourth)

growing season but were not different for the 1988 (fifth)

Table 2.--Subterranean clover yields fig/ha) in the spring on dtrerent cultural treatments

Treatment 1984 1985

Control 252 l

YCXU

1986 1987 1988 1989 Average

l l

437 452 380

Disk 4188

Chemical 3953

‘No measurement made.

4419 4910 4051 3457 3216 4040

4483 4910 3314 1841 2239 3457

Table 3.--Chemical content (mg/g) of immature and mature pine needles (1987)

Growth Nitrogen Phosphorus Potassium

Stage Loblolly Slash Loblolly Slash Loblolly Slash

Immature 16.6 a’ 12.1 a 3.0 a 2.4 a 10.6 a 8.7 a

Mature 13.2 b 9.7 b 1.7 b 1.5 b 4.6 b 4.0 b

*Different letters in same column denote significance at the 0.05 level.

1 8
growing season (table 4). Nitrogen tram&cation from the

lower needles possibly occurred prior to the May sampling

but not by the January sampling. Nitrogen content of

loblolly pine needles was higher during both sample years

when compared to slash pine needles. Phosphorus and

potassium contents of the needles were higher in the upper

third compared to the lower third of the crown for both

species in both years.

Although high yields of subterranean clover occurred on

the tame pasture paddocks with the disk and chemical

cultural treatments, nitrogen contents of pine foliage were

apparently not affected prior to the 1988 growing season

(table 5). However, nitrogen contents of needles were higher

in both the disk and chemical treatments compared to the

control treatment after the 1988 growing season @year-old

trees). Loblolly pines in subterranean clover pasture had the

highest nitrogen content in the needles with the chemical

cultural treatment that exceeded amounts measured in either

the disk or control treatments. Both disk and chemical

treatments were similarly higher in nitrogen than the control

for slash pine. Apparently, nitrogen fixation by the

subterranean clover was sufficient to be incorporated into the

soil and translocated to the pine needles during the fifth

growing season. This higher nitrogen uptake by pines should

eventually increase tree growth similar to results reported in

North Carolina using lespedeza (Schoeneberger and

Jorgensen 1989).

Phosphorus contents of the needles were not different

among treatments in the 4-year-old loblolly pine or the Syear-old slash pine (table 5). On the other hand, both the

1988 loblolly and 1987 slash pine needles were different

among cultural treatments with the control higher in slash

pine and the chemical treatment higher in loblolly pine.

Except for the 1988 loblolly pine f&liar analyses, potassium

content of needles was higher on the control treatment than

on either the disk or chemical cultural treatments. Possibly

potassium in subterranean clover was not being recycled

sufficiently to be translocated to the pines. Apparently,

nitrogen and phosphorus were sufficiently cycled on the

cultural treatments for absorption and translocation by the

pines; hence, higher foliar nutrient contents on the disk and

chemical treatments compared to the control treatment.

CONCLUSIONS

High yields of subtl:rranean clover for forage are possible

on tame pastures in the South where cultural treatments

control the warm season grasses. Also, subterranean clover

fixes nitrogen sufficiently to be absorbed and used by

southern , pines. The soil nitrogen apparently increases

through nitrogen fixation by the cool season legume

subterranean clover, but the nitrogen is absorbed and

translocated by the pines only after several years of nitrogen

fixation and accumulation in the soil. Immature, actively

growing pine needles during spring have higher nitrogen,

Table 4. --Chemical content (mg/g) of pine foliage from direrent location

Nitrogen Phosphorus Potassium

Crown Loblolly Slash Loblolly Slash Loblolly Slash

Location 1987 1988 1987 1988 1987 1988 1987 1988 1987 1988 1987 1988

Upper third 16.7a’ 16.1a 11.3a ll.2a 2.8a 1.4a 2.la l.la 9.3a 5.9a 7.9a 5.2a

Lower third 13.lb 15.9a lO.Sb 12.2a 1.9b 1.3b 1.7b l.Ob 5.9b 4.8b 4.8b 4.Ob

*Different letters in same column denote significance at the 0.05 level.

Table 5.--Chemical content (mglg) of pine foliage from diflerent cultural treatments for subterranean clover

oasture

Cultural

treatment

Nitrogen Phosphorus

Loblolly Slash Loblolly Slash

1987 1988 1987 1988 1987 1988 1987 1988

Potassium

Loblolly Slash

1987 1988 1987 1988

Control 14.2a’ 13.4~ 11.3a 9.8b 2.4a 1.3b 2.0a l.la

Disk 14.8a 16.3b ll.Oab 12.2a 2.3a 1.4b 1.9ab l.Oa

Chemical 15.6a 18.4a 10.3b 13.la 2.3a 1.4a t.9b l.la

*Different letters in same column denote significance at the 0.05 level.

8.3a 5.6a 7.5a 5.3a

7.2b 5.2a 5.8b 4.2b

7.3b 5.2a 5.7b 4.4b

19
phosphorus, and potassium contents than mature needles.

Needles from the upper crown generally have higher nutrient

contents than lower crown needles. Loblolly pines

apparently assimilate nitrogen more efficiently than slash

pines.

LITERATURE CITED

Date, R. A. 1970. Microbiological problems in the

inoculation and nodulation of legumes. Plant and Soil 32:

703-725.

Hagedorn, C.; Ardahl, A. H.; Materon, L. A. 1983.

Characteristics of Rhizobium trij3lii populations associated

with subclover in Mississippi soils. Journal of the Soil

Science Society of America 47: 1148-l 152.

Hagedom, C.; Watson, V. H.; Knight, W. E. 1980. Forage

legumes in a forested environment. In: Child, R. C.;

Byington, E. K., eds. Southern forest range and pasture

symposium; 1980 March 13-14; New Orleans, LA: 143-

145.

Haines, S. G.; DeBell, D. S. 1979. Use of nitrogen-fixing

plants to improve and maintain productivity of forest

soils. In: Proceedings, Impact of intensive harvesting on

forest nutrient cycling symposium; 1979 August 13-16;

Syracuse, NY. Syracuse, NY: State University of New

York, College of Environmental Science and Forestry:

279-303.

John, M. K. 1970. Calorimetric determination of phosphorus

in soil and plant materials with ascorbic acid. Soil Science

109: 214-220.

Jorgensen, J. R. 1985. Sericea influences early survival,

growth, and nutrition of sand pine. Gen. Tech. Rep. SO54. New Orleans, LA: U.S. Department of Agriculture,

Forest Service, Southern Forest Experiment Station; 363-

368.

Pearson, H.A. 1983. Forest grazing in the southern United

States. In: Hannaway, D. B. ed. Oregon State University

College of Agricultural Sciences Symposium Series No.

2 -- Foothills for food and forests. Beaverton, OR:

Timber Press: 247-260.

Pearson, H. A.; Baldwin, V. C.; Barnett, J. P. 1990. Cattle

grazing and pine survival and growth in subterranean

clover pasture. Agroforestry Systems 10: 161-168.

Pearson, H.A.; Bamett, J.P. 1984. Agroforestry--

subterranean clover and pines. Establ. Prog. Rep.

FS-SO-1701-3.24. New Orleans, LA: U.S. Department of

Agriculture, Forest Service, Southern Forest Experiment

Station. 43 p.

Powers, R. F.; Van Gunt, D. L.; Townsend, R. F. 1981.

Ammonia electrode analysis of nitrogen in microKjeldah1

digests of forest vegetation. Communications, Soil

Science Plant Annals 12: 19-30.

Schoeneberger, M. M.; Jorgensen, J. R. 1989. Long-term

benefit of lespedeza on loblolly pine growth. Gen. Tech.

Rep. SO-74. New Orleans, LA: U.S. Department of

Agriculture, Forest Service, Southern Forest Experiment

Station; 477-481.

20
A Method for the Dekction of Trace

Quantities of Ethylene in the Atmosphere

Lewis R. Brown, Gerard S. Pabst, Jr.,

James L. Spencer, Jr., and Ted H. Filer, Jr.

INTRODUCTION II.

Ethylene is a natural product of metabol@m in plants and

is considered a plant hormone. When a plant is stressed,

however, ethylene production increases significantly. Since

stress ethylene is produced prior to the visual manifestations,

of stress, its early detection could make it possible to

eliminate the cause of the stress, thereby reducing damage to

the plant.

The most wmmon method of detecting ethylene in the

atmosphere is by gas chromatography (GC), and this method

has been employed in our laboratory for several studies

involving the microbial production of ethylene. However, all

these studies were conducted using closed vessels and, as a

consequence, the ethylene concentration in the atmospheres

reached levels easily detected by GC. The sample size

employed for the GC analyses was 100 pL, and with this

sample size, a concentration between 0.13 and 1 ~1 was

detectable using a thermal conductivity detector.

Concentrations of ethylene of the above magnitude would not

be expected in a field situation nor would adapting a gas

chromatograph for field use seem practical.

This paper describes the evaluation of two methods

amenable to field use to detect low levels of ethylene in the

atmosphere.

METHOD ONE

An attempt was made to develop an ethylene detection

system based on the reaction between ethylene and bromine

water. This reaction decolorizes the bromine water and can

be conveniently monitored by spectroscopic methods.

Saturated bromine water, containing 3.35g bromine per 1OOg

water at 25 “C, was obtained from American Scientific

Products, Inc. A Bausch and Lomb Spectronic 20

Spectrophotometer was employed for measuring light

transmittance.

Saturated bromine water was diluted 1:33 with’distilled

water, and samples were analyzed for percentage of light

transmission at various wavelengths. Representative data are

given in figure 1. This experiment was repeated using a

1: 100 dilution of saturated bromine water in distilled water,

and representative data are given in fig. 2.

Lewis R. Brown, Gerard S. Pabst, Jr., and James Spencer, Jr., are

at Department of Biological Sciences, Mississippi State University,

Starkville, MS; Ted H. Filer, Jr., formerly plant pathologist at the

Southern Hardwoods Laboratory, Southern Forest Experiment

Station, USDA Forest Service, Stoneville, MS 38776.

Figure 1. -Spectrophotometric observations of l/33 dilutions

of bromine water using a wide range phototube

and plter.

aao- * tc

aoQ-

** *t*

%clr

4w400- *8=*

40 ma 00.0 at26 ea.0 07.6 *a

PuoontD

Figure 2.--Spectrophotometric observations of l/loOdilutions

of bromine water using a wide range phototube

and jilter.

Six ml of a 1:33 dilution of saturated bromine water in

distilled water was pipetted into each of a number of

spectrophotometer cuvettes, the light transmission measured

(450 nm), and the contents transferred to a serum-stoppered,

16 x 100 ml glass test tube. A known amount of ethylene

was injected into each tube, the contents of the tube mixed

on a vortex mixer for 30 seconds, and the light transmission

of the bromine water again measured. Representative

results, as shown in fig. 3, indicate that the intensity of the

color of the bromine water is reduced in proportion to the

amount of ethylene injected into the tube.

Other tests were conducted by bubbling different amounts

of air that varied in ethylene concentration through different

concentrations of bromine water. The major problem

encountered was that air without ethylene decolorized the

bromine water, albeit at a rate considerably slower than that

caused by ethylene. This problem was overcome by

accounting for the decolorization caused by a given volume

of air. It was found also that light accelerated decolorixation

slightly. Shielding the bromine water from light easily

overcame this problem.

While the system did work, it was found that at very low

levels of ethylene, where large volumes of air must be passed

through the bromine water, the problem of decolorization by

the air alone caused major problems. The controls necessary
*

1

* * 220- * MO- *

** *

%a a0 02 04 2s w ia

Fwcent Tranrmittancr

Figure 3.--Spectrophotometric observations of l/33 dilutions

of bromine water at 450 nm afler injection of

ethylene.

to obtain accurate results seemed to make the system

impractical for routine use.

Obviously, the degree of decolorization is dependent upon

(1) the concentration of the bromine water, (2) the volume of

bromine water employed, and (3) the amount of ethylene

added. It is realized, of course, that materials other than

ethylene will decolorize bromine; in fact, any gaseous olefin

will do this. The probability of the presence of other

interfering gaseous compounds in the atmosphere being

monitored is sufficiently remote and should not be a major

problem. Even if such compounds did get into the

atmosphere, they would cause a “false positive” reading,

thereby indicating “stress” in the plants where, in fact, this

might not be the case. The above situation is preferred to

one where a false negative would fail to indicate stress even

though it were present.

METHOD TWO

As stated earlier, gas chromatography is an accurate and

reliable method of determining ethylene in air, but

unfortunately, the concentration of ethylene expected is

below the limits of detection of the gas chromatograph.

Therefore, it was necessary to concentrate the ethylene in a

sample in order to detect it by GC. Recently, Supelco, Inc.,

began marketing a thermal desorption unit (TDU) that makes

collection of trace contaminants in air practicable. In the

present case, the ethylene is adsorbed onto

CarbotrapKarbosieve contained in a small glass column,

with the subsequent elution (thermally) of the gas from the

column in the TDU.

This instrument thermally desorbs the gas and delivers it

directly into the gas chromatograph for conventional

detection.

Collection of the Sample. Samples were collected using

an E. I. DuPont De Nemours and Co., Inc., Permissible Air

Sampling Pump, Model P-4000. The pump is set for the

desired airflow; the 4 mm I.D. Carbotrap/Carbosieve 200

desorption tube (Supelco, Inc., 2-2042) is inserted into a

rubber tube connected to the pump, and the pump turned on.

Once the sample has been collected, the desorption tube is

removed from the rubber tubing and placed in a carrying vial

(Supelco, Inc., 2-0375).

Thermal Desorption Unit and Gas Chromatograph.

The Thermal Desorption Unit is illustrated in figure 4 and is

connected to a Fisher Model 1200 Gas Partitioner.

Settings on the TDU. The TDU set for use with the

GC should have a helium gas flow of 60 psi with the tube

heat selector switch (fig. 4) set on automatic, the tube

heat timer set for 10 minutes, and the tube heat set at 330

T.

Settings on the Gas Partitioner. The GC should have

the following settings and conditions. The helium gas flow

should be maintained above 15 psi to maintain the cutoff

relay (this is necessary because the column is connected

directly to the TDU transfer line). The column is a lo-foot,

l/&inch stainless steel column packed with 100/120 mesh

Carbosieve S-II. The column temperature is 150 “C. The

Bridge current is 175 mA with an attenuation of lx. The

Fisher Recordall, Model 5000 is connected to the GC and is

set to record at 0.25 cm/min at 0.001 volts full scale.

Desorption of Ethylene Rapped on Desorption tibes.

The desoqition tube is placed in the tube desorption

chamber, the valve arming button is pressed, and the

valve handle moved to the heat position.

Determination of Ethylene. To determine the volume

of ethylene injected directly into the TDU, an empty

desorption tube was placed in the tube desorption chamber of the TDU, the valve arming button was depressed

and the sample injected through the tube desorption

.ehamber injection port. Immediately (within 10 seconds)

the valve handle was turned to heat.

To develop a curve for ethylene, 5, 10, 25, 50, and 100

~1 samples of ethylene gas mixture were made up to a total

volume of 100 ~1 with air and injected into the TDU as

described above. Ethylene was detected by comparison of

retention time of the unknown with the retention time of a

known sample of ethylene. Quantification was accomplished

by comparison of the area under the peak with the area under

the peak of a known amount of ethylene. The results are

shown in fig. 5 by the solid dots.

Detection of Ethylene Trapped on Desorption Tubes.

With desorption tubes attached to the DuPont air pump,

various amounts of an air mixture containing ethylene were

passed through the desorption tube. The tubes were then

desorbed by placing a tube into the desorption chamber of the

TDU, depressing the valve arming button, and immediately
Tube Heat

SetpAnt Cc&d

I

VO nt Thumbwtwel ‘9. -PC

‘Tube Heat

seleotof switch

Vahm Arming ELIton

Valve Handle ’

TutzeDesotption /

chamber

,

Tube Desorptbn Chamber r

Injection Port L!

m--

Figure 4.--Diagram of the thermal desolption unit.

13

12 n Direct injection

11

10

i

X Tube injection

9 0.7

8

7

8

35 69 144

30 40 50 80 70 80 90 100110120130140150150

Area (mm2)

Figure S.--Comparison of directly injected ethylene with thermally desorbed ethylene.

23
moving the valve handle to the heat position. After

desorption is indicated on the GC recorder, the valve handle

was moved to the load position for subsequent tubes to be

desorbed.

The results of desorption of the ethylene from the tubes

and the resultant detection of the ethylene are shown by x’s

fig. 5. As may be observed, the results of this experiment

e essentially identical to those achieved by injecting

.,ylene directly into the TDU, thus indicating that, within

he limits of detection, all of the ethylene trapped on the

iesorption tube was recovered.

While the system worked well, very low concentrations

If ethylene in air could not be detected due to (1) the lack of

sensitivity of the thermal conductivity detector and (2) the

ow breakthrough volume for the desorption tubes. The

smallest amount of ethylene detectable using the TDU was 1

11, and the breakthrough volume for the desorption tubes was

100 ml/min for 8 minutes or 10 ml/min for 2 hours. Under

these conditions, the ethylene had to be in a concentration of

at least 0.8 ppm or 800 ppb. The sensitivity can be

increased by at least a hundredfold through the use of a

flame ionization detector, making it possible to detect 8 ppb.

The thermal desorption unit is designed in such a way as

to make it possible to focus the ethylene from the 4 mm

(I.D.) collection tube onto a smaller (2 mm I.D.) desorption

tube. It was found that the ethylene from three 4-mm

collection tubes could be focused onto a single 2-mm tube,

thereby making it possible to detect 0.26 ppm with the

thermal conductivity detector. The flame ionization detector,

therefore, should be able to detect 2.6 ppb.

ACKNOWLEDGMENTS

This project was supported in part by US Department of

Agriculture, Forest Service.

24
Optimization of Graphite Furnace

Atomic Absorption Technique for

Determination of Rubidium in Southern Pine

Beetle, Dendroctonus frontalis, for

Mark-Recapture Studies

W.T. Thoeny, A.E. Tiarks, and M.L. Elliott-Smith

Little is known about the dispersal of the southern pine

beetle, Dendroctonus frontalis Zimm. (SPB) and the role it

plays in the population dynamics of this important pest of

southern pines. Despite the importance of scolytid bark

beetles, few mark-recapture studies have been conducted with

any species. This lack of information is partly due to the

lack of a suitable marking technique for these small insects.

Moore and Taylor (1976) demonstrated a 32p marking

technique for the SPB, but this method has been abandoned

due to safety and environmental concerns. The use of trace

elements, particularly rubidium (Rb), as internal markers for

insects has been widely adopted (Bridges and others 1989 and

references therein). Labelling of phytophagous insects has

commonly been accomplished by incorporating trace elements

into artificial diets (Stimmann and others 1973) or by

application and translocation through host plants (Van

Steenwyk and others 1978, Bridges and others 1989).

Analyses by atomic absorption spectroscopy have

demonstrated that addition of low levels of Rb to larval diet

of several insect species does not affect longevity, fecundity,

or behavior.

Rubidium occurs naturally at low levels in soils, and

natural background levels are normally low in native flora

and fauna. Previous rubidium-marking experiments have

established a criterion for the level of Rb necessary for the

insect to be considered a marked individual (Stimmann

1974). This standard based on the mean background Rb

level plus 3 SD results in a 99percent confidence limit that

a marked insect did not belong to the natural population

(Bain and Engelhardt 1987).

The inherently low background Rb levels and small size

of the SPB necessitate the use of graphite furnace technology.

With the advent of the platform furnace technique and

control of experimental conditions, the graphite furnace has

become a convenient analytical tool, suitable for routine

analysis of inorganics, although little has been published on

the determination of Rb (Slavin and others 1983). During

graphite furnace analysis, desolvation of the sample,

dissociation from the matrix, and generation of analyte

ground state atoms occur sequentially during predetermined

drying, pretreatment, and atomization steps. Before analyzing

W.T. Thoeny, is former research entomologist, A.E. Tiarks is soil

scientist, and M.L. Elliott-Smith is biological laboratory

technician, at Southern Forest Experiment Station, USDA Forest

Service, Pineville, LA 71360.

samples containing complex unknown compositions, it is

essential to determine whether the recommended standard

conditions for the element in question are appropriate, or

whether alterations are necessary for optimum performance

(Perkin-Elmer 1985). Grobenski and others (1983) reported

on optimum conditions for Rb analysis and determined a

detection limit of 5 pg, with no ionization effect. They

recommended a thermal pretreatment temperature of 800 “C

and a fast heating atomization step to 2,000 “C for routine

analysis with the L’vov platform in pyrolytically coated

graphite tubes. The purpose of this study was to establish

optimum operating conditions for Rb determination of

unmarked SPB by atomic absorption spectroscopy (AAS)

using a graphite furnace.

MATERIALS AND METHODS

Adult beetles were obtained from naturally infested

loblolly pines, Pinus taeda L., within the Kisatchie National

Forest in central Louisiana. Infested bark samples were

placed into rearing containers, and emerging beetles were

collected for analysis. Beetles were killed by freezing and

dried for 24 hours at 70 “C before determining dry weights.

Fifty beetles were placed in 8-ml borosilicate glass vials

along with 1.0 ml reagent nitric acid (HNQ) and heated at

100 “C for 4 hours until the samples cleared and diluted to

25 ml with deionized water.

NBS pine needle standards (ref. mat. 1575) were prepared

by ashing 0.5 g of needle sample in a muffle furnace at 500

“C and placing 0.02 g of ash into lOO-ml volumetric flasks.

Preliminary results indicated that perchloric acid was

interfering with Rb analysis, so ash was diluted to 100 ml

with 0.2 percent HCl in deionized water and 0.2 ml HNOs

or with both 0.2 ml HNO, and 2 ml HClO,.

Samples were analyzed by atomic absorption spectroscopy

using a Perkin-Elmer 2100 with an HGA 700 graphite

furnace and AS-70 autosampler (Perkin-Elmer

Manufacturers, Norwalk, Connecticut). An electrodeless

discharge lamp (wavelength 780 nm) was used to detect

rubidium. Samples were atomized on L’vov platforms within

pyrolitically coated graphite tubes. Rubidium concentration

was determined by the area under the peak of the AAS

signal.

Optimum pretreatment temperature was determined by

setting the dry and atomization parameters to the optimum

values taken from the recommended conditions (Perkin-Elmer

1985). Sample aliquots (20 pL) of RbCl sample solution (10

pg/L) and SPB-acid matrix solution were analyzed at thermal

pretreatment temperatures of 200, 400, 600, 800

‘(recommended condition), 900, 1,000, 1,200, and 1,400 “C.

Optimum atomization temperature was determined by

setting the dry and pretreatment parameters to the optimum

recommended conditions and analyzing aliquots of RbCl

sample solution atomized at 1,000, 1,200, 1,400, 1,600,

1,700, 1,800, 1,900 (recommended condition), 2,000,2,100,

and 2,200 “C. Based on these results, the SPB-acid matrix

25
solution was analyzed at atomization tempera&res from

1,600 - 2,200 “C at 100” increments.

In someinstances, standards that are sufficiently similar

to the sample solution are difficult to prepare because of the

presence of matrix materials or solids (Perkin-Elmer 1985).

The standard additions technique can often be used to correct

for these nonspectral interferences by determining the

concentration of the analyte. Standard additions were

conducted with the SPB-acid matrix solution and NBS pine

needle samples in HNO, or in HNO, plus HClO,.

The standard additions. technique was used to check for

nonspectral interferences by adding increasing volumes of a

standard to a constant volume of unknown. Standard

additions were conducted using SPB-acid matrix solution,

ashed pine needle samples in HNOr, or ashed pine needle

samples in HNO, plus HClO, as the unknown. A five-PL

aliquot of the unknown was added to the furnace followed by

a 15-PL aliquot of blank plus standard. The amount of

standard was increased from 0 to 10 PL in 1-PL increments.

Linear regression analysis was used to find the relationship

between Rb added as RbCl and absorbance.

It is often necessary to add a chemical matrix modifier

that allows higher pretreatment temperatures in order to

remove matrix compounds that interfere with the analyte

signal. Little has been published on the determination of Rb

with the graphite furnace, and Slavin (1984) does not include

established matrix modifier recommendations for Rb.

However, in order to determine if matrix interferences

existed, two commonly used matrix modifier compounds

were evaluated. A 4- percent solution of ammonium

phosphate (NH,H,PO,) and a l-percent solution of magnesium

nitrate (Mg(NO,),) were evaluated for matrix modifier benefit

with NBS pine needle standard samples in HN03 and HNO,

plus HClO,.

RESULTS AND DISCUSSION

Optimum thermal pretreatment temperatures for Rb in

both the RbCl sample and the SPB-acid matrix sample were

determined to be the recommended standard condition of 800

“C (fig. 1). Although the Rb signal did not decline

significantly at 900” or 1,000”, Grobenski and others (1983)

reported minor losses at 900”. Because of this, a

pretreatment temperature of 800 “C will be used for

analyzing Rb in SPB during our research. An atomization

temperature of 2,000” was determined to be the optimum

condition for Rb analysis in both the RbCl sample and the

SPB-acid matrix sample (fig. 2). This 100” increase in

temperature resulted in a significant increase in Rb signal

from the recommended condition of 1,900” and was also

recommended by Grobenski and others (1983).

When possible, standards should be made up in a matrix

similar to the samples to minimize the possibilities of

nonspectral interferences. in some instances, standards that

are sufficiently similar to the sample solution are difficult to

prepare because of the presence of matrix materials or solids

0.25 .

RbCl

4 ;I;. Beetle m&t-lx

4 . .

0.10 -

0.05 .

0.00 v

0 200 400 600 000 loo0 1200 1400

TNERUAL PRETREATMENT TEYPEBAlWtE, “C

Figure 1. --Optimization ofpretreatment temperature for RbCl

sample and digested southern-pine-beetle-acid

mat& sample by atomic absorption spectroscopy

with a graphite furnace.

1000 1200 1400 1600 1800 2ooo 2200 2400

ATQYlZATlON TEMPERATURE. %

Figure 2.--Optimization of atomization temperature for RbCl

sample and digested southern-pine-beetle-acid

matrix sample by atomic absorption spectroscopy

with a graphite sueace.

(Perkin-Elmer 1985). In our case, insufficient knowledge

about the constituents of the beetle matrix and the materials

that may cause interferences in measuring Rb prevented us

from duplicating the sample matrix. The possibilities of

nonspectral interferences were examined by checking the

recovery of added standard to a constant volume of unknown.

We found a linear relationship between the combined

absorbance of the sample and the increasing volume of

standard solution indicating interferences are not a problem

(fig. 3). The intercepts of the lines are close to the

concentration of the sample when no Rb was added, showing

that any interferences are consistent across the Rb

concentrations.

However, the solution used to dissolve the needle ash had

a major influence on the recovery of added Rb. When the

ash was in 0.2 percent HN4, the recovery of added Rb was

89 perc.ent. When the solution was 0.2 percent HNO, plus

26
Beetle Matrix

9.0 . .

NBS Pine Needle Matrix

4.0 8.0 8.0

Rb Standard added, pL

Figure 3.--Standard additions technique with RbC1 standard

solution (20&L) to southern-pine-beetle-nitricacid matrix sample, NBS standard pine-needlenitric-acid mat& sample, and NBS standardpineneedle-nitric-plus-perchloric-acid matrix sample.

2 percent HClO,, the recovery dropped to 41 percent. The

presence of perchloric acid in the sample reduced recovery

by half. Rubidium perchlorate (RbClO,) decomposes when

heated (Weast 1975), and some Rb is evidently lost in one

of the earlier drying or pretreatment steps when HClO, is

added.

The concentration of nitric acid also affected the recovery

of Rb from the beetle matrix. Because the sample was

brought to volume with water rather than nitric acid, the

recovery of added Rb was only 67 percent. Thus, the kind

and concentration of the acid used to dilute the samples are

critical. The standards and samples should be made in the

same solution, and any dilutions of the samples or standards

need to be made in that solution and not water.

There was no improvement of the Rb signal with the

addition of ammonium phosphate as a matrix modifier.

Although there was a slight improvement with magnesium

nitrate, contamination problems with this compound negated

any advantage.

LITERATURE CITED

Bain, L.J.; Engelhardt, M. 1987. Introduction to probability

and mathematical statistics. Boston: Duxbury Press. 565

P*

Bridges, J.R.; Thoeny, W.T. ; Tiarks, A.E. 1989. Technique

for studying bark beetle dispersal. In: Payne, T.L.;

Saarenmaa, H., eds. Integrated control of scolytid bark

beetles. Proceedings 18 International Congress

Entomology; April 10-14; Vancouver, Canada.

Blacksburg, VA: Virginia Polytechnic Institute and State

University Press: 307-3 19.

Grobenski, Z.; Weber, D.; Welz B.; Wolff, J. 1983.

Determination of caesium and rubidium by flame and

furnace atomic-absorption spectrometry. Analyst 108:

925-932. “’

Moore, G.E.; Taylor, J.F. 1976. Tagging of southern pine

beetle with phosphorous 32. Environmental Entomology

5: 1065-1067.

Perkin-Elmer. 1985. Techniques in graphite furnace atomic

absorption spectrophotometry. Ridgefield, CT: PerkinElmer Corp. 224 pp.

Slavin, W. 1984. Graphite furnace AAS. A sourcebook.

Norwalk, CT: Perkin-Elmer Corp. 229 p.

Slavin, W.; Camick, G.R.; Manning, D.C.; Pruszkowska,

E. 1983. Recent experiences with the stabilized

temperature platform furnace and Zeeman background

correction. Atomic Spectroscopy 4(3): 69-86.

Stimmann, M.W. 1974. Marking insects with rubidium:

imported cabbageworm marked in the field.

Environmental Entomology 3: 327-328.

Stimmann, M.W.; Wolf W.W.; Berry, W.L. 1973. Cabbage

loopers: biological effects of rubidium in the larval diet.

Journal of Economic Entomology 66: 324-326.

Van Steenwyk, R.A.; Ballmer, G.R. ; Page, A.L. ; Reynolds,

H.T. 1978. Marking pinkbollworm with rubidium. Anna1

of the Entomological Society of America 71: 81-84.

Weast, R.C. 1975. Handbook of chemistry and physics.

Cleveland, OH: CRC Press.

27
Chemical Stimulation-of Host Tree

Antibiosis for Southern Pine Beetle,

Dendroctonus frontalis Zimm., Suppression

M. C. Miller, T. G. Rials, and D. N. Kinn

INTRODUCTION

The southern pine beetle (SPB), D-8

Zimmermann, is responsible for most insect-caused mortality

of timber in the South. Periodic epidemics produce timber

and pulpwood losses valued in the millions (Price and

Doggett 1978). Current methods for slowing or stopping

SPB spots--cut and leave, use of insecticides, pile and bum,

or intensive salvage (Swain and Remion 1981)--are limited by

cost of chemicals, labor and equipment, or environmental

concerns about pesticides (Billings 1980). Due to the

potential hazard to humans, livestock, beneficial insects, fish,

and wildlife (Bennett and Ciesla 1971), there will likely ,be

future restrictions on use of chemicals. Alternative methods

for protecting high value trees from SPB in an economical

and environmentally sound manner are needed.

Induced host plant resistance may have potential as an

alternative method for SPB suppression. Although resistance

to pests and pathogens has been induced by chemical

treatment of some plants, a response against insects has not

previously been reported in treated trees (Stipes 1988). A

formulation of 32.7 percent sodium N-methyldithiocarbamate

(SMDC) in 99.9 percent dimethylsulfoxide (DMSO) (4: 1 v/v)

applied by hack and squirt appears effective against SPB

(Roton 1987). This formulation induces a hypersensitive

response in which long, vertical, necrotic lesions are formed.

The response may change monoterpene composition, does not

kill trees when properly applied, and is apparently not

harmful to natural enemies (Roton 1987). SMDC is a soil

fumigant for control of fungi, bacteria, nematodes, weeds,

and soil insects (Thomson 1986). DMSO is an agricultural

solvent, penetrant, carrier, and antiviral agent (Smale 1969).

The method of introducing the chemical into the target

tree may influence treatment efficacy and acceptance.

Infusion is less contaminating than spraying, requires less

labor and equipment, and relies instead on atmospheric

pressure and the tree’s uptake/translocation capability (Stipes

1988). The research described here shows the residual life

of SMDC and tests the ability of SMDC +DMSO infusion to

inhibit SPB brood production. Also reported here is a

preliminary evaluation of SMDC+DMSO infusion on SPB

ratio of increase (Thatcher and Pickard 1964), a measure of

the success of SPB development in individual trees.

MATERIALS, METHODS, AND RESULTS

Residue Tests. In the fall of 1986, active SPB spots in

Stewart County, Georgia, were used to evaluate residual

activity of the test compound’. Four replications, each of

32.7 percent SMDC in DMSO (4: 1 v/v) in hacks, bark hacks

only, and no treatments (control), were applied to loblolly

pines (Pinus rue&z L.). The chemical (5.0 - 10.0 ml) was

applied to 2.5 cm long by 2.0 cm deep axe frills made

around the circumference of the tree at 1.0 m above the

ground and bridged by 2 to 4 cm of undamaged bark.

Uninfested trees were sampled 1.0 m above the infusion’ ”

point, at midbole, and 1.0 m below the live crown. The

outer xylem, phloem, and bark were split off from small

portions of the trunk at the designated height. The bark was

peeled away, phloem separated from xylem and cut into

small pieces. The upper 1.0 cm of xylem was turned into

wood shavings. Samples were immediately processed,

frozen, and stored in freezers.

Extracts were made from the samples for analysis by gas

chromatography (GC) for methylisothiocyanate (MIT), the

breakdown product of SMDC. A 5-10 g sample of xylem

shavings or phloem bits sat overnight covered with water at

room temperature in tared liquid scintillation vials. The

aqueous portion was salted, 5.0 ml of ethyl acetate added,

and then gently shaken for an hour. The organic layer

containing MIT was drawn off and dried with sodium sulfate.

GC analysis was performed on a capillary column at 180 “C

with a nitrogen-phosphorous detector at 275 “C. Residual

chemical was quantified by comparison of integrator counts

of samples to counts from an analytical standard curve.

Total nanograms of MIT was obtained by linear regression

of emergence on dose per application point and expressed as

ppm MIT, sample dry weight.

In spring 1987, eight pines were treated with chemical

and sampled for residues as in 1986. Samples were taken 1,

4, 7, and 14 days posttreatment. The test was repeated in late

summer, deleting sample day 4 and adding day 21.

In spring 1988, six trees were treated with chemical. One

tree was intensively sampled at 3 heights (low, mid, top) as

described. Three replicates at each height of phloem strips

and drilled xylem shavings were removed and placed on dry

ice. Day 1 samples were taken at 1, 5, 10, and 24 hours.

Subsequent samples were taken on day 4 and weeks 1, 2, 4,

8, 10, 12, 14, and 20. In addition, infusion sites were

sampled on weeks 2, 4, 8, 10, 12, and 14. All residues

were determined as described above.

M.C. Miller is research entomologist, T.G. Rials is research

physical scientist, and D.N. Kinn is research entomologist at

Southern Forest Experiment Station, USDA Forest Service,

Pineville, LA 71360.

‘Berisford, C. W. and M. J. Dalusky. 1989. Efficacy of

systemic toxicants for southern pine beetle control. Final Report,

U.S. Department of Agriculture, Forest Service, SFES

Cooperative Agreement #19-87-097. 11 p., 2 tables, 9 figures.

1989.

28
Results. The 1988 SMDC+DMSO mobility study (fig..

1) confirmed results from 1986 and ‘1987. No residues were

detected above the low height samples. Residual MIT,

detected in the xylem at 1.0 m above the application point at

5 hours posttreatment, was undetectable by day 28. Ploem

residues were detected at 1 .O m’ at lO;hours, peaked at 24

hours, then tailed off. No residues were detected from

application sites after day 21 through day 140. The streaking

effect on the xylem extended to midbole by day 14 and into

the upper bole by week 4. The absence of residues after 21

days suggests that the infused formulation may metabolize

within, or volatilize from, the tree..’

Efficacy tests with hanging bolts, 1987. Loblolly pines

at the head of active SPB infestations (spots) were treated in

Camp Livingston, Catahoula Ranger District, Kisatchie

National Forest, Louisiana. From 19

chemical were placed in horizontal, 1.0 - 5.0 mm deep X 75

mm long, axe frills, 1.0 m above the ground. Six unattacked

trees were felled on the same day 1.3, 12, 18,20, 27.5, and

28 months. posttreatment and each cut into four 150 cm

lengths. Four bolts, each from the 12-, 20-, and 27.5month

trees were moved to Dawson County, Georgia, and, with

. control bolts, were hung from trees at the front of an active

SPB spot (Berisford and others 1980). Trees supporting

bolts were baited with frontalure to ensure attack. Bolts

from the remaining trees and an untreated control tree were

installed at the advancing edge of a SPB spot in Rapides

Parish, Louisiana.

When SPB became pupae or callow adults, 41.0 cm

sections of these bolts were caged to collect emerging

beetles. When emergence ceased, gallery lengths were

measured and oviposition estimates (eggs/m’) calculated

(Foltz and others 1976).

Rotonicide

3 0

Xylem: m Low Phloem: m Low

25’ r

20’

1:

t : + less than detectable level

t

t:

.2 .4 1 4 7 14 28 56 70 84 98 140

Sample day

Figure l.--Residues of SMDC (MIT) detected in the xylem

and phloem of treated pines at 1 .O m above the

infusion point, spring -fall 1988.

Results. In Louisiana, brood emergence from &ted

bolts was 58 percent less than controls.

was 54 percent less emergence from

support trees (fig. 2). There was only

emergence from treated tree bolts. Estimated egg densities

were similar in treated and check bolts, but fewer eggs were

found in treated and check bolts than in support trees (fig.

2). There was less survival in treated bolts (fig. 3) and less

survival with longer treatment-attack intervals. The Georgia

replicates were misplaced, and it was not possible to analyze

the data due to lack ,of experimental error. However, the

emergence counts from each treatment were similar to those

seen in the Louisiana test. .4_

There was a consistent reduction in SPB emergence in

Louisiana. Apparently SMDC+DMSO raduoes brood

production indirectly by eliciting a hypersensitive response. g

Fewer SPB emerged from trees treated from 1.3 to 28

months before felling. Therefore, the induced response can

last more than 2 years. Lower survival in check bolts than

support trees is probably due to desiccation of cut bolts.

Dose response/efficacy tests. Tests were conducted in

active SPB spots consisting of 12, to 27 infested trees in the

Bienville Ranger District, Bienville National Forest,

Mississippi. From November 16-18, 1988, five unattacked

trees at the head of each of six active SPB spots were

randomly assigned to four treatments and a control.

Chemical (3.0, 6.0,9.0, or 12.0 ml) or water (12.0 ml) were

ireatment

Figure 2. --So#hern pine beetle development: estimated

oviposition and emergence counts from bolts from

treated trees, untreated control bolts, and from

bolts taken from support trees. Gates = + 2 SE.

29
m

0

Su+yep$tt W&k 1.3 18 2 8

Months post-SMDCJDMSO

treatment

Figure 3.--Percent survival of southern pine beetle in bolts

taken from suppon trees, untreated check trees,

and SMDC + DMSO-treated. Gates = + 2 SE.

pipetted into horizofual axe frills, as described above. On

February 21, 1989, frontalure was placed on trees to ensure

SPB.attack.

Bark samples (ca. 30 cm long) were removed from the

circumference of the bole at 3.7 and 6.1 m when SPB

reached the pupa/callow adult stage. Eight disks (94 cm3

were removed from each 30<m long sample for radiography,

each placed in a rearing cup, and emerging SPB were

counted. Data were analyzed for each sample height with

polynomial regressions of emergence count means and

weighted with the reciprocal of the variance. Significant

regression of emergence on dose per application point was

plotted to produce a dose-response line with 95 percent C.I.

Five loblolly pines (Pinus taeda L.) in Camp Livingston,

Catahoula Ranger District, Kisatchie National Forest,

Louisiana, were treated on May 23, 1989. The bark was

shaved smooth, and 2.2-cm holes were drilled on 15.2&m

centers around the circumference of each tree. Coiled

TampaxR tampons were pushed into the holes and tamped

even with the bark surface. Each tree received either 2.5,

5.0, or 10.0 ml SMDC+DMSO/hole injected through the

center of the coiled plugs. There were two 10.0 ml/hole

treatment trees in each block, control trees received 10 ml of

water/hole, and infusion sites were wrapped with two layers

of duct tape. The same method was used to infuse trees at

the head of active southern pine beetle infestations of 40 or

more trees on the Yellowpine Ranger District, Sabine

National Forest, Hemphill, Texas, on May 30, and on the

Neches Ranger District, Davy Crockett National Forest,

Ratcliff, Texas, from June 5 - September 30, for a total of

nine replicated trials. If SPB did not attack by posttreatment

day 14, aggregation pheromone (frontalin: a-pinene 1:2) was

applied to ensure attack. The data were processed as

described above for the Mississippi studies.

across doses and heights was fairly constant, the consistency

of the response improved as dosage increased. The residuals

of the observed and predicted values showed that the variance

of the data decreased with increasing dose. Emergence

counts were lower, and the range of the data was narrower

from 6.0 and ‘12.0 ml/hack treatments compared with the

check and other treatment trees.

In Louisiana and Texas, the regression lines for

emergence from both heights were not significant when all

plots were analyzed. However, when the data were

tabulated, emergence was found to be high from all trees

from the first three plots, in which beetle mass attack

occutied 7 days posttreatment. In six later blocks, mass

attack occurred between 14 and 21 days. In these blocks at

the upper height, there was no significant dose-response

regression. However, at the lower height, there was a

significant decreasing linear response (Prob. >F = 0.0001,

rZ = 0.84) as dose increased. Confidence intervals (95

percent) showed significant reductions with increased dose.

At the lO.O-ml infusion rate there was a 9spercent average

reduction in SPB emergence (fig. 4).

Preliminary ratio of increase tests. Bolts (91.5 cm)

were cut at 4.6 m from trees in two blocks installed on the

Neches Ranger District, Davy Crockett National Forest,

Ratcliffe, Texas,on June 6, 1989. Portions (41.0 cm) were

excised from the larger bolts qd placed in rearing cans.

Successful SPB attacks were identified and counted

(Thatcher and Pickard 1964) on four bark disks (94 cm3 cut

from the lower portion of each bolt. The bark surface area

of each bolt was measured, and all data were converted to

counts per 0.1 m*. The ratio of increase (RI) was calculated

for each bolt using the total emergence from each bolt and

the average number of successful SPB attacks from the four

disk subsamples. The RI was derived from the formula (SPB

2.5 8.0

Dose (ml / infusion alto)

Figure 4.--Linear regression of SPB emergence against

SMDC + DMSO applied by the tampon in a hole

method in six replicated in East Texas. The’center

line is the regression surrounded by 95-percent

C.I.

Results. There were no significanct regression lines for

emergence from Mississippi tests. While the mean response

3 0
emergence) / (2 * number successful attacks) (Thatcher and DISCUSSION AND CONCLUSIONS

Pickard 1964). Average SPB brood emergence and RI were

tabulated.

Results. The ratio of increase for untreated east Texas

SPB infested trees in July and August is 0.4: 1 (table 1). The

RI from treated trees is from 10.5 to 1Cfold less than that

of untreated infested trees, and corresponds with a 90 to 97

percent reduction in SPB emergence (table 1).

Infrared spectroscopy. The observed synergism of the

SMDC +DMSO system prompted speculation concerning

possible chemical reaction between the components. To

resolve this question, the infrared spectum of an aged,

SMDC+DMSO mixture was collected, and a direct

subtraction of the DMSO spectrum performed. Infrared

spectra of newly made SMDC and DMSO, of

SMDC+DMSO, and of 3 month SMDC+DMSO mixtures,

were collected with a Nicolet Fourier Transform Infrared

Spectrophotometer (Model DXB-20). Samples were prepared

as a smear on a KB’ pellet and data collected over 10 scans

at a resolution of 4 cm-‘.

Results. When the resulting difference spectrum is

compared with that of a fresh SMDC solution over the region

1,900 cm-’ to 900 cm-’ (fig. 5), some differences in peak

intensities are readily apparent. However, the frequencies of

the absorption bands are esentially identical. The slight shift

to a higher frequency of the C =S peak (1,300 cm-‘) in the

DMSO mixture may indicate some associative phenomenon

between the components. There is no evidence to suggest

formation of any new compound in the neat mixture.

6

I

Figure 5.--Comparison of the injkared spectrum over the

region 1 ,!XXI cm-’ to 900 cm-’ oj CA) afresh SMDC solution,

and(B) an aged, SMDC + DMSO mixture minus DMSO

(d@erence spectrum).

In Georgia, mobility testing showed little or no movement

of MIT above the lower bole, and then only at very low

levels. Residues peaked within 3 days of application and

were only present in the lower bole for 3 weeks. The

residue studies do not support a SMDC +DMSO translocation

hypothesis.

Table 1 .--Ratios of increase and emergence/O.lm’ after

treatment with various doses of SMDC+DMSO

Dose

Ratio of Emergence

Increase /O.lm* Percent

Mean n Mean Change

0 0.42:1 1 26.83

2.5 0.04:1 2 1.08 -95.97

5.0 0.04:1 2 2.12 -89.86

10.0 0.03:1 4 0.77 -97.13

The induced response is at least partially responsible for

inhibiting SPB brood development. Efficacy is a function of

the application method and dose. In Mississippi, there was a

nonsignificant reduction in emergence. In Texas, comparable

doses applied with a tampon in a hole significantly reduced

emergence. The Louisiana hanging bolt studies, showing

reduced emergence, are supported by the Texas results.

Treated trees can affect SPB emergence up to 28 months

after infusion. Girdling of the tree by SPB during mass

attack soon after treatment reduced efficacy.

SPB development is reduced by SMDC+DMSO infusion.

The pitch-soaked lesion response is involved in inhibiting of

SPB brood development. Direct toxicity of the mixture of

active ingredients may not be the main source of mortality.

The small amount of chemical infused into the host tree, the

lack of mobility, and the short-term residues, suggest

stimulation of defensive chemical production. The induced

resinosis and streaking of the xylem appear to be moisture

dependent. The tearing effect was present continuously

under high moisture conditions in Louisiana and after heavy

rainfall in Mississippi. Results are more effective as the

period between SMDC+DMSO infusion and beetle attack

lengthens.

Lack of available moisture for translocation of the

chemical or early attack on the tree by SPB before

development of pitch-soaked lesions may affect resinosis and

length of lesions. Others have observed that residual MIT

production from SMDC fumigation of transmission poles

varies widely in different buffer solutions (Miller and Morrell

1989). Two of us (Miller and Kinn, unpublished) observed

the absence of resinosis in trees treated under drought

conditions and the start of resinosis 2 months later shortly

after a period of heavy rainfall in the summer of 1988.

31
Residual MIT volatilizes at fungitoxic concentrations when

wetted (Zahora and Morrell 1988). This suggests that in the

living tree an active ingredient might remain immobile, or

cells might not be activated, until sufficient moisture is

available. The range of effects of residual MIT on living

trees is not known.

Infusion of SMDC +DMSO may be an alternative to other

suppression tactics. The hazard to the applicator and the user

appears to be low. The LD,, of SMDC is 820 mg/kg, much

less toxic than either Lindane, Chlorpyrifos, or Fenitrothion

(Thomson 1986). DMSO assists in translocation of several

insecticides, but did not markedly lower LD,, values of

insecticides used against boll weevil (Moore and others

1970). Oligman (1965) found no skin penetration of

fluorescein with less than 30 percent DMSO. DMSO may

penetrate animal skin in concentrations above 40 percent

(Moore and others 1970). It also has extremely low toxicity

to humans, animals, and plants (Anon. 1984), and is

exempted from tolerances on raw agricultural commodities

when applied with certain pesticides (Federal Register 1983).

DMSO toxicity is so low that it is measured in grams/kg

(Wong and Reinertson 1984).

Summer is the least favorable time for SPB development,

with RI in East Texas for 1960-62 remaining at less than 1: 1

from June - October, and an average RI of 0.4 for June -

July (Thatcher and Pickard 1964). Roton (1987) found that

most associated insects were not affected by SMDC +DMSO

treatment and a few increased significantly in treated trees.

Our results also show that SMDC+DMSO infusion may

supplement seasonal SPB mortality, especially during

midsummer months. Observations of control and treated

trees suggest that, though the RI during midsummer may be

0.42: 1, SMDC+DMSO infusion lowers this by 10.5- to 14-

fold (table 1). Therefore, induced resistance may be able to

play a role in suppression of breakouts from summer cut-andleave sites. If SMDC+DMSO infusion reduces RI in

individual trees in infestations by a factor of 10, then

collapse of treated spots would be expected. The treatment

might also be of value from fall through spring, when ratios

of increase are normally high.

This method does not rapidly reduce SPB. Studies on

application methods are needed to improve consistency of

results. The physiological mechanisms being stimulated and

the necessary seasonal effects need to be identified to

determine the biochemical basis for toxicity, the optimum

time for application, and the greatest impact on beetle

populations.

LITERATURE CITED

Anon. 1984. Dimethyl sulfoxide (DMSO) for pesticide

formulations [Leaflet No. 326-221. [Place of publication

unknown]: Crown Zellerbach Chemical Products

Division.

Bennett, W. H.; Ciesla, W. M. 1971. The southern pine

beetle, [Leaflet No 491. Washington, DC: U.S.

Department of Agriculture, Forest Service, Southern

Forest Experiment Station. 8 p.

Berisford, C. W.; Brady, U. E.; Mizell, R. F.; [and others].

1980. A technique for testing insecticides for long-term

prevention of bark beetle attack. Journal of Economic

Entomology 73: 694-697.

Billings, R. 1980. Direct Control. Chapter 10. In: Thatcher,

R. C.; Seamy, J. L.; Coster, J. E.; Hertel, G. D., eds.

The southern pine beetle. Tech. Bul. 1631. Washington,

DC: U.S. Department of Agriculture, Forest Service. 126

Fedtral Register. 1983. Rules and Regulations. 48(236):

54818.

Foltz, J. L.; Mayyasi, A. M.; Hain, F. P.; [and others].

1976. Egg-gallery length relationship and within-tree

analyses for the southern pine beetle, Dendroctonus

frontalis (Coleoptera: Scolytidae). Canadian Entomology

Journal 108: 341-352.

Miller, D. B.; Morrell, J. J. 1989. Stability and

methylisothiocyanate production of 12 potential solid

fumigants for controlling wood decay. Holzforschung 43:

191-194.

Moore, R. J., Jr., Taft, H. M.; Payne, L. B. 1970.

Dimethyl sulfoxide as a possible synergist for selected

insecticides against boll weevil. Journal of Economic

Entomology 63: 1342-1343.

Oligman, A. M. 1965. Topical pharmacology and toxicology

of dimethyl sulfoxide - Part I. Journal of the American

Medical Association 193: 140-148.

Price, T. S.; Doggett, C. 1978. A history of the southern

pine beetle outbreaks in the southeastern United States.

Macon, GA: Georgia Forestry Commission. 31 p.

Roton, L. M. 1987. Promising treatment for southern pine

beetles. American Papermaker. October: 30-32.

Smale, B. C. 1969. DMSO, agricultural solvent, penetrantcarrier, antiviral agent. The Sulfur Institute Journal 5:

2-6.

Stipes, R. J. 1988. Glitches and gaps in the science and

technology of tree injection. J. Arboric. 14: 165-172.

Swain, K. M.; Remion, M. C. 1981. Direct control methods

for the southern pine beetle. Agric. Handb. 575.

Washington, DC: U.S. Department of Agriculture. 15 p.

Thatcher, R. C.; Pickard, L. S. 1964. Seasonal variations in

activity of the southern pine beetle in East Texas. Journal

of Economic Entomology 57: 840-842.

Thomson, W. T. 1986. Agricultural chemicals - Book III.

Miscellaneous chemicals; fumigants, growth regulators,

repellents, and rodenticides. Fresno, CA.: Thomson

Publishing. 187 p.

Wong, L. K.; Reinertson, E. L. 1984. Clinical

considerations of dimethyl sulfoxide. Iowa State

Veterinarian 46: 89-95.

Zahora, A. R.; Morrell, J. J. 1988. Decomposition of

methylisothiocyanate in Douglas-fir heartwood. Forest

Products Journal 38: 46-52.

32
Development of Analytical Methods for Escort

Herbicide in Forest Environment Samples

Joseph B. Fischer and Jerry L. Michael

INTRODUCTION

The USDA Forest Service laboratory in Auburn,

Alabama, is engaged in research to determine the

environmental fate and ecosystem impacts of forestry

herbicides. Much of the effort is associated with the conduct

of field dissipation studies in which herbicides are applied to

forest sites and then monitored over time in a variety of

environmental matrices (soil, water, and plant tissue). While

some methodology does exist for both sampling and

analytical procedures, the available methods have often been

developed in an ideal laboratory setting and are not directly

suitable for forest environmental samples. Often, new

methods must be developed to solve site-specific problems.

This paper presents work completed to date on soil and water

method development for a relatively new forestry herbicide,

Escort.

Escort is the DuPont trade name for a formulation of

metsulfuron methyl, a herbicide of the sulfonyl urea family,

whose chemical name is methyl 2-[[[[(4-methyoxy-6-methyl1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl] benzoic

acid. Sulfonyl urea herbicides are much less stable

chemically than other classes (like the phenoxy acids 2,4-D

and 2,4,5-T) and break down rapidly following application.

This makes them environmentally preferable, but it also

presents challenges for storage and handling of field samples

collected for environmental fate studies. Analytical methods

for these herbicides must incorporate measures to minimize

their decomposition between the time they are collected in

the field and the time they are analyzed in the laboratory.

DEVELOPMENT- OF A HIGH PERFORMANCE

LIQtJlD CHROMATOGRAPHIC (HPLC)

SEPARATION FOR METSULFURON METHYL

Metsulfuron methyl is similar chemically to sulfometuron

methyl (trade name Oust), another sulfonyl urea herbicide

previously studied in our laboratory. DuPont’s published

method for sulfometuron methyl (Zahnow 1985) utilized the

Tracer 965 photoconductivity detector together with normalphase HPLC on a bare silica column. We found this detector

to be extremely temperamental and unsuitable for routine

use. The hydrocarbon-based mobile phase proved to be

incompatible with our Waters WISP autoinjectors,

necessitating manual injections on round-the-clock shifts.

The chromatographic system was itself unstable, requiring

Joseph B. Fischer is chemist and Jerry L. Michael is research

ecologist at Southern Forest Experiment Station, USDA Forest

Service, Auburn, AL 36849.

constant recalibration for changing retention times and

responses,and periodic column rejuvenation whenever the

system “crashed.” We developed a more dependable

autosampler-compatible reversed-phase. HPLC separation for

sulfometuron methyl using the ultraviolet absorbance

detector, which proved highly successful (Wells and Michael

1987). These conditions were therefore used as the starting

point for development of a metsulfuron methyl separation.

Successive refinements of these conditions produced the

following mobile phase/column combination, which is

optimized for peak shape and retention time:

Column: Supelco LCS-DB, 150 mm x 4.6 mm I.D.,

5 urn diameter spherical porous particles

Mobile phase: 30:70 (v/v) Acetonitrile/H,O (0.1 M H,PO,,

pH 2.0)

Flow rate: 1.0 ml/min at 25 “C

Detection: U.V. Absorbance at 224 nm wavelength.

The optimum wavelength for detection was determined

from the ultraviolet spectrum of metsulfuron methyl in the

mobile phase. Under these conditions, metsulfuron methyl

elutcd at 6.5 minutes (fig. 1). The detection limit was 0.8 ng

injected on-column, based on a minimum peak height five

times the baseline noise level. Detector response was linear

up to 2,000 ng injected.

DEVELOPMENT OF AN ANALYTICAL METHOD

FOR METSULFURON METHYL IN WATER

Our method for analysis of metsulfuron methyl in water

samples is derived from the method developed in our

laboratory for sulfometuron methyl (Wells and Michael

1987). It is based on the principle of solid phase extraction

(SPE). In SPE, a dilute solution of the analyte is passed

through a column of adsorbent particles whose surface has a

higher affinity for the analyte than does the solvent. The

analyte is immobilized on the adsorbent, then released again

by elution with a small volume of a more powerful solvent.

In reversed-phase SPE, the analyte is nonpolar, the solvent

is polar (usually water), and the adsorbent is nonpolar

(usually silica gel derivatized with long-chain alkyl silane

groups of up to 18 carbon atoms). Elution of the analyte is

achieved with a less polar solvent, such as methanol.

Since metsulfuron methyl is a weak acid (pKa = 3.3), its

ionization had to be suppressed in order to make it

sufficiently nonpolar for reversed-phase SPE. This was

accomplished by acidifying the water sample to pH 2.0 with

85 percent phosphoric acid. Elution from the SPE column

was effected by acetonitrile or methanol, but the resulting

concentrates were unstable, probably due to traces of

phosphoric acid coeluted with the metsulfuron methyl. A

better elution buffer was found to be 30:70 (v/v)

acetonitrile/H,O (pH 7.0/w 0.1 M potassium phosphate),

33
Metsulfuron Methyl Standard

k

Spiked Well Water Extract

20 ng injection 20 ppb Metsulfuron Methyl

Figure 1 .--LC chromatograms of metsulfuron methyl.

which stabilized the extracts and was compatible with the

HPLC mobile phase. Due to the extreme sensitivity reported

for one aquatic species to metsulfuron methyl, the lowest

possible detection limit was desired. This was done by

maximizing the size of the water samples (to 500 ml) and

minimizing the volume of the final eluted extract (to 5 ml).

Sulfonyl urea herbicides are known to be hydrolytically

unstable in acid solution (E. I. DuPont de Nemours and Co.,

Inc. 1984). Samples of surface water taken from a treatment

site in Florida had a pH value of 4.2. In order to determine

the stability of metsulfuron methyl in field water samples of

varying pH, a set of distilled water samples was buffered

with 0.1 M potassium phosphate to pH 4.0, 5.0, and 6.0,

then spiked with known amounts of metsulfuron methyl and

stored at 35 “C. Five replicate samples at each pH level

were analyzed every few days to monitor the percent age of

the original spike remaining. The results are plotted in fig.

2 . Hydrolysis was rapid at pH 4.0 and pH 5.0 and

appreciable even at pH 6.0. It was therefore deemed

necessary to add buffer to the sample containers in the field

to stabilize the water samples as they were collected.

Accordingly, a mixture of 0.04 M KH2P0,, and 0.06 M

K,HPO,, which produces a pH of 7.0, was added in dry

form to each 1 liter field container. The containers were

frozen as soon as possible and kept frozen during transport

and storage.

Since the water samples are adjusted to pH 2.0 to

suppress their ionization prior to SPE, a recovery study was

done to determine the effect of time at this pH on

metsulfuron methyl recovery. A set of spiked water samples

was adjusted to pH 2.0 and allowed to stand on the lab bench

at 25 “C for periods of 1, 2, and 3 hours before being

adsorbed on SPE columns and eluted with pH 7.0 buffer.

Significant loss was observed even after 1 hour (fig. 3)

reaching 15 percent by 3 hours. This loss was minimized by

performing the SPE adsorption and elution steps as quickly

as possible after acidification (less than 1 hour if possible)

and by recording the time at pH 2.0 so that a correction

factor could be applied if necessary. Prompt elution gave a

recovery of 98.7 percent.

A test was conducted to determine if acidification to pH

2.0 was really necessary to get maximal adsorption of

metsulfuron methyl on the Cle-SPE columns. A set of 500

ml water samples was buffered to pH 7.0 with 0.1 M

potassium phosphate, then spiked with 40 ppb each of

metsulfuron methyl. The pH of each spiked solution was

then adjusted with 85 percent H,PO, to values ranging from

pH 2.0 to pH 7.0. These pa-adjusted water samples were

then adsorbed and eluted from the SPE columns. Some of

the columns were prewashed with methanol followed by pH

2.0 water and postwashed with pH 2.0 water. Some were

prewashed with methanol and pH 2.0 water but not

postwashed. Some were prewashed with methanol and pH

LOS

Percent

ReCQVerY

0.0 L81’ I

0 IO II 10 21 30

Day* .t 36 OC

-f+ PH 4.00 LOG Y - -0.864X * 1.2418 - 0.899

+

pti 6.00 LOQ Y . -0.0621x l 1.9816 1’2 - 0.977

-I- pti 6.00 LOQ Y . -0.0000x l 1.9864 R2 - 0.966

Figure 2.--Efect of water sample pH on metsulfiron methyl

recovery.

‘O”*

Figure 3.--Eflect of time at pH 2.0 on metsulfiron methyl

recovery.

34
7.0 buffered water with no postwash. The results are

graphed in fig. 4. In all cases but one, the recoveries were

in the range of 95-99 percent. Apparently, metsulfuron

methyl is sufficiently insoluble in water that it adsorbs to the

C,,-SPE columns even without ion-suppression by acid. The

0.1 M potassium phosphate buffer in the water samples may

also contribute to suppression of ionization of metsulfuron

methyl by the “salting-out” effect.

The final method is outlined as follows:

(1) Collect the water sample in the field from a well or an

automatic streamflow sampler (ISCO), add 0.1 M potassium

phosphate buffer to adjust the pH to 7.0,‘and freeze as soon

as possible.

(2) Thaw the sample just prior to analysis. Measure the

sample volume with a graduated cylinder.

(3) Precondition an SPE column containing 1000 mg of C,,-

derivatized silica gel adsorbent (Raker #7020-7) by washing

with 5 ml methanol followed by 10 ml of 0.1 M potassium

phosphate buffer (pH 7.0).

(4) Pass the sample (500 ml) through an SPE column at a

rate of 4 drops per second or less, controlled by adjusting the

vacuum applied to the column.

(5) Elute the column with 4.5 ml 30:70 (v/v)

acetonitrile/H,O) (ph 7.0/w 0.1 M potassium phosphate

buffer). Make up to exactly 5.0 ml with buffer. Mix

thoroughly.

(6) Transfer the sample to an autosampler vial, filtering

through a Millipore Millex-SR membrane filter if cloudy.

(7) Inject 10 ~1 on the HPLC. Quantitate against external

standards injected before and after every fourth sample.

900

- 2 2 4 6 6 7

pli of Water SampI*

* Prawaeh and po.tw..h with pH 2.0 w.te,.

0 Prewalh with pH 2.0 water, no PoatwaM

m Pr.wa.h with pH 7.0 bulfer. no poatwaah.

Figure 4.--Metsulfuron methyl SPE recovery vs. pH of water

extracted.

Many samples of water from ponds and shallow wells at

treatment sites are not clear, but instead tinted amber with

dissolved organic matter. This complex polymeric material,

principally humic and tannic acids, co-adsorbs and co-elutes

with metsulfuron methyl from the solid phase adsorbent,

competing for adsorption sites and reducing recovery. It also

interferes with the HPLC separation, reducing the signal-tonoise ratio and lengthening equilibration time between

injections. At the Florida treatment site, this has limited the

overall concentration factor we can reliably use to 100: 1 (500

ml water sample concentrated to 5 ml final extract) and the

amount of extract we can safely inject on the HPLC system

to 10 ~1. This has made our practical detection limit in

water about 1 ppb.

We have experimented with adding small amounts of

divalent metal salts to “humifled” water samples to

precipitate the organic material prior to SPE. Lead (II)

acetate (0.01 M) was very effective in precipitating organic

material from unbuffered water, producing a clear

supernatant and a flocculent brown precipitate that could be

removed by suction filtration through glass fiber paper.

Unfortunately, when this treatment was applied to water

buffered with 0.1 M potassium phosphate, a grey precipitate

of lead phosphate formed that interfered with filtration and

left the organic material in solution. This treatment may

prove more effective when applied to herbicides that do not

require buffering for field stabilization.

DEVELOPMENT OF AN ANALYTICAL METHOD

FOR METSULFURON METHYL IN SOILS

In environmental fate studies, it is necessary to sample

soil at different depths to detect movement of the herbicide

underground. In our laboratory, a method has evolved for

collecting and processing soil samples that has proven

effective for other herbicides (Michael and Neary 1988). A

2-inch I.D. PVC pipe is driven into the ground to a

prescribed depth, then removed and frozen for transport to

the laboratory. The frozen pipe is cut into 4-inch (lo-cm)

sections with a band saw. Each section is thawed, extruded,

and spread out to dry on aluminum foil. When dry, each

soil sample is screened to remove roots and pebbles, then

refrozen in a zip-lock bag. The dry soil is passed through a

14-channel soil splitter several times until a representative

sample of suitable size for analysis is obtained. The

extraction of metsulfuron methyl proceeds as follows:

(1) A 50.0-g sample of the soil is weighed into a 250-m&

polypropylene, widemouth, screw-capped jar.

(2) A lOO-ml portion of extracting buffer solution [50:50

(v/v) methanol/H,0 (pH 7.0/w 0.1 M potassium phosphate)]

is added, and the mixture is shaken for 10 minutes on a table

shaker.

(3) The mixture is centrifuged at 3,000 rpm for 10 minutes,

and the supematant is decanted.

35
(4) Steps 2 and 3 are repeated, and the supernatants are

combined and suction-filtered through glass fiber paper (1

micron, GF/B, Whatman).

(5) The filtrate is rotary evaporated under reduced pressure

on a warm water bath (45-50 “C) to remove the methanol

component.

(6) The aqueous residue is extracted and eluted by SPE in the

same manner as the water samples.

Unfortunately, polymeric materials also coextract with

metsulfuron methyl from soils, and present serious problems

in surface soils with high organic matter content. Soils low

in organic matter give metsulfuron methyl recoveries of

about 90 percent, whereas high organic soils can give

recoveries as low as 70-80 percent. The detection limit is

about 20 ppb for high organic soils.

Soils cannot be buffered as can water samples, and many

forest soils have pH values around 4-5. We therefore ran an

experiment to test the effect of soil drying time at room

temperature on metsulfuron methyl recovery. A large sample

of surface soil @H 4.3) was tested to determine its field

water capacity (43 percent w/w) and then was air-dried and

subdivided into test samples using a soil splitter. These

samples were individually treated with water to bring them

to 25 percent, 50 percent, and 75 percent of their field

capacity, then spiked with metsulfuron methyl and allowed

to air-dry for 1, 2, and 4 days before being extracted and

analyzed (fig. 4). We observed an appreciable drop in

metsulfuron methyl recovery over the first 2 days of drying

with a less rapid decline thereafter. This corresponds to

visual observations that drying was complete by 2 days and

-suggests that hydroIysis is indeed taking place while the-soil

is damp. We are presently trying to modify our soil workup

to eliminate the drying step and obtain a representative soil

sample without splitting.

CONCLUSIONS

We have developed workable methods for analysis of

metsulfuron methyl (Escort) herbicide in environmental

samples of water and soil from forestry test sites. Detection

limits are about 1 ppb in water and 20 ppb in soil. Measures

for minimizing hydrolysis prior to analysis have been

established for water and are under development for soil.

&extraction of dissolved organic matter remains a problem,

especially for high organic soils.

LITERATURE CITED

E . I. DuPont de Nemours and Co., Inc. 1984. Technical

aspects of DuPont sulfonylurea herbicides for industrial

weed control. DuPont Technical Report, Wilmington,

DE: 10 pp.

Michael, J. L.; Neary, D. G. 1988. Environmental fate of

sulfometuron methyl in southern forest watersheds. In

Proceedings, Southern Weed Science Society. 41: 219.

Wells, Martha J. M.; Michael, Jerry L. 1987. Reversedphase solid-phase extraction for aqueous environmental

sample preparation in herbicide residue analysis. Journal

of Chromatographic Science. 25(8): 345-350.

Zahnow, E.W. 1985. Analysis of the herbicide sulfometuron

methyl in soil and water by liquid chromatography.

Journal of Agriculture and Food Chemistry 33: 479-483.

36
Evaluation of Worker Respiratory Exposure

to Herbicide Residues in Prescribed

Fire Smoke: A Preliminary Report

scenario. The area monitors were placed in zones of high

smoke concentrations by research personnel to measure

herbicide and “total” smoke particulate matter concentrations

under a worst-case operational scenario.

C. K. McMahon and P. B. Bush

RESULTS

INTRODUCTION

Occupational safety and health concerns have been raised

in a number of southern States by workers conducting

prescribed bums on forest lands treated with herbicides.

Primary concern has focused on speculation that hazardous

levels of herbicide residues are present in the smoke near

breathing zones of forest workers. This speculation is fueled

by fire hazard caution statements found on product labels and

material safety data sheets. While these cautions are

appropriate in connection with fires near herbicide storage

sites, they were not intended to apply to fires following

application on a forest site. In these cases, on a given acre,

only a few ounces or pounds of active ingredient are spread

over several tons of ground litter and forest vegetation,

which becomes the dominant fuel for the prescribed fire.

Modeling assessments (Dost 1982, U. S. Department of

Agriculture 1989) coupled with laboratory experiments

(McMahon and others 1985, McMahon and Bush 1986) have

shown that the herbicide risk to forest workers is

insignificant, even if the fire occurs immediately after

herbicide application. However, no field studies have been

conducted to confirm these findings.

METHODS

To bridge that gap, a field validation study was conducted

in Georgia in August-October 1988 to measure breathing

zone concentrations of smoke particles, herbicide residues,

and carbon monoxide (CO) on 14 operational site-preparation

prescribed fires; Smoke was monitored on sites treated with

labeled rates of imazapyr, triclopyr, hexazinone, and

picloram (table 1) and burned operationally within 30-169

days after herbicide application. Tract size ranged from 6 to

380 acres (table 2). Prebum herbicide residues were

estimated by analyzing samples of standing hardwood foliage,

ground litter, and surface soil (table 3).

Personal monitors and area monitors employing glass

fiber tilters and polyurethane foam collection media were

developed and validated for use in this study. A complete

description of the monitors and analytical methods has been

previously reported (McMahon and Bush 1990a, McMahon

and Bush 199Ob). The personal monitors were worn by

forest workers to measure herbicide and “respirable” smoke

particulate matter concentrations under a normal operational

No herbicide residues were detected in the smoke sampled

from the 14 fires conducted in this study. This included a

total of 48 personal monitors worn by forest workers, 22

personal monitors worn by research personnel, and 70 area

monitors. The sensitivity of the monitoring methods were in

the 0.1 to 4.0 pg/m3 range, which is several hundred to

several thousand times below any known herbicide inhalation

risk level.

The monitoring of particulate matter and CO on these

tires marks the first time worker exposure to these smoke

constituents has been measured in the South. As expected,

concentrations were highly variable depending on fire

conditions and the location (upwind vs. downwind) of the

personnel. Worker respirable (2.3 pm particle cutpoint)

particulate matter concentrations ranged between 248 and

3,723 pg/m3 with a mean of 1,429 gg/m3. Exposure times

depended on fire size and ranged from 1.2 to 6.3 hours with

a mean of 2.8 hours (table 4). Area monitor total particulate

concentrations ranged between 2,000 and 45,000 pg/m3 with

a mean of 8,311 pg/m3. Based on the experiences of the

research personnel who deployed these monitors, the area

concentrations often exceeded tolerable long-duration

working conditions. The respiratory distress and eye

irritation associated with the higher values (above 8,000

pglm3) would prompt most workers to retreat to areas of

lower smoke concentration after only a few minutes

exposure. At this time, no occupational limits or guidelines

have been established for worker exposure to either

respirable or total smoke particulate matter.

Worker exposure to CO ranged from less than 6 to 23

ppm/hr while working on the fires. These values are well

below the Occupational Safety and Health Administration

(OSHA) permissible exposure limit (PEL) for CO of 35

ppm/hr, when normalized to an 8-hour workshift.

In summary, the findings of this study support the “no

significant herbicide risk” findings of the earlier modeling

studies. In addition, the information on smoke particulate

matter and carbon monoxide will be useful to those beginning

to address other questions of worker safety and risk from

exposure to forest fire smoke. These topics are discussed in

more detail in the project final report (McMahon and Bush

1990b).

C.K. McMahon is project leader at Southern Forest Experiment

Station, USDA Forest Service, Auburn, AL 36849; P.B. Bush is

at Cooperative Extension Service, University of Georgia, Athens,

GA 30602.

37
Table 1. --Herbicide formulations, active ingredients (ai), and common names of

herbicides monitored in a smoke study in Georgia, August-October

1988

Formulation Active ingredient Common name

Pronone 10G’

Granular herbicide

10 percent ai

soil active

Velpar ULW+

Granular herbicide

75 percent ai

soil active

Garlon 4t

Liquid herbicide

61.6 percent ai

44.3 percent aer

foliar active

Arsenal++

Applicators

Concentrate

Liquid herbicide

53.1 percent ai

43.3 percent ae

foliar and

soil active

Tordon KSt

Liquid herbicide

24.0 percent ai

20.8 percent ae

foliar and

soil active

hexazinone (3-cyclohexyl6-(dimethylamino)-l-methyl

1,3,5-triazine-2,4(1H, 3H)-dione)

hexazinone (3-cyclohexyl6-(dimethylamino)-l-methyl1,3,5-triazine-2,4(1H, 3H)-dione)

Triclopyr (3,5,6-trichloro-2-

pyridinyloxyacetic acid),

butoxyethyl ester

Imazapyr (2-[4,5dihydro-4-

methyi-4-( 1-methylethyl)-5-oxolH-imidazol-2-yl]3-pyridine

carboxylic acid), isopropylamine salt

Picloram (4-amino-3,5,6-

trichloropiclinic acid)

potassium salt

Hexazinone

Hexazinone

Triclopyr

Imazapyr

Picloram

* Trademark, Pro-Serv Inc.

+ Trademark, E.I. duPont de Nemours and Company, Inc.

* Trademark, DowElanco

* ae= acid equivalent

‘+ Trademark, American Cyanamid Company

*+ Trademark, DowElanco

38
Table 2.--Prescribed Jire study areas and treatments in 1988

Fire

no. Location

Acres Herbicide treatment

burned formulation and rate

Date of

herbicide

application

(4

Bum Interval *

date @YS)

(B) (B-4

Herbicide ai’

monitored in smoke

1 0

11

W-4)

W4

12(C)

13(A)

13(B)

1 4

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbtidge, GA

Bainbridge, GA

Harris Cty, GA

Harris Cty, GA

Meriweather

Cty, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

Bainbridge, GA

42 Pronone lOG, 25 Ib/ac 5120188 8129188 101 Hexazinone

I Garlon 4, 3.5 qts/ac l/29/88 8130188 3 2 Triclopyr

13 Accord, 2 qt/ac; Garlon 4, 2 qtlac 7129188 8130188 3 2 Triclopyr

8 Accord, 5 qt/ac 718188 9113188 61 None

42 Arsenali, 1 ptlac; Accord, 3 pt/ac 717188 9114188 6 9 Imazapyr

3 2 Arsenal, 1 pt/ac; Garlon 4, 3 pt/ac 812188 9115188 4 4 Triclopyr

200 Velpar ULW, 4.7 Ib/ac 4/13/88 9127188 167 Hexazinone

380 Velpar ULW, 4.7 Ib/ac 4113188 9128188 168 Hexaxinone

320

6

42

48'

48W

6

Velpar ULW, 4.7 Ib/ac

Arsenal 3 pt/ac*

Pronone lOG, 25 Ib/ac

Arsenal, 3 pt/ac*

Arsenal, 1.5 pt/ac

Arsenal, 1 pt/ac

Garlon 4, 2 qt/ac, Tordon K , 2 qt/ac

Garlon 4, 2 qt/ac, Tordon K , 2 qt/ac

No herbicides applied

4113188

9125188

5120188

7112188

7112188

7112188

7121188

7121188

___

9129188

10125188

10/25/88

10126188

10126188

10126188

: 10126188

10/26!88

10127188

169

3 0

158

106

106

106

9 7

9 7

__

Hexaxinone

I~zmyr

Hexazinone

Inwwr

I-wyr

I~znvr

Triclopyr & Picloram

Triclopyr & Picloram

None

* ai = active ingredient. Except for triclopyr and picloram, smoke monitoring procedures only allowed sampling for one herbicide when tank mixtures

were used. Glyphosate (Accord) was not included in the monitoring program for this study.

’ All present treatments were with Arsenal Applicators Concentrate formulation.

* Three-pint rate applied under an experimental use permit; for research purposes, on sites 10 acres or less. Under normal use, the maximum label rate

for Arsenal Applicators Concentrate is 2.5 pints/acre.

r Three sub-plots, 10, 15, and 23 acres respectively.

tt Two sub-plots, 24 acres each.

39
Table 3.--Herbicide’ residues in preburn site samples’

Hardwood

foliage

Ground

litter

Surface

soil

a. Hexazinone residues

Fire #l 101 DAT’

Fire #7 & #g 167 DAT

Fire #9 169 DAT

b. Imazvuvr residues

Fire #5 69 DAT

Fire #lO 1 DAT ’

Fire #lO 30 DAT

Fire #lZABC 106 DAT

c. Triclonvr residues

Fire #2 32 DAT

Fire #3 32 DAT

Fire #6 44 DAT

Fire #13AB 97 DAT

d. Picloram residues

Fire #13AB 97 DAT

0.08 f 0.50

< 0.05

0.12 + 0.11

0.06 + 0.04

< 0:05

c 0.05

CO.5 CO.5

4.6 + 3.2 4.1 * 4.0

1.5 * 1.2 2.1 f 2.6

c 0.5 <OS

21.0 f 23.7

9.9 + 9.8

10.6 + 5.9

8.4 + 13.2

11.2 & 10.1

4.3 * 2.3

2.8 f 1.7

1.6 f 1.1

1.14 f 1.19 0.35 f 0.28

0.24 f 0.20

0.58 f 0.54

0.25 f 0.21

<0.5

< 0.5

<0.5

<0.5

0.16 + 0.11

0.43 f. 0.60

0.21 f 0.29

0.54 f 0.43

0.21 f 0.22

* Values in mglkg. Average + standard deviation, n=6. Detection limits for

hexazinone, triclopyr, and picloram, 0.05 mg/kg; for imazapyr 0.5 mg/kg. In those cases

where a sample was below the detection level, one-half the detection level was used in

computing the average value.

+ DAT = Days after herbicide treatment.

* Fire # 10 burned 30 DAT, however, site samples were also taken 1 DAT.

Table 4.--Smoke respirable particulate matter concentrations and exposure

times for forest workers (by jre size)

Particulate matter &g/m3)

Low High Mean f S.D.

Exposure time

Hours f S.D.

Small fires (a ac av) 405 2,312 1,118 + 625 1.4 + 0.2

Fires 2,3,4,10,14

Medium fires (42 ac av)

Fires 1,5,6,11,12,13 694 2,375 1,573 + 498 2.3 + 0.4

Large fires (300 ac av)

Fires 7,8,9 248 3,723 1,596 f 1,140 4.7 + 0.8

All fires 248 3,723 1,429 f a01 2.8 f 1.8

Particle 50 percent cutpoint, 2.3 urn (aerodynamic diameter).

40
Dost, F.N. 1982. Combustion of herbicides. Unpublished

report prepared for Bonneville Power Administration.

Available from Author, Department of Agricultural

Chemistry, Oregon State University, Corvallis, OR. 24 p.

McMahon, C.K.; Bush, P.B. 1986. Emission from burning

herbicide treated forest fields -- a laboratory approach. In:

Proceedings, 79th annual meeting of Air Pollution

Control Association; 1986 June 22-27; Minneapolis, MN.

Pittsburgh, PA: Air Pollution Control Association. 2 p.

McMahon, C.K.; Bush, P.B. 1990a. A low cost sampler for

monitoring worker exposure to herbicide residues in

forest fire smoke. In: Proceedings, 1990 EPAIAWMA

international symposium on measurement of toxic and

related air pollutants; 1990 April 30-May 4; Pittsburgh,

PA: Air and Waste Management Association. 498-505.

McMahon, C.K.; Bush, P.B. 199Ob. Final Report.

Evaluation of worker respiratory exposure to herbicide

residues in the smoke from prescribed fires in the South.

FS-SO-4105-1.21 NAPIAP Project SO-30. Available from

Author, 60 p.

McMahon, C.K.; Clements, H.B.; Bush, P.B. [and others].

1985. Pesticides released from burning treated wood. In:

Donoghue, Linda R.; Martin, Robert E., eds.

Proceedings of the eighth conference on fire and forest

meteorology; 1985 April 19-May 2; Detroit, MI.

Bethesda, MD: Society of American Foresters: 145-152.

U. S. Department of Agriculture. 1989. Final environmental

impact statement: vegetation management in the Coastal

Plain/Piedmont. Vol. II. Mgt. Bull. RS-MB-23. Atlanta,

GA: US Department of Agriculture, Forest Service,

Southern Region. 462 pp.

41
NMR Experiments for Elucidation

of the Structure of Flavanoid

Oligomers: Some Examples

Richard W. Hemingway, Jan P. Steynberg, and Seiji Ohara

INTRODUCTION

The phenomenal advance in our understanding of the

chemistry of condensed tannins and related flavanoid

derivatives over the past 10 years is largely due to new NMR

experiments made possible by modem superconducting FT

spectrometers. For example, it was only 10 years ago, in the

Alexandria Forestry Center, coincident with Lawrence

Porter’s work in New Zealand, that 13C-NMR spectra of

condensed tannin polymers extracted from conifer tree barks

were recorded (Karchesy and Hemingway 1980, Czochanska

and others 1980). These spectra contain a vast quantity of

information about the structure of flavanoid polymers (1)

(fig. 1) including the hydroxylation pattern of the A- and Brings, the stereochemistry of the chain extender units, the

stereochemistry of the terminal units, and an approximation

of the number average molecular weight of the polymers (fig.

1) (Porter and others 1982, Newman and others 1982).

However, new two-dimensional experiments are so powerful

that few studies of flavanoid oligomers or their derivatives

are now based on a simple ‘H- or proton decoupled i3C-NMR

experiments. The objective of this report is to present some

examples of how modem NMR experiments have been used

to determine the structure of flavanoid oligomers in hopes

that they might be of use to others addressing structural

problems in the Southern Station.

RESULTS AND DISCUSSION

“C-NMR and the Stereochemistry of the Heterocyclic

Ring in Profiietinidins. Although 13C-NMR has been

widely used in the study of the procyanidins (polymers based

on 3,5,7,3’,4’-pentahydroxyflavans; i.e. (l)), it has been

largely ignored in study of the profisetindins (polymers based

on 3,7,3’,4’-tetrahydroxyflavans). Jan Steynberg has made

a series of profisetinidins during his studies in Pineville (fig.

2) (Steynberg and others, in preparation-b). These

compounds have made possible a study of the influence of

stereochemistry on the chemical shifts of carbons in the

heterocyclic rings of these compounds (Steynberg and others,

in preparation-a). Even a dimeric compound contains five

chiral centers, so definition of the relative stereochemistry by

NMR methods is very important. The absolute

Richard W, Hemingway is project leader at Southern Forest

Experiment Station, USDA Forest Service, Pineville, LA 71360;

Jan P. Steynberg is visiting scientist from Department of

Chemistry, University of the Orange Free State, Bloemfontein,

South Africa; Seiji Ohara is visiting scientist from the Forestry and

Forest Products Research Institute, Tsukuba, Ibaraki, Japan.

stereochemistry can then normally be defined from CD

spectra, which establish the absolute stereochemistry at the

C-4 position:

The relative stereochemistry at C-2 and C-3 is readily

apparent in ‘3C-NMR spectra because of the large downfield

shift of the C-2 carbon signal in 2,3-tram isomers such as (2)

or (3) figure 2, in comparison to the chemical shift of C-2 in

the 2,3-cis isomers such as (4) or (5) (See fig. 3-5). In

contrast to the *‘C-NMR spectra of procyanidins (l), spectra

of the 2,3-trans profisetinidins such as (2) or (3) also reveal

the relative stereochemistry at C-4 by the large downfield

shift of the C-4 signal in the 2,3-tram3,4-cis isomers.

Definition of the stereochemistry of the terminal units is also

straightforward from the 13C-NMR spectra (fig. 5) where the

2,3-tram isomers show resonances for C-2 and C-3 at about

82 and 68 ppm, whereas these carbons of 2,3-cis terminal

units appear at about 79 and 66 ppm, respectively. One of

the primary advantages of 13C-NMR is that plant extracts

containing proanthocyanidins can be easily surveyed for

hydroxylation pattern and stereochemistry by 13C-NMR

without the necessity of making or isolating individual

compounds. Another advantage is that we can learn

something about the conformational properties of these

compounds in the free phenolic state. The spectrum of the

2,3-truns-3,4-tran isomer (2) is a case in point (fig. 3).

Doubling of the C-2 carbon resonance is considered to be

due to two rotational isomers, and integration of these signals

provides an estimate of the relative proportions of each. We

can also see doubling of the C-4 carbon of the C-ring.

Because of symmetry (fig. 6) with respect to this carbon in

the two rotamers, we might attribute doubling of this carbon

signal to differences in the proportions of A and E

conformers between the two rotamers (fig. 7).

‘H n.0.e. Difference Spectra for Proof of the Location

of Substitution and evidence for Rotational Isomerism in

a Trhner of Miied Stereochemistry. The great power of

‘H n.0.e. difference experiments is demonstrated in its

application to determine the structure of a trimer of mixed

stereochemistry that was isolated from Guamuchil bark

(Steynberg and others, in preparation-a). A ‘H-nmr spectra

showed that this trimer contained both a 2,3-tram3,4-trans

fisetinidiol unit and a 2,3-cis-3,4-cis ent-epifisetinidol unit

substituted at either C-6 or C-8 of a terminal 2,3-cis

epicatechin unit [i.e., (6)]. Assignment of the location of

substitution was possible from consideration of n.0.e.

enhancements from the 5-OMe and 7-OMe of the D-ring

(fig. 8). Since only the 7-OMe would result in enhancement

of the 4-H of both rings C and I, the 2,3-trans-3,4-tram unit

with J2,3 and J3,4 both about 10 Hz must be the unit at C-8

and the 2,3-cis-3,4-cis unit with coupling constants of J2,3 =

3.0 and J3,., = 5.0 Hz must be at C-6 of the D-ring.

This same experiment also provides evidence on the

rotational mobility of the two profisetinidin units in this

trimer. The 2,3-cis-3,4-cis unit substituted at C-6 of ring D

rotates freely about the interflavanoid bond as evidenced by

the n.0.e. enhancement of the 5-H, proton from both 5-

4 2
OH

Figure 1 .--i?zejirst “C-NMR spectrum recorded for a tannin extract from

a conifer tree bark.

w”gfo” HOgio”

OH OH

(2) 1,1,1 (4) 11181

(3) -

(5) -

Figure 2.--Structures of profisetinidins illustrating variations

in stereochemistry.

OMe,,, and 7-OMe,,. For this to occur, there must be

comparatively free rotation about this bond. In contrast, the

2,3-trans-3,4-truns unit at C-8 of the D-ring does not appear

to rotate freely as suggested from the absence of an n.0.e.

enhancement between the 7-OMe,, and the 5-H,,. The

freedom of rotation observed for methyl ether acetate

derivatives of the 2,3-cis-3,4-cis profisetinidin units is most

unusual.

C-H HETCORR Experiment to Assign Carbon Signals

in the Propyl Chain and Nonatrione Ring System of a

Diarylpropanol-Catwhiqic Acid Dimer. During Seiji

Ohara’s year of study in Pineville, he made an interesting

compound (7) that suggests stereospecific intra- and

intermolecular reactions of catechin in base (fig. 9) (Ohara

and Hemingway in press). There are many instances in

which it is difficult if not impossible to assign carbon signals

when these spectra are considered alone. The complexity of

the proton decoupled 13C-NMR spectrum of the methyl ether

derivative of Ohara’s compound (8) is a good example (fig.

10). However, the Hetcorr experiment (fig. 11) solves most

of these problems. This connectivity experiment allows us

to determine what protons are attached to what carbons. B y

combining information on the chemical shifts and coupling

constants obtained from the proton spectra with chemical

shifts of the attached carbons one can usually assign the

spectra and solve the structure. For example, connectivity

between the propyl 1-H at 4.06 with the carbon signal at

49.63 and the 2-H at 5.20 with the carbon signal shifted far

downfield at 94.71 is clear from this spectrum. The C-3

carbon is assigned to the signal at 28.1 by its connectivity

with the double doublets centered at 2.90 and 3.10 ppm. In

addition, most of the carbons of the nonatrione ring C system

are readily assigned from consideration of the Hetcorr

experiment. The proton signal at 3.32 ppm assigned to 1-H

was correlated with the carbon signal at 66.63, and the

43
Figure 3.--“C-NMR spectrum of 2,3-trans-3,4-trans (2).

O h

OH

OH

OH

I

Figure 4.--“C-NMR spectrum of 2,3-trans-3,4-cis (3).

44
O H

O H

(4) 1,111

(5) -

Figure 5.--“C-NMR spectrum of a mixture of 2,3- cis -3,4- cis with

epicatechin and catechin terminal units (4) or (5).

Figure &--Two rotameric for& of a proJisetinidin.

t

Figure 7.--A and E conformers of a procyanidin, catechin-(4a -- > 8)-catechin.

45
OMe

3.38 5-OMe(D)

----4-

3.47 7-OMe(D)

---v6.82

OMe

Figure %--An n.0.e difference experiment showing the location of substitution of

2,3-tram and 2,3-cis projsetinidin units on a trimer.

Home& “o&; no& O H O H O H

O H

O H

‘~<,O,- 0

Ii R?., -

0”; ‘: o*’ ..OH i b.. . . . . . .-

Figure 9.--Stereoselective base-catalyzed reactions of (+)-catechin.

46
1,,,,,,,,,,,, 200 180 160 140 ,,, I,, r,,,,,, 1 I

120 100 80

, mI

w 40

Figure IO.--“C-NMR spectrum of the methyl ether derivative of the main reaction

product of base-catalyzed reactions of catechin at 40 “C.

Figure 11 .--l&e “C-‘H Hetcorr spectrum of (8).

multiplet at ppm that is clearly 7-H is correlated with the

close signal at 67.03. The broad signal at 3.50 ppm assigned

to 5-H is correlated with the carbon signal at 48.2. Similar

correlations for the 6 and 8 carbons permitted assignment of

all carbon signals except the carbonyls.

CONCLUSIONS

These examples are but a few of the experiments that are

required to obtain proof of the structure of flavanoid

oligomers by NMR methods. Other ‘H n.0.e. difference

experiments are useful for assignment of the conformational

features of these compounds and are described more fully by

Ferreira and Brandt (1989). A recent book (Martin and

Zektzer 1988) provides very readable additional details on

the nature of these experiments and their application to the

elucidation of structure of complex organic compounds.

LITERATURE CITED

Czochanska, Z.; Foo, L.Y.; Newman, R.H.; Porter, L.J.

1980. Polymeric proanthocyanidins. Stereochemistry,

structural units, and molecular weight. Journal of

Chemical Society, Perkin Trans. 1: 2278.

Ferreira, D.; Brandt, E.V. 1989. New NMR experiments

applicable to structure and conformational analysis. In:

Hemingway, R. W.; Karchesy, J.J.; Branham, S.J.; eds.

Chemistry and significance of condensed tannins. New

York: Plenum Press: 153-175.

4 7
Karchesy, J.J.; Hemingway, R.W. 1980. Loblolly pine bark

polyflavanoids. Journal of Agricultural and Food

Chemistry. 28(2): 222.

Martin, G.E.; Zektzer, AS. 1988. Two-dimensional NMR

methods for establishing molecular connectivity. New

York: VCH Publishers. 508 pp.

Newman, R.H. ; Porter, L.J. ; Foo, L.Y. ; [and others]. 1982.

High resolution 13C-NMR studies of proanthocyanidin

polymers. Magnetic Resonance Chemistry 25: 118.

Ohara, S.; Hemingway, R.W. Condensed tannins: The

formation of a diarylpropanol-catechinic acid dimer from

base-catalyzed reactions of (+)-catechin. Journal of Wood

Chemistry and Technology. (In press)

Porter, L.J.: Newman, R.H.: Foo. L.Y. land othersl. 1982.

Polymeric procyanidins. ‘3C-NMR studiks of

procyanidins. Journal of Chemical Society, Perkin Trans.

1 :1217.

Steynberg, J.P. ; Hemingway, R. W. ; Brandt, E.V. ; Ferreira,

D. [In preparationla. Condensed tannins: 13C-NMR

studies of profisetinidins.

Steynberg, J.P.; Ohara, S.; Hemingway, R.W. [and others].

[In preparationlb. Condensed tannins: isolation of 2S,3Sprofesetinidins from Guamuchil, and observations on their

synthesis and biogenesis.

4 8
Principles and Applications of

Relaxation Spectroscopy

Timothy G. Rials

INTRODUCTION

Amorphous polymers can readily be classified into two

distinct groups according to their mechanical behavior.

Some polymers like polystyrene and poly(methy1

methacrylate) are rigid, glassy plastics at room temperature,

whereas others are flexible, rubbery materials such as

polybutadiene and polyisoprene. However, if polystyrene is

heated to about 125 “C, it exhibits typical rubbery behavior,

and when polybutadiene is cooled in liquid nitrogen, it

becomes rigid and glassy. The temperature at which this

change in properties occurs is known as the glass transition

temperature, T,, a primary relaxation process in polymers,

and involves large-scale molecular motion (Seymour and

Carraher 1988, Rosen 1987). Consequently, whether a

polymer exhibits rubbery or glassy properties depends on the

relative position of its application temperature to its TB.

This primary relaxation process ultimately determines the

service conditions of a given polymer, but these materials

typically exhibit at least one other secondary relaxation

process below To. Glassy state relaxations are attributed to

motions of groups of atoms smaller than those necessary to

produce a glass- to-rubber transition and can significantly

influence mechanical properties. As an example,

polycarbonate (bullet-proof glass) exhibits a strong relaxation

process below room temperature that is not present in brittle

plastics like polystyrene. In addition, relaxation processes

may arise as a consequence of multiple phases (e.g., crystallinity) or multiple components (e.g., polymer blends,

copolymers, fiber composites), providing an approach to

modifying the mechanical properties of a material.

Therefore, interpretation of relaxation properties at the

molecular level is of great scientific and practical importance

in understanding the mechanical behavior of polymers.

Several excellent reviews of relaxation properties in relation

to the molecular structure of polymers are available (Aklonis

and MacKnight 1983, Nielsen 1974, Ferry 1980, Murayama

1987).

The study of molecular motion in polymeric materials has

been led by nuclear magnetic resonance spectroscopy;

however, recent years have seen the appearance of several

new analytical techniques including dynamic mechanical

analysis and, more recently, dielectric analysis. The recent

acquisition of these two instruments at the Forest Products

Utilization Research Laboratory has greatly expanded the

polymer characterization capability in the Southern Station.

A brief introduction to these new techniques in terms of their

Timothy G. Rials is research physical scientist at Southern Forest

Experiment Station, USDA Forest Service, Pineville, LA 71360.

principles of operation, as well as exemplary applications, is

presented in the following discussion.

FUNDAMENTALS AND INTERPRETATION

Dynamic relaxation experiments rely on the fundamentally

different response of a spring and a dashpot to a sinusoidally

applied stress (Aklonis and MacKnight 1983). The response

of an ideally elastic material (the spring) is exactly inphase

with the applied stress, whereas a purely viscous material

(the dashpot) lags behind by a 90” phase angle. In the more

realistic case of a viscoelastic material, the response lags

behind by some intermediate phase angle, 6, since a portion

of the energy associated with that stress is stored through an

elastic response, whereas another portion is lost due to

molecular friction. From this information, the complex

response can then be resolved into its individual components.

For example, from the sinusoidal mechanical wave applied

in dynamic mechanical analysis, the parameters derived

include the storage modulus (E’), the loss modulus (El’), and

the dissipation factor (tan 6). To a first approximation,

dielectric analysis presents an analogous situation with the

exception that an electrical force, rather than a mechanical

deformation, is applied. The input and output for these

methods are compared in table 1.

Table 1 .--Comparison of dynamic mechanical and dielectric

analysis

Dynamic mechanical Dielectric

Parameter themud analysis thermal analysis

Input Stress Voltage

output Strain Current

Properties

Elastic component Storage modulus (E’) Dielectric const. (E’)

Viscous component Loss modulus (E”) Dielectric loss (E”)

Other Tan delta (E”/E’) Tan delta (E”/E’)

Ionic conductivity

In general, relaxation spectroscopy focuses on defining

the variation in the individual response mechanisms with

temperature, as illustrated through the following example.

Figure 1 shows the variation in the dynamic mechanical

properties, Log E’ and tan 6, for poly(methy1 methacrylate),

a typical amorphous polymer, over the temperature range -50

“C to 160 “C. At low temperatures, a modulus of about 10”

dynes/cm2 is observed, which is typical of a glassy polymer.

A precipitous drop in the modulus to about 106 dynes/cm2 is

encountered around 110 “C to 130 “C along with a sharp

peak in tan 6. This relaxation process corresponds to the Tg

of PMMA and is indicative of large-scale molecular motion.

An additional relaxation process is centered around 50 “C in

the tan 6 spectrum, but is accompanied by a much smaller

decline in modulus. This secondary relaxation, termed the

D relaxation, arises from the motion of the -C02CH3 side

groups. It is important to note that the time scale of the

experiment can influence the relaxation behavior in a manner

49
APPLICATIONS OF RELAXATION SPECTROSCOPY

The Wood Composite. Because of the complexity of

wood at the macroscopic level, it is convenient to overlook

the fact that the woody cell wall is itself a unique composite

consisting of three structural polymers. The system is

generally described as a fibrous, cellulosic component

embedded in an amorphous matrix of hemicellulose and

lignin (Erins and others 1976); however, very little

information is available on the in situ morphology (i.e., the

extent of molecular mixing) of the blend of polymers

comprising the amorphous element of the cell wall. Dynamic

mechanical analysis has recently been used to address this

question by evaluating the influence of moisture on the

viscoelastic response of wood (Kelley and others 1987).

The presence of a low molecular weight species often

serves to “plasticize” an amorphous polymer, lowering the

energy requirements of chain mobility (Rudin 1982).

Consequently, the precise temperature at which the primary

dispersion occurs can be expected to vary with moisture

content as is illustrated in figure 2. At low moisture content,

the glass transition temperature of lignin (ol) and

hemicellulose (02) are barely detectable, well above 100 “C,

and are indistinguishable from each other. The plasticizing

efficiency of water, however, varies considerably for the two

relaxations. Whereas the lignin transition declines to 60 “C

as moisture content rises to 10 percent and remains constant,

the hemicellulose transition continues to decline to

subambient temperatures with increasing moisture content.

The resolution of two separate Tg values confirms the

immiscibility of the two amorphous wood polymers, lignin

and hemicellulose. Of particular interest is the observation

that hemicellulose exhibits a subambient glass transition at

relatively low moisture contents, whereas lignin remains

glassy even at high moisture contents. The combined

rubbery and glassy response may contribute to the overall

toughness of the composite system in a manner similar to

rubber-toughened epoxies. In light of the importance of

moisture on the processing of wood and pulp fibers, the

recognition of this viscoelastic response may have significant

implications in the production of wood composites.

Wood Fiber/Polymer Composites. The utilization of

wood as a reinforcing fiber in thermoplastic polymers has

recently received considerable attention (Takase and Shiraishi

1989, Dalvag and others 1985, Woodhams and others 1984).

The strength and toughness of this natural fiber offer a

number of advantages over conventional reinforcements, but

present the problem of limited compatibility with commodity

thermoplastics such as polyethylene or polypropylene. The

incompatible nature of the two components results in an

inadequate interfacial region that prohibits the successful

transfer of stresses to the load-beating fiber. Thus, much of

the research effort in this area has focused on fiber

modification reactions aimed at enhancing the quality of the

fiber/polymer interface. Dynamic mechanical analysis

provides a convenient technique to study the extent of

t

,

0. I

t

101

-50 -20 IO 40 70 loo 130 160

Figure 1 .--The eflect of temperature on the dynamic

mechanical properties (Log E’ and tan 6) of

poly(methyl methacrylate).

similar to temperature. That is, a viscoelastic material will

become more glasslike as the frequency is increased and the

time-dependent response mechanisms are unable to respond

in time. Consequently, the typical glassy modulus of the

polymer may be observed even at temperatures well above its

glass transition temperature. The complete characterization

of a material’s relaxation properties must, then, include both

time and temperature as variables.

As mentioned earlier, dielectric relaxation spectroscopy is

similar in principle to dynamic mechanical analysis;

however, it is phenomenologically distinctive (Aklonis and

MacKnight 1983, Hedvig 1977). This technique is

concerned with the time/temperature dependence of the

dielectric constant and, therefore, focuses on dipolar (e.g.,

hydroxyl or carbonyl) relaxation. Dielectric analysis

provides a wider frequency range and eliminates sample

restrictions, making it an extremely versatile, stand-alone,

analytical tool (Day 1988, Grentzer and Leckenby 1989). In

addition, dielectric analysis is often more sensitive to glassy

state relaxations and, therefore, provides a complementary

technique to dynamic mechanical experiments. Because of

the sensitivity of these methods to a number of different

types of molecular motions, transitions, and relaxation

processes, dynamic mechanical and dielectric analyses find

applications in a wide variety of polymeric systems.

5 0
IO 0

i;;#yy/~o:~:~~~~

I

I 3

.I50 -50 5 0 IS0 -150 -50 50 150

Temperature (“C) Temperature (“C)

Figure 2.--l& injluence of moisture on the dynamic mechanical response (a) log E’ and (6) tan 6 of

spruce wood. Moisture contents are: 5 percent (‘J, 10 percent (---), 20percent (-J,

and 30 percent (1.

interaction between the individual components of the material

and has recently been applied to wood fiber/polypropylene

composites (Krebaum and others 1990).

Figure 3 shows the variation in the storage modulus (E’)

and tan 6 with temperature for several polypropylene

composites containing 50 wt. percent wood fiber. The

materials were obtained by disk-refining red oak chips and

include the control fiber, a common titanate coupling agent

modification, and modification with a long chain fatty amine.

There are two events common to all three spectra, the most

notable being the melting transition of polypropylene (a

semicrystalline polymer) at high temperature (Quirk and

Alsamarraie 1989). Interestingly, the melting temperature

(T,,,) is depressed from 158 “C for the titanate-modified fiber

to 150 “C for the fatty amine modification and the

unmodified fiber composites, indicating more favorable

- 0.27

- 0.24

- 0.21

- 0.18

4,

- 0.15 -

5

- 0.12

- 0.09

- 0.06

Figure 3.--fie variation in storage modulus (log E’) and tan 6 with temperature of polypropylene

composites containing 50 wt. percent of (A) unmodijied, (+)titanate mod@ed, and (v)

fatty amine modeed, red oakjiber.

51
interaction for the latter two systems. Also common to all

three spectra is the broad relaxation centered around 80 “C.

The exact origin of this process is unclear at the moment, but

may tentatively be assigned to the melting of small, imperfect

crystalline domains. Of particular interest is the low

temperature region of the tan 6 curve where there is an

additional relaxation peak that is unique to the fatty amine

fiber composite and the unmodified fiber composite. This

new transition is attributed to the wood fiber/polypropylene

interfacial region and suggests improved compatibility

between components.

From the observed T,,, depression and the intensity of the

low temperature transition, the degree of interaction can be

ranked as follows: TITANATE MODIFIED <

UNMODIFIED < FA7TY AMINE MODIFIED. It is

interesting to note that the impact strength of the composites,

although exhibiting only small differences, followed a similar

trend.

Phenolic Resin Cure. The curing analysis, of

thermosetting polymers presents a unique challenge due to

the associated phase change from liquid resin to vitrified

polymer. Although dynamic mechanical analysis has

contributed extensively to this area of research (Aronhime

and Gillham 1984), this method suffers in that some type of

support (e.g., glass cloth) is required for the prepolymer. A

relatively new version of the older dielectric analysis,

microdielectric spectroscopy addresses this shortcoming.

This technique incorporates new microsensor technology that

can directly evaluate liquid samples and also provides the

luxury of remote data acquisition. As a result, it affords a

potentially powerful method for the study of thermosets such

as phenol-formaldehyde polymers (Rials, in press).

The dielectric response as a function of temperature is

compared with the reaction exotherm obtained by differential

scanning calorimetry in figure 4. In this case, the dielectric

data are plotted as ionic conductivity that is related to the

mobility of ions in the medium and is, therefore, inversely

proportional to viscosity. As the resin is heated, the

conductivity increases rapidly until a slight slope change is

observed at 65 “C, corresponding to the calorimetrically

observed reaction onset. A conductivity peak, corresponding

to the minimum viscosity, is reached at about 110 “C

followed by a plateau region to about 140 “C. Interestingly,

it is not until the DSC exotherm nears completion that a

rapid decrease in conductivity occurs, indicative of increased

crosslinking and network development. The crosslinking

reaction exhibits a final advance as the experimental

temperature exceeds that of the network polymer’s T, around

180 “C.

Microdielectric analysis of phenol-formaldehyde polymers

yields information that is readily interpreted in very practical

terms. Cure parameters such as viscosity minima, onset of

reaction, rate of cure, and completion of reaction are easily

extracted from the data. In addition, this technique has

demonstrated considerable sensitivity to chemical structure

variables relating hydroxymethyl phenol content to residual

Temperature (“C)

Figure 4.--Comparison of the dielectric response (log

conductivity) with the curing exotherm from

dtgerential scanning calorimetry of a phenolformaldehyde resin.

cure and illustrating the impact of unsubstituted para

positions on the overall chemorheological response of the

resin. In view of this capability, as well as the potential to

collect data from remote environments, microdielectric

spectroscopy can be expected to become as routine an

analytical method as the other relaxation techniques.

CONCLUSIONS

Dynamic mechanical analysis and dielectric analysis have

proved to be very useful in the study of polymer relaxation

properties. In large part, their relatively rapid rise in

popularity can be attributed to two unique features. First,

these two analytical methods fill a critical void in terms of

experimental time scale between transient experiments like

creep and stress relaxation, and high frequency methods like

nuclear magnetic resonance spectroscopy. The frequency

range where they are applied allows the detection of largescale molecular motion, as well as smaller, glassy state

relaxation events. Perhaps the greatest strength of these

techniques, however, lies in their unique capacity to relate

molecular motion to polymer properties of immediate,

practical significance.

The foundation of relaxation spectroscopy has been firmly

established in the realm of polymer science, but its

availability has helped to expand its scope of application to

disciplines as diverse as food science and ceramics. The

extension of dielectric analysis (and particularly, dynamic

mechanical analysis) into wood science was inevitable as the

consideration of wood as a polymeric composite has become

increasingly common. As has been demonstrated above,

understanding the viscoelastic behavior of wood may

significantly impact the production of composite products like

particleboard and fiberboard. A look to the future suggests

an increased presence of synthetic polymers, beyond phenolic

52
resins, as novel thermoplastic composites are investigated.

Given the demonstrated utility of both dynamic mechanical

and dielectric analysis in studying the inherent properties of

the components and the structural factors of the systems,

their contributions to the science and technology of wood

utilization will unquestionably increase.

LITERATURE CITED

Kelley, S.S.; Rials, T.G.; Glasser, W.G. 1987. Relaxation

behaviour of the amorphous components of wood. Journal

of Materials Science. 22: 617-624.

Krebaum, L.; Rials, T.G.; Hemingway, R. W. 1990. Final

Report, Cooperation with Dr. Laurence J. Krebaum.

Murayama, T. 1987. Dynamic mechanical properties. In:

Kroschwitz, J.I., ed. Polymers: an encyclopedic

scourcebook of engineering properties. New York: John

Aklonis, J.J.; Ma&night, W.J. 1983. Introduction to

polymer viscoelasticty. 2nd ed. New York: John Wiley

& Sons. 295 p.

Aronhime, M.T.; Gillham, J.K. 1984. The transformation of

liquid to amorphous solid. Effect of reaction mechanism

on the time to vitrify for linear and network

polymerization. Journal of Coatings Technology.

56(718): 35-47.

Dalvag, H.; Klason, C.; Stromvall, H.-E. 1985. The

efficiency of cellulosic fillers in common thermoplastics.

Part II. Filling with processing aids and coupling agents.

International Journal of Polymeric Materials. 11: 9-38.

Day, D.R. 1988. Dielectric properties of polymeric

materials. Cambridge, MA: Micromet Instruments, Inc.

51 p.

Erins, P.; Cinitec, V.; Jakobsons, M.; Gravitis, J. 1976.

Wood as a multicomponent, crosslinked polymer system.

Applied Polymer Symposium. 28: 1117-l 138.

Ferry, J.D. 1980. Viscoelastic properties of polymers. 3rd

ed. New York: John Wiley & Sons. 220 p.

Grentzer, T.; Leckenby, J. 1989. The theory and practice of

dielectric analysis. American Laboratory. 21(l): 82.

Hedvig, P. 1977. Dielectric spectroscopy of polymers. New

York: John Wiley & Sons. 255 p.

Wiley & Sons: 176-206.

Nielsen, L.E. 1974. Mechanical properties of polymers and

composites. New York: Marcel Dekker, Inc. 175 p.

Quirk, R.P.; Alsamarraie, M.A.A. 1989. Physical constants

of poly(propylene). In: Brandrup, J.; Immergut, E.H.,

eds. Polymer handbook. 3rd ed. New York: John Wiley

& Sons: V/27-V/34.

Rials, T.G. Cure analysis of phenol-formaldehyde resins by

microdielectric spectroscopy. [In press]. In: Glasser, W.

G.; Sarkanen, S. eds. Viscoelasticity of biomaterials.

American Chemical Society.

Rosen, S.L. 1987. Fundamental principles of polymeric

materials. New York: John Wiley & Sons. 346 p.

Rudin, A. 1982. The elements of ‘polymer science and

engineering. New York: Academic Press. 433 p.

Seymour, R.B.; Carraher, C.E., Jr. 1988. Polymer

chemistry: an introduction. New York: Marcel Dekker,

Inc. 688 p.

Takase, S.; Shiraishi, N. 1989. Studies on composites from

wood and polypropylene. Journal of Applied Polymer

Science. 37: 647-659.

Woodhams, R.T.; Thomas, G.; Rodgers, D.K. 1984. Wood

fibers as reinforcing fillers for polyolefins. Polymer

Engineering and Science. 24( 15): 1166- 117 1.

53
Use of Gel Permeation Chromatography

in the Development of Resin Adhesives

Chung-Y. Hse

In a crude sense, gel permeation chromatography (GPC)

is a molecular sieving process in which the polymer

molecules are separated as a function of their size in

solution. This separation technique provides the fastest and

most accurate method for determining the molecular weight

distribution of a polymer. Since the product uniformity,

process characteristics, and physical properties of most

polymers are related to the molecular weight (number

average M, and weight average MJ and molecular weight

distribution (M,,,/M,J, the importance of this analytical

determination can readily be seen.

The application of GPC in resin research at the Pineville

laboratory began in 1967 with laboratory-assembled

instruments (fig. 1). A typical chromatogram of a phenolic

resin by this homemade instrument is shown in figure 2.

The commercial GPC instrument was purchased in the early

1970’s. The capability of GPC for polymer characterization

has improved substantially in recent years with new

instrumentation, techniques, and data manipulation. The

Figure l.--Front view of laboratory assembled GPC. El&ion Volume (ml)

Chung-y. Hse is principal wood scientist at Southern Forest

Experiment Station, USDA Forest Service, Pineville, LA 71360.

Elution Volume (x 10 ml)

Figure 2.-&l permeation chromatograms of the NaOH

catalyzed phenol formaldehyde resins at van’ous

reaction times.

resolution of phenolic species by GPC has improved

significantly as shown in figure 3.

Over the years at the Pineville laboratory, GPC has been

used in studies of resin formulation, characterization,

molecular blending, resin storage and stability, fractionation

and separation, and copolymerization. At present, GPC is

used routinely to define molecular distribution profiles of

resins formulated in the laboratory. The usefulness of gel

permeation chromatography in the development of resin

adhesives can be illustrated by the following examples.

To modify the urea formaldehyde (UF) resin formulation

from the conventional two-stage reaction (i.e., alkaline pH to

initiate the reaction and acidic pH to promote condensation)

to a single acidic reaction without first using an alkaline

catalyst, GPC was used to follow the reaction (fig. 4). The

large fraction of sample eluted between 21 and 29 mL (upper

chromatogram) indicated that strong acidic conditions favored

condensation. On the other hand, the strong signal between

30 and 39 mL (lower chromatogram) showed that alkaline

conditions enhanced methyl01 formation. The high methyl01

content in resins made under alkaline conditions as compared

to acidic conditions was also detected by 13C-NMR analysis

(Hse and others, in preparation). In general, the

chromatogram can be divided into two groups of peaks. The

peaks between 39 and 40 mL elution volume are those of

urea or mono-methyl01 urea, whereas the first negative peak

between 41 and 43 mL is due to formaldehyde and water in

the sample. These identifications were made by comparing

GPC chromatograms of UF resins before and after addition

of urea, formaldehyde, and water to the sample. Other peaks

20 min. I40 min. 120 min. 1 180 min.

--L-LLLLY JlAJL& 4 0 50 30 40 5020 30 40 5020 30 40 50

Figure 3 .--Gel permeation chromatograms of pherwlic resins

forflakeboard at various reaction times.

54
A pH1.2 F/U 2.5II

20 30 40 50

PH4.8

pH W-5.0

Elution Volume (ml) Elution Volume (ml)

Figure 4-421 permeation chromatograms of UF resins

reacted at different pH and F/U ratios.

evident at early stages of reaction (i.e., those between elution

volumes 30 and 39 mL) may be dimers, trimers, and

tetramers of polymethylol urea as indicated by previous

workers (Dankelman and others 1982; Dunky and Lederer

1982; Matsuzaki and others 1980, Tsuge 1980). These peaks

can be integrated together (group A) as an estimate of the

amount of low molecular weight products. The peaks at low

elution volumes (i.e., 21 to 29 mL) can be integrated

together (group B) to represent a measure of the high

molecular weight products obtained from condensation

reactions.

The composition of low and high molecular weight

products is summarized in table 1. The proportion of high

molecular weight products increased substantially as the

reaction pH decreased. Furthermore, the F/U molar ratios

interacted with reaction pH to effect resin molecular weight.

At acidic pH, the high molecular weight products increased

as F/U ratio decreased; while, at alkaline pH, little difference

was evident between the high or low molecular weight

products at various F/U ratios. It should be noted, however,

the molecular weight buildup was so fast for the resin made

with an F/U ratio of 2.5 at strong acidic condition (i.e., pH

1) that an insoluble gel formed early, so control of the

reaction was not practical.

The analysis of the molecular distribution profiles

suggested that a very different resin was produced under

strong acidic conditions because a high proportion of large

molecular fraction was formed so quickly (i.e., 30 minutes)

at such a mild temperature (i.e., 55 “C), yet the resins were

extremely stable. NMR later confirmed the formation of a

high percentage of uron derivatives that were not in the

conventional UF resin. Furthermore, 2-hour desicator tests

indicated that UF resin formulated with strong acidic pH also

resulted in significantly lower formaldehyde emission even

though the bonding strength was slightly lower than the

conventional UF resin (Hse and others, in preparation).

Preparative GPC is being used to fractionate the resins. It is

expected that the IR and NMR studies of these fractions

should provide more detailed structures of the resins.

The condensation polymers of phenol and formaldehyde

as shown in figure 3 and UF as shown in figure 4 are the

two most important wood adhesives. Phenolic resins have

proven performance in producing exterior quality composites,

whereas low cost UF resins have performed well in interior

applications. Many users of wood adhesives have no doubt

speculated on the advantages to be gained by blending

phenolic and urea resins in searching for lower cost phenolic

adhesives or for upgrading performance and controlling

formaldehyde emission of UF adhesives. Efforts in

Table 1 .--Eflect of reaction conditions on the proportion of low and high

molecular weight species

Resin pH Low molecular High molecular

F/U ratio weight products Weight products

--_------------_-percent _____-__________

A 1.0 3.0 54.7 45.3

B 1.0 3.5 78.2 21.8

C 1.0 4.0 88.8 11.2

D 4.8 2.5 70.5 29.5

E 4.8 3.0 79.8 20.2

F 4.8 3.5 88.2 11.8

G 4.8 4.0 92.3 7.7

H 8.0-5.0 2.5 94.8 5.2

I 8.0-5.0 3.0 96.2 3.8

J 8.0-5.0 3.5 97.0 3.0

K 8.0-5.0 4.0 96.1 3.9

55
developing these types of resin systems have not been

successful due to the basic incompatibility of the curing

properties of the two resins. Phenolic resins are normally

cured at alkaline pH, whereas UF resins are cured at acidic

pH. Therefore, in order to develop practical usage, it is

necessary to combine urea to phenol-formaldehyde polymers

as true co-polymers. Again, fractionation by preparative

GPC was used to great advantage in examining the cocondensation of the model reaction.

Figure 5 shows the effect of reaction pH on the molecular

weight distribution profile of the reaction products between

4-hydroxylbenzyl alcohol (PMP) and urea flomita and Hse,

1989) at the molar ratio of PMP/U = 1. It is obvious that

the stronger the acidic condition, the shorter the reaction time

to produce condensates. At pH 2.0, for instance, the oily

reactant consisting mainly of hydroxybenzylurea began to

separate from the water layer within 10 minutes. (The

changes of gel permeation chromatography of the reactants

reacted 30 minutes at pH 2.0 as affected by molar ratio of

PMP/U are shown in figure 6.) Increased molar ratio (i.e.,

2.0 and 4.0) accelerated the formation of the high molecular

weight portion. Examination with NMR in a previous study

(Tomita and Hse 1989) showed that the formation of the

condensates involved co-condensation and self-condensation,

with co-condensation between p-methyl01 group and urea

prevailing against self-condensation of phenol,

On the other hand, at lower molar ratios (i.e., 1.0 and

OS), GPC showed three separate peaks in the lower

molecular weight region. The fractions of these three peaks

were collected by preparative GPC. The chromatogram of

each fraction is shown in figure 7. Again, the fractions were

studied extensively with IR and NMR (Tomita and Hse

1989). The results revealed that peak 1 was crystallized 4-

p-methylophenol

$

p. methylophenol

PMP/U=O.PAJ

321

p::p

32

pM&

I I

ElutiZn Volum”e (ml)

Figure 6.--Gel permeation chromatograms of the reactants

from p-methylolphenol and urea at various molar

ratios (PMPLJ). Reaction condition: 8.5 “C, pH

2.0, 30 min.

4 5 60 4 5 6 0

Elution Volume

Figure 5.--Gel permeation chromatograms of the reactant

jkom urea and p-methylolphenol at the molar

ratio of PMPIU = 1 and 85 “C, under van’ous

pH levels.

JLPeak 1

Elution Volume (ml)

Figure 7.--Gel permeation chromatograms of molecular

fractions isolated by preparative GPC.

hydroxybenzyl urea, peak 2 was crystallized N,N’-Bis(C

hydroxybenzyl) urea, and peak 3 was amorphous solid of

Tris(4-hydroxybenzyl) urea.

The foregoing examples have clearly shown the utility of

gel permeation chromatography as a separation technique for

previously hard-to-analyze phenolic and UF materials. The

effectiveness of GPC in wood adhesive research will grow

56
continuously, particularly in combination with other

analytical methods such as NMR, DSC, and FTIR, etc. The

ability of GPC to measure and evaluate the molecular weight

distribution of polymer species should lead to better process

control and more uniform products. It is expected that socalled “fingerprints” of wood adhesives may one day be

realized for quality assurance, even though the wood

adhesive is often noted for its complexity.

LITERATURE CITED

Dankelman, W.; Daemen, J. M. H.; de Brett, A. J. J.; [and

others]. 1982. Angewande Makromolekulare Chemie 54:

187-201.

Dunky, M.; Lederer, K. 1982. Angewande Makromolekulare

Chemie 102: 199-213.

Hse, C. Y.; Xia, Z. Y.; Tomita, B. [In preparation]. Effects

of reaction pH on properties and performance of ureaformaldehyde resins.

Matsuzaki, T.; Inoue, Y.; Ookubo, T. [and others]. 1980.

Journal of Liquid Chromatography 3(3): 353-365.

Tom@ B.; Hse, C. Y. 1989. Multi-polymer wood adhesive

system to enhance adhesion and reduce bonding costs.

New Orleans, LA: U.S. Department of Agriculture,

Forest Service, Southern Forest Experiment Station; final

report FS-SO-4701-109. 107 p.

Tomita, B.; Hse, C. Y. [In preparation]. Co-condensation of

urea with methylolphenols in acidic conditions.

Tsuge, M. 1980. Journal of Thermoset Resins (Japan). l(2):

31-45.

57
Condensed Tannins: Must Enzymes Control the

Stereochemistry in Profisetinidin Biosynthesis?

Jan P. Steynberg and Richard W. Hemingway

INTRODUCTION

The condensed tannins are polymeric flavan-3-01s that are

found in the fruits, leaves, bark, and occasionally wood of a

broad spectrum of plants (Hemingway and others 1989).

Depending upon the plant species, these compounds vary in

their hydroxylation patterns (for example, 3,5,7,3’,4’-

pentahydroxyflavans as in conifer tree barks or 3,7,3’,4’-

tetrahydroxyflavans as in wattle tree heartwood),

stereochemistry (for example, predominantly 2,3-c&3,4-

trans in conifer tree barks or mixtures of 2,3-traw3,4-trans

and 2,3-trans-3,4-cis in wattle tree heartwood), as well as

molecular weight distribution. The most common naturally

occurring profisetinidins are polymers made up of 2R,3S-(-)-

fisetinidol(1) as chain extenders with 2R,3S-( +)-catechin (2)

as chain terminators.

Much effort has recently been devoted to a better

understanding of enzymic pathways leading to condensed

tannins, but debate continues (Stafford 1983, Platt and others

1984) regarding the exact nature of proanthocyanidin

biosynthesis. Good progress has been made in understanding

of the individual enzymic steps up to C-15 (monomer) level

(Lewis and Yamamoto 1989, Stafford 1989), and more

evidence for flavan-3,4-diols as the precursors to

proanthocyanidins has been obtained (Stafford and others

1985). The ease with which these intermediates polymerize

under very mild conditions has in some instances led to

abandonment of hypotheses that invoke enzyme mediation in

the polymerization process (Roux and others 1980, Roux and

Ferreira 1982a, Roux and Ferreira 1982b). In contrast, our

recent characterization of novel 2,3-cis-3,4-cis-profisetinidins

(Steynberg and others, in preparation), as well as efforts to

synthesize these compounds biomimetically, argue strongly

in favor of the idea that the whole process of synthesis from

the initial precursor to the ultimate polymer must be under

enzymic control.

RESULTS AND DISCUSSION

The basic chemistry of the profisetinidin-rich wattle

tannins extracted from Acacia mearnsii has been extensively

studied by Professor Roux’s group in South Africa, and this

has led to successful industrial application of these tannins

(Roux and others 1980, Roux and Ferreira 1982a, Roux and

Ferreira 1982b). Much of the success obtained in these

Jan P. Steynberg is visiting scientist from Department of

Chemistry, University of the Orange Free State, Bloemfontein,

South Africa; Richard W. Hemingway is project leader at Southern

Forest Experiment Station, USDA Forest Service, Pineville, LA

71360.

efforts can be related to the development of “biomimetic”

synthesis, where these oligomers can be synthesized by mild

acid-catalyzed condensation between naturally occurring

flavan-3,4-diols and flavan-3-01s. This leads to the formation

of an interflavanoid bond by the attack of the highly

nucleophilic A-ring of catechin on the acid-mediated 4-

carbocation of mollisacacidin (fig. 1).

As part of a search for North American sources of tannins

suitable for leather manufacture, we investigated the bark

tannins of a Mexican tree, (Phytecellobium duke) (Steynberg

and others, in preparation). Two novel 2,3-cis-3,4-cis

profisetinidin dimers (3) and (4) dominated the dimeric

fraction (fig. 2). The structural elucidation of these unique

compounds relied mainly on physical data obtained by

powerful n.m.r. techniques such as selective decoupling and

n.0.e. difference spectroscopy that again stresses the need to

have modem n.m.r. instrumentation available in a study of

relatively complex molecules like tannins. Diagnostic 13C

resonances indicated 2,3-cis stereochemistry for both

extender units as well as for one of the terminal units. The

magnitude of the heterocyclic ring proton coupling constants

for the methyl ether acetate derivatives, assisted by ‘Hdecoupling experiments and n.0.e. difference spectra, not

only confirmed the relative stereochemistry at C-2 and C-3

(2,3-cis in the chain extender units and either 2,3-trans or

2,3-cis, respectively, in the bottom units), but also indicated

2,3-cis-3,4-cis relative stereochemistry for the upper units.

Absolute stereochemistry at the interflavanoid bond was

derived from a positive Cotton effect in the CD-spectra of

both dimers. The absolute stereochemistry of the bottom unit

in (3) was evident from optical rotational measurements of

the terminal flavan-3-01 obtained after interflavanoid bond

cleavage. Collectively, these data confirmed the suggested

structures (3) and (4).

O.lNHCI H

1) R=h l-l-fieetinidol

2) R-(m (+I-catechin

06,

OH

Tetramera

Oligomere

Figure 1 .--Biomimetic synthesis of profisetinidins.

5 8
Figure 2.--Novel all-&s proflsetinidin dimers from

Guamuchil.

Not only do these dimers contribute to the rare class of

2,3-cis profisetinidins, but in all known proanthocyanidins,

there has been only one other natural product isolated with a

2,3-cis-3,4-cis stereochemistry (Foo 1987). Attempts to

obtain these compounds via acid-catalyzed condensation

(Roux’s methodology) was severely limited in two ways. A

2,3-cis mollisacacidin is extremely rare, having been isolated

in only very small quantities from Acacia mearnsii (Drewes

and Ilsley 1969) and is not easily obtainable by synthesis.

Additionally, 2,3-cis-flavan-3,4-dials will undergo

stereospecific reaction to yield only 3,4-trans products when

exposed to mild acid-catalyzed condensation reaction

conditions (Botha and others 1981). Therefore, an obvious

hypothesis for how these all-cis compounds might be formed

was to suggest that they originate from a tran.s-cis

profisetinidin precursor by epimerization at C-2. All tannin

chemists agree that any suggested biochemical conversions

should be demonstrated to be possible on solid chemical

grounds, so we attempted this synthesis.

Epimerizations of (+)-catechin and (-)-epicatechin have

been successfully accomplished by treatment with either

strong base or high temperature (fig. 3a) (Foo and Porter

1983). In these reactions, opening of the pyran ring to a

quinone-methide intermediate (4) destroys the absolute

stereochemistry at C-2. Ring closure via Re- as well as Siface attack of the A-ring oxygen nucleophile on the quinonemethide results in reaction products with both 2R and 2S

absolute stereochemistry. However, the ease with which

protisetinidins undergo pyran ring isomerization to

phlobatannins restricts approaches to use of epimerization

reactions in oligomeric compounds. Here the terminal unit’s

A-ring hydroxyl acts as a competing nucleophile in the ring

closure step to produce a different pyran ring with

“inversion” of the A- and B-rings (fig. 3b) (Steynberg and

others 1988). Therefore, tetra-O-methyl catechin (5) was

reacted with mollisacacidin to synthesize selective protected

dimers (6) and (7) suitable for these attempts (fig. 4). By

protecting the bottom unit’s hydroxyls as methyl ether

derivatives, not only are pyran ring isomerizations prevented,

but also the bottom unit is protected from unwanted

epimerization.

Although it was possible to epimerize the trans-tram

derivative (6) at C-2 by heating it in ethanol under pressure

Figure 3a.--Quinone-methide intermediates in projisetinidin

synthesis.

Figure 3b.--Quinone-methide intermediates in proflsetinidin

synthesis.

at 180 “C, all efforts to epimerize the trans-cis dimer (7)

resulted only in recovery of the starting compound. This

might be explained in terms of a stereospecific ring closure

directed by the bulky 4-substituent occupying an axial

orientation as is the case with the trans-cis dimer (7). In the

quinone-methide intermediate originating from this

compound, orientation of the quinone above the mean plain

of the A-ring, a conformation required for attack that will

lead to epimerization, may be very unfavorable due to severe

steric interaction between the quinone and the bulky quasiaxial 4-substituent. The ar-orientation of the 4-substituent

(below the mean plane of the A-ring) in the trans-trans dimer

(6) makes such stereocontrol impossible in this case and

explains why (6) does epimerize under high temperature

conditions.

CONCLUSIONS

It is most unlikely that the all-cis profisetinidins isolated

from Guamuchil are produced in the plant from 2,3-

trans-3,4-cis precursors such as fisetinidol-(4l.I --+ 8)-catechin

5 9
nign t01

treatabel

strong base

treatmt

151 (6)

Figure 4.--Attempts to synthesize the all-cis profisetinidin dimers via epimerization of a suitable precursor.

or fisetinidol-(48 -, 8)-epicatechin by an epimerization at C-2

because frsetinidol-(48 --, 8)-tetra-O-methyl catechin cannot

be epimerized to the 2,3-cis-3,4-cis ent-epifisetinidol-(48 --+

8)-tetra-O-methyl catechin. These results together with the

facile formation of phlobatannins suggest that the 2,3-cis

stereochemistry must be established prior to the formation of

the flavan-3,4-diol precursor. Therefore, enzymatic control,

both in the formation of a 2S,3S precursor and

stereoselective formation of the 48 interflavanoid bond seems

to be required in the biogenesis of these compounds.

LITERATURE CITED

Botha, J.J.; Ferreira, D.; Roux, D.G. 1981. Synthesis of

condensed tannins. Part 1. Stereoselectivity and

stereospecific syntheses of optically pure 4-arylflavan-3-

ols, and assessment of their absolute stereochemistry at

C-4 by means of circular dichroism. Journal of the

Chemical Society, Perkin Transactions 1: 1235.

Drewes, S.E., Ilsley, A.H. 1989. Isomeric leucofisetinidins

from Acacia meamsii. Phytochemistry 28: 1039-1042.

Foo, L.Y. 1987. A novel pyrogallol A-ring proanthocyanidin

dimer from Acacia melanoxylon. Journal of the Chemical

Society, Chemical Communications 3: 236.

Foo, L.Y.; Porter, L.J. 1983. Synthesis and conformation of

procyanidin diastereoisomers. Journal of the Chemical

Society, Perkin Transactions 1: 1535.

Hemingway, R.W. ; Karchesy, J.J. ; Branham, S.J., eds.

1989. Chemistry and significance of condensed tannins.

New York: Plenum Press. 553 p.

Lewis, N. G.; Yamamoto, E. 1989. Tannins - their place

in plant metabolism. In: Hemingway, R.W., Karchesy,

J.J.; Branham, S.J., eds. Chemistry and significance of

condensed tannins. New York: Plenum Press: 23-46.

Platt, R.V.; Gpie, C.T.; Haslam, E. 1984. Biosynthesis of

flavan-3-01s and other secondary plant products from

(2S)-phenylalanine. Phytochemistry. 23: 2211-2217.

Roux, D.G.; Ferreira, D. 1982a. The direct biomimetic

synthesis, structure, and absolute configuration of angular

and linear condensed tannins. Fortschritte der Chemie

Organ&her Naturstoffe 4 1: 48.

Roux, D.G.; Ferreira, D. 1982b. Structure and function in

biomimetic synthesis of linear, angular, and branched

condensed tannins. Reviews in Pure & Applied Chemistry

54: 2465.

Roux, D.G.; Ferreira, D.; Botha, J.J. 1980. Structural

considerations in predicting the utilization of tannins.

Journal of Agriculture and Food Chemistry 28: 216-222.

Stafford, H.A. 1983. Enzymic regulation of procyanidin

biosynthesis: lack of flav-3-en-3-01 intermediate.

Phytochemistry. 22: 2643-2650.

Stafford, H.A. 1989. The enzymology of proanthocyanidin

biosynthesis. In: Hemingway, R. W.; Karchesy, J. J.;

Branham, S. J., eds. Chemistry and significance of

condensed tannins. New York: Plenum Press: 47-70.

Stafford, H.A.; Lester, H.H.; Porter, L.J. 1985. Chemical

and enzymatic synthesis of monomeric procyanidins

(leucocyanidinsor3’,4’,5,7-tetrahydroxyflavan-3,4-diols)

from (2R,3R) dihydroquercetin. Phytochemistry. 24: 333.

Steynberg, J.P.; Burger, J.F. W.; Young, D.A. [and others].

1988. Novel base-catalyzed rearrangements of (-)-

fisetinidol-( +)-catechin profisetinidins with 2,3-tram-3,4-

cis flavan-3-01 constituent units. Journal of the Chemical

Society, Chemical Communications 6: 1055.

Steynberg, J.P.; Stynberg, E.C.; Ohara, S. [and others]. [in

preparation]. Natural 2S,3S,4R-profisetinidins and

observations on their synthesis and biogenesis. [in

preparation].

60
Can Wood Extractives be Used as

Wood Protectants?

C. A. McDaniel and Billie S. Dunn

INTRODUCTION

It is estimated that the cost of the control and prevention

of subterranean termites and the repair of damage they cause

is approximately 1.5 billion dollars per year in the United

States (Ebeling 1975). This estimate is from data in the

1960’s and, because of inflation, the present cost is probably

much higher.

Investigators have shown that certain woods demonstrate

a natural resistance to termite attack (Beal and others 1974;

Carter 1972, 1974; Carter and de Camargo 1983; Carter and

Huffman 1982; Carter and Smythe 1972, 1974; Carter and

Stringer, 1976; Carter and others, 1972, 1976; Smythe and

Carter, 1970a, 1970b). Our investigations involve the

isolation and identification of the antitermitic compounds

extracted from the heartwood of these resistant woods, as

they present possible environmentally safe alternatives to

currently used synthetic wood preservatives (McDaniel 1989;

McDaniel and others, in press).

METHODS AND MATERIALS

Heartwoods from species that have previously shown

resistance to termite attack in laboratory tests are ground into

sawdust and exhaustively extracted with a ternary solvent

mixture consisting of 54:44:2 acetone:hexane:water (v:v:v).

The sawdust is bioassayed before and after solvent extraction

in order to insure that the antitermitic activity has been

removed from the wood into the solvent extract. The total

extract is chemically fractionated into acidic, phenolic, and

neutral fractions by successively washing an ether solution of

the extract with 4N sodium bicarbonate, followed by a 1N

solution of sodium bicarbonate, followed by a 1N solution of

sodium hydroxide.

Since antitermitic extracts typically contain a mixture of

compounds, some of which have antitermitic activity while

others do not, separation must be achieved into pure

compounds that may then be bioassayed individually. These

separations are achieved by a variety of chromatographic

methods including thin-layer chromatography, column

chromatography, and primarily by high performance liquid

chromatography (HPLC) . Most of our separations are

achieved on a Perkin-Elmer 3B Liquid Chromatograph using

a reverse-phase HPLC on a Whatmann M-9 ODS-E

semipreparative column. A typical solvent system employed

is a 60:40 (v:v) acetonitrile:water mixture with a HewlettC.A. McDaniel is principal scientist and Billie S. Dunn is physical

science technician at Southern Forest Experiment Station, USDA

Forest Service, Gulfport, MS 39505.

Packard 1037A refractive index detector monitored with a

Hewlett-Packard 3392A integrator. The fractions are

collected, partitioned into methylene chloride, and the solvent

removed in vacua at 40 “C. Purities and component

identities are primarily determined by capillarilly gas

chromatography-mass spectrometry (GC-MS) using a

Hewlett-Packard 5890-A GC interfaced to a 5970 Mass

Selective Detector, a 9000-216 computer, and a 9133 disk

drive.

Bioassays. After sufficient quantities of materials have

been collected in acceptable purities (> 95 percent) the

isolates are bioassayed for efficacy as wood protectants. The

materials are vacuum-impregnated into either sweetgum

Liquidambar styracijlua L. or southern pine (Pinus spp.)

blocks according to ASTM methodology (1976) at

concentrations of 1 percent and 0.5 percent by mass. In this

way, the compounds responsible for antitermitic activity can

be assessed.

Two protocols are employed in laboratory bioassays: the

no-choice test and the choice test.

In the no-choice test, a mixture of ‘sand:vermiculite:water

(1: 1:0.4 by volume) is placed into a cylindrical plastic test

container that measures 4.0 cm high by 5.3 cm in diameter.

A treated wooden block is carefully weighed and buried in

the test chamber, and 100 worker termites of the eastern

subterranean termite species Reticulitermes jlavipes are then

added. Blocks treated with solvent alone serve as controls.

After 4 weeks, the block is removed, ovendried at 60 “C for

4 hours, and reweighed. It is also visually inspected, and a

block rating is assigned (table 1) based on weight loss and

the extent of termite damage. Surviving termites are

counted, ‘and their physical condition is noted.

The choice test is similar except that two test chambers

connected by a plastic foraging tube 0.5 X 10 cm are used.

The treated wooden block is placed into one chamber along

with the termites, and an untreated wooden block is placed

in the second chamber. The termites then have an alternative

habitat and food supply available to them, and they may

avoid the treated block without facing starvation, and

therefore, any repellent or antifeedant activity may be

evaluated. As with no-choice tests, the blocks and termites

are examined at the end of a 4-week period.

Table 1. --Grading scale for vacuumimpregnated wood

Number Physical

assigned appearance Dry weight loss

percem

0 no attack apparent 0 - 2.0

1 surface nibbling 2.1 - 5.0

2 moderate attack 5.1 - 20.0

3 heavy attack 20.0 - 50.0

4 very heavy attack >50.1

61
Those compounds that exhibit antitermitic activity in

laboratory bioassays are vacuum-impregnated into either

Table 2.--Toxicity to Reticulitermes flavipes of

stakes of sweetgum measuring 15.5 X 0.75 X 3.2 cm or into

cellulose pads treated at 0.5 percent

with some heartwood-extracted

sweetgum blocks measuring 2.3 X 3.9 X 0.75 cm, which are

then stapled onto untreated stakes having the above

dimensions. The blocks are employed in situations involving

materials

Sample extract Percent Survival

minor components where difficulties arise in isolating

sufficient materials to treat entire stakes. The stakes and/or

blocks are impregnated at levels of 1.0 and 0.5 percent by

mass and field-tested for 1 year. The rating system

previously described for laboratory tests (table 1) is used to

evaluate the test materials at 6 months and at 1 year.

The following isolates have been field-tested: cr-terpineol

(Port-Orford-cedar) , cedrol (eastern redcedar), and

catalpalactone (southern catalpa). Sesquiterpenoids available

from other sources that have been tested are: citral,

citronellal, geraniol, nerolidol, and famesol. Current

materials in field tests are: eastern redcedar leaf oil,

occidentalol, occidol, and eudesmol isomers, all from

northern white cedar, and finally, an acelylated derivative of

a wood waste product from Hercules Chemical Corporation

called Vinsol.

RESULTS AND DISCUSSION

For all wood species examined, the preliminary tests on

cellulose pads (some of which are shown in table 2) indicated

that the acidic and phenolic fractions possess little or no

antitermitic activity. The hydrocarbon sesquiterpenes and

terpenes likewise were found to be innocuous, or nearly SO,

to termites at the levels tested. The activity was found to be

in oxygenated terpenes and sesquiterpenes. Results of some

of these tests are shown in table 3.

Port-Orford cedar

neutral extract

sesquiterpene hydrocarbons

cr-terpineol

85 percent cr-cadinoll

15 percent torreyol

T-muurolol

Southern catalpa

neutral extract

phenolic extract

catalponol

epicatalponol

catalponone

catalpalactone

Western redcedar

neutral extract

acidic extract

0 (2 days)’

0 (1 day)

0 (1 day)

0 (2 days)

0 (4 days)

0 (IO days)

95

0 (7 days)

0 (21 days)

0 (3 days)

0 (28 days)

0 (2 days)

92

phenolic extract

‘Days to mortality

100

Table 3.--Antitemtitic activity towards Reticulitermes flavipes of some heartwood extracted

materials in susceptible woods at 0.5 percent

Sample Percent survival No choice

extract choice (block rating) (block rating)

Western redcedar

neutral extract 85 (0) 50 (1)

phenoli extract 73 (1) 87 (2)

acidic extract 92 (2) 95 (2)

methyl thujate 82 (1) 84 (2)

Southern catalpa

catalponol * 82 (2)

epicatalponol *

72 (2)

catalpononet * 68 (2)

catalpalactone *

0 (0)

Eastern redcedar

total extract

cedrol

t26 (1) 85 (2)

*

50 (1)

Port-Orford-cedar

ar-terpineol

* Test not performed.

t Pathogenic fungus present in test container.

*

0 (0)

62
But this cellulose pad test bioassay was found not to be a

reliable indicator for our purposes. It indicated toxicity, but

did not show which compounds would protect woods from

attack, the main thrust of our investigation. Vacuumimpregnating the test materials into susceptible woods and

bioassay of this treated wood were chosen as the indicators

for this purpose. Some results are shown in table 4. The

results of these bioassays sometimes differed from the earlier

pad bioassay. Catalponol, for example, showed greater

toxicity in pad tests than did catalpalactone; but catapalactone

protected wood from attack better than did catalponol, its

epimer epicatalponol, or the sesquiterpene acetone,

catalponone.

Our field bioassays (table 4) indicate that the laboratory

bioassays are reliable, but also that, in the extended

weathering and higher termite pressure of field conditions,

higher levels of treatment are probably necessary. Future

studies will reflect treatment levels at or above those found

naturally in resistant heartwoods.

Table of susceptible woods in a woodland

environment by treatment with heartwood-extracted

materials at 1 .O percent

Sample extract Rating f SC (10 replicates)

Port Orford-cedar

total extract 0.56 + 0.88

o-terpineol 0.56 f 1.33

Eastern redcedar 0.78 f 1.20

cedrol 0.11 f 0.33

Southern catalpa

total extract 0.25 f 0.70

catalpalactone 0.11 f 0.33

LITEKATURE CITED

American Society for Testing and Materials. 1976. Standard

method of testing wood preservatives by laboratory soilblock

cultures. Designation D-1413-76, In: Book of ASTM

Standards, Part 22, Philadelphia, PA: 424-433.

Beal, R. H.; Carter, F. L.; Southwell, C. R. 1974. Survival

and feeding of subterranean termites on tropical woods. Forest

Products Journal. 24(8): 44-48.

Carter, F. L. 1972. Effects of host constituents on termite

feeding and survival. Problem Analysis FS-SO-2205-2.

Gulfport, MS: U.S. Department of Agricnlture, Forest Service,

Southern Forest Experiment Station. 238~.

Carter, F. L. 1974. Effects of host constituents on termite

feeding and survival. .Problem Analysis FS-SO-2205-2.

Gulfport, MS: U.S. Department of Agriculture, Forest Service,

Southern Forest Experiment Station. 9 p.

Carter, F. L.; Amburgey, T. L.; Manwiller, F. G. 1976.

Resistance of 22 southern hardwoods to wooddecay fungi and

subterranean termites. Wood Science. 8(4): 223-226.

Carter, F. L.; de Camargo, C. R. R. 1983. Testing

antitermitic properties of Brazilian woods and their extracts.

Wood and Fiber Science. 15(4): 350-35,7.

Carter, F. L.; Huffman, J. B. 1982. Termite responses to

wood and extracts of melaleuca. Wood Science. 14(3): 127-

133.

Carter, F. L.; Smythe, R. V. 1972. Extractives of

baldcypress, black walnut, and redwood and survival of the

eastern subterranean termite, Reticulitermesflavipes. Annals of

the Entomological Society of America. ‘65(3): 686-689.

Carter, F. L.; Smythe, R. V. 1974. Feeding and survival

responses of Reticulitermes flavipes (Kollar) to extractives of

wood from 11 coniferous genera. Holzforschung. 28: 41-45.

Carter, F. L.; Stringer, C. A. 1976. Influence of extractives

of selected American hardwoods on ‘feeding response and

survival of Reticulitermes flavipes (Kollar). First Progress

Report FS-SO-2205-9.527. Gulfport, MS: U.S. Department of

Agriculture, Forest Service, Southern Forest Experiment

Station. 7 p.

Carter, F. L.; Stringer, C. A.; Smythe, Richard V. 1972.

Survival of six colonies of Reticulitetmes flavipes on

unfavorable woods. Annals of the Entomological Society of

America. 65(4): 984-985.

Ebeling, W. 1975. Wood-destroying insects and fungi. In:

Urban Entomology. Berkeley, CA: University of California,

Division of Agricultural Science. 394 p.

McDaniel, C. A. 1989. Major termiticidal components of

heartwood of Port-Orford-cedar, Chamaecyparis lawsoniana

(A. Murr.) Par1 Material und Organismen 24(l): 1-15.

McDaniel, C. A.; Klocke, J. A.; Balandrin, M. F. [In

press]. Major antitermitic wood extractive components of

eastern redcedar, Juniperus virginiana. Material und

Organismen.

Smythe, R. V.; Carter, F. L. 1970a. , Feeding responses to

sound wood by Coptotermes formosanus, Reticulitermes

pavipes, and R. virginicus (Isoptera: Rhinotermitidae). Annals

of the Entomological Society of America. 63(3): 841-847.

Smythe, R. V.; Carter, F. L. 1970b. Survival and behavior

,of three subterranean termite species in sawdust of eleven wood

species. Annals of the Entomological Society of America.

63(3): 847-850.

63
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Rials, Timothy G., ed. 1994. Symposium on current research in the

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