Diagnostics, Prevention and Therapy of Fish Diseases and Intoxications

DIAGNOSTICS, PREVENTION AND THERAPY OF FISH DISEASES AND INTOXICATIONS

RESEARCH INSTITUTE OF FISH CULTURE AND HYDROBIOLOGY VODŇANY CZECHOSLOVAKIA

 Manual for Internati International onal Training Course Course on Fresh-Wa Fresh-Water ter Fish Diseases Diseases and   Intoxications:  Intoxications: Diagnostics, Diagnostics, Prophylaxis Prophylaxis and Therapy Therapy

1991 Edited by: Z. Svobodová, B. Vykusová Research Institute of Fish Culture and Hydrobiology Vodňany, Czechoslovakia Vydal: Výzkumný ústav rybáský r ybáský a hydrobiologický, hydrobiologický, Vodňany. Rok Ro k vydání 1991. Redakce: MVDr. Zdeňka Svobodová, DrSc., Ing. Blanka Vykusová. Obálka: J. Vinklát. Tisk: AP Písek. ISBN 80-901087-0-9 Acknowledgements

The editors are grateful for effort donated by all co-authors who prepared this manual. Individual chapters of manual were compile co mpiledd by the following authors: Kolář P. Vladík P.

(2.1.1) (2.1.2)

State Veterinary Institute, Inst itute, České Budéjovice Dyková I. Ergens R. Lom J. Moravec F.  Našincová V. Scholz T.

(2.1.4) (2.1.4) (2.1.4) (2.1. 4) (2.1.4) (2.1.4) (2.1.4)

Parasitological Institute of o f Czechoslovak Academy of Sciences, České Budějovice Máchová J. Řehulka J. Svobodová Z. Tesarčík J. Vykusová B.

(3, 5) (2.1.3, 2.2.5) (1, 2.2.1, 2.2.2, 2.2.6, 3, 4, 5, 6) (2.2.1, 2.2.3, 2.2.4) (3, 6)

Research Institute of Fish Culture and Hydrobiology, Vodšany

RESEARCH INSTITUTE OF FISH CULTURE AND HYDROBIOLOGY VODŇANY CZECHOSLOVAKIA

 Manual for Internati International onal Training Course Course on Fresh-Wa Fresh-Water ter Fish Diseases Diseases and   Intoxications:  Intoxications: Diagnostics, Diagnostics, Prophylaxis Prophylaxis and Therapy Therapy

1991 Edited by: Z. Svobodová, B. Vykusová Research Institute of Fish Culture and Hydrobiology Vodňany, Czechoslovakia Vydal: Výzkumný ústav rybáský r ybáský a hydrobiologický, hydrobiologický, Vodňany. Rok Ro k vydání 1991. Redakce: MVDr. Zdeňka Svobodová, DrSc., Ing. Blanka Vykusová. Obálka: J. Vinklát. Tisk: AP Písek. ISBN 80-901087-0-9 Acknowledgements

The editors are grateful for effort donated by all co-authors who prepared this manual. Individual chapters of manual were compile co mpiledd by the following authors: Kolář P. Vladík P.

(2.1.1) (2.1.2)

State Veterinary Institute, Inst itute, České Budéjovice Dyková I. Ergens R. Lom J. Moravec F.  Našincová V. Scholz T.

(2.1.4) (2.1.4) (2.1.4) (2.1. 4) (2.1.4) (2.1.4) (2.1.4)

Parasitological Institute of o f Czechoslovak Academy of Sciences, České Budějovice Máchová J. Řehulka J. Svobodová Z. Tesarčík J. Vykusová B.

(3, 5) (2.1.3, 2.2.5) (1, 2.2.1, 2.2.2, 2.2.6, 3, 4, 5, 6) (2.2.1, 2.2.3, 2.2.4) (3, 6)

Research Institute of Fish Culture and Hydrobiology, Vodšany

Ruprich J. (4) Institute of Hygienic and E Epidemiology, pidemiology, Brno Hybková M. (5) Hygienic Station, České Budějovice Hejtmánek M. (5) University University of Chemistry and Technology, Technology, Praha Pr aha Last but not least, thanks are expressed to the staff who technically realized the manual: Z. Adámek, R. Berka, J. Máchová, M. Pečená, V. Sochor, and B. Vykusová.

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CONTENTS 1. Introduction 2. Fish diseases 2.1 Diagnostics of fish diseases 2.1.1 Viral diseases 2.1.2 Bacterial diseases 2.1.3 Fungal diseases 2.1.4 Parasitic diseases 2.2 Prevention and therapy of fish diseases 2.2.1 General principles of prevention 2.2.2 General principles of therapy 2.2.3 Viral diseases 2.2.4 Bacterial diseases 2.2.5 Fungal diseases 2.2.6 Parasitic diseases 3. Intoxications of fish 3.1 Aetiology of fish intoxications 3.2 Diagnostics of fish intoxications 3.3 Prevention of fish intoxications 4. Diseases of alimentary origin 5. Control of hygienic quality of fish from point of view of foreign substances content 6. Haematological examination of fish

1. INTRODUCTION At present, qualitative aspects of aquatic environment and within these the relating health conditions of fishes are the limiting factors of further development in freshwater fisheries and aquaculture. It is the reason why Food and Agriculture Organization of the United Nations focusses its attention on these problems. In 1991, FAO has offered to organize and international training course devoted to this field in Czechoslovakia. Working out and publishing a manual for participants of such course has been decided as an important integral part of the prepared course. The presented manual is primarily focussed on participants of the FAO International Training Course on Fresh-Water Fish Diseases and Intoxications: Diagnostics, Prophylaxis and Therapy. It is believed this manual can be utilized even by other  users engaged in the field of fish diseases and intoxications. Text of this manual was  prepared by top Czechoslovak authors who are world-wide known as experts in these fields. Manual doesn't substitute a scientific publication on this topic; it is only a basic informative compilation covering up-to-date knowledge in this field. The most of the text is devoted to disease diagnostics and intoxications of fish. Some methods of  haematological examination are included into diagnostic methods, too. Extensive part of text is devoted to basic principles of prevention and therapy of fish. Prevention of  diseases and intoxications is a basic presupposition for healthy environment and successful fish farming. This presupposition is created particularly through emphatic  prevention of surface waters pollution because there is very close relation between the quality of environment (quality of surface waters) and animal health. This is the reason why an extent text in manual is focussed on problems of surface waters  pollution and partly on quality of applied feeds, too. A quality of final product, i.e. the fish for human consumption, is closely connected with quality o f aquatic environment and of applied feeds. One chapter deals with these problems, i.e. hygienic and health acceptability of fish. Last but not least, the basic principles of fish therapy are  presented in manual, too. Also this field is an important part of the group of problems to be solved. In fact, it is the last opportunity of fish stock protection and in many cases the decisive opportunity.

2. FISH DISEASES 2.1 Diagnostics of Fish Diseases 2.1.1 Viral Diseases

(P. Kolář) Diagnostics of viral diseases in fish can be considered as falling in two major groups:  

 basic special

The basic methods include:   

clinical examination  patho-anatomic examination laboratory examination - isolation of viruses on cell cultures.

The special methods include:    

immunofluorescence methods (IF) immunoperoxidase method electron microscopy ELISA

Sending fish samples to laboratory for virological examination

Virological examination can be made in live fish only. Individual fish are chosen for  examination from among those which - still alive though with symptoms of disease gather close to the water surface or at the inlets or outlets. The water for the transport of the fish must be cooled with natural ice and, if possible, there should be a supply of  air or oxygen. If the live fish cannot be sent as described, the dead fish for the laboratory examination should be taken as early as possible and covered by green  plants; they may also be wrapped in wet towels or may be put into packages through which air should get inside (hence, not PVC). The fish should always be sent by messengers. Usually it is necessary also to take water samples according to pertinent instructions. In cases of infectious diseases the sample package usually contains 5–10 fish. Fish sampling for virological examination

The fish to be used for virological examination should preferably be alive. If the fish are dead there is much less chance to detect the virus agents. Aids and instruments for  the sampling must all be sterile. Samples are taken from 5–10 fish. In fry up to a size of 5 cm the whole body makes a sample, though, in fact, it seems better to remove the head and the tail just behind the anus after disinfecting the body surface by means of  70 % alcohol (the disinfection markedly reduces the possibility of bacterial

contamination). In the larger fish the body cavity is opened by incisions starting about 1 cm before the anus. The first incision is perpendicular to the lateral line and the other runs along the ventral line of the body cavity. A flap of body wall is thus  produced; hold it with pean tweezers or surgical forceps, lift it, and continue the incision along the lateral line and the ventral line up to the gill arches. Turn the whole flap and expose the organs. Disinfect the body surface with 70 % alcohol along the incisions. Parenchymatous organs are sampled for further examination, including the liver, spleen, fore and rear kidney; pyloric caecaes with the pancreas are also sampled in the case of trout. Also sampled are the eggs, sperm and sometimes the swim bladder. Isolation of viruses on cell cultures

Isolation of viruses on cell cultures is among the basic methods in studying and diagnosing the viral diseases of fish. Several stable cell lines have been prepared for  this purpose. These are: FMH - cell line from the caudal part of the minnow  Pimephales promelas RTG-2- cell line from the testes of the rainbow trout Oncorhynchus mykiss PG - cell line from the gonads of the pike  Esox lucius CHSE - cell line from the embryonal cells of the salmon Oncorhynchus tschawytscha Cell line cultivation

The media. All cell cultures are cultivated and maintained on media with 10 % of phoetal calf  serum (growth) and with 1 % of phoetal calf serum (maintenance). Composition: MEM Eagle Phoetal calf serum Sodium hydrogen carbonate 7.5 % NaHCO 3 Penicillin-potassium salt + streptomycin 500 I.U. +500 mg Amphotericin B

Growth medium Maintenance medium 90.0 ml 10.0 ml 1.0 ml (2.0)

99.0 ml 1.0 ml 1.0 ml (2.0)

1.0 ml

1.0 ml

0.1 – 0.2 ml up to 0.5 ml

0.1 – 0.2 ml up to 0.5 ml

Every batch of the phoetal calf serum must be tested for cytotoxicity. The store solution of amphotericin is prepared by diluting the content of one phial (50 mg) in 50 ml of distilled water. The media are replaced if the following criteria are met:

a. when the cells have produced a compact monolayer: the growth medium is replaced by the maintenance medium,  b. when there is a great decrease in the pH, assessed by the colour of the medium, c. when fungal contamination occurs and the Roux flask cannot be directly eliminated. If the cell lines are maintained in thermostats at temperatures specific of each line, it will suffice to replace the media after 1 to 10 days. After about 30 days the lines must  be trypsinated again. Trypsination The Difco certified trypsin 1:250 (Detroit, Michigan USA) is used for the trypsination. For use it is prepared at a 0.16 % concentration in combination with 0.02 % versene in PBS (pH 7.2–7.4). Store solution of trypsin 10.0 % Trypsin Difco 10.0 g Glucose 5.0 g PBS up to 100 ml All is stirred in a beaker with a magnet for 10 minutes, using an solenoid stirrer. Then the mixture is centrifuged at 2000–3000 r.p.m., the supernatant is filtered and frozen. Store solution of versene (Chelaton 3): Sodium chloride Pottasium chloride Sodium hydrophosphate Na 2HPO4 Potassium dihydrophosphate KH 2PO4 Distilled water Chelaton III

8.0 g 0.4 g 0.06 g 0.06 g to 1000 ml 0.2 g

After dissolving, sterilize the solution in autoclave at 1.5 atm for 30 min. For  trypsination, always prepare a fresh solution by mixing 25.0 ml of store solution of  versene - 0.4 ml of store solution of trypsin. Trypsination procedure Maintain the trypsinating solution at 20°C. After sucking off the growth medium or  maintenance medium, use the MEM Earle solution with 1 % NaHCO 3 and antibiotics (100 I.U. penicillin and 100 μg streptomycin in 100 ml of the cell rinsing medium) to rinse the cell growth in the Roux flask. After rinsing pour the rinsings off and cover  the cell monolayer with the trypsinating solution. Leave the cells in direct contact with the trypsin for 30 to 90 seconds (depending on cell quality and type) and pour the solution off. To inspect the process of further “dry” trypsination, watch the Roux flask  against light (the growth inside will have a milky colouring) or under inversion microscope (cytoplasmic membranes will become distinct and cells will successively round). Having shaken the cells from the walls of the Roux flask by patting the flask 

against the hand, resuspend them in 5 ml of growth medium and loosen them by careful pipetting. Counting the cells. To count the cells in the suspension, put 1 ml of suspension into a test tube, add one drop of 0.5 % crystal violet and stirr for 2 min. Take one drop of this volume and put it in the Bürger cell. Count only the healthy unstained cells. Conversion formula: number of cells in 1 large square (arithmetic mean for 10 squares) × 10 000 × amount of stained cell suspension (in the actual case 1).

The Roux flasks for cultivation of the parental line are normally stocked at a rate of  150–250 thousand cells per 1 ml of medium the Leighton or Müller tubes for infection with the tested suspensions are stocked with 300–400 thousand cells per 1 ml. It is necessary that the monolayer should grow over the whole area of the tube in 24–72 hours. Technique of cultivating cell cultures with the fish tissue suspension being examined

1/ Use a mortar to homogenize the material being examined together with some sea sand. Resuspend the homogenate by means of isotonic phosphate buffer (PBS pH 7.3) or in serum-free culture medium (Earle, Eagle MEM-TM SEVAC VI) at a dilution ratio of 1:10. Earle solution in ten-fold concentration: Sodium chloride NaCl Potassium chloride KCl Magnesium sulphate MgSO4.7H2O Sodium phosphate NaH2PO4.2H2O Unhydrous calcium, chloride CaCl 2 (may be replaced by CaCl 2.6H2O

68.0 g 4.0 g 2.04 g 1.5 g 2.0 g 3.94 g

- add the calcium chloride to the solution when all the above mentioned chemicals are completely dissolved, or preferably dissolve it separately in distilled water and add to the solution) Glucose 10.0 g Phenol red. 0.4 % 15.0 ml Distilled water to a volume of 1000 ml Filter the solution through filter paper. Add 2.0 ml chloroform p.a. to the whole volume and store the solution at 4°C. Sterilize by filtering. Autoclave the diluted working solutions at 1 atm for 20 min. Phosphate buffer (PBS), pH 7.2–7.4: Sodium chloride NaCl 9.2 g Sodium hydrophosphate 1.2 g

 Na2HPO4.12H2O Sodium dihydrophosphate  Na2PO4.H2O Distilled water

0.115 g to a volume of 1000 ml

2/ Centrifuge the suspension prepared in this way (3000 r.p.m., 10 min, 20°C). 3/ Pour the supernatant into a test tube, add an antibiotic solution (1000 i.u. penicillin,  potassium salt and 1000 μg streptomycin). Thoroughly shake the test tube. Check  sterility on bacteriological media. After five minutes of exposure, dilute part of the starting material (10 %) ten times, using PBS, to get a final dilution ratio of 1:100 in another test tube. The prepared suspensions can be frozen at minus 15°C to minus 25°C and stored frozen until they are examined. 4/ Put each inoculum diluted 1:10 and 1:100 (volume of 0.1–0.2 ml per 1 test tube) into three test tubes with the grown cell cultures 24 to 48 h old; before doing that,  pour off the growth medium with 10 % of phoetal calf serum and rinse the grown test tube cell culture twice with a serum-free medium containing 1 % NaHCO 3. 5/ Leave the absorption to go on for 30–60 min at 17 – 22°C. 6/ Having poured the inoculum off, use serum-free medium with NaHCO 3 to rinse the test tubes with initial suspension diluted 1:10 and 1:100 and cover the cell cultures in the tubes with 1–2 ml of medium containing 1% phoetal calf serum. 7/ Maintain the infected cultures at a temperature of 15°C for 5–6 days. Inspect the test tubes every day. 8/ If the cytopathic effect (CPE) occurs, identify the virus by immunofluorescence methods (IF), immunoperoxidase test of ELISA. 9/ If no CPE occurs or if the CPE cannot be safely determined, perform two successive passages with both concentrations after prior freezing and thawing of the suspension. Watch for 5–6 days. 10/ If CPE occurs during the watching period, identify the virus by methods chosen from among the following: IF, ELISA, electron microscopy, immunoperoxidase test. Immunofluorescence methods (IF)

Microscope slides sized to fit into the Leighton test tubes are used for IF diagnosis. The number of cells in the cultures on the slides is the same as in the virus isolations, i.e. 300–400 thousand in 1 ml of medium. Having poured off the growth medium, rinse the slide cell cultures twice in a routine way and infect them with the material  being examined, 0.1 ml of the material being applied to each culture. Leave the adsorption to continue for 45 hours and then add 1.5 ml of maintenance medium to the test tubes. Always take the second or third passage for IF examination. Stop the infection in the test tubes when the first visible changes occur on the cell monolayer. 1/ Indirect IF technique for antigen detection

Carefully remove the infected slides from the Leighton tubes and mark them with the code of the sample being examined. Rinse them in IF buffer, dry them thoroughly at laboratory temperature and fix them in cooled acetylene at a refrigerator temperature (+4°C) for 10 minutes. In this stage the preparation can be interrupted and the slides may be maintained at minus 20°C until examination. In the moist chamber, cover the slides with a specific rabbit hyperimmune serum against the respective virus, the serum having been diluted according to box titration tested in advance. Leave the reaction of fixation of the antigen with the antibody to go on for 30 minutes. The temperature of this reaction depends on the characteristics of  the virus under study. Then pour the hyperimmune serum off, rinse the slides and dip them in IF buffer three times, 10 minutes each time. Remove the slides from the  buffer, cover them with a layer of SwAR/FITC conjugate (swine antibody against the FITC specific rabbit serum) and leave them so for 30 minutes. Then rinse the slides again and dip them three times in IF buffer, 10 minutes each time. Without drying, apply onto them 1 drop of glycerine (9 parts of IF buffer + 1 part of glycerine). Put a micro cover slip on top of the slide. Watch the sample under IF microscope at a wavelength of 510 nm, using the G 247 filter (Fluoval IF microscope, Carl Zeiss Jena, Germany) 2/ Direct IF technique for antigen detection The technique differs from the indirect IF in that the FITC is directly fixed to the specific hyperimmune serum against the respective virus. Generally it is claimed that the direct IF has a lower sensitivity and specificity. In both the direct and indirect IF the preparations can be examined immediately or can  be stored in the refrigerator to examine them the following day. Buffer for IF  NaH2PO4.H2O (or KH2PO4)  Na2HPO4.12H2O  NaCl Distilled water

0.39 g 2.69 g 8.5 g ad 1000 ml

Preparing the hyperimmune serum To have a sufficient amount of the virus with which the hyperimmune serum is  prepared, propagate the serum in 6 passages on the respective cell line on which the greatest cytopathic effect is recorded. Freeze and thaw the virus suspension several times, then centrifuge it at 6000 r.p.m. for 10 minutes in a precooled centrifuge. Check the suspernatant by the IF method and administer it to 3 rabbits. Immunization scheme: 1st treatment: 3 ml of mixture of virus and complete Freund's adjuvant aa, divided for  i.m. administration at several sites.

2nd treatment: 2 ml of mixture administered i.m. 3 weeks later. 3rd treatment: 1 ml of virus supernatant administered i.v. three weeks after the 2nd treatment. Collect blood by intracardial punction into test tubes the following week. Leave the  blood to coagulate, separate the blood clot from the walls of the cells, cut the coagulum, and centrifuge the blood at 2000 r.p.m. for 20 minutes. Pipette the serum, distribute it in 2 ml amounts into ampoules and, if necessary, store at minus 25°C for  long. The quality of the serum should be tested by box IF titration. Immunoperoxidase method

Viruses can also be detected in the infected cell cultures by the direct immunoperoxidase method. It is an advantage of this method that no immunofluorescence microscope is needed: an optical microscope will easily do the job. The principle of the test, whose practical application depends on the producer, is the reaction of the enzymatic constituent of the conjugate, i.e. peroxidase + the rabbit antibodies against the respective virus, with the substrate. In the positive case, when there is a strong bond between the antigen (virus) and the enzyme-labelled antibody, this bond is visualized by the staining agent, which reacts with the enzyme. The infected cells have clusters of dark brown granules in their cytoplasm. The colour  intensity and the number of positive cells are directly proportional to the length of  incubation and to the infectivity of the initial organ suspension. Electron microscopy

Electron microscopy can be used with advantage when there is a need for rapid tentative detection of viruses from isolates on cell cultures and for classification of the viruses in different virus groups. 1. Method of negative staining - is successful at a virus concentration of about 1.10 5 TCID in infected cell culture: Centrifuge 0.5–0.1 ml of cultivation medium from infected cell culture at 1000 r.p.m. for 10 minutes. Apply a drop of supernatant on the electron-microscope screen, coated in advance with the formvar film. Suck the drop off with filter paper after several seconds and apply the staining agent (2 % ammonium molybdate) onto the screen. Suck the staining agent off and examine the screen under an EM. 2. Method of ultrathin slices

- not suitable for rapid detection but provides perfect material for documentation: Pour the cultivation medium off the infected cell culture, fix the culture by a layer of  3 % glutaraldehyde in PBS (pH 7.2) for 1 hour. Scrape the cells off the glass, centrifuge them and fix them with 1 % OsO 4. Dehydrate the cells in acetone in a decreasing series of 100 %, 90 %, 60 %, 30 % for  30 minutes to 1 hour (depending on the size of the pellet). Encast the cells in Durkupan ACM. Contrast the ultrathin slices by means of a solution of uranyl acetate and citrate. 3. Immune electron microscopy method - a method of higher sensitivity than the negative staining method. Inactive the respective hyperimmune rabbit serum at 56°C for 30 minutes before use and dilute it in PBS 1:20, 1:40, 1:80, 1:100, 1:400. Mix each of these dilutions with the same volume of the sample being examined and incubate the mixture at 37°C for  1–1.5 hours. After incubation, apply a drop of the sample onto the electron microscope screen coated in advance with a formvar film. Remove the sample drop  by means of filter paper after several seconds and apply a drop of staining agent (2% ammonium molybdate) onto the screen. Suck the staining agent off and examine the sample under EM: evaluate the presence and size of virus clusters (immunocomplexes). ELISA

This method is used a. for determination of the amount of antibodies and/or presence of antibodies  b. for determination of the amount of antigen and/or presence of antigen This is only a description of the principle. Its practical application depends on the  producers of the kits. Indirect ELISA for antibody detection

Double antibody sandwich ELISA technique to detect antigens (viruses)

1/Antigen is adsorbed onto bottom of the pit

1/Antibodies are adsorbed onto bottom of the pit

2/Fixing the specific antibodies onto adsorbed antigen

2/Fixing the antigen onto adsorbed antibodies

3/Fixing the enzymelabelled antiglobulin

3/Fixing the specific enzymelabelled antibodies against

4/Adding substrate

4/Adding substrate

The positive reaction will manifest itself in the colour of the pit: colour intensity is directly proportional to the amount of the antibodies or antigen present there. Important viral diseases of freshwater fishes

There are about 50 fish diseases that are regarded as being of virus origin. Seventyfive percent of these diseases affect freshwater fishes, twenty-five percent the marine fishes. Fish viruses fall within the following families:

ds-DNA-pelleted ds-DNA-naked es-RNA-naked es-RNA-pelleted ds-RNA-naked

-

 Herpesviridae  Iridoviridae  Adenoviridae Caliciviridae  Rhabdoviridae

Orthomyxoviridae  Birnaviridae

Rheoviridae

The following viral diseases cause significant economic losses in intensive fish culture: Channel catfish virus (CCV)

Virus of the family  Herpesviridae, causing acute disease in the channel catfish ( Ictalurus punctatus). The young channel catfish are killed by the disease. The virus is endemically spread in the U.S.A. Diagnosis: a. clinical: the affected fish are lethargic, show bilateral exophthalmus, markedly dark body colour, pale gills, haemorrhages in the skin and on fin bases;  b.  patho-anatomic: changes as above plus haemorrhages in the liver and kidneys; ascites of the body cavity;

c. laboratory: direct isolation of virus from organ suspension on cell lines coming from the catfish (cell line BB). Incubation at 30°C. Positive finding manifests itself as early as 16 hours after infection by the formation of  syncytia and by successive lysis of the cells. Infectious pancreatic necrosis (IPN)

The IPN virus ( Birnaviridae) is responsible for a highly contagious disease affecting the fry and the young fish (not older than 20 weeks). The losses may amount up to 100 %. The condition usually has an acute course with a high mortality. In the older  fish the course of the disease is usually inapparent. The disease occurs in Europe,  North America and Asia. Diagnosis: a. clinical: dark colour of the body, especially the dorsal part, arched fore part of   body, haemorrhages in the skin and on fin bases;  b.  patho-anatomic: changes as above plus haemorrhages in the pyloric region, the intestine without food is filled with yellow catarrhal exudate. Adult specimens are usually free of patho-anatomic changes. Such fish constitute a significant group of virus carriers; c. laboratory: direct: virus isolation and identification in acute stage of  infection, indirect: detection of antibodies. Stable cell lines RTG-2 and PG, electron microscopy, immunofluorescence, immunoperoxidase test and ELISA are the best for the isolation and identification of  the virus. The IPN virus exerts a marked cytopathic effect on the cell lines. Antibodies can be detected by ELISA. Spring viraemia of carp (SVC)

This disease affects carp. Its clinical manifestation is most marked in spring at water  temperatures below 15°C. The mortality may be up to 90 % under experimental conditions. Poor state of nourishment of the fish and decreased immunity worsen the course of the disease. The disease occurs in Europe. Diagnosis: a. clinical: dark colour of the dorsal region, incoordination of motion, ataxia, slowed respiration, intermittent muscular twitchings. Enlarged and reddened  belly (ascites) is a typical sign. The anal papilla is swollen, often discharging  pseudofaeces. Bilateral exophthalmus, haemorrhages on the skin and gills;  b.  patho-anatomic: changes as above plus marked petechial haemorrhages on the heart, kidneys, in the caudal part of the swim bladder and/or in muscle. The internal organs are usually oedematous and the gills are anaemic. Fish with inapparent clinical infection become virus vectors;

c.

laboratory: direct: virus isolation indirect: detection of antibodies.

and

identification

The causative agent of the disease,  Rhabdovirus carpio, belongs to the group of  Vesiculoviruses . It readily propagates on the FHM and CHSE stable cell lines. It exerts a marked cytopathic effect. The virus can be detected by immunofluorescence, electron microscopy or by the immunoperoxidase test. Antibodies can be detected by ELISA. Viral haemorrhagic septicaemia (VHS)

An acute disease of salmonids. It is endemically spread in Europe. Its causative agent is a virus of the family Rhabdoviridae, subgroup  Lyssaviruses. The mortality is up to 80 %. Diagnosis: a. clinical: there are three forms of the course of the disease. Fish may be affected by any of them separately or by all of them in succession. These are: acute form with necroses in the haematopoetic organs (kidneys, pancreas), chronical form characterized by apathy, anaemia, exophthalmus, and nerve form with ataxia, swimming in spiral, high mortality.  b.  patho-anatomic: in the acute stage of the disease the pathognomic findings include haemorrhages on the palate of the oral cavity, on fin bases and on the gills, and also in the skeletal muscles, on the serosae, swim bladder, visceral fatty tissue, in the heart and sometimes in the liver. The findings are recorded on the background of haemorrhagic diathesis; in the chronic stage of the disease the important signs include a very dark body colour, exophthalmus, anaemic gills, and petechial haemorrhages dispersed over the organs of the abdominal cavity; in the nerve stage, besides the exophthalmus and dark body colour, there is a marked swelling of the kidneys; c. laboratory: direct: virus isolation and identification. The VHS virus readily propagates on the RTG-2, CHSE, PG and FHM cell lines at 15°C. Immunofluorescence techniques can be used for the identification. Infectious haematopoetic necrosis (IHN)

This is an acute virus disease whose causative agent belongs to the family  Rhabdoviridae. The disease affects young populations of salmonids with an up to 100  percent mortality. IHN occurs in North America and Japan. Diagnosis: a. clinical: an abrupt increase in mortality is a typical sign. The fish alternate lethargy with excess spontaneous activity. In the final stage the fish have a marked dark colour, show bilateral exophthalmus and ascites of the body

cavity, and secrete strings of pseudofaeces. There are haemorrhages on the gills and fin bases;  b.  patho-anatomic: besides the changes described above, there are petechial haemorrhages in the haematopoetic organs, which are anaemic. The intestine is usually free of digested food. Necroses are visible in the spleen and kidney. Pathognomic necroses can be seen in the stratum compactum of the digestive tract. Older fish are more resistant and individuals with clinically inapparent infection become virus carriers; c. laboratory: direct: virus isolation and identification. IHN virus readily propagates on the FHM, RTG-2 and CHSE cell lines at temperatures between 13 and 18°C. CPE manifests itself as rounded cells and lysis. The virus is identified by means of immunofluorescence. Recommended literature Ahne W., Wolf K. (1980): Viruskrankheiten der Fische. In: Reichenbach-Klinke H.H. Krankheiten and Schädigungen der Fische. Gustav Fischer Verlag, Stuttgart,  New York, 56–106. Hornych M., Pospíšil Z., Tománek J. (1986): Diagnostics of IPN in trout by immunofluorescence. Med. Vet., Praha, 31, 55–64 (In Czech). Pospíšil Z., et al. (1986): Isolation and identifications of IPN virus in trout. Med. Vet., Praha, 31, 103–112 (In Czech). Pospíšil Z., et al. (1987): Epizootology of IPN in salmonids. Veterinářstvi, 37, 489–  490 (In Czech). Pospíšil Z., Tománek J. et al. (1991): Diagnosis and prevention of infectious  pancreatic necrosis in salmonids. Research Institute of Fish Culture and Hydrobiology, Vodňany, pp. 15. Rodák L., et al. (1986): Diagnostics of spring viraemia of carp by ELISA method. Med.Vet., Praha, 31, 505–512 (In Czech). Rodák L., et al. (1988): Enzyme linked immunosorbent assay detection infection  pancreatic necrosis virus. Fish. Dis., 11, 225–235. Tománek J., et al. (1988): Causes of swimming bladder inflammation in carp. Veterinářství, 38, 76–77, (In Czech). 2.1.2 Bacterial Diseases

(P. Vladík) Most of the bacteria associated with fish diseases are naturally saprophytic organisms, widely distributed in the aquatic environment. Comparatively few species are classified as true obligate pathogens. Both groups of organisms may be present on the external body surface or in the tissues of apparently healthy fish and their pathogenic role will only manifest itself as a consequence of stress.

Fish presented for bacteriological examination fall into two categories (diseased fish, healthy fish), although the dividing line is not always obvious. If clinically diseased fish are subjected to bacterial examination, the effort is focused either on the detection of the causative agent in external lesions such as dermal changes, changes in the gills and so on, or on the examination of the internal organs, especially kidneys, if there is suspicion of a septicaemic disease. If the presence of a specific germ (e.g. vectorship) is to be eliminated or the bacteriological examination is performed for another reason, such bacteriological examination is mainly focused on the kidneys and/or other   parenchymatous tissues. Methods of examination

1/ Wet preparations These are useful for the rapid presumptive diagnosis of myxobacterial infections, including columnaris, cold water or bacterial gill diseases. Myxobacteria ( Flexibacter  spp.) can be seen as long slender rods exhibiting a characteristic flexile motility. 2/ Stained smears Gram, Giemsa and Ziehl-Neelsen stains are commonly used. Though the detection of   bacteria in tissues may be conducive to presumptive diagnosis, confirmation by culture is required in the majority of cases, only in the case of diagnosis of bacterial kidney disease (BKD) in salmonid fish are stained smears used for diagnosis. The  presence of small, strongly Gram-positive bacilli in kidney smears is considered diagnostic for infection with Renibacterium salmoninarum. 3/ Isolation of bacteria There are essentially two major approaches: the use of selective media or the use of  non-selective media. Our laboratory prefers the non-selective media because such a method of bacteriological examination provides a more complete picture of the  bacterial status of the fish. The majority of the bacterial pathogens require no special media for isolation, Tryptone Soya Agar (TSA) and the Blood Agar (BA) being regarded as sufficient; in some cases we can also use other media, e.g. Endo Agar. The exception are  Myxobacteria, which require comparatively low levels of both nutrients and agar. Cytophaga Agar (ANACKER, ORDAL 1959), composed of (in g  per litre of distiled water) tryptone 0.5, sodium acetate 0.2, beef extract 0.2 and agar  0.9, provides a suitable medium for the isolation of freshwater strains.  Renibacterium  salmoninarum is an extremely fastidious organism. For its isolation there exist a number of modifications of the ORDAL, EARP (1956) medium. Commonly used is the KDM-2 medium after EVELYN (1977), which contains (in g per litre of distilled water) peptone 10, yeast extract 0.5, cysteine HCl 1.0, agar 15.0; it is supplemented with foetal calf serum (10 % v/w).  Mycobacteria spp. can be isolated on any of the glycerol or egg-based media commonly used for this group of organisms (e.g. the Lowenstein-Jensen medium, Dorset medium etc.). Incubation temperatures of 18–  23°C are generally used for isolation and visible growth may be expected to appear  within 48 hrs in most cultures.  R. salmoninarum may require 6 weeks and  Mycobacteria 3 weeks.

Myxobacterial infections

Although the term  Myxobacteria is commonly used to describe the group of gliding  bacteria associated with a number of fish diseases, all the pathogens belong to the genus  Flexibacter . The organisms can be recognized by the characteristic yellow, spreading colonies and gliding motility on low nutrient media (ANACKER, ORDAL 1959). Further identification is seldom required because it would need a special  bacteriological techniques.  Flexibacter columnaris is pathogenic only at water  temperatures above 15°C.  Flexibacter psychrophila causes disease at temperatures  below 12°C. Aeromonas infections

Aeromonas infections of freshwater fish may, in the essence, be divided into those caused by the motile species (septicaemia) and those caused by the non-motile species (furunculosis, carp erythrodermatitis). Present characteristics of the genus Cells straigth, rod shaped with rounded ends to coccoid. Gram-negative, generally motile (Table 1), only one species is nonmotile (Table 2). Facultative anaerobes. Metabolism of glucose is both respiratory and fermentative. Table 1: Differentiation between motile aeromonads  A.hydrophila  A.caviae  A.sobria

Esculin hydrolysis Growth in KCN broth L-histidine, L-arginine

+ + +

+ + +

-

+ + + + + +

+ + + -

+ + d + +

Fermentation of:

Salicin Sucrose Manitol Acetoin from glucose Gas from glucose H2S from cysteine

Oxidase positive. Catalase positive. Resistant to vibriostatic agent 0/129. Optimum temperature range 22–28°C.

Table 2: Differentiation between non-motile subspecies of  A. salmonicida

Brown pigment Indol production Esculin hydrolysis Growth in KCN broth L-histidine, L-agrinine

subssp.

subssp.

subssp.

 salmonicida

achromogenes

masoucid 

+ + -

+ -

+ + -

+

-

+

d + + -

d + -

d + + + + +

Utilization of:

L-arabinosa Fermentation of: Salicin Sucrose Manitol Acetoin from glucose Gas from glucose H2S from cysteine Pseudomonas infections

 Pseudomonas fluorescens and  Pseudomonas putida are considered as casual

 pathogens responsible for bacterial septicaemia of fish, which as a rule cannot be distinguished from aeromonas-induced septicaemia occurring in fish stocks exposed to different stresses. Present characteristic of the genus (Table 3) Straight or slightly curved rods. Aerobic, with a strictly respiratory type of  metabolism with oxygen as the terminal electron acceptor; in some cases nitrate can  be used as an alternative electron acceptor. Oxidase and catalase positive. The fish  pathogens belong to the group of rRNA group I.

Table 3: General characteristics of species 1–5 of  Pseudomonas (sec.I)  Ps. aeruginosa  Ps. fluorescens

Pyocyanin production Pyoverdin production Oxidase Levan from glucose Gelatin liquefaction Starch hydrolysis Lecithinase Growth at 4 °C Growth at 41 °C Denitrification Arginine dihydrolase

+ +/+ + + + +

+/+ + + + + + +

 Ps. putida

+ + + +

Edwardsiella tarda and Edwardsiella ictaluri infections

 Edwardsiella tarda causes gas-filled malodorous lesions in the muscle tissue of  channel catfish (MEYER, BULLOCK 1973).  Edwardsiella ictaluri causes intestinal

disease in the catfish (HAWKE 1979). Table 4: Differentiation within the genus Edwardsiella  E. tarda

Indole production H2S production (TSI) Motility Malonate utilization Fermentation of: D-manitol Sucrose Trehalose L-arabinose Tetrathionate Reduction

most strains biogroup 1 + +

 E. hoshinae  E. ictaluri

-

-

+

-

-

-

+ -

+ -

+ +

-

-

+ + +

+ + + -

-

+

-

-

-

Present characteristics the genus (Table 4) Motile peritrichously flagellated rods, not encapsulated. Gram negative, facultative anaerobes. Oxidase negative. Catalase positive. Nitrates are not reduced.

Fermentation of glucose with gas. H 2S is produced abudantly from TSI agar, but  poorly producing biotypes may occur. Indole is produced. Manitol is not fermented by most strains. Enteric redmouth disease (Yersinia ruckeri infection)

Enteric redmouth is an economic ally important disease of salmonid fish especially in  North America. Present characteristics of the genus (Table 5) Straigth rods to coccobacilli. Endospores are not formed. Gram negative. Non-motile at 37°C, but motile with peritrichous flagella when grown below 30°C, except for  Y.  pestis which is always non-motile. Facultatively anaerobic, having both respiratory and fermentative type of metabolism. Oxidase negative, catalase positive. Nitrate is reduced to nitrite. Glucose and other carbohydrates are fermented with acid  production but little or no gas. Phenotype characteristics are often temperaturedependent and a greater number of characteristics are usually expressed by cultures incubated at 25–29°C rather than at 35–37°C. Table 5: General characteristics of the species of Yersinia

Indol production Methyl red Voges-Proskauer Simmons citrate Urea hydrolysis Lysin decarboxylase Arginin dihydrolase Ornitine decarboxylase D-glucose/acid/ Fermentation of: Sucrose Lactose Manitol

1 +/+ + + +

2 + + + + +

3 + + + + +

4 + + + +

5 + + +

6 + + +

7 + +/+ +

+ +

+ +

+ +

+

+

+

+

1 - Y. enterocolitica, 2 - Y. frederiksenii, 3 - Y. intermedia, 4 - Y. kristensenii, 5 - Y. pestis, 6 - Y. pseudotuberculosis, 7 - Y. ruckeri.

Vibrio infections

Vibrio anguillarum is a pathogen of marine fish and eels and a major cause of disease

in fish culture. Several other species not properly described are also pathogenic for  fish. Present characteristics of the genus Gram negative straigth or curved rods. Motile. Do not form endospores. Chemoorganothrops and facultative anaerobes capable of respiratory and fermentative metabolism. Most are oxidase positive. All utilize D-glucose as a sole principal source of carbon and energy. Most species require 2–3 % NaCl or seawater base for optimum growth. Characteristics of Vibrio parahaemolyticus: Gram negative curved rods. Endospores not formed. Swarming on solid complex media negative. Non-pigmented. Arginin dihydrolase negative. Oxidase positive. Reduction of nitrate positive. Not producing gas from glucose. Voges-Proskauer  (acetoin production) negative. Growing at 40°C. Sucrose not fermented. D-gluconate  positive. Bacterial kidney disease (R. salmoninarum infection)

 Renibacterium salmoninarum is an obligate pathogen of the subfamily Salmoninae (salmon, trout and char) of the family Salmonidae, which produces a slowly

developing chronic infection characterized by enlarged gray-white necrotic abscesses  primarily in (but not restricted to) the kidney. The bacterium belongs to the group of regular, non-sporing Gram negative rods (Section 14, Bergey s Manual 1986). Present characteristics of the genus Short Gram-positive rods. Non-capsulated. Non-motile. Endospores not formed. Aerobic. No acid production from sugars. Catalase positive. Oxidase negative. Grows very slowly at 5 and 22°C. No growth at 37°C. Cysteine required for growth. Streptococcal fish infections

A less common bacterial disease of fish. HOSHINA et al. (1958) isolated Streptococcus faecalis from dying juvenile rainbow trout. Streptococcal sepsis, accompanied by exopthalmus (the so-called pop-eye) in rainbow trout was described  by BARHAM et al. (1979). Streptococcal infections in the channel catfish were described by PLUMB et al. (1974). Characteristics of the genus Cells spherical or ovoid, occurring in pairs or chains when grown in liquid media. Endospores are not formed. Gram positive. Most are facultatively anaerobic. Catalase

negative. Metabolism is fermentative. Growth on solid media is enhanced by addition of blood, serum or glucose. Serological identificatin is important for determination of haemolytic streptococci. This is a method of classification suitable for use in animals and humans. On the other  hand, many so-called viridizing and non-haemolytic streptococci lack the existing group antigens, so they are very difficult to classify. Streptococcus faecalis belongs to Lancefield serological group D, whose latest

denomination is Enterococci (Table 6). Table 6: Differentiation between enterococci S. faecalis S. faecium

Hydrolysis of esculine Ammonia from arginine Reduction of potassium tellurit Fermentation of: L-arabinose Melecitose Melibiose Sorbitol Sorbose

S. avium

S. gallinarum

+ +

+ +

+ -

+ d

+

d

d

-

+ + -

d + d +

+ + d + +

+ d + + -

Mycobacterial infections of fish

Mycobacterial infections (mycobacterioses) have so far been observed in more than 150 species of marine and freswater fish (NIGRELLI, VOGEL 1963). Glycerol or egg  based media, commonly used for these group of organism, serve for their isolation.  Mycobacterium fortuitum and  Mycobacterium marinum are isolated most commonly. Identification of Mycobacteria is best carried out by a specialist laboratory. Characteristics of the genus Slightly curved or straight rods, mycelium-like growth may occur but on slight disturbance it usually becomes fragmented into rods or coccoid elements. Acidalcohol fast. Non-motile. Growth slow. Easily visible colonies produced in 5 days to 3 weeks. Incubation temperature for fish pathogens 25°C. Identification requires special  bacteriological techniques. Recommended literature Anacker R.L., Ordal E.J. (1959): Studies on the myxobacterium Chondrococcus columnaris. I Serological typing. J. Bacteriol., 78, 25–32.

Barham W.T., Schoonbee H., Smit G.L. (1979): The occurence of Aeromonas and Streptococcus in rainbow trout, Salmo gairdneri Richardson. J. Fish Biol., 15, 457–460. Evelyn T.P.T. (1977): An improved growth medium for the Kidney Disease  bacterium and some notes on using the medium. Bulletin de l'Office International des Epizooties. 87, 511–513. Hawke J.P. (1979): A bacterium associated with disease of pond cultured channel catfish, Ictalurus punctatus. J. Fish. Res. Bd. Can., 36, 1508–1512. Hoshina T., Sano T., Morimoto Y.A. (1958): A streptococcus pathogenic to fish. J. Tokyo Univ. Fish, 44, pp. 57. Krieg N.R., Holt J.G. (ed.) (1984): Bergey's Manual of Systematic Bacteriology. Vol. 1. Meyer F.P., Bullock G.L. (1973): Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus punctatus). Appl. Microbiol., 25, 155–156.  Nigrelli R.F., Vogel H. (1963): Spontaneous tuberculosis in fishes and other cold blooded vertebrates with special reference to Mycobacterium fortuitum Cruz from fish and human lesions. Zoologica, New York, 48, 130–143. Ordal E.J., Earp B.J. (1956): Cultivation and transmission of etiological agent of  kidney disease in salmonid fishes. Proc. Soc. Exp. Biol. Med. 92, 85–88. Plumb J.A. et al. (1974): Streptococcus sp. from marine fishes along the Alabama and northwest Florida coast of the Gulf of Mexico. Trans. Amer. Fish. Soc., 103, 358–361. Sneath P.H.A, Mair N.S., Sharpe M.E., Holt J.G. (ed.) (1986): Bergey's Manual of  Systematic Bacteriology. Vol. 2, pp. 1599.. 2.1.3. Fungal Diseases

(J. Řehulka) Moulds, which cause mycoses, are microscopic organisms producing filamentous coatings on various substrates. Basically, moulds are fungi, which are heterotropic organisms nutritionally dependent on organic substrate. They are classified as a separate phyllum, Mycota, of thalloid plants (Thallophyta ). It is still widely believed that mould infestation of fishes is largely a secondary  phenomenon. However, there is evidence that fungi may affect healthy fish in certain circumstances. From the viewpoint of the fish pathologist, there are mycoses that hinder the function of organs and kill the fish on mass and also mycoses that successively deprive the fish body of its natural strength, bringing it up to a cachectic state.

I. Determining moulds and filamentous fungi parasitizing freshwater and marine fishes: laboratory methods

1. Methods of isolation Mycological examination is an integral part of the complex of examinations of health of the fish and is done more or less simultaneously with the bacteriological examination. Semiparasitic and parasitic fungi are obtained fresh from the host by taking an inoculum from the suspect tissue onto agar medium, using a scorched and cooled snare or dissecting needle. If mycelium is present in the tissue, the sample can  be surface-disinfected to prevent secondary contamination by airborne spores. The disinfection is done by dipping in 1% formaldehyde for 1 to 5 minutes. Then the sample is transferred to 70% alcohol and finally to sterile water in which it is throughly rinsed. When isolating an actual species it is also possible to hold the sample under a fast stream of water to wash the secondary infection off. 2. Methods of cultivation a) Cultivation The purpose of cultivation is to obtain pure cultures for studying the fungus thalli, their variability and conditions of growth, and for investigating the whole ontogenesis. The cultivation is usually done on synthetic agar media. By the cultivation, an individual is left to produce its progeny which is then regarded as a cultured strain. Differences between strains of the same species are physiological (e.g.  production of enzymes or toxins) a may also be morphological (e.g. different colours, the appearance of the fungal growth etc.). On the culture medium the strain produces monospore or multispore colonies which fuse to form a growth. A colony consists of  surface and substrate mycelium and these forms may differ on different substrates.  b) Culture media Besides the common bacterial culture media such as the blood agar or Ordal's agar,  parasitic fish fungi can also be cultivated on media of natural substrates. Of these, we recommend the sweet wort agar (1000 ml of wort diluted to half the concentration, 20 g of agar), and of the synthetic media were recommend Sabouraud's agar (40 g glucose, 10 g peptone, 1000 ml distilled water, 15 g agar), Sabouraud's preserving agar for collection cultures (30 g peptone, 1000 ml distilled water, 20 g agar) or the Czapek agar (3 g NaNO3, 1 g KH2PO4, 0.5 g KCl, 0.5 g MgSO 4.7H2O, 0.01 g FeSO4.7H2O, 30 g saccharose, 15 g agar, 1000 ml distilled water). These media are suitable for morphological study of the mycelium and the sporulation organs. Lownutrient media are suitable for maintaining the strains in collection cultures. c) Inoculation Perfect cleanness must be maintained in the rooms where the inoculation is  performed. The table on which the work is done should be wiped with a cloth soaked in a disinfectant (ajatin); 4% formaldehyde or 3% chloramin with detergent should be used when the work is finished (preferably before leaving the lab). When the Petri dishes are to be opened, the lids should be lifted with care. Small containers (test

tubes) are opened when necessary just for a short while, the necks of the containers should be heated before and after transferring the inoculum, and the cotton wool stoppers should be singed on surface. Straight or bent hollow dissecting needles are used for reinoculation of moulds and filamentous fungi. The spores must be transferred together with the mycelium to avoid sterility of the strain. The inoculated test tubes and Petri dishes are put in thermostat at a room temperature. d) Preserving the cultures To continuously maintain the fungus cultures, reinoculate them twice or three times annually. The fast growing species need reinoculation every other month, others once in 4–6 months. Low temperatures (4–10°C) slow down growth and allow the cultures to survive up to a year without reinoculation. Abundantly sporulating fungi are reinoculated by transferring the spores with a snare. In the case of fungi with poor  sporulation, culture cutouts with agar are transferred. Bent inoculating needles made of a thick wire are suitable for this purpose. 3) Methods of preparation Moulds and filamentous fungi should first be evaluated for their general habit by examination under binocular lens or, just slightly enlarged, under a microscope,  preferably with phase contrast. Preparations that serve for detailed study may be native, semipermanent or permanent. a) Native preparations are examined in a drop of water. They are stained with either  an aqueous solution of neutral red (dilution 0.01 %) or methylene blue (0.01 %). These dyes stain the plasm. To stain the cell wall, use aqueous solution of cresyl blue or a solution of Congo red in ammonia (3 g Congo red per 100 ml H 2O plus an addition of 2 ml ammonia).  b) Chloral hydrate, lactophenol or lactofuchsin are used for better clarification of the object. Lactophenol is the best: methylene blue is added to it to enhance its colour, and part of mycelium with the reproduction organs is transferred to. it by means of a dissecting needle. The preparation treated in this way is covered with a micro cover  slip and is carefully heated over moderate flame to let the air bubble out; during this it is carefully compressed with the dissecting needle. Lactophenol composition: pure crystalline phenol 20 g, lactic acid 20 g, glycerol 40 g, distilled water 20 g. The  preparation is stored in a brown flask. c) Glycerol gelatine after Kaiser is used for the permanent preparations: distilled water 42 ml, glycerol 30 ml, gelatine 7 g, phenol 1 g. Acetone lacquer or synthetic lacquer can be used for the framing. DuNoyer's cement is also suitable (heat 20 g anhydrous lanolin over a sand bath for 30 min, then continue the heating while mixing the lanolin with 80 g rosin which is added in lumps). A thick wire bent into a triangle can be used to apply the cement: the wire, heated to a high temperature, is laid onto the surface of the cement which melts and can be easily applied onto the edges of the cover slip.

II. The special part Oomycetosis

Affections caused by fungi of the Oomycetes class are among the most widely known and most widespread mycoses of fishes. The characteristic trait by which the Oomycetes are distinguished from other classes of fungi is the presence of motile spores (zoospores) with two flagella (feather and smooth), which develop in the zoosporangia. In a large part of  Oomycetes this is the main mode of nonsexual reproduction; the other modes are through chlamydospores and gemmae. Union of the nuclei of immobile gametes produces a thick-walled spore (oospore). Oomycetes also differ from the majority of fungi by having cellulose in their walls. Besides that, most of the Oomycetes have a diploid nucleus when they are in a vegetative stage. All Oomycetes produce filaments called hyphae which, unlike in the higher fungi, have a limited number of septa. A grouping of hyphae makes a mycelium. The Oomycetes class breaks into four orders:  Legenidiales, Perenosporales,  Leptomitales and Saprolegniales . Of these, the genus  Pythium of the order   Perenosporales, the genus  Leptomitus of the order  Leptomitales and eight genera of  the order  Saprolegniales (Achlya, Aphhanomyces, Calyptralegnia, Dictyuchus,  Leptolegnia, Pythiopsis, Saprolegnia, Thraustotheca ) are considered to be parasites of  fishes. The most widespread species parasitizing fish include representatives of the genera Achlya and Saprolegnia , which will be treated in detail below. The species of the genus Saprolegnia (Fig. 1) have two kinds of zoospores. The  primary zoospore, having the shape of a grain with flagella on the end, encysts short after leaving the zoosporangium. Then the cyst may die and produce a mycelium or  form a secondary kidney-shaped zoospore with lateral flagella: the feathery one will  point forward and the smooth one will point back. The secondary zoospore lives longer and also forms a cyst. The cyst may proliferate owing to mycelium formation, or may produce a secondary zoospore.

Fig. 1: Developmental cycle in Saprolegnia: 1 - mycelium, 2 - gemmae, 3 zoosporogenesis, 4 - zoosporangia, 5 - primary zoospore, 6 - encysted spora, 7 - secondary zoospore, 8 - proliferating zoospore, 9 - oosporogenesis, 10 meiosis, 11 - antheridium, 12 - antheridial cell, 13 - oogonium, 14 - oospore There are no freely flowing primary zoospores in the fungi of the genus  Achlya; secondary oocysts emerge from the cyst and appear at the sporangium opening. In species of the genus  Aphanomyces the cysts are produced in the same way as in  Achlya but unlike the latter, the former have much thinner hyphae and the zoospores in their zoosporangium are arranged in a single line. Fish infestation by Saprolegnia , saprolegniasis, occurs in all types of waters all over  the world and is one of the most widespread diseases of inland fishes both in ponds and in water courses. The disease breaks out where pathogenous strains are present: absence of saprolegniasis is ascribed to the absence of pathogenous strains. The spores most easily and most frequently penetrate into the fish body when surface of  the skin or gills is damaged (mechanically or by a parasitary or bacterial infection) and the fish is weak, unable to produce substances that would protect it. In salmonids the susceptibility to saprolegniasis is induced by exposure to stress: stress raises the corticosteroid level in the blood plasma, thus suppressing the inflammatory reaction and boosting protein catabolism, regulated by the corticosteroids. In the final stage, this results in protein deficit, which in turn is conducive to atrophy of the skeletal muscles and suppression of collagen synthesis. Lack of collagen leads to poor  regeneration of lesions on the skin. The Pacific salmon has a high level of  corticosteroids in the blood plasma when it migrates and reaches sexual maturity.

Laboratory diagnosis of the order  Saprolegniales is performed after routine isolation on common agar media (blood agar, Ordal's agar). A bacterium-free pure fungus culture is obtained by the method of repeated tapping, preferably onto an agar  medium with pea extract or onto sweet wort agar. The species are best identified in hemp water after incubating the culture at 18–20°C. Another procedure that can be used is to take a piece of mycelium from the diseased fish or eggs and put it in sterile Petri dishes with sterile distilled water where sterilized halved hemp seeds are added. The fungi start growing two to three days later. Washing and transfer in sterile water  are repeated many times to successively obtain a more or less bacterium-free macro culture. The fungus species are then diagnosed according to the morphology of the hyphae and the reproductive organs. The saprolegnia are fixed by means of a mixture of 90 % alcohol (94 parts) and 40 % formaldehyde (6 parts). Depending on the degree of infestation, the affected fish stop taking food, lose their  escaping reflex and stay close to the shore. They carry white-grey woll-like fungus growths of different sizes, prominent on their body surface. New growths may be difficult to distinguish; the older ones are usually grey-green. The transparent mycelium with a large amount of sporangia can be seen under the microscope. The genus can be identified according to the morphology of the sporangia. When the fungus is removed the primary injury appears underneath. Its margins are dark red and in an advanced stage of the disease the area under the fungal growth is necrotic. The form and location of the fungus may be specific, e.g. in the Atlantic salmon affected  by UDN (head); in some fishes it may depend on the sex or may occur with Staff's disease, which sometimes affects the olfactory pits. A reliable histological determination of the fungus hyphae is by staining after Grocott and by the PAS method. Ten percent neutral formol would suffice to fix the tissue for the histological examination. The inflammatory response is surprisingly weak in saprolegniosis: muscle is infiltrated by the lymphocytes and histiocytes and there are proliferated monocytes in the blood; the inflammatory cellulization may be completely absent. Differential diagnosis is necessary in the early stage of the disease when the symptoms may be the same as those of pox. Branchiomycosis

Branchiomycosis is a much feared fungal disease of fishes almost all over the world, especially on carp farms. The disease occurs most frequently in the warm climatic regions. Branchiomycosis occurs in two types: 1.  branchiomycosis of carp and/or tench, crucian carp and sticklebacks (causative agent: Branchiomyces sanguinis). 2.  branchiomycosis of pike and tench (causative agent:  Branchiomyces demigrans). The two species can be distinguished from each other by their morphological traits and by peculiarities of their development. These can be characterized as follows:

 Branchiomyces sanguinis (Fig. 2) is generally located in the blood vessels of the gill

arch and gill filaments. The diameter of the hyphae is 8–20 μm, the thickness of the hypha wall is 0.2 μm and the diameter of the spore is 5–9 μm.  Branchiomyces demigrans produces hyphae which are able to penetrate into the gill

filaments and spread on their surface. The diameter of the hyphae usually is 13–14 μm and may be up to 22–28 μm at the end of the hypha. The thickness of the wall is 0.5–0.7 μm and the diameter of the spore is 12–17 μm.  B. demigrans differs from B. sanguinis by having thicker-walled hyphae and by being

able to proliferate from the blood wessels to the adjacent tissue of the gills. The spectrum of hosts harbouring these fungi has recently been extended and includes also the salmonids. Laboratory diagnosis is based on the examination of the gill filaments by the compression method. Fungal hyphae are visible in the blood vessels of the gills at a 150-fold magnification under the microscope. Spores can also be identified when the diseases are in its acute stage: tear the affected tissue into pieces on the microscope slide and examine it in a drop of 50 % glycerol solution in water or alcohol. The  branchiomycetes are also well discernible in preparations from dead fish bodies decomposed by rot. For isolation and cultivation, scrape part of the affected tissue on a watchglass with water, pour it into a centrifuging test tube with water and centrifuge for 3 minutes. Pour the supernatant out of the test tube, pour new distilled water in and centrifuge again. Wash the material again until the liquid is clear. Then use a  pipette to transfer the sediment onto the microscope slide and, under the microscope, separate the hyphae of the mycelium from the rest of the tissue. Transfer the mycelium again into the test tube with water and centrifuge it. Put the washed hyphae in a 2 % formaldehyde solution, wash in water again, sow them on the Sabouraud's medium, 0.5 % semiliquid agar or blood agar and cultivate them at 20–22°C.

Fig. 2: Branchiomycosis of fish: A - branching hyphae of the fungus Branchiomyces sanguinis, B - fungus hyphae spreading in the branchial blood circulation, C -

congested and poor-supply areas arise on the gill filaments, D - parts of the gill filaments necrotize, E - necrotic the gill filaments fall The rise and course of the disease depend on factors that underline them; water  temperature is one of the factors that play the most important part. The disease breaks out most frequently when the water temperature is above 20°C. The infection comes via the gills where the spores are intercepted. The digestive tract is another infectious route. The spores produce hyphae which proliferate in the  branchial blood vessels. The disease may take a peracute course which is very rapid and leads to the blocking of a number of blood vessels: the fish die of suffocation. In the case of an acute course of the disease, necrotic processes take place in the gills, the blocking of the blood vessels is not so extensive but when the water is poor in oxygen the fish may die on mass. The chronic stage of the disease is characterized by the shedding of the necrotic parts of the gill filaments and by a regeneration process accompanied by hyperplasia of the stratified cuboidal epithelium. In the early stages of the disease, dark red strips can be seen on the gill filaments when the fish are examined; these strips signal the blocking of the blood vessels by the hyphae of the fungi. Later these strips are dirty grey. In an advanced stage of the disease the gill tissue disintegrates and the necrotic parts are shed; this is accompanied  by secondary development of the saprolegnia. The fish killed by the disease are usually found dead in the morning. The fish that withstood the disease have gaps in their gill filaments. Differential diagnosis should be performed to eliminate sanguinicolosis,  branchionecrosis and intoxications caused by environmental factors. Ichthyophonosis

This genus comprises two species:  Ichthyophonus hoferi and  I. gasterophilum. From the viewpoint of pathogenicity,  I. hoferi (Fig. 3) is more important as the causative agent of ichthyophonosis (ichthyosporidiosis). The disease has until now been diagnosed in more than 80 fishes among which sea fishes prevail. Most of the affected freshwater fishes belong to the salmonids. Microscopic appearence of the organism is dependent on its stage of development. The stages include (1) spore at “resting” stage, (2) germinating spore, (3) hyphal stage. It is believed that there are two forms of  Ichthyophonus, both belonging to one genus. One of them is known as the “salmon” form, occuring in freshwater and cold preferring sea fishes: this form is characterized by its ability to produce long tubulose germ hyphae. The other is called the “aquarium fish” form, typical of the tropical freshwater fishes. This form is completely devoid of hyphae.

Fig. 3: Developmental cycle of Ichthyophonus hoferi: 1–5 - development of  “daughter” spores, 7–11 - development of resting spore from the “daughter” spore, 12–19 - development of resting spore by fragmentation For mycological diagnosis in the lab, the affected organs should be cultivated on gelatine or in fish broth. After cultivation the fungus germinates in 1 to 7 days. Fine filaments free of septa with conical ends are sent forth: the conical ends, filled with protoplasm, become round and are throttled off to produce globose or cystose formations. In old cultures, large resting-stage formations (usually unable to germinate in artificial media) appear  on the ends of the filaments. In 60 days the fungus culture produces in the medium a growth 5–12 mm in size. The fungus grows at temperatures between 3 and 20°C, ten degrees being the optimum. The disease is introduced in the fish culture environment by the latently sick fish: they may shed the fungus from the attacked skin or the infection may spread from their  urinary tract if their kidneys are invaded. Decaying bodies of the fish killed by the disease may also be a copious source of infection. The infection is contracted via the digestive tract. The clinical signs depend on the intensity of invasion of the different organs. Rainbow trout shows inappetence and inco-ordination of motion in swimming in the early stages; later the flanks darken and finally the dark colour spreads over the whole body. Sometimes the fins disintegrate and may fall off: the affected fins are frayed and white-rimed. If the liver, kidney and spleen are invaded, they enlarge and so does the whole belly of the fish; the eyes often bulge and erode.

The symptoms that accompany severe infection of the kidneys and liver include exophthalmus, distended scales and accumulation of exudate in the body cavity. If the skin is invaded, ulcers develop. No organ is safe from the disease; generally it can be said that tissues abundantly supplied with blood are most vulnerable (heart, spleen). The organisms elicit a severe focal granulomatous response which replaces much of  the organ it occupies. Masses of epithelioid cells surround the developing and germinating spores. The whole granuloma is bounded by a thin connective tissue capsule. Foreign body giant cells may rarely be present. In differential diagnosis, ichthyophonosis must be distinguished from bacterial diseases accompanied by a chronic proliferative granulomatous response - especially from the mycobacteriosis of aquarium fishes. Imprints of the affeced tissue or  histological slices are stained after Ziehl-Neelsen: if acid resistant rods are identified, the ailment is mycobacteriosis (tuberculosis). In a certain stage the disease may look  like myxosporidiosis, bacterial disintegration of the fins or deficit of vitamin C and tryptophan. In cases of dermal defects in salmonids, bacteriological or histological examination should be performed to exclude haemophilosis and furunculosis. Moniliales

 Moniliales, also known under their former name,  Hyphomycetes - philamentous fungi,  belong to the group of imperfect fungi ( Fungi imperfecti).

Most of the  Moniliales are outside the range of potential parasites of fishes, though in recent years researchers have studied them with increased attention from the point of  view of their possible primary pathogenicity. Some species, especially  Exophiala  salmonis, Exophiala pisciphila and Ochroconis tschawytschae, are particularly suspected in this respect. These fungi can be generally characterized by their forming either sterile mycelium or mycelium with conidiophores on which conidia (spores developing in nonsexual reproduction) are produced. They are classified by morphology, colour and distribution of the spores and hyphae and morphology, colour  and distribution of the spores and hyphae and also by the mode of conidium  production by conidiogenous cells. The genus Exophiala Fungi of this genus produce conidia on the end of the conidiogenous cells called anellide. The important fish pathogens of this genus include  E. salmonis and  E.  pisciphila.  E. salmonis (Fig. 4) produces grey-brown colonies 5–8 mm in size on the Czapek-

Dox agar at 25°C in 14 days. It does not grow at 37°C. The anellideconidia are yellow cinnamon in colour and cylindrical in shape, with a rounded distal end and a flat outgrowth on the proximal end. The number of septa is 0 to 2. The septated conidia are 3x11 μm in size and the size of the nonseptated conidia is 3x5 μm. The sporulation is good on grain and maize agar and/ or completely absent on the Sabouraud agar.

 E. pisciphila (Fig. 5) produces dark grey to dark olive colonies, 25–28 mm in size,

under the same cultivation conditions on potato agar with dextrose and on the grain agar. Like  E. salmonis, it fails to grow at 37°C. The anellideconidia are yellow cinamon in colour and round in shape with a rounded distal end and cut outgrowth. The septated conidia are 2×5 μm in size. Sporulation is good on potato agar with dextrose and grain agar and bad on the Sabouraud's agar.

Fig. 4: Exophiala salmonis: A-C - conidial apparatus, D - conidia  E. salmonis and  E. pisciphila can be isolated with success on the above-mentioned

media and also on the Ordal's and blood agars. The best medium for their cultivation is sweet wort agar. The epizootics caused by  E. salmonis, recorded in Salvelinus namaycush, showed symptoms such as ataxia, whirling, exophthalmus and led to death.

Fig 5.: Exophiala pisciphila: A-C - conidial apparatus, D - conidia, various strains On histological examination, there was chronic granulomatous response with foreign  body giant cells. The pathogenic action of  E. pisciphila was demonstrated in a number  of hosts, especially in aquarium fishes. The pathogen invades all internal organs,  particularly the swim bladder. The sick fish refuse food, keep aloof, stagger, and the final symptoms before death include ascites, distended scales and exophthalm us. Differential diagnosis should exclude mycobacteriosis. The genus Ochroconis Species of this genus produce ellipsoid and cylindrical conidia with wide rounded ends. Ochroconis tschawytschae was isolated from two fishes and also from the kidneys of  Oncorhynchus kisutch yearlings. Olive-coloured colonies 30 to 40 mm accross will

grow on potato agar with dextrose at 25°C in three weeks. As distinct from other  species of the genus Ochroconis tschawytschae produces small needle-like colonies with three septa ovoid in shape. The genus Fusarium  Fusarium tricinctum (Fig. 6): Aerial mycelium sparse to floccose, white becoming

carmine red to purple on the surface of the agar. Microconidia ovate to pyriform with a minute apiculum; some later become 1 septate. Microconidia are formed initially from simple lateral conidiophores bearing one or two simple cylindrical to subulate  phialides. Macroconidia are falcate or more strongly curved and with a well marked foot cell. Sporodochia are pale to orange and formed of a plectenchymatous stroma covered with a palisade of short profusely branched conidiophores each branch of  which terminates in 2–3 phialides. Chlamydospores are intercalary, singly or in chains, or occasionally terminal on short lateral branches.

Fig. 6: Fusarium tricinctum: Conidia and conidiophores This species has recently been isolated from the swim bladder of an intensively farmed rainbow trout. The fish invaded by this fungus swim with difficulty because their swim bladders are filled with a clear or slightly haemorrhagic exudate. Coelomycetosis

This class is characterized by their conidia's being produced in several types of  fruiting bodies, chief among which are picnidia, acervuli and stromata.  Phoma herbarum produces hyaline unseptated conidia 4.5 x 2.5 μm in size, oval to

cylindrical in shape, in single or compound picnidia. Cultivation on artificial media must be performed in order to identify the species. All standard media will do, including their variations. Ailment caused by this fungus has so far been recorded in the fishes of the genus Oncorhynchus and occurs mainly in specimens up to an age of 100 days. The infection starts in the swim bladder, which is ascribed to the fact that the disease is contracted by the fry during the first filling of the bladder: spores may get in together  with the air. In cases of spontaneous infection the fish have an enlarged anus with haemorrhages and their belly is laterally constricted. Petechial haemorrhages can also  be seen on the caudal peduncle and in the dorsal and ventral part of the body. The invaded fish usually swim on their side with their caudal fin bent down. The yearlings' swim bladder is filled with a liquid and older specimens have a liquid also in their  stomach. In the early stage of the infection, mycelium of  P. herbarum can be found inside the swim bladder. The mycelium may later completely fill the swim bladder  and proliferate into the internal organs.

In conclusion it should be noted that the aetiological spectrum of the diseases of the swim bladder of salmonids includes another six fungus species which have been isolated recently. These are  Acremonium koliense, Tolypocladium inflatum, Alternaria consortiale, Fusarium avenaceum, Volutella salmonis, Pyrenochaeta accicola

Recommended literature Amlacher E. (1986) : Taaschenbuch der Fischkrankheiten. VEB Gustav Fischer  Verlag Jena.  Neish G.A., Hughes G.C. (1990) : Fungal diseases of fishes. T.F.H. Publications, Inc., Ltd. Wolke R.E. (1975) : Pathology of bacterial and fungal diseases affecting fish. In: The Pathology of Fishes (ed by W. E. Ribelin and G. Mikagi), pp, 33–116. The University of Wisconsin Press, Madison, Wisconsin. 2.1.4 Parasitic Diseases A) PARASITIC PROTOZOA

(J. Lom, I. Dyková) 1. Introduction into the study of protozoan parasites

Discovered already by Leeuwenhoek in late 17th century, protozoa count today about 50 000 species, living in water and soil habitats and also as parasites of animals. Many of them are serious pathogens and several are known as true pests in freshwater  or marine aquaculture and in breeding of ornamental fishes. Protozoa represent a vast assemblage of essentially unicellular organisms, in which a single cell is capable of all life functions executed in higher organisms by special organs. In the system of living organisms they belong to the kingdom  Protista. Protozoa grouped into several phyla,  Flagellates (phylum  Mastigophora), Opalines (Opalinata) ,  Amoebae (Rhizopoda), Coccidia and  Piroplasmia (Apicomplexa),  Microsporidia (Microspora), Myxosporidia (Myxozoa) and Ciliates (Ciliophora) include agents of serious diseases of fishes. This chapter will supply a short overwiev on protozoans adapted to the life inside or  on the surface of the body of fishes. Emphasis is laid on presentation of most important representatives of various groups of protozoa infecting fishes and on their  differentiation. More detailed information on taxonomy, biology and relations to the host can be found in special monographs (see references). Examining fish for protozoan parasites includes inspection of blood; external surface (skin, fins, nasal pits and gills); internal organs (liver, spleen, kidney, gonads, brain, heart, swimbladder and its rete mirabile, gall and urinary bladders, digestive tract and their content) and musculature.

For an exact, reliable determination of the protozoans, in addition to fresh mounts, and histological sections, special techniques must be applied. They comprise selected staining and silver impregnation methods (Giemsa, Klein), nuclear staining (Feulgen),  photomicrography of fresh and stained material and special techniques for  characterizing of myxosporean spores. In order to reveal the true prevalence and intensities of infections fish must be examined as soon as possible after capture. Careful handling during collection and transport minimizes the loss of ectoparasites. To obtain first overall picture of the  protozoan fauna the fish sample should comprise at least 10 specimens. 2. Practical key for the determination of fish protozoa in fresh material

A.

Protozoa can be detected as macroscopical whitish aggregations from tiny dots to cyst-like structures of several mm or even cm in size. On the skin, or gills, or on the internal organs or in their tissue. Determination is only possible by use of a compound microscope.

A.1.

Protozoa visible as tiny dots on the body surface and gills. Under the microscope the dot proves to be one or several large (up to 1 mm) slowly rotating cells, uniformly covered with synchronously beating cilia. Next to large ones, there may be small ones of different size. Their cytoplasm is full of granules and contains a large central macronucleus .............................. Ciliates of the genera  Ichthyophthirius (freshwater) of Cryptocarion (marine)

A.2.

The dot-, nodule-, or cyst-like structures are composed of a mass of  small, uniform, refractile bodies (spores or oocysts).

A.2.a

The spores, typically 7–20 μm in size, most commonly have 2 (some genera possess 1, 4, 5, 6 or 7) refractile bodies (polar capsules) at their anterior pole ..............................  Myxosporea The spores, typically 3–10 μm in size, are usually ellipsoidal or  oviform and have a faintly visible vacuole at their posterior end .............................. Microsporidia The spores, typically 3–7 μm in size, are spherical and contain a large centrally located globular refractile inclusion .............................. Dermocystidium

A.2.b

A.2.c

A.2.d

The protozoa are spherical or ellipsoidal bodies of about 10–20 μm size, each containing four ellipsoidal bodies each of which contains 2 slender cells. Whitish nodules within the body organs are not sharply delimited .............................. Coccidian oocysts

B. B.1.

Protozoa detectable only under the compound microscope. Protozoa infecting the surface (skin, fins, nasal pits, or gills).

B.1.a

Protozoa that move.

B.1.a.1 Cells up to 15 μm in size, possessing 2 flagella, moving with jerky, creeping motion or swimming spirally forward .............................. flagellates (Cryptobia, Ichthyobodo)

B.1.a.2 Cells 20 μm and larger, either covered uniformly with cilia or with only several ciliary belts or circular ciliary wreaths. They move directly forward, glide over the surface, or roll on the spot .............................. Ciliates B.1.a.3 Cells with amoeboid movement and changes of body shape (about 20 μm in size) ..............................  Amoebae B.1.b Sessile or motionless protozoa attached to the surface. B.1.b.1 Small transparent, pyriform cells not exceeding 15 μm in size .............................. Attached  Ichthyobodo B.1.b.2 Pyriform or sac like cells, 30–300 μm in size, their cytoplasm yellowish or greenish and containing many refractile granules .............................. Parasitic dinoflagellates B.1.b.3 Rounded cells 40–60 μm in size, with rows of cilia; with a transparent cystic envelope .............................. Encysted ciliates (e.g., Amphileptus) B.1.b.4 Cells 40–100 μm in size, with cytoplasm dark due to refractile granules, and with bundles of tubules with knoblike ends protruding from their surface .............................. Suctorian ciliates B.1.b.5 Goblet-like or cylindrical cells of about 40–90 μm in length, each with a wide free end encircled by wreaths of beating cilia. The cells may contract a little .............................. Sessiline peritriches B.1.b.6  Protozoa of 40–60 μm in size, hidden within shells which often have an ornamental surface pattern .............................. Thecate amoebae B.2.  Protozoa infecting the lumen of the intestine and/or the tissue of the intestinal wall. B.2.a  Myxosporea (see A.2.a); Microsporidia (see A.2.b); coccidian oocysts (see A.2.d), which are as a rule encased with an amorphous mass of yellowish materia (yellow body); or amoebae .............................. (see B.1.a.3) B.2.b Cells up to 15 μm in size, with up to 8 flagella, moving about with a  jerky motion or swimming directly forward .............................. Flagellates B.2.c

Spindle-shaped cells, of about 30–140 μm in size, uniformly covered with cilia, with both ends pointed and with sluggish movement .............................. Protoopalina B.2.d Ciliated cells of another shape, up to about 120 μm in length .............................. Ciliates B.3.  Protozoa infecting the urinary tract (from renal tubules of kidney to urinary bladder). B.3.a Plasmodial cells, 10 μm - 5 mm in size, with a variety of cell inclusions and surface outgrowths. .............................. Myxosporean trophozoites, mostly with spores (see A.2.a) B.3.b  Microsporidia (see A.2.b)

B.3.c

Motile cells of 40–120 μm in size, with ciliary wreaths .............................. .............................. Endocommensal Endocommensal trichodinids t richodinids

B.4.

 Protozoa  Protozoa infecting the gall bladder  Myxosporea  Myxosporea (see B.3.A) and  Microsporidia  Microsporidia (see A.2.b)

B.5.

 Protozoa  Protozoa infecting all tissues of the t he fish body .............................. ..............................  Microsporidia  Microsporidia (see A.2.b)  Myxosporea  Myxosporea (see B.3.a), or coccidians coccidians

B.6.

(see A.2.d).  Protozoa  Protozoa infecting the blood.

B.6.a

Slender cells, typically 10–15 μm long, moving with a wriggling or  undulating motion, with 1 or 2 flagella .............................. Kinetoplastid flagellates

B.6.b

Cells of about 3–15 μm in size, of amoeboid amoebo id shape, displaying displaying a twitching motion on the t he spot .............................. Developmental Developmental stages of some myxosporeans.

3. Flagellates (Phylum Mastigophora)

Trophozoites move by means of one to many flagella. They have one nucleus, or  exceptionally more monomorphic nuclei. Phylum includes free-living and many  parasitic organisms of different groups (six classes), autotrophic forms with chloroplasts and heterotrophs without chloroplasts. They reproduce by binary fission. Dinoflagellates grouped by botanists with algae and by zoologists with the protozoa represent large class of flagellates, 60% are free living and photosynthetizing, many are nonphotosynthetic or parasitic. Trophozoites have two unequal flagella, one transversal, within an equatorial groove, the other longitudinal within a longitudinal groove. Most dinoflagellates adapted to the parasitic way wa y of life live as ectoparasites.  Piscinoodinium  Piscinoodinium pillulare (Fig.7) is a dangerous ectoparasite in aquaria and fish

cultures in tropical as well as in the temperate zone. It appears to be non-specific, infecting indiscriminately various fishes. Invades skin, fins and gills. The pyriform, sack-like or in grown state subspherical trophont is up to 160 μm long and appears yellowish-brown. After a period of growth of about 6 days at 25°C grown trophont drops of the host's surface, sinks to the bottom, rounds off, becomes a tomont and without secreting a common envelope, divides successively into 64 or o r 128 small cells. They divide again to produce 128 or 256 cells which differentiate into gymnospores. Pathogenicity is high. In heavily infected aquarium fish, infection symptoms are signs of discomfort, a golden, velvety hue on the body surface, spreading opercule, folding of fins and eventually emaciation.  Amyloodinium  Amyloodinium occelaltum is a marine counterpart of  Piscinoodinium,  Piscinoodinium, Crepidoodinium Crepidoodinium cyprinodontum has been known thus far only from the gills of 3 species of  cyprinodontid cyprinodontid fishes. f ishes. A dubious genus Oodinioides with a sole so le species O.vastator  was

described as an ectoparasite on the gills and skin of freshwater and marine fishes of  various families.

The name “Oodinium” still survive among ichthyopathologists and aquarium hobbyists, although it is in fact the name of a different dinoflagellate genus ectoparasitic on marine invertebrates, not fishes. T r y p a n o s o m a t i d s occuring in fish are currently assigned to the sole genus Trypanosoma , which includes 184 species in fishes. They have single flagellum which extends free or is attached to the body as the undulating membrane (its end extends free anteriorly) and a single, small disc-shaped kinetoplast. All are parasitic and are transmitted exlusively by leeches. The life cycle in vertebrate host and vectors consist of a series of morphological changes comprising shifts in position of the kinetosomes and kinetoplast in relation to the nucleus and anterior end of the cell, it involves trypomastigote, epimastigote, sphaeromastigote and promastigote stages. Depending on the phase of infection at which the fish is examined, one finds young, slender or large adult forms only, or  intermediate ones. A mix of forms is most likely to be result of sequential leech feeds. Trypanosomes make themselves apparent by their vigorous movement in fresh blood mounts, observed under the compound microscope at 100x magnification. If present, trypanosomes are found in Giemsa stained blood smears. The infections of extremely low intensity can be detected using hematocrit centrifuge techniques. At present about 100 named species of fish trypanosomes are defined and many of them may be synonymous. A due characterization of a fish trypanosome species existing or to be described, requires: requires: 1) a detailed morphological analysis (shape, structure and position of the nucleus, shape and position of kinetoplast, subsurface striation in adult form, arrangement of the undulating membrane and free flagellum, cytoplasmic granules (Fig. 7); 2) morphometric evaluation; 3) ascertainment of pleomorphism in the course of infection; 4) observation on host specificity and 5) observation on the sequence of  stages in the leech vector.  Ichthyobodo  Ichthyobodo necator  (syn. Costia necatrix ), (Fig. 7) is a dangerous ectoparasite of 

 practically all freshwater fish causing c ausing mortalities in young fish and fish with lowered resistance. The free non-feeding form has a flat, slightly asymmetrical, oval body, 10x5 μm; it is strongly convex dorsally and slightly concave ventrally. It swims in a hesitant, spiral way. A longitudinal groove traverse the hinder 2/3 of the ventral surface near its right margin. Two unequal flagella extend from the flagellar pocket. There is a small contractile vacuole close to the pocket, the cytostome opens at the  border of the pocket. Centrally located nucleus, nucleus, up to 2.5 μm has a large nucleolus. nucleolus. The feeding of attached form, fixed onto the epithelial cells, is highly modified. The cell furls in a pyriform shape, the flagella pointed off the fish surface. No digestive vacuoles are formed. Cryptobia branchialis (Fig. 7) has been reported from the gills of an enormous

number of hosts in Europe, Asia, North America and Africa. It abounds in organically  polluted waters, feeding on bacteria and detritus particles. C. branchialis is dropshaped, rounded anteriorly and tapering posteriorly, size range is 12–22×3.5–4.5 μm. At the side of the flagellar pocket is a contractile vacuole. The recurrent flagellum adheres to the cell surface along a wavy line. C. branchialis has been often found in enormous numbers on the gills of its freshwater hosts and acused of causing disease,  but the adherence adherence of the recurrent flagella inflicts no damage to the epithelial epithelial cells and

reasons for the massive occurrence may be debilitated condition resulting in decreased repellent repellent ability abilit y of gill surface. Trypanoplasma species, especially T. salmositica, T. boreli and T. bullocki are known

to cause disease and mortalities in many fish species, both feral and cultured ones. They have a wavy undulating membrane, much wider than in Cryptobia, which differ  mainly in being transmitted by a leech and in undergoing a series of regular  morphological changes during their life cycle. From the flagellar pocket at the anterior apex of the body extends a short anterior free flagellum and a long recurrent flagellum, which is attached to the body along its left, foldlike outstretched border  with which it forms a striking undulating membrane. A single, large, elongated, densely staining kinetoplast extends from the apex of the cell where its pointed end subtends the flagellar kinetosome. The rounded nucleus lies opposite to the kinetoplast in the left border of the dorsolateral groove. During the progress of  infection in the fish trypanoplasms manifest a marked pleomorphism, but unlike in fish trypanosomes it is difficult to characterize the shape differences in forms from early and late infection stages in more precise terms. The validity of many if not most species can be questioned due to great uniformity of alleged species. An adequate characterization of  Trypanoplasma species requires inclusion of essentially similar  features as those outlined for trypanosomes (Fig. 7). The best known species from freshwater fishes are: T. boreli, T. keysselitzy, T. tincae, T. carassii .  Hexamita salmonis (Fig. 7) is a common intestinal endocommensal of salmonids

sometimes turning into a serious pathogen. The body is ellipsoidal e llipsoidal or eggshaped, eggshaped, 7–14 x 3–10 μm. Ellipsoidal nuclei have large nucleoli. Length of flagella is one and half  the body length. The flagellates swim quickly with slight wavering forward. The transmission occurs via cysts or trophozoites which may survive for some time in water. The cysts are ovoidal 10x7 μm, with the flagella divided inside in two. Although the parasite is quite common, the t he disease is rare in natural natur al conditions conditions and in cultured fish it only causes disease when the host's vitality is adversely affected by some predisposing factors. Fig. 7: A - Piscinoodinium pillulare, cell organisation of a trophont attached to host cell (h); B - Measurements in Trypanosoma: a-body length (the actual length of the curved body, to be measured midway between the body sides, a'-midpoint of the nucleus to kinetoplast midpoint, a''-midpoint of the nucleus to the anterior body end (nuclear index), b-greatest body width, d-length of the free end of the flagellum, edistance of the midpoint of the kinetoplast to the posterior end, f-midpoint of the nucleus to midpoint of the kinetoplast; C - Ichthyobodo necator, ventral view; D lateral view; E - Cryptobia branchialis; F - Measurements in Trypanoplasma: a-body length, b-greatest body width, c-length of the anterior flagellum, d-length of the free end of the recurrent flagellum, e-distance of the midpoint of the kinetoplast to the anterior end; G - Hexamita salmonis.

4. Amoebae (Phylum Rhizopoda)

Amoebae are mostly difficult to determine. Their cell has a simple structure, the  plasmalemma is naked or with an external test. Trophic stages move using  pseudopodia or a simple protoplasmic flow. Flagella when present are usually restricted to developmental or sexual stages. The diagnostic features include primarily the locomotive form and behaviour, presence of flagellated stages, cyst structure and nuclear division patterns (stained cells are needed). There are very few specific endocommensals of fish, such as species of the genera  Entamobea or  Schizamobea ,

which under certain conditions can turn in true parasites. All other amoebae infecting fish are amphizoic species, i.e., free-living forms which can under certain conditions colonize the fish. Amphizoic species are found especially in families  Hartmanellidae,  Mayorellidae, Acanthamoebidae and Vahlkamphiidae (Fig. 8). 5. Coccidia (Phylum Apicomplexa)

Fish-infecting coccidia, members of the order  Eimeriida are exclusively intracellular  tissue parasites, located in 1the host tissue cell within a parasitophorous vacuole. Their life cycle comprises asexual phase, i.e., merogony, sexual phase, i.e., gamogony and sporogony. The latter produces the infective stages, oocysts, which contain sporocysts with sporozoites. The oocyst and sporocyst structure is of primary importance in distinguishing between genera and species and in practical determination. Practical key for determination of fish-infecting coccidians 1. a) Oocyst with 4 “naked” sporozoites, without sporocysts, which develops in submembrane position .............................. Cryptosporidium  b) Oocyst with 2 sporocysts ..............................  Isospora c) Oocyst with 4 sporocysts .............................. 3 d) Oocyst with 8 sporocyst .............................. Octosporella (Fig. 8) 2. a) Sporocyst with 1,2 or many long knob-bearing projections on their surface .............................. Calyptospora (Fig. 8)  b) Sporocyst without projections .............................. 3 3. a) Sporocyst with one pole bearing a special structure (Stieda body) - pointed, collar - or knob-like .............................. 4  b) Sporocyst without a Stieda body, with wall composed of 2 valves adhering together along a longitudinal suture line (which is sometimes difficult to see in the light microscope .............................. Gousia (Fig. 8) c) Sporocyst crystal-like, composed of 2 hexagonal valves adhering along a transverse suture line .............................. Crystallospora (Fig. 8) 4. a) Merogony and gamogony in submembrane position, sporogony intracellular  .............................. Epieimeria  b) Development as a rule intracellular, if not, so unlike the above pattern .............................. Eimeria (Fig. 8) 6. Microsporidia (Phylum Microspora)

Microsporidia are widely distributed parasites of various teleosts in freshwater, estuarine and marine habitats. They may seriously endanger whole stocks of feral fishes as well as cultured ones. Microsporidia are strictly intracellular. In some species, the infected cell becomes hypertrophic (up to several mm) offering space to a mass of proliferating parasites, and is called the xenoma. The unicellular spores have an imperforate one-piece wall, contain one sporoplasm and an elaborate hatching apparatus where an extrudible hollow polar tube serves for the injection of the sporoplasm into the host cell (Fig. 8). Most microsporidian stages reach very small

dimensions up to about 5 μm, which has always made their study rather difficult;  plasmodial stages are up to 30 μm. The developmental cycle includes a proliferative  phase (merogony or schizogony) producing a great number of parasites and sporogony which gives rise to mature spores (in most species of uniform shape, in some species macro- and microspores are produced (Fig. 8). In some genera mature spores are grouped together within an envelope (sporophorous vesicle or SPV), which originally covered the spore-producing (i.e., sporogonial) plasmodium. The diagnostic characters are spore structure, presence or absence of SPVs and diplokary, details of  merogony and sporogony and structure of xenoma if present. Practical key for determination of microsporidian genera 1. a) Spores are formed in SPVs localized directly in the tissue; there is no xenoma (do not take for xenoma a parasite focus encapsulated by connective tissue of the host .............................. 2  b) Spores are formed in a xenoma, huge or small, appearing mostly as small whitish nodules or corpuscles .............................. 4 2. a) SPVs free in the tissue .............................. 3  b) Clusters of SPVs within another envelope of parasite origin, the sporophorocyst wall (not to be confused with connective tissue cell capsule) ..............................  Heterosporis

3. a) SPVs with a firm wall contain 8 spores .............................. Thelohania  b) SPVs with a firm wall contain 6 to about 200, mostly many spores .............................. Pleistophora 4. a) The xenoma is a hypertrophic ganglion cell, spores are of two kinds .............................. Spraguea  b) There are two kinds of xenomas: small compartmentalized ones (up to 200 μm) and huge undivided ones (up to 4 mm) ..............................  Ichthyosporidium c) Xenomas of still another character .............................. 5 5. a) Xenomas form whitish nodules in the musculature, consisting of numerous interlocked xenomas, spores with a conspicuous inclusion in the posterior vacuole .............................. Tetramicra  b) Xenomas stay single, spores without such an inclusion .............................. 6 6. a) Dot-like xenomas, sporogonial plasmodia produce several spores in direct contact with the host cell cytoplasm .............................. 7  b) Dot-like or larger xenomas; one to many spores are produced within membrane walled SPVs .............................. 8 7. a) Xenomas in the liver ..............................  Microgemma  b) Xenomas in the gills and on the surface of visceral organs ..............................  Nosemoides

8. a) Dot-like xenomas in the gills or digestive tract; one to several spores in the SPV .............................. Loma

8. b) Dot-like to peas-sized xenomas subcutaneously and in various body organs .............................. Glugea Fig. 8: A - Entamoeba ctenopharyngodoni - trophozoite; B - Hartmanella vermiformis - trophozoite; C - Vexillifera bacillipedes - trophozoite; D - Acanthamoeba  polyphaga-cyst; E - Vahlkampfia debilis - cyst; F - Goussia carpelli; G - Calyptospora funduli; H - Crystallospora crystalloides; I - Octosporella notropis; J - Eimeria rutili; K - Diagrammatic representation of a microsporidian spore, e-exospore, enendospore, c-cell membrane of the sporoplasm(s), n-nucleus, v-posterior vacuole, aanchoring disc or polar sac; p-polaroplast, pt-polar tube; L - Microsporidian spores as seen in fresh state, a-Glugea destruens; b-Pleistophora elegans; c-microspore, dmedium spore, e-macrospore of P.priacanthicola; f-various spores of Glugea machari; g-spores of Microsporidium rhabdofilia

Within the genus, specific determination is also based on spore morphology, e.g., on their size and shape, on the size and shape of the large posterior vacuole and number  of polar tube coils. Among the microsporidians known for their pathogenic effect, is  Pleisthophora hyphessobryconis pervading musculature of many species of ornamental fishes and inflicting high mortalities.  P. oolytica develops within oocysts of  Esox lucius and several other fish.  P. ovariae is a serious pathogen in cultures of bait minnows in the USA ( Notemigonus chrysoleucas). Glugea hertwigi elicits massive formation of large xenomas in the body of smelts of the genus Osmerus, resulting in mass fish kills. 7. Myxosporidia (Phylum Myxozoa)

Among the large number of myxosporeans there are many serious pathogens of  commercially important fishes. They are parasites of organ cavities and tissues of fish. In salmonid cultures, heavy losses have been inflicted until recently by  Myxosoma cerebralis and still are by Ceratomaxa shasta or by the enigmatic PKX (the agent of   proliferative kidney disease). Myxosporidia are characterized by spores composed of  several cell transfigured into 2 to 7 spore shell valves, 1 to 2 amoeboid infective germs (sporoplasms) and 2 to 7 polar capsules. The latter contain an extrudible filament of anchoring function. During the life cycle, some cells (the generative ones) are enclosed within others (somatic cells). Myxosporean spores may be of various shape and structure. Spore dimensions mostly fall within the range of 10 to 20 μm. The trophic (or vegetative) stages, or trophozoites, vary greatly in dimensions and shape. There are macroscopic plasmodia up to several mm in size, some species form small plasmodia only about 10 μm in size and produce a single spores. Since vegetative stages offer mostly no features important for classification, the taxonomy is  based solely on the shape and structure of the spore. Practical key for determination of myxosporean genera (PC - polar capsule; exclusively marine species are marked with an asterisk) 1. a) Mature spore contains only one PC .............................. 2  b) Two PCs per spore (one of them can be much smaller than the other one) .............................. 7 c) Three PCs per spore .............................. * Trilospora d) Four PCs per spore .............................. 24 e) Five PCs per spore .............................. * Pentacapsula f) Six PCs per spore .............................. * Hexacapsula g) Seven PCs per spore .............................. * Septemcapsula 2. a) Spores pyriform, drop-like or elongated ellipsoidal .............................. 3  b) Spores are more or less spherical, with the sutural line difficult to see .............................. 6 c) Spores have a rounded ellipsoidal body with a large, sac-like and curved extension .............................. * Auerbachia 3. a) The suture line of the two shell valves is sigmoidal .............................. *Coccomyxa  b) The suture line of the two shell valves is straight .............................. 4

4. a) Spores without a bifurcated caudal process .............................. 5  b) Spores with a bifurcated caudal process ..............................  Phlogospora 5. a) Spores with PC discharging apically and axially .............................. Thelohanellus

 b) Spores with PC discharging subapically and to the side. ..............................  Neothelohanellus

6. a) Spores with three shell valves (two small ones and one large) difficult to discern in light microscope; exclusively histozoic .............................. * Unicapsula  b) Spores with two shell valves adhering along a delicate sinuous suture line (exclusively coelozoic) .............................. * Globospora 7. a) PCs located each separately in the ends of a spindle -shaped or elongated spore .............................. 8  b) PCs not terminally in the opposing ends, but also not close to each other  .............................. 10 c) PCs close to each other .............................. 16 8. a) The polar filament within the capsule is a thin spirally round tube of about the same thickness all its length through .............................. 9  b) Polar filament strongly tapers from its base to the tip; within the PC it does not form a regular coil, being rather folded and bent over several times .............................. .............................. * Sphaeromyxa 9. a) Spores have as a rule more or less pointed ends ..............................  Myxidium  b) Spores are mostly ellipsoidal, with rounded or bluntly pointed ends and almost spherical PCs ..............................  Zschokkella 10. a) Elongated spores with central, transversal sutural line; PCs near opposite ends discharge laterally ................................... * Fabespora  b) Rounded or ovoid spores with PCs set widely apart in the sutural plane .............................. 11 c) Spherical, rounded or pyramidal spores have PCs in a plane perpendicular to the sutural line which is mostly sinuous .......................... 12 11. a) Spores spherical or subspherical; in marine fishes .............................. *Ortholinea  b) Spores ovoid; flattened parallel to the sutural plane; in freshwater fishes .............................. * Neomyxobolus 12. a) Shell valves without projections .............................. 13  b) Shell valves with various projections .............................. 14 13. a) Spores spherical of subspherical .............................. * Sinuolinea  b) Spores inversely pyramidal or triangular and with rounded outlines .............................. * Myxoproteus 14. a) Spores spherical, each shell valves bears in about its center a hollow, mostly horn-like projection .............................................. * Davisia

 b) Spores inversely pyramidal with tapering end extending backwards; at the anterior surface of each valve, a wing-like projection is attached .............................. * Bipteria see also .............................. * Paramyxoproteus c) In addition to valvular projections, a stiff, keel-like membrane runs sidewise along the sutural line .............................. 15 15. a) Valvular projections wing-like as in Bipteria .............................. * Neobipteria  b) Valvular projections keel-like, meridionally along midline of the valves .............................. * Schulmania c) Valvular projections in form of thickenings covering the spore apex and slightly unstuck off its surface .............................. * Noblea 16. a) Spores elongated, asymmetrical, curved and very thin-walled ..............................  Parvicapsula

 b) Spores essentially bilaterally symmetrical .............................. 17 17. a) The two spherical PCs are tandem in position and at distance from the anterior  end; two fine projections at both posterior and anterior end of the spindle shaped spore ..............................  Neohenneguya  b) PCs are set in plane essentially perpendicular to the sutural plane .............................. 22 18. a) Large spherical PCs in the center of the spore which is triangular in sutural view .............................. Wardia  b) PCs more or less close to the anterior apex of the spore .............................. 19 19. a) Spores without any projections or veils .............................. 20  b) Spores with projections or veils .............................. 21 20. a) Spores spherical or almost spherical .............................. Sphaerospora  b) Spores elongated in the direction perpendicular to the sutural plane, sometimes arcuate; the depth of the shell valve may reach the dimensions of the sutural diameter ..............................  Leptotheca c) Spores crescent-shaped, extremely elongated in the direction perpendicular to the suture line .............................. Ceratomyxa 21. a) Subspherical spores are enveloped in a large membranaceous veil .............................. * Palliatus  b) Triangular spores are laterally drawn into wing-like membranous projections .............................. * Alatospora c) Same as above, but the projections are doubled to parachute-like pockets .............................. * Pseudoalatospora d) Spores spindle-shaped, with a pair of long posterior projections .............................. * Myxobilatus e) Spores spherical to bullet-like with numerous thread- or pin-like projections .............................. Hoferellus 22. a) Spores without projections .............................. 23  b) Spores with projections .............................. 24

23. a) The sutural line straight .............................. Myxobolus  b) The sutural line strongly sinuous .............................. Spirosuturia 24. a) A single caudal projection .............................. Unicauda  b) Two caudal, often slightly divergent projections ..............................  Henneguya c) Two caudal, projections extend in opposite directions  Dicauda d) Four posterolateral projections .............................. Tetrauronema e) Two projections extend laterally from one side of the posterior end of the spore .............................. Laterocaudata f) The valves of broadly triangular spores are drawn into filamentous processes on each side, each pair being connected by a filament .............................. Trigonosporus

25. a) Four shell valves ..............................  Kudoa  b) Two shell valves .............................. 26 26. a) Spores spherical .............................. Chloromyxum  b) Spores almost spherical, with one or two caudal projections .............................. Caudomyxum

c) Spores spindle-shaped, with two caudal projections ..............................  Agarella d) Spores elongated, asymmetrical, curved and thin-walled ..............................  Neoparvicapsula

Presently, there are about 1100 myxosporean species described from fish. Unfortunately, in many descriptions, the spores have very often been quite inadequately characterized. For the proper description of myxosporean species fresh spores have to be observed. To make an accurate diagnosis we have to take measurements according to diagram in Fig. 9. In spores with polar capsules situated at one end, the length (L) is the distance between the apex and the posterior (opposite) end. In Bipolaria, it is the distance between the two opposite ends of the spore. Perpendicular to the length, there is the width (W) which is measured in the plane of  the suture, and the thickness (T) measured in the plane perpendicular to the sutural  plane of the spore. There is the guideline for description of new myxosporean species  by Lom 1989 (see references). Some of the most serious pathogens of commercially important fishes are:  Myxobolus cerebralis (syn.  Myxosoma cerebralis) is the agent of the “whirling

disease” of salmonid fry affecting head and vertebral column cartilage. Spores are extremely variable, oval to circular in front view (L 7.4–9.7 × W 7–10 × T 6.2 – 7.4 μm). The development up to mature spores takes about 2 month at 19°C. Depending on water temperature, clinical signs (“black tail”) appear 2 to 8 weeks after exposure, abnormal tail behaviour, the “whirling” appears later, 2 to 3 month p.i. Chronic infection may be manifested in the second year by cachexia, listlessness and head deformation. Head and gill arch cartilage are sites of predilection. Light infections can  be revealed by recovery of spores after the host tissue has been digested. Trypsinization may be preceded by decalcification. Sphaerospora renicola , the best known species of the genus Sphaerospora is widely distributed in intensive cultures of  Cyprinus carpio (Europe, Israel). Disporic

 pseudoplasmodia are located in the lumen of renal tubules, spores are subspherical (L 7.3 x W 7.2 μm), with ovoid polar capsules (PC) (2.8 x 2.3 μm) and 4 filament turns oriented perpendicularly to the PC. S. renicola may be a serious pathogen because of  extrasporogonic developmental sequences which elicit among others swimbladder  inflammation in one-year-old carp in its 3–4 month of age. Fig. 9: Methods of measurement of myxosporean spores of various genera: A and B Myxobolus in frontal and side sutural view; C and D - Henneguya in frontal and side view; E and F - Myxidium in frontal and side view; G and H Chloromyxum in side or  sutural (G) and frontal (H) view; I - Kudoa in apical view; J - Kudoa in one of the  possible side views which is the diagonal one. Measurement of the polar capsule is indicated in A: L - length of the spore, W - width of the spore, T - thickness of the spore; in spores with caudal appendages such as Henneguya, Al - length of the caudal appendage, Tl - total length of the spore.

“P K X” is the abbreviation for the still undetermined agent of proliferative kidney disease (“P K D”) of salmonid fingerlings. The agent is a myxosporean trophozoite (not specific for its salmonid hosts) which elicits an intense defence reaction of the

host, the intersticial nephritis. Annual losses in the UK alone were estimated at 640 000 £ in the early eighties. Ceratomyxa shasta is a dangerous pathogen of North American west-coast salmonids causing serious losses in cultured and wild salmonid populations. C. shasta infects all

layers of the entire digestive tract wall, but can also be disseminated in other organs. Small disporic pseudoplasmodia area 13 x 19 μm in size, spores (L 6–8 x W 14–17 μm) are strongly arched, with broadly rounded ends, smooth valves and PC 2.2 μm in diameter. The mode of infection is still obscure. Exposure to contaminated bottom sediments or to contaminated water induces infection. 8. Ciliates (Phylum Ciliophora)

Ciliates are highly organized protists with the cell covered by cilia arranged in rows or  kineties. In some groups the number of cilia is reduced or they may be secondarily absent. Ciliates show nuclear dualism, having as a rule generative diploid micronuclei and vegetative polyploid macronuclei. The buccal apparatus serves for ingestion of   particular food. Only several groups of ciliates became secondarily astome, feeding  by pinocytosis. They divide by transverse binary fission, sexual process is conjugation. Ciliates living in and/or on fishes range from completely harmless ectocommensals (e.g.,  Erastophrya) to most noxious parasites as e.g.,  Ichthyophthirius or  Tetrahymena . Manifestations of their pathogenic action are most varied at the tissue and organ levels. The mechanisms of the damage are irritation of the surface cells in extremely heavy growth of ectocommensals and surface cell destruction, penetration into deep tissue layers and ingestion of the cell debris thus produced in e.g., Tetrahymena or  Ichthyophthirius infections. Gill and skin tissue destruction is then necessarily reflected by the debilitated or moribund condition of the fish. Ectoparasitic ciliates include species that are the most common parasites of fishes (Trichodina ). Although in free water trichodinids are common and form sometimes dense populations, morbidity due to Trichodina is rather an exception. In fish confined to ponds, tanks and aquaria, trichodiniasis is a frequent problem. Much more so, the ciliates Chilodonella and Ichthyophthirius are rather rare in fish in free waters,  but require a lot of attention to prevent losses in freshwater fish culture, especially in young fish. Their determinal economic impact is enhanced by their lack of host specificity and cosmopolitan distribution. For a proper identification all important ciliate features must be observed in live as well as stained specimens. Thus in addition to fresh mounts, slides stained with nuclear stains and impregnated with Klein's method or with protargol are necessary. Genus  Balantidium includes 10 species of endocommensal ciliates with cystosome at the base of anteriorly located vestibulum. Their body is uniformly covered with ciliary rows. They are endocommensals which can turn into histophagous parasites capable of abrading the epithelium, penetrating into the subepithelial intestinal layer  and feeding on the tissue cells. Balantidia destroy the epithelium, cause desqúamation, extensive ulceration, and when deep in the tissue, they elicit granuloma formation. The losses caused by  B. ctenopharyngodoni (Fig. 10) were recorded in 1 to 3 year old grass carp (Ctenopharyngodon idella).

Chilodonella piscicola (Fig. 10) is one of the most dangerous ectoparasites which

lives on gills and skin of practically all freshwater fish and also in estuarine and  brackish waters. The body is asymmetrically oval, with a notch in the posterior  margin, 55 x 43 μm (range 30–80 x 20–62 μm). In the right ciliary band there are mostly 8 to 11 kineties, in the left ciliary band, 12 to 13. Chilodonella hexasticha differs from the preceeding in the absence of a notch at the

 posterior body margin, in less numerous and more loosely spaced kineties (Fig. 10) and in smaller body size (30–65 x 20–50 μm). Its biology and pathogenicity are the same. It appears that C. piscicola tends to infect rather the fingerlings while C. hexasticha prevails on older fish. Both species may occur on the same host. Chilodonellas seem to be of a great ecological adaptability, proliferating both in cold and warm water. Under conditions which favour their proliferation, chilodonellas may cover the body surface in a contiguous layer. They disintegrate the surface tissue by means of their oral cytoskeletal armament and feed on the cell debris. Pathological manifestations may vary depending on the intensity of infection. Gill disintegration and necrosis renders the gills nonfunctional, fish lose osmotic balance and die.  Ichthyophthirius multifiliis (Fig. 10) is a commonly distributed dangerous ectoparasite

in freshwater fish culture, known as an agent of “white spot disease”. It belongs to Ophryoglenina , a suborder of large ciliates with polymorphic life cycle. Their buccal cavity opens at the bottom of a deep depression called prebuccal area, which is densely covered with arched anterior ends of kineties of the right body side. Close to the buccal cavity lies a lens-like refractile organelle. The life cycle of   I. multifiliis comprises a small migratory stage in search of a host, the theront (Fig. 10); the feeding and growing stage in the skin or gills of fish, is the trophont (Fig. 10) with a characteristic, large horse shoe-shaped macronucleus. After it has reached a certain size it escapes from the host and encysts on a convenient substrate as a tomont. Within its cyst, it divides by a series of 10 to 11 divisions to produce small tomites. They break through the cyst wall to become theronts again. The duration of the life cycle stages, their size and number depend on the ambient temperature. During the  period of growth in the host's tissue, the trophont reaches the size up to 1 mm, increasing its volume about 3000 times. Due to the growing trophont volume the microcirculatory disorders and associated necrotic changes are enhanced.  I. multiphiliis feeds on the cell debris, forms small cavities beneath the epithelial layer. Both in gills and skin the regressive changes of the epithelium are the most deleterial. Economic importance of ichthyophthiriasis is manifested not solely by direct losses. In fish culture, it is primarily the reduced increment. P e r i t r i c h s are ciliates characterized by inverted bell-shaped, conical or  cylindrical body with the ciliature encircling the apical pole. The antapical pole is equipped with holdfast organelles, a scopula or adhesive disc. Somatic ciliature is reduced. S e s s i l i n a, which include genera  Ambiphrya, Apiosoma, Riboscyphidia, Epistylis,  Propyxidium, Vorticella, Zoothamnium and Carchesium are attached as a rule  permanently to the substrate with specialized area called scopula. The scopula either  directly adheres to the substrate often being cemented with a sticky substance, or it secretes a stalk. The stalk may bear one ciliate or if branched, it may bear many

ciliates sometimes forming colonies. For a reliable description of sessiline peritrichs, observation of living, non-fixed ciliates is absolutely necessary, in order to record the true body shape, as well as morphology of the peristomial region. The body of   peritrichous ciliates is generally uniform and all structural peculiarities are helpful in characterization. The morphology of the stalk, scopula and telotrochs (migratory stages) is most important. Essentially, sessiline peritrichs found in fish are ectocommensals or symphorionts that use their hosts as a living moving substrate to settle where they gain access to a convenient source of food particles - organic debris and water-born bacteria. They are specifically adapted to the life on the surface of  certain species of fishes. M o b i l i n a are constantly moving peritrichous ciliates which can attach temporarily to the substrate by means of a slightly concave adhesive disc reinforced by a system of skeletal elements of proteinaceous character. The disc can contract and its function is reminiscent of a sucker. They do not form cysts, the transmission is direct by swimming ciliates. Fish are invaded by members of a single family - Trichodinidae . All trichodinids are essentially commensals, five genera occur in fish. Ectozoic species use their host as a convenient substrate upon which they glide and to which they temporarily attach. They feed on waterborne particles and bacteria as well as detritus particles from the fish surface. Trichodinids never occur in larger amounts on a fish in good health condition. In a fish debilitated by some other factors or on young fry, the trichodinids can massively proliferate, and then they behave like serious  parasites. Fig. 10 - Balantidium ctenopharyngodoni; A - Chilodonella piscicola, ventral side; B the cyst of C. piscicola; C - kineties on the ventral side of C. hexasticha; D Ichthyophthirius multifiliis, a grown trophont in ventral view; E - I. multifiliis, a theront in ventral view; F - Diagnostic features of trichodinid ciliates: skeletal parts of  the adhesive disc, s - rods in the border membrane, r- -radial pins, nu - number of  radial pins per denticle, d - denticles, dd - diameter of the denticulate ring, da diameter of the adhesive disc; G - horse-shoe shaped macronucleus of trichodinids with three possible positions of micronucleus; H - a denticle of Trichodina: t - length of thorn, c - width of the central part, b - length of blade, e - length of denticle; I shape of denticles in Trichodinella.

The taxonomy of trichodinids is based on the structure of the buccal ciliature and on the appearance of the adhesive disc and numbers and size of its constituents. All these features can be revealed only by the silver impregnation technique of Klein.

Key to the genera occuring in fish: 1.a) The adoral ciliary spiral makes two and half to three turns .............................. Vauchomia

 b) The adoral spiral makes one turn of slightly less or more (330° to 540°) .............................. 2 c) The adoral spiral makes one half to three quarters of a turn (an arch of 150° to 290°) .............................. 3 2.a) The denticles have well developed both thorns and blades .............................. Trichodina

 b) The blades of denticles are stunted ..............................  Hemitrichodina 3.a) The denticles have well developed thorns .............................. 4  b) The thorns are stunted to form short crooks or platelets .............................. Trichodinella

c) No thorns at all, central part indistinct, blades triangular ..............................  Dipartiella

4.a) The centrifugal projections of denticles (blades) are attached to the central part almost perpendicularly and the denticles are interlocked only by their central conical  Paratrichodina  parts ..............................  b) Blades extend from the central part obliquely backwards; denticles are interlocked by central parts and by anterior projections of blades fitting into corresponding notches in blades of the preceding denticles .............................. Tripartiella

For specific determination, the appearance of structures revealed by Klein's technique is most important, as well as the measurements of constituents of the adhesive disc (see Fig. 10). Size of denticles is measured according to Fig. 10. The diameter of the macronuclear horse-shoe is measured after Fig. 10. In Trichodinella, the thorn is reduced to a short crook. Recommended literature Arthur J.R., Lom J. (1984): Trichodinid protozoa (Ciliophora: Peritricha) from freshwater fishes of Rybinsk Reservoir, USSR. J. Protozool. 31, 82–91. Canning E.U., Lom J. (1986): The Microsporidia of vertebrates. Academic Press, pp. 289. Dyková I., Lom J. (1983): Fish Coccidia: An annotated list of described species. Folia Parasitol. 30, 193–208. Halliday M.M. (1976): The biology of Myxosoma cerebralis: the causative organism of whirling disease of salmonids. J. Fish Biol. 9, 339–357. Lom J. (1979): Biology of the trypanosomes and trypanoplasms of fish. In: Lumsden W.H.R. and Evans D.A. (Eds.). Biology of the Kinetoplastida, Academic Press, London - New York - San Francisco, 269–337.

Lom J. (1981): Fish invading Dinoflagellates: a synopsis of existing and newly  proposed genera. Folia Parasitol. 28, 3–11. Lom J., Arthur J.R. (1989): A guideline for the preparation of species descriptions in Myxosporea. J.Fish Dis. 12, 151–156. Mitchell L.G. (1977): Myxosporida. In: J.F.Kreier (Ed.), Parasitic protozoa, Vol. 4, 115–154. Academic Press. Page F.C. (1976): An illustrated key to freshwater and soil amoebae. Freshwater  Biological Assoc. Publ. No 34, Titus Wilson and Son, Kendal, England, pp. 155. Popovský J., Pfiester L.A. (1990): Dinophyceae (Dinoflagellida). In: Süsswasserflora von Mitteleuropa. Band 6. Gustav Fisher Verlag, Jena, Stuttgart, pp. 272. Reichenbach-Klinke H.H. (1980): Krankheiten und Schädigungen der Fische. Gustav Fisher Verlag, Stuttgart, New York, pp. 472. Roberts R.J. (Ed.) (1978): Fish pathology. Bailliere Tindall. London, pp. 318. Scheubel J. (1973): Die sessilen Ciliaten unserer Süsswasserfischen unter besonderer  Berücksichtigung der Gattung Apiosoma Blanchard. Zool. Jb. Syst. 100, 1–63. Upton S.J., Reduker D.W., Current W.L., Duszynski D.W. (1984): Taxonomy of   North American fish Eimeriidae. NOAA Technical Report NMFS 11, (U.S. Department of Commerce, National Marine Fisheries Service), pp. 18. B) PARASITIC METAZOA

(F. Moravec, R. Ergens, V. Našincová, T. Scholz) In addition to protozoans, an important role among the agents of fish diseases is  played by multicellular or metazoan parasites. These belong to several very different groups that include enormous numbers of species occurring in fishes both as ectoparasites and endoparasites, attacking practically all organs and tissues of the host's body. In view of a considerable morphological diversity of metazoans it is by far impossible to deal with these parasites in more detail in this very short review and, therefore, their correct identification will require the use of special literature. Only the most important groups of metazoans parasitic in fishes have been included. The body size of metazoan parasites may be very different, ranging from less than one millimetre up to several tens of centrimetres and, therefore, most of them can be observed with the naked eye. Nevertheless, the use of binocular microscope for fish examinations is quite necessary. Live and/or freshly caught (dead) fish are preferable. In general, the detailed examination of a small number of fish (10–15 of each species) is sufficient to recognize the parasitological situation in the locality. Following the external examination of the body surface, fins, gills and nasal cavity, the fish body is dissected and individual organs and tissues (e.g. digestive tube, liver, urinary bladder, kidneys, gall-bladder, swim-bladder, mesenteries, eyes, brain, pieces of muscles) are thoroughly examined for the presence of endoparasites.

In general, external parasites can be kept in clean freshwater for a few hours without serious damage, but all internal macro-parasites should be kept in saline and fixed within a few minutes to one hour. The most commonly used fixatives for parasites are 70 % ethanol or 2–4 % formalin. Trematodes and cestodes are usually fixed slightly  pressed between a glass slide and a coverslip. Delicate organisms and semimicroscopic forms (e.g. some monogeneans) need special techniques to reduce damage and prevent loss. There are many special procedures of preparing the  parasites for their subsequent microscopical examination. For example, the trematodes and cestodes are usually stained with carmine, nematodes cleared in glycerine or lactophenol, etc. The metazoan parasites of fishes belong mostly to organisms loosely referred to as worms ( Platyhelminthes, Nematoda, Acanthocephala, Hirudinea), less often to crustaceans and molluscs. Platyhelminthes - flatworms

They tend to be flat, although many of them are fusiform or even filiform. They may  be segmented or unsegmented and they all are equipped with characteristic attachment organs. Key to classes of  Platyhelminthes 1 Ectoparasites; one posterior attachment organ, with one or more pairs of median hooks and varying numbers of marginal hooks and/or clamps ..............................  Monogenea

- Endoparasites; attachment organs not as above .............................. 2 2 One attachment organ, armed with hooks and/or suckers (some without this organ). Gut absent. Body ribbon-like, segmented or unsegmented .............................. Cestoda

- Two sucker-like attachment organs. Gut present, usually bifurcated. Body unsegmented, flat to fusiform … Trematoda Class Monogenea

The monogeneans (Fig. 11) are hermaphroditic flatworms parasitic mostly on or in fishes and some other aquatic or amphibious cold-blooded vertebrates, occasionally on aquatic invertebrates. They are most commonly found in the gill chamber, on skin, in buccopharyngeal cavity, or in other organs communicating directly or indirectly with the exterior (nostrils, cloaca, urinary bladder, etc.). Their development is without intermediate hosts. The chief attachment organ (opisthaptor) is posterior, more or less discoid, muscular, provided with paired or unpaired anchors and a number of  marginal hooks or with muscular suckers or clamps without or with supporting sclerites and anchor complexes. Accesory adhesive organs may be present in form of  armed plaques, lappets or appendices.

Fig. 11: The representatives of some families of the Monogenea. A - Dactylogyridae; B,C - Ancyrocephalidae; D - Tetraonchidae; E - Gyrodactylidae; F - Diclobothriidae; G - Mazocraeidae; H - Diplozoidae; I - Discocotylidae; J - Octomacridae. (After  Gussev and Bychowsky, 1957). Anterior adhesive organ (prohaptor) may be represented by so-called head organs, which are made up of distended cephalic gland ducts opening along the lateral margins of the anterior extremity, occasionally by glandular structures often accompanied by paired or unpaired suckers, paired pseudosuckers or sucking grooves. Almost all the previous authors, including the most recent ones, have placed great emphasis on the external morphology, particularly on the sclerotized parts of the body such as opisthaptoral anchors, clamp sclerites, copulatory organ, etc. These hard parts are, of course, of special value as taxonomic criteria. The internal anatomy,  particularly that of the gonads, is also of great taxonomic value, although not much attention has been paid to it by the majority of helminthologists. The class Monogenea is usually divided into two subclasses.

Key to subclasses of  Monogenea 1 Opisthaptor with 1–3 pairs of anchors and with 7–8 pairs of marginal hooks, without clamps .............................. Polyonchoinea - Larval opisthaptor mostly with 5 pairs of marginal hooks. Opisthaptor of the adult is developed as suckers or clamps supported by sclerites or a combination of clamps and anchor complexes .............................. Oligonchoinea The most frequent  Polyonchoinea infecting freshwater fishes are represented by numerous genera and species of 4 families (3 orders). Key to orders and families of  Polyonchoinea 1 (6) Oviparous 2 (5) Opisthaptor with 7 pairs of marginal hooks and with 1–2 pairs of anchors ..............................Order  Dactylogiridea 3 (4) Opisthaptor with 7 pairs of marginal hooks and with 1 pair of anchors ..............................Family Dactylogiridae 4 (3) Opisthaptor with 7 pairs of marginal hooks and with 2 pairs of anchors ..............................Family Ancyrocephalidae 5 (2) Opisthaptor with 8 pairs of marginal hooks and with 2 pairs of anchors .............................. Order  Tetraonchidea Family Tetraonchidae 6 (1) Viviparous ..............................Order  Gyrodactylidea Family Gyrodactylidae The most frequent Oligonchoinea infecting freshwater fishes are represented by some genera and species of 5 families (2 orders). Key to orders and families of Oligonchoinea 1 (2) Opisthaptor with 3 pairs of sessile suckers, each provided with a large curved sclerite ending in a hook and a prominent opisthaptoral appendix bearing 3 pairs of anchors..............................Order  Diclobothriidea Family Diclobothriidae 2 (1) Opisthaptor with 4 or more pairs of clamps and with 1–2 pairs of anchors on terminal lappet..............................Order  Mazocraeidea 3 (4) Opisthaptor with 4 pairs of clamps and with 2 pairs of  anchors..............................Family Mazocraeidae 4 (3) Opisthaptor with 4 pairs of clamps and with 1 pair of anchors. 5 (6) Two adults permanently fused in form of letter..............................Family  Diplozoidae

6 (5) Each adult exists individually 7 (8) Testes numerous, gill parasites of  Salmonidae ..............................Family  Discocotylidae

8 (7) Testis one, gill parasites of  Cyprinidae..............................Family Octomacridae Class Cestoda

Tapeworms (Fig. 12) represent a platyhelminth group with exclusively endoparasitic  pattern of life. They mostly parasitize in the intestine of vertebrates. Their extreme adaptations to endoparasitism resulted in complete loss of digestive system and an increase in reproductive capacity that often staggers the imagination. A total of 4.000 species of cestodes have been described; many of them are parasitic in fishes either as adults or as larvae. Although considerable variation of morphology occurs between different orders of  tapeworms, there are underlying similarities that unite the orders into the class Cestoda. The tapeworm body usually consists of a chain of segments called proglottids, each of  which contains one or more sets of reproductive organs. The proglottids are continuously produced by a process of asexual budding. The entire body formed by  proglottids is called a strobila, and a segmented strobila is said to be polyzoic. In some groups, particularly in fish cestodes, the body consists of a single segment, and is then said to be monozoic. At the anterior end a holdfast organ or scolex is usually found, that is the principal means of locomotion of these animals. Depending on the group, the scolex may be provided with suckers, grooves, hooks, spines, glandular  areas, or combination of these. All fish cestodes are hermaphroditic and their life cycle includes at least one intermediate host, mostly invertebrates (oligochaetes, copepods, amphipods, etc.). Key to orders of fish cestodes 1 (2) Strobila with no internal segmentation; one set of reproductive organs present ..............................Caryophyllidea 2 (1) Strobila with internal segmentation; more than one set of reproductice organs  present. 3 (4) Scolex with no true suckers, bothria, bothridia, or tentacles; no external segmentation .............................. Spathebotriidea 4 (3) Scolex with one of the holdfast types listed above (3); external segmentation usually distinct. 5 (6) Scolex with two bothria .............................. Pseudophyllidea

6 (5) Bothria absent; scolex armed with four rounded suckers .............................. Proteocephalidea Short survey of the orders of fish cestodes with brief characterization of some important representatives. Caryophyllidea

Tapeworms with nonsegmented body containing only one set of reproductive organs. The scolex is simple, has shallow groowes or loculi, shallow suckers, or is frilled. It is never armed with hooks. Life cycle of all species involves a freshwater oligochaete as the intermediate host. Caryophyllideans are parasites of freshwater teleosts, especially Cypriniformes (mainly in Palaearctic Eurasia and America) and Siluriformes (in Asia). Caryophyllaeus fimbriceps is an important parasite of carp ( Cyprinus carpio) cultures

in eastern Europe and the USSR. This species, about 15 mm long, is typified by the shape of scolex armed with deep grooves. As a consequence of introduction of the species  Khavia sinensis, another carp parasite, to many European countries, C.  fimbriceps is now rather rare and does not represent a veterinary problem. Some other  representatives of the genus Caryophyllaeus have been recorded in siluroid fishes in India and southeast Asia (Java).  Khavia sinensis is larger (length of body up to 120 mm) than C. fimbriceps and its

scolex bears deeper incisions forming festoon-like shape of scolex. It occurred originally only in east Asia. Owing to the import of herbivorous fishes, particularly of  grass carp (Ctenopharyngodon idella , it has been spread to many European countries and most parts of the USSR. Several species of the genus  Lytocestus have been hitherto described from catfishes, mainly Clarias batrachus, in India, southeast Asia and Africa. Members of this genus are characteristic by the presence of undifferentiated scolex and the absence of  vitelline follicles in postovarian zone. Caryophyllidean cestodes with scolex often constricted off from body and parasitizing siluroid fishes in Africa, mostly those of the genus Synodontis , belong to the genus Wenionia. Spathebothriidea

Parasites of teleost fishes, both marine and freshwater. Cestodes bearing undifferentiated, funnel-shaped or hollow, cup-like organ. External segmentation is absent, but there are several serially arranged sets of male and female reproductive systems. Cyathocephalus truncatus, a common parasite of salmonid and some other freshwater 

fishes in Europe, the USSR and North America, has scolex with funnel-shaped apical adhesive organ. Strobila flat, with 20 to 45 sets of reproductive organs. Intermediate hosts are amphipods.

 Pseudophyllidea

Members of this large order are parasites of fish, amphibians, reptiles, birds, and mammals. Their scolex has dorsal and ventral depressions called bothria, which are quite agile and serve as holdfast and locomotor organs. The life cycle involves a crustacean first intermediate host and mostly fish second intermediate host. Larval stages called plerocercoids of   Ligula intestinalis, up to several tens of  centimetres long, are common in many freshwater fishes, particularly cyprinids. Plerocercoids, localized in body cavity, are rather pathogenic for their fish hosts and can cause even their death. Representatives of the genus  Diphyllobothrium are cosmopolitan parasites of reptiles, marine and terrestrial mammals, and birds. Scolex has narrow and deep bothria, and distinct neck. Plerocercoids of some species, including the species  D. latum,  parasitizing humans, are commonly found in flesh of coregonid, perciform, and other  freshwater fishes. In Asian teleosts, members of the genus Senga are often recorded. They are typified  by the rectangular scolex, with bilobed apical disc bearing many small hooks on margins. Eggs are anoperculate. The species  Bothriocephalus acheilognathi is a dangerous parasite of carp in fish culture, imported from Far East to many countries in Europe, America and Africa. Scolex of this tapeworm is elongate, with apical disc and narrow, very deep bothria. External metamerism complete, length of strobila up to 15 cm. Uterine pores median. Copepods serve as the first and the only intermediate hosts of the parasite. Triaenophorus nodulosus and T. crassus are common, circumboreal parasites of pike ( Esox spp.). Plerocercoids are parasitic in internal organs and muscles of many

species of freshwater fishes, mainly perciforms and salmonids. Adults and larvae from fishes are characteristic of the presence of four trident-shaped hooks on scolex and two shallow botria.

Fig. 12: A - Khawia sinensis (order Caryophyllidea), total view; B, G Bothriocephalus acheilognathi (Pseudophyllidea), scolex and gravid segment; C, I Proteocephalus neglectus (Proteocephalidea), scolex and mature segment; D - Ligula intestinalis, plerocercoid (Pseudophyllidea), anterior end; E - Cyathocephalus truncatus (Spathebothriidea), scolex; F - Triaenophorus nodulosus (Pseudophyllidea), scolex; H - Bothriocephalus claviceps (Pseudophyllidea), mature segment.  Proteocephalidea

Members of this order are easily recognized by the presence of a scolex with simple suckers and follicular, usually lateral vitellaria. Most of them are parasites of  freshwater fishes. Tapeworms of the genus Gangesia have rounded scolex with rostellum armed with one or two circles of hooks. They parasitize on siluroid fishes in Europe and Asia. The genus  Proteocephalus comprises many parasites of freshwater fishes, amphibians and reptiles. Scolex unarmed, with four simple suckers, sometimes with fifth apical organ.  P. ambloplitis is commonly found in many species of freshwater fishes in  North America;  P. neglectus is a veterinary important parasite of trout in Europe;  P. exiguus parasitizes coregonid fishes in Europe and Asia.

Class Trematoda

Fish trematodes (Fig. 13) are endoparasitic. They possess bilaterally symmetrical, dorsoventrally flattened, unsegmented bodies, usually oval or lanceolate. The majority of trematodes fall into the range of 1–30 mm. Trematodes are commonly equipped with two attachment organs, the oral sucker at or near the body's anterior end and the ventral sucker, or acetabulum, the position of which varies from near-anterior to near posterior, with all position in between. Some species lack one of suckers or even both. Most fish trematodes are hermaphroditic. The life cycles of trematoda involve more than one (often three) hosts and include several morphologically different developmental stages. The first intermediate hosts are usually molluscs, which release motile larval stages - cercariae. Freshwater fish may serve as the second intermediate hosts of the next developmental stages (metacercariae) or as definitive hosts. Metacercariae are encysted in various inner  organs and tissues, adults are localized mostly in the alimentary system, less often in excretory or blood-vascular systems and in body cavities. The trematode fauna of freshwater fish is diverse. Moreover, the taxonomic treatment of the class Trematoda has been frequently reassessed as new information becomes available, particularly that concerned with life cycles and larval stages. The most complete information on fish trematodes gives Yamaguti (1971). Some more important representatives parasitizing freshwater fish, listed according to families, are mentioned below. ADULT TREMATODES  Bucephalidae

Gasterostomatous trematodes without oral sucker, with rhynchus as adhesive organ at anterior end. Pharynx and oesophagus present. Intestine simple, saccular; no acetabulum. Members of the genera  Busephalus, Bucephalopsis, Rhipidocotyle,  Prosorhynchus are common parasites of freshwater fish. Sanguinicolidae

Trematodes of lanceolate body shape, living in blood-vascular system; usually without suckers. Pharynx absent, intestine X- or H-shaped. Thin-shelled eggs, without operculum, sometimes with lateral projection ( Sanguinicola). Some species can cause serious damage to fish in aquaculture.  Monorchiidae

Parasites of marine, sometimes freshwater, fish. Trematodes from freshwater fish are small, about 1 mm in length, with elongate body covered with spines. Acetabulum in anterior half of body. Genital pore submarginal. Caeca short, testis double ( Paleorchis), or caeca long and only one testis present ( Asymphylodora). Gorgoderidae

Trematodes with smooth body divided into a narrow forebody and large hindbody. Suckers well-developed, pharynx absent, caeca reaching near to posterior extremity. Fish gorgoderids, belonging mainly to the genus  Phyllodistomum, are localized in urinary bladder.  Allocreadiidae

 Nearly exclusively parasites in digestive tract of freshwater fish. Trematodes with elongate, mostly unarmed body. Oral sucker without appendages ( Allocreadium, Orientocreadium ) or with typical muscular appendages ( Bunodera, Bunoderina, Crepidostomum ). Acetabulum in anterior half of body. Digestive system welldeveloped; caeca long, terminating near posterior extremity. Opecoelidae

Trematodes resembling in adult morphology members of the family  Allocreadiidae, with small body, less or more elongate, usually unarmed. Parasitic in digestive tract of  marine and freshwater fish ( Nicolla, Helicometra, Sphaerostomum, Plagioporus). LARVAL STAGES (metacercariae)  Bucephalidae

Metacercariae of several genera ( Bucephalus, Bucephalopsis, Rhipidocotyle,  Prosorhynchus) are commonly found encysted on gills, in skin, fins, eyes, brain, subcuticular tissue, and muscles of freshwater fish. Metacercariae are morphologically similar to adults, parasitizing predatory fish.  Diplostomatidae

Body divided into two parts; forebody leaf-shaped, hindbody considerably smaller. Oral sucker small, terminal, anterior end provided sometimes with two  pseudosuckers. Acetabulum postequatorial, holdfast organ situated in posterior part of  forebody. Metacercariae motile, unencysted, localized in eyes ( Diplostomum, Tylodelphys ), or encystated in brain, skin, muscles and other organs of many freshwater fish ( Neodiplostomum, Posthodiplostomum). Some representatives ( Diplostomum spp.) are serious pathogens causing even death of highly infected fish. Strigeidae

Metacercariae of tetracotyle type are encysted in musculature, body cavity and on surface of various inner organs. Cysts oval to pear-shaped. Suckers well-developed,  pseudosuckers usually present. Holdfast organ postacetabular. Important genera  parasitizing fish are Apharyngostrigea, Cotylurus and Apatemon. Clinostomatidae

Free or encysted metacercariae of the genera Clinostomum and  Euclinistomum are localized in body cavity, muscles or inner organs of fish. Body elongated, with both

suckers in anterior third of body. Pharynx absent. Caeca reaching near to posterior  extremity, usually with lateral diverticula. Opisthorchidae

Metacercariae elongate, encysted in muscles of mainly cyprinid fish. Suckers relatively large, acetabulum near middle of body. Caeca long, reaching near posterior  extremity. Large excretory bladder filled with numerous granules. Adults of some species (Opisthorchis felineus, O. viverrini, Clonorchis sinensis ) are important human liver parasites. Man acquires infection by eating raw fish flesh with encysted metacercariae.

Fig. 13: A - A generalized trematode: a - acetabulum, c - caecum, cr - cirrus, d vitelline ducts, esv - external seminal vesicle, ex - excretory vesicle, f - vitelline follicles, isv - internal seminal vesicle, L - Laure s canal, o - oral sucker, oe oesophagus, oo - ootype, ov - ovary, p - prostate, ph - pharynx, px - prepharynx, sr -

seminal receptable, t - testis, u - uterus, vd - vas deferens, ve - vas efferens; B-D Metacercariae: B - Opisthorchis viverrini, C - Cotylurus pileatus, D - Diplostomum sp.; E-I - Adult trematodes: E - Bucephalus polymorphus, F - Sanguinicola inermis, G - Phyllodistomum elongatum, H - Crepidostomum farionis, I - Orientocreadium  batrachoides.  Heterophyidae

Encysted metacercariae with two poorly developed suckers. Oral sucker provided sometimes with spines ( Centrocestus, Phagicola, Pygidiopsis ); acetabulum often modified and armed with numerous spines and sclerites ( Heterophyes, Metagonimus,  Haplorchis, Stellantchasmus). Metacercariae encysted mostly in muscles, sometimes in gills, fins or under scales of many freshwater fish. Adults of some species have  been found in man. Nematoda - roundworms

The nematodes (Fig. 14) represent a large group of metazoan invertebrate animals. They are noted for the elongate, cylindrical body covered with strong protecting cuticle that usually has fine transverse striations. The mouth opening is usually surrounded by lips and cephalic papillae, sometimes a sclerotized buccal capsule occurs. The digestive tube is formed by the oesophagus, often subdivided in two distinct parts, the intestine and the rectum. The sexes are separated. The male tail is sometimes provided with caudal alae, genital papillae and usually one or two sclerotized spicules are present. They are free-living or parasitic in animals a nd plants. From about 16,000 described species, about 40 % are animal parasites.  Nematodes belong to the most frequent parasites of freshwater fishes, some of them  being the agents of serious fish diseases. In addition to adult forms, some nematode species occur in fishes only as larvae. The definitive hosts of these species are then various piscivorous vertebrates (fishes, reptiles, birds, mammals), whereas the fish harbouring these larvae serves as the intermediate or the paratenic host. The development of fish nematodes is either direct or, more frequently, with the  participance of an obligate intermediate host (usually an invertebrate). Key to subclasses of  Nematoda parasitic in vertebrates 1 Eggs with plug at either pole; males with one spicule or spicule absent; oesophagus cylindrical or with oesophageal glands free in pseudocoel stichosome; phasmids absent.............................. .............................. Adenophorea - Eggs without polar plugs, rarely operculate at one pole, or hatching in uterus; males usually with two spicules, exceptionally spicule one or absent; oesophagus never in form of stichosoma; plasmids present .............................. Secernentea  Adenophorea

are represented in fishes only by one order   Enoplida; only its superfamily Trichuroidea includes adults from fishes, while  Dioctophymatoidea contains some genera ( Dioctophyma, Eustrongylides) parasitic in fishes only as larvae.

Key to families of Trichuroidea 1 Complete digestive tract present, posterior region of female cylindrical ..............................Capillariidae - Digestive tract incomplete; posterior region of female expanded to form vesicle ..............................Cystoopsidae  Pseudocapillaria salvelini is a frequent intestinal parasite of salmonids and cottids in

the Holarctic Region. Intermediate or paratenic hosts are various oligochaetes. Schulmanela petruschewskii is parasitic in the liver of many species of freshwater  fishes of different families in Europe. It is a serious parasite of pond-reared carp and rainbow trout. Oligochaetes serve as intermediate hosts. Cystoopsis acipenseris is  parasitic in cysts under the skin of sturgeons in Palaearctic Eurasia and North America. Its intermediate hosts are gammarids. Secernentea

include 3 orders containing the species parasitizing freshwater fishes. Key to orders of Secernentea 1 Oesophagus with bulb; male with reduced caudal papillae; generally only one spicule; body short and stout.............................. .............................. Oxyurida (Only a few species are known to occur in freshwater fishes in tropical Asia and South America). - Nematodes lacking most of the above characters ..............................2 2 Anterior extremity triradiate (except in some Seuratoidea ); head end with three lips or lips absent; oesophagus not divided into muscular and glandular   parts.............................. .............................. Ascaridida - Anterior extremity bilaterally symmetrical; head end with two lateral lips or lips reduced or absent; oesophagus divided into anterior muscular and posterior  glandular parts.............................. Spirurida Key to superfamilies of  Ascarida from freshwater fishes 1 Head end with well developed lips, sometimes separated by interlabia; oesophagus simple and cylindrical or terminated by ventriculus; precloacal sucker absent in male; genital papillae numerous .............................. Ascaridoidea - Lips on head strongly reduced or absent; oesophagus simple and cylindrical, or  divided into two parts having or not having same diameter; ventriculus absent;  precloacal sucker may be present in male; genital papillae not numerous ..............................Seuratoidea

Superfamily Ascaridoidea  Raphidascaris acus in noted for the presence of the posterior ventricular appendix.

Adults are parasitic in the gut of pike and some other predatory fishes in the Holarctic Region. Larvae occur encysted in many other fish species.  Hysterothylacium bidentatum is an intestinal parasite of sturgeons of Palaearctic Eurasia. It possesses the posterior venticular appendix and the anterior intestinal caecum. Goezia ascaroides is parasitic in the stomach of European catfish. Its cuticle is armed with transverse rows of minute spines. Superfamily Seuratoidea  Paraguimperia tenerrima of the family Quimperiidae is an intestinal parasite of 

European eels. Other congeneric species occur in eels in India and North America. Cucullanus truttae of the family Cucullanidae is a frequent intestinal parasite of  salmonids of the Holarctic Region. Its intermediate hosts are lampreys. Many congeneric species occur in various fishes in other regions. Key to superfamilies of Spirurida from freshwater fishes 1 Pseudolabia always absent; oesophagus divided into muscular and glandular   portions or muscular throughout … 2 - Head end with pseudolabia, sometimes rudimentary; muscular and glandular   portions of oesophagus well differentiated ..............................3 2 Buccal capsule well developed, orange-brown; parasites of digestive tract ..............................Camallanoidea - Buccal capsule reduced or absent (except for  Anguicolla), colourless. Not parasitic in digestive tract .............................. Dracunculoidea 3 Caudal alae in male absent. Pseudolabia almost absent .............................. ..............................Thelazioidea - Caudal alae in male present. Pseudolabia present..............................4 4 Pseudolabia small, not covering entire cephalic surface. Collarette absent .............................. Habronematoidea - Pseudolabia large, massive. Body cuticle immediately behind pseudolabia often expanded to form collarette.............................. .............................. Physalopteroidea Superfamily Camallanoidea Camallanus lacustris is noted for a complex orange-brown capsule formed by two

lateral valves and provided with two large tridents. It is a frequent intestinal parasite of many fish species (mainly percids) in Europe. Intermediate hosts are copepods. Procamallanus spiculogubernaculus with a simple buccal capsule is a common parasite of some catfishes in India.

Superfamily Dracunculoidea  Philometra ovata is a common parasite of some cyprinids in Europe. Large females

occur in the abdominal cavity, whereas small males are found on the swimbladder. It develops through copepods.

Fig. 14: Nematoda. A, B - nematode morphology, generalized and diagramatic (A male, B - female; abbreviations: a - anus, bc - buccal capsule, co - cloacal opening, cp - caudal papillae, go - glandular oesophagus, i - intestine, mo - muscular oesophagus, nr - nerve ring, o - ovary, s - spicules, t - testis, u - uterus with eggs, v - vagina, vu vulva); C-P - anterior and posterior body ends (C, D - Pseudocapillaria, D, K, O Raphidascaris, E, L, P - Spinitectus, F - Camallanus, G, H - Anguillicola, I Philometra, J, M - Skrjabillanus). Skrjabillanus tincae is parasitic in mesenteries and kidneys of tench in Europe. These

are very delicate, thin and long nematodes; its intermediate hosts are branchiurids.

 Anguillicola crassus is a danger parasite of the swimbladder of eel which was recently

introduced into Europe from East Asia. Its intermediate hosts are copepods and ostracods. Superfamily Thelazoidea  Rhabdochona denudata of the family Rhabdochonidae is a frequent intestinal parasite

of some European cyprinids. Its intermediate hosts are mayflies. Superfamily Habronematoidea Cystidicola farionis of the family Cystidicolidae is a pathogenic parasite of the

swimbladder of various Holarctic salmonids. Its intermediate hosts are gammarids. Spinitectus inermis of the same family is parasitic in the stomach of European eels. Its  body is provided with rings of cuticular spines. Many congeneric species occur in fishes in other continents. Intermediate hosts are various crustaceans and aquatic insects. Superfamily Physalopteroidea  Pseudoproleptus notopteri of the family Physalopteridae is parasitic in the stomach of  the featherback,  Notopterus notopterus, in India. Acanthocephala - spiny-headed worms

Acanthocephalans (Fig. 15) belong to one class and they are all parasitic. The body shows three externally recognizable regions: the trunk, the neck and the retractable  proboscis armed with a set of posteriorly pointing hooks the number, size and arrangement of which are of taxonomic importance. The proboscis functions to anchor the worm in place more or less permanently, by penetrating the host's intestinal wall. The trunk is a sac-like structure, subcylindrical, usually with  pseudosegmentation. The digestive tract is absent. The sexes are separate. Their  development involves an invertebrate intermediate host. Many acanthocephalan species of different genera and families are parasitic in both marine and freshwater fishes all over the world. The forms parasitizing freshwater  fishes belong to 3 orders. Key to orders of freshwater fish acanthocephalans 1 Cement glands syncytial; cuticle nuclei giant..............................2 - Cement glands in form of several compact formations; cuticle nuclei usually small.............................. Palaeacanthocephala 2 Trunk with cuticular spines.............................. Gyracanthocephala - Trunk without cuticular spines.............................. Neoacanthocephala Order Gyracanthocephala

 Pallisentis ophiocephali belonging to the family Quadrigyridae parasitizes mainly fishes of the family Channidae in tropical Asia. It develops probably through

copepods. Order  Neoacanthocephala  Neoechinorhynchus rutili of the family Neoechinorhynchidae is parasitic in many fish

species of different families (mainly cyprinids) in Palaearctic Eurasia. Its development is through ostracods. Order  Palaeacanthocephala  Acanthocephalus anguillae of the family  Echinorhynchidae is a frequent parasite of 

cyprinids and some other fishes in Europe. Its intermediate host is a freshwater isopod  Asellus aquaticus.  Pomphorhynchus laevis of the family  Pomphorhynchidae is a common parasite of 

cyprinids and other fishes in Europe. Its intermediate hosts are various species of  freshwater gammarids.

Fig. 15: A-E, I - Acanthocephala (A-C - Pallisentis, male; D - Acanthocephalus, E, I  Neoechinorhynchus); F-H - Mollusca (glochidium of Unio); J-L - Hirudinea (J, K Acanthobdella, L - Piscicola). Crustacea - crustaceans Species parasitic on fishes are found in higher as well as in lower classes of  Crustacea (Fig. 16). They occasionally undergo structural modifications of various degree called forth by the adaptation to parasitism which involve fusion (partial or complete) of  segments of various parts of the body and reduction and change in the limbs. In addition a progressive development of attachment organs, simplifications of external morphology (development of various hornlike, rootlike and lobed processes, etc.) occur. This review will take in turn three major divisions of crustaceans.

Key to major crustacean groups 1 Entire dorsal surface of body divided into many narrow segments, tagmata poorly developed; parasite immovably attached to external surface, buccal or branchial cavity of fish.............................. Isopoda - Dorsal surface of body with reduced segmentation and well developed tagmata, or  unsegmented.............................. 2 2 Pair of compound eyes; body covered anteriorly with dorsal shield; main attachment organs suckers of modified first maxillae; parasite capable of movement over host surface.............................. Branchiura - Compound eyes absent; double median eye present or absent; shape of body varied;  parasites anchored and sessile, attached more or less firmly to surface on fish, or  able to move over its surface.............................. Copepoda Copepoda

Undoubtedly the largest group of  Crustacea parasitic on fishes, Copepoda have among them species showing various stages of adaptation to parasitism. At one end of  the adaptive range are those ectoparasites whose structure has not yet been greatly affected by their association with the host. Some of them are freely mobile, able to swim and to change one fish host for another. At the other end of the range there are completely sessile endoparasites, sometimes with greatly reduced appendages and shapeless, sac-like bodies. There are known some 1,700 species of copepods parasitic on fishes. A large  proportion of them belongs to the suborder  Siphonostomatoidea (75%), some 20% to  Poecilostomatoidea, and only about 5% to Cyclopoida. The members of the first two major taxa are predominantly marine, whereas those of the third one are exclusively freshwater. Copepods infest the skin, gills, nasal cavities, buccal cavities, and the fins of infected fishes. Their development is mostly straight, without a change of hosts. Massive infestation with copepods, particularly with  Ergasilus, Lernaea and Salmincola often cause fish epizootics and mortality. The copepods parasitizing freshwater fishes belong to three suborders, distinguishable from one another mainly by their mouth parts. Key to suborders 1 Mouth forming short, subcylindrical tube, sometimes situated at apex of more or  less prominent cone; mandible rod-shaped, with flat distal part usually, though not invariably, bearing row of teeth.............................. Siphonostomatoidea - Mouth and mandible not as above..............................2 2 Mouth not forming tube, more or less gaping; mandible falcate.............................. Poecilostomatoidea

- Mouth not forming tube, more or less gaping; mandible small, with unciform, unarmed distal segment.............................. Cyclopoida Suborder Cyclopoida

This suborder contains many species parasitic in fishes; practically all species are freshwater. Most of them belong to the family Lernaeidae; it is rather evenly divided into two groups of species, the first of them not overly modified and still retaining most, or at least some, of their segmentation, the second highly metamorphosed and usually possessed of a more or less well developed holdfast as an attachment organ.  Lamproglena pulchella and some 20 other congeneric species are parasitic on the gills

of freshwater teleosts in the old World. The segmentation of their body is partly retained. The above species is a frequent parasite of many species of cyprinids and  pike in Europe and Central Asia.  Lernea cyprinacea is a representative of the genus including about 40 species

 parasitizing freshwater teleosts in Europe, Africa and North and South Americas. The metamorphosed female has a diminutive, semispherical cephalothorax followed by a well developed holdfast consisting of simple or dividing branches. Males are not  parasitic. The anterior part of the parasitic female is embedded in musculature of the fish, its posterior part is protruding. Penetrating deep into the tissues of the fish and developing within them a large holdfast, it causes extensive tissue damage and has  been known to bring about mass kills of valuable fish stocks. Suborder  Poecilostomatoida This suborder contains many species and genera representing a very wide range of  adaptations to parasitic life, beginning with forms which are only slightly modified from the ancestral free-living condition, and ending with forms strongly metamorphosed under the impact of parasitism. Both ectoparasites and endoparasites are found among them. Most of them are small and tend to be more or less mobile and less prone to cause extensive tissue damage.  Ergasilus sieboldi is a widely distributed parasite of gills of many European

freshwater teleosts, causing frequently problems in some pond-reared fishes,  particularly tench. It belongs to the family  Ergasilidae distributed in various continents. Only the female is parasitic. The entire life cycle and the process of  mating occurs in water, before the females find and settle on their hosts. Suborder Siphonostomatoida It comprises a significant majority of copepods parasitic on fishes, infecting them in  both freshwater and marine habitats. Their general diagnosis is very difficult because of the great morphological variety. Salmincola salmoneus is a member of the family  Lernaeopodidae comprising about 250 species distributed in the fresh waters in the northern hemisphere and in the oceans. The genus Salmincola contains some 17 species parasitic on salmonids and their relatives. The above species is parasitic on the gills of salmons.

Fig. 16: Crustacea. A - Ergasilus sieboldi, B - Lamproglena pulchella, C - Salmincola salmonea, D - Lernaea cyprinacea, E, F - Ichthyoxenus amurensis, G, H - Argulus coregoni.  Branchiura

A rather small group, comprising fewer than 150 species grouped in 5 genera; as many as 100 of them belong to the genus  Argulus which has a world-wide distribution, with species in both marine and freshwater habitats. They are part icularly important in freshwater aquaculture, where their harmful impact upon fish can assume devastating proportions. Although unequivocally parasitic, they are capable or  spending prolonged periods without a host. They mate free-swimming and deposit eggs in characteristic clusters on various submerged objects. They are also capable of  moving from fish to fish. Most species are only loosely specific and may be found on the skin and gills of many fresh water fish species.  Argulus foliaceus is one of the most distributed species of the genus, occurring in Europe, Palaearctic Asia and some other continents, parasitizing there many fish species.

 Isopoda

Parasitic isopods found on fishes all belong to the suborder  Flabellifera and are found through the world, virtually in all habitats, throughout by far the greatest number are marine. They are well adapted to their parasitic way of life and can cause considerable damage to the host tissues at the infection site. They are important parasites in certain freshwater environments of South America and Africa, where they infect fishes used locally for food. Isopods associated with the external surfaces of fish sometimes produce gall-like depressions in the skin and muscles of the body wall. Others live in the buccal or   branchial cavities.  Ichthyoxenus amurensis penetrates the skin, entering the body cavity in the region of 

the pectoral fins of some freshwater cyprinids in the Far East. It feeds on blood. Hirudinea -

leeches

Leeches (Fig. 15) are annelid worms with more or less uniformly metameric bodies. Only about a quarter of leech species are parasitic, their parasitism being of a temporary, intermittent character. Although they vary quite widely in their  morphology, they are always large enough to be observed with the naked eye, narrowly oval or lanceolate in outline, usually more or less dorsoventrally flattened and provided with anterior and posterior suckers; the latter is always larger than the former and fairly regularly discoid. The body is always transversely striated, the grooves dividing it into very definite annuli, three of which usually correspond to a single segment. The most important anatomical feature of leeches is their alimentary canal whose capacity is enlarged by numerous diverticula; their number and arrangement are important taxonomic characters. Unfortunately, they often require serial sectioning for accurate examination. Leeches from fishes have been reported as feeding on host's blood, body fluids or ingesting tissue particles. Key to subclasses of  Hirudinea 1 Anterior end of body with ventral bristles. Anterior sucker absent or if present, its  posterior lip weakly developed.............................. Acanthobdelliones - Anterior end of body without ventral bristles. Anterior sucker with well developed  posterior lip.............................. Hirudiniones Subclass Acanthobdelliones This subclass is represented only by two species,  Acanthobdella peledina and  Paracanthobdella livanowi, belonging to two monotypic families differing from each other mainly by the presence or absence of the anterior sucker. Both species are  parasitic on the skin of various salmonids of the Palaearctic Asia and, in the first species, also of Europe. Subclass Hirudiniones

This subclass is represented by some 400 species of which members of the families  Piscicolidae and Glossiphonidae , including several genera, are parasitic on fishes.  Piscicola geometra with the usual body length 15–30 mm is a widespread freshwater 

 parasite of many fish species of different families and orders of the Palaearctic Region. It is usually found on the body surface, fins and sometimes in buccal and  branchial cavities. Cystobranchus mamillatus is, as the foregoing species, a member of  Piscicolidae. It is

a specific parasite of burbot of the Palaearctic Region; it is found in the branchial cavity and on gills.  Placobdella sp. of the family Glossiphonidae has been reported from some freshwater  fishes ( Anabas, Channa, Puntius, Tilapia, Trichogaster ) from Thailand. Congeneric

species occur only on freshwater fish (but also on aquatic birds, reptiles and amphibians). Mollusca - molluscs

The freshwater bivalve moluscs of the family Unionidae, during their early development, pass through a larval form known as the glochidium (Fig. 15). Hatching out from eggs incubated between the gill lamellae of the parental individual, glochidia are expelled into the surrounding water, where they can survive for a limited period of  time. Glochidia are miniature bivalves, the margins of their shells equipped with sharp teeth; a filamentous tentacle is situated between the valves. To survive, the glochidium, which is often narrowly host specific, must find a fish. Glochidia become encysted on gills, fins and skin of the fish where the young molluscs undergo extensive development and the definitive shell replaces the early larval valves. Upon maturation, which takes three weeks to several months depending on the water  temperature and certain other conditions, they fall to the substrate as young mussels. If the fish gills are attacked by a very large number of glochidia, the fish may suffer  from respiratory difficulties. The escape of glochidia from their cysts leaves open wounds that are subject to fungal or microbial infections. For identification, glochidia should be removed from fish gills, fins and skin without any squashing or deformation of their shape, because size and shape are distinctive characters of species. The size of glochidia of a given species of mussel may vary with the habitat; the glochidia infesting river and lake fishes tend to be slightly larger  than those infesting small stream fishes. However, since the pre-parasitic and parasitic stages of many species of glochidia are nearly equal in size, the identification made can thus be ascertained by comparing the size of glochidia from gravid mussels of  known species collected from the same area of study. Recommended literature Anderson R.C., Chabaud A.G., Willmott S. (eds.) (1974–1983): CIH Keys to the nematode parasites of vertebrates. Nos. 1–10. Commonwealth Agricult. Bureaux, Farnham Royal, Bucks, England.

Bauer O.N. (ed.) (1985): Key to parasites of freshwater fishes of the USSR fauna, Vol. 2. Parasitic Metazoa, Pt. 1. Nauka, Leningrad, pp. 424. (In Russian.) Bauer O.N. (ed.), 1987: Key to parasites of freshwater fishes of the USSR fauna, Vol. 3. Parasitic Metazoa, Pt. 2. Nauka, Leningrad, 583 pp. (In Russian). Crompton D.W.T., Nickol B.B., (1985): Biology of the Acanthocephala. Cambridge Univ. Press, Cambridge, London, New York, New Rochelle, Melbourne, Sydney,  pp. 519. Fernando C.H., Furtado J.I., Gussev A.V., Hanek G., Kakonge S.A., (1972): Methods for the study of freshwater fish parasites. University of Waterloo, Canada, Biology Series No. 12, 76 pp. Margolis L., Kabata Z. (eds.) (1984–1989): Guide to the parasites of fishes of Canada, Pts. i, II, III. Can. Spec. Publ. Fish. Aquat. Sci. 74, 101–107. Yamaguti S. (1971): Synopsis of digenetic trematodes of vertebrates. Part I, II. Keigaku Publishing Co., Tokyo, pp. 1074 + 349 Plts. 2.2 Prevention and Therapy of Fish Diseases 2.2.1 General Principles of Prevention

(J. Tesarčík, Z. Svobodová) The ways of prevention and contingently of medical treatment of fish are very specific and often different from those in warm-blooded animals. They require a thorough knowledge of the environment of fish. Preventive arrangements are consisting of complicated set of treatments elaborated on the base of a good knowledge of the aetiology of disease and a host (fish) biology. It concerns the elimination or restriction of infection (invasion) sources and the possibilities of its further expansion likewise the enhancement of condition of fish organism in the way to be able to withstand the infection (invasion). The prevention is of basic importance in diseases elimination. No specific therapeutics were developed for a number of  diseases up to now and the result of the application of effective, experimentally verified medicaments, is often reversely affected by the operational conditions and/or  the technology of rearing. The medical treatment becames economically unrenumerative in this way. In addition, some treatments cannot be performed in certain periods, e.g. in growing season, during the wintering, or in some fish culture units (e.g. large ponds). That is why it is much more important to prevent from the diseases than to recover  them. The effective preventive treatments are to be applied above all in specialized fish culture units with closed warm water system, in early fish fry rearing, hatcheries, trout farms, wintering ponds and storage reservoirs. Generally accepted and effective principles are as follows:

a) Providing water sources free of pathogens

Underground waters are the most suitable water sources free of pathogens. These sources are limited both for trout farms and hatcheries and for other special fish culture units at present. The surface water from rivers and channels is used as the source of inflow water in most cases. In these situations, suitable filters can partially reduce the numbers of invasion stages of parasites in inflow water, above all when supplying smaller reservoirs with intensive culture. Bars are usually placed before these filters to separate rough particles. Sand filters are consisted of a set of  sedimentation divisions terminated by filter with fibre and sand. These type of filters catch above all the heavier parasite stages unable to move actively (e.g. spores). Lower efficiency is registered in elimination of moving parasites like e.g. infusorians. The water from the pond with fish stock is quite unsuitable for these purposes (esp. as the source of inflow water for trout farms, hatcheries and units for early fish fry stages). Chemical treatment of inflow water is an emergency arrangement with often undesirable parallel affects. Disinfection of the water entering fish culture units by UV radiation is not still an usual way although it can be considered as the simple method how to destroy viruses, bacteria and moulds germs. Since the inflow water  from rivers and channels is slightly turbid and contents a number of suspended solids and dissolved compounds, the disinfective efficiency of UV radiation is markedly reduced in these situations. It is very profitable to supply the individual ponds and/or reservoirs independently, not throughflowly. The water from each pond or reservoir should be drained separately and should not flow into any other. Especially quarantine ponds and other  reservoirs can be separated by this way. b) Protection from the transfer of pathogens

This principle means above all the transfer of pathogens by uncontrolled transport of  fish and spawns. The transport of fish with unknown health condition is to be avoided in principle. All transported fish are to be accompanied by veterinary certificate confirming that fish were examined before transporting them, they are healthy and originate from the environment in which no important transfer diseases appear. The list of these diseases is precisely stated in veterinary instructions. Except of the internal survey for each country also the list of diseases stated in international codex is obligatory for veterinary service. This list is currently specified with the development of diagnostic methods and improvement of knowledge about individual fish diseases. Some viral and bacterial diseases can be transfered also by spawns. Their transport must be completed by the same veterinary certificate like fish transport from this reason. Fish introduced from other territories must be subjected to quarantine for one year  regardless if native or extraneous species. The duration of quarantine can be  prolongated e.g. in the case of fish imported from abroad until the period of 3 years. Prolongated period of quarantine is of special importance especially in spawners  predestined for further reproduction of imported species.

The selfsustaining in stock production in individual farms and similar organizations is a significant way of prevention from dissemination of fish diseases. Only fish  previously examined, free of diseases and relevantly treated by medicinal baths are to  be stocked into ponds and fish culture units. The stocking of fry originating from semi-artificial and artificial spawning not contacted with fish of higher age categories also minimizes the danger of infection. The prevention from introduction of coarse fish into ponds and fish culture units is the other important arrangement protecting the stock against transfer of pathogens. These fish are above all the source of ectoparasites, dangerous especially in the period of  decreased resistence of fish. Except of this they can transfer also some other   pathogens which can result in heavy losses in important fish species. Adequate bars and filters can serve for prevention from coarse fish penetration. The protection of piscivorous birds to step into fish culture units (esp. trout farms) is the prevention limiting the expansion of some fish diseases. Protective nets are used to prevent the birds from running in. The numbers of piscivorous birds are regulated in localities where overpopulated. Preventive control of snails ( Lymnaea sp.) as intermediate hosts of some fish parasites can be performed by biological (introduction of black carp -  Myelopharyngodon  piceus or 3-years-old tench Tinca tinca), mechanical (placing nets in the inflow),  physical (drying and freezing of the bottom) and chemical (application of  molluscocides) ways. Safe and harmless removing of dead fish is a significant way how to prevent from further transfer of fish pathogens. Fresh or slightly decayed dead fish are decontaminated in the nearest veterinary facility. Lower masses of dead fish are to be  burnt or burried into deep pits (aprox. 2 m) in distance of at least 20 m from the pond  bank. The bottom of this pit and dead fish must be covered by burnt or chlorinated lime. The layer of at least 60 – 80 cm of the soil must cover the content of a pit. c) Disinfection of ponds, fish culture units and equipment; winter freezing and summer drying of ponds

Disinfection is of a big importance in prevention and elimination of fish diseases. Preventive disinfection protects the fish stocks against pathogens. Hygiene of  environmental conditions for fish is improved by this way. Focal disinfection is  performed for control of the focus of dangerous fish disease.  Natural physical phenomena are fully used for disinfection in intensive fish culture due to their ecomical convenience. It concerns the drying and freezing of the pond  bottom. The most of pathogens die after perfect drying of the pond bottom when its relative moisture had dropped on 10 – 15 %. The perfect freezing of the wet places and sun radiation (above all by its UV rays) have a very favourable effect in our  conditions. The influence of these natural physical phenomena is exploited by summer drying and winter freezing of water reservoirs (ponds). Summer drying is a radical, long-term intervention during which all pathogens are controlled due to the  perfect drying of the pond bottom. The aim of winter drying is to destroy the  pathogens by freeze. It safely leads to destruction of leeches ( Piscicola geometra),

fish lice ( Argulus sp.), predatory larvae of water insects, eggs and spores of parasites and also other pathogens. Employing the natural ways for disinfection has an disadvantage in usually long-term duration (a number of months up to one year). Chemical disinfection is an effective way of prevention from and/or suppressing of  fish diseases. Usually accessible disinfective preparations are used in fish culture (e.g.  burnt lime, chlorinated lime, nitrogen lime, natrium hydroxide, potassium  permanganate, formaldehyde, Chloramine, Chlorseptol, Jodonal etc.). Burnt lime is mostly employed for disinfection of the bottom of ponds and reservoirs in the dose of  2.5 – 3 t.ha -1, or chlorinated lime in the dose of 0.5 – 0.6 t.ha -1. In case of  myxosporoses, nitrogen lime (5 t.ha -1, or 0.5 kg.m-2) is to be applied. Immediately after fishing out the pond, the disinfection of fishing pit, pond ditches and muddy wet  places is performed on large ponds where the whole-surface bottom disinfection is not  possible. 5% water solution of formaldehyde, chlorinated lime (200 – 400 mg.1 -1), 0.5 % water solution of natrium hydroxide, Chloramine and chlorseptol (30 g.1 -1) or other  disinfectants can be used for treatment of concrete channels, troughs and other  arrangements employed for fish culture. The same disinfectants and concentrations are to be used for the treatment of the equipment. Potassium permanganate (5 g.l -1), Jodonal (2.8 – 4.5 ml.l -1) and other disinfectants can be also employed for these  purposes. d) Optimalization of environmental conditions

The optimalization of natural environmental conditions is the main pre-condition how to ensure the good health condition of stock during the rearing period. The following  principles must be ensured: 







optimal water quality without stressing physico-chemical effects. Keeping the oxygen concentration on optimal level and protection against water pollution are of special importance, maximum development of natural food resources by the adequate interventions, feeding fish by supplementary feed mixtures in sufficient amount and quality (the attention should be paid on the quality of individual feed components and biofactors), basic preventive arrangements protecting the early developmental stages and young fish from bacteria and protozoans, including sufficient amount of natural food of appropriate size and species composition, responsible establishment of maximum stocking density. Inadequately high stocking density results in stress behavior, worsened condition and resistence, and makes the expansion of diseases easier. The stocking density is of special importance in trout farming and fish culture in special intensive units (but also in ponds),  prevention from stress situations evoked by other factors, above all manipulation during fishing out, transport and long-term storage.

e) Regular control of health condition and preventive treatment of fish

Preventive control of health condition is to be carried out in hatcheries and early fry rearing units twice a week, and in highly productive intensificated ponds, trout farms

and fish culture units with recycling warmed water weekly. Other stocks (esp. in usual  pond culture) are investigated monthly. Health condition of fish is always to be controlled before fishing out, transporting fish and stocking. Preventive treatment can be suggested on the base of investigational results. This treatment is performed above all by the application of medicaments into the water environment and feeding by medicated feeds. More detailed principles of  this type of treatment are presented in Chapter 2.2.2. f) Other preventive principles

The ways of prevention from individual, most important viral, bacterial, fungal and  parasitic diseases are described in adequate individual chapters. Specific, very effective way of prevention from diseases is the vaccination of fish. Vaccines against following relevant viral and bacterial diseases are recently tested with different success: CCV, IPN, SVC, VHS, IHN, furunculosis, ERM, and vibriosis. Individual vaccines are applied intraperitoneally, perorally or in the form of   bath. Peroral application or bath are most suitable ways from the point of view of fish culture practice. Also vaccines against some other fish diseases including parasitoses are currently developed. Recommended literature Bauer O.N., Musselius V.A., Strelkov Ju.A. (1981): Diseases of pond fish. Izd. Legkaja i piščevaja promyšlennost, Moskva, pp. 318 (In Russian). Ellis A.E. (1988): Fish vaccination. London, Academic Press, pp. 255. Lucký Z. (1978): Veterinary care in fish culture. ÚVO, Pardubice, pp. 205 (In Czech). Prost M. (1989): Fish diseases. Warszawa, PWRiL, pp. 460 (In Polish). Reichenbach-Klinke H.H. (1980): Krankheiten und Schãdigungen der Fische. Gustav Fischer Verlag, Stuttgart, New York, pp. 472. Roberts R.J. (1989): Fish pathology. Ballière Tindall, pp. 467. Schãperclaus W. et al. (1979): Fischkrankheiten. Akademie-Verlag, Berlin, pp. 1089. Svobodová Z., Zajíček J. (1989): Control of fish protozoases. In: Lom J., Dyková I.: Protozoal parasites of important fishes. SZN, Praha, pp. 102 (In Czech). 2.2.2 General Principles of Therapy

(Z. Svobodová) Fish are subjected to therapy in those cases when a disease is so developed that the life or performance of the fish is immediately endangered or expected to be

endangered in the subsequent period. Therapeutic treatment should be regarded as emergency measure resorted to when prevention has failed. The therapeutic treatments may be as follows: a. application of therapeutic substances and preparations to the aquatic environment (therapeutic baths for fish and eggs)  b. administration of therapeutic substances in feed c. administration of therapeutic substances via a probe d. administration of therapeutic substances by means of injections Application of therapeutic substances and preparations to the aquatic environment (therapeutic baths for fish and eggs)

Therapeutic substances are put into water to control ectoparasitic, fungal and bacterial diseases of the body surface and the gills. In some cases the therapeutic baths can also  be used (after absorption of the active substances via the skin) for controlling the causative agents of internal diseases. According to the lenght of exposure, the therapeutic baths are subdivided as follows:   

immersion baths (up to 5 minutes) short-term baths (5 minutes to 2 hours) long-term baths (2 hours to several days)

The long-term baths also include the treatment, with therapeutic substances, of whole fish culture reservoirs and ponds. A list of preparations and substances most frequently used for the different types of baths is given in Table 7. Table 7: Chemical substances used for therapeutic baths of fish Type of therapeutic bath immersion short-term long-term lysol NaCl malachite green lime milk formaldehyde trichlorphon KMnO4 malachite green acriflavin ammonia and malachite green antibiotics trypaflavin and formaldehyde Metronidazol malachite green NaCl CuSO4.5H2O KMnO4 formaldehyde KMnO4 General principles of therapeutic baths for fish To perform the therapeutic baths effectively and to avoid losses of the fish, a number  of general principles must be respected, including:

a) The state of health of the fish stock must be continuously monitored so that the most effective therapeutic bath can be promptly chosen and applied: fish in an advanced phase of a disease are exhausted and weak and can be easily killed by exposure to the drug in the bath.  b) The results of examination of the fish serve as a basis for determining the type of  therapeutic bath. Most of the therapeutic preparations are toxic to the fish at higher  concentrations, so the instructions have to be strictly adhered to. The substances and  preparations used for the baths must be fresh, packed in original containers. The dose to be used in the bath must be accurately calculated to avoid poisoning the fish by overdosage, or to avoid a poor effect if the dose is too low. If the instructions state a range of doses between two limits, then the lower amount is given to the weakened fish and the higher one to fish in good condition. The drugs must have been dissolved  before application to the water; the application itself is performed by spraying over  the water surface. With the substances and preparations used for long-term therapeutic treatment of fish in reservoirs and ponds, there should be a satisfactory difference  between the lethal concentration (LC) for the causative agent of the disease and the LC for the fish: the therapeutic index* is to be at least 4 or above 4, 10 at the maximum. These therapeutic means must be readily soluble in water and must easily  break down. c) Fresh and uncontaminated water must be used to prepare the solution for the bath. The physico-chemical characteristics of the water influence the effectiveness of the therapeutic substances and preparations and also their toxicity to the fish. The most important water characteristics include temperature, pH, concentration of organic substances, acid capacity (alkalinity), ∑ Ca + Mg and others. d) A tolerance test must have been conducted before any bath. The tolerance test is a  bioassay on several fish to see the safety or harmfulness of the therapeutic bath for the fish stock to be treated under the existing conditions. e) The therapeutic baths themselves are carried out in all-glass tanks, fibre-glass tubs, vats, fibre-glass plastic troughs, in concrete or earth storage basins or straight in the  ponds. It is also possible to subject the fish to short-term therapeutic baths in the transport boxes during shipment if the shipment time is the same as, or shorter than, the recommended exposure time. The fish should have been given no feed before an immersion bath or a short-term  bath to avoid increased need for oxygen (for example, one to three feedings are skipped on the trout farms). Fish exposed to long-term baths, with several days' exposure times, have to be fed with supplementary feeds. Emergency scenarios must  be prepared for the prevention of possible accidents: water aeration facilities must be ready for use, or precautions should be made for promptly removing the fish from the  bath and putting them in fresh (preferably flowing) water, or an emergency inlet of  clean and safe water must be available for fast dilution of the bath solution. The tanks or reservoirs with the therapeutic solutions should never be overstocked: the fish must have enough space to move freely and the solution must get to every spot on the body surface of each fish. A 100-litre bath will accommodate 30 kg of fish at the maximum and the bath solution is as a rule replaced after treating 5–10 sets of fish.

* The therapeutic index says how many times the given substance's LC for fish is higher than that for the causative agent of the disease. For long-term baths straight in the pond, the substance or preparation is either applied in a single batch into the inlet or may be evenly distributed over the water surface in the pond. For the whole period of treatment the flow of water through the pond must  be stopped and warning plates should be placed around it. Residues of the therapeutic substance must have completely disappeared before water is allowed to flow through the pond again. Treatment of the whole pond is seldom resorted to: it is carried out when the fish are in acute danger. It is a problem with such large scale baths that together with the causative agents of the disease the drug used in the bath also kills the organisms in the food chain, thus reducing the nourishing capacity of the pond. f) When the treatment is finished the fish should be removed from the bath and put into clean (preferably flowing) water. If the treatment was performed in a whole pond, the inlet source must be strong enough to allow for rapid dilution of the bath solution. All regulations and standards regarding surface water quality conservation must be respected in discharging the used therapeutic solution outside the fish culture facility. In the majority of cases the used solutions are disposed of outside the aquatic environment: for example, they are left to seep into the ground in places free of the danger of penetration into surface or underground waters. g) The effectiveness of the therapeutic baths must be checked by macro- and microscopic examination of 5 fish at the minimum from each pond or tank after the rinsing of the treated fish in clean water. This must be done immediately after the  bath, within one day of the termination of the bath at the latest. h) It is a general principle that market fish should not be treated by therapeutic baths 14 days before shipment to the market. Treatment of market fish in malachite green  bath must be avoided for 6 months before assumed time of consumption. i) All labour safety precautions must be taken during the treatment of fish by therapeutic baths. A survey of the most important chemicals and preparations used in the therapeutic baths of fish. Preparing and performing the baths

Sodium chloride (NaCl) is widely used in fish culture for parasite control during the rearing of the fish from the earliest stages of the fry up to the market fish. As the difference between the lethal concentrations of sodium chloride to fish and parasites is not very large, it is necessary during the treatment to stick to the general principles, especially the instructions concerning the tolerance tests. Zinc-coated containers should never be used for the NaCl baths. Sodium chloride is largely used in the form of short-term baths which are fairly effective in the control of the species of the genera Cryptobia, Ichthyobodo, Chilodonella, Trichodina and Trichodinella, and somewhat less effective in the control of the species of the genera  Dactylogyrus, Gyrodactylus, Piscicola, Argulus , and in the cases of the fungal diseases. The salt bath is prepared by dissolving 10 to 30 g NaCl in one litre of water. The exposure time is 15 to 30 minutes. If necessary, the salt bath may be repeated in the

majority of species. In the early stages of the fry, treated at a water temperature of 20 to 25°C, good results are obtained at a concentration of 10 g per litre and at an exposure time of 30 minutes. In cyprinid culture, NaCl concentration of 20 g per litre is used at an exposure time of 15 minutes for the treatment of weaker fry. Baths of the same characteristics may also be used for the treatment of salmonids. Stronger fry and older fish of the cyprinid group may be treated with success by a bath at a concentration of 30 g per litre for 25 to 30 minutes. It should be taken into account that at water temperatures below 5°C the effectiveness of the salt baths is substantially reduced. Sodium chloride may also be used for long-term baths (concentration of 1–2 g per litre, exposure time 1–2 days) in cases of occurrence of chilodonellosis in fish kept in storage ponds or provisional handling ponds in autumn or spring. Formaldehyde is distributed in the form of 36–38% aquatic solution. The chemical to  be used for parasite control in the fish must be a clear solution free of   paraformaldehyde sediment (white sediment on the bottom). During the bath itself, the main factor to be taken into account (among the factors underlying the effectiveness and toxicity of the bath) is water temperature. The market fish may be treated with formaldehyde bath 14 days before delivery to the market at the latest. Formaldehyde is largely used for the short-term baths to control pests of the genera Cryptobia, Ichthyobodo, Chilodonella, Trichodina, Trichodinella, Dactylogyrus, Gyrodactylus , and the fungal diseases.

The concentration of formaldehyde in the bath depends on water temperature. At water temperatures up to 10°C the concentration is 0.25 ml of 36–38 % aqueous solution per litre, at 10–15°C it is 0.20 ml per litre, and at a temperature above 15°C it is 0.17 ml per litre. The time of exposure is 30 to 60 minutes. For example, a concentration of 0.25 ml per litre and exposure time of 30 minutes at a water  temperature of 25°C are recommended for the treatment of the early fry stages of  cyprinids and catfish. Long-exposure formaldehyde baths can be used in the same cases of long-exposure  NaCl baths, the concentration of formaldehyde (36–38% aqueous solution) being 0.025–0.030 ml per litre. The solution is unrepeatedly applied to the water inlet and there is no time limit of exposure. Malachite green is deep green in colour, readily soluble in water. Exposure to a therapeutic malachite green bath without prior tolerance test may kill a whole stock. Hence, every new batch of the chemical must be tested for toxicity to fish and effectiveness of parasite control before it is used for the treatment. Some limits apply to the use of malachite green in fish culture: for example, it should not be used for the treatment of the fish later than 6 month to delivery to the market (the hygiene aspect) and at the recommended concentrations and exposure times it should not be used for  the treatment of the early stages of the fry (the fish safety aspect). Malachite green is used either for the short-exposure baths or, more frequently, for  long-exposure baths, especially for the control of  Ichthyophthirius multifiliis and also in the cases of occurrence of the species of the genera Cryptobia, Ichthyobodo, Trichodina, Trichodinella, Chilodonella and for treatment of fish against the fungal diseases. To control the fungal diseases, it is also possible to use immersion baths in malachite green (66.7 mg per litre, exposure for 10 to 30 seconds). Recently very

good results have been recorded with the use of a combined malachite green and formaldehyde bath (exposure for 2 or 6 hours). The short-term malachite green bath uses a concentration of 6.7 mg per litre and an exposure time of 1 to 1.5 hours. At water temperatures of up to 10°C it can be  performed in different types of reservoirs but when the temperature is higher it can only be done in ponds or tanks that can be drained and filled again in 30 minutes. For the long-term malachite green bath of cyprinids, the chemical is applied at a concentration of 0.5 mg per ml, after accurate calculation of the amount of water in the reservoir or tank. For salmonids the concentration is 0.15 to 0.20 mg per 1. Upon the application of malachite green and its thorough distribution throughout the tank, the water flow is stopped and aeration is provided. Twenty-four hours later the bath is replaced: the tank is drained, clean water is left to flow through it for an hour, the tank  is filled again to the same level as before and another dose of the chemical is applied. All this is done six times. For the combined malachite green and formaldehyde bath, the water in the tank or   pond should contain 0.25 mg malachite green and 0.125 ml of 36–38 % aqueous solution of formaldehyde per litre. In fibre-glass troughs the exposure time is 2 hours (with aeration provided) and in the storage ponds 6 hours. In practice this is done as follows: the pond is drained to contain half as much water as normally, the flow is adjusted to a rate at which the water is replaced in 6 hours, and the calculated amount of solution is slowly added to the water inlet. Six hours later the flow is increased to speed up the diluting process and to increase the amount of water in the tank or pond to the normal level. When fish are treated for ichthyophpthiriasis in laminated plastic troughs, it is recommended to repeat the combined bath twice or three times in one week. Substances and preparations containing copper are used for the therapeutic baths of  fish, though they have a toxic action in the aquatic environment. CuSO 4.5H2O is used most frequently for the control of some fungal, parasitic and bacterial diseases of fish. At the present time its use is limited to the control of flexibacteriosis of the gills in the salmonids. It is used in the form of immersion bath (concentration 0.5 g per litre, exposure for 1 min) and good results are also obtained when the chemical is applied to a flow-through tank. Another substance used for the parasite-control treatment of fish is copper in the form of oxychloride [3Cu(OH) 2.CuCl2.H2O]. It is used for short-term baths when the fish are found to harbour species of the genera Cryptobia, Trichodina, Trichodinella and Chilodonella . This substance is also a good molluscocide, used to control aquatic molluscs, especially those of the genus  Lymnea, which are intermediate hosts of the causative agent of serious fish parasitoses. Preparations based on copper oxychloride include Kuprikol 50, which contains 47.5 % of the active ingredient at the minimum. It is used at a concentration of 30–70 mg per litre for 15–30 min for the treatment of  common carp and grass carp. With other fishes the therapeutic dose of Kuprikol 50 is at the level of lethal concentrations. To kill the water molluscs, Kuprikol 50 is used at a rate of 15–30 kg per ha (if the average depth of the pond is 1 m).

The therapeutic efficiency of CuSO 4.5H2O and copper oxychloride, as well as their  toxicity to fish, is significantly influenced by the physical and chemical properties of  the water. Trichlorphon is used for long-term baths for cyprinids. For salmonids it is very  poisonous, so it cannot be used for therapeutic baths in these species. The  preparations on trichlorphon basis, used for the baths, are distributed under the brand names Masoten, Neguvon, Dipterex, Soldep and others. When used for fish parasite control, the trichlorphon-based preparations are applied to  ponds or other fish culture facilities (tanks, troughs) at a single dose. The minimum exposure is 48 hours. The parasites on the invaded fish are immobilized during the first day of treatment. In 24 hours the intensity of invasion is considerably reduced and the percentage of immobilized parasites highly increases, in 48 hours the treatment results in a negative parasitological finding. Preparations on the basis of  trichlorphon can only be applied to ponds and other facilities with perfectly tight outlet systems. During the treatment and as long as the residues of trichlorphon and its metabolite dichlorvos remain in the water, the flow through the pond must be stopped and there must be a warning plate on the pond dam. The water flow through the pond may be resumed 2 to 3 days after getting a negative result of the bioassay: the time of   persistence of the action of trichlorphon and its metabolites is determined by bioassay on daphnias. The average persistence time of these harmful substances in the pond is 1–2 weeks at a water temperature of about 20°C and water pH of 7–8, and 2–3 months in winter, when the water temperature and the pH are low. Long-continued exposure of pond water to trichlorphon and its metabolite, dichlorvos, kills the majority of the natural food for the fish. Owing to this, full-value feeds must be administered until the natural food organisms develop again in the pond. Two weeks must have elapsed from the day of getting a negative result of the test on daphnias,  before the fish may be taken from the treated pond for human consumption. The rates of administration of Soldep, containing about 25 % trichlorphon, can be used as an example. Soldep at a concentration of 1–2.10 -3 ml.litre-1, i.e. 10–20 litres per ha at an average pond depth of 1 m, is used to kill the species of the genera  Dactylogyrus, Gyrodactylus, Piscicola and  Argulus, and is also partly effective in the control of the genus  Ergasilus. At the same concentration, Soldep also kills the intermediate hosts (Cyclops, Mesocyclops) of some fish parasites, e.g. the tapeworm  Bothriocephalus acheilognathi.

The rates of administration of other organo-phosphorus preparations is proportional to the content of the active ingredient, trichlorphon. The use of trichlorphon-based  preparations in ponds must always be well-thought and should only be resorted to when the fish stock is exposed to immediate danger. Ammonia is used in combination with trypaflavin (acriflavin) in the form of  immersion baths to kill the pests of the genera  Dactylogyrus, Gyrodactylus and  Diplozoon; the same bath may also be used when species of the genera Trichodina, Trichodinella and Chilodonella are found in the fish. The ammonia and trypaflavin  baths are prepared from a store solution, which consists of 100 parts of 10 % NH 4OH and one part of 2.5 % aqueous solution of trypaflavin. The solution for the bath itself  is prepared by diluting the store solution with water at a rate of 1:1000. The time for  which the fish are left in the bath depends on water temperature. At temperatures up

to 12°C the exposure time is 2.5 min, at temperatures above 12°C (up to 20°C) the fish are treated for only 1.5 min. No baths are performed at temperatures above 20°C. Owing to the toxicity of ammonia to fish at higher water temperatures and water pH, the use of ammonia and trypaflavin baths has been much less frequent in recent years. Acriflavin (trypaflavin) is a brown-red crystalline powder soluble in water. The recommended therapeutic acriflavin concentrations are several times lower than the lethal concentrations to fish (the therapeutic index is about 5). For this reason, acriflavin baths can be regarded as comparatively safe to fish. Acriflavin is used in the form of long-term baths (concentration of 10 mg per litre, exposure for 10 hours), most commonly in aquarium fish culture: owing to the long exposure time, these  baths are not very common in fish farming. Acriflavin controls protozoan parasites of  fish and bacterial diseases on the surface of the fish body. German authors recommend to use long-term acriflavin baths at a concentration of 3 mg per litre (exposure time 12 hours-repated three times) for the control of local flexibacterioses in trout, eel and carp. Lime milk is prepared by dissolving 2 g of newly burnt lime in one litre of water. It is used in the form of immersion baths to kill  Piscicola geometra. Carp fry are exposed to this bath for 5 seconds. For cachectic stock fish after poor hibernation the exposure time is 10 seconds and for stock carp in good condition, and for older carp, the exposure time ranges from 15 to 20 seconds. Lime bath is not recommended for fish with sensitive gills (pike, trout). Lysol is a disinfectant aqueous solution of cresol with potassium soap. It is used at a concentration of 2 ml per litre in the form of immersion baths (5–15 seconds) to control the species of the genera  Argulus and Piscicola. Lysol is not recommended for   brood fish of salmonids. Potassium permanganate is used in the form of immersion baths (1 g per litre, 30–45 seconds), short-term baths (0.1g per litre, 5–10 min; 0.01 g per litre, 60–90 min) as well as long-term baths for the control of fungal diseases, parasites (when protozooses occur) and bacterial diseases. A potassium permanganate bath at a concentration of 1 g per litre for 150 second was tested with good results for the control of  Eudiplozoon nipponicum in higher age categories of carp. This bath cannot be used at temperatures higher than 10°C. Long-term treatments are performed in storage ponds or other   ponds for easy fish handling; the concentration is 0.3 to 0.6 mg KMnO 4 per litre of  water, exposure time 12 hours. The therapeutic doses of potassium permanganate are very close to the lethal concentrations to fish, so the treatment must be performed very carefully, especially with the aquarium fishes. It should be borne in mind that in summer when the water is warm these baths may be dangerous to fish. When brood fish are handled, local injuries on their bodies are treated with a pledget or sponge soaked with potassium permanganate. Antibiotics are recommended to be used in the form of therapeutic baths to control  bacterial diseases of the skin and gills of fish. These baths are used mainly in aquaristics and today also in rearing young stages of fish in special fish culture facilities. Before the treatment, the antibiotic must be well determined as to its  performance in the control of the bacteria responsible for the disease the fish suffer 

from. The therapeutic doses of antibiotics are in the order of tens of mg per litre at long-term baths and in the order of hundreds of mg at short baths. Entizol, whose active ingredient is metronidazol, can be used for baths at a concentration of 4 mg per litre for 2–3 days. Metronidazol is absorbed via the gills and produces in the blood a therapeutically effective concentration to kill parasitic  Flagellata, e.g. the genus  Hexamita. The bath is particularly suitable for the treatment of aquarium fishes. The method of treatment using a temporary increase in water temperature is  performed by successively increasing the temperature of the water with invaded fish to 31–32°C for 3 days and then reducing the temperature again to the starting level. The fish stock gets rid of the infection and acquires an appreciable level of immunity. In fish culture practice this method is used to control ichthyophthiriasis mainly in aquarium fishes and in special warm-water fish facilities. In the rearing of the early stages of cyprinids and catfish, warming is the only efficient and practically applicable method of ichthyophthiriasis control. Therapeutic baths of the eggs

Malachite green, formaldehyde and sodium chloride are most frequently used in fish culture practice for the control of the fungal and bacterial diseases of fish eggs. Malachite green bath provides a good treatment of the eggs of carp, tench, sheatfish,  pike, whitefish and salmonids; its concentrations range between about 5 and 10 mg  per litre and exposure times are 5 to 30 minutes once to twice daily. Malachite green is not used for the treatment of the eggs of herbivorous fishes: formaldehyde is better  for this purpose, its concentration being 0.05 to 0.35 ml per litre and exposure time 10 minutes once in two hours. Formaldehyde bath can also be used for the treatment of  other fishes eggs. Salmonid and whitefish eggs may also be subjected to an immersion  bath of sodium chloride at a concentration of 20–50 g per litre. Acriflavin (500 mg per  litre, 20–30 minutes) is also recommended for these fishes. Besides these traditional  preparations, combined-action iodine-detergent, Jodonal preparations such as e.g. Wescodyne or Incodyne have recently been used on an increasing scale: these  preparations control fungi and bacteria as well as the virus diseases of fish eggs. Administration of therapeutic substances in feed

Administration of drugs contained in feed is now practiced increasingly frequently in all types of fish culture. This approach is advantageous, hence promising, mainly from the point of view of fish farm operation. With cyprinids, the stock must have  been attracted and concentrated, as far as possible, around the feeding places, and habituated to the administered feed, before the treatment itself can be started. Administration of the same feed as normally, but containing the drugs, may be  performed when there is plenty of oxygen in the water and the fish take the feed greedily. In larger water reservoirs it is difficult to habituate the fish to regular  feeding, especially in those reservoirs where a larger amount of natural food is available. With salmonids it is very easy to administer drugs with feeds. Before the treatment it is recommended to skip one feeding to be sure the fish will take the medicated feed as soon and as greedily as possible. The disandvantage is that the diseased fish take successively decreasing amounts of the feed offered to them.

Heavily infected or invaded individuals do not take food at all, so the treatment has no effect on them. The therapeutic drugs are administered either as medicated granulated feeds or are admixed to the feeds straight on the fish farm. In the medicated granulated feeds the drug is incorporated in the pellets. The pellets are hard but they soften and swell in contact with water, where they remain compact for 12 hours. Four medicated feeds are available in Czechoslovakia at present. These are: VR (formerly called Karpex; the 5 kg packages distributed through pharmacies are called Rupin). Composition: chloramphenicol palmitate 2.173 g, vitamin A 50 000 i.u., vitamin D 3 25 000 i.u., methylene blue 0.3 g, saccharin 0.05 g, anise oil 0.4 g, stabilizers and obduction substances 59.68 g, wheat flour added to make 1 kg. Indication: treatment of carp for erythrodermatitis, possibly also to control other   bacterial diseases in cyprinids. Administration: administered in the feeding place at a rate of 15 g per 1 kg of the weight of the stock per one feeding. The treatment is repeated 4 to 8 times in an interval of 2 to 3 days, depending on water temperature (two-day intervals are used when the temperature is above 20°C). The feeding must always be adjusted so as to let the fish consume the preparation within 12 hours of administration. VR-NeO (small packages are labelled Rupin-NeO). Composition: oxytetracyclin 1.33 g, neomycin 0.67 g, methylene blue 0.3 g, feed flour to 1 kg. Indication: infectious diseases of cyprinids caused by oxytetracyclin- and neomycinsensitive germs. Administration: administered in the same way as VR. Taenifugin carp Composition: niclosamide piperazine salt 7 g, obduction, auxiliary and appetizing substances 46.25 g, ground limestone 100 g, wheat flour to 1 kg. Indication: bothriocephalosis, caviosis and caryophyleosis of cyprinids,  proteocephalosis of rainbow trout. Administration: Taenifugin carp is used in any season of the year when the fish take food. It is administered in the usual feeding places either once or repeatedly in 48 hours to allow a maximum number of the fish to take the pellets. The amount given to the fish should be equal to 1–2 % of the weight the fish stock has at the time of  treatment. The efficiency of the treatment must be checked by a parasitological examination of the guts 2–4 days after the treatment. If live tapeworms are found to occur again, the treatment should be repeated. Chronicin salm Composition: chloramphenicol 30.0 g, potassium propionate 100 g, feed flour to 1 kg. Indication: furunculosis of salmonids. Administration: Offer the medicated feed in the place where the common feed is normally given, do this daily for 7 days. The amount administered should correspond

to 1 % of the weight of the stock at the time of administration. The administered dose of about 30 mg per 1 kg of fish weight is to provide the fish with an effective chloramphenicol level for 24 hours. Other drugs administered in feed include, in particular, various antibiotics, sulphonamides, furazolidon, carboneum tetrachloratum, Entizol and others. These are admixed into the feeds just before administration. The best cyprinid feeds to carry the drugs are wheat groats or wheat flour high in gluten; for salmonids the best feed for  such purposes is ground spleen or ground beef. Antibiotics administered in feeds are used for the control of the bacterial diseases of  fishes. Chloramfenicol has been the most widely used antibiotic: it has a wide spectrum of action but at the same time a number of adverse side effects, as demonstrated in recent studies. Chloramfenicol is administered at a rate of 40–60 mg  per kg of live weight of the fish for 10–14 days. Its use is now declining, owing to the mentioned side effects. The best antibiotic for the treatment of each particular disease should preferably be selected on the basis of the results of antibiotic sensitivity tests in the pathogenic bacteria. Sulphonamides can also be used with success for the treatment of bacterial diseases of  fish, especially furunculosis in salmonids. They are administered in feed at a rate of  0.1–0.25 g per 1 kg of live weight of the fish for 8 days. Furazolidon has been tested with success in the control of the bacterial diseases (especially furunculosis of salmonids and erythrodermatitis of carp) and parasitic diseases of fishes (particularly hexamitosis of salmonids and partly also coccidiosis in carp). To treat the fish suffering from bacterial diseases, furazolidon is added to the feed at a rate of 0.1–0.2 g per 1 kg of the live weight of the fish and is administered for 8 days. For the control of hexamitosis of trout the rate is 0.5 g per 1 kg of the weight of the feed and the administration is continued for 10–14 days. Carboneum tetrachloratum (CCl4) is used for the control of the most widespread spiny headed worm,  Neoechonorhynchus rutili. Equal parts of CCl4 and paraffin oil are added to dry feed (groats) and the rate of administration (in single treatment) is 0.5 ml CCl4 per 1 kg of live weight. Entizol (active ingredient metrinidazol) is a good therapeutic preparation to treat hexamitoses at a rate of 0.25 g per 1 kg of feed, the treatment being continued for 3 days. Administration of therapeutic substances via a probe

This method of drug administration is resorted to in exceptional cases to treat limited numbers of fish, e.g. for the control of bothriocephalosis and caviosis in the brood fish at sites with the occurrence of these diseases, before the brood fish are transported to another area or country. The therapeutic substance, e.g. nitrosamine piperazine salt, is dissolved in semiliquid starch gel, which is prepared by boiling about 60 g of food starch (Solamyl) in 1 litre of water. In cyprinids the drug is administered via a thickwalled elastic hose of plastic material, connected with a syringe. The hose is introduced along the central longitudinal axis of the upper palate. The moment when

the hose hits the pharyngeal teeth can be clearly identified (by feeling the mild stroke). At this moment the hose should be inserted, with slight twisting, between the  pharyngeal teeth and the crushing plate. The hose should be pushed in very lightly and only to a depth where it opens into the gullet (Fig. 17), and then follows the administration of the drug.

Fig. 17: Administration of drugs via a probe Administering therapeutic substances by injection

In the past the injection method of administration of therapeutic substances was used on mass mainly in the treatment of stock carp. Intraperitoneal administration (into the  body cavity) was used mainly with chloramphenicol and later also with the vaccine against spring viraemia (prevention of the disease). However, mass use of these treatments is now becoming less common because of the great laboriousness and of  the frequent mechanical injuries and stresses. The therapeutic and preventive substances are administered in feed, as far as possible.  Nevertheless, injection treatment will continue to be practiced in small groups of fish, especially the brood fish. Brood fish may receive in this way, for example, different antibiotics, vaccines, sexual hormones (in the prespawning period) and other  substances; T-globulin injections are used in Poland to increase non-specific resistance of brood fish and their progenies. The drug or sexual hormone is injected into the body cavity (intraperitoneal administration) or into the muscle (intramuscular  administration). For the intraperitoneal injection, the site where the needle is to be injected is on the left side of the fish body at the point of intersection of two fictitious lines, the first starting at the base of the pectoral fin and runing along the longitudinal axis of the  body and the other starting at about the centre of the pelvic fin and running  perpendicularly to the first one (Fig. 18). The angle at which the needle is introduced

into the body is also important. In the scaleless fish it should be 20 to 30 degrees, in scaly fish it should be 10 to 15 degrees, the needle passing between two successive scales (Fig. 18). The drug flows easily from the needle introduced in the body wall, visible blotches occur under the skin. For the intramuscular administration to the carp, the site of injection is on the left flank 1 to 2 cm behind the fore end of the dorsal fin and 3 to 4 cm below it (Fig. 18). With other fishes the injection site is on the boundary between the first and second third of the body, 2 to 3 cm below the upper line. The needle and the injection site should be wiped with a pledget or sponge, dipped in 1 % solution of potassium  permanganate.

Fig. 18: Administration of drugs by injection. A - Injection site for administration of  the drug into the muscle; B - into the body cavity; C - the angle at which the needle is introduced into the body of scaly fish; D - scaleless fish. Recommended literature Aldermann D.J. (1985): Malachite green: a review. J. Fish Dis., 8, 289–298. Herwig N. (1979): Handbook of drugs and chemicals used in the treatment of fish diseases, Charles C. Thomas, Illinois, USA, pp, 272.

Kouřil J. et al. (1991): Antiparasitic and antifungal baths for the early fry of common carp, phytophageous fishes and sheatfish. Research Institute of Fish Culture and Hydrobiology, Vodňany, pp. 8. Prost M. (1989): Fish diseases. Warszawa, PWRiL, pp. 460 (in Polish). Reichenbach-Klinke H.H. (1980): Krankheiten und Schãdigungen der Fische. Gustav Fischer Verlag, Stuttgart, New York, pp. 472. Roberts R.J. (ed) (1989): Fish pathology. Ballière Tindall, pp. 467. Schãperclaus W. et al. (1979): Fischkrankheiten. Academie-Verlag, Berlin, pp. 1089. Svobodová Z., Faina R. (1989): Application of Soldep preparation in fish culture. Edice Metodik, VÚRH Vodňany, No.12, pp. 15 (In Czech). Svobodová Z., Faina R., Vykusová B. (1985): Application of Kuprikol 50 preparation in fish culture. Edice Metodik, VÚRH Vodňany, No. 19, pp. 10 (In Czech). Tesarčík J., Rajchard J. (1983): Veterinary preparations in fish culture. Edice Metodik, VÚRH Vodňany, No. 11, pp. 11 (in Czech). ON 46 6809 Antiparasitic and antimycotic bath of fish. ÚNM, Praha, 1983, pp. 14 (In Czech). 2.2.3 Viral Diseases

(J. Tesarčik) Prevention and therapy are stated for the following major viral diseases of freshwater  fishes: Channel catfish virus (CCV)

Prevention efforts are focused on avoiding infection of the rearing facility through strict veterinary controls on the transfers of brood material. The eggs must come from infection-free environments. Eggs at the eye point stage are preventively subjected to Jodonal bath (concentration of 2.8 – 6.7 ml per 1 litre, exposure for 5 minutes) or to a Wescodyn R bath (concentration 50 mg per 1 litre, exposure for 10 minutes). The channel catfish fry are closely checked for health mainly during the early stage of  their life until they reach a weight of 10 g. When an infection breaks out, the infected stock must be isolated, subjected to laboratory examination and - in the event of mass death - safely disposed of. Keeping the fish at a water temperature below 19°C for 24 hours may result in substantial reduction of losses. Upon mechanical cleaning, the infected environment must be disinfected with chlorinated lime (concentration of 200  – 400 ml per litre, exposure for 12 hours). All stress situations are to be avoided. Fish vaccination provides specific prevention of CCV.  No therapy has been developed as yet. Secondary bacterial infections can be controlled by trypaflavine, chlorampfenicol or oxytetracycline baths.

Infectious pancreatic necrosis (IPN)

Prevention relies on strict veterinary controls imposed on all t ransfers of eggs and fish to uncontaminated fish culture facilities and the flowing waters that feed the rearing  ponds. The young fry are best reared from the own stock of each farm and best kept in spring water. The start of intensive fish feeding is a critical period: the fish have to be carefully habituated with no abrupt switchover. The preventative practices also include the general principles of hygiene and disinfection. The regular virological examination of individual trout farms is important for prevention from this disease. Fingerlings at the age of 2–4 weeks and 8–10 weeks is to subject to the examination. The possibilty of this virus registration is the highest in these periods. Fish vaccination provides specific prevention of IPN.  No therapy at a significant efficiency level has been developed as yet. The mortality of the affected stock can be reduced by adding polyvinylpyrrolidone iodine to the feed  pellets at a dose of 1.6 – 1.9 g per 1 kg of the live weight of the fish per day for 15 days. This treatment can only be effective if started on time. Spring viraemia of carp (SVC)

Virus of SVC is spread in all fish culture facilities in our country. Prevention is to be tended to enhance the non-specific resistance of fish. Fish must be reared by welltested methods, stress should be excluded, fish stock should be optimalized, plenty of  natural food and quality replacer pellets should be provided and the fish being reared must be kept under strict health control. It should be combined with preventative antibiotic and chemotherapeutic treatment of the fish and with the control of the causative agent through the disinfection of the fishing gears, transport containers and small reservoirs (storage ponds, wintering tanks) and waterlogged places. Ponds, reservoirs and tanks are disinfected by means of burnt lime applied at a rate of 2.0 to 2.5 tonnes per ha. Apparently diseased fish should be eliminated and suspected stocks should be reared in small ponds away from the rest of the fish farm's stock. Fish vaccination provides specific prevention of SVC. A vaccine has been developed in Czechoslovakia; it was administered intraperitoneally on an experimental scale. From the point of view of fish farming practice, the most promising method would be the administration of the vaccine in food; this method is being currently finished in Czechoslovakia. There is no therapy to kill Rhabdovirus carpio. Antibiotics (chloramphenicol, neomycine) or chemotherapeutics (sulphonamides), all administered in feed, are used for the prevention of secondary bacterial infections. Medicated VR or VR-Neo feeds can also be used with advantage. Viral haemorrhagic septicaemia (VHS)

Prevention relies on preventing the introduction of the causative agents into the fish culture facilities or to the watershed; this is done through strict veterinary controls on any transfers of eggs and stock fish. As to the general preventive measures, emphasis is laid (mainly in intensive culture facilities) on feed quality, copious supply of  vitamins, hygiene of the environment and regular veterinary checkups. It is

recommended to treat the eggs by Thiomersal, Merthiolat and Bactosept baths (concentration of 0.2 mg) per litre, exposure for 10 minutes). Burnt lime (1 kg per  m2), calcium cyanamide (1 kg per m2), chlorinated lime (2 kg per m2) or sodium hydroxide (2 g per litre) can be used for the disinfection of the rearing facilities and the appliances used. Fish vaccination is a specific prevention of VHS. There is no known effective therapeutic means to control the disease. In is recommended to give the fish easily digestible feed (spleen) enriched with vitamins A, B1, B 2, D and E during the critical period. Good response is also gained when the fish are given the Meso-Inosit vitamin mix at a rate of 350 mg per 1 kg of feed pellets for several days at a reduced water temperature. Infectious haematopoetic necrosis (IHN)

It is possible to provide prevention of the disease by applying the common measures normally taken in cases of any infection. The only effective measure is to keep the stocks away from the causative agent of the disease. It is recommended to use the following practice to prevent the disease: rear the fry at a temperature of 15°C at the minimum for 1 month after hatching; then transfer the fry to normal conditions with a temperature of about 10°C. As to the eggs, it is useful to subject them to regular  Jodonal baths (concentration of 4.27 ml per litre, exposure time 10 minutes) or to Wescodyn R bath (concentration of 76 mg per ml, exposure for 10 minutes). Fish vaccination provides specific IHN prevention.  No therapeutic method with a specific IHN control activity is currently available. Pike fry rhabdovirus (PFR)

Prevention is based on adherence to hygienic principles and implementation of  infection control measures; this includes, in particular, the care of the cleanness of all gears and tools and fish culture facilities; it is also necessary to prevent water from dubious sources from entering into the fish culture environment. For prevention it is recommended to dip the eggs in the eye point stage in a Jodonal solution at a concentration of 2.85 ml per litre for 10 minutes or in Wescodyn R at a concentration of 50 mg per litre for 10 minutes. Swim bladder inflammation

Prevention is focused on timely identification of the foci of the infection and on adherence to the veterinary regulations (the same as for SVC). As to the general  preventive measures, plenty of fresh natural food and replacer feeds should be  provided mainly to carp fry. Burnt lime can be used at a dose of 2.5 tonnes per hectare for the disinfection of the fish rearing environment.  No specific-action therapy has yet been developed. Secondary bacterial infections which complicate the cases can be controlled by timely administration of antibiotics or chemotherapeutics. The therapeutic intervention can only alleviate the course of the infection and reduce the mortality of the fish. Medicated VR or VR-Neo feeds can be used with advantage, both therapeutically and preventively.

Pox of carp

Prevention relies on the elimination of the factors underlying the occurrence of the disease. When selecting the brood fish the individuals with blisters on the body are eliminated. Lime is applied if the water in the pond or in the feed source is acid. Ectoparasites, especially the fish lice, are strictly controlled. Indirect treatment can be performed in the pond stocked with diseased fish: apply 50 kg of burnt lime per 1 ha several (three to four) times with an interval of three to four  days between each two treatments. Give plenty of feed enriched with vitamins, minerals and trace elements, in case also with chloramphenicol to the diseased fish. Papillomatosis of eel

Prevention relies on the veterinary checking of the eels during the first and second year after stocking (this can only be done in fishponds); if positive cases are reported, the import of elvers from the infected areas should be reduced. The import of elvers from localities free of this disease is the basic preventative pressuposition. In practice, as a rule, no preventative measures are taken. The fish showing symptoms of the disease are eliminated from breeding and the infected individuals are killed and  burnt. A description has been published concerning the treatment of fish infected with this disease, using quinine sulphate at a concentration of 60 mg per litre for several hours (quinine sulphate bath). Recommended literature Ahne W. (ed) (1980): Fish diseases. Spring Verlag, Berlin, Heidelberg, New York,  pp. 252. Ahne W. (1985): Virusinfektionen bei Fischen: Ãtiologie, Diagnose and Bekampfung. Zentbl. Vetmed., B, 32, 237–264. Ellis A.E. (1988): Fish vaccination. London, Academic Press, pp. 255. Lucký Z. (1986): Diseases of important fishes. SPN, Praha, pp. 201 (In Czech). Pospíšil Z., Tománek J. et al. (1991): Diagnosis and prevention of infections  pancreatic necrosis in salmonids. Research Institute of Fish Culture and Hydrobiology, Vodňany, Czechoslovakia, pp. 15. Prost M. (1989): Fish diseases. Warszawa, PWRil, pp. 460 (In Polish). Reichenbach-Klinke H.H. (1980): Krankheiten und Schädigungen der Fische. Gustav Fischer Verlag, Stuttgart, New York, pp. 472. 2.2.4 Bacterial Diseases

(J. Tesarčík)

Prevention and therapy are stated for the following major bacterial diseases of  freshwater fishes: Furunculosis

Prevention in flowing waters relies on water purity and sufficient oxygen concentration. Organic pollution of the trout and grayling waters must be as low as  possible. Watersheds exposed to the danger of occurrence of furunculosis should be stocked with species less susceptible to the infection (rainbow trout) or with salmonids at a younger age (fry), which are slightly more resistant. Under such circumstances, the number of fry (fish) for the stocking is usually reduced, sometimes even to half the normal level. If the disease breaks out, the dead fish are taken out of  the water and safely disposed of (burnt or burried together with a powerful dissinfectant-chlorinated lime or burnt lime). In salmonid culture, the prevention relies on protecting the stock against the introduction of the disease with fish transferred from infected flowing waters or from fish farms where the disease occurs. Immediately after fertilization, or at the eye-point stage, the eggs should be treated with an acriflavine solution at a concentration of 0.5 g per litre for 20 minutes. The  preventative measures recommended for flowing waters must be implemented even more strictly in salmonid rearing operations where the intensity of the culture is much higher. Fish vaccination provides specific prevention of furunculosis. Drugs to control the disease are administered either for prevention or for therapy; in  both cases they are added to the food (ground spleen - admixed before administration;  pellets - medication during the manufacturing process). These drugs may include  powerful antibiotics, sulphonamides, furazolidone; the medicated feed Chronicin salm is also suitable. Burnt lime (dose of 2.5 to 3.0 tonnes per ha) is used for the disinfection of the rearing facilities (small ponds). Fishing gears and tools are disinfected with a potassium permanganate (KMNO 4) solution at a concentration of  10 g per litre. Carp erythrodermatitis (CE)

As to the available preventive measures, it is necessary first of all to handle the fish very carefully during fishing to avoid any skin injury. Try also to avoid injury during the transportation of the fish to longer distances and during stocking. Maintain the fish in good condition and in a good state of nourishment above all by support of natural food sources development in ponds. Antibiotics and chemotherapeutics may also be used for prevention. The causative agent is very sensitive to antibiotics and sulfonamids so mass administration is the preferred route of introducing these substances in the fish body;  parenteral treatment and baths are less frequent. Chloramphenicol is the most frequently used antibiotic: it is incorporated in medicated feeds, e.g. the VR brand. Oxytetracycline and neomycine may also be used: these are incorporated in the VR NeO medicated feed. Burnt lime (2.5 tonnes per ha) or chlorinated lime (500 kg per  ha) are used for the disinfection of the fish culture facilities (ponds). A 10 % solution of the lime milk is used for the disinfection of the gears and tools.

Enteric redmouth disease (ERM)

Strict veterinary controls of transferred fish, and strict quarantine, are the most effective preventive measures. Infected fish farms are allowed to sell no other brood material but eggs, which must have been thoroughly treated with iodine preparations (e.g. Wescodyne R bath) at a concentration of 50 mg per litre for 5 – 10 minutes. Literary sources say that this is sufficient exposure to reliably kill the causative agent of the disease. Vaccination provides a specific prevention of enteric redmouth disease: the immobilized vaccine is applied in the form of bath. ERM therapy is based on the administration of oxytetracycline or on a combination of  sulphonamides with chloramphenicol. Thimethoprin combined with sulphadiazine (e.g. the Duon speciality) is considered as the most effective of the recently developed  preparations. Fish therapists in some countries outside Czechoslovakia use oxolinic acid and flumequin, which are even more powerful. All these preparations are administered in feed. The problem with the treatment is that it usually has to be repeated. Vibriosis

Prevention of this disease relies on direct veterinary inspection of transported fish, on strictly implementing hygienic precautions in fish culture, on bacteriological analysis of the feeds prepared from sea fish, and on respecting the general conditions of the control of bacterial diseases, including quarantine, disinfection, safe disposal of  diseased fish and others. Vaccination of the fish provides specific prevention of  vibriosis. The preferred therapy is the administration of sulphonamides or furazolidone in feed. Bacterial kidney disease

Prevention of this disease requires strict quarantine of the fish brought from sources not subjected to fish health control. If eggs are to be transported they must first be  bathed in sodium methiolate at a concentration of 0.2 g per litre for 10 minutes. Jodanal can also be used (concentration of 4.3 ml per litre, exposure for 10 minutes). The eggs may be treated in this way either immediately after fertlization or later, just  before transportation, when they are at the eye-point stage. Where the disease persists for a long time, sulphonamides are preventively administered in feed. If the disease occurs, the dead fish, or the whole affected stock, must be safely disposed of (in a rendering plant). Erythromycine, administered in feed at a rate of  100 mg per 1 kg of the fish live weight for 21 days, gives the best results in the treatment of the fish suffering from this disease. Mycobacteriosis

Prevention is based on good hygiene of fish culture. This involves regular cleaning of  the tanks and avoidance of overstocking. Plenty of varied food should be available to the fish to maintain them in good condition. Stress situations (control fishing, transportation) must be minized and long-term weakening of the fish (caused e.g. by

inadequate temperatures or physico-chemical properties of water) must be avoided. Suspect individuals should be immediately removed. When fish are purchased and especially when they are imported from a foreign country, the supplier should be required to provide a veterinary certificate proving that the fish came from a diseasefree environment. Nevertheless, in spite of the certificate, the purchased fish must spend some time in quarantine. Medicamentous treatment in advanced stages of mycobacteriosis usually fails to bring longer-lasting results. However, longer exposure to streptomycin baths in an earlier  stage of the ailment can stop the disease: add 50 to 300 mg of the drug per 1 litre of  water and leave the fish in the bath for 4 to 6 days. In cases of mass infection it is recommended to remove and destroy all the fish stock and the plants and disinfect the tank and everything inside it (preferably using preparations containing active chlorine). Myxobacteriosis

This is a group of diseases of which the following three are the most serious: a) Columnarosis Prevention of this disease involves careful handling of the fish during fishing, grading and transport. Ectoparasite control is another important factor of prevention. In salmonid culture it is useful to reduce water temperature to a level at which the development of the disease is curbed. Therapy based on baths is most frequently used in salmonids and in aquarium fishes, using the advantage of the fish being kept in small containers. Immersion bath is used for the treatment of salmonids, the fish being dipped in a solution of blue vitriol (CuSO4.5H2O) at a concentration of 0.5 g per litre for 1 minute. Malachite green is also a good chemical for immersion baths (concentration of 66.7 mg per litre, exposure time 10 to 30 seconds). Sulphonamides are administred in feeds to the fish suffering from columnarosis. Longer-term baths are used in aquarium fish culture, e.g. chloramfenicol at a concentration of 60 mg per litre for six days.  b) Cytophagosis The therapy is about the same as with columnarosis, except for the recommended reduction of water temperature: for the treatment of cytophagosis it is recommended to increase water temperature. An increase in water temperature above 15°C will suffice to arrest the disease. However, this treatment is difficult to provide in salmonids in many cases. c) Bacterial gill disease Prevention of this disease relies on maintaining good hygienic and breeding conditions. The food must contain plenty of vitamins of group B. Trout of the critical size have to be reared in clean water (preferably spring water). The size of the stock  must be adjusted according to water purity and water inflow rate. All debris and dirt must be removed from the rearing tanks (especially the residues of food and the

excrements). A small amount of cod liver oil may be added to the feed replacer if it is too dusty. Stocks in chronically diseased rearing facilities are preventively treated in critical  periods of the year, or soon after the first signs of the disease occur. The treatment rests in short term or dipping (immersion) baths; during the short term bath an aeration device must be used to maintain sufficient oxygen in the water for the fish. Regularly performed baths in substances containig active chlorine (e.g. chloramine in concentration of 20 mg.l-1 for 20 min.). Immersion baths in blue vitriol (CuSO 4.5H2O) give good therapeutic results (concentration of 0.5 g per litre, exposure time 1 to 1.5 minutes). Malachite green is another good chemical for short-term bath treatment. Also sulphonamids and chinolone chemotherapeutics respectively can be applied by  peroral way. Recomended literature Lucký Z. (1986): Diseases of important fishes. SPN, Praha, pp. 201 (in Czech). Prost M. (1989): Fish diseases. Warszawa, PWRiL, pp. 460 (In Polish). Reichenbach-Klinke H.H. (1980): Krankheiten und Schädigungen der Fische. Gustav Fischer Verlag, Stuttgart, New York, pp. 472. Roberts R.J. (ed) (1989): Fish pathology. Ballière Tindall, pp. 467. Schäperclaus W. et al. (1979): Fischkrankheiten. Akademie-Verlag, Berlin, pp. 1089. Tesarčík J. Rajchard J. (1983): Veterinary preparations in fish culture. Edice Metodik, VÚRH Vodňany, No. 11, pp. 11 (In Czech). 2.2.5 Fungal Diseases

(J. Řehulka) Oomycetosis (primarily caused by Achlya and Saprolegnia)

The best of the therapeutic substances is malachite green B (oxalate compound), active even at a very low concentration. The amount used for the dipping treatment is 66.7 mg per one litre of water, the exposure time being 10–30 seconds. For a short  bath with an exposure time of 1–1.5 hours the concentration is also 6.67 mg of MG  per one litre of water. For a long-time bath (6 days), the concentration must be much smaller, 0.2 to 0.5 mg per one litre of water. The effectiveness of the long-term bath depends on water quality. The fish usually have to undergo the bath again three days later. This approach is suitable for the treatment of fish in large storage basins or  tanks. For aquarium fishes and salmonids it is recommended to use the lowest active concentration of malachite green, 0.15 mg per litre. Market fish should be free of malachite green treatment 6 months before going to the market. The eggs of salmonids can be subjected to preventive and therapeutic treatment at a rate of 5 mg MG per one litre of water for an hour twice a week. This is

done without handling the eggs, the store solution being left to drop into the water that flows to the tank. Saprolegniasis of fish can also be controlled by short potassium permanganate baths. The concentration of the chemical in the bath is 0.01 g per 1 litre of water and exposure time is 1 to 1.5 hours. Branchiomycosis

Preventive treatment is conducted in ponds where branchiomycosis permanently occurs in July and April, when burnt lime powder is applied onto the water surface at a rate of 50–100 kg per hectare. It is recommended in this period to increase the rate of flow through such ponds to improve oxygen content. It is also advisable to avoid  putting too much organic matter in the pond through intensive organic manuring and intensive breeding of water fowl. Burnt lime should be used at a rate of 2.5 t per  hectare to dissinfect the bottom. If the disease occurs in its acute stage, stop feeding the fish, increase the flow through the pond and treat the water surface with chlorinated lime at a rate of 10–15 kg per ha (at an average pond depth of 1 m) three times a week at the maximum. When the branchiomycosis is over it is recommended to administer medicated feed (with antibiotics) to prevent secondary bacteriosis of the gills. Ichthyophonosis

 No therapy is available as yet. Prevention is based on thorough veterinary inspection of the fish rearing facilities and quality control of the feed on trout farms where sea fish are administered. The preventive sanitation measures include pasteurization of   potenntially infected fish flesh. The reservoirs infested with the fungus are disinfected with burnt lime at a rate of 2.5 t per hectare. Aquarium tanks are drained from above and the bottom and walls are dissinfected with potassium permanganate at a rate of 1 g per 100 litres of water for 90 minutes. Moniliales

In exophialosis prevention, increased attention should be paid to the quality of food especially the tubificids, which are vectors of  Exophiala pisciphila to aquarium fishes. The most vulnerable of the aquarium fishes is  Hemmigramus pulcher . In ochroconiosis neither prevention nor effective therapy are available yet. In fusariosis  prevention and therapy are similar to those in coelomycetosis. Coelomycetes

Prevention of this disease is based on maintaining the water at an optimum quality. In  places where it occurs the fungi must have been killed in the reservoirs or tanks (by disinfection repeated twice or three times with 20-day intervals between the treatments) before stocking and during the rearing. The disinfectant suitable for the treatment of tanks without fish include a solution of sodium hypochloride with a 2 % content of active chlorine, or a 2 % formaldehyde solution, the exposure time being 20–30 minutes.

Recommended literature Amlacher E. (1986) : Taaschenbuch der Fischkrankheiten. VEB Gustav Fischer  Verlag Jena.  Neish G.A., Hughes G.C. (1990): Fungal diseases of fishes. T.F.H. Publications, Inc., Ltd. Wolke R.E. (1975): Pathology of bacterial and fungal diseases affecting fish. In: The Pathology of Fishes (ed by W.E. Ribelin and G. Mikagi), 33–116. The University of Wisconsin Press, Madison, Wisconsin. 2.2.6 Parasitic Diseases

(Z. Svobodová) The methods of prevention and therapy of important parasitic diseases are described as follows. Protozooses

1. Fish diseases caused by ectoparasitic Protozoa Ichthyobodosis Prevention of ichthyobodosis should be realized with absolutely full care. The main effective rules of prevention are: a) To use the source of water free of parasites and their cysts. The underground water is the most suitable source of water free of parasites.  Nevertheless, these sources are very limited even for trout farms, hatcheries and/or  other special fish culture facilities. In most cases, water from streams is used as a source of inlet water. In such cases, suitable filters can free the inlet water from infectious stages of parasites prior the water enters smaller tanks with intensive fish culture. A water from ponds stocked with fish is absolutely unsuitable to be used in trout farms, hatcheries and/or rearing facilities with early stages of fishes.  b) The stocking of healthy fish Only well examined fish, free of ichthyobodosis and treated by antiparasitic bath (in case of necessity) should be stocked into ponds and fish rearing facilities. c) Creation of presuppositions for good performance and health conditions of fish. It refers particularly to the optimum quality of water without physical and chemical stressing effects. It is necessary to avoid the stress effects caused by other factors too, namely by inconsiderate handling with fish. Through appropriate culture management, the maximum development of natural food should be gained. The supplementary feed must be applied in sufficient quantity and quality. Abundant

natural food of suitable species and size composition is a basic preventive measure in rearing the early stages of fry. The maximum level of stock density must be calculated carefully. Unreasonably dense stock in trout ponds and/or raceways, in fry reared in special culture facilities, in any other fish production units, and in ponds too, leads to stressing situations, worsens the nutritional state of fish, decreases the resistance and opens the door to infections. d) Regular control of health condition of fish A preventive control of health condition of fish reared in hatcheries and in special fish culture facilities (in case of early fry rearing) must be realized twice a week, in trout farms once a week. A control of health condition must be preformed just prior the fishing, the fish transportation and/or stocking the fish. Therapy - Many antiparasitic baths and their modifications have been proposed as a therapeutic measure. The effectiveness of baths is not absolute; some flagellata survive in folds of epithelium which creates new presupposition for infection. Basicaly, short-term baths in sodium chloride and/or formaldehyde are used for  ichthyobodosis control treatment in early stages of cyprinids and European catfish, reared in facilities utilizing the heated effluents. In salmonid fish culture, a long-term  bath in malachite green is recommended in most cases from practical aspects. In cyprinids and salmonids, a two-hour- and/or six-hour-long combined bath in malachite green and formaldehyde can be used too. A long-term bath in acriflavine is recommended in aquaria fish culture for ichthyobodosis control. Cryptobiosis Abundant natural food of suitable species and size composition preventing the condition impairing of fish is a basic preventive measure in rearing the early stages of  fry and preventing the protozoal infection. Optimum quality of water is an another  important aspect. In case of cryptobiosis, a frequent sludge removing from rearing tanks is a necessary measure preventing the increase in organic substances concentration and level of organic detritus in water. To control the dense growths of  Cryptobia on the body and the gill surface, a decrease of organic load and of organic detritus (by frequent sludge removing from rearing tanks) together with improving the quality of nutrition (by abundant natural food) is mostly sufficient. In case this measurement is not effective, the same baths as in ichthyobodosis can be applied. Ichthyophthiriosis Prevention is based both on the stocking of healthy fish and the use of water free from  parasite infectious stages. In special fish culture facilities with heated water, in trout culture, and in special wintering ponds, inlet water from lower layers and/or from retention reservoirs without fish stock should be used. In case of inlet water from streams, water must be filtered through gravel-sand filters. If diseased fish have appeared in fish culture facilities or ponds, tanks and pond bottom must be completely dried after fishing and disinfected with calcium oxide (2.5 t.ha -1) and/or calcium hypochlorite (0.5 – 0.6 t.ha -1). The fishing equipment contacted with water and/or fish

from infected environment, must be thoroughly dried. It is important to take into mind when controlling ichthyophthiriosis, the encysted parasites (tomonts) can survive in aquatic environment 1 week in water temperature 20°C and 2 weeks in low temperatures. After this period, aquatic environment is free from parasites. The creation of optimum living conditions is another important presupposition ensuring the good health state of stocks in course of their culture. In case of ichthyophthiriosis  prevention, a respective good nutritive and health state of fish is a presupposition together with appropriate density of stock, particularly in early fry rearing and in wintering ponds. Unreasonable dense stock enables the spreading of infection. When fish are kept in good condition, parasites survive in minimum numbers. In weakened fish, in decrease of their resistance and/or in increase of water temperature (which is common in spring period in wintering ponds), parasites reproduce intensively. Therapy of ichthyophthiriosis is difficult. Due to immersing into the surface tissues of  the fish body, parasites are more or less protected against the antiparasitic baths. A method of temporary increase of water temperature and antiparasitic bath can be used in therapeutic treatment of ichthyophthiriosis. Method of temporary increase of water  temperature to 31 – 32°C for 3 days is well proved in aquaria fish culture and in special fish culture facilities utilizing the heated water. Antiparasitic baths must be used in period when adult trophonts leave the surface of host to produce a cyst. Baths are effective to these free stages of parasite only. Most frequently, long-term bath in malachite green is applied. Based on specific culture conditions, short-term baths in malachite green can be used too. Recently, a repeated combined bath in malachite green and formaldehyde for 2 hours has been proved with success in aquaria and troughs, and for 6 hours in earth ponds and holding ponds. Chilodonellosis Prevention of chilodonellosis is based in creating the optimum conditions of fish culture and in preventing the transfer of causative parasite. To prevent the infestation of rearing tanks, the diseased fish are not allowed to stock and gravel-sand filters are recommended to install on inlets to special fish culture facilities. Optimum quality of  water is important as well; low content of organic substances and high content of  oxygen in water is unfavourable for parasite. An appropriate nutrition of fish,  particularly prior the wintering, is an important preventive measure. Therapy of chilodonellosis is realized through several antiparasitic baths which, unfortunately, are not very effective in certain cases. Short-term baths in sodium chloride, formaldehyde, malachite green and bath in Kuprikol 50 as regards to carp and grass carp can be mentioned. As to long-term baths suitable for chilodonellosis control, bath in malachite green (fish kept in holding ponds) and long-term bath in sodium chloride and formaldehyde can be applied. A long-term bath in acriflavine is used in aquaria fish culture. Trichodinosis Prevention is identical with measure for skin and gill protozooses. Therapy is identical with chilodonellosis.

2. Fish Diseases Caused by Endoparasitic Protozoa Trypanoplasmosis, trypanosomosis Prevention is based in diminishing and control of leeches occurrence. A good condition of fish prior the wintering is important to diminish the losses caused by  blood flagellata. Effective therapy has not been worked out yet. Hexamitosis Prevention - To prevent the transfer of cysts into the fish culture facility via both infected fish and inlet water is one of the most important measures. Regular control of  health state of fish and microscopic examination of fish excreta (once in a week in case of salmonid fry rearing) is a necessity. In case of positive findings, a preventive dose of furazolidon (0.3 g per 1 kg of feed) is applied daily for 14 days. Appropriate hygienic conditions and sufficient nutrition of fish belong to important preventive measures in hexamitosis control. Therapy - furazolidon in feed can be used successfuly for therapeutic treatment. The Polish preparation Entizol, applied in feed, is also a very effective drug. This  preparation can be used in form of long-term bath as well. Coccidiosis Prevention is based on oocysts and sporocysts (occurring on the bottom of ponds and holding ponds) control. Drying the bottom and its disinfection is very effective. Regular wintering, drying and disinfection of muddy parts of nursery ponds with  burnt lime (2.5 t.ha -1) and/or chlorinated lime (0.5 – 0.6 t.ha -1) is an effective method of prevention. To prevent the transfer of parasite from older classes of fish and/or  from coarse fish to carp fry is an another important element of prevention. In natural spawning of carp, brood fish should be fished out as soon as possible from spawning  ponds. In infected cyprinid culture, supplementary feeds must be applied frequently to  prevent the fry to feed on benthos due to abundant population of coccidia oocysts. Therapy - peroral application of furazolidon in dose 0.3 g per 1 kg of feed for 5 days is used in carp fry rearing. Myxosporeosis Prevention is focussed on regular veterinary examination of fish and on selection of  fish with positive findings out of culture. Most of species of freshwater myxosporea have a very resistant spores, some of them are adapted to temporal drying-up. In natural environment, spores keep their viability for 6 months to 2 years based on different conditions. Soon or later, spores sediment on the bottom, and mud and  benthos create a source of infestation. Due to turbulent stream movement, spores are transferred to other localities. Spores can be distributed by fish-consuming birds too (spores were not damaged in bird's digestive tract).

Myxosporeosis control must be focussed on spores as the infectious stage, the only able element in the developmental cycle. Experiments with myxosporeosis therapy failed yet so the spores control is the only measure. Pond bottom disinfection through summer drying and chemical substances application is very important. Optimum method of prevention includes the drainage of pond in the autumn period and application of disinfective substance on the wet bottom. Afterwards, pond is filled with water only to cover the bottom, and disinfective substance is let to act for 3 weeks. Then the pond is flushed-through, drained and dried through the winter period. It is recommended to repeat this process once again in spring time, and then the pond can be filled with water. Calcium cyanamide in dose 0.5 kg per 1 m 2 of bottom area is advised as an optimum disinfection. Therapy of myxosporeosis is not worked out yet. Myxobolosis Prevention is focussed on regular veterinary control of fish health state and on control of spores, as it is mentioned in myxosporeosis prevention. Rearing the fry till the age of 2 to 3 months in plastic troughs and/or concrete tanks with inlet of suitable water is another measure in prevention. After this period, fry can be stocked in earth ponds. In  ponds permnanently watered from naturally disease-infected localities, rearing the fry till the age of 2 to 3 months is not realized. Therapy of myxobolosis is not worked out yet. Metazooses

Monogeneosis Prevention is based on excluding the parasites transfer from culture environment via  both diseased fish and inlet water including the infectious  Dactylogyrus and  Eudiplozoon larvae. Natural spawning should be restricted and artificial propagation of fish should be utilized which excludes the contact of brood fish and fry. It must be avoided to stock the infected fish into the rearing facilities. Each fish species must be stocked as a single age-class category into ponds. In nursery ponds, inlet water should  be free from infected larvae, i.e. source of this water is out of reservoirs stocked with the same fish species. Abundant natural food and supplementary feeding the fry with quality feeds is another preventive measure in carp fry rearing. In such case, 6 cm  body length of fry is reached as soon as possible, and in this stage, the fry is relatively resisting to  Monogenea (particularly to  Dactylogyrus vastator ). Till this moment, a regular frequent examination of health state of fish is necessary. Therapy. Short-term formaldehyde bath in the course of re-fishing the fish, and longterm bath of cyprinids in trichlorphone-based preparations diluted directly in pond, is an effective therapeutic method in dactylogyrosis and gyrodactylosis. It is necessary to note that trichlorphone application in ponds is followed by intensive reduction in natural food abundance. It is therefore necessary to feed the fish intensively with high-quality feeds till the time of re-development of natural food. In case of lower  intensity of carp fry infection in nursery ponds, a standard measure to improve the culture environment and health state of fish is applied instead of trichlorphone

application. Inlet of oxygen-rich water is increased, rotten vegetation is removed, and fry is fed intensively. Short-term bath in sodium chloride can be mentioned too, nevertheless it is a less used treatment method with no extra effectivity. Ammonia and trypaflavine immersion bath is too risky in conditions of higher temperature and pH of water. Potassium permanganate bath in concentration 1 g.1 -1 for 150 sec is used as therapeutic method in eudiplozoonosis. This bath is recommended for older carp and it is effective up to water temperature 10°C. Cestodoses This group of diseases is represented mainly by the following five parasitoses: Khawiosis Prevention is based on careful veterinary control of transported fish and fish reared in own culture facilities. The preventive feeding with medicated feed Taenifugin-carp is applied in case of low intesity infection in fry and stock carp. This preventive feeding is obligatory in case of transportation of fish from infected localities to areas free of   parasite. As to antiparasitic treatment in brood fish, piperazine salt of niclosamide is applied in starch gel through probe. When khawiosis is registered in growing period, intensive feeding can prevent the fish to consume the tubificids with developmental stages of parasite. Direct control of intermediate host tubificids is not realised in our  conditions. Abundance of tubificids is controlled through pond reclamation, winter  and/or summer drying of pond bottom, and bottom disinfection (2.5 t.ha -1 of burnt lime or 0.5 – 0.6 t.ha -1 of chlorinated lime). This is also a method to control the occurrence of  Khawia sinensis eggs. Therapy. Single or repeated application of medicated feed Taenifugin-carp is proved as a therapeutic treatment. Caryophyllaeosis Prevention is identical as to khawiosis. Therapy is identical as to khawiosis. It can be applied in pond culture only. Bothriocephalosis Prevention is also identical as to khawiosis. A preventive control of intermediate hosts is not realized. Only in extreme mass occurrence of bothriocephalosis, application of  trichlorphone-based preparations (e.g. Soldep in dose 10 l.ha -1 for a water column 1 m on average) is used to control the intermediate hosts in a reservoir stocked with fish. Therapy is identical as to khawiosis. Proteocephalosis

Prevention is applied in cage culture of rainbow trout only. It is identical with  preventive measure in khawiosis and bothriocephalosis. First of all, it refers to careful control of health state of fish, prevention the spreading of parasitosis into other  localities, and preventive feeding the rainbow trout with feed medicated with  piperazine salt of niclosamide. Therapy. Single or repeated feeding the trout with feed medicated with piperazine salt of niclosamide in dose 5 g per 1 kg is applied as a therapeutic measure. Triaenophorosis Prevention is based on excluding the infected pike from inlet water entering the trout culture. Therapy is not practised. Trematodoses Prevention is based on regular examination of health state of fish. In more intensive infestation, interruption of developmental cycle of parasite is realized by means of  control of intermediate hosts. In diplostomosis and postodiplostomosis, occurrence of  fish-consuming birds as definite hosts should be restricted in intensive fish culture. This can be done through a control of gull eggs, a use of nets over the trout raceways etc. Snails are controlled by biological, physical and/or chemical method. No any method itself is sufficiently effective. To interrupt the developmental cycle of   parasite, individual methods must be combined. In herbivorous fish fry rearing in  ponds with intensive occurrence of snails, careful drying and winter freezing of pond  bottom is recommended. Afterwards in spring period, a pond is partly filled with water. After leaving the bottom mud, pond snails ( Lymnaea stagnalis) are controlled  by means of chemicals. The preparations with copper as an active substance (e.g. Kuprikol 50 in dose to 30 kg.ha -1 for a water column 1 m) are effective to snails. Other molluscocides (e.g. based on tin) are tested too. The pond is filled with water  through a sieve (mostly made from synthetic web called Uhelon) installed directly on an inlet pipe. The sieve must be regularly controlled and cleaned. This is a very simple way preventing the snails to reach the pond. These mechanical, physical and chemical methods of snail control can be suitably combined with biological method. In such case, 500 – 600 individuals per 1 ha of two-year-old carp (over 500 g) and/or  three-year-old tench are stocked into pond. This stock frequently consumes small  juvenile snails. In the USSR, black carp is stocked in water bodies to control large quantities of intermediate host snails. Fry of herbivorous fish fed in troughs for  several days prior the stocking is stocked into ponds at the turn of June and July. In this period, the stock carp and tench are fed with highly effective feed rich in animal  protein to prevent the losses. Therapy is not yet realized in practice. Acanthocephalosis Prevention is based on regular examination of health state of stocked fish and even fish from flowing waters from which the ponds are fed. A main aim of prevention is

to prevent the introduction of parasites. Careful drying and/or disinfection of pond  bottom with 2.5 t.ha -1 of burnt lime or 0.5 – 0.6 t.ha -1 of chlorinated lime applied immediately after the fishing, controls the eggs and intermediate hosts. Recently an increase in pond fish culture intensity seems to be an important limiting factor of carp neoechynorhynchosis occurrence. It was recognized that an occurrence of  intermediate hosts is very sporadic in intensively managed ponds with dense stocks. Therapy was worked out against neoechinorhynchosis of carp. Carboneum tetrachloratum and paraffin oil in the same ratio is applied in a single dose 1 ml per 1 kg of fish in feed. Fish Diseases Caused by Parasitic Crustaceans This group of diseases is represented mainly by the following parasitoses: ergasilosis, lernaeosis and argulosis. Prevention is based on the excluding infected fish, both from culture and coarse water, to enter the fish culture facilities. Water sources used to supply the fish culture facilities must be under strict control. Thorough drying and/or disinfection of pond  bottom with burnt lime or chlorinated lime is a method controlling the eggs and larvae of parasites. Therapy of ergasilosis and lernaeosis is very difficult. Long-term bath in preparations  based on trichlorphone proved to be only temporary effective on nauplia and copepodite stages under higher water temperature (20°C); the same bath is uneffective on adult parasites if applied under lower water temperature. Immersion bath in lysol is recommended for re-fished fish. Long-term bath in preparations based on trichlorphone can be applied directly in pond to cyprinids as argulosis treatment. Hirudineosis Prevention - disinfection of pond bottom with burnt lime (2.5 t.ha -1) controls the occurrence of leeches ( Piscicola geometra). To prevent the entering of infected coarse fish into the fish culture environment is also an effective method. Another way of   prevention consists of creation the unsuitable conditions for leeches in pond which means an eradication of hard littoral vegetation, stones and tree branches serving as a shelter for leeches and/or a substrate for cocoons with eggs. Therapy is applied primarily in cyprinids. A single application of trichlorphone-based  preparation directly into pond is an effective method of leeches control. Blue vitriol (CuSO4.5H2O) in dose 0.05 mg.1 -1, i.e. 0.5 kg.ha -1 of pond for water column 1 m on average, applied 3 – 4 times repeatedly in a two- to four-week period is recommended as a control of leeches in carp ponds by some authors. An immersion lime and/or lysol  bath, and as the case may be, short-term bath in sodium chloride can be used for  leeches control in re-fished fish. It must be taken into mind, that leeches drop off from fish after bath and they are able to recover again in suitable conditions. The salt bath is less effective. From brood fish, the leeches can be removed mechanically by  pincette. Recomended literature

Bauer O.N., Musselius V.A., Strelkov Ju.A. (1981): Diseases of pond fishes. Izd. Legkaja i piščevaja promyšlennost, Moskva, pp. 318 (In Russian) Lucký Z. (1986): Diseases of important fishes. SPN, Praha, pp. 201 (In Czech). Prost M. (1989): Fish diseases. Warszawa, PWRiL, pp. 460 (In Polish). Reichenbach-Klinke H.H. (1980): Krankheiten und Schädigungen der Fische. Gustav Fischer Verlag, Stuttgart, New York, pp. 472. Schäperclaus W et al. (1979): Fischkrankheiten. Akademie-Verlag, Berlin, pp. 1089. Svobodová Z., Zajíček J. (1989): Control of fish protozoases. In: Lom J., Dyková I. Protozoal parasites of important fishes. SZN, Praha, pp. 102 (In Czech). ON 46 6809 Antiparasitic and antimycotic bath of fish. ÚNM, Praha, pp. 14 (In Czech).

3. Intoxications of Fish (Z. Svobodová, B. Vykusová, J. Máchová) 3.1 Aetiology of Fish Intoxications The following physico-chemical changes in the aquatic medium are most frequently recorded as the immediate causes of fish injury and intoxication in fish culture  practice. Water temperature

Fish are poikilothermic animals, which means that their body temperature is the same as, or 0.5 to 1°C above or below, the temperature of the water they live in. The intensity of the metabolism of the fish is closely associated with water temperature: the higher the water temperature (the closer to the optimum values), the higher the metabolic rate. This fully applies to the warm-water fishes. The cold-water fishes, e.g. salmonids, whitefish, or burbot, have a different type of metabolism: their metabolism continues at comparatively low temperatures, whereas at high water temperatures, usually above 20°C, they lose much of their activity and eat much less food. Water  temperature also has a great influence on the rise and course of a number of fish diseases. The immunity system of the majority of fishes shows optimum function at water temperatures about 15°C. In their original area, fish easily tolerate seasonal change in temperature, e.g. a decrease to 0°C in winter and increase to 20–30°C (depending on species) in summer  under Central European conditions, but these changes should not be abrupt. Temperature shock occurs if the fish are put into a new environment in which the temperature is 12°C colder or warmer (8°C in the case of salmonids). Such fish die, showing symptoms of paralysis of the respiratory and cardiac muscles with early fry  problems may even arise when the difference in temperature is as low as 1.5 – 3°C. If  fish are fed and then abruptly transferred to water colder by 8°C or more, their  digestion process will slow down or stop. The food will remain undigested or halfdigested in the digestive tract and the gases produced bloat the fish which lose balance and die. In carp, given high-nitrogen food (natural food, high-protein pellets), abrupt transfer to much colder water highly increases the level of ammonia nitrogen in the  blood serum and the decrease in metabolic rate reduces ammonia elimination through the gills. This leads to ammonia autointoxication and death. Great progress has recently been recorded in warm water fish culture. Water  temperature control offers good conditions for optimally encouraging the fish to grow and to utilize their growth potential for maximum weight gains. Water pH

The pH levels optimum to the fish range from 6.5 to 8.5. Levels of pH above 9.2 and  below 4.8 damage and kill salmonids (mainly brown and rainbow trout) and pH values above 10.8 and below 5.0 may be fatal to cyprinids (especially carp and tench). Salmonids, compared with cyprinids, are more vulnerable to high pH and more

resistant to low pH. American char is especially resistant to low pH: it tolerates pH levels as low as 4.5–5.0. Low water pH most frequently occurs in spring when the snow thaws, mainly in peat  bog areas. High water pH in turn, occurs in eutrophicated reservoirs (ponds) where the green plants (the blue-green algae, green algae and higher aquatic plants) consume too much CO 2 for their intensive photosynthetic assimilation. This affects the neutralizing capacity of the water and increases its pH to 9.0–10.0. Water pH also changes when acids, hydroxides or other acid or alkaline substances leak into the water courses, ponds or lakes. To defend itself against the effect of a low or high water pH, the fish body produces an increased amount of mucus on the skin and on the inner side of the gill covers. Extremely high or low pH values cause damage to the tissues, especially the gills, and haemorrhages may occur on the gills and on the lower part of the body. Excess amounts of mucus, often containing blood, are observed post mortem on the skin and gills. The mucus is dull-coloured and watery. Water pH also has a significant influence on the toxic action of a number of other  substances (ammonia, sulphane, cyanides, toxic metals and others) on the fish. Oxygen

Oxygen enters the water in diffuse form from the air and also through the  photosynthetic assimilation of aquatic plants. On the other hand, oxygen is removed  by anaerobic decomposition of organic substances, by the oxidation of some organic compounds and through the respiration of the organisms present in the water. The concentration of the oxygen dissolved in water can be expressed in mg per litre or as  percentage of saturation. Water temperature, atmospheric pressure and contents of  salts dissolved in water have to be taken into account when the values in mg per litre are converted to saturation % or vice versa. Different fishes have different requirements for the concentration of oxygen dissolved in water. Salmonids are especially particular about oxygen in the water they live in: for them the optimum concentration is 8–10 mg per litre, and if the level declines  below 3 mg per litre they show symptoms of suffocation. Cyprinids are less demanding: they feel best in water containing 6–8 mg per litre and show symptoms of  suffocation when oxygen concentration falls to 1.5–2 mg per litre. Oxygen demand in fish also depends on water temperature, pH, CO 2 level, stress, metabolic rate and other factors. The major criteria in oxygen demand in fish include temperature, average individual weight and total weight of the fish per unit volume of  water. Oxygen demand increases at a higher temperature and higher total weight of  fish per unit volume of water (e.g. an increase in water temperature from 10 to 20°C doubles the oxygen demand). Oxygen demand significantly declines with increasing individual weights. In carp this decline may be expressed by the following indices: yearling = 1, two-year-old carp = 0.5 – 0.7, market carp = 0.3 – 0.4. Significant differences in oxygen demand are also recorded between different species. Putting coefficient 1 as expressing oxygen

demand in common carp, the converted values for some other fishes will be as follows: trout 2.83, peled 2.20, pike-perch 1.76, roach 1.51, sturgeon 1.50, perch 1.46,  bream 1.41, pike 1.10, eel 0.83, tench 0.83. The factor most frequently responsible for decreases in oxygen concentration in water  (oxygen deficiency*) is water pollution with organic substances (waste waters from agriculture, food industry, public sewage). The organic substances decompose in water and take from the water the oxygen they need for the decomposition. The concentration of organic substances in water and their capacity of taking oxygen from the water are evaluated by means of the chemical oxygen demand (COD) and  biochemical oxygen demand for five days (BOD 5). The COD level, determined by the Kubela method and considered as optimum for cyprinids in pond or river waters (CODMn), is up to 20–30 mg O 2 per litre, and the optimum BOD5 for cyprinids is up to 8–15 mg O2 per litre, depending on the intensity of the culture. For salmonids the respective levels are up to 10 mg O 2 per litre and up to 5 mg O2 per litre. In winter, fish are most frequently killed by suffocation in polluted storage ponds and in summer this most frequently happens in polluted water courses at a high temperature and low flow rate. In severely eutrophicated ponds, oxygen deficiency often occurs in summer early in morning as a result of increased oxygen consumption for the bacterial decomposition of organic substances and for dissimilation in aquatic  plants. In highly nutritive ponds (e.g. the sewage ones) with a constant inflow of  decomposed organic substances, oxygen deficiency is also caused by excess development of zooplankton: the zooplankton itself needs plenty of oxygen and, in addition, its feeding pressure considerably suppresses oxygen producers (phytoplankton). * Oxygen deficiency is the additional oxygen concentration needed to reach saturation Oxygen deficiency elicits symptoms of suffocation and fish of different species successively die, depending on how much oxygen each species needs. Fish exposed to oxygen deficit do not take food, move near the water surface, gasp for air (cyprinids), gather at the inflow (in ponds), are torpid, fail to react to irritation, lose escaping reflex and die. The major pathologico-anatomic changes include a very pale skin colour, congested to the cyanotic gills, the gill fringes stuck together, small haemorrhages in the front chamber of the eye and in the skin of the gill covers. In the majority of predatory fishes the mouth is spasmodically open and the opercles of the gills loosely stick out. Damage caused to fish by too much oxygen in water is seldom encountered. This may happen, for example, when fish are transported in polythene bags with oxygen atmosphere. The oxygen saturation of water critical to fish is 250 to 300 %. The fish may be injured at saturation values above this level: the gills of the affected fish have a conspicous light red colour and the ends of the gill fringes fray. When such fish are used for stocking they suffer from secondary fungus infestation and some of them may die.

Ammonia

Ammonia pollution of water courses, ponds and lakes may be of organic origin (public sewage, focal and large-scale agricultural pollution, biochemical reduction of  nitrates and nitrites contained in water) or of inorganic origin (industrial waste waters from gas works, coking plants and generator stations). In water or in biological liquids, ammonia is present either in its molecular (nondissociated) form (NH 3) or in the form of the ammonia ion (dissociated) (NH 4+). The ratio of these two forms depends on the pH and on water temperature (Table 8). For the toxic action it is important that the cell wall is comparatively insoluble to the ammonia ion (NH 4+), but molecular ammonia (NH3) penetrates the tissue barriers very easily, so it is toxic to fish. Under normal conditions there is an acidobasic balance on the tissue barriers. If  this balance is broken, the side on which the pH is lower intercepts molecular  ammonia. This explains how molecular ammonia passes from water through the epithelium of the gills to the blood and also how it passes from the blood to the tissues. Ammonia possesses special affinity for the brain: this is why nervous symptoms are so strong in cases of ammonia intoxication of fish. Water quality monitoring in water courses, lakes and fish culture facilities includes determination of total ammonia. From the viewpoint of toxic action it is important to know the concentration of nondissociated toxic ammonia (NH 3). This is determined from the measured values of total ammonia (NH 4++NH3), temperature (T) and water   pH, using the formula:

or the values are read from a table 8 compiled from calculations on the basis of that formula. Besides water temperature and pH, the factors that influence ammonia toxicity include the concentration of oxygen dissolved in water. The lower the oxygen concentration in water the greater the toxicity of ammonia (Fig. 19).  Nondissociated ammonia is highly to very highly toxic to fish. The LC 50 value, determined in the acute toxicity test, ranges between 1 and 1.5 mg NH 3 per litre in cyprinids and between 0.5 and 0.8 mg NH 3 per litre in salmonids. The maximum admissible ammonia (NH3) concentration is 0.05 mg per litre for cyprinids and 0.0125 mg per litre for salmonids. Table 8 : Dependence of NH 3 content (as % of total ammonia) on water pH and temperature  pH 7.0 7.2 7.4 7.6 7.8

t °C 0 0.082 0.13 0.21 0.33 0.52

5 0.12 0.19 0.30 0.48 0.75

10 0.175 0.28 0.44 0.69 1.09

15 0.26 0.41 0.64 1.01 1.60

20 0.37 0.59 0.94 1.47 2.32

25 0.55 0.86 1.36 2.14 3.35

8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 11.0

0.82 1.29 2.02 3.17 4.93 7.60 11.53 17.12 24.66 34.16 45.12 56.58 67.38 89.16

1.19 1.87 2.93 4.57 7.05 10.73 16.00 23.19 32.37 43.14 54.59 65.58 75.12 92.32

1.73 1.73 2.71 2.71 4.23 6.54 9.98 14.95 21.79 30.36 41.17 52.59 63.74 73.59 81.54 94.62

2.51 2.51 3.91 3.91 6.06 6.06 9.28 13.95 20.45 28.95 39.23 50.58 61.86 71.99 80.29 86.59 96.26

3.62 5.62 8.63 13.02 19.17 27.32 37.33 48.56 59.94 70.34 78.98 85.63 90.42 97.41

5.21 8.01 12.13 17.95 25.75 35.46 46.55 57.99 68.63 77.62 84.60 89.70 93.24 98.21

The first symptoms of ammonia intoxication include slight restlessness, accelerated respiration, the fish stay close to the water surface, intoxicated cyprinids gasp for air, the restlessness increases, motion accelerates, respiration becomes irregular, and then follows the excitation stage. The fish intensely react to outside stimuli, lose balance, leap above the water surface and their muscles twitch in tonic-clonic spasms. The affected fish lie on one side and keep the mouth and gill covers wide open in spasms. Then follows a short period of apparent recovery. The fish return to normal gait and look slightly restless. This stage is later replaced by another severe excitation; the  body surface becomes becomes pale and the fish die.

Fig. 19: If there is not enough oxygen in the water, nondissociated ammonia kills the fish: fatal cases; + waters with high concentration of nondissociated ammonia in which no cases of injury to fish did occur in March to April; xxx - lethal  boundary of nondissociated nondissociated ammonia The skin of ammonia-intoxicated fish is light coloured, covered with plenty or excess of mucus. In some cases there are small haemorrhages, mainly at the bases of the  pectoral fins f ins and in the t he fore chamber of o f the t he eye. The gills are heavily congested and filled with plenty of mucus; the gills of fish exposed to high ammonia concentrations

may slightly to severely bleed. Intense production of mucus can be observed on the inner side of the gill covers, mainly on the end flap of the gill cover arch. The organs inside the body cavity are congested and parenchymatous, showing dystrophic alterations. In recent years, great losses in carp culture have been caused by the so-called toxic necrosis of the gills. The factors responsible for the rise of this disease include ammonia intoxication at which the ammonia level in the blood is highly increased. Ammonia is, at the same time, the final product of nitrogen metabolism in carp and most of it is eliminated through the gills to the water. If this elimination is reduced for  some reason or another (high water pH, oxygen deficit, damaged gills etc.), the ammonia level in the blood is highly increased, causing a condition called autointoxication of the fish. A very interesting case of autointoxication of carp yearling (C 1) with extremely high ammonia N levels in the blood serum was diagnosed after a fter the transfer of the fish from the pond to well water in large aquarium tanks. Part of the fish caught and transferred during the forenoon exhibited typical symptoms of ammonia intoxication the following morning. These symptoms included great restlessness, accelerated respiration, leaping above the water surface, inco-ordination of motion, and tonicclonic spasms of the muscles. The skin of the affected fish was light in colour, the gills were heavily congested, dark red and oedematously swollen (the swelling was  particularly severe on the edges of the gill g ill fringes). The histopathological changes in the gills corresponded with what was described with toxic necrosis of carp gills. The digestive tract of the fish with severe symptoms of intoxication was filled with undigested food. On the other hand, fish that had evacuated (faeces on the bottom of  the tank, gut almost empty), were free of symptoms of toxic damage. The average  blood serum level of o f ammonia N in the fish with symptoms of intoxication was 3054 (2400 – 3600) μg per 100 ml of serum, whereas in the fish free of such symptoms the ammonia level was 825 (750 – 900) μg per 100 ml of serum. In the affected fish the autointoxication, caused by an enormous increase in the ammonia a mmonia N level in the t he blood serum, was probably due to the persistence and absorption of the digesta (natural food, high-protein feed pellets) in the digestive tract of the carp exposed to stress (transport, reduced oxygen level during the transport, water temperature reduced by about 5°C). Considering this case of ammonia autointoxication of carp, some other unexplained abrupt deaths of fish may be ascribed to a similar cause. Such events may happen mainly on carp farms with a high intensity of feeding high-nitrogen feeds if the fish are exposed to stresses such as abrupt oxygen deficit, abrupt change in water  temperature and others. Nitrites, nitrates

 Nitrites as a rule accompany nitrates and ammonia nitrogen in surface waters but their  concentrations are low because of their low stability. They readily oxidize or are readily reduced, both chemically and biochemically. Nitrates are the final product of  the decomposition of organic nitrogen compounds in aerobic medium. They are  present at small concentrations in all surface waters. There is almost no nitrate interception in the soil, so nitrates are washed down to water courses, ponds and

lakes. The main source of nitrate pollution of surface waters is the application of  nitrogenous fertilizers and manure to farm soil. so il.  Nitrites have a variable toxicity to fish: the variability depends on a number of  internal and external factors (fish species and age, water quality and others). The importance and role of many of the factors are continuously checked and reviewed. Different authors often assert contradictory conclusions and still fail to offer a definitive explanation of either the mechanism of nitrites toxic action on fish or the involvement of different environmental factors. According to the latest findings, nitrite ions get into the fish bodies with the help of  chloride cells and the main route of their penetration is via the gills. In the blood, nitrites are bound by haemoglobin, giving rise to methaemoglobin: this reduces the oxygen-transporting capacity of the blood. The increase in the amount of  methaemoglobin manifests itself as the brown colour of the blood and gills. If the amount of methaemoglobin in the blood does not exceed 50 % of the total haemoglobin, the fish usually survive. If the fish have more methaemoglobin in their   blood (70–80 %) they become torpid, and with a further increase in the methaemoglobin level they lose orientation and are unable to react. Nevertheless, in spite of all that, the fish may survive because the erythrocytes in their blood contain the enzyme reductase which converts methaemoglobin to haemoglobin. This process can return haemoglobin to its normal level in 24–48 hours if the fish are put into nitrite-free water. A great differentation in nitrite toxicity to fish kept in water having different  properties is asserted by LEWIS and MORRIS (1986). In I n their investigations the 96h LC50 for rainbow trout ranged from 0.24 to 12.20 mg per litre, depending on chloride content in the diluting water (in the given case the chloride content in water ranged from 0.35 to 40.9 mg per litre). The effect of chlorides on nitrite toxicity is so significant that the results of tests conducted without recording chloride concentrations in water cannot be compared with other tests.  Nitrite toxicity is also influenced influenced (reduced) by hydrogen carbonates, potassium, sodium, calcium and other ions, but the effect is not so great as that of chlorides. Among these ions, those of potassium are most significant; the importance of sodium and calcium is lower. These monovalent ions compete with nitrites in their   penetration into the fish body body and inhibit the passage of nitrites nitrites through the gills. The pH value was also considered to be important for nitrite toxicity: pH and temperature control the balance between NO 2- and nondissociated HNO 2 and it was  believed that the amount of nitrites in the blood plasma of fish depends on the  penetration of o f nondissociated HNO2 to the fish body through the gills. Nevertheless, the results of later experiments denied these theories and demonstrated that within the acidity /alkalinity/ range normally encountered in waters the pH is not so important for the toxicity of nitrites. Another factor that influences nitrite toxicity is the amount of dissolved oxygen in combination with water temperature. This is associated with the fact that fish need more oxygen when their blood contains methaemoglobin which cuts the oxygencarrying capacity of the blood.

Long exposure to sublethal concentrations of nitrites causes no great damage to the fish. Concentrations corresponding to 20–40 % of the levels that have a lethal action on the fish may slightly depress growth but no serious damage has ever been recorded. For estimating the safe nitrite concentration in each particular case it is recommended to monitor the weight ratio of chloride and nitrite concentrations. These ratios (mg.1 -1 C1- : mg.1-1 N-NO2-) are recommended to be around 17 for rainbow trout and around 8 for fish of low economic importance. As to nitrates, their toxicity to fish is very low. Toxic action of nitrates is only recorded when their concentration is above 1000 mg per litre. 80 mg per litre is considered to be the maximum admissible nitrate concentration for carp and 20 mg for rainbow trout. In surface waters and in fish farming facilities where the water  contains plenty of oxygen with no danger of denitrification (i.e. conversion of NO 3- to  NO2- and then to elementary nitrogen or N 2O and NO), it is not so important to monitor the concentrations of nitrates. Sulphane (hydrogen sulphide)

Sulphane occurs in organically polluted waters where proteins decompose. Sulphane is also present in industrial waste waters (from metallurgical and chemical works, effluents from pulp plants, tanneries and other plants). Sulphane is highly to very highly toxic to fish. The lethal concentrations to different fishes range from 0.4 mg H2S per litre (salmonids) to 4 mg per litre (crucian carp, tench and eel). The toxicity of sulphane decreases with increasing water pH (nondissociated toxic H 2S changes into the less toxic HS - ions). The concentration of nondissociated sulphane is determined from the measured total sulphane values (HS - + H2S + S2-) and from the  pH value of the water. The formula is:

where τ = activity coefficient depending on the ionic strength of water. For natural water it is ± 0.92. Carbon dioxide

Carbon dioxide is dissolved in water in its molecular state; only 10 % of it forms carbonic acid H2CO3. These two forms in which carbon dioxide occurs constitute what is called free CO2. The ionic forms, i.e. fixed carbon dioxide, are represented by the hydrogen carbonate and carbonate ions (HCO 3- and CO32-, respectively). Their   presence in the aquatic medium is important for the neutralization capacity of water. The amounts of CO2 present in flowing surface waters are typically in the order of mg  per litre, seldom above 20 to 30 mg per litre. In stagnant surface waters the CO 2 levels are stratified because of phytoplankton photosynthetic assimilation: the upper strata usually have less free CO 2 than the lower strata. It may happen in the surface strata that all the free CO2 may be consumed for photosynthesis and the water pH may thus

increase above 8.3. Common underground waters usually contain several tens of mg of free CO2 per litre. The action of carbon dioxide is either direct or indirect. The indirect action of both free and bound CO 2 is exerted on the fish through its influence on water pH. A direct adverse effect takes place when there is an excess or lack of free CO 2. In low-oxygen waters in which intensive microbial processes take place when the fish are stored or  transported, and also when poorly aerated ground waters are used, high concentrations of free CO2 may be a danger to the fish. In such cases the fish are unable to excrete as much CO2 as they should, their blood loses its acidobasic balance, and acidosis develops. CO 2 and O2 exchange in the blood is limited, the fish accelerate their  respiration, become restless, stagger, and may die. Twenty mg free CO 2 per litre is considered the maximum permissible free CO 2 concentration for trout and 25 mg free CO2 per litre is the maximum for carp (if the acid capacity is 0.5 mmol per litre at a  pH of up to 4.5). The sensitivity of fish to free carbon dioxide declines with increasing acid capacity of water. However, what happens much more frequently is a lack of free carbon dioxide in water. Carbon dioxide deficiency occurs when too much free CO 2 is consumed in the  phytoplankton photosynthetic assimilation, when the water used in thermal power   plants is artificially softened, when water is aerated more intensively than needed and in other cases. Free carbon dioxide concentrations below 1 mg per litre break the acidobasic balance in the fish bodies and cause alkalosis. A lack of free carbon dioxide is particularly dangerous for cyprinid fry when they pass from endogenous to exogenous nutrition. Cyprinid fry breathe through their body surface and are unable to regulate their acidobasic balance by respiration. Low partial pressure of free CO 2 in water is conducive to a high CO 2 output from the body, to alkalosis and finally to death. If the fry of cyprinids suffer from free CO 2 deficiency, they gather close to the water surface and exhibit symptoms of suffocation though the concentration of  oxygen in the water is high enough. Chlorine

Active chlorine* enters into the water courses, lakes and ponds with the effluents discharged from the textile and paper plants. Chlorine and the coumpounds that release active chlorine are used as dissinfectants in both human and veterinary medicine. Chlorinated lime serves for total dissinfection of pond bottoms (application rate of 600 kg per ha), storage ponds and other facilities for fish culture and transport. If fish suffer from a disease of the gills it is recommended to spread chlorinated lime on the surface of the pond at a rate of 10–15 kg per ha (if the average depth of the  pond is 1 m). Overdosage or improper handling of chlorine or chlorine-releasing compounds may damage or kill the fish. Fish may also be damaged by chlorine if  retailers keep them in tanks with intensive flow of chlorinated tapwater which contains 0.05 to 0.3 mg active chlorine per 1 litre. Active chlorine is very toxic to fish. Its toxicity largely depends on water temperature: for example, an active chlorine concentration of 3.5 mg per litre has a sublethal action on carp at a water temperature of 3–7°C but when carp are exposed to the same concentration of active chlorine at a temperature of 15–20°C they die in 1 to 2 hours.

Generally it can be said that long exposure to active chlorine concentrations of 0.04 to 0.2 mg per litre are toxic to the majority of fishes. Active chlorine may affect the fish both locally (the skin and gills) and systemically (when chlorine is absorbed into the blood). The systemic affection manifests itself  mainly as nervous disorders. The clinical symptoms of chlorine intoxication include great restlessness, leaping above the water surface, cramps, lying on the side and spastic movements of the mouth, fins and tail. The spasms of the mouth hinder  respiration, the fish suffocate, fall into depression and die. The skin and gills of the intoxicated fish are covered with a thick layer of mucus and if the concentration of  active chlorine is very high the gills are congested and may bleed. The body surface of the poisoned fish is pale and the margins of the gill fringes and fins are covered with a grey-white coating. Histopathologically, there is a marked dystrophy, necrobiosis to necrosis, with desquammation of the respiratory epithelium of the gills and the epidermis of the skin. * Active chlorine means all forms of chlorine that oxidize iodides into iodine in acid medium (molecular chlorine, hypochlorites, chloramines, ClO 2) Cyanides

As far as cyanides occur in waters, they are not of natural origin: they come from different industrial effluents and/or from plating shops and from thermal processing of  coal. Cyanides may be present in waters either as simple compounds (nondissociated HCN, simple CN- ions) or as complex compounds (e.g. complex iron, cobalt, nickel cyanides and others). Simple cyanides are very toxic up to extremely toxic to fish. The lethal concentrations for the majority of fishes range between 0.03 and 0.5 mg per  litre. The toxicity of these substances is conditioned by the pH value of the water: if  the water pH is low the proportion of nondissociated HCN increases and so does the toxicity (Table 9). Cyanide toxicity is also markedly enhanced by increasing water  temperature and decreasing concentration of oxygen dissolved in water. In complex cyanides the toxicity varies considerably with their ability to split off HCN. For  example, the complex iron cyanides are of low to very low toxicity to fish but the complex cyanides of zinc, cadmium, copper and mercury are highly toxic. The concentrations of different cyanide compounds proposed as the maximum admissible levels in fish culture range between 0.002 and 0.02 mg per litre. Table 9: The dependence of HCN content (% of simple cyanide content) on water pH  pH 7 8 9 10 11

% HCN 100 93 60 10 2

% CN0 7 40 90 98

The mechanism of the toxic action of cyanides is based on the inhibition of the respiratory enzymes (cytochromoxidase). This blocks the transfer of oxygen from the  blood to the tissues, damages tissue respiration and leads to tissue asphyxia. The clinical symptoms of the cyanide poisoning of fish include laboured respiration, nervous disorders and a long agony. If the fish are transferred to clean water while they are in the stage of loss of balance they will recover in 1 to 2 hours. The characteristic features of the patho-anatomic finding in cases of cyanide poisoning include cherry red colour of the gills and sometimes also the presence of transsudate with an admixture of blood in the body cavity. Metals and metal salts

Trace quantities of metals present in waters are of natural origin. If waters are  polluted with metals at greater concentrations, the metals can be traced back to ore mining and processing, to smelting plants, rolling mills plants for the surface treatment of metals, film, textile and leather industries and other sources. Atmospheric  precipitation polluted by fumes generated by the burning of fossil fuels and by the exhausted gases of motor vehicles may be another source. The mechanism of the toxic action of metals on fish varies. Most of the metals have a great affinity for amino acids and the SH groups of proteins: as such, they act as enzymic poisons. The toxicity of metals to fish is significantly influenced by the form of their occurrence in water. Inorganic or organic insoluble or hardly soluble complexes are usually less toxic than simple ions. The adverse action of metals is  particularly strong in the early stages of development of the fish. Another important adverse property of many metals is their ability of amply accumulating in the sediments and in the aquatic flora and fauna (biological accumulation). This property is quantitatively described by the accumulation (concentration) coefficient whose values range from several hundred to hundreds of thousands. Mercury, selenium and some other metals have a particularly high bioaccumulating capacity. Hence, the concentration of these metals in water does not provide a true indication of the actual over-all pollution of the aquatic medium: it is better to use the content of metals in the sediments and especially in the bodies of predatory fishes, which are the final link in the food chain in the aquatic medium. The metals of the highest ichthyotoxicological importance include aluminium, chromium, iron, nickel, copper, zinc, arsenic, cadmium, mercury and lead. Aluminium The toxicity of aluminium to fish depends, to a considerable extent, on the physicochemical properties of the water and particularly on its pH, underlying the form of  occurrence of aluminium: at a higher water pH there is a higher concentration of the dissolved forms and a higher toxicity of aluminium to fish. The concentration of the dissolved forms of aluminium is also great when the pH is very low, about 4. In aluminium toxicity tests, rainbow trout fry were exposed to different aluminium concentrations at pH 7. A concentration as low as 0.52 mg aluminium per litre was recorded to markedly reduce the growth of these fish. When a still lower  concentration, 0.05 mg per litre, was tested no adverse effect was exerted on the

growth of rainbow trout fry, so it can be regarded as safe. A mass kill of maraena and  peled fry, reared in water clarified by means of aluminium sulphate, can be mentioned as an example of practical experience. Aluminium concentration in that water was up to 0.3 mg per litre and the pH value was between 7 and 7.5. All the fry of maraena and peled died in about 10-14 days of hatching. Chromium In surface waters, chromium can be present in oxidation states III and VI. Chromium compounds in oxidation state III are more toxic to fish and other aquatic organisms than are those in oxidation state VI. According to the LC 50 for different fishes, chromium compounds in oxidation state III can be included among substances of a high toxicity to fish (LC50 2 to 7.5 mg per litre) and chromium compounds in oxidation state VI can be included among substances of medium toxicity (LC50 35 to 75 mg per litre). The toxicity of chromium compounds to fish is also considerably influenced by the physico-chemical properties of water, especially by the pH value and the concentrations of calcium and magnesium. At a low pH and at high concentrations of calcium and magnesium the toxicity of chromium to aquatic organisms is low. At acute intoxications by chromium compounds, the body surface of the fish is covered with mucus, the respiratory epithelium of the gills is damaged and the fish die with symptoms of suffocation. Fish suffering from chronic chromium intoxication accumulate an orange yellow liquid in their body cavity. Iron In surface waters, iron occurs in oxidation state II (soluble compounds) or oxidation state III (mostly insoluble compounds). The ratio of these two forms of iron depends on oxygen concentration in water, on water pH and on other chemical properties of  the water where the fish are kept. Cases of damage caused to fish by iron compounds may occur in low-oxygen waters of a low pH where the iron is present mainly in the form of soluble compounds. On the gills of the fish, showing an alkaline reaction, iron in oxidation state II oxidizes into insoluble compounds of oxidation state III, which cover the gill fringes and hinder respiration. At a low water temperature and in the  presence of iron, iron-depositing bacteria will propagate at a high rate on the gills and contribute to the oxidation of iron compounds in oxidation state II. Their filamentous colonies cover the gills: first they are colourless but later the deposited iron colours them brown. The precipitated iron compounds and tufts of the iron bacteria reduce the gill area available for respiration, damage the respiratory epithelium and may choke the fish. Like on the gills of adult fish, iron compounds act on the eggs which die of  the lack of oxygen. The lethal concentration of iron for fish is not easy to determine because it depends on the physico-chemical properties of the water. It is generally required in cyprinid culture that the concentration of the soluble forms of iron should not be greater than 0.2 mg per litre; for salmonids this limit is 0.1 mg per litre.  Nickel  Nickel can get into the surface waters with the effluents from the plants where metals are surface-treated. Nickel compounds are of medium toxicity to fish. In short

exposure, the lethal concentration is between 30 and 75 mg per litre. Like the toxicity of other metals, the toxicity of nickel compounds to aquatic organisms is markedly influenced by the physico-chemical properties of water. For example, in low-calcium and low-magnesium waters the lethal concentrations of nickel compounds for the stickle-back were below 10 mg per litre. In such cases nickel is highly toxic to fish. After intoxication with nickel compounds, the gills of the poisoned fish are filled with slime and are dark red in colour. Copper  Though copper is highly toxic to fish, its compounds are used in fish culture and fisheries as algicides and as substances that are useful in the prevention and therapy of  some fish diseases. The physical and chemical properties of water exert a strong influence on the toxicity of copper to fish. In water containing organic substances at a high concentration copper produces insoluble complex compounds. In high-pH water  it produces low-solubility compounds of alkaline nature (hydroxides), and in waters of a high acid capacity (alkalinity) copper precipitates as low-soluble or insoluble cupric carbonate. For compounds that are reluctant to dissolve or are insoluble it is difficult to penetrate into the fish body, so their toxicity to fish is low. It will be a good example to compare the different LC50 levels for carp as recorded during 48 hours exposure to CuSO 4.5H2O in pond water [ph 7.6, acid capacity (up to a pH of  4.5) 2.2 mmol per litre, COD Mn 32 mg O2 per litre] and in well water [pH 6.2, acid capacity (up to a pH of 4.5) 0.6 mmol per litre, COD Mn 2.2 mg O2 per litre]: in the  pond water the LC50 was 8.1 mg per litre and in well water it was 1.5 mg per litre. The highest copper concentration in water still admissible from the viewpoint of the safety of fish ranges from 0.001 to 0.01 mg per litre, depending on the physical and chemical properties of water and on the species of the fish. The characteristic clinical symptoms of fish intoxication with copper and copper compounds include laboured  breathing and, in cyprinids, gasping for air on the water surface. The typical pathoanatomic finding includes a large amount of mucus on body surface, under the gill covers and in the gills. Acute copper intoxication can be diagnosed on the basis of a chemical analysis of the gills in which the concentration of copper is much greater  than in other parts of the body of the fish. The gills of fish caught in waters free of  copper contamination contain up to 10 mg of copper per 1 kg of dry matter. Zinc Zinc intoxication of fish is most frequently encountered in trout culture. Rainbow trout and brown trout, and especially their fry, are extremely sensitive to zinc and its compounds. The lethal concentrations are around 0.1 mg per litre in salmonids (some authors even assert a level of 0.01 mg per litre) and 0.5 to 1.0 mg per litre in cyprinids. Resistance to zinc compounds increases with age. The toxicity of zinc to fish is influenced by the chemical characteristics of water. The clinical symptoms and  patho-anatomic picture of the zinc intoxications in fish are similar to those in exposure to copper. The best prevention of the frequent intoxications with zinc compounds in trout culture is to avoid using galvanized pipes for the supply of water  and to avoid using galvanized containers and equipment.

Arsenic Arsenic as a rule occurs in water in oxidation state V but some of it may also be  present there in non-stable forms, i.e. in oxidation state III. Like with mercury,  biological activity may lead to the formation of organic methyl derivatives of arsenic. The main sources of arsenic pollution in surface waters include industrial effluents e.g. from tanneries, ore processing plants and dyestuff production plants. Arsenic is able to cumulate in large quantities in sediments on the bottom of water courses and reservoirs and in the aquatic organisms. Arsenic compounds in the third oxidation state (arsenites) are absorbed fairly fast into the fish body and are more toxic than arsenic compounds in oxidation state V (arsenates). According to lethal concentrations during 48 hours of exposure of different fishes, diarsenic trioxide, for  example, can be included among substances of a medium to high toxicity to fish. Lethal concentrations are between 3 and 30 mg per litre. Cadmium Cadmium in waters accompanies zinc but its concentrations are much lower. The cadmium present in surface waters may be either dissolved or undissolved. As to the dissolved forms, those which may be poisonous to fish include the simple ion and the different inorganic and organic complex ions. Apart from the toxic action similar to that of other poisonous metals (damage to the central nervous system and  parenchymatous organs), cadmium - though in trace amounts - may have some specific effects if exposure to its action is long. Chief among the specific effects are those exerted on the reproduction organs. An adverse influence of long exposure to cadmium upon the maturation, hatchability and development of larvae in rainbow trout was recorded at concentrations as low as 2 to 20 mg per litre. Cadmium toxicity declines with increasing contents of calcium and magnesium in water. For salmonids the highest admissible cadmium concentration in water is 0.0002 mg per litre and for  cyprinids 0.001 mg per litre. Mercury Mercury is carried to the aquatic medium mainly with industrial effluents and atmospheric precipitation. Unpolluted waters permanently contain a trace amount of  mercury which does not exceed 0.1 μg per litre. Mercury concentration determined in surface waters is not a true measure of either the actual amount of mercury or the mercury pollution of the aquatic medium: mercury  passes from water to the sediments on the bottoms of water courses, lakes and reservoirs and accumulates there mostly in the sulphide form. Elementary mercury and its organic and inorganic compounds undergo methylation (a process induced by the activity of microorganisms) in the bottom sediments. The toxic products of this methylation (methyl mercury) enter the food chains and accumulate in increased amounts in aquatic organisms. Mercury penetrates into the fish bodies with food via the alimentary tract and the other routes are through the gills and skin. Absorption from the alimentary tract has  proved to be of the greatest importance in mercury accumulation process: evidence is  provided by the results of investigation at sites in the drainage area of the Berounka

river in Central Bohemia. The total mercury content in the flesh of fish in these localities is about 10 times that recorded in the food organisms. This coefficient of  cumulation can be compared with the food efficiency coeficient of the fish living in open waters and feeding on the aquatic invertebrates. Of the other aquatic organisms in the drainage area of the Berounka river, the greatest mercury levels were recorded in leeches: this can be ascribed to their exclusively predatory mode of feeding. With their wide distribution in different types of waters, leeches ( Helobdella stagnalis) may also be considered as good indicators of mercury contamination of the aquatic medium. Fish as the final link in the food chain in water contain the largest amounts of  mercury. The issue of mercury in the aquatic medium is important not only from the environmental and hygienic viewpoints but also from the viewpoint of fish culture. It has been demonstrated that mercury compounds damage some important tissues and organs in the fish body and may also have a harmful effect on fish reproduction. At very low concentrations they reduce the vitality of spermatozoa, depress the  production of eggs and affect the survival rate of fecundated eggs and fry. Acute lethal concentratios of inorganic mercury compounds range from 0.3 to 1.0 mg per  litre for salmonids and from 0.2 to 4.0 mg per litre for cyprinids, depending on the  physical and chemical properties of the water. The acute lethal concentrations of  organic mercury compounds are 0.025 to 0.125 mg per litre for salmonids and 0.20 to 0.70 mg per litre for cyprinids. For salmonids the highest admissible concentration of  mercury in the form of inorganic compounds is about 0.001 mg per litre and for  cyprinids about 0.002 mg per litre. For fish in general, the highest admissible concentration of mercury in organic compounds is claimed to be 0.0003 mg per litre. Lead A significant source of the lead contamination of atmospheric water, and thereby also surface waters, are the exhausted gases of motor vehicles which contain the products of decomposition of tetraethyl lead. In the aquatic medium, lead largely accumulates in bottom sediments where the lead content is about four orders larger than in water. Lead toxicity to fish and other aquatic organisms is significantly influenced by water  quality: it depends on the solubility of lead compounds and on the concentration of  calcium and magnesium in water. The water solubility of lead compounds declines with increasing acid capacity (alkalinity) and pH value of the water. Further, the toxicity of lead is known to decline with increasing calcium and magnesium concentrations in water. The acute toxic and lethal concentrations in different types of  water range between 1 and 10 mg per litre for salmonids and between 10 and 100 mg  per litre for cyprinids. Acute lead intoxication is characterized, first of all, by damage to the gill epithelium: the affected fish are killed by suffocation. The characteristic symptoms of chronic lead intoxication include changes in the blood picture, with severe damage to the erythrocytes and leucocytes, degenerative alterations of the  parenchymatous organs and damage to the nervous system. The highest admissible lead concentration in water is 0.004 to 0.008 mg per litre for salmonids and 0.07 mg  per litre for cyprinids.

Phenols

Phenols penetrate into surface waters with industrial effluents, especially the waste waters from the thermal processing of coal, from petroleum refineries, from the  production of synthetic fabrics and other industrial segments. Analytically, phenols are either monobasic (phenol, cresol, naphtol, xylenol) or polybasic (pyrocatechol, resorcine, hydroquinon, pyrogalol, floroglucin). Surface waters may also contain badly smelling chlorophenols, which arise as a result of the chlorination of phenols. The highest concentrations admissible from the point of view of fish culture are 0.001 mg per litre for phenol, 0.003 mg per litre for cresol, 0.004 mg per litre for resorcine, and 0.001 mg per litre for hydroquinon. Concentrations of 0.1 mg of phenols per litre of water and 0.02 mg of chlorophenols  per litre of water are high enough to cause changes in the sensory properties of fish flesh. Long exposure to a concentration of 0.2 mg of phenols per litre of water was observed to drive the fish out of the drainage area of the polluted water course. According to the lethal concentrations to fish, the different phenol compounds will rank as follows: hydroquinon (0.2 mg per litre), naphthols (2 to 4 mg per litre),  phenol, cresol, pyrocatechol and xylenol (2 to 20 mg per litre), resorcine and  pyrogalol (10 to 50 mg per litre), floroglucin (400 to 600 mg per litre). Phenols are typical nerve poisons which damage the central nervous system. The clinical signs of intoxication are characterized by great restlessness, increased irritability, leaping over the water surface, loss of balance and muscular spasms. The post-mortem findings include conspicuous whitening of the water surface, amply covered by mucus; exposure to high temperatures cause haemorrhages on the lower  side of the body. Long exposure to low concentrations leads to dystrophic to necrobiotic changes in the brain, parenchymatous organs, circulation system and gills. Polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) have recently become a very important factor of  environmental pollution. PCBs are among organic compounds of the highest stability. Their solubility in water is low but they readily dissolve in nonpolar solvents and fats. A mixture of PCBs isomers with different ingredients is used in the electrotechnical industry (fillings of power capacitors and high-voltage transformers), mechanical engineering (incombustible liquids for heat transfer, fillings of hydraulic equipments and lubricants for compressors) and in the chemical industry (production of synthetic varnishes, dyestuffs and plastics). The widespread brandnames of PCBs include Delor  (CSFR), Aroclor (USA), Clophen (Germany), Kaneclor (Japan), and Savol and Sovtol (USSR). As a response to many warning signals, drawing attention to the environmental hazards and excess spread of the PCBs, the production of   polychlorinated biphenyls has been successively restricted since 1971. In surface waters, PCBs occur at concentrations from 1.10 -8 to 1.10-4 mg per litre. Polychlorinated biphenyls have a high capacity of accumulation in the bottom sediments and in the aquatic organisms in which the accumulation coefficient is 10 3 to 105.

PCBs are very toxic to extremely toxic to fish, mainly their young developmental stages. Their 48 hours LC50 are below 1 mg per litre. As to the mechanism of the toxic action of PCBs, these substances have been found to adversely interfere with the enzyme systems of the microsomal fraction of the liver. If  fish are exposed for long to low sublethal PCBs levels the contaminants accumulate in their bodies and cause, mainly in the fry, deformations of the skeleton, damage to the skin and fins (the fins disintegrate), damage to the parenchymatous organs, mainly liver (hypertrophy, local dystrophic, necrobiotic to necrotic changes), damage to the gonads and subsequent great losses during hatching, high mortality of early fry and increased occurrence of different deformations in the early fry. The highest admissible PCBs concentrations in water range from 1.10 -6 to 5.10-6 mg  per litre for salmonids and from 2.10-6 to 1.10-5 mg per litre for cyprinids. Lower  admissible concentration levels are recommended during hatching and rearing of the early stages of the fry. Tensides

Tensides are either natural or synthetically produced surfactants used in many segments of the economy. They are traditionally used for their detergent and cleaning action and also for their emulsifying, dispersing and other effects. From the chemical point of view, tensides are either ionic (liable to electrolytic dissociation) or nonionic (not dissociating in water). Ionic tensides are subdivided into anion-active (dissociating to surface-active anion and inactive cation), cation-active (dissociating to surface-active cation and inactive anion), and ampholytic (assuming either anion-active or cation-active properties, depending on ambient conditions). The anion-active tensides are ionic tensides most widely used in industry. As to the mechanism of the toxic action of tensides on fish and other aquatic organisms, it is stated in literature that besides the toxicity ensuing from the chemical structure of the tenside, its physico-chemical efect also contributes to the harmful action. The surface tension of the water decreases and this leads to hydration and to enlargement of the cell volume. At lower tenside concentrations this enlargement is reversible. High concentrations suppress metabolic processes in the cells. Long exposure damages the cells which necrotize in the later stages. These changes manifest themselves mainly in impairment of the respiratory epithelium of the gills. In addition, exposure of fish to some tensides changes the activity of the respiratory enzymes, especially cytochromoxidase. Tensides also damage the protective layer of  mucus on the skin: the layer loosens and the infection resistance of the fish decreases. Sublethal tenside concentrations also damage the eggs and sperm. The toxicity of tensides to fish is influenced by a number of biotic and, especially, abiotic factors. The age of the fish is a particularly important biotic factor. During embryonal and larval development, the sensitivity of fish to tensides is sometimes greater by an order, in comparison to the juvenile and adult stages. Of the abiotic factors, the tensides molecular structure and the physico-chemical properties of water exert the greatest influence on their toxicity. The results of investigation of 

the relationship between toxicity and molecular structure indicate, for example in linear alkylbenzene sulphonates, that toxicity to fish markedly increased with the length of the chain. Similar dependence of toxicity on chain length was observed in other tensides. Among the physico-chemical properties of water, calcium and magnesium concentrations have the greatest influence on tenside toxicity and some influence is also exerted by the pH: toxicity declines with increasing calcium and magnesium concentrations in water. Where cation-active and anion-active tensides are contained in the waste waters the toxicity is much lower. This is due to the formation of insoluble complex between the anion-active and a nd cation-active tensides. The acute toxicity of tenside t ensidess varies greatly great ly with the species of the fish. Nevertheless, Nevertheless, these substances and preparations are highly toxic to fish in the majority of cases, the 48-hour LC50 ranging between 1 and 10 mg per litre. A minor part of the tenside group can be classified as medium toxic substances (48-hour LC50 between 10 and 100 mg per litre) down to substances of very low toxicity (48-hour LC50 to 10 000 mg per litre). No significant differences in toxicity to fish were recorded between the anion-active, cation-active and nonionic tensides t ensides.. Tenside intoxication of fish cause, in particular, damage to the respiration epithelium of the gills (enlargement and vacuolation, dystrophic to necrobiotic changes in cells). Hence, the clinical signs of intoxication include respiration disorders (accelerated respiration; cyprinids gasp for air on the water surface) and later by depression. The characteristic findings in the patho-anatomic picture are an increased amount of  mucus on the skin and in the gills and congestion to oedematous swelling of the gill apparatus. The mucus easily falls off the t he body surface and gills. Pesticides

The use of pesticides has considerably increased (both the total quantity and the spectrum of pesticides used). At present, pesticides are among the main factors responsible for intoxication of fish. At lower concentration they may not alvays have a direct effect on the fish stock but may exert a long-continued adverse effect on the earliest developmental stages of the fish. Besides these acute and chronic direct effects, importance is also attached to their indirect action. Inexpert application of herbicides or algicides to the water or  contamination of surface waters with these chemicals may kill the aquatic plants and algae. The decomposition of this organic matter requires plenty of dissolved oxygen and takes it from the water. This leads to oxygen deficit and the fish may die of  suffocation. Another very serious indirect consequence of pesticide contamination of  the aquatic medium is the reduction or complete destruction of the natural food base for the fish. Many of the organisms the fish feed on are much more sensitive than the fish themselves. The LC50 levels of o f organo-phosphorus pesticides, determined during the acute toxicity test, may be a good example: for common carp the LC50 is 545 mg of Soldep per litre of water and for  Daphnia magna it is 0.0002 to 0.001 mg per litre (the active ingredient of Soldep is 25 % trichlorphon). Besides their active ingredient, pesticides introduce in the surface waters a number of  other chemicals which may sometimes be much more toxic to fish than the active ingredient itself.

When a pesticide penetrates into the aquatic medium the active ingredient undergoes chemical and biological decomposition. In some cases the decomposition products may be more toxic to the fish than is the original active ingredient. For example,  biological oxidation of parathion gives rise to paraoxon, which is more toxic; decomposition of trichlorphon produces more toxic dichlorvos and the like. From this it follows that absence of a pesticide in water cannot guarantee the absence of the harmful products of its decomposition. Some pesticides are used in fish culture and water management to kill the aquatic  plants (Gramoxone S, Reglone). Trichlorphone-based Trichlorphone-based organo-phosphorus pesticides, e.g. Soldep, Masoten, Neguvon and others, serve to reduce the coarse  Daphnia zooplankton, to counter the oxygen deficit in the pond, to kill predatory cyclopids  before stocking the pond with the sac fry of fishes, to control the parasites that vex cyprinids, and for other useful purposes. Pesticides based on copper oxichloride may  be used for antiparasitic treatment treat ment of the fish, for the control of water wat er slugs and to kill water bloom. bloom. However, in the majority of cases pesticides cause damage to the fish. The most dangerous of them are those based on chlorohydrocarbons, organic phosphorus compounds, carbamates and thiocarbamates, carboxylic acid derivatives, substituted urea, triazines and diazines, synthetic pyrethroids, metallic compounds and others. Pesticides based on chlorohydrocarbons These pesticides act as nerve poisons. They are highly to extremely toxic to fish (48hour LC50 > 10 mg per litre). The clinical signs of fish intoxication by pesticides on the basis of  chlorohydrocarbons include excitation, followed by a long stage of depression. There is no specific patho-anatomic picture in these cases of intoxication; dystrophic alterations are recorded in the liver and kidneys. Pesticides based on organo-phosphorus compounds compounds The mechanism of the toxic action of pesticides containing organo-phosphorus compounds upon fish has the same pattern as their toxic action on homoiothermic animals. Some hydrolytic enzymes, particularly part icularly acetylcholine hydrolase, are inhibited. inhibited. The degree of inhibition of cerebral acetylcholine hydrolase in fish varies with the organo-phosphorus organo-phosphorus substances by which w hich the inhibition is caused. Phenitrothion-based Phenitrothion-based  pesticides reduce the enzymes activity to 60 %, dichlorvosdichlorvos- and imidane-based  pesticides cause a reduction as great as to t o leave only 22 % of physiological act ivity. The toxicity of these preparations to fish also varies: according to the values of 48h LC50 they are included among substances of medium to very high toxicity to fish (91  – 100 mg per litre). Salmonids are very sensitive to pesticides containing organic  phosphorus  phosphorus compounds. The typical typical sign of fish intoxication with organo-phosphorusorgano-phosphorus based pesticides is the darkening of body surface at the onset of inco-ordination inco-ordination of  motion. The patho-anatomic picture of such intoxications is characterized by much mucus on the body surface and in the gills, great congestion of the gills and dot-like haemorrhages in the gills when the pesticides concentration is high.

The water fleas  Daphnia magna are most sensitive to organic phosphorus pesticides in water. According to the values of 48h LC50 these t hese substances substances are extremely extr emely toxic to fish. It is interesting to note that the water fleas were found to be as sensitive to trichlorphon and dichlorvos as the gas chromatograph.  Daphnia magna can be regarded as the most reliable indicator of pollution of the aquatic medium with organo-phosphorus organo-phosphorus pesticides. Pesticides based on carbamates and thiocarbamates Carbamate- and thiocarbamate-based preparations inhibit the activity of the acetylcholine hydrolase. Unlike in the fish poisoned by organo-phosphorus compounds, the inhibited enzyme soon regenerates in cases of carbamate and thiocarbamate intoxication. The toxicity levels of these substances to fish vary from low to wery high toxicity (48h LC50 ranging from 1 to 1000 mg per litre). The clinical and patho-anatomic picture of intoxication of fish with carbamate-based  pesticides are not not specific. Pesticides based on carboxylic acid derivatives der ivatives A number of these pesticides are based on phenoxyacetic acid. The main representative of this group is 2-methyl-4-chlorphenoxyacetic acid (MCPA). Most of  the MCPA-based products are of low to medium toxicity to fish (the 48h LC50 ranges from 10 to 1000 mg per litre). The clinical signs of intoxication are mostly characterized by the stages of depression. The patho-anatomic picture of fish intoxication with these herbicides is not very pronounced. Pesticides based on substituted urea Herbicides formulated on the basis of substituted urea are of low to high toxicity to fish (the 48h LC50 ranges from 1 to 1000 mg per litre). The clinical symptoms of  intoxication are not specific and include restlessness, irregular respiration, incoordination of motion and a long period of agony. The patho-anatomic picture is characterized by increased amount of mucus on the darkened body surface, hyperemia of the gills and presence of a small amount of transsudate in the body cavity of the fish. Pesticides based on diazines and triazines Triazine-based pesticides are of medium to high toxicity to fish (48h LC50 ranging from 1 to 100 mg per litre). The clinical signs of fish intoxication with these chemicals are largely characterized by depression stages. Formation of transudate in the body cavity and in the digestive tract is an especially characteristic pathoanatomic sign, mainly in rainbow trout. The presence of transudates causes a marked enlargement of the body cavity; in rainbow trout it was conducive to a rupture of the  body wall in some cases. cases. Diazine-based herbicidal preparations are less toxic to fish than are triazine-based  preparations. Most of the former are of low to very low toxicity to fish (48h LC50 ranging from 100 to 10 000 mg per litre). The clinical course of intoxication is characterized by stages of depression. depression. The T he patho-anatomic picture is not specific. specific.

Pesticides based on synthetic pyrethroids With their 48h LC50, these pesticides rank among substances of high toxicity (up to 10 mg per litre) to extreme toxicity (less than 0.1 mg per litre) to fish. The clinical signs of intoxication are not specific and include respiration disorders. The most conspicuous patho-anatomic change is the presence of a small amount of transsudate in the body cavity. Pesticides based on metal compounds These include, first of all, fungicides formulated on the basis of copper, mercury and aluminium. In the majority of cases, the toxicity to fish and the clinical and pathoanatomic symptoms correspond to those recorded in fish intoxicated with the respective metals. Petroleum and petroleum products

Petroleum and the products of its processing are responsible for most of the recent  pollution of surface and underground waters. Over 1970 to 1990 these materials were responsible for the majority of water pollution accidents that happened worldwide. These accidents were not caused by problems in sewage water treatment plants: most of them were due to negligent storage and handling, transport accidents, defects in equipment and the like - hence, all can be ascribed, either directly or indirectly, to the human factor. However, petroleum and products of its processing also penetrate into the aquatic medium with waste waters. Petrochemical industry is responsible for most of the  pollution. Other important sources of pollution include engineering and metallurgical works and the car and truck repair and service shops. Most of these sources have been  polluting waters for a long time.  None of the petroleum processing products will readily solve in water. There are large differences between petroleum and its different products as to their toxicity to fish. Most of them have 48h LC50 values between 0.5 and 200 mg per litre. Toxicity varies with the chemical composition of the different products, with the water solubility of  the different petroleum hydrocarbons, with the degree of emulsification and other  factors. It is generally asserted that the lighter petroleum fractions (kerosene, petrol) are much more toxic to fish than the heavy fractions (oils). There are also differences in the sensitivity to petroleum and its products in different fishes. The fry of the  predatory fishes (asp, pike-perch, trout) show the greatest sensitivity to petroleum  products. When petroleum products penetrate to rivers or ponds they spread on the surface, thus reducing (especially in stagnant waters) the passage of oxygen from the air to water. In cases of pollution of flowing waters the pollutant does not form a compact layer on the water surface: the petroleum products emulsify, the gills of the fish are mechanically contaminated and their respiration capacity is reduced. Petroleum  products also contain various highly toxic substances, soluble in water, which  penetrate into the bodies of the fish and cause straight intoxication. These include the

naphthenic acids which are acute nerve poisons and are able to kill fish at concentrations as low as 0.03 to 0.1 mg per litre. Symptoms of disturbances of the function of the nervous system and respiration  prevail in cases of acute fish intoxication by petroleum and products of o f its processing. The main clinical symptoms include great restlessness, accelerated respiration, loss of   balance (the fish lie on the side), loss of reactivity to irritation, irr itation, depression, depression, slow-down of respiratory movements, and death. The scales of the dead fish are dull in colour and are covered with mucus, the skin shows local congestion, the epidermis breaks and peels off and surface wounds may occur in some cases. Damage to the cornea of the eyes may lead to blindness. The gills suffer from severe dysthrophic changes and necrosis and there may also be  proliferation of the respiratory epithelium and hyperthrophy of the mucus cells. Longcontinued exposure to petroleum at low concentrations is conducive to severe degenerative necrobiotic changes in the kidneys of the fish and in the eggs. The dead fish have a petroleum smell and taste. Toxicity is not the only danger associated with petroleum and the products of its  processing: the aquatic ecosystems and fish farming are badly affected by the oil smell and taste of the water and of organisms that live in such water. For this reason, sensory assessment is preferred to toxicological analyses in determining the highest admissible amounts of petroleum petro leum and petroleum products that can be contained in water. For the different substances derived from petroleum, the highest admissible concentrations range from 0.002 to 0.025 mg per litre. Dyes

Chemical dyestuffs have also been attracting toxicologists attention in recent years. They are present in the waste waters discharged from textile plants, food plants and  paper mills. Though the waste waters containing dyes are very conspicuous even e ven at very great dilutions they seldom cause severe damage to the fish. The toxicity of dyes depends on the physico-chemical composition of the water. Where the water contains much organic matter the dyes are bound to these organic substances and their toxicity decreases. The mechanism of the toxic action of dyestuffs on fish is not direct in the majority of  cases. If the water is polluted with organic dyes, the increase in the content of  organics leads to oxygen deficit. Other dyes increase or decrease water pH. Still others, e.g. anilline, act as methaemoglobin poisons and as cancerogenic substances. The acute toxicity of different dyestuffs to fish considerably varies. Most of the dyes are among the substances of low to wery low toxicity to fish (48h LC50 ranging from 100 to 10 000 mg per litre). This group includes colouring agents used in the food industry and selected organic dyestuffs. Another group, including e.g. acriflavin, rhodamin and also anilline and methylene blue, comprises substances of medium toxicity to fish. Their LC50 values at 48h exposure range from 10 to 100 mg per litre. The group of dyes of very high toxicity to t o fish includes, for example, malachite green.

The clinical symptoms of fish intoxication by the different dyes are not specific. The  patho-anatomic changes that signal such intoxication intoxication may include a change of body colour by the dyestuffs tested, and the organs or gans inside the fish body may also take t ake on an intensive colouring, e.g. from malachite green. Phytoplankton toxins

Increasing eutrophication of surface waters brings mass development of   phytoplankton and higher aquatic plants. This raises water pH to levels above 10 and a nd the breakdown of the mass of dead phytoplankton and higher aquatic plants causes oxygen deficit. Further, some representatives of these aquatic organisms produce substances (toxins) that may affect not only fish but also farm animals and man.  Microcystis, These include, in particular, the blue green algae of the genera  Microcystis,  Aphanizomenon  Aphanizomenon and  Anabaena. An endotoxin, having the nature of cyclic aeruginosa. a.  polypeptides,  polypeptides, was isolated from the alga Microcystis aeruginos In exposed fish the action of the products of these aquatic organisms (blue green algae) increases thiaminase activity and reduces thiamine content in organs and tissues; this leads to B 1 avitaminosis. The toxic products get into water during the  period of development of water wat er bloom (algae) and particularly part icularly when the water wat er bloom dies and breaks down. These toxins penetrate into the fish body through the gills and  body surface. Part of it is also ingested with food. The clinical symptoms of  intoxication include a damage to the central nervous system. First there is increased restlessness and accelerated respiration, followed by inco-ordination of motion, then the fish lie flat on the bottom and die. The major patho-anatomic signs include haemorrhages on the skin and gills and in the internal organs. Some phytoplankton representatives have been found to generate hydroxylamine as a  product of their metabolism. The occurrence of hydroxylamine in the heavily eutrophicated waters of some ponds is also supported by a high concentration of  organic substances which reduces the oxide reduction potential of the environment, thus allowing the hydroxylamine to accumulate. This is why the highest hydroxylamine concentrations in surface waters are usually recorded during the  periods of mass decomposition of the water bloom (blue-green algae), when the concentrations may reach toxic levels for a short time. Hydroxylamine is highly toxic to fish, its LC50 in acute exposure being less than 10 mg per litre (in sensitive fishes it may be as low as about 1 mg per litre). The toxic action of this substance causes severe methaemoglobinaemia and damage to the central nervous system. Though the cases of damage to fish are seldom ascribable to the action of phytoplankton toxins, this danger should not be underrated, especially in the t he warm regions. 3.2 Diagnostics of Fish Intoxications It is a difficult and complicated task to diagnose fish poisoning because the mortality is often recorded with a delay and the fish and water are not sampled in time. In such cases the patho-anatomic changes in the fish are obscured by the starting post-mortem changes and the toxic substances that poisoned the fish have been carried away with the water flow. Hence, it is necessary necessary to use all the available information informat ion and possible analytic methods to detect the cause of the death of the fish and aquatic invertebrates. The analytical work should start from examining the anamnestic data and performing

the physico-chemical and hydrobiological analyses of the water, and if necessary also the bottom sediments and periphytes and then the fish themselves should be examined. Bioassay for water toxicity is an important item in the diagnosis of fish  poisoning.  poisoning. Examination in situ

When the fish are observed to exhibit strange behaviour, or to die, the following major steps should be taken on the spot. a. Define the area where the fish die or change their behaviour.  b. Catch the damaged or newly killed fish and deliver them to veterinary examination. c. Record the state of zooplankton, phytoplankton and benthos. d. Take water samples for hydrochemical analysis (part of the analyses and measurements must be done on the spot: O 2, temperature, transparency, smell and the like), and for the bioassay for acute toxicity. e. Draw a chart with an indication indicat ion of the affected area, with the sites of water and sediment sediment sampling. sa mpling. Fill in the form on the in-situ examination. If it is suspected that the fish might die or change their behaviour owing to the application of a chemical in the vicinity of the affected water course or pond or lake, detailed information should be gathered, concerning the time and method of  application and about the kind and amount of the chemical applied. An example of  notes serving as document on local inquiry in cases of abrupt change of behaviour or  mortality of fish. 1. 2. 3. 4. 5. 6. 7. 8.

Day, hour and place of investigation Persons involved in investigation  Name and address address of the organization that suffered loss loss (owner of the facility)  Name or another denomination of the water course or reservoir where fish were killed Species and age of the fish that occur in affected water wat er course or reservoir  The killed fish (number, ( number, species, age) Clinical signs and macroscopic changes in the fish with changed behaviour  Present local state of health of o f the fish in the t he given water course or reservoir  9. Status of  killed zooplankton  phytoplankton killed  benthos killed aquatic plants killed

yes

no

yes yes yes

no no no

10. Possible sources of pollution that might be associated with mortality of the fish 11. Preliminary estimate of the t he damage (lenght or area of o f water course or reservoir  affected) 12. Total weight of the t he dead species of the fish

13. Place, hour of sampling, designation of samples water  sediments  periphytes fish to be examined:   

newly killed with clinical symptoms with no signs of intoxication or disease

14. Determination of water quality in situ (water temperature, colour, transparency, smell, concentration of dissolved oxygen, ammonia concentration and other criteria) 15. Opinion of participants of local investigation 16. Signatures of local investigation participants If the fish are killed by the application of a chemical in the vicinity of a water course or reservoir (e.g. a pesticide), the following data should also be included in the document on local inquiry: 1. Day and hour when a crop was treated 2. Chemical used (trade name, name and content of active ingredient, application rate, concentration) 3. Method of application: 4. Local denomination of the plot, species of crop treated and area under the crop treated, distance from water course or reservoir  5.  Name and address of the employer of the person who did the application 6. Weather that prevailed when the field was treated (wind direction, windspeed, rain, cloudiness) and records on rains in the period between application and fish death Map: A brief chart of situation, indicating the area where fish died and the places where water samples were taken.

A - Fenolana plant  part of river where fish died

Water sampling sites: 1. 2. 3. 4. 5. 6.

 place of the greatest kill - 10.00 h September 20th 60 m downstream of the mouth of sewer - 10.15 h in the place of the mouth of sewer - 10.30 h 40 m upstream of the mouth of sewer - 10.45 h about 3 km downstream of the mouth of sewer - 11.45 h about 6 km downstream of the mouth of sewer - 12.30 h

Hydrochemical examination

Choice of the right sampling sites and the right water sampling method is the main  prerequisite for successful work, so they must be given maximum attention. In flowing waters, water sampling sites are laid out as follows: a. in the place where fish die or have strange behaviour   b. upstream of the place of the kill: 50 to 100 m downstream of the mouth of pollution source the place where the pollution source joins a water course stocked with fish, 30 to 50 m upstream of the place of the mouth of the pollution source, c. downstream of the place of the kill: in places where the first signs of unusual  behaviour of the fish are observed. Places downstream of the place of the kill often have to be determined by calculation: the front line of the polluted area can be calculated from the time of leakage of the toxic pollutant and the flow rate of the water course. o o

o

In reservoirs and fishponds, the places of water sampling should be specified according to the actual situation; in some cases the samples have to be taken from different heights of the water column. Special sampling bottles (e.g. the Hrbáček  displacement bottle, Fig. 20) are used to take water samples from different depths and from just above the bottom. The samples are poured in clean 1- to 2-litre bottles. It is not recommended to fill the  bottle with water near the shore: the samples are as usual taken 1–2 m off shore. From near the bottom, the water samples must be taken with care not to raise mud and other  sediments. For maximum objectivity of the data on the water examined, the time  between sampling and analysis must be as short as possible. The samples are shipped with advantage in thermally insulated bags. In the laboratory the bottles with the samples should be placed in refrigerators and kept at 3–4°C. in thermally insulated  bags. In the laboratory the bottles with the samples should be placed in refrigerators and kept at 3–4°C. However, all this may not be sufficient for some water  components. In such cases the samples must be analyzed as soon as they are collected, or may be preserved with a small amount of preservative. Detailed data on the  preservation and treatment of the samples are given in the Table 10.

Table 10: Preservation and treatment of water samples Water  Characteristics

Preservative (amount per 1 litre of sample)

Method of sample treatment

Temperature

-

Measure during sampling

Colour

-

1. Determine during sampling 2.Store at 4°C - determine within 24 h

Transparency

-

Odour

-

 pH

-

Oxygen capacity

-

Dissolved oxygen Chemical a) 1 ml H2SO4 oxygen demand (CODMn,CODCr )  b) store at 4°C Biochemical oxygen demand (BOD5) Ammonia

-

2.Determine in 24 hours (laboratory determination) Identify some smells during sampling (e.g.chlorophenol), others in 24 h at the maximum 1.Determine during sampling 2. Collect into oxygen bottle, determine within 24 h 1.Determine during sampling 2.Collect into oxygen bottle, determine within 24 h Collect into oxygen bottles, fix after  collecting Determine as soon as possible after  sampling, in 24 h at the maximum Maintain at 4°C, process in 24 h

a) 1 ml H2SO4

1.Determine during sampling

 b) 3 ml CHCl3

2. Store at 4°C - determine within 24 h 3.Determine after preservation

 Nitrates, nitrites a) 1 ml H2SO4

1.Determine on sampling day 2.Maintain at 4°C - -determine in 24 h 3.Determine after preservation Collect into brown bottle, determine immediately after sampling

Chlorine Cyanides

1. Determine during sampling (field determination)

solid NaOH up to  pH 11 at the minimum

Determine immediately, several hours after sampling at the maximum

Copper, zinc

5 ml HNO 3

Cannot be preserved if sample contains cyanides

Iron (total)

5 ml HNO3

Completely filled bottles: prevent contact with air 

Phenols

1.Determine within 24 h  NaOH added to reach pH 11 (about 2.Determine after preservation 4 g per litre) 3.No phenol preservation needed at  phenol levels above 150 mg per litre

Tensides: anion active cation active nonionic Petroleum and its products

3 ml CHCl3 -

Collect to glass bottles, determine within 72 h

The collected sample size is 1 to 5 litres, depending on the  pollution. Use glass bottles (never use polythene bottles). Determine as soon after sampling as possible

Fig. 20: Hrbáček's displacement-type bottle for water sampling to determine the concentration of dissolved oxygen The basic hydrochemical analysis includes the determination of the colour, odour, pH, acid capacity (alkalinity), concentration of dissolved oxygen, chemical oxygen demand (COD), biochemical oxygen demand (BOD 5), ammonia, nitrites, nitrates,  phosphates and total phosphorus. The method of determination of other chemicals is chosen on the basis of the results of local investigation (anamnestic data) so as to make it possible, from the results of the investigation, to identify the causes of the mortality or damage of the fish. When assessing the results of the physico-chemical analysis of water samples as to the causes of mortality, the parameters should not be evaluated in isolation: a complex approach has to be used. The toxicity of the different chemicals and products to fish and aquatic invertebrates is influenced by the nature of the aquatic environment as a whole.

Hydrochemical examination of the water is performed at site during the local investigation and also in chemical laboratories. In the field analyses, the Combi kit,  produced by the Central Laboratories of the Fish Culture and Hydrobiology Research Institute at Vodnany, Czechoslovakia, can be used with advantage. The Combi kit allows to determine the following: water transparency (using the Secci disc), concentration of dissolved oxygen, pH, acid capacity (alkalinity) and total concentrations of ammonia and phosphates in water. The physico-chemical characteristics of the water are determined according to Czechoslovak Standard ČSN 83 0530 Chemical and Physical Analysis of Surface Water, Parts 1–50, chlorine level is determined on the basis of ČSN 83 0520 Physicochemical Analysis of Drinking Water, Part 18. The amount of metals contained in water is measured by the atomic absorption spectrophotometry method (AAS). The gas and liquid chromatography methods are used for the determination of organic compounds, e.g. the active ingredients of pesticides, tensides, organic dyes, PCBs and others. Examination of bottom sediments

In justified cases, bottom sediments may be sampled, apart from collecting the samples of water. This is usually the case when there is suspicion that a water course or reservoir is polluted with petroleum products, metals, pesticides and some other  substances which accumulate in the bottom sediments. There is no standardization of sediment sampling: local conditions must always be respected. The main principle is to take the samples mainly from the top layer of the sediments, to take them at several sites within the locality investigated, and to mix the samples for analysis. The bottom sediments are analyzed by modifications of the methods used for determination of pollutants in waters and other (mainly biological) materials. Hydrobiological examination

Hydrobiological examination of water is very important for the diagnosis of  intoxication of fish and lower aquatic organisms. This examination includes evaluation of the qualitative and quantitative structure of the lower aquatic organisms and recording of the damage these organisms might suffer, and of the changes in the  behaviour of the fish, or their death. Exposure to a specific group of poisons can be estimated from the changes in the composition of the biocoenosis after the intoxication. For example, crustaceans and insect larvae are very sensitive to insecticides, aquatic plants are sensitive to herbicides, algae react sensitively to algicides etc. In cases of accidents on water courses and in reservoirs, aquatic invertebrates are usually the first indicator of pollution of the aquatic environment, and the fish follow afterwards. This is especially characteristic of the pollution of  water courses and reservoirs with pesticides and some metals. Substances of surface activity (tensides) exhibit about the same toxicity to fish and to aquatic invertebrates. On the other hand, fish are the main indicators of pollution in the cases of accidents at which the water courses or reservoirs are polluted with organic substances.

Examination of periphytes

Periphytes form a mat consisting of an association of aquatic organisms (bacteria, fungi, algae, protozoans, bryozoans, rotifers and others) growing on, or attached to, surfaces such as the stems of higher aquatic vegetation, stones, structures built in water, immersed logs and the like. In surface waters, periphytes are important as food for many water animals, including fish. They make a significant contribution to the self-cleaning processes and their quality and quantity indicate the average quality of  water at the given site: short-term and insignificant changes in water quality usually have an influence on the association of organisms making up the periphytic mat. This is of great importance in water analyses. At accidents, mostly characterized by a short toxic wave killing the free-living organisms and carrying them away, the damaged  periphytes may provide information on the past wave as well as on the quality of  water in the preceding period. Periphyte analyses are an important part of the over-all examination of water quality. Their importance is particularly high in places with flowing water where the analyses of single samples from single sampling sites usually fail to provide representative results. Periphyte samples are usually obtained by  plucking them off the different surfaces by means of a pair of long tweezers. A scraper held on a long handle will be of help in greater depths. The periphytes attached to underwater structures of concrete or other materials can be easily scraped  by means of a laboratory brush (normally used in washing laboratory glassware) on a long pole: the material attached to the bristles of the brush are then put into the sampling phial, using a pair of tweezers. In the laboratory the periphyte samples are subjected to hydrobiological analysis in normal microscopic preparations, the organisms present in the samples are identified and their number is estimated by means of an estimation scale. The procedure is described in Czechoslovak Standard CSN 83 0532 Biological Analysis of Surface Water (its Part 5 Periphyton Determination). Biological assay for water toxicity testing

Examination of the source and cause of an accident that happens in a water course or  reservoir includes, besides hydrochemical and hydrobiological analyses, also a  bioassay for water toxicity. The water used for the bioassay is taken from part of the water sample (free of preservatives) sent to laboratory for physico-chemical analysis. The aquatic organisms serving for the assay include aquarium fishes, especially  poecilia reticulata, and aquatic invertebrates, especially the water fleas of the genus Daphnia, which are among the aquatic organisms of the highest sensitivity to the majority of pollutants. As to  poecilia reticulata, it is not an extremely sensitive fish  but its advantage like in the majority of aquarium fish is its easy availability all the year round. The procedure of the bioassay for water toxicity is as follows: Ten water fleas (Daphnia) or two or three aquarium fish ( Poecilia reticulata) are put in 100-200 ml of  water sample. If the water samples are large enough, the assay is done simultaneously on both organisms. Parallel to the tested sample, the same number of organisms are  put in uncontaminated water for control. The best containers are, by our experience, crystallizing dishes where the water column is shallow, allowing sufficient oxygen to diffuse from air to the water. The behaviour and state of the organisms are monitored during the bioassay, which may last 24 to 48 hours.

If the result of the assay is negative (the behaviour of the fish or water fleas does not differ from the control), it may be assumed with some probability that the tested water  sample does not contain toxic substances at harmful or lethal concentrations. If the testing organisms change their behaviour or die, physico-chemical and/or other  methods should be immediately started to find the cause of intoxication. Though the use of the bioassay for water toxicity testing cannot result in a direct identification of the cause of the accident or the mortality of the fish and other aquatic organisms, it can be of much help in the diagnostic process. Other aquatic organisms, e.g. rainbow trout, common carp or the cyclopes, may also be used for the bioassays,  but these are not available all the year round, and rainbow trout and common carp have the additional disadvantage of needing more water in the sample and the water  has to be aerated. It is a general feature of all water toxicity bioassays that their  methodology is considerably variable: in any particular case the method must be chosen with respect to the actual conditions and to the equipment and capacity of the laboratory. Examination of the fish

The number of the fish that have to be examined and sent to diagnostic examination is not always the same. If there is any suspicion that fish might have been poisoned or  suffocated by water pollution, 3 to 5 fish of each of several species (those most frequently occurring among the killed or dying fish) are taken as samples. If such a situation occurs in a reservoir (pond) with a single-species stock, the number of fish in the sample should be 5 to 20, depending on weight, age and other circumstances. Success of the examination depends on the state of the fish. It is of no use to send fish that start decaying or even are decayed. The sample fish should best be delivered alive, with clinical symptoms of damage. If this is impossible, it is absolutely necessary that the fish be fresh and intact when they reach the lab. They should never   be sent in water in which they died. The fish sent for general examination should be free of preservatives (formalin, spirit or others) because such substances would make the diagnosis impossible. In those cases when the fish are only sent for chemicotoxicological examination for the contents of metals and residues of pesticides and other pollutants, it is recommended to send the samples frozen. Fish that have just died or those with clinical symptoms of damage are subjected to detailed health examination to eliminate diseases of infection or invasion type. If such diseases are eliminated, it is necessary to specify the cause of the abrupt change in the environment that caused the death of the fish or the change in their behaviour. The clinical symptoms given in the document from the local investigation are evaluated in the first stage of the examination, then follows the patho-anatomic dissection, in  justified cases the organs and tissues of the fish are subjected to chemicotoxicological examination, and further examinations are performed if necessary. Chemico-toxicological examination of the organs and tissues is one of the most objective, and also most difficult, methods of diagnosis of intoxication. In the case of  suspicion of intoxication by methals or in the inspection of the content of metals in fish as human food, the chemico-technological examination has its justified position. Such examinations are also performed when phenols and pesticides may be involved,

and if certain conditions are met, chemico-toxicological analysis may be of help in diagnosing ammonia intoxiacation of fish. On the other hand, in diagnosis of  intoxications of fish by some substances (chlorine, sulphane, cyanides) this method cannot be used. The presence of metals in fish organs and tissues is determined by the atomic absorption spectrophotometry (AAS). An increase in the concentration of metals is most frequently recorded in the parenchymatous organs and in the gills of the fish. For example, acute copper intoxication can be diagnosed on the basis of chemical analysis of the gills, where the concentration of the metal increases several times. Copper content in the gills of the fish in water not contaminated with copper is up to 10 mg per 1 kg of dry matter. Fish intoxication with pesticides based on organo-phosphorus compounds can be diagnosed either by direct determination of these substances or their metabolites in the organs and tissues or by indirect determination, based on the inhibition of  acetylcholine hydrolase mainly in the brain of the fish. The same method is applicable to the diagnosis of fish intoxication by pesticides on the basis of carbamates. Diagnoses of the intoxication of fish by other pesticides and also other organic compounds are based on the determination of these substances in the organs and tissues of the fish by gas chromatography methods. Another chemico-toxicological method used in the diagnoses of intoxication is the detection of phenols in the fish. The chemicals are detected in the flesh and skin of the fish by the photometric method and by means of 4-amino-antipyrin (Czechoslovak  Standard CSN 83 0530, part 33) after repeated distilling of the tissue used for  analysis. Ammonia intoxication of the fish can be diagnosed, if some conditions are maintained (fish blood and brain sampling during intoxication or immediately after the death of  the fish, performance of the analysis within 48 hours at the maximum of freezestorage of the samples), by determining ammonia nitrogen in the blood serum and  brain of the fish. The level of ammonia nitrogen in the blood serum and the fish brain homogenate is best determined by the Blood Ammonia Test kit, made by HYLAND. However, ammonia nitrogen level in the serum and brain of the fish varies with metabolic rate and increases under the action of the various adverse factors, especially O2 deficiency. Owing to these circumstances it is impossible to diagnose ammonia intoxication of fish by merely determining the ammonia N level in the grain or in the  blood serum: the final diagnosis must be based on a thorough quality examination of  the water and detailed examination of the state of health of the fish. The presence of petroleum and its products in fish can be easily detected by changes in sensory characteristics: the “petroleum smell” can be detected at concentrations of  an order as low as 10 2 mg per litre of water. Contamination of aquatic medium with  phenols and chlorophenols can be detected in the same way at or above the respective concentrations of 0.1 mg per litre and 0.02 mg per litre. This method of detection needs no sophisticated laboratory equipment, though its sensitivity compares favourably with that of chemico-toxicological examination.

3.3 Prevention of Fish Intoxications General principles of preventing fish poisoning

All persons who handle substances that may pollute the environment should regularly check their equipment and take measures to prevent accidents: this is a moral obligation, which is in many countries supported by law. Strict discipline and high responsibility in handling such substances provide the most effective, most readily available and cheapest prevention. The primary measure that can save surface water from pollution is the building of  treatment plants, both for the treatment of sewage and industrial effluents. In  principle, any used water must have been treated before it is discharged back to the environment. An important role in the protection of the aquatic medium is also played  by the so-called biological ponds which are able to intercept and eliminate mainly the organic pollutants generated in agricultural production (livestock fattening and rearing facilities, including those for waterfowl) and in the food industries (slaughterhouses,  poultry processing plants and others). To a lesser degree these biological ponds may intercept and remove the sewage from residential areas, if such waste waters are not  polluted with petroleum products, PCB, pesticides and other dangerous contaminants. In industrial production, especially in those industries where there is a danger of  leakage of petroleum products and other very toxic substances which do not easily yield to biological degradation, it is necessary to build special leak-proof pits to  protect water sources against contamination. Much attention must also be paid to some technological processes in agriculture. Particularly great dangers are involved in spray application of chemicals, including fertilizers and pesticides. Any direct drift of the chemicals to water areas during application must of course be avoided and precautions should be taken to prevent later  washing of the chemicals with rainwater down to the rivers and ponds. Such  precautions are incorporated in technological regulations for application (no chemical sprays in rain or wind and 24 hours before the rain is expected etc.) and they are also reflected in some principles of soil cultivation: grassy strips around water reservoirs, the use of adequate crops and adequate systems of tillage in exposed fields, and some other pollution-control practices. It is required by current regulations that any newly developed chemical must have undergone a complex of toxicological testing and evaluation before it is introduced in  practice. Its biological degradability, acute and chronic toxicity and/or teratogenicity, mutagenicity and the like should be known. Priority should be given to substances of  a high selectivity, low active concentration, low toxicity and easy degradability. The natural environment should not be contaminated with toxic substances of low degradability. The toxicity of substances, preparations and effluents to the aquatic organisms is influenced, first of all, by their nature (e.g. chemical composition, water solubility,  pH), sensitivity of the fish on which these substances act (salmonids are most sensitive, cyprinids are somewhat more resistant, older fish are more resistant than younger fish; an important role is also played by the momentary condition and state of 

health of the fish, and in early fry also by the state of their feeding) and, last but not least, by the nature of the aquatic medium (water temperature, pH, content of  dissolved oxygen and the like). Evaluation of the chemicals, preparations and effluents

For the fish culturist, the most important trait of any new product or chemical is its toxicity to aquatic organisms. Toxicity tests are carried out, in the essence, at three levels: a. at the level of cells and tissues  b. at the level of organisms (individuals) c. at the level of biocoenoses (communities) The cell- and tissue-level tests are often used for theoretical explanation of findings obtained in trials conducted at the organisms level. Good reproducibility is their  advantage, but unfortunately, the results obtained in vivo often considerably differ  from those in vitro. Tests at the level of biocoenoses offer the advantage of the toxic actions being studied in the natural environment or on a model which can simulate reality with good authenticity. However, biocoenosis-level trials have drawbacks: the changes in the composition of the biocoenosis may not always be a consequence of  direct toxic action on a given species but may be due to a disturbance in the food chain or other factors; the reproducibility of such tests is often very limited because it is impossible in authentic natural environment to prepare exactly the same conditions as in the preceding test. Most of the trials, especially the acute toxicity tests, are performed at the organisms' level. Though some reproducibility problems and risks are involved in applying their  results to actual natural conditions, these tests represent a practical compromise, acceptable to all: the fish culturist, conservationist, technologist and economist. Distinction is drawn between acute toxicity and chronic toxicity; hence, substances,  preparations and/or waste waters (effluents) are subjected to acute toxicity tests and chronic toxicity tests. Almost all these tests are carried out at the organisms level. Acute toxicity tests Determination of the acute toxicity of chemical substances, preparations and effluents to aquatic organisms is among the main duties of the producers. The majority of the acute toxicity tests are performed on fish and some aquatic invertebrates. Different methods of toxicity tests have been standardized in different countries. Some methodical procedures are recognized internationally, including the most widespread standards issued by the International Standards Organization (ISO), the OECD methodical manuals and some others. In determining the acute toxicity to aquatic organisms, the following ISO methodical manuals are most widely used: 

ISO 6341 Water quality - Determination of the inhibition of the mobility of   Daphnia magna Straus (Cladocera, Crustacea) issued in 1982.

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ISO 7346/1 Water quality - Determination of the acute lethal toxicity of  substances to a freshwater fish [ Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 1: Static method of 1984. ISO 7346/3 Water quality - Determination of the acute lethal toxicity of  substances to a freshwater fish [ Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) ] - Part 2: Semi-static method of 1984. ISO 7346/3 Water quality - Determination of the acute lethal toxicity of  substances to a freshwater fish [ Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae )] - Part 3: Flow-through method of 1984. ISO 5664 - Inhibition of algal growth.

It follows from this survey that the basic tests include the acute toxicity tests on the highly sensitive water flea  Daphnia magna and on the aquarium fish  Brachydanio rerio. The standard also allows to use some other species, e.g.  Poecilia reticulata,  pimephales promelas, Oryzias latipes and others. The basic data are those on LC50 for a period of 24, 48, 72 or 96 hours. The representatives of primary producers used for the acute toxicity tests include, first of all, those of algae. Apart from these standard methods of toxicity testing, laboratories worldwide use many modified toxicological methods. In using the modified methods, the laboratories respect the actual natural and economic conditions prevailing in each country, in each  particular region. Some countries have standardized the methods of determining the tests of acute toxicity of waste waters to freshwater fishes (e.g. the USA, UK), others have introduced special tests for the determination of the toxicity of pesticides to fishes (e.g. Japan, the UK). The national methodologies differ in the test organisms used, in the time of exposure, in the water used for dilution, but in the majority of  cases the main output is the LC50 value as the most accurately determinable quantity. The division standard currently used in Czechoslovakia, ON 46 6807 Acute Toxicity Test on Fish and Other Aquatic Organisms, also derives from the above-mentioned ISO and OECD standards. In the test methodology (2 levels: the orientation test and  basic test), as well as in the selection of the test organisms (the basic test organisms are  Daphnia magna and  Poecilia reticulata and the auxiliary ones include common carp, rainbow trout,  Brachydanio rerio,  Rasbora heteromorpha, Cyclopidae, Tubificidae and also the larvae of the amphibians,  Xenopus laevis and the green frog ( Rana temporaria), which serve in special tests. The selection of these aquatic organisms for the acute toxicity tests should be adjusted to the purpose for which the tested substances are evaluated. For example, when evaluating a substances or preparation to be used in fish culture or for direct application to the aqautic environment, toxicity tests will have to be performed on all the above-mentioned representatives of aqautic fauna. On the other hand, for  classification of the tested substances, e.g. newly developed preparations to be introduced in practical use, the basic evaluation will be sufficient (compulsory tests on  Daphnia magna and  Poecilia reticulata) . Tests on  Rasbora heteromorpha is required in chemicals to be exported. In accordance with the internationally agreed methods, the Czechoslovak division standard, ON 46 6807, recommends to use artificially prepared dilution water. Further, it recommends to use a reference substance, i.e. a control preparation

analyzed for LC50 alongside the tested substance. The changes in the LC50 of the standard reflect the variability in the circumstances in which the trials are Performed and in the condition of the organisms tested. K 2Cr 2O7 is the most frequently used reference substance. According to the level of 48h LC50, determined on the basis of ON 46 6807, the tested substances (preparations) are classified (in Czechoslovakia) into the following toxicity classes: 0123456-

substances of almost no toxicity: substances in which the 48h LC50 is higher than 10 000 mg per litre, substances of very low toxicity: substances in which the determined 48h LC50 is between 1000 and 10 000 mg per litre, substances of low toxicity: substances in which the determined 48h LC50 is between 100 and 1000 mg per litre, substances of medium toxicity: substances in which the determined 48h LC50 ranges between 10 and 100 mg per litre, substances of high toxicity: substances in which the determined 48h LC50 is between 1 and 10 mg per litre, substances of very high toxicity: substances in which the determined 48h LC50 ranges from 0.1 to 1 mg per litre, substances of extreme toxicity: substances in which the determined 48h LC50 is smaller than 0.1 mg per litre.

Any tested sample (substance, preparation) is definitively included in the appropriate toxicity class according to its 48h LC50 level for the most sensitive organism tetsed. The acute toxicity test also yields data on the following parameters, included in the result: 

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LC5 (concentration that kills 5 % of the individuals within the given period of  time), formerly also referred to as the minimum lethal concentration; LT50 (the time within which half of the individuals are killed at the given concentration of the substance), also referred to as mean killing time; Dependence of the mean killing time on the concentration of the substance tested (the toxicity curve); Description of the behaviour of fish and amphibian larvae during the test, obvious changes found in the fish by the patho-anatomic dissection, and changes in the aquatic invertebrates.  Poecilia reticulata, Brachydanio rerio and  Rasbora heteromorpha are tested for the intake of usual food when the test is finished.

Chronic toxicity tests The maximum concentration of substances in water still admissible for the fish (MAC) serves as a basis for evaluation of the water that flows to the fish culture facilities, for diagnostic analyses when the causes of damage or mortality are sought, or for permitting the discharge of effluents, containing different contaminants, to the

rivers or ponds. The MAC levels are determined in the chronic toxicity tests. A standard procedure of a chronic toxicity test for examination of toxicity in different substances, products and effluents was proposed, for example, by Lesnikov (1976). By Lesnikov the MAC is a concentration of a substance or its metabolites in waters at which a permanent exposure has no adverse effect on: a. the hydrochemical regime of water courses, lakes and ponds and on the microorganisms,  b.  primary production in the above-mentioned water bodies, c.  plankton food organisms, d. the fish (including the eggs and fry in larval development as well as fish in higher age categories) and also the market value of the fish (the hygienic aspect). The MAC methodical manuals recommend first to perform acute toxicity tests, then tests of detoxication of the substances and their metabolites into safe products, and finally, long-term observations, using the results of acute toxicity tests and detoxication tests. The resultant MAC should then be determined according to a  parameter which is adversely affected at the lowest concentration of the substance or  its metabolite. Materials of high stability (96 % decomposition into non-toxic compounds in summer lasting longer than half a year) with a high accumulation capacity and with a MAC below 0.0001 mg per litre are regarded as particularly dangerous. Salmonids, especially one-year-old rainbow trout, are most frequently used for the chronic toxicity tests. The main parameters examined when the long-term toxicity test is being finished include the state of nourishment of the fish, the individual and total gain of the fish, the change in the sensory assessment of the flesh and the degree of  accumulation of toxic substances in the fish body. The supplemental parameters include the behaviour of the fish during the test, the patho-anatomic and histo pathologic picture, the physiological, biochemical and haematological changes in the fish after the termination of the test. Together with the basic parameters under study, the histologic examination of the fish organs and tissues after the long-term tests is among the most important criteria of evaluation, because its results usually reduce the MAC of the substances and products determined according to the basic parameters. These principles are also fully applied in the methodical manual Determination of the Maximum Admissible Concentrations of substances in Water from the Viewpoint of  Fish Culture, issued by the Research Institute of Fish Culture and Hydrobiology, Vodňany, in 1989. According to this manual, the chronic toxicity test is to last 90–100 days and the test is performed on the fry of common carp and rainbow trout. A comparatively stable concentration of the tested substance is obtained by putting the fish into a new fresh bath of the toxic substance every day. After computation and evaluation of all the results of the chronic toxicity test, the concentration which, compared with the control group, did not cause any significant changes in the experimental fish is denoted as the maximum admissible concentration (MAC). of the aquatic invertebrates, the chronic toxicity tests are conducted mainly on the water fleas Daphnia magna. All the individuals of this species used in the test must be of the same age (3–7 days), produced in synchronized culture. Both the basic

 parameters (survival, release of the young from the germ sac, viability of the young, changes in the biomass) and supplemental parameters and criteria (behaviour of the water fleas, symptoms of death, state of the gonads and contents of the germ sac, filling of the intestine and the colour of the intestinal contents, body colour, amount and colour of the fat droplets) are evaluated during the course of the test. A detailed description of this method is given in the OECD standard. Bêsides the chronic toxicity test, water fleas are also subjected to the reproduction toxicity test, based on the examination of the reproduction capacity of several generations of the tested water fleas, kept in a solution of the substance being tested. The evaluated parameters include the release of the young, the number of the young, their survival, and the ability of parthenogenetic reproduction. Other toxicity tests Efforts to replace the lengthy and costly chronic toxicity tests by other tests, which would be both fast and sensitive, have intensified in recent years. The use of cell cultures is one of the promising methods. These tests are based on the observation of  the toxic action straight on the primary cell cultures from the different fish tissues or  on stable cell lines (e.g. FMH, PG, RTG-2 and others). At present they are being elaborated on. The method of embryolarval toxicity tests is also being examined (the technique after  Birge et al. is regarded as an ISO method). The influence of toxic substance is monitored until the yolk sac is exhausted. Unfortunately, the first results of  experiments conducted in our laboratories do not confirm a high sensitivity of this technique; it is rather comparative with the acute toxicity tests. To reach a greater  accuracy, the technique was extended to include a fasting test. This provided a slight improvement of sensitivity but in spite of this improvement the technique cannot replace the traditional chronic toxicity test. In this case, the time of killing of the experimental fish is evaluated in comparison with the control fish. Another interesting method is the bacterial bioluminiscence inhibition technique (the Microtox test: ISO Standard N110/1988, draft elaborated in France). Of course, this technique also has to be adjusted to local conditions. Degradability of substances in aquatic environment

Besides toxicity, another important criterion of evaluation of substances and products is their biological degradability in the aquatic medium. It may be evaluated by determining the biological degradability itself (the non-specific method) or by determining the time of persistence of the residues of the tested substances in the aquatic medium (specific method). Biological degradation means a sequence of processes taking place in removing the organic substances by micro-organisms. The degradation involves both dissimilation and assimilation processes. Czechoslovak ichthyotoxicologists, conservationists and water management experts widely use the technique proposed by pitter (1974) as a standard test of biological

degradability of organic substances. This is a single-act kinetic test performed in an open system. The decrease of the amount of the tested substance is measured by the determination of the chemical oxygen demand (COD Cr ), total organic carbon (C org), or   by specific reactions. The results are compared with a blank test and with the degradability of a standard substance. Environmental importance is attached not only to the degree of decomposition but also to the rate at which the substance is degraded. For practical reasons it is recommended to indicated biological degradability as  percentage of removal of CODCr  or Corg in the days of incubation. When the results of biological degradability tests are applied to actual natural conditions, it should be taken into account that the test simulates the conditions existing in the sewage plants. A number of other factors (temperature, microbiological life, pH, amount of dissolved oxygen in water and others) influence the rate of  degradation of the tested substances under natural conditions, outside the sewage treated plants. Nevertheless, it generally holds that a material of good biological degradability in non-adapted activated sludge is also most likely to decompose well under natural conditions. The techniques of determining the residues of the various pollutants and their  metabolites in the aquatic medium are not easy to perform; for this reason primary attention is focused on really toxic substances and those which are not readily degradable. The residues are determined by chemical analyses (metals, DDT and its metabolites, HCH, PCB, triazines and others) and also by bioassays for toxicity testing (e.g. test on  Daphnia magna serves to determine the residues of organo phosphates). Legislation

Today special legislation is being developed in each country where due efforts are taken to maintain and improve the state of the aquatic environment. In addition to this, international agreements are being prepared and signed to extend the possibilities of  conservation of aquatic resources and natural environment as a whole (e.g. agreements on the Baltic Sea). The legislation of every country should take into account the adverse aspects of the action of various chemicals, products, waste waters and solid wastes on the aquatic medium. The duties of the producers and users of such substances as well as those of  the producers of the wastes must be clearly defined, including the penalties entailed  by violation or circumventing the legislation, strict and regular inspection, and emphasis on personal responsibilities. A whole complex of mutually related  preventive measures should be developed and introduced in practice to reduce the number of accidents in water courses, to eliminate the sources of pollution and to minimize the consequences of accidents on the aquatic biocoenoses. Recomended literature Alabaster J.S., Lloyd R. (1980): Water quality criteria for freshwater fish. Butterworths. pp. 297.

Baier Ch., Hurle K., Kirchhoff J. (1985): Datensammlung zur Abschätzung des Gefãhrdungspotentials von pflanzenschutzmittel-Wirkstoffen für Gewãsser. Verlag paul parey, Hamburg, Berlin, pp. 294. Horáková M., Lischke p., Grünwald A. (1989): Chemical and physical metods of  water analyses, SNTL, ALFA, Praha, pp. 389 (In Czech). Hrbáček J. et al. (1974): Limnological methods. SPN, Praha pp. 208 (In Czech). Lesnikov L.A. (1979): Elaboration of standards of acceptable concentration of   pollutants in the water of fish culture units, Sbornik naučnych trudov, GosNIORCH, Leningrad, 144, pp. 3–41 (In Russian). Lewis W.M., Morris D.P.(1986): Toxicity of nitrite to fish: A review. Trans. Amer. Fish. Soc., 115, No. 2, 183–195. Liebmann H. (1960): Handbuch der Frischwasser-und Abwasserbiologie. VEB Gustav Fischer Verlag, Jena, pp. 1149. Lukjanenko V.I. (1967): Fish toxikology. Piščevaja promyšlennost, pp. 216 (In Russian). Marchetti R. (1965): Critical review of the effects of synthetic detergents on aquatic life. Stud. Rev. Gen. Fish. Coun. Medit., No. 26, pp. 25. Mattheis Th., Taege M. (1982): Datensammlung der Grenzkoncentrationen von Schadstoffen für die Fischproduction. II. Tenside. Institut Für Binnefischerei, Berlin, pp.54. Mattheis Th., Sommer M. Ch, Grahl K. (1984); Datensammlung der  Grenzkoncentrationen von Schadstoffen für die Fischprodution. III. Polychlorierte Biphenyle. Institut für Binnenfischerei, Berlin, pp. 58. Pitter p. (1974): Design of standard test for assessment of biological degradability of  organic substances. Vod. Hospod., B, 24, 247–250 (In Czech). Pitter P. (1981):The effect of pH, temperature and conductivity on relative share of  different forms of ammonium nitrogen in water. vod. Hospod., B, 31, 217–219 (In Czech). Pitter P. (1990): Hydrochemistry. SNTL, Praha, pp. 565 (In Czech). Schäperclaus W. et al. (1979): Fischkrankheiten. Akademie-Verlag, Berlin, pp.1089. Schreckenbach K. (1982): Die bedeutung von Umweltfaktoren bei der  Fischproduction in Binnengewässer. Mh. Vet. Med., 37, 220–230. Svobodová Z., Máchová J. (1985): Causes, diagnostics and prevention of fish intoxication. ÚVO, Pardubice, pp. 136 (In Czech). Svobodová Z., Vykusová B. (1991): Determining the maximum admissible concentrations of substances in water from the point of view of fish culture

requirements. Research Institute of Fish Culture and Hydrobiology, Vodňany, pp. 39 Svobodová Z. et al. (1987): Toxicology of water animals. SZN, Praha, pp. 231 (In Czech). Vámos R., Szöllözy G. (1974): There is not a danger of ammonia intoxication of fish if there is enough oxygen in water. Halázat, 20, No. 4, 124 (In Hungarish). Revised report on fish toxicity testing procedures. FAO-EIFAC, Rome, Techn. Paper, 24, 1983, pp. 37. Wasserschadstoff-Katalog. Institut für Wasserwirtschaft, Berlin, 1981. ČSN 83 0520 Fyzikálně chemický rozbor pitné vody. ÚNM, Praha, 1979 ČSN 83 0530 Chemický a fyzikálni rozbor povrchové vody, část 1 – 50, ÚNM, Praha, 1979 ČSN 83 0532 Biologický rozbor povrchové Vody, část 4–6, ÚNM, Praha, 1979 ISO 6341 Water quality-Determination of the inhibition of the mobility of Daphnia máagna Straus (Cladocera, Crustacea), 1982. ISO 7346/1 Water quality-Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) Part 1: Static method of 1984. ISO 7346/2 Water quality-Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] Part 2: Semi-static method of 1984. ISO 7346/3 Water quality-Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] Part 3: Flow-through method of 1984. ISO 8692 Water quality freshwater algal growth inhibition the with Scenedesmus subspicatus and Selenastrum capricornutum, 1989.

4. Diseases of Alimentary Origin (Z. Svobodová, J. Ruprich) Mycotoxicoses

Mycotoxicoses that occur in animals are caused by administration of feeds affected by toxinogenic fungi and contaminated with their toxic metabolites. Fungi living on the dead plant substrate are most frequently responsible for such disturbances. These are fungi of the saprophytic group, comprising many species of the genera  Aspergillus,  Fusarium and others. Aflatoxicosis

Aflatoxicosis is a disease of domestic animals sensitive to the toxic action of the metabolites of the fungi  Aspergillus flavus, Aspergillus parasiticus and some others. These fungi grow best on plants containing oil. However, a fungus present on the feed may not always produce a toxic metabolite, an aflatoxin: aflatoxin production as well as the growth of the fungi themselves are influenced by a number of environmental factors (humidity, temperature and others) of the place where the feed is stored. Aflatoxins have a high thermal stability, withstanding temperatures up to 300°C. There are various kinds of aflatoxins of which aflatoxin B 1 has the greatest toxicity. Aflatoxins action is mainly hepatotoxic: they damage the liver of the animals affected. For some animals, long-continued administration of sublethal doses of aflatoxins has a cancerogenic effect. Of all fishes, rainbow trout is most vulnerable to the cancerogenic action of  aflatoxins. Tumorous nodes develop on the surface as well as inside the liver of  rainbow trout given these substances for several months. The nodes are gray-yellow, tough when palpated, well supplied with blood, and on section they look like connective tissue. Large tumours occur in brood trout old 3–6 years: these fish often have metastases in the kidneys, spleen, gut, gonads and other organs. The tumours may be as large as a chestnut. The big ulcers are palpable through the body wall. They often compress the heart and cause disorders in blood circulation. Ascites often occurs and organs often coalesce. The hepatomata are of the nature of hepatocellular  carcinoma. Literature provides evidence that aflatoxin B 1 also has a strong acute hepatotoxic action on rainbow trout fry. Feed containing about 5 μg aflatoxin B 1 per 1 kg killed 75–100 % of a rainbow trout fry stock in a short time. The liver of the affected rainbow trout was pale, with heavily injected blood vessels and with degenerative changes. Hepatotoxic action (circulation disorders in parenchymatous organs, represented by venostasis, reactive processes around intrahepatal bile ducts, dystrophic alterations of liver cells) prevails in the effect of aflatoxins on carp. Prevention of aflatoxicosis is based on thorough inspection of the peanuts and meals, inspection of produced feeds, and cool and dry storage of all feedstuffs, especially those for salmonids, with particular emphasis on the feeds for rainbow trout. For the rainbow trout the highest admissible amount of aflatoxin B 1 in feed is 0.1 μg per 1 kg and for the remaining fishes 5μg per 1 kg.

From the point of view of the hygiene of human food, the maximum tolerable B 1 aflatoxin concentration is 5 μg per 1 kg of fish flesh. Fusariotoxicosis

This ailment is caused by fungi of the genus  Fusarium. The production of these moulds toxic metabolites is influenced by the composition of the substrate parasitized  by them, and by the environmental conditions.  Fusarium fungi frequently attack  fodder cereals during the growing season and also infest stored fodder grain and feeds  produced by grain processing. The fungi produce fusariotoxins, mainly T 2 toxin (12,13-epoxytrichothecen) while parasitizing these substrates. Carp exhibits the greatest sensitivity to this toxin. At a 72 hour exposure, the lethal doses are 0.46 mg  per 1 kg live weight for carp and 6.1 mg per 1 kg of live weight for rainbow trout. Carp orally treated with lethal doses of T 2toxin exhibit depression, poor reactivity to stimulation, accelerated respiration, dark body surface, the fish refuse food, excrete undigested food, later replaced by long strings of white mucus. The fish keep close to the water surface and gasp for air. Then follow ataxia, inco-ordination of motion, the fish stop moving and die. The patho-anatomic picture includes dark body surface and marked changes on the gills (oedematous swelling and marbling). Inside the body cavity the blood vessels are heavily injected, parenchymatous organs change colour  and size, the mucous membranes of the digestive tract are inflamed with dots of  haemorrhages. Long-continued administration of T 2 toxin together with other  fusariotoxins at sublethal concentrations to carp causes depression, the fish excrete half-digested food, reduce their food intake and show much reduced weight gains. Determination of mycotoxins in feeds and in fish as human food

1/ Sampling A high importance is attached to the taking of samples for the detection of  mycotoxins. If a feed was produced from contaminated raw materials, its contamination may be uniform, but if the substrate itself is infested by toxinogenic microscopic fungi it often happens that the contamination is of focal nature, with mycotoxin concentrations sometimes higher by an order in the foci than in the rest of  the substrate. This of course must be taken into account in the sampling. As a  practical solution, an average sample is taken, having a final weight of 1 kg. The whole sample is homogenized and the weight of its portion used for analysis is 20–  100 g (most frequently 50 g). 2/ Analytical methods In principle, there are several different techniques available for the analysis of  mycotoxins. Because of the importance of aflatoxins, further text will deal with this group of mycotoxins. Biological determination There are a number of organisms on which the toxicity of mycotoxins can be tested. However, the method is not very accurate and usually fails to identify actual mycotoxins. Tests are most frequently conducted on micro-organisms such as

 Bacillus brevis or  Bacillus megaterium, on aquatic organisms such as  Artemia salina,

and sometimes also on higher organisms, e.g. chick embryos, ducklings and others. Chromatographical determination Chromatography is among the most frequently used techniques and among the most accurate ones. The main chromatographic techniques include thin layer  chromatography (TLC, HPTLC), high performance liquid chromatography (HPLC) and gas chromatography (GC). Of these, TLC is employed in the majority of cases  because it is simple and requires no hi-tech equipment. Where great accuracy is required, instrumentalized HPTLC, HPLC and GC are of help, but they need costly equipment. Immunochemical determination Immunochemical techniques were developed for fast tentative determination of some mycotoxins. The principle of these techniques is that the sample mycotoxin and standard mycotoxin are left to compete for a bond to the specific antibody. The mycotoxins are low-molecular organic compounds and do not possess the nature of  antigen. However, if they are bound to a suitable proteinaceous carrier they have the nature of an antigenous determinant against which the immunized organism produces antibodies. The mycotoxin then can react with them as a haptene. The substances used most frequently for labelling in the immunochemical determination are enzyme (EIA, ELISA) or radioisotope (RIA). Such techniques are now commercially available e.g. for the determination of aflatoxin B1, ochratoxin A and T 2 toxin (TRANSIA, France), aflatoxin B1 and ochratoxin A (RIEDEL-DE HAEN, Germany) etc. Determining Aflatoxin B1, B2, G1 and G2 by the HPTLC Method

The method is designed for quantitative determination of aflatoxin and is based on the HPTLC technique with visual or densitometric evaluation. Material: Methanol, chloroform, acetone, diethylether (peroxide-free), petrolether, all of a p.a.  purity grade. Sulphuric acid, trifluoroacetic acid, sodium chloride, anhydrous sodium sulphate, distilled water and other ingredients. Chromatographic HPTLC Merck (5547) plates or others of adequate properties. Developing chamber for vertical development of TLC plates. Pipettes, glas capillaries, atomizer and other aids. Densitometer. Preparation of sample: 1. Homogenize 50 g of sample in a mixture of 125 ml of methanol and 12.5 ml distilled water for 3–5 min. 2. Add 37.5 ml distilled water and homogenize for another 3–5 min. 3. Filter the material through a medium-density filter (Filtrak, 389). The filtration can be encouraged by an addition of fused quartz.

4. Mix 70 ml of filtrate with 55 ml of distilled water in a separating funnel and dissolve 5 g of NaCl in the mixture. Add 50 ml petrolether or diethylether and shake for 3–5 min. Leave the layers to separate and use the lower layer for  further work. In the case of fat samples the cleaning can be repeated. 5. Having discharged part of the material from the separating funnel, add 20 ml distilled water to the aqueous (lower) phase and then add 80 ml chloroform. Shake for 3–5 min. 6. Discharge part of chloroform extract and repeat extraction with 40 ml chloroform. 7. Dry the combined chloroform extracts by means of anhydrous sodium sulphate and evaporate them on a vacuum evaporator at a temperature of 60°C at the maximum until they are dry. 8. Dissolve the evaporation residue in a final volume of 0.5 ml of chloroform and use for HPTLC. Ten microlitres of extract represents 0,4 g of sample. Chromatographic separation: If extracts are contaminated, subject them first to purification straight on the chromatographic plate. Mark the start 5 cm from the end on the HPTLC plate about 12 cm high. Apply to the start the individual aflatoxins standard (mixed standard of  100 picogrammes of each aflatoxin in 1 microlotre) in amounts of 100, 500 and 1000  pg, together with the sample in amounts of 1, 5 and 10 microlitres (0.04, 0.2 and 0.4 g of sample per 1 spot). It is advantageous to repeat the application of sample with an addition of standard (internal standard). Develop the chromatogramme in pure diethyl ether in the direction from the start to the nearest border of the plate in a saturated chamber. After development, cut off the washed impurities about 1 cm above the start, turn the plate and develop in the reverse direction in a mixture of chloroform + acetone + water (88 + 12 + 0.2) in a saturated chamber. Chromatogramme evaluation: Evaluate the chromatogramme in long-wave UV light (366 nm). Aflatoxins fluoresce  blue and green. The positions of the spots must be identical with the standards. Determine the quantity tentatively by visual comparison of fluorescence intensity with the standards. Use densitometer (e.g. CAMAG TLC SCANNER II) for accurate evaluation. In visual evaluation, the sensitivity of determination is about 1μg per 1 kg of sample. With the use of the densitometer (employing a greater number of  standards) the sensitivity is about 0.1 μg per 1 kg of sample. The recovery is around 90 %. Aflatoxin identification:

1. To be able to exclude the presence of aflatoxins, spray the chromatogramme with a 20 % solution of sulphuric acid in water. Aflatoxin spots fluoresce yellow when sprayed. If there is no yellow fluorescence, there are no aflatoxins in the material tested. 2. To be able to perform the positive identification, derivatize the aflatoxins in the sample by means of trifluoroacetic acid. The reaction produces hydroxy derivatives of aflatoxins B1 and G1 which move less than the original aflatoxins on the chromatogramme. Hence, for comparison, new

chromatographic separation is performed after the derivatization. Details are given e.g. in FAO Publication 14/10 (1990), Manuals of Food Quality, 10, Training in Mycotoxins Analysis. Ceroid degeneration of liver

This ailment occurs in intensive fish culture (especially in salmonids) where the feeding is not much varied. Rainbow trout is particularly vulnerable. As known, unlike other salmonids, rainbow trout shows no great food preference and is able for  long to consume food of low value and poor quality, even containing rancid fats. Wild rainbow trout does not deposit store fat in liver but rainbow trout in intensive culture, given low-value pellets, suffers from considerable adiposis of the liver. As fat gathers in the liver cells, there is a considerable increase in the amount of unsaturated fatty acids which are very susceptible to self-oxidation and to the formation of ceroid. Ceroid is a brown-yellow pigment, produced as a result of self-oxidation of  unsaturated fatty acids accumulated in the liver of the fish. Ceroid accumulation in the liver cells leads to a serious specific disease of the rainbow trout: ceroid liver degeneration. It occurs most frequently in those fish  populations which are given poor quality feed pellets containing fish and meat-and bone meals high in rancid fats. Such pellets contain large quantities of unsaturated fatty acid peroxides which destroy vitamins A and E in the fish bodies. (These two vitamins, especially vitamin E, are strong natural antioxidants and, under normal conditions, provide significant protection of the fatty acids of cell membranes). The ceroid degeneration syndrome is characterized by very conspicuous clinical symptoms. The first of them are dark pigmentation, anaemia and inappetence of the affected fish: this may easily occur even in fish showing a good state of nourishment. The liver is enlarged, sand yellow in colour, with a positive ceroid finding in the hepatocytes. The gall bladder is filled with transparent fluid, the gut is grey-white, its wall is thin and sagged. The stomach is empty or may contain a small amount of  transparent whitish fluid. The gill system is markedly anaemic, which is associated with the considerable changes that occur in the blood picture; these changes are characterized by a reduction of the erythrocyte count and corpuscular haemoglobin. As a result, the growth of the fish is retarded and their mortality is high. Prevention of this disease includes through veterinary inspection of the feed pellets, inspection of feed freshness, adequacy of feed, storage and of the feed administration technique. Feed inspection includes determination of three important parameters: fat acid number as a measure of the hydrolytic breakdown of the fat, peroxide number, and 2-thiobarbituric acid number. The values of these parameters should not exceed the following levels: fat acid number 45 mg KOH per 1 g, peroxide number 0.30 % iodine, 2-thiobarbituric acid number 1.7 % absorbance of one-percent solution. Preventive liver examination and haematological investigation of rainbow trout may also be of help in recognizing the first signs of damage and in preventing the disease from spreading. In the early stages the disease can be countered by stopping the administration of the faulty pellets and by replacing them by a sound food fortified

with vitamin E (fresh beef spleen has been found to be the best for this purpose). Vitamin E is administered in the form of germ oil with vitamin E forte (at a rate of 10 ml per 1 kg of feed). In the Rt 1 category (rainbow trout yearling), such a feed is administered every other day, in the Rt 2 category (two-years-old rainbow trout) the feed is administered once in three days until the fish are restored to health. At the  present time the rainbow trout pellets are stabilized by antioxidant supplementation (e.g. with ethoxycholine-based preparations). Feed safety inspection by determining the fat acid number, peroxide number and 2-thiobarbituric acid number

In intensive fish culture, the point always is to evaluate the state and quality of the fat contained in the feed pellets. The samples must be taken so as to be representative of  the average quality of the feed batch inspected. They have to be kept in the dark and cold to prevent changes (deviations from the original state) and the laboratory analysis should be performed within as short a time as possible. The acid number, the peroxide value and the 2-thiobarbituric acid number are used for  evaluating the state and quality of fat. Fat is extracted by means of chloroform for the above-mentioned methods: grind the  pellets but do that with care not to generate heat. Mix 50 g of the ground material with 70 ml of chloroform. After 2 hours of frequent stirring, filter the mixture through filter   paper into a beaker and leave the chloroform to evaporate in a fume chamber  overnight. Then dry the material in a drier at a temperature of up to 40°C. For  determination of the peroxide value and the 2-thiobarbituric acid number, do the extraction at a room temperature to avoid further oxidation or loss of volatile oxidation products. Before the analysis, carefully melt the extracted fat on water bath and stirr it with a glass rod. The acid number of fat expresses the consumption of KOH (in mg) for the neutralization of free fatty acids contained in 1 g of fat. Weigh 30 to 50 mg of fat and add to it, at a 1:1 ratio, 20 ml of alcohol ether (alcohol denaturated with 1 % benzine), neutralized with 0.1 M KOH to phenolphthalein (use about 1 ml of one-percent solution of phenolphthalein in alcohol per 100 ml of alcohol ether and titrate until first  pink colouring occurs). Titrate against a white background of 0.1 N KOH. The acid number (A) of fat in mg KOH per 1 gramme has the following formula:

where a = consumption of 0.1 N KOH t = weighed amount of fat in g (number having 4 decimal places) The peroxide value expresses the amount of iodine (in grammes) released from  potassium iodide by the peroxide compounds contained in 100 g of fat. Filter the fat dissolved in chloroform into dried and weighed Erlenmayer flasks. Dissolve the fat (0.5 to 0.9 g), weighed with an accuracy of 4 decimals, in 50 ml of a mixture of acetic

acid and chloroform (3:2). Add 1 ml of saturated solution of potassium iodide, close the flask and put it in refrigerator. Five to ten minutes later add 50 ml distilled water  cooled to 4°C, thoroughly shake the flask (0.5 min), add 1 ml of 1% starch and titrate with 0.002 N sodium thiosulphate until original colour is obtained (when starch is added the solution becomes blue). The peroxide value (per), expressed as the % of iodine, has the following formula:

Where V1 VO K P

= = = =

consumption of 0.002 N sodium thiosulphate in ml consumption of 0.002 N sodium thiosulphate in blank sample sodium thiosulphate factor (1) weighed amount of fat in g

For purposes of international expression in mE per 1 kg of fat, the amount of   peroxides, calculated by the formula 800 In the formula, a  N P

= = =

should be divided by 8.

consumption of Na2S2O3 in ml normality of this solution Weight of fat in g.

The 2-thiobarbituric acid number offers a good method to study the early stages of  rancidification of fats and fat-containing feeds, as far as these fats contain polyenic fatty acids. To determine the 2-thiobarbituric acid number by the direct method without distilling, weigh 30–50 mg of fat in a volumetric flask and dissolve it in 5 ml chloroform. Add 10 ml of 20 % solution of trichloroacetic acid in 2-propanol (isopropyl alcohol) and 10 ml of saturated solution of 2-thiobarbituric acid in 2-propanol (shake 1 g of acid with 100 ml of isopropanol and leave to stand overnight to let the solution saturate). Warm to 60°C and keep that temperature for 24 hours, then measure the intensity of  colouring at 530 nm. Amyl alcohol can be used instead of 2-propanol: in such a case, warm the test material to 120°C and maintain the temperature for an hour. The calibration curve is drawn with the help of tetraethoxypropane (malondialdehyde diacetal), which hydrolyzes into malondialdehyde. Pipette 0.2 to 1.6 ml of 10 -4 M solution of tetraethoxypropane in 40 % alcohol and then follow the procedure as in the determination itself. Prepare the 10-5 M solution by 10 -4 dilution at a ratio of 1:9. The 2-thiobarbituric acid number is expressed as malondialdehyde per 1 g of fat (μmol MDA per 1 g). Damage caused to fish by high-mercury feed

such damage may be caused



 

When excess seed grain treated with mercury-based fungicides is administered to fish, When feed pellets containing high-mercury fish meal are administered to fish, When feed pellet protein is replaced by high-mercury ingredients.

The administration of high-mercury feeds to fish affects the hygienic value of the fish flesh. Mercury compounds have also been found to damage some fish organs and tissues. Mercury accumulates in the fish bodies and is strongly fixed to the SH-groups of amino acids. Evidence that mercury fixed in the organs and tissues of fish  practically fails to be eliminated from the body is provided by the results of many experiments. It is therefore important for prevention to check for mercury content the feeds and feed mixture pellets intended for fish. The maximum admissible amount is 0.1 mg of total mercury per 1 kg of feed. The technique of feed checking for mercury content is shown in detail in the chapter on the health safety of fish flesh from the viewpoint of contaminants content. Recommended literature Halver J.E. (1976): Aflatoxicosis and advetitionis toxins for fish. Fish Patol., 10, 199–  219. Galaš V.T., Ivanova N.S. (1978): About the necessity of research of fish mycotoxicoses. Ryb. Choz., CNNTEIRCH, Moskva, 4, 17 (In Russian). Ruprich J., Piskač A. (1983): Treatment, purification and identification of some types of aflatoxins. Veterinářstvi, 33, 555–557 (In Czech). Řehulka J. (1990): Effect of hydrolytically changed and oxidized fat in dry pellets on the health of rainbow trout, Ocorhynchus mykiss (Richardson). Aquaculture and Fisheries Management, 21, 419–434. Svobodová Z. et al. (1987): Toxicology of  water animals. SZN, Praha, pp. 231 (In Czech). Zanini E. et al. (1974): Riserche sulla possibilita di inquinomento dietetico de mercurio in piscicoltura. Riv. Ital. Piscic. Ittiopatol., 9, 7–12. Manuals of Food Quality Control. 10. training in Mycotoxins Analysis. 14/10, FAO Rome 1990, pp. 113.

5. Control of Hygienic Quality of Fish from Point of View of Foreign Substances Content (J. Máchová, Z. Svobodová, M. Hrjtmánek, M. Hrbková) Health safety inspection of fish flesh: contaminants (Hg, pb, cd, PCB, DDT, and others) levels

The presence of contaminants, including sediments, in rivers, lakes and ponds, and generally the water quality in each particular area or place, are among the important factors that influence the health of fish and the quality (in hygiene terms) of the fish flesh. To provide safe and good-quality food for the people, all meat, including fish flesh, must be evaluated for what is called hygienic quality. The concept expressed by this term comprises several aspects: - Physiological action - total level of microbi - nutritive value - chemical changes of components - energy value - sensory assessment - chemico-toxicological analysis From the point of view of surface water pollution, it is most important to evaluate the results of sensory assessment of the fish flesh and the result of the chemicotoxicological analysis, which is, at the same time, indicative of contamination of the aquatic medium. The need to analyze food for contaminants arose from the tragical consequences of  consumption of highly contaminated food. For example, a disease called Minimata occurred in Japan in the 1960's. The disease affected people's nervous system, induced mental disorders, paralysis of the limbs, lips and tongue, inco-ordination of  motion, tremor of the hands, disturbed vision, and insomnia. The disease was found to have been caused by the consumption of fish whose flesh contained about 20 mg menthylmercury per 1 kg. Another example, again from Japan, is the disease called itai-itai (ouchi-ouchi), which caused pain in the abdominal area and limbs, deformation of the bones, decalcification and fractures. This disease was caused by the presence of cadmium used for the irrigation of rice and soya. Still another disease, called “oil diseases” or “yusho”, was caused by table oil contaminated with PCB (ca. 0.5 g per litre). Another important factor that encouraged analysts to study food contamination was the availability of new analytical methods, which enable reliable detection of even very low contaminants concentrations in various materials, including food.

The worst food contaminants include those of a high toxicity and poor degradability (or no degradability at all). Chief among them are metals and chlorinated carbohydrates, so it is also in this report that attention is paid to them. We also mention and describe here the substances that are most frequently the cause of  accidents in surface waters and which intensively change the sensory properties of the fish flesh, e.g. petroleum and its products. Metals

Mercury Mercury gets into water mainly with industrial effluents and atmospheric precipitation and very quickly passes into the bottom sediments. It accumulates there, usually in sulphite form. Elementary mercury and its organic and inorganic compounds are liable to methylation. The toxic products of this methylation (methylmercury) enter  the food chains and accumulate in the aquatic organisms. Owing to the high accumulation capacity of mercury (the accumulation coefficient reaches the level of  104), its content successively increases with increasing levels in the food chain: the highest levels are recorded in the fish. In Czechoslovakia, the maximum admissible concentration (MAC) of total mercury is 0.1 mg per kg of flesh in the non-predatory fishes and 0.6 mg per 1 kg of flesh in the  predatory freshwater fishes. In the USA, Canada, Austria, Switzerland, Germany, Israel, Poland and Cyprus, the maximum admissible mercury concentration in the fish flesh is 0.5 mg Hg per 1 kg, in France 0.5 mg per 1 kg with a tolerance up to 0.7 mg, in the USSR 0.6 mg per kg, in the U.K. 0.5 to 0.7 mg per kg, in Italy 0.7 mg per kg, in Sweden, Finland and the Netherlands 1.0 mg per kg, and in Norway 1.5 mg per kg. FAO/WHO expert group on contaminants recommended that 0.3 mg per person be the still tolerable maximum mercury intake of mercury; methylmercury should not represent more than 0.2 mg within the maximum tolerable amount. Mercury determination in biological material Put 10 g of sample to a beaker and mineralize it by boiling for 10–20 min in a mixture of 11 ml of nitric acid and sulphuric acid (ratio 10:1) under a powerful reverse water  cooler to prevent leakage of vapours. When 15–20 min elapses, cool the clear yellow solution, put it in a measuring vessel and add distilled water. The determination of the amount of mercury in the mineralization product is performed by flameless atom absorption spectrophotometry (the AAS “cold steam” method). Mercury may also be determined on the TMA 254 instrument (trace mercury analyzer), developed at the University of Chemical Technology in Prague and  produced in Czechoslovakia. The results obtained by the two methods compare well with each other.

Both the methods have a sensitivity of 0.001 mg per 1 kg. Lead In the aquatic medium, lead accumulates mainly in the bottom sediments where its level is usually four orders higher than in the water. Like mercury, lead is able, through the action of some micro-organisms, to produce organic methyl derivatives which accumulate in the aquatic organisms. However, as distinct from mercury, lead was not observed to accumulate in fish. The highest admissible lead concentration in the fish flesh is 1 mg per kg in Czechoslovakia. In other European countries the limit lead concentrations in fish flesh range from 0.3 mg per kg (Denmark) to 2.0 mg per kg (the Netherlands). Cadmium In waters, cadmium is accompanied by zinc; it is also contained in industrial effluents. Waters that wash phosphate fertilizers from farm land are also a significant source of  cadmium contamination. Like lead, cadmium was not found to significantly accumulate in aquatic organisms. In Czechoslovakia the maximum admissible concentration in fish is 0.05 mg per kg of fish flesh and 0.5 mg per kg in fish entrails. In other European countries this level ranges from 0.05 mg per kg (Denmark, the  Netherlands, Great Britain) to 0.2 mg per kg of fish flesh (USSR). According to the FAO/WHO Expert Commission on Contaminants, the still tolerable cadmium intake  per one person should not be higher than 400 to 500 microgrammes. Determination of metals in biological materials

Sample mineralization: The so-called “dry method” is used. Dry a weighed porcelain or quartz dish to a constant temperature and weigh fresh tissue (about 10 g of muscle or 5 g of liver) in the dish. Dry the samples to constant weight at 105°C and determine dry matter content. Moisten the dry sample with 5 % NH 4 NO3 p.a., dry it at 105°C and leave the dry sample to incinerate on an electrically heated plate. Use a platinum rod to carefully crush the carbonized sample and incinerate it in the Mufle oven at a temperature of 45°C (for cadmium or lead determination) or 550°C (chromium determination) at the maximum. The ash obtained should be white to greyish in colour. If it is not, moisten it again with NH4 NO3, dry it and incinerate repeatedly until the ash is white or greyish. Dissolve the ash in a mixture of HCl and HNO 3 and evaporate it on water bath until it is almost dry. Dissolve the evaporation residue in 10 ml HNO3 and put the solution into measuring beakers (volume 25 to 10 ml), using distilled water. If part of the evaporation residue remains on the dish, warm it carefully and then put all the sample into the measuring vessel. Blank determination is performed in the same way as with all the other samples (incineration in the Mufle oven for about and hour). The concentration of metal in the mineralization product is determined by atom absorption spectrophotometry.

A quicker and simpler method of sample mineralization is to use a mineralizer. The Research Institute of Fish Culture and Hydrobiology at Vodňany has a mineralizer  made by the Austrian company PAAR, which breaks the sample by means of a microwave system at a high pressure (80 barr) and at a temperature of about 300°C. Limits of  determination:

Cd Cr Pb

0.5 mg per 1 kg 2.5 mg per 1 kg 5.8 mg per 1 kg

Chlorinated carbohydrates

Pesticides based on DDT and HCH Pesticides based on chlorinated carbohydrates, including for example DDT, HCH and HCB, are among substances of a high cumulation capacity and low biological degradability. This is the reason why their use is strictly limited today; the use of  DDT, for example, has been forbidden in Czechoslovakia since 1975. Nevertheless, residues of these substances and their metabolites are still often recorded in biological materials, owing to these pesticides poor degradability. Contents of these substances must be continuously monitored in various materials. They are lipophilous, accumulating in fats. Owing to this, the highest levels of residues occur in the high-fat fishes, e.g. eel. The following hygienic limits, applied in Germany, are used for evaluation of the residues of these substances recorded in freshwater fish: Σ DDT (DDT + DDE + DDD) HCB τ-HCB α + β-HCH

2.0 mg per 1 kg of flesh 0.05 mg per 1 kg of flesh 0.2 mg per 1 kg of flesh 0.05 mg per 1 kg of flesh

Polychlorinated biphenyls Polychlorinated biephenyls are among the highest-stability compounds. They are used as part of the filling of power condensers, fillings in hydraulic equipments, are contained in lubricants, synthetic lacquers, dyes and plastics. The common brand names include Delor, Aroclor, Clophen, Kaneclor, Savol, Softol and others. As a rule, numbers are added to the brand names to indicate the number of chlorine atoms (e.g. Delor 103 is a low-chlorine substance, Delor 106 is a high-chlorine substance). High-chlorine substances have a higher stability in natural environments than are PCBs with a low content of chlorine. Owing to the warning data on the harmful effect of chlorinated biphenyls, the  production of these substances was considerably restricted in Czechoslovakia in 1971 and in 1984 it was copletely forbidden. Nevertheless, like in the case of chlorinated carbohydrates, residues of polychlorinated biphenyls are still encountered. In each  particular locality, their content in the fish depends mainly on the species of the fish, especially on the content of fat in their flesh. Therefore, high concentrations of PCBs

residues occur in high-fat fishes such as the eel in which the fat content is as great as 20–30 %, unlike in the low-fat fishes, e.g. pike or perch having less than 1 % of fat in their muscle. The hygienic concentration limit of PCBs level in fish is 0.5 mg per kg in the edible  portion in Czechoslovakia, 2.0 mg per kg of flesh in the USA, Canada and Germany. The tolerated maximum daily PCBs intake is 1 μ of PCBs per 1 kg of human body weight. Determination of polychlorinated biphenyls (PCBs) and chlorinated pesticides in fish flesh, sediments and water

Chlorinated carbohydrates are extracted by organic solvents, the extracts are purified on Florisil column and with sulphuric acid. Gas chromatography with an EC detector  is used for the determination itself. The results of PCBs determination are expressed as the sum of Delor 103 and Delor 106 and the results of determination of chlorinated carbohydrates are determined as the sum of HCB, α-HCH, β-HCH, τ-HCH, p,p'-DDE,  p,p'-DDD, p,p'-DDT. Chlorinated carbohydrates are extracted from fish flesh and bottom sediment samples as follows: Weigh about 5 g of homogenized sample on analytical balance and extract it with acetone. Put the extract in distilled water and shake it into petrolether. Dry the  petrolether layer with sodium sulphate, concentrate it and clean it on a Florisil column. Depending on the nature of the sample, chlorinated carbohydrates are extracted from water samples by shaking 1 to 3 litre of water with petrolether. Shake the petrolether  extract with anhydrous sodium sulphate, discharge it via an anhydrous sodium sulphate column, evaporate it to a small volume and clean it on a Florisil column. Use acetone to displace the intercepted chlorinated carbohydrates. Evaporate the acetone until the extract is dry and purify the extract again on a Florisil column or by means of  concentrated sulphuric acid. The use of PCBs sorption on solid sorbent, e.g. small PRESEP columns (Separon S6 × 18), followed by washing with acetone, is also applicable for PCBs determination in water. To determine chlorinated carbohydrates on gas chromatograph, the following conditions have to be provided: 1.PCBs: The column: length 2 m, inner diameter 3 mm, filling 5 % DOW 200 on Varaport 30 carrier. Carrier gas: nitrogen. Operational temperatures: injector 240°C, column 225°C, detector 280°C. Limits of determination: fish flesh and sediment: 0.004 mg per kg D 103 and D 106; water: 0.01 μg per 1 litre D 103 and D 106. 2. Chlorinated pesticides:

The column: length 1.5 – 2 m, inner diameter 2–3 mm, filling: mixture of phases 3 % OV 17, 7.5 % OF 1, 3 % XE 60 at a ratio of 2:2:1, on Chromaton N-AW-DMCS as carrier. Carrier gas: nitrogen. Operational temperatures: injector 235°C, column 210°C, detector 250°C. Limits of determination: fish flesh and sediment: HCB, α-HCH, τ-HCH: 0.05 μg per 1 kg, β-HCH, DDE, DDD, DDT: 0.1μg per 1 kg; water: HCB, α-HCH, τ-HCH: 0.0002 μg per 1 litre, β- HCH, DDE, DDD, DDT: 0.0005 μg per 1 litre. Petroleum and the petroleum products

Petroleum and the products of its processing (gasoline, kerosene, diesel oil, mineral oils) are a group of substances responsible for an increasing extent and proportion of  environment pollution. They are dangerous because of their toxicity to the aquatic organisms, but may also badly affect fish farming because even the presence of a very small amount of these substances in water (petroleum 0.025 mg per 1 litre, gas oil 0.01 mg per 1 litre, kerosene 0.01 mg per 1 litre, gasoline 0.002 mg per 1 litre) can spoil the sensory characteristics of fish flesh. The flesh gets a disagreeable "petrol smell" and its taste is spoiled. To remove the bad taste and smell, the fish must have  been kept in clean water for several weeks before consumption; some sources even assert that these substances' half-life in the fish body is 400 to 700 days. Phenols

Rivers, lakes and ponds may be polluted with phenols carried to the water with industrial effluents, especially the waste waters generated during the thermal  processing of coal, effluents from petroleum refineries, from synthetic fabrics  production plants and from other industrial operations. When petroleum and its products get into contact with chlorine, offensively smelling chlorophenols develop, which induce sensory changes in the fish flesh even at very low concentration (0.02 mg per 1 litre). Phenols themselves cause sensory changes in fish flesh at and above a concentration of 0.1 mg per 1 litre. Sensoric evaluation of fish flesh

Fish flesh is sensorically assessed according to Czechoslovak Standard CSN 46 6802 Marketed Freshwater Fish. The smell and taste of the fish are assessed in cases of fish flesh contamination with petroleum, its products and phenols. The flavour (smell) is first assessed in raw flesh as soon as the viscera are removed. Then the samples are  boiled in steam (not by immersing them in water) with no ingredients, in a closed container on a water bath for 30 to 40 min. Every sample is boiled separately. When the required time elapses, the samples are evaluated as to their flavour and taste. The sensory assessment should be performed by at least three persons with good senses of taste and smell. During work, the assessors are not allowed to smoke or eat other kinds of food.

Recommended literature Cibulka et al. (1991): Lead, cadmium and mercury transfer in biosphere.Academia, Praha, in press (In Czech) Halm J. (1980): Blei-, Cadmium-, Arsen- and Zinkgehalte von Fischen aus unbelasten and belasten Binnengewassern. Fleischwirtschaft, 60, 1076–1083. Matyas Z. (1989): Hygiene and technology of frozen and fish products. SPN, Praha,  pp. 53 (In Czech).  Nuorteva p. (1991): Bioaccumulation of mercury. Department of Environmental Conservation University of Helsinky, pp. 30 Schuler W., Brunn H., Manz D. (1985): Pesticides and PCBs in fish from the Lahn river. Bull. Environ. Contam. Toxicol., 34, 608–616. Svobodová Z. et al. (1987): Toxicology of water animals. SZN, Praha, pp. 231 (In Czech).

6. Haematological Examination of Fish (Z. Svobodová, B. Vykusová) Analysis of the peripheral blood of fish serves for diagnostic purposes; apart from this main purpose, it is also used at present to examine the effect of toxic substances on the fish, to evaluate the condition of the fish, to evaluate the non-specific resistance of  different fish breeds and strains and of the brood fish, to assess the suitability of feeds and feed mixture pellets, to evaluate the effect of stress situations etc. The recommended methods described here include only well-tested and well-proven  procedures and techniques of the determination of the different haematological  parameters. The values of the different haematological parameters are significantly influenced by endogenous and exogenous factors, so it is not easy to determine their   physiological range. Hence, the haematological and biochemical data given in the different chapters should only serve for rough orientation. Blood sampling

Blood is sampled for ichthyohaematological examination as soon as the fish are taken out of the environment in which they have lived. The main criteria for selection a  particular method of sampling include the size of the fish, the amount of blood needed and the further fate of the fish caught for different examinations. 1/ Blood sampling in fish fry Blood from fry at an individual weight of at least 8 grammes can be sampled by the methods of cardiopunction, using a glass capillary long about 200 mm whose inner  surface is lined with a fine film of heparin before use. Lift the fish, fixed head down, to the level of the eyes and apply the tip of the capillary at an angle of about 60° (in relation to the longitudinal axis of the fish body) about 1-2 mm cranially from the mid  point, which is the point of intersection of the longitudinal axis of the body with the line connecting the cranial edges of the base of both pectoral fins. At this point the carp fry have the so-called stigma: a shallow, usually pigmented depression in the skin up to 1 mm wide. Now drive the tip of the heparinized blood-collecting capillary quickly through the body wall to the pericardium and further to the heart (Fig. 21). Blood, appearing in the capillary, is the most reliable evidence that one of the cavities of the heart has been hit. When the collection is finished, pull the tip of the capillary out of the wound, hold the capillary in horizontal position and then turn it upside down several times to let the blood mix well with the anticoagulant mixture. In heavier fish fry, for example at a weight of about 20 g, the blood collecting capillary may be replaced by a dry and clean heparinized injection needle. Such needles are driven into the heart in the same way as the glass capillary. The cardiopunction technique can also be used with older fish: this is so in those cases when the fish can be killed. 2/ Collecting blood from fish weighing above 200 g, including brood fish

In these fish, the best method is that of collecting blood by punction of the caudal  blood vessels (Fig. 22). On the caudal peduncle ventral side the unpaired dermal scale is removed in caudal direction from the anal fin base. Within the central plane, about 1 cm caudally from the anal fin, a sufficiently long needle is introduced, firmly held on the cone of a heparinized disposable syringe, into the fish body in a craniodorsal direction at an angle of 45°. The described method is fully recommendable, mainly in larger series of blood collection; collection of 2 ml of blood from cyprinids weighing above 1000 g involves no risk of loss of the treated specimens.

Fig 21: Blood sampling in fish fry by the method of cardiopunction using a 200 long glass capillary.

Fig. 22: Blood sampling in fish of more than 200 g weight by the method of punction of caudal blood vessels. 3/ Stabilization of blood Aqueous solution of heparin sodium salt is the only product used for stabilization of  the fish blood. One ml of this aqueous solution contains 5000 I.U. of heparin sodium salt. 0.01 ml (about one drop) of the aqueous solution of heparin suffices to stabilize 1 ml of fish blood: the substance is left to dry on the inner surface of the test tube or  flask (bublet) and the blood is collected in the test tube or bublet afterwards. A slight overdosage of heparin does not produce changes in the blood cells of the fish. Determination of the parameters of the red blood picture

1/ Erythrocyte count (Er, RBC)

The erythrocyte count in fish blood is determined in heparinized blood diluted by the Hayem solution at a ratio of 1:200. The solution has the following composition: mercury dichloride HgCl2 - sublimate sodium sulphate Na2SO4 sodium chloride NaCl distilled water

2.5 g 25 g 5g ad 1000 ml

The flask (bublet) method after Bürker is used for the dilution of the blood. The blood is diluted in special glass bublets or in penicillin phials (volume 15 to 25 ml). First, a special pipette is used to put an accurate amount of 4975 μl of Hayem solution (filtered before use) into the bublet or phial; then 25 μl of heparinized blood is added, using a flushing micropipette. The micropipette is rinsed several times by repeatedly sucking the solution, the bublet is closed with a rubber stopper and its contents are stirred by circling motion for 2 to 3 min. A dropper or a Pasteur pipette is used to fill Bürker's counting cell with the diluted blood. The red blood cells are counted in 20 rectangles, regularly distributed over the whole lattice of the counting cell. The counting is usually done at a 200-fold magnification. The resultant counted amount of erythrocytes is then reduced 100 times and the resultant value is the number of erythrocytes in T.1 -1 (Tera = 1012). The erythrocyte count can still be determined by the traditional method after 24 hours of storage of  heparinized blood at a temperature of up to 4 °C. Colorimetric methods of determining the erythrocyte count in the blood of fish are introduced at present. The colorimetric method of determining the erythrocyte count after Pawinski, compared with the traditional method, is simpler, more rapid, less laborious, does not bear a subjective error, has a greater reproducibility, and is suitable for series determinations. The disadvantage is that its use in ichthyotoxicology is limited, especially in the study of the action of substances that increased the mean corpuscular volume. The Pawinski solution having the following composition is used in the colorimethric method of determining the erythrocyte count in the blood of fish: anhydrous sodium sulphate Na 2SO4 sodium sulphate decahydrate Na 2SO4. 10H20 sulphosalicylic acid distilled water

26.7 g or  67.9 g 2.0 g ad 1000 ml

Blood is collected by means of a heparinized needle and dropped onto a watchglass from where 20 μl of blood is transferred by means of a rinsing micropipette into 10 ml of the Pawinski solution and is thoroughly stirred but not shaken. Sample extinction is measured against distilled water in a measuring cell (1 cm), using a filter at a wavelength of 600 nm. The erythrocyte count is read from the calibration curve drawn on the basis of parallel measurements of erythrocyte counts by the colorimetric method after Pawinski and by traditional method, using the Bürker counting cell. As far as possible, the measurement should be done within the whole variation range of  erythrocytes in the blood of several tens of fish (30–40 fish usually suffice). An

example of calibration curve is show in Fig. 23. When using the colorimetric method of determining the erythrocyte count, the blood sample should be diluted with the Pawinski solution straight on the spot of field examination, as soon as the blood is collected. The determination of extinction should be performed within 20 minutes to 4 hours after collecting the blood.

Fig. 23: Relationship between extinction (E) determined by the colorimetric technique after Pawinski and the erythrocyte count (Er) determined by means of the Burker  counting cell In the healthy carp, the erythrocyte count ranges from 1.1 to 1.8 T.1 -1. In rainbow trout this range is from 0.80 to 1.50 T.1 -1 of blood. 2/ Haemoglobin (Hb) The cyanohaemiglobin method is used for determination of haemoglobin in the blood of fish. The principle of the method is that haemoglobin is released from the erythrocytes and transferred to cyanohaemiglobin by means of a transformation solution; the cyanohaemiglobin is then determined photometrically. Solution after van kampen and Zijlster can be used as the transformation solution. Its composition is as follows:  potassium ferricyanide K 3 [Fe(CN) 6]  potassium cyanide KCN  potassium dihydrophosphate KH2PO4 distilled water

0.20 g 0.05 g 0.14 g ad 1000 ml

The transformation solution is stored in a dark reagent bottle in a refrigerator and is replaced every 3–4 weeks. If left to freeze, the transformation solution is destroyed. For the analysis of the blood to determine the haemoglobin, about 7 ml (or 5 ml) of  the transformation solution is measured and poured into a test tube, a rinsing pipette is

used to add 25 μl (or 20 μl) of fresh collected heparinized blood and the contents of  the test tube are stirred immediately. The examination of heparinized blood for  haemoglobin content should be performed within 24 hours after blood collection at the latest, if the blood is stored at a temperature up to 4 °C. The conversion of  haemoglobin into cyanohaemiglobin is rapid: the data can be read from the  photocolorimeter after 3 minutes. The cyanohaemiglobin colouring remains stable for  24 hours at the minimum. The sample extinction measurement itself is performed in a 1 cm cell at a wavelength of 540–546 nm against the transformation solution. The haemoglobin content is determined from the calibration curve. The calibration curve is drawn in the usual way, using the cyanohaemiglobin standard and the transformation solution. Haemoglobin content in blood is expressed in g per litre. In healthy carp and trout its level ranges within the approximate limits of 60 and 100 g per litre. 3/ Haematocrit value (Hk, PCV) The haematocrit value expresses the corpuscular volume in relation to the total volume of blood. In ichthyohaematology, heparinized capillaries 7.5 cm long are exclusively used for the determination of the haematocrit value. The freshly collected non-stabilized or heparinized blood (which may be left to stand at a temperature of up to 4 °C for 4 hours after collection at the maximum) is sucked into the capillaries to about ⅔ of their height and the clean end is sealed over a burner. Then the capillaries are put into the centrifuge (speed 14 000 r.p.m.) and are left to be centrifuged for 3 minutes. After centrifuging, the haematocrit percentage is directly read on the haematocrit meter which is a part of the haematocrit centrifuge set. The percent value obtained in this way is multiplied by coefficient 0.01 and the resultant value is the PCV in 1.1-1. Owing to its simpleness and accuracy, determination of the haematocrit has become one of the basic examinations of the red part of the blood of fish. The physiological PCV values range from about 0.28 to 0.40 1.1 -1 in carp and from 0.30 to 0.45 1.1 -1 in rainbow trout. 4/ Basic erythrocyte data and their calculation Mean corpuscular volume (MCV) The value of the mean corpuscular volume can be calculated from the haematocrit value (PCV), expressed in 1.1 -1, and from the erythrocyte count (Er), expressed in T.1 1 . The following formula is used for this calculation:

The value of the mean corpuscular volume, expressed in fentolitres (f1), ranges from 200 to 300 fl in healthy carp and from 350 to 400 fl in healthy rainbow trout. Mean corpuscular haemoglobin (MCH)

Mean corpuscular haemoglobin expresses the average haemoglobin concentration in individual erythrocytes and is given in picogrammes - pg (10 -12 g). It is calculated from the haemoglobin value in g.1 -1 and from the erythrocyte count (Er) in T.1 -1 according to the following formula:

In carp, the optimum value of mean corpuscular haemoglobin ranges from 50 to 60  pg, in rainbow trout from 65 to 75 pg. Mean corpuscular haemoglobin concentration (MCHC) The mean corpuscular haemoglobin concentration expresses the concentration of  haemoglobin in unit volume of erythrocytes. It is calculated from the haemoglobin value (Hb) in g.1 -1 and from the haematocrit value (PCV), expressed in 1.1 -1, according to the following formula:

In healthy carp the value of the mean corpuscular haemoglobin concentration ranges from 0.20 to 0.26 1.1 -1, in healthy rainbow trout from 0.17 to 0.20 1.1 -1. Determination of the parameters of the white blood picture

1/ Leucocyte count (Leuco) The leucocyte count in fish is determined in heparinized blood, diluted with a solution after Procházka and Škrobák at a ratio of 1:200. The Procházka-Škrobák solution has the following composition: sodium chloride NaCl sodium sulphate Na2SO4 sodium monohydrophosphate dodecahydrate  Na2HPO4.12H2O  potassium dihydrophosphate KH2PO4 formaldehyde 37%  brilliant cresil blue distilled water

3.88 g 2.50 g 2.91 g 0.25 ml 7.50 ml 0.10 g ad 1000 ml

Fresh (newly prepared) solution cannot be used for the determination itself: the solution must have been left to stand for about 2 weeks and should be filtered before use. The flask method after Bürker is used for diluting the blood. Special glass flasks (bublets) or penicillin phials (volume about 15 to 25 ml) are used for the dilution:

first, 4975 μl of the Procházka-Škrobák solution is put in the flasks or phial by means of a special pipette and then 25 μl of heparinized blood is added by means of a rinsing micropipette. The solution is then sucked repeatedly several times to rinse the micropipette, the flask is closed by means of a rubber stopper and its contents are stirred by a cycling motion for 2–3 minutes. A dropper or a Pasteur pipette is then used to fill Bürker's counting cells with the diluted blood. The leucocytes are counted in 100 large squares. The counting is usually done at a 200-fold magnification. The total number of leucocytes, counted in the 100 large squares, is multiplied by 0.5 to give the leucocyte count, expressed in G.1 -1 (G-giga = 109). The leucocyte count can still be determined after 24 hours of storage of heparinized blood at a temperature of  up to 4 °C. The range of variation of the physiological values is very wide: in carp from 10 to 80 G.1-1 and in rainbow trout from 10 to 60 G.1 -1. 2/ The leucocrit value (Lc, BC) The leucocrit value expresses the leucocyte volume in relation to the total volume of   blood. The leucocrit value is determined in heparinized microcapillaries simultaneously with the determination of the haematocrit value. The thickness of the leucocyte layer is measured under microscope using an eyepiece micrometer with a 60-fold magnification. Like the haematocrit value, the leucocrit value must also be determined within 4 hours after collection and storing heparinized blood at a temperature of up to 4 °C. The technique of leucocrit value determination has until now been only developed for  carp. The physiological values of leucocrit in carp range from 0.002 to 0.01 1.1 -1. 3/ Differential leucocyte count (leucogramme) Leucocytes include agranulocytes and granulocytes. The main distinguishing mark is the absence or presence of the differently coloured granules in the cytoplasm of these cells. Agranulocytes comprise lymphocytes and monocytes whose cytoplasm contains no granules, except sporadic azure grains which may occur in part of these leucocytes. The group of granulocytes comprises leucocytes whose cytoplasm usually contains a large amount of fine or coarser granules which differ from one another in staining capacity, i.e. ability to absorb either basic or acid colouring agents, or both. Depending on staining capacity, the granulocytes may be basophile, eosinophile or  neutrophile. Besides the cytoplasm, the nucleus is another feature by which these cells are differentiated. The basic distinguishing traits are the shape of the nucleus, its size and internal structure. Large and compact nuclei are typical of agranulocytes. The nuclei of granulocytes are generally smaller, elongate in shape, and often divided into  parts, or segments, connected by a thin strip of nuclear chromatin. As to the internal structure and density of the nucleus, lymphocytes rank first: they contain a dense and fine network of nuclear matter, the nuclei are compact and dense, giving a high degree of blue-mauve basic shade. On the other hand, the nuclei of granulocytes usually have a weaker basic tint. The size of the lymphocytes ranges between 7 and 9 μ. Monocytes in size of 15 to 18 μ or even more in exceptional cases are among the largest cellular elements of the

 blood of fish. The size of the neutrophile granulocytes ranges between 5 and 10 μ. Eosinophile granulocytes are cells whose sizes range from 8 to 12 μ. Basophile granulocytes have a size about 10 μ. The technique of determining the leucogramme of the blood of fish is as follows: the main point is to prepare the blood smear from the native blood immediately after  sampling. Method of preparing the smears is pictured in Fig. 24. The ground cover slip of the Bürker counting cell may be used as the smearing slip. The slides for the blood smears should have been washed with soap and water, rinsed and left in a chromium-sulphur bath. Pliers are used to remove the slides from the  bath; then the slides are rinsed under flowing water and put into an alcohol bath where they are left for 12 hours at the minimum. Then they are taken from the bath and dried with a clean washed cloth. These slides are labelled as “fit for haematological use”. The produced blood smears are left to dry. The fish blood smears should be stained as soon as possible: within four hours at the maximum from making the smears. For   panoptic staining of the blood smears for the purpose of determining the leucogramme, the Pappenhaim staining method is used. The technique is as follows: cover the smear with about 30 drops of the May-Grünwald stain. Leave the stain to act for 3 minutes (in this stage the cells are only fixed with absolute alcohol of the staining mixture). After 3 minutes apply onto the May-Grünwald stain about 25 drops of neutral distilled water (its pH must be neither higher nor lower than 7) which,  producing whirls, mixes with the alcohol of the mixture. In this way an aqueous solution is produced, having its own staining capacity. This stage of staining lasts two minutes. Then the staining solution is poured off the smears which, still wet, are then covered by a prepared solution of the Giemsa-Romanowski staining agent at a dilution ratio of 1:40 (1 part of stain, 40 parts neutral distilled water). It is mainly the cell nuclei that are stained during this stage, lasting 20 to 30 minutes. When 20 minutes elapse, the staining solution is removed from one (any) smear which is rinsed under flowing water and the staining effect is inspected at a small magnification under  a microscope while the smear is still wet. If the cytoplasm of the cells is pink and the nuclei are blue, the staining is finished. Now the solutions are poured off all the remaining smears which are then rinsed under a stream of water and left to air-dry in vertical or slanting position. Microscopic analysis is done in oil immersion under a strong light, preferably at a 1000- to 1500-fold magnification. The slide with the smear is driven to move along a meander-like path under the microscope (Fig. 24). The leucocytes found during this process are classified and are recorded in a suitable table or fed to a digital computer. When the 200th cell is entered, the percentual  proportions of the different kinds of leucocytes are determined. If accurate data on the total number of leucocytes are available, the leucogramme data may help also to  provide valuable information on the absolute numbers of the different kinds of  leucocytes.

Fig. 24: Method of preparing the blood smears. Direction of smearing and examination. The average leucogramme data (%) on carp and rainbow trout are shown here as examples: total lymphocytes monocytes neutrophile granulocytes eosinophile granulocytes  basophile granulocytes

common carp 92 3

rainbow trout 93 3

4

4

1

0

0

0

Determining the values of selected biochemical characteristics in blood plasm

The collected heparinized blood must be centrifuged in cold conditions as soon as  possible. If a cooled centrifuge is not available, a centrifuge with replaceable rotor can  be used instead, the rotor having been cooled in a refrigerator before use. Owing to a lack of stability of the values of some parameters (ALT activity, glucose concentration) it is recommended to perform the determination as soon as possible after centrifuging the non-clotting blood (10 min, 100 G), or to store it at 4 °C or in frozen condition in microtubes with a seal.

Table 11. International system of SI units in ichthyohaematology on the example of  normal values in the blood and blood plasm (pl) of carp Haematological parameter

variation range

SI units

Aminotransferase (AST)-pl. Aminotransferase (ALT)-pl.

0.20–1.50 0.05–0.32

μcat.1 -1 μcat.1 -1

Ammonia -pl. Total protein (TP)-pl.

200–800 20–40

μmol.1-1 g.1-1

Erythrocytes (Er,RBC) Glucose - pl.

1.1–1.8 3–10

T.1 -1 mmol.1 -1

Glutamate dehydrogenase (CDH) - pl. Haematocrit value (Hk, PCV)

0.35–0.75 0.28–0.40

μcat. 1 -1 l.1 -1

Haemoglobin (Hb) Mean corpuscular haemoglobin (MCH)

60–100 50–60

g.1 -1 pg

Cholesterol - pl.

1.5–12

mmol.1 -1

Lactic acid (lactate) - pl.

0.7–2.2

mmol.1-1.

Lactate dehydrogenase, (LDH) - pl. Leucocytes (Leuco)

3.5–9.5 10–80

μcat.1 -1 G.1 -1

Leucogramme: - lymphocytes

76–97.5

%

- monocytes - neutrophile granulocytes

3–5 2–10

% %

- eosinophile granulocytes - basophile granulocytes

0–1 0–0.5

% %

Leucocrit value (LK, BC) Total lipids (TL) - pl.

0.002–0.01 2–10

1.1 -1 g.1-1

1–3

mmol.1-1

Mean corpuscular volume (MCV)

200–300

fl

Mean corpuscular concentration (MCHC)

0.20–0.26

1.1 -1

1–4

mmol1.1-1

Urea -pl.

Triacylglycerols - pl.

haemoglobin

Values of total protein (TP), total lipids (TL), glucosis, triacylglycerol, cholesterol, urea, ammonia, lactic acid, methaemoglobine, enzymes and other parameters are determined in blood plasm of fish. Analytic kits from producers Lachema Brno, Boehringen Mannheim, Hyland and others can be used for determinations. Orientation physiological ranges of these parameters are presented in table 11.