Carbamazepine-mediated pro-oxidant effects on the unicellularmarine algal species Dunaliella tertiolecta and the hemocytesof mussel Mytilus galloprovincialis

Pinelopi Tsiaka • Vasiliki Tsarpali •

Ioanna Ntaikou • Maria N. Kostopoulou •

Gerasimos Lyberatos • Stefanos Dailianis

Accepted: 23 July 2013 / Published online: 4 August 2013

� Springer Science+Business Media New York 2013

Abstract This study investigates the pro-oxidant behavior

of the antiepileptic drug carbamazepine (CBZ) on the mar-

ine algal species Dunaliella tertiolecta and the immune

defense-related hemocytes of mussel Mytilus galloprovin-

cialis. A phytotoxicity test, performed in a first step, showed

a significant inhibition of the growth rate and the chlorophyll

alpha (Chl-a) content in algae after exposure for 24 h to

different concentrations of CBZ (1–200 mg L-1). On the

other hand, the increased levels of lipid peroxidation prod-

ucts, such as MDA, measured in 24 h CBZ-treated cells

were attenuated with time (48–96 h), followed by a signif-

icant recovery of both the algal growth rate and the Chl-acontent in all cases. The latter could be related to the con-

comitant enhancement of total carotenoids in CBZ-treated

algae with time, which in turn could protect algal growth and

survival against CBZ-induced oxidative stress. On the other

hand, the increased levels of cell death, superoxide anions

(�O2-), nitric oxides (NO, in terms of nitrites, NO2

-) and

MDA content observed in mussel hemocytes exposed to

environmentally relevant (0.01–1 lg L-1) and/or higher (10

and 100 lg L-1) concentrations of the drug, clearly indicate

the ability of CBZ to induce oxidative effects on cells of non-

target species, such as mussels, affecting thus their overall

health status. The significant relationships occurred among

the tested biological parameters in both bioassays, further

reinforce CBZ-mediated pro-oxidant effects on species,

widely used in ecotoxicological and toxicological studies

and provide a more comprehensive view on its environ-

mental fate and ecotoxicological risk evaluation.

Keywords Carbamazepine � Dunaliella �Hemocytes � Lipid peroxidation � Oxidative stress-

related effects � Toxicity

Introduction

In recent years, increasing awareness of the adverse envi-

ronmental effects of pharmaceutical compounds (PhCs) has

been observed, since their excretion and environmental

release could pose a toxicological risk to non-target

organisms (Al Aukidy et al. 2012). Regarding their clas-

sification as a new class of environmental pollutants

(Zuccato et al. 2000), more than 80 PhCs have been

detected in the aquatic environment, originating mainly

from irrigation of treated wastewater, leaching from waste

disposal sites and septic tanks, as well as from treatment

plants effluents disposal (Dietrich et al. 2002; Richardson

and Ternes 2011). Due to their metabolic stability and

incomplete degradation (Rizzo et al. 2010), PhCs as well as

P. Tsiaka � V. Tsarpali � S. Dailianis

Section of Animal Biology, Department of Biology, School of

Natural Sciences, University of Patras, 26500 Patras, Greece

I. Ntaikou

Institute of Chemical Engineering Sciences, Foundation of

Research and Technology Hellas (ICEHT/FORTH), 10 Stadiou

st., Platani, 26504 Patras, Greece

M. N. Kostopoulou

Department of Marine Sciences, School of the Environment,

University of the Aegean, University Hill, 81100 Mytilene,

Greece

G. Lyberatos

School of Chemical Engineering, National Technical University

of Athens, Zografou Campus, 15780 Athens, Greece

S. Dailianis (&)

Section of Animal Biology, Department of Biology, Faculty of

Sciences, University of Patras, 26500 Patras, Greece

e-mail: sdailianis@upatras.gr

123

Ecotoxicology (2013) 22:1208–1220

DOI 10.1007/s10646-013-1108-3

their metabolites may interfere with molecules, cells and

organs of aquatic organisms, thus being a potent threat for

their health status (for a review see Fent et al. 2006).

Among PhCs, carbamazepine (CBZ) is one of the most

consumed (annual consumption rate about 2,235,000

pounds worldwide) antiepileptic drugs (Zhang et al. 2008).

Despite its beneficial use for the control of a variety of

mental disorders, due to its ability to block sodium chan-

nels of excitatory neurons (RxList 2006; Garcia-Morales

et al. 2007), its presence into the aquatic environment has

been reported as an anthropogenic marker of urban pollu-

tion (Clara et al. 2004). In particular, Ferrari et al. (2003)

ranked CBZ as the highest-risk compound for aquatic

environments when compared to other PhCs, such as dic-

lofenac and clofibric acid, and it has been classified as

‘‘R52/53 Harmful to aquatic organisms and may cause

long term adverse effects in the aquatic environment’’

(European legislation on the classification and labeling of

chemicals, 92/32/EEC), since it is present in municipal

sewage-treatment plant (STP) effluents, surface waters and

even in seawater, mainly due to its low (below 10 %) STP

removal efficiency (Chen et al. 2006; Zhang et al. 2008)

and incomplete biodegradation in both water bodies (An-

dreozzi et al. 2002; Stamatelatou et al. 2003; Zhou et al.

2009). High CBZ levels (6 300 ng L-1) were detected in

WWTP effluents (Ternes 1998), as well as in surface

waters (1,075 ng L-1) in Berlin, Germany (Heberer et al.

2002), while CBZ levels in municipal WWTP effluents

from different Greek cities, such as Iraklion and Ioannina,

were up to 1 lg L-1 (for further details see Ferrari et al.

2003; Paxeus 2004; Zhang et al. 2008; Kosma et al. 2010).

Different species and cellular types are characterized by

different sensitivity, metabolic pathways and protective

responses against PhCs (Schmidt et al. 2011), a fact that

should be taken into account when performing toxicolog-

ical and ecotoxicological studies. Motivated by the need to

investigate the concentration range at which a chemical

produces adverse effects on organisms (Jos et al. 2003), a

lot of in vitro studies have been performed in the last

decade, in order to assess the environmental risk of CBZ on

aquatic biota, including fish, algal species, crustaceans,

molluscs and many others (for a review see Huerta et al.

2012). However, the majority of ecotoxicological studies

were focused on the mortality endpoints and only a few

reported CBZ-mediated pro-oxidant effects on aquatic

species, such as algae and marine bivalves (Gagne et al.

2006b; Martin-Diaz et al. 2009; Vernouillet et al. 2010;

Contardo-Jara et al. 2011; Vannini et al. 2011).

Given that PhCs may affect the health status of species

from different trophic levels (Sacan and Balcioglu 2006;

DeLorenzo and Flemming 2008), the present study inves-

tigated CBZ-mediated pro-oxidant effects on two distinct

biological models, the unicellular marine algal species

Dunaliella tertiolecta, widely used as a bioassay organism

for seawater toxicity tests (APHA, Awwa, WEF 1998;

Reish and Lemay 2008), and hemocytes of mussel Mytilus

galloprovincialis, widely used in ecotoxicological and

toxicological studies for investigating cellular responses

against non-self substances (Borenfreund and Puerner

1985; Carballal et al. 1997; Olabarrieta et al. 2001; Cao

et al. 2003; Bouki et al. 2013; Toufexi et al. 2013). Spe-

cifically, the present study investigated not only the con-

centration range at which CBZ alters algal growth and

chlorophyll alpha (Chl-a) content within algal cells, but

also its ability to cause oxidative stress-related events, such

as lipid peroxidation. Moreover, since the role of antioxi-

dant compounds in green algae is of great concern, the

amount of total carotenoids was estimated for the first time,

revealing thus their important role during algal growth

and survival, under CBZ-mediated stress conditions. In

parallel, taking into account that the immune system of

molluscs, commonly linked with hemocytes’ immunosur-

veillance, is a major target for PhCs (Canesi et al. 2007;

Contardo-Jara et al. 2011; Gust et al. 2012, 2013), CBZ-

mediated cytotoxic (in terms of cell death) and oxidative

effects (in terms of superoxide anion and nitric oxide

production, as well as lipid peroxidation content) on pri-

mary culture of mussel hemocytes were investigated.

Finally, a linear correlation analysis (Pearson test,

p \ 0.05) between parameters measured in algae and

mussel hemocytes was performed, in order to elucidate any

relationship among CBZ-mediated biological responses in

algae and/or mussel hemocytes, providing thus a more

comprehensive view on CBZ pro-oxidant behavior, envi-

ronmental fate and eco-toxicological risk evaluation.

Materials and methods

Chemicals and reagent

CBZ and sulfanilic acid were purchased from Sigma-

Aldrich Chemical Co. (St. Louis, MO, USA). Nitroblue-

tetrazolium (NBT), neutral red, N-(1-Napthyl)ethylene-

diamine, sodium nitrite, phosphoric acid, fetal calf serum

(FCS), penicillin G, streptomycin, gentamycin, amphoter-

icin B were purchased from Applichem (Darmstadt,

Germany). Leibovitz L-15 medium was purchased from

Biochrom A.G. (Berlin, Germany). All other reagents and

solvents used were of the highest analytical grade and

purity.

Toxicity tests with the use of D. tertiolecta

CBZ-mediated toxic effects on D. tertiolecta were inves-

tigated according to well-known protocols and guidelines

Carbamazepine-mediated pro-oxidant effects 1209

123

(OECD 1984; NIWA 1998). In brief, D. tertiolecta (strain

CCAP 19/6B, from Scottish Marine Institute, Oban, Argyll,

Scotland) was grown in f/2 medium without Si (24 ± 1 �C,

pH 8.3 ± 0.3, salinity 35 %), under constant illumination

(4 300 lux). At late logarithmic phase, a proper amount of

culture (1x104 cells mL-1) was transferred to conical

sterilized flasks, containing f/2 medium (final volume

200 mL) and finally exposed to different concentrations of

CBZ (0, 1, 10, 50, 100 and 200 mg L-1) for 96 h, under

constant values of pH, temperature and salinity, as men-

tioned above. A stock solution of CBZ was prepared in

0.1 % v/v DMSO and maintained in the dark, during the

experimental procedure. Each solution was filtered through

a 0.22 lm filter before being used. CBZ concentrations

used in the present study (1–200 mg L-1) were similar to

those previously used for estimating the toxic effects of the

drug on green algae and other species (Jones et al. 2002;

Ferrari et al. 2003; Jos et al. 2003; DeLorenzo and Flem-

ming 2008; Zhang et al. 2012).

Every 24 h, the cell number was counted, using a

Neubauer hemocytometer, while the growth (l) and the

inhibition rate (%I) were determined according to the

guideline OECD 201 (1984). In addition, although previous

studies showed that DMSO, at a final concentration of

0.1 % v/v, showed no inhibitory effects on algal growth

(Kim et al. 2007; Vernouillet et al. 2010), cell number and

l values were also estimated in DMSO-treated algae. The

aforementioned procedure was repeated 4 times and the

obtained results are the mean ± SD from 4 measurements.

In parallel, the amount of total carotenoids and Chl-a, as

well as lipid peroxidation products (in terms of malondi-

aldehyde equivalents), were determined in algal cultures

exposed to each concentration of the drug tested.

Determination of lipid peroxidation in green algae D.

tertiolecta

Lipid peroxidation was determined by measuring the for-

mation of thiobarbituric acid reactive substances, quanti-

fied as malondialdehyde (MDA) equivalents, according to

the method described by Nikookar et al. (2005). In brief, an

appropriate volume of each culture, containing 106 cells,

was collected every day. Each sample was centrifuged at

4,0009g for 10 min, the supernatant was removed care-

fully and 5 mL of 0.1 % TCA were finally added to the cell

pellets. Thereafter, samples were sonicated for 60 s (model

W-220F, 30 s 9 2, with 15 s time interval) and equal

volumes of 0.5 % TBA, in 20 % TCA solution, were

finally added. After incubation at 95 �C for 30 min, sam-

ples were cooled to room temperature, centrifuged at

10,0009g for 10 min and finally measured spectrophoto-

metrically at 532 nm and 600 nm (correct errors caused

from non-specific turbidity). A molar absorption co-

efficient (e = 1.5 9 105 M-1 cm-1) (Wills 1969) was used

for the determination of the concentration of MDA. The

results, expressed as nmol MDA per 106 cells, are

mean ± SD from 4 independent measurements in each case.

Determination of total carotenoids

Total carotenoids were determined according to the method

initially described by Shaish et al. (1992) and further

modified by Garcia-Gonzalez et al. (2005). In brief, a

culture aliquot of 1 mL (containing known number of

cells) was centrifuged at 1,0009g for 15 min and the cell

pellet was extracted with 3 mL of ethanol:hexane (2:1,

v/v). Thereafter, 2 mL of water and 4 mL of hexane were

added, the mixture was thoroughly mixed, vigorously

shaken and finally centrifuged at 1,2009g for 10 min. The

hexane layer was separated and its absorbance at 450 nm

was determined: A450 9 (25.2) equals to lg of carotenoids

in sample. The results, expressed as pg carotenoids per cell

(based on the known number of cells, previously measured

in each sample), are mean ± SD from 4 independent

measurements in each case.

Determination of Chl-a

Chl-a was determined according to Aminot and Ray (2000).

In brief, every 24 h, a culture aliquot of 10 mL was filtered

through glass microfiber filters (Whatman GF-F, pore size

0.7 lm) and filters were kept at -21 �C till extraction.

Extraction of chlorophyll was performed with 10 mL of

90 % v/v acetone, at 4 �C for 5 h. Extracts were separated by

centrifugation at 1,3509g for 15 min and absorption was

measured at 630, 647, 664 and 750, using acetone as blank.

Chl-a was calculated according to the equation:

CChl�a ðlg mL�1Þ ¼ ½ð11:85 � ðA664 � A750Þ � 1:54

� ðA647 � A750Þ � 0:08 � ðA630

� A750Þ� � Vextr=Vsample ð1Þ

and expressed as pg of Chl-a per cell (mean ± SD from 4

independent measurements in each case).

Toxicity tests with the use of mussel hemocytes

Mussel collection and handling

Mussels of the species M. galloprovincialis (5–6 cm long)

were collected from a mussel farm located to the north side

of Korinthiakos Gulf (Gulf of Kontinova, Galaxidi,

Greece), since this area, being far from urban/anthropo-

genic activities, is characterized as unpolluted (Grintzalis

et al. 2012). Mussels were transferred to the laboratory and

handled under appropriate conditions. Specifically, mussels

1210 P. Tsiaka et al.

123

were acclimated without feeding in static tanks, containing

aerated (dissolved oxygen 7–8 mg L-1 and 35 % salinity),

recirculated with UV-sterilized and filtered artificial sea-

water (ASW) for 7 days at 15 �C. After the end of the

acclimation period, mussels were fed daily with approxi-

mately 30 mg of dry-microencapsules/mussel (Myspat,

Inve AquacultureNV, Belgium).

Mixed primary culture of hemocytes

Although the hemolymph of mussels contains two basic

cell types, granulocytes (basophilic and acidophilic) and

hyalinocytes (Carballal et al. 1997), a mixed primary cul-

ture of mussel hemocytes is widely used, in order to

investigate the cellular responses under short-term expo-

sure conditions (e.g. 1 h). This is because of hemocytes

ability to promptly respond to a variety of toxic com-

pounds, revealing thus alterations in both their viability and

a battery of biological stress indices (Olabarrieta et al.

2001; Dailianis 2009; Banakou and Dailianis 2010; Chat-

ziargyriou and Dailianis 2010; Vouras and Dailianis 2012;

Bouki et al. 2013).

Primary cultures of mussel hemocytes were prepared

according to the method described by Cao et al. (2003).

Specifically, hemolymph extracted from the posterior

adductor muscle from 10 mussels with a sterile 1 mL syringe

(equipped with an 18G1/200 needle), containing 0.1 mL of

Alseve buffer (ALS buffer; 60 mM glucose, 27.2 mM

sodium citrate tribasic, 9 mM EDTA, 385 mM NaCl, pH 7

and 1,000 mOsm L-1), was centrifuged at 1509g for 15 min

at room temperature (20 ± 3 �C) and the cell pellet was re-

suspended in Leibovitz L-15 medium supplemented with

350 mM NaCl, 7 mM KCl, 4 mM CaCl2, 8 mM MgSO4,

40 mM MgCl2, 10 % v/v FCS, 100 U mL-1 penicillin G,

100 lg mL-1 streptomycin, 40 lg mL-1 gentamycin,

0.1 lg mL-1 amphotericin B (pH 7 and 1,000 mOsm L-1).

The cell culture medium was filtered (through 0.45 lm fil-

ters), cells were counted in a Neubauer haemocytometer and

re-suspended to obtain a concentration of 106 cells mL-1.

Primary cultures of hemocytes were kept at 15 �C for at least

1 h before being used for the experiments. In all cases, the

experimental procedure was performed only in case that the

cell viability, previously estimated by the use of Eosin

exclusion test, was at least 95 %.

Hemocytes exposure condition

Approximately 500 lL of cell suspension (106 cells mL-1)

were incubated for 1 h (at 15 �C) with different concen-

trations of the drug (CBZ at final concentrations of 0.01,

0.1, 1, 10, 100 and 200 lg L-1, from a stock solution,

freshly prepared in 0.01 % v/v of DMSO and maintained in

the dark) and/or DMSO at a final concentration of 0.01 %

v/v, which is considered a low dose, having no effects

(Contardo-Jara et al. 2011). Although there are no data

concerning the levels of free and protein serum-binding

CBZ, which could clearly indicate the effective concen-

trations of CBZ (for more details see Kramer et al. 2012

and references inside), the selected CBZ concentrations

were close to those found in the aquatic environment

(Ferrari et al. 2003) and similar to those previously used for

investigating CBZ effects on tissues and cells of aquatic

species, such as mussels (Canesi et al. 2007; Contardo-Jara

et al. 2011; Gust et al. 2012).

After the end of the experimental procedure, cell via-

bility, superoxide anions (�O2-), nitrites (NO2

-) and lipid

peroxidation content (in terms of malondialdehyde equiv-

alents) were estimated.

Neutral red uptake determination in hemocytes of mussels

Estimation of the uptake of the cationic dye neutral red

(NR) was assessed as reported by Dailianis (2009). In brief,

CBZ-treated cell suspension was centrifuged at 1509g and

the supernatant was removed carefully. The packed cells

were re-suspended in ALS buffer and maintained in the

dark (1 h at 4 �C), in order for the cells to adhere to the

walls. Thereafter, the non-adherent cells were removed

carefully and 500 lL of ALS, containing 0.004 % w/v NR,

were added. After incubation for 2 h, in order to allow

uptake of the dye, cells were centrifuged at 1509g for

10 min and washed twice with ALS. Afterwards, the dye

was extracted from intact cells with an acetic acid–ethanol

solution (1 % v/v acetic acid and 50 % v/v ethanol) and

the absorbance was determined spectrophotometrically

(Perkin-Elmer 551) at 550 nm. The results are mean ± SD

from 6 independent experiments and are expressed as

OD550nm per milligram of protein, as determined in each

sample by an ultrasensitive hydrophobic method (Georgiou

et al. 2008), with the use of known concentrations of

bovine serum albumin (BSA).

Detection of superoxide anions in hemocytes of mussels

Superoxide anions (�O2-) were detected according to the

method described by Pipe et al. (1995), with the use of

nitroblue tetrazolium (NBT). Those anions were primarily

chosen to be measured, due to their enhancement/generation

during the induction of the respiratory burst in mussel

hemocytes, as well as their involvement in the induction of

oxidative stress via the production of hydrogen peroxide

(H2O2), hydroxyl (OH�) and peroxynitrite (ONOO-) radi-

cals (Hermes-Lima et al. 2001). Briefly, CBZ-treated

hemocytes (primary suspended in L-15 modified medium,

containing 1 mg mL-1 of NBT, plus different concentra-

tions of CBZ), were centrifuged at 1509g (10 min, at 4 �C)

Carbamazepine-mediated pro-oxidant effects 1211

123

and washed with 300 lL TBS (0.05 M TRIS/HCl buffer, pH

7.6, containing 2 % NaCl), in order to remove extracellular

NBT. Hemocytes were then fixed with 300 lL of 70 %

methanol for 10 min and centrifuged at 1509g (10 min, at

4 �C). Cells were air-dried for 5 min at room temperature

(20 ± 3 �C) and 1 mL of extraction fluid (2 M KOH-

DMSO) was finally added. After solubilization for 30 min,

samples were measured spectrophotometrically at 620 nm.

The results, expressed as OD620nm mg-1 protein, are

mean ± SD from 6 independent experiments in each case.

Determination of nitric oxide (in terms of nitrites)

in hemocytes of mussels

Although the original NO content is represented by the sum

of nitrite (NO2-) and nitrate (NO3

-), the measurement of

NO2- steady-state (stationary) levels with the Griess

reaction (Green et al. 1982) has been commonly used in

order to estimate indirectly NO generation in activated

cells (for more details see Tafalla et al. 2002; Dailianis

2009; Vouras and Dailianis 2012; Bouki et al. 2013).

Briefly, the CBZ-treated cell suspensions were centrifuged

(1509g, 10 min, at 4 �C), the supernatant was removed

carefully and 500 lL of 1 % sulfanilic acid in 5 % phos-

phoric acid were added. After incubation for 10 min at

20 ± 3 �C, 500 lL of 0.1 % v/v N-(1-Napthyl)ethylene-

diamine in 5 % phosphoric acid were added, incubated for

15 min and finally the optical density at 540 nm was

measured (spectrophotometer Perkin Elmer 551). The

molar concentration of NO2- in each sample was deter-

mined from standard curves generated using known con-

centrations of sodium nitrite (1–100 lM NaNO2). The

results, expressed as nmol NO2- mg-1 protein, are

mean ± SD from 6 independent experiments in each case.

Estimation of lipid peroxidation in hemocytes of mussels

Lipid peroxidation was quantified as malondialdehyde

(MDA) equivalents, according to the method described by

Chatziargyriou and Dailianis (2010). In brief, CBZ-treated

hemocytes were centrifuged at 150 x g (10 min, at 4 �C), the

supernatant was removed carefully and the packed cells were

mixed with 1 mL of trichloroacetic acid (TCA)-thiobarbi-

turic acid (TBA)-HCl (15 % w/v TCA, 0.375 % w/v TBA in

HCl 0.25 N). After vortexing for 5 s, butylated hydroxytol-

uene (BHT) at final concentration of 0.02 % w/v was finally

added, in order to prevent further peroxidation of lipids.

Samples were incubated at 90–100 �C for 15 min and cooled

at room temperature (20 ± 3 �C). Thereafter, the samples

were centrifuged at 1,0009g for 10 min and were measured

spectrophotometrically at 535 nm. A molar absorption

co-efficient (e = 1.5 9 105 M-1 cm-1) (Wills 1969) was

used for the determination of MDA concentration. The

results, expressed as nmol MDA mg-1 protein, are

mean ± SD from 6 independent experiments in each case.

Statistical analysis

The estimation of the IC50 endpoint in algae was performed

with the use of probit analysis (p \ 0.05, IBM SPSS 19 Inc.

software package). After checking for homogeneity of the

variance (Levene’s test of equality of error variances), the

significant differences among the biological parameters

measured in CBZ-treated algae and/or hemocytes of mussels

were tested, with the use of Mann–Whitney U test

(p \ 0.05). Significant alterations in the growth rate (l)

observed in algae treated with each concentration of CBZ for

a period of 24, 48, 72 and 96 h were tested with the use of the

Friedman test (p \ 0.05). Simple linear correlation (Pearson

test, p \ 0.05) analysis was conducted with the mean values

of each parameter tested (N = 5 in each case) for investi-

gating significant relationships among the biological

responses obtained in both algae and mussel hemocytes.

Results

Growth rate inhibition in the CBZ-treated green algae

Dunaliella tertiolecta

According to the results of the present study, a significant

inhibition (%I) of algae growth was observed at 24 h of

exposure (24 h IC50 = 53.2 mg L-1) for all concentrations

tested (Fig. 1A; Table 1A, B). The exposure of algae for

48, 72 and 96 h to CBZ showed a significant attenuation of

the CBZ ability to inhibit their growth (Fig. 1B–D), a fact

that was reinforced by the concomitant recovery of algal

growth rate (l), at levels almost similar to those obtained in

both control and DMSO-treated cells (Table 1A), as well

as by the increase of the IC50 values with time (Table 1B).

Lipid peroxidation in the CBZ-treated green algae

Dunaliella tertiolecta

Significantly elevated levels of MDA were observed in

cells treated with different concentrations of CBZ for 24,

48, 72 and 96 h (Fig. 2A–D). Specifically, CBZ at con-

centrations higher than 50 mg L-1 (100 and 200 mg L-1)

caused a significant increase of MDA content after expo-

sure of 24 h (Fig. 2A). On the other hand, despite the fact

that MDA levels measured in all cases showed a time-

dependent attenuation, cells treated with CBZ at concen-

trations higher than 10 mg L-1 (at 48 and 72 h) and/or

50 mg L-1 (at 96 h) showed significantly elevated MDA

levels, compared to the respective values obtained in

control cells in any case (Fig. 2B–D).

1212 P. Tsiaka et al.

123

Total carotenoids and Chl-a content in the CBZ-treated

green algae Dunaliella tertiolecta

The exposure of the green algae to different concentrations

of CBZ showed significant alterations in the levels of

carotenoids with time. Specifically, algae treated for 24, 48,

72 and 96 h with CBZ concentrations almost higher than

10 mg L-1 (50, 100 and 200 mg L-1) showed increased

levels of carotenoids (Fig. 3A–D). On the other hand, algal

cells treated for 24 h with each concentration of CBZ,

0

20

40

60

80

100

120

1 10 50 100 200

% I

24h

CBZ [mg/L]

0102030405060708090

100

1 10 50 100 200

% I

48h

CBZ [mg/L]

0

10

20

30

40

50

60

70

1 10 50 100 200

% I

72h

CBZ [mg/L]

0

5

10

15

20

25

30

35

1 10 50 100 200%

I 96

h

CBZ [mg/L]

aef bgh

ceh dfg

a

a

a

a

ab cd

ac

abcd

bd

A B

C D

Fig. 1 Inhibition of Dunaliella tertiolecta growth rate (%I), after

exposure to different concentrations of carbamazepine (CBZ) for

(A) 24, (B) 48, (C) 72 and (D) 96 h. In each case, values that share the

same letter represent significant difference from each other (Mann–

Whitney U test, p \ 0.05)

Table 1 CBZ effects on (A) cell number (cells/mL 9 104) and algal growth rate (l values in the parenthesis), as well as (B) CBZ inhibitory

concentration (24, 48, 72 and 96 h IC50 values)

A Incubation time (h)

24 48 72 96

Control 2.38 ± 0.14 (0.86 ± 0.06) 5.25 ± 0.87 (0.82 ± 0.08) 12.30 ± 1.50 (0.83 ± 0.04) 24.85 ± 1.76 (0.80 ± 0.02)

DMSO-treated cells 2.29 ± 0.15 (0.83 ± 0.07) 5.20 ± 0.43 (0.82 ± 0.04) 12.01 ± 1.60 (0.83 ± 0.00) 23.50 ± 2.50 (0.79 ± 0.03)

CBZ (mg L-1)

1 2.08 ± 0.14 (0.73 ± 0.07) 4.11 ± 0.34 (0.71 ± 0.04)a 8.71 ± 0.10 (0.72 ± 0.05) 24.25 ± 2.02 (0.80 ± 0.02)a

10 1.63 ± 0.18 (0.48 ± 0.11)*a 3.77 ± 1.21 (0.64 ± 0.07) 7.67 ± 1.00 (0.68 ± 0.08) 23.84 ± 2.42 (0.79 ± 0.03)a

50 1.63 ± 0.18 (0.48 ± 0.11)*ab 3.56 ± 1.38 (0.60 ± 0.21) 9.33 ± 3.18 (0.73 ± 0.11)a 19.79 ± 5.27 (0.74 ± 0.06)b

100 1.11 ± 0.13 (0.10 ± 0.11)*abc 2.92 ± 1.09 (0.51 ± 0.20)*a 7.14 ± 2.69 (0.64 ± 0.12)*b 16.88 ± 3.34 (0.70 ± 0.05)c

200 1.08 ± 0.14 (0.07 ± 0.13)*ab 2.17 ± 1.30 (0.33 ± 0.28)* 5.19 ± 3.51 (0.50 ± 0.21)*a 14.31 ± 5.97 (0.65 ± 0.13)*b

B Exposure period (h) IC50 (mg L-1)

24 53.2 (ND)

48 147.4 (93.7–332.8)

72 235.8 (159.4–660.2)

96 295.6 (209.5–711.6)

Values with * in each column differ significantly from control and/or DMSO-treated cells in each case (Mann–Whitney U test, p \ 0.05). Values in each row that

share the same letter are significantly different from each other (Friedman test, p \ 0.05)

IC50 values and confidence intervals (lower and upper bound values within parenthesis), as obtained by probit analysis, p \ 0.05, N = 4

Carbamazepine-mediated pro-oxidant effects 1213

123

showed a significant reduction of Chl-a (Fig. 4A), which

was followed by a significant increase when CBZ con-

centrations were higher than 10 mg L-1 (at 72 and 96 h)

(Fig. 4B–D).

Neutral red uptake in CBZ-treated hemocytes

of mussels

Since the neutral red uptake (NRU) provides a quantitative

estimation of cell viability, the results of the present study

showed a gradual decrease of hemocytes’ viability after

exposure to elevated levels of CBZ for 1 h (Fig. 5).

Hemocytes treated with 0.01 % DMSO showed negligible

(p [ 0.05) changes in cell viability, as well as in the rest of

the biological responses tested, compared to those occurred

in control cells (Table 2). Cells treated with CBZ at con-

centrations ranging from 0.01 to 10 lg L-1 showed up to

50 % reduction of their viability, while cells treated with

100 lg L-1 showed even higher levels of cell death (60 %

of mortality). According to the latter, further analysis of

biological responses was carried out by exposing hemo-

cytes to 0.01, 0.1, 1 and 10 lg L-1 of CBZ.

Oxidative effects of CBZ (in terms of �O2-, NO2

-

and MDA) on hemocytes of mussels

CBZ-treated hemocytes showed increased levels of �O2- in

any case, compared to those obtained in control and

DMSO-treated cells (Fig. 6A; Table 2). Similarly,

significantly elevated NO2- steady-state (stationary) levels

and MDA content were observed in cells treated with

different concentrations of CBZ (0.01. 0.1, 1 and

10 lg L-1), compared to those occurred in control and/or

DMSO-treated cells, in any case (Fig. 6B–C).

Correlations between biological parameters tested

In order to elucidate any relationship among CBZ-mediated

biological responses in algae and mussel hemocytes, a linear

correlation analysis (Pearson test, p \ 0.05, N = 5) was

performed (Table 3). According to the results, NRRT values

were negatively correlated to the levels of both �O2- and

NO2-, as well as to MDA content measured in hemocytes of

mussels. Similarly, the levels of either �O2- or NO2

- showed

a positive correlation with the levels of MDA (Table 3A).

On the other hand, Chl-a levels measured in 24 h CBZ-

treated algae showed a significantly negative correlation

with all other parameters tested (%I, carotenoids, MDA

content) (Table 3B). The growth rate inhibition (%I) cal-

culated for CBZ-treated algal cells showed a positive cor-

relation with the levels of MDA measured in all cases (24,

48, 72 and 96 h), while there was a strong positive corre-

lation among MDA with carotenoids in cells treated with

CBZ for 24, 72 and 96 h (Table 3B). Moreover, the MDA

content showed a positive correlation to Chl-a (at 72 and

96 h exposure period), while a strong positive correlation

was obtained among carotenoids with Chl-a in algae

treated with CBZ for 72 h (Table 3B).

0,00

0,20

0,40

0,60

0,80

1,00

1,20

0 1 10 50 100 200n

mo

l MD

A/1

06ce

llsCBZ [mg/L]

24hA B

C D

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0 1 10 50 100 200

nm

ol M

DA

/106

cells

CBZ [mg/L]

48h

0,00

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

0 1 10 50 100 200

nm

ol M

DA

/106

cells

CBZ [mg/L]

72h

0,000,010,010,020,020,030,030,040,040,050,05

0 1 10 50 100 200

nm

ol M

DA

/106

cells

CBZ [mg/L]

96h

*bcd

*b*a

*de

*ae

*c

**a

*

*a

*

*a

*bd *ce

bad abcd

c

abc de

Fig. 2 Determination of lipid

peroxidation (in terms of MDA

equivalents) in the green algae

Dunaliella tertiolecta, after

exposure to different

concentrations of

carbamazepine (CBZ) for

(A) 24, (B) 48, (C) 72 and

(D) 96 h. Asterisk indicates

significant difference from

control in each case. In each

case, values that share the same

letter represent significant

difference from each other

(Mann–Whitney U test,

p \ 0.05)

1214 P. Tsiaka et al.

123

Discussion

CBZ-mediated effects observed in both primary cell cul-

ture of mussel hemocytes and the unicellular marine algal

species D. tertiolecta could provide a useful tool for the

understanding of mechanisms involved in cellular respon-

ses, which represent a key level of organism and/or pop-

ulation health status. Despite the absence of data

concerning the antioxidant responses (including enzymatic

systems), which could elucidate the role of CBZ as a pro-

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 1 10 50 100 200p

g/c

ell

CBZ [mg/L]

24hA

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 1 10 50 100 200

pg

car

/cel

l

CBZ [mg/L]

48hB

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

0 1 10 50 100 200

pg

car

/cel

l

CBZ [mg/L]

72hC

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0 1 10 50 100 200

pg

car

/cel

l

CBZ [mg/L]

96hD

* *cd

*bf*cg

*a

*ef

*defg *

*bc *bdf

*ac *bd

*ace

*ae

abab

c

abcd

cdab

Fig. 3 Determination of

carotenoids in the green algae

Dunaliella tertiolecta, after

exposure to different

concentrations of

carbamazepine (CBZ) for

(A) 24, (B) 48, (C) 72 and

(D) 96 h. Asterisk indicates

significant difference from

control in each case. In each

case, values that share the same

letter represent significant

difference from each other

(Mann–Whitney U test,

p \ 0.05)

0,0

2,0

4,0

6,0

8,0

10,0

12,0

pg

/cel

l

CBZ [mg/L]

72 hC

*adg*be

*cfg

defabc

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

pg

/cel

l

CBZ [mg/L]

96hD

ab

*adf

cde

*beg

*cfg

0,0

0,5

1,0

1,5

2,0

2,5

pg

/cel

l

CBZ [mg/L]

24 hA

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

0 1 10 50 100 200 0 1 10 50 100 200

0 1 10 50 100 200 0 1 10 50 100 200

pg

/cel

l

CBZ [mg/L]

48 hB

*****

Fig. 4 Chlorophyll alpha (Chl-

a) content in the green algae

Dunaliella tertiolecta after

exposure to different

concentrations of

carbamazepine (CBZ) for

(A) 24 h, (B) 48, (C) 72 and

(D) 96 h. Asterisk indicates

significant difference from

control in each case. In each

case, values that share the same

letter represent significant

difference from each other

(Mann–Whitney U test,

p \ 0.05)

Carbamazepine-mediated pro-oxidant effects 1215

123

oxidant, the present study clearly showed that environ-

mentally relevant concentrations of CBZ could enhance

oxidative stress-related effects on mussel hemocytes. On

the other hand, given that a lot of testing chemicals, such as

CBZ, could bind to serum and proteins of the culture

medium (Kramer et al. 2012), which in turn could decrease

the free or effective concentration of the drug, CBZ-med-

iated effects on algal cells were investigated after their

exposure to CBZ concentrations, higher than those found in

the environment. The latter should be taken into account

when performing in vitro studies in order to assess the

possible biological effects of chemicals and their environ-

mental risk.

CBZ-induced effects on the green algae Dunaliella

tertiolecta

Since phytotoxicity tests based on growth inhibition, as

well as the amount of chlorophyll a, are widely used for

testing the adverse effects of chemicals, the decreased

values of algal growth rates and/or Chl-a measured in algal

cells, treated with different concentrations of CBZ at least

after 24 h of exposure, clearly indicate the CBZ toxic

potency. These findings are in accordance with previous

studies performed in algae and species from various taxa,

such as bacteria and invertebrates (Ferrari et al. 2003;

Nalecz-Jawecki and Persoone 2006; Reish and Lemay

2008; Zhang et al. 2012).

Although there is evidence that PhCs, such as CBZ and

chloramphenicol, can inhibit Chl-a synthesis and the algal

growth rate (reviewed by Zhang et al. 2012), probably via

their interference with the synthesis of protochlorophyll and

its subsequent conversion to chlorophyll, the CBZ ability to

inhibit algal growth rate and Chl-a in algal cells via the

enhancement of oxidative stress-related effects, such as

lipid peroxidation, could not be excluded. In fact, increased

levels of lipid peroxidation was observed in rainbow trout

Oncorhynchus mykiss primary hepatocytes and the cnidar-

ians Hydra attenuate (Quinn et al. 2004; Gagne et al.

2006a), but CBZ ability to induce lipid peroxidation prod-

ucts in algal cells is reported for the first time. It seems that

the enhancement of lipid peroxidation in algae could be due

to their ability to take up CBZ in a time-dependent manner

(Andreozzi et al. 2002) as well as its oxidative biotrans-

formation, which could lead to oxidative stress-related

products, such as lipid peroxides (Vernouillet et al. 2010).

The attenuation of CBZ toxic effects with time, as

indicated by the concomitant recovery of both the algal

growth rate and Chl-a in algal cells, is of great concern.

Although the decrease of Chl-a is commonly considered as

a reliable indicator of pollutant toxicity (Wang and Free-

mark 1995), there is evidence for the enhancement of its

levels in Dunaliella sp. and other species tested after

exposure to pro-oxidants (El-Sheekh et al. 2003; Ma-

nankina et al. 2003; Nikookar et al. 2005). Since the

implication of CBZ in biochemical processes that could

lead to the induction of oxidative stress-related effects on

marine algae is merely known, the present study showed

for the first time that the attenuation of CBZ inhibitory

potency with time could be related with the induction of

protective mechanisms against CBZ-induced oxidative

effects. In fact, although Zhang et al. (2012) reported a

significant increase of antioxidant enzymes activity in algal

species Scenedesmus obliquus and Chlorella pyrenoidosa,

after exposure to different concentrations of CBZ

(0.5–10 mg L-1), in order to protect cells from the oxi-

dative effects induced by the excessive production of free

radicals, such as .O2-, the results of the present study

showed for the first time the important role of other pro-

tective mechanisms, such as carotenoids increase, against

CBZ-mediated oxidative stress.

The role of antioxidant molecules in green algae is of

great concern and the significant increase of total carote-

noids in CBZ-treated algal cells could reveal their impor-

tant role during algal growth and survival, under CBZ-

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

0 0.01 0.1 1 10 100

OD

550

nm

/mg

pro

tein

CBZ [µg/L]

* * ** *

Fig. 5 Evaluation of cell viability (Neutral red uptake) in hemocytes

treated with different concentrations of carbamazepine (CBZ 0.01,

0.1, 1, 10 and 100 lg L-1) for 1 h. Asterisk indicates significant

difference from control in each case (Mann–Whitney U test,

p \ 0.05)

Table 2 Neutral red uptake (NR), superoxide anions (.O2-), nitrite

(NO2-) and lipid peroxidation content (in terms of MDA equivalents)

in hemocytes of mussels exposed to DMSO 0.01 % v/v, in relation to

control values

NR* .O2-** NO2

-*** MDA****

Control 1.19 ± 0.22 1.06 ± 0.43 0.45 ± 0.06 0.12 ± 0.02

DMSO-treated cells 1.05 ± 0.31 0.97 ± 0.39 0.51 ± 0.07 0.13 ± 0.02

The results are mean ± SD from at least 4 independent experiments in each case

* OD550nm/mg protein

** OD620nm/mg protein

*** nmol NO2-/mg protein

**** nmol MDA/mg protein

1216 P. Tsiaka et al.

123

mediated stress conditions. Given that Dunaliella sp.

accumulates large amounts of total carotenoids and espe-

cially b-carotenes (accounted for more than 80 % of total

carotenoids) (Del Campo et al. 2007), their enhanced levels

in CBZ-treated cells could be linked with growth rate and

Chl-a recovery with time. In specific, total carotenoids can

react with lipid peroxidation products, in order to terminate

chain reactions, and/or quench singlet oxygen and free

radicals, possibly generated within cells, thus serving as a

protective mechanism of the photosynthetic apparatus

(Telfer 2002) and the concomitant algal growth and sur-

vival. Although the latter was further reinforced by the

significant relationships occurred among lipid peroxidation

products and/or the inhibition rate (%I) with the levels of

carotenoids occurred in CBZ-treated algal cells, thus

indicating the important role of low molecular weight

antioxidants such as carotenoids against oxidative stress

(Salguero et al. 2003; Nikookar et al. 2005), more studies

are needed in order to elucidate the role of non enzymatic

and enzymatic antioxidant mechanisms in algal cells faced

with agents, such as CBZ, that limit their growth and

survival.

Cytotoxic and oxidative stress-related effects of CBZ

on mixed primary culture of mussel hemocytes

The cytotoxic and oxidative stress-related effects observed

in CBZ-treated hemocytes of mussels clearly indicate the

ability of some PhCs to affect the immune system of non-

target organisms, such as molluscs (Canesi et al. 2007;

Contardo-Jara et al. 2011; Gust et al. 2012). It seems that

CBZ-mediated oxidative stress-related effects could be due

its ability to stimulate the phagocytic process in hemocytes

of mussels (Gagne et al. 2006b). Specifically, since the

CBZ uptake by aquatic organisms is well known (Quinn

et al. 2008; Vernouillet et al. 2010; Contardo-Jara et al.

2011), it seems reasonable to suggest that CBZ accumu-

lation within lysosomes could lead to lysosomal membrane

impairment and cell death, as previously mentioned (Jos

et al. 2003; Martin-Diaz et al. 2009; Parolini et al. 2011).

The intralysosomal environment is a site of oxyradical

production (Winston et al. 1991; Livingstone 2001) and the

release of hydrolytic enzymes into the cytosol could induce

oxidative effects, such as peroxidation of membrane lipids,

as reported in the present study.

Although the induction of lipid peroxidation in tissues of

mussels and fish exposed to CBZ is well documented

(Gagne et al. 2006a, 2006b; Martin-Diaz et al. 2009; Li

et al. 2010), the present study showed for the first time the

enhancement of free radicals, such as �O2- and NO2

- within

mussels hemocytes, after exposure to environmentally rel-

evant concentrations of CBZ. Those radicals could lead to

the formation of peroxynitrite (ONOO-), which is highly

toxic for cells, because of its strong oxidizing properties

towards proteins and non-protein thiols, deoxyribose, and

membrane phospholipids (Xia and Zweier 1997).

00,5

11,5

22,5

33,5

4

OD

620n

m/m

g p

rote

in

CBZ [µg/L]

**

*

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

nm

ol N

O2- /m

g p

rote

in

CBZ [µg/L)

* * **

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0 0.01 0.1 1 10 0 0.01 0.1 1 10

0 0.01 0.1 1 10nm

ol M

DA

/mg

pro

tein

CBZ [µg/L]

** * *

A B

C

Fig. 6 Determination of (A) superoxide anions (�O2-), (B) nitrites

(NO2-) and (C) MDA content in hemocytes treated with sub-lethal

concentrations of carbamazepine (CBZ 0.01, 0.1, 1 and 10 lg L-1)

for 1 h. Asterisk indicates significant difference from control in each

case (Mann–Whitney U test, p \ 0.05)

Carbamazepine-mediated pro-oxidant effects 1217

123

Regarding that phagocytosis can promote the respiratory

burst process (Arumugam et al. 2000), the CBZ-mediated�O2

- and NO2- generation could be merely due its ability

to enhance respiratory burst enzymes activity, such as

NADPH oxidase and NO synthase. This suggestion is

based on CBZ ability to interact directly with the adenylyl

cyclase (AC) enzyme, and/or with membrane receptors

coupled to the AC/cAMP system (Montezinho et al. 2007;

Martin-Diaz et al. 2009), as well as the important role of

the aforementioned signaling molecules to the induction of

respiratory burst process in hemocytes of mussels, as

recently mentioned (Dailianis 2009; Banakou and Dailianis

2010; Vouras and Dailianis 2012). Although the latter was

further reinforced by the significant relationships between

cytotoxic and oxidative stress-related parameters tested in

CBZ-treated hemocytes, more studies are needed for elu-

cidating the involvement of CBZ to signaling cascades,

commonly linked with immune response mechanisms.

Conclusion

The present study clearly showed the CBZ pro-oxidant

behavior in the marine species D. tertiolecta and the

immune defence-related hemocytes of mussel M. gallo-

provincialis, widely used in ecotoxicological and toxico-

logical studies. Algal growth and survival could be related

with the enhancement of total carotenoids, such as b-

carotenes, against CBZ-mediated adverse effects. More-

over, the effects of environmentally relevant concentrations

of CBZ on the immune system of mussels reveal its

potential environmental risk, since immunosurveillance is

commonly linked with the health status of these organisms.

In general, further studies (both in vivo and in vitro) with

the use of different non-target organisms and different

cellular types could reveal the adverse environmental

impact of PhCs in aquatic species.

Acknowledgments This study was supported by the annual

research budget sanctioned to the Section of Animal Biology by the

University of Patras, Greece.

Conflict of interest The authors declare that they have no conflict

of interest.

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