Isotopic Techniques in Plant, Soil, and Aquatic Biology Series Editors Eldor A. Paul Jerry M. Melillo Department of Crop and Soil Science The Ecosystems Center Michigan State University Marine Biological Laboratory East Lansing, Michigan Woods Hole, Massachusetts Carbon Isotope Techniques Review Board C. V. Cole D. A. Crossley, Jr. Natural Resource Ecology Laboratory Institute of Ecology Colorado State University University of Georgia Fort Collins, Colorado Athens, Georgia Carbon Isotope Techniques Edited by David C. Coleman Brian Fry Department of Entomology The Ecosystems Center University of Georgia Marine Biological Laboratory Athens, Georgia Woods Hole, Massachusetts ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto This book is printed on acid-free paper. @ Copyright © 1991 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Cataloging-in-Publication Data Carbon isotope techniques / edited by David C. Coleman, Brian Fry. p. cm. - (Isotopic techniques in plant, soil, and aquatic biology series) Includes index. ISBN 0-12-179730-9 (hardcover)(alk. paper) ISBN 0-12-179731-7 (paperback)(alk. paper) 1. Stable isotope tracers. 2. Biology-Technique. 3. Carbon- -Isotopes. I. Coleman, David C, date. II. Fry, Brian. III. Series. QH324.3.C37 1991 574.19'285-dc20 90-25392 CIP PRINTED IN THE UNITED STATES OF AMERICA 91 92 93 94 9 8 7 6 5 4 3 2 1 Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. L. A. Bjelk (101), Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695 T. W. Boutton (155, 173, 219), Department of Rangeland Ecology and Man- agement, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843 D. C. Coleman (3), Department of Entomology, University of Georgia, Athens, Georgia 30602 F. T. Corbin (3, 101), Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27695-7627 J. R. Ehleringer (187), Department of Biology, University of Utah, Salt Lake City, Utah 84112 J. N. Gearing (201), Maurice - Lamontagne Institut, Fisheries and Oceans Canada, Mont-Joli, Quebec G5H 3Z4, Canada K. M. Goh (125, 147), Department of Soil Science, Lincoln College, Canter- bury, New Zealand E. G. Gregorich (77), Land Resource Research Centre, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario K1A 0C6, Canada D. Harris (39), Department of Crop and Soil Science, Michigan State Univer- sity, East Lansing, Michigan 48824 J. Kummerow (11), Department of Biology, College of Sciences, San Diego State University, San Diego, California 92182-0057 A. E. McElroy (109), Environmental Sciences Program, University of Massa- chusetts at Boston, Harbor Campus, Boston, Massachusetts 02125 ix x Contributors T. J. Monaco (101), Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695 E. A. Paul (39), Department of Crop and Soil Science, Michigan State Univer- sity, East Lansing, Michigan 48824 A. M. Pregnall (53), Biology Department, Vassar College, Poughkeepsie, New York 12601 P. J. H. Sharpe (245), Biosystems Research Division, Department of Industrial Engineering, Texas A&M University, College Station, Texas 77843 R. D. Spence (245), Division of Science and Engineering, University of Texas of the Permian Basin, Odessa, Texas 79762 R. P. Voroney (77), University of Guelph, Ontario Agricultural College, De- partment of Land Resource Science, Guelph, Ontario NIG 2W1, Canada F. R. Warembourg (11), Centre d'Ecologie Fonctionnelle et Evolutive, Centre Nationale de la Reserche Scientifique, CNRS Centre L. Emberger, F-34033 Montpellier Cedex, France J. P. Winter (77), University of Guelph, Ontario Agricultural College, Depart- ment of Land Resource Science, Guelph, Ontario NIG 2W1, Canada Preface Historically, isotopes have played an important role as diagnostic tools in natural or human-modified experiments throughout a wide range of biologi- cal studies. As various disciplines have matured and instrumentation has become more sophisticated and readily available, a range of opportunities for use of stable isotopes has been added to the impressive repertoire of radioiso- topes that have been in general use since the 1960s. When we were asked to edit a book on carbon isotopes in plant, soil, and aquatic biology, our response was enthusiastic. We felt there was a real need for a user-oriented book that would be of interest to a wide audience. Our authors have written explicitly for the advanced undergraduate or graduate student, as well as any well-rounded generalist scientist who has not previously used radioisotopes or stable isotopes in his or her research. This book is meant for frequent use; its most suitable habitat is a laboratory bench or laboratory desk. It is designed for easy perusal as people plan laboratory or field research. Perhaps at no time in the history of biology has there been such a profusion of new techniques and tools developed for analytical purposes. This is most evident at the subcellular, cellular, tissue, and organ levels, since a wide variety of molecular genetic and biochemical techniques have come into use. Of perhaps equal intensity, but less evident in popular science publications (e.g., Science, Scientific American), has been a merger of research objectives in the disciplines of physiological ecology and ecosystems studies. The main impetus has come from the revelation that "warm season" plants with the 4C pathway (or Hatch-Slack pathway, with 4C compounds as principal compo- nents of intermediary metabolism) have a significantly different content of 13C than do "cool season" plants with the 3C (or Calvin-Benson) pathway. Impli- cations of this basic separation on a physiological basis are extended and amplified through entire ecosystems, from plants into soils, and in aquatic systems, as noted in several of the chapters in our book (see especially chapters by Boutton and Ehleringer). XI xii Preface For finer levels of resolution, particularly necessary when initial starting materials are considerably diluted (e.g., see Chapter 5 by Voroney et al. and Chapters 8 and 9 by Goh on long-term soil studies, or see Chapter 6 by Corbin et al. and Chapter 7 by McElroy on aspects of herbicides and environmental toxicology) it is advisable to measure constituents labeled with the radioisotope 14C. This isotope also has been introduced by humankind via nuclear weapons tests (cf. Goh) and by cosmic rays, and is traceable with a variety of techniques. What we, the editors, find both challenging and exciting is that the ap- proaches contained herein enable workers from diverse backgrounds to bring their analytical tools to bear on a range of whole-system problems. For example, who would have suspected that physiological ecologists, experi- menting on plant translocation of photosynthates, would have anything in common with air pollution researchers? Thus short-term changes in key plant physiological parameters (such as stomatal conductance, carbon exchange rates, and phloem transport of carbon in shoots and roots) can be assessed using the short-lived gamma-emitting isotope nC (half-life = 20.3 minutes), as noted by Spence and Sharpe. We welcome any readers' suggestions and comments about additional areas of interest, or possible changes and additions to existing chapters and protocols. David C. Coleman Brian Fry Introduction and Ordinary Counting as Currently Used D. C. Coleman F. T. Corbin Department of Entomology Department of Crop Science University of Georgia North Carolina State University Athens, Georgia 30602 Raleigh, North Carolina 27695 I. INTRODUCTION A. Units of Measure Natural radioactivity cannot be increased or decreased by any ordinary process. The activity of a quantity of radioactive material is the number of nuclear disintegrations that occur in unit time. The unit of activity was described originally as the curie and was defined as the radioactivity in one gram of radium. The value of the curie thus was dependent upon experimen- tal measurement and was finally stabilized at 3.7 X 1010 disintegrations per second (DPS). The SI (Systeme Internationale) unit of radioactivity, the Becquerel (Bq), is the preferred unit for current publication in professional journals. Also the unit value of the Becquerel (1 Bq = 1 DPS) is the desired number to follow in calculations, in preference to the microcurie (1 //Ci = 37,000 DPS). Some of the more common terms in current use on product labels and in technical literature and the appropriate conversion factors are listed in Table 1. B. Specific Activity An accurate analysis of the amount of radioactive substances in soils and plants requires that the radiolabel be described as a function of concentra- tion. Specific activity is the rate of decay per unit mass of an element and is usually listed as mCi/mmol, or kBq/mmol. The maximum specific activity, in which every molecule of a substance contains the radiolabel, is seldom CARBON ISOTOPE TECHNIQUES 3 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 4 D. C. Coleman and F. T. Corbin Table 1 Units of Radioactivity and Corresponding DPM Values DPS DPM (disintegrations (disintegrations Unit Fraction of unit per second) per minute) Curie (Ci) 10° 3.7 X 1010 2.22 X 1012 Milli-Curie (mCi) lO"3 3.7 X 107 2.22 X 109 Micro-Curie (//Ci) 10"6 3.7 X104 2.22 X 106 Nano-Curie (nCi) 10"9 3.7 X 101 2.22 X 103 Becquerel (Bq) 10° 1 60 Kilo-Becquerel (kBq) 103 1X103 60X103 Mega-Becquerel (MBq) 106 1X106 60 X 10« Note: SI notation uses Becquerels exclusively. attained in actual practice; usually only one or two radioactive atoms per 106 atoms total are needed in radiotracer work. The constant, A, is known as the decay constant and is expressed in units of time. The rate of decay is proportional to the number of radioactive atoms present. 1. Example Calculation Carbon (14C) is continually produced in the upper atmosphere by cosmic radiation and is relatively constant in nature. What is the maximum specific activity of K£4C0 ? One mole contains 6.023 X 1023 radioactive carbon 3 atoms (Avogadro's number). Specific activity = AN . .. (0.00012 yr)(6.023 X 10 23) 0 n Specific activity = ec) (365 days)(24 hr)(60 min)(60 S Specific activity = 62 Ci/mol where λ is the decay constant and N is the number of radioactive carbon atoms in 1 mole. C. Radiation Detection There are several radiation-counting instruments that can be employed for measuring the radioactivity in samples containing carbon-14. 1. Gas Counters Particle counters for measurement of radioactive emissions were designed by Rutherford and Geiger and later extensively developed by Geiger and Müller. Similar counters are in use today and the current sophisticated 1. Introduction and Ordinary Counting as Currently Used 5 microprocessor-controlled Imaging Proportional Counters and thin layer chromatography (TLC) scanners operate on the basic principles of the early instruments. Operation is based on the phenomenon that large numbers of ions are formed during the passage of charged particles, such as alpha and beta emissions, through a gas. The number of ions produced by a beta particle is not very large, but when a high voltage (1000 volts) is connected in series with the counter, a cascade effect results, which amplifies the original current, and the resultant current can be detected with ease and quantified. 2. Scintillation Counters Experiments by Becquerel on the fluorescence of substances during expo- sure to X rays resulted in one of the early ways of detecting nuclear particles. Flashes of light, or scintillations, occur in certain crystals, such as naphtha- lene and anthracene, when they are exposed to nuclear particles. Light is emitted with frequencies characteristic of the atoms of the crystal, but this process had only limited use for many years because the light intensities of each scintillation were too low for accurate measurements. In recent years, the photomultiplier tube and associated circuitry have served to multiply the photoelectric currents from scintillators by a large factor. These tubes can be incorporated into amplifying and counting circuits for counting particles up to a million per second. 3. Liquid Scintillation Counting The most frequently used method for counting is a liquid scintillation counter (LSC) (Fig. 1). This device measures photons given off by photosen- sitive chemicals (fluors) in the liquid scintillation "cocktail" (a mixture of Fig. 1 A representative, modern "bench-top" liquid scintillation counter, complete with printer. (Photo courtesy of Beckman Instruments, Fullerton, California.)