Pro-oxidant potency of clothianidin in rainbow trout

The choice of trout was based on earlier evidence that this fish is highly sensitive to environmental changes (24) and can serve as a reliable bioindicator of acute and chronic toxicity (25). We used 48 10-month-old specimens (weighing 139.02±15.26 g and 19.23±1.68 cm long), purchased from a local breeder (Tunc Alabalık-Sanliurfa, Birecik, Turkey) and acclimatised to laboratory conditions for two weeks. They were held in 150 L water tanks with aerated tap water (under the 12/12 h dark/light cycle) and fed daily with commercial trout food (Optima trout-Skretting, Muğla, Turkey) at a rate of 2 % of their body weight throughout acclimatisation and exposure to clothianidin, except for the last 24 h before killing and immediate dissection.

The physico-chemical properties of water were as follows: temperature 16.96±0.32 °C, pH 7.5±0.34, dissolved oxygen 8.5±0.67 mg/L, and conductivity 731.25±14.34 μS/cm. These were maintained and checked twice daily with a multi-parameter water meter (84051 AZ Combo Water Meter-pH/COND./SALT/TDS/D.O, AZ Instrument Corp., Taichung City, Taiwan) before and after renewal of test solution (42 measurements in total).

The experimental protocols were authorized by the local Ethics Committee of Animal Experiments of Gaziantep University, under protocol No. 92 and decision No. 2019/13.

Following acclimatisation, the trout were divided into four groups of 12. One served as control and other groups were exposed to three sublethal and environmentally relevant concentrations of analytical standard (≥98 % purity) clothianidin (PESTANAL^TM, MilliporeSigma Supelco, Munich, Germany) falling within reported ranges in water sources (2, 26, 27, 28), namely 3, 15, and 30 μg/L. To obtain desired concentrations in the water tanks, clothianidin was dissolved in test water to prepare stock solutions and then diluted further as described elsewhere (29). These concentrations were checked and maintained daily through renewal bioassays.

Subacute toxicity was determined at 7-day sampling intervals (on days 7, 14, and 21).

We determined oxidative stress through its adverse effects on lipids (lipid peroxidation) and proteins (protein carbonylation), activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and the level of glutathione (GSH) as radical scavenger (30, 31, 32, 33), while the selection of the heart and spleen tissues in which they were measured was based on their role in general fitness and adaptation to environmental conditions (heart) and the function of the immune system (spleen).

At the end of each exposure period, four randomly selected fish from each tank were anaesthetised with tricaine mesylate (MS-222, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and euthanised by spinal cord section. Heart and spleen were carefully removed, washed with ice-cold physiological saline (0.59 % NaCl), weighted, and stored at -80 °C until analysis. The weights of each heart and spleen were used to calculate heart and spleen somatic indexes (HSI and SSI) according to following formula:

Frozen tissue samples were homogenised with 0.1 mol/L pH 7.4 phosphate buffer saline (PBS) (1:5 w/v, 0.2 g tissue/1 mL buffer) and centrifuged at 9744 g and 4 °C for 30 min (Hettich Universal 320R, Sigma-Aldrich) to obtain post-mitochondrial supernatants.

MDA, GSH, and protein carbonyl were analysed with respective commercial enzyme-linked immunosorbent assay (ELISA) kits (Fish Malondialdehyde ELISA Kit, Cat. No E0017Fi; Fish Glutathione ELISA Kit, Cat. No EA0037Fi; Fish Protein Carbonyl ELISA Kit, Cat. No E0112Fi; Bioassay Technology Laboratory, Shanghai, China) using the quantitative sandwich method according to the manufacturer’s instructions. Optical density was measured at 450 nm with a Thermo Scientific Multiscan GO microplate reader (Thermo Fisher Scientific Oy, Ratastie, Finland) and the quantity calculated from the standard curve. The standard curve range for MDA was 0.05–30 nmol/mL, for protein carbonyl 1–400 ng/mL, and the sensitivity for GSH was 0.25 nmol/mL.

SOD, CAT, GPx, and AChE activities and protein levels in the supernatants were determined spectrophotometrically (Shimadzu UV Mini-1240, Shimadzu Corporation, Duisburg, Germany). SOD activity was analysed as described by McCord and Fridovich (35) through iodonitrotetrazolium chloride (INT, Sigma-Aldrich) reduction monitored at 505 nm. The assay mixture was as follows: 0.01 mol/L phosphate buffer (pH 7.0), 80 U/L xanthine oxidase (Sigma-Aldrich), 0.05 mmol/L xanthine (Sigma-Aldrich), 0.025 mmol/L INT, and 1 mL of tissue homogenate.

GPx activity was measured by monitoring dismutation of t-butylhydroperoxide (Sigma-Aldrich) at 340 nm (36). The assay mixture contained 0.5 mol/L Tris buffer (pH 8.0), 0.1 mol/L GSH, 10 U/mL of glutathione reductase (Sigma-Aldrich), 2 mmol/L NADPH (Sigma-Aldrich), 7 mmol/L t-butylhydroperoxide, 660 μL of distilled water, and 1 mL of tissue homogenate.

CAT activity was measured through the degradation rate of hydrogen peroxide at 230 nm in a reaction mixture of 1 mol/L Tris buffer (pH 8.0), 10 mmol/L H ₂O ₂, 30 μL distilled water, and 20 μL tissue homogenate (36).

AChE activity was measured according to Ellman et al. (37) by recording absorbance at 412 nm in a reaction mixture of 0.1 mol/L pH phosphate buffer (pH 8.0), 0.015 mol/L acetylthiocholine iodide (Sigma Aldrich), 8.52 mmol/L ethopropazine (Sigma Aldrich), and 0.01 mol/L 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Sigma-Aldrich).

Tissue protein levels were determined according to the method described by Lowry et al. (38) using bovine serum albumin (Sigma Aldrich) as standard.

All data were analysed using the SPSS v. 22 software (SPSS Inc., Chicago, IL, USA). Their distribution was checked for normality (Q-Q plot) and homogeneity of variance (Levene’s test), respectively. Intergroup differences were assessed using one-way analysis of variance (ANOVA) followed by post-hoc least-significant difference test (LSD) (p<0.05). The strength of association between variables was tested with Pearson’s correlation analysis. Results are given as means ± standard error.

Heart and spleen somatic indices did not significantly differ from control on days 7 and 14 of exposure (Table 1). Significant changes were observed at 30 μg/L on day 21. Heart somatic index increased 45 % and spleen somatic index decreased 55 % (p<0.05).

In heart tissue, clothianidin exposure resulted in a 48 % reduction (p<0.01) in tissue protein level on day 21, while no significant changes were observed on days 7 and 14. A similar decreasing trend was observed for spleen tissue, with the highest reduction rate of 42 % and 47 % on days 14 and 21 (p<0.05) (Table 2).

GSH levels did not change significantly on days 7 and 14, but increased 216 % on day 21 day at 30 μg/L in heart tissue (p<0.01). In spleen tissue, the only significant change was the 90 % increase at the 30 μg/L concentration on day 14 (p<0.01) (Table 2).

Protein carbonyl levels showed an increase of 70 %, 69 %, 184 % in heart tissue on respective days 7, 14, and 21 of exposure to clothianidin at 30 μg/L (p<0.05). In spleen tissue, the same concentration increased protein carbonyl levels 32 % and 56 % on days 14 and 21 (p<0.01) (Table 2).

In heart tissue, MDA levels increased 150 %, 81 %, and 371 % at the highest pesticide concentration on days 7, 14, and 21 (p<0.05). In spleen tissue, MDA levels significantly increased on days 14 and 21, 54 % and 63 %, respectively (p<0.01) (Table 2).

Figure 1 shows changes in antioxidant enzyme activities in heart and spleen tissues. SOD sharply increased (198 %) only on day 21 in heart tissue, regardless of clothianidin concentration (p<0.01). In spleen, it increased earlier at all tested concentrations, and the highest increase was 123 % (p<0.01).

Figure 1

Similarly, heart CAT activity increased on day 21, reaching the maximum of 165 % (r=0.795, p<0.01). Spleentissue activity statistically rose at 30 μg/L on day 14 and at all tested concentrations on day 21 (max. 168 %, p<0.05).

GPx heart tissue activity also waited to significantly rise until day 21 at the highest clothianidin concentration (max. 194 %, p<0.01). In spleen tissue, antioxidant response to the highest concentration came earlier to reach 118 % and 44 % increase on days 14 and 21, respectively (p<0.01).

AChE activities started to significantly drop only at 30 μg/L on days 14 and 21 in heart tissue (25 % and 44 %, respectively, p<0.01) and on day 21 in spleen (27 %, p<0.05).

Table 3 shows Pearson’s correlation coefficients for all heart and spleen tissue parameters. Protein carbonyl and GSH levels and all antioxidant activities positively correlated with MDA in both tissues (p<0.01). AChE activity negatively correlated with MDA levels (p<0.01) only in heart tissue.