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TECHNICAL REPORTS
Heavy Metals in the Environment

Treatment of Contaminated Soil with Phosphorus and Manganese Oxide Reduces Lead Absorption by Sprague–Dawley Rats

^a National Risk Management Research Laboratory, USEPA, Cincinnati, OH 45224
^b Comparative Toxicology Laboratories, Kansas State Univ., Manhattan, KS 66506
^c Dep. of Soil Science, Faculty of Agriculture, Univ. of Peradeniya, Peradeniya, Sri Lanka

^* Corresponding author (ganga{at}ksu.edu)

Received for publication April 15, 2002.

	ABSTRACT

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This study was conducted to determine the extent of Pb absorption into young rats (Rattus norvegicus var. Sprague–Dawley) fed untreated Pb-contaminated soil or Pb-contaminated soil treated with two different sources of P and P + Mn oxide. Data were compared from an in vitro, physiologically based extraction test (PBET) with the animal data to support the validity of the in vitro test to assess bioavailable Pb from a treated Pb-contaminated soil. Soil with a total Pb concentration of 2290 mg kg^-1 was used. Rats were fed 19 different test diets for 21 consecutive days. The test diets represented 95 g AIN93G rat meal kg^-1 diet with varying proportions of silica sand or soil to provide low, medium, or high doses of Pb from either Pb acetate, treated, or untreated soil. Blood, liver, kidney, and bone Pb concentrations were examined. For all four tissues, Pb concentrations for the Pb acetate groups were significantly higher than concentrations for all the soil groups. In general, either triple superphosphate (TSP) or phosphate rock (PR) treatments resulted in significant reductions in tissue Pb concentrations compared with untreated soil. Blood and kidney Pb concentrations for the PR + Mn oxide group were significantly lower than those of the PR group at the low and high doses. Relative bioavailability of Pb, as measured in all tissues, was significantly reduced when comparing untreated with amended soil. Correlation between the in vitro and in vivo tests, based on bone and liver tissue, showed that the in vitro test is successful at predicting Pb bioavailability.

Abbreviations: BAF, bioavailability factor • CRYP, cryptomelane • PBET, physiologically based extraction test • PR, phosphate rock • RBA, relative bioavailability • RR, relative response • TSP, triple superphosphate

	INTRODUCTION

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SOILS CONTAMINATED with toxic elements at concentrations higher than regulatory limits pose significant health and environmental risks. Regulatory limits designed to protect exposed individuals from soil contamination are generally based on the total concentration rather than the bioavailable fraction. The environmental risk of a toxic element, however, is related to its bioavailability (Oliver et al., 1999). Chemical or in situ stabilization of soil Pb using P and other soil amendments focuses on reducing its environmental risk by chemically altering soil Pb to less bioavailable forms. Once stabilization is achieved via chemical alteration, the next challenge is assessing the changes in environmental risk associated with soil Pb. In the absence of human studies, animal model studies or procedures have been used for evaluating changes in soil Pb bioavailability. Bioavailability of Pb in soils, as well as ores and mine waste materials, has been evaluated extensively in rat (Rattus spp.) (Davis et al., 1993; Dieter et al., 1993; Freeman et al., 1992, 1994, 1996; Schoof et al., 1995) and swine (Sus spp.) (Casteel et al., 1997).

Rats, rabbits (Oryctolagus spp.), or young swine have been used as the animal model for studies of gastrointestinal function of children. There is, however, no validated animal model for experimental uses in measuring bioavailability for children. Each animal model is expected to respond uniquely to absolute Pb absorption (i.e., oral uptake versus intravenous dosing), compared with children. It is expected that differences observed within a model would be observed with the other models but the magnitude of the differences may vary between models. Thus, any of the animal models could be used to evaluate treatment effects but the magnitude of the treatment effect in the animal model and its relationship to the human models would be an unknown.

Water, soil, dust, air, and food are the major exposure pathways of Pb to humans. The two main routes of intake of Pb-containing materials are through the gastrointestinal tract (ingestion) and lungs (inhalation). For the purpose of this study, Pb ingestion is considered as the major route of Pb intake. Measurements for the oral bioavailability of Pb from contaminated soils for rats are commonly taken by orally exposing rats daily to soil Pb and a soluble Pb salt for two to four weeks, and then analyzing Pb concentrations in different tissues, such as blood, bone, liver, and kidney (Freeman et al., 1994; Schoof et al., 1995). Relative bioavailability of soil Pb is then estimated by comparing tissue concentrations resulting from the different soils or forms of Pb relative to the soluble Pb salt. In most animal studies the mean particle size of test soils used to feed test animals was either 250 or 10 µm. A particle size of 100 µm is the upper size limit for the particles reported to adhere to the hand of a child (Duggan et al., 1985, Chaney et al., 1989). However, it is difficult to collect adequate quantities of the 100-µm fraction from soils, and the 250-µm fraction is most often used to approximate the particle size distribution ingested by children.

Lead phosphates, and in particular pyromorphites, are one of the most stable forms of Pb in soils under a wide range of environmental conditions (Nriagu, 1973, 1974; Lindsay, 1979). Cotter-Howells et al. (1994) and Nriagu (1974) have observed pyromorphite as a common weathering product of Pb compounds in mine waste materials and in urban and roadside soils at typical soil P concentrations. Experimental evidence supports the hypothesis that Pb phosphates can form rapidly when adequate Pb and phosphate are present in aqueous systems (Ma et al., 1994a,b; Zhang and Ryan, 1999a,b). In vivo (animal feeding) studies have indicated that Pb availability in mammalian gastrointestinal systems is dependent on the form and relative dissolution rates of Pb solids (Ruby et al., 1993, 1996). Formation of pyromorphites on addition of apatite or soluble inorganic P amendments was observed in Pb-contaminated soil materials (Laperche et al., 1996; Cotter-Howells and Caporn, 1996; Hettiarachchi et al., 2001; Ryan et al., 2001). Reduced plant Pb uptake was also observed on apatite and/or triple superphosphate addition to Pb-contaminated soils (Laperche et al., 1997; Brown et al., 1999; Hettiarachchi and Pierzynski, 2002).

In addition to the formation of insoluble Pb compounds as a means of reducing Pb bioavailability, adsorption is another potentially important process controlling the bioavailability of Pb in soils. Less attention has been given to manganese (Mn) oxides, even though they are known to adsorb Pb more strongly than any other metal (hydr)oxides. A laboratory incubation study revealed that reductions in bioavailable Pb in stomach-phase extractions (from physiologically based extraction test; PBET) ranged from 15 to 41% and 23 to 67%, on addition of P or P + Mn oxide (cryptomelane), respectively, in five metal-contaminated soils or mine spoils compared with the unamended control, indicating that the addition of both Mn oxide and P together is more effective than addition of P alone (Hettiarachchi et al., 2000). Greenhouse experiments using sudax [Sorghum vulgare (L.) Moench] and Swiss chard [Beta vulgaris (L.) Koch] revealed that the addition of P and/or Mn oxide significantly reduced bioavailable Pb, as measured by the PBET, in three Pb-contaminated soils compared with the control even after continual P removal through plant growth (Hettiarachchi and Pierzynski, 2002).

The PBET procedure has been validated for use in determining bioavailability of Pb in untreated contaminated soils with animal feeding studies done with weanling rats (r² = 0.93 for the stomach phase and blood Pb concentrations; Ruby et al., 1996) and young swine (r² = 0.85 for the stomach phase and blood Pb concentrations; Medlin, 1997). However, for amended soils literature comparing the PBET data with an appropriate animal model is lacking. In particular, no studies report on the validation of PBET data against animal feeding data in which soils were treated in an attempt to reduce Pb bioavailability. Therefore, animal feeding studies would be useful to further verify estimated reductions in soil Pb bioavailability as indicated by PBET before adapting this technology to remediate Pb-contaminated soil in residential areas. Further, changes measured by the animal studies can be compared with that measured by PBET.

The present study was conducted to determine the extent of absorption of Pb into the blood, liver, kidney, and bone of young rats (Rattus norvegicus var. Sprague–Dawley) fed Pb-contaminated soil treated with two different sources of P and P + Mn oxide. Further, this study compares PBET data with animal data to provide support for the validity of the PBET to assess bioavailable Pb from treated Pb-contaminated soil.

	MATERIALS AND METHODS

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Soil and Soil Treatments
A metal-contaminated soil having a total Pb concentration of 2290 mg kg^-1, collected from a vacant lot directly adjacent to an abandoned Zn and Pb smelter in Joplin, MO, was used. Selected chemical properties of the soil are given in Table 1 ; this was one of five soil samples used by Hettiarachchi et al. (2000)(2001). Cryptomelane (K₂Mn₈O₁₆, potassium manganese oxide; Joint Committee on Powder Diffraction File no. 20-908) was selected as the representative Mn oxide. It was prepared according to the procedure described by Hettiarachchi et al. (2000). Soil treatments included an unamended control; 5000 mg of P kg^-1 either as triple superphosphate [TSP, a common P fertilizer comprised mainly of monocalcium orthophosphate, Ca(H₂PO₄)₂·H₂O] or phosphate rock [PR, comprised mainly of fluoroapatite, Ca₁₀(PO₄)₆F₂]; and 5000 mg of Mn oxide kg^-1 + 5000 mg of P kg^-1 as either TSP or PR. These treatments were a subset of those described in Hettiarachchi et al. (2000). Treatments were evenly applied to the materials in plastic containers and thoroughly mixed. Deionized water was added to bring the samples up to a gravimetric water content of 20% and were then again thoroughly mixed. Predetermined amounts of CaO were added to all samples, except for the control and PR, 24 h after P amendment addition to increase the soil pH. Treated soil samples were incubated for 2 wk at 20% gravimetric moisture content and 25°C. At the end of the incubation period, soils were air-dried and sieved using a 250-µm screen before mixing with the standard rat diet.

View this table: [in this window] [in a new window]   Table 1. Selected properties of test soil material (250-µm fraction) before treatment applications.

Diet Preparation and Analysis
The basal diet consisted of 950 g fiber-free rat feed (fiber free AIN93G, TestDiet; Purina, Richmond, IN) plus 50 g inert material kg^-1. The inert material was varying proportions of silica sand (<250 µm) and the contaminated soil (treated or untreated) to give the desired Pb dose. For the Pb acetate diets, Pb acetate dissolved in deionized water was added to the silica, mixed well and dried at 400°C in an oven before mixing with the AIN93G rat feed. To prepare the diets, a small amount of AIN93G was first combined with the Pb acetate–silica or soil–silica and mixed until the mixture became homogeneous. This mixture was then added to the remaining AIN93G feed in small amounts with mixing until homogenous. The whole mixture was then placed in a cleaned V-shaped mixer and mixed again for approximately 2 h. Nineteen different AIN93G meal–soil–silica or AIN93G–silica with Pb acetate diets were prepared separately by mixing soil–silica or Pb acetate–silica with AIN93G feed following the same procedure mentioned above. All nineteen diets are listed in Table 2 . Diets were then doubled-bagged, secured with a cable tie, and stored at 4°C until fed. Subsamples from each diet mixture were digested in triplicate using 4 M HNO₃ (trace-metal grade) for 4 h in a water bath kept at 85°C. Filtered digestions were analyzed for Pb by inductively coupled plasma–atomic emission spectrometry (ICP–AES). These concentrations were used to calculate the actual dietary Pb ingestion.

A total of 152 male Sprague–Dawley rats, 12 wk of age at the beginning of the experimental diet feeding (approximately 8 rats per group), were fed the test diets for 21 consecutive days. Rats were assigned to different diet groups randomly, and the average body weight of animal per group was approximately similar for all 19 groups at the beginning of the feeding study. Feed consumption was monitored throughout the study. Weight gain was recorded approximately once every 5 d. The animals were checked for morbidity or mortality daily. Clinical signs of toxicity such as weight loss (rapid or gradual), inappetance, ruffled fur, hunched posture, diarrhea or constipation, tremors, and slow, shallow, or labored breathing were watched for daily. The Pb doses administered were lower than those thought to induce clinical signs of toxicity and no such effects were noted during the study.

Tissue Collection, Preparation, and Analysis
After 21 d on test diets, animals were anesthetized and sacrificed. Blood samples were collected into test tubes containing sodium heparin and gently mixed to prevent clotting before storing at less than -20°C. Whole liver and kidneys were collected, weighed, and stored in plastic tubes or vials at less than -20°C until processing. Entire femurs from each rat were also collected and similarly stored.

Blood samples were sonified using sonic dismembrator (Fisher Scientific, Pittsburgh, PA) with a stainless steel probe to rupture any clots and to assure homogeneity and dispersion. Blood samples were then diluted with a mixture of 5 g kg^-1 Triton X-100 (Sigma-Aldrich, St. Louis, MO), 2 g kg^-1 NH₄H₂PO₄, and 2 g kg^-1 concentrated HNO₃ and analyzed for Pb in duplicate using a graphite tube atomizer (GTA) connected to an atomic absorption spectrometer (AAS).

Liver samples were homogenized separately using a tissue homogenizer (BIOSPEC Products, Bartlesville, OK) with stainless steel rotary blades before weighing and collection of representative subsamples for microwave digestion. Before digestion, bone samples were scraped using a dissecting knife with a stainless steel blade to remove any residual soft tissues. Tissue samples (liver, kidney, or bone) were digested using concentrated HNO₃ (trace metal grade) in a CEM programmable laboratory microwave oven (CEM Corporation, Matthews, NC) (EPA Method 3051; USEPA, 2001). The digests were then brought to volume and the Pb concentration was measured in duplicate or triplicate using ICP–AES or GTA–AAS. Additionally, blanks and quality control samples were included in each digestion and analysis procedure. The duplicate or triplicate results were then averaged and used for statistical analysis. Lead concentrations in kidney, liver, and bone were expressed on a fresh-weight basis.

In Vitro Bioaccessible and Bioavailable Lead
Bioavailable Pb in the 250-µm size fraction of the untreated and treated soil samples was determined by a modified PBET procedure (Ruby et al., 1996). The gastric solution was prepared by adding 5 g of pepsin (activity of 800–2500 units mg^-1), 2 g of anhydrous citric acid, 2 g of DL-malic acid, 1.68 mL of DL-lactic acid (Sigma, St. Louis, MO), and 2 mL of glacial acetic acid (Fisher Scientific) to 4 L of deionized water. Variable amounts of trace-metal-grade, concentrated HCl were added to the gastric solution to ensure a pH of 2.0.

One hundred milliliters of gastric solution prewarmed to 37°C was combined with 1 g of test material (250-µm fraction) in a 125-mL wide-mouth HDPE bottle that was covered with a cap containing a rubber septum. The headspace was replaced with argon (Ar) gas, and the bottles were shaken for 1 h in a chamber maintained at 37°C. The pH was maintained at 2.0 ± 0.2 throughout this period. After 1 h, a 10-mL aliquot of gastric solution was removed for analysis using a 10-mL disposable syringe attached with a cellulose acetate membrane filter. This sample represents the stomach phase of the PBET procedure. This aliquot was replaced by 10 mL of fresh gastric solution at 37°C. The contents of the bottle were brought to pH approximately 6.5 by adding a 15-cm-length of dialysis tubing (100000 MWCO, Spectra/Por cellulose ester membrane tubing; Spectrum Laboratories, Rancho Dominguez, CA) containing 2.5 g of NaHCO₃. The tubing was removed after 30 min, and 0.175 g of bile extract (Porcine, 8008-63-7; Sigma) and 0.05 g of pancreatin (Porcine Pancreas-4X 8049-47-6; Sigma) were added. The cap was replaced and the head space was replaced again with Ar. The sample was shaken for another 1 h at 37°C. Another 10 mL of aliquot was removed for analysis as before. This sample represents the intestinal phase of the PBET procedure.

A drop of concentrated HNO₃ (trace-metal grade) was added to each aliquot to prevent precipitation of metals. Extracts were stored at 4°C until analysis using ICP–AES. Samples were run in duplicate. Quality assurance–quality control consisted of check samples and/or blanks for each extraction. The bioavailability factor in the soil sample was calculated as either the Pb extractable in the stomach-phase solution as a percentage of the total Pb in the soil (BAF_s, Table 1), or as the Pb extractable in the intestinal phase solution as a percentage of the total Pb in the soil (BAF_i).

Statistical Analysis
Data in soil-dose groups were analyzed using the PROC GLM procedure in the Statistical Analysis System (SAS Institute, 1997). Significant differences were determined at P < 0.05 using LSMEANS. Linear and nonlinear curve fitting were performed using the PROC REG and PROC NLIN procedures in the Statistical Analysis System. Significant differences between the slopes of lines or the plateaus from nonlinear equations were determined with a two-tailed t test (Steele and Torrie, 1980)

	RESULTS AND DISCUSSION

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Clinical Observations
No treatment-related toxicity, morbidity, or mortality were observed. Several rats in different diet groups died of renal calculi-related kidney failure, but this was not related to treatment.

Body Weights and Feed Consumption
There were no significant effects of diet on body weight, body weight gain, and feed consumption (data not shown). A reduction in feed consumption for rats affected by renal calculi was observed 1 to 2 d before their deaths.

Lead Absorption
Lead absorption in rats is known to be less than occurs in humans. Therefore, this study used young rats as suggested by Schoof et al. (1995) to maximize Pb absorption. Moreover, a diet low in fiber was used because fiber can inhibit Pb absorption. Ingested Pb may dissolve in the stomach (depending on particle size, form, accessibility, and kinetics of dissolution) and be absorbed into the blood stream. Absorbed lead may then be transported to body tissues or be excreted by various routes. Urinary excretion accounts for 95% of excreted absorbed Pb with the rest being lost via bile, sweat, and milk. Lead that remains in the body distributes to three pools: blood, soft tissues, and skeleton. The largest pool is skeletal Pb, and it is inactive physiologically. Lead in blood and soft tissues is associated with biological effects (National Academy of Science, 1980). Under stress, however, some Pb may be released from the skeleton to the blood. In this study we examined the Pb concentrations in four major body components: blood, liver, kidney, and bone.

Blood Lead Concentrations
The blood Pb concentrations detected in various rat treatment groups are shown in Fig. 1 . Mean group blood Pb concentration increased with increasing dose level for all treatments. Increasing blood Pb concentrations were not proportional to increased dose levels, indicating that the relationship between blood Pb and dose was nonlinear. It is also apparent from Fig. 1 that the plateaus for soil Pb dose response curves were different than the plateau for PbOAc. This observation is in agreement with previous studies (Freeman et al., 1992; Polák et al., 1996). The fractional absorption decreased as Pb dose increased regardless of the source of Pb. Further, the fractional absorption was lower for soil than for the PbOAc curve, resulting in lower plateau values for soil dose response curves than the plateau for PbOAc dose response curve (Fig. 1a). This being the case it would appear that the difference in response between PbOAc and soil Pb is a function of source characteristics not animal physiology.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Dose–response curves for blood Pb concentrations (a) for Pb acetate and untreated soil groups and (b) for soil groups. Correlations were run using data from individual animals. To reduce the number of data points on this graph, only the mean Pb concentration and dose from each group were plotted.

Mean blood Pb concentrations for the Pb acetate dose groups were significantly higher than the respective blood Pb concentrations for all the soil Pb dose groups. Of greatest interest in this study were the effects of soil amendment on Pb bioavailability as compared with the unamended soil, and the effects of Mn oxide + P addition compared with addition of P alone. The soil treatment effects on blood Pb concentrations are shown in Fig. 1b. Table 3 shows mean blood, liver, kidney, and femur Pb concentrations for the groups receiving soil. Mean blood Pb concentrations ranged from 83 to 134 µg L^-1 for the untreated soil diet and from 52 to 120 µg L^-1 for the P- and/or Mn oxide–treated diets (Table 3). When the soil was treated with P as either TSP or PR, significant reductions in blood Pb concentration compared with the untreated soil were found for TSP and PR at the medium dose, and for PR at the high dose. Blood Pb concentrations for the PR + cryptomelane (CRYP) group were significantly lower than the blood Pb concentrations for the PR group at the low and high dose levels, indicating that PR with cryptomelane was more effective in reducing Pb bioavailability than was PR alone. The general behavior of TSP with cryptomelane was similar to that of PR with cryptomelane; however, the reductions in blood Pb concentrations were less and not always significantly different than those with the corresponding TSP-alone treatment.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Dose–response curves for kidney Pb concentrations (a) for Pb acetate and untreated soil groups and (b) for soil groups. Correlations were run using data from individual animals. To reduce the number of data points on this graph, only the mean Pb concentration and dose from each group were plotted.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Dose–response curves for liver Pb concentrations (a) for Pb acetate and untreated soil groups and (b) for soil groups. Correlations were run using data from individual animals. To reduce the number of data points on this graph, only the mean Pb concentration and dose from each group were plotted.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Dose–response curves for bone Pb concentrations (a) for Pb acetate and untreated soil groups and (b) for soil groups. Correlations were run using data from individual animals. To reduce the number of data points on this graph, only the mean Pb concentration and dose from each group were plotted.

[1]

where a is intercept, b is plateau, and f is a curve-fitting parameter, or the linear equation:

[2]

where c is intercept and m is slope of the curve. The exponential curve allowed evaluation of data that appeared to reflect some degree of saturation (plateau) in response, while the linear curve allowed fitting of data that did not show evidence of a plateau response. The equation that yielded a clearly superior coefficient of determination (r²) was selected for curve fitting. If both equations fit the data approximately equally well, the linear model (simpler) was selected. The exponential model was best for the blood data and a linear model was best for the kidney, liver, and bone data. All correlations were significant at P 0.05 with r² values ranging from 0.2 to 0.9, while 17 of 24 correlations had r² > 0.6.

This study supports the finding of Polák et al. (1996) that RBA for blood was dose dependent; thus, calculating a single RBA value for blood is not possible. Schoof et al. (1995) used a blood Pb concentration of 60 µg L^-1 as a response to calculate RBA value because 60 µg L^-1 was approximately twice the detection limit for Pb in blood. In our study this blood Pb concentration was approximately 10 times the instrument detection limit and was used to calculate the RBA in a similar fashion. The RBA of soil Pb relative to Pb acetate was 44% at a blood Pb concentration of 60 µg L^-1 (Fig. 1a). This response was induced by a Pb intake of 0.79 mg (kg d)^-1 as untreated soil, but only 0.35 mg (kg d)^-1 as Pb acetate. Similarly, Schoof et al. (1995) assessed the RBA of Pb in soil from a former smelter site in Sandy, Utah that had a total Pb concentration of 2090 mg kg^-1. Sprague–Dawley rats were fed either soil Pb or Pb acetate in a purified diet at four different doses for 31 consecutive days. The RBA of soil Pb in their study was 41% at a blood concentration of 60 µg L^-1. Freeman et al. (1992) evaluated the RBA of Pb from Butte, Montana mine waste soils (810 or 3908 mg Pb kg^-1) in rats. Their RBA was 20% based on blood data, nearly half of that reported by Schoof et al. (1995) and this study. Unpublished X-ray absorption spectroscopy work from our laboratory revealed that most of the Pb in the untreated soil was present as adsorbed Pb and Pb carbonate, whereas test soils used by Freeman et al. (1992) contained considerable amounts of anglesite, a Pb sulfate mineral (28 and 53% of total Pb). It is likely that anglesite has restricted Pb solubility in the digestive tract compared with adsorbed Pb or Pb carbonate. Differences in absorption could be further complicated by physiological factors of the animals, such as age, weight, diet, feed consumption pattern, nutritional status, and genetic differences.

The RBA cannot be calculated in this fashion at the higher doses (e.g., at the plateau response) for the untreated soil and treated soils. The "relative bioavailability" (or, more accurately, relative response, RR) values from the blood data also were calculated by dividing the plateau value for a given dose–response curve for the diets containing soil by the plateau value of the PbOAc curve and multiplying by 100 (Table 4) . This allowed us to obtain one RR value for each curve. Relative response values from blood data for all treated soils (P or P + Mn oxide) were significantly lower than the untreated soil (Table 4). Moreover, P + Mn oxide treatments produced significantly lower RR values than either P source alone, supporting our previous observations using PBET data (Hettiarachchi et al., 2000). Several mechanisms could contribute to this reduction, including enhanced sorption of Pb onto Mn oxide surfaces in the presence of P; formation of insoluble Pb phosphates on the surfaces of Mn oxides; and reductive dissolution of Mn oxide in the acidic stomach followed by precipitation of P, Pb, and/or Mn as stable compounds. Davis et al. (1993) attempted to relate the Pb mineralogy of mine waste to low blood Pb levels observed in children living in Butte, Montana. They found through electron microprobe analysis that most of the Pb in these soils was present as Mn and Pb oxides (24%), Pb phosphates (23%), Pb oxide (12%), and Fe and Pb oxides (9%). Moreover, they hypothesized that soluble Pb-bearing phases, such as Pb oxides, converted to less soluble phosphate and ferromanganese solid solutions. Finally, they concluded that the bioavailability of Pb from these mine waste materials was possibly limited by the presence of Pb phosphates and ferromanganese oxides.

Despite the inconsistencies observed for the RR or RBA for the different exposure matrices (blood, bone, liver, and kidney), Table 4 clearly shows that soil Pb is less bioavailable than PbOAc and that P treatment significantly reduced the bioavailability of soil Pb regardless of tissue type. Further, it shows that P plus Mn oxide treatment was equally or more effective in reducing bioavailability and again that was true for all tissue types.

To develop a single bioavailability estimate for use in the Integrated Exposure Uptake Biokinetic (IEUBK) model results, Casteel et al. (1997) suggested a method for calculating a "best estimate." Later they renamed it to "point estimate" (unpublished report). Based on feeding studies done by swine they calculated a point estimate by a weighted average of RBA across all tissue types. Blood was given a greater weighting as they had multiple measurements of blood Pb in each pig over time versus a single, terminal measurement for other tissues and clinical relevance of blood Pb concentration in humans. We have calculated a point estimate by giving equal weight to RBA values and RR of all four tissue types and values were 33.5, 27.5, 25.5, 23.8, and 22% for control, TSP, PR, TSP + CRYP, and PR + CRYP soils, respectively. According to Stanek and Calabrese (1995), a child with pica behavior would ingest 10 g soil per day for those days with pica behavior. Therefore, our higher Pb dose levels (approximately 5 mg Pb kg^-1 d^-1) correspond to a pica child's daily Pb intake (assuming soil intake was 10 g d^-1 by a 10-kg child and soil Pb concentration of 5000 mg kg^-1). This level of Pb exposure can be considered as the worst-case scenario and our calculated point estimate using the RR for blood may only be applicable to those situations.

Correlation between In Vitro Bioaccessible Lead and In Vivo Data
Correlations between the in vivo (animal) model and in vitro model were evaluated between RBA and RR values calculated for each tissue separately (Table 4) and BAF_s or BAF_i values calculated for the same soil samples using in vitro bioaccessibility data. The calculated BAF_s values were 30.7% for untreated soil, 18.9% for TSP-treated soil, 18.2% for PR-treated soil, 17.9% for TSP + CRYP–treated soil, and 14.2% for PR + CRYP–treated soil. The calculated BAF_i values were 11.2% for untreated soil, 7.9% for TSP-treated soil, 9.4% for PR-treated soil, 5.5% for TSP + CRYP–treated soil, and 5.7% for PR + CRYP–treated soil. Figure 5 shows the relationships between the RBA calculated from each tissue and BAF_s.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Correlations between relative response (RR) or bioavailability (RBA) of Pb as determined by blood, kidney, liver, and bone dose–response curves and the bioavailability factor (BAFs) as determined from soil samples by the stomach phase of the physiologically based extraction test (PBET).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Correlations between relative response (RR) or bioavailability (RBA) of Pb as determined by blood, kidney, liver, and bone dose–response curves and bioavailability factor (BAFi) as determined from soil samples by the intestinal phase of the physiologically based extraction test (PBET).

Previous work has shown BAF values to decrease for Pb-contaminated soils on treatment with P or P + Mn oxide (Hettiarachchi et al., 2000, 2001). Based on in vitro model, those studies showed that combination of P (either TSP or PR) and Mn oxide is more effective in reducing "bioavailable Pb" as measured by in vitro model than either of materials alone for five different contaminated soils and mine wastes materials. While the data from this current study generally support the results from previous studies, they do not provide simple and straightforward answers to sort treatment efficacies. Further, reduced plant Pb uptake was observed with TSP and TSP and Mn oxide in most soil materials tested while effects of PR and PR and Mn oxide treatments on plant Pb uptake were minimal (Hettiarachchi and Pierzynski, 2002). Lack of effects on plant Pb uptake observed for PR treatments in our previous studies may be due to the fact that reactions between soil Pb and PR occur in the in vitro or in vivo model systems rather than in soil. The studies of Ruby et al. (1996) and Medlin (1997) were more successful in establishing a significant relationship between PBET results and an animal model. These latter two studies did not use contaminated soils that had been amended to reduce Pb bioavailability, had a larger number of data pairs in their correlations, and had soils with a wider range of Pb bioavailability, thus improving their chances of finding significant correlations. Overall, the issue of using PBET results to verify changes in Pb bioavailability after the addition of soil amendments remains inconclusive.

	ACKNOWLEDGMENTS

 
The assistance of Shajan Mannale and Shawn Taylor in collecting and preparing the animal fluids and tissues is gratefully recognized.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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