Multiple Biomarker Responses in Female Catsh (Clarias Gariepinus) Exposed to Acetaminophen

Acetaminophen is one of the most commonly detected Analgesics and pain killer drug in freshwater environments. This study evaluated the possible multi-toxic effects of environmentally relevant concentrations (15.5, 25.5, 35.5 and 45.5µg/L) of acetaminophen in Clarias gariepinus sh exposed for 28 days using multiple biomarkers. Hepatosomatic index (HIS) and condition factor (K) of acetaminophen–exposed group were not different from the control. The superoxide dismutase (SOD) activity increased signicantly at 15.5 and 35.5µg/L and Catalase (CAT) activity in all acetaminophen-exposed groups barely showed an upward trend. The concentration of glutathione S-transferase (GST) activities was not different from the control. Glutathione peroxidase (GPx) activities increased at all concentrations when compared to the control group. There were general inhibitions of Acetylcholinesterase (AChE) activities in all exposed groups including the control. Total antioxidant capacity (TAC) increased signicantly at 25.5 and 45.5µg/L and Interluekin-6 (IL-6) showed non-signicant increases in all exposed concentrations. Acetaminophen exposure caused non-signicant increases in the activities of C reactive protein (CRP). White blood cells (WBC) and lymphocytes (LYM counts) were signicant reduced. Acetaminophen induced signicant changes in hormones of the hypothalamic-pituitary-gonad (HPG) axis (17β-Estradiol and Testosterone) and vitellogenin (Vtg) synthesis at 45.5µg/L. Histopathological alterations in the liver was evident of apoptotic hyperplasia, sinusoidal congestion and necrosis of the hepatocytes and was concentration dependent. Acetaminophen exposure to the sh gills enhanced the fusion and shortening of some laments, hyperplasia of the epithelia gills cells, aneurism, congestion and epithelia rupture of the gills. Gonad examination showed acetaminophen exposure triggered the occurrence of intersex in 25.5, 35.5 and 45.5µg/L. The collaborative biomarkers used in this study showed the multiple impacts of acetaminophen on the physiology of C. gariepinus. Multivariate statistical analysis indicated that sh in the control groups exhibited a distinctly response from the acetaminophen-exposed sh and that over 95% of the biomarkers signicantly contribute to discriminate between the acetaminophen-exposed sh and the control group. Our research provides evidence supporting the use of multiple-biomarker approach to evaluate the health status of C. gariepinus in future studies.

A logarithmic dilution series was used for the range-nding assay. For the de nitive test, stock solutions of the required concentrations were prepared. 35.5g acetaminophen powder was dissolved in 1 L of distilled water. The stock was then diluted serially and the required concentrations were used for the de nitive test and prepared every 48hrs.

Measurement Of Biomarkers
Endpoints used to assess sublethal toxicity of acetaminophen include alterations in physiological condition indices (condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI)), Haematological alterations (Full Blood count), reproductive alterations (testosterone, 17β-Estradiol and plasma vitellogenin (VTG), histopathological alterations (gills, liver and gonad (for post-juveniles) and alterations in oxidative stress and antioxidant defence markers (SOD/Catalase, GST, Glutathione peroxidase (GPx), Acetylcholinesterase, Total Antioxidant Capacity(TAC)) and immunological (IL-6, CRP). After 28 days of exposure, the sh were anaesthetized with benzocaine 1%. Blood was taken by the perforating the genital opening with a heparin-coated 21 gauge ×0.5in needle, attached to a 2 ml syringe of steroid hormones. The sh were put down by medullar section. The liver, gills and gonads were used for histopathological analysis 2.4.1 Biomarkers Of Oxidative Stress 2.4.1.1 Superoxide dismutase activity (SOD) SOD activity in the supernatant was evaluated by the procedure of Magnani et al. 2000. SOD activity was measured by its ability to stimulate pyrogallol autoxidation. The pyrogallol autoxidation in the case of EDTA at pH 8.2 is 50%. Blood was centrifuged for 10 min at 3500 rpm to discharge the plasma. The centrifuged erythrocytes were made up of 2.0 ml of distilled water (40C) and disparate for 15 min. The assay was performed by applying 50 µl of supernatant to a clean tank. 1ml of SOD assay buffer was applied to the cuvette, followed by 1ml of SOD chromogenic solution added to the cuvette and combined for 1min. Absorbance was assessed at 420 nm immediately and after 2 min. The activity is expressed in SOD units per mg of tissue protein.

Catalase Activity
Catalase was determined by measuring the H 2 O 2 substrate remaining after the decomposition of hydrogen peroxide to H 2 O and O following the method of Ding et al., 2000. The assay reaction was performed at room temperature (25 0 C). 25ul blood agar was introduced into a test tube followed by 75ul assay buffer. Another 25ul of substrate was added, mixed and incubated for 15minutes, and then 825ul of the stop solution was introduce into the mixture and disparate. 10ul aliquot of the mixture was removed and introduced into another test tube. 1ml of the chromogen reagent was added and mixed thoroughly and allowed to cool for 15 minutes for colour development. The resultant colour is read at 520nm.

Glutathione Peroxidase Activity (Gpx)
Glutathione peroxidase activity in plasma was determined by the oxidation of Glutathione (GSH) by cumene hydroperoxides. The oxidized glutathione was reduced in the presence of glutathione reductase (GR) and NADPH. In this reaction the NADPH is oxidized to NADP + and the absorbance was measured at 340nm.The enzyme activity was expressed in µmol H 2 O 2 min-1 mg of protein −1 .
2.4.1.4. Total Antioxidant Capacity (Tac) TAC was determined by the reducing potential of the plasma to covert Cu 2+ to Cu + and the ion absorbance was measured at 450nm. Fish blood samples were centrifuge at 3000 x g for 12 minutes at 4 0 C and the plasma and aliquot were stored at -70 0 C. 10µL serum and 190 µL Dilution buffer were introduced to a 96-well microplate and read at 450nm. 50µL of chromogen were added to each well and incubated for 5minutes (25 0 C) and absorbance read for the second time at 450nm. CRP was determined using a quantitative sandwich enzyme immunoassay technique (ELISA) (Kindmark, 1972). Before assay, all reagents and samples were maintained at 25 0 C. Blood samples of the acetaminophen-exposed sh and the control were diluted 1:1000 by adding 5µl of samples to obtain 495µl of sample diluent. 10µl of Standard, diluted samples and control were added to the wells; 100 µl HRP-Conjugate solution was introduced into every well except blank wells; Air bubbles were drawn out from the liquid by tapping the holder, and mix properly and incubated for 60 minutes at 25 0 C. The liquid was removed from the wells and wells were rinsed 3times with 300ul of 1x buffer and plotted on absorbance paper. A solution of TB substrate was added to the well and placed in the oven for 15 minutes. Absorbance was read at 450 nm within 15 minutes after adding Stop Solution.

Il-6 Activity
Plasma IL-6 intensity was calculated using ELISA with a reactivity of 12.5 pg/mL using the accessible IL-6 ELISA sh kit as instructed by the producer.

Determination of Vitellogenin Elisa
Assessment of plasma VTG was determined using ELISA kit (Bioassay Technology Laboratory, China). The plates were supplied pre-coated with VTG sh antibodies. 40µl sh plasma (VTG) introduced into the plate binds to the antibodies coated with the well. 50µl biotinylated sh VTG is added and binds to the sample VTG followed by the addition of 50µl Streptavidin-HRP to the solution and incubated for 60 minutes at 37 0 C. Plates were rinsed 5 times with buffer followed by the addition of 50µl of substrate A and B simultaneously and incubated for 10 minutes at 37 0 C. 50µl of stop solution added and observed a colour change from blue to yellow. The optical density was read at 450nm.

17β-estradiol And Testosterone
The levels of testosterone (T) and 17β-estradiol (E2) in test sh were determined using Enzyme Immunoassay (EIA) kits (Cayman Chemical Company, USA). Blood samples were obtained using method employed by Alohan et al, (2014). Plasma samples were taken after the blood samples were spun at 7,000 g for 10 minutes. The plasma samples were aliquoted and kept at -20°C until they were analyzed. Methylene chloride was used to extract E2 from the aliquot of plasma, whereas diethyl ether was used to extract T. Under a nitrogen stream, the extracts were evaporated. By vortexing, the dry extract was reconstituted in 500 mL Enzyme Immuno-Assay (EIA) buffer. E2 and T were made in duplicate and measured. EIA measured the E2 and T levels using the method of the Azuadi et al. (2013) and absorbance was read at 405 nm.

Determination of Acetylcholinesterase
Sample preparation of blood sample was diluted 40-fold in Assay Buffer. 10ul of samples was introduced into a 96 well microplate. The blank well contained 200up of distilled water. 160ul assay buffer was introduced to the sample wells followed by 20ul ACHE chromogen. The absorbance was read after 2 minutes at 413NM and the absorbance was recorded. 10ul of ACHE Substrate was added, disparate and let to stand for 10min. The absorbance was read again at 412nm and the initial absorbance was recorded and values of acetylcholine calculated. "Organs were harvested and rinsed in saltwater to take out the blood and xed in Bouins's xative for 24 hours. The organs were dehydrated in a graded series of alcohol (ethanol) and xed in para n wax (M.P. 58-680c). Alterations caused by acetaminophen exposure in the organs were analyzed and photographed under photomicroscope along with control group. Tissue samples were sent to the Faculty of Basic Medicine, Department of Anatomy, University of Benin, for nal histological processing and preparation (HE staining) for light microscopy analysis".

Statistical Analysis
Statistical calculations were carried out with GraphPad Prism 8 software. Data were presented as mean ± Standard error of mean (SEM). The parameters were calculated using one-way ANOVA followed by the Dunnett's multi-comparison test was used to denote the signi cant differences between acetaminophen concentrations and the control treatment. Differences were considered signi cant at p≤0.05. Due to the large number of biomarkers analysed, Principal component analysis (PCA) was used to correlate Acetaminophen-exposed sh with the control group with the responses of biomarkers.

Physiological Condition Indices
The Fish morphometric indices (K and HIS) were not signi cantly affected (p>0.05) by the exposure to acetaminophen ( Fig. 1a & b), but lead to slight increases in all exposed groups as the condition factors of the shes increased by 24.14, 6.89, 3.

Oxidative Stress Biomarkers
The SOD enzyme activities were signi cantly increased (p<0.05) in in all acetaminophen-exposed groups compared to the control. The signi cant differential increases of 189. 29 The measured GST activities of sh are shown in (Fig. 2C). Acetaminophen exposures did not cause any noticeable change (p>0.05) in GST activities and were found to uctuate as GST activities were reduced in 15.50 and 35.50µg/L by 21.00 and 14.28% and increased in 25.50 and 45.50µg/L by 28.57 and 50.0% increase respectively (Fig. 2C).
Fish exposed to acetaminophen revealed a remarkable reduction in GPx activity compared to the control (Fig. 2D). The differential decrease in the GPx did not show a dose-response pattern with 74 Acetaminophen caused an increase in TAC activities in all exposed sh compared to the control. Signi cant increases were observed at 25.5 and 45.5µg/L (Fig. 3B).

Neurotransmitter
In term of AChE induction, there were no signi cant increases (p>0.05) in the acetaminophen treated groups compared to the control. Extraneous increases in AChE activities in 15.50, 35.50 and 45.50 were 08.09, 11.51 and 7.80% respectively and a non-signi cant reduction of 06.38% in 25.50µg/L (Fig. 3A).

Immunological Markers
Immunological analysis of IL-6 demonstrated that the exposure to acetaminophen caused non-signi cant increases of 21.02, 6.25, 34.09 and 21.02% at 15.50, 25.50, 35.50 and 45.50µg/L (Fig. 3C). C reactive protein activities were signi cantly increased (p<0.0001) in all acetaminophen exposed groups compared to the control (Fig. 3D)

Haematological Parameters
There were increases in WBC's of acetaminophen-exposed sh relative to the control. Increases of 101. HCT. Acetaminophen caused increases in MCV, MCH in all exposed groups while the MCHC count was not affected. PLT increased in all exposed sh is predicted to decrease at 15.50µg/L. Lymphocyte increased by 81.49, 90.34 and 48.03% in sh exposed to 15.50, 35.50 and 43.50 µg/L, while decreases in the community exposed to 25.50 µg/L were observed.

Reproductive Biomarkers
Only sh subjected to 43.5µg/L acetaminophen showed statistically signi cant increased (p<0.05) VTG levels than control sh. The levels of testosterone were signi cantly reduced in all acetaminophenexposed sh by 19.71, 13.84 and 37.12% in 25.50, 35.50 and 45.50µg/L (Fig. 4). Acetaminophen exposure caused 17β-Estradiol increases in all exposed group compare to the control. Fish from 35.5µg/L acetaminophen-exposed group had signi cantly lower level of 17β-Estradiol (p < 0.05) compared to controls. The levels of 17β-Estradiol in exposed sh increased by 41.58, 33.22, 30. Acetaminophen exposure caused histopathological alterations in the gill of exposed sh (Fig. 5). The common changes observed on the gills of acetaminophen-exposed sh were huge elevation of the lamellar epithelium, edema of the lamellae, blood congestion, exfoliated epithelium of lamellae, an increase of mucosal cells, lamellar disorganization, hypertrophy and hyperplasia of the epithelial cell which resulted in incomplete merging of the secondary lamellae and a reduced water space at 45.50, 35.50 and 25.50µg/L. Hemorrhagic telangiectasia, fatty lobes, biliary atresia, hepatic brosis, congestion, Kupfer cells, apoptotic hepatocytes, portal tract edema, single-cell necrosis, sclerosis cholangitis, biliary cirrhosis, coagulative necrosis, in ammatory cells in ltration characterized the gills of sh exposed to 15.50µg/L.

Liver
The alterations in the liver of acetaminophen-exposed C. gariepinus are shown in Fig. 6. Fish in the control group showed normal hepatic structure and parenchyma. The most frequent alterations found in the liver of acetaminophen-exposed sh were single-cell necrosis, hyperplasia, sinusoidal congestion, degeneration of the hepatocyte, sclerosis cholangitis, coagulative necrosis, focal necrosis, thickening of the tunica adventitia and portal tract edema. The severity of these alterations increased with increasing concentration. Hepatocellular carcinoma, dilation of central vein, cavernous hemangioma, biliary cirrhosis, hepatic steatosis, focal nodular hyperplasia, dilation of central vein, hepatic oedema, Portal tract edema, thickening of the tunica adventitia characterized the liver of sh exposed to 35.50 and 25.50µg/L (Fig. 6). Hemorrhagic telangiectasia, biliary atresia, apoptotic hepatocytes, portal tract edema, biliary cirrhosis, coagulative necrosis, in ammatory cells in ltration were common alterations observed in 15.50µg/L.

Gonads
Acetaminophen exposure caused histological changes in the gonads of the acetaminophen-exposed cat sh (Fig. 7). Ovarian examination of cat sh and the corresponding changes were observed at various concentrations of acetaminophen. There were no reported alterations in the gonad of sh gonads in the control group (Figure 7a) as the photomicrograph showed oocytes with perinuecleolar phase (Primary growth oocytes). Acetaminophen exposure caused several alterations in the exposed groups compared to controls, degenerative nucleus; degenerative follicles were noticed in the ovarian structure in the experimental groups. In the 15.50µg/L group, histopathological alterations were observed at minimum degree and include disintegrative follicles, necrosis and the destruction of muscles bundles (Figure 7b). An increase in the primary oocyte numbers and yellow patches was observed at 25.50µg/L (Figure 7c). The female gonad exposed to 35.50µg/L showed alterations which include increased atretic follicles, vascuolarized oocytes, and necrosis of the oocytes, the appearance of fat deposit (Yellow patches), degenerative nucleus and ovarian hyperplasia (Figure 7d). Disintegration in ovarian structures was observed for the highest concentration. (Figure 7d). Others include large occurrence of atretic follicles, yellow patches, necrosis and vascuolarized oocytes.

Multivariate analysis
The results of the PCA of the sh exposed to vary concentrations of acetaminophen plotted against the sh in the control are shown in Figure 9. There was a clear distinction between the sh in the control on the left site of the plot and the Acetaminophen-exposed sh on the right side of the plot. Figure 9 also reveals that the pattern of most biomarkers in acetaminophen-exposed-sh is strongly correlated with in increasing concentration.

Effect of Acetaminophen on Physiological Condition Indices and Oxidative stress biomarkers
Exposure to acetaminophen at environmentally relevant concentrations had distinct effects in female post-juvenile Clarias gariepinus. Morphometric analyses demonstrated that the HIS and K of the acetaminophen-exposed sh did show any signi cant change and this was similar to the report of Stancova et al. (2014) who observed no changes in growth parameters of Tench (Tinca tinca) exposed to a combination of ibuprofen, diclofenac, and carbamazepine for 35 days. Acetaminophen signi cantly reduced gonadosomatic index of exposed female C. gariepinus and agrees with the report of Mills et al. 2011 who showed a signi cant decrease in the gonadosomatic index of marine sh Cunner (Tautogolabrus adspersus) exposed to an anticancer drug, Anastrozole.
The biotransformation pathway of APAP has showed that the toxicity is as a result of the formation of the very active metabolite N-acetyl-p-benzo-quinone Imine (NAPQ1) by cytochrome P450s (Macherey, 2015; Ramos-Tavar and Muriel, 2019) when consumed at a non-pharmacologic doses. At the high doses, the pathways for the transformation of APAA (sulfation and glucuronidation) becomes saturated and a portion of APAP is excreted in the parent form leading to excess NAPQ1 which further reacts with other protein groups to form protein adducts (McGill et al., 2013) leading to oxidative stress and mitochondrial dysfunction resulting in the production of free radicals.
The increased SOD enzyme activities must have been triggered by the presence of acetaminophen which produced reactive oxygen species (ROS) (Halliwell and Gutteridge, 1999). As the levels of ROS increases, the biological system creates a rst-line defence mechanism by altering the behaviours of SOD (Roberts and Oris, 2004;Bagnyukova et al., 2006). Increased SOD activity is an indication of increased antioxidant status that attempts to neutralize the ROS effect (Kurutas, 2016). The elevated CAT activity in our study might be as a result of the functioning of the defence mechanism, which counteracts the oxidative stress induced by acetaminophen metabolism in exposed cat sh (Li et al., 2010;Shukla et al., 2017). Acetaminophens have been reported to disturb the redox status of the organism (Trachootham et al. 2008) and CAT level is an excellent marker of protein oxidation and lipid peroxidation (Bohn, 2019).
Several authors hitherto detailed the signi cance of GST, GPx, and GSH in averting cellular destruction (Livingstone, 2001;Guiloski et al., 2015). The depletions in GPx activities observed in acetaminophenexposed sh may have been a sign of the induced distress in the sh and agrees with the ndings of Guiloski et al. (2015) who reported a decline in GPx activities in gonads of H. malabaricus exposed to dexamethasone.
GST is a phase II detoxifying enzyme that catalyzes the conjugation of the reduced form of glutathione to xenobiotics for detoxi cation (Stancova et al., 2017). GST is known to be the rst line of defence preventing oxidative stress damage, decomposition of superoxide radicals and hydrogen peroxide before interacting to form reactive hydroxyl, which has many unfavourable biological consequences when present in high concentrations (Kaur et al., 2017). The increase in GST enzyme activity in exposed cat sh may have represented the occurrence of a defensive mechanism to prevent the impact of acetaminophen, as indicated by Sayeed et al. (2003).
The increase in TAC activity in exposed sh could have been triggered by the oxidative stress in the exposed sh, which may be due to an increase in antioxidant activity employing a cellular defensive mechanism to achieve homeostasis and, in some way, adaptation in the management of antioxidant stress (  Increased C-reactive protein (CRP) in all acetaminophen-exposed sh may have resulted from the entrance of pro-in ammatory cytokines into the circulatory system of the exposed sh due to the presence of acetaminophen (Gani et al. 2009). Our study revealed that acetaminophen induces the expression of CRP in exposed sh, which may be an indication that the synthesis of the CRP is part of the host defence reaction to the toxic effect of Acetaminophen. In the recent past, the innate immune system of sh has drawn attention and it's considered to be crucial in primary defence and adaptive immunity in sh (Whyte, 2007). Acetaminophen exposure caused elevated hepatocyte injuries as seen in the histopathological analysis of the liver and the increased level of CRP shows the protective function (Sproston and Ashworth 2018). Similarly, Ghosh et al. (1993) and Kodama et al. (2004) documented a rise in CRP activity in sh exposed to Carbaryl and formalin.

Immunological Biomarkers Alteration
Interleukin-6 (IL-6) is one of the most pleiotropic cytokines due to its function in both innate and adaptive immune responses and other physiological processes (Varela et al. 2012). Our study showed increased IL-6 activity in the acetaminophen-exposed sh. This increase may be an indication that IL-6 returned the exposed shes to a homeostatic condition and it has been stated that IL-6 runs to regulate the magnitude of tissue in ammatory responses (Gabay, 2006, Hong et al., 2013. Interleukin (IL)-6 is developed at the site of in ammation and plays a signi cant role in the acute phase response as de ned by a variety of diagnostic and biological characteristics, such as the production of acute-phase proteins (Gabay, 2006).
IL-6 is an in ammatory cytokine that is freed from monocytes, lymphocytes at the sites of tissue injury and may have been freed from the severe liver, gill injuries observed in this study (Gani et al., 2009).
Several authors (Kopf et al. 1994, Xing et al. 1994, Romani et al., 1996, Ruzek et al., 1997, Gabay, 2006 have noted that in chronic disorder, usually evidenced by immune stressors such as chronic intracellular infection and tumours, IL-6 not only induce acute phase reactions but also perform an important role in producing cellular immune responses to affected cells and mucosal humoral responses to re-infection.

Neurotransmitter Biomarker
The inhibition of AChE in all Acetaminophen-exposed sh suggests an adverse consequence in cholinergic neurotransmission, and probably in nervous and neuromuscular function (Ribeiro et al., 2017). The normalization of the enzyme activity in the exposed sh compared to the control can be due to the compensatory response of the sh cell to the toxic effects induced by acetaminophen exposures.
This study has shown the susceptibility of AChE activities in plasma of acetaminophen-exposed C. gariepinus (Mdegela et al., 2010). These results indicate that C. gariepinus is a model organism for pollutants monitoring and evaluation (Mdegela et al., 2006) and the activity of AChE in C. gariepinus is a prospective diagnostic tool for environmental pollution assessment of anticholinesterases.

Haematological Indices
There were increased responses in most haematological parameters of exposed sh except MCV and MCH after the 28 days exposure period. Acetaminophen-exposed sh showed an increase in WBC counts which indicate immune and protective response (Saravanan et al., 2011) to the presence of acetaminophen. The increased WBC counts show that C. gariepinus develop a protective mechanism to control the toxic stress caused by acetaminophen exposure (Mohammod Mostakim et al., 2015).
Similarly, ibuprofen and clo bric acid increased WBCs in exposed C. carpio and Cirrhinus mrigala (Saravanan et al., 2011. Endocrine-disrupting compounds (EDCs) disrupt hormonal pathways in some organisms, negatively in uencing reproductive performance (Martin and Voulvoulis, 2009). There was an anti-androgenic effect observed in all acetaminophen-exposed sh as a result of the increased aromatase activity which facilitated the transformation of Testosterone to Estradiol and therefore leads to a reduction of Testosterone (Fenske andSegner 2004, Baumann, 2012). There was a concentration-dependent increase in 17β-estradiol with the corresponding reduction of T. These alterations in Estradiol concentration and aromatase activity are signi cant as they can lead to the alterations in reproduction (Flippin et al. 2007; Han et al. 2010). Similarly, the report of Guiloski et al. (2015) showed a reduction in testosterone levels when male sh Hoplias malabaricus were exposed to anti-in ammatory drugs diclofenac and dexamethasone. Guiloski et al. (2017) revealed that the anti-androgenic impact of acetaminophen in male sh Rhamdia quelen, as the reduction in testosterone levels and a corresponding increase in the volume of 17β-estradiol were found in sh exposed to higher concentrations of acetaminophen.

Histopathology Biomarkers
Histological changes are associated with complex biochemical and physiological responses to any stressor (Lushchak et al. 2018). Tissue histology is seen as a sign of exposure to contaminants and is an essential apparatus for assessing pollution levels, especially for short-term exposure and prolonged effects ( The liver is primarily involved in metabolism, detoxi cation, storage, and removal of xenobiotics and their metabolites (Mela et al. 2007, Alimba et al. 2019. Liver morphology of acetaminophen-exposed sh showed several alterations which were concentration-dependent. Degenerated vacuolar, Kuffer cells activation, hepatocellular necrosis, single-cell necrosis, coagulation necrosis, focal necrosis, necrosis with in ammatory cells, spotty necrosis, necrosis characterized by hyperplasia with loss of cellular details and necrosis of hepatocytes characterized the liver. Matos et al. 2007;Sepici-Dinçel et al. 2009;Banaee, 2013 "reported similar histopathological alterations in the liver tissue of O. niloticus and C. carpio exposed to sub-lethal concentrations of carbaryl and cy uthrin, respectively". Acetaminophen cause liver injury in animals in large concentrations leading to increased liver enzymes (Kumar et al. 2004, Guiloski et al. 2017).
Gonad histological analysis proved useful in understanding and evaluating the impacts of possible endocrine-disrupting compounds on aquatic organisms (Leino et al. 2005). Acetaminophen-exposed female sh showed several altered ovarian structure with patches of degenerative follicles, follicular atresia, necrosis and degenerative nucleus. Atresia follicles were lower in lower concentrations of acetaminophen exposure. Increased follicle atresia in the ovaries of estrogen-exposed sh have been

Multivariate Statistical Analysis
Cazenave et al., 2009 highlighted the signi cant use of arrays of biomarkers when assessing the biological effects in polluted environments, since one marker may not show the true status of exposed organisms. Beliaeff and Burgeot (2002) have stressed the need for researchers to carefully select suitable fusion of markers that can yield data about universal detrimental environmental effects. The statistical analyses of our results distinctly con rm that the use of a single biomarker analysis was not enough to detect alteration induced by acetaminophen in the in exposed sh. Nevertheless, the assessment of multiple-biomarkers in physiological, biochemical, oxidative, reproductive, immunological and neurological activities in the C. gariepinus validate to visibly differentiate between the control groups and the acetaminophen-exposed groups. Multivariate statistical analysis ( Figure 9) indicated that sh in the control groups exhibited a distinctly response from the acetaminophen-exposed sh and that over 95% of the biomarkers signi cantly contribute to discriminate between the acetaminophen-exposed sh and the control group. Figure 9 denotes that acetaminophen-exposed sh may have undergone several stresses, possibly due to exposure to different acetaminophen concentrations.

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The consideration of multiple biomarkers in sh for the evaluation of single pharmaceutical will be a useful tool in assessing the effects of pharmaceuticals in exposed sh. The multiple biomarker approach in this study gives an insight to the capacity of APAP to cause multiple changes from cellular to organismal level and reduce the possibility of biasness in single biomarker evaluation. The multiple biomarkers provided more information on the overall health status of the APAP-exposed sh which include the induction of oxidative stress, activation of the immune response system, alteration of the haematological pro le and oxidative damage in many tissues. The studied organs (gills, liver and gonads) provided better information on the hepatotoxicity, neurotoxicity and immunomodulatory effect of APAP at varying concentrations. Our study has showed that multiple biomarkers evaluation revealed consistent evidence on the health status of the sh and provides better vigorous information obvious in justifying the multiple effects of APAP previously misjudged and documented. The multivariate analysis further justi es the use of multiple biomarkers in potential unknown impact of APAP in exposed sh. We therefore recommend that the antioxidant activities (SOD, TAC), immmunomodulators (IL-6, C-RP), haematological (WBC, LYM, PLT), intersex in gonad and hormones of the HPGH axis (testosterone and 17β-Estradiol) may be used as indicators for the evaluation of sh tness and toxicological impacts of PPCPs.

Declarations Ethical Approval
The use of sh for this study was authorized by the Animal Experimentation Ethics Committee of the University of Benin, Faculty of Life Sciences and all protocols followed the International Guidelines for Animal Use. Box and whisker plots of Acetaminophen-exposed sh and control group. Plot shows Minimum to Maximum values of oxidative biomarkers responses in cat sh (A) SOD (B) CAT (C) GST activity (D) GPx.
Values represent mean ± standard error of each concentration. Asterisk (*) shows the signi cant difference between the exposed group and the control *p<0.05, **p<0.01(n=7).