Influence of Different Rates of Plant-Based Compost on Clay Soil Metal Behavior and Human Health Risk Assessment in Moringa oleifera Leaf Biomass

An investigation of the impact of adding plant-based organic compost to clay soil from a Moringa oleifera farm focusing on the metal content, bioavailability, and accumulation of nutrients in M. oleifera leaves was conducted. Clay soil was mixed with 15%, 30%, 45% and 60% plant-based organic compost (by volume) in 20 cm wide, 2 L pots. Moringa oleifera plants were planted in four replicates of each treatment and control group. Results revealed that the addition of compost significantly (P < 0.05) altered the concentration of metals in the soil. Correspondingly, accumulation of nutrients in M. oleifera leaves increased with the addition of compost to the soil, except for cobalt and chromium. Trace elements had minimal bioavailability in the amended soils, and their presence in the leaves was lower than the permissible trace metal levels in food. The 30% combination had the highest concentration of calcium (45 042.5 mg/kg), magnesium (17430.0 mg/kg) and phosphorous (8802. 5 mg/kg) in M. oleifera leaves. The study concluded the addition of compost improved bioavailability of nutrients in the soil and their concentration in M. oleifera leaves. The target hazard quotients for heavy metals was less than one, indicating that M. oleifera leaf biomass harvested from soil amended with plant-based compost is safe for human consumption. These results serve as guidelines for recommended organic certification requiremets where plant-based compost is often used in the fast-growing herbal industry.


Introduction
Moringa oleifera L. commonly known as "horseradish," "drumstick," or "miracle tree" is a soft-stem plant in the monogeneric Moringaceae family (Gopalakrishnan et al. 2016).Native to the Himalayan Mountains of India, Pakistan and Nepal (Leone et al. 2015), it also grows naturally in Namibia, Angola, Kenya, Ethiopia and Egypt (Konmy et al. 2016).Moringa oleifera is well-known for its properties affect production and nutrient content of M. oleifera (Grosshagauer et al. 2021).Artificial fertilizers have been used to increase the production and nutrient content of M. oleifera (Sarwar et al. 2017).Despite increasing growth, artificial fertilizers may result in the accumulation of heavy metals in plant leaves (Chibuike and Obiora 2014).Furthermore, as consumers gradually embrace a heathier lifestyle, demand for organically grown food has steadily increased (Meemken and Qaim 2018).Thus, many M. oleifera farmers are avoiding the use of artificial fertilizers.
Compost is an organic fertilizer that supports sustainable agriculture.It improves the physiochemical, organic matter, and nutrient content of the soil (Adugna 2016).Organically grown plant products may contain higher levels of antioxidants, minerals, and dry matter, and have less chemical residues (Crinnion 2010).Studies perfomed in Nigeria revealed that organically grown M. oleifera plants performed better in terms of biomass and the nutritious status compared to the control and nitrogen (N), phosphorous (P), and K (NPK) fertiliser treatments (Adebayo et al. 2017).Use of compost in the production of M. oleifera improved the nutritional content of the plant and also helped prevent soil degradation (Ngakou et al. 2020).Studies conducted in South Korea revealed that a combination of general compost and NPK improved the biochemical content of M. oleifera (Sarwar et al. 2020).Organic soil amendments widely used by farmers are generally from livestock manure.However, previous research has revealed that improperly treated and utilized livestock manure may pose a health risk to the environment due to microbial contamination and addition of some chemicals to animal feed (Kumar et al. 2013).Foodborne pathogenic bacteria such as Salmonella and Escherichia coli were detected in agricultural soils and in vegetables grown with animal manure application (You et al. 2006).In addition, compost obtained from sewage sludge and livestock excreta can contain traces of heavy metals (Liu et al. 2006).
Compost obtained from plant-based material has been used as a fertilizer in organic farming, as it contains little to no heavy metals (Bożym 2017) and fecal coliforms.Plantbased compost also suppressed plant diseases caused by the pathogenic formae speciales Fusarium oxysporum (Yogev et al. 2006).Compost is used for soil amendment programs to reduce heavy metal contamination (Huang et al. 2016).Soil amendments are essential in M. oleifera farms around South Africa (Mashamaite et al. 2021), as approximately 50% of the land is deemed less than suitable for M. oleifera production based on climate and soil physiochemical properties (Tshabalala et al. 2020).This study focused on organic soil amendments to improve the nutrient content of M. oleifera from the community farm in Hammanskraal, Gauteng, South Africa.Soils in this area pose a challenge to M. oleifera growth as they are prone to water logging.
The soil also cracks when dry, exposing M. oleifera tubers to frost during winter.Plant-based compost amendments are expected to improve soil physiochemical properties and nutrient quality of M. oleifera.Limited scientific literature on the use of plant-based compost to increase the nutrient quality of M. oleifera leaves grown in clay soil exists.In addition, no human health risk assessment has been carried out on the potential hazard associated with consuming compost grown M. oleifera products.

Plant Material, Treatments and Sowing
Moringa oleifera seeds and the clay soil used were obtained from the community farm in Hammanskraal, Gauteng, South Africa.Topsoil (0-15 cm) was collected using a spade, air-dried, homogenized, and sieved (0.25 μm mesh) to remove gravel and debris.Seeds were primed by soaking in water for 24 h and subsequently planted into germination trays containing potting soil and watered every two days for a period of four weeks.Four weeks old seedlings exhibiting uniform optimum growth were then transferred into 20 cm diameter, 2 L plastic pots containing clay soil as a control and clay soil amended with four levels (85% soil: 15% compost; 70% soil: 30% compost; 55% soil: 45% compost and 40% soil: 60% compost) of organic plant-based compost as treatments in four replicates each.The percentage of each compost-soil combination was calculated by the volume of the total mixture.Plants were watered with tap water twice a week, after which plant growth was monitored for six months.Treatment percentages followed those used by Vouillamoz and Milke (2001) and Zheljazkov and Warman (2004).Plants were grown in the greenhouse at the School of Animal, Plant and Environmental Sciences (APES), University of the Witwatersrand under ambient temperature.

Sample Preparation
All leaves in each replicate were harvested and washed, and both leaf and soil samples were air-dried under the shade, ground and sieved.Processed samples were stored in polyethylene bottles.Soil pH and electrical conductivity (EC) were determined by a pH and conductivity meter after suspending soil in deionized water at a ratio of 1: 2 soil (20 ± 0.001 g) and deionized water (40 mL), respectively.

Microwave Sample Digestion and Metal Content Analysis
A microwave digester (Anton Paar, Switzerland) was used for digestion.A mass of 0.1 ± 0.001 g sieved soil (0.25 μm mesh) was weighed into a Teflon microwave digestion vessel, after which 9 mL of concentrated HCl, 3 mL concentrated HNO 3, 1 mL concentrated HF and 6 drops of concentrated H 2 O 2 were added.Digestion was then performed at 200 °C for 30 min.To a mass of 0.1 ± 0.001 g dried leaf powder in a Teflon microwave digestion vessel was added 8 mL of concentrated HNO 3 and 2 mL of concentrated H 2 O 2 .Microwave digestion was carried out for 30 min.Each sample was digested in triplicate.
The digested solution for both soil and leaf samples was transferred into a 50 mL volumetric flask, and the solution was mixed with deionized water.Digested samples were then filtered, and dilutions were made before analyses of the metal content using inductively coupled plasma -optical emission spectroscopy (ICP-OES) (Spectro Genesis, Spectro, Germany).A carbon (C), hydrogen (H), N and sulphur (S) analyzer (CHNS) was used to determine the percentages of each element.

BCR Sequential Extraction of Metals
Bioavailability of metals in the soil was determined by slightly changing the modified BCR sequential extraction method of Alan and Kara (2019).The procedure was applied to 1 ± 0.001 g of each soil sample placed in 50 mL centrifuge tubes, and each soil sample was replicated thrice.The procedure is summarized as follows.
In the first step (exchangeable fraction), 40 mL of 0.11 molL − 1 acetic acid was used to extract free and acid-soluble fractions bound to carbonates.The sample was shaken at 30 ± 10 rpm for 16 h at room temperature, 25 ± 1 °C.The extract was separated from the solid by centrifuging at 3000 rpm for 20 min, collected and stored in polyethylene bottles at 4 °C.Washing was performed with 20 mL of deionized water, and the mixture was shaken for 15 min and subsequently centrifuged at 3000 rpm.The supernatant was discarded.The second step focused on the reducible fraction, i.e., extraction of metals bound to Fe and Mn oxides.To the residue from step one, 40 mL of freshly prepared 0.5 molL − 1 hydroxy ammonium chloride (NH 2 OH.HCL) at pH 1.5 (adjusted by adding 25 ml of 2 molL − 1 HNO 3 ) was added.The mixture was shaken for 16 h at room temperature.Separation of the extract and washing were performed as described in step one.In the third step (oxidizable fraction, bound to sulfides and organic matter), 10 mL of 8.8 molL − 1 of concentrated H 2 O 2 of pH 2-3 (pH adjusted by adding HNO 3 ) was added to the step 2 residue.The mixture was digested for an hour at room temperature.Digestion was continued for another hour by heating the vessel at 85 °C in a water bath until the residue was reduced to 2-3 mL.Afterwards, 50 mL of 1 moL − 1 ammonium acetate (C 2 H 7 NO 2; pH 2 adjusted by HNO 3 ) was added, and the mixture was shaken for 16 h at room temperature.Extraction and washing of the residue was carried out as described in step 1. Residue was subjected to aqua regia digestion.Total metal content was analyzed using the (ICP-OES) (Spectro Genesis, Spectro, Germany).

Data Analysis
All measurements were performed in triplicate, and the data was subsequently analyzed using one-way analysis of variance (ANOVA) with the SAS statistical package version 17.0.Significance within the means was compared using Fisher's test.

Human Health Risk Assessment
The Target Hazard Quotient (THQ) estimates the possible carcinogenic risk associated with the consumption of M. oleifera leaf biomass and was determined by the equation: where EF is the exposure frequency (365 days/year); ED is the exposure duration in a lifetime; DIM is the daily metal intake; RfD is the reference oral dose of metals in mg/kg/ day with values of 1, 0.02, 0.001, 0. Al and Fe, were slightly more in clay soil, and the addition of plant-based compost reduced the presence of these elements.High levels of metals (e.g.Fe concentration of 28629.2mg/kg) were also detected in compost by Manungufala et al. (2008).The high increase in P in the soil upon the addition of compost was due to the high P levels in the plant compost, consistent with the findings of Verma et al. (2013) where compost increased the solubility and concentration of P in the soil.Thus, the addition of compost to clay soil generally increases the concentration of metals present in high amounts in compost.Different letters in each row indicate significant difference (P < 0.05).

Total Concentrations of Metals in Dried M. oleifera Leaves
Metal concentrations of dried M. oleifera leaves are shown in Table 2. Concentrations of macro-and micronutrients in M. oleifera leaf powder varied significantly (P < 0.05) with the addition of compost to the soil, with the exception of Co and Cr (Table 2).Variation was also noted within different levels of plant-based compost (Table 2).Similarly, compared with plants of other compositions, leaf biomass of plants grown in clay soil amended with 30% compost had the highest concentrations of metals, approximately 50%.Interestingly, the high concentration in 30% compost was attributed to major nutrients such as Ca, Mg, P and S. Adebayo et al. ( 2017) reported an increase in M. oleifera leaf metal content upon the addition of different forms of compost, which was similar with our current findings.Clay soil amended with 45% compost contained more Zn.Sodium was highest in the 30% and 45% clay soil compost amendments and leaves from clay soil amended with 60% compost contained more Fe.However, the concentration of Al in the leaves was greatest in the control.This could be due to the fact that addition of compost to soil reduces exchangeable Al and its concentration in plants (Ho et al. 2022).These

Soil pH, Electrical Conductivity, and Carbon and Nitrogen Content
The pH of the control clay soil was 7.68 while that of the compost was 8.01 (Table S1).However, the addition of compost to the soil slightly reduced the pH (Table S1).Similarly, the EC of the compost (249.67 µS/cm) was greater than that of the control clay soil (14.62 µS/cm) (Table S1).The total C and N contents in the soil increased with the addition of compost, as expected (Hu et al. 2018).Although the C and N contents increased simultaneously, the C: N decreased with the addition of compost to the soil.A C: N lower than 20 for all the soil amendments indicated very stable organic matter (Ammari et al. 2012) and the ability of the plants to assimilate N.These values are ideal for nutrient uptake, as N can be assimilated only when the C: N value is less than 20 (Khwairakpam and Bhargava 2009).High soil electrical conductivity (EC) has been known to increase absorption and accumulation of metals in plant shoots (Salimi et al. 2012;Pakade et al. 2013).Addition of compost to clay soil increased the conductivity because of increased cation exchange capacity (CEC) (Sung et al. 2011).Duong et al. ( 2012) also discovered that addition of compost to clay soil increased EC and N availability to wheat plants in comparison with unamended clay soil.

Total Metal Concentration in Clay Soil Amended with Plant-Based Compost
Metal concentrations in clay soil amended with compost are presented in Table 1.Plant-based compost had a higher mean concentration of Zn, K, P, S, Na and Ca than clay soil (Table 1).This is expected because compost is made up of plant material, and these are major nutrients found in plants (Hocking 1994).On the other hand, the concentrations of metals needed in trace amounts by plants, such as  (2015).Furthermore, Ca is highly soluble in water (Van Herck et al. 2000) and acid, as indicated by its high concentration in the acid-soluble fraction (Figure S1 (a), facilitating plant uptake and leading to high Ca concentrations in the leaves.Jakubus ( 2016) suggested different soil compositions have an effect on the release and phytoavailability of metals.Additionally, organic amendments influence the content and mobility of metals in the soil and the uptake of nutrients by plants (Herencia et al. 2008).The current study affirms these findings.

Assessment of Possible Adverse Health Effects Posed by Daily Intake of M. oleifera
The daily intake of the metals found in the present study is of paramount importance for preventing cumulative toxicity (Grosshagauer et al. 2021).The DIM results (Table S2) indicate that the Al, Fe, Mn and Zn DIM are below the recommended daily intake.However, the Co and Cr concentrations (Table S2) were above the recommended daily intake limits.High levels of Cr could be attributed to the fact that South Africa geologically has high Cr deposits (Nuapia et al. 2018).The concentration of Cr in M. oleifera leaf powder from farms around South Africa reported by IDC (2019) was also above the permissible limit of Cr concentration in plants.However, further investigations are needed to determine the sources of Co and Cr in the present study, as the addition of compost did not significantly increase their bioavailability in the soil or their accumulation in M. oleifera biomass (Figure S1 (a), Table 2).
The calculation of health risk assessments for all consumed products is of paramount importance because foodborne toxicity is a global crisis (Areo and Njobeh 2021).
variations in uptake of metals by M. oleifera plants grown in different soil amendments reflect the ability of plant-based compost to alter soil physiochemical properties, which in turn alters metal uptake by roots (Celik et al. 2004).The addition of compost to clay soil improves aeration, water holding capacity and cation exchange capacity (CEC) and thus improves the uptake of metals (Omara et al. 2022).Haouvang et al. (2017) reported that high levels of compost in the soil may lead to toxicity due to overaccumulation of metals such as Fe and Mn.

Studying Metal Bioavailability in Clay Soil Amended with Compost
Results for metal speciation in the clay soil and in the plantbased compost treatments are summarized in Figure S1.Increases in the exchangeable forms of Ca, K, Mg, Mn, Na, P and Zn increased with the addition of compost to the soil; however, there was no significant difference (P > 0.05) in the different soil to compost ratios.Addition of compost increased the availability of Al and Fe by decreasing the residual fraction and increasing the oxidizable fraction.No significant difference (P > 0.05) was detected among the clay soils, and no significant differences were detected for Cr or Co.The calcium exchangeable fraction was greater in the 30% compost clay-soil amendment, while the Mg and Mn concentrations were greater in both the 15% and 30% treatments.The potassium exchangeable fraction was greatest in the 15% compost clay-soil treatment group (Figure S1).Sungur et al. (2014) used sequential extraction to reveal that some metals are present in the soil but are not accessible to plants.Similarly, in the present study, the Al, Fe and Zn concentrations were high in the soil but low in M. oleifera leaves (Tables 1 and 2).This could be due to the high soil pH, which increases the ability of Al to bind to plants, decreasing the availability of Al (Zhao and Shen 2018).high concentrations in the M. oleifera biomass studied are major nutrients needed for humans and animals.The concentrations of these nutrients significantly increased with the addition of compost to the soil.However, the addition of plant-based compost did not significantly increase the availability or accumulation of trace metals such as Cr and Co, which are needed at relatively low concentrations for plant growth.Thus, plant-based compost is recommended for increasing essential elements in the soil and M. oleifera leaves and for suppressing trace and/or heavy metals that could be present in the soil.In this study, it was discovered that addition of 30% plant compost to clay soil gave the highest amount of major nutrients of Ca, K, Mg, P and S.This composition is therefore recommended for improved nutrients in M. oleifera leaves at the M. oleifera farms grown in clay soils.In addition, the nutrient concentration of M. oleifera leaves grown under plant-based compost soil amendments is recommended for use as a nutrient supplement for both humans and animals.Metals such as Al, Co, Cr, Fe, Mn and Zn are known to have toxic effects on the human body; therefore, caution must be taken when consuming leaf powder.The noncarcinogenic test based on the THQ values confirmed that M. oleifera in all the soil amendments in the present study was safe for consumption.However, comprehensive studies are needed before plant-based In addition, heavy metal contamination has always been a major factor compromising the quality and safety of herbal plants (Okem et al. 2014).Figure 1 shows the noncarcinogenic THQs for Al, Co, Cr, Fe, Mn and Zn.The THQs for the individuall tested metals for both males and females in all soil types were less than one [Fig. 1 (a), (b)].This indicates that daily intake of each substance may not lead to a health risk.This further confirmed that organic-plant-based compost is a safe way to improve the growth of M. oleifera because it limits the bioavailability of heavy metals in the soil and their accumulation in the leaves (Olaniran et al. 2013).The aluminum THQs decreased with the addition of plant-based compost.Similar results were reported by Ghanim et al. (2023), where the addition of compost to the soil reduced the THQs of heavy metals in pea plants.

Conclusion and Implications of Using Plant-Based Compost for Clay Soil Amendments in Growing M. oleifera Plants
The overall outcome of the study affirms that the addition of plant-based compost to clay soil increases the content and availability of metals in M. oleifera leaf biomass.The essential nutrient elements Ca, K, Mg, P and S found at composts are used to increase the nutrient content of M. oleifera biomass, in order to avoid increasing the concentration of trace elements above the maximum permissible limits.
Funding Open access funding provided by University of the Witwatersrand.

Declarations
Declaration of Conflict of Interest All the authors of the paper declare that there are no conflicts of interest.
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Fig. 1
Fig. 1 (a and b) Target hazard quotients (THQ) for male and female for Moringa oleifera leaf biomass harvested from clay soil amended with different levels of plant-based compost

Table 1
(Ptistišek et al. 2001)g kg − 1 , mean ± SD, n = 3) in clay soils amended with different levels of plant-based organic compost Zinc is also known to be largely bound to silicate in the soil(Ptistišek et al. 2001).Higher concentrations of Cu and Mn in the residual fraction indicate the immobile state of these metals, and the same trend was reported by Sahito et al.

Table 2
Metal concentration (mg kg − 1 , mean ± SD, n = 3) of Moringa oleifera leaves harvested from clay soil amended with different levels of plant-based organic compost Different letters in each row indicate significant difference (P < 0.05)