Histopathological alterations are considered dependable biomarkers of stress and are widely used as indices for pesticide toxicity in fish (van der Oost et al., 2003). Several experiments indicated that pesticides caused hepatotoxicity, hematotoxicity (Kalender et al. 2010; Lu et al. 2018), neurotoxicity (Das and Mukherjee 2000), genotoxicity (Giri et al. 2002), and nephrotoxicity (Al-Attar 2010) in different animal models. In this study, the EB-medication at the recommended dose caused about 2% mortalities, 25% reduced feed intake, and 6.52% reduction in biomass on day 21 of EB-dosing, which increased proportionately in a dose-dependent manner and interfered with various organs of O. niloticus similar to earlier studies (Julinta et al. 2020a,b; Singha et al. 2022). The histopathological observations of this study suggested a direct but dose-dependent relationship between EB-medication and the functioning of various vital organs and ultimately the safety of O. niloticus. As the most common route of pesticide intoxication is ingestion, the toxicants are absorbed and guided by the blood circulation to the liver for transformation and/or storage. The kidneys and liver are relatively vulnerable to toxic injuries as they are exposed directly to the blood plasma (van der Oost et al. 2003).
In the present study, the major kidney histopathological change due to EB-dosing was renal tubular epithelial degeneration and this process led to tissue necrosis at the higher doses. Tubular degeneration may occur in the form of vacuolization, the ragged appearance of the epithelial lining, atrophied epithelium, and necrosis (Heikal et al. 2012). Tubular vacuolization and necrosis perceived in the kidneys might be the consequence of the failure of ion pump transport of tubular cells and the inability of renal cells to cope with functional disorders incited by EB. Inflammation of renal tubules observed in the present study could be ascribed to disruption of cell volume and ion homeostasis by EB, possibly by increasing ion permeability and obstructing energy production, thus leading to ATP depletion. The EB-dosing also caused fragmented and shrunken glomeruli with dilated Bowman’s space in the 2.5X − 10X groups. Similar to this study, degeneration of renal tubules, inflammation, vacuolation in renal tubular cells, and widened tubular lumen were documented in different fish species subjected to varied pesticides (Ullah and Zorriehzahra 2015; Faheem and Lone 2017). The dilation of Bowman’s space suggested an increase in the filtration rate and a probable mechanism of counteracting toxicant stress. The observed mild but dose-dependent increase in the histopathological changes during the EB-dosing and post-EB dosing periods possibly indicated an impairment of the kidney function. The tolerability of O. niloticus to the recommended EB dose (1X) even during the extended dosing period was confirmed by the lack of major changes in the histological sections of the kidney. This study, in general, brought into light the renal toxicity induced by EB, which was found to be significant at the higher doses. The pathological alterations observed in the kidney of O. niloticus could be attributed to internal exhaustion as a consequence of the interaction between EB and renal tissue. These alterations could be understood as a defense mechanism against exposure to EB-medication and could be reversible. Contrarily, no histopathological observations were noted in EB-treated Atlantic salmon Salmo salar smolts (Stone et al. 2002).
The fish liver is the most delicate organ and hepatocyte alterations due to drugs may be useful biomarkers, as they occupy 85% of the teleost’s liver volume (van der Oost et al. 2003). Also, the liver is vulnerable to toxicants due to a large blood supply and its role in metabolism (Roberts 2012). Stressor-associated alterations of hepatocytes may be found in the nucleus or the cytoplasm or both. In this study, the histopathology of the liver of O. niloticus fed the EB-diets (1X − 10X) showed mild to severe glycogen-type vacuolation, indicating the loss of cytoplasmic hepatic glycogen as an early toxic response. The hepatocytes were vacuolated and lost the usual polyhedral shape. Such direct toxic effects may disturb the detoxification or cleansing mechanisms of the liver. Besides, all EB-dosed groups documented varying degrees of cytoplasmic vacuolation and degeneration of the hepatocytes similar to what Bowker et al. (2013) observed in O. mykiss fingerling fed 150 µg EB/kg biomass/day or other pesticides and toxicants (El-Murr et al. 2015; Lu et al. 2018). The existence of glycogen-type vacuolar degeneration of hepatocytes may be a consequence of extreme exertion required by the liver to get rid of EB-toxicant during the process of detoxification. Nevertheless, vacuolar degeneration is a revocable injury, and cells can revert to normal functions or homeostasis when the stress is removed (Roberts 2012). However, the recuperation of cells rests on the severity and duration of exposure to the stressors. The general feature of the liver of EB-dosed fish was the enhancement of the degree of structural heterogeneity with the increasing doses. Contrarily, during the gross necropsy or histopathological examination, no pathological signs of EB-toxicity were identified in S. salar and O. mykiss (Roy et al. 2000; Stone et al. 2002). Like several other insecticides, EB may critically affect the mitochondrial membrane transport system of hepatocytes (Gokcimen et al. 2007). In our previous study, the EB-dosed O. niloticus had significantly higher ALT and AST levels on day 7 of EB-dosing than the control group (Julinta et al. 2020b; Singha et al. 2022) and these changes were consistent with the injury to the hepatic tissues of the EB-dosed groups.
In fish, the first organ to interact with medicated feeds is the intestine and the wide microvilli structure results in a large surface area that is well suited for absorption. The elements absorbed through the intestine are dispersed to the liver via the portal vein, as over 75% of the total blood flow to the liver comes from the intestines (DeSesso and Jacobson 2001). Therefore, the intestinal and liver metabolism can alter the oral absorption of a drug (Zhang and Benet 2001). The control fish intestine was characterized by the existence of well-differentiated enterocytes with many absorptive vacuoles and goblet cells. On the other hand, all EB-dosed groups documented dose-dependent inflammation, loss of absorptive vacuoles, necrotized intestinal villi, mucinous degeneration, necrotized absorptive region, and degeneration of the epithelial layer in the intestine. Similarly, the intestine of Oreochromis spp. subjected to varied pesticides revealed mucinous degeneration, epithelial degeneration, inflammatory cell infiltration in submucosal edema, and alterations in the intestinal wall (Soufy et al. 2007; El-Murr et al. 2015). The degenerative changes observed in the different intestinal layers may be due to a reduction in oxygen supply to the tissue (Miller and Zachary 2017). The 5X and 10X groups recorded swelled lamina propria, which may be the direct effect of higher EB-doses for a longer period, which may reduce the lamina propria facilitated contractile activity of the intestine.
The degeneration of the epithelial layer and inflammation in the intestine of EB-dosed O. niloticus indicated a failure in the functional integrity of the intestinal mucosal epithelial cells and the harmonized regulation of the mucus layer, the intercellular tight junction, epithelial cells, and the host’s immune responses (Dharmani et al. 2009). The histological examination also revealed necrotized intestinal villi and absorptive region, mucinous degeneration, and loss of absorptive vacuoles in EB-dosed groups, suggesting a breach of the first line of defense. The direct EB-toxicity might have caused intestinal irritation and destruction of the mucous membrane. The histopathological variations observed in different intestinal layers of the present study may be due to the direct consequence of EB. The results revealed that the recommended dose of EB can cause apparent pathological changes in the intestine of O. niloticus during extended feeding. Though the nervous system is the target of EB-toxicity (USDA 2010), the neurotoxic nature of EB was not attempted in this research. Yet our earlier study (Singha et al. 2022) established the neurotoxic potential of EB. Overall, the EB-dosing experiments in healthy O. niloticus suggested the hepatotoxic, nephrotoxic, and intestinal toxic potential of EB in a dose-dependent manner similar to several earlier studies (Julinta et al. 2020a,b; Singha et al. 2022).