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Microbiology and Tumorbiology Center, MTC Karolinska Institutet Stockholm, Sweden Assessing the toxic impact of chemicals using bacteria Jenny Gabrielson Stockholm 2004 All published papers were reproduced
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Microbiology and Tumorbiology Center, MTC Karolinska Institutet Stockholm, Sweden Assessing the toxic impact of chemicals using bacteria Jenny Gabrielson Stockholm 2004 All published papers were reproduced with the permission of the publisher Published and printed by Karolinska University Press Box 200, SE Stockholm, Sweden Jenny Gabrielson, 2004 ISBN Allting har sin tid. Dansa har sin tid arbeta har sin tid älska har sin tid. Predikaren 3:1-8 Abstract Abstract There is a considerable backlog in the testing of new chemical compounds for their ecotoxic properties, mostly due to the lack of appropriate testing methods. In addition, there is a vivid debate today about the test endpoint for many of the existing methods. This thesis describes the Microbial Assay for Risk Assessment, MARA, which is a new method for ecological risk assessment and toxicity testing. MARA is based on the simultaneous reading of growth inhibition of eleven microbial strains, exposed to a concentration gradient of the tested chemical compound. The eleven strains have been selected so that different strains exhibit different sensitivities to chemicals. A growth inhibition pattern the toxic fingerprint can be thus detected on the microplate in which the test is performed. The toxic fingerprint, rather than the eleven individual growth inhibitory concentrations, is the test result. It can be compared to toxic fingerprints from other tested chemicals in a database and thereby generate more information regarding the type of toxic effect and species specificity of the tested compound than a single-value result would on its own. The toxic fingerprint for two groups of chemicals, disinfectants and chlorophenols, were compared. It was concluded that MARA can differentiate between different types of toxic effects and also between chemicals with similar molecular structure. MARA is comparable to other bacteria based tests both regarding sensitivity and reproducibility. The toxic fingerprint is heavily dependent on the selection of the strains on which the assay is based. They must give a diversified answer to large groups of chemicals, and as bacteria have a larger genetic diversity than higher organisms, it would plausibly be advantageous with a high genetic diversity among the strains. It was shown that the strains should preferably belong to different genera, but not necessarily to different phyla, to yield the most differentiated response to different chemicals. In order to make MARA as user-friendly as possible, a new method for detection of microbial growth/inhibition of growth in microplates using a flat-bed scanner was developed. The results obtained with the scanner were highly correlated to results obtained with a spectrophotometer, the classical device used for instant quantification of microbial growth, when using TTC (triphenyl tetrazolium chloride, tetrazolium red) or MTT (chelating tetrazole) as growth indicators. v Sammanfattning Det föreligger en avsevärd eftersläpning i arbetet med att testa nya kemikaliers ekotoxiska egenskaper, i huvudsak beroende på bristen på lämpliga testmetoder. Dessutom pågår en livlig debatt om vilken parameter man bör studera i de tester som används. Denna avhandling beskriver ett Mikrobiellt Test för RiskBedömning (MARA), en ny metod för ekologisk riskbedömning. MARA är baserat på mätning av tillväxtinhibering av elva mikrobiella stammar som utsätts för en koncentrationsgradient av ett kemiskt ämne. De elva stammarna har valts så att de skall uppvisa olika känslighet för olika kemikalier. Ett inhibitionsmönster det toxiska fingeravtrycket kan avläsas på mikroplattan som testet utförs i. Det är det toxiska fingeravtrycket snarare än de elva enskilda inhiberingsvärdena som är slutresultatet. Det kan jämföras med toxiska fingeravtryck från andra kemikalier i en databas och således kan ett mer informativt svar erhållas än om resultatet bestått av ett enda värde. De toxiska fingeravtrycken från två kemikaliegrupper, desinfektionsmedel och klorfenoler, har jämförts. Det konstaterades att MARA kan skilja mellan olika typer av toxiska effekter och även mellan kemikalier med likartade molekylära strukturer. MARA är jämförbart med andra liknande tester m a p såväl känslighet som reproducerbarhet. Urvalet av stammar i MARA påverkar i högsta grad de toxiska fingeravtrycken. Stammarna måste ha varierande känslighet för olika kemikalier, och emedan bakterier har en större genetisk variation än högre organismer torde en hög genetisk diversitet vara önskvärd. Det konstaterades att stammarna i MARA med fördel bör plockas från olika genus, men inte nödvändigtvis från olika fyla, för att största möjliga känslighetsvariation skall erhållas. För att göra MARA så användarvänligt som möjligt har en ny metod för mätning av mikrobiell växt/inhibering av växt i mikroplattor m h a en skanner utvecklats. Plattorna skannades och ett speciellt utvecklat datorprogram användes sedan för att mäta växten genom att analysera bilderna. Resultaten som erhölls med skannern visade hög korrelation med resultat som erhållits med en spektrofotometer, vilken är den klassiska metoden för momentan avläsning av mikrobiell tillväxt, om TTC (trifenyltetrazoliumklorid, tetrazoliumrött) eller MTT (kelaterande tetrazol) användes som tillväxtindikatorer. vi List of publications List of publications This thesis is based on the following papers, which in the text will be referred to by their roman numerals Paper I Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates Jenny Gabrielson, Mark Hart, Anna Jarelöv, Inger Kühn, Douglas McKenzie, Roland Möllby Journal of Microbiological Methods, : Paper II A microplate based Microbial Assay for Risk Assessment (MARA) and (eco)toxic fingerprinting of chemicals Jenny Gabrielson, Inger Kühn, Patricia Colque-Navarro, Mark Hart, Aina Iversen, Douglas McKenzie, Roland Möllby Analytica Chimica Acta, 485 (2003) Paper III Bacterial diversity related to the toxic impact of chemicals Jenny Gabrielson, Inger Kühn, Ruth de Karzow, Terry Dando, Roland Möllby Submitted Paper IV Microbial Arrays and Pattern Recognition for analysis of toxicity of chemicals Inger Kühn, Jenny Gabrielson, Ruth de Karzow, Patricia Colque-Navarro, Roland Möllby Submitted vii Abbreviations used in the thesis ARDRA BTB DGGE ECVAM FAME ICCVAM IC 50 LC 50 LOEL MARA MEIC MIC MTC MTT NCIMB NOEC QSAR PLFA RISA SIR TGGE T-RFLP TTC Amplified Ribosomal DNA Restriction Analysis Bromothymol Blue Denaturating Gradient Gel Electrophoresis European Centre for Validation of Alternative Methods Fatty Acid Methyl Ester Interagency Coordinating Committee on the Validation of Alternative Methods the Concentration at which 50% Inhibition is obtained the Concentration at which 50% Lethality is obtained Lowest Observed Effect Level Microbial Assay for Risk Assessment Multicentre Evaluation of In vitro Cytotoxicity Minimal Inhibitory Concentration Microbial Toxic Concentration 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide National Collections of Industrial, Food and Marine Bacteria No Observed Effect Concentration Quantitative Structure-Activity Relationships Phospholipid Fatty Acid Ribosomal Intergenic Spacer Analysis Substrate Induced Respiration Temperature Gradient Gel Electrophoresis Terminal Restriction Fragment Length Polymorphism Tetrazolium red, 2,3,5-triphenyl tetrazolium chloride viii Abbreviations used in the thesis Contents ABSTRACT SAMMANFATTNING LIST OF PUBLICATIONS ABBREVIATIONS USED IN THE THESIS FOREWORD V VI VII VIII XI INTRODUCTION 12 AIMS OF THE STUDY 2 ECOTOXICITY TESTING 3 ALTERNATIVE METHODS TO ANIMAL TOXICITY TESTING 6 EFFECTS OF TOXIC CHEMICALS ON BACTERIA 8 READING OF MICROBIAL GROWTH 11 TETRAZOLIUM THEORY 12 IMAGE ANALYSIS 14 CALCULATION OF INHIBITION VALUES 16 LYOPHILISATION 18 BACTERIAL TAXONOMY AND PHYLOGENY 20 BIODIVERSITY 22 A COMPARISON OF THE TOXIC EFFECT ON A MIXED AND SINGLE SPECIES COMMUNITY 26 RESULTS AND FUTURE PERSPECTIVES 28 ABSTRACTS OF INCLUDED PAPERS 29 CONCLUSIONS 31 ix REFERENCES 32 x Foreword Foreword I would like to thank each and everyone who helped me during these years to finish my PhDwork colleagues, family and friends. To name and express my gratefulness to all of you would require another thesis. This study has been performed with financial support from the European Union s Fifth Framework Program, Project No. MAS3-CT , The Swedish National Board for Laboratory Animals, Vinnova (project number ) and The Swedish Animal Welfare Agency (project number ). xi Introduction Introduction There is an emerging need for fast, cheap and simple ecotoxicity tests. The number of chemicals used in our society is constantly increasing, and for most chemicals nothing or very little is known about their effects when being discharged into nature. Unanimous reports about damages to the environment due to toxic chemicals are regularly found in the media. There are a number of (eco)toxicity tests in use today, most of them animal based. A system based on microbes would be an ideal model for use within an assay designed to assess potentially toxic compounds. In contrast to multi-cellular eukaryotic organisms, bacteria have rapid rates of growth and reproduction. Furthermore, there are a large number of individuals within a given population limiting the effects of cell-to-cell variability s, the use of naturally occurring strains has no ethical ramifications, and they interact rapidly with their environment. Bacteria-based tests are currently available, but they tend to utilise a single strain. The correlation of the toxicity of a chemical, as shown by its inhibitory effect upon a single bacterial strain, with overall environmental toxicity is tenuous, and has a potential for creating inaccurate and unnecessary responses. The use of a multiple strain assay, using metabolically and genetically diverse bacteria, could potentially provide a much more accurate and appropriate tool for the screening of chemicals with unknown ecotoxic properties. Figure 1 MARA-plate with a seven step concentration gradient of H 2 O 2. One microbial strain in each of columns 1-11, column 12 is a control without microbes. H 2 O 2 is added with the highest concentration in row H and the lowest in row B. Row A is the control. The dots seen on the plate indicate microbial growth. The toxic fingerprint is marked in the figure. In the present thesis a Microbial Assay for Risk Assessment, MARA, has been developed (Paper II). The idea is to expose at least eleven microbial strains, lyophilised in a microplate, to a concentration gradient of the chemical to be tested. Since the different strains exhibit different sensitivities to chemicals, a growth pattern the toxic fingerprint can be seen on the plate (Figure 1). Our hypothesis is that the toxic fingerprint is unique for each chemical and that it is indicative for the mode of toxicity of the chemical. The importance of having a wide phylogenetic diversity among the strains in MARA for getting the optimal fingerprint was assessed in Paper III. In Paper IV the concept of toxic fingerprint was further tested and was proven to be successful. To be able to read the result of the assay in a simple way, a suitable system for capturing data using a flat-bed scanner was developed (Paper I). The plates were scanned and a software program developed in-house was used to analyse microbial growth. An appropriate growth indicator, such as tetrazolium red (TTC), was added to facilitate the reading. 2 1 Aims of the study Similar tests have been developed elsewhere, e g a toxicity test based on algae in microplates (3) or a test for detection of the antimicrobial activity of plant extracts (4). In both tests, microorganisms are exposed to a concentration gradient of a sample to be tested in the same way as in MARA. The major novelties with MARA are 1) The toxic fingerprints. It is not the inhibition values of a specific strain that give the result but rather the combination of the inhibition values from multiple strains that forms the toxic fingerprint. This toxic fingerprint can be compared to toxic fingerprints from other chemicals. In cases when the result is preferred as a single value, such as when comparing the result from MARA to that from other tests, either the mean inhibition value or the inhibition value of the most sensitive strain can be used. 2) The possibility to build a database of the toxic fingerprints. In this way more information can be retrieved from each test. If the toxic fingerprint of a tested chemical is similar to that of a tested chemical with known (eco)toxic effects it may also have a similar type of toxic effects. At this time, this correlation is just a hypothesis though. 3) The simple reading of the result. A flat-bed scanner can be found in almost every office these days. Together with the MARA-software it takes less than 24h from the start of the test to the completed result. Only one of those hours is working time, the rest is incubation time. 4) A new way to calculate growth inhibition was developed, the Microbial Toxic Concentration (MTC), which is based on the comparison of the areas under and above the growth inhibition curve. Aims of the study The aim of this study was to develop a cheap and simple (eco)toxicity test based on growth inhibition of microorganisms in a microplate. The hypothesis was that since different bacteria have different sensitivities towards different toxic chemicals, unique toxic fingerprints from the chemicals would be obtained from the test. In order to get the unique fingerprints, a wide diversity among the bacteria in the test is desirable and thus a study on the importance of phylogenetic diversity was needed. In addition, a method for capturing data by computerised reading of bacterial growth would enhance the possibilities for the test to reach outside the sphere of microbiological laboratories. 2 Ecotoxicity testing Ecotoxicity testing Every day of our lives we meet a large amount of anthropogenic substances. Most of them pass unnoticed, some of them affect us temporarily (like ethanol) and others again have long-term harmful influence on our bodies even though they do not constitute any acute risk (like e g mercury). All these chemicals also pass through the environment: some of them without making any harm, some of them making temporary lesions and some of them making irreversible damages. It is obvious that efforts must be made to minimise the use of such chemicals that may damage the environment or our health. But to make those efforts, knowledge must first be gained about the effects of different chemicals. The only known way to find out about the hazardous effects is The ecotoxicological to make assays on living materials, traditionally whole animals. methodology include as well During the past years there has been a vivid debate about the cell- and organophysiological experiments in right of the animals what gives the right to mankind to make painful tests of our innovations on innocent animals? As a the laboratory as field studies and mathematical result of this debate, a number of in vitro-tests on cell-lines, models. primitive animals and microorganisms have been developed. Much work has been done, and it has been proven that these National Encyclopedia alternative tests may give results just as valid as tests on whole animals (5),(6). See also next chapter, Alternative methods to animal toxicity testing. New chemicals that are released on the European market today must be tested on fish, Daphnia (a crustacean) and algae according to the OECD Test Guidelines. These tests are about to be the worldwide standard for ecotoxicity testing. In special cases, or when the standard tests give doubtful results, other tests such as tests on activated sludge and multi-species tests are performed. If satisfying results are still missing, further assays such as tests on mesocosms (see next paragraph) may be necessary. These tests are very costly though and the chemical must be valuable for the manufacturer to proceed to this stage. (Alf Lundgren, National Chemicals Inspectorate (Kemikalieinspektionen), personal communication) Tests for assessing the ecotoxic effects of a chemical have evolved later than tests for human health. The ideal test from an ecologist s point of view would be a lab-scale ecosystem (a so called micro- or mesocosm) where the fate of contaminants can be monitored. A number of such studies have been presented, but the amount of information retrieved from a mesocosm is enormous and is very difficult to interpret (7). Further, the knowledge of complex ecological systems is still limited and this makes the interpretation of data even more difficult. Criticism against the use of mesocosms has been expressed regarding the limited value of a test; even if full knowledge can be gained about the effects in this specific ecosystem not much can be said about the effects under other circumstances. Kraufvelin (8) showed that the repeatability of results from mesocosms is at an unacceptably low level and it would be unrealistic to make any predictive validations based on these data. On the other hand, Hall and Giddings (9) has shown that concentrations of a chemical exceeding the critical level found in single-species tests has no or little effect in an aquatic ecosystem. To make a full assessment of a chemical it would thus probably be necessary with a combination of the different testing methods. Much research is 3 Ecotoxicity testing needed though before a satisfying level of knowledge is reached regarding how to interpret the results, and before such knowledge is gained deficient but efficient methods must be used. Most ecotoxicity tests used today are based on one single species. This is of course much simpler and cheaper than creating a complex mesocosm. Several studies (10),(11), have shown that a combined set of single species tests can give NOEC (No Observed Effect Concentration)-values in the same order of magnitude as a model ecosystem. It has been found though that singlespecies tests often give somewhat higher NOEC-values than multi-species tests and thus rather underestimate the risk of a chemical. There are a number of plausible explanations to this; variability of data collected in the test systems, lack of knowledge about the most appropriate test methods and times, extrapolation from individual to population-level endpoints etc (11),(9). It has often been suggested that a battery of different single-species tests would make a good ecotoxicity test (12),(13),(14). Care must be taken though when selecting the species to be included in a battery. Henschel et al (15) found that of salicylic acid, paracetamol, clofibrinic acid and methotrexate tested with three acute standard tests alone (algae, Daphnia and fish), ecotoxic potentials were underestimated for all tested substances but salicylic acid. Microorganisms, such as bacteria, are very suitable for ecotoxicity testing. Their small size makes it possible to make assays on millions of individuals simultaneously and thereby avoid uncertainties due to individual variations. As bacteria have short generation times, sometimes as short as 20 minutes, test times can be reduced considerably compared to tests on larger organisms. Bacteria are easy and cheap to handle, and staff without any advanced training are generally able to perform the tests. They incorporate toxicants in their metabolism faster than higher organisms do. Last, but not least, there are no ethical problems connected to the use of microorganisms in toxicity testing. (16),(17) Microorganism based ecotoxicity testing has been explored both commercially and in academic research. The most commonly used commercial test is Microtox, a test based on
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