Fish Maintenance. Juvenile Clarias gariepinus (mean length = 10.73 ± 1.34 cm; mean weight = 12.34 ± 3.44 g) were purchased from Farm Project, Faculty of Agriculture, University of Benin, Nigeria, and maintained in a 160 L plastic tank for two weeks according to OECD guidelines for fish acute toxicity testing (OECD 2019) at the Laboratory for Ecotoxicology and Environmental Forensics at the University of Benin, Nigeria. Fish were fed with a pelleted commercial diet (Durante Fish Feed, Durante Fish Industries Limited, having 45% crude protein and 3000 Kcal/Kg metabolizable energy) to satiation once daily until 24 hours before exposure. Daily measurements of water physicochemical parameters, including temperature (26.64 ± 0.09°C), dissolved oxygen (6.18 ± 0.55 mg/L), and pH (6.74 ± 0.05), were taken. All experimental procedures were carried out in conformity with the relevant norms and standards established by the Research Ethics Committee of the College of Medicine of the University of Benin, approval number (CMS/REC/2020/34), and conducted following ARRIVE guidelines.
Chemical Exposures. Chemical exposure was conducted through a static renewal system for 96 hours, following OECD guidelines for fish acute toxicity testing (OECD 2019). Throughout the experimental period, the tanks were aerated continuously, and 70% of the water was replaced daily through siphoning. The treatment groups consisted of four concentrations of imidacloprid: 100, 130, 160, and 190 mg/L using a commercially available insecticide, Imi Force® (Jubaili™), containing an imidacloprid concentration of 200 g/L, and a control group. The treatment concentrations were achieved by mixing 10, 13, 16, and 19 mL of the Imi Force® insecticide in 20 L of water to get the required imidacloprid concentrations of 100, 130, 160, and 190 mg/L, respectively. To maintain a constant concentration, imidacloprid was added to each tank following the daily water exchange of 70%. To ensure the desired concentration of imidacloprid, water samples were collected from each tank daily during the exposure period, and their concentration was determined by GC-MS analysis. No insecticide or chemical was added to the control group. The concentrations and duration were selected based on previous studies and a range-finding test. Each group was comprised of two replicates (n = 6). The fish were not fed during the exposure period. Mortalities were counted daily for LC50 determination.
Behavioral Alterations and Mortality. The fish were inspected multiple times daily during the exposure period to record behavioral changes and clinical diagnostic signs through direct observation, according to OECD guidelines for fish acute toxicity testing (OECD 2019). The toxicological endpoint of this study was mortality. Mortality was recorded daily when the fish failed to respond to gentle prodding. The dead fish were removed immediately.
Sampling. All fish were euthanized after 96 hours of exposure to a lethal dose of benzocaine (0.5 g L− 1; Sigma-Aldrich). Samples were selected randomly from each aquarium (n = 2). To create a homogeneous mixture, the whole fish tissue (0.1 g) was ground in a mortar and pestle with 0.9 mL of ice-cold, distilled water. The mixture was then centrifuged at 3000 x g for 20 minutes at 4°C to obtain the supernatant, which was then used for additional analysis.
Determination of AST. AST activity was measured according to the method described by Reitman and Frankel (1957), using pyruvic acid as standard. The supernatant was treated with 2, 4-dinitrophenyl hydrazine solution and then 0.4 N sodium hydroxide after being incubated at 37°C for 1 hour. A spectrophotometer was used to measure the color intensity developed after 10 minutes at 520 nm, with the amount of oxaloacetate produced serving as an index of the AST level. The final estimation was determined using the AST calibration curve.
Determination of ALT. ALT activity was measured according to the method described by Reitman and Frankel (1957), with pyruvic acid as the standard. After incubating at 37°C for 1 hour, the supernatant was treated with 2, 4-dinitrophenyl hydrazine solution and then 0.4 N sodium hydroxide. The color intensity developed after 10 minutes was measured at 520 nm in a spectrophotometer, with the amount of pyruvate formed serving as an index of the ALT level. The final estimation was determined using the ALT calibration curve.
Determination of Total and Conjugate Bilirubin. Randox reagents from Randox Labs Ltd., Antrim, United Kingdom, were purchased for total and conjugate bilirubin determination. The bilirubin levels were determined using the Jendrassik and Grof (1938) method. This method is based on the reaction between bilirubin and diazotized sulphanilic acid in the presence of hydrochloric acid and sodium nitrite, which generates a pink azobilirubin complex. The unconjugated bilirubin requires an accelerator (caffeine) to form the azobilirubin complex, while the conjugated bilirubin combines directly with the diazotized sulphanilic acid. The pink acid azobilirubin can be converted into blue azobilirubin by using an alkaline (sodium) tartrate reagent. To measure the levels of total and conjugate bilirubin, the optical density of the colored complex was determined using a spectrophotometer at 580 nm and 540 nm, respectively.
Determination of Amylase. The Bernfeld (1955) method was used in determining amylase activity. A substrate solution containing 1% starch and 1mL of 0.1M phosphate buffer was mixed with 0.5 mL of the supernatant and incubated at 40 oC for 10 minutes. The reaction was stopped by adding 2 mL of 3,5-dinitrosalicylic acid, followed by 2 mL of distilled water. The test tubes were then heated in a boiling water bath for 5 minutes and cooled. A blue-black color was produced using a 3,5-dinitrosalicylic acid reagent, and the intensity of the color was measured with a spectrophotometer. The amount of hydrolyzed starch was proportional to the decrease in color intensity.
Determination of Lipase. The Bier (1955) method was used to measure lipase activity by examining the breakdown of triacylglycerols, diacylglycerols, and monoacylglycerols into free fatty acids in an olive oil emulsion. First, 500 mL of a 1% polyvinyl alcohol solution was mixed with 5 mL of 0.1 N HCl and heated for 1 hour at 75–85°C. The mixture was then cooled, filtered, and adjusted to pH 8.0 with 0.1 N NaOH. Next, pure olive oil was added to an aliquot of the solution to create a substrate concentration of 0.1 M, and the mixture was emulsified for 5 minutes. The reaction mixture was composed of 1 ml of the emulsified substrate in polyvinyl alcohol solution, 0.5 ml of McIlvaine buffer at pH 8 (prepared from 0.1 M citric acid and 0.2 M bisodium phosphate), and 0.5 ml of enzyme extract. The reaction mixture was incubated for 4 hours at 37°C, and then 3 mL of a 1:1 ethanol-acetone solution was added to stop the reaction and break the emulsion. The reaction mixture was titrated with 0.01 M NaOH after adding a few drops of 1% phenolphthalein in ethanol. Porcine type-II pancreatic lipase was used as a standard, and lipase activity was defined as the hydrolysis of 1.0 microequivalent fatty acids from triacylglycerols in 1 hour at pH 7.7 and 37°C.
Determination of E2 and Testosterone. The levels of 17β-estradiol and testosterone were quantified by the enzyme-linked immunosorbent assay (ELISA) method using the commercial kit (Cayman Chemical, USA). For all samples, the ELISA was conducted in duplicate, and a separate standard curve was generated for each ELISA plate. To validate the levels of the steroids, various serial dilutions of the sample were prepared and run in parallel to the relevant standard curve.
Determination of GST. The activity of GST was determined using the method of Habig and Jakoby (1981). The assay involved the use of 2 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 2 mM reduced glutathione (GSH) in 0.1 M potassium phosphate buffer at pH 7.0. The absorbance was measured for 2 minutes at 340 nm and at 30°C to obtain better detection using a microplate reader. To determine the enzyme activity, a standard unit (U) of GST was defined as the amount of enzyme that catalyzes the conjugation of 1 µmole of substrate per minute.
Determination of CA 19 − 9. The CA 19 − 9 level was determined according to Minamide, Hosoi, and Yanagi (2000). A volume of 100 µl of each sample was added to an equal volume of biotinylated monoclonal antibody against CA 19 − 9. The mixture was incubated for 30 minutes at room temperature. After washing the plate, streptavidin-peroxidase conjugate was added and incubated for another 30 minutes. Finally, the plate was washed, and the substrate solution was added. After 10 minutes of incubation at room temperature, the reaction was stopped, and the absorbance was measured at 450 nm using a microplate reader. The concentration of CA 19 − 9 was determined by comparing the absorbance of each sample with a standard curve prepared using known concentrations of CA 19 − 9.
Determination of CRP. CRP was determined according to the method described by Lowry et al. (1951). The supernatant was first diluted in phosphate buffer to a concentration of 1:10,000. Then, 0.2 ml of this dilution was mixed with 0.1 ml of antiserum against CRP and incubated for 2 hours at 37°C. After this, 0.1 ml of a 1% solution of staphylococcal protein A was added to the mixture and incubated for another hour at 37°C. The mixture was then centrifuged, and the pellet was washed with 0.9% saline solution. Finally, the pellet was dissolved in 0.5 ml of 0.1 N NaOH, and the absorbance was measured at 280 nm. The amount of CRP in the sample was determined by comparing the absorbance of the sample with the absorbance of a known amount of CRP that was precipitated under the same conditions. The concentration of CRP was expressed in milligrams per liter (mg/L).
Determination of TAC. The TAC was measured using the Ferric Reducing Ability of Plasma (FRAP) assay as described by Teimouri et al. (2019). First, 10 µL of the sample was added to 200 µL of freshly prepared FRAP reagent, which consisted of 10 mM 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl, 20 mM ferric chloride (FeCl3), and 300 mM acetate buffer (pH 3.6). The reaction mixture was incubated for 6 minutes at 37°C, and the absorbance was measured at 593 nm using a spectrophotometer. The FRAP reagent alone was used as a blank control, and the absorbance of each sample was compared to a standard curve of known concentrations of FeSO4.
Determination of CAT. The Beer and Sizer (1952) method was used in determining the CAT levels. Hydrogen peroxide was used as the substrate, and the decrease in its concentration was monitored spectrophotometrically at 240 nm in phosphate buffer (50 mmol, pH 7.0) at 22°C. The CAT activity was defined as the amount of enzyme that catalyzes the decomposition of 1 µmol of H2O2 per minute.
Determination of GPx. GPx activity was determined using the method of Hafemann, Sunde, and Hoekstra (1974). The assay mixture contained 0.1 M phosphate buffer (pH 7.0), 1 mM NaN3, 1 mM reduced glutathione (GSH), 0.15 U/mL glutathione reductase, 0.5 mM NADPH, and 0.25 mM cumene hydroperoxide. The reaction was initiated by adding 50 µL of the supernatant to the mixture, and the decrease in absorbance at 340 nm was monitored for 5 minutes at room temperature. The activity of GPx was calculated from the rate of decrease in absorbance. One unit of GPx activity was defined as the amount of enzyme that catalyzed the oxidation of 1 µmol of NADPH per minute.
Determination of SOD. The SOD was determined according to McCord and Fridovich (1969). The assay mixture contained 0.05 M carbonate buffer (pH 10.2), 0.1 mM EDTA, and 0.02 mM epinephrine. The reaction was initiated by adding the supernatant, and the absorbance at 480 nm was recorded every 15 seconds for 2 − 3 minutes. One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%.
Determination of T3, T4, and TSH. The quantification of T3, T4, and TSH levels was carried out using an ELISA kit (Millipore-Sigma, USA) following the manufacturer's instructions. Briefly, 100 µL of standards and supernatant were added to the wells of a microtiter plate coated with monoclonal antibodies specific to T3, T4, or TSH. The plate was then incubated for 2 hours at room temperature on an orbital shaker. After washing the plate three times with wash buffer, 100 µL of a biotinylated detection antibody was added to each well, and the plate was incubated for an additional 1 hour at room temperature. After another round of washing, 100 µL of streptavidin-horseradish peroxidase conjugate was added to each well, and the plate was incubated for 30 minutes at room temperature. Following a final washing step, 100 µL of a substrate solution was added to each well, and the plate was incubated in the dark for 10 − 15 minutes. The enzymatic reaction was stopped by adding 100 µL of stop solution, and the absorbance was measured at 450 nm using a microplate reader. The concentration of T3, T4, and TSH in each sample was calculated based on the standard curve generated using the standards provided in the kit.
Statistical Analysis. LC50 was calculated with probit analysis using Microsoft Excel 2016. Statistical analyses were conducted with GraphPad Prism (version 5.01). All data were analyzed using a single factor one-way analysis of variance (ANOVA), followed by Dunnett’s posthoc test. Statistical significance was determined at P < 0.05.