- Email: [email protected]
Accumulation of heavy metals by flounder, Platichthys flesus (Linnaeus 1758), in a heterogeneously contaminated nursery area
Marine Pollution Bulletin 49 (2004) 1109–1126 www.elsevier.com/locate/marpolbul
Baseline
Edited by Bruce J. Richardson
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. 42, 703– 704).
Accumulation of heavy metals by flounder, Platichthys flesus (Linnaeus 1758), in a heterogeneously contaminated nursery area C. Vinagre *, S. Franc¸a, M.J. Costa, H.N. Cabral
*
Instituto de Oceanografia, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Estuaries and associated coastal waters across the world are among the most modified and threatened aquatic environments (e.g. Suchanek, 1994; Vernberg, 1997; Goksoyr et al., 1997). The Douro estuary, in Northern Portugal, is a heavily modified urban estuary, receiving untreated sewage from over one million people, as well as industrial discharges. Despite public health concerns, this estuarine system has never been subject to an ecotoxicological study. Industrial discharges into estuaries have been the main source of heavy metals contamination with several sub-lethal and lethal effects being reported for estuarine species, mainly in North European and North American estuaries (e.g. Amiard et al., 1982; Haedrich and Haedrich, 1974; Amiard-Triquet et al., 1986; Long et al., 1995). Some of the most common effects of this type of pollution are physiological disturbances such as enzymatic and hormonal alterations (Amiard et al., 1982; Amiard-Triquet et al., 1986).
* Corresponding authors. Tel.: +351 21 750 08 26; fax: +351 21 750 00 09. E-mail addresses: [email protected] (C. Vinagre), [email protected] (H.N. Cabral).
Flatfish are particularly vulnerable to sediment contamination, through direct contact with the substrate, benthic prey ingestion, as well as sediment particle ingestion, especially among juveniles (Moles et al., 1994). The flounder Platichthys flesus (Linnaeus, 1758), an abundant and commercially important flatfish in the Douro estuary, has been used in several monitoring programs since the 1970Õs (Bos, 1999) and was thus chosen for this study. The aim of the present study was to evaluate heavy metal contamination of both sediments and flounder throughout the Douro estuary and adjacent coastal area. In addition, the accumulation of heavy metals in fish of different length groups was assessed. Flounder were collected during bimonthly sampling surveys performed between November 2000 and March 2002, in 12 sampling stations along the Douro estuary and 6 sites in the adjacent coastal waters (Portugal) (Fig. 1), using a 12 m otter-trawl with 18 mm mesh size (stretched mesh). Trawls were towed during daylight, in neap tide, at a constant speed of 2.5 knots, and lasted 15 min. The distance travelled in each tow was determined based on GPS and the headline length used as a measure of width in the swept area calculations. Depth, temperature and salinity were measured with an YSI 600 XLM probe near the sediment. Fish abundance was
1110
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
42ºN
18
Atlantic Ocean
40ºN
Portugal
Douro estuary
38ºN
Porto
17 16
12 11 15 14
8ºW
10ºW
10
8
9 Gaia
6ºW
6 7
5
4
13 3 2 N
Dam
1
1Km
Section 3
Section 2
Section 1
Fig. 1. Location of the sampling areas in the Douro estuary and adjacent coastal areas.
expressed as density (number of individuals per 1000 m2). All flounder caught were identified, measured (total length with 1 mm precision) and weighed (wet weight with 0.01 g precision). In March 2002, sediment samples were collected, using a van Veen grab, for heavy metal determination. The 18 sites were clustered into three estuarine sections, according to environmental conditions and human disturbance: upper estuary (site 1 to 6), lower estuary (site 7 to 12) and adjacent coastal area (site 13 to 18) (Fig. 1). Three sediment samples were collected in each site. Liver and muscle samples were collected from flounder specimens of two length classes, corresponding to different age groups, according to Vinagre (2002): the <180 mm class (including fish from the 0-group and the 1-group) and the P180 mm class (including fish from the 2+group). For each section and length class, tissue samples of nine individuals were collected for heavy metal determination. Sediment samples were dried to constant weight at 80 C and 1 g was extracted twice with 10 mL of HNO3/HCl (3:1 v/v) at 130 C (Otte, 1991). The procedure was then repeated and the two extracts added together. The metal concentration in the solutions was determined by atomic absorption spectrometry (Perkin Elmer 4000). Standard additions and sludge reference materials were used (EC standard CRM 145 and 146). The biological samples were dried to constant weight at 60 C and the percentage of water in the tissues determined. Two grams of dry tissue were subsequently di-
gested in a mixture of nitric and perchloric acid (suprapure quality; 9:1) as described by Julshmamn et al. (1982). The metal concentrations were determined by atomic spectrometry on a Perkin Elmer 4000 spectrometer. Standard curves were used for the determination of Cu and Zn, whereas standard addition procedures were employed for the calculation of Cd and Pb. A one-way ANOVA was performed in order to evaluate the differences in sediment contamination between the three sections considered, for each heavy metal. The differences in contamination of flounder of different length groups and in different sections of the estuary and adjacent coast were evaluated by a two-way ANOVA. For both analyses, Tukey a posteriori tests were performed whenever the null hypotheses were rejected. A significance level of 0.05 was considered in all test procedures. Sediment analysis revealed that the most contaminated area was the lower estuary, adjacent to the second largest Portuguese urban concentration (the Porto-Gaia agglomerate) (section 2, Table 1). Cadmium was the only metal that was below the detection level of the method throughout the estuary and in the adjacent coastal area. Copper, lead and zinc were present throughout the estuary and in the adjacent coastal area, with significantly higher levels in the lower estuary (section 2) (Table 1). Sediment contamination levels found in the Douro estuary and adjoining coastal area are low when compared to some estuarine and coastal areas
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
1111
Table 1 Summary of environmental conditions, P. flesus density and heavy metal contamination for each estuarine and coastal section
Environmental variables Depth (m) Sediment type Water temperature (C) (summer) Water temperature (C) (winter) Salinity (&) (summer) Salinity (&) (winter) Biological data P. flesus density (ind.1000 m2)a Heavy metals (mg kg Cu Cd Pb Zn a b
1
Section 1: Upper estuary
Section 2: Lower estuary
Section 3: Coastal area
7 coarse sand and gravel 20.5 (1.4) 10.3 (0.2) 4.0 (4.5) 1.7 (0.9)
5 fine sand and mud 18.9 (0.9) 12.2 (1.0) 29.2 (6.6) 14.1 (10.9)
12 fine sand 18.1 (0.1) 14.1 (0.1) 35.5 (0.1) 35.2 (0.2)
0.62 (1.56)
1.20 (8.08)
0.05 (0.11)
4.4 (2.3) <1 6.0 (1.2) 21.7 (9.9)
50.5 (9.3) <1 65.8 (14.9) 168.8 (32.5)
5.0 (3.5) <1 7.2 (4.3) 34.5 (22.6)
b
dry weight)
Mean density over a 2 year period of bimonthly sampling surveys. Maximum density registered between brackets. Mean values from the sampling stations included in each section. Standard deviation between brackets.
of North Europe. Berge and Brevik (1996) found much higher concentrations of Pb and Cu in Kristiansandfjord, Norway, while Beyer et al. (1996) found considerably higher concentrations of Cu and Cd in Sorfjorden, Norway. All values recorded for the Douro estuary and adjoining coastal area were under the ‘‘maximum permissible concentration’’ defined by Crommentuijn et al. (2000) in an environmental quality standards study. Cd and Pb values were all under the ‘‘negligible concentration’’ defined by Crommentuijn et al. (2000), yet Cu and Zn concentrations recorded in the lower estuary were above the referred ‘‘negligible concentration’’. When compared to the sediment contamination guidelines proposed by Long et al. (1995) only cadmium was under the ‘‘ERL (effects range low)’’ reference concentration throughout the estuary and in the adjacent coastal area. Copper, lead and zinc concentrations in the Douro were below the ‘‘ERL’’ in the upper estuary (section 1) and in the adjacent coastal area (section 3), but were above the ‘‘ERM (effects range median)’’ reference in the lower estuary (section 2). The results from the heavy metal analysis in flounder tissues revealed differential contamination in the muscle and liver. Copper concentrations in liver were considerably higher than those found in muscle tissue (Table 2, F = 115.9; p < 0.05). This is valid for most teleosts (Neff, 2002). Various authors have also noted that fish muscle usually contains less than 2 mg kg1 (dry weight) copper, even in top predatory fish (Hall et al., 1978; Sidwell et al., 1978; Neff, 2002). In the present study, several fish recorded cooper concentrations in muscle that exceeded this value. Yet the values recorded for Douro flounder were considerably lower than those found in flounder from polluted areas such as Hardangerfjord, Norway (Julshmamn and Grahl-Nielsen, 1996) and the Seine
estuary, France (Miramand et al., 2001). Neff (2002) also stated that liver copper concentrations in fish are rarely higher than 10 times the concentration in muscle, yet flounder from the Douro exhibited liver concentrations generally more than 10 times higher than those found in muscle (Table 2). High concentrations in liver are difficult to explain but may be related to copper concentrations in the diet (Neff, 2002). Cadmium values were higher in the liver (Table 2, F = 11.4; p < 0.05) than in the muscle, as observed by Julshmamn and GrahlNielsen (1996) in the same species. The values recorded were much lower than those found in heavily polluted areas (e.g. Julshmamn and Grahl-Nielsen, 1996). All values were under the maximum load permitted in fish for human consumption by the European Union, which is 0.05 mg kg1 (wet weight) (Regulation N221/2002/ CE). The concentration of lead in the liver and muscle of flounder was similar (Table 2, F = 1.5; p > 0.05). Neff (2002) concluded that lead concentrations in fish muscle are usually under 1 mg kg1 (dry weight), which is similar to the concentration of this metal in the liver. In the present study all samples analysed both from muscle and liver had lead concentrations above the 1 mg kg1 (dry weight) value, yet all values were below the maximum permitted in fish for human consumption by the European Union, which is 0.2 mg kg1 (wet weight) (Regulation N221/2002/CE). The values recorded were also much lower than that found in flounder from other polluted areas (e.g. Julshmamn and Grahl-Nielsen, 1996). Zinc showed considerably higher accumulation in the liver (Table 2, F = 128.9; p < 0.05). This is in agreement with results obtained by Miramand et al. (1991) for Mullus barbatus (Linnaeus, 1758) and Julshmamn and Grahl-Nielsen (1996) for P. flesus. Zinc concentrations
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Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
Table 2 Mean concentration of heavy metals in liver and muscle of flounder (standard deviation between brackets) mg kg1 dry weight
mg kg1 wet weight
Tissue
Section
Length group (mm)
Cd
Pb
Zn
Cu
Cd
Pb
Zn
Liver
1
<180
51.38 (8.39)
0.88 (0.05)
2.82 (0.19)
110.67 (6.83)
0.70 (0.11)
0.01 (0.00)
0.04 (0.00)
1.51 (0.09)
Liver
1
P180
43.21 (14.31)
0.66 (0.06)
2.98 (0.30)
115.82 (3.65)
0.59 (0.19)
0.01 (0.00)
0.04 (0.00)
1.58 (0.05)
Liver
2
<180
40.50 (8.01)
1.00 (0.15)
8.90 (3.10)
146.00 (7.12)
0.55 (0.09)
0.01 (0.00)
0.12 (0.04)
1.99 (0.08)
Liver
2
P180
49.18 (16.77)
2.05 (0.25)
3.32 (1.98)
135.54 (3.29)
0.67 (0.23)
0.03 (0.00)
0.05 (0.03)
1.84 (0.04)
Liver
3
<180
41.62 (5.11)
0.69 (0.05)
4.91 (0.50)
171.10 (8.07)
0.57 (0.14)
0.01 (0.00)
0.07 (0.02)
2.33 (0.09)
Liver
3
P180
58.84 (32.11)
1.27 (0.71)
3.95 (0.53)
175.93 (76.58)
0.80 (0.24)
0.02 (0.01)
0.05 (0.01)
2.39 (1.04)
Muscle
1
<180
1.39 (0.71)
0.26 (0.03)
2.16 (0.43)
25.62 (1.82)
0.02 (0.01)
0.00 (0.00)
0.03 (0.01)
0.32 (0.02)
Muscle
1
P180
4.04 (5.16)
1.00 (1.23)
4.04 (1.78)
23.85 (8.34)
0.05 (0.06)
0.01 (0.01)
0.05 (0.02)
0.29 (0.11)
Muscle
2
<180
1.30 (0.07)
0.52 (0.27)
3.46 (0.25)
43.39 (9.21)
0.02 (0.00)
0.01 (0.00)
0.04 (0.00)
0.54 (0.24)
Muscle
2
P180
1.87 (1.13)
0.39 (0.04)
2.58 (0.52)
15.70 (5.33)
0.02 (0.01)
0.00 (0.00)
0.03 (0.01)
0.20 (0.07)
Muscle
3
P180
2.09 (1.61)
0.52 (0.24)
3.79 (0.37)
20.48 (6.20)
0.03 (0.02)
0.01 (0.00)
0.05 (0.00)
0.25 (0.08)
Cu
were considerably lower than reported for Pleuronectidae by Sidwell et al. (1974) and for P. flesus in other polluted areas (Amiard et al., 1980; Julshmamn and Grahl-Nielsen, 1996; Miramand et al., 2001). No tissue accumulation difference was detected for Cu, Cd and Pb (F > 1.5, p > 0.5) between the area sections where flounder were caught. It would be expected that if flounder concentrate in the lower estuary and that is the most contaminated area, then they should bioaccumulate more of these metals than flounder from lesser polluted areas of the estuary. Our data reveal that although flounder juveniles seem to concentrate in selected areas of the estuary they must disperse frequently to other parts of the estuarine nursery. Accumulation differences were detected between the sections of the estuary only for zinc, (F = 3.6, p < 0.05). Post-hoc tests revealed that flounder from the adjoining coastal waters (section 3) accumulated more zinc than those caught within the estuary (Table 2). Several field and experimental studies have demonstrated that the amount of zinc accumulated in various tissues by fish is mostly independent of the concentrations in the rearing medium (Eisler and Gardner, 1973; Pentreath, 1977; Van Hoof and Van San, 1981). No accumulation differences were detected between length groups of flounder for all heavy metals (F < 0.2.4, p > 0.05). Several authors have noted that
the concentrations of heavy metals in P. flesus varies with the length of the fish (Hardisty et al., 1974; Phillips, 1977; Jensen and Cheng, 1987). The fact that these differences were not detected in the Douro estuary could be due to the fact that the flounder population in this estuary is composed mainly by young fish and that flounder from the P180 group varied only between 180 mm and 230 mm. Also very few flounder smaller than 100 mm were caught. Therefore, the length spectrum analyzed may not have been wide enough for the detection of metals accumulation differences. This study shows that P. flesus juveniles concentrate in the most polluted areas of the estuary but also disperse along a wider area and it is therefore important to continue to monitor levels of contamination in this important species.
Acknowledgments This study was developed in the scope of the ERIC Project ÔEffects of river inflow changes on the fish communities of the Douro, Tagus and Guadiana estuaries and adjoining coastal areas. Ecological and socio-economical predictions.Õ (PDCTM/C/MAR/15263/1999), financially supported by ÔFundac¸a˜o para a Cieˆncia e a TecnologiaÕ.
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
References Amiard, J.C., Amiard-Triquet, C., Metayer, C., Marchand, J., 1980. Etude du transfert de Cd, Pb, Cu et Zn dans les chaıˆnes trophiques ne´ritiques et estuariennes I. Etat dans lÕestuaire de la Loire (France) au cours de lÕe´te 1978. Water Research 14, 665–673. Amiard, J.C., Amiard-Triquet, C., Denuit, C., 1982. Variations de lÕactivite´de quelques enzymes de lÕhemolymphe et de lÕhe´patopancre´as de Carcinus maenas in situ et expe´rimentalement sous lÕeffect de polluants me´talliques (Cd, Pb, Cu et Zn). In: Indices biochimiques et milieux marins, Publi. CNEXO (Actes Coloq.), 14, 385– 398. Amiard-Triquet, C., Amiard, J.C., Ferrand, R., Andersen, A.C., Dubois, M.P., 1986. Disturbance of a met-enkephalin-like hormone in the hepatopancreas of crabs contaminated by metals. Ecotoxicology and Environmental Safety 11, 198–209. Beyer, J., Sandvik, M., Hylland, K., Fjield, E., Egaas, E., Aas, E., Skare, J.U., Goksoyr, A., 1996. Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed to polluted sediments in Sorfjorden, Norway. Aquatic Toxicology 36, 75–98. Berge, J.A., Brevik, E.M., 1996. Uptake of metals and persistent organochlorines in crabs (Cancer pagurus) and flounder (Platichthys flesus) from contaminated sediments: mesocosm and field experiments. Marine Pollution Bulletin 33 (1–6), 46–55. Bos, A.R., 1999. Aspects of the life history of the European flounder (Pleuronectes flesus L.1758) in the tidal river Elbe. PhD Thesis, University of Hamburg, Germany. Crommentuijn, T., Sijm, D., de Bruin, J., van den Hoop, M., van Leeuwen, K., van de Plassche, E., 2000. Maximum permissible and negligible concentrations for metals and metalloids in the Netherlands, taking into account background concentrations. Journal of Environmental Management 60, 121–143. Eisler, R., Gardner, G.R., 1973. Acute toxicity to an estuarine teleost of mixtures of cadmium, copper and zinc salts. Journal of Fish Biology 5, 131–142. Goksoyr, A., Beyer, J., Egaas, E., Grosvik, B.E., Hylland, K., Sandvik, M., Skaare, J.U., 1997. Biomarker responses in flounder (Platichthys flesus) and their use in pollution monitoring. Marine Pollution Bulletin 33 (1–6), 36–45. Haedrich, R.L., Haedrich, S.O., 1974. A seasonal survey of the fishes in the Mystic river, a polluted estuary in downtown Boston, Masachusetts. Estuarine and Coastal Marine Science 2, 59–73. Hall, R.A., Zook, E.G., Meaburn, G.M., 1978. National Marine Fisheries Service Survey of Trace Elements in the Fishery Resource. US Dept. Commerce NOAA Tech. Rep. NMFS SSRF-721. Hardisty, M.W., Huggins, R.J., Harter, S., Sansbury, M., 1974. Ecological implications of heavy metals in fish from the Severn estuary. Marine Pollution Bulletin 5, 12–15. Jensen, A., Cheng, Z., 1987. Statistical Analysis of trend monitoring data of heavy metals in flounder (Platichthys flesus). Marine Biology Bulletin 18 (5), 230–238. 0025-326X/$ - see front matter 2004 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2004.08.021
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Julshmamn, K., Ringdal, O., Slinning, K.-E., Braekan, O., 1982. Optimization of the determination of selenium in marine samples by atomic absorption spectrometry: comparison of a flameless graphite furnace atomic absorption system with a hydride generation atomic absorption system. Spectrochim. Acta 37B, 473–482. Julshmamn, K., Grahl-Nielsen, O., 1996. Distribution of trace elements from industrial discharges in Hardangerfjord, Norway: A multivariate data analysis of saithe, flounder and blue mussel as sentinel organisms. Marine Pollution Bulletin 32, 564– 571. Long, E.R., MacDonald, D.D., Smith, S.L., Calder, F.D., 1995. Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. Environmental management 19, 81–97. Miramand, P., Lafaurie, M., Fowler, S.W., Lemaire, P., Guary, J.C., Bentley, C., 1991. Reproductive cycle and heavy metals in the organs of red mullet, Mullus barbatus (L.) from the north western Mediterranean. The Science of Total Environment 103, 47–56. Miramand, P., Guyot, T., Rybarczyk, H., Elkaim, B., Mouny, P., Dauvin, J.C., Bessineton, C., 2001. Contamination of the biological compartment in the Seine estuary by Cd, Cu, Pb and Zn. Estuaries 24 (6B), 1056–1065. Moles, A., Rice, S., Norcross, B.L., 1994. Non avoidance of hydrocarbon laden sediments by juvenile flatfishes. Netherlands Journal of Sea Research 32 (3/4), 361–367. Neff, J.M., 2002. Bioaccumulation in Marine Organisms, first ed. Elsevier, Oxford. Otte, M.L., 1991. Heavy metals and arsenic in vegetation of saltmarshes and foodplains. PhD Thesis, Vrije University, Amsterdam, Netherlands. Pentreath, R.J., 1977. Radionuclides in marine fish. Oceanographic Marine Biology Annual Revue 15, 365–460. Phillips, D.J.H., 1977. The use of biological indicator organisms to monitor trace metals pollution in marine and estuarine environments—a review. Environmental pollution 13, 282– 317. Suchanek, T.T., 1994. Temperate coastal marine communities: biodiversity and threats. Am. Zool. 34, 100–114. Sidwell, V.D., Loomis, K.J., Foncannon, P.R., Buzzell, D.H., 1978. Composition of the edible of raw (fresh or frozen) crustacean, finfish and mollusks. III. Microelements. Marine Fisheries Review 40 (9), 1–20. Van Hoof, F., Van San, M., 1981. Analysis of copper, zinc, cadmium and chromium in fish tissues. A tool to detect metal caused fish kills. Chemosphere 10, 1127–1135. Vernberg, W.B., 1997. Introduction: an overview of the effects of urbanization on estuaries: the land-estuarine interface. Journal of Experimental Marine Biology and Ecology 213, ix–x. Vinagre, C., 2002. Ecologia dos Pleuronectiformes do estua´rio do Douro e zona costeira adjacente. Degree Thesis. Faculty of Sciences, University of Lisbon, Portugal.
Baseline
Edited by Bruce J. Richardson
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. 42, 703– 704).
Accumulation of heavy metals by flounder, Platichthys flesus (Linnaeus 1758), in a heterogeneously contaminated nursery area C. Vinagre *, S. Franc¸a, M.J. Costa, H.N. Cabral
*
Instituto de Oceanografia, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Estuaries and associated coastal waters across the world are among the most modified and threatened aquatic environments (e.g. Suchanek, 1994; Vernberg, 1997; Goksoyr et al., 1997). The Douro estuary, in Northern Portugal, is a heavily modified urban estuary, receiving untreated sewage from over one million people, as well as industrial discharges. Despite public health concerns, this estuarine system has never been subject to an ecotoxicological study. Industrial discharges into estuaries have been the main source of heavy metals contamination with several sub-lethal and lethal effects being reported for estuarine species, mainly in North European and North American estuaries (e.g. Amiard et al., 1982; Haedrich and Haedrich, 1974; Amiard-Triquet et al., 1986; Long et al., 1995). Some of the most common effects of this type of pollution are physiological disturbances such as enzymatic and hormonal alterations (Amiard et al., 1982; Amiard-Triquet et al., 1986).
* Corresponding authors. Tel.: +351 21 750 08 26; fax: +351 21 750 00 09. E-mail addresses: [email protected] (C. Vinagre), [email protected] (H.N. Cabral).
Flatfish are particularly vulnerable to sediment contamination, through direct contact with the substrate, benthic prey ingestion, as well as sediment particle ingestion, especially among juveniles (Moles et al., 1994). The flounder Platichthys flesus (Linnaeus, 1758), an abundant and commercially important flatfish in the Douro estuary, has been used in several monitoring programs since the 1970Õs (Bos, 1999) and was thus chosen for this study. The aim of the present study was to evaluate heavy metal contamination of both sediments and flounder throughout the Douro estuary and adjacent coastal area. In addition, the accumulation of heavy metals in fish of different length groups was assessed. Flounder were collected during bimonthly sampling surveys performed between November 2000 and March 2002, in 12 sampling stations along the Douro estuary and 6 sites in the adjacent coastal waters (Portugal) (Fig. 1), using a 12 m otter-trawl with 18 mm mesh size (stretched mesh). Trawls were towed during daylight, in neap tide, at a constant speed of 2.5 knots, and lasted 15 min. The distance travelled in each tow was determined based on GPS and the headline length used as a measure of width in the swept area calculations. Depth, temperature and salinity were measured with an YSI 600 XLM probe near the sediment. Fish abundance was
1110
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
42ºN
18
Atlantic Ocean
40ºN
Portugal
Douro estuary
38ºN
Porto
17 16
12 11 15 14
8ºW
10ºW
10
8
9 Gaia
6ºW
6 7
5
4
13 3 2 N
Dam
1
1Km
Section 3
Section 2
Section 1
Fig. 1. Location of the sampling areas in the Douro estuary and adjacent coastal areas.
expressed as density (number of individuals per 1000 m2). All flounder caught were identified, measured (total length with 1 mm precision) and weighed (wet weight with 0.01 g precision). In March 2002, sediment samples were collected, using a van Veen grab, for heavy metal determination. The 18 sites were clustered into three estuarine sections, according to environmental conditions and human disturbance: upper estuary (site 1 to 6), lower estuary (site 7 to 12) and adjacent coastal area (site 13 to 18) (Fig. 1). Three sediment samples were collected in each site. Liver and muscle samples were collected from flounder specimens of two length classes, corresponding to different age groups, according to Vinagre (2002): the <180 mm class (including fish from the 0-group and the 1-group) and the P180 mm class (including fish from the 2+group). For each section and length class, tissue samples of nine individuals were collected for heavy metal determination. Sediment samples were dried to constant weight at 80 C and 1 g was extracted twice with 10 mL of HNO3/HCl (3:1 v/v) at 130 C (Otte, 1991). The procedure was then repeated and the two extracts added together. The metal concentration in the solutions was determined by atomic absorption spectrometry (Perkin Elmer 4000). Standard additions and sludge reference materials were used (EC standard CRM 145 and 146). The biological samples were dried to constant weight at 60 C and the percentage of water in the tissues determined. Two grams of dry tissue were subsequently di-
gested in a mixture of nitric and perchloric acid (suprapure quality; 9:1) as described by Julshmamn et al. (1982). The metal concentrations were determined by atomic spectrometry on a Perkin Elmer 4000 spectrometer. Standard curves were used for the determination of Cu and Zn, whereas standard addition procedures were employed for the calculation of Cd and Pb. A one-way ANOVA was performed in order to evaluate the differences in sediment contamination between the three sections considered, for each heavy metal. The differences in contamination of flounder of different length groups and in different sections of the estuary and adjacent coast were evaluated by a two-way ANOVA. For both analyses, Tukey a posteriori tests were performed whenever the null hypotheses were rejected. A significance level of 0.05 was considered in all test procedures. Sediment analysis revealed that the most contaminated area was the lower estuary, adjacent to the second largest Portuguese urban concentration (the Porto-Gaia agglomerate) (section 2, Table 1). Cadmium was the only metal that was below the detection level of the method throughout the estuary and in the adjacent coastal area. Copper, lead and zinc were present throughout the estuary and in the adjacent coastal area, with significantly higher levels in the lower estuary (section 2) (Table 1). Sediment contamination levels found in the Douro estuary and adjoining coastal area are low when compared to some estuarine and coastal areas
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
1111
Table 1 Summary of environmental conditions, P. flesus density and heavy metal contamination for each estuarine and coastal section
Environmental variables Depth (m) Sediment type Water temperature (C) (summer) Water temperature (C) (winter) Salinity (&) (summer) Salinity (&) (winter) Biological data P. flesus density (ind.1000 m2)a Heavy metals (mg kg Cu Cd Pb Zn a b
1
Section 1: Upper estuary
Section 2: Lower estuary
Section 3: Coastal area
7 coarse sand and gravel 20.5 (1.4) 10.3 (0.2) 4.0 (4.5) 1.7 (0.9)
5 fine sand and mud 18.9 (0.9) 12.2 (1.0) 29.2 (6.6) 14.1 (10.9)
12 fine sand 18.1 (0.1) 14.1 (0.1) 35.5 (0.1) 35.2 (0.2)
0.62 (1.56)
1.20 (8.08)
0.05 (0.11)
4.4 (2.3) <1 6.0 (1.2) 21.7 (9.9)
50.5 (9.3) <1 65.8 (14.9) 168.8 (32.5)
5.0 (3.5) <1 7.2 (4.3) 34.5 (22.6)
b
dry weight)
Mean density over a 2 year period of bimonthly sampling surveys. Maximum density registered between brackets. Mean values from the sampling stations included in each section. Standard deviation between brackets.
of North Europe. Berge and Brevik (1996) found much higher concentrations of Pb and Cu in Kristiansandfjord, Norway, while Beyer et al. (1996) found considerably higher concentrations of Cu and Cd in Sorfjorden, Norway. All values recorded for the Douro estuary and adjoining coastal area were under the ‘‘maximum permissible concentration’’ defined by Crommentuijn et al. (2000) in an environmental quality standards study. Cd and Pb values were all under the ‘‘negligible concentration’’ defined by Crommentuijn et al. (2000), yet Cu and Zn concentrations recorded in the lower estuary were above the referred ‘‘negligible concentration’’. When compared to the sediment contamination guidelines proposed by Long et al. (1995) only cadmium was under the ‘‘ERL (effects range low)’’ reference concentration throughout the estuary and in the adjacent coastal area. Copper, lead and zinc concentrations in the Douro were below the ‘‘ERL’’ in the upper estuary (section 1) and in the adjacent coastal area (section 3), but were above the ‘‘ERM (effects range median)’’ reference in the lower estuary (section 2). The results from the heavy metal analysis in flounder tissues revealed differential contamination in the muscle and liver. Copper concentrations in liver were considerably higher than those found in muscle tissue (Table 2, F = 115.9; p < 0.05). This is valid for most teleosts (Neff, 2002). Various authors have also noted that fish muscle usually contains less than 2 mg kg1 (dry weight) copper, even in top predatory fish (Hall et al., 1978; Sidwell et al., 1978; Neff, 2002). In the present study, several fish recorded cooper concentrations in muscle that exceeded this value. Yet the values recorded for Douro flounder were considerably lower than those found in flounder from polluted areas such as Hardangerfjord, Norway (Julshmamn and Grahl-Nielsen, 1996) and the Seine
estuary, France (Miramand et al., 2001). Neff (2002) also stated that liver copper concentrations in fish are rarely higher than 10 times the concentration in muscle, yet flounder from the Douro exhibited liver concentrations generally more than 10 times higher than those found in muscle (Table 2). High concentrations in liver are difficult to explain but may be related to copper concentrations in the diet (Neff, 2002). Cadmium values were higher in the liver (Table 2, F = 11.4; p < 0.05) than in the muscle, as observed by Julshmamn and GrahlNielsen (1996) in the same species. The values recorded were much lower than those found in heavily polluted areas (e.g. Julshmamn and Grahl-Nielsen, 1996). All values were under the maximum load permitted in fish for human consumption by the European Union, which is 0.05 mg kg1 (wet weight) (Regulation N221/2002/ CE). The concentration of lead in the liver and muscle of flounder was similar (Table 2, F = 1.5; p > 0.05). Neff (2002) concluded that lead concentrations in fish muscle are usually under 1 mg kg1 (dry weight), which is similar to the concentration of this metal in the liver. In the present study all samples analysed both from muscle and liver had lead concentrations above the 1 mg kg1 (dry weight) value, yet all values were below the maximum permitted in fish for human consumption by the European Union, which is 0.2 mg kg1 (wet weight) (Regulation N221/2002/CE). The values recorded were also much lower than that found in flounder from other polluted areas (e.g. Julshmamn and Grahl-Nielsen, 1996). Zinc showed considerably higher accumulation in the liver (Table 2, F = 128.9; p < 0.05). This is in agreement with results obtained by Miramand et al. (1991) for Mullus barbatus (Linnaeus, 1758) and Julshmamn and Grahl-Nielsen (1996) for P. flesus. Zinc concentrations
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Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
Table 2 Mean concentration of heavy metals in liver and muscle of flounder (standard deviation between brackets) mg kg1 dry weight
mg kg1 wet weight
Tissue
Section
Length group (mm)
Cd
Pb
Zn
Cu
Cd
Pb
Zn
Liver
1
<180
51.38 (8.39)
0.88 (0.05)
2.82 (0.19)
110.67 (6.83)
0.70 (0.11)
0.01 (0.00)
0.04 (0.00)
1.51 (0.09)
Liver
1
P180
43.21 (14.31)
0.66 (0.06)
2.98 (0.30)
115.82 (3.65)
0.59 (0.19)
0.01 (0.00)
0.04 (0.00)
1.58 (0.05)
Liver
2
<180
40.50 (8.01)
1.00 (0.15)
8.90 (3.10)
146.00 (7.12)
0.55 (0.09)
0.01 (0.00)
0.12 (0.04)
1.99 (0.08)
Liver
2
P180
49.18 (16.77)
2.05 (0.25)
3.32 (1.98)
135.54 (3.29)
0.67 (0.23)
0.03 (0.00)
0.05 (0.03)
1.84 (0.04)
Liver
3
<180
41.62 (5.11)
0.69 (0.05)
4.91 (0.50)
171.10 (8.07)
0.57 (0.14)
0.01 (0.00)
0.07 (0.02)
2.33 (0.09)
Liver
3
P180
58.84 (32.11)
1.27 (0.71)
3.95 (0.53)
175.93 (76.58)
0.80 (0.24)
0.02 (0.01)
0.05 (0.01)
2.39 (1.04)
Muscle
1
<180
1.39 (0.71)
0.26 (0.03)
2.16 (0.43)
25.62 (1.82)
0.02 (0.01)
0.00 (0.00)
0.03 (0.01)
0.32 (0.02)
Muscle
1
P180
4.04 (5.16)
1.00 (1.23)
4.04 (1.78)
23.85 (8.34)
0.05 (0.06)
0.01 (0.01)
0.05 (0.02)
0.29 (0.11)
Muscle
2
<180
1.30 (0.07)
0.52 (0.27)
3.46 (0.25)
43.39 (9.21)
0.02 (0.00)
0.01 (0.00)
0.04 (0.00)
0.54 (0.24)
Muscle
2
P180
1.87 (1.13)
0.39 (0.04)
2.58 (0.52)
15.70 (5.33)
0.02 (0.01)
0.00 (0.00)
0.03 (0.01)
0.20 (0.07)
Muscle
3
P180
2.09 (1.61)
0.52 (0.24)
3.79 (0.37)
20.48 (6.20)
0.03 (0.02)
0.01 (0.00)
0.05 (0.00)
0.25 (0.08)
Cu
were considerably lower than reported for Pleuronectidae by Sidwell et al. (1974) and for P. flesus in other polluted areas (Amiard et al., 1980; Julshmamn and Grahl-Nielsen, 1996; Miramand et al., 2001). No tissue accumulation difference was detected for Cu, Cd and Pb (F > 1.5, p > 0.5) between the area sections where flounder were caught. It would be expected that if flounder concentrate in the lower estuary and that is the most contaminated area, then they should bioaccumulate more of these metals than flounder from lesser polluted areas of the estuary. Our data reveal that although flounder juveniles seem to concentrate in selected areas of the estuary they must disperse frequently to other parts of the estuarine nursery. Accumulation differences were detected between the sections of the estuary only for zinc, (F = 3.6, p < 0.05). Post-hoc tests revealed that flounder from the adjoining coastal waters (section 3) accumulated more zinc than those caught within the estuary (Table 2). Several field and experimental studies have demonstrated that the amount of zinc accumulated in various tissues by fish is mostly independent of the concentrations in the rearing medium (Eisler and Gardner, 1973; Pentreath, 1977; Van Hoof and Van San, 1981). No accumulation differences were detected between length groups of flounder for all heavy metals (F < 0.2.4, p > 0.05). Several authors have noted that
the concentrations of heavy metals in P. flesus varies with the length of the fish (Hardisty et al., 1974; Phillips, 1977; Jensen and Cheng, 1987). The fact that these differences were not detected in the Douro estuary could be due to the fact that the flounder population in this estuary is composed mainly by young fish and that flounder from the P180 group varied only between 180 mm and 230 mm. Also very few flounder smaller than 100 mm were caught. Therefore, the length spectrum analyzed may not have been wide enough for the detection of metals accumulation differences. This study shows that P. flesus juveniles concentrate in the most polluted areas of the estuary but also disperse along a wider area and it is therefore important to continue to monitor levels of contamination in this important species.
Acknowledgments This study was developed in the scope of the ERIC Project ÔEffects of river inflow changes on the fish communities of the Douro, Tagus and Guadiana estuaries and adjoining coastal areas. Ecological and socio-economical predictions.Õ (PDCTM/C/MAR/15263/1999), financially supported by ÔFundac¸a˜o para a Cieˆncia e a TecnologiaÕ.
Baseline / Marine Pollution Bulletin 49 (2004) 1109–1126
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