Heavy metals in crustaceans from the Iberian deep sea plain

Trace metals (Cd, Cu, Pb and Zn) were analysed in different well-defined mixed copepod and euphausiid samples as well as in individually analysed decapods collected during a cruise on board “RV Walther Herwig III” to the Iberian Deep Sea Plain in March and April, 2002. We found a substantial interspecific heterogeneity with partially high mean Cd levels in the decapods: 1.9 mg kg-1 dry weight in samples of the genus Sergia; 6.1 mg kg-1 in Acanthephyra pelagica (Risso, 1816) and Acanthephyra acanthithelsonis Bate, 1888; 10.7 mg kg-1 in Bentheogennema intermedia (Bate, 1888); 14.9 mg kg-1 in Benthesicymus iridescens (Bate 1881) and 16.3 mg kg-1 in Systellaspis debilis (A. Milne Edwards, 1881). The agreement of these high Cd concentrations with those reported for polar crustaceans, and Cu concentrations (17–56 mg kg-1) that indicate that the total metabolic requirements have probably not been met, support the hypothesis that the frequently reported Cd-anomaly in polar crustaceans might be extended to other relatively deep ocean waters like the Iberian Deep Sea Plain and that this might be related to a corresponding Cu deficiency. Zn concentrations in decapods from this study (52–80 mg kg-1) are within the worldwide range, which indicates that decapods are able to regulate their Zn body concentrations. Our preliminary data on mixed zooplankton samples suggest that Pb concentrations far below 1 mg Pb kg-1 might serve as a regional background value for comparison in biomonitoring studies. Results for Cd, Cu and Zn are largely within the reported range, especially for data from the Central and Southern North Sea.

incorporated in organisms, because this is more dependent on species-specific uptake and detoxification mechanisms and metabolic requirements than on the concentration in the soluble phase ( Rainbow, 1988;Depledge, 1989;Ritterhoff and Zauke, 1998).As a result, we can frequently find different species of zooplankton with greatly varying metal concentrations in the same body of ocean water (Petri and Zauke, 1993;Ritterhoff and Zauke, 1997d;Kahle and Zauke, 2003).
In order to differentiate human impact from natural variability, knowledge of background concentrations of metals and their fluctuations in biomonitor organisms is essential as well as a thorough understanding of accumulation and detoxification strategies.These depend on various aspects, including the biological species and element considered, the applied exposure regime, cation homeostasis mechanisms, life-history status, spatial and temporal scales and other factors (Rainbow and White, 1989;Zauke and Petri, 1993).Studies on crustacean zooplankton from polar regions have revealed particularly high Cd concentrations (Petri and Zauke, 1993;Bargagli et al., 1996;Ritterhoff and Zauke, 1997d), suggesting that probably no global background values can be defined.Instead, regional background values should be established in order to follow possible future trends of metals in biomonitors.
That zooplankton play an important role in the biogeochemical cycling of metals in marine systems in general is well known, especially regarding particle-reactive metals in the water column (e.g.Fowler and Knauer, 1986;Fisher et al., 1991;Lee and Fisher, 1994).Since this was established, several attempts have been made to explicitly use macroand meso-zooplankton organisms as biomonitors for assessing the bioavailability of elements in marine systems, covering a variety of spatial and temporal scales (e.g.Zauke et al., 1996a;Ritterhoff and Zauke, 1997d;Kahle and Zauke, 2003).This is largely due to their worldwide presence, their major role in the food webs and their high contribution to the zooplankton biomass in marine systems.
Classically, studies on metals in biota are related to the species level because this level is implicitly regarded as highly relevant for describing the processes of metal metabolism.This is certainly true in basic physiological studies.For applied studies, however, we propose to select appropriate study units based on a straightforward calibration procedure, involving assessing toxicokinetic parameters (BCFs, rate constants), clarification of uptake, elimination and storage mechanisms and validation of invariable BCFs or rate constants under different exposure conditions (see discussions in Phillips and Rainbow, 1989;Rainbow and White, 1989;Petri and Zauke, 1993;Rainbow, 1993;Viarengo and Nott, 1993;Zauke et al., 1995;Zauke et al., 1996b).This calibration may eventually lead to considering levels below the biological species or to welldefined collectives of organisms above the species level (Zauke et al., 1996a).
Recently, an increasing body of information on metals in marine zooplankton has become available for specific regions such as the North Sea, the Nordic Seas and the Antarctic Ocean.In several studies unexpectedly high Cd concentrations in polar invertebrates were related to effective but unselective uptake mechanisms which had evolved due to potential metabolic deficiencies in Cu (Petri and Zauke, 1993;Bargagli et al., 1996;Demoreno et al., 1997;Bustamante et al., 1998 ).However, for areas such as the Iberian Deep Sea Plain recent information on metals in zooplankton is largely lacking.
Since early papers reported similarly high Cd concentrations in the decapod Systellaspis debilis from the East Atlantic Ocean (Leatherland et al., 1973;Ridout et al., 1985;Ridout et al., 1989) in comparison to polar regions (see above), we want to provide more detailed information on different decapods from the Iberian Deep Sea Plain to evaluate whether the "Cd-anomaly" is a phenomenon characteristic of polar waters or also common in other deep ocean waters.In addition, mixed meso-zooplankton collectives will be considered as the basis for future biomonitoring assessments, especially in comparison with the North Sea and polar waters.

Sampling and sample preparation
Samples were collected from FRV "Walther Herwig III" (cruise 238, March 15 to April 05, 2002) in two regions of the Iberian Deep Sea Plain at 11 stations (area B 45°50'N, 16°00'W -46°10'N, 17°30'W, area East-B 46°00'N, 13°00'W, see Fig. 1).Zooplankton samples were taken with a vertically towed plankton net (the so called 'Helgoländer Planktonnetz', a net originally used to catch ichthyoplankton, with a mesh size of 500 µm and a diam-eter of 1 m) which was hauled at 0.2 m sec -1 from depths of 100 and 800 m respectively.Decapod samples originate from catches with an Agassiz trawl (AGT) operated at depths of 4400 to 4800 m for 4 to 5 h.This trawl was used because the cruise was principally aiming at radio ecological investigations in the marine benthic system.Most probably, many specimens of decapods were caught accidentally at intermediate water depths when hauling the AGT, but more appropriate gear such as the rectangular midwater trawl (RMT) was not available.
On board the ship zooplankton from the 'Helgoländer Planktonnetz' was transferred to polyethylene buckets and kept alive in sea water collected from depths > 500 m for at least 1 to 3 h to allow for defecation.We assume complete defecation within this period as gut transit times in copepods are less than 0.5 h at 18°C (Reinfelder and Fisher, 1991).The animals were then sorted, still alive, under a binocular microscope, briefly rinsed with double-distilled water, dried on good-quality filter paper and stored at -18°C in Eppendorf caps (2 ml, polypropylene).In order to meet the minimum requirement of several milligrams dry weight required for further analysis, samples could only be sorted down to a group level (giving samples of small calanoid copepods, larger copepods of the family Euchaetidae and finally Euphausiacea), since more detailed biological assessment procedures were not applicable.This is mainly due to the small size of organisms and the relative species richness, which is comparable to that of the southern North Sea (see Zauke et al., 1996a), but contrasts with polar waters, where sorting of cope-pods and euphausiids down to the species level is no problem (Kahle and Zauke, 2003;Ritterhoff and Zauke, 1997d).
To assure the quality of the sampling procedure, we followed the guidelines set up in Zauke et al. (1996a) and Zauke and Petri (1993).The risk of contamination was largely reduced by keeping the animals in sea water or on high-quality filter paper whenever possible.

Analytical procedures
Upon arrival in the laboratory in Oldenburg, the frozen crustacean samples were subjected to freezedrying for 48 h to remove remaining water (LYOVAG GT2, Leybold Heraeus).Then the samples were homogenised using a small boron carbide mortar and pestle to avoid losses of biomass.Aliquots of about 10 mg dried material were digested for 3 h at 80°C with 100 µl HNO 3 (65 %, suprapure, Merck) in tightly closed 2-ml Eppendorf reaction tubes (Clason and Zauke, 2000).The digests were made up to 2 ml volume with double distilled water.
Metal determinations in biological samples were performed using a Varian SpectrAA 880 Zeeman instrument and a GTA 110 graphite tube atomiser with Zeeman background correction according to Clason and Zauke (2000) and Kahle et al. (2003).Ashing and atomisation temperatures were 600 and 1800°C for Cd, 1000 and 2200°C for Pb, 800 and 2300°C for Cu.For Cd and Pb, palladium and magnesium nitrate modifiers were applied.Zn was assayed using an air-acetylene flame (SpectrAA-30, deuterium background correction) and a manual micro-injection method (100 µl sample volume).All metal concentrations in biological tissues are reported in mg kg -1 (µg g -1 ) dry weight (DW).
Quality assurance was performed in line with German GLP regulations (Anonymous, 1999), using the following documented criteria: stability of instrumental recalibration, precision of parallel injections (normally showing a coefficient of variation of 1 to 5 %) and analytical blanks (also reflecting the digestion procedure).The precision and validity were evaluated using three certified reference materials which were randomly allocated within the determinations (see Table 1).Limits of detection were calculated according to Büttner et al. (1980).

Statistical procedures
The hypothesis of normal distribution was tested using the Lilliefors test (α = 0.01) provided in SYS-TAT for Windows (Version 10, Wilkinson, 2000).This program was also used to compute means and 95% confidence intervals (Wilkinson and Engelman, 2000) of metal concentrations in independent subsamples (viz.bulk samples of meso-zooplankton or specimens of decapods from the different sampling stations, regarded as measurement units).Thus, the experimental unit is the total study area within the Iberian Deep Sea Plain.
Further statistical evaluation was performed with BMDP Dynamics (Release 8.0 for Windows 2000, XP) using one-and two-way analysis of variance with data screening (Dixon, 1992).Firstly, global null hypotheses (equality of means between the taxa investigated) were tested either by classical ANOVA (assuming equality of variances) or by non-classical Welch Test (not assuming equality of variances).The adequate procedure was selected after testing equality of variances using the Levene Test.Null hypotheses were rejected at 95% significance level (P < 0.05).Secondly, heterogeneity was analysed in more detail using the Student-Newman-Keuls Multiple Range Test (NK) (α = 0.05).The robust NK procedure involves an adjusted significance level for each group of ordered means (Dixon, 1992;p. 585).BMDP outputs do not include values for the test statistic but only provide graphical information (that is, means which do not differ significantly are joined to groups by vertical bars).The advantage of this procedure is that results are readily available, in contrast for example to outputs of pairwise t-tests (adjusted to multiple comparisons), which would likewise have been adequate.

Quality assurance
The analysed values obtained for reference materials are in most cases in good agreement with the certified values (Table 1), especially regarding lobster hepatopancreas (TORT 2) and mussel tissue (CRM 278).Measured concentrations for cod muscle (CRM 422) are well below our calculated limits of detection, with the sole exception of Zn; the net result is that no relevant contamination has occurred even at low concentrations.Nevertheless, we report the measured values in Table 1 for comparison with the certified values, with the exception of Cd.Limits of detection proved to be adequate for the range of metal concentrations found in this study for marine crustaceans.

Interspecific heterogeneities
Results for metal concentrations in various crustaceans are shown in   2).
The highest concentrations of Cd were found for the decapods Bentheogennema intermedia, Benthesicymus iridescens and Systellaspis debilis (group 3) and the lowest concentrations (group 1) for euphausiids, both collectives of copepods and the decapods Sergia spec.and Acanthephyra pelagica / A. acanthithelsonis, for which results were pooled due to their rare occurrence.However, we found a substantial overlap of the NK groups, most probably due to the large intraspecific heterogeneities in the decapods.A fine separation of groups was also detected by the NK procedure for Zn with the highest values in both copepod collectives (group 2) and the lower values in the euphausiids and the decapods (group 1).For Cu and Cd no further interspecific hetero-  geneity could be discerned: regarding Pb, due to the generally low concentrations close to the limit of detection and regarding Cu, most probably due to the large intraspecific heterogeneity in the decapods analysed.The large confidence intervals obtained for the decapod samples not only reflect a high natural variability of single organisms but may also be caused by the bad state of many animals caught with the AGT.Since many trace metals are stored in detoxified form in certain organs like the hepatopancreas of decapods (Viarengo and Nott, 1993), damage to the relevant body part can result in decreasing and varying whole-body concentrations.However, since better material could not be collected during this cruise, we regarded this circumstance as acceptable in order to evaluate one of our main hypotheses regarding the Cd-anomaly (see below).Furthermore, determinations relying on single organisms, as in the case of decapod samples, are influenced by the high natural variability of metals in individual organisms due to varying life-history stages (such as body length) or the sex of the individuals ( Rainbow, 1988;Ridout et al., 1989;Zauke and Petri, 1993;Ritterhoff and Zauke, 1997c).
An a posteriori quality control of sample collection according to Zauke and Petri (1993) can be inferred from our results compiled in Table 2.A successful quality control of the analytical procedure (see previous section) is probably not the most crucial feature in determining metal concentrations in organisms from remote areas.In contrast to the widely accepted concept of "good laboratory practice", there are no straightforward protocols available for quality control of sampling and sample preparation on board ship.Therefore we can only present arguments based more or less on plausibility.Regarding individual organisms collected on one particular cruise, it is very likely that a possible contamination would affect all samples (e.g.due to contaminated water or dust or equipment), or some individual organisms (not species or measurement units) by chance, e.g.due to residues of paints or grease.Pb concentrations close to the limit of detection in almost all cases, as well as the pronounced interspecific heterogeneity found for Cd (starting at low concentrations in euphausiids), demonstrate that on board contamination was very unlikely in this study, as in previous investigations (Petri and Zauke, 1993;Zauke et al., 1996a;Ritterhoff and Zauke, 1997d).

Hypothesis of a Cd-anomaly and Cu-deficiency inferred from literature data
Heavy-metal concentrations in various marine pelagic crustaceans and in zooplankton reported in the literature are compiled in Table 4. High Cd concentrations found in this study (Table 2) for Systellaspis debilis are in good agreement with data reported for the same species from the North East Atlantic ranging from 5.7 to 32 mg Cd kg -1 .Conversely, our values found for Acanthephyra pelagica and A. acanthithelsonis which show similar metal concentrations, clearly exceed the ranges reported for Acanthephyra exima and A. purpurea.
Our results for Cd in decapods coincide with the Cd-anomaly reported for many invertebrates from polar waters, which has been intensively discussed in the literature (Petri and Zauke, 1993;Zauke and Petri, 1993;Bargagli et al., 1996;Demoreno et al., 1997;Ritterhoff and Zauke, 1997d;Bustamante et al., 1998).Since these regions are considered to be rather isolated, high Cd concentrations in Antarctic crustaceans have not been related to anthropogenic influences but are regarded as indications of a Cu deficiency (Petri and Zauke, 1993;Zauke and Petri, 1993).It has been hypothesised that a potential copper deficiency might be related to an increased uptake of Cd due to insufficient selectivity of the uptake process for the essential element Cu.Theoretical considerations have suggested metabolic requirements for decapods from temperate waters with about 7 to 15 mg kg -1 of Cu being sufficient for enzymatic requirements and 30 to 40 mg kg -1 meeting the total metabolic (enzymatic and haemocyanin component) demand (Rainbow, 1993).Thus, some of the decapod species analysed from polar regions (Table 4), and even more those from the Iberian Deep Sea Plain (Table 2), might be suffering Cu deficiencies in the sense that their total metabolic requirements have probably not been met.
Zn concentrations in decapods of this study (Table 2) are within the range reported for comparable organisms in the literature (Table 4).These results support the conclusion of Rainbow and White (1989) that decapods are able to regulate their Zn body concentrations, probably leading to wholebody concentrations that are comparable worldwide.Pb in decapods is generally low, mostly below 1 mg kg -1 and at times close to the limit of detection.
Our results for mixed copepods and copepods of the family Euchaetidae are largely within the report-ed range for Cd, Cu and Zn, especially where data from the Central and Southern North Sea are concerned.Only Cd concentrations reported from the Weddell Sea (Table 4) and from the Northern North Sea (11 mg kg -1 ; Zauke et al., 1996a) are distinctly higher.The latter were attributed to upwelling phenomena in this region.For Pb, we found distinctly lower concentrations in copepods from the Iberian Deep Sea Plain, probably indicating some regional background value.This idea is supported by the following arguments: Pb mainly enters the marine system by atmospheric deposition, so that the Pb concentration in the soluble phase is highest in the euphotic zone, decreasing with increasing depths, in contrast to other trace metals, where depth distributions are closely related to those of nutrients.This is the reason for increased Cd availabilities in upwelling areas (see above).Moreover, Pb concentrations in seawater were found to be higher in parts of the North Sea surrounded by highly industrialised areas (40-60 ng l -1 , OSPAR Commission, 2000b) compared to the open Atlantic (5-20 ng l -1 , OSPAR Commission, 2000c) with predominant west winds coming from the west Atlantic, or compared to the rather isolated Arctic Ocean (4-15 ng l -1 , OSPAR Commission, 2000a).This might account for observed regional differences of Pb in copepods and perhaps in decapods.
Even if Cu requirements for copepods are considered to be similar to those of decapods (which has to be proven in future studies) they must be lower, because copepods, and also some hyperiid amphipods like Themisto, lack haemocyanin (Gruner et al., 1993).This might also help to explain the observed low Cu concentrations in copepods as opposed to decapods.
Our data for euphausiids in the Iberian Deep Sea Plain (Table 2) are largely within the reported range for Cu, Pb and Zn.Only for Cd do we find somewhat increased values compared to northern waters, but much lower ones compared to the Weddell Sea (Table 4).Only limited information is available from our study on pelagic amphipods of the Iberian Deep Sea Plain, namely the ranges 4.1 to 31.0 mg Cd kg -1 ; 4 to 17 mg Cu kg -1 ; 1.0 to 2.6 mg Pb kg -1 and 26 to 260 mg Zn kg -1 .These values are again within the reported range for other pelagic amphipods (Table 4).These observations probably  indicate a more general occurrence of accumulation strategies (see arguments given above for decapods) -especially for Cd, for which efficient detoxification mechanisms have been described for Themisto libellula and Themisto abyssorum from the Greenland sea, suggesting that Cd-binding proteins like metallothionein may play a major role in this process (Ritterhoff and Zauke, 1998).Despite the large intraspecific variability observed for individually analysed decapods, our study provides further evidence that the frequently reported Cd-anomaly in polar crustaceans might be extended to other deeper ocean waters like the Iberian Deep Sea Plain and that this might be related to a corresponding Cu deficiency.This could be the consequence of efficient uptake mechanisms for Cu which cannot discriminate between the two elements.To test this hypothesis, further experimental studies are required, investigating in more detail, for example, the role of metal-binding proteins in this process.Our preliminary data on mixed zooplankton samples suggest that Pb concentrations below 1 mg Pb kg -1 might serve as a regional background value for comparison in biomonitoring studies.This should be further evaluated by toxicokinetic bioaccumulation studies on board ship as previously reported for polar waters (Ritterhoff andZauke, 1997a, 1997b;Kahle andZauke, 2002a, 2002b).

TABLE 1 .
-Quality assurance using certified reference materials randomly allocated within the determinations.Values are means ± 95% CI [mg kg -1 DW].Numbers of independent determinations: a 7; b 10; c 11; d 16; others: 12; limits of detection (calculated as 2.Cu and Zn due to the results of the Welch or F statistics, but not for Pb (Table3).Subsequent analyses with the Student-Newman-Keuls Multiple Range Test revealed different groups for Cd and Zn but not for Cu and Pb (Table