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Lab Report: Lab Rotations (Spring 2007) in the laboratory of Prof. Dr. Angela Koehlerunder the supervision of Dr. Katja Broeg and Sonja Einsporn, PhD;,Alfred Wegener Institute for Polar and Marine Research (AWI)Am Handelshafen 12, 27570 Bremerhaven, Germany
IMPACT OF METAL POLLUTION IN LIVER TISSUES OFCORKWING WRASSE FISH
(SYMPHODUS MELOPS L.) 
ATCELLULAR LEVEL
ByKedar Ghimire
 
Jacobs University Bremen
 
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de
2
Structural differences in organelles and its consequences in the livertissues of Corkwing Wrasse fish
(Symphodus melops L.)
sampledfrom differently polluted coastal sites of Norway
Kedar Ghimire,
Jacobs University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen,Germany
Wrasse
(
Symphodus melop 
s
L
.)
is an important marine species for monitoring theenvironmental and health effects of contamination in North Sea. Due to the toxicsubstances like PAH (polycyclic aromatic hydrocarbons), biocide(C-Treat 6), TBT etcreleased by aluminium smelters; metal contamination of coastal water due to coppermines; the habitats of this fish have been negatively effected. Many of these fishes havebeen found to be effected with various diseases that directly affects the vital metabolicorgans of the body like the liver hinting to the fact that the situation of life forms in theseareas are in peril. Through this study, we have attempted to explore the liver tissues(hepatocytes) from various wrasse samples living in metal (copper) contaminated sitesand reference sites and make a comparable analysis of the structural and functionalchanges observed in the cell organelles at electron microscope level. We conclude that Cucontamination is harmful and it affects the cell organelles in liver tissues of Wrasse indifferent ways.Keywords:
Hepatocytes, lipid, copper, metallic crystals, metabolism, glycogen, electronmicroscopy
Abbreviations:
TBT: TributyltinPAH: Polycyclic aromatic hydrocarbonEM: Electron microscopy
Introduction:
Wrasse is an interesting fish specieswhose gender changes from female to maleduring the life time (a protogyn) (Broeg et al,2007). It has a flat body structure. Specificchemical impacts are expected to changemorphology and consequently, the function ofits organs. Increasing frequencies oftoxipathic lesions and liver tumors have beenreported in other fish from areas with chemicalimpact of pollution
(Gardner et al., 1991; Koehler et al., 1992; Johnson et al., 1993; Stein et al., 1990; Stentiford et al., 2003)
. Wefear Wrasse can be another such victim.Fish are poikilothermic vertebrates sothey change their metabolism according to thetemperature variations throughout the yearand all those changes are reflected in the liver.Fish are highly susceptible to environmentalvariations and respond sensitively to pollutantsthan other various mammals (Munsi andDutta, fish morphology, 1996). The liver of thewrasse has many digestive and storagefunctions. Liver cells secrete bile whichemulsifies fat and helps change the acidic pHof stomach into neutral pH of the intestine. Bilecollects in the bile capillaries, which then unite,forming bile ducts. The bile canaliculus is astructure formed by grooves on the contactsurface of adjacent liver cells, i.e. the dilatedintercellular space between adjacenthepatocytes. Bile forms in these canaliculi andthen flows into small ducts, and finally intolarger hepatic ducts.Figs. 1.2 and 1.1 in the next pagesshow a liver tissue with a normal nucleus,plenty of glycogen granules, lot of vesicles,lysosome and plenty of mitochondria. It shouldbe noted that the liver is the major site for Cuexcretion (in the bile) in vertebrates. Whilecopper is an endocrine disrupter in the aquaticanimals and has a number of neuro-endocrineeffects in vertebrates (Handy, 2003).
 
The fish were sampled from fivedifferent fjord sites in Norway. Site 1 was
 
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de
3
SalvØy, considered to be an outer referencesite on the west side of Karmoy. Site 2 wasVisnes- a highly copper and zinc contaminatedsite on the west side of Karmoy. In this site,both tailings and slag was dumped too. Site 3was FØrlandsfjorden- an extremely shelteredfjord representing the inner part of the fjordsystem, with small boat traffic and some smallfarms that drain to the fjord with vast amountof mussels found along the shores of the fjord.Site 4 was Bokn- a reference site in theexposed part of the fjord system. Site 5 wasHØgevarde- a site just north of the PAHdischarge from the Alumina smelter inKarmsund. Other discharge there consisted ofbiocide, TBT. Among these sites, our studyfocused on site 2 and 3. Site 2 was influencedby an old copper mine which was inproduction 1865-94 and a new production fora few years until 1965. The area closest to theold mine had no sign of life. The sea waterwas exposed to the metals mainly copper andzinc from the fillings and the run off from land.Station 3 was our reference site.We used the methods of microscopicanalysis at light and EM level in our study toobserve structural changes in the liver tissuesand the consequences of these changestowards the physiology and adaptation ofWrasse.
Materials and Methods:
Our samples were embedded in theyear September 2001 and were preservedsafely.For electron microscopy, afterembedding liver tissues into epoxy resin, amicrotome
(Model Leica EM UC6)
equippedwith a diamond knife was used to cut first verythin sections for examining under lightmicroscope
(Zeiss Axioskop)
. The settings ofmicrotome was speed (mm/s) =1 andfeed/nm=500. For this, three samples (Nr. 2,3, 4) from station 2 and two samples (Nr. 1, 5)from station 1 were chosen. Five slides fromeach sample were prepared. Among them,two of each sample were stained with TolueneBlue 0.5% in Na
2
CO
3
. Toluene blue wasfiltered and the tissue sections were stainedfor 1-2 minutes in it. We had used variationsfor this process. Two slides for each samplewere prepared. One sample was heated(Stuart SB 300 heater) to magnitude 2 andwas stained for 2 minutes. The other samplewas stained for 1 minute with heat magnitudeof 3. The sections were then washed with dist.water and dipped in ethanol for dehydrationand quickly taken out and dried. After lightmicroscopic analysis, it was found thatsamples heated at 2 and stained for 2 minutesproduced better results and weresubsequently used.After light microscopy, theblock sections were marked after consideringtheir special characteristics to observe underEM. These marked sections were prepared inblock removing other unnecessary areas withblade. Then the microtome
(Model Leica EM UC6)
was used to prepare ultra sections forEM. The settings of 1 mm/s speed and 60feed/nm was used for this purpose. The slicedsections were placed in small grids carefully.Then staining was performed. First, thesample was stained with Uranyl acetate for 5minutes, and then washed thoroughly withddH
2
O. Then the sample was again stained inlead citrate for exactly 1 minute and washedthoroughly well and let to dry for a night. Fewequivalent samples were not stained so thatcomparable analysis could be done betweenstained (contrasted) and unstained(uncontrasted) sections of the same region.
Results:
We tried to see and note thedifferences in structure and consequently infunction of cellular organelles of Corkwingwrasse fish from metal sites and referencesite. The differences between normal andpollution-effected tissues will be discussed infull detail in the discussion. Our results couldbe explained through various EM images ofthe liver tissue of Corkwing Wrasse fish.
Overview 
Fig 1.
Transmission EM Overview of the tissuefrom station 2 (polluted site) at 3000 × magnification
of 00

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