Metal pollution results in many dangerous consequences to the environment and human health due to the bioaccumulation in their tissues. The present study aims to measure the bioaccumulation factor of the Manganese (Mn) heavy metal in Biomphlaria alexandrina snails' tissues and water samples. The present results showed the concentration of Mn heavy metal in water (87.5 mg/l) and its bioaccumulation factor in Helisoma duryi tissue was higher than that in tissues of Physa acuta and B. alexandrina snails. Results showed that 87.5 mg/l Mn concentration had miracidial and cercaricidal activities. Also, this concentration decreased the mean total number of the hemocytes after exposure for 24h or 48h, while increased both the mean mortality and phagocytic indices of the hemocytes of exposed snails. It caused alterations in the cytomorphology of the hemocytes of exposed snails after 24 or 48h, where, the granulocytes had irregular cell membrane, and forming pseudopodia. Besides, both levels of Testosterone (T) and Estradiol (E) were increased after exposure to 87.5mg/l Mn metal compared to the control group. Also, it increased MDA (Malonaldehyde) and TAC (Total antioxidant capacity) contents, while, decreased SOD (superoxide dismutase). Besides, it caused great histopathological damages in both hermaphrodite and digestive glands, represented in the degeneration of the gonadal, digestive, secretory cells and the connective tissues. Therefore, B. alexandrina might be used as sensitive bio-indicator of pollution with Mn heavy metal to avoid ethics rules; beside they are easily available and large in number.
Research Article
Biomphalaria alexandrina snail as a bio-indicator of pollution with Manganese metal and its effect on physiological, immunological, histopathological parameters and larvicidal potencies
https://doi.org/10.21203/rs.3.rs-2123811/v1
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published 18 Sep, 2023
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Manganese metal
B. alexandrina
Bioaccumulation factor
Larvicidal
Hormonal alterations
Histopathology.
Environmental metal pollution represents a dramatic problem in the world (Salih et al. 2021). The heavy metals are natural parts of the earth’s crust, but the higher levels of these metals could threat the environment and human biological system (Ugbaja et al. 2020; Briffa et al. 2020). Since 1940s, the rate of these heavy metal were increased worldwide due to industrialization and urbanization (Ali et al. 2019).
Heavy metals are classified as dangerous pollutants as they when swallowed or inhaled into organisms bodies it bioaccumulated in their systems causing biological and physiological complications (Briffa et al. 2020). Some heavy metals are essential elements for life with small traces (Ni, Cu, Zn & Cr) (Dhiman and Pant 2021). Heavy metals, such as, manganese (Mn), zinc (Zn), nickel (Ni), copper (Cu), cadmium (Cd), chromium (Cr), lead (Pb), and iron (Fe) have high levels of toxicity and ability for bioaccumulation and combination into the aquatic food chain subsequently reaching in the aquatic ecosystems (Kibria et al. 2016).
Heavy metals pollution of the aquatic environment caused severe damages to fish tissues and hence can affect human health (Ali et al. 2019). Manganese (Mn), is an essential metal required for enzymes and cofactors but its higher concentration could cause several neurological disorders (Schmidt and Husted 2019)
Environmental biomonitoring could help to elucidate the harmful effects of the heavy metal to the biological systems of the surrounding organisms (Morais et al. 2022). Bioindicators are a group of species that reflect the natural state of the environment including biotic or abiotic changes in the habitat (McGeogh 1998). Several aquatic species have been used as bioindicators for the chemical pollution (Ibrahim and Sayed 2019). Mollusks are widely used as bioindicators and biosensors of heavy metal poisoning due to their rapid accumulation of these toxic pollutants, widespread and have a moderate life span (Abd-Allah et al.; 1997; Ugbaja et al.; 2020). These aquatic invertebrates could replace the higher vertebrate in the ecological studies because of the ethical rules and the low cost (Ibrahim et al. 2018; Dhiman and Pant 2021).
Biomphalaria alexandrina snails the intermediate host for schistosomiasis mansoni in Egypt, that are widely distributed throughout the Nile River (Ibrahim and Sayed 2019; Morad et al. 2022). Where, Schistosomiasis can affect at least 236.6 million people in 2019 (WHO 2022). B. alexandrina can serve as a good and sensitive bioindicator of heavy metal pollution (Habib et al. 2016). These snails proved to be a good bioindicator because of their adult and embryonic phases could reflect the pollution by both physical and chemical agents (Morais et al. 2022).
Therefore, the objective of the present study is to measure the concentration of the Manganese heavy metal in water samples and Biomphlaria alexandrina tissues and calculation of the bioaccumulation factor. Then study its concentration in water on survival and infection rates of B. alexandrina with Schistosoma mansoni miracidia, and its cercaricidal activities. Also, elucidating its immunotoxicity, endocrine disruption, oxidative stress biomarkers and histopathological effects on tissues of these snails.
A) Field study
1. Snail samples and water collection:
Snails were collected from different irrigation canals in Abo-Rawash, Giza governorate. It was placed in numbered plastic aquaria with water from these canals. Also, water samples were collected in sterilized one-liter polyethylene bottles below the water′s surface about 30 cm (Kaufmann et al. 1988). Both samples were transported in an ice box to the laboratory for analysis within 12 hours.
2. Estimation of heavy metals in snails soft tissue and water samples:
Determination of heavy metals in water samples and snail's tissues were performed by using Atomic Absorption Spectrophotometry (AAS) in Environmental Research Laboratory, Theodor Bilharz Research Institute (TBRI) according to the method of (Abdel Kader et al. 2016).
- Analysis of heavy metals in water: For the analysis of total heavy metals, water samples (200 ml) were digested with 5 ml of acidified concentrated nitric acid (HNO3), on a hot plate and filtered by Whatman No. 42 filter paper and made up the volume to 50 ml by double distilled (ddH2O) (Shaaban et al. 2017)
- Analysis of heavy metals in snail tissues: About 0.01 gm of the snail's soft tissues were separated from their shells, oven dried at 50°C, and then digested in 1ml of conc. HNO3 at 70°C for 2h. The digested samples were then diluted with 5 ml ultrapure deionized water for analyzing heavy metals (Federici et al. 2007).
- The Bio accumulation factor (BAF), which is the ratio of the chemical concentration in an organism or biota to the concentration in water (Gobas and Morrison 2000), was calculated as follow:
Bio accumulation factor (BAF) = concentration of the metal in snail tissues (mg/g dry weight)/ concentration of the metal in water (mg/l).
BAF<1 indicates no contamination, 1>BAF≤10, the snail is tolerant and BAF>10, a hyper-accumulator (Ávila et al. 2017).
B) Lab study:
1. Snails:
Biomphalaria alexandrina (Ehrenberg, 1831) snails (9- 10 mm) were maintained in Medical Malacology Laboratory, Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Snails were kept in plastic aquaria (16 x 23 x 9 cm) with dechlorinated aerated tap water (10 snails/ L), pH: 7± 0.2 and temperature (25± 2 Cº). Oven dried lettuce leaves, blue-green algae (Nostoc muscorum) and Tetramin were provided to aquaria for feeding and 30 mg/l calcium carbonate (CaCO3) for snails' shell and fecundity (Eveland and Haseeb 2011).
2. Manganese (Mn) and exposure conditions (Mn):
A stock solution (1000 mg/l) of manganese (as MnCl2·4H2O, Fisher Scientific, Fair Lawn, NJ, USA) was made in distilled water.
C) Bioassays:
1- Miracidicidal and cercaricidal activites:
Five ml of 87.5 mg/l of Mn solution was mixed with 5ml of water containing about 100 freshly hatched miracidia or cercariae. As well as, another 10 ml of dechlorinated tap water containing 100 freshly hatched miracidia or cercariae were kept as a control. After intervals of 30, 50, 70, 90, 110, and 130 min, the alterations in movement of miracidiae and cercariae were observed under a dissecting microscope (Eissa et al. 2011).
2- Survival and infection rates:
Laboratory juveniles B. alexandrina snails (4-7 mm) were exposed individually to 4-6 freshly hatched S. mansoni miracidiae for 24h and then transferred to separate aquaria. From day 21 post-exposure, each snail was tested weekly for the shedding of cercariae by exposing it to fluorescent light in 1 ml of water for 1 hours at 25°C (Hung et al. 2015).
3- Toxicity on snails:
Snails were subjected to 87.5 mg/l of Manganese solution either for 24h or 48h followed by one day of recovery. For each exposure time, 30 adult snails (9–12 mm in shell diameter) were used in three replicates, each of 10 snails/ liter. The control group was also set up in triplicates using only distilled water (WHO 1965).
3.1. Hemolymph collection and Light microscopy preparation:
The hemolymph of the living snails from each group was collected according to (Nduku and Harrison 1980). Total hemocytes count was done by a Bürker- Turk hemocytometer (Der Knaap et al. 1981). Blood smears were done as monolayers to show different shapes of the hemocytes (Ibrahim et al. 2018). To measure the phagocytic index, hemocyte suspension of 100 μl collected from each specimen was smeared on glass slides and incubated with activated charcoal particles for 1 h at 37 °C in a humid chamber for cell adherence. The phagocytic index was calculated as per the following formula:
Also, to measure the mortality index cells were treated with 50 μl of 0.25 % trypan blue dye solution for 5 minutes. Cells that have taken up the dye are dead. The percentage of blue-stained cells represents a mortality index (Guria 2018):
3.2. Tissue Preparation
From each treated group and the control group, soft tissues of snails were withdrawn from the shell using a forceps, weighed (1g tissue/10 ml phosphate buffer) and then homogenized by a glass Dounce homogenizer. The homogenates were centrifuged at 3,000 rpm for 10 min and the supernatants were stored at –80 C° until used.
a- Investigation of testosterone and Estradiol hormones:
Hormone concentrations (T and E) were assayed for all groups according to the manufacturer instructions of T EIA kit (Enzo Life Science, Michigan, USA, ADI-900-065) and E EIA kit (Cayman Chemical Company, Michigan, USA, item no. 582251).
b- investigation of the antioxidant responses SOD, the oxidative stress marker (Malondialdhyde, MDA) and Total antioxidant capacity:
The supernatant of the soft tissue homogenate from each group was used. Biodiagnostic kits (Biodiagnostic Dokki, Giza, Egypt) were used for the determination of SOD (Damerval et al. 1986). Malondialdhyde (lipid peroxide) was done according to (Ohkawa et al. 1979), and Total antioxidant capacity was estimated by kit (Cat. No. TA 2513) (Koracevic et al. 2001).
3.3. Histological studies:
Sections of the digestive and hermaphrodite glands of B. alexandrina snails of either control or exposed groups were done and stained with hematoxylin and eosin according to (Mohamed and Saad 1990).
4- Statistical Analysis:
The median lethal and lethal concentration values were analyzed by Probit facility (Finney 1971) using the statistical program SPSS version 20 (SPSS, Inc., Chicago, IL) for windows. Student’s t-test was used for comparing the means of experimental and control groups (Murray 1981).
The present results showed that Mn heavy metal could accumulate in the tissue of B. alexandrina, P. acuta and H. duryi snails. The bioaccumulation of Mn heavy metal in H. duryi tissue was higher than that in tissues of P. acuta and B. alexandrina snails (Table 1).
| Snails | Conc in tissue (ppb) | Conc in water (ppb) | Bioaccumulation factor |
|---|---|---|---|
| Biomphalaria alexandrina | 0.231 | 0.0875 | 2.64 |
| Physa acuta | 0.289 | 0.0875 | 3.302 |
| Helisoma duryi | 0.3114 | 0.0875 | 3.558 |
The present results showed that Mn heavy metal (87.5 mg/l) has miracidial and cercaricidal activities, where after 80 min all miracidiae had died compared to 35% deaths in control group (Fig. 1). Also, all cercariae had died after 130 min of exposure compared to 25% of control group.
The present results showed that after exposure to 87.5 mg/l Mn heavy metal for 24h, the survival rate was significantly decreased, while the infection rate was increased. After exposure for 48h, both survival and infection rates were significantly decreased (Fig. 2).
The present results showed that Mn heavy metal (87.5mg/l) significantly decreased the mean total number of the hemocytes (p˂0.05) after exposure for 24h or 48h comparing with B. alexandrina control group. While, it significantly increased both the mean mortality index and the mean phagocytic index of the hemocytes of these snails (Table 2).
| Groups | Hemocytes/mm3 ±S.D | % Reduction | Phagocytic index | Mortality index |
|---|---|---|---|---|
| Control | 648.33 ± 22.5 | 44.2 | 10 | |
| 24h | 416.66 ± 28.86 ** | 35.73 | 72.4 | 50 |
| 48h | 258.3 ± 38.18 * | 60.15 | 84.25 | 81.5 |
* significant different from control at p < 0.05
Examination of the hemocytes' monolayers showed the presence of three shapes of hemocytes in the normal control group: A) small hemocytes; B) granulocytes and c) hyalinocytes. After exposure to Mn heavy metal (87.5mg/l) for 24h, the granulocytes had irregular cell membrane, forming pseudopodia and had numerous granules. The nucleus of some hyalinocytes shrank and some had two nuclei divided in the same cell (Fig. 3, D, E, F). After exposure for 48h, some granulocytes had an irregular cell membrane with incomplete cell division, numerous granules and forming pseudopodia. Hyalinocytes had a shrunk nucleus; other had two nuclei with irregular outer membrane (Fig. 3, G, H, I).
The present results showed that both levels of Testosterone (T) and Estradiol (E) were significantly increased (p < 0.05) after exposure to Mn heavy metal (87.5mg/l) for 24h or 48h compared with control group (Table, 3).
| Groups | Testesterone (nmol/L) | Estradiol (pg/ml) |
|---|---|---|
| Control | 18.6 ± 0.2 | 96.8 ± 4.3 |
| 24 | 29.1 ± 0.1* | 250 ± 4.2* |
| 48 | 36.3 ± 0.6** | 450 ± 5.4** |
Exposure of B. alexandrina snails to Mn heavy metal (87.5mg/l) either to 24h or 48h exhibited a significant increase (p < 0.05) in MDA and TAC contents compared with the control group. On the other hand, tissue SOD was significantly (p < 0.05) decreased after exposure in a time dependent manner (Table, 4).
| Parameters | MDA(nmol/g. tissue) | SOD (U/g. tissue) | TAC (mM/l) |
|---|---|---|---|
| Control | 10.1 ± 0.12 | 9.13 ± 0.1 | 1.505 ± 0.4 |
| 24h | 12.12 ± 0.3* | 5.2 ± 0.2* | 1.88 ± 0.21 |
| 48h | 15. 7 ± 0.13* | 4.9 ± 0.32* | 2.3 ± 0.13* |
The hermaphrodite gland of control B. alexandrina is composed of the male reproductive cells and the female oogenic cells. After exposure to Mn heavy metal (87.5mg/l) either for 24h or 48h, there were great damages in these gonadal cells including degeneration of sperms, some ova, oocytes and spermatocytes. Also, Also, the connective tissue between the acini was raptured and the vacuoles were increased (Fig. 4; A, B and C).
The tubules of the digestive gland of normal B. alexandrina snails were lined by two types of cells; the digestive and secretory cells and in between there is the connective tissue. After exposure to Mn heavy metal (87.5mg/l) either for 24h or 48h, there were great deleterious rupture and degeneration of the digestive cells and the secretory cells. Also, the vacuoles and lumen inside the tubules were increased and the connective tissue was dissolved and ruptured (Fig. 4; D, E and F).
Heavy metals pollution has gained a great attention in researches because of its dangerous consequences. The present results showed that the concentration of Mn heavy metal in water was 87.5 mg/l. This obtained concentration is higher than the allowable FAO limits for Manganese metal (0.20 mg/l) (Simmons 2000). The present results showed that Mn heavy metal could accumulate in the tissue of B. alexandrina, P. acuta and H. duryi snails. The bioaccumulation of Mn heavy metal in H. duryi tissue was higher than that in tissues of P. acuta and B. alexandrina snails. These results were in a good accordance with (Abdel Kader et al. 2016) who found that P. acuta and H. duryi were the highest snails that could accumulate the heavy metals in their tissue and reasoned the high variation in BAF values depend on the type of the snail and the metal. The present results showed that BAF is higher than one in the three snails type and this indicated that these snails were tolerant (Liao et al. 2003; Ávila et al. 2017; Kachenga et al. 2020).
The present study elucidated the effect of 87.5 mg/l Mn exposure on different parameters of B. alexandrina snails. Results showed that this concentration of Mn (87.5 mg/l) has miracidial and cercaricidal activities, where after 80 min all miracidiae had died and 130 min of exposure all cercariae had died. Cercariae and miracidiae might be used as excellent indicator organisms for heavy metal pollution (Morley et al. 2003b). (Cross et al. 2001) studied the effect of 4 mg/l manganese in artificial seawater on the cercariae of the marine trematode Cryptocotyle lingua and showed that this concentration induced reductions in cercarial quality which might lead to failure of transmission of this disease and concluded that the metal pollution could alter parasite populations and communities. Also, (Morley et al. 2003a) found that the survival of S. mansoni exposed to Cd/Zn mixtures was significantly reduced at concentrations of 10 mg/l or higher. The authors reasoned this reduction on the effect of these metals on the enzymatic function of miracidia and leading to their death. These results could explain the decrease in the survival and the infection rates of adult snails in the present study. Where, after exposure of snails to 87.5 mg/l Mn heavy metal for 24h, the survival rate was significantly decreased, while the infection rate was increased. While, after exposure to 87.5 mg/l Mn heavy metal for 48h, both survival and infection rates were significantly decreased. Similarly, (Morley et al. 2003a) found that the survival rate of the infected Lymnaea peregra and L. stagnalis snails was severely reduced by exposure to Cadmium metal.
Snails have an open circulatory system which contains the hemolymph and it acted as the defense system against the foreign particles (Baroudi et al. 2020). The present results showed that Mn heavy metal (87.5mg/l) significantly decreased the mean total number of the hemocytes after exposure for 24h or 48h comparing with B. alexandrina control group. This decrease could be due to the sharing of these hemocutes in the repairing the damages occurred by Mn in both hermaphrodite and digestive glands (Esmaeil 2009; Ibrahim et al. 2018).
The main cellular component of the snails immune system is the hemocytes that is responsible for phagocytosis (Baroudi et al. 2020). The present investigation showed that Mn heavy metal (87.5mg/l) significantly increased both the mean mortality index and the mean phagocytic index of the hemocytes of exposed snails. The increase in the mortality index is indication of the Cell death or apoptosis (Guria 2018). This increase depends on the nature and the type of the stressors (Ibrahim and Sayed 2020).
Three types of hemocytes were found in smears of B. alexandrina. The more reactive cell were known as granulocytes, followed by small undifferentiated and then hyalinocytes that were found at the site of wounds (Ibrahim and Abdel-Tawab 2020). After exposure to Mn (87.5mg/l) for 24h, the granulocytes had irregular cell membrane, forming pseudopodia and had numerous granules. The nucleus of some hyalinocytes shrank and some has two nuclei divided in the same cell. After exposure for 48h, some granulocytes had an irregular cell membrane with incomplete cell division, numerous granules and forming pseudopodia. Hyalinocytes had a shrunk nucleus; other had two nuclei with irregular outer membrane. (Guria 2018) concluded that the alterations in the cytomorphology of hemocytes of Lamellidens marginalis (Bivalvia: Eulamelli branchiata) due to the toxic effect of lead (Pb) heavy metal. The formation of pseudopodia of the granulocyte after exposure was away to remove the foreign materials by the phagocytic process (Ibrahim et al. 2022).
Exposure to the heavy metals causes deleterious health effects in the liver, kidney, brain, reproductive system and other body systems. It impaired the hormonal regulation, steroidogenesis, gametogenic process where it is known as endocrine-disruptors (Verma et al. 2018). The present results showed that both levels of Testosterone (T) and Estradiol (E) were significantly increased after exposure to Mn heavy metal (87.5mg/l) compared with control group.
The present study showed that exposure of B. alexandrina snails to Mn heavy metal (87.5mg/l) either to 24h or 48h exhibited a significant increase in MDA and TAC contents, while, SOD was decreased compared with the control group. (Khalil et al. 2017) conclude that, the altered activities of SOD, CAT, GPx, GST and GR) and MDA levels could be useful biomarkers of water pollution with the heavy metals. These results in accordance with (Siwela et al. 2010) who reported that the bioaccumulation of heavy metals in Lymnaea natalensis tissues increased malondialdehyde (MDA) levels, while decreased SOD and catalase (CAT) activities and reasoned these alterations due to the metal induced oxidative stress. Also, (Atailia et al. 2016) concluded that exposure of the terrestrial land snail Helix aspersa to metals under field and laboratory conditions caused changes in the non-enzymatic and enzymatic oxidative biomarkers, where it increased activity of catalase and lipid peroxidation, and the decrease of GST activity and GSH level.
The present results showed that exposure of B. alexandrina snails to Mn heavy metal (87.5mg/l) either for 24h or 48h caused great histopathological damages in both hermaphrodite and digestive glands. The damages in the gonadal cells included degeneration of sperms, some ova, oocytes and spermatocytes with vacuoles and ruptured connective tissue between the acini. Also, there were great deleterious rupture and degeneration of the digestive cells and the secretory cells. Also, the vacuoles and lumen inside the tubules were increased and the connective tissue was degenerated.
These histopathological alterations might be related to the direct toxic effects of the heavy metals on the edible organs of target animals like fish (Elwasify et al. 2021), where the metal caused vacuolated hepatocytes, dilated central vein, compressed blood sinusoids, and congestion were seen in fish liver. Similarly, (Abdel-Tawab et al. 2021), reported that Cerium oxide nanoparticle synthesized with Moringa oleifera seeds at concentration 314.5 mg/l caused great damages in both hermaphrodite and digestive glands.
Therefore, B. alexandrina could be used as sensitive heavy metal pollution bioindicators as they are large in number, easy to adapt and manipulate under controlled conditions.
Ethical Approval
Snails are not subject to the ethics committee
Consent to Participate and Publish
All authors whose names appear on the submission made substantial contributions to the conception of the work, analysis, interpretation of data, approved the version to be published.
Author Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Amina Mohamed Ibrahim], [Rania Gamal Taha] and [Ahmed Abd-Elsalam Abdel-Haleem]. The first draft of the manuscript was written by [Amina Mohamed Ibrahim] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Competing Interests
The authors have no relevant financial or non-financial interests to disclose
Availability of data and materials
All date that are presented in the manuscript are available
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