Heavy Metals in Soil and Salad in the Proximity of Historical Ferroalloy Emission ()
1. Introduction
Metal contamination of the environment raises concern for the possible impact on human health, and the occurrence of heavy metals in soils, of both natural and anthropogenic origin, is well-recognized as a potentially important source of human exposure [1,2]. Once contaminated, soils typically remain contaminated for protracted periods of time because of sorption of metals onto soil particles and limited mobility. Data have shown that increased soil levels of metals like chromium (Cr), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni) [3], and arsenic (As) [4] are associated with neurodevelopmental effects in children, presumably due to increased metal exposure from hand-to-mouth behavior and mouthing of hands and objects contaminated with soil particles.
Where metals in soil are increased by anthropogenic disposal and emission, the measure of metal concentrations provide useful information on potential human cumulative exposure. Estimates of cumulative exposure are intended to integrate a variety of different processes affecting exposure over extended time periods. Lifetime cumulative exposure is particularly relevant for evaluating health risks from heavy metals, since it may help predict cumulative neurotoxic effects. Prolonged metal exposure spanning neurodevelopmental periods may also increase the risk of neurodegenerative conditions in old age [3]. Metals are naturally present in soil in relation to the soil parent material, and may be modified by both natural (e.g., flooding, volcanic eruptions, forest fires) and anthropogenic processes. Anthropogenic processes recognized as potential sources of soil contamination with heavy metals include: 1) agricultural activities with the use of metal-containing fertilizers, pesticides, sewage sludge, and irrigation water; 2) emissions from energy and fuel production activities; 3) mining and smelting operations, such as tailing, smelting, refining and transportation; 4) vehicle traffic and combustion of petroleum fuels containing metal additives; 5) emissions from waste incineration; and 6) metal recycling operations like scrap melting [5].
The accumulation of heavy metals in agricultural soils, including home vegetable gardens may be of particular concern since consumption of vegetables grown in metal contaminated soils may pose health risks for the population residing in these areas [6,7]. While essential trace metals such as Cu, Cr, Ni, Mn, fluorine (F), molybdenum (Mo), selenium (Se) and zinc (Zn) are necessary for plant growth and/or human nutrition at low levels, they may also be toxic to both animals and humans at high exposures. Other trace elements, for example As, Pb, cadmium (Cd), and mercury (Hg), may also inadvertently enter the food chain and pose health risks to humans and animals [8].
The uptake of metals from soil into plants is affected by soil chemistry, metal speciation (i.e., inorganic and organic complexation), and molecular transport and storage processes in plants [9]. These processes can be summarized in terms of metal bioavailability, which reflects the fraction of a metal in soil that is available for uptake into a plant. Some plants, such as Thlaspi rotundifolium, Brassica juncea, Festuca arundinacea, Helianthus annuus, and Medicago sativa can hyper-accumulate metal ions because of specialized mechanisms of absorption and transport of internal ions. These plants can tolerate high concentrations of toxic metals in soil, and may also have potential for phyto-remediation in contaminated soils [9,10]. However, edible plants grown in contaminated soils may also accumulate elevated levels of metals that may, when consumed, increase exposures to humans. For example, crops like lettuce, spinach, carrot, radish, zucchini have been shown to accumulate increased levels of potentially toxic metals such as Mn, Pb, Fe, Zn, Cu, etc. when grown in soils contaminated from sewage sludge [11,12], mine wastes [13], and application of livestock and poultry manures [14].
The ferroalloy industry produces various metal alloys used in iron smelters, resulting in increased atmospheric release and deposition of potentially toxic metals like Mn, Pb, Fe, Zn, Cu, etc. Few data are available on soil accumulation of these metals from ferroalloy and smelting activity, the human absorption through locally cultivated vegetables, and the potential impacts on human health. Manganese raises particular concern because of neurotoxic effects resembling Parkinson’s Disease that have been observed in exposed workers to airborne concentrations above 1 mg/m3 [15]. An epidemiological study in the province of Brescia, Italy, showed an increased prevalence of parkinsonian disturbances in the vicinities of ferro-manganese plants in the area of Valcamonica [16]. The Standardized Morbidity Ratios were positively associated with the Mn levels in deposited dust, originating from atmospheric deposition from ferroalloy plant emissions. Levels of Pb, Fe, and Zn were also elevated in deposited dust in the same area [17].
Here, we investigated metal concentrations in soil and locally cultivated edible plants in the Province of Brescia, Italy, in locales with prolonged histories of ferroalloy plant activity. This assessment was part of an extended epidemiological study on health outcomes as a function of metal exposure in three different age groups of residents in the area of Valcamonica: adolescents, elderly and pregnant mothers. The region of Garda Lake was identified as local reference area because of the absence of major industrial emissions, and based on lower levels of metals in settled dust [17]. The specific aims of this study were to determine: 1) if the levels of Mn and other metals in the soil and vegetables from home gardens in Valcamonica differed from those located in the reference area; 2) if the levels of metals in soil and vegetables from the home gardens in Valcamonica were inversely related to the distance from the point emitting sources; and 3) if there was a correlation between the concentration of Mn and other metals in soil and vegetables from the same home gardens. This study is part of a large on-going investigation in which we have conducted detailed exposure assessment of metal concentrations in airborne particles, deposited dust, soil, diet and several biomarkers of resident children, in addition to a comprehensive assessment [18] of neurobehavioral motor and cognitive functions in children as potential health impacts of metal exposure.
2. Materials and Methods
Concentrations of Mn, Fe, Zn, Cu were measured in cultivated edible plants and in the adjacent soils from local vegetable gardens of subjects recruited into the larger epidemiological study noted above. Subjects (n = 82) were selected for the present study from the larger cohort (n = 322 subjects) based on response to a dietary survey indicating that their families cultivated and consumed home grown vegetables. Subjects were provided verbal and written description of the study and requirements, and in turn provided written consent to participate as approved by the Ethical Committee of the Public Health
Figure 1. Study location: the target area of Valcamonica, and the reference area of Garda Lake in the province of Brescia, Italy, with the location of the previously and currently operating ferroalloy plants.
Agency of Brescia, Italy. Recruitment and enrolment procedures of participants are described in a separate publication [19].
3. Study Area
Valcamonica is a valley of the Italian pre-Alps, located in the province of Brescia that has been the operational site of three 85 ferroalloy plants for about a century until 2001. Ferroalloy plants were located from South to North in the villages of Darfo (operational period 1930-1995), Breno (operational period 1902-2001), and Sellero (operational period 1950-1985). The reference area of Garda Lake has no history of 90 metallurgic industry activity (Figure 1). More detailed information on the study sites and the relative concentration of metals in outdoor settled dust have been previously published [16,17]. Vegetable and soil metal measurements were performed in 82 home vegetable gardens, 59 of them 95 were located in the Valcamonica region and 23 in the reference area of Garda Lake.
4. Soil Measurement
The metal content in the soil of the vegetable gardens was directly analyzed by using a portable instrument based on X-Ray Fluorescence (XRF) (Thermo Scientific Niton, model XLt) equipped with GPS geo-referencing capability. The instrument was kept steadily on top of the soil surface, with an x-ray exposure and x-ray fluorescence signal collection time of approximately 100 seconds. One to four randomly distributed readings of surface soil, depending on the size of the garden, were taken per garden to obtain an average value for each garden. In addition, one representative sub-surface soil reading was taken from each garden after removing surface soil to a depth of 10 cm. A GPS reader yielded geo-referencing of each measurement to calculate the distance of each point from the ferroalloy plant exposure point source; i.e., GPS coordinates for each measurement were used to calculate an average distance of each garden from the ferroalloy plant.
Soil pH was determined in a 1:2.5 soil/water suspension to allow more accurate comparisons among the gardens. A pH meter fitted with a glass electrode was used according to the Italian official methodology [20].
In order to intercalibrate the portable XRF soil metal measurements, and to determine the association between total soil metal levels and the chemically labile fraction of metals in soil, a sub-set of 23 randomly selected soil samples were analyzed by ICP-OES after sequential extraction using a modified BCR procedure [21]. Briefly, surface soil samples (~100 g) were collected, dried in an oven (65˚C) for 24 h to a constant dry weight, sieved to 150 μm, and a ~1 g ± 10% sub-sample taken for sequential extraction and analyses. Soil samples were extracted first with 0.11 mol·L–1 acetic acid (fraction 1, exchangeable and weak acid soluble metals), second with 0.5 mol L–1 hydroxylammonium chloride (fraction 2, reducible metals), third with repeated extraction with H2O2 (fraction 3, oxidizable metals), and finally with 7.5 N HNO3 (fraction 4, remaining strong acid extractable metals). The concentrations of Cu, Fe, Mn, Zn in the extracts were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES) using a Perkin-Elmer Optima 4300 DV Series instrument. Parallel analyses of standard reference material BCR 483 (sewage sludge amended soil certified by Community Bureau of Reference) was used to evaluate extraction efficiency and analytical accuracy [22,23]. The analytical detection limit for Cu Fe, Mn, and Zn were 0.01, 0.68, 0.025, and 0.020 ug/mL for the analyzed extract, respectively. The analytical reproducibility averaged 9% (RSD) based on triplicate processing and analyses of selected soil samples. Analytical accuracy averaged 88% (range 80% - 107%) of expected values in BCR fractions 1 - 3 for certified metals, based on repeated triplicate processing and analyses of SRM BCR 483.
5. Vegetable Sampling and Analysis
Two kinds of the most commonly grown leafy vegetables in local gardens were selected: lettuce (Lactuca sativa) and chicory (Chichorium spp., also called Radicchio). Throughout the time of sample collection, which occurred from April to October, vegetables were collected at a normal harvestable and edible stage and were representative of the crop. Three vegetable leaves were collected from 3 plants into polyethylene bags and stored at 4˚C for 24 hours in the laboratory before processing for metal concentration analysis.
The edible parts of each vegetable sample were carefully washed with MQ water to eliminate any possible external contamination due to superficial airborne-deposited particles or residual surface dirt. Then they were air-dried in a fume hood at room temperature (approximately 25˚C for 24 h).
The three leaves of each plant were ground and a quantity of 10 mg was taken from the mixture and digested in a polyvinyl-fluoride test-tube with 1 ml of concentrated nitric acid for 48 hours in a fume hood at room temperature.
Metal levels in plant digestated were analyzed at the Laboratory of Chemistry for Technology, University of Brescia, with a spectrometer based on Total Reflection X-Ray Fluorescence (TXRF) (Bruker S2 Picofox, air cooled, Mo tube, Silicon-Drift Detector, operating values 50 kV and 1000 μA), using an acquisition time of 600 seconds. The concentration of Mn, Zn, Cu, Fe, and Pb was determined on the basis of the known content of Ga, which was used as an internal standard. TXRF is a non destructive methodology that provides efficient multielemental identification and quantification of elements in samples for human environmental exposure assessment. TXRF is both rapid and sensitive (to the ppb level), and is not affected by complex sample matrices [24]. Detection/quantitation limit, accuracy, and precision of Picofox have been reported in literature [25]. A significant correlation has been demonstrated between the TXRF techniques and the other standard methods for metal analysis such as AAS and ICPMS [26].
6. Statistical Analysis
Since most of the metal concentrations in plants and soils showed a skewed distribution, we used empirical quartiles to summarize the data and non-parametric statistics (Wilcoxon Mann-Whitney U Test) were applied to compare the results of soil and vegetable measures between the exposed and reference areas. Wilcoxon signed rank test was used to compare surface soil and sub-surface soil (10 cm depth) metal levels. For the same reason Kendall’s Tau was used to measure the association between the concentration of metals in soil and in vegetables. Statistical analyses and graphics were made with R 2.10.1 [27].
7. Results
7.1. Inter-Comparison between XRF and ICP-OES
Analyses of a subset of soil samples (n = 23) from the Valcamonica and Garda Lake study sites were performed using a BCR sequential extraction method and ICP-OES analyses, in order to 1) validate the portable field XRF analyses that were used at all study sites, and 2) to evaluate the relationship between total soil metal levels and the chemically labile fraction of metals in soil (defined here as the sum of extractable metals in BCR fractions 1 + 2). While the sequential BCR extraction method does not directly indicate bioavailability of metals in soil, metals that are chemically labile would be expected to reflect increased bioavailability as well.
A significant association was observed between both the near total BCR extractable metal levels (sum fractions 1 – 4) and the chemically labile metals fractions (BCR fractions 1 + 2) with soil metal levels measured by XRF. For example, near total Mn levels measured with the BCR and ICP-OES were highly significantly correlated with Mn levels measured by XRF (XRF Mn = 1.33*OES Mn – 71.8, R = 0.830, p < 0.0001). Similarly, chemically labile Mn levels measured with the BCR and ICP-OES (BCR fractions 1 + 2) were also significantly correlated with Mn levels measured by XRF (XRF Mn = 1.43*OES Mn – 14.7, R = 0.826, p < 0.0001). Importantly, a highly significant relationship resulted also between near total soil Mn (sum fractions 1 – 4) and the chemically labile Mn in soil (fractions 1 + 2), as determined by the BCR sequential extraction method and ICP-OES (labile Mn = 0.9141*total Mn – 30.86, R = 0.990, p < 0.0001).
Highly significant relationships exist between soil measurements by ICP-OES and XRF (both in ppm units) for the other metals as well: for Fe near total BCR and OES vs XRF, XRF Fe = 2.36*OES Fe + 5360, R = 0.772, p < 0.0001; for labile Fe by OES (BCR F1 + F2) vs XRF, XRF Fe = 13.8*OES Fe + 10660, R = 0.772, p < 0.0001; for near total Fe by OES vs labile Fe by OES, labile Fe = 0.145*total Fe – 257, R = 0.876, p < 0.0001. For Zn near total BCR and OES vs XRF, XRF Zn = 1.056*OES Zn + 25.3, R = 0.902, p < 0.0001; for labile Zn by OES (BCR F1 + F2) vs XRF, XRF Zn = 1.25*OES Zn + 60.5, R = 0.843, p < 0.0001; for near total Zn by OES vs labile Zn by OES, labile Zn = 0. 773*total Zn – 17.9, R = 0.977, p < 0.0001. For Pb near total BCR and OES vs XRF, XRF Pb = 0.893*OES Pb + 2.11, R= 0.774, p < 0.0001; for labile Pb by OES (BCR F1 + F2) vs XRF, XRF Pb = 1.52*OES Pb + 5.24, R = 0.719, p < 0.0001; for near total Pb by OES vs labile Pb by OES, labile Pb = 0.487* total Pb + 2.86, R = 0.894, p < 0.0001.
7.2. Comparison of Metals in Soil and Vegetables in the Two Areas
Table 1 reports the results of soil measurements. Average metal concentrations were significantly higher in Valcamonica compared to Garda Lake for Mn (p < 0.0001), Pb (p = 0.04), and Fe (p < 0.0001) in surface soil, and for Mn (p = 0.0002), Pb (p = 0.03), and Fe (p <