## 4 Discussion  
Industrialization, urbanization, and agricultural production represent important sources of extraneous chemicals, including xenobiotic compounds, for living organisms. Figure 4: Erythrocyte osmotic fragility (hemolysis) curves obtained from bovine and equine blood samples exposed to Thiaclopird at 30 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, Thiacloprid at 60 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, Quaternium-15 at 0.1 mg/L, and Quaternium-15 at 1 mg/L. Xenobiotics, in particular pesticides and their careless use, can negatively affect the environment and animal and human health [34]. To the authors' knowledge, this is the first study assessing the effects of Quaternium-15 and Thiacloprid on erythrocytes parameters, including EOF in horse and cattle blood, through in vitro whole blood exposure. Despite being preliminary, the current study emphasizes the concept that all blood cells are an excellent target for evaluating the effects of xenobiotics. The investigation of tissue, including blood, in animal species, represents a valuable tool to assess their health status as well as to investigate the relationship between blood properties and habitat, as well as the species' ability to adapt to the environment. The evaluation of haematologic variables is one of the most common investigations performed in clinical practice. Within the haematologic profile, erythrocyte variables represent a valuable tool for the assessment of the severity of many pathologic conditions. According to the results gathered in the current study, a decrease in RBC, Hct, Hgb, and MCV was observed both in blood samples from horses and cows exposed to Quaternium-15 and Thiacloprid. According to previous studies, the decrease of RBC and related erythrocyte parameters (i.e., Hgb, Hct, and MCV) is mainly caused by the ability of these xenobiotics to interfere with the biosynthesis of haemoglobin in the bone marrow, thus causing a reduction in the life length of circulating erythrocytes [35; 36]. The decrease in RBC could also be due to the lipid peroxidation of their cell membrane and to the destruction of these cells already in the bone marrow [37]. There is also evidence that exposure to xenobiotics can affect their ability to incorporate iron and its destruction within erythrocytes [38]. In particular, decreases in Hgb, Hct, and MCV also appear to be induced by oxidative stress in erythrocytes, resulting in an increased production of ROS that can oxidise certain macromolecules such as phospholipids, DNA, and proteins [39]. Additionally, the flexibility of the erythrocyte membrane exposed to xenobotic compounds could be compromised. Therefore, tests of the integrity and elasticity of the RBC membrane have value for the assessment of the proper function of RBC. One method used to determine the membrane elasticity of RBC is the EOF test. The degree of resistance of RBC to lysis, as a result of a decrease in the NaCl concentration of their environment, is the basis of the EOF test [21]. Osmotic fragility is widely used both in the human and veterinary field to elucidate mechanisms of the influence of different factors on the osmotic properties of RBC membranes, such as shear stress and mechanical hemolysis, drugs [40], temperature [41], ultrasound effects and irradiation [42], physical exercise [43], peripartum and neonatal period [44]. As animals enrolled in the study lived in the same area of Sicily, an influence of pasture diet can be excluded. Additionally, although no environmental pollution problems have been documented in the area where the animals live, we cannot exclude the presence of other pollutants in that area. The EOF is important for assessing cell permeability and is considered a valuable biomarker for oxidative membrane damage caused by xenobiotics [45]. As the concentration of NaCl increased, an increase in haemolysis was noted in horse and bovine blood samples exposed to higher levels of Quaternium and Thiacloprid.