Bio-accessibility of Trace Metals Using Chemical Fractionation and In Vitro Extraction Methods
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1 Bio-accessibility of Trace Metals Using Chemical Fractionation and In Vitro Extraction Methods Naomi Waissman Assadian 1 and Juan Pedro Flores Margez 2 Abstract Rural and urban border communities at Paso del Norte Region are exposed to heavy metals from industry, atmospheric fallout from smelting, combustion of petroleum products, and wastewaters for agricultural use. Chemical fractionation methods to determine the bioavailability/bioaccessibility of metals in soils, particularly under alkaline conditions have been inconsistent to poor indicators of predicting trace metal accumulation in plants. Consequently, risk assessment of contaminated soil to animals and humans has been limited. In vitro gastrointestinal (IVG) and physiologically based extraction test (PBET) methods may provide better estimates of trace metal bioavailability and human risk. The objective of this study was to compare chemical fractionation methods with emerging IVG PBET methods to predict the bio-accessibility of cadmium (Cd), chromium (Cr), nickel (Ni) and lead (Pb) in moderately alkaline soils with and without biosolids amendments in the Juarez Valley of Mexico. The exposure pathway for this study is; untreated effluents for irrigation - metal contaminated soil oat forage sheep. Selected trace metals in soil and biosolids were chemically extracted using total and sequential metal extraction protocols, total acid extraction in plant tissue and in sheep tissues (muscle, liver, and kidneys). Soil and plant tissue were re-extracted using IVG and PBET methods. Ongoing results shows that chemical fractionation of selected trace elements in soil did not reflect metal accumulation in oat forage or in sheep kidney, liver, or muscle tissues. Bio-available metal concentrations using in vitro methods with metal accumulation in plant and sheep tissues were compared. Validity of the various chemical extraction methods will depend on how closely analytical results predict metal accumulation in plant and sheep tissues. 1 Texas A&M Research & Extension Center 1380 A&M, El Paso, TX, n-assadian@tamu.edu 2 Universidad Autonoma de Ciudad Juarez, Chihuahua, Mexico. Av.Henry Dunant 4016, juflores@uacj.mx 1
2 Introduction There is a persistent perception that metal deposition on soils along the Texas/Mexico border from smelting activities, industry and wastewater reclamation compromises food safety, environmental integrity, and public health. Recalcitrant metals may have long-term ramifications on agro-ecosystems and on crop products. Local consumption of crop and/or animal products may increase the metal exposure of urban border populations. There has been limited risk assessment of heavy metal mobility into the local agricultural food chain based on the bioavailability of metals in soils using sequential extraction protocols. Emerging in vitro models and assessment techniques show promise in predicting metal exposure, but are based on bioaccessibility rather than bioavailability. Bioavailability in this study was defined as the fraction of trace elements that are in soluble and exchange phases in soil and are readily released from the solid phase of the soil. Bioaccessibility was defined as the metal fraction in soil that is soluble in the gastroinstinal environment and available for absorption. Objective Identify rapid, simple, and predictive techniques to predict animal (sheep) and potential human risks to recalcitrant trace elements in moderately calcareous soils. 2
3 Materials and Methods The exposure pathway for this study was: untreated effluents for irrigation soil incorporated biosolids - metal contaminated soil oat forage sheep in a replicated field study (a one-way layout) established in the Ejido San Isidro, Juarez Valley, Chihuahua, Mexico. Field soil was a Caseta clay (Typic Torrifluvent) irrigated with wastewater, and amended with LS biosolids at rates of 0, 25, and 50 Mg ha -1 on a dry weight basis. Ammonium sulfate at 120 kg N ha -1 was the control. Treatments were incorporated into the top 15 cm of 15 m x 20 m plots. Forage oats (Avena sativa L.) were planted to plots and grazed by five Pelibuey for each soil treatment (total of 20 sheep). An additional three sheep were sacrificed prior to sheep grazing on plots. Irrigation water was collected 8 times, soil collected 4 times, oat plants sampled two times, and sheep were sacrificed after 206 d of grazing. The soluble fraction of Cd, Cr, Ni and Pb were analyzed in irrigation water; total and available metal fractions were analyzed in soil and biosolids; total metal fractions were analyzed in plant and sheep tissues (muscle, liver, and kidneys). Sequential protocols for bioavailability (soluble and exchangeable soil fractions) followed Ramos et al. (1994) and total metal extraction followed USEPA Method 3050B using inductively coupled plasma spectroscopy (ICP). Soil samples were re-extracted to determine bioaccessibility using a modified IVG method without salts (Morrison, 2003), a modified PBET method (Fendorf et al., 2004), and in vitro digestion using sheep rumen combined with alfalfa (Dr. Clint Loest, New Mexico State University, Animal Science Laboratory) using ICP. A mass balance of metals in the agro-system was calculated on estimated irrigation supply and average forage consumption by sheep. 3
4 Results and Discussion Metal Bio-availability Bioavailable concentrations of wastewater and biosolids Cd, Cr, Ni, and Pb were low (Figures 1 and 1A). Based on 10 cm of irrigation wastewater and a biosolids loading rate of 50 Mg ha -1, bioavailable metal inputs to field soil were < 0.5 kg ha -1, for anyone metal species and accounted for < 5% of bioavailable metals in field soil. In biosolids amended soil, bioavailable Cr concentrations exceeded those of Cd, Ni, and Pb and were > 32 kg ha -1, in the top 15 cm of the soil profile (Fig. 2). Available chromates are rare in soil, but are known to be stable under alkaline oxidizing conditions. Bioavailable concentrations of Cd, Ni, and Pb were < 9.0 kg ha Cd Cr Ni Pb Concentration (ug / L) 20 0 Aug28 Sep8 Sep28 Oct25 Mar2 Apr4 Apr19 Apr30 May5 Irrigation Date ( ) Figure 1. Maximum values of heavy metals observed in irrigation water along the experimental period at the Juarez Valley
5 60 Concentration (mg kg -1 ) Total Bioavailable Cd Cr Ni Pb Figure 1A. Selected metals in lime stabilized biosolids. 5
6 In oat tissue, total Cd concentrations were < 0.5 mg kg -1 on a dry weight basis from forage harvested from plots, as expected (Fig. 3). Despite bioavailable Cr in soil, oat tissue levels of Cr were < 2 mg kg -1. Plant uptake and translocation of Cr is known to be low and about 10% of the bioavailability. Total Ni and Pb oat tissue concentrations ranged from 2 to 8 mg kg -1. Sauerbeck (1991) reported 0.7, 0.5, 3, and 2.5 mg kg -1 d.w. of Cd, Cr, Ni and Pb in oat leaves which are similar ranges found in our experiment (Fig. 3). Given an average forage yield of 864 kg ha-1 harvest -1, oat forage removed < kg ha -1 for each of the metal species, which was 0.02% of bioavailable soil metals. However, our data suggests that soil particles on forage surface may also increase metal exposure to sheep (data not shown). 6
7 Total metal concentrations in tissues from sheep grazing on forage oats was < 1 mg kg -1, except for Ni that was < 13 mg kg -1 (Fig.4). Cadmium is a cumulative poison often found in kidney and liver tissue, but had not accumulated in these sheep tissues. Low Pb absorption should not have interfered with Fe metabolism. Both Cr and Ni are essential for animals. Nickel is an integral part of urease and Cr participates in mammalian glucose metabolism. Given that sheep consumed 1.5 kg forage (dry) day -1, trace element deficiencies may develop. Metal Bio-accessiblity Selected In Vitro methods were easy to follow and rapid. Soil that was also digested with alfalfa in sheep rumen and saliva used a more complicated procedure that may be more challenging to transfer. Results suggested that soil incorporation of biosolids had no impact or interaction with bioavailable-bioaccessibility methods regardless of metal species. Results from bioaccessibility methods were significantly different from those using bioavailable metal fractionation, with the exception of Ni (p<0.05) (Fig. 5). The magnitude and differences in bioavailability-accessibility were dependent on metal species. Cadmium, Cr, Ni and Pb concentrations were below method detection limits when digested in sheep rumen. 7
8 8
9 No single analytical method best described metal concentration in sheep tissues (Figs. 4 and 5). The modified PBET method was close to predicting Cd and Cr concentrations measured in sheep tissues. Bioavailable metal fractionation best predicted Ni and Pb in sheep tissue. The In Vitro method using HCl also closely predicted Ni concentrations in sheep tissue. The field experiment reflected commercial operations rather than a controlled animal feeding experiment. Oat forage was also supplemented with alfalfa hay. The test animal was a ruminant rather than a monogastric. These experimental conditions likely affected the metal exposure pathway and the goodness of analytical methods to predict metals in sheep tissue. Conclusions Field soil rather than wastewater or LS biosolids was the primary source of bioavailable metals. Soil Cd, Cr, Ni, and Pb concentrations were within conventional ranges. Alkaline field soil (reduced metal bioavailability), forage oats (Cr exclusion), and sheep digestion (Pb exclusion) were barriers preventing metal exposure of Bioavailable Cd, Cr, Ni, and Pb to grazing sheep. Limited Cr mobility in the food chain may create Cr deficiencies. There were likely several metal exposure pathways to sheep under field conditions and affected by grazing management. No single analytical method based on soil samples predicted selected trace elements in sheep tissues. 9
10 Bibliography Fendorf, S, M.J. La Force, and G. Li Temporal changes in soil partitioning and bioaccessibility of arsenic, chromium, and lead. J. Environ. Qual. 33: Morrison, A.L An examination of the chemistry and oral bioaccessibility of bas metal smelter slags in North Lake macquarie, NSW. Graudate School of the Environment, Macquaire University. Ramos, L., M. Hernandez, and M.J. Gonzalez Sequential fractionation of copper, lead, cadmium and zinc in soils from or near Donana Nacional Park. J. Environ. Qual. 23: Sauerbeck, D.R Plant, element and soil properties governing uptake and availability of heavy metals derived from sewage sludge. Water, Air, and Soil Pollution : Acknowledge Appreciation is expressed to the USDA Rio Grande Basin Initiative , and to the Center for Border Health Research (CBHR), Paso del Norte Health Foundation, El Paso, TX. 10
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