Environ. Sci. Technol. 2003, 37, 3399-3404
Elucidating the Routes of Exposure
for Organic Chemicals in the
Earthworm, Eisenia andrei
TJALLING JAGER,* ,†
ROEL H. L. J. FLEUREN, ‡
ELBERT A. HOGENDOORN, § AND
GERT DE KORTE §
Department of Theoretical Biology, Vrije Universiteit
Amsterdam, de Boelelaan 1085, NL-1081 HV Amsterdam,
The Netherlands, and Laboratory for Ecotoxicology and
Laboratory for Organic-Analytical Chemistry, National
Institute of Public Health and the Environment (RIVM),
P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands
Earthworms take up organic compounds through their
skin as well as from their food, but the quantitative contribution
of each route is unclear. In this contribution, we
experimentally validate an accumulation model containing
a separate compartment for the gut. Uptake from the
gut is modeled as passive diffusion from the dissolved
phase in the gut contents. For the experiments, we exposed
Eisenia andrei in artificial soil spiked with tetrachlorobenzene,
hexachlorobenzene, and PCB 153. Apart from the standard
accumulation and elimination experiments, we ligatured the
worm (using tissue adhesive) to prevent feeding. Model
fits were good, thus supporting the validity of the model. The
contribution of the gut route increased with increasing
hydrophobicity of the chemical, and for PCB 153 the gut
route clearly dominated. Despite the importance of the gut
route, the final steady-state body residues did not
exceed equilibrium partitioning predictions by more than
25%. Rate constants for exchange across the skin and the
gut wall could be separately identified. The rate constant
across the skin decreases with K ow but was generally
higher than data derived from water-only exposure. The
relationship with hydrophobicity was less clear for the rate
constant across the gut wall.
Earthworms are able to take up organic chemicals through
their skin (1) as well as from their food (2). However, the
quantitative contribution of each route remains unclear. As
earthworms regularly consume soil, it is difficult to study
both routes in isolation in a relevant experimental setup.
Earthworms can be exposed in water alone (3), but the
relevance of this setup for porewater uptake from soil is not
obvious. More work has been done in this area for sediment
organisms, showing that ingestion is an important pathway
for very hydrophobic chemicals such as pyrene and dioxins
(4, 5). For earthworms, Belfroid et al. (6) predicted, on the
* Corresponding author: e-mail: email@example.com; telephone:
+31 20 444 7134; fax: +31 20 444 7123.
Vrije Universiteit Amsterdam.
Laboratory for Ecotoxicology, RIVM.
Laboratory for Organic-Analytical Chemistry, RIVM.
basis of model extrapolations, that food uptake becomes an
important exposure route for very hydrophobic chemicals
(log K ow > 5). It seems to be a generally held opinion that
feeding on soil can lead to the invalidation of equilibrium
partitioning (EP), which is why additional safety factors were
prompted in European risk assessment guidelines (7).
In most case, uptake from food is modeled by simply
adding uptake routes (8), but in a previous contribution (9),
we proposed a more mechanistic accumulation model. Based
on the work of Gobas et al. (10, 11), the model includes a
separate compartment for the gut contents and a closed mass
balance. The mechanism for uptake from the gut is likely to
be the same as for uptake across the skin (i.e., passive
diffusion). This assumption is strongly supported by experimental
evidence as derived for intact goldfish (10), isolated
gut segments of catfish (12), and humans (13). For earthworms,
the validity of this assumption was indicated,
although a proper validation was impossible because the
physiological data regarding the feeding process were lacking
(9). Most routine studies with earthworms are carried out
with the compost worm (Eisenia andrei/fetida) in an artificial
soil medium (14). It is for this system that the essential feeding
parameters have recently been identified (15) including gut
loading, gut retention time, and digestion efficiency.
In this study, we set out to validate the accumulation
model with three organic compounds [tetrachlorobenzene
(TeCB), hexachlorobenzene (HeCB), and PCB 153] in artificial
soil. A series of experiments was performed with these
chemicals, starting with a straightforward accumulation and
elimination phase. Subsequently, soil from these experiments
was reused to see whether the bioavailable phase had
been altered, as indications of depletion have been observed
(1). Finally, an accumulation experiment was performed
with worms sealed with a tissue adhesive, thus
preventing feeding. This procedure was pioneered by Vijver
et al. (16) to demonstrate uptake routes for heavy metals.
Other forms of ligaturing have been applied to demonstrate
that pesticides are mainly taken up through the skin in wateronly
exposure (1) and that calcium is mainly taken up from
the diet (17). However, this study is to our knowledge the
first to separate exposure routes for organic chemicals in a
The data from all these experiments are used together to
fit the accumulation model and to identify the rate constants
for uptake through the skin and from the gut. Furthermore,
the model can shed light on the central questions: which
exposure route dominates and does feeding lead to body
residues exceeding the predictions made by equilibrium
Exposure Media and Spiking Procedure. Artificial soil was
used for the experiments (14). The water content was brought
to 40% (weight basis, water/dry medium) with a lutetium
solution (Lu, hydrated chloride salt, purity 99.9%, Alfa Aesar,
Karlsruhe, Germany) to obtain a nominal concentration of
15 mg/kg dwt. The Lu is used as a nonassimilated tracer to
compare the feeding activity to earlier experiments (15). After
the soil was wetted, it was thoroughly mixed and stored in
closed plastic containers at 5 °C for 1 week prior to spiking
with organic chemicals. After storage of the containers, the
pH (KCl) of the soil was 5.0. 1,2,3,4-Tetrachlorobenzene and
hexachlorobenzene were obtained from Riedel de Haën,
Seelze, Germany (99% purity, Pestanal); PCB 153 was
synthesized at the IRAS, Utrecht, The Netherlands (99%
10.1021/es0340578 CCC: $25.00 © 2003 American Chemical Society VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3399
Published on Web 07/02/2003
The spiking procedure for these compounds was adapted
from Northcott and Jones (18). Because we needed to spike
4kg dwt of medium, we had to do the procedure in steps
(dilution spike). First, the chemicals needed to achieve a
nominal concentration of 10 mg/kg dwt for each chemical were
dissolved in 100 mL of acetone (pro analysis). Wet soil (1 kg)
was placed in a kitchen blender, and the acetone solution
was slowly added while mixing. Mixing continued for several
minutes (stopping a few times to crush aggregates with a
spatula). The spiked soil was left in a fume cabinet overnight
after which the acetone and also most of the water had
evaporated. Next, one-fifth of the spiked soil was put in the
blender with 900 g wwt of uncontaminated medium and the
water needed to restore the 40% water content. This was
mixed for several minutes, stopping a few times to prevent
the medium from overheating and crushing the aggregates.
This entire procedure was followed five times until the entire
medium was spiked. The moisture content was checked by
oven drying at 80 °C and was 41%. The fraction organic matter
(F om) in the soil was 10.5% (loss on ignition). To allow
equilibration, the medium was stored at 10 °C in glass jars
for 1 week before animals were introduced. One day before
animals were introduced, soils were transferred to the test
temperature of 20 °C.
Test Animals and Experimental Setup. Sub-adult earthworms
(E. andrei), were taken from mass cultures at RIVM
(Bilthoven, The Netherlands). The animals weighed between
200 and 300 mg wwt. First, the animals were allowed to evacuate
their gut contents by keeping them on moist filter paper for
24hat20°C. Next, the animals were transferred to plastic
containers with 175 g wwt of uncontaminated medium (four
worms per container), and the containers were placed at 20
°C, covered by a black plastic pot to minimize disturbance.
The animals were left to acclimatize for 1 week under these
conditions. After this, they were exposed to the chemicals in
1-L glass jars using 250 g wwt of spiked medium and four
animals per jar. Several jars were used for the determination
of the feeding activity (see Supporting Information).
For the accumulation experiment, the worms were
recaptured following exposure (0, 1, 2, 3, 5, 7, 14, and 21 d)
and placed in a Petri dish on moist filter paper for 24 h at
20 °C. After this period, worms were packed in aluminum
foil and frozen at -20 °C. Soil samples were taken and frozen
in glass jars at -20 °C (four samples at t ) 0 and two
samples at days 14 and 21). At t ) 14, three additional jars
were emptied, and the worms were recaptured. The worms
were transferred to 250 g wwt of uncontaminated medium
and allowed to eliminate for 2, 5, and 11 d. The bioavailability
of the chemicals may change during the experiment, which
is why the spiked soil from the four jars emptied at t ) 14
was reused. Fresh worms were taken from the culture, placed
on wet filter paper for 24 h and subsequently on uncontaminated
medium for another 24 h, and then introduced
in the reused soil. These worms were recaptured after 1, 3,
7, and 11 d.
For the ligaturing experiment, worms were taken from
the culture and allowed to empty their gut for 24 h on moist
filter paper. Their anterior end was ligatured using a tissue
adhesive (Indermil, from Loctite Ireland Ltd.), conforming
to the procedure by Vijver et al. (16). Gluing turned out to
be difficult for this species because the worms were irritated
by the procedure and excreted coelomic fluid, which
interfered with the setting of the glue. As exposure time, 0,
1, 2, 3, 5, 7, and 14 d were employed using five worms per
jar with 250 g wwt of soil. After exposure, the worms were
recaptured and placed individually in a Petri dish with moist
filter paper overnight. Only the worms that did not evacuate
any solids were used for chemical analysis. For t ) 0, five
worms were taken; on days 1 and 5, three worms had been
successfully exposed; on day 2, only one worm was exposed
(after longer periods, all worms excreted solid materials and
Analysis of Organochlorine Compounds. For the sample
pretreatment of soil, 10 g wwt was mechanically shaken during
10 min in a glass tube with 25 mL of acetone. Next, 50 mL
of light petroleum (a mixture of saturated alkanes with a
boiling point of 40-60 °C) were added, and the contents
were mechanically shaken for 20 min. After centrifugation
(5 min. at 3000 rpm), the liquid phase was transferred into
a shaking funnel. The remaining part was extracted again
following the same procedure, and the liquid was transferred
to the funnel. After the addition of 500 mL of water, the funnel
was manually shaken for 1 min. The aqueous phase was
discharged, and the upper layer was extracted once more for
1 min with 500 mL of water. The light petroleum phase was
passed through a funnel with about 10 g of anhydrous sodium
sulfate and concentrated to a volume of 10 mL.
For analysis of the worms, a sample of approximately 1
g was placed into a glass tube and weighed. After addition
of 100 µL of the internal standard (approximately 100 ng of
[ 13 C 12]-PCB 153), 9 mL of isopropyl alcohol and 10 mL of
cyclohexane were added, and the mixture was macerated
with an ultra-speed homogenizer for 2 min. Next, 10 mL of
water was added, and the mixture was macerated again for
1 min. The phases were separated by centrifugation for 10
min at 3000 rpm. The upper organic layer was transferred
through a funnel with sodium sulfate to a Kuderna-Danish
evaporation apparatus by means of a pasteur pipet. The
remaining part of the sample was macerated again for 1 min
with 10 mL of a mixture of 2-propanol-cyclohexane (13:87,
v/v). After centrifugation, the upper layer was added to the
first extract, and sodium sulfate was rinsed with 5 mL of
cyclohexane; the organic layer was concentrated to 1 mL.
For cleanup (fat destruction), the extract was brought onto
a chromatography column filled with 0.5 g of silica gel
impregnated with sulfuric acid (100 g of silica heated for 4
hat200°C and 43.5 mL of concentrated sulfuric acid mixed
by rotating for 12 h). Next, 5 mL of hexane was passed through
the column, and the organic solvent was collected in a
calibrated glass tube and brought to a volume of 5 mL.
For instrumental analysis with GC/MS operating in the
electron impact (EI) mode, 1 mL of extract was transferred
into an autosampler vial, and 10 µL of pentachlorobenzene
(PeCB, 1 µg) was added as internal standard. A total of 1.5
µL was on-column injected into a fused silica DB-5MS
capillary column coated with 5% cross-linked 5% phenyl
methyl siloxane with a length of 30 m × 0.25 mm i.d. and a
film thickness of 0.25 µm. Helium was applied as carrier gas
at a flow of 1 mL/min. Quantification was done using the
internal standard PeCB for calibration and, in case of worm
samples, the isotope PCB 153 to correct for losses during
sample pretreatment. The average recoveries performed at
levels between 2 and 10 µg/g ranged between 83 and 103%
with SD below 9% (n ) 4 for each analyte-matrix combination).
The Model. The model is set up as a three-compartment
model with a closed mass balance (Figure 1). Diffusion (the
two-way arrows) and advection (one-way arrows) are the
basic transport processes. The model has been described
earlier (9), and the full model formulation is available in the
Supporting Information. Each compartment is assumed to
be well-mixed and of constant volume. Although the gut is
probably better reflected by a plug-flow reactor (19), the
simpler mixed compartment serves as an approximation (11).
The chemicals will be taken up into the tissue of the worm
from the outside soil as well as from the gut contents; both
processes are modeled as passive diffusion from the dissolved
The diffusion gradient between soil or gut contents and
worm tissue is defined by the concentrations in both
3400 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 15, 2003
FIGURE 1. Schematic representation of the accumulation model.
compartments and the partition coefficient between organic
matter (OM) and earthworm tissue (K ws in kg OM/kg wwt). The
K ws can be viewed as the ratio of the bioconcentration factor
with water (BCF in L/kg wwt) and the organic matter specific
sorption coefficient (K om in L/kg OM). When multiplied by the
soil concentration on OM basis, K ws would reflect the
equilibrium body residue of a nonfeeding worm. The OMworm
partition coefficient is the same for soil to worm as
from gut contents to worm. However, the magnitude of the
gradient differs between these two uptake routes because
the chemical concentration as well as the F om in the gut
contents differs from that in soil (due to selective feeding,
compaction, and OM digestion) (10, 11). The net result of
the two uptake routes depends on the kinetics of the various
transport processes, and the steady-state body residue in a
feeding earthworm will thus end up somewhere between
equilibrium with the soil and equilibrium with the gut
In this study, we chose to ignore degradation in the gut
and biotransformation as removal processes. This is acceptable,
given the very short gut retention time (Table 1) and
lack of indications for biotransformation of chlorobenzenes
and PCBs in earthworms (20, 21). However, the analysis
results prompted us to include a first-order loss term for the
soil (k d). Supported by pilot calculations (9), instantaneous
chemical equilibrium between solid and water phases is
assumed. Several adaptations to the previous model (9) are
made. First, compaction of the gut contents is included,
meaning that the gut volume decreases as food is absorbed
(15). Also, a slightly different formulation for the F om in the
gut is taken as average of F om determined in ingesta and
egesta instead of egesta only.
Model Fitting. The model equations are implemented in
matrix form in MatLab Version 6.1 (The Mathworks, Inc.)
and solved with the matrix exponential function. For each
chemical, we have five data sets (soil concentrations, two
accumulation phases, one elimination phase, and accumulation
in glued worms) that must be described by the
same model and with the same parameter values. Therefore,
all data sets must be fitted simultaneously. This is accomplished
by defining a likelihood function on the basis of
the sums of squares (SSQ) from the model fits on each data
set assuming normally distributed data (22):
L(θ|data) ∝ ∏ SSQ(θ; data i ) -n i/2
where θ is the entire set of parameters, and n i is the number
of points in data set i. Different likelihood functions may be
multiplied, so in this way we end up with one expression for
the overall likelihood of the model parameters given all five
data sets. For the gluing experiment, the number of worms
that were successfully glued was taken as a weight coefficient
in the SSQ. The overall log-likelihood function is maximized
by a Nelder-Mead simplex search in MatLab, yielding
maximum-likelihood (ML) estimates of the parameters. The
likelihood function is also used to construct confidence
intervals by calculating the profile likelihood (23), which is
more realistic for small data sets than standard asymptotic
procedures based on large-sample theory.
Even though the gut retention time is only 2.9 h in feeding
worms, 24 h depuration on filter paper is insufficient to
remove all of the gut contents. However, a longer depuration
could lead to bias as also chemicals will be lost from the
tissues. We corrected the modeled concentration for the
remaining gut contents using an estimate of the remaining
fraction (F rem, Table 1, see Supporting Information). The ML
estimates are used to estimate the net chemical assimilation
efficiency (AE), the biota-soil accumulation factor (BSAF),
and the deviation from EP. The AE can be calculated from
the uptake flux from the gut contents into the worm tissues
and the chemical flux with feeding (see Supporting Information).
BSAF (in kg OM/kg wwt) is calculated from the modeled
concentration in the worm (C w in mg/kg wwt) att ) 21, the
concentration in the soil (C s in mg/kg dwt), and the F om in soil:
BSAF ) C w (21)F om (soil)
C s (21)
The magnitude of the BSAF cannot be directly interpreted
in relation to EP as we did not measure porewater concentrations
and lipid content of the worms. However, the
deviation from EP can be assessed in an indirect manner by
comparing C w(21) to equilibrium estimates based on the
concentration in soil and the modeled concentration in the
gut (C g):
C w (EP, soil) ) K ws C s (21)
F om (soil)
C w (EP, gut) ) K ws C g (21)
F om (gut)
In these equations, we use the parameter estimate for K ws
(resulting from the model fit) for the EP estimates. The worm
can come to equilibrium with the soil (eq 3) or the gut
contents (eq 4) or end up somewhere between (in a
nonequilibrium steady state).
To obtain a confidence interval on these derived results,
we applied a random parameter search. Parameters were
randomly drawn from log-uniform distributions. When the
likelihood of these parameters (eq 1) was not significantly
lower than the ML estimate, the parameter combination was
stored. Random parameters were drawn until 200 acceptable
parameter combinations were obtained. For each parameter
combination in this set, the BSAF, AE, and deviation from
EP were calculated. The maximum and minimum values serve
as confidence intervals.
Results and Discussion
General Observations. We want to apply the parameter
values for the feeding activity, as observed previously (Table
1), to our current experiments. For this reason, we first
confirmed that these values are indeed representative for
this study (see Supporting Information). The worms increased
approximately 10% in weight during the 21-d exposure; the
potential effects on accumulation kinetics are expected to
be small and were ignored.
The initial concentration in the soil was 5.8 mg/kg dwt for
TeCB, 7.2 for HeCB, and 6.8 for PCB 153. These were lower
than the nominal 10 mg/kg dwt, presumably due to losses when
evaporating the acetone. However, the homogeneity of the
VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3401
TABLE 1. Fixed Parameters for the Feeding Process (15)
symbol description unit value SE
F ege egested feces as fraction of body weight kg dwt/kg wwt 0.12 0.012
F rem fraction of gut contents remaining after 24 h depuration kg dwt/kg dwt 0.056 0.021
F sel selectivity for OM in diet (-) 2.1 0.053
F dig digestion efficiency of OM (-) 0.35 0.033
F com factor by which gut contents are compacted (-) 1.09 0.011
T ret gut retention time h 2.9 2.5-3.3 a
F solids fraction solids in worm kg dwt/kg wwt 0.14 0.0013
90% probability interval.
TABLE 2. Parameter Estimates and Derived Measures, Resulting from the Model Fits a
parameter unit TeCB HeCB PCB 153
Chemical Properties and QSAR Estimations
log K ow 4.64 (25) 5.73 (25) 6.92 (26)
K ws estimated from QSARs (20, 27) kg OM/kg wwt 0.12 0.20 0.33
Estimated Model Parameters
K ws (first accumulation exp) kg OM/kg wwt 0.11 [0.077, 0.13] 0.20 [0.19, 0.22] 0.24 [0.23, 0.27]
K ws (second accumulation exp) kg OM/kg wwt [-] b 0.16 [0.15, 0.18] 0.16 [0.13, 0.21]
rate constant skin (k s) d -1 0.74 [0.42, 2.1] 0.30 [0.22, 0.43] 0.027 [0.020, 0.037]
rate constant gut wall (k g) d -1 0.27 [0, +∞] 0.43 [0.18, 0.72] 0.16 [0.13, 0.20]
degradation and/or volatilization (k d) d -1 0.0065 [0.0051, 0.0077] 0.0055 [0.0051, 0.0058] 0.0062 [0.0058, 0.0066]
BSAF at t ) 21 kg OM/kg wwt 0.12 [0.072, 0.13] 0.23 [0.21, 0.26] 0.30 [0.25, 0.36]
c lip 7.1 14 18
assimilation efficiency (maximum) % 10 [2.6 × 10 -4 , 50] 26 [0.19, 42] 16 [12, 22]
deviation from EP with soil % 7.3 [0,18] 13 [1.0, 23] 24 [18, 27]
deviation from EP with gut contents % -14 [-21, 0] -7.1 [-21, -1.9] -3.3 [-6.4, -0.3]
Maximum-likelihood estimates with 95% likelihood-based confidence intervals. K ws is the OM-worm partition coefficient. b Assumed the
same value as in the first accumulation phase. c Calculated, assuming a lipid content of 1% (20) and a factor of 1.7 between organic carbon and
FIGURE 2. Model fits for the different accumulation and elimination experiments (top) and the modeled uptake fluxes from soil and gut
contents, with 95% confidence intervals (bottom). The EP prediction marks the estimated body residue for a worm in equilibrium with
spiking was acceptable as the standard deviations were
4-5% (n ) 4) of the average value. The soil concentrations
appeared to decrease some 10% in the course of the exposure
experiment. It is striking that the rate constants for this
disappearance (k d, Table 2) are practically identical for all
three chemicals. The reasons for this disappearance have
not been investigated but could include the formation of
3402 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 15, 2003
resistant fractions (24), degradation, or volatilization.
The accumulation model can adequately describe the data
from the different experiments simultaneously (Figure 2) with
only five free parameters (see Table 2), an average of one
parameter per data set. Only the data for TeCB are rather
scattered (especially in the first accumulation phase). In most
cases, the parameters are accurately identified by the data,
evidenced from the tight 95% likelihood-based confidence
intervals (Table 2). However, it should be noted that these
confidence intervals do not include the uncertainty in the
feeding parameters (Table 1). Only for TeCB, the poor fit
results in ill-defined estimates. The good fit for the other
compounds is consistent with the assumption that gut uptake
is mediated through passive diffusion from the dissolved
phase in the gut (10, 11).
Ligaturing the earthworm allows to isolate exchange across
the skin in a soil situation. Even though the worms survive
for several days without problems, the treatment is stressful.
The stress is indicated by the coelomic fluid, expelled by all
the worms in reaction to the glue. Expelling fluid is a normal
response of E. andrei to rough handling and has no lasting
effects on their health. Other species (e.g., the genus
Lumbricus) do not have this response, but E. andrei was the
species for which the feeding parameters were available
(Table 1). The ligatured worms may show deviating behavior
in soil, which can also bias chemical uptake. Nevertheless,
the difference between glued and intact worms is remarkably
small for both chlorobenzenes, providing some reassurance
that the earthworms are sufficiently active to take up the
chemicals through the skin.
Exposure Routes and Assimilation Efficiency. The estimated
uptake fluxes clearly show an increase of the
importance of the gut in the total uptake with increasing
hydrophobicity (Figure 2). Furthermore, the net fluxes
decrease in time as the animal is approaching steady state.
Figure 2 shows that for TeCB the skin is probably the most
important route, although the large confidence intervals
preclude firm conclusions. For HeCB, both routes are
approximately equally important, but for PCB 153, the gut
route is truly the dominant exposure route. Although we do
not agree with the approach taken by Belfroid et al. (6), our
conclusion is comparable: the gut begins to become an
important route for chemicals with a log K ow above approximately
5 and dominates above 6. This is also reflected
in the deviation from EP with soil, which increases with K ow
from 7 to 24%. However, the confidence intervals are quite
large, and only the deviation for PCB is clearly higher than
0%. Similarly, the deviation from EP with the gut contents
decreases with increasing K ow so that for PCB 153 the tissue
residues are nearly in equilibrium with the gut contents.
The parameter estimates also allow calculation of the net
chemical AE from the estimated fluxes in the model. As shown
previously (9), the net AE depends on time; therefore, only
the maximum is given here. No general trend with K ow is
observed, although a trend may be obscured by the large
Partition Coefficients. The OM-worm partition coefficient
(K ws) was accurately predicted by the QSARs for wormwater
(20) and organic carbon-water partitioning (27),
assuming a factor of 1.7 between organic carbon and organic
matter (Table 2). Note that K ws in this case is a model
parameter; the final body residue in steady state depends on
the kinetics and lies between the extremes: equilibrium with
the soil or the gut contents (eqs 3 and 4). The actual BSAF
is thus a secondary result (eq 2) and is slightly higher than
the K ws, indicating that the concentrations in the worms
exceed equilibrium with the soil. The BSAFs after lipid and
carbon normalization are larger than unity, reflecting that
sorption increases less with K ow (27) than does bioconcentration
The K ws in the reused soil was generally somewhat lower
than the value in the initial accumulation phase. Only for
TeCB, a higher K ws was estimated in the reused soil. As we
judged this behavior to be an artifact (because of the scatter
in the initial accumulation phase), we chose to use only one
K ws for both accumulation experiments with this chemical.
For PCB 153, we saw a clear decrease in K ws for the reused
soil, showing that bioavailability had declined over 2 weeks.
This difference could not be modeled as a depletion of the
FIGURE 3. Rate constants for exchange across the skin and across
the gut wall vs log K ow. Error bars represent 95% likelihood-based
confidence intervals. The broken line indicates elimination rates
for earthworms in water only (3).
bioavailable phase, as mentioned in the Introduction.
Unfortunately, we cannot offer a convincing explanation for
this phenomenon, but some form of sequestration (either
autonomous or caused by the worms or their feces) remains
In the model, we assume the same partition coefficient
(K ws) for exchange from soil to worm (via porewater) as from
gut contents to worm (via the gut fluid). However, gut fluids
differ from porewater in that they include secretions from
the worm to aid digestion. Mayer et al. (28) have shown that,
in marine deposit feeders, these secretions include surfactants
that also act to solubilize organic contaminants above levels
expected in seawater. It may appear that the action of gut
fluids invalidates the hypothesis of passive diffusion via a
water phase. However, we do not believe this to be the case
as there is strong support for a diffusion-driven uptake (see
Experimental Section), even though surfactants facilitate this
transport (13). Furthermore, although secretions with surfaceactive
properties will increase the dissolved chemical concentration,
they cannot increase the fugacity gradient.
Because the gut fluid becomes less polar than water due to
these secretions, the chemical has less urge to flee to the
earthworm’s tissues. The gut fluid-worm partition coefficient
is decreased by the same factor as the solubility is increased,
leading to the same net uptake. The same conclusion was
reached by Lu et al. (29). We therefore believe that gut
secretions act mainly on the gut rate constant (k g) and not
on the OM-worm partition coefficient (K ws).
Rate Constants. The rate constants for exchange across
the skin (k s) and the gut wall (k g) could be separately identified
(Table 2). Only for TeCB, a confidence interval for the gut
rate constant cannot be made. There is a value that has the
highest likelihood, but very high and very low values are not
significantly worse (apparently exchange across the skin is
so rapid that the gut route cannot compete). The rate
constants for chemical exchange are shown in Figure 3, which
also gives a fit on data for the elimination from worms in a
water-only situation (3). Comparison to literature data for
elimination rates in soil is not useful as reported constants
will always reflect the total elimination flux through all routes.
The skin rate constant (k s) for TeCB is quite comparable to
the water-only data, but our value for HeCB is much higher.
This is striking given the fact that, in a water-only situation,
the contact between worm and water is likely to be more
intensive. Furthermore, rate constants for passive diffusive
exchange are expected to decrease with K ow with a slope
close to 1 (on log scale) in this K ow range because for these
compounds the diffusion across a stagnant water layer is
rate-limiting (30). However, for the rate constant across the
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skin, we find a slope that is less steep than for water-only
exposure (-0.63 vs -1.3). An explanation may be derived
from the earthworm’s physiology. In a soil situation, earthworms
lose 10-20% of their body weight in moisture each
day due to their respiratory system, which requires the
maintenance of a moist outer surface (31). In water-only
exposure, the water loss will likely be much less. The water
losses need to be replenished, requiring water transport
across the skin (also advectively transporting the chemical).
This process may explain a higher exchange rate in soil than
in water and the different relationship with K ow.
The relationship between the rate constant across the gut
wall (k g) and K ow cannot be properly assessed because of the
large confidence interval for TeCB (Figure 3). However, given
the tight confidence intervals for the other chemicals and
judging from the maximum likelihood estimate of TeCB, it
appears that k g is actually quite constant over the studied
K ow range (Figure 3). Again, what we expected was a decrease
with a slope around unity. It is possible that surfactants in
the gut facilitate the transport across the stagnant water layer,
thus influencing the relationship with K ow. This was also
proposed for the absorption of lipids in the gut, a process
following passive diffusion but with bile salts enhancing the
Consequences for EP in Risk Assessment. To our
knowledge, this is the first time that, in earthworms, the
uptake of organic chemicals from soil through the skin has
been separated from uptake resulting from feeding on soil
particles. The importance of the gut route increases with
increasing hydrophobicity, and very hydrophobic chemicals
(log K ow > 6) will mainly be absorbed from the gut contents.
There is some additional uptake as a result of feeding on soil,
but the deviation from EP with the soil is less than a factor
of 1.3, which is well within the accuracy of risk assessment
applications. The general fear that feeding leads to the
invalidation of EP is thus unwarranted.
The model presented here can adequately describe the
experimental data, and the results are consistent with the
diffusion mechanism for gut uptake (10, 11). However, in
view of the small deviations, risk assessment can rely on EP,
and specific modeling of the gut compartment is usually not
necessary. Nevertheless, this model may be useful for specific
cases, especially when the worms are not feeding on soil
alone but on a diet that is specifically contaminated (e.g.,
manure from farm animals treated with pharmaceuticals or
pesticide residues in leaf litter). In these situations, soil
concentrations along with EP are insufficient to predict body
We thank the Laboratory for Inorganic Chemistry at RIVM
for performing the lutetium measurements in soil and worms
and the Institute for Risk Assessment Sciences (IRAS, Utrecht)
for kindly providing the organic chemicals. Furthermore, we
thank Martina Vijver for demonstrating the ligaturing procedure,
Rob Baerselman for support in the experiments, and
Willie Peijnenburg and Joop Hermens for reviewing drafts of
Supporting Information Available
Model equations and data on checking the validity of the
feeding parameters of Table 1. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Received for review January 20, 2003. Revised manuscript
received May 7, 2003. Accepted May 12, 2003.
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