Dietary Selenium reduces the toxic effect of Mercury on different organs (Brain, Gills, Kidney and Liver) of Rohu fish (Labeo rohita)

Because of improbably high partiality with antioxidant selenium to mercury, selenium isolates mercury and reduces its biological handiness to organisms. The present study investigates the effect of mercury (Hg) and selenium (Se) on the fingerling of Labeo rohita. The various sub-lethal concentrations of Hg i.e. 0.125μg/g, 0.250μg/g, and 0.500μg/g was used and incorporated in diet with a commercial diet (fish meal 40%, soya bean 33%, 3 % of vitamins premix, mineral premix and oil each and rice polish 18%). To understand how selenium reduces the toxic effect of mercury, fingerlings were exposed with 6ug/g Se singly and combined with doses of Hg. The effect of these heavy metals was observed after 24, 48, 72, and 96 hours in different organs of (Brain, Gills, Kidney and Liver) of Labeo rohita. The organs of exposed rohu showed significant changes in their microscopic anatomy in comparison to control. Prominent changes were observed in Hg treatments and found embody shrinkage of capillary, and dilation of the hollow lumen. In addition to the aforementioned changes vacuolation, peeling, hydropic swelling, and hyaline degeneration of hollow epithelium were also observed. Cysts and hamorrhage developed additionally seem in the organs of fish. Length of exposure seems to own a profound impact on organs because the increase in length of exposure enhanced the severity of histopathological damages. However, combined doses of metals cause reduction in the toxicity of mercury results in decrease damage in the shrinkage of capillary and dilation of the hollow lumen. During histological study vacuolation, hydropic swelling, and hyaline degeneration of hollow epithelium were also reduced. In Hg, the lethal concentration (LC50) was 0.374ug/g while when Hg and Se were combined, LC50 drops to 0.491ug/g. Results of this study suggest that exposure to Se helps to scale back the impact of mercury on mortality and different organs of Labeo rohita fingerling. Copyright © 2021 The Authors. Production and hosting by Department of Life Sciences, University of Management and Technology is licensed under a Creative Commons Attribution 4.0 International License


INTRODUCTION
Fish shows almost sixteen percent of the universal population's intake of animal protein and six percent of all protein used. Fish is a healthy and fair diet because it contains a high level of omega-3 fatty acids and macromolecules that the physical body needs to remain energetic and functioning. Fish culture provides an ample opportunity to produce wonderful quality food for utilization by masses which is free from contagion [1]. Due to violation of an existing set of rules and regulations there is a continuous rise in the concentration of contagions in the freshwaters because of household squanders, bug sprays and herbicides, nourishment handling waste, toxins domesticated animals tasks, unstable natural mixes, substantial metals, concoction waste and others [2]. Among natural contagions, substantial metals are a matter of grave concern due to their potential dangerous impact and capability to bio-aggregate in aquatic environments [3]. The pollution inside the water ambiance with substantial metals has become an overall issue from the previous few years as a result of their indestructible and a deadly impact on living organisms [4]. Due to its possible hazards to higher biological process levels, mercury has attracted greater attention. Methyl mercury is predominant in fish organs and is biologically available in greater concentrations than inorganic Hg. The high degree of mercury (Hg) in aquatic living beings, primarily fish, correlates with nursing environmental and human concern [5]. Hg reflection in fish is inclined by fish age [6], Hg concentrations in water food contagion, chemical, organic and objective processes in water environment, and seasonal alterations [7,8]. In fish, as higher vertebrates, the excretory organ plays out an important role in balancing the water and other solutions ultimately maintaining the internal environment stablility. The excretory organ secretes nitrogen-containing waste products from digestion, for instance, carbamide and creatinine. Following exposure of fish to compounds like pesticides, histological changes are visible in animal tissue and blood [9]. The fish is very sensible to the external environment and sensible measures should be taken to give compound checking of the getting from its usage.. Histopathological studies can be used for watching the effects of maritime pollutions particularly considerable metals [10]. The toxic nature of total mercury (Hg) and methylmercury(MeHg) was observed via histopathological studies of living things which identify the degree of cell damage. [9]. Selenium is a basic element for living organisms and has been seemed to overcome the toxic effects of mercury and other heavy metals like arsenic, cadmium, and probably lead [11]. Selenium is incorporated into the twenty initial amino corrosive L-selenocysteine that could be a constituent of selenoproteins [12]. Selenium sensitizes the gathering and harmfulness of mercury in oceanic living beings in a very intricate manner. In some marine well evolved creatures, demethylation of the alkyl group of mercury within the liver prompts arrangement of insoluble mercury selenium giving ascend to a 1:1 molar proportion Mercury: Selenium [13]. It has been projected that selenium detoxifies MeHg by shaping edifices containing the two elements are present at equimolar proportion. It has been well documented that metallic element mercury selenide (HgSe) has been found within the liver of marine well-evolved creatures and seabirds. This element is believed to be a dormant consequence of the detoxification procedure in these marine creatures [14]. The objective of the present study was to determine the Hg toxicity and its effect on Labeo rohita species and the combined effect of Hg and Se as well. The study helps how the L. rohita species respond to different level of heavy metal (Hg).

Sample Collection and Maintenance
The fingerlings of Labeo rohita, were obtained from a fish farm settled some 40 kilometres from the city (latitude 31˚58 N, longitude 74˚13'E), on the North certain GT road, Manawa.
The collected fish were raised in a polyculture of major carps under well-established procedures and practices. The fishes were transported in air-packed polythene bags from the farm. The fish were then given a shower in 0.1% methylene blue before moving them to the aquaria. This prophylactic action was done to keep the fish away from any infection from bacteria that can badly influence fish. Prophylaxis was followed by the transfer of fish in neat aquaria supplied with dechlorinated water. Artificial aeration was provided to the fish [15]. The fish were fed ad libitum with commercial fish feed.

Experimental Plan
After acclimatization, fish were placed in the glass aquariums. Glass aquarium was filled with 15 litters of water about ¾ of the capacity of each aquarium. The water was replaced with fresh clean water once a day. Fish was weighed in gm and measured in cm and then randomly distributed in each aquarium 25 fishes per aquarium. Fish was then exposed to three sub-lethal concentrations of mercuric chloride i.e. 0.125µg/g, 0.250µg/g, and 0.500µg/g respectively with a commercial diet for 24, 48, 72, and 96 hours. Then commercial diet was mixed with both HgCl2 and Se i.e. (0.125+6 µg/g, 0.250+6 µg/g and 0.500+6 µg/g). The test concentrations were restored after every 24 hours by cleaning and replacement of dechlorinated water. The above protocol was repeated after every 24 hours. The fish of each aquarium was fed thrice a day. After each exposure, the fish specimens were measured, weighed, and dissected to obtain organs (Brain, Kidney, Gills and Liver) for histological processes. The fish was anesthetized immediately to reduce stress. The Clove oil (National Chemicals, Pakistan) was use as anaesthesia [16]. The fish was put in the solution for 3 to 5 minutes nonetheless, time period of anaesthesia could be changed according to the age and size of the fish.

Dissection and Preparation of Organs
The lower abdomen of the fish was cut from the posterior to the anterior end by a sharp surgical scalpel and scissors. Kidney tissue was removed with small scissors and freed from any extraneous tissue. The kidney of fish was removed and weighed on an electric balance. Examination of the microanatomy of tissue was started with surgery of animal Brain, Kidney, Gills and Liver. The tissues were preserved in one hundred millilitres of 100% unbiased buffered formol which is four percent aldehyde in phosphate-buffered brackish mixture until processed for histological examination. The embedding was done to pieces of approximately 1 cm 3 before loading the cassettes for histological processing. The sample-loaded cassettes were passed for dehydration of the tissues in the following pattern of alcohol (30% → 50% → 70% → 90%). The sample-loaded cassettes were passed for dehydration of the tissues in following pattern of alcohol (30% → 50% → 70% → 90%). The samples were cleared in two applications of xylene followed by applications of molten wax (58˚C) for impregnation. The resultant tissues were then placed in a very mold containing additional liquefied wax (embedded) and allowed to cool down until wax hardened. Tissues were then removed from the cassettes and molded with molten paraffin wax at 58˚C melting point, for making blocks. The blocks were attached to the cassettes, trimmed, frozen and sectioned [17].

Microtome and Staining
The tissues were cut at 3-4 micrometer thickness by a rotating mechanized microtome (Shandon, Thermo, CD-2235) fixed with microtome blade. The strips of segments were shaped and extended in warm water. The slides were mounted with egg albumen, following the placement of tissues on the slide. Slides were then placed under warm water letting the wax to melt and fix tissue on the slide.
Hematoxylin and fluorescent dye (Hematoxylin & Eosin Stain) is the most typically used light weight mark in microscopic anatomy & histology. Hematoxylin, a dyestuff, mark nucleus indigo as a result of its resemblance to nucleic acids within the cell centre; eosin, a bitter stain, dyes the protoplasm crimson. The mandatory portion was photographed by using a digital camera mounted on a Leica DM-500 microscope.

Estimation of lethal concentration (LC50)
Keeping in view the scope of the study it was necessary to estimate LC50 for the selection of different dosages of mercury for further initiatives in these studies. LC50 test (24,48,72, and 96 hours) based on fish mortalities when fishes were exposed to known toxicant concentration sequence and then its comparison with parallel control that did not receive any toxicant. During this cycle, the fish was not fed [18].
Seven groups of fish were exposed to increased concentrations of HgCl2. An appropriate quantity of HgCl2 was added to the required lethal concentration based on the understanding that there would be no mortality up to 0.125 µg/g. But almost all the fishes died within 96 hours at 0.500 µg/g exposure to Hg [19].

Statistical analyses
Statistical analysis was performed by using SPSS (Ver. 19). LC50 was detected by probit analysis using Statplus 5.

Results
The percentage survival rate and lethal concentration were assessed. No mortality was observed in the control group but due to increases in Hg survival rate decreases from 24 h to 96 h. When Se had added in all Hg concentrations the survival rate increases when Hg concentration increases from 24 h to 96 h. LC50 and LC90 were also high in Hg groups but when Se was added lethal concentration increases. This shows that the addition of Se reduces the effect of Hg in feed (Table 1). In the brain, the cell was observed with no change in all-time duration in control while granular cell loss, purkinje cells started aggregation and neutrophil cells loss were observed in 24 h exposure time. In 48 h, the presence of neural cells, pyramidal cells, and nissl substances were observed. In 72 h, delicate chronic changes were discovered within the neural cells, Gangrene of neurons, living hydrops, and congestion of neural cells were noticed. In 92 h, initiation of the degeneration of neural cells and protoplasm vacuolation occurs. The severity was observed in brains cell when Hg concentration increases. When Se was added with Hg, improvement was observed in all brains cell (Fig 1a). In the kidney, no change was observed in the tissue after 24, 48, 72, and 92 h with any treatment. In 24 h, slight inflation was observed in the area between the capillary vessel and capsule glomeruli while in 48 h, shrinkage was observed. After 72 h, numerous degenerative changes were observed in some areas i.e. tubular epithelium with desquamation in tubes, Hyaline degeneration, hydropic swelling, vacuolization, and necrosis. In 92 h, shrinkage of the glomerulus, shortening and narrowing of lumen space and reduction of renal cell number count. But when both metals were exposed to fish, some minor changes were observed but the improvement was observed in low Hg concentration but in high concentration Hg cause extreme shrinkage of capillary, capillary and membrane was conjointly exaggerated, Gangrene and pycnosis was ascertained in animal tissue lining, epithelium of kidney tubules were extremely denatured (Fig 1b). In gills, the tissue did not display any change after 24, 48, 72 and 92 hours of zero dose. In 24 h, exposure the changes in the gill histology was not very conspicuous. In 48 h, Eubacteria cholerae showed deformities within the gills. The gill filaments degenerated and sphacelus. In 72 h, cellular damage was observed within the gills in conditions of animal tissue propagation. In 92 h, gills abnormalities increased which incorporated dysplasia of animal tissue cells, a mixture of derivative lamellae, enlivening of the lamellar epithelial hankie, blood obstruction, proliferation of animal tissue cells of primary and secondary lamellae, and sphacelus. The increased effects on these cells were observed with respect to increase in Hg concentrations. When Hg was mixed with Se, cell growth was observed normal in low Hg concentrations. Disruption of cell damage was observed in high concentrations but not as much as Hg (Fig 1c). In the liver, no change was observed in the tissue after 24, 48, 72, and 92 hours in the control group. In 24 h, few mirror changes were revealed like degradation of viscous parenchyma cells and viscous cells did not create distinct lobules. In 48 h, there was structural harm to the central vein, busted and irregular viscous plate, granulation in living substance, necrosis, vacuolization, and disruption of hepatocytes. In 72 h, deterioration, mortification of the hepatocytes, vacuoles degenerated within the hepatocytes, occlusion formation in central veins, and dilation occurred. In 92 h, tissue displayed several alterations such as mild necrosis, pyknosis, cytoplasmic degeneration, and infiltration of leukocytes. In high concentrations of Hg, extreme alterations were observed in nuclear structures like nuclear degradation. The structure of the liver was completely denatured; the cytoplasm appeared degraded with the appearance of adverse necrosis and pyknosis. When Se was mixed with Hg, improvements were observed in cells with respect to Hg alone (Fig 1d).

DISCUSSION
The present study was very useful in understanding how to reduce the mercury level in the organs of fish (Labeo rohita) by the use of selenium.. During the investigation, mercury was combined with selenium and showed minor alterations in organs as compared to those groups which were solely exposed to mercury.
It has been conjointly ascertained that if the concentration of mercury is incredibly high within the tissue, it should cause severe structural harm [20]. The current study revealed that Hg change the structure of the brain, gills, kidney, and liver even when they are in low concentrations but denaturation occurs when they are in high quantity. Similar findings with dilation, oedema, and enlarged nuclei of nephritic tubules were additionally reported within the excretory organ of Mugil auratus [21]. [22] also studied that toxicity of HgCl2 in fish excretory organ varies from slight disruption of cannular cells to swelling in cells with delicate to high dose in major carps. They also reported a similar effect of HgCl2 on the liver of C. mrigala [23] also, study the effect of Hg on Danio rerio and observed a negative impact on growth and survival. All the fishes died within 96 hours at high exposure of Hg [19]. The LC50 determines the population died in the presence of xenobiotics. Our study shows that LC50 of Hg is 0.374 ug/g and LC90 is 0.704 ug/g. [24] have reported that LC50 of Cd for zebrafish (Danio rerio) after 96 h exposure was 9.68 mg/L. So far, 96 h LC50 of Cd showed considerable differences among fish species. [28] studied that when total Hg and MeHg levels increases, 40 % accumulation increases in liver of Brosme brosme as compared to normal fish. [29] reported that when Hg exposed to fish for 96 h, it cause tissue anomalies like blood congestion in gills, swelling of liver, lesion in kidney, vacuolation and exfolitation. Many studies were conducted to estimate the effect of Hg in different organs of fish [30] i.e. gills [31][32][33], liver [34][35][36][37], kidney [38][39][40], and brain [41,42].
5. The present study was to observe the effect of Se on Hg toxicity in Rahu fingerling. [25] have reported that the kidney of Trichomycterus brasiliensis treated with selenium was inappropriate shape, there was an increase in the number of tubule cells which ultimately reshaped normally. The Se reduces the negative impact on Danio rerio in the presence of Hg [23]. [26] Studied that Se improves the bioavailability and intercept the selenium-dependent enzymes that's why Se can use to minimize the toxicity of Hg. [27] also studied that high the concentration of Hg weekly food intake exceeds in Hippoglossus hippoglossus and cause severe damage but Se reduce the toxic effect of MeHg. Our study indicated that Se when mixed with Hg reduces the LC50 and LC90 up to 0.491 and 0.798, respectively. This shows that in the presence of Se more Hg concentration was required to eliminate 50% population of Rohu fingerling. [43] studied the ratio of Hg and Se, in commercial diet of fish was very crucial in safety of ecosystem. Similar studied were conducted to overcome the Hg toxicity with help of Se [44][45][46][47][48][49][50] 6. CONCLUSION In view of present findings, it is concluded that mercury at sub-lethal concentrations caused considerable histological damages in different organs in L. rohita. Mercury entered in fish caused severe injury to the cellular structural integrity in organs of L. rohita. However, once the fish were exposed to Se the tissue recovers and damage to organ cells decreased gradually. Our findings reveal that selenium plays an important role in reducing the toxic metal in fishes and can play an important role in mending this prevalent and dominant problem of the fisheries sector. In future, more investigation is required to understand the importance of selenium in fisheries sector. Current histopathological investigations suggest that selenium can help in reducing the toxic level of mercury and other heavy metals. Nonetheless, further studies need to be undertaken to clarify exactly the toxicity of mercury to fish and counteracting effects of selenium in neutralizing the toxicity of heavy metals.