Histopathological changes in Melanopsis praemorsa exposed to imidacloprid and the protective effect of ascorbic acid

Abstract

The widespread use of neonicotinoid pesticides, particularly imidacloprid, poses significant risks to aquatic ecosystems. This study investigates the histopathological effects of sublethal concentrations of imidacloprid (4.016 µg/L, 40.16 µg/L, and 80.32 µg/L) on the hepatopancreas and mantle tissues of Melanopsis praemorsa and evaluates the protective role of ascorbic acid. Snails were exposed to imidacloprid, with and without ascorbic acid, under laboratory conditions for 7, 14, and 21 days. Histopathological analysis revealed severe tissue damage, including vacuolation in digestive cells, hypertrophy of basophilic cells, lipid accumulation, and necrosis in the hepatopancreas. In the mantle, epithelial desquamation, muscle fiber atrophy, and increased lipid vacuoles were observed. However, ascorbic acid mitigated these effects, reducing the severity of tissue damage. The extent of histopathological alterations correlated with imidacloprid concentration and exposure duration. These findings suggest that M. praemorsa can serve as a bioindicator for pesticide pollution, and ascorbic acid may play a protective role against oxidative damage in aquatic organisms.

Introduction

The rapid growth of the global population and the increasing demand for food have necessitated the widespread use of pesticides to maximize agricultural yields and minimize crop losses caused by pests and diseases. However, improper pesticide application can lead to environmental contamination, posing serious toxicological risks to ecosystems and human health^1–3. Pesticides can enter aquatic environments directly through application or indirectly through soil runoff and leaching, threatening the biodiversity of freshwater ecosystems⁴. Additionally, these contaminants may accumulate in the food chain, ultimately affecting human health^5,6.

Among the various classes of pesticides, neonicotinoid insecticides have gained prominence due to their high efficacy, systemic action, and long-lasting effects. These neuroactive insecticides are extensively used to control sucking and boring pests in crops such as cereals, vegetables, tea, and cotton. However, their prolonged environmental persistence and high mobility raise concerns about their unintended impact on non-target organisms^7–9.

Imidacloprid [1-(6-Chloro-3-pyridinylmethyl)-N-nitroimidazolidin-2-ylideneamine] is one of the most widely used neonicotinoid insecticides globally, first approved for agricultural use in 1994¹⁰. The U.S. Environmental Protection Agency classifies imidacloprid under toxicity class II/III, indicating moderate to low acute toxicity¹¹. The widespread use of neonicotinoid pesticides, particularly imidacloprid, poses significant risks to aquatic ecosystems. As a systemic insecticide, imidacloprid is highly water-soluble (0.610 mg/L at 25 °C) and persists in soil for months to years (half-life: 9–1250 days), enabling its pervasive infiltration into aquatic environments via runoff, leaching, and atmospheric deposition^12,13. Global monitoring studies have detected imidacloprid in surface waters at concentrations exceeding ecotoxicological thresholds, with reported levels reaching up to 320 µg/L in agricultural regions of the United States and Europe^1,14,15. Such concentrations are particularly alarming given the insecticide’s high toxicity to non-target aquatic invertebrates, including crustaceans, insects, and mollusks, even at sublethal doses^8,13. Imidacloprid’s environmental persistence and mobility contribute to bioaccumulation in aquatic organisms, disrupting food webs and cascading through ecosystems. For instance, declines in insectivorous bird populations have been linked to neonicotinoid-induced reductions in aquatic insect prey¹⁵. Freshwater mollusks, due to their sedentary nature and filter-feeding behavior, are especially vulnerable to chronic exposure, leading to impaired physiological functions, oxidative stress, and histopathological damage^16,17. Regulatory agencies, including the U.S. EPA and European Food Safety Authority, have flagged imidacloprid’s risks to aquatic life, prompting restrictions in some regions^7,9. Despite these concerns, gaps remain in understanding its sublethal effects on keystone molluskan species and potential mitigation strategies.

Imidacloprid, a widely used neonicotinoid, acts as an agonist of nicotinic acetylcholine receptors (nAChRs), causing their persistent activation and disrupting synaptic transmission. In insects, this leads to neuronal overexcitation, paralysis, and death. However, similar mechanisms also affect non-target aquatic organisms, where imidacloprid can cause sublethal effects such as behavioral, physiological, and reproductive impairments, ultimately threatening population stability⁴.

Ascorbic acid, commonly known as vitamin C, is an essential antioxidant that plays a critical role in neutralizing reactive oxygen species (ROS) and reducing oxidative stress^18,19. It is known to enhance cellular defense mechanisms, protect against oxidative tissue damage, and aid in detoxification processes²⁰. Given its antioxidant properties, ascorbic acid has been investigated for its potential protective effects against various environmental toxicants, including pesticides.

Histopathological studies are particularly useful in evaluating tissue-level alterations caused by contaminants and have been widely applied to assess the toxicological impacts of pesticides and heavy metals in aquatic organisms^4,16,21,22. Several studies have documented histopathological changes induced by acute and chronic pesticide toxicity in gastropods^23–26. However, research specifically examining the histopathological effects of imidacloprid in freshwater snails remains limited¹⁷.

Freshwater mollusks are recognized as highly sensitive to environmental pollutants and are widely employed as reliable bioindicators in aquatic contamination assessments^27,28. Their remarkable biodiversity, sedentary lifestyle, and capacity to bioaccumulate toxicants enable them to provide critical insights into the sublethal and chronic impacts of pollutants. M. praemorsa was selected as a bioindicator species owing to its wide distribution in Turkey, high reproductive capacity, sensitivity to pollutants, and ease of sampling^29,30. Accordingly, this study aimed to (i) investigate the histopathological effects of sublethal concentrations of imidacloprid on the hepatopancreas and mantle tissues of M. praemorsa, (ii) evaluate the protective role of ascorbic acid against imidacloprid-induced damage, and (iii) assess the potential of this species as a bioindicator for pesticide pollution in aquatic ecosystems.

Materials and methods

The M. praemorsa specimens used in this study were collected in May 2023 from an unpolluted water source near Yuvacık Town on the Diyarbakır-Bismil highway (37°48′8.51″N 40°25′14.05″E). They were transported to the laboratory in ventilated plastic containers, half-filled with water and containing aquatic plants to maintain natural habitat conditions. To ensure experimental consistency, only individuals of similar size (shell height: 10.1 ± 2 mm) were selected. The snails underwent a 30-day acclimation period in a controlled laboratory environment with a 16 h/8 h light–dark photoperiod, a constant temperature of 22 ± 1 °C, and an aerated aquarium. During this period, the samples were fed daily with Lactuca sativa leaves (specially produced in laboratory conditions without pesticides) and water quality was ensured by regular cleaning and oxygenation with an air compressor^31,32. The physico-chemical parameters of the tap water used in the experiments are presented in Table 1.

Table 1.

Physico-chemical characteristics of aquarium water prepared under laboratory conditions.

Physico-chemical Parameters	Value
pH	7.92
Conductivity (µS/cm)	353 µS/cm
Calcium (mg/L)	67.5 mg/L
Nitrate (mg/L)	2.85 mg/L
Nitrite (mg/L)	0.23 mg/L
Chloride (mg/L)	0.15 mg/L
Iron (mg/L)	0.61 mg/L

Acute toxicity tests were conducted in 5 L glass jars containing 2 L of dechlorinated tap water, with 30 healthy snails of uniform size per replicate. The snails were not fed during the test. Imidacloprid stock solutions were prepared using commercially available analytical-grade imidacloprid (35 g/L active ingredient; CAS 138261-41-3, purity ≥ 98%). Certified reference materials were not available for direct quantification, but all working solutions were prepared gravimetrically using a calibrated analytical balance (Mettler Toledo ME204, ± 0.1 mg precision) and volumetric glassware (Class A). Imidacloprid stock solutions were prepared gravimetrically using a calibrated analytical balance (Mettler Toledo ME204, ± 0.1 mg precision) and volumetric glassware (Class A). Analytical verification (e.g., HPLC/GC-MS) was not performed, but nominal concentrations were validated through adherence to OECD Guidelines for Chemical Testing³³ and APHA³⁴, protocols. Snails were exposed to nominal concentrations of 0 (control), 100, 150, 225, 338, 510, 760, and 1140 μg/L, with each dose increasing by 50% from 100 μg/L.

Mortality was recorded at 24, 48, 72, and 96 h, and deceased individuals were removed.

For the acute toxicity test, snails were exposed to concentrations of 0 (control), 100, 150, 225, 338, 510, 760, and 1140 μg/L. These concentrations were selected by applying a stepwise 50% increase starting from 100 μg/L. The 96-h median lethal concentration (LC₅₀) was determined to be 401.6 μg/L based on probit analysis³⁵. Sublethal concentrations were set at 80.32, 40.16, and 4.016 μg/L (1/5, 1/10, and 1/100 of the LC₅₀ value, respectively). Snails were divided into eight groups (30 individuals each) and placed in glass jars containing 2 L of aerated, settled water. The experimental groups were as follows: Group I: Control (no imidacloprid or ascorbic acid), Group II: Control + 7.5 mg ascorbic acid, Group III: 4.016 μg/L imidacloprid, Group IV: 4.016 μg/L imidacloprid + 7.5 mg ascorbic acid, Group V: 40.16 μg/L imidacloprid, Group VI: 40.16 μg/L imidacloprid + 7.5 mg ascorbic acid, Group VII: 80.32 μg/L imidacloprid, Group VIII: 80.32 μg/L imidacloprid + 7.5 mg ascorbic acid.

The experiment was conducted for 7, 14, and 21 days under semi-static conditions (daily water renewal)³³. All experiments included triplicate exposure groups (n = 30 snails per replicate) and negative controls (imidacloprid-free and ascorbic acid-free conditions) to account for baseline variability. Physicochemical parameters (pH, temperature, dissolved oxygen) were monitored daily using calibrated instruments (Hanna HI98194 multiparameter meter), with deviations outside ± 1 °C or ± 0.5 pH units triggering corrective actions. Semi-static exposure conditions minimized potential losses due to degradation or adsorption. While analytical verification (e.g., HPLC/GC–MS) was not performed, nominal concentrations were validated through rigorous adherence to OECD Guidelines for Chemical Testing³⁴ and protocols for aquatic toxicity assays³³.

To evaluate the histopathological effects of imidacloprid and the protective role of ascorbic acid, six snails from each group were anesthetized with 50 mg/L MS-222 (3-aminobenzoic acid ethyl ester methane sulfonate salt; Sigma) at each time point. Hepatopancreas and mantle tissues were then dissected for histopathological examination. Tissue fixation, dehydration, and embedding followed standardized protocols³⁶. Paraffin blocks were sectioned at 4–5 µm thickness using a Leica RM2235 rotary microtome, calibrated weekly to ensure consistency. Hematoxylin–Eosin (H&E) staining was performed under controlled conditions, with fresh staining solutions prepared daily to avoid cross-contamination. Slides were independently evaluated by two researchers to confirm scoring consistency, and discrepancies were resolved via consensus. Microscopic examinations utilized an Olympus BX51 light microscope equipped with a DP71 digital camera. The microscope was calibrated using a stage micrometer, and imaging parameters (e.g., magnification, light intensity) were standardized across all samples.

Histopathological scoring followed a validated semi-quantitative system^21,22. Two independent researchers scored the samples, resolving discrepancies through consensus: Absent (−): No detectable changes, Mild (+): Lesions affecting < 25% of tissue (e.g., minor vacuolation, slight epithelial desquamation), Moderate (+ +): Lesions affecting 25–50% of tissue (e.g., widespread vacuolation, moderate lipid accumulation), Severe (+ + +): Lesions affecting > 50% of tissue (e.g., extensive necrosis, tubule lumen collapse). Three non-overlapping fields per tissue section were analyzed at 40 × magnification, and inter-observer variability was assessed using Cohen’s kappa coefficient (κ = 0.85, indicating strong agreement).

Results

In the control group, the digestive gland was composed of oval-shaped tubules surrounded by loose connective tissue and hemolymphatic spaces. The tubule epithelium consisted of two distinct cell types: digestive cells and basophilic cells. Digestive cells exhibited a cuboidal to cylindrical morphology and contained cytoplasmic secretory granules of varying sizes. Basophilic cells were triangular in shape and characterized by the presence of yellow, refractile granules. No histopathological alterations were detected in the hepatopancreas of either the control or ascorbic acid control groups (Figs. 1A, 2A).

Fig. 1.

Histological structure of M. praemorsa hepatopancreas in control (Ascorbic acid and imidacloprid free medium) on day 21 (A), snails exposed to 4.016 μg/L Imidacloprid on day 21 (B), snails exposed to 40.16 μg/L Imidacloprid on day 21 (C), snails exposed to 80.32 μg/L Imidacloprid on day 21 (D). (HA hemolymphatic area, BC basophilic cell, DC digestive cell, YG yellow granule, A ameobocytes, N necrosis, LV lipid vacuole). H&E.

Fig. 2.

Histological structure of M. praemorsa hepatopancreas in ascorbic acid control (Imidacloprid free medium) on day 21 (A), snails exposed to 4.016 μg/L Imaclidoprid + 7.5 mg ascorbic acid on day 21 (B), snails exposed to 40.16 μg/L Imidacloprid + 7.5 mg ascorbic acid on day 21 (C), snails exposed to 80.32 μg/L Imidacloprid + 7.5 mg ascorbic acid on day 21 (D). (HA hemolymphatic area, DC digestive cell, BC basophilic cell, YG yellow granule, LV lipid vacuole, A ameobocytes). H&E.

Snails exposed to 4.016 µg/L imidacloprid showed mild histopathological alterations on days 7 and 14, while more severe changes were observed by day 21 (Table 2). These included vacuolation in digestive cells, atrophy in connective tissue, expansion of tubule lumens, and enlargement of hemolymphatic spaces. Lipid vacuoles and amoebocyte infiltration also increased significantly (Fig. 1B). In the 4.016 µg/L imidacloprid + ascorbic acid group, hypertrophy of basophilic cells and an increase in lipid vacuoles were observed by day 21, but overall histopathological changes were milder compared to the imidacloprid-only group (Fig. 2B).

Table 2.

Qualitative evaluation of Lesions in the hepatopancreas following exposure to imidacloprid and imidacloprid + ascorbic Acid.

Histopathological evaluation	Days	Imidacloprid and Imidacloprid + Ascorbic acid exposure Groups
		I	II	III	IV	V	VI	VII	VIII
Swelling and growth in basophilic cells	7	−	−	+	−	+	−	+	+
	14	−	−	+	−	+ +	−	+ +	+
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Lipid vacuoles accumulation	7	−	−	+	−	+	−	+ +	−
	14	−	−	+	−	+ +	+	+ +	+
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Atrophy of connective tissue between tubules	7	−	−	−	−	+	−	+	+
	14	−	−	+	−	+ +	−	+ +	+ +
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Distortionin hemolymphatic areas	7	−	−	−	−	+	−	+	−
	14	−	−	+	−	+	−	+ +	+
	21	−	−	+	−	+ +	+	+ + +	+ +
Expansion of tubule lumen	7	−	−	−	−	−	−	+	+
	14	−	−	−	−	+	+	+	+
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Amebocyte accumulation	7	−	−	−	−	+	−	+	−
	14	−	−	+	−	+ +	−	+ +	+
	21	−	−	+	−	+ +	+	+ + +	+ +
Increased dark granules	7	−	−	−	−	+	−	+	−
	14	−	−	+	−	+ +	−	+ +	+
	21	−	−	+	−	+ +	+	+ + +	+ +
Vacuolization in digestive cells	7	−	−	−	−	+	−	+	+
	14	−	−	+	−	+ +	+	+ +	+ +
	21	−	−	+	−	+ +	+	+ + +	+ +

Absent (−), Mild (+), Moderate (+ +), Severe (+ + +).

Following a similar pattern, snails exposed to 40.16 µg/L imidacloprid exhibited moderate histopathological changes on days 7 and 14, with more pronounced alterations by day 21. These included increased vacuolation in digestive cells, atrophy of connective tissue and basophilic cells, and further expansion of hemolymphatic spaces. There was also a notable accumulation of lipid vacuoles, dark granules, and amoebocytes (Fig. 1C). In the 40.16 µg/L imidacloprid + ascorbic acid group, hemolymphatic spaces continued to enlarge, and lipid vacuoles and dark granules increased, but the severity of tissue damage remained lower than in the imidacloprid-only group (Fig. 2C).

The most severe histopathological alterations were observed in snails exposed to 80.32 µg/L imidacloprid. On days 7 and 14, tissue changes were relatively mild; however, by day 21, digestive cells showed extensive vacuolation and necrotic changes, while connective tissue atrophy became more pronounced. Hypertrophy in basophilic cells was advanced, and tubule lumens and hemolymphatic spaces exhibited significant expansion. There was also a marked increase in lipid vacuoles and amoebocytes (Fig. 1D). In the 80.32 µg/L imidacloprid + ascorbic acid group, an increase in dark granules and lipid vacuoles was detected, but the overall severity of damage was reduced compared to the imidacloprid-only group (Fig. 2D).

In the control group, the mantle was covered by a single-layered ciliated cylindrical epithelium. The tissue contained muscle fibrils, lipid vacuoles embedded in connective tissue, mucus cells, pigment cells, and protein cells^22,37,38. No histopathological changes were observed in the mantle of either the control or ascorbic acid control groups (Figs. 3A, 4A). Histopathological changes in the mantle followed a similar pattern to those observed in the hepatopancreas, with severity increasing over time and with higher imidacloprid concentrations.

Fig. 3.

Histological structure of M. praemorsa mantle tissue in control (Ascorbic acid and imidacloprid free medium) on day 21 (A), snails exposed to 4.016 μg/L Imidacloprid on day 21 (B), snails exposed to 40.16 μg/L Imidacloprid on day 21 (C), snails exposed to 80.32 μg/L Imidacloprid on day 21 (D). (E epithelium, MF, muscle fibril, C sil, MC mucus cell, LV lipid vacuole, PH pigment cell, YG yellow granule, N necrosis). H&E.

Fig. 4.

Histological structure of M. praemorsa mantle in ascorbic acid control (Imidacloprid free medium) on day 21 (A), snails exposed to 4.016 μg/L Imaclidoprid + 7.5 mg ascorbic acid on day 21 (B), snails exposed to 40.16 μg/L Imidacloprid + 7.5 mg ascorbic acid on day 21 (C), snails exposed to 80.32 μg/L Imidacloprid + 7.5 mg ascorbic acid on day 21 (D). (E epithelium, MF, muscle fibril, MC mucus cell, LV lipid vacuole, PC pigment cell, N necrosis). H&E.

Snails exposed to 4.016 µg/L imidacloprid showed mild alterations on days 7 and 14, which progressed to significant histopathological changes by day 21, as detailed in Table 3. These included epithelial desquamation, muscle fiber atrophy, an increase in lipid vacuoles, and dark granule accumulation within the lipid vacuoles (Fig. 3B). In the 4.016 µg/L imidacloprid + ascorbic acid group, an increase in mucus cells and lipid vacuoles was noted, but histopathological alterations were less severe compared to the imidacloprid-only group (Fig. 4B).

Table 3.

Qualitative evaluation of Lesions in the Mantle following exposure to imidacloprid and imidacloprid + ascorbic acid.

Histopathological evaluation	Days	Imidacloprid and Imidacloprid + Ascorbic acid exposure Groups
		I	II	III	IV	V	VI	VII	VIII
Desquamation in epithelial cells	7	−	−	−	−	+	−	+	+
	14	−	−	+	−	+ +	+	+ +	+
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Increase in lipid vacuoles	7	−	−	+	−	+	−	+ +	+
	14	−	−	+ +	−	+ +	−	+ +	+
	21	−	−	+ +	+	+ + +	+ +	+ + +	+ +
Atrophy of muscle cells	7	−	−	−	−	+	−	+	−
	14	−	−	+	−	+ +	+	+ +	+
	21	−	−	+ +	+	+ +	+	+ + +	+ +
Increase in pigment cells	7	−	−	−	−	+	−	+	+
	14	−	−	+	−	+ +	−	+ +	+
	21	−	−	+	−	+ +	+	+ + +	+ +
Increase mucus cell	7	−	−	−	−	+	−	+	−
	14	−	−	+	−	+ +	+	+ +	+
	21	−	−	+	−	+ +	+	+ +	+ +

Absent (−), Mild (+), Moderate (+ +), Severe (+ + +).

Snails exposed to 40.16 µg/L imidacloprid exhibited progressive histopathological damage, with more pronounced alterations by day 21. These included increased epithelial desquamation, atrophy and necrosis of muscle fibers, and a significant increase in pigment cells and lipid vacuoles (Fig. 3C). In the 40.16 µg/L imidacloprid + ascorbic acid group, the number of mucus cells and lipid vacuoles increased, but the overall tissue damage was less severe (Fig. 4C).

Exposure to 80.32 µg/L imidacloprid resulted in the most severe histopathological changes. On days 7 and 14, alterations were moderate; however, by day 21, extensive epithelial desquamation, severe atrophy in muscle fibers, and advanced necrosis were observed (Fig. 3D). In the 80.32 µg/L imidacloprid + ascorbic acid group, epithelial desquamation, mucus cell proliferation, and lipid vacuole accumulation were present but at reduced levels compared to the imidacloprid-only group (Fig. 4D).

As with the hepatopancreas, imidacloprid exposure led to significant pathological lesions in the mantle, which increased in severity with higher concentrations and longer exposure times. However, ascorbic acid co-treatment reduced the extent of tissue damage.

Discussion

The toxic effects of any compound on an organism can be evaluated by observing the histopathological changes it induces³⁹. The histological examination of M. praemorsa tissues demonstrated that imidacloprid caused significant structural deterioration even at the lowest concentration, while ascorbic acid mitigated these toxic effects. The results of toxic effects observed in the hepatopancreas and mantle tissues are consistent with studies on other snail species exposed to heavy metals and pesticides^{16,22–24,36,40,41}.

In mollusks, the hepatopancreas is considered the primary target organ for evaluating the effects of various xenobiotics^9,42. Beyond its role in digestion and nutrient storage, the hepatopancreas is crucial for the accumulation, detoxification, and excretion of toxins. This is primarily due to its function as the main organ of biotransformation in snails. One of the most common lesions in the hepatopancreas of pesticide-exposed snails is lipid accumulation, resulting from abnormal lipid storage. Lipid accumulation is a frequent cellular response to toxic compounds, particularly in metabolically active organs such as the hepatopancreas^9,41. Increased waste activity in snails may be linked to aging and stressed digestive cells⁴³ or directly related to the intensity of oxidative damage^44,45. It can lead to the accumulation of lipofuscin aggregates and degeneration in digestive tubules²⁶, as well as breakdown of digestive tubules and basal membranes, lumen disruption, and vacuolation in the cytoplasm¹⁷. Reactive intermediates entering the cell can inhibit protein synthesis, damage cell membranes, and ultimately result in necrosis⁹.

The histopathological changes observed in the hepatopancreas, including swollen pyramidal basophilic cells, an increased number of pyknotic cells, lipid accumulation, and hemolymphatic space enlargement, align with findings from previous studies on pesticide- and heavy metal-exposed snails^{16,22,25,37,40,46,47}.

In M. praemorsa, imidacloprid exposure led to significant histopathological alterations in the mantle, including muscle fiber atrophy, lipid accumulation, increased mucus and pigment cells, and epithelial necrosis. These findings are consistent with histopathological changes observed in gastropods under stress conditions^17,22,48. Increased mucus production is one of the first responses of gastropods to mechanical or chemical stressors⁴⁹. The severity of mantle damage increased with imidacloprid concentration and exposure duration, whereas histopathological lesions were notably less severe in groups treated with ascorbic acid.

Pesticide-induced toxic effects are known to be associated with an increase in reactive oxygen species (ROS). Ascorbic acid is believed to act as an antioxidant, neutralizing ROS and reducing oxidative stress^4,19. Several studies have demonstrated that ascorbic acid is an effective protective agent against tissue damage and toxicity caused by environmental pollutants in various organisms^50,51.

Imidacloprid acts primarily as an agonist of nicotinic acetylcholine receptors (nAChRs), causing persistent receptor activation and neuronal overstimulation¹¹. In non-target aquatic organisms, this leads to excessive calcium influx, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS)^4,17. The resulting oxidative stress in turn leads to lipid peroxidation, protein oxidation, and DNA damage^5,8. These oxidative imbalances disrupt cellular integrity, leading to necrosis and degeneration in metabolically active tissues such as the hepatopancreas and mantle^8,16,17,33. Ascorbic acid exerts a protective effect by directly scavenging ROS, stabilizing cell membranes, and restoring endogenous antioxidant enzyme activities (such as catalase and superoxide dismutase)^18,50, thereby mitigating imidacloprid-induced histopathological alterations³³.

Conclusion

This study demonstrated that sublethal exposure to imidacloprid causes progressive, dose- and time-dependent histopathological damage in the hepatopancreas and mantle tissues of M. praemorsa. Co-treatment with ascorbic acid markedly reduced the severity of these lesions, indicating its antioxidant protective role against oxidative stress–induced cellular injury. The findings suggest that M. praemorsa is a suitable bioindicator species for monitoring pesticide pollution in freshwater ecosystems. Overall, this research contributes to a better understanding of neonicotinoid toxicity in non-target aquatic organisms and supports the potential use of antioxidant supplementation as a mitigating strategy against pesticide-induced damage.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Birgül Otludil and Lokman Damar All authors commented on previous versions of the manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Since a common invertebrate species was used in this study, animal ethics committee approval was not required.