Comprehensive Assessment of Lead Bioaccumulation in Helix aspersa (Müller, 1774), Snails: A Study of Histopathological and Biochemical Impacts

Soil contamination has been a prominent focus of research and efforts to address it for the past two decades (Byrne, 2021). Soils concentrate all emitted pollutants, including metallic elements (Facchinelli et al., 2001). The presence of heavy metals in soils is particularly problematic due to their non-biodegradability compared to organic pollutants and their toxicity (Olaniran et al., 2013). They have a toxicological impact on plants, daily consumer products and humans (Gove et al., 2001). These pollutants are considered hazardous to the environment due to their persistence and ability to bioaccumulate in living organisms (Ali et al., 2019)
       
In both aquatic and terrestrial habitats, invertebrates have long been used to evaluate the quality of ecosystems. as members of terrestrial ecosystems, snails can integrate many sources of pollution (soil, atmosphere, vegetation) via several routes (digestive, respiratory and/or cutaneous). Small grey snails (H. aspersa) are important ecological indicators of metal and organic pollution in the terrestrial environment, using biomarkers as a natural biological tool to signal pollution and harmful chemical bioavailability. They are complementary tools for assessing ecosystem quality (McCarthy and Shugart, 1990). As a result, they enable early detection of physiological dysfunctions in individuals, identifying the harmful effect before it becomes visible (Van der Oost et al., 2003).
       
H. aspersa has been widely used as a biological model to evaluate the toxic effects of different soil pollutants (Gimbert et al., 2006, De Vaufleury et al., 2006, Druart et al., 2011). Therefore, H. aspersa is used in the present study to evaluate the lead accumulation in the foot and hepatopancreas of the juvenile snails under controlled laboratory conditions. Furthermore, the biochemical composition (proteins, carbohydrates and lipids) in the two considered organs are examined with histological study of the hepatopancreas.

Following ISO (2018) criteria, a semi-static ecotoxicity test was conducted During the period from March to April 2022, in the Animal Biology Laboratory of the Department of Natural and Life Sciences at the University of Skikda. Juvenile snails were exposed to lead-contaminated soil at varying concentrations (100, 500, 1000 and 1500 μg/g of soil). The snails were placed in polystyrene boxes with 100 g of dry soil covering the bottom, which was then moistened to 50% of its water-holding capacity. The specimens were divided into two groups: control groups and groups treated with the tested concentrations, each group with 10 individuals. The soil, the sole source of contamination, was renewed once a week throughout the test duration. After the experimental period, the snails were euthanized and dissected. The hepatopancreas and foot tissues were collected for lead and metabolite assessment and sections of the hepatopancreas were prepared for histological examination.
 
Lead analysis
 
The hepatopancreas and foot tissues were individually placed in screw-cap tubes and then dried in the oven at 80°C for 24 hours. Subsequently, the dried fragments were weighed and 4 ml of 65% nitric acid were added to each tube. The tubes were carefully sealed and placed in the oven at 60°C for 72 hours to complete tissue digestion under pressure. Following digestion, each sample was diluted to a volume of 19 ml with distilled water and stored at 4°C until analysis (Cœurdassier, 2001). Metal concentrations in the various samples were determined using inductively coupled plasma atomic emission spectrophotometry (ICP-AES), In the analysis laboratory of the SONATRACH Skikda oil refining complex.
 
Metabolites extraction and measurement
 
The technique described by Shibko et al., (1966) was used to extract metabolites (carbohydrates, lipids and proteins) from the tissues investigated. Carbohydrates were measured using the method proposed by Goldsworthy et al., (1972) and proteins that of Bradford (1976).
 
Histological study
 
For the histological study of hepatopancreatic fragments, the tissues of the control and treated groups, were fixed in 10% formalin for 24 hours, dehydrated in increasing alcohol baths and then embedded in a paraffin block. Sections of 5ìm thickness were made using a microtome (Leitz, Germany). These sections were stained with hematoxylin and eosin (HandE) following the criteria of Martoja and Martoja, (1967). Finally, they were observed and photographed using a light microscope.
 
Statistical analysis
 
All the results were expressed as mean ± SE and analyzed using Student’s t test with the Minitab program (version-15) comparing each treated group with the control. The significance levels of p≤0.05 was considered.

Gastropod mollusks are well-known for their ability to accumulate heavy metals, including Cd, Cu, Pb and Zn. By virtue of this property, snails have been used as bioindicators of heavy metal contamination (Beeby and Richmond, 2002; Viard et al., 2004; Notten et al., 2005). Most gastropods acquire critical or hazardous heavy metals through food consumption and absorption via the digestive epithelium or the skin (Marigómez et al., 1998). Once they pass through various biological barriers like skin and digestive epithelium, metallic elements circulate within the organism via the hemolymph and distribute among various organs where they are stored. Heavy metals are considered truly toxic agents, disrupting certain enzymatic systems and metabolic and physiological activities in both humans and animals (Nedjoud et al., 2016).
 
Lead levels in the hepatopancreas and foot
 
After two months of therapy, the data presented in Fig 1 illustrate the progression of lead concentration in the hepatopancreas and foot of the treated groups, revealing a significant difference (P≤0.001) between the concentrations of the control group and the treated groups with different doses. On the other hand, it is evident that lead levels in both the organs tend to increase in a dose-dependent manner. However, the highest concentration was recorded in the hepatopancreas compared to the foot. Following the exposure of a population of juvenile H. aspersa to soil contaminated with increasing doses of lead, a non-essential element for living organisms, the accumulation and toxic effects of this metal in the hepatopancreas and foot of the snails were demonstrated (Carbone and Faggio, 2019). Further, the data revealed that lead levels in hepatopancreas and foot increased in a dose-dependent manner in the treated snails under the laboratory conditions. The high concentrations of lead in the snails’ organs indicate significant metal accumulation due to the ingestion of contaminated soil particles and/or the diffusion of the metal through the epidermal epithelium of the foot during the experiment. Additionally, the lead levels in the hepatopancreas of H. aspersa was found to be substantially higher than those in the foot.
 

 
Effect of lead on hepatopancreas and foot biochemical parameters
 
Protein levels
 
The lead-contaminated snails exhibited a dose-dependent increase in protein level compared to control animals. However, even at lower dose, this rise becomes considerable in the hepatopancreas (Fig 2). Across all species investigated thus far, the hepatopancreas consistently contains the highest concentrations of Cd, Pb and Zn (Cooke et al., 1979, Dallinger and Wieser, 1984). It appears that the digestive system is also implicated in the storage of metals, with the foot serving as a site of transient accumulation in connection with cutaneous absorption. (Dallinger and Wieser, 1984, Chabicovsky et al., 2004). The metals are subsequently redirected to the hepatopancreas, either definitively for excretion over varying periods or temporarily. In the presence of lead, the evolution of total protein levels in the two organs of treated snails increase in a dose-dependent way. These findings are congruent with those of (Nedjoud et al., 2016) following the findings of (Besnaci et al., 2016), who demonstrated that total protein level has significantly increased after 28 days of treatment of snails with metal dust and those of exposing an adult population of H. aspersa to iron oxide nanoparticle toxicity. This phenomenon could be considered an early biomarker of exposure to chemical contaminants. This increase in proteins may be explained by the accumulation of Pb in the tissues. Proteins are primarily involved in cell structure and can also be bound to toxins, serving as transport proteins (Cui et al., 2010). Metals can bind to proteins that require a metal ion as part of their structure (hemoglobin, hemocyanin, etc.) and to transport/store proteins that are crucial for the control of metal homeostasis or detoxification., binding to certain metals, especially heavy metals, more or less precisely (lead-binding proteins in some species) (Cœurdassier, 2001).
 

 
Carbohydrate levels
 
In comparison to the carbohydrate levels in the control groups, the carbohydrate levels in all four contaminated groups are considerably lower at all tested concentrations and also in both the organs examined in the present study (Fig 3).  These results are consistent with those of El Wakil and Radwan, (1991). Decrease in carbohydrate levels under the influence of metal stress implies a disturbance of carbohydrate metabolism (Nzengue, 2008). Eissa et al., (2002) reported that the harmful effect of chemical compounds could be attributed to increased energy use and/or altered cell organelles (of treated snails) and may interfere with protein synthesis. This drop in carbohydrates might be attributed to the oxidation of the proteins of metal ions, leading to the release of aldehydes and hydrogen peroxide (El Wakil and Radwan, 1991).
 

 
Lipid levels
 
Increasing the concentration of lead has led to a reduction in lipid levels in both examined tissues compared to the control group. The lowest concentration reached was 1.9 and 2.2 μg/mg at 1500 μg/g in the foot and hepatopancreas, respectively, while in the control group, the concentration was 5.45 and 7.8 μg/mg in the foot and hepatopancreas, respectively. The statistical analysis showed significant differences between the concentrations (1500, 1000 and 500 μg/g) and the control group, while there were no significant differences between the concentration of 100 and the control group (Fig 4). These results are supported by the findings of Padmaja and Rao (1994) and they are of the view that after carbohydrates, lipids are the primary energy source provided to tissues when needed. According to Nzengue (2008), metals such as iron and copper have been widely used as initiators of lipid oxidation.
 

 
Histological examination of the hepatopancreas
 
Fig (5A) shows the histology of the hepatopancreas of control snails, which displays a digestive epithelium composed of lobules that create a collection of acini bound together by connective tissue. The epithelium is made up of three types of cells: digestive cells, excretory cells and calcium cells. Lead particles cause histological alterations (Fig 5B) in the group treated with 100 μg/g, demonstrating excretory cell hypertrophy and tubule clustering as a result of acini lumen constriction. Similar changes were also observed in the group treated with 500 μg/g of lead (Fig 5C), along with enlargement of hemolymphatic gaps between tubules, degeneration of the acini’s basement membrane and an inflammatory appearence of the tissue. These alterations are more severe in the treated groups with 1000 and 1500 ìg/g (Fig 5D and E), with inflammatory infiltrates and cellular debris throughout the tissue and necrosis affecting the connective tissue and digestive tubule membranes.
 

       
Earlier observations by Zaldibar et al., (2007) also revealed the structural changes in the hepatopancreas (Zaldibar et al., 2007), who showed a high relative number of calcium cells and hypertrophy of intercellular spaces in the digestive gland of terrestrial snails under chemical stress (metallic or otherwise). Studies by Besnaci et al., (2016) demonstrated undeniable tissue alterations in the digestive gland and kidney of H. aspersa in response to the toxicity of iron oxide nanoparticles at the investigated doses (1, 2 and 3 mg of flour) administered through digestion, leading to structural alterations like enlargement of hemolymphatic spaces between tubules, inflammatory infiltrates and cellular necrosis. The degree and frequency of reported lesions varies depending on the organ and the species under consideration and they are more obvious at greater doses. (Adams et al., 2018; Adams et al., 1990; Świergosz-Kowalewska et al., 2007).
       
These changes might be caused by lipid disturbances, in which free radicals generated cause structural and functional abnormalities in the cell, as well as membrane permeability associated to the creation of lipid peroxides (Lawton and Donaldson, 1991). All components can be damaged, including lipids, proteins and the membrane (Halliwell and Chirico, 1993), affecting DNA and causing pathologies (Curtin et al., 2002).

In conclusion, this study demonstrates that juvenile H. aspersa snails are sensitive to the presence of lead particles in the soil. We have shown unambiguously that the snail is capable of accumulating the lead in hepatopancreas and foot, with a higher accumulation in the hepatopancreas. The biochemical composition is affected by lead exposure, with a significant increase in proteins and a decrease in carbohydrate and lipid levels. The histological examination of the hepatopancreas confirms the metal accumulation with important qualitative alterations even at the lowest tested concentration.
       
These findings emphasize the possible impact of lead on snails while also raising concerns about the consequences on other creatures in the environment. The findings also highlight the need of monitoring heavy metal pollution in the ecosystem, as well as possible consequences for biodiversity and ecological health. On the other hand, further research is needed to understand the long-term effects of lead exposure on snails and its potential consequences in the ecosystem. This research adds to the expanding body of knowledge on heavy metal contamination and its effects on the ecosystem, offering important data for risk assessments and environmental conservation initiatives.

SN, FB and BF designed the study, FA and BS conducted data analyses and wrote the manuscript. LM performed light microscopy experiments. MS and SL helped in writing the manuscript and conducted data analyses.

Researchers Supporting Project number (RSP2024R112), King Saud University, Riyadh, Saudi Arabia.

The conducted research is not related to either human or animal use.

All the data is available within the manuscript.

The authors declare no conflicts of interest.