Novel synthesis of an iron oxalate capped iron oxide nanomaterial: a unique soil conditioner and slow release eco-friendly source of iron sustenance in plants

Abstract

Iron (Fe) is a vital plant-derived micronutrient in the human diet. Fe availability in soil largely depends on the pH and leaching behaviour of the soil. Although common salts (FeSO₄) and chelates (EDTA) of Fe ensure high availability of the nutrient, they often interfere with P availability in the soil. Considering such disadvantages of the well-known Fe sources, we attempted to evolve efficient Fe₃O₄ nanomaterials that are independent of soil reaction (i.e. pH) and do not prevent P solubility in soil. The present investigation resulted in a novel, green and an easy pathway of large-scale synthesis of orthorhombic Fe–oxalate capped-Fe-oxide (Fe₃O₄) (OCIO) nanomaterial with a prolific agricultural applicability. This nanomaterial did not affect the growth of beneficial soil bacteria and had no phytotoxic effects on seed germination. The Fe release profile from the OCIO was uniform at different pH (4 to 9) conditions due to its exceptional H⁺ ion scavenging quality. Significantly higher P availability was recorded in aqueous and soil media treated with OCIO as compared to FeSO₄ and Fe–EDTA. Additionally, application of OCIO@10–20 mg kg⁻¹ considerably increased organic C, N, P, and enzyme activity in soil. Furthermore, the OCIO dramatically recovered Fe deficiency, maintained steady P availability, and stabilized pH in poorly fertile soil which promoted healthy growth and productivity of tomato.

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Introduction

A large portion of the world's population suffers from iron deficiency and plants are the prime contributor of iron in the human diet.¹ Fe deficiency is one of the most prominent nutritional disorders in many economically important crops.^2,3 The most easily detectable symptom of Fe deficiency in plants is the yellowing of leaves, technically known as leaf chlorosis. Although iron is the fourth most abundant element on earth, its bioavailability is noticeably low in the soil. Various factors like high soil pH, ‘iron-insufficient’ plant species, predominance of bicarbonates, and abiotic stress reduce the bioavailability of Fe in the soil. Primarily, Fe is held on the organic inorganic interfaces in soils.⁴ The solubility of this element in soils is determined by Fe(OH)₃, Fe₃(OH)₈ (ferric hydroxide) or by FeCO₃ (siderite) depending on the prevailing oxidation state in the soil.⁵ The dissolution–precipitation dynamics of ferric oxides in aerated soils largely governs Fe solubility, which is highly pH dependent. For example, the Fe³⁺ precipitation in soil increases by 1000 folds for each unit increase in soil pH.⁵ Plants can absorb iron as ferrous iron (Fe²⁺). However, the Fe²⁺ iron is readily oxidized in soil and transforms into plant-unavailable ferric (Fe³⁺) form when soil pH is greater than 5.3.⁶ Under such situations, if soluble Fe salts (e.g., FeSO₄) are applied to correct Fe deficiency, they rapidly precipitate as amorphous Fe(OH)₃ and decrease Fe availability over time.⁴

Various iron complexes conjugated with ligands (EDTA, DTPA and EDDHA) are used as slow releasing iron fertilizer to improve iron availability in the soil. Although these complexes are greener and rather more efficient compared to FeSO₄ fertilization, the impending global demand for iron in soil has prompted intense research on the development of various types of sustainable and cost effective iron source.⁷ Incidentally, the rapidly emerging nanotechnology may render smart and effective Fe fertilizers, although the outcome of their environmental exposure raises several concerns.⁸ Some studies showed that Fe-NPs released Fe²⁺/Fe³⁺ ions and Fe–OH groups are formed on their surfaces in aqueous solutions.^9,10 In a more recent study, Zhou et al. demonstrated that although iron oxide nanomaterials enhanced organic C and N availability in soil it substantially affected P availability.⁸ On the other hand, Fe oxide nanomaterials acidified soils and reduced N availability significantly.⁸

Herein, we present an efficient, sustainable, cost effective, novel, and green procedure for large scale synthesis of selective orthorhombic iron(oxalate) capped Fe(0) [Fe(ox)–Fe(0)] nanomaterial without the use of high temperature calcination. The novelty of the synthesis and applicability of this nanomaterial has been claimed for intellectual protection under the law (application no. 201631010727). Interestingly, the transformation of Fe(0) to Fe₃O₄ in the synthesized [Fe(ox)–Fe(0)] nanomaterial was observed in water after stirring the reaction mixture at room temperature for 14 h. The oxidized orthorhombic iron oxalate capped Fe₃O₄ nanomaterial [hereafter OCIO] was further characterized by different physical methods such as FTIR, XRD, FESEM, and TEM. Owing to the biocompatibility and non-toxic nature, the synthesized material can be potentially used in many in vivo applications (magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, drug delivery, hyperthermia etc.).¹¹ In addition the material has demonstrated a unique dye degrading property.¹² Subsequently, the OCIO was applied in soil at various concentrations to evaluate their influences on soil quality. We also conducted a few lab scale mimic experiments to define the underlying mechanism of the nanomaterials induced changes in soil properties. As a final point, we assessed the ability of the synthesized OCIO to overcome Fe deficiency in soil in regard to plant growth.

Results and discussion

Characterization and large scale applicability of the synthesized nanomaterial

Powder XRD and FT-IR analysis. The FTIR spectrum of all the synthesized materials were recorded and plotted in Fig. 1a. The carboxylate coordination mode can be determined from the corresponding position and separation of ν(COO–) bands, Δ, in the 1300–1700 cm⁻¹ region. Generally, Δ > 200 cm⁻¹ signified a unidentate ligand and a bidentate ligand was represented by Δ < 110 cm⁻¹; while an intermediate Δ represented a bridging ligand. The FTIR spectra of Fe(C₂O₄)·2H₂O showed peaks at 1640 and 1362 cm⁻¹ for typical metal carboxylate, in which oxalic acid is acting as a bidentate ligand. The other two peaks at 1320 and 820 cm⁻¹ were due to the C–O and C–C stretching vibration of coordinated oxalic acid in Fe(C₂O₄)·2H₂O. The comparative FTIR spectra of synthesized [Fe(ox)–Fe(0)] and Fe(C₂O₄)·2H₂O clearly showed the occurrence of iron oxalate in synthesized [Fe(ox)–Fe(0)] as both spectra clearly matched with each other. On the other hand, FTIR spectrum of [Fe(ox)–Fe₃O₄] nanomaterial showed peaks at 1412 and 1655 cm⁻¹ due to the oxalate coordination to metal oxide and two other peaks at 415 and 563 cm⁻¹ were due to Fe–O symmetric bending vibration and Fe–O–Fe stretching. However, [Fe(ox)–Fe(0)] material was competent in the reduction of methylene blue to its corresponding leuco methylene blue¹³ and on completion of the reaction the black material transformed into brown because of the oxidation of corresponding Fe(0) into its oxide (Fig. 1b). The XRD spectrum of synthesized [Fe(ox)–Fe(0)] confirmed the presence of orthorhombic Fe(C₂O₄)·2H₂O, as the peaks in XRD were in good agreement with the reported XRD pattern of Fe(C₂O₄)·2H₂O (Fig. 1c).^12,14 However, we failed to detect the presence of Fe(0) in the synthesized material from the XRD spectrum as the peaks for Fe(ox) and Fe(0) overlapped each other. The XRD pattern of oxidized material showed the co-existence of both orthorhombic iron oxalate and magnetite Fe₃O₄ because the peak position and intensity were vividly indicating their (orthorhombic Fe(C₂O₄)·2H₂O and Fe₃O₄) occurrence (JCPDS card 85-1436) (Fig. 1d).¹⁵

Fig. 1 Comparative FT-IR spectrum of Fe(ox)–Fe(0) and Fe(ox)–Fe₃O₄ (OCIO) with iron oxalate complex (a); Fe(ox)–Fe(0) promoted reversible methylene blue to leuco methylene blue redox reaction (b). XRD spectrum of Fe(ox)–Fe(0) (c) and OCIO (d).

Morphology, surface and elemental analysis. The surface morphology of the synthesized nanomaterial was analyzed through HR-SEM (Fig. 2a). The porous morphology of the OCIO was confirmed by the HR-SEM image. In addition, the EDS analysis of the selected region of the OCIO nanomaterial confirmed the presence of carbon, oxygen, and iron in 17.5, 56.1, and 26.4% respectively (Fig. 2b). We were also interested to identify the Fe content in the OCIO and performed the UV-Vis titration method¹⁶ with 1,10-phenanthroline after digesting the sample with HCl. The result showed 28.6 wt% of iron content in the material. Moreover, the surface characteristic of the synthesized nanomaterial was analyzed through the nitrogen adsorption–desorption isotherm and the pore size distribution of the synthesized material (Table 1S in the ESI†). We recorded 55.2 m² g⁻¹ surface area of OCIO and observed porous nature of the nanomaterial from BET analysis (Fig. 1S in the ESI†), which was consistent with the morphology revealed from HR-SEM and HR-TEM images (Fig. 2c). Interestingly, the HR-TEM images (Fig. 2c and d) explained that the synthesized OCIO had ribbon like structure with a size range of 10–100 nm in length and 1–5 nm in breadth. Moreover, the SAED pattern confirmed the 111 and 200 planes of the Fe₃O₄ (Fig. 2d). Furthermore, the particle size distribution analysis confirmed that the majority of the particles in the OCIO materials were within the range of 2–5 nm (Fig. 2e).

Fig. 2 HR-SEM image (a); EDAX (b); HR-TEM image (c); HR-TEM image and SAED (d); and particle distribution analysis (e) of OCIO nanomaterial.

Oxidation states of iron: X-ray photoelectron spectral analysis. The valance of iron was important to interpret the changes in soil properties. Hence, X-ray photoelectron spectrometry (XPS) was carried out to analyze the chemical composition and status of Fe–C–O in the OCIO. The XPS (ESCALAB 220i) measurement was performed on the sample with Al α X-ray source; the energy calibration was made against the C 1s peak. As shown in Fig. 3a, the XPS scan spectra of Fe–C–O exhibits distinct C_1s and O_1s peaks at 283.77 eV and 531.9 eV respectively, which confirms the presence of oxalic acid in the synthesised nanomaterial. It has been previously reported that Fe_2p3/2 for Fe₃O₄ does not have a satellite peak, which was also confirmed (Fig. 3b). The peak positions of Fe_2p3/2 and Fe_2p1/2 are 710.86 eV and 724.74 eV respectively clearly match for Fe₃O₄. Another peak was observed at 56.1 eV for Fe_3p. The Fe_2p3/2 peak for Fe₃O₄ was deconvoluted into Fe²⁺ and Fe³⁺ peaks. It is known that stoichiometric Fe₃O₄ can be expressed to FeOFe₂O₃, the ratio of Fe²⁺ : Fe³⁺ should be 1 : 2. In our case, the results of the deconvoluted peaks give Fe²⁺ : Fe³⁺ = 0.31 : 0.69.^17,18 This value satisfies the stoichiometric values with a negligible analytical error (SD = 0.1).

Fig. 3 Full scan XPS plot for OCIO (a) and XPS spectra of deconvoluted Fe₃ p_1/2 and Fe₃ p_3/2 (b).

Large scale synthesis and economical possibility. Here, we report the synthesis of iron(oxalate) capped Fe(0) [Fe(ox)–Fe(0)] nanomaterial from the aqueous phase room temperature reduction of FeSO₄·6H₂O with sodium borohydride (NaBH₄) in the presence of oxalic acid. After the completion of reaction, [Fe(ox)–Fe(0)] the nanomaterial was kept under stirring at 50 °C for 14 h to get the brown oxidized [Fe(ox)–Fe₃O₄, OCIO] nanomaterial. However, the final processing costs of the catalyst must fall within a reasonable budget to develop the nanomaterial for large scale industrial application. To our delight, OCIO was successfully synthesized up to 500 g scale in a single batch without compromising any reduction in its conversion as compared to the small scale reaction. Generally, market prices of iron chelates (Fe–EDTA or Fe–EDDHA) are considerably high (INR 200–300 or 3.07–4.61 $) which negates their use by farmers in developing nations. Whereas the synthetic cost of 100 g of OCIO nanomaterial is found to be INR 111.0 (1.6 $, 1.5 euro), suggesting a better cost effective synthetic route of material as compared to iron-chelates.

Effect on soil bacterial diversity

The data on effects of OCIO on total bacterial growth along with responses of nitrogen fixing bacteria and phosphate solubilizing bacteria to OCIO exposure are presented in Table 2S,† Fig. 4a and b respectively. Significantly high bacteria count was recorded in OCIO treated soil as compared to the control (Table 2S†) (P = 0.002). This may be due to a steady Fe sustenance contributed by the OCIO material. In addition, antagonistic effect on two well characterized beneficial soil microorganisms (Rhizobium sp. and Serratia marcescens) was tested. The role of Rhizobium in N fixation and S. marcescens in P solubilization has been well documented by other workers.^19,20 Moreover, the authenticity of the germplasm of these species have been thoroughly verified in our previous study.²¹ Therefore, these two species were an easy and dependable choice as models for this experiment. This property is important to evaluate the quality of the synthesized material from an ecological viewpoint.²² Generally, the zone of inhibition in growth medium is the indicator of fatal impact on microbial colonies for any material (Fig. 4a and b). Fascinatingly, no zone of inhibition was noticed even after 72 hours of incubation in the medium. This result ensured the eco-friendly nature of the synthesized OCIO. In addition, the plate count of colony forming units of both NFBs and PSBs was highly encouraging in OCIO treated soil. As such, soluble iron (Fe II) has a significant role in promoting growth of N-fixing bacteria like Bacillus and Klebsiella.²³ Our results are in good agreement with previous findings.¹²

Fig. 4 Anti-bacterial assay of the synthesized compound on N-fixing (Rhizobium sp.) (a) and P-solubilizing (Serratia marcescens) (b) soil bacteria; the germination index (GI), relative seed germination (RSG), relative root growth (RRG) of V. radiata (c) and V. mungo (d) seeds treated with OCIO, FeSO₄, Fe–EDTA and Fe–oxalate.

Phytotoxicity: seed germination assay

Fig. 4c and d represent data on the effect of photoactivity sources of Fe on seed germination of two pulse crops. Seed germination and root elongation measurement are widely used in phytotoxicity assay because of simplicity, low cost, sensitivity, and authenticity in regard to reactive chemicals.^24,25 Significantly high GI was recorded in OCIO treated seeds of Vigna radiata and V. mungo as compared to the other sources of Fe (FeSO₄, Fe–oxalate, and Fe–EDTA) (LSD = 1.33; 1.26) (Fig. 4c and d). Concurrently, RSG and RRG were substantially higher in OCIO treated seeds than Fe–EDTA and FeSO₄ (LSD: RSG = 0.82; RRG = 0.93) (Fig. 4d). Contrary to our results, Temsah and Joner reported high phytotoxicity of zero valent iron (Fe⁰) nanoparticles.²⁶ However, such results were found only at an abnormally high dose (1000 to 5000 mg kg⁻¹) of Fe⁰ nanoparticles; in fact, 100% seed germination was observed with low dose (100–200 mg kg⁻¹) in their research. Predominance of Fe⁰ may create anoxic condition through their reductive reactions, which is unhealthy for plants and soil bacteria.²⁶ Hence, such toxic impacts could be avoided using the OCIO in our study. Generally, the inhibitory effects of Fe on root growth and germination of plant seeds is highly pronounced at low pH (2–3) condition.²⁷ In all probability, the unique buffering property of the OCIO (explained in the following section) helped to maintain a neutral pH of the aqueous medium that could minimize the probability of excessive Fe solubility and phytotoxicity.

Influence of the OCIO on pH and Fe release: a unique buffering capacity

We were curious to validate the relationship of pH and Fe release from OCIO through a simple lab-scale experiment with aqueous solutions of different pH (Table 3S† and Fig. 5a–c). The pH change of the solution after treating with all three materials at different pH was monitored. In case of OCIO, strongly acidic solutions (pH 4 and 5) rapidly shifted towards near neutral value (pH – 4 to 6.54; pH – 5 to 6.6), while the pH of both the neutral and alkaline solutions increased marginally (pH – 7 to 7.17, pH – 8 to 8.13; pH – 9 to 8.6) over time (Table 3S† and Fig. 5a). On the other hand, rapid acidification of all the solutions was noted with Fe(EDTA) and Fe(C₂O₄) treatments. In both the cases, the higher amount of H⁺ in solution was recorded because of either ionization of the ligand or ionization of water.⁶ However, contribution of ligand ionization towards enhancing H⁺ concentration is negligible because both EDTA and oxalate should have protonated into their corresponding weakly dissociating carboxylic acid during the release of iron from their corresponding complexes (Fig. 5c). Therefore, the possible Fe^II ion promoted ionization of water enhanced the concentration of H⁺ and thereby decreased the pH of the solution. On the other hand, pH of all the solutions were found to increase over time (72 hours) when OCIO was added to the solutions, which may be ascribed to the abstraction of H⁺ by both polymeric iron oxalate template and Fe₃O₄ nanomaterial present in the material.

Fig. 5 Effect on pH shift (a) and Fe release (b) from OCIO, Fe–EDTA and Fe–oxalate; the proposed mechanism of H⁺ ion scavenging property of OCIO (c); differences in phosphorous solubility profile from KH₂PO₄ and its interaction with Fe release from OCIO, FeSO₄, Fe–EDTA, and Fe–oxalate (d).

The iron release profile for all the three materials was checked after treating the material at different pH (Table 3S† and Fig. 5b). In acidic condition (pH = 4), all the three material showed almost similar Fe release profile. In acidic pH, the H⁺ ion should bind with the corresponding ligand in all the three materials and protonate the carboxylate into the corresponding carboxylic acid and subsequently release the Fe in a similar fashion. However, in neutral condition (pH = 7), the amount of Fe release from the material was in the order of OCIO > EDTA > oxalate, which indicated different behavior of the materials in absence of H⁺ ion. While, in alkaline condition (pH = 9), the amount of Fe released from OCIO nanomaterial was significantly higher than the other two complexes, which further suggested the indispensability of H⁺ ion for release of Fe from EDTA and oxalate complexes. However, the nature of Fe release from OCIO was independent of solution pH and suggested that nano dimension of iron, present in both polymeric iron oxalate and Fe₃O₄, was loosely bound and thus enhanced the solubility of iron in all the tested conditions (Fig. 5b).

P and Fe release and their interaction with pH in aqueous medium

We conducted a small experiment to investigate the underlying mechanisms of high P availability in presence of OCIO (Fig. 5d). Phosphate release from KH₂PO₄ in aqueous medium was significantly higher in presence of OCIO as compared to FeSO₄, Fe–EDTA, and Fe–oxalate after 24 hours. Phosphate release gradually increased over time with OCIO, while it substantially reduced in presence of FeSO₄, Fe–EDTA, and Fe–oxalate after 21 days. Moreover, pH of the medium did not alter in OCIO containing solution, whereas, substantial reduction in pH was observed in presence of FeSO₄, Fe–EDTA, and Fe–oxalate (Fig. 5d). Furthermore, the Fe availability dramatically increased from 69.4 mg l⁻¹ to 268.5 mg l⁻¹ after 21 days in the OCIO containing solution. Although the Fe release in OCIO solution was lower than FeSO₄ solution, it was substantially higher than Fe–EDTA and Fe–oxalate solutions (Fig. 5d). Hence, the unique pH balancing ability coupled with slow Fe release pattern probably resisted the formation of insoluble Fe₃(PO₄)₂ in OCIO containing solution, which is an obvious phenomenon in an abundance of FeSO₄ or Fe–EDTA.

Impact of the OCIO on physico-chemical properties of soil: changes in pH, bulk density (BD), and water holding capacity (WHC), total organic C (TOC), available N, available P, DTPA extractable Fe, and urease and phosphatase activity

A typical alluvial soil was incubated with different levels of the synthesized material (OCIO) for 90 days. The inherent physico-chemical properties of the soil are presented in Table 4S.† Incorporation of OCIO in soil@10 mg kg⁻¹ significantly reduced bulk density (BD) of the soil from 1.38 ± 0.01 g cc⁻¹ to 1.29 ± 0.01 g cc⁻¹ after 90 days (Table 1) (P_0.05 = 0.000, LSD_day = 0.004, LSD_treatment = 0.01). Concurrently, increment in water holding capacity of soil was significant due to OCIO application in different concentration (Table 1). Such increment was highest in 10 mg kg⁻¹ application of OCIO followed by 20 mg kg⁻¹ and 50 mg kg⁻¹ doses (P_0.05 = 0.000; LSD_treatment = 0.22). The pH of the treated soil increased with time due to application of OCIO, while the soil pH substantially reduced due to application of FeSO₄ application (Table 1). The soil pH increased by 1.03–1.04 folds to 5.67–5.70 as compared to the initial value (5.5 ± 0.1) under different concentrations of OCIO over 90 days (Table 1). In general, pH change towards “point zero charge” (neutrality), induce agglomeration of nanoparticles due to reduction in electrostatic repulsive forces between particles.²⁸ As such, increase in agglomeration leads to enhanced water retention in soil through enlargement of soil pores.²⁹ Here, the reduction in BD indicates increment in voids in the soil (Table 1). Therefore, the increment in pH probably induced dispersion of the oxalate capped OCIO in soil, which in turn, increased water retention capacity of the soil through enhancement of pore spaces. Interestingly, we detected strong correlation between pH, BD, and WHC of the soil treated with different doses of OCIO during 90 day [r: pH–BD = 0.997 (P_0.05 = 0.000); pH–WHC = 0.998 (P_0.05 = 0.000); BD–WHC = 0.999 (P_0.05 = 0.000)].

Table 1 Impact of the OCIO on changes in pH, bulk density (BD), water holding capacity (WHC), and total organic C (TOC) of soil^a

Attributes	Days	Treatments
		T1	T2	T3	T4	T5	T6	T7	T8	T9	T10	T11
a T1 = control, T2 = OCIO10 mg kg^−1, T3 = OCIO20 mg kg^−1, T4 = OCIO50 mg kg^−1, T5 = Fe–EDTA10 mg kg^−1, T6 = Fe–EDTA20 mg kg^−1, T7 = Fe–EDTA50 mg kg^−1, T8 = Fe–oxalate10 mg kg^−1, T9 = Fe–oxalate20 mg kg^−1, T10 = Fe–oxalate50 mg kg^−1, T11 = FeSO4.
pH	0 d	5.51 ± 0.02	5.52 ± 0.01	5.51 ± 0.01	5.5 ± 0.01	5.45 ± 0.01	5.48 ± 0.02	5.43 ± 0.02	5.43 ± 0.01	5.43 ± 0.01	5.46 ± 0.01	4.5 ± 0.09
	45 d	5.48 ± 0.02	5.61 ± 0.01	5.64 ± 0.01	5.62 ± 0.05	5.43 ± 0.01	5.45 ± 0.02	5.44 ± 0.04	5.41 ± 0.01	5.44 ± 0.03	5.45 ± 0.01	4.33 ± 0.01
	90 d	5.52 ± 0.02	5.67 ± 0.01	5.7 ± 0.03	5.69 ± 0.05	5.45 ± 0.01	5.55 ± 0.01	5.51 ± 0.03	5.44 ± 0.02	5.49 ± 0.02	5.5 ± 0.02	4.3 ± 0.02
BD (g cc^−1)	0 d	1.42 ± 0.01	1.38 ± 0.01	1.41 ± 0.01	1.40 ± 0.01	1.38 ± 0.01	1.41 ± 0.01	1.40 ± 0.01	1.38 ± 0.01	1.41 ± 0.01	1.41 ± 0.01	1.41 ± 0.01
	45 d	1.39 ± 0.01	1.35 ± 0.01	1.38 ± 0.01	1.35 ± 0.01	1.36 ± 0.01	1.40 ± 0.01	1.38 ± 0.01	1.39 ± 0.01	1.40 ± 0.01	1.40 ± 0.01	1.40 ± 0.01
	90 d	1.42 ± 0.01	1.29 ± 0.01	1.34 ± 0.01	1.33 ± 0.01	1.41 ± 0.01	1.44 ± 0.01	1.45 ± 0.01	1.42 ± 0.01	1.44 ± 0.01	1.45 ± 0.01	1.43 ± 0.01
WHC (%)	0 d	68.02 ± 0.41	74.45 ± 0.57	73.05 ± 0.67	72.05 ± 0.48	62.39 ± 0.62	62.56 ± 0.4	61.94 ± 0.9	60.34 ± 0.65	60.57 ± 0.8	62.04 ± 0.14	63.03 ± 0.47
	45 d	68.71 ± 0.43	75.48 ± 0.36	75.31 ± 0.51	74.93 ± 0.4	58.18 ± 0.34	59.72 ± 0.26	58.23 ± 0.48	57.07 ± 0.46	58.49 ± 0.26	58.09 ± 0.15	54.92 ± 0.3
	90 d	71.93 ± 0.5	80.18 ± 0.15	78.11 ± 0.43	77.4 ± 0.21	56.27 ± 0.53	57.43 ± 0.37	56.17 ± 0.63	54.11 ± 0.7	54.86 ± 0.29	55.77 ± 0.25	58.94 ± 0.61
TOC (%)	0 d	0.48 ± 0.01	0.5 ± 0.02	0.52 ± 0.02	0.55 ± 0.01	0.43 ± 0.02	0.44 ± 0.01	0.45 ± 0.01	0.44 ± 0.01	0.45 ± 0.02	0.45 ± 0.01	0.48 ± 0.03
	45 d	0.64 ± 0.02	0.89 ± 0.01	0.9 ± 0.01	0.84 ± 0.02	0.64 ± 0.01	0.69 ± 0.02	0.62 ± 0.01	0.61 ± 0.01	0.6 ± 0.02	0.58 ± 0.02	0.73 ± 0.01
	90 d	0.73 ± 0.02	1.14 ± 0.02	1.12 ± 0.02	1.08 ± 0.02	0.78 ± 0.04	0.76 ± 0.01	0.74 ± 0.05	0.71 ± 0.02	0.64 ± 0.02	0.64 ± 0.02	0.60 ± 0.01

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	pH	BD	WHC	TOC
P value	P0.05 = 0.000	P0.05 = 0.000	P0.05 = 0.000	P0.05 = 0.000
LSD	LSD(d) = 0.022	LSD(d) = 0.004	LSD(d) = 0.12	LSD(d) = 0.02
	LSD(t) = 0.04	LSD(t) = 0.01	LSD(t) = 0.22	LSD(t) = 0.04

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The inherent TOC in the soil was low (Table 4S†). Overall, the application of OCIO at different concentrations significantly enhanced TOC level in soil. After 90 days the TOC gain in Fe–oxalate treated soil ranged from 1.42 to 1.61 folds as compared to the initial value. Similarly, TOC gain was also marginal due to Fe–EDTA application (1.64–1.81 folds). Whereas TOC increased by 1.96 to 2.28 folds in OCIO treated soils (Table 1). This indicated a synergistic impact of the OCIO on soil organic C, which may be due to the unique oxalate–Fe-oxide combination in our material. At the end of the study period, the TOC level in soil under various treatments was in the order: OCIO_{10 mg kg⁻¹} > OCIO_{20 mg kg⁻¹} > OCIO_{50 mg kg⁻¹} > Fe–EDTA_{10 mg kg⁻¹} > Fe–EDTA_{20 mg kg⁻¹} > Fe–EDTA_{50 mg kg⁻¹} > Fe–oxalate_{10 mg kg⁻¹} > Fe–oxalate_{20 mg kg⁻¹} = Fe–oxalate_{50 mg kg⁻¹} > FeSO_{4 50 mg kg⁻¹} (P_0.05 = 0.000, LSD_treatment = 0.04, LSD_day = 0.02). Generally, iron oxide nanoparticles being highly reactive in soil readily release Fe²⁺ or Fe³⁺ ions.^8,30 Interestingly, such release of Fe ions could enhance C levels in soil through formation of soluble complexes with the soil organic matter molecules.³¹

The N availability remarkably increased under OCIO application during the study period (Table 2). Significantly, high N availability in soil was recorded for OCIO_{10 mg kg⁻¹} followed by OCIO_{20 mg kg⁻¹} and OCIO_{50 mg kg⁻¹} (P_0.05 = 0.000; LSD_day = 3.24; LSD_treatment = 6.19). However, such improvement in N availability was not observed with Fe–EDTA, Fe–oxalate, and FeSO₄ application in various concentrations (Table 2). Concurrently, we recorded significantly higher urease activity in OCIO_{10 mg kg⁻¹} followed by OCIO_{20 mg kg⁻¹} and OCIO_{50 mg kg⁻¹} as compared to the Fe–EDTA, Fe–oxalate and FeSO₄ treatments (P_0.05 = 0.000; LSD_treatment = 0.07). Interestingly, there was a strong positive correlation (r = 0.97; P = 0.000) between N availability and urease activity in soil treated with different doses of OCIO. This indicated that the increment in N availability was mainly due to enhancement of urease activity in soil. Generally, iron is an important co-factor for several soil enzymes.³² Although iron-based nanoparticles induce toxicity through generation of reactive oxygen species (ROS), chemically stable Fe₃O₄ have no ecotoxicity.³² On the other hand, predominance of Fe²⁺ in soil solution generally leads to acceleration in NO₃⁻ reduction through activation of N reducing bacteria (Bacillus sp.) which may induce denitrification loss of N in certain conditions.^23,33 Such possibilities are highly obvious when FeSO₄ is used as fertilizer. Interestingly, the oxalate capping in our material should reduce the pace of NO₃⁻ reduction in soil because the Fe release from OCIO was significantly slower than FeSO₄ (Fig. 5d) which warrants in-depth research in future for improving bio-compatibility of OCIO. Hence, the highly oxidized crystals in our material render greater stability in the soil environment, which in turn, might have induced urease activity.

Table 2 Impact of the OCIO on changes in available N, available P, DTPA extractable Fe, urease, and phosphatase activity in soil^a

Attributes	Days	Treatments
		T1	T2	T3	T4	T5	T6	T7	T8	T9	T10	T11
a T1 = control, T2 = OCIO10 mg kg^−1, T3 = OCIO20 mg kg^−1, T4 = OCIO50 mg kg^−1, T5 = Fe–EDTA10 mg kg^−1, T6 = Fe–EDTA20 mg kg^−1, T7 = Fe–EDTA50 mg kg^−1, T8 = Fe–oxalate10 mg kg^−1, T9 = Fe–oxalate20 mg kg^−1, T10 = Fe–oxalate50 mg kg^−1, T11 = FeSO4.
Avl N (mg kg^−1)	0 d	282.7 ± 3.3	287.6 ± 7.03	284.4 ± 5.6	282.53 ± 4.3	280 ± 5.4	284.66 ± 8	289.33 ± 6.1	275.67 ± 8.6	280 ± 4	280.67 ± 8	283.6 ± 1.6
	45 d	291.07 ± 4.7	381.27 ± 2.7	372.87 ± 6.7	374.73 ± 9.1	279.53 ± 9.6	284.2 ± 7.3	284.67 ± 3.4	275.35 ± 4.9	278.15 ± 3.2	279.05 ± 8.4	297.27 ± 2.4
	90 d	299.47 ± 3.2	387.33 ± 3.2	384.53 ± 6.5	381.73 ± 9	290.26 ± 9	281.87 ± 1.6	287.47 ± 2.9	284.7 ± 8.1	275.3 ± 8	281.9 ± 4	280 ± 8
Avl P (mg kg^−1)	0 d	46.05 ± 0.63	49.64 ± 0.87	46.98 ± 0.79	46.23 ± 0.98	43 ± 0.85	42.16 ± 1.09	42.69 ± 0.55	40.77 ± 0.63	38.37 ± 0.8	40.76 ± 0.96	44.15 ± 0.54
	45 d	51.91 ± 0.47	74.92 ± 0.41	72.97 ± 0.87	78.76 ± 0.94	42 ± 0.71	41.73 ± 0.77	39.79 ± 0.84	39.57 ± 0.71	37.83 ± 0.35	38.76 ± 0.59	42.54 ± 0.87
	90 d	58.73 ± 0.86	105.54 ± 1.3	91.77 ± 1.5	88.28 ± 0.81	45.18 ± 0.89	45.24 ± 0.85	45.75 ± 0.43	42.28 ± 0.82	41.94 ± 0.77	42.09 ± 0.64	40.12 ± 0.93
Urease (μg g^−1 h^−1)	0 d	16.08 ± 0.05	18.81 ± 0.01	17.98 ± 0.01	17.65 ± 0.03	16.89 ± 0.04	16.45 ± 0.23	16.61 ± 0.04	16.51 ± 0.03	16.08 ± 0.7	16.23 ± 0.05	16.34 ± 0.02
	45 d	16.29 ± 0.07	29.78 ± 0.07	28.15 ± 0.06	23.39 ± 0.05	16.97 ± 0.07	18.03 ± 0.11	16.95 ± 0.05	16.8 ± 0.06	17.13 ± 0.05	16.37 ± 0.08	16.38 ± 0.06
	90 d	18.89 ± 0.02	32.36 ± 0.09	31.38 ± 0.03	30.99 ± 0.07	20.71 ± 0.03	20.11 ± 0.04	19.54 ± 0.11	19.11 ± 0.05	18.6 ± 0.18	17.57 ± 0.14	19.41 ± 0.02
Phosphatase (μg g^−1 h^−1)	0 d	7.9 ± 0.15	12.82 ± 0.5	11.89 ± 0.12	10.65 ± 0.09	8.1 ± 0.04	7.96 ± 0.77	7.95 ± 0.66	7.95 ± 0.14	7.75 ± 0.34	7.3 ± 0.32	8.09 ± 0.06
	45 d	7.2 ± 0.39	19.8 ± 0.19	19.56 ± 0.39	17.54 ± 0.27	8.18 ± 0.49	8.08 ± 0.34	8.11 ± 0.35	8.08 ± 0.09	7.83 ± 0.05	7.44 ± 0.06	7.8 ± 0.11
	90 d	8.86 ± 0.11	27.5 ± 0.16	24.14 ± 0.41	22.85 ± 0.11	8.56 ± 0.29	8.24 ± 0.05	8.32 ± 0.04	8.36 ± 0.11	7.94 ± 0.04	7.62 ± 0.02	7.4 ± 0.2
Fe (mg kg^−1)	0 d	124 ± 2.1	136.25 ± 1.4	134 ± 2.6	136.1 ± 2.7	119.4 ± 1.8	121.5 ± 1.1	122.8 ± 1.4	123.5 ± 0.9	120.8 ± 1.4	124.68 ± 0.9	168.95 ± 1.8
	45 d	126.05 ± 2.04	151.5 ± 2.3	154.9 ± 1.4	138.2 ± 1.9	92.05 ± 1.1	93.4 ± 1.3	95.5 ± 1.01	97.2 ± 1.1	92.59 ± 1	100.14 ± 0.9	196.9 ± 1.6
	90 d	128.4 ± 1.21	173.88 ± 1.07	179.1 ± 1.01	183.1 ± 5.1	69.4 ± 1.2	71.5 ± 1.1	73.9 ± 1.1	74.27 ± 0.9	77.05 ± 3.5	78.2 ± 1.9	174.9 ± 1.3

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	Avl N	Avl P	Urease	Phosphatase	Fe
P value	P0.05 = 0.000	P0.05 = 0.000	P0.05 = 0.000	P0.05 = 0.000	P0.05 = 0.000
LSD	LSD(d) = 3.24	LSD(d) = 0.12	LSD(d) = 0.03	LSD(d) = 0.07	LSD(d) = 0.43
	LSD(t) = 6.19	LSD(t) = 0.20	LSD(t) = 0.07	LSD(t) = 0.14	LSD(t) = 0.83

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The test soil in our experiment was acidic in nature (Table 4S†). In such condition, Fe availability is generally enhanced and can considerably precipitate P and renders high P deficiency in soil.³⁴ This can be evidenced from the effects of Fe–EDTA, Fe–oxalate, and FeSO₄ on soil P (Table 2). In contrast, significant increment in P availability was recorded with OCIO_{10 mg kg⁻¹} application followed by OCIO_{20 mg kg⁻¹} and OCIO_{50 mg kg⁻¹} (P_0.05 = 0.000; LSD_treatment = 0.20). Moreover, Fe availability in soil significantly increased after 90 days due to incorporation of OCIO@50, 20 and 10 mg kg⁻¹ respectively. On the other hand, phosphatase activity was highest with 10 mg kg⁻¹ application of OCIO followed by 20 and 50 mg kg⁻¹ application as compared to other treatments (P_0.05 = 0.000; LSD_treatment = 0.14).

Thus, the result suggested that there was a dose dependent relationship between P availability, phosphatase activity, and Fe availability in OCIO treated soil. Briefly, higher dose induced Fe richness in soil but reduced P availability and phosphatase activity and vice versa. Interestingly, the correlation statistics also suggested strong positive correlation between available P and phosphatase activity in OCIO treated soil (r = 0.99; P value = 0.000), while Fe availability were negatively correlated with both available P and phosphatase (Fe–P: r = −0.99; P = 0.000 and Fe–phosphatase: r = −0.99; P = 0.000). Previously, Zhou et al. showed reduction in P availability in soil due to incorporation of iron oxide (FeO, Fe₂O₃, and Fe₃O₄) which suppressed the acid phosphatase activity.⁸ However, they did not mention the dose dependency of such a phenomenon. In contrast, He et al. proposed that Fe₃O₄ nanoparticles provide beneficial nutrients to soil bacteria which, in turn, greatly stimulate enzyme production in soil.³² Moreover, Fe³⁺ and Fe²⁺ were in 2 : 1 ratio in our material. Therefore, Fe³⁺ predominance in soil was likely to be higher than Fe²⁺ that is known to generate oxidative stress.³⁵ Thus, the chance of inhibition in bacterial proliferation was less and application of OCIO at a low dose (10 mg kg⁻¹) was beneficial with respect to P and Fe nutrition in soil.

In nature, Fenton-like system can be established due to presence of oxalic acid, Fe, and sunlight.³⁶ The use of oxalic acid in the synthesis process ensured the oxalate capping over Fe₂O₃ in our material. Therefore it can be speculated that sunlight exposure of OCIO in agricultural fields should create a photo-Fe–oxalate system which is highly efficient in degrading toxic organic pollutants like pentachlorophenol.^36,37 Interestingly, in this study the OCIO exhibited a slow but steady Fe release profile (Fig. 5) mainly due to the oxalate capping; which presumably should establish a homogenous photo-Fe–oxalate system where Fe exist in a soluble form.³⁶ As a result, the photoactivity of the material is not likely to interact with Fe bioavailability in soil. Such multifarious traits of OCIO can enhance the value of the material in practice.

Fe and P enrichment in nutrient poor soil: deficiency recovery potential

The changes in nutrient levels in the leached soil are presented in Fig. 6a–c. Significant rise in soil pH was observed under OCIO, whereas, slight reduction in soil pH was recorded under Fe–EDTA, Fe–oxalate, and FeSO₄ (P_0.05 = 0.000; LSD = 0.13). Moreover, remarkable gain in available P, phosphatase, and Fe availability occurred in OCIO treated soil as compared to the initial value (Fig. 6a). Interestingly, we recorded significantly higher uptake of P and Fe in tomato plants treated with OCIO as compared to others (Fig. 6c). Interestingly, the plants under OCIO treatment did not show leaf chlorosis as noted for FeSO₄ treated plants (Fig. 6b). Interveinal chlorosis is a definite symptom of Fe deficiency.³⁸ Moreover, we recorded a significantly high tomato yield in OCIO treated plants (Fig. 6c). This signifies that the synthesized material was not only efficient in recovering Fe and P fertility in soil but also greatly facilitated plant growth and productivity. On the whole, the results of soil and plant experiments indicated that the efficiency of OCIO was highest when applied@10 mg kg⁻¹ (i.e. 22 kg ha⁻¹). Therefore, 22 kg ha⁻¹ can be recommended as optimum dose for field application of OCIO. However, long term and in depth confirmatory field experiments are needed to be conducted in future in different soil types before the final recommendation.

Fig. 6 Changes in pH, avl P, phosphatase activity, Fe content in nutrient poor soil under various treatments (a); Fe-deficiency symptoms (chlorosis) in tomato leaves (b); effects of OCIO, FeSO₄, Fe–EDTA, and Fe–oxalate on chlorophyll content, yield and uptake of P and Fe in tomato grown in nutrient poor soil (c).

Experimental

Synthesis of the Fe–(ox)–Fe₃O₄ (OCIO)

In a typical procedure, 34.7 g of FeSO₄·6H₂O and 9.45 g of oxalic acid was added in 400 ml of distilled water to prepare a solution in a 1 l beaker and stirred for 20 minutes. Thereafter, a solution of 30 g of NaBH₄ was prepared in 100 ml distilled water and added drop wise in the former solution under vigorous stirring. While adding NaBH₄, the color of the solution slowly turns into yellow then green and finally black iron nanomaterials begin to appear in the solution. After completion of the reaction, the reaction mixture was kept under stirring at 50 °C for 14 h. Consequently, the black solution slowly turns yellow-brown and finally about 42 g of brown material was collected after centrifuging and oven drying at 80 °C for 8 h.

Experimental plan to assess the applicability of OCIO

The impact of the synthesized OCIO on soil health and plant growth was examined through three major experiments. At first, we assessed the impact of the selected concentrations of OCIO on beneficial soil bacteria and germination potential of plant seeds to assess their ecotoxicity. Secondly, we performed lab scale experiments in aqueous medium to understand the behavior of the OCIO with respect to varied chemical status (pH), the Fe release mechanism, and their role in phosphorus solubilization. Thirdly, we evaluated the impact of various levels of OCIO on the physico-chemical properties of soil in comparison with Fe–EDTA, Fe–oxalate, and FeSO₄. Moreover, the efficiency of OCIO to sustain plant growth in Fe deficient soil was evaluated. The minute details of these experiments are given in the following sections.

Experiment-I – effects on soil beneficial bacteria and seed germination

The effects of the OCIO on growth of beneficial soil bacteria N-fixing (NFB) and P-solubilizing (PSB) were assessed. The test was conducted by disc diffusion method against one NFB (Rhizobium sp.) and one PSB (Serratia marcescens).³⁹ In addition, colony growth of NFBs and PSBs in treated soil samples was analyzed respectively in Burk's and Pikovskaya's media.⁴⁰

10 seeds of V. radiata and V. mungo were placed on tissue papers in Petriplates. Concurrently, 2 mg of OCIO, FeSO₄, Fe–EDTA, Fe–oxalate were mixed with 10 ml deionizer water in sealed containers and sonicated for 10 minutes. Afterwards, the resultant mixtures were applied to the seeds of both species. Later, the inoculated seeds were maintained in dark at 25 °C. The number of germinated seeds and length of roots and shoots were measured after 48 hours of incubation. The germination index (GI), relative root growth (RRG), and relative seed germination (RSG) were calculated following Karak et al.,⁴¹ as below:

Experiment II – Fe release profile of OCIO in aqueous medium of various pH and the effect on P solubility

10 ml solutions of various ranges of pH (pH 4, 5, 6, 7, 8, and 9) were prepared with the help of an acid (HCL) and a base medium (NH₄OH) respectively in Erlenmeyer flasks and 0.1 g OCIO, Fe–EDTA, Fe–oxalate, and FeSO₄ added separately in the flasks. Then, the flasks were kept in a mechanical shaker set at 120 rpm for 72 hours and the change in pH and Fe release of different solutions was recorded at 12, 24, 48, and 72 h by following standard methods.^42,43

In a similar study, the mechanistic evidence of the influence of OCIO on time dependent bioavailability of P and Fe was estimated in an aqueous medium. An identical amount (10 g) of the synthesized compounds [OCIO, Fe–EDTA, Fe–oxalate, and FeSO₄] and one easily soluble salt of P (KH₂PO₄) were mixed with de-ionized water (100 ml) in Erlenmeyer flasks and placed in a mechanical shaker at 120 rpm for 21 days. The filtered portion of the suspension were analyzed periodically (24, 48 hours, 7, 14, and 21 days) for pH and available P following standard methods.⁴² The Fe release profile was estimated in ICP-OES by following APHA.⁴³

Experiment III – changes in soil quality and nutrient recovery potential of OCIO in Fe deficient soil

Representative and composite soil samples were collected from a typical alluvial soil (order: typic endoaquept) from a nearby area (Napaam, Tezpur, Assam). Subsequently, the soil samples were air dried, freed from plant parts, and sieved through a 2 mm sieve. Then, the prepared soil samples were separately poured in uniform quantity (2 kg) in burnt earthen pots and treated with various concentrations of the synthesized compounds, FeSO₄, Fe–EDTA, and Fe–oxalate with a control as shown below:

This study was conducted for 90 days with three identical replicates for each of the above treatments and the soil was sampled periodically at 0, 45, and 90 days for analysis.

The temporal changes in the physico-chemical properties (pH, water holding capacity (WHC), bulk density (BD), total organic carbon (TOC), available nitrogen (avl N), and available phosphorus (avl P)) of the soil were analyzed by following Page et al.⁴² In addition, the activity of vital soil enzymes (urease and phosphatase) was analyzed following standard methods.^44,45 Moreover, the DTPA extractable Fe was analyzed in ICP-OES following standard method.⁴⁶

A pot experiment was carried out to evaluate the competence of the synthesized compound to correct Fe deficiency in a Fe deficient alluvial soil. Such deficiency was created by leaching with deionizer water for 96 h. At this stage, the Fe content was below detection limit and the pH of soil was 6.3. Subsequently, 1 kg of this soil was poured separately into burnt earthen pots and the whole set was replicated thrice; treated with uniform quantity (50 mg kg⁻¹) of OCIO, Fe–EDTA, Fe–oxalate, and FeSO₄. Thereafter, tomato (Badshah F1 hybrid) seedlings (3–4 leaf stage) were planted to each pot and the plant response was assessed. The Fe deficiency was monitored on the basis of leaf chlorosis symptoms. The crop was harvested after 60 days; changes in pH, avl P, Fe, and phosphatase activity in soil were analyzed following the methods stated earlier. Finally, the chlorophyll content, tomato yield (g per plant), and P and Fe uptake in plants was measured following established methods.^47,48

Statistical analysis

One-way and two-way analysis of variance (ANOVA) was performed for eco-toxicity and soil quality experiments respectively. The least significance test (LSD) was also carried out to detect the relative efficiency between different treatments. Moreover, Pearson's correlation analysis was performed for the soil studies conducted under Experiment-III in addition to one-way and two-way ANOVA.

Conclusion

A novel and easy large-scale synthesis route for iron oxalate capped iron-oxide (OCIO) nanomaterial was developed avoiding high temperature calcination. The material was synthesized with a specific goal to utilize it as a soil conditioner that renders optimum iron nutrition to plants in varied kind of soil reaction and fertility status. The major findings of this work can be summarized as:

(1) The OCIO material did not show any inhibitory effects on growth of beneficial soil bacteria (NFBs and PSBs). Moreover, profuse germination of OCIO treated black gram and green gram seeds not only confirmed the ecofriendly property of OCIO but also showed high promise of the material as a prolific plant growth promoter.

(2) OCIO possesses a unique H⁺ scavenging property which facilitates to rectify aberrant pH thereby assuring a steady iron release. This quality also indirectly helps to sustain phosphorous solubility in soil.

(3) 10 and 20 mg kg⁻¹ of OCIO significantly improved soil health (N, P, organic C status, and urease and phosphatase activity) as compared to Fe–EDTA and FeSO₄. The present results clearly indicated that a dose of 22 kg ha⁻¹ of OCIO should be optimum for field application. Fascinatingly, the material recovered iron and phosphorous availability in a nutrient-starved soil and sustained plant growth potential.

Authors' contribution

PD carried out the field experiments, done the soil and plant analyses, and written the MS. PD and KS prepared and characterized the nanomaterial and KS specifically made some graphics and partly written the MS. NH helped in the microbial study. SD assisted in the germination experiment and prepared the graphics from that experiment. PB did the statistical analyses, discussed the results and made the overall graphics. SAP, HK, and MIK performed the XPS analysis, discussed the results, and partly wrote the MS. SP conceptualized and supervised the work related to chemical synthesis of the nano-material, analyzed the data and discussed the chemical aspects of the nano-material, and wrote the manuscript. SSB conceived the whole study, designed experiments, supervised the study, analyzed data and wrote the manuscript.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra18840k

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