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Aquatic Toxicology 101 (2011) 146–154

Contents lists available at ScienceDirect

Aquatic Toxicology

journa l homepage: www.e lsev ier .com/ locate /aquatox

ource and impact of lead contamination on �-aminolevulinic acid dehydratasectivity in several marine bivalve species along the Gulf of Cadiz

. Companya, A. Serafima, B. Lopesa, A. Cravoa, J. Kalmanc, I. Ribac, T.A. DelValls c, J. Blascob,. Delgadod, A.M. Sarmientoc,d, J.M. Nietod, T.J. Shepherde, G. Nowell e, M.J. Bebiannoa,∗

CIMA, University of Algarve, Campus de Gambelas, 8005-139 Faro, PortugalInstituto Ciencias Marinas Andalucía (CSIC), Campus Río San Pedro, 11510 Puerto Real, Cádiz, SpainCátedra UNESCO/UNITWIN/WiCop, Department of Physical-Chemistry, Faculty Marine and Environmental Sciences, University of Cádiz, 11510 Puerto Real, Cádiz, SpainDepartment of Geology, University of Huelva, Avda Fuerzas Armadas s/n, 21071 Huelva, SpainDepartment of Earth Sciences, University of Durham, Science Laboratories, Durham DH1 3LE, United Kingdom

r t i c l e i n f o

rticle history:eceived 1 June 2010eceived in revised form7 September 2010ccepted 21 September 2010

eywords:eadead isotopesLADivalvesontaminationulf of Cadiz

beria

a b s t r a c t

Coastal areas and estuaries are particularly sensitive to metal contamination from anthropogenic sourcesand in the last few decades the study of space–time distribution and variation of metals has been exten-sively researched. The Gulf of Cadiz is no exception, with several rivers draining one of the largestconcentrations of sulphide deposits in the world, the Iberian Pyrite Belt (IPB). Of these rivers, the Guadi-ana, one of the most important in the Iberian Peninsula, together with smaller rivers like the Tinto andOdiel, delivers a very high metal load to the adjacent coastal areas.

The purpose of this work was to study the source and impact of lead (Pb) drained from historical oractive mining areas in the IPB on the activity of a Pb inhibited enzyme (�-aminolevulinic acid dehydratase,ALAD) in several bivalve species along the Gulf of Cadiz.

Seven marine species (Chamelea gallina, Mactra corallina, Donax trunculus, Cerastoderma edule, Mytilusgalloprovincialis, Scrobicularia plana and Crassostrea angulata) were collected at 12 sites from Mazagón,near the mouth of the rivers Tinto and Odiel (Spain), to Cacela Velha (Ria Formosa lagoon system, Portu-gal). Lead concentrations, ALAD activity and lead isotope ratios (206Pb/204Pb, 207Pb/204Pb and 208Pb/204Pb)were determined in the whole soft tissues.

The highest Pb concentrations were determined in S. plana (3.50 ± 1.09 �g g−1 Pb d.w.) and D. trun-culus (1.95 ± 0.10 �g g−1 Pb d.w.), while M. galloprovincialis and C. angulata showed the lowest Pb levels(<0.38 �g g−1 Pb d.w.). In general, ALAD activity is negatively correlated with total Pb concentration. How-ever this relationship is species dependent (e.g. linear for C. gallina ALAD = −0.36[Pb] + 0.79; r = 0.837; orexponential for M. galloprovincialis ALAD = 2.48e−8.3[Pb]; r = 0.911). This indicates that ALAD activity has

considerable potential as a biomarker of Pb and moreover, in marine bivalve species with different feedinghabits. Lead isotope data showed significant seasonal and spatial changes in bivalve isotopic compositionreflecting seasonal and geographic differences in bioaccumulation. Within the study area, Pb can be mod-elled as a mixing between geogenic Pb and mine-related, discharges of Pb from the IPB. For some sitesat the mouth of the Guadiana River, the bivalves show contamination from other anthropogenic sources,such as leaded boat/aviation fuel and/or leaded paint. Finally, the study demonstrates convincingly the

speci

need to consider species-

. Introduction

The Gulf of Cadiz is located at the SW margin of the Iberianeninsula and the metal concentrations of its coastal areas aretrongly influenced by discharges from the Guadiana, Tinto, Odielnd Guadalquivir rivers. Though the freshwater discharges of these

∗ Corresponding author. Tel.: +351 289 800 900; fax: +351 289 800 069.E-mail address: mbebian@ualg.pt (M.J. Bebianno).

166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2010.09.012

fic variation when using bivalve ALAD activity as a biomarker for Pb.© 2010 Elsevier B.V. All rights reserved.

rivers are relatively minor, their metal loads are exceptionally high.This is because they drain the Iberian Pyrite Belt (IPB), the largestconcentration of massive sulphide deposits in the world (Leistel etal., 1998), where weathering and extensive mining activities haveled to river waters with enhanced metal concentrations (average

values of 285 mg l−1 Fe, 110 mg l−1 Zn, 3.4 g l−1 sulphates in theOdiel River; 5 g l−1 Fe, 70 mg l−1 Zn, 23 g l−1 sulphates in the TintoRiver, etc. (Sarmiento et al., 2009a) and acidity (pH < 3)). The trans-ference of acidity and toxic metals to the Tinto–Odiel estuarinesystem and the Gulf of Cadiz has been the subject of numerous

R. Company et al. / Aquatic Toxicology 101 (2011) 146–154 147

F o PadI o Ant

iaOo

tm1oiatmtib1ao2ta2

ctrolG

ig. 1. Sampling sites along the Gulf of Cadiz. (1) Mazagón (beach), (2) Canal Santnternacional, (8) Vila Real de Santo António (city), (9) Molhe, (10) Vila Real de Sant

nvestigations as discussed and summarised by Olías et al. (2006)nd Sarmiento et al. (2009b). Pollution levels are so extreme in thediel and Tinto rivers that the Huelva Estuary is considered as onef the most polluted aquatic systems in the world.

Several studies correlate the high contents of toxic metals inhe Gulf of Cadiz area with biochemical effects due to metal accu-

ulation in fish and bivalve molluscs (Rodriguez-Ariza et al., 1992,993; Sarasquete et al., 1997; Funes et al., 2006). Lead (Pb) is onef the most toxic metals to marine environments due to its abil-ty to mimic biologically important metals, mainly calcium, ironnd zinc. It is able to bind to and interact with the same pro-eins and molecules as these metals but, after displacement, those

olecules function differently and fail to carry out the same reac-ions. �-Aminolevulinate dehydratase (ALAD, EC 4.2.1.24) is anmportant cytosolic and non-limiting enzyme involved in the hemeiosynthetic pathway, and is expressed in all tissues (Wetmur,994). Lead is well-known for its ability to inhibit this enzymend the effects on ALAD have been reported for several aquaticrganisms such as fish (Conner and Fowler, 1994; Schmitt et al.,002; Costa et al., 2007), amphibians (Arrieta et al., 2000), rep-iles (Overmann and Krajicek, 1995) and, more recently, for marinend freshwater bivalves (Company et al., 2008; Kalman et al.,008).

The Gulf of Cadiz is responsible for 5–10% of fish and bivalveatches of Spain and Portugal, and therefore it is of great impor-

ance to understand the degree of Pb contamination in this coastalegion and to study the effects of bioaccumulated Pb in the marinerganisms. Thus, the main purpose of this work was to study theevels and effects of Pb in several bivalve species collected along theulf of Cadiz. The impact of lead in these species was determined

re, (3) Punta Umbria, (4) La Antilla, (5) Isla Cristina, (6) Desembocadura, (7) Ponteónio (beach), (11) Manta Rota (beach) and (12) Cacela Velha (Ria Formosa).

by the activity of the enzyme �-aminolevulinic acid dehydratase(ALAD). Lead isotope ratios were also determined to identify theprincipal sources of lead in the coastal waters of the Gulf of Cadizand the influence of seasonal changes and bivalve feeding habitson the bio-accumulation of lead.

2. Materials and methods

2.1. Study area

To quantify the effects of lead pollution on marine bivalves, 12sites were selected along an 80 km coastal section of the Gulf ofCadiz; from Mazagón beach in the east (Spain) to Cacela Velha inthe west (Portugal) (Fig. 1). Sampling was carried out during twoseasons; Spring 2005 and Winter 2006. This provided contrastingflow and water conditions for assessing seasonal variation. In situwater measurements at each site (temperature, salinity, pH anddissolved oxygen) were measured using a WTW MultiLine F/SET-3probe (Table 1). Temperatures ranged from 16.7 ◦C to 20.8 ◦C inSpring and 11.6 ◦C to 14.4 ◦C in Winter. Salinity remained rela-tively constant (34.7 ± 0.2‰) for the eastern sites (Sites 1–6) butmore variable for open water sites west of the Guandiana (Sites9, 12) for the same season. For sites within the more geographi-cally enclosed Guadiana Estuary (Sites 7, 8), the significantly lower

salinities reflect the mixing of fresh and marine waters. pH valueswere fairly constant for all sites (8.40 ± 0.32), contrasting with dis-solved oxygen values that were consistently higher for sites eastof the Guadiana (12.99 ± 1.56 mg L−1 O2) than those west of theGuadiana (8.37 ± 0.62 mg L−1 O2).

148 R. Company et al. / Aquatic Toxic

Table 1Water temperature, salinity, pH and dissolved oxygen during Spring 2005 and Win-ter 2006.

Site Season Temperature (◦C) Salinity (‰) pH DissolvedO2 (mg/L)

1 Spring 19.52 34.9 8.14 11.002 Spring 20.2 34.8 8.22 12.283 Spring 19.76 34.8 8.30 13.944 Spring 19.15 34.7 8.28 12.895 Spring 19.32 34.7 8.33 13.036 Spring 19.29 34.3 8.33 15.84

7Spring 20.75 24.4 8.21 11.94Spring 18.2 19.0 8.25 8.20Winter 11.6 23.0 8.56 9.64

8Spring 18.8 28.4 8.31 7.64Winter 12.2 36.5 8.66 8.78

9Spring 16.7 33.3 8.04 8.30Winter 12.4 33.6 8.88 8.04

10 Winter 12.8 38.0 8.94 7.53

2

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2

2

sawcm�

2

sfdwp452t2tftaio

−1 −1

11 Winter 14.1 37.5 8.00 8.15

12Spring 27.4 36.7 8.25 8.90Winter 14.4 37.8 9.09 8.49

.2. Sampling and analytical methods

.2.1. Sampling

.2.1.1. Bivalves. The bivalves selected for study (Chamelea gallina,actra corallina, Donax trunculus, Cerastoderma edule, Mytilus gal-

oprovincialis, Scrobicularia plana and Crassostrea angulata) wereollected either by dredging (Spanish coast) or manual sam-ling (Portuguese coast). Approximately 30 organisms per species,er season were collected at each site. After removing sedimentesidues, the organisms were depurated for 48 h in an aerated waterank (≈20 l) to eliminate their digestive contents. Following depu-ation, 15–20 organisms from each site were dissected, individuallyrozen in liquid nitrogen and stored at −80 ◦C until required forLAD and Pb analysis. The remaining organisms (10) were freezeried at −20 ◦C for 48 h for Pb isotope analysis.

.3. Analytical methods

.3.1. Total Pb concentrationsTotal Pb concentrations were determined on a homogenised

ub-sample of the selected bivalves by graphite furnace atomicbsorption spectrophotometry (PerkinElmer) after wet digestionith nitric acid. Analytical precision and accuracy were routinely

hecked by analysis of certified oyster tissue standard referenceaterial (SRM 1566b - NIST). Lead concentrations are reported asg g−1 Pb dry weight.

.3.2. ALAD activityALAD activity (EC 4.2.1.24) was determined on the same sub-

amples as prepared for Pb determination using a method modifiedrom the European standardized method for the determination ofelta-ALAD activity in blood (Berlin and Schaller, 1974). Briefly, thehole soft tissues of bivalves were homogenized with 0.1 M phos-hate buffer (pH 6.6) and centrifuged at 10,000 rpm for 15 min at◦C. The resulting supernatants were separated into 5 aliquots of0 �l each. 200 �l of phosphate buffer was added to 2 aliquots and00 �l of ALA-reagent (containing �-aminolevulinic acid) addedo the remaining 3 aliquots. The mixtures were incubated forh at room temperature and afterwards 750 �l of the precipita-

ion reagent (containing trichloroacetic acid) was added, mixed

or 30 min and then centrifuged at 2500 × g for 5 min. 500 �l ofhe resulting supernatant was transferred to a plastic cell (1 ml)nd mixed with 500 �l of the Ehrlich chromogenic reagent andncubated for 15 min at 25 ◦C. The UV absorbance of the amountf porphobilinogen (PBG) was determined in the bivalve sam-

ology 101 (2011) 146–154

ples and blanks at 550 nm. The activity of ALAD was expressed inng PBG min−1 mg−1 total protein.

2.3.3. Lead isotopesTwenty eight bivalve samples were prepared for isotope analy-

sis using conventional nitric acid dissolution techniques followedby bromination of the lead and standard separation on anionexchange columns. The freeze dried, depurated bivalves in eachbulk sample were ground, homogenised and a representative sub-sample taken to yield approximately 50–100 ng Pb for analysis.Routine laboratory blanks using comparable resin columns werebetter than 0.1 ng Pb. Blank corrections for the bivalves were thusconsidered to be very small.

Isotope compositions were measured on a Thermo Nep-tune ICP-multi-collector mass spectrometer and are reported as208Pb/204Pb, 207Pb/204Pb, 206Pb/204Pb, 208Pb/206Pb and 207Pb/206Pbratios. For each analytical session, multiple analyses of theinternational Standard Reference Material NBS981 gave the fol-lowing values of external reproducibility, where uncertainties areexpressed as 2 standard deviations. Session A (n = 12): 206Pb/204Pb16.9405 ± 0.0007; 207Pb/204Pb 15.4973 ± 0.0009; 208Pb/204Pb36.716 ± 0.004. Session B (n = 15): 206Pb/204Pb 16.9405 ± 0.0010;207Pb/204Pb 15.4973 ± 0.0010; 208Pb/204Pb 36.716 ± 0.003. Datawere corrected for mass discrimination using thallium as aninternal standard (Ketterer et al., 1991).

2.3.4. Statistical analysisThe Pb concentration and ALAD activity in bivalves were

expressed as average ± standard deviation (SD). Statistical analy-sis of the data was performed using the STATISTICA 6.0 softwarepackage (StatSoft, USA). Normality of data was verified using theKolmogorov–Smirnov test, and the homogeneity of variance of thedata was checked by the Bartlett’s test. Post hoc comparison testsfor means were made using the Duncan’s test. Regression analyseswere also applied between the concentrations of Pb and ALAD. Thelevel of significance was set at 0.05.

3. Results

3.1. Pb concentrations and ALAD activity in different bivalvespecies

Lead concentrations and ALAD activity are presented in Table 2.The consistently highest Pb concentrations were observed inC. gallina (0.61–1.58 �g g−1 Pb d.w.) (Sites 1–6) and S. plana(1.55–3.50 �g g−1 Pb d.w.) (Site 7). By comparison the low-est overall Pb values were recorded for M. galloprovincialis(0.04–0.38 �g g−1 Pb d.w.) (Sites 7–9 and 12) and C. angulata(0.03–0.12 �g g−1 Pb d.w.) (Sites 7 and 12). For M. galloprovincialis,Pb concentrations were significantly higher during the wet season(Winter 2006) compared to the dry season (Spring 2005) and showa decreasing gradient from the Guadiana estuary (Site 7) westwardsto the Ria Formosa lagoon system (Site 12). The lowest measuredPb value was for C. edule (0.02 ± 0.01 �g g−1 Pb d.w.) (Site 12).

ALAD activity in the different bivalve species was ingeneral inversely correlated with Pb levels in the sameorganism (Table 2). The lowest ALAD activity levels werefor bivalves from Sites 7 and 8, collected in Winter 2006

(M. galloprovincialis 0.10–0.16 ng PBG min mg prot ; S. plana0.15 ng PBG min−1 mg prot−1). By contrast, Sites 7 and 8 sampled inSpring 2005 recorded the highest ALAD activities (M. galloprovin-cialis 1.70 and 1.94 ng PBG min−1 mg prot−1 respectively); implyinga strong seasonal influence.

R. Company et al. / Aquatic Toxicology 101 (2011) 146–154 149

Table 2Total lead concentration (�g g−1) and ALAD activity (ng PBG min−1 mg prot−1) for whole tissue samples according to bivalve species, season and sampling sites (Gulf of Cadiz).

Species Season Site Pb (�g g−1) ALAD (ng PBG min−1 mg prot−1)

C. gallina Spring 1 1.58 ± 0.04 (a) 0.25 ± 0.044 (bc)C. gallina Spring 2 0.94 ± 0.01 (b) 0.32 ± 0.04 (bc)C. gallina Spring 3 0.84 ± 0.04 (c) 0.55 ± 0.08 (ab)C. gallina Spring 4 0.68 ± 0.01 (d) 0.51 ± 0.06 (ab)C. gallina Spring 5 0.61 ± 0.02 (e) 0.54 ± 0.22 (ab)C. gallina Spring 6 0.64 ± 0.02 (e) 0.67 ± 0.11 (a)C. gallina Winter 11 0.41 ± 0.01 (f) 0.18 ± 0.06 (c)

M. galloprovincialis Spring 7 0.04 ± 0.02 (b) 1.70 ± 0.65 (ab)M. galloprovincialis Winter 7 0.38 ± 0.13 (a) 0.10 ± 0.02 (c)M. galloprovincialis Spring 8 n.d. 1.94 ± 0.40 (a)M. galloprovincialis Winter 8 0.32 ± 0.05 (a) 0.16 ± 0.02 (c)M. galloprovincialis Spring 9 n.d. 2.00 ± 0.46 (a)M. galloprovincialis Winter 9 0.29 ± 0.04 (a) 0.19 ± 0.05 (c)M. galloprovincialis Spring 12 0.22 ± 0.08 (ab) 0.86 ± 0.31 (bc)M. galloprovincialis Winter 12 0.20 ± 0.06 (ab) 0.26 ± 0.15 (c)

C. angulata Spring 7 0.03 ± 0.02 (b) 1.02 ± 0.20 (ab)C. angulata Winter 7 0.10 ± 0.02 (a) 0.47 ± 0.10 (b)C. angulata Spring 12 0.12 ± 0.04 (ab) 1.11 ± 0.41 (a)C. angulata Winter 12 0.08 ± 0.02 (ab) 0.42 ± 0.04 (c)

S. plana Spring 7 1.55 ± 0.49 (b) 0.36 ± 0.10 (a)S. plana Winter 7 3.50 ± 1.09 (a) 0.15 ± 0.02 (b)

C. edule Spring 7 1.46 ± 0.02 (a) 0.31 ± 0.114 (a)C. edule Winter 12 0.02 ± 0.01 (b) 0.52 ± 0.15 (a)

D. trunculus Spring 3 1.95 ± 0.10 (a) n.d.D. trunculus Spring 6 0.79 ± 0.02 (b) 0.70 ± 0.06 (a)D. trunculus Winter 10 0.13 ± 0.03 (c) 0.59 ± 0.04 (b)

0.22 ± 0.02 (c) 0.46 ± 0.18 (b)

0.41 ± 0.03 0.12 ± 0.09

n istically different (p > 0.05).

3

cHlMsf(dW

3

asc

Springy = 1.16e

R = 0.859

Wintery = -1.14x + 0.58

R = 0.7940.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0

Pb (µg/g)

AL

AD

(ng

PB

G/m

in/m

g p

rot)

Fg

D. trunculus Winter 11

M. corallina Winter 10

.d. – not detected; values followed by the same letter (in parentheses) are not stat

.2. Relationship between Pb levels and ALAD activity

A robust inverse relationship between ALAD activity and Pb con-entration was confirmed by regression analysis (Figs. 2 and 3).owever, the ALAD–Pb relationship is strongly influenced by bio-

ogical factors associated with individual species. In C. gallina and. galloprovincialis for example, although ALAD activity decreases

ignificantly with increasing levels of Pb, the relationship is dif-erent for each species, reflecting differences in feeding behaviorFig. 2). Also, the inverse relationship ALAD–Pb is strongly seasonependent, reflecting a change in Pb bioavailability from Spring tointer (Fig. 3).

.3. Lead isotopes

Lead isotope analyses for the bivalves are detailed in Table 3nd displayed graphically in Figs. 4–7 to show the relation-hips between isotopic composition, bivalve species, seasonalhanges, geographic location and source apportionment. All data

Fig. 3. Seasonal variation between ALAD activity (ng PBG min−1 mg prot−1) and Pbconcentrations (�g g−1 d.w.) in bivalves collected during Spring 2005 and Winter2006 (Gulf of Cadiz). (�) M. galloprovincialis; (©) C. gallina; (�) C. angulata; (�) M.corallina; (�) S. plana; (�) C. edule; (♦) D. trunculus.

A

y = 1.01eR = 0.782

0.0

0.2

0.4

0.6

0.8

1.0

0 0.5 1 1.5 2

Pb (µg/g)

AL

AD

(ng

PB

G/m

in/m

g p

rot)

B

y = 2.48eR = 0.830

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0 0.1 0.2 0.3 0.4 0.5

Pb (µg/g)

AL

AD

(ng

PB

G/m

in/m

g p

rot)

ig. 2. Relationship between ALAD activity (ng PBG min−1 mg prot−1) and Pb concentrations (�g g−1 d.w.) for different bivalve species (Gulf of Cadiz). (A) C.gallina; (B) M.alloprovincialis.

150 R. Company et al. / Aquatic Toxicology 101 (2011) 146–154

Table 3Location, season, species and lead isotope composition of bivalves, Gulf of Cadiz.

Site Location Season Bivalve 206/204Pb 2SE 207/204Pb 2SE 208/204Pb 2SE

1 Mazagon beach Spring 2005 C. gallina 18.2125 0.0007 15.6334 0.0007 38.2592 0.00192 Canal Santo Padre Spring 2005 C. gallina 18.2183 0.0014 15.6336 0.0015 38.2657 0.00442 Canal Santo Padre Spring 2005 M. corallina 18.2255 0.0010 15.6326 0.0010 38.3036 0.00283 Punta Umbria Spring 2005 C. gallina 18.2315 0.0013 15.6349 0.0015 38.2839 0.00314 La Antilla Spring 2005 C. gallina 18.2437 0.0018 15.6317 0.0017 38.2953 0.00435 Isla Cristina Spring 2005 C. gallina 18.279O 0.0019 15.6313 0.0017 38.3443 0.00405 Isla Cristina Spring 2005 M. corallina 18.2844 0.0022 15.6301 0.0020 38.3487 0.00566 Desembocadura Spring 2005 C. gallina 18.2984 0.0019 15.6310 0.0022 38.3641 0.00436 Desembocadura Spring 2005 M. corallina 18.3282 0.0006 15.6323 0.0007 38.3953 0.00197 Ponte Internacional Spring 2005 C. edule 18.2798 0.0008 15.6303 0.0008 38.3832 0.00217 Ponte Internacional Spring 2005 M. galloprovincialis 18.1808 0.0009 15.6284 0.0012 38.2312 0.00257 Ponte Internacional Spring 2005 S. plana 18.1548 0.0008 15.619O 0.0009 38.1804 0.00267 Ponte Internacional Spring 2005 C. angulata 18.2388 0.0021 15.6304 0.0019 38.3375 0.00478 VRSA city Spring 2005 M. galloprovincialis 18.1879 0.0009 15.6311 0.0009 38.2443 0.00249 Molhe Spring 2005 M. galloprovincialis 18.1231 0.0016 15.6212 0.0020 38.1751 0.0032

12 Cacela Velha Spring 2005 M. galloprovincialis 18.2518 0.0008 15.6347 0.0008 38.2318 0.002512 Cacela Velha Spring 2005 C. angulata 18.1962 0.0010 15.6273 0.0011 38.1502 0.0027

7 Ponte Internacional Winter 2006 M. galloprovincialis 18.3058 0.0011 15.6382 0.0010 38.3933 0.00247 Ponte Internacional Winter 2006 C. angulata 18.3310 0.0018 15.6373 0.0015 38.4136 0.00388 VRSA city Winter 2006 M. galloprovincialis 18.2465 0.0010 15.6392 0.0011 38.3392 0.00259 Molhe Winter 2006 M. galloprovincialis 18.2392 0.0015 15.6416 0.0011 38.3446 0.0041

10 VRSA beach Winter 2006 D. trunculus 18.2282 0.0017 15.6262 0.0015 38.2792 0.003610 VRSA beach Winter 2006 M. corallina 18.3021 0.0007 15.6351 0.0006 38.3731 0.001611 Manta Rota beach Winter 2006 Donax trunculus 18.2172 0.0022 15.6518 0.0019 38.3803 0.004712 Cacela Velha Winter 2006 M. galloprovincialis 18.2715 0.0050 15.6445 0.0043 38.3671 0.012712 Cacela Velha Winter 2006 C. angulata 18.2612 Cacela Velha Winter 2006 C. edule 18.24

Key: VRSA Vila Real de Santo Antonio; SE: standard error.

18.20

18.22

18.24

18.26

18.28

18.30

18.32

654321Sites

20

6P

b/2

04

Pb

2 σ error bar

Fig. 4. Variation in the isotopic composition of lead (206Pb/204Pb) for bivalve speciesat the same site. Sites 2, 5 and 6 (Spring 2005): (�) C. gallina; (�) M. corallina; Site 12a(Spring 2005) and Site 12b (Winter 2006): (�) M. galloprovincialis; ( ) C. angulata.

Fig. 5. Spatial variation in the isotopic composition of lead (206Pb/204Pb) for C. gal-lina, Gulf of Cadiz (Spring 2005). Sites numbers as for Fig. 1.

36 0.0022 15.6331 0.0017 38.3185 0.004835 0.0014 15.6409 0.0012 38.3454 0.0032

are reported as ratios with respect to 204Pb. Analytical uncertaintiesare shown as error bars (see figure captions for details).

At Sites 2, 5 and 6, the 206Pb/204Pb ratio for C. gallina is con-sistently lower than for M. corallina. Likewise, M. galloprovincialisand C. angulata show similarly contrasting 206Pb/204Pb ratios in thesame site (Site 12) for different seasons (Fig. 4).

Compelling evidence for a systematic change in the isotopiccomposition of lead along the Spanish coast during Spring (i.e. thedrier season) is provided by C. gallina. This shows a westerly trendof increasing 206Pb/204Pb ratios from Sites 1 to 6 (Fig. 5).

4. Discussion

4.1. Species variation

Marine bivalves are in general filter-feeders and therefore havethe capacity to absorb metallic elements from food and water,and also from ingested, inorganic particulate matter (El-Sikaily etal., 2004 Full Text via CrossRef | View Record in Scopus | CitedBy in Scopus (8) Özden et al., 2008). In fact, marine bivalves arewell established as bioindicators for monitoring the concentrationof metals in seawater. This study focused on seven of the mostrepresentative bivalves along the Gulf of Cadiz to study the lev-els and sources of Pb and the activity of �-aminolevulinic aciddehydratase (ALAD) as a lead bioindicator. Pb content varied sig-nificantly among the different species due to their specific habitatand feeding behavior. In general, higher Pb concentrations wereobserved in sediment burying species like S. plana, C. edule, D. trun-culus and C. gallina, compared to those living attached to rockysubstrates (M. galloprovincialis and C. angulata) (Table 2) show-ing the capacity of fine sediments to retain metal contaminantsand that particulate Pb may be an important determinant of con-

tamination for these bivalves. Nevertheless, Pb concentrations inbivalves collected along the Gulf of Cadiz influenced by the IberianPyrite Belt are not exceptionally high and in general lower thanPb levels found for the same species in other geographic locations.The levels of Pb in C. edule collected from two Moroccan Atlantic

R. Company et al. / Aquatic Toxicology 101 (2011) 146–154 151

Fig. 6. Seasonal changes in the lead isotope composition of bivalves along the Gulf of Cadiz. (�) M. galloprovincialis (Spring 2005); (©) M. galloprovincialis (Winter 2006); (�)C D andu Herrer

liflSaHshac

Ft2oapaaf2

. angulata (Spring 2005); (�) C. angulata (Winter 2006). Area SD is the field for AMnpublished data INTERREG-UTPIA project). Area HL is the field for AMD for the Lasefer to the sample sites shown in Fig. 1.

agoons were 25-fold higher than those in the present study, rang-ng from 8.1 to 37.7 �g g−1 Pb d.w. (Cheggour et al., 2001). Also,or S. plana, much higher Pb levels were found in organisms col-ected from a heavily contaminated estuary in Bilbao (Northwestpain) (87–198 �g g−1 Pb d.w.) associated with several industrialctivities that release metallic wastes (Ruiz and Saiz-Salinas, 2000).igher Pb levels were found in M. galloprovincialis collected at 6

ites along the Galician coast and Gulf of Biscay (Spain) (20-foldigher) (Besada et al., 2008), and in several locations along the Adri-tic Sea (Orescanin et al., 2006). Only for C. gallina and D. trunculusollected along the Turkish coast (Sea of Marmara) were Pb levels

ig. 7. Lead isotope trends and principal lead sources for marine bivalves alonghe Gulf of Cadiz. Filled circles refer to bivalves for Sites 7–12 (Portugal Winter006); open circles refer to bivalves for Sites 7–9 and 12 (Portugal Spring 2005);pen triangles refer to bivalves for Sites 1–6 (Spain Spring 2005); half filled circlesre marine sediments from the Gulf of Cadiz (unpublished data INTERREG-UTPIAroject). Point ‘a’ is M. galloprovincialis at Site 9 (Spring 2005); point ‘b’ is S. planat Site 7 (Spring 2005); point ‘c’ is D. trunculus at Site 11 (Winter 2006). Areas SDnd HL as for Fig. 6. Arrow labelled ‘leaded petrol’ is the projected trend to the fieldor European leaded petrol pre-1999 (data from Monna et al., 1997 and Haack et al.,004).

sulphide ores of the Rio Tinto and Sao Domingos mines (data from Marcoux, 1998;rias and El Lagunazo mines (unpublished data INTERREG-UTPIA project). Numbers

considerably lower (0.161–1.342 and 0.292–1.321 �g g−1 Pb d.w.respectively) than those reported for the Gulf of Cadiz (Özden etal., 2008).

Concerning ALAD activity for bivalves collected along the Gulfof Cadiz, values ranged from 0.10 to 2.00 ng PBG min−1 mg prot−1,which is very low compared to what is typically found in verte-brate organisms by comparable methods (e.g. in the blood, liverand kidneys of Halobatrachus didactylus ALAD activity was 7.54,37.7 and 75.4 ng PBG min−1 mg prot−1 respectively) (Campana etal., 2003). This is related to the general lack of respiratory pig-ments in most bivalve species (van Holde et al., 1992). While itis generally accepted that bivalves do not contain any respira-tory pigments, the presence of hemocyanin has been documentedin primitive bivalve species (Protobranquia) such as Acila cas-trensis, Cyclocardia ventricosa, Solemya velum, Nuculana hamata,Nucula proxima, Nucula sulcata, Nucula hanleyi, Nucula nucleus,Yoldia limatula, Yoldia thraciaeformis, and Yoldia eightsi (Morse etal., 1986; Mangum et al., 1987; Toulmond et al., 1987; Terwilligeret al., 1988; Herskovits et al., 1990; Taylor et al., 1995; Angeliniet al., 1998; Bergmann et al., 2007). The ALAD enzyme cat-alyzes the condensation of two molecules of 5-aminolevulinicacid (ALA) to form one molecule of the monopyrrole porpho-bilinogen (PBG) in the heme biosynthetic pathway (Wetmur,1994) and is strongly inhibited by Pb exposure (Kelada et al.,2001). The range of ALAD activity was similar, however, to thatfound for other bivalves such as Corbicula fluminea collectedalong the Guadiana River (0.030–1.754 ng PBG min−1 mg prot−1)(Company et al., 2008) and C. gallina collected along the coast ofSpain (0.2–0.7 ng PBG min−1 mg prot−1) (Kalman et al., 2008) usingcomparable methods. Although the ALAD activity in bivalves is rel-atively low and Pb levels are not exceptionally high in this study,the strong negative relationship between ALAD activity and Pb

observed in C. gallina and M. galloprovincialis (Fig. 2) confirms theinhibiting effect of lead. This is true for all species; either individu-ally or combined (Fig. 3) and supports the recent work of Companyet al. (2008) and Kalman et al. (2008). Since the collection of largenumbers of different bivalve species is not always feasible and prac-

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ical in field studies, it is reasonable to reduce the sampling effort byelecting the best bio-indicator species; taking into account avail-bility/occurrence and biological characteristics. From our studiese propose that the mussel M. galloprovincialis and the clam C.

allina are the most suitable bio-indicator species to evaluate Pbcotoxicity and ALAD response in estuarine and/or coastal environ-ents. This is based on their wider spatial distribution compared to

ther bivalves and their ability to accumulate large concentrationsf Pb. By virtue of their habits, mussels are more representative ofocky substrates and artificial constructions (harbours, marine con-tructions, etc.) while clams are representative of sandy substratesbeaches, etc.).

Regarding the variation of Pb isotopes between species anmportant conclusion and one that has a major bearing on thenterpretation of the bivalve lead isotope data, is the systematicifference in isotope composition between M. corallina and C. gal-

ina collected at the same time and same location (Fig. 1). At Sites, 5 and 6, the 206Pb/204Pb ratio for C. gallina is consistently lowerhan for M. corallina (Fig. 4). These differences can be attributed toifferent feeding mechanisms. C. gallina lives in the substrate and

s recognized as a deposit feeder or grazer, whereas M. corallinas a filter feeder and utilises suspended particulate matter (SPM).f the exposure to lead were controlled solely by lead in solutionne would expect little difference in isotope composition betweenpecies. This is not the case. The large differences in 206Pb/204Pbuggest that the substrate (food source) used by C. gallina is iso-opically distinct from the SPM (food source) used by M. corallina.upport for this assertion is provided by lead isotope data for theerman Bight where Hinrichs et al. (2002) have shown strong

sotopic contrasts between marine sediment and suspended par-iculate matter.

At Site 2, the 206Pb/204Pb ratio for C. gallina (18.218) is similar tohe mean 206Pb/204Pb ratio (18.192 ± 0.007) reported for sulphideres of the Rio Tinto mining district (Marcoux, 1998). This is in goodgreement with the site location (Fig. 1) being within the plume ofetal-contaminated water discharged into the Gulf of Cadiz by thediel and Tinto rivers (Braungardt et al., 2003; Nieto et al., 2007).

n contrast, the high 206Pb/204Pb ratio for M. corallina (∼18.33) atite 6 (Table 3) closely matches the geogenic signature (206Pb/204Pb8.35–18.38) for marine sediments sampled along the south Por-uguese coast (Unpublished data INTERREG-UTPIA Project). Thus, C.allina and M. corallina are bioaccumulating different proportionsf the total geogenic–anthropogenic lead flux and should be inter-reted accordingly. Albeit based on limited analyses, M. galloprovin-ialis and C. angulata show similarly contrasting 206Pb/204Pb ratiosFig. 1, Site 12). Changes in the source and degree of lead pollutions inferred from isotope ratios must therefore be treated as speciespecific. The potential for discriminating between different foodources provides crucially important information for the biokineticodelling of metal bioaccumulation (Wang and Rainbow, 2008).

.2. Spatial variation

The spatial variation of Pb is closely linked to the metal dis-harges of the Tinto and Odiel Rivers in the east (Spain) and theuadiana River in the west (Portugal). This is especially apparent

or C. gallina and M. galloprovincialis which show high Pb levels nearhe river mouths (Sites 1, 2 and 7), decreasing progressively withistance away from these source (Table 2).

The westerly trend of increasing 206Pb/204Pb ratios from Site 1o 6 observed for C. gallina is consistent with an increasing frac-

ion of bioaccumulated, geogenic lead. Fig. 5 is thus interpreteds a mixing array between mine-related lead entering the marinenvironment at Huelva and geogenic lead characteristic of marineediments between the Guadiana and Odiel–Tinto estuaries. Dataor C. gallina, at sites further west, are lacking but other bivalves (S.

ology 101 (2011) 146–154

plana, M. galloprovincialis, C. angulata) display a range of 206Pb/204Pbratios that are characteristic of AMD discharges into the Guadianaas determined from river water analyses and analyses of the fresh-water bivalve Corbicula fluminea (Company et al., 2008). AlthoughC. fluminea shows large seasonal changes in isotopic composition,the values for Spring 2005 (206Pb/204Pb 18.196–18.252) are in verygood agreement with those for S. plana, M. galloprovincialis andC. angulata (206Pb/204Pb 18.155–18.252) sampled at Sites 7 and12. The discharge of anthropogenic lead into the Gulf of Cadiz bythe Guadiana in the west and Odiel–Tinto rivers in the east, fromabandoned metalliferous mines in the Iberian Pyrite Belt, appeartherefore to control the spatial variation in bivalve lead isotopecomposition along the coast.

4.3. Seasonal variation

The interpretation of seasonal variation in Pb along the coastof the Gulf of Cadiz is difficult due to the absence of represen-tative sampling of species in both seasons. In M. galloprovincialisand D. trunculus for example, Pb levels were consistently higherin winter at all sites, while for C. angulata no seasonal effect wasevident. Higher Pb levels observed in organisms during the wetseason are often attributed to a significant increase of run-off con-tributions from adjacent arable land and roads. Seasonal variationsin the metal content of bivalves are also dependent on physiologicalprocesses, especially those linked to reproductive activity. Duringgametogenesis, gonads represent an additional, temporary metal-storage tissue. Most bivalves have episodes of gametes emissionduring the warmer months (spring and summer), with a subse-quent elimination of metals via this process (Cheggour et al., 2001).The case for seasonal variation is strongly supported however byseasonal changes in ALAD activity and the negative relationshipwith Pb (Fig. 3). As noted above, seasonal variability in ALAD activ-ity was observed for C. fluminea (Company et al., 2008) suggestingthat the degree of enzyme inhibition depends not only on Pbaccumulated in their tissues but also on biotic parameters (espe-cially temperature) and animal physiology (reproductive cycle andmetabolism).

A case for seasonal variation in the isotopic composition oflead for the marine bivalves is not as clear cut as for C. flu-minea (Company et al., 2008) partly because there are fewer datapoints for individual species per season. However, there are sev-eral interesting indicators. C. angulata at Sites 7 and12 and M.galloprovincialis at Sites 7, 9, 10 and 12, show significant sea-sonal changes in isotopic composition (Fig. 6). The 207Pb/204Pb,206Pb/204Pb ratios for M. galloprovincialis and C. angulata in Spring2005 are shifted to higher ratios in Winter 2006. This may be dueto variation in the timing of winter precipitation influencing whenAMD is released into the rivers, or it could be a result of changingwater management strategies under drier climatic conditions (seebelow). Nevertheless, for whatever reason, they display an isotopicshift between seasons. The slight displacement of values to higher206Pb/204Pb ratios away from the AMD fields for São Domingos andLas Herrerias-El Lagunazo is due to the mixing with a third compo-nent ‘geogenic’ lead (see Fig. 7). To fully verify a seasonal periodicityone would require more continuous monitoring. This would alsopermit the rate of environmental change to be determined andallow comparison with the biokinetics of metal elimination in thespecies under consideration (Wang and Rainbow, 2008). A lack ofseasonal data for bivalves along the Spanish section of the coastprecludes comparison with the Portuguese sites.

4.4. Source apportionment for lead

Considering all the data, bivalve lead isotope compositionsdefine two arrays; a Winter array and a Spring array (Fig. 7).

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uring Spring 2005 when river flows were low, the isotopic com-ositions of marine bivalves are distributed along a linear binaryrray between the field for Rio Tinto- São Domingos sulphide oresnd recent marine sediments (Marcoux, 1998; unpublished dataNTERREG-UTPIA Project). For the Guadiana, mine pollution is dom-nated by AMD discharges from the historic São Domingos mine,

hereas the Odiel and Tinto rivers are dominated by AMD dis-harges from mines in the Rio Tinto district. The bivalve isotope datare therefore consistent with the mixing of an anthropogenic mine-elated lead and a predominantly background, geogenic lead. Theecond array, displaced to higher 207Pb/204Pb, 206Pb/204Pb ratios,orresponds to the same Portuguese sites but sampled when waterow in the Guadiana was higher (Winter 2006). Unfortunately,omplementary samples for the Spanish coastal sites were notaken. Unlike the Spring array, the Winter array projects into thesotopic field for AMD discharges from the less important miningrea of Las Herrerías (unpublished data, INTERREG-UTPIA Project).mong the streams affected by AMD in the Lower Guadiana catch-ent, two have been identified as of greater importance for water

uality in the Guadiana - the Cobica stream in Spain that drainshe mines of El Lagunazo and Las Herrerías, and the São Domingostream in Portugal that drains the São Domingos mine (Delgado etl., 2009). As proposed for the freshwater bivalve data, the switchrom a São Domingos source to a Las Herrerías source is probably aombination of water flow and water management (Company et al.,008). AMD salts stored in the mine waste at Las Herrerías duringry periods are not flushed into the Guadiana marine environmentntil rainfall and water flow permit. Being within the Guadianaatershed one would not expect to see the Las Herrerías isotope

ignature in bivalves influenced by the Odiel and Tinto rivers. Weonclude therefore that although lead isotope compositions forarine bivalves are linked to water flow, the crucial biomonitoring

ata with respect to one species that would unequivocally con-rm a seasonal shift along the Gulf of Cadiz has still to be acquired.he overall evidence for seasonal trends based on several speciess however strong, consistent and supportive.

Three bivalves do not conform to the above arrays. S. plana (SiteSpring 2005) and M. galloprovincialis (Site 9 Spring 2005) have

nomalously low 207Pb/204Pb (<15.635) and 206Pb/204Pb (<18.170)atios while D. trunculus (Site 11 Winter 2006) has 207Pb/204Pb15.652) and 206Pb/204Pb (18.217) ratios that are anomalously highFig. 7). These compositions plot well outside the potential sourcesor anthropogenic mine pollution and geogenic lead in the Loweruadiana catchment. The low ratios for S. plana and M. galloprovin-ialis (Points a and b, Fig. 7) appear to be displaced towards theeld for tetra-ethyl lead; the lead additive to petrol that has noween phased out in Western Europe. Isotope data for countriestill marketing leaded petrol (e.g. Algeria, Morocco and Tunisia)ave not been published. However if one assumes similarity withestern Europe (Monna et al., 1997; Haack et al., 2004) then

he bivalve compositions are displaced in the correct sense anduggests contamination with pre-1999 leaded petrol (207Pb/204Pb∼15.49–15.54)) and 206Pb/204Pb (∼16.54–17.00). Site 7 is immedi-tely below the International Bridge across the Guadiana and mayherefore be a response to tetra-ethyl lead from current or pre 1999ehicular traffic using this route, or from re-suspended contami-ated estuarine sediments caused by heavy river traffic. Site 9, byontrast, is adjacent to the breakwater (or mole) across the Guadi-na estuary. In this instance, the lead may be derived from leadeduel still used by power boats and high performance marine crafthich increase in numbers as the tourist season begins. Site 11

Point c, Fig. 7) is more problematic and, pending a replicate analy-is, we are unable to account for this sample lying well outside theeneral field for the marine bivalves.

With hindsight, the decision to sample several bivalve speciesith different feeding habits, at different seasons and sites, with

logy 101 (2011) 146–154 153

potentially multiple sources of lead was overambitious. Also, due totechnical constraints it was not possible to sample all species dur-ing the winter season at all sites. As a result, interpretation was verychallenging. In future, the experimental design needs to be morefocused, by limiting biological variability (i.e. selecting the mostappropriate bio-indicator species) and carrying out more contin-uous seasonal monitoring. Furthermore, the experimental designcould be significantly improved by collecting organisms from a Pb-free control site to establish basal ALAD activity for the speciesunder consideration.

5. Conclusions

Given the evidence for intense, mine-related metal dischargesinto the Gulf of Cadiz by the Odiel, Tinto and Guadiana rivers, leadlevels in several bivalve species collected along the Gulf are notparticularly high when compared to other polluted coastal envi-ronments (e.g. Morocco, NW Spain). Absolute lead concentrationsare strongly influenced by species (mainly habitat, food source andreproductive cycle). The significant negative relationship betweenALAD activity and lead concentration confirms the use of ALAD asa biomarker for lead exposure in marine bivalves, although sea-sonal effects need to be taken into consideration when assessing thedegree of enzyme inhibition. Variation in 207Pb/204Pb 206Pb/204Pbratios indicates significant seasonal and spatial changes in bivalveisotopic composition reflecting seasonal and geographic differ-ences in bioaccumulation. Furthermore, the analyses provide aninsight into different food sources and reinforce the importance ofconsidering species-specific factors when using ALAD activity as abiomarker for lead. Within the study area, Pb can be modelled as amixing between geogenic Pb and anthropogenic, mine-related Pbfrom the IPB. For some sites at the mouth of the Guadiana River,the bivalves show minor contamination from leaded boat/aviationfuel and/or leaded paint.

Acknowledgements

The present research was carried out in the framework of anINTERREG III Programme between Portugal and Spain that wasfinancially supported by the EU (UTPIA/SP5.P9/02-INTERREG IIIA)and Centre for Marine and Environmental Research, University ofAlgarve (CIMA).

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