Inhibition of Nickel (II) Mobility in Theobroma cacao L. Seedlings Using Zeolite 5A

Abstract

In search of efficient solutions for the treatment of contaminated soils and in favor of the sustainable development of agriculture, this work aimed at developing an efficient method that helps to directly overcome the contamination by nickel in soils and Theobroma cacao L. seedlings. In this study, the genotypes ICS-39, CCN-51, and TSH-1188, which are high-yielding varieties in South America, were studied. The compound used as an adsorbent was commercial zeolite 5A. The zeolite 5A and soil samples were analyzed by X-ray diffraction, Raman microscopy, chemical analysis, electron microscopy techniques, and atomic absorption spectroscopy. This last technique was used for quantitative determination of Ni concentrations in seedlings. Zeolite 5A presented a high adsorption efficiency (95%) among the studied cacao genotypes, making this material a viable adsorbent and inhibitor agent of Ni. In addition, zeolite 5A was found to be not chemically harmful to the plant morphology (root and height), as demonstrated using statistical analysis. Finally, the Ni mechanism was described based on zeolite 5A physicochemical properties, suggesting that this material has remarkable soil remediation application.

1. Introduction

Cacao is a commercial crop from the tropical Amazon, whose commerce is vital to the economy of many nations, in addition to serving as a raw ingredient in industrial operations [1]. Cacao is considered a luxury commodity in the coffee business since it is not often necessary for nourishment; nonetheless, it is highly important for the economies of developing nations (mostly in South America and Africa) because it has helped build very strong and growing economies, which is key for cacao-producing countries [1]. The African continent produces the largest quantity of cacao beans (76% of global output), followed by the Americas (19%) and Oceania (5%). Ecuador, Brazil, and Peru stand out in the production of cacao beans in America. The latter is the third largest producer in the area and the world’s second largest exporter of organic cacao, trailing only the Dominican Republic [2]. In this context, it is critical to research and evaluate cacao bean production, employing a variety of techniques and approaches to increase quality and maintain continuous output.

It is important to note that the quality of cacao is crucial for the cacao industry, so analyzing the effects of heavy metals in these products is critical because, as stated in [3], the elements commonly known as toxic metals affect the quality of foodstuffs. It should be mentioned that this problem has been studied for more than 30 years. For example, there are several works that report the effect of Pb, As, Cd, and Cu on the quality of cacao-based final products such as candies and chocolates [4,5]. In reference [6], a complete analysis of the parameters and factors that determine the quality of cacao in food is carried out, where the content of toxic elements in the cacao bean and in its final products is taken as an important parameter.

In terms of cacao cultivation, several strategies have been investigated, but the most popular incorporate fertilizers and bioremediating chemicals that minimize heavy metal concentration, for example, the application of organic and inorganic calcium supplements in the cacao plant to reduce cadmium mobility [2]. Furthermore, nickel (Ni⁺²) concentrations of around 3.8 mg kg⁻¹ were reported in several varieties of cacao-based chocolates, according to [7]. In this regard, it is critical to investigate the impact of heavy metals on cacao bean products and to seek alternatives to decrease the detrimental effects they can have on human and animal health. As mentioned in [8], Ni, Cd, and Pb are the metals with the highest presence in cacao-derived products and the consequences of these on health are reported in reference [9], e.g., causing dermatitis, headaches, and gastrointestinal and respiratory manifestations.

In 1988, Ni⁺² was added to the list of heavy metals as an important microelement for plants [10]. When it accumulates in large amounts it becomes phytotoxic, preventing seedling growth, i.e., it reduces production and leaf expansion, etc. However, it is important to note that at low quantities, this microelement is required to metabolize the urease found in the leaves; otherwise, a buildup of this molecule might induce seedling mortality [11]. It is found in modest amounts in cacao, but it should be noted that this metal, along with iron and cobalt, is one of the most plentiful on the soil surface. To date, neither the Codex Alimentarius nor the European Commission have established maximum permissible levels of Ni⁺² and lead concentrations in cacao and its derivatives.

One of the major issues that has developed in relation to soil pollution is the presence of trace elements such as Pb⁺², Cu⁺², Cd⁺², and Ni⁺². This work focuses on finding a solution to soil pollution caused by Ni, as it is one of the heavy metals with little mention in the literature [9,12]. Physical and chemical cleanup options are available. We must keep in mind that remediation strategies are defined as procedures that follow a certain protocol and combine one or more approaches to treat existing contamination [13], which means that we may utilize several strategies such as pollutant destruction or modification, extraction or separation, and isolation or immobilization of the contaminant. The many remediation approaches have different advantages, and depending on the kind of polluted system, the cost, the impacts on the environment, and the method of application, some drawbacks exist. For example, biological remediation techniques are extremely effective, ecologically friendly, and do not require constant monitoring, and many do not produce side effects; however, they have a slow remediation time [14]. By comparison, physical remediation is easier to apply, is typically the most commonly used for soil remediation, has a low cost, and does not change the stability or composition of the soil [15]. However, in certain circumstances these techniques create waste after treatment, which raises costs (mainly in applications of contaminated water remediation). Finally, thermal remediation is extremely efficient at burning, dissolving, or melting pollutants, but has a high cost [16].

Zeolites are a class of crystalline compounds that are typically hydrated or possess cations (placed outside their cages), generating regular three-dimensional patterns. They can also interchange additional structural cations without affecting their crystalline structure. However, depending on the type of zeolite being examined, they are also capable of reversibly gaining and/or losing water molecules that are often held in the cavities or bigger channels [17]. The uses of these materials, both natural and manmade, have enabled numerous sorts of scientific advancements, including their employment in heavy metal adsorption, soil improvement, and air filtration, and as molecular sieves for oil refining [18,19,20]. Zeolites in general include porous channels and holes that allow molecules to adsorb within them, preventing bigger molecules from passing through. Thus, they have the property of a molecular sieve, which favors their employment in several sectors of agriculture. A systematic review of the natural zeolite, clinoptilolite, as a compound that can be used as an antioxidizing, detoxifying, and anti-inflammatory supplement was performed in reference [21], while reference [22] investigated the effects of clinoptilolite as a supplement in cows, pigs, and sheep; results showed an increased milk production in cows and improved reproductive performance in pigs. Reference [23] investigated the effects of zeolite NaA as a feed supplement, and results showed an improvement in weight and a decrease in feed cost. The adsorption capacity of the zeolites Linde Type A (LTA) and Faujasita (FAU) was analyzed with respect to the divalent heavy metals Pb⁺², Cu⁺², and Ni⁺², determining that the adsorption of Ni⁺² was influenced by the shape of the zeolitic structure, whereas the adsorption mechanism of Pb⁺² and Cu⁺² was mostly influenced by the cation exchange within the structure [24].

Based on the above, we may conclude that different techniques can be used in different media depending on the pollution to be addressed. The process involved in contaminated soil remediation is the adsorption, absorption, and/or degradation of certain pollutants to lower their concentration or transform them into compounds that are less damaging to the environment. Many publications address the use of inorganic materials in the remediation of pollutants in soils. These materials are an effective, efficient, and simple approach to address the contamination problem, and are the most widely employed for contaminant adsorption [20,25]. However, it should be highlighted that, as far as we are aware, very little work has been done in cacao plantations [8]. Therefore, our work attempts to look at this issue and provide a unique and novel viewpoint on the rehabilitation of contaminated soils with agricultural potential in a nation where agricultural exports are highly significant. Natural zeolites are a type of an inorganic compound that have been used for a long time since they are known to be useful as soil conditioners with fertilizer potential due to their natural adsorbent properties. Furthermore, their implementation and use are not expensive. These compounds are frequently found in nature, so do not require any transformation, as is the case with the usage of clinoptilolite in agriculture [26]. Scientists have concentrated on the application of synthetic zeolites for environmental remediation since they can not only take advantage of their qualities, but also change and enhance parameters that allow them to have more usable properties. In addition, their synthesis in a laboratory does not incur significant expenses and the synthesis time is short [27].

In this study, it was established using X-ray diffraction and Raman microscopy techniques that zeolite 5A is a material with a three-dimensional structure made up of TO₄-type tetrahedra, where T can be either Si or Al. Through Rietveld refinement, it was also demonstrated that zeolite 5A is a hydrated material. Scanning electron microscopy and energy dispersive X-ray spectroscopy (EDS) mapping was also performed before and after the experiment to verify the integrity of zeolite 5A, and it was noticed that this compound is homogeneously disseminated in the substrate employed, with Si and Al being the compounds that can be detected in the substrate, indicating the homogeneous presence of zeolite in the substrate. Furthermore, a quantitative and qualitative examination was undertaken regarding the experimental design to determine the impact of zeolite 5A on the growth and development of seedlings of Theobroma cacao L., including the height, root length, and concentration of Ni⁺². These data that revealed a statistically significant difference in the seedlings subjected to treatment compared to an untreated control.

2. Materials and Methods

2.1. Zeolite 5A Adsorbent

The material utilized was commercial synthetic zeolite 5A from Sigma-Aldrich laboratories, which was used without further purification. Moreover, an amount of roughly 105 g of the product was utilized for the dosage experiments (including repetitions). The indicated amount accounts for five repetitions for each treatment, although at the end of the experiment, only three replicates were considered for seedling sacrifice and analysis.

2.2. Physical Characterization of the Adsorbent and Soils

The adsorbent sample was evaluated using X-ray diffraction on an Empyrean diffractometer equipped with CuK_α radiation (λ = 1.5406 Å), 40 kV, and 30 mA; the powder sample was analyzed using a Bragg–Brentano optical setup consisting of a fixed radius goniometer. The sample composition was determined using Matchv3 software [28] and the crystallographic information file (CIF) #2102128 [29]. Furthermore, the Rietveld method was used for refinement with the FullProf Suite software (Gif sur Yvette Cedex, France, version January 2021). The soil samples were subjected to X-ray diffraction analysis using an Empyrean diffractometer with CuK_α radiation (λ = 1.5406 Å), 40 kV, and 30 mA; the diffractometer was operated with a Bragg–Brentano optical arrangement. The sample was identified using Matchv3 software and crystallographic information file (CIF) #9009666 [30] for Bragg peak identification of samples V1-V3 and #1011176 for V4 [31].

The substance was studied using the micro-Raman spectroscopy approach on a high-resolution Raman Renishaw spectrometer. The measurement was carried out in the range of 0 to 3000 cm⁻¹ using a laser with a wavelength of 785 nm, which corresponded to the infrared spectrum; also, the laser power employed was 41.4 mW with a percentage of 50%, and the spectrum was compared with the literature [32].

The morphology, size, and content of the powders were determined using a TESCAN LYRA3 high-resolution scanning electron microscope equipped with a FEG type electron source and an Oxford EDS detector (Tescan Brno s.r.o., Brno, Czech Republic). Secondary electron imaging and atomic element mapping were obtained jointly utilizing a 15 kV accelerating voltage and a 9 mm working distance.

2.3. Sampling of Soils

Soil samples were collected at the Instituto de Cultivos Tropicales (ICT) Juan Bernito station, located in Tarapoto, Peru (18 M 352141.71 E 9281002.18 S). Approximately, 800 kg of acid soil (ultisol) [33] was collected from various sites around the station and cleansed of any leaf litter, organic materials, or other factor that might impact the soil composition. Following that, the soil samples were transferred to the laboratory, where they were ground and sieved (with a sieve diameter of 2 mm), and the soil physicochemical parameters were measured according to the previously described protocol [34]. According to [35], Cacao cultivation requires a clay loam soil that provides sufficient moisture for cacao in extremely dry or desert locations, such as in African countries. The ideal pH range for cacao cultivation is between pH 6.0 and 7.5, and according to Paredes et al. [36], the pH for effective cacao development ranges from 6.0 to 6.5, providing high yields, while cacao is adaptable to pH ranges ranging from 4.5 to 8.5 [37].

Once the extraction point for the experiment was established, the soil was homogenized to guarantee that the substrate to be created would be the same for each pot subjected to the subsequent treatments, and for this purpose, samples were obtained for structural characterization from four pots chosen at random: V1, V2, V3, and V4, in order to verify that the composition was homogenous in all the pots utilized, with V1 and V2 soil samples taken at random. V1 and V2 soil samples were taken at the beginning and V3 and V4 soil samples taken at the end of the experiment (with 1 and 2 g of zeolite 5A, respectively), in order to verify the integrity and composition of the soil throughout the experiment.

2.4. Sampling of Cacao’s Fruits

Fruit samples were collected from the central part of the trees available at the ICT for the different genotypes used (ICS-39, CCN-51, and TSH-1188) of each cacao tree. For the extraction and germination of cacao seeds, mature, well-constituted cobs located in the upper third of the tree (recommended) were chosen. Then, the seeds located in three-quarters of the cob (useful seeds) were extracted and subsequently cleaned of the mucilage that covered them with sawdust by simply rubbing. Cacao germination was performed carefully, avoiding the exposure of seeds to contaminants. Seeds were arranged on a carpet of wet sawdust as a “bed” and then covered with banana leaves to maintain humidity, according to reference [36]. As part of the pre-germination process, the seeds were exposed to a 2 g L⁻¹ antifungal solution, commercial parachupadera (Flutalonyl 0.10 mg kg⁻¹ + Captan 0.64 mg kg⁻¹), to prevent the seed from being affected by any fungus, insect, and/or external agent that could affect its growth.

2.5. Determination of the Physical and Chemical Properties of the Soil

The analysis of physical and chemical properties was performed in the analytical laboratory of plants and soils at ICT, see

Table 1. Chemical and physical characterization of the substrate. (mean ± sd).

Chemical Analysis
pH	CaCO3 (%)	O.M. (%)			N (%)	P* mg kg−1			K* mg kg−1		E.C. dS cm−1
4.3 ± 0.1	<0.3	0.81 ± 0.04			0.04 ± 0.01	1.65 ± 0.02			24 ± 3		0.08 ± 0.01
CEC	E.C.E.C.	Ca			Mg	K			Na		Al+3
cmol kg−1
8.6 ± 0.3	4.6 ± 0.1	1.62 ± 0.02			0.58 ± 0.01	0.06 ± 0.01			0.34 ± 0.03		2 ± 0.1
Physical analysis (%)
Sand			Silt				Clay			B. S.
59.7 ± 4.36			8.3 ± 0.4				32.04 ± 1.21			30.26 ± 1.42
Ni Concentration mg kg−1
Ni Concentration Initial				Ni Concentration before treatment				Ni Concentration after treatment
1.83 ± 0.04				70.21 ± 1.91				25.34 ± 0.82

*P and K indicates phosphorus and potassium concentration, respectively. Organic matter (O.M.), cation exchange capacity (C.E.C.), effective cation exchange capacity (E.C.E.C.), electrical conductivity (E.C.), base saturation (B.S.). From the results of the soil characterization, it can be noted that the B.S. is less than 50%, which indicates that it is a very acid soil, and the percentage of carbonates (CaCO₃) in it suggests that the soil requires liming.

, following the protocols recommended by [38,39].

2.6. Substrate Contamination with Ni⁺²

In reference [40], it is mentioned that Ni⁺²-contaminated soil presents a threshold concentration of 100 mg kg⁻¹. On the other hand, according to [41], it is mentioned that the normal amount of Ni⁺² concentration in soils varies between 5 and 500 mg kg⁻¹ (in normal soil conditions), which has its origin in soils such as those derived from limestone or acid igneous rocks containing <50 mg kg⁻¹ and those derived from clayey sediments containing from 5 to more than 500 mg kg⁻¹ Ni⁺². Other authors, such as [42], reported an average concentration value of 22 mg kg⁻¹ Ni⁺² for tropical Asian paddy soils, while Alloway [40], citing [43], reported a value of 25 mg kg⁻¹ Ni⁺² for soils prevalent in various parts of the world; in [34], a study was conducted where Ni⁺² concentrations were determined in the San Martin-Peru region at values of 1.64–13.69 mg kg⁻¹. In the present work, 7.2 kg of substrate per pot was used to which 50 mL of an aqueous solution of 0.14 M NiCl₂•6H₂O was added, mixing it uniformly until the solution was homogeneous in the substrate. The process of Ni homogenization in the substrate consisted of the following steps: Once 50 mL of the contaminant solution was added to the 7.2 kg of substrate, the soil was mixed for approximately 30 min, wetting the entire substrate. Then, it was left to dry. The washing cycle began with the wetting and uniform mixing of the substrate with distilled water, since this process guarantees that the Ni cations, due to their mobility in the substrate, are uniformly distributed in the whole volume of the substrate; thereafter, it is left to dry in the open air and then the process was repeated three times. This process ensured that the Ni⁺² cations were mobilized uniformly throughout the substrate contained in the pot. Initially, the Ni⁺² concentration contained in the substrate was 1.83 mg kg⁻¹, according to the values reported by [34], but after contamination, the Ni⁺² concentration was 70 mg kg⁻¹.

2.7. Implementation of a Suitable Space for Cacao Planting

According to [36], the cultivation space (nursery) should be the most feasible, taking into account numerous characteristics such as illumination, quantity of shadow, and terrain topography. Because these conditions impact its growth throughout the trial period, it is also crucial to provide it with the appropriate care, such as weed extraction and dead seed separation, and to maintain it free of pests and fungus that may impair the plant’s normal growth. The experiment was conducted as a completely randomized design in a split-plot arrangement with five replications (Figure 1), where the plots considered (A and B, respectively) were the control (substrate with Ni⁺² only) and the treatment (substrate with the presence of zeolite 5A). Seedlings were grown for three months in a greenhouse protected from rain with a protective mesh that provided 50% shade. During the experiment, the substrate of each pot was moistened to 90% of its field capacity, as recommended [44]. It should be mentioned that at the end of the experiment, only 3 seedling samples of each genotype were taken for the corresponding analysis.

2.8. Preparation and Application of Zeolite 5A in Pots

The zeolite 5A was weighed in an analytical balance in doses of 1, 2, and 4 g, respectively, for each genotype used; in addition, the number of repetitions for each dose was considered. In reference [45], it is mentioned that zeolite can be used as a slow-release fertilizer and soil improver. The purpose of the application of zeolite 5A as a heavy metal inhibitor is that this compound has contact with the root part of the plant, so it was applied in the pot in a small hole around the seed. This was mixed with soil in a ratio of 1 to 2 (see Figure 2) because the seed is very sensitive to chemical compounds and that direct exposure can be harmful. Because of that, it was decided to place the seed on a thin layer of soil between the zeolite 5A and the root of the seed, hoping that the root, with the passage of time and greater consistency, will have contact with the compound. For its proper growth, the cacao seed should be placed in the substrate so that only half of it is covered.

2.9. Statistical Analysis

For the analysis of the different variables used in this work, Minitab^® Statistical Software version 2021 [46] and a statistical analysis of variance (ANOVA) with Tukey’s comparison method (p ≤ 0.05) were used to define the statistical significance of the treatment used with respect to the control samples. Among the morphological variables analyzed were height, root size, and Ni⁺² concentration in the aerial part of the seedling (stem and leaves).

By means of the analysis of variance, the significance of the treatments was identified by means of Tukey’s comparative analysis for each factor evaluated. For groups that did not show significant differences in the statistical analysis, the same letter was assigned, and the null hypothesis was accepted, while for groups that were significantly different, different letters were assigned, and the null hypothesis was rejected. For this experiment, we considered the three genotypes ICS-39, CCN-51, and TSH-1188 which were labeled as G1, G2, and G3, respectively; similarly, for the statistics we compared the factors of Dose and Genotype interaction for the variables analyzed for the different D0, D1, D2, and D3, as shown in

Table 2. Labels used for the description of the treatments on each genotype.

Genotype	G1		G2		G3
	ICS-39		CCN-51		TSH-1189
Dose of Zeolite (g)	D0	D1		D2		D3
	0	1		2		4

.

2.10. Ni⁺² Determination Using Atomic Absorption Spectroscopy

The aerial and root parts of the sacrificed samples were separated at the end of 90 days; then they were disinfected in a 0.5% HCl solution for 30 s, and finally the plant parts were placed in manila envelopes to be dried in an oven at 60 °C. Next, the aerial and root parts were weighed to calculate the dry matter. After that, they were crushed in a mill and passed through a 20 mm sieve, and finally they were stored for later analysis. After crushing all the plant parts, a quantity of 0.40 to 0.50 g was weighed in a digester tube, 8 mL of nitric acid was added, and they were taken to the digester block for a period of approximately 10 h at 130 °C; after the digestion process, each sample was diluted in a 50 mL flask with distilled water. The Ni⁺² concentration was analyzed in an Varian 55B atomic absorption spectrophotometer [44,47].

3. Results and Discussion

3.1. Characterization by X-ray Diffraction and SEM Microscopy

3.1.1. Soil Samples

As mentioned in Section 2.3, soil samples were structurally characterized by X-ray diffraction, obtaining similar diffractograms, as shown in Figure 3, presenting a trigonal structure with space group P312 and lattice parameters a = b = 4.91 Å, c = 5.40 Å for V1-V3 and a = b = 4.90 Å, c = 5.40 Å for V1 according to the CIFs found (Section 2.2).

Furthermore, we also corroborated the peaks identified in the works of [48,49], as shown in Figure 2. The results V1-V4 corresponded to the soil samples used to perform the experiment, where the presence of silica was observed in its entirety. In addition, they confirmed the integrity of the used substrate, which was not affected during the experiment.

3.1.2. Zeolite 5A

At the time of performing the Rietveld refinement, it was taken into account that the sample corresponds to a hydrated zeolite phase; therefore, the contribution of oxygen corresponding to the water molecules (H and O) was added due to the significant impact of the tetrahedra formed by the T-O-T bonds, which increased the intensity of the peaks between the angles of 20° and 40°, and a better refinement was obtained [50]. The mean crystallite size was 85 nm. Figure 4 shows a magnification of the region corresponding to the interval from 25 to 35° where the contribution of intensities of the oxygens can be observed. Figure 5 represents the zeolite 5A structure before and after Rietveld refinement.

3.1.3. Raman Spectroscopy Analysis

The Raman spectrum showed the characteristic vibrational modes for zeolite 5A located in the region from 100 to 1200 cm⁻¹; see Figure 6.

Table 3. Active vibrational modes identified for zeolite 5A.

Band Assignment	Raman Shift (cm−1)
Double ring	248 283 337 412 699
Molecular Bonding T-O-T	491
Molecular T-O	971 1038 1113

summarizes the main Raman modes ranging from 248 to 412 cm⁻¹, corresponding to the nodes caused by the rings of zeolite 5A (Figure 5). These frequencies do not change with respect to the Si/Al ratio, while the strong peak corresponding to 491 cm⁻¹ is linked to the stretching modes corresponding to the T-O-T bonds (four Si₂Al₂O₄ ring) [51], and the peak at 699 cm⁻¹ is related to the two-ring configuration of the zeolite 5A, as seen in Figure 5. In addition, we found three Raman bands corresponding to 971, 1038, and 1113 cm⁻¹, which were assigned to the T-O bonds.

3.1.4. SEM Analysis (Zeolite 5A Integrity in Soils)

Integrity of zeolite 5A (Z5A) and soils was analyzed was SEM. Figure 7a–e depict the featured morphology for the cubic Z5A structure, which agrees with the refined structure in Figure 4. When analyzing soil samples containing Z5A at selected doses (Figure 8), it is seen in

Table 4. Quantitative atomic composition found in soil samples. Errors represents ±0.1 of estimated values.

Sample	Al (%wt)	Si (%wt)	O (%wt)	Fe (%wt)	Ti (%wt)
V1	15.3	25.9	51.1	6.8	0.9
V2	15.1	22.2	57.3	4.8	0.6
V3	12.5	22.6	60.1	3.9	0.9
V4	13.9	25.1	55.4	4.9	0.7
V5	20.6	22.1	57.4	-	-

that the atomic ratio between Al and Si varies compared to that of pure Z5A, indicating the presence of other natural elements such as Fe, Na, and Ti, as proven by chemical and X-ray diffraction analyses. Irregular morphologies were observed in the EDS mapping image for V samples, and it was not possible to observe the cubic regular forms for Z5A. This last result indicates that Z5A integrates to the soils forming a homogenous mixture, which favors Ni²⁺ adsorption, as discussed in Section 3.2.3.

3.2. Analysis of Morphological Variables and Concentration of Ni⁺²

In the figures presented in the next subsections, the colors used for the graphs correspond to the different doses of zeolite 5A applied to genotypes G1, G2, and G3, i.e., red (D1), green (D2), and blue (D3). By means of ANOVA analysis, statistically significant differences (p < 0.05) were observed in the interaction Dose × Genotype (D×G) in terms of the analyzed variables such as height, root, and heavy metal concentration in each seedling. Above each histogram, the letters a, b, c, and d denote the groups that had statistical significance among themselves, within each treatment. For detailed analysis, please see the Supplementary Material section (Tables S1–S18).

For seedlings that were neither exposed to the contaminant nor to the treatment, the morphological parameters on average for each genotype were the following: 29.47 ± 0.92 cm, 26.47 ± 0.98 cm, and 27.57 ± 2.13 cm for the heights of genotypes G1, G2, and G3, respectively; while for the length of roots, the average was 33.83 ± 5.86 cm for G1, 31.23 ± 3.44 cm for G2, and 29.13 ± 2.21 cm for G3.

3.2.1. Height

To determine the height of the cacao seedlings, a morphological monitoring was carried out every 15 days; Figure 9 shows the average height for each genotype 90 days after the start of the experiment. As shown, significant differences were found (p < 0.05) in terms of the factors evaluated with respect to height and the interaction of the dose with respect to each genotype where zeolite 5A was applied. The treatments applied with a dose of 1 g (D1) for genotypes G2 and G1 with a mean height of 13.63 ± 0.12 cm and 12.67 ± 0.06 cm were compared to the negative control with Ni⁺², which had an average height of 10.02 ± 0.03 and 10.17 ± 0.02 cm, respectively. Therefore, it can be inferred that this dose is optimal. For the application of D2, height averages of 14.12 cm for G1 and 6.12 cm for G3 were obtained, so the optimal dose in this case was D2 for G1.

Finally, regarding the treatments with D3 for G1 and G3, G3 showed greater growth than the negative control. Figure 9 can be interpreted as follows: It was determined that for treatment D1 (red color) there was a significant difference with respect to the height variable for G1 and G2. Similarly, the significance of treatments D2 and D3 was corroborated.

3.2.2. Root

For the evaluation of root length for each treatment, a mean of 3.71 ± 0.01 cm and 4.23 ± 0.2 cm was obtained for G1 and G2, while their negative controls were 2.13 ± 0.15 cm and 3.05 ± 0.15 cm respectively. For the treatment with D2 in G1 and G3, an average length of 4.43 ± 0.05 cm and 2.63 ± 0.3 cm was obtained, and the treatment for G2 was significant. On the other hand, for D3 in G1 and G3, average lengths of 5.43 ± 0.05 cm and 3.05 ± 0.15 cm were obtained, which were significant with respect to the negative control, as shown in Figure 10.

When Ni⁺² concentration in the soil is high, it tends to accumulate in the roots, and therefore cause the essential displacement of other essential elements for plant development when compared to non-exposed seedlings. Some cases have been reported where Ni⁺² excess affects the homeostasis of ions circulating in the plasma membrane, which in turn causes a decrease in the mobility of solutes such as phosphates and nitrates, affecting photosynthesis and cellular respiration [52].

As shown in Figure 10, the average root length varies between 2 and 5 cm, which is a very small size compared to a seedling in normal growth, whose length ranges between 25 and 35 cm. This is mainly due to the availability of Al⁺³ in the substrate due to the acid medium, which causes a negative effect in terms of stem development on root size, as will be seen later [53]. As shown in the soil analysis in Table 1, this is because of the ratio (Ca + Mg + K)/Al is <1, which is an ideal value, and the contributions of Al and Si by the zeolite 5A are probably responsible for root growth.

As mentioned in previous paragraphs, the availability of Al⁺³ in the substrate has an influence on the growth of the root part of the seedling, especially considering that the compound used is an aluminosilicate. This is because the substrate has an acid pH, which favors a better mobility of Ni⁺² due to the abundance of H⁺. As Ni⁺² and Al⁺³ become available, they occupy ion exchange sites in the substrate, generating a deficiency in essential micronutrients such as Fe, Mg, and K, resulting in a reduction in root length.

3.2.3. Ni⁺² Concentration in Seedlings

It should be first considered that the initial Ni concentration in the Juan Bernito station within the ICT was 1.83 mg kg⁻¹, and in reference [34] it is mentioned that in the San Martin region Ni⁺² concentrations vary between 1.64 and 13.69 mg kg⁻¹. The Ni⁺² concentrations in the substrate decreased from 70.21 to 25.34 mg kg⁻¹, 90 days after contamination. By only observing the variation in the mentioned concentration, it can be supposed that part of this variant concentration was adsorbed by zeolite 5A and by the seedling.

Table 5. Ni⁺² concentrations with respect to the dry matter obtained from the aerial part of each seedling.

Dose	Genotype	Dry Matter	Ni Concentration without Treatment (µg g−1) ±5.77 × 10−4	Ni Concentration with Treatment (µg g−1)
D1	G1	0.659	0.0832	0.0194 ± 1 × 10−4
D2		0.711	0.0831	0.0075 ± 5.77 × 10−5
D3		0.661	0.0831	0.0042 ± 2.65 × 10−4
D1	G2	0.725	0.0830	0.026 ± 1 × 10−3
D2	G3	0.863	0.0832	0.0373 ± 1 × 10−4
D3		0.393	0.0831	0.0095 ± 3.21 × 10−3

shows the Ni⁺² concentrations obtained at the end of 90 days for each genotype under each dose applied with respect to the dry matter obtained from the aerial part of each seedling.

It can be seen that the seedlings subjected to treatments with D1 have a high significance with respect to the seedlings with purely Ni⁺², as shown in Figure 11. This denotes the high capacity of zeolite 5A to adsorb Ni⁺², whose mean concentrations are between 0.0194 ± 1 × 10⁻⁴ (G1) and 0.026 ± 1 × 10⁻³ µg g⁻¹ (G2) compared to the 0.0832 ± 5.77 × 10⁻⁴ µg g⁻¹ presented by the Ni⁺² control.

The significance of the D2 treatments with respect to the G1 and G1 genotypes can also be noted, where the average concentrations in the seedlings were around 0.0373 ± 1 × 10⁻⁴ µg g⁻¹ for D2 × G3 and 0.0075 ± 5.77 × 10⁻⁵ µg g⁻¹ for D2 × G1; this can be contrasted with the adsorption and absorption percentages in Table N3, with zeolite 5A being a good adsorbent. Regarding the concentrations, we can say that by means of the ionic exchange processes that occur in the substrate mixed with zeolite 5A, the Ni⁺² will have a greater mobility. We must also consider that, in this medium, Ni⁺² forms complexes such as NiSO₄ in the presence of sulfate ions present in the substrate either due to remaining fertilizers or organic matter.

Moreover, the osmotic process of the plant involves the exchange of H⁺ and OH^- ions, as well as the absorption of micronutrients necessary for the plant such as Ca⁺², K⁺, and Mg⁺², in such a way that ionic equilibrium is maintained, so that an electrical balance of ions is also maintained. As there is a high concentration of Ni⁺², there is a large quantity of excess Ni⁺² ions, and zeolite 5A, having a Si/Al ratio equal to 1, is a hydrophilic compound, as demonstrated by the Rietveld method; hence, it hydrates in the presence of water molecules and retains them in its central cavity (which is larger). Similarly, the Ca⁺² cations present in the zeolite 5A are polarized, generating an electrical imbalance within the zeolitic structure, and generating the Ni adsorption. As a consequence, the quantity of Ni absorbed by the seedling through the root is lower than the concentration of this metal in the substrate.

Table 6. Percentages of absorption and adsorption of Ni by the seedling and zeolite 5A.

Dose	Genotype	Dry Matter	Absorption *	Adsorption **
D1	G1	0.659	23.47%	76.53%
D2		0.711	9.11%	90.89%
D3		0.661	5.14%	94.86%
D1	G2	0.725	31.44%	68.55%
D2	G3	0.863	44.92%	55.07%
D3		0.393	11.39%	88.60%

Percentage of Ni adsorbed by seedling (*); Percentage Ni adsorbed by zeolite 5A (**).

shows the percentages of adsorption and absorption of Ni by the zeolite 5A and the seedling; this amount was obtained by taking the amount of Ni contained in the respective seedlings contaminated only with Ni for each genotype as a reference. Thus, it showed an increasing proportion between the dose and the amount adsorbed. In addition, it can also be noted that for each treatment applied to each genotype there is a close relationship between the percentage of Ni absorption by the seedling with respect to the absorption capacity of heavy metals. This is consistent with [44], who reports that genotype G1 is a variety with a low capacity for absorption of heavy metals, which for this work was approximately 9% and 5% for doses D2 and D3, respectively. By comparison, genotypes G2 and G3 have the highest percentages of Ni absorption by the seedling, of 31% and 44% for doses D1 and D2, respectively.

Figure 12 shows the zeolite 5A dependence on the Ni absorption and adsorption for genotype G3, while for G1 only the adsorption percentages for D1 and G3 have the same tendency. The higher the dose, the higher the Ni adsorption, and the mentioned doses were optimal for each genotype. It should be mentioned that in this work only Ni was studied because this metal is the most abundant ion in the substrate used for substrate remediation. However, it cannot be discounted that the proposed material also acts on the adsorption of other heavy metals, since in [24] it is mentioned that zeolites have the chemical affinity to adsorb other divalent cationic metals such as Pb⁺² and Cu.

3.2.4. Mechanism of Ni Adsorption

To understand the mechanism of Ni adsorption within the substrate, several factors must be considered, for example, the physical characteristics of the soil such as pH, cation exchange capacity, B.S., and the availability of ions within the composition of the substrate. Considering that the soil has a pH of 4.3, a B.S. of 30.26, and E.C. of 0.08 dS/cm, it can be concluded that the substrate used is very acidic, so the quantity of acid cations (H⁺ and Al⁺) is abundant in comparison to other elements such as Ca, Mg, K, and Na, and organic matter of 0.81 [54].

Using this as a starting point, we can confirm that the substrate had a high concentration of acid cations prior to being contaminated with Ni⁺², which is a significant explanation of the mechanism since the medium was “wrapped” and rich in H⁺ and Al⁺, which will encourage the predicted cationic exchange.

But how exactly does it work? Since we know that Si-O and Al-O tetrahedra form zeolite 5A structure (see Figure 5), and because it is probably for Al⁴⁺ ions to be substituted by Al³⁺ because they have a certain similarity in terms of their ionic radius and charge; in other cases, the new configuration forms the same structures as the initial substituted tetrahedra in zeolite 5A.

The oxygen atoms can be shared between two of these units, that is, one of the vertices of the tetrahedron is shared, as shown in Figure 13, and it is common to find these ions in soils since these ions are acidic components of the soil. Thus, the zeolite 5A is in contact with this medium (homogeneous substrate) and the Al⁺³ (pink spheres) ions replace the Al⁺⁴ (purple spheres) ions, therefore causing Ca⁺² (light blue spheres) replacement by Ni⁺² (green spheres) cations via cation exchange favored by the zeolite 5A structure [18] (see Figure 13).

Unfortunately, due to the large amount of substrate used in each treatment, the stoichiometric variation in the zeolite 5A could not be measured using physical methods; nonetheless, the dosages employed in the treatments revealed a satisfactory Ni⁺² adsorption capability under the circumstances tested. It is crucial to note that the mode of application of the chemical is significant, as described in Section 2.8, since optimal dosages of zeolite 5A for the treatments were identified in this work, and doses greater than those mentioned might be harmful to the growth of the cacao seedling.

4. Conclusions

Commercial zeolite 5A phase was characterized by the Rietveld method and Raman microscopy. It was demonstrated that zeolite 5A was hydrated and the simulated structure was obtained. The integrity of zeolite 5A in soils before and after remediation experiments was evaluated by SEM microscopy and quantitative EDS mapping. These results suggest that zeolite 5A was homogenously distributed in the substrates. Morphological parameters (root and height) were not significantly affected by increasing zeolite 5A doses. The efficiency removal was demonstrated to increase with adsorbent dose and was the highest in G1. The results showed that zeolite 5A was able to immobilize high Ni concentrations contained in the substrate. Adsorption percentages of 91% and 95% were obtained for doses of 2 and 4 g applied to genotype ICS-39 (G1), respectively, which shows that this genotype has a low Ni absorption capacity. By comparison, for genotype CCN-51 (G2), it was observed that the optimum zeolite 5A dose was 1 g, thus obtaining an adsorption of 69%. Finally, for genotype TSH-1188 (G3), the optimum dose was the application of 2 g. The pH of the initial solution (4.3) is a key factor affecting the adsorption properties of Ni since it influences the electrostatic interactions between the adsorbate and the adsorbent. The electronic imbalance occurring in the substrate between Al species favored Ca replacement by Ni cations, hence favoring a cation exchange mechanism between the zeolite 5A structure and Ni. These novel findings indicate that zeolite 5A is a strong candidate for Ni remediation in soils and Theobroma cacao L.