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Drug Chem Toxicol, Early Online: 1–9

! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2015.1029048

RESEARCH ARTICLE

Acute and chronic ecotoxicological effects of four pharmaceuticals

drugs on cladoceran Daphnia magna
Laira L. Damasceno de Oliveira1,2, Sara Cristina Antunes3,4, Fernando Gonçalves2,3, Odete Rocha5, and
Bruno Nunes2,3
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1
Post Graduate Program in Environmental Engineering Sciences, Engineering School of São Carlos, University of São Paulo, São Carlos, SP, Brazil,
2
Department of Biology, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal, 3Centre for Environmental and Marine Studies
(CESAM), University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal, 4Department of Biology, Faculty of Sciences, University of Porto,
Porto, Portugal, and 5Department of Ecology and Evolutionary Biology, Federal University of São CarlosSão Carlos, SP, Brazil

Abstract Keywords
The occurrence of pharmaceuticals in the aquatic environment has received increasing Acetaminophen, aquatic biota,
attention in recent years, as concerns have risen about their environmental persistence, chlorpromazine, diclofenac sodium,
biological activity and different effects toward nontarget organisms. Considering the ecotoxicity, propranolol, non-target
magnitude of concentrations (ng L1 to mg L1) and their often-specific modes of action, the organisms
assessment of physiological responses of exposed aquatic biota may provide significant
information regarding the potential ecological consequences of exposure to these contam- History
inants. The present study intended to assess the acute and chronic effects of four
For personal use only.

pharmaceuticals: acetaminophen, chlorpromazine, diclofenac sodium and propranolol in the Received 4 July 2014
cladoceran species Daphnia magna. Parameters such as immobility, total of offspring and Revised 19 February 2015
rate of population increase were analyzed. Results of acute exposures showed a consider- Accepted 10 March 2015
able variability of toxicity among pharmaceuticals, with the following ranking of toxicity: Published online 13 April 2015
diclofenac (EC50 ¼ 123.3 mg L1)5propranolol (EC50 ¼ 5.531 mg L1)5acetaminophen
(EC50 ¼ 2.831 mg L1)5chlorpromazine (EC50 ¼ 1.805 mg L1). The chronic toxicity data showed
the exertion of reproductive adverse effects. The compounds chlorpromazine and propranolol
caused a significant decrease in fecundity, and the rate of population increase parameter
suffered a significant decrease from 0.33 mg L1 to 0.128 mg L1 onwards, respectively. The
levels of exposure to which our test organism was acutely and chronically exposed were above
those already reported in the wild. Nevertheless, the extensive production, prescription and
release of pharmaceuticals drugs will continue to grow in the future, and consequently their
loadings to the environment can result in potential long-term ecological risks to aquatic biota.

Introduction sublethal concentrations. This chronic modality of exposure

can result in the irreversible alteration of key physiological
Pharmaceuticals such as analgesics, antibiotics, antiepileptics,
processes of individual organisms (Minguez et al., 2014).
antidepressants, b-blockers, blood-lipid regulators and contra-
Despite their overall ecotoxicological importance, little is
ceptives, are used in large quantities leading to their
known about possible counterparts of human target receptors/
appearance in the environment (Massarsky et al., 2011).
biomolecules of these compounds in many vertebrates, and
These xenobiotics are designed to comply with a set of pre-
even more so in invertebrates. Additionally, for some
requisites, such as a specific mode of action, pharmacological
pharmaceuticals, the specific mode of action is not well-
effectiveness, being slowly metabolized to act for longer
known, and often not only one, but different modes of action
periods. However, slow metabolism can also imply environ-
can be present.
mental persistence, when these substances reach the distinct
The ecotoxicological assessment of pharmaceuticals has
ecosystems (Brandão et al., 2013). Pharmaceuticals can thus
been mostly based on short-term experiments performed by
exert adverse effects on non-target organisms in many
standard tests according to existing guidelines (e.g. OECD)
different ways, potentially during their entire life cycle at
using test organisms from different trophic levels such as
algae, zooplankton, other invertebrates and fish (Kim et al.,
Address for correspondence: Laira L. Damasceno de Oliveira, 2009; Li, 2013). However, chronic toxicity data, or the
Universidade de São Paulo, Escola de Engenharia de São Carlos,
Avenida Trabalhador São-carlense, 400, São Carlos, SP, Brazil. Tel: bioaccumulation potential of pharmaceuticals in biota and
+00551633518384. E-mail: lairaoliver@yahoo.com.br food webs, are largely unknown. Considering that exposure to
 2 L. L. D. de Oliveira et al.
pseudopersistent substances (the degradation rates equals
their input into the environment) such as pharmaceuticals is
likely to occur for the entire life cycle of aquatic organisms, it
is of fundamental importance to address their chronic toxicity,
and the consequent effects in long term traits, such as growth
or reproduction rates.
The purpose of this study was to assess the acute and
chronic effects of four pharmaceutical drugs from different
pharmacotherapeutic classes: acetaminophen (analgesic, anti-
pyretic), chlorpromazine (antipsychotic), diclofenac sodium
(anti-inflammatory) and propranolol (antihypertensive), on
the standard freshwater species of cladoceran Daphnia
magna. This species is a common invertebrate biological
model that plays an important role in aquatic food webs by
serving as an intermediate between primary producers and
higher trophic levels (Flaherty & Dodson, 2005).
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/17/15
Acetaminophen (APAP, N-acetyl-p-aminophenol, para-
cetamol) is one of the world’s most widely sold non-
prescription drug, and is used for the relief of fevers and
headaches, being frequently detected in the aquatic environ-
ment (Antunes et al., 2013). These wide uses lead to its
classification as a class 2 compound – priority pharmaceut-
icals (Voogt et al., 2009). The presence of acetaminophen has
been reported in significant amounts; sewage treatment plant
(STP) effluents have loads of paracetamol from 20 ng L1
(Roberts & Tomas, 2006) to 4.3 mg L1 (Gomez et al., 2006),
and natural North American waters are also contaminated by
For personal use only.
this drug in levels up 10 mg L1 (Kolpin et al., 2002).
Contamination by paracetamol was also shown to occur in the
UK, since concentrations above 65 ng L1 were reported in
the Tyne River, UK (Roberts & Thomas, 2006).
Chlorpromazine has been detected at concentrations from 5
to 364 ng L1 in wastewater treatment plant influents from
psychiatric hospitals (Yuan et al., 2013), and below to
1 ng L1 in surface waters in UK (Roberts & Bersuder, 2006),
and from 0.9 to 2.6 ng L1 in Tajo River, Spain (Fernandéz
et al., 2010). Despite the environmental occurrence, no
toxicological classification was reported in pharmaceutical
databases. Diclofenac has been reported as one of the three
most frequently detected drugs in aquatic ecosystems (Santos
et al., 2010) and was classified as class 1 – High-Priority
Pharmaceuticals (Voogt et al., 2009). The presence of
diclofenac sodium has been reported to occur in relatively
high concentrations in sewage treatment plant effluents and
surface water up to 4.7 mg L1 and 1.2 mg L1, respectively,
and even in groundwater and drinking waters at concentra-
tions up to 380 ng L1 and below or just above 1–7 ng L1,
respectively (López-Serna et al., 2013; Vulliet et al., 2011).
Propranolol is a drug whose environmental presence requires
attention, since its PEC/PNEC ratio evidently exceeds 1, the
consensually adopted threshold that indicates potential envir-
onmental concerns (Ågerstrand & Rudén, 2010). Information
about the photodegradation of propranolol has been evaluated
as having a half-life of 16.8 days (Cleuvers, 2005).
Propranolol has been detected in STP effluents (Zhou et al.,
2009) at concentrations from 16 to 388 ng L1 and in surface
waters at levels up to 510 ng L1 and below 37 ng L1,
respectively (Kim et al., 2009). This pharmaceutical has also
been detected in hospital effluents at concentrations that can
reach 6.5 mg L1 (Lin & Tsai, 2009).
Drug Chem Toxicol, Early Online: 1–9
Material and methods
Test organism and culture conditions
Monoclonal cultures of Daphnia magna (clone A sensu Baird
et al., 1989) were maintained in the laboratory for several
generations in synthetic hard water medium –ASTM (ASTM,
1980; USEPA, 2002) – supplemented with an organic additive
(suspension extracted from Ascophyllum nodosum).
The cultures were maintained under a temperature of
20 ± 2  C and a photoperiod of 16L:8D and were regularly
renewed using neonates from 3rd to 5th broods. The
organisms were fed thrice weekly, with a ration of
3.0  105 cells m L1 day1 of the algae Pseudokirchneriella
subcapitata. This microalga was cultured in nonaxenic batch
cultures with Woods Hole MBL medium under the same
laboratory conditions described above for Daphnia magna.
Algae were regularly harvested while still in the exponential
growth phase (5–7 days old) and inoculated in fresh medium.
All experiments were initiated with neonates (524 hold), born
between the 3rd and 5th broods, derived from a healthy parent
stock.
Chemicals
All pharmaceutical drugs were purchased from Sigma
Aldrich, with purities 98%: acetaminophen (CAS: 32113-
41-0), chlorpromazine (CAS: 69-09-0), diclofenac sodium
(CAS: 15307-79-6) and propranolol (CAS: 318-98-9).
Acute toxicity tests
Independent experiments were used to assess the acute
toxicity of the four pharmaceutical drugs: acetaminophen,
chlorpromazine, diclofenac sodium and propranolol to
Daphnia magna. Methods for acute toxicity tests were
performed in agreement with standard protocols (ASTM,
1997; ISO, 1996; OECD, 2000), under the same laboratory
conditions described for rearing procedures.
The acute assays were conducted in glass tubes containing
10 mL of test solution and 10 mL of clean ASTM medium
(negative control). Twenty animals (524 h old) divided into
four groups of five animals per treatment concentration were
used. For all acute tests, a geometric range of 6–7 test
concentrations was obtained by diluting different pharma-
ceuticals with culture medium. Range-finding bioassays were
carried out to set appropriate dilutions to obtain EC50 values
with the best confidence interval. Final nominal concentra-
tions to be used in acute tests were: 1.2–9.0 mg L1 for
acetaminophen; 0.50–3.14 mg L1 for chlorpromazine; 52.0–
155.3 mg L1 for diclofenac sodium and 4.0–11.9 mg L1 for
propranolol. The test organisms were not fed during the
experiment, and after 48-h tests the immobile/dead organisms
were observed, counted and registered.
Chronic toxicity tests
Daphnia magna chronic assays were conducted based on the
agreement with standard protocols (ASTM, 1997; ISO, 2000;
OECD, 1998), for 21 days under the same temperature and
photoperiod regimes described for rearing procedures.
A semi-static design was employed using 10 randomly
 DOI: 10.3109/01480545.2015.1029048 Effects of pharmaceuticals drugs on D. magna 3
Figure 1. Percentage of immobilized organ-
isms of Daphnia magna exposed to different
concentrations of pharmaceuticals
(acetaminophen, chlorpromazine, diclofenac
and propranolol), after 48 hours exposure.
The EC50 values were calculated based on
mortality values, and corresponding 95%
confidence intervals (IC95%) were included.
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selected, individualized animals (524 h old) assigned to the Results

control (clean ASTM medium) and different concentrations
Figure 1 shows the results obtained in the acute toxicity tests,
tested (see Figure 1 for more information). Test vessels
indicating the calculated 48 h EC50 and their respective
consisted of glass beakers containing 50 ml of test solution.
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confidence intervals (CI95%) for the four pharmaceutical

Daphniids were transferred to newly prepared pharmaceut-
compounds tested. It was possible to observe a clear dose–
icals dilutions every other day, and were daily fed with their
response relationship, since increasing concentrations of the
respective P. subcapitata ration (3.0  105 cells m L1 day1).
studied pharmaceuticals caused increased immobility (%
For 21 days, the animals were checked daily for mortality and
immobility organisms). The data obtained for the different
reproductive state and, if neonates had been released, they
drugs tested showed an important variability, with the
were counted and immediately discarded. The following life-
following ranking of relative toxicity: chlorpromazine
history parameters were registered: mortality and total of
(EC50 ¼ 1.81 mg L1)4acetaminophen (EC50 ¼ 2.83 mg L1)
offspring. Survival and fecundity estimates were also used to
4propranolol (EC50 ¼ 5.53 mg L1)4diclofenac (EC50 ¼
compute the rate of population increase (r), which was
123.3 mg L1). Daphnia magna showed more sensitivity to
iterated from the Euler–Lotka equation:
X chlorpromazine when compared to the other pharmaceuticals
1¼ 1x mx ermx tested, with the lowest value of EC50 of 1.81 mg L1.
Additionally, the calculated EC10 values also showed a
where r is the rate of population increase (day1), x is the age similar trend (1.069 mg L1 for chlorpromazine;
class in days (0, . . . , n), lx is the probability of surviving to 1.88 mg L for acetaminophen; 4.34 mg L1 for propranolol;
1

age x, and mx is the fecundity per female at age x. Standard
errors for r were estimated using the jack-knifing technique
described by Meyer et al. (1986), thus allowing hypothesis
testing on this demographic parameter.
Statistical analysis
EC50 and EC10 (only for acute assays) values and corres-
ponding 95% confidence limits for the acute toxicity (immo-
bile organisms) and chronic tests (total offspring) for each
pharmaceutical drug were determined by nonlinear regression
analysis, fitting a logistic equation to the data using the
technique of least squares. The software Statistica 11.0
(Tulsa, OK) was used for this purpose. A one-way analysis of
variance (ANOVA), followed by a Dunnett test (if applicable),
was applied to each endpoint of the chronic assay to assess
statistical differences between the different pharmaceuticals
concentrations and the control (p50.05).
and 99.20 mg L1 for diclofenac). Calculated EC10 values
were considered as starting points for choosing the nominal
concentrations to be used during the chronic tests, in order to
avoid considerable mortality of tests organisms during
exposures.
The chronic toxicity data showed the existence of repro-
ductive adverse effects for chlorpromazine and propranolol
(Figure 2). These two pharmaceuticals caused a significant
decrease in the production of offspring when compared to the
control (one way ANOVA: MS ¼ 8626.5; F[4, 43] ¼ 15.71;
p50.001 and MS ¼ 6784.8; F[3, 33] ¼ 70.33; p50.001),
respectively. A reproductive EC50 of 0.285 mg L1 was
found for chlorpromazine, and an EC50 of 0.132 mg L1 was
found for propranolol. Survival and fecundity were integrated
in the intrinsic rate of population increase, which showed a
significant population decrease of D. magna for chlorpro-
mazine in a concentration of 0.33 mg L1 and for propranolol
in a level of 0.128 mg L1 onwards.
 4 L. L. D. de Oliveira et al. Drug Chem Toxicol, Early Online: 1–9
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Figure 2. Life history responses of Daphnia magna exposed for 21 days to acetaminophen, chlorpromazine, diclofenac and propranolol (x-axis, in
mg L1). Error bars correspond to standard error and * represents statistically significant differences (Dunnett test, p  0.05) between the different
pharmaceuticals concentrations and the control.
 DOI: 10.3109/01480545.2015.1029048
Differently to what was observed for chlorpromazine and
propranolol, the rate of population increase for acetamino-
phen no alterations was observed, however in the lowest
tested concentration (0.2 mg L1) a significant increase was
observed (one-way ANOVA: MS ¼ 0.118; F[6,63] ¼ 331.758;
p50.001). However, no significant reproductive impairment
was observed in the remaining concentrations for diclofenac
sodium, considering total offspring (one-way ANOVA,
MS ¼ 1154.085; F[5,50] ¼ 2.660; p ¼ 0.033) and rate of popu-
lation increase parameters (one-way ANOVA, MS ¼ 0.00187;
F[5,54] ¼ 1.410; p ¼ 0.235, respectively).
Discussion
Acetaminophen
Acetaminophen is consensually considered a toxic compound
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in higher doses, which in vertebrates (namely mammals) may
cause hepatotoxicity leading to liver failure and death (Hadi,
2013). In therapeutic doses, acetaminophen is predominantly
eliminated from vertebrates by conjugation (sulphation and
glucuronidation) (Xu et al., 2008). A small amount is partially
metabolized by the cytochrome P450 system, which leads to
the formation of N-acetyl-p-benzoquinoneimine (NAPQI).
NAPQI is detoxified by conjugation with glutathione (GSH)
and excreted as the cysteine or mercaptopurine acid conjugate
(Kavitha et al., 2011). However, in cases of excessive dosages
of acetaminophen, the highly reactive and electrophilic
For personal use only.
NAPQI can accumulate and, exert multiple toxic effects,
such as covalent modifications of thiol groups on cellular
proteins (Xu et al., 2008), elimination of ATP synthesis, DNA
and RNA damage, leading to membrane failure and
centrilobular necrosis of hepatocytes.
Toxicity of acetaminophen has been documented in
different animals and humans (Dave & Herger, 2012; Li,
2013; Xu et al., 2008). The comparison of acetaminophen
EC50 for Daphnia magna (2.831 mg L1) with previously
calculated values for other species can result in large
differences being reported (Nunes et al., 2014). Despite the
intrinsic differences observable among species, the import-
ance of EC50 values is the possibility of establishing direct
comparisons in terms of acute stress, and sensitivity towards a
given compound. It is thus possible to conclude that D. magna
was more sensitive than phylogenetically distinct aquatic test
organisms such as Scenedesmus subspicatus (EC50 of
134 mg L1), Lemna minor (EC50 of 429.9 mg L1),
Photobacterium phosphoreum (Microtox test, EC50 of
331 mg L1), Hydra vulgaris (410 mg L1), and Dugesia
japonica (150.8 mg L1) (Kuhn et al., 1989; Li, 2013; Nunes
et al., 2014). This was also verified for crustacean species,
such as Moina macrocopa (48 h LC50 of 56.34 mg L1; Kim
et al., 2012) and Hyalella azteca (72 h LC50 of 7.7 mg/kg;
Gómez-Oliván et al., 2012).
The obtained 48 h EC50 is comparable to the 24 h EC50
reported by Dave & Herger (2012) for Daphnia magna.
However, the calculated values are slightly below the reported
literature values, showing that important factors such as
culture and experimental conditions (feeding, temperature,
photoperiod, medium and hardness of water) can have a
decisive contribution to the variability of results. Illustrative
cases were show by Kuhn et al. (1989) and Kim et al. (2007),
Effects of pharmaceuticals drugs on D. magna 5
with values of EC50 for D. magna of 9.2 and 11.85 mg L1,
respectively, significantly above those calculated for this
present study, but in the same order of magnitude. These
results show that this cladoceran can be a sensitive species for
acetaminophen effects, being an important test organism in
ecotoxicity assays to determine the effect of pharmaceutical
drugs. However, acute ecological risks posed by acetamino-
phen may be negligible, since the environmental concentra-
tions already reported are generally several orders of
magnitude lower than the effective concentrations used.
Some authors reported the presence of acetaminophen in
STP effluents at concentrations below to 20 ng L1 (Roberts
& Tomas, 2006) to 4.3 mg L1 (Gomez et al., 2006), up to
10 mg L1 in natural waters in USA (Kolpin et al., 2002), and
above 65 ng L1 in the Tyne River, UK (Roberts & Thomas,
2006). Even considering that acetaminophen can remain in
bodies of water without degradation for 15 days (Lin & Tsai,
2009), the reported adverse effects were only found for high
levels of contamination that are not likely to be attained under
realistic conditions. Consequently, our data concerning acute
toxicity of this substance is of poor ecological relevance, but
very important regarding the protection of wildlife, not
presenting considerable risks to aquatic organisms.
Concerning chronic toxicity data, the pharmaceutical para-
cetamol in tested concentrations (0.2–1.25 mg L1) did not
cause population effects for the studied species. However,
Nunes et al. (2014) observed that, in concentrations from
4.0 mg L1 to 17.8 mg L1, this pharmaceutical caused signifi-
cant decrease in the rate of population increase in D. magna and
D. longispina, respectively. Crustacean species seem particu-
larly sensitive to acetaminophen exposure, since this compound
appears to be involved in endocrine disruption, by interference
with moulting process (Fanjul-Moles & Gonsebatt, 2011;
Rodrı́guez et al., 2007). The alkyphenolic structure of
acetaminophen be may be responsible for this effects, since
this pharmaceutical belongs to a group of compounds well-
known for their endocrine effects in crustaceans (Rodrı́guez
et al., 2007), which exhibit anti-ecdysteroidal activity
(LeBlanc, 2007). Though, further studies are needed about
the potential endocrine disruption of paracetamol and their
long-term ecological consequences on aquatic biota, including
crustaceans. As shown by Kim et al. (2012), for concentrations
above (0.64 to 51.50 mg L1) those used in our study no
reproduction related effects were reported for D. magna.
However, despite the low possibility of occurrence of different
effects in the reported levels, the impacts of acetaminophen on
survival and reproduction of non-target organisms, exposed via
environment, is a possibility.
Chlorpromazine
Chlorpromazine (CPR) was the first modern antipsychotic
drug released in 1952 and approved by the Food and Drug
Administration in 1954 (Kane & Correll, 2010). In the human
body, CPR is metabolized to phase I and phase II metabolites,
some of them being responsible for the pharmacological
activity. However, it was shown that mutations in the human
cytochrome P450 2D6 isoenzyme lead to the excretion of un-
metabolized chlorpromazine (Zhou, 2009). Despite being
generally safe under therapeutic regimes, toxicity against
 6 L. L. D. de Oliveira et al.
aquatic organisms has been reported for several standard test
organisms. The acute EC50 values obtained for chlorpromaz-
ine (1.805 mg L1) with D. magna are slightly above those for
aquatic organisms such as the duckweed plant Lemna minor
(48-h EC50 value of 0.92 mg L1), the protozoan Spirostomum
ambiguum (24-h LC50 value of 0.5 mg L1) and the fresh-
water goldfish Carassius auratus (96-h EC50 value of
0.32 mg L1) (Li et al., 2008; Nałecz-Jawecki et al., 2008).
Other studies about chlorpromazine ecotoxicity are of diffi-
cult extrapolation and comparison to our results because the
endpoints are very different (Arkhipova et al., 2006; Hui-qin
et al., 2011; Munro et al., 1986; Rihel et al., 2010). The work
conducted by Arkhipova et al. (2006) with the species
terrestrial pulmonate mollusk Helix lucorum showed that a
prolonged (chronic) treatment with this compound led to a
significant increase in the pneumostoma closure time, as well
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as changes in motor behavior, with a decrease in the rate of
locomotion. However, these effects were only observed at
higher levels of chlorpromazine compared with the present
study. The developmental neurotoxicity on zebrafish (Danio
rerio) embryos and larvae were also affected by chlorpro-
mazine exposure (5 mg L1), as demonstrated by Hui-qin
et al. (2011). In addition to this parameter, Selderslaghs et al.
(2013) showed that these antipsychotics affected the swim-
ming activity of larvae and also decreased the activity of
zebrafish embryos, eleutheroembryos and larvae.
Nevertheless, in our study chlorpromazine provoked
For personal use only.
significant population effects, with dose-dependent reductions
in the total of offspring and rate of population increase
(Figure 1). According to Trautwein & Kümmerer (2012), only
a limited amount of data on the environmental fate and effects
of chlorpromazine is now available. However, considering the
lack of studies about its mode of action and population effects
in crustaceans, our results are extremely significant, since
they show the potential of chlorpromazine to exert ecological
effects. Despite the low ecological relevance of the chlorpro-
mazine range level used in our study, the potential long-term
ecological risks to aquatic biota should be considered because
no one can anticipate the levels of this compound in the wild.
Diclofenac
Diclofenac is a potent nonsteroidal anti-inflammatory drug
(NSAID) that has been used in the treatment of pain,
inflammation and joint stiffness. Similar to all NSAIDs,
diclofenac acts by reversible or irreversible inhibition of one or
both isoforms of the cyclooxygenase enzymes (COX-1 and
COX-2) involved in the synthesis of different prostaglandins
from arachidonic acid (Vane & Botting, 1998). Despite not
being entirely clear for the majority of ecologically relevant
organisms, this pharmaceutical is considered to be one of the
most relevant compounds that may exert toxic effects on non-
target organisms belonging to different biological organiza-
tional levels (Cleuvers, 2003; Ferrari et al., 2003). Diclofenac
EC50 values for D. magna showed that, in comparative
terms, this species can be less sensitive than other standard
tests such the phytoplanktonic species Pseudokirchneriella
subcapitata (Quinn et al., 2011), the diatom Desmodesmus
subspicatus (Cleuvers, 2004), the macrophyte species Lemna
minor (Quinn et al., 2011) and the bacteria Vibrio fischeri
Drug Chem Toxicol, Early Online: 1–9
(Zhang et al., 2008). This was also observed for species of
crustaceans, such as Brachionus calyciflorus, Ceriodaphnia
dubia and Thamnocephalus platyurus and fish Danio rerio
(Ferrari et al., 2003). On the contrary, D. magna is less
sensitive to diclofenac exposure than the crustacean Moina
macrocopa (Lee et al., 2011). The observed acute EC50 for
D. magna, 123.3 mg L1, was about two orders of magnitude
above the data published by Cleuvers (2003) (68 mg L1).
In chronic toxicity studies, no significant reproductive
impairment was observed for organisms exposed to diclofe-
nac, considering total of offspring and rate of population
increase parameters. However, the work conducted by Sarma
et al. (2013) showed that the rate of population increase for
the rotifer Plationus patulus and the cladoceran Moina
macrocopa was negatively affected by diclofenac at the
levels from 1.56 to 25 mg L1. As reported by Lee et al.
(2011), diclofenac exposure led to significant reduction in the
number of young per female, number of young per brood and
the population growth rate of D. magna, in concentrations
(0.93–25 mg L1) lower than those used in our study.
According to Cleuvers & Heinrichs (2008) and Lee et al.
(2011), the chronic EC50 values obtained for D. magna were
50.5 and 23.8 mg L1, respectively, which are several orders
of magnitude higher than those detected in the aquatic
environment; therefore, direct impact due to this pharma-
ceutical is not expected to occur in the wildlife. Thus, further
studies about adverse effects on aquatic non-target species are
needed for a better understanding about the consequences of
exposure to diclofenac in aquatic ecosystems.
Propranolol
Beta-blockers, including propranolol, act through the com-
petitive inhibition of b-adrenergic receptors, which are
involved in many physiological functions, including the
regulation of heart rate and oxygen, vasodilatation and
bronchodilation in mammals (Fent et al., 2006). Propranolol
is a nonselective b-adrenergic receptor antagonist that func-
tions as a 5-hydroxytryptamine (5-HT) serotonin receptor
antagonist in vertebrates (Alexander & Wood, 1987). As
aquatic invertebrates can have adrenergic receptors that are
distinct from mammals, different potential impacts on these
organisms may be expected. Data from literature show that
propranolol, of all the b-blockers investigated, is the most
toxic to aquatic organisms (Fent et al., 2006; Küster et al.,
2009). The potential adverse ecological effects of propranolol
have been studied on aquatic organisms such as cladocerans
species Daphnia lumholtzi and Ceriodaphnia dubia (Huggett
et al., 2002); macrophyte Lemna minor (Cleuvers, 2003), fish
Oryzias latipes (Kim et al., 2009) and mussel Mytilus
galloprovincialis (Franzellitti et al., 2011). The acute EC50
for propranolol (5.531 mg L1) are close to the ones already
reported in the literature for other crustacean species, namely
Daphnia pulex (Lilius et al., 1995), for the diatom species
Desmodesmus subspicatus, and the planarian Dugesia japon-
ica (Cleuvers, 2005; Li et al., 2013). Alternatively, D. magna
was less sensitive than other species, such as protozoan
Spirostomum ambiguum (EC50 of 1.77 mg L1), crustacean
Ceriodaphnia dubia (EC50 of 1.51 mg L1) and Brachionus
calyciflorus (EC50 of 2.59 mg L1), and larvae of the fish
 DOI: 10.3109/01480545.2015.1029048
Danio rerio (EC50 of 2.48 mg L1) (Calleja et al., 1994;
Ferrari et al., 2004; Nałecz-Jawecki et al., 2008; Sun et al.,
2014). The obtained results for EC50 are one order of
magnitude lower than those obtained for macrophytes (e.g.
Lemna minor; Cleuvers, 2005), bacteria (e.g. Vibrio fischeri;
Ferrari et al., 2004), other crustaceans (e.g. Hyalella azteca
and Thamnocephalus platyurus; Hugget et al., 2002; Calleja
et al., 1994, repectively), and fish (e.g. Oryias latipes; Kim
et al., 2009). Propranolol EC50 values reported in the
literature for D. magna covers a high range from 1.6 to
15.9 mg L1 (Calleja et al., 1994; Huggett et al., 2002; Lilius
et al., 1995). Considering the levels of propranolol required
for exertion of acute effects in aquatic biota, it is not likely
that this substance can compromise the survival of D. magna.
Moreover, the mechanism of action of propranolol and its
metabolites in invertebrates has not been investigated so far.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/17/15
Thus, differences in receptor types could potentially be
mediating the differential toxic response, but no data have
been published so far for lower organisms (Fent et al., 2006).
However, one must not discard the potential exertion of
chronic effects by propranolol, resulting in deleterious long-
term effects. This study showed that the pharmaceutical
propranolol caused a significant decrease in fecundity and
the rate of population increase for D. magna. Likewise,
Dzialowski et al. (2006) reported significant decreases in other
physiological parameters, such as metabolic and heart beating
rates that resulted in significant reductions in dry body mass,
For personal use only.
thus reflecting growth impairment. Similarly, Campbell et al.
(2004) and Stanley et al. (2006) reported decreased heart rate in
D. magna exposed to different concentrations of propranolol
(29.6 mg L1 and 2.97 mg L1, respectively). Dzialowski et al.
(2006) suggests that, due to the depression of heart rate caused
by blockade of b-receptors by propranolol, a direct conse-
quence is decrease in metabolism. Several other studies
(Ericson et al., 2010) also demonstrated alterations in the
metabolism of aquatic organisms caused by propranolol. This
compound caused a reduction of metabolism in Baltic Sea blue
mussels, Mytilus edulis trossulus, reflected by significant
reduction in growth, in the abundance of byssus threads, and in
byssus strength. Other effects, such as increased respiration
and excretion rates, were also observed (Oskarsson et al., 2014)
for this mussel species. Similar results were obtained for other
aquatic organisms, since exposure to propranolol affected the
metabolism of Chironomus riparius, causing a decrease in
growth and biomass, as demonstrated by López-Doval et al.
(2012). The effects of propranolol on aquatic organisms
(specifically, the Mediterranean mussel Mytilus galloprovin-
cialis) are related to the interference in the cAMP dependent
pathway, which can explain disorder in neuro-endocrinological
metabolic functions that are vital for this organism (Franzellitti
et al., 2011). According to these authors, the changes in cAMP
levels in Mytilus galloprovincialis were correlated with
changes in PKA activities, which decreased in digestive
glands, but increased in mantle/gonadal tissue. This work
successfully verified that even the low concentrations of
b-blockers already reported for the aquatic environment can
impact mollusks physiology. However, considering that
b-blockers may cause different impacts in reproduction,
growth and heart rate/metabolism in some species, along
with the lack of studies that explain these putative toxicity
Effects of pharmaceuticals drugs on D. magna 7
mechanisms and mode of action in crustaceans, it is only
possible to suggest the occurrence of a similar pathway in
D. magna that justifies the obtained reproductive results.
Organisms such as Ceriodaphnia dubia and Hyalella
azteca (Huggett et al., 2002) showed similar responses after
long-term exposure to propranolol, with decreases in repro-
duction at concentrations of 0.25 and 0.1 mg L1, respect-
ively. This latter study also reported reproductive impairment
caused by this compound in vertebrates (fish Oryias latipes)
through non-significant decreases in egg production, in
growth rate, and in sex steroid hormonal levels. However,
according to the criteria proposed by the authors, this type of
biphasic dose-response relationship has previously been
observed in adrenergic receptors, and exposure to low
treatment levels can result in opposite responses of those
registered for higher concentrations, that are mediated via the
alteration of hormonal levels.
Conclusions
– Despite the somewhat high levels of acetaminophen that
were selected for acute and chronic exposures (consid-
erably higher than those reported for aquatic ecosystems),
it was not possible to observe effects on the parameters
studied in D. magna.
– The results obtained for chlorpromazine and propranolol
showed to be more significant, because of the impact on
the parameters survival and reproduction of D. magna.
However, it must be emphasized that the levels of
chlorpromazine that caused deleterious effects were in
the order of mg L1, while the concentrations detected in
most freshwater ecosystems are in ng L1.
– Exposure to propranolol yielded similar results, since the
observed alteration only occurred for high levels of
exposure, that are not likely to be attained in the wild.
Some studies showed the potential of propranolol to
cause adverse effects, at least in some species and for the
low concentrations of b-blockers already reported for the
aquatic environment.
– The results obtained for diclofenac allowed concluding
that this drug was the most harmless among those
studied, since the environmental levels of this pharma-
ceutical do not pose a direct potential threat to aquatic
wildlife. Levels of exposure to which organisms were
acutely and chronically exposed, and were able to cause
significant effects, were several orders of magnitude
higher than those already reported in the wild.
Declaration of interest
The authors acknowledge the Brazilian Research Council
(CNPq) for the financial support provided for this research
project (No. 245436/2012-0). This work was supported by
European Funds through COMPETE and by National Funds
through the Portuguese Science Foundation (FCT) within
project PEst-C/MAR/LA0017/2013.
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