Toxicological Approach for Assessing the Heavy Metal Binding Capacity of Soils

By Feng, Nan Dagan, Roi; Bitton, Gabriel

A toxicological approach was taken to determine the heavy metal binding capacity of soils. A soil heavy metal binding capacity (SHMBC) methodology was developed and was based on the use of the MetPLATE(TM) toxicity test kit, a bioassay that is specific for heavy metal toxicity. SHMBC test is based on the heavy metal binding capacity (HMBC) concept that has been considered in the assessment of the metal binding capacity of surface waters (Huang et al., 1999) and solid wastes landfill leachates (Ward et al., 2005). SHMBC is the ratio of the EC^sub 50^ of an added metal in a soil sample divided by the EC^sub 50^ of a metal in a reference soil (clean Ottawa sand). A higher SHMBC value indicates higher metal binding to soil and lower bioavailability and potential toxicity to the test bacteria. Five soils (two sandy soils, two organic soils and a clay soil) were used to determine their binding capacity towards Cu, Zn, and Hg, using the developed SHMBC test. The test measured the ability of the solids to reduce metal bioavailability and toxicity. SHMBC was highest for the clay soil and lowest for the sandy soils. The potential application of this relatively rapid (a few hours) test to predict metal toxicity to terrestrial plants is discussed.

Keywords Metals in soils, adsorption, toxicity testing, bioavailability, MetPLATE

Introduction

Soils are widely used as receptacles for organic and inorganic contaminants such as metals. They are useful in mitigating the impact of contaminants on groundwater resources and surface waters. Soils are impacted by several types of toxic wastes, including industrial wastes, biosolids, mining, construction and demolition wastes. Toxic metals in the applied wastes have attracted the attention of regulatory agencies because they can be transported to groundwater or taken up by agricultural crops, leading to concerns over human and animal health. Toxic metals tend to bind to soils, thus becoming less available to the biota and to roots of agricultural crops (Adriano, 1986; Salomons, 1995). Metal phytoavailability (i.e., availability to plants) is controlled by several factors, which include metal species, soil characteristics (e.g., pH, clay type and content, organic matter content, moisture content) and duration of contact between soil and metals (Naidu et al., 2003; Weng et al., 2002). Moreover, bioavailability of metals in soils also depends on the type of clay and organic matter (Lock and Janssens, 2001). Soil amendment with clay minerals (e.g., bentonite, zeolite), iron oxides (e.g., goethite, hematite), and phosphate fertilizers was effective in reducing metal availability to wheat (Triticum aestivum) (Usman et al., 2005).

Metal binding and immobilization in soils involves several mechanisms such as adsorption, ion exchange, complexation by humic substances, and precipitation reactions (Weng et al., 2002). Sequential extraction procedures provide a good indication of metal partitioning in soils. They involve a range of chemical reagents that extract different metal fractions (soluble, exchangeable, carbonate-bound, oxide/hydroxide-bound, organic matter-bound, and residual fractions) in soils (Balasoiu et al., 2001; Smith et al., 1999; Tack and Verloo, 1995; Tessier et al., 1979; Yong et al., 2001). It is generally agreed that the soluble and, potentially, the exchangeable fractions are available to plants and the biota, and that the total concentration of metals in soils does not indicate their availability to plants (Adriano, 1986). It was reported that phytoavailability may vary among different soils contaminated with the same total metal concentration, suggesting that the soil matrix plays an important role in phytoavailability and, ultimately, phytotoxicity (Naidu et al., 2003).

The metal fractionation chemical procedures need, however, to be complemented with toxicity testing to obtain information about the biological activity of metals in soils. Some investigators have used both chemical and toxicological approaches to assess the bioavailability of metals in solid matrices (Kong and Bitton, 2003; Schultz et al., 2004; De Vevey et al., 1993).

We report here the development of a relatively rapid test to assess the heavy metal binding capacity (HMBC) of five soils. The test is based on the use of MetPLATE(TM) , a bioassay that responds specifically to heavy metal toxicity (Bitton et al., 1994). The test compares the relative toxicity of a metal in a given soil to metal toxicity in a reference soil (Ottawa sand).

Materials and Methods

Soils Used

Three soil types were used to assess their capacity to bind metals such as copper, zinc and mercury. Two sandy soils were collected from the top 4 feet at two different sites and were chosen because they are representatives of the soils prevailing in North Central Florida. An organic soil (organic soil 1) was collected from the first few top inches along Hogtown Creek in Gainesville, FL. The second organic soil (organic soil 2) was a top soil purchased from a local landscaping store. The Georgia clay soil (top 20 cm) was collected in Atlanta, Georgia. Table 1 shows some characteristics of the soils under study. Ottawa sand, due to its low ability to bind metals, was selected as a reference soil.

Methodology for Assessing Soil Heavy Metal Binding Capacity (SHMBC)

Briefly, the test consists of adding metal-laden solutions to soils under study, allowing the mixtures to reach equilibrium, separating the solid phase from the pore water by centrifugation, and assaying for metal toxicity of the soil extracts. A similar methodology was used for the Ottawa sand, which serves as a reference soil.

Soils were first screened (sieve # 16; 1.19 mm particles) and homogenized. Subsequently, serial dilutions of metal-spiked solutions were prepared in moderately hard water (78 mg/L Ca, 24 mg/ L Mg, pH = 6.8) for soil spiking. The Cu, Zn and Hg concentration range of the metal-spiked solutions is shown in Table 2. The solutions were labeled A, B, C, D, and E. A sixth solution, labeled F, was not spiked with metals and served as the negative control for the soil. Five other metal-spiked solutions were added to Ottawa sand (reference soil) and were labeled A^sub o^, B^sub o^, C^sub o^, D^sub o^, and E^sub o^. A sixth solution, labeled F^sub o^, was prepared without any metal added. 20 rnL of each solution was added to 5 g of soil or Ottawa sand in 50-mL Erlenmeyer flasks. The flask were covered with parafilm and placed on a shaker at 300 rpm for 4 hours. After shaking, the soils were centrifuged at 10,000 rpm for 15 minutes. The metal toxicity of the soil extracts was assayed with MetPLATE (see http://www.ees.ufl.edu/homepp/bitton/ for more information), a microbial test which responds specifically to heavy metal toxicity. The MetPLATE assay was carried out according to Bitton et al. (1994). Following rehydration of the MetPLATE(TM) bacterial reagent, 0. 1 mL of bacterial suspension was added to 0.9 mL of the soil extracts in small culture tubes. The tubes were vortexed and placed in an incubator at 35[degrees]C for 90 minutes. Following incubation, 0.2 mL of the content from each tube was transferred to a 96-well microplate. 0.1 mL of rehydrated MetPLATE chromogenic substrate was added to each well. The plate was shaken gently and returned to the incubator until a purple color developed (after about 1 hr) in the negative controls (solutions F and F^sub o^). The absorbance was determined by a microplate spectrophotometer (Maxline Microplate Readers, Molecular Devices, Sunnyvale, CA) at 570 nm. All HMBC tests were run in triplicate and 3 MetPLATE toxicity tests were run for each HMBC test.

Table 1

Soils characteristics

Table 2

Metal concentration ranges of the solutions used to spike soils

Regression analysis was used to determine the EC^sub 50^ for both the soil under study and Ottawa sand, the reference soil. The SHMBC was determined by dividing the EC^sub 50^ for the soil sample by the EC^sub 50^ for the metal in the reference soil, Ottawa sand.

Results and Discussion

Soil heavy metal binding capacity (SHMBC) was determined for five soils sampled in Florida and Georgia. Three metals (Cu, Zn, and Hg) were tested for their binding to the soils. Table 3 shows the EC^sub 50^s (expressed as metal added in mg/kg soil) of the three metals in the different soils under study. The Ottawa sand (reference soil) extracts were quite toxic, with EC^sub 50^S of 1.1 mg/kg for Cu, 0.9 mg/kg for Zn, and 1.5 mg/kg for Hg, indicating that the Ottawa sand displayed a relatively low binding capacity for metals, thus justifying its selection as a reference soil.

It is worth mentioning that the higher the EC^sub 50^, the lower the toxicity of the soil extracts, indicating that the metal was bound by the soil and, thus, unavailable to the test organisms. The Georgia clay soil displayed the highest EC^sub 50^s (i.e., lowest toxicity to MetPLATE) for the three metals tested. Among the five soils under study, the EC^sub 50^s, expressed as metal added in mg/ kg, varied between 19.1 and 416 for Cu, between 14.3 and 296.9 for Zn, and between 8.4 and 448.4 for Hg (Table 3).

The soil heavy metal binding capacity (SHMBC) for the three metals and five soils tested is shown in Figure 2. As regard their binding capacity towards the three metals, the soils were classified in the following order: Georgia clay soil > organic soils > sandy soils

The organic (humic substances) and inorganic (clay minerals) colloidal particles in soils generally play a significant role in binding metals. Georgia clay soil contains 21% clay and thus displayed the highest binding capacity towards all three metals. Similarly, the organic soils (1 & 2) showed much higher SHMBCs than the sandy soils, due to their much higher organic matter content (12.2-18.8%) (Figure 2). Thus, the higher the SHMBC, the higher is metal binding to the soils.

Figure 1. Soil HMBC (SHMBC) methodology.

Table 3

Table 3 EC^sub 50^s (metal added in mg/kg soil), as determined by MetPLATE, of water extracts from five soils and Ottawa sand

The HMBC concept, as reviewed by Bitton et al. (2005), was previously used to assay metal bioavailability in surface waters (Huang et al., 1999) and municipal landfill !cachates (Ward et al., 2005). The present research shows the first application to date of this bioassay to soils. It is a relatively rapid methodology to assess metal bioavailability in soils. This methodology is based on toxicity testing of soil extracts with MetPLATE, a test specific for heavy metal toxicity (Bitton et al., 1994). We need to know if this test can predict metal uptake and subsequent toxicity to plants (Feng, Boularbah and Bitton, unpublished results). Culture studies with plants show that metal uptake by plants varies with the type of soil, with the uptake being higher in sandy than in clay soils, which generally display a higher metal binding capacity (Naidu et al., 2003). For example, terrestrial plants should be protected from Cu toxicity at the benchmark concentration of 100 mg/kg (Will and Suter, 1995). However, no phytotoxicity was found in Australian orchard soils with a range of copper concentrations of 11 to 320 mg/ kg (Merry et al., 1983). Cu and Cr toxicity to barley was lower in a spiked natural forest soil (82.8 sand, 9.2% silt, 8.0% clay, 3.8% organic matter content) than in an artificial sandy soil (100% sand; pH 7.80, 0.27% organic matter). (Ait Ali et al., 2004). Around a Peruvian copper mine, phytoxicity was found to be higher in soils with low organic matter (Bech et al., 1997). These findings confirm that total soil metal concentrations do not give an indication of metal bioavailability and phytotoxicity, with the soil matrix playing an important role in metal toxicity. Our findings, using a bacterial toxicity test, confirm that, at least for Cu, Zn and Hg, the SHMBC for clay and organic soils is much higher than for sandy soils.

Figure 2. SHMBC for three metals (Cu, Zn, Hg) and five soils.

Conclusion

This new technique, based on a toxicological bioassay, shows a novel approach to evaluate heavy metal binding to soils and, hence, bioavailability. We have shown that the soil metal binding capacity (SHMBC) varies with the type of soil, with clay and organic soils displaying a higher metal binding than sandy soils. This relatively rapid test could be used in a number of applications, mainly ecological risk assessment. The SHMBC test could also be used to assess the suitability of soils to receive metallic wastes. The test could simulate more realistic conditions by determining the SHMBC of metal-spiked soils, which have been subsequently “aged” for a few weeks or a few months. Moreover, it would be valuable to run SHMBC tests in parallel with metal uptake by terrestrial plants to determine their application in assessing phytoavailability and potential for phytotoxicity. More research should be conducted to validate the results of the SHMBC method.

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NAN FENG, ROI DAGAN, AND GABRIEL BITTON

Laboratory of Environmental Microbiology and Toxicology, Department

of Environmental Engineering Sciences, University of Florida, Gainesville,

FL1USA

This work was funded by the National Science Foundation (grant # BES-9906060).

Address correspondence to Dr. Gabriel Bitton, Laboratory of Environmental Microbiology and Toxicology, Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL 32611, USA. E-mail: gbitton@ufl.edu

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