Artículos

 

Fe, Zn, Cu and Cd concentrations in the digestive gland and muscle tissues of Octopus vulgaris and Sepia officinalis from two coastal areas in Portugal

 

Concentraciones de Fe, Zn, Cu y Cd en la glándula digestiva y los tejidos musculares de Octopus vulgaris y Sepia officinalis de dos zonas costeras de Portugal

 

Joana Raimundo*, Patricia Pereira, Carlos Vale and Miguel Caetano

 

^* IPIMAR. Institute for Fisheries and Sea Research Av. Brasilia, 1449-006 Lisbon, Portugal. * E-mail: joanar@ipimar.pt

 

Recibido en junio de 2003;
aceptado en abril de 2004.

 

Abstract

Concentrations of Fe, Zn, Cu and Cd were measured in the mantle, arm and digestive gland of Octopus vulgaris (Cuvier, 1797) and in the mantle and digestive gland of Sepia officinalis (Linnaeus, 1758) captured at two sites on the Portuguese coast during 2001. The most abundant element was Zn, reaching 121 µg g^-1 in muscle tissues; Fe and Cu presented a similar range of concentration, 5.4-81 µg g^-1 and 3.3-72 µg g^-1, respectively; and Cd varied between 0.010 and 3.3 µg g^-1. Metal concentrations in the digestive gland were two and three orders of magnitude higher than those recorded in the mantle and arm. The relationship between metal concentration and body weight was found mainly in the digestive gland and rarely in the mantle and arm. The Cd:Zn and Cd:Cu ratios were particularly high in the digestive gland of the two species captured at the most contaminated site and presented positive linear relationships with the body weight. This suggests a progressive accumulation of Cd in the digestive gland of octopus and cuttlefish with growth, in comparison to the essential elements Zn and Cu.

Key words: metals, digestive gland, cephalopods, Portugal.

 

Resumen

Se midieron las concentraciones de Fe, Zn, Cu y Cd en el manto, el brazo y la glándula digestiva de Octopus vulgaris (Cuvier, 1797) y en el manto y la glándula digestiva de Sepia officinalis (Linnaeus, 1758), capturados en dos zonas de la costa portuguesa durante 2001. El elemento más abundante fue Zn, alcanzando 121 µg g^-1 en los tejidos musculares; Fe y Cu presentaron un intervalo de concentración similar, 5.4-81 µg g^-1 y 3.3-72 µg g^-1, respectivamente; y Cd varió entre 0.010 y 3.3 µg g^-1. Las concentraciones de metales en la glándula digestiva fueron dos y tres órdenes de magnitud superiores que los registrados en el manto y brazo. La relación entre la concentración de metal y el peso corporal se encontró principalmente con la glándula digestiva y raramente con el manto y brazo. Las razones Cd:Zn y Cd:Cu fueron particularmente altas para la glándula digestiva de las dos especies capturadas en la zona más contaminada y presentaron una relación lineal con el peso corporal. Esto sugiere una acumulación progresiva de Cd en las glándulas digestivas del pulpo y la sepia con el crecimiento, en comparación con los elementos esenciales Zn y Cu.

Palabras clave: metales, glándula digestiva, cefalópodos, Portugal.

 

Introduction

Metal concentrations in cephalopods from several regions are well documented (e.g., Smith et al., 1984; Miramand and Bentley, 1992; Bustamante et al., 2000). Most of these studies have highlighted the ability of these organisms to concentrate Zn, Cu and Cd in the digestive gland even in environments of low metal contamination. This organ has a major physiological function in the digestive process of cephalopods, supplying most of the digestive enzymes and storing nutrients and trace elements (Bustamante, 1998). The accumulation of non-essential metals in marine organisms, like Cd, is probably related to efficient sequestration and detoxification mechanisms (Rainbow et al., 1990). One well-known detoxification strategy of marine invertebrates involves the binding of trace metals to metallothioneins (Bebianno and Langston, 1991; Viarengo and Nott, 1993). These molecules have not been found in cephalopods (Bustamante et al., 2002), but proteins with higher molecular weight (>70 kDa) have been reported as potential binding sites for Cd in the digestive gland of cephalo-pod species (Tanaka et al., 1983; Finger and Smith, 1987; Castillo et al., 1990).

Essential elements (e.g., Zn and Cu) are regulated in organisms by homeostatic mechanisms (Langston et al., 1998), although non-essential metals (e.g., Cd) may occasionally substitute them (Zauke and Petri, 1993). These interactions between metals have been assessed through metal-metal correlations (Smith et al., 1984) and by the ratios Cd:Cu and Cd:Zn (Bustamante et al., 1998). Ratios have been determined in the digestive gland of Benthoctopus thielei, Eledone cirrhosa, Graneledone sp., Octopus vulgaris and Sepia officinalis and higher ratios interpreted as competition and/or substitution of essential metals by Cd (Miramand and Guary, 1980; Miramand and Bentley, 1992; Bustamante et al., 1998).

Octopus vulgaris (common octopus) and Sepia officinalis (cuttlefish) are important fishery resources in several coastal regions. These species are voracious cephalopods with sedentary habits (Boletzky, 1983; Mangold, 1983). The Portuguese coastal area presents different metal concentrations in seawa-ter, seston and mussel tissues between the northwestern and southern coasts (Vale et al., 1985; Caetano and Vale, 2003). The present work reports the concentrations of Fe, Zn, Cu and Cd in the muscle tissues and digestive gland of O. vulgaris and S. officinalis captured on the northwestern and southern coasts of Portugal. The Cd:Cu and Cd:Zn ratios were examined in organisms at different growth stages from both areas.

 

Materials and methods

Sampling and sample preparation

The species O. vulgaris and S. officinalis were captured at site A (northwest coast) and at site B (south coast) during 2001 (fig. 1). Twelve octopuses were obtained at each site, while thirty-one cuttlefish were collected from site A and eight from site B. The organisms were immediately frozen (-25°C) on board in individual plastic bags. In the laboratory, organisms were weighed, measured (mantle length) and sexed. Each organism was considered individually. Muscle tissues (arm and mantle) without skin and the digestive gland were removed from the octopuses, and the mantle and digestive gland from cuttlefish. In order to avoid contamination from other organs, only the inner part of the digestive gland was sampled.

Analytical procedure

Tissue samples were freeze-dried, homogenized and digested with HNO₃ and H₂O₂ according to the method described in Ferreira et al. (1990). Concentrations of Fe, Zn, Cu and Cd were determined either by flame atomic absorption spectrophotometry (Perkin Elmer Analyst 100) or graphite furnace atomic absorption spectrophotometry (Perkin Elmer 4110ZL). All labware was cleaned with HNO₃ and HCl and rinsed with Milli-Q water. The accuracy of the analytical procedure was assessed by the analysis of international standard reference materials DORM-1, DOLT-2 and TORT-1 (National Research Council of Canada). For all metals investigated, the obtained and certified values were not statistically different (P < 0.01). Blanks and standard reference materials were run together with samples. All the results are given in micrograms of metal per gram of dry weight tissue (µg g^-1 dw). Detection limits were 4.2 µg^-1 for Fe, 0.50 µg^-1 for Zn, 1.2 µg^-1 for Cu and 0.010 µg g^-1 for Cd. Precision errors were 2%, 1%, 8% and 2% for Fe, Zn, Cu and Cd, respectively.

Range and median were obtained for each element. Statistical analysis was performed using the non-parametric Mann-Whitney and Kruskal-Wallis tests, and a two-tailed Student t-test.

 

Results

Relationship between mantle length and body weight

The number of individuals, mantle length and body weight of O. vulgaris and S. officinalis specimens collected at sites A and B are presented in table 1. Octopuses captured at the two sites were similar in length (P < 0.05) and weight (P < 0.05), while cuttlefish from site A showed higher mantle length and weight than those from site B (P < 0.05). The length of all the octopuses analyzed was highly correlated to weight (r² = 0.85) (fig. 2). Despite the differences in length and weight of cuttlefish from both sites, these parameters were also highly correlated (r² = 0.91).

Metal concentrations in mantle and arm

The concentrations of Fe, Cu, Zn and Cd in the mantle and arm of O. vulgaris and in the mantle of S. officinalis are presented in table 2. The most abundant element was Zn, reaching 121 µg g^-1; Fe and Cu presented a similar range of concentration, of 5.4-81 µg g^-1 and 3.3-72 µg g^-1, respectively; and Cd varied between 0.010 and 3.3 µg g^-1. In most cases, the concentrations in these tissues were not correlated with sex (P < 0.05) or body weight (P < 0.05). Exceptions were found at site B: Cu (µg g^-1) in the octopus arm increased with weight (g) (y = 0.038 x - 5.4, r² = 0.62); and Zn levels in the cuttlefish mantle decreased with weight (y = -0.12 x + 121, r² = 0.79). Metals in muscle tissues differ between the two sites. The concentrations of Cd in octopus and cuttlefish muscle tissues were higher at site A (P < 0.05). The concentrations of Cd in the mantle of octopus from site A (median =1.8 µg g^-1) were notably higher in comparison to values of other mantle and arm samples (max. 0.81 µg g^-1). That value exceeds the safety limit (1.0 µg g^-1dw) for human consumption (Journal of EU Communities, 2001). The level of Cd in mantle of cuttlefish from both sites was below this limit (site A median = 0.16 µg g^-1 and site B median = 0.025 µg g^-1). Enhanced values at site A were also observed for Fe in octopus mantle (P < 0.05). The Cu concentrations in octopus arm and cuttlefish mantle were higher at site B (P < 0.05). The Zn concentrations in the tissues analyzed were similar at both sites (P < 0.05).

Metal concentrations in the digestive gland

The concentrations of Fe, Cu, Zn and Cd in the digestive gland of O. vulgaris and S. officinalis are presented in table 3. At both sites, the digestive gland of octopus presented higher levels of Fe and lower values of Zn than those of cuttlefish. Most octopus specimens captured at site A showed higher Cd concentrations than cuttlefish. Cadmium was particularly high in the digestive gland of both octopus (median = 185 µg g^-1) and cuttlefish (median = 137 µg g^-1) from site A, when compared to values from site B (32 and 35 µg g^-1, respectively). In contrast, median Cu concentrations in the digestive gland of octopus and cuttlefish from site A were lower than those from site B (601 and 998 µg g^-1 for octopus, and 293 and 2765 µg g^-1 for cuttlefish, respectively). Zinc presented a similar pattern: median concentrations for octopus were 616 and 2541 µg g^-1 at sites A and B, respectively, and for cuttlefish they were 907 and 2754 µg g^-1. The concentrations of Zn and Cu in the digestive gland of octopus and cuttlefish from site A were poor although significantly correlated to body weight; smaller organisms presented higher concentrations (fig. 3). A positive correlation between Cd and body weight was found only in the digestive gland of octopus from site A. No significant correlations between Fe and weight were obtained. At site B, no correlations were found between metal concentrations in the digestive gland and body weight of the two species.

 

Discussion

The results obtained in this study revealed extremely high concentrations of Cd in the digestive glands of O. vulgaris and S. officinalis captured on the northwest coast of Portugal. These values exceeded levels reported for the same species in the Mediterranean Sea and North Atlantic (Miramand and Guary, 1980; Miramand and Bentley, 1992), and for other cephalopods species, namely Eledone cirrhosa (Miramand and Bentley, 1992), Loligo opalescens (Martin and Flegal, 1975) and Nototodarus gouldi (Finger and Smith, 1987). The concentrations in the mantle and arm were much lower, indicating that Cd, as well as Fe, Cu and Zn are preferentially accumulated in the digestive gland. This partition is in line with other findings, where the digestive gland of several cephalopod species reveals considerably higher accumulation of metals than muscles, branchial hearts, gills, digestive tract, kidney, genital tract, skin and shell (Miramand and Guary, 1980; Miramand and Bentley, 1992). On the basis of Cd concentrations and weight of the octopus and cuttlefish tissues analyzed from both sites, one may conclude that more than 90% of the total body Cd is sequestrated in the digestive gland. This estimation, which agrees with calculations presented in other studies (Bustamante, 1998; Bustamante et al., 2002), emphasizes the capacity of the digestive gland to accumulate Cd. Although this capacity has been observed in cephalopods from areas with reduced levels of contamination (Bustamante et al., 1998), higher Cd levels in the digestive gland of species captured in contaminated areas (Bustamante et al., 2000) suggest that accumulation in this organ is also related to the environment.

The levels of Cd in the digestive gland and muscle tissues of octopus and cuttlefish from the northwest coast (site A) were one order of magnitude higher than those from the south coast (site B). This contrast seems related to the differences of Cd availability in the waters of both sites. In fact, water quality surveys in 2000 and 2001 along the Portuguese coast revealed higher concentrations of Cd in the northwestern coastal waters than in the southern ones (Caetano and Vale, 2003). As octopus and cuttlefish have sedentary habits (Boletzky, 1983; Mangold, 1983), presumably specimens captured at site A were exposed to higher levels of Cd during their life span than those at site B. Higher accumulation of Cd in the digestive gland of specimens from site A appears, therefore, to reflect this environmental exposure; however, enhanced values in the mantle and arms indicate that the sequestration capacity of that organ did not avoid the transfer of Cd to muscle tissues.

The relationship between metal concentrations and body weight was rarely found for mantle and arm. This lack of relationship was also observed for Zn, Cu and Cd in other octopus species (Bustamante et al., 1998). In the case of the digestive gland of octopus and cuttlefish, negative correlations between metal concentration and body weight were more recurrent at site A for Zn and Cu. A positive relationship was also found between the Cd in the digestive gland and the body weight of octopus at site A, where exposure to higher levels of Cd is considered. This reinforces the key function of the digestive gland in the accumulation and/or detoxification of Cd. In these type of mechanisms, similarities in chemical behaviour based on electron configuration between Cd and essential elements like Zn and Cu may be significant (Smith et al., 1984; Miramand and Bentley, 1992). In order to examine the sequestration of Cd in the digestive gland with individual growth and its interaction with Zn and Cu, ratios of Cd:Zn and Cd:Cu (on molar basis) were plotted against body weight (fig. 4). Two clear-cut aspects may be discerned: the digestive gland of octopus and cuttlefish from site A (contaminated site) exhibited Cd:Zn ratios (median 0.19 and 0.11 nmol g^-1, respectively) and Cd:Cu ratios (median 0.15 and 0.23 nmol g^-1, respectively) one to two orders of magnitude higher than Cd:Zn ratios (median 0.0067 and 0.019 nmol g^-1, respectively) and Cd:Cu ratios (median 0.0043 and 0.0058 nmol g^-1, respectively) from site B; both Cd:Zn and Cd:Cu ratios of site A increased linearly with growth. This increase suggests that Cd was progressively sequestered in the digestive gland of cephalopods, possibly competing with Zn and Cu for the binding-sites of the molecular structures. In contrast, the metal ratios in the mantle and arm were not related to body weight, as a result of the high Cd storage capacity of the digestive gland.

 

Acknowledgements

The authors wish to thank our colleagues Joao Pereira, Pedro da Conceigao, Manuel Sobral and Isabel Sobral, and the anonymous reviewers for their helpful comments to improve the manuscript. This work was supported by the FCT Project PLE-14 and the QCA III Project-MARE, 22-05-05-FDR-00005.

 

References

Bebianno, M.J. and Langston, W.J. (1991). Metallothionein induction inMytilus edulis exposed to cadmium. Mar. Biol., 108: 91-96.         [ Links ]

Boletzky, S.V. (1983). Sepia officinalis. In: P.R. Boyle (eds.), Cephalopod Life Cycles, Species Accounts. Vol. I. Academic Press, UK, pp. 335-364.         [ Links ]

Bustamante, P. (1998). Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs. Thesis, University of La Rochelle, pp. 290.         [ Links ]

Bustamante, P., Cherel, Y., Caurant, F. and Miramand, P. (1998). Cadmium, copper and zinc in octopus from Kerguelen Islands, Southern Indian Ocean. Polar Biol., 19: 264-271.         [ Links ]

Bustamante, P., Grigioni, S., Boucher-Rodoni, R., Caurant, F. and Miramand, P. (2000). Bioaccumulation of 12 trace elements in the tissues of Nautilus Nautilus macromphalus from New Caledonia. Mar. Pollut. Bull., 40(8): 688-696.         [ Links ]

Bustamante, P., Cosson, R.P., Gallien, I., Caurant, F. and Miramand, P. (2002). Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations. Mar. Environ. Res., 53: 227-241.         [ Links ]

Caetano, M. and Vale, C. (2003). Trace-elemental composition of seston and plankton along the Portuguese coast. Acta Oecol., 24: S341-S349.         [ Links ]

Castillo, L.V., Kawagushi, S. and Maita, Y. (1990). Evidence for the presence of heavy metal binding proteins in the squid, Onychoteuthis borealijaponica. In: R. Hirano and I. Hanyu (eds.), The Second Asian Fisheries Forum. Manila: Asian Fish. Soc., pp. 991.         [ Links ]

Ferreira, A.M., Cortesão, C., Castro, O. and Vale, C. (1990). Accumulation of metals and organochlorines in tissues of the oyster Crassostrea angulata from the Sado estuary. Sci. Total Environ., 97/98: 627-239.         [ Links ]

Finger, J.M. and Smith, J.D. (1987). Molecular association of Cu, Zn, Cd and ²¹⁰Po in the digestive gland of the squid Nototodarus gouldi. Mar. Biol., 95: 87-91.         [ Links ]

Journal of EU Communities (2001). Resolution No. 466/2001, pp. 113.         [ Links ]

Langston, W.J., Bebianno, M.J. and Burt, G.R. (1998). Metal handling strategies in molluscs. In: W.J. Langston and M.J. Bebianno (eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, UK, pp. 220-283.         [ Links ]

Mangold, K. (1983). Octopus vulgaris. In: P.R. Boyle (eds.), Cephalopod Life Cycles, Species Accounts. Vol. I. Academic Press, UK, pp. 335-364.         [ Links ]

Martin, J.H and Flegal, A.R. (1975). High copper concentrations in squid livers in association with elevated levels of silver, cadmium and zinc. Mar. Biol., 30: 51-55.         [ Links ]

Miramand, P. and Guary, J.C. (1980). High concentrations of some heavy metals in tissues of the Mediterranean octopus. Bull. Environ. Contam. Toxicol., 24: 783-788.         [ Links ]

Miramand, P. and Bentley, D. (1992). Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel. Mar. Biol., 114: 407-414.         [ Links ]

Rainbow, P.S., Phillips, D.J.H. and Depledge, M.H. (1990). The significance of trace metal concentrations in marine invertebrates. A need for laboratory investigation of accumulation strategies. Mar. Pollut. Bull., 21(7): 321-324.         [ Links ]

Smith, J.D., Plues, L., Heyraud, M. and Cherry, R.D. (1984). Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Mn, Pb and Zn, and the radionuclides ²¹⁰Pb and ²¹⁰Po in the digestive gland of the squid Notodarus gouldi. Mar. Environ. Res., 13: 55-68.         [ Links ]

Tanaka, T., Hayahi, Y. and Ishizawa, M. (1983). Subcellular distribution and binding of heavy metals in the untreated liver of the squid, comparison with data from the livers of cadmium and silver exposed rats. Experientia, 39: 746-748.         [ Links ]

Vale, C., Ferreira, A.M., Cortesao, C., Castro, O.G. and Mendes, M.C. (1985). A mussel watch in the Portuguese coast: 1984. ICES, C.M. 1985/E: pp. 18.         [ Links ]

Viarengo, A. and Nott, J.A. (1993). Mini-review. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol., 104C: 355-372.         [ Links ]

Zauke, G.P. and Petri, G. (1993). Metal concentrations in Antarctic crustacean: The problem of background levels. In: R. Dallinger and P.S. Rainbow (eds.), Ecotoxicology of Metals in Invertebrates. Lewis, London, pp. 73-101.         [ Links ]