New Insights Into Hematological, Serum Biochemical and Histopathological Toxicity of Bisphenol a on Bighead Carp (Aristichthys Nobils) Under Long-Term Exposure

Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide and is frequently used in dental sealants, water bottles, food and beverage packaging. Due to persistant application, BPA has become potential threat to variety of organisms including public health. In this study for the rst time 80 bighead carps were randomly placed in different four groups (A-D). Fish in groups (B-D) were treated with BPA 60 days while sh in group A served as control group. Body weight, absolute and relative weight of different visceral organs of sh exposed to higher concentrations (1500 µg/L) of BPA decreased signicantly (p < .05). Results on proximate analysis showed signicantly decreased in crude proteins, lipid contents and moisture contents in muscles while increased ash contents. Red blood cells count, hemoglobin concentration, lymphocytes and monocytes were signicantly decreased while leukocytes counts and neutrophil counts was signicantly increased in treated sh. Results on different serum biochemistry parameters like serum albumin and total proteins decreased signicantly (p < .05) while alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), urea creatinine, glucose, cholesterol and lactate dehydrogenase (LDH) increased signicantly (p < .05) in treated sh. Histopathological ailments like pyknosis, degeneration glomeruli, increased Bowman’s space, ceroid formation in kidneys while ceroid formation, hemorrhages, pyknosis, karyorrhexis, karyolysis, binucleated hepatocytes, nuclear hypertrophy and eccentric nuclei in liver were observed in treated sh. Histological observation of ndifferent sections of brain of treated sh exhibited degenerated neurons in cerebellum, lipofuscin deposition, microgliosis, necrotic neurons, inammatory cell and severe hemorrhage. Results on light microscopic observation of different sections of heart of bighead carp revealed necrosis, inammatory reaction, neutrophilic myocarditis and hemorrhages. In conclusion, it is suggested that BPA induces adverse effects on physical, blood-biochemical parameters and histopathological changes in multiple visceral tissues of exposed sh.


Introduction
Over the past few decades in addition to the pathogenic risk of microbes, the indiscriminate application of synthetic chemicals in agriculture, aquatic life, industries, veterinary practice, protection of environment and to improve public health has become serious threat ( Bio-monitoring and epidemiological reports have shown that bisphenol A is frequently used in paints, protective coatings, mechanical parts and as a liner in plastic food and beverage containers (Prins et  2020) in sh has been well documented. But to the best of our knowledge, the toxicity of BPA on fresh water sh particularly in Bighead carp is scanty. Therefore, in present study, we attempted to evaluate the toxicity of BPA at sublethal concentrations in bighead carp (Aristhicthys nobilis).

Materials And Methods
Toxicant and chemicals hematological parameters including red blood cells counts and total and differential white blood cell counts (Islam et al., 2019) while hematocrit %, hemoglobin quantity and total proteins were measured according to earlier protocols (Hussain et al., 2019; Ghaffar et al. 2020) at days 15, 30, 45 and 60 of the experiment.

Serum biochemical studies
For different serological parameters, serum was separated from the blood of each sh placing on ice at different experimental intervals at days 15, 30, 45 and 60 of the experiment. Various serum biochemical parameters including ALT, AST, ALP, LDH, urea, creatinine, gulucose, cholesterol and triglycerides were measured using commercially available kits (Randox company Pvt.) using a chemistry analyzer (Randox company Pvt.).

Statistical analysis
Data collected during the tral were presented as mean ± S.E. All the collected data in each group was normally distributed and statistical analysis was carried out by one-way analysis of variance (ANOVA) using IBM SPSS statistics (version 20). The difference in mean values (mean ± S.E) of body weight, organ weight, hematological parameters and serum biochemistry of control and treated groups was conducted by using post hoc Tukey's test at p < 0.05.

Physical parameters
The results revealed that the body weight of sh exposed to higher concentrations (1500 µg/L) of BPA decreased signi cantly (p < .05) compared to control group at day 60 of the experiment ( Table 1). The absolute weight of visceral organs of sh such as liver, kidnyes and gills increased signi cantly at higher concentrations (1500 µg/L) of BPA (Table 1). The relative weight of liver, kidnyes and gills increased signi cantly (p < .05) at higher concentrations (1500 µg/L) of BPA while non-signi cat difference was recorded in relative weight of brain as compared to untreated control sh (Table 2).

Proximate Analysis
Proximate analysis of A.nobilis revealed that crude protein and lipid contents signi cantly decreased in muscles of sh exposed to higher concentrations (1000 µg/L and 1500 µg/L) at days 45 and 60 of the study. Results showed that the moisture contents also decreased signi cantly in muscles of sh exposed to higher concentrations (1000 µg/L and 1500 µg/L) at day 60 of experiment. Ash contents increased signi cantly in sh meat exposed to higher concentrations (1000 µg/L & 1500 µg/L) at day 45 and 60 of the experiment (Table 6).

Hematological and serum analysis
Results of different hematological parameters of blood cells of sh exposed to various levels of bisphenol A are presented in table 2. The sh exposed to 1000 µg/L and 1500 µg/L bisphenol A showed signi cantly decreased in red blood cells count at days 45 and 60 of trial. The hemoglobin concentration was signi cantly decreased in sh exposed to higher concentrations (1000 µg/L and 1500 µg/L) of bisphenol A at days 45 and 60 of the study as compared to control sh. The sh exposed to 1000 µg/L and 1500 µg/L of bisphenol A showed that differential leukocytes counts were signi cantly higher (neutrophilic leukocytosis) at days 30, 45, and 60 of experiment (Table 3). Results showed that the lymphocytes and monocytes were signi cantly decreased in sh exposed to 1000 µg/L and 1500 µg/L bisphenol A at days 45 and 60 of the experiment in comparison to untreated control sh. The pack cell volume of blood was also signi cantly decreased in sh exposed to higher concentrations (1000 µg/L and 1500 µg/L) of bisphenol A at day 45 and 60 of the experiment.
The results on different serum biochemical parameters in sh exposed to various levels of bisphenol A are presented in table 4. Serum albumin quantity and serum total protein signi cantly decreased in sh exposed to 1000 µg/L and 1500 µg/L of bisphenol A at days 45 and 60 of the trial in comparison to unexposed sh. The quantity of serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and serum alkaline phosphatase (ALP) increased signi cantly in liver tissues of sh exposed to higher concentrations (1000 µg/L and 1500 µg/L) of bisphenol A at days 45 and 60 of trial ( Table 3). The serum triglycerides were signi cantly increased in sh exposed to 1000 µg/L and 1500 µg/L at days 45 and 60 of trial. The quantity of urea and creatinine signi cantly increased in kidneys of sh exposed to 1000 µg/L and 1500 µg/L bisphenol A at days 45 and 60 of trial as compared to control sh. The quantity of glucose signi cantly increased in sh exposed to 1000 µg/L and 1500 µg/L at days 45 and 60 of the trial. The cholesterol and lactate dehydrogenase signi cantly increased at days 45 and 60 of the trial in sh exposed to 1000 µg/L and 1500 µg/L bisphenol A compared to control sh.

Histopathology
Results on intensity/severity of different histopathological changes if various tissues of sh exposed to various levels of bisphenol A are presented in the table 5. Various sections of gills of fish in groups C-D showed severe histopathological abnormalities such as lamellar disorganization, necrosis of lamellar pillar, lamellar atrophy, disruption of primary lamellae, curling of secondary lamellae, fusion of lamellae, severe congestion and degenration in cartilaginous cores and telangiectasia. Curling and uplifting arrangements in epithelial cells of secondary lamellae were frequently observed in these groups after day 45 and 60 of the experiment (Fig. 1). Mild to moderate histopathological abnormalities like pyknosis, degeneration glomeruli, congestion, increased Bowman's space, atrophic cells, edema, degeneration of tubular epithelium, aggregation of melanomacrophages and atrophy of the lumen of renal tubules were observed in kidneys of treated groups (Fig. 2). Moderate histopathological changes, such as deterioration of glomerulus, increased bowmens space and necrosis of tubular cells were evident in the kidneys of fish in group B at days 45 and 60 of the experiment. Moderate to sever histopathological abnormalities in liver sections including congestion, ceroid formation, hemorrhages, pyknosis, karyorrhexis, karyolysis, binucleated hepatocytes, nuclear hypertrophy and eccentric nuclei, vacuolar degeneration were observed in different at days 45 and 60 of the experiment (Fig. 3). Sever histopathological abnormality of liver in fish (A.nobilis) exposed to 1000 µg/L and 1500 µg/L concentrations exhibited degeneration and vacuolar degeneration, karyorrhexis, karyolysis at days 45 and 60 of the experimental study. Microscopic observation of different section of brain of untreated brain tissues showed no pathological changes while different microscopic changes including intracellular edema, congestion, necrosis of neurons and cytoplasmic vacuolization were observed at days 45 and 60 of the experiment. Histopathological analysis of different sections of brain of bighead carp exposed to higher concentrations of BPA exhibited degenerated neurons in cerebellum, lipofuscin deposition, microgliosis, necrotic neurons, in ammatory cell and severe hemorrhage at days 45 and 60 of the trial (Fig. 4). Mild to moderate similar histopathological changes in various sections of brain of sh exposed to 1000 µg/L BPA were observed at days 45 and 60 of the experiment. Histopathological observation of different sections of heart of bighead carp exposed to higher levels of BPA (1000 µg/L and 1500 µg/L) showed neutrophilic in ltration, necrosis, in ammatory reaction, edema, neutrophilic myocarditis, hemorrhages and deposition of brin were observed (Fig. 5). Vaious histopathological ailments in intestine such as extensive vacuolation of enterocytes, in ammatory response, congestion, necrosis and sporadic hemorrhages were observed in bighead carp exposed to BPA (1000 µg/L and 1500 µg/L) at days 45 and 60 of the trial.

Discussion
Bisphenol A is a commercially used chemical, an additive in the production of polycarbonate plastics as a developing agent in the manufacturing of thermal paper and epoxy resins. Bisphenol A is also present in dental sealants, water bottles, and baby bottles, paper coatings, adhesives, ame retardants, food, and beverage packaging (Staples et al., 1998). Bisphenol A is one of the highest volume chemicals produced worldwide and its demand is increasing due to the ever-increasing demand and production of plastic products. Since aquatic environments are the ultimate sink of all anthropogenic chemicals, aquatic animals including sh are often exposed to these chemical compounds (Routledge et al., 1998, Metzler andErica, 2001). In the present study, Absolute organ weight increased and relative organ weight of all of the visceral organs (Gills, Liver, and kidney (Table: 1, 2) except the brain decreased in sh. Many reports on the relative organ weight of different vertebrates exposed to different toxicants are available in previous studies but scanty of information available about bisphenol A effect on absolute and relative organ weight of freshwater sh. In previous research work, similar results showed like decrease in the relative weight of visceral organs (liver, kidneys) was observed in sh (Gaffar et al. . Furthermore, it has been reported that bisphenol A induces adverse effects on serum biochemical index in adult sh, leading to a defect in growth performance and sh health (Wang et al. 2016). In the present study, serum biochemical parameters like ALT, AST, and ALP increased signi cantly in treated sh in association to stress induced by bisphenol A. Serum biochemical parameters like serum albumin quantity and serum total protein decreased in the present investigation. However, increased glucose, cholesterol, and lactate dehydrogenase level were observed due to stress conditions in treated sh. Serum creatinine and uric acid are essential factors for muscle and purine metabolism for renal safety and kidney function (Hamed and Tawwab, 2017). Urea and creatinine levels were also increased in the liver and kidney which indicated that disturbance in ltration mechanisms and damages of kidneys and liver tissues of sh exposed to bisphenol A in the current experiment. Many previous reports are also available in other species exposed to bisphenol A. Previously, abnormal liver, kidney enzymes, an increase in hepatic enzymes as ALT, ALP, AST, abnormal urea ad creatinine, fatty liver disease, edema, vacuolation of hepatocytes, abnormal structure of cells, degeneration of structural protein due to increase in hepatic In the current study, histopathological responses of the sh indicate the degree of damage caused by BPA to the liver of ah (A.nobilis). In this present research work, histopathological lesions in liver tissues of sh were congestion, decreased cytoplasmic space, vacuolar degeneration, increased sinusoidal space, karyolysis of hepatocytes, and necrosis exposed to the higher concentration of BPA. Similar results are available in previous other species of aquatic organisms like ruptured central vein, lipids like vacuolization, macrophage, and lymphocytes in ltration, ruptured and degenerated hepatocytes in Ctenopharyngodon Idella (Faheem et al., 2017), seabream (Carnevali et al., 2017) exposed to sublethal concentration of BPA. The current study suggests that bisphenol-A is capable of causing damage to vital organs (brain, gills, lungs, and liver) of sh at biologically appropriate concentrations, contributing to altered rates of enzymes that could potentially affect sh health and reproduction. If these sh with high BPA load are routinely eaten by humans may also cause similar health problems. In the current study, kidneys of bisphenol A treated sh also showed microscopic lesions as edema, ceroid formation, glomerular degeneration, Bowman's space, congestion atrophy of tubules, and atrophy of lumen of renal tubules. However, similar results as necrosis, vacuolation, aggregation of melanomacrophages, degeneration, blood congestion, cellular rupture, nuclear hypertrophy degeneration, pyknotic nucleus, and reduction of lumen were observed in other species of sh like Heteropneustes fossilis ( and deposition of brin were observed in heart of big head carp exposed to different concentrations of bisphenol A. In one of the previous reports, calci c aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration were con rmed by histopathology for high-level of BPA exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood ow) were observed in zebra sh (Brown et al 2019) exposed to bisphenol A. Few reports are available on histological changes of heart in sh exposed to bisphenol A. However, many reports of heart histology are available on rats. Potential Toxic Effect of

Conclusion
The results showed that BPA at sublethal concentration changes the hematological and biochemical parameters of sh, A.nobilis, and these parameters can be used to detect adverse effects of BPA in aquatic environments and to determine the physiological condition of sh. Histopathological studies are therefore conducted to con rm the degree of damage in vital organs of sh especially the liver. In contrast, the introduction of such compounds into rivers should be restricted, although carps (A.nobilis) are natural inhabitants of freshwater environments and are desired species in countries like Pakistan as food. The ndings of this research work have indicated that environmental contaminants probably had adversely affected the proximate composition of sh meat in A.nobili. However, more research work is required to verify these ndings Declarations Con ict of interest: All the authors carefully read the paper and declare that they have no nancial/personal con ict of interests. Funding: This research work was not funded any national and international funding agency and is part of PhD thesis.
Availability of data and materials: The datasets used and/or analysed during the current study are available from the rst author on reasonable request (Rabia Akram).