Zinc (Zn) deficiency is one of the most widespread micronutrient disorders among different crops (Westfall et al., 1971; Romheld and Marschner, 1991) affecting the quantity and the nutritional quality of the harvest (Cakmak et al., 1997). This fact is of special importance because Zn deficiency in humans has a high prevalence in the world (an estimated 31% in the global population). In total, 1.4% (0.8 million) of deaths worldwide were attributable to zinc deficiency: 1.4% in males and 1.5% in females (World Health Organization, 2002).

Zinc synthetic chelates such as ZnEDTA are the common source used to alleviate Zn deficiency of crops. Synthetic chelates application, regarded as the most effective source of plant micronutrients (Alloway, 2008) is limited to cash crops because of its high price. Synthetic chelating agents are so persistent in the environment that they can largely contribute to accelerating the movement of Zn and other metals (Álvarez et al., 1996; Li and Shuman, 1997; Rodella and Chiou, 2009) in soil profiles that may produce accumulations of trace elements in ground water and over the years, Zn deficiency (Mikkelson and Brandon, 1975). Zinc natural organic complexes such as Zn-Lignosulfonate (ZnLS) forms another group of Zn fertilizers that are more biodegradable and less expensive than synthetic chelates because they are by-products from the manufacturing of paper pulp through sulphite process. Lignin polymers became water-soluble by the introduction of sulfonic acid groups. The presence of a considerable number of anionic groups including sulfonic, carboxylic acid and phenolic hydroxyl groups (Askvik et al., 1999) which can form coordinated bonds with metals converts the LS into an appropriate micronutrient’s complexing agent. In addition, the utilization of LS prevents the environmental impact of the black liquor of the paper industry due to the LS removed from it (González et al., 1989). Composition and properties of LS depend on the kind of wood source, the procedure used to obtain the paper pulp and the process of treatment of the black liquor. Therefore, the composition of the final product presents a wide variability. In order to improve properties required in final applications and to reduce the impurity content, the LS are treated physical and chemically. It is important to understand the relationship between the modification reactions and the characterization of LS. The molecular weight, the presence and the amount of functional groups could modify the efficacy of the fertilizers based on LS as a complexing agent. In a previous work, Benedicto et al. (2011) studied Zn Complexing Capacity (ZnCC) of six ZnLS complexes employed in this work and their reactivity vs. pH. This work continues the physical-chemical characterization of six LS complexes, by studying their reactivity vs. different soil components.

In 2015, more than 25% of the Zn complexes sold in the Spanish market used the LS as the complexing agent (Sánchez Jiménez and Lucena, 2015). European regulation allows its application in fertirrigation, by foliar spray and recently, direct application to soil. Several authors tested the efficiency of ZnLS complexes in hydroponic culture conditions, in soil conditions or by foliar applications (Álvarez et al., 1996; Moraghan, 1996; Gangloff et al., 2002; Álvarez-Fernández et al., 2007; Gonzalez et al., 2008; Martín-Ortiz et al., 2009; Benedicto et al., 2011).

In most cases the LS are less effective (Mortvedt and Gilkes, 1993) than chelates because of its known weak bonds and low complex stability. Lindsay and Schwab (1982) proposed a mechanism for the action of iron chelates in soils in which the chelating agent works ideally as a shuttle iron transporter between soil and plant roots. This mechanism was later referred to as the “shuttle effect” (Lucena, 2003) but nothing has been yet demonstrated. An attempt to explain zinc chelates and zinc lignosulfonates (LS) reactions in soil, iron theoretical model (Lucena, 2006) was adapted for Zn in this work.

At the present time, ⁶⁷Zn is a tracer tool to evaluate Zn uptake and distribution behavior in plants (Benedicto et al., 2011; McBeath et al., 2013). The ⁶⁷Zn is a natural stable isotope (abundance: 4.1%), so it can be safely used with high flexibility in experimental designs and field studies. Moreover, zinc isotope amounts can be quantified by ICP-MS, and after mathematical deconvolution (Benedicto et al., 2011), Zn from the fertilizers (Zn_Fer) and Zn from natural sources such as the soil or the seeds (Zn_Nat) can be determinated in each plant organ or soil fraction. Schenkeveld et al. (2014) suggested that tracer studies with isotopically labeled iron chelates would provide more understanding of the actual effectiveness of the shuttle mechanism in soil-plant systems. In this way, the theoretical iron model would be renewed and adjusted.

The aims of this work were first, to compare modified and non-modified ZnLS complexes efficiency in respect to ZnEDTA in hydroponics and soil conditions, and secondly, to evaluate EDTA shuttle effect and ZnLS complexes direct effect in soil and their influence in navy bean zinc nutrition.

The hydroponic experiment showed that Zn amounts in plants treated with ZnLS complexes were higher than in Control plants, which indicates that modified and non-modified ZnLS complexes are able to provide Zn to navy bean plants applied in nutrient solution (Table 3). Other authors have obtained similar results for corn, wheat and navy bean (Martín-Ortiz et al., 2009; Benedicto et al., 2011).

With respect to reactivity of modified and non-modified ZnLS complexes vs. soil components (Figure 1), around 70% of soluble Zn remained in solution for peat and Ca montmorillonite and 55% for illite while only 20% remained for dolomite and small amounts for lime. Modified and non-modified ZnLS complexes showed the same behavior with respect to the reactivity with different soil materials, which means this property does not depend on the wood sources or LS modifications. However, Benedicto et al. (2011) determined the stability of ZnLS complexes used in this work, as a function of pH in the presence of Ca and all the products revealed to be almost 100% soluble at pH below 7.5. This study shows the interaction with different soil materials affects the Zn solubility and it decreases as the soil pH increases. Therefore, taking into account only these results in calcareous soil conditions, where is common the zinc deficiency, the zinc LS would tend to stay retained in soil and not soluble for the plant taking up (Álvarez et al., 2001). However, the availability of this retained fraction on plant nutrition has not been yet elucidated.

The ⁶⁷Zn tracer technique allowed monitoring Zn from the fertilizer in soil experiments and distinguishing from the native Zn contained in the soil. A combination of ⁶⁷Zn isotope and mathematical deconvolution was an important tool to evaluate the efficacy between different treatments to correct zinc deficiency.

There were significant differences among ZnEDTA and ZnLS complexes for Zn_Fer and Zn_Nat content in navy bean leaves at 21 DAT for the first soil experiment (Table 4). ZnEDTA showed the highest concentration for Zn_Fer, Zn_Nat and Zn_Total. Rico et al. (1996) obtained similar results for wheat (Zn_Total). Álvarez and Rico (2003) as well as Prasad and Sinha (1981) suggested the diffusion as the principal mechanism which contribute to plant nutrition when stable chelates as ZnEDTA are applied to calcareous soils. Singh et al. (1986) also observed the ZnEDTA efficiency for navy bean plants in calcareous soil and argued that chelating agents enhance the uptake of Zn by increasing the rate of diffusion in the immediate vicinity of plant roots.

The aim of LS chemical modification is to improve physical and chemical properties, especially zinc complexing capacity, and hence, increase LS efficiency. ZnLSEO and ZnLSEU presented the highest amounts of Zn_Total in leaves for the hydroponic assay (Table 3). We selected ZnLSEO for the first soil experiment because of its highest content in carboxylic groups would ensure higher zinc complexing capacity and thus, more efficient zinc nutrition for the plants. However, ZnLSEO didn’t show better results than non-modified ZnLS complexes (Table 4). Benedicto et al. (2011) concluded that a high zinc complexing capacity did not necessarily imply a high Zn supply to plants in hydroponic assays. Moreover, ZnLSEO pH was 6.8 (Table 1), the soil pH was 7.9 and the nutrient solution used for the irrigation had a pH between 8.0 and 8.2. Benedicto et al. (2011) observed that ZnLSEO solubility decreased almost 20% at pH 8.5 in calcareous conditions. Thus, almost 80% of ZnLSEO remained soluble in the calcareous soil and its efficiency was lower than non-modified ZnLS.

Taking into account those results, a second soil experiment was designed and different doses (1.5, 2.3, 4.6 μmol ⁶⁷Zn per pot) of non-modified ⁶⁷ZnLS (ZnLSE and ZnLSS) were compared respect to ⁶⁷ZnEDTA (2.3 μmol ⁶⁷Zn per pot).

In this last soil experiment, ZnEDTA showed, as the first experiment, the highest content of Zn_Total in leaves (Figure 2). Moreover, for soluble Zn_Fer and Zn_Nat in soil, ZnEDTA showed higher contents than ZnLS complexes (Figure 3) in pots with and without plants. This was an expected result for Zn_Fer caused by the high solubility of the chelates but not for Zn_Nat, as it was almost three times larger than control. These results are apparently contradictory with theoretical models. According to Lindsay (1979), once ZnEDTA releases the zinc to the plant, EDTA should primarily chelate calcium and not zinc. This was also confirmed by López-Rayo et al. (2015) that obtained the lowest ZnEDTA presence in oxidizing conditions (pe + pH: 18) when its behavior was modeled using the software VMINTEQ 3.0. The pH decrease at the rhizosphere may explain this apparent contradiction. EDTA tends to complex zinc instead of calcium ought to the rhizosphere pH can differ in two units less than the bulk soil (Neumann and Römheld, 2012). Furthermore, soluble Zn_Nat amounts in pots with plants were four times larger than pots without plants. It is clear that roots play an essential role in the solubility of Zn in soil. Respect LS interactions with other nutrients, no differences were observed for micronutrients (Supplementary Tables S4–S8) but total phosphorous concentration decreased in leaves. This possible antagonism between phosphorous and zinc is surely explained by the precipitation of Hopeite [Zn₃(PO₄)₂.4H₂O] in the soil (Lindsay, 1979) formed by binding of the soluble zinc LS and soil phosphates in that pH condition.

Figure 4 shows the proposed shuttle model for zinc chelates in calcareous soil. Zinc chelate (ZnY) applied to a calcareous soil, dissociates to attain equilibrium with Zn²⁺ and Y (1). The roots may take Zn²⁺ and Y, the chelating agent, dissolves native zinc from the soil, chelates it and carries it to the roots by diffusion (2), and the process begins again. This is the “Shuttle effect.” Also, ligand Y can chelate another cations present in soil, e.g., Ca²⁺, Fe³⁺, Fe²⁺, Cu²⁺, Mn²⁺ (3). Ligand Y can be retained by the soil surface too (4), or can react with the solid phases and extract more native zinc from the soil. Our results attempt to prove this mechanism for ZnEDTA, using ⁶⁷Zn for this task.

Thus, this theoretical model explains ZnEDTA mechanism in the soil experiments. Steps 2 and 5 are carried out in pots experiments with plants and ZnEDTA provide Zn_Nat to the plants as follow:

Regarding LS, although ZnLSE3 and ZnLSE2 demonstrated to be as efficient as ZnEDTA to provide Zn_Fer to the navy bean plants (Figure 2), the third dose is statistically the best to obtain the similar results to ZnEDTA fertilization (Table 6). Moreover, significant differences for Zn_Fer in leaves between treatments were observed (Table 6) and ZnLS from eucalyptus wood showed to be more efficient than from spruce. It is clear that Zn_Total contents for plants treated with ZnLS were lower than with ZnEDTA. The likely explanation is that shuttle effect and isotopic exchange are occurring to a higher extent with EDTA than with complexes. Lignosulfonate complexes are less stable and mobile than chelates in soil because of their weak bonds and high molecular weight. Therefore, ZnLS complexes do not provide extra Zn_Nat as synthetic chelate and the schematic representation for LS (Figure 4) only would show reactions 1, 3, and 4. While ZnEDTA was the most efficient treatment in providing Zn_Total to the navy plants, eucalyptus lignosulfonate at the maximum dose (ZnLSE3) was the most efficient treatments to supply Zn_Fer. Further, the average molecular weight of eucalyptus lignosulfonates (LSE) is four times less (Table 1) than spruce lignosulfonate (LSS), hence LSE mobility is faster than LSS. Based on our results, an equal or double dose of ZnLSE applied in soil would provide similar Zn_Fer content in leaves to ZnEDTA.

Other authors have indicated that LS are more reactive than chelates and remain retained in soil surfaces when they were applied as a zinc source, allowing zinc to be available but non-soluble, to plant requirements (Rico et al., 1996; Álvarez et al., 2001; Álvarez and Gonzalez, 2006). In this work, we observed similar soluble Zn_Fer contents for ZnLS as ZnEDTA and better Zn_Fer availability for ZnLS than for the synthetic chelate at the highest dose, independently of the lignosulfonate source.

According to Table 7, navy bean fertilized with the lowest dose of ZnLSE took up similar Zn_Fer percentage than plants fertilized with ZnEDTA. Moreover, plants fertilized with ZnLSE took up around 4% Zn_Fer more than plants fertilized with ZnLSS. It is noticeable that while ZnLSE tends to improve the Zn_Fer distribution mainly in the plant, ZnLSS tends to remain available in soil. Chelation by low-molecular-weight organic substances is a factor that strongly affects the concentration of micronutrient cations in the soil solution and their transport to the root surfaces by mass flow and diffusion. In nutrient solution experiments, the rate of uptake of metal cations from metal–organic complexes is lower than from free cations and decreases with the size of the organic ligand (Rengel, 2012). In this case, molecular weight of spruce lignosulfonate is four times larger than eucalyptus ones, so, its mobility in the rhizosphere by diffusion may not be favored. Benedicto et al. (2011) have obtained similar results for ⁶⁷Zn foliar applications with LS.

In relation to the results obtained in this work, ZnEDTA is the most efficient fertilizer in providing zinc to the navy bean plants (Phaseolus vulgaris L c. v. Negro Polo) not only Zn_Fer but also extra Zn_Nat, as consequence of a shuttle effect and an isotope exchange. Furthermore, a double dose of non-modified ZnLS complex coming from eucalyptus woods, applied in calcareous soil would provide the similar Zn_Fer content in leaves to ZnEDTA. Moreover, ZnLSE would supply similar soluble Zn_Fer content in soil to ZnEDTA and higher available Zn_Fer than ZnEDTA, which allow the plants to take zinc along the entire growth process. Finally, ZnLSE complex is an eco-friendly and low cost option to the synthetic chelates to provide zinc to navy bean plants that grown in calcareous soil.

AB carried out the lignosulfonate characterization and the hydroponic experiments with the subsequent analysis and statistical analysis. MC carried out the soil experiments, analyzed the plant and soil material, did the mathematical deconvolution and performed the statistical study. LH-A and JL conceived the study. MC wrote the manuscript together with the revision of JL. JL designed the manuscript and supervised all the experimental work presented. All the authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.