Inorganic Contaminants Evaluation in Tuberous and Leafy Vegetables Consumed in Paraíba Valley, Brazil

The aim of the present study is to investigate the aluminum, cadmium and lead concentrations in vegetables such as lettuce, potatoes, carrots, beets and cassava often consumed in human diet. Calcination and acid digestion methods were used to prepare the samples. The Spike test has shown that the methods were viable for almost all samples. The recovered analyte percentage ranged from 88.0% to 108.7% in the acid digestion, and from 55.1% to 109.6% in the calcination method. Comparative analyses conducted in lettuce samples have shown lead concentrations 26.4 ± 2.6 mg kg in electrothermal atomic absorption spectrometer (ETAAS) and 33.3 ± 0.3 mg kg -1 in inductively coupled plasma optical emission spectrometer (ICP-OES). The herein presented results allow saying that the concentrations of the elements analyzed in each sample were above the limit set by the Brazilian Ministry of Health and within the tolerance limit set by the Codex Alimentarius. Keywords— Al, AAS, Cd, Pb, food.


I. INTRODUCTION
Metal elements may affect the environment, as well as humans, in different ways, depending on their availability and essentiality; it happens due to their physical and chemical features. Some elements such as B, Ca, Fe, Mg, Mo, among others, are essential at concentrations between micro-and milligrams per day [1;2;3]. On the other hand, elements such as Al, Cd and Pb do not present known biological function and are well-known for their toxicity when they are inhaled or ingested in excess [4][5][6][7][8][9]. Aluminum is seen as non-essential. In addition, there is evidence that the Al 3+ ion may be linked to health issues such as Alzheimer's disease, and that it may exacerbate intoxications in people with impaired renal function [7;10]. The Al 3+ may be incorporated to the tissue of vegetables cultivated in soils showing high bauxite content; thus, it is a contamination liability and, consequently, a Public Health concern [7; 8;9]. It is known that cadmium (Cd) mobility and toxicity mainly depend on its oxidation state. Thus, Cd may become bioavailable and harm the environment, as well as humans [3;11]. Therefore, this element has been investigated to help better understanding its toxicity mechanisms and setting threshold concentrations that cannot cause any ty pe of harm in case of exposure [12;13]. The particulate lead, which is transported in the form of oxides, may be aspirated due to its wide distribution in the atmosphere [6;11;14]. Whereas the gastrointestinal Al absorption is influenced by several factors, mainly age and diet [7;9]. The lead toxicity results from its capacity to interact with the essential metals of the main metabolic pathways, mainly with Zn, Ca and Fe [2;9]. Paraíba Valley is a São Paulo State region located on the triple border between Minas Gerais, São Paulo and Rio de Janeiro states. This region has been the target of several technical-scientific investigations focused on assessing and warning about the impact of the regional development on the quality of life of humans and on the environment [4;15;16]. Okada et al. (1997) Salazar et al. (2006) have investigated the concentration of oligoelements (Cu, Fe and Zn) in samples comprising vegetables consumed in Lorena County (SP). However, the way the samples were prepared did not allow validating the analytical procedures, thus compromising data reliability. Nonetheless, they could see that the investigated samples showed indications that the concentrations of some elements were above the tolerance limit set by the legislation [17]. Salazar et al. (2011) have found passive Ni contamination in soil samples and in vegetables consumed in Lorena and Taubaté counties (SP), after they properly proposed and validated simplified analytical calcination and acid digestion procedures. According to the authors, the contamination results from the inappropriate use of fertilizers presenting trace element concentration as an attempt to remediate the presence of this micronutrient [16]. In light of the foregoing, the investigation and monitoring of different elements and contaminants are demanding and increase researchers' interest in better understanding their toxicity mechanisms, as well as their interest in environmental and sanitary sensing. Therefore, the aim of the current study was to investigate the Al, Cd and Pb concentrations in vegetables often consumed in Paraíba Valley region, mainly in Taubaté and Lorena counties (SP).

Sampling, reagents and solutions
The sampling consisted of lettuce (Lactuca sativa), beet (Beta vulgaris), potato (Solanum tuberosum), carrot (Daucus carota) and cassava (Manihot utilissima) samples purchased in street markets and supermarkets in Taubaté and Lorena counties; the tuberous samples comprised roots and skin, whereas the leafy samples comprised leaves and stems. All samples were fresh and showed no signs of putrefaction [16;18]. The solutions were prepared by employing analytical degree reagents, ultrapure water obtained from using Milli-Q (Millipore Corp, de Billerica, MA, EUA) water system at 18.2 MΩ cm resistivity, nitric and chloridric acid distilled in quartz sub-boiling (Milestone, Sorisole, Italy). To prevent contamination, the vials, glassware and polypropylene materials were washed and soaked in 10% v v -1 HNO3 and fully washed with deionized water. All the chemical reagents were of the P.A. degree. The mineral acids used were of the Dinâmica brand. The metallic patterns used, with 1-mg mL -1 concentration, were of the SpecSol brand with NIST traceability, Reagent organic solvents and other chemical reagents of the Vetec brand [18].

Sample preparation
First, each vegetable sample was washed in distilled water to remove soil and other relevant debris. Subsequently, they were brushed with polypropylene bristles and washed in deionized water [16]. The lettuce shoot was carefully cut along with the stalks of each leaf, except for the main leafstalk, which was discarded along with the roots. The potato, beet, carrot and cassava root samples were grated in a polypropylene grater and placed in porcelain capsules. Then, the capsules holding the samples were taken to the oven at 120 ± 5°C, for 24 h, for dehydrated mass obtainment [18]. After the dehydration process was complete, the samples were placed in polypropylene beakers covered with PVC film and stored in a desiccator. Calculations were made for each dried sample analyzed in the current study in order to determine how much of these samples represented, in weight, the corresponding fresh samples (eq.1): Wherein Mf is the mass of the fresh sample (g), Ms is the weighted mass of the dry sample (g) and % Moist is the mean moisture content of the sample. The samples were prepared by using adaptations of the procedures developed by Wieteska et al. (1996) for plant sample preparation via acid digestion and calcination; then, they were subjected to further determination through ETAAS or FAAS [19].

Calcination method
Firstly, the calcination of dehydrated samples was carried out by weighing approximately 0.5 g of each vegetable in porcelain crucibles and, then, covering them 19. The crucibles were transferred to a muffle, which was heated at 500°C for 2 h; the heating was done at 12°C min -1 , with 20-minutes threshold each, at 100, 200, 300 and 400°C. The calcination residues were digested in 2.0 mL of deionized H2O, 0.5 mL of HCl and 1.0 mL of concentrated HNO3. The crucibles were covered with watch glass and heated on a plate, for 30 min, in sand bath, in order to assure better heating and reflux control to prevent analyte loss [16]. After the digestion and cooling were complete, the entire digestion system was properly rinsed with deionized water, the crucible solutions were quantitatively transferred to 50.0 mL flasks and supplemented with deionized H2O.

Acid Digestion method
A 0.5 g of each sample was weighed in 50 mL glass beakers. Next, 5.0 mL of an acidic mixture composed of HNO3 and HClO4 (4:1 v/v) and of 2.0 mL of deionized H2O were pipetted into each beaker. Then, each beaker was covered with watch glass and transferred to sand bath in order to allow mild digestion, as recommended when one works with this type of acid mixture. The digestion time ranged from 1 to 2 hours; the system solution was kept in continuous reflux until the digestion produ ct became as clear as possible [19]. After the digestion and cooling processes were complete, the crucible solutions were quantitatively transferred to 50.0 mL flasks and supplemented with deionized H2O. The Al, Cd and Pb determination values and the Mf of the vegetable samples were used to calculate the metal mass per fresh sample mass, through Equation 2: (2) Wherein the parameter "m" is the metal concentration per fresh sample mass (mg Kg -1 or g Kg -1 ); "C" is the metal concentration in the solution (mg L -1 or g L -1 ); "Mf" is the fresh sample mass (g); and "f" is the unit conversion factor. Addition and analyte recovery tests were used to assess result accuracy by adding 2.0 μg L -1 of Cd standard, 10 μg L -1 of Pb standard and 30mg L -1 of Al standard to 0.5 g of dehydrated samples. Interlaboratory analyses of lettuce samples digested through calcination and acid digestion were referred to inductively coupled plasma optical emission spectrometry (ICP-OES) in order to give greater reliability to the spike and recovery tests performed in the current study [16;18;20;21].

Instrumentation
All atomic absorption measurements were performed in the Perkin Elmer; Model: AAnalyst 800, using a deuterium lamp (D2) for background correction. The aluminum was determined through FAAS (flame atomic absorption spectrometry). The electrothermal atomic absorption spectrometry (ETAAS) system was used to determine Cd and Pb [21]. The herein used experimental conditions are described in Tables 1 and 2.

Validating the sample preparation procedures
The methods adopted in the current study were feasible when the recovered analyte rates ranged from 95.9% to 108.8% in the acid digestion, and from 102.7% to 122.9% in the calcination, for Al. When applying the t-test and the F-test (p <0.05) it was verified there are statistically significant differences in results obtained for Al in carrot and cassava or Cd in potato and beet, Pb in beet and carrot. However, it is possible to verify that the repeatability oscillated in methodological terms and by the analyzed element. Tables 4 and 5 show the recovery results of the calcination and acid diges tion procedures, respectively. The low lead recovery in the calcination method may be attributed to a possible Pb entrainment during the heating ramp, whereas the low cadmium recovery in the acid digestion method may due to a possible reaction of the HClO4-derived chlorine, which generated volatile chlorides [14]. The interlaboratory analyses conducted in the lettuce sample showed Al concentration 19.3 ± 0.3 mg kg -1 when it was determined through FAAS, and 18.2 ± 0.2 mg kg -1 when it was determined through ICP-OES. The addition and recovery tests, as well as the interlaboratory analysis, showed similar results, fact that made it possible using the sample preparation procedures to determine Al. The interlaboratory analyses conducted in the lettuce digests showed Pb concentration 26.4 ± 0.3 g kg -1 when it was determined through ETAAS, and 18.2 ± 0.2 g kg -1 , when it was determined through ICP-OES. The results of the interlaboratory analysis made it possible using the acid digestion methodology and reinforced the results found in the analyte addition and recovery test used to determine Pb. With respect to Cd, it was not possible comparing the techniques, since the analyte was below their detection limit (LOD <0.001 mg L -1 ). The fact that Cd was not detected through ICP-OES may be explained according to the techniques. The ETAAS techniques show higher sensitivity and, consequently, better detection and quantification limits than the ICP-OES ones [8;12].  This scenario and the concern about controlling the contamination and setting safe exposure levels can be seen in other countries, as shown in Table 7.

IV. CONCLUSION
All vegetable samples investigated in the present study showed Al, Cd and Pb levels above those allowed by the legislation. The most worrisome results refer to the Cd levels in the lettuce and cassava plants from Lorena County, whose contents were more than five and eight times higher than those allowed by the legislation, respectively. All samples showed Pb concentrations higher than those specified by the Ministry of Health. However, the Cd and Pb levels were within the tolerable daily intake limit set by the Codex Alimentarius. Yet, these results may work as an indication of environmental contamination in these counties, since the Al levels in the vegetables were above the values allowed by the Brazilian Ministry of Health and by international bodies. The values found in the current study represent the total concentration of the herein investigated elements. Chemical speciation studies should be conducted in order to assess the impact caused by the daily intake of these elements, as well as to determine their bioactivity and interaction mechanisms in the human body.