Toxicology Letters 172 (2007) 137–145
Chronic toxicity of ibuprofen to Daphnia magna: Effects on life
history traits and population dynamics
Lars-Henrik Heckmann
, Amanda Callaghan
, Helen L. Hooper
, Richard Connon
,
Thomas H. Hutchinson
, Steve J. Maund
, Richard M. Sibly
a
University of Reading, School of Biological Sciences, Environmental Biology,
Philip Lyle Building, Reading RG6 6BX, United Kingdom
b
AstraZeneca Global Safety, Health & Environment, Brixham Environmental Laboratory,
Devon TQ5 8BA, United Kingdom
c
Syngenta Crop Protection AG, 4002 Basel, Switzerland
Received 8 April 2007; received in revised form 4 June 2007; accepted 5 June 2007
Available online 14 June 2007
Abstract
The non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) is a widely used pharmaceutical that can be found in several
freshwater ecosystems. Acute toxicity studies with Daphnia magna suggest that the 48 h EC
50
(immobilisation) is 10–100 mg IB l
−1
.
However, there are currently no chronic IB toxicity data on arthropod populations, and the aquatic life impacts of such analgesic drugs
are still undefined. We performed a 14-day exposure of D. magna to IB as a model compound (concentration range: 0, 20, 40 and
80 mg IB l
−1
) measuring chronic effects on life history traits and population performance. Population growth rate was significantly
reduced at all IB concentrations, although survival was only affected at 80 mg IB l
−1
. Reproduction, however, was affected at lower
concentrations of IB (14-day EC
50
of 13.4 mg IB l
−1
), and was completely inhibited at the highest test concentration. The results
from this study indicate that the long-term crustacean population consequences of a chronic IB exposure at environmentally realistic
concentrations (ng l
−1
to
g l
−1
) would most likely be of minor importance. We discuss our results in relation to recent genomic
studies, which suggest that the potential mechanism of toxicity in Daphnia is similar to the mode of action in mammals, where IB
inhibits eicosanoid biosynthesis.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Invertebrate; Stress response; Fecundity; Reproduction; Mode of action; NSAID
1. Introduction
The release of human and veterinary pharmaceuti-
cals into the environment has been the focus of recent
∗
Corresponding author. Tel.: +44 118 378 4426;
fax: +44 118 931 0180.
E-mail address:
(L.-H. Heckmann).
1
Present address: University of California, School of Veterinary
Medicine, Department of Anatomy, Physiology and Cell Biology,
Davis, CA 95616, USA.
research and review programmes (
). In
Europe, several over-the-counter drugs like acetylsali-
cylic acid, ibuprofen (IB) and paracetamol are consumed
in amounts of over 100 t per year (
2005; Zwiener and Frimmel, 2000
). IB is not fully
metabolised by humans and may, therefore, enter the
sewage system as the parent compound or metabolites
(
). Treatment in modern sewage treat-
ment plants appears to eliminate the vast majority of IB
and its metabolites with degradation by >95% reported
in the literature (
). In surface water
0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:
138
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
monitoring programmes, IB has been detected at the
ng l
−1
to
g l
−1
range in various parts of the world
(
Andreozzi et al., 2003; Han et al., 2006; Kolpin et al.,
2002; Metcalfe et al., 2003; Stumpf et al., 1999
IB is a non-steroidal anti-inflammatory drug
(NSAID) that has relatively high mobility in aquatic
environments, but a low persistence compared to other
pharmaceuticals (
). The half-life of IB
has been estimated to be 32 days in the field (
). Maximum concentrations of IB in UK surface
waters have been found to be 5
g l
−1
with an esti-
mated risk quotient (RQ) of 0.01 (
Previously,
rather conservative RQ above 1 that was based on no
elimination of the active compound, and a predicted envi-
ronmental concentration of IB. Both of these RQ-values
are based on data from the few acute studies that have
been published on the cladoceran crustacean Daphnia
magna Straus, which report that the 48 h EC
50
for immo-
bility is in the region of 10–100 mg IB l
−1
2003, 2004; Halling-Sørensen et al., 1998; Han et al.,
2006; Heckmann et al., 2005
). However, there are no
published data on the chronic population effects of IB on
D. magna or other arthropod species. In one of the more
extensive studies investigating the effects of pharmaceu-
ticals on planktonic communities,
revealed that a mixture of IB (0.6 mg l
−1
), fluoxetine
(1.0 mg l
−1
) and ciprofloxacin (1.0 mg l
−1
) decreased
the diversity of a zooplankton community in a 35-
day microcosm experiment, concurrently increasing the
overall abundance of a few species. Other non-arthropod
studies show that IB, acetylsalicylic acid and paraceta-
mol had no effect on the cnidarian Hydra vulgaris at
concentrations up to 1 mg l
−1
following 7 days exposure
(
). However, 1 mg IB l
−1
was suffi-
cient to reduce the growth of duckweed Lemna minor by
25%, with a 7-day EC
50
of 4 mg IB l
−1
). Conversely, growth of the cyanobacteria Syne-
chocystis sp. was stimulated at 10
g IB l
−1
(
At present, there is limited ecotoxicological informa-
tion available in the literature regarding the potential
chronic impact of pharmaceuticals on aquatic organ-
isms and ecosystems (
); although the few reported chronic-effect stud-
ies indicate that IB probably has very little impact on
aquatic environments (
Han et al., 2006; Pascoe et al.,
2003; Pomati et al., 2004; Pounds et al., submitted
for publication; Richards et al., 2004
). Here, we report
the chronic effects of IB on the life history traits and
population dynamics of D. magna following a 14-day
exposure. Furthermore, we suggest a potential mecha-
nism of toxicity for this NSAID based on recent genomic
studies.
2. Materials and methods
2.1. Test species
D. magna (clone type 5-IRCHA), originally obtained from
the Water Research Centre, Medmenham, UK, was used for this
study. For an outline of the life history of D. magna and full
information on culturing conditions see
2.2. Test chemical
The NSAID IB is widely used in the treatment of rheumatic
disorders, pain and fever due to its analgesic, antipyretic and
anti-inflammatory properties. IB was obtained as a sodium
salt (C
13
H
17
NaO
2
) from Sigma–Aldrich (CAS no. 31121-93-4:
batch no. 64K0892). In principle, IB is a fatty acid and, thus,
virtually insoluble at low pH, but fully ionized at pH 7 and
above (
). In the case of IB sodium
(IB-Na), the parent compound dissociates to form C
13
H
17
O
2
−
and Na
+
at pH 7 and above. Here, we refer to concentrations
of the NSAID based on the molecular weight of the pharma-
cological active ingredient IB.
2.3. Experimental design
Concentrations of IB were selected based on preliminary
acute and chronic toxicity test results. Initially, a standard acute
test method (
) was used to determine the toxicity
of IB to our laboratory clone. The 48 h EC
50
for immobilisa-
tion was estimated as 108 mg IB l
−1
(
Subsequently, populations were exposed to eight IB concentra-
tions ranging from 10 to 160 mg IB l
−1
for 12 days (
), using an experimental design similar to the one
described below. This pilot study revealed that reproduction
was markedly reduced above 10 mg IB l
−1
, and that survival
was unaffected at concentrations up to 40 mg IB l
−1
with no
survivors above 80 mg l
−1
. Accordingly, we chose the concen-
trations 20, 40 and 80 mg IB l
−1
and a dilution water control for
the current study. Each treatment was replicated four times and
assigned to a randomised block design. During the first 24 h of
the test, each replicate consisted of 310 fourth brood neonates
(<24 h old) that were held in a glass cylinder (height 13 cm;
internal diameter 8.5 cm) with a 200
m mesh covering the
bottom. This cylinder was contained within a larger glass ves-
sel (height 22 cm; internal diameter 18.5 cm; thickness 5 mm)
(Harzkristall GmbH, Derenburg, Germany) with a clear plastic
lid, containing 5 l of reconstituted water (
with or without the addition of IB-Na. After 24 h exposure, 10
neonates were transferred to the larger vessel, and the cylin-
der containing the remaining 300 neonates was removed. These
daphnids were analysed for differences in gene expression (not
reported here). The 10 transferred neonates were observed over
the following 13 days to assess the effects of IB on popula-
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
139
tion growth rate (PGR), reproduction, survival, somatic growth
and population size structure. The latter two parameters were
derived from measurements of individual’s body surface area,
captured by digital imaging using the method and settings of
. There was no feeding during the first day
(24 h), but, subsequently, all populations were fed daily with
equal amounts of green algae Chlorella vulgaris var viridis
(equivalent to 0.25 mg carbon per day on day 2; 0.50 mg car-
bon per day from days 3–9; 0.75 mg carbon per day on days 10
and 11; and 1.00 mg carbon per day on days 12 and 13). The
test vessels were kept in a 20
± 1
◦
C temperature-controlled
room with a light:dark regime of 16:8 h with lighting provided
by a 70 W, cool white fluorescent tube, situated 10 cm directly
above the test vessels.
2.4. Physicochemical conditions
During the test, the water was aerated regularly (2 h per
day) via a glass tube (length 20 cm, internal diameter 3 mm)
submerged 3 cm below the water surface. Temperature was
monitored daily having a mean of 19.5
± 0.2
◦
C S.E. (n = 60)
throughout the 14-day exposure. Dissolved oxygen (DO),
conductivity and pH were monitored on days 2, 4, 8 and
14 (
). Conductivity increased with IB concentration
(P < 0.05) following the dissociation of sodium ions from
IB-Na. However, the difference in conductivity between the
control and 80 mg IB l
−1
was less than 5% throughout the
experiment. There was a consistent increase in both DO and
pH in all treatments until day 8, most likely the result of
increased photosynthesis in the test medium from the daily
addition of C. vulgaris. By day 14, DO and pH levels had
decreased in the control and the 20 and 40 mg IB l
−1
treat-
ments, but remained elevated at 80 mg IB l
−1
where algae had
accumulated (
To quantify IB, 1.5 ml was sampled from each replicate on
days 1, 2, 4, 8 and 14, and stored at
−20
◦
C. Subsequently,
IB was quantified at 217 nm by UV-spectrophotometry using
a Shimadzu UV-1201 (Shimadzu, Europa GmbH, Duisburg,
Germany) following the method of
. All the
measured IB concentrations were within
±20% of the nominal
concentration during the experiment, except on day 14 where
the measured concentration of IB was >25% less than the no-
minal concentration in one of the treatments (
). This
was due to a slight reduction (average 16%) in the measured
concentration of the highest treatment on day 14 compared
with day 1 (
2.5. Data analysis and statistical methods
All data, except PGR and pH, were log
10
-transformed.
Two-way ANOVAs were performed in Minitab
®
Release 14.1
(Minitab Inc., State College, PA, USA). Results on all data
verified that no block effect was present. Differences between
treatments for water chemical parameters and biological end-
points were analysed in SPSS 12.0.1 for Windows (SPSS,
Chicago, IL, USA) using regression analysis, and one-way
ANOVA together with Tukey’s honestly significant difference
for post hoc comparisons. Equality of variance was tested
Table 1
Water chemistry and quantification of ibuprofen
Day 1
Day 2
Day 4
Day 8
Day 14
Dissolved oxygen (mg l
−1
)
Control
–
8.15
± 0.03
a
8.45
± 0.03
a
9.03
± 0.09
a
7.83
± 0.13
a
20 mg IB l
−1
–
8.18
± 0.03
a
8.48
± 0.03
a
9.23
± 0.09
ab
8.18
± 0.24
a
40 mg IB l
−1
–
8.15
± 0.03
a
8.48
± 0.03
a
9.08
± 0.02
ab
8.08
± 0.28
a
80 mg IB l
−1
–
8.10
± 0.00
a
8.50
± 0.00
a
9.43
± 0.10
b
9.48
± 0.15
b
Conductivity (
S cm
−1
)
Control
–
421
± 0.9
a
426
± 1.2
a
423
± 1.3
a
417
± 1.6
a
20 mg IB l
−1
–
427
± 0.8
b
431
± 0.6
b
428
± 0.5
b
422
± 0.5
b
40 mg IB l
−1
–
431
± 0.3
c
437
± 0.5
c
432
± 0.8
c
427
± 0.8
c
80 mg IB l
−1
–
442
± 0.3
d
447
± 0.9
d
444
± 0.9
d
435
± 0.9
d
pH
Control
–
7.72
± 0.01
a
7.99
± 0.02
a
8.32
± 0.06
a
7.74
± 0.06
a
20 mg IB l
−1
–
7.73
± 0.00
a
8.03
± 0.02
a
8.52
± 0.05
b
8.00
± 0.16
a
40 mg IB l
−1
–
7.73
± 0.01
a
8.03
± 0.02
a
8.45
± 0.01
a
7.96
± 0.18
a
80 mg IB l
−1
–
7.72
± 0.01
a
8.06
± 0.01
a
8.66
± 0.02
b
8.83
± 0.04
b
Nominal concentration (mg IB l
−1
)
Measured concentration (mg IB l
−1
)
Control
n.d.
n.d.
n.d.
n.d.
n.d.
20
20.8
± 0.04
18.5
± 0.40
18.0
± 0.85
18.6
± 0.84
18.1
± 0.95 (18.2 ± 0.41)
40
41.8
± 0.29
37.4
± 0.23
37.3
± 0.98
38.5
± 0.75
36.3
± 1.15 (37.3 ± 0.57)
80
80.5
± 1.11
72.4
± 0.73
76.2
± 1.87
78.0
± 2.80
67.3
± 4.01 (72.7 ± 1.93)
Values are mean
± S.E. (n = 4); n.d. signifies “none detected”. Values in parantheses represent the time weighted average (TWA) concentrations
following 14 days of exposure. Different letters within the same day signify a significant difference (P < 0.05).
140
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
using Levene’s test. For all tests, a significant level of 5% was
applied.
EC
x
values for reproduction were estimated from the rela-
tionship between number of juveniles and the IB concentration
by fitting an exponential decay model:
R
E
= R
C
e
−aIB
(1)
where R
E
is the reproduction in the exposed treatment, R
C
the control reproduction (total number of juveniles), a the
decay constant and IB represents the tested concentration of IB.
PROC NLMIXED (
) was used to fit the
data to the model and provide estimates and 95% confidence
limits for the EC
x
values.
PGR was based on manually counting of population num-
bers and estimated as:
PGR
= log
e
(
N
t
/N
0
)
t
(2)
where N
0
is the initial number of individuals at time 0 and N
t
is
the final number of individuals t days later. Positive values of
PGR indicate a growing population, PGR = 0 indicates a sta-
ble population and negative PGR values indicate a population
in decline and headed toward probable extinction (
3. Results
3.1. Somatic growth
Somatic growth, measured as body surface area,
was significantly and strongly positively correlated
(r
131
= 0.855; P < 0.001) with age in the control popu-
lations (data not shown). Interestingly, somatic growth
of the founder members of the populations increased sig-
nificantly with increasing IB concentrations from day 8
and onwards (
). Initially, the increase was only
significant at 20 mg IB l
−1
, but on days 12 and 14, a sig-
nificantly larger body surface area was also evident at
40 and 80 mg IB l
−1
, respectively (
). On day 14,
daphnids from the treated populations were on average
22%–29% larger than control daphnids.
Fig. 1. Somatic growth of founding Daphnia magna measured
as body surface area during a 14-day exposure to ibuprofen
(n = 23–40, mean
± S.E.). Letters a, b and c signify a significant
difference (P < 0.05) between control and 20 mg IB l
−1
; control and
20–40 mg IB l
−1
and control and 20–80 mg IB l
−1
, respectively.
3.2. Reproduction
The most significant effects observed were on repro-
duction, realised through delayed onset and reduced
fecundity (the latter supported by observations of egg
abortions). The day of first reproduction was delayed
significantly at 40 mg IB l
−1
), and total repro-
duction and number of offspring per female was
significantly reduced in all the IB treatments with
cessation at 80 mg IB l
−1
). There was a sig-
nificant and strongly negative concentration-dependent
relationship (r
15
=
−0.985, P < 0.001) between total
reproduction and IB (data not shown) with an estimated
14-day EC
10
and EC
50
of 2.04 mg IB l
−1
(95% C.L.
1.62; 2.46) and 13.4 mg IB l
−1
(95% C.L. 10.7; 16.2),
respectively. However, extrapolations based on these
EC
x
values should be treated with caution, especially
EC
10
, as both estimates are below the tested concentra-
tions.
Table 2
Reproduction and survival of Daphnia magna following a 14-day exposure to ibuprofen
Control
20 mg IB l
−1
40 mg IB l
−1
80 mg IB l
−1
Time of first reproduction (d)
10.8
± 0.25
a
10.8
± 0.25
a
12.3
± 0.25
b
n.a.
Total reproduction
290
± 10.9
a
127
± 24.4
b
28.5
± 4.91
c
0
± 0
d
Offspring/female
31.3
± 0.74
a
12.7
± 2.44
b
28.5
± 0.49
c
0
± 0
d
Survival (%)
92.5
± 2.50
a
100
± 0
a
100
± 0
a
75.0
± 6.45
b
Values are mean
± S.E. (n = 4). Different letters signify a significant difference (P < 0.05); n.a. signifies “not applicable”.
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
141
3.3. Survival
There was no effect of IB on survival at 20 and
40 mg IB l
−1
, but significant mortality was apparent at
80 mg IB l
−1
). Previous results on D. magna
indicated that total mortality occurs at and above
80–100 mg IB l
−1
after 12 days exposure (
) demonstrating a threshold-like relationship
between IB and survival.
3.4. Population dynamics
PGR was significantly reduced in all the IB treatments
compared with the control, and followed a significant and
strongly negative linear relationship (
). PGR was
positive at 0, 20 and 40 mg IB l
−1
, but at 80 mg IB l
−1
populations were in decline due to failure to reproduce
and reduced survivorship.
Individuals were classified into three size classes,
corresponding to neonates, juveniles and adults, to eval-
uate how population size structure was affected by IB
(
). Ibuprofen significantly affected all the treated
populations, and at 80 mg IB l
−1
the populations con-
sisted entirely of adults. The relative percentage of
adults in the population increased more than 2-fold at
20 mg IB l
−1
and almost 5-fold at 40 mg IB l
−1
; and the
percentage of juveniles was more than 14-fold lower at
40 mg IB l
−1
compared with control populations (
These results show that IB had a targeted effect on
Fig. 2. Population growth rate (PGR) of Daphnia magna follow-
ing a 14-day exposure to ibuprofen (n = 4, mean
± S.E.). The line
represents a significant concentration-dependent linear relationship
(r
15
=
−0.945, P < 0.001). Different letters signify a significant dif-
ference (P < 0.05).
Fig. 3. Distribution of Daphnia magna populations in three size classes
following a 14-day exposure to ibuprofen (n = 4, mean
± S.E.). Differ-
ent letters within size classes signify a significant difference (P < 0.05).
reproduction. The changes to population size structure
appeared to be induced by delayed reproduction and
reduced fecundity (
), rather than by increased
juvenile mortality.
4. Discussion
The results from this study indicate that the long-
term population consequences of a chronic IB exposure
at environmentally realistic concentrations (ng l
−1
to
g l
−1
) would most likely be of minor importance
(see also overview of invertebrate chronic-effect data in
). However, no definite conclusion can be made on
the risks of environmentally relevant IB concentrations
before potential effects following multi-generational
exposure have been assessed. In support of our results,
induced egg abortion and reduced PGR has also been
reported previously in D. magna exposed chronically
to a metabolite (o-hydroxyhippuric at 10 mg l
−1
) of the
NSAID acetylsalicylic acid (
although the parent compound had no impact at the same
concentration (
). A recent study
on killifish (Oryzias latipes) revealed that, although
reproduction was delayed following a 6-week chronic
exposure to
g l
−1
levels of IB, total reproduction of
killifish did not differ between treatments (
). Whereas, an in vitro study revealed endocrine
disruption at sub-molar IB concentrations decreasing
cortisol production by approximately 40% in interre-
nal cells of rainbow trout Oncorhynchus mykiss; which
potentially may impair overall stress response (
). Thus, clearly more chronic studies
need to be conducted on aquatic organisms to assess the
ecological risks of IB and NSAIDs in general.
In mammals, IB and related NSAIDs are known to
interrupt the production of various eicosanoids, mainly
by inhibiting the cyclooxygenase (COX) pathway, which
142
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
Table 3
Overview of invertebrate chronic-effect data on ibuprofen
Test species
Endpoint
Exposure range
(mg IB l
−1
)
Effect concentration
(mg IB l
−1
)
Reference
Hydra vulgaris (Cnidarian)
Reproduction (bud
formation)
0.01–10
7d NOEC = 10
Hydra vulgaris (Cnidarian)
Survival
0.01–10
7d NOEC = 10
Planorbis carinatus (Mollusc)
Growth
0.32–5.36
21d NOEC = 1.02
Pounds et al. (submitted for publication)
21d LOEC = 2.43
Planorbis carinatus (Mollusc)
Reproduction
0.32–5.36
21d NOEC = 2.43
Pounds et al. (submitted for publication)
21d LOEC = 5.36
Planorbis carinatus (Mollusc)
Survival
0.32–5.36
21d NOEC = 5.36
Pounds et al. (submitted for publication)
21d LOEC > 5.36
Daphnia magna (Crustacean)
Reproduction
0.1–80
21d NOEC = 20
Daphnia magna (Crustacean)
Reproduction
20–80
14d EC
10
= 2.04
(1.62; 2.46)
This study
14d EC
50
= 13.4
(10.7; 16.2)
Daphnia magna (Crustacean)
Survival
20–80
14d NOEC = 20
This study
14d LOEC = 80
Daphnia magna (Crustacean)
Population growth
rate (PGR)
20–80
14d NOEC < 20
This study
14d LOEC = 20
a
Values are measured concentrations (with the exception of
). Numbers in parantheses signify the lower and upper 95% confidence
limits.
is one of the three major pathways involved in eicosanoid
biosynthesis. All eicosanoids derive from a common pre-
cursor, arachidonic acid (an omega-6 fatty acid), which
is converted into different eicosanoids through either
the COX pathway (e.g. prostaglandins); the lipoxyge-
nase (LOX) pathway (e.g. leukotrienes and lipoxins); or
the cytochrome P450 epoxygenase pathway (e.g. epoxy-
eicosatrienoic acids) (
). Eicosanoids act as
autocrine or paracrine signallers (also referred to as local
hormones) mediating proximal cell responses to stimuli.
They differ from hormones in that the compound is usu-
ally broken down quickly, and, thus, their effects are
normally local (i.e. within the same tissue from which
they were synthesised). In mammals, eicosanoids oper-
ate as important regulators of inflammation, ion flux
and neural and reproductive function, and several stud-
ies reveal that they also play vital roles in reproduction,
immune response and temperature regulation of insects
(reviewed in
Rowley et al., 2005; Stanley-Samuelson,
). Recently, a mechanism corresponding to the
mammalian eicosanoid biosynthesis was proposed in the
coral Plexaura homomalla (
). This
indicates that the NSAID mode of action in invertebrates
could be similar to that of mammals, which is further sup-
ported by evidence of inhibition of eicosanoid generation
by NSAIDs in a wide range of invertebrate species (e.g.
). We have recently provided gene
expression data, which suggests an IB concentration-
dependent induction of the expression of the D. magna
ortholog of Leukotriene B4 12-hydroxydehydrogenase
(
). The translated enzyme is
known to metabolise Leukotriene B4 in eicosanoid
biosynthesis (
). Interestingly,
Leukotriene B4 has been shown to have an important
role in regulating yolk uptake during oogenesis in insects
Fig. 4. Simplified overview of eicosanoid biosynthesis based on
the present knowledge from mammalian models. Arachidonic acid
originates from different phospholipids (e.g. diacylglycerol), and is
then further metabolised through one of the three shown pathways:
cyclooxygenase, lipooxygenase and cytochrome P450 epoxygenase.
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
143
). This apparent link between
eicosanoids and reproduction may help to explain the
effects of IB on reproduction in D. magna observed
in this study. But further work is needed to confirm
the genomic observations preferably including tran-
scriptomics, proteomics and enzyme activity assays.
Moreover, linking the impact of NSAIDs on mammals
and invertebrates should be approached with caution till
more evidence exists.
The IB concentrations tested here (
≤80 mg l
−1
) had
little effect on survival, demonstrating the low acute
toxicity of IB. This is similar to the majority of phar-
maceuticals, with the exception of, e.g. anti-depressive
drugs (
Fent et al., 2006; K¨ummerer, 2004
). Through-
out the duration of the experiment, Daphnia populations
were fed an equal daily ration of algae, and based on the
results and observations on somatic growth, there did
not seem to be any major differences in energy intake.
However, one could argue that the observed increase in
somatic growth was an artefact of increased availabi-
lity of algae due to fewer or no offspring being present
amongst IB treated populations. Compared to the other
treatments on day 14, the steep increase in somatic
growth at 80 mg IB l
−1
) could well be influenced
by a greater per capita availability and intake of algae.
But individuals exposed to 20 mg IB l
−1
had an increased
somatic growth compared with the controls on day 8.
At this time, there was a similar survival within the two
treatments, and no offspring had yet been produced. Con-
sequently, increased somatic growth was probably not
caused by an increasing amount of available food/energy,
but may be interpreted as a direct effect of IB. The effects
on somatic growth and survival, together with a strong
negative impact of IB on reproduction support the “Prin-
ciple of Allocation”, where the amount of energy that an
individual can invest in maintenance, growth and repro-
duction is limited and highly dependent on energy input
(
). Thus, the quenching of repro-
duction would liberate energy to be invested in somatic
growth given that maintenance and energy input remain
constant.
In previous acute immobility studies on D. magna,
has stated that the IB mode of
action is acting non-specific by narcosis or baseline
toxicity (sensu
). We have clearly
shown that there is a very specific chronic effect of IB
on reproduction. But why is such a high IB concen-
tration necessary to cause any effect in Daphnia; and
is the mode of action truly non-specific especially in
the light of our recent molecular evidence? In mam-
mals, at the molecular level, IB reversibly inhibits the
enzymatic activity of COX through competing with its
substrate, arachidonic acid, for the catalytic sites of COX
(
gest that high effect concentrations in fish may be due to
inefficient IB inhibition of teleost COX. Based on evi-
dence from invertebrates, one could hypothesize that the
eicosanoid precursor arachidonic acid is very abundant,
which would require high IB concentrations to pro-
duce any effect. For instance, Weinheimer and Spraggins
(reviewed in
) report that up to 8% of
the dry mass of the coral P. homomalla are eicosanoids.
Such high content of eicosanoids may be extraordinary,
but if these concentrations are directly in proportion to
their precursor arachidonic acid, and if a similar level is
found in Daphnia, it would explain why such high IB
concentrations are needed to cause an effect in Daphnia.
A high body content of eicosanoids could also imply
a high availability of the major enzymes (e.g. COX or
LOX) involved in eicosanoid biosynthesis. This is the
situation in humans where COX-1 (the only COX iso-
form present in invertebrates) is constitutively expressed
in almost all tissues (e.g.
Further work is currently underway to assess the global
expression profile of Daphnia and attempt to identify the
key genes involved in stress responses to IB. We hope
this work will shed further light on the effects of IB on
eicosanoid biosynthesis and reproduction in Daphnia.
As a concluding remark, and as encouraged by
other investigators (
), further exper-
imentation should focus on invertebrate genomic and
other molecular toxicology studies, taking care to
consider quality control (
and
references herein). This could reveal similarities with
the genomic effects of pharmaceuticals in mammalian
systems, which could have major consequences for the
future testing of pharmaceuticals by, e.g. reducing the
need for vertebrate models such as the use of fish in
environmental safety assessments. Additionally, further
chronic studies on pharmaceuticals are recommended at
the population or community level to increase our under-
standing of the long-term impact of pharmaceuticals in
aquatic environments.
Conflict of interest
The authors state that they have no competing finan-
cial or other interests that could inappropriately influence
the current study.
Acknowledgements
We gratefully acknowledge the financial support
of AstraZeneca, Syngenta, NERC (project NER/
144
L.-H. Heckmann et al. / Toxicology Letters 172 (2007) 137–145
D/S/2002/00413 “The population and molecular stress
responses of an ecotoxicology indicator species”) and
The Research Endowment Trust Fund of the University
of Reading. We would also like to thank Paul Henning
Krogh for statistical assistance on EC
x
modelling, and
two anonymous reviewers for their valuable comments
on the manuscript.
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