Nanoparticle characterization
Magnetite nanoparticles have been synthesized biologically using the laurel plant (Laurus nobilis). In an aqueous colloidal solution, the stability and formation of magnetite nanoparticles were confirmed by using UV-Vis spectral analysis. As a result of UV-Vis measurements of the obtained nanoparticle, the highest peak value was found in the wavelength range of 235–250 nm which represents the surface plasmon resonance of the magnetite NPs (Fig. 1). The higher absorbance between 200–250 confirms the formation of magnetite nanoparticles (Yew et al. 2016).
Identification of the possible biomolecules responsible for stabilization and capping of nanoparticles were carried out by FT-IR spectra of synthesized magnetite nanoparticles. The peaks correlated with magnetite nanoparticles are shown in Fig. 2. FT-IR was used to examine the presence of biomolecules responsible for magnetite nanoparticle synthesis. According to the FT-IR spectrometer results, the peaks around 590–610 cm− 1 could be assigned to the Fe–O stretching vibrational mode of Fe3O4. The peak occurring at 3308 cm− 1 can be referred as -OH band groups and to the traces of molecular water and the molecules come from the laurel extract. In the study of Souza et al. (2019), it was mentioned that the Fe–O stretching vibration parallel to the c-axis modes corresponds to the bands at 445–690 cm− 1. In the same study, it was also mentioned that the band near 690 cm− 1 might refer to iron hydroxide structures such as amorphous goethite, defective hematite (proto/hydrohematite), and errihydrite and is recurrent in hematite spectra. Chernyshova et al. (2007) referred to same bands for Fe-O stretching also added that the band around 3500 cm− 1 could be associated to the surface hydration layer.
According to the Zeta size analysis results, the size of the magnetite nanoparticles’ average was determined as 108.5 nm (Fig. 3a). The presence of the extra hydrated layers attached to the surface cause the difference in hydrodynamic size which usually occurs greater than the actual size measurements of the Fe3O4 NPs. Approximate size, surface morphology, shape and elemental composition were defined using scanning electron microscopy (SEM) (Fig. 3b) and EDS (Fig. 3c). SEM analysis showed that nanoparticles were spherical in shape and have 247 nm mean size. Dispersion/aggregation behavior of nanoparticles is difficult to determine through dried solutions from microscopic data from like SEM. This situation usually causes a difference between SEM and Zeta size results for nanoparticle size. EDS results showed that the green synthesized nanoparticle contained 51% Fe.
ICP-MS results showed there was 590 mg Fe in 1 kg magnetite NP sample. According to ICP-MS results the Fe content in 2000 mg mL− 1 (highest applied concentration) was 1.18 mg while this content was 0,059 mg for 10 mL of growth medium.
Toxicological Effects Of Magnetite Nanoparticles
Growth Parameters
L. minor plants treated with 0.1, 1, 10, 100, 1000 and 2000 mg L− 1 magnetite nanoparticles for 15 days. The frond number, colony number, dry weight test results were examined for the physiological effects of the biological synthesis product magnetite nanoparticle on duckweed.
At the end of treatment, a significant decrease was observed in the number of leaves (frond number) at 100 mg L− 1 and higher concentrations (Fig. 4a). In case of the number of colonies, the lowest treatment concentration (0.1 mg L− 1) stimulated the colony development (16%) while the highest two concentrations 1000 and 2000 mg L− 1 caused a significant decrement in the number of colonies around 19 and 39%, respectively, compared to the control group (Fig. 4b).
Iron oxide magnetite nanoparticles can function as a plant growth stimulator (Shao et al. 2022). Blinova et al. (2017) revealed that at concentrations ≤ 100 mg L− 1 magnetite nanoparticles with numerous hydrochemical compositions were not toxic to duckweed (L. minor) in waters. Similar results were obtained in the studies with Lemna gibba (Barhoumi et al. 2015) and Chlorella vulgaris (Chen et al. 2012; Barhoumi and Dewez, 2013) which showed that low toxic effect at concentrations higher than 100 mg L− 1. According to previous studies, chemically synthesized magnetite nanoparticles in concentrations less than 100 mg L− 1 did not pose a threat to aquatic vegetation. In another study, the internalization and distribution of magnetite nanoparticles in the plants and their effects on plant growth were studied. The results demonstrated that magnetite nanoparticles stimulated alfalfa and soybean growth in 50 and 100 mg L− 1 concentrations. The stimulations were found related to citric acid coating of the nanoparticles (Iannone et al. 2021).
In the current study, the results illustrated that green synthesized Fe3O4 NPs are 10 times less toxic (threshold concentration 1000 mg L− 1) when compared to chemically synthesized Fe3O4 NPs which are toxic in the concentrations ≤ 100 mg L− 1 in waters (Barhoumi et al. 2015, Chen et al. 2012; Barhoumi and Dewez, 2013))
Photosynthesis And Mass Accumulation
Photosynthesis counts as a good method to assess the overall performance of a plant since it is the only energy entry point in plants (Kalaji et al. 2014). Also, it functions as a sensor for interpreting plant physiology and metabolism. Thus, to exhibit the effect of stress factors in the plant, photosynthetic pigments and activity are commonly used (Rastogi et al., 2017).
There were no negative impact of magnetite NP treatment on photosynthetic pigment content. Chlorophyll a/b ratio (Fig. 5a) which is an important parameter for toxicity on pigments, maintained similar to control level. This result indicated that magnetite NP has no hazardous effect on pigment system. Magnetite NP treatment did not cause any change in total carotenoids content (Fig. 5b) which are also important parameters to measure plant response to stress treatments (Rmiki et al., 1999). As photosynthetic accessory pigments and the components of antioxidant metabolism, their level and integrity were not damaged nor induced by magnetite NP treatment. These results, also are consistent with data from oxidative stress parameters (Fig. 7a,b).
In the study with Hordeum vulgare, the moderately increased pigment content up to 250 mg L− 1 was observed, then gradual a reduction with the enhanced concentrations of NPs was recorded (Tombuloglu et al., 2019). Furthermore, in Citrus maxima plants, as compared with control chlorophyll content was considerably enhanced by 50 mg L− 1 g-Fe2O3 NPs treatment (23.2%) whereas 100 mg L− 1 g-Fe2O3 NPs remarkably reduced chlorophyll content (Hu et al. 2017). Likewise, iron oxide NPs increased the chlorophyll level in soybean (Ghafariyan et al. 2013).
The photosynthetic pigment content maintenance as control group level was not reflected by dry weight of plants. The lower concentrations of magnetite NPs (0.1 and 1 mg L− 1) induced mass accumulation in L. minor plants, indicating that an induction in biosynthesis reactions, however, dry mass of treated plants significantly decreased after 100 mg L− 1 and higher concentrations in comparison to control. As parallel with the decrement of the number of the leaves from 100 mg L− 1, dry mass accumulation was blocked by the treatments higher than 100 mg L− 1. As a border of the inhibition of biosynthetic reactions it seems 100 mg L− 1 is a prominent concentration for water plants. Overall, it seems, photosynthetic pigments were not affected other biosynthesis reactions are suppressed by high magnetite concentrations. The increased dry weight of plants treated with the 0.1 and 1 mg L− 1 concentrations could be a result of Lemna's ability to accumulate metals. In resemblance to the these results, in the study of Horvat et al. (2007), dry weight of the L. minor plants that were treated with was increased after the treatments with Pb, Mn, Ni, Zn and Fe due to Lemna's ability to accumulate metals.
Hydrogen Peroxide Content And Lipid Peroxidation
The magnetite NP toxicity is prominent for 1000 and 2000 mg L− 1 concentrations showed by hydrogen peroxide content (Fig. 7a). As an oxidative stress indicator of cellular metabolism, enhanced H2O2 content in the highest two concentrations point out that increase in oxidative stress level in cells of L. minor fronds. This result was confirmed by data of Superoxide anion accumulation experiments which displayed cell injury in 1000 and 2000 mg L− 1 concentrations of magnetite NP (Fig. 8). Although oxidative stress and resulting injury were obvious for 1000 and 2000 mg L− 1 concentrations of magnetite NPs, necrosis on fronds were more prominent for 2000 mg L− 1 of magnetite NP application. These results are in accordance with malondialdehyde (MDA) production which is significantly increased in only for 2000 mg L (30%) (Fig. 7b). It may be speculated that the higher concentrations of treatments may cause oxidative stress in cells, but plant membranes could be protected up to 2000 mg L− 1 by antioxidant mechanisms to defend cell integrity.
MDA is a universally acknowledged lipid peroxidation (oxidation stress) biomarker. Polyunsaturated fatty acids form MDA after ROS peroxidation. Formed MDA reacts with TBA and creates a red-coloured TBA-MDA adduct from lipid peroxidation. The more colorization indicates more production of TBARS which means higher lipid peroxidation (Tsikas et al., 2017). Souza et al. (2019) reported, different iron oxide nanoparticles (akaganeite predominance + hematite) caused a dose dependent lipid peroxidation in the plant increased with rising iron oxide NP concentration for all iron-based nanoparticles tested.
The treatment to Brassica napus with different concentrations of iron oxide NPs caused a significant increase at the accumulation of H2O2 in the study of Palmqvist et al. (2017). The obtained results in the current study, showed similarity for H2O2 and MDA contents which statistically significant difference was observed after 1000 mg L− 1 treated plants.
Iron release from magnetite NPs might trigger Fenton reactions which forms hydroxyl radicals with H2O2. Through a Fenton’s reaction, H2O2 reaction with Fe2+ produces hydroxyl radicals while with Fe3+ generates the superoxide anion (Jalali et al. 2017). Accordingly, to the present study’s results, the high H2O2 content for the concentrations above 1000 mg L− 1 might resulted in higher superoxide anion accumulation.