Environmental Pollution 220 (2017) 567e576

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Combined effects of chlorpyriphos, copper and temperature on

acetylcholinesterase activity and toxicokinetics of the chemicals in the
earthworm Eisenia fetida*
Agnieszka J. Bednarska a, *, Maciej Choczyn

ski b, Ryszard Laskowski b, Marcin Walczak b
a
Institute of Nature Conservation, Polish Academy of Sciences, Mickiewicza 33, 31-120 Krako
w, Poland
b
w, Poland
Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krako

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

Article history: In polluted environments organisms are commonly exposed to a combination of chemicals with different
Received 14 June 2016 modes of action, and their effects can be additionally modified by natural abiotic conditions. One possible
Received in revised form mechanism for interactions in mixtures is via toxicokinetics, as chemicals may alter the uptake, distri-
30 September 2016
bution, biotransformation and/or elimination of each other, and all these processes can be affected by
Accepted 2 October 2016
Available online 12 October 2016
temperature. In this study, the effect of temperature (T) on the toxicokinetics of copper (Cu) and
chlorpyriphos (CHP), applied either singly or in binary mixtures, was studied in the earthworm Eisenia
fetida. The experiments were conducted at 10 or 20
C and the earthworms were exposed to environ-
Keywords:
Mixture
mentally realistic concentrations of Cu and/or CHP for 16 d, followed by a depuration period of 4 d in
Metal uncontaminated soil. The earthworms were sampled for body Cu and/or CHP concentrations and
Organophosphate insecticide acetylcholinesterase (AChE) activity measurements. The CHP degradation rate in the soil was substan-
Kinetics tially higher at 20
C and in soil treated with Cu. The significant (p < 0.05) inhibition of AChE activity in
Invertebrate the earthworms exposed to CHP was found. The effect of Cu was significant only at p < 0.1. No synergistic
effect of the parallel CHP and Cu exposure was found. Four days after transferring the earthworms to
uncontaminated soil, the AChE activity recovered to the level observed in control animals. The tem-
perature effect on the toxicokinetic parameters was more pronounced for CHP than for Cu. In the case of
CHP, the assimilation rate constant (kA) was significantly higher at 20
C than at 10
C, both in CHP-only
and CHP þ Cu treatments. A similar trend was found for the elimination rate constant (kE), but the
difference was statistically significant only for non-Cu treatments. In the case of Cu, the general trend of
higher kA and kE at 20
C and in the absence of CHP was observed.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction mechanism for interactions in chemical mixtures is via tox-

In polluted soils, earthworms are exposed to many different
potentially toxic chemicals. These chemicals may interact with each
other and affect their toxicity to an organism. The problem of
mixture toxicity and the interactive effects of toxic chemicals and
natural stressors (e.g. temperature) was identified at least 30 years
ago (Cooney et al., 1983) and has been extensively reviewed over
the past few years (Van Gestel et al., 2010; Holmstrup et al., 2010;
Laskowski et al., 2010; Bednarska et al., 2013). The possible
*
This paper has been recommended for acceptance by Dr. Chen Da.
* Corresponding author. Institute of Nature Conservation, Polish Academy of

w, Poland.
Sciences, Mickiewicza 33, 31-120 Krako
E-mail address: bednarska@iop.krakow.pl (A.J. Bednarska).
icokinetics, as chemicals may alter the uptake, distribution,
biotransformation and/or elimination of each other (Spurgeon
et al., 2010). Many toxicokinetic experiments have been conduct-
ed to understand the uptake and loss of different metals (e.g. Smith
et al., 2010; Li et al., 2009; Nahmani et al., 2009; Vijver et al., 2005;
Giska et al., 2014) and organic pollutants (Belfroid et al., 1994) by
different species of earthworms, including Eisenia fetida. However,
the effects of chemicals with different modes of action (i.e. a metal
and a pesticide) on each other's toxicokinetics has rarely been
studied for terrestrial invertebrates.
The study by Lister et al. (2011) conducted on Lumbricus ter-
restris L. exposed to a binary mixture of a metal (nickel) and an
organophosphate insecticide (chlorpyrifos) showed that Ni uptake
followed the same pattern as in the case of exposure to the single

http://dx.doi.org/10.1016/j.envpol.2016.10.004
0269-7491/© 2016 Elsevier Ltd. All rights reserved.
568 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
chemical. However, this was not the case for chlorpyrifos (CHP)
which showed a faster rate of uptake and elimination and a slightly
higher equilibrium concentration in the mixture. On the other
hand, Zhou et al. (2012) demonstrated that the uptake of Cu was
completely stopped in E. fetida by the presence of glyphosate. Sig-
nificant interactions between metals and insecticides were also
found in invertebrates other than earthworms. Bednarska and
Kaszowska (2014) showed that the temporal pattern of internal
Ni concentration in the ground beetle Pterostichus oblongopunctatus
depended on the concentration of CHP in its food. Nickel's tox-
icokinetics were similar for the Ni-only exposure and in a mixture
with low CHP concentration (10 mg kg1), but was different for Ni
mixed with high CHP concentration (30 mg kg1). At a high con-
centration, CHP decreased the accumulation of Ni, but no CHP effect
was found in the elimination phase when the animals were
switched to uncontaminated food (Bednarska and Kaszowska,
2014). On the other hand, Broerse and Van Gestel (2010)
observed that, at non-lethal concentrations, CHP increased the
elimination rate of Ni in the collembolan Folsomia candida.
Not only chemical factors may interact with each other, but also
the simultaneous exposure to variable natural physicochemical
parameters may cause organisms to respond in an unexpected way.
Physicochemical parameters of the environment, such as tempera-
ture, moisture or pH, may interact with chemicals either directly, for
example by changing their bioavailability, or indirectly by changing
the organism's biology and/or behaviour (Holmstrup et al., 2010).
The temperature-dependent differences in kinetic parameters have
been found for Cd in the collembolan Orchestella cincta (Janssen and
Bergema, 1991), and for Ni in P. oblongopunctatus larvae (Bednarska
et al., 2011), but to our knowledge the effects of temperature on the
toxicokinetics of metals and pesticides in their binary mixtures have
not been studied to date.
In our study copper (Cu) and the organophosphate insecticide
chlorpyrifos (CHP) were used as they have different modes of action,
and the co-occurrence of these pollutants is highly probable in
arable soils, especially in vineyards where CHP and Cu are sprayed as
a conventional protection of the vines against pests (Schreck et al.,
2012). Copper is an essential element but can be toxic if accumu-
lated in excess (Holmstrup et al.,1998). The toxicity of Cu seems to be
mostly due to free Cu ions combining with proteins and altering
their physiological functions. Moreover, excessive amounts of Cu
ions can react with thiol groups of membrane proteins, negatively
affecting membrane functioning or even destroying them
(Holmstrup et al., 1998). Copper was found to be regulated in Eisenia
fetida (Spurgeon and Hopkin, 1999) and E. andrei (Peijnenburg et al.,
1999) and almost constant Cu body concentrations over a range of
soil concentrations were found for Lumbricus rebellus and Dendro-
drilus rubidus by Morgan and Morgan (1988). Chlorpyrifos is one of
the most widely used and best-studied pesticides in terms of its
effects on soil invertebrates in the laboratory and in the field (Ja €nsch
et al., 2006). The rapid accumulation and elimination of CHP has
been found in L. terrestris (Lister et al., 2011), providing the evidence
for the metabolism of this pesticide by earthworms. The mode of
action of CHP is through acetylcholinesterase inhibition at the syn-
aptic junction, thus acetylcholinesterase (AChE) activity was chosen
as a biomarker in our study. To check the effect of temperature on the
toxicokinetics of Cu under the exposure to CHP and vice versa, we
exposed E. fetida to the chemicals, applied either singly or in a binary
mixture, at two temperatures: 10 and 20
C.
2. Materials and methods
2.1. Animals and experimental design
The earthworms, Eisenia fetida, were obtained from the Institute
of Industrial Organic Chemistry, Pszczyna Branch (Poland), and
were kept at constant temperature of 20 ± 1
C in the OECD soil
mixed with seasoned cow manure and dry shredded straw. Before
starting the experiment, the earthworms were acclimatised for
24 h to the experimental temperatures (T ¼ 10 or 20
C) and OECD
soil. The animals were not fed during the experiment. Tox-
icokinetics (TK) of copper and chlorpyriphos and the activity of
acetylcholinesterase were followed at the two temperatures in the
following combinations (nominal concentrations of chemicals in
mg/kg dry weight soil followed by temperature): 0 Cu þ 0 CHP,
10
C (C10); 0 Cu þ 0 CHP, 20
C (C20); 0 Cu þ 5 CHP, 10
C (CHP10);
0 Cu þ 5 CHP, 20
C (CHP20); 200 Cu þ 0 CHP, 10
C (Cu10); 200
Cu þ 0 CHP, 20
C (Cu20); 200 Cu þ 5 CHP, 10
C (CuCHP10); 200
Cu þ 5 CHP, 20
C (CuCHP20), five replicates each. The soil for all the
CHP treatments was prepared 48 h before starting the experiment.
CHP (min. 98% technical, Cheminova, Denmark) was added to the
OECD dry soil as an acetone (Ac) solution, mixed and left for 24 h
under a fume board to let the acetone evaporate. Two additional
acetone-only controls were run at each temperature: C10 þ Ac and
C20 þ Ac. Twenty four hours before starting the experiment an
aqueous solution of CuSO4 (CuSO4  5 H2O, POCh S.A., Poland) was
mixed with soil of all copper treatments. The remaining treatments
received at the same time 3.15 mM H2SO4 (min. 95%, POCh S.A.,
Poland) to obtain equimolar concentrations of sulphate ions in all
treatments. The soil was brought to 43.5% of its water holding ca-
pacity in all the treatments.
Ten adult earthworms were randomly selected for each replicate
and placed in a 0.5 L plastic container filled with 650 g wet soil (50 g
dry soil per earthworm). The containers were covered with perfo-
rated lids to achieve adequate ventilation while preventing exces-
sive water evaporation during the experiment. Every second day
the containers were weighed and the lost moisture was supple-
mented with distilled water. The experiment was run in darkness.
The earthworms were exposed to contaminated soil for 16 d
(uptake phase) after which they were transferred for 4 d to un-
contaminated soil (the elimination phase, in literature described
also as the decontamination phase). One earthworm was sampled
per replicate after 0.5, 1, 2, 4, 8, and 16 d since the start of exposure,
and 0.5, 1, 2, and 4 d since starting the elimination phase. Addi-
tionally, five earthworms per temperature were sampled right
before starting the experiment (time 0). The earthworms were
collected after gently shaking a container until an earthworm
appeared on the soil surface. Single animals were transferred with a
spatula to individually marked plastic sample tubes and killed by
freezing at 20
C for 30 min. Then, the earthworms were prepared
for further analyses (see below). Soil for chemical analysis was
sampled before starting the experiment (time 0) and 6 h after
finishing the exposure phase, one sample per replicate. Soil sam-
ples, 65 g wet weight, were placed in 50 mL hermetic plastic tubes
and frozen at 20
C until analysis.
2.2. Tissue sampling
After removal from the freezer, each earthworm was flushed
with a cooled (6e8
C) 0.02 M phosphate buffer (pH 7.4) to clean
the surface from remnant soil particles and de-freeze the tissues.
Then, the animals were weighed to the nearest 0.1 mg (AS 160/C/2
Radwag, Poland) and dissected under the stereo-microscope. The
first 8 body segments (anterior part including the pair of cerebral
ganglia) were cut off and, after removing the contents of alimentary
tract by flushing with the phosphate buffer, were placed in a plastic
tube, frozen in liquid nitrogen and stored at 70
C for further
acetylcholinesterase (AChE) activity measurements. The remaining
body part was divided into two sections by cutting it after the 48th
segment (hence, the second part consisted of the middle 40
 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
segments). Both sections were dissected longitudinally and the
contents of the alimentary tract was removed by flushing with the
phosphate buffer. Each part was placed in a separate Eppendorf
tube and frozen at 20
C for further analyses of CHP and Cu
concentrations in the middle and posterior part of the body,
respectively. The sample processing time, between de-freezing an
earthworm and freezing the dissected fragments back, was
2e4 min for the first 8 segments and 9e15 min for the remaining
two body parts.
2.3. Acetylcholinesterase activity analysis
The samples were homogenised on ice in 0.02 M phosphate
buffer (pH 7.4) with 0.1 % Triton X-100 (Sigma-Aldrich, Germany), at
1:4 tissue:buffer ratio (w:v), using a mechanical homogeniser (PRO
200, Bioeko, Poland). The homogenates were centrifuged at 4
C for
15 min at 15,000g (centrifuge MPW-350R, MPW MED Instruments,
Poland). The supernatant from each sample was used for AChE
activity and protein content analyses.
Acetylcholinesterase activity was measured according to the
modified method by Ellman et al. (1961) on 96-well plates (Sar-
stedt, USA), using the mQuant spectrometer (Bio-TEK Instruments,
USA). The final reaction mixture contained 5 mL of sample, 180 ml of
0.02 M phosphate buffer (pH 7.4) and 10 mL of 0.01 M DTNB (5,50 -
dithiobis(2-nitro-benzoic acid); Sigma-Aldrich, Germany) solution
in 0.1 M Tris-HCl (pH 8.0, Sigma-Aldrich, Germany). The reaction
was started by adding 5 mL of 0.1 M acetylthiocholine iodide ((2-
mercaptoethyl) trimethylammonium iodide acetate; Sigma-
Aldrich, Germany) as a substrate. Absorbance at 405 nm was then
determined in 36 s intervals over 3 min. The reaction mixture with
phosphate buffer (0.02 M, pH 7.4) was used as a negative control.
The mean readings of three replicates per sample were taken for
the calculation of AChE activity. AChE activity was expressed in
nmol acetylcholine hydrolised per min per mg protein (nmol min1
mg protein1).
2.4. Protein analysis
Protein content in the same homogenate as used for the AChE
analysis was assessed using Bradford's reagent (Sigma, USA;
Bradford, 1976). The absorbance was measured at 592 nm, using
bovine serum albumin (BSA standard; Sigma, USA) as a standard
(Technical Bulletin, Sigma).
2.5. Chemical analyses
All sampled individuals were analysed for both CHP and Cu
concentrations. Soil samples were analysed for actual CHP con-
centration right before the start of the experiment (d 0) and after
completing the exposure phase, i.e. after 16 d. The total Cu con-
centration in the soil was analysed before the start of the
experiment.
2.5.1. Copper
The samples of the posterior body part were placed into glass
sample tubes, oven-dried at 105
C for 12 h and weighed to the
nearest 0.1 mg (AS 160/C/2 Radwag, Poland). The average sample
dry weight (±SD) was 11.5 ± 3.4 mg. The samples were then
digested in 2 mL of concentrated HNO3 (65%, INSTRA-Analysed;
Baker, Germany) at a temperature gradually increasing from ca.
20
C (overnight) to ca. 100
C, with final evaporation at
120e130
C. The samples were evaporated to ca. 0.3e0.5 mL and
the remaining solution, after cooling down to room temperature,
was made up to 5 mL with deionized water, vortexed, and 12 h later
569
decanted to plastic tubes, sealed and stored in a fridge until anal-
ysis. Copper concentration in the samples was analysed with a
graphite furnace AAS (Perkin-Elmer AAnalyst 800) and expressed
in mg kg1 dry body mass. To check the analytical precision, three
blanks and three samples of reference material (fish liver - Certified
Reference Material Dolt-4 Dogfish Liver, National Research Council of
Canada) were run with the samples.
Approximately 1 g dry soil was digested in 10 mL of HNO3 (65%,
INSTRA-Analysed; Baker, Germany) in 50 mL Erlenmeyer flasks,
increasing the temperature gradually from ca. 20
C (overnight) to
150
C. The excess of the digestive mixture was evaporated and the
samples were then resuspended to 25 mL with deionized water,
stored overnight, decanted to plastic sample tubes and stored in a
refrigerator until analysis. Copper concentration was measured on
flame AAS (Perkin-Elmer AAnalyst 200) and expressed in mg kg1
dry mass. Three blank samples and three samples of certified
reference material (Sand 1, CRM048-50 g, Sigma-Aldrich, USA)
were run in parallel.
The measured copper concentrations in the certified materials
were always within ±7% of the certified values. The results were not
corrected for recovery.
2.5.2. Chlorpyriphos
The middle body part of each earthworm was first cut into
smaller pieces with a scalpel and then homogenised in an Eppen-
dorf tube with a pestle in 0.3e0.6 mL acetonitrile (POCh S.A.,
Poland) for 3e5 min. The homogenate was made up to 1 mL with
acetonitrile, which was also used to flush tissue remnants from the
pestle. The homogenate was quantitatively transferred to the SPE
cartridge (ISOLUTE-NH2, 100 mg, 1 mL) and flushed two times with
1 mL of acetonitrile:toluene 3:1 (v:v) mixture. The SPE cartridges
were previously conditioned with 1 mL of acetonitrile and 1 mL of
acetonitrile:toluene, 3:1, respectively. Then, the cartridge was dried
under vacuum and the sample was collected in a 3 mL glass sample
tube and evaporated to dryness at 40
C in the stream of nitrogen.
After complete evaporation, the residues were dissolved in 200 mL
of ethyl acetate (POCh S.A., Poland). The solution was transferred to
a GC autosampler vials equipped with 250 mL glass adapters. The
analyses were performed by gas chromatography-mass spectrom-
etry (GC-MS, Clarus 600, Perkin Elmer). To check the CHP recovery,
five samples of the middle parts of the unexposed earthworms
were spiked with 50 mL of CHP solution to obtain concentrations of
approximately 5 mg kg1. These samples, together with three
samples uncontaminated with CHP, were processed according to
above-mentioned procedure. The percentage of recovery was
85 ± 15 (average ± SD). The analytical results for the CHP-exposed
earthworms were not corrected for recovery. The cartridges with
tissues after extraction were then dried for 6 d at room tempera-
ture. Then, the tissues were removed from the cartridges and
weighed on a laboratory balance (Sartorius M2P, Germany) to the
nearest 0.001 mg. CHP concentration was expressed in mg kg1 dry
tissue mass.
Approximately 0.5 mg dry soil was homogenised/extracted with
1 mL acetone (POCh S.A., Poland) in Eppendorf tube using a vortex
mixer. After centrifugation at 1000g for 10 min, 0.5 mL of extract
was transferred to autosampler vials. The procedure was repeated
twice and the combined volumes of extracts were analysed by the
GC-MS system. The recovery for the soil samples, calculated as an
average value (±SD) of three samples previously spiked with CHP at
concentrations of 1, 4 and 6 mg kg1, was 96 ± 0.4%. The analytical
results were not corrected for recovery. Limits of detection (LOD)
for the earthworm and soil samples were 0.12 and 0.51 mg kg1,
respectively and was calculated from calibration curve, using
standard deviation of intensity intercept line.
570 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
2.6. Statistical analysis
Before data analysis, the control treatments were tested for
solvent effects. As no acetone effect was found, all the controls were
pooled. The combined effect of CHP, Cu, temperature and time
(sampling day) on AChE activity was tested with multiple ANOVA,
including all factors and their interactions, separately for the
exposure and elimination phase. Nonsignificant interactions were
removed consecutively from the model until only those significant
at p  0.05 remained. As residuals did not follow normal distri-
bution (p  0.05), the AChE values were log-transformed to fulfil
the normality assumption.
The pattern of change in internal copper concentrations (CI) over
time (t) was described for all Cu-treated earthworms (i.e. Cu10,
Cu20, CuCHP10 and CuCHP20) with a one-compartment model
(Skip et al., 2014):
for the uptake phase (t < tc):
kA  
CI ðtÞ ¼ CI0 $ekE $t þ CEu 1  ekE $t
kE
and for the elimination phase (t > tc):
kA  
CI ðtÞ ¼ CItc $ekE $ðttc Þ þ CEd 1  ekE ðttc Þ
kE
where:
kA   (kCHP ¼ 0.0238 d1, t50 ¼ 29 d), and at 20
C (CuCHP20) the
CItc ¼ CI0 $ekE $tc þ CEu 1  ekE $tc
kE
The symbols used in the model and the units of model param-
eters were as follows: kA e the assimilation rate constant [day1], kE
e the elimination rate constant [day1], tc e the time of changing
the soil from contaminated to uncontaminated (here: 16th d of the
experiment) [days], CI0e the initial body metal concentration at
t ¼ 0, given in the model explicitly as the average Cu concentration
measured in ten individuals before starting the exposure (Day 0)
(7.99 mg kg1 dw), CEu and CEd e the exposure concentration in soil
(measured) in the uptake and elimination phase, respectively [mg
kg1 dw].
The pattern of change in CHP concentrations over time was
described for CHP10, CHP20, CuCHP10 and CuCHP20 with a similar
set of equations with the following modifications:
(1) To incorporate CHP degradation, which was expected to
depend on temperature and/or simultaneous Cu exposure,
CEu in all equations was replaced with CEu$e(kCHP$t), where
kCHP stands for treatment-specific CHP degradation rate as
different degradation rates were used depending on tem-
perature (10 or 20
C) and Cu treatment (0 or 200 mg kg1).
(2) As the initial CHP concentration in earthworms was assumed
to be null, CI0 was set to zero.
(3) Because the soil was not contaminated with CHP during the
elimination phase, CEd was set to zero.
The kinetic parameters kA and kE were obtained by simulta-
neously fitting the equations to the data from both experimental
phases using the Marquardt procedure. After fitting the initial
model, data points with the studentised residuals greater than 3
were excluded as outliers and the final model was fitted. All the
parameters were checked for significance using 95% confidence
intervals. The confidence intervals around the estimated parame-
ters were also used to compare the treatments.
The estimated TK parameters for Cu were used to calculate
bioaccumulation factors (BAF ¼ kA/kE) and theoretical equilibrium
Cu concentrations in the earthworms at t∞, Ceq ¼ CEu$(kA/kE).
All statistical analyses were performed using Statgraphics
Centurion XVII (Statpoint Technologies, Inc.). Data are reported as
average ± SD unless stated otherwise.
3. Results
The measured Cu concentration in contaminated soil,
197.8 ± 9.48 mg kg1, was in good accordance with the nominal
concentration (200 mg kg1). The soil not treated with Cu had a
very low content of this element: 1.33 ± 0.45 mg kg1. These two
concentrations were used in the Cu TK modelling as concentrations
of the metal in soil for the uptake phase (CEu) and elimination phase
(CEd), respectively.
The measured concentration in CHP-contaminated soil at the
start of the experiment (t0), i.e. 48 h after adding CHP to the soil,
was 4.59 ± 0.96 mg kg1 (91.8% of the nominal concentration
5 mg kg1), and at the end of the uptake phase (t16) were as follows
(in mg kg1): CHP20, 4.43 ± 1.25; CHP10, 4.15 ± 0.62; CuCHP10,
3.14 ± 0.35; CuCHP20, 2.50 ± 0.50. Thus, the lowest degradation
rate was found in CHP10, with 96.4% of the initial content still
present in soil after 16 d; the estimated degradation constant kCHP
was 0.0023 d1, and the half-life (t50) - 305 d. The next slowest
degradation was found in CHP20: 90.3% of CHP remained in the soil
at t16, resulting in kCHP ¼ 0.0064 day1 and t50 ¼ 108 d. The pres-
ence of Cu in the soil clearly accelerated the degradation rate of
CHP: at 10
C (CuCHP10) 68.3% CHP remained after 16 d
degradation was the fastest, leaving only 54.4% of initial CHP con-
tent after 16 d (kCHP ¼ 0.0380 d1, t50 ¼ 18 d). These large differ-
ences between treatments in the CHP degradation rates meant that
the actual exposure of the earthworms to the insecticide decreased
with time at different rates among the treatments. Therefore, the
treatment-specific degradation rates were incorporated into the TK
models as described in the Methods section. No mortality was
recorded during the experiment.
3.1. Acetylcholinesterase activity
Multiple ANOVA revealed a significant effect of CHP (p ¼ 0.029)
and time of exposure (p ¼ 0.0097) on AChE activity in the uptake
phase. Neither Cu nor temperature affected AChE activity (p ¼ 0.98
and p ¼ 0.75, respectively). A significant interaction between time
and CHP (p ¼ 0.044) indicated that the CHP effect on AChE activity
changed over time. Also, a significant interaction between CHP and
Cu (p ¼ 0.0016) was found. Due to the significant effect of time and
two significant interactions, further multiple ANOVA was per-
formed separately for each sampling day, with all the remaining
factors (CHP, Cu, T) and their interactions. The analysis revealed
that the CHP decreased AChE activity 12 h after starting the expo-
sure (p ¼ 0.047), with no other factor or interaction being signifi-
cant. None of the factors was significant on d 1, 2, 4 and 8, and on
16th d a significant (p ¼ 0.001) decrease in AChE activity was found
again in the CHP-treated earthworms (Table 1). At d 16, AChE ac-
tivity was also lower in Cu-treated worms at p ¼ 0.079 (Table 1). No
temperature effect was found and no significant interaction be-
tween any of the factors was detected (p > 0.1) at the end of the
uptake phase.
Multiple ANOVA for AChE activity in the elimination phase
revealed the following variables and interactions being significant:
CHP (p ¼ 0.01), time (p ¼ 0.04), CHP  Cu (p ¼ 0.040) and CHP  T
(p ¼ 0.0002). Again, to see if AChE activity remained significantly
decreased in CHP treatment throughout the elimination phase, the
multiple ANOVA for each sampling day was performed separately.
The results showed that the negative effect of CHP on AChE activity
 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576 571

disappeared at the last day of the elimination phase (p ¼ 0.52).

chlorpyriphos for 16 d in the artificial OECD soil) and after switching for 4 d to the uncontaminated OECD soil (elimination phase, starting after day 16); the treatment symbols stand for: C e control for a specific temperature, CHP
Average (±standard deviation) activities of acetylcholinesterase (AChE) in nmol min1 mg protein1 measured in Eisenia fetida sampled at different time points during the uptake phase (earthworms exposed to copper and/or

18.05 (N ¼ 5)
10.7 (N ¼ 5)
6.85 (N ¼ 5)
6.40 (N ¼ 5)
6.50 (N ¼ 5)
6.56 (N ¼ 5)

5)
5)
5)
5)
None of the other experimental factors or their interactions were

¼
¼
¼
¼
significant at the last day of the experiment.

(N
(N
(N
(N
7.54
8.42
8.20
8.00
CuCHP20
3.2. Toxicokinetics of copper and chlorpyriphos

±
±
±
±
±
±

±
±
±
±
52.3
59.5
62.4
64.5
51.6
41.8

52.6
38.2
44.2
50.4
Copper accumulated during the exposure in all Cu treatments
(Cu10, Cu20, CuCHP10 and CuCHP20). The one-compartment TK
models explained between 24.8 and 50.6% of the total temporal-
13.78 (N ¼ 5)
11.34 (N ¼ 5)

10.72 (N ¼ 5)

10.62 (N ¼ 5)
9.26 (N ¼ 5)
7.05 (N ¼ 5)

9.12 (N ¼ 5)

6.57 (N ¼ 5)
6.73 (N ¼ 5)
9.35 (N ¼ 5)
plus-individual variance in Cu concentrations (Fig. 1), with model
parameters being statistically significant in all treatments: the
asymptotic 95% confidence intervals did not cover 0 in any case. In
general, the assimilation (kA) and elimination (kE) rates were
±
±
±
±
±
±

±
±
±
±
Cu20

higher at 20
C and in CHP-free treatments (Table 2), but neither

59.6
47.4
51.1
56.8
55.7
48.8

59.7
52.0
55.8
47.7
temperature nor CHP effects were statistically significant (i.e. the
confidence intervals for the estimated parameters overlapped
13.39 (N ¼ 5)
11.35 (N ¼ 5)
16.86 (N ¼ 5)
11.76 (N ¼ 5)

11.44 (N ¼ 5)

46.78 ± 8.61 (N ¼ 5)
3.55 (N ¼ 5)

36.9 ± 4.99 (N ¼ 5)
41.3 ± 6.54 (N ¼ 5)

53.6 ± 9.65 (N ¼ 5)
between treatments). The only significant difference was found
for kA between Cu20 and CuCHP10 (Table 2). Within each tem-
perature the estimated bioaccumulation factors and equilibrium
Cu concentrations were almost identical in treatments, with or
CHP20

without CHP: Cu10 BAF ¼ 0.12, Ceq ¼ 23.9 mg kg1 vs. CuCHP10
±
±
±
±
±
±
54.3
42.4
55.1
55.9
43.4
42.7

BAF ¼ 0.11, Ceq ¼ 20.9 mg kg1; Cu20 BAF ¼ 0.15,

Ceq ¼ 28.8 mg kg1 vs. CuCHP20 BAF ¼ 0.16, and
Ceq ¼ 30.7 mg kg1.
63.7 ± 16.98 (N ¼ 8)

11.38 (N ¼ 7)

16.09 (N ¼ 7)
18.54 (N ¼ 7)
10.66 (N ¼ 7)
12.99 (N ¼ 7)

14.49 (N ¼ 6)
5.79 (N ¼ 7)

8.68 (N ¼ 7)
7.13 (N ¼ 7)
6.43 (N ¼ 7)

The toxicokinetic models for CHP generally fitted the data

better than the models for Cu, with R2 between 64.6% and 86.0%
and all parameters significant at p  0.05 (Fig. 2). The temperature
effect on the TK parameters was more pronounced for CHP than
±
±
±
±
±
±

±
±
±
±

for Cu, and kA was significantly higher at 20
C than at 10
C both

58.7
52.0
58.5
60.3
67.4
61.4

54.8
55.8
53.3
57.3
C20

in CHP-only exposed earthworms and in those exposed to both

chemicals combined (Table 2). Similar differences between tem-
14.18 (N ¼ 5)
13.77 (N ¼ 5)

11.72 (N ¼ 5)

54.21 ± 6.52 (N ¼ 5)

55.1 ± 10.29 (N ¼ 5)
6.96 (N ¼ 5)
8.91 (N ¼ 5)

9.05 (N ¼ 5)

57.1 ± 9.73 (N ¼ 4)

54.0 ± 7.30 (N ¼ 5)

peratures were found for kE, but the confidence intervals did not
overlap only for Cu-free treatments. Additional contamination of
the soil with Cu substantially decreased kA and kE for CHP, with
statistically significant differences found at 20
C.
CuCHP10

±
±
±
±
±
±

The low degradation rate of CHP in the CHP10 treatment (see

53.4
51.9
51.0
54.2
64.9
48.6

above) resulted in a nearly classic TK curve. Regardless of unex-

pectedly high CHP concentrations observed at the 8th d of
exposure, the fitted TK curve indicated fast accumulation of the
13.03 (N ¼ 5)

16.25 (N ¼ 5)
13.58 (N ¼ 5)

13.31 (N ¼ 5)

10.07 (N ¼ 5)

11.31 (N ¼ 5)
e chlorpyriphos at 5 mg kg1, Cu e copper at 200 mg kg1, numbers e temperature in
C.

9.86 (N ¼ 5)

6.97 (N ¼ 5)

9.36 (N ¼ 5)

6.16 (N ¼ 5)

pesticide in the first 4 d followed by approximately stable con-

centration up to the end of the uptake phase. TK of CHP in the
presence of Cu was similar within temperatures, but lower CHP
concentrations and a slight decrease in concentration during the
±
±
±
±
±
±

±
±
±
±
Cu10

61.6
54.4
51.3
55.0
56.4
52.9

45.2
46.7
49.6
54.2

uptake phase was seen due to the higher CHP degradation rate in
the treatments with Cu. At 20
C the highest concentrations of
CHP were already reached within the first 2 d of exposure in both
12.86 (N ¼ 5)
15.38 (N ¼ 5)
11.47 (N ¼ 5)
11.25 (N ¼ 5)
10.76 (N ¼ 5)

16.51 (N ¼ 5)

14.03 (N ¼ 5)

CHP-only and CHP þ Cu-exposed earthworms, and were clearly

5.96 (N ¼ 5)

8.67 (N ¼ 5)
4.96 (N ¼ 5)

higher than in the corresponding treatments at 10
C (Fig. 2). Due

to the fast degradation rate of CHP, and thus the rapidly
decreasing exposure concentration, a decrease of internal CHP
CHP10

±
±
±
±
±
±

±
±
±
±

concentration was observed already at 20
C in the uptake phase,

48.6
45.8
53.3
67.1
60.0
48.4

55.7
45.6
45.9
49.7

and was particularly noticeable in CuCHP20. Nevertheless, the

CHP concentrations in earthworms at the end of the uptake phase
were still slightly higher at 20
C than 10
C (Fig. 2). No bio-
61.1 ± 13.32 (N ¼ 8)

7)
7)
7)
7)
7)
7)

11.30 (N ¼ 7)

10.62 (N ¼ 7)
13.47 (N ¼ 5)
7.20 (N ¼ 7)
¼
¼
¼
¼
¼
¼

accumulation factors or equilibrium concentrations were calcu-

(N
(N
(N
(N
(N
(N

lated for CHP, as these make no sense for fast-degrading

15.15
10.92
14.48
20.04
16.03
12.63

chemicals such as organic insecticides.

Treatment

±
±
±
±
±
±

±
±
±
±
61.0
52.9
53.3
60.3
54.0
62.2

49.3
44.9
54.5
59.5

4. Discussion
C10

The study revealed a significant decrease in AChE activity in

Time [days]

earthworms exposed to an environmentally realistic concentra-

tion of CHP in the soil at both the tested temperatures. The
Table 1

16.5

moderate copper effect, at p ¼ 0.079, was visible only on 16th d of

0.5

16

17
18
20
0

1
2
4
8

exposure. Four days after moving to uncontaminated soil, the

572 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576

80 80
Cu10 (R2=33.2%) Cu20 (R2=50.6%)
60 60

Cu (mg/kg)

Cu (mg/kg)
40 40

20 20

0 0
0 4 8 12 16 20 0 4 8 12 16 20
time (days) time (days)

80 80
CuCHP10 (R 2=24.8%) CuCHP20 (R2=29.9%)
60 60
Cu (mg/kg)

Cu (mg/kg)
40 40

20 20

0 0
0 4 8 12 16 20 0 4 8 12 16 20
time (days) time (days)

Fig. 1. Copper toxicokinetics in the earthworms Eisenia fetida at 10 and 20
C in soil contaminated either with copper (Cu) only or with a combination of copper and chlorpyriphos
(CuCHP); after 16 d of exposure, the animals were transferred to uncontaminated soil. Small crosses indicate data points excluded from the model (studentised residuals >3 in the
preliminary fit).

Table 2 only once - before the start of the experiment. The water-soluble
Assimilation rates (kA) and elimination rates (kE) of the one-compartment tox- fraction of Cu in soil was not followed in the uptake phase, so we
icokinetic models for copper (Cu) and chlorpyriphos (CHP) in Eisenia fetida, with
confidence intervals (in italics); L stands for the lower band and H for the higher
cannot exclude the possibility of bioavailability changes over time.
band of the 95% confidence interval. On the other hand, Lu et al. (2009) showed that aging (3e56 days)
had little effect on Cu bioavailability to earthworms. Also, a single
Treatment kA kA L kA H kE kE L kE H
application of CHP, resulting in the concentration of ca. 5 mg kg1
Cu toxicokinetics soil, is environmentally realistic, as typical agricultural soil appli-
Cu10 0.026 0.014 0.038 0.213 0.118 0.309
cations of CHP result in soil surface residues of 0.3e32 mg kg1
Cu20 0.041 0.026 0.056 0.282 0.185 0.380
CuCHP10 0.014 0.006 0.021 0.128 0.050 0.206 (Racke et al., 1994). The concentration of chlorpyrifos at 5 mg kg1
CuCHP20 0.024 0.011 0.037 0.155 0.065 0.245 in OECD soil did not affect growth but had some negative effect on
CHP toxicokinetics the fecundity of the earthworm Eisenia fetida after 8 weeks of
CHP10 1.264 0.902 1.625 0.758 0.540 0.976
exposure (Zhou et al., 2007), which, however, was not confirmed in
CHP20 4.915 3.690 6.140 1.750 1.294 2.206
CuCHP10 0.734 0.516 0.952 0.526 0.353 0.699
another study by the same research team (Zhou et al., 2011).
CuCHP20 1.863 1.514 2.213 0.849 0.669 1.030 Chlorpyriphos is a relatively non-persistent broad-spectrum
insecticide, with a surface soil half-life ranging from one to 24
weeks, depending on soil moisture, microbial activity, organic
AChE activity recovered to the level observed in the control matter content and temperature (Odenkirchen and Eisler, 1988).
earthworms. Both kA and kE for Cu were higher at 20
C and in non- Hence, the degradation rates observed in our study agree in most
CHP treatments but only the difference between Cu20 and cases with those reported in the literature. The only exception was
CuCHP10 was significant. In the case of CHP toxicokinetics, kA was the CHP10 treatment with t50 ¼ 305 d, but it must be kept in mind
significantly higher at 20
C in both treatments. A similar trend for that the artificial OECD soil was used in the study, hence the mi-
CHP kE was found, but the difference was statistically significant crobial degradation was probably very low. Because the experiment
only for non-Cu treatments. was performed in darkness, there was also no photodegradation.
The temperature on the CHP degradation rate was confirmed in our
study by almost 3 times lower half-life at 20
C than at 10
C in CHP-
4.1. Justification of the concentrations used and the CHP only treatments. The presence of Cu additionally decreased the CPF
degradation rate half-life substantially: from 305 to 29 d at 10
C, and from 108 to
18 d at 20
C. The earlier study showed that degradation rate of CHP
The background level of Cu in soils all over the world ranges in wastewater was stimulated by the application of Cu at 5 mg L1,
between 14 and 109 mg kg1 and is closely associated with the increasing the degradation constant from 0.0542 h1 to 0.091 h1
parent material and soil formation processes, but high Cu concen- in the control (Khalid et al., 2016). Also, Rafique et al. (2016) found
trations up to 1500 mg kg1 in agricultural soils and 4000 mg kg1 that Cu increased the rate of CHP photodegradation in soil six-fold,
in soils polluted by industrial sources can be found (Kabata- whereas the microbial degradation increased this rate two-fold. As
Pendias, 2011). Thus, the total concentration of 200 mg kg1 soil the most plausible mechanism for the positive effect of Cu on CHP
used in our study is within the range of environmentally realistic degradation, both Khalid et al. (2016) and Rafique et al. (2016)
levels for moderately polluted areas. As the total Cu concentration indicated the fact that Cu ions influence the activity of soil micro-
in the soil does not change with time, it was analysed in the soil organisms that degrade the pesticide.
 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
CHP10 (R =69.6%)
CHP (mg/kg)
CHP (mg/kg)
time (days)
CuCHP10 (R2=64.6%)
CHP (mg/kg)
CHP (mg/kg)
time (days)
Fig. 2. Chlorpyriphos toxicokinetics in earthworms Eisenia fetida at 10 and 20
C in soil contaminated either with chlorpyriphos (CHP) only or with a combination of copper and
chlorpyriphos (CuCHP); after 16 d of exposure, the animals were transferred to uncontaminated soil. Small crosses indicate data points excluded from the model (studentised
residuals >3 in the preliminary fit).
4.2. Comparison of internal levels of chemicals found in E. fetida
with other studies/species
The theoretical equilibrium Cu concentrations in the earth-
worms calculated in our study range from 21 to 31 mg kg1 dry
weight. Because the metal kinetic parameters depend on the
exposure concentration (Bednarska et al., 2015; Lock and Janssen,
2001), the comparison of our results with published data is diffi-
cult, as different authors have used different exposure concentra-
tions. Moreover, the use of different equations to describe metal TK
by other authors complicates a comparison of the results. The
comparable study on E. fetida, although with fixed initial body Cu
concentration and conducted over a different time scale (42 d of
exposure) and soil type (field soil contaminated with 161 mg Cu
kg1 dry weight), was performed by Nahmani et al. (2009). The
equilibrium Cu concentration of 26.5 mg kg1 dry weight reported
in that work, based on data for the whole body mean values, is
within the rage found in our study. The values of kA and kE obtained
in our study and by Nahmani et al. (2009) were within a factor of 2
for earthworms exposed at a constant temperature of 20
C. On the
other hand, up to ca. 3-fold higher Cu concentration (60 mg kg1)
than in our study was found in a similar species, E. andrei, exposed
to 192 mg Cu kg1 dry weight of sandy forest soil for 4 weeks at
15
C (Svendsen and Weeks, 1997). However, in contrast to our and
Nahmani et al. (2009) studies, Svendsen and Weeks (1997) pro-
vided E. andrei with food during the exposure, so the Cu found in
the food (81 mg kg1 dry weight) also contributed to the exposure
and, thus, to the total body burden.
The quantitative comparison of internal CHP concentrations
found in our study with published data for earthworms is even
more difficult than in the case of Cu due to different degradation
rates of CHP under different experimental conditions, which were
not taken into account in earlier studies. Neither Lister et al. (2011)
nor Spurgeon et al. (2011) included the degradation rate of CHP in
their toxicokinetic models for earthworms. Although Lister et al.
(2011) reported the predicted ‘equilibrium’ concentration for CHP
in L. terrestris of approximately 10 mg kg1 and 5 mg kg1 in
earthworms exposed to CHP at either 50 mg kg1 or 25 mg kg1,
573
C HP20 (R =83.6%)
time (days)
CuCHP20 (R =86.0%)
time (days)
being reached after approximately 2.5 d, we did not calculate Ceq
for our data as such calculations do not make much sense for fast-
degrading chemicals. We found the highest maximal mean internal
CHP concentration in the uptake phase, 14.1 mg kg1, in CHP20
treatment after 2 d of CHP exposure; the lowest e in earthworms
exposed to CHP-only at 10
C, where 6.8 mg kg1 was reached at
day 16. At the end of the exposure period (day 16), the internal CHP
body concentrations were: 6.7 ± 1.8, 6.8 ± 2.6, 9.2 ± 5.5 and
7.0 ± 2.5 mg kg1 for CHP10, CuCHP10, CHP20 and CuCHP20
respectively. For comparison, a tissue concentration of ca.
10 mg kg1, with differences of up to a factor of 5 between the
lowest and highest individual values within time points, was found
for L. rubellus exposed to much higher CHP concentrations, i.e.
25 mg kg1, for 7 d at 12 ± 2
C (Spurgeon et al., 2011). Spurgeon
et al. (2011) concluded that an inherent inter-individual variation
in pollutant handling is a characteristic of earthworms under many
exposure scenarios.
4.3. Toxicokinetics of Cu and CHP at different temperatures
Although Cu accumulated in the exposed earthworms during
the uptake phase, the internal body concentration decreased
rapidly after moving the animals to the uncontaminated soil.
Spurgeon and Hopkin (1999) also found that Cu-exposed E. fetida
was able to remove an excess of the metal efficiently. However,
metal kinetics can be temperature-dependent, resulting in different
kA and/or kE at different temperatures, as partly shown in our
studies: both kA and kE were higher at 20
C than at 10
C within
both the CHP and non-CHP treatments (even if the 95% confidence
intervals for the estimated parameters overlapped for these treat-
ments). Similarly, Janssen and Bergema (1991) found out that
O. cincta increased Cd accumulation and excretion rates at 20
C
compared to 10
C, resulting in similar body burdens at both
temperatures. In contrast to O. cincta, in Platynothrus peltifer only
the assimilation rate increased with temperature, while the elimi-
nation rate was not temperature-dependent, resulting in higher
internal concentrations at 20
C than at 10
C (Janssen and
Bergema, 1991). The estimated equilibrium Cu concentrations in
574 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
our studies, similar to the study on P. peltifer (Janssen and Bergema,
1991), were higher in earthworms exposed at 20
C than at 10
C, so
although both kA and kE for Cu seem to increase with temperature
in E. fetida, the increase in kA was not fully compensated for by the
increase in kE.
CHP was rapidly accumulated by the earthworms, and the
fastest accumulation was seen at 20
C, especially in the CHP-only
treatment, where the maximum concentration was reached already
after 2 d of exposure. The elimination rate was also fast, and after
4 d of elimination its concentration dropped to 0.2 (at CHP10) e 0.7
(at CuCHP10) mg kg1. A high rate of CHP elimination was found
also in L. terrestris (Lister et al., 2011). In general, in our experiment
the temperature effect on kinetic parameters was more pro-
nounced for CHP than for Cu, as statistically significant differences
between the TK parameters at different temperatures were found
for CHP.
4.4. Toxicokinetics of Cu under the exposure to CHP and vice versa
We did not find a significant effect of CHP on the Cu kinetic
parameters for E. fetida, although kA and kE were visibly higher at
Cu-only than in mixture treatments at both 10
C and 20
C.
Similarly, in L. terrestris exposed to a mixture of 70 mg kg1 Ni and
25 mg kg1 CHP, the patterns of metal uptake were not statistically
different between the single and mixture exposures, but a signifi-
cant difference between the single-chemical and mixture treat-
ments was found for CHP (Lister et al., 2011). Also, in our study
additional contamination of soil with Cu decreased kA and kE for
CHP, with statistically significant differences found at 20
C. On the
other hand, Zhou et al. (2012) demonstrated that the uptake of Cu
by E. fetida was completely stopped by the presence of glyphosate
(GPS) which, similarly to CHP, is also an organophosphorus com-
pound, but used as a broad-spectrum systemic herbicide, not as an
insecticide. A further study indicated that GPS could reduce the
bioavailability of Cu in the soil because of its strong chelating effects
(Zhou et al., 2013).
4.5. Acetylcholinesterase
The important issue when comparing the toxicokinetics of CHP,
either at different temperatures or in the presence of copper, is that
measurement of the parent compound may not be a good indicator
of the toxicodynamics of this pesticide. This is because CHP itself
has lower acetylcholinesterase inhibitory activity than its primary
metabolite, chlorpyriphos-oxon (Monnet-Tschudi et al., 2000).
Although Lister et al. (2011) confirmed the presence of the
chlorpyrifos-oxon in L. terrestris exposed to 25 or 50 mg CHP kg1,
the detected concentrations in worms were ca. 1000-fold lower
than the parent molecule, making detailed appraisal of CHP-oxon
toxicokinetics impossible. Therefore, the specific effect of CHP on
AChE activity was followed over time, rather than the pattern of
chlorpyriphos-oxon, to assess the effects of exposure to this
insecticide. This revealed that the exposure of E. fetida to the real-
istic CHP concentration in soil negatively affects AChE activity. The
CHP effect on AChE activity varied over time. First, a moderate yet
significant decrease was noted already after 12 h exposure but then
no effect was recorded up to the 8th d. The significant decrease in
AChE activity, in comparison with the control, appeared again on
day 16. In fact, due to the specificity of the classic TK experiment we
cannot exclude that the effect appeared between the 8th and 16th
d of exposure, when no animals were sampled. In the earthworm
Pheretima peguana exposed to soil spiked with CHP at concentra-
tions 0.1, 1, 10 and 100 mg kg1 at 25
C, the observed dose-
dependent inhibition of AChE was greater after 7 than 14 d of
exposure (Muangphra et al., 2015). The exposure of E. andrei to
another organophosphate insecticide, pirimiphos-methyl, in OECD
soil also caused a significant inhibition of AChE activity in a dose-
dependent manner, and the highest inhibition of AChE activity
was achieved 6 d after exposure to the highest dose (2 mg kg1 soil)
of pirimiphos-methyl, and after 15 d of exposure a slight increase in
the activity was recorded (Velki and Hackenberger, 2013a). On the
other hand, in the earthworm Aporrectodea caliginosa exposed to
nominal concentrations of either 1 or 10 mg kg1 for 3 and 21 d, the
inhibition of AChE was more pronounced after 21 d of exposure
(Sanchez-Hernandez et al., 2014). Thus, the effect of OPs on
earthworms can differ between species and depends on the
exposure level and time of exposure. Species also differ greatly in
the baseline AChE activity, which was found to be 51.5 ± 4.6 to
54.2 ± 4.5 nmol min1 mg1 protein for E. andrei (Velki and
Hackenberger, 2013a), but 593 ± 174 nmol min1 mg1 protein
for A. caliginosa (Sanchez-Hernandez et al., 2014). In our study,
AChE activity in unexposed E. fetida at 20
C was
52.0e67.4 nmol min1 mg protein1 e very similar values to those
obtained by Velki and Hackenberger (2013a) for the closely related
species, E. andrei. However, because different authors measured
AChE activity in different parts of the earthworms (whole earth-
worms were used by Velki and Hackenberger (2013a), wall muscle
tissues by Sanchez-Hernandez et al. (2014) and we used the ante-
rior part of the body) the direct comparison of the baseline AChE
activity between species is difficult.
Knowledge about the recovery of enzyme activity after exposure
to OPs is important for predicting the period in which, after the
exposure to an insecticide, changes in the organism can still be
detected (Velki and Hackenberger, 2013b). The recovery of enzyme
activity after exposure to OPs is usually slow, relying on reactivation
and/or de novo biosynthesis of new enzyme particles after elimi-
nation of the insecticide from the body (Aamodt et al., 2007). In our
study, however, the recovery of AChE activity was fast, as already
4 d after transferring to the clean soil the previously exposed
earthworms had AChE activity similar to the control animals. This
was not the case in the study by Aamodt et al. (2007) on E. fetida, in
which cholinesterase activity displayed an extremely slow recovery
to normal activity after the OP-mediated inhibition e it only started
to recover 3 weeks after the exposure. However, the authors
exposed the earthworms for 48 h to an extremely high CHP con-
centration, 240 mg kg1 soil, which is 48 times higher than that
used in our study, causing drastic inhibition of the enzyme (Aamodt
et al., 2007).
Studies involving three stressors with different modes of action
are exceptional (Heugens et al., 2006), and to our knowledge no
previous studies have investigated the effects of a natural stressor
(e.g., temperature) in combination with binary mixtures of chem-
icals with different modes of action (e.g. a metal and a pesticide) on
AChE activity. In aquatic species, a combination of OPs (carbofuran,
dichlorvos or malathion) with metals (arsenic or copper) had a
synergistic effect on the inhibition of AChE activity in the micro-
crustacean Tigriopus brevicornis (Forget et al., 1999) after 96 h of
exposure. We did not find any synergistic effect of Cu and CHP on
AChE in our study. In general, the CHP  Cu interaction observed in
full ANOVA for the uptake phase is most probably an artefact
stemming from another significant interaction with time e as
shown in the analyses performed for each sampling day separately,
no significant interactions were found throughout the experiment.
A moderate Cu effect on AChE activity was noted only at 16th d of
exposure; indeed, as can be seen in Table 1, AChE activity was lower
in Cu treatments at both temperatures compared to the control.
Nevertheless, at the end of the exposure phase, AChE activity in
treatments with CHP-only and with a mixture of CHP and Cu were
almost identical. Although Forget et al. (1999) found synergistic
effects of Cu and OP pesticides in T. brevicornis, Yologlu and Ozmen
 A.J. Bednarska et al. / Environmental Pollution 220 (2017) 567e576
(2013) did not observe any effect on AChE in the earthworm
L. terrestris when copper oxychloride (a fungicide) and methyl
parathion were applied in a mixture in apricot orchards. Thus, the
interactions between pesticides and metals can be highly
unpredictable.
5. Conclusions
The study showed that CHP at realistic concentration in soil
inhibits AChE activity in E. fetida. Nevertheless, throughout most of
the exposure phase, except for the immediate effect observed 12 h
after starting the exposure, the effect on AChE activity remained
non-significant and appeared again, much more pronounced, at the
end of exposure (day 16). One of the possible explanations of this
delayed effect of CHP on AChE can be the increasing accumulation
of the metabolite, chlorpyriphos-oxon, over time, which has a
higher AChE inhibiting activity than CHP itself (Monnet-Tschudi
et al., 2000). Therefore, for proper assessment of the CHP effect
on AChE, longer exposure periods should be used in future studies,
with more frequent sampling during the uptake phase to assess
when exactly AChE activity drops below the normal level.
The direct Cu effect on AChE activity was moderate, although
noticeable. In contrast to our expectations that the additional
exposure to copper contamination would amplify the CHP effects,
this was not the case for AChE activity. However, spiking the soil
with Cu at 200 mg kg1 substantially increased the CHP degrada-
tion rate in the soil, which decreased the exposure of the earth-
worms to CHP over time. The effect of Cu treatment and
temperature on CHP half-life showed that at any particular initial
CHP concentration in the soil, the actual exposure of soil-inhabiting
animals to the insecticide largely depends on environmental con-
ditions. The decreasing exposure, due to CHP degradation in the
soil, influenced the internal CHP concentration in earthworms
which, after an initial increase, decreased with time even during
the exposure phase. Because the Cu effect on the CHP degradation
rate was so strong, it would be interesting to test more concen-
trations of Cu and CHP in binary mixtures since their effect on AChE
activity can be dose-dependent, as observed for Pterostichus
cupreus by Jensen et al. (1997). Also, the observed effects of Cu on
CHP toxicokinetics call for testing more Cu concentrations in
combination with different CHP treatments, especially that con-
centrations over five times higher than the one tested herein are
known from agricultural areas.
Acknowledgements
The study was supported by the Jagiellonian University grant DS
758.
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