Download Effects of Pharmaceuticals on Aquatic Invertebrates. Part II: The

Arch. Environ. Contam. Toxicol. 52, 163–170 (2007)
DOI: 10.1007/s00244-005-7190-7
Effects of Pharmaceuticals on Aquatic Invertebrates. Part II: The Antidepressant
Drug Fluoxetine
Gerrit Nentwig
Department of Aquatic Ecotoxicology, J.W. Goethe-University Frankfurt am Main, Siesmayerstrasse 70, D-60054 Frankfurt, Germany
Received: 27 July 2005 /Accepted: 2 May 2006
Abstract. Fluoxetine, a selective serotonin reuptake inhibitor
antidepressant and high-prescription-volume drug, is excreted
unchanged or as a glucuronide from the human organism.
Little is known about its fate in sewage treatment plants. Effects of fluoxetine on life-cycle parameters of the midge
Chironomus riparius, especially development (mean emergence time), emergence, sex ratio, and fecundity, were assessed, as well as effects on reproduction of the oligochaete
Lumbriculus variegatus and of the freshwater mudsnail
Potamopyrgus antipodarum. Due to the moderate lipophilic
properties of the compound with a log POW of 4.05, C. riparius
and L. variegatus were exposed to fluoxetine via spiked artificial sediment at a nominal concentration range between 0.15
and 5.86 mg/kg (dry weight). Additionally, a test was
performed exposing P. antipodarum via water in a nominal
concentration range between 0.64 and 400 lg/L. As endpoints,
emergence rate and timing, sex ratio, clutch numbers and
clutch size of the midges, the number of worms in the oligochaete test, as well as the number of embryos in the snail test
were monitored. For C. riparius, no clear substance-related
effects were observed; for L. variegatus, the results indicated a
slight increase in reproduction, which was statistically significant at nominal fluoxetine concentrations of 0.94 and 2.34
mg/kg. In P. antipodarum, the antidepressant reduced reproduction significantly. No observed effect concentration
(NOEC) and 10% effect concentration (EC10) were determined
to be 0.47 and 0.81 lg/L, respectively, based on measured
fluoxetine concentrations in water. These low values indicate
that P. antipodarum and possibly other aquatic mollusks are
sensitive to fluoxetine and that the drug might pose a risk to
gastropod populations in the field.
During the last 10–15 years, an increasing number of pharmaceutical residues have been detected in the aquatic environment (Sattelberger 1999; Ternes 1998; Tixier et al. 2003).
Correspondence to: Gerrit Nentwig; email: [email protected]
-frankfurt.de
This is of concern because some residues have been detected
in drinking water (Ternes 2000). Although most of these
compounds occur in surface waters in the nanogram-per-liter
range, hazards for aquatic biota cannot be excluded because of
high biological activity. This has been demonstrated for
endocrine active pharmaceuticals such as ethinylestradiol,
which already affects aquatic wildlife at concentrations between 1 and 10 ng/L (Jobling et al. 2004) and particularly fish
populations, even in the subnanogam-per-liter range (Young
et al. 2002). Because the active ingredients of many drugs are
quite stable, they persist in the environment and are hardly
changed during sewage treatment. Such is the case for
the antiepileptic agent carbamazepine, of which only 7% is
eliminated by sewage treatment, and the lipid-lowering agent
clofibric acid, persistent with a half-life-time (DT50) greater
than 365 days (Ternes 1998). Despite the widespread occurrence of pharmaceuticals, chronic ecotoxicological effect data
are sparse (Kolpin et al. 2002; Ternes 1998; Tixier et al. 2003).
In the present study, part of a project assessing the environmental impact of a range of pharmaceuticals, the effects of
the antidepressant drug fluoxetine on the freshwater mudsnail
Potamopyrgus antipodarum, the nonbiting midge Chironomus
riparius, and the oligochaete Lumbriculus variegatus were
assessed. Ecotoxicological research on drugs such as fluoxetine is important because they are prescribed in large amounts.
Schwabe and Paffrath (2004) reported that 23.1 million Defined Daily Doses were prescribed in 2003 in Germany,
reflecting a total amount of 4.62 tons. In the United States,
fluoxetine was widely prescribed after its introduction in 1988
under the brand names of Prozac and Sarafem. It was soon
advertised in the lay press and, according to Olfson et al.
(1998), is often used without any medical necessity, as a
lifestyle drug. Medawar (1994) stated that annual sales of
Prozac exceeded 1.2 billion US dollars in 1994. Prescription
amounts continued to increase after less costly generic products became available (Schwabe and Paffrath 2004) and it is
often used for the treatment of maladies other than depression
(Barondes 1994). Fluoxetine is excreted 20–30% unchanged;
the rest is excreted as fluoxetine glucuronide and norfluoxetine, the active metabolite (Hartke and Mutschler 1993). The
parent compound can be reactivated in wastewater treatment
plants by cleavage of the glucuronides (Mçhle et al. 1999).
Fluoxetine has previously been detected in the environment.
G. Nentwig
164
Metcalfe et al. (2003) found quantities up to 0.099 lg/L in the
Little River (Canada) below a sewage treatment plant. Kolpin
et al. (2002) reported fluoxetine levels up to 0.012 lg/L in US
streams. Weston et al. (2001) estimated fluoxetine concentrations of 0.54 lg/L in sewage treatment plant effluents.
Because of its serotonergic action, fluoxetine can influence
the reproductive behavior of mollusks (Hecker 2004). Several
studies have shown that spawning and oocyte maturation is
directly controlled by serotonin in several taxa of this phylum
(Fong et al. 1994; Fong 1998; Hirai et al. 1988, Krantic et al.
1991, Ram et al. 1993). Exogenous application of serotonin as
well as fluoxetine to Dreissena polymorpha induced spawning
(Fong et al. 1993, 1996, 1998; Ram et al. 1993). The lowest
observed effect concentration (LOEC) of 0.155 mg fluoxetine/
L for male mussels and 1.55 mg/L for female mussels has been
reported (Fong 1998).
We assessed the effects of fluoxetine on the mudsnail
P. antipodarum to investigate whether representatives of
different mollusk classes react in a similar way. Recent reports have dealt with the effects in bivalve mollusks. Mudsnails have proved to be sensitive test organisms in several
studies (Duft et al. 2003a, 2003b; Schulte-Oehlmann 1997;
Schulte-Oehlmann et al. 2001). Additionally, Brooks et al.
(2003) detected effects of fluoxetine on the midge Chironomus tentans. For this reason we assessed effects via sediment
exposure on the sediment-dwelling organisms Chironomus
riparius and Lumbriculus variegatus. Because sediments can
serve as a repository for many toxic compounds (Fiedler and
Rçsler 1993; Loeffler 2003), this study was designed to
investigate whether representatives of the benthic biological
community are affected by fluoxetine when exposed via
spiked sediments. Because fluoxetine has a log POW of 4.05,
its water solubility is not high. Therefore, accumulation in the
sediment is likely.
Materials and Methods
Test Substance
Fluoxetine (CAS 54910-89-3) was purchased as fluoxetine hydrochloride from Alltech Associates Inc. (State College, PA, USA).
Relevant physical and chemical properties are as follows: chemical
purity >99%, water solubility = 38.4 mg/l at 25C, log KOW = 4.05,
and vapor pressure = 8.9E-007 mm Hg (25C).
The compound exerts its effects by raising the serotonin level by
means of inhibiting neuronal and muscle 5-hydroxytryptamine (5-HT)
receptors, thereby increasing the serotonergic synaptic action. It is
mainly used for depression treatment and against premenstrual disorders (Wong et al. 1995). Because the drug also has tranquillizing
and mood-lightening effects, it is often used for the treatment of
nongenuine depression (Olfson et al. 1998).
Test Organisms
All tests were conducted as part of a diploma thesis (Hecker 2004). The
organisms used were the same as described by Oetken et al. (2005).
P. antipodarum is a small freshwater snail with shell heights up to 6
mm. The snails feed on plants and detritus. Due to parthenogenecity,
European populations consist almost exclusively of females.
The midge, C. riparius, usually breeds within 24 h after emergence.
Females extrude gelatinous egg clutches containing approximately
400 eggs into the water and the larvae hatch after 2–4 days. After four
instar stages, they molt to a pharate pupa and emerge.
The oligochaete L. variegatus is a common subject for sediment
toxicity tests (Egeler et al. 1997; Leppnen and Kukkonen 1998;
Phipps et al. 1993; West and Ankley 1998) and thus recommended by
the American Society for Testing and Materials (ASTM) as a standard
organism to be used in bioaccumulation and sediment studies (ASTM
1995).
Fifty-Six-Day Water Test with P. antipodarum
Sexually mature snails of the prosobranch P. antipodarum (shell
height >3.7 mm) were exposed to fluoxetine via water at a nominal
concentration of 0.64, 3.2, 16, 80, and 400 lg/L. For each concentration and the control, three replicates were used. The experiment
was conducted in a semistatic system with complete renewal of the
test medium every 48 h. The snails were kept in 1-L Erlenmeyer
flasks at 16 € 1C and a light–dark rhythm of 16:8 h. All snails were
fed with ground TetraPhyll daily ad libitum. At the beginning of the
experiment (day 0), 80 snails per replicate were exposed to fluoxetine
in the test vessels. The number of embryos (differentiated by individuals with and without a shell) of 20 individuals per replicate in the
brood pouch of each maternal snail was determined at days 14, 21, 28,
and 56. To count the embryos, the maternal snails were anesthetized
in MgCl2 (2.5%), and the shell was broken (for details, cf. Duft et al.
2003a, 2003b).
Twenty-Eight-Day Sediment Tests with C. riparius and
L. variegatus
The sediment tests were performed according to the OECD Guideline 218 (OECD 2004) except that kaolin was not mixed into the
sediment and ground leaves were used instead of peat moss as
carbon sources. Quartz sand was purchased from Quarzwerke Frechen (Germany). The grain size of test sediments was as follows:
90–125 lm, 1%; 125–180 lm, 27%; 180–250 lm, 57%; 250–
355 lm, 14%; 355–500 lm, 1%. For the Lumbriculus assay, 1.6%
(dry weight [dw]) pulverized alder leaves (Alnus glutinosa) were
used as the carbon source. In the experiment with C. riparius, 0.5%
(dw) pulverized leaves of stinging nettle (Urtica dioica) and alder
(Alnus glutinosa) were added. Thus, it was not necessary to feed the
animals during the experiments. The total content of organic carbon
in the sediment, per beaker, was 1.36% for L. variegatus and 0.85%
for C. riparius. In both assays, the light regime was 16:8 h light:dark
(light intensity 500–1000 lux) and the temperature was 20 € 1C.
For the life-cycle test with C. riparius, the artificial sediment was
spiked with fluoxetine in the following concentrations: 0.15, 0.38,
0.94, 2.34, and 5.86 mg/kg, expressed on a dry-weight basis. The
concentrations are referred to as treatment 0.15, 0.38, 0.94, 2.34, and
5.86, respectively. The experiments were conducted in 600-ml glass
beakers measuring 9 cm in diameter. The beakers contained 1 cm of
artificial sediment corresponding to a total of 100 g dw, covered
with 400 ml reconstituted water (Oetken et al. 2005). As fluoxetine
is easily soluble in water, no solvent was needed for spiking. A
stock solution of 4.91 mg fluoxetine hydrochloride in 100 ml deionized water was prepared. From this solution, the necessary
amount of fluoxetine was removed and diluted in 30 ml reconstituted water. The sediment was soaked with this solution and left
overnight to dry. After complete drying, 400 ml reconstituted water
were added without disturbing the sediment. Due to the low vapor
pressure of fluoxetine, it is unlikely that a significant amount of the
165
Effects of Fluoxetine on Aquatic Invertebrates
Table 1. The 56-day reproduction test with P. antipodarum
Measured fluoxetine concentrations
(lg/L)
Nominal fluoxetine concentrations
0h
24 h
48 h
72 h
Recovery after 72 h exposure (%)
Control
3.2
16
80
400
2
3
12
65
363
n.d.
2
12
66
317
n.d.
2
11
55
341
n.d.
2
11
58
345
—
62.5
68.8
72.5
86.3
Norfluoxetine (lg/L)
—
n.d.
n.d.
n.d.
13.0
Measured concentrations of fluoxetine and norfluoxetine directly and 24, 48, and 72 h after a change of test medium [detection limit in water:
1 lg/L; therefore, the lowest concentration (0.64 lg/L) was not detectable (n.d.)].
Data from Hecker (2004).
substance evaporated while drying. All tests were run with four
replicates, including a control. In each beaker, a glass Pasteur pipette
was fixed 2–3 cm above the sediment layer for gentle aeration. After
the end of the equilibration period, 20 first-instar larvae were placed
randomly in each test beaker. Emergence and sex ratio were recorded daily and emerged adults were removed. All midges from
one treatment were collected in a glass aquarium in which a water
beaker was placed. The number of clutches and the clutch size
(number of eggs per clutch) were counted as additional end points
next to the percentage of emerged midges, the mean emergence time
(EmT50) (i.e., the time at which 50% of the midges emerged), and
the sex ratio.
The reproduction test with L. variegatus was conducted using 500ml glass beakers that measured 8 cm in diameter and were covered by
a plastic screw cap (Oetken et al. 2005). Each beaker was filled with
40 g dw artificial sediment and 200 ml reconstituted water. In this
experiment, the same fluoxetine concentrations were used as in the
experiment with C. riparius. Ten worms of the same developmental
status were randomly inserted in each test beaker. For aeration, a glass
Pasteur pipette was fixed with the plastic cap 0.5 cm above the sediment layer. At the end of the test, the worms were removed from the
sediment and the number of worms as well as their biomass (dry
weight) were recorded as end points.
ment samples were centrifuged; the samples were then mixed with
100 ml methanol and extracted in an ultrasonic bath for 10 min. The
pore water was discarded.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 4.0 for
Windows (GraphPad Software, San Diego, CA). The no observed
effect concentration (NOEC)/LOEC values were determined by
analysis of variance (one-way analysis of variance [ANOVA]) followed by DunnettÕs post hoc test. In case of non-Gaussian distribution,
a Kruskal–Wallis test with DunnÕs post hoc test was used. Effects
concentrations (EC(x)) concentrations were calculated using a
LogNorm or Weibull nonlinear regression model (Kusk 2003).
Differences in EmT50 values were analyzed according to Sprague and
Fogels (1977).
Results and Discussion
Chemical Analysis
Chemical Analysis
The chemical analysis was performed by Medizinisches Labor Bremen GmbH. Water samples were taken directly after placing the snails
in fresh test medium and 24, 48, and 72 h after changing the test
medium. They were shipped to the analyzing laboratory in glass
bottles without being frozen. Samples were mixed with the internal
standard Perphenazin, then diluted in a 1:8 ratio and injected into a
high-performance liquid chromatography (HPLC) system. The analytes were separated using a HPLC column (Chromolith Speed ROD
C18 5 lm, 50 · 4.6 mm;, VWR/Merck). The mobile phase, a mixture
of acetonitrile and 5 mM acetic acid (36:64 v/v), was adjusted with
ammonia to pH 3.9. The flow rate was 1 ml/min. Subsequently, the
analyte was analyzed with a tandem mass spectrometer (ABI 4000
Mass Spectrometer; Applied Biosystems) turbo ion spray interface
using a positive multiple reaction monitoring (MRM) mode. At a
retention time of 2 min, separation of the fluoxetine molecule ion
occurred at 310.1 m/z and at 148.1 m/z for the fluoxetine fragment ion.
Given a retention time of 1.8 min, the values were 296.1 m/z and
134.1 m/z, respectively. In the analysis, the fragment ions were detected and quantified. Validation data from the laboratory were as
follows: In a linear array of 5–500 ll/min, the detection limit was
1 lg/L for water samples and sediment extracts. The serial precision
was 11.4% and the recovery rate was 111%. Water samples were
filtered before analysis and directly measured. Before analysis, sedi-
Table 1 summarizes the measured fluoxetine and norfluoxetine concentrations in the P. antipodarum assay directly after
the changing and spiking of the test medium (0 h) and 24, 48,
and 72 h after a water change. Because the detection limit was
1 lg/L, water from the 0.64-lg/L exposure group was not
analyzed. The data show that concentrations did not vary
exorbitantly during the cycle. Thus, snails were exposed to a
relatively constant concentration of fluoxetine in the assay.
The rate of recovery after an exposure of 72 h was between
62.5% (3.2 lg/L) and 86.3% (400 lg/L). Due to a mean fluoxetine recovery rate of 73.4% in water, the concentration of
the lowest treatment can be estimated at 0.47 lg/L. Table 2
shows that the recovery rates of fluoxetine are low when
sediment was used in the tests (C. riparius assay). The concentration in the water samples of the treatments varied from
<1 to 130 lg/L. The fluoxetine concentration of the sediment
samples ranged from 53 to 1120 lg/kg. Thus, between 18.1%
(2.34 mg/kg) and 35.3% (0.15 mg/kg) of the nominal concentrations could be detected analytically.
On day 28, a decrease of fluoxetine in both water and
sediment samples associated with a slight increase of the main
metabolite norfluoxetine was observed. Measured initial fluoxetine concentrations between 8.7% and 16.8% were still
G. Nentwig
166
Table 2. Life-cycle test with C. riparius
Day 0
Nominal concentrations (mg/kg)
Measured concentrations in sediment (mg/kg)
Control
0.15
0.38
0.94
2.34
5.86
Measured concentrations in water (lg/l)
Control
0.15
0.38
0.94
2.34
5.86
Day 28
FLX
% of nominal
NFL
0.001
0.053
0.091
0.217
0.423
1.12
—
35.3
23.9
23.1
18.1
19.1
n.d.
n.d.
0.003
0.004
0.009
0.037
n.d.
n.d.
3.00
9.00
29.0
130
—
—
—
—
—
—
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
FLX
n.d.
0.013
0.045
0.158
0.278
0.734
n.d.
n.d.
n.d.
1.00
6.00
18.0
NFL
n.d.
n.d.
0.003
0.009
0.017
0.063
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Detected concentrations of fluoxetine (FLX) and its metabolite norfluoxetine (NFL) in water and sediment at day 0 and day 28 (detection limit:
1 lg/L in water and 1 lg/kg dw in sediment; n.d. = not detectable).
Data from Hecker (2004).
present in the sediments. The low recovery might be due to
strong adsorption or covalent binding between substance and
sediment. If such a covalent binding did occur, the bound part
of the analyte would not have been detected in the analysis by
the applied extraction method.
Fifty-Six-Day Water Test with P. antipodarum
Potamopyrgus antipodarum has been shown to be a suitable
test organism for toxicity testing (Duft et al. 2003a, 2003b;
Oetken et al. 2005; Schulte-Oehlmann 1997). In this investigation, the snails were used to evaluate the reproductive toxicity of fluoxetine in an aqueous exposure assay. From the
beginning of the assay, almost all snails exposed to the highest
level of fluoxetine were immobile on the bottom of the test
beaker. A small percentage of the snails recovered and began
grazing on food after a few days. Up to 70 of the 80 snails per
replicate remained immobile, and at day 56, 100% mortality
occurred in all replicates exposed to the highest fluoxetine
level. The embryos in the dead snails, if present, were counted.
At the first evaluation on day 14, a significant reduction of the
mean embryo number was detected at nominal concentrations
of 80 and 400 lg/L, following a concentration response curve
(Figure 1A). This reduction was detected over the whole
exposure time. At day 56, the three highest fluoxetine treatment levels significantly reduced reproduction in P. antipodarum (Figure 1C). The number of embryos without a shell
was already significantly reduced at measured fluoxetine
concentrations of 11.5 lg/L (Figure 1B, p < 0.001) and
2.25 lg/L (Figure 1D, p < 0.001) at days 28 and 56, respectively, resulting in a NOEC of 0.47 lg/L and an EC10 of
0.81 lg/L, based on measured concentrations, at day 56.
Therefore, the endpoint—embryos without a shell—was more
sensitive to fluoxetine than the total number of embryos.
Selective serotonin reuptake inhibitors (SSRIs) increase the
action of the neurotransmitter serotonin (5-HT) by raising
serotonin concentrations in the synaptic cleft. A range of effects on aquatic invertebrates, mainly mollusks and arthropods,
caused by 5-HT and antidepressants has been reported (Avila
et al. 1996; Fong et al. 1998). Fong (1998) performed laboratory experiments with the zebra mussel Dreissena polymorpha to determine the effects of some antidepressants. After
fluoxetine application, spawning was observed in both sexes.
The LOEC was 155 lg/L for male mussels and 1.5 mg/L for
female mussels. Honkoop et al. (1999) confirmed these results
for a marine bivalve species, Macoma balthica, at a fluoxetine
concentration of 1 mg/L. In gastropods, spawning was not
reported after application of fluoxetine, but a SSRI-induced
cilia-driven rotational behavior of embryos in the freshwater
gastropod Physa elliptica was observed at nominal fluoxetine
concentrations of 309 lg/L and 3.09 mg/L (Uhler et al. 2000).
Avila et al. (1996) found an increased metamorphosis success
in laboratory cultures of the nudibranch Hermissenda crassicornis at serotonin concentrations of 1.76 and 17.6 mg/L. EC10
and NOEC values determined here show that effects of fluoxetine can occur at much lower concentrations than have
previously been reported.
Twenty-Eight-Day Sediment Test with C. riparius
As shown in Table 3, fluoxetine had no effect on emergence
except at the highest exposure level (5.86 mg/kg dw). Brooks et
al. (2003) evaluated the potential aquatic toxicity of fluoxetine
using the arthropods Ceriodaphnia dubia and C. tentans.
Fecundity of the crustacean was decreased at a concentration of
223 lg/L fluoxetine. In a 10-day sediment toxicity test with
C. tentans, a LC50 of 15.2 mg/kg dw was found (Brooks et al.
2003). A significant reduction in the growth of C. tentans occurred already at a LOEC of 1.3 mg/kg. If the reduction in
emergence of C. riparius is due to fluoxetine, the LOEC found
167
Effects of Fluoxetine on Aquatic Invertebrates
B
day 28 total number of
embryos (mean ± SD)
25
20
15
10
5
0
C
0.64 3.2
16
80 400
fluoxetine concentrations [µg/L]
25
day 56 total number of
embryos (mean ± SD)
15
10
5
0
C
0.64 3.2
16
80
400
fluoxetine concentrations [µg/L]
D
day 56 number of embryos
without shell (mean ± SD)
C
day 28 number of embryos
without shell (mean ± SD)
A
20
15
10
5
0
20
Fig. 1. The 56-day reproduction
test with P. antipodarum. Total
number of embryos (A, C) and
number of embryos without a shell
in the snails (B, D) after 28 and 56
days (mean € SD). Asterisks
indicate significant differences
compared to the control (Kruskal–
Wallis test with DunnÕs post
hoc test: *p < 0.05, **p < 0.01, ***p
< 0.001; C = control). Data from
Hecker (2004)
15
10
5
0
C
0.64 3.2
16
80
400
fluoxetine concentrations [µg/L]
C
0.64 3.2
16
80
400
fluoxetine concentrations [µg/L]
Table 3. Life-cycle test with C. riparius
Nominal concentrations
(mg/kg)
Emergence (%)
EmT50
males (days € SD)
females (days € SD)
Clutch size
(No.of eggs/clutch € SD)
No. of
clutches
Control
0.150
0.380
0.940
2.34
5.86
100
92.5
95.0
96.3
95.0
87.5
15.7
16.5
16.7
16.1
16.9
16.4
18.6
18.3
18.8
18.3
19.3
18.5
316
416
397
363
241
515
16
16
12
13
19
18
€
€
€
€
€
2.89
5.77
4.79
5.77
11.9*
€
€
€
€
€
€
0.090
0.230
0.170
0.150
0.220
0.130
€
€
€
€
€
€
0.210
0.170
0.130
0.330
0.180
0.130
€
€
€
€
€
€
214
246
243
123
192
194**
Mean emergence (€ SD), sex-specific mean emergence times (EmT50), clutch size (eggs per clutch), and numbers for all treatments (one-way
ANOVA with DunnettÕs post hoc test, *p < 0.05; **p < 0.01).
Data from Hecker (2004).
in our experiment would be 1.12 mg/kg, based on measured
values, and thus at a comparable concentration to that reported
by Brooks et al. (2003) for C. tentans. Regarding the EmT50 of
both males and females, no differences between the control and
the treatments were observed. Egg clutches exposed to the
highest fluoxetine concentration contained significantly more
eggs than the control (Table 3). However, because this effect
occurred at only one treatment level, the data do not allow
conclusions to be drawn about possible effects of fluoxetine on
reproduction in C. riparius. The increased number of eggs at
the highest exposure level could be due to an enhanced egg cell
proliferation rate. Williams and Herrup (1988) showed that
fluoxetine could accelerate cell proliferation. Therefore, the
increased clutch size could be due to fluoxetine. Further
experiments at higher concentrations would be necessary to
confirm a potential reduced emergence and increased clutch
size. However, higher sediment concentrations of fluoxetine
than employed in our study are beyond environmental
relevance.
Twenty-Eight-Day Sediment Test with L. variegatus
Fluoxetine effects on reproduction in L. variegatus resemble
an inverted U-shaped concentration–response curve (Table 4
and Figure 2). Starting at the lowest fluoxetine concentration,
the total number of worms increases, compared to the control.
For the treatments 0.94 mg/kg dw and 2.34 mg/kg dw, significantly more worms were found (18.8 and 16.5, respectively). At the highest exposure level, the mean number of
worms was lower (15.3). The mean number of juvenile worms
showed the same tendency as the total numbers, with significantly more worms than at 0.94 mg/kg dw. This could indicate
a stimulating effect on reproduction of L. variegatus at low
concentrations of fluoxetine, possibly caused by an increased
division rate in the worms. However, because the reproduction
rate in the control is low, this potential effect needs verification.
The observation could be explained either by a specific
effect on reproduction or by an unspecific stimulation of
G. Nentwig
168
Table 4. Number of worms in the 28-day sediment test with L. variegatus
No. of worms
Nominal concentrations (mg/kg)
Total
Control
0.150
0.380
0.940
2.34
5.86
11.3
15.5
14.3
18.8
16.5
15.3
€
€
€
€
€
€
1.26
1.73
2.63
1.89**
4.93*
0.957
Adult worms
Juvenile worms
7.75
4.75
6.00
2.50
6.75
6.00
3.50
10.8
8.25
16.3
9.75
9.25
€
€
€
€
€
€
3.20
1.71
2.16
1.73**
2.36
1.16
€
€
€
€
€
€
4.44
3.40
4.79
3.09**
7.14
2.06
A
25
day 28 total number of
worms (mean ± SD)
Data given as mean € SD. One-way ANOVA with DunnettÕs post hoc test: *p < 0.05; **p < 0.01.
Data from Hecker (2004).
20
L. variegatus. This might be due to a higher reproduction rate
with exclusively asexual reproduction under laboratory conditions (Brust et al. 2001). Accelerated proliferation could lead
to more and faster division of the worms. Because the test did
not match the validity criterion of a minimum of 20% reproduction in the controls, proposed by Egeler et al. (2005), the
results need confirmation in further experiments. In these assays, use of the sediment mixture proposed by Egeler et al.
(2005) should be considered. When repeating the experiments,
mixtures with and without kaolin should be compared to
determine the influence of kaolin on the bioavailability of
fluoxetine.
15
10
5
0
C
day 28 number of juvenile
worms (mean ± SD)
B
0.15
0.38
0.94
2.34
5.86
fluoxetine concentrations [mg/kgdw]
20
15
10
5
0
C
0.15
0.38
0.94
2.34
5.86
fluoxetine concentrations [mg/kg dw]
Fig. 2. The 28-day reproduction test with L. variegatus. Total number
of worms (A) and number of juvenile worms (B) after 28 days
(mean € SD). Asterisks indicate significant differences compared to
the control (one-way-ANOVA with DunnettÕs post hoc test: *p < 0.05,
**p < 0.01; C = control). Data from Hecker (2004)
physiological parameters in the sense of a general stress response. In the latter case, fluoxetine would not directly affect
reproductive mechanisms, but would constitute a stressor for
which L. variegatus compensates with increased reproduction.
This explanation is not likely due to the declining reproduction
rate at the highest fluoxetine concentration. Because annelids
have serotonergic neurons (Hessling et al. 1999), an increased
serotonin level might have specific effects on the test organism. Furthermore, because serotonin acts as a mitogenic factor,
fluoxetine might lead to an enhanced proliferation rate in
Environmental Risk Assessment of Fluoxetine
The European Agency for the Evaluation of Medicinal Products (EMEA) has proposed a scheme for environmental risk
assessment (ERA) for pharmaceuticals (EMEA 2005), based
on a two-phased, tiered assessment concept. Phase I consists of
a crude initial predicted environmental concentration assessment of the substance in surface water (PECsw). If the PECsw is
below 0.01 lg/L, no testing or evaluations will be required
unless the compound exhibits specific modes of action such as
endocrine activity. If the PECsw is above 0.01 lg/L, a Phase II,
Tier A environmental effect analysis should be performed.
Metcalfe et al. (2003) investigated surface water of the Little
River (Canada), measuring mean fluoxetine concentrations of
0.099 lg/L near a sewage treatment plant. Kolpin et al. (2002)
reported a median fluoxetine concentration of 0.012 lg/L in
American streams, indicating that the threshold value for
Phase II analyses according to EMEA (2005) is exceeded by
fluoxetine.
In the present study, the ratio between predicted environmental concentration and predicted no effect concentration
(PEC/PNEC) is 1.48, indicating a risk to the population level.
The calculation is founded on an EC10 of 0.81 lg/L (based on
measured concentrations) for reproduction in P. antipodarum
with an assessment factor of 100 (EU 2003) and using the
median fluoxetine concentrations of 0.012 lg/L from Kolpin
et al. (2002) as the PEC. The use of the assessment factor 100
according to EU (2003) is warranted because all test organisms
in the present study represent one trophic level (consumer).
Based on this calculation, it is likely that the drug poses a risk
to the survival of gastropod populations in the field. Sebastine
and Wakeman (2003) quoted a PEC/PNEC ratio of 14.2 for
fluoxetine, one order of magnitude higher. This supports the
Effects of Fluoxetine on Aquatic Invertebrates
evaluation of fluoxetine as an environmentally relevant substance. Significant effects on reproduction as a populationrelevant end point occurred at a measured concentration of
2.25 lg/L (nominal concentration = 3.2 lg/L). In the field,
other stress factors act on a population, and in this way, toxic
compounds might affect wildlife populations at even lower
concentrations than in the laboratory.
Acknowledgments. We thank Gabi Elter for excellent technical
assistance, the Center of Environmental Research (ZUF) at the University Frankfurt am Main for financial support, and two anonymous
reviewers for their valuable comments. Many thanks also to Karen
Nelson for the linguistic revision of this manuscript.
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