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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 CITATIONS 434 READS 2,915 5 authors, including: Some of the authors of this publication are also working on these related projects: invasive goby View project Investigation of the host parasite relationship between trout and Tetracapsuloides bryosalmonae including environmental stressors View project Patricia Burkhardt-Holm University of Basel 148 PUBLICATIONS 3,100 CITATIONS SEE PROFILE Thomas Wahli Universität Bern 104 PUBLICATIONS 2,223 CITATIONS SEE PROFILE All content following this page was uploaded by Thomas Wahli on 05 May 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. 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 Ó 1999 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 Ó 1999 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. 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