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37
Abstract
A validated method is needed to measure reductions of in
vitro bioaccessible (IVBA) Pb in urban soil remediated with
amendments. This study evaluated the eect of in vitro extraction
solution pH and glycine buer on bioaccessible Pb in P-treated
soils. Two Pb-contaminated soils (790–1300 mg Pb kg−1), one from
a garden and one from a city lot in Cleveland, OH, were incubated
in a bench scale experiment for 1 yr. Six phosphate amendments,
including bone meal, sh bone, poultry litter, monoammonium
phosphate, diammonium phosphate, and triple superphosphate,
were added to containers at two application rates. Lead IVBA was
assessed using USEPA Method 1340 and three modied versions
of this method. Modications included using solutions with pH
1.5 and 2.5 as well as using solutions with and without 0.4 mol
L−1 glycine. Soil amendments were ineective in reducing IVBA
Pb in these soils as measured by pH 1.5 with glycine buer. The
greatest reductions in IVBA Pb, from 5 to 26%, were found using
pH 2.5 extractions. Lead mineral results showed several soil
amendments promoted Pb phosphate formation, an indicator
of remediation success. A signicant negative linear relationship
between reduction in IVBA Pb and Pb-phosphate formation
was found only for pH 2.5 without glycine extraction solution.
A modied USEPA Method 1340 without glycine and using pH
2.5 has the potential to predict P soil treatment ecacy and
reductions in bioavailable Pb.
Phosphorus Amendment Ecacy for In Situ Remediation of Soil
Lead Depends on the Bioaccessible Method
John F. Obrycki, Nicholas T. Basta,* Kirk Scheckel, Brooke N. Stevens, and Kristen K. Minca
D management recommendations for
Pb-contaminated urban soils is necessary to address
public questions regarding best practices for using
urban soils (Kim et al., 2014). Bioaccessible Pb, dened as
the potential for a substance to interact and be absorbed by
an organism, is commonly determined to evaluate potential
exposure risk to Pb-contaminated urban soil. Bioaccessible Pb
is determined by in vitro gastrointestinal (GI) soil extraction
methods (Scheckel et al., 2009; Scheckel et al., 2013). Adding
phosphates to Pb-contaminated soils oers one management
technique for reducing Pb bioaccessibility as measured by in
vitro bioaccessibility tests (Basta et al., 2001; Brown et al., 2007;
Scheckel et al., 2013; Zia et al., 2011). Phosphate soil amend-
ments can react with soil Pb to form Pb-phosphate minerals and
lower Pb solubility. A common urban soil contaminant from Pb
paint, cerrusite (PbCO3), readily dissolves in the human GI tract
and subsequently has a high Pb bioaccessibility (Casteel et al.,
2006). Treatment of cerrusite containing soil with phosphate
amendment has been shown to form insoluble pyromorphite,
which does not completely dissolve in the GI tract (Scheckel et
al., 2013). Because Pb-phosphates have reduced bioaccessibility
relative to some Pb mineral forms in the soil, Pb-phosphate for-
mation can be used as an indicator of remediation success.
Amending soil with phosphate is not always eective in reduc-
ing Pb bioaccessibility (Ruby et al., 1999; Ryan et al., 2004). e
soil Pb may be in a relatively insoluble and unavailable form, such
as the Pb mineral form galena (PbS). Amendment eectiveness
depends on amendment P form, soil Pb form, and soil condi-
tions, such as pH. Soluble P can interact with soil Pb (Basta and
McGowen, 2004) to reduce Pb mobility. Soluble Pb can react
with insoluble amendment P located on soil or amendment sur-
faces (Basta et al., 2005). e ineectiveness of phosphate for
immobilizing Pb has recently been reviewed by Scheckel et al.
(2013).
In vitro GI methods are commonly used to measure in vitro
bioaccessible (IVBA) soil Pb. Several in vitro methods have been
used to evaluate IVBA Pb in contaminated soils and soil-like
material (Drexler and Brattin, 2007; Juhasz et al., 2013; Koch
et al., 2013; Scheckel et al., 2013; Zia et al., 2011). Soil IVBA
Abbreviations: GI, gastrointestinal; IVBA, in vitro bioaccessible; MAP,
monoammonium phosphate.
J.F. Obrycki, N.T. Basta, B.N. Stevens, and K.K. Minca, School of Environment and
Natural Resources, 210 Kottman Hall, 2021 Coey Rd., The Ohio State Univ.,
Columbus, OH 43210; K. Scheckel, USEPA, National Risk Management Research
Lab., Land Remediation and Pollution Control Division, Cincinnati, OH 45224-1701.
Assigned to Associate Editor Benny Chefetz.
Copyright © 2015 American Society of Agronomy, Crop Science Society of America,
and Soil Science Society of America. 5585 Guilford Rd., Madison, WI 53711 USA.
All rights reserved.
J. Environ. Qual. 45:37–44 (2016)
doi:10.2134/jeq2015.05.0244
Received 28 May 2015.
Accepted 24 Aug. 2015.
*Corresponding author (basta.4@osu.edu).
Journal of Environmental Quality
SOIL IN THE CITY
SPECIAL SECTION
Core Ideas
• The ability of soil amendments to reduce IVBA Pb depended on
the in vitro method.
• Modied USEPA Method 1340 may predict bioaccessible Pb in
P-amended soil.
• Soil amendments were largely ineective in reducing IVBA Pb
using USEPA Method 1340.
Published January 11, 2016
38 Journal of Environmental Quality
Pb can vary between GI methods because of dierent pH and
extraction solution conditions. More Pb is extracted using meth-
ods with lower extraction solution pH, such as pH 1.5, compared
with methods with higher pH, such as pH 2.5 (Attanayake et al.,
2014; Brown et al., 2004; Scheckel et al., 2013; USEPA, 2007a).
Lead compounds are more soluble at lower pH values compared
with circumneutral values (Hettiarachchi and Pierzynski, 2004).
In vitro GI methods are commonly used to measure bioaccessible
Pb in soil amended with phosphates.
However, there is not an in vitro method, including the
standard USEPA Method 1340 (USEPA, 2013), validated for
use with P-treated soils. e USEPA Method 1340 uses 0.4
mol L-1 glycine (C2H5NO2) solution adjusted to pH 1.5 using
hydrochloric acid (HCl). During the development of USEPA
Method 1340, extraction solution pH values of 1.5 and 2.5
were investigated using unamended soils. It is unclear which in
vitro extraction, pH 1.5 or 2.5, is most appropriate to evaluate
Pb bioaccessibility for P-treated soils (Scheckel et al., 2009; Zia
et al., 2011). e USEPA Method 1340 may not be appropriate
for P-treated soils because previous research indicated the pre-
dicted reduction in IVBA Pb using the in vitro method under-
estimated the reduction in swine Pb uptake (Ryan et al., 2004).
For example, 1% P-treated Joplin soils aged for 32 mo resulted in
10% reduction in IVBA Pb using the pH 1.5, 0.4 mol L−1 glycine
extraction. e same soil sample showed an approximately 35%
reduction to the same control soil when a pH 2.5, 0.4 mol L−1
glycine solution was used (Ryan et al., 2004). A similar reduction
of 25% was found using a pH 2.5 solution with other organic
acids as buers (Hettiarachchi et al., 2001). e 1% P-treated
Joplin soil reduced relative bioavailability of Pb 71% (Ryan et al.,
2004). e pH 2.5, 0.4 mol L−1 glycine extraction yielded results
(i.e., 35% reduction), closer to the observed 71% reduction in
relative bioavailability Pb using the in vivo swine model.
e presence of pH buers within the extraction solution
can increase IVBA Pb. Organic acids, including the amino
acid glycine, chelate metals (Furia, 1968), including Pb (Banu
et al., 2012). Using a pH 2.0, 0.4 mol L−1 glycine extraction, a
1% P-treated soil showed 25% reduction in IVBA Pb compared
with the control soil (Ryan et al., 2004). However, using a pH
2.0 extraction solution without glycine buer for the same soil
showed a relative reduction in IVBA Pb of 60% (Yang et al.,
2001). In vitro extraction pH and glycine buer can have a pro-
found eect on IVBA Pb in amended soil. e objective of this
study was to evaluate the eect of in vitro method extraction
solution pH and glycine buer on bioaccessible Pb in P-treated
urban soils.
Materials and Methods
Two soils were selected to represent a historical garden area
(G) and an urban vacant city lot (L). Soils were selected with
Pb concentration above the USEPA screening level for bare soil
areas that will be used by children of >400 mg kg−1 (Table 1).
e soils represented two important types of contaminated sites
encountered in urban areas, a garden and a vacant lot, where
phosphate amendments would likely be considered as a reme-
diation strategy. Surface soils (0–15.24 cm) were collected from
each site, air-dried, and sieved to 2 mm. e soils from the two
sites were separately homogenized by rotating within closed
20-L buckets. Soils were weighed using the air-dried mass into
1.5-L plastic containers for laboratory incubations. For G soils,
500 g of air-dried soil were used in each incubation container;
for L soils, 300 g of air-dried soil were used in each incubation
container. e experimental design included two soils (G and L),
six soil amendments, two application levels (high and low), and
three replicates for each soil-amendment combination (Table 2),
resulting in 72 1.5-L plastic incubation containers.
Amendments were selected to include phosphate fertilizers
used in previous P-Pb solubility studies (Scheckel et al., 2013).
e amendments included the partially soluble phosphate fer-
tilizers bone meal (Scotts Miracle-Gro), sh bone (K. Scheckel,
personal communication), and poultry litter (S. Albro, personal
communication). Readily soluble forms of phosphate fertilizers
used were diammonium phosphate (USA/Interplast Corp.),
monoammonium phosphate (MAP) (USA/Interplast Corp.),
and triple superphosphate (Espoma Co.).
Variability in the P:Pb molar ratios occurred due to variabil-
ity in Pb contamination among the soil pots. e amended soils
were incubated at room temperature (approximately 20–28°C)
in a laboratory from September 2012 until November 2013. e
14-mo incubation time was longer than most previous short-term
studies (3 mo) (Scheckel et al., 2013) to ensure adequate time for
reaction between soil Pb and amendment. Incubation contain-
ers were watered every other day with 18 MW deionized water
to achieve 30% gravimetric water content as determined before
the start of the experiment. Gravimetric water content of 30%
was selected to maintain wet aerobic conditions. Porewater pH
(ibault and Sheppard, 1992; omas, 1996) and Mehlich-3
P (Mehlich, 1984) (Table 2) were evaluated 1 mo aer the start
of soil incubation. Organic carbon (Heanes, 1984) and texture
(Kilmer and Alexander, 1949) were evaluated on the control
soils before the start of the incubations.
Aer the conclusion of the incubation, soil was subsampled
from each container and sieved to <250 mm. Soils were analyzed
for total soil Pb using USEPA Method 3051A (USEPA, 2007b).
Briey, 0.5 g of <250 mm sieved soil was placed in 55-mL Teon
vessels. Samples were digested in a CEM-MARS (CEM Corp.)
using 9.0 ± 0.1 mL of trace metal–grade HNO3 (CAS 7697-37-
2, Fisher Scientic) and 3.0 ± 0.1 mL of trace metal–grade HCl
(CAS 7647-01-0, Fisher Scientic). ree replicates for each soil
type–treatment combination were analyzed. Eight replicates of
the unamended G and L samples were digested to estimate total
Pb in the control soils. Soil extracts from the total digest and
Table 1. Selected properties for the control soils used in this study.
Soil properties Garden site City Lot
pH 6.95 6.75
Organic C, g kg−1 8.0 9.0
Texture loamy sand sandy loam
Mehlich-3 P, mg kg−1 100 110
Total Pb, mg kg−1 911 ± 8.4 807 ± 4.2
pH 1.5 with glycine IVBA† Pb(%) 79 ± 1.0 85 ± 1.0
Total As, mg kg−1 25.1 ± 0.2 12.5 ± 0.1
Total Cd, mg kg−1 2.3 ± 0.0 2.6 ± 0.0
Total Fe, mg kg−1 17,954 ± 79.5 15,572 ± 72.4
Total Zn, mg kg−1 438 ± 3.3 448 ± 3.3
† In vitro bioaccessible.
Journal of Environmental Quality 39
the in vitro extractions were analyzed using inductively coupled
plasma optical emission spectrometry (Varian 720, Varian, Inc.).
Determination of Pb Bioaccessibility by In Vitro
Gastrointestinal Extractions
Samples were analyzed using four in vitro extractions:
USEPA Method 1340 (USEPA, 2013) and three variations of
this method with modied pH and glycine content. Extraction
solution pH values of 1.5 and 2.5 were selected because previ-
ous research indicated the pH 2.5 solution may be better able
to detect reductions in IVBA Pb aer P treatment. Extraction
solutions were produced with and without 0.4 mol L-1 glycine to
determine the eect of the buer on IVBA Pb. e four extrac-
tion solutions evaluated were: pH 1.5 with 0.4 mol L-1 glycine
(Method 1340), pH 1.5 without glycine, pH 2.5 with 0.4 mol
L-1 glycine, and pH 2.5 without glycine.
For the 0.4 mol L-1 glycine-containing extractions, deionized
water was mixed with 60.06 g glycine (Fisher Scientic, CAS
56–40–6) and placed in a covered 2-L volumetric ask. is
ask was placed in a 37°C cabinet overnight. Trace metal–grade
HCl and 37°C deionized water were added to bring the solution
to a volume of 2 L. e trace metal–grade HCl adjusted the nal
solution to pH 1.5 ± 0.05 and 2.5 ± 0.05, respectively. For the
nonglycine solutions, 37°C deionized water was adjusted to pH
1.5 ± 0.05 and pH 2.5 ± 0.05, respectively, using trace metal–
grade HCl.
For each in vitro extraction, all soil type–treatment combina-
tions had three replications. Four subsamples of the G control
soil (each subsample was approximately 50 g) were analyzed
from the bulk container of G control soil, and four subsamples
of the L control soil (each subsample was approximately 50 g)
were analyzed from the bulk container of L control soil. Sample
duplicates, duplicate matrix spikes (1000 mg L-1 Pb), blanks,
blank spikes (1000 mg L-1 Pb), and a standard reference mate-
rial, NIST 2711a Montana II Soil, were included in the analysis
as part of quality assurance and quality control measures.
e in vitro extraction process consisted of mixing 1.00 ±
0.01 g of <250 mm sieved soil and 100 ± 0.5 mL of extracting
solution. Samples were placed in 125-mL plastic bottles, sealed,
and secured on a rotating shaker in a 37°C cabinet (Nelson et
al., 2013). Rotations were maintained at 30 ± 2 rotations per
minute. Samples were pH adjusted to 1.50 ± 0.05 or 2.50 ± 0.05
pH at 5 min using 25% trace metal–grade HCl. Samples were
checked and pH adjusted at 30 min. Aer 60 min of rotating,
samples were removed, nal pH was recorded, and samples were
passed through 0.45-mm lters. Final solution pH stayed within
±0.25 pH units of the initial extraction solution pH for all four
in vitro extractions. is met the USEPA 1340 requirement
of the nal solution pH being ±0.5 pH units from the starting
solution pH (USEPA, 2013). Laboratory quality assurance and
Table 2. Overview of study amendments, P content, rate, soils, amount of amendment added, amended soil P:Pb molar ratio, soil pH, and Mehlich-3 P.
Amendment† Amendment P
content Rate Soil‡ Amendment
added§ P:Pb¶ Soil pH Mehlich-3 P
mg kg−1 g molar ratio mg kg−1
BM 40 low G 9.1 4.9 6.4 229
40 low L 11.2 8.6 6.8 335
40 high G 30.2 17.4 7.5 353
40 high L 37.2 30.5 7.5 548
FB 109 low G 3.4 5.0 5.8 233
109 low L 4.2 9.2 6.8 291
109 high G 11.5 17.3 6.1 360
109 high L 14.1 33.6 6.9 545
DAP 202 low G 1.8 4.9 6.2 616
202 low L 2.2 7.6 6.9 239
202 high G 5.9 16.0 6.0 1671
202 high L 7.3 29.0 6.8 1076
MAP 229 low G 1.6 5.1 5.7 736
229 low L 1.9 7.4 6.4 1262
229 high G 5.2 16.1 6.0 1959
229 high L 6.4 29.3 6.4 3680
TSP 202 low G 1.8 5.1 6.3 588
202 low L 2.2 8.4 6.4 1143
202 high G 5.9 17.1 5.7 1624
202 high L 7.3 30.1 5.4 2978
PL 11 low G 32.9 5.1 7.2 397
11 low L 40.6 8.9 7.9 644
11 high G 109.8 16.0 8.2 840
11 high L 135.0 28.9 7.9 1654
† BM, bone meal; DAP, diammonium phosphate; FB, sh bone; MAP, monoammonium phosphate; PL, poultry litter; TSP, triple superphosphate.
‡ G, Garden; L, City Lot.
§ Air-dried mass.
¶ Calculated comparing the amount of P added to the soil and the total Pb content in the soil.
40 Journal of Environmental Quality
quality control measures procedures were followed for each of
the extractions, including using duplicates, blanks, blank spikes,
and matrix spikes. All deionized water used in extractions was
18 MW.
Statistical analysis, including ANOVA tests, matched pairs t
test, and linear regression, were conducted using Minitab (v. 16).
Analysis of variance tests used a post hoc Tukey’s honest signi-
cant dierence (HSD) test with a P value of 0.1. is P value was
used to better capture the variability of the data and to highlight
potential P amendment trends to be evaluated in future stud-
ies. In vitro bioaccessibility for each treatment was calculated
by dividing the in vitro extractable Pb concentration (mg kg-1)
by the total Pb (mg kg-1) measured by USEPA Method 3051A.
For evaluating treatment ecacy, percentage reductions in IVBA
Pb between treated and control for each extraction were calcu-
lated by taking the control soil IVBA Pb minus the treated soil
IVBA Pb. For linear regression, percentage reduction in IVBA
Pb between treated and control soil was calculated by taking the
control soil IVBA Pb minus the treated soil IVBA Pb, and this
quantity was divided by the control soil IVBA Pb.
Pb Mineral Identication by X-ray Absorption Fine
Structure Spectroscopy
X-ray absorption ne structure spectroscopy was used
to evaluate Pb mineral forms using the Materials Research
Collaborative Access Team at the Advanced Photon Source of
the Argonne National Laboratory. Mineral identication meth-
ods are described in Minca et al. (2013). Lead minerals were
quantied using linear combination tting against known Pb
mineral standards. R-factors were reported to indicate variation
that occurred between the standards and the samples.
Various Pb standards were used as reference spectra, including
mineral sorbed Pb [Pb-ferrihydrite, Pb-kaolinite, Pb-goethite,
Pb-gibbsite, Pb-birnessite, and Pb-montmorillonite in which
each mineral was equilibrated with Pb(NO3)2 at pH 6 for a target
surface loading of 2500 mg kg−1 aer dialysis], organic bound
Pb [Pb fulvic acid and Pb-humic acid as reagent-grade organic
acids equilibrated with Pb(NO3)2 at pH 6 for a target loading
of 1500 mg kg−1 aer dialysis and reagent-grade Pb acetate, Pb
cysteine, and Pb citrate], Pb carbonate (Smithsonian Natural
History Minerals Collection specimens of cerussite, hydrocerus-
site, and plumbonacrite with X-ray diraction verication), PbO
(massicot and litharge), Pb phosphates [chloropyromorphite,
hydroxypyromorphite, Pb3(PO4)2, PbHPO4, and Pb sorbed
to apatite at pH 6 and surface loading of 2000 mg kg−1], and
other Pb minerals (leadhillite, magnetoplumbite, plumboferrite,
plumbogummite, plumboyarosite, anglesite, and galena from the
Smithsonian Natural History Minerals Collection with X-ray
diraction verication). All reference spectra were collected in
transmission mode with dilution calculations determined by
XAFSMass (Klementiev, 2012) mixed in binder and pressed
into a pellet. ese spectra were acquired on the same beamline
with identical scan parameters simultaneously with a Pb metal
foil for calibration but on separate occasions to the samples.
Within linear combination tting the sum was not forced
to 1, but the results were normalized to 1 (or 100%) at the end.
e tting required the value for each reference to vary between
0 and 1 so there would be no negative values inuencing the
tting. e R-factor was reported for each analysis. e R-factor
is a measure of the mean square sum of the mist at each data
point: R-factor = sum[(data-t)2]/sum[data2].
Results and Discussion
Determination of Pb Bioaccessibility by In Vitro
Gastrointestinal Extractions
Extractable Pb using USEPA Method 1340 from refer-
ence soil NIST 2711a was 1137 ± 19 mg Pb kg−1 soil. is was
within the range recommended by USEPA Method 1340, which
reports an average of 1114 mg Pb kg−1 soil (980–1249 mg Pb
kg−1 soil 99th percentile prediction interval). Extractable P from
2711a using USEPA Method 1340 was 493 ± 10 mg P kg−1 soil.
Extractable P was 889 ± 9.9 mg P kg−1 from control soil L and
508 ± 4.2 mg P kg−1 for control soil G. Treated soils had extract-
able P ranging from 816 to 7488 mg P kg−1 and a mean of 2857
± 210 mg P kg−1.
Blank spike samples were consistent throughout the four
extractions, with a Pb spike recovery of 103 to 110%. e spike
recovery was within the range recommended by USEPA Method
1340 (85–115%). Matrix Pb spike recoveries at pH 1.5 with and
without glycine (88–102%) were acceptable. e matrix spiked
recoveries ranged from 68 to 90% at pH 2.5 with glycine and
from 22 to 50% at pH 2.5 without glycine. e dierence in the
spike recovery for the pH 2.5 solutions could be due to glycine
chelating Pb or Pb being sorbed to the soil surface. Matrix spikes
were applied to the same treatment subsamples across the four
extractions. is means that glycine and small uctuations in
extraction solution pH were potential explanatory factors when
comparing spiked recovery for a given soil sample at pH 2.5
without glycine and at pH 2.5 with 0.4 mol L−1 glycine.
Bioaccessible Pb determined by the four in vitro extractions
for the G and L soils are presented in Fig. 1. Amendments low-
ered IVBA Pb relative to the controls more frequently in the
pH 2.5 extractions than in the pH 1.5 extractions for both soils.
None of the amendments resulted in Pb IVBA reductions using
the pH 1.5 with 0.4 mol L−1 glycine solution. e ability of soil
amendment to reduce IVBA Pb varied with in vitro method and
soil amendment. A greater number of amendments reduced Pb
IVBA on the L than the G site (Fig. 1).
Table 3 provides an expanded look at the pH 2.5 extraction
solution data to highlight the variation in IVBA Pb reductions
across treatments (P < 0.10). e P treatments consistently
reduced Pb IVBA using the pH 2.5 extractions on the city lot
site. Ten of the 12 treatments demonstrated at least a 10% reduc-
tion in Pb IVBA using either of the two pH 2.5 extractions.
e largest reduction in IVBA Pb (26%) occurred in the “MAP
high” treatment on the city lot using the pH 2.5 with glycine
extraction. e “MAP high” amendment did not demonstrate
this same eectiveness on the garden soil. On the garden soil,
none of the amendments in any of the extractions demonstrated
a greater than 10% reduction in Pb IVBA compared with the
control soil.
e IVBA Pb depended on extraction pH and glycine con-
tent of the in vitro method when comparing the four extractions
by soil (Table 4). e dierences in IVBA Pb due to soil type are
much smaller than the dierences in IVBA Pb due to extraction
method (i.e., the in vitro method used is more important than
Journal of Environmental Quality 41
the soil). e IVBA Pb for both soils decreased in the following
order: pH 1.5 with glycine > pH 1.5 without glycine > pH 2.5
with glycine > pH 2.5 without glycine (P < 0.001). Analysis of
variance with a post hoc Tukey HSD test found statistically sig-
nicant dierences among the four extractions when controlling
for soil type. Within the same pH level, the pH 1.5 with glycine
IVBA Pb was 15 to 20% greater than the pH 1.5 without glycine.
At pH 2.5, the glycine-containing extractions’ IVBA Pb values
were 30% greater than the extraction solution without glycine.
When comparing dierent pH values for the same extractions
(glycine vs. nonglycine), the IVBA Pb between pH 1.5 and pH
2.5 both with glycine was 35 to 40% greater at pH 1.5. Without
glycine, the pH dierence accounted for IVBA Pb values that
were 50% higher at pH 1.5 compared with pH 2.5. Glycine
may be better able to chelate Pb at pH 2.5 because glycine has
an acid dissociation constant of 2.34. ese results support pre-
vious ndings regarding pH 2.5 with glycine showing greater
treatment eects than pH 1.5 with glycine (Ryan et al., 2004).
Fig. 1. Extractable Pb for four in vitro extractions of the
Garden (A) and City Lot (B) sites. * Treatment in vitro
bioaccessible (IVBA) Pb lower than the control soil IVBA Pb
within each soil and each extraction at the p = 0.05 signi-
cance level. † Treatment IVBA Pb lower than the control soil
IVBA Pb within each soil and each extraction at the p = 0.10
signicance level. BM, bone meal; DAP, diammonium phos-
phate; FB, sh bone; MAP, monoammonium phosphate; PL,
poultry litter; TSP, triple superphosphate.
Table 3. Reduction in mean percentage in vitro bioaccessible Pb between control and P-treated soils for pH 2.5 extraction solutions.†
Treatment‡ Garden City Lot
Glycine No glycine Glycine No glycine
BM low – – 11 ± 2% –
BM high – – 10 ± 2% 9 ± 1%
FB low – 8 ± 1% – 10 ± 1%
FB high – 9 ± 0.5% – 8 ± 1.5%
DAP low – – 9 ± 2.5% –
DAP high – – 19 ± 1% 16 ± 1%
MAP low – 5 ± 1% 11 ± 4% 7 ± 1%
MAP high – 8 ± 2% 26 ± 3% 18 ± 1%
TSP low – – 13 ± 0.5% 6 ± 1%
TSP high 5 ± 1% – – 13 ± 1%
PL low – 5 ± 0.5% 13 ± 1% 5 ± 1%
PL high 5 ± 1% – 14 ± 2% 7 ± 1%
† Percentage dierence calculated as Mean Control IVBA Pb minus Treatment IVBA Pb. Values listed are those that were signicant at P < 0.10 for each
extraction ANOVA.
‡ BM, bone meal; DAP, diammonium phosphate; FB, sh bone; MAP, monoammonium phosphate; PL, poultry litter; TSP, triple superphosphate.
42 Journal of Environmental Quality
e control soil IVBA was lowest in a pH 2.5 without glycine or
other organic buer solutions.
Pb Mineral Identication by X-ray Absorption Fine
Structure Spectroscopy Results
Spectroscopy results indicated that both control soils
were predominantly mineral-sorbed or organic-bound Pb
(Table 5). Similar amounts of mineral-sorbed (62 vs. 55%)
and organic-bound Pb (29 vs. 31%) were in the control soils
(Table 5). Neither control soil contained Pb-phosphates. e
Pb-phosphate category included Pb pyromorphite, Pb-sorbed
Ca-phosphate, and Pb3(PO4)2. Figure 2 expands on the data
presented in Table 5 by providing the proportion of these three
Pb-phosphate types by treatment and location. As compared
with the control soils, the phosphate amendments increased
Pb-phosphate formation between 6 and 32% for both soils (Fig.
2). Lead phosphate formation ranged from 6 to 27% on the
Garden site and from 8 to 32% on the City Lot site. e high P
application rate led to 10 to 15% greater Pb-phosphate forma-
tion when compared with the low application rate for the same
amendment. Pyromorphite, a more insoluble Pb-phosphate
(Scheckel et al., 2013), was not formed consistently across all
treatments. At the Garden site, the greatest pyromorphite for-
mation was 7% within the Diammonium Phosphate High treat-
ment. At the City Lot site, the “sh bone high” treatment led to
a 16% increase in pyromorphite over the control soil.
ese pyromorphite formation rates are comparable to other
studies that have used similar P treatments and achieved 1 to 16%
pyromorphite formation (Scheckel et al., 2013). is formation
can be increased to 40% if phosphoric acid is used as a treatment
(Juhasz et al., 2014; Scheckel et al., 2013). Juhasz et al. (2014)
included a soil with similar Pb mineral forms compared with the
vacant lot and garden soils in this current study and achieved
Table 4. Percentage in vitro bioaccessible Pb compared with total soil
Pb for each soil location and each extraction.
Soil location Extraction Mean IVBA ± SE‡
Garden† pH 1.5 with glycine 88 ± 1%
pH 1.5 no glycine 69 ± 1%
pH 2.5 with glycine 50 ± 1%
pH 2.5 no glycine 17 ± 1%
City Lot† pH 1.5 with glycine 81 ± 1%
pH 1.5 no glycine 66 ± 1%
pH 2.5 with glycine 47 ± 1%
pH 2.5 no glycine 17 ± 1%
† Calculated as in vitro bioaccessible Pb divided by the total soil Pb and
expressed as a percentage.
‡ Statistically signicant at the P < 0.001 level when the four extractions
are compared within soil location. This analysis did not compare
between the Garden and City Lot soils.
Table 5. Lead mineral types identied in control and treated Garden and City Lot soil using X-ray absorption ne structure spectroscopy.
Location Amendment† Mineral sorbed Organic bound Pb carbonate Pb-phosphate sum‡ R-factor§
———————————————— % ————————————————
Garden control 55 31 14 0 0.002
BM low 60 30 5 6 0.004
BM high 58 25 5 11 0.003
FB low 56 24 4 16 0.003
FB high 54 11 8 27 0.005
DAP low 48 23 18 11 0.003
DAP high 48 22 5 24 0.003
MAP low 48 39 4 10 0.002
MAP high 47 22 7 24 0.009
TSP low 53 21 15 11 0.003
TSP high 51 20 5 24 0.003
PL low 54 24 9 13 0.004
PL high 59 20 3 19 0.004
City Lot control 62 29 9 0 0.002
BM low 52 28 9 11 0.002
BM high 40 23 13 25 0.003
FB low 52 17 10 21 0.006
FB high 47 6 17 30 0.005
DAP low 44 35 10 10 0.002
DAP high 56 13 11 21 0.004
MAP low 27 37 18 18 0.003
MAP high 24 24 20 32 0.007
TSP low 28 36 28 8 0.002
TSP high 37 31 12 20 0.004
PL low 44 36 7 12 0.004
PL high¶
† BM, bone meal; DAP, diammonium phosphate; FB, sh bone; MAP, monoammonium phosphate; PL, poultry litter; TSP, triple superphosphate.
‡ Column includes sum of pyromorphite, Pb3(PO4)2, and Pb-sorbed Ca-phosphate. Data for these three mineral types are shown in Fig. 3.
§ The R-factor is a measure of the mean square sum of the mist at each data point. R-factor = sum[(data-t)2]/sum[data2].
¶ Not determined.
Journal of Environmental Quality 43
43% pyromorphite formation aer phosphoric acid treatment.
is current study did not use phosphoric acid because it is
unlikely to be adopted as a publicly used treatment due to the
potential hazards involved with applying concentrated acid to
residential soils.
e Pb-phosphate formation could not be explained solely by
the variability in the P:Pb molar ratios for the incubation (Table
2). Although L soils had consistently higher P:Pb molar ratios,
the Pb-phosphate formation was not consistently higher across
all treatments. A matched-pairs analysis of Pb-phosphate forma-
tion for the 11 treatments evaluated on both soils found the L
soils did not have consistently higher Pb-phosphate formation
(P = 0.136). e average Pb-phosphate formation in L soils was
approximately 2.8% higher, with a 95% condence interval of
6.69% higher to 1.05% lower. Several other factors, such as incu-
bation pH, extraction solution pH, and soil mineralogy, could be
combined in future studies to better explain how P treatments
react with in situ Pb.
e in vitro results were compared with the percentage
Pb-phosphate formation to evaluate if in vitro solutions could
predict Pb-phosphate formation and, in turn, remediation suc-
cess (Fig. 3). Using the pH 2.5 without glycine extraction data,
we found a linear relationship between the reduction in IVBA
compared with the percentage Pb-phosphate formation (P <
0.001). e percentage Pb-phosphate formation was not related
to IVBA Pb as determined by the other three extractions (Fig.
3). ese results indicate that a pH 2.5 solution without glycine
may be used as a predictor of Pb-phosphate content in soils.
is extraction has the potential to be used in one of two ways.
First, site assessments could incorporate a pH 2.5 extraction
without glycine as an assessment of treatment ecacy. Second,
assessments could use this extraction as a pretreatment test.
is could improve amendment recommendations because if
Pb-phosphate levels are found to be low, there may be a potential
for Pb-phosphate to form during remediation. If Pb-phosphate
levels are found to be higher, adding phosphates to the soils may
not yield additional Pb-phosphate formation. ese results from
this study need to be rigorously tested before widespread adop-
tion for site assessment and remediation.
Soil Management Considerations and Recommendations
Six phosphate amendments showed mixed results regarding
IVBA Pb and soil amendment ecacy. e ability of soil amend-
ments to reduce IVBA Pb depended on the in vitro method.
Soil amendments were ineective in reducing IVBA Pb in these
two urban soils when using USEPA Method 1340. However, P
treatments were more eective when evaluated using modica-
tions of USEPA Method 1340. e greatest number of reduc-
tions in IVBA Pb were found at pH 2.5 without glycine buer.
Reductions in bioaccessible Pb from soil treatment ranged from
5 to 26% for the pH 2.5 extractions. Lead mineral identication
data indicated phosphate treatments were eective in forming
Pb phosphates.
e soils in this study contained Pb at concentrations of 790
to 1300 mg Pb kg−1. ese concentrations are found in urban
soils of industrial cities. Managing these soils gains additional
Fig. 2. Percentage Pb-phosphate formation by Pb-phosphate
type for the Garden (A) and City Lot (B) sites. Control soil had 0%
Pb-phosphates. † Not determined. BM, bone meal; DAP, diammonium
phosphate; FB, sh bone; MAP, monoammonium phosphate; PL,
poultry litter; TSP, triple superphosphate.
Fig. 3. Relationship between in vitro bioaccessible Pb and the amount
of Pb associated with the sum of Pb-phosphate minerals. The percent-
age in vitro bioaccessible (IVBA) reduction was the dierence in IVBA
treatment and IVBA control divided by the IVBA control. X, percent-
age IVBA reduction expressed as a percentage and negative sign
indicates treatment reduced IVBA Pb; Y, percentage Pb-phosphate
sum. (A) pH 1.5 with 0.4 mol L−1 glycine. Regression equation: y =
0.0269x + 17.5; r2 = 0.0016. (B) pH 1.5 without glycine. Regression
equation: y = 0.0630x + 18.1; r2 = 0.0032. (C) pH 2.5 with 0.4 mol L−1
glycine. Regression equation: y = −0.1532x + 15.8; r2 = 0.0622. (D) pH
2.5 without glycine. Regression equation: y = −0.2176x + 11.97; r2 =
0.4165. *** Signicant at the p < 0.001 level.
44 Journal of Environmental Quality
importance because the Centers for Disease Control recently
changed the denition of elevated blood Pb level from 10 to 5
mg dL−1 (CDC, 2012). It is likely this change will lower the soil
screening level below 400 mg Pb kg−1 soil (Henry et al., 2015).
is will result in a much greater demand for bioaccessibil-
ity Pb testing of many more urban soils that exceed the lower
soil screening level. Without an accepted method for screening
P-treated soils and no recommended concentration threshold
for what constitutes a P-treated soil, how will researchers, city
ocials, and the general public evaluate Pb-contaminated soil
remediation eorts? Research is needed to validate an in vitro
method accurate for measuring reductions in IVBA Pb. is
research must include a comprehensive in vitro–in vivo correla-
tion showing the in vitro method is an accurate predictor of in
vivo Pb uptake.
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