Soil Biology & Biochemistry 35 (2003) 313–322
www.elsevier.com/locate/soilbio
Feeding activity of the earthworm Eisenia andrei in artificial soil
Tjalling Jager*, Roel H.L.J. Fleuren, Willem Roelofs, Arthur C. de Groot
Laboratory for Ecotoxicology, National Institute of Public Health and the Environment (RIVM), P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands
Received 3 June 2002; received in revised form 19 September 2002; accepted 29 September 2002
Abstract
Quantitative information on the feeding activity of earthworms is scarce but this information is valuable in many eco(toxico)logical
studies. In this study, the feeding activity of the compost worm Eisenia andrei is examined in artificial soil (OECD medium), with and
without a high-quality food source (cow manure), and at two temperatures (10 and 20 8C). Methods are provided to estimate the most
important parameters: gut load, selection of organic matter (OM), digestion efficiency, compaction, gut retention time, and fraction of
manure in the diet. Lanthanides (Lu and Tm) were successfully used as inert markers in soil and manure, and we applied Bayesian statistics to
analyse the data and fully capture the compounded uncertainty in the parameter estimates. Results show that the compost worm does not feed
on soil indiscriminately but is able to select an OM-enriched diet from apparently homogeneous OECD medium. When manure is present on
the soil surface, approximately three-quarters of the diet still consists of soil particles. The gut load of the worms was approximately 10%
(dwt gut/wwt empty worm), varying little with the treatments. Unfortunately, the digestion efficiency could only be reliably estimated at
20 8C, and was approximately 40%. Temperature clearly affected feeding as a 108 temperature decrease nearly doubled the gut retention time
(from 2.9 to 5.5 h), which corresponds to a two-fold decrease in feeding rate. The present data may be used to interpret toxicity and
accumulation studies with E. andrei in OECD medium. However, care must be taken, as it seems possible that feeding is influenced by the
size of the worm and subtle differences in experimental set-up.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Earthworms; Eisenia andrei; Feeding activity; Digestion; Gut load; Gut retention time
1. Introduction
Earthworms play a vital role in many soil ecosystems,
but quantitative information on their feeding habits is
scarce. Nevertheless, this information is particularly valuable for studying earthworm energy budgets (Bolton and
Phillipson, 1976), nutrient cycling in ecosystems (Whalen
et al., 1999), dung removal from pastures (Hendriksen,
1991), and the uptake of chemicals by earthworms from
their food (Jager, 2003). Because much of the earthworm’s
activity is located below the surface, it is difficult to observe
and quantify feeding without disturbing the organisms. It
may seem that earthworms just feed indiscriminately on soil
particles at a fixed rate, but a few examples illustrate that the
situation is more complex. Firstly, earthworms are able to
select a particular fraction from the soil matrix that is more
* Corresponding author. Present address: Department of Theoretical
Biology, Vrije Universiteit, FALW, De Boelelaan 1085, NL-1081
HV Amsterdam, The Netherlands. Tel.: þ 31-20-444-7134; fax: þ 31-20444-7123.
E-mail address: [email protected] (T. Jager).
organic than the bulk soil (Bolton and Phillipson, 1976),
moreover, the exact preference seems to be species specific
(Piearce, 1978). Secondly, many earthworm species prefer
high-quality food sources like leaf litter or manure.
However, even when such a preferred food source is
present, the diet will still consist of an appreciable amount
of mineral soil (Barley, 1958; Hendriksen, 1991), possibly
because of the mechanical grinding action of the mineral
particles (Schulmann and Tiunov, 1999). As a third
example, the retention time of materials in the gut may
depend on whether the animal is feeding or making new
burrows (Barley, 1958; Parle, 1963).
The set of parameters necessary to describe the
feeding activity depends on the subsequent use of the
data. The following set is needed to describe feeding in
an extended bioaccumulation model for chemicals (Jager,
2003): gut load, digestion efficiency, selection of organic
matter (OM) from soil, weight decrease of the gut
contents due to digestion and absorption of food
(compaction), gut retention time, and the fraction of a
specific food source in the diet. Digestion efficiency and
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
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T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
OM selection may be determined from measuring carbon
in the crop contents and faeces (Morgan and Morgan,
1992), gut load by weighing the faeces produced on
starvation (Hartenstein et al., 1981). A dynamic approach
is, however, needed to determine the retention time.
Simple methods relate the defecation rate to the weight
of the gut contents (Barley, 1958; Bolton and Phillipson,
1976) or follow faecal production (egestion) of recognisable ingested material (Curry and Bolger, 1984;
Hartenstein et al., 1981). Others apply inert markers;
i.e. materials that are not assimilated by earthworms but
can be easily measured like chromic oxide (Hendriksen,
1991). In this study, rare earth metals (lanthanides) are
proposed. Lanthanides have no known biological function
and have already been successfully applied as inert
markers in studies with ruminants (Ellis, 1968) and fish
(Austreng et al., 2000). They can be added to the soil or
a food source in solution (e.g. as chloride salts), thereby
allowing homogeneous distribution and likely preventing
irritation (which could lead to shorter retention times;
Parle, 1963). These compounds are easily measured by
ICP-MS in low concentrations, and have very high
sorption coefficients in soils (Jones, 1997) and sediments
(Weltje et al., 2002), making them unavailable for uptake
into the earthworm’s tissues (Helmke et al., 1979).
In the present study, several methods are combined to
obtain a complete set of parameters for the compost worm
(Eisenia andrei) in artificial soil (OECD, 1984). The feeding
activity is quantified in the presence and absence of a food
source (ground cow manure), and at two temperatures (10
and 20 8C). Although this system is not really representative
for soil ecosystems, it may act as a model system. Artificial
soil and ground cow manure are highly homogeneous and
the combination with E. andrei is recommended for routine
testing of chemicals (OECD, 1984). If quantification of the
feeding habits is not possible in this simplified system, it
will certainly fail for more field-relevant situations.
2. Material and methods
The symbols that are used in this study are explained in
Table 1, along with their units.
2.1. Exposure media and acclimatisation of earthworms
Artificial soil (OECD medium) was prepared according
to OECD (1984). The water content was brought to 40%
(l water/kg dry medium). After wetting the soil, it was
stored in closed plastic containers at 5 8C for 4 – 6 weeks,
prior to the experiments. After storage, the pH (KCl) of the
OECD medium was 5.0. Cow manure was oven dried,
ground (0.5 mm), and brought to 100% water content
(l water/kg dry manure). The fraction OM (Fom) in the
OECD medium was 11% and in cow manure 52%
(determined as described in Section 2.2).
Sub-adult earthworms (E. andrei, weighing between 200
and 300 mg), were taken from mass cultures at our
laboratory. First, the animals were allowed to evacuate
their gut contents by keeping them on moist filter paper for
24 h at 20 8C. Subsequently, the animals were transferred to
plastic containers with 175 g of unspiked OECD medium.
Three animals were used per container, and the containers
were placed at 10 and 20 8C, covered by a black plastic pot
to minimise disturbance. For the animals that were fed with
manure, ground cow manure was wetted and added several
times (ad libitum conditions) in a small hole at the soil
surface. The animals were left to acclimatise for approximately 1 week under these conditions after which they were
used for the experiments described below.
2.2. Determination of carbon in the crop
and the anterior gut
Per treatment, six containers were used (three animals
each). After 1 week under the test conditions, animals were
killed by brief immersion in hot tap water. Crop contents
(representative for ingesta) as well as gut contents from the
posterior part of the gut (last cm of the gut, representing
egesta) were removed by dissection and weighed separately
into aluminium cups for carbon analysis. Crop contents of
three animals were combined in one cup; contents from the
posterior part of the gut were analysed individually when
possible (values were averaged afterwards). Additionally,
samples of soil and manure were taken. All samples were
freeze dried before carbon analysis (elemental analyser by
Fisons Instruments Model EA 1108, Rodana, Italy). Carbon
levels were translated to Fom by multiplication with 1.7.
The digestion efficiency of OM after gut passage (Fdig)
may be directly calculated from the Fom in ingesta and
egesta
Fdig ¼
Fom-ingesta 2 Fom-egesta
Fom-ingesta
ð1Þ
This equation is sufficiently accurate as long as the digestion
efficiency is not too high and the Fom of the ingesta is low.
Otherwise, digestion and absorption of OM will result in a
significant weight decrease of the gut contents (compaction), thereby affecting Fom of the egesta (which are
effectively concentrated). This effect is usually ignored in
feeding studies, and, therefore, we need to derive a new
equation for digestion, accounting for compaction. If we
assume that the weight decrease of the gut contents during
gut passage is only due to digestion of OM, we obtain the
following relation for the compaction factor (Fcom)
Fcom ¼
ingestion rate ðkg=hÞ
1
¼
egestion rate ðkg=hÞ
1 2 Fdig Fom-ingesta
ð2Þ
The validity of this relationship between digestion and
compaction was verified using the data of Dickschen and
Topp (1987) who followed feeding of Lumbricus rubellus
T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
on a diet of leaf litter in the absence of soil medium.
Although this study provides an excellent way to check our
assumptions, the use of a litter-only diet leads to very high
compaction rates, irrelevant for field situations. When (low
OM) soil is part of the diet, compaction is likely to be
(much) less important (Bolton and Phillipson, 1976;
Piearce, 1972).
When compaction is significant, the measured Fom in the
egesta should satisfy the following equation
Fom-egesta ¼ Fcom ð1 2 Fdig ÞFom-ingesta
ð3Þ
This equation could also be verified on the basis of the data
from Dickschen and Topp (1987). Eqs. (2) and (3) can be
rearranged to free Fdig; leading to an estimate of digestion
on the basis of the measured OM content in ingesta and
egesta, and accounting for the effects of compaction
!
Fom-egesta
1
Fdig ¼ 1 2
ð4Þ
Fom-ingesta 1 2 Fom-egesta
The measured carbon content in the crop can also be used to
derive a selectivity factor (Fsel) for OM. This factor is
defined as the ratio of the organic carbon content in the crop
and the soil, and gives an indication of the ability of
earthworms to select an enriched fraction from the soil
matrix.
2.3. Weight of the faeces on starvation
Per treatment, four containers were used (three animals
each). After 1 week of acclimatisation, the animals were
placed in a petri dish with wet filter paper to evacuate their
gut contents (24 h at 20 8C). Animals as well as their faeces
were weighed (faeces were collected after drying the entire
petri dish overnight at 80 8C). The animals were transferred
to clean filter paper and left for another 24 h. Again, worms
and faeces were weighed. An estimate of the growth rate
could be made as the worms were also weighed before the
acclimatisation.
Under constant environmental conditions, the weight of
the gut contents appears to be a fixed fraction of the body
weight (Bolton and Phillipson, 1976; Hartenstein et al.,
1981; Curry and Bolger, 1984). The weight fraction of
faeces (Fege) was taken as the total dry weight of gut
contents evacuated in 48 h, divided by the worm wet weight
after 24 h. The body weight after 24 h was used, as the
worms seemed to lose more weight between 24 and 48 h
than could be attributed to gut contents alone (presumably
water loss from the tissue). Furthermore, the fraction of the
gut contents remaining in the worm after 24 h starvation
(Frem) was calculated from the faecal weights.
The fact that compaction of the gut contents may occur
during gut passage also has consequences for the measurements of the gut load. Weighing the faeces provides us with
an estimate of the gut load after compaction. When
compaction of the gut contents is not negligible, the faecal
315
weight (Fege) cannot be related directly to feeding rates.
Instead, the weight of the gut contents before compaction
(Fing) is required, which can be calculated as the product of
Fege and Fcom. The relative weight of the gut contents
present in a living worm (Fgut) is calculated as the average
of Fing and Fege. Body growth is not problematic for this
assessment, as long as the gut load remains a constant
fraction of the body weight.
2.4. Experiments with inert markers
Pilot experiments showed that E. andrei does not
appreciably absorb lanthanides from OECD medium. This
is true for the naturally present lanthanides as well as spiked
salts of lutetium (Lu) and thulium (Tm). Lu and Tm were
selected as these have very low background concentrations
in OECD medium (approx. 40 mg kg21
dwt). Animals taken
directly from the culture may behave differently in the
initial phase following transfer to OECD medium. Therefore, all animals were pre-treated on this medium (Section
2.1) before transfer to medium spiked with lanthanides. Per
treatment, 11 containers were used (three animals each).
Lu and Tm were purchased as hydrated chloride salts
(purity 99.9%) from Alfa Aesar (Karlsruhe, Germany).
Chemicals were dissolved in tap water and added to the dry
medium (Lu to soil, Tm to cow manure) to achieve nominal
concentrations of 15 mg kg21
dwt. The medium was immediately mixed with a high-speed blender for several minutes.
The spiked OECD medium was stored in closed plastic
containers at 5 8C for 4 – 6 weeks, prior to the experiments to
achieve equilibrium in the soil system. The spiked cow
manure was stored at 5 8C for 3 days only, to minimise
fungal and bacterial growth.
After the acclimatisation period, the animals were
transferred to similar containers, containing OECD medium
spiked with Lu. The soils had been moved to test
temperatures 1 day before the animals were introduced,
and for the treatments with food, 3 gwwt of Tm-spiked cow
manure was added to the soil surface. After an exposure
period (0, 1, 2, 3, 5, 7, 9, 15, 24 and 48 h), the animals were
recollected, rinsed in tap water, weighed and frozen at
2 20 8C (including their gut contents).
Four samples of soil and three of manure were taken to
check the spiked concentrations. An additional container
with earthworms was used to check whether the worms
assimilated the chemicals. These worms were exposed for
48 h under the test conditions, and allowed to evacuate their
gut contents for 48 h on moist filter paper at 20 8C (the filter
paper was changed after 24 h).
Before analysis, the frozen earthworms, soil and
manure samples were freeze dried for 2 days. An acid
digestion of the samples was performed as described
earlier (Janssen et al., 1997a,b), including procedural
blanks for contamination in the digestion procedure, and
for the proper functioning of the microwave oven. In
order to check the digestion procedure, the certified
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T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
reference material NIST 2710 ‘Montana soil’ (Garthersburg, MD, US) was used. Because Lu and Tm were not
certified in this material, Cd was used as a target
compound for the digestion efficiency (under the
assumption that this compound is representative for the
spiked Lu and Tm). The recovery for cadmium was
within the certified range indicating that the digestion
was complete. The digests were analysed for Lu and Tm
on an ICP-MS (Hewlett Packard 4500plus, Avondale,
PA, US). Standard addition experiments in the different
matrices digests showed excellent recovery of these
elements (100 ^ 5%) indicating that interferences did not
occur during final analysis. Limits of determination were
15 and 3 mg kg21 for Lu and Tm, respectively.
Table 1
Summary of the parameters used in this study (symbols, explanation and
units)
2.5. Models used to analyse experiments with inert markers
Fgut
After the earthworm is transferred to the spiked soil,
the concentration of Lu in the worm will linearly
increase until the entire gut is filled with Lu-spiked
medium (assuming perfect ‘plug-flow’ conditions). While
the animal is replacing clean soil with spiked soil in its
gut, the total concentration in the worm (including gut
contents) is given by
Fing
Cw ðtÞ ¼
Qf t Fsel Cs
Ww Fsolids þ Fgut Ww
ð5Þ
Symbols
Explanation
Unit
Cf
Cs
Cw
Concentration of marker in food
Concentration of marker in soil
Concentration of marker in total
worm (incl. gut contents)
Compaction factor of gut contents
during gut passage
Digestion efficiency of OM during
gut passage
Egested faeces (after compaction), fraction
of empty worm wwt
Fraction of specific food source
in diet
Gut weight, fraction of empty
worm wwt
Ingested weight (gut weight fraction
before compaction)
Fraction OM
Fraction of total gut contents,
remaining after 24 h starvation
Factor by which ingesta contain
more OM or Lu than
bulk soil
Fraction solids in empty worm
(wet-dry weight ratio)
Time needed before worm starts
to feed on surface
Retention time of material in
the gut
Feeding rate
Worm wet weight
mg kg21
dwt
mg kg21
dwt
mg kg21
dwt
Fcom
Fdig
Fege
Ffood
Fom
Frem
Fsel
Fsolids
Tlag
Tret
where t is the exposure time (other symbols explained in
Table 1). Because the worm may select specific fractions
of the soil, a selectivity factor for Lu is included (Fsel).
The wwt/dwt ratio of the worm (Fsolids) enters the
equation because Fgut is expressed on the basis of worm
wet weight while Cw is measured on a dry weight basis.
The Fsolids was determined by comparing the weight of
empty worms before and after freeze-drying.
The feeding rate (Qf) can be written as a function of the
gut weight before compaction (Fing Ww) and the retention
time of the gut contents (Tret) as
Qf ¼
Fing Ww
Tret
ð6Þ
After replacing Eq. (6) in Eq. (5), the full concentration
equation becomes
Fing
t
Cw ðtÞ ¼
F C min
;1
ð7Þ
Tret
Fsolids þ Fgut sel s
The construction at the right-hand side of the equation (with
the minimum of t/Tret and 1) ensures that the uptake is linear
until the retention time is reached, after which no further
increase of the total concentration in the worm takes place.
Growth is not problematic for this assessment, as long as the
gut load remains a constant fraction of the body weight (Cw
is weight-based, just as Fing and Fgut).
In two of the four treatments, Tm-spiked manure is
given at the soil surface. The earthworms will feed on
Qf
Ww
(wt fraction)
(wt fraction)
kgdwt kg21
wwt
(wt fraction)
kgdwt kg21
wwt
kgdwt kg21
wwt
(wt fraction)
(wt fraction)
(wt fraction)
kgdwt kg21
wwt
h
h
kgdwt h21
kgwwt
the preferred food source, but will also include the Luspiked soil in their diet. We, therefore, have two uptake
curves for these treatments, which need to be analysed
simultaneously. Two equations similar to Eq. (7) are
needed, but we need to add a parameter that divides the
ingestion into food and soil (Ffood). For the marker in soil,
this implies multiplication of the right-hand side of Eq. (7)
with a factor 1 2 Ffood. The Tm equation is given by Eq.
(7), multiplied with Ffood and lacking a selectivity factor
(the worm is not expected to select for Tm-enriched
fractions in its food). We observed that, although the Lu
concentration immediately increases after transfer to spiked
medium, the Tm concentration only starts to increase after
several hours. This behaviour is probably caused by the
stress of handling, particularly affecting feeding from the
soil surface, where the food is located. The equation for Tm,
therefore, also includes a lag time (Tlag).
Eq. (7) contains many parameters, which implies that the
measured marker concentrations against the time are not
sufficient to identify them all. However, we have prior
information on several of the parameters: Cs and Cf were
measured, as well as Fsolids. Knowledge about Fing and Fgut
is available from the determination of Fege and Fcom
T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
(Section 2.3). These measurements are however, also
uncertain, and we want to include this uncertainty in the
estimation of the other parameters. Furthermore, when food
is present, several parameters are common to the models for
Lu and Tm: Ffood is part of both model equations, as well as
the retention time of the gut contents. Moreover, the
retention time in the experiments with added manure is
likely to be the same as on OECD medium only, as retention
time seems to be independent of food type (Hartenstein et al.,
1981).
A Bayesian statistical framework provides a straightforward approach for the analysis of these data. In a
Bayesian framework, parameters are characterised by a
probability distribution (representing the ‘degree of
believe’ about the parameter’s value). Before analysing
new data, the a priori knowledge about a parameter is
summarised in a so-called prior distribution. This prior
distribution is multiplied by the parameter information
contained in the data (the likelihood), to obtain the
posterior distribution from which statistical inference can
be made. The measurements from the previous experiments (Section 2.2 and 2.3) are considered prior
information for the analysis of the marker data; the
parameters that are truly unknown receive uniform (noninformative) prior distributions (Table 2). For a lucid
introduction to Bayesian methods in ecology, see Ellison
(1996).
When the data are normally distributed, the likelihood
of the parameters is related to the sum-of-squares (SSQ),
resulting from the fit of the model to the data (Box and
Tiao, 1992). Here, we expect multiplicative errors and,
therefore, chose to log-transform the data before
Table 2
List of parameters included in the Bayesian analysis and their prior
distributions (symbols explained in Table 1)
Symbols
Prior information
General parameters
Cs
Measured: 13.8 (s.e. 1.06,
n ¼ 4) mg kg21
dwt
Cf
Measured: 11.6 (s.e. 0.272,
21
n ¼ 3) mg kgdwt
Fsolids
Measured: 0.14 (s.e. 0.0013,
n ¼ 9)
OECD-soil only
See Table 3 (temperature dependent)
Fcom
See Table 3 (temperature dependent)
Fege
Fsel
Non-informative
Tret
Non-informative
Manure added to surface
See Table 3 (temperature dependent)
Fcom
See Table 3 (temperature dependent)
Fege
Fsel
Non-informative
Tret
Posterior from OECD (temperature
dependent)
Ffood
Non-informative
Tlag
Non-informative
Distribution
Student-t
Student-t
None (constant)
None (constant)
Student-t
Uniform
Uniform
None (constant)
Student-t
Uniform
Custom
Uniform
Uniform
317
calculating the SSQ (thus assuming a log-normal
distribution of the data). The resulting posterior probability of the total set of m parameters (u1…um), given
the observed n data points, is calculated by
pðu1 …um ldataÞ / ½SSQðu1 …um ; dataÞ2n=2
m
Y
pðui Þ
ð8Þ
i¼1
where p(ui) stands for the prior probability distribution of
the parameter ui. Multiplication of the likelihood (a
function of the parameters and the data) with the prior
knowledge thus gives us the joint posterior distribution of
the parameters. We can now search for the parameter
values that maximise the posterior probability (simplex
search).
The model of Eq. (7) is highly non-linear because of the
thresholds times (Tret and Tlag), hampering the estimation of
confidence intervals (Klepper and Bedaux, 1997). Therefore, Eq. (8) was evaluated numerically on a regular
parameter grid. From this joint distribution, probability
distributions of the individual parameters were determined,
which were summarised with a 90% posterior probability
interval. For the studies with food, the posterior distributions of Tret from the experiments without food at 10 and
20 8C were used as prior information. The entire analysis
was programmed in MatLab 6.1; a list of all prior
distributions is given in Table 2.
3. Results
3.1. Selection, digestion and compaction
The dissection and subsequent determination of carbon
in crop contents and posterior gut provides estimates for
selection (Fsel, ratio of Fom in crop and soil), digestion (Fdig,
Eq. (4)), and compaction (Fcom, Eq. (2)), summarised in
Table 3. Most samples showed a clear difference between
the amount of carbon in crops and posterior gut. The data at
20 8C are quite consistent: when no food is present, the
digestion of OM is approximately 35% but when the worms
are fed manure, digestion was higher (46%). This difference
was, however, not significant because of the large variation
in the data. At 10 8C the data are less consistent, firstly
because there are less data points (several samples were lost
in the analysis), and secondly, several of the data lead to
unrealistic estimates and appear to be outliers.
The current study shows compaction Eq. (2) to be
appreciable, and diet specific: 9% for OECD medium and
18% when fed manure (at 20 8C). On the basis of the carbon
content in the crop, the worms appear to select a fraction of
the soil that is more than two times as organic as the bulk
soil. When manure is present on the soil surface, the Fom in
the ingesta results from the soil as well as the manure, and,
therefore, no estimation of selectivity is possible.
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T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
Table 3
Summary of the parameters (symbols explained in Table 1) and the resulting values for the different treatments (n.a. ¼ not applicable). Each value is derived
from n pooled samples of three worms
Symbols
Fsel
Fdig
Fcom
Fege
Frem
r2adja
Fsel
Tret
Ffood
Tlag
a
OECD 10 8C
OECD 20 8C
Manure 10 8C
Selection, digestion and compaction (mean with s.e. or individual data)
n¼3
n¼6
n¼3
2.3, 3.4, 1.7
2.1 (0.053)
n.a.
0.35, 0.51, 0.048
0.35 (0.033)
20.18, 0.24, 0.30
1.08, 1.20, 1.01
1.09 (0.011)
0.96, 1.09, 1.08
Gut load (mean with s.e.)
n¼4
n¼4
n¼4
0.096 (0.0043)
0.12 (0.012)
0.082 (0.0042)
0.074 (0.012)
0.056 (0.021)
0.21 (0.031)
Inert markers (best estimate with 90% probability region)
n ¼ 11
n ¼ 11
n ¼ 20
0.99
0.99
0.88/99
1.6 (1.3–2.2)
1.7 (1.4–2.3)
0.94 (0.57– 1.4)
5.6 (4.5–7.5)
2.9 (2.5–3.3)
5.4 (4.3–6.5)
n.a.
n.a.
0.35 (0.28– 0.42)
n.a.
n.a.
2.3 (2.0–2.5)
Manure 20 8C
n¼5
n.a.
0.46 (0.067)
1.18 (0.038)
n¼4
0.098 (0.010)
0.13 (0.025)
n ¼ 22
0.94/0.93
1.0 (0.72–1.6)
2.9 (2.4–3.1)
0.22 (0.16–0.31)
1.0 (0.96–1.5)
Adjusted r 2 of the model fit after log-transformation, for manure treatment separate r 2 values for Lu and Tm, respectively.
3.2. Gut load
On OECD medium, the growth over the acclimatisation
period was 1 and 4% (20 and 10 8C, respectively), and on
manure 35 and 16% (20 and 10 8C, respectively). The
egested dry weight of faeces was approximately 10% of the
fresh body weight of the worm (Fege, Table 3). There is no
significant difference between the treatments, although there
is a tendency for a slight decrease with temperature and with
the addition of manure. The fraction of the gut contents
remaining in the worm after 24 h starvation (Frem) appears
to be related to the treatment. As can be seen from Table 3,
the remaining fraction is larger when the worms are fed
manure, and larger at lower test temperatures (the only
Fig. 1. Concentration of lanthanides in the earthworms (including gut contents) against time. Filled symbols are Lu from soil, open symbols Tm from manure,
the line is the highest probability fit. Triangles are the concentrations in worms after 48 h starvation.
T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
significant differences are between Frem of manure at 10 8C
versus both OECD-only treatments).
3.3. Inert markers
Concentrations of the lanthanides in the worms (including gut contents) are shown in Fig. 1, together with the fits of
Eq. (7). The model fits are acceptable (adjusted r 2 . 0.88
after log-transformation), although the variation in the data
from worms that were fed manure is clearly higher than in
soil only. The lanthanides appear to function well as inert
markers, judging from the fact that the concentration in the
worms does not increase further after the retention time.
After 48 h exposure and 48 h starvation on filter paper, the
concentration of Lu in the worm samples is 4– 11% of the
concentration in worms with gut contents, and for Tm this
was even lower (1 – 3%).
The model parameters are quite accurately identified in
the Bayesian fitting procedure; the results are shown in
Table 3 as the highest-probability estimate with 90%
probability intervals. The earthworms are clearly selecting
an Lu-enriched diet from OECD medium, independent of
the test temperature (Fsel is 1.6 and 1.7, somewhat less than
for OM). A clear lag time can be observed before the worms
start feeding on the surface, which is larger at 10 8C (2.3 vs.
1.2 h). The selectivity factor (Fsel) from soil is lower when
manure is present than in the OECD-only experiment. The
worms are thus selecting less Lu-enriched soil fractions
when a preferred food source is available. Despite the
preference for manure, this matrix makes up less than half of
the total diet: 0.22 at 20 8C, and 0.34 at 10 8C (probability
intervals overlap).
The retention time of the gut contents (Tret) is
approximately two times higher at 10 than at 20 8C
(approximately 5.5 vs. 2.9 h). The posterior distribution of
retention time for the OECD-only experiments was used as
prior information for the experiments with manure (Table 2).
This was helpful, as feeding activity was more variable
when a food source was present in the system (the data from
the manure-fed worms did not provide clear information on
the retention time). As a result, the resulting posterior
distribution for retention time from the manure data was
very similar to those of the OECD-only experiments
(Table 3).
4. Discussion
4.1. Selection, digestion and compaction
The dissection of E. andrei turned out to be rather
difficult because of the small size of the tested sub-adults.
Furthermore, not all animals had material in the last part of
their gut, and some of the crop contents could not be
properly removed. An additional difficulty was the loss of
samples at 10 8C and the apparent outliers. For this reason,
319
the discussion will focus on the results at 20 8C. In general,
the variation in the data is large, precluding conclusions on
differences between the treatments. This variation partly
reflects inter-individual variation in feeding pattern and
physiology, but is also caused by the form of Eq. (3). An
increase in digestion does not lead to a proportional
decrease of Fom in egesta because digestion also increases
compaction (Eq. (2)), especially when Fom of the ingesta is
high. Small measurement errors can therefore, be magnified
in the estimates of digestion and compaction. The observed
digestion efficiencies around 40% are much higher than
values predicted earlier for E. andrei (Jager, 2003). They are
also much higher than values reported for the geophageous
Aporrectodea rosea (2%; Bolton and Phillipson, 1976), and
are more in line with values given for A. longa and L.
terrestris (30 – 40%, Morgan and Morgan, 1992). These
species are larger and have a much higher gut retention time
(20 h for L. terrestris, Parle, 1963).
Compaction is often ignored in earthworms feeding on
soil (Piearce, 1972), but was demonstrated to be up to a
factor of 4 when worms were kept on a litter-only diet
(Dickschen and Topp, 1987). In the present study,
compaction was less extreme, though still considerable
(9% in OECD medium, 18% with manure). One should note
that the estimates of digestion efficiency and compaction are
not independent, as discussed above.
The measured carbon content in the crop suggests
selectivity for OM of a factor of 2.1 on OECD medium.
However, it is not clear whether the carbon measured in the
crop and in the gut is derived from the diet only; it may
include secretions from the organism itself (from the
calciferous glands, mucus and digestive enzymes), or
contamination of the samples with coelomic fluid. The
exact origin of the carbon in the gut requires further
elaboration, but for now we will assume that the main
contribution is from the diet.
4.2. Gut load
Weighing the faecal production proved to be very simple
to perform on a routine basis although it is likely to
underestimate the faecal output, as it is difficult to collect all
of the faeces from the filter paper. Here, the faeces produced
over 48 h are taken as the total output, but even then, the
worm may not be completely clean. In most bioaccumulation studies, 24 h is taken to evacuate the gut contents.
However, the data in Table 3 show that at least 6 –21% of
the gut contents remains after this period, and worse, that
this fraction may depend on the treatment. It is for this
reason that several authors propose methods with a longer
duration (Denneman, 1994; Pokarzhevskii et al., 2000),
which decreases the bias from remaining gut contents but
may also lead to substantial elimination of the chemical
from the tissues. Stafford and McGrath (1986) proposed to
correct for remaining soil in the worm by measuring the
acid-insoluble residue in the worm and comparing it to
320
T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
the soil. However, the validity of this approach is
questionable, given the fact that the earthworm is selecting
a particular fraction from the total soil (which may differ in
insoluble residues and in chemical content from the bulk
soil).
The relative egested weight (Fege) is quite constant
around 10% (dwt/wwt worm, Table 3). This fraction seems
to be lower when food is present, but this difference may
also be caused by an increased compaction in the gut (at
least at 20 8C, Table 3). Hartenstein et al. (1981) found a
much higher effect of temperature and addition of manure
on E. fetida which, given the close relation of this species
with E. andrei, is remarkable. The reasons remain unclear,
but may be related to differences between the studies in the
quality of the soil and manure.
The addition of manure allows the worms to grow
considerably, but OECD soil still contains sufficient
nutrition to sustain them throughout the experiment. The
total amount of OM digested when fed manure can be
calculated from the parameters and is 1.4 times larger than
on OECD soil only (at 20 8C). However, the quality of the
OM may also have led to the differences in growth.
4.3. Inert markers
The model fits in Fig. 1 clearly indicate the usefulness of
the lanthanides Lu and Tm as markers of the feeding
activity, and support the assumption of plug flow conditions
in the gut. Nevertheless, some Lu and, to a lesser degree, Tm
is remaining in the worm after 48 h of starvation. It is
possible that this represents soil particles still left in the gut,
or sorption to the outer skin of the worm, but some
assimilation cannot be ruled out. Compared to simpler
methods, the use of markers like Lu and Tm has the
advantage that these compounds are unlikely to irritate the
worm (and thus influence retention), provide a dynamic
picture of feeding (and are thereby able to test the plug-flow
assumption, and show lag times), and allow quantification
of feeding from multiple sources (here from soil and from
manure). The Bayesian framework allows accurate parameter estimations, but the computations cannot be done by
standard statistical packages, and are quite time consuming.
However, this framework lends itself readily to the use of
existing (uncertain) information in the analysis of new data
(Ellison, 1996).
The selection of Lu from soil is less than estimated on the
basis of the carbon measured in the crop contents (Fsel,
Table 3). Even though the variation in both values is large
enough to ignore this difference, it is conceivable that Lu is
also partly sorbed to clay minerals and is, therefore, not
entirely representative for OM selection (although the
affinity for kaolinite clay is not very high at this pH;
Coppin et al., 2002). However, if not all of the carbon in the
crop is derived from the soil (Section 4.1), these two figures
may be more consistent. Using a marker that is more
specifically bound to OM may resolve this matter
(Kukkonen and Landrum, 1995).
The retention times (Tret) observed, are quite comparable
to the estimates made by Hartenstein et al. (1981), also
showing a clear effect of temperature. As the gut load hardly
depends on temperature, the feeding rate (Eq. (6)) of the
worms will be nearly halved at 10 8C, compared to 20 8C.
The manure data do not seriously affect the estimate of the
retention time, although there is a slight shift to lower
values. Care must be taken in applying the estimates for
retention time directly to other studies, as it seems likely
that retention is directly related to the length of the gut. A
large difference between adults and juveniles was indeed
found in a polychaete (Ahrens et al., 2001), in contrast,
Hartenstein et al. (1981) showed no influence of size in E.
fetida. The data collected by Bolton and Phillipson (1976)
show slightly higher retention times for adult A. rosea,
compared to juveniles, and suggest that temperature
influences gut retention for juvenile worms more than for
adults.
The data for the manure-fed worms are clearly more
variable than for the worms kept in OECD medium only
(Fig. 1). It is likely that the worms do not feed continuously
on a mix of soil and manure but alternate between feeding
on soil and manure (as indicated by the co-varying data of
Lu and Tm in Fig. 1). The worms also use the soil in a
different way when manure is present as the selection factor
is reduced to around unity. Possibly, the worms now feed on
soil indiscriminately as a better source of nutrition is
available, and soil only serves to add mineral particles to the
ingesta (Schulmann and Tiunov, 1999). Even though
the worms were acclimatised to a situation with manure
on the surface, it still took them several hours to start
feeding after transfer to the spiked media (but ingestion of
soil started immediately). This lag time (Tlag) was nearly
twice as long at 10 8C.
Despite the high variation, the fraction of manure in the
diet (Ffood) is quite accurately fixed at 0.22 at 20 8C and 0.34
at 10 8C. These figures are, however, not consistent with the
measured Fom in the crop contents (data not shown). If we
can assume that no selection of OM from soil occurs, the
Fom data in the crop indicate a fraction of manure in the diet
of approximately 0.5. This discrepancy could point at the
presence of carbon in the crop, derived from various
secretions by the worm. On the other hand, the carbon
content in the crop and in the faeces were determined after 1
week exposure to manure, whereas the Tm-spiked manure
was only a few days old and included a small amount of
chloride (the counter-ion in the metal salt). Possibly, the
chloride and the age of the manure play a role in its quality
for the worm and may thus influence the diet composition.
Hendriksen (1991) observed variation in gut load and
retention time with dung age for L. festivus. These
observations stress the sensitivity of the feeding process to
all kinds of factors. Care should thus be taken to perform
T. Jager et al. / Soil Biology & Biochemistry 35 (2003) 313–322
the estimation of digestion and gut load, and the marker
experiments under the exact same conditions.
5. Conclusions
In this study, the feeding activity of the compost worm E.
andrei is examined, and methods are provided to estimate
the physiological parameters. Gut load was derived from
weighing the faecal output; selection, digestion and
compaction from measuring carbon in the gastro-intestinal
tract. Lanthanides (Lu and Tm) are successfully used as
inert markers and provide estimates of the gut retention time
and the fraction of manure in the diet. The methods and
models applied in this study may also be used for
experiments with more field-relevant worm-soil combinations in ecological studies.
This study clearly shows that the compost worm does
not feed on soil indiscriminately but is able to select an
OM-enriched diet from apparently homogeneous OECD
medium. Even when manure is present, a large part of
the diet still consists of soil particles. The gut load is not
significantly affected by the presence of manure or
temperature, but the gut retention time nearly doubles by
a 108 temperature decrease. The present data may be
used to aid the interpretation of routine studies with E.
andrei in OECD medium (e.g. how chemical exposure
via ingestion is affected by temperature and providing
manure). Especially, these data can be used to parameterise bioaccumulation models that include the feeding
process. However, care must be taken in using these
data, as feeding activity may be influenced by subtle
differences in experimental set-up (e.g. the age of the
manure used as feed).
Acknowledgements
The authors would like to thank the department of
inorganic analytical chemistry at the RIVM (especially Rob
Ritsema and Carlo Strien) for the measurements of the
lanthanides and carbon. Furthermore, we are grateful to
Lennart Weltje, Willie Peijnenburg, Kees van Leeuwen,
Joop Hermens, and two anonymous reviewers for valuable
comments on drafts of this paper.
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