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Heavy metal concentrations in molluscs from the Atlantic coast of southern Spain
Chemosphere 59 (2005) 1175–1181 www.elsevier.com/locate/chemosphere
Heavy metal concentrations in molluscs from the Atlantic coast of southern Spain Jose´ Usero, Jose´ Morillo *, Ignacio Gracia Department of Chemical and Environmental Engineering, University of Seville, Camino de los Descubrimientos s/n, 41092 Seville, Spain Received 18 April 2004; received in revised form 23 November 2004; accepted 29 November 2004
Abstract Trace metals were determined in the two most abundant species of bivalve molluscs along the Atlantic coast of southern Spain (Donax trunculus and Chamelea gallina) and in the sediments where they live. The results show that the area near the mouth of the Huelva estuary is where the highest metal concentrations are found in sediments and in the two bivalve species. This is not surprising, considering that the Huelva estuary is the mouth of the Tinto and Odiel rivers, which have one of the highest levels of metal pollution of all the rivers of Europe. The two species of bivalves have different amounts of metals in their tissues. The concentrations of Cr, Cu, Pb, Zn, As and Hg in D. trunculus were significantly higher (p < 0.05) than in C. gallina; however, C. gallina contained more Ni and Cd. In both species the most abundant elements were Cu and Zn, while Hg showed the lowest values. There is a significant correlation (p < 0.05) for concentrations of Cu, Pb, Zn and Hg in D. trunculus and C. gallina relative to their concentrations in surface sediments. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Trace heavy metals; Bivalve molluscs; Donax trunculus; Chamelea gallina; Marine sediments
1. Introduction From an environmental point of view, the coastal zone can be considered as the geographic space of interaction between terrestrial and marine ecosystems that is of great importance for the survival of a large variety of plants, animals and marine species (Castro et al., 1999). Metal pollution of the coastal environment continues to attract the attention of environmental researchers (Shulkin et al., 2003). The coastal zone receives a large amount of metal pollution from coastal towns, indus-
*
Corresponding author. Tel.: +34 954 487 276. E-mail address: [email protected] (J. Morillo).
trial dumps and rivers. Pollution by heavy metals is a serious problem due to their toxicity and their ability to accumulate in the biota (Islam and Tanaka, 2004). Therefore, a determination of metal concentrations in organisms should be part of any assessment and monitoring program in the coastal zone. The area studied—the southern Spanish Atlantic coast—is around 300 km long and runs from the Portuguese border (in Huelva province) to Algeciras Bay (in Cadiz province). The main source of pollution on this coast are inputs from the Huelva estuary, where the Tinto and Odiel rivers discharge after crossing the Iberian Pyrite Belt, a region that has been a rich source of minerals and metals from time immemorial (Morillo et al., 2002). Both rivers are acidic and contain large
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.089
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amounts of metals from erosion and mining activity. In addition, sewage from the city of Huelva and waste from industrial estates with numerous plants in the area are discharged into the Huelva estuary. This study looked at two bivalve mollusc species, Donax trunculus and Chamelea gallina, which are the most abundant along the entire Atlantic coast of southern Spain and are also highly appreciated for human consumption in Spain. D. trunculus, commonly known as the coquina clam or wedge shell, has a trapezoidal shell nearly twice as long as wide. It is brownish yellow with somewhat darker bands and thin radial lines and is slightly purple inside. C. gallina is triangular in shape, whitish in colour (sometimes brownish or greyish) and has about 80 thin, somewhat irregular concentric ribs. The primary purpose of this study was to obtain quantitative information on the concentration of trace metals in two bivalve species from the Atlantic coast of southern Spain. The results reported here will provide valuable information on heavy metal pollution along the Atlantic coast of southern Spain. We also studied the relation between metal content in the two species and in the sediments they inhabit.
2. Materials and methods 2.1. Organisms Along the southern Spanish Atlantic coast there are only 11 areas where collection of the species under study is permitted for human consumption. We took samples in each of these 11 areas (Fig. 1). Samples were taken during 3 days in October 2003, a month in which the two species have no reproductive activity. The individuals were selected for a standard shell size (30 ± 5 mm for C. gallina and 35 ± 5 mm for D. trunculus). Most of the adult population of both species in the area studied is found within these size ranges, which are larger than the minimum commercial size in Spain. At each station 30 individuals of each species were collected to prepare a pooled sample to reduce individual variations in heavy metal concentrations (Daskalakis, 1996; Szefer et al., 1997). After collection, the clams were allowed to flush out undigested matter in filtered seawater from the sampling sites for 24 h (Sokolowski et al., 2002). The soft tissues of 30 individuals from each location and species were
Fig. 1. Study area showing the location of the sampling sites.
J. Usero et al. / Chemosphere 59 (2005) 1175–1181
carefully removed by shelling the bivalves with a plastic knife; they were then freeze-dried (Chiu et al., 2000) and ground to a fine powder in a mortar before analysis (Ruelas-Inzunza and Pa´ez-Osuna, 2000). The resulting powder underwent microwave acid (HNO3 Suprapur) digestion (Camusso et al., 2001). This technique is less complicated and less time-consuming than conventional dissolution methods. High-pressure digestion bombs (CEM 3010, consisting of a body made of a specific microwave-transparent polymer with a Teflon cup and cover) were used for dissolving samples. Following acid digestion, all the samples were analysed for eight elements by atomic absorption spectrometry (AAS). Zn and Cu were determined in an air–acetylene flame (Perkin–Elmer 2380 with double beam and deuterium background corrector). Cr, Ni, Cd, and Pb were analysed in a graphite furnace (Perkin– Elmer 4110 ZL with Zeeman background corrector) with an autosampler. The following matrix modifiers were used: (NO3)2Mg for Cr and (NO3)2Mg and PO4H2NH4 for Cd and Pb. No matrix modifier was used with Ni. The hydride-generation technique was used to determine As, employing a Perkin–Elmer MSH-10 connected to a Perkin–Elmer 2380 spectrophotometer. Prior to As analysis, an aliquot of the digested sample was treated with potassium iodide and ascorbic acid to convert the As(V) to As(III). Hg concentrations were measured by cold vapour atomic absorption spectrophotometry with a flow injection system (FIMS-400) and autosampler (AS-90), both from Perkin–Elmer. To compare the total metal content at the different sampling sites, the metal pollution index (MPI) was used, obtained with the equation (Usero et al., 1997): MPI ¼ ðCf 1 Cf 2 . . . Cf n Þ1=n where Cfn = concentration of the metal n in the sample. 2.2. Sediment Core samples were taken from the same points where the molluscs were collected using polyvinyl chloride (PVC) corers. The top 20 cm of sediment were taken for samples. Six replicates were collected at each station. For different types of sediments, five sampling units sufficed to make a representative mixed sample (Bervoets and Blust, 2003). The corers were immediately sealed and stored at 4 °C until arriving at the laboratory. In the laboratory the cores were extruded and sectioned. The first 15-cm section of each core was used in this study. Sections were air-dried (Thomas et al., 1994) and sieved with a 63-lm nylon mesh, and the fraction <63 lm was chosen for chemical analysis (Salomons and Fo¨rstner, 1984; Friedler et al., 1994). A pooled sample was prepared for each station by mixing
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and homogenising the <63 lm of sediment from the six cores taken at each station. The samples underwent acid digestion (HNO3–HClO4) in an automatic microwave digestion system and their metal content was analysed by AAS in the same way as the organisms were analysed. Cr, Ni, Cu, Pb and Zn were determined in an air–acetylene flame (Perkin–Elmer 2380). Cd was analysed in a graphite furnace (Perkin–Elmer 4110 ZL). Cold-vapour and hydride-generation techniques were used for analysis of Hg and As, respectively. 2.3. Reagents and quality assurance The nitric acid and perchloric acid used for digestion were Suprapur (Merck). All other reagents used for testing were of analytical reagent grade (Merck). Stock solutions (Merck) of 1000 mg l1 with certificates of analysis traceable to NIST of the different elements analysed were used to prepare the calibration standards. All the analyses were performed within the laboratoryÕs updated rigorous quality control system (International Standard Organization ISO/IEC 17025, 1999). The digestion and analytical procedures were checked by analysis of standard reference materials in every batch digestion (sediment: CRM-277, Community Bureau of Reference; bivalves: CRM 278R, mussel M. edulis). Replicate analysis of these reference materials showed good accuracy, with metals recovery rates between 94% and 105% for mussels and between 92% and 97% for sediment. Precision was verified by analysing a replicate sample in every batch digestion. The percentage relation between the difference of the two values obtained and the mean value of these was less than 10% in most cases, and the precision was, therefore, considered satisfactory. The potential contamination of samples was evaluated analysing one acid blank in every batch. The results of the blanks were always below the methodÕs detection limit. Detection limits (calculated on the basis of 10 determinations of the blanks as three times the standard deviation of the blank) in the sediment tests were: 0.01 mg kg1 for Hg, 0.02 mg kg1 for Cd, 0.2 mg kg1 for As, 0.5 mg kg1 for Zn, 1.0 mg kg1 for Ni and Cu, and 2.0 mg kg1 for Pb and Cr. The following detection limits were found for the bivalves: 0.01 mg kg1 for Hg, 0.02 mg kg1 for Cr and Cd, 0.06 mg kg1 for Ni and Pb, 0.2 mg kg1 for As, 0.5 mg kg1 for Zn and 1.0 mg kg1 for Cu. 2.4. Statistical analysis StudentÕs t-test was used to study differences in metal concentrations between C. gallina and D. trunculus. A linear regression model was used to test the relations between the metal concentrations in the bivalve molluscs and the sediments.
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3. Results and discussion
3.2. Metal concentrations in organisms
3.1. Metal concentrations in sediments
The two species of bivalves have different amounts of metals in their tissues (Tables 2 and 3). The concentrations of Cr, Cu, Pb, Zn, As and Hg were significantly higher (p < 0.05) in D. trunculus than in C. gallina. On the other hand, the Ni and Cd levels in C. gallina were significantly higher than in D. trunculus. Some papers have already shown that different species of bivalves have different capacities for accumulating metals (Villar et al., 1999; Jeng et al., 2000). The most abundant elements in D. trunculus were Cu and Zn. The Cu levels in this species can be considered high if we compare them with those usually found for this element in other bivalve studies (Widdows et al., 1997; Baraj et al., 2003; Paulson et al., 2003). However, the Zn concentration in D. trunculus was similar to what is found in the literature (Usero et al., 1997; Cheggour et al., 2001; Shulkin et al., 2003). In contrast to D. trunculus, C. gallina presented higher Zn concentrations than those of Cu. It is common to find higher concentrations of Zn than Cu in bivalve studies (Szefer et al., 1997; Wong et al., 2000). In order of significance, the Cu and Zn concentrations were followed by As. The element presenting the lowest mean concentrations in both species is Hg. Besada et al. (2002) reported similar concentrations of this element in bivalves from the Spanish north-Atlantic coast. The concentrations of most of the metals in the two bivalves studied varied notably depending on the location of the sampling sites. Tables 2 and 3 show that for both species the MPI (metal pollution index) increased considerably at points 3, 4 and 5. This increase was especially marked in the case of metals of pyritic origin, such as Cu, Pb, Zn and As. These points are located near the mouth of the Huelva estuary, where the Tinto
The sampling points in the area around the mouth of the Huelva estuary (3–5) showed the highest concentrations of Cu, Cd, Pb, Zn, As and Hg (see Table 1). This is not surprising, considering that the Huelva estuary is the mouth of the Tinto and Odiel rivers, which have one of the highest levels of metal pollution in Europe (Elbaz-Poulichet et al., 1999). In contrast, the lowest levels of the metals mentioned above were found at points 9, 10 and 11. The concentrations of Cr and Ni at the different sampling points showed no differences worthy of mention. The levels of these metals were similar to those obtained by other authors in non-polluted sediment (Salomons and Fo¨rstner, 1984; Ruiz, 2001).
Table 1 Metal concentrations (mg kg1 dry mass) in sediments Sampling site
1 2 3 4 5 6 7 8 9 10 11
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
33 28 22 18 21 20 23 21 13 10 30
13 11 5 5 3 3 8 10 7 6 9
23 35 42 92 45 40 15 19 6 7 7
0.38 0.36 0.53 0.72 0.35 0.37 0.30 0.26 0.29 0.30 0.27
9 13 16 46 31 28 12 12 4 2 4
105 172 289 460 338 301 87 77 32 29 18
19 24 42 102 72 68 21 17 4.6 3.6 3.5
0.18 0.16 0.22 0.41 0.29 0.24 0.17 0.21 0.13 0.11 0.12
Table 2 Metal concentrations (mg kg1 dry mass), mean values, metal pollution index (MPI), percentage of moisture and mean bio-sediment accumulation factor values (BSAF) in D. trunculus Sampling site
1 2 3 4 5 6 7 8 9 10 11 Mean BSAF
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.91 1.24 1.12 1.35 1.30 1.05 0.84 0.34 2.11 1.50 1.42 1.2 0.07
1.10 1.56 1.34 1.54 1.25 1.66 0.51 0.45 1.59 0.74 1.46 1.2 0.22
104 126 325 383 285 134 160 187 86 74 60 175 7.61
0.20 0.16 0.15 0.20 0.24 0.15 0.21 0.19 0.18 0.18 0.19 0.19 0.54
2.1 1.1 9.0 9.5 9.0 1.2 2.4 2.3 1.1 1.5 1.1 3.6 0.28
131 113 150 152 134 74 126 99 56 74 63 107 1.27
7.4 6.2 12.1 11.7 9.5 4.9 6.8 9.7 7.0 10.1 8.4 8.5 0.81
0.11 0.07 0.20 0.25 0.12 0.15 0.11 0.09 0.06 0.08 0.07 0.12 0.59
MPI
Moisture (%)
2.9 2.6 4.7 5.4 4.5 2.6 2.8 2.4 2.5 2.5 2.4
81.7 82.2 80.7 81.5 82.9 80.3 81.6 79.7 82.1 79.7 80.4
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Table 3 Metal concentrations (mg kg1 dry mass), mean values, metal pollution index (MPI), percentage of moisture and mean bio-sediment accumulation factor values (BSAF) in C. gallina Sampling site
1 2 3 4 5 6 7 8 9 10 11 Mean BSAF
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.33 0.24 0.36 0.80 0.95 0.84 0.67 0.51 0.78 1.05 1.22 0.70 0.04
1.59 2.12 2.23 2.22 1.92 2.11 1.93 1.98 1.97 1.62 1.41 1.9 0.33
9.2 11 41 90 55 36 29 48 31 32 33 38 2.15
0.34 0.32 0.33 0.35 0.29 0.34 0.33 0.30 0.38 0.36 0.34 0.33 0.97
0.74 0.82 1.36 1.92 1.54 1.51 1.05 1.14 1.15 1.37 1.36 1.3 0.17
64 73 66 92 85 79 63 71 68 74 61 72 1.06
5.3 6.2 5.7 8.1 6.3 6.1 5.4 8.3 6.2 6.4 6.1 6.4 0.59
0.03 0.04 0.06 0.19 0.04 0.02 0.03 0.04 0.03 <0.01 <0.01 0.05 0.20
and Odiel rivers discharge and both rivers are highly polluted by metals. 3.3. Human consumption There is specific legislation for bivalve molluscs in Europe (European Communities, 2001), which establishes the maximum permissible concentration for three metals (Pb: 1.5 mg kg1, Cd: 1.0 mg kg1 and Hg: 0.5 mg kg1, wet mass). If we convert the data in Tables 2 and 3 to express them in terms of wet mass and compare this with the European legislation, we note that all the samples of C. gallina have values below the legal limits. This is not true for D. trunculus, however. The samples taken at points 3, 4 and 5 showed concentrations of 1.7, 1.8 and 1.5 mg kg1 of Pb (wet mass), respectively. When comparing with the legal limit, we must take into account measurement error, which is established on the basis of expanded uncertainty. One way of estimating the expanded uncertainty is to multiply by 2 the relative standard deviation calculated by the Horwitz equation. This procedure gives the following concentration ranges: 1.7 ± 0.5, 1.8 ± 0.5 and 1.5 ± 0.4 mg kg1 of Pb (wet mass) for points 3, 4 and 5, respectively. The values obtained by subtracting the uncertainty from reported concentrations (1.2, 1.3 and 1.1 mg kg1) are not above the maximum permitted by European legislation. We cannot, therefore, conclude, with absolute certainty, that the samples are not fit for consumption. Only if the lowest value of the range is greater than the maximum level in the legislation is it certain ‘‘beyond reasonable doubt’’ that the sample concentration of the analyte is greater than required by the legislation. There is also legislation in other countries regulating the maximum concentrations of Pb, Cd and Hg. For
MPI
Moisture (%)
1.4 1.5 2.1 3.3 2.4 2.1 1.8 2.1 2.0 1.8 1.8
80.6 81.0 80.4 79.9 81.2 81.2 78.8 81.9 79.5 80.0 81.4
example, the Australia New Zealand Food Standards Code (Abbott et al., 2003) also limits the Hg concentration in molluscs to 0.5 mg kg1. However, for Cd and Pb it sets a limit of 2.0 mg kg1 (wet mass), higher than the European Union legislation. 3.4. Bioconcentration of metals To evaluate the efficiency of metal bioaccumulation in the two mollusc species, we calculated the biosediment accumulation factor (BSAF), which is defined as the ratio between the metal concentration in the organism and that in the sediment (Lau et al., 1998; Szefer et al., 1999). Cu and Zn are the metals with the highest mean BSAF for the two species studied (Tables 2 and 3). The metal with the lowest BSAF is Cr. Finally, if we compare the two species studied, we can conclude that D. trunculus has a greater capacity for metal bioaccumulation than C. gallina since, with the exception of Ni and Cd, D. trunculus attains higher mean BSAF values. 3.5. Relations between metals in organisms and sediments There is a significant relation (p < 0.05) for concentrations of Cu, Pb, Zn and Hg in D. trunculus and C. gallina relative to their concentrations in surface sediments (Table 4). The correlation coefficient obtained for As was positive but with a low level of confidence (p > 0.05). Coefficients close to zero or negative were found for Cr, Ni and Cd, which indicate that the amount of Cr, Ni and Cd in the sediment is not directly reflected in the tissues of D. trunculus and C. gallina. This can be explained if we consider that concentrations of Cr, Ni and Cd are low in sediments, probably lower
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Table 4 Correlation coefficients between metal concentrations in sediments and in D. trunculus and C. gallina Mollusc
D. trunculus C. gallina
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.42 0.39
0.33 0.39
0.85** 0.72*
0.09 0.11
0.72* 0.69*
0.65* 0.79**
0.27 0.35
0.85** 0.85**
*p < 0.05; **p < 0.01.
than the threshold below which these organisms are able to regulate the accumulation of metals in their bodies. For metals with significant correlations (p < 0.05) we calculated the linear regression between the concentrations obtained in the bivalves (y values) and those in the sediments (x values), both expressed as dry mass. The elements can be ordered as follows in terms of the value of their slope: Cu (3.7) > Hg (0.58) > Pb (0.19) > Zn (0.15) in D. trunculus; Cu (0.6) > Hg (0.49) > Zn (0.05) > Pb (0.02) in C. gallina. The slope is significantly higher than 1 for Cu in D. trunculus, this suggests that the bioavailability of this metal is disproportionately increased with a degree of Cu enrichment of the sediments (Szefer et al., 1999). We also observe that the slopes obtained for D. trunculus are greater than those for C. gallina, from which we deduce that C. gallina is less affected by metal pollution than D. trunculus.
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Heavy metal concentrations in molluscs from the Atlantic coast of southern Spain Jose´ Usero, Jose´ Morillo *, Ignacio Gracia Department of Chemical and Environmental Engineering, University of Seville, Camino de los Descubrimientos s/n, 41092 Seville, Spain Received 18 April 2004; received in revised form 23 November 2004; accepted 29 November 2004
Abstract Trace metals were determined in the two most abundant species of bivalve molluscs along the Atlantic coast of southern Spain (Donax trunculus and Chamelea gallina) and in the sediments where they live. The results show that the area near the mouth of the Huelva estuary is where the highest metal concentrations are found in sediments and in the two bivalve species. This is not surprising, considering that the Huelva estuary is the mouth of the Tinto and Odiel rivers, which have one of the highest levels of metal pollution of all the rivers of Europe. The two species of bivalves have different amounts of metals in their tissues. The concentrations of Cr, Cu, Pb, Zn, As and Hg in D. trunculus were significantly higher (p < 0.05) than in C. gallina; however, C. gallina contained more Ni and Cd. In both species the most abundant elements were Cu and Zn, while Hg showed the lowest values. There is a significant correlation (p < 0.05) for concentrations of Cu, Pb, Zn and Hg in D. trunculus and C. gallina relative to their concentrations in surface sediments. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Trace heavy metals; Bivalve molluscs; Donax trunculus; Chamelea gallina; Marine sediments
1. Introduction From an environmental point of view, the coastal zone can be considered as the geographic space of interaction between terrestrial and marine ecosystems that is of great importance for the survival of a large variety of plants, animals and marine species (Castro et al., 1999). Metal pollution of the coastal environment continues to attract the attention of environmental researchers (Shulkin et al., 2003). The coastal zone receives a large amount of metal pollution from coastal towns, indus-
*
Corresponding author. Tel.: +34 954 487 276. E-mail address: [email protected] (J. Morillo).
trial dumps and rivers. Pollution by heavy metals is a serious problem due to their toxicity and their ability to accumulate in the biota (Islam and Tanaka, 2004). Therefore, a determination of metal concentrations in organisms should be part of any assessment and monitoring program in the coastal zone. The area studied—the southern Spanish Atlantic coast—is around 300 km long and runs from the Portuguese border (in Huelva province) to Algeciras Bay (in Cadiz province). The main source of pollution on this coast are inputs from the Huelva estuary, where the Tinto and Odiel rivers discharge after crossing the Iberian Pyrite Belt, a region that has been a rich source of minerals and metals from time immemorial (Morillo et al., 2002). Both rivers are acidic and contain large
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.089
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amounts of metals from erosion and mining activity. In addition, sewage from the city of Huelva and waste from industrial estates with numerous plants in the area are discharged into the Huelva estuary. This study looked at two bivalve mollusc species, Donax trunculus and Chamelea gallina, which are the most abundant along the entire Atlantic coast of southern Spain and are also highly appreciated for human consumption in Spain. D. trunculus, commonly known as the coquina clam or wedge shell, has a trapezoidal shell nearly twice as long as wide. It is brownish yellow with somewhat darker bands and thin radial lines and is slightly purple inside. C. gallina is triangular in shape, whitish in colour (sometimes brownish or greyish) and has about 80 thin, somewhat irregular concentric ribs. The primary purpose of this study was to obtain quantitative information on the concentration of trace metals in two bivalve species from the Atlantic coast of southern Spain. The results reported here will provide valuable information on heavy metal pollution along the Atlantic coast of southern Spain. We also studied the relation between metal content in the two species and in the sediments they inhabit.
2. Materials and methods 2.1. Organisms Along the southern Spanish Atlantic coast there are only 11 areas where collection of the species under study is permitted for human consumption. We took samples in each of these 11 areas (Fig. 1). Samples were taken during 3 days in October 2003, a month in which the two species have no reproductive activity. The individuals were selected for a standard shell size (30 ± 5 mm for C. gallina and 35 ± 5 mm for D. trunculus). Most of the adult population of both species in the area studied is found within these size ranges, which are larger than the minimum commercial size in Spain. At each station 30 individuals of each species were collected to prepare a pooled sample to reduce individual variations in heavy metal concentrations (Daskalakis, 1996; Szefer et al., 1997). After collection, the clams were allowed to flush out undigested matter in filtered seawater from the sampling sites for 24 h (Sokolowski et al., 2002). The soft tissues of 30 individuals from each location and species were
Fig. 1. Study area showing the location of the sampling sites.
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carefully removed by shelling the bivalves with a plastic knife; they were then freeze-dried (Chiu et al., 2000) and ground to a fine powder in a mortar before analysis (Ruelas-Inzunza and Pa´ez-Osuna, 2000). The resulting powder underwent microwave acid (HNO3 Suprapur) digestion (Camusso et al., 2001). This technique is less complicated and less time-consuming than conventional dissolution methods. High-pressure digestion bombs (CEM 3010, consisting of a body made of a specific microwave-transparent polymer with a Teflon cup and cover) were used for dissolving samples. Following acid digestion, all the samples were analysed for eight elements by atomic absorption spectrometry (AAS). Zn and Cu were determined in an air–acetylene flame (Perkin–Elmer 2380 with double beam and deuterium background corrector). Cr, Ni, Cd, and Pb were analysed in a graphite furnace (Perkin– Elmer 4110 ZL with Zeeman background corrector) with an autosampler. The following matrix modifiers were used: (NO3)2Mg for Cr and (NO3)2Mg and PO4H2NH4 for Cd and Pb. No matrix modifier was used with Ni. The hydride-generation technique was used to determine As, employing a Perkin–Elmer MSH-10 connected to a Perkin–Elmer 2380 spectrophotometer. Prior to As analysis, an aliquot of the digested sample was treated with potassium iodide and ascorbic acid to convert the As(V) to As(III). Hg concentrations were measured by cold vapour atomic absorption spectrophotometry with a flow injection system (FIMS-400) and autosampler (AS-90), both from Perkin–Elmer. To compare the total metal content at the different sampling sites, the metal pollution index (MPI) was used, obtained with the equation (Usero et al., 1997): MPI ¼ ðCf 1 Cf 2 . . . Cf n Þ1=n where Cfn = concentration of the metal n in the sample. 2.2. Sediment Core samples were taken from the same points where the molluscs were collected using polyvinyl chloride (PVC) corers. The top 20 cm of sediment were taken for samples. Six replicates were collected at each station. For different types of sediments, five sampling units sufficed to make a representative mixed sample (Bervoets and Blust, 2003). The corers were immediately sealed and stored at 4 °C until arriving at the laboratory. In the laboratory the cores were extruded and sectioned. The first 15-cm section of each core was used in this study. Sections were air-dried (Thomas et al., 1994) and sieved with a 63-lm nylon mesh, and the fraction <63 lm was chosen for chemical analysis (Salomons and Fo¨rstner, 1984; Friedler et al., 1994). A pooled sample was prepared for each station by mixing
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and homogenising the <63 lm of sediment from the six cores taken at each station. The samples underwent acid digestion (HNO3–HClO4) in an automatic microwave digestion system and their metal content was analysed by AAS in the same way as the organisms were analysed. Cr, Ni, Cu, Pb and Zn were determined in an air–acetylene flame (Perkin–Elmer 2380). Cd was analysed in a graphite furnace (Perkin–Elmer 4110 ZL). Cold-vapour and hydride-generation techniques were used for analysis of Hg and As, respectively. 2.3. Reagents and quality assurance The nitric acid and perchloric acid used for digestion were Suprapur (Merck). All other reagents used for testing were of analytical reagent grade (Merck). Stock solutions (Merck) of 1000 mg l1 with certificates of analysis traceable to NIST of the different elements analysed were used to prepare the calibration standards. All the analyses were performed within the laboratoryÕs updated rigorous quality control system (International Standard Organization ISO/IEC 17025, 1999). The digestion and analytical procedures were checked by analysis of standard reference materials in every batch digestion (sediment: CRM-277, Community Bureau of Reference; bivalves: CRM 278R, mussel M. edulis). Replicate analysis of these reference materials showed good accuracy, with metals recovery rates between 94% and 105% for mussels and between 92% and 97% for sediment. Precision was verified by analysing a replicate sample in every batch digestion. The percentage relation between the difference of the two values obtained and the mean value of these was less than 10% in most cases, and the precision was, therefore, considered satisfactory. The potential contamination of samples was evaluated analysing one acid blank in every batch. The results of the blanks were always below the methodÕs detection limit. Detection limits (calculated on the basis of 10 determinations of the blanks as three times the standard deviation of the blank) in the sediment tests were: 0.01 mg kg1 for Hg, 0.02 mg kg1 for Cd, 0.2 mg kg1 for As, 0.5 mg kg1 for Zn, 1.0 mg kg1 for Ni and Cu, and 2.0 mg kg1 for Pb and Cr. The following detection limits were found for the bivalves: 0.01 mg kg1 for Hg, 0.02 mg kg1 for Cr and Cd, 0.06 mg kg1 for Ni and Pb, 0.2 mg kg1 for As, 0.5 mg kg1 for Zn and 1.0 mg kg1 for Cu. 2.4. Statistical analysis StudentÕs t-test was used to study differences in metal concentrations between C. gallina and D. trunculus. A linear regression model was used to test the relations between the metal concentrations in the bivalve molluscs and the sediments.
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3. Results and discussion
3.2. Metal concentrations in organisms
3.1. Metal concentrations in sediments
The two species of bivalves have different amounts of metals in their tissues (Tables 2 and 3). The concentrations of Cr, Cu, Pb, Zn, As and Hg were significantly higher (p < 0.05) in D. trunculus than in C. gallina. On the other hand, the Ni and Cd levels in C. gallina were significantly higher than in D. trunculus. Some papers have already shown that different species of bivalves have different capacities for accumulating metals (Villar et al., 1999; Jeng et al., 2000). The most abundant elements in D. trunculus were Cu and Zn. The Cu levels in this species can be considered high if we compare them with those usually found for this element in other bivalve studies (Widdows et al., 1997; Baraj et al., 2003; Paulson et al., 2003). However, the Zn concentration in D. trunculus was similar to what is found in the literature (Usero et al., 1997; Cheggour et al., 2001; Shulkin et al., 2003). In contrast to D. trunculus, C. gallina presented higher Zn concentrations than those of Cu. It is common to find higher concentrations of Zn than Cu in bivalve studies (Szefer et al., 1997; Wong et al., 2000). In order of significance, the Cu and Zn concentrations were followed by As. The element presenting the lowest mean concentrations in both species is Hg. Besada et al. (2002) reported similar concentrations of this element in bivalves from the Spanish north-Atlantic coast. The concentrations of most of the metals in the two bivalves studied varied notably depending on the location of the sampling sites. Tables 2 and 3 show that for both species the MPI (metal pollution index) increased considerably at points 3, 4 and 5. This increase was especially marked in the case of metals of pyritic origin, such as Cu, Pb, Zn and As. These points are located near the mouth of the Huelva estuary, where the Tinto
The sampling points in the area around the mouth of the Huelva estuary (3–5) showed the highest concentrations of Cu, Cd, Pb, Zn, As and Hg (see Table 1). This is not surprising, considering that the Huelva estuary is the mouth of the Tinto and Odiel rivers, which have one of the highest levels of metal pollution in Europe (Elbaz-Poulichet et al., 1999). In contrast, the lowest levels of the metals mentioned above were found at points 9, 10 and 11. The concentrations of Cr and Ni at the different sampling points showed no differences worthy of mention. The levels of these metals were similar to those obtained by other authors in non-polluted sediment (Salomons and Fo¨rstner, 1984; Ruiz, 2001).
Table 1 Metal concentrations (mg kg1 dry mass) in sediments Sampling site
1 2 3 4 5 6 7 8 9 10 11
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
33 28 22 18 21 20 23 21 13 10 30
13 11 5 5 3 3 8 10 7 6 9
23 35 42 92 45 40 15 19 6 7 7
0.38 0.36 0.53 0.72 0.35 0.37 0.30 0.26 0.29 0.30 0.27
9 13 16 46 31 28 12 12 4 2 4
105 172 289 460 338 301 87 77 32 29 18
19 24 42 102 72 68 21 17 4.6 3.6 3.5
0.18 0.16 0.22 0.41 0.29 0.24 0.17 0.21 0.13 0.11 0.12
Table 2 Metal concentrations (mg kg1 dry mass), mean values, metal pollution index (MPI), percentage of moisture and mean bio-sediment accumulation factor values (BSAF) in D. trunculus Sampling site
1 2 3 4 5 6 7 8 9 10 11 Mean BSAF
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.91 1.24 1.12 1.35 1.30 1.05 0.84 0.34 2.11 1.50 1.42 1.2 0.07
1.10 1.56 1.34 1.54 1.25 1.66 0.51 0.45 1.59 0.74 1.46 1.2 0.22
104 126 325 383 285 134 160 187 86 74 60 175 7.61
0.20 0.16 0.15 0.20 0.24 0.15 0.21 0.19 0.18 0.18 0.19 0.19 0.54
2.1 1.1 9.0 9.5 9.0 1.2 2.4 2.3 1.1 1.5 1.1 3.6 0.28
131 113 150 152 134 74 126 99 56 74 63 107 1.27
7.4 6.2 12.1 11.7 9.5 4.9 6.8 9.7 7.0 10.1 8.4 8.5 0.81
0.11 0.07 0.20 0.25 0.12 0.15 0.11 0.09 0.06 0.08 0.07 0.12 0.59
MPI
Moisture (%)
2.9 2.6 4.7 5.4 4.5 2.6 2.8 2.4 2.5 2.5 2.4
81.7 82.2 80.7 81.5 82.9 80.3 81.6 79.7 82.1 79.7 80.4
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Table 3 Metal concentrations (mg kg1 dry mass), mean values, metal pollution index (MPI), percentage of moisture and mean bio-sediment accumulation factor values (BSAF) in C. gallina Sampling site
1 2 3 4 5 6 7 8 9 10 11 Mean BSAF
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.33 0.24 0.36 0.80 0.95 0.84 0.67 0.51 0.78 1.05 1.22 0.70 0.04
1.59 2.12 2.23 2.22 1.92 2.11 1.93 1.98 1.97 1.62 1.41 1.9 0.33
9.2 11 41 90 55 36 29 48 31 32 33 38 2.15
0.34 0.32 0.33 0.35 0.29 0.34 0.33 0.30 0.38 0.36 0.34 0.33 0.97
0.74 0.82 1.36 1.92 1.54 1.51 1.05 1.14 1.15 1.37 1.36 1.3 0.17
64 73 66 92 85 79 63 71 68 74 61 72 1.06
5.3 6.2 5.7 8.1 6.3 6.1 5.4 8.3 6.2 6.4 6.1 6.4 0.59
0.03 0.04 0.06 0.19 0.04 0.02 0.03 0.04 0.03 <0.01 <0.01 0.05 0.20
and Odiel rivers discharge and both rivers are highly polluted by metals. 3.3. Human consumption There is specific legislation for bivalve molluscs in Europe (European Communities, 2001), which establishes the maximum permissible concentration for three metals (Pb: 1.5 mg kg1, Cd: 1.0 mg kg1 and Hg: 0.5 mg kg1, wet mass). If we convert the data in Tables 2 and 3 to express them in terms of wet mass and compare this with the European legislation, we note that all the samples of C. gallina have values below the legal limits. This is not true for D. trunculus, however. The samples taken at points 3, 4 and 5 showed concentrations of 1.7, 1.8 and 1.5 mg kg1 of Pb (wet mass), respectively. When comparing with the legal limit, we must take into account measurement error, which is established on the basis of expanded uncertainty. One way of estimating the expanded uncertainty is to multiply by 2 the relative standard deviation calculated by the Horwitz equation. This procedure gives the following concentration ranges: 1.7 ± 0.5, 1.8 ± 0.5 and 1.5 ± 0.4 mg kg1 of Pb (wet mass) for points 3, 4 and 5, respectively. The values obtained by subtracting the uncertainty from reported concentrations (1.2, 1.3 and 1.1 mg kg1) are not above the maximum permitted by European legislation. We cannot, therefore, conclude, with absolute certainty, that the samples are not fit for consumption. Only if the lowest value of the range is greater than the maximum level in the legislation is it certain ‘‘beyond reasonable doubt’’ that the sample concentration of the analyte is greater than required by the legislation. There is also legislation in other countries regulating the maximum concentrations of Pb, Cd and Hg. For
MPI
Moisture (%)
1.4 1.5 2.1 3.3 2.4 2.1 1.8 2.1 2.0 1.8 1.8
80.6 81.0 80.4 79.9 81.2 81.2 78.8 81.9 79.5 80.0 81.4
example, the Australia New Zealand Food Standards Code (Abbott et al., 2003) also limits the Hg concentration in molluscs to 0.5 mg kg1. However, for Cd and Pb it sets a limit of 2.0 mg kg1 (wet mass), higher than the European Union legislation. 3.4. Bioconcentration of metals To evaluate the efficiency of metal bioaccumulation in the two mollusc species, we calculated the biosediment accumulation factor (BSAF), which is defined as the ratio between the metal concentration in the organism and that in the sediment (Lau et al., 1998; Szefer et al., 1999). Cu and Zn are the metals with the highest mean BSAF for the two species studied (Tables 2 and 3). The metal with the lowest BSAF is Cr. Finally, if we compare the two species studied, we can conclude that D. trunculus has a greater capacity for metal bioaccumulation than C. gallina since, with the exception of Ni and Cd, D. trunculus attains higher mean BSAF values. 3.5. Relations between metals in organisms and sediments There is a significant relation (p < 0.05) for concentrations of Cu, Pb, Zn and Hg in D. trunculus and C. gallina relative to their concentrations in surface sediments (Table 4). The correlation coefficient obtained for As was positive but with a low level of confidence (p > 0.05). Coefficients close to zero or negative were found for Cr, Ni and Cd, which indicate that the amount of Cr, Ni and Cd in the sediment is not directly reflected in the tissues of D. trunculus and C. gallina. This can be explained if we consider that concentrations of Cr, Ni and Cd are low in sediments, probably lower
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Table 4 Correlation coefficients between metal concentrations in sediments and in D. trunculus and C. gallina Mollusc
D. trunculus C. gallina
Element Cr
Ni
Cu
Cd
Pb
Zn
As
Hg
0.42 0.39
0.33 0.39
0.85** 0.72*
0.09 0.11
0.72* 0.69*
0.65* 0.79**
0.27 0.35
0.85** 0.85**
*p < 0.05; **p < 0.01.
than the threshold below which these organisms are able to regulate the accumulation of metals in their bodies. For metals with significant correlations (p < 0.05) we calculated the linear regression between the concentrations obtained in the bivalves (y values) and those in the sediments (x values), both expressed as dry mass. The elements can be ordered as follows in terms of the value of their slope: Cu (3.7) > Hg (0.58) > Pb (0.19) > Zn (0.15) in D. trunculus; Cu (0.6) > Hg (0.49) > Zn (0.05) > Pb (0.02) in C. gallina. The slope is significantly higher than 1 for Cu in D. trunculus, this suggests that the bioavailability of this metal is disproportionately increased with a degree of Cu enrichment of the sediments (Szefer et al., 1999). We also observe that the slopes obtained for D. trunculus are greater than those for C. gallina, from which we deduce that C. gallina is less affected by metal pollution than D. trunculus.
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