Journal of Fish Diseases 1999, 22, 2534

Histopathology in fish: proposal for a protocol to assess

aquatic pollution
D Bernet1, H Schmidt1, W Meier1, P Burkhardt-Holm1,2 and T Wahli1
1 Centre for Fish and Wildlife Health, Institute of Veterinary Pathology, University of Berne, Berne, Switzerland
2 Interdisciplinary Centre for General Ecology, University of Berne, Berne, Switzerland

Abstract

Water pollution induces pathological changes in

fish. As an indicator of exposure to contaminants,
histology represents a useful tool to assess the degree
of pollution, particularly for sub-lethal and chronic
effects. However, a standardized method for the
description and assessment of histological changes,
mainly for use in freshwater fish, is still lacking. In
this paper, the present authors propose a standardized tool for the assessment of histological findings
which can be applied to different organs. The
methodology is based on two factors: (1) the
extension of a pathological change is rated with a
`score value'; and (2) the pathological importance of
this alteration is defined as an `importance factor'.
The sum of the multiplied score values and
importance factors of all diagnosed changes results
in different indices. With these indices, statistical
analysis can be carried out. Assessment methods for
the gills, liver, kidney and skin are described.
Introduction

The discharge of industrial, agricultural and

domestic waste water into the environment results
in the pollution of aquatic systems. Fish are often
exposed to highly contaminated water, especially in
areas where the dilution rate of waste water is low.
This has adverse effects, particularly when con-

Correspondence D Bernet, Centre for Fish and Wildlife

Health, Institute of Veterinary Pathology, University of Berne,
Laenggass-Strasse 122, CH-3012 Berne, Switzerland

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25

taminants: (1) are not or only slightly decomposable; (2) exhibit a high biological effectiveness;
(3) possess a high potential for accumulation; and
(4) influence each other in a synergistic or additive
way in the case of multiple contaminants. In fish,
water pollution can lead to different changes
ranging from biochemical alterations in single cells
up to changes in whole populations.
In the 1990s, the concept of biomarkers has
become increasingly established (Hinton & Lauren
1990; McCarthy & Shugart 1990; Huggett,
Kimerle, Mehrle & Bergman 1992; Myers, Johnson, Olson, Stehr, Lomax, Horness, Anulacion,
Willis, Collier, McCain, Stein & Varanasi 1994).
According to Huggett et al. (1992), the most
common usage of the term biomarker has been
for biochemical, physiological or histological indicators of either exposure to or the effects of
xenobiotic chemicals at the sub-organismal or
organismal level.
The advantage of histopathology as a biomarker
lies in its intermediate location with regard to the
level of biological organization (Adams, Shepard,
Greeley, Jimenez, Ryon, Shugart & McCarthy
1989). Histological changes appear as a mediumterm response to sub-lethal stressors, and histology
provides a rapid method to detect effects of
irritants, especially chronic ones, in various tissues
and organs (Johnson, Stehr, Olson, Myers, Pierce,
Wigren, McCain & Varanasi 1993). The exposure
of fish to chemical contaminants is likely to induce
a number of lesions in different organs (Sindermann 1979; Bucke, Vethaak, Lang & Mellergaard
1996). Gills (Mallatt 1985; Poleksic & MitrovicTutundzic 1994), kidney (Oronsaye 1989; Bucher
& Hofer 1993), liver (Hinton & Lauren 1990;

Journal of Fish Diseases 1999, 22, 2534

Myers et al. 1994; ICES 1997) and skin (Vethaak

1994) are suitable organs for histological examination in order to determine the effect of pollution.
These organs are primary markers for aquatic
pollution: gills and skin exhibit large surfaces which
are in direct and permanent contact with potential
irritants. Furthermore, both organs have mucous
cells. As thoroughly reviewed by Shephard (1994),
mucus plays an important role in disease resistance
against pathogens and toxic substances, as well as a
wide range of other functions. The liver plays a key
role in metabolism and subsequent excretion of
xenobiotics and is also the site of vitellogenin
production. This protein is induced by endogenous
oestrogens and is normally only detectable in
females. As vitellogenin is induced even in males
by an increasing number of man-made compounds
which mimic the effects of oestrogens, the liver is of
additional interest for the investigation of environmental impacts (Jobling & Sumpter 1993). The
kidney is important for the maintenance of a stable
internal environment with respect to water and salt,
excretion, and partially, for the metabolism of
xenobiotics.
In marine ecosystems, there exist some wide-scale
national and regional monitoring programmes
designed to assess the influence of environmental
pollution on histological features in fish (Susani,
Mearns & Long 1986; ICES 1989; Johnson, Stehr,
Olson, Myers, Pierce, McCain & Varanasi 1992;
Johnson et al. 1993; Myers, Stehr, Olson, Johnson,
McCain, Chan & Varanasi 1993; Bylund &
Lonnstrom 1994; ICES 1997). Neoplasms and
pre-neoplastic lesions, which are common findings
in bottom-dwelling fish from polluted areas, play a
major role in these monitoring programmes. These
lesions are specific, meaning that statistical analyses
have revealed an association between lesions and
exposure to irritants such as polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs), DDT, dieldrin and chlordanes (Johnson
et al. 1993; Myers et al. 1994; Vethaak 1994).
There is some evidence that neoplasms in young
fish living in contaminated areas are not as frequent
as in older fish and that young fish are at a
significantly higher risk of suffering from nonneoplastic lesions (Myers, Olson, Johnson, Stehr,
Hom & Varanasi 1992). With a quantification of
such non-neoplastic lesions, it might be possible to
obtain an obvious link between the degree of
pollution and lesions. Therefore, these lesions
might represent earlier indications for environmen 1999
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D Bernet et al. Proposal for a histopathological assessment protocol

tal pollution than neoplastic lesions, but are less

well described.
The above studies pointed out the importance
of standardizing techniques such as sampling,
handling samples, preservation and laboratory
investigations. Standardized methodologies for
histological techniques were described by (Bucke
1989, 1994). Since there is not yet any standardized method for describing and assessing histological lesions in freshwater fish, a comparison
between examinations dealing with the same organ
is not possible. Furthermore, a quantitative
comparison between various organs cannot be
made because many changes are considered to be
organ-specific alterations, and therefore, are not
generally applicable.
The present authors describe an assessment tool
which (1) is applicable on any given organ, (2) leads
to a standardized quantification, (3) allows the
possibility of legitimate comparison between different studies, and (4) with restrictions, between
different organs as well. This tool should lead to a
better understanding of the significance of histological findings after contaminant exposure.
Proposed methodology

Histological description
For each organ investigated, the respective pathological changes are classified into five reaction
patterns. These patterns represent a slight modification of the classification of Takashima & Hibiya
(1995), and are also in accordance with the
recommendations of Sindermann (1979), who
proposed this classification for the histopathological
assessment of experimental studies. A similar
categorization is found in the National Oceanic
and Atmospheric Administration (NOAA) quality
assurance programme on marine fish histopathology (Susani et al. 1986), where, among others, the
organs included in the present study were examined.
Each reaction pattern includes several alterations
which concern either functional units of the organ
(e.g. epidermal and dermal parts of the skin) or an
entire organ. An example is given in Table 1.
Reaction pattern 1 (rp1): circulatory disturbances
Circulatory disturbances result from a pathological condition of blood and tissue fluid flow. Fluid
content alterations in tissues related to inflamma-

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Journal of Fish Diseases 1999, 22, 2534

Table 1 Histopathological assessment tools for four fish organs (i.e. gills, liver, kidney and skin). An importance factor (worg rp alt)
ranging from 1 to 3 is assigned to every alteration: it is composed of the respective organ (org), the reaction pattern (rp) and the
alteration (alt)*
Reaction pattern

Functional unit
of the tissue

Gills
Circulatory disturbances
Regressive changes

Epithelium

Supporting tissue

Progressive changes

Epithelium
Supporting tissue

Inflammation

Tumour

Kidney
Circulatory disturbances
Regressive changes

Tubule

Glomerulus

Interstitial tissue

Progressive changes

Tubule
Glomerulus

Interstitial tissue
Inflammation

Tumour

Liver
Circulatory disturbances

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Alteration

Importance
factor

Score
value

Index

Haemorrhage/hyperaemia/aneurysm
Intercellular oedema
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Rupture of the pillar cells
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Hypertrophy
Hyperplasia
Hypertrophy
Hyperplasia
Exudate
Activation of RES
Infiltration
Benign tumour
Malignant tumour

wGC1 = 1
wGC2 = 1
wGR1 = 1
wGR2 = 1
wGR3 = 1
wGR4 = 2
wGR5 = 2
wGR6 = 3

aGC1
aGC2
aGR1
aGR2
aGR3
aGR4
aGR5
aGR6

IGC

wGR7 = 1
wGR8 = 1
wGR9 = 1
wGR10 = 2
wGR11 = 2
wGR12 = 3
wGP1 = 1
wGP2 = 2
wGP3 = 1
wGP3 = 2
wGI1 = 1
wGI2 = 1
wGI3 = 2
wGT1 = 2
wGT2 = 3

aGR7
aGR8
aGR9
aGR10
aGR11
aGR12
aGP1
aGP2
aGP3
aGP4
aGI1
aGI2
aGI3
aGT1
aGT2

Haemorrhage/hyperaemia/aneurysm
Intercellular oedema
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Hypertrophy
Hyperplasia
Hypertrophy
Hyperplasia
Thickening of Bowman's capsular membrane
Hypertrophy
Hyperplasia
Exudate
Activation of RES
Infiltration
Benign tumour
Malignant tumour

wKC1 = 1
wKC2 = 1
wKR1 = 1
wKR2 = 1
wKR3 = 1
wKR4 = 2
wKR5 = 2
wKR6 = 3
wKR7 = 1
wKR8 = 1
wKR9 = 1
wKR10 = 2
wKR11 = 2
wKR12 = 3
wKR13 = 1
wKR14 = 1
wKR15 = 1
wKR16 = 2
wKR17 = 2
wKR18 = 3
wKP1 = 1
wKP2 = 2
wKP3 = 1
wKP4 = 2

aKC1
aKC2
aKR1
aKR2
aKR3
aKR4
aKR5
aKR6
aKR7
aKR8
aKR9
aKR10
aKR11
aKR12
aKR13
aKR14
aKR15
aKR16
aKR17
aKR18
aKP1
aKP2
aKP3
aKP4

wKP5 = 1
wKP6 = 2
wKI1 = 1
wKI2 = 1
wKI3 = 2
wKT1 = 2
wKT2 = 3

aKP5
aKP6
aKI1
aKI2
aKI3
aKT1
aKT2

Haemorrhage/hyperaemia/aneurysm

wLC1 = 1

aLC1

IGR

IGP

IGI

IGT
IG.
IKC
IKR

IKP

IKI

IKT
IK.
ILC

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Journal of Fish Diseases 1999, 22, 2534

Table 1. Continued
Reaction pattern

Functional unit
of the tissue

Regressive changes

Liver tissue

Interstitial tissue

Bile duct

Progressive changes

Liver tissue
Interstitial tissue
Bile duct

Inflammation

Tumour

Skin
Circulatory disturbances
Regressive changes

Epidermis

Basement membrane
Dermis

Progressive changes

Epidermis

Dermis
Inflammation

Tumour

Alteration

Importance
factor

Score
value

Intercellular oedema
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Vacuolar degeneration
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Hypertrophy
Hyperplasia
Hypertrophy
Hyperplasia
Hypertrophy
Hyperplasia
Wall proliferation of bile ducts or ductules
Exudate
Activation of RES
Infiltration
Benign tumour
Malignant tumour

wLC2 = 1
wLR1 = 1
wLR2 = 1
wLR3 = 1
wLR4 = 2
wLR5 = 2
wLR6 = 3

aLC2
aLR1
aLR2
aLR3
aLR4
aLR5
aLR6

wLR7 = 1
wLR8 = 1
wLR9 = 1
wLR10 = 2
wLR11 = 2
wLR12 = 3
wLR13 = 1
wLR14 = 1
wLR15 = 1
wLR16 = 2
wLR17 = 2
wLR18 = 3
wLP1 = 1
wLP2 = 2
wLP3 = 1
wLP4 = 2
wLP5 = 1
wLP6 = 2

aLR7
aLR8
aLR9
aLR10
aLR11
aLR12
aLR13
aLR14
aLR15
aLR16
aLR17
aLR18
aLP1
aLP2
aLP3
aLP4
aLP5
aLP6

wLI1 = 1
wLI2 = 1
wLI3 = 2
wLT1 = 2
wLT2 = 3

aLI1
aLI2
aLI3
aLT1
aLT2

ILI

wSC1 = 1
wSC2 = 1
wSR1 = 1
wSR2 = 1
wSR3 = 1
wSR4 = 2
wSR5 = 2
wSR6 = 3
wSR7 = 2
wSR8 = 1
wSR9 = 1
wSR10 = 1
wSR11 = 2
wSR12 = 2
wSR13 = 3
wSP1 = 1
wSP2 = 2

aSC1
aSC2
aSR1
aSR2
aSR3
aSR4
aSR5
aSR6
aSR7
aSR8
aSR9
aSR10
aSR11
aSR12
aSR13
aSP1
aSP2

ISC

wSP3 = 1
wSP4 = 2
wSI1 = 1
wSI2 = 1
wSI3 = 2
wST1 = 2
wST2 = 3

aSP3
aSP4
aSI1
aSI2
aSI3
aST1
aST2

Haemorrhage/hyperaemia/aneurysm
Intercellular oedema
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Defect
Architectural and structural alterations
Plasma alterations
Deposits
Nuclear alterations
Atrophy
Necrosis
Hypertrophy
Hyperplasia
Hyperplasia of mucous cells
Hypertrophy
Hyperplasia
Exudate
Activation of RES
Infiltration
Benign tumour
Malignant tumour

Index

ILR

ILP

ILT
IL.

ISR

ISP

ISI

IST
IS.

*Abbreviations: (G) gills; (L) liver; (K) kidney; (S) skin; (C) circulatory disturbances; (R) regressive changes; (P) progressive changes; (I) inflammation; and
(T) tumour. The alterations per reaction pattern are numbered beginning with 1. The score value is aorg rp alt. The composition corresponds to that of the
importance factor. The score value has to be rated for every alteration by histopathological assessment with a score ranging from 0 to 6. Iorg rp is the reaction
index of an organ, Iorg. the organ index. The sections of the table in italics are examples of the addition of supplementary alterations according to the
specific needs of a study or an investigator. However, these are not considered for the calculation of the indices.
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Journal of Fish Diseases 1999, 22, 2534

tory processes (e.g. exudate) are considered in

reaction pattern 4. The alterations included here
are:
1 Haemorrhage/hyperaemia/aneurysm: blood leaking from blood vessels (haemorrhage), congestion of
blood in an organ caused by venous as well as
arterial processes (hyperaemia), and well-outlined
dilations of arterial blood vessels (aneurysm).
2 Intercellular oedema: stagnant tissue fluid which
has leaked from capillaries into tissue.
Reaction pattern 2 (rp2): regressive changes
Regressive changes are processes which terminate
in a functional reduction or loss of an organ. These
involve atrophy, degeneration (malformation or
dysfunction of cellular structures as a result of cell
damage) and necrosis. This reaction pattern
involves the following alterations:
1 Architectural and structural alterations: changes
in tissue structure as well as in shape and
arrangement of cells.
2 Plasma alterations: changes in cellular plasma
caused by hyaline droplets (granular degeneration),
colloidal droplets (colloid degeneration), degenerative fatty vacuolization or hydropic glycogen
droplets (glycogen degeneration), calcareous degeneration, and thickening of the fine fibres of
connective tissue (hyaline degeneration).
3 Deposits: intercellular accumulations of substances primarily caused by degenerative processes.
4 Nuclear alterations: changes in the nuclear
shape and structure of chromatin (e.g. karyopyknosis and karyorrhexis).
5 Atrophy: reduction in number and volume of
cells and/or a decreasing amount of intercellular
substances.
6 Necrosis: morphological state of a cell or a tissue
which appears after irrevocable loss of cell function.
Reaction pattern 3 (rp3): progressive changes
Progressive changes are processes which lead to
an increased activity of cells or tissues. Typical
lesions are:
1 Hypertrophy: enlargement of cell volume or
tissue without increase in cell number.
2 Hyperplasia: enlargement of tissue or organ by a
greater number of cells without change in volume of
the cells.
Reaction pattern 4 (rp4): inflammation
Inflammatory changes are often associated with
processes belonging to other reaction patterns (e.g.
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D Bernet et al. Proposal for a histopathological assessment protocol

oedema). Therefore, it is often difficult to attribute

inflammatory changes to one single reaction
pattern. Hence, the present authors use the term
`inflammation' in a very strict sense:
1 Exudate: fluid containing a high protein
concentration, and a large amount of cellular debris
exuded from blood and lymph vessels.
2 Activation of the reticuloendothelial system
(RES): hypertrophy of the RES, which consists of
endothelial cells and macrophages that line small
blood vessels.
3 Infiltration: leucocytes penetrating the walls of
blood vessels and infiltrating the surrounding tissue.
Reaction pattern 5 (rp5): tumour (neoplasm)
A tumour is an uncontrolled cell and tissue
proliferation (autonomous proliferation). Tumours
are divided into two classes:
1 Benign tumours: differentiated cells which
replace or displace the original tissue; these tumour
cells resemble the cells of the normal tissue.
2 Malignant tumours: poorly differentiated,
rapidly multiplying cells which invade and destroy
resident tissues; metastasis may be observed.
Individual description parameters
In addition to alterations within the reaction
patterns described, it is possible to designate
individual description parameters (see Table 1).
With these parameters, organ-specific lesions can be
shown in an explicit way. For the index calculations, these will not be considered since the changes
are already covered by the standardized expressions
(alterations) within the respective reaction pattern
as described above.
Histological evaluation
Importance factor (w)
The relevance of a lesion depends on its pathological importance, i.e. how it affects organ function
and the ability of the fish to survive. This is taken
into account by an importance factor assigned to
every alteration listed in the histological description.
The alterations are classified into three importance factors:
1 minimal pathological importance, the lesion is
easily reversible as exposure to irritants ends;
2 moderate pathological importance, the lesion is
reversible in most cases if the stressor is neutralized;
and

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Journal of Fish Diseases 1999, 22, 2534

Table 2 An example of lesion indices in one fish in which four organs were investigated
Reaction pattern
Organ

rp1

org1
org2
org3
org4
S

Iorg1
Iorg2
Iorg3
Iorg4
I.rp1

rp2
rp1
rp1
rp1
rp1

Iorg1
Iorg2
Iorg3
Iorg4
I.rp2

rp3
rp2
rp2
rp2
rp2

3 marked pathological importance, the lesion is

generally irreversible, leading to partial or total loss
of the organ function.
Score value (a)
Every alteration is assessed using a score ranging
from 0 to 6, depending on the degree and extent of
the alteration: (0) unchanged; (2) mild occurrence;
(4) moderate occurrence; and (6) severe occurrence
(diffuse lesion). Intermediate values are also
considered.
Mathematical calculation of lesion indices
Using importance factors and score values, four
different indices can be calculated (Table 2).
If the lesions within one organ only are studied,
the two following indices are applicable:
1 Organ index (Iorg.)

where: org = organ (constant); rp = reaction pattern; alt = alteration; a = score value; w = importance factor.
This index represents the degree of damage to an
organ. It is the sum of the multiplied importance
factors and score values of all changes found within
the examined organ. A high index indicates a high
degree of damage. Calculating the organ index
allows a comparison between the degree of damage
of the same organ in different individuals.
2 Reaction index of an organ (Iorg rp)

where: org, rp = constant (for abbreviations, see

organ index formula).
The quality of the lesions in an organ is expressed
by the reaction index. It is calculated by the sum of
the multiplied importance factors and score values
of the alterations of the corresponding reaction
pattern. The sum of the five reaction indices of an
organ is equivalent to the organ index (Iorg.).
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Iorg1
Iorg2
Iorg3
Iorg4
I.rp3

rp4
rp3
rp3
rp3
rp3

Iorg1
Iorg2
Iorg3
Iorg4
I.rp4

rp5
rp4
rp4
rp4
rp4

Iorg1
Iorg2
Iorg3
Iorg4
I.rp5

S
rp5
rp5
rp5
rp5

Iorg1.
Iorg2.
Iorg3.
Iorg4.
Tot-I

Respective reaction indices of an organ (Iorg rp)

from different individuals can be compared.
The following two indices can be applied if
several organs of a fish are examined:
3 Total index (Tot-I)
(for abbreviations, see organ index formula).
This index represents a measure of the overall
health status based on the histological lesions. It is
calculated by adding up all organ indices of an
individual fish. As the total index is calculated in
the same way for every fish, a comparison between
individuals is possible.
4 Total reaction index (I.rp)

where rp = constant (for abbreviations, see organ

index formula).
This index represents the quality of the histological lesions in all examined organs of an individual
fish. It is the sum of the corresponding reaction
indices of all examined organs of a fish. Using this
index allows a comparison between different
individuals.
Mathematical calculation of prevalences
Beside the indices calculated by extent (score
value) and pathological importance (importance
factor) of lesions, a further point of interest is the
prevalence of histopathological features. The prevalence of every alteration listed here can be
calculated as the percentage occurrence of an
alteration within all animals of a sample. This
allows an estimation of the occurrence of alterations
in an examined stock or a population.
Guidelines for field sampling

Along with pollution, several other exogenous

conditions may influence the histopathological
features of an organ. To minimize the histological

Journal of Fish Diseases 1999, 22, 2534

changes which are caused by variables other than

irritants, a standardized selection of the sampled
material must be taken into account. Some
variables which could affect the histological appearance are briefly considered below. These reflect the
present authors' experience and are in accordance
with the recommendations of ICES (1997). For a
thorough interpretation of histopathological results,
it is most important to take into account all these
features.

ence. As a compromise, it is recommended to

sample fish of a standardised size range within one
species.

Sample size

Sampling season

The sample size is a very important factor in every

histopathological monitoring programme. However, there is no absolute recommendation for an
optimal sample size because the latter varies
according to the objectives of a study. If it is the
determination of whether a pathological lesion is
present within a population which is intended,
statistical requirements must be fulfilled (95%
confidence of detection of a certain disease
prevalence in a population). If the aim is to
determine whether histopathological changes in
animals at polluted sites differ significantly from
those in fish from an unpolluted site, the required
sample size depends on the detectable differences
between the sites: the smaller the difference, the
larger the required sample size for a statistical
verification of this difference (e.g. in a x2 test).
Tables to determine the required sample size are
given in standard statistical texts.

Seasonality may play an important role in many

pathological conditions of fish and can be explained
by the influence of temperature e.g. on the biology
of the causative agent or the immune system of
poikilothermic animals or by the role of hormonal
variations in disease susceptibility.

Species

Discussion

The sensitivity to pollutants, as well as the

pollution-induced histopathological features, may
vary within a wide range depending on the species
(e.g. Braunbeck, Burkhardt-Holm, Gorge, Nagel,
Negele & Storch 1992). Therefore, a comparison of
results from different sites should be based on
samples from the same species.

Many publications have addressed the issue of

induction of histological lesions by irritants. However, the methods for evaluating histopathological
lesions are rather divergent. In some publications,
lesions have only been described morphologically
(Mitz & Giesy 1985; Hinton, Lantz, Hampton,
McCuskey & McCuskey 1987). In these studies,
the effect of water pollution has been correlated
with the abundance of lesions found in an organ.
Other studies concentrated on a few alterations in
an organ and assessed their extent by using a scale.
This allowed statements on abundance and intensity of lesions (Couillard, Berman & Panisset
1988; Bucher & Hofer 1993; Haaparanta, Valtonen & Hoffmann 1997). However, the use of
different methods and assessment scales as well as
the inclusion of different histological changes has

Age
The age of all fish should be recorded since the age
of stocks will strongly determine the range and
nature of pathologies (e.g. neoplasms are significantly more frequent in older fish). The determination of the age can be done by reading of scales,
otoliths or interopercular bones. However, these
techniques are time consuming and need experi 1999
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D Bernet et al. Proposal for a histopathological assessment protocol

31

Sex and stage of sexual maturity

Sex and stage of sexual maturity should be noted
since both factors are known to potentially
influence the histological appearance of certain
organs.

Migration
Migrations during the life-cycle (e.g. for spawning),
but also quick flight reactions to a short-time
pollution peak (Triebskorn, Kohler, Honnen,
Schramm, Adams & Muller 1997), can affect the
distribution of diseased fish within a geographical
region. To allow a comparison of samples from
different sites, it is recommended that sampling
within the same season is performed, preferably
when fish are on their primary resident feeding
grounds.

Journal of Fish Diseases 1999, 22, 2534

always made it difficult to compare different

studies. Therefore, a standardized assessment method is urgently needed.
The standardized assessment method proposed in
the present study allows the quantification of organ
damage, including the extent and pathological
importance of changes. Different indices can be
calculated which characterize a histology-based
health status at different levels: indices of the
organism (Tot-I), of an organ (Iorg.) and of the
reaction pattern (Iorg rp and I.rp). The indices
represent the degree of damage. However, these also
indicate the significance of the lesions. The degree
and significance of damage are important since
these allow the assessment of the suitability of an
organ or a lesion as an indicator for water pollution.
With the quantification of the lesions, statistical
evaluation becomes practicable. The corresponding
indices can be compared more easily than morphological descriptions of pathological changes.
Although histopathology is a descriptive science
and the assessment of the lesions will always depend
on the investigator's experience and interpretation,
the tool allows a more reliable comparison of
different studies.
However, a direct comparison of the organ
indices (Iorg.) within the same fish to quantify the
impact of an irritant on the respective organ is not
possible. Since the number of functional units is not
identical in all organs, the attainable maximum
values for the organ indices differ. To overcome this
problem, an indirect comparison of the organ
indices (Iorg.) with those of an unaffected control
group of fish from an unpolluted water system is
necessary. The higher the proportional difference of
Iorg. to the corresponding value in the unpolluted
group, the more damage has been done to the
affected organ.
Efforts to standardize the judgement of histopathological lesions have already been made. There
is agreement on the classification of liver alterations
in regional and national surveillance projects for the
assessment of the influence of contamination in
coastal and estuarine waters on marine bottomdwelling fish (Johnson et al. 1992; Myers et al.
1993; ICES 1997). The following classification has
been accepted: neoplasms, foci of cellular alterations, unique (specific) degenerative lesions, general
(non-specific) necrotic/degenerative changes and
non-neoplastic proliferative lesions. Additionally,
vascular abnormalities and anomalous storage
conditions have been recommended as diagnostic
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32

D Bernet et al. Proposal for a histopathological assessment protocol

criteria by ICES (1997). In contrast, lesions in

kidney are categorized as necrosis, proliferation and
sclerosis, as described by the National Benthic
Surveillance Project on the Pacific (Myers et al.
1993) and the North-east Coast (Johnson et al.
1992). Thus, differing histological evaluations of
the two organs prevent a direct comparison of the
degree and significance of an organ damage. By
introducing reaction indices (Iorg rp), as proposed in
the present study, a comparison is possible at least
between corresponding reaction patterns of the
same organ in different fish.
A further advantage of categorizing histopathological lesions lies in the possibility of assessing
which organs have been damaged and to what
extent changes have been induced. This is an
important prerequisite for surveillance and monitoring projects. However, it must be appreciated
that the categorization of pathological findings
results in a simplification, particularly because the
method has to be applicable to different organs.
Therefore, a thorough morphological description of
the lesions is essential. Individual description
parameters can be added to the assessment method
which, although not considered for the calculation
of the indices (see Table 1), allow additional and
more detailed evaluations according to the specific
needs of an investigator.
Recently, modern diagnostic methodologies for
the detection of exposure to contamination have
been well established for the liver. Most of these
biomarkers are designed to detect cellular/subcellular (e.g. lysosomal membrane stability), or
biochemical and molecular responses [e.g. enzyme-altered foci (G6PDH), proliferating cell
nuclear antigen (PCNA), CYP1A and DNA
adducts]. These are early indicators of biological
damage and some can be induced experimentally
within a few days (Collier & Varanasi 1991; Stein,
Collier, Reichert, Casillas, Tom & Varanasi 1992).
Although Myers et al. (1994) have shown in fish
that a significant correlation between biochemical
changes and histological lesions in the liver can be
drawn, these early response biochemical markers
cannot totally substitute the assessment of histopathological lesions as a marker for chronic
exposure to pollution. Therefore, in many regional
and national programmes, cellular/sub-cellular and
biochemical biomarkers, as well as histopathology,
are included in a decision-tree-type model (Adams
et al. 1989; ICES 1997; Triebskorn et al. 1997).
For monitoring of early changes, cellular/sub-

Journal of Fish Diseases 1999, 22, 2534

cellular and biochemical biomarkers are used,

followed by histopathology which partly considers
endpoint effects. These considerations further
support the importance of a standardized assessment tool for histopathological lesions, as described
in the present study.
Acknowledgments

We thank Dr D. Bucke, Consultant Fish and

Shellfish Pathologist, Weymouth, UK, and Dr E.
Staub, Swiss Agency for Environment, Forests and
Landscape (BUWAL), Berne, Switzerland, for
helpful suggestions. Dr A. Hemphill improved the
English. This study was supported by grants from
the Swiss National Science Foundation (3145894.95), BUWAL, the Inspectorate of Fisheries
of Berne, and the Water and Soil Protection
Laboratory of the District of Berne (GBL).
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