*Article* **Nematicidal Activity and Phytochemistry of Greek Lamiaceae Species**

#### **Nikoletta G. Ntalli 1,\*, Efstathia X. Ozalexandridou 2, Konstantinos M. Kasiotis 3, Maria Samara <sup>1</sup> and Spyros K. Golfinopoulos 2,4,\***


Received: 23 June 2020; Accepted: 30 July 2020; Published: 1 August 2020

**Abstract:** Natural pesticides are in the forefront of interest as ecofriendly alternatives to their synthetic ancestors. In the present study, we evaluated the nematicidal activity of seven Greek Lamiaceae species and discerned among principal components for activity according to GC-MS analysis. Care was taken that all botanicals used were easily prepared without employing elaborate procedures and toxic solvents. We established the *in vitro* EC50 values of the hydrosols of *Origanum vulgare* L., *Mentha piperita* L. and *Melissa o*ffi*cinalis* L. and the water extracts of *Origanum vulgare*, *Thymus vulgaris* L., *Thymus citriodorus* (Schreb), *Rosmarinus officinalis* (Spenn), and *Ocimum basilicum* L. against *Meloidogyne javanica* (Treub) and *Meloidogyne incognita* (Kofoid & White). Furthermore, we amended nematode-infested soil with powdered leaves and flowers of *O. vulgare* to assess for efficacy. According to *in vitro* studies, the most active botanical preparations against both nematode species was *O. vulgare*, as regards its hydrosol and water extract. *Thymus citriodorus* was proved very potent against *M. javanica*, provoking 100% paralysis at 4 μL/mL after 96 h, but was only nematostatic against *M. incognita* since the second-stage juveniles (J2s) recovered movement 48 h after immersion in test solutions. Interestingly, *O. vulgare* was also proved nematicidal in pot bioassays but at test concentrations over 50 g/kg was phytotoxic for tomato plants. According to GC-MS analysis, the principal components sustaining activity of *O. vulgare* are carvacrol and thymol. The nematicidal activity of *O. vulgare* seems promising in the forms of essential oil leftovers (i.e., hydrosol), self-prepared water extract that can be of consideration as α "basic substance", and powder for soil amendment.

**Keywords:** natural substances; nematicidal; root-knot nematodes; oregano; soil amendments; basic substances

#### **1. Introduction**

Nematodes are among the most complex and numerous organisms on the planet. The name Nematoda, or Nematelminthes, is derived from the Greek word "νη´μα" (thread or threadworms). They belong to the kingdom Animalia, phylum Nematoda [1]; and after arthropods, they form the second most numerous group of Metazoa. They have the form of a worm, with a cylindrical, elongated body and a circular cross-section. Their diffusion on earth is wide due to their ability to adapt easily

due to their inner and outer morphology [2]. Nematode species that cause damage to cultivated plant species are called plant-parasitic nematodes. Root-knot nematodes (*Meloidogyne* sp.) cause considerable damage to more than 5000 plant species and use their stylets to feed on the roots of the plants. [3]. *Meloidogyne incognita, M. javanica*, and *M. arenaria* infect *Solanaceae* and *Malvaceae*, have broad host ranges, and are in the list of the most economically damaging root-knot nematodes [4]. On the other hand, tomato is one of the most significant crop hosts of *Meloidogyne* spp. and in the presence of disease complexes with soil pathogens the subsequent damages may even lead to total crop loss [5].

Plant nematodes are not controlled using just one method but are tackled by a combination of methods in the context of an integrated system for management of harmful organisms [6]. Although many synthetic pesticides have been used in the past as chemical nematicides, only a few are still authorized according to European legislation [7]. This fact creates an urgent need for discovering less toxic and environmentally friendly substitutes for commercial use [8]. The use of plant secondary metabolites, as well as the reutilization of culture's debris can potentially constitute a powerful weapon for plant protection. Efforts for a transition to an environmentally friendly crop protection pave a new way towards pest control. The essential oils (EOs) of aromatic plants are used in many fields because of their antimicrobial, antifungal, antioxidant, and antibacterial activities [9]. The biological activity of EOs is related to their chemical composition, which is influenced by the specific climatic, seasonal, and geographic conditions affecting the aromatic species from which EOs derive [10]. It is accepted that the EOs exhibit efficacy against insects [11] and nematodes [12]. We have previously shown that the EOs of the family Lamiaceae, including the species *Mentha pulegium* L., *Origanum vulgare*, *Origanum dictamnus* L. and *Melissa o*ffi*cinalis* display powerful *in vitro* nematicidal activity [13]. In another study using material from different botanical families, a consistent suppression of *M. incognita* population on tomato roots was evident after soil drench treatments with water emulsions of EOs from *Schinus molle* L., *Cinnamomum camphora* L., *Eugenia caryophillata* L., *Cinnamomum zeylanicum* (J. Presl), and *Citrus aurantium* L. [14]. Likewise, the EOs of *Eucalyptus citriodora* Hook, *Eucalyptus globulus* (), *Mentha piperita*, *Pelargonium asperum* (L'Hér), and *Ruta graveolens* L. were proved to be nematicidal against *M. incognita* [15], while *Dysphania ambrosioides* L., *Filipendula ulmaria* L., *Ruta graveolens*, *Satureja montana* L. and *Thymbra capitata* L. EOs revealed EC50 values lower than 0.15 μL/mL against root-knot nematodes according to Faria et al. [16]. The bioactive EOs' principal components are the terpenes, playing an essential protective role for the plants [17]; their significant synergistic and/or antagonistic activities compose the overall efficacy against target organisms [18,19].

According to SANCO draft working document 10472, reduced data requirements are described for plant protection products made from all edible parts of plants of human and animal feed. The document refers to plant extracts made with water or ethanol from parts of plants currently authorized as herbal drugs according to European Pharmacopoeia. This manufacturing process (e.g., crushing, drying, and water and/or ethanol extraction) is not considered to modify the toxicity or ecotoxicity profiles [20,21]. Moreover, the regulation 1107/2009 (EC2009) contains some facilitation for "low-risk active substances" and for "basic substances", that is, "nematicidal recipes" prepared by the farmer with low risk of harmfulness for soil, water, air, plants, or animals [22]. In this frame, water extracts prepared from *O. vulgare* and *T. citriodorus* may plausibly be developed as "low-risk plant protection products" or "basic substances".

The aim of this study was to examine the nematicidal effect of botanical extracts that are easily prepared from seven Greek Lamiaceae species without employing elaborate procedures and toxic solvents. Interestingly, we used *T. citriodorus* in a recent study to test for nematicidal activity against *M. incognita* and *M. javanica*, but with a different extraction protocol [23], thus herein we evaluate any subsequent differences in activity. Specifically we study the *in vitro* nematicidal activity of the hydrosols of *Origanum vulgare*, *Mentha piperita,* and *Melissa o*ffi*cinalis* and the water extracts of *Origanum vulgare*, *Thymus vulgaris*, *Thymus citriodorus*, *Rosmarinus o*ffi*cinalis* and *Ocimum basilicum* against *Meloidogyne javanica* and *Meloidogyne incognita*. Furthermore, we evaluate *in planta* the most

effective botanical species, i.e., *O. vulgare*, in the form of powdered leaves and flowers amended into soil that has been artificially infested by nematodes and assess for efficacy. The outcomes of the study are self-prepared water-based extracts that can be of consideration as "basic substances" or "low-risk" active ingredients for plant protection products, as well as soil bio-amendment practices that can plausibly be integrated into IPM schemes and/or organic farming.

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

#### *2.1. Aromatic Materials*

The aromatic materials tested for the nematicidal activity against *M. javanica* and *M. incognita* include: (a) hydrosols obtained as aquatic leftovers after EO obtainment from Clevenger distillation of *Origanum vulgare*, *Mentha piperita*, and *Melissa o*ffi*cinalis* from the Aitheria Company based in Velvento, Kozani, Greece, and (b) water extracts from powdered plant parts of *Origanum vulgare*, *Thymus vulgaris*, *Thymus citriodorus*, *Rosmarinus o*ffi*cinalis*, and *Ocimum basilicum* obtained from the Ethericon Greek Herbs company based in Larisa, Greece.

#### *2.2. Plant Extraction*

The extraction of plant powders was performed with the Sonicator Branson 2510 ultrasound device. Initially, 5 g of plant residue powder was weighed and placed in a beaker with 25 mL of water, forming a ratio of 1/25 (*w*/*v*). The mixture was carefully stirred in order to ensure even distribution and placed in the Sonicator device. Afterwards, distilled water was added to the volume needed to surpass the surface of the solvent in the beaker, and the mixture was sonicated for 15 min. Finally, the water extract was obtained by filtering through cotton (filtering had no effect on the recovery of the constituents assessed by GC-MS). To proceed to GC-MS chemical analysis, water extracts and hydrosols were subjected to subsequent extraction with two portions of petroleum ether (25 mL each). The combined organic phases were dried (MgSO4), evaporated to dryness using N2 stream, filtered with Chromafil syringe Nylon filter (0.22 μm, Macherey Nagel GmbH & Co. KG, Düren, Germany), and injected into the GC-MS system.

#### *2.3. Nematode Culture*

A population of *M. javanica* and another of *M. incognita* were obtained upon a single eggmass per species and were reared on tomato (*Solanum lycopersicum* L.) cv Belladonna, a variety that is particularly sensitive to root-knot nematodes. The tomato plants used for the development of the population of root-knot nematodes were maintained in a growth chamber at 25–28 ◦C, 60% relative humidity and 16 h photoperiod in plastic plant pots (18 cm diameter) containing a 10:1 (*v*/*v*) mixture of peat and pearlite. These conditions remained stable at these levels throughout the whole duration of the experiments. Plants used for inoculations were 7 weeks old, at the five-leaf stage. After 40 days, the plants were uprooted, and the roots were washed to remove any soil residue and cut into 2 cm pieces. The roots were placed in a solution of 1% sodium hypochlorite, and the suspension was shaken for 5 min. Then, it was washed in running water through nested sieves of a 250 and 38 μm cross-section, and the eggs of the nematodes were recovered and finally transferred in Baermann modified funnels at 28 ◦C [24]. The water suspension with the nematode eggs was placed in filter paper inside a sieve (2 mm hole size) and secured in a plastic tray with distilled water. All second-stage juveniles (J2s) hatching in the first 3 days were discarded. After an additional 24 h, J2s were collected and used in the experiments.

#### *2.4. J2 Paralysis*

Appropriate amounts of hydrosol and extract solutions were used in order to achieve the concentration range for EC50 calculation. The solutions were mixed with the suspension of nematodes, in the wells of a polystyrene plate of 96-well plates at a ratio 1:1 (*v*/*v*), and the final volume per well

was 140 μL. Each test solution was 2 × so as to reach the expected concentration after mixing with the nematode suspension.

Five replicates were performed on five concentration levels covering the range of 25 to 200 μL/mL for all treatments. Regarding the best effective ones, which were the water extracts of *O. vulgare* and *T. citriodorus,* the EC50 values were finally established using the test concentration range of 3.9, 7.8, 15.6, 31.2, and 62.5 μL/mL. Distilled water was used as control, and the nematode number per experimental well was 15–20 J2s. The plates were covered with a lid, so as to prevent evaporation which would differentiate final test concentrations, and intermediate wells were used to immerse J2 in water so as to control cross-contamination between treatments due to the volatility. The plates were placed in a chamber with stable conditions at 28 ◦C. J2 paralysis was assessed by observation in a reverse microscope (Euromex, Holland) at 40× zoom at the timepoints of 24, 48, and 96 h after establishment of the experiment. The J2s were classified into two categories: motile and paralyzed. Paralysis assessed after 96 h of immersion was characterized as death if J2s never regained motility after significantly augmenting the water volume in immersion solutions for an additional 24 h.

#### *2.5. Soil Amendment with* O. vulgare *Powder for* M. incognita *Control and Phytotoxicity to Tomato Plants*

The sandy loam soil (18% clay, 22% silt, 60% sand), with pH 6.5, 3.3% organic carbon, and 1.9 mg g−<sup>1</sup> total N, was collected from a noncultivated field of the Benaki Phytopathological Institute. Initially, it was sieved through a 3 mm sieve and partially air dried overnight; then, a mixture with sand at a ratio of 2:1 was prepared to form the hereafter referred to soil. Six plastic bags represented the experimental treatments, 1 kg of soil each receiving a nematode inoculation of 2500 J2s per kg. After appropriate mixing and overnight incubation at room temperature, according to Ntalli et al. [23], the plastic bags were spiked with appropriate amounts of *O. vulgare* powder to reach the test concentrations of 1, 5, 10, 50, and 100 g kg−<sup>1</sup> soil. A water control was also included in the experiment. Seven-week-old tomato plants, cv. Belladonna, were transplanted into the treated soil, separated in five different pots containing 200 g of soil each, and the bioassay was kept in an incubator at 27 ◦C and 60% relative humidity at a 16 h photoperiod for 40 days. Every pot received 20 mL of water every 3 days for 40 days; afterwards, plants were uprooted and gently washed. Shoots were separated from roots and the latter were stained with acid fuchsin, according to Byrd et al. [25], and the following parameters were assessed: (a) *M. incognita* females per g of root at 10× magnification, (b) fresh stem weight, and (c) fresh root weight. The experiment was performed twice, and the treatments were arranged in a completely randomized design with five replicates.

#### *2.6. Gas Chromatography–Mass Spectrometry Analyses of Water Extracts (WEs) and Hydrosols (Hs)*

The GC-MS analysis was conducted on a Chromtech Evolution 3 MS/MS triple quadrupole mass spectrometer built on an Agilent 5975 B inert XL EI/CI MSD system that was operated in full scan data acquisition mode, within the mass range from m/z 50 to 500. Samples were injected with a Gerstel MPS-2 autosampler using a 10 μL syringe. Component separations were performed on the chromatographic column HP-5 ms Ultra-Inert (UI), with a length of 30 m, inner diameter (ID) of 0.25 mm, and film thickness of 0.25 μm (J&W Scientific, Folsom, CA, USA). Helium (99.9999% purity) was used as the carrier gas at a flow rate of 1.2 mL min−1. The column oven temperature program initiated from 45 ◦C and stayed there for 1 min before increasing at a rate of 5 ◦C min−<sup>1</sup> to 250 ◦C, where it stayed for 5 min. The transfer line, manifold, and source of ionization temperatures were 300, 40 and 230 ◦C, respectively. The electron multiplier voltage was set at 2000 V. The total GC analysis time was 47 min. Identified peaks in GC-MS (triplicate analysis) were verified by matching the acquired mass spectra with those in the commercial library of NIST 08.

#### *2.7. Statistical Analysis*

Concerning the effect of the hydrosols and the aquatic extracts on J2 mobility, the experiments were repeated five times for each concentration level on an experimental project of completely randomized groups. The experiment was conducted twice.

For each paralysis test, the analysis of all data was correlated to time. The average of the two temporal repetitions for each experiment is presented, as the correlated analysis of variability did not show a significant interaction between the interventions and the execution time of the experiments.

For the statistical analysis and since the paralysis in the carrier did not differ from that measured in water, the paralysis data for both experiments were expressed as percentage rates of the paralysis values corresponding to the water control according to Schneider Orelli's equation [26]: Paralysis increase % = ((paralysis % during treatment − paralysis % in the water control)/(100 − paralysis % in the water control)) × 100.

Following the immersion in hydrosol and extract solutions, a variability analysis (ANOVA) was performed. Then the log-logistic equation of Seefeldt et al. [27] for the calculation of the values EC50 was used, according to the following equation: Y = C + (D − C)/{1 + exp[b (log(x) − log(EC50))]} where C is the lower limit, D is the upper limit, b is the slope of line in value EC50, and EC50 is the concentration of the hydrosol or aquatic extract required for the 50% increase of paralyzed J2s compared with those of the water control.

In the particular regression equation, the concentration of the hydrosol or extract (μg/mL) was the independent factor (*x*), and J2 immobility (percentage increase as compared to the water control) the dependent factor (*y*).

Concerning the pot bioassays, the means were averaged over experiments, since ANOVAs showed no significant treatment between runs. Statistical analysis was performed using SPSS 20 (IBM, Armonk, NY, USA). Both ANOVA and Duncan's test were set at *p* ≤ 0.05.

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

#### *3.1. In Vitro Nematicidal Activity*

Among all the seven species tested for their nematicidal activity, only *O. vulgare* and *T. citriodorus* have nematicidal activity at the concentration range tested (3.9 to 200 μL/mL). Table 1 presents the effect on the paralysis of J2s after their immersion for 24, 48, and 96 h in test solutions. Paralysis reported after 96 h was irreversible in all cases. In most cases, the rate of J2 paralysis was proportionate to the increase of concentration and time of immersion in test solutions. Instead, plant extracts of *M. piperita*, *M. o*ffi*cinalis*, *T. vulgaris*, *R. o*ffi*cinalis*, and *O. basilicum* did not show activity in the test concentration range (25 to 200 μL/mL)*. Thymus citriodorus* water extract (WE) was the most effective against *M. javanica*, and the observed paralysis for J2s immersed at 3.9 μL/mL was 100% at 96 h after establishment of the experiment. In contrast, *T. citriodorus* was not found equally active on *M. incognita* since paralysis obtained at 24 h was reversible 48 h after establishment of the experiment.

In the same context, previously we studied *T. citriodorus* WE obtained with a different extraction protocol applying lower eluent volume 1/10 (*w*/*v*), and the EC50/48h values were calculated to be 84.19 and 61.97 μL/mL against *M. incognita* and *M. javanica*, respectively [23]. In this study, *T. citriodorus* was extracted with water, using a ratio of 1/25 (*w*/*v*), and the respective EC50/48h value against *M. javanica* was lower than the value previously reported; meanwhile it was found nematostatic against *M. incognita*. Consequently, it appears that the solvent volume affects extraction efficiency, influencing the equilibrium constant of the analytes' partitioning between the two phases [28]. In this case, the higher amount of solvent seems to yield higher extraction recoveries of the particular nematicidal components (i.e., a more concentrated extract, not affected under these conditions by the dilution), leading to higher activity against *M. javanica*.

In fact, the WE of *O. vulgare* was the best effective against *M. incognita*, followed by *T. citriodorus*. Interestingly, *O. vulgare* hydrosol (H) only achieved paralysis against *M. javanica*, but was found inactive against *M. incognita*. *Origanum vulgare* WE achieved better paralysis of *M. incognita* than *T. citriodorus* did. It should be noted that there was no cross-contamination between the treatments due to volatility, as the mobility of the J2s in the peripheral wells, where J2s were immersed in water, did not change.

**Table 1.** EC50 (μL/mL) values of water extracts (WE) and hydrosols (H) against *M. incognita* and *M. javanica*, calculated after immersion of second-stage juveniles (J2s) in test solutions for 24, 48, and 96 h. The test concentrations used for the H of *O. vulgare* were 25 to 200 μL/mL, while for the water extracts of *O. vulgare* and *T. citriodorus* 3.9, 7.8, 15.6, 31.2, and 62.5 μL/mL.


EC50 values were not calculated (na) they were outside the test concentration range.

#### *3.2. In Planta Nematicidal Activity and Secondary E*ff*ects on Tomato Plants*

When different *O. vulgare* powder quantities were used in the pot bioassay to assess for efficacy, a clear dose–response relationship was established and efficacy was evident at 5 g/kg of soil (Figure 1) both in terms of root galls and female counts. Considering stem and root weights, no statistical differences were evident until the test concentration of 50 g/kg of soil, which was proved to be phytotoxic for the tomato plants.

**Figure 1.** Tomato stem and root weights (g), as assessed after treatment with *O. vulgare* powder for *M. incognita* control in pot bioassays 40 days after experiment establishment (**A**). Root galls and female counts per g of tomato root 40 days after experiment establishment (**B**). The data are means of ten replicates with standard deviations. Means followed by the same letter are not significantly different according to Duncan's test (*p* ≤ 0.05). Within each graph, letters correspond to statistical differences amongst same pattern bars.

#### *3.3. Chemical Composition Analysis of Water Extracts and Hydrosols of Lamiaceae Species—Nematicidal Activity Implication*

The GC-MS chemical analysis of the petroleum ether extracts of the WE and hydrosols unveiled several substances. More specifically, numerous constituents related to EOs of the respective species were identified. According to the GC-MS, the principal components in respective extracts were as follows: thymol in *T. vulgaris* WE (89.15%), carvacrol in *O. vulgare* WE (86.77%), eugenol and linalool in *O. basilicum* WE (50.75 and 34.45%, respectively), carvacrol in *O. vulgare* H (93.00%), levomenthol in *M. piperita* H (54.35%), and thymol in *M. o*ffi*cinalis* H (39.43%) (Table 2). Therefore, the predominant detections in the most active species (*Thymus* spp. and *O. vulgare*) were carvacrol and thymol. Carvacrol in *O. vulgare* WE and H displayed a profound difference in abundance from thymol.

Previously, we have demonstrated the significant individual nematicidal activity of carvacrol, along with its synergic potency with other terpenes, against *Meloidogyne* sp. [13,18]. Similarly, carvacrol has been proved to be a nematicidal component against *M. incognita* by others [29]. Interestingly, although *O. vulgare* H was richer in terms of number of constituents compared to its WE, it exhibited higher EC50 values—a fact that reveals the complexity of bioactivity interactions amongst plant secondary metabolites within an extract.

In specific, based on the abundances of the key constituents of the GC-MS chromatograms (same plant material quantity used), the H contains higher concentrations of carvacrol and thymol than the WE; however, the WE is more potent than the H. Likewise, *M. piperita* H did not exhibit significant activity, although it yielded numerous ingredients considered active on *Meloidogyne* sp., like pulegone and geraniol [13,18].

*Lamiaceae* species' EOs, extracts, hydrosols, and contained constituents are reported to exhibit nematicidal activity [13,30–34]. It is worth mentioning that, in this work, emphasis was given to the volatile and semivolatile constituents of the specific species, since many of these constituents display nematicidal activity. Nevertheless, it is expected that some of the semipolar and polar liquid-chromatography-amenable compounds (such as phenolic acids and flavonoids) can contribute to the nematicidal properties exhibited in this work. Therefore, the enhanced activity of *O. vulgare* WE and *T. citriodorus* WE might be attributed to the potential high content of phenolic acids (such as rosmarinic acid and oleanolic acid) that exhibit nematicidal properties [32,35,36] and other phenolic compounds, including their glycosides and hexosides.


Chemical and relative composition of water extract (WE) and hydrosol (H) of Lamiaceae species

 \*.

**Table 2.**

findings are indicated.

#### **4. Conclusions**

The Mediterranean basin constitutes a chemical arsenal owing to its wealth of self-sown, aromatic plants which could be used for the development of "low-risk" plant protection products and "basic substances". As the EO manufacturing industry grows, producers are imposing plans, strategies, and technology for waste management and reutilization. The reutilization of this waste for the production of a new generation of nematicides constitutes an utterly successful practice not only for plant protection but also for the environment. Our results show that the easily prepared water extracts of *T. citriodorus* and *O. vulgare*, along with the distillation waste of *O. vulgare*, can be alternatives for the control of *Meloidogyne*. Additionally, plant powder of these two species incorporated into nematode-infested soil blocks infestation augmentation and can thus be an additional practice for farmers to incorporate into an integrated nematode management frame.

**Author Contributions:** Conceptualization, N.G.N.; methodology, N.G.N., E.X.O., K.M.K. and M.S.; software, N.G.N. and K.M.K.; validation, N.G.N., E.X.O., K.M.K. and S.K.G.; formal analysis, N.G.N. and K.M.K.; investigation, N.G.N., E.X.O., K.M.K. and M.S.; resources, M.S.; data curation, N.G.N. and K.M.K.; writing—original draft preparation, N.G.N. and S.K.G.; writing—review and editing, N.G.N., K.M.K. and S.K.G.; supervision, N.G.N. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** The authors are grateful to T. Koufakis and AGRIS SA for providing seeds and seedlings. We are also grateful to Aitheria Company, based in Velvento, Kozani, Greece, and Ethericon Greek Herbs Company, based in Larisa, Greece, for the kind offer of aromatic material.

**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/).

## *Review* **Zeolites Enhance Soil Health, Crop Productivity and Environmental Safety**

**Mousumi Mondal 1, Benukar Biswas 1, Sourav Garai 1, Sukamal Sarkar 1,2, Hirak Banerjee 1, Koushik Brahmachari 1, Prasanta Kumar Bandyopadhyay 3, Sagar Maitra 4, Marian Brestic 5,6,\*, Milan Skalicky 6, Peter Ondrisik <sup>7</sup> and Akbar Hossain 8,\***


Czech University of Life Sciences Prague, Kamycka 129, 165 00 Prague, Czech Republic; skalicky@af.czu.cz <sup>7</sup> Department of Environment and Zoology, Slovak University of Agriculture, Nitra, Tr. A. Hlinku 2, 949 01 Nitra, Slovakia; peter.ondrisik@uniag.sk


**Abstract:** In modern days, rapid urbanisation, climatic abnormalities, water scarcity and quality degradation vis-à-vis the increasing demand for food to feed the growing population necessitate a more efficient agriculture production system. In this context, farming with zeolites, hydrated naturally occurring aluminosilicates found in sedimentary rocks, which are ubiquitous and environment friendly, has attracted attention in the recent past owing to multidisciplinary benefits accrued from them in agricultural activities. The use of these minerals as soil ameliorants facilitates the improvement of soil's physical and chemical properties as well as alleviates heavy metal toxicity. Additionally, natural and surface-modified zeolites have selectivity for major essential nutrients, including ammonium (NH4 +), phosphate (PO4 <sup>2</sup>−), nitrate (NO3 <sup>−</sup>), potassium (K+) and sulphate (SO4 <sup>2</sup>−), in their unique porous structure that reduces nutrient leaching. The slow-release nature of zeolites is also beneficial to avail nutrients optimally throughout crop growth. These unique characteristics of zeolites improve the fertilizer and water use efficiency and, subsequently, diminish environmental pollution by reducing nitrate leaching and the emissions of nitrous oxides and ammonia. The aforesaid characteristics significantly improve the growth, productivity and quality of versatile crops, along with maximising resource use efficiency. This literature review highlights the findings of previous studies as well as the prospects of zeolite application for achieving sustenance in agriculture without negotiating the output.

**Keywords:** soil amelioration; resource use efficiency; water conservation; nutrient retention; heavy metal toxicity

#### **1. Introduction**

The increasing pressure of the population leads to a higher food demand, and at least 50% more food production is required to meet the demand of people by 2050, without any

**Citation:** Mondal, M.; Biswas, B.; Garai, S.; Sarkar, S.; Banerjee, H.; Brahmachari, K.; Bandyopadhyay, P.K.; Maitra, S.; Brestic, M.; Skalicky, M.; et al. Zeolites Enhance Soil Health, Crop Productivity and Environmental Safety. *Agronomy* **2021**, *11*, 448. https://doi.org/10.3390/ agronomy11030448

Academic Editors: Nikolaos Monokrousos and Efimia M. Papatheodorou

Received: 4 February 2021 Accepted: 24 February 2021 Published: 28 February 2021

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scope of horizontal land diversification [1,2]. Therefore, intensive agricultural practices in food and nutritional security force the use of irrational chemical inputs, water and heavy machinery. More than two-thirds of the renewable water resources are exclusively used by agricultural activities, resulting in uneven water sharing with the other sectors [2–4]. Furthermore, the consequences of intensive practices are the degradation of soil and water qualities, such as depletion of soil organic carbon and inherent soil nutrient status, heavy metal contamination and residual fertilizer and/or pesticide mixing with groundwater visà-vis surface water resources, that dwindle crop productivity and ultimately the per capita food grain availability [5]. Long-term intensive farming activities make the agricultural land unproductive, resulting in low soil retention capacity. The most important element, nitrogen, is widely used in agricultural systems, although its use efficiency in nitrogenous fertilizers rarely exceeds 50% as it is mostly lost through denitrification, leaching and volatilisation [6]. Moreover, irrational application of nitrogenous fertilizers facilitates easy NO3 <sup>+</sup> discharge from soil to groundwater, causing negative anthropogenic impacts on the groundwater quality and public health hazards such as methemoglobinemia, cancer of digestive organs, eutrophication in water bodies and production of greenhouse gases such as nitrous oxide (N2O) through the denitrification process [7–10]. Phosphate (PO4 3+) is another major nutrient in fertilizer, also responsible for eutrophication in water bodies [11]. Therefore, soil nutrient retention is a major concern in modern agriculture to account for maximum nutrient use efficiency, improve the soil nutrient status and prevent groundwater contamination [12–14]. Nutrient use efficiency and better plant growth are highly related to soil's physical and chemical properties. In this context, the application of soil amendments, more particularly natural or organic amendments, has great importance for the long-term reclamation of soil's physicochemical properties [15–17]. Zeolites are naturally occurring, alkaline-hydrated aluminosilicates with more than 50 different forms [18,19] and a wide range of applications such as soil-binding agents and nutrient supplements for animal and aquatic lives. Additionally, they can be used as heat storage materials and solar refrigerators, both absorber and adsorber; ion-exchanging elements; molecular sieving agents; and catalysing agents in various chemical reactions [20,21]. In agriculture, the importance of zeolites has been realised to a greater extent with their varying applicability (Figure 1) [20]. Natural zeolites are being considered as good soil ameliorating substances, having good water and nutrient holding capacity (WHC); it improves infiltration rate, saturated hydraulic conductivity, cation exchange capacity, and prevents water losses from deep percolation [22–26]. Moreover, zeolites could be used as fertilizer and chelating agent [27]. Zeolites minimize the rate of nutrient release from both organic and inorganic fertilizers and enable better nutrient availability throughout the crop growth stages [27]. The improvement of the wide range of agronomic and horticultural crops in respect to growth, yield and quality traits with the application of zeolites has been well reported by various researchers [28–33]. Additionally, zeolite can effectively absorb heavy metals such as cadmium (Cd), lead (Pb), nickel (Ni), anions like chromate (CrO4 <sup>2</sup>−) and arsenate (AsO4 <sup>−</sup>3), and organic pollutants such as volatile organic compounds (VOCs) including benzene, toluene, ethylbenzene, and xylene (BTEX) from soil or water body [34–36]. Acknowledging all the aforesaid advantages, the applications of zeolites in the agricultural research field have been widely gained importance since the last two decades (Figure 2), evidenced by the chronological ascending trend of the publication rate accessed from "Scopus" online database with the keywords of "zeolit", "soil remediation", "water retention", "nutrient retention", "crop production" and "heavy metal toxicity". Several earlier findings reported the applicability of zeolites on soil properties along with water and nutrient retention capacity, crop yield and heavy metal toxicity. Therefore, it is high time to give importance to zeolites application in agricultural activities and this review article gives a comprehensive assessment on the sources of zeolites, their structure and properties, and wide application in agriculture with the special consideration of soil properties, resource conservation, pest management, pollution control and crop productivity.

**Figure 1.** Multidimensional Uses of Zeolites in Agriculture.

**Figure 2.** The Trend of Annual Publications on Zeolite Applications in Agriculture for the Last Two Decades. Source: Scopus Preview [37].

#### **2. Origin, Structure and Properties of Zeolites**

The word zeolites refer to 'boiling stones' because of their ability to froth when heated to about 200 ◦C. The first time, the mineral zeolites are identified by a Swedish mineralogist Alex Fredrik Cronstedt in 1756 [38]. However, zeolites production was started commercially in the 1960s [38]. China contributes ~75% market share of total zeolites production, followed by Korea (8%), the United States (3%), and Turkey (2%) [39]. In India, the maximum zeolitic enriched soil is found in the state of Maharashtra followed by Karnataka, Gujrat, Andhra Pradesh and West Bengal (Figure 3).

**Figure 3.** Distribution of Zeolitic Soil in India. Modified from Bhattacharyya et al. [40].

Structurally, zeolite is comprised of aluminosilicate (AlO4 and SiO4) tetrahedrons, joined into three-dimensional frameworks and seems like a honeycomb structure (Figure 4) [41]. The cages in the porous structure of zeolite are approximately 12 Å in diameter, interlinked through the channels of 8 Å diameter, includes 12 tetrahedrons rings [42]. Depending on the minerals, the pores are interlinked to form long wide channels which facilitate easy molecular movement into and out of the zeolite structure. The negative charge of aluminum ions in the zeolite structure is balanced by positively charged cations.

**Figure 4.** The Tetrahedral Framework of Clinoptilolite Zeolite. Modified from IZA [43].

The general empirical formula that refers to a zeolite structure is M2nO. Al2O3. xSiO2. yH2O. M refers to any alkali or alkaline earth cation; the valence of the cation is indicated by n, x ranges between 2 and 10, and y ranges between 2 and 7, with structural cations comprising Si2+, Al3+ and Fe3+, and exchangeable cations K+, Na+ and Ca2+ [44]. The

spacious porous structure with large channels in zeolite structure makes it unique in nature as compared to other silicate minerals [45]. Natural zeolites are loaded with aforesaid cations with various considerable properties such as higher cation exchange capacity (CEC) than normal soil, ranges between 100 and 200 centimol (+) kg−<sup>1</sup> [46], free water storage within their structural channels, and also have a great ability of ion adsorption in large surface area. Zeolites can adsorb or exchange various cations viz. strontium (Sr) and cesium (Cs); heavy metals like zinc (Zn), cadmium (Cd), lead (Pb), manganese (Mn), nickel (Ni), chrome (Cr), iron (Fe), and copper (Cu) [34]; anions such as chromate (CrO4 2+) and arsenate (AsO4 3+) [35]; and numerous organic pollutants mentioned earlier [36]. Other useful physical and chemical properties of zeolites include high void volume (~50%), low density (2.1–2.2 g/cm3), excellent molecular sieving properties and high cation selectivity exclusively for ammonium, potassium, and cesium ions [40]. Physical characteristics of some naturally occurring zeolites are summarized in Table 1. In respect to pore diameter Zeolites have been classified by Flanigen [47], viz. (i) Small-pore (0.3–0.45 nm diameter with 8 rings), (ii) Medium-pore (0.45–0.6 nm diameter with 10 rings), (iii) Large-pore (0.6–0.8 nm diameter with 12 rings), and (iv) Extra-large pore zeolites (0.8–1.0 nm diameter with 14 rings).

**Table 1.** Physical Characteristics of Some Naturally Occurring Zeolites.


#### **3. Impacts of Zeolite Application in Agriculture**

*3.1. Improvement of Soil Physical Properties*

Soil physical properties include bulk density, particle density, aeration, soil porosity, water holding capacity in which bulk density is the basic soil property that influences the total porosity and topsoil stability [48]. The application of zeolites in light texture soil reduces the bulk density that modifies the water holding capacity and soil air porosity [49]. However, total porosity is not influenced significantly [49]. In a previous study, Xiubin and Zhanbin [3] opined the natural zeolite mainly mordenite with less than 0.25 mm size to the fine-grained calcareous loess which had low WHC. Result revealed that after 25 h of water addition to treated and normal soils, the zeolites applied soil resulted in higher

water content (Figure 5). They also reported that water holding capacity in zeolites treated soil increased 0.4–1.8% in drought condition while 5–15% in normal situation as compared to non-treated soil.

**Figure 5.** Soil Water Content as Influenced by Zeolitic Soil. Modified from Xiubin and Zhanbin [3].

In another study, the effect of modified Ca+2 type zeolite on sand dune soil was determined where irrigated was given with saline water. Sand dune soil samples were treated with the three different rates of zeolite i.e., 5 kg m−2, 1 kg m−<sup>2</sup> and no zeolite (control) and irrigated with seawater diluted to electrical conductivity (EC) levels of 3 and 16 deciSiemens per metre (dSm−1). Results showed that soil with 5 kg zeolites m−<sup>2</sup> enhanced soil water as well as salt content, accounting for 20 and 1.4% higher than no application of zeolite [16]. The concentration of cations namely Ca2+, K+, Na+, Mg2+ is increased with the increasing soil salinity. The findings were attributed to the fact that zeolite increases the cation exchange capacity, and subsequent cations holding on the surface soil, and release them at the expense of salts in the saline water [22]. Thus, the low salt accumulation in subsurface soil facilitates low salt stress on plants and creates a better environment for plant growth. Lowering of particle size with the application of zeolites in sandy soil might be another reason for higher water holding capacity. Higher pore volumes in zeolites facilitate greater water holding in their structures [49]. Such structures are not damaged by water particles during surface evaporation and/or reabsorption. Zeolites may be considered as the permanent water reservoir. Retention of soil moisture in longer duration, particularly during dry periods helps to mitigate drought-induced abiotic stresses and enable plants to withstand in dry spell; zeolites also facilitate to rapid rewetting and the lateral water spreading throughout the root zone during the time of irrigation that reduces the timing of water application [41]. Soil amelioration with zeolites increases the water availability to plants by 50% [42]. Application of zeolite @ 10 g kg−<sup>1</sup> soil could maintain maximum water percentage (8.4%) at field capacity and delay in permanent wilting point in sandy loam soils [50]. Al–Busaidi et al. [16] reported that the existence of fine particles and micropores in zeolites slowed down the deep percolation of soil water. The infiltration rate is inversely proportional to zeolites application (Figure 6) indicating the higher soil water residence and subsequent restriction in nutrient and salt leaching. Xiubin and Zhanbin [3] observed that the mixing of zeolites with fine grain calcareous loess soil increased the infiltration rate by 7–30% and 50% in a gentle and steep slope respectively. Furthermore, run-off and subsequent soil erosion were reduced with the zeolites application and the sedimentation also found to be decreased by 85% and 50% in a gentle and steep slope respectively. Interestingly, a combination of zeolites and selenium application check the water deficit oxidative damages in plants [51]. Colombani et al. [52]

quantified the changes in flow and transport parameters induced by the addition of zeolites in a silty-clay soil and reported that NH4 <sup>+</sup> enriched zeolites enhanced the capacity of water retention in silty-clay soil, thus diminishing the water and solute losses. Maximum irrigation water productivity (0.81 kg m<sup>−</sup>3) under limited irrigation supply was registered with the supplemental application of zeolites (21% ww−1) along with urea, while the minimum water productivity (0.48 kg m<sup>−</sup>3) was observed under full irrigation supply and exclusive urea application [53]. Bernardi et al. [54] also observed that concentrated zeolites as a sand-soil amendment increase at least 10% of soil-water retention and 15% of available water to plants. Zeolite increases the periods between the starting of rainfall and runoff occurrence. Rainfall intensity with 10 mm per hr. results in the beginning of runoff within 15 min in normal soil while in zeolites (20%) treated soil runoff starts after 30 min of rainfall occurrence [55].

**Figure 6.** Soil Infiltration Rate as Influenced by Different Rates of Zeolite Application. Modified from Al-Busaidi et al. [16].

Zeolites help to improve the water-stable aggregates in soil. As per example, nano zeolite with 30% concentration increased the mean weight diameter of water-stable aggregates by 0.735 mm [56]. With the use of this property, Moritani et al. [57] reported that the incorporation of 10% artificial zeolites in sodic soils resulted in improved wet aggregate stability ranged between 22.4% and 59.4% depends on the soil textural classes. Cario et al. [58] categorized the soil with average assessment ranking 'good' and 'excellent' in terms of water-stable aggregates and degree of soil aggregation in Vertisols and they showed the application of zeolite along with chemical fertilizers or organic manure (Zeolite @ 7.5 t h−<sup>1</sup> + sugarcane filter cake @ 22.5 t h<sup>−</sup>1) improved the soil properties from good to excellent. Sepaskhah and Yousefi [59] conducted an experiment to justify the effect of various rate of calcium-potassium zeolite on the pore velocity of water in the soil they observed higher pore water velocity (35 and 74%) with the application of 4 and 8 g zeolite kg−<sup>1</sup> soil respectively. Changes of soil physical properties with the Zeolite application in thin (heavy) textured medium-thin textured, and medium coarse (light) textured soil was observed by Gholizadeh-Sarabi and Sepaskhah [60] reported that in fine and medium texture soil, zeolites application at the rate of 4 and 8 g kg−<sup>1</sup> of soil at the low salinity level (0.5 and 1.5 dS m−1) and 16 g zeolites kg−<sup>1</sup> soil at the high salinity level (3.0 and 5.0 dS m−1) increased saturated hydraulic conductivity significantly while in coarse texture soil similar rate of zeolites application reduced the saturated hydraulic conductivity considerably. They also assumed that zeolites application in the heavy (clay loam) and medium-textured

soil (loam) changed the shape and size of the soil pores and resulted in an improvement of soil structure and the water movement in these soils. Zeolites application alleviates the adverse effect of salinity on hydraulic conductivity and thus it would prevent waterlogging in heavy and medium soil textures. In case of sandy soils, zeolites addition would be appropriate to decrease the hydraulic conductivity and the transferability of water that results in low deep percolation and loss of soil water. However, Razmi and Sepaskhah [61] reported that the application of zeolite (8 g kg<sup>−</sup>1) in silty clay soils significantly improved the hydraulic conductivity. They also established that the soil treated with zeolite resulted in 50% less crack depth in dry puddled soil with pre-application of zeolites in comparison to no zeolites application. A similar observation was also recorded after the first and second irrigation in puddled condition (Figure 7).

**Figure 7.** Effect of Zeolite on Crack Depth in Puddled Transplanted Rice. Modified from Razmi and Sepaskhah [61].

Furthermore, the sorptivity of clay-loam soil was reduced with a higher rate of zeolites application as reported by Gholizadeh-Sarabi and Sepaskhah [60]. However, a contrasting result was observed in the case of sandy-loam and loamy soil. Proper use of water is the immediate need in agriculture to ensure food security with available water resources; hence, technologies that enhance water use efficiency are being widespread. The aforesaid discussions indicate that zeolites addition positively influence the inter-particle porosity as well as total porosity, bulk density, hydraulic conductivity, infiltration rate, and cation exchange capacity of soil that ultimately accelerates the soil water content. Additionally, the open pore network channels into zeolites structure mainly play the significant roles' in water retention. The summarization of zeolitic impacts to the wide range of soils in Table 2 indicates that the use of zeolites as a soil ameliorant would be a welcome strategy in agriculture.


**Table 2.** Physical Properties of Soils as Influenced by Zeolites Application.

#### *3.2. Nutrient Retention*

Zeolites positively influence the physical, chemical, and biological properties of soil directly or indirectly which in turns improves the nutrient dynamics as well as nutrient retention capacity. Zeolitic minerals have high CEC which attributes to high NH4 <sup>+</sup> sorption selectivity as a consequence of the electrostatic attraction between positively charged NH4 <sup>+</sup> and negatively charged sites in zeolite structure [63,64]. The effective diffusion coefficient was around 4–5 × 10–12 m2 <sup>s</sup>−<sup>1</sup> for ammonium and sodium ions respectively in clinoptilolite [65,66]. The adsorption capacity of zeolites for these ions is determined by isotherms and kinetics and this adsorption property is used for various purposes such as wastewater treatment, heavy metal removal. Clinoptilolite generally exhibits a high selectivity for NH4 <sup>+</sup> ion, having theoretical CEC of 2.16 cmol (+) kg−<sup>1</sup> [67]. I on adsorption efficiency of zeolites are mainly depends of the factors like mass, particle size, initial concentration of cations of model solution, contact time, temperature and pH [68,69]. Additionally, modification of zeolites surface with strong acids accelerates the cation sorption capacity [70]. The modification of natural zeolites includes pretreatment by grinding and sieving, mixing with sodium salt and finally, calcinations makes a change in the pore size and surface area of zeolites, and thereby the ammonium ion uptake is increased [71].Soil application of zeolites in combination with chemical fertilizers reduces nitrogen leaching [72–74] and volatilization [75–77] slows down the mineralization process and subsequent reduction in greenhouse gases (GHGs) emission [78], and retards the nutrients release into soil solution [79,80]. In the incubation studies, researchers had clearly seen the difference in the ammonia loss with chemical fertilizers and chemical fertilizers with zeolite and reported low ammonia losses when fertilizer applied with zeolite [81,82]. Omar et al. [83] proved the significant improvement in soil exchangeable ammonium retention by 40–50% in zeolite treated soil. The leaching reduction of NH4 <sup>+</sup> and NO3 − from different nitrogenous fertilizer with the application of zeolite is depicted in Table 3. The zeourea and nano-zeourea contain 18.5% and 28% of N respectively and capable to release N up to 34 and 48 days, respectively, while from conventional urea the N releases within 4 days after application [84]. The reason behind this may be the urease activity is significantly reduced by zeolite application that lowers the nutrient release from fertilizer [85]. The slow-released nature of fertilizer helps to release their nutrient contents gradually and to coincide with the nutrient requirement of a plant [86].

**Table 3.** Leaching Reduction Percent of NH4 <sup>+</sup> and NO3 − from Different Nitrogenous Fertilizer with the Application of Zeolite.


\* With the unit of t ha−1; † With the unit of mg kg−1; ‡ With the unit of mg L−1.

Urea saturated zeolite chips have also been developed elsewhere. Piñón-Villarreal et al. [92] experimented to assess the leaching loss from urea ammonium nitrate solution (UAN32) where 443 mg total N was present per liter of solution. They observed that 82% reduction in leaching loss happened from the pure clinoptilolite zeolite loaded column in comparison to the column of loamy sand. In a sorption experiment, Piñón-Villarreal et al. [92] reported more than 90% NH4 <sup>+</sup> absorption by zeolite incorporated soil in initial several minutes. Very small particle size with a greater surface area of zeolitic minerals accelerates the stabilization of exchange equilibrium in only a few hours. Zeolite minerals also protect the

conversion of NH4 <sup>+</sup> to NO3 <sup>+</sup> through the nitrification process. The latter is more prone to leach out into the soil and facilitates to groundwater contaminations [59]. The small pores in zeolite crystal lattice structure (4–5 Å) in which cations like ammonium can easily adsorb, do not give access to the nitrifying microorganisms into the pores [93]; thus, nitrification does not take place easily in zeolites treated soil. One of the most usefulness of zeolite is utilized in compost making a way to convert agricultural farm waste into valuable organic amendments. However, a significant amount of N losses take place during the time of composting [94]. In an experiment, Ramesh and Islam [95] confirmed that the application of 14–21% zeolite in fresh manure resulted in low ammonium loss. Zeolite also could absorb volatile substances such as acetic acid, butanoic acid, skatole and isovaleric acid and also could effectively control the odor released during composting [96,97].

The extent of reduction in total nitrogen and even phosphorus losses with the application of zeolite into organic manure was successfully reported by Murnane et al. [98]. The reason behind the low N losses from manure is the high specific selectivity of zeolites to ammonium (NH4 +) that helps in holding this ion during volatilization. Moreover, the existences of small internal channels protect NH4 <sup>+</sup> from rapid nitrification by microbes [99]. Interestingly, zeolites not only help to protect the N loss but also reduces P leaching; however, it helps in reducing NO3 − leaching greater than P leaching [53,100,101]. Being alkaline in nature and the presence of negative charges, zeolite ameliorated soil improves soil P availability through lowering of soil acidity, soil exchangeable Al, and Fe [101–103]. These help in less P fixation by metal oxyhydroxides. Moreover, zeolites supplementation triggers more P uptake by enhancing the exchange- induced dissolution mechanisms as follows [102]:

$$\text{RP}(\text{rock phosphate}) + \text{NH}\_4^+ + \text{zolite} \rightarrow \text{Ca} - \text{zeolite} + \text{NH}\_4^+ + \text{H}\_2\text{PO}\_4^- \tag{1}$$

In this reaction, released Ca is adsorbed on the zeolite surface due to high CEC and as a result, more rock phosphate will be dissolved with lowering Ca2+ activity in the solution. This system releases the NH4 <sup>+</sup> and PO4 <sup>3</sup><sup>−</sup> ions. The addition of clinoptilolite zeolites with a 75% recommended rate of fertilizers showed comparable total and available P with the existing recommended dose without any zeolite application [31]. In this experiment, the addition of clinoptilolite zeolites also helped to reduce Al as well as soil acidity that resulted in low P fixation to soil colloid. A similar trend of observation was recorded by Zheng et al. [104], accounted for 14.1% higher available P with the application of zeolite relative to non-zeolite treatment. Antoniadis et al. [105] also reported an increase in P recovery efficiency of 4.02% due to zeolite application in acidic soil as compared to no zeolite application. The slow-release nature of zeolite in P release was observed by Bansiwal et al. [106] resulted in the continuous phosphate release even after 1080 h of continuous percolation from zeolite loaded modified phosphorous surface, while within only 264 h phosphate from potassium dihydrogen phosphate (KH2PO4) was exhausted.

Rather than N and P zeolites have strong selectivity on K<sup>+</sup> than Na+, Ca2+, and Mg2+ that makes it difficult to remove K<sup>+</sup> from exchange sites, facilitating greater absorption of K+ by plant root hairs through the ion exchange within root and zeolite [107]. The losses of K<sup>+</sup> by surface runoff and groundwater leaching can be reduced by supplementing the zeolites as slow-release fertilizer [108]. For example, the application of zeolites in municipal compost to investigate the K<sup>+</sup> release pattern resulted in six times less leaching loss from the zeolitic compost as compared to normal compost [109]. Additionally, Williams and Nelson [110] observed that in a soil-less medium K<sup>+</sup> saturated clinoptilolite recorded 23% less leaching of K<sup>+</sup> over-controlled control substrate. Moraetis et al. [109] reported that there was 18-fold increase in bioavailable K when zeolites were added through kinetic experiment to the soil-compost mixture, suggesting high potassium affinity in the soilcompost-zeolite mixture. Zeolite is considered as nano-enhanced green application as it adsorbs molecules at relatively low pressure [111,112]. Zeolite coated fertilizers have higher potential in water absorption and retention, and this coating materials retard the nutrient release rate from soil applied fertilizers, especially in sandy and sandy loam soil [113]. Similar nutrient retention ability of zeolites in secondary nutrients such as S was registered by Li and Zhang [114] who revealed that after leaching with 50 pore volumes, 85% of the pre-loaded SO4 2+ remained on the zeolite modified S fertilizer. Moreover, the initial SO4 2+ concentration in the leachate of S-loaded surfaced modified zeolite was found to be lowered, in comparison with the non-zeolitic sulfur sources. In addition to clinoptilolite, nano-zeolite based S fertilizer is also comprised of epistilbite zeolite. The findings from an experiment conducted by Thirunavukkarasu and Subramanian [115] exhibited that SO4 2+ was available even after 912 h of continuous percolation from S loaded modified nano-zeolite, while SO4 2+ from (NH4)2SO4 was depleted within 384 h. The presence of a huge number of channels, pores, and cages in the structure of the zeolite which helps in holding the SO4 2+ tightly might be the reason behind the slow release of this secondary nutrient from surface modified nano-zeolite [115].

The increase in micronutrient use efficiency with zeolites supplementation was also registered in previous literatures [33,116–118]. Sheta et al. [116] reported the ability of five natural zeolites and bentonite minerals to adsorb and release of zinc and iron as natural zeolites have a greater affinity to these micronutrients. Iskander et al. [117] found 74.7% and 84.63% are readily extractable by DTPA (diethylene-triamine pentaacetic acid) extractant (0.005 M DTPA + 0.01 M CaCI2 + 0.1 M triethanolamine, adjusted to pH 7.30) after three successive extractions of Zn and Mn, respectively and rest were retained by zeolite. Yuvaraj and Subramaniannano [119] reported that nano-zeolite adsorbed more Zn and the adsorption rate obtained with the nano-zeolite appeared to be efficient adsorbents for Zn. They also observed that ZnSO4 released the Zn up to 200 hours whereas micronutrients from nano-zeolite were releasing even after 800 h (Figure 8). The better availability of micronutrients in soil with zeolite application ultimately facilitates to greater micronutrients contents in plants. Ozbahce et al. [33] resulted in significantly higher Zn, Mn and Cu content in bean leaves with the maximization of zeolite application up to 90 kg ha−<sup>1</sup> (Figure 9). From the above-mentioned discussions, it can be concluded that the zeolite application accelerates the availability of primary, secondary and micronutrients in soils and subsequent plant uptake (Figure 10), and its application is most significant in arid and semi-arid regions that suffer from high water and nutrient scarcity all-time.

**Figure 8.** Zn Release Pattern with Time Duration as Influenced by Nano Zeolite Application. Modified from Yuvaraj and Subramaniannano [119].

**Figure 9.** Micronutrient content in bean leaves with different levels of zeolite application (Z0: 0; Z30: 30; Z60: 60; Z90: 90; Z120: 120 t ha−1); within treatments, different letters indicate significant differences at *p* ≤ 0.05(otherwise statistically at par); error bars represent the least significant difference value. Modified after Ozbahce et al. [33].

**Figure 10.** Effectiveness of Zeolite on Water and Nutrient Retention in Soil. Modified from Nakhli et al. (2014).

#### *3.3. Environmental Impact*

The addition of zeolites increases the C sequestration and subsequent soil C stock as compared to untreated soil [120,121]. According to Aminiyan et al. [56], application of zeolite (30%) along with crop residues (5%) to wheat could maintain the highest amount of organic carbon in light and heavy fractions. Soil organic matter even in the light fraction is highly correlated with N mineralization and subsequent soil management practices. The light fraction of soil organic matter (SOM) is not only sensitive to changes in management practices but also correlates well with the rate of N mineralization. Periodical measurements of N2O and N2 emissions in fields from the applied cow urine or potassium nitrate (KNO3) each at 200 kg N ha−<sup>1</sup> with and without the addition of zeolite (clinoptilonite) showed that zeolite significantly lowered the total N2O emissions by 11% from urine treated soils.

Specific channel size enables zeolite to act as molecular gas sieves. Wang et al. [122] recommended the use of zeolite as an amendment to reduce GHGs emission from duck manure as they found almost 27% of GHG emissions reduction from zeolite treated soil than no zeolite application. Additionally, low NO3 − and PO4 <sup>2</sup><sup>−</sup> leaching from zeolite amended soil helps to prevent groundwater pollution as well as surface water contamination and subsequent eutrophication [59]. They claimed that the better retention of anions in zeolite structure might be the reason for less leaching loss. Zeolite prevents rapid mineralization by preventing the entry of nitrifying bacteria into its structure and thus reduces the emission of N2O [99].

#### *3.4. Slow Release of Herbicides*

Being porous in nature along with a well-ordered structure, zeolites are considered as potential substances for storage and release of organic guest molecules. The most hydrophobic solid form of zeolite 'ZSM 5' adsorbs triazine group of herbicides in the compartmentalized intra-crystalline void space and release them slowly [123]. Furthermore, ZSM-5 was found to be restricted to the mobility of post-emergence herbicide such as paraquat [124,125]. Humic acid zeolites act as a sorbent of the herbicides belongs to the phenylurea group [126]. Clinoptilolitic turf has the potential to remove atrazine from soil and water [127,128]. Application of 2, 4–D herbicide along with zeolites results in a gradual temporal release pattern and keeps the active ingredient of herbicide in upper 0–5 cm of soil layer [129,130]. This slow-release nature of herbicide when used with zeolites improves the herbicide efficiency to control the weed floras and the prolonged effect of herbicide keeps the weed-free crop field throughout the entire crop weed competition period. Zeolite-rich nanocapsule is used as an herbicide carrier, adsorbent and retaining agent [130]. A longer retention period of zeolite added herbicide on weed leaves helps in maximizing the efficacy of the herbicidal mode of action. Interestingly, the synergistic effect between zeolite-loaded catalysts with isoproturon accelerates the visible light absorption and moreover better adsorption of recalcitrant molecules by the porous structure of zeolites [131].

#### *3.5. Remediation of Contaminated Soil*

Heavy metals induced soil pollution is one of the major concerns in modern agriculture. The anthropogenic activities of human, rapid industrialization and injudicious use of fertilizers without proper precaution make the soil toxic with heavy metal contamination. The solubility of heavy metal in soil is depending on complex chemical degradation and numerous factors. Among them, low soil pH is one of the major determining factors. In an acidic environment, oxides of iron, aluminum and manganese are slowly solubilized, and the primary and secondary minerals release the heavy metal into soil [132]. Soil sorption capacity is another determining factor for the retention of heavy metal ions. The ongoing concern in relation to the purity of the soil and the need to restore its original properties forced us to seek new and alternative ways of soil cleansing. Zeolite additions increase the soil pH significantly which facilitates to the heavy metal adsorption on its surface; thus, the solubility and bioavailability of heavy metals are ultimately reduced [133]. Chen et al. [134] observed that the cadmium and lead accumulation in wheat is significantly reduced with soil application of zeolite in soil. Moreover, it has been well reported that the clinoptilolite zeolite effectively controlled the heavy metal solubility including cadmium and lead up to 72% and 81% respectively [135,136]. However, this area of research needs extensive studies to find out heavy metal-specific appropriate dose and methods of zeolite application [85].

#### *3.6. Wastewater Treatment*

Industrial development with fast urbanization produces large quantities of wastewater that contains heavy metals, oils and organics that badly affect the aqueous environment [137]. Various efficient techniques such as solvent extraction, ion exchange and

adsorption are often used to remove those contaminants. Among them, the use of zeolites as adsorbents is most popular due to low-cost involvement, eco-friendly and poses good selectivity for toxic cations [138]. It also prevents the generation of new waste materials [139]. Furthermore, zeolites more specifically clinoptilolite could adsorb dyes, humic acid, phenols and phenol derivatives from the water body [140–142]. The clinoptilolite is mostly effective against metallic cations such as Al3+, Cd2+, Cu2+, Ni2+, Pb2+, and Zn2+ from copper mine wastewater [143]. The selectivity by clinoptilolite for heavy metals following the order: Pb2+> Cd2+> Cu2+> Co2+> Cr3+> Zn2+> Ni2+ [144]. The most advantage of clinoptilolite use in wastewater treatment is it can adsorb the heavy metals at a wide range of temperature (25–60 °C), pH (1–4) and different agitation speed (0, 100, 200, 400 rpm) [145]. The greater surface area along with high cation exchange capacity makes zeolite as a good adsorbent of cations [142]. The ability of heavy metals uptake by clinoptilolite zeolite was investigated by Baker et al. 2009 and opined the high selectivity of zeolite for the discharge of Pb2+ (98%), followed by Cr3+, Cu2+ and Cd2+ with 96% selectivity within 90 min. Morkou et al. (2015) [146] reported that wastewater nutrients can be recycled and used for microalgal and cyanobacterial biomass production by using zeolite as a medium.

#### *3.7. Crop Management Practices*

Zeolites have been used in a wide range of field crops production such as rice (*Oryza sativa* L.), corn (*Zea mays* L.), wheat (*Triticum aestivum* L.), potato (*Solanum tuberosum* L.), soybeans (*Glycine max* L.), and other upland crops in all types of soil to improve their productivity, water, and nutrient use efficiency (NUE), also maintaining the soil ecology and environment [83,147,148]. In an experiment, Chen et al. [29] estimated the effect of different rates of zeolite in combination with different N levels on transplanted rice and concluded that the highest yield was achieved consistently when rice plant was treated with a maximum dose of N (157.50 kg ha<sup>−</sup>1) along with zeolite supplementation (15 t ha−1), accounting 14.90% higher than the exclusive application of N. They also revealed that yield attributing characters namely effective tillers per plant, number of grains per panicle, grain filling percentage, and 1000-grain weight were positively influenced by the higher dose of N; however, zeolite consistently increased the number of effective tillers (Figure 11). A possible explanation of these results is the slow-release characteristics of zeolite amendment that makes the essential plant nutrients available throughout the crop growth within 0–30 cm soil depth. Furthermore, the supplementary application of zeolite significantly influenced the quality traits like protein content and tasting score of rice but did not influence the head rice recovery and chalkiness of rice grain [29].

**Figure 11.** Effect of Zeolite Application on Tillering Pattern of Rice. Modified from Chen et al. [29].

In another experiment, Zheng et al. [32] evaluated the effect of zeolite application on rice under limited water condition and they confirmed that the zeolite treatment (15 t ha<sup>−</sup>1) improved the LAI, transpiration rate and stomatal conductance (Figure 12). They observed that chalky rice rate and chalkiness were decreased by 29.6% and 41.2% respectively in zeolite treated plants as compared to the non-zeolite control. There was no significant difference in zeolite application on the starch viscosity properties. As rice quality is thought to be determined both genetically and environmentally, any improvements with zeolite application may result from better nitrogen and water availability to plants. The better crop performance and N partitioning in different parts of the rice plant with higher levels of zeolite application were depicted by Wu et al. [149]. Kavoosi et al. [150] resulted in both rice grain and straw yield increment with the application of zeolite at a certain level and thereafter decreased (Figure 13).

**Figure 12.** Effect of zeolite application on LAI, Tr, Sc and Chalkiness of rice. Tr: Transpiration rate (mmol m–2 s–1); Sc: Stomatal conductance (mol m–2 s–1); Z0: No Zeolite; Z15: Zeolite at 15 t ha−1. Within treatments, different letters indicate significant differences at *p* ≤ 0.05(otherwise statistically at par). Modified from Zheng et al. [32].

**Figure 13.** Effect of zeolite application on grain and straw yield of rice (Z0: 0; Z8: 8; Z16: 16; Z24: 24 t ha<sup>−</sup>1). Within treatments, different letters indicate significant differences at *<sup>p</sup>* <sup>≤</sup> 0.05(otherwise statistically at par). Modified from Kavoosi [150].

According to Wu et al. [151], the zeolites amendment significantly improved the root characteristics in terms of root length, dry weight, root diameter and volume, total root surface area, root bleeding intensity in rice plant over no zeolite application. Developed root traits may enhance nutrient transportation from the root to the above-ground parts and result in higher biomass and grain yield [152]. In previous studies, researchers confirmed that additional zeolites supply maximized the leaf area index (LAI) as well as leaf SPAD values and photosynthetic efficiency in rice plant, which might be attributed to its better ammonium retention capacity and slow-release nature that increase the better N availability to plants [53,72]. In a lowland rice production system, Sepaskhah and Barzegar [153] established the positive correlation between zeolites application and N retention in the upper soil profile. This higher availability favours better N uptake and subsequently higher nitrogen use efficiency (Figure 14). Zeolites induced rice cultivation resulted in greater apparent N recovery (65%) while 40% recovery was observed in exclusive N fertilization [150,154].

**Figure 14.** Nitrogen use Efficiency of Rice with Different Rates of Zeolite Application (Z0: 0; Z5: 5; Z10: 10; Z15: 15 t ha<sup>−</sup>1). Modified from Chen et al. [29].

Zheng et al. [104] evaluated the consequence of zeolite and phosphorus applications in rice under different irrigation regimes and resulted in 15.2% higher water use efficiency (WUE) as well as greater leaf and stem P concentration by 20.3% and 32.7% respectively than no-zeolite control. The better water use efficiency may be attributed to higher soil water retention in the porous structure of zeolites and thus better water availability to plant [3,53]. Additionally, restriction in deep percolation and leaching beyond the crop root zone in zeolite loaded soil are major reasons for better water use efficiency [23,155].

The application of zeolite in maize cultivation was reported by Malekian et al. (2011) [156] who opined that maize plants resulted in better response to zeolite when used as a fertilizer carrier at the rate of 60 g kg−<sup>1</sup> of soil. The application of clinoptilolite zeolite (CZ) with a 75% recommended dose of fertilizer resulted in significantly similar cobs yield in maize as compared to the full recommended dose of fertilizer [31]. A similar trend of observation was recorded regarding dry matter production and nutrient uptake, especially N and K uptake. It is possible due to the higher cation exchange capacity and affinity of CZ to NH4 <sup>+</sup> and K<sup>+</sup> ions. More specifically, reduced nitrification, prevention of leaching and volatilization by inhibiting ureolytic activity of microorganisms in the presence of CZ facilitate better nutrients availability [157]. Moreover, the cation selectivity of the CZ in the order to K+> NH4 +> Na+> Ca2+> Mg2+ supports to the aforesaid observation [31,158]. Increased nitrogen-use efficiency with the application of zeolites and ensured good retention of soil-exchangeable cations, available P and NO3 − within the soil have been found by Rabai et al. [159] in maize cultivation. Low fertilizer requirement with zeolites application not only gives a similar yield but also reduces the environmental pollution in respect to nitrous oxide emission, with maintaining the economic viability. Andronikashvilf et al. [147] also suggested that the zeolite application facilitates a reduction in the recommended dose of fertilizer by 25% and maintains a positive effect for 2–3 years in upland crops production systems.

In high saline condition zeolite amendment in soil responded well in Barley crop and it was reported that zeolite at 5% level produced taller plants; accumulated maximum plant biomass and more grain yield over 1% and no zeolite application [16]. Similarly, in alkaline condition soil application of zeolites for French bean (*Phaseolus vulgaris* L.) cultivation maximized the nutrient accumulation in plant tissues. Additionally, better crop performance as well as greater water use efficiency, water productivity and crop yield were recorded from the zeolites treated plots [154]. Usually, the higher Na<sup>+</sup> content in alkaline and saline soils disturbs the soil nutritional balance and osmotic regulations in plant tissues. Zeolite provides additional Ca2+ cations in the soil to reduce the Na+/Ca2+ ratio. The provision of Ca2+ from zeolite in the growing media would alleviate the toxic Na<sup>+</sup> ions accumulation and helps in the improvement of soil structure by aggregating the soil particles [16].

Not only in cereals and pulses zeolites have significant importance in oilseed crops. An additional supply of 10-ton zeolites ha−<sup>1</sup> with recommended fertilizer significantly increased the seed and oil yield in safflower, accounted for 2.7 and 9.38 t ha−<sup>1</sup> respectively [160]. Zahedi et al. [161] evaluated the effects of zeolite and selenium applications on some agronomic traits of three Canola cultivars under drought stress. They opined that stem diameter significantly decreased due to water stress, while the application of zeolite along with selenium improved stem diameter may be attributed to better water and nutrients availability from zeolites induced soil. They reported that 10 t zeolite ha−<sup>1</sup> significantly improved the growth, yield attributes, and yield (Figure 15). They also observed reduced N leaching along with higher water holding capacity and CEC in alkaline soil when supplemented with 10 t zeolite as compared to normal soil. The oil yield and oil qualities such as palmitic acid, Oleic acid, Linoleic acid, Linolenic acid and Erucic acid of canola significantly improved with zeolite application (15 t ha−1) rather than no zeolite use [28].

**Figure 15.** Effects of zeolite on some agronomic traits in canola. SD—Stem diameter (mm); NS— Number of siliquae; LS—Length of siliqua (cm); SS—Seeds per siliqua; TW—Test weight (g); SY— Seed yield (t ha—1). Adapted and modified from Zahedi et al. [162].

From an experiment, the findings recorded by Ozbahce et al. [162] revealed that application of 60 t zeolite ha−<sup>1</sup> along with proper irrigation and nutrient management, potato yielded (39.1 t ha<sup>−</sup>1) maximum tubers (Figure 16). They also recorded superior crop performance even under limited water supply when treated with zeolite while non-zeolite traditional practices sharply decreased the tuber yield. The interaction of zeolites and irrigation regimes was found to be significant for tuber weight, tuber diameter and crude protein percentage.

**Figure 16.** Effect of Zeolite Rates Eighth Different Levels of Irrigation on Tuber Yield of Potato (Z0: 0; Z30: 30; Z60: 60; Z90: 90; Z120: 120 t ha—1) Within treatments, different letters indicate significant differences at *p* ≤ 0.05(otherwise statistically at par). (Adapted and modified from Ozbahce et al. [163]).

The effectiveness of zeolite on Peppermint (*Mentha piperita* L.) cultivation was reported by Ghanbari and Ariafar [30]. They opined that zeolite treatment significantly improved the fresh and dry leaf weight of mint and the highest value of fresh dry leaf weight was observed in 2.5 g zeolite application per kg of soil even under—water scare situation. They also observed that drought intensity was decreased with increasing the zeolite application. In 30% field capacity, zeolite application maximized the leaf dry weight from 18.54 to 32.76 g and fresh leaf weight from 41.7 to 67.14 g. Interestingly, zeolite helps to keep the essential components of mint oil such as menthol, menthone, methyl acetal, menthofuran and palegone [163,164]. Actually, these essential components are adversely affected by drought and salinity stress whereas, zeolites consist of alkali and alkaline materials and crystalline alumino—silicate which act as a water reservoir in their internal surface area during drought situation [165,166].

Numerous scientific reports were also concluded that significant positive influence on cocoa fruiting [167], eye numbers in potato tubers [160], pod and siliqua number in pulses and oilseeds [33,168], and overall development of soybean, sweet potato, wheat, bean and safflower with the application of soil—applied zeolites [160–171]. The use of Clinoptilolite rich tuff as soil conditioner was found to be effective to improve the productivity of wheat, eggplant, carrots, and apples by 13–15%, 19–55%, 13–38% and 63% respectively [172]. Not only in field crops or vegetables, zeolite induced soil significantly improved the production as well as qualitative traits of mycelium mushroom [173]. The treatment with 30% zeolite + 70% urea resulted in a positive effect on the microbiological community in spring barley, soybean and maize [174]. Andronikashvili et al. [175] opined that the introduction of

clinoptilolite containing tuffs into soils enhanced the soil microbial population viz. bacteria, fungi and actinomycetes.

Another interesting dimension of zeolite application was introduced by the National Aeronautics and Space Administration (NASA), which developed a special type of clinoptilolite loaded plant growth media including synthetic apatite, dolomite, and several essential trace nutrients mainly for vegetable production (10% higher than non—zeolite application) in space missions, known as 'zeoponic' [176]. Life support system for regenerating and recycling the air, water and food are essentially required for the long duration Mars mission and only the growing of plants could be fulfilled this aim. The ultimate objective of zeoponic research is to develop a solid substrate that can supply all essential macro and micronutrients slowly for a long duration in a space habitat. In an experiment, Gruener et al. [176] resulted in higher biomass accumulation, root and leaf development and nutrient uptake by radish when cultivated in zeoponic as compared to normal soil. Rodriguez—Fuentes et al. [177] reported that root architecture, plant growth and yield of different vegetables, spices and strawberries, were significantly improved by zeoponic substrates without further fertilization. Researchers confirmed that the native clinoptilolite in zeoponic acts as a good source of N and K as the clinoptilolite cations are exchanged for NH4 <sup>+</sup> and K+ ions [102]. Additionally, apatite and dolomite dissolution supplies Ca2+ into soil solution. This Ca2+ rich solution removes the NH4 <sup>+</sup> and K<sup>+</sup> ions from zeolite exchange complex and makes them more available to plants [176]. Sometimes, nitrifying bacteria are supplemented to zeoponic substrates prior to plant growth to augment the nitrification process [178]. Since most zeolites are advantageous in the growth and development of crops, however, erionite (one type of zeolite) was found to be detrimental to the proper growth of plants [179]. Therefore, the selection of an appropriate form of zeolites should be taken into consideration.

#### *3.8. Used as a Pesticide*

Zeolites that contain silica gel and alumina silicate crystals have been successfully tested against some stored grain pests such as lesser grain borers (Rhyzopertha dominica), rice weevils (Sitophilus oryzae), and saw—toothed grain beetles (Oryzaephilus surinamensis) [180]. Natural zeolites application at the rate of 50 g kg−<sup>1</sup> of maize grain were also found to be effective against maize weevil (*Sitophilus zeamais*) in accordance with Haryadi et al. [181]. Clinoptilolite was successfully investigated on organic oilseed rape fields against the pollen beetle (*Meligethes* sp.). Daniel et al. [182] observed that under dry and sunny weather condition, pollen beetles were significantly reduced by 50 to 80% with zeolite application while in rainy weather zeolite did not perform against pollen beetles. Zeolites loaded organophosphorus compound was used with success against the *Aedes aegyptii* [183]. Clinoptilolite is gaining importance as possible sorbents because it acts as a slow—release carrier and retard water contamination [184]. Clinoptilolite riched metalaxyl application on turfgrass against *Phythium* sp. resulted that the active ingredient of fungicide was prevented from groundwater contamination by clinoptilolite zeolite [185]. Actually, the adsorption of pesticide molecules is happened due to polar chemical bonds with the external surface of the microporous zeolitic minerals [108]. Additionally, the dusting of natural zeolites has been successfully tested to control the aphid population in fruit orchard [186]. Moreover, in herbicide application, pest control and in nano—sensing for pest detection, the nano—porous zeolites have been implicated as nano—capsules [187–189]. Stadler et al. [190] examined the insecticidal effect of nanostructured zeolites on two stored—grain insect species, *S. oryzae* and *R. dominica*, and found 80–100% mortality rate within 14 days after application to wheat grain. In this regard, natural zeolites may provide a cheap and reliable alternative to commercial insecticides in pest management. The insecticidal efficacy of natural zeolite on different stored grain pests is summarized in Table 4. Additional research is needed to investigate the mode of action, non—target toxicity, and the potential use in integrated pest control strategies.


**Table 4.** Efficacy of Natural Zeolites on Stored—Product Pests.

#### *3.9. Mycotoxin Control*

The use of aluminosilicates such as zeolites has emerged as a mycotoxin—binding agent in the feed and food industry to effectively adsorb mycotoxin [194]. Clinoptilolite has the capacity to adsorb aflatoxins by chelating of the β—dicarbonylmoiety in aflatoxin with uncoordinated metal ions [195]. There are some well—established criteria to evaluate the function of any binding additive, such as low inclusion rate, stability over a wide range of pH, huge capacity, and affinity to absorb various concentrations of mycotoxins [194]. The supplementation of mycotoxin binders in contaminated foods has been suggested as the most advantageous dietary approach to lower the mycotoxins efficacy [196]. Hydrated sodium calcium alumino—silicates—zeolite powder (HSCAS) has been identified as "aflatoxin—selective clay", but it does not adsorb other mycotoxins such as cyclopiazonic acid which may coexist with aflatoxin [197] while responses seem to be dose—dependent [198]. Parlat et al. [199] observed that clinoptilolite could successfully minimize the effects of aflatoxin in quail. Natural zeolites with high clinoptilolite content (over 80%) effectively adsorbed aflatoxin B1, aflatoxin B2, and aflatoxin G2 [200]. On the contrary, surface modified zeolites with NH4 <sup>+</sup> showed very well adsorption of ochratoxin A, T—2 toxin, zearalenone and aflatoxin B1 [201]. According to Adamovic et al. [202], the application of zeolites at 2 g kg−<sup>1</sup> of silage accelerates the fermentation and reduction of T—2 toxin, mould and zearalenone. Zeolites application as mycotoxin binder is impressive against aflatoxicosis, however, their effectiveness against trichothecenes, zearalenone and ochratoxin is restricted. At the same time, these compounds show high inclusion rates for vitamins and minerals, which are considered as one of the major disadvantages [203].

#### **4. Limitation of Zeolites**

Rather than the huge applicability of zeolites in agriculture, it should be considered that the zeolites are not without disadvantages. The fine—grained synthetic zeolites are highly dispersive in nature which creates worrisome problems during their use. After mining the usable form of natural zeolites is obtained via isolation procedures like crushing and pellet generation while the application of the synthetic form of zeolites are limited into hard, wear—resistant granular forms. The practical use of granular zeolites is not yet discovered [204]. The distribution of the zeolites sources is very limited such asthezeolitic soil is confined to only 1% of the total geographic area of India and more than 50% of natural zeolites are produced in China among all over the world [40] that may increase the price and the gap between demand and supply. Therefore, the uninterrupted availability of zeolites for farming purposes in worldwide is another major constraint.

#### **5. Future Scope**

The significant application of zeolites in agricultural activities has been well established by various researchers. However, systematic and comprehensive efforts are further needed for future research, including (a) precision mapping of the available zeolite deposits in each country, (b) determination of the physical stability of zeolites in various agro-climatic conditions, (c) economically viable organo-zeolitic manure or fertilizer development, (d) evaluation of the risk of leaching of a toxic surfactant that is loosely attached to the zeolite surface, (e) assessment of the long-term impact of zeolite application on rhizospheric microflora and fauna, (f) understanding of the mechanisms of zeolite-mediated heavy metal stabilisation in contaminated soil and (h) development of zeolite-rich herbicides to minimise the residual risk hazard.

#### **6. Conclusions**

In the situation of rapid urbanisation and over-increasing population where resources are limited, there is no choice for us but to depend on agricultural productivity. In this context, various researchers suggest that farming with zeolites may be an option to improve soil's physical environments in terms of decreasing bulk density, increasing total porosity and increasing water-holding capacity. Furthermore, the existence of open networks in the zeolite structure leads to the formation of new routes for water movement, subsequently improving the infiltration rate and saturated hydraulic conductivity. Zeolites also show a strong affinity to various essential nutrient ions by modifying their surface chemistries using cationic surfactants, multifunctional adsorbents that have the capacity to trap anions and non-polar organics. Thus, the application of zeolite-loaded fertilizer improves the nutrient retention in soil and releases nutrients slowly throughout the crop life; otherwise, rapid mineralisation would take place, leading to nutrient loss. Zeolites are very much effective in remediation of heavy metal toxicity and wastewater treatment, and they could help to improve soil's biological properties. Zeolite application in space missions as zeoponic substrates opens a new dimension of zeolites. The aforesaid positive impacts ultimately enhance crop growth, productivity and even quality attributes of various agronomic and horticultural crops. The higher input use efficiency significantly reduces greenhouse gas emissions and energy involvement and facilitates better carbon sequestration. However, the impact of zeolite application varies with the agro-climatic location, the nature of zeolites, their availability and application strategies, and soil textural classes. Further studies are needed to identify zeolite resources and the long-term impact on the soil environment and to develop new, cost-effective zeolite-based nutrient resources for sound agricultural practices.

**Author Contributions:** Conceptualization, M.M.; B.B.; and S.G.; writing—original draft preparation, M.M.; S.G.; S.S.; H.B.; and A.H.; writing—review and editing, B.B.; S.M.; K.B.; P.K.B.; H.B., A.H., M.S., P.O.; and M.B.; funding acquisition, P.O., M.S., A.H. and M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the 'Slovak University of Agriculture', Nitra, Tr. A. Hlinku 2,949 01 Nitra, Slovak Republic under the project 'APVV—18—0465 and EPPN2020—OPVaI—VA— ITMS313011T813'.

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

#### **References**


## *Article* **Water Table Fluctuation and Methane Emission in Pineapples (***Ananas comosus* **(L.) Merr.) Cultivated on a Tropical Peatland**

**Wendy Luta 1, Osumanu Haruna Ahmed 1,2,3, Latifah Omar 1,2,\*, Roland Kueh Jui Heng 2,3,4, Liza Nuriati Lim Kim Choo 5, Mohamadu Boyie Jalloh 6, Adiza Alhassan Musah <sup>7</sup> and Arifin Abdu <sup>8</sup>**


**Abstract:** Inappropriate drainage and agricultural development on tropical peatland may lead to an increase in methane (CH4) emission, thus expediting the rate of global warming and climate change. It was hypothesized that water table fluctuation affects CH4 emission in pineapple cultivation on tropical peat soils. The objectives of this study were to: (i) quantify CH4 emission from a tropical peat soil cultivated with pineapple and (ii) determine the effects of water table depth on CH4 emission from a peat soil under simulated water table fluctuation. Soil CH4 emissions from an open field pineapple cultivation system and field lysimeters were determined using the closed chamber method. High-density polyethylene field lysimeters were set up to simulate the natural condition of cultivated drained peat soils under different water table fluctuations. The soil CH4 flux was measured at five time intervals to obtain a 24 h CH4 emission in the dry and wet seasons during low- and high-water tables. Soil CH4 emissions from open field pineapple cultivation were significantly lower compared with field lysimeters under simulated water table fluctuation. Soil CH4 emissions throughout the dry and wet seasons irrespective of water table fluctuation were not affected by soil temperature but emissions were influenced by the balance between methanogenic and methanotrophic microorganisms controlling CH4 production and consumption, CH4 transportation through molecular diffusion via peat pore spaces, and non-microbial CH4 production in peat soils. Findings from the study suggest that water table fluctuation at the soil–water interface relatively controls the soil CH4 emission from lysimeters under simulated low- and high-water table fluctuation. The findings of this study provide an understanding of the effects of water table fluctuation on CH4 emission in a tropical peatland cultivated with pineapple.

**Keywords:** drained peat; greenhouse gas; global warming; organic soil; pineapple; water table

**Citation:** Luta, W.; Ahmed, O.H.; Omar, L.; Heng, R.K.J.; Choo, L.N.L.K.; Jalloh, M.B.; Musah, A.A.; Abdu, A. Water Table Fluctuation and Methane Emission in Pineapples (*Ananas comosus* (L.) Merr.) Cultivated on a Tropical Peatland. *Agronomy* **2021**, *11*, 1448. https:// doi.org/10.3390/agronomy11081448

Academic Editors: Nikolaos Monokrousos and Efimia M. Papatheodorou

Received: 22 April 2021 Accepted: 10 June 2021 Published: 21 July 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/).

#### **1. Introduction**

Drained peatlands worldwide emit approximately 2 Gt of carbon dioxide (CO2) through microbial peat oxidation or peat fires representing 5% of all anthropogenic greenhouse gas (GHG) emissions [1]. Carbon dioxide emitted from peatlands has been implicated in the ongoing global warming debate [2]. Unlike CO2, which is cycled and released into the atmosphere, methane (CH4) is emitted mostly from agricultural activities [3]. Methane contributes to significant anthropogenic GHG and the concentration of CH4 is on the increase [4,5]. The pathways of CH4 emissions are through aerobic and anaerobic microbial respiration, root respiration, peat oxidation, nitrification, and denitrification although the determinant factors which affect CH4 emissions are land-use type [6], peat type [7], photosynthetic activities [8], and water table fluctuation [9]. Carbon (C) is transformed and stored in different pools within the C cycle through, for example, burning of fossil fuels or decomposition of soil organic matter in the form of C gases into the atmosphere, whereas photosynthesis locks atmospheric C in plant tissues and deposition of organic-rich sediments on the ocean floor locks C in geologic rocks and sediments.

The increasing interest in reducing CH4 emissions to meet global temperature targets is because of the short atmospheric life span of CH4 which is approximately 10 years, but CH4 has relatively high global warming potential [10]. Current and future regional and global CH4 budgets and mitigation strategies require better quantitative and processbased understanding of CH4 sources, pathways, and removals under climate and land-use change [11]. According to Leifeld [12], peatland rewetting is a cost-effective measure to curb GHG emissions, however, increasing water table depth increases CH4 emissions [13]. Lowering peatland water tables increases peat decomposition rates because of enhanced microbial degradation of organic matter [14]. It must be stressed that the understanding of soil C flux based on studies conducted in boreal and temperate peats is not fully applicable to tropical peatland because of differences in environmental factors, peat soil properties, peat temperature, peatland-use practices, vegetation composition and structure, and microbial diversity and population.

Tropical peatlands are commonly developed for agriculture. Huang et al. [15] reported that agricultural productivity in low latitudes (tropical and semi-tropical) are likely to decline due to climate change which affects world food security and farm incomes because most developing countries, including Malaysia, are located in lower latitude regions. Falling farm incomes will increase poverty and reduce households' ability to invest for a better future [16]. According to Melling et al. [17], peat soil reclamation for agriculture involves drainage which is characterized by lowering water table and soil compaction to aerate crop root zones. Drainage of tropical peatland may cause loss of soil C reserve. Drainage via lowering of water table could change peatlands from being a C sink to a C source because drainage reverses the C flux into net CO2 emissions [18]. The decomposition of organic materials and microbial activities releases CO2, CH4, organic acids, and organic particulates. The rate of C loss is related to the increased intensity of dry and wet periods. The resultant extreme water table fluctuation could affect the amount and nature of aerobic and anaerobic peat material, which subsequently affect the decomposition of peat material, microbial activity, and the crop growth. A study had revealed that the CH4 emissions from drained tropical peatland for pineapple (*Ananas comosus* (L.) Merr.) cultivation was lower than those emitted from bare peatland and bare peatland fumigated with chloroform [19]. In this study, our approach is to estimate CH4 emissions from tropical peatland cultivated with pineapple under fluctuating water table. Pineapple could absorb CO2 for photosynthesis to produce carbohydrates in plant tissues. The emissions of CH4 could be reduced through maintaining ground water level because the ground water level below the surface alters the CH4 dynamic by weakening the potential for CH4 production and increasing the potential for CH4 oxidation in the upper peat layers [17,20]. Considering the potential importance of tropical peatlands in the global CH4 budgets [21], it is essential to understand the effects of water table fluctuation on CH4 emissions from tropical peatlands cultivated with, for example, pineapples.

There is lack of standard procedures to measure CH4 emissions in tropical peatlands as reported by Ahmed and Liza [19]. Couwenberg [18] and Burrows et al. [22] suggested that GHG emissions should be measured on the soil surface using a closed chamber method [23,24]. Greenhouse gas monitoring which is conducted using the closed chamber method is limited in space (few cm2) and time (few minutes). To date, there is limited information on CH4 emissions from peatlands which are cultivated with pineapples that are relatively tolerant to peatlands' acidity. According to a study conducted by Raziah and Alam [25], the contribution of pineapples cultivated on tropical peatlands to CH4 emissions is important because 90% of pineapples are grown on peatlands of Malaysia. In this study, it was hypothesized that peatland water table fluctuation will affect the emission of CH4 in pineapple cultivation on tropical peatlands. This assumption is premised on the fact that peatland water table fluctuation could minimize the CH4 emissions through suppression of the anaerobic decomposition of organic matter (reduction of CO2 by H2) after which it is affected by the balance of CH4 production and oxidation. Also, regulating water table level could control the peatland water temperature because increasing soil temperature leads to an increase in CH4 emission.

The research questions that were addressed in this study were: (i) does water table depth affect CH4 emission from tropical peatlands cultivated with pineapples? and (ii) what is the amount of CH4 emitted from tropical peatland which are cultivated with pineapple in relation to simulated water table fluctuation? The quantification of CH4 emission was carried out in the dry season (July and August 2015) and wet season (September and December 2015) to take into account the effects of temperature. Warm peatlands transformed soil organic C from a C sink to a C source [26] with well-drained soils releasing CO2 to the atmosphere [27]. However, the decomposition of organic matter and peatland temperature in relation to water table fluctuation and CH4 emission from a tropical peatland cultivated with pineapples have scarcely been explored. Thus, this study was carried out to: (i) determine the effects of water table depth and CH4 loss from a tropical peatland cultivated with pineapples; (ii) quantify CH4 loss in a tropical peatland under simulated water table fluctuation; and (iii) determine the effects of water table fluctuation on soil temperature during CH4 emission.

The implication of regulating water table level as an approach to minimizing CH4 emission from a tropical peat soil cultivated with pineapples is an attempt to hinder CH4 emission or consumption. It is well known that the pathways of CH4 emission are diffusion, ebullition, and plant-mediated transport. This study focuses on the loss of CH4 from a tropical peatland cultivated with pineapples because different vegetation growing on the same peatland results in differences in CH4 emission or consumption. According to Hu et al. [28], under forest vegetation, soil served as a net sink of CH4, whereas maize field (*Zea mays* L.) was essentially CH4 neutral, and a paddy field was a net source of CH4 diffused to the atmosphere. The findings suggested that the water table fluctuation has significant effects on CH4 emission apart from the different crop-mediated transport. Most of the crop mediated transport are focused on the aerenchyma and not on the crassulacean acid metabolism plants such as pineapple. Aerenchyma is a type of plant that has porous root tissue, particularly well developed in wetland plants, which enables diffusive flux of gases from above-ground tissues to root tips [29]. Owing to this, most of the CH4 emission studies on drained peatlands are limited to rice, soybean, and sago [23,30] with little exploration focus on the pineapple [19]. Our approach was not only limited to determining the effects of water table fluctuation on CH4 emitted from a tropical peatland cultivated with pineapples, but it was also focused on the measurements of soil CH4 emission. This study also provides information on the mechanism of CH4 emission from different water table depth and the amount of CH4 emission from dry and wet seasons. This study partly shows that appropriately reclaimed land use on tropical peatlands favours low CH4 emission, and benefits pineapple planters, economy, society, and the environment.

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

#### *2.1. Site Description for Soil Methane Emission from Field Cultivated with Pineapples*

The study was carried out to quantify C losses in the form of CO2 (data on CO2 emissions have been published in 2017) and CH4 in a tropical peatland subjected to water table fluctuation. The study was carried out under field conditions and simulated lysimeter at the Malaysian Agricultural Research and Development Institute (MARDI), Sesang, Saratok, Sarawak, Malaysia (Figure 1). The study area of 387 hectares (ha) was located on the logged-over forest with a flat topography of 5 to 6 m above the mean sea level. Based on the Von Post scale of H7 to H9 [31], the peatland is classified as well decomposed dark brown to dark coloured sapric peat with a strong smell and the thickness of 0.5 to 3.0 m. The average temperature of the area ranges from 22.1 to 31.7 ◦C while the relative humidity of the area ranges from 61% to 98% humidity with annual mean rainfall of 3749 mm. From November to January (wet season), the monthly rainfall is greater than 400 mm, whereas the mean rainfall during the dry season particularly in July is 189 mm [19]. The area of the peatland cultivated with *Moris* pineapple was 0.21 hectares (Figure 2). The Moris pineapple were planted in two rows with a planting distance of 30 cm × 60 cm × 90 cm, and the pineapples were managed according to the standard agronomic practices for pineapple management on tropical peat soils. Soil CH4 flux measurements were carried out using the closed chamber method [32] on a 10 m × 10 m plot with five replications. The study was carried in the dry (July and August 2015) and wet (September and December 2015) seasons.

**Figure 1.** Location of study site in Sesang, Saratok, Sarawak, Malaysia.

**Figure 2.** Study site of peatland cultivated with *Ananas comosus*.

#### *2.2. Establishment of Lysimeters for Methane Estimation under Simulated Water Table Fluctuation*

Ten cylindrical field lysimeters made from high-density polyethylene (HDPE), 0.56 m in diameter and 0.97 m in height, were set up to simulate the natural condition of drained tropical peats (Figure 3). The size of the lysimeters used in this study was designed to ensure satisfactory growth and development of the pineapple. The lysimeters were equipped with a water spillage opening which was attached to clear tubes mounted on the outside of the vessel to regulate and monitor water level. Each lysimeter was filled with peat soil up to 0.90 m depth (Figure 3). Water loss from the soil was replenished by showering each lysimeter with rainwater. The amounts of the rainwater added were based on the volume of the fabricated lysimeter and the mean annual rainfall at Saratok, Sarawak, Malaysia. The lysimeters with the peat soil were left in the open field for five months (January to June 2015) to equilibrate. During the modification of the lysimeter, clear tubing and water spillage openings were attached to one side of the lysimeter to regulate and monitor the water level. Before the lysimeter was filled with peat soil, a polyvinyl chloride (PVC) pipe was installed vertically onto the soil to enable the bailer to reach the bottom of the lysimeter (Figure 3). The water table in the lysimeter was controlled by draining excess water through the water spillage opening or watering the peat soil with rainwater to the desired water table depth to simulate the effects of drainage and rainfall. During rainy days, the lysimeter was covered with a plastic cover to maintain a consistent water level. The depth of the water table in the lysimeter was controlled at 0 m and 0.9 m from the soil surface to represent the driest (low water table) and wettest (high water table) months, respectively.

**Figure 3.** Fabricated field lysimeter made from high density polyethylene.

#### *2.3. Estimation of Soil Methane Emission during Water Table Fluctuation*

Soil CH4 emissions from the field and lysimeters were measured using the closed chamber method [32]. The CH4 emissions from peatland were quantified using gas chromatography (Agilent 7890A) equipped with a thermal conductivity detector (TCD). The chambers were placed vertically on the soil surface between pineapple plants. The CH4 emissions measurements were carried out on the daily basis of dry and wet months, before total draining of the plot, at 2–4 h intervals over 2–3 days duration to reflect the total of CH4 losses through the soil surface. The size of the closed chamber was 20 cm × 20 cm × 20 cm and made up of acrylic (Figure 4). The top of the chamber was fitted with two sampling ports plugged with a rubber septum for gas sampling and thermometer installation, respectively (Figure 4). A battery-operated fan was also attached to the chamber to allow equilibrium gas pressure in and outside the closed chamber (Figure 4). The chamber was covered with a reflective aluminium foil to minimize the effect of temperature differences within and outside the chamber. The headspace samples of 20 mL were extracted from the chamber at 1, 2, 3, 4, 5 and 6 min using a polypropylene syringe equipped with a three-way stopcock. The extracted CH4 gas was transferred to a 10 mL vacuum vial bottle by a double-ended hypodermic needle to be quantified using gas chromatography (Agilent 7890A, Agilent Technologies Inc., Wilmington, DE, USA) equipped with a flame ionization detector (FID). The values obtained were averaged and converted into units of t ha<sup>−</sup>1yr−1.

The CH4 flux was calculated from the increase in the chamber concentration over time using the chamber volume and soil area covered, using the following equation:

$$\text{Flux} = [d(\text{CH}\_4)/dt] \times PV/ART$$

where *d*(CH4)/(*dt*) is the evolution rate of CH4 within the chamber headspace at a given time after putting the chamber into the soil, *P* is the atmospheric pressure, *V* is the volume headspace gas within the chamber, *A* is the area of the soil closed by the chamber, *R* is the gas constant, and *T* is the air temperature [24,32].

The CH4 flux was measured in the early morning I (06:00 to 06:35), afternoon (12:00 to 12:35), evening (18:00 to 18:35), midnight (00:00 to 00:35) and early morning II (06:00 a.m. to 06:35 a.m.) to obtain a 24 h of CH4 emissions. The 24 h measurement was carried out to meet the gas flux measurement requirement based on the procedure described by Ahmed and Liza [19]. The flux measurements were carried out in July and August 2015 for the

dry season and in September and December 2015 to represent the concentrations of CH4 emitted in the wet season. Soil temperature at 6 cm depth were measured at the same time of the CH4 flux measurement using a digital thermometer. Rainfall distribution data was collected from a portable weather station (WatchDog 2900ET, Spectrum Technologies Inc., Plainfield, IL, USA) installed at the experimental site. Although CH4 fluxes were only monitored for two cycles for each weather season and results obtained might not be conclusive enough to confirm the findings on the effect of water table fluctuation on CH4 emission, it must be emphasized that time allocated for soil CH4 emission determination per sample was the limitation of this present study. This is because increasing the number of gas flux monitoring cycles are costly and time consuming. For example, a minimum retention time of 6 min is required for a gas sample analysed using gas chromatography, and the total samples for each CH4 flux monitoring cycle were 450 per month.

**Figure 4.** A closed chamber system to estimate soil methane emission from tropical peatland.

#### *2.4. Statistical Analysis*

Fluctuation of water table in relation to CH4 emission was tested using analysis of variance (ANOVA) and means of the water table fluctuations in triplicates were compared using Duncan's new multiple range test (DNMRT) at *p* ≤ 0.05. The relationships between CH4 flux and soil temperature were analyzed using Pearson correlation analysis. The statistical software used for this analysis was the Statistical Analysis System (SAS) Version 9.3.

#### **3. Results**

*3.1. Soil Methane Emissions from Peat Soils Grown with Pineapple under Open Field Cultivation System in the Dry and Wet Seasons*

Soil CH4 emissions from tropical peat soils cultivated with pineapples in the dry and wet seasons are presented in Figures 5 and 6, respectively. During the dry season (July and August 2015), the soil CH4 emission showed no specific trend with the time of sampling but CH4 emissions were higher at midnight (Figure 5). In July 2015, the CH4 emissions was generally similar, whereas soil CH4 emissions were lower in the early morning I, afternoon, evening, and early morning II than at midnight in August 2015. Compared with the wet season, soil CH4 emissions were lower in the afternoon and at midnight during the gas flux monitoring in September and December 2015, respectively (Figure 6).

Averaged soil CH4 emissions over 24 h from a drained peat soil cultivated with pineapples throughout the dry (July and August 2015) and wet (September and December 2015) seasons are presented in Figure 7. Soil CH4 emissions were higher in September 2015 but emissions were lower in July, August and December 2015. However, soil CH4 emissions in July, August and December 2015 were similar.

**Figure 5.** Soil CH4 emissions (at different times of the day) from a tropical peatland cultivated with pineapples in the dry season (July and August 2015). Error bars represent standard error and soil mean fluxes with different letters and noted by prime are significantly different using Duncan's new multiple range test (DNMRT) at *p* ≤ 0.05.

**Figure 6.** Soil CH4 emissions (at different times of the day) from a tropical peatland cultivated with pineapples in the wet season (September and December 2015). Error bars represent standard error and soil mean fluxes with different letters and noted by prime are significantly different using DNMRT at *p* ≤ 0.05.

**Figure 7.** Averaged soil CH4 emissions over 24 h from a tropical peat soils cultivated with pineapple throughout the dry (July and August 2015) and wet (September and December 2015) seasons. Error bars represent standard error and soil mean fluxes with different letters are significantly different using DNMRT at *p* ≤ 0.05.

Throughout the CH4 flux monitoring, soil temperature was statistically similar during the dry and wet seasons irrespective of sampling time (Table 1). Also, there was no significant correlation between CH4 emission and soil temperature (Table 1).

**Table 1.** Relationship between soil CH4 emission and soil temperature from a peat soil cultivated with pineapples throughout the dry and wet seasons in 2015.


Mean values with same letters within the same column are not significantly difference between means using DNMRT at *p* ≤ 0.05. Top value indicates Pearson's correlation coefficient (r), whereas the bottom values indicate probability level at 0.05 (*n* = 600).

#### *3.2. Soil Methane Emissions from Peat Soils Cultivated with Pineapples in Lysimeters under Simulated Water Table Fluctuation in the Dry and Wet Seasons*

Soil CH4 emission varied with time of sampling throughout the wet and dry seasons under low and high water table fluctuations (Figure 8a,b). At low water table during the dry season (Figure 8a), soil CH4 emissions decreased from early morning I to early morning II in July 2015, whereas CH4 emissions were higher in the afternoon but lower at midnight in August 2015. However, at low water table during the wet season (Figure 8a), soil CH4 emissions were higher in the evening but lower in the early morning II in September 2015, whereas in December 2015, CH4 emission decreased from early morning I to evening, followed by an increase at midnight, after which the CH4 emission decreased until early morning II. Compared with lysimeters subjected to a high water table (Figure 8b), soil CH4 emissions in the dry season were higher at midnight and early morning II in July and

August, respectively. However, at high water table during the wet season (Figure 8b), soil CH4 emission was higher at noon in September 2015, whereas CH4 emissions decreased from early morning I to evening, after which the CH4 emission increased until early morning II.

(**b**)

**Figure 8.** Soil CH4 emissions (at different times of the day) from a peat soil grown with pineapples in lysimeters under simulated water table fluctuation (**a**) low water table and (**b**) high water table throughout the dry (July and August 2015) and wet (September and December 2015) seasons. Error bars represent standard error and soil mean fluxes with different letters and noted by prime, asterisk, and double prime are significantly different using DNMRT at *p* ≤ 0.05.

Averaged soil CH4 emissions over 24 h under different water table depth varied in the dry and wet seasons (Figure 9). At low water table (0.9 m from the soil surface), averaged soil CH4 emissions were higher in the wet season (December 2015) but lower throughout the monitoring period in July, August and September 2015. Conversely, at high water table (0 m from the soil surface), averaged soil CH4 emissions were higher in the dry season (July 2015) but emissions were lower in August and September 2015. However, throughout the dry and wet seasons, averaged soil CH4 emissions were significantly higher under the low water table compared with that of the high water table (Figure 10).

**Figure 9.** Averaged soil CH4 emissions over 24 h from a peat soil cultivated with pineapples in lysimeters under low and high water tables throughout the dry (July and August 2015) and wet (September and December 2015) seasons. Error bars represent standard error and soil mean fluxes with different letters and noted by prime are significantly different using DNMRT at *p* ≤ 0.05.

**Figure 10.** Averaged soil CH4 emissions over 24 h from a peat soil grown with pineapple in lysimeters under low and high water table. Error bars represent standard error and soil mean fluxes with different letters are significantly different using DNMRT at *p* ≤ 0.05.

Throughout the dry and wet seasons, soil temperature was statistically similar irrespective of water table and sampling time (Table 2). Moreover, there was no significant correlation between soil temperature and CH4 emission (Table 2). This observation is consistent with the results obtained from the soil CH4 measurement from open field pineapple cultivation (Table 1).

Compared with the peat soils grown with pineapple under open field cultivation system, averaged soil CH4 emissions from pineapples in lysimeters subjected to water table fluctuation were significantly higher throughout the dry and wet seasons in 2015 (Figure 11).


**Table 2.** Relationship between soil CH4 emission and soil temperature from lysimeters cultivated with pineapples under simulated water table fluctuation throughout the dry and wet seasons in 2015.

Mean values with same letters within the same column are not significantly difference between means using DNMRT at *p* ≤ 0.05. Top value indicates Pearson's correlation coefficient (r), whereas the bottom values indicate probability level at 0.05 (*n* = 1200).

**Figure 11.** Averaged soil CH4 emissions from peat soils grown with pineapples under open field cultivation system and lysimeters subjected to water table fluctuation in the dry and wet seasons. Error bars represent standard error and soil mean fluxes with different letters and noted by prime are significantly different using DNMRT at *p* ≤ 0.05.

#### **4. Discussion**

*4.1. Soil Methane Emissions from Peat Soils Grown with Pineapple under Open Field Cultivation System in the Dry and Wet Seasons*

Differences in soil CH4 emission across time (early morning, afternoon, evening and midnight) from pineapple cultivated peat soils could be attributed to the microbial structure in the peat soil that controls the balance between CH4 production and CO2 conversion and vice versa by methanogenic bacteria and methanotrophs under anaerobic and aerobic conditions [33], respectively, throughout the dry and wet seasons. Peat soils become net source of CH4 when CH4 production by methanogenic bacteria surpasses consumption by methanotrophic bacteria [34]. Moreover, soil CH4 fluxes are regulated by oxygen supply and availability of labile carbon, where methanogenesis is predominant under anaerobic conditions. Also, soil CH4 emissions might have been affected by the transportation of CH4 by molecular diffusion through the aerobic layer of the peat soils, and through ebullition in the form of bubbles at the peat water table interface [35–37].

Although the averaged soil CH4 emissions were not affected by the flux monitoring period throughout the dry (July and August 2015) and wet (December 2015) seasons, the higher CH4 emission particularly in September 2015 during the wet season was because of the higher rainfall received at the experimental site amounting to 72 mm compared with the lower rainfall received in July (29 mm), August (52 mm) and September (69 mm) 2015 [38]. This result suggests that higher CH4 is emitted under anaerobic and waterlogged conditions. The waterlogged condition of the peat soil in September 2015 might have favoured the thriving of methanogenesis bacteria under anoxic conditions, thus causing higher soil CH4 emission. This result also corroborates previous work by Furukawa et al. [20] and Inubushi et al. [30], who reported that the increase in soil CH4 emission is due to high rainfall.

Although soil CH4 emission was affected by the time of sampling, the insignificant correlation between soil CH4 emission and soil temperature regardless of seasons (dry and wet period) suggest that CH4 emission was not affected by soil temperature due to the moderate soil temperature fluctuation during CH4 flux measurement. The peat soil temperature ranged between 25 to 31 ◦C during the CH4 sampling (Table 1).

#### *4.2. Soil Methane Emissions from Peat Soils Cultivated with Pineapples in Lysimeters under Simulated Water Table Fluctuation in the Dry and Wet Seasons*

Similar to the pineapple cultivated under an open field system, differences in soil CH4 emission across time from field lysimeters subjected to water table fluctuation (low and high water table) relates to the microbial structure in the peat soils, particularly the methanogenic and methanotrophic microorganisms because these organisms control CH4 production and consumption. Also, soil CH4 release might have been influenced by the collapse of peat pores (during the soil excavation and setting up of the lysimeters) that affected CH4 transportation through molecular diffusion, and subsequent soil subsidence in the lysimeters due to water table fluctuation.

In this present study, there was a discrepancy on the averaged soil CH4 emissions from lysimeters under low and high water tables in the dry and wet seasons (Figure 9). The findings reported higher soil CH4 emissions both under low and high water table in the wet (December 2015) and dry (July 2015) seasons, respectively. Moreover, averaged soil CH4 emission under a low water table were higher compared with that of high water table. These observations were not in agreement with the general belief that soil CH4 emission increases with a higher water table. There are no specific reasons that explain the anomaly from the findings obtained. However, the inconsistency of soil CH4 emissions from peat the soils suggest that the factor controlling CH4 emission from the lysimeters could be attributed to the fluctuation of the water table at the soil–water interface. The water table level and its fluctuation at the soil–water interface may have altered the intensity and duration of CH4 production and oxidation processes throughout the dry and wet seasons.

The results on the insignificant correlation between soil temperature and CH4 emission from lysimeters under simulated water table fluctuation irrespective of seasons was consistent with that reported for CH4 measurement under an open field pineapple cultivation system. These observations are further supported by the fact that CH4 emission was not affected by soil temperature because of the moderate soil temperature fluctuation (24.7 to 32.7 ◦C) during CH4 flux measurement. This finding was in agreement with the study by [39,40] who reported that temperature changes had minimal effects on CH4 emission from cultivated peatlands.

It is also possible that soil CH4 from lysimeters and under open field pineapple cultivation was released from non-microbial production of CH4 sources particularly humic acids and lignin [41]. The emission of non-microbial CH4 may have occurred under moderate temperature fluctuations of the tropics because of the high amount of organic matter, humic acids, fulvic acids, lignin, humin and carbohydrate in peat soils [42–44]. In this study, the lower soil CH4 emission from peat soils grown with pineapple under an open field cultivation system compared with that of CH4 emission from the lysimeters throughout the dry and wet seasons (Figure 11) could be attributed to pineapple fertilization. Compound NPK fertilizers containing ammonium were applied to pineapple at 3, 6 and 9 months after planting in June, September and December 2015, respectively. The compound fertilizers might have increased nitrate content in the peat soils because nitrification increases with peat oxidation. The lower CH4 emission due to pineapple fertilization relates to the availability of electron acceptors namely nitrate which inhibits CH4 production [45].

Nitrate is water soluble and leaches to the water table interface leading to decreased CH4 production in anaerobic condition. Also, water table fluctuation (50 to 70 cm from the soil surface) and lateral water movement in the peat soil (open field cultivation system) might have affected the balance between CH4 production and consumption by methanogenic bacteria and methanotrophs. Water table depth affects the soil CH4 emissions because it determines the depth of the oxic or anoxic boundary and redox level within the soil. By contrast, water table fluctuation at the soil–water interface, transportation of CH4 through molecular diffusion through the aerobic peat layer and ebullition at the peat water table interface relatively explains the higher CH4 emission from lysimeters under simulated water table fluctuation.

#### **5. Conclusions**

Soil CH4 emission throughout the dry and wet seasons under open field pineapple cultivation and from lysimeters subjected to water table fluctuation were not affected by soil temperature but emissions were influenced by the balance between methanogenic and methanotrophic microorganisms controlling CH4 production and consumption, CH4 transportation via molecular diffusion through peat pore spaces, and non-microbial CH4 production sources in peat soils namely humic acids and lignin. Although it is generally believed that a high water table increases soil CH4 emission, findings from the study suggest that water table fluctuation at the soil-water interface relatively controls the soil CH4 emission from lysimeters under simulated low and high water table fluctuations. The outcome of this present study demonstrated that soil CH4 emission throughout the dry and wet seasons under an open field cultivation system was affected by the availability of nitrate electron acceptors from pineapple fertilization, which restrict CH4 production, thus leading to lower soil CH4 emission compared with that of CH4 emissions from lysimeters. However, the limited number of CH4 flux monitoring throughout the dry and wet seasons (July, August, September and December 2015) may not be conclusive enough to confirm the findings from the study. Thus, a long-term CH4 flux monitoring period is required to confirm the findings obtained because rainfall distribution, microbial population, chamber humidity and headspace temperature may influence CH4 emission and the outcome of the study. The findings of this study provide an understanding on the effects of water table fluctuation on CH4 emissions in a tropical peatland under pineapple cultivation.

**Author Contributions:** W.L. was responsible for investigation, writing, and original draft preparation. L.N.L.K.C., R.K.J.H. and M.B.J. were responsible for data analysis and visualization. O.H.A. was responsible for supervising, funding acquisition, project administration, experimental methodology, editing, and reviewing. L.O. was responsible for data arrangement, conceptualization, reviewing, and editing the second draft. A.A., A.A.M. were also involved in funding acquisition and project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Long-term Research Grant Scheme (LRGS) and Translational Research Grant Scheme (TRGS) vote number 6300914 from the Ministry of Higher Education Malaysia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors gratefully acknowledge the financial support provided through Putra Grant UPM. Appreciation also goes to Malaysia Agricultural Research and Development Institute (MARDI) Saratok, Sarawak, Malaysia, for the collaborative research.

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

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