In recent decades, the impact of pesticides on the environment is becoming a major problem worldwide. The continuous use of pesticides is burden on the soil ecosystem and causes deterioration in its health, along with potential consequences on soil-inhabiting invertebrates, which are indicators of soil quality. Therefore, more and more detailed ecotoxicological data are needed to better understand its actual threats as pesticide use is unlikely diminish in the near future (Gunstone et al. 2021). Up to date, the ecotoxicological impacts of avermectins against land gastropods have been rarely studied. This prompted us to study the lethal and sub-lethal toxicity of ABM against T. pisana snails.
In current study, the acute toxicity data obviously showed that ABM has lethal action against T. pisana snails. Regardless to the route of administration (contact or dietary exposure), the obtained data are in a good agreement with previous results in which ABM has lethal toxic action against different land gastropod species; T. pisana (Gad et al. 2016; Radwan 2016), Massylaea vermiculata (Syn: Eobania vermiculata) (Gabr et al. 2006; Kandil et al. 2014; Abdelgalil et al. 2018; Hussein and Sabry 2019), Monacha obstructa (Gabr et al. 2006; Kandil et al. 2014) and Deroceras reticulatum (Airey et al. 1989). On the other hand, ABM at 0.2% spray has high potential usefulness in protecting rape seedlings from the slug, Arion lusitanicus, but non-lethal to the animal (Kozlowski et al. 2010).
It is well known that pesticide sub-lethal toxicity is measured using molecular and cellular endpoints, which are also used to assess modes of action, metabolic pathways and detoxification mechanisms (van der Oost et al. 2003; Moreira et al. 2020). The digestive gland (hepatopancreas) is the main target for the toxic effects of xenobiotics, such as pesticides, that play crucial role in the accumulation, metabolism and detoxification as well as the biosynthesis of energetic macromolecules for different essential functions in molluscs (Dallinger et al. 2002). Therefore, changes in the digestive gland biochemical parameters as biomarkers due to the sub-lethal doses of the compound intoxication have been widely utilized as an indicator to assess the toxic action of xenobiotics on snails. In order to get insights into sub-lethal effects on the survivor's snails that exposed to 20% and 60% LD50 ABM doses in our study, the energy reserves (glycogen, lipids and proteins) and enzyme activities (GST, γ-GT, LDH, AST and ALT) as usual biomarkers were assessed in T. pisana snails.
The role of biomolecules include lipids, carbohydrates and proteins are critical in triggering different types of biochemical, physiological and behavioral responses in living organisms (Yazdani et al. 2013). These bioenergetics parameters have been suggested as useful biomarker to detect the deleterious effects and toxicological mechanisms induced by environmental pollutants. Few studies have been done to evaluate the adverse effects of pesticides including ABM on the energy reserves of land snails (Radwan et al. 2008; Radwan and Mohamed 2013; Kandil et al. 2014). Therefore, this negative impact on the energy reserves as a result of pesticide exposure needs more investigations.
Lipids play a very important role in the normal functioning of cells. They not only act as a highly reduced form of energy storage, but also play a close role in the structure of cell membranes and organelles found in cells (Kandil et al. 2014). In this investigation, total lipids were significantly decreased in the survivors of ABM- treated snails with sub-lethal doses. The decreased level of lipids after treatment may be ascribed to the impairment of lipid biosynthesis, metabolism and/or utilization as an energy source for surviving under stressful conditions (Radwan et al. 2008; Shaurub and Aziz 2015). In agreement with our results, Megahed et al. (2013) noticed that total lipids significantly decreased in hemolymph of treated 4th instar larvae of Spodoptera littoralis with ABM, emamectin and spinosad at 24, 48 and 72 h.
Glycogen is an important component of living cells and a source of energy for animals. In our study, there were significant decreases in the glycogen contents of the survivors of T. pisana snails throughout the ABM-treatment periods. This depletion indicating animals are utilizing their energy reserves to cope with toxic stress (Tendulkar and Kulkarni 2012) or for increased rate of glycogen breakdown "glycogenolysis" (Ansaldo et al. 2006). The aforementioned findings are in coincidence with those of Riaz et al. (2019) who showed that the glycogen contents were significantly deceased in 4th and 6th larval instars of two geographically distinct Trogoderma granarium field populations exposed to LC20 of ABM, emamectin and spinosad alone and in various combinations.
Protein is an important organic constituent of animal tissue. It plays an important role in energy metabolism. Protein regulates the process of interaction between intra and extra cellular media (Remia et al. 2008). In the present study, decreases in total proteins of the survivor's snails exposed to 20 % LD50 of ABM were observed, however, total proteins were increased in the survivor's snails exposed to 60 % LD50 compared to the control. The obtained data clearly indicate that the changes in the content of proteins depends on the sub-lethal dose used. The increase in total proteins could be elucidated by increased the protein synthesis of animal in response to this stress. On the other hand, the decrease in total proteins under pesticide exposure could be due to the formation of lipoproteins usage to repair the damage of cells and/or for straight usage by cells for energy demands (Padmaja and Rao 1994; Radwan et al. 2008).
Our data confirmed the results of Kandil et al. (2014), where total protein levels in, M. vermiculata and Monacha obstructa were increased when the snails exposed to ABM as a contact poison. A single dose of 0.25 LD50 ABM significantly decrease the total proteins in male albino rats (El- Shafey et al. 2011). Moreover, Al-Kahtani (2011) showed that total protein levels in various organs and/or tissues in the tilapia fish (Oreochromis niloticus) decreased after exposure to 20 µg/L ABM for up to 96 h.
The GST enzyme is a part of the detoxification pathway II via conjugation of xenobiotics and/or endogenous compounds with glutathione (GSH) (van der Oost et al. 2003). Our data clearly indicate that ABM induced increment in GST activity of the survivors exposed snails throughout the experimental period. These data suggest that the elevation of antioxidant protection is associated with increased production of oxygen-free radicals, which can enhance antioxidant activity to prevent oxidative stress and protect cells from damage (Elia et al. 2007). An increase in GST activity is also detected in response to pollutants e.g., pesticides, resulting from their detoxification via the formation of glutathione conjugates (Saravana Bhavan and Geraldine 2000). Similar to our investigation, the activity of this enzyme was increased in the same snail species treated with 1/20 LC50 ABM for 2 weeks of exposure (El-Gendy et al. 2019). Enhancement of GST activity in the snail, Physa acuta treated with ABM during the periods of 12- 48 h exposure was also observed (Ma et al. 2014).
Among the enzymes commonly used to assess hepatic function, γ-GT is considered a reliable biomarker that is closely associated with the identification of damage caused by oxidative stress (O¨zer et al. 2008). This enzyme plays a central role in the re-synthesis of glutathione. In addition, Lee et al. (2004) suggested that it is inversely proportional to the levels of many other antioxidants. It is conceivable that the pro-oxidation effect of γ-GT activity is usually balanced by its established role in facilitating the uptake of precursors by the cell to promote the re-synthesis of GSH. Thereby allowing the rebuilding of cellular antioxidant defenses (Banerjee et a1. 1999). In the current study, a significant increase in γ-GT activity in the survivor's snails was noticed due to their treatment with the two sub-lethal of ABM doses. This enzyme elevation may be attributed to the significant tissue injury provoked by pesticides, even at low doses. These results are in line with Khaldoun-Oularbi et al. (2017) who recorded that ABM caused an increase in the activity of γ-GT in male and female rats, Rattus norvegicus at 14, 28 and 42 days. Likewise, there were significant increases in γ-GT activity after the isolated rat hepatocytes exposed to 10 and 100 µM of ABM, for 30, 60 and 120 min as compared to respective control (El-Shenawy 2010).
One of the ways for assessing the integrity of cell membranes is to determine the activity of LDH, an enzyme present in all organs and tissues (Kending and Tarloff 2007). LDH is an enzyme shared in the induction of anaerobic metabolism, and its assessment can be used for understanding the energy production in organisms that can occur either aerobically or anaerobically (Müller et al. 2012). In our study, ABM at sub-lethal doses caused a marked increase in the activity of LDH of the treated survivors snails, which indicates its ability to change the permeability of cell membranes, causing cell death, since an increased leakage of LDH into the serum indicates membrane degradation (Amin and Hamza 2005). Thus, it is considered a good biomarker for cell and membrane damage. The increasing energy demand of the organism during pesticide stress is achieved by using carbohydrates as the main and immediate source of energy (Umminger 1977). This may be accompanied by increased LHD levels as result of the role of LDH in converting the pyruvate into lactate. Previous studies recorded the enhancement of LDH activity in ABM-treated rats (El-Shenawy 2010; Mossa et al. 2017). However, ABM recorded no significant decrease in the LDH activity of hamsters uninfected and infected with Schistosoma mansoni (El-Kabbany et al. 2017).
Among the hepatocellular injury markers, transaminases enzymes ALT and AST are probably the most commonly used in both clinical diagnosis and research involving liver damage. Both enzymes are not only found in liver cells, but also in many body organs. Of the two, ALT is predominantly present in the cytosol of the liver and is present elsewhere at low concentrations and is therefore thought to be more specific for hepatic damage. Our results indicated that the activity of AST and ALT decreased in the survivor's snails treated with sub-lethal doses of ABM. The decrease in AST and ALT could indicate tissue damage in the snails as a result of the presence of ABM in their tissues. Thus, biochemical disorders and lesions of tissue and cellular functions can occur when the activity of both enzymes are deviate from the normal range (Radwan et al. 1992). In the literature, the effect of ABM on the AST and ALT activities fluctuate among activation, inhibition, and no effect. Previous studies reported that the activity of AST decreased in treated peach fruit fly, Bactrocera zonata than the untreated ones after treatment with ABM (Biomectin®) and spinosad (Tracer®), while the activity of ALT increased after treatment with Biomectin® and decreased after treatment with Tracer® compared to controls (Farag et al. 2017). Enhancement in ALT and AST activities were recorded in M. vermiculata and T. pisana after treatment with 0.1, 0.2 and 0.5 of LD50 ABM for 24 and 72 h (Hamza et al. 2020). No significant differences in the AST and ALT activities after treatment of hamsters uninfected and infected with Schistosoma mansoni with ABM were observed (El-Kabbany et al. 2017).