Use of the Physiologically Based Extraction Test for the Assessment of Bioaccessibility of Toxic Metals in Vegetables Grown on Contaminated Soils

the range of 0.4-114.4 mg/kg for Cu, 0.1-2.4 mg/kg for Cd, and 0.8-137 mg/ kg for Zn, but there was little or no bioaccessibility in the intestinal phase. Cr and Pb were non-detectable in either the gastric or intestinal phase. Conclusions. Although the level of metals (Cd, Cu and Zn) available for absorption in the gastric phase were found to be low in most of the vegetables studied, the experiment revealed that metals are likely released in the human gut after intake of vegetables grown on contaminated soil. Competing Interests. The authors declare no competing financial interests.


Introduction
In recent years, the attention of researchers has focused on the bioaccumulation of potentially toxic metals due to increasing human activities resulting in extensive contamination of urban surface soils worldwide. 1 These metals present significant risks to human health if ingested indirectly through inadvertent ingestion of soil (especially the hand to mouth behavior of children) or transferred through the food chain by consumption of vegetables grown on these soils. 2, 3 Metals in soil can bioaccumulate in plants and get transferred to the food chain, causing adverse health effects in humans. Metals such as cadmium (Cd), chromium (Cr) and lead (Pb) are of most concern because they are toxic to plants and animals, even in small concentrations. Zinc and copper however, are essential trace metals for plants and animals, but can be dangerous at high concentrations. 4 In the assessment of the environmental and human health risks of toxicity from metals, total concentration alone is considered an overestimation of the amount absorbed through oral ingestion. A better estimation of the oral exposure risk to humans depends on measuring the bioaccessible fraction. 5 Bioaccessibility can be estimated by a method where metals are solubilized from a sample under simulated gastrointestinal conditions and thus become available for intestinal absorption in the human body, i.e. by entering the blood stream. 6 Several in vitro methods have been developed for bioaccessibility studies, but the physiologically based extraction test (PBET) is widely known and applied as an in vitro digestion test. [7][8][9][10][11] It is composed of the successive Transfer factor Zinc digestion of gastric and intestinal phases, each one carried out using simulated human conditions such as pH, enzymes and temperature. 12 The PBET uses a two-stage enzymolysis procedure to assess the in vitro potential for metals to be released in the human gut and hence be absorbed into the blood stream and transported through the body to vital organs. 13 Research has been carried out on the total concentration of potentially toxic metals in both soils and vegetables, but little research been done or published on the assessment of the environmental health risks to humans from ingestion of soil or vegetables, and most specifically, the oral bioaccessible fraction in developing countries such as Nigeria and other African countries. Therefore, research of this nature is needed. The aim of the present research is to determine the oral bioaccessibility of potentially toxic metals in edible parts of vegetables grown on contaminated soils using the physiologically based extraction test.

Instruments and Reagents
The analysis of the total and bioaccessible concentrations of soils and vegetable extracts was carried out using a flame atomic absorption spectrophotometer ( The pseudo-total metal concentration was determined by digesting with aqua regia (3 hydrochloric acid: 1 HNO 3 ) on a hot plate. One gram of the soil was digested with 20 ml aqua regia for 2 hours. After cooling, the digests were filtered into 50 ml flasks and stored in plastic bottles prior to analysis.

Planted Vegetables
Seeds of tropical vegetables-waterleaf (talinum triangulare), spinach (basella alba), pumpkin (telfairia occidentalis), okro (abelmolschus esculentus) and celosia (celosia argentea) obtained from the National Horticultural Research Institute, Ibadan, Nigeria were pre-germinated in a nursery and the seedlings were transplanted after two weeks into individual plastic pots containing contaminated soil samples at the greenhouse of the botanical garden of the University of Lagos, Nigeria. Approximately 8 weeks after planting with periodic watering and monitoring, mature vegetables were harvested, rinsed with tap water followed by distilled water to wash off soil particles, then taken to the laboratory to be oven dried at 60 ± 2 o C for 48 hours, pulverized and stored in a refrigerator for further analysis.

Plant Digestion
One g of each portion of vegetable leaves was weighed and digested with 10 ml of concentrated nitric acid.
The digestion was carried out for 2 hours, after which it was allowed to evaporate to near dryness. The residue was then taken up to 25 ml with 1 m HNO 3 and allowed to cool. After cooling, the sample was filtered with Whatman filter paper (11 cm) into a 25 ml volumetric flask and brought to volume.

Physiologically Based Extraction Test
The PBET consists of two sequential extraction processes, gastric and intestinal digestion, each one carried out by employing simulated human conditions (i.e., enzymes, pH and temperature), according to the method proposed by Ruby et al. 17 In the gastric phase, the sample was subjected to a solution containing pepsin, malate, citrate, acetic acid and lactic acid at pH 1.8 (adjusted with 12 m hydrochloric acid), while the intestinal phase was treated with pancreatin and bile salts to pH 7.0 (adjusted with saturated sodium bicarbonate solution). Both phases were subjected to the human body temperature of 37 0 C. 13

Results
The total metal concentrations in the different soil samples are presented in Table 1. High concentrations of metals were found in soil samples B, C, D, E, but not in sample A.

Cadmium
The solubility of cadmium was observed to be highest for celosia, 16 to 109% in the gastric phase, followed by waterleaf (26.2 to 50%) and pumpkin 41.4%, while spinach had only 17% solubility. For the intestinal phase, bioaccessibility was observed for celosia only (12%). The percentage of Cd found in the residual phase was up to 50% in spinach, 45% in celosia, 64.5% in waterleaf and 109% in pumpkin.

Zinc
The bioaccessibility in the gastric phase was 6 to 28% for pumpkin, 10 to 59.8% for waterleaf, 23 to 66% for celosia, and 27% for spinach, while 7.4 to 16.1% was solubilized in the intestinal phase. However, most of the concentrations of zinc in the plants were found not to be available for absorption, but rather remained in the residual fraction, 29 to 116% in pumpkin, 18 to 74% in waterleaf, 12.7 to 68.5% in celosia, 53 to 72% in okro, and 33 to 56% in spinach.

Chromium and Lead
It was observed from the results that Cr and Pb were not bioaccessibile to the plants in either of the two phases.

Conclusion
The results of this study suggest that metals taken up by the vegetables was available for absorption (solubilized) in the gastric extraction phase rather than in the intestinal phase. The level of metals absorbed in the gastric phase were found to be relatively low. This study illustrates that metals can be solubilized in the human gut when taken in through consumption of contaminated vegetable plants, although in low concentrations. Bioaccumulation of these metals could take place over time in the human system through continuous consumption of contaminated vegetables, possibly leading to deleterious health effects in humans.