Seasonal variation of bioaccumulation in Engraulis encrasicolus (Linneaus, 1758) and related biomarkers of exposure

Page 1

Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]

Contents lists available at SciVerse ScienceDirect

Ecotoxicology and Environmental Safety

0147-65

http://d

$The

Departmn Corr

E-m

Pleasbiom

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

Frontier Article

Seasonal variation of bioaccumulation in Engraulis encrasicolus(Linneaus, 1758) and related biomarkers of exposure$

Chiara Copat a,b, Maria Violetta Brundo c, Giovanni Arena a, Alfina Grasso a, Gea Oliveri Conti a,Caterina Ledda a, Roberto Fallico a, Salvatore Sciacca a, Margherita Ferrante a,n

a Department of Hygiene and Public Health ‘‘G.F. Ingrassia’’, University of Catania, Via Santa Sofia 87, 95123 Catania, Italyb Ketos Scientific Association, Viale Mario Rapisardi 220, 95100 Catania, Italyc Department of Biology ‘‘Marcello La Greca’’, University of Catania, Via Androne 81, 95124 Catania, Italy

a r t i c l e i n f o

Article history:

Received 12 July 2012

Received in revised form

3 September 2012

Accepted 6 September 2012

Keywords:

Bioaccumulation

Biomarkers

Fish

Mediterranean sea

Environmental health

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.09.006

work described in the manuscript was entire

ent of Hygiene and Public Health ‘‘G.F.Ingra

esponding author. Fax: þ39 095 378 2177.

ail address: [email protected] (M. Ferrante).

e cite this article as: Copat, C., et al.,arkers of exposure. Ecotoxicol. Envi

a b s t r a c t

In the last years, the development of new techniques and the increase of sophisticated assays has

improved the environmental monitoring programs by using fish as bioindicators of marine ecosystems,

applying a multibiomarkers approach.

The aim of this study was to assess the contamination level of the Catania Gulf through

bioaccumulation and contaminant exposure response in Engraulis encrasicolus (Linnaeus, 1758). Heavy

metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were analyzed

in muscle tissue of the species. Furthermore we evaluated biomarkers of exposure to such contami-

nants: the metallothioneins (MTs), the ethoxyresorufin-O-deethylase (EROD) and stress protein or

‘‘heat shock proteins’’ (HSP70).

In total, three sampling of 30 specimens each were performed during 2011. Comparison between

classes of contaminants was performed using Student’s t-test for paired samples

Data showed that: the populations of E. encrasicolus accumulated a certain amount of metals, PAHs

and organochlorine compounds in their tissues, that, however, did not exceed the legal limits set by

Regulation 1881/2006 for contaminants in fish muscle; essential metals accumulate more during the

reproductive periods of the species; the accumulation of heavy metals and PAHs are more closely

related to natural and anthropogenic events; the presence of PCBs, although in traces, is the

confirmation that banned compounds persist in the environment for several years; the levels of

contaminants found in the tissues of the species do not seem to be likely to cause toxic effects both in

the studied fish species.

In conclusion, environmental risk assessment came out in this study for the Catania Gulf, showed a

positive picture, although the contaminants detected are related to a certain degree of anthropic

impact, and should be constantly monitored to ensure quality standards of the studied area.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

In recent years the development of new techniques andincreasingly of sophisticated assays has improved the environ-mental monitoring plans through the use of bio-indicator species(Al-Yakoob et al., 1993; Andral et al., 2004; Bayarri et al., 2001;Coelhan et al., 2006; Copat et al., 2012a; de Andrade et al., 2004;Dural et al., 2006; Perugini et al., 2009). The use of biomarkers,working closely with a targeted approach to the study of ‘‘health’’of an ecosystem and its components, evaluate the ‘‘early adverse

ll rights reserved.

ly financed from funds of the

ssia’’, University of Catania.

Seasonal variation of bioaccron. Saf. (2012), http://dx.d

effects’’ caused by contaminants on organisms directly in theirnatural environment (Bucheli and Fent, 1995; Depledge, 1994;Doherty et al., 2010; Fasulo et al., 2010; Flammarion et al., 2002;Fossi et al., 2000; Linde-Arias et al., 2008; van der Oost et al.,2003).

The mere presence of chemical contaminants in a segment ofthe aquatic ecosystem, does not indicate itself harmful effects, butaccording to van der Oost et al. (2003) it is necessary to establishconnections between the bioaccumulation levels, and the ‘‘earlyadverse effects’’.

Fish have been considered good bioindicators of aquaticecosystems in environmental quality assessment (Whitfield andElliott, 2002), and analysis of levels of contaminants in theirtissues and their biomarkers exposure is one of the main objec-tives in environmental biomonitoring programs (Fossi, 1998).

umulation in Engraulis encrasicolus (Linneaus, 1758) and relatedoi.org/10.1016/j.ecoenv.2012.09.006

Page 2

Fig. 1. Study area.

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]2

E. encrasicolus, perfectly reflects the bioavailability of contami-nants in its trophic niche, with the exception of those subject tobiomagnification such as mercury, which reaches very highconcentrations in other pelagic species (Falco et al., 2006;Marti-Cid et al., 2008). Furthermore, this fish species is amongthe most important from an economic point of view in the Sicilianfisheries, representing 5.67 percent of total seafood production(ISTAT, 2000).

The study area chosen for monitor the marine environmentstatus is the Gulf of Catania, an inlet of the Ionian Sea on theeastern coast of the Italian island of Sicily. The area coversapproximately 300 km2 and it lies between Cape Mulini(371 42,871N, 0151 13,183E) to the north and by Cape Campolato(371 14,569N, 0151 15,430E) to the south. The north-central costalarea consisting of volcanic rocks originated by Mt. Etna activity,instead, the southern coast, consisting of sedimentary soils,originated by the floods of the Simeto river and its maintributaries. This area receives a large amount of urban wastewaterand treated water from a variety of sources: light industry, foodprocessing industries, greenhouses and farms. Till date, fewstudies have provided information about the marine pollutiongrade of this area (Copat et al., 2012b; Storelli and Marcotrigiano,2005; Tigano et al., 2009), revealing a generalized low contam-ination of heavy metals in muscle tissue of E. encrasicolus,S. pilchardus and P. sanguinolentus, and M. barbatus.

Thus, the present study was aimed to evaluate the possibleseasonal variation of muscle bioaccumulation of the species E.

encrasicolus (Linnaeus, 1758), by analyzing essential and nonessential metals, polycyclic aromatic hydrocarbons (PAHs) andpolychlorinated biphenyls (PCBs) dioxin-like. Furthermore, inliver samples we evaluated biomarkers of exposure to thesecontaminants, such as: the metallothioneins (MTs), the heatshock proteins (HSP70) and the ethoxyresorufin O-deethylase(EROD).

2. Materials and methods

2.1. Sampling

During 2011, a total of 90 E. encrasicolus specimens were collected in the Gulf

of Catania (Fig. 1), respectively 30 in February, 30 in June and 30 in September. The

last two sampling periods correspond to the reproductive period of the species.

Adult fish samples were caught from a local fishing boat, where fish liver and

muscles were excised, transported in dry ice to the laboratory where they were

stored at �80 1C until analysis.

2.2. Heavy and essential metals

Using an heated mixture of strong acids, 1 g of muscle tissue per fish was

mineralized in a microwave system (ETHOS TC MILESTONE). The method for

animal tissue, provided from the instrument digestion cookbook Milestone,

requires a digestion solution prepared with 6 mL of HNO3 65 percent (Carlo Erba

Chemicals) and 2 mL of H2O2 30 percent (Carlo Erba Chemicals) with a 50 min

operation cycle at 200 1C. After mineralization the samples were made up to

20 mL with the addition of ultra pure water (Merck), they were then divided into

two aliquots each of 10 mL: one for Hg measurement and the second for the other

metals. Blanks, standards and samples for Hg analysis were oxidized with 1 mL of

potassium permanganate 5 percent (KMnO4), to obtain the conversion of organic

Hg into inorganic Hg and then the excess of permanganate was reduced with 1 mL

of hydroxylamine hydrochloride (NH2OH �HCl) 1.5 percent (final volume of

12 mL). An ICP-MS Elan-DRC-e (PERKIN ELMER) was used for the quantification

of As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Se, V and Zn, applying UNI EN

ISO 17294–2:2005 method Hg was analyzed with a FIAS 100 (PERKIN ELMER)

using the cold vapor capture technique, applying UNI EN 1483. Standards for the

instrument calibration were prepared on the basis of the mono-element certified

reference solution AAS Standard (Merck). Suitability of the methods with these

matrices were checked by analysis of NIST standard reference material Lake

Superior fish 1946, processed with a mean recovery between 75 percent and 120

percent of the available certified values. The estimated method detection limits

are (mg/kg w.w.): As 0.013, Ca 1.15, Cd 0.0003, Co 0.001, Cr 0.003, Cu 0.346,

Please cite this article as: Copat, C., et al., Seasonal variation of bioacbiomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.d

Fe 0.050, Hg 0.001, K 1.15, Mg 1.54, Mn 0.055, Na 1.74, Ni 0.001, P 1.87, Pb 0.001,

Se 0.002, V 0.002, Zn 1.09.

2.3. Polycyclic aromatic hydrocarbons (PAHs)

PAHs extracted and analyzed with an in-house method (Conti et al., 2012)

included naphthalene (NA), acenaphthylene (ACY), acenaphthene (AC), fluorene

(FL), fhenanthrene (PHE), anthracene (AN), fluoranthene (FA), pyrene (PY), benzo a

antrachene (BaA), chrysene (CH), benzo b fluoranthene (BbF), benzo k fluor-

anthene (BkF), benzo a pyrene (BaP), dibenzo ah antrachene (DB a,h A), benzo g h I

pyrene (B g,h,i P), indeno 123-cd pyrene (I 1,2,3-cd P). For the analysis, a sample of

about 1 g of muscle tissue was weighed with an analytical balance (Mettler Toledo

AT 104, USA), homogenized, and sonicated with a solution of dichloromethane:a-

cetone 1:1 (v:v) using a sonicator (ISCO, Italy). The extract obtained was

concentrated with a stream of nitrogen and loaded into a Varian Bond Elut C18

cartridge (12 mL), previously conditioned with 5 mL of dichloromethane, 5 mL of

isopropanol and 5 mL of distilled water. The collection vial was cleaned with 8 mL

of 1:1 (v:v) dichloromethane:acetone and always loaded on the cartridge. The

eluates were again concentrated in a stream of nitrogen, 1 mL of acetonitrile (ACN)

was added, and analysis was performed by high performance liquid chromato-

graphy (HPLC) Prostar Varian, USA. The analytical method provided a mobile

phase consisting of 1:1 (v:v) H2O/ACN for 5 min, which achieved 100 percent ACN

in 10 min with a flow of 1.5 mL/min. The ultraviolet determination was performed

at 255 nm, while the fluorescent detection was conducted at six different

wavelengths of excitation and emission (Table 1). Suitability of the methods

was checked by recovery tests of fortified fish samples (1 mg/mL) that showed

mean recovery between 85 percent and 120 percent of the individual PAHs. The

estimated detection limits for all PAHs analyzed is 0.002 (mg/kg w.w).

2.4. Polychlorinated biphenyls (PCBs)

For the determination of PCBs was used an in-house method. Suitability of the

method was checked by analysis of NIST standard reference material Lake

Superior fish 1946, processed with a mean recovery between 75 percent and

120 percent for the available certified values. 1 g of each muscle sample was

digested with 20 mL of HCl (Carlo Erba Chemicals) for 24 h. Then, 20 mL of 3:1

(v:v) dichloromethane-hexane solution was added to this liquid fraction and the

cumulation in Engraulis encrasicolus (Linneaus, 1758) and relatedoi.org/10.1016/j.ecoenv.2012.09.006

Page 3

Table 1Excitation/emission time program for fluorescence detector.

Time interval (min) Excitation (nm) Emission (nm)

0–17.9 250 360

17.9–20.5 375 425

20.5–24.8 335 440

24.8–33.5 350 430

33.5–38.0 305 500

38.0–45.0 250 360

PP

bS

eV

Zn

31

18

73

45

0.0

05

70

.00

3

0.4

34

70

.07

6

0.3

38

70

.08

4

8.9

6

72

.03

28

64

74

50

0.0

12

70

.00

6

0.6

85

70

.13

8

0.3

15

70

.05

7

27

.6

78

.80

29

41

75

50

0.0

2

70

.01

0

0.3

01

70

.04

9

0.0

74

70

.03

7

19

.4

79

.29

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]] 3

PCBs extraction was performed with separator funnel in twice. The extract was

collected in flasks, dried in rotavapor (Buchi-Heating Bath B-490) till a 2 mL of

volume and purified in florisil 12 mL cartridge with hexane, previously condi-

tioned as per the attached guidelines. The collected extract was again dried in

rotavapor and with a stream of nitrogen till a total dryness, and recovered with

hexane up 1 mL volume. A gas chromatography GC2010-ECD Schimadzu was used

to perform analysis of the PCBs dioxin-like (81S; 77S; 123S; 118S; 114S; 105S;

126S; 167S; 156S; 157S; 169S; 189S). The estimated detection limits for all PCBs

analyzed is 0.002 (mg/kg w.w).

Mg

Mn

Na

Ni

79

76

1

41

3

74

2.8

0.5

75

70

.12

3

11

75

73

88

0.0

66

70

.03

2

49

63

7

60

4

71

49

1.1

06

70

.40

7

16

84

75

15

0.1

26

70

.04

6

35

14

10

57

0

79

9.2

0.6

69

70

.28

8

18

18

75

11

0.3

56

70

.17

2

2.5. Immunoblotting

MTs, HSP70 and EROD determination in liver was carried out by Western

blotting as previously reported (Tigano et al., 2009). Briefly, the fish tissues were

weighed, homogenized 1:10 (w/v) in a lysis buffer (Tris–HCl 40 mM, EDTA 25 mM,

0.2 percent SDS, pH 7.4) containing 1/100 (v/v) protease inhibitors (Sigma) and

centrifuged. Total protein concentration in the supernatant was determined

according to the Bradford method (Bradford, 1976). 30 mg of protein/lane was

analyzed by minigel SDS-PAGE and transferred to a nitrocellulose membrane

using Transblot (Biorad). The proteins levels were measured by incubating

nitrocellulose membranes overnight at 4 1C with mouse monoclonal primary

antibodies anti-MTs (1:500, QED Bioscience 5 Inc.) anti-HSP70 (1:1000, Abcam),

and anti CYP1A1 (1:500, Abcam) respectively. The complex protein-primary

antibody was detected using a HRP-conjugated Ig-G anti-mouse secondary anti-

body (1:5000, Abcam) by chemiluminescent method. Quantitative measurements

were performed by densitometry analysis of the X-ray films with the Scion Image

(4.03) program, expressing values as arbitrary densitometric units (A.D.U.).

ng

pe

rio

ds

(N¼

30

).

Hg

K

0.1

44

70

.06

5

45 7

0.0

39

70

.03

1

45 7

0.0

65

70

.02

5

52 7

2.6. Statistical analysis

Statistical analysis was carried out with Student’s t-test for paired samples,

applying po0.05 as the minimum level of significance, using the statistical

software package SYSTAT, version 9 (Systat Inc., Evanston, IL, USA).

Ta

ble

2M

ea

nco

nce

ntr

ati

on

(mg

/kg

w.w

.)a

nd

sta

nd

ard

de

via

tio

no

fm

eta

lsin

the

thre

esa

mp

li

As

Ca

Cd

Co

Cr

Cu

Fe

Feb

6.6

3

72

.02

62

5

73

27

0.0

05

70

.00

3

0.0

12

70

.00

3

0.8

9

70

.17

0.6

9

70

.13

7.9

8

74

.39

Jun

e5

.41

71

.39

94

5

72

84

0.0

07

70

.00

5

0.0

22

70

.00

6

1.1

3

70

.32

1.8

8

70

.90

12

.3

75

.08

Se

p5

.28

71

.75

84

9

72

32

0.0

05

70

.00

3

0.0

18

70

.01

1

0.3

3

70

.31

0.7

9

70

.24

17

.4

79

.41

3. Results and discussions

The results obtained on heavy metals bioaccumulation(Table 2) show some statistically significant differences, both inrelation to the different periods of the year as well as differentstages of the life cycle of the species.

Concentrations of metals such as Ca, Co, Cu, Fe, K, Mg, Mn, Na,Se and Zn, were significantly higher in June (po0.05 for Ni,po0.01 for Fe, po0.001 for Ca, Co, Cu, Mg, Mn, Na, Se, Zn) andSeptember (po0.01 for Ca, po0.001 for Co, Fe, Mg, Na, K, Ni, Zn )(reproductive period) compared to those found in February (nonreproductive period) (Table 2). This is due to the increase inmetabolic demand during the reproductive phases, that mayfacilitate the absorption of some essential metals (Dragun et al.,2007; Marijic and Raspor, 2010; Riggio et al., 2003).

Among essential metals, it was possible to conduct a compar-ison with the literature data only for Zn, Fe and Mn: Znconcentrations found by others authors (Papetti and Rossi,2009; Topcuoglu et al., 2002; Turan et al., 2009; Turkmen et al.,2008; Yildirim et al., 2009), are in line with concentrations foundin February, but significantly lower than those found in repro-ductive periods; Fe and Mn concentrations are lower than thosefound by other authors (Topcuoglu et al., 2002; Turan et al., 2009;Turkmen et al., 2008).

Please cite this article as: Copat, C., et al., Seasonal variation of bioaccumulation in Engraulis encrasicolus (Linneaus, 1758) and relatedbiomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.doi.org/10.1016/j.ecoenv.2012.09.006

Page 4

Ta

ble

3M

ea

nco

nce

ntr

ati

on

s(m

g/k

gw

.w.)

an

dst

an

da

rdd

ev

iati

on

so

fP

AH

sin

the

thre

esa

mp

lep

eri

od

s(N¼

30

).

PA

Hs

NA

AC

YA

CFL

PH

EA

NFA

PY

Ba

AC

HB

bF

Bk

FB

aP

DB

ah

AB

gh

iPIP

P1

6IP

A

Feb

1.1

76

70

.73

2

0.7

57

70

.42

3

0.5

61

70

.25

8

0.8

22

70

.49

7

0.1

03

70

.08

6

0.1

04

70

.04

4

0.4

20

70

.28

3

0.0

07

70

.00

9

0.0

18

70

.01

9

0.0

05

70

.00

7

0.0

01

a

70

.00

1a

0.0

04

70

.00

5

0.0

02

70

.00

3

0.0

21

70

.03

3

0.0

01

a

70

.00

1a

0.0

09

70

.00

7

4.0

11

72

.43

3

Jun

e1

.07

4

70

.26

0

0.6

75

70

.13

3

0.2

94

70

.45

2

0.8

02

70

.49

0

0.2

95

70

.13

2

0.1

33

70

.07

4

0.7

68

70

.11

6

0.0

06

70

.00

7

0.0

12

70

.00

9

0.0

02

70

.00

3

0.0

02

70

.00

7

0.0

03

70

.00

4

0.0

02

70

.00

2

0.0

08

70

.01

5

0.0

03

70

.00

4

0.0

02

70

.00

3

4.0

82

72

.05

6

Se

p1

.09

8

70

.08

2

0.6

56

70

.29

1

0.3

52

70

.18

8

0.3

15

70

.26

3

0.7

68

70

.34

7

0.7

68

70

.24

9

0.8

84

70

.14

9

0.0

02

70

.00

1a

0.0

10

70

.00

8

0.0

02

70

.00

3

0.0

02

70

.00

1a

0.0

02

70

.00

1a

0.0

02

70

.00

3

0.0

02

70

.00

1a

0.0

02

70

.00

2

0.0

01

a

70

.00

1a

4.8

68

71

.61

0

a0

.00

1v

alu

eco

rre

spo

nd

sto

the

ha

lfm

eth

od

de

tect

ion

lim

it.

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]4

Analysis of fish sampled in winter showed a greater accumula-tion of As, Hg and V (Table 2), compared to the June (po0.05 forAs, po0.01 for V, po0.001 for Hg) and September ones (po0.05for As, po0.001 for Hg, V), and concentrations we found arehigher than those shown in literature for the same species (Falcoet al., 2006; Marti-Cid et al., 2007; Papetti and Rossi, 2009; Sepeet al., 2003). This is probably due to the volcanic origin of thesemetals (Bundschuh et al., 2011; Di Leonardo et al., 2006;Hernandez and Rodriguez, 2012; Nagai et al., 2006), since in theMediterranean basin there are numerous underwater volcanoes,especially in the Ionian and Tyrrhenian Seas (Di Leonardo et al.,2006), and the Gulf of Catania is located at the base of the biggestactive European volcano, the Mt. Etna. However, among thesemetals, only Hg has a limit of 0.5 mg/kg set by the EuropeanRegulation 1881/2006 for muscle fish tissue, and our results arelower (Table 2). Furthermore, during the winter, the speciesstudied migrates to deeper and offshore waters, where thebioavailability of volcanic contaminants increases.

The other analyzed metals do not show significant seasonaldifferences in concentrations. Comparing our data with literatureinformation for the same species it arises that: mean values of Cd,Co and Pb (Table 2), in all seasons, are in agreement withconcentrations found by other authors (Falco et al., 2006; Marti-Cid et al., 2007; Papetti and Rossi, 2009; Sepe et al., 2003;Topcuoglu et al., 2002; Turan et al., 2009; Turkmen et al., 2008;Yildirim et al., 2009); Ni mean values of fish sampled in Februaryand June are on in line with literature data, except mean value ofSeptember (Table 2), that is slightly higher (Papetti and Rossi,2009; Turan et al., 2009; Turkmen et al., 2008; Yildirim et al.,2009); Cu mean values (Table 2), are in agreement with literaturedata (Topcuoglu et al., 2002; Turkmen et al., 2008), except for Cudetected by Papetti and Rossi (2009) in samples of E. encrasicolus

caught in the Tyrrhenian coast of Southern Lazio. Instead, wefound Cr concentrations (Table 2) higher than those found inliterature (Sepe et al., 2003; Topcuoglu et al., 2002; Turan et al.,2009; Turkmen et al., 2008; Yildirim et al., 2009), although theyare not significantly higher to assume a particular contaminationof the examined study area. Among those heavy metals, only Cdand Pb, have the limits set by EC Regulation no. 1881/2006 formuscle fish tissue, of 0.10 mg/ kg and 0.30 mg/ kg, respectively.Again, concentrations we found do not exceed the above limits.

Data concerning single polycyclic aromatic hydrocarbons(PAHs) have shown, for low molecular weight PAHs, higherbioaccumulation of PHE, AN and FA in the summer period(po0.001; Po0.01; po0.05), while, some high molecular weightcompounds, have a greater bioaccumulation in winter, in parti-cular BaA, CH, BkF, DbahA and IP (po0.05).

It is known that PAHs originate from potentially incompletecombustion of fossil fuels, combustion of biomass, municipalwaste, from accidental spills of hydrocarbons, from the decom-position of organic matter and the vessel traffic (Guo et al., 2006,2007; Liu et al., 2005). But, it must be required to consider alsoseasonal variations due to rainfall, chemical and physical watervariations, and changes in annual emissions of anthropogenicPAHs, which together result in change of bioavailability. Theincreased presence of low molecular weight PAHs in summer iscertainly a consequence of increased ship traffic, while theincreased availability of high molecular weight PAHs in thewinter period is probably given by the greater water hydrody-namic that promotes, with the mixing of seabed, the ascent of theheavier compounds and PAHs bound with sediments (Colomboet al., 2005; Kannan et al., 2005). Although there are significantseasonal variations for the single compound, the statisticalcomparison of the SPAHs do not show significant seasonaldifferences. PAHs data reported in literature for the same speciesand for other species in different areas, seem to indicate a degree

Please cite this article as: Copat, C., et al., Seasonal variation of bioaccumulation in Engraulis encrasicolus (Linneaus, 1758) and relatedbiomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.doi.org/10.1016/j.ecoenv.2012.09.006

Page 5

Ta

ble

4M

ea

nco

nce

ntr

ati

on

(pg

/gT

EQ

)a

nd

sta

nd

ard

de

via

tio

no

fP

CB

sd

iox

in-l

ike

inth

eth

ree

sam

pli

ng

pe

rio

ds

(N¼

30

).

PC

Bs

dio

xin

-lik

e

81

S7

7S

12

3S

11

8S

11

4S

10

5S

12

6S

16

7S

15

6S

15

7S

16

9S

18

9S

PP

CB

Feb

0.0

677

0.0

54

0.0

107

0.0

22

0.0

047

0.0

08

0.0

557

0.0

59

0.0

317

0.0

36

0.0

587

0.0

91

0.1

207

0.1

95

0.0

107

0.0

19

0.0

057

0.0

03

0.0

037

0.0

02

0.0

027

0.0

02

0.0

057

0.0

06

0.3

647

0.2

59

Jun

e0

.05

87

0.0

23

0.0

297

0.0

38

0.0

01

a7

0.0

01

a0

.05

37

0.0

45

0.0

117

0.0

23

0.0

227

0.0

22

0.1

007

0.2

43

0.0

01

a7

0.0

01

a0

.00

1a7

0.0

01

a0

.00

1a7

0.0

01

a0

.00

1a7

0.0

01

a0

.00

1a7

0.0

01

a0

.27

07

0.2

58

Se

p0

.05

87

0.0

42

0.0

227

0.0

27

0.0

01

a7

0.0

01

a0

.15

07

0.0

56

0.0

057

0.0

10

0.0

667

0.0

38

0.1

627

0.2

26

0.0

607

0.1

45

0.0

307

0.0

11

0.0

147

0.0

29

0.0

01

a7

0.0

01

a0

.01

17

0.0

09

0.5

767

0.3

32

a0

.00

1v

alu

eco

rre

spo

nd

sto

the

ha

lfm

eth

od

de

tect

ion

lim

it.

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]] 5

Please cite this article as: Copat, C., et al., Seasonal variation of bioaccbiomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.d

of contamination similar to ours (Bordajandi et al., 2004; Llobetet al., 2006; Perugini et al., 2007). The EC Regulation 1881/2006sets a limit of 5 mg/kg in the muscle of fish only for benzo(a)pyr-ene, and our seasonal values are lower than the above limit(Table 3).

As well as for SPAHs, distribution of SPCBs dioxin-like showedno significant difference between the periods analyzed, and meanvalues are lower than the limit set by the EC Regulation 1881/2006 for fish muscle (Table 4).

For SPCBs dioxin-like, data we obtained are in line or lowerthan those reported in literature for the same species (Coelhanet al., 2006; Moon et al., 2009; Naso et al., 2005; Perugini et al.,2004) and for other fish species (Bocio et al., 2007; Bordajandiet al., 2004; Burreau et al., 2006; Johnston et al., 2002;Kucuksezgin et al., 2001; Llobet et al., 2007; Storelli et al., 2011).

In addition, data arose from biomarkers of exposure, do notseem to highlight toxic levels of contaminants in fish.

In fact, the MTs were found uniformly represented in the threeanalyzed groups, with concentrations increasing respectively inFebruary, June and September (Fig. 2a). The higher concentrationof MTs in the reproductive periods of the species is probablyrelated, rather than the need to detoxify metals, to their functionin ion transport of essential metals, especially Zn and Cu thatcontribute to the development of oocytes (Riggio et al., 2003).In fact, although the MTs have the ability to bind a large number ofheavy metals by facilitating their transportation and their excre-tion, representing a significant biomarkers of exposure to this classof contaminants (Berthet et al., 2005; Kayaalti et al., 2011; Knapenet al., 2007; Mohamed et al., 2008; Sinaie et al., 2010), these non-enzymatic proteins are constitutively expressed, because theirfunction to bind essential metals present, participating activelyin their homeostatic regulation (Kayaalti et al., 2011).

Fig. 2. Box-plot of biomarkers expression in liver of E. encrasicolus. The values are

expressed in arbitrary densitometric unit A.D.U. (y-axis), and show the trend in

the different sampling periods.

umulation in Engraulis encrasicolus (Linneaus, 1758) and relatedoi.org/10.1016/j.ecoenv.2012.09.006

Page 6

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]6

The HSP70 were low in the specimens sampled in February,while in those sampled in June and September were found weaklyexpressed, probably due to increased in water temperature(Ivanina et al., 2009) in summer (Fig. 2b). The heat shock proteinsHSP 70 prevent the aggregation and/or degradation of otherproteins (Buckley et al., 2006; Lapointe et al., 2011; Parsell andLindquist, 1993) and these two responses have the effect ofrestricting and compensate possible changes in protein resultingfrom the heat, that reduces the initial phase of the translocationprocess and promotes protein degradation (Buckley et al., 2006;Lapointe et al., 2011).

The enzyme 7-etossiresorufina O-dietilasi (EROD), was low inall sampled groups (Fig. 2c); this result is indicative of a lowcontamination from PAHs and organochlorine compounds in theGulf of Catania, and strengthens the evidence that the content andthe induction activity of CYP1A in fish in the presence of thosecompounds is characterized by a response directly proportional todose (Stegeman and Hahn, 1994; van der Oost et al., 2003).

4. Conclusions

In summary, data from this research show that:

�

Pb

The populations of E. encrasicolus accumulate a certain amountof metals, PAHs and organochlorine compounds in theirtissues; nevertheless, the legal limits set by Regulation 1881/2006 for contaminants in fish muscle, were not exceeded.

� The essential metals accumulate more during reproductive

periods.

� The accumulation of other metals and PAHs is more related to

natural and anthropogenic changes in different seasons.

� The presence of PCBs, although in trace amounts, confirms that

those compounds banned since 40 years persist in theenvironment.

� The levels of contaminants found in the tissues of the species

does not seem to be likely to cause toxic effects.

In conclusion, the analysis of the offshore environmental riskassessment in the Gulf of Catania, showed a positive picture,although the contaminants detected are related to a certaindegree of human influence, and should be constantly monitoredto ensure quality standards of the area under consideration. In aprevious study of this area, we carried out a preliminar investiga-tion on some metals concentrations (Hg, Cd, Cr, Pb) in the samepelagic species and in S. pilchardus, showing comparable results(Copat et al., 2012b). Furtheremore Tigano et al. (2009) revealedPb concentrations higher than the EC regulation value in ademersal fish from two costal locations of the area examined,paired with DNA damage. The same for Pb, Cd and NA concentra-tions in some abiotic samples, highlighting a slight costal envir-onmental degradation due to the higher antropic impact.

Therefore, it will certainly be interesting to include, in a futureenvironmental monitoring in the Gulf of Catania, fish species witha longer average life and a different trophic niche, but still kind ofhigh economic importance, in order to more accurately define andcharacterize the degree of pollution of the area, not only for thehealth of the sea, but above all, for the risk to human health.

References

Al-Yakoob, S., Saeed, T., Al-Hashash, H., 1993. Polycylic aromatic hydrocarbons inedible tissue of fish from the Gulf after the 1991 oil spill. Mar. Pollut. Bull. 27,297–301.

Andral, B., Stanisiere, J.Y., Sauzade, D., Damier, E., Thebault, H., Galgani, F., Boissery,P., 2004. Monitoring chemical contamination levels in the Mediterraneanbased on the use of mussel caging. Mar. Pollut. Bull. 49, 704–712.

lease cite this article as: Copat, C., et al., Seasonal variation of bioaciomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.d

Bayarri, S., Baldassarri, L.T., Iacovella, N., Ferrara, F., di Domenico, A., 2001. PCDDs,PCDFs, PCBs and DDE in edible marine species from the Adriatic Sea. Chemo-sphere 43, 601–610.

Berthet, B., Mouneyrac, C., Perez, T., Amiard-Triquet, C., 2005. Metallothioneinconcentration in sponges (Spongia officinalis) as a biomarker of metalcontamination. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 141, 306–313.

Bocio, A., Domingo, J.L., Falco, G., Llobet, J.M., 2007. Concentrations of PCDD/PCDFsand PCBs in fish and seafood from the Catalan (Spain) market: estimatedhuman intake. Environ. Int. 33, 170–175.

Bordajandi, L.R., Gomez, G., Abad, E., Rivera, J., Del Mar Fernandez-Baston, M.,Blasco, J., Gonzalez, M.J., 2004. Survey of persistent organochlorine contami-nants (PCBs, PCDD/Fs, and PAHs), heavy metals (Cu, Cd, Zn, Pb, and Hg), andarsenic in food samples from Huelva (Spain): levels and health implications.J. Agric. Food Chem. 52, 992–1001.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein–dye binding.Anal. Biochem. 72, 248–254.

Bucheli, T.D., Fent, K., 1995. Induction of cytochrome P450 as a biomarker forenvironmental contamination in aquatic ecosystems. Crit. Rev. Environ. Sci.Technol. 25, 201–268.

Buckley, B.A., Gracey, A.Y., Somero, G.N., 2006. The cellular response to heat stressin the goby Gillichthys mirabilis: a cDNA microarray and protein-levelanalysis. J. Exp. Biol. 209, 2660–2677.

Bundschuh, J.Litter, M.I., Parvez, F., Roman-Ross, G., Nicolli, H.B., Jean, J.S., Liu, C.W.,Lopez, D., Armienta, M.A., Guilherme, L.R., Cuevas, A.G., Cornejo, L., Cumbal, L.,Toujaguez, R, 2011. One century of arsenic exposure in Latin America: a reviewof history and occurrence from 14 countries. The Science of the totalenvironment.

Burreau, S., Zebuhr, Y., Broman, D., Ishaq, R., 2006. Biomagnification of PBDEs andPCBs in food webs from the Baltic Sea and the northern Atlantic Ocean. Sci.Total Environ. 366, 659–672.

Coelhan, M., Strohmeier, J., Barlas, H., 2006. Organochlorine levels in edible fishfrom the Marmara Sea, Turkey. Environ. Int. 32, 775–780.

Colombo, J.C., Cappelletti, N., Lasci, J., Migoya, M.C., Speranza, E., Skorupka, N.,2005. Sources, vertical fluxes, and accumulation of aliphatic hydrocarbons incoastal sediments of the Rio de la Plata Estuary, Argentina. Environ. Sci.Technol. 39, 8227–8234.

Conti, G.O., Copat, C., Ledda, C., Fiore, M., Fallico, R., Sciacca, S., Ferrante, M., 2012.Evaluation of heavy metals and polycyclic aromatic hydrocarbons (PAHs)in Mullus barbatus from Sicily Channel and risk-based consumption limits.Bull. Environ. Contam. Toxicol. 88, 946–950.

Copat, C., Bella, F., Castaing, M., Fallico, R., Sciacca, S., Ferrante, M., 2012a. Heavymetals concentrations in fish from Sicily (Mediterranean Sea) and evaluationof possible health risks to consumers. Bull. Environ. Contam. Toxicol. 88,78–83.

Copat, C., Maggiore, R., Arena, G., Lanzafame, S., Fallico, R., Sciacca, S., Ferrante, M.,2012b. Evaluation of a temporal trend heavy metals contamination inPosidonia oceanica (L.) Delile, (1813) along the western coastline of Sicily(Italy). J. Environ. Monit. 14, 187–192.

de Andrade, V.M., de Freitas, T.R., da Silva, J., 2004. Comet assay using mullet(Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection ofgenotoxic pollutants in aquatic environment. Mutat. Res. 560, 57–67.

Depledge, M.H., 1994. The rational basis of the use of biomarkers as ecotoxicolo-gical tools. In: Fossi, M.C., Leonzio, C. (Eds.), Nondestructive Biomarkers inVertebrates. Lewis, Boca Raton, FL, pp. 271–295.

Di Leonardo, R., Tranchida, G., Bellanca, A., Neri, R., Angelone, M., Mazzola, S., 2006.Mercury levels in sediments of central Mediterranean Sea: a 150þ year recordfrom box-cores recovered in the Strait of Sicily. Chemosphere 65, 2366–2376.

Doherty, V.F., Ogunkuade, O.O., Kanife, U.C., 2010. Biomarkers of Oxidative Stress andHeavy Metal Levels as Indicators of Environmental Pollution in Some SelectedFishes in Lagos, Nigeria. American-Eurasian J. Agric. Environ. Sci. 7, 359–365.

Dragun, Z., Raspor, B., Podrug, M., 2007. The influence of the season and the bioticfactors on the cytosolic metal concentrations in the gills of the European chub(Leuciscus cephalus L.). Chemosphere 69, 911–919.

Dural, M., Goksu, M.Z., Ozak, A.A., Derici, B., 2006. Bioaccumulation of some heavymetals in different tissues of Dicentrarchus labrax L, 1758, Sparus aurata L,1758 and Mugil cephalus L, 1758 from the Camlik lagoon of the eastern coastof Mediterranean (Turkey). Environ. Monit. Assess. 118, 65–74.

Falco, G., Llobet, J.M., Bocio, A., Domingo, J.L., 2006. Daily intake of arsenic,cadmium, mercury, and lead by consumption of edible marine species. J.Agric. Food Chem. 54, 6106–6112.

Fasulo, S., Mauceri, A., Maisano, M., Giannetto, A., Parrino, V., Gennuso, F., D’Agata,A., 2010. Immunohistochemical and molecular biomarkers in Coris julisexposed to environmental contaminants. Ecotoxicol. Environ. Saf. 73,873–882.

Flammarion, P., Devaux, A., Nehls, S., Migeon, B., Noury, P., Garric, J., 2002.Multibiomarker responses in fish from the Moselle River (France). Ecotoxicol.Environ. Saf. 51, 145–153.

Fossi, M.C., 1998. Biomarkers as diagnostic and prognostic tools for wildlife riskassessment: integrating endocrine-disrupting chemicals. Toxicol. Ind. Health.14, 291–309.

Fossi, M.C., Casini, S., Savelli, C., Corbelli, C., Franchi, E., Mattei, N., Sanchez-Hernandez, J.C., Corsi, I., Bamber, S., Depledge, M.H., 2000. Biomarkerresponses at different levels of biological organisation in crabs (Carcinusaestuarii) experimentally exposed to benzo(alpha)pyrene. Chemosphere 40,861–874.

cumulation in Engraulis encrasicolus (Linneaus, 1758) and relatedoi.org/10.1016/j.ecoenv.2012.09.006

Page 7

C. Copat et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]] 7

Guo, Z., Lin, T., Zhang, G., Yang, Z., Fang, M., 2006. High-resolution depositionalrecords of polycyclic aromatic hydrocarbons in the central continental shelfmud of the East China Sea. Environ. Sci. Technol. 40.

Guo, Z., Lin, T., Zhang, G., Zhang, Z., Hao, Y., Fang, M., 2007. The sedimentary fluxesof polycyclic aromatic hydrocarbons in the Yangtze River Estuary coastal seafor the past century. Sci. Total Environ. 386, 33–41.

Hernandez, H., Rodriguez, R., 2012. Geochemical evidence for the origin of vanadiumin an urban environment. Environ. Monit. Assess. 184, 5327–5342.

ISTAT, 2000. Statistica sulla pesca, caccia e zootecnia. Alba Grafica s.p.a., Roma.Ivanina, A.V., Taylor, C., Sokolova, I.M., 2009. Effects of elevated temperature and

cadmium exposure on stress protein response in eastern oysters Crassostreavirginica (Gmelin). Aquat. Toxicol. (Amsterdam, Netherlands) 91, 245–254.

Johnston, T.A., Fisk, A.T., Whittle, D.M., Muir, D.C., 2002. Variation in organo-chlorine bioaccumulation by a predatory fish; gender, geography, and dataanalysis methods. Environ. Sci. Technol. 36, 4238–4244.

Kannan, K., Johnson-Restrepo, B., Yohn, S.S., Giesy, J.P., Long, D.T., 2005. Spatial andtemporal distribution of polycyclic aromatic hydrocarbons in sediments fromMichigan inland lakes. Environ. Sci. Technol. 39, 4700–4706.

Kayaalti, Z., Aliyev, V., Soylemezoglu, T., 2011. The potential effect of metallothio-nein 2A–5A/G single nucleotide polymorphism on blood cadmium, lead, zincand copper levels. Toxicol. Appl. Pharmacol. 256, 1–7.

Knapen, D., Reynders, H., Bervoets, L., Verheyen, E., Blust, R., 2007. Metallothioneingene and protein expression as a biomarker for metal pollution in naturalgudgeon populations. Aquat. Toxicol. (Amsterdam, Netherlands) 82, 163–172.

Kucuksezgin, F., Altay, O., Uluturhan, E., Kontas, A., 2001. Trace metal andorganochlorine residue levels in red mullet (Mullus barbatus) from the easternAegean, Turkey. Water Res. 35, 2327–2332.

Lapointe, D., Pierron, F., Couture, P., 2011. Individual and combined effects of heatstress and aqueous or dietary copper exposure in fathead minnows (Pime-phales promelas). Aquat. Toxicol. (Amsterdam, Netherlands) 104, 80–85.

Linde-Arias, A.R., Inacio, A.F., de Alburquerque, C., Freire, M.M., Moreira, J.C., 2008.Biomarkers in an invasive fish species, Oreochromis niloticus, to assess theeffects of pollution in a highly degraded Brazilian River. Sci. Total Environ. 399,186–192.

Liu, G.Q., Zhang, G., Li, X.D., Li, J., Peng, X.Z., Qi, S.H., 2005. Sedimentary record ofpolycyclic aromatic hydrocarbons in a sediment core from the Pearl RiverEstuary, South China. Mar. Pollut. Bull. 51, 912–921.

Llobet, J.M., Falco, G., Bocio, A., Domingo, J.L., 2006. Exposure to polycyclicaromatic hydrocarbons through consumption of edible marine species inCatalonia, Spain. J. Food Prot. 69, 2493–2499.

Llobet, J.M., Falco, G., Bocio, A., Domingo, J.L., 2007. Human exposure to poly-chlorinated naphthalenes through the consumption of edible marine species.Chemosphere 66, 1107–1113.

Marijic, V.F., Raspor, B., 2010. The impact of fish spawning on metal and proteinlevels in gastrointestinal cytosol of indigenous European chub. Comp. Bio-chem. Physiol. C Toxicol. Pharmacol. 152, 133–138.

Marti-Cid, R., Bocio, A., Llobet, J.M., Domingo, J.L., 2007. Intake of chemicalcontaminants through fish and seafood consumption by children of Catalonia,Spain: health risks. Food Chem. Toxicol. 45, 1968–1974.

Marti-Cid, R., Llobet, J.M., Castell, V., Domingo, J.L., 2008. Dietary intake of arsenic,cadmium, mercury, and lead by the population of Catalonia, Spain. Biol. Trace.Elem. Res. 125, 120–132.

Mohamed, S., Kheireddine, O., Wyllia, H.M., Roquia, R., Aicha, D., Mourad, B., 2008.Proportioning of biomarkers (GSH, GST, Ache, Catalase) indicator of pollutionat Gambusia affinis (Teleostei fish) exposed to cadmium. J. Environ. Res. 2,177–181.

Moon, H.B., Kim, H.S., Choi, M., Yu, J., Choi, H.G., 2009. Human health risk ofpolychlorinated biphenyls and organochlorine pesticides resulting from sea-food consumption in South Korea, 2005-2007. Food Chem. Toxicol. 47,1819–1825.

Nagai, M., Saitoh, J., Ohno, H., Hitomi, C., Wada, M., 2006. Pentavalent vanadium atconcentration of the underground water level enhances the sweet taste senseto glucose in college students. Biometals 19, 7–12.

Please cite this article as: Copat, C., et al., Seasonal variation of bioaccbiomarkers of exposure. Ecotoxicol. Environ. Saf. (2012), http://dx.d

Naso, B., Perrone, D., Ferrante, M.C., Bilancione, M., Lucisano, A., 2005. Persistentorganic pollutants in edible marine species from the Gulf of Naples, SouthernItaly. Sci. Total Environ. 343, 83–95.

Papetti, P., Rossi, G., 2009. Heavy metals in the fishery products of low Lazio andthe use of metallothionein as a biomarker of contamination. Environ. Monit.Assess. 159, 589–598.

Parsell, D.A., Lindquist, S., 1993. The function of heat-shock proteins in stresstolerance: degradation and reactivation of damaged proteins. Annu. Rev.Genet. 27, 437–439.

Perugini, M., Cavaliere, M., Giammarino, A., Mazzone, P., Olivieri, V., Amorena, M.,2004. Levels of polychlorinated biphenyls and organochlorine pesticides insome edible marine organisms from the Central Adriatic Sea. Chemosphere 57,391–400.

Perugini, M., Visciano, P., Giammarino, A., Manera, M., Di Nardo, W., Amorena, M.,2007. Polycyclic aromatic hydrocarbons in marine organisms from the AdriaticSea, Italy. Chemosphere 66, 1904–1910.

Perugini, M., Visciano, P., Manera, M., Zaccaroni, A., Olivieri, V., Amorena, M., 2009.Levels of total mercury in marine organisms from Adriatic Sea, Italy. Bull.Environ. Contam. Toxicol. 83, 244–248.

Riggio, M., Filosa, S., Parisi, E., Scudiero, R., 2003. Changes in zinc, copper andmetallothionein contents during oocyte growth and early development of theteleost Danio rerio (zebrafish). Comp. Biochem. Physiol. C Toxicol. Pharmacol.135, 191–196.

Sepe, A., Ciaralli, L., Ciprotti, M., Giordano, R., Funari, E., Costantini, S., 2003.Determination of cadmium, chromium, lead and vanadium in six fish speciesfrom the Adriatic Sea. Food Addit. Contam. 20, 543–552.

Sinaie, M., Bastami, K.D., Ghorbanpour, M., Najafzadeh, H., Shekari, M., Haghparast,S., 2010. Metallothionein biosynthesis as a detoxification mechanism inmercury exposure in fish, spotted scat (Scatophagus argus). Fish Physiol.Biochem. 36, 1235–1242.

Stegeman, J.J., Hahn, M.E., 1994. Biochemistry and molecular biology of mono-oxygenases. In: Malins, D.C., Ostrander, G.K. (Eds.), Aquatic toxicology:molecular, biochemical, and cellular perspectives. Lewis NW, USA, pp. 87–206.

Storelli, M.M., Barone, G., Storelli, A., Marcotrigiano, G.O., 2011. Levels andcongener profiles of PCBs and PCDD/Fs in blue shark (Prionace glauca) liverfrom the South-Eastern Mediterranean Sea (Italy). Chemosphere 82, 37–42.

Storelli, M.M., Marcotrigiano, G.O., 2005. Bioindicator organisms: heavy metalpollution evaluation in the Ionian Sea (Mediterranean Sea–Italy). Environ.Monit. Assess. 102, 159–166.

Tigano, C., Tomasello, B., Pulvirenti, V., Ferrito, V., Copat, C., Carpinteri, G., Mollica,E., Sciacca, S., Renis, M., 2009. Assessment of environmental stress inParablennius sanguinolentus (Pallas, 1814) of the Sicilian Ionian coast.Ecotoxicol. Environ. Saf. 72, 1278–1286.

Topcuoglu, S., Kirbasoglu, C., Gungor, N., 2002. Heavy metals in organisms andsediments from Turkish Coast of the Black Sea, 1997–1998. Environ. Int. 27,521–526.

Turan, C., Dural, M., Oksuz, A., Ozturk, B., 2009. Levels of heavy metals in somecommercial fish species captured from the Black Sea and Mediterranean coastof Turkey. Bull. Environ. Contam. Toxicol. 82, 601–604.

Turkmen, A., Tepe, Y., Turkmen, M., 2008. Metal levels in tissues of the Europeananchovy, Engraulis encrasicolus L., 1758, and picarel, Spicara smaris L., 1758,from Black, Marmara and Aegean seas. Bull. Environ. Contam. Toxicol. 80,521–525.

van der Oost, R., Beyer, J., Vermeulen, N.P., 2003. Fish bioaccumulation andbiomarkers in environmental risk assessment: a review. Environ. Toxicol.Pharmacol. 13, 57–149.

Whitfield, A.K., Elliott, M., 2002. Fishes as indicators of environmental andecological changes within estuaries: a review of progress and some suggestionfor the future. J. Fish Biol. 61, 229–250.

Yildirim, Y., Gonulalan, Z., Narin, I., Soylak, M., 2009. Evaluation of trace heavymetal levels of some fish species sold at retail in Kayseri, Turkey. Environ.Monit. Assess. 149, 223–228.

umulation in Engraulis encrasicolus (Linneaus, 1758) and relatedoi.org/10.1016/j.ecoenv.2012.09.006