Influence of Cyanotoxins in Histology of Carassius Carassius

N. Medja,1 E. Panariti2, S. Duro2, A.Striniqi1 Department of Biology , University of Shkodra, Albania 2 Agricultural University of Tirana, Faculty of Veterinary Medicine, Department of Veterinary Public Health
1

Abstract The aim of this study was to find out how crude extract of cyanobacteria can influence larval development of carassius on the basis of embryo-larval toxicity test and histological changes of liver of larvae carassius exposed 30 days to the crude extract of cyanobacteria with the cumulative concentration 9.0 g l-1 (medium concentration of the extract) and 0.9 g l-1 (low concentration of the extract) of microcystins LR, RR and YR. The experiments were finished after 30 days. Evaluation of the tests was based on the OECD guideline for testing chemicals, direction 210 from 1992. Liver sections were stained with haematoxylin-eosin and using light microscopy. The extract with medium concentration caused an increase in malformed and dead larvae. The extract with low concentration caused an increase in dead larvae. Vacuolar dystrophy of hepatocytes accompanied by damage of nuclei (pyknosis,) were found in the group exposed to the low concentration of the extract. Focal necroses and dystrophic changes of hepatocytes with vacuolization and nuclei damage (pyknosis, karyolysis,) were found in the group exposed to the medium concentration of the extract. The degree of damage depended on the concentration of the extract. Keyword: Hepatocyte, cyanotoxins, malformations, fish, embryo-larval toxicity test, Introduction Cyanotoxins produced by cyanobacteria pose an environmental problem and influence the health status of both human and aquatic organisms. The most common cyanotoxins are hepatotoxins. Many authors examined the histopathological findings and the mechanism of influence of microcystins. Deformation of hepatocytes is the most pronounced effect (Falconer and Yeung 1992). Falconer and Yeung (1992) concluded that the mechanism of microcystin toxicity to the hepatocyte is through cytoskeletal damage leading to loss of cell morphology, to cell adhesion and finally cellular necrosis. Toxicity of microcystin LR in vivo primarily consists in the hepatocellular deformation inducing degenerative changes of the tissue (Eriksson et al. 1989). Recently, research into this area has also been aimed at the evaluation of effects of cyanotoxins to the early life stages of organisms. The effect of microcystins and the crude extract of cyanobacteria on the development of fish and amphibians were studied (Oberemm et al. 1997, 1999) without description of histopathology of fish embryos and larvae. The level of dissolved microcystins in the Shkodra lake was measured in south coast where untreated waste water are deposited.. The level of dissolved microcystins in the Shkodra lake was measured at IPH Tirane. The results of embryo-larval tests in carassius exposed to crude extract of cyanobacteria containing the known amount of microcystins are presented in this study. Concentrations of microcystins were chosen to compare the results with literature and to be compared with the level of dissolved microcystins in natural waters.

Materials and Method The carassius eggs were obtained by artificial reproduction at the fishery in Laknas (Albania). Fertilised and unsticked carassius eggs were divided into three groups till 8 hours from fertilisation, each containing two hundred eggs. The eggs were incubated in glass vials containing 1 L of water. The water was changed every 8 hours including the crude extract of cyanobacteria to keep the concentration of crude extract of cyanobacteria. The conditions in baths were as follows water temperature 21-22 C, dissolved oxygen 75-100%, i.e. 5.5-10 mgL-1 and pH was 8-9. The larvae were fed by commercial food Artemia Premium since the 5th day. Feeding was performed before 20-30 min of every water changing intervals. Tests were performed with the crude cell extract obtained from field samples of water bloom (Shkodra lake). Water samples contained the planktonic species M. aeruginosa (90%),

Microcystis ichtyoblabe (4%) and Aphanizomenon flos-aquae (2%). The samples were collected from surface water bloom (0.3 m depth) and concentrated by plankton net 25 m. The samples were stored frozen at -20 C. The concentration of microcystins was determined by HPLC according to the method described by Lawton et al. (1994). Total microcystin concentration (MC) was 1 056.2 gg-1 dry weight in the biomass. To obtain the crude extract, the material was ultrasonicated for 7 min. and was centrifuged for 20 min at 4 500 rpm. Reextraction was done twice by standard water. The final concentration of hepatotoxic microcystins in the crude extract used for exposure was17.3 gL1 (4.8 gL1 of microcystin YR, 9.59 gL1 of microcystin LR, 2.9 gL-1 of microcystin RR). Experimental treatments The crude extract of cyanobacteria with known amount of microcystin LR (5 and 0.5 gL-1) was added to the eggs in two concentrations: the first with 0.5 gL-1 of microcystin LR (low concentration of the extract) the second with 5 gL-1 of microcystin LR (medium concentration of the extract). The controlled eggs were incubated in toxic free water. The cumulative amount of microcystins was 9 and 0.9 gL-1, respectively. Tests were finished after 30 days. Evaluation of the tests was based on the OECD Direction 210 from 1992. During the test we observed: the time of start and the end of hatching numbers of larvae hatching each day numbers of malformed larvae After finishing the tests, we evaluated: cumulative mortality numbers of healthy fish at the end of the test average total length and body mass (the average total length was determined in 10 larvae and body mass in 20 larvae). Histology Five fish from each group were killed, immediately fixed in Bodian solution and processed using standard methods for histology. Tissue sections were stained with haematoxylin-eosin and cells were detected with H&E test. All sections were examined using light microscopy. Liver tissues were examined. Results Larvae hatched during three days. In the control group, the majority of larvae hatched in the second day. In the groups with medium and low concentrations of the extract it was similar, the majority of larvae hatched in the first day. Total numbers of hatched larvae were 186 in the control, 187 in the group with low concentration of the extract and 186 in the group with medium concentration of the extract. Results of tests with egg hatching is presented in Table 1. Malformed and dead larvae (numbers of malformed and dead larvae are presented in Table 2). In the control group 5 malformed larvae were found (2.68% from 186 hatched larvae), 8 in the group with low concentration of the extract (4.27% from 187 hatched larvae) and 10 in the group with medium concentration of the extract (5.37% from 186 hatched larvae) during the experiment. 15 larvae died during the experiment in the control group (8.06% from 186 hatched larvae), 21 in the group with low concentration of the extract (11.23% from 187 hatched larvae) and 36 in the group with medium concentration of the extract 19.35% from 186 hatched larvae). Cumulative mortality (cumulative mortality is presented in the Table 2). In the control group 171 larvae survived. In the group with low concentration of the extract 166 larvae survived and in the group with medium concentration of the extract were 150 surviving larvae. Average total

length and body mass of surviving larvae (no significant differences in the average total length and body mass were found, see Table 2).

Table 1. Egg hatching and malformations Concentration the extract of Start of hatching End of hatching Numbers of hatched larvae for a day 3rd day medium low control 58 58 58 102 102 102 156 121 60 4th day 28 65 125 5th day 2 1 1 5,37 4,27 2,68 Percentage of malformed larvae

Table 2. Fry measurements and survival Concentration of the extract medium low control Histology No changes in liver were found in the control group. Vacuolar dystrophy of hepatocytes with damage of nuclei (pyknosis) was found in the group exposed to low concentration of the extract. These changes were found in all sampled larvae. Focal necroses and dystrophic changes of hepatocytes with vacuolisation and nucleic damage pyknosis, were found in the group exposed to the medium concentration of the extract. These changes were found in all sampled larvae. Discussion The concentrations of crude extract of cyanobacteria (5 and 0.5 gL-1 of microcystin LR) had effects even at lower concentrations, in particular, in the group exposed to the medium concentration of the extract. Cumulative mortality was higher and the number of malformed individuals was increased. Neither the total length nor the body weight of larvae was significantly altered. Histopathological changes of liver in our study were similar to the changes described in various papers in young and adult fish. Rodger et al. (1994) described the histopathological changes of brown trout (Salmo trutta) associated with the death of water blooms of Anabaena flos-aquae. The changes in liver were characterised by confluent necrosis showing cellular degeneration and loss of obvious cell boundaries. Pyknosis and karyorrhexis of hepatocytes was obvious. Similar changes in liver have been described in different fish species by other authors. Carbis et al. (1996) detected histopathological changes in the gills, in liver and kidney of carp exposed to microcystins by gavage, immersion and Cumulative mortality (%) 25 17 14,5 Average total length (mm SD) 13,872,09 14,211,41 14,562,1 Average total body mass (mg SD) 34,1715,3 29,412,11 30,8112,48 Percentage of dead larvae (%) 19,35 11,22 8,06

intraperitoneal administration. Intraperitoneal inoculation caused necrosis or dose depended degeneration. Gavaging caused changes in the histopathology of the liver and gills. Cellular degeneration and necrosis occurred in the liver, gills and kidneys when carp were introduced to a tank containing 1.7 gml-1 of microcystins. Carbis et al. (1997) studied carps exposed to Microcystis aeruginosa at Lake Mokoan (Australia). The total concentration of the microcystins was approximately 4.0 gg-1 of the lyophilised scum material. During February, March and April the liver histology was characterised by cytoskeletal collapse, cytoplasmic vacuolization, pyknosis, chromatin margination, eosinophilia and widespread hepatocyte atrophy, particularly in areas close to the arterial blood supply in about 66% of the carp examined. We detected damage of liver in fish The degree of damage depended on the concentration of the extract.

Conclusion In conclusion we can say that the medium and low concentrations of the extract corresponds with the level of dissolved microcystins in lake water. Then it could negatively influence the tissue of larvae of various fish species in natural waters. References Carbis, C. Rawlin, G. Mitchell, G. Anderson, J. Mccauley, I. 1996: The histopathology of carp, Cyprinus carpio, L., exposed to microcystins by gavage, immersion and intraperitoneal administration. J Fish Dis 19: 199-207 Carbis, C. Rawlin, G. Grant, P. Mitchell, G. Anderson, J. Mccauley, I. 1997: A study offeral carp, Cyprinus carpio L., exposed to Microcystis aeruginosa at Lake Mokoan, Australia, and possible implications for fish health. J Fish Dis 20: 81-91 Eriksson, J. Paatero, G. Meriluoto, J. Codd, G. Kass, G. Nicotera, P. Orrenius, S. 1989: Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin. Exp Cell Res 185: 86-100 Falconer, I. Yeung, D. 1992: Cytoskeletal changes in hepatocytes induced by Microcystis toxins and their relation to hyperphosphorylation of cell proteins. Chem Biol Interact 81: 181196 Lawton, L. Edwards, C. Codd, G. 1994: Extraction and high-preformance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst 119, 1525-30. Rodger, H. Turnbull, T. Edwards, C. Codd, G. 1994: Cyanobacterial (blue-green algal) bloomassociated pathology in brown trout, Salmo trutta L., in Loch Leven, Scotland. J Fish Dis 17: 177-181 Oberemm, A. Becker, J. Codd ,G. Steinberg, C. 1999: Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environm Toxicol 14, 77-87. Oberemm, A. Fastner, J. Steinberg, C. 1997: Effects of microcystin LR and cyanobacterial crude extracts on embryo-larval development of zebrafish (Danio rerio). Water Res 31, 29182921. OECD guideline for testing of chemicals, 210,1992,p18.

Photo 1 Vacuolar dystrophy of hepatocytes with pyknosis and karyolysis of nuclei in liver of carassius with low concentrationof the extract (H&E 400).

Photo 2. Liver of carassius with medium concentration of the extract. Perivascular focal necrosis (H&E 400)

Ndikimi i Cyanotoxins n histology e Carassius Carassius N. Medja, 1 E. Panariti2, S. Duro2, A.Striniqi1 1 Departamenti i Biologjis, Universiteti i Shkodrs, Shqipri 2 Universiteti Bujqsor i Tirans, Fakulteti i Mjeksis Veterinare, Departamenti i Shndetit Veterinar Publik

Abstrakt Qllimi i ktij studimi ishte pr t gjetur se si ekstrakt i vrazhd i cyanobacteria mund t ndikoj n zhvillimin larvor of carassius mbi bazn elarvor embrionit test toksiciteti dhe ndryshimet histologjike e mlis larvat carassius ekspozuar 30 dit pr t ekstrakt i vrazhd i cyanobacteria me prqendrim kumulative 9.0 pesh trupore l-1 (prqendrimi t mesme i ekstrakt) dhe 0.9 pesh trupore l-1 (prqendrimi i ult i ekstrakt) e microcystins LR, Rr dhe yr. Eksperimentet jan prfunduar pas 30 ditsh. Vlersimi i testeve u bazuar n udhzimin e OECD pr testimin e kimikate, drejtimin e 210 nga 1992. Seksionet mlis jan ngjyrosur me hematoksilin t eosin dhe prdorimin e mikroskopit t lehta. Ekstrakti me koncentrim t mesme shkaktuar nj rritje n larvat keqformuar dhe t vdekur.Ekstrakti me koncentrim t ult t shkaktuar nj rritje n larva t vdekur. Distrofi Vacuolar e hepatocytes shoqruara me dme t brthamave simbolit pyknosis), jan gjetur n grupin e ekspozuar ndaj prqendrimit t ult t ekstrakt. Necroses fokale dhe ndryshimet dystrophic of hepatocytes me vacuolization dhe brthamat demin e pyknosis, karyolysis), jan gjetur n grupin e ekspozuar ndaj prqendrimit t mesm t ekstrakt. Shkalla e dmtimit varet nga prqendrimit t ekstrakt. Keyword: Hepatocyte, t cyanotoxins, t keqformime, peshk, embrion-larvor test toksiciteti, t Hyrje Cyanotoxins prodhuara nga cyanobacteria paraqesin nj problem t mjedisit dhe t ndikoj n statusin shndetsor t organizmave t dy t njeriut dhe ujore. The cyanotoxins m t zakonshme jan hepatotoxins. Shum autor ekzaminuar gjetjet histopathological dhe mekanizmin e ndikimit t microcystins. Deformimi i hepatocytes sht efekti m e theksuar (Falconer dhe Yeung 1992). Falconer dhe Yeung (1992) arriti n prfundimin se mekanizmi i toksicitetit microcystin n hepatocyte sht me an t dmtimit cytoskeletal q on n humbjen e morfologjis celular, me aderimin e qelizs dhe n fund nekrotizues qelizore. Toksiciteti i microcystin LR n vivo kryesisht konsiston n deformim hepatocellular inducing ndryshimeve degjeneruese e indeve simbolit Eriksson et al. 1989). Koht e fundit, hulumtimet n kt fush ka qen gjithashtu pr qllim vlersimin e efekteve t cyanotoxins n fazat e hershme t jets s organizmave. Efekti i microcystins dhe ekstrakt i vrazhd i cyanobacteria mbi zhvillimin e peshkut dhe amfibve jan studiuar (Oberemm et al. 1997, 1999) pa prshkrimin e histopatologjin e embrioneve t peshkut dhe larva. Niveli i microcystins tretur n liqenin e Shkodrs sht matur n bregdetin e jugut ku uji trajtohet mbeturina jan depozituar .. Niveli i microcystins tretur n liqenin e Shkodrs sht matur n ISHP Tiran. Rezultatet e embrion-larvor testeve n carassius ekspozuar pr nxjerrjen e paprpunuar t cyanobacteria prmban shumn e njohur e microcystins jan paraqitur n kt studim. Prqendrimet e microcystins jan zgjedhur pr t krahasuar rezultatet me literatur dhe q t krahasohet me nivelin e microcystins tretur n ujrat natyrore.

Materialet dhe Metoda Vezt carassius jan marr nga riprodhimit artificial n peshkimin n Laknas (Shqipri). Fertilised dhe unsticked vez carassius u ndan n tre grupe deri 8 or nga fekondimit, secili prmban dyqind vez. Vezt jan inkubohet n shishe qelqi q prmbajn 1 L uj. Uji sht ndryshuar do 8 or duke prfshir ekstrakt i vrazhd i cyanobacteria pr t mbajtur prqendrimin e ekstrakt i vrazhd i cyanobacteria. Kushtet n banjot ishin si m posht temperaturn e ujit 21-22 C, oksigjen tretur 75-100%, dmth 5,5-10 MGL-1 dhe pH ishte 8-9. Larvat u ushqyer nga komercial Premium ushqimit artemia q nga dita e 5. Ushqyerit sht kryer para 20-30 min e do uji ndryshon intervale. Testet jan kryer me ekstrakt celular bruto t marra nga mostrat e ujit n terren lulzim (Shkodr liqeni). Mostrat e ujit prmbante aeruginosa specieve planktonic M. (90%), t Microcystis ichtyoblabe (4%) dhe Aphanizomenon Flos-Aquae (2%). Mostrat jan mbledhur nga t ujit siprfaqsor lulzim (0.3 m thellsi) dhe koncentruar me plankton 25 m neto. Mostrat jan ruajtur ngrir n -20 C. Prqendrimi i microcystins u prcaktua nga HPLC sipas metods s prshkruar nga Lawton et al. (1994). Total prqendrimi microcystin (MC) ishte 1 056.2 pesh trupore g-1 pesh e that n biomass. Pr t marr ekstrakt t paprpunuar, materiali u ultrasonicated pr 7 min. dhe u centrifugohen pr 20 min n 4 rpm 500. Re-nxjerrjes sht br dy her nga uji standarde. Prqendrimi i fundit i microcystins hepatotoxic n ekstrakt bruto prdoren pr was17.3 pesh trupore ekspozimit L-1 (4.8 pesh trupore L-1 e microcystin yr, 9,59 pesh trupore L-1 e microcystin LR, 2.9 pesh trupore L-1 e microcystin RR). Trajtimet eksperimentale Ekstrakti i vrazhd i cyanobacteria me sasi t njohur t microcystin LR (5 dhe 0.5 pesh trupore L-1) sht shtuar n vez n dy prqendrimet: i pari me 0,5 pesh trupore L-1 e microcystin LR (prqendrimi i ult i ekstrakt) t e dyt me 5 pesh trupore L-1 e microcystin LR (prqendrimi t mesme t ekstrakt). Vezt e kontrolluara u inkubohet n uj toksike t lir. Shuma kumulative e microcystins ishte 9 dhe 0.9 pesh trupore L-1, respektivisht. Testet jan prfunduar pas 30 ditsh. Vlersimi i testeve u bazuar n Urdhresn OECD 210 nga 1992. Gjat testit kemi vrejtur: koha e fillimit dhe n fund t vizim T numrat e vizim larvat do dit T numrat e larvat keqformuar Pas prfundimit t testeve, ne vlersohet: Vdekshmria kumulative T numrat e peshkut t shndosh n fund t testit Gjatsia mesatare e prgjithshme dhe n mas t trupit (gjatsia mesatare e prgjithshme ishte i vendosur n 10 larvat dhe mas trupore n 20 larvat). Histologji Pes peshk nga secili grup u vran, t fiksuara menjher n zgjidhjen Bodian dhe t prpunuara duke prdorur metoda standarde pr histology. Seksionet e indeve u ngjyrosur me hematoksilin t eosin dhe qelizat jan zbuluar me H & prov E. T gjitha pjest ishin shqyrtuar me

mikroskop me drit. Indet e mlis jan ekzaminuar. Rezultatet Larvat e elur gjat tri ditve. N grupin e kontrollit, shumica e larvat e elur n ditn e dyt. N grupet me prqendrime t mesme dhe t ult t ekstrakt ajo ishte e ngjashme, shumica e larvat elur n ditn e par. Numri total i larvat komplote ishin 186 n kontrollin, 187 n grup me prqendrim t ult t ekstrakt dhe 186 n grup me prqendrim t mesm t ekstrakt. Rezultatet e testeve me vizim vez sht paraqitur n Tabeln 1. Larvat e shformuar dhe t vdekur (numri i larvave keqformuar dhe t vdekur jan paraqitur n Tabeln 2). N grupin e kontrollit 5 larvat shformuar u gjet (2,68% nga 186 larvat komplote), 8 n grup me prqendrim t ult t ekstrakt (4.27% nga 187 larvat komplote) dhe 10, n grup me prqendrim t mesm t ekstrakt (5.37% nga 186 larva hatched) gjate eksperimentit. 15 larvat vdiq gjate eksperimentin n grupin e kontrollit (8.06% nga 186 larvat komplote), 21 n grupin me prqendrim t ult t ekstrakt (11,23% nga 187 larvat komplote) dhe 36, n grup me prqendrim t mesm e 19.35 pr qind ekstrakt nga 186 elur larvat). Vdekshmria kumulative (vdekshmria kumulative sht paraqitur n tabeln 2). N grupin e kontrollit 171 larvat mbijetuar. N grupin me t prqendrimit t ult t 166 larvae ekstrakt mbijetuar dhe n grup me prqendrim t mesm e ekstraktit ishin 150 larvae mbijetuar. Gjatsia mesatare e prgjithshme dhe n mas trupi i mbijetuar larvat e (pa dallime t mdha n gjatsi mesatare t prgjithshme dhe n mas t trupit u gjetn, shih tabeln 2).

Tabela 1. Vizim vez dhe keqformime Prqendrimi i Start ekstrakt t Fundi vizim e Numrave vizim e larvat komplote pr nj prqindje t prditshm t larvat keqformuar 3 dit 4 dit 5 dit t mesme 58 102 156 28 2 5,37 i ult 58 102 121 65 1 4,27 kontrolluar 58 102 60 125 1 2,68 Tabela 2. Matjet e skuqura dhe mbijetess Prqendrimi i vdekshmris ekstrakt kumulative (%) gjatsia mesatare e prgjithshme (mm SD) Mesatarja e prgjithshme n mas trupit (mg SD) Prqindja larvat vdekur (%) mesme 25 13,87 2,09 34,17 15,3 19,35 ult 17 14,21 1,41 29,4 12,11 11,22 Kontrolli 14,5 14,56 30,81 2,1 12,48 8,06 Histologji Nuk ka ndryshime n mli jan gjetur n grupin e kontrollit. Distrofi

Vacuolar of hepatocytes me dmtimin e brthamave simbolit pyknosis) u gjet n grupin e t ekspozuar t prqendrimit t ult t ekstrakt. Kto ndryshime jan gjetur n t gjitha larvat kampionuar. Necroses fokale dhe ndryshimet dystrophic of hepatocytes me vacuolisation dhe pyknosis nukleik dmit, jan gjetur n grupin e ekspozuar ndaj prqendrimit t mesm t ekstrakt. Kto ndryshime jan gjetur n t gjitha larvat kampionuar. Diskutim Prqendrimet e ekstrakt i vrazhd i cyanobacteria (5 dhe 0.5 pesh trupore L-1 e microcystin LR) pati efekte edhe n prqendrime t ulta, n veanti, n grupin e ekspozuar ndaj prqendrimit t mesm t ekstrakt. Vdekshmria kumulative ishte m i lart dhe numri i individve t keqformuar u rrit. As Gjatsia totale dhe as pesha e trupit t larvs u ndryshua n mnyr t konsiderueshme. Ndryshimet Histopathological e mlis n studimin ton ishin t ngjashme me ndryshimet e prshkruara n gazeta t ndryshme n peshk t rinj dhe t rritur. Rodger et al.(1994) prshkroi ndryshimet histopathological e kafe troft simbolit Salmo trutta) lidhur me vdekjen e blooms me uj t Anabaena Flos-Aquae. Ndryshimet n mli jan karakterizuar nga nekroz bashkohet tregon degjenerim qelizore dhe humbjen e kufijve t qelizave t dukshme. Pyknosis dhe karyorrhexis i hepatocytes ishte e qart. Ndryshime t ngjashme n mli jan prshkruar n specie t peshkut t ndryshme nga autor t tjer. Carbis et al. (1996) zbuluar ndryshime histopathological n gush, n mli dhe veshka e krapit t ekspozuar ndaj microcystins nga, zhytje gavage dhe administrimin e SHQ. Nekroza SHQ inokulimit shkaktuar ose doz varej degjenerim. Gavaging ndryshimet e shkaktuara n histopatologjin e mli dhe gush. Degjenerimi Cellular dhe nekroz ndodhur n gusha, mlin dhe veshkat kur krap u futur n nj tank prmban 1.7 pesh trupore ml-1 i microcystins. Carbis et al. (1997) ka studiuar carps ekspozuar ndaj aeruginosa Microcystis n liqenin Mokoan (Australi). Prqendrimi i prgjithshm i microcystins ishte afrsisht 4.0 pesh trupore g-1 e materialit shkums liofil. Gjat shkurt, mars dhe prillit histology mlis u karakterizua nga kolapsi cytoskeletal, t vacuolization cytoplasmic, t pyknosis, t margination chromatin, t eosinophilia dhe atrofi prhapur hepatocyte, veanrisht n zonat afr furnizimin me arterial t gjakut n rreth 66 pr qind t krapit ekzaminuar. Ne zbuluar dmtimin e mlis n peshk Shkalla e dmit varej nga prqendrimit t ekstrakt. Prfundim N prfundim mund t themi se prqendrimet t mesme dhe t ult t ekstrakt korrespondon me nivelin e microcystins tretur n uj t liqenit. Ather ajo mund t ndikoj negativisht n indet e larvave t llojeve t peshkut t ndryshme n ujrat natyrore. Referencat Carbis, C. Rawlin, G. Mitchell, G. Anderson, J. McCauley, I. 1996: The Histopatologjia e krapit, e PANARITI carpio, L., t ekspozuar ndaj microcystins me zhytje gavage dhe administrimin e SHQ. Peshku J Dis 19: 199-207

Carbis, C. Rawlin, G. Grant, P. Mitchell, G. Anderson, J. McCauley, I. 1997: Nj studim offeral krapi, PANARITI carpio L., t ekspozuar ndaj aeruginosa Microcystis n liqenin Mokoan, Australi, dhe implikimet e mundshme pr shndetsor peshk. J Peshku Dis 20: 81-91 Eriksson, J. Paatero, G. Meriluoto, J. Codd, G. Kass, G. Nicotera, P. Orrenius, S. 1989: Riorganizimi i shpejt microfilament detyruar n hepatocytes miu izoluara nga microcystin t LR, nj toksina ciklike peptide. Res Cell exp 185: 86-100 Falconer, I. Yeung, D. 1992: Ndryshimet n Cytoskeletal hepatocytes detyruar nga toksina Microcystis dhe lidhja e tyre me hyperphosphorylation e proteinave celular.Kim Biol Interact 81: 181-196 Lawton, L. Edwards, C. Codd, G. 1994: Nxjerrja dhe t mesme-preformance metod t lngshme kromatografi ne faze pr prcaktimin e microcystins n ujrat para dhe t trajtohen. Analist 119, 1525-1530. Rodger, H. Turnbull, T. Edwards, C. Codd, G. 1994: Cyanobacterial (blujeshile algal) bloomassociated patologji n kafe Troft, Salmo trutta L., n liqen Leven, Skoci. Peshku J Dis 17: 177-181 Oberemm, A. Becker, J. Codd, G. Steinberg, C. 1999: Efektet e toksinave cyanobacterial dhe ekstrakte ujore t paprpunuar cyanobacteria n zhvillimin e peshkut dhe amfibve. Environm Toxicol 14, 77-87. Oberemm, A. Fastner, J. Steinberg, C. 1997: Efektet e microcystin LR dhe ekstrakte cyanobacterial paprpunuar pr-fjetur zhvillimin e embrionit zebrafish simbolit Danio rerio). Uji Res 31, 2918-2921. Udhzues OECD pr testim t kimikateve, 210,1992, t p18.

Foto 1 distrofi Vacuolar e hepatocytes me pyknosis dhe karyolysis e brthamave n mli e carassius me concentrationof ult ekstrakt ( H & E 400).

Foto 2. Mlia e carassius me t mesme prqendrimi i ekstrakt. Perivaskulare qendrore nekroz ( H & E 400)