Biochemical And Histopathological Responses Of Biomphalaria Alexandrina To RIPEX (plant Growth Regulator)Ⅱ
Jun 02, 2023
3 Results
3.1 RIPEX toxicity
The toxicity results of RIPEX to Biomphalaria alexandrina after 1 day indicated that the values of LC50 and LC90 were 39.9 5 and 73.6 µL/L, respectively. Te LC10 and LC20 sublethal concentrations were 8 and 16 µL/L, respectively (Table 1 and Fig. 1).
3.2 Effect of RIPEX on the oxidative stress markers
The exposure to RIPEX affected the activities of SOD and GST and the levels of MDA in the digestive gland of B. Alexandrina (Table 2). Snails exposed to RIPEX at 8 µL/L showed an insignificant increase (p=0.6) in the activity of SOD compared to the control snails. However, B. alexandrina snails exposed to 16 µL/L for 7 days exhibited a significant reduction in SOD activity (p=0.05). GST activity significantly increased (p≤0.001) after exposure to both concentrations. The highest increase was noted in snails exposed to 16 µL/L for 1 day (39.7±3.2 U/mg) and the highest value at the concentration 8 µL/L was (33.7±1.7 U/mg) at 7 days, compared with the controls at 17.1±3.6 U/mg (after 1 day) and 22.6±1.3 U/ mg (after 7 days).
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Dependent on time intervals, exposure for 7 days caused a significant increase (p=0.003). The exposure to diferent concentrations of RIPEX caused a significant increase (p≤0.04) in the levels of MDA. The concentration of 8 µL/L increased the MDA level (45.06±4.1) compared to the control (16.4±2.1). Dependent on the time of exposure, MDA levels were 45.06±4.1and 56.1±0.7 nmol/g at the concentrations, 8 and 16 µL/L, respectively, compared to 13.5±0.7 nmol/g for the control (Table 2).
3.3 Effect of RIPEX on the activity of steroid hormones
Exposure to 16 µL/L caused a significant increase in testosterone concentration (52.6±2.5 nmol/L) (p=0.002) compared with control (34.6±2.3 nmol/L). While the concentration of 8 µL/L RIPEX caused a significant increase (p=0.005) in testosterone after 3 days of exposure compared with 16 µL/L. Dependent on the time intervals, there is no significance except the significant decrease in testosterone concentration which occurred at the 7 days (19.6±2 nmol/L) of exposure (p=0.003) compared with 3 days (47.3±6 nmol/L). Regarding estradiol, there was a significant increase (p=0.003) in the hormone concentration after 3 days of exposure to 16 µl/L (73.6±3.5 pg/mL compared with the control (43.3 pg/mL) and 7 days of exposure to 8 µL/L (56.3±1.5 pg/mL compared with the control (50.3±5.6 pg/mL) (Fig. 2, b).
3.4 Effect of RIPEX on immune potential enzymes
The exposure of B. Alexandrina to two concentrations of RIPEX caused a significant increase in the activity of the MPO enzyme (Fig. 3a) dependent on the time of exposure at 3 days (p=0.004) and 7 days (p=0.01). Exposure to 8 µL/L RIPEX, significantly increased the activity of MPO (55.3±4.8 mU/mg protein (p=0.004) compared to 15.4±1.2 mU/mg for the control. While 16 µL/L exposed snails the activity was (53.03±2.3 mU/mg protein and 73.9±2.8 mU/mg protein, p≤0.05). Also, the concentration of 16 µL/L caused a significant increase (p=0.05) in the activity when compared with 8 µL. Both concentrations (8 and 16 µL/L) significantly increased (p≤0.009) the activity of ADA when compared with the control (Fig. 3b) at all time points. The increase of the activity at 16 µL/L concentration. Dependent on the time intervals, the exposure for 7 days recorded a significant increase when compared with 1 day and 3 days (p≤0.05). The values of activity at7 days were 27.03±14 and 74.3±1.9 U/mg protein
3.5 Differential hemocyte count
Examination of hemolymph samples from B. alexandrina snails indicated three morphologically distinct types of hemocytes; hyalinocytes, round small, and spreading hemocytes. Hyalinocytes are characterized by their circular shape, clear, dense cell membrane, and large nucleus-to-cytoplasm ratio. It is represented by a high count in control (56.3±0.5, 1 day). Small round cells are intermediate cells with few cytoplasmic granules and no pseudopodia. Spreading hemocytes have diferent sizes, with a small nucleus and large cytoplasmic granules. They are phagocytic cells because they can form few and short-spreading pseudopodia. The exposure to the two concentrations of RIPEX increased the hemocyte count, and a significant increase was observed in the granulocytes of snails exposed to 8 µL/L (p=0.02) and 16 µL/L (p=0.006) for 1, 3, and 7 days compared to the control. (Table 3). The treatment caused some morphological abnormalities in hemocytes such as hyalinocytes with filopodial elongation, deformed granulocytes with vacuoles, and spreading filopodia (Fig. 4).
3.6 Effect of RIPEX on the histopathology of the digestive glands and ovotestis
The digestive glands of B. Alexandrina are composed of tubules, and each tubule consists of one layer of columnar epithelial cells differentiated into digestive and secretory cells, which are arranged around a central lumen (Fig. 5a). Te tubules are linked together by connective tissue. RIPEX exposure caused some alterations, such as degeneration in the connective tissue. Snails exposed to 8 µL/L showed the presence of cellular blebs, which indicate the death of cells and clogging of the lumen. Undigested food was observed (Fig. 5b–d). Snails exposed to 16 µL/L, exhibited degeneration of the lined cells with some pathological signs such as vacuolation and dilation of the lumen. The tubule became damaged, deformed, and necrotic (Fig. 5e, f).
Ovotestis of control B. alexandrina snails consist of several acini joined by connective tissue. Each acinus is lined with germinal epithelium composed of diferent stages of spermatogonia and oogonia. Tey differentiates into primary, secondary, and one or two mature oocytes (oogonia). Matured sperms are found in groups inside the acinus (Fig. 6a). Te histological structure of the ovotestis of exposed snails changed after 7 days of exposure to RIPEX. In snails exposed to 8 µL/L, an increasing number of sperms, necrotic sperms, and atrophy were observed. Deformation of male or female gametocytes and degeneration of acini were also abundant. Also, the exposure to 16 µL/L made the acini more degenerated and necrosis was increased (Fig. 6b–e).
4 Discussion
There is extensive use of PGRs in agricultural practices. These substances have numerous deleterious efects on living organisms. The present study aimed at assessing the toxicological impacts of RIPEX (an organophosphorus pesticide containing ethephon containing 48% ethephon as the active ingredient plus 52% inert ingredients). Toxicity assays of RIPEX with B. Alexandrina indicated that the 1-day LC50 and -LC90 values were 39.95 and 73.6 µl/L, respectively.
These results were supported by [54] who demonstrated the EC50 value for ethephon against Daphnia magna embryos (125 mg/L after 48 h of exposure). Tis pesticide seems more toxic to Biomphalaria than other organophosphorus compound formulations such as chlorpyrifos (LC50 9.6 mg/L; [30] and profenofos (LC50 1.10 mg/L; [10]).
A homeostasis exists between the generation of oxygen free radicals and the production of antioxidant molecules under normal physiological conditions [23]. An imbalance between the antioxidant and oxidant systems is known as oxidative stress [32]. In both eukaryotes and prokaryotes, superoxide dismutase (SOD) and glutathione S-transferases are crucial antioxidant enzymes that may remove too much oxygen from free radicals and shield the organism from oxidative damage [34, 38].
They also participate in the detoxification of several xenobiotics [8, 15]. The present study revealed that exposure of B. alexandrina snails to sublethal concentrations of RIPEX 48% EC disturbed the antioxidant activity of the exposed snails. Acute exposure (24 h) tends to reduce overall SOD and GST activities in snails, followed by an increase after 3 days of exposure and a sharp inhibition after 7 days of exposure.
MDA levels were significantly higher in exposed snails compared to controls after all exposure periods. These results suggest a state of oxidative stress in snails induced by RIPEX exposure. The snails experienced a sudden decrease in enzyme activities after 1 day of exposure and attempted to compensate by overproducing enzymes after 3 days of exposure. However, by the 7th day of exposure, snails were unable to overcome the excess production of reactive oxygen species, as manifested by the increase in MDA levels (lipid peroxidation), leading to an inhibition of antioxidant enzyme production. MDA is among the major impacts of lipid peroxidation and has the power to disrupt ion exchange in the cell membrane, modify ion permeability, and influence enzyme function [9].
The mechanism of RIPEX 48% EC-induced oxidative stress in Biomphalaria may differ from other organophosphorus pesticides. It caused enzyme inhibition only after 7 days of exposure. However, chlorpyrifos, for example, caused an increase in SOD activity and a reduction in GST activity in B. Alexandrina shortly after 1 day of exposure [30]. Testosterone and estradiol play a crucial role in the reproduction of B. alexandrina snails [20, 45]. Changes in these hormones can be used as an endpoint of chemical toxicity [14]. RIPEX 48% EC caused a hormonal disruption in B. alexandrina snails.
Snails exposed to 16 µL/L showed a significant increase in testosterone after 3 days of exposure. However, RIPEX decreased the hormone levels in snails exposed to 8 µL/L after 1 and 3 days of exposure. About estradiol, the hormone tends to increase in exposed snails. However, the most prominent increase was observed in snails exposed to 16 µL/L after 3 days of exposure. RIPEX may therefore have an endocrine-disrupting effect on B. alexandrina snails. Similar findings were also reported by Ibrahim and Hussein [30], who reported a significant increase in the levels of both testosterone and estradiol after exposure of B. Alexandrina to sublethal concentrations of chlorpyrifos 48% EC. Other agrochemicals, however, such as the oxyfluorfen (24% EC) herbicide, reduced testosterone and 17-estradiol levels in B. Alexandrina after two weeks of exposure [31]. Myeloperoxidase (MPO), which converts H2O2 to hypochlorous acid (HOCl), a potent antibiotic, is frequently regarded as the cornerstone of ROS-mediated cytotoxicity, and there is strong evidence that molluscan hemocytes have MPO activity comparable to that of mammalian phagocytes [22, 44, 49]. Adenosine deaminase (ADA) plays an important role in the immune systems of vertebrate and invertebrate animals, including Biomphalaria snails [51].
Its deficiency is linked to combined immunodeficiency disease [25]. Exposure of B. Alexandrina to 8 and 16 µL/L RIPEX caused a significant increase in the activities of MPO and ADA enzymes in the digestive glands of snails after 3 and 7 days of exposure. The increase in enzyme activity seems time- and concertation dependent. The digestive glands are the primary site for the metabolism of chemical contaminants in gastropods [46]. The increase in these enzymes suggests an immunological response of B. Alexandrina to RIPEX toxicity since MPO, for example, can convert pollutants to free radicals via single electron abstractions [41], which can result in redox-cycling events that produce oxyradicals and possibly initiate lipid peroxidation [36].
Snails primarily rely on cell-mediated cytotoxicity to get rid of invading objects [5, 53]. The most well-studied defensive cells in snails are hemocytes. Hemocytes progress from small, rounded, agranular cells to large, granular, spreading cells via intermediate phases [6, 53]. Granular hemocytes mostly phagocytose foreign substances [28]. The exposure to two concentrations of RIPEX 48% EC increased the hemocyte count. Te hyalinocytes appeared with vacuoles in samples collected from exposed snails. A significant increase was noted in granulocytes, indicating an increase in the snail’s cellular immune response to challenge RIPEX toxicity. Ibrahim and Hussein [30] showed that exposure of B. Alexandrina to chlorpyrifos had negative impacts on the morphology and total hemocyte count of exposed snails.
Furthermore, light microscopy revealed that some hyalinocytes had shrunken nuclei, incomplete cell division, and pseudopodia formation. These efects on the hemocyte count and morphology of B. Alexandrina have a negative implication on the snails’ immunity, as these organisms depend largely on hemocytes for their protection against foreign encounters. Histopathological investigations of the digestive glands of B. Alexandrina showed that RIPEX exposure for 7 days caused some pathological alterations such as the degeneration of connective tissue, cellular blebbing, and the degeneration of lined cells in the lumen. The digestive tubules became damaged, deformed, and necrotic. Histopathology is an important endpoint in assessing the pathological changes in aquatic animals exposed to contaminants [33].
The high sensitivity of the digestive glands is directly attributed to its role in homeostasis, contaminant uptake, digestion, metabolism, and the detoxification process [24]. The observed efects may be due to direct accumulation of the RIPEX in the digestive gland cells or indirectly via oxidative damage caused by reactive oxygen species (ROS) production. Indeed, extreme oxidative stress has been linked to tissue damage involving a variety of physiological processes and environmental variables, including necrotic and apoptotic cell death [19]. Moreover, exposure to two diferent concentrations of RIPEX for 7 days altered the histological structure of ovotestis with an increase in the number of necrotic sperms, atrophy, deformation of male or female gametocytes, and degeneration of the acini. These changes are likely to reduce the reproductive potential of snails and, therefore, their fitness in the aquatic system.
5 Conclusion
Sublethal concentrations of RIPEX 48% EC had a multifaceted effect on Biomphalaria alexandrina snails. In snails, chronic exposure to RIPEX reduced the production of the antioxidant enzymes SOD and GST while increasing MDA production, indicating an oxidative stress state. Furthermore, it had an endocrine-disrupting effect on snails, as evidenced by an overall increase in steroid hormone levels. The snail's immune system appears to be vulnerable to RIPEX exposure. It caused a significant increase in the activity of MPO and ADA enzymes in snail digestive glands after 3 and 7 days of exposure, as well as a significant increase in granulocytes in exposed snail groups. Pathological abnormalities in exposed snails’ digestive glands and ovotestis were also signs of histopathological changes caused by RIPEX exposure. These findings show that RIPEX hurts aquatic fauna, including B. Alexandrina. Tus, Biomphalaria snails can be used as bioindicators for environmental pollution with plant growth regulators.
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