Procurement of experimental fish
A total of 120 C. gariepinus 16 to 40 cm SL and 200 to 250 bodyweights were procured from Freedom Fisheries Ltd., University Market Road, Nsukka, Enugu State, Nigeria, and immediately transported to the Fisheries Wet Laboratory, Department of Zoology, and Environmental Biology, University of Nigeria, Nsukka. The fishes were disinfected with 0.05% potassium permanganate (KMnO4) for 2 min to avoid any dermal infections, later acclimatized for 2 weeks in plastic tanks of 300 liter (L) capacity. They were fed daily with food (Coppens commercial feed) containing 40% crude protein. Food, fecal matter, and other waste materials were siphoned off and water changed daily to reduce ammonia content in the water. Dead fishes were also removed with forceps to avoid possible deterioration of the water quality. During acclimatization, the water was changed after 48 h with well aerated tap water.
Procurement of the test compound
A commercial formulation of lambda-cyhalothrin (600 gl−1) with batch number 160227 marked by Amanik Agro Investment Limited Lagos, Nigeria, was purchased at Ogige Local Market Nsukka, Enugu State, Nigeria.
Experimental design for sublethal exposure
The experiment consisted of 120 fish assigned into four treatments of 0.00, 2.5 × 10−4 μg/L, 5.0 × 10−4 μg/L, and 6.25 × 10−4 μg/L (A-D), each treatment consisted of 30 fishes with three replicates (10 fishes per replicate) (Fig. 1). Each tank contained 10 L dechlorinated tap water with 10 fishes. Fish maintained in dechlorinated tap water served as the control treatment (A) while the three other treatments were exposed to water containing 2.5 × 10−4 μg/L (B), 5.0 × 10−4 μg/L (C), and 6.25 × 10−4 μg/L (D) of LCT corresponding to 1/20, 1/10, and 1/5 of the 96 h LC50 value that were determined after the acute toxicity assay. The exposure lasted for a period of 28 days during which the fish were fed with small quantity of feed approximately 1% of total body weight about an hour before the test solution were renewed daily. The feeding was to avoid catabolism and subsequent mortality. On each sampling day (7, 14, 21, and 28), three to five fishes from each of the treatment groups including the control were sacrificed after anesthetizing with tricaine methanessulfonate (MS 222) to minimize stress. Blood samples were collected (through caudal alteration) for antioxidant enzyme test while gonads were harvested for histological studies. After the end of the sublethal exposure, the remaining fish in each of the concentrations were withdrawn from the exposure of the chemical and were placed in chemical-free water after which further observation were made after 7 days of the withdrawal.
Lipid peroxidation
LPO were examined by measuring malondialdehyde (MDA) formation as described by Sharma and Krishna-murti (1968) method. The MDA activity was expressed as nmol/protein after calculating with:
$$ LPO=\frac{OD\times 1000}{156\times mg\ protein} $$
Catalase activity
Catalase activity was examined as described by Acbi (1984) method. The CAT activity was expressed as U/mg protein after calculating with:
$$ CAT=\frac{\Delta OD\ of\ Test\times total\ volume\times 1000}{43.6\times 0.05 ml\ sample\times mg\ protein} $$
Superoxide dismutase
SOD activity was determined by measuring the inhibition of autoxidation of adrenaline at pH 10.2 at 30 °C as described by Misra and Fridovich (1972) method.
The SOD activity was expressed in U/mg protein after calculating with:
$$ SOD=\frac{\Delta OD\ of\ control- OD\ of\ test\ }{\Delta OD\ of\ control\ x\ mg\ protein}\times 100 $$
Assay for glutathione peroxidase
The activity of GPx was determined by monitoring the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase. The specific activity was determined using the extinction coefficient 6.22 mM cm−1 (Lawrence & Burk, 1976).
Glutathione reductase
GR was estimated by measuring the rate of conversion of NADPH using the method of Tayarani, Cloez, and Bourne (1989). Specific activity was expressed as μmol NADPH oxidized min−1 mg−1 protein, taking motor extinction coefficient of NADPH as 6300 m−1 cm−1 after calculating with:
$$ GR=\frac{\Delta OD\ change/\min \times 6.3\ }{mg\ of\ protein}\times 100 $$
Histological study of the gonad
At days 1, 14, and 28, fish were sampled from control and each treatment. The Gonad (testis and ovary) were harvested through dissection, preserved in 10% phosphate-buffered formalin for 24 h, dehydrated by a series of graded concentration of ethanol solution, embedded in paraffin, and sectioned at 5 μm thick using microtila. Tissue sections were routinely processed and stained with hematoxylin and eosin (H&E), examined by binocular microscopy while the photomicrographs were captured using motic camera (Robert, 2001).
Statistical analysis
Data were analyzed using Statistical Packages for Social Sciences (SPSS) version 20.0 (IBM Corp, Armonk, USA) and Statplus v5.9.8 (AnalystSoft Inc., Walnut, Canada). Two-way analysis of variance (ANOVA) was used to compare concentration of lambda-cyhalothrin and duration of exposure dependent effects. The means were partitioned using DMRT (Duncan multiple range test). Level of significance was set at p < 0.05.