Toxopathological Studies on the Effect of Formalin …. Appl. Environ. Biol...The present study aimed to investigate the effect of therapeutic doses of formalin and copper sulphate

J. Appl. Environ. Biol. Sci., 3(6)7-20, 2013

© 2013, TextRoad Publication

ISSN: 2090-4215 Journal of Applied Environmental

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Corresponding Author: Dr. Wael G. Nouh, Department of pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Tel: 00201068384900 Fax: 0020552283683

Email: [email protected]

Toxopathological Studies on the Effect of Formalin and Copper Sulphate in Tilapia as A Commonly Used Disinfectant in Aquaculture

Wael G Nouh1 and Ahmed G Selim2

1Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Egypt

2Consultant of Clinical Pathology,Vet. Clinic, Faculty of Veterinary Medicine, Zagazig University, Egypt

ABSTRACT

The effect of specific/therapeutic dose of disinfectants (formalin and copper sulphate) was studied on 180 apparently healthy Nile tilapia through hematological, biochemical, histopathological and electron microscopic examinations. Tilapia exposed to formalin (25mg/l, gp 2), clinically, no mortality was detected throughout the period of the treatment. During the first three days post treatment, the exposed Tilapia nilotica showed accelerated opercular beats while grossly, a copious mucus secretion covering the skin and gills with congested liver, kidneys and spleen were seen. Histopathologically, one days post-treatment, congestion was evident in all examined organs. Five and ten days post-treatment, degeneration, leukocytic infiltration and focal depletion in the lymphoid tissue were noticed. Tilapia exposed to copper sulfate (2mg/l, gp 3), clinically, the mortality rate was 5% throughout the period of treatment and the exposed Tilapia nilotica showed unbalanced fast movement and gasping during the first three days post treatment. Grossly, the exposed tilapia showed numerous hemorrhagic areas on the body along the period. Histopathologically, one and five days post-treatment, the gills showed telangiectasis and focal hyperplasia in the secondary gill lamellae and the other organs were congested and suffered muscular hyaline degeneration, vacuolar degeneration in the hepatocytes, tubular nephrosis and proliferation of splenic MMC.Ten days post-treatment, the gills exhibited hyperplasia in the secondary lamellae, the muscles and liver showed degeneration and proliferation of melanomacrophages while the kidney showed vacuolation in the renal epithelium with focal depletion of hematopoietic tissue. The ultrastuructural changes in the hepatocytes and renal epithelium of tilapia exposed to formalin and copper sulphate were mentioned and fully discussed. It could be concluded that, although therapeutic dose of disinfections is efficient against many fish

infection, their use should be done under strict regulation to avoid its pathological side effect KEYWORDS:Pathology, formalin, copper sulphate, tilapia, aquaculture.

INTRODUCTION

Fish are persistently bathed in potential pathogens, including bacteria, fungi and parasites. Among

them, parasitic diseases in fish are most frequently caused by protozoa. Most protozoan infections are comparatively easy to control using common fishery chemicals, such as copper sulfate, formalin or potassium permanganate, methylene blue, brilliant green, parasite green, malachite green and trichlorforn [1]. In the aquaculture industry, copper sulfate is used as an algaecide and as a therapeutic chemical for various ectoparasitic and bacterial infections [2, 3,]. Copper sulfate also inhibits growth of bacteria such as Salmonella spp., Pasteurella spp., Vibrio spp., Streptococcus spp., Aeromonas spp., Pseudomonas spp. and Edwardsiella spp. [2]. Copper ions are quite toxic to fish at various functional levels when environmental concentrations are increased [4].Formaldehyde is used in aquaculture to control bacterial, fungal and parasitic diseases, demonstrating efficacy in short term baths [5]. Formalin efficient to treat white spot disease (ichthyophthiriasis) caused by Ichthyophthiriusmultifiliis[6], and effective for the control of ectoparasitic diseases in Oreochromis niloticus[7].The TLm24h value (LC50) for copper sulfate is 4.0 ppm in mirror carp (Cyprinuscarpio), 10.0 ppm in goldfish, 4.4 ppm in rainbow trout and 4.0 ppm in flounder (Paralichthysolivaceus), the TLm24h value (LC50) in the guppy was 1.17 ppm [8]. Formalin was more toxic to blue spottedcorydora LC50 (I) 50.75 mg.L-1 than to trairão (H. lacerdae) larvae with LC50 (I) of 2.02 Ml.L-1.However, the formaldehyde is a mutagen and carcinogen even at low concentrations and presented toxicity in a variety of organisms. In aquatic organisms, deleterious effects were observed on the concentration of 1 mg.L-1 for fish and 5 mg.L-1

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Nouh and Selim, 2013

for micro crustaceans and algae [8, 9]. The effects of these products in fish can be manifested at various levels of biological organization, including physiological dysfunction, structural changes in organs and tissues and behavioral changes that lead to impaired growth and reproduction[10].Fish has a great potential to serve as sensitive indicators, signaling exposure and understanding the toxic mechanisms of stressors in aquatic ecosystems. The implications of interrelated toxic effects of disinfectants on the survival, histology, physiology, biochemical constituents and behavior are less frequently studied.

The present study aimed to investigate the effect of therapeutic doses of formalin and copper sulphate on Oreochromis niloticus with special reference to gross, histopathological, ultra structural and hematological as well as the biochemical alterations .

MATERIALS AND METHODS

Experimental fish: A total number of 180 Tilapia nilotica (Oreochremis niloticus) were collected alive from Abbassa

fish farms, Abou-Hammad, Sharkia. They were of both sexes with body weight of 57.6 ± 5 gm and apparently healthy. They transport in a sterile water tanks to the lab and kept for two weeks in nine glass aquaria (70 × 60 × 50 cm) under observation for acclimatization. The aquaria were continuously supplied with chlorine free water and the water was renewed daily. The water quality monitored had a pH of 5.2 ± 0.26, dissolved oxygen showed values of 6.94 ± 0.37 mg.L-1, the temperature presented values of 27. 1 ± 0.11° C, the conductivity 318.42 ± 16.93mS.cm-1 and ammonia of 0.011 mg.L-1, and none of these variables were restricted to the maintenance of fishes in this condition, remaining within the range recommended by [11].The temperature was adjusted thermostatically by using heater (type CMI, Germany) and continuous aeration using electric air pumping compressors.The fish were fed on a balanced diet along the period of the experiment.

Experimental design:

A total of 180 apparently healthy Nile tilapia were divided into 3 equal groups (60 fish/ group). Each group was subdivided into 3 replicates, each replicate fish was allocated in glass aquarium (70 × 60 × 50 cm). The first group served as control without any disinfectant. The second and the third groups exposed to specific/therapeutic dose of disinfectant as a definite treatment where the second group exposed to formalin (25mg/l) and the third group exposed to (2mg/l) copper sulfate concentration (total alkalinity/100). All experimentally fish were subjected for hematological, biochemical, histopathological and electron microscopic examination at 1, 5 and10 day post exposure to each disinfectant.

Clinical signs and gross lesions:

Clinical sings were monitored throughout the study. Fish were observed for morbidity and mortality at least twice daily. At necropsy, the gross lesions were recorded in gills, liver, kidney, spleen and muscle of experimented fish.

Hematological and serum biochemical parameters:

Two blood samples were collected from the fish groups at 1, 5 and 10 days from the beginning of the experiment (FBE) by cutting the tail, each blood sample was divided into two portion. The first portion (1 ml) was collected in vacutainer tubes containing EDTA as anticoagulant for hematological studies. Erythrocytes and leukocytes counts were carried out by standard clinical method [12, 13]. Hematocrit (PCV) value was measured [14], while hemoglobin concentration was performed [15]. The second portion was collected in a plain centrifuge tubes and serum was separated and used for determine the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), the levels of total protein (TP), albumin (Alb), blood urea nitrogen (BUN)creatinine (CR) and glucose using anAuto Clinical Analyzer, Hitachi Model 7150 (HitachiLtd., Tokyo, Japan). Histopathological technique:

Tissue specimens were taken from the gills, liver, kidneys, spleen and muscle of experimented fish (groups 1 & 2&3)at 1, 5 and 10 days of experiment and fixed in 10% phosphate buffer formalin. Processed routinely and blocked in paraffin then, five micron thick paraffin sections were prepared and stained with hematoxylin and eosin, (H&-E) [16]. Electron microscopy:

Specimens from the liver and kidneys were fixed in 6.25 % cacodylate buffer gluteraldehyd followed by 1% osmium tetraoxide.After dehydration, the specimens were embedded in poly-ethylene

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capsules containing the embedding mixture (Epon mixture and hardener ).Ultra-thin sections were prepared and stained by uranyl acetate and lead citrate [17]. Statistical analyses:

The obtained data were statistically analyzed by t-test using SAS statistical software computer program [18].

* Significant (P < 0.05). ** Highly Significant (P < 0.01).

RESULTS

Pathological findings:- Group 1(control): Showed no apparent behavioral or pathological changes. Group 2(formalin treated):

Clinically, the exposed Tilapia nilotica showed accelerated opercular beats during the first three day post treatment followed by dark skin and impairment of swimming and sinking of the fish to the bottom of the aquarium were evident till the end of the experiment. No mortality was detected throughout the period of the treatment.

Grossly, the experimental fish showed copious mucus secretion covering the skin and gills, congested liver, Kidneys and spleen during the first three days post treatment, latter on the liver and kidneys were pale in color.

Histopathologically, one day post-treatment the gills revealed congestion and hyperplasia in the epithelium of the secondary lamellae[Fig.1]. The muscles exhibited intermuscular edema and congestion. The hepatopancreas showed swollen and vacuolated hepatocytes with basophilic cytoplasm and congestion of sinusoids[Fig.2]. The posterior kidneys and spleen showed congestion, edema with focal activation of the lymphoid follicles and proliferation of MMC [Fig.3].

Five days post-treatment, the gills revealed hyperplasia in the epithelium of the secondary lamellae with mononuclear leukocytes in the gill arch and lamellae. The muscles showed edema and suffered hyaline degeneration [Fig.4]. The hepatopancreas showed aggregation of MMCs, vacuolar degeneration. The posterior kidneys showed hyaline degeneration and epithelial cast with vacuolization of renal tubular epithelium. In the spleen, edema in the subcapsular area was seen together with focal depletion in the lymphoid tissue and atrophy in some melanomacrophage centers.

Ten days post-treatment, the gills showed focal hyperplasia in the epithelium of the secondary lamellae. Interlamellar spaces of the primary lamellae were infiltrated with mononuclear leukocytes. The muscles exhibited intermuscular edema and focal hyaline degeneration in the muscle bundles. The liver showed focal area of vacuolation in the hepatopancreas. The kidneys showed focal tubular nephrosis of renal tubules[Fig.5]. In the spleen, focal depletion in the lymphoid tissue was noticed.

Electron microscopic findings: The hepatocytes one day post treatment, revealed swelling of the mitochondria with loss of the

matrix and cristae [Fig.6]while 5 day showed dilatation as well as vesiculation of RER cisternae and decrease in glycogen granules were seen. By ten day of treatment, the nuclear size was reduced and the heterochromatin was condensed with decrease of glycogen granules and increase in fat globules [Fig.7].

The kidneys, one day post treatment, revealed marked increase in the mitochondria of the tubular epithelium with condensation of the nuclear heterochromatin while 5 day showed mitochondrial degeneration with loss of the matrix and cristae as well as fragmentation and vesiculation of rough endoplasmic reticulum cisternae. By ten day of treatment small vacuoles were noticed in the cytoplasm with loss of peroxisomes, ribosome and condensation of nuclear chromatin[Fig.8]. Group 3 (Copper sulphate treated):

Clinically, the exposed Tilapia nilotica showed unbalanced fast movement and gasping during the first three days post treatment. The activity of fish was reduced and skin darkening with loss reflex action were seen till the end of the experiment. The mortality rate was 5% throughout the period of treatment.

Grossly, the exposed Tilapia nilotica showed numerous hemorrhagic areas on the body along the period of treatment. The gills appeared pale with slight erosion on the free portion. Paleness of the liver and distention of gallbladder were also noticed. The kidneys were slightly enlarged and congested.

Histopathologically, one and five days post-treatment,the gills showed telangiectasis and focal hyperplasia in the secondary gill lamellae[Fig.9]. The muscles showed congestion and hyaline

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degeneration. The hepatopancreas revealed vacuolar degeneration in the hepatocytes, congestion and perivascular melanomacrophages were evident [Fig.10]. The kidneys showed congestion of intertubular capillaries and perivascular edema and tubular nephrosis. In the spleen, congestion and Proliferation of MMCwere observed[Fig.11].

Ten days post-treatment, the gills exhibited hyperplasia in the secondary lamellae.The muscles suffered intermuscular edema and focal hyaline degeneration and melanomacrophages were seen among the muscle bundles[Fig.12]. The liver showed edema, hyperplasia in the bile ducts and focal aggregations of melanomacrophages. The kidney showed vacuolation in the renal epithelium with focal depletion of hematopoietic tissue.

Electron microscopic findings: The hepatocytes one day post treatment, revealed mild vesiculation of SER & RER with

mitochondrial swelling while 5 day showed increase in the lipid droplets. Lysosomal activities was evident with marked swelling and vesiculation of RER and loss of some ribosomes[Fig.13]. By ten day of treatment, mitochondrial swelling was evident in addition to condensation of nuclear heterochromatin. Marked decrease in glycogen together with increase lipid droplets, vacuolation and cavitation of cytoplasm were evident[Fig.14].

The kidneys one day post treatment revealed mitochondrial degeneration in the tubular epithelium with partial to complete loss of the matrix and cristae while 5 day showed dilatation and vesiculation of rough endoplasmic reticulum cisternae that changed into circular arrays. By ten day of treatment numerous fat globules were noticed in the cytoplasm with loss of peroxisomes, ribosome and glycogen[Fig.15].

Fig.1: Gill, 1 day post-treatment of formalin, showing congestion and hyperplasia in the epithelium of the secondary lamellae . H & E stain x 250.

Fig.2: Hepatopancreas, 1 day post-treatment of formalin, showing swollen and vacuolated hepatocytes

with basophilic cytoplasm and congestion of sinusoids.H & E stain x 250.

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Fig. 3: Spleen, 1 day post-treatment of formalin, showing edema, focal activation of the lymphoid follicles and proliferation of MMC . H & E stain x100.

Fig. 4: Muscles, 5 days post-treatment of formalin, showing edema and suffered hyaline degeneration.

H & E stain x 250.

Fig. 5:Kidney, 10 day post-treatment of formalin, showing focal tubular nephrosis ofrenal tubules. H & E stain x250.

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Fig. 6: Hepatocytes, one day post treatment of formalin, showing swelling of the mitochondria with loss of the matrix and cristae. Uranyl acetate and lead citrate, x 25000.

Fig. 7: Hepatocytes, ten day post- treatment of formalin, showing reduced nuclear size with condensed

heterochromatin, decrease of glycogen granules and increase of fat globules. Uranyl acetate and lead citrate, x 25000.

Fig. 8: Renal epithelium, ten day post- treatment of formalin, showing small vacuoles were noticed

inthe cytoplasm with loss of peroxisomes, ribosome and condensation of nuclear chromatin. Uranyl acetate and lead citrate, x 35000.

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Fig. 9: Gill, 5 days post-treatment of Copper sulphate, showing telangiectasis and focalhyperplasia in

the secondary gill lamellae .H & E stain x 250.

Fig. 10:Hepatopancreas, 5 days post-treatment of Copper sulphate, showing vacuolar degeneration in

the hepatocytes and perivascular melanomacrophages. H & E stain x 250.

Fig. 11: Spleen, 5 days post-treatment of Copper sulphate, showing congestion and proliferation of

MMC. H & E stain x100.

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Fig. 12:Muscles, 10 days post-treatment of Copper sulphate, showing intermuscular edema and focal

hyaline degeneration and melanomacrophages. H & E stain x250

Fig. 13: Hepatocytes, 5 day post-treatment of Copper sulphate, showing lysosomal activities with

marked swelling and vesiculation of RER and loss of some ribosomes. Uranyl acetate and lead citrate, x 35000.

Fig. 14: Hepatocytes, ten day post-treatment of Copper sulphate, showing mitochondrial swelling,

decrease in glycogen together with vacuolation and cavitation of cytoplasm. Uranyl acetate and lead citrate, x 25000.

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Fig. 15: Renal epithelium, ten day post-treatment of Copper sulphate, showing numerous fat globules in the cytoplasm with loss of peroxisomes, ribosome and glycogen. Uranyl acetate and lead citrate, x 35000.

Haematobiochemical findings: Exposure of fish to formalin and copper sulphate affect hemogram and some biochemical profiles,

such changes were statistically analyzed and tabulated (Tables1and 2).

Table (1): Some hematological parameters of Oreochromis niloticus after treatment with formalin (25mg/l) and copper sulphate (2mg/l) for 10 days in comparison to the control

(Mean ± SE). Substance Time of

sampling RBCs

×106/µl Hb

gm/dl PCV

% WBCs ×103/µl

Formalin Control 2.58 0.10

9.49 0.40

26.30 1.11

26.20 1.12

1day * 2.20 0.13

** 6.20 0.37

** 20.00 1.20

* 21.41 1.39

5 day 2.32 0.12

8.50 .36

25.50 1.03

25.16 1.21

10 day 2.43 0.13

9.28 0.51

26.10 .44

25.30 1.52

Copper sulphate Control 2.58 0.10

9.49 0.40

26.30 1.11

26.20 1.12

1day ** 2.10 0.13

** 5.25 0.32

** 16.40 0.98

** 21.34 1.28

5 day 2.34 0.13

8.78 0.48

25.34 1.39

24.43 1.35

10 day 2.51 0.13

9.02 0.45

25.70 1.24

25.36 1.27

* Significant (P < 0.05). ** Highly Significant (P < 0.01). Table (2): Some biochemical parameters of Oreochromis niloticus after treatment with formalin

(25 mg/l) and copper sulphate (2 mg/l) for 10 days in comparison to the control (Mean ± SE).

Substance Time of sampling

Total protein g/dl

Albumin g/dl

Glucose g/dl

AST U/L

ALT U/L

UREA mg/dl

Creatinine mg/dl

Formalin Control 3.20 ± 0.13

1.80 ± 0.07

88.3 ± 3.53

213.7 ± 8.55

106.10 ± 4.24

13.86 ± 0.55

1.13 ± 0.05

1day * 2.69

± 0.15

* 1.53

± 0.08

** 110.14 ± 6.01

** 258.69 ± 12.93

** 152.13 ± 8.37

** 20.41 ± 1.12

** 2.22

± 0.12 5 day 2.88

± 0.17 1.79

± 0.11 95.14 ± 5.71

235.28 ± 14.12

118.55 ± 7.11

15.81 ± 0.95

1.31 ± 0.08

10 day 2.94 ± 0.18

1.77 ± 0.11

92.13 ± 5.53

222.23 ± 13.33

112.14 ± 6.73

14.98 ± 0.90

1.22 ± 0.07

Copper sulphate Control 3.2 ± 0.13

1.8 ± 0.07

88.3 ± 3.53

213.7 ± 8.55

106.10 ± 4.24

13.86 ± 0.55

1.13 ± 0.05

1day ** 2.55

± 0.16

** 1.42

± 0.09

** 122.15 ± 7.70

** 279.12 ± 17.58

** 177.17 ± 11.16

** 22.09 ± 1.39

** 2.19

± 0.14 5 day 2.81

± 0.15 1.58

± 0.09 98.70 ± 6.22

240.19 ± 13.21

120.53 ± 6.63

15.15 ± 0.83

1.27 ± 0.07

10 day 2.88 ± 0.18

1.67 ± 0.11

90.13 ± 5.68

235.09 ± 14.81

122.08 ± 7.69

15.13 ± 0.95

1.31 ± 0.08

* Significant (P < 0.05). ** Highly Significant (P < 0.01).

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DISCUSSION

The reaction and survival of aquatic animals depend not only on the biological state of the

animals and physico-chemical characteristics of water but also on kind, toxicity, type and time of exposure to the toxicant [19]. The Oreochromis niloticus in our study showed accelerated opercular beats during the first three days post treatment followed by dark skin and impairment of swimming and sinking of the fish to the bottom of the aquarium till the end of the experiment. Similar behavioural manifestations of acute toxicity like copious mucus secretion, loss of scales, grouping, loss of equilibrium and mortality rate 6.66 were observed in Esomusdanricus exposed to 5.5 mg/l copper[20]. Loss of swimming performance was also observed in brown trout, Salmotrutta exposed to sub lethal concentration of copper [21]. [8]evaluate the toxicity of 1.17 ppm of copper sulfate in the guppy (Poeciliareticulata), fish exhibited abnormal behavior such as erratic swimming and loss of equilibrium and became lethargic with high mortality. The ornamental fish, bluespotted coridora (Corydorasmelanistius) exposed to formalin concentrations showed two patterns of behavior during the first 24 hours. Fishes exposed to higher concentrations(50,100,150,200and 250mg/l) presented agitation, followed by erratic swimming and positioning on the water surface immediately after the addition of the substance. While the fishes submitted to the lower concentrations (3,6,12 and 25 mg/l) remained standing, making small movements and resting at bottom of containers [10]. Toxicity levels of therapeutic compounds (formalin, potassium permanganate, copper sulfate, acetic acid, and hydrogen peroxide ),used in aquaculture may vary depending on fish size and species, water quality, concentration of the compound, and duration of exposure[22].

The TLm24h value (LC50) for copper sulfate is 4.0 ppm in mirror carp (Cyprinus carpio), 10.0 ppm in goldfish, 4.4 ppm in rainbow trout and 4.0 ppm in flounder (Paralichthy solivaceus) [Heo,1997], the TLm24h value (LC50) in the guppy was 1.17 ppm [8]. Formalin was more toxic to bluespottedcorydora LC50 (I) 50.75 mg.L-1 than to trairão (H. lacerdae) larvae with LC50 (I) of 2.02 Ml.L-1 [23]. However, each species responds differently to the action of a substance, therefore the importance of conducting toxicity tests with different organisms is recommended. [5], evaluated the efficacy of formalin to control ectoparasites of fish "bullseye" Sphoeroidesannulatus, determining CL 50-96h (I) of 1095 mg.L-1 (30 min) and 972 mg.L-1 (60 min) and 79 mg.L-1 (72h), this last level showing similar toxicity to bluespottedcorydora in 96 hours. [24], determined the LC 50-96h (I) of 429.68 mg.L-1 of formaldehyde for tilapia (Oreochromis niloticus). According to [25], the formaldehyde presented more toxic activity for temperate fish in concentration range of 15 to 30 mg.L-1. For rainbow trout (Oncorhynchus mykiss) the LC50 48h of formalin concentration of 6.1 mg.L-1 was determined.

As the alkalinity of water increases, the concentration of copper ions in solution decreases. Consequently, a therapeutic level of copper in water with high alkalinity would be lethal to fish in water with low alkalinity [26]. The toxicity of free copper ions is well known. Accumulation of copper has also been seen in gill, kidney and liver tissue from the brown bullhead catfish (Ameiurusnebulosus) [27], bluegill (Lepomismacrochirus) [28] and Rainbow trout (Oncorhynchus mykiss) [29]. The organs of aquatic animals accumulate copper, which may cause morphological alterations and change certain physiological processes [30]. Copper have cytotoxic and genotoxic effects [31]. The uptake of waterborne copper in fish occurs primarily through the gills [32]

Therefore, histopathological changes in fish tissue can be used as a tool to detect the direct toxic effects of chemicals in target organs [33], because they reflect the damage caused by duration and intensity of exposure to toxic element and the tissue adaptive capacity [34]. Histopathologically, the gill showed congestion and hyperplasia in secondary lamellae with filling of the interlamellar spaces, intravascular hemolysis with vacuolation and necrosis of hepatocytes and renal tubules.There was depletion of hematopoietic tissues in the kidneys and spleen .The degenerative and proliferative changes in the gills of the exposed fish could be a body response against copper sulphate and formalin.The observed muscle necrosis resulted from the ability of copper to precipitate in the soft tissue and proliferation of melanomacrophage between the muscle bundles and inside the spleen could play a role in the clearance of the muscles from this compound. According to [1], the toxicity of formaldehyde is the cause of pathological damage in the gills, causing a gill dysfunction, osmoregulatory and respiratory imbalance and mortality. This is due to the fact that the gill epithelium is the primary contact surface, by having a large surface area, with the external environment, became a target of the pollutants present in the water [35]. This epithelial hyperplasia with filling of the interlamellar spaces is described in several fish in response to a variety of agents[36, 37, 38], like a primary strategy for fish protection. [39], mentioned that, juvenile rainbow trout, Oncorhynchus mykiss, exposed to 20 or 100 µgl(-1) of Cu (as CuSO(4)) revealed hyperplasia, aneurisms, and necrosis in the secondary lamellae of the gills.[8], evaluate the toxicity of copper sulfate in the guppy

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(Poeciliareticulata). Fish subjected to an acute toxicity 1.17 ppm reveled severe hyperplasia and exfoliation of the epithelial cells of gill lamellae.[40], stated that jundia fingerlings treatment formalin (0.2 ml/litre),showed hyperemia and hyperplasia of gill cells . The study showed that the employed therapeutic treatments can cause adverse changes to the gill tissue. In rainbow trout (Oncorhynchus mykiss), copper sulfate causes lumen closure of renal tubules as a result of epithelial edema and causes structural damage to the epithelial cells of renal tubules and parenchymal cells in the kidney [2].[23], also found hepatocytes disarray for larvae of Hopliaslacerdae submitted to the concentration of formaldehyde (7 ml.L-1) after 96 hours of exposure. [10], reported that ornamental fish, bluespottedcoridora (Corydorasmelanistius) exposed to formalin concentrations showed disorganization of liver arrangement, and necrosis in kidney, similar result recorded by [39, 41, 42]. These histopathological changes in varies internal organ including liver, kidney and spleen attributed to copper concentration in these organ [29]. Copper is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS) [43, 44, 45].

The ultracellular changes, in both hepatocytes and renal epithelium revealed decrease of glycogen granules and increase of lysosomes as well as lipid droplets. An increase in nuclear heterochromatin with degeneration in the mitochondria and endoplasmic reticulum were seen. This observation was in agreement with the previous hematological parameters where the serum protein was decrease. Moreover, the ultra structural modification observed in rough endoplasmic reticulum suggested an impairment of synthetic and secretory cell activity. The damage in the mitochondria suggested an explanation for the altered glycolytic and Kreb’s cycle enzymes which is leading to changes in cell permeability followed by influx of copper and formalin into the cells and subsequent loss of mitochondrial function [46]. The increase in lipid might related to toxic or hypoxic condition by copper and formalin .The increase number of lysosomes was also developed after chronic and subacutedosing of copper and formalin especially in liver and kidney [41], and the alteration may represent a cellular response to sublethal injury which could induce cytoplasmic vacuolation and liver dysfunction . These results are in agreement with [47, 48, 49].

The hematological parameters in Oreochromis niloticus exposed to copper sulphate and formalin revealed a significant decrease of RBCs, Hb and PCV at 1day of the experiment.These findings were confirmed histopathologically by the renal and splenic depletion of hematopoietic tissue and hepatic intravascular erythrocytichemolysis.This type of anemia could attributed to the effect of copper sulphate and formalin on the erythrocytes as a result of prooxidative effect [20] and tubular nephrosis that leading to drop in erythropoietin production and attenuation of erythropoiesis [50] .These data were in agreement with Srivastava et al., 2009 who study the effects of four days therapeutic bath of formalin (F) on catfish( Heteropneustesfossilis),0.25 ml F/litre water revealed that the total RBC count, Hb, PCV, MCHC and MCH values showed a significant decrease after four days bath. [51], mentioned that on heavy metal stress induced by lead and copper in rainbow trout (Oncorhynchus mykiss), erythrocyte number (RBC), hemoglobin (Hb), and hematocrit value were decreased. On the other hand [52], stated that, oral supplementation of copper sulfate solution at 120 mg Cu/L or 300 mg Cu/L, respectively. No significant changes were observed in hematological parameters. The decrease of total leukocytes and elevated blood glucose level in our study could be attributed to the generalized stress response leading to an increase in the pituitary internal activity with an increase in the secretion of corticosteroid [53]. [54], mentioned that plasma glucose, and plasma cortisol levels were elevated in trout exposed to 26.9 µg Cu/L.[55], stated that, Nile tilapia exposed to varying concentrations (0.1, 0.5 and 1.0 mg/litre) of a mixture of formalin, malachite green and methylene blue (FMC) for 1, 10 and 60 min. treatment of fish with FMC elicited marked elevations of plasma glucose. A significant decrease in total protein and albumin of experimented fish with a significant increase in ALT and AST were detected at first day of our experiment .These biochemical changes were carried out due to the histopathological alterations in the hepatic parenchyma. Similar findings were reported by [56], who stated that excessive copper ingestion in rat the ALT level in serum was increase. The increased level of urea in our study could be due to the gill dysfunction[10, 39].The increase level of creatinine in our study attributed to the microscopical changes in the kidneys which affected on its functions.[57], reported that, the level of serum creatinine may be an indicator for kidneys dysfunctions.

It could be concluded that, Copper sulphate and formalin are found to be highly toxic to tilapia nilotica(Oreochromus nilotucus) that reflected to the gross, histopathological and hematological as well as biochemical findings in fish even in therapeutic doses.To reduce the risk and maximize the medical efficiency, additional studies about the therapeutic level of copper sulfate and formalin are necessary to show how much copper sulfate and formalin are required for treatment of each disease in different species of fish under specific water condition.

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REFERENCES

1. Reardon, I. S. and R. M. Harrell, 1990. Acute toxicity of formalin and copper sulfate to striped

bass fingerings held in varying salinities. Aquaculture, 87: 255–270. 2. Heo, G. J., 1997. Antibacterial efficacy and safety of copper sulfate pentahydrate to cultured fish.

Korean J. Vet. Res., 37:203–212. 3. Straus, D. L. and C. S. Tucker, 1993. Acute toxicity of copper sulfate and chelated copper to

channel catfish, Ictalurus punctatus.J. World Aquacult. Soc., 24: 390–395.

4. Moore, J. and S. Ramamoorthy,1984.Heavy metals in natural waters.Applied Monitoring and Impact Assessment,. Springer-Verlag, New York,

5. Fajer-Ávila, E., I. Parra, G. Zarate, R. Arce, J. Ramirez and M. Lozano, 2003. Toxicity of formalin to bullseye puffer fish (Sphoeroidesannulatus - Jenyns, 1843) and its effectiveness to control ectoparasitas. Aquaculture, 223:41-50.

6. Kinnunen, P., M.Rahkonen, H. Mykrä and E. Valtonen,2005. Treatment of ichthyophthiriasis after malachite green. II. Earth ponds at salmonid farms. Dis. Aquat. Org., 66: 15–20.

7. Badran, A., S. Aly, A. Abdel-Aal, 1996.Studies on skin parasitic diseases of hybrid tilapia. Assiut Vet. Med. J., l35(70):163-175.

8. Park, K., and G. Heo, 2008.Acute and Subacute Toxicity of Copper Sulfate Pentahydrate (CuSO45·H2O) in the Guppy (Poeciliareticulata) J. Vet. Med. Sci., 71(3): 333–336.

9. Srivastava, S., A.Gupta, S.Chaturvedi, A. Abhinavand R. Singh, 2009. Effects of short-term therapeutic bath of malachite green and formalin on certain hematological parameters of afreshwater catfish, (Heteropneustes fossilis) (Bloch).Journal of Experimental Zoology, India. 12(1):79-83.

10. Santos, R., H. Dias and R. Fujimoto,2012.Acute toxicity and histopathology in ornamentalfish amazon bluespottedcorydora (Corydorasmelanistius) exposed to formalin. An. Acad. Bras.Cienc., 84 (4):1001-1007.

11. ABNT.AssociacaoBrasileira De NormasTecnicas, 2006.NBR 15350.Ecotoxicologiaaquática –Toxicidadecrônica de curtaduração – Método de ensaio com ouriço do mar(Echinodermata: Echinoidea).

12. Nutt, M. and C. Herrick, 1952. A new diluent for counting the red and white cells of chickens.Poult. Sci., 31: 335.

13. Soliman, M.K., 1986. Blood and Body Fluid.2nd Ed., Fac. Vet.Med., Press.University of Alexandria.

14. Feldman, B., J. Zinkl and N. Jain, 2000. Schalm,s Veterinary Hematology . 5th Ed., Lippincott Williams and Wilkins. A Wolterscompany .Philadeia, Baltimore, Naw York, London, Buenos Aires, Hong Kong, Sydney, Tokyo.

15. Drabkin, D.L., 1949. Standardization of hemoglobin measurements. Am. J. Med. Sci., 210-217.

16. Bancroft, T.D., A. Stevens and D. Turner, 1996.Theory and Practice of histological technique.4th edition.Churchill, Livingeston, New York, London, San Francisco, Tokyo.

17. Weakly, B.1981.A Beginner,s Handbook in Biological Transmission Electron Microscopy. Churchill, Livingeston Co., London.

18. SAS Institute.2003.SAS Statistical guide for personal computers ,6thed.Cary, Nourth Carolina.

19. Brungs, W., J. McCormick, T. Neiheisel, C. Spehar and G.Stokes,1977. Effects of pollution on freshwater fishes. J. W.P.C.F, Washington DC.,49: 1425-1493.

20. Vutukuru,S., C. Suma, K. Madhavi, S. Juveria, J. Pauleena, J. Raoand Y. Anjaneyulu,2005. Studies on the Development of Potential Biomarkers for Rapid Assessment of Copper Toxicity to Freshwater Fish using Esomusdanricus as Model. Int. J. Environ. Res. Public Health, 2(1):63-73.

21. Beaumont, M., P. Butler and E.Taylor, 2000. Exposure of brown trout, Salmotrutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential. Aquat.Toxicol., 51(2): 259-72.

22. Taylor, P. and R.Glenn, 2008.Toxicity of five therapeutic compounds on juvenile salmonids.NorthAmerican Journal of Aquaculture, 70(2): 175-183.

18


23. Cruz, C., R. Fujimoto, R. Luz, M. Portella and M. Martins, 2005. Toxicidadeaguda e histopatologia do fígado de larvas de trairão (Hopliaslacerdae) expostas à soluçãoaquosa deformaldeído a 10%. Pesticidas: ecotoxicol. emeioambiente, 15: 21-28.

24. Macniven, A. and D. Little, 2001. Development and evaluation of a stress challenge testing methodology for assessment of Nile tilapia (Oreochromis niloticus, Linn.) fry quality. Aquacul.Res., 32: 671-679.

25. Tisler, T., and J. Koncan, 1997. Comparative assessment of phenol, formaldehyde and industrial wastewater to aquatic organisms. Earth and Environmental Science, 97: 315-322.

26. Lauren, D. and D. McDonald, 1986. Influence of water hardness, pH, and alkalinity on themechanisms of copper toxicity in juvenile rainbow trout, Salmogairdneri. Can. J. Fish Aquat. Sci., 43: 1488–1496.

27. Brungs, W., E. Leonard and J. McKim, 1973. Acute and long-term accumulation of copper by the brown bullhead, Ictalurusnebulosus. J. Fish Res. Bd. Can., 30: 583–586.

28. Benoit, D. A., 1975. Chronic effects of copper on survival, growth, and reproduction of the bluegill, Lepomismacrochirus. Trans. Am. Fish Soc., 104: 353–358.

29. Clearwater, S., S. Baskin, C. Wood and D.Mcdonald, 2000.Gastrointestinal uptake and distribution of copper in rainbow trout. J. Exp. Biol., 203: 2455–2466.

30. Shaw, B., G. Al-Bairuty and R. Handy, 2012. Effects of waterborne copper nanoparticles and copper suphate on Rainbow trout, (Oncorhynchus mykiss): physiology and accumulation.Aqua.Toxicol.,117: 90-101.

31. Hernandez, P., C. Undurraga, V. Gallardo1, N. Mackenzie1, M. Allende and A. Reyes,2011.Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae.Biol. Res., 44: 7-15.

32. Kamunde, C., M. Grosell, D.Higgs and C. Wood,2002. Copper metabolism in actively growing rainbow trout (Oncorhynchusmykiss):interactions between dietary and waterborne copper uptake. J. Exp. Biol., 205: 279–290 .

33. Schwaiger, J., U. Wanke, S. Adam, M. Pawert, W. Honnen and R. Triebskorn, 1997. The use of histopathological indicators to evaluate ontaminant-related stress in fish.J .Aquat.Ecosyst. Stress and Recov., 6: 75-86.

34. Ferreira, M., P. Antunes, O. Gil, C. Vale and M. Reis-Henriques, 2004. Organochlorine contaminants in flonder (Platichthysflesus) and mullet (Mugilcephatus) from Douro estuary, andtheir use as sentinel speiceis for environmental monitoring. Aquat.Toxicol., 69: 347-357.

35. Wong, C.K. and M.Wong , 2000. Morphological and biochemical changes in the gills of Tilapia (Oreochromismossambicus) to ambient cadmium exposure. Aquat.Toxicol., 48: 517-527.

36. Pane, E., A. Haque, G. Goss and C. Wood, 2004. The physiological consequences of exposure to chronic, sublethal waterborne nickel in rainbow trout (Oncorhynchus mykiss): exercise vs resting physiology.J. Exp. Biol., 7:1249-61.

37. Karan, V., S. Vitorović, V. Tutundzićand V. Poleksić, 1998.Functional enzymes activity and gill histology of carp after copper sulfate exposure and recovery. Ecotoxicol. Environ. Saf., 40(1-2):49-55.

38. Khangarot, B. and D. Tripathi, 1991.Changes in humoral and cell-mediated immune responses andin skin and respiratory surfaces of catfish, Saccobranchusfossilis, following copper exposure.Ecotoxicol. Environ. Saf., 22(3):291-308

39. Al-Bairuty, G.,B.Shaw, R. Handy andT.Henry, 2013.Histopathological effects of waterbornecopper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchusmykiss).Aquat. Toxicol.,126:104-15.

40. Carneiro, P., S.Cirio and M.Schorer, 2006. Pathological study of jundia (Rhamdiaquelen)fingerlings experimentally infected with Ichthyophthirius multifilis and submitted to conventional treatments. Arch.Vet.Sci., 11(1): 33-38.

41. Wan, X., Y.Li and X. Luo, 2007.Curcumin attenuated the lipid peroxidation and apoptotic liverinjury in copper-overloaded rats.Zhonghua.Er. Ke.Za.Zhi., 45(8):604-8.

19


42. Hebert, C.,1993.NTP technical report on the toxicity studies of Cupric Sulfate (CAS No. 7758-99-

8) Administered in Drinking Water and Feed to F344/N Rats and B6C3F1 Mice.Toxic. Rep. Ser., 29:1-D3.

43. Bopp, S., H. Abichtand K. Knauer, 2008.Copper-induced oxidative stress in rainbow trout gill cells.Aquat.Toxicol., 86(2):197-204.

44. Sampaio, G., D. Boijink, E. Oba, R. Santos, L. Kalinin and T. Rantin, 2008. Antioxidant defensesand biochemical changes in pacu (Piaractusmesopotamicus) in response to single and combined copper and hypoxia exposure. Comp. Biochem. Physiol. C. Toxicol. Pharmacol.,147(1):43-51.

45. Eyckmans, M., N. Celis, N. Horemans, R. Blustand G. De Boeck, 2011.Exposure to waterborne copper reveals differences in oxidative stress response in three freshwater fish species.Aquat. Toxicol.,103(1-2):112-20.

46. Liu, T.andX. Bai, 2005.Effect of formaldehyde on energy metabolism in postnatal rat cortex neurons in culture.Wei. Sheng.Yan.Jiu., 34(3):275-7.

47. Cikmaz, S., T. Kutoglu, M. Kanter and R. Mesut, 2010.Effect of formaldehyde inhalation on rat livers: a light and electron microscopic study.Toxicol. Ind. Health, 26(2):113-9.

48. Bansal, N., V. Uppaland D. Pathak, 2011.Toxic effect of formaldehyde on the respiratory organs of rabbits: a light and electron microscopic study.Toxicol. Ind. Health, 27(6):563-9.

49. Waldemarin, D., R. Alves, M.Beletti, F. Rantinand A. Kalinin, 2012.Copper sulfate affects Nile tilapia (Oreochromisniloticus) cardiomyocytes structure and contractile function. Ecotoxicology, 21(3):783-94.

50. Horiguchi, H., H. Ternishi, K. Niiya, K. Aoshima, T. Katoh, N. Sakurayawa and M. Kasuy, 1994. Hypoproducton of erythropoietin contributes to anemia in chronic cadmium intoxication.Clinical study on Itaiitai disease in Japan. Arch. Toxciol., 68: 632-636.

51. Ates, B., I. Orun, Z. Talas, G. Durmazand I. Yilmaz, 2008.Effects of sodium selenite on some biochemical and hematological parameters of rainbow trout (Oncorhynchu smykissWalbaum, 1792) exposed to Pb2+ and Cu2+.Fish Physiol. Biochem., 34(1):53-9.

52. Kvietkauskaite, R., A. Dringeliene, A. Markevicius, A. Siaurysand J. Acaite, 2004.Effect of low copper exposure on the antioxidant system and some immune parameters.Vet. Hum. Toxicol., 46(4):169-72.

53. Salonius, K., and G. Iwama,1993 .Effect of early rearing environment on stress response, immune function and disease resistance in juvenile Coho ( Oncorhynchus kisutch) and Chinook salmon (O.Tahawytscha).Can. J. Fish. Aqua.Sci., 50: 759-766.

54. Dethloff, G., D. Schlenk, S. Khan and H.Bailey, 1999.The effects of copper on blood and biochemical parameters of rainbow trout (Oncorhynchus mykiss).Arch. Environ. Contam. Toxicol.,36(4):415-23.

55. Yildiz, H. and S. Pulatsu, 1999. Evaluation of the secondary stress response in healthy Nile tilapia (Oreochromisniloticus) after treatment with a mixture of formalin, malachite green and methyleneblue.Aquacult. Res.,30(5): 379-383.

56. Li, Y., X.Wang, Q. Nin and X. Luo, 2008.Excessive copper induces hepatocyte apoptosis and affects Bax and Bcl-2 expression in rat liver.Zhongguo.Dang.Dai.Er. Ke.Za. Zhi.,10(1):42-6.

57. Lockart, W. and D. Metner, 1984. Fish serum chemistry and a pathology tool. Contaminant Effection Fisheries, 16: 73-86.

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Toxopathological Studies on the Effect of Formalin …. Appl. Environ. Biol...The present study aimed to investigate the effect of therapeutic doses of formalin and copper sulphate