United States
Environmental
Protection Agency
Formation of plumbojarosite (PLJ) reduces bioavailability of soil-borne lead (Pb)
Ranju Kama1, Matt Noerpel1, Clay Nelson2, Brittany Elek3, Karen Herbin-Davis3, Gary Diamond4,
Karen Bradham2, Kirk Scheckel1, David J. Thomas3
1. Center for Environmental Solutions & Emergency Response, Cincinnati, OH; 2. Center for Environmental Measurement & Modeling; 3. Center for Computational Toxicology & Exposure, Research
Triangle Park, NC, Office of Research and Development, U.S. Environmental Protection Agency; 4. SRC. North Syracuse, NY	David J. Thomas-
a.qov: 919-541-4974
Abstract
Methods
Results
Exposure to Pb during early life has long-lasting adverse effects on health. Ingestion of Pb-contaminated soil is a
major route for exposure of children to this toxic metal. Soil remediation procedures that alter physiochemical
properties of soil-borne Pb can limit exposure by reducing gastrointestinal Pb uptake. A novel approach for
remediation of soil Pb uses addition of iron (Fe) sulfate and application of heat to promote formation of PLJ, a
poorly soluble Pb-Fe sulfate compound. Here, two Pb-contaminated soils and samples of a low-lead soil spiked with
various Pb compounds (i.e., carbonate, chloride, phosphate, or sulfate) were treated to convert native Pb species to
PLJ. We used a mouse assay to examine tissue Pb distribution after ingestion of diets amended with untreated or
treated soils. Bone and blood Pb levels were determined to evaluate uptake across the gastrointestinal barrier. For
both Pb-contaminated soils and all Pb compounds, bone and blood Pb levels were significantly lower (P<0.001,
student t-test) in mice that consumed diets amended with treated soils than in mice that consumed diets amended
with untreated soils. After treatment, estimated relative bioavailability (RBA) of Pb in both soils and for all Pb
compounds were reduced by more than 90% compared to RBA estimates for untreated soils or compounds. X-ray
absorption spectroscopy was used to determine Pb species in soil-amended diets and in feces excreted by mice
consuming these diets. Treatment of Pb-contaminated soils or Pb compounds consistently converted more than
90% of all Pb species in these materials to PLJ. Speciation of Pb in feces from mice fed diets containing soils or Pb
compounds treated to promote PLJ formation found no evidence that ingested PLJ underwent chemical
transformation during transit of the gastrointestinal tract. This evidence suggests that formation of PLJ could be an
effective strategy to reduce the RBA of Pb in soil and minimize this medium's role as a source of exposure to Pb in
young children. (This abstract does not represent U.S. Environmental Protection Agency policy.)
Background
Exposure of children to Pb has profound and long-lasting health effects1. Extensive release of Pb into the
environment has resulted in widespread and persistent contamination of urban soil and dust with this toxic metal2.
Although Pb levels in some media (e.g., air and food) have declined in recent decades34, soil and dust Pb levels
remain elevated. As a consequence, Pb in soil and dust have emerged as significant sources of Pb exposure in
one-to-six year-old children5. Therefore, reducing exposure of children to Pb through soil or dust ingestion is an
important public health goal6.
Removal of Pb-contaminated soil and its replacement with uncontaminated soil has been a common and effective
approach to reduce Pb exposure in children7-8. However, soil removal and replacement are expensive and
complicated procedures that can be difficult to implement in some settings. If removal and replacement of
contaminated soil are not feasible, an alternative approach is to reduce the bioavailability of Pb present in soil and
dust. Operationally, bioavailability is defined as the amount of a contaminant absorbed into the body following skin
contact, ingestion, or inhalation. In children, age-dependent hand to mouth activity creates a unique and significant
pathway for ingestion of Pb in soil and dust9. Reducing the bioavailability of Pb in soil or dust ingested by hand to
mouth transfer can have the salutary effect of reducing the internal dose of this toxic metal.
The bioavailability of Pb is a function of physical and chemical properties of the matrix in which it is ingested and
can vary from 0 to 100%. One approach to reducing the bioavailability of Pb is in situ solidification and stabilization
to reduce the solubility of Pb in soil1011. Thus, addition of phosphorus as phosphate (P) to Pb-contaminated soil can
promote formation of stable and relatively insoluble Pb-P species. Although Pb-P interactions can reduce soil Pb
bioavailability12-14, the limitations of this approach and its applicability to a variety of soil types have not been
systematically evaluated. As part of an effort to explore novel options for stabilization of soil Pb, we have evaluated
the effect of formation of plumbojarosite, an insoluble iron-sulfate mineral, on the bioavailability of soil Pb. Here, we
used adult female mice as the test species in assays to estimate Pb bioavailability in treated and untreated soils15.

t
t t t t
Test materials - Soil 1 originated in an orchard where lead arsenate had been used as a herbicide. Soil 2 was
collected from a former mining site. A soil with low Pb content was spiked with Pb minerals for bioavailability
studies. All test soils were sieved to produce a < 250 |jm fraction for testing. Pb (lead acetate trihydrate, Sigma-
Aldrich, St. Louis, MO) was used as the reference compound in all studies.
Soil treatments - In soils 1 and 2 and in Pb-mineral spiked soils, formation of plumbojarosite, a poorly soluble Pb-
Fe sulfate compound, was promoted by addition of iron (Fe) sulfate and application of heat for 8 or 67 hours.
Test Diets - Test materials, (untreated or treated soils, Pb acetate) were incorporated by the vendor (Dyets,
Bethlehem, PA) into powdered AIN-93G purified rodent diet. This diet meets the nutritional requirements of rapidly
growing immature rodents.
Mouse origin and maintenance - Four week-old female C57BL/6 mice (Charles River) were acclimated for 12 to
14 days in a 12 hour light—12 hour dark photocycle at 20-22°C with free access to rodent diet and tap water before
use in the mouse assay.
Mouse assay - In this assay, the bioavailability of Pb in a test
material (e.g., soil or mineral) was calculated using data on Pb levels
n bone and blood of mice that consumed AIN-93G rodent diet that
contained the test material. The diagram shows assay design. Mice
were placed in metabolic cages (3 per cage) on the morning of day 1
with free access to amended AIN-93G rodent diet and drinking water.
Daily food and water consumption were measured and a cumulative
feces sample was collected for each cage. Mice were euthanized by
C02 anesthesia on day 9. Pooled heparinized blood samples were
collected for Pb analysis. Mouse carcasses were defleshed by
dermestid beetles; defleshed skeletons were pooled by cage for Pb
analysis.
Lead analysis - Pooled skeletons from each cage were homogenized by freeze grinding. Bone samples were
digested in ultra-high purity nitric acid in a closed vessel microwave reaction system. Digested samples were diluted
to 5 to 10% nitric acid with deionized water. Total Pb in acid-digested bone samples were determined by Inductively-
Coupled Plasma-Mass Spectrometry (X-Series II ICP/MS, Thermo Scientific). Pb in blood samples was routinely
measured electrochemically by anodic stripping voltammetry (LeadCare Ultra, Magellan Diagnostics, North
Billerica, MA). In some cases, blood Pb levels were determined using ICP-high resolution MS. Quality control
samples were analyzed with each digestion batch and included reagent blanks, blank spikes, matrix spikes, and a
matrix-matched NIST SRM.
Pb speciation analysis - Pb species in samples of diets amended with test materials and cumulative feces
samples were determined by X-ray absorption spectroscopy at the DuPont-Northwestern-Dow Collaborative Access
Team Sector 5, beam line 5BM-D, at the Advanced Photon Source of the Argonne National Laboratory, Lemont, IL.
Data analysis -Tissue-specific RBAs (bone, blood) were estimated for treated and untreated soils and for treated
and untreated soils spiked with Pb minerals. RBA was estimated as the tissue dose ratio (TDR) for the test material
TM (e.g. soil or mineral) and reference material (RM, Pb acetate):
RBA = TDRti/TDRrm
where the TDR is the ratio of the Pb concentration (mg/kg total skeleton or mg/L blood) to the cumulative Pb dose
(mg) for the study.
Confidence limits on each RBA were estimated based on Fieller's Theorem for estimating confidence limits on the
ratio of means. A point estimate for the RBA was calculated as the average of tissue-specific RBA values.
Confidence intervals on the point estimate were estimated from Monte Carlo simulation of the probability
distributions of each tissue-specific RBA.
Figure 1 - Effect of
treatment on estimates
of relative bioavailability
(RBA) for Pb in two soils.
Soil 1 (upper panel) and
soil 2 (lower panel)
tested in untreated state
or after 8 or 67 hours of
treatment to promote
plumbojarosite formation.
Mean estimates shown
with upper and lower
95% confidence intervals
shown for RBA estimates
based on bone or blood
tissue Pb levels. RBA
point estimates are
means of tissue data
RBA estimates.
Figur.
r f f

t + + 1
[ f +
Figure 3 - Contribution
of Pb species in diet (D)
and feces (F) of mice
that consumed diets
amended with soil 1 or
soil 2. Soils tested in
untreated (U) or treated
(T) forms. % of each Pb
species present shown.
PLJ - plumbojarosite;
OH-Cer - cerussite; PbS
- Pb sulfate
Figure 2 - Effect of treatment on estimates of relative bioavailability (RBA)
for Pb in soils spiked with Pb minerals. RBA estimates shown for untreated
and treated soils spiked with (a) Pb carbonate, (b) Pb chloride, (c) Pb
phosphate, and (d) Pb sulfate. Results shown for soil spiked with authentic
plumbojarosite (e). Mean estimates with upper and lower 95% confidence
intervals shown for RBA estimates based on bone or blood tissue Pb
levels. RBA point estimates are means of tissue data RBA estimates.
Figure 4
Figure 4 - Contribution
of Pb species in diet (D)
and feces (F) of mice
' that consumed mineral-
amended diets Minerals
tested in untreated (U) or
treated (T) forms. % of
each Pb species present
shown. Legends as in
Figure 3; Pyr
pyromorphite.
Conclusions and future directions
1.	Treatment of Pb-contaminated soils to promote plumbojarosite formation results in substantial reductions (>90%) in estimates of relative
bioavailability of Pb in these soils. The magnitude of reduction in the % relative bioavailability is not strongly affected by the length of
time used for soil treatment.
2.	For a range of Pb minerals commonly found in contaminated soils, treatment to promote plumbojarosite formation markedly reduces
estimated relative bioavailability. For all minerals, estimates of relative bioavailability for treated soils are similar to those obtained for
authentic plumbojarosite.
3.	Determination of Pb species in diets amended with untreated and treated soils and minerals shows that treatment results in near
quantitative conversion of native Pb species to plumbojarosite.
4.	Determination of Pb species in feces collected from mice that consumed diets amended with untreated and treated soils and minerals
shows that ingested plumbojarosite transits the gastrointestinal tract without alteration.
5.	The high efficiency of conversion of soil Pb species to plumbojarosite coupled with evidence of low relative bioavailability for Pb ingested
in this form suggests that remediation of soils by plumbojarosite formation in situ may be a valuable tool to reduce exposure of children
to this toxic metal. Optimization of soil treatment procedures and studies of efficacy of treatment under field conditions are needed to
validate this new approach.
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The views expressed in this presentation are those of the presenters and do not necessarily reflect the views or policies of the US EPA

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