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Biochemical Ecotoxicology Principles and Methods 1st
Edition Francois Gagne Digital Instant Download
Author(s): Francois Gagne
ISBN(s): 9780124116047, 0124116043
Edition: 1
File Details: PDF, 5.07 MB
Year: 2014
Language: english
Biochemical
ECOTOXICOLOGY
Principles and Methods
This page intentionally left blank
Biochemical
ECOTOXICOLOGY
Principles and Methods
FRANÇOIS GAGNÉ
Senior Research Scientist
Biochemical Ecotoxicology at Environment Canada
Québec, Canada
With Additional Contributions From
CHANTALE ANDRÉ
JOËLLE AUCLAIR
ÉMILIE LACAZE
BRIAN QUINN
AMSTERDAM BOSTON HEIDELBERG LONDON

NEW YORK OXFORD PARIS SAN DIEGO
SAN FRANCISCO SINGAPORE SYDNEY TOKYO
Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier
32 Jamestown Road, London NW1 7BY, UK
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Copyright r 2014 Elsevier Inc. All rights reserved.
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by any means electronic, mechanical, photocopying, recording or otherwise without the prior written
permission of the publisher.
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Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions
or ideas contained in the material herein.
Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug
dosages should be made.
British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress
ISBN : 978-0-12-411604-7
For information on all Academic Press publications

visit our website at elsevierdirect.com
Printed and bound in United States of America
14 15 16 17 10 9 8 7 6 5 4 3 2 1
CONTENTS
List of Contributors xiii

Dedication xv
Preface xvii
Acknowledgments xix
Introduction xxi
1. Quantitative Assessments of Biochemical Analyses 1

1.1. General Overview 1
1.2. Replication in Data Analysis 5
1.3. Replication of the Experiment 6
1.3.1. Calibration or Standardization 7
1.3.2. Using Specific and Generic Standards 8
1.4. Reference Substances 10
1.5. Defining Detection Limits in the Absence of Analytical Standards 10
1.6. Reproducibility 12
1.6.1. Reproducibility at the Calibration Level 12
1.6.2. Reproducibility at the Level of the Experiment 13
1.7. Using Reference Material to Standardize Methodologies 13
1.8. Normalization Approaches 15
1.8.1. Residual Extraction Method 16
1.8.2. Stepwise Addition Method 18
References 19
2. Tissue Preparation and Subcellular Fractionation Techniques 21

2.1. Types of Homogenization Buffers 22
2.1.1. pH 22
2.1.2. Ion Chelators 24
2.1.3. Antioxidants 24
2.1.4. Proteases Inhibitors 24
2.1.5. DNase/RNase Inhibitors 25
2.1.6. Osmolarity 26
2.1.7. Cryoprotective Agents 26
2.2. Homogenization Methods 27
2.2.1. Teflon Tissue Pestle 27
v
vi Contents
2.2.2. Polytron 28
2.2.3. Ultrasounds 28
2.2.4. FreezeThaw 29
2.2.5. Subcellular Fractionation by Differential Centrifugation 29
2.2.6. Conservation of Biological Samples for Biomarkers 31
References 31
3. Preparation and Maintenance of Live Tissues and Primary Cultures

for Toxicity Studies 33
Contributed by Brian Quinn
3.1. General Introduction 33

3.2. Reagents Required and Solution Preparation 35
3.3. Procedure 37
3.3.1. Animal Collection and Maintenance 37
3.3.2. Preparation of Primary Cell Cultures 38
3.4. Application to Toxicity Testing 41
3.4.1. Cytotoxicity Evaluation 41
3.4.2. Chronic Effects (Biomarker Expression) 44
3.5. Calculations/Data Handling 45
3.5.1. Calculating Cell Density and Viability Using the Neubauer Hemocytometer 45
References 45
4. Measuring Effects at the Gene Transcription Level 49

Contributed by Chantale André
4.1. Introduction 49
4.2. Material 53
4.2.1. Reagents 53
4.2.2. Equipment 54
4.3. Procedure 54
4.3.1. Choice of Detection Chemistry 54
4.3.2. Primer Design 55
4.3.3. Selecting the Target Nucleotide Sequence (Target and Reference Genes) 55
4.3.4. Preparation of Nucleic acid Template 56
4.3.5. Assay Optimization 62
4.4. Data Analysis-Relative Quantification with Reference Gene Normalization 69
References 72
Web Resources 73
Contents vii
5. Metal Metabolism and Detoxification 75

Contributed by Joëlle Auclair
5.1. Intracellular Free Metal Availability 77

5.1.1. Introduction 77
5.1.2. Reagents 77
5.1.3. Procedure 77
5.1.4. Data Calculation 78
5.2. Subcellular Metal Binding Fractions 78
5.2.1. Reagents and Equipment 79
5.2.2. Procedure 79
5.2.3. Data Analysis and Calculation 80
5.3. Metallothioneins 80
5.3.1. Silver Saturation Assay 80
5.3.2. Spectrophotometric Assay 82
5.4. Enzyme Immunoassay 84
5.4.1. Reagent 84
5.4.2. Procedure 85
5.4.3. Data Calculation and Analysis 86
5.5. Heat Shock Protein 70 Detection and Quantification
by Western Blot Analysis 86
5.5.1. Reagents and Materials 86
5.5.2. Procedure 91
5.5.3. Detection-Colorimetric Alkaline Phosphatase Activity 95
5.5.4. Troubleshooting 97
5.5.5. Case Study 99
References 100
6. Oxidative Stress 103

6.1. Antioxidant Enzymes 104
6.1.1. SOD 104
6.1.2. Peroxidase 106
6.1.3. Catalase 107
6.1.4. Measurement of Antioxidant Capacity 109
6.1.5. Lipid Peroxidation (Oxidative Damage) 110
6.1.6. Measurement of Age-Related Pigments and Lipofuscins 111
6.2. Applications 113
References 115
viii Contents
7. Xenobiotic Biotransformation 117

7.1. Xenobiotic Biotransformation 117
7.2. Generic Assay for Cytochrome P450 119
7.2.1. Reagents 119
7.2.2. Procedure 120
7.3. Cytochrome P4501A1 and Benzo(a)Pyrene Hydroxylase Activities 120
7.3.1. Reagents 121
7.4. Cytochrome P4503A4 Activity 122
7.4.1. Reagents 122
7.5. Glutathione S-Transferase Activity 123
7.5.1. Reagents 124
7.6. Uridine Diphosphate-Glucuronyl Transferase Activity 124
7.6.1. Reagents 125
7.7. Sulfotransferases 126
7.7.1. Reagents 126
7.8. Multidrug Resistance Glycoproteins 127
7.8.1. Reagents 127
References 129
8. Cellular Energy Allocation 131

8.1. Energy Reserves 132
8.1.1. Total Carbohydrates 132
8.1.2. Lipids 133
8.1.3. Analysis of Lipid Profiles by 1H NMR 135
8.1.4. Protein Content 136
8.1.5. Mitochondria Electron Transport Activity and
Temperature Dependence 141
Contents ix
8.1.6. Cytochrome c Oxidase Activity 142

References 144
9. Neuroendocrine Disruption 145

9.1. Vitellogenin Evaluation as a Marker of Estrogenicity 146
9.1.1. Reagents 147
9.1.4. Precautions 148
9.2. Estradiol-17β or Steroid Binding Sites 150
9.2.1. Reagents 150
9.3. Fluorometric Assay for Catecholamines and Indolamines 152
9.3.1. Reagents 152
9.3.2. Biogenic Amine Extraction 153
9.3.3. Determination of Indolamines (Serotonin) 153
9.3.4. Determination of 5-HIAA 154
9.3.5. Determination of Adrenaline and Dopamine 154
9.4. Competitive Immunoassays for Dopamine and Serotonin 154
9.4.1. Reagents and Solutions 155
9.5. Monoamine Oxidase Activity 158
9.5.1. Reagents 158
9.5.3. Data Analysis 159
9.6. Neurotransmitter Directed Adenylate Cyclase Activity in Synaptosomes 160
9.6.1. Reagents 160
9.7. Acetylcholinesterase 162
9.7.1. Reagents 162
9.8. Steroid Analysis by High-Performance Thin Layer Chromatography 164
9.8.1. Reagents 164
x Contents
9.9. Evaluation of Glutamate and γ-Aminobutyrate 166

9.9.1. Determination of Glutamate Decarboxylase Activity 166
9.9.2. Determination of Endogenous GABA 167
9.9.3. Detection of GABA 168
References 169
10. Genotoxicity 171

Contributed by Émilie Lacaze, Sylvie Bony and Alain Devaux
10.1. Introduction 172

10.2. Alkaline Precipitation Assay 172
10.2.1. Reagents 173
10.2.3. Data Handling and Calculation 174
10.3. Comet Assay 174
10.3.2. Reagents and Materials 176
10.3.4. Positive Controls 182
10.3.5. Data Calculation and Interpretation 182
10.3.6. Advantages and Disadvantages 185
10.4. DNA Content Variation in Cells 186
10.4.3. Procedure and Data Collection 187
10.5. Micronuclei 188
10.6. DNA Synthesis and Catabolism 190
10.6.1. Dihydrofolate Reductase 190
10.6.2. Aspartate Transcarbamoylase Activity 191
10.6.3. Xanthine Oxidoreductase (XOR) 193
References 195
11. Biomarkers of Infection and Diseases 197

11.1. Biomarkers of Exposure 199
11.2. Lysozyme Activity 200
11.2.1. Reagent 200
Contents xi
11.3. NO Synthase Activity 201

11.3.3. Data Calculation and Expression 202
11.4. Cyclooxygenase Activity 202
11.5. DNA Nitrosylation 204
11.5.1. Reagent 204
References 206
12. Descriptive Statistics and Analysis in Biochemical Ecotoxicology 209

12.1. Descriptive Statistics—Back to Basics 210
12.2. What Should be Done with Outliers and Extreme Data? 214
12.3. Univariate Statistics 216
12.3.1. Hypothesis Testing 216
12.3.2. Correlations 218
12.3.3. ANCOVA 221
12.4. Multivariate Statistics 222
12.4.1. Factorial Analysis 222
12.4.2. Discriminant Function Analysis 224
12.5. Artificial Neural Networks 226
References 229
13. Biomarker Expression and Integration 231

13.1. Toxicity Endpoints 233
13.1.1. Threshold Concentrations 233
13.1.2. Effects Concentration by Linear DoseResponse
Relationships 234
13.1.3. Method of Variation 235
13.2. Integrated Biomarker Index 242
13.2.1. Scale of Classification Method 242
13.2.2. Rank-Based Approach 244
13.2.3. Finding the Normal or Natural Range of Biomarkers 246
References 248
Index 249
LIST OF CONTRIBUTORS
Chantale André
Emerging methods, Aquatic Contaminants Research Division, Environment Canada,
Montréal, Québec, Canada
Joëlle Auclair
Sylvie Bony
Ecole Nationale des Travaux Publics de l’Etat France
Alain Devaux
Ecole Nationale des Travaux Publics de l’Etat France
François Gagné
Émilie Lacaze
Institut Armand-Frappier-INRS, Laval, Québec, Canada
Brian Quinn
University of West Scotland, Scotland, UK
xiii
DEDICATION
I dedicate this book to Lucie, my life-long companion, for her unconditional support
in the many projects I undertake. I also dedicate this book to my daughters Geneviève,
Catherine and Valérie for providing me with reasons to fight for the protection of the
environment and to my parents for their support during my studies in biochemistry and
thereafter. I also dedicate this book to my mentors Prof Francine Denizeau (in memoriam)
and Dr Christian Blaise for their guidance, foresights and for opening my eyes to the
science of ecotoxicology.
François Gagné
January 2014
xv
PREFACE
The science of ecotoxicology is the study of contaminant exposure and its effects on
all organisms in the biosphere, including humans. This discipline started in the late
1960s and was defined as the study of the contamination of the biosphere and the
resulting toxic effects to all life forms. The scope of ecotoxicology is quite broad
because it deals with the studies of the fate and effects of contaminants from the
molecular level to the ecosystem. To address this complexity, ecotoxicology was subdi-
vided into three phases or pillars: exposure and bioavailability of contaminants, toxicity
evaluation of substances with their mixtures, and biomarkers to determine the long-
term risk of pollution in organisms and corresponding populations. This relatively new
science was further complicated by the observation that not only chemical agents are
at play but the presence of physical (radiation, temperature variations) and biological
agents (infectious diseases) are also involved. Hence, there is a need to understand the
cumulative contribution of these agents to organism health and the maintenance of
populations. In addition, the advent of new exotic products such as nanotechnology,
climate change, and landscape transformation will bring new types of toxic interactions
that could overwhelm the ability of organisms to cope with existing environmental
stressors such as preypredation changes, eutrophication, food availability, water levels
(and quality), and seasonal temperature changes.
Biomarkers are broadly defined as a measured variable that results in or follows the
interactions of (toxic) agents with a molecular or physiological target. It is generally
recognized that fundamental interactions occur at the biochemical level in cells pro-
vided that this interaction forms the basis of initiating toxic events in organisms. Some
biomarkers are more specific (more closely related) to the toxic agents so they can be
used in testing schemes to identify the contribution or the cumulative effects of vari-
ous toxic agents. These types of biomarkers are particularly valuable since organisms
are seldom exposed to one compound or toxic agent at a time. Indeed, organisms are
usually exposed to low concentrations of hundreds and perhaps thousands of chemicals
in their lifetime. Biochemical markers could also determine some biochemical changes
in organisms that could lead to deleterious effects on organism health in the long
term. To provide a clear representation for the reader, the occurrence of high lipids
(cholesterol) and oxidative stress (inflammation) in the plasma is associated with the
degeneration of blood vessels and related diseases in humans. Hence, the measure of
plasma lipids or cholesterol represents a biomarker of risk in developing a vascular
pathology in time. In other words, a biomarker could represent a biochemical
xvii
xviii Preface
condition that could lead to damage at a higher level of biological organization. In

order to predict effects at the level of organism’s health and maintenance of popula-
tion, we need to understand how a biochemical change resonates at a higher level of
biological organization. Biochemical markers could contribute to this understanding
and find their use and place in ecotoxicology.
When I started as an experimental scientist, many techniques were not always read-
ily available in the laboratory. We (my colleagues and myself) often had to develop and
adapt methods from those reported in the primary literature. We also had to examine
hypotheses on issues related to a particular biochemical effect of given pollutants to a
physiological target in organisms under budget and time constraints. As a preliminary
approach we sometimes first used generic and cost-effective biomarkers to determine
whether a type of effect occurred in exposed organisms before using more sophisti-
cated and expensive techniques or approaches. We quickly came to realize that these
preliminary testing tools were of great interest to laboratories with tight budgets
(which is often the case for most laboratories) or located in remote areas. We discov-
ered that not only highly sophisticated techniques are valuable in ecotoxicology but
generic ones as well. Another advantage of generic assays, which are based on a partic-
ular and fundamental property of the biomarker, is their potential adaptability to be
used in new species. These experiences made me decide to produce a book of meth-
ods in biochemical ecotoxicology that illustrates both generic and highly sophisticated
and species-specific biomarkers that are accessible to many laboratories with budget
and other constraints. We also realized that biochemical markers should stand the test
of time, be cost-effective, simple to use, and accessible by science laboratory students
without major investment. In this respect, this book will assist the experimenter in
ecotoxicological sciences to quickly start biochemical-based assays on various organ-
isms. Our laboratory receives many requests by graduate students, young scientists
beginning in this field, and other researchers getting acquainted with a new area of
research to learn these techniques. Each chapter of this book could be considered
comprehensive on its own and follows a common structure to enable the reader to
quickly learn and try the assay. Each chapter has a brief introduction showing the con-
text and principle of the assays, a section on reagents and solutions required for the
procedure, data handling, and references. In some instances, case studies or examples
are provided to demonstrate how the methods can be used. I am confident that this
book will be well appreciated by both researchers and students who need quick and
direct guidance in performing biochemical assays in ecotoxicology.
François Gagné
Montréal, January 2014
ACKNOWLEDGMENTS
The principal author is very grateful to the contributions of Dr. Brian Quinn for the
preparation and use of cell cultures in toxicity investigations in Chapter 3, Dr. Émilie
Lacaze, Sylvie Bony and Alain Devaux for the contribution on the COMET assay in
Chapter 10, Chantale André for her contribution on gene expression assays using
quantitative reverse-transcriptase polymerase chain reaction in Chapter 4, and to Joëlle
Auclair for her contribution on protein transfer and detection techniques (Western
blots) in Chapter 5 for the detection of protein-based markers.
François Gagné
xix
INTRODUCTION
Biochemical ecotoxicology is the study of the effects of contaminants in ecosystems

using biochemical methods. The scope of this discipline is vast and complex because it
implicates exposure to low concentrations of mixtures in the long term (chronic),
encompassing most of the life stages of organisms. Although the toxicity of chemicals
mostly depends on the physicochemical properties of chemicals, the resulting effects
will depend on the lifestyle of the organisms, such as nutritional status, reproduction
activity, preypredation pressures, and habitat characteristics. The advent of climate
change adds another level of complexity to predicting the toxic outcomes of pollutants,
which is a challenge for the risk evaluation community. The biochemical approaches in
ecotoxicology have the advantage of finding early warning signals at the molecular level,
which can lead to effects at higher levels of biological organization. The interactions of
chemicals with biochemical targets in cells or tissues that could lead to altered function
represent a special type of “biomarker” in toxicology. There are many definitions pro-
posed in the literature for biomarkers, but we think this one best reflects the biochemi-
cal markers: the term biomarker is used in a broad sense to include almost any
measurement reflecting an interaction between a biological system and an environmen-
tal agent, which may be of chemical, physical, or biological origin [1].
This book presents practical methods and approaches used in the field of biochemical
ecotoxicology. It is destined for laboratory investigators involved in environmental toxi-
cology investigations for the protection of wildlife and aquatic ecosystems against pollu-
tion and other stressors such as climate changes. As explained previously, biomarkers are
defined as measures highlighting an interaction between a xenobiotic and other stressors
(e.g., temperature) and a biological target in the physiological sense [2]. Biomarkers are
also determined at different levels of biological organization starting at the molecular,
subcellular (organites), cellular, organ/tissues, organism, and population levels
(Figure 1). Often called the ecotoxicology continuum, biochemical ecotoxicology is
mainly focused at the molecular and subcellular levels and the conditions or criteria by
which a molecular toxic interaction resonates at a higher level of biological organiza-
tion. For example, the expression of genes involved in the biotransformation of xeno-
biotics that leads to increased activity of a given enzyme, which in turn leads to
potentially toxic protein or DNA adducts. Changes at the molecular level are dynamic
and occur rapidly in low timescales (seconds to minutes and up to many days), while
effects at the tissues/individual level are manifested generally at later times (hours and
days to years). In some cases, biochemical alterations take place at the early stages of
xxi
xxii Introduction
Individual/
population
Complexicity
Systemic
effects/health
Cells
(altered cellular
function)
Organites/
Subcellular function
(altered function)
Molecular
(primary interaction)
Minutes Hours Days Weeks Months Year

Time scale
Figure 1 Levels of biological organization in the manifestation of toxicity.
pathogenesis, but the observed effects are not always irreversible, hence their usefulness
in prevention. In other cases, responses observed at the biochemical level (i.e., gene
expression changes) do not imply an impact on the organism’s health status and perfor-
mance, hence their lower “physiological or ecological” relevance. Endless arguments
about which levels are more physiologically or ecologically relevant for ecotoxicology
studies are considered useless and outdated. It depends on the questions being asked in a
given study and the context of pollution studies. The science of ecotoxicology is hierar-
chized, i.e., one compartment is a part of another compartment at a higher level of bio-
logical organization. It is generally accepted that an impact at any level is likely to
produce changes at a higher level provided the intensity of the responses and/or dura-
tion of effects are sufficient. It is a question of knowing when an alteration at one level
(e.g., enzyme inhibition) will translate to an effect at the next level of complexity (e.g.,
altered metabolic pathway from an inhibited enzyme). From the risk assessment per-
spective, biomarkers that provide information on health status toward survival, growth,
and reproduction are particularly relevant for the protection of population toward pollu-
tion and other stressors involved in these times of climate change.
Investigators in ecotoxicology are often confronted to examine or work with all
types of species from which no genetic information or specific antibodies or other
probes are (yet) available. They have to fall back on generic tests, which focus on spe-
cial biochemical properties of the biological target. Indeed, modern and state-of-the-
art techniques are highly dependent on the information of the test species under
Introduction xxiii
investigation (availability of specific gene sequences or antibodies). In the absence of

such information with new species, the investigator has to rely on “generic” biomar-
kers, which take into consideration a particular property of the analyte, e.g., high cal-
cium and phosphate content (vitellogenin), heat-stable metal binding proteins
(metallothioneins), or the capacity to catalyze a given chemical reaction (enzymes) [3].
Although these methods are sometimes less specific and not as sensitive as immu-
noassays or quantitative PCR techniques, generic tests are an ideal choice for screening
and exploration studies because they are more accessible (cheap, quick, and require
only basic equipment) compared to the more sophisticated modern assays. These tests
“stand the test of time” because of their ability to be used on various species, in dif-
ferent laboratories, and under budget constraints. For example, the acetylcholinester-
ase assay based on the dithionitrobenzoate (DTNB) thiol reactive reagent has been
actively used since 1961 [4]. Indeed, not all laboratories have the financial capacity
or infrastructure to purchase highly specialized equipment. Sometimes an investiga-
tor needs to explore whether an ecotoxicological problem occurs at a given site
without making a major investment. For example, the evaluation of the estrogenicity
of a municipal effluent outfall in a river could be easily verified by measuring vitel-
logenin (egg yolk protein, which is regulated by estradiol-17β) in the plasma of fish
or the gonad tissues in mussels using a generic assay based on the alkali-labile
method [2,5]. This assay will require only basic equipment (centrifuge and spectro-
photometer) and is based on a cheap and accessible inorganic phosphate assay.
Hence, the use of cheap and rapid methods to explore a specific topic or examine a
hypothesis could be valuable when human and financial resources are limited. This
is especially useful for remote laboratories or laboratories with limited financial
capacity. That is not to say that if resources are available, high-throughput screening
tools by DNA microarrays or proteomics, for example, could be chosen for bio-
marker mining and discovery.
This book offers a balance between generic biochemical techniques and highly spe-
cific high-throughput techniques such as quantitative polymerase chain reaction
and enzyme-linked immunoassays. We think that this book will introduce novice and
more experienced scientists to techniques that are accessible to all laboratory sizes and
budgets. This work will provide a way for investigators to quickly start biochemical
assays in an ecotoxicology laboratory. Guidelines in the application of biochemical
markers in research and monitoring studies are also provided that will give insights on
quality control and assurance aspects of studies using biomarkers. This book was cre-
ated with these principles in mind to enable investigators to quickly start biochemical
ecotoxicity assessments in their respective laboratories. It is destined to be found
on both the laboratory bench and office desk. Each chapter begins with a brief
xxiv Introduction
introduction to set the context of the proposed analyses with a viewpoint on the rele-
vant biomarkers. The general structure of the chapters will follow a simple, direct, and
comprehensive layout: introduction and principle, reagents and materials, procedure,
and data calculation. In some cases, some chapters will end with a case study to pro-
vide examples. We are confident that this book on principles and methods of bio-
chemical ecotoxicology will help initiate real-life experiments in biochemical
ecotoxicology.
François Gagné, Environment Canada
Chantale André, Environment Canada
Joëlle Auclair, Environment Canada
Émilie Lacaze, INRS-Institut Armand Frappier
Brian Quinn, University of the West Scotland
REFERENCES
[1] Benford D, Hanley AB, Bottrill K, Oehlschager S, Balls M, et al. Biomarkers as predictive tools in
toxicity testing. ATLA 1999;28:11931.
[2] Gagné F, Blaise C. Review of biomarkers and new techniques for in situ aquatic studies with bivalves.
Environmental toxicity testing. New York: Blackwell Publishing; 2005, 205228.
[3] Gagné F, André C, Blaise C. Increased vitellogenin gene expression in the mussel Elliptio complanata
exposed to estradiol. Fresenius Environ Bull 2005;14:8616.
[4] Ellman GL, Courtney KD, Andres Jr V, Featherstone RM. A new and rapid colorimetric determina-
tion of acetylcholinesterase activity. Biochem Pharmacol 1961;961:8895.
[5] Verslycke T, Vanderbergh GF, Versonnen B, Arijs K, Janssen CR. Induction of vitellogenesis in
17alpha-ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison. Comp
Biochem Physiol 2002;132:48392.
CHAPTER 1
Quantitative Assessments of
Biochemical Analyses
François Gagné
Chapter Outline
1.1 General Overview 1
1.2 Replication in Data Analysis 5
1.3 Replication of the Experiment 6
1.3.1 Calibration or Standardization 7
1.3.2 Using Specific and Generic Standards 8
1.4 Reference Substances 10
1.5 Defining Detection Limits in the Absence of Analytical Standards 10
1.6 Reproducibility 12
1.6.1 Reproducibility at the Calibration Level 12
1.6.2 Reproducibility at the Level of the Experiment 13
1.7 Using Reference Material to Standardize Methodologies 13
1.8 Normalization Approaches 15
1.8.1 Residual Extraction Method 16
1.8.2 Stepwise Addition Method 18
References 19
1.1 GENERAL OVERVIEW

In ecotoxicological studies, the early biological (toxic) effects of chemicals are
determined by the use of biochemical markers. Biomarkers rely on chemical
analyses of biological macromolecules such as deoxyribonucleic acids, polypep-
tides/proteins, and membrane assemblages. The ability of proteins to catalyze
biochemical reactions (enzymes) is noteworthy and constitutes an important
part of quantitative biochemistry. The chemical analyses are identical to quanti-
tative analysis of chemicals in the classic sense (i.e., evaluation of mercury by
cold vapor atomic absorption spectrometry) but differ because the analysis is
done in relation to a given biological function that often involves highly com-
plex molecules (i.e., mercury binding thiol-rich proteins such as metallothio-
neins, MTs). As another example, the quantitative evaluation of serotonin, a
Biochemical Ecotoxicology r 2014 Elsevier Inc.

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2 Biochemical Ecotoxicology
derivative of the amino acid tryptophan, by liquid chromatography with elec-

trochemical detection is an example of chemical analysis, but the interaction of
serotonin on membrane receptors in nerve cell membrane is biochemical
because it intervenes in complex biochemical interactions with macromole-
cules. These assays are practiced in the context of hypothesis testing, i.e., in
studies to determine negative interactions, if any, of exposure to a given xeno-
biotic to a physiological function in the exposed organisms or cells. Moreover,
test organisms in the real world are genetically heterogeneous and under
various habitat pressures such as nutritional stress, temperature, climate varia-
tions, and reproductive cycle. This brings more interindividual variability in
the measures [1]. This variation is added to the variation of the method of
biochemical analysis. It is therefore essential to determine both the variation
induced by the method of analysis and interindividual variation if one wants to
highlight toxicological effects. Hence, in the effort to better understand the
variability of biological responses, we must acquire a good understanding on
the life cycle of the organism and seek the normal range of responses of the
biomarker over time (seasonal variability) in a given age range [2]. However,
the knowledge of all these aspects might be difficult when dealing with a new
species not before examined. The understanding of the “normal” range of
responses of biomarkers actually represents an important aspect of ecotoxicol-
ogy research and monitoring.
Studies in biochemical ecotoxicology bring about new methodologies, new
versions, or adaptations of existing methods. In most cases, the methods were
developed for mammals where modifications and adaptations are made when
applied to a new species. Moreover, there is a big step between a newly devel-
oped methodology for a given research study and for a method for monitoring
purposes. Many biomarkers are often measured by many means (methods),
each having their advantages and caveats. For example, methods developed for
a given research project require different types of validation than monitoring
types of studies. Some of these aspects will be discussed in this chapter. For
example, the family of heavy metal binding proteins, including MTs, is
involved in the sequestration and protection of toxic metals and they are deter-
mined by many methodologies such as the silver or cadmium or mercury satu-
ration assays, pulse polarography, enzyme-linked immunoassays, by mRNA
determination using quantitative polymerase chain reaction (qPCR), high-
pressure liquid chromatography (HPLC), and gel electrophoresis. Each of these
Quantitative Assessments of Biochemical Analyses 3
methodologies has advantages and caveats in respect to rapidity, cost-effectiveness,

sensitivity, specificity, and reproducibility. It is needless to argue that only one
methodology should be used for a given project. Many of these assays are
partially quantitative where the relative levels of the biomarker are determined
in contrast to the absolute amount. In addition, most biomarkers require nor-
malization against biomass, which is also determined by a variety of approaches.
For example, the biomarker could be normalized against wet or dry tissue
weights or total protein or DNA contents. For mRNA determinations by
qPCR, the levels of mRNA are usually normalized against total mRNA levels
or with the levels of housekeeping genes provided they are stable in the condi-
tions of the experiment (which is often not the case). These aspects will be dis-
cussed in detail in Chapter 4. Hence, biomarkers are not only determined by
numerous methodologies, but their normalization could be determined by a
variety of methods as well. This introduces another level of complexity for
quantitative assessment of biomarkers between laboratories and projects. From
a monitoring perspective, the use of certified reference material for biomarkers
to determine the precision and trueness of the method is lacking in most cases.
These materials are used to validate the various laboratories to produce similar
results, especially in the context of long-term temporal and spatial surveys.
Nevertheless, some precautions or steps could be undertaken to improve the
reproducibility and reliability of quantitative biomarker assessments in a research
setting [3]. This chapter consists of guidelines to assist the laboratory investiga-
tor (or research assistants, students, etc.) in generating sound bioanalytical data
and in establishing the context and limits of the analyses. As discussed earlier,
the MT biomarker will be used as an example model for clarity purposes for
the reader. The scope of this chapter is not only to provide quality control and
assurance notions in a strict sense but to adapt them in a research context in
biochemical ecotoxicology studies. The principle of quality control and assur-
ance for standardization are similar but not identical for standardization meth-
ods in monitoring, which makes use of certified reference material [4].
Notwithstanding, good laboratory practices should always be practiced in the
most rigorous fashion possible.
In a research laboratory, various steps are taken to secure and validate the data
when developing a methodology and generating data (Table 1.1). These criteria
will be explained in this chapter. First, a laboratory notebook should accompany
the experimenter at all times where all relevant information is duly noted on a
Table 1.1 Levels of Control for the Generation of Biomarker Data

Control Level Parameters Comments
1. Type of project Objectives Replication To be included in the

Hypothesis Standard solutions laboratory notebook
Protocols Instrument readings
Secure rough data
2. Replication Analysis Replication used
Experiment Determine the
repeatability of the
experimental design
3. Variability Method of analysis Determine the
Biological variability variation of the method
and the biological
variation
(interindividual)
4. Validation Correspondence Determine the constant Find the linear range of
between the biomarker slope between the the rate of change of
and means of biomarker and biomass the biomarker with the
normalization evaluation rate of change in the
biomass
5. Reproducibility Calibration External standard Induction of the
(standardization) Internal standard biomarker with a
Determine the stability (matrix effects) known and previously
of the signal of a Positive control (with a reported inducer of the
standard corrected known inducer) biomarker
against a blank Quality control chart of Determine the
Stability of a reference the standard signal in method’s trueness
material at each time of time in time and with
analysis of the Confirm the stability of different analyst,
biological samples the biomarker signal instruments, and
(composite sample) over time using reagents
different reagents/ A mean to validate that
standards preparations the method used was
and even with different always correct in
analyst by including the quantifying the
analysis of the same biomarker in time
sample at each sample
batch
6. Data archives Store all data in a safe Hard drives Secure data for
place: rough and Networks long-term use
compiled data should
be included
day by day basis. Moreover, bioanalytical data should be protected and well iden-
tified in the storage media. It is important to safely store the data and identify the
quality control measures from which the data were generated: the protocol, the
composition, and date of preparation of blanks, standards, type of samples, and
replication of the analysis. First, archives call for laboratory notebooks where all
the methodological and procedural details including any small modifications, if
present, are found. Replication and the method used for calibration/standardiza-
tion should always be noted in the log book for the production of any (semi-)
quantitative bioanalytical data. Details on the experimental design used and the
variation of the methodology with its limit of detection should be included as
well. In a research laboratory, it is possible that specific standards are lacking. In
this situation, recommendations are proposed to provide some extent of valida-
tion for the method of analysis. Finally, elements of quality control proposed in
situations were certified reference material for biomarkers in a given test species.
These elements will permit the undertaking of spatial or temporal surveys in
ecotoxicological investigations.
1.2 REPLICATION IN DATA ANALYSIS

In all research activities, replication is required at both the analysis and experi-
mental steps. Each blank, standard (if available), and sample should be analyzed
using three replicates with the instrument. In some instances, when resources are
limited or the method was proved to be very reliable (like a pH or weight mea-
surements), the samples could be analyzed once, but one test sample in the lot is
analyzed four to eight times to have at least one good estimate of the method’s
variability. The number of replicates depends on the intrinsic variation of the
methodology, type of instrument, and the biological variability. Replication at
the analysis step permits the definition of the variation of the analytical method
or the precision. The experimental precision consists of the dispersion of the data
around a central attractor such as the mean or median. Precision is usually
expressed as the coefficient of variation of the mean ((standard deviation/
mean) 3100) for normally distributed data. To obtain good estimates of preci-
sion, the sample must be analyzed several times (ideally four to eight times,
depending on the intrinsic variation of the assay) at least once during the analysis
batch. The number of samples should take into account the number of indivi-
duals involved in a given study in an attempt to determine the sensitivity of the
assay since the biological responses should produce changes that are above the
variation of the method. In other words, the intraindividual variation (variation of
the biomarker during analysis) should be lower than the interindividual variation
to obtain a significant effect between groups of individuals.
At the level of biomarker data, care should be taken during the sequence of
analysis of the blank, standards, and samples by the instrument to control for
any drift in the performance of the instrument. With microplate readers, read-
ings are taken within seconds depending on the technology, thus the variation
of measurement within the microplate is expected to be negligible. This is not
necessary for absorbance readings given the changes in absorbance are obtained
by the ratio in the light intensity of the instrument in the presence (Is) and
absence (Io) of the sample (log Is/Io as defined by BeerLambert’s law). If the
readings are repeated after 30 or 60 min, the rough data could differ between
reading times because the performance of the instrument could vary in time
(e.g., the lamp energy could vary in times and have major effects on fluores-
cence readings or change in a photomultiplier tube efficiency for luminescence
readings). Hence, there is a need to include blanks and standards for each
sequence analysis. Some instruments are equipped with internal reference,
which directs correction of the data after each reading by the software.
Variations in the efficiency of an instrument are more important with
instruments that require samples to be determined over minutes to hours of
analysis time. For example, if the performance of the instrument changes by
25%, then this could introduce important variability in the data. In these cases,
it is recommended to measure the blank and standards at the beginning of the
analysis, measure 510 samples, and measure again the blank and one standard
and so forth. With this approach, one could control for any drifts of the instru-
ment performances. The instrument’s reading efficacy depends on the type of
instrument (HPLC columns and detectors, fluorescence or luminescence read-
ers) and is characterized as change in (sample signalblank signal)/concentra-
tion of the standard sample. This metric is important if measures are made on
different instruments across laboratories. Indeed, the efficacy of the instrument
changes in time and with different instruments.
1.3 REPLICATION OF THE EXPERIMENT

The notion of replication should be extended at the level of the experiment
itself, especially in a research context. This approach differs from the context of
routine analysis where a sample is analyzed and compared with a standard and
certified reference material in a similar matrix. Take a study consisting of the
evaluation of MT in mussel populations at two sites (one contaminated by
metals and one pristine). To start, eight individuals were collected for the
determination of MT in the digestive gland (equivalent to the liver) at each
site. The MT assay was practiced in duplicate for each individual. In theory, if
the method is reproducible and precise, on the order of 5% or less, then the
required number of individuals would depend on the interindividual variability
(biological variation). If the interindividual variability is at 30% (coefficient of var-
iation of 30%), then the mean level of MT should be higher than the mean 1 30%
value to detect a difference. If the sample size is sufficient to appraise the state of
the population (we assume for simplicity that the gender, size, and age of sampled
mussels are constant) then the repetition of this experiment should provide similar
results. If not then the N 5 8 individuals was not representative of the local popula-
tion and more individuals should be determined. Usually the experiment is
repeated three times to definitely ensure the replication number was enough. In
other words, we have to make sure the differences in MT levels between the two
sites are not the result of method variation or insufficient sample size. We must
determine the number of replicates for analysis (which depends on the methodol-
ogy used) and the number of individuals (sample size), which depends on the bio-
logical variation. The latter case is a question of power analysis, that is, the number
of replicates required for a significant effect above a threshold, usually . 30% of
the mean, since the normal biological variation is usually in this order. When the
sample size is not sufficient, repeating the experiment will provide confirmation if
the observed effects are real or not.
1.3.1 Calibration or Standardization

The methodology used should include analytical blank and standard samples
for calibration. In addition, the reading of the test sample (unknown sample)
should be within the readings of the blank and standards where a linear rela-
tionship exists. For example, if the absorbances of the blank and highest stan-
dard are 0.01 and 0.22, respectively, then the sample absorbance should be
included within this interval. The blank is defined as the matrix in which the
analysis is performed without the biomarker (or very low levels depending on
the type of the assay) of interest. Standardization of the method supports two
roles: (1) quantitative determinations, and (2) the establishment of the limit of
detection of the methodology. The limit of detection is defined two ways:
theoretical and operational. The theoretical limit of detection of the biomarker

is the value corresponding on the mean signal 12 3 SD of the blank. For
example, if the blank samples give a mean of 0.1 6 0.02 then the theoretical
detection limit is 0.1 1 (2 3 0.02) 5 0.14. Hence, there is a need to have repli-
cation during analysis. The operational limit of detection corresponds to the
lowest measured standard value. This definition is more conservative and
depends on the linear relationship between the analytical signal and the con-
centration of standard used. Since these definitions are different, it is important
to identify which one is used.
1.3.2 Using Specific and Generic Standards

When a specific standard for a given species is lacking, it is possible to use a
standard from another species provided the biomarker shares similar properties
in each species. For example, rabbit MT standards could be used to standardize
the assay dealing with other species such as fish, mussels, or rats. The standard
addition approach, where the standard is directly added to the unknown sam-
ples, could be used to account for unsuspected matrix effects that could arise in
samples from other species. In this case, these data could be expressed in an
equivalent amount of added standard. For the MT example, MT levels in the
digestive gland of mussels using rabbit MT would be expressed as nanomoles of
rabbit MT equivalent/biomass of the digestive gland.
Standards can be added externally in a separate tube without the test sample
or directly in the test sample. When the method is impervious to interferences
from the complex biological matrix, an external calibration curve could be
produced where pure standards are serially diluted in the assay buffer. In this
situation the data point starts at the origin (Figure 1.1A) and the presence of
sample does not contribute to the signal or is effectively removed by an analyti-
cal blank. The concentration of the test sample is then calculated by the linear
regression line: Analytical Signal (y) 5 value (b) 1 Slope (a) 3 Standard concen-
tration (x)-x 5 (y 2 b)/a. This procedure holds if the presence of the biologi-
cal matrix does not interfere in any way with the analytical signal y.
When the biological matrix could produce interfering effects (i.e., decreasing
or increasing the analytical signal), the standard curve is constructed in the presence
of the test sample (Figure 1.1B). In this situation the absence of the added standard
yields a signal (interference) and changes in the analytical signal output (the slope is
either lower or higher). In this situation, the complexity of the matrix introduces a
(A) 12
10
Analytical signal
8
6
y = –0.19 + 2.14X
4
0
0 1 2 3 4 5 6
Standard
(ng/mL)
(B) 12
y = 1.04 + 1.9x
10
Analytical signal
0
0 1 2 3 4 5 6
Standard added
(ng/mL)
Figure 1.1 Typical calibration curve between the analytical signal and the concentration of the standard.
An external calibration curve is shown in A and an internal calibration curve using the standard addition
method in B.
bias toward the analytical signals. In this case the standards are added directly in a
constant volume of the test sample. The concentration of the analyte in the test
sample is then extrapolated by giving a value of 0 to the analytical signal (y), for
example: 0 5 1.04 1 1.9 3 (standard concentration) - standard concentration
(x) 5 j(21.04/1.9)j. This procedure, albeit more robust, requires the construction
of a standard addition in each test sample, which can become time-consuming.
When the volume of the test sample is limited, one standard addition could be
added to one of the test samples. The sample concentration is then calculated as
follows: concentration of sample 5 [Analytical Signal (sample 2 blank)/Analytical

Signal (spiked sample 2 blank)] 3 added standard concentration. The standard
addition method represents a convenient means to control for matrix interference
or when this effect in uncertain (e.g., when working with novel organisms). If no
interference exists then the standard addition method will produce results similar
to the external standard curve; in other words, if the slopes are equal then there is
no need to proceed with the standard addition method. This verification could be
done at the start of the project when dealing with a new biological matrix such as
different organs/tissues or new species.
1.4 REFERENCE SUBSTANCES

When no standard exists for a given species and when no other standard exists
from other species, the methodology could be validated by using a reference
toxic substance known to induce the biomarker of interest. The ratio between
the biomarker level of the control organism and biomarker level of the induced
organism should be the same between batches of analysis. This is particularly
important when the analyses are performed at different times and in separate
batches. For example, MT could be induced in the liver of fish exposed to cad-
mium or zinc (0.5 mg/L in water) for 96 h at 15 C. The ratio of MT levels
between the control and the exposed fish could be used as a reference point.
The liver samples could be stored at 2 85 C as reference samples and they
could be tested at each batch of analysis in time.
1.5 DEFINING DETECTION LIMITS IN THE ABSENCE

OF ANALYTICAL STANDARDS
As described previously, the operational detection limit is defined as the
smallest standard used during calibration or the concentration producing a
signal $ 2 3 the SD of the analytical blank for the theoretical limit of detec-
tion. For the latter, a signal above this threshold usually is statistically significant
at the p , 0.05 level. When analytical standards are available the former
method is the preferred procedure. In a research context, standards are some-
times not commercially available and the production of standards is outside the
reach of the laboratory. In this situation, we can establish another operational
detection limit of the methodology. Let’s come back to the MT in the digestive
gland example (eight mussels from a reference and metal-contaminated site).
(A) 9
Detection limit of y
8
Detection limit of y and x
7 Detection limit of x
(arbitrary units/mL)
Biomarker signal
6
5
4
3
2
1
0
0 1 2 3 4 5 6 7
T otal proteins
(mg/mL)
(B) 10
8
(arbitrary units/mL)
Biomarker signal
6
Optimal zone
of quantification
4
0
0 2 4 6 8 10
Total proteins added
(mg/mL)
Figure 1.2 Defining an operational limit of detection without standards. An operational (lower) limit
of detection could be determined between the biomarker signal and the normalization endpoint,
here as total proteins (A). The upper limit of quantification of the biomarker and the normalization
method should also be known (B).
We could prepare a representative homogenate extract of the digestive gland and

run the assay on serial dilutions of the homogenate. The ratio between MT levels
normalized against total proteins should remain constant throughout dilutions and
the sample could be used within this boundary. When approaching the limit of
detection of either MT or protein assessment methods, the slope between total
MT or proteins will change (Figure 1.2A). This imposes a limit of detection of the
method that is based on the slope between the biomarker and biomass endpoint.
When the limit of detection is reached for either the biomarker or the normalizing
assay, the slope between the biomarker and its normalization unit will change, as
shown in Figure 1.2A. In this example, the operational detection limit of the
methodology will depend on which method the limit of detection reached; for
limit of detection x, it is reached first then the detection limit is 3 (total pro-
teins 3 axis), which corresponds to the biomarker signal of 1.5 on the y-axis. For
the limit of detection y, it is reached when the detection limit is 2 for total proteins
(x-axis) corresponding to a biomarker signal of 1 on the y-axis. This approach gives
the boundary of the minimal amount of biomass (in protein units in the example)
required to measure the biomarker.
At the other side of the limit of detection, the upper limit of quantification
should also be determined to discover the maximal amount of biomass that can
be tested for the biomarker (Figure 1.2B). For example, the upper boundary
would correspond to 6 mg/mL total proteins with a biomarker of 8 arbitrary
units. Indeed, using a highly concentrated homogenate extract could over-
whelm the reagent capacity of the methodology or introduce new interferences
or matrix effects leading to altered changes in the slope of the biomarker with
its biomass. Hence, the experimenter should define the upper and lower limits
of the assay when standards are absent to determine the biomarker in the
region where the slope between the biomarker and biomass unit is invariant, as
depicted in Figure 1.2B.
1.6 REPRODUCIBILITY
There is a distinction between the methodological and the experimental or
biological reproducibility. The methodological reproducibility is characterized
by the capacity of producing constant data regardless of “when and where” the
assay is practiced. Indeed, a reproducible method will produce similar data
independent of the analyst or technician performing the data, the time and
period of the analysis, and the instrument in a given laboratory.
1.6.1 Reproducibility at the Calibration Level

The slope between the analytical signal and the concentration of a standard should
be constant regardless of the time and where the analysis was produced. Moreover,
the signal difference between the blank and the standard should be relatively
constant for a given instrument. These criteria are closely related to the stability of
the biomarker and the reagents used to quantify it at the day of analysis. It is
important to mark in the laboratory notebook the date of preparation of the
reagents and standards, commercial origin, and the lot number when possible.
1.6.2 Reproducibility at the Level of the Experiment

As already mentioned in this chapter, the experiment should be repeated a
number of times to confirm whether the produced data lead to similar results
when possible, for example, if the ratio of the mean value for MT from the
metal-contaminated site/mean value from the reference site is 4 for a given
experiment. Repeating this experiment should give a similar ratio, i.e., within
the normal variation of the methodology. If the ratio differs markedly (more
than the interindividual variability) then this could indicate a problem with the
biomarker analysis or the execution of the exposure experiment.
1.7 USING REFERENCE MATERIAL TO STANDARDIZE METHODOLOGIES

It is recommended that all the analyses are made during the same day or analy-
sis batch in order to eliminate temporal drift of the assay methodology.
To control for these methodological drifts (related to the method’s trueness), a
reference material could be produced in-house, which consists of preparing a
similar sample and storing separate aliquots at 285 C. At the given day of
analysis, an aliquot of the reference material is taken for biomarker determina-
tions. At each time of analysis, the reference material should always give the
same biomarker value, i.e., within the confidence interval or normal variation
of the methodology. The reference material approach consists of producing our
own in-house material. In some cases a certified reference material could be
produced by a national laboratory using strict procedures, but these have yet
to be available for biomarkers in ecotoxicology. For example, if the MT bio-
marker in the digestive gland is to be examined at 10 sites (8 mussels per site),
then a pool of digestive glands (1050 mussels) could be prepared, stored in
separate aliquots, and used as the reference material. Of course, the digestive
gland extracts would be prepared using the same conditions such as tissue
grinding apparatus, homogenizing steps, temperature, and buffer. The reference
material would be used at each day or time of analysis. If it takes one day of
analysis time per site, then the project will require 10 separate periods of
6,0
Mean
5,5 Standard deviation
Outlier
Standard or reference material

(Signal sample - signal blank
5,0
or Biomarker unit/biomass)
data (suspect)
4,5
4,0
+1 x standard deviation
3,5
3,0 Mean (2.6)
2,5
2,0
1,5 –1 x standard deviation
1,0
1 2 3 4 5 6 7 8 9 10
Time of analysis
Figure 1.3 Quality control chart illustrating a biomarker from a prepared reference material or a
fixed concentration standard determined through 10 days of analysis. These data should be distrib-
uted around the mean value (filled line) with SD (dotted lines). Data value outside the SD is consid-
ered a suspect outlier and should be either removed from the data set or reanalyzed after making
the appropriate revisions of the method.
analysis. For each period of analysis, the reference material is measured (in qua-
druplicate) where it should give a value within the normal variation of the
methodology. The reference material approach is used to track the time at
which the measurement was done and determine the trueness of the method
in time [5]. This approach enables one to track the performance of the method
of analysis in time and in different laboratories. The second option consists of
using a standard from another species that is commercially available to ensure
the difference between the standard and blank is constant in time. A quality
control chart could be produced to identify which samples or day of analysis
yield data outside the normal variation of the method. In Figure 1.3, each data
point is either a fixed standard corrected against a blank or a normalized bio-
marker value (biomarker unit/biomass unit) that was analyzed at each period.
The SD of the data points corresponds to the methodological variation.
The method should always give, in principle, the same value, i.e., a value
within the normal variation of the method (within the SD or the 95% confi-
dence interval). The true “normal” variation of the method in Figure 1.3 is
depicted as the solid line with the corresponding SD (dotted lines) for all analy-
sis time. At time 6, the obtained data are outside the upper SD of the method
and considered an outlier or suspect. Ideally all the samples and standards
(blanks) should be reanalyzed after rechecking/re-preparing the reagents or
bringing the proper corrections of the methodology. If this is not possible, then
the data from all samples analyzed at that time should be removed from the
dataset. In some instances (for exploratory purposes), a correction factor could
be used to bring down the mean data value at time 6 to the mean value of
the method (solid line) for all the samples analyzed at time 6. The correction
factor is derived as follows: mean value at suspect time/mean value of the end-
point: 4.9/2.6 5 1.88. Hence, all the biomarker values obtained at day 6 are
divided by 1.88. However, this approach should not be applied as a final
measure of the biomarker at this time but only as a preliminary view of the
biomarker response after correcting for methodological drift. This is especially
important when each time period corresponds to a different treatment group
or study site or time survey. Some laboratories take the samples randomly and
sometimes blindly (the sample identity is hidden and replaced randomly by
numbers) to alleviate potential artifacts and subjectivity.
The use of quality control charts determines the performance of the method-
ology over different times (and different laboratories). When the production of
an in-house reference material is not possible (low availability of biological mate-
rial or test species), the mid standard value corrected against the blank could be
used. This approach is related to the precision of the method at each time of
analysis. In most cases, values outside 23 the SD are considered an outlier or
aberrant value. In conclusion, when the assays are performed at different times,
which involve different preparations of reagents and standards for calibration, the
use of a reference material is recommended. This procedure establishes the true-
ness of the method, which is the normal variation of the methodology applied at
different times regardless of the analyst, reagent, and calibration used.
1.8 NORMALIZATION APPROACHES

Most biochemical endpoints (enzyme activity, particular protein, metabolite,
etc.) are normalized against a measure of biomass such as dry or wet weight
of the biological samples, total proteins, or DNA content. With plasma or sera,
the biomarker values are also expressed in concentration (e.g., μg/L or nmol/
mL). For tissues, normalization is achieved by simply dividing the endpoint
value concentration by the biomass value. When the analyte value is small
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*** START OF THE PROJECT GUTENBERG EBOOK THE MISSING

CHUMS ***
THE HARDY BOYS
THE MISSING CHUMS
By FRANKLIN W. DIXON
Author of
The Hardy Boys: The Tower Treasure
The Hardy Boys: The Secret of the Old Mill
ILLUSTRATED BY
Walter S. Rogers
NEW YORK
GROSSET & DUNLAP
PUBLISHERS
Made in the United States of America

Copyright, 1928, by
GROSSET & DUNLAP, Inc.
All Rights Reserved
The Hardy Boys: The Missing Chums

HE CRAWLED ON HANDS AND KNEES.
CONTENTS
I. The Three Strangers
II. Quick Thinking
III. A Shady Trio
IV. The Send-Off
V. No Word from the Chums
VI. Missing
VII. Wreckage
VIII. The Strange Letter
IX. Blacksnake Island
X. The Boy on the Deck
XI. The Island
XII. Into the Cave
XIII. The Four Men
XIV. The Storm
XV. A Startling Announcement
XVI. The Alarm
XVII. Capture
XVIII. Back to the Cave
XIX. Separated
XX. Seizing the Boats
XXI. At the Island
XXII. The Chase
XXIII. Home Again
THE HARDY BOYS:
THE MISSING CHUMS
CHAPTER I
The Three Strangers
"You certainly ought to have a dandy trip."
"I'll say we will, Frank! We sure wish you could come along."
Frank Hardy grinned ruefully and shook his head.
"I'm afraid we're out of luck. Joe and I may take a little trip later on,
but we can't make it this time."
"Just think of it!" said Chet Morton, the other speaker. "A whole
week motorboating along the coast! We're the lucky boys, eh, Biff?"
Biff Hooper, at the wheel of his father's new motorboat, nodded
emphatically.
"You bet we're lucky. I'm glad dad got this boat in time for the
summer holidays. I've been dreaming of a trip like that for years."
"It won't be the same without the Hardy Boys," returned Chet. "I
had it all planned out that Frank and Joe would be coming along
with us in their own boat and we'd make a real party of it."
"Can't be done," observed Joe Hardy, settling himself more
comfortably in the back of the boat. "There's nothing Frank and I
would like better—but duty calls!" he exclaimed dramatically,
slapping himself on the chest.
"Duty, my neck!" grunted Frank. "We just have to stay at home while
dad is in Chicago, that's all. It'll be pretty dull without Chet and Biff
around to help us kill time."
"You can put in the hours thinking of Biff and me," consoled Chet.
"At night you can just picture us sitting around our campfire away up
the coast, and in the daytime you can imagine us speeding away out
over the bounding main." He postured with one foot on the gunwale.
"A sailor's life for me, my hearties! Yo, ho, and a bottle of ink!"
The boat gave a sudden lurch at that moment, for Biff Hooper had
not yet mastered the art of navigation and Chet wavered
precariously for a few seconds, finally losing his balance and sitting
down heavily in a smear of grease at the bottom of the craft.
"Yo ho, and a bottle of ink

And he nearly fell into the drink,"
chanted Frank Hardy, as the boys roared with laughter at their

chum's discomfiture.
"Poet!" sniffed Chet, as he got up. Then, as he gingerly felt the seat
of his trousers: "Another pair of pants ready for the cleaners. I ought
to wear overalls when I go boating." He grinned as he said it, for
Chet Morton was the soul of good nature and it took a great deal
more than a smear of grease to erase his ready smile.
The four boys, Frank and Joe Hardy, Chet Morton and Biff Hooper, all
chums in the same set at the Bayport high school, were out on
Barmet Bay in the Envoy, the Hooper motorboat, helping Biff learn
to run the craft. Their assistance consisted chiefly of mocking
criticisms of the luckless Biff's posture at the helm and sundry false
alarms to the effect that the boat was springing a leak or that the
engine was about to blow up. Each announcement had the effect of
precipitating the steersman into a panic of apprehension and
sending his tormentors into convulsions of laughter.
Biff had made good progress, however, as he had been with the
Hardy boys on previous occasions in their own motorboat, the
Sleuth, and he had picked up the rudiments of handling the craft. He
was anxious to be a first-rate pilot before starting up the coast on
his projected trip with Chet Morton the following week. He had an
aptitude for mechanics and he was satisfied that he would have a
thorough understanding of his boat by the time they were ready to
start.
"If the coast guards find two little boys like you roaming around in a
great big motorboat they're likely to give you a spanking and send
you back home," laughed Frank. "I'll bet you'll be back in Bayport
inside of two days."
"Rats!" replied Chet, inelegantly, if forcefully. "If our grub holds out
we'll be away more than the week."
"There's no danger of that. Not with you along," Joe remarked, and
deftly dodged a wad of waste that Chet flung at him. Chet Morton's
enormous appetite was proverbial among the chums.
"Just sore because you can't come along with us," Chet scoffed.
"You know mighty well that the two of you would give your eye-
teeth to be on this trip. Oh, well, we'll tell you all about it when we
get back."
"A lot of comfort that will be!"
"A leak!" roared Chet suddenly, pounding Biff on the back. "The boat
has sprung a leak. Get a pail!"
"What!" shouted Biff, in alarm, starting up from the wheel. Then, for
the fifth time that afternoon, he realized that he had been fooled
and he sank back with a look of disgust on his face.
"Some time that boat will spring a leak and I won't believe you," he
warned, settling down to his steering again.
"I'll be good," promised Chet, sitting down and looking out over the
bay. "Say, there's a big brute of a motorboat coming along behind
us, isn't it?"
"I'll say she's big," Frank agreed, looking back. "I don't remember
ever having seen that boat around here before."
"Me neither," declared Joe. "I wonder where it came from."
The strange craft was painted a dingy gray. It was large and
unwieldy and did not ride easily in the water. Although that boat was
some distance in the wake of their own craft the boys could
distinguish the figures of three men, all seated well up toward the
front. Biff glanced back.
"It's a new one on me," he said. "I've never seen it before."
"Sure has lots of power, anyway," Chet commented. The roar of the
engine could be plainly heard across the water. In spite of its clumsy
appearance, the big boat ploughed ahead at good speed, and, as Bill
had the Envoy, his craft, throttled down, the second boat was slowly
overtaking them.
"Let's wait till they get abreast of us and give them a race," Chet
suggested.
"Not on your life," objected Biff. "I'm only learning to run this tub
and I'm not in the racing class yet. Besides, there are too many
other boats out in the bay this afternoon. I'd be sure to run into one
of them."
The boys watched as the other craft overtook them. The big
motorboat ploughed noisily ahead, keeping directly in their wake.
"I wonder if the man at the helm is asleep," said Frank. "He doesn't
seem to be making any attempt to pull over."
"He's awake, all right," declared Chet. "I can see him talking to the
man beside him. He won't run us down. Don't worry—not with
Captain Hooper at the helm, my hearties!"
The roaring of the pursuing craft suddenly took on a new note and
the big boat seemed to leap out of the water as it increased its
speed and bore rapidly down on the Envoy. Spray flew about the
heads of the helmsman and his two passengers and a high crest of
foam rose from either side of the bow. Biff Hooper shifted the wheel
slightly and the Envoy veered in toward the shore. To the surprise of
the boys, the other boat also changed its course and continued
directly in their wake.
"The idiots!" exclaimed Biff.
"I don't get the idea of this at all," muttered Frank Hardy to his
brother. "What are they following us so closely for?"
Joe shrugged. "Probably just trying to give us a scare."
The other boat was now almost upon their craft. It nosed out to the
right and drew alongside, coming dangerously close. The boys could
see the three men clearly and they noticed that all three scrutinized
them, seeming to pay particular attention to Chet and Biff.
The men were unsavory looking fellows, unshaven, surly of
expression. The man at the helm was sharp-featured and keen-eyed,
while the other two were of heavier build. One of the pair wore a
cap, while the other man was bare-headed, revealing a scant thatch
of carroty hair so close-cropped that it seemed to stick out at all
angles to his cranium. This man, the boys saw, nudged his
companion and pointed to Biff, who was too busy at the helm of his
own craft to notice.
"Not so close!" yelled Chet, seeing that the other boat was running
broadside in dangerous proximity to the Envoy.
In reply, the man at the helm of the other craft merely sneered and
brought his boat in until the two speeding launches were almost
touching sides.
"What's the idea?" Joe shouted. "Trying to run us down?"
Biff Hooper shifted the wheel so that the Envoy would edge away
from the other boat, and in this effort he was successful, for a gap
of water was soon apparent between the speeding craft. But in
escaping one danger he had risked another.
Two sailboats that had been flitting about Barmet Bay that afternoon
were racing with the wind, and they now came threshing along with
billowing canvas, immediately into the course of the motorboat. Biff
had seen the sailboats previously and had judged his own course
accordingly, but in his efforts to get away from the mysterious
launch he had unwittingly maneuvered the Envoy into such a
position that a collision now seemed inevitable.
The sailboats seemed to loom right up before him, not more than a
hundred yards away. They were racing close together, one boat but
a nose in the lead. They were scudding along with the wind at high
speed and the motorboat roared down upon them.
Biff Hooper bent desperately over the helm. He was so close that no
matter which way he turned it seemed impossible that he could miss
one or the other of the sailboats. If he turned to the right he would
crash into them head-on; if he turned to the left he would run before
them and a general smash-up might be the result.
The men in the sailboats were also aware of their danger.
The boys had a glimpse of one man waving his arms. One of the
boats veered out abruptly and the yardarm swung around. The
sailboat was lying directly in the path of the Envoy.
The roaring of the engine, the threshing of the sails, the warning
shouts of the boys, all created a confusion of sound. The white sails
seemed to loom high above the speeding boat. A hideous collision
appeared to be inevitable.
CHAPTER II
Quick Thinking
Every second was precious.

Frank Hardy realized the full extent of their peril and in the same
moment he realized the only way of averting it.
Without a word he sprang toward the helm, brushing Biff Hooper
aside. In this emergency, Biff was helpless. Swiftly, Frank bore down
on the wheel, bringing the boat around into the wind. At the same
time, he opened up the throttle so that the Envoy leaped forward at
her highest speed.
The motorboat passed just a few inches in front of the bow of the
first sailboat; so close, Chet Morton said afterward, that he "could
count every stitch on the patch in the sailcloth." But the danger was
not yet over. There was still the other sailboat to be considered. It
was pounding along immediately ahead of them; the man at the
tiller was making frantic efforts to get out of the way, but the danger
lay in the fact that in trying to guess the possible course of the
Envoy he might make a false move that would have him shoot
directly across its path.
Frank swung the helm around again. Once more, the Envoy veered
to the left so sharply that a cloud of spray drenched the boys.
Another shift of the wheel and the motorboat zig-zagged safely past
the sailboat and on out into open water.
Not one of the boys had uttered a word during this. They had been
tense and anxious, but now that the peril of a smash-up had been
averted, they sank back with sighs of relief.
"I sure thought we were headed for Davy Jones' locker that time!"
breathed Chet.
Biff Hooper looked up at Frank.
"Thanks," he said. "I'd have never got out of that mess if you hadn't
taken the wheel. I was so rattled that I didn't know what to do."
"After you've run the boat a few more weeks you'll get so used to it
that it'll be second nature to you. But that sure was a tight squeeze,"
Frank admitted. "It mighty near meant that you wouldn't have had
any motorboat left to go on that trip with."
"It mighty near meant that we wouldn't have been left to make the
trip at all," Chet declared solemnly. "What say we go home? I've had
enough excitement for one day."
"It's beginning to rain, anyway," Biff remarked, glancing up at the
sky. "I guess we may as well go back."
The sky had clouded over in the past hour and the eastern sky was
black, while scurrying masses of ragged clouds flew overhead before
the stiffening wind. A few drops of water splashed into the boat,
then came a gust of rain, followed by a light shower that passed
over in a few minutes. The big motorboat that had crowded them
had disappeared.
"A real storm coming up," Frank said. "Let's make for the
boathouse."
The Envoy headed for Bayport.
"I'd like to tell those three fellows in that other boat what I think of
them," declared Biff. "They got us into that jam. They were crowding
me so close that I didn't have a chance to keep an eye on the
sailboats."
"I still can't see why they drew up alongside," Joe observed. "They
seemed mighty inquisitive. Gave us all the once-over."
Chet offered a solution.
"Perhaps they thought we were some one else and when they found
out their mistake they went away."
"But they didn't go away," Frank pointed out. "They kept crowding
us over. And one of them pointed at Biff."
"At me?"
"Yes."
"I didn't notice that."
"He seemed to recognize you and was pointing you out to the other
men."
"Well, if he recognized me I can't return the compliment. I never
saw any of them before in my life."
"He was probably pointing you out as a unique specimen," ventured
Joe, with a grin. "Probably those fellows are from a museum, Biff.
They'll likely make an offer for your carcass after you're dead and
they'll have it stuffed and put it on display in a glass case. That's
why they were so interested."
Joe's suggestion elicited warm words from Biff and a friendly
struggle ensued. Inasmuch as Biff Hooper was the champion boxer
and wrestler of Bayport High, Joe was at a disadvantage, and paid
for his derogatory remarks by being held over the side by the scruff
of the neck and given a ducking until he pleaded for mercy.
By the time the boys reached Bayport it was raining heavily, and
after leaving the Envoy in the boathouse they raced up the street to
the Hardy boys' home. The barn in the back yard was a favorite
retreat of the chums and there they spent many of their Saturday
afternoons. The barn was fitted up as a gymnasium, with parallel
bars, a trapeze, boxing gloves and a punching bag, and was an ideal
refuge on a rainy day. The thrilling experience with the sailboats and
the mystery of the strange motorboat were soon forgotten.
Phil Cohen and Tony Prito, school chums of the Hardy boys, drifted
in during the afternoon, as well as Jerry Gilroy and "Slim" Robinson.
This comprised the "gang," of which the two Hardy boys were the
leading spirits.
Frank and Joe Hardy were the sons of Fenton Hardy, an
internationally famous detective. Mr. Hardy had been for many years
a detective on the New York police force, where he was so
successful that he went into practice for himself. His two sons
already showed signs of inheriting his ability and in a number of
instances had solved difficult criminal cases.
The first of these was the mystery of the theft of valuable jewels and
bonds from Tower Mansion, an old-fashioned building on the
outskirts of Bayport. How the Hardy boys solved the mystery has
already been related in the first volume of this series, entitled, "The
Tower Treasure."
In the second volume, "The House on the Cliff," the Hardy boys and
their chums had a thrilling experience in a reputedly haunted house
on the cliffs overlooking Barmet Bay. This was the starting point of
an exciting chase for smugglers, in which the Hardy boys came to
the rescue of their father after undergoing many dangers in the cliff
caves.
The third volume of the series, "The Mystery of the Old Mill," which
precedes the present book, relates the efforts of the Hardy boys to
run to earth a gang of counterfeiters operating in and about Bayport
and their efforts to solve the mystery surrounding an abandoned mill
in the farming country back of Barmet Bay.
Frank Hardy, a tall, dark-haired boy of sixteen, was a year older than
his brother Joe, and usually took the lead in their exploits, although
Joe was not a whit behind his brother in shrewdness and in
deductive ability.
Mrs. Hardy viewed their passion for detective work with considerable
apprehension, preferring that they plan to go to a university and
direct their energies toward entering one of the professions; but the
success of the lads had been so marked in the cases on which they
had been engaged that she had by now almost resigned herself to
seeing them destined for careers as private detectives when they
should grow older.
Just now, however, detective work was farthest from their thoughts.
Frank and Tony Prito were engaged in some complicated maneuvers
on the parallel bars, Joe was taking a boxing lesson from Biff
Hooper, and Phil Cohen was trying to learn how to walk on his
hands, under the guidance of Jerry Gilroy and Slim Robinson.
As for Chet Morton, the mischief-maker, he was sitting on the
window-sill, meditating. And when Chet Morton meditated, it usually
meant that a practical joke was in the offing.
"I'll bet you can't 'skin the cat' on that trapeze, Jerry," he called out
suddenly.
Jerry Gilroy looked up.
"Skin the cat?" he said. "Of course I can."
"Bet you can't."
"Bet I can."
"Can't."
"Can."
"Do it, then."
"Watch me."
As every boy knows, "skinning the cat" is an acrobatic feat that does
not necessarily embrace cruelty to animals. Jerry Gilroy was not
unjustly proud of his prowess on the trapeze and Chet Morton's
doubt of his ability to perform one of the simplest stunts in his
repertoire made him resolve to "skin the cat" as slowly and
elaborately as lay within his power.
He grasped the trapeze bar with both hands, then swung forward,
raising his feet from the floor, bending his knees. Chet edged
forward, presumably to get a better view of proceedings, but at the
same time he tightened his grip on a long, flat stick that he had
found by the window ledge.
Jerry slowly doubled up until his feet were above his head,
immediately below the bar, and then commenced the second stage
of the elaborate back somersault, coming down slowly toward the
floor. At this juncture the rear of his trousers was presented as a
tempting mark to the waiting Chet. This was the stage of the feat for
which the joker had been waiting and he raised the flat stick,
bringing it down with a resounding smack on his human target.
There was a yelp of pain from Jerry and a roar of laughter from
Chet. Doubled up on the bar as he was, Jerry could not immediately
regain the floor, and Chet managed to belabor him twice more
before the unfortunate acrobat finally found his footing. There he
stood, bewildered, rubbing the seat of his trousers, with a rueful
expression on his face, while Chet leaned against the wall, helpless
with laughter.
The other boys joined in the merriment, for they had stopped to
witness the incident, and after a while Jerry achieved a wry smile,
although he looked reflectively at his tormentor as though
wondering just what form his revenge should take.
No one enjoyed Chet Morton's practical jokes more than he did
himself. He whooped with laughter, wiped the tears from his eyes,
and leaned out of the window, spluttering with mirth.
"Oh, boy!" he giggled. "The expression—on your—face—!" Then he
was away again, leaning across the window-sill weakly, shaking with
laughter.
Jerry Gilroy tiptoed quietly up behind him. A quick movement and he
lowered the window until it was against Chet's back.
The practical joker suddenly stopped laughing, and turned his head.
"Hey! What's the matter?" he inquired.
He was pinned down by the window and he could not see Jerry
picking up the flat piece of board that had been the instrument of
torture a few minutes previously. But a suspicion of the truth came
to him, and a roar of laughter from the other boys warned him that
vengeance was due.
It came.
Smack!
Chet Morton wriggled and squirmed, but he was pinned helplessly by
the weight of the window against his shoulders, and he presented a
more tempting target for Jerry's ministrations with the flat stick, and
a more stationary target as well, than Jerry had presented for him.
Smack! Smack! Smack!
He roared with pain and, helpless as he was, danced vainly on the
floor in his efforts to escape. Jerry Gilroy belabored him across the
rear with that stinging stick until his desire for revenge had been
fully satisfied, while the other boys howled with glee at the manner
in which the tables had been turned.
Finally, when Jerry tossed the flat stick away and joined the others in
their laughter, Chet managed to raise the window and escape.
"Can't see what you're all laughing at," he grumbled, as he sat down
carefully on a near-by box. Then he rose hurriedly and rubbed the
tender spot.
"He laughs best who laughs last," quoted Jerry Gilroy.
"Guess I've got to get home," announced Biff, a moment later, and
soon he and the others were on their way, dodging through the rain.
Then Frank and Joe put the barn in order and went into the house.
They felt particularly carefree and never dreamed of the news they
were to hear or of how it was to affect them and their chums.
CHAPTER III
A Shady Trio
"I am sure my man is in Chicago. I know for a fact that he went
West, and the Windy City would naturally be his hiding place."
Fenton Hardy tapped the library table reflectively with a pencil. Mrs.
Hardy put aside the magazine she had been reading.
"Are you going to follow him?"
"I'll trail him right to the Pacific Coast if necessary."
Frank and Joe Hardy, who had been standing by the window,
disconsolately watching the rain streaking down the pane, looked
around.
"Who is he, dad?" asked Frank.
"One of the cleverest and most daring bank robbers in the country.
I've been after him for almost a year now and it's only been within
the last few weeks that I've ever come anywhere near catching
him."
"What's his name?"
Fenton Hardy laughed. "I've made you curious, eh? Well, this chap
has about a dozen names. He has a new alias every week, but so far
as the police are concerned he's known as Baldy Turk, because he's
as bald as an egg. He and his gang held up a bank in a small New
Jersey town about a month ago and got away with over ten
thousand dollars in broad daylight. That's how I managed to get
trace of him again. Even the police didn't know Baldy Turk was
mixed up in the affair because he was wearing a wig that day, but
he double-crossed one of the members of his gang out of his share
in the loot."
"And that fellow told the police," ventured Joe.
Mr. Hardy shook his head.
"Not the police. He didn't dare go near them because he was wanted
for two or three robberies himself. But he came to me and tipped me
off as to where Baldy Turk could be found. He wanted revenge. I
went to New York, where Baldy was in hiding; but evidently some of
his friends knew I was on his trail and he disappeared before I could
lay my hands on him."
"Where did he go then?" asked Frank, with interest.
"He hid out on Long Island for a while, but I managed to pick up the
trail again and went after him, but he was too smart for me. He got
away in a fast automobile and took a couple of shots at me in the
bargain. I managed to get the number of the car and traced it to
Manhattan and later found that Baldy Turk had left the East
altogether. He bought a ticket to Cleveland, doubled back to Buffalo
and managed to shake me off."
"What makes you think he is in Chicago?"
"Because another member of his gang went to Chicago just a week
ago. So I imagine Baldy Turk was to meet him there. In any case,
Chicago is a thieves' paradise, so it seems logical that Baldy Turk
would make for there."
"And you're going after him! Gee, I wish I could go," declared Joe.
Fenton Hardy smiled.
"It's no job for a boy," he said. "Baldy Turk is a bad man with a gun.
If I ever do find him it will take some maneuvering to get the
handcuffs on him, I'll tell you."
"You'll be careful, won't you, Fenton," said Mrs. Hardy anxiously. "I'm
always frightened whenever I know you're after one of these
desperate criminals."
"I'll be as careful as I can, Laura," promised her husband; "but in my
business I have to take chances. Baldy Turk knows I'm after him and
he doesn't mean to be caught if he can help it. He or any of the men
in his gang would shoot me on sight. There's a standing reward of
five thousand dollars out for Baldy and, besides, the Bankers'
Association have promised me a handsome fee if I can get him
behind the bars and break up the gang."
"I won't rest easy in my mind until you're back home safe," Mrs.
Hardy declared.
"Don't worry about me," replied her husband, going over to her and
patting her shoulder reassuringly. "I'll get back safely all right, and
Baldy Turk will be in jail if I have to chase him all over the States.
The boys will look after you while I'm away."
"You bet we will!" Frank promised.
"I'm sorry it keeps you from going on that motorboat trip with Chet
and Biff," Mr. Hardy remarked. "Perhaps you can arrange another
jaunt after I come back."
"We're not worrying about that, dad. We don't mind staying at
home."
"That's the spirit," approved their father.
"When do you leave?" Frank asked.
"I'm waiting for a letter from a friend of mine in Chicago. If he writes
as I expect he will write, I should be away by the day after to-
morrow."
"Then let Baldy Turk watch his step!" observed Joe.
"We'll both have to watch our step," answered Mr. Hardy, smiling. "If
I don't get him, he'll probably get me."
"Well, I'm betting on you."
Mrs. Hardy shook her head doubtfully, but said nothing. She knew
that her detective husband had escaped death at the hands of
desperate criminals many times in the course of his career and there
seemed to be no reason why he should not bring Baldy Turk to book
just as he had captured many other notorious criminals in the past;
but this time she had a vague premonition of danger. She knew that
her husband would laugh at her fears if she expressed them, so she
remained silent.
The rain had stopped, as Frank noticed when he glanced out the
window again.
"It's clearing up. What say we go out for a spin, Joe?"
"Suits me."
"Let's go."
"Don't be late for supper," warned Mrs. Hardy, as the boys started
out the door.
"We'll be in time," they promised, and the door closed behind them.
The Hardy boys went out to the shed where they kept their
motorcycles. Both Joe and Frank had machines, given to them by
their father, and in their spare time they spent many hours speeding
about the roads in and around Bayport.
Their native city had a population of about fifty thousand people and
was on the Atlantic coast, on Barmet Bay. There were good roads
along both northern and southern arms of the bay, besides the State
highway and the numerous country roads that led through the
farming country back of Bayport.
Chet Morton, whose father was a real estate dealer with an office in
the city, lived on a farm some distance off the road along the north
arm of the bay, Chet making the daily journey to school and back in
a roadster that had been given to him by his father. Chet was as
proud of his roadster as the Hardy boys were proud of the
motorboat that they had bought from the money they had received
as reward for solving the Tower Mystery.
"Where shall we go?" asked Joe, as the Hardy boys rode out of the
lane.
"Let's go to the Morton farm and see Chet."
"Good idea. I wonder if he's able to sit down yet," replied Joe,
alluding to Chet's practical joke earlier in the day.
The motorcycles roared and spluttered as the boys sped along the
gleaming pavements of the city. They rode through the main streets,
threading their way easily through the traffic until at last they were
at the outskirts of Bayport. Finally they left the city behind and
reached the road leading toward the Morton farm. The leaves of the
trees were still wet with rain and the luxuriant grass by the road-side
glistened with the heavy drops. The air was cool and sweet after the
storm. The roads had dried quickly, however, and the boys
experienced no inconvenience.
They reached the Morton farmhouse in good time and Chet's sister,
Iola, answered their knock. Iola was a pretty girl of about fifteen,
one of the few girls at whom Joe Hardy had ever cast more than a
passing glance. He lowered his eyes bashfully when she appeared in
the doorway.
"Chet just left in the car about ten minutes ago," she said smilingly,
in answer to their inquiry. "It's strange you didn't meet him."
"He probably went by the other road. We'll catch up to him."
"Won't you come in?"
"N-no thanks," stammered Joe, blushing. "Guess we'll be going."
"Oh, do come in," said Iola coaxingly. "Callie Shaw is here."
"Is she?" Frank brightened up at this intelligence, and at that
moment a brown-eyed, dark-haired girl about his own age appeared
in the hall.
"Hello!" she called, smiling pleasantly, and displaying small, even
teeth of a dazzling whiteness.
"Let's go," muttered Joe, tugging at Frank's sleeve. He was incurably
shy in the presence of girls, especially Iola.
But Frank did not go just then. He chatted with Callie Shaw for a
while, and Iola tried to make conversation with Joe, whose answers
were mumbled and muttered, while he inwardly wished he could talk
as freely and without embarrassment as his brother. At length Frank
decided to go and Joe sighed with relief. The girls bade them good-
bye after again urging them to come inside the house, and the boys
departed.
"Whew!" breathed Joe, mopping his brow. "I'm glad that's over."
Frank looked at him in surprise.
"Why, what's the matter? I thought you liked Iola Morton."
"That's just the trouble—I do," answered Joe mysteriously, and
Frank wisely forbore further inquiry.
They mounted their motorcycles again and rode down the lane, out
to the road. Hardly had they gone more than a few hundred yards,
however, than Frank suddenly gestured to his brother and they
slowed down.
Pulled up beside the road was an automobile, and as the boys drew
near they saw that three men were in the car. The men were talking
together and they looked up as the boys approached.
Something in the attitude of the trio aroused Frank's suspicions, and
this prompted him to ride slower. There seemed no apparent reason
why the men should have pulled their car up beside the road, for
they were not repairing a breakdown and they were still a little
distance from the lane leading to the Morton farmhouse. Then, as
the motorcycles slowly passed the car and the three men sullenly
regarded the two boys, Frank suppressed an exclamation of surprise.
The three men in the car were the three men who had pursued the
boys in the motorboat earlier in the day!
Frank and Joe drove past, conscious of the scrutiny of the unsavory
trio in the automobile. The men did not speak, although Frank
noticed that one of them drew his cap down over his eyes and
muttered something to one of his companions.
When they had gone by, Joe glanced back. The man were paying no
further attention to them, but were leaning close together, evidently
having resumed their interrupted conversation. There was something
stealthy and secretive in their demeanor that was far from
reassuring.
"Did you recognize them?" asked Frank, when they were out of
earshot.
"I'll say I did! The same gang that followed us in the motorboat."
"I wonder what they're up to."
"Up to no good, by the looks of them."
"That's a queer place to park their car—so close to the Morton farm,
too."
"They look like a bad outfit to me," remarked Joe.
"I'd like to know more about them. There was something funny
about the way they chased us in the boat. And don't you remember
how closely they looked at Chet and Biff? It seems funny to see
them hanging around the farm."
"Well, they haven't done us any harm. I suppose it's none of our
business—but I'd sure like to know what their game is. Let's find
Chet and tell him."
They increased their speed and before long overtook Chet Morton on
the shore road. But Chet laughed at their fears.
"You're too suspicious," he said. "They had probably just stopped to
fix a tire when you came along. However, we'll go back to the farm
and see if they're still on hand."
But when the boys drove back to the Morton farm they found that
the mysterious trio in the automobile were no longer in sight.
CHAPTER IV
The Send-Off
On Monday, Chet Morton and Biff Hooper set out on their motorboat
trip up the coast. They were well equipped with provisions and
supplies and had been up since six o'clock that morning getting the
boat in readiness.
The Hardy boys went down to the dock to bid them good-bye, and
although they chaffed the adventurers and laughed with them,
neither Frank nor Joe could repress the disappointment they
naturally felt at being unable to go with their chums.
Chet was busy stowing away the last of the provisions and Biff was
tuning up the engine when the Hardy boys arrived. In a few minutes
Tony Prito, at the helm of his own motorboat, arrived on the scene
with Jerry Gilroy and Phil Cohen. Then, down the dock, came
tripping Iola Morton and Callie Shaw.
"Hail, hail, the gang's all here!" roared Chet, when he saw them.
"Oy, what a fine day you pick for your trip!" exclaimed Phil Cohen,
looking up at the clouds. For the sky was overcast and there was no
sun.
"That's all right," answered Chet. "We made up our minds to start
to-day and we'd start if there was a thunderstorm on."
"Brave sailors!" mocked Callie Shaw, with a smile.
"How long will you be away?" shouted Frank.
"Until the grub runs out."
"That should be about next December," ventured Iola. "It looks to
me as if you have enough provisions there to last you a year."
"Not with Chet Morton on the trip, we haven't," grunted Biff Hooper,
looking up from the engine. "We'll be lucky if it lasts us a week. I've
seen him eat before."
"I'll do my share," Chet promised modestly.
"We should have had the City Band down to give you a proper send-
off," Joe Hardy remarked.
"It doesn't matter. We'll forgive you this time. But be sure and have
the band here to welcome us when we come back."
"You'll be back by to-morrow night," declared Iola. "I know you!
Why, I'll bet you'll both be scared green when darkness comes on.
One night will cure you of sleeping in the open."
"Rats!" replied Chet good-naturedly. "I'm not afraid of the dark."
"Cut out the jawing and let's get started," said Biff Hooper. "No use
hanging around here. Are you ready?"
"All set!"
"Let's go then. Good-bye, everybody."
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Introduction to Proteomics Principles and Applications

Biochemical Methods A Practical Approach 1st Edition

Principles of Biochemical Toxicology Fourth Edition John

Statistical Bioinformatics For Biomedical and Life Science

Fundamentals of Ecotoxicology Third Edition Newman

Biochemical Engineering and Biotechnology 1st ed Edition

Ecotoxicology of Explosives 1st Edition Geoffrey I.

Francois Ozon 1st Edition Edition Thibaut Schilt

With Additional Contributions From

AMSTERDAM BOSTON HEIDELBERG LONDON

Copyright r 2014 Elsevier Inc. All rights reserved.

British Library Cataloguing-in-Publication Data

Library of Congress Cataloging-in-Publication Data

For information on all Academic Press publications

Printed and bound in United States of America

List of Contributors xiii

1. Quantitative Assessments of Biochemical Analyses 1

2. Tissue Preparation and Subcellular Fractionation Techniques 21

3. Preparation and Maintenance of Live Tissues and Primary Cultures

3.1. General Introduction 33

4. Measuring Effects at the Gene Transcription Level 49

5. Metal Metabolism and Detoxification 75

5.1. Intracellular Free Metal Availability 77

6. Oxidative Stress 103

7. Xenobiotic Biotransformation 117

8. Cellular Energy Allocation 131

8.1.6. Cytochrome c Oxidase Activity 142

9. Neuroendocrine Disruption 145

9.9. Evaluation of Glutamate and γ-Aminobutyrate 166

10. Genotoxicity 171

10.1. Introduction 172

11. Biomarkers of Infection and Diseases 197

11.3. NO Synthase Activity 201

12. Descriptive Statistics and Analysis in Biochemical Ecotoxicology 209

13. Biomarker Expression and Integration 231

condition that could lead to damage at a higher level of biological organization. In

Biochemical ecotoxicology is the study of the effects of contaminants in ecosystems

Minutes Hours Days Weeks Months Year

investigation (availability of specific gene sequences or antibodies). In the absence of

1.1 GENERAL OVERVIEW

Biochemical Ecotoxicology r 2014 Elsevier Inc.

derivative of the amino acid tryptophan, by liquid chromatography with elec-

methodologies has advantages and caveats in respect to rapidity, cost-effectiveness,

Table 1.1 Levels of Control for the Generation of Biomarker Data

1. Type of project Objectives Replication To be included in the

1.2 REPLICATION IN DATA ANALYSIS

1.3 REPLICATION OF THE EXPERIMENT

1.3.1 Calibration or Standardization

theoretical and operational. The theoretical limit of detection of the biomarker

1.3.2 Using Specific and Generic Standards

follows: concentration of sample 5 [Analytical Signal (sample 2 blank)/Analytical

1.4 REFERENCE SUBSTANCES

1.5 DEFINING DETECTION LIMITS IN THE ABSENCE

We could prepare a representative homogenate extract of the digestive gland and

1.6.1 Reproducibility at the Calibration Level

1.6.2 Reproducibility at the Level of the Experiment

1.7 USING REFERENCE MATERIAL TO STANDARDIZE METHODOLOGIES