Iraqi Journal of Veterinary Sciences

Introduction

Copper is an essential trace element that has beneficial roles in organisms, it is required for connective tissue and hemoglobin synthesis, cellular metabolism, and respiration, as well as to it is roles in the action of enzymes such as tyrosinase, copper-zinc superoxide dismutase and cytochrome c oxidase, which are involved in critical of growth and maturation (1,2). Copper inter aquatic environment industrial activity, herbicide, supplemented fish diet as well as it considered an essential component in some chemotherapeutic agent as copper sulfate, it is widely used in fish culture as one of the broad chemotherapeutic agents against bacterial, fungal, parasitic diseases it will be used at concentration 0.3-2 mg/L (3,4), in addition it has bio-effects and ability to reduce the negative effects of free radical oxygen, stimulated antioxidant activity and reduce adverse effects of toxic agent as nano-zinc oxide in C. carpio (5,6).

The permissible limited concentration of Cu ion in Iraqi water environment is 0.05 mg/l. As pollution concentration is 0.199 mg/l, a high concentration of Cu inters the body through the digestive tract, gills, and accumulation in the tissue lead to abnormal locomotor responses and adverse pathological effects in fish and other aquatic organisms (7-10). Delahaut et al. (11) reported the significant effects of Cu in gill electrolytes and cause reduction in sodium ions and cause, an imbalance of osmoregulation in C. carpio and cause damage to gill, kidney, liver, and spleen architecture (12), blood lysis, and anemia in Clarias lazera, and enzyme activity disturbances(13,14). Although the toxic effects of copper sulfate are more clearly in the gill, liver, and kidney, it has mild effects on the brain and causes histopathological alteration in the telencephalon, mesencephalon layers, and cerebellum. Boareto et al. (15) revealed the neurotoxicity effects of copper in Oreochromis niloticus, which cause central nervous system injuries and reduces acetylcholine activity in the brain. Recently study demonstrated the toxic effects of copper on the lateral line, loss of sensation, and loss of olfactory function even at low concentrations ≥20 parts per million and for short periods of about 3 hours (16). As the result of acute toxic effects of Cu on freshwater fish even at low concentrations (17), that the toxicity of copper sulfate in the brain will be unknown, this study was performed to demonstrate the histopathological alterations of lethal concentration during short time exposure in the gill, brain and spinal cord in C. carpio.

Material and methods

Ethical approve

Scientific Ethical Committee on Animal Experimentation at College of Veterinary of Medicine, University of Mosul, UM.VET.2021.049.

Experimental animals

Seventy-two C. carpio (18,19) weight of 200±10 gm, were obtained from the hatchery in Erbil governorate, and transported to the fish laboratory in College of Agriculture at the University of Mosul. The fish were left in the tank with aerated, dechlorinated water during the acclimation period and fed with commercial feed. during the experiment period, the light cycle was regular at 12 hours light/ 12 hours dark, and dissolved oxygen 5 mg/l, the pH 7.5, and the temperature was continued at 20±1.5ºC.

Determine lethal concentration of CuSO₄

After one week, expanding acclimation to laboratory circumstances and determining the lethal and median lethal concentration of copper sulfate, fish were randomly divided into four groups. Each sex fish was placed at 70 L of aerated water and treated with stock variable concentration of CuSO₄∙5H₂O 0, 2.5, 5, 10 mg/L for 24 hours according to the Trevan method (20). The behavior and clinical signs were reported, and dead, poisoned fish were removed immediately from the aquarium to collect the gills, brain, and spinal cord, sample. At the end of the experiment 24 hours, fish from the control group were treated with general anesthesia MS-222 at a concentration of 150 mg/l (21) to collect the samples from gills, brain, and spinal cord.

Microscopic examination

Gills and Brain were fixed with 10% formalin for 48 hours. For dehydration, the sample treated with series ethanol xylene was used for sample clearance then embedded in the paraffin and sectioning at 5μm then staining with routine stained Hematoxylin &Eosin.

Semi-quantities score scheme (SSS)

The semi-quantitative scheme was employed for determining the severity and degree of histopathological changes in the gills. This system involves (score value, importance factor, alteration index and reaction pattern indices). Bernet et al. (22) briefly explain the SSS. The histopathological alteration was an evaluation for each ten gills filament from blinded slide has score value (SV; 0, 2,4,6), the histopathological alteration ranging from circulatory disturbances, inflammatory reaction, regressive and progressive alteration all these alterations take importance factor (IF) ranging from 0-3, (0 mean reversible alteration and minimal importance) while, (3 mean irreversible alteration and minimal importance).The formula for calculating the Alteration Index (AI) was multiplying the score value by the importance factor, AI= SV×IF (22). Calculating the gills index (GI) is by summing all AI- related to each category.

Statistical analysis

The data of this study was statistical analysis by T test and Chi² square in the (P≤0.05). Both these analyses were completed using (23).

Results

Lethal concentration experiment: The result of this experiment was reported the lethal concentrations of CuSO₄at 10 mg/l led to killing the fish, and mortality was 100% during 24 hours, while the concentration at five mg/l was the median lethal concentration which led to killing 50% of treated fish as in figure 1.

Figure 1: Standard curve for LC₅₀ of copper sulfate (mg/L)

Clinical signs and fish behavior

The fish treated at a variable concentration of CuSO₄ exhibited variable nervous and respiratory signs according to the duration time of exposure. After half hour from fish treated with 10 mg/l, fish had abnormal respiration there was an increase and then a reducation in the opercula frequency, excessive mucus secretion appeared as thick thread (Figure 2) with abnormal and gasping swimming and fish were excited and exhibited nervous signs at the last time of exposure with up-down swimming, then theyed stay at the bottom and died.

Figure 2: C. carpio exposed to lethal concentration of CuSO₄ 10 mg/l for three-hour show thick thread mucus secretion (red row)

Gross lesion

Excessive gill mucus secretion with CuSO₄ precipitated on the gill and body at a concentration 10 mg/L and less precipitation at 5mg/l with pale patches at the apex of secondary gill filaments with hemorrhage figure 3.

Figure 3: C. carpio exposed to lethal concentration of CuSO₄ 10 mg/l for three-hour show pale patches at the apex of secondary gill filaments (red row) and congestion (white row).

Semi-quantities score scheme

The histopathological alteration in the gill was estimated as semi- quantities exploration revealed significant alteration in both, treatments. The pathological lesion was classified into three categories. In the Circulatory alteration the statistical analysis revealed there was only a high significance (P≤0.05) in the mean of hyperemia in the gill of fish treated with CuSO₄ for three hours, this significance was detected in the progression alteration in the hypertrophy features in the epithelial, undifferentiated and chloride cells of gills in the fish treated for three hour and only hypertrophy of pillar cells was significant in the gills of fish treated for 2 hours in addition the third categories was a regressive alteration and the means of pathological lesions was significantly in both damage apex of primary gills filaments and lamellae necrosis in fish treated for three hours, table (2).

Table 1: Histopathological alteration in the gill of fish treated with lethal concentration of CuSO₄ 10 mg/l during 2 and 3 hours

The pathological alteration for each category was calculated as gills pathological index, which statistically revealed that fish treated for 3 hours is higher significant (P≤0.05) for both progressive and regressive indexes in contrast to circulatory and cellularity reaction figure 4.

Figure 4: Histogram for gills indexes.

Microscopic examination

The microscopic study of gills in the control fish shows the typical structure as in figure 5 in contrast to treated group, the primary lesions of fish gill which exposed to 10mg/l for 2 hour of CuSO₄ which represented by curling of the secondary gill filaments,, edema, lifting of the epithelial cells of secondary gill filaments figure 6, sever infiltration of mononuclear cell led to complete adhesion between secondary gill filaments with hypertrophy of mucus cells and vacuolar degeneration of undifferentiated cells figure 7, the lesion are more sever in the gills of fish exposed to lethal concentration for 3 hour represented by hyperemia in the arterial primary gill filaments figure 8 with abnormal shape of chloride cells with sever infiltration of inflammatory cells, hyperemic capillaries in the secondary gills filaments with vacuolar degeneration of pillar cells figure 9, edema and lifting epithelial cells of the secondary gills filaments and destruction the apex of primary gill gills filaments figure 10 and necrosis figure 11, microscopic examination of the gill arch revealed there was sever hyperemia with infiltration of inflammatory cells figure 11.

Figure 5: Microscopic examination of normal gill architecture in C. carpio, shows inter lamellar space (red-two head), secondary gill filament (red dot row), piller cells (black row), and mucus cells (white row). H&E. 270x.

Figure 6: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 2 hour shows curling of the secondary gill filaments (black row), edema (green row), and lifting of the epithelial cells of secondary gill filaments (red row). H&E. 40x.

Figure 7: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 3 hour shows sever infiltration of mononuclear cell (yellow star) led to complete adhesion between secondary gill filaments (two head yellow row) with hypertrophy of mucus cells (black row), and vacuolar degeneration of undifferentiated cells (red row). H&E. 48x.

Figure 8: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 3 hour shows hyperemia in the arterial primary gill filaments (red row). H&E. 4x.

Figure 9: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 3 hour shows abnormal shape of chloride cells (yellow row) with sever infiltration of inflammatory cells (yellow star), hyperemic capillaries in the secondary gills’ filaments (green row) with vacuolar degeneration of pillar cells (black row), edema (red row) with lifting epithelial cells (red dot row). H&E. 40x.

Figure 10: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 3 hour shows destruction the apex of primary gills filaments (yellow star). H&E. 40x.

Figure 11: Microscopic examination of gill in fish exposed to lethal concentration 10 mg/l for 3 hour shows necrosis and dead gills. H&E. 13x.

Figure 12: Microscopic examination of gill arch in fish exposed to lethal concentration 10 mg/l for 3 hour shows sever hyperemia (yellow star) with infiltration of inflammatory cells (red row). H&E. 16x.

The effects of CuSO₄ in the central nervous system were clearly by microscopic examination in the brain and spinal cord. There was an infiltration of inflammatory cells in the brain with hemorrhage figure 13, hyperemia, vasogenic edema, with gliosis as in figure 14 and figure 15. In the spinal cord, the histopathological alteration distinguished as the infiltration of inflammatory cells with edema around neuronal bodies and tissue figure 16.

Figure 13: Microscopic examination of brain in fish exposed to lethal concentration 10 mg/l for 2 hour shows infiltration of inflammatory cells in the brain (black row) and hemorrhage (red row). H&E. 80x.

Figure 14: Microscopic examination of brain in fish exposed to lethal concentration 10 mg/l for 2 hour shows hyperemia (red star), vasogenic edema (red row) with gliosis (red dot row). H&E. 140x

Figure 15: Microscopic examination of brain in fish exposed to lethal concentration 10 mg/l for 3 hour shows sever vasogenic edema (red row) with gliosis (red dot row). H&E. 240x.

Figure 16: Microscopic examination of spinal cord in fish exposed to lethal concentration 10 mg/l for 3 hour shows infiltration of inflammatory cells (red star) edema around neuronal bodies (red row) and in the tissue (red dot row). H&E, 40X.

Discussion

Copper is essential elements for all organisms (24,25), copper sulfate is generally used in aquaculture as a chemotherapeutic agent. However, it is more toxic to C. carpio (26), so this study determined the LC₁₀₀ of CuSO₄ in C. carpio is 10 mg/L, and LC₅₀ is 5mg/L. This result does not agree with the results of (27), who reported the lethal concentration of CuSO₄ in C. carpio was 0.45 mg/L during 96 hours also, Nekoubin et al. (28) reported the LC₅₀ is 2.422 mg/L in the grass carp Ctenopharyngodon idella, however the LC50 of CuSO₄ in freshwater fish variable from 0.30-7.57 ppm (29) These variable results may be related to fish species with different sensitivity to toxic material, age, size, physiological state, food habitat, water quality, and experimental conditions (30,31). The mortality occurred in the first hours of the experiments. This result comes with other results were got by Marinovic et al. (32), the death may result from direct effects of a toxic compound or indirect by forming an unconducive medium for the fish, the mucogenesis and increased mucus secretion from the gill may lead to decrease oxygen intake and cause respiratory disturbances under this condition fish may stress and exhibit abnormal behavior (32).

Histopathological alteration of gill architecture is considered a promising biomarker for lacking quality of fish environment (32-34) because it has direct contact with the aquaculture environment and has a specialized structure as thin epithelial cells with large particular sizable area. It is biological functions and ion balance. These alterations are common and represent general responses to variable stress such as season, the quantity of water, and infectious and non-infectious diseases. Thus, the semi-quantitive system score is a benefit for describing and analysis of the pathological alteration is considered a good indicator of the severity of lesions (11). Previous studies suggested the toxic, pathological effects of CuSO₄ in gills involve hypertrophy or hyperplasia in chloride cells, mucus and pillar cells, edema, hemorrhage, clubbing gill arch with thickening of primary and secondary lamellae led to fusion and dead tissue and necrosis. As a result, all of the lesions discovered in fish exposed to toxicants for a short period of 3 hours (35) and are likely to impair the respiratory, secretory, and excretory functioning of fish gills. Thirumavalavan (36) found that glucose and lactic acid levels in the blood of freshwater fish Catla catla exposed to copper sulfate were elevated. This elevation in glucose and lactic acid led to turnover cell respiration and caused an increase in anaerobic respiration and decreased in aerobic respiration. This is one of the causative agents for histopathological alteration in the gills and other organs. Also, a copper ion can share a sodium channel leading to competitive sodium ion uptake in the gill and affecting the Na⁺-K⁺/ATPase activity and disturbances the K and Ca ions balance. This is the key to cell injury. The alteration in the gill structure comes mainly from two hypotheses: firstly it considers a defense mechanism (hypertrophy and hyperplasia)and increased respiratory surface with increased mucus secretion act as physical capture and reduce uptake xenobiotic, these lesions are reversible, while the second hypothesis considers the gill alteration as a pathological mechanism which are irreversible as disturbances in the blood circulation and necrosis (32).BOARETO et al. (15), Al-Bairuty et al., (37) and Sharma et al., (38) reported that copper sulfate is neurotoxic so this study suggested the toxicity of CuSO₄ in the brain and spinal cord which reported histopathological lesions represented by circulatory disturbances and infiltration of inflammatory cells. The neural tissue is vulnerable to oxidative damage which is the most well-known mechanism associated with copper ion toxicity, gliosis, and lacked Nissl substances with glycolysis leads to mitochondrial and microsomal dysfunctions (39-41).

Conclusion

This study suggested that copper sulfate is one of the chemotherapeutic agents and is commonly used in aquaculture. However, it should be used at a limited concentration with duration time because it is very toxic to fish, leading to respiratory disturbances, and abnormal fish behavior with histopathological alteration in both gill and CNS. This study suggested that gills are more susceptible to toxicity than CNS.

Acknowledgment

 All thankfully for both members of Colleges of Veterinary Medicine and Agriculture, University of Mosul.

Conflict of interest

No conflict of interest.

1. Copper sulfate is one of the important chemotherapeutic agents in fish culture.
2. Although Copper is essential elements for alive cell and tissue it is very toxic.
3. It should be used as limited concentration and limited duration period.
4. High concentration in short time of exposure lead to toxic, pathological effects on fish (behavior and clinical signs with histopathological alteration).

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