Pedosphere 24(6): 743–752, 2014

ISSN 1002-0160/CN 32-1315/P

°c 2014 Soil Science Society of China
Published by Elsevier B.V. and Science Press

A Hierarchic Method for Studying the Distribution of

Phenanthrene in Eisenia fetida∗1

SHI Zhi-Ming, XU Li and HU Feng∗2

College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095 (China)
(Received December 12, 2013; revised July 15, 2014)

ABSTRACT
The distribution of heavy metals in earthworms has been widely studied, highlighting the importance of the fate of these metals.
However, little information is available on the distribution of hydrophobic organic contaminants (HOCs) within earthworms. The aim
of this study was to propose a hierarchic method to study the distribution of phenanthrene (PHE), a typical HOC, in Eisenia fetida
at several levels: sub-organism (pre-clitellum, clitellum and post-clitellum), tissue (body wall, gut and body fluid) and subcellular
(intracellular and extracellular fractions). Earthworms were incubated in the soils amended with low (LC, 10 mg kg−1 ) and high
concentrations (HC, 50 mg kg−1 ) of PHE and sampled at different time intervals. At the sub-organism level, the distribution of PHE
was homogeneous among the sub-organism fractions in the LC treatment but heterogeneous in the HC treatment and gradually reached
the following form of post-clitellum ≈ clitellum > pre-clitellum. The uptake and elimination kinetics of PHE in the sub-organism were
well described by a one-compartment model. At the tissue level, the concentration of PHE followed the order of gut > body fluid >
body wall; while at the subcellular level, the concentration of PHE in the extracellular fraction was 1.23 to 4.68 times higher than
that in the intracellular fraction. Therefore, the simple circulatory system of earthworms may account for the PHE distribution at the
sub-organism level. Partition pathways (passive diffusion) of PHE between the body wall, body fluid and gut as well as the processes
of PHE entrance into the inner cellular compartment via passive diffusion were experimentally supported.
Key Words: earthworm, passive diffusion, subcellular level, sub-organism level, tissue level

Citation: Shi, Z. M., Xu, L. and Hu, F. 2014. A hierarchic method for studying the distribution of phenanthrene in Eisenia fetida.
Pedosphere. 24(6): 743–752.

INTRODUCTION substrate parameters and the concentrations in whole

Earthworms are the most important soil fauna, are
found in a wide range of soils, and may represent 60%–
80% of the total soil biomass (Saint-Denis et al., 1999;
Contreras-Ramos et al., 2008). They are also an im-
portant food source for many animals, such as moles,
badgers and thrushes. Therefore, predators may en-
counter toxic effects via the food chain (second poi-
soning) from the consumption of contaminated worms
(Jager, 1998). Due to their critical role in terrestrial
ecosystems, earthworms have been considered to be
a typical organism for studying the effects of chemi-
cals on terrestrial invertebrates by the Organization
for Economic Co-operation and Development (OECD,
1998) and the International Organization for Standar-
dization (Zheng et al., 2008). Accordingly, a standard
protocol for earthworms toxicity testing has been de-
veloped and has been successfully and widely used (Shi
et al., 2007; Lee et al., 2008). However, these toxicity
tests have mainly focused on the relationship between
earthworms, the general toxicity symptoms and spe-
cific effects, ignoring the behavior of pollutants inside
of earthworms (Felten and Emmerling, 2009; Marques
et al., 2009; Wu et al., 2011).
In fact, understanding the inner behavior, such as
the internal distribution of pollutants within earth-
worms, is urgent and important because 1) any com-
prehensive pollution bio-monitoring and risk assess-
ment practice should ideally explore the fate and the ef-
fects of the environmental pollutants at all levels of bi-
ological organization; 2) the information could furnish
clues to the pollutant immobilization/detoxification
strategies adopted by the organisms and to the range
of sub-lethal effects to which they may be subjected
(Morgan and Morgan, 1990); and 3) important eco-
toxicological information can be acquired from the
analysis of specific tissues in a bioaccumulation species
rather than the whole organism (Simkiss and Taylor,
1981; Bengtsson et al., 1983).
Currently, the study of pollutant distribution in

∗1 Supported by the National Natural Science Foundation of China (No. 41101292).

∗2 Corresponding author. E-mail: fenghu@njau.edu.cn.
744
earthworms has focused on heavy metals, and great
progress has been made. These studies indicate that
metals are not homogeneously distributed among the
major earthworm organs/tissues; on the contrary, they
are discretely compartmentalized within certain tissues
and cells, such as chloragogenous tissues (Morgan and
Morgan, 1990; Honeycutt et al., 1995; Li et al., 2009).
Moreover, at a subcellular level, the total metal bur-
den in earthworms has been partitioned into different
fractions, such as metal-sensitive fractions (MSF), bio-
logically detoxified metal (BDM) and trophically avai-
lable metal (TAM), which is related to risk assessment
and food chain security (Wallace et al., 2003; Wal-
lace and Luoma, 2003; Vijver et al., 2004). Further-
more, to some extent, these studies also clarify earth-
worm detoxification and metal tolerance mechanisms
(Yu and Lanno, 2010). These findings highlight the
importance of the distribution behavior of pollutants
within earthworms. However, little is known about the
behavior and fate of the distribution of hydrophobic or-
ganic contaminants (HOCs) within earthworms.
Among HOCs, PAHs are an important class. Phe-
nanthrene (PHE) is a lower molecular weight PAH
and is one of PAHs with the simplest chemical struc-
tures, with 3 fused benzene rings (de Bruyn et al.,
2012). PHE level in contaminated sites is relatively
high and was reported to be 8 mg kg−1 out of to-
tal PAHs of 90 mg kg−1 in a sediment (Hwang and
Cutright, 2002). Therefore, PHE is most often selected
as a typical species to study relevant and important is-
sues (Liste and Alexander, 2000; Cheema et al., 2010;
Khan et al., 2012).
On the basis of these findings, we used PHE as a
typical representative for PAHs and Eisenia fetida as
a typical earthworm to investigate PHE distribution
patterns inside earthworms at three fractionation le-
vels: sub-organism, tissue and subcellular. We believe
that the results may offer initial reference materials
for a detailed understanding of the fate and behavior
of organic pollutants in soil and earthworms.
MATERIALS AND METHODS
Test organisms and soil
Earthworms Eisenia fetida were purchased from a
commercial supplier. Before the experiment, the adult
earthworms were cultivated in the mixtures of potting
compost soil and cow dung in the dark at 22 ◦ C for 2
weeks.
Yellow-brown soil was sampled from Nanjing City,
Jiangsu Province in China. The top 20-cm layer was
Z. M. SHI et al.
manually collected and transferred to the laboratory
after removing the vegetation. The soil was air-dried,
ground and passed through a 2-mm sieve. The soil had
a pH of 6.12, total organic carbon content of 15.3 g
kg−1 and cation exchange capacity of 20.6 cmol kg−1 ,
with 24.7% clay, 13.4% sand, and 61.9% silt.
According to the concentration of PHE in real en-
vironments, soil samples were artificially spiked with
PHE (purity ≥ 97%, FLUKA Co., Germany) by adding
an acetone (analytically pure) solution to produce
nominal concentrations of 10 and 50 mg kg−1 PHE
(based on the dry mass of soil) (Hwang and Cutright,
2002). The samples were stored in open containers in
a fume hood until all of the acetone evaporated (which
was confirmed by checking for the absence of odor) and
were then aged in sealed glass bottles in the dark at
room temperature (22 ± 1 ◦ C) for 2 weeks.
Experimental design
The experiment consisted of two treatments: low
(10 mg kg−1 ) and high concentrations (50 mg kg−1 )
of PHE with four replicates. To investigate the PHE
distribution at three levels (sub-organism, tissue and
subcellular), the experiment was carried out three in-
dependent times.
One hundred grams of PHE-spiked soil were ad-
justed to a 50% water-holding capacity, placed in a
glass beaker (200 mL) with a foil cover and pre-in-
cubated for 14 d in the dark at room temperature
(22 ± 1 ◦ C). Adult earthworms (each 550–600 mg, wet
weight) with well-developed clitellum were chosen ran-
domly and placed on a moist filter to depurate their
gastrointestinal tract for 24 h (Arnold and Hodson,
2007). After rinsing with deionized water, four worms
were placed on the surface of the soil in each beaker. To
prevent the escape of the earthworms, the room was il-
luminated constantly (Vijver et al., 2007). Preliminary
studies showed that the photolysis loss of PHE in the
soil surface (< 5%) can be ignored. Soil moisture was
checked and kept at the initial level. After 1, 3, 5, 7
and 14 d of exposure, the earthworms were recollected
by hand. The earthworms were allowed to void their
gut contents on moist filter paper for 24 h and were
then washed and reweighed. Following this, the earth-
worms were immediately dissected to study tissue level
distribution of PHE.
The same experimental design was carried out two
more times to study the PHE distribution at the sub-
organism and subcellular levels. The earthworms were
frozen using liquid nitrogen and were stored in an
ultra-low temperature freezer at −70 ◦ C for the sub-
DISTRIBUTION OF PHENANTHRENE IN EARTHWORM
organism and subcellular distribution studies.
Sub-organism, tissues and subcellular fractionation
Sub-organism fractionation. On the basis of
their external morphology, the earthworms were gro-
uped into three fractions (from the anterior to poste-
rior): the pre-clitellum, clitellum, and post-clitellum
(Tewatia, 2007). Briefly, the earthworms were first
scarified in liquid nitrogen and were then rapidly cut
with a blade into the three parts before they thawed:
the portion anterior to the clitellum is the pre-clitellum
(segments 1–25), the middle section is the clitellum
(segments 26–31), and the rest of the worm is the post-
clitellum region. The body segments were stored at
−70 ◦ C until analysis.
Tissue fractionation. The earthworm tissues
were classified into three categories (from the exterior
to interior): body wall, gut and body fluid (coelomic or
extracellular fluid) (Honeycutt et al., 1995). Briefly, the
earthworms were placed into vials containing 3 mL of
50% ethanol to extrude their body fluids and sacrifice
the animals. The dead earthworms were then placed
in a waxen dish and fixed with dissecting pins, and
the gut was carefully removed from the body wall u-
sing a blade before they softened. The body fluid, gut
and body wall fractions were weighed and stored in
an ultra-low temperature freezer at −70 ◦ C for further
analysis.
Subcellular fractionation. Earthworms used to
investigate subcellular distribution were divided into
two groups: a 10 000 × g pellet, mainly consisting of
the outer cell fractions (granules, tissue fragment, cell
membrane), and a 10 000 × g supernatant, consisting
of the inner cell fractions (cytosol and microsomal,
e.g., organelles) after centrifuging at 10 000 × g for 30
min at 4 ◦ C using a Sigma 3K 30 preparative ultra-
centrifuge (Sigma, German) (Vijver et al., 2004). Brie-
fly, the earthworms were thawed, placed in a 10 mL
glass tube, and homogenized by a tissue homogenizer
(DY89-2, Ningbo Scientz Biotechnology, Ltd., China)
in 5 mL of ice-cold Tris-HCl buffer (0.01 mol L−1 , pH
7.0) for 5 min at 1 500 r min−1 (Li et al., 2009). The
homogenates were then centrifuged at 10 000 × g for
30 min at 4 ◦ C. Subsequently, the supernatant was
poured into another tube. The pellet and supernatant
fractions were freeze-dried using ALPHA 1-2 LD plus
(Sigma, German) and then stored in the ultra-low tem-
perature freezer at −70 ◦ C for further analysis.
PHE extraction and determination
After pre-treatment, the solid fractions (sub-orga-
nism, subcellular, and gut and body wall fractions)
745
were weighed, crushed and ground using a pestle and
mortar and were mixed with 3 times their weight of an-
hydrous sodium sulfate to form a fine powder. The con-
centrations of PHE in the solid fractions were analyzed
using a modified ultrasonic extraction method. The po-
wder was transferred into a 40-mL brown glass flask
(Aglient, USA) to which 20 mL of dichloromethane was
added. The flasks were placed in an ultrasonic bath
with ice water for 30 min, mechanically shaken on a
vortex for 60 s, and sonicated again for 30 min. The
extracts were separated from the power by centrifuga-
tion at 3 500 × g for 15 min. The process was repe-
ated twice. The extracts were combined and cleaned
up on a 3-g of silica gel column and eluted with 11
mL of acetone and hexane acetone (1:1, v/v). The
eluted solvents were then evaporated, dissolved in 2
mL of methanol and passed through a 0.22-µm Teflon
filter to be analyzed on a high-performance liquid chro-
matography (HPLC) system (Contreras-Ramos et al.,
2006; Contreras-Ramos et al., 2008). For quality con-
trol, unspiked earthworm fractions were analyzed using
the same procedures.
The body fluid was diluted with 50% ethanol to 20
mL, passed through a 0.22-µm Teflon filter to remove
the particulate matter, and then analyzed on an HPLC
system with no further treatments.
Treated earthworm fraction extracts were deter-
mined for PHE using a reverse-phase HPLC (Shi-
madzu LC-20AT, Japan; C18 column, 4.6 mm × 250
mm) with an ultraviolet detector (Shimadzu SPD-20A,
Japan). The mobile phase was methanol with a flow
rate of 1 mL min−1 , and chromatography was per-
formed at 30 ◦ C. PHE were detected at 254 nm, and
the injection volume of the sample was 20 µL. The
PHE retention time under the liquid chromatography
condition was approximately 4.600 min. The detection
limit was 6.4 ng g−1 . PHE was quantified to external
standards for PHE. Average recoveries of PHE based
on the spiked earthworm fraction samples were 94.1%–
103.7% for sub-organism fractions (n = 7), 91.2%–
102.7% (n = 6) for tissue fractions, and 96.3%–108.4%
(n = 6) for subcellular fractions. All relative standard
deviations (RSD) were below 5%.
Statistical analysis and modeling
All data were processed using OriginPro 8.0 and
Microsoft Excel software packages. Data were tested
for normality and homogeneity of variances by using
Kolmogorov-Smirnoff and Levene’s tests, respectively.
When necessary, the variables were logarithmic-trans-
formed to achieve equality of variances. The PHE con-
centration in earthworms was assessed using a two-
746 Z. M. SHI et al.

way analysis of variance (exposure time and concen- results were stable with a small relative standard de-
tration levels) followed by Tukey’s post hoc compari- viation, indicating that these fractionation strategies
sons for testing the difference between the treatments were valid.
at P < 0.05.
TABLE I
To acquire the toxicokinetic constants, a first-order
one-compartment model (Eq. 1) was applied at the Mass distribution of earthworms at different fractionation levels
sub-organism level. In this study, each sub-organism
Fractionation level Fraction Proportiona)
fraction was considered to be a compartment, an as-
%
sumption also made in other similar studies (Li et al.,
Sub-organism Pre-clitellum 25.11±0.33b)
2009; Spurgeon et al., 2011). Clitellum 15.69±0.43
Post-clitellum 59.20±0.57
k1 Cs
Cw (t) = (1 − e−k2 t ) (1) Tissue Body wall 49.85±0.33
k2 Gut 30.11±0.33
Coelomic fluid 20.04±0.05
where Cw (t) is the internal concentration of PHE in Subcellular 10 000 × g pellet 23.86±1.13
the sub-organism fractions at exposure time t (d), k1 10 000 × g supernatant 76.14±1.13
is the uptake rate constant (g soil g−1 worm d−1 ), k2 a) Based on wet weight for sub-organism level and on dry weight
is the elimination rate constant (d−1 ), and Cs is the for tissue and subcellular levels.
exposure concentration of PHE in soil (mg kg−1 ). b) Means±standard deviations (n = 54, 125 and 80, respectively,

for sub-organism, tissue and subcellular levels).

RESULTS AND DISCUSSION
Mass distribution of earthworms at different fraction-
ation levels
The mass distribution proportion of each fraction
was analyzed, and the results are shown in Table I. The
Concentration and distribution of PHE in sub-orga-
nism fractions of earthworms
The concentrations of PHE in the sub-organism
fractions of earthworm increased with exposure time
(Fig. 1). At the low concentration (Fig. 1a), the PHE

Fig. 1 Concentrations and distribution proportions of phenanthrene (PHE) in the sub-organism fractions of earthworms grown in
the soil spiked with low (a and c) and high (b and d) concentrations of PHE. Vertical bars indicate standard deviations of the means
(n = 4). Bars with the same letter are not significantly different for each sampling time using the analysis of variance at P < 0.05.
DISTRIBUTION OF PHENANTHRENE IN EARTHWORM 747

concentrations in the different sub-organism fractions in the whole body. This implies that uptake from the
showed no significant difference. This may due to the soil by earthworms is an equilibrium partition process
following reasons. First, the contact of the fractions driven by fugacity. No significant differences were ob-
with soil is even, and each fraction has the same access served in the uptake and elimination fluxes between
to PHE. According to the models developed by Bel- different fractions within an exposure level, but cor-
froid et al. (1995) and Jager et al. (1998), the domi- responding parameter values increased with elevated
nant uptake for PHE is passive diffusion through body exposure level. In principle, k1 and k2 are constants
wall. Second, earthworms have a very simple circula- (Nahmani et al., 2009). One explanation for the k1 vari-
tory system with two blood vessels (ventral and dorsal ation is that the nominal exposure concentration did
blood vessels), which run the length of the body (Tewa- not reflect the real bio-available fractions for contami-
tia, 2007), and pollutants that enter into earthworms nants in the soil, especially for HOCs for the aging
can be transported and distributed quickly and evenly effect. The reason for the variation in k2 is that k2 is a
throughout the whole body by the circulatory system property of the organism and earthworms have a capa-
(Lee et al., 2008). Third, the body fluid fills the space city to accumulate PHE. Only when the body burden
between the earthworm body wall and gut; therefore, exceeds their capacity, earthworms quickly eliminate
body fluid containing PHE may provide another expla- excess PHE.
nation for this homogeneous distribution.
TABLE II
However, with the increased body burden of PHE
in earthworms approaching the steady state, at the Uptake and elimination kinetics parametersa) estimated from
first-order one-compartment for phenanthrene (PHE) at sub-
high concentration (Fig. 1b), the PHE concentrations
organism level of earthworm
were significantly different among the sub-organism
fractions in the following order of post-clitellum > cli- PHE Sub-organism k1 k2 R2b)
level level
tellum > pre-clitellum (at 5 d of exposure). The PHE
concentrations finally reached a distribution of the fo- mg kg−1 g soil g−1 worm d−1 d−1
10 Anterior 0.35 0.03 0.89
llowing form of post-clitellum ≈ clitellum > pre-clite- Clitellum 0.34 0.01 0.93
llum (at 7 and 14 d exposure), which may be due to Posterior 0.40 0.04 0.94
approaching the steady state. The activity of earth- 50 Anterior 1.32 0.59 0.78
worms was reduced and constrained within a small Clitellum 1.31 0.40 0.91
Posterior 1.40 0.43 0.85
space, resulting in less PHE intake. Moreover, the cli-
a) k is the uptake rate constant based on PHE concentration in
tellum and posterior fractions contain the chlorago- 1
dry soil and k2 is the elimination rate constant.
genous tissue, a liver-type tissue surrounding the ali- b) Coefficient of determination.
mentary canal, which may be related to toxin accumu-
lation and excretion (Franchini and Marchetti, 2006; Concentration and distribution of PHE in tissue frac-
Arnold et al., 2008). tions of earthworms
The distribution proportions of PHE in the sub-
organism fractions (Fig. 1c and d) were relatively stable The concentration of PHE in earthworm tissue
and were ordered over the entire course of the expe- fractions may reflect the extent of organic entrance
riment as follows: post-clitellum (58%–72%) > pre- into the different body parts from the soil (Ma et al.,
clitellum (18%–24%) > clitellum (12%–19%). This or- 2012). As shown in Fig. 2, the overall concentrations
der was positively related to the mass distribution of of PHE in the gut were higher than those in the body
earthworms (Table I). wall and body fluid over the whole course of the expe-
The uptake and elimination rate constant of the riment and at the two exposure levels of the pollutant.
sub-organism fractions were also estimated and are This result was consistent with a previous study by our
given in Table II. The first-order uptake kinetic model group (Ma et al., 2012). However, the concentrations
fit well (R2 = 0.78–0.94). Although there were only in the body wall and body fluid showed different pat-
two exposure levels in the study, body burdens and terns. At the low concentration (Fig. 2a), the 5- and 7-d
PHE concentrations in different fractions correlated PHE concentrations in the body wall were significantly
positively with the PHE concentration in the soil. This lower than those in the body fluid; at the high concen-
is consistent with the results reported by Jager et al. tration (Fig. 2b), 7- and 14-d PHE concentrations in
(2000), who found that the residues of pyrene, another the body wall also significantly lower than those in the
PAH species, increased with concentration exposure body fluid.
748 Z. M. SHI et al.

Fig. 2 Concentrations and distribution proportions of phenanthrene (PHE) in the tissue fractions of earthworms grown in the soil
spiked with low (a and c) and high (b and d) concentrations of PHE. Vertical bars indicate standard deviations of the means (n =
4). Bars with the same letter are not significantly different for each sampling time using the analysis of variance at P < 0.05.

The distribution proportions of PHE in each tissue
fraction of earthworm were calculated and are shown
in Fig. 2c and d. The distribution proportions of PHE
in the body fluid were relatively stable (15%–22%) and
were lower than those in both the body wall and gut
over the entire course of the experiment and at the
two exposure levels of the pollutant. Moreover, the
proportions in the body wall and gut showed an in-
verse relationship. Within the tested concentrations of
the pollutant (10 and 50 mg kg−1 ), the proportions in
the body wall were higher (47.5% at 10 mg kg−1 and
45.2% at 50 mg kg−1 ) than those in the gut (36.3%
and 36.7%, respectively) at 1 d. This may be because
the earthworms needed to adapt to the spiked soil with
restrained bioactivity; therefore the partition between
body wall and gut was small. When adaptation was
finished, the partitioning ability was enhanced, resul-
ting in more PHE in the body wall entering into the
gut fraction. At a low concentration (Fig. 2c), the pro-
portion in the body wall (22%–47%) was lower than
that in the gut (35%–54%). This is because the uptake
of PHE by earthworms has not reached a steady state,
whereas the partition between the soil and body wall
is smaller than that between the body wall and gut.
With the uptake approaching steady state (Fig. 2d),
the proportions of PHE in the body wall and gut also
approached equilibrium (38%–43% and 34%–47%, re-
spectively).
Earthworms may take up organic chemicals thr-
ough two pathways: passive epidermal uptake, in which
only compounds dissolved in pore water or weakly as-
sociated with the surfaces of particulate matter are
available, and dietary uptake. However, the contribu-
tions of the two routes are different; for hydrophobic
chemicals that have a logKow < 5 (Kow is the octanol-
water partition coefficient), epidermal uptake is the
dominant route (Belfroid et al., 1995; Jager, 2004). Ad-
ditionally, some researchers believe that earthworms
take up organic chemicals mainly through sequential
partition processes between soil solution-earthworm
tissue-gut contents (Jager, 1998, 2004; Jager et al.,
2003). These authors clarified how and how much or-
ganic pollutants entered into the earthworms but did
not illustrate what happened to the pollutants inside
the earthworms. As PHE (logKow = 4.46) has a mode-
rate hydrophobicity, the dietary uptake route is mini-
mal; PHE should, therefore, mainly accumulate in the
body wall. However, our study showed that PHE not
only accumulated in the body wall but also in the
gut and body fluid. Moreover, the concentrations of
DISTRIBUTION OF PHENANTHRENE IN EARTHWORM 749

PHE in the gut and body fluid were even higher than
those in the body wall at some time points, implying
that the PHE in the body wall was translocated into
the body fluid and gut. Furthermore, because PHE is
a hydrophobic organic pollutant and the body fluid
links the body wall and gut, it is reasonable to con-
clude that the PHE distribution within earthworms is
similar to PHE entrance into earthworms, which occurs
through a series of passive diffusion and partition pro-
cesses. The factor determining the PHE concentration
in these fractions may be the chemical composition of
earthworms, such as their lipid content. The total lipid
content was determined gravimetrically from aliquots
of hexane extracts after evaporation to dryness (Ma et
al., 1995; Ma et al., 1998). The average lipid content
in the body wall and gut were 5 and 13 g kg−1 on a
fresh weight basis, respectively.
On the basis of the results mentioned above, we
believe that at the tissue level, the entire pathway of
PHE entrance and partitioning within earthworms un-
derwent the following processes: PHE in the soil pore
water passively diffused into the body wall, the water
was further partitioned into the body fluid, and the wa-
ter finally entered into the gut, sequentially, resulting
in an equilibrium state between these phases.
Concentration and distribution of PHE in subcellular
fractions of earthworms
In Fig. 3, the uptake of PHE from spiked soil by the
earthworm subcellular fractions is shown as a function
of exposure time. The concentrations of PHE in diffe-
rent subcellular fractions increased with exposure time.
At the low concentration (Fig. 3a), the PHE concentra-
tion in the pellet gradually increased from 92.4 to 371.8
mg kg−1 (on a dry weight basis) at 7 d and then slightly
decreased to 352.1 mg kg−1 at 14 d, while it increased
from 19.8 to 108.7 mg kg−1 in the supernatant during
the whole exposure time. PHE underwent a similar up-
take pattern at the high concentration (Fig. 3b): the
PHE in the pellet rose from 436.7 to 888.6 mg kg−1
and then slightly dropped to 790.3 mg kg−1 , while it
rose from 155.9 to 639.7 mg kg−1 in the supernatant.
During the entire course of the experiment, the
PHE concentrations in the pellet were higher than
those in the supernatant, mostly because the pellet
fraction had a relatively high lipid content that easily
captured hydrophobic chemicals. PHE is a hydropho-
bic organic chemical with a low solubility (1 mg L−1 )
that is readily partitioned into the solid phase (pellet)
in a solid-solution system (pellet-supernatant system),

Fig. 3 Concentrations and distribution proportions of phenanthrene (PHE) in the subcellular fractions of earthworms grown in the
soil spiked with low (a and c) and high (b and d) concentrations of PHE. Vertical bars indicate standard deviations of the means
n = 4). Bars with the same letter are not significantly different for each sampling time using the analysis of variance at P < 0.05.
750
which has a higher content of lipid than that of super-
natant (Gao et al., 2011).
However, the concentration pattern was different at
different time points and concentration exposures. We
further calculated the subcellular concentration factor
(SCF, dimensionless) of PHE (Table III). We defined
SCF = Cpel. /Csup. , where Cpel. and Csup. are the PHE
concentrations (on a dry weight basis) in the pellet
and supernatant, respectively. Because SCF is inde-
pendent of the PHE concentration in soil and the pel-
lets mainly consist of outer cell fractions (granules,
tissue fragment, cell membrane), whereas the super-
natant consists of inner cell fractions (cytosol, micro-
somal organelles), the SCF may reflect the process of
PHE entrance into cells. The SCF of PHE decreased
with prolonged exposure time, except at 7 d of ex-
posure; the SCF went from 4.68 to 3.34 at the low
concentration and from 2.80 to 1.23 at the high con-
centration. This clearly indicates that PHE was gra-
dually partitioned into the inner cell (10 000 × g su-
pernatant) from the outer cell (10 000 × g pellet) and
finally reached a partitioning equilibrium. Moreover,
the partition ability originates from the PHE concen-
tration gradient, which is consistent with passive diffu-
sion (Lodish et al., 2008). Although some researchers
have argued that hydrophobic organic pollutants often
accumulate largely via passive transport and diffusion
across membrane barriers (McCarty et al., 2011), as far
as we know, no direct evidence supports these opin-
ions. Our study may therefore constitute direct evi-
dence supporting these models.
TABLE III
Subcellular concentration factors (dimensionless) of phenan-
threne (PHE) in earthworms grown in the soil spiked with low
(LC) and high concentrations (HC) of PHE
Exposure time LC HC
d
1 4.68±0.75a) 2.80±0.37
3 4.24±0.55 2.29±0.22
5 3.67±0.72 1.64±0.24
7 4.58±0.65 1.57±0.13
14 3.23±0.32 1.23±0.15
a) Means±standard deviations (n = 4).
The distribution proportion of PHE in each earth-
worm subcellular fraction was calculated and is shown
in Fig. 3. At the low concentration, the proportions
of PHE in the pellet at 7 d of exposure slowly de-
creased from 60.9% to 51.9% (Fig. 3c). The propor-
tion of PHE in the pellet was higher than that in the
supernatant because PHE uptake did not reach equi-
librium between the subcellular fractions. At the high
Z. M. SHI et al.
concentration (Fig. 3d), the PHE proportion in pellets
decreased faster than that at the low concentration at
the same periods, and reached distribution equilibrium
at 14 d of exposure. Although the PHE concentration
in the pellet was higher than that in the supernatant,
the proportion of PHE in the pellet was lower than that
in the supernatant, which is due to the relatively larger
supernatant fraction accumulating greater amounts of
PHE (Table I). These results indicate that the parti-
tion between subcellular fractions is a dynamic process,
and the final distribution pattern may be determined
by the whole body burden of PHE.
On the basis of these results, we conclude that the
entrance of PHE into the inner cell may follow the fol-
lowing pathway: PHE in the extra-cellular fraction was
dissolved in the cell membrane lipid fraction and then
partitioned into the cell solution through passive dif-
fusion. These results are new, as the subcellular PHE
distribution in earthworms had not previously been re-
ported.
CONCLUSIONS
To the best of our knowledge, little information
on the internal distribution of HOCs in earthworms is
available. In the present study, it is explorative to take
PHE as a representative HOC to study the distribution
of organic pollutants in earthworms at different frac-
tionation levels. The analysis of PHE in earthworms
at different fractionation levels yielded valuable data
that differed from the results of traditional earthworm
bioaccumulation tests, which only focused on the con-
centrations in the whole earthworm. This study pro-
vided a hierarchic method (from sub-organism to the
subcellular level) for understanding the behavior and
fate of organic pollutants in earthworms.
The distribution pattern of PHE in earthworms
varied with the exposure time and exposure levels of
PHE, indicating that the distribution of PHE in earth-
worms is dynamic and heterogeneous. Overall, at diffe-
rent fractionation levels, the distribution of PHE ex-
hibits orderliness in the different fractions.
The earthworm circulatory system might be re-
sponsible for the distribution of PHE in the sub-or-
ganism, the transportation of PHE through the body
wall-body fluid-gut and the transportation of PHE
from the outer cell to inner cell via passive diffusion,
which further contributes to our knowledge of the fate
and behavior of PAHs in earthworms.
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