Squire, S., Scelfo, G.M., Revenaugh, J., Flegal, A.R., 2002.Decadal trends of silver and lead contamination in San FranciscoBay surface waters. Environmental Science and Technology 36, 2376–2379.

Stephenson, M.D., Leonard, G.H., 1994. Evidence for the decline ofsilver and lead and the increase of copper from 1977 to 1990 in the

coastal marine waters of California. Marine Pollution Bulletin 28, 148–153.

Webb, N.A., Wood, C.M., 1998. Physiological analysis of the stressresponse associated with acute silver nitrate exposure in freshwaterrainbow trout (Oncorhynchus mykiss). Environmental Toxicology andChemistry 17 (4), 579–588.

0025-326X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2005.09.009

Temporal and spatial variation of trace metals in clamsTivela mactroidea along the Venezuelan coast

Juan A. Alfonso *, Jose A. Azocar, John J. LaBrecque, Zully Benzo, Eunice Marcano,Clara V. Gomez, Manuelita Quintal

Instituto Venezolano de Investigaciones Cientıficas (IVIC), Centro de Quimica, Apartado 21827, Caracas 1020A, Venezuela

Bivalves have been extensively used as bioindicators toassess coastal aquatic environments, since they can accu-mulate trace metals and other substances. In recent years,the abundance of clams, Tivela mactroidea, along the coastof Venezuela has increased greatly. Only a few studies havebeen reported on trace metals in bivalves from Venezuela,however, such studies have not dealt with temporal varia-tions, have only considered a limited number of elements,and have mainly been undertaken in restricted area, nearthe mouth of the Tuy River (State of Miranda). Thus, con-sidering the wide extension of the Venezuelan coast and thefact that this species is widely consumed by local popula-tions, a more complete study on trace metal contaminationwas conducted.

Samples of T. mactroidea were collected from marinesediments at six sampling sites (Fig. 1), two in the stateof Falcon, two in the state of Miranda and two in the stateof Anzoategui. Sampling took place once a month betweenDecember 2002 and November 2003 at each site. The exte-rior (shells) of the clams were rinsed with seawater to elim-inate marine sediments before being transferred to 1 l widemouth bottles filled with distilled deionized water. After thebottles were closed, they were placed in a cooler with ice fortransportation to the laboratory.

It is well known that the accumulation of heavy metals inbivalves is affected by age, size and weight of the clams (Cainand Luoma, 1990; Wang and Fisher, 1999; Rainbow et al.,2000; Ruelas-Inzunza and Paez-Ozuna, 2000). As these fac-tors were not the objective of our study, they were kept asconstant as possible, in order to minimize their influence

on the measured concentrations. Each sample consisted ofa pool of at least 50 clams (T. mactroidea) 2–4 cm in size.

After a few days in the refrigerator the clams opened bythemselves. The complete soft tissues inside the shells wereremoved and washed with distilled deionized water to sep-arate the soft clam tissue from any marine sediment andother foreign material. The tissues were transferred to clean1 l beakers and washed several times with distilled deion-ized water until the solution was clear. After discardingthe excess liquid, the beakers were placed in an oven at65 �C until most of the liquid evaporated, then the temper-ature was increased to 80 �C for 24 h and then the sampleswere cooled in desiccators. The dried soft clam tissue wasground into a fine powder with an agate mortar and pestleand well mixed to prepare a homogenous sample.

Portions of the dried soft clam tissues (1 g) were dis-solved in 8 ml of concentrated super pure nitric acid(Merck) by heating on a hot plate at about 70 �C forapproximately 8 h. When the solution cooled, 2 ml ofhydrogen peroxide (30%) (Merck) was added and heatedagain at 70 �C for another 4 h. Finally, the resulting solu-tion was diluted to 50 ml with distilled deionized water.Duplicate digestions were performed for each sample.

The samples were then analysed by inductively coupledplasma optical emission spectroscopy (ICP-OES) using aPerkin–Elmer, Optima 3000. Determination of Ba, Cd,Co, Cr, Cu, Ni, Sr, Ti, V and Zn was carried out in theautomatic mode, with background correction employingthe ICPWinLab Optima 3000 software package. Thefollowing wavelengths were used: 455.409 nm for Ba,226.502 nm for Cd, 228.616 nm for Co, 205.552 nm forCr, 324.754 nm for Cu, 231.604 nm for Ni, 421.552 nmfor Sr, 337.280 nm for Ti, 292.402 nm for V and202.548 nm for Zn. A comparison of the trace metals deter-mination in mussel tissue reference materials NIST-2976

* Corresponding author. Present address: Centro de Quimica, 8424 NW56th Street, Suite CCS 00204, Miami, FL 33166, USA. Fax: +58 212 5041511.

E-mail address: jalfonso@ivic.ve (J.A. Alfonso).

Baseline / Marine Pollution Bulletin 50 (2005) 1713–1744 1723

and NIST-2977 with the certified, recommended andinformation values is presented in Table 1. It can be seenthat determined values were very similar to the certifiedand recommended values, thus the ICP-OES method (LaB-recque et al., 2004a) employed can be considered accurate.Standard deviations (1r) of the element determinations(n = 6) were small, indicating good precision.

Mean, minimum, maximum and standard deviation val-ues of the trace metal concentrations in soft clam tissuesfrom the six sampling sites are presented in Table 2. Themean concentration values for Cd, Cr, Cu, Ni V and Znfound in the sites 3 and 4 are within the range of valuesdetermined one year earlier for the same species of clamat the same sampling area: the state of Miranda (LaBrecqueet al., 2004b). No known values have been reported for Ba,Co, Ti and Sr for this species in Venezuela. A comparison ofthe two studies indicates that the temporal difference had alower impact on the elemental concentrations than the spa-tial difference between the three sampling areas.

A one way ANOVA was performed in order to evaluatethe statistical significance between the sites, for each metalconsidered. Tukey a posteriori tests were performed when-

ever the null hypotheses were rejected. Statistical analyseswere carried out using Statistica 6.0 software (StatSoft,USA). In the last column of Table 2, the p-values fromthe ANOVA-test are listed. Significance levels were fixedat p < 0.05 on a 95% confidence level, p-values of elementswith significant concentration differences between the sam-pling sites are presented in boldface. The discussion ofpossible environmental and/or anthropogenic sourcesresponsible for these differences will be focused on theseelements.

The mean concentrations are generally higher at sites 3and 4 (state of Miranda) for all determined elements withsignificant differences between sites, with the exception ofthe elements Ba, V and Zn. The higher concentrations inthe sampling sites of the state Miranda were not unex-pected, considering the fact that these sites are located westof the mouth of the Tuy River, which has a plume knownto move in a north-westerly direction and receives wastewater effluents from the metropolitan area of Caracas, acity of approximately 6 million inhabitants, via the Guaireriver. Most of this waste water undergoes only minortreatment. In addition, the Tuy river drainage basin, of

Fig. 1. A partial map of the Venezuelan coast showing the sampling sites, states and rivers.

Table 1Comparison of the determined values (n = 6) in mussel tissue standard reference materials NIST-2976 and NIST-2977 in this study with the certified (CV),recommended (RV) and information (IV) values

Element NIST-2976 NIST-2977

Reference material values (lg/g) This study (lg/g) Reference material values (lg/g) This study (lg/g)

Ba – – 4.7 (IV) 4.32 ± 0.27Cd 0.82 ± 0.16 (CV) 0.809 ± 0.054 0.179 ± 0.003 (CV) <dlCo 0.61 ± 0.02 (RV) 0.594 ± 0.021 0.48 ± 0.13 (RV) <dlCr 0.50 ± 0.16 (RV) <dl 3.91 ± 0.47 (RV) 3.92 ± 0.07Cu 4.02 ± 0.33 (CV) 4.08 ± 0.32 9.42 ± 0.52 (CV) 9.42 ± 0.13Ni 0.93 ± 0.12 (RV) <dl 6.06 ± 0.24 (CV) 6.78 ± 0.49Sr 93 ± 2 (RV) 91.05 ± 1.93 69.3 ± 4.2 (CV) 67.39 ± 3.41V – – 1.1 (IV) 1.06 ± 0.05Zn 137 ± 13 (CV) 134.8 ± 5.0 135 ± 5 (RV) 129.5 ± 5.8

<dl = Below detection limited.

1724 Baseline / Marine Pollution Bulletin 50 (2005) 1713–1744

approximately 6600 km2, covers an active industrial andagricultural area, which also affects water quality (Jaffeet al., 1995).

The mean concentrations of V and Ba were significantlyhigher in sites 1 and 2. This coastal area contains veryextensive petroleum refining and transportation activitiesand is located near of the mouth of the Unare River, whichflows along the one of the most important oil producingareas for Venezuela. Vanadium is typically associated tooil (Macıas-Zamora et al., 1999; Agusa et al., 2004) andBa has been related to the use of barite (BaSO4) in drillingmuds (Sharma et al., 1999). A good correlation betweenlocation of oil and gas wells and high Ba concentrationshas been reported in the south Texas Bays (Holmes et al.,1974). Thus, the present results are consistent with theuse of V and Ba as possible markers for oil transportationand exploitation activities.

In contrast to the others elements with significant differ-ences between sites, the difference in the mean Zn concen-trations between the three sampling areas was negligible.Some mussel species have been found to regulate the levelof this biologically essential element (Phillips, 1985;

Malinovskaya and Khristoforova, 1997; Gunther et al.,1999), if the concentration in the aquatic environment doesnot exceed a certain level. As a consequence of this effect, adirect relationship between Zn concentration in the envi-ronment, on the one hand, and in a biomonitor, on theother hand, would be impossible to observe.

The relatively large variations occurring within one pop-ulation, expressed by high standard deviations and largevariations between minimum and maximum values inTable 2 are not unexpected, because of the complex rela-tionships between environmental concentrations and bio-accumulation. Ba, Sr, V and Zn concentrations in clamsdisplayed very little variation during the year. Fig. 2 showsthe temporal variation of Cd, Co, Cr, Cu, Ni and Ti con-centrations in clams from the sampling sites 1, 3 and 5.In this figure, results below the lower limit of detection ofthe analytical method were plotted as half the detectionlimit value. The results clearly show that for each metalthere is a similar temporal variation pattern. The maximumconcentrations observed in this study were as follows:for Cd (4.96 ± 0.16 lg/g), Co (8.79 ± 0.45 lg/g), Cr(6.08 ± 0.18 lg/g) and Cu (32.57 ± 0.97 lg/g) during

Table 2Mean, minimum, maximum and standard deviation values of the trace element concentrations in dried soft clam tissue from the different sampling sitesduring this temporal study (n = 12) along the Venezuelan coast

Element (lg/g) Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 p

Ba 4.22 3.64 2.32 2.66 2.51 1.852.10–6.52 1.97–6.00 1.13–3.62 0.88–8.17 0.77–5.51 0.63–3.79 <0.00011.15 1.13 0.88 1.89 1.73 1.02

Cd 1.14 1.35 1.37 1.47 1.53 1.020.50–3.67 0.50–3.71 0.50–4.58 0.50–4.96 0.50–4.65 0.50–3.67 0.24460.97 1.06 1.22 1.34 1.46 0.97

Co 5.02 5.32 4.96 5.24 5.47 4.912.30–8.36 2.18–8.79 2.98–8.68 3.13–8.40 3.08–10.88 3.22–8.18 0.11881.80 1.89 1.46 1.63 2.24 1.57

Cr 2.32 3.02 3.08 3.38 2.51 2.540.50–6.08 0.50–5.43 1.31–5.67 1.59–5.77 0.50–5.11 0.50–4.58 0.0180

1.43 1.73 1.42 1.41 1.49 1.28

Cu 10.11 9.98 19.71 18.14 16.97 15.445.36–16.94 5.19–16.73 12.54–32.57 13.17–27.82 9.94–25.09 10.44–26.88 <0.00013.15 3.30 5.93 3.97 4.39 5.08

Ni 12.33 13.86 14.06 13.88 11.79 12.037.72–16.90 7.70–25.04 9.35–25.97 8.43–23.11 6.52–17.25 6.03–20.28 0.0029

2.78 5.34 4.36 3.79 3.40 5.00

Ti 11.41 12.42 36.53 45.25 25.10 20.942.00–78.79 2.00–77.23 5.64–133.65 5.63–228.36 2.00–125.17 5.15–100.34 <0.000121.35 20.76 36.99 63.83 33.27 26.27

V 3.78 3.59 2.53 1.72 1.64 1.921.39–5.96 0.50–6.13 0.50–3.83 0.50–4.18 0.50–3.00 0.50–3.12 <0.00011.51 1.67 1.17 1.16 0.88 0.75

Zn 117.79 112.66 119.64 102.93 126.33 98.9594.55–138.58 82.93–137.04 87.79–150.81 84.06–116.54 91.52–148.20 73.77–136.38 <0.000114.23 17.53 20.13 10.14 16.76 16.92

Sr 63.47 63.54 89.06 79.23 63.49 52.9640.89–100.97 42.47–78.57 60.30–128.54 52.07–130.77 48.89–92.12 28.86–82.11 <0.000116.36 10.94 22.24 23.00 12.22 13.99

Baseline / Marine Pollution Bulletin 50 (2005) 1713–1744 1725

February–March, for Cr (6.07 ± 0.18 lg/g) in May, andfor Ni (25.97 ± 2.31 lg/g) and Ti (228.36 ± 5.31 lg/g) inAugust and May, respectively.

Exogenous factors, such as the changes in salinity dur-ing the year depending on rainfall and river freshwaterinput, contribute to seasonal variations (Wagner andBoman, 2004). It has also been reported that one of themost important endogenous factors is the effect of thereproductive cycle on metal accumulation (Coimbraet al., 1991). Unfortunately, the previous reported studiesutilising T. mactroidea have not included information onthe variability in metal concentration during the year,impeding comparative studies, and there is no precise infor-mation of reproductive cycles. However, our results suggestthat the endogenous factors have a higher impact onannual variation than the exogenous factors along the Ven-ezuelan coast. Further investigation is necessary to under-stand the dynamics of bioaccumulation and to interpretthe annual variations observed.

Although average content of trace metals measured inthe present study are within the range with those foundby other authors, the highest concentrations observed for

Cr are greater than the legal limit (5 lg/g dry weight) forthat metal in molluscs/shellfish according to the Foodand Agricultural Organization of the United Nations(Wagner and Boman, 2004; FAO-UN, 1982), indicatingthat these clams constitute a health risk in respect to theCr metal concentration. Finally, our results suggest theimportance of studying the temporal variation of metalaccumulation by clams and other bivalves.

Acknowledgements

We thank Carlos Bastidas and Melanio Sulbaran fortheir assistance.

References

Agusa, T., Kunito, T., Tanabe, S., Pourkazemi, M., Aubrey, D.G., 2004.Concentrations of trace elements in muscle of sturgeons in the CaspianSea. Marine Pollution Bulletin 49, 789–800.

Cain, D.J., Luoma, S.N., 1990. Influence of seasonal growth, age andenvironmental exposure on Cu and Ag in a bivalve indicator, Macoma

balthica, in San Francisco Bay. Marine Ecology-Progress Series 60,45–55.

0

2

4

6

8

Cr (

µg/

g)

0

40

80

120

160

Ti (µ

g/g)

0

10

20

30

40

dec jan feb mar apr

may jun julau

gse

p oct nov

Cu

(µg/

g)

SITE.1 SITE.3 SITE.5

0

10

20

30

Ni (

µg/

g)

0

1

2

3

4

5

Cd

(µg/

g)

0

4

8

12

dec jan feb mar apr

may jun julau

gse

p oct nov

Co

(µg/

g)

SITE.1 SITE.3 SITE.5

Fig. 2. Annual variation of Cd, Co, Cr, Cu, Ni, and Ti concentrations in Tivela mactroidea from sampling sites 1, 3 and 5, between December 2002 andNovember 2003.

1726 Baseline / Marine Pollution Bulletin 50 (2005) 1713–1744

Coimbra, J., Carraca, S., Ferreira, A., 1991. Metals in Mytilus edulis fromthe Northern Coast of Portugal. Marine Pollution Bulletin 22, 249–253.

FAO-UN, 1982. Appendix V: median international standards 1982, Inter-national standards for trace elements in fish and molluscs. homepage:<http://www.waterboards.ca.gov/programs/smw/docs/9597/appen-dix_v.pdf> (accessed February 2005).

Gunther, A.J., Davis, J.A., Hardin, D.D., Gold, J., Bell, D., Crick, J.R.,Scelfo, G.M., Sericano, J., Stephenson, M., 1999. Long-term bioaccu-mulation monitoring with transplanted bivalves in the San Franciscoestuary. Marine Pollution Bulletin 38, 170–181.

Holmes, C.W., Slade, E.A., Mickerran, C.J., 1974. Migration andredistribution of zinc and cadmium in marine estuarine system.Environmental Science and Technology 8, 254–259.

Jaffe, R., Leal, I., Alvarado, J., Gardinali, P., Sericano, J., 1995. Pollutioneffects of the Tuy River on the Central Venezuelan Coast: Anthropo-genic organic compounds and heavy metals in Tivela mactroidea.Marine Pollution Bulletin 30, 820–825.

LaBrecque, J.J., Benzo, Z., Alfonso, J.A., Cordoves, P.R., Quintal, M.,Gomez, C.V., Marcano, E., 2004a. The determination of selected traceelements in raw clams and commercial clam meats from the State ofMiranda (Venezuela) employing ICP-OES, GF-AAS and WD-XRF.Atomic Spectroscopy 25 (1), 112–124.

LaBrecque, J.J., Benzo, Z., Alfonso, J.A., Cordoves, P.R., Quintal, M.,Gomez, C.V., Marcano, E., 2004b. The concentrations of selectedtrace elements in clams, Tivela mactroidea along the Venezuelan coastin the state of Miranda. Marine Pollution Bulletin 49, 659–667.

Macıas-Zamora, J.V., Villaescusa-Celaya, J.A., Munoz-Barbosa, A.,Gold-Bouchot, G., 1999. Trace metals in sediment cores from theCampeche shelf, Gulf of Mexico. Environmental Pollution 104,69–77.

Malinovskaya, T.M., Khristoforova, N.K., 1997. Characterization ofcoastal waters of the south Kuril Islands by the trace element contentof indicatory organisms. Russian Journal of Marine Biology 23,212–218.

Phillips, D.J.H., 1985. Trace metals in bivalve molluscs from Thailand.Marine Environmental Research 15, 215–234.

Rainbow, P.S., Wolowicz, M., Fialkowski, W., Smith, B.D., Sokolowski,A., 2000. Biomonitoring of trace metals in the Gulf of Gdansk, usingmussels (Mytilus trossulus) and barnacles (Balanus improvisus). WaterResearch 6, 1823–1829.

Ruelas-Inzunza, J.R., Paez-Ozuna, F., 2000. Comparative bioavailabilityof trace metals using filter-feeder organisms in a subtropical coastalenvironment (Southeast Gulf of California). Environmental Pollution107, 437–444.

Sharma, V.K., Rhudy, K.B., Koening, R., Vazquez, F.G., 1999. Metals insediments of the Upper Laguna Madre. Marine Pollution Bulletin 38,1221–1226.

Wagner, A., Boman, J., 2004. Biomonitoring of trace elements inVietnamese freshwater mussels. Spectrochimica Acta Part B 59,1125–1132.

Wang, W.X., Fisher, N.S., 1999. Delineating metal accumulation path-ways for marine invertebrates. Science of the Total Environment 237–238, 459–472.

0025-326X/$ - see front matter � 2005 Published by Elsevier Ltd.

doi:10.1016/j.marpolbul.2005.09.006

PCDD and PCDF intake through consumption of locallyproduced seafood by Venice lagoon residents:

Elements for risk management

R. Miniero a,*, G. Ceretti b, E. Cherin b, E. Dellatte a, S. De Luca a, F. Ferri a,A.R. Fulgenzi a, F. Grim b, N. Iacovella a, A.L. Iamiceli a, A.M. Ingelido a,

P. Vio b, A. di Domenico a,*

a Toxicological Chemistry Unit, Italian National Institute for Health, 00161 Rome, Italyb Department of Prevention, Veneto Region, 30123 Venice, Italy

In recent years, a number of research programs havebeen carried out on contamination in the Venice lagoon(Fig. 1), focusing on the determination of high concernmicro-contaminants in several environmental matrices,including edible organisms such as clams (Tapes sp.) andmussels (Mytilus galloprovincialis); and the assessment oflagoon resident exposure to these contaminants (di Dome-nico et al., 1997a, 1998a; Miniero et al., 2003). The pres-ence of micro-contaminants in the lagoon is due to a

many-decade-old large industrial settlement (Porto Marg-hera) and the century-old city of Venice, both of whichhave released their wastes into lagoon waters throughindustrial and urban effluents.

Data on trace organic contaminant presence in the differ-ent lagoon matrices is essential to perform a risk assessmentand to take risk management actions. For example, for thedevelopment of risk management protocols for the contam-inants of concern, knowledge of their baseline or back-ground values in biota is an essential requirement. Forexample, following the 1999 case of dioxin contaminationof foodstuffs in Belgium, an evaluation of the risk associatedwith dioxin and dioxin-like PCBs intake due to food con-sumption was produced by the EU/EC Scientific Committee

* Corresponding authors. Tel.: +39 6 49902826; fax: +39 6 49387139(R. Miniero).

E-mail addresses: roberto.miniero@iss.it (R. Miniero), addeke@iss.it(A. di Domenico).

Baseline / Marine Pollution Bulletin 50 (2005) 1713–1744 1727