Science of the Total Environment 609 (2017) 715723

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Effects of exposure to pharmaceuticals (diclofenac and carbamazepine)

spiked sediments in the midge, Chironomus riparius
(Diptera, Chironomidae)
Elena Nieto a, Carmen Corada-Fernndez b, Miriam Hampel c, Pablo A. Lara-Martn c,
Paloma Snchez-Argello d, Julin Blasco a,
a
Instituto de Ciencias Marinas de Andaluca (ICMAN-CSIC), Campus Universitario Rio San Pedro, 11519 Puerto Real, Spain
b
Facultad de Ciencias del Mar y Ambientales, Universidad de Cdiz (UCA), Campus Universitario Ro San Pedro, 11510 Puerto Real, Spain
c
Instituto Universitario de Investigacin Marina (INMAR), Campus de Excelencia Internacional del Mar (CEIMAR), Universidad de Cdiz, Av. Repblica Saharaui s/n, 11510 Puerto Real, Cdiz, Spain
d
Laboratorio de Ecotoxicologia, Departamento de Medio Ambiente, INIA, Ctra, A Corua km 7, 28040 Madrid, Spain.

H I G H L I G H T S G R A P H I C A L A B S T R A C T

The effect of sediment sorbed pharma-

ceuticals (diclofenac and carbamaze-
pine) has been assessed
Chironomus riparius, rst instar stage
has been exposed in a long-term
experiment- to pharmaceutical spiked
sediments
Growth, development, sex-ratio and
emergence have been selected as end-
points
Adverse effects of both pharmaceuticals
have been reported at the exposure con-
ditions
Chronic exposure tests are useful to as-
sess the environmental risk of these
compounds

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

Article history: Human and veterinary pharmaceuticals and degradation products are continuously introduced into the environ-
Received 17 April 2017 ment. To date, there is a lack of information about the effects of pharmaceuticals in spiked toxicity tests with non-
Received in revised form 19 July 2017 target organisms. In this study, we have evaluated the effects of exposure to two common pharmaceuticals in the
Accepted 19 July 2017
midge Chironomus riparius in spiked sediment experiments. The selected pharmaceuticals are the nonsteroidal
Available online xxxx
anti-inammatory drug (NSAID): diclofenac (DF) and the anti-depressant drug carbamazepine (CBZ). In order to as-
Keywords:
sess the effects of the pharmaceuticals, a chronic toxicity test with the midge was carried out. The endpoints survival,
Pharmaceuticals growth and developmental stage by means of biomass, were measured after 10 days, and emergence rates and sex-
Diclofenac ratio (male/female) were measured after 21 days of exposure. Signicant mortality was observed in organisms at day
Carbamazepine 10 with a 40% of larvae surviving in the highest exposure concentration of CBZ. DF decreased the emergence ratio
Sediment toxicity with respect to the controls in organisms exposed at concentrations of 34.0 gg1 whereas CBZ reduced the growth
Chronic toxicity of the midges (30,6% with respect to the control) and induced a signicant change in sex-ratio at concentrations of
Chironomus riparius 31.4 gg1. The results obtained in the present study indicate possible adverse effects on aquatic invertebrates,
which should be taken into account for environmental risk assessment of pharmaceutical compounds in sediments.
2017 Elsevier B.V. All rights reserved.

Corresponding author at: Instituto de Ciencias Marinas de Andaluca (ICMAN-CSIC), Spain.

E-mail address: julian.blasco@csic.es (J. Blasco).

http://dx.doi.org/10.1016/j.scitotenv.2017.07.171
0048-9697/ 2017 Elsevier B.V. All rights reserved.
716 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723
1. Introduction
Due to globally increasing consumption, human and veterinary
pharmaceuticals are being found in several environmental matrices in
the majority of countries around the world (Daneshvar et al., 2010;
Sim et al., 2010; Osorio et al., 2012; Writer et al., 2013; Valds et al.,
2014). The occurrence of pharmaceuticals in the environment has
only relatively recently become an issue of concern, with the develop-
ment of sensitive analytical tools that made possible the detection at
low concentrations. This occurrence has been acknowledged as poten-
tially hazardous to ecosystems (Kolpin et al., 2002; Fent et al., 2006).
Although these compounds are being detected at concentrations in
the range of ngL1 to gL1 (Farr et al., 2001; Sacher et al., 2001;
Gros et al., 2006; Martnez Bueno et al., 2007; Lara-Martn et al.,
2014), the occurrence in the aquatic environment can affect water qual-
ity and potentially impact drinking water supplies, ecosystems and
human health (Yuan et al., 2009; Sirs and Brillas, 2012), particularly
since pharmaceutical compounds are designed to have a specic acute
effect in the patient. However, little is known about the potential chron-
ic effects on wildlife. In order to assess the potential effects that the oc-
currence of pharmaceuticals could have on wildlife, we have selected
two of the most commonly found pharmaceuticals in water bodies:
the non-steroidal anti-inammatory drug (NSAID) diclofenac (DF), as
well as the anti-epileptic and mood stabilizing drug carbamazepine
(CBZ). Due to previously observed effects in non-target organisms
(Ericson et al., 2010; Oviedo-Gmez et al., 2010; Lee et al., 2011; Schmidt
et al., 2011), DF has already been included in the EU watch list of sub-
stances as possible candidate to be part of the priority list of substances
(Directive 2013/39/EU, 2017). Likewise, CBZ has been proposed as
marker for anthropogenic contamination (Clara et al., 2004) due to its
poor elimination rate in waste water treatment plants (WWTPs) and
its frequent occurrence in aquatic environments (Gros et al., 2009).
Once discharged into the sewage system, mainly by excretion, these
compounds generally reach WWTPs where they are partly or entirely
removed. For CBZ, the average removal rate in WWTP is low and varies
between 7 and 8% (Heberer, 2002a; Clara et al., 2004), whereas for DF
the removal rate is around 2239% (Paxus, 2004; Bendz et al., 2005).
Concentrations of these compounds have been analyzed in WWTP
efuents with values comprised between 0.01 and 5.92 gL 1 and
0.10 and 0.8 gL1, for DF and CBZ, respectively (llers et al., 2001;
Martnez Bueno et al., 2007; Gros et al., 2009). In freshwater rivers, DF
and CBZ have been found at concentration ranges between 0.02 and
5.45 gL1 and 0.001 and 3.7 gL1, respectively (llers et al., 2001;
Drillia et al., 2005; Gros et al., 2009). However, only little information
is available about the concentration levels of these pharmaceuticals in
sediments. Concentrations of 2.68 and 8 ngg1 have been reported
for CBZ and DF, respectively in river sediments (Lfer and Terner,
2003; Silva et al., 2011), and Vazquez-Roig et al. (2010) reported CBZ
concentrations from 1.81 to 6.85 ngg1 in sediments of marsh areas.
Aquatic ecosystems near to municipal and industrial efuent dis-
charges generally present sediments that contain a great variety of
chemical contaminants (Liber et al., 1996). Sediment sorbed contami-
nants in general can affect benthic organisms via interstitial and overly-
ing water (Gilroy et al., 2012) as well as through ingestion of sediment
particles (Soares et al., 2005a, 2005b). Although not being lethal, an or-
ganism's ability to function normally in an ecosystem may be impaired
at sublethal contaminant concentrations (Gerhardt et al., 2002). The
freshwater macroinvertebrate Chironomus riparius, is a common
midge in freshwater ecosystems and plays an important role in the
aquatic food chain being a major food source for both sh and other
macroinvertebrates (Lee and Choi, 2007). It is highly sensitive to the
presence of environmental contaminants and is commonly employed
in standard tests to assess the toxicity of sediment-associated contami-
nants (ASTM, 2005). C. riparius larvae pass through four instars, which
are dened as developmental stages of arthropods between each
moult, until sexual maturity is reached (Allaby, 2006). Thus, the juvenile
and pupal stages are spent in the sediments feeding on organic detritus
making them a relevant stage for assessing sediment contamination.
Towards the end of the fourth instar, the larvae cease feeding and pupa-
tion begins (Benoit et al., 1997). The emergence occurs immediately
after pupae swim to the water surface (Environment Canada, 1997)
being this development stage an useful endpoint to determine effects
in the development of the organisms. Test procedures with C. riparius
are widely standardized (ASTM, 1997, 2005; OECD, 2004a, 2004b,
2010) and the organism has been widely used in short-term assess-
ments of contaminated sediments, mainly due to its easy culture and
handling under laboratory conditions and the fact that it remains in
close contact with the sediment during larvae development (Benoit
et al., 1997).
In the past years, the number of studies that have reported the neg-
ative effects of pharmaceutical compounds on benthic macroinverte-
brates from contaminated sediments employing midges has increased
signicantly (Oetken et al., 2005; Nentwig, 2007; Snchez-Argello
et al., 2009; Gilroy et al., 2012; Lpez-Doval et al., 2012; Heye et al.,
2016) underlining the rising concern about the presence of this kind
of compounds in the environment by the general population and regu-
latory organisms. Among the analyzed responses in these studies, the
authors observed decrease of emergence, reduced growth, increase of
the biomass and increase of the female/male ratio in spiked sediment
exposure experiments at low concentrations with different
pharmaceuticals.
On the background of the current information on the effects of sed-
iment associated pharmaceuticals, this study aims to address current
knowledge gaps related to the sublethal effects of sediment sorbed
pharmaceuticals on non-target organisms. For this purpose, we have se-
lected the freshwater midge, C. riparius, due to its high sensitivity and
the fact that it undergoes part of its life-cycle within the sediment and
two model pharmaceutical compounds (DF and CBZ) on the basis of
the high consumption rates and presence in WWTP efuents (Kolpin
et al., 2002; Roberts and Thomas, 2006; Carballa et al., 2008). The expo-
sure route was through spiked sediments and the analyzed responses
included: survival, growth (biomass) and development stage after
10 days of exposure, as well as emergence rate, cumulative emergence
and sex-ratio (male/female) at days 1521, of the surviving organisms.
This study aims to contribute to ll the gap of information about the im-
pact of these two pharmaceuticals sorbed on sediments for their consid-
eration in sediment quality guidelines.
2. Material and methods
2.1. Chemicals
DF (CAS no. 15307-86-05) and CBZ (CAS no. 298-46-4) were pur-
chased from Sigma-Aldrich (St. Louis, USA).
2.2. Organisms
C. riparius were obtained from our laboratory culture at the facilities
of the Institute of Marine Sciences of Andalusia following the recom-
mendations described by OECD guidelines 218 and 219 (OECD, 2004a,
2004b) and Snchez-Argello et al. (2005). Briey, C. riparius were allo-
cated in glass aquaria with dechlorinated water, under static ow and
gentle aeration, a photoperiod of 16:8 h (light:dark) and a temperature
of 20 1 C. Egg masses were placed in a glass beaker and green algae
(Chlorella vulgaris) were added ad libitum. After hatching (23 days),
b48 h old larvae were transferred to 25 L glass aquaria containing
sandy sediment. On the 7th day, larvae were removed and placed into
a clean rearing container which was covered with plastic mesh to
avoid escape of the emerging adults. Commercial sh food, Tetramin,
was added to the culture chamber three times a week. Water was re-
placed weekly. Under these conditions adult emergence occurred on
the 1315th day. Emerging adults were left in the rearing aquaria to
 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723
permit mating and oviposition (23 days). After removal of adults by as-
piration, egg ropes were collected and then used to initiate the next
generation.
2.3. Sediment spiking
Sediments were collected from a pristine site in the Guadalete river
(364754.93N-51953.74W). Sediment was transported to the labo-
ratory and stored at 4 C in the dark until spiking. Before spiking, the
sediment was sieved through a 2 mm mesh and homogenized. A por-
tion of the sediment was removed to be used in the physicochemical
characterization (humidity 13.3%; loss of ignition 3.2% and organic car-
bon 1.10%). Humidity was measured on basis of the UNE 77311 proce-
dure (UNE, 77311, 2000), organic carbon according to Gaudette et al.
(1974) modied by El Rayis (1985) and loss of ignition was estimated
in a mufe furnace at 550 C and gravimetric determination. Sediment
was then frozen in liquid nitrogen over 24 h to remove indigenous mac-
rofauna. Sediment was spiked adapting the method described by Gilroy
et al. (2012) with 0.2% (v/v) methanol or DMSO in the case of DF and
CBZ, respectively. A varying volume of each solution was added in in-
creasing concentrations of each compound to achieve the targeted sed-
iment exposure concentrations (0.12; 1.2; 12.0; 30.0 and 60 gg1).
Sediments were mixed three times a day during 72 h, allowed to rest
for further 7 days at room temperature and stored at 4 C until the be-
ginning of the experiment, in order to provide an adequate mixing
while minimizing the biodegradation of the compounds (ASTM, 2004).
2.4. Experimental design
Bioassays were carried out adapting the standard procedure ASTM E
1706-05 for long-term sediment toxicity tests. After 10 days survival,
growth and developmental stage, and during day 15 to day 21, adult
emergence and sex-ratio were assessed. For each compound, seven con-
ditions (sediment control + solvent control + ve concentrations), and
six replicates (three for survival, growth and development stage and
three for adult emergence and sex-ratio) were used in each test. On
the day before the start of the experiment, 80 g wet weight (w/w)
of spiked sediment and subsequently 320 mL of dechlorinated
water were added to each beaker. After a period of 24 h to allow
the settling of the sediment, twenty larvae of C. riparius (b3-d old)
were added to each replicate. Overlying water was renewed every
day substituting 90% of the total volume with dechlorinated water.
The tests were performed at 20 1 C with a photoperiod of 16:8
(light: darkness). Midges in each beaker were fed daily with 1.5 mL
of a 4 gL 1 sh food akes suspension. Dissolved oxygen, pH and
conductivity of the overlying water were measured at the beginning
and end of the experiment and twice a week in at least two replicates
per treatment.
2.5. Evaluation of selected toxicity endpoints
Larval survival, growth and developmental stage were determined
on day 10. Sediments of three replicates were passed through a 300
m sieve to retain the larvae. Surviving animals were removed, counted
and collected for estimation of developmental stage and growth as dry
weight (DW). For determination of DW, the larvae were dried at 60 C
for 24 h and weight estimated with a precision balance Mettler AE166
( 0.1 mg). Developmental stage was determined by measuring the
width of the cephalic capsule and the rst segment with a Nikon SMA
800 stereomicroscope. From day 15 onwards, adult emergence and
sex-ratio (dened as n males/n females) were recorded daily in the re-
maining three replicates according to the procedures described by
OECD (2010).
717
2.6. Sediment extractions
For the determination of effective exposure concentrations, a sedi-
ment sample from each concentration was collected at the beginning,
after 10 days of exposure and at the end of the experiment, and stored
at 20 C until determination. Analysis of pharmaceuticals in sediment
samples was performed by pressurized liquid extraction (PLE) followed
by solid-phase extraction (SPE) and ultra-performance liquid
chromatography-triple quadrupole mass spectrometry (UPLC-QqQ-
MS/MS). Briey, pharmaceuticals were extracted from sediments by
PLE using water at 100 C and 1500 psi. Subsequently, the aqueous ex-
tracts were puried and pre-concentrated according to the method re-
ported by Baena-Nogueras et al. (2016). SPE was carried out using
200 mg Oasis HLB cartridges activated with 8 mL of metanol and 8 mL
of Milli-Q water at a ow rate of 1 mL min1. Subsequently, 30 mL of
aqueous extracts were passed through the cartridges at the same ow
and the cartridges were rinsed with 8 mL of Milli-Q water and dried
using a vacuum for 20 min in order to remove the excess of water.
The analytes retained were eluted from the HLB sorbent with 10 mL of
methanol at a ow rate of 1 mL min1. Methanolic extracts were evap-
orated to dryness under a nitrogen current and nally, samples were
reconstituted with 1 mL methanol-water (25:75, v/v). Prior to injection,
extracts were ltered using PTFE lters (0.45 m from Teknokroma Cor-
poration, Spain).
Separation, identication and quantication of the compounds were
carried out using UPLC-QqQ-MS/MS (Bruker EVOQ Elite system) with
autosampler and C18 analytical column of 100 mm 2.1 mm and 2
m particle size. Calibration curves were constructed for target com-
pounds in the range of 0.1100 gL1. The compounds were identied
by comparison of their retention times of two transitions of each analyte
(one for quantication and one for conrmation) and their ion ratio. In-
ternal standards were added to the vials before injection to correct pos-
sible uctuations in the MS signal. The method limits of detection
(mLOD) were below 1 ngL1 for the target compounds. All the data
were processed using peak areas with Bruker MS Workstation. Recover-
ies were 86 and 101%, for DF and CBZ, respectively.
2.7. Risk assessment of pharmaceuticals
Environmental risk for sediment sorbed pharmaceuticals (diclofenac
and carbamazepine) was assessed using the Risk Quotient (RQ) based
on the ratio between predicted environmental concentration (PEC)
and predicted no effect concentration (PNEC).
PEC
RQ
PNEC
We have employed the most sensitive measured endpoint of
Chironomus riparius test to estimate PNEC (Trombini et al., 2016). The
RQ for a mixture of both pharmaceuticals can also be calculated as the
sum of PEC/PNEC. We applied an assessment factor (100) in the RQ
quantication as chronic toxicity endpoints (NOEC) were used (ECB,
2003)
Xn
PECi
RQmixture
i1
PNECi
For mixtures, risk levels were established as: low (0.010.1), medi-
um (0.11.0) and high (N 1) (Hernando et al., 2006).
2.8. Statistical analysis
Data analysis was carried out by means of the statistical software
package IBM SPSS Statistics Package 21.0. The Levene test was rst ap-
plied to evaluate the homogeneity of variances of the results. Signicant
differences were determined by one-way ANOVA (with treatment as a
718 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723

factor) at a signicance level lower than 0.05 (p b 0.05). When signi-

cant differences were found for the factor treatment, the Dunnett's
test (signicance, p b 0.05) was used as post hoc comparison between
controls and treatment.

3. Results

3.1. Exposure assessment

Nominally targeted sediment concentrations for both DF and CBZ

were 0.12; 1.2; 12.0; 30 and 60 gg1. Measured concentrations at
the start of the experiment are displayed in Table 1.
Initial exposure concentrations of the spiked sediments ranged be-
tween 0.1 and 34.04 and 0.2 and 31.4 gg1 for DF and CBZ, respective-
ly. During the experiment, sediment concentrations decreased with
exposure time, reaching nal concentrations ranges between (n.d and
2.4) for DF and (n.d and 6.3 gg1) for CBZ.

3.2. Effect assessment

Due to the lack of signicant differences in the selected endpoints

between sediment control and solvent control, only the sediment con-
trol was considered for statistical analysis. In both sediment and solvent
controls, survival was N 70%, as recommended by ASTM E 1706-05 for
test acceptability in all assays.
At day 10, 92 and 40% of larvae had survived in the highest exposure
concentration of DF and CBZ, respectively (Fig. 1). Growth was signi-
cantly reduced after exposure to the highest concentration of CBZ in
30.65% with respect to the control (Fig. 2). With regard to the develop-
mental endpoint instar measured by means of mean head capsule width
SD, all surviving organisms exposed to DF and CBZ were found within
the range of the fourth instar. However, although not statistically signif-
icant, a decrease in head capsule and 1st segment widths could be ob-
served with exposure to increasing concentrations of DF (Table 1).
Changes in population dynamics could take more relevance with the
results obtained for CBZ in this study with respect to the endpoint sex- Fig. 1. Percentage of survival (mean SD) of Chironomus riparius after 10 days of exposure
ratio. After exposure to high concentrations of this compound, the pro- to diclofenac (DF) and carbamazepine (CBZ) in spiked sediments. Measured
portion of males observed increased (Fig. 3). On the contrary, DF did not concentrations at the beginning of the test are presented.
change the sex-ratio in exposed organisms signicantly (Fig. 3). Regard-
ing the results obtained after 21 days of exposure, emergence rates were exposed to 31.4 gg1 CBZ compared to the controls indicating a rela-
signicantly lower in organisms exposed at 34.0 gg1 DF (p = 0.01) tive increase in the abundance of male over female individuals (Fig. 5).
compared with the control (Fig. 3). Emergence started on day 15 in sed-
iments spiked with DF and CBZ. Percentages of cumulative emergence 3.3. Risk assessment
varied between 23% and 87% in the case of DF where at 34.0 gg1 a
signicant difference was found with respect to the control (p = To calculate the RQ for DF and CBZ, the selected concentration corre-
0.003) (Fig. 4). The sex-ratio was statistically greater in organisms sponding to the highest concentration where no signicant differences
were reported to respect to the control was used. For DF and CBZ,
these values are close to 30.0 gg1 (the second highest tested concen-
Table 1
Mean values (SD) of head capsule width and 1st segment width (mm) of Chironomus tration). Instead of use PEC, we have employed the measured environ-
riparius after 10 days of exposure to diclofenac (DF) and carbamazepine (CBZ). mental concentration (MEC) reported by Ebele et al. (2017) where the
current state of knowledge about global pharmaceutical levels has
Measured concentrationa Instar Head capsule width 1st segment width
(g g1) (mm) (mm)
been reviewed. The concentrations selected for DF and CBZ were 57
and 33 ngg1, respectively, corresponding to worst case scenarios re-
DF
Control 4th 0.54 (0.01) 0.80 (0.06)
ported in Ebele et al. (2017). The RQ for DF and CBZ were 0.19 and
0.1 4th 0.54 (0.07) 0.77 (0.06) 0.11, respectively and for the mixture 0.30, indicating medium risk for
0.7 4th 0.52 (0.06) 0.75 (0.06) both compounds or its mixture.
11.1 4th 0.51 (0.06) 0.77 (0.05)
27.8 4th 0.45 (0.07) 0.72 (0.07)
4. Discussion
34.0 4th 0.44 (0.06) 0.73 (0.07)

CBZ The freshwater midge C. riparius has been frequently employed to

Control 4th 0.42 (0.05) 0.63 (0.09)
evaluate the effects of sediment sorbed contaminants in non-target or-
0.2 4th 0.43 (0.05) 0.62 (0.09)
1.6 4th 0.42 (0.05) 0.62 (0.09) ganisms. Also in the case of pharmaceuticals as environmental contam-
13.6 4th 0.42 (0.05) 0.63 (0.10) inants, the number of studies reporting dened endpoints in spiked
38.7 4th 0.43 (0.05) 0.61 (0.10) sediments using benthic invertebrates (Oviedo-Gmez et al., 2010;
31.4 4th 0.41 (0.05) 0.62 (0.10) Mndez et al., 2013; Maranho et al., 2014, 2015) and more specically
a
Data correspond 0 day. midges (Oetken et al., 2005; Snchez-Argello et al., 2005, 2009;
 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723 719

Fig. 2. Growth of larvae measured as mean dry weight (d.w SD) and mean values of percentage of increase with respect to sediment control (d.w.%) after 10 days of exposure to
diclofenac (DF) and carbamazepine (CBZ). Asterisks indicate signicant differences between means compared with sediment control (p b 0.05). Measured concentrations at the
beginning of the test are presented.

Nentwig, 2007; Pry et al., 2008; Gilroy et al., 2012; Lpez-Doval et al., to be addressed urgently to prevent potential adverse effects of this
2012) as non-target organisms, are increasing. However, there is still kind of compounds at community level. For this reason, we performed
an important knowledge gap about the effects of hundreds of pharma- a spiked sediment exposure test in order to evaluate the possible effects
ceuticals and derivates on aquatic and benthic organisms which have of two pharmaceutical compounds in the midge C. riparius. The selected

Fig. 3. Mean values (SD) of emergence ratio for Chironomus riparius after 21 days of exposure at diclofenac (DF) and carbamazepine (CBZ). Asterisks indicate signicant differences
between means compared with sediment control (p b 0.05).
720 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723
Fig. 4. Cumulative emergence curves (%) of midges exposed at diclofenac (DF) and
carbamazepine (CBZ) in spiked sediment. Asterisks indicate signicant differences
between means compared with sediment control (p b 0.05).
compounds belong to two important groups of prescription drugs: anti-
epileptics and mood stabilizing drugs (carbamazepine) and nonsteroi-
dal anti-inammatory drugs, NSAIDs (diclofenac). The selected end-
points included mortality as well as several developmental endpoints
such as growth (biomass), developmental instar, emergence rate and
Fig. 5. Mean values (SD) of sex-ratio (n males/n females) for Chironomus riparius after 21 days of exposure at diclofenac (DF) and carbamazepine (CBZ). Asterisks indicate signicant
differences between means compared with sediment control (p b 0.05).
sex-ratio (male/female). The obtained results were compared, when
data were available, with those obtained for other benthic invertebrates.
4.1. Exposure
For all concentrations, except the highest, targeted and measured
values are satisfactorily similar. However, the highest targeted spiked
exposure concentration was not achieved in both cases, DF and CBZ,
presenting instead values similar to the next lower concentration. Dif-
ferences in targeted and measured concentration in spiking procedures
can be due to binding of the compound to glass surfaces, evaporation
processes, biological and chemical degradation or sub-optimal extrac-
tion efciency as it has been also observed by other authors (Ramil
et al., 2010; Lpez-Doval et al., 2012) as well as to the different sorption
behavior of the selected compounds onto the sediment and its charac-
teristics to achieve the steady-state of the water-sediment system, as
for example in a study carried out by Buser et al. (1998), who showed
negligible adsorption of DF onto sediment particles in laboratory
experiments.
A clear continuous decrease of the sediment exposure concentra-
tions was reported, reaching at the end of the experiment values be-
tween 2.4 for DF and 6.3 gg1 for CBZ in the highest exposure
concentrations. Biological or chemical degradation of the compounds
as explanation for the decrease in exposure concentrations can rather
certainly be ruled out: CBZ has been found to be persistent in soils, bio-
solids and soil-biosolid mixtures with no degradation observed over
60 d (Monteiro and Boxall, 2009) and Lfer et al. (2005) reported
high persistence of CBZ in sediment with values of N365 d (DT90). In
the case of DF, a half-live of 20.44 d was reported by Qin et al. (2015)
in agricultural soils irrigated with reclaimed water. These residence
times, although not determined in freshwater sediments, suggest that
the concentration of these compounds decreased along the experiment
due to other reasons than degradation and can be rather contributed to
their polar or moderately polar character, evidenced by the octanol-
water partition coefcients of 1.51 and 1.90 for CBZ and DF, respectively
(Scheytt et al., 2005) being desorbed to the overlying water. In our ex-
periment, the exposure water was renewed daily, leading probably to
the leaching of the compounds from the sediment into the water col-
umn where a further degradation could also be possible. Our experi-
ments aimed to simulate the effects of punctually contaminated
sediment under the conditions of non-continuous contaminant input.
For the assessment of continuously contaminated sediments, water
change frequency has to be reconsidered in the case of polar compounds
envisaging a compromise between avoiding the leaching of the
 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723
compound and the wellbeing of the exposed population in the test.
Within this context, the biologic activity of benthic organisms (e.g.
burrowing) in the sediments can further alter certain processes such
as sorption or desorption of compounds (Timmermann et al., 2011;
Chen et al., 2016). In the case of more polar compounds, bioturbation
could increase desorption from the sediment facilitating the transfer
into the overlying water and thus increasing bioavailability and the
risk of water contamination (Goedkoop and Peterson, 2003).
4.2. Effect assessment
Gilroy et al. (2012) reported a survival N 72.5% in Chironomus dilutus
exposed to CBZ in a range of concentrations between 0.1 and 100
gg1, being the exposure concentrations slightly higher than the re-
ported in our study. Maranho et al. (2014) reported signicant mortal-
ities in the marine polychaete Hediste diversicolor exposed to 0.050.5
gg1 CBZ even if the exposure concentrations were much lower. Pre-
viously, Oetken et al. (2005) reported no acute toxicity in C. riparius ex-
posed to CBZ spiked sediments in a series of tests at CBZ concentrations
of 0.1; 0.2; 0.7 and 2.9 gg1 indicating that CBZ seems not to be acute-
ly toxic at environmentally relevant concentrations. These different re-
sponses of invertebrates to stress agents are related to the taxonomy
and phylogeny (Holan et al., 2016; Malaj et al., 2016).
With respect to our results obtained for DF, Lpez-Doval et al. (2012)
observed a survival between 70 and 90% in C. riparius larvae exposed
during 10 days to 0.3300 gg1 to indomethacin, belonging like DF
also to the group of NSAIDs. On the other hand, Oviedo-Gmez et al.
(2010) observed that sediment sorbed DF produced oxidative stress in
H. azteca exposed to 46.7 gkg1 however without being signicantly
lethal. Maranho et al. (2015) did not observe any signicant mortality in
the amphipod A. brevicornis exposed to ibuprofen, another NSAID for
10 days.
The endpoint growth is an environmentally very relevant and easily
measurable indicator of changes due to sediment contamination.
Changes in growth are ecologically very relevant since they can mani-
fest at population level through effects on reproductive output on the
longer term (Sibley et al., 1997). In our experiments, we observed sig-
nicant changes in growth measured by biomass (somatic growth)
(dry weight) in those organisms exposed to the highest tested CBZ con-
centration with respect to the control (Fig. 2). Although not signicant,
there is a continuous decreasing trend in growth of the organisms with
increasing exposure concentrations of both of the pharmaceuticals, CBZ
and DF. The difference in the case of CBZ, against organisms from the
control experiment is the biggest, with those from the increasing expo-
sure concentrations progressively being smaller. For DF, all exposed or-
ganisms are bigger than the control organisms suggesting the
stimulation of the larval growth, with the highest relative increase in
growth occurring at the lowest DF exposure concentration and succes-
sively decreasing with the concentration (Fig. 2). A similar observation
was made by Gilroy et al. (2012) who found a growth occasionally
greater than controls in Chironomus dilutus but exposed in this case to
56.6 gg1 d.w. of CBZ. Sibley et al. (1997) suggested that in larvae of
C. tentans, a closely related species of the family Chironomidae, the or-
ganisms should have a minimum weight of approx. 0.5 mg dry
weight/individual for emergence to take place. According with this sug-
gestion, in our study, the organisms exposed to the selected pharmaceu-
tical compounds reach that estimated weight being comprised between
1.4 and 2.0 mg and 1.7 and 2.4 mg for DF and CBZ, respectively.
With regard to the developmental stage, morphometry is a widely
used tool in the determination of insect instars (Daly, 1985). The dimen-
sions most commonly used to determine the number of larval instars is
the width (Ecole, 1999; Silva et al., 2008) from ventral view. In our ex-
periments, the organisms exposed to DF and CBZ had reached the fourth
instar independently from the compound and the exposure concentra-
tion applied. In this context, the emergence ratio for CBZ was comprised
around 0.5 for all treatments including the control, whereas for DF all
721
treatments resulted in an emergence rate of about 0.9 except for the
highest concentration where the emergence rate decreased signicant-
ly to 0.2 (Fig. 3) In agreement, the total emergence of adults (%) de-
creased with the highest DF concentration (Fig. 3). Oetken et al.
(2005) observed a signicant and concentration dependent decrease
of emergence with a negative impact of CBZ on emergence of midges
at sediment concentrations N 0.07 gg1. These authors also observed
a blockade of pupation in CBZ-exposed C. riparius larvae such that they
survived up to 4 weeks without undergoing pupation until they nally
died. The suppression of pupation in C. riparius under CBZ exposure re-
fers to a specic mode of action, most probably some interference with a
physiologic pathway rst activated in this life stage or to some modula-
tion of endocrine functions (Oetken et al., 2005). Similar ndings have
been made by Oropesa et al. (2016) who observed that chronic expo-
sure to CBZ signicantly decreased the reproductive output and the
number of molts of D. magna at 200 g/L. CBZ induced the production
of male offspring (12 1.7%), in a concentration independent manner,
acting as a weak juvenile hormone analog. Further, CBZ showed to be
toxic to daphnid embryos through maternal exposure interfering with
their normal gastrulation and organogenesis stages but not producing
direct embryo toxicity. No information on effects of emergence rate in
C. riparius has been found for treatment with DF.
Other authors have previously associated effects between the rela-
tionships growth-emergency. At ecological level, larval growth retarda-
tion and low emergence rate, could, in a longer term, lead to reduced
reproductive success and consequently reduction in abundance in natu-
ral populations (Liber et al., 1996; Benoit et al., 1997). Changes in
growth induced by stress can be used to make meaningful predictions
regarding reproduction and population dynamics (Sibley et al., 1997).
These authors observed that a reduction in the growth during larval
stages in C. tentans is associated with a proportional decline in repro-
ductive output of adult females.
Effects on sex-ratio may affect reproduction and therefore, popula-
tion dynamics, even more in the context of chronic exposure that affects
successive generations of organisms (Snchez-Argello et al., 2009). Re-
cent experiments showed reduction of development rate and fecundity
(egg ropes/female) when C. riparius was exposed to 100 g/L of CBZ for-
tications of reclaimed water (Snchez-Argello et al. personal
communication).
In our experiments, we have assessed several endpoints throughout
the life cycle of the midge, C. riparius exposed to sediments spiked with
two different pharmaceutical compounds. At our assayed concentra-
tions, we observed an increase of mortality for CBZ and a reduction in
the emergence rate for DF. CBZ also induced a signicant change in
the sex-ratio, increasing the number of males with respect to females.
CBZ, as well as other antidepressants have been previously related
with endocrine disrupting activity in human (Isojrvi et al., 2004;
Lfgren et al., 2006; Lekiewicz et al., 2008) as well as environmentally
exposed non-target organisms (Hampel et al., 2014; Fong and Ford,
2014; Hazelton et al., 2014). This fact is of great environmental rele-
vance, as changes in natural proportions between male and female
can have important consequences in population in the longer term.
4.3. Risk assessment
To establish the risk associated to the occurrence of pharmaceuticals
in the sediment involve the need to have a wide database about pres-
ence and effect. However, our results can be considered only as a pre-
liminary assessment in relation to the risk associated to DF and CBZ in
sediments. One of the limitations is that only one species C. riparius
has been considered for the calculation and, on the other hand, the da-
tabase about quantication of these compounds in sediments is limited.
Nevertheless, the use of chronic instead of acute toxicity data gives ad-
ditional value from the point of view of ecological relevance (ECB,
2003). Our results showed higher risk than those found by Trombini
et al. (2016) for the species Tisbe battagliai using acute toxicity test
722 E. Nieto et al. / Science of the Total Environment 609 (2017) 715723
and an assessment factor (AF) = 1000, according to ECB (2003) guide-
lines. The use of RQ can be employed also for priorization of emerging
organic contaminants (EOCs) individually or such as mixture of com-
pounds, because exposure and effect are taken in account together.
Although there is still an urgent need to establish links between the
observed effects in laboratory exposure experiments and their impact
on ecosystem level, the present study contributes to the understanding
on the possible chronic effects of the selected pharmaceuticals, DF and
CBZ at low concentrations. Our results indicate that their presence in
sediments may produce changes in the survival and development of
C. riparius which in a long term could have important ecological implica-
tions in natural populations.
5. Conclusions
Our spiked sediment experiments with DF and CBZ employing the
midge C. riparius have shown that exposure to these compounds may
produce negative effects in survival and growth and provoke certain
changes in emergence and sex-ratio. The C. riparius bioassay is suitable
for the quality assessment of sediments contaminated by pharmaceuti-
cals compounds. The employed endpoints are considered useful to indi-
cate changes in population structure and size. Therefore, the obtained
results should be taken into account for threshold settings and quality
guidelines. Nevertheless, further research should be done to assess the
environmental risk of these emerging contaminants in benthic organ-
isms that inhabit in the sediments at population level to guarantee the
protection of ecosystems.
Acknowledgements
Authors would like to thank to Dr. A.M. Arias for the Chironomus lar-
vae image employed in the graphical abstract and the Spanish Ministry
of Economy and Competitiveness for its nancial support through the
project SCARCE (Consolider-Ingenio 2010 CSD2009-00065), NET-
SCARCE (CTM2015-69780-REDC) and the PIE 20143E072 (CSIC). M.H.
is supported by a Ramn y Cajal contract (RYC-2012-12217) from the
Spanish Ministry of Economy and Competitiveness (MINECO). Ministry
of Economy and Competitiveness and European Regional Development
Fund (ERDF).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.07.171.
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