Chemosphere 86 (2012) 1072–1078

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Chemosphere
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The effects of aging time on the fraction distribution and bioavailability of PAH
Lili Ma, Jing Zhang, Lisi Han, Weiming Li, Li Xu, Feng Hu, Huixin Li ⇑
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China

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

Article history: Understanding the effects of aging time on the fraction distribution and bioavailability of PAH, such as
Received 4 June 2011 phenanthrene (PHE) and pyrene (PYR), has considerable benefits for risk assessment, food security and
Received in revised form 27 November 2011 remediation strategies for contaminated soil. The results of the present study show that the proportion
Accepted 28 November 2011
of the desorbed PHE decreased from ca. 82% at day 0 to ca. 65% at day 150. In addition, non-desorbed
Available online 9 January 2012
PHE increased from ca. 18% at day 0 to ca. 31% at day 150, whereas the changes of desorbed and non-
desorbed PYR showed no significant trend during this aging period. The proportion of desorbed PYR
Keywords:
was lower than that of PHE, whereas the opposite occurred with the non-desorbed fraction. After
Phenanthrene
Pyrene
150 d of aging, the proportion of bound residues (PHE and PYR) increased significantly with the cultivat-
Aging time ing time from ca. 0.2% to ca. 4.7% and ca. 0.1% to ca. 1.2% for PHE and PYR, respectively. In addition, the
Fraction distribution bioavailability of PAH (PHE and PYR) to earthworms was also assessed over 0–150 d. The results showed
Bioavailability that the uptake rate and bioconcentration factor (BCF) of pollutants by earthworms displayed the follow-
ing biphasic character: a rapid decrease over the first 15 d followed by a slow decrease over the next
135 d. Moreover, the earthworm uptake rate of PHE was greater than that of PYR throughout the incuba-
tion period, indicating that PHE has a higher bioavailability than PYR. In addition, the positive correlation
between the uptake rate of earthworms and PAH extractability suggested that a three-step extraction is a
reliable approach to predict PHE bioavailability in soil. However, a limit was observed for PYR.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction of their toxic, mutagenic and carcinogenic properties, PHE and

When organic pollutants enter into soil, they undergo a number
of processes such as volatilization, leaching, chemical and biologi-
cal degradation. Additionally, sorption and transportation pro-
cesses may cause these chemicals to go into the soil matrix or
mineral particles, combine with organic substances in the soil or
be restrained in the micropore, becoming a non-extractable sol-
vent or ‘‘irreversibly bound’’ (Macleod and Semple, 2003). How-
ever, with the exception of microbial degradation, organic
pollutants may undergo mainly sorption and transportation pro-
cesses (Li et al., 2007). These pollutants may stay in soil for a long
time, resulting in potential health or biological risks (Wang and
Zhu, 2003). Therefore, the extractable or total amount of PAH
may be of limited utility when assessing their environmental sig-
nificance. Instead, the fraction distribution and availability of these
contaminants are the most important indices for risk assessment.
PHE and PYR are 2 out of 16 PAH on the EPA’s priority list that
are widely found in the environment (Gana et al., 2009). Because
⇑ Corresponding author. Tel./fax: +86 025 84395374.
E-mail addresses: malifeng19830615@126.com (L.L. Ma), nancy-zj@163.com
(J. Zhang), hanlisiwawawa@sina.com (L.S. Han), 2010103100@njau.edu.cn (W.M. Li),
xuli602@njau.edu.cn (L. Xu), fenghu@njau.edu.cn (F. Hu), huixinli@njau.edu.cn
(H.X. Li).
PYR have received great attention in environmental problems.
The forms of organic contaminants in soil have been previously
reported (Lesan and Bhandari, 2004; Strynar et al., 2004). Macleod
and Semple (2003) observed a significant decrease in PYR in the
extractable fraction using a 14C-radiolabeling technique, whereas
the bound residual fraction increased as a long soil-PYR contact
time. However, in this study, the PAH in soil were separated into
three fractions: desorbing, non-desorbing, and bound residual frac-
tions using the following sequential extraction mass balance ap-
proach (Gao et al., 2009): hydroxypropyl-b-cyclodextrin (HPCD),
dichloromethane:acetone (1:1, v:v), and sodium hydroxide-dichlo-
romethane extractions, respectively.
Because the earthworm has relatively high body-lipid content
and a range of PAH detected in its tissues, it is widely used as an
indicator for determining the ecological risks of soil (OECD, 2000).
Numerous extraction techniques have been developed to predict
the bioavailability of PAH to earthworms (Hawthorne et al., 2000;
Reichenberg and Mayer, 2006). Recently, an HPCD aqueous solution
extraction was developed and applied to assess the bioavailability
of PAH, including naphthalene (Patterson et al., 2004), phenan-
threne (Allan et al., 2006), pyrene (Doick et al., 2006), B[a]P (Reid
et al., 2000b), linear alkyl benzenes (Dew et al., 2005), hexadecane
and field-aged PAH contaminated sediments (Cuypers et al., 2002).
Moreover, Reid et al. (2000b) demonstrated that PAH extractability

0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.11.065
 L.L. Ma et al. / Chemosphere 86 (2012) 1072–1078
with HPCD was closely related to PAH mineralization by microor-
ganisms. Thus far, little is known about the relationship between
the different morphology of aged PAH and PAH bioavailability to
earthworms.
The objectives of the present study are: (1) to measure the frac-
tion distribution of PAH using a ‘‘3-step’’ sequential extraction; (2)
to evaluate the uptake rate and BCF of earthworms using the Soxh-
let extraction method; and (3) to explore the relationship between
the fraction distribution and PAH bioavailability (PHE and PYR).
2. Materials and methods
2.1. Soil
Sand loam soil (0–20 cm) was collected from Rugao City, Jiang-
su Province, China. Its physical and chemical properties are pre-
sented in Table 1.
Soil pH and organic matter were determined using a calibrated
pH electrode in water (1:2.5, soil:water) and the K2Cr2O7 oxidation
method, respectively. Soil CEC was measured by the CH3COONH4
method, and clay content analyses were determined by sedimenta-
tion using 40 g of air-dried soil. The total N and P contents were
determined using the Kjeldahl method and H2SO4–H2O2–HF diges-
tion, respectively.
2.2. Earthworms
An adult Eisenia fetida earthworm (obtained from the Nanjing
Special Animal Factory, China) is defined to have an average weight
of 0.25–0.30 g and well-developed clitella. These earthworms were
laboratory-bred in soil at 20 °C in the dark and fed on cattle man-
ure. The soil moisture was maintained at 70% of the water holding
capacity using tap water. All earthworms were allowed to acclima-
tize to the laboratory conditions for at least 2 weeks before they
were used in experiments.
2.3. Chemicals
Phenanthrene (purity P 97%, FLUKA company), pyrene
(purity P 98%, Alfa Aesar Company), hydroxypropyl-b-cyclodex-
trin (HPCD, purity P 98%, Shandong Zibo Health Fine Chemical
Co., Ltd.), methanol (HPLC pure), NaN3 (purity P 98%, Nanjing Sang
Hing Biotechnology Co., Ltd.), and other commonly used reagents
were of analytical grade.
2.4. Soil spiking
Soil was air-dried and passed though a 2 mm sieve. A total of
2.4 kg of soil was evenly placed in 48 narrow-mouth 100-mL glass
bottles (50.0 g of soil per bottle) and immediately sterilized using
2.5 M rad c-irradiation with a 60Co source to minimize microbial
activity (Sabate et al., 2006). Under sterile conditions, PHE and
PYR dissolved in acetone were added drop-wise to the soil. The
mixture was shaken well for even distribution and evaporated
overnight to produce the initial PHE and PYR concentrations of
50 mg kg1 and 100 mg kg1 (dry mass of soil), respectively. Sterile
deionized water was added to maintain a moisture content of 70%
Table 1
Physiochemical properties of the soil.
Soil type pH Organic Total N Total P CEC
carbon (g kg1) (g kg1) (cmol kg1)
(g kg1)
Sand loam soil 7.55 5.42 0.65 0.66 7.2
1073
water holding capacity (WHC), which is suited for the incubation of
earthworms. After sterilization, the bottles were tightly capped
with bottle caps inside aluminum foil (the gap between the bottle
mouth and cap was sealed with Paraffin) and stored in the dark at
room temperature (22 ± 1 °C) to allow for aging. Each treatment
was performed in quadruplicate. Treatments were also destruc-
tively sampled and analyzed at 0, 15, 30, 60, 90 and 150 d. At each
time point, soil sterility was confirmed by the lack of clones on
trypticase-soy agar.
2.5. Bioavailability experiments
At 0, 15, 30, 60, 90 and 150 d after spiking, a sub-sample (3.0 g)
was collected to determine the concentration of pollutants, and the
remaining soil was transferred into a 150-mL Erlenmeyer flask to
cultivate the earthworms. Six earthworms of similar age and size
were put into an Erlenmeyer flask and sealed with foil that had
eight pinholes for ventilation. Sterility was no longer maintained,
and no additional food was provided for the earthworms. There
were 288 individuals in total. After 7 d of incubation in the dark-
ness at 22 ± 1 °C, the earthworms were removed from the Erlen-
meyer flask, rinsed with distilled water, and placed on moist
filter papers for 24 h to evacuate their guts. Prior to Soxhlet extrac-
tion, earthworms were frozen in liquid nitrogen, mixed with anhy-
drous sodium sulfate three times their weight, and subsequently
crushed using a pestle and mortar. The ground earthworm tissues
were then packed on filter paper and stored at 4 °C for extraction.
2.6. PAH extraction from soil: The 3-step sequential extraction
A 3-step sequential extraction method (Gao et al., 2009) was
used to extract different morphological PAH in soil.
2.6.1. Desorbed fraction
A mild extraction technique was adapted from the methods de-
scribed by Reid et al. (2000b) and Cuypers et al. (2002). Soil (3.0 g)
was placed in a 25-mL centrifuge glass tube, and 15 mL of
70 mmol L1 HPCD extraction solution containing 0.05% NaN3
was added. Tubes were then closed, shielded from light and shaken
horizontally at 150 rpm. At intervals corresponding to the total
extraction times of 24, 60, and 240 h, tubes were centrifuged in a
Beckman J2-21 centrifuge for 25 min at 2000 rpm to separate soil
from the aqueous extraction solution. Fresh mild extraction solu-
tion (15 mL) was added to soil after 24, 60, and 240 h. The super-
natant was liquid–liquid extracted three times using 10 mL of
dichloromethane. Organic phases were dehydrated by percolation
through Na2SO4 anhydride, combined, and concentrated to near
dryness in a rotary evaporator at 45 °C. The residue was then
reconstituted in 5 mL of methanol. Before HPLC analysis, the solu-
tion was passed through a 0.22 lm Teflon filter to remove particu-
late matter.
2.6.2. Non-desorbed fraction
After 240 h of mild extraction, the soil was dried at 37 °C, a
10 mL dichloromethane and acetone mixture (1:1, v:v) was added
for extraction by ultrasound for 10 min, and this process was re-
peated 4 times. After extraction, the resulting liquids were com-
bined, evaporated, and dissolved as described above.
2.6.3. Bound residue fraction
After obtaining both desorbed and non-desorbed fractions,
10 mL of 2 mol L1 NaOH was added to each vial and sealed under
Clay
content N2 atmosphere. The vials were closed and subsequently heated at
(g kg1) 100 °C for 2 h. The aqueous fraction was obtained by centrifugation
110.4 in a Beckman J2-21 centrifuge at 2000 rpm for 25 min, acidified
with 6 mol L1 HCl to pH < 2, and liquid–liquid extracted three
1074 L.L. Ma et al. / Chemosphere 86 (2012) 1072–1078
times with 10 mL of dichloromethane. The organic fractions were
evaporated and subsequently dissolved as described above.
2.7. PAH extraction from earthworms via the Soxhlet extraction
method
To carry out Soxhlet extraction, ground worm tissues were
placed into a cellulose extraction thimble (25 mm  80 mm,
Whatman International, Maidstone, England) and extracted for
10 h with 100 mL of dichloromethane and hexane (1:1, v:v; HPLC
grade) in Soxhlet extractors at 5–6 min per cycle. The extracts were
concentrated to near dryness in a rotary evaporator at 45 °C, and the
remaining residue was reconstituted in 5 mL methanol.
2.8. Analytical methods
The HPLC system used was equipped with a UV-visible detector
(LC-20AT, SPD-20A/20AV) and a reverse-phase C18 column
(4.6  250 mm). The column, with a pore size of 12.0 nm, was pur-
chased from the SHIMADZU Corporation. The packing materials
were composed of totally porous, high purity spherical silica parti-
cles. These silica particles were chemically bound to octadecylsilyl
(ODS) groups and thoroughly end-capped. The column was in-
stalled in a Column heater (QT-330) with a constant temperature
of 30 °C. Methanol (100%) was used the eluent solvent at a flow
rate of 1.0 mL min1 under isocratic conditions. Detection was car-
ried out at 254 nm, and the injection volume of the sample was
20 ll. The recovery rates of phenanthrene and pyrene are 93.1%
and 89.49%, and the relative standard deviations (RSD) are 3.34%
and 4.05% (n = 5), respectively. Additionally, the detection limits
are 44.1 pg and 50.2 pg, respectively.
2.9. For the explanation of earthworm uptake rate, BCF
The earthworm uptake rate refers to the amount of pollutants
absorbed and utilized by the earthworm divided by the original
amount of pollutants.
The earthworm bioaccumulation factor (BCF) was calculated as
the ratio between the concentrations of PAH measured in earth-
worms and the concentration of the same chemical in the soil at
different aging stages. The BCF reflects the level of hydrophobic or-
ganic contaminants entering in earthworms from the soil by
assigning access.
2.10. Statistical analysis
The data were analyzed using the SPSS program (Standard Ver-
sion 13.0, SPSS Inc.). The relationships between the earthworm up-
take rate, BCF on PAH (PHE and PYR) and aging time were tested
using the analysis of variance (ANOVA). The probability threshold
used for statistical significance was P < 0.01.
3. Results and discussion
3.1. The effect of aging time on the fraction variations of PHE and PYR
The quantity of the desorbed PAH (PHE and PYR) extracted was
high and progressively decreased during the following extractions.
The desorption percentage of PHE decreased generally over 0–
150 d, whereas the reverse was observed for non-desorbed
fractions (Fig. 1). The desorbed fraction of organic pollutants is typ-
ically the most bioavailable portion. Therefore, this decrease indi-
cates a decrease in the availability of PHE, which is consistent
with the uptake rate and the BCF of earthworms (posterior results)
as aging time prolonged. Because the microcosms were sterilized
and sealed during the incubation, the microbial degradation and
volatility of PAH could be ignored, and the sorption of PAH to the
glass wall of microcosms was observed to be negligible, indicating
that the organic substance of soil (SOM) may control and restrain
the behavior of the organic pollutant in soil (Alexander, 2000).
And the decrease in the desorbed PAH and bioavailability after
aging is closely related to the SOM. During the aging process, the
molecules of the organic pollutant go into the soil matrix, in which
the smallest microorganism, organization or higher biology may
not enter easily, making it harder for the pollutant to be extracted
with solvent (Hatzinger and Alexander, 1995; Lin and Pan, 2006).
The transfer of easy desorbing fractions to non-desorbing and
bound residues results in the apparent phenomenon that the first
extractability and bioavailability of the compounds are further de-
creased and the last two fractions increased (Kristensen et al.,
2001; Reeves et al., 2001; Sverdrud et al., 2002; Sun and Li, 2005).
As shown in Fig. 1, the rate of decrease in the desorption
percentage of PHE was quick during the first 15 d and evidently
slowed during the following 135 d. After 15 d of aging, the percent-
age of the desorbed fraction declined by 10.5%. However, from 15
to 150 d, the desorbed fraction only declined by 6.9%, which is con-
sistent with the increase in the percentage of the non-desorbed
fraction. After 15 d of aging, the percentage of the non-desorbed
fraction increased by 9.7%. However, from 15 to 150 d, the non-
desorbed fraction only increased by 3.1%. Aging of compounds in
soil occurs due to sorption in ‘rubbery’ and ‘glass-like’ regions of
SOM via isocratic diffusion or partitioning coupled with hole-filling
mechanisms. Rubbery-phase partitioning occurs initially. It is char-
acterized by rapid and no solute competitive sorption within soft
organic matrices such as humic or fulvic acids. Subsequently, a
slower and solute competitive sorption phase of kinetic diffusion
into condensed (thermodynamically glassy) microcrystalline voids
can commence over a longer period of time (Gunasekara and Xing,
2003; Henny and Noort, 2003).
Generally, the percentage of the desorbed fraction of PYR was
lower than that of PHE at each aging stage, which was inversely re-
lated to the non-desorbed fraction (Figs. 1 and 2). However, the
change of the desorbed and the non-desorbed fractions of PYR did
not show an obvious trend with aging time. HPCD is a cyclic oligo-
saccharide with a hydrophilic shell and a toroidal-shaped apolar
cavity. This oligosaccharide can form water-soluble inclusion com-
plexes by incorporating PAH in its cavities. A prerequisite for the
formation of the inclusion complexes is that the size and geometry
of the target molecule (PAH) and the HPCD cavity are complemen-
tary. Although this is the case for low molecular weight PAH, it can
be calculated that high molecular weight PAH are too large to fit in
the HPCD cavity. Wang and Brusseau (1995) calculated the sizes of
PHE (width–length: 0.58–0.78 nm) and PYR (width–length: 0.71–
Phenanthrene
Distribution percentage (%)
100
90
80
70
60
0 15 30 60 90 150
Aging time (d)
desorbing fraction non-desorbing fraction
bound residue fration
Fig. 1. The effect of aging time on the fraction variations of phenanthrene.

Pyrene
Distribution percentage (%)
100
90
80
70
60
0 15 30 60
Aging time (d)
desorbing fraction non-desorbing fraction
bound residue fraction
Fig. 2. The effect of aging time on the fraction variations of pyrene.
0.89 nm) and determined that PHE fit into the HPCD cavity com-
pletely (diameter–depth: 0.75–0.78 nm), whereas PYR can only be
partially included. The calculations carried out by the authors dem-
onstrate that the HPCD provides sufficient sorption capacity for the
extraction of bioavailable PHE. However, the HPCD is limited for
bioavailable PYR. Reid et al. (2000b) reported that KHPCD values
for high molecular weight PAH do not increase with molecular
weight as significantly as Kom and Koc values. Gao et al. (1998) also
reported that the compatibility degree decided the inclusion’s for-
mation and stability first, followed by molecular hydrophobic.
These results of PYR are due to the incompatibility of the PYR
molecular structure with the cyclodextrin cavity and the sorption
of PYR by soil humus which contains a hydrophobic group partially
included in the HPCD cavity.
Compared to the desorbing and non-desorbing PAH fractions,
the percentage of the PAH-bound residues was less than 5%, irre-
spective of PHE and PYR (Figs. 1 and 2). At day 0, the bound resid-
ual fraction of PHE or PYR accounted for a percentage of less than
0.2%. After 150 d, the bound residual fraction of PHE accounted for
4.72%, whereas that of PYR accounted for 1.21%. The percentage of
PHE and PYR showed an increasing tendency, which was consistent
with the study conducted by Christopher et al. (2003). The increase
might because that other PHE or PYR fractions partially convert
into the bound residue locked in the micropores of soil particles
or embedded in the reticular formation of humus with aging,
making it hard to be desorbed. The decreases in the desorbing
and non-desorbing PAH fractions over 0–150 d shown in Figs. 1
and 2 support this hypothesis. However, Gao et al. (2009) reported
that the concentrations of bound PAH residues generally increased
over 0–12 weeks and decreased at 12–16 weeks and suggested
that the bound residues may be conditionally released into the soil
environment.
3.2. The effects of aging time on the bioavailability of PHE and PYR
After cultivation for 7 d, the average weight loss rate of the
earthworms exposed to fresh spiked-soil was higher than aging
soil (Table 2), suggesting that the toxicity of fresh soil to earth-
worms was greater than that of aging soil. The average weight loss
rate of PHE was larger than that of PYR, suggesting that the toxicity
of 50 mg kg1 PHE to earthworms was greater than the toxicity of
100 mg kg1 of PYR. Other studies reported that the earthworm
mortality rate was up to 13.3% when exposed to 50 mg kg1 PHE
after being incubated for 7 d. Additionally, no significant difference
was observed for the earthworm mortality rate when exposed to
500 mg kg1 PYR for 7 d of cultivation compared with that of the
control (Song et al., 2003). The difference between the toxic effects
L.L. Ma et al. / Chemosphere 86 (2012) 1072–1078 1075
of PHE and PYR was related to their solubility in aqueous solutions
of soil (PHE: 1.3 mg L1, PYR: 0.03 mg L1), and greater solubility of
PHE caused greater toxic effects than that of PYR, even the concen-
tration of PYR was higher than PHE in the soil (Song et al., 2003).
With the increase in the soil-pollutant contact time, the earth-
worm uptake rate and BCF of both PHE and PYR decreased
(Figs. 3 and 4). This decrease may be a result of the aging process
reducing the extractability and availability of PAH by a progressive
increase of soil sorption. More molecular contaminants gradually
migrated from the point of easy desorption to difficult desorption.
Furthermore, PAH diffused into micro- and nanopores within the
90 150
solid phases of the soil, enhancing the diffusion limits of the uptake
process (Alexander, 2000; Gevao et al., 2000; Reid et al., 2000a,b;
Contreras-Ramos et al., 2008). The earthworm uptake rates of
PHE and PYR were 10.6% and 6.5% at 0 d and decreased to 6.1%
and 4.5% after 15 d, respectively. After 150 d of aging, the earth-
worm uptake rates of PHE and PYR were 2.8% and 2.4%, respec-
tively, which were 3.3% and 2.1% lower than that at 15 d of
aging. This was similar to Gao’s work that the BCF of some organic
pollutants such as hexachlorobenzene (Gao, 2009), HCH, o,p-DDT,
o,p-DDE, p,p-DDT and p,p-DDE in earthworms were quickly re-
duced from 0–15 d, and showed the slow decreasing trend over
the following 165 d (Gao et al., 2007, 2008, 2009). These confirmed
that the effects of aging on the bioavailability of pollutants are
mainly concentrated in the initial stages and weaken in the later
aging period, which is consistent with the aging rate of the
pollutants in soil.
The uptake rate of earthworms for PHE was higher at day 0, and
showed significant differences (P < 0.01) compared to later aging
times. However, there was no significant difference (P < 0.01) be-
tween the adjacent aging times after 60 d (Fig. 3). In addition, there
were no significant differences (P < 0.01) between 15 and 30 d of
aging time. With the exception of there being no obvious differ-
ence (P < 0.01) between 90 and 150 d for PYR, there was a signifi-
cant difference (P < 0.01) before an aging time of 90 d. The BCF for
PHE and PYR were also observed to be higher at day 0, with signif-
icant differences between day 0 and other aging days (P < 0.01).
BCF showed significant differences (P < 0.01) for PHE and PYR be-
fore 30 d and after 60 d of aging time. However, there were no sig-
nificant differences (P < 0.01) at 15 and 30 d (Fig 4). Slizovskiy and
Kelsey (2010) reported that the bioaccumulation of pollutants de-
clined as the aging of p,p0 -DDE and anthracene increased from 3 to
203 d. Moreover, tissue concentrations between treatments at each
time before 103 d were statistically declined (p < 0.05) and de-
creased slowly from 103 to 203 d. Compared to later aging soil,
the two indexes of fresh spiked-soil were also observed to increase,
indicating that PAH were more easily enriched in the organism at
the start of incubation. Morrison et al. (2000) also reported that
hydrocarbons that were added to soil for aging a long time are
far less available than those freshly added.
The earthworm can accumulate organic compounds from both
aqueous and solid phases of soil via two differing uptake routes
(Gevao et al., 2000). The first route involved simple diffusion across
the epidermis of the earthworm, which can be described by equi-
librium assignment and diffusion models. This uptake is facilitated
by the hydrophobic property of PAH, resulting in a partitioning of
soil-associated PAH in lipid-rich earthworm tissues. The second
route involved the diffusion of contaminants across the gastroin-
testinal tract and enrichment in the earthworm tissues, which oc-
curs when PAH are ingested together with soil (Krauss et al., 2000;
Stroo et al., 2000).
With the aging time, the earthworm uptake rate of PHE de-
creased from 10.6% to 2.8%, whereas the uptake rate of PYR only
decreased from 6.5% to 2.4%, and the BCF of PHE decreased from
4.5 to 1.5, whereas PYR decreased from 2.7 to 1.1, indicating that
the bioavailability of PHE was greater than that of PYR. Based on
1076 L.L. Ma et al. / Chemosphere 86 (2012) 1072–1078

Table 2
Survival (%) and average weight loss (%) of earthworms after 7 d of cultivation at different aging times.

Treatment The change of biomass (%) Different aging time (d)

0 15 30 60 90 150
Phenanthrene Survival rate 80.0 100 100 93.3 100 100
Average weight loss 25.1 17.7 14.1 16.5 17.0 15.2
Pyrene Survival rate 100 96.7 100 100 100 96.7
Average weight loss 21.2 15.0 13.5 13.4 13.6 12.2

absorbed by earthworm through diffusion models. On the contrary,

phenanthrene pyrene the highly fat-soluble PYR was easily adsorbed and combined with
Uptake rate of earthworm (%)

12.0 the clay composition, making it difficult to be desorbed and

showed poor bioavailability to earthworm. Besides, this may also
9.0 be related to the molecular diameter of PHE and PYR. Cuypers
et al. (2002) calculated the molecular diameter of different PAH
6.0 rings and found that PAH molecular diameter of 2–6 rings in-
creases from 0.5 nm to 0.9 nm, making it increasingly more diffi-
cult to enter into organisms and accumulated in earthworms.
3.0
This phenomenon suggested that PAH with higher molecular
weights, longer molecular diameters and larger Kow values were
0.0 more recalcitrant and difficult to be desorbed in soil and utilized
0 15 30 60 90 150
by earthworms (Gao et al., 2009; Sabate et al., 2006). In present
Aging time (d)
study, the earthworm uptake rate and BCF of both PHE and PYR
Notes: the uppercase letters represent phenanthrene.
were relatively higher when exposed to fresh contaminated soil
the lowercase letters represent pyrene. may due to the sandy loam soil having low organic matter and clay
Fig. 3. The effects of aging time on the earthworm uptake rate of phenanthrene and
content, which leads to a relatively small sorption capacity of PAH
pyrene. and easily be absorbed by earthworms.

3.3. The effects of aging time on the correlation between the

earthworm uptake rate (y) and the extraction ratio (x) of PAH
phenanthrene pyrene
6.0
Linear correlation results showed that the earthworm uptake
rate and the extraction percentage of desorbed PHE fractions were
4.5 positively correlated (Table 3). The correlation coefficient reached
0.95, indicating that the HPCD-extraction technique can be used
BCF

3.0
1.5
0.0
0 15 30 60 90
Aging time (d)
Notes: the uppercase letters represent phenanthrene.
the lowercase letters represent pyrene.
Fig. 4. The effects of aging time on the BCF of phenanthrene and pyrene.
these results, the major uptake route by earthworms may depend
on the properties of the compound, especially its octanol-water
partition coefficient (Kow) (Kaag et al., 1997; Matscheko et al.,
2002). Eggleton and Thomas (2004) found that the Kow of com-
pounds was the key factor impacting the bioavailability of com-
pounds. PHE was determined to have a significantly lower Kow
than PYR (PHE, Kow = 2.82  104; PYR, Kow = 1.51  105). There-
fore, PHE is easily desorbed from the soil system and easily
to estimate aged PHE bioavailability from soil. Moreover, the earth-
worm uptake rate was observed to be negatively correlated with
the proportion of non-desorbed PHE, resulting in a correlation
coefficient of 0.89. Furthermore, for bound residues, the correlation
coefficient was 0.64. These results illustrate that the last two mor-
150
phologies were more difficult for the earthworms to absorb and
can also estimate earthworm accumulation of different aged PHE.
Tang et al. (1998) reported that earthworms ingest soil particles di-
rectly and can potentially take in contaminants that are considered
non-bioavailable to other organisms.
No significant correlation was determined for PYR, indicating
that the HPCD extraction method for analyzing PYR bioavailability
is defective. Lv et al. (2011) reported that the HPCD extraction of
3-ring PAH had a significantly positive correlation with accumula-
tion in earthworms (r2 = 0.79), whereas there was no correlation
for a greater number of rings including 4-ring, 5-ring, and 6-ring.
Although Cuypers et al. (2002) thought that the HPCD solution
could be a good simulation for the bioavailability of PAH with dif-
ferent number of rings in soil, the results obtained from that meth-
od do not match with ours. In the correlation analysis, Cuypers

Table 3
The correlation between the earthworm’s uptake rate (y) and extraction rate (x) for phenanthrene and pyrene during the aging process.

PAH The earthworm uptake rate and extraction rate Correlation Correlation coefficient
Phenanthrene The uptake rate and proportion of the desorbing fraction y = 43.83x  25.51 R2 = 0.95
The uptake rate and proportion of the non-desorbing fraction y = 0.53x + 19.91 R2 = 0.89
The uptake rate and proportion of bound residues y = 1.36x + 8.06 R2 = 0.64
Pyrene The uptake rate and proportion of the desorbing fraction y = 0.16x  6.54 R2 = 0.08
The uptake rate and proportion of the non-desorbing fraction y = 0.08x + 6.44 R2 = 0.02
The uptake rate and proportion of bound residues y = 3.30x + 5.67 R2 = 0.80
 L.L. Ma et al. / Chemosphere 86 (2012) 1072–1078
et al. (2002) compared the HPCD extraction amount with microbial
degradation in soil. However, here HPCD was used to evaluate
earthworm accumulation. PAH with a greater number of rings
may be directly mineralized or either metabolized or degraded as
the substrate by microorganisms and difficult in entering the
bodies of earthworms through the cell membrane due to their
complex molecular structures, relative large molecular quantities.
One evaluation method is based on the bioavailability of bacteria
degradation, whereas the other is based on the bioavailability of
earthworm accumulation, which results in differences between
the results. Meanwhile, Hickman and Reid (2005) found no rela-
tionship between HPCD-extractable PHE concentrations and accu-
mulation by Lumbricus rubellus. This result was different with ours
may because of the different earthworm species we used, and
Vander Wal et al. (2004) argued that it was to obtain a better cor-
relation, based on different worm species and set of compounds.
Besides, our study focuses on the correlation between the different
aged and morphological PAH and earthworm uptake rates.
4. Conclusions
Using a sequential extraction mass balance approach, the PAH
in soil were separated into three fractions. More than 95% of each
PAH tested was extractable, and the desorbed and non-desorbed
fractions were always observed to dominate in soils. The portions
of bound PAH residues were much lower (less than 5%). The des-
orbed PHE was observed to decrease as it penetrated into soils
due to the aging phenomena, whereas the non-desorbing PHE in-
creased. The desorbing percentage of PHE was greater than PYR,
whereas the opposite trend was observed for non-desorbing PHE
throughout the experiment period, indicating that PHE is easily
desorbed from soil compared to PYR. The bioaccumulation of
PAH (PHE and PYR) in earthworms declined drastically during
the early aging period and subsequently slowed down, which is
consistent with the aging rate of PAH in soil. The earthworm up-
take rate of PHE was higher than that of PYR because PHE has a
lower Kow than PYR, indicating that PHE has a greater bioavailabil-
ity compared to PYR. The utility of the three-extraction technique
for the prediction of PHE availability to earthworms has been val-
idated to be feasible over an extended period of 150 d. However,
this technique has limitations with PYR.
Acknowledgements
This research was financially supported by The National Natural
Science Foundation of Youth Fund (No. 41101292), Environmental
public welfare project (No. 201009032) and the Priority Academic
Program Development of Jiangsu Higher Education Institutions
(PAPD). We thank Shanmei Wu, in Berkeley, University of
California for language improvement.
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