Toxicological evaluation of engineered layered double hydroxide nanomaterials to Biomphalaria alexandrina snails: a study on the mechanism of action

Layered double hydroxide (LDH) nanomaterials have recently become immense research area as it is used widely in industries. So, it's chance of their release into natural environment and risk assessment to non-target aquatic invertebrate increasing. So, the present study aimed to synthesize and conrme the crystalline formation of Co-Cd-Fe LDHs and Co-Cd-Fe /PbI 2 (LDH), and then to investigate the toxic impact of the two LDH on the adult freshwater snails (Biomphalaia alexandrina). Results showed that Co-Cd-Fe /PbI 2 LDH has more toxic effect to adult Biomphalaria than Co-Cd-Fe LDHs (LC 50 was 56.4 mg/l, 72 h of exposure). The effect of LC 25 (117.1 mg/l) of Co-Cd-Fe LDHs exposure on the embryo; showed suppression of embryonic development and induced embryo malformation. Also, it showed alterations in the tegmental architectures of the mantle-foot region of B. alexandrina snails as declared in scanning electron micrograph. Also, exposure to this sub lethal concentration caused abnormalities in hemocyte shapes and up-regulated IL-2 level in soft tissue. In addition, it decreased levels of non-enzymatic reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), caspase-3 activity and total protein content in signicant manner. While, glutathione S-transferase (GST) activity was not affected by LDH exposure. It caused histopathological damages in both the hermaphrodite and digestive glands. Also, it has a genotoxic effect that was conrmed by alkaline comet assay. The results from the present study indicated that LDH has risk assessment on aquatic B. alexandrina snails and that it can be used as a biological indicator of water pollution with LDH


Introduction
Nanomaterials have been applied in many biomedical researches due to their unique optical, electronic and magnetic characteristics. (Bazrafshan et al., 2017;Corsi et al., 2018;Tarafdar et al., 2013). Layered double hydroxide (LDH) is one of two dimensional layered inorganic nanomaterials. It is one of various cheap nanoparticles bearing positive charge (Thomas and Daniel 2019, Qu et al., 2016. Recently, Layered materials have been extensively used in the application of catalysis, polymer nanocomposites and sensors (Zhao et al., 2007;Manzi-Nshuti et al., 2009;Han et al., 2011), in medicine and pharmacy (Ladewig et al., 2009;Choi and Choy, 2011). Additionally, it is used as fertilizers, herbicides, growth regulators and in removing environmental chemical pollution (Li et al., 2016;Peligro et al., 2016;Daniel and Thomas, 2020;Benício et al., 2020). Their unique uses in many applications depending on the host molecule. These have been attributed to their exchange capacity of anionic and capability to accommodate in the interlayer region different types of functional anions/molecules (metals, halocomplexes, polymers, proteins, drugs, etc.). The wide spread utilization of LDHs may lead to increase the chance of their release into the aquatic ecosystem, which has not been investigated. Toxicological evaluation of LDH has been gained environmental and human health care, since it may cause a negative impact to non-target aquatic fauna.
B. alexandrina snails are widely accepted invertebrate models to study the toxicity and toxico-kinetic of inorganic nanomaterial for aquatic ecosystem (Kaloyianni et al., 2020;Oliveira-Filho et al., 2017). It is the intermediate host of Schistosoma mansoni. it is wildly disseminated throughout tropical and subtropical highly polluted canals and in the Nile River (DeJong et al. 2001). Biomphalaria characterized by their availability, easy way for collection, Acclimate to laboratory conditions, sensitivity to water and chemical pollutant. All the previous characters nominate it to use as laboratory monitoring in ecotoxicological studies and for analyzing multiples biomarkers. (Duft et al., 2007;OECD, 2016;Oliveira-Filho et al., 2017;Ruppert et al., 2017). Many studies in immunology, reproductive and developmental biology used Biomphalaria as paradigm ( Boisseaux et al., 2017;Khangarot and Das, 2010;Pirger et al., 2018). Nanomaterials (NMs) such as carbon nanotubes, silver nanoparticles has potential effects to B. alexandrina as conducted in many studies (Moustafa et al. 2018). The toxicity of NMs has been attributed to reactive oxygen species (ROS) generated that subsequently by lipid peroxidation, DNA and protein damage (Caixeta et al. 2020).
The present study focused on the toxicity of LDHs to B. alexandrina, how it affects their biological processes, and to donate well knowledge about biological behavior and risk assessment of Co-Cd-Fe LDHs in aquatic environments. solution was stirred for 24h. A pH10 of the reaction is adjusted by using sodium hydroxide solution. At pH 10, the solution was divided into two solutions; one of them is stirred for 24h and the second one is put in the autoclave for 3h. A washing process using DI water is carried out for the resulting precipitate to reduce the pH to 7. Finally, the product is dried at 80˚C for one day.

T-LDHs /PbI 2 NC
In a general synthesis technique, in-situ growth of the metal cations, typically, NaOH (5M) in 200ml of distilled H 2 O is prepared. Another solution of Fe(NO 3 ) 3 .9H 2 O (0.1M), Co(NO 3 ) 21 .6H 2 O (0.1M), Cd(NO 3 ) 2 .4H 2 O (0.1M), and 2.5g PbI 2 was prepared. This later solution was stirred for 24h. A pH10 of the reaction is adjusted by using the sodium hydroxide solution. After reaching pH 10, the solution was remained under continuous stirring for 24h. A washing process using DI water is carried out for the resulting precipitate to reduce the pH to 7. After washing, a drying process is carried out at 80˚C for one day.

Characterization Of Ldh
The XRD patterns of , Co-Cd-Fe LDH, and Co-Cd-Fe LDH/PbI 2 NC were obtained by Philips1 X`Pert 1 -MRD1 X-ray diffraction (λ CuKa = 0.15418 nm). Samples morphology is investigated using a 1 eld-emission1 1scanning electron11 microscope (FESEM, TEM, Zeiss SUPRA/55VP with GEMINI/ column). (Fourier Transform Infrared Spectroscopy (FTIR) was performed by A Shimadzu1-FTIR-3401-Jasco1 spectrometer to obtain the important functional groups of the samples. Finally, the optical absorbance behaviors of the products are investigated by Lambda 900-UV/Vis/IR Perkin Elmer spectrophotometer up to 1200 nm.

Study Snails Source And Maintenance
Adult B. alexandrina, snails (8-10 mm in diameter; 0.26g weight) have been obtained from Theodor Bilharz Research Institute (TBRI), (Giza, Egypt). Snails were transferred to Medical Malacology Lab and kept in plastic tank with dechlorinated aerated tap water (10 snails/ L) with a photoperiodicity of 12hr.
light/12 hr. dark cycle, a temperature of 25 ±3 °C and fed on oven dried lettuce leaves (1gm/ 10 snails) and Tetramin. The tank water was changed every three days. For collecting egg masses, pieces of polyethylene sheets (5 × 10 cm) were used (OECD, 2016).

Acute toxicity test in adult B. alexandrina
The toxicity of the two layered material Co-Fe-Cd and Co-Fe-Cd /PbI 2 LDH against adult mature snails (10-12 mm) were determined. Stock solution of two layered material was prepared using dechlorinated tap water (1000 mg/ L). A series of concentrations was prepared to calculate LC 50 and LC 90 at laboratory temperature (22-25ºC). Three replicates were conducted for each concentration and the control group (30 snails per experimental group). 72 hour after, the snails were transferred from the exposure concentrations, and maintained in dechlorinated tap water for another 24 hours of recovery. Mortality percent of snails were recorded and lethal concentration and slope values were analyzed by Probit analysis (WHO 1965).

Embryo-toxicity test.
According to Rapado et al., (2011) pieces of polyethylene sheets (5 × 10 cm) containing egg clutches (100 eggs) were collected for the embryotoxicity assay. The egg masses were transferred to Petri dishes contains LC 25 of LDH for 24h, subsequently washed with ltered and dechlorinated water (pH 7.0). Seven days after exposure, the embryos were examined for unviability (malformed embryos or dead) by stereomicroscope. Another egg clutches was transferred to dechlorinated aerated tap water as a control. Assays were performed in triplicate.

Scanning Electron Microscope of the mantle-foot region
The mantle foot regions of snails were separated under a stereomicroscope. Then, the specimens were xed, dehydrated, critically dried and coated as recommended by Ibrahim and Abdel-Tawab (2020 Nduku and Harrison, 1980, the hemolymph was collected from the snail heart by insertion a capillary tube into the snail shell that is directly over the heart. 10 µl of hemolymph was spared on a glass slide to prepared hemocytes monolayers and leave to air-dry for 15 min at laboratory temperature. Hemocytes were xed with 100% methanol for 5 min and then stained with 10% Giemsa stain (Aldrich) for 20 min, then examined under the light microscope. This assay was done in triplicate for each group. Morphological changes observed were classi ed.

Measurement of IL-2 level and Caspase-3 activity
IL-2 in tissue homogenate was measured by enzyme linked immunosorbant assay (ELISA). Cytokine levels were determined by commercially available ELISA kits for IL-2 (OptEIA™ Kits; BD Biosciences). The depth of the color can then be measured spectrophotometrically at appropriate wave length. The intensity of colored end product provided a measure of the cytokine concentration (Hemdan et al. 2007). Caspase-3 activity was determined according to Bonomini et al., 2004. The released p-nitroaniline (pNA) moiety concentration was measured colorimetrically at 405 nm.

Tissue preparation for oxidant/antioxidant biomarker and biochemical studies
The snail soft tissues were removed from the exposure group and the control one, weighted, and then homogenized in ice cold, twice-distilled water using a glass Dounce homogenizer. The supernatants were separated using high speed centrifuged (3000 rpm for 10 min) and stored at − 80 °C until used.

Oxidant/antioxidant defense biomarker:
These biomarkers have been measured in the supernatant of the tissue homogenate for LDH exposure group and control one. The enzymatic responses SOD, CAT and GST were measured according to Aebi 1984, (Mannervik and Guthenberg 1981). While, non-enzymatic responses GSH, was determined according to the method of Ellman, 1959. For biochemical Studies the snails' total protein was done according to the method of Gornall et al., 1949. All parameter determined using biodiagnostic kits (Biodiagnostic Dokki, Giza, Egypt). 4.6. Genotoxicity: 4.6.1 Detecting of DNA single strand breaks (Comet assay) DNA single strand damage of snails exposed to LC 25 of LDH for 48 h was detected by single cell gel assay as previously described by Singh et al., 1988 andGrazeffe et al., 2008. 4.7. Histological evolution of the gland After two weeks of exposure and recovery, adult B. alexandrina snails (8-10 mm) were selected randomly and dissected. The digestive and hermaphrodite gland were removed, and xed in Bouin's solution. The glands dehydrated, embedded in para n wax. then, the both sectioned and stained with hematoxylin and eosin (Mohamed and Saad 1990). The digestive and hermaphrodite gland were examined by light microscopy for any alteration in compared to control snails 5. Statistical Analysis Data analysis were performed by t-test to determine the signi cant difference between exposure and control group and expressed as mean ± SME of mean (Graph Pad Prism 6.04 software). The lethal concentration (LC 10 , LC 25 , LC 50 , and LC 90 ) values, slope and respective 95% Con dence limit (CL) of LC 50 was calculated by Probit analysis (Finney 1971 The FTIR charts of (Co-Cd-Fe) LDH and its composite are displayed in Fig. 1A (A, B), and Table 1. After combination of PbI 2 , there are red shifts in absorption bands and some peaks changed in intensity and other broads Fig. 1A (B).
Their crystal sizes were calculated using Scherrer's relation [R]. The mean size was ~23.5 nm. In addition to their average microstrain value was ~ 0.7% and its dislocations density was 0.0018 that evaluates the density of defects and the quality of the crystal. This result gives a re ection to high quality of the synthesized LDH crystal.

Morphological properties
The morphological properties were examined through FESEM and TEM, at g. 1C (A, B). The morphology of Co-Cd-Fe LDH was characterized with the agglomeration of the particles which have plate like morphology [r]. This behavior was similar for all hydrotalcite prepared by co-precipitation method. TEM clari ed the plate like of LDH layers and proved the morphology of the LDH.

Toxic impact of LDH on adult B. alexandrina
In the present study Co-Cd-Fe LDHs and Co-Cd-Fe LDHs /PbI 2 (LDH) was tested for its toxic effect against B. alexandrina. Snails were exposed to different concentrations of Co-Cd-Fe LDHs and Co-Cd-Fe LDHs /PbI 2 (LDH) for 72 h of exposure followed by another 24 h for recovery. Probit analysis showed that the LC 50 and LC 90 of Co-Cd-Fe LDHs were 147.7 and 205.9, respectively. While Co-Cd-Fe /PbI 2 LDH showed more toxic effect, LC 50 and LC 90 were 56.4 and 95.3 mg/l ( Table 2).

Embryo-toxicity
The results of the LDH embryotoxicity are illustrated in g. 2. Exposure to Co-Cd-Fe LDHs caused suppression of embryonic development, embryo malformation and accumulation of LDH NPs in the eggclutches ( g. 2B).

Effect of LDH on B. alexandrina ultrasturacture:
The scanning electron micrographs of the soft part of Biomphalaria alexandrina snails showing the normal foot plantaris with notable surface fold and covered with ne and smooth cilia (Fig. 3A), and tegmental surface of mantle with microvilli and ne spines (Fig. 3D). Following the exposure to LC 25 , the foot cilia became tangled, adherent, and ultimately peeled off ( g. 3A and 3B). Also, the tegmental surface of mantle became rough, most microvilli completely destroyed, nipples and erosion ( Fig. 3E and   3F).

Impact of LDH on hemocytes of B. alexandrina
In control group, microscopical examinations of B. alexandrina hemocytes showed three types of cell that differentiated morphologically. The rst type is hyalinocytes; the second is granulocytes (spreading hemocytes), and the third is round small (undifferentiated) (Fig. 4A, 4B, 4C). After exposure to the LDH at sub lethal concentration (LC 25 ), hyalinocytes nucleus showed shrinkage and others had two separate nuclei; also, aggregations of hyalinocytes were more evident after exposure to LC 25 . While, granulocytes having irregular cell membrane, aggregate and formed either pseudopodia or lopodia (Fig. 4D, 4E). 6.6. In uence of LDH on IL-2 level and caspase-3 activity In the present study, there are a marked increase in expression of IL-2 in LDH exposure group (p< 0.001) in compared to non-exposure one ( g. 5B). While, caspase-3 activity was slightly increased (p < 0.05) ( g. 5A).

Impact of LDH on oxidant/antioxidant defense biomarker and biochemical studies:
In the present study, Exposing of snails to LC 25 of LDH induced signi cant decreased (p<0.001) in SOD and CAT (p<0.01) activity compared to the non-exposer group (control), ( g. 6A and 6B) with no change in GST activity (P 0.05), ( g. 6C). Concomitantly, a signi cant decrease of GSH levels and total protein content (p<0.001) in tissue homogenate was observed in LDH exposure group compared with their timematched controls ( g. 6D and 6E).

In uence of LDH on DNA
The present results showed that the olive tail moment (OTM) of snails subjected to sub lethal concentrations of LDH was highly increased (p < 0.01) than control snails ( g. 7A and 7B) 6.9. Impact of LDH on digestive and hermaphrodite gland of B. alexandrina Examination of the histological sections through digestive gland showed many tubular glands with single layer of secretory cells (SC) and digestive cells (DC) (Fig. 8A). Treatment these snails with LC 25 of LDH, showed rupturing, vacuolation and a signi cant increase in the number of SC. also, the lumen (L) increased, most of the DC and SC were degenerated and ruptured while the tubular glands lose their con rmed shape (Fig. 8B). Meanwhile, the histological sections of B. alexandrina snails of the control group through the hermaphrodite gland revealed female oogenic cells with normal oocytes and mature ova and male reproductive cells with normal spermatocytes, and sperms (Fig. 8C). The treatment of snails with a dose of LC 25 caused degenerations and destruction of some oocytes, mature ova, spermatocytes and sperms (Fig. 8D).

Discussion
Layered Double Hydroxide (LDH) gains signi cant attention in life science applications due to their extremely governable synthesis and high biocompatibility. But, few studies highlight toxicity and toxicokinetic of LDH. In the present study, we engineered Co-Cd-Fe LDH, and T-LDH/PbI 2 NC and its toxicological impact was evaluated. The results of Co-Cd-Fe LDH characterization by XRD were matched with a usual LDH with crystallinity mean size ~23.5 nm (Lu et al., 2015;Mohamed et al. 2018). Also, their FTIR spectra were similar to that previously recorded Mohamed et al. 2018;Parida and Mohapatra 2012). While, FSEM and TEM clari ed the plate like of LDH layers (Tedim et al. 2011;Chen et al. 2017).
It was proven that LDH has toxic impact against human cell line (Choi et al., 2007) and green algae Scenedesmus quadricauda (Ding et al., 2018). In the present study, Co-Cd-Fe LDH, and T-LDH/PbI 2 NC showed toxic effect against B. alexandrina and T-LDH/PbI 2 NC more toxic to adult Biomphalaria than Co-Cd-Fe LDHs with LC 50 of 56.4 mg/L. Enzymatic (GST, SOD and CAT) and non-enzymatic (GSH) antioxidant markers play a vital role in protection the organisms from oxidative stress and suppression of its cellular damage as it reduce and converted H 2 o 2 and superoxide anion radical. While, GSH act as a reducing agent in conjugation with xenobiotics (Pena-Llopis et al. 2001). Disturbance of oxidant/antioxidant system has been the main toxic impact induced by NMs in snails. LDH increased the ROS production and subsequently altered the enzymatic and non-enzymatic antioxidant enzyme, such as SOD,CAT, GSH (Ali, 2014b;Ali et al., 2012;Bao et al., 2018). In addition, this reduction can be elucidated to the direct combination of metal with active site of enzyme and its bio-transformation. The present data showed signi cant decrease in SOD, CAT, GSH and this in agreement with Gnatyshyna et al., 2020 as non-enzymatic marker activity decreased in Lymnaea stagnalis after exposure to Cu, Zn, Cd and Thiocarbamate. Also, a decrease of catalase activity was seen previously in snail exposed to herbicides (Bhagat et al. 2016). In addition, exposure of B. alexanderina snails to ZnONPs showed signi cant inhibition of GSH and CAT ( (Fahmy et al. 2014). In contrast with our result, Atli and Grosell, 2016 reported that exposing L. stagnalis to only the highest concentrations of Cu caused an increase in antioxidant enzyme.
Exposed snails showed no signi cant variation in GST activity compared to control. This nding agreed with those obtained by Sánchez-Marín et al., 2020 as they indicated that this enzyme is not activated in response to Organophosphate ame retardants, tris (1,3-dichloro-2-propyl) phosphate in mussels Also, the present study declared a marked decrease in the total protein content (p< 0.001) compared with controls. Fahmy et al., 2014, recorded a signi cant decrease in B. alexanderina snail protein content after exposure to ZnONPs. Whatever, activity of antioxidant marker differed depending upon the tissue type and metal concentrations and animal species. SOD and CAT activity in Daphnia magna exposed to Cd and Cu varied according to metal concentration. Also, L. natalensis snails collected from polluted dams in Zimbabwe showed variation in SOD and CAT activity (Siwela et al., 2010) . In addition, Achatina fulica showed reduction in CAT and SOD activities after exposure to Cd and Zn. This variation could be attributed to the excess production of ROS (Chandran et al., 2005). Similarly such enzyme reductions were also observed in the present study in response to LDH exposure.
A slightly increase of caspase-3 activity was detected as unspeci c response to LDH stress. Its elevation was observed previously in apoptotic cells (Elmore, 2007;Florentin and Arama, 2012), and this elevation may be due to cytological changes in the digestive gland of LDH-stressed snails (Zaldibar et al., 2007a(Zaldibar et al., , 2007bHödl et al. 2010;Benito et al., 2017). Previously, increasing in caspase-3 activity has been detected in L. stagnalis in response to pollutant stress ( (Gnatyshyna et al. 2020). Also, caspases-3 levels increased in Helix aspersa snails after exposure to iron oxides nanoparticles (Sidiropoulou et al. 2018) In the declared data, LDH at sub lethal concentration (LC 25 ), caused abnormalities in hyalinocytes and granulocytes shapes as nucleus shrinkage, divided to two separate nuclei, aggregate or formed pseudopodia. The immuno-cell responses and molecular aspects in B. alexandrina snails considered as important biomarkers of exposure to environmental pollutants (Mohamed 2011). Biomphalaria snails immunology can be attributed to hemocyte which are the critical line of cellular defense (Larson et al. 2014), where, they contributed in many defense mechanism against several pathogen as it is responsible for the phagocytosis, cytotoxic reactions (Fried 2016) and release soluble compounds including agglutinins and antimicrobial peptides (Ottaviani, 2006;Mitta et al., 2000).
Chronic exposure of the T. pisana to Ag NPs caused alterations in hemocytes, such as micronuclei, binucleated cell and kidney-like nuclei (Radwan et al. 2019). Also, B. glabrata exposed to CdTe quantum dot showed altered hemocytes binucleates, micronuclei, and apoptosis (de Vasconcelos Lima et al. 2019). Cell-cell aggregation was considered as an immunological response for host defense. Cellular aggregation of the invertebrates' hemocytes prevented the accidental blood loss by the formation of a biological plug at the site of the wound and resisted the entry of pathogenic microorganism (Guria et al. 2016). Hughes et al., 1990 andOttaviani et al., 1993 were detected cytokine-like molecules in marine and freshwater mollusks. IL-2 was one of the cytokines assayed. It is responsible for phagocytosis and provokes the strongest response in the synthesis of biogenic amines, nitric oxide (NO) or oxygen radicals (Ottaviani et al. 1995a, b). In the present study, there are a marked increase in expression of IL-2 in LDH exposure group (p< 0.001) in compared to non-exposure one. (IL)-2-like peptide were also detected in sea mussel which may be involved in the regulation of responses to different types of stress (Cao, 1998;Barcia et al., 1999).
On the level of DNA damage, as an important biomarker of NM toxicity in snails. Comet assay is a sensitive tool to detect DNA damages like DNA single-strand breaks (SSBs) . The present results showed that the olive tail moment (OTM) of snails exposed to sub lethal concentrations was increased than control snails. This in agreement with (Ibrahim and Ghoname 2018) who demonstrated that the OTM of snails exposed to LC 10 (27.5 mg L -1 ) or LC 25 (32.4 mg L -1 ) of the aqueous leaves extract of Anagalis arvensis was signi cantly higher than the control group. Such genotoxic effects might be due to either oxidation of DNA bases or covalent binding to DNA resulting in strand breaks. Some recent studies link DNA SSBs in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics (Lee and Steinert, 2003). Exposure to inorganic nanomaterial as Ag NPs, CuO NPs, IONPs, MgO NPs, TiO2 NPs, and ZnO NPs induced genotoxic effects in snails (Caixeta et al. 2020).
The embryotoxicity observed after exposure has been attributed to ROS production, oxidative stress and damage. Also, penetration of LDH NPs to gelatinous capsule and cross the egg membrane reduce essential growth metabolism aspects, changes in its permeability, consuming energy for the development and nally interrupting the mechanics of hatching (de Chavez and de Lara, 2003;de Vasconcelos Lima et al., 2019).
Our result in agreement with Besnaci et al., 2016, who state morphological changes and precipitation of Fe 2 O 3 NPs in the egg mass. Also, morphological abnormality and hatchability hinder was seen in B. pfeifferi embryos following exposure to curcumin-nisin polylactic acid NPs for 96 h. In addition, Hydrophilic nanosilica induced embryotoxic effects in B. alexandrina snail at concentration 590 ppm for 6 h and 980 ppm for 48 h ( (Attia et al. 2017). Similarly, the growth and hatching rate reduction was seen in B. glabrata embryos exposed to CdTe NPs for 24 h (Vasconcelos -Lima et al., 2019). In contrast, dimer captosuccinic acid (DMSA)-functionalized Fe 2 O 3 NPs did not induce embryo mortality, morphological alterations and hatching inhibition due to their physical properties and limited internalization in the eggclutches (Oliveira-lho et al., 2016). LDH posed a signi cant suppression in the growth of S. quadricauda algae after 72 h of incubation and a complete growth inhibition (100%) at higher LDH concentration. LDH had a higher inhibitory effect to growth than the other NPs (Ding et al., 2018).
In the present study, the foot and mantle of Biomphalaria alexandrina snails showed bioaccumulation of LDH in its surface and some morphological disturbances after the exposure to LDH for 24 h followed by 24h recovery as was detected by scanning electron microscope. LDH can interact, accumulated in foot and digestive gland of snails, and distributed to the mantle. Both Ag NPs and CuO NPs accumulation in mantle, foot and digestive gland of B. aeruginosa (Bao et al., 2018;Oliver et al., 2014;Croteau et al., 2014;Ma et al., 2017). Also, NMs possessed a highly adhesive property to a cell membrane therefore, it could affect the membrane structures and its macromolecules (Rasel et al. 2019). In addition this damage in ultrastructure could lead to snail death (Ibrahim and Abdel-Tawab 2020).
The deformation declared in the hermaphrodite gland of B. alexandrina histological sections after exposure to LC 25 of LDH was accompanied with a great damage in the gonadal cells where degenerations of some mature ova, spermatocytes, oocytes and sperms. Also, the connective tissue was dissolved and replaced by vacuoles. Saad et al. (2019) reported similar histological alterations in the hermaphrodite glands of B. alexandrina snails treated with copper oxide nano-composite (CuO NC), where, the ova and sperms degenerated and there were loss in the connective tissues between acini (Saad et al. 2019). The exposure of the snail to LDH may be lead to metabolic changes, destruction of gametogenic cells and damage of hermaphrodite glands which possibly resulting from a decrease in tissue proteins, apoptosis, or degeneration of cells of these vital organs (Omobhude et al. 2017).
The digestive gland was the main organ analysed in studies concerning oxidative stress induced by NM due to its higher accumulation capacity and role in the metal detoxi cation. Exposing of the digestive gland of B. alexandrina snails to LC 25 of the LDH, showed signi cant increase in the number and degeneration of the SC. The DC ruptured and vacuolated in addition, the tubular glands lose their con rmed shape. In like manner, Saad et al. reported histological alterations in the digestive gland of Coelatura aegyptiaca following treatment with ZnONPs for 6 consecutive days, where, there were gradual hypertrophy and hyperplasia in the glandular cells (Fahmy and Sayed 2017).

Conclusion
The data of the current study consider, the rst toxicological evaluation of LDH nanomaterial on freshwater snail B. alexandrina. In light of the above, LDH induce disturbance in both enzymatic and nonenzymatic antioxidant marker in the tissues of Biomphalaria following exposure to sublethal concentration, suppression the embryonic development. It caused alteration in mantle foot ultrastructure, immune response, histopathology of gland, and nally, genotoxic effect. This result re ects the possible ecological implications of LDH release in aquatic ecosystems and its risk assessment to aquatic invertebrate.

Declarations
Funding: Not applicable Con icts of interest/Competing interests: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
Availability of data and material: The data that supports the ndings of this study are available in the material of this article     Effect of Co-Fe-Cd LDH on the levels of enzymatic and non-enzymatic parameters and total protein in soft tissue of Biomphalaria alexandrina snail. All values presented as Mean ± SE. *,**,*** Signi cant difference as compared to control (P < 0.05, P < 0.01, P < 0.001 ).

Figure 7
Page 25/25 Light micrograph shows the extent of DNA migration by comet assay. (7A) Control B. alexandrina; (7B) snails exposed to sublethal concentration of Co-Fe-Cd LDH for 48 h with high DNA migration (p < 0.01) than control snails.