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Concentrations of metals and metallothioneins in Mytilus galloprovincialis along the south coast of Portugal
Marine Pollution Bulletin
Edited by D. J. H. Phillips margin bordering the Atlantic Ocean (Fig. 1). Apart from the Guadiana River, two major aquatic systems exist: the River Arade; and the largest lagoon of the Portuguese coast, the Ria Formosa (Bebianno, 1995). In the Algarve there is an important fish, shellfish and tourism industry, the total catch representing around 20% of all Portuguese fisheries. The region of the Algarve is also of high environmental and economic value. However, the coastline is suffering from environmental degradation which is starting to have economic impacts on shellfish production and the tourist industries. The coastline has been under constant urban pressure during the last fifteen years due to tourist development. The population, which is concentrated in the coastal zone with a density of about 167 inhabitants km -2, increases more than 10-fold over the summer, which aggravates the effects from the paucity of sewage treatment facilities. Sewage discharges have been identified as one of the most important sources of pollution loading on the Algarve Coast. Some of the streams, rivers and estuarine systems along the coast also contribute anthropogenic inputs. One aspect of environmental degradation is pollution from metals which are persistent and are bioaccumulated by marine organisms, with serious public health implications (Phillips and Rainbow, 1993). Methods of biomonitoring metal loads have been evolved using organisms which are sedentary, have a
The objective of BASELINE is to publish short communications on different aspects of pollution of the marine environment. Only those papers which clearly identify the quality of the data will be considered for publication. Contributors to Baseline should refer to 'Baseline---The New Format and Content' (Mar. Pollut. Bull. 24, 124). Marine Pollution Bulletin, Vol. 34, N o . 8, pp. 666--671, 1997
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© 1997 E l s e v i e r Science L t d All ri ght s reserved. P r i n t e d in Great Britain 0025-326X/97
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Concentrations of Metals and Metallothioneins in Mytilus galloprovincialis along the South Coast of Portugal M. J. BEBIANNO and L. M. MACHADO Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal The Algarve Coast of southern Portugal bordering the Atlantic Ocean between 37035 ' and 36°58'N, and 7025' and 9°00'W is 135 km long from its eastern limit with Spain, marked by the Guadiana River, to the western
River
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SPAIN
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/
Vila Real de Sic Antbnio
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Ria Formosa
4
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Sagres
ili:diii~i; Fig.
666
1 Sampling
sites
along
the
Algarve
Coast.
i
Volume 34/Number 8/August 1997 wide geographical distribution and accumulate metals in a manner that reflects the ambient conditions. Bivalve molluscs, and in particular Mytilus spp., have been successfully utilized for coastal water quality monitoring. The 'Mussel Watch Program' (Goldberg et al., 1978) is based on the chemical analysis of contaminants in the soft tissues of individuals. One important modification to conventional programmes of measuring contaminants directly in the whole soft tissues is to measure other compounds which also reflect levels of contamination. These techniques may be more sensitive than measuring the contaminants directly in the tissues (Viarengo, 1985). An example is provided by the detoxification strategy in marine organisms of producing low molecular weight metal binding proteins known as metallothioneins (Bebianno and Langston, 1991, 1992; Bebianno et al., 1992; Walkes and Goering, 1992; Roesijadi, 1994). Measurements of the levels of these proteins provide an accurate indication of subtle environmental increases in metal contamination. Following the principle of the Mussel Watch Program (Goldberg et al., 1978) the purpose of this study was to use the mussel Mytilus galloprovincialis Lmk. from the Algarve Coast as a biomonitor to identify areas contaminated by heavy metals. In addition to measuring the concentrations of Cd, Cu, Fe, Mn, Ni and Zn, metallothionein levels were determined in the whole soft tissues of the mussels. The latter have been utilized as a specific biochemical indicator for the early detection of potential detrimental effects induced by metal contamination. Nineteen sites were selected along the Algarve Coast (Fig. 1) for the sampling of M. galloprovincialis. Thirty mussels of similar length (45-55 mm, 1.08-2.66 g dry weight) were collected from the same intertidal depth during a spring tide, in April and May 1994. All the animals were in the pre-spawning period, with 91% of them in Maturation State III (Lubet, 1959; Lucas, 1965). In the laboratory, all mussels were depurated for 24 h to eliminate gut contents, prior to their analysis for metals and metallothionein. The animals were carefully opened by cutting the adductor muscle and the soft tissues were dissected out. The tissues were pooled in groups of five individuals, after discarding the shell, byssus and cavity liquor. Each pooled sample was weighed and homogenized in three volumes of 0.02 M Tris-HCl buffer (pH 8.6) in an ice-bath. Subsamples were taken for the determination of wet/dry weight ratios. The samples were dried and digested in concentrated nitric acid, following the procedure described by Bryan et al. (1985). Metal concentrations were determined using an atomic absorption spectrophotometer (VARIAN-SpectrAA20). Analysis of the TORT I lobster hepatopancreas reference material (National Research, Canada) were carried out following the same sample treatment, to validate the metal analysis (Table 1). The concentrations of all metals are expressed in I.tg g - ~ dry weight of soft tissue.
TABLE 1 Comparison of metal concentrations(lagg- l dry weight)in standard reference materialTORT 1 certifiedby the National ResearchCouncil
of Canada, and analyticalresults from the current study. Metal Cd Cu Zn
Certified values
Our values (n = 10)
26.3-/-2.1 439+22 177+10
26.98+0.01 449+0.01 160:~-0.7
Aliquots of the homogenate (3 to 5 ml) were centrifuged at 30 000 g for 1 h at 4°C. The supernatant (cystosol) was separated from the pellet, heated at 80°C for I0 min to precipitate the high molecular weight proteins denatured by heat, and subsequently centrifuged at 30 000 g for 1 h at 4°C. Aliquots (50 to 250 ~1) of the heat-denatured cytosol were taken for the quantification of metallothioneins (MT) by differential pulse polarography as described by Bebianno and Langston (1989), with a polarograph Methrom 646 and using a MT rabbit standard (Sigma). Metallothionein concentrations were determined as mg g - t dry weight of homogenized tissue. The parametric Tukey Test was used to identify differences between a specific pair of treatment means for heavy metal and metallothionein concentrations. Statistical treatments also included a numeric classification method, Cluster Analysis (CA) and an ordination method, Principal Component Analysis (PCA). In the application of the CA, the method known as UPGMA (Unweighted Pair-Group Method Arithmetic Averages) was utilized. For the PCA, a correlation coefficient matrix was used instead of the original data. CA was applied in the Q (grouping the variable stations according to metal concentrations) and R (grouping objects, namely metals or MT, according to the variable stations) modes for heavy metals; and in the R mode for MT, Cd, Cu and Zn concentrations. PCA was only applied to the heavy metal concentrations. Both CA and PCA were performed with the NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System) software. Metal concentrations (means i standard deviations) in the whole soft tissues of M. galloprovincialis collected along the southern coast of Portugal are presented in Table 2. Cadmium concentrations in the mussels ranged from 1.3 to 3.1 lag g - l dry weight, the highest concentrations being found at stations 5, 16 and 17. These concentrations were significantly different from all the other sampling points (p < 0.05). In comparison to the other metal concentrations determined in mussels of the Algarve, copper levels did not show much spatial variability (4.8-7.0 lag g-l). However, mussels from station 19 (which was located in one of the major estuaries directly influenced by mining effluents) exhibited the highest copper concentrations, and these were significantly different from the concentrations at the other sampling points (p < 0.05). 667
Marine Pollution Bulletin TABLE 2 Metal concentrations (means 4- standard deviations; lag g - 1 dry weight) in the whole soft tissues of Mytilus galloprovincialis from the Algarve Coast. Stations 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19
Cd
Cu
Fe
Mn
Ni
Zn
2.3-t-0.1 2.3::t:0.1 2.0+0.0 2.0:t:0.1 3.1+0.7 2.2+0.2 2.1 +0.1 2.2+0.1 2.3+0.2 1.9+0.4 1.34-0.2 1.45:0.0 1.6+0.2 1.3+0.1 1.6+0.1 2.44-0.6 2.74-0.3 2.1+0.1 2.24-0.4
5.0-I-0.4 5.0+0.2 5.3+0.3 6.14-1.1 6.84-0.8 6.8+0.6 5.75:0.3 5.54-0.2 4.84-0.5 5.9+0.7 5.1 +0.4 5.54-0.6 5.94-0.6 5.7+0.6 5.1 +0.1 5.95_ 1.7 6.1+0.9 5.74-0.4 7.04-0.2
200+147 95+3 131+9 1974-20 1884-76 1844-63 2094-17 764-4 964-3 994-10 724-4 784-5 2944-49 904-4 974-4 1414-58 1174-19 1164-13 1124-38
5.7+3.6 3.6-t-0.3 4.04-0.4 6.04-0.5 15.54-4.3 13.94-5.4 6.94-0.8 3.84-0.2 4.2+0.5 3.74-0.5 3.44-0.8 3.6+0.4 7.94-0.7 5.94-0.5 3.14-0.4 5.54-1.7 5.0+0.7 5.04-1.1 10.24-1.1
0.44+0.02 0.374-0.03 0.554-0.01 0.58 4-0.09 0.774-0.24 0.62+0.12 0.57 4-0.03 0.544-0.06 0.59+0.05 0.664-0.06 0.37+0.06 0.47+0.03 0. 544-0.08 0.40+0.03 0.44+0.03 0.49 4-0.08 0.444-0.14 0.45+0.01 0.42+0.07
206-t-101 199+25 1864-21 244+ 13 3984-147 2924-148 204+26 215+7 2294-30 1964-71 2624-10 2854-49 2294- 32 3984-120 316+90 200+60 361+134 212-1-22 2004-8
Iron concentrations ranged from 72 to 294 p.g g - t and Mn concentrations from 3.1 to 15.5 gg g - l . Iron and Mn concentrations levels exhibited a similar spatial distribution along the Algarve coast. The application of the Tukey Test to these results suggested that Fe concentrations at stations 4-7 and 13 were significantly different from the others (p < 0.05). Similarly, mussels from stations 4.7 and 19 exhibited Mn concentrations which were significantly different from those of the other sampling points. Nickel concentrations obtained for the mussels sampled ranged from 0.37 to 0.77 gg g-1. The highest Ni concentrations were detected in the intermediate part of the coast (between stations 3 and 10, and at station 13). In contrast to Cu concentrations, Zn concentrations in the mussels showed greater variability (186398 gg g - l ) , although no significant differences were detected between the sampling points (ANOVA, p < 0.05). These results are related to the heterogeneity of the variances, a problem known as heteroscedasticity (Hernandez et al., 1992; Zar, 1996). The CA in Q mode for all metal concentrations (Fig. 2) showed that the mussels were divided into two main groups. The first of these included samples 1 to 10 and the second, samples 11 to 19. The CA results were confirmed by the application of PCA (Fig. 3). PCI represented 45% of the total variation, while PC2 included about 24%. The PC1 component can be interpreted in items of the similarity between sampling locations and their influence by freshwater or seawater. The PC2 component is related to the geographical distribution of the sampling stations. The spatial variation of metallothionein concentrations in the mussels collected is presented in Fig. 4. The metallothionein concentrations ranged from 3.9 to 668
-1.0 i
-0.5 i
0t
0.5
1.0
r-- z
8 14 12 15 17
i
V-c:]9
Fig. 2 Cluster Analysis: Q mode dendogram with UPGMA method.
1.0
[]
% 0.5
-3
[]
i0
-2
[]
-D.
[]
I
I
I
1
2
3
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Fig. 3 PCA analysis of metal concentrations in mussels.
Volume 34/Number 8/August 1997
N
L12 °
t
4 0
Lagos
7
I8
19
5 aro
Sagres
Atlantic Ocean
18 CabedeStaiMarla10
2~}Km
Fig. 4 The spatial distribution of metallothionein concentrations (means+standard deviations, mg g - i dry weight) in the whole soft tissues of Mytilus galloprovincialis along the Algarve
coast.
13.0 mg g - t dry weight. Concentrations in the mussels from station 9 were significantly lower than those of mussels from stations 1, 2 and 16 (p < 0.05). The application of CA to MT, Cd, Cu and Zn concentrations (the metals that are able to induce MT synthesis) revealed that MT concentrations are better correlated with Cd and Cu (r--0.164) levels than with Zn concentrations (r = 0.072; see Fig. 5). The data for metal concentrations reveal a significant increase of metal levels in zones directly affected by urban areas and industrial effluents. Metal concentrations in mussels were also higher in areas directly influenced by freshwater inputs, including the River
0.0 I
0.1 i
0.2 !
0.3 t
0.4 t
Cd
MT Zn Fig. 5 Cluster Analysis in the R mode with UPGMA method and correlation coefficients for Cd, Cu, Zn and metallothionein.
Arade (stations 5-7); the coastal lagoon known as the Ria Formosa (stations 16 and 17); and the estuary of the River Guadiana (station 19). The highest metal concentrations were obtained along the River Arade (station 5) at a site which is affected by sewage and industrial inputs. Mean metal concentrations in the mussels studied here are compared with values from the literature in Table 3. Cd and Zn concentrations were mostly higher than those obtained by Vale et al. (1985) for the same area 10 years ago, and this could be related to the recent industrial and tourism development. However, Fe, Mn and Ni concentrations exhibited a significant decrease over the last 10 years. The reduction in Fe and Mn concentrations observed in this study may be related to a significant decrease in the rainfall observed over recent years, with a consequent decrease in land runoff. The metal concentrations found were in general similar to those obtained by other authors for the same species collected in other geographical areas. The application of the CA and PCA to metal concentrations in the soft tissues of M . galloprovincialis showed a significant difference between mussels from the west coast (stations 1 to 10) and the east coast (stations 11 to 19). With the exception of Zn, mussels collected from the west coast showed higher metal concentrations than those from the east coast. Zn concentrations exhibited less consistent variations. Similar results were obtained by Hernandez et al. (1992). Apart from anthropogenic sources, there could 669
Marine Pollution Bulletin
be other effects responsible for changes in metal concentrations, such as the natural upwelling which is known to occur in this area. Metallothionein concentrations have been utilized as a biomarker for the early detection of metal contamination. Metallothionein concentrations from 2-3 mg g have been proposed as background levels against which metal contamination should be assessed (Bebianno and Langston, 1991, 1992). The application of Cluster Analysis techniques (Fig. 5) supported results obtained in laboratory studies, where correlations were evident between metaUothionein and Cd concentrations in M. edulis and M. galloprovincialis (Bebianno and Langston, 1991, 1992), and between metallothionein and Cu concentrations in M. galloprovincialis (Viarengo et al., 1980; Viarengo, 1985). This indicates that metallothionein concentrations in mussels could be a useful early warning signal of Cd and Cu contamination. A similar pattern of metallothionein induction was observed for the oyster (Crassostrea virginica), where Cd appeared to be the metal responsible for the changes in metallothionein concentration (Roesijadi, 1994).
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Bargagli, R., Baldi, F. and Leonzio, C. (1985) Trace metal assessment in sediment, molluscs and leaves in the Bay of Follonica (Italy). Marine Environmental Research 16, 281-300. Bebianno, M. J. (1995) Effects of pollutants on the Ria Formosa Lagoon. The Science of the Total Environment 171, 107-115. Bebianno, M. J. and Langston, W. J. (1989) Quantification of metallothioneins in marine invertebrates using differential pulse polarography. Portugalice Electrochimica Acta 7, 59-64. Bebianno, M. J. and Langston, W. J. (1991) Metallothionein induction in Mytilus edulis exposed to cadmium. Marine Biology 108, 91-96. Bebianno, M. J. and Langston, W. J. (1992) Metallothionein induction and cadmium binding in Mytilus galloprovincialis. Comparative Biochemistry and Physiology 103C, 79-85. Bebianno, M. J., Langston, W. J. and Simkiss, K. (1992) Metallothionein induction in Littorina littorea (Mollusca: Prosobranchia) on exposure to cadmium. Journal of the Marine Biological Association UK 72, 329-342. Bryan, G., Langston, W., Hummerstone, L. and Burt, G. (1985) A guide to the assessment of heavy metal contamination in estuaries using biological indicators. Marine Biological Association UK (Occasional Publication) 4, 92. Cortesao, C., Mendes, R. and Vale, C. (1986) Metais pesados em bivalves e sedimentos na Ria Formosa Algarve. Bol. INIP 14, 3-28. Davies, I. M. and Pirie, J. M. (1980) Evaluation of a Mussel Watch project for heavy metals in Scottish coastal waters. Marine Pollution Bulletin 11, 261-263. Gault, N. F., Tolland, E. L. and Parker, J. G. (1983) Spatial and temporal trends in heavy metal concentrations in mussels from northern Ireland coastal waters. Marine Biology 77, 307-316. Golderg, E. D., Bowen, V. T., Farrington, J. W., Harvey, G., Martin, J. H., Parker, P. L., Risebrough, R. W., Robertson, W., Schneider, E. and Gamble, E. (1978) The Mussel Watch. Environmental Conservation 5, 101-125. Hamilton, E. I. (1991) Metals in Mytilus edulis from the northern coast of Portugal. Marine Pollution Bulletin 22, 249-253. Hernandez, H., Pastor, A., Gisber, M., Ansuategui, J. and Serrano, R. (1992) Biomonitoring of heavy metal distribution in the western Mediterranean area of Spain. Marine Pollution Bulletin 24, 512-515. Lobel, P. B., Belkhode, S. P., Jackson, S. E. and Longerich, H. P. (1990) Recent taxonomic discoveries concerning the mussel Mytilus. Archives of Environmental Contamination and Toxicology 19, 508512.
Volume 34/Number 8/August 1997 Lubet, P. (1959) Recherche sur le cycle sexuel et l'rmission des gam&es chez les pectinidaes (mollusques bivalves). Revue des Travaux Institute des P~ches Maritimes 23, 387-548. Lucas, A. (1965) Recherche sur la sexualit6 des mollusques bivalves. Bulletim Biologique de la France et de la Belgique 29, 115--217. Martincic, D., Stoeppler, M. and Branica, M. (1987) Bioacummulation of metals by bivalves from the Limski Kanal (North Adratic Sea). IV. Zinc distribution between Mytilus galloprovincialis, Ostrea edulis and ambient water. The Science of the Total Environment 60, 143-172. Phillips, D. J. H. and Rainbow, P. S. (1993) Biomonitoring of Trace Aquatic Contaminants. Elsevier Science, Barking. Roesijadi, G. (1994) Behaviour of metallothionein-bound metals in a natural population of an estuarine mollusc. Marine Environmental Research 38, 147-168. Rogrrio, M. and Vale, C. (1984) Geographic variation of heavy metal contents in mussels (Mytilus galloprovincialis) from the coastal area adjacent to the Tagus Estuary. ICES C.M. 1984/E:38, International Council for the Exploration of the Seas, Paris.
UNEP. (1994) Final reports on research projects dealing with the effects of pollutants on maine organisms and communities. United Nations Environment Programme, MAP Tech. Reports Ser., 80, 2538. Vale, C., Ferreira, A. M. Cortesho, C. Barros, M. C., Castro, O. G., Mendes, R. A. (1985) Mussel Watch in the Portuguese coast, 1984, ICES C.M., 1985/E: International Council for the Exploration of the Seas, Paris, 18, 12. Viarengo, A. (1985) Biochemical effects of trace metals. Marine Pollution Bulletin 16, 153-158. Viarengo, A., Pertica, M., Mancin¢lli, G., Zanichi, G. and Orunesu, M. (1980) Rapid induction of copper-binding proteins in the gills of metal exposed mussels. Comparative Biochemistry and Physiology .6712, 215. Walkes, M. P. and Goering, P. L. (1992) Metallothionein and other cadmium-binding proteins: recent developments, ed. L. J. Mannett, pp. 263-270. American Chemical Society, Washington, DC. Zar, J. H. (1996) Biostatistical Analysis, 3rd Edn. Prentice-Hall Inc., London.
Marine Pollution Bulletin. Vol. 34, No. 8, pp. 671-674, 1997 © 1997ElsevierScienceLtd All rights reserved. Printed in Great Britain 002Y-326X/97 $17.00+0.00
are consumed domestically. In recent years, the amount of fish in the Bay has diminished considerably (Munoz, 1991). The Bay receives water from numerous inflowing rivers that drain approximately 17000 km: of watershed. Manila Bay receives significant discharges of domestic and industrial wastes (Chua et al., 1989), and the area bordering the Bay has undergone extensive industrial development and rapid urbanization. Thus, inputs of chemical pollutants such as chlorinated hydrocarbons and heavy metals may have changed the Bay's geochemistry and affected the quality of the local coastal environment. In an earlier study of Manila Bay sediments (Prudente et al., 1994), the vertical distribution patterns of metals suggested that inputs from anthropogenic sources are increasing over time. In view of this, it was deemed necessary to survey the levels of heavy metals in biota from the Bay. Presently, no detailed studies exist on metal concentrations in commercial species of fish from Manila Bay, despite the fact that fish are an important component of the diet in the region. Analyses of 13 elements are presented here and their concentrations in the whole bodies of 16 fish species are discussed. The metal concentrations detected in this study were compared with values from other regions in an effort to determine the degree of contamination in the study area. Seventeen species of fish deemed commercially important in the Philippines were examined. Of these, nine are demersal, while the other eight species are pelagic (Table 1). Fish samples were purchased directly from the fishermen based in the ports of Coastal Road (Paranaque) and Naic (Cavite) in April 1994 and March 1996 respectively. Samples were stored in polyethylene bags at - 2 0 ° C and brought to Japan for analysis. In order to obtain representative samples, composites of slipmouth, crevalle, sardine, mackerel, mullet and perch were prepared by pooling together two or three small
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Metal Levels in some Commercial Fish Species from Manila Bay, the Philippines MARICAR PRUDENTE*, EUN-YOUNG KIMt, SHINSUKE TANABEt§ and RYO TATSUKAWA:~ *College of St Benilde, De La Salle University System, Taft Avenue, Manila 1004, Philippines t Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan ~Kochi University, Akebono 2-5-1. Kochi 780, Japan
Pollution of marine ecosystems by heavy metals is of environmental concern worldwide. Domestic sewage, industrial effluents, combustion emissions, mining operations and metallurgical activities are among the sources of anthropogenic metal inputs. Although heavy metals in trace concentrations are normal constituents of marine organisms, at high levels they are potentially toxic and may disrupt the biological activities of aquatic ecosystems. The ability of heavy metals to be concentrated in the organs of marine organisms accounts for their toxicity and also poses a direct threat to both the aquatic biota and man (Watling, 1983). Manila Bay, a semi-enclosed marine inlet of the South China Sea, is an economically important area in the Philippines. Fish, shellfish and aquaculture products from the Bay are exported abroad, but larger quantities
§Corresponding author.
671
Edited by D. J. H. Phillips margin bordering the Atlantic Ocean (Fig. 1). Apart from the Guadiana River, two major aquatic systems exist: the River Arade; and the largest lagoon of the Portuguese coast, the Ria Formosa (Bebianno, 1995). In the Algarve there is an important fish, shellfish and tourism industry, the total catch representing around 20% of all Portuguese fisheries. The region of the Algarve is also of high environmental and economic value. However, the coastline is suffering from environmental degradation which is starting to have economic impacts on shellfish production and the tourist industries. The coastline has been under constant urban pressure during the last fifteen years due to tourist development. The population, which is concentrated in the coastal zone with a density of about 167 inhabitants km -2, increases more than 10-fold over the summer, which aggravates the effects from the paucity of sewage treatment facilities. Sewage discharges have been identified as one of the most important sources of pollution loading on the Algarve Coast. Some of the streams, rivers and estuarine systems along the coast also contribute anthropogenic inputs. One aspect of environmental degradation is pollution from metals which are persistent and are bioaccumulated by marine organisms, with serious public health implications (Phillips and Rainbow, 1993). Methods of biomonitoring metal loads have been evolved using organisms which are sedentary, have a
The objective of BASELINE is to publish short communications on different aspects of pollution of the marine environment. Only those papers which clearly identify the quality of the data will be considered for publication. Contributors to Baseline should refer to 'Baseline---The New Format and Content' (Mar. Pollut. Bull. 24, 124). Marine Pollution Bulletin, Vol. 34, N o . 8, pp. 666--671, 1997
Pergamon
© 1997 E l s e v i e r Science L t d All ri ght s reserved. P r i n t e d in Great Britain 0025-326X/97
$17.00 + 0.00
PII: S0025--326X(97)00036-2
Concentrations of Metals and Metallothioneins in Mytilus galloprovincialis along the South Coast of Portugal M. J. BEBIANNO and L. M. MACHADO Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal The Algarve Coast of southern Portugal bordering the Atlantic Ocean between 37035 ' and 36°58'N, and 7025' and 9°00'W is 135 km long from its eastern limit with Spain, marked by the Guadiana River, to the western
River
N T V~:i~! !:!:i: ~:~ kV: x, V2 2
SPAIN
ii!iii!i!i!iCL!ili!! Monte CI6rigo
/
Vila Real de Sic Antbnio
River Arade 13 .._14
Ria Formosa
4
iii!!iiiiii?;idii:ii:ili~iliiiiiiiiiiii;i?i ii )iiljilii!iiiiiii!:? ili l ¸
}:>i:i:}¸:::I:;i }:Q ::~.:ii::::}::i::?: /:d::i:?: :<:::!:!.:kd : >: • :>: :, .: : :U ::::V:U::::::hV::52h:: L: ~:L:k:qV::::/~::::kU~~ ~ ~
Sagres
ili:diii~i; Fig.
666
1 Sampling
sites
along
the
Algarve
Coast.
i
Volume 34/Number 8/August 1997 wide geographical distribution and accumulate metals in a manner that reflects the ambient conditions. Bivalve molluscs, and in particular Mytilus spp., have been successfully utilized for coastal water quality monitoring. The 'Mussel Watch Program' (Goldberg et al., 1978) is based on the chemical analysis of contaminants in the soft tissues of individuals. One important modification to conventional programmes of measuring contaminants directly in the whole soft tissues is to measure other compounds which also reflect levels of contamination. These techniques may be more sensitive than measuring the contaminants directly in the tissues (Viarengo, 1985). An example is provided by the detoxification strategy in marine organisms of producing low molecular weight metal binding proteins known as metallothioneins (Bebianno and Langston, 1991, 1992; Bebianno et al., 1992; Walkes and Goering, 1992; Roesijadi, 1994). Measurements of the levels of these proteins provide an accurate indication of subtle environmental increases in metal contamination. Following the principle of the Mussel Watch Program (Goldberg et al., 1978) the purpose of this study was to use the mussel Mytilus galloprovincialis Lmk. from the Algarve Coast as a biomonitor to identify areas contaminated by heavy metals. In addition to measuring the concentrations of Cd, Cu, Fe, Mn, Ni and Zn, metallothionein levels were determined in the whole soft tissues of the mussels. The latter have been utilized as a specific biochemical indicator for the early detection of potential detrimental effects induced by metal contamination. Nineteen sites were selected along the Algarve Coast (Fig. 1) for the sampling of M. galloprovincialis. Thirty mussels of similar length (45-55 mm, 1.08-2.66 g dry weight) were collected from the same intertidal depth during a spring tide, in April and May 1994. All the animals were in the pre-spawning period, with 91% of them in Maturation State III (Lubet, 1959; Lucas, 1965). In the laboratory, all mussels were depurated for 24 h to eliminate gut contents, prior to their analysis for metals and metallothionein. The animals were carefully opened by cutting the adductor muscle and the soft tissues were dissected out. The tissues were pooled in groups of five individuals, after discarding the shell, byssus and cavity liquor. Each pooled sample was weighed and homogenized in three volumes of 0.02 M Tris-HCl buffer (pH 8.6) in an ice-bath. Subsamples were taken for the determination of wet/dry weight ratios. The samples were dried and digested in concentrated nitric acid, following the procedure described by Bryan et al. (1985). Metal concentrations were determined using an atomic absorption spectrophotometer (VARIAN-SpectrAA20). Analysis of the TORT I lobster hepatopancreas reference material (National Research, Canada) were carried out following the same sample treatment, to validate the metal analysis (Table 1). The concentrations of all metals are expressed in I.tg g - ~ dry weight of soft tissue.
TABLE 1 Comparison of metal concentrations(lagg- l dry weight)in standard reference materialTORT 1 certifiedby the National ResearchCouncil
of Canada, and analyticalresults from the current study. Metal Cd Cu Zn
Certified values
Our values (n = 10)
26.3-/-2.1 439+22 177+10
26.98+0.01 449+0.01 160:~-0.7
Aliquots of the homogenate (3 to 5 ml) were centrifuged at 30 000 g for 1 h at 4°C. The supernatant (cystosol) was separated from the pellet, heated at 80°C for I0 min to precipitate the high molecular weight proteins denatured by heat, and subsequently centrifuged at 30 000 g for 1 h at 4°C. Aliquots (50 to 250 ~1) of the heat-denatured cytosol were taken for the quantification of metallothioneins (MT) by differential pulse polarography as described by Bebianno and Langston (1989), with a polarograph Methrom 646 and using a MT rabbit standard (Sigma). Metallothionein concentrations were determined as mg g - t dry weight of homogenized tissue. The parametric Tukey Test was used to identify differences between a specific pair of treatment means for heavy metal and metallothionein concentrations. Statistical treatments also included a numeric classification method, Cluster Analysis (CA) and an ordination method, Principal Component Analysis (PCA). In the application of the CA, the method known as UPGMA (Unweighted Pair-Group Method Arithmetic Averages) was utilized. For the PCA, a correlation coefficient matrix was used instead of the original data. CA was applied in the Q (grouping the variable stations according to metal concentrations) and R (grouping objects, namely metals or MT, according to the variable stations) modes for heavy metals; and in the R mode for MT, Cd, Cu and Zn concentrations. PCA was only applied to the heavy metal concentrations. Both CA and PCA were performed with the NTSYS-PC (Numerical Taxonomy and Multivariate Analysis System) software. Metal concentrations (means i standard deviations) in the whole soft tissues of M. galloprovincialis collected along the southern coast of Portugal are presented in Table 2. Cadmium concentrations in the mussels ranged from 1.3 to 3.1 lag g - l dry weight, the highest concentrations being found at stations 5, 16 and 17. These concentrations were significantly different from all the other sampling points (p < 0.05). In comparison to the other metal concentrations determined in mussels of the Algarve, copper levels did not show much spatial variability (4.8-7.0 lag g-l). However, mussels from station 19 (which was located in one of the major estuaries directly influenced by mining effluents) exhibited the highest copper concentrations, and these were significantly different from the concentrations at the other sampling points (p < 0.05). 667
Marine Pollution Bulletin TABLE 2 Metal concentrations (means 4- standard deviations; lag g - 1 dry weight) in the whole soft tissues of Mytilus galloprovincialis from the Algarve Coast. Stations 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19
Cd
Cu
Fe
Mn
Ni
Zn
2.3-t-0.1 2.3::t:0.1 2.0+0.0 2.0:t:0.1 3.1+0.7 2.2+0.2 2.1 +0.1 2.2+0.1 2.3+0.2 1.9+0.4 1.34-0.2 1.45:0.0 1.6+0.2 1.3+0.1 1.6+0.1 2.44-0.6 2.74-0.3 2.1+0.1 2.24-0.4
5.0-I-0.4 5.0+0.2 5.3+0.3 6.14-1.1 6.84-0.8 6.8+0.6 5.75:0.3 5.54-0.2 4.84-0.5 5.9+0.7 5.1 +0.4 5.54-0.6 5.94-0.6 5.7+0.6 5.1 +0.1 5.95_ 1.7 6.1+0.9 5.74-0.4 7.04-0.2
200+147 95+3 131+9 1974-20 1884-76 1844-63 2094-17 764-4 964-3 994-10 724-4 784-5 2944-49 904-4 974-4 1414-58 1174-19 1164-13 1124-38
5.7+3.6 3.6-t-0.3 4.04-0.4 6.04-0.5 15.54-4.3 13.94-5.4 6.94-0.8 3.84-0.2 4.2+0.5 3.74-0.5 3.44-0.8 3.6+0.4 7.94-0.7 5.94-0.5 3.14-0.4 5.54-1.7 5.0+0.7 5.04-1.1 10.24-1.1
0.44+0.02 0.374-0.03 0.554-0.01 0.58 4-0.09 0.774-0.24 0.62+0.12 0.57 4-0.03 0.544-0.06 0.59+0.05 0.664-0.06 0.37+0.06 0.47+0.03 0. 544-0.08 0.40+0.03 0.44+0.03 0.49 4-0.08 0.444-0.14 0.45+0.01 0.42+0.07
206-t-101 199+25 1864-21 244+ 13 3984-147 2924-148 204+26 215+7 2294-30 1964-71 2624-10 2854-49 2294- 32 3984-120 316+90 200+60 361+134 212-1-22 2004-8
Iron concentrations ranged from 72 to 294 p.g g - t and Mn concentrations from 3.1 to 15.5 gg g - l . Iron and Mn concentrations levels exhibited a similar spatial distribution along the Algarve coast. The application of the Tukey Test to these results suggested that Fe concentrations at stations 4-7 and 13 were significantly different from the others (p < 0.05). Similarly, mussels from stations 4.7 and 19 exhibited Mn concentrations which were significantly different from those of the other sampling points. Nickel concentrations obtained for the mussels sampled ranged from 0.37 to 0.77 gg g-1. The highest Ni concentrations were detected in the intermediate part of the coast (between stations 3 and 10, and at station 13). In contrast to Cu concentrations, Zn concentrations in the mussels showed greater variability (186398 gg g - l ) , although no significant differences were detected between the sampling points (ANOVA, p < 0.05). These results are related to the heterogeneity of the variances, a problem known as heteroscedasticity (Hernandez et al., 1992; Zar, 1996). The CA in Q mode for all metal concentrations (Fig. 2) showed that the mussels were divided into two main groups. The first of these included samples 1 to 10 and the second, samples 11 to 19. The CA results were confirmed by the application of PCA (Fig. 3). PCI represented 45% of the total variation, while PC2 included about 24%. The PC1 component can be interpreted in items of the similarity between sampling locations and their influence by freshwater or seawater. The PC2 component is related to the geographical distribution of the sampling stations. The spatial variation of metallothionein concentrations in the mussels collected is presented in Fig. 4. The metallothionein concentrations ranged from 3.9 to 668
-1.0 i
-0.5 i
0t
0.5
1.0
r-- z
8 14 12 15 17
i
V-c:]9
Fig. 2 Cluster Analysis: Q mode dendogram with UPGMA method.
1.0
[]
% 0.5
-3
[]
i0
-2
[]
-D.
[]
I
I
I
1
2
3
[]
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Fig. 3 PCA analysis of metal concentrations in mussels.
Volume 34/Number 8/August 1997
N
L12 °
t
4 0
Lagos
7
I8
19
5 aro
Sagres
Atlantic Ocean
18 CabedeStaiMarla10
2~}Km
Fig. 4 The spatial distribution of metallothionein concentrations (means+standard deviations, mg g - i dry weight) in the whole soft tissues of Mytilus galloprovincialis along the Algarve
coast.
13.0 mg g - t dry weight. Concentrations in the mussels from station 9 were significantly lower than those of mussels from stations 1, 2 and 16 (p < 0.05). The application of CA to MT, Cd, Cu and Zn concentrations (the metals that are able to induce MT synthesis) revealed that MT concentrations are better correlated with Cd and Cu (r--0.164) levels than with Zn concentrations (r = 0.072; see Fig. 5). The data for metal concentrations reveal a significant increase of metal levels in zones directly affected by urban areas and industrial effluents. Metal concentrations in mussels were also higher in areas directly influenced by freshwater inputs, including the River
0.0 I
0.1 i
0.2 !
0.3 t
0.4 t
Cd
MT Zn Fig. 5 Cluster Analysis in the R mode with UPGMA method and correlation coefficients for Cd, Cu, Zn and metallothionein.
Arade (stations 5-7); the coastal lagoon known as the Ria Formosa (stations 16 and 17); and the estuary of the River Guadiana (station 19). The highest metal concentrations were obtained along the River Arade (station 5) at a site which is affected by sewage and industrial inputs. Mean metal concentrations in the mussels studied here are compared with values from the literature in Table 3. Cd and Zn concentrations were mostly higher than those obtained by Vale et al. (1985) for the same area 10 years ago, and this could be related to the recent industrial and tourism development. However, Fe, Mn and Ni concentrations exhibited a significant decrease over the last 10 years. The reduction in Fe and Mn concentrations observed in this study may be related to a significant decrease in the rainfall observed over recent years, with a consequent decrease in land runoff. The metal concentrations found were in general similar to those obtained by other authors for the same species collected in other geographical areas. The application of the CA and PCA to metal concentrations in the soft tissues of M . galloprovincialis showed a significant difference between mussels from the west coast (stations 1 to 10) and the east coast (stations 11 to 19). With the exception of Zn, mussels collected from the west coast showed higher metal concentrations than those from the east coast. Zn concentrations exhibited less consistent variations. Similar results were obtained by Hernandez et al. (1992). Apart from anthropogenic sources, there could 669
Marine Pollution Bulletin
be other effects responsible for changes in metal concentrations, such as the natural upwelling which is known to occur in this area. Metallothionein concentrations have been utilized as a biomarker for the early detection of metal contamination. Metallothionein concentrations from 2-3 mg g have been proposed as background levels against which metal contamination should be assessed (Bebianno and Langston, 1991, 1992). The application of Cluster Analysis techniques (Fig. 5) supported results obtained in laboratory studies, where correlations were evident between metaUothionein and Cd concentrations in M. edulis and M. galloprovincialis (Bebianno and Langston, 1991, 1992), and between metallothionein and Cu concentrations in M. galloprovincialis (Viarengo et al., 1980; Viarengo, 1985). This indicates that metallothionein concentrations in mussels could be a useful early warning signal of Cd and Cu contamination. A similar pattern of metallothionein induction was observed for the oyster (Crassostrea virginica), where Cd appeared to be the metal responsible for the changes in metallothionein concentration (Roesijadi, 1994).
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Bargagli, R., Baldi, F. and Leonzio, C. (1985) Trace metal assessment in sediment, molluscs and leaves in the Bay of Follonica (Italy). Marine Environmental Research 16, 281-300. Bebianno, M. J. (1995) Effects of pollutants on the Ria Formosa Lagoon. The Science of the Total Environment 171, 107-115. Bebianno, M. J. and Langston, W. J. (1989) Quantification of metallothioneins in marine invertebrates using differential pulse polarography. Portugalice Electrochimica Acta 7, 59-64. Bebianno, M. J. and Langston, W. J. (1991) Metallothionein induction in Mytilus edulis exposed to cadmium. Marine Biology 108, 91-96. Bebianno, M. J. and Langston, W. J. (1992) Metallothionein induction and cadmium binding in Mytilus galloprovincialis. Comparative Biochemistry and Physiology 103C, 79-85. Bebianno, M. J., Langston, W. J. and Simkiss, K. (1992) Metallothionein induction in Littorina littorea (Mollusca: Prosobranchia) on exposure to cadmium. Journal of the Marine Biological Association UK 72, 329-342. Bryan, G., Langston, W., Hummerstone, L. and Burt, G. (1985) A guide to the assessment of heavy metal contamination in estuaries using biological indicators. Marine Biological Association UK (Occasional Publication) 4, 92. Cortesao, C., Mendes, R. and Vale, C. (1986) Metais pesados em bivalves e sedimentos na Ria Formosa Algarve. Bol. INIP 14, 3-28. Davies, I. M. and Pirie, J. M. (1980) Evaluation of a Mussel Watch project for heavy metals in Scottish coastal waters. Marine Pollution Bulletin 11, 261-263. Gault, N. F., Tolland, E. L. and Parker, J. G. (1983) Spatial and temporal trends in heavy metal concentrations in mussels from northern Ireland coastal waters. Marine Biology 77, 307-316. Golderg, E. D., Bowen, V. T., Farrington, J. W., Harvey, G., Martin, J. H., Parker, P. L., Risebrough, R. W., Robertson, W., Schneider, E. and Gamble, E. (1978) The Mussel Watch. Environmental Conservation 5, 101-125. Hamilton, E. I. (1991) Metals in Mytilus edulis from the northern coast of Portugal. Marine Pollution Bulletin 22, 249-253. Hernandez, H., Pastor, A., Gisber, M., Ansuategui, J. and Serrano, R. (1992) Biomonitoring of heavy metal distribution in the western Mediterranean area of Spain. Marine Pollution Bulletin 24, 512-515. Lobel, P. B., Belkhode, S. P., Jackson, S. E. and Longerich, H. P. (1990) Recent taxonomic discoveries concerning the mussel Mytilus. Archives of Environmental Contamination and Toxicology 19, 508512.
Volume 34/Number 8/August 1997 Lubet, P. (1959) Recherche sur le cycle sexuel et l'rmission des gam&es chez les pectinidaes (mollusques bivalves). Revue des Travaux Institute des P~ches Maritimes 23, 387-548. Lucas, A. (1965) Recherche sur la sexualit6 des mollusques bivalves. Bulletim Biologique de la France et de la Belgique 29, 115--217. Martincic, D., Stoeppler, M. and Branica, M. (1987) Bioacummulation of metals by bivalves from the Limski Kanal (North Adratic Sea). IV. Zinc distribution between Mytilus galloprovincialis, Ostrea edulis and ambient water. The Science of the Total Environment 60, 143-172. Phillips, D. J. H. and Rainbow, P. S. (1993) Biomonitoring of Trace Aquatic Contaminants. Elsevier Science, Barking. Roesijadi, G. (1994) Behaviour of metallothionein-bound metals in a natural population of an estuarine mollusc. Marine Environmental Research 38, 147-168. Rogrrio, M. and Vale, C. (1984) Geographic variation of heavy metal contents in mussels (Mytilus galloprovincialis) from the coastal area adjacent to the Tagus Estuary. ICES C.M. 1984/E:38, International Council for the Exploration of the Seas, Paris.
UNEP. (1994) Final reports on research projects dealing with the effects of pollutants on maine organisms and communities. United Nations Environment Programme, MAP Tech. Reports Ser., 80, 2538. Vale, C., Ferreira, A. M. Cortesho, C. Barros, M. C., Castro, O. G., Mendes, R. A. (1985) Mussel Watch in the Portuguese coast, 1984, ICES C.M., 1985/E: International Council for the Exploration of the Seas, Paris, 18, 12. Viarengo, A. (1985) Biochemical effects of trace metals. Marine Pollution Bulletin 16, 153-158. Viarengo, A., Pertica, M., Mancin¢lli, G., Zanichi, G. and Orunesu, M. (1980) Rapid induction of copper-binding proteins in the gills of metal exposed mussels. Comparative Biochemistry and Physiology .6712, 215. Walkes, M. P. and Goering, P. L. (1992) Metallothionein and other cadmium-binding proteins: recent developments, ed. L. J. Mannett, pp. 263-270. American Chemical Society, Washington, DC. Zar, J. H. (1996) Biostatistical Analysis, 3rd Edn. Prentice-Hall Inc., London.
Marine Pollution Bulletin. Vol. 34, No. 8, pp. 671-674, 1997 © 1997ElsevierScienceLtd All rights reserved. Printed in Great Britain 002Y-326X/97 $17.00+0.00
are consumed domestically. In recent years, the amount of fish in the Bay has diminished considerably (Munoz, 1991). The Bay receives water from numerous inflowing rivers that drain approximately 17000 km: of watershed. Manila Bay receives significant discharges of domestic and industrial wastes (Chua et al., 1989), and the area bordering the Bay has undergone extensive industrial development and rapid urbanization. Thus, inputs of chemical pollutants such as chlorinated hydrocarbons and heavy metals may have changed the Bay's geochemistry and affected the quality of the local coastal environment. In an earlier study of Manila Bay sediments (Prudente et al., 1994), the vertical distribution patterns of metals suggested that inputs from anthropogenic sources are increasing over time. In view of this, it was deemed necessary to survey the levels of heavy metals in biota from the Bay. Presently, no detailed studies exist on metal concentrations in commercial species of fish from Manila Bay, despite the fact that fish are an important component of the diet in the region. Analyses of 13 elements are presented here and their concentrations in the whole bodies of 16 fish species are discussed. The metal concentrations detected in this study were compared with values from other regions in an effort to determine the degree of contamination in the study area. Seventeen species of fish deemed commercially important in the Philippines were examined. Of these, nine are demersal, while the other eight species are pelagic (Table 1). Fish samples were purchased directly from the fishermen based in the ports of Coastal Road (Paranaque) and Naic (Cavite) in April 1994 and March 1996 respectively. Samples were stored in polyethylene bags at - 2 0 ° C and brought to Japan for analysis. In order to obtain representative samples, composites of slipmouth, crevalle, sardine, mackerel, mullet and perch were prepared by pooling together two or three small
Pergamon
PII: S0025--326X(9"/)00035--0
Metal Levels in some Commercial Fish Species from Manila Bay, the Philippines MARICAR PRUDENTE*, EUN-YOUNG KIMt, SHINSUKE TANABEt§ and RYO TATSUKAWA:~ *College of St Benilde, De La Salle University System, Taft Avenue, Manila 1004, Philippines t Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan ~Kochi University, Akebono 2-5-1. Kochi 780, Japan
Pollution of marine ecosystems by heavy metals is of environmental concern worldwide. Domestic sewage, industrial effluents, combustion emissions, mining operations and metallurgical activities are among the sources of anthropogenic metal inputs. Although heavy metals in trace concentrations are normal constituents of marine organisms, at high levels they are potentially toxic and may disrupt the biological activities of aquatic ecosystems. The ability of heavy metals to be concentrated in the organs of marine organisms accounts for their toxicity and also poses a direct threat to both the aquatic biota and man (Watling, 1983). Manila Bay, a semi-enclosed marine inlet of the South China Sea, is an economically important area in the Philippines. Fish, shellfish and aquaculture products from the Bay are exported abroad, but larger quantities
§Corresponding author.
671