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Heavy metal concentrations in Squilla mantis (L.) (Crustacea, Stomatopoda) from the Gulf of Cádiz
Environment International 28 (2002) 111 – 116 www.elsevier.com/locate/envint
Heavy metal concentrations in Squilla mantis (L.) (Crustacea, Stomatopoda) from the Gulf of Ca´diz Evaluation of the impact of the Aznalcollar mining spill J. Blasco*, A.M. Arias, V. Sa´enz Departamento Oceanografia, Instituto de Ciencias Marinas de Andalucı´a (CSIC), Campus Universitario Rı´o San Pedro, 11510 Puerto Real, Ca´diz, Spain Received 30 June 2001; accepted 7 February 2002
Abstract After the Aznalco´llar mining spill (25th April 1998), considerable social concern arose amongst the inhabitants of the SW Iberian Peninsula concerning the consumption of local seafood. Squilla mantis was collected in four regions of the Gulf of Ca´diz with a dual objective: to analyze the heavy metal levels for human consumption and as part of biomonitoring program. Heavy metal concentrations (Fe, Mn, Zn, Cu, Cd and Pb) were analyzed in soft tissues and cuticle. The highest values were found in the soft tissues for zinc, copper and cadmium and in the cuticle for iron, manganese and lead. The mean copper concentration in the soft tissue, corresponding to the edible part, was 27.1 mgg 1 wet weight. Approximately 80% of stations showed values higher than 20 mgg 1 wet weight of copper, the Spanish legal limit for the concentration of this metal in the crustacean for human consumption. For Zn and Cu no significant differences were found between regions, probably related with the capacity for regulation of S. mantis. The highest values found for copper in the Gulf of Ca´diz compared to other areas is likely to be related with contamination from terrestrial mining activities (copper and pyrites) in the region, dating back to the times of Tartessians and Romans, rather than the effects of mining spill which was shown not to create any significant increases in heavy metal concentrations of organisms of the Guadalquivir River or the adjacent coastal area. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Heavy metals; Mining spill; Crustacean; Squilla mantis; The Gulf of Ca´diz
1. Introduction The use of aquatic organisms to establish geographical and temporal variations in the bioavailabilities of heavy metals in coastal and estuarine waters is well established (Phillips, 1980; Phillips and Rainbow, 1993). Some crustaceans (particularly decapods) are large and easily identified and, from this point of view, could be considered as suitable candidates to be used as biomonitors. Nevertheless, their use has been criticised because they regulate the tissue concentrations of trace elements such as copper, manganese and zinc to approximately constant concentrations over a wide range of metal availabilities (Rainbow and Phillips, 1993). Besides their potential employment as biomonitors, several species of crustaceans are included in the diet of coastal inhabitants and the concentrations of heavy metals consid-
* Corresponding author. Tel.: +34-9568-32612; fax: +34-9568-34701. E-mail address: [email protected] (J. Blasco).
ered safe for human consumption in such seafood are regulated by legislation (BOE, 1991). After the Aznalco´llar mining spill in southern Spain, considerable social concern arose amongst the inhabitants of the SW Iberian Peninsula concerning the consumption of local seafood. For this reason, an intensive biomonitoring program was carried out at several sampling stations in the estuary of the river Guadalquivir (Blasco et al., 1999; Sun˜er et al., 1999) and in the Gulf of Ca´diz. One species collected in the Gulf of Ca´diz was a benthic crustacean, the mantis shrimp Squilla mantis (Stomatopoda). This species is common in the diet of local inhabitants and its fishery has considerable economic importance. The Gulf of Ca´diz receives effluent from the Guadiana and the Guadalquivir Rivers, with the Tinto and Odiel Rivers also representing significant sources of heavy metals. Important mines and relatively big cities and industries are situated in the drainage basin of these fluvial systems, and are responsible for metal loadings in the continental shelf waters of the Gulf of Ca´diz (Elbaz-Poulichet and Leblanc, 1996).
0160-4120/02/$ – see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 2 ) 0 0 0 1 4 - 4
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J. Blasco et al. / Environment International 28 (2002) 111–116
The aim of this study was to improve the available knowledge on the background concentrations of heavy metals in S. mantis, and to investigate the impact of the mining spill of Aznalco´llar on these concentrations, particularly with respect to safe levels for human consumption.
2. Materials and methods Samples of S. mantis were collected using commercial trawlers at 14 sampling stations subdivided into four regions
(1 –4) located in the area of the fishery of this species in the Gulf of Ca´diz, Spain (Fig. 1). Specimens were kept at 4 C until transported to the laboratory, where they were dissected into cuticle and soft tissues. The samples from each station generally consisted of about 15 specimens which were pooled in three groups of five individuals, providing 5, 9, 13 and 7 pooled samples from regions 1 to 4, respectively. The mean carapace length within each pool ranged between 26 and 35 mm. Cuticle and soft tissues were freeze dried in a VIRTIS lyophiliser, then crushed and homogenised to a fine powder in an agate bowl mill with a
Fig. 1. Map of sampling stations and delimited regions in the Gulf of Ca´diz fisheries of S. mantis.
J. Blasco et al. / Environment International 28 (2002) 111–116 Table 1 Certified and measured values of iron, manganese, zinc, copper, cadmium and lead in standard reference materials (expressed as microgram per gram dry weight) Metal
Certificate values DORM1 TORT1
Our values DORM1
TORT1
Fe Mn Zn Cu Cd Pb
63.6 ± 5.3 1.32 ± 0.26 21.3 ± 1.0 5.22 ± 0.33 0.086 ± 0.012 0.40 ± 0.12
54.3 ± 2.4 1.44 ± 0.19 20.3 ± 0.7 5.32 ± 0.29 0.082 ± 0.010 0.61 ± 0.14
176.4 ± 8.1 20.2 ± 0.5 171 ± 8 417 ± 13 26.2 ± 1.5 12.8 ± 3.2
186 ± 11 23.4 ± 1.0 177 ± 10 439 ± 22 26.3 ± 2.1 10.4 ± 2.0
The values are mean and 95% confidence intervals (n = 10).
Planetary Mono Mill (Pulverissette 6, Fristch). Samples were digested according to the procedure of Amiard et al. (1987). In summarized way, ca. 0.1 g of lyophilised crushed and homogenised tissues were digested with 1 ml HNO3 Suprapur (Merck) at 95 C for 1 h in a heater block. The resultant solutions were diluted to a known volume with Milli-Q water and the heavy metals Cd and Pb were analysed by graphite furnace atomic absorption spectrophotometry (GFAAS) (PE 4100 ZL), and flame AAS (PE 3110) for Fe, Mn, Cu and Zn. The results are expressed as microgram per gram dry or wet weight. The analytical procedure was checked using reference material (DORM1 and TORT1 of NRC Canada). The results are shown in Table 1. All statistical analyses, including regression analysis, ANOVA and ANCOVA, were carried out using the software package Statgraphic plus for Windows (Statgraphics, 1998). Data were log transformed before statistical treatment.
3. Results Table 2 gives the mean and range of concentrations of heavy metals (Fe, Mn, Zn, Cu, Cd and Pb) in the soft tissues and cuticle of all the S. mantis collected in the Gulf of Ca´diz. The highest values were found in the soft tissues for zinc, copper and cadmium and in the cuticle for iron, manganese and lead. The ratio [M]soft body/[M] cuticle ranged between 3.4 for Cd and 0.13 for Mn. In the case of
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lead, for 50% of the sampling stations, concentrations in cuticle and soft tissues of S. mantis were beneath detection levels ( < 0.05 mg g 1 dry weight). Iron, manganese, zinc, copper and cadmium concentrations in the soft tissue and the cuticle of S. mantis from the four regions stations of the Gulf of Ca´diz are reported in Figs. 2 and 3. Correlation analysis for tissue heavy metal concentration against carapace length, firstly for whole data set and secondly for data for each separate zone, showed that in both the total soft tissues and the cuticle a significant and positive linear correlation was reported for iron, while copper showed against length ( P < .05) in the cuticle only. In consequence, in order to establish differences between mean tissue metal concentrations of S. mantis from the different zones, ANOVA analysis was carried out for manganese, zinc and copper concentrations in the total soft tissues, and for cadmium and manganese in the cuticle. The remaining metal concentrations in the tissues were analysed by ANCOVA, where the carapace length was introduced as a covariate. In the total soft tissues (Fig. 2), Fe and Cd concentrations showed significant differences between zones ( P < 0.05). The lowest values were found in region one. For the cuticle (Fig. 3), only manganese showed a significant difference between zones, with the highest concentrations in zone 1. A multiple range test (LSD) was carried out to determine which zones were statistically different from each other. Thus for iron in the soft tissue, zone 1 was significantly different to zones 2 and 3, and zone 4– 3. In the case of cadmium, zone 1 was significantly different from the rest. For manganese in the cuticle, the multiple range test showed significant differences between zones 1 and 2, 2 and 4, and 2 and 3 at the P < 0.05 significance level. The mean concentrations were similar for copper and zinc in the soft body (136 and 135 mgg 1 dry weight, respectively) and the body concentration for Zn ranged between 64 and 104 mgg 1 dry weight. Iron and copper showed the highest values in the cuticle (105 and 84.8 mgg 1 dry weight, respectively). The mean copper concentration in the soft tissue, corresponding to the edible part, was 27.1 mgg 1 wet weight. Approximately 80% of stations showed values higher than 20 mgg 1 wet weight
Table 2 Heavy metal concentrations (means and ranges) in the soft tissues and cuticle (n = 14) crustacean S. mantis collected in the Gulf of Ca´diz Tissue Soft tissues Mean Range Cuticle Mean Range
Fe
Mn
Zn
Cu
Cd
Pb
77.89 36.70 – 133.14
4.24 0.54 – 7.18
134.97 118.36 – 154.84
135.47 60.24 – 202.41
1.66 0.91 – 2.13
0.19 < 0.05 – 0.84
104.90 55.25 – 207.22
32.40 13.59 – 55.31
48.36 33.65 – 73.61
84.85 58.20 – 102.89
0.49 0.31 – 0.67
0.33 < 0.05 – 2.65
* Results are expressed as microgram per gram dry weight.
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centrations of zinc in decapods typically lie in the range 50 – 120 mg Zn g 1 dry weight (White and Rainbow, 1982). This is the same as the range that we have reported in the studied species. The restricted range of Zn concentrations found and the lack of significant differences between zones indicate that S. mantis could, therefore, carry out Zn regulation in the soft tissues, in a similar way to that reported in the palaemonid decapod
Fig. 2. Heavy metal concentrations (means and standard deviations) in the soft tissues of S. mantis collected in four regions in the Gulf of Ca´diz. Results are expressed as microgram per gram dry weight.
of copper, the Spanish legal limit for the concentration of this metal in the crustacean for human consumption.
4. Discussion Information on the concentrations of trace metals in stomatopod crustaceans is scarce. Regulated body con-
Fig. 3. Heavy metal concentrations (means and standard deviations) in the cuticle of S. mantis collected in four regions in the Gulf of Ca´diz. Results are expressed as microgram per gram dry weight.
J. Blasco et al. / Environment International 28 (2002) 111–116
Palaemon elegans and the crab Carcinus maenas. (Rainbow, 1998). Although the net result is the same in both these species, the mechanisms involved in the homeostasis of Zn concentration differ between these decapods. P. elegans matches the rate of zinc uptake with the rate of zinc excretion (White and Rainbow, 1982; Rainbow and White, 1988), while C. maenas has such a low rate of zinc uptake rate under most zinc availabilities that body zinc concentrations remain unaltered (Rainbow, 1998). To establish the mechanism for any such zinc regulation in S. mantis, it will be necessary to carry out kinetic accumulation studies. With regard to copper, the variation in soft tissue copper concentrations in S. mantis was greater than for zinc, although no significant differences were present between zones and regulation of soft tissue copper concentrations could also be occurring. In decapods, the situation concerning the regulation of copper is less clear than for zinc, given the lack of a convenient radiotracer to allow investigation of the kinetics of accumulation (Rainbow, 1998). Molar ratios of copper and zinc were ca. 1000:1000. This ratio satisfies the theoretical Zn and Cu requirements of crustaceans (Zauke and Petri, 1993). In the case of Cd, the ratio Cd/ Cu (4.0:1000) was of the same order as that found for other marine crustaceans (Rainbow, 1998). Although size may affect the accumulated concentration in organisms (Phillips, 1980), we only found statistically significant relationships in the cases of at P < .05, except for copper in the cuticle, and iron in both cuticle and total soft tissues. This fact may be a consequence of the small size range of the crustaceans analysed. The concentrations of iron and manganese in the cuticle were higher than in the soft tissues and may be a consequence of the adsorption of these metals onto the crustacean exoskeleton This process has been highlighted previously for manganese in coastal decapods (Bryan and Ward, 1965). The positive correlation between manganese in the soft tissues and cuticle could indicate that the accumulation in this species, as in other coastal decapods, increases greatly if the conditions promote the deposition of manganese onto the cuticle (Rainbow, 1988), whereas for iron the behaviour is the opposite. In both cases, a significant amount of heavy metals on the exoskeleton is in an adsorbed form (unpublished data). Samples of this species previously collected in this area (Establier, 1975) showed Cd values around 2.5 –3.0 mgg 1 dry weight. These values are higher than in our study (Table 2), but the differences may be due to the smaller size of the specimens collected, analytical methods employed and/or the variation of levels of metal contamination over time. Iron and manganese showed higher values in this species than in another stomatopod, S. oratoria (Eisler, 1981) while the Zn concentration reported was similar. Copper levels exceeded the statutory limit of 20 mg g 1 wet weight and the Spanish authorities prohibited their sale.
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Other crustacean species (Palaemon longirostris and Uca tangeri) collected in the Guadalquivir River estuary in the same period after the Aznalco´llar mining spill, also exceeded the legal limits. Nevertheless, the prawn Melicertus kerathurus collected in the adjacent coastal area had copper concentrations in the range 6.7 –11.0 mg g 1 wet weight (Blasco et al., 1999). Specimens of S. mantis collected in the Mediterranean Sea (Tarragona and Alicante coasts) had lower concentrations (13.5 ± 3.0 and 17.0 ± 4.0 mg g 1 wet weight, respectively) (Rodrı´guez, personal communication). The higher values found in S. mantis from the Gulf of Ca´diz area could be related with the effect of variables such as season, size, reproduction/moult stage and also with ambient levels of bioavailable copper. With relation to the impact of the mining spill on the Guadalquivir River estuary and coastal adjacent area, the main input of heavy metal involved zinc. Thus, a 20-fold increase of Zn concentrations in the local sediment compared to background levels in unpolluted sediment has been reported (Palanques et al., 1999). This metal was bioavailable to local aquatic organisms for a significant increase of accumulated Zn concentrations in the oyster Crassostrea angulata has been reported (Blasco et al., 1999). Other heavy metals discharged in the aquatic environment as consequence of the toxic spill were retained within a temporary dam at Entremuros (before the confluence with the estuary). The copper levels in the estuary of the Guadalquivir River are traditionally high and green-sick (copper contaminated) oysters have been known for 30 years to be present along the banks at the mouth of the river (Establier and Pascual, 1974). The mining activity and the use of the compounds which containing this metal for fumigation activities (Querol et al., 1999) are the main sources. Analysis of heavy metal levels in the plume of Guadalquivir River estuary and the adjacent coastal area produced no evidence of the impact of mining spill after 6 months (Achterberg et al., 1999). This fact was attributed to human intervention and natural removal processes. The metal concentrations reported in S. mantis from the Gulf of Cadiz could be related to contamination from terrestrial mining activities (copper and pyrites) in the Gulf of Ca´diz present since the times of the Tartesians and Romans. Strong heavy metal anomalies have been detected on the continental shelf near the mouths of the Guadiana and Tinto-Odiel Rivers and on the continental slope (Palanques et al., 1995). A metal-enriched seawater plume entering the western Mediterranean Sea through the Gibraltar Strait originates in the Tinto estuary (Elbaz-Poulichet and Leblanc, 1996; Van Geen et al., 1997). Recently, Elbaz-Poulichet et al. (2001) have identified the occurrence of a metal enriched water mass in the Gulf of Ca´diz (Spanish Shelf Water, SSW), which proves that the rivers draining the Iberian Pyrite Belt and additionally the Guadalquivir River are the ultimate source of metal. In consequence, the levels reported in S. mantis probably reflect the contamination levels in the Gulf of Ca´diz resulting from the high metal
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fluxes from the Tinto and Odiel River estuaries and therefore cannot be directly and exclusively attributed to the Aznalco´llar mining spill.
5. Conclusions S. mantis appears to regulate soft tissue concentrations of zinc and its pattern of accumulation of this metal appears, therefore, very similar to those of decapods. However, studies of kinetic accumulation will be necessary to establish mechanisms involved in any such regulation. Copper levels in this species are high in the study area in comparison with other sites, this being related to background levels in this region resulting from inputs originating in the Rio Tinto sulphide mining district and not from the impact of the Anzalco´llar mining spill. The use of S. mantis for biomonitoring programs might not be adequate. Nevertheless, the risk of copper concentrations exceeding the maximum allowed under Spanish legislation for human consumption of seafood will require the determination of its levels periodically. An ecotoxicological assessment of heavy metals, especially for copper, may be necessary to establish the relation between body loads of contaminants and potential toxic effects, and in this way be used to derive tissue quality values.
Acknowledgments We wish to thank Prof. P.S. Rainbow for revising the manuscript and giving helpful comments, Mr. T. Ransome for language revision, Ms. I. Ferna´ndez and Ms. F. Osta for their technical assistance, and Mr. J. Santana for his collaboration in the sampling. This research was supported by the CSIC and Junta Andalucı´a (Oficina Te´cnica Corredor Verde).
References Achterberg EP, Braungardt C, Morey NH, Elbaz-Poulichet F, Leblanc M. Impact of Los Frailes mine spill on riverine, estuarine and coastal waters in Southern Spain. Water Res 1999;33:3387 – 94. Amiard JC, Pineau A, Boiteau HL, Metayer C, Amiard-Triquet C. Application of atomic absorption spectrophotometry using Zeeman effect, to the determination of eight trace elements (Ag, Cd, Cr, Cu, Mn, Ni, Pb and Se) in biological materials. Water Res 1987;21:693 – 7. Blasco J, Arias AM, Sa´enz V. Heavy metals in organisms of the River Guadalquivir estuary: possible incidence of Aznalco´llar disaster. Sci Total Environ 1999;242:249 – 59. BOE. Lı´mites de contenidos de metales pesados y me´todos analı´ticos para
la determinacio´n de metales pesados para productos de la pesca y la acuicultura. Ministerio de Sanidad y Consumo 1991;195:27153 – 5. Bryan GW, Ward E. The absorption and loss of radioactive and non radioactive manganese by the lobster, Homarus vulgaris. J Mar Biol Assoc U K 1965;45:65 – 95. Eisler R. Trace metal concentrations in marine organisms. New York: Pergamon, 1981 (687 pp.). Elbaz-Poulichet F, Leblanc M. Transfert de me´taux d’une province minie`re a` l’ocean par des fleuves acides (Rio Tinto, Espagne). C R Acad Sci Paris 1996;322:1047 – 52. Elbaz-Poulichet F, Morley NH, Beckers JM, Nomerange P. Metal fluxes through the Strait of Gibraltar: the influence of the Tinto and Odiel rivers (SW Spain). Mar Chem 2001;73:193 – 213. Establier R. Concentracio´n de cadmio en organismos marinos de la costa sudatla´ntica espan˜ola. Inf Te´cn Inst Inv Pesq 1975;26:1 – 26. Establier R, Pascual E. Estudio de cobre, manganeso, hierro y zinc en ostiones (Crassostrea angulata) del Golfo de Ca´diz. Inv Pesq 1974;38: 371 – 84. Palanques A, Dı´az JI, Farran M. Contamination of heavy metals in the suspended and surface sediment of the Gulf of Cadiz (Spain): the role of sources, currents, pathways and sinks. Oceanol Acta 1995;18: 469 – 77. Palanques A, Puig P, Guille´n J, Querol X, Alastuey A. Zinc contamination in the bottom and suspended sediments of the Guadalquivir estuary after the Aznalcollar spill (south-western Spain). Control of hydrodynamic processes. Sci Total Environ 1999;242:211 – 20. Phillips DJH. Quantitative aquatic biological indicators: their use to monitor trace metal and organochlorine pollution. London: Applied Science Publisher, 1980 (487 pp.). Phillips DJH, Rainbow PS. Biomonitoring of trace aquatic contaminants. Barking: Elsevier, 1993 (371 pp.). Querol X, Alastuey A, Lo´pez-Soler A, Plana F, Mesas A, Ortiz L, Alzaga R, Bayona JM, Rosa J. Physico-chemical characterisation of atmospheric aerosols in a rural area affected by the Aznalcollar toxic spill, southwest Spain during the soil reclamation activities. Sci Total Environ 1999;242:89 – 104. Rainbow PS. The significance of trace metal concentrations in decapods. Symp Zool Soc. 1988;291 – 313. Rainbow PS. Phylogeny of trace metal accumulation in crustaceans. In: Langston WJ, Bebianno M, editors. Metal metabolism in aquatic environment. London: Chapman & Hall, 1998. pp. 285 – 319. Rainbow PS, Phillips DJH. Cosmopolitan biomonitors of trace metals. Mar Pollut Bull 1993;26:591 – 601. Rainbow PS, White SL. Comparative strategies of heavy metal accumulation by crustaceans: zinc, copper and cadmium in a decapod, an amphipod and a barnacle. Hydrobiologia 1988;174:245 – 64. Statgraphics. Statgraphic plus for Windows 3.3 statistical software package. Manugistics, MD (USA), 1998. Sun˜er MA, Devesa V, Mun˜oz O, Lo´pez F, Montoro R, Arias AM, Blasco J. Total and inorganic arsenic in the fauna of the Guadalquivir estuary: environmental and human health implications. Sci Total Environ 1999;242:261 – 70. Van Geen A, Adkins JF, Boyle EA, Nelson CH, Palanques A. A 120 yr record of widespread contamination from mining of the Iberian pyrite belt. Geology 1997;25:291 – 4. White SL, Rainbow PS. Regulation and accumulation of copper, zinc and cadmium by the shrimp Palaemon elegans. Mar Ecol: Prog Ser 1982;8:95 – 101. Zauke GP, Petri G. Metal concentrations in Antarctic crustacea: the problem of background level. In: Dallinger R, Rainbow PS, editors. Ecotoxicology of metals in invertebrates. Boca: Lewis Publisher, 1993. pp. 73 – 101.
Heavy metal concentrations in Squilla mantis (L.) (Crustacea, Stomatopoda) from the Gulf of Ca´diz Evaluation of the impact of the Aznalcollar mining spill J. Blasco*, A.M. Arias, V. Sa´enz Departamento Oceanografia, Instituto de Ciencias Marinas de Andalucı´a (CSIC), Campus Universitario Rı´o San Pedro, 11510 Puerto Real, Ca´diz, Spain Received 30 June 2001; accepted 7 February 2002
Abstract After the Aznalco´llar mining spill (25th April 1998), considerable social concern arose amongst the inhabitants of the SW Iberian Peninsula concerning the consumption of local seafood. Squilla mantis was collected in four regions of the Gulf of Ca´diz with a dual objective: to analyze the heavy metal levels for human consumption and as part of biomonitoring program. Heavy metal concentrations (Fe, Mn, Zn, Cu, Cd and Pb) were analyzed in soft tissues and cuticle. The highest values were found in the soft tissues for zinc, copper and cadmium and in the cuticle for iron, manganese and lead. The mean copper concentration in the soft tissue, corresponding to the edible part, was 27.1 mgg 1 wet weight. Approximately 80% of stations showed values higher than 20 mgg 1 wet weight of copper, the Spanish legal limit for the concentration of this metal in the crustacean for human consumption. For Zn and Cu no significant differences were found between regions, probably related with the capacity for regulation of S. mantis. The highest values found for copper in the Gulf of Ca´diz compared to other areas is likely to be related with contamination from terrestrial mining activities (copper and pyrites) in the region, dating back to the times of Tartessians and Romans, rather than the effects of mining spill which was shown not to create any significant increases in heavy metal concentrations of organisms of the Guadalquivir River or the adjacent coastal area. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Heavy metals; Mining spill; Crustacean; Squilla mantis; The Gulf of Ca´diz
1. Introduction The use of aquatic organisms to establish geographical and temporal variations in the bioavailabilities of heavy metals in coastal and estuarine waters is well established (Phillips, 1980; Phillips and Rainbow, 1993). Some crustaceans (particularly decapods) are large and easily identified and, from this point of view, could be considered as suitable candidates to be used as biomonitors. Nevertheless, their use has been criticised because they regulate the tissue concentrations of trace elements such as copper, manganese and zinc to approximately constant concentrations over a wide range of metal availabilities (Rainbow and Phillips, 1993). Besides their potential employment as biomonitors, several species of crustaceans are included in the diet of coastal inhabitants and the concentrations of heavy metals consid-
* Corresponding author. Tel.: +34-9568-32612; fax: +34-9568-34701. E-mail address: [email protected] (J. Blasco).
ered safe for human consumption in such seafood are regulated by legislation (BOE, 1991). After the Aznalco´llar mining spill in southern Spain, considerable social concern arose amongst the inhabitants of the SW Iberian Peninsula concerning the consumption of local seafood. For this reason, an intensive biomonitoring program was carried out at several sampling stations in the estuary of the river Guadalquivir (Blasco et al., 1999; Sun˜er et al., 1999) and in the Gulf of Ca´diz. One species collected in the Gulf of Ca´diz was a benthic crustacean, the mantis shrimp Squilla mantis (Stomatopoda). This species is common in the diet of local inhabitants and its fishery has considerable economic importance. The Gulf of Ca´diz receives effluent from the Guadiana and the Guadalquivir Rivers, with the Tinto and Odiel Rivers also representing significant sources of heavy metals. Important mines and relatively big cities and industries are situated in the drainage basin of these fluvial systems, and are responsible for metal loadings in the continental shelf waters of the Gulf of Ca´diz (Elbaz-Poulichet and Leblanc, 1996).
0160-4120/02/$ – see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 2 ) 0 0 0 1 4 - 4
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J. Blasco et al. / Environment International 28 (2002) 111–116
The aim of this study was to improve the available knowledge on the background concentrations of heavy metals in S. mantis, and to investigate the impact of the mining spill of Aznalco´llar on these concentrations, particularly with respect to safe levels for human consumption.
2. Materials and methods Samples of S. mantis were collected using commercial trawlers at 14 sampling stations subdivided into four regions
(1 –4) located in the area of the fishery of this species in the Gulf of Ca´diz, Spain (Fig. 1). Specimens were kept at 4 C until transported to the laboratory, where they were dissected into cuticle and soft tissues. The samples from each station generally consisted of about 15 specimens which were pooled in three groups of five individuals, providing 5, 9, 13 and 7 pooled samples from regions 1 to 4, respectively. The mean carapace length within each pool ranged between 26 and 35 mm. Cuticle and soft tissues were freeze dried in a VIRTIS lyophiliser, then crushed and homogenised to a fine powder in an agate bowl mill with a
Fig. 1. Map of sampling stations and delimited regions in the Gulf of Ca´diz fisheries of S. mantis.
J. Blasco et al. / Environment International 28 (2002) 111–116 Table 1 Certified and measured values of iron, manganese, zinc, copper, cadmium and lead in standard reference materials (expressed as microgram per gram dry weight) Metal
Certificate values DORM1 TORT1
Our values DORM1
TORT1
Fe Mn Zn Cu Cd Pb
63.6 ± 5.3 1.32 ± 0.26 21.3 ± 1.0 5.22 ± 0.33 0.086 ± 0.012 0.40 ± 0.12
54.3 ± 2.4 1.44 ± 0.19 20.3 ± 0.7 5.32 ± 0.29 0.082 ± 0.010 0.61 ± 0.14
176.4 ± 8.1 20.2 ± 0.5 171 ± 8 417 ± 13 26.2 ± 1.5 12.8 ± 3.2
186 ± 11 23.4 ± 1.0 177 ± 10 439 ± 22 26.3 ± 2.1 10.4 ± 2.0
The values are mean and 95% confidence intervals (n = 10).
Planetary Mono Mill (Pulverissette 6, Fristch). Samples were digested according to the procedure of Amiard et al. (1987). In summarized way, ca. 0.1 g of lyophilised crushed and homogenised tissues were digested with 1 ml HNO3 Suprapur (Merck) at 95 C for 1 h in a heater block. The resultant solutions were diluted to a known volume with Milli-Q water and the heavy metals Cd and Pb were analysed by graphite furnace atomic absorption spectrophotometry (GFAAS) (PE 4100 ZL), and flame AAS (PE 3110) for Fe, Mn, Cu and Zn. The results are expressed as microgram per gram dry or wet weight. The analytical procedure was checked using reference material (DORM1 and TORT1 of NRC Canada). The results are shown in Table 1. All statistical analyses, including regression analysis, ANOVA and ANCOVA, were carried out using the software package Statgraphic plus for Windows (Statgraphics, 1998). Data were log transformed before statistical treatment.
3. Results Table 2 gives the mean and range of concentrations of heavy metals (Fe, Mn, Zn, Cu, Cd and Pb) in the soft tissues and cuticle of all the S. mantis collected in the Gulf of Ca´diz. The highest values were found in the soft tissues for zinc, copper and cadmium and in the cuticle for iron, manganese and lead. The ratio [M]soft body/[M] cuticle ranged between 3.4 for Cd and 0.13 for Mn. In the case of
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lead, for 50% of the sampling stations, concentrations in cuticle and soft tissues of S. mantis were beneath detection levels ( < 0.05 mg g 1 dry weight). Iron, manganese, zinc, copper and cadmium concentrations in the soft tissue and the cuticle of S. mantis from the four regions stations of the Gulf of Ca´diz are reported in Figs. 2 and 3. Correlation analysis for tissue heavy metal concentration against carapace length, firstly for whole data set and secondly for data for each separate zone, showed that in both the total soft tissues and the cuticle a significant and positive linear correlation was reported for iron, while copper showed against length ( P < .05) in the cuticle only. In consequence, in order to establish differences between mean tissue metal concentrations of S. mantis from the different zones, ANOVA analysis was carried out for manganese, zinc and copper concentrations in the total soft tissues, and for cadmium and manganese in the cuticle. The remaining metal concentrations in the tissues were analysed by ANCOVA, where the carapace length was introduced as a covariate. In the total soft tissues (Fig. 2), Fe and Cd concentrations showed significant differences between zones ( P < 0.05). The lowest values were found in region one. For the cuticle (Fig. 3), only manganese showed a significant difference between zones, with the highest concentrations in zone 1. A multiple range test (LSD) was carried out to determine which zones were statistically different from each other. Thus for iron in the soft tissue, zone 1 was significantly different to zones 2 and 3, and zone 4– 3. In the case of cadmium, zone 1 was significantly different from the rest. For manganese in the cuticle, the multiple range test showed significant differences between zones 1 and 2, 2 and 4, and 2 and 3 at the P < 0.05 significance level. The mean concentrations were similar for copper and zinc in the soft body (136 and 135 mgg 1 dry weight, respectively) and the body concentration for Zn ranged between 64 and 104 mgg 1 dry weight. Iron and copper showed the highest values in the cuticle (105 and 84.8 mgg 1 dry weight, respectively). The mean copper concentration in the soft tissue, corresponding to the edible part, was 27.1 mgg 1 wet weight. Approximately 80% of stations showed values higher than 20 mgg 1 wet weight
Table 2 Heavy metal concentrations (means and ranges) in the soft tissues and cuticle (n = 14) crustacean S. mantis collected in the Gulf of Ca´diz Tissue Soft tissues Mean Range Cuticle Mean Range
Fe
Mn
Zn
Cu
Cd
Pb
77.89 36.70 – 133.14
4.24 0.54 – 7.18
134.97 118.36 – 154.84
135.47 60.24 – 202.41
1.66 0.91 – 2.13
0.19 < 0.05 – 0.84
104.90 55.25 – 207.22
32.40 13.59 – 55.31
48.36 33.65 – 73.61
84.85 58.20 – 102.89
0.49 0.31 – 0.67
0.33 < 0.05 – 2.65
* Results are expressed as microgram per gram dry weight.
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centrations of zinc in decapods typically lie in the range 50 – 120 mg Zn g 1 dry weight (White and Rainbow, 1982). This is the same as the range that we have reported in the studied species. The restricted range of Zn concentrations found and the lack of significant differences between zones indicate that S. mantis could, therefore, carry out Zn regulation in the soft tissues, in a similar way to that reported in the palaemonid decapod
Fig. 2. Heavy metal concentrations (means and standard deviations) in the soft tissues of S. mantis collected in four regions in the Gulf of Ca´diz. Results are expressed as microgram per gram dry weight.
of copper, the Spanish legal limit for the concentration of this metal in the crustacean for human consumption.
4. Discussion Information on the concentrations of trace metals in stomatopod crustaceans is scarce. Regulated body con-
Fig. 3. Heavy metal concentrations (means and standard deviations) in the cuticle of S. mantis collected in four regions in the Gulf of Ca´diz. Results are expressed as microgram per gram dry weight.
J. Blasco et al. / Environment International 28 (2002) 111–116
Palaemon elegans and the crab Carcinus maenas. (Rainbow, 1998). Although the net result is the same in both these species, the mechanisms involved in the homeostasis of Zn concentration differ between these decapods. P. elegans matches the rate of zinc uptake with the rate of zinc excretion (White and Rainbow, 1982; Rainbow and White, 1988), while C. maenas has such a low rate of zinc uptake rate under most zinc availabilities that body zinc concentrations remain unaltered (Rainbow, 1998). To establish the mechanism for any such zinc regulation in S. mantis, it will be necessary to carry out kinetic accumulation studies. With regard to copper, the variation in soft tissue copper concentrations in S. mantis was greater than for zinc, although no significant differences were present between zones and regulation of soft tissue copper concentrations could also be occurring. In decapods, the situation concerning the regulation of copper is less clear than for zinc, given the lack of a convenient radiotracer to allow investigation of the kinetics of accumulation (Rainbow, 1998). Molar ratios of copper and zinc were ca. 1000:1000. This ratio satisfies the theoretical Zn and Cu requirements of crustaceans (Zauke and Petri, 1993). In the case of Cd, the ratio Cd/ Cu (4.0:1000) was of the same order as that found for other marine crustaceans (Rainbow, 1998). Although size may affect the accumulated concentration in organisms (Phillips, 1980), we only found statistically significant relationships in the cases of at P < .05, except for copper in the cuticle, and iron in both cuticle and total soft tissues. This fact may be a consequence of the small size range of the crustaceans analysed. The concentrations of iron and manganese in the cuticle were higher than in the soft tissues and may be a consequence of the adsorption of these metals onto the crustacean exoskeleton This process has been highlighted previously for manganese in coastal decapods (Bryan and Ward, 1965). The positive correlation between manganese in the soft tissues and cuticle could indicate that the accumulation in this species, as in other coastal decapods, increases greatly if the conditions promote the deposition of manganese onto the cuticle (Rainbow, 1988), whereas for iron the behaviour is the opposite. In both cases, a significant amount of heavy metals on the exoskeleton is in an adsorbed form (unpublished data). Samples of this species previously collected in this area (Establier, 1975) showed Cd values around 2.5 –3.0 mgg 1 dry weight. These values are higher than in our study (Table 2), but the differences may be due to the smaller size of the specimens collected, analytical methods employed and/or the variation of levels of metal contamination over time. Iron and manganese showed higher values in this species than in another stomatopod, S. oratoria (Eisler, 1981) while the Zn concentration reported was similar. Copper levels exceeded the statutory limit of 20 mg g 1 wet weight and the Spanish authorities prohibited their sale.
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Other crustacean species (Palaemon longirostris and Uca tangeri) collected in the Guadalquivir River estuary in the same period after the Aznalco´llar mining spill, also exceeded the legal limits. Nevertheless, the prawn Melicertus kerathurus collected in the adjacent coastal area had copper concentrations in the range 6.7 –11.0 mg g 1 wet weight (Blasco et al., 1999). Specimens of S. mantis collected in the Mediterranean Sea (Tarragona and Alicante coasts) had lower concentrations (13.5 ± 3.0 and 17.0 ± 4.0 mg g 1 wet weight, respectively) (Rodrı´guez, personal communication). The higher values found in S. mantis from the Gulf of Ca´diz area could be related with the effect of variables such as season, size, reproduction/moult stage and also with ambient levels of bioavailable copper. With relation to the impact of the mining spill on the Guadalquivir River estuary and coastal adjacent area, the main input of heavy metal involved zinc. Thus, a 20-fold increase of Zn concentrations in the local sediment compared to background levels in unpolluted sediment has been reported (Palanques et al., 1999). This metal was bioavailable to local aquatic organisms for a significant increase of accumulated Zn concentrations in the oyster Crassostrea angulata has been reported (Blasco et al., 1999). Other heavy metals discharged in the aquatic environment as consequence of the toxic spill were retained within a temporary dam at Entremuros (before the confluence with the estuary). The copper levels in the estuary of the Guadalquivir River are traditionally high and green-sick (copper contaminated) oysters have been known for 30 years to be present along the banks at the mouth of the river (Establier and Pascual, 1974). The mining activity and the use of the compounds which containing this metal for fumigation activities (Querol et al., 1999) are the main sources. Analysis of heavy metal levels in the plume of Guadalquivir River estuary and the adjacent coastal area produced no evidence of the impact of mining spill after 6 months (Achterberg et al., 1999). This fact was attributed to human intervention and natural removal processes. The metal concentrations reported in S. mantis from the Gulf of Cadiz could be related to contamination from terrestrial mining activities (copper and pyrites) in the Gulf of Ca´diz present since the times of the Tartesians and Romans. Strong heavy metal anomalies have been detected on the continental shelf near the mouths of the Guadiana and Tinto-Odiel Rivers and on the continental slope (Palanques et al., 1995). A metal-enriched seawater plume entering the western Mediterranean Sea through the Gibraltar Strait originates in the Tinto estuary (Elbaz-Poulichet and Leblanc, 1996; Van Geen et al., 1997). Recently, Elbaz-Poulichet et al. (2001) have identified the occurrence of a metal enriched water mass in the Gulf of Ca´diz (Spanish Shelf Water, SSW), which proves that the rivers draining the Iberian Pyrite Belt and additionally the Guadalquivir River are the ultimate source of metal. In consequence, the levels reported in S. mantis probably reflect the contamination levels in the Gulf of Ca´diz resulting from the high metal
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fluxes from the Tinto and Odiel River estuaries and therefore cannot be directly and exclusively attributed to the Aznalco´llar mining spill.
5. Conclusions S. mantis appears to regulate soft tissue concentrations of zinc and its pattern of accumulation of this metal appears, therefore, very similar to those of decapods. However, studies of kinetic accumulation will be necessary to establish mechanisms involved in any such regulation. Copper levels in this species are high in the study area in comparison with other sites, this being related to background levels in this region resulting from inputs originating in the Rio Tinto sulphide mining district and not from the impact of the Anzalco´llar mining spill. The use of S. mantis for biomonitoring programs might not be adequate. Nevertheless, the risk of copper concentrations exceeding the maximum allowed under Spanish legislation for human consumption of seafood will require the determination of its levels periodically. An ecotoxicological assessment of heavy metals, especially for copper, may be necessary to establish the relation between body loads of contaminants and potential toxic effects, and in this way be used to derive tissue quality values.
Acknowledgments We wish to thank Prof. P.S. Rainbow for revising the manuscript and giving helpful comments, Mr. T. Ransome for language revision, Ms. I. Ferna´ndez and Ms. F. Osta for their technical assistance, and Mr. J. Santana for his collaboration in the sampling. This research was supported by the CSIC and Junta Andalucı´a (Oficina Te´cnica Corredor Verde).
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