**Smart Fertilizers and Innovative Organic Amendments for Sustainable Agricultural Systems**

Editors

**Maria de la Luz Mora Cornelia Rumpel Marcela Calabi-Floody**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Maria de la Luz Mora Center of Plant-Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Bioresource Nucleus, Universidad de la Frontera Chile

Cornelia Rumpel CNRS, Institute of Ecology and Environmental Sciences, UMR 7618 (CNRS, Sorbonne U, UPEC, INRAE, IRD) France

Marcela Calabi-Floody Center of Plant-Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Bioresource Nucleus, Universidad de la Frontera Chile

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Agronomy* (ISSN 2073-4395) (available at: https://www.mdpi.com/journal/agronomy/special issues/smart fertilizers sustainable-agricultural).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-2762-8 (Hbk) ISBN 978-3-0365-2763-5 (PDF)**

Cover image courtesy of Cornelia Rumpel

© 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.

## **Contents**



## **About the Editors**

**Maria de la Luz Mora** leads an interdisciplinary research group (Plant–Soil Interaction and Plant Biotechnology) and contributes significantly to plant production, especially in the cereal, meat, and milk industries in southern Chile. Dr. Mora is the author and co-author of more than 190 scientific publications with contributions of international researchers, doctorate students, and post doctorates. In addition, she has lead and participated in several International Cooperation Programs with Research Centers of excellence. She has been the Director of the Scientific and Technological Bioresources Nucleus (Bioren-Ufro) since 2009. During her career, she has directed several National and Co-International projects. She was the creator of the Doctorate Program in Sciences of Natural Resources, and she also has advised about 20 PhD students. She has been working on projects that contribute to the knowledge to understand the coupled C-N-P cycles to contribute to food security and production.

**Cornelia Rumpel** is a soil biogeochemist working for the French National Research Center (CNRS) at the Institute of Ecology and Environment in Paris, France. Her work is concerned with the dynamics of organic matter at the molecular scale and the biogeochemical cycling of carbon, nitrogen, and phosphorus in natural and managed ecosystems. She focuses on temperate and tropical environments and has contributed to the change of several paradigms. Her recent work focuses on the development of innovative agroecological strategies to increase soil carbon sequestration. She has supervised 18 PhD students to successful completion of their thesis, has published more than 200 papers in international peer reviewed journals, and is listed as a highly cited researcher.

**Marcela Calabi-Floody** is an interdisciplinary researcher with a strong vision on development and innovation (R&D&i), with expertise in nanotechnology, soil–organic matter interactions, and smart biofertilizer generation. She works in La Frontera University at the Scientific and Technological Bioresource Nucleus (BIOREN) in the field of nanotechnology associated with C sequestration and smart fertilizer development. In addition, she has specialized in nanotechnological and the biotechnological management of agricultural wastes (development, revaluation, and reuse) in order to improve its utilization as C sequestration matrix in soils. Dr. Calabi-Floody has directed and co-directed more than 18 national and international research projects, generating a strong collaboration network. With this research network, she has generated more than 25 high-level scientific articles published in international journals, and disseminated the results at national and international conferences.

## **Preface to "Smart Fertilizers and Innovative Organic Amendments for Sustainable Agricultural Systems"**

Sustainable agricultural practices are needed to provide food security for a growing global population. Food production is usually associated with high nutrient inputs in the form of mineral fertilisers. Since the beginning of agriculture, such practices have led to soil degradation and the release of environmental contaminants. This book focuses on innovations in organic and inorganic fertiliser production. We have compiled studies presenting smart fertilization strategies. The idea for this book originated from the Chilean–French collaboration of the three guest editors during the MEC fellowship of Dr. Cornelia Rumpel at the Universidad de la Frontera in Temuco (Chile). The editors thank all authors for their excellent contributions

> **Maria de la Luz Mora, Cornelia Rumpel, Marcela Calabi-Floody** *Editors*

## *Editorial* **Closing Biogeochemical Cycles and Meeting Plant Requirements by Smart Fertilizers and Innovative Organic Amendments**

**María de la Luz Mora 1, Marcela Calabi-Floody 1,2,\* and Cornelia Rumpel 1,3,\***


### **1. Meeting the Growing Food Demand of the Global Population: Challenges for Sustainable Agriculture**

Expansion of farmland with food production as a major service has been largely associated with conversion of natural ecosystems like the Amazon and Savanna into new agricultural land [1]. It has resulted in the large scale modification of natural landscapes and ecosystems, altering important climate interactions, such as surface moisture and microbial diversity, and strongly impacting greenhouse gas (GHG) emissions [2]. Indeed, current agricultural activities are the main contributors to GHG emissions, representing globally 20% of the annual atmospheric emissions [3]. They are largely driven by land use change and decoupling of biogeochemical cycles leading to land degradation, and soil organic matter loss since the introduction of agriculture [2,4]. Agriculture contributes to GHG emissions by releasing CO2, N2O and CH4. The agricultural sector is the main contributor to N2O and CH4 emissions, which have 260 and 40 times greater global warming potential than CO2. In particular, N fertilizers are important N2O sources. Nowadays, it has become clear that agriculture is not only causing climate change by GHG emission, but that it is also highly vulnerable to climate change, which is threatening crop yields in many parts of the world [5]. Moreover, the green revolution in the 1960s, which strongly reformed and increased agricultural production, also had many adverse effects on the environment [6], including soil acidification and pollution of waterways through export of mineral fertilizers not taken up by plants [7,8]. The use of agrochemicals to fight weeds and pests led to biodiversity loss, and highlighted the need for an agroecological transition and a second green revolution. An additional challenge is the necessity to achieve food security of a growing global population [9].

As the earth surface covered by agricultural land (more than 40% of the total Earth surface) cannot be expended anymore [2], sustainable intensification of production on the existing area, while reducing mineral agrochemical use and adapting to climate change, is a great challenge for landowners, scientists and politicians. Recently, it has been suggested that increasing carbon storage in soils could be a solution to mitigate and to adapt to climate change while at the same time supporting agricultural production to increase food security [10,11]. Such a solution can only be brought to scale if region specific solutions adapted to pedoclimatic and socioeconomic conditions are employed [12]. One aspect of sustainable solutions is the replacement of mineral fertilizers by innovative strategies to enhance plant growth. In the light of circular economy, it was suggested that smart fertilization strategies could be based on the transformation of organic wastes from agricultural systems into innovative organic amendments [13,14] and/or carrier materials

**Citation:** Mora, M.d.l.L.; Calabi-Floody, M.; Rumpel, C. Closing Biogeochemical Cycles and Meeting Plant Requirements by Smart Fertilizers and Innovative Organic Amendments. *Agronomy* **2021**, *11*, 1158. https://doi.org/ 10.3390/agronomy11061158

Received: 29 April 2021 Accepted: 3 June 2021 Published: 5 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for beneficial microorganisms and/or enzymes [15]. While improving nutrient availability, fertilization strategies based on organic materials may be an avenue towards sustainable intensification.

Therefore, it is imperative to concentrate efforts to develop innovative fertilizers through biotechnological approaches with or without use of beneficial microorganisms that allow their viability in the soil in the face of an established microbiome. Innovative organic amendments with low GHG emission potential should be designed to foster terrestrial C sequestration and stabilization, capable of mitigating the environmental impact caused by the agricultural sector, while at the same time increasing soil health and adapting to climate change. For this Special Issue, we invited contributions dealing with innovations in organic and inorganic fertilization strategies in order to improve agricultural yields, while at the same time reducing negative externalities and increasing the soils' organic matter contents. It contains 11 articles reporting (1) the effects of new fertilization strategies on soils and plants, (2) characteristics of innovative organic amendments and (3) system innovations based on organic fertilization and waste recycling.

### **2. Smart Fertilizer Development and Their Effect on Soils and Plants**

As the main limiting factor for food production is N and P availability, huge amounts of conventional N and P-fertilizer are applied per year, which often lead to environmental damage [16]. Especially N-fertilizer application at levels exceeding plant requirements leads to significant environmental consequences due to N losses, such as: nitrate (NO3 −) leaching, ammonia (NH3) volatilization, and nitrous oxide (N2O) emission [8,17–19]. The N losses by urea application is one of the main problems in agricultural systems, where leaching of NO3 − is one of the most important loss pathways due to its high mobility [20]. Incrocci et al. [21] used a (bio)technological approach to develop an innovative controlledrelease polyurethane-coated urea fertilizer, which could considerably reduce the N leaching in tomato cultivations. Unlike N, phosphorus (P) is mainly fixed in the soil systems. Therefore, the efforts to improve P use efficiency are focusing on favoring slow release and preventing P fixation in the soil. In this context, Shafi et al. [22] reported that the incorporation of humic acid in combination with chemical P fertilizer can prevent the P fixation in calcareous soil, thus improving crop yield and wheat (*Triticum aestivum* L.) plants' P uptake. Teles et al. [23] developed a new P fertilizer with slow solubility through the partial acidification of rock phosphates, incorporating zeolite and pillared clay into partially acidulated phosphates with high adsorption characteristics. The mechanism of P release is based on the saturation the acidic sites of the clay materials before adsorption. These saturated sites may act as a vehicle for slow and gradual dissolution into soil solution. This strategy seems to be highly promising as it was able to compete with conventional fertilizers.

Mineral fertilization may also lead to soil acidification thereby increasing aluminum (Al3+) toxicity [7,24,25]. In this context, Vega et al. [26] studied the beneficial effects of silicon application for mitigating Al3+ toxicity in sensitive barley cultivars. Their findings revealed that silicon fertilization could increase the resistance of barley to Al3+ toxicity by regulating the metabolism of phenolic compounds with antioxidant and structural functions.

A liquid biofertilizer combined with a microbial consortium was evaluated by Yousef et al. [27]. These authors used a bacteria and fungi consortium containing *Bacillus circulans*, *B. poylmyxa*, *B. megatherium*, *Candida* spp., and *Trichoderma* spp. and studied its combined effect with liquid biofertilizer on Jew's mallow plant production. They concluded that combined application of inorganic NPK plus biofertilizer is most beneficial to increase growth, yield, and nutrient accumulation of Jew's mallow plants.

### **3. Innovative Organic Amendments**

In order to move to carbon neutrality in agricultural production in agreement with sustainable development goals and global governmental treaties to minimize climate change, different initiatives were launched, such as "4 per Thousand (4p1000)" at the COP21 in Paris, or the Agenda 2030 of the United Nations [28]. Recently, FAO and the Global Soil Partnership (GSP) established "RECSOIL: Recarbonization of global soils" as an initiative to implement the soil organic carbon (SOC) agenda by using the best tools and technologies available [29]. Organic amendments may be a keystone to increase SOC sequestration and provide food security through soil quality improvement. Reuse of organic waste and their transformation into organic amendments is a sustainable strategy that farmers need to apply and scientists have the challenge to innovate. In this context, Emadodin et al. [30] proposed jellyfish application as an organic soil amendment able to allow enhanced seedling growth and establishment of ryegrass on sand dune soil even under water scarcity conditions.

Under a circular economy approach, black soldier flies have the capacity to transform anthropogenic organic wastes into nutritious insect biomass (frass). The fertilizer potential of frass was studied by Klammsteiner et al. [31], who reported that it may serve as a valuable alternative to mineral fertilizers with beneficial effects on plant growth and not impairing the hygienic properties of soils. In addition, Dulaurent et al. [32] investigated the effect of earthworms (*Lumbricus terrestris* L.) on nutrient uptake and crop growth in the presence of frass from mealworm (*Tenebrio molitor* L.). Their study showed a synergistic effect between earthworms and frass on soil fertility and that earthworms thus may improve the efficiency of frass as an organic fertilizer.

Composting, vermicomposting and biochar production using organic wastes are known sustainable practices, which convert these raw materials into valuable organic amendments. Organic amendments may be used individually or in mixture to improve soil carbon sequestration and fertility at the same time [13,33,34]. Aubertin et al. [35] addressed the effect of weathering on biochar-compost mixture properties, their biological stability and their effect on plant growth after soil addition. They were able to show that weathering changed synergistic effects of biochar compost mixtures in terms of carbon sequestration potential and biomass production.

### **4. System Innovations Based on Organic Fertilization and Waste Recycling**

Finally, in order to integrate sustainable agricultural practices, system innovations based on agroecological approaches and waste recycling must be employed. In this context, use of legumes as cover crops in agricultural rotations may reduce the production costs associated with the use of mineral N fertilizers, and also result in environmental benefits. In order to optimize biomass productivity, biological nitrogen fixation, and transpiration efficiency, Berriel et al. [36] evaluated the application of two *Crotalaria* species, specifically *C*. *juncea* and *C*. *spectabilis* grown under extreme environmental conditions with the finality to maximize their beneficial attributes, while minimizing water consumption through high transpiration. Their results showed that the *C*. *spectabilis* has advantages as legumes cover crop over *C*. *juncea*, in terms of transpiration as indicated by a 13C isotopic analyses.

On the other hand, the chemical fertilizers dependence and/or its coming shortage is a global concern and a huge challenge in terms of food security [15,37]. Thus, the circular economy approach suggests that agricultural systems must become more efficient and favor reuse of their waste as fertilizers in order to reduce external inputs. In this context, recycling of fishpond sediments may be an alternative to reduce the reliance on synthetic fertilizers, due to its high nutritional value [38]. Da et al. [39] studied organic fertilizers based on a composted mixture of 30% of the Pangasius catfish pond sediment and 70% of agricultural waste in cucumber vegetable production. With this strategy, they reduced mineral fertilizer use by up to 75%. Therefore, their results provide evidence that systeminherent organic amendments can be integrated in fish–vegetable farming to provide a more diversified production system with tangible environmental benefits and potentially improved farm income.

The papers presented in this Special Issue indicate that there are multiple ways to increase the production efficiency of agriculture and to reduce external inputs using smart fertizers and innovative organic amendments. We suggest that such strategies should be scaled up to achieve sustainability in agriculture through waste recycling aiming a circular approach to close biogeochemical cycles.

**Funding:** The authors thank the Chilean government for providing a MEC grant Nº MEC80180025. M.C.F. acknowledges funding from Chilean CONICYT (National foundation for Science and Technology) for the financial support under CONICYT-FONDECYT Regural project Nº 1201375, and M.L.M. thanks FONDECYT Regular Nº 1181050. We also acknowledge ECOSSUD-CONICYT C13U02 for their financial support to encourage collaboration between French and Chilean research groups.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This Special Issue was launched during the sabbatical of C.R. at the Universidad de la Frontera (Temuco, Chile).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


*Article*

## **Innovative Controlled-Release Polyurethane-Coated Urea Could Reduce N Leaching in Tomato Crop in Comparison to Conventional and Stabilized Fertilizers**

**Luca Incrocci 1, Rita Maggini 1,\*, Tommaso Cei 1, Giulia Carmassi 1, Luca Botrini 1, Ferruccio Filippi 1, Ronald Clemens 2, Cristian Terrones <sup>2</sup> and Alberto Pardossi <sup>1</sup>**


Received: 28 September 2020; Accepted: 17 November 2020; Published: 20 November 2020

**Abstract:** Large amounts of fertilizers are being used in agriculture to sustain growing demands for food, especially in vegetable production systems. Soluble fertilizers can generally ensure high crop yields, but excessive leaching of nutrients, mainly as nitrate, can be a major cause of water pollution. Controlled-release fertilizers improve the nutrient use efficiency and lower the environmental hazard, usually without affecting the production. In this study, an innovative controlled-release coated urea fertilizer was compared to conventional nitrogen (N) fertilizers and a soluble ammonium-based fertilizer containing a nitrification inhibitor, in a round table tomato cultivation. Both the water and N balance were evaluated for each treatment, along with the yield and quality of the production. The experiment was repeated in three different seasons (spring, autumn and summer-autumn) in a glasshouse to prevent the effect of uncontrolled rainfall. The results indicated that N leaching decreased by increasing the percentage of coated urea. The application of at least 50% total N as coated urea strongly reduced N leaching and improved N agronomic efficiency in comparison with traditional fertilizers, ensuring at the same time a similar fruit production. Due to reduced leaching, the total N amount commonly applied by growers could be lowered by 25% without detrimental effects on commercial production.

**Keywords:** nitrogen fertilizer; nitrification inhibitor; nitrogen leaching; nitrogen use efficiency; 3,4-dimethylpyrazole phosphate (DMPP)

### **1. Introduction**

With the rapid increase of the global population, agriculture is required to satisfy the consequent boost in food demand worldwide. For example, in 2013 the production of primary foodstuffs such as wheat and maize reached 713 and 1018 millions of metric tons, respectively, and it has been estimated that in 2050 the world requirement will be 85% higher than in 2013 [1]. Along with water, considerable amounts of fertilizers have been thus far applied to raise the yield of agricultural crops. Nitrogen (N) is the main plant macronutrient and its concentration in natural soils is often deficient to ensure adequate plant growth and crop yield [2], eventually leading to high rates of N fertilization. Over a four-decade period from 1961 to 2013, the world consumption of N fertilizers has increased from 11.3 Tg N/year to 107.6 Tg N/year [3]. The use of fertilizers has especially increased in the intensive vegetable crop production system [4–6]. In China, N fertilization for the cultivation of vegetables exceeds 1000 and 3000 kg N/ha year in the open-field and greenhouse conditions, respectively. In the same country, in 2008, 17% of the national input of N fertilizers was devoted to the vegetable cropping system [7].

The conventional fertilizers that are commonly applied by growers are highly soluble salts and are liable up to 70% N losses due to volatilization and leaching [8]. These processes have two main undesirable effects: (1) a poor fertilization efficiency because the nutrient element is driven off the root zone, making it unavailable to the plant; (2) a harmful impact on the environment, due to either greenhouse gas emissions or surface water pollution by eutrophication. Nitrogen is commonly applied as nitrate ion, or it is quickly oxidized to this form through nitrification by soil microorganisms. The supply of different N forms or the nitrification process can cause hazardous volatilization losses as ammonia, N monoxide or other N oxides that could contribute to the greenhouse effect. In addition, nitrate ion is not retained by the soil and is easily leached [6,9].

Nitrogen leaching is generally more severe with intensive greenhouse cultures than with open-field crops, as plant growth is faster under controlled conditions and N fertilization represents an effective and low-cost practice to increase the production yield [6]. In fact, several authors drew attention to the occurrence of eutrophication and water pollution in the main European districts for protected vegetable crop production, such as Spain [10], Italy [11], The Netherlands [12], Poland [13] and Greece [14]. The environmental impact associated with nitrate leaching has become a major concern all over the world. In Europe, this has led to the introduction of the Nitrates Directive [15], to limit N pollution and improve water quality. According to the directive, the Nitrate Vulnerable Zones (NVZs) are land areas where drainage water from agricultural crops can cause contamination of larger water bodies by excess nitrate [16]. Hence, the limitation of fertilizers application in agriculture represents an effective measure to counteract nitrate pollution of surface water [17]. With N overfertilization, nitrate ion can also accumulate in the edible parts of several food crops [18]. Human intake of nitrate with the diet has been related to gastric cancer [19–21] and has directed the European Union toward a restriction to the nitrate content in food as a safety measure for the consumer [22].

Based on the above considerations, many efforts have been made to rationalize N fertilization. The application of enhanced efficiency fertilizers is a functional approach to achieve this purpose by limiting nutrient amounts in soils and at the same time reducing both N leaching and N volatilization losses. Enhanced efficiency fertilizers can be divided into three subgroups [23]: (i) slow-release fertilizers, (ii) stabilized fertilizers, (iii) controlled-release fertilizers. Slow-release fertilizers contain low solubility N compounds that become available to plants only after microbial degradation. Stabilized fertilizers contain chemical inhibitors, which slow down or stop biological processes. These include urease inhibitors that hinder the hydrolysis of urea by urease enzyme, or nitrification inhibitors such as dicyandiamide (DCD), or 3,4-dimethylpyrazole phosphate (DMPP), which prevent the oxidation of ammonium ion [24]. Controlled-release fertilizers are made of an inner core and an outer layer. The former is a water-soluble fertilizer such as urea, ammonium nitrate or potassium nitrate; the latter is a coating material such as sulfur, an alkyd- or polyurethane-like resin, a thermoplastic polymer or a mineral-based inorganic material [25]. Controlled-release fertilizers can also be made by the combination of sulfur-coated urea with an additional polymer or resin coating [26,27].

Two main limitations to the use of controlled-release fertilizers are their relatively high cost, and the difficulty to develop an adequate coating for irregularly shaped fertilizers or highly soluble compounds such as urea. The controlled-release fertilizer used in this study consisted of polyurethane-coated urea granules and was manufactured using an innovative polymer coating patented technology (E-MAX) that can be employed in combination with many types of fertilizers, including hygroscopic compounds or irregularly shaped materials. The release mechanism of coated fertilizers is based on the osmosis phenomenon produced by the diffusion of water through the coating, which leads to the solubilisation of the inner fertilizer. Water transfer through the coating layer is the rate determining step and depends on the chemical structure of the polymer, the thickness of the coating layer and the temperature. Therefore, for a given polymer with a fixed thickness, the release rate should be temperature-dependent and should be assessed through the temperature regime experienced by the coated fertilizer [28,29]. Thus, the release of nutrients into the soil could be predicted and controlled over time. With the E-MAX coating technology, the thickness of the polymer layer is well below 100 μm; the coating material is evenly spread and fixed on the whole surface of discrete 2- to 4-mm-diameter particles, degrades slowly and is essentially inert in the soil after the nutrient has been released. The work was aimed at evaluating: (i) the release curve and the effectiveness of the polyurethane-coated urea in relation to the plant N requirements in different climate growing conditions; (ii) the effect of this controlled-release fertilizer on N leaching and on the yield and quality of a soil greenhouse tomato cultivation in comparison with fertilization techniques that employ a nitrification inhibitor or soluble salts.

### **2. Materials and Methods**

Although the controlled-release fertilizer used in this study was being developed and marketed mainly for open field application, three experiments were carried out in a glasshouse at the University of Pisa on round-table tomato plants (cultivar Hybrid F1 "OPTIMA"). The use of a greenhouse equipped with lysimeters allowed for the prevention of the negative effects of uncontrolled rainfall events and made possible to easily collect, measure and analyse water drainage and N leaching, thus enabling reliable computations of both water and N balance in the different treatments. The present study was focused on the time interval of N release by the coated urea (3–4 months) rather than to the long-term agronomical effects of the treatments. Therefore, the growing period lasted from the transplanting to the harvesting of the third or fourth truss and was shorter than that of a typical greenhouse cultivation of tomato, which is generally conducted until the ripening of the fifth or sixth truss. However, the growing conditions of the experiments closely resembled those of a real cropping system and enabled the evaluation of the yield and quality of the production.

### *2.1. Experimental Design*

Three experiments were performed under different growing conditions: Experiment 1 (spring 2015), Experiment 2 (autumn 2015) and Experiment 3 (summer–autumn 2016). In all the experiments, either the stabilized or the controlled-release fertilizer were compared with a conventional treatment (CON). The distinct N treatments and fertilizer addition programs are detailed in Table 1. The same total N dose was applied in all the treatments using different N sources: (i) the inorganic salts potassium nitrate KNO3, calcium nitrate Ca(NO3)2, ammonium nitrate NH4NO3 and ammonium sulphate (NH4)2SO4; (ii) a stabilized fertilizer containing 26% total N (7.5% as nitrate and 18.5% as ammonium), with the addition of DMPP as a nitrification inhibitor (ENTEC® 26:0:0 Nitrogen-Phosphorus-Potassium, EuroChem Agro, Cesano Maderno, Italy); (iii) an innovative controlled-release fertilizer, manufactured using the E-MAX coating technology and consisting of granules of urea fertilizer coated by a permeable and very thin polyurethane layer (Agrocote® Max; ICL Specialty Fertilizers, Heerlen, The Netherlands; Patent EP 2672813 B1). The stabilized and controlled-release fertilizers will be hereafter indicated as DMPP and CU, respectively.

The total N dose was adapted to the different climate conditions of each experiment and, as plant growth is normally slower in autumn, N fertilization was necessarily lower in Experiment 2 than in Experiment 1 (300 kg/ha against 360 kg/ha) to prevent excess leaching. A reduced total N application in the cold season is consistent with the growers' common practice. For this reason, a similar absolute amount of CU or DMPP applied as base fertilization corresponded to a different percentage of total N. For example, Table 1 shows that the DMPP dose tested in Experiment 1 was 72 kg N/ha (20% of total N) and was comparable to the DMPP amount applied in Experiment 2 (75 kg N/ha; 25% of total N). Both the N dose and the N percentage are reported in Table 1 for each fertilizer.



10

### *Agronomy* **2020**, *10*, 1827

For both conventional and stabilized fertilizers, the high solubility limited the amount that could be applied as base fertilization to 75 kg N/ha, to prevent detrimental salinity effects on the crop. In contrast, with the controlled-release CU fertilizer higher doses could be applied, up to 270 kg N/ha. The outcome of Experiment 1 was used to tune the conditions of the subsequent trial and, as in spring no significant effect was observed on the production with the CU20 treatment, higher CU doses were employed in autumn. In addition, at least 75 kg N/ha was applied in all the treatments as calcium nitrate. This amount was never decreased in the three experiments, to allow a sufficient calcium supply to the plants and ensure a correct calcium nutrition, preventing the occurrence of the blossom-end rot. For the CUred treatment, which employed a reduced N dose, the above amount of Ca(NO3)2 represented 33% of total N applied (Table 1).

In all the experiments, both N and water balance of the tomato culture were evaluated for each fertilization treatment. Inside the greenhouse, the plants were grown in lysimeters to enable reliable determination of water and N status in the growing system. Each lysimeter hosted four plants and consisted of a 200 L plastic tank (75 × 53 cm, height 50 cm), containing 20 L (5 cm) pumice layer at the bottom to ensure correct drainage. The pumice layer was topped off with 160 L sandy soil and peat (40 cm depth; 60:40 volume ratio; 1.2 kg/L specific weight). Along with the results of soil analyses, the main climatic parameters of the three experiments are reported in Table 2. The greenhouse heating guaranteed a minimum inner air temperature of 12.5 ◦C. Global radiation, air temperature, soil temperature at 15 cm depth and relative humidity (RH) were recorded every ten minutes by a climate station equipped with three different probes for soil temperature, connected to a database (Econorma, Treviso, Italy). The recorded values were used to calculate the cumulative radiation and the average daily values of RH, soil temperature and air temperature. The cumulative soil temperature was obtained by the sum of the values of daily average soil temperature recorded in each experimental period.

In each experiment, a zero-N fertilization treatment with the same levels of the other nutrients was also included for the assessment of N use efficiency. Although this is normally the control treatment in agronomic experiments, the main goal of the present study was to evaluate the effect of different fertilization strategies on the reduction of N leachate as compared with conventional fertilization. For this reason, the conventional treatment rather than the zero-N treatment was regarded as the control in our experiments.

After transplanting and until the end of the experimental period, each treatment was fertigated with nutrient solution to ensure a proper supply of all the macro- and micronutrients to the plants. Along with N, all the treatments of the three experiments received the same total amounts of phosphorus (P) and potassium (K), which were 1.4 and 12 g/plant, respectively (equivalent to 96 kg/ha P2O5 and 433 kg/ha K2O). These P and K doses are commonly used by the greenhouse growers in Italy and were either applied as base fertilizers or supplied by fertigation. The total calcium supply ranged from 60 mg/L (that is the concentration in the irrigation water) to 150 mg/L. The latter value was reached only when calcium nitrate was used as a N source to prevent the occurrence of the blossom-end rot. The concentrations of the other elements in the nutrient solution were the following (mg/L): Mg 30; Na 230; Cl 320; Fe 2; B 0.27; Cu 0.24; Zn 0.29; Mn 0.55 and Mo 0.05. Depending on the treatment and on the phenological phase, different amounts of inorganic N fertilizers were added when necessary to the nutrient solution (Table 1) to achieve the same final N dose in each treatment. Specifically, NH4NO3 was supplied from transplanting until the blooming of the second truss, Ca(NO3)2 was employed until the ripening of the first truss and KNO3 was added during the ripening stage, until the end of the experiment. The irrigation was generally applied twice a day, according to the climate conditions and the canopy development, in the same amount for all the treatments investigated.

The tomato plantlets were transplanted at the stage of six-seven true leaves, which in the three experiments corresponded to a different plant age (50–30 days), depending on the thermal growing conditions. Similarly, the end of the experimental period corresponded to the harvest of the fourth truss in Experiment 1 or to the harvest of the third truss otherwise.

**Table 2.** Climate and soil parameters measured in the three experiments. Temperature, humidity and radiation are reported as the average values inside the greenhouse during the whole experimental period. Soil parameters are reported as the initial values immediately before the beginning of each experiment.


Mean values ± standard deviation. n = 5 in Experiment 1; n = 3 in Experiment 2 and Experiment 3.

### *2.2. Analyses of Water, Soil, CU Granules and Plant Tissue Samples*

The average values of the climate parameters (RH, air and soil temperature, cumulated global radiation) were recorded daily. Nitrogen was contained as urea in CU granules and in different chemical forms in water, soil and tissue samples. A summary of N determinations and analytical assays used can be found in Table 3.

Due to the autumn climate conditions, in both Experiments 2 and 3 the growing cycle was longer than in spring, while the crop evapotranspiration and the plant growth were strongly reduced. Therefore, an increase of the water collection period was necessary to maintain the same number of drainage samplings as Experiment 1. The cumulated drainage water was sampled from each container every 7–10 days in Experiment 1 and every 13–15 days in Experiment 2 and Experiment 3. The water samples were filtered on Whatman qualitative filter paper and analysed for the concentrations of nitrate (salicylic acid method) [30]; ammoniacal N (indophenol method) [31] and ureic N (enzymatic assay using a commercial kit; Megazyme International, Wicklow, Ireland). All the absorbance measurements were carried out using a Lambda35 UV-vis double beam spectrophotometer (Perkin Elmer, Waltham, MA, USA).

The soil samples were dehydrated at 40 ◦C in a ventilated oven and sieved to separate intact CU granules. The dried soil samples were extracted with water, 1 M KCl, 0.5 N NaHCO3 at pH 8.5 or 1 N CH3COONH4 at pH 7.0, respectively, for the spectrophotometric determinations of nitrate [30], ammoniacal N [31] and available P [32] and for the assessment of exchangeable K by atomic absorption

spectroscopy (AAS) [33]. In all cases, a 1:2 w/v extraction ratio was used. The total organic matter and the other soil parameters reported in Table 2 were assessed according to official methods the Italian Ministry of Agriculture and Forestry [34].


**Table 3.** Analytical assays used to determine nitrogen concentration in samples of water, soil, coated urea fertilizer and plant tissues in the three experiments.

The N amount retained by the coated urea granules was determined in all the CU treatments. At the beginning of each experiment, 2 g aliquots of the coated fertilizer were wrapped in net fabric before application to each lysimeter. Every 30 days during the cultivation period (for Experiments 1 and 2) and at the end of the cultivation period (for all three experiments), the wrappings were removed from the soil to collect the residual granules, which were gently washed with distilled water, oven-dried at 70 ◦C and powdered with mortar and pestle. The powder was dispersed into 200 mL distilled water and the filtered solution was analysed for the concentration of urea. For each cultivation period, the N release by the coated fertilizer was evaluated by the difference between the initial and final ureic-N amounts in the net-wrapped granules.

All the plant samples were dried in a ventilated oven at 70 ◦C till constant weight and ground in a mill to a fine powder. The crop yield was determined as the number and fresh weight of the fruits, which were picked weekly and divided into marketable and nonmarketable categories. To evaluate the quality of the production, four fruits from different plants were collected from each lysimeter in the middle of the harvesting period and were homogenized in a mixer. Part of each homogenized sample was oven-dried for dry matter determination; the remaining material was centrifuged, and the resulting juice was analysed for pH, EC, total soluble solids (determined by refractometry and expressed as ◦Brix) and total titratable acidity (determined by acid-base titration with 0.1 M sodium hydroxide and expressed as g citric acid in 100 mL juice). The shoot dry biomass production was determined at the end of each experiment. All the dry tissue samples were analysed for their contents of nitric, ammoniacal and reduced N, as described previously for soil samples.

### *2.3. Calculation of N and Water Balance Sheet and N Use E*ffi*ciency*

A balance sheet for both water and N was computed for each treatment and experiment. Water evapotranspiration was calculated as the difference between water supply and water drainage (both measured); the leaching fraction was computed as the ratio between water drainage and water supply. The computation of N balance was based on the available amount during cultivation (initially contained in the soil or supplied through fertilization) and the amount that was actually removed

(leached or absorbed by the plants) or remained in the soil at the end of the experiments. The amounts of fertilizers were weighed using a technical balance with 0.1 g precision and 1.0 kg/ha was cautiously assumed as the standard deviation for the total N applied. Soil mineral N was evaluated as the sum of nitric and ammoniacal N (Table 3) and was assessed both at the beginning (prior to base fertilization) and the end of each experiment. The total N amount of the system at the end of the experiment (N output) was evaluated as the sum of the N fractions that were absorbed by the plants, were lost by leaching, remained in the soil as mineral N or remained in the CU granules as residual urea. The final amount of urea in the soil was negligible (less than 0.1 mg/kg), due to both the controlled release by the CU fertilizer and to the fast leaching and mineralization processes that urea undergoes in soils [35]. The total N amount available during the growing period (N input) was calculated as the sum of the initial mineral amount in the soil and the amount applied with fertilizers, both as base fertilization and with fertigation (total N supplied). Based on the results of the zero-N treatments, some nitrogen use efficiency (NUE) indexes were calculated according to [36,37], using the following formulas:

Agronomic Efficiency (AE) = (Y − Y0)/F

Partial Factor Productivity (PFP) = Y/F

Apparent Recovery Efficiency by difference (REC) = (U − U0)/F

Physiological Efficiency (PE) = (Y − Y0)/(U − U0)

where Y and Y0 (g/m2 on a fresh weight basis) are the tomato yields with and without N fertilization, respectively; F is the total N supplied (g N/m2) and U and U0 (g N/m2) are the N contents in fruits with and without N fertilization, respectively.

### *2.4. Statistical Analysis*

A completely randomized design was adopted. As the statistical variability of the data was initially unknown, in Experiment 1 five replicates (lysimeters) were prudentially arranged. Based on the results of the first experiment, the number of replicates could be reasonably reduced to three in the subsequent trials to obtain an adequate statistical discrimination and limit the cost of data collection. Each replicate consisted of four tomato plants. The collected data were tested for normality and homoschedasticity by means of the Shapiro–Wilk's and Levene's test, respectively. The data were subjected to one-way ANOVA and the mean values were compared by Tukey test using the Statgraphics Plus 5.1 software (StatPoint, Inc., Herndon, VA, USA).

### **3. Results**

For all the experiments, Table 4 reports the water balance, Table 5 shows the biomass and N distribution in different plant tissues and Table 6 reports the data concerning the yield and quality of the tomato production obtained with the different treatments. The N balance for the three experiments is reported in Table 7. Table 8 reports the NUE indexes that were calculated from the tomato yield (Y0; kg/m2 on a fresh weight basis) and the N content of fruits (U0; g N/m2) obtained without N fertilization (zero-N treatment).

In all the treatments, only negligible amounts of urea and ammonium (0–0.08 g N/m2) were detected in the drainage water. Thus, N leached from the soil was almost completely in the form of nitrate ion.

In Experiment 1, the water balance was similar for the CON1, DMPP20 and CU20 treatments, while a higher water drainage and leaching fraction along with a lower evapotranspiration were observed for CU40 and CU75-1 treatments (Table 4). Both the dry biomass and the N concentration in the tissues were affected by N fertilization. Compared with CON1, all the treatments except CU20 increased the dry biomass of fruits. In addition, both CU40 and CU75-1 increased the fruit N

concentration (Table 5). However, apart from slight differences in the number of fruits, the distinct treatments had no significant effect on the tomato yield or quality (Table 6).


**Table 4.** Effect of different fertilization strategies on the water balance.

Mean values ± standard deviation. In each experiment, different letters within the same column identify a significant difference (*p* < 0.05), according to Tukey test following one-way ANOVA. Mean values without any letters are not significantly different. CON: conventional treatment; DMPP: treatment with stabilized fertilizer; CU: treatment with coated urea.

**Table 5.** The influence of different fertilization strategies on the distribution of biomass and nitrogen in tomato tissues.


Mean values ± standard deviation. For each column in each experiment, different letters identify a significant difference (*p* < 0.05), according to Tukey test following one-way ANOVA. Mean values without any letters are not significantly different. CON: conventional treatment; DMPP: treatment with stabilized fertilizer; CU: treatment with coated urea.


**Table 6.** The influence of different fertilization strategies on yield and quality of the tomato crop.


 parameter experiment, identify significant (*<sup>p</sup>* 0.05), according Tukey following one-way Mean values without any letters are not significantly different. CON: conventional treatment; DMPP: treatment with stabilized fertilizer; CU: treatment with coated urea. FW: fresh weight; EC: electrical conductivity.



Nitrogenbalancefordifferentfertilizationstrategiesinthethreeexperiments.

 **error**




Mean values ± standard deviation. For each parameter, different letters identify a significant difference (*<sup>p</sup>* < 0.05), according to Tukey test following one-way ANOVA. CON: conventionaltreatment; DMPP: treatment with stabilized fertilizer; CU: treatment with coated urea. I = A + B + C; O = E + F + G + H; Δ = O − I; Relative error = Δ/O.


**Table 8.** Nitrogen use efficiency indexes calculated from the data collected in the three experiments.

Mean values ± standard deviation. For each index and each experiment, a different letter indicates a significant difference, according to Tukey test following one-way ANOVA (*p* < 0.05). Mean values without any letters are not significantly different. Y0: tomato yield; U0: nitrogen content in fruits; AE: agronomic efficiency; PFP: partial factor productivity; REC: Apparent recovery efficiency by difference; PE: physiological efficiency; FW: fresh weight; CON: conventional treatment; DMPP: treatment with stabilized fertilizer; CU: treatment with coated urea. The values of Y0 (kg FW/m2) and U0 (g N/m2)used for the calculations were, respectively 1.74 ± 0.11 and 1.73 ± 0.15 in Experiment 1; 1.00 ± 0.09 and 0.76 ± 0.08 in Experiment 2; 0.90 ± 0.07, and 0.65 ± 0.05 in Experiment 3.

Concerning the N balance (Table 7), the total N plant uptake was lower in CON1 than the other treatments. The coated fertilizer (CU40 or CU75-1) was able to reduce N leaching by about 55% or 80% as compared to the control. The same effect was observed also for the DMPP20 and CU20 treatments, although to a lower extent (about 24% reduction). The soil contained always more mineral N at the end of the experiment than at the beginning, especially with the CU treatments that decreased N loss by leaching. However, in all the treatments the N output was higher than the N supplied. The REC index was significantly higher with the CU40 and CU75-1 treatments than with the control, while no significant difference was observed for AE and PFP (Table 8).

In Experiment 2, the water balance for the DMPP25 treatment was similar to that of the control. In contrast, both CU treatments exhibited the highest leaching fraction and the lowest evapotranspiration. Moreover, the CU75-2 produced the highest water drainage (Table 4). The different fertilizers affected the distribution of both dry matter and N content among plant organs, although the dry biomass of the whole plants remained generally unchanged (Table 5). The best results for yield and fruit quality were obtained with the CU75-2 treatment (Table 6). With the CU fertilizer, the total N plant uptake resulted similar to that of the control, but higher than that of the DMPP25 treatment. In addition, the coated fertilizer reduced N leaching, determined higher values of all the agronomical indexes and, in contrast with the outcome of Experiment 1, resulted in a lower final content of mineral N in the soil compared with the other treatments. At the end of Experiment 2, about 14% ureic N was still retained by the coated fertilizer (Tables 7 and 8).

The analysis of the CU granules during and at the end of the growing period gave similar results in both Experiments 1 and 2 (Figure 1). The N release into the soil by the CU fertilizer was temperaturerather than time-dependent and the whole set of data was fitted by an exponential-type function of the cumulative daily average soil temperature (thermal sum, X) with excellent correlation (r2 = 0.99, n = 30). Nevertheless, for N release values below 80%, the relationship could be well described (r2 = 0.95; n = 18) by the linear function (data not shown):

$$\% \text{ N release} = \text{3 } + \text{ 0.05203 } \times \text{\textdegree}$$

**Figure 1.** Percentage of N released by the granules of coated urea (CU) in Experiments 1 and 2, as a function of the cumulative daily average soil temperature (thermal sum).

In Experiment 3, the CUred treatment exhibited a similar water balance as the control (Table 4) and produced similar fruit biomass and yield, without affecting the quality parameters or the N content of the fruits (Tables 5 and 6). The amount of N leached was almost 3-fold lower with the coated fertilizer and a decrease was also observed in both plant N uptake and soil mineral N at the end of the experiment. The analysis of the CU granules recovered at the end of the trial revealed that 11% ureic N had not been released into the soil. As in Experiment 2, higher values of AE, PFP and PE indexes were obtained with the coated fertilizer (Table 8).

### **4. Discussion**

All the treatments received the same amount of irrigation water, apart from low dissimilarities due to unavoidable inefficiencies in the irrigation system. The maximum differences in water supply were only 1.2% in Experiment 1, 3.0% in Experiment 2 and 1.5% in Experiment 3. Moreover, the leaching fraction was never lower than 13% (Table 4), which is indicative of a correct irrigation regime. With the only exception of the CUred treatment, all the treatments within the same experiment received the same total N amount.

### *4.1. E*ff*ect on the Crop (Yield and Quality)*

Table 6 shows that in all the experiments the use of the DMPP fertilizer did not significantly affect the tomato yield compared with the control treatment. In contrast, both the CU40 and CU75-1 treatments in Experiment 1 improved the fruit amount and the CU75-2 treatment in Experiment 2 improved both the yield and the tomato quality. Although the differences were not always significant, at the highest urea doses we observed an increasing trend in all the parameters of fruit production in both Experiments 1 and 2. In each experiment, the different treatments did not affect the dry matter percentage of the fruits (Table 6) and the dry weight of the whole plants was also generally unaffected (Table 5). On the other hand, a different weight distribution among plant organs was observed with the different fertilizers; in Experiment 1, the leaf dry biomass was higher for the CON1 than for the

high dose CU treatments, and the same behaviour was observed in Experiment 2, where also the N concentrations of leaf and stem tissues were higher for CON2 than for the CU75-2 treatment (Table 5). This outcome indicated a lower vegetative vigour for the CU-treated plants, which could be due to a reduced initial soil N availability and was consistent with a significantly lower evapotranspiration and a higher leaching fraction than those of the control and DMPP treatments (Table 4).

On the other hand, in Experiment 1 the application of coated urea at low concentration (CU20) produced a similar effect as DMPP20; although both treatments significantly lowered N leaching (Table 7), they determined an increase in leaf dry biomass and N concentration compared with CON1 (Table 5). Nitrogen is the main constituent responsible for vegetative growth and top dressing was initially applied as NH4NO3 with both treatments (Table 1). Hence, this outcome suggested that the plants vegetative behaviour was not effectively limited, due to a ready N availability in the root zone at the beginning of the growing period. In agreement with our findings, it was reported that in tomato high N levels increased plant vigour and delayed flower and fruit formation [38]. Similar results were reported also for different vegetable species, such as zucchini [39].

A limitation of plant vigour by the CU fertilizer was observed also in Experiments 2 and 3. Compared with CON2, the CU75-2 treatment increased both yield and fruit size and determined a similar N uptake; to a lesser extent, the same behaviour was observed also for the CU50 treatment, thereby suggesting that application of the coated fertilizer did not affect the plants ability to take up N from the soil. In Experiment 3, the reduction of the total N dose determined a strong decrease of N leaching compared with the control; thus, despite a slightly lower N uptake, the CUred treatment did not have any effect on the production (Tables 6 and 7).

### *4.2. N Use E*ffi*ciency and Agronomical Implications*

In Experiment 1, all the values of the agronomical indexes were higher than the other trials, probably due to high light intensity conditions (approximately 5-fold higher than in Experiment 2) and high fruit yield during the spring season. In agreement with this outcome, [37,40] found that the REC index, which denotes the crop ability to absorb N from the soil, could be increased in processing tomato by good climatic conditions, since the crop could use more efficiently the N fertilizer available. Moreover, the lower ratio between crop N uptake and N supply that occurred in Experiments 2 and 3 could have contributed to reduce the NUE indexes as compared with Experiment 1. Several authors [37,41,42] reported that the NUE starts to decline when the N supply exceeds the crop N requirement. In all the experiments, the physiological index PE was not influenced by the type of fertilizer that was supplied to the plants (Table 8), indicating that the distinct treatments did not affect the physiological processes of N uptake and use. On the other hand, except for the CU20 treatment, in all the experiments the REC index was higher with CU than with the other fertilizers. A similar trend was observed in Experiments 2 and 3 for AE and PFP. The substantial increase of the agronomical indexes observed with the coated fertilizer can be explained by a higher fruit yield (Table 6), and in Experiment 3, by the reduction of the total N dose (Table 1). Several authors [7,43,44] reported NUE data for distinct vegetable cropping systems, either under greenhouse or in open-air conditions. With a fertilizer dose below 500 kg N/ha, the literature values of REC for greenhouse tomato ranged from 0.21 to 0.33 [7], which is in good agreement with those reported in Table 8 for Experiments 2 and 3. It was found that, along with yield and quality, the NUE was improved in potato fertilized with controlled release urea [45]. Similar results were obtained in wheat [46] and rice [47].

One possible drawback of CU application is the time gap between N release and N plant uptake [26,27]. Generally, the controlled release fertilizers are characterized by a release period, that is the time interval necessary for a fertilizer granule to release 80% of the inner nutrient at a fixed temperature (21 ◦C or 25 ◦C). Our study showed that the N release by the CU fertilizer was positively correlated with the cumulative daily average soil temperature (thermal sum) rather than with the time elapsed from transplanting both in spring and in autumn (Figure 1), despite the daily average temperature increased during the growing cycle in Experiment 1 and followed the opposite

trend in Experiment 2. As expected, the crop development and the N uptake were also increased by higher temperatures in all the treatments. Therefore, the application of the CU fertilizer enabled us to effectively meet the plants nutritional needs, and our results demonstrated that the CU fertilizer could be used as the predominant N source, with simplification of the fertigation programs. However, to prevent a possible yield reduction due to calcium disorder (blossom-end rot), about 25–33% of the total N crop requirements should be beneficially satisfied by the application of calcium nitrate [48].

### *4.3. E*ff*ect on the Environment (N Leaching)*

Compared with the conventional treatment, the use of DMPP fertilizer reduced N leaching only in Experiment 1 (Table 7), even though the nitrification inhibitor was expected to be less effective at higher temperature [49]. However, some authors [50] reported that the inhibiting efficiency of DMPP is modulated by several soil parameters acting simultaneously.

Both in Experiments 1 and 2, a lower evapotranspiration was observed for the high-dose CU treatments than for the other treatments. Because of similar irrigation, this was associated with higher values of water drainage and leaching fraction (Table 4). However, the CU treatments determined a lower N leaching (Table 7), in agreement with studies on several species, such as potato and corn [51], bell pepper [52] and rice [53]. This outcome suggested that CU application was effective in limiting N losses into drainage water. Following a similar trend with this result, a recent life cycle assessment (LCA) study on the impact of N fertilizers on the environment [8] reported the use of alternative coated N fertilizers as an effective strategy to reduce water pollution by eutrophication.

A reduced N loss by leaching with the CU fertilizer suggested the possibility to decrease the N dose commonly applied by growers. This hypothesis was tested in Experiment 3, where the CUred treatment employed -25% total N compared to the conventional fertilization. The data proved the effectiveness of the CU fertilizer, which enabled to decrease N leaching by about 65% (Table 7) without appreciable differences in tomato yield or quality (Table 6). Moreover, the results of Experiment 3 confirmed that with the CUred treatment, the combined effects of lower N supply and lower N loss allowed for the saving of considerable amounts of fertilizer, improving both economic costs and environmental impact. Specifically, in Experiment 3 the amount of fertilizer that could be saved with no influence on the production was up to 114.8 kg N/ha, that is about 30% of total N normally applied in tomato culture.

Concerning the N balance, our results showed that in Experiment 2, the plant growth was lower than expected, due to unexpectedly low light intensity in the autumn season (Table 2). In consequence, N input was higher than N output with both the stabilized and the coated fertilizer. On the other hand, both in Experiments 1 and 3, N input was always lower than N output, with a difference ranging from 24.0 to 43.4 kg N/ha. However, it is worth noting that the computation of N input reported in Table 7 did not include the N supply from soil organic matter mineralization during the growing period. This contribution could be estimated as 23 kg N/ ha in Experiment 1 and 21 kg N/ha in Experiment 3, based on literature data for mineral N release in different types of soils [54]. By adding the estimated amounts to the N input, the overestimation of N output resulted well below 5% for all the treatments.

### **5. Conclusions**

This study confirmed the effectiveness of the CU fertilizer in reducing N leaching from the soil both in spring and autumn growing cycles. At the same time, the results showed that with CU application both tomato yield and quality were maintained or even improved compared with conventional or stabilized fertilizers. Therefore, the CU treatments could satisfy the plants N requirement, preventing at the same time excess concentration of the element in the root zone. This outcome is consistent with the expected performance of controlled-release fertilizers, which should match the nutritional needs of plants better than the soluble or stabilized fertilizers, by providing a gradual N release in the soil. In contrast, with both the CON and DMPP treatments, the high availability of soluble N in the soil promoted vegetative behaviour, with a consequent increase in water use and a possible blooming delay. The experiments indicated that N leaching could be effectively decreased by increasing the percentage of coated fertilizer and that the decrease of N leaching ranged from 9 to 28% of total N applied.

Further work (specifically, a proper validation trial) is needed to extend the results obtained in the greenhouse to the open field growing conditions. The main outcome of this study was that the limitation of N losses achieved using the coated fertilizer enabled a reduction of N application by 25% as compared with the growers' practice, without detrimental effects on the tomato production.

**Author Contributions:** Conceptualization, L.I., R.C. and A.P.; methodology, L.I. and R.C.; investigation, T.C. and L.B.; data curation, T.C., L.B., G.C. and F.F.; writing—original draft preparation, R.M., L.I. and C.T.; writing—review and editing L.I., R.M., A.P. and C.T.; visualization, R.M. and L.I.; supervision, L.I. and R.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors wish to thank Chingoileima Maibam for the final English editing of the text.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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*Article*

## **Application of Single Superphosphate with Humic Acid Improves the Growth, Yield and Phosphorus Uptake of Wheat (***Triticum aestivum* **L.) in Calcareous Soil**

**Muhammad Izhar Shafi 1,**†**, Muhammad Adnan 2, Shah Fahad 3,4,\*,**†**, Fazli Wahid 2, Ahsan Khan 5, Zhen Yue 6, Subhan Danish 7,\*, Muhammad Zafar-ul-Hye 7, Martin Brtnicky 8,9,10 and Rahul Datta 8,\***


Received: 7 July 2020; Accepted: 11 August 2020; Published: 19 August 2020

**Abstract:** In calcareous soil, the significant portion of applied phosphorus (P) fertilizers is adsorbed on the calcite surface and becomes unavailable to plants. Addition of organic amendments with chemical fertilizers can be helpful in releasing the absorbed nutrients from these surfaces. To check out this problem, a field experiment was conducted for two years to determine the effect of P fertilizers and humic acid (HA) in enhancing P availability in soil and their ultimate effect on growth, yield and P uptake of wheat in calcareous soils. The experiment was comprised of five levels of P (0, 45, 67.5, 90 and 112.5 kg P2O5 ha<sup>−</sup>1) as a single superphosphate (SSP) and 2 levels of locally produced humic acid (with and without HA) arranged in a two factorial randomized complete block design (RCBD) with three replications. Wheat plant height, spike length, number of grains per spike, 1000-grain weight, grain, straw and biological yield were significantly improved by the addition of HA with SSP. Very often, the performance of 67.5 kg P2O5 ha−<sup>1</sup> with HA were either similar or better than 90 or even 112.5 kg P2O5 ha−<sup>1</sup> applied without HA. Post-harvest soil organic matter, AB-DTPA extractable and water-soluble P, plant P concentration and its uptake were also significantly improved by the addition of HA with SSP compared to sole SSP application. It was evident that P efficiency could be increased with HA addition and it has the potential to improve crop yield and plants P uptake in calcareous soils.

**Keywords:** calcareous soil; humic acid; phosphorus uptake; single superphosphate; wheat

### **1. Introduction**

Soil fertility and crop productivity are closely related to three main components of soil ecosystems: the bio-available soil nutrients, soil microbiota and organic matter content [1–5]. Phosphorus is 2nd most yield limiting nutrient after nitrogen in agricultural production across the world [6,7]. Phosphorus (P) plays many key functions in plant life especially in the storage and transfer of energy, photosynthesis, respiration, cell division, and enlargement. Plants take P in H2PO4 <sup>−</sup> or HPO4 <sup>−</sup><sup>2</sup> forms from the soil solution. Application of phosphatic fertilizers in a balanced amount and at the correct time with good application techniques and management methods has good impacts on crop yield. However, responses to fertilization can be species and variety-dependent, which greatly influences nutrient accumulation and utilization in the plant [8,9]. Organic Fertilizer addition to soil increases risk of xenobiotic contamination [10–13].

Phosphorus deficiency is often a yield-limiting factor in agricultural soils, particularly in those having high carbonate contents, which reduces phosphorus solubility. In these conditions, achieving a target crop productivity generally demands the use of higher fertilizer rates as a way to account for that increased inefficiency. Besides being expensive [14], use of supra-optimum rates of chemical fertilizers in recent years has been frequently pointed as the reason behind in reduction in organic substances found within the soil [15]. Moreover, excessive usage of chemical fertilizers in agriculture has caused environmental issues like biological processes, physical destruction of the soil and nutritional imbalances [16]. In addition, research shows that increased corn and bean overall yields and quality can be obtained by using organic and chemical fertilizers simultaneously, which in turn aids to reducing the use of chemical fertilizers and improving soil health and overall sustainability [17].

Several studies reported that plant growth and development are greatly related with the movement of specific organic fractions present in both the soil solutions (known as dissolved organic matter) and soil matrix (soil organic matter). These fractions have been defined as humic substances and include humic acids, fulvic acids and humin [1,18]. The advantageous activities of these humic substances in relation to crop production has been attributed to two main effects [19,20]: the indirect effect on soil properties and fertility, related to the ability of these substances to form complexes or chelates with soil metals [14], which impacts soil structure, texture and nutrients availability [19]. While the direct regulating growth effect on plant hormones such as auxin, ethylene, nitric oxide, cytokinins, abscisic acid and reactive oxygen species [20].

Humic acid (HA) is an active ingredient of humus that can play an essential role in improved soil health and plant growth. Physically, it provides good soil structure and enhances the water holding capacity of the soil; biologically it enhances the growth of beneficial soil organisms, while chemically it acts as an adsorption and retention complex for inorganic plant nutrients [21]. Humic acid cannot be only found in soils, but also in peats, rivers, oceans and lignitic coals and can result from the biological decomposition and of organic matter. Humic substances can change the unavailable elements into available forms and can rupture Fe or Al bonded P in acidic soils and Ca in calcareous soils, rendering more soil P to be available for plant uptake. Humic acid can make complexes with Na, K, Mn, Zn, Ca, Fe, Cu and with a variety of other elements to overcome a particular element shortage in the soil. Thus, under certain conditions, the use of HA and its concomitant stimulating effect on various crops has received considerable attention [22]. Chemical composition of HA varies depending on the source and edaphoclimatic conditions where it was formed, but average HA composition contains 51–57% organic C, 4–6% N and 0.2 to 1% P that can be used both for plant nutrition and for improving soil physicochemical and biological parameters [21]. Additionally, HS was found to have a marked effect on the emergence of lateral roots, and the hyper induction of sites for lateral root emergence [23]. Research shows that the effect of Humic substances (HS) on plant growth depends on the source, concentration and molecular weight of humic fractions [24,25]. That is why the present study was

conducted to evaluate the role of locally produced HA in enhancing P availability and wheat growth in calcareous soil amended with different P levels as SSP.

### **2. Materials and Methods**

### *2.1. Experimental Procedure*

A field study was conducted over two years, to investigate the effect of different levels of single superphosphate applied alone and in combination of HA on growth, yield and phosphorus uptake by wheat crop at the Research Farm of the University of Agriculture, Peshawar-Pakistan. The experimental farm is located at 34.01◦ N latitude, 71.35◦ E longitude at an altitude of 350 m above sea level in Peshawar valley. The soil in the experimental site was a silt loam, alkaline calcareous, low in organic matter and P contents (Table 1) and was also highly responsive to P application [26]. Treatments included the application of P at 0, 45, 67.5, 90 and 112.5 kg P2O5 ha−<sup>1</sup> applied as Single Superphosphate (SSP)without and with the HA addition (i.e., 0 and 5 kg HA ha−1). Humic acid was extracted from brown coal collected from Hyderabad, Pakistan in the laboratory of Soil and Environmental\_Sciences, The University of Agriculture Peshawar, Pakistan by following the procedure of Hai and Mir [27]. The experiment was arranged as a two factorial [5 (P levels) and 2 (HA levels)] randomized complete block design (RCBD) with 3 replications. The required P fertilizer along with half recommended levels of N (120 kg ha<sup>−</sup>1) and a full dose of K as 60 kg K2O ha−<sup>1</sup> were applied as urea and sulphate of potash respectively, before planting. The remaining half rate of N was applied with the first irrigation. Wheat (*Triticum aestivum* L.) variety "Atta Habib" having a seed rate of 100 kg ha−<sup>1</sup> was sown through handrill with 30 cm row spacing in a plot size of 4 m × 5 m. All the agronomic practices and manual weed control were followed as per the standard advised procedure for all treatments uniformly. Plants were harvested at physiological maturity. The field was irrigated as per crop requirements.


**Table 1.** Characteristics of composite soil of the area and humic acid used in the experiment.

Data on plant height was determined by taking the height of five randomly selected plants from soil surface to the tip of each plant at physiological maturity in each treatment plot and then averaged. To determine the spike length, five spikes were randomly selected in each plot and their length was measured from base of rachis to the tip of uppermost spikelet. After that the spikes were threshed individually to determine grains per spike and their mean were taken as a grains spike−1. Thousand grains weight was recorded by counting and weighing thousand grains randomly taken

from each treatment plot. Grain yield was recorded after threshing of plants taken from central four rows in each treatment and then converted into kg ha−<sup>1</sup> by using the following formula:

$$\text{Grain yield} \,(\text{kgha}^{-1}) = \frac{\text{Grain yield in kg obtained from Arvested rows} \times 10000 \,\text{m}^2}{(\text{Row length} \times \text{Row spacing in meter} \times \text{No. of rows})}$$

Biological yield (BY) was measured by weighing the entire harvested crop (un-threshed crop i.e., both grain and straw) in each treatment. The BY was then converted in to kg ha−<sup>1</sup> with the following formula:

$$\text{Biological yield } (\text{kgha}^{-1}) \xrightarrow{\text{Biological yield in kg obtained from Arvested rows} \times 10000 \text{ m}^2} \text{ (1)}$$

Straw yield was calculated by subtracting grain yield from biological yield.

### *2.2. Samples Collection and Physicochemical Analysis*

Soil samples were taken from (0–30 cm) of the soil used in the field experiment and were prepared for some physical and chemical analysis. Soil properties changes with land use [28–30]. The whole plant samples (shoots + grains) were randomly taken from each treatment plot after harvesting and were dried at 70 ◦C until constant mass weight. Then, dried samples were grounded to pass a 1-mm screen, as suggested by Weidhuner et al. [30–32] and samples were thoroughly mixed and stored for analysis. Soil EC and pH were quantified in 1:5 soil water suspensions by the procedure of Rhoades [33] and Thomas [34], respectively. Organic matter (O.M.) contents in soil were determined by dichromate oxidation as described by Nelson and Sommers [35], AB-DTPA extractable and water-soluble phosphorus concentration was measured by the standard procedures of Soltanpour and Schwab [36]. The soil was also analyzed for lime content by adopting the procedure of Loeppert and Suarez [37] while soil texture was measured by the procedure of Gee and Bauder [38]. Plant P concentration and its uptake by wheat were determined by the protocol of Jones et al. [39]. Characterization of soil and HA is provided in Table 1.

### *2.3. Statistical Analysis*

Data were subjected to two way ANOVA analysis for the significance of treatment effects and means were separated using the least significant difference (LSD) test with significance set at *p* ≤ 0.05 using Staistix 2000 statistical package [40].

### **3. Results**

### *3.1. Plant Height (cm)*

Application of different levels of P and HA produced significantly (*p* ≤ 0.05) taller wheat plants than the plots where SSP without HA was applied. When averaged across the P levels, HA produced 84 cm taller plants as compared to 82 cm without HA. When average across the HA levels, the application of P levels at 90 kg P2O5 ha−<sup>1</sup> produced maximum taller plants of height 88 cm and the decrease in plant height at a higher level of P indicates that 90 kg P2O5 ha−<sup>1</sup> is the optimum dose in the given soil conditions. The interactive effect of HA and P levels was also significant. Maximum plant height 89 cm was observed where 90 kg P2O5 ha−<sup>1</sup> with 5 kg HA ha−<sup>1</sup> was applied, whereas a minimum of 77 cm was observed in control.

### *3.2. Spike Length (cm)*

Spike length as influenced by the different levels of P and HA is given in Table 2. When averaged across HA, different levels of SSP significantly increased the spike length as compared to control. On average the maximum spike length (10.77 cm) was recorded in the treatments applied with

112.5 kg P2O5 ha−<sup>1</sup> which was statistically higher than the spike length obtained at 67.5 and 45 kg P2O5 ha−<sup>1</sup> but not significant from the plots treated with 90 kg P2O5 ha−1. Application of HA also increased the spike length of wheat plants significantly, but the interactive effect of P and HA was non-significant with a range from 9.75 cm in control to 10.89 cm observed in treatments P at 90 kg P2O5 ha−<sup>1</sup> with 5 kg HA ha<sup>−</sup>1.

### *3.3. Grains Spike*−*<sup>1</sup>*

Data showed that both different doses of SSP and HA had a significant effect on the number of grains spike−<sup>1</sup> (Table 2). The number of grains spike−<sup>1</sup> significantly increased with each level of SSP over control. On average maximum, 58 grains spike−<sup>1</sup> were observed in plots treated with 112.5 kg P2O5 ha−<sup>1</sup> SSP that was statistically similar to those obtained at 90 kg P2O5 ha−<sup>1</sup> SSP, whereas a minimum of 51 grain spike−<sup>1</sup> was recorded in control. Application of HA also showed significant results with maximum grains spike−<sup>1</sup> (56) in the plots receiving 5 kg HA ha−<sup>1</sup> dose which was statistically higher than the plots treated without HA, while the interactive effect was non-significant with a maximum number of 60 grains spike−<sup>1</sup> obtained from the plots treated with 90 kg P2O5 ha−<sup>1</sup> with the application of HA.

### *3.4. 1000-Grain Weight*

Table 2 shows the 1000-grain weight as influenced by the application of different levels of SSP with and without HA. The data revealed the increase in grain weight with increasing levels of P fertilizer and HA. The non-significant interaction revealed that HA increased the grain size irrespective of P levels. Similarly, when averaged across the HA, the application of 112.5 P2O5 ha−<sup>1</sup> produced heavier grains amounting 46.05 g per 1000 seeds which was 25.64% heavier than control. When averaged across the P levels, application of HA produced heavier grains of 42.61 g which were statistically higher than the grain weight of the plots which receive no HA.

### *3.5. Grain Yield (kg ha*<sup>−</sup>*1)*

The addition of HA and P levels significantly increased the grain yield over control. Application of HA showed an additional advantage over the sole application of SSP by increasing the grain yield from 3% in control to 16% at 90 kg P2O5 ha−<sup>1</sup> suggesting an increase in P use efficiency with the application of HA with each increment in P level (Table 2). When averaged across HA, grain yield of 2947 kg ha<sup>−</sup>1was recorded in the plots treated with 90 kg P2O5 ha−<sup>1</sup> which was statistically higher than the grain yield obtained at 112.5, 67.5 and 45 kg P2O5 ha−<sup>1</sup> indicating that the 90 kg P2O5 ha−<sup>1</sup> could be the optimum level in the prevailing soil and climatic conditions for the wheat crop. The HA also showed significant results with grain yield of 2540 kg ha−<sup>1</sup> over no HA application with grain yield of 2338 kg ha<sup>−</sup>1.


 with same letters are not significantly different from each other. The ±values represent stander error of the mean (*<sup>n</sup>* = 3) while, ns stands for non-significant difference at *p* ≤ 0.05.

### *Agronomy* **2020** , *10*, 1224

32

### *3.6. Straw Yield (kg ha*<sup>−</sup>*1)*

Wheat straw yield as influenced by different levels of SSP with and without HA is presented in Table 2. With an increase in P level, the straw yield significantly increased over control except for 67.5 and 90 kg P2O5 ha−<sup>1</sup> which were statistically similar. The maximum straw yield of 4549 kg ha−<sup>1</sup> was recorded from the treatments applied with 112.5 kg P2O5 ha−<sup>1</sup> which was 71% higher than control. This increase signifies the function of P in crop growth and productivity in the tested soil and climatic conditions, however, the role of HA was found non-significant. The interactive effect of P levels and HA was also non-significant with maximum straw yield of 4607 kg ha−<sup>1</sup> was observed in the plots treated with 112.5 kg P2O5 ha−<sup>1</sup> and HA, while minimum (2534 kg ha<sup>−</sup>1) was in case of control.

### *3.7. Biological Yield (kg ha*<sup>−</sup>*1)*

Both the P levels, HA and their interaction significantly (*p* ≤ 0.05) increased the biological yield of wheat (Table 2). The interaction of P levels with HA exhibited that biological yield increases with increasing application of P but HA application further intensify such improvement. On average, each increment of P produced higher biological yield than the preceding lower dose indicating the role of P in increasing the crop growth in the given soil and climatic conditions. The maximum biological yield (7227 kg ha−1) was recorded in the plots treated with 112.5 kg P2O5 ha−<sup>1</sup> that was statistically higher than the plots applied with 90, 67.5 and 45 kg P2O5 ha−<sup>1</sup> (Table 3 and Figure 1A). The data regarding biological yield also revealed that the increase over control with 90 kg P2O5 kg ha−<sup>1</sup> and HA was 64% which was close to 62% increase observed with 112.5 kg P2O5 ha−<sup>1</sup> applied without HA suggesting that P application dose could be reduced with HA (Figure 1B). Similarly, when averaged across the P levels, the HA application produced 6322 kg biological yield ha−<sup>1</sup> on dry weight basis which was significantly higher than the plots which does not receive HA advocating the increasing role of P with HA. A remarkable percent increase of 6.2%, 5.2%, 7.9% and 5.4% in biological yield was observed with the addition of HA and P levels over the sole application of P levels.


**Table 3.** Effect of different levels of phosphorus and humic acid on post-harvest soil properties of wheat under field conditions of Peshawar, Pakistan.

The mean while tanders Means with same letters are not significantly different from each other. OM and WSP stand for organic matter and water soluble P respectively. The ± values represent stander error of the mean (*n* = 3).

**Figure 1.** Comparative increase in biological yield of wheat after the application of SSP with and without HA; observed values (**A**) and increase over control (**B**).

### *3.8. Harvest Index (%)*

Application of different P levels significantly affect the harvest index of wheat crop, where the HA addition and interaction of P levels and HA non-significantly affect the harvest index of wheat plants (Table 2). When averaged across the HA levels, maximum harvest index of 43% was observed in the plots that received 90 kg P2O5 ha<sup>−</sup>1, whereas lowest harvest index of 36% was noted in plots that receive 112.5 kg P2O5 ha−<sup>1</sup> in the form of SSP.

### *3.9. Plant Phosphorus Concentration (g kg*<sup>−</sup>*1)*

Both P levels and HA showed significant effects and increased plant phosphorous concentration (Table 2). The application of 112.5 kg P2O5 ha−<sup>1</sup> enhanced the plant shoots [P] in leaves with a value of 3.3 g kg−<sup>1</sup> which was statistically higher than other treatments. On average maximum plant [P] of 3.1 g kg−<sup>1</sup> was observed in the plots applied with 5 kg HA ha−<sup>1</sup> which was statistically higher than 2.9 g kg−<sup>1</sup> observed in plots that receive no HA. The interactive effect of P levels with HA was non-significant with maximum P concentration of 3.4 g kg−<sup>1</sup> in plots where 112.5 kg P2O5 ha−<sup>1</sup> was used with HA, while the minimum of 2.5 g kg−<sup>1</sup> was noted in control plots.

### *3.10. Phosphorous Uptake by the Plant (kg ha*<sup>−</sup>*1)*

Regarding the P-uptake of wheat plants, the addition of different levels of SSP with HA showed superior results over sole SSP application (Table 2). When averaged across the HA, maximum P uptake of 24.30 kg ha−<sup>1</sup> was noted in plots that were treated with 112.5 kg P2O5 ha<sup>−</sup>1, followed by 21.41 kg ha−<sup>1</sup> in plots received 90 kg P2O5 ha<sup>−</sup>1, whereas, the minimum was noted in control plots. When averaged across P levels, the maximum P uptake of 19.97 kg ha−<sup>1</sup> which was 13% higher than uptake in plots treated without HA (Figure 2A). Regarding different P levels, addition of HA increases the P uptake from 10.3 to 21.2% as compared to plots where P levels were applied without HA. The interaction of P levels and HA was non-significant with the maximum uptake of 25.50 kg ha<sup>−</sup>1observed with 112.5 kg P2O5 ha−<sup>1</sup> applied with HA. The total uptake of P at 90 kg P2O5 ha−<sup>1</sup> applied with HA (23.33 kg ha−1) was similar to uptake of 23.10 kg ha−<sup>1</sup> at 112.5 kg P2O5 ha−<sup>1</sup> without HA suggesting the increase in P use efficiency and reduction in P requirements for optimum crop growth and yield. Similarly, the percent increase over control with HA-SSP at 67.5 kg ha−<sup>1</sup> was 90.5% that was more than the percent increase over control at 90 kg SSP alone (75.4%) confirming the fact that P use efficiency increased with HA application (Figure 2B).

**Figure 2.** Comparative increases in plant P uptake after the application of SSP with and without HA; observed values (**A**) and Increase over control (**B**).

### *3.11. Soil Organic Matter*

Soil organic matter (SOM) content was significantly affected by P levels, HA and their interaction. Treatments receiving SSP and HA had significantly (*p* ≤ 0.05) higher organic matter than sole SSP levels at each increment of P levels from 45 to 112.50 kg P2O5 ha−<sup>1</sup> (Table 3). When averaged across P levels, the HA had 1.11% organic matter which was significantly higher than 0.95% calculated for plots received no HA. Similarly, when averaged across the HA, the maximum soil organic matter contents of 1.16% was observed in plots treated with higher doses of SSP of 112.5 kg P2O5 ha−<sup>1</sup> that was statistically identical to1.13% observed in the plots treated with 90 kg P2O5 ha−<sup>1</sup> but higher than other treatments and control. The interactive effect of HA and SSP was significant with the highest organic matter contents of 1.24% in plots treated with 112.5 kg P2O5 ha−<sup>1</sup> and HA, and the lowest of 0.82% was noted in control. It was evident from the interaction (HA\*P) that SOM increased with the application of HA regardless of P rate except in control and plots treated with 45 kg P2O5 ha−<sup>1</sup> where SOM content was similar for with and without HA treated plots. The organic matter content observed for 112.50 kg P2O5 ha−<sup>1</sup> without HA was at par to 67.5 kg P2O5 ha−<sup>1</sup> with HA which were significantly lower than plots amended with 90 kg P2O5 ha−<sup>1</sup> with HA. Similarly, OM observed at 45 kg P2O5 ha−<sup>1</sup> with HA was statistically similar to that observed for 67.5 kg P2O5 ha−<sup>1</sup> without HA. These finding suggested that, HA application can preserved soil organic matter content when applied with SSP.

### *3.12. AB-DTPA Extractable Phosphorus*

Results showed that treatments receiving HA + SSP had significantly (*p* ≤ 0.05) more soil AB-DTPA extractable P than SSP alone at each increment of P levels from 45 to 112.50 kg P2O5 ha−<sup>1</sup> (Table 3). When averaged across the P levels, HA-SSP produced 7.61 mg P kg−<sup>1</sup> which was significantly higher than 6.12 mg P kg−<sup>1</sup> observed in no HA plots. Addition the SSP with HA increased the post-harvest soil AB-DTPA P over alone SSP levels with a mean value of 24.3%. Similarly, when averaged across the fertilizer HA, the AB-DTPA extractable P increased with each increment of P. The mean maximum AB-DTPA ext. P of 8.68 mg kg−<sup>1</sup> was recorded in treatments applied with 112.5 kg P2O5 ha−<sup>1</sup> that was statistically higher than the phosphorous content of other treatments. The minimum AB-DTPA ext. P with a value of 5.05 mg kg−<sup>1</sup> was observed in control. The interactive effect of SSP and HA was also significant showing increases with an increase in P levels. The maximum AB-DTPA ext. P of 9.23 mg kg−<sup>1</sup> was recorded in plots receiving 120 kg P2O5 ha−<sup>1</sup> as HA + SSP while the minimum of (4.20 mg kg<sup>−</sup>1) was recorded in control.

### *3.13. Water-Solublephosphorus*

Analysis of variance (ANOVA) revealed that P levels, humic acid and their interaction significantly affected soil water soluble P. Water-soluble phosphorus significantly (*p* ≤ 0.05) increased with HA and P levels (Table 3). HA treated plots, when averaged across P levels, maintained higher water-soluble P of (0.201 mg kg<sup>−</sup>1) than plots receiving no HA which had 0.165 mg kg−1water-soluble P, on an average basis. The percent increase in water-soluble phosphorous with HA+SSP levels over respective sole P levels ranged from 6.7% in control to 34.3% in treatments receiving 112.5 kg P2O5 ha−<sup>1</sup> suggesting higher release from HA+SSP than commercially available SSP. The interactive effect of HA and SSP was significant with maximum water-soluble P of (0.266 mg kg<sup>−</sup>1) observed in 112.5 kg P2O5 ha−<sup>1</sup> as HA-SSP while a minimum of (0.135 mg kg−1) was recorded in control. The significant interaction of HA\*P demonstrated that, WSP increased with increasing P level, however, this increase was more in plot treated with 5 ton HA ha−<sup>1</sup> compared to control HA at respective P levels. Furthermore, with respect to WSP the response of 45 kg P2O5 ha−<sup>1</sup> with HA was significantly better than 67.5 kg P2O5 ha−<sup>1</sup> without HA and the performance of 67.5 kg P2O5 ha−<sup>1</sup> with HA was at par to 90 kg P2O5 ha−<sup>1</sup> without HA. It was also evident that 90 and 112.5 kg P2O5 ha−<sup>1</sup> with HA performed similar which was significantly superior than 112.5 kg P2O5 ha−<sup>1</sup> without HA. Thus, it can be deduced that, HA enhances P availability in soil amended with P as chemical fertilizers.

### **4. Discussion**

Application of P levels with humic acid (HA) produced significantly taller wheat plants than sole P application at each increment of P levels from 45 to 112.50 kg P2O5 ha−1, whereas the interactive effect of P and HA was also significant. The increase over control with 67.5 kg P2O5 kg ha−<sup>1</sup> and HA was 10.4% which was close to 11.7% increases observed with the application of 90 kg P2O5 ha−<sup>1</sup> alone indicating that P application dose could be reduced with HA. Such differential increases revealed that P use efficiency was increased with HA over sole application of P as commercial SSP fertilizer and as such could reduce the farmer input cost without compromise on yield and crop productivity. These results revealed that the linear increase in plant height could be achieved with P increased levels and HA applications. Ahmad et al. [26] also suggested that plant height can be increased with a higher P rate application. Khattak and Dost [41] also reported the increase in plant height with HA which has suggested that the application of HA with different fertilizers may cause beneficial effects on plant growth and nutrients uptake. This could be associated with the capability of HA to improve the biochemical environment of soil by promoting soil enzymatic activities, microbial activities and population, cation exchange capacity and water retention of soil that ultimately enhance the plant growth and nutrients uptake. These results were also in accordance with Tahir et al. [42] who reported that the application of HA significantly improved the plant height. The application HA with SSP improved the quality of wheat produced as indicated by grain size and weight. Combine application of SSP and HA produced higher 1000-grain weight than SSP alone at each P level, while their interaction remained non-significant at *p* ≤ 0.05. These results advocated the role of P in increasing the size and quality of seed in the present study. These results were similar to the study of Kaleem et al. [9] who reported that maximum phosphorus dose enhanced the number of grains spike−1, tillers number, thousand-grain weights and grain yield due to the highest accumulation of photosynthates in the plants and increased grain ripening which resulted in heavier grains. The results were also in close consistency with the findings of Ibrahim et al. [43] who stated that wheat 1000 grains weight could be increased significantly with the combined application of chemical and organic fertilizers. Wheat spike length and grains per spike showed almost similar trend and an increase was observed with increasing P and HA levels. Addition of HA with all P levels showed increased spike length except 112.5 kg P2O5 ha−<sup>1</sup> which showed a decrease of 1.11%. This negative increase may be associated with the imbalance in plant nutrients caused by increased concentration of phosphorus. It is an established fact that higher doses of one nutrient can have detrimental effect on the absorption of others and as such reduce the crop performances. The results also depicted that balance ratio of P fertilizers is essential to

obtain higher yield of wheat against the common farmer's practice in the area who do not bother to keep in mind the balance of different fertilizers at the time of sowing [9]. These improvements revealed that application of P with HA improved the storage of photosynthates in the plants. This accumulation of photosynthates in plants enhances the enzymatic, microbial and catalytic activities in plants and thus produces higher grains spike<sup>−</sup>1, grain yield, straw yield and biomass as well [9]. Results regarding the grain yield showed that the percent increases of 41.7% with HA and 67.5 kg P2O5 ha−<sup>1</sup> is closely resembled to 40.9 and 51% obtained with SSP alone at 90 and 112.5 kg P2O5 ha<sup>−</sup>1, respectively, revealing a reduction in P requirements and increase in P fertilizer use efficiency with the addition of HA. A similar effect was noted by Khattak and Dost [41] who stated that the combined use of fertilizers and HA could increase the yield of different crops and reduce the crop fertilizer requirements without compromise on yields and quality. The substantial increase in wheat yield over control with SSP and HA suggested its potential use as an effective P fertilizer. The increase in grain yields with P levels is a well-established fact in P deficient soils. The HA would have increased the P availability by making its soluble complexes and stimulating plant growth [24] as well as by providing a good physicochemical and biological environment of the soils to the plants [21]. Proliferation of rhizobacteria also played an imperative role in better uptake of phosphorus when applied in the presence of organic carbon [44–47]. Improvement in straw and biological yield revealed that a significant increase in straw and biological yield could be obtained by the application of phosphorus and HA [27]. The increase in growth parameter and yield may be related with the stimulating effect of P and HA on plant growth by the assimilation of major and minor elements, enzyme activation and/or inhibition, changes in membrane permeability, protein synthesis that ultimately enhances the biomass or biological production [48,49]. The results were also in consistence with Sarir et al. [50] and Sharif et al. [51] who reviewed that HA application can improve biological yield up to a prominent level. Data regarding the P concentration in plant shoots suggested that the application of SSP with HA increased the plant P concentration as 3.5, 10.4, 6.9 and 6.3% with 45, 67.5, 90 and 112.5 kg P2O5, respectively, over the same levels of SSP alone indicating that HA increased the P use efficiency and uptake of plants. The results of the study were similar to the study of Khattak and Dost [41] who reported that HA increased the plant growth and nutrients uptake capability through the improvement of soil enzymatic system and microbial activities and population ultimately making the soil conditions favourable for plant uptake. The results of the study can also be supported by the results of Majumdar et al. [52] who stated that the application of rock phosphate mixed with different organic manures could increase P concentration in plants significantly. The higher P concentration in plant leaves with the application of HA along with SSP was in line with the study of Cooper et al. [53] and Atiyeh et al. [54]. They stated that the addition of HA in the soils enhanced the root growth as well as the proliferation, branching, and initiation of root hairs and thus able the roots towards more nutrient capturing and increase nutrient concentrations in the plants. A lot of studies showed an increase in root length, root number and root branching with the application of HA. However, the increase in root growth is generally more noticeable than shoot growth [55]. Pettit [56] also reported a prominent increase of root initiation and increased root growth with the application humic and fulvic acids to the soil ultimately increasing the nutrient concentrations in plant tissues. The uptake of P indicated that the observed increases in wheat growth and yield with SSP levels and HA in the present study. The increase in P uptake with increasing P levels is an accepted fact [57] and the additional advantage with HA is in consistence with the findings of Erdal et al. [55] who stated a prominent accumulation of nutrients in the plant with the application of organic materials along with mixing of inorganic fertilizers. These results can also be supported by Sharif et al. [51] who reported that P and N uptake could be increased with the addition of P fertilizers (SSP) along with organic materials. The results regarded the soil organic matter contents indicated that both SSP and HA could improve the soil organic matter content. The improvement in the organic matter could be attributed to higher biomass and bumper root growth and as such more leftover fraction as also indicated by close resemblance between plant biomass and soil organic matter. These results are in line with the findings of Sharif et al. [51] who reported that the use of inorganic fertilizers along with organic

fertilizers (humic acid and FYM) increased soil organic matter content. Similar findings were reported by Tamayo et al. [58] and Han et al. [59], who stated an increase in soil organic carbon with the use of chemical fertilizer along with the organic. The data regarding the AB-DTPA extractable P revealed that the increase over control with 67.5 kg P2O5 kg ha−<sup>1</sup> and HA was 83% which was more than 70% increases observed with 90 kg P2O5 ha−<sup>1</sup> applied alone suggesting that P application dose could be reduced with the HA. The results of the study are similar to the findings of [60] who reported that the application of HA to acidic and alkaline soil decreases the P complex formation and dissolves the insoluble and unavailable P thus enhance the availability of phosphorus to the plants. Similar results were reported by Sharif et al. [51] and Majumdar et al. [52] who stated that phosphorus concentration could be increased with the application of rock phosphate (RP) along with the mixing of different organic materials. An increase in water-soluble P is related to the accessibility of phosphorus added to the soil as well as with HA which decreases the P fixation and provides more water-soluble P for plants. These results were in uniformity with the findings of several researchers [26,61–65]. They reported that the supplementation of P from various P sources including rock phosphate and HA application increased the soil solution P whereas the high pH and lime contents in calcareous soils reduced it by making its insoluble complexes. Like other organic materials, the HA make soluble complexes with P and increase its concentration in the soil solution. However, plant and microbial exudates neutralized the rhizosphere by producing (H+) as a result of cation uptake can increase the availability of P in the soil.

### **5. Conclusions**

Application of P as SSP fertilizer and humic acid (HA) significantly improved wheat growth, yields, P uptake and post-harvest soil AB-DTPA extractable and water-soluble P contents. The interactive effect of P and HA were found significant for plant height, grain and biological yield and soil AB-DTPA extractable P and K. The significant superiority of SSP and HA over sole SSP at almost every application rate suggested improvement in P fertilizer use efficiency with HA. The grain yield obtained at 67.5 kg P2O5 ha−<sup>1</sup> with HA was statistically comparable to 112.5 kg P2O5 ha−<sup>1</sup> applied as commercial SSP suggested that the input expenditures of fertilizer may be reduced up to 50% by combine application of chemical fertilizers with HA. Similarly, HA with SSP maintained higher AB-DTPA extractable, water-soluble P, soil organic matter contents, plant P concentrations and P uptake over commercial SSP in the soil, which further signifies the importance of HA in enhancing P availability.

**Author Contributions:** Formal analysis, A.K.; Investigation, S.F.; Methodology, F.W.; Project administration, Z.Y.; Software, S.D. and R.D.; Supervision, Z.Y.; Validation, S.D.; M.Z.-u.-H.; and M.B.; Writing—original draft, M.A.; Writing—review & editing, M.I.S., S.F., M.B. and R.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** "This work was supported by the 1. Program for "Shandong Provincial Natural Science Foundation of China (ZR2018BC012)". 2. Project of Technology Agency of the Czech Republic "TH02030169".

**Acknowledgments:** The authors would like to acknowledge the financial support of the Shandong Provincial Natural Science Foundation of China for this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Solubility and E**ffi**ciency of Rock Phosphate Fertilizers Partially Acidulated with Zeolite and Pillared Clay as Additives**

### **Ana Paula Bettoni Teles, Marcos Rodrigues and Paulo Sergio Pavinato \***

College of Agriculture Luiz de Queiroz—ESALQ-USP, Av. Pádua Dias, 11, Piracicaba-SP 13418-900, Brazil; ana.bettoni@usp.br (A.P.B.T.); rodrigues.m@alumni.usp.br (M.R.)

**\*** Correspondence: pavinato@usp.br; Tel.: +55-19-3417-2136

Received: 24 April 2020; Accepted: 24 June 2020; Published: 27 June 2020

**Abstract:** Soluble phosphates are the most common sources currently used in crop production in tropical soils; however, they present low efficiency and are more expensive than natural rock phosphates. The objective was to develop new phosphate fertilizers with slow solubility through the partial acidification of rock phosphates (RPs), incorporating materials with adsorption characteristics to favor slow dissolution and prevent phosphorus (P) fixation in the soil. Three rock phosphates, Araxá (ARP), Bayovar (BRP) and Morocco (MRP), were evaluated at two acidulation levels (25 and 50% Ac.) and two additives; pillared clays (PILC) and zeolites (Zeo), plus triple superphosphate (TSP) and a control (nil-P). The soil diffusion was evaluated in concentric rings in Petri dishes. Solubility was evaluated in leaching columns and sampled in layers from surface for P forms in the soil profile. The relative agronomic efficiency (RAE) was evaluated in maize. Greater diffusion was provided by TSP, followed by BRP and MRP both with 50% Ac. + Zeo, and MRP with 50% Ac. + PILC. Percolated P was more pronounced under TSP, followed by RPs (BRP and MRP) with 50% Ac. + Zeo. BRP and MRP + 50% Ac. were the most promising sources with RAE above 74% compared to TSP.

**Keywords:** phosphorus sources; P solubilization; P acidulation; relative agronomic efficiency

### **1. Introduction**

Phosphorus (P) plays an important role in plant metabolism, since it is involved in processes such as cell energy transfer, respiration and photosynthesis [1], making it an essential and irreplaceable element. Plants absorb P from the soil solution as phosphate ions, mainly H2PO4 − [2]. However, soils usually have low levels of plant-available P, especially in tropical regions. This is a result of adsorption and precipitation reactions, and its high affinity with soil constituents [3]. Given this limited P availability, agricultural production is highly dependent on the use of fertilizers.

Phosphate fertilizers are produced from rock phosphate (RP), a natural non-renewable resource. About 80% of the RP mined annually is used for fertilizer production and, considering the current level of consumption, it is expected that reserves will vanish in three centuries time [2,4]. The possibility of exhaustion of this resource may compromise global food production [5]. The most used phosphate sources in agriculture are those that are highly water soluble, with fast dissolution in the soil which favors precipitation and adsorption. Approximately three days after the application of these sources in the soil, a large part of their P (more than half in some cases) is transformed into non-labile forms [6,7], substantially reducing their efficiency when applied to crops. Therefore, the future availability of P depends on the development of new technologies or soil management practices to improve its efficiency.

Considering this reference to the motivation to improve P efficiency, partial acidulation of RP is a technological development already in existence [8] that can be of help. In the processing of partially acidulated phosphates, a small amount of sulfuric or phosphoric acid reacts with RP in order to breakdown part of the hydroxyapatite (insoluble P) into monocalcium phosphate (soluble P), and thereby obtain a fast dissolution product [9]. Moreover, the incorporation of minerals with high phosphate adsorption capacity and/or high cation exchange capacity (CEC), such as pillared clays (PILC) and zeolites, into partially acidulated phosphates shows promise to improve agronomic efficiency [10,11]. The premise is that the initial P release will saturate the acidic sites of the PILC before adsorption, which act as a vehicle for slow and gradual dissolution into soil solution. Furthermore, when calcium (Ca) from RP is released after dissolution, it can be held by negative charges of the PILC or zeolites, favoring the solubilization of acid-unreacted RP, which also prevents Ca-P retrogradation [10,11].

Thus, we hypothesized herein that new phosphate fertilizers with slower and more synchronized solubility according to plant demand are more efficient than completely acidulated commercial sources. This study aimed to develop new crop efficient sources of phosphate fertilizers with gradual solubility using partial acidulation (25 and 50% of total solubilization) in the following distinct rock phosphates—Araxá (ARP), Bayovar (BRP) and Morocco (MRP)—and also adding high reactivity minerals such as pillared clay (PILC) and zeolites (Zeo), thus enabling the evaluation of the potential pH change in the soil, soil P diffusion; P solubilization and P agronomic efficiency.

### **2. Materials and Methods**

### *2.1. Phosphate Rocks and Additives*

Two high-reactivity sources of RP (Bayovar from Peru and BG4 from Morocco) and one low-reactivity source (Araxá from Brazil) were used as raw material for the production of partially acidulated phosphate fertilizers. Their chemical parameters and composition and total P2O5, 2% citric acid soluble P (PCA 2%) and water soluble concentrations of each RP are presented in Table 1. Additionally, zeolites and pillared clay (PILC) were used as additives in the phosphate fertilizers for improving the dissolution, also characterized in Table 1.

**Table 1.** Chemical characteristics of the rock phosphates (RPs) used and total chemical composition by X-ray fluorescence, cation exchange capacity (CEC) and maximum P adsorption capacity (MPAC) of pillared clays and zeolites.


<sup>1</sup> Determined according to the USDA-NRCS [12]; <sup>2</sup> determined according to Farkaš et al. [13]; <sup>3</sup> determined according to Alvarez et al. [14].

Natural zeolite, a mineral composed of intertwined tetrahedra, was obtained from Slovakia, provided by the Celta Brazil company. The zeolites were ground and passed through a 60 mesh sieve to increase the specific surface area. The clays used for pillarization came from a natural rock rich in montmorillonite (bentonite), supplied by Bentonit União Nordeste S.A., commercially known as Brasgel. For the pillarization process, the bentonite was finely milled, fractionated and purified in order to eliminate its coarser fractions and decrease its impurities. Purification of the material was carried out by removing iron oxides following the dithionite-citrate-bicarbonate method [15]. Next, the material

was dispersed with Na2CO3 0.1 g L−<sup>1</sup> solution with continuous stirring for approximately 12 h and fractionated by sedimentation to obtain the clay fraction (<2 μm).

The purified clay was pillared based on the methodology described by Narayanan and Deshpande [16]. The pillaring solution was obtained by using constant dripping of 0.4 mol L−<sup>1</sup> NaOH in a solution of 0.2 mol L−<sup>1</sup> AlCl3·6H2O under constant stirring at room temperature. At the end of the drip, the solution remained under stirring for a period of 15 h, with the first two hours at a temperature of 60 ◦C and the remaining time at room temperature (25 ◦C). For clay intercalation, the pillaring solution was then dripped at a maximum rate of 5 mL min−<sup>1</sup> in a clay suspension of 1% (*w*/*w*), under vigorous stirring. After this, the material was continuously stirred for a period of 20 h. The resulting product was washed with deionized water to remove all free chlorine, oven dried at 60 ◦C and calcinated at 350 ◦C. In order to confirm pillarization, the final product was submitted to X-ray diffractometry (XRD), in a Miniflex II Desktop X-Ray Diffractometer Rigaku apparatus, with CuKα radiation, using the powder blade method. When saturated with sodium, the natural clay had a basal spacing of 1.4 nm. After the intercalation of aluminum polyhidroxication (pillarization) and calcination at 350 ◦C, it was observed that the clay reached a basal space of 1.8 nm, evidencing that the pillarization had been effective (Figure 1).

**Figure 1.** X-ray diffractograms of natural clay and pillared clay after calcination at 350 ◦C.

### *2.2. Fertilizer Production*

Both minerals, PILC and zeolites, were mixed with each RP in a proportion of 9% of the final volume, followed by partial acidification with sulfuric acid corresponding to 25 and 50% of the proportional commercial acidulation of soluble phosphates, such as simple superphosphate. The resulting mixture was cured for seven days, then oven dried, ground and passed through a 60 mesh sieve and granulated in a wet dish granulator, which consisted of the application of a dextrin-based binder solution (10% *w*/*v*) to the dry blend to obtain the granules. The final products were oven dried at 45 ◦C until reaching a constant weight and then standardized into granules of 2–3 mm diameter.

### *2.3. Chemical Analysis*

Twelve new products were obtained with the combinations mentioned above, which were characterized in terms of total P2O5 extracted with concentrated nitric acid + hydrochloric acid, and P2O5 soluble in water (H2O), soluble in neutral ammonium citrate + water (NAC + H2O) and soluble in 2% citric acid (PCA), according to the methodologies of the Brazilian Ministry of Agriculture, Livestock and Supply [17] (Table 2).

**Table 2.** Characterization for the total P2O5, water soluble, neutral ammonium citrate + water (NAC + H2O) and 2% citric acid (PCA 2%) soluble P of phosphate fertilizers partially acidulated plus the additives pillared clay (PILC) and zeolite (Zeo).


ARP = Araxá rock phosphate; BRP = Bayovar rock phosphate; MRP = Morocco rock phosphate.

### *2.4. X-Ray Di*ff*raction Analysis*

All the final products (phosphate fertilizers) were characterized in terms of the mineralogical composition and structural changes that occurred in the RPs after acidification by means of X-ray diffraction (XRD). For this purpose, the powder blade method was used and the diffractograms were generated using the Miniflex II Desktop X-Ray Diffractometer Rigaku, with CuKα radiation, with an analysis interval of 5◦ to 60◦ 2θ.

### *2.5. Phosphorus Di*ff*usion in Petri Dishes*

Phosphorus diffusion from fertilizer granules was evaluated in plastic Petri dishes (8.6 cm in diameter and 1.1 cm tall) containing 78 g of dry soil with four replications, following a methodology described by Degryse and McLaughlin (2014) [18]. The soil was a loamy sand Hapludox [19] or Latossolo Vermelho Amarelo distrófico according to the Brazilian classification system [20]. In order to increase the soil base saturation to 70%, CaCO3 and MgCO3 were applied at a ratio of 3:1, respectively. The soil properties after liming are listed in Table 3.

Soil in Petri dishes was moistened with deionized water up to 60% of the soil water holding capacity. Plates were then sealed with plastic film, covered with aluminum foil and left to equilibrate the soil solution for 24 h at 26 ◦C. The next day, they were opened and a granule of each fertilizer, corresponding to each treatment (Table 2) containing about 5.0 mg P, was placed exactly in the center of the plate, and lightly pressed into the soil. Next, they were again sealed with plastic film and covered with aluminum foil to prevent water loss and light incidence. They were then incubated at a temperature of 26 ◦C, for five weeks. One no-P fertilizer treatment and one treatment containing triple superphosphate (TSP) were also incubated as controls. Each treatment was replicated four times.

After incubation, Petri dishes were dismantled and the soil was sampled in concentric circles around the granule. The radii of the soil layers sampled were 0–7.75; 7.75–13.5; 13.5–25.5 and 25.5–43 mm, starting from the center (granule). Samples were dried at 40 ◦C and sieved (<2 mm) to determine the total and available P and the pH in water (ratio 1:10). Total P was determined by acid digestion with H2SO4 + H2O2, following the methodology proposed by Olsen and Sommers [21]. Available P was determined by anion exchange resin (membranes), following the first step of the procedure proposed by Hedley et al. [22]. Concentration of P in the extracts was colorimetrically determined by the blue-molybdate method [23].


**Table 3.** Chemical, physical and mineralogical characteristics of the soil used in diffusion study.

MPAC = maximum phosphorus adsorption capacity; CBD = citrate-bicarbonate-dithionite; Fed and Ald = iron and aluminum, respectively, extracted by the dithionite-citrate-bicarbonate method; Feox and Alox = iron and aluminum, respectively, extracted by the acid ammonium oxalate method; BS = base sum; CEC = cation exchange capacity; V = base saturation.

### *2.6. Soil Columns P Solubilization*

Fertilizer solubility was evaluated in leaching thermoplastic acrylic columns with an internal diameter of 2.1 cm and a height of 25 cm. Nylon caps were fitted to the bottom of the column with a hole in the center where plastic hoses were attached to collect leaked water. Above each column, 300 mL bottles were attached, adapted to drip water constantly, controlling the flow. Fifty grams of dry soil were added to each column equivalent to reaching up to nearly 10 cm in height in the column. Original soil properties are presented in Table 4. Next, it was saturated with distilled water. Subsequently, fertilizers corresponding to each treatment were added at 100 mg P per column to the top soil surface. The treatments evaluated herein were: T1–12 (Table 2) as well as pure phosphates ARP(T13); BRP(T14); MRP(T15); TSP(T16) and a control (T17). Deionized water was percolated through the columns at a rate of 20 mL day−<sup>1</sup> in the first 25 days. After that, the same amount was used every three days until 60 days had elapsed. The content of P was determined in leachates by the blue-molybdate method, following Murphy and Riley [23].



MPAC = maximum P adsorption capacity; CBD = citrate-bicarbonate-dithionite; Fed and Ald = iron and aluminum, respectively, extracted by the dithionite-citrate-bicarbonate method; Feox and Alox = iron and aluminum, respectively, extracted by the acid ammonium oxalate method; OM = organic matter; BS = base sum; CEC = cation exchange capacity; m = aluminum saturation; V = base saturation.

At the end of the incubation period, the columns were disassembled and the soil sampled in the following layers of 0–1; 1–2; 2–3; 3–6 and 6–10 cm, starting from the top. Samples were oven dried at 40 ◦C and sieved (<2 mm) and chemical P fractionation was performed according to the methodology proposed by Hedley et al. [22], with modifications by Condron et al. [24]. The last extractor (0.5 mol L−<sup>1</sup> NaOH), because of lack of interest for our purpose, was skipped. The P concentration in extracts was determined by the blue-molybdate method [23]. The compartments estimated with the respective fractions were as follows: labile, which includes the inorganic P extracted by anion exchange resin (PiAER) plus inorganic and organic P extracted by 0.5 mol L<sup>−</sup><sup>1</sup> NaHCO3 (PiBIC and PoBIC); moderately labile, which includes the inorganic and organic P extracted by 0.1 mol L−<sup>1</sup> NaOH (PiHid0.1 and PoHid0.1) more inorganic P extracted by HCl (PiHCl); and non-labile, composed of the residual acid digestion (Presidual).

### *2.7. Plant Growth-Pot Experiment*

The agronomic efficiency of the phosphate fertilizers generated in our lab was evaluated in a greenhouse pot study using a maize (*Zea mays* L.) hybrid 2B587 from Dow Seeds as a test crop. Soil and treatments used here were the same from the previously mentioned column P solubilization test. The experimental design was completely randomized with four replicates, in plastic pots with 3 L capacity, coated with plastic bags containing 3 kg of soil.

Phosphate treatments were added at the rate of 60 mg kg−<sup>1</sup> soil, based on the total P content of each fertilizer. The basic sowing fertilization for all pots consisted of 20 mg kg−<sup>1</sup> of N as ammonium nitrate (32% N) and 60 mg kg−<sup>1</sup> of K2O as polyhalite (14% of K2O) in a uniform hand mixture in the total soil volume. Thirty milliliters per pot of micronutrients solution were added containing: 0.81 mg kg−<sup>1</sup> B (H3BO3 p.a.), 1.56 mg kg−<sup>1</sup> Fe (Fe (NO3)2.9H2O p.a.), 3.66 mg kg<sup>−</sup><sup>1</sup> Mn (MnSO4.H2O p.a.), 4.0 mg kg−<sup>1</sup> Zn (ZnSO4.7H2O p.a.), 1.33 mg kg<sup>−</sup><sup>1</sup> Cu (CuSO4.5H2O p.a.) and 0.15 mg kg<sup>−</sup><sup>1</sup> Mo ((NH4)6Mo7O24.4H2O p.a.). Twenty days after sowing, this was complemented with 40 mg kg<sup>−</sup><sup>1</sup> of N as ammonium nitrate solution and another 30 mL per pot of the same micronutrient solution.

Each pot was sown with five maize seeds, later leaving the two best plants growing for 45 days. At the end, maize shoots and roots were harvested. Roots were washed in distilled water. Both plant parts were oven dried at 65 ◦C until constant dry mass (DM). After determining the DM of the shoot and root, the tissue was ground to determine the foliar P content through nitric-perchloric digestion [25] and to estimate accumulated total P.

The agronomic efficiency of the phosphate fertilizers was estimated in relation to the high water solubility commercial source (TSP), and therefore named, relative agronomic efficiency (RAE), obtained from the following equation:

$$RAE\_{\bar{i}} = (\Upsilon\_{\bar{i}} - \Upsilon\_{\bar{0}}\mathcal{Y}\_{TSP} - \Upsilon\_{\bar{0}}) \, \, ^\*100 \tag{1}$$

where *Yi* is the DM produced by source *i*, *YTSP* the DM produced by the commercial source (TSP) and *Y*<sup>0</sup> is the DM produced by the control treatment (no P addition).

### *2.8. Statistical Analysis*

All data were submitted to normality analysis (Shapiro–Wilk test) and homoscedasticity (Barlett's test) at 5% of error probability and then to variance analysis (ANOVA). The Scott–Knott test at 5% was used for comparisons between treatments. Statistical analysis was performed using the ExpDes statistical package [26] in the R computational statistical environment [27].

### **3. Results and Discussion**

### *3.1. Mineralogical and Structural Changes in Fertilizers*

X-ray diffraction was performed to visualize changes in the mineral structure and its arrangement in RPs after partial acidification and incorporation of PILC and zeolites. Notably, in pure phosphates (ARP, BRP and MRP), there was a dominance of apatite, with some quartz present only in MRP. After acidulation, the intensity of the apatite peaks and the appearance of calcium sulfates, such as gypsum and bassanite, were observed. In RPs treated by 25% acidulation, the decrease in apatite peaks was less intense than those treated by 50% acidulation. Furthermore, calcium sulfate peaks

became more intense under the highest acidification level, as a consequence of the sulfuric acid reaction (Figure 2). When the reaction was 100%, it was described as follows:

$$\text{Ca}\_{10}(\text{PO}\_{4})\_{6}\text{F}\_{2} + \text{7H}\_{2}\text{SO}\_{4} + 6.5\text{H}\_{2}\text{O} \rightarrow \text{3Ca}(\text{H}\_{2}\text{PO}\_{4})\cdot\text{H}\_{2}\text{O} + \text{7CaSO}\_{4}\cdot\text{1}\text{2H}\_{2}\text{O} + 2\text{HF} \tag{2}$$

When the amount of acid used was not sufficient to react 100% of the apatite, we obtained the so-called partially acidulated phosphates [9], as obtained in this study, and the apatite peaks did not disappear completely. Aside from this, the presence of zeolites or PILC as additives did not interfere in the phosphate peaks presented in diffractograms (Figure 2).

**Figure 2.** X-ray diffractograms of pure and acidulated RP (25 and 50% ac.) with the incorporation of pillared clay (PILC) or zeolite (Zeo), powder blades. ARP = Araxá Rock Phosphate; BRP = Bayovar Rock Phosphate; MRP = Morocco Rock Phosphate; PILC = pillared clay; Zeo = zeolite. Identified minerals: Ap = apatite; Qz = quartz; Gy = gypsum; Ba = bassanite.

The BRP diffractogram showed apatite peaks of lower crystallinity when compared to other pure phosphates (ARP and MRP). This is evidenced by wider peaks and lower intensity due to the occurrence of isomorphic substitutions of phosphate by carbonate in the mineral structure [28]. As a consequence, BRP was more susceptible to solubilization, with apatite peaks almost disappearing after its partial acidification, even at the lowest rate (25% ac.) (Figure 2). These results are in accordance with Mattiello et al. [28] and Santos et al. [29], who both studied the generation of phosphate fertilizers from the acidic residues of the metallurgical industry and different RPs, including BRP and ARP, and observed the greater vulnerability of BRP to acidulation when compared to other non-reactive RPs such as ARP.

According to Dorozhkin [30], several factors may influence the solubility of apatite and among them the most relevant are the composition of the rock, the particle size and the strength and composition of the acid used to solubilize. In this study, the smaller amount of sulfuric acid used (25%) was enough to alter the crystalline structure of all RPs, generating calcium sulfates (gypsum and bassanite) and transforming part of the apatite into more soluble forms (e.g., monocalcium phosphate). The presence of more soluble forms of P in these partially acidulated products was detected by the greater solubility in water (Table 2) when compared to the pure RPs, whose solubility in water was nearly zero (Table 1). Thus, even under less acidulation, increments in agronomic efficiency according to the structural changes promoted in relation to the pure phosphates were expected and will be discussed below.

### *3.2. Phosphorus Di*ff*usion*

### 3.2.1. Changes in Soil pH by Phosphate Fertilizers

All treatments, including TSP, increased the pH around the granule in relation to control (Figure 3). In the first layer, where the effect was more pronounced, the greatest increases were observed under TSP and almost all reactive RPs (T5, T6, T7, T9, T11 and T12), with the exception of treatments 8 and 10. Therefore, the capacity of these fertilizers to change the soil pH does not seem to be related to the presence of PILC and zeolites in their formulation, but to the higher solubility of phosphates. In corroboration, Cesar [31], when evaluating the diffusion of P from several phosphate sources in two contrasting textured tropical soils, observed that all phosphate sources, including pure TSP and those associated with BRP, were able to increase the pH close to the fertilizer granule.

According to Hettiarachchi et al. [32], after soluble or partially soluble phosphate fertilizer deposition in the soil, the granules' first action is to absorb water. This water moves towards the pores of the granules predominantly by capillarity flow and vapor transfer, and from there a series of reactions begins and one of the first items to change is the pH. Commonly, it is expected that partially acidulated phosphates and TSP decrease soil pH near the granule because of the acidic nature of their saturated solution [33], and the displacement of H<sup>+</sup> from the surface of the colloids to the soil solution caused by the increase in Ca concentration [34]. This acidifying effect of the dissolution of fertilizers containing monocalcium phosphate has already been reported in other studies. Lombi et al. [35], studying the lability, mobility and solubility of different phosphate fertilizers in calcareous and non-limestone soils, observed a significant decrease in soil pH up to a distance of 13.5 mm from the granule. Similar results were reported by them in another study carried out only on calcareous soils [36]. Silva [37] evaluated the diffusion of P from traditional fertilizers with reduced solubility in Entisol and observed that all fertilizers containing monocalcium phosphate were able to decrease the soil pH near the application point. Nascimento et al. [38] when studying the diffusion of P from calcium, magnesium and ammonium phosphates in soils of Brazil (Ultisol) and the United States (Mollisol), observed that calcium phosphate (TSP) was able to decrease the pH in all situations; however, this decrease was more significant in the soil with an initial pH of 8.0 (alkaline). In addition to the acidic characteristics of this source and the displacement of H<sup>+</sup> from the CEC to the soil solution induced by the increase in Ca concentration, the authors explain, based on the work of Cerozi and Fitzsimmons [39], that phosphate ions present three protonation constants (pH 2.1, 7.2 and 12.6). Thus, when the phosphate ion is added to soils with a pH higher than 7.2, there is a tendency for this ion to deprotonate, i.e., to donate H<sup>+</sup> to the solution, leading to a decrease in soil pH.

**Figure 3.** Soil pH as function of fertilizer source and soil layer after five weeks of incubation. Mean values followed by the same letter in the same layer do not differ by *t*-test (Scott-Knott, P < 0.05). T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4= ARP+Zeo+25% Ac.; T5=BRP+PILC +50% Ac.; T6=BRP+PILC +25% Ac.; T7=BRP+Zeo+50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; T13 = TSP (triple superphosphate); T14 = control. ARP = Araxá rock phosphate; BRP = Bayovar rock phosphate; MRP = Morocco rock phosphate; PILC = pillared clay; Zeo = zeolite.

Giving due credit to substantial evidence that the soil pH around the granule would decrease with the dissolution of phosphate fertilizers, the contradictory results observed here can be explained by the specific adsorption reactions of P on the surface of Fe and Al oxides, which potentially released OH− into the soil solution [40]. It is important to highlight that the majority of the soils in which there was a decrease in pH around the granules were calcareous or alkaline soils, with distinct characteristics from our study. Thus, the contrasting results can be attributed to these differences, since in soils with high pH, the precipitation of P with Ca becomes one of the main mechanisms of P immobilization [32], which does not release OH− into the soil solution, as is the case with adsorption to Fe and Al oxyhydroxides [40].

### 3.2.2. Available and Total P after Fertilizer Diffusion

TSP was the source with the highest soil available P content up to 25.5 mm from the granule (Figure 4a). Partial acidulated fertilizers (50% ac.) produced from BRP and MRP containing PILC and zeolites in the formulation (T5, T7, T9 and T11) also presented superior values of available P in the first layer (<7.75 mm) compared to other sources. According to Williams [41], soil P movement depends, among other factors, on the composition of the fertilizer granule. Thus, in the second layer (7.75–13.5 mm), fertilizers from BRP and MRP containing zeolite resulted in higher P resin contents when compared to the same phosphates containing PILC, within the same level of acidulation (T5 < T7; T6 < T8; T9 < T11; T10 < T12). In the third layer (13.5–25.5 mm), this behavior was observed only in phosphates produced from BRP at 50% acidulation (T5 < T7) (Figure 4a). The highest P diffusion under

products containing zeolite is attributed to its lower MPAC (31 mg kg−1) when compared to PILC (5527 mg kg<sup>−</sup>1). In the case of products containing PILC, the P released by acidulation may possibly have bound to its clay acidic sites, inhibiting P movement in the soil.

**Figure 4.** Soil available P (resin) (**a**) and total P content (**b**) in samples at distinct distances from the fertilizer granule application (radii of 0–7.75, 7.75–13.5, 13.5–25.5, and 25.5–43 mm) after five weeks of incubation. Mean values followed by the same letter for each soil layer do not differ by *t*-test (Scott–Knott, P < 0.05). Percent distribution of available P (**c**) and total P (**d**) from fertilizer in each layer (% PfS1–4) calculated according to the equation proposed by Lombi et al. [35]: %PfSi = [(Pf)Si \* Wi/Σi = 1 − 4((Pf)Si \* Wi], where i is the layer of the petri dish (1 to 4); (Pf)Si is the content of available or total P as a function of the fertilizer addition; and Wi the mass of soil in a particular layer. (Pf)Si was calculated by subtracting the mean of the control treatment of the other treatments with fertilizers. T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac. TSP = triple superphosphate; CONT = control; ARP = Araxá rock phosphate; BRP = Bayovar rock phosphate; MRP = Morocco rock phosphate; PILC = pillared clay; Zeo = zeolite.

No difference in available P was observed between control (T14) and the sources produced (T1–T12) in the last layer (25.5–43 mm), except for treatments T1, T4 and T7 whose differences are most likely due to small analytical variations (Figure 4a). Hardly any P from fertilizers would reach this distance and still remain available due to their strong interactions with the soil clay minerals [18]. In general, the phosphates produced from ARP with 50% ac. containing PILC or zeolites (T1 and T3) provided the lowest levels of available P up to a distance of 13.5 mm when compared to BRP and MRP, at the same level of acidulation (T5, T7, T9 and T11). However, within the less acidulated group (25%

ac.), there were no major differences between them (T2, T4, T6, T8, T10 and T12), especially in the first layer (0–7.75 mm) (Figure 4a).

All the RPs with 50% ac. presented higher available P up to 13.5 mm from the granule when compared with 25% ac. Apatite is the main mineral present in RPs (Figure 2), and its dissolution is facilitated in acidic medium [42]; thus, the more H<sup>+</sup> enters the system, the greater its dissolution. More than 65% of the available P from our fertilizers was restricted to the first layer (<7.75 mm), similar to TSP although a completely soluble source (Figure 4c). In general, the available P in soil decreased gradually with the distance from the granule. Overridingly, in the literature, there are reports of small movements of P from phosphate fertilizers in soil [18,35,36,43,44], confirming that only a small portion of the soil (few millimeters) is actually influenced by P fertilizers.

The results of total P content in each distance from the granule represent the diffusion of P from fertilizers in five weeks of incubation (Figure 4b). In general, the phosphate sources that resulted in the lowest total P in the first layer (<7.75 mm), regardless of the control, were also the ones that resulted in the highest P content in the adjacent layer (7.75–13.5 mm) (T7, T11 and TSP). More than 90% of total P from phosphate sources derived from ARP at both acidulation levels (T1–T4) and from reactive RP (BRP and MRP) with 25% ac. (T6, T8, T10 and T12) was restricted to the first 7.75 mm from the granule. The source with the greatest diffusion was TSP, even though more than 60% remained close to the granule (<7.75 mm) (Figure 4d).

In general, partially acidulated phosphates containing zeolite promoted more total P diffusion when compared to the same phosphates containing PILC, at the same level of acidulation (T1 < T3; T2 < T4; T5 < T7; T6 < T8; T9 < T11; T10 < T12) (Figure 4d). As already mentioned, this greater diffusion with zeolites is attributed to its smaller MPAC. Possibly, P released from the dissolution of RPs + PILC was potentially bound to the acidic sites of its own clay, limiting P movement in the soil. Silva [37] evaluated the diffusion of P from reduced solubility phosphate fertilizers in Cerrado soil using the SEM-EDXA (Scanning Electron Microscope with Energy Dispersive X-ray Analyzer) technique to determine the elemental composition of the granules before and after a period of soil contact. The results show that approximately 50% of P remained within the fertilizer granule even after five weeks of incubation in most of the evaluated products. He also verified the presence of ions such as Ca, Fe and Al in the constitution of these granules after the incubation, which may explain their small dissolution.

A certain level of soil moisture is fundamental to adequate fertilizer dissolution. However, once the fertilizer granules are in contact with the soil, two forces will regulate P availability; firstly, the water flows towards the granule by negative osmotic potential, carrying with it numerous chemical species such as Ca2<sup>+</sup>, Al3<sup>+</sup>, Fe2<sup>+</sup>, Mg2<sup>+</sup>, etc. This explains the presence of several elements inside the granules after incubation which were not part of their original composition, as observed by Silva [37]. Secondly, dissolved elements from the granule move to outside the surrounding areas of lower concentration. At this time, due to the high affinity of P with various metals, insoluble compounds such as P-Al and P-Fe precipitates, for example, may form [32,37], which justifies the weak diffusion and great permanence of P close to the fertilizer granule (<7.75 mm). Another fact is the movement of the companion ion [45], in this case especially Ca. For more intense P diffusion into the soil solution, more Ca dissolution and movement is required, preferably outside the granule region, which was not intensified in our study because of the static incubation, without solution flow, in agreement with the results already reported by Silva [37].

Moreover, the movement of P depends on fertilizer characteristics, such as the size of their particles/granules and their composition, and a series of soil properties, such as compaction, moisture level and mineralogical composition [41]. Thus, Benbi and Gilkes [46] studied the movement of P from TSP in two soils with high and low MPAC. After four weeks of fertilizer application, the added P was retained up to 80 mm away from the fertilizer granule in both soils. Within this 80 mm boundary, they also observed that P retention occurs in three different zones; one refers to the local of fertilizer granule, another one to the region next to the granule where precipitation and adsorption reactions

predominate near the maximum limits, and the last is the most external where P is adsorbed to the soil at lower levels than its MPAC. These different zones of P accumulation are in agreement with the restricted available P diffusion observed here in our study.

### *3.3. Solubilization of Fertilizers in Soil Columns*

### 3.3.1. Leaching Potential

The presence of P in leached solution was not detected in the control treatment. Moreover, no P was detected in the leached solutions containing pure RPs (ARP, BRP and MRP), which testifies to the zero water solubility of these sources (Table 1). Therefore, it is assumed that all the P contained in the leached water derives from the lab treatments applied to these RPs (Figure 5).

**Figure 5.** Phosphorus leaching from phosphate fertilizers over time (**a**) and accumulated leached P after 60 days of incubation (**b**). Mean values followed by the same letter do not differ by *t*-test (Scott–Knott, P < 0.05). T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; ARP = Araxá rock phosphate; BRP = Bayovar rock phosphate; MRP = Morocco rock phosphate; TSP = triple superphosphate; CONT = control. PILC = pillared clay; Zeo = zeolite.

According to the acidulation level and additives incorporated in each source evaluated herein, distinct amounts of P were detected in the leachate through the soil columns over the 60 days' incubation period (T1–T12) (Figure 5a). In total, five groups were identified; the first group, involving treatments T2, T4, T6 and T10, was classified as insoluble phosphates, meaning that these treatments were not able to promote leaching of P sufficient to differentiate from pure RPs and control, varying from 1.2 to 3.5 mg P per column over the 60 days' incubation period. The second group, consisting of treatments T1, T3, T8 and T12, represented fertilizers with reduced solubility, sufficient to be different from the insoluble ones, with the P leached ranging from 7.4 to 10.0 mg P per column. The third group consisted of treatments T5 and T9, whose leaching was 18.0 and 17.5 mg of P per column, respectively, and the fourth group, composed of treatments T7 and T11, registered 25.2 and 23.0 mg of P per column, respectively. These last two groups, involving treatments with high reactive RPs, are potentially viable alternatives for overcoming the totally soluble sources due to the slower dissolution of P into solution. TSP, the one with the highest P loss in leached solution (64 mg of P per column), constituted the fifth and last group (Figure 5b).

According to the P solubilization and leaching patterns observed for all the fertilizers evaluated here (except for the insoluble ones—group 1), it is possible to identify two distinct phases (Figure 5a). The first phase consisted of the first 10 days, when more than 50% of P had already leached. From the 10th day onwards, there was a significant decrease in the P content in the leaching solution, comprising a second leaching phase. These two phases are explained by the high P affinity to the soil constituents. When released from fertilizer, the P will potentially bind to the surface of Fe and Al oxides. Initially, the soft energy of these bonds still allows for P percolation through the profile (first phase). Subsequently, the concentration of P in leachate decreases due to the increase in energy ("aging") of P linkage (bidentate and binucleate bonds), inhibiting P leaching in the solution [44].

Clearly, the acidulation levels were the major factors responsible for the dissolution of RPs and, consequently, for the differences in P levels detected in leachate (Figure 5b). However, the presence of PILC restricted the leaching of P, mainly in the treatments from reactive RPs (T5, T6, T9 and T10), due to its high MPAC (5527 mg kg<sup>−</sup>1) when compared to treatments with Zeolite in the same RPs (T7, T8, T11 and T12).

### 3.3.2. Phosphorus Lability

For better comprehension of the P dynamics and its accumulation in soils, sequential extraction with distinct strength solutions has become a fundamental tool [47–50]. The "P fractionation" procedure allows for evaluating the forms and distribution of this nutrient in the soil according to the fractions extracted, as well as the fate of P applied via fertilizers, in order to identify changes in soil nutrient dynamics. All the fertilizers studied, including pure RPs, were able to increase the labile P fractions in the soil profile after 60 days of incubation. In the first layer (0–1 cm) ARP + PILC + 50% ac. (T1) provided the highest levels of labile P when compared to other sources, in the following decreasing sequence: T1 > TSP = T3 = T5 = T9 > T2 = T4 = T7 = T9 > T6 = T10 > T8 > T12 > T14 = T15 > T13 > T17 (Figure 6a and Appendix A). In deeper layers, in general, sources with 50% ac. recorded the highest increases in soil labile P pool, but were much less expressive than in the 0–1 cm layer (Figure 6b–e).

There was great accumulation of moderate labile P throughout the profile under our fertilizer sources (T1–T12), detaching the first layer (0–1 cm), where it represented more than 88% of the total P (Figure 6a and Appendix A). In general, it was observed that 50% ac. resulted in higher contents of P extracted by 0.1 mol L−<sup>1</sup> NaOH and lower contents of P extracted by 1 mol L−<sup>1</sup> HCl when compared to 25% ac. (Appendix C). Based on these observations, it is clear that P solubilized from fertilizers was rapidly bound to mineral compounds in our test soil. Thus, its availability over time will be compromised by the strength of the reaction (monodentate, bidentate and/or binucleate bonds). Nevertheless, the sources produced from the same RPs containing incorporated PILC had a higher content of P extracted with 0.1 mol L−<sup>1</sup> NaOH than those containing zeolites at the same level of acidulation (25 or 50% ac.) (Appendix C). This is mainly due to the presence of AlOH and a

AlOH2 groups in PILC that are able to adsorb a large amount of P (PILC MPAC = 5527 mg kg<sup>−</sup>1) [51]. Some plant species are capable of acquiring P from this moderately labile inorganic P fraction via different mechanisms such as mycorrhizal association or P-solubilizing rhizosphere exudates [52,53].

**Figure 6.** Labile, moderately labile and non-labile P pools in different soil layers of the column, 0–1 (**a**), 1–2 (**b**), 2–3 (**c**), 3–6 (**d**) and 6–10 cm (**e**), in response to phosphate fertilizers application after 60 days. Mean values followed by the same letter do not differ by *t*-test (Scott–Knott, P < 0.05). ns, not significant. T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; T13 = ARP (Araxá rock phosphate); T14 = BRP (Bayovar rock phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple superphosphate); T17 = control. PILC = pillared clay; Zeo = zeolite.

In the first soil layer (0–1 cm), the total P content ranged from 2.007 (T15) to 13.242 (T6) mg kg−<sup>1</sup> (Figure 6a). The residual fertilizer granules were homogenized to the soil at this layer when sampling, justifying this large amount of total P. According to Silva [37], after a short time in contact with the soil, approximately 50% of the P remains inside the fertilizer granule. The permanence of P within or surrounding the granule, as observed in this study, was due to the incomplete dissolution of the partially acidulated phosphates, proved by the significant participation of P extracted by HCl, which refers to Ca-phosphates (Appendix C). The formation of insoluble compounds such as P-Al and P-Fe or the adsorption of P onto the surface of Fe and Al sesquioxides also contributed to this accumulation, evidenced by the Pi fraction extracted by 0.1 mol L−<sup>1</sup> NaOH (Appendix C). Under TSP, although a soluble source, P accumulation was also observed in this top layer due to its high affinity to the surface of Fe and Al sesquioxides. The retrogradation process (P-Ca) also restricted the movement of P from TSP, but to a lesser extent than the other sources (Figure 6a, Appendix C).

The non-labile P pool was the least influenced by our treatments (Figure 6). When analyzing the participation of each compartment in the total P in our soil, it was observed that under no fertilizer (control), the non-labile P represented the greatest part of the total P throughout the profile (74–89%). Several studies in tropical soils similarly reported this expressive proportion of non-labile P due to the high energy binding between phosphate and functional groups of Fe and Al sesquioxides [47–49,54–56]. Although there was an accumulation of non-labile P in the 0–1 cm layer under fertilizer application, its proportion to the total P was very small (3.2–13.4%) compared to other layers evaluated, and the sources that provided the highest accumulations were those produced from ARP (T1–T4), given its much lower reactivity compared to other sources (BRP and MRP) (Figure 6a). For other layers, non-labile P participation varied from 25 to 44.3% of the total P, but was not clearly influenced by any specific source/treatment (Figure 6b–e).

### *3.4. Agronomic E*ffi*ciency*

At maize harvest (45 days after sowing), plants had typical symptoms of P deficiency under control (T17) and under pure RP treatments ARP (T13) and MRP (T15). Reduced growth and purplish, dark brown or dried leaves were the main symptoms observed (Appendix E). The difference in development between treatments was expressive, plants that received TSP looked healthy and better than other treatments, but all the partially acidulated phosphate sources with the incorporation of PILC and zeolites were able to promote greater maize growth than control and pure RPs.

Pure sources of ARP (T13) and MRP (T15) did not provide sufficient P for maize plants to express their initial growth potential, showing performances similar to the control for all the parameters evaluated (shoot and root DM, and accumulated P in shoot and root), resulting in very low RAE (0.8 and 10.6%, respectively) (Table 5). Therefore, the use of these RPs for direct application as fertilizer is not feasible. Partial acidulation (25 or 50% ac.) and incorporation of PILC or zeolites into their formulations resulted in significant increases in all plant parameters, with RAE ranging from 26.8 (T2) to 85.4% (T11).

The pure source BRP (T14) was able to differentiate from the control in plant growth parameters, with an RAE of 36.6% (Table 5). When BRP was acidulated by 25% (T6 and T8), it was not enough to significantly increase plant response. Similar results were also detected for ARP and MRP under 25% ac. (T2, T4, T10 and T12). However, it is worth mentioning that there was a physical difference between these fertilizers when BRP was applied in the powder/bran form, the same way that it is commercialized, and the phosphates that received 25% acidulation were applied as granules. The higher the phosphate contact with the soil, i.e., the greater its specific surface area, the greater its dissolution due to the higher contact of phosphate with the H<sup>+</sup> protons present in the soil [57–60].

Among the lab fertilizers produced in our study, treatments T5 and T11 were the ones that provided RAE nearly similar to TSP (89 and 85%, respectively). Both received 50% acidulation, but T5 was produced from BRP with PILC and T11 was produced from MRP with zeolites. Treatments T7 (BRP + Zeo + 50% Ac.) and T9 (MRP + PILC + 50% Ac.) also resulted in good RAE indexes (74 and 76%, respectively). In view of these results, 50% acidulation may be a profitable alternative to improve fertilizer efficiency when using sedimentary RPs for crop production. Otherwise, additives PILC and zeolite do not seem to be effective in increasing P agronomic efficiency (Table 5).


**Table 5.** Dry matter yield and accumulated P content in shoot and root of maize under phosphate sources partially acidulated with incorporation of pillared clays or zeolites.

Means followed by the same letter in the column do not differ from each other by the Scott–Knott test at 5% probability. RAE = relative agronomic efficiency. T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; T13 = ARP (Araxá rock phosphate); T14 = BRP (Bayovar rock phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple superphosphate); T17 = control. PILC = pillared clay; Zeo = zeolite.

Numerous published research studies on the effectiveness of partially acidulated phosphate fertilizers found conflicting results [57,61–66]. This is attributed to differences in the physical form of application (powder versus granular), the products used for acidulation, the type of soil used to test the fertilizers and the doses tested [57,67]. From our study, it is possible to confirm that the type of RP used as raw material for the production of a partially acidulated phosphate also influences its effectiveness.

In fact, our previous incubation experiments (diffusion and solubilization) showed better results from sources containing zeolites (greater P diffusion and solubilization). This high P availability of fertilizers containing zeolite reflected in higher RAE only for MRP under 50% ac. (T11). In sources under 25% ac., the difference between zeolite and PILC products in similar RP sources was negligible. However, as solution soluble P can be easily adsorbed in a short period of time, the slower solubilization under the presence of PILC or even zeolite may play an important effect for better plant P utilization over time.

We confirmed here the very low diffusion of P in the soil. Therefore, we can say that the phosphate fertilizer placement can strongly influence its agronomic efficiency, and application techniques should be considered when thinking about improving the phosphate fertilizer use efficiency. A study conducted by Nkebiwe et al. [68], summarizing current techniques for N and P fertilizer placement in soil, showed that overall, fertilizer placement led to 3.7% higher yield, 3.7% higher nutrient concentration and 11.9% higher nutrient content in above-ground parts than fertilizer broadcast in soil surface. In fact, understanding the dynamics of P when fertilizers are applied in soil by different placement strategies and the use of new technologies may help to utilize P more efficiently.

### **4. Conclusions**

The highest values of relative agronomic efficiency (>74%) were obtained with 50% acidulation of reactive RPs from Peru (BRP) and from Morocco (MRP) (T5, T7, T9; T11). Thus, these sources can be considered as being better suited alternatives for overcoming high solubility sources when searching for a product with more gradual P release into the soil. In the same trend, fertilizers produced from BRP and MRP with 50% acidulation containing zeolite in the formulation (T7 and T11) provided the highest diffusion and percolation of P in the soil profile, although still much lower than TSP. Otherwise, even zeolite and PILC seem not effective in increasing P agronomic efficiency.

All the fertilizers were able to increase the labile and moderately labile P fractions in the soil profile after 60 days of incubation. However, in the top layer, close to the fertilizer (0–1 cm), the sources containing PILC with 50% acidulation provided higher labile P contents when compared to zeolite, at the same level of acidulation and even RP source (T1 > T3, T5 > T7, T9 > T11). TSP was the most effective in percolating P in the soil profile, even in labile or moderate labile pools.

**Author Contributions:** Conceptualization, A.P.B.T. and P.S.P.; methodology, A.P.B.T., M.R. and P.S.P.; data curation, A.P.B.T.; statistical analysis, M.R.; writing—original draft preparation, A.P.B.T.; writing—review and editing, P.S.P. and M.R.; validation, A.P.B.T., M.R. and P.S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors are grateful to Coordination for the Improvement of Higher Education Personnel (CAPES), which supported the scholarship to the first author.

**Conflicts of Interest:** The authors declare no conflicts of interest.

### **Appendix A**


**Table A1.** Labile, moderately labile and non-labile P pools in the surface soil layer of the column in response to phosphate fertilizer application after 60 days.

Mean values followed by the same letter do not differ by t-test (Scott-Knott, P < 0.05). ns, not significant. T1 = ARP + PILC + 50% Ac.; T2 = ARP + PILC + 25% Ac; T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; T13 = ARP (Araxá rock phosphate); T14 = BRP (Bayovar rock phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple superphosphate); T17 = control. PILC = pillared clay; Zeo = zeolite.




phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple

superphosphate);

 T17 = control. PILC = pillared clay; Zeo = zeolite.




T3 = ARP + Zeo + 50% Ac; T4 = ARP + Zeo + 25% Ac.; T5 = BRP + PILC + 50% Ac.; T6 = BRP + PILC + 25% Ac.; T7 = BRP + Zeo + 50% Ac; T8 = BRP + Zeo + 25% Ac; T9 = MRP + PILC + 50% Ac.; T10 = MRP + PILC + 25% Ac.; T11 = MRP + Zeo + 50% Ac.; T12 = MRP + Zeo + 25% Ac.; T13 = ARP (Araxá rock phosphate); T14 = BRP (Bayovar rock phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple superphosphate); T17 = control. PILC = pillared clay; Zeo = zeolite.

### **Appendix D**


**Table A4.** Residual fraction (Presidual) of P in the soil, considered non-labile, in different layers of the columns according to the sources of phosphate fertilizers.

Means followed by the same letter in the column do not differ from each other by the Scott-Knott test at 5% probability. ns = not significant. T1 = ARP + PILC + 50% ac.; T2 = ARP + PILC + 25% ac.; T3 = ARP + Zeo + 50% ac.; T4 = ARP + Zeo + 25% ac.; T5 = BRP + PILC + 50% ac.; T6 = BRP + PILC + 25% ac.; T7 = BRP + Zeo + 50% ac.; T8 = BRP + Zeo + 25% ac.; T9 = MRP + PILC + 50% ac.; T10 = MRP + PILC + 25% ac.; T11 = MRP + Zeo + 50% ac.; T12 = MRP + Zeo + 25% ac.; T13 = ARP (Araxá rock phosphate); T14 = BRP (Bayovar rock phosphate); T15 = MRP (Morocco rock phosphate); T16 = TSP (triple superphosphate); T17 = control. PILC = pillared clay; Zeo = zeolite.

### **Appendix E**

**Figure A1.** Maize plants at harvest (45 days growth) showing the differences between treatments. (**A**) 1 = Control (T17); 2 = ARP (T13); 3 = ARP + PILC + 25% ac. (T2); 4 = ARP + PILC + 50% ac. (T1);

5 = TSP (T16). (**B**) 1 = Control (T17); 2 = ARP (T13); 3 = ARP + Zeo + 25% ac. (T4); 4 = ARP + Zeo + 50% ac. (T3); 5 = TSP (T16). (**C**) 1 = Control (T17); 2 = BRP (T14); 3 = BRP + PILC + 25% ac. (T6); 4=BRP+PILC +50% ac. (T5); 5=TSP (T16). (**D**) 1= Control (T17); 2=BRP (T14); 3=BRP+Zeo+25% ac. (T8); 4 = BRP + Zeo + 50% ac. (T7); 5 = TSP (T16). (**E**) 1 = Control (T17); 2 = MRP (T15); 3 = MRP + PILC + 25% ac. (T10); 4 = MRP + PILC + 50% ac. (T9); 5 = TSP (T16). (**F**) 1 = Control (T17); 2 = MRP (T15); 3 = MRP + Zeo + 25% ac. (T12); 4 = MRP + Zeo + 50% ac. (T11); 5 = TSP (T16). ARP = Araxá rock phosphate; BRP = Bayovar rock phosphate; MRP = Morroco rock phosphate; TSP = triplo superphosphate; PILC = pillared clay; Zeo = zeolite.

### **References**


*e Contaminantes [O*ffi*cial Analytical Methods Manual for Correctives, Inoculants, Substrates and Contaminants]*; MAPA: Brasília, Brazil, 2007. (In Portuguese)


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