The Barbus meridionalis petenyi Heck. is a typical benthophagous fish, feeding on the zoobenthos and plant components and thanks to its sensitivity to the changes in surrounding medium is an ideal object for indication of the health of aquatic ecosystems. The Teleostei fish liver is susceptible to numerous, both toxic and metabolic, disturbances.
Liver pieces of 40 individuals collected from Trebeništa Reservoir in July 1999, were excised and processed for standard histopathological analysis. The obtained results revealed pathological changes in the liver tissue including parenchyme cell necrosis associated with hemorragia.Barbus meridionalis petenyi Heck. tipična je bentofagna riba koja se hrani zoobentosom i dijelovima biljaka, a zbog svoje osjetljivosti na promjene u okolišu idealan je objekt za određivanje zdravlja vodenih ekosustava. Jetra riba koštunjača podložna je mnogobrojnim toksičnim i metaboličkim promjenama.
U srpnju 1999. prikupljeni su iz akumulacije Trebeništa uzorci jetre 40 riba koji su obrađeni standardnom histopatološkom analizom. Prikupljeni podaci otkrili su patološke promjene u tkivu jetre, uključujući nekrozu parenhimskih stanica povezanu s hemoragijom

G. Kostoski

L. Velkova-Jordanoska

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original scientific paper / izvorni znanstveni radHISTOPATHOLOGICAL ANALYSIS OF LIVERIN FISH (BARBUS MERIDIONALIS PETENYIHECKEL) IN RESERVOIR TREBENI[TALIDIJA VELKOVA-JORDANOSKA & GOCE KOSTOSKIPSI Hydrobiological Institute,Naum Ohridski 50, 6000 Ohrid, R. Macedonia(E-mail: lidvejo@yahoo.com)Velkova-Jordanoska, L. & Kostoski, G.: Histopathological analysis of liver in fish (Barbusmeridionalis petenyi Heckel) in reservoir Trebeni{ta. Nat. Croat., Vol. 14, No. 2., 147–153, 2005,Zagreb.The Barbus meridionalis petenyi Heck. is a typical benthophagous fish, feeding on the zoobenthosand plant components and thanks to its sensitivity to the changes in surrounding medium is anideal object for indication of the health of aquatic ecosystems. The Teleostei fish liver is susceptibleto numerous, both toxic and metabolic, disturbances.Liver pieces of 40 individuals collected from Trebeni{ta Reservoir in July 1999, were excised andprocessed for standard histopathological analysis. The obtained results revealed pathological chan-ges in the liver tissue including parenchyme cell necrosis associated with hemorragia.Key words: histopathology, liver, necrosis, hemorragiaVelkova-Jordanoska, L. & Kostoski, G.: Histopatolo{ka analiza jetre riba (Barbus meridionalispetenyi Heckel) iz akumulacije Trebeni{ta. Nat. Croat., Vol. 14, No. 2., 147–153, 2005, Zagreb.Barbus meridionalis petenyi Heck. tipi~na je bentofagna riba koja se hrani zoobentosom i dije-lovima biljaka, a zbog svoje osjetljivosti na promjene u okoli{u idealan je objekt za odre|ivanjezdravlja vodenih ekosustava. Jetra riba ko{tunja~a podlo`na je mnogobrojnim toksi~nim i meta-boli~kim promjenama.U srpnju 1999. prikupljeni su iz akumulacije Trebeni{ta uzorci jetre 40 riba koji su obra|enistandardnom histopatolo{kom analizom. Prikupljeni podaci otkrili su patolo{ke promjene u tkivujetre, uklju~uju}i nekrozu parenhimskih stanica povezanu s hemoragijom.Klju~ne rije~i: histopatologija, jetra, nekroza, hemoragijaINTRODUCTIONHistopathological biomarkers can be indicators of the effects on organisms ofvarious anthropogenic pollutants and are a reflection of the overall health of the en-tire population in the ecosystem. The alterations in cells and tissues in vertebrateNAT. CROAT. VOL. 14 No 2 147¿153 ZAGREB June 30, 2005Croatian Natural History Museum, Demetrova 1, Zagreb, Croatiafish are recurrently used biomarkers in many studies, but such changes occur in allvertebrates and invertebrates inhabiting aquatic basins. Histopathological biomar-kers embody tissue lesions arising as a result of a previous or current exposure ofthe organism to one or more toxins. Well-documented lesions based on experimen-tal data in liver, ovary, skeleton system and skin have been used as biomarkers todate (HINTON et al., 1985).Histopathological biomarkers are closely related to other biomarkers of stresssince many pollutants have to undergo metabolic activation in order to be able toprovoke cellular change in the affected organism. For example, the mechanism ofaction of several xenobiotics could initiate the formation of a specific enzyme thatcauses changes in metabolism, further leading to cellular intoxication and death, ata cellular level, whereas this manifests as necrosis, i. e. histopathological biomarkeron a tissue level. As well as from chemical insult, histopathological lesions mayarise from infectious diseases and parasites, provoking necrotic and degenerativealterations to which the organism responds with an inflammatory, defensive reac-tion (VELKOVA-JORDANOSKA, 2002; ROGANOVIC-ZAFIROVA et al., 2003). An increasednumber of macrophagic aggregates can be found in the liver, kidney and spleen infish exposed to chemical pollutants, bacteria, fungi or parasites (ROGANOVIC-ZA-FIROVA & JORDANOVA, 1998).The liver plays a key role in the metabolism and biochemical transformations ofpollutants from the environment, which inevitably reflects on its integrity by creat-ing lesions and other histopathological alterations of the liver parenchyma or thebile duct (ROBERTS, 1978).Trebeni{ta is a small natural reservoir of the Livoiska River, part of the water-shed of Lake Ohrid, with very small dimensions: length of 200 m, width 80 m anddepth from 2 to 6 m (Fig. 1).Lake Ohrid and its watershed have been under strong anthropogenic influencefrom the use of various fertilizers, herbicides and insecticides leading to gradualbut crucial modifications not only of the coastline but also of the lake as a whole(CHADO, 1974). Another type of anthropogenic impact is manifested by pollution ofthe tributaries where there is an inflow of wastewater from household and indus-trial facilities. Fecal pollution, detergents and other chemical waste material, andfine alluvium from the Sateska River are undesirable in Lake Ohrid archaic lacus-trine ecosystem (OCEVSKI et al., 1977).The ecological effects of pollutants in aquatic ecosystems and their bioavail-ability and toxicity are closely related to species distribution, both in the solid andthe liquid phase of the aquatic ecosystem. Pollutants are transferred to the plank-ton, aquatic plants, mollusks and fish. A wide range of microscopic and macro-scopic animals and plants live in and on bottom sediments of the aquatic ecosys-tems, and a great number of these organisms ingest organic matter from thesesediments (MOSISCH & ARTHINGTON, 2000).Young fish and larvae of Barbus meridionalis petenyi Heck. in Lake Ohrid hide inplaces close to the bottom and at a depth of about 10 cm. Chironomid larvae arefound in their gut content, then copepods and harpacticids, as well as chlorophytes,148 Velkova-Jordanoska, L. & Kostoski, G.: Histopathological analysis of liver in fish in reservoir Trebeni{tamany diatoms and cyanophytes of the plant species. According to this, their nutri-tion is of a mixed character, with numerous animal elements (TOCKO, 1987). TheBarbus species is a typical benthophagous fish feeding on the zoobenthos and plantcomponents in the course of the autumn. Every natural community of the bottom,mainly the larvae, is included in the Barbus nutrition (MORAVEC, 1997).MATERIALS AND METHODSIn this study livers of 40 individuals of barbel (Barbus meridionalis petenyi Heck.)were examined. Fishes were collected during July 1999 from Trebeni{ta Reservoir.Each fish was dissected according to the following protocol: measurement of thefish length, dissection by opening the abdominal cavity, determination of the sex,dissection of the digestive tract and taking the liver in the end. Tissue specimensNat. Croat. Vol. 14(2), 2005 149Fig. 1. Investigated locality – Trebeni{ta Reservoir.with diameter of 3–5 mm were fixed in 10% formalin and processed according to astandard paraffin procedure. The 5 m paraffin sections were stained with theHemalaun & Eosin method and approximately 2–4 sections of each individual fishwere analyzed by light microscope. The prevalence of registered histopathologicalchanges was calculated as the percent of affected fishes.Materials for zooplankton investigations were collected in July 1999, fixed in 4%formalin and counted under microscope.The aim of these investigations was to perform a histological analysis of liver tis-sue in barbel in Trebeni{ta Reservoir, to represent a reliable indicator of the ecologi-cal condition of the aquatic ecosystem and the possible negative impact of the sur-rounding environment.RESULTS AND DISCUTIONZooplankton investigations were scheduled in Trebeni{ta Reservoir, zooplanktonconstituting important components for monitoring water quality and the changesthat occur in it. The following species were registered in the investigation period:Phylum ROTIFERAPolyarthra vulgarysTrichocerca (D) tigrisPhylum, Subphylum CRUSTACEAClass BranchiopodaOrder CladoceraDaphnia sp.Leidigia leidigiBosmina longirostrisMacrotrix laticornisChidorus sphaericusSubclass CopepodaOrder CyclopoidaCyclops vicinusMesocyclops leuckartiInvestigations of the qualitative composition of zooplankton have revealed thesparse presence of the species in Trebeni{ta Reservoir, probably pointing to certainnegative processes (SERAFIMOVA-HADZISCHE, 1957, 1975; KOSTOSKI, 1998).The direct acute effect of exposure to insecticide is a rapid decrease in many spe-cies of zooplankton. The alteration in crustacean species composition was probablydue to variation in susceptibility to the direct toxic effect of insecticide (WENDT--RASCH et al., 2003). These results provided knowledge about the direct and indirecteffect of pesticide stress on the ecosystem level. They also showed that there was a150 Velkova-Jordanoska, L. & Kostoski, G.: Histopathological analysis of liver in fish in reservoir Trebeni{tavariation in sensivity between different species of zooplankton under natural con-ditions and thus exemplify the necessity of multispecies approaches in the risk as-sessment of pesticides (FRIBERG-JENSEN et al., 2003).In arid periods with a small quantity of rainwater and high evaporation in thesummer months due to high temperatures, a rapid reduction of the water quantityin Trebenista Reservoir occurs. In some cases fish pestilence was evidenced, proba-bly as a result of the increased water temperature, intensified eutrophication pro-cesses and hypoxic conditions in the aquatic environment. There were no dead in-dividuals in the samples caught in this reservoir, which indicates their health wasgood in morphological terms. Nevertheless, the microscopic analysis of the histo-logical preparations indicated presence of hemorrhaging in the hepatocellular pa-renchyma (Fig. 2a); moreover the pigment hemosiderin was evidenced, probably asa result of internal bleeding in the hepatic tissue of barbel (Fig. 2b). Apart fromthese changes, there was also the presence of parasites, most probably of the classNematodes (Fig. 2c). Approximately 22.5 % of all barbels collected at Trebeni{taReservoir showed evidence of hemorragia, and 17.5 % showed the presence of thepigment hemosiderin in liver tissue. Prevalence of parasite infestation was only 10%. Hemosiderin in the liver is not suprising as it represents a product of hemoglo-Nat. Croat. Vol. 14(2), 2005 151Fig. 2. Liver lesions in barbel (Barbus meridionalis petenyi Heck.) from Trebeni{taReservoir (H&E, x 100). a. hemorragia in the liver tissue associated with parenchymecell necrosis; b. the pigment hemosiderin in hepatocellular parenchyma (arrows);c. presence of parasite in hepatocellular parenchyma (probably Nematode).bin degradation that has been filtered out by the lymphoid-macrophage system(KHAN et al., 1994).Recent evidence indicates that increases in oxidative stress and associated bio-chemical alterations could be a toxic mechanism in fish exposed to pulp mill effluent(OAKES & VAN DER KRAAK, 2003). Also, high amounts of ions (especially nitrogenand potassium) in mining wastewater have drastically changed the water chemistryin lakes, and affected species composition, the food chain structure and fish health.Despite high fish population densities, histology of liver and gills indicates unfa-vorable environmental conditions for individual fish in lakes receiving mining ef-fluents (TKATCHEVA et al., 2002).All these findings imply that certain negative processes are taking place in Tre-beni{ta Reservoir, not only for the fish population, but also most probably for theother vertebrate and invertebrate forms inhabiting it. These negative impacts espe-cially accelerate in the droughty periods of arid and hot summers, when fish pesti-lence has been evidenced in the reservoir. Certain localities of Ohrid Lake and itswatershed display loading with contaminants from the ground, especially in thecourse of the summer period (MITIC et al., 2002). 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Histopatološka analiza jetre riba (Barbus meridionalis petenyi Heckel) iz akumulacije Trebeništa

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