A study on influence of superparamagnetic iron oxide nanoparticles (SPIONs) on green gram (Vigna radiata L.) and earthworm (Eudrilus eugeniae L.)

The seeds of Vigna radiata were surface sterilized with 2 % sodium hypochlorite solution and were soaked overnight in distilled water. The soaked seeds were again surface sterilized before use.

The impact analysis of the SPOINs on Vigna radiata was carried out for a study period of 6 days. The SPIONs sample were diluted with distilled water for the hydroponic culturing to a working concentrations of 1 μg ml⁻¹, 10 μg ml⁻¹ and 20 μg ml⁻¹. The seeds were allowed to germinate and grown in-vitro using culture tubes containing SPIONs with no other nutrient. Each culture tubes were introduced with pretreated seeds containing a M-shaped paper inserts (Whatmann filter paper) in order to keep them in contact with the SPIONs solution. The seed germination and growth were observed for the study concentrations in triplicates with distilled water as control.

The germinated seeds were checked for their root and shoot length on the 3^rd and 6^th day. The total weight of the plants on the 6th day was measured to evaluate their biomass.

2 g of the plant was crushed with 15 ml of distilled water using mortar and pestle. The puree was filtered using Whatmann Filter paper. The filtrate was used to assess the presence of the following phytochemical test.

2.4.4.1. Test for carbohydrates

2.4.4.2. Test for proteins

2.4.4.3. Test for lipids

2.4.4.4. Test for tannins

2.4.4.5. Test for saponins

2.4.4.6. Test for alkaloids

2.4.4.7. Test for flavonoids

2.4.4.8. Test for phenols

2.4.4.9. Test for cardiac glycosides

The root tips, the basal region of shoot and the leaf apex of the plants after 6 days treatment were subjected to Prussian blue staining to identify iron elements present in the tissues. Equal volumes of freshly prepared 2% Potassium ferrocyanide and 2% Hydrochloric acid were mixed together to get Prussian blue staining buffer and the above mentioned plant parts were soaked in staining buffer separately for 15 min (Wei et al 2015). After incubation, they were rinsed in distilled water and thin sections of the sample was cut using a blade & scalpel and fixed on a glass slide to observe under Light microscope at 10 X magnification.

SPIONs treated plants were rinsed in distilled water, dried and digested with 2 ml of nitric acid to obtain its acidic extract. The obtained extract was heated to 120 °C for 30 min and subjected for ICP–OES analysis to detect iron (Agilent ICP-OES VDV 51009) (Geisler-Lee et al 2012).

The earthworm, Eudrilus eugeniae were collected from SS vermicompost, Tambaram, Chennai and were kept in manure rich soil. Prior to the nanoparticle exposure, they were allowed to adapt to the soil environment for 2 days. The moisture content was maintained and the Earthworms were fed with powdered cow dung every 24 h (Samrot et al 2017, Samrot et al 2019d). The earthworms were pretreated as mentioned earlier (Samrot et al 2017).

The pretreated earthworms were exposed to different concentrations of SPIONs (10 mg, 20 mg and 30 mg) added to soil, as per the same procedures followed in our earlier reports on gold nanoparticle by Samrot et al (2018c). A count of 20 earthworms was introduced to each of the study concentration. On the 10th day, the earthworms from each concentration was subjected to histology and ICP–OES analysis. Similar protocol was trialed for all the SPIONs concentration to observe the impact on reproduction for a period of 30 days (Samrot et al 2017). Phenotypic changes in appearance and behavioral patterns were observed for each set of earthworms and compared to the control.

On the 10th day, foregut, midgut and hindgut region of three earthworms from each concentration was cut using a surgical blade. Tissues were preserved in 10 % formaldehyde solution, dehydrated in ethanol and then fixed in wax. Sections were made using microtome and H & E staining was performed. Sections were observed under microscope and histological images were documented.

After the study for 10 days, one earthworm from each concentration were dissected, digested in nitric acid (Samrot et al 2017, Samrot et al 2019b) and was subjected to elemental analysis for the presence of Fe using ICP-OES.

The absorbance property of the synthesized SPIONs can be understood on exposure to UV-Visible radiations. From the results, it is observed that the synthesized SPIONs show maximum absorbance near 420 nm followed by a blunt peak in the range of 450 nm–540 nm (figure 1). Kumar et al (2015) has stated that the absorption peak for Fe₃O₄ appeared at 492 nm while Samrot et al (2017) reported peak absorbance near 250–260 nm for SPIONs, these discrepancies in the absorption spectra could be due to the varying size and composition of SPIONs as a contributing factor.

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Using SEM, the topographical view was visualized at the magnification of 185.93 KX where the synthesized SPIONs were in the size range of 14.53 nm to 20.54 nm (figure 2). Samrot et al (2019c) showed in his earlier discussions the size of SPIONs produced using ammonia as reducing agent was around 25 nm, whereas the use of a different reducing agent in this case Cobalt chloride in NaOH did contribute to the change in size of the nanoparticle formation. EDX results affirmed the elemental composition of the synthesized SPIONs which showed the presence of Fe and O.

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TEM images revealed the aggregation of synthesized SPIONs with particles appearing roughly spherical shaped in the size range between 15 nm – 20 nm. These results on size and distribution supplemented with the SEM results (figure 3). The smaller size of SPIONs were evenly reported where Justin et al (2018) stated SPIONs of size 15 nm and Samrot et al (2019c) detailed the SPIONs produced of 20 nm in size when NaOH was used as the reducing agent.

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The 3D illustrative image of the synthesized SPIONs was seen with the help of AFM which recorded the size to be around 15 nm, which further supported the TEM and SEM results (figure 4).

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The particle stability detected through the charge distribution on the surface of the synthesized SPIONs exhibited the zeta potentials as −15 mV (figure 5). The values such as these indicate that the particles are stable with negative surface charge. Nanoparticles are said to be stable when their zeta potential values are greater than +25 mV or less than −25 mV(Justin et al 2018).

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From the obtained records of XPS, the binding energy of Fe was recognized at 710 eV and the binding energy of CO and O was at 285.0 eV and 533.0 eV respectively (figure 6), which was showing it to be Fe₃O₄. Samrot et al (2018d) has also reported the characteristics of Fe₃O₄ as the binding energy at 711.8 eV, FeO at 709.6 eV and Fe at 706.7 eV. By fitting the XPS data in Shirley background subtraction using the CASAPEAK software, the highresolution spectrum of C 1s, O 1s, Fe 2p3/2 and Fe 2p1/2 were represented.

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The XRD diffractogram with sharp peaks signify high crystalline nature of the synthesized particle. The diffraction peaks of 2θ with the planes at (220), (311),(400), (511), and (440) figure 7 corresponds to cubic spinel arrangement of Fe₃O₄ (JCPDS 85-1436). This is more comparable with the XRD data of SPIONs in our earlier report (Samrot et al 2018d).

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Using the below Debye–Scherrer equation, the crystallite size was calculated.

$$\begin{eqnarray*}&&{\rm{D}}=\displaystyle \frac{{\rm{K}}\lambda }{\beta \,\mathrm{Cos}\,\theta }\end{eqnarray*}$$

where K is known as constant which is equal to 0.90, θ represents the Bragg angle, λ represents the wavelength of the incident x-ray used (λ = 0.1540 nm), β is Full Width at Half Maximum (FWHM) of the sharp peak recorded from the XRD analysis. The high intensity sharp peaks were considered in order to calculate the average crystallite size. It was found to be 11.97 nm. The maximum diffraction peak at plane (311) with FWHM of 0.521° corresponded to crystallite size of 15.98 nm. This further supports the results of AFM (15 nm) which is a 3D imaging. Thus the size of each crystal or grain of SPIONs was understood from XRD.

The effect of SPIONs on root and shoot development was examined by measuring their growth in centimeter (cm). Vigna radiata plants were measured on 3rd and 6th day. The minimal concentration (1 μg ml⁻¹ and 10 μg ml⁻¹) exposure has led to biostimulation in treated plants showing increased growth in root and shoot compared to control, expressing a maximum growth of 13:10.4 (shoot : root) by 1 μg ml⁻¹ treated plants (figures 8 and 10(a)). And also, optimistic impact as numerous rootlets development was evident with 1 μg ml⁻¹ and 10 μg ml⁻¹ treated Vigna radiata which is on par with the results of Shankramma et al (2016) who stated an enhanced growth in iron oxide treated Solanum lycopersicum owing to biomineralization and has also identified the deposition of iron oxides on the root hairs, root tips and nodal regions of treated plants. While the prolonged exposure (4–6 days) at the minimal concentration (1 μg ml⁻¹ and 10 μg ml⁻¹) did not influence the growth of treated plants effectively as compared to the control which showed progressive growth (figures 9 and 10(b)). However, there was reduction in root development in 20 μg ml⁻¹ exposed groups. Similar report has been discussed by Bombin et al (2015), where they studied the iron oxide nanoparticles (Fe₂O₃) with different charge coatings on Arabidopsis thaliana to hinder the root and shoot growth with reduced pollen viability, distressing its reproducing ability. Conversely, Iannone et al (2016) has exposed citric acid coated Fe₃O₄ nanoparticles on Triticum aestivum under hydroponic condition and reported null effect of Fe₃O₄ on plant growth and germination but reported an increased activity of antioxidant enzymes. Lin and Xing (2007) experimented with zinc oxide nanoparticle in higher plants and concluded with termination of root elongation in radish, grape and ryegrass.

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The plantlets of Vigna radiata were checked for their biomass content soon after the treatment with SPIONs and it was found that the plants treated with 1 μg ml⁻¹ and 10 μg ml⁻¹ of SPIONs showed maximum biomass of 0.426 g/plant and 0.53 g/plant respectively when compared to the control (0.358 g ml⁻¹) (figure 11). This could be due to the growth stimulation factor of SPIONs in treated Vigna radiata. Wang et al (2015) reported that nano-ferric oxides at 20 mg l⁻¹ on Citrullus lanatus showed increased soluble protein and chlorophyll contents. It could possibly infer that iron oxide nanoparticles at low concentrations are likely to promote the quality of the treated plants conversely the effect is stumpy at higher concentrations (20 μg ml⁻¹) on treated Vigna radiata plantlets. From the observations and results, it can be deduced that the quality of the plants were affected by the presence of SPIONs. Ghodake et al (2011) also stated a comparable result where cobalt and zinc oxide nanoparticles have inhibited the root elongation in Allium cepa through bioaccumulation causing severe hazardous effect at cellular and chromosomal levels. Although Rui et al (2016) have supported that the use of iron oxide nanoparticles as iron fertilizers on Arachis hypogaea, a crop sensitive to iron deficiency had evidently showed increased root length, plant height, higher biomass, phytohormone contents and antioxidant enzyme activity, the prolonged exposure and long term usage may lead to risk associated with biomagnification, which are needed to be studied in field conditions.

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The phytochemical analysis of treated Vigna radiata indicated the presence of carbohydrates, protein, lipids, flavonoids, saponins, glycosides and the absence of phenols, tannins and alkaloids were observed (table 1).

Accumulation of iron oxide particles was observed in all the test groups, where higher concentrations were found in the cortex of roots and stem (figures 12 and 13(b)–(d)), iron oxide particles were even found inside the leaves (figure 14). Prussian blue staining has been used to identify the uptake of iron by plants (Rios et al 2016). It is obvious from the SPIONs accumulated in tissues of plants might pave way for bioaccumulation.

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The incorporation of iron oxide into the plant tissues were further confirmed by ICP-OES. The study detected the highest iron content of 1.59 mg l⁻¹ in 20 μg ml⁻¹ treated plants, followed by 10 μg ml⁻¹ having 1.21 mg l⁻¹ and 1 μg ml⁻¹ with 0.76 mg l⁻¹ while the control having 0.705 mg l⁻¹ of Fe (figure 15). The presence of Fe in control must be the natural mineral composition in the seeds. Raju et al (2016) have also used Vigna radiata as the plant model to study the effect of iron oxide nanoparticles and detected the uptake of iron oxide nanoparticles by the plant tissue through ICP-MS analysis.

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Due to the accumulation of SPIONs in the epithelium of earthworms, the color of the Earthworm was found to get darker with increasing concentrations but no mortality was observed in the Earthworms, Samrot et al (2017, 2018b) also reported similar observations when earthworms were exposed to magnetite and gold nanoparticles. The accumulation of nanoparticles has also affected the reproduction rate of earthworms. The reproduction rate was highly affected as the concentration increased table 2. Gold nanoparticles and silver nanoparticles were also found to reduce the number of earthworms after exposure. (Samrot et al 2018c, 2018e).

The histology images have revealed adverse effects of SPIONs caused on the tissues of earthworms. Impact of SPIONs in the gut region was evidenced by disintegration of the gut and lipofuscin deposits (figure 16). The outer structure of earthworm revealed drastic changes (figure 17). As the concentration of the Earthworms increased, erosion in circular and longitudinal muscle was observed (figures 16–18). And a complete destruction of gizzard area was also seen (figure 18(d)). Similar effects were observed in earthworms exposed to iron oxide, silver and gold nanoparticles causing disintegration of gut and other adverse effects in the Earthworms (Schlich et al 2013, Samrot et al 2018c, Samrot et al 2018e, ). The effect of carbon nanotubes along with sodium pentachlorophenol caused DNA and membrane damage on earthworms (Yang et al 2017).

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Accumulation of SPIONs into the treated earthworms can be witnessed with increase in concentration of nanoparticle. Accumulation of SPIONs in the control earthworms may be due to the nanoparticles present in the soil naturally (Samrot et al 2017). But the increased accumulation with increase in concentration (figure 19) explains that the effects noted are due to the nanoparticle ingestion.

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