tozoan ectoparasite Ichthyophthirius multifiliis within the gill epithelia (Figure 7). As in brown trout, the histological appearance of gill tissue in exposed loach.
75
Journal of Aquatic Ecosystem Stress and Recovery 6: 75–86, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
The use of histopathological indicators to evaluate contaminant-related stress in fish Julia Schwaiger1 , R¨udiger Wanke2 , Stefan Adam3 , Michael Pawert3 , Wolfgang Honnen4 & Rita Triebskorn3 1
Laboratory for Fish Pathology, Steinseestr. 32, 81671 M¨unchen, Germany; 2 Institute of Veterinary Pathology, University of Munich, Veterin¨arstr. 13, 80539 M¨unchen, Germany; 3 Zoological Institute, University of T¨ubingen, Dept. Physiological Ecology, Auf der Morgenstelle 28, 72076 T¨ubingen, Germany; 4 Steinbeis-Transfer-Centre Reutlingen, Applied and Environmental Chemistry, Alteburgstr. 150, 72762 Reutlingen, Germany Received 14 August 1997; accepted in revised form 15 September 1997
Key words: biomarker, environmental pollution, fish disease, histopathology, morphometry
Abstract As a component of a large research programme to evaluate the effects of contaminants on fish health in the field, histopathological studies have been conducted to help establish causal relationships between contaminant exposure and various biological responses. Brown trout (Salmo trutta f. fario) and loach (Barbatula barbatula) were exposed to water diverted from polluted streams under semi-field conditions at various times during the year. The histopathological studies revealed seasonal differences in the types and severity of organ lesions between fish of the two streams. Both toxicant-induced alterations and organ lesions resulting from natural stressors (physicochemical and limnological water parameters) and secondary stress effects of pollution (diseases) could be detected. In evaluating the general health of experimental and control fish, the use of histopathological studies is recommended for making more reliable assessments of biochemical responses in fish exposed to a variety of environmental stressors. Stereological analysis provides quantitative data on pathological lesions which helps to establish correlation with other biomarkers thereby increasing the probability of identifying cause (stressor) and effect (biomarker) relationships.
Introduction One of the main areas of emphasis in current biomarker research is the application and validation of markers to assess toxic effects of aquatic pollutants in the field. The aim of a current research programme is to characterize the chemical impacts of two experimental streams on brown trout (Salmo trutta f. fario) and loach (Barbatula barbatula) by the application of a variety of biochemical and cytological markers as well as histopathological investigations. Comprehensive chemical analysis of water and sediment of two streams in southwest Germany, Kr¨ahenbach (A) and K¨orsch (K), revealed different contamination patterns. Some of the biomarker responses, such as the expression of stress proteins belonging to the Hsp70 group, as
well as ultrastructural findings have been shown to correspond to the different degrees of pollution (Triebskorn et al., 1997, this issue). The present paper focuses on histopathological investigations which have been proved to be a sensitive tool to detect direct toxic effects of chemical compounds within target organs of fish in laboratory experiments (Wester & Canton, 1991; Schwaiger et al., 1992, 1996) and in field investigations (Hinton et al., 1992; Teh et al., 1997). In addition to assessing toxic effects, a further aim of the histopathological investigations was to identify histopathological alterations which may occur from environmental stressors other than contaminants (Teh et al., 1997) and which are known to occur as a consequence of adverse environmental conditions (Snieszko, 1974; M¨oller, 1985; Anderson,
76 1990). Such alterations which occur in fish living in polluted environments are in most cases described as “pollutant-associated” rather than “pollutant-induced” (Overstreet, 1988). Histopathological investigations have the capacity to differentiate between organ lesions induced by diseases and other environmental factors from those lesions due to pollutant exposure. Histopathological responses were quantified using semiquantitive as well as stereological (morphometric) procedures to compare the severity of lesions in fish exposed to the more polluted stream (K¨orsch) to individuals of the moderately polluted Kr¨ahenbach system. Furthermore, quantitative differences between fish exposed to the two rivers and controls were evaluated. The histopathological findings are discussed in context to physicochemical and limno-chemical data of the two experimental streams, as well as to other related biomarker responses, described by Triebskorn et al. (1997, this issue).
Materials and methods Experimental design From June 1995 until September 1996, a total of 9 semi-field (stream bypass) experiments were conducted. At various seasons of the year, juvenile brown trout from a hatchery and adult loach captured in the field by electrofishing were exposed to water from the two experimental streams (Kr¨ahenbach (A) and K¨orsch (K)). After capture, loach were maintained in the laboratory for 4 weeks until they were subjected to experiments. The individual exposure periods varied between 6 and 21 weeks. Exposure of the fish took place in 250-L aquaria connected to the rivers. A flowthrough exposure system was used with an exchange rate of water of 250 l/h. The bypass exposure simulated the field situation with regard to the chemical impact of water and sediment as well as the physicochemical and limno-chemical characteristics of the two streams. Control fish were maintained in flow-through aquaria in chlorinated tap water (15.8 mg Cl /l). All fish were fed a commercial ration (ALMA, Botzenhard GmbH, Kempten, F.R.G.) once per day (2% of body weight) during the exposure periods. Table 1 provides an overview on the experiments performed and the fish treatment groups subjected to histological investigations.
Chemical, physicochemical and limno-chemical investigations Comprehensive chemical analysis of water and sediment of the two experimental streams Kr¨ahenbach (A) and K¨orsch (K) was carried out as described by Triebskorn et al. (1997, this issue). Water quality was characterized by higher pesticide levels in the water of the river K¨orsch with heavy metal and PAH concentrations being similar. Sediments in the K¨orsch system contained elevated levels of heavy metals, PAHs and pesticides while loading in the Kr¨ahenbach system was less dramatic. In Table 2 the average concentrations of chemicals in the two rivers during the experimental periods are presented. Detailed contaminant data are shown by Triebskorn et al. (1997, this issue). Control water did not contain detectable amounts of pesticides and PCBs. Contamination with heavy metals and PAHs was negligible (information was kindly provided by the Stadtwerke T¨ubingen). The physicochemical characteristics of water in the two experimental streams such as pH, oxygen saturation, conductivity, hardness and water temperature, were analyzed once a month throughout the exposure period. Limno-chemical investigations were also conducted which included quantification of nitrogen bound in ammonia (NH4 -N), in nitrite (NO2 -N), and in nitrate (NO3 -N), as well as analysis on chloride (Cl ) and the biological oxygen demand (BOD5 ). Water quality of control aquaria was checked repeatedly (four times) during each of the experiments. The temperature of the control water as well as the light:dark regimen were adapted continously to simulate field conditions. As shown in Table 3 the two experimental streams clearly differed from each other with regard to physicochemical and limno-chemical characteristics. The annual means of most of the parameters, including Cl , NH4 -N, NO2 -N, NO3 -N, conductivity, biological oxygen demand (BOD5 ), and the annual water temperature were higher in the K¨orsch system than in the Kr¨ahenbach. Both rivers proved to be similar with regard to mean annual pH values, hardness, and oxygen saturation. Pathological examinations Complete necropsies were performed on 140 fish exposed in the bypass systems and on 66 control fish. Fish were killed under anesthesia (0.1% benzocaine) by decapitation. Tissue specimens of the anterior and posterior kidney, liver, and gills of each individual
77 Table 1. Fish groups subjected to histopathological investigations, exposure periods and sampling times Experiment
Season
Exposure time
Code no.
Month of sampling
Experimental groups (number of fish)
(weeks)
Control
Kr¨ahenbach (A)
K¨orsch (K)
October December July July September
13 21 12 6 20
6 6 8 8 8
6 6 8 8 8
8 8 8 8 8
October December September September
13 15 12 12
8 6 8 8
8 8 8 8
8 8 8 8
Brown trout 05F1 07F1 14F3 14F4 16F3 Loach 05S1 07S2 16S3 16S4
Table 2. Groups of chemicals analyzed once per month in water and sediment of Kr¨ahenbach and K¨orsch Parameter
Kr¨ahenbach (A) Water (g/l)
Pesticides PCBs PAHs Heavy metals
0.01 0.00 0.06 86.25
Data are given as means
0.02 0.00 0.02 76.96
K¨orsch (K))
Sediment (g/kg) 6.5 29.3 3628 50561
8.2 40.3 2396 19076
Water (g/l) 0.17 0.00 0.11 119.41
0.15 0.00 0.04 73.89
Sediment (g/kg) 25.6 102.5 12177 346564
22.8 81.5 4458 123688
standard deviations.
were fixed for two days in buffered formalin (10%) and routinely embedded in paraffin wax for light microscopy. Sections were cut at 3 m and stained with hematoxylin and eosin (H&E). Periodic acid schiff reaction (PAS) was used to identify glycoproteins, and Masson’s trichrome stain was applied to demonstrate connective tissue. A section of the liver samples were fixed in Schaffer solution (Romeis, 1989), processed for paraffin embedding, sectioned at 3 m, and stained with carmine according to Best (1906) to detect glycogen deposits within the hepatocytes. Histopathological sections were read during the course of each experiment. Alterations were evaluated semiquantitatively by ranking the severity of tissue lesions. Ranking was: grade 1 = no pathological alterations, grade 2 = focal mild to moderate changes and grade 3 = extended severe pathological alterations. This ranking was used to establish an overall assessment value of the histopathological lesions for each organ of each individual fish. On the basis of these
data, mean assessment values (MAV) of organ lesions were calculated for each treatment group. In a limited number of fish (n = 4 per experimental group, i.e., A, K, control) from the October experiments, additional stereological investigations were conducted in order to quantify the histopathological alterations in the kidney and the liver. The organ was embedded as a whole in paraffin and a series of histological sections were cut at a nominal thickness of 3 m and stained with H&E. From each series at least one complete section was selected for morphometric evaluation using a Videoplan image analysis system (Zeiss-Kontron, Germany) coupled to a microscope via a color video camera. Measurements were made on images displayed on a color monitor. The crosssectional area of altered tissue as well as the total area of the histological section was planimetrically determined. For calibration, an object micrometer (Zeiss, Germany) was used. From the area density which was calculated as the ratio of the sum of cross-sectional
r
78 Table 3. Annual mean values of water parameters determined once per month in Kr¨ahenbach, K¨orsch and Control (tap) water Parameter
Control water
Temperature ( C) pH Oxygen saturation (%) Conductivity (S/cm) Cl (mg/l) NH4 + -N (mg/l) NO2 -N (mg/l) NO3 -N (mg/l) BOD5 (mg/l) Hardness (dGH)
8.51 95.25 504 22 0.38 0.01 4.7 0.58 16
#
0.17 2.95 63 4.3 0.38 0.01 3.6 0.18 0.71
Kr¨ahenbach (A) 7.77 8.38 92.08 735 19.88 0.02 0.01 5.26 1.32 19.0
4.66 0.29 4.39 63 2.55 0.01 0.01 5.43 0.55 3.76
K¨orsch (K) 10.89 8.18 93.08 955 137.83 1.87 0.23 9.50 4.60 13.67
4.26 0.12 8.34 151 222.95 3.72 0.15 9.91 2.35 2.36
p < 0.05 (K water versus A water); data are given as means standard deviations. # water temperature was continuously adapted to the field situation.
areas of altered tissue to the sum of section area the volume density of altered tissue was estimated according to the principle of Delesse (Weibel, 1979).
ations, however, which were quantified by morphometric evaluations did not differ significantly between exposed fish and controls (Table 4).
Statistical evaluation
Kidney Except for occasional slight hyaline droplets within single tubular epithelial cells, controls of both fish species did not show any morphological alterations of the kidney. Kidney lesions of brown trout exposed to K¨orsch water showed a completely different pattern of alterations when compared to renal changes found in individuals from the Kr¨ahenbach. As shown in Figure 1 the MAVs for kidney alterations, on the basis of semiquantitative evaluation of findings, were higher in fish exposed to K¨orsch water compared to Kr¨ahenbach water. The severe changes in the K¨orschgroups were most distinct during the warmer seasons and displayed all morphological characteristics of the Proliferative Kidney Disease (PKD), such as a granulomatous nephritis, accompanied by degenerative and necrotic changes of hematopoietic cells and of excretory renal tissue, as well as the occurence of so-called PKX-cells within the damaged areas (Figure 4). Stereological investigations, carried out on four individuals of each group during the October sampling, revealed a more than 50% reduction in the volume density of tubules in the kidney for K¨orsch water exposed trout compared to controls (Table 4). Kidney alterations of brown trout exposed to the moderately polluted Kr¨ahenbach, as well as loach from both sites did not show seasonal variations concerning the degree of lesions (Figure 1). These consisted primarily of vacuolation, hypertrophy, and single cell necrosis of tubu-
Means standard deviation (SD) were calculated for each experimental group, and data were analyzed for significance of differences between control and exposed groups according to the Mann-Whitney U-Test for independent samples with p < 0.05 set as statistical significance.
Results Histopathological responses In brown trout and loach, a variety of histopathological changes were found in the organs examined. The severity and frequency of organ lesions was found to be more pronounced in trout exposed to K¨orsch river water than in fish exposed to both Kr¨ahenbach water and controls, as evidenced by statistical evaluation of semiquantitative histopathological (Figures 1–3) and morphometric data (Table 4). In loach, however, no distinct differences between fish exposed to the two experimental streams could be observed with regard to the type, severity, and frequency of histopathological alterations. However, histopathological findings in kidney and gills of experimental fish could be distinguished from the slight tissue lesions observed in control individuals. Liver alter-
79 Table 4. Morphometric data on kidney and liver alterations in brown trout and loach; No. of fish examined per group: n = 4 Treatment
Parameter
Control
05F1
Volume fraction of tubules in the kidney (%)
27.15 4.37 (22.18 – 34.00)
05F1
Volume fraction of inflammatory altered tissue in the liver (%)
1.08 1.09 ( 0 – 2.46)
Volume fraction of parasitic affected tissue in the liver (%)
4.27 2.60 (0 – 6.93)
Kr¨ahenbach (A)
Brown trout
25.53 5.76 (19.80 – 34.70)
0.23 0.30 (0 – 0.73)
K¨orsch (K) 13.00 6.01# (6.25 – 21.37)
1.70 0.99# (0 – 2.52)
Loach 05S1
p < 0.05 ( K/A versus Control; shown in parentheses.
#
K versus A) data are given as means
8.28 8.99 (0 – 23.09)
6.02 7.40 (0 – 18.60)
standard deviations; minimum and maximum values are
Figure 1. Mean assessment values (MAV) of kidney alterations in brown trout and loach calculated per fish group on the basis of semiquantitative data. p < 0.05 ( K/A versus Control; # K versus A); data are given as means standard deviations.
lar epithelial cells and occasional dilation of kidney tubules with exfoliation of epithelial cells and accumulation of proteinaceous material within tubulus lumina (Figure 5).
Liver Liver tissue of brown trout exposed to control water was characterized by the presence of a high amount of glycogen within irregular shaped vacuoles of liver cells. Predominantly in the liver of brown trout exposed
80
Figure 2. Mean assessment values (MAV) of liver alterations in brown trout and loach calculated per fish group on the basis of semiquantitative data. p < 0.05 ( K/A versus Control; # K versus A); data are given as means standard deviations.
to K¨orsch water, a distinct reduction of glycogen deposits within the hepatocytes occurred (Figure 6, see also Triebskorn et al., 1997, this issue). Furthermore, multifocal inflammatory processes within the liver parenchyma could also be observed (Figure 6). In general, the MAVs for liver alterations, on the basis of semiquantitative evaluations of both findings, were higher in fish exposed to K¨orsch water compared to Kr¨ahenbach water (Figure 2). Fractional volume of inflammatory altered tissue in the liver was highest in individuals exposed to K¨orsch water followed by controls and fish exposed to Kr¨ahenbach water (Table 4). In loach from both exposure sites, as well as in control fish, a varying degree of glycogen depletion was observed. Moderate to extreme inflammatory fibrotic liver changes due to a manifestation of a nematode parasite (Raphitascaris) could also be detected in experimental fish as well as in controls. Stereological quantification of the volume fraction of altered liver tissue due to Raphitascaris demonstrated extreme individual variations relative to the degree of parasitic infections
in both control and exposed fish (Table 4). Occasionally, the percentage of parasitically affected tissue within the liver exceeded 20%. Gills As indicated by the mean assessment values for gill lesions (Figure 3), in the majority of the experiments these histopathological alterations in brown trout were most prominent in individuals exposed to the water of the higher polluted river. These lesions consisted primarily of focal proliferation of primary and secondary lamellae epithelial cells (Figure 7), occasionally resulting in a fusion of adjacent secondary lamellae. Furthermore, mucous cell hyperplasia and, occasionally, hydropic degeneration of respiratory epithelial cells could be observed (Figure 7). Slight hyperplastic changes were also found in a few control fish. Severe degenerative and necrotic changes of gill epithelia and inflammatory reactions could be detected at varying degrees in both test fish and controls from the July and September experiments. These changes were always
81
Figure 3. Mean assessment values (MAV) of gill alterations in brown trout and loach calculated per fish group on the basis of semiquantitative data. p < 0.05 ( K/A versus Control); data are given as means standard deviations.
accompanied by an extreme manifestation of the protozoan ectoparasite Ichthyophthirius multifiliis within the gill epithelia (Figure 7). As in brown trout, the histological appearance of gill tissue in exposed loach was characterized by focal hyperplasia of epithelial and mucous cells. To a minor degree, these findings could also be observed in a few control fish. In addition, a manifestation of the ciliate ectoparasite Trichodina sp. could be detected exclusively in test fish. The MAVs based on semiquantitative evaluation of both the frequency and severity of gill lesions and the degree of parasite manifestation did not show distinct differences between the fish exposed to the two polluted streams (Figure 3).
Discussion As part of a comprehensive research programme, this study emphasizes the use of histopathological investigations for assessing the effects of contami-
nated streams on the health of two fish species. The histopathological responses in brown trout clearly differentiate between fish exposed to the moderately polluted stream (Kr¨ahenbach) from those individuals of the more polluted river (K¨orsch). Organ lesions in control fish were restricted to slight background alterations within kidney, liver and gills, except for one group which demonstrated severe parasitic damage of the gills. These results are consistent with the findings of Triebskorn et al. (1997, this issue) who investigated relationships between stress protein levels and ultrastructural lesions in organs in fish under similar exposure regimes. In loach, histopathological findings did not reflect the different levels of contaminant loading between the two test streams. Furthermore, some morphological alterations were also observed in control individuals. However, differences were observed between controls and exposed fish with regard to the type and severity of lesions. For example, in controls, kidney lesions consisted of slight to moderate hyalin droplets
82
Figure 4. Renal tissue of brown trout (A) without pathological alterations, (B) showing an almost complete loss of renal tubules, necrotic changes and PKX-cells (arrow) due to Proliferative Kidney Disease; original magnification 480 , H&E stain.
within epithelial cells of the tubules, whereas in individuals exposed to both contaminated systems, severe tubulonephrotic changes were the predominant finding. In contrast, the degree of hepatic alterations due to the parasite Raphitascaris was similar in controls and exposed loach. The types of histopathological lesions observed in this study indicate that fish are responding to both direct toxicant effects of contaminated water and sediment and secondary stress effects caused by factors such as disease and parasitism. This later situation most likely occurs because contaminant stressors can weaken fish (Rice et al., 1996; Anderson & Zeeman, 1995) rendering them more susceptible to mortality from numerous causes (Shul’man, 1974). An example of direct toxic effects are tubulonephrotic changes which are known to be indicators of renal toxicity (Maxie, 1985). Tubular degeneration and dilation, proteinaceous casts and cellular debris within the tubular lumina are frequently observed as a consequence due to heavy metal exposure, e.g., cadmium and lead (Goyer, 1971; Gill et al., 1989; Goering et al., 1993). Tubulonephrotic alterations have also be induced by triazine herbicides (Fischer-Scherl et al., 1991). In brown trout exposed to the more polluted stream (K¨orsch), these nephrotic
lesions were not as severe as kidney alterations due to Proliferative Kidney Disease (PKD), which is induced by a myxosporean parasite (El-Matbouli et al., 1992). This later condition suggests that the immune system of the fish may have been compromised by contaminant exposure. Histopathological liver alterations in brown trout, such as a marked glycogen depletion and multiple foci of inflammation, were most pronounced in fish from the more contaminated system but were also found in individuals from the Kr¨ahenbach and in controls. Glycogen depletion as well as inflammation within the liver tissue has been reported also for redbreast sunfish collected from contaminated and reference sites. As also found in our studies, lesions were more severe in fish from the contaminated sites (Teh et al., 1997). A loss of glycogen within hepatocytes has been suggested to be an early toxic response (Hinton & Laur´en, 1990), induced in fish by various compounds such as organotins (Schwaiger et al., 1996). According to Thomas (1990), the glycogen depletion of hepatocytes observed in the present study could be interpreted as a nonspecific response to stress. Various adverse stimuli might induce endocrine changes leading to metabolic effects such as the reduction of glycogen deposits.
83
Figure 5. Renal tissue of loach (A) without pathological alterations, (B) showing severe vacuolization and hypertrophy of tubular epithelial cells (arrow), (C) showing distinct dilation of tubules (arrow), and accumulation of proteinaceous material within tubulus lumina (arrowhead); original magnification 300 , H&E stain (A,B), PAS stain (C).
A long-term mobilization of energy reserves in turn might contribute to an increased susceptibility to disease (Wedemeyer et al., 1976). In loach, no distinct differences could be observed between controls and exposed fish relative to glycogen content of hepatocytes. Furthermore, the similar frequency and severity of the liver alterations due to Raphitascaris in loach exposed to contaminated water and in controls leads to the assumption that the fish had already been infected by the parasite in their natural habitat before they were captured and subjected to our experiments. Gill lesions in brown trout and loach such as focal hyperplasia and slight degenerative changes of epithelial cells were in most cases more pronounced in individuals exposed to contaminated water than in controls. Hyperplasia of undifferentiated epithelial cells is known to be a chronic, non-specific alteration, which can be caused by a variety of unrelated insults (Hin-
ton & Laur´en, 1990) such as heavy metals (Randi et al., 1996), excessive ammonia (Wedemeyer et al., 1976), or protozoan ectoparasites (Rodgers & Gaines, 1975). Severe necrosis and inflammation of gill tissue, occasionally found in exposed brown trout and controls, was always associated with a manifestation of the ciliatic parasite Ichthyophthirius multifiliis. Gill alterations in loach were in general accompanied by a parasitic infection with Trichodina sp.. The fact that lesions caused by ectoparasites can often mimic alterations due to direct contaminant exposure (Hinton et al., 1992) indicates the importance of additional parasitological investigations. Studies which attempt to establish cause-and-effect relationships under field conditions are limited because of the complexity of polluted aquatic environments (Malins et al., 1988). The two streams investigated in this study can be distinguished with regard to chemical pollution. Furthermore, these systems are differentiated relative to limnological parameters such as mean annual temperature and organic loading. Regarding the histopathological alterations in brown trout from the K¨orsch, the relative high annual water temperature in this system and its possible interactions with water quality and the pathogenic responses of fish (Overstreet, 1988) might be of special aetiologic significance. For many parasitic diseases there is a conditional dependence on organic pollution (Svobodov´a et al., 1993), temperature, and other environmental factors. For example, outbreaks of Proliferative Kidney Disease are usually associated with high water temperatures and organic load of the water (El-Matbouli et al., 1992), and this situation usually occurs during the summer months in fish exposed to the highly contaminated stream (K¨orsch). In loach distinct differences relative to the quality and severity of lesions were only observed between controls and exposed fish. This species, in contrast to trout, lives in close contact to the sediment, the later in the K¨orsch system being more contaminated by heavy metals, PAHs, and pesticides than in the Kr¨ahenbach. Despite differences in contaminant loading between the two experimental streams no differences were observed between individuals from the two sites. Based on these findings it appears that loach might not be as sensitive an indicator species for water contamination as brown trout. One reason for this might be that loach were captured in the field and therefore may have been exposed to chemical toxicants prior to our experiments leading to adaptive mechanisms such as enhanced detoxification processes
84
Figure 6. Hepatic tissue of brown trout (A) without pathological alterations (note vacuolation of hepatocytes, characteristic for glycogen accumulation), (B) showing complete depletion of glycogen deposits and focal inflammation (arrow); original magnification 480 , H&E stain.
(Svobodov´a, 1993) or a stimulation of the immune system (Weeks et al., 1992). Another reason for the less sensitive histopathological response of loach could be that in comparison to brown trout, this cyprinid fish species is less susceptible to toxic effects of heavy metals such as cadmium (Norey et al., 1990), and in general can better tolerate high organic load and elevated temperatures of the water. The fact that the chronic sensitivity differs greatly among species and life stages (Overstreet, 1988; Geyer et al., 1993) as well as the origin of experimental fish groups should be taken into account when chosing appropriate indicator species for histopathological bioindicator studies. In the present study, morphometric analysis of major lesion types was performed to quantify the degree of alterations in exposed and non-exposed individuals. Stereologic methods should be included in histopathological studies to compare the frequency and severity of lesions in exposed fish and reference individuals. The use of morphometric and stereologic procedures to quantify morphological findings was also recommended by Hinton et al. (1992). Stereological analysis provides quantitative data on pathological findings and thereby helps to establish correlations or causal relationships with other biomarkers.
The assessment of the health status of both exposed and control fish by histopathological investigations proved to be important to avoid possible misinterpretation of various other biomarker responses. Several biochemical markers, for example, are usually analyzed within homogenates of entire organs without taking into consideration the amount of parasites or parasiteassociated lesions within the tissues analyzed. The fact that some types of histopathological lesions could also be detected in controls should also be considered. The background knowledge concerning the control condition is essential in interpretating biomarker responses in fish because for many of them, control values are used as reference. Histopathological alterations and biochemical markers such as stress proteins and metabolic enzymes generaly reflect stress conditions in fish induced by a broad range of environmental factors (Adams, 1990; Thomas, 1990; Hinton et al. 1992; Sanders, 1993). Our results indicate that in addition to contamination, the limno-chemical and physical characteristics of the two experimental streams as well as various infectious pathogens, may play an important role in the development and persistence of organ lesions. Because of the complex relationship of aquatic toxicants, natural
85
Figure 7. Gill tissue of brown trout (A) without pathological alterations, (B) showing focal proliferation (arrowhead), and (C) vacuolization of epithelial cells (arrowhead), (D) Ichthyophthirius multifiliis within the gill tissue (arrow); original magnification 480 , H&E stain.
environmental factors, and the occurrence of infectious pathogens, multidisciplinary approaches are required. In accordance with findings of other similar studies (Hinton & Laur´en, 1990; Hinton et al., 1992; Teh et al., 1997) histopathological investigations appear to be valuable tools to detect effects of various aetiologies and contribute to the understanding of the nature of stress responses at lower levels of biological organization.
Acknowledgements For excellent histotechnical assistance we are grateful to Renate Deffner and for maintaining the exposure systems we thank Michael Schramm. Special thanks to Gregor Lindermayr and Hermann Ferling for helping with the statistical analysis and the graphs of data. Parts of this study were presented at the Seventh Annual Meeting of SETAC-Europe, Amsterdam, The Netherlands, April 6–10, 1997.
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