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

Toxicity and histopathological effects of tributyltin chloride (TBT) were studied in early life stages of minnows Phoxinus phoxinus. Eggs and yolk sac fry (newly hatched larvae) were exposed in a static-renewal procedure to aqueous TBT concentrations ranging from 0.82 to 19.51 micrograms/L for 3 to 10 days at 16 degrees C and 21 degrees C, respectively. Aqueous TBT concentrations were determined by capillary GC-FPD and revealed a concentration decrease during the static phase. TBT exposure led to mortality, behavioral, gross morphological and histopathological effects. In larvae, increased mortality, deformation of body axis, paralysis and opaque eyes occurred at 4.26 micrograms/L TBT and higher both in the embryonic-larval and larval exposure. Histological changes were evident at initial TBT concentrations of 0.82 up to 19.51 micrograms/L, and were more pronounced after embryonic-larval exposure than after larval exposure. Degenerative alterations occurred in skin, skeletal muscle, kidney, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. Hydropic vacuolation of the cytoplasm and, in more pronounced cases, irreversible nuclear alterations such as pycnosis, karyorrhexis and karyolysis were also evident. Exposure to 0.82 micrograms/L TBT resulted in alterations in skin, muscle and kidney, with greater effects occurring at 21 degrees C than at 16 degrees C. Toxicity was significantly reduced in the presence of sediment. The observed histopathological effects suggest that early life stages of fish may be negatively affected in environments that are considerably polluted by TBT.

Using a static-renewal procedure, effects of triphenyltin chloride (TPT) on hatching, survival, and morphology were assessed in early life stages of European minnows Phoxinus phoxinus. Embryonic-larval exposure at 16 and 21 degrees C, and larval exposure at 16 degrees C were compared. In the embryonic-larval exposure at 16 degrees C, hatching was delayed and hatching success decreased at 15.9 micrograms/L. Mortality increased at > or = 3.9 micrograms/L TPT, and complete mortality occurred after 7 and 9 days at 15.9 and 5.1 micrograms/L, respectively. Mortality was higher at 21 degrees C that at 16 degrees C. Triphenyltin was more toxic to fish in larval stages. The induced effects were dose related, mortality increased at 1.8 microgram/L after 3 days, and was total after 5 days at 10.6 micrograms/L. In all high TPT exposures, larvae developed skeletal malformations (bent tails), showed impaired swimming behavior or paralysis, and eyes became opaque. Marked histopathological alterations were found. Degenerative hydropic vacuolation of the cytoplasm were evident in skeletal muscles, skin, kidneys, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. In severe cases, nuclear changes including pycnosis and karyorrhexis occurred. The observed toxicity of TPT was similar to that of tributyltin, but TPT acted more selectively on the lens and CNS, whereas other tissues were less affected. The study indicates that Phoxinus phoxinus larvae are negatively affected at peak TPT concentrations found in polluted environments.

Young rainbow trout were fed diets containing different combinations of ascorbylmonophosphate (vitamin C) and all-rae-alpha-tocophcryl acetate (vitamin E) for 31 weeks. The fish fed a diet deficient in both vitamins exhibited a high mortality and were anaemic after 8-12 weeks. Histopathological examination revealed a severe muscular dystrophy and splenic haemosiderosis. Fish fed a diet deficient in vitamin C but high in vitamin E developed the typical signs of vitamin C deficiency after 16-20 weeks, ineluding reduced growth rate, haemorrhages and gill alterations as well as severe deformations and fractures of the vertebral column. A diet deficient in vitamin E but high in vitamin C led to splenic haemosiderosis after 20 weeks. At the end of the experiment, this group showed signifieantly decreased haematocrit, haemoglobin content and red blood cell numbers and increased spontaneous erythrocyte haemolysis. In a seeond experiment, older tish of the same origin were fed diets eontaining high or low levels of ascorbyl-monophosphate and all-rae-alphatocopheryl acetate, respectively, for 23 weeks. The results were qualitatively the same as in the first experiment, but the onset of mortality in fish fed the diet deficient in both vitamins occurred later (weeks 18-23) and deformations of the vertebral eolumn were absent. Haematocrit. haemoglobin content and red blood eell numbers were significantly decreased in the group fed the diet deficient in both vitamins and erythrocyte haemolysis was increased in both groups receiving a vitamin E free diet, the diet deficient in both vitamins having a more pronounced effeet. The results of these two experiments indicate that there is an interrelation between vitamins C and E in rainbow trout. Possible mechanisms are discussed.

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Malachite green, the drug most commonly used against ichthyophthiriosis, is suspected to be carcinogenic and mutagenic. Therefore, the use of this d e is restricted or rohibited in several countries.Ichthyophthirius multifilis (tomont, cyst and tomite) was investigated: Amphotericin B, chloramphenicol, chlortetracycline, dimetridazole, formalin, furazolidone, oxytetracycline and sulfachlorpyrazine, and as controls malachite green and mixtures of malachite green and formalin. In vitro, Arnphothericin B, chlortetracycline, formalin and mixtures of malachite green and formalin showed the same potential as malachite green. Furazolidone inhibited the development of theronts but was relatively ineffective a ainst emerged theronts. Little or no parasiticidal effect could be attributed to chloramphenicol, iimetridazole, oxytetracline or sulfachlorpyrazine. The substances, which had proved all highly effective in vitro, were subsequently examined in vivo in a bath treatment and/or using medicated diets. Thereby successful treatment was achieved only with malachite green and mixtures of malachite green and formalin. All other substances were either ineffective or their use resulted in increased mortality ( formalin).

lmmunohistochem~stry and virus isolation were compared for their ability to detect viral haemorrhagic septicaemia vlrus (VHS) in experimentally ~nfected ralnbow trout Oncorhynchus mykiss.The f~s h were divided into 3 g]-oups (I to Ill), and infected, respectively, by bath challenge w~t h 102, 103 ', and 10"CID50 ml-l water of a VHS virus strain serologically sllnilar to reference straln F1 The cumulative mortality in Groups I to I11 was 44, 64, and 96%, respectively, at 14 d post infection (p.i.). Statistical comparison of the results from all groups showed that virus isolation was significantly more sensitive than im~nunohistochemistry ( p < 0.05). The same result was obtained by separate comparison of Groups 1 and 11, but there was no significant difference between the 2 methods for Group 111 (10' TCID;,,,). Immunohistochemically. virus antigens were detected early (2 to 4 d ) in endothelial cells linlng venules and sinusoids and in the haematopoietic cells in the head kidney, a s well as in interstitial macrophages and melanomacrophages; they were detected subsequently in hepatocytes (4 d p-i.) and exocrine pancreatic cells (6 d p i.). Presence of virus was accompanied by cell degeneration and necrosis from 4 d p.i. in all positive organs. These findings show that virus cultivation is the most sensitlve method for detection of VII-us, although immunohistochem~stry may represent an adjunct to diagnosis of acute VHS virus infections. The main advantage of ~mmunohistochemistry is the possibility of simultaneous demonstration of VII-us and morphological changes, making it a valuable tool for pathogenesis studies.