Relationship between Pb relative bioavailability and bioaccessibility in phosphate amended soil: Uncertainty associated with predicting Pb immobilization efficacy using in vitro assays

Abstract

In this study, an in vitro in vivo correlation (IVIVC) between Pb in vitro bioaccessibility (IVBA) and relative bioavailability (RBA) was explored to determine whether the efficacy of Pb immobilization in phosphate amended soils could be predicted using an in vitro approach. Mining/smelting impacted soil from Broken Hill, Australia (582–3536 mg/kg of Pb in the <250 μm soil particle fraction) was amended with Phosphoric Acid (PA), Mono Ammonium Phosphate (MAP) or Triple Super Phosphate (TSP) at Pb:P molar ratios of 1:1–1:5. Pb speciation in pre- and post-treated soil was assessed using X-ray Absorption Spectroscopy (XAS), Pb IVBA was measured using the Solubility Bioaccessibility Research Consortium (SBRC) assay (gastric and intestinal phases), and Pb RBA was determined in mice using blood Pb concentration as the bioavailability endpoint. XAS analysis revealed a 3.75–6.00 fold increase in the weighted % of Pb phosphates in soil containing >1000 mg/kg Pb while treatment effect ratios of 0.89–0.99 (SBRC-G), 0.09–0.71 (SBRC-I) and 0.27–0.80 (RBA) were observed in PA amended soil (Pb:P = 1:5). Although significant (p < 0.05) correlation were obtained between Pb RBA and IVBA (%) determined using SBRC-G (r = 0.64) and SBRC-I (r = 0.67), the strengths of the relationships were weak (r² = 0.41–0.45). This research highlights the complexities associated with the prediction of Pb RBA in phosphate amended soil.

Graphical Abstract

1. Introduction

Lead (Pb) exposure is a significant global concern due to its negative impact on neurological and cognitive development in children (Bihaqi, 2019; Mason et al., 2014; Chiodo et al., 2004; Jusko et al., 2007; Lanphear et al., 2005; Surkan et al., 2007). Main routes of Pb exposure in humans include ingestion (soil/surface dust, food, water), inhalation (ambient particulate matter or dust) and dermal absorption (Chen et al., 2019; Bradham et al., 2018; Karna et al., 2018; Kastury et al., 2018a, Kastury et al., 2018b; Khalili et al., 2019). A recent multimedia modelling analysis revealed that ingestion is the most important pathway for Pb exposure in the sensitive population (e.g. children <6 years old) (Zartarian et al., 2017). Immobilization of Pb via phosphate treatment is an in situ remediation strategy that has been documented to reduce Pb exposure in humans, swine, minipig, rats and mice (Chen et al., 2019; Bradham et al., 2018; Karna et al., 2018; Li et al., 2017; Juhasz et al., 2014; Brown et al., 2007; Casteel et al., 2006; Marschner et al., 2006; Ryan et al., 2004; Hettiarachchi et al., 2003; Brown et al., 2003; Maddaloni et al., 1998;). Phosphate amendments may promote the formation of Pb-phosphate species (e.g. pyromorphites and tertiary Pb phosphates), which exhibit low solubility in the acidic conditions of the stomach, thereby limiting absorption in the small intestines (Hettiarachchi et al., 2001; Lindsay, 1979; Ma et al., 1995; Nriagu, 1973; Scheckel et al., 2009). It has also been suggested that if Pb-phosphates are not formed in situ, they may form in vivo following solubilization of Pb and phosphate in the stomach and reaction in the small intestines (Juhasz et al., 2014).

Exposure may be assessed in vivo using Pb relative bioavailability (RBA; the fraction of soil-borne Pb absorbed into the systemic circulation following comparison to Pb absorbed from a Pb acetate reference dose) or by in vitro Pb bioaccessibility (IVBA) (the fraction of soil-borne Pb that is dissolved in simulated gastro-intestinal [GI] solutions) (USEPA, 2012). When assessing the efficacy of immobilization strategies, a reduction in Pb exposure may be expressed as the treatment effect ratio (TER), which is calculated by dividing Pb RBA or IVBA in treated soil by that in untreated soil. A value ≥1 signifies that the amendment was not successful at reducing Pb RBA or IVBA, while a TER < 1 indicates the occurrence of Pb immobilization. A range of TER values have been reported in the literature (0.03 to 1.16), with treatment efficacy dependent on phosphate source, soil pH, Pb concentration and competing ions (Juhasz et al., 2014; Scheckel et al., 2013).

Although the assessment of Pb RBA is the preferred method for predicting phosphate treatment efficacy, due to expense and ethical concerns, IVBA approaches are often used (Scheckel et al., 2013; Tang et al., 2004). A number of studies have reported the efficacy of phosphate amendments for reducing Pb IVBA (Bosso et al., 2008; Brown et al., 2007; Juhasz et al., 2014; Juhasz et al., 2016; Moseley et al., 2008; Scheckel et al., 2005), however, there is conjecture as to the most appropriate approach for predicting immobilization efficacy. While IVBA assays have been validated for the prediction of Pb RBA showing strong in vitro in vivo correlation (IVIVC) (e.g. Relative Bioaccessibility Leaching Procedure [RBALP] (Bannon et al., 2009; Drexler and Brattin, 2007), and relative bioaccessibility using the intestinal phase of the SBRC assay (Juhasz et al., 2009)), research into the development of an IVIVC to predict the efficacy of phosphate amendments for reducing Pb RBA in soil is limited. This is particularly important given that the USEPA does not recommend the use of the RBALP for predicting Pb RBA in phosphate amended soil (USEPA, 2012). Consequently, several researchers have suggested that a higher gastric phase pH (2.0–2.5) may be required because lower TER values are observed with increasing pH (Brown et al., 2003; Brown et al., 2007; Chaney et al., 2011; Obrycki et al., 2016). However, owing to a weak IVIVC at pH 2.5 (r² = 0.09) and the possibility of pyromorphite formation in vitro, Scheckel et al. (2005) contended that IVBA assays should not be undertaken at a pH > 1.5 and that the presence of Pb-phosphates in soil, using synchrotron based X-ray Absorption Spectroscopy (XAS) techniques, should be employed instead. Additionally, Bradham et al. (2018) reported that changes in Pb speciation may occur during the transit of amended soil in vivo, recommending the use of mouse bioassays. An alternative approach was proposed by Juhasz et al. (2016) who suggested that the TER using intestinal phase Pb IVBA of the SBRC or In Vitro Gastrointestinal (IVG) assay may be used to predict Pb RBA TER in phosphate amended soil (r² = 0.83 and 0.89 for SBRC and IVG respectively). However, the robustness of this relationship was questioned by Li et al. (2017) due to the small sample size (n = 6).

This study was conducted to explore an IVIVC between Pb RBA and IVBA. In order to achieve this aim, mining/smelting impacted soil from Broken Hill was treated with phosphate and the Pb RBA and IVBA was assessed. This data, in conjunction with data from Juhasz et al. (2016), was used in linear regression models to investigate if a predictive relationship exists between Pb RBA and IVBA for assessing the efficacy of phosphate amended soils.

2. Materials and methods

2.1. Soil collection and physico-chemical characterization

Twelve top soils (0–20 cm) were collected from Broken Hill, Australia (BHK1-BHK12), dried at 40 °C and sieved to <2 mm soil particle fraction. Samples were collected predominantly along King Street (Fig. S1), located in the southern part of the Broken Hill urban area. Sampling points were approximately 0.1 to 1.5 km away from the Line of Lode, which is the location of the historic ore body where mining/smelting activities have occurred. A subsample from each soil was sieved to <250 μm to obtain the incidentally ingestible fraction. To determine the pseudo-total elemental concentrations, 0.10 g of the <2 mm and the <250 μm soil particle size fractions were pre-digested overnight in 5 mL aqua-regia (70% HNO₃: 37% HCl = 1:3) (n = 3), followed by digestion in a MARS-6 microwave (CEM) using USEPA method 3051 (1998). Dissolved metal(loid)s were separated from solid residues by filtration (0.45 μm filter) and stored at 4 °C until analysis using ICP-MS following EPA method 6020A (1998). Water holding capacity, total organic carbon content (LECO TrueMac CNS) and pH was measured using the <2 mm soil particle size fraction (soil:water = 1:5 (m/v)) (n = 3).

2.2. Phosphate treatment

Four samples were selected (BHK5, BHK6, BHK10 and BHK11) for phosphate treatment to reflect a wide range of Pb concentrations exceeding the health based investigation level in residential areas (HILa) nominated by the National Environmental Protection Measure (NEPM) for the Assessment of Site Contamination, Australia (NEPM, 2013). Three sources of phosphates were utilized in this study: Phosphoric Acid (PA), Mono Ammonium Phosphate (MAP) and Triple Super Phosphate (TSP). Each treatment was applied at three application rates based on a Pb:P molar ratio of 1:1, 1:2.5 and 1:5. Because the aim of this study was to develop an IVIVC by combining the IVBA, RBA and TER results from this study to that reported in Juhasz et al. (2014), method of phosphate amendment, ageing period and the highest application rate in this study (Pb:P = 1:5) was kept similar to that described in Juhasz et al. (2014). Additionally, two lower Pb:P ratio (e.g. 1:1 and 1:2.5) was utilized in order to identify if a lower P application rate can achieve the same Pb immobilization efficacy so that adverse environmental effects due to P run-off may be minimized. PA and MAP were dissolved in MilliQ water and added to soil (350 g) to achieve 60% water holding capacity. During addition, soil was mixed for 10 min to ensure even distribution. TSP was ground, sieved to <53 μm, added to dry soil and rotated end-over-end (30 rpm) overnight. Subsequently, MilliQ water was added to achieve 60% water holding capacity with amended soils stirred for an additional 10 min. Amended soils were aged for two weeks (22 ± 2 °C) according to methods outlined in Juhasz et al. (2014), dried at 40 °C and the pH measured. In order to remove (excess) unreacted phosphate, soil was leached with 250 mL of natural rainwater collected from rooftop into rain water tanks, which was approximately one year’s worth of rainfall in Broken Hill according to the Bureau of Meteorology, Government of Australia (2018). Soils were re-dried at 40 °C, rotated end-over-end overnight to ensure homogeneity and the <250 μm particle size fraction recovered by sieving. Metal(loid) concentration in treated soil was determined by digesting samples (n = 2) using the methods described above.

Lead and Fe speciation was determined in pre- and post-treated soil (<250 μm soil particle size fraction) by X-ray absorption spectroscopy (XAS) using MRCAT beamlines 10-ID at the Advanced Photon Source of the Argonne National Laboratory, US (Kropf et al., 2010; Segre et al., 2000). Further details regarding XAS analysis can be found in the Supporting Information.

2.3. Assessment of Pb bioaccessibility and relative bioavailability

Lead IVBA was assessed in the <250 μm soil particle size fraction using both the gastric and intestinal phases of the Solubility Bioaccessibility Research Consortium (SBRC) assay according to Kelley et al. (2002). Percent IVBA was calculated using Eq. (1).

$$\mathit{\text{Pb}}\mathit{\text{bioaccessibility}}\left(\mathrm{\%}\right)=\left(\frac{\mathit{\text{SBRC}}-\mathit{G}\mathit{\text{or}}\mathit{\text{SBRC}}-\mathit{I}\mathit{\text{Pb}}(\mathit{\text{mg}}/\mathit{L})}{\mathit{\text{Total}}\mathit{P}\mathit{b}\left(\mathit{\text{mg}}/\mathit{\text{kg}}\right)}\right)\mathit{\text{x}}100$$ (1)

where: SBRC-G = concentration of Pb (mg/L) in solution following gastric phase extraction of amended or unamended Pb-contaminated soil, SBRC-I = concentration of Pb (mg/L) in solution following intestinal phase extraction of amended or unamended Pb-contaminated soil, total Pb = Concentration of Pb in contaminated soil (mg/kg) used in the in vitro assay.

In vivo studies utilized female Balb/C mice (aged 4–6 weeks) with Ethical and experimental approval given by the South Australian Health and Medical Research Institute Animal Ethics Committees (application number SAM268). Animal care was compliant with the Standard Operating Procedures of the South Australian Health and Medical Research Institute, and the Guidelines for the Care and Use of Laboratory Animals (Clark et al., 1997). In vivo Pb RBA was assessed in PA amended (Pb:P molar ratio of 1:5) and unamended soils using methods detailed in Smith et al. (2011). Briefly, a single soil suspension (20–108 μg Pb in 180 μL of Pb free water) was administered via gavage to fasting mice (20–25 g). Blood samples (~0.5 mL) were collected by cardiac puncture and stored at 4 °C until analysis by ICP-MS. Prior to analysis, blood was diluted ten-fold with biological diluent solution (1-butanol, 20 g/L; EDTA, 0.5 g/L; Triton X-100, 0.5 g/L; NH₄OH, 10 g/L) (Agilent Technologies, 2006). Lead bioavailability was assessed using pharmacokinetic analysis encompassing area under curve (AUC) following zero correction and dose normalization. The AUC for a Pb acetate oral dose was used for calculating Pb RBA in phosphate amended and unamended soil.

$$\mathit{\text{Pb}}\mathit{\text{RBA}}\left(\mathrm{\%}\right)=\left(\frac{\mathit{\text{AUC}}\mathit{\text{oral}}-\mathit{\text{soil}}}{\mathit{\text{AUC}}\mathit{\text{oral}}-\mathit{\text{Pb}}\mathit{\text{acetate}}}\times \frac{\mathit{D}\mathit{\text{oral}}-\mathit{\text{Pb}}\mathit{\text{acetate}}}{\mathit{D}\mathit{\text{oral}}-\mathit{\text{soil}}}\right)\mathit{\text{x}}100$$ (2)

where: AUC oral-soil = area under the Pb blood concentration time curve for an oral Pb-contaminated soil dose, AUC oral-Pb acetate = area under the Pb blood concentration time curve for an oral Pb-acetate dose, D oral-Pb acetate = dose of orally administered Pb acetate (mg/kg), D oral-Pb soil = dose of orally administered Pb in contaminated soil (mg/kg).

Treatment effect ratio (TER) for Pb IVBA (%) and Pb RBA (%) was calculated using Eq. (3)

$$\mathit{\text{TER}}=\frac{\mathit{\text{Pb}}\mathit{\text{IVBA}}\mathit{\text{or}}\mathit{\text{RBA}}\mathit{\text{in}}\mathit{\text{amended}}\mathit{\text{soil}}}{\mathit{\text{Pb}}\mathit{\text{IVBA}}\mathit{\text{or}}\mathit{\text{RBA}}\mathit{\text{in}}\mathit{\text{unamended}}\mathit{\text{soil}}}$$ (3)

2.4. Statistical analysis

For each soil, the difference in Pb concentration among the <250 μm soil particle size fractions (3 phosphate sources and 3 application rates) was determined using One-way ANOVA (α = 0.05). The reduction in Pb IVBA (gastric and intestinal phases) and Pb RBA between the amended and unamended soils was assessed using the t-test (α = 0.05).

During the assessment of IVIVC, data from Juhasz et al. (2014) and Juhasz et al. (2016) was used in conjunction with the data from this study because the former two studies were conducted according to the same methodologies in relation to phosphate amendment (Pb:P = 1:5) and the assessment of Pb RBA and IVBA. The strength of the relationship between the gastric and intestinal phases of IVBA and RBA, as well as between IVBA TER and RBA TER was assessed using coefficients of correlation (Pearson r). Linear regression was used to generate the line of best fit and r² value was used to measure the goodness of fit.

2.5. Quality assurance and quality control

Trace grade acids and a standard reference material: National Institute of Standards and Technology (NIST) 2710a were utilized during microwave digestion of soil samples. The accuracy of the aqua-regia digestion method was confirmed by a quantitative average recovery ± SEM of 5218 ± 84 mg/kg (94.5%) from NIST 2710a (certified total 5520 ± 30 mg/kg). During the analysis of metals from soil and blood, blanks, duplicates, check value recoveries and spikes were used. Blanks were below limit of detection (0.10 μg Pb/L) and the average recovery of spiked Pb (n = 16) was 94.2%. The average deviation from check value recoveries (n = 51) and duplicates (n = 16) were 4.36% and 4.28% respectively.

3. Results and discussion

3.1. Soil physico-chemical properties

Table S1 details the concentrations of trace and major elements in the <2 mm soil particle fraction of BHK soils with the concentration of Pb ranging from 215 ± 0.9 mg/kg (BHK8) to 8036 ± 651 mg/kg (BHK1). The screening level for Pb in residential area in Australia : Health based Investigation Level (HIL)a is 300 mg/kg according to the NEPM (2013). Apart from BHK7 and BHK8, which were the furthest away from the line of lode (Fig. S1), all other samples exceeded the HILa by a factor of 1.43 (BHK9) to 26.7 (BHK1). Among other notable co-contaminants, zinc (Zn) (HILa: 7400 mg/kg) and manganese (Mn) (HILa: 3800 mg/kg) screening levels were also exceeded in BHK1, while the concentration of cadmium (Cd) in BHK1 (18.1 mg/kg) and BHK3 (18.2 mg/kg) approached the HILa of 20 mg/kg.

Table 1 details the concentrations of trace and major elements in the <250 μm soil particle size fraction of the BHK soils. The concentration of Pb in this particle size fraction ranged from 267 ± 14 (BHK8) to 9930 ± 468 mg/kg (BHK1; Table 1), showing an enrichment of up to 1.42 fold (BHK11) compared to the <2 mm fraction. Four soils (BHK5, BHK6, BHK10 and BHK11) were selected for phosphate amendment studies to reflect a range of Pb concentration (582–3536 mg/kg) that were above the HILa value prescribed by NEPM (2013). The pH range of the selected four soils was 6.5 (BHK5) to 7.3 (BHK11), while organic carbon content ranged from 0.2 to 1.8%. In these soils, the concentration of P ranged from 260 ± 28 mg/kg (BHK6) to 1015 ± 14 mg/kg (BHK5), while the concentration of Ca ranged from 6100 ± 0.3 mg/kg (BHK5) to 42,900 ± 1.0 mg/kg (BHK11). BHK11 was treated with cracker dust to suppress Pb mobilization via aeolian transport (remediation trial of the central southern urban area of Broken Hill), which may explain the elevated Ca concentration. Iron (Fe) concentration was higher in BHK10 (35,800 mg/kg) than the other three samples included in the phosphate amendment study (23700–24,500 mg Fe/kg). Although the source of this variation in Fe concentration was not clear, it was within the range of the overall variability of the twelve soils sampled from King street (Table 1). Divalent cations, such as Ca, Fe and Mg, may have significant effect on Pb bioavailability, presumably due to competition between divalent metal cations during absorption (Gulson et al., 2018).

Table 1.

Average concentrations of trace and major elements (mean ± SEM) in the <250 μm particle fraction (n = 3).

	Trace element concentration (mg/kg)			Major element concentration (mg/kg)
	Pb	Mn	Zn	Al	Ca	Fe	K	Mg	P
BHK1	9930 ± 468	10,200 ± 300	7081 ± 221	17,700 ± 600	23,400 ± 400	23,700 ± 1300	4000 ± 100	3700 ± 100	14.2 ± 5.5
BHK2	3670 ± 178	3500 ± 200	3140 ± 139	23,200 ± 800	16,700 ± 900	30,600 ± 1500	7000 ± 200	5600 ± 300	808 ± 1.0
BHK3	6416 ± 453	4500 ± 400	5236 ± 514	28,700 ± 2600	27,400 ± 2200	31,300 ± 3000	7000 ± 400	6500 ± 500	1184 ± 105
BHK4	2107 ± 66	1800 ± 100	1844 ± 115	19,600 ± 1700	16,300 ± 700	24,100 ± 1600	5000 ± 100	5100 ± 300	535 ± 9.2
BHK5	3536 ± 51	2300 ± 200	3202 ± 231	20,500 ± 900	18,200 ± 200	24,500 ± 300	5200 ± 300	6200 ± 100	1015 ± 14
BHK6	582 ± 12	700 ± 100	540 ± 13	13,500 ± 400	6100 ± 300	23,700 ± 600	4800 ± 200	4200 ± 200	260 ± 28
BHK7	316 ± 10	700 ± 100	389 ± 2.7	24,300 ± 200	1400 ± 100	33,100 ± 1000	4400 ± 100	2900 ± 100	176 ± 3.2
BHK8	267 ± 14	1500 ± 400	456 ± 78	17,200 ± 100	21,700 ± 600	24,700 ± 300	5500 ± 100	2900 ± 100	357 ± 42
BHK9	452 ± 18	1000 ± 100	369 ± 11	16,500 ± 300	8000 ± 200	24,500 ± 200	4900 ± 100	3300 ± 100	186 ± 1.4
BHK10	1572 ± 16	1600 ± 100	1722 ± 26	21,400 ± 400	10,300 ± 100	35,800 ± 600	5700 ± 100	7600 ± 200	516 ± 9.6
BHK11	1097 ± 15	1500 ± 100	948 ± 25	17,000 ± 100	42,900 ± 1000	23,700 ± 400	4300 ± 100	4600 ± 100	419 ± 32
BHK12	1312 ± 42	1800 ± 200	1201 ± 1.0	16,000 ± 100	12,200 ± 100	38,000 ± 3400	7400 ± 200	5000 ± 100	477 ± 16

Lead (Table 2) and Fe speciation (Table S2) was similar across the four samples selected for phosphate amendment studies, with Pb predominantly sorbed to mineral oxides (45–58%), bound to organic matter (11–21%) or present as plumbojarosite (23–26%) and Pb-phosphates (5–12%). Fe was present as ferrihydrite (30–34%), goethite (11–15%), magnetite (0–12%) pyrrhotite (5–15%) and in clay minerals (31–47%). Although Pb and Fe speciation may significantly affect its bioavailability, and in turn Pb bioavailability, there was no major difference in the speciation among the four samples prior to phosphate amendment.

Table 2.

Change in Pb speciation (weighted %) in the < 250 μm soil particle fraction as a result of phosphoric acid treatment at a molar ratio of Pb:P = 1:5.

		Pb speciation (weighted %)					R- factor
		Pb bound to clay or mineral oxides	Organic bound Pb	Anglesite	Plumbojarosite	Pb phosphate (pyromorphite + Pb3(PO4)2
BHK5	Pre-treatment	58	13	0	24	5	0.0066
	Post-treatment	17	58	0	0	25	0.0237
BHK6	Pre- treatment	49	16	0	23	12	0.0600
	Post- treatment	31	69	0	0	0	0.1388
BHK10	Pre- treatment	57	11	0	26	6	0.0111
	Post- treatment	42	0	22	0	36	0.0415
BHK11	Pre- treatment	45	21	0	23	12	0.0222
	Post- treatment	0	0	21	0	45	0.0230

Changes in Pb speciation in PA amended soil are detailed in Table 2. The formation of Pb phosphates is considered to occur within a pH range of 3–9 but may be optimized by lowering soil pH (Karna et al., 2018; Scheckel et al., 2013). Therefore, when PA was added, the decrease in pH was expected to promote the formation of Pb-phosphates. BHK5 exhibited lower buffering capacity compared to the other three samples, evidenced by a decrease in soil pH from 6.5 to 5.3 after the addition of phosphate amendments, while the change in pH in the other three soils was <1 unit. The decrease in pH may have resulted in the desorption of mineral sorbed Pb (Pb oxides and adsorbed to clays) and the dissolution of plumbojarosites, in favor of the formation of Pb-phosphates and re-sorption of Pb onto organic matter. In contrast, a smaller change in the weighted percentage of Pb sorbed to oxides and clays was observed in BHK10, presumably because of a small change in pH (from 7.2 to 6.9) as a result of the PA amendment. However, an increase in the weighted percentage of Pb phosphates was observed in the PA amended soil (36%) compared to BHK10 pre-amendment.

In BHK6 and BHK11, pH was not significantly affected by PA amendment (change from 6.9 to 6.5 and from 7.2 to 7.3 respectively). Although, it is not clear why Pb phosphates in unamended BHK6 were destabilized after PA application, the high buffering capacity of BHK11 may have caused localized pockets of low pH in BHK11 when PA was added, resulting in Pb desorption from oxides and clays. Compared to BHK6, a higher concentration of P was added to BHK11, which may have shifted the equilibrium towards the formation of Pb-phosphates in BHK11.

The two factors that govern the formation of Pb phosphates are the solubility of Pb and P (Scheckel et al., 2013). When both of these elements are in solution, the reaction to form chloropyromorphite may occur instantaneously (Scheckel and Ryan, 2002). However, when a soluble source of P is used, Pb dissolution from soil may be considered the rate limitation step (Ryan et al. 2001). Hence, the short ageing time (<1 month) utilized in this study to keep the incubation conditions similar to Juhasz et al. (2014) for the investigation of IVIVC may limit the extrapolation of the results observed in this study to a real environmental scenario. However, Scheckel and Ryan (2004) demonstrated that the reaction to form Pb phosphates may complete within a short time frame (e.g. within 3 months) in a field trial using TSP and PA, which suggests that because water soluble phosphate sources are used, Pb phosphate formation obserevd in this study using a short ageing period may not be significantly different from a field trial, although longer ageing time may be required to confirm the reproducibility of the results.

3.2. Pb IVBA in untreated BHK soils

Gastric and intestinal phases of the SBRC assay (SBRC-G and SBRC-I respectively) were utilized to assess Pb IVBA in BHK5, BHK6, BHK10 and BHK11 prior to the addition of phosphate amendments. Lead IVBA following SBRC-G extraction ranged from 67.4 ± 0.5% (BHK11) to 88.4 ± 4.9% (BHK10). Despite exhibiting a wide range, there was no significant difference among the SBRC-G Pb IVBA values for unamended soils (ANOVA, α = 0.05). Presumably, this was due to the similarity in Pb speciation (Table 2), which influences the extent of Pb solubilization in vitro at pH 1.5 (Scheckel et al., 2013). Yang and Cattle (2015) reported Pb IVBA values ranging from 23.7 to 89.3% in Broken Hill topsoil, with an average value of 61.2 ± 14.0% using the RBALP assay (SBRC-G). The wider Pb IVBA range reported by Yang and Cattle (2015) may be attributed to a more dispersed soil collection area, (southern and northern region of Broken Hill urban area), with soils containing greater variation in Pb speciation (e.g. PbS, PbSO₄, PbCO₃).

When in vitro assays were modified to reflect SBRC-I phase conditions, Pb IVBA in the unamended soil was reduced by 4.19 (BHK11; 16.1 ± 0.9%) to 16 fold (BHK6; 4.96 ± 0.03%). The decrease in Pb IVBA may be attributed to Pb precipitation and/or re-adsorption onto soil particles resulting from the increase in pH from 1.5 to 7.0 (Juhasz et al., 2009; Li et al., 2018; Smith et al., 2011). During the increase in pH to 7.0, oversaturation of hydrolyzed Fe species may occur, resulting in the precipitation of amorphous Fe phases (Mercer and Tobiason, 2008), onto which Pb may co-precipitate (Smith et al., 2011). Lower Pb IVBA was observed in BHK6 presumably due to the presence of higher Fe concentrations compared to Pb in this soil.

3.3. The effect of phosphate amendment on Pb IVBA

Following phosphate amendments, BHK5, BHK6, BHK10 and BHK11 were aged for 2 weeks, leached in natural rainwater, re-sieved and the total elemental concentration in the <250 μm soil particle size fraction was assessed. While some variability in Pb concentration was observed, values were within 10% of those determined pre-treatment.

When Pb IVBA, determined using the SBRC-G phase in phosphate amended soils, was compared to the values obtained for unamended soils, no significant decrease (p > 0.05) was observed for BHK10 for either phosphate source or application rate (TER ranging between 0.93 and 1.08, Fig. 1). Although a 12.5 to 14.8% decrease in Pb IVBA was observed in BHK6 treated with MAP and TSP (Pb:P = 1:5) (TER 0.88 and 0.85), the reduction was not significant, which may be attributed to the high variability in Pb IVBA results obtained for the unamended soil. For PA and TSP treated BHK5 and MAP treated BHK11, significant reductions in SBRC-G Pb IVBA (p < 0.05) were observed (10.3, 9.6 and 9.5% reductions respectively with TERs of 0.96, 0.93 and 0.98).

Fig. 1.

TER of Pb IVBA (gastric phase ■ and small intestinal tract phase ■) determined using SBRC assay in the phosphate amended soils (BHK5, BHK6, BHK10 and BHK11). Phosphoric Acid (PA), Mono-Ammonium Phosphate (MAP) and Triple Super Phosphate (TSP) were applied at three application rates based on Pb:P molar ratio of 1:1, 1:2.5, 1:5 in the < 250 μm soil particle fraction.

According to Scheckel et al. (2005), the main factor influencing Pb IVBA results in phosphate amended soil was the gastric phase pH, where lower TER may result from using a pH >1.5 (e.g. TER was 0.80–1.01 at pH 1.5, TER was 0.63–1.02 at pH 2.0 and TER was 0.35–0.86 at pH 2.5. The overall modest decrease in Pb IVBA observed in this study is similar to values reported in Scheckel et al. (2005) using a pH of 1.5. Although Pb-phosphates are considered to exhibit low solubility in the acidic conditions of the stomach (Scheckel et al., 2013), the results of SBRC-G IVBA extractions suggest that the majority of Pb species were solubilized in vitro, presumably due to the low pH, including Pb-phosphates that were formed in situ following the addition of phosphate amendments. It is possible that Ca may also be incorporated into the Pb-phosphate structure which may result in destabilization of Pb-phosphates formed in situ (Mavropoulos et al., 2002) and solubilization under gastric phase conditions.

In contrast to the modest or no significant decrease in the SBRC-G phase, Pb IVBA TER decreased in the SBRC-I phase in most phosphate amended soils, with the greatest reduction being evident for the highest phosphate application rate (from 0.09 [BHK5] to 0.71 [BHK6] at a Pb:P molar ratio of 1:5) (Fig. 1). The decrease in Pb IVBA when assessed using the SBRC-I phase was, in part, a result of precipitation/reabsorption of Pb and co-precipitation with Fe that was previously discussed for unamended samples. Additionally, during gastric phase extraction, Pb solubilization increased its availability to react with P once the pH was increased to 7 (i.e. where H₂PO₄⁻¹ dominates) following transition to the intestinal phase. This may have caused the formation of Pb-phosphates in vitro and lowered the TER in phosphate amended soils.

The results of this study demonstrated that the highest application rate of Pb:P = 1:5 was the most effective in all four samples. The greatest reduction occurred using PA amendment, followed by TSP, while MAP amendment was able to reduce Pb IVBA in BHK5, BHK10 and BHK11 (Table S3). The higher efficiency of PA may be attributed to the greatest reduction in pH, which is thought to promote the formation of Pb phosphates (Scheckel et al., 2013). Although the ideal molar ratio of Pb:P required for pyromorphite formation is 3:5; it was suggested in Scheckel et al. (2013) that higher P application may be necessary to compensate for the presence of other cations that may compete for sorption sites with Pb (e.g. Ca, Al, Mg). Comparison of the reduction in Pb IVBA in the SBRC-I stage showed that utilizing a molar ratio of 1:5 or higher may be necessary for the stabilization of Pb in Broken Hill soils and that PA amendment may be the most effective, followed by TSP.

Although studies investigating the efficacy of Pb immobilization using phosphate amendment using intestinal phase extraction are limited, reduction of Pb IVBA has previously been reported by Tang et al. (2004) and Juhasz et al. (2016). In a recent study, Juhasz et al. (2016) reported SBRC-I phase TER of 0.0005–0.55 when Pb contaminated soils (smelting, mining and shooting range impacted) were amended with PA or Rock Phosphate (RP). The range of TER reported for the same soils during intestinal phase extraction using PBET, IVG, Unified Bioaccessibility Research Group of Europe Method (UBM) and Deutsches Institut für Normung (DIN), were in the range of <0.003 to 0.84 (Juhasz et al., 2016). Speciation analysis of the post-assay residuals identified that a fraction of Pb-phosphates, formed in situ (e.g. hydroxypyromorphite or chloropyromorphite), became solubilized in vitro during gastric phase extraction and subsequently were converted into Pb-phosphate species during the intestinal phase extraction. Additionally, Juhasz et al. (2014) demonstrated that Pb-phosphates may solubilize in the stomach and reform in the small intestines when phosphate amended soils were assessed using an in vivo mouse model. Therefore, the lowest TER values obtained during the SBRC-I phase for each sample (i.e. PA amendment at Pb:P molar ratio = 1:5) were selected for further in vivo studies.

3.4. The effect of phosphate amendment on Pb RBA

To investigate the relationship between IVBA and Pb RBA outcomes in phosphate amended soils, unamended and PA amended samples (Pb:P = 1:5) were assessed using a mouse model with Pb RBA determined using blood Pb concentration as the bioavailability endpoint. Lead RBA in unamended soil ranged from 16.1 ± 1.2% (BHK6) to 54.4 ± 6.5% (BHK10) (Fig. 2). Bioavailability studies using mining/smelting impacted soils have reported low Pb RBA values (<30%) because of the presence of poorly soluble Pb species, for example, PbS, hydroxypyromorphite or Pb-phosphates (Juhasz et al., 2014; Ruby et al., 1996; Schoof et al., 1995). Because of the similarity in Pb speciation among the four unamended soils, the most likely contributor to Pb RBA variability is competition between divalent cations for absorption into the systemic circulation. The absorbtion of Pb²⁺ from the intestines into blood is thought to be mediated by Divalent Metal Transporter 1 (Elsenhans et al., 2011) and calcium binding proteins (e.g. calbindin) (Bressler et al., 2007). Therefore, the presence of elevated concentrations of divalent cations, particularly Zn, Ni and Ca, may lower absorption of Pb into blood (Gulson et al., 2018). Table 1 illustrates that among the four soils, BHK10 contained the lowest concentration of Ca, which offered the least competition for Pb absorption (i.e. high Pb RBA of 54.4 ± 6.5%), whereas BHK11 contained the highest concentration of Ca which may be responsible for the lower Pb RBA of 23.1 ± 5.3%. Similarly, compared to the Pb concentration, BHK6 contained the highest concentration of Ca + Fe, resulting in the lowest Pb RBA (16.1 ± 1.2%).

Fig. 2.

Comparison of Pb relative bioavailability (RBA) using the blood Pb concentration in BALB/c mice following gavage in the unamended (■) and PA amended (■) samples using a molar ratio of Pb:P = 1:5. Significant reduction in Pb RBA between the amended and unamended samples using t-tests are indicated as * = p < 0.05.

Although Pb RBA was reduced in all soils as a result of PA amendment, the reduction was significant in BHK5 and BHK10 (p < 0.05), which may be attributed to the 5 and 6 fold increase in Pb-phosphate formation compared to the unamended counterpart. Pb RBA was reduced 3.6 fold in BHK5 (from 42.5 ± 6.1% to 11.8 ± 1.6%) and 2.5 fold in BHK10 (from 54.4 ± 6.5% to 21.8 ± 4.2%), with TERs of 0.27 and 0.41 respectively. Although a 44.7% decrease in Pb RBA was observed in BHK6 (from 16.1 ± 1.2% to 8.9 ± 2.5%, TER 0.55), the reduction was not significant (p > 0.05), which may be attributable to the high variability associated with Pb RBA assessment in the amended sample. In spite of a 3.8 fold increase in the weighted percentage of Pb-phosphates in BHK11, a modest 1.2 fold reduction in Pb RBA was observed (from 23.1 ± 5.3% to 18.6 ± 0.8%; TER: 0.81). As mentioned previously, BHK11 contained elevated Ca concentration (42.9 g/kg), which was 2.3 to 7.0 fold higher than the other soils. Table S3 details the molar ratio of (Pb + Ca):P that were observed in the <250 μm soil particle size fraction after phosphate application and leaching with rainwater. Although the ratio of (Pb + Ca):P in BHK5, BHK6 and BHK10 ranged from 0.26 to 0.52, that in BHK11 was 0.06. As mentioned previously, high Ca concentrations may reduce Pb absorption due to competition for uptake via the divalent cation transporter 1 (Gulson et al., 2018). Therefore, it is likely that Ca in BHK11 may have reduced Pb RBA similarly in both the amended and unamended soils, minimizing the effect of added phosphate. BHK11 was sampled in an area where cracker dust had been applied for soil capping (personal communication), which has been reported as an effective strategy to suppress Pb exposure in Broken Hill top soil (Yang and Cattle, 2017). Cracker dust is a by-product of crushed volcanic rocks (e.g. granite and basalt) and has been characterized as “lead-poor”, with an expected lifespan of 100 years if not disturbed. Although this study included only one sample that was impacted by cracker dust, the results warrant further investigation into the optimum rate of phosphate application for different regions of Broken Hill based on soil physico-chemical properties. Conceivably, the effectiveness of soil capping as a risk management strategy may be enhanced by phosphate treating cracker dust, or any similar soil capping material before capping, in order to promote the formation of poorly soluble Pb species in situ over time.

3.5. Correlation between Pb RBA and IVBA in phosphate amended soil using SBRC assay

Lead immobilization in soil using phosphate amendments may be a cost-effective and simple strategy for minimizing childhood Pb exposure compared to soil removal and capping (Henry et al., 2015; Hettiarachchi and Pierzynski, 2004; Udeigwe et al., 2011). However, owing to the heterogeneity of soil chemistry (e.g. soil pH, Pb mineralogy, cation concentrations), site specific strategies may be required which involve investigation into the suitability of phosphate source and application rates using bench top studies and in vivo bioassays (Scheckel et al., 2013). Using IVBA to investigate treatment efficacy may be a cost effective and rapid measure, reducing the number of bioassays required for each site. Although the SBRC-G phase (otherwise known as RBALP), may be used to predict Pb RBA in unamended Pb-contaminated soil due to a strong IVIVC, its capacity to predict Pb RBA in phosphate amended soil is currently not well-established. Scheckel et al. (2005) demonstrated that above pH 1.5, the presence of excess phosphate may induce the formation of pyromorphites in vitro. Conversely, Juhasz et al. (2014) demonstrated that when phosphate amendments and Pb-contaminated soil were gavaged sequentially, pyromorphite may form fortuitously in vivo (in the small intestines) following solubilization of Pb and P in the stomach. Due to a strong correlation between Pb IVBA TER (using the intestinal phase of SBRC and IVG assays) and Pb RBA TER (using an in vivo mouse model using blood Pb as an endpoint), (r² = 0.83 and 0.89 respectively), Juhasz et al. (2016) proposed that the efficacy of phosphate amendments for reducing Pb RBA could be predicted using IVBA TER. However, a limitation of the study of Juhasz et al. (2016) was the small data set (n = 6) used for IVIVC analysis.

In order to investigate whether the relationship described in Juhasz et al. (2016) holds with additional contaminated soils, Pb IVBA and RBA data from the four PA amended soils this study and the six RP and PA amended soils in Juhasz et al. (2014) and Juhasz et al. (2016) were combined and the relationship between in vivo and in vitro data was compared. The soils used in the studies by Juhasz et al., 2014, Juhasz et al., 2016 varied from the present study in terms of source, Pb concentration, speciation and the amendments applied, which are detailed in Juhasz et al. (2014). Briefly, the sources of Pb contamination in these soil samples were smelting (PP2: pH 7.4, 1738 ± 27 mg/kg of Pb), non-ferrous slag application (SH15: pH 6.9, 656 ± 82 mg/kg of Pb) and shooting range (SR01: pH 5.7, 8709 ± 298 mg/kg of Pb). Pb species in PP2 were distributed between mineral sorbed (55%) and organic bound Pb (45%), while Pb in SH15 was distributed between mineral sorbed (52%) and Pb-phosphates (48%). In contrast, Pb in SR01 existed as PbO (77%), Pb carbonates (11%) and mineral sorbed Pb (13%). Each soil in Juhasz et al., 2014, Juhasz et al., 2016 was amended with PA and rock phosphate. Comparing the paired in vivo and in vitro (SBRC-G and SBRC-I) values from Juhasz et al., 2014, Juhasz et al., 2016 to that generated in this study was feasible as the method of phosphate treatment and the assessment of Pb IVBA and RBA were identical these three studies.

Fig. 3a shows a significant correlation between Pb RBA (%) and Pb IVBA (%) as determined using SBRC-G (coefficients of correlation, r = 0.64 with the 95% confidence interval between 0.02 and 0.91, p = 0.04). Similarly, the correlation between Pb RBA (%) and Pb IVBA (%) determined using SBRC-I (Fig. 3b) was significant (r = 0.67 with the 95% confidence interval between 0.08 and 0.92, p = 0.03). As illustrated by the 95% confidence intervals in Fig. 3a and b, the range of predicted Pb RBA (%) values using SBRC-G and SBRC-I values of 64.8% and 1.37% in BHK5 would be 14.0–14.8% (mean RBA of 14.4%) and 9.63–9.73% (mean RBA of 9.71%) respectively, while the observed Pb RBA was 11.8% (Table S4). A stronger correlation was observed when the relationship between Pb RBA and Pb IVBA (SBRC-G) TER were plotted (Fig. 3c, r = 0.71 with the 95% confidence interval between 0.14 and 0.93, p = 0.02).

Fig. 3.

Relationship between Pb IVBA using SBRC assay (gastric phase: ● and intestinal phase: ■) and Pb RBA using the blood Pb concentration in BALB/c mice following gavage. (a) and (b) depicts the relationship between Pb IVBA (%) and Pb RBA (%), (c) and (d) depicts the relationship between Pb IVBA TER and RBA TER. Paired Pb IVBA and RBA data from four PA amended soils (n = 4) from this study were combined with three soils, each amended with RP and PA (n = 6) from Juhasz et al., 2014, Juhasz et al., 2016. The in vitro in vivo relationship is indicated by the solid black line and described by the equation, while the r² value demonstrates the strength of the relationship. The vertical error bars represent SEM in RBA.

Fig. 3a–d suggests the existence of a correlation between Pb RBA and SBRC assay, which can be used to predict Pb RBA in phosphate amended soil using the SBRC assay. However, the coefficient of determination (r²) using both % bioaccessibility and TER approaches may be argued to be weak (r² of 0.4–0.5), suggesting that between 40 and 50% of the variations in Pb RBA may be predicted by IVBA. The most likely reason for the high uncertainty indicated by the low r² values is the heterogeneity of soil chemistry and phosphate sources (e.g. Pb speciation, soil pH, type of phosphate amendment, impact of phosphate amendment on soil pH, presence of elements/phases competing for phosphate). For example, the predicted Pb RBA in BHK5 and BHK6 using the equations displayed in Fig. 3a (Table S4) were similar (14.4% and 15.1% respectively), however, observed Pb RBA was lower in BHK6 (8.9%) than BHK5 (11.8%), which most likely resulted from the differences in Pb and Fe + P concentration in BHK6. Similarly, average Pb RBA predicted in the PA and RP treated SH15 was similar (12.6% and 11.0%), however, Pb RBA in the PA treated SH15 was approximately half of that in RP treated SH15. This observed difference in Pb RBA between the two treatments using the same sample may be attributed to the fact that liquid source of phosphate is more effective in immobilizing Pb than less soluble RP counterpart, a phenomenon that is not reflected by the in vitro assay.

Furthermore, the addition of the paired values of this study to that in Juhasz et al. (2016) generated a weaker overall r² value using SBRC-G phase [from 0.84 using SBRC-G (%) in Juhasz et al., 2016 to 0.41, from 0.6 SBRC-G TER to 0.45]. Although the overall r² value using SBRC-I phase improved using % bioaccessibility (from 0.23 to 0.45), it decreased when the TER approach was used (from 0.83 to 0.43). Additionally, the relationship appeared to be driven by two data points exhibiting the lowest Pb RBA and SBRC-G TER, while the remaining 8 data points had similar SBRC-G TER, ranging between 0.8 and 1.0. Particularly, the two lowest data points were derived from shooting range soils, where highly soluble forms of Pb (e.g. 77% PbO and 11% Pb carbonates) dominate the Pb speciations, while the other six samples were dominated by mineral sorbed and organic bound Pb, plumbojarosites or Pb phosphates which exhibit lower bioavailability. It is therefore conceivable that when Pb RBA values using soils from different sites are compared, the influence of soil chemistry and Pb absorption in vivo in phosphate amended soil may not be replicated in vitro.

As reviewed in Henry et al. (2015), the criteria that may be applied for an initial assessment of an IVIVC suggested by Wragg et al. (2011) include an r value of >0.8 and a slope between 0.8 and 1.2. In this regard, despite exhibiting a significant correlation, the equations reported in Fig. 3 does not meet these suggested criteria. The uncertainty of using IVIVC which is conducted at pH > 1.5 has been previously expressed in a critical review by Scheckel et al. (2013). This study demonstrates that predictive capability of immobilization efficacy using equations derived from IVIVC at pH 1.5 (RBALP or the gastric phase of SBRC assay) may also be problematic. Therefore, detection of Pb phosphate species using synchrotron X-ray based spectroscopic methods, followed by in vivo investigations may be the best predictor of Pb immobilization efficacy, pending further research using larger studies.

4. Conclusion

This study highlights the uncertainty associated with predicting Pb RBA in phosphate amended soil using IVBA owing to the complexities associated with Pb phosphate formation in situ and in vivo. Because of an indiscriminant Pb solubilization in the gastric phase at pH 1.5, the effect of soluble vs non-soluble sources of phosphate was not observed in vitro. Complexity was also evident in the stability in Pb-phosphates formed in situ during solubilization in the gastric phase extraction in vitro, which may not necessarily be reflected in vivo. In addition to soil chemistry, biological factors that impact the absorption of Pb in vivo (e.g. competition between Pb and cations that is not reflected in vitro) were not observed in vitro and may complicate IVIVC. Although this study highlights that a correlation may exist between Pb RBA and IVBA, weak r² value in the equation reported in this study limits the application of equations derived in this study in predicting Pb RBA. Additionally, the short period of ageing [e.g. two weeks incubation with phosphate amendment in this study and four weeks in Juhasz et al., 2014], may not reflect the longer term Pb immobilization efficacy. However, in vitro studies may be applied to optimize the phosphate application rates in a particular soil using the same source of phosphate, since the factors that affect Pb immobilization in vitro is constant in this case. Further investigation using larger studies with different animal models and end points (e.g. Pb concentration in liver, kidney and bones) are urgently required to elucidate the impact of the aforementioned factors in predicting Pb RBA in phosphate amended soils.