The Handbook of Histopathological Practices in Aquatic Environments: Guide to Histology for Environmental Toxicology

The Handbook of Histopathological Practices in Aquatic Environments

Guide to Histology for Environmental Toxicology

Pedro M. Costa

Table of Contents

Cover image

Title page

Copyright

Foreword

Preface

Acknowledgments

List of Abbreviations and Acronyms

Chapter 1. Introduction

Abstract

1.1 A Brief Account on the History and Purpose of Histology and Histopathology in Aquatic Organisms

1.2 Histopathological Traits as Biomarkers in Marine Environmental Science

1.3 Target Species and Organ: The Importance of Understanding Toxicological Pathways

1.4 Histopathology Links Many Levels of Biological Organization: The Systems Biology Approach

1.5 How to Use This Book

Chapter 2. Fundamental Concepts

Abstract

2.1 Procedural Overview

2.2 Basic Equipment and Material

2.3 Important Considerations on Solvents and Reagents

2.4 Safety Precautions

Chapter 3. Sample Preparation

Abstract

3.1 Tissue Preparation and Fixation

3.2 Paraffin-Embedded Samples

3.3 Procedures for Resin Embedding

Chapter 4. Staining Protocols

Abstract

4.1 General Histological Stains

4.2 Histochemistry of Lipids and Sugars

4.3 Peptide Histochemistry

4.4 Histochemistry of Metals, Metalloids, and Metallic Compounds

4.5 Stains for Microorganisms

4.6 Polychrome Stains

4.7 Techniques for Fluorescence Microscopy

4.8 Immunohistochemistry

Chapter 5. Microphotography and Image Processing: Creating Artwork

Abstract

5.1 Background

5.2 General Considerations on Digital Images and File Types

5.3 Image Resolution and Print Size

5.4 Color and Grayscale

5.5 Correcting Color and Shading

5.6 Post Hoc Image Corrections

Chapter 6. Identification of Major Histopathological Traits

Abstract

6.1 Understanding Deviations From Normal Histological Features in Aquatic Animals

6.2 Inflammation and Circulatory Disorders

6.3 Diagnosing Cell Death

6.4 Degenerative Disorders

6.5 Regressive Alterations

6.6 Cell Proliferation

6.7 Preneoplasms and Neoplasms

6.8 Symbionts, Parasites, and Commensals

Chapter 7. Scoring and Data Processing

Abstract

7.1 Fundamental Principles of Histopathological Scoring

7.2 Quantitative Histopathology

7.3 Semiquantitative Histopathology

7.4 Experimental Design and Statistical Processing of Data

Chapter 8. Common Problems and Troubleshooting

Abstract

8.1 Histological Artifacts and Misdiagnosis

8.2 Technical Problems in Sample Processing

Appendix A. Fixative Solutions

A.1 Bouin’s Solution

A.2 Carnoy’s Fluid

A.3 Castel’s Fixative

A.4 Davidson’s Fixative

A.5 Glutaraldehyde Fixative (Standard)

A.6 Neutral-Buffered Formalin

A.7 Osmium Tetroxide

A.8 Paraformaldehyde Fixative (PFA)

A.9 Zenker’s Fixative

Appendix B. Dyes

B.1 Alcian Blue

B.2 Aniline Blue

B.3 Aqueous Picric Acid

B.4 Biebrich’s Scarlet–Acid Fuchsin

B.5 Carbol Fuchsin

B.6 Crystal Violet (Hucker–Conn’s)

B.7 DAPI Nuclear Stain

B.8 Eosin (Alcoholic)

B.9 Fisher’s Coomassie Blue

B.10 Hematoxylin (Alum–Based)

B.11 Methylene Blue

B.12 Neutral Red

B.13 Nuclear Fast Red

B.14 Paragon

B.15 Reynolds’ Lead Citrate

B.16 Rubeanic Acid

B.17 Schiff’s Reagent (for Periodic Acid–Schiff’s Procedure)

B.18 Toluidine Blue

B.19 Uranyl Acetate

B.20 Weigert’s Iodine

B.21 Weigert’s Iron Hematoxylin

B.22 van Gieson’s Dye

Appendix C. Accessory Solutions

C.1 Acid Alcohol

C.2 Cacodylate Buffer

C.3 Gum Arabic, Mounting Medium

C.4 Histological Adhesive

C.5 Phosphate Buffer (PB) and Phosphate-Buffered Saline (PBS)

C.6 Phosphotungstic/Phosphomolybdic Acid

C.7 Scott’s Tap Water Substitute

C.8 Sodium Ethoxide or Methoxide (Epon Removal)

C.9 Trypsin Solution

Appendix D. Rapid Protocols

D.1 Hematoxylin & Eosin Staining

D.2 Rapid Protocol for Fluorescence Immunohistochemistry

D.3 Basic Preparation of Samples for TEM

Appendix E. Accessory Tables

E.1 Gay–Lussac’s Table for Ethanol Dilutions

E.2 Ultramicrotome Section Thickness Color Reference Chart

References

Index

Copyright

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Foreword

Manu Soto, Prof. Cell Biology, Plentzia Marine Station (PiE-UPV/EHU), University of the Basque Country, Spain

Toxicology has been defined as the corpus of science devoted to the study of the harmful effects produced by chemical compounds on living organisms. This definition became wider some decades ago with the aim of integrating toxicology and ecology in what is called ecotoxicology or environmental toxicology. There are only slight differences between both words. Ecotoxicology intends to link the effects of pollution at different levels of biological complexity, from molecular to population level passing through cell and tissue levels, while environmental toxicology is more focused on the effects produced at the individual level and below. In any case, the tissue interface is placed in the crossroad between all the functional levels mentioned beforehand. Thus, the use of histological approaches to study the normal status and the altered structure of given tissues of aquatic animals affected by disease (histopathology: histo, tissue; pathos, disease; logos, study) has been continuously increasing in the last years and has become a common endpoint in (eco)toxicity bioassays and (active and passive) biomonitoring programs. The histopathological evaluation of the effects produced by toxic substances or pathogens gives information on the target cells and/or tissues of animals affected by those compounds or their derivatives or environmental stressors (not necessarily chemicals), and can be very useful to make a diagnosis and to determine the severity and progress of the disease.

A routine evaluation can include the assessment of anatomic/morphological alterations under the microscope in fixed (or nonfixed) samples, embedded in resin or paraffin, and sections stained with different dyes, including the most conventional one, hematoxylin/eosin (H/E). Moreover, results obtained using special stains and fluorochromes, immunohistochemistry, in situ hybridization, or ultrastructural investigations can be included to determine the elemental composition of cells and tissues (i.e., proteins, enzyme activities, lipids, sugars, pigments, carbohydrates, polysaccharides, metals/metalloids, DNA, RNA, etc.) and their normal or altered functionality. I have to admit that a subjective component exists in the art of histopathology that might affect the reliability of histological data. Such skew, however, is meaningless when an adequate specimen preparation (practical skills and precision) and/or sufficient diagnostic or interpretive expertise are achieved. In order to obtain relevant results, therefore, not only high-quality samples/slides are needed, but also, in a further step, accurate image processing is needed to produce high-quality artwork. In this framework, this Guide for Environmental Toxicologists aims to draw attention to a sort of technical constraint that can affect histopathology results. Finally, histopathology is entering the era of high-throughput technologies with the production of massive quantitative and semiquantitative data based on morphometry/stereology and de visu observations, respectively. This huge amount of data generated by the combination of quantitative analysis and modern microscopy is rapidly permeating histopathology and requires a complementary deep statistical knowledge, and previous accurate, statistically significant, and biologically relevant experimental setups either for lab experiments or field works. All these issues, methodological and conceptual, are to be taken into account, and in fact, are reviewed in the present handbook, in order to help in producing an expert judgment based on histopathology for environmental health assessment.

Preface

Histopathology is, by all means, a discipline that results from the fusion between histology and pathology that, in spite of sharing many technical aspects with the former, holds quite distinct objectives, assumptions, and validation criteria from purely descriptive microanatomy. The growing interest in aquatic animal histopathology—both marine and freshwater, on account of aquaculture, species plus stock conservation and, very importantly, the environmental monitoring of pollutants—calls for expeditious methods for sample preparation and accurate identification of histopathological traits. In addition, the integration of aquatic animal histopathology within biomonitoring programs for environmental quality and toxicity testing of novel and classical substances is calling for more quantitative methods and tighter validation that, altogether, greatly differ from standard pathology. However, the readers should be warned that, when setting forth the writing of the Handbook, it has never been my intention to produce a thorough book on histotechnology, as there are plenty of solid references on the subject prepared by far better specialists. The Handbook is, as its name implies, a hands-on guide, as comprehensive as possible, on the theory and practice of histopathological techniques to be applied on such a diversified group of animals that each major taxon would justify a book on its own.

Histopathology of aquatic organisms is particularly challenging. To begin with, the variability between major animal taxa renders the objective identification of histopathological traits remarkably difficult, and it also generates potentially inconsistent nomenclature. This is especially true for nonvertebrate organisms, for which detailed histological studies are scarcer. On the other hand, histopathologists of aquatic organisms would likely benefit from a concise and specific guide to their craft as opposed to relying on scattered, gray, and often aged literature while spending years compiling amendments for protocols that needed adaptation from biomedical practice and even plant histology. In its turn, toxicological sciences hold their very own set of procedures and requirements that generate a whole different genre of histopathological assessment. In fact, as every toxicologist is aware of, diagnosing exposure to hazardous chemicals is particularly complicated, at least because the etiological agent of disease is, in essence, invisible, and even when it can be quantified the link between its presence and deleterious effects is neither obvious nor specific. As a consequence, determining cause–effect relationships in toxicological sciences requires quite particular interpretation of data and experimental design.

Altogether, this work started out as a compilation of protocols, improvements, and hints that resulted from a histopathology laboratory’s everyday work along many years. From this point of view, this work is meant to be just what the title implies: a handbook of routine techniques, recipes, troubleshooting, and clues to aid researchers, students, and technicians dealing with histopathology of aquatic animals, with emphasis on aquatic environmental toxicology. Most importantly, this book has been devised as a starter’s guide and is organized with that intention: it begins with basic concepts, from general theory to technical considerations, and then advances through histopathological assessment protocols and their validation. More elaborate techniques, such as cryotomy, electron microscopy, or confocal microscopy deserve entire books on their own and will only very briefly be dealt with here. Finally, easy-to-find recipes and hints are included in the text and the reader should refer to them when looking for information on staining protocols. It must be mentioned at this point that the histopathologist’s work is laborious and that patience and persistence are qualities just as needed as a steady hand and getting theory straight.

Unavoidably, this book is incomplete, for it is far from being able to include every technique or address every issue related to histology and histochemistry of aquatic organisms. Furthermore, the histological world is vast and every corner of biology, from animal to plant, from terrestrial to aquatic, has its own premises. Here, the motto lies within aquatic zoology, even though the basic techniques may apply just as well to other fields. Very importantly, this book intends to provide an overview of acknowledged and potential applications of histopathology in state-of-the-art science where aquatic organisms are targeted, from passive biomonitoring to the more complex and integrative systems biology approaches that stand on the frontline of biology as a science for the 21st century. The overall approach was simple to devise in concept albeit complicated in practice: instead of endeavoring the impossible work of describing histopathological features for all major aquatic taxa, the basic concepts that are common to almost every animal group are explained and illustrated. I hope this work meets its purpose at least by supplying the needed basis to set off new histologists, while keeping organized and ready-to-use well-established techniques to those who already retain expertise. This book is thus not a histopathological atlas, nor could it be, considering the biodiversity of aquatic animals. Instead, basic notions will be debated, hopefully aiding to place beginners on the right track toward the accurate detection, identification, and quantification of histopathological traits, as well as avoiding misdiagnosis, with emphasis on aquatic environmental toxicology and ecotoxicology.

Acknowledgments

I could not begin this book without placing a word of acknowledgment and appreciation to all those who directly or indirectly contributed to this book. I must thus express my gratitude to all my colleagues, mentors, and students, with whom I learned so much. Among these I am especially grateful to M. Diniz, who first introduced me to aquatic animal histopathology many years ago, M.H. Costa, M. Martins, J. Lobo, A.P. Matos, F. Carrapiço, L. Ascensão, A.P. Rodrigo, C. Martins, C. Gonçalves, N. Cuevas, M. Larguinho, and T. Neuparth; as well as A. Silva, S. Pereira, J. Santos, S. Carreira, J. Raimundo, J. Ramos, S. Caeiro and C. Sousa Reis (with whom I began working as a marine biology researcher, still as an undergraduate) and others to whom I must apologize for not being listed here, which includes reviewers and editors of this and other manuscripts, who certainly contributed to my development as a histopathologist. A kind word of appreciation must also be given to Elsevier staff for their support throughout this enterprise. A very special acknowledgment is also given to Professor Manu Soto for writing the foreword to this book. Finally, I must also acknowledge Fundação para a Ciência e Tecnologia (Portugal) for the support while preparing this book, through the grants IF/00265/2015 and UID/MAR/04292/2013.

List of Abbreviations and Acronyms

% m/v Percentage of solute mass of solute per total volume of solution, considering that 1 mL of pure water weighs 1 g. For example, 1% m/v sodium chloride (aqueous) means 1 g of the salt per 100 mL of solution.

% v/v Percentage of solute volume per total volume of solution. As an example, 1% ethanol v/v (aqueous) means 1 mL of ethanol per 100 mL of solution (i.e., 1 mL+99 mL of water).

Ahr Aryl hydrocarbon receptor

AIF Apoptosis-inducing factor

Arnt Aryl hydrocarbon receptor nuclear translocator

B[a]P Benzo[a]pyrene

BPDE Benzo[a]pyrene diol epoxide

BSA Bovine serum albumin

CYP1A Cytochrome P450 1A

DAPI 4′,6-diamidino-2-phenylindole (fluorochrome used as nuclear stain)

DPI Dots-per-inch (image resolution unit)

EDC Endocrine disruptor chemical

EDTA Ethylenediaminetetraacetic acid (used as buffer substance and anticoagulant)

ELISA Enzyme-linked immunosorbent assay

EM Electron microscopy

EROD Ethoxyresorufin O-deethylase

ETC Electron transport chain

FAA Formalin–alcohol–acetic acid (fixative solution)

FISH Fluorescent in situ hybridization

GA Glutaraldehyde

GFP Green fluorescent protein (fluorochrome)

GPx Glutathione peroxidase

GR Glutathione reductase

GSH Glutathione (reduced)

GSSG Glutathione (oxidized)

GST Glutathione S-transferase

H&E Hematoxylin and eosin stain

HSP Heat shock protein

IF Immunohistochemistry for fluorescence microscopy

Ig Immunoglobulin

IHC Immunohistochemistry

LRW London Resin White (embedding medium)

MFO Mixed-function oxidase

MMA Melanomacrophage aggregate (a more up-to-date designation for MMC)

MMC Melanomacrophage center

o/n Overnight

PAS Periodic acid–Schiff’s stain

PCD Programmed cell death

PCNA Proliferating cell nuclear antigen

PFA Paraformaldehyde

PKD Proliferative kidney disease

RER Rough endoplasmic reticulum

RES Reticuloendothelial system

ROS Reactive oxygen species

SEM Scanning electron microscopy

SER Smooth endoplasmic reticulum

SOD Superoxide dismutase

TBT Tributyltin

TCDD Tetrachlorodibenzodioxin

TEM Transmission electron microscopy

Tris Tris(hydroxymethyl)aminomethane (buffer substance)

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling (enzymatic method for in situ localization of apoptotic cells)

UV Ultraviolet light

XRE Xenobiotic response element

Chapter 1

Introduction

Abstract

The merger between pathology and histology gave birth to histopathology as a strong diagnosis tool for toxicologists, with the ability to act as a hinge between sub- and supraindividual levels of biological organization. The advent of environmental toxicology, from the 1960s onwards, turned histopathology into an important tool to monitor and understand the effects of pollutants onto aquatic organisms, in spite of many challenges in the identification and quantification of true toxicopathic effects in such diverse taxonomic groups. Although the bulk of technical achievements in histological practices were devised a century or more ago, the same basic principles are just as valid today and are steering histopathology of aquatic animals toward a wide range of applications, from biomonitoring programs to its integration within systems toxicology approaches.

Keywords

Histology; histopathology; cytopathology; aquatic organisms; pollutants; ecotoxicology; environmental toxicology; systems toxicology

1.1 A Brief Account on the History and Purpose of Histology and Histopathology in Aquatic Organisms

Histopathology is a powerful acknowledged tool with a wide range of applications within almost every domain of life sciences. It derives from the merging of two fields of expertise: histology (the study of living tissue) and pathology (the study of changes caused by an agent of disease to an organism). Histopathology thus permits the identification of the alterations to the normal state of living tissues and potentially their etiological (causative) agent that cannot be detected or confirmed with the naked eye. Whereas pathology needs little introduction, for it is as old as medical sciences; perhaps not many people know that, roughly a hundred years ago or more, histology was already a mature field of science on its own. One may recall that the Nobel Prize in Physiology or Medicine was attributed to Ramón Y Cajal in 1906, following the pioneering works on neurosciences, most of which involved histology. At this time, microphotography was a distant perspective but drawings and schematics made by early histologists, some of whom were rather talented science artists, provided descriptions of the microanatomy of numerous organisms, many of which are still acknowledged to the present day. Interestingly, aquatic animal histology is nearly as old as histology itself. Among the early studies can be found, inclusively, the very first descriptions of the cephalopod digestive gland, made public by the German Biologist J. Frenzel in 1886, which are accepted as reference science to this very day (Fig. 1.1).

Figure 1.1 Frenzel’s (1886) detailed histological drawings of the cephalopod digestive gland (Sepia spp.). Source: From Frenzel, J.H., 1886. Mikrographie der mitteldarmdrüse (leber) der mollusken. Erster theil. Allgemeine morphologie und physiologie des drüsenepithels. Nova Acta Ksl. Leop.-Carol. Deutsch. Akad. Naturf. 48, 81–296.

In another example, Keys and Willmer (1932) first described the chloride cells in the gills of teleost fish, in yet another beautiful set of handmade detailed drawings that still constitute valid knowledge as well (Fig. 1.2). The advent of microphotography, already well known after the mid-20th century onwards, broke new ground in microscopy science, even though, by that time, histology was already beginning to lose its role as an end in itself in favor of its use as a routine means for biomedical diagnostics, albeit not so much in environmental science. Still, and perhaps not surprisingly, most, if not all, the basic histological techniques still in use today were generated before that period, when histotechnologists were developing the theory and practice of fixatives, mordants, and dyes. Believe it or not, selecting the adequate paraffin for each climate was once a serious craft on its own, particularly in the first half of the 20th century. A second revolution occurred with the introduction of digital microphotography, by the beginning of the 21st century. From this milestone onward, taking a micrograph no longer needed to be a time-consuming, well-planned, and carefully executed ritual, which meant that larger batches of micrographs could now be produced and analyzed routinely. This innovation greatly contributed to generalize histopathology and helped bring it into the realm of more expeditious quantitative analyses, and was therefore of particular usefulness for environmental toxicologists and ecotoxicologists. This does not mean, however, that histopathology in ecotoxicology and related research started with digital microphotography. Far from it, in fact.

Figure 1.2 The original drawing from Keys and Willmer (1932), and respective caption, first describing chloride cells in fish gills—a histological trait with which fish histologists and histopathologists are quite familiar. Source: Reprinted with permission from Keys, A., Wilmer, E.N., 1932. Chloride secreting cells in the gills of fishes with special reference to the common eel. J. Physiol. 76, 368–378. Wiley Publishers Inc. (©1932 The Physiological Society).

Notwithstanding, it took time since the 1960s—when the pioneer works by Carson (1962) and Patterson (1965) on pesticide and Pb contamination of the environment, respectively, gave birth to environmental toxicology as a scientific discipline—to start the routine employment of histopathology as a tool in the field of research. Most notoriously, it started with aquatic animals, especially fish, in part due to the acknowledgment that the ultimate fate of pollutants is the aquatic milieu, in part due to the ease of collecting and rearing such ecologically relevant organisms for biomonitoring and substance testing. As such, the application of histopathological practices in these organisms rose gradually from the late 1970s to the 1990s, when some of the first reference works highlighting the benefits of the approach were made public (see, e.g., Wester and Vos, 1994).

Technical developments like digital microphotography, however, also brought the disadvantage of hasty and careless artwork, if not also misguided interpretation of histopathological traits, due to the trend to produce the highest possible amount of data in the least amount of time. Similarly, automated tissue processors and other assets for the batch processing of samples brought advantages and disadvantages. Ideally, the histopathologist should technically be a good histologist, which means that he or she should master the theory and practice, which requires hands-on expertise in all steps of the procedures before letting machines do most of the bench work. Furthermore, and what is perhaps both a challenge and a highlight of histopathology, every histopathologist should be a keen physiologist, pathologist, zoologist, toxicologist, and otherwise holder of good knowledge pertaining to any other branch of biological sciences that may be required to interpret the findings.

Aquaculture, fisheries biology, aquatic ecotoxicology and even the growing deployment of fish as models in biomedical research (i.e., the zebrafish, Danio rerio, and its laboratory strains) have been pushing aquatic animal histopathology frontwards (concerning fish, see, e.g., the review by Bolis et al., 2001). However, nonmammalian histology is hindered by scarcer reference works on histopathological traits, consensual terminology, and even proper methodology, which is even more critical with respect to marine invertebrates. In this case, it is quite common for the researcher to search and dwell in old histological reference works (if indeed are there any) just to identify some strange structure inside some mollusk’s digestive gland. This may, of course, bring about problems of nomenclature, doubts whether a lesion or alteration is or is not a sample-processing artifact, etc.

Ecotoxicology and environmental toxicology, in particular, have been responsible for bringing much emphasis to aquatic animal histopathology. In fact, quite a few decades-long biomonitoring programs have relied on histopathology of fish and mollusks as a major endpoint, such as the United Kingdom’s Clean Seas Programme and the BEST (Biomonitoring of Environmental Status and Trends) program enforced by the United States Geological Survey (USGS). Altogether, biomonitoring and aquaculture have relied on histopathology from a veterinarian point of view, seeking various alterations and their etiological agents such as contaminants, water warming and acidification, parasites, or feed. In any case, the basic methodological tools are similar and so are their assets and constraints. Regardless of the latter, histopathology holds the grand advantage of providing a direct assessment of the organism’s actual health status. The readers may refer, for instance, to the excellent and timeless reviews by Mallatt (1985), van der Oost et al. (2003), and Au (2004), who debate the importance and overall strategies of histopathological biomarkers in environmental monitoring of aquatic ecosystems, albeit alerting also for the risks of misdiagnosis resulting from inexperience and lack of sufficient background information.

1.2 Histopathological Traits as Biomarkers in Marine Environmental Science

Aquatic ecosystems tend to be the ultimate reservoir of pollutants, whether contamination results from direct input of wastewaters or from diffuse sources like rivers, atmosphere, and runoffs from urban settlements and agricultural grounds, to state a few examples. Overall, areas adjacent to waterbodies continuously endure strong anthropogenic pressure. These pressures and the importance of these areas for human settlement and activities thus dictate the need to safeguard environmental quality in all its aspects, from public health to the protection of aquatic resources. It is important, though, to make a distinction between contamination and pollution: while the first refers to the rise of levels of toxicants above the background the latter occurs when these levels are indeed causing adverse effects to the biota. These two concepts are linked to two others that need definition as well: hazard, which is the ability to induce adverse effects, and risk, which is the probability of a given substance to cause deleterious effects under certain circumstances of concentration and bioavailability. Altogether, these concepts are linked to the essential principle with which the Swiss-German alchemist Theophrast von Hohenheim, better known as Paracelsus (1493–1541), founded toxicology: it is only the dose what separates benefit from poison. Environmental toxicologists and ecotoxicologists must thus keep these concepts in mind in order to reach the most important and most challenging goal: causation. Establishing a positive link between an environmental stressor and adverse effects to the biota is in fact far from linear and it is acknowledged to rely on a multiple-evidence approach, similar to what has been proposed by epidemiologists to associate a cause to human disease at the population level (see Text Box 1.1). Even though it is not an absolute requirement that all assumptions are met, failing to meet one or more such assumptions will unavoidably weaken the drawing of conclusive cause–effect relations.

Text Box 1.1

What is needed to establish cause–effect relationships between environmental toxicants and adverse effects? Epidemiology may provide the basic assumptions to address causality:

• Consistency of association

• Done-responsiveness

• Time-responsiveness

• Biological plausibility

• Experimental validation

As for biomedical diagnosis, the relative expediteness of histological tools has not been overlooked by environmental scientists dedicated to environmental monitoring (or, better put, biomonitoring, since the analyses of living organisms is at stake), conservation biology, and even zootechnologists. To this is added the fact that pathology and histopathology provide a more direct diagnosis of the condition of a tissue or organ than, for instance, measuring the number of copies of a given gene transcript or the concentration of a given metabolite. This is of particular relevance in any situation where there is no predetermined link between a biomarker and an etiological agent of disease.

In environmental toxicology and ecotoxicology, a biomarker is defined as a subindividual response or effect that may act as an early warning of exposure to toxicants, i.e., any nonacute changes to the natural status of individuals that could be related to chemical aggression. van Gestel and van Brummelen (1996) justly included histological alterations in the lot of biomarkers, together with biochemical, physiological, and morphological responses compliant with the concept (from which behavior changes were, perhaps rather unjustly, excluded). The same authors provide perhaps the first clear distinction between biomarker, bioindicator species (which is a term more or less interchangeably used as sentinel organism) and ecological indicator, by defining a bioindicator as a species whose presence, absence, or altered behavior may indicate pressure onto an ecosystem, whereas an ecological indicator should be a change in ecosystem functioning (e.g., affecting biodiversity and food webs) or, to simplify, a concept that applies to supraindividual levels similarly as a biomarker applies to the subindividual. Histopathological traits, therefore, fall within the biomarker category, since they apply to the individual and may only through somewhat rough extrapolations provide an insight into population dynamics or ecosystem functioning. In general practice, ecotoxicologists analyze one or a range of biomarkers in a selected bioindicator species, whose relevance (ecological as well as economical), together with availability, ease to process, and sensitivity toward stressors, dictate its value in biomonitoring. In other words, the three concepts reflect three different levels of organization: individual, population, and ecosystem. However, the frontier between the terms is merely artificial, since it applies to whatever a researcher actually measures and not necessarily to what is means, biologically, as all levels of organization are evidently interlinked. Likewise, the common distinction between biomarkers of exposure, effect, and susceptibility may have a rather difficult, fuzzy definition (see, e.g., Martín-Díaz et al., 2004). Biomarkers of exposure can be regarded as quantifiable interactions between toxicants and organisms, for example, a metabolite or a specific gene transcript, elicited by exposure. Biomarkers of effect are most often considered signs of underlying changes in the organisms’ health. Finally, biomarkers of susceptibility reflect changes in the inherent ability to cope with stressors. These definitions are neither unambiguous nor specific and depend a lot on interpretation of findings per se. Hence, these definitions are not often employed. Also, as debated later in this book, histopathological traits are seemingly unspecific to a given toxicant (xenobiotic, if considering a purely exogenous, nonbiotic, nature of the substance), which further complicates the issue of using them as biomarkers. It must also be noted that ecotoxicology and environmental toxicology may not necessarily be terms as overlapping as may be expected, because addressing the effects, responses, and mechanisms without fully considering their effective ecological consequences (i.e., environmental relevance) does not contribute to place the eco before toxicology even if the substance is an acknowledged pollutant. As a consequence, the choice and interpretation of biomarkers may considerably differ between the two subsciences.

There are several approaches to address histopathology. The most common is qualitative. Under this perspective, the researcher or technician provides a report without any attempt to render the results in quantifiable measures, thus purely descriptive. This approach meets perfectly its purpose when the aim is to describe new pathological features with detail, which mandates high-quality histology and accurate terminology to produce a solid reference work. Also, it is acceptable to complement other analyses, such as biochemical or molecular biomarkers of disease or exposure to toxicants (e.g., Costa et al., 2010b), especially when a reduced number of specimens is available. As a rule of thumb, a minimum of three individuals per condition (i.e., three true biological replicates, not samples from the same individual, which is commonly referred to as pseudoreplication), are needed in order to provide minimally consistent conclusions in a study that is purely qualitative. Nonetheless, histopathology should try, whenever possible and as much as possible, to be quantitative, thus requiring larger numbers of samples, which will be addressed specifically in Chapter 7, Scoring and Data Processing. Also, the number of specimens to be analyzed is drastically increased if sampling is stratified, for instance, per gender, age, sexual maturation, and other factors that will certainly modulate the condition of animals as well as their sensitivity to external aggressors. Unfortunately, these factors tend to be overlooked in both field and laboratory work either by negligence or by logistical constraints. In addition, it is mandatory to acknowledge that disclosing whether a set of alterations is indeed caused by toxicological insult implies the understanding that such an assessment is always comparative toward a basal (normal) state, since even healthy organisms present histopathological traits. This means that all studies need an adequate control or at least a reference situation for comparison, as it is often called in field situations where an ideal control is unachievable. Unfortunately, there are some studies that use time-zero animals as controls in bioassay approaches. However, these animals are essentially nontested subjects that merely reflect the rearing conditions and not the bioassay conditions per se. Even though it is of importance to sample time-zero animals, at least to infer on the general condition before the beginning of the experiment, it must be acknowledged that there are many histopathological conditions that may derive from the experimental set-up itself. Among these there are included alterations to lipid and glycogen storage in livers with growth and feeding regime, for instance, changes in gill epithelial cells to cope with changes in oxygenation and salinity, even if minute, etc. It is thus critical to include and sample control animals throughout the bioassay as for any other experimental condition.

1.3 Target Species and Organ: The Importance of Understanding Toxicological Pathways

An important issue that applies to either field-sampling or experimental bioassays concerns the choice of target organism and organ or tissue. There are five main factors that should be taken into consideration when selecting the target species: (1) ecological relevance, (2) availability, (3) sensitivity to aggression, (4) previous information on physiology and microanatomy, and (5) experimental and sampling logistics. Researchers focusing on field sampling–based research (which may be called passive biomonitoring) must also consider the mobility of the species, since a wild animal reflects exposure throughout its life cycle and not just the conditions of its current location. As such, sampling migratory animals render establishing conclusive cause–effect relationships particularly difficult. It is also reasonable to consider the economical value of the species, if the study is aimed at stock assessment and conservation. On the other hand, extrapolation towards human health via consumption of affected animals must be taken very cautiously or avoided unless an adequate epidemiological approach can back such conclusions.

Fish and bivalves, both marine and freshwater, have, by far, received the major focus. Besides their importance for ecosystems and human populations, fish are vertebrates, which means that they share many common features with their mammalian fellows. Therefore, many histological and histopathological features can be extrapolated, to some extent. On the other hand, the growing interest in the zebrafish model is also leading to increased knowledge on fish microanatomy. However, the impressive variety of fish, which constitute, by a long range, the most abundant and diverse group of vertebrates, suggests that interpolations between piscine species need some caution. Also, differences between freshwater and marine animals imply specific interpretation of findings, not only regarding osmoregulatory organs, such as kidney and gills, but also the distinct sensitivity of the two groups regarding environmental changes, since marine environments are more stable, which means that the evolution of marine species (and this does not, of course, apply only to fish), tended to be more markedly directional. In other words, freshwater and estuarine animals are adapted to their naturally labile environments, which may render them phenotypically more plastic to resist anthropogenic pressure as well. Although gathering almost all advantages stated previously for the purpose of biomonitoring, many fish species, especially marine, are not confined to a single habitat along their life cycle. In many coastal species, younglings tend to be schooled in shallower waters, often confined, like estuaries; whereas adults migrate to deeper areas. In addition to such vertical movements, adults may perform horizontal migrations as well, in search of feeding and breeding grounds. This may constrain the association between local impacts and pathological traits. However, some benthic species may be more loyal to their habitats. This is one of the reasons why flatfishes (Teleostei: Pleuronectiformes) have been receiving growing attention for the biomonitoring of estuaries and other priority areas. In fact, the first work (and indeed, one of the very few) that conclusively established a link between organic carcinogens, polycyclic aromatic hydrocarbons (PAHs) in particular, and neoplasms in aquatic wildlife resulted from extensive passive sampling of English sole, Pleuronectes vetulus, from impacted areas on the Pacific coast of the United States, assisted by sediment analyses, thorough toxicopathological screening, laboratory assays, and statistical modeling (Myers et al., 2003). Note that throughout the book I will use the linguistically incorrect term fishes when referring to multiple species of fish, as acknowledged by ichthyologists. Flatfishes also offer the advantage of close contact with sediments, which effectively constitute the main storage of hydrophilic and hydrophobic contaminants, in particular those sediments that are enriched with organic matter and fine particles. Also, the relatively sluggish growth and slow maturation of flatfishes increase their appeal in assessing long-term chronic consequences of exposure to pollutants in a given area. In freshwater ecosystems, salmonids like the rainbow trout (Oncorhynchus mykiss) and cyprinids (like carps), cichlids (such as tilapias), catfishes (Siluriformes), basses (Micropterus spp., for instance), and even eels (which are catadromous but spend most of their life cycle in rivers) have most often been targeted as sentinels, regardless of being native or not. Overall, the diversity of freshwater organisms tends to reflect the high biogeographical diversity of freshwater basins. Still, in many cases, nonnative species are chosen for their abundance and availability of preceding information.

The growing availability of fish species from aquaculture facilities, in addition to laboratory breeding (as for the zebrafish) is also appealing to researchers in the field determined in performing bioassays in situ (field) and ex situ (under controlled laboratory environment) as an alternative or a complement to passive biomonitoring. A wide range of marine fish, like sea basses, breams, and even flatfish, are nowadays available from closed-cycle hatcheries for commercial and research purposes and have been widely taken advantage of by researchers in the field to perform all sorts of toxicological bioassays, with various endpoints being addressed, histopathology included. Freshwater species like the rainbow trout have been one of the most important models contributing to the advancement of fish histopathology in environmental toxicology and ecotoxicology. Researchers recurring to these species profit from aquaculture facilities (for species that are produced as meal or ornament) and ease of rearing and even breeding many freshwater species in the laboratory (much simpler if compared to marine species). As a consequence, cyprinids like carps and tilapias, plus the rising model Japanese medaka (Poecilidae: Orizias latipes) have also earned their place in preferential test organisms. As a general perspective, the deployment of aquaculture and laboratory breeding in toxicological testing has greatly contributed to advances in the knowledge of pathological traits, physiology, and responses to toxicants, as well as contributing to widen genomic annotation. It must be stated though, that much pathological and physiological knowledge has been imported from veterinary science associated with fish farming. Not surprisingly, researchers tend to choose these organisms over field-collected native animals for the purpose of conducting bioassays (Fig. 1.3).

Figure 1.3 Two simple fish bioassay arrangements. On the left, a system designed for the testing of contaminated estuarine sediment onto aquaculture-brood juveniles of the flatfish Solea senegalensis (Pleuronectiformes: Soleidae), ≈8 cm standard length, equipped with water recirculation and constant aeration. On the right, a testing system for adult laboratory-strain zebrafish (≈2.5 cm standard length), conceived for the testing of waterborne contaminants. In both cases, the tanks