Polarographic Oxygen Sensors Aquatic and Physiological Applications Edited by E. Gnaiger and H. Forstner With 142 Figures Springer-Verlag Berlin Heidelberg New York 1983 Dr. ERICH GNAIGER Dr. HELLMUTH FORSTNER Institut fUr Zoologie Abteilung Zoophysiologie Universitat Innsbruck Peter-Mayr-StraBe 1 a A-6020 Innsbruck, Austria ISBN-13: 978-3-642-81865-3 e-ISBN-13: 978-3-642-81863-9 DOl: 10.1007/978-3-642-81863-9 Library of Congress Cataloging in Publication Data. Main entry under title: Polarographic oxygen sensors. Bibliography: p. Includes index. I. Oxygen - Analysis. 2. Polarograph and polarography. I. Gnaiger, E. (Erich), 1952-. II. Forstner, H. (Hellmuth), 1935--. QP535.0IP58. 1983. 574.19'214. 82-19419. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use a fee is payable to 'Verwertungsgesellschaft Wort', Munich. © by Springer-Verlag Berlin Heidelberg 1983. Softcover reprint of the hardcover lst edition 1983 The use of registered names, trademarks, etc. In this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. 2131/3130-543210 Preface: A Biologist's View Es liegt ein tiefes und griindliches Gliick darin, daf> die Wissenschaft Dinge ermittelt, die stand halten und die immer wieder den Grund zu neuen Ermittlungen abgeben: - es konnte ja anders sein! Friedrich Nietzsche Die frohliche Wissenschaft Molecular oxygen comprises about 20% of our atmosphere, but less than 5% of this amount is dissolved at equilibrium in water. As a con sequence of this low solubility in seawater, in freshwater and in aqueous body fluids, living cells are subjected to a universal problem of low oxygen availability. Thus biologically relevant measurement of dis solved oxygen extends from a cellular scale where sensors with diame ters of less than 10-6 m are employed, to the oceanic scale where in situ measurements are performed at depths beyond 104 m. This book covers the wide spectrum of aquatic and physiological applications of polarographic oxygen sensors. It is intended as a basic introduction for the student, and as a readily available compilation of detailed information for the specialist. Assessments of various methods for monitoring aquatic environments and physiological processes aid in overcoming practical problems frequently encountered in the labo ratory and in the field. Concomitant with the provision of experi mental guidelines, topics of bioenergetics are addressed on the basis of respiratory oxygen exchange and related physiological mechanisms. A respiratory physiologist would specify polarographic oxygen sen sors as perfect oxygen conformers, their respiratory rate being linearly dependent on Po, . He would find that their oxygen consumption is proportional to the area of their oxygen-transducing membrane, with a QIO of 1.4 and a constant of proportionality (about 2 nmol O2 h-1 mm-2) which might be typical of, for example, fish eggs. Although the increasing unpredictability of their oxygen consumption with in creasing age would be understandable, he might be surprised that, des pite their vulnerability to desiccation, oxygen consumption remained the same in air as in water. In these respects polarographic oxygen sen sors are not different from organisms, except that microsensors con sume oxygen proportional to their diameter instead of area. But do we know that microorganisms do not behave Similarly? These apparent phenomena are explained on the basis of physico chemical principles in Part I. Why is this understanding so important? Since oxygen concentration per unit partial pressure (co /PO = solubility) is low in water, small concentration changes beco~e appar- VI Preface: A Biologist's View ent as large changes of POl' signalled by the sensor. This feature pre destines polarographic oxygen sensors for aquatic application: The sensor is 20 to 30 times more sensitive to respiratory rates in water than in the same volume of air. The operational principle of polarographic oxygen sensors is based on oxygen diffusion to the polarized cathode, where oxygen reduc tion (in micromoles O per second) generates the electrical signal (in 2 amperes). According to Faraday's law the ratio is calculated as 2.591 /lmol O2 S-1 A-I (:::: 9.328 nmol O2 h-1 /lA-I). Variations in the construction of polarographic oxygen sensors are mainly related to the problem of how oxygen diffuses to the cathode. In this respect the actual design of every sensor necessarily involves a compromise, since optimising particular functions, such as sensitivity and response time, detracts from others, such as stability and stirring requirements. The optimal design therefore depends upon the application. The many new commercial oxygen sensors that have come onto the market during the past few years include, to some extent, original ideas, or are merely copies of existing sensors. For instance, the in formation that a polarographic oxygen sensor for marine applications (InterOcean Systems, San Diego) is virtually the same sensor at a price six times that of the original (Ingold, CH) probably comes too late for some purchasers. Proficiency in solving respirometric problems facilitates research into the physiological mechanisms and functional interpretations of gas exchange. With this in mind, respirometric and in situ monitoring methods, of which the polarographic oxygen sensor forms an integral part, are outlined in the detail that is of practical importance but usually neglected (Parts II and III). Methods related to studies of oxygen exchange are also dealt with. Responses to environmental variables and to toxicological or pharmacological agents, metabolic patterns and biological rhythms can be resolved by automatic long term monitoring of oxygen consumption with polarographic oxygen sensors. In combination with simultaneous measurements of, e.g., locomotory activity, ventilatory rates, chloroplast migration, biomass production and notably heat dissipation such investigations can be most fruitful. It is noteworthy that bioenergetics and the recognition of oxygen have a common root in Lavoisier's ingeneous concept of combustion and his classical experiments on direct and indirect calorimetry. Keep ing in line with the extension of the direct and indirect calorimetric approach to the bioenergetics of aquatic animals, the thermodynamic interpretation of oxygen consumption in aquatic organisms is revised in an Appendix and more closely aligned to the needs of ecological energetics. Several case studies illustrate the practical application of the methods and point out concurrent conceptual advances. Preface: A Biologist's View VII The respirometers employed in these studies are no longer simply closed or open systems. Further differentiation has given rise to specific types such as the "rubber mask", "twin-flow" or "slurp gun" respirometers. Various types have invaded lakes, coastal regions and even the deep sea, yet respirometers are still most common in the shallow, constant temperature water baths on the bench. Restocking with new variations and their association with other instumental groups seems rewarding, especially in some laboratories where respiro meters have become fossilized or extinct. Previous summaries of the applications of polarographic oxygen sensors in medicine and cellular physiology can be found in Kessler M. et al., eds. (Oxygen Supply. Urban Schwarzenberg, Mtinchen-Berlin Wien, 1973) and Fatt I. (Polarographic Oxygen Sensors. CRC Press, Cleveland, 1975). For an extended theoretical discussion the reader is referred to Hitchman L.M. (Measurement of dissolved oxygen. Wiley, New York and Orbisphere Laboratories, Geneva, 1978). We hope that the joy and benefits of interdisciplinary communica tion as experienced by the editors of this book may be shared by our readers. Ideas and technological advances in one field may unex pectedly provide the key to solving problems in other apparently un related disciplines (compare e.g. Chaps. 1.4 and III.2, or p. 117 and 247). Thus perhaps even those readers whose special knowledge or special needs are neglected or inadequately dealt with may find some inspiration. The plan for this book originated during a workshop held at the Institut ftic Zoophysiologie der Universitat Innsbruck, Austria, in October 1978. Several methodological developments and the editorial work were supported by the Fonds zur F6rderung der wissenschaft lichen Forschung in Osterreich, projects no. 2919, 2939, 3307 and 3917 (Univ. Prof. Dr. W. Wieser, Univ. Innsbruck, principal investiga tor), by the Forschungsfdrderungsbeitrag der Vorarlberger Landesre gierung and by a British Council Scholarship at the Institute for Marine Environmental Research, Plymouth, England. I thank all con tributors for their encouraging cooperation. I want to express my special gratitude to Mrs. J. Wieser for her experienced help in improv ing the English style of several manuscripts, and I thank my colleagues and friends for their support and fruitful comments. Innsbruck-Plymouth Erich Gnaiger Contributors You will find the addresses at the beginning of the respective contributions Baldwin, R.J. 298 Hitchman,M.L. 18,31 Bals, I. 102 Johansen, K. 127 Baumgard, H. 37 Knapp, W. 195 Bridges, C.R. 219 Lomholt, J.P. 127 Broda, H. 190 Lilbbers, D.W. 37 Bucher, R. 66 Mickel, TJ. 81,184 Bilhler, H. 76 Newrkla, P. 274 Childress, J.J. 81 Ott, J. 285 Dalla Via, G.J. 202 Pamatmat, M.M. 167 Fatt, I. 234 Quetin,L.B.81,176,184 Forstner, H. 90, 111,321 Revsbech, N.P. 265 Gnaiger, E. 31,134,245,321, Schweiger, G. 190 334,337,352 Schweiger, H.G. 190 Golterman, H.L. 346 Smith, K.L., Jr. 298 Grabner, W. 86 Svoboda, A. 285 Hale, J.M. 3,73,102 Wolff, D. 190 Contents Part I Principles of Sensor Design and Operation Chapter I.l Factors Influencing the Stability of Polarographic Oxygen Sensors J.M. Hale (With 2 Figures) ................. 3 Chapter 1.2 Calibration and Accuracy of Polarographic Oxygen Sensors M.L. Hitchman (With 2 Figures) ............. 18 Chapter 1.3 A Thermodynamic Consideration of Permeability Coefficients of Membranes M.L. Hitchman and E. Gnaiger (With 1 Figure) ... 31 Chapter 1.4 Microcoaxial Needle Sensor for Polarographic Measurement of Local O Pressure in the Cellular 2 Range of Living Tissue. Its Construction and Properties H. Baumgartl and D.W. LUbbers (With 13 Figures) 37 Chapter 1.5 Electrolytes R. Bucher ........................... 66 Chapter 1.6 The Action of Hydrogen Sulfide on Polarographic Oxygen Sensors J.M. Hale ............................ 73 Chapter 1.7 A Double-Membrane Sterilizable Oxygen Sensor H. BUhler (With 1 Figure) ................. 76 Chapter 1.8 Construction of a Polarographic Oxygen Sensor in the Laboratory T.J. Mickel, L.B. Quetin, and J.J. Childress (With 2 Figures) ....................... 81 X Contents Chapter 1.9 A Polarographic Oxygen Sensor Designed for Sewage Work and Field Application W. Grabner (With 1 Figure). . . . . . . . . . . . . . .. 86 Chapter 1.10 Electronic Circuits for Polarographic Oxygen Sensors H. Forstner (With 8 Figures) . . . . . . . . . . . . . .. 90 Chapter 1.11 The Application of a Microprocessor to Dissolved Oxygen Measurement Instrumentation I. Bals and J.M. Hale (With 3 Figures) ......... 102 Part II Laboratory Applications of Polarographic Oxygen Sensors Chapter ILl An Automated Multiple-Chamber Intermittent-Flow Respirometer H. Forstner (With 7 Figures) ............... 111 Chapter 11.2 The Application of Polarographic Oxygen Sensors for Continuous Assessment of Gas Exchange in Aquatic Animals J.P. Lomholt and K. Johansen (With 4 Figures) ... 127 Chapter 11.3 The Twin-Flow Microrespirometer and Simultaneous Calorimetry E. Gnaiger (With 20 Figures) ............... 134 Chapter 11.4 Simultaneous Direct and Indirect Calorimetry M.M. Pamatmat (With 3 Figures) ............ 167 Chapter 11.5 An Automated, Intermittent Flow Respirometer for Monitoring Oxygen Consumption and Long-Term Activity of Pelagic Crustaceans L.B. Quetin (With 5 Figures) ............... 176 Chapter 11.6 Sealed Respirometers for Small Invertebrates L.B. Quetin and T.J. Mickel (With 3 Figures) .... 184 Chapter 11.7 A Method for the Simultaneous Long-Term Recording of Oxygen Evolution and Chloroplast Migration in an Individual Cell of Acetabularia H.G. Schweiger, H. Broda, D. Wolff, and G. Schweiger (With 3 Figures) .............. 190 Contents XI Chapter 11.8 A Respirometer for Monitoring Homogenate and Mitochondrial Respiration W. Knapp (With 2 Figures) ................ 195 Chapter 11.9 Bacterial Growth and Antibiotics in Animal Respirometry GJ. Dalla Via (With 3 Figures) .............. 202 Chapter 11.1 0 Po and Oxygen Content Measurement in Blood Sa~ples Using Polarographic Oxygen Sensors C.R. Bridges (With 4 Figures) ............... 219 Chapter 11.11 Determination of the In Vivo Oxygen Flux into the Eye I. Fatt (With 8 Figures) ................... 234 Part III Field Applications of Polarographic Oxygen Sensors Chapter 111.1 In Situ Measurement of Oxygen Profiles in Lakes: Microstratifications, Oscillations, and the Limits of Comparison with Chemical Methods E. Gnaiger (With 11 Figures) ............... 245 Chapter III.2 In Situ Measurement of Oxygen Profiles of Sediments by Use of Oxygen Microelectrodes N.P. Revsbech (With 5 Figures) ............. 265 Chapter III.3 Methods for Measuring Benthic Community Respiration Rates P. Newrkla (With 7 Figures) ................ 274 Chapter I1I.4 In Situ Measurement of Community Metabolism in Littoral Marine Systems A. Svoboda and J. Ott (With 11 Figures) ....... 285 Chapter I1I.5 Deap-Sea Respirometry: In Situ Techniques K.L. Smith, Jr. and R.J. Baldwin (With 10 Figures) 298 Appendix A Calculation of Equilibrium Oxygen Concentration H. Forstner and E. Gnaiger ................ 321 AppendixB Calculation of Po in Water Equilibrated with a Mixture of Room 2A ir and Nitrogen E. Gnaiger (With 1 Figure) ................ 334