Chemosphere 184 (2017) 197e206

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Chemosphere
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Potential value of phosphate compounds in enhancing immobilization

and reducing bioavailability of mixed heavy metal contaminants in
shooting range soil
B. Seshadri a, b, N.S. Bolan a, b, *, G. Choppala c, A. Kunhikrishnan d, P. Sanderson a, b,
H. Wang e, f, L.D. Currie g, Daniel C.W. Tsang h, Y.S. Ok i, G. Kim j
a
Global Institute for Environmental Research, ATC Building, Faculty of Science and Information Technology, The University of Newcastle, University Drive,
Callaghan, NSW 2308, Australia
b
Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), P.O. Box 486, Salisbury, SA 5106, Australia
c
Southern Cross GeoScience, Southern Cross University, Lismore, New South Wales, Australia
d
Department of Agro-Food Safety, National Institute of Agricultural Science, Wanju, Jeollabuk-do 55365, Republic of Korea
e
School of Environment and Chemical Engineering, Foshan University, Foshan, Guangdong 528000, People’s Republic of China
f
Key Laboratory of Soil Contamination Bioremediation of Zhejiang Province, School of Environmental and Resource Sciences, Zhejiang A & F University,
Lin’an, Hangzhou, Zhejiang 311300, People’s Republic of China
g
Fertilizer and Lime Research Centre, Massey University, Palmerston North, New Zealand
h
Department of Civil and Environmental Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
i
Korea Biochar Research Center & Department of Biological Environment, Kangwon National University, Chuncheon 200-701, Republic of Korea
j
Department of Civil and Environmental Engineering, Hannam University, Republic of Korea

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Compared various P sources for their

effectiveness in the immobilization of
metals.

The bioavailability of mixed contam-
inants in a shooting range soil was
examined.

Bioavailability was examined using
plant growth and earthworm toxicity
experiments.

The solubility of P compound impacts
the immobilization of metals.

P-induced metal immobilization re-
sults from adsorption and precipita-
tion reactions.

a r t i c l e i n f o a b s t r a c t

Shooting range soils contain mixed heavy metal contaminants including lead (Pb), cadmium (Cd), and
Article history: zinc (Zn). Phosphate (P) compounds have been used to immobilize these metals, particularly Pb, thereby
Received 3 March 2017
reducing their bioavailability. However, research on immobilization of Pb’s co-contaminants showed the
Received in revised form
26 April 2017
relative importance of soluble and insoluble P compounds, which is critical in evaluating the overall
Accepted 30 May 2017 success of in situ stabilization practice in the sustainable remediation of mixed heavy metal contami-
Available online 5 June 2017 nated soils. Soluble synthetic P fertilizer (diammonium phosphate; DAP) and reactive (Sechura; SPR) and
unreactive (Christmas Island; CPR) natural phosphate rocks (PR) were tested for Cd, Pb and Zn immo-
bilization and later their mobility and bioavailability in a shooting range soil. The addition of P

* Corresponding author. Global Centre for Environmental Remediation, Univer-

sity of Newcastle, NSW 2308, Australia.
E-mail address: Nanthi.Bolan@newcastle.edu.au (N.S. Bolan).

http://dx.doi.org/10.1016/j.chemosphere.2017.05.172
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
198 B. Seshadri et al. / Chemosphere 184 (2017) 197e206

Keywords:
compounds resulted in the immobilization of Cd, Pb and Zn by 1.56e76.2%, 3.21e83.56%, and 2.31e74.6%,
Heavy metals respectively. The reactive SPR significantly reduced Cd, Pb and Zn leaching while soluble DAP increased
Bioavailability their leachate concentrations. The SPR reduced the bioaccumulation of Cd, Pb and Zn in earthworms by
Rock phosphate 7.13e23.4% and 14.3e54.6% in comparison with earthworms in the DAP and control treatment, respec-
Leaching tively. Bioaccessible Cd, Pb and Zn concentrations as determined using a simplified bioaccessibility
Shooting range soil extraction test showed higher long-term stability of P-immobilized Pb and Zn than Cd. The differential
effect of P-induced immobilization between P compounds and metals is due to the variation in the
solubility characteristics of P compounds and nature of metal phosphate compounds formed. Therefore,
Pb and Zn immobilization by P compounds is an effective long-term remediation strategy for mixed
heavy metal contaminated soils.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction 2001) prescribed this practice for Pb remediation in shooting

Metal mining and smelting activities, and indiscriminate
disposal of agricultural and industrial wastes have resulted in the
pollution of terrestrial and aquatic environments with heavy
metals (Raicevic et al., 2009; Sun et al., 2010). For instance, shooting
ranges and base-metal tailings are a major source of mixed-
contamination (co-contamination) of heavy metal(loid)s
including cadmium (Cd), copper (Cu), lead (Pb), antimony (Sb), and
zinc (Zn) (Lafond et al., 2014; Sanderson et al., 2012).
Mixed heavy metal(loid) contamination from bullets is a major
issue in shooting range soils. In United States, 0.8 million tons
(approx.) of Pb was used in the production of bullets annually in the
late 1990s (USEPA, 2001). Consequently, there was Pb contamina-
tion at several civilian and military shooting ranges across the
country (Hardison et al., 2004). These soils may also be enriched
with other metal(loid)s such as arsenic (As), antimony (Sb), cad-
mium (Cd), copper (Cu), nickel (Ni) and zinc (Zn), which are minor
components of bullets (Dermatas et al., 2006; Evangelou et al.,
2012; Johnson et al., 2005; Hockmann et al., 2015). Nearly 200 kg
of Pb bullets may reside at any time in Australian soils before
management practices remove bullets from the soil (Sanderson
et al., 2012). These bullets are subject to weathering in the soil
and release of contaminants into the soil profile. High concentra-
tions of these metal(loid)s are toxic to both humans and animals.
Lead has no known biological function and in humans, the devel-
oping fetus and children are more sensitive to high levels of Pb
exposure than adults due to neurological effects of Pb exposure,
which may cause learning disabilities, attention deficit disorders,
lowered IQ, and anti-social behavior (ATSDR, 2007; Park et al.,
2011).
Increased awareness on the hazards of heavy metal(loid)s has
led to the need to observe with stricter environmental regulations
and improve management approaches to reduce their hostile
environmental and health effects (Cao et al., 2003). The immobili-
zation of metal(loid)s contaminated soils using inorganic and
organic amendments is a sustainable remediation technique to
reduce their mobility and bioavailability (Kumpiene et al., 2008;
Sanderson et al., 2011). Phosphate compounds are known to be
effective in the immobilization of heavy metal(loid)s including Pb,
Cd and Zn (Bolan et al., 2003; Cao et al., 2009; Park et al., 2011).
Precipitation of metal phosphates is considered as the main process
behind the immobilization (Basta and McGowen, 2004).
For example, Hettiarachchi et al. (2001) and Cao et al. (2004)
demonstrated that phosphate rock (PR) reduced bioavailable Pb
through the formation of pyromorphite-like minerals. Similarly, a
study by Melamed et al. (2003) indicated up to 60% of Pb trans-
formation from labile to residual fraction compared to the control,
which occurred in the presence of PR and phosphoric acid (H3PO4).
Previously, the U.S. Environmental Protection Agency (USEPA,
ranges.
Bolan et al. (2003) examined the influence of P on the immo-
bilization and bioavailability of Cd in a range of variable charge soils
spiked with Cd. They suggested that rise in pH and improvement in
surface charge was mainly responsible for Cd immobilization,
thereby resulting in the P-induced mitigation of Cd phytotoxicity.
Similarly, McGowen et al. (2001) applied high amounts of DAP
(2300 mg P kg1) to a smelter-contaminated soil and observed
enhanced immobilization of Cd, Pb and Zn in the soil. Recently,
Valipour et al. (2016) reported significant decline in the availability
of Pb and Cd after the application of triple super phosphate (TSP),
but they found mobilization of Ni. Interestingly, the immobilization
of metals was relatively ineffective when phosphate rock was
applied to calcareous soils, which can be attributed to the poor
reactivity of apatite.
Immobilization of metal(loid)s in polluted soil or water is
known to be comparatively economical and highly effective, when
suitable P compounds are used (Ma et al., 1993; Wu et al., 2013;
Sanderson et al., 2015a,b). For instance, some studies showed that
the stabilization of soil contaminated with lead in situ with the help
of P compounds required just $9e55/Mg soil compared to signifi-
cantly higher cost ($282e1080/Mg soil) involved in the removal
and land disposal of top 60 cm of contaminated soil (Berti and
Cunningham, 1997; Chen et al., 2007; Ma and Rao, 1997). Howev-
er, there are some apprehensions on the stability of the immobi-
lized metal(loid)s in the long term, concerning the proven
efficiencies of P treatments (Stanforth et al., 2005; Kumpiene et al.,
2008). For example, a long-term study on the effect of soil organic
acids in rhizosphere showed potential liberation of Pb from pyro-
morphite (Debela et al., 2010).
In the case of Cd contamination in soil, the presence of iron and
aluminium in soil suspensions influences Cd3(PO4)2 solubility,
which may sometimes be too high to control Cd concentration in
those suspensions (Kuo, 1986; Soon, 1981). Gao et al. (2011)
observed an immediate increase in Cd concentration when phos-
phate is applied and elevated Cd accumulation in wheat may be
attributed to the competition between Zn and Cd for adsorption
into plants. Richards et al. (1998) observed no evidence of enrich-
ment of Cd either in soil or crop after 29 years of phosphorus
application. However, in a field study of 7 years (2002e2009)
conducted by Grant et al. (2013) revealed that Cd in phosphate
fertilizer increases Cd availability for plant uptake and Cd accu-
mulation in plants is affected by soil characteristics and environ-
mental conditions. There is discrepancy in the results related to Cd
availability in long term P applied soil. Scheckel et al. (2013)
recently review Pb immobilization in soil, including stability of
pyromorphite and effect on co-contaminants.
While most researches showed the prospective influence of P
compounds in the immobilization of individual metals in soils
 B. Seshadri et al. / Chemosphere 184 (2017) 197e206 199

(Hong et al., 2010; Park et al., 2011), studies are limited in terms of
mixed heavy metal(loid)s contaminated soils. Hence, this research
focused on evaluating the immobilization and subsequent mobility
and bioavailability of Cd, Pb and Zn in a shooting range soil. This
study also reports on the stability of Cd, Zn and Pb immobilization
by both synthetic and natural P compounds as observed in an
extended period of 3 years.
2. Materials and methods
2.2. Immobilization of metals
The soil samples (300 g each) were taken in 500 mL glass bottle
and were treated with various P compounds at 600 mg P/kg soil
concentration, and incubated for 30 days. Soil was maintained at
60% of water holding capacity. After incubation, NH4NO3 extract-
able Cd, Pb and Zn concentrations were analysed with an ICP-OES.
Phosphate-induced immobilization of Cd, Pb and Zn was deter-
mined based on the decrease in NH4NO3-extractable metal con-
centration as shown in Eq. (1).

2.1. Soil sample and phosphate compounds

NH4NO3 extractable metal in ðcontrol  sampleÞ

Immobilized metal ð%Þ ¼  100 (1)
NH4NO3 extractable metal in control

The soil sample with mixed metal contaminants was collected
from a shooting range in South Australia, dried at room tempera-
ture (25  C), and then sieved (<2 mm) (Table 1). The soil sample
(Mottled, Mesotrophic, GreyeYellow Kandosol) was also analysed
for various properties including texture, organic matter, pH, elec-
trical conductivity (EC), and total and NH4NO3-extractable metal(-
loid) concentrations. Exactly, 0.5 g of air-dried soil was digested
with 5 mL of aqua regia in Teflon microwave soil digest vessels, to
determine the total heavy metal content. For the measurement of
extractable metal(loid) concentration, 5 g of soil sample was
extracted with 12.5 mL of 1 M NH4NO3 solution for 2 h in an end-
over-end shaker and extractable Cd, Pb and Zn concentrations were
analysed with Inductively Coupled Plasma-Optical Emission Spec-
troscopy (ICP-OES, Agilent).
Both soluble synthetic and insoluble natural P compounds were
used in this study (Table 2). The P compounds include soluble dia-
mmonium phosphate (DAP) and insoluble Sechura (SPR - from Peru)
2.3. Column leaching
Soil samples incubated with DAP or SPR and also P-untreated
control soil were filled into a column of 3 cm diameter and a
height of 7.5 cm; the bulk density used was 1.4 g/cm3. For leach-
ing, 1 mM CaCl2 solution was used and a peristaltic pump was
employed to maintain the flow rate at 20 mL/h. The leachates
were collected from the columns as two aliquots per pore volume
(10 mL) after leaching up to 10 pore volumes. The leachates were
analysed for Cd, Pb, Zn and P concentration by ICP-MS (Agilent).
Mobility of Cd, Pb and Zn was described as percentage of mass
leached with respect to the total metal mass in soil (Eq. (2)). The
mass of leached metal was calculated by multiplying metal con-
centration in leachate with the volume of the leachate collected.
The mass of the metal in the soil was calculated by multiplying
metal concentration in the soil with the mass of the soil used in
the leaching experiment.

Metal concentration in leachate  leachate volume

Metal Mobility ð%Þ ¼  100 (2)
Metal concentration in soil  soil mass

and Christmas Island (CPR - from Australia) phosphate rocks (PR). 2.4. Bioavailability of immobilized metals using earthworms
Water-soluble and citric-soluble P concentrations of P amendments
were analysed as described by Braithwaite et al. (1990). The pre-incubated soils (100 g) with DAP or SPR and also P-
untreated control soil were used in this experiment. Before
exposing the adult earthworms to test soils, they were washed and
Table 1
Properties of soil and phosphate compounds used. Means with the same letter depurated for 24 h. The mature earthworms (ten Eisenia fetida)
within a row are not significantly different at p < 0.05 by Duncan’s multiple range were weighed and cultured at 18  C in the presence of continuous
test. (DAP ¼ Diammonium phosphate; SPR ¼ Sechura Phosphate Rock; light; the culturing was carried out in three replicates. The earth-
CPR ¼ Christmas Island Phosphate Rock). worms were again depurated for 24 h at the end of two weeks, and
Properties Shooting range DAP SPR CPR weighed into 75 mL of glass tubes; 5 mL of HNO3 was added to each
soil glass tube. Subsequently, the tubes were heated first at 75  C for
pH 6.21d 9.46a 8.43b 7.21c 30 min and then at 100  C for 30 min. The samples were diluted
Organic carbon (mg/kg) 2243a e 123.3b 87.23b with Milli-Q water after digestion and Cd, Pb and Zn concentrations
Total P (g/kg) 0.57d 223a 113b 97.6c were analysed using ICP-OES. An automated total organic carbon
Citric P (g/kg) e 202 75.9 6.79
analyser (Model 1010, O.I. Analytical) was used to determine dis-
Total Cd (mg/kg) 224a 2.34c 12.3b 3.45c
NH4NO3-Cd (mg/kg) 15.3a 1.86b 1.75b 0.13c solved organic carbon (DOC) in the soil samples. Bioavailability of
Total Pb (mg/kg) 9876a e 3.42b 1.23b Cd, Pb and Zn was expressed as percentage of metal mass taken up
NH4NO3-Pb (mg/kg) 528a e 0.76b e by earthworm with respect to the total mass in soil (Eq. (3)).
Total Zn (mg/kg) 654a 3.45c 7.87b 11.23b
NH4NO3-Zn (mg/kg) 56.7a 3.34b 0.86c 0.97c
200 B. Seshadri et al. / Chemosphere 184 (2017) 197e206

Metal concentration in earthworm  earthworm biomass

Bioavailability ð%Þ ¼  100 (3)
Metal concentration in soil  soil mass

2.5. Plant bioavailability of immobilized metals
Air-dried soil samples (400 g) were weighed and mixed with
DAP or SPR (600 mg P/kg) and placed into 600 mL plastic pots. P-
untreated control soil samples were also used. Soils were allowed
to equilibrate for 2 weeks at 70% WHC. Seeds of Indian mustard
(Brassica juncea) were sown directly into the soils (5 seeds per pot),
and the pots were thinned to 2 samplings per pot a week after
germination. Mustard was allowed to grow for 6 weeks in a glass
house, with natural sunlight providing a photoperiod of 12e14 h.
The temperature was maintained at 28  C. Three replications were
used and the pots were watered regularly. The shoots and roots
were measured individually for their dry weights after washing
with Milli-Q water and drying in an oven. Cadmium, Pb and Zn
concentrations of shoots and roots were analysed separately using
concentrated nitric acid. The plant digests were diluted with Milli-
Q water, filtered with a syringe filter and analysed for metals by ICP-
OES. The digestion was also carried out for blank and standard
samples. Phytoavailability of Cd, Pb and Zn was expressed as per-
centage of metal mass taken up by the plant with respect to total
metal mass in soil per each pot (Eq. (4)) (Cao et al., 2009).
Translocation coefficient (Tl) ¼ Shoot concentration/Root
concentration (6)
where, the soil and plant concentration are expressed in mg/kg on a
dry weight basis.
2.6. Long-term stability of immobilized metals
Soil samples pre-incubated with DAP or SPR and also P-un-
treated control soil for 30 days and 3 years were analysed for
bioavailability using NH4NO3 extraction and bioaccessibility using
simplified physiological-based extraction technique (SBET) metal
concentrations. For SBET analysis, 1 g of soil (<250 mm) was mixed
with 100 mL of glycine extracting solution (pH ¼ 1.50) in a 125 mL
wide mouth high density polyethylene bottle. The bottles were
shaken in end-over-end extractor at 30 ± 2 rpm for 1 h in 37  C
room to simulate conditions and residence time of solid material
within the stomach of mammals. Metal bioaccessibility was
expressed as percentage of metal mass extracted by SBET with
respect to the total mass in soil (Eq. (7)) (Cao et al., 2009). The mass
of metal extracted by SBET was calculated by multiplying metal

Metal concentration in shoot  shoot biomass

Phytoavailability ð%Þ ¼  100 (4)
Metal concentration in soil  soil mass

The effect of P compounds on the bioavailability of Cd, Pb and Zn concentration in the extract with the volume of SBET extract. The
in the naturally contaminated soil was evaluated by calculating the mass of the metal in the soil was calculated by multiplying metal
transfer of metal(loid)s (Eq. (5)) and translocation (Eq. (6)) concentration in the soil by with the mass of the soil used in SBET
coefficients. analysis.

Transfer coefficient (Tf) ¼ Plant tissue concentration/Soil

concentration (5)

Metal concentration in solution  solution volume

Bioaccessibility ð%Þ ¼  100 (7)
Metal concentration in soil  soil mass

Table 2
Effect of phosphate amendments on mobility, bioavailability, phytoavailability and bioaccessibility of Cd, Pb and Cd. Means with the same letter within a column for each metal
are not significantly different at p < 0.05 by Duncan’s multiple range test. (DAP ¼ Diammonium phosphate; SPR ¼ Sechura Phosphate Rock; CPR ¼ Christmas Island Phosphate
Rock).

Heavy metal P treatment Immobilization Mobility (%) Bioavailability (%) Phytoavailability (%) Bioaccessibility (%)

Cadmium Control 15.4a 5.72a 2.35a 12.3a 56.7a

DAP 8.65c 4.21b 1.02b 7.81b 21.7c
SPR 12.2b 5.18a 2.18a 11.2a 32.7b
CPR 14.6a 5.36a 2.42a e e
Lead Control 5.12a 1.26a 1.26a 3.89a 35.5a
DAP 0.52c 0.08c 0.12c 0.45c 13.3c
SPR 1.87b 0.37b 0.67b 1.01b 22.1b
CPR 4.72a 1.12a 1.28a e e
Zinc Control 9.82a 4.27a 3.23a 7.65a 45.2a
DAP 2.73c 1.20c 2.01b 4.35b 16.7c
SPR 4.52b 2.11b 2.45b 6.75a 23.6b
CPR 8.72a 3.87a 3.26a e e
 B. Seshadri et al. / Chemosphere 184 (2017) 197e206 201

2.7. Statistical analysis influence the extent of P induced metal immobilization. Dissolution
of P from insoluble P compounds such as PR is an important factor
Three independent samples were used for all soil and P com- in controlling metal concentration because P should remain in soil
pounds analyses including pH, organic matter, and total metal(loid) solution to react with metals in aqueous phase (Ma et al., 1997).
and P contents. The immobilization, leaching, earthworm, and
plant growth experiments were conducted using three replications. 3.3. Leaching of metals
To test for significant differences between the metal(loid)s and P
compounds, statistical comparisons were made using analysis of The concentrations of Cd, Pb and Zn in the leachate were higher
variance (ANOVA) in Predictive Analytics SoftWare (PASW) statis- in the soluble DAP treatment than in the SPR treatment and control.
tics, release version 18.0.0 (SPSS, Inc., 2009, Chicago, IL). Duncan’s However, only a small fraction of the total metal (0.0049e0.015%)
multiple range test was used to compare the means of the treat- was leached from the soluble DAP treatment (Table 3). Metal
ments, variability in the data was expressed as the standard devi- mobility was lower in SPR than DAP treatment and the effect was
ation and a p < 0.05 was considered to be statistically significant. more pronounced in the case of Cd and Pb than Zn (Table 2).
Compared to the control, insoluble SPR treatment reduced total Cd,
3. Results and discussion Pb and Zn leaching by 43.2, 39.1 and 11.5%, respectively. A similar
study with the addition of bone meal to soil, showed a substantial
3.1. Soil and P compounds characterization reduction in release of Pb to solution (Sneddon et al., 2006). They
attributed this decrease to the formation of a sparingly soluble Pb
The various physicochemical properties of soils are presented in phosphate phase within the soil. Hong et al. (2010) found that
Table 1. The texture of soil was sandy loam. The pH of soil was phosphate materials such as fused phosphate and super phosphate
slightly acidic which may facilitate P solubilisation from insoluble alleviated Cd extractability from soils, where rise in soil pH and
PR used for metal immobilization. Ammonium nitrate extractable negative charge was linked to Cd immobilization rather than Cd-P
Cd, Pb and Zn concentrations were 4.5, 7.8 and 11.2% of total metal, precipitation.
respectively, indicating the relative difference in the bioavailability As expected, the leachate concentration of P was much higher in
of these co-contaminants. the soluble DAP than SPR treatment (Table 2). Although P com-
The P compounds vary in total and soluble P contents (Table 2). pounds were shown to be effective for the immobilization of metals
The solubility of P compounds is the primary factor in immobilizing in soils, there is a possibility of P leaching and the may result in
metals from contaminated soils (Ma and Rao, 1997). Based on the eutrophication of water bodies (Chen et al., 2007). The leachate P
2% citric acid solubility test, while SPR is considered ‘reactive’ concentration for SPR treated soil was negligible compared to DAP
(75.1% citric P) and CPR is ‘unreactive’ (2.35% citric P) (Hedley and treatment. The average P concentration in the leachate up to 10
Bolan, 1997). The effectiveness of PR as a P source for plant up- pore volumes for the soluble DAP treatment was 0.56 mg/L -
take and metal immobilization depends on both the solubility and exceeding the threshold concentration of 0.05 mg/L (ANZECC,
particle size (Szilas et al., 2008). 1992). Therefore, evaluation of P leaching under field conditions
is essential to avoid possible secondary contamination of P while
using soluble P sources for heavy metal immobilization (Cao et al.,
3.2. Immobilization of metals
2003).
The NH4NO3-extractable Cd, Pb and Zn concentrations were
3.4. Earthworm bioavailability of metals
lower with amendment by P compounds, which indicates that
metal immobilization was induced by those P compounds. DAP, SPR
Although there was no mortality of earthworms, some weight
and CPR immobilized Cd by 76.2, 35.5, and 4.61%, Pb by 83.5, 47.4
reduction in earthworms was noticed in both P treated and un-
and 7.42%, and Zn by 83.5, 51.2 and 4.35%, respectively (Table 2). The
treated soils. The weight of the earthworms in soluble DAP treated
water soluble DAP was the most effective in the immobilization of
soil was lower in comparison with SPR and control treatments,
all 3 metals. The immobilization of metals was significantly higher
which may be due to the higher accumulation of metal(loid)s in
in reactive SPR than unreactive CPR amended soil. The metal sol-
those earthworms. Bioaccumulation of Cd, Pb and Zn in earth-
ubility from both contaminated soils and the P compounds
worms was the highest in soluble DAP treated soil, but did not show
significant difference for Cd and Pb compared to the control
Table 3 (Table 2). Bioaccumulation of Cd, Pb and Zn in SPR amended soil as
Biomass and Cd, Pb and Zn concentrations in shoots and roots of mustard (expressed calculated using Eq. (3) was 0.018, 0.009 and 0.034%, respectively
on a dry weight basis) grown in shooting range soil with or without P treatments.
which was significantly lower than the other treatments (Table 2).
Means with the same letter within a row are not significantly different at p < 0.05 by
Duncan’s multiple range test. (DAP ¼ Diammonium phosphate; SPR ¼ Sechura
Reactive SPR treatment reduced earthworms’ Cd, Pb and Zn loading
Phosphate Rock). by 17.8, 32.56 and 11.2%, respectively compared to the control.
Darling and Thomas (2005) noticed that Pb bioaccumulation by
Parameter Control DAP SPR
earthworm, Lumbricus terrestris was higher in the presence of sol-
Dry weight (g/pot) 2.15c 13.4a 9.78b uble lead acetate trihydrate than less soluble lead carbonate. Ownby
Shoot Cd (mg/kg) 45.6a 12.4b 18.7b
Root Cd (mg/kg) 213a 34.6b 45.7b
et al. (2005) noticed that in a Pb and Zn mixed metal(loid)
Shoot Pb (mg/kg) 178a 23.2b 34.5b contaminated soil, while soluble triple super phosphate decreased
Root Pb (mg/kg) 897a 123c 176b Pb bioavailability to earthworm, the Pb bioavailability on PR appli-
Shoot Zn (mg/kg) 198a 43.4b 53.5b cation was not reduced which may be attributed to the competition
Root Zn (mg/kg) 675a 134c 231b
of Zn with Pb towards low levels of P released by PR. Sanderson et al.
Transfer factor (Tf) for Cd 0.951a 0.154b 0.204b
Translocation factor (Tl) for Cd 0.214b 0.358a 0.409a (2014) examined ecotoxicity of shooting range soils after chemical
Transfer factor (Tf) for Pb 0.091a 0.012b 0.018b stabilization with a range of amendments, including P. Soft Rock
Translocation factor (Tl) for Pb 0.198a 0.189a 0.196a phosphate was found to significantly reduce earthworm weight
Transfer factor (Tf) for Zn 1.03a 0.204b 0.353b loss, and mortality in the most contaminated soil. The uptake of Pb
Translocation factor (Tl) for Zn 0.293a 0.324a 0.232a
was greatly reduced by soft rock phosphate in three soils, however
202 B. Seshadri et al. / Chemosphere 184 (2017) 197e206
the uptake of Zn and Cu increased. Zn was more readily uptaken by
earthworms relative to total concentration than Pb.
In DAP amended soil, while NH4NO3-extractable metal concen-
tration decreased compared to the control, water-extractable metal
concentration was the highest among the treatments. Direct diffu-
sion from soil solution to earthworm is the primary reason for the
metal accumulation in earthworms (Spurgeon and Hopkin, 1999).
Addition of DAP to soil not only increases DOC, but also improves the
concentration of water-extractable metals, thereby resulting in
greater metal accumulation in earthworms in soluble DAP treated
soil. It has been reported that acidification caused by nitrification in
DAP and competition of P for sorption sites are likely to increase
DOC in solution (Giesler et al., 2005). Additionally, the activity of
earthworms in the soil has been reported to increase the pH of the
soils, enhance DOC and increase the water soluble, exchangeable
and carbonate fraction of metal(loid)s (Wen et al., 2004).
3.5. Plant bioavailability of metals
In the shooting range soil, mustard plants showed symptoms of
necrosis and stunting in the absence of P amendments, thereby
showing the effects of heavy metal toxicity (Tarradellas et al., 1996;
Chen et al., 2007). Both soluble DAP and reactive SPR treatments
significantly increased shoot and root weight. Plant dry weight
increased by 560 and 367% for DAP and SPR, respectively compared
to the control (Table 3), which is attributed to both increased supply
of P and decreased bioavailability of heavy metals. Chen et al.
(2007) noticed that the addition of P amendments to a Pb
contaminated soil increased the shoot and root biomass of cauli-
flower (Brassica oleracea L.), which was attributed to the rise in P
nutrition and reduction in Pb toxicity. In a plant growth study using
soybeans by Cao et al. (2009), they applied PR and phosphoric acid
to a soil contaminated with mixed metals (Cu, Pb and Zn) and
observed higher above-ground biomass. However, the soil treated
70
60
50
SBET (%)
40
30
20
10
0
Fresh Aged
Cd
Fig. 1. Effect of phosphate compounds on SBET of Cd, Pb and Zn in the shooting range soil. (S ¼ soil alone (control); DAP ¼ soil þ DAP; SPR ¼ soil þ SPR; CPR ¼ soil þ CPR;
Fresh ¼ incubated with P for 30 days; Aged ¼ incubated with P for 3 years). Means with the same letter within a category are not significantly different at p < 0.05 using Duncan’s
multiple range test. Each value represents the mean of three replicates with standard deviation shown by error bars. (DAP ¼ Diammonium phosphate; SPR ¼ Sechura Phosphate
Rock; CPR ¼ Christmas Island Phosphate Rock).
with PR only showed no increase in biomass.
Soluble DAP and reactive SPR decreased metal concentration of
mustard by 56.7 and 34.4% for Cd, 78.9 and 56.7% for Pb and 39.2
and 23.4% for Zn, respectively compared to the control (Table 3).
The decrease in metal concentration resulting from P addition
could be attributed to increased biomass (i.e. dilution effect) and
decreased bioavailability of metals. Cao et al. (2009) observed that
an increase in the biomass can offset the reduction in metal con-
centration, thereby maintaining the same phytoavailability. Cad-
mium, Pb and Zn phytoavailability in soluble DAP amended soil was
1.24, 0.23 and 22.34%, which was the lowest and attributed to the
significant low concentration of metals in biomass. Both DAP and
SPR decreased the transfer coefficient (Tf) for all 3 metals, but there
was no effect of P amendments on translocation coefficient (Tl)
values for these metals. It has often been found that immobilization
of metals using soil amendments results in a decrease in their
concentrations in soil solution thereby decreasing metal transfer
coefficients. Phosphorus application was found to be effective in
remediating metal contaminated sites, especially in the presence of
vegetation which is essential for containing metal contaminants
(i.e. phytostabilization) (Bert and Cunningham, 2000; Bolan et al.,
2013a).
3.6. Long-term stability of immobilization
Compared to fresh soils, the incubated soils (for three years)
showed significantly lower ammonium nitrate extractable metal
concentration, especially in Zn and Pb (Fig. 1). For example, in the
case of DAP treatment, NH4NO3 extractable Cd, Pb and Zn con-
centration decreased from by 23.4, 67.8 and 54.7%, respectively,
after 3 years (Fig. 1). Cao et al. (2009) observed lower metal solu-
bility due to natural attenuation of metals as a result of aging in
contaminated soil.
Simplified Bioaccessibility Extraction Test (SBET) is being used
Control
DAP
SPR
CPR
Fresh Aged Fresh Aged
Pb Zn
 B. Seshadri et al. / Chemosphere 184 (2017) 197e206
to estimate the amount of contaminants absorbed via the ingestion
of soils and other similar media (Hu et al., 2011; Denys et al., 2012).
Phosphate amendments decreased bioaccessibility of metals as
measured by SBET. Bioaccessibility of Cd, Pb and Zn in the control
soil was 34.5, 56.7 and 76.3%, respectively. Reactive SPR and DAP
reduced bioaccessible Cd by 40.2 and 60.5%, Pb by 67.1 and 78.9%
and Zn by 35.6 and 56.5% compared to the control, respectively. The
effect of P amendments on bioaccessibility was higher in the case of
Pb and Zn than Cd, which may be because of the formation of
insoluble Pb and Zn phosphates (Cao et al., 2009). Although some
studies have shown that Cd solubility can be controlled using
Cd3(PO4)2 in P amended soils (Hong et al., 2010; Santillan-Medrano
and Jurinak, 1975; Street et al., 1978), the high solubility of
Cd3(PO4)2 is a disadvantage in terms of controlling Cd concentra-
tion in soils. Therefore, the chances are minimal for this solid phase
to influence Cd immobilization (Bolland et al., 1977; Soon, 1981).
Similar to bioavailable metal concentration, the bioaccessibility also
decreased with aging especially in the case of Pb and Zn. Tang and
Yang et al. (2012) demonstrated the effectiveness of soluble P
amendment in the long-term (over 5 years post-treatment) for
reduction in Pb bioaccessibility, leachability, and phytoavailability
by formation of lead phosphate.
In a study by Hao et al. (2010), application bone char to soil (1, 2
and 5% wt/wt) significantly reduced the concentration of bio-
accessible Pb in soil, after 3 month incubation. From the results,
they recommended that 1% bone char addition was adequate for
the precipitation of pyromorphite. Scheckel et al. (2005) observed
METAL IMMOBILIZATION
Adsorption Precipitation
PHOSPHATE
COMPOUNDS
Fig. 2. Schematic diagram illustrating the value of phosphate compounds on the immobilization of mixed contaminants.
203
increased precipitation of pyromorphite because of the extraction
procedures used in bioaccessibility tests such as physiologically
based extraction test (pH 2) and SBET induced the precipitation of
pyromorphite. Cao et al. (2009) also showed that SBET extraction
solution can influence higher dissolution of soil Pb and P in PR to
allow more Pb phosphate formation, due to high acidity (pH 1.5) in
the SBET solution. Hence, reduction in Pb bioaccessibility and long-
term stability of Pb contaminated soil can be effectively carried out
in the presence of both PR and soluble P.
3.7. Mechanisms for phosphate-induced immobilization of metals
Phosphate compounds including phosphates rocks (PRs) are
known to both release and also adsorb heavy metals, thereby acting
as a source and sink of heavy metals, respectively. Mobilization and
immobilization are the processes that enables PRs to be source and
sink, correspondingly. Direct adsorption/precipitation of metals by
P compounds, precipitation as a result of PR’s liming ability and
metal adsorption induced by phosphates (anions) are shown to be
the main pathways of metal immobilization by P compounds
(Fig. 2).
Direct adsorption of metals: Apatite mineral and the effect of pH
on the order of heavy metal adsorption by apatite mineral has been
studied by many researchers (Chen et al., 1997a). For instance, Zn
(Brudevold et al., 1963), Cd (Middelburg and Comans, 1991), Sr
(Lazic and Vukovic, 1991), and Ni and Cu adsorbed to hydroxyap-
atite surfaces has been observed (Misra et al., 1975). Similarly, in a
Acidification Liming effect
204 B. Seshadri et al. / Chemosphere 184 (2017) 197e206
study by Seaman et al. (2001), they showed a decrease in concen-
tration of several metals as a results of apatite addition in
contaminated pond sediments. Suzuki et al. (1981) demonstrated
that Ca2þ exchange of the metal cations from the apatite particle in
the soil solution as the main mechanism of metal adsorption onto
apatite. Studies have confirmed this mechanism for Cu and Zn (Cao
et al., 2004, 2009).
Similarly, Mignardi et al. (2012) examined the effect of phos-
phate on metals (Cd, Cu, Pb and Zn) in mine wastes and attributed
ion exchange and metal complexation onto the phosphate grains
surface as the mode of immobilization of those metals. However, Xu
et al. (1994) and He et al. (2013) concluded that co-precipitation
and surface complexation as the most significant mode of adsorp-
tion for Cd (Eq. (8)), while solid-state diffusion and ion exchange
were considered potential contributors of hydroxyapatite related
adsorption process.
Ca10 ðPO4 Þ6 ðOHÞ2 þ xCd2þ /ðCdx Ca10x ÞðPO4 Þ6 ðOHÞ2 þ xCa2þ
(8)
Phosphate-Induced Metal Adsorption: Anion-induced metal
adsorption has been observed for a number of cations (Bolan et al.,
2013b). For example, Naidu et al. (1994) showed that the presence of
H2PO 2
4 and SO4 ions increased Cd
2þ
adsorption by an Oxisol and a
Xeralf significantly. In a subsequent study by Bolan et al. (1999b),
they observed significant increases in Cd2þ adsorption in two
variable-charge soils when P addition was incrementally increased
(Bolan et al., 1999b). Other similar studies showed that the presence
of Al and Fe oxides with P enhanced the adsorption of Zn2þ, Cd2þ or
Cu2þ by variable charge components (Agbenin, 1998; Barrow, 1987;
Bolland et al., 1977; Krishnamurti et al., 1999) thereby increasing
surface negative charge.
There are many processes that can favour adsorption of metals
in soils in the presence of H2PO 4 such as, (i) rise in negative charge
(ii) co-sorption of metal and H2PO 4 as an ion pair; and (iii) surface
complex formation of metal on the P compound. The amount of
surface charge acquired through specific adsorption depends on
the nature of anion adsorbed, pH and electrolyte concentration of
the solute, which can also lead to increase in surface negative
charge (Eq. (9)) (Bolan et al., 1999a).
FeOOHðsÞ þ /HPO2  
4 4FeOHP4 þ OH (9)
Subsequently, Bolan et al. (1999b) achieved a significant corre-
lation between the increase in Cd2þ adsorption and the rise in
negative charge because of H2PO 4 addition. Interestingly, Cd
2þ
occupied just a minor portion of the increased negative charge. The
results also indicated that there the competition towards adsorption
sites was dominated by Ca2þ compared to Kþ, confirming the ob-
servations by Boekhold et al. (1993). Helyar et al. (1976) and Bolland
et al. (1977) proposed that the increased adsorption of Ca2þ and
Zn2þ onto H2PO 4 -treated gibbsite and goethite may be attributed to
the surface complex formation. Xu et al. (1994) concluded that
surface complexation as one of the mechanisms involved in the
adsorption of metals (Cd and Zn) by hydroxyapatite (Xu et al., 1994).
Precipitation of Metals: Immobilization of metals such as Zn and
Pb is predominantly governed by the precipitation as metal phos-
phates. The application of P is a widely accepted approach for
managing metal-contaminated soils because of the very low solu-
bility of the metallic phosphates, over a broad pH range. Ford and
Sparks (2000) used X-ray absorption fine structure spectroscopy
(XFAS) to study the precipitation processes and observed that the
precipitation on the surface may occur even when solution con-
centration is under-saturated with respect to homogeneous pre-
cipitation of pure metal precipitate phase.
There are many studies showing the precipitation Pb phosphate
minerals. The reaction of P with Pb is predominantly by the for-
mation of the highly insoluble pyromorphites [Pb5(PO4)3(OH, Cl, F)]
(Nriagu, 1974; Bolan et al., 2003; Stanforth and Qui, 2001; Cao et al.,
2003). In the presence of sufficient P, Pb and Cl, chloropyr-
omorphite is formed instantly according to Eq. (10).
5Pb2þ þ 3H2 PO 
4 þ Cl ¼ Pb5 ðPO4 Þ3 ClðsÞ þ 6H
þ
(10)
Laperche and Traina (1998) suggested two processes for the
reaction of dissolved Pb with apatite. (i) Dissolving of hydroxyap-
atite (HA, Ca10(PO4)6(OH)2) enables the reaction of Pb with apatite
(Eq. (11)), followed by precipitation (Eq. (12)) of pure hydroxypyr-
omorphite (Pb10(PO4)6(OH)2) as defined by Ma et al. (1993). (ii) Ca
in the apatite can be replaced by Pb (Eq. (13)). Hence there is
possible formation of Ca-Pb-apatite firstly by dissolving HA and
then via. coprecipitation of mixed apatites. Lead immobilization as
pyromorphite is mainly influenced by the dissolution of apatite. For
instance, research by Laperche et al. (1997) and Zhang et al. (1997)
showed that the pyromorphite formation was limited in alkaline
soils because of limited dissolution of PRs.
þ 
Ca10 ðPO4 Þ6 ðOHÞ2 þ 14HðaqÞ /10Ca2þ
ðaqÞ þ 6H2 PO4 ðaqÞ þ 2H2 O
(11)

10Pb2þ
ðaqÞ þ 6H2 PO4 ðaqÞ þ 2H2 O/Pb10 ðPO4 Þ6 ðOHÞ2ðsÞ (12)
 
Ca10 ðPO4 Þ6 ðOHÞ2ðsÞ þ xPb2þ / Cað10xÞ Pbx ðPO4 Þ6 ðOHÞ2ðsÞ
þ xCa2þ
(13)
In most arable soils, the formation of pyromorphite is restricted
because of the ability of these soils to readily adsorb both metal
ions and H2PO 4 , thereby resulting in low concentrations of these
ions in soil solution. Hashimoto et al. (2009) found organically
complexed Pb limited transformation to pyromorphite in phos-
phate amended soils. Pb solubility in firing range soils has also been
suggested to be controlled by desorption reaction from iron oxy-
hydroxides (Stanforth and Qui, 2001; Sanderson et al., 2015a,b).
However, under acidic and reducing conditions, some metal ion
adsorbing components (e.g. ferromanganese oxyhydroxides) and
H2PO 4 are prone to undergo dissolution, thereby increasing their
concentrations and subsequently favouring metal phosphates
precipitation, in most geological environments.
Acidification of P fertilizer and liming potential of PRs: When
soluble P fertilizers such as DAP dissolve in soil solution they
release phosphoric acid which dissociates into phosphate ions and
acidic hydrogen ions. The oxidation of ammonium to nitrate in DAP
fertilizer also results in the release of acidic hydrogen ions. These
acidic hydrogen ions promote the solubilisation of metal ions such
as Cd, Pb and Zn, thereby facilitating their subsequent precipitation.
In the case of natural P fertilizers such as PRs, in addition to sup-
plying P and Ca, they can also have a liming value. The liming action
of PRs can occur through two processes. Firstly most PRs contain
free calcium carbonate (CaCO3) which can act as a liming agent in
its own right. Secondly, the dissolution of PRs in soils consumes
acids and thereby reduces the soil acidity.
Apart from the new solid phases formed as discussed above, the
liming potential of hydroxyapatite can also encourage metal
immobilization as a result of metal oxy-hydroxides formation
(Basta et al., 2001; Chen et al., 1997b; Ma and Rao, 1997). The ability
of PRs to neutralize acids with their liming potential is well docu-
mented, especially in the case of alleviating acid mine drainage
 B. Seshadri et al. / Chemosphere 184 (2017) 197e206
(Frazer, 2001). The another benefit of PR in the mitigation of acid
mine drainage with PRs is their ability to reduce the solution
concentration of metals through precipitation and ion exchange
reactions (Feng et al., 2000).
4. Conclusions
Through this study, it is clear that the soluble and insoluble P
compounds are effective in the immobilization of metals, especially
Cd, Pb and Zn. The variation in the solubility of the P compounds
and the nature of metal phosphate compounds formed influenced
the differential effect of P-induced immobilization between P
compounds and metals. Nevertheless, surplus amount of soluble P
compound can cause eutrophication of water bodies. Moreover,
soluble P compounds increase water extractable metal concentra-
tion by increasing DOC. Therefore, care should be taken while
applying soluble P compounds by evaluating the potential adverse
effects of the compound in the environment. Through this study, it
is evident that both soluble and insoluble P compounds have
shown to achieve long-term stabilization of Pb and Zn as measured
by bioavailable (NH4NO3 extractable) and bioaccessible (SBET)
metal concentrations.
Acknowledgements
Cooperative Research Centre sponsored this study for Contam-
ination Assessment and Remediation of the Environment (CRC
CARE), Australia in collaboration with University of South Australia.
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