Bioaccumulation potential of heavy metals in leaves of Amaranthus hybridus L.) and Telfaria occidentalis Hook. f. from selected vegetable farms within Akure metropolis

Bioaccumulation potential of heavy metals in leaves of Amaranthus hybridus L.) and Telfaria occidentalis Hook. f. from selected vegetable farms within Akure metropolis.

^*1Olajire Ayodele Gbaye, ²Olufemi Olasoji Olalowo and ³Foluso Ologundudu

^1,2,3Department of Biology, School of Sciences, Federal University of Technology, Akure, Ondo State, Nigeria. ^* E-mail: gbayejire@yahoo.com, Phone: +2347055496607

Abstract

Anthropogenic activities and indiscriminate waste disposal constitutes a threat to the environment. The bioaccumulation potential of copper (Cu), zinc (Zn), lead (Pb), Manganese (Mn) and Cadmium (Cd) were investigated in two leafy vegetables; Amaranthus hybridus L (Green Amaranth) and Telfaria occidentalis Hook. f. (Fluted Pumpkin) and in the soil of selected vegetable farms within Akure metropolis, Ondo State, Nigeria. This is with the aim of determining the concentration of heavy metals in these leafy vegetables and in their corresponding soil and also to determine their safety levels relative to the tolerable limits recommended for heavy metals by World Health Organization (WHO) and Food Agricultural Organization (FAO). Vegetable and soil samples were collected randomly from selected sampling sites, prepared and digested according to standard protocols recommended by the American Public Health Association. The concentrations of heavy metals present in the vegetable and soil samples were determined using Atomic Absorption Spectrophotometer (AAS). Findings from this study revealed that the concentration of Cu, Zn, Mn and Cd in both Fluted Pumpkin and Green Amaranth were below the permissible limit recommended by WHO/FAO. The concentration of Pb in Amaranthus hybridus L. and Telfaria occidentalis Hook. f. sampled from the markets are 0.390-0.400 mg/kg respectively while their concentration in vegetables sampled from farms are 0.000-0.600 mg/kg and 0.000-0.590 mg/kg respectively. Hence, the concentration of Pb. In both vegetable species sampled from selected markets and vegetable farms were above the threshold of 0.300 mg/kg recommended by WHO/FAO. However, the concentrations of heavy metals in the soil of vegetable farms were below the standard limits recommended by WHO/FAO and National Environmental Standard and Regulation Enforcement Agency (NESREA).

Keywords: Anthropogenic, bioaccumulation, permissible limits, safety levels, threshold.

1.0 Introduction

There had being an alarming issue of pollution by heavy metals because of their toxicity for plant, animal, humans and their lack of biodegradability (Li et al., 2006, Jang et al.,2006, Zhuang et al., 2009). Soil is the primary reservoir of heavy metals in the atmosphere, hydrosphere and biota, and thus plays a fundamental role in the overall metal cycle in nature (Cao et al., 2010). Heavy metals in soil pose potential threats to the environment and can damage human health through various absorption pathways such as direct ingestion, dermal contact, diet through the soil–food chain, inhalation, and oral intake (Lu et al., 2011; Komarek et al., 2008; Park et al., 2004; Al-Saleh et al., 2004). Vegetables are important edible crops and are an essential part of the human diet. They are rich in nutrients required for human health, and are an important source of carbohydrates, vitamins, minerals, and fibers (Yang et al., 2009; Hu et al., 2013). The uptake and accumulation of heavy metals in vegetables is influenced by some factors such as atmospheric deposition, concentration of heavy metals in the soil, nature of soil on which the vegetables are grown, degree of maturity of the plant at the time of harvest, physiochemical properties of the soil and the species of the vegetable (Ismail et al., 2005; Sharma et al., 2006). Vegetables play important roles in our daily diet as economic crops. However, various human activities such as mining, industrial processing like smelting, pesticides, automobile exhausts and fertilization, especially the huge annual applications of organic livestock manure, are causing elevated heavy metal concentrations in the environment (Zhuang et al., 2009; Cao et al.,

2010; Zheng et al., 2010; Shi et al., 2011). Heavy metal toxicity had received attention globally due to its neurotoxicity, carcinogenicity and several other imparts arising from their consumption even at low concentration (Satharawa et al., 2004). Chronic intakes of heavy metals have damaging effects on human beings and other animals (Zheng et al., 2007; Lai et al., 2010; John and Andrew, 2011). For example, Cr, Cu and Zn can cause non-carcinogenic hazardous such as neurologic involvement, headache and liver disease, when they exceed their safe threshold values (US EPA, 2000). There is also evidence that chronic exposure to low doses of cancer-causing heavy metals may cause many types of cancer. For example, Park et al. (2004) found increased lifetime risk of lung cancer death resulted from occupational exposure to dusts and mists containing hexavalent chromium. Dietary cadmium intake due to the consumption of environmentally contaminated rice and other foods was associated with an increased risk of postmenopausal breast cancer (Hiroaki et al., 2013). Acute and chronic arsenic exposure could also cause numerous human health problems. These included dermal, respiratory, cardiovascular, gastrointestinal, hematological, hepatic, renal, neurological, developmental, reproductive, immunological, genotoxic, mutagenetic, and carcinogenic effects (such as liver cancer) (Kapaj et al., 2006; Lin et al., 2013). Vegetable species differ widely in their ability to take up and accumulate heavy metals, even among cultivars and varieties within the same species (Zhu et al., 2007; Samuel et al 2012). Alexander et al. (2006) reported that Pb significantly accumulated in lettuce and onion, while Cd accumulated to the greatest extents in spinach and lettuce. Yang et al. (2009) found that Chinese leek, and carrot had higher Cd concentrations in their edible parts than radish, cucumber, and tomato. Säumel et al. (2012) reported that Zn concentrations in green beans, tomato, potato, kahlrabi, and carrots were significantly lower than the concentrations in leafy vegetables. Cd accumulation in vegetable species decreased in the order of leafy vegetables > solanaceous vegetables > root vegetables > allimus vegetables > melon vegetables > legumes vegetables (Yang et al., 2010).There are elevated levels of heavy metal in soils in many areas of the world, especially in developing countries and regions. In some mining and smelting areas in China, although the agriculture soils are contaminated with heavy metals, the farmers cannot afford to leave farmland fallow for remediation because the demand and pressure to produce foodstuffs and vegetables are so high. The selection and breeding of crop and vegetable species or cultivars that have low heavy metal accumulation, without having to leave the farmland fallow, seems to be a suitable method to reduce the adverse health effects of heavy metals (Xin et al., 2010; Yang et al., 2010; Ding et al., 2013). There have been some studies that have focused on Cd contaminated soil and the selection of vegetable species or cultivars with low heavy metal accumulation (Dunbaret et al., 2003; Yang et al., 2009;2010) However, very few studies have investigated multiple heavy metals (Pb, Cd,Cu, Zn, and As) in soils. Fluted pumpkin leaves are used as food supplement, it contains adequate vitamins A and C which help in enhancing good sight and prevention of scurvy (Alegbejo, 2012). Green Amaranth contains appreciable amount of proteins, fat, fibre, carbohydrate, vitamins, amino acids and low level of toxicants (Akubugwo, 2007).

2.0 Materials and Method

 2.01. Study area: The study area is Akure, Ondo State, Nigeria (7⁰16¹ 4’N latitude, 5⁰14¹  41^’E Longitude. It experiences warm humid tropical climate with average rainfall of about 1500mm/annum. Annual mean temperature range between 21.4 and 31.1^oC. Mean annual relative humidity is about 77.1% (Nigerian Metrological Agency Report, 1980-2007).

2.02. Sample collection: 2 kg each of vegetable samples (Amaranthus hybrdus L. and Telfaria occidentalis Hook. f. were randomly selected from three different markets within Akure metropolis (Isinkan, Odopetu and NEPA) and Vegetable farms in Araromi, Owode and Army Barracks respectively for Green Amaranth while Fluted pumpkin was collected from Ala, Army barracks 2 and 3. The collected samples were separately packed in polythene bags, labelled and transported to the Research Laboratory, Department of Biology, Federal University of Technology, Akure, Ondo State, Nigeria. 5 kg of soil samples were randomly collected with the aid of a soil auger at 20 cm depth from five different points in each of the Vegetable farms. The soil samples were separately packed in polythene bags, labelled and transported to the Research Laboratory, Department of Biology, Federal University of Technology, Akure, Ondo State, Nigeria, for further analysis.

2.03. Sample preparation: vegetable samples were separately chopped into smaller pieces and washed with distilled water to get rid of sand particles. The samples were oven dried at 80oC for 8 hours. The dried vegetable samples were later grounded into fine powder with the aid of a Binatone blender (BLG-555) and stored in a polythene bag for further treatment. Soil samples were air dried, passed through a 2-mm sieve, and analyzed for the physico-chemical parameters according to AOAC (1998).

2.04. Digestion of samples: Vegetable samples were digested following the procedure established by American Public Health Association (APHA, 1992). 2 g of dried vegetable sample was transferred into a digestion tube and 40 ml of concentrated nitric acid (HNO₃) and perchloric acid (HClO₄) with 3:1 were added to the sample. The sample was later digested at 120^oC for 2 hours until the solution became transparent. The sample was then cooled and diluted with 0.1 M HNO₃. The solution was filtered into a 25 ml volumetric flask through Whatmann No 1 filter paper. The digested samples were stored in screwed sample bottles. I g of air-dried soil samples were weighed and digested with HNO₃ and HCl (1:3). The mixture was heated on a sand bath and the digested samples were treated with 20 ml of 10%  HNO_3,  filtered and made up to standard volume in a 25 ml volumetric flask.

2.05. Determination of heavy metal concentration: Heavy metal contents (Pb, Mn, Zn, Cu, Cd) in vegetable and soil samples were determined using Atomic Absorption Sectrophotometer (AAS Buck Scientific 210 VGP) according to Doherty et al., 2012.

2.06. Measurement of physicochemical properties of the soil

2.06.1. Soil pH: The method described was used to determine the pH of the soil. 10 g of 2 mm mesh sieved air-dried soil sample was transferred into a 100 ml beaker and 20 ml of distilled water was added and allowed to stand for 30 mins. The mixture was stirred occasionally with a glass rod. The pH meter was calibrated with pH 7.0 and pH 4.0 buffer solutions. The electrode meter was then inserted into the partly settled soil suspension solution and the pH of the soil sample was taken.

2.06.2. Soil moisture content: The moisture content was determined using oven drying method. Clean and dry glass dishes were weighed with Mettler balance and their respective weights were recorded (W₁). About 2 g of each of the soil sample was weighed separately into respective glass dishes (W₂). The soil was evenly spread and transferred into a desiccator. The samples were transferred into an oven at 105^oC for 4 hours until a constant weight (W₃) was achieved (AOAC, 1980). The percentage moisture content was then determined using the formula below;

% moisture = Loss of weight                                                                                                                        weight of sample

2.06.3. Organic matter content: Organic matter content was determined following established protocols. The soil sample was sieved with a 2 mm mesh. 1 g of the soil sample was weighed and transferred into 250 ml conical flask. 10 ml of 0.167 M of Potassium Heptaoxodichromate VI (K₂Cr₂O7) was added to the soil and swirled to mix. 20 ml of concentrated Tetraoxosulphate (VI) acid (H₂SO₄) was rapidly added, shaken and allowed to stand for 30 mins. The mixture was diluted with 100 ml of distilled water and later titrated with 0.5M Iron II Sulphate (FeSO4).

Quality Assurance: Appropriate safety measures and quality assurance procedures were followed to ensure the reliability of the test results.

2.7: Statistical Analysis: All data were statistically analyzed using the statistical package for Social Sciences (SPSS Version 20.0). Statistical means were separated using Tukey’s test (P≤0.05).

3.0 Results and Discussion

3.01. Concentration of heavy metals in vegetables sampled from selected markets within Akure metropolis: There were variations in the concentration of heavy metals in the Green Amaranth (Amaranthus hybridus L.) sampled from the four markets (Oba, Isinkan, Odopetu and NEPA). Cu, Zn and Mn were detected in all the Green Amaranth sampled from the markets (Table 1). However lead (Pb) was not detected in samples from Isinkan market (0.000mg/kg). The concentrations of Cu, Zn, Mn and Pb in Green Amaranth from the markets were all below the permissible limit recommended by WHO/FAO. Food safety is known to be one of the major public concerns in almost every society (D’mello, 2003). The concentration of Pb observed in Fluted pumpkin samples sourced from markets and vegetable farms varied between 0.300-0.590 mg/kg and 0.400-0.430 mg/kg respectively (Table 2). The result of this study is below the concentration of Pb reported by Atayese et al., 2009 in Green Amaranth (68-151mg/kg). This was attributed to the fact that most of the vegetable farms were located at 5, 10 15 and 20 metres away from three major highways in Lagos state. Pb pollution has been shown to be commensurate with population and vehicular density (Afolami et al., 2010).          The concentration of Cu in Green Amaranth and Fluted Pumpkin sampled from the various markets varied between 0.100-0.576 mg.kg and 0.140-0.626 mg.kg respectively. Similarly, its concentration in selected vegetable farms varied between 0.19-0.36 mg.kg and 0.28-0.61 mg/kg respectively (Table 3). However, these concentrations are higher than the findings of Divrikil et al., 2006 and Ozcan, 2004, who reported Cu concentrations of 0.002 mg/kg and 0.0081 mg/kg respectively for Indian Basil. According to Doherty et al., 2012, the difference in concentrations of Cu in different sampling sites observed in their study was attributed to the different cultivation methods employed during the cultivation of vegetables. The concentration of Cu in this study was below the safe limit of 73 mg/kg set by WHO/FAO. The concentration of Cd in Fluted Pumpkin sampled from vegetable farms and markets ranged between 0.003-0.010 mg/kg and 0.000-0.0100 mg/kg respectively (Tables 3 and 4). Various sources of environmental contamination such as pesticides and fertilizer have been reported as sources of Cd in vegetables which include 0.090 mg/kg for Fluted Pumpkin by Sobukola et al. (2010), 0.049 mg/kg for Lettuce by Muhammad et al. (2008). The concentration of Cd in both vegetables were below the safe limit (0.10 mg/kg) recommended by WHO/FAO. Vegetable species differ widely in their ability to take up and accumulate heavy metals, even among cultivars and varieties within the same species (Zhu et al., 2007; Saumel et al., 2012). It has been reported that Cd uptake and accumulation in leafy vegetables are greater than in non-leafy vegetables (Yang et al., 2009; 2010). In this study, significant differences were observed in heavy metal concentration of selected vegetables samples from markets and farms. However, Pb and Cd concentrations were significantly higher than Cu and Zn in this study. These was probably because Cu and Zn were the essential elements for vegetable growth and were readily accumulated in roots and transported to aerial parts (Zhou et al., 2015); while Pb and Cd were toxic elements not required for vegetable growth, hence their transport and uptake to aerial parts of plant was limited (Liu et al., 2007; Yang et al., 2009). Generally, Cu and Zn which are important nutrients for humans are considered a much lower health risk to humans than Pb and Cd and As (Alexander et al., 2006). Poor health can be caused by a lack of these required elements, but excessive ingestion can also have adverse effects on human health (Hu et al., 2013; Rahman et al., 2014). However, continuous monitoring is required because of the harmful effect of Cd. The highest concentration of Zn in Green Amaranth sourced from markets and vegetable farms were 4.690 mg/kg and 1.680 mg/kg respectively while the highest concentration of Fluted Pumpkin sourced from the markets and farms was 2.140mg/kg and 2.900 mg/kg respectively. However, the concentration of this heavy metal is below the safe limit of 100 mg.kg set by WHO/FAO. Similarly, Subukola et al. (2010) reported a lower Zn concentration in Bitter leaf (0.011 mg/kg), Water leaf (0.07 mg/kg) and Cabbage (0.05 mg/kg). Regular consumption of these vegetables may assist in attaining the recommended dietary allowance for Zn (15 mg/day for men and 12 mg/day for women) and also prevent the adverse effect of Zn deficiency which could result in retarded growth and delayed sexual maturation (Barminas et al., 1998). The concentration of Mn was higher in the two vegetable species compared to other heavy metals. This may be due to the natural availability of Mn in leaves of vegetables. The highest concentration of Mn reported for both Green Amaranth and Fluted Pumpkin samples from the markets are 3.310 mg/kg and 5.296 mg/kg while its concentration from vegetable farms are 2.05 mg/kg and 6.06 mg/kg. This findings is not in agreement with the report of Mubofu et al., 2012 in African Spinach and Pumpkin leaves sourced from Kariako Market, Dar es Salaam, Tanzania. The concentration of heavy metals (mg/kg) in the soil of Green Amaranth farm did not vary significantly (p<0.05). Mn was observed to have the highest concentration across the various farm with 0.946mg/kg (Owode), 2.295 mg/kg (Araromi) and 1.936 mg/kg (Army Barrack 1). Cd was reported to have the least concentration across the farms (Table 5). The concentration of heavy metals in all the soil samples were below the standard limit for soil recommended by National Environmental Standards and Regulation Enforcement Agency (NESREA, 2007) and WHO/FAO, 2001. This is in agreement with the findings of Al jassir et al., 2005; who reported that leafy vegetables accumulate higher metal contents than others. Similar trends were observed in the soil of Fluted Pumpkin farms with concentration range of 0.03-0.05 mg/kg (Cu), 0.110-0.240 mg/kg (Zn), 0.310-3.773 mg/kg (Mn), 0.050-0.150 mg/kg (Pb) and 0.00-0.018 mg/kg (Cd). However, it was observed that the concentration of Mn in the soil of Army Barack 3 farm was slightly lower than in Fluted Pumpkin leaves (2.236 mg/kg) (Table 6). The soil is a major reservoir of heavy metals from which plants take up these metals. The concentration of heavy metals in the soil observed in this study were less than the concentration of vegetables cultivated. Also, leafy vegetables grow faster with high transpiration rate thus metal uptake is enhanced resulting in the translocation from the root to other vegetable tissues (Luo et al., 2011). Plant species vary in their capacity to remove and accumulate heavy metals (Singh et al., 2010). Uptake of metals by plants is affected by several factors including the soil type, pH of soil, organic matter content, redox potential, cation exchange capacity, surface area and texture of soil particles, presence and concentration of foreign ions. With reference to plant species, metal tolerance may result from two basic strategies; metal exclusion and metal accumulation (Haiyan et al., 2008). In this study, soil physiochemical properties of the tested vegetables varied between the various studies locations. Organic matter content (4.52%) and pH of soil (10.36) respectively were significantly higher in Amaranthus hybridus L. (Table 7) in Owode, Araromi and Army Baracks 1 than in the soil of Telfaria occidentalis HOOK.  f. farms within Akure metropolis (Table 8). This is probably due to industrial activities in the area than in Army Baracks 2 and 3 which is more residential. This could also be attributed to the prevalence of metal scrap deposition by the artisans located in the Mechanic village which could probably lead to increase in the organic matter content of the soil. Obi-Iyeke et al., 2016 an increase in cd, Cu and Ni in different edible vegetables along with its soils on which they were grown in Amukpe and Kokori areas of Delta state stemming from oil pollution/spillage resulting in environmental degradation that have a significant effect on their farming activities. Particle size composition of the soil of Telfaria occidentalis HOOK. f. from selected farms were higher than in Amaranthus hybridus L.

Conclusion: concentrations of heavy metals in the soil of vegetable farms were below the standard limits recommended by WHO/FAO and National Environmental Standard and Regulation Enforcement Agency (NESREA). However, the concentration of Pb. in both vegetable species sampled from selected markets and vegetable farms were above the threshold of 0.300 mg/kg recommended by WHO/FAO. Hence, there should be continuous monitoring of heavy metals in vegetables consumed by the populace in order to safeguard their health. Awareness about the health implications of consuming such vegetables should be carried out by appropriate authorities since such samples were also considered as bio indicators of environmental pollution.