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See	discussions,	stats,	and	author	profiles	for	this	publication	at:	https://www.researchgate.net/publication/227741664
Histopathology	in	fish:	Proposal	for	a	protocol
to	assess	aquatic	pollution
Article		in		Journal	of	Fish	Diseases	·	December	2001
DOI:	10.1046/j.1365-2761.1999.00134.x
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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 standar-
dized 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-
taminants: (1) are not or only slightly decom-
posable; (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 & LaureÂn
1990; McCarthy & Shugart 1990; Huggett,
Kimerle, Mehrle & Bergman 1992; Myers, John-
son, 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 in-
dicators 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 medium-
term 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 (Sinder-
mann 1979; Bucke, Vethaak, Lang & Mellergaard
1996). Gills (Mallatt 1985; Poleksic & Mitrovic-
Tutundzic 1994), kidney (Oronsaye 1989; Bucher
& Hofer 1993), liver (Hinton & LaureÂn 1990;
Journal of Fish Diseases 1999, 22, 25±34
Correspondence D Bernet, Centre for Fish and Wildlife
Health, Institute of Veterinary Pathology, University of Berne,
Laenggass-Strasse 122, CH-3012 Berne, Switzerland
25
Ó 1999
Blackwell Science Ltd.
Myers et al. 1994; ICES 1997) and skin (Vethaak
1994) are suitable organs for histological examina-
tion 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 environ-
mental 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 &
LoÈnnstroÈm 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 non-
neoplastic 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-
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 standar-
dized method for describing and assessing histo-
logical lesions in freshwater fish, a comparison
between examinations dealing with the same organ
is not possible. Furthermore, a quantitative
comparison between variousorgans 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 dif-
ferent studies, and (4) with restrictions, between
different organs as well. This tool should lead to a
better understanding of the significance of histolo-
gical findings after contaminant exposure.
Proposed methodology
Histological description
For each organ investigated, the respective patho-
logical changes are classified into five reaction
patterns. These patterns represent a slight modifica-
tion 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 histopathol-
ogy (Susani et al. 1986), where, among others, the
organs included in the present study were exam-
ined.
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 patholo-
gical condition of blood and tissue fluid flow. Fluid
content alterations in tissues related to inflamma-
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
26
Ó 1999
Blackwell Science Ltd.
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
Alteration Importance
factor
Score
value
Index
Gills
Circulatory disturbances Haemorrhage/hyperaemia/aneurysm wGC1 = 1 aGC1 IGC
Intercellular oedema wGC2 = 1 aGC2
Regressive changes Epithelium Architectural and structural alterations wGR1 = 1 aGR1 IGR
Plasma alterations wGR2 = 1 aGR2
Deposits wGR3 = 1 aGR3
Nuclear alterations wGR4 = 2 aGR4
Atrophy wGR5 = 2 aGR5
Necrosis wGR6 = 3 aGR6
Rupture of the pillar cells
Supporting tissue Architectural and structural alterations wGR7 = 1 aGR7
Plasma alterations wGR8 = 1 aGR8
Deposits wGR9 = 1 aGR9
Nuclear alterations wGR10 = 2 aGR10
Atrophy wGR11 = 2 aGR11
Necrosis wGR12 = 3 aGR12
Progressive changes Epithelium Hypertrophy wGP1 = 1 aGP1 IGP
Hyperplasia wGP2 = 2 aGP2
Supporting tissue Hypertrophy wGP3 = 1 aGP3
Hyperplasia wGP3 = 2 aGP4
Inflammation Exudate wGI1 = 1 aGI1 IGI
Activation of RES wGI2 = 1 aGI2
Infiltration wGI3 = 2 aGI3
Tumour Benign tumour wGT1 = 2 aGT1 IGT
Malignant tumour wGT2 = 3 aGT2
IG.
Kidney
Circulatory disturbances Haemorrhage/hyperaemia/aneurysm wKC1 = 1 aKC1 IKC
Intercellular oedema wKC2 = 1 aKC2
Regressive changes Tubule Architectural and structural alterations wKR1 = 1 aKR1 IKR
Plasma alterations wKR2 = 1 aKR2
Deposits wKR3 = 1 aKR3
Nuclear alterations wKR4 = 2 aKR4
Atrophy wKR5 = 2 aKR5
Necrosis wKR6 = 3 aKR6
Glomerulus Architectural and structural alterations wKR7 = 1 aKR7
Plasma alterations wKR8 = 1 aKR8
Deposits wKR9 = 1 aKR9
Nuclear alterations wKR10 = 2 aKR10
Atrophy wKR11 = 2 aKR11
Necrosis wKR12 = 3 aKR12
Interstitial tissue Architectural and structural alterations wKR13 = 1 aKR13
Plasma alterations wKR14 = 1 aKR14
Deposits wKR15 = 1 aKR15
Nuclear alterations wKR16 = 2 aKR16
Atrophy wKR17 = 2 aKR17
Necrosis wKR18 = 3 aKR18
Progressive changes Tubule Hypertrophy wKP1 = 1 aKP1 IKP
Hyperplasia wKP2 = 2 aKP2
Glomerulus Hypertrophy wKP3 = 1 aKP3
Hyperplasia wKP4 = 2 aKP4
Thickening of Bowman's capsular membrane
Interstitial tissue Hypertrophy wKP5 = 1 aKP5
Hyperplasia wKP6 = 2 aKP6
Inflammation Exudate wKI1 = 1 aKI1 IKI
Activation of RES wKI2 = 1 aKI2
Infiltration wKI3 = 2 aKI3
Tumour Benign tumour wKT1 = 2 aKT1 IKT
Malignant tumour wKT2 = 3 aKT2
IK.
Liver
Circulatory disturbances Haemorrhage/hyperaemia/aneurysm wLC1 = 1 aLC1 ILC
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
27
Ó 1999
Blackwell Science Ltd.
Table 1. Continued
Reaction pattern Functional unit
of the tissue
Alteration Importance
factor
Score
value
Index
Intercellular oedema wLC2 = 1 aLC2
Regressive changes Liver tissue Architectural and structural alterations wLR1 = 1 aLR1 ILR
Plasma alterations wLR2 = 1 aLR2
Deposits wLR3 = 1 aLR3
Nuclear alterations wLR4 = 2 aLR4
Atrophy wLR5 = 2 aLR5
Necrosis wLR6 = 3 aLR6
Vacuolar degeneration
Interstitial tissue Architectural and structural alterations wLR7 = 1 aLR7
Plasma alterations wLR8 = 1 aLR8
Deposits wLR9 = 1 aLR9
Nuclear alterations wLR10 = 2 aLR10
Atrophy wLR11 = 2 aLR11
Necrosis wLR12 = 3 aLR12
Bile duct Architectural and structural alterations wLR13 = 1 aLR13
Plasma alterations wLR14 = 1 aLR14
Deposits wLR15 = 1 aLR15
Nuclear alterations wLR16 = 2 aLR16
Atrophy wLR17 = 2 aLR17
Necrosis wLR18 = 3 aLR18
Progressive changes Liver tissue Hypertrophy wLP1 = 1 aLP1 ILP
Hyperplasia wLP2 = 2 aLP2
Interstitial tissue Hypertrophy wLP3 = 1 aLP3
Hyperplasia wLP4 = 2 aLP4
Bile duct Hypertrophy wLP5 = 1 aLP5
Hyperplasia wLP6 = 2 aLP6
Wall proliferation of bile ducts or ductules
Inflammation Exudate wLI1 = 1 aLI1 ILI
Activation of RES wLI2 = 1 aLI2
Infiltration wLI3 = 2 aLI3
Tumour Benign tumour wLT1 = 2 aLT1 ILT
Malignant tumour wLT2 = 3 aLT2
IL.
Skin
Circulatory disturbances Haemorrhage/hyperaemia/aneurysm wSC1 = 1 aSC1 ISC
Intercellular oedema wSC2 = 1 aSC2
Regressive changes Epidermis Architectural and structural alterations wSR1 = 1 aSR1 ISR
Plasma alterations wSR2 = 1 aSR2
Deposits wSR3 = 1 aSR3
Nuclear alterations wSR4 = 2 aSR4
Atrophy wSR5 = 2 aSR5
Necrosis wSR6 = 3 aSR6
Basement membrane Defect wSR7 = 2 aSR7
Dermis Architectural and structural alterations wSR8 = 1 aSR8
Plasma alterations wSR9 = 1 aSR9
Deposits wSR10 = 1 aSR10
Nuclear alterations wSR11 = 2 aSR11
Atrophy wSR12 = 2 aSR12
Necrosis wSR13 = 3 aSR13
Progressive changes Epidermis Hypertrophy wSP1 = 1 aSP1 ISP
Hyperplasia wSP2 = 2 aSP2
Hyperplasia of mucous cells
Dermis Hypertrophy wSP3 = 1 aSP3
Hyperplasia wSP4 = 2 aSP4
Inflammation Exudate wSI1 = 1 aSI1 ISI
Activation of RES wSI2 = 1 aSI2
Infiltration wSI3 = 2 aSI3
Tumour Benign tumour wST1 = 2 aST1 IST
Malignant tumour wST2 = 3 aST2
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.
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
28
Ó 1999
Blackwell Science Ltd.
tory processes (e.g. exudate) are considered in
reaction pattern 4. The alterations included here
are:
1Haemorrhage/hyperaemia/aneurysm: blood leak-
ing 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), degenera-
tive fatty vacuolization or hydropic glycogen
droplets (glycogen degeneration), calcareous degen-
eration, and thickening of the fine fibres of
connective tissue (hyaline degeneration).
3 Deposits: intercellular accumulations of sub-
stances primarily caused by degenerative processes.
4 Nuclear alterations: changes in the nuclear
shape and structure of chromatin (e.g. karyopykno-
sis 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.
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 calcula-
tions, 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 patholo-
gical 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 impor-
tance 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
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
29
Ó 1999
Blackwell Science Ltd.
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 pat-
tern; alt = alteration; a = score value; w = impor-
tance 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.).
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 histolo-
gical 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 pre-
valence 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
Table 2 An example of lesion indices in one fish in which four organs were investigated
Reaction pattern
Organ rp1 rp2 rp3 rp4 rp5 S
org1 Iorg1 rp1 Iorg1 rp2 Iorg1 rp3 Iorg1 rp4 Iorg1 rp5 Iorg1.
org2 Iorg2 rp1 Iorg2 rp2 Iorg2 rp3 Iorg2 rp4 Iorg2 rp5 Iorg2.
org3 Iorg3 rp1 Iorg3 rp2 Iorg3 rp3 Iorg3 rp4 Iorg3 rp5 Iorg3.
org4 Iorg4 rp1 Iorg4 rp2 Iorg4 rp3 Iorg4 rp4 Iorg4 rp5 Iorg4.
S I.rp1 I.rp2 I.rp3 I.rp4I.rp5 Tot-I
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
30
Ó 1999
Blackwell Science Ltd.
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 appear-
ance 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.
Sample size
The sample size is a very important factor in every
histopathological monitoring programme. How-
ever, 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.
Species
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, GoÈrge, Nagel,
Negele & Storch 1992). Therefore, a comparison of
results from different sites should be based on
samples from the same species.
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 signifi-
cantly more frequent in older fish). The determina-
tion of the age can be done by reading of scales,
otoliths or interopercular bones. However, these
techniques are time consuming and need experi-
ence. As a compromise, it is recommended to
sample fish of a standardised size range within one
species.
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.
Sampling season
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.
Migration
Migrations during the life-cycle (e.g. for spawning),
but also quick flight reactions to a short-time
pollution peak (Triebskorn, KoÈhler, Honnen,
Schramm, Adams & MuÈller 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.
Discussion
Many publications have addressed the issue of
induction of histological lesions by irritants. How-
ever, 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 in-
tensity of lesions (Couillard, Berman & Panisset
1988; Bucher & Hofer 1993; Haaparanta, Valto-
nen & Hoffmann 1997). However, the use of
different methods and assessment scales as well as
the inclusion of different histological changes has
Journal of Fish Diseases 1999, 22, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
31
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Blackwell Science Ltd.
always made it difficult to compare different
studies. Therefore, a standardized assessment meth-
od 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 morpho-
logical 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 histo-
pathological 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 bottom-
dwelling fish (Johnson et al. 1992; Myers et al.
1993; ICES 1997). The following classification has
been accepted: neoplasms, foci of cellular altera-
tions, 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
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 histopatho-
logical 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 mon-
itoring 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/subcel-
lular (e.g. lysosomal membrane stability), or
biochemical and molecular responses [e.g. en-
zyme-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 histo-
pathological 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, 25±34 D Bernet et al. Proposal for a histopathological assessment protocol
32
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Blackwell Science Ltd.
cellular and biochemical biomarkers are used,
followed by histopathology which partly considers
endpoint effects. These considerations further
support the importance of a standardized assess-
ment 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 (31-
45894.95), BUWAL, the Inspectorate of Fisheries
of Berne, and the Water and Soil Protection
Laboratory of the District of Berne (GBL).
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