## 2 Materials and Methods  
From 10 horses (Sella Italiana breed, aged 4-5 years) and 10 cows (Simmental breed, aged 3-4 years) living in the same area of Sicily, Italy (38\({}^{\circ}\)00\({}^{\prime}\)49\({}^{\prime\prime}\) N 15\({}^{\circ}\)25\({}^{\prime}\)18\({}^{\prime\prime}\) E, 80 m above sea level), blood samples were collected using 5 mL vactainer tubes containing ethylenediaminetetraacetic acid (EDTAA) upon the owner's informed consent. Before starting the study, horses and cows were subjected to clinical examination, routine haematology, and biochemistry analyses to assess their health status. Whole blood samples collected from horses and cattle were divided into 5 aliquots of 1 mL each (one aliquot represents the control blood sample which was not exposed to xenobiotics, the other aliquots were exposed to Quaternium-15 and Thiacloprid at two different concentrations for each xenobiotic). Thiacloprid (purity 99.9%) was obtained from Sigma-Aldrich, Cesko, Czech Republic, and was dissolved in distilled water. The whole blood collected from horses and cattle was exposed for 24 h at 37 \({}^{\circ}\)C to Thiacloprid at a concentration of 30 \(\mathrm{\SIUnitSymbolMicro mL}\) and 60 \(\mathrm{\SIUnitSymbolMicro mL}\), according to a previous study carried out on mammalian species [31]. The Quaternium-15 (hexamethylenettermine chloroallyl chloride) concentrations to which whole blood was exposed were 0.1 mg/L and 1 mg/L, according to its presence in the environment as previously demonstrated [27]. The Quaternium-15 was obtained from Alfa Aesar GmbH & Co. KG A Johnson Matthey Company, Karlsruhe, Germany. The haematological profile was investigated using an automated hematology analyzer (HeCo Vet C; SEAC, Florence, Italy). Specifically, the values of red blood cell (RBC), haematocrit (Hct), haemoglobin (Hgb), mean corpuscular volume (MCV), white blood cell (WBC), and platelets (PLT) were assessed. Moreover, a manual analysis was performed on all whole blood samples to evaluate cell morphology. Specifically, two peripheral blood smears were performed for each sample, and after air drying, the obtained slides were stained through Dif-Stain kit (Titolchimica srl, Rome, Italy). The same laboratory professional later performed the microscopic analysis of blood films by using an optical microscope (Nikon Eclipse e200; Nikon Instruments Europe BV, Amsterdam, The Netherlands). For the determination of the erythrocyte osmotic fragility (EOF) test, a sodium chloride (NaCl) solution was prepared as previously described [33; 34]. Briefly, 10 plastic tubes without anticoagulant or other additives (Nunc, Thermo Scientific, Wyman Street, Waltham, MA, USA) were prepared with 3 mL of serially increasing concentrations of NaCl solution (0.0% up to 0.9%) at pH 7.4, checked by a pH meter (HI 3220, Hanna Instruments, Milan, Italy). An aliquot of 0.05 mL of whole EDTA blood was transferred to each tube. After gentle mixing, the tubes were maintained at room temperature (26-27 \({}^{\circ}\)C) for 30 min, mixed once more, and centrifuged at 327 g for 15 min. The supernatant was transferred to a glass cuvette, and the concentration of haemoglobin in the supernatant solution was assessed by employing an automated analyzer UV Spectrophotometer (Slim SEAC, Florence, Italy) at 540 nm. For each tube, haemolysis was expressed as a percentage, with a 100% maximum after haemolysis in straight distilled water (0.0% NaCl concentration). The percentage of haemolysis was calculated according to the formula [33]:  
\[\text{Haemolysis (\%): = (OD of test/OD of distilled water) \times 100}\]  
where OD is optical density.  
The EOF curve was obtained by plotting the percentage of haemolysis at each saline concentration.