Abstract
Introduction
Essential oils in plants possess many properties such as antimicrobial activity and antioxidants for protection of plants against insects.1,2 Industrialization and commercialization of essential oils have resulted in their wide array of use such as manufacture of cosmetics, perfumes, pharmaceuticals, tooth pastes, mouthwashes, sweets, tobacco, scented soaps, fringe medicine, and in the production of wine.1,3 Furthermore, some components of essential oils have found applications as chiral accessories in the synthesis and transformation of organic structures that are of economic benefits. 4 This implies that production of essential oils in the world would continue to expand immensely in the near future as a result of a surge in the number and preferences of consumers. It is estimated that the worldwide demand for essential oils would reach a total cost of US$ 5 trillion by 2050. 5
Therefore, a highest degree of interdisciplinary investigation on essential oil plants is required for expanded production and high-quality products. 4 It is noteworthy of the fact that the production of essential oils does not necessarily only depend upon the plant metabolism and structural growth processes of the synthesizing tissue, but also on the entire plant physiology. 4 The synthesis and collection of essential oil producing structures are located near the surfaces of the glandular trichomes and secretory canals of the plants. 6 The quality and quantity of essential oils produced by plants depends on various environmental factors which are either physical or chemical. 6 The physical factors include temperature, soil type, drought and wind while the chemical stress comprises of salinity, pH, organic matter, and toxins. 6
Among essential oils producing plants is the vetiver (Vetiveria zizanioides (L.) Nash. Syn. Chrysopogon zizanioides (L.) Roberty; family Poaceae) that has been widely used in top-ranking perfumery and cosmetics due mainly to its persistent scent. 7 It has also been used in food production as a flavor additive, for example; ice cream and beverages. 7 Furthermore, the essential oils from this plant have been found to possess anticancer, antibacterial, antifungal, and anti-inflammatory activities. 8 Essential oils producing plants can be contaminated with heavy metals existing in the air, soil, and water due to anthropogenic activities such as mining, agriculture, and industrial activities that produce plant protective agents.9,10 This can take place during developmental stages of the plant. The occurrence of heavy metals such as Ni, Cu, Mn, Zn, Pb, and As in the environment has seen drastic increase in recent years due to rapid growth of industrialization. 11 Low concentrations of Cu, Mn, Ni, and Zn play an important biological role but elevated levels of these metals can be toxic to plants. 9 Cu and Zn are essential micronutrients required by plants for normal development and metabolism. These metals take part in cell wall metabolism, are dominant components in regulatory proteins, electron transporters in photosynthesis, and respiration processes, serve as cofactors for diverse enzymes and are responsible for the biosynthesis of a variety of plant hormones. 9 However, elevated concentration of these metals in plant tissues can bring about undesirable effects such as inhibition of plant growth and development, induces production of toxic reactive oxygen species and destroys cell membranes. 9 On the other hand, heavy metals such as Pb and As are regarded as nonthreshold elements, which means that they may be toxic to humans at any concentration level. 12 The toxicity of Pb and As to plants comprises disruption of photosynthesis and water balance. 5
Therefore, excess concentrations of these metals can affect the quality and quantity of essential oils. 13 In addition, the uptake of these toxic elements by plants can be harmful to animals when such plants are consumed. 13 Metals cannot be biodegraded and therefore their removal requires their immobilization or conversion into a less toxic form. 14 The application of chelators such as ethylene diamine tetra-acetic acid (EDTA) has been reported to enhance bioavailability, uptake, and translocation of metals from the soil. 15 EDTA assists in effective phytoextraction of heavy metals by maintaining adequate heavy metal concentrations in soil solution. 16 Pb uptake by Chinese brake ferns was enhanced after application of EDTA and its translocation from roots to shoots was also increased. 17 In another study, concentrations of arsenic in the shoots of Dixie silver-back fern (Pityrogramma calomelanos) doubled after amendment the nutrient solution with EDTA. 18 The use of microorganisms such as arbuscular mycorrhizal fungi (AMF) has also been reported to influence the uptake of metals due to the close interaction of AMF with plant roots to create a rhizosphere community. 19 AMF achieves this by providing an uninterrupted link between soil and roots through establishment of enormous root system with extensive branching resulting in enhanced efficiency in metal uptake and translocation. 20 For example, an AMF called Glomus mosseae has been reported to increase metal bioavailability through mineral solubilization and promotes root to shoot translocation. 21
In view of the intricacy of the organic components of essential oils and their intrinsic biological distinction, it becomes apparent that their safety and quality should be evaluated. 10 While most of the studies carried out before in Botswana have looked at characterization and structure elucidation of essential oils originating from plants cultivated in Botswana, less emphasis has been put on heavy metal content of essential oils.22,23 Therefore the aim of the present investigation is to determine heavy metal content in essential oil extracts and establish the effect of such metals on the yields and quality of essential oils obtained from vetiver grass cultivated in mine tailings. The impact of soil amendment from EDTA and microbes on the yield and quality of essential oils will also be investigated.
Materials and Methods
Description of the Mine Tailings Sampling Location and Pot Preparations
The tailings used in the experiment were collected from the Bamangwato Concessions Limited mine which has been mining and smelting Cu and Ni since 1960 and is situated between longitude 27°47′East and 27°53′ East and latitude 22°55′ South 22°00′South in Selibe-Phikwe, Botswana (Figure 1). Tailings were collected in the dumpsite with no vegetation. Mine tailings were collected, air dried and sieved to pass through 5 and 2 mm mesh for cultivation of plants and chemical analysis, respectively. The tailing's properties and the heavy metal contents are presented in Table 1. The tailings are highly acidic, sandy loam with very low organic matter and nutrient content and have high amounts of Cu, Ni, and As. The cow manure used in the experiment was obtained from local farm that contain 3.8% N, 34% organic carbon, and pH 7.9. The 10 L plastic pots with 28 cm top diameter, 24 cm base diameter, and 22.5 cm height were used for the experiments under greenhouse conditions. Plastic pots were filled with 8 kg tailings, or a mixture of mine tailings + cow manure (MTCM) before mycorrhiza inoculation and plant establishment.
(A) Map of Africa indicating location of Botswana and (B) map of Botswana showing mine tailings sampling site in Selibe-Phikwe town and (C) the location of the mine tailings in Selibe-Phikwe town.
Physicochemical Properties, Total and Water-Soluble Heavy Metal Content (Mean ± SD) of Mine Tailings, Cow Manure and Mixture Mine Tailings and Cow Manure.
Means within the same column followed by the same letter(s) are not significantly different at 5% level based on Tukey's test. ns (P > .05); P < .05 (*significant); P < .01 (**highly significant); P < .001 (***very highly significant).
Abbreviations: ns, nonsignificant; SD, standard deviation per treatment.
Description of the Treatments and Plant Culture
A 2-factor experiment was established on the response on vetiver to mycorrhiza inoculation and EDTA amendments in a heavy metal contaminated MTCM. There are 2 levels of mycorrhiza (−AM and + AM) and 3 types of EDTA application consisting of 6 treatments: T1—MTCM (without AMF and EDTA); T2—MTCM with 5 mmol EDTA split application; T3—MTCM with 5 mmol EDTA single-dose application; T4—MTCM with AMF alone; T5—MTCM with AMF and 5 mmol EDTA split application; T6—MTCM with AMF and 5 mmol EDTA single-dose application. The single dose was applied 20 weeks after transplanting while for the split dose the first dose was applied 20 weeks after transplanting at 5 mmol/kg of soil and second dose 22 weeks after transplanting at 5 mmol/kg soil. Harvesting took place at week 24 after transplanting. Cow manure equivalent to 10% of the total weight of soil was mixed thoroughly before potting. The slips of the vetiver were obtained from seedlings grown under greenhouse conditions with 70% light penetration at the Environmental Remediation and Management Laboratory of the Botswana International University of Science and Technology in Palapye. Palapye has a climatic condition composed of a hot and rainy season with subtropical thunderstorms from November to March; a cool dry season from April to August; and a hot season with minimum precipitation from September to November. The average temperature in Palapye is 20.1°C and the annual rainfall of about 424 mm. The experiment was conducted from October 2018 to June 2019. The slips of the vetiver were selected for visual uniformity. These vetiver slips were cleaned with tap water and surface sterilized before replanting at the center of each pot. In each pot was planted 1 slip of 15 cm from the base of the roots while the roots were trimmed to 3 cm. The slips were surface sterilized with 10% chlorox for 30 min and washed thoroughly with sterile distilled water before planting. AMF were applied to the mine tailings and their effect on the yield of essential oils was determined. For the mycorrhiza treatments, 5g of sterile (121 °C, 30 min for 3 consecutive days) or nonsterile AM inoculants (MYKOVAM®, National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Banos, Philippines) were mixed with the soil at the center of pot just below the roots of the vetiver slips of the without AMF and with AMF treatments, respectively. Three replicates were used for each treatment laid out in a complete randomized design. The plants were irrigated regularly and monitored for survival and growth inside the shade house (70% exposure). To assess the mycorrhizal infection, a portion of the fresh roots from each plant (0.2 g) was subjected to trypan blue staining before the microscopic examination. 24 The colonization rate on the roots by AMF was quantified using the gridline intersection method. 25 The shoots and the remaining roots were oven-dried at 70 °C for 3 days, dry weights were recorded and ground for chemical analysis.
Plant Sampling and Chemical Analysis
During the 40-weeks cultivation period, vetiver grasses were periodically monitored for survival and growth. At harvest, the shoots were excised, and the roots were carefully separated from the soil, and their fresh weights were determined. The roots and all the shoots were then air dried for 3 days, weights were recorded and the air dried plant materials were used for extraction of essential oils. A portion of roots and shoots (5 g each) were oven dried at 70°C for 3 days and ground for chemical analysis. For the chemical analysis of plant tissues, 1.00 g samples were digested using a nitric acid and hydrogen peroxide mixture in an open vessel at 120 °C for a minimum of 4 h and analyzed for heavy metals using inductively coupled plasma mass spectrometry (ICP-MS)
Oil Extraction and Characterization
The dried vetiver grass roots samples were crushed with a blender and weighed to around 50 g. The samples respectively were then dissolved with 2L water in a distillation flask and subjected to ultrasonication at the following optimum parameters: duration 25 min; temperature 70 °C; high power (005) and high frequency. This was followed by hydro distillation (HD) for 5 h. 26 After collection of the distillate, the distillate was introduced to liquid–liquid extraction method: water was decanted by separation of water from essential oil using 5 mL pentane/dichloromethane thrice consecutively. Then half a spatula sodium sulfate was put in essential oil pentane/dichloromethane mixture to dry the possible remaining water. To finally get the essential oil, pentane/dichloromethane was evaporated at its boiling point of 40 °C.
Soil Sampling and Chemical Analysis
Representative soil samples from each pot were collected after harvest of the grass. The samples were passed through a 2 mm sieve, air dried, and stored at 4 °C until chemical analysis. The soil pH and electrical conductivity (EC) were measured in water suspension (1:2) after shaking for 1 h. Total heavy metal contents were measured after wet acid digestion in an open vessel at 120 °C for 6 h using HNO3: HCl mixture (1:3) in a hot plate followed by quantification using ICP-MS. 27 The available fraction of heavy metal in soil was assessed by determining the water soluble (WS) and ammonium acetate exchangeable. Soil samples were shaken with distilled water (1:100) for 2 h at room temperature to obtain a WS fraction. The exchangeable fraction (Exch) was determined by using 1N ammonium acetate (pH 7) at 1:100 ratio for 2 h at room temperature. The WS and the Exch fractions were analyzed for different heavy metals using ICP-MS. Samples were analyzed in duplicates. Control soil was collected from the noncontaminated site near the University Research center. The control soil and the tailings were sterilized for 3 consecutive days at 121 °C for 1 h at 15 psi.
Statistical Analysis
The data were analyzed using a 2-factor analysis of variance (ANOVA) in completely randomized design (CRD) using IBM SPSSv25 (IBM, 2017) statistical software. All data are presented as mean values with standard deviation (mean ± SD, n = 3). When significant differences were observed, Tukey's test was performed for treatment mean comparison at 5% level of significance. Each parameter was analyzed separately per soil treatment.
Results and Discussion
Physicochemical Properties and Heavy Metal Content of the Mine Tailings
The mine tailings were highly acidic, sandy loam with very low organic matter and nutrient content and contained high amounts of various heavy metals (Table 1). The soil pH plays a pivotal role on soil biogeochemical processes such as biodegradation of organic pollutants, soil enzyme activities, precipitation or dissolution of organic matter, heavy metals and microbial ecophysiological indicators. 28 The pH has a huge impact on the soil biophysicochemical characteristics that influence plant tissue development and growth as well as biomass yield. 29 In addition, soil pH dictates the fate of chemicals in the soil matrix which has an effect on nutrient bioavailability for plant growth, distribution, and removal of toxins in the environment. 28 In the current study, the mine tailings were highly acidic with pH as low as 3.72 (Table 1). Cow manure was mixed with the mine tailings so as to provide nutrients to plants grown in the mine tailings. The pH of cow manure was in the neutral region at a value of 7.9 (Table 1). After mixing cow manure with the mine tailings the pH of the mixture was measured to be 6.24, which was weakly acidic (Table 1). Cow manure contained organic matter of 34% compared to only 3.21% of a mixture of MTCM. This implied the obvious high content of carbon in cow manure compared to the untreated mine tailings. Organic matter plays a vital role in the release of nitrate used for plant growth and development. 30 The presence of microorganisms such as AMF boosts processes such as nitrogen-fixing, nitrate reduction, and nitrification which in turn accelerate nitrogen cycling. 31 AMF has also been found to promote plant nutrient content through transfer of phosphorus (P) captured from soil in exchange for carbon (C) acquired from photosynthate. 31 The EC of the mine tailings was determined in the range of 982 to 2482 μS/cm (Table 1). Vetiver grass has been found to thrive in soils with high salinity (with EC ∼ 4800 μS/cm). 30 Therefore the electrical conductivities of the mine tailings were lower than the maximum values at which vetiver grass can still grow and flourish. The EC of the mine tailings was about 3 times higher than that of cow manure. Both mine tailings and a mixture of MTCM contained the largest concentrations of Cu and Ni which did not come as a surprise since the mine tailings are from a copper and nickel mine (Table 1). Copper concentrations of 1632.24 and 1489.23 mg/kg in the mine tailings and mixture of cow manure and mine tailings, respectively, were the highest of all the metals investigated. The concentrations of Ni and Cu determined in the mine tailings were higher than the World Health Organization (WHO) set permissible limits of 50 and 100 mg/kg in soil, respectively. 32 On the other hand, the concentrations of Pb, Zn, and Mn were lower that the WHO set permissible limits of 100, 300, and 2000 mg/kg, respectively. 32 As concentration was twice the maximum acceptable limit for As (20 mg/kg) in agricultural soils as proposed by the European Union. 33 Similarly, the WS concentrations of Cu and Ni in mine tailings and MTCM were the highest compared to all the other metals studied. It is anticipated that elevated levels of heavy metals in mine tailings would accelerate the production of essential oils due to heavy metal stress resulting in the release of secondary metabolites in the form of essential oils.
Growth and Dry Matter Yield of Vetiver Grass
Vetiver grass was able to grow and survive in Selibe-Phikwe Cu and Ni mine tailings amended with AMF and/or EDTA in the presence of cow manure. The percentage of the mycorrhiza was significantly affected by the introduction of EDTA under both split-dose and single-dose application (Table 2). Under split application, the percentage of mycorrhizal roots was lowered by 3 times compared to the control (T1) while single-dose application resulted in no mycorrhizal roots detected. Previous studies have shown that EDTA is bound to inhibit mychorrizal roots because of its antifungal activity. 34 In contrast, single application of AMF (T4) resulted in a significant increase of mycorrhizal roots by a magnitude of over 10 times compared to control (T1). In addition, percentage of mycorrhizal roots still increased 7 times after application of both EDTA and AMF (T5), however, this increase was lower than that of single application of AMF, an indication that EDTA has antifungal activities. The shoots dry matter yield was raised by either split or single application of EDTA, a single application of AMF or a combined application of EDTA and AMF. The split and single application of EDTA both caused minimal increases in the shoot dry matter yield over the control (T1). On the other hand, mycorrhizal inoculation alone (T4) led to the highest dry matter yield of the shoot of magnitude of 1.5 times higher than the control. The single and split combined application of EDTA and AMF enhanced the shoot dry matter yield relative to when EDTA was used alone in both split and single applications. Similarly, application of AMF alone or in combination with EDTA for both split and single application exhibited enhanced root dry matter yield compared to application of EDTA alone. Mycorrhizal inoculation resulted in an increase in the root dry matter by 1.5 times over the control (T1) while EDTA alone caused a decrease in the root dry matter yield. The results of the shoot and root dry matter yields demonstrate the effectiveness of AMF to improve and optimize nutrient uptake. 35 AMF does this by enabling the root system to reach a large volume of soil nutrients by extending the root zone and consequently promoting plant growth. 36 AMF has also been found to increase the soil volume accessible for phosphorus (P) acquisition and its effective transport to plant roots in soils highly contaminated with heavy metals, resulting in enhanced plant growth and nutrition.37,38 This is supported by the results in Table 2, whereby the dry matter yield for shoots and roots increased by 45.5% and 51.9%, respectively, after treatment with AMF (T4) compared to the control (T1).
Mycorrhizal Infection and Dry Matter Yield (Mean ± SD) of Chrysopogon zizanioides Grown in Cu-Ni Mine Tailings With Cow Manure as Affected by Mycorrhizal Inoculation and EDTA.
Means within the same column followed by the same letter(s) are not significantly different at 5% level based on Tukey's test. nonsignificant (P > .05); P < .05 (*significant); P < .01 (**highly significant); P < .001 (***very highly significant).
Abbreviations: AMF, arbuscular mycorrhizal fungi; EDTA, ethylene diamine tetra-acetic acid; SD, standard deviation per treatment.
Heavy Metal Concentration in the Roots of Vetiver Grass
Results of accumulated Cu, Ni, Pb, Zn, Mn, and As in the roots of vetiver grass after application of either AMF or EDTA or both are presented in Table 3. The results showed that single application of EDTA (T3) resulted in enhanced Cu uptake of over 2.3 times (316.8% increase) over the control (T1) by the roots compared to split application of EDTA where an increase of only 4.7% was observed. Application of AMF alone and in combination with EDTA also enhanced Cu uptake by the roots of vetiver grass. Application of AMF alone (T4) caused more increase in Cu uptake by the roots compared to when combined with EDTA (T5 and T6). A similar trend was observed for Ni where application of either AMF or EDTA or their combination resulted in enhanced Ni, Mn, and Zn uptake by the vetiver grass roots. However, application of AMF alone (T4) led to significant increase in the uptake of Ni (108.0% increase) compared to single application of EDTA (T3, 42.6% increase). The same was observed for Zn where treatment with AMF (T4) induced more Zn uptake of 31.1% compared to 19.8% increase for single treatment with EDTA (T3). Plants such as vetiver grass have devised some mechanisms to mitigate and tolerate the severity of elevated metal concentrations in their roots. 39 The uptake of heavy metals from the soil by the plant roots and their translocation to shoots depend largely on the availability of metals in soluble form for their efficient phytoextraction. 40 EDTA has been reported to be an efficient phytoextractant of metals in contaminated soils. 40 In a study by Chen et al, 41 vetiver grass cultivated in soil treated with EDTA was found to successfully enhance phytoextraction of Pb, Cu, Zn, and Cd by 98%, 54%, 41%, and 88%, respectively. They found that soil amendment using EDTA enhanced the bioavailability and improved the uptake of Cu and Pb by the roots and their subsequent translocation to the shoot. In another study by Roongtanakiat et al, 42 the application of EDTA to the soil was found to enhance the concentration and uptake of Zn, Mn, and Cu by vetiver grass. In a recent study by Rathika et al, 43 application of EDTA showed enhanced Pb uptake by Brassica juncea by 60.2 mg/g compared to untreated soil (10.0 mg/g). EDTA has been found to modify metal bioavailability resulting in enhanced phytoextraction efficiency and subsequent uptake and translocation in shoots. 42
Heavy Metal Concentration (Mean ± SD) in the Roots of Chrysopogon zizanioides Grown in Cu-Ni Mine Tailings With Cow Manure as Affected by Mycorrhizal Inoculation and EDTA.
Means within the same column followed by the same letter(s) are not significantly different at 5% level based on Tukey's test. ns (P > .05); P < .05 (*significant); P < .01 (**highly significant); P < .001 (***very highly significant).
Abbreviations: AMF, arbuscular mycorrhizal fungi; EDTA, ethylene diamine tetra-acetic acid; MTCM, mine tailings + cow manure; ns, nonsignificant; SD, standard deviation per treatment.
Previous studies have shown that mycorrhiza in the roots could strengthen the plant's ability to uptake and translocate essential elements, water, and even tolerate hostile environmental conditions.44,45 Plant roots and mycorrhiza work in a symbiotic strategy whereby plant root network and activities support the flourishing microbial community that assist various biogeochemical processes in the rhizosphere and help improve plant growth cultivated in stressful conditions such as those with high metal loading.37,46 Mycorrhizal colonization of plant roots has been found to increase root surface area for essential elements uptake due to extrametrical fungal hyphae that can extend some distance into the soil and uptake nutrients with concomitant transport of heavy metals in the host root. 37 The secretion of secondary plant metabolites includes such metabolites as organic acids and biosurfactants that can influence the biogeochemical processes in the plant roots. 46 The plant provides the microbial community with soluble carbon sources while the mycorrhiza provides the plant with enhanced ability to uptake water and essential elements from the soil. 46 The mycorrhiza can achieve this through several mechanisms plant through several mechanisms such as secretion of secondary metabolites that alter various biogeochemical processes in the rhizosphere and help improve plant growth cultivated in stressful conditions such as those with high metal loading. 46 Therefore, AMF can enhance the effectiveness of plants such as vetiver grass for phytoremediation applications whereby the mycorrizae and not the roots are the major routes of nutrient uptake by the plants. 37 The inoculation of plant roots suing AMF has demonstrated its efficient ability to enhance phytoaccumulation of heavy metals in plants. 47 Lastly, AMF has also been found to alleviate metal stress in plants through a mechanism that involves membrane transporters in AMF arbuscules that may transport metals to the interfacial matrix and their subsequent integration into the plant. 3
Yields of Essential Oils Extracted From Vetiver Grass
It is worth noting that vetiver grass roots essential oils are not easily extractable since they are found in concealed root tissues that are not easily accessible. 48 In addition, the presence of high percentage of large molecular weight organic components (ie sesquiterpenes) with low vapor pressure make the extraction process even more difficult and time consuming. 48 To be successfully extracted, the essential oil has to be extracted by a relatively fast process to improve diffusion of the oil from inside fibrous root tissues outward and to the surface. Ultrasonic-assisted HD (US-HD) was employed in this study across all sample treatments at optimized parameters to crush the cell wall (cause cell lysis) or increase the diffusion rate and thus allowing the oil to be exposed out of the oil glands/sacs resulting in a short extraction time. 49 US-HD proved to be efficient in this study, since more constituents including most valuable constituents (those with high boiling points-late eluters, i.e; vetivone) in some treatments were reported at shorter extraction time; 5 h of extraction as compared to literature which need 24 h in HD to achieve similar results. 50 US-HD also provides better yield, the percentage yield across all treatments ranged between 0.26% and 0.94% which is a good yield when compared to other extraction methods (Table 4). It is reported that essential oil production reaches a peak after at least 2 growing seasons whereas the harvest was carried out after a single growing season in the present study. 51 Similar studies employing such methods as steam distillation usually produce around 0.3 to 1.0wt% on dry root basis of oil.52,53 The extracted vetiver grass roots oil retained its viscosity property and was light brown in color which is classified as a regular quality while clear yellow color normally indicates premium quality.52,54 The freshly extracted vetiver grass essential oil had an earthy woody and sweet odor which correlates well with related research. 55 The presence of heavy metals in the soils have been reported to enhance the yield of essential oils by plants such as vetiver grass. 56 The increase in percentage yield of essential oil by plants growing in heavy metal polluted sites has been attributed to the plant's response to heavy metal stress resulting in increased release of secondary metabolites such as essential oils.56,57 The mine tailings demonstrated high concentrations of metals.
Average Yields (Mean ± SD) of Essential Oils Extracted From Vetiver Grass Roots Grown in Cu-Ni Mine Tailings Amended With Cow Manure and EDTA and Inoculated with AMF.
Means within the same column followed by the same letter(s) are not significantly different at 5% level based on Tukey's test. ns (P > .05); P < .05 (*significant); P < .01 (**highly significant); P < .001 (***very highly significant).
Abbreviations: AMF, arbuscular mycorrhizal fungi; EDTA, ethylene diamine tetra-acetic acid; MTCM, mine tailings + cow manure; ns, nonsignificant; SD, standard deviation per treatment.
In the present study, the control treatment that was free from heavy metals presented the lowest yields of essential oils since vetiver grass did not have any metal stress as a stimulating factor to trigger it to produce more essential oil. A similar study by Sulastri et al 56 reported an increase in the essential oil content by up to 101.5% from vetiver grass grown in soils spiked with 100 ppm of Cd compared to untreated soils. In another study by Prasad et al, 57 application of 50 ppm of Cd to healthy soils resulted in an increase of 33.33% of essential oils recovered from plants grown in such soils. The adsorption of high concentrations of heavy metals by essential oil producing plants has also been reported to affect the chemical composition of essential oils. 58 For example, different concentrations of Pb affected the yield and chemical composition of essential oil and phytoaccumulation of garden mint whereby carvone as major constituent of the essential ranged from 39.3% for plants grown in healthy soils (control) to 90% for plants cultivated in soils with concentrations of Pb. 59 The concentrations of heavy metals established in the mine tailings were quite high and this resulted in heavy metal stress experienced by vetiver grass. 60 There are various ways through which plants defend themselves against high concentrations of toxic heavy metals which includes generation of metal binding proteins such as phytochelatins-oligomers of gluthione that is responsible for heavy metal detoxification. 39 One way through which plants alleviate heavy metal stress is through the release of secondary metabolites such as essential oils. 39 This is one way through which plants growing in heavy metal polluted sites are able to tolerate high concentrations of such metals in the soil. The release of phenolic compounds is caused by the over-expression of the biosynthesis of phehylpropanoid enzymes route. 15 This is brought about by heavy metals via upregulating the functions of key biosynthetic enzymes such as Shikimate dehydrogenase, glucose-6-phosphate dehydrogenase, and cinnamyl alcohol dehydrogenase. 61 There are other studies that have successfully demonstrated the ability of vetiver grass to tolerate heavy metals, acidity, salty soils, and agrochemicals. 62 In a study by Maleki et al, 58 garden mint grown on soil contaminated with high concentrations of Pb (about 9000 mg/kg) was able to produce essential oil that is 10 times higher than that of the control.
The effect of EDTA on essential oil production by vetiver grass was also studied. The application of EDTA (split application) to the mine tailings resulted in significant increase in the yield of essential oils from 0.26% (C) to 0.95% (T2) (Table 4). The application of EDTA to the mine tailings further increased the percentage yield of essential oils by up to 0.95% compared to the mine tailings without EDTA (T1, 0.86%). EDTA, as an excellent chelating agent is known to strongly bind heavy metals in the soil and enhance their translocation from the plant's roots to the shoots and thereby increasing heavy metal stress in plants. 41 Consequently, the heavy metal stress would lead to excretion of secondary metabolites such as essential oils resulting in their increased yield. 57 For example, addition of EDTA to the soils resulted in a tremendous increase in Pb concentrations in the roots of vetiver grass reaching high levels of 2280 mg/kg after application of 5.0 mmol/kg of EDTA relative to only 556 mg/kg without EDTA amendment. The availability of EDTA molecules improves the metal extraction from exchangeable sites and eventually creating soluble metal-EDTA complexes. 63 However chelating metal ions expose plants to toxic metal stress which triggers the plant to produce more of certain components including phenolic components.15,53 Therefore, this indicates the intensification of heavy metal stress to plants upon application of soil amendments such as EDTA that chelate heavy metals and make them easily adsorbed into plant tissues. The heavy metals stress was strengthened by increased bioavailability of heavy metals as a result of application of EDTA leading to production of secondary metabolites such as essential oils as a defence mechanism to survive the heavy metals stress. 56
Microbes (arbuscular mycorrhizal) symbiosis has exhibited varying effects on the secondary metabolites inclusive of essential oil quality and quantity. 64 Essential oils yields from plants grown in mine tailings amended with AMF (T4, 0.89%) were significantly higher than yields in healthy soils (C, 0.26%). The essential oil yields in the AMF amended mine tailings (0.89%) did not show significance difference from that of the untreated mine tailings (T1, 0.86%). However, this small increase in essential oil yield in soils amended with AMF result from the fact that mycorrhization enables the root system to utilize large volume of soil by elongating the root zone thus accessing soil pores that the root hairs cannot reach. 36 This means that heavy metals in the soil have access to a large area of the root system resulting in high levels of metal stress in plants that compels plants to devise mechanisms of alleviating this heavy metal stress. The effect of microbes on metal uptake by vetiver grass has also been carried out by Pripdeevech et al, 65 whereby essential oil yield of 0.27% was achieved from vetiver grass grown in soils amended with microbes compared to 0.18% in normal soils. The high essential oil yields in microbes-amended soil have been postulated to be due to the possibility of biotransformation of the essential oil precursors by the microbes. In a similar study, tissue culture cleansed (bacteria and fungus free) and noncleansed vetiver grasses were cultivated for 5 months in pots with identical conditions except for sterilized soil for cleansed vetiver grass and unsterilized soil for noncleansed vetiver grass. 66 The essential oil yield of 0.35% was attained for noncleansed plants compared to 0.02% for plants free from microbes. Various studies have confirmed the effectiveness of microbes in increasing production of essential oil and improving essential oil composition.37,67 Moreover, essential oil yield and chemical composition is dependent on many factors such as genetic and evolution, climate conditions, photoperiod, and nutrients. 68 Two factor ANOVA in CRD showed that statistically, the percentage yields across all the treatments are very highly significant (P < .001).
Heavy Metals in Essential Oils Extracted From Vetiver Grass Roots
For vetiver grass to be successfully credited for their use in the perfumery and food production, vetiver grass extracts must be free from any toxic substances that may have adverse health effects to consumers. However, vetiver grass has great capability to absorb large concentrations of heavy metals, especially Pb, Zn, and Cu, in their roots. 69 This ability by vetiver grass to accumulate heavy metals in their roots may lead to cross-contamination of CZ oils with heavy metals. 69 Essential oils of vetiver grass grown on heavy metal contaminated mine tailings extracted by US-HD was further analyzed for heavy metals content. Vetiver grass grown in healthy soil (control, C) and in soils with the following treatments; T1,T2, T3, T4, T5, and T6 showed no noticeable traces of heavy metals in their essential oils (Table 5). This buttresses the assertion that essential oils producing glands in vetiver grass roots are in concealed and inaccessible tissues leading to recovery of micro volumes of oils extracted. It was then decided to mix the extracts from all 6 extractions to make a pool treatment. The results from cumulative treatments showed contamination from heavy metals such as: Cu (0.150 mg/kg), Ni (0.063 mg/kg), Pb (0.022 mg/kg), As (0.003 mg/kg), Zn (0.109 mg/kg), and Mn (0.043 mg/kg) (Table 5). As expected, the concentration of Cu in the essential oils was the highest compared to other heavy metals analyzed. Similar studies employing HD have found no heavy metals in vetiver grass essential oil even though high concentrations of heavy metals were determined in plant tissues. 53 The set permissible limits for heavy metals in plants as per WHO are 1.63, 10.0, 10.0, 20.0, and 50.0 mg/kg for Ni, As, Pb, Cu, and Zn, respectively.70,71 Even though these are the set permissible limits for heavy metals in plant tissue and not essential oil, it was established that concentrations of all heavy metals determined in essential oils were lower than the set permissible limits. It is important to note that even though the mine tailings retained high concentrations of heavy metals (up to 1632.24 mg/kg Cu), the occurrence of heavy metals in the extracted essential oils was low. Similar studies carried out previously have also reported similar results of low or undetectable heavy metals in the essential oils extracts. In a study by Kunwar et al, 72 undetectable levels of Cu and Cd were established for essential oils obtained from O. basilicum grown in soils treated with 270 to 700 mg/kg and 6 to 30 mg/kg of Cu and Cd, respectively. Lal et al 73 also reported very low concentrations of heavy metals Cr (0.14 ppm), Ni (0.10), and Pb (0.04 ppm) in essential oils extracts whereby the concentrations were found to be even lower than the set critical limits for food. 73 Zheljazkov et al 2 suggested in their study that aromatic crops could be cultivated on heavy metal polluted sites without running the risk of metal transfer from the soil to the essential oil. The concentrations of heavy metals in the present study were below the threshold limit in the essential oil extracts, thereby posing no threat if used in the perfumery and cosmetics production. There are many contributing factors to this observation such as plant species and environmental conditions. However, it should be noted that the continuous buildup of heavy metals in soil may result in the concentrations of heavy metals in vetiver grass exceeding permissible limits.
Heavy Metal Concentrations (Mean ± SD) in the Essential Oils Extracted From Vetiver Grass Grown in Cu-Ni Mine Tailings Amended With Cow Manure and EDTA and Inoculated With AMF.
Abbreviations: AMF, arbuscular mycorrhizal fungi; EDTA, ethylene diamine tetra-acetic acid; HM, heavy metal; MTCM, mine tailings + cow manure; PT, pool treatment; SD, standard deviation per treatment.
Conclusions
In summary, we have shown the phytoremediation capability of vetiver grass cultivated in mine tailings and its ability to produce more essential oils under heavy metal stress. The concentrations of Ni and Cu determined in the mine tailings were higher than the WHO set permissible limits while As concentration was twice the maximum acceptable limit for As in agricultural soils as proposed by the European Union. Essential oil yields 0.86% were obtained from vetiver grass cultivated in mine tailings compared to only 0.26% in healthy soil. The large concentrations of heavy metals As, Cu, Mn, Ni, Pb, and Zn in the mine tailings resulted in increased yield of essential oils due to the plant's response to heavy metal stress that led to production of secondary metabolites in the form of essential oils. Addition of either EDTA or AMF to the mine tailings also increased the essential oil yields due to the chelating effect of EDTA and mychorrhization by AMF. EDTA complexed heavy metals and brought them into solution form and increased heavy metal uptake from the soil. This raised the heavy metal stress level of the vetiver grass and subsequently increased excretion of secondary metabolites in the form of essential oils. Mine tailings amended with EDTA resulted in a further increase of 0.95% yield of essential oil compared to 0.86% and 0.26% for un-amended mine tailings and healthy soil, respectively. Similarly, mine tailings inoculated with AMF demonstrated increased essential oil yield of 0.89% compared to untreated mine tailings (0.86%) and healthy soil (0.26%). Furthermore, mine tailings treated with both EDTA and AMF revealed increased essential oil yield (0.94%). It is important to note that treatment of mine tailings with EDTA slightly increased essential oil yield (0.95%) compared to treatment with AMF (0.94%). The essential oil extracts were found to be free from heavy metal pollutants, making them useful for various applications without further purification or treatment. However, the long-term effect of heavy metals on essential oil producing plants should be kept in check to keep the heavy metals in the oils within permissible limits.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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