Awi Labreport-kedar

Lab Report: Lab Rotations (Spring 2007) in the laboratory of Prof. Dr. Angela Koehler under 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 OF CORKWING WRASSE FISH (SYMPHODUS MELOPS L.) AT CELLULAR LEVEL

By Kedar Ghimire Jacobs University Bremen

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Structural differences in organelles and its consequences in the liver tissues of Corkwing Wrasse fish (Symphodus melops L.) sampled from differently polluted coastal sites of Norway Kedar Ghimire, Jacobs University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany Wrasse (Symphodus melops L.) is an important marine species for monitoring the environmental and health effects of contamination in North Sea. Due to the toxic substances like PAH (polycyclic aromatic hydrocarbons), biocide(C-Treat 6), TBT etc released by aluminium smelters; metal contamination of coastal water due to copper mines; the habitats of this fish have been negatively effected. Many of these fishes have been found to be effected with various diseases that directly affects the vital metabolic organs of the body like the liver hinting to the fact that the situation of life forms in these areas are in peril. Through this study, we have attempted to explore the liver tissues (hepatocytes) from various wrasse samples living in metal (copper) contaminated sites and reference sites and make a comparable analysis of the structural and functional changes observed in the cell organelles at electron microscope level. We conclude that Cu contamination is harmful and it affects the cell organelles in liver tissues of Wrasse in different ways. Keywords: Hepatocytes, lipid, copper, metallic crystals, metabolism, glycogen, electron microscopy Abbreviations: TBT: Tributyltin PAH: Polycyclic aromatic hydrocarbon EM: Electron microscopy

Introduction: Wrasse is an interesting fish species whose gender changes from female to male during the life time (a protogyn) (Broeg et al, 2007). It has a flat body structure. Specific chemical impacts are expected to change morphology and consequently, the function of its organs. Increasing frequencies of toxipathic lesions and liver tumors have been reported in other fish from areas with chemical impact of pollution (Gardner et al., 1991; Koehler et al., 1992; Johnson et al., 1993; Stein et al., 1990; Stentiford et al., 2003). We fear Wrasse can be another such victim. Fish are poikilothermic vertebrates so they change their metabolism according to the temperature variations throughout the year and all those changes are reflected in the liver. Fish are highly susceptible to environmental variations and respond sensitively to pollutants than other various mammals (Munsi and Dutta, fish morphology, 1996). The liver of the wrasse has many digestive and storage

functions. Liver cells secrete bile which emulsifies fat and helps change the acidic pH of stomach into neutral pH of the intestine. Bile collects in the bile capillaries, which then unite, forming bile ducts. The bile canaliculus is a structure formed by grooves on the contact surface of adjacent liver cells, i.e. the dilated intercellular space between adjacent hepatocytes. Bile forms in these canaliculi and then flows into small ducts, and finally into larger hepatic ducts. Figs. 1.2 and 1.1 in the next pages show a liver tissue with a normal nucleus, plenty of glycogen granules, lot of vesicles, lysosome and plenty of mitochondria. It should be noted that the liver is the major site for Cu excretion (in the bile) in vertebrates. While copper is an endocrine disrupter in the aquatic animals and has a number of neuro-endocrine effects in vertebrates (Handy, 2003). The fish were sampled from five different fjord sites in Norway. Site 1 was

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SalvØy, considered to be an outer reference site on the west side of Karmoy. Site 2 was Visnes- a highly copper and zinc contaminated site on the west side of Karmoy. In this site, both tailings and slag was dumped too. Site 3 was FØrlandsfjorden- an extremely sheltered fjord representing the inner part of the fjord system, with small boat traffic and some small farms that drain to the fjord with vast amount of mussels found along the shores of the fjord. Site 4 was Bokn- a reference site in the exposed part of the fjord system. Site 5 was HØgevarde- a site just north of the PAH discharge from the Alumina smelter in Karmsund. Other discharge there consisted of biocide, TBT. Among these sites, our study focused on site 2 and 3. Site 2 was influenced by an old copper mine which was in production 1865-94 and a new production for a few years until 1965. The area closest to the old mine had no sign of life. The sea water was exposed to the metals mainly copper and zinc from the fillings and the run off from land. Station 3 was our reference site. We used the methods of microscopic analysis at light and EM level in our study to observe structural changes in the liver tissues and the consequences of these changes towards the physiology and adaptation of Wrasse.

Materials and Methods: Our samples were embedded in the year September 2001 and were preserved safely. For electron microscopy, after embedding liver tissues into epoxy resin, a microtome (Model Leica EM UC6) equipped with a diamond knife was used to cut first very thin sections for examining under light microscope (Zeiss Axioskop). The settings of microtome was speed (mm/s) =1 and feed/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 from each sample were prepared. Among them, two of each sample were stained with Toluene Blue 0.5% in Na2CO3. Toluene blue was filtered and the tissue sections were stained for 1-2 minutes in it. We had used variations for this process. Two slides for each sample were prepared. One sample was heated (Stuart SB 300 heater) to magnitude 2 and was stained for 2 minutes. The other sample was stained for 1 minute with heat magnitude

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of 3. The sections were then washed with dist. water and dipped in ethanol for dehydration and quickly taken out and dried. After light microscopic analysis, it was found that samples heated at 2 and stained for 2 minutes produced better results and were subsequently used. After light microscopy, the block sections were marked after considering their special characteristics to observe under EM. These marked sections were prepared in block removing other unnecessary areas with blade. Then the microtome (Model Leica EM UC6) was used to prepare ultra sections for EM. The settings of 1 mm/s speed and 60 feed/nm was used for this purpose. The sliced sections were placed in small grids carefully. Then staining was performed. First, the sample was stained with Uranyl acetate for 5 minutes, and then washed thoroughly with ddH2O. Then the sample was again stained in lead citrate for exactly 1 minute and washed thoroughly well and let to dry for a night. Few equivalent samples were not stained so that comparable analysis could be done between stained (contrasted) and unstained (uncontrasted) sections of the same region.

Results: We tried to see and note the differences in structure and consequently in function of cellular organelles of Corkwing wrasse fish from metal sites and reference site. The differences between normal and pollution-effected tissues will be discussed in full detail in the discussion. Our results could be explained through various EM images of the liver tissue of Corkwing Wrasse fish.

Overview

Fig 1. Transmission EM Overview of the tissue from station 2 (polluted site) at 3000 × magnification

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Fig 1.1 Transmission EM Overview of the liver tissue from station 3 (reference site) at 12000 × magnification

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Fig3. Transmission EM of a mitochondrial overview in section of liver tissues from station 3 at 20000× magnification

Figs. 4, 4.1, 5, 6, 7, 8, 9, 10 under the same heading as fig. 2 could be found in appendix 1.

Observations lysosome

on

mitochondria

and

Fig 2. Normal mitochondria seen in liver tissues from station 3 at 12000 × magnification

Observations on nucleus Endoplasmic Reticulum

and

Fig 11. EM of Rough endoplasmic reticulum from station 2 at 20000 × magnification

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Observations on metal deposits in tissues and bile canaliculus

Fig 12. Assumed metal deposits inside the cells from station 2 at 7000× magnification

Fig 18. Black deposits in intracellular space at 20000× from station 2.

The black deposits in figure 12 are assumed to be pieces of metallic elements. It was seen at random places and not uniformly.

Figs. 13, 15, 16, 16.1, 17 and 21 under the same heading as fig. 12 could be found in appendix 1.

Fig 14. Bile canaliculi with black deposits in the surrounding along intracellular pathways at 4400 × magnification from the station 2 metal site.

Fig19. Assumed metal deposits in Bile canaliculus with lipids alongside at 3000× magnification, (station 2- nC)

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Discussion:

Fig 20. Black deposits in the intercellular space near bile canaliculi at 3000× magnification- noncontrasted image (station 3)

Observations on lipid and glycogen present in tissues

Fig 22. Glycogen penetrating lipid and mitochondria attached to lipid from liver tissues at station 2 at 30000× magnification Fig 23, 24, 25 and 26 under the same heading as fig. 22 could be found on the appendix 1.

Aqueous Cu has been reported to accumulate in several tissues like gill, kidney and liver during chronic exposure and there is lesser accumulation in muscle (Handy, 2003). The metal site at Visnes is a chronic exposure to fish since there is an abandoned mine. In such type of exposure, it had been found fish have more time to down regulate Cu uptake through the gills and distribute newly acquired copper to the liver for excretion to minimize the toxological effects of copper (Grosell et al., 1996, 1997, 1998). It is also known that fish try to adjust to the metal exposure by initiating complex physiological adjustments like increased oxygen consumption, increasing neutrophils, altered immunity, increasing ionic regulation and altered cellularity (Handy, 2003). Copper demonstrates a high affinity for thiol groups and is therefore capable of severely disrupting many metabolic functions in the cell (Hultberg et al., 1998). Fig 1.2 (see Appendix 1) is a transmission electron micrograph which shows a clear overview of the wrasse liver tissue at 3000× magnification. The section was from station 3 which consisted of our reference site. Lysosome engulfing a lipid molecule could be seen. A normal nucleus with general lipid droplets was seen. No abnormalities were seen in the cells from the liver tissues of Wrasse from reference site as expected. Fig 1.1 shows another section from the station 3 at higher magnification of 12000×. A noticeable observation was that lots of vesicles could be seen, implicates that the cell was quite active with all types of intra-cellular activities going on and consequently, should be a very healthy cell. Mitochondria seemed to be coupled with the lipid droplet. Compared to cells from reference site, fig 1 shows EM of the liver tissue from site 2metal contaminated sites. No. of glycogen granules was much higher than those seen in the reference site. It can be inferred that there is at least some disruption in gluconeogenesis due to which glycogen couldn’t convert sufficiently into glucose. Carattino et al. (2004) had shown that Cu significantly inhibits glucose-6-phosphate dehydrogenase activity in-vitro in their study on effects of Cu on metabolism through pentose phosphate pathway in toad ovaries. Cu has also been

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found to interfere with the glycolytic pathway (Strydom et al., 2006). Glycogen granules were seen attached to the periphery of the lipid molecules. The reason for this close association could be that glycogen as a polymer of glucose can form covalent bonds with the fatty acid chains in lipid. Fatty acids are made by repeatedly joining together the two-carbon fragments found in acetyl-CoA and then reducing the (-CO-) part of the molecule to (-CH2-). In this way, the hydrocarbon chain can become the hydrophobic, energy storing part of the fatty acid. The number of lipid molecules is considerably high in comparison to cells from reference site. This clearly hints towards some negative effects on the reactions catalyzing lipid break down. In reverse, there is increased importance of the catabolism of lipids in such cells so that they wouldn’t accumulate to harmful level. Lipid peroxidation in response to copper exposure has been reported in freshwater crab (Vosloo et al, 2002). In our images, lysosome was seen attached to the lipid and could be in the process of degrading lipids. Lipid peroxidation is considered as a measure of oxidative stress and general stress thus is an indicator of fish health as a whole (Marcogliese et al, 2005). A suspected transport of black deposits (possibly metal crystals) was seen at 3000 M denoted in the figure as SP. An increased stimulation of ROS production by metals may lead to an imbalanced oxidative stress condition in fish that may result in physiological alterations (Sies, 1993, Paris-Palacios et al., 2000 and Varanka et al., 2001). Huge lipids were seen in sections from polluted sites. Figure 2 and figure 3 shows an image of the tissue at 12000 M from station 3, showing normal mitochondria (1-5 µm) with parallel cristae. There was no sign of any precipitates in these mitochondria. Lysosome seemed to be in pearl structure and ER was dilated. Again, a lot of vesicular activity was seen around. In contrast to these, EM of liver tissues from polluted sites (fig 4) showed elongated mitochondrias, which seem to be damaged and totally irregular in shape. A considerable amount of precipitate was seen inside the mitochondria. The parallel cristae structure wasn’t seen but cristae seemed to be damaged at various places in the mitochondria as it was seen at random positions within the mitochondria.

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Figure 4.1 (reference site) shows the presence of lipid alongside mitochondria. The lipids also were seen to be degraded by lysosome. Figure 5 shows a non contrasted image of a cluster of lipids attached to mitochondria, this combination of mitochondria and lipid structure hints towards a possible interaction between mitochondria and lipid for degradation of lipids. Eugene P. Kennedy and Albert Lehninger had already demonstrated in 1948 that enzymes of fatty acid oxidation in animal cells are located in the mitochondrial matrix. It is known that A-Methylacyl-CoA racemase, found in both mitochondria and peroxisomes, is required for the metabolism of isoprenoid compounds, e.g. cholesterol to bile acids, and other methyl branched lipids. So, this could well be happening in case of fish as well. Mitochondria could be well affected due to metal deposits and thus might not be functioning properly to produce enzymes for lipid oxidation. Also in the same image, faults and cuts on lipids could be seen where metal crystals had aggregated. This could be seen as the harsh physical effects of metal crystals on the cellular organelles. Definitely, metal precipitates seem to affect the cell organelles chemically as well as physically. Figure 6 shows glycogen filled lysosome and high amount of glycogen all around. It shows that there has been obstruction in the pathway of conversion of glycogen. The cell seems to be very active as a lot of lysosome and vesicles were seen. Again, an elongated and irregularly shaped mitochondria could be seen in the figure. In figure 7, lysosome degrading lipid and a lot of black metal crystals were seen. However, in fig. 6, lysosome seemed to have weaker membrane structure since the membrane lining looked very irregular compared to normal lysosome. It had been already shown that the lysosomal membrane stability of wrasse was impaired at the sites of PAHs and organic contamination (Einsporn and Koehler, 2007) so it seems metallic pollution results in same, but could be in lesser extent as PAH and organic contamination are harsh and more effective than simple metals like copper. The interior of the lysosomes (pH 4.8) is more acidic than the cytosol (pH 7). The lysosome single membrane stabilizes the low pH by + pumping in H from the cytosol, and also protects the cytosol and the rest of the cell,

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from the degradative enzymes within the lysosome. Fig. 8 clearly shows glycogen and ribosome, ribosome attached to endoplasmic reticulum. Glycogen was seen to be of two types. Both bigger than ribosome in size, and were stained differently. One was darkly stained electron dense granules while the other was lightly stained. Glycogen appeared as electron-dense rosettes, termed alpha particles. However, glycogen-rich and glycogen-poor liver is differentiated by no. of alpha particles rather than in their size. It is noticeable that some particles of glycogen seem to have combination of both light and dark areas. This could either be due to complex formation of glycogen with ribosome or it is also known that alpha glycogen contains beta particles within itself. And the alpha particles additionally contain various enzymatic proteins involved in the synthesis of glycogen and hence they are called glycosomes (Rybicka, 1996). The shape of black particles in this reference site image seemed to be different from an uncontrasted image from metal site (fig. 26). It is hard to distinguish between ribosomes and glycosomes because both types of organelles appear in U/Pb stained sections, as 20-30 nm particles. Also both could attach to ER membranes and cytoskeletal components (Hesketh and Pryme, 1991). Fig. 9 shows an uncontrasted image of lysosome degrading lipid at 12000×. Fig. 10 (12000×) shows bulged mitochondria in contrast to the normal mitochondria in liver tissues of reference site (Fig 2, 12000×). A huge difference in size could be seen between mitochondrias from polluted and unpolluted sites. In polluted site, mitochondria were considerably swelled with cristae at random positions. Again, we saw certain amount of granules like precipitates in the mitochondria. We propose the swelling of mitochondria is directly related to the effect of precipitates on the mitochondria. In figure 11, the mitochondria seemed to be damaged with almost no cristae. The mitochondria are greatly enlarged and are filled with concentric cristae, which is abnormal. Also, the mitochondrial matrix appeared to be inexistent. It is well known that

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mitochondria are sensitive to cellular stress and have a pivotal role in the initiation of programmed cell death. It could be proposed that the structural change in the mitochondria begins with the degradation of cristae due to metal pollution. In fig. 12 and fig. 14, dense metal deposits could be seen near bile canaliculi. The tissue sections were from copper polluted site. It is of specific observation that the density of the black deposits is around bile canaliculi and metal crystals could be seen attached to the grooves of bile canaliculus. This could well be the export and elimination pathway for these precipitates from the fish through bile canaliculi. There is less possibility for these deposits to be background staining or dirt particles because it can be seen clearly that it is not uniform throughout the tissue and localized in certain specific areas. If it had been the remains of staining procedure, it should well have been seen all around the tissue. Also, the sections were dead so there is no reason that the dirt, if it was, should be attached to the grooves of bile canaliculus instead of any other parts of tissue. But if these were metal deposits, it makes perfect sense they were being eliminated while the samples were taken and prepared for EM in 2001 and remained there frozen. Fig. 15 (see appendix I) shows the same at 12000×. Our evidence is strongly supported by Fig. 16 (see appendix I), 16.1(see appendix I) and 20 and 21(see appendix I). These were not stained before electron microscopy and were noncontrasted images but they still showed black particles on the canaliculus and intercellular space. Also evident was the fact that the cells in direct contact with bile canaliculus showed more particles than the secondary cells after them and the particles were polarized at one side of bile canaliculus, (see figure 12, 14, 15). In fig. 13 (see Appendix I); unlike in the middle of the cell, the lining of the tissue was filled with black deposits. It could be assumed that these were metal crystals but it couldn’t be confirmed whether it is copper or the residues of uranyl citrate and lead acetate, since this was a contrasted image stained with these two compounds. Numerous lipid droplets were seen and this section was from polluted site. Fig. 17 (see appendix I) and 18 shows black deposits passing through an intercellular

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space presumably. When closely observed, the black deposits are minute rectangular shaped crystalline structures with sharp edges. These prove that they are metallic crystals. Presence of abnormally accumulated metal crystals show detoxification function of liver has been impaired. Fig. 22 shows us the glycogen enclosed with lipid. Both dark and light granules of glycogen can be seen. It was interesting to see mitochondria blocking the way for glycogen to come out of the lipid droplet. It might well be that this is one of the ways through which glycogen make their way inside lipid and are later trapped. Similar case was seen in fig 24. Mitochondria squeezed between lipids, limiting the movement of glycogen. The occurrence of glycogen inclusions in the liver hints that the metal deposits could be causing biochemical stress in the fish from polluted areas. The accumulation of glycogen in hepatocytes could result in type IV glycogen storage disease (amylopectinosis) (Sherlock and Dooley, 1997; Peplow and Edmunds, 2005). Fig. 23 is a wonderful case showing the two types of glycogen, dark and lighter in color complexion. On liver, glycogen appears as electron dense rosettes, which are alpha particles so we assumed the darker particles to be alpha glycogen. The structural backbone of ER could be seen clearly with ribosomes. It could be inferred that glycogen particles could have also formed a stable complex with ribosomes. Figures 25 and 26 show lipid droplets in tissues from metal site at 12000 ×. Also evident are the lipids being degraded by lysosome selectively. Generally, Lysosomes were seen to degrade lipids with black metal deposits before others. In Cu-loaded animals there is overburden on biliary excretion pathways. It is typified by the sub-cellular localization of Cu in non-cytosolic fractions, especially lysosomes (Klaverkamp et al., 1991). This is clearly seen in fig 26, which is an uncontrasted image of wrasse liver exposed to metal site. To summarize, the study was successful to illustrate that various cell organelles could be affected due to physical and chemical toxicity of metal pollution

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especially copper. Very important organelles like the mitochondria which are the power house of cells were found to have irregular structures, excess of glycogen was seen and amount of lipid droplets were by far, in lot more amount than a normal cell should have. On the other hand, due to the small scale and time of this lab study, many observations could not be decided conclusively. In future, Autometallography should be performed at the light and electron microscope levels to provide information on the intracellular distribution of metals as well as evidence of different responses to metal accumulation. By checking the pH in and around lysosome, it could be found out whether they had weak membrane structure in real since a considerable variation from pH 4.8 means the proton pumps and chloride ion channels on the membrane to maintain the pH is not functioning well, which is related to damages in membrane of lysosome. The effects of structural differences in mitochondria should be studied thoroughly and quantitative analysis should be done on the unknown effects on ATP production due to loss of cristae seen on affected mitochondria.

Acknowledgements I would like to thank Ute Marx (Alfred Wegener Institute) for her technical assistance in electron microscopy and Sonja Einsporn, PhD. and Dr. Katja Broeg for their guidance and supervision during the experiments.

References: Broeg, K., Kaiser, W., Bahns, S., Koehler, A.(2007)The liver of wrasse-morphology and function as a mirror of point source chemical impact. Marine environmental research. 14th international symposium pollutant responsed in marine organisms. May,6-9 2007, Brazil CARATTINO MD, PERALTA S, PÉREZ-COLL C, NAAB F, BURLÓN A, KREINER AJ, PRELLER AF and FONOVICH DE SCHROEDER TM (2004) Effects of longterm exposure to Cu2+ and Cd2+ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: Possible role as biomarker for Cu2+ toxicity. Ecotoxicol. Environ. Saf. 57 311-318 C Strydom, C Robinson, E Pretorius, JM Whitcutt, J Marx, and MS Bornman (2006) The effect of selected metals on the central metabolic pathways in biology: A review. ISSN 0378-4738 = Water SA Vol. 32 No. 4

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Einsporn S. and Koehler A., (2007) Lysosomal changes in Wrasse and in blue mussel from differently polluted norwegian fjord sites (2007), Ecotoxicology

metal pollution controls in a smelter by using metallothionein and other biochemical responses in fish. In: M.C. Newman and A.W. McIntosh, Editors, Metal Ecotoxicology—Concepts and Applications, Lewis Publishers Ltd., Chelsea, MI, USA (1991), pp. 33–64

Grosell, M.H., Boëtius, I., Hansen, J.M. and Rosenkilde, P., 1996. Influence of pre-exposure to sublethal levels of copper on 64Cu uptake and distribution among tissues of the European eel (Anguilla anguilla). Comp. Biochem. Physiol. Part C 114, pp. 229–235

Marcogliese DJ, Brambilla LG, Gagne F, Gendron AD (2005) Joint effects of parasitism and pollution on oxidative stress biomarkers in yellow perch Perca flavescens. Diseases of aquatic organisms. Vol 63: 77-84

Grosell, M.H., Hogstrand, C. and Wood, C.M., 1997. Copper uptake and turnover in both Cu acclimated and non-acclimated rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 38, pp. 257–276. M.H., Hogstrand, C. and Wood, C.M., 1998. Renal Cu and Na excretion and hepatic Cu metabolism in both Cu acclimated and non acclimated rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 40, pp. 275–291. Handy D. R. (2003) Chronic effects of copper exposure versus endocrine toxicity: two sides of the same toxicological process? CBP. Part A 135 25-38. HULTBERG B, ANDERSSON A and ISAKSSON A (1998) Alterations of thiol metabolism in human cell lines induced by low amounts of copper, mercury or cadmium ions. Toxicol. 126 203-212. J E Hesketh and I F Pryme (1991) Interaction between mRNA, ribosomes and the cytoskeleton. Biochem. J. 277 (1–0) J.F. Klaverkamp, M.D. Dutton, H.S. Majewski, R.V. Hunt and L.J. Wesson, Evaluating the effectiveness of

Munshi J.S., Dutta H.M. (1996) Fish Morphology: Horizon of New Research, CRC press Peplow D, R Edmonds, 2005. The effects of mine waste contamination at multiple levels of biological organization. Ecological Engineering, 2005 (Vol. 24) (No. 1/2) Rybicka KK (1996) Glycosomes- the organelles of glycogen metabolism. Tissire Cell 28:253 Sherlock S. and Dooley J.S. Diseases of the Liver and Biliary System, 10th ed. Blackwell science, Oxford Sies H. (1994) Oxidative stress: oxidants and antioxidants, Experimental Physiology 82.2 pp 291-295 Vosloo A., Aardt van W.J., Mienie, L.J. (2002) Sublethal effects of copper on the freshwater crab Potamonautes. Comp. Biochem and Physiology 695-702

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APPENDIX 1, Lab rotation III and IV

Figures included in the discussion

Fig 1.2 Overview of the liver tissue from station 3 (reference site) at 3000 × magnification

Fig 15. Bile canaliculi with black crystal deposits at 12000× magnification from metal site at station 2.

Fig 13. The bile duct with a black metal lining throughout edges at 3000× magnification, from metal site.

Fig 16. Bile canaliculi with black crystal deposits at 20000× magnification from station 2 (uncontrasted image)

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Fig 16.1. Metal deposits in the bile canaliculi at 12000× magnification from station 2 (uncontrasted image)

Fig 21. Assumed metal crystals from station 2 at 12000× magnification (non contrasted image)

Fig 17. Black deposits along an intracellular space at 12000 × magnification from station 2

Fig 23. Glycogen around lipid and ER at 12000 × magnification (station 2)

L represents lipid while G shows glycogen granules.

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Fig 24. Glycogen around lipid and at 20000 × magnification (station 2)

Fig26. Lipid filled with metal crystals being degraded by lysosome at 12000× magnification from station 2 (non contrasted image)

Fig 25. Lipids being degraded by lysosome in liver tissues at 12000× magnification from station 2 (uncontrasted image- metal site)

Fig 4. EM of Elongated mitochondria from station 2 at 12000×.

Fig. 4 shows a section of liver of Wrasse which was collected from station 2- the metal polluted sites. Unusually long, stretched mitochondria were found. A lot of glycogen was seen at most of the places.

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Fig 4.1. Lysosome in liver tissues degrading lipids from station 3 at 12000 ×

Fig 6. Mitochondria and lysosome from station 2 at 12000×

Fig 5. EM of Mitochondria in an uncontrasted image, from station 2 at magnification 12000× magnification

Fig7. Lysosome filled with black deposits from station 2.

Mitochondria can be seen squeezed between lipid droplets. Faults on lipids could also be seen where black deposits had accumulated.

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Fig 8. Ribosome, glycogen from liver tissues from station 3 at 20000 × magnification

Fig9. Lysosome degrading lipid in uncontrasted TM image from station 3 at 12000 × magnification

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Fig 10. An uncontrasted image of mitochondria and lipid in liver tissues from metal site at 12000 × magnification