Assessment on the Toxic Effects of Chemically Synthesized SPIONs against Model Organisms

The remarkable physicochemical properties of nanomaterials have attracted researchers due to the numerous applications in the ﬁ eld of chemistry, biology, and physics. Despite the various applications, superparamagnetic iron oxide nanoparticles (SPIONs) are harmful to living organisms and to the environment as they are released without any safety testing. In this study, SPIONs were synthesized and further characterized. The aim of the study was to examine the toxicity of synthesized SPIONs against animal models: Zebra ﬁ sh — Danio rerio , Earthworm — Eudrilus eugeniae , and Drosophila — Drosophila melanogaster through histology using Hematoxylin – Eosin and Prussian Blue staining. The accumulation of SPIONs was further quanti ﬁ ed by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Through histology images, it was observed that the SPIONs had caused damages and a lower concentration of 0.001 – 0.002 μ g/ml of metal accumulation was detected in the ICP-MS analysis.


Introduction
New horizons of technical advancement have been opened with the help of nanotechnology [1].There are some promising applications of nanoparticles in nanomedicine, wastewater treatment, etc. [2][3][4][5][6][7].Magnetism is one of the numerous magnificent qualities of nanoparticles that fascinates researchers of both material sciences and biosciences [8][9][10].Research is being conducted on the applications of magnetic nanoparticles in many fields [11][12][13][14], which include improving cell seeding and distribution in tissue engineering scaffolds, targeted imaging, and therapy [15,16]; targeted imaging and therapy [17]; improving anticancer drug efficiency by amplifying reactive oxygen species stress [18]; drug delivery applications [13]; magnetic resonance imaging [19]; magnetically induced local hyperthermia [20][21][22]; wastewater treatment [23][24][25][26][27][28][29][30], etc. Superparamagnetic Iron Oxide Nanoparticles (SPIONs) act as carriers for drug targets as they obey to an external magnetic field [31].They can deliver biotherapeutics by targeting the ligands or stimulator-sensitive moieties that are responsive to external stimuli, such as light, ultrasounds, or magnetic fields [32].They help in the implementation of drugs directly to the target area with lower systemic concentration [33].SPIONs are also employed in the diagnosis of a variety of diseases.Intravenous injection of folate-tagged SPIONs in an antigen-induced arthritis model improved macrophage endocytosis in vitro and produced hypointense signals in affected joints; it may also be used in X-ray imaging [34,35].
Despite the advantages, biocompatibilities and toxicities of iron oxide nanoparticles raise real concerns [36,37].When iron oxide nanoparticles were injected into a rat model, it was claimed that they created oxidative stress, lowering the antioxidant capability of the blood cells [38,39].It has been reported that nanoparticles induce oxidative stress, which affects cell signaling [40].SPIONs are reported to cause oxidative stress responses such as inflammation, damage to the membrane, denaturation of protein, mitochondriamediated apoptosis, lipid peroxidation, genotoxicity, etc. [41,42].Low level of oxidative stress activates the genes responsible for transcription defense through transcription factor, leading to the activation of inflammation and apoptosis, and necrosis [43].The impact of iron oxide nanoparticles on cellular systems, like vascular systems, including blood cells, fibroblast, stromal cells, reproductive cells, lung cells, liver cells, kidney cells, and cerebral cells, have also been reported earlier [44].Iron oxide nanoparticles induces genotoxicity in intratracheally instilled mouse lungs, and the inflammatory responses lead to oxidative and lipid peroxide-related DNA adduct formations [45][46][47][48].The CCK-8 and lactate dehydrogenase assays can be used to determine cytotoxicity [49], and Comet reaction can be used to determine the genotoxicity in biological tissue [50].Various functional groups can be added to SPIONs to minimize the adverse biological effects [51].Mice, Drosophila [46], earthworms [52], fishes [53][54][55][56], and even its embryos [36,57] are some of the model organisms previously reported for studying the toxicological effects of iron oxide nanoparticles.The objective of this investigation is to explore the histopathological impact of SPIONs in three different animal models: zebrafish (Danio rerio), earthworms (Eudrilus eugeniae), and Drosophila (Drosophila melanogaster).The study aims to assess the harm and accumulation of SPIONs resulting from their administration to these animal models.This research provides insights into the potential applications of SPIONs in fields such as drug delivery and X-ray imaging.

Journal of Nanomaterials
Previous studies have demonstrated that SPIONs with varying physiochemical properties can exhibit either lower cytotoxicity or toxicity that is dependent on the dosage, particularly at dosages of 100 g/ml or higher [58].2.2.Synthesis of SPIONs.SPIONs were synthesized using magnetic field-mediated reaction method [35].Accordingly, 0.1 g of ferric chloride hexahydrate (FeCl 3 •6H 2 O) and 0.1 g of ferrous sulfate heptahydrate (FeSO 4 •7H 2 O) were separately dissolved in 1 ml of nitrogenized double distilled water and then mixed together for 5 min.500 µl of nitrogenized iron salt solution was then added to 500 µl of tetramethylammonium hydroxide (C 4 H 13 NO) and kept in vortex for 5 min.The obtained black precipitate was then washed in 1 ml of acetone and dried in hot air oven at 60°C for 30 min.The pellet was then dissolved in 700 µl of formaldehyde solution (CH 2 O), and 300 µl of nitrogenized H 2 O was added dropwise with simultaneous vortex, which was followed by 500 µl formaldehyde solution.The obtained solution was then kept in the magnetic field for 2 days and was then dispersed in formaldehyde solution.The nanoparticles were then washed four to five times in nitrogen-purged double distilled water prior to toxicological experiments.Journal of Nanomaterials (Bruker, Dimension icon model, Germany) to examine the confinement of the nanoparticle to the three coordinates as 3D images, X-ray Diffraction (XRD) by Smartlab X-ray Diffractometer (Rigaku Corporation, Japan) to check the crystallinity and Vibrating Sample Magnetometer (VSM) (LakeShore Co. Ltd., Lake Shore 7407, Westerville, Ohio, USA) with a maximum magnetic field of 2.5 T and dynamic moment range of 1 × 10e −6 -10e 3 emu to check the magnetization property.

Characterization of
2.4.Feed Preparation.Feed was prepared separately for D. rerio, E. eugeniae, and D. melanogaster.Stock solution of 0.010 mg/10 ml of SPIONs were prepared using distilled water and used in this study against earthworm and zebrafish.For study against Drosophila, we used different concentrations of SPIONs ranging from 0.002 g/1 ml to 0.005 g/1 ml.
2.4.1.D. rerio Feed Preparation.Micro pellet feed was soaked in different concentrations of SPIONs suspension and incubated at room temperature until all the nanoparticles were absorbed into the feed.It was then dried in a hot air oven at 40°C overnight, after which the nanoabsorbed feed was kept at the room temperature for 15 min to cool and then stored for further use.

E. eugeniae Feed Preparation. Dried cow dung was fed
to the E. eugeniae.The stock SPIONs solution was suspended in the deionized water in different concentrations and were sprayed onto the cow dung and dried, which was then stored for further use.
2.4.3.D. melanogaster Feed Preparation.25 g of jaggery was crushed and boiled in 250 ml of water until it was dissolved completely.25 g of semolina was added to the previously prepared solution, which was followed by the addition of 10% agar.About 1.9 ml of propionic acid was added to the prepared porridge and thoroughly stirred while it was still hot.The porridge was then transferred to 5 beakers (50 ml each).Stock SPIONs of varied concentrations were added to the heated mixture and thoroughly stirred.10 ml of each was poured into separate test tubes while the porridge was still  for feeding and the study for three generations, i.e., larva, pupa, and adult fly, each of which were collected for analysis.
The administered feed details are given in Table 3.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
and Staining of Organisms.The organisms after the study period, were subjected to histopathological staining, for which they were preserved in 10% formaldehyde solution.
Hematoxylin-Eosin (H&E) staining was done to detect changes in the tissue of the organism, according to the procedure followed by Samrot et al. [59] and Cardiff et al. [60].Prussian Blue staining was also done to examine the iron accumulation inside the tissue [61][62][63].Organisms from each concentration were subjected to acid digestion [64] and analyzed using ICP-MS analysis (Agilent Technologies, 7700 series, Santa Clara, CA, USA).10-25 nm (Figure 1), which was also confirmed by TEM images (Figure 2).Samrot et al. [59] also reported the size of SPIONs to be around 25 nm, synthesized using ammonia as a reducing agent.In earlier reports, Samrot et al. [65] synthesized SPIONs which ranged from 9 to 25 nm.SPIONs synthesized using precursor solutions such as FeCl 3 and FeCl 2 were found to be 40-45 nm in size [66].In a study by Sathya et al. [67], the size of iron oxide nanoparticles synthesized using a magnetic stirrer and ultrasound method ranged from 20 to 90 nm.The 3D structure of the synthesized SPIONs was confirmed by AFM analysis (Figure 3).It was confirmed that the particles were spherical and well dispersed.According to the XRD pattern (Figure 4), the synthesized SPIONs were in the inverse spinel structure.Shukla et al. [68] reported that the iron oxide nanoparticles exhibited peaks at 30.1, 35.5, 42.6, 53.6, 57.0, and 62.8 assigned to the diffraction plane of the spinel structured magnetite nanoparticles (220), (311), ( 400), ( 422), (511), and (440), respectively.VSM measurements were done to estimate the magnetization and coercivity of the synthesized SPIONs.The magnetization curves clearly indicated the superparamagnetic behavior of the synthesized SPIONs (Figure 5).

3.2.
Histology.H&E stain is usually used for the identification of different kinds of cells and its pattern, shape, and structures, whereas Prussian Blue staining is performed to identify the accumulation of iron as it can form ferric ferrocyanide complex by producing blue color in the tissue [69,70].Intestines of D. rerio were stained using H&E following exposure to different concentrations of SPIONs (Figure 6).Lower concentrations of SPIONs did not show any significant impact, whereas exposure of higher concentrations caused erosion in the goblet cells in the intestinal area and on the intestinal wall of D. rerio.Tissues stained using Prussian Blue (Figure 7) revealed the accumulation of iron oxide nanoparticles inside the tissues.It was accumulated in and around the intestinal walls.Samrot et al. [59] earlier reported that the metal nanoparticles impacted the rate of reproduction.The exposure of SPIONs had a negative impact on the tissue of E. eugeniae as well, which was seen in the histology images.The earthworm's exterior structure was altered significantly.As the concentration of SPIONs increased, the circular and longitudinal muscles were found to be degraded in addition to the gizzard area (Figure 8).E. eugeniae treated with different concentrations of SPIONs, and control were also studied using Prussian Blue staining (Figure 9), which showed the accumulation of SPIONs and is believed to be the reason for damaged circular muscle and epidermal erosion.In earlier reports, it has been evidenced that exposure of Fe 2 O 3 onto the earthworm, significantly decreased its growth and rate of reproduction [72,73].Samrot et al. [52] also reported erosion of epithelium, fibrosis of the circular muscle, and gut disintegration of earthworms on exposure to magnetite nanoparticles.
The histology images of D. melanogaster exposed to different concentrations of SPIONs.Figures 10-15 depicted visible distortion in the waxy cuticles of larvae at lower concentrations, whereas degradation at higher concentrations was also noticed (Figure 10).The deposition of iron in the larvae of D. melanogaster in Figure 11 is visualized using Prussian Blue staining.The images show the accumulation of SPIONs in the larvae's anterior and in the midguts.The mouth, spiracles, and trachea in the anterior and the ovary and gonads in the posterior of the larvae have been found to be degraded at higher concentrations.
The histology images of the D. melanogaster pupa stained with H&E are shown in Figure 12.The images do not show any damages after being exposed to lower concentrations, such as 0.002 g : 50 ml and 0.003 g : 50 ml of SPIONs : feed ratio but higher concentrations, such as 0.004 g : 50 ml and 0.005 g : 50 ml, exhibited erosion of the puparium, anterior, and posterior regions and severe damages in the tracheal region.The Prussian Blue staining of the pupa illustrated the accumulation of the iron (Figure 13).No visible accumulations were observed at lower concentrations, but at higher concentrations, the posterior region of pupa was accumulated with iron nanoparticles.Raj et al. [74] reported that a high dose of silver nanoparticles at the early stages of development could negatively impact the behavior and metabolism of D. melanogaster.Adult D. melanogaster did not show any considerable impact at the lower concentration but revealed the formation of lipofuscin-like granules in the abdominal region.But whereas at higher concentrations, the head, thorax, and abdomen were entirely distorted (Figure 14).The iron accumulation is visible in the histology images (Figure 15) stained by Prussian Blue.Abdominal region of adult D. melanogaster also showed lipofuscin-like granules responsible for the erosion in gut area.The thorax and abdomen were entirely degraded at the higher concentrations.Ong [75] revealed that the survival rate of D. melanogaster fed with silver nanoparticles decreased significantly when compared to control flies.

ICP-MS Analysis.
The concentration of SPIONs in the aqueous solution of acid-digested organisms from each concentration was studied using ICP-MS analysis [76].The control samples showed no substantial deposition, albeit a small amount (0.001-0.002 μg/ml) was detected, which can be attributed to the presence of metal in the organism's natural habitat.Figures 16 and 17 shows the metal accumulation inside the bodies of D. rerio, E. eugeniae, and different stages of D. melanogaster, which was supported by histology images as well.

Conclusion
SPIONs have been synthesized and characterized using SEM, TEM, AFM, XRD, and VSM in this study.The toxicity studies of SPIONs were carried against D. rerio, E. eugeniae, and the different stages of D. melanogaster and histology studies were carried out by staining the tissues with H&E and Prussian Blue.
The exposure of nanoparticles onto the organisms were depicted through histology studies, and metal accumulation was investigated by ICP-MS analysis.The synthesized SPIONs had considerable damages in the organisms which are suspected to be toxic.12 Journal of Nanomaterials

FIGURE 17 :
FIGURE 17: ICP-MS analysis for different stages of Drosophila.

TABLE 3 :
Dietary ratio provided to Drosophila melanogaster.

TABLE 1 :
Dietary ratio provided to Danio rerio.
2.5.Feeding the Organisms.Four different concentrations of feed were given to D. rerio, E. eugeniae, and D. melanogaster.2.5.1.Feeding D. rerio.After a month-long acclimation, D. rerio were bifurcated into five different fish bowls, among which one bowl was considered as control.The administered feed details are given in Table1.2.5.2.Feeding E. eugeniae.E.eugeniae were introduced to the soil containing cow dung in a plastic tub and allowed to acclimate for a week.The earthworms were fed dried cow dung at regular intervals.In order to ensure proper growing circumstances for the earthworms, the moisture conditions in the box were also checked regularly.The administered feed details are given in Table2.