Toxicity Testing Using Microorganisms Volume II Editors Bernard J. Dutka Head, Microbiology Laboratories Section National Water Research Institute Department of the Environment Canada Centre for Inland Waters Burlington, Ontario, Canada Gabriel Bitton Professor Department of Environmental Engineering Sciences University of Florida Gainesville, Florida Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRCPress Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2019 by CRC Press © 1986 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. 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Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-0-367-25253-3 (hbk) ISBN 13: 978-0-429-28681-0 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com THE EDITORS Gabriel Bitton is a professor at the Department of Environmental Engineering Sciences at the University of Florida, Gainesville, Fla. He has extensively published in the field of environmental microbiology and virology and has authored or edited four books in his research area. He has participated in several national and international committees on public health issues. Dr. Bitton is the past National Chairman of the Division of Environmental and General Applied Microbiology of the American Society for Microbiology. He is an Associate Editor for Toxicity Assessment: An International Quarterly. Bernard J. Dutka is Head of the Microbiology Laboratories Section of the National Water Research Institute, Canada Centre for Inland Waters, Department of Environment, Burlington, Ontario, Canada. He has written or co-written over 120 papers and reports on such topics as coliform index, fecal sterol-bacterial relationships, sediment microbiology, microbial toxicity screening tests, and membrane filter methodology, has edited the book Membrane Filtration: Applicationsf Techniques, and Problems, and has co-edited three other books. Mr. Dutka is former Chairman of the ASTM subcommittee D19.24 on water microbiology, international convenor of two ISO working groups, and a member of the AWWA microbiological problems committee. He is also co-Chairman of the Symposium Committee which organizes the biennial International Symposium on Toxicity Testing Using Bacteria, and co-editor of Toxicity Assessment: An International Quarterly. CONTRIBUTORS Harvey Babich Dickson Liu Senior Research Associate Environmental Contaminants Division Rockefeller University National Water Research Institute Laboratory Animal Research Center Burlington, Ontario, Canada New York, New York Tamar Barkay Betty H. Olson U.S. EPA Professor of Civil Engineering Sabine Island Program in Social Ecology Gulf Breeze, Florida University of California at Irvine Irvine, California Gabriel Bitton Professor Department of Environmental Engineering O.F. Shearer Sciences Research Assistant University of Florida Program in Social Ecology Gainesville, Florida University of California at Irvine Pierre Couture Irvine, California INRS-Eau University of Quebec St.-Foy, Quebec, Canada Guenther Stotzky Professor Bernard J. Dutka Department of Biology Head, Microbiology Laboratories Section New York University Analytical Methods Division New York, New York National Water Research Institue Burlington, Ontario, Canada John D. Walker Geoffrey M. Gadd Senior Scientist Department of Biological Sciences Office of Toxic Substances University of Dundee U.S. Environmental Protection Agency Dundee, Scotland Washington, D.C. Ben Koopman Associate Professor Paul T.S. Wong Department of Environmental Engineering Great Lakes Fisheries Research Branch Sciences Canadian Center for Inland Waters University of Florida Burlington, Ontario Gainesville, Florida Canada TABLE OF CONTENTS Chapter 1 Introduction and Review of Microbial and Biochemical Toxicity Screening Procedures...................................................................................................................................... 1 Gabriel Bitton and Bernard J. Dutka Chapter 2 Environmental Factors That Affect the Utility of Microbial Assays for the Toxicity and Mutagenicity of Chemical Pollutants.................................................................................9 Harvey Babich and Guenther Stotzky Chapter 3 Toxicity Screening Using Fungi and Yeasts............................................................................43 Geoffrey M. Gadd Chapter 4 Toxicity Screening Using Phytoplankton.................................................................................79 Paul T.S. Wong and Pierre Coutre Chapter 5 Toxicant Screening in Wastewater Systems..........................................................................101 Ben Koopman and Gabriel Bitton Chapter 6 Toxicity Testing in Soil Using Microorganisms...................................................................133 Tamar Barkay, Betty H. Olson, and O.F. Shearer Chapter 7 Biodegradation Tests.................................................................................................................157 Dickson Liu Chapter 8 A U.S. EPA Perspective on Ecotoxicity Testing Using Microorganisms........................175 John D. Walker Index 187 Volume II 1 Chapter 1 INTRODUCTION AND REVIEW OF MICROBIAL AND BIOCHEMICAL TOXICITY SCREENING PROCEDURES G. Bitton and B. J. Dutka TABLE OF CONTENTS I. Introduction.........................................................................................................................2 II. Effects of Toxicants on Microorganisms.........................................................................2 III. Biochemical Tests.............................................................................................................3 A. Enzymes..................................................................................................................3 B. ATP Assays...........................................................................................................3 IV. Bacterial Tests...................................................................................................................3 A. Assays Based on Bacterial Luminescence........................................................4 B. Assays Based on the Measurement of Growth Inhibition, Respiration, and Viability of Bacterial Cells...........................................................................4 C. “Ecological Effect” Assays................................................................................4 V. Algal Tests..........................................................................................................................4 VI. Fungi and Yeast Bioassays...............................................................................................5 VII. Other Approaches.............................................................................................................5 A. Microcosms...........................................................................................................5 B. Microcalorimetric Techniques............................................................................6 VIII. Conclusions........................................................................................................................6 References 7 2 Toxicity Testing Using Microorganisms I. INTRODUCTION With the increased world-wide industrialization over the past 25 years, and with the concomitant higher demand for chemicals, both the developed and developing nations face increasing ecological and toxicological problems from the release of toxic contaminants to the environment. In response to these expanding stresses on the environment and in the belief that there is no single criterion by which to adequately judge the potential hazard (either to the environment or man) of a given substance,1 a multitude of biological assay procedures have been developed, proposed, and used to assess toxicant impacts.2 3 Due to our newly acquired awareness of the long-term effects of chemicals discharged into receiving waters, research efforts are being directed at short-term bioassay tests in an attempt to alert monitoring agencies as well as dischargers of toxic conditions.4'8 As industrial pollutants and toxicants such as herbicides, insecticides, fertilizers, and car exhaust fumes affect aquatic biota systems at different levels and in many ways, it is acknowledged that the battery approach utilizing several different short-term biological tests would be preferred in any monitoring scheme. In some studies, investigators9 have employed a battery of ecological and health effect tests to estimate the toxicity and mutagenicity of industrial effluents. In general there are two main groups of toxicity screening tests: in vitro “health effect” tests and “ecological effect” tests. “Health effect” toxicity tests are based on the use of subcellular components (e.g., enzymes, DNA, RNA), isolated cells (e.g., cell cultures, red blood cells), tissue sections, or isolated whole organs.10 12 These tests consist of determining cell viability (vital staining- dye inclusion test, plating efficiency, colony formation), cell reproduction, or macromole- cular biosynthesis.1012 “Ecological effect” tests are conducted to measure mainly the acute toxicity of chemicals to aquatic organisms representing various trophic levels of the food chain. These tests help in the estimation of chemical toxicity in natural and man-modified ecosystems. Bacteria, algae, zooplankton, benthic invertebrates, and fish have been used in these tests.1315 Bacteria and enzymes may be exposed to a wide range of toxic, organic, and inorganic compounds in natural waters, soil, and in sewage treatment processes. The toxicity of the compounds depends on environmental parameters as well as on the microorganism or enzyme systems being tested. The compounds may be metabolically altered to nontoxic metabolites or may exert a direct toxic action on microbial populations. Bacteria also may be subjected to synergistic or antagonistic effects between components of toxicant mixtures. In sewage treatment plants, toxicants may cause shifts in microbial populations, and this may adversely affect the operation of the plant.16 The effect of toxicants on waste treatment processes will be reviewed in Chapter 5 by Koopman and Bitton. Toxicant action is concentration dependent. For example, phenol can be metabolized at low concentrations but becomes toxic at higher concentrations. Toxicant action also depends on the presence of other chemicals in solution.17 The purpose of this chapter and book is to survey the literature on microbial and enzymatic tests which are used to screen for chemical toxicity in the research laboratory or in the aquatic system, and to present in detail some of the more commonly used microbial toxicity screening procedures. II. EFFECTS OF TOXICANTS ON MICROORGANISMS There are many proposed mechanisms by which toxicants inhibit and eventually kill bacteria.18 Toxicants may cause damage to the genetic material or may lead to protein denaturation, e.g., halogens. They may also disrupt bacterial cell membranes (e.g., phenol Volume II 3 and quaternary ammonium compounds), the result of which is the leakage of DNA, RNA, proteins, and other organic materials. Certain toxic chemicals may displace cations (e.g., Na+, Ca+) from adsorption sites on the bacterial cell, e.g., acids and alkalis. A more subtle action of toxic pollutants is their ability to block bacterial chemoreceptors19 which may lead to the inhibition of organic decomposition and self-purification processes in sewage treatment plants and in waters receiving fecal material.20 It is believed that one of the most important effects of the toxic action of chemicals on bacteria is on enzyme activity.21 However, in any toxicity study, one must also take into account the physico­ chemical factors (presence of other cations, pH, oxidation-reduction potential, temperature, organic matter, clay minerals, etc.) that control the toxic action towards microorganisms.22 23 Chapter 2 (Babich and Stotzky) focuses on the effect of abiotic factors on toxicant impact. The impact of toxicants on bacterial cells may be measured via biochemical tests which include measurement of enzyme activity, ATP content, and bioluminescence. Some bio­ chemical indicators (e.g., ATP, lipopolysaccharides, muramic acid) have been used for the determination of microbial biomass in environmental samples.24 We will now briefly review the major categories of tests which are used or could potentially be used in toxicity assays. III. BIOCHEMICAL TESTS A. Enzymes Since enzymes drive numerous key metabolic reactions in microbial, plant, and animal cells, their inhibition could be the underlying cause of toxicity to the cells. Thus, numerous studies have been carried out to test the effect of toxic pollutants upon enzyme activity, although most of them dealt with dehydrogenase enzymes. The latter catalyze the oxidation of substrates by transfer of electrons through the electron transport system (ETS), which consists of a complex chain of intermediates (flavoproteins, cytochromes, etc.) which trans port electrons from the nutrient source to 02, the final electron acceptor.25 Specific dyes can be used as indicators of ETS activity. They act as artificial hydrogen acceptors and they change color upon reduction. Thus, the activity may easily be measured with the aid of a spectrophotometer. The most widely used indicator dyes are methylene blue, triphenyltetrazolium chloride (TTC), tetrazolium blue, rasazurin, and 2-(p-iodophenyl)- 3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT). Toxicity tests based on the reduction of these indicator dyes, as well as on other enzymatic assays (e.g., ATPases, esterases, ureases), are described in Chapter 3 of Volume I. B. ATP Assays Adenosine triphosphate (ATP) is a product of catabolic reactions, common to all protists and animal and plant cells. Since ATP is rapidly destroyed after cell death, one then has an ideal means of distinguishing between live and dead cells. The basic assay consists of measuring the light emitted following the reaction of firefly luciferin with ATP. This reaction is catalyzed26 by luciferase and Mg2+. Brezonik and Patterson27 first proposed the use of ATP in toxicity testing in activated sludges. This was further explored by other investigators (see Chapter 3 of Volume I for more details). IV. BACTERIAL TESTS Bacteria are involved primarily in the mineralization of organic substrates and in the recycling of mineral nutrients. Their activities are essential to self-purification processes in aquatic environments. They have relatively short life cycles and respond rather quickly to changes in the environment. They are stable and easily maintained at low cost. Relatively large numbers of cells are exposed to the toxicant under study. These characteristics make bacteria suitable for rapid screening of toxicants in natural waters. The various bacterial