Cadmium affects toxicokinetics of pyrene in the collembolan Folsomia candida.

Abstract

Since toxicity is time dependent, short-term toxicity tests may overlook mixture effects, because chemical accumulation within an organism takes time. We therefore studied the effects of cadmium on the toxicokinetics of pyrene and its metabolites in the soil-dwelling collembolan Folsomia candida exposed through Lufa 2.2 soil. Single pyrene was rapidly taken up and steady state was reached within the 337-h exposure period. Simultaneous exposure to cadmium significantly decreased the pyrene uptake and elimination rate, resulting in a prolonged half life. Kinetics of the first phase metabolite OH-pyrene was also significantly influenced by cadmium. Cadmium increased the hydroxylation rate of pyrene but slowed down its further metabolization, again resulting in a prolonged half life. We showed that pyrene accumulation and metabolization are significantly influenced by the presence of cadmium. Our results suggest that mixture effects may be dependent on exposure time.

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Cadmium affects toxicokinetics of pyrene in the collembolan
Folsomia candida
Mieke Broerse•Hilde Oorsprong•
Cornelis A. M. van Gestel
Accepted: 30 November 2011/Published online: 11 February 2012
? The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract
toxicity tests may overlook mixture effects, because
chemical accumulation within an organism takes time. We
therefore studied the effects of cadmium on the toxicoki-
netics of pyrene and its metabolites in the soil-dwelling
collembolan Folsomia candida exposed through Lufa 2.2
soil. Single pyrene was rapidly taken up and steady state
was reached within the 337-h exposure period. Simulta-
neous exposure to cadmium significantly decreased the
pyrene uptake and elimination rate, resulting in a pro-
longed half life. Kinetics of the first phase metabolite OH-
pyrene was also significantly influenced by cadmium.
Cadmium increased the hydroxylation rate of pyrene but
slowed down its further metabolization, again resulting in a
prolonged half life. We showed that pyrene accumulation
and metabolization are significantly influenced by the
presence of cadmium. Our results suggest that mixture
effects may be dependent on exposure time.
Since toxicity is time dependent, short-term
Keywords
aromatic hydrocarbons ? Biotransformation ? Soil
arthropods ? Mixtures
Uptake and elimination kinetics ? Polycyclic
Introduction
When exposed to chemicals, the extent to which an
organism may be affected is determined by several
aspects that influence the bioavailability, uptake, internal
distribution and eventually the concentration and, hence
the effect of the chemical at the site of action within the
body. If a chemical is taken up by an organism, exposure
time is a very important factor to reach an internal effect
concentration. When for a given chemical the uptake is
slow, but still exceeds the excretion/detoxification rate, the
organism will slowly accumulate the chemical, which
eventually may lead to toxic effects. For such chemicals
the lower exposure concentrations cause no or little acute
or short-term toxicity, but may be harmful upon long-time
exposure. When studying the (toxico) kinetics, the organ-
ism usually is exposed to a substrate (e.g. water, food, soil)
contaminated with only the chemical of interest (see e.g.
Van Brummelen and Van Straalen 1996; Dı ´ez-Ortiz et al.
2010). In reality organisms however, are usually subjected
to a broad variety of different chemicals. Once inside the
body, different chemicals may be dealt with differently.
This is especially true for organic chemicals and metals.
Organic chemicals, like Polycyclic Aromatic Hydrocar-
bons (PAHs), generally distribute over different tissues
based on their chemical properties (e.g. molecular size,
lipophilicity) and are eliminated through metabolism
(phase 1 and 2) or excretion of either the parent compound
or the metabolites (Svendsen et al. 2011). Metals can be
essential or non-essential for the organism and biochemical
pathways have evolved to regulate body concentration of
essential metals. Non-essential metals, however, may use
some of these pathways and are also taken up actively
(Luoma and Rainbow 2008).
PAHs are present in crude oil and formed as by-product
of the combustion of organic materials, and therefore are
found throughout the environment. Due to their great
binding affinity to soil organic matter these lipophillic
chemicals may reach concentrations in soil that are con-
siderably higher than elsewhere in the environment (Van
M. Broerse (&) ? H. Oorsprong ? C. A. M. van Gestel
Department of Animal Ecology, Faculty of Earth and Life
Sciences, VU University Amsterdam, De Boelelaan 1085,
1081 HV Amsterdam, The Netherlands
e-mail: miekebio@gmail.com
123
Ecotoxicology (2012) 21:795–802
DOI 10.1007/s10646-011-0839-2

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Brummelen and Van Straalen 1996). Pyrene is one of the
most abundant of the PAHs and always present in PAH
mixtures (Jongeneelen 2001; Levin 1995). Pyrene is con-
sidered to be non-carcinogenic and its toxicity is believed
to be mainly through non-polar narcosis (Jensen and
Sverdrup 2003). In Eukaryotes, detoxification usually
occurs in two phases. In the first phase (phase 1) the
cytochrome P450 enzyme system introduces a functional
group, such as hydroxyl, to the non-polar compound. In
phase 2, detoxification enzymes such as glutathione
S-transferase attach a large polar water-soluble moiety to
phase 1 metabolites to promote excretion and elimination
(Brown et al. 2004). Besides detoxification, biotransfor-
mation may produce metabolites that are more toxic than
the parent compound (Di Giulio et al. 1995; cited by
Akkanen and Kukkonen 2003). Thus biotransformation not
only enhances the number of potentially toxic compounds,
but may also change the mode of toxic action (Droge et al.
2006). Cadmium (Cd) is a widespread environmental pol-
lutant due to anthropogenic activities such as mining and
fossil fuel combustion. It is one of the most toxic metals,
and negative effects may be found already at relatively low
concentrations (Luoma and Rainbow 2008). It is non-
degradable and readily accumulates in organisms (Janssen
et al. 1991; Crommentuijn et al. 1994). Cd has been clas-
sified as class 1 human carcinogen (IARC 1993) due to
multifactorial mechanisms, such as accumulation of DNA
damage due to inhibition of DNA repair enzymes (Huff
et al. 2007; and see also Ragunathan et al. 2010 for ref-
erences). PAHs and metals usually co-occur in soil and
their joint adverse effects in a mixture are usually consid-
ered independent within an organism, since they act dif-
ferently (e.g. Price et al. 2002). However, this is an
assumption and nothing is known about possible interac-
tions between these chemicals in soil invertebrates.
In this study we investigated if simultaneous exposure to
a non-essential metal (Cd) influences the uptake, metabo-
lism and/or the elimination rate of pyrene in the springtail
Folsomia candida (Collembola) when exposed to pyrene in
Lufa 2.2 soil with and without the presence of cadmium.
Collembolans are an integral part of soil ecosystems,
playing an important role in the functioning of the soil
ecosystem, and are vulnerable to soil contaminants
(Fountain and Hopkin 2001).
Materials and methods
Test design
To derive uptake and elimination rate constants for both
pyrene and OH-pyrene, F. candida was exposed to soil
contaminated with pyrene (nominal concentration of 40 lg
Pyr/g dry soil). To assess the effect of cadmium on pyrene
toxicokinetics, animals were also exposed to the mixture of
pyrene and cadmium (nominal concentration of 40 lg Pyr/
g dry soil ? 100 lg Cd/g dry soil). In addition, animals
were exposed to single cadmium (nominal concentration of
100 lg Cd/g dry soil). All exposures lasted for 337 h
(uptake phase). Pyrene and cadmium concentrations were
based on previous studies and never exceeded the 28-day
LC50values for pyrene and Cd (e.g. Droge et al. 2006;
Sørensen and Holmstrup 2005; Van Gestel and Hensbergen
1997; Van Gestel and Van Diepen 1997). Springtails were
sampled at different times during the uptake phase (see
Table 1 for sampling times). At the end of the exposure
period (t = 337 h) all remaining springtails were trans-
ferred to clean soil (elimination phase) and detoxification
of F. candida was followed for 216 h (see Table 1 for
sampling times). During the uptake phase and the elimi-
nation phase, duplicate test containers were sampled at
each sampling time. Animals were analysed for pyrene,
OH-pyrene and other metabolites at each sampling, while
soils samples were analysed for pyrene and Cd at the start
and end of the uptake phase (see Table 1).
Test soils
A natural soil from Germany, LUFA—Speyer soil 2.2
(Lufa 2.2) was used. This sandy soil has a pH(H2O )of
approximately 5.8 and contains 3.9% organic matter and
5.1% clay. All test soils were prepared by adding deionized
Table 1 Outline of sampling times (in hours) for the uptake and elimination kinetics study with pyrene in Folsomia candida in Lufa 2.2 soil,
with and without simultaneous exposure to cadmium
Treatment Uptake phaseElimination phase
Hours:048 2436 48 72146 241 337337 344360384 408 482553
Pyrene (± Cd)XXXXXXXXXXXXXXXX
CdXX
ControlsXXXXX
Soil analysesXXXX
The table shows moments (X) at which duplicate soil and animal samples were taken for the different treatments
796 M. Broerse et al.
123

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water to obtain a soil moisture content of 22% (w/w) (50%
of the Water Holding Capacity). After adding water or Cd
solution (CdCl2
left to equilibrate for 4 weeks before further treatment (at
20 ± 1?C). As pyrene (CAS 129-00-0, Riedel-de Hae ¨n;
99 ? %) is poorly water-soluble, it was dissolved in ace-
tone (Fluka 99.5% Chemie GmbH) before adding to the
soil. All soils, except for the water control, were treated
with acetone with or without (solvent control) pyrene, by
submerging 10% of the soil, per treatment, with acetone.
After 24 h equilibration, the acetone was allowed to
evaporate for 2 days in a fume cupboard. Dried soil was
mixed in with the remainder of the soil and re-moistened to
22% (w/w) by adding deionized water. For all treatments
and controls glass test containers (100 ml) were filled with
20 g wet soil and closed with a lid.
Additionally, duplicate jars for soil analyses contained
40 g wet soil. Pyrene-treated soils were protected from
light by covering with aluminum foil prior to the experi-
ment to avoid photochemical transformation of the parent
compound.
.H2O, Aldrich; [98% A.C.S.), soils were
Uptake and elimination tests
F. candida was cultured in the laboratory at 20 ± 1?C with
a 12:12 h light:dark regime, using plastic containers with a
moist bottom of plaster of Paris mixed with active coal.
Animals were age synchronized by allowing individuals
from the culture to lay eggs for 2 days in separate con-
tainers. Springtails were fed ad libitum with commercial
dried baker’s yeast (Oetker) during synchronization and
experiment.
Twenty 30 ± 1 day-old age-synchronized springtails
were introduced into each test container at the start of the
experiment. Twice a week test containers were opened for
aeration and compensation of water loss. To derive kinetics
parameters, two test containers per treatment were sampled
at each sampling time (Table 1) by adding 33 ml water to a
test container, stirring gently and transferring all soil, water
and springtails into a glass beaker, repeating this procedure
three times. Living springtails came floating to the water
surface and were gently transferred to a dry bottom of
plaster of Paris to get rid of moist and soil particles before
being weighed (Mettler Toledo UMT2, ultramicrobalance;
accuracy 0.1 lg). If total weight of all springtails per test
container was less than 1 mg, animals from the duplicate
test containers were pooled. Springtails were stored at
-80?C before further analyses.
Chemical analysis
Soil moisture content was determined by drying soils for
48 h at 60?C. For pH measurements dried soil samples
were shaken with a 0.01 M CaCl2 solution for 2 h at
200 rpm, at a solution:soil (w/w) ratio of 5:1. After sedi-
mentation of soil particles, pH was measured using a
Consort p907 pH meter.
Total Cd concentrations were determined by digesting
dried soil samples in a 2:6:2 mixture of HNO3 (65%,
Riedel-de Hae ¨n), HCl (37%, Baker) and demineralised
H2O using a microwave (CEM MARS 5). This digest was
diluted to 25 ml with demineralized water and analysed by
flame Atomic Absorption Spectrophotometry (AAS) (Per-
kin Elmer AAnalyst 100). The certified reference material
ISE-989 (River Clay, Wageningen Evaluating Programs)
was used to determine the accuracy of the analytical pro-
cedure, which was within performance acceptance limits
(\4% deviation).
Measurement of total Cd concentrations in the animals
was done by three runs of mini-destruction of freeze-dried
springtails in a block heater with HNO3? HNO4(Ultrex 2
(71%) and Ultrex; 7:1). After evaporation to dryness, res-
idues were taken up in 300 ll 0.1 M HNO3and analysed
by graphite furnace AAS (Perkin Elmer 5,100). The ref-
erence material Dolt-2, certified by the National Research
Council of Canada as reference material, was used to
determine the accuracy of the analytical procedure, which
was within 10% of the certified value.
For determining actual pyrene concentrations in the soil,
we followed the method described by Leon Paumen et al.
(2008) using Soxhlet extraction followed by high perfor-
mance liquid chromatography (HPLC). The HPLC con-
sisted of a Vydac RP 18 201TP column with a Vydac
201GD RP-18 guard column (Alltech, Breda, The Neth-
erlands), a Jasco FP-1520 fluorescence detector (Jasco,
Essex, UK), and a Gynkotek UVD320s ultraviolet diode-
array detector (Gynkotek, Germering, Germany).
To measure pyrene and its metabolites (OH-pyrene and
five conjugates), springtails were pottered with a Teflon
pestle with Tris buffer (pH 8.7) and carbazole as an internal
standard and stored for 30 min. at -80?C. After 5 min. of
sonication, 400 units of proteinase K solution was added
and the sample was vortexed before being incubated at
38?C for 2 h. Ethanol amended with ascorbic acid was
added and samples were centrifuged at 12,000 rpm, after
which the debris were left in the Eppendorf tubes while
liquid was transferred into brown HPLC vials. Superna-
tants were measured by HPLC applying an elution gradient
as described by Stroomberg et al. (2004) using the same
equipment as mentioned for soil analysis and with fluo-
rescence detection at kex/em= 346/384 nm.
Data analyses
Pyrene kinetics in the Collembola were described using a
one-compartment model. This model considers the animal
Cadmium affects toxicokinetics 797
123

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as a homogeneous compartment with single uptake and
elimination rates (e.g., Janssen et al. 1991; Stroomberg
et al. 2004). The model parameters and corresponding
standard errors were estimated by fitting the following
equations simultaneously using non-linear regression in
SPSS15.0:
Uptake kinetics (for t B te):
?
And elimination kinetics (for t[te):
Q t ð Þ ¼ k1=k2? C ? 1 ? e?k2?t
? 1 ? e?k2?ðt?teÞ
Q t ð Þ ¼ k1=k2? C ? 1 ? eð?k2?tÞ
?
??
? k1=k2? C
??
where: Q(t) = pyrene concentration in the organism at
time (t) (lg/g fresh body weight), C = pyrene exposure
concentration (lg/g dry soil), k1= uptake rate (g dry soil/g
fresh body weight, hour), k2= elimination rate constant
(per hour), (t) = time (hour), te= time at which animals
were transferred to clean soil (337 h).
The same model was also fit to model kinetics of OH-
pyrene formation and elimination in the animals.
To compare pyrene and OH-pyrene kinetics parameters
for exposures with or without Cd a generalized likelihood
ratio test was performed (Sokal and Rohlf 1995).
Bioaccumulation factors (BAF) were derived by divid-
ing k1by k2. The chemical half lifes of pyrene and OH-
pyrene in the springtails were calculated using ln2/k2.
All calculations were performed using SPSS version
15.0.
Results
Soil moisture contents, pH and chemical concentrations
Test soils had moisture contents between 19.1 and 21.3%
and an average pH(CaCl2) of 5.22 ± 0.12 (SD; n = 48).
Actual pyrene concentrations in the soil were lower than
nominal ones (27.0–30.2 lg pyrene/g dry soil) while for
Cd concentrations this was the opposite (116–130 lg Cd/g
dry soil), as shown in Table 2. Pyrene concentration in the
soil of the pyrene-only treatment remained fairly constant
throughout the uptake phase, while in the treatment with
Cd, pyrene concentration showed a decrease of 28%
after 337 h (Table 2). To calculate kinetics parameters, the
average pyreneconcentrations
26.0 ± 4.9 lg Pyrene/g dry soil (SD; n = 4) were used for
the single pyrene and pyrene with Cd exposure, respec-
tively. In the uptake tests with cadmium, average Cd
concentration in the test soil was 123 lg/g dry soil.
of26.6 ± 4.5and
Toxicokinetics
Not all springtails survived until the end of the experiment,
with mortality being most visible in the Cd treatments both
with or without pyrene. Unfortunately, we did not quantify
mortality. In the control and single pyrene treatments
[90% of the F. candida survived until they were sacrificed
for measuring internal chemical concentrations.
Cd concentrations in the animals did not differ between
treatments with and without pyrene and were 59–75 lg Cd/g
dry body weight after the 337-h uptake phase and
15–16 lg Cd/g dry body weight at the end of the elimi-
nation phase. Figures 1 and 2, respectively show the
internal pyrene and OH-pyrene concentrations (lg/g fresh
weight) in the Collembola, without or with Cd in the soil.
The corresponding toxicokinetics parameters are given in
Table 3. Single pyrene was rapidly taken up by the
springtails and steady state was well reached within the
337-h uptake period (after approx. 100 h). In the mixture
with Cd, pyrene uptake and elimination were significantly
Table 2 Pyrene and cadmium concentrations in Lufa 2.2 soil (in lg/g
dry weight) at the start and end of the uptake phase during the tox-
icokinetic test with Folsomia candida in Lufa 2.2 soil (±SD; n = 2)
Treatment Uptake (0 h)
(lg/g dry soil)
Uptake (337 h)
(lg/g dry soil)
Pyrene27.0 (7.5)26.2 (2.1)
Pyrene ? Cd30.2 (n = 1) 21.8 (0.9)
Cd116 (0.65) 130 (5.36)
Fig. 1 Uptake and elimination of pyrene in Folsomia candida
exposed to Lufa 2.2 soil treated with approximately 26.5 lg
pyrene/g dry soil, with (open symbols) or without (closed symbols)
approximately 123 lg Cd/g dry soil. Lines represent fit of the first-
order one-compartment kinetics model
798 M. Broerse et al.
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slower (likelihood ratio test: Xdf
(Table 3), and led to more pyrene accumulation in the
springtails and a prolonged elimination half life. BAF
values were estimated at 14.3 and 16.3 g dry soil/g fresh
body weight for exposures without and with cadmium,
respectively (Table 3). Pyrene hydroxylation into OH-
pyrene was even more influenced by the presence of Cd,
resulting in a very long half life (173 h) for OH-pyrene in
the springtails. Cd was accumulated in both treatments, i.e.
with and without pyrene with on average 67.0 ± 10.9 lg
Cd/g dw after 337-h and 15.8 ± 0.45 lg Cd/g dw in the
animals at the end of the elimination phase (t = 553 h). In
addition to OH-pyrene several possible phase 2 metabolites
were measured, as shown in Fig. 3. We named them after
their retention times and expressed the amounts as area/
body weight units, since neither chemical identification nor
quantification were done. Internal concentrations of all
phase 2 metabolites showed an increase in the uptake
phase, followed by a decline during the elimination phase.
2
=1[3.84; P\0.05)
For two metabolites (21.9 and 28.3), the increasing and
decreasing trends were different between exposures with or
without Cd. For the other metabolites, trends seemed not
affected by cadmium.
Discussion
We demonstrated that pyrene accumulation and metaboli-
zation in the collembolan F. candida is significantly
influenced by the presence of cadmium.
We estimated an uptake rate for pyrene of 0.458 g/g,
hour with an elimination rate constant of 0.032 per hour for
single pyrene exposure. It is difficult to compare the pyrene
kinetics found in this study with other studies as test
organism, exposure medium as well as exposure route
differed greatly. The estimated uptake rate differed from
those found by Jonsson et al. (2004), exposing sheephead
minnows to contaminated seawater. They found that,
depending on the exposure concentration, the uptake rate
was 5.375 l/g, hour (low) and 4.83 l/g, hour (high). Both
uptake rates are much faster than for F. candida, which is
not surprising since the units are very different. Elimination
rate constants (0.037 and 0.049 per hour, respectively) did
not differ much from the values found in our study. This
suggests that the uptake route through water is different
than for soil, but internal elimination might follow the
same pathway in some organisms, corresponding with a
similar excretion rate. This similarity in elimination rate
constants was also found for the elimination of benzo(a)-
pyrene in Porcellio scaber of 0.046 per hour (Van Brum-
melen and Van Straalen 1996) and for pyrene in Eisenia
andrei of 0.032 per hour (Jager et al. 2000).
We found a significant influence on pyrene kinetics in the
presence of Cd with more pyrene being accumulated and a
prolonged half life. In this study we aimed at minimizing
toxic effects by using sublethal concentrations. Neverthe-
less, we observed increased mortality in the treatments with
cadmium. Even though the measured cadmium concentra-
tion in soil was somewhat higher than anticipated, it did not
exceed 28-day LC50values found in other studies (e.g.
Sørensen and Holmstrup 2005; Van Gestel and Hensbergen
Fig. 2 Development in time of internal OH-pyrene concentrations in
Folsomia candida exposed to pyrene in Lufa 2.2 soil for treatments
with (open symbols) and without (closed symbols) Cd (see Fig. 1 for
kinetics of pyrene). Lines represent fit of the first-order one-
compartment kinetics model
Table 3 Uptake rate (k1) and elimination rate constant (k2) (with s.e.) for pyrene and OH-pyrene in Folsomia candida following exposure to
pyrene in Lufa 2.2 soil with or without cadmium
TreatmentPyrenePyrene ? CdX2; P
OH-pyreneOH-pyrene ? CdX2; P
k1(g/g, h)
k2(per h)
BAF (g/g)
0.458 (0.046) 0.309 (0.024)98.7; P\0.001
4.10; P\0.05
0.014 (0.003) 0.023 (0.005)10.0; P\0.01
0.446; n.s0.032 (0.003)0.019 (0.002) 0.009 (0.002)0.004 (0.001)
14.316.3
t1/2(h) 21.7 36.577 173
Differences were determined by comparing uptake and elimination kinetics applying generalized likelihood ratio tests (X2at 1 df; n.s. means not
significantly different)
Cadmium affects toxicokinetics799
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1997; Van Gestel and Van Diepen 1997) with Askov (1.6%
organic carbon) and artificial soils (variable organic matter
contents).Also,internalCdconcentrationsintheanimalsdid
notexceedtheinternalEC50fortheeffectonreproductionof
F. candida reported by Van Gestel and Mol (2003) after
4 weeks of exposure. It therefore remains unclear why
mortality was increased in the treatments with cadmium.
Our results showed an interaction between pyrene and
Cd that may have consequences for their combined toxic-
ity. There is a lack of information about pyrene uptake and
elimination rate constants in the presence of Cd in litera-
ture. In addition, the literature data on combined effects of
PAHs and metals are not conclusive. Depending on the
combination of metals and PAHs, the exposure medium,
the test organism, test duration and the studied endpoint,
results range from additive, independent or antagonistic to
synergistic mixture effects (e.g. Gust 2006; Shen et al.
2006). As shown for nickel and chlorpyrifos (Broerse and
Van Gestel 2010a, b) toxicity is a dynamic process and
mixture interactions may change over time. The presence
of Cd may enhance pyrene toxicity by increasing accu-
mulation. In addition, accumulation of the OH-pyrene
metabolite was faster in the presence of Cd, probably
because further metabolization was stagnated, resulting in a
prolonged half life of OH-pyrene. Inhibition of metabolism
has been shown to increase the bioconcentration of a
variety of organic chemicals (Barron 1990). The mecha-
nism behind interaction between Cd and pyrene might lie
in the fact that both chemicals create oxidative stress.
PAHs are known for enhancing the production of reactive
oxygen species (ROS) like hydrogen peroxide (Gastaldi
et al. 2007). Although Cd is not a redox metal (Satarug
et al. 2003), it is believed to cause oxidative stress through
the Fenton reaction producing radical species that might
initiate lipid peroxidation (Banni et al. 2009). This was
confirmed by a microarray study, showing the up regula-
tion of transcripts encoding for monooxygenase and short-
chain dehydrogenase (Nota et al. 2009). Cd can be
sequestered by reduced glutathione (GSH) and/or metal-
lothioneinto preventits adverse interactionwith
conjugate (21.4)
0
2
4
6
8
10
12
0 200400600
time (hour)
area/weight (mg)
pyrene
pyrene + Cd
conjugate (21.9)
0,0
0,5
1,0
1,5
2,0
2,5
0 200400 600
time (hour)
area/weight (mg)
pyrene
pyrene + Cd
conjugate (24.3)
0
1
2
3
4
5
6
0 200400 600
time (hour)
area/weight (mg)
pyrene
pyrene + Cd
conjugate (26.2)
0,0
0,5
1,0
1,5
2,0
2,5
0 200 400600
time (hour)
area/weight (mg)
pyrene
pyrene + Cd
conjugate (28.3)
0,0
0,5
1,0
1,5
2,0
0200400 600
time (hour)
area/weight (mg)
pyrene
pyrene + Cd
Fig. 3 Internal pyrene
conjugate concentrations in time
in Folsomia candida exposed to
pyrene in Lufa 2.2 soil for
treatments with (open symbols)
and without (closed symbols)
Cd (see Fig. 1 for kinetics of
pyrene). The different figures
relate to different pyrene
conjugates; see text for further
information. Since no
quantification of the
concentrations was possible,
amounts of the different
metabolites are expressed as
units (of the HPLC readings)
per fresh body weight of the
animals
800M. Broerse et al.
123

Page 7

biomolecules (Wang et al. 2009). GSH and other thiols
play a critical role in scavenging ROS. When exposed to
both PAHs and Cd, GSH and other thiols might deplete,
indirectly elevating ROS levels (Martelli and Moulis 2004;
cited by Wang et al. 2009). Cd may also delay further
metabolization of the phase 1 products of pyrene, by a
depletion of possible conjugates. In the second phase,
reactive phase 1 metabolites are conjugated with chemicals
like glutathione or glucuronic acid (Baird et al. 2005; Ja-
koby and Ziegler 1990), which also may be involved in
scavenging and detoxifying free Cd. The observed delay in
further metabolization of OH-pyrene in the presence of Cd
might be due to this.
We conclude that pyrene accumulation and metaboli-
zation in the collembolan F. candida was significantly
influenced by the presence of cadmium. Since toxicity is a
dynamic process, mixture effects may be dependent on
exposure time. Further research is needed to underpin the
actual mechanisms responsible for our findings and the
possible consequences for toxicity.
Acknowledgments
with Cd measurements. This study was supported by the EU Inte-
grated project NoMiracle (Novel Methods for Integrated Risk
assessment of Cumulative Stressors in Europe; http://nomiracle.jrc.it)
contract No. 003956 under the EU-theme ‘‘Global Change and Eco-
systems’’ topic ‘‘Development of risk assessment methodologies’’,
coordinated by Dr. Hans Løkke at NERI, DK-8600 Silkeborg,
Denmark.
We wish to thank Rudo Verweij for assisting
Open Access
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tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
This article is distributed under the terms of the
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