Metal concentrations in the tissues of the hydrothermal vent mussel Bathymodiolus: Reflection of different metal sources

Marine Environmental Research 95 (2014) 62e73

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Metal concentrations in the tissues of the hydrothermal vent mussel Bathymodiolus: Reflection of different metal sources Andrea Koschinsky a, *, Matteo Kausch a,1, Christian Borowski b a b

Jacobs University Bremen, School of Engineering and Science, P.O. Box 750561, D-28725 Bremen, Germany Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D-28359 Bremen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 December 2009 Received in revised form 23 April 2013 Accepted 16 December 2013

Hydrothermal vent mussels of the genus Bathymodiolus are ideally positioned for the use of recording hydrothermal fluxes at the hydrothermal vent sites they inhabit. Barium, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Pb, Sr, and U concentrations in tissue sections of Bathymodiolus mussels from several hydrothermal fields between 15
N and 9
S at the Mid-Atlantic Ridge were determined and compared to the surrounding fluids and solid substrates in the habitats. Elements generally enriched in hydrothermal fluids, such as Fe, Cu, Zn, Pb and Cd, were significantly enriched in the gills and digestive glands of the hydrothermal mussels. The rather small variability of Zn (and Mn) and positive correlation with K and earth alkaline metals may indicate a biological regulation of accumulation. Enrichments of Mo and U in many tissue samples indicate that particulate matter such as hydrothermal mineral particles from the plumes can play a more important role as a metal source than dissolved metals. Highest enrichments of Cu in mussels from the Golden Valley site indicate a relation to the 400
C hot heavy-metal rich fluids emanating in the vicinity. In contrast, mussels from the low-temperature Lilliput field are affected by the Fe oxyhydroxide sediment of their habitat. In a comparison of two different sites within the Logatchev field metal distributions in the tissues reflected small-scale local variations in the metal content of the fluids and the particulate material. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Bathymodiolus mussels Hydrothermal vent Heavy metals Tissue partitioning Seawater Sediment Mid-Atlantic ridge

1. Introduction Marine invertebrates, such as bivalves, are exposed to a range of concentrations of dissolved and particulate trace metals in their environment. Uptake and accumulation of these metals in different body tissues depends on a number of factors, such as metal speciation and bioavailability, and different strategies of the organisms to handle metal exposure, including storage by metalbinding proteins or excretion (Deplede and Rainbow, 1990). In contrast to the large amount of data on trace metals in coastal mussels and their relationship to the geochemical environment (e.g., Burger and Gochfeld, 2006; Goldberg et al., 1978; O’Connor and Lauenstein, 2006), only a few studies regarding metal concentrations in hydrothermal vent Bathymodiolus have been carried out (e.g., Smith and Flegal, 1989; Rousse et al., 1998; Kádár et al., 2006a,b, 2007; Colaço et al., 2006; Cosson et al., 2008). In these

* Corresponding author. Tel.: þ49 421 200 3567. E-mail address: [email protected] (A. Koschinsky). 1 Present address: Cradle to Cradle Products Innovation Institute, San Francisco, USA. 0141-1136/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2013.12.012

studies metal concentrations in the soft tissue, subdivided into different tissue parts, were investigated. The concentrations in the gills were generally found to be increased with respect to the remaining tissue. Just like for mussels from non-hydrothermal environments, differences in tissue metal concentrations of the mussels were suggested to be due to differences in the fluid (physico-)chemistry of their environment (Rousse et al., 1998). Mussels from different hydrothermal vent sites show differences in their metal enrichments, reflecting differences in environmental parameters (Cosson et al., 2008). However, the interpretation of metal accumulations in the tissues may not be very straightforward because hydrothermal Bathymodiolus are thought to possess special adaptations that help them deal with high environmental metal loads (Smith and Flegal, 1989; Rousse et al., 1998; Geret et al., 1998; Hardivillier et al., 2004; Kádár et al., 2006a,b; Hardivillier et al., 2006). This may lead to ambient metal concentrations being reflected in the organic tissue in a different manner than in their littoral relatives. Also, the fact that Bathymodiolus mussels can live mixotrophic (i.e., they take up nutrients from bacterial endosymbionts in their gills and somtetimes also by feeding) may have an impact on metal bioaccumulation. Thus, there is the need to extend corresponding studies on hydrothermal mussels.

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Comparing data from the work that so far has been carried out on metal accumulations of Bathymodiolus mussels (Smith and Flegal, 1989; Rousse et al., 1998; Kádár et al., 2005, 2006a,b; Colaço et al., 2006; Cosson et al., 2008) is hampered by the fact that these studies partly differ in setup, method, species and metals investigated. Smith and Flegal (1989) found that the As, Fe, Se and Zn concentrations in the Bathymodiolus thermophilus specimens they analyzed were comparable to those found for coastal Mytilus edulis in other studies, despite the elevated concentrations found in their habitats. Similarly, Kádár et al. (2006a) found that the concentrations of Al, Cd and Co they measured in Bathymodiolus azoricus specimens were partially below those reported for nonhydrothermal mussels from contaminated sites. They also found highly significant differences between the metal concentrations of B. azoricus sampled from different, geochemically distinct sites. These results were essentially confirmed in a comparative study of metal bioaccumulation in B. azoricus in different Mid-Atlantic Ridge (MAR) vent fields, with indications that also within a single vent field, mussels taken at different sites reflect the conditions specific for this location (Cosson et al., 2008). In a similar approach, shrimps from hydrothermal and estuarine environments were found to reflect local peculiarities (Gonzales-Rey et al., 2008). As to be expected, in none of the studies were the relationships between metal accumulation in the mussel tissue and the environmental parameters straightforward. Furthermore, when comparing studies on Bathymodiolus mussels, it has to be taken into account that even between species of the same genus, metal accumulations may vary. Here we present data for metal concentrations in different tissue sections of Bathymodiolus sampled from several hydrothermal locations at the Mid-Atlantic Ridge, which represent a wide range of very specific conditions including different potential metal sources, such as sediment, fluids, and sulfide particles. The chosen sites are the ultramafic-hosted Logatchev hydrothermal field at 15
N on the Mid-Atlantic Ridge (MAR), the Golden Valley diffuse-flow hydrothermal site in the area of basalt-hosted hot vents at 5
S on the MAR and the Lilliput hydrothermal field, a low-temperature diffuse-flow field at 9
S on the MAR. Tissue sections (gills, digestive gland, foot) were analyzed for Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Pb, Sr, Zn, and U. The goal of this study was to assess the role of different dissolved and particulate metal sources in various hydrothermal settings for the bioaccumulation in the tissues of Bathymodiolus, while evaluating the possibilities and challenges of using metal accumulations in the mussel tissues as proxies for hydrothermal metal fluxes in the seawater-fluid mixing zone. While previous studies on metal accumulations in Bathymodiolus species of the Mid-Atlantic Ridge were, to our knowledge, carried out largely on B. azoricus, this is the first substantial set of data for B. puteoserpentis from Logatchev and two closely related Bathymodiolus species that were recently discovered at 5
S and 9
S on the southern MAR (van der Heijden et al., 2012).

2000; Petersen et al., 2009). The vent field is ultramafic-hosted, its endmember fluid chemistry is well known and the fluid composition has been stable for more than 10 years (Schmidt et al., 2007; Schmidt et al., 2011). Bathymodiolus puteoserpentis mussels and diffuse hydrothermal vent fluids were sampled from the hydrothermal vent complexes Irina II and Quest. Irina II is a 15-m high and 50-m wide unsedimented mound of ultramafic rock talus with an active black smoker complex on its top exiting 340
C hot fluids (Fig. 1a). The chimney complex is surrounded by many sites of diffuse outflow and extensive mussel beds (Fig. 1a,b). Quest is a 10 m wide “smoking crater” complex (Fig. 1c) that vents fluids of up to 347
C (measured in 2007, unpublished data) from small but vigorously active chimneys sitting on hydrothermal sediment, serpentinite and chimney talus. The smoking crater is surrounded by silicified crusts at its rim and larger patches of hydrothermal sediment in the wider periphery. A number of fissures in the rim crust release seawater-diluted fluids supporting patches of B. puteoserpentis. Animals and fluids used in this study were collected from a 1  2 m wide mussel patch bordering a sedimentary setting some 20 m apart from the smoking crater (Fig. 1d). The basalt-hosted 5
S vents are characterized by several scattered smoker complexes discharging fluids of up to 407
C and neighboring sites with diffuse outflow (Haase et al., 2007; German et al., 2008; Koschinsky et al., 2008). Bathymodiolus sp. A and diffuse fluids were collected in the Golden Valley vent site that is marked by a large mussel bed stretching for some 30 m in a 4-m deep and up to 5-m wide basaltic fissure (Fig. 1e). Golden Valley did not harbor focused emanations of hot fluids at the times of sampling in 2005, and the in situ temperatures in the mussel bed were around 3.9
C. However, sampling of basalts in the mussel bed by an ROV in 2009 created an exit for black smoke indicating that hot fluids are present in only a few centimeters below the basaltic surface. The 9
S area is also basalt-hosted and several low-temperature vent fields of various sizes have been discovered. The Lilliput field is located at 9
33’S in a large young lava flow which is partly covered by hydrothermal Fe-oxide/hydroxide sediments. The Lilliput fauna is strongly dominated by small Bathymodiolus sp. B (90% < 30 mm shell length) aggregating on pillows and around lava cracks suggesting a recent reactivation of the hydrothermal fluid flow (Haase et al., 2009). The samples for this study were collected discretely from various spots on or between pillows that were covered with thick orange-red Fe oxyhydroxide layers (Fig. 1f). Sampling of mussels at all sites was complimented by sampling diffuse hydrothermal fluids with the ROV-operated KIPS fluid sampling system (Garbe-Schönberg et al., 2006). Sediments were collected with pushcorers handled by the ROV Quest (MARUM) at sites Quest and Lilliput.

2. Materials and methods

Mussel specimens were kept in chilled seawater and either dissected alive soon after recovery and the dissected material was separately frozen at 20
or animals were entirely frozen, transported on dry ice to the home laboratory and dissected after thawing. After opening the shells, the byssus thread was carefully removed to avoid contamination (see Gundacker, 1999; Kádár et al., 2006b). Tissue fractions of entirely thawed animals were further processed immediately after dissection. Gills, digestive glands and foot tissues from adult specimens from Irina II, Quest and 5
S were analyzed individually. In the small Lilliput specimens, only the gills provided enough material for individual analyses, while analyses of foot and digestive gland were performed with material pooled from four animals. The tissue samples were again frozen, lyophilized in a dry-freezer for 24 h and homogenized in a quartz mortar prior to digestion.

2.1. Sampling and sample-sites The material used in this study was collected along the MAR in the Logatchev hydrothermal vent field (LHF) in 3000 m water depth at 15
N and in the vent fields Golden Valley in 3000 m water depth at 5
S and Lilliput in 1500 m at 9
S using the ROVs Quest, Jason II and Kiel 6000 during cruises M64/1 in 2005, M68/1 in 2006, Atalante-Leg2 MARSUED IV in 2008 and M78/2 in 2009. The LHF provides excellent opportunities for studying different metal sources in hydrothermal mussels because it harbors a variety of focused and diffuse vents with various fluid flow conditions and well described biological assemblages (Gebruk et al.,

2.2. Sample preparation

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A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

Fig. 1. (a) Irina II sulfide mound in the Logatchev field, 15
N, showing intense colonization by Bathymodiolus puteoserpentis; (b) Diffuse hydrothermal outflow at the base of Irina II, where the mussels for this study where sampled; (c) Smoking crater Quest in the Logatchev field; (d) Sediment site close to crater Quest, from where mussels and sediments were taken for this study; (e) Bathymodiolus mussel beds sitting on relatively fresh lava in a fissure of the Golden Valley at 5
S; (f) Beds of juvenile Bathymodiolus mussels on Fe oxyhydroxide crusts in the low-temperature Lilliput field at 9
S.

2.3. Sample analysis Approximately 0.25 g of each prepared mussel tissue sample was weighed into the Teflon-bombs of a Picotrace digestion system. In each digestion series, NIST SRM 2976 mussel tissue standard reference material and a method blank were included. All acids used were of Suprapure grade (Merck). 5 ml of 65% HNO3 were added to each vessel and the acid was left to react with the organic material at room temperature for about 2 h, followed by the addition of 1 ml of suprapure H2O2. Then the Picotrace system was set to 75
C for about 24 h. After addition of 5 ml of 70% HClO4, the samples were allowed to react for another 3e4 h before the pressure plates were mounted and the system was set to heat up to 175
C over 2 h and to hold at that temperature for 12 h. Finally the acid was evaporated followed by two further evaporations at 170
C after the addition of 5 ml of 30% HCl. Following the last evaporation, the samples were taken up in 0.5 M HNO3, transferred into acid cleaned PET-bottles, diluted gravimetrically to a total mass of 10 g,

yielding dilution factors of the order of 40, and refrigerated until analysis. All samples were spiked with 10 ppm of Y as internal standard prior to analysis. The analyses of major and minor metals in the digested tissue material were carried out with a Spectro AS 500 ICP-OES by triplicate measurements and external calibration method. The accuracy of the method was validated by the determination of NIST SRM 2976 mussel tissue standard reference material. Recoveries were generally within 10% of the referenced values, suggesting that the proposed method was feasible. The elemental concentrations in the method blanks were below the detection limit, except for Ca and K, for which, however, the concentrations in the blank lay several orders of magnitude below all sample values. Trace metals including Ba, Co, Pb, Cd, Mo, and U were measured by ICP-MS (Perkin Elmer 500 DRCe). Measured Co, Cd, and Pb data of the NIST SRM 2976 standard matched the certified values very well (4%). Ba, Mo and U are not certified, but ICP-MS data of two Mo isotopes agree well with each other and with our ICP-OES data

A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

for Mo (with a deviation of <9% for Mo concentrations >10 mg/kg). For Ba, data of the two measured isotopes Ba-135 and Ba-137 agree with a deviation of <6%. For verification of the U data, soil reference material was used. Diffuse hydrothermal fluid samples were analyzed on board after filtration (0.2 mm pore size) by voltammetry after UV digestion using a Metrohm 757 VA Computrace, or by photometry (Fe). Cu, Zn, Cd, and Pb were analyzed by anodic stripping voltammetry in an acetate buffer medium, while Mn was determined by anodic stripping voltammetry in an alkaline ammonia buffer solution. Iron was determined as a ferroin complex with 1,10-phenanthroline in a pH after reduction to Fe(II) with ascorbic acid. Hydrothermal sediment samples were analyzed by ICP-OES and ICP-MS after acidpressure digestion with a mixture of Suprapure (Merck) HCl, HNO3 and HF. 3. Results Metal concentrations in gills, digestive glands and foot tissues were analyzed in a total of 27 individuals (Fig. 2), with reported values referring to the dry weight of the tissue sample. Gills and digestive glands showed for most metals higher concentrations than foot tissues. The metal concentrations within the tissue fractions varied between locations and also between individuals within the same sites. However, all metal concentrations measured in this study fell within the ranges previously observed in mussels from other vent sites (Table 1) as there is also considerable variability among the limited amount of published data. Most other publications concentrated on a very limited number of elements and only very little if any information at all exists on the bioaccumulation of Cr, Mo, Pb, and U in Bathymodiolus. 3.1. Metal partitioning between tissue types Fig. 2 depicts the average distributions between the tissues for individual elements. For the heavy metals, the most highly enriched elements are Fe (up to 3300 mg/kg) and Cu (up to 1300 mg/kg), followed by Zn (up to 320 mg/kg), Mo (up to 123 mg/ kg) and Pb (up to 43 mg/kg). The concentrations of Cr, Cd, Co, Mn and U were mostly <10 mg/kg in all tissues. The highest enrichments of Cu, Zn and Pb were found in the gills. Fe concentrations were usually highest in the digestive glands, except for animals from Golden Valley. Also for Co, values in the digestive glands appear to be higher than in the other tissues, however, as all values were consistently <1 mg/kg, Co will not be discussed any further. Zn concentrations vary less between different tissues and sites compared to most other heavy metals and partly resembles rather the behavior of the major elements and Sr. This is noteworthy because chalcophile elements are usually enriched in both hydrothermal fluids and particles. Bioaccumulated Zn should therefore come from the same environmental source as the other chalcophile elements Fe, Cu, and Cd. Manganese was generally low in all tissues (around 5 mg/kg) except for significantly higher values in the gills of the mussels from Golden Valley. Chromium (between <1 and 4 mg/kg) seemed to follow the trend of Fe, while for Cd the variability was larger (between <1 and 26 mg/kg) and highest concentrations were found either in the gills or the digestive glands. Molydenum, an element which is depleted in hydrothermal fluids but may be enriched in sulfide particles, was found to be strongly enriched in the gills and digestive glands of mussels from Lilliput and Golden Valley. Similarly, U, which is also mostly depleted in hydrothermal fluids compared to seawater, often showed higher concentrations in digestive gland and gill than in foot. Especially for the Lilliput mussels, there was an apparent

65

correlation of high Fe concentrations with enrichments of Cr, U, and Mo in the digestive gland. The elements of the alkaline/alkaline-earth metals group were lowest in the digestive glands and somewhat higher in the gills than in the foot. Calcium, Mg, and Sr show correlated differences between the vent sites in the data sets of the gills. Barium (<1e 11 mg/kg) and Sr (8e52 mg/kg) are trace components, while Ca, Mg, and K are major components in the range of a few thousand mg/kg. Except Ba, these elements have their highest concentrations in the gills and the lowest in the digestive gland. 3.2. Differences of metal accumulations between the areas In the following, differences between hydrothermal vent sites will be highlighted (Fig. 2). At Logatchev Irina II, Fe concentrations in the gills and digestive gland were significantly higher than in all other tissues of all other sites including the other Logatchev site Quest. This was also observed for Zn in the gills, and Cd in the glands and foot. Quest mussels have by far the highest Pb concentrations, especially in the gills, and highest Ba concentrations. In both Logatchev sites, the Cu concentrations were at the lower end of the range in all tissues. Golden Valley mussels showed high Cu values in all tissues and their gills had the highest Cu values measured. In these mussels, Zn was also slightly enriched, while Fe contents were comparably low. In Lilliput animals, the digestive glands were characterized by highest contents of Cd, Mn, Sr, and Ca. In Lilliput animals, the digestive glands were characterized by relatively high Fe, Cr, and Cd contents, and also Cu and Mo were higher in the gills and glands of the Lilliput mussels than of the Logatchev mussels. 3.3. Relationships between elements Although the number of analyzed animals was rather small due to sampling constraints, several relationships between two or more elements could be observed. For the heavy metals, there is no single dominating relationship with any other element. This is demonstrated here for the element pairs FeeCu and FeeZn (Fig. 3); the tissues mostly have either high concentrations of Fe, or of the other elements. Breakdown of the bulk data set into tissue types indicates that values with high to very high Fe contents at low Cu and Zn contents originate from digestive glands, while data with high Cu and Zn contents at lower to moderate Fe concentrations refer to the gills. The values of the foot are generally at the lower end for all elements. For the alkaline/alkaline earth metal group, there is a clear positive correlation between the different elements, as shown for the examples of CaeSr, CaeMg, and KeMg in Fig. 3. Interestingly, also Zn shows a positive trend with Mg (Fig. 3) and Ca, which is not observed for any other heavy metal. These relationships seem to be valid irrespective of the tissue type (except for ZneMg in foot tissue). 4. Discussion 4.1. General element partitioning The partitioning behavior of chalcophile elements in the Bathymodiolus puteoserpentis mussels studied in this report mostly matches what has previously been observed for B. azoricus (Table 1). While the general enrichment of chalcophile elements (Cu, Cd, Zn, Pb, and Fe) in the gills and digestive gland is well established, there seem to be local differences in the partitioning between these two tissues. Interestingly, Zn concentrations in the mussel tissues vary over a smaller range than the other chalcophile elements in this and in

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A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

Fig. 2. Average concentrations and standard deviations for elements measured in the different mussel tissues from the four different hydrothermal locations.

A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

67

Fig. 2. (continued).

other studies and show a stronger relationship to K, Mg, and Sr (Fig. 3), than to Cu, Cd, and Fe in many sample groups. Also compared to other studies (Table 1), Zn concentrations are in a comparable range in contrast to most other elements such as Cu and Fe, which may vary over orders of magnitude between study sites. However, the Zn and Cu concentrations in the different sources are both highly variable in the sediment, sulfide structures and fluids (Table 2). Together with a distribution pattern of Zn comparable with Mg and K, this could be seen as a certain biological regulation of the accumulation of the essential element Zn. Similarly, Mn concentrations do not display significant differences between tissue types and locations, although Mn concentrations are highly variable in hydrothermal fluids in a range of up to mg/kg. This may hint at a similar regulation mechanism for the micronutrient Mn as for Zn, or at the absence of Mn-rich particles at most hydrothermal sites, as will be discussed in more detail in paragraph 4.3. Support for a possible bioregulation of Zn (and Mn) accumulation by the mussels comes from studies on littoral mussels. Klerks and Fraleigh (1997, and references therein) discuss the different behavior of Zn compared to other elements in the context of regulation of internal Zn levels by the mussels and missing correlation of Zn concentrations and water levels in other studies were

mentioned. Experiments with the mussel Perna perna gave similar Zn and Mn concentrations in the soft tissues of experimentally contaminated mussels as of the reference mussels from their natural unpolluted habitat (Bellotto and Miekeley, 2007). Even for these mussels from a habitat that is very different from the hydrothermal habitat investigated in our study, the average Zn and Mn concentrations were in a similar range as in our study (Zn 119 mg/kg, Mn 11 mg/kg). The authors also explain the behavior of the two elements by their essential function and strong regulation mechanisms, which are apparently efficient even in highly contaminated environments. Correspondingly we conclude, that such regulation mechanisms may partly explain the Zn and Mn distributions in hydrothermal mussels. As for the other, non-chalcophile elements, for Cr, which is typically not strongly enriched in hydrothermal fluids, no clear accumulation patterns (except for higher values in Lilliput mussels) can be observed, probably reflecting a rather low content of Cr in the environment. As U and Mo are mostly even depleted in hydrothermal fluids compared to ambient seawater (see Table 2a), their partial enrichment in the gills and digestive gland can only be attributed to either inclusion of particulate sulfides (especially for Mo, see Section 4.3), or to uptake from ambient seawater, as these

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A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

Table 1 Comparison of average metal concentrations (in mg/kg dry weight in the soft tissue) in gill and digestive gland tissues of Bathymodiolus puteoserpentis and Bathymodiolus sp. mussels from this study with data for other Bathymodiolus species from other publications. Hydrothermal site

Species

Tissue

Cd

Cr

Cu

Fe

Pb

Mn

Logatchev, Irina II Logatchev, Irina II Logatchev, Quest Logatchev, Quest 5
S, Golden Valley 5
S, Golden Valley Lilliput Lilliput Snake Pit* Snake Pit* Menez Gwen Menez Gwen Menez Gwen Menez Gwen Menez Gwen Menez Gwen Menez Gwen Rainbow Rainbow Rainbow Rainbow Rainbow Rainbow Rainbow Rainbow Lucky Strike Lucky Strike Lucky Strike Lucky Strike Lucky Strike Galápagos Rift Galápagos Rift

B. puteoserpentis B. puteoserpentis B. puteoserpentis B. puteoserpentis Bathymodiolus sp. Bathymodiolus sp. Bathymodiolus sp. Bathymodiolus sp. B. puteoserpentis B. puteoserpentis B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. azoricus B. thermophilus B. thermophilus

Gills Dig. gland Gills Dig. gland Gills Dig. gland Gills Dig. gland Gills Other organs Gills Other organs Gills Dig. gland Gills Dig. gland Whole tissue Gills Other organs Gills Dig. gland Gills Dig. gland Gills Whole tissue Gills Gills Dig. gland Gills Whole tissue Gills Dig. gland

2.8 11.1 6.5 1.4 9.7 1.3 2.3 7.7 63.9 9.0 1.2 2.3 3.2/7.4 4.0/2.3

0.77 1.52 1.13 1.11 1.46 0.96 2.37 3.97 2.35 0.26 1.05 0.34

757 1495 361 403 352 196 280 830 37459 5871 233 124 183/206 198/200

6.8 1.2 29.7 6.6 10.9 1.4 1.6 10.3 408 10.8 16.3 17

4.8 4.3 6.7 6.7 15.2 3.8 3.2 4.5 102.5 10.1 5.1 2.8 6.3/4.8 3.8/2.1

1.4 1.3 1.8 1.5

4.66 2.56

187 23 148 30 352 53 290 91 975 135 124 24 57/130 30/172 47 22 41 424 20 52 11 67 18

A A B B

47.2/17.6

80/109 70 52 250 226

120 4240 1231 2066 1854

21 5.9

Mo 1.0 1.4 4.2 1.2 36.7 7.1 72.0 86.6

25.4 16.5 9.1 8.1

4000 2700 361/488

9.5/7.2

600 310 948

111 26

131 159

Zn

References

187 75 118 80 169 104 126 65 57090 1781 162 77 168/197 82/111 111 40 180 229 94 106 45 106 80

This study This study This study This study This study This study This study This study Demina and Galkin (2008) Demina and Galkin (2008) Demina and Galkin (2008) Demina and Galkin (2008) Cosson et al. (2008) Cosson et al. (2008) Colaço et al. (2006) Colaço et al. (2006) Kádár et al. (2007) Demina and Galkin (2008) Demina and Galkin (2008) Cosson et al. (2008) Cosson et al. (2008) Colaço et al. (2006) Colaço et al. (2006) Kadar et al. (2006) Kádár et al. (2007) Cosson et al. (2008) Colaço et al. (2006) Colaço et al. (2006) Kádár et al. (2006a,b) Kádár et al. (2007) Smith and Flegal (1989) Smith and Flegal (1989)

140 1977/768 555 201 500 400 217 61

Note *: Data for Snake Pit come from only 1 sample.

two elements have rather high concentrations in deep seawater (8 mg/kg Mo and 4 mg/kg U, Table 2a) compared to other elements. The positive correlation of alkaline-earth metals in the mussel tissues (Fig. 3) is consistent with relationships in freshwater mussel species, in which a comparative accumulation of alkaline-earth metals was observed and Ca concentration was shown to be a highly significant predictor for the concentrations of Mg, Sr, and Ba (Jeffree et al., 1993). The similar trends of this element group (together with K) in the hydrothermal mussels can only partly be explained by their coherent geochemical behavior. While Ba is mostly enriched in hydrothermal fluids, K, Ca and Sr can be enriched (such as in the Logatchev fluids, Table 2a) or depleted (such as the Sisters Peak fluid, Table 2a) compared to ambient seawater; in contrast, Mg is always depleted and often even zero in hot endmember fluids, due to the quantitative removal as Mg hydroxide (e.g., Von Damm, 1995). Apart from the dissolved contents, formation of sulfates such as anhydrite (CaSO4, controlling Ca and partly Sr concentrations by Sr substituting Ca in the crystal lattice) and barite (BaSO4, strongly limiting Ba solubility) must be considered. Probably this element group is also regulated by biological functions, as Ca and Mg are involved in numerous metabolic processes on both cellular and inter-cellular level and they are also known to interact and control each other in living beings (Bara et al., 1993). Apart from the direct role of the dissolved and particulate metals, biological factors may play an additional important role in trace metal accumulations. E.g., Colaço et al. (2006) have discussed the good metal-binding properties of bacterial exopolysaccharides and further transformation within the cell. Also the symbiotic bacteria in the gills can influence metal concentrations and uptake by the mussel (Kádár et al., 2006a,b). Significant concentrations of

Fe, Pb, Cr, and other metals were measured in endosymbionts and lysosomes of B. azoricus, with conclusions for a special significance of large lysosomal bodies in metal sequestration and detoxification (Kádár et al., 2006). Hence, bioaccumulation of trace metals by hydrothermal mussels is defined by the sum of a number of abiotic and biological parameters of the environment, which may interact with each other (Demina and Galkin, 2008). Abiotic parameters reflect the specific physicochemical environment of the hydrothermal habitats, including concentrations and chemical speciation of metals, metal distribution between dissolved and particulate forms, the substrate the mussels grow on, temperature, and pH. 4.2. Patterns of metal accumulations in Bathymodiolus mussels in relationship to the geochemical environment Despite the complexity of geoebio interactions in hydrothermal habitats, certain relationships between the specific environment of four hydrothermal sites investigated here, and metal accumulations in tissues of Bathymodiolus puteoserpentis (Irina II and Quest in the Logatchev field) and two unidentified Bathymodiolus species closely related to B. puteoserpentis in the Golden Valley (5
S) and Lilliput (9
S) fields could be observed. High concentrations especially of the chalcophile elements Fe, Cu, Zn, Pb, and Cd in gills and viscera of the Logatchev and Golden Valley mussels can be put into relation with the hot endmember fluid character of these fields, as chalcophile elements are generally enriched in hot hydrothermal fluids (Von Damm, 1995). In the Logatchev field, the endmember fluid is about 350
C hot and more enriched in alkaline and earthalkaline elements but less enriched in most heavy metals than

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Fig. 3. Scatter plots demonstrating the relationships of the transition metals Fe, Cu, and Zn, and the alkaline earth and alkali metals Ca, Mg, Sr, and K, subdivided into data for gills, digestive glands, and foot.

the 400
C hot fluids from the smoker Sisters Peak, which is located at a distance of about 70 m from the Golden Valley mussel field (Table 2a). In both fields, the mussel habitats comprise dilute versions of hot endmember fluid, with the hot fluids being closer by and diffuse fluids being warmer at Logatchev than at Golden Valley, where only 3e4
C were measured. Correspondingly, trace metal concentrations in the diffuse fluids of Golden Valley are mostly lower than in those of the two sites at Logatchev. Still, concentrations of all metals, except Fe and Zn, are higher in the gills of the mussels from Golden Valley than from both Logatchev sites. In the digestive gland and foot, only Cu is significantly higher in Golden Valley mussels than in Logatchev mussels. Apparently, the metal accumulations in the mussels reflect the nature of the hot endmember fluid, although the mussels live only adjacent to the black smokers in the mixing zone characterized by diffuse fluid

flow, oxidation of metals and gases and precipitation of hydrothermal minerals. This points to the important role of mineral particles for trace metal accumulation by the mussels, as will be discussed in more detail below. A comparison of data from the two sites in the Logatchev field also indicates slight difference in metal accumulations, namely high Fe and Cd contents in the digestive gland and higher Fe, Zn and earth-alkaline concentrations in the gills of Irina II mussels compared to Quest mussels. As the mussel beds at the Irina II sulfide complex are located directly at the base of the structure, here the impact of sulfide particles appears to be stronger than at site Quest. Here the mussels were taken from the surface sediment, which according to its composition with very high Fe and Cu concentrations (Table 2b) comprises mostly plume fallout from the nearby smoking crater Quest. The role of different plume particles at both sites will be discussed in Section 4.3.

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Table 2 Composition of potential dissolved (a) and particulate (b) metal sources for metal accumulations in the investigated mussels; empty spaces indicate no data. a) Hydrothermal fluids and seawater (as concentrations in the fraction <0.2 mm, as defined by the filters used) Sample location

Sample/source description Data source

Lilliput

Haase et al., 2009, this study Schmidt et al. (2007)

Ca (mg/kg)

23

Cd (mg/kg)

Cu (mg/kg)

Fe (mg/kg)

0.02

0.4e1.8

Up to 2400

0.14

Up to 41 80

Schmidt et al. (2007), this study Schmidt et al. (2007)

4100

1160

3.6

This study

5

416

<0.02

Koschinsky et al. (2008) 480 This study

<5

700 428

<0.02

Cr (mg/kg)

K Mn (mg/kg) (mg/kg)

Mg (mg/kg)

2e58

Mo (mg/kg)

Pb (mg/kg)

7.8

<0.1e0.4 7.6

3

9e42

2

3

72

3

20

1

Sr U (mg/kg) (mg/kg)

7.5

Zn (mg/kg)

7

6.5

2800

135,000

936

0.4

257

0.16

22,000

190,000

290

39,000

0

<0.1

<0.1

<1

422

<1

1366

Fe (%)

K (%)

Mn Mg (%) (mg/kg)

Mo Pb (mg/kg) (mg/kg)

Sr U Zn (mg/kg) (mg/kg) (mg/kg)

1.4

18,600

0

0.4

29

11.2

0.7

2300

0.1

6.9

3

Up to 23

26

87

4.5

0.1

10,000

8

<0.02

7.9

4

<0.1

1290

b) Solid substrates Sample location

Sample/source description Data source

M68/1 e 39ROV1, semilithified Fe-oxyhydroxide crusts Lilliput M68/1 e 41ROV1, semilithified Fe-oxyhydroxide crusts Logatchev, Quest MSM04/3 e 259ROV21, hydrothermal surface sediment Logatchev, Quest MSM04/3 e 259ROV18, hydrothermal surface sediment Logatchev, Irina II Sulfide mound with black smoker chimneys Lilliput

Ba Ca (%) (mg/kg)

Cd Cr Cu (mg/kg) (mg/kg) (mg/kg)

This study

39

0.82

20

46.8

258

0.94

80

0.6

262

5.6

59

This study

36

0.71

27

42.6

474

0.62

159

1.1

248

10.2

75

This study

917

0.49

<1

71

43,625

20.6

1194

0.81

13

198

190

4.84

671

This study

339

0.39

<1

51

29,000

20.5

1739

0.81

40

343

82

14.2

600

35e50%

17.7e24.3

Kuznetsov et al., 2006

0.03e0.19 <10e13

0.12

0.04e0.31

200

0.08e0.36 %

A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

Diffuse hydrothermal fluid (5e16
C) Logatchev, Irina II Diffuse hydrothermal fluid (up to 30
C) Logatchev, Quest Diffuse hydrothermal fluid (up to 16
C) Logatchev Hot endmember fluid (360
C) 5
S, Golden Valley Diffuse hydrothermal fluid (3e4
C) Hot hydrothermal 5
S, Sisters Peak endmember fluid (400
C) Background deepsea water 5
S

Ba (mg/kg)

A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

The Lilliput hydrothermal field is the only site, where direct influence of hot, metal-rich hydrothermal fluids can be excluded, as only low-temperature diffuse venting with low dissolved trace metal concentrations (Table 2a) has been observed in this area (Haase et al., 2009). Still, metal concentrations in the mussel tissues are in a similar range as for the mussels from the other three areas. Here, the mussels seem to be influenced by the thick coatings of Fe oxyhydroxide, which represent a kind of semi-consolidated sediment (see sediment data in Table 2b and Section 4.3). Investigation of size-dependent variations of symbiosis and filter-feeding to B. azoricus (Martins et al., 2008) indicated a strong dependence on filter-feeding for small mussels, while in contrast the energy gain of larger mussels was significantly controlled by the metabolic activity of the chemosynthetic endosymbionts. Since the Lilliput mussels were all very small (around 1 cm shell length), this may possibly explain the high metal concentrations in the Lilliput mussels, since they might be strongly dependent on filter-feeding of particulate matter. 4.3. The role of particulate matter for metal uptake The mixing zones of the hydrothermal fluid and ambient seawater are characterized by precipitation of metal sulfides, sulfates, and partly oxides. Considering the rather low dissolved metal concentrations of diffuse fluids in our study areas, which are partly close to ambient seawater values (Table 2a), an important impact of the particulate matter on metal uptake in the mussel habitats can be anticipated. In the warm, diffuse fluid that the vent mussels are exposed to most of the chalcophile elements will have precipitated as sulfide minerals, or Fe as Fe oxyhydroxide with other metals sorbed in the case of the Lilliput field. Also in other studies, the potential importance of the particulate metal fraction including metal sulfides, Fe oxides and sulphates for metal accumulation in mussel tissue has been demonstrated (Kádár et al., 2005; Sarradin et al., 2008). In these studies, the particulate load of many metals was shown to be higher than the corresponding load of dissolved metals, which was attributed to sulfide precipitation and metal and sulfide oxidation in the mixing zone. Typical particulate matter found in the vent habitats were crystalline particles settling from the hot smokers, such as sphalerite, pyrite, chalcopyrite, sphalerite, barite, and aluminum silicates. Demina and Galkin (2008) found sulfides of iron and zinc in the gill tissues of a Bathymodiolus puteoserpentis mussel from the field Snake Pit, which showed unusually high metal concentrations of Fe, Zn, and other metals. Colaço et al. (2006) also found such hydrothermal mineral fragments in the stomachs of B. azoricus and other hydrothermal vent species, confirming that the high particle load in the fluid-seawater mixing zone comprises an important source of trace metals for the potential uptake by filtering organisms. Once ingested, these tracemetal rich minerals would pass through the entire digestive process, leading to the enrichment of the metals in different tissue sections. It can be assumed that the gills are more susceptible towards dissolved metals (Bustamante et al., 2002), whereas the inner organs accumulate metals through the ingestion of small particles, as proposed for coastal mussels. In this case the peak metal concentrations in the digestive glands, as observed for Fe, Ba, and partly Pb, Mo, Cd, Cr, and U may signify dominant metal uptake by particles. In the Logatchev field, the gray smoke at Irina II is dominated by wurzite (Zn oxide) or Pb-rich sphalerite (Zn sulfide) and pyrrhotite (Fe sulfide), while in the black smoke mostly Cu sulfides were found (Schmidt et al., 2007). At site Quest, Cu sulfides with minor amounts of Fe and Zn sulfides dominate in the black smoke (Schmidt et al., 2007), which is reflected by much higher Cu concentrations (up to 4.3%) compared to Zn (0.06%, Table 2b) in the

71

sediment sampled at site Quest. These local differences in particulate matter agree very well with a higher enrichment of Fe and Zn in mussels from Irina II compared to mussels at Quest, while Cu concentrations are in a similar range at the two sites. The high Pb and Ba concentrations in the mussels from site Quest may be related to Pb and Ba contents in the range of a few hundred mg/kg in the sediment at this location (Table 2b). In the 5
S field, where the Golden Valley mussel field is located, filtered particles were mostly found to consist of a FeeCueZn sulfide matrix, in which often native sulfur and anhydrite were intergrown (Klevenz et al., 2011). The importance of anhydrite in this system (Schmidt et al., 2010) may explain why Sr and Ca concentrations in gills of the mussels from Golden Valley are higher than at the other sites. Geochemical analysis of particulate matter from the fluid-seawater mixing zone of the Logatchev and 5
S vent fields accompanied by geochemical modeling (Klevenz et al., 2011) indicated the presence of more Cu-rich particles closer to the hot fluid emanations, while with higher degrees of dilution more Ferich particles were found. Correspondingly, FeeCu ratios in the gill tissues may reflect the degree of mixing of hot fluid and seawater, from which the metals originate. The low-temperature venting style and low sulfide and trace metal concentrations (except Fe, which may represent colloidal Fe oxyhydroxide; Table 2a) make sulfide particles an improbable metal source at Lilliput. Here, high Fe concentrations in the digestive gland accompanied by the highest Cr, Mo, and U values (which as anionic species in seawater preferentially sorb on FeOOH phases; Koschinsky et al., 2003) of the four sites indicate that particulate Feoxyhydroxide material contributes significantly to the metal loads of the mussels. High Fe, Mo, (Sr) and low Zn, Pb, Ba, and Mn concentrations of the Lilliput sediment compared to the Quest sediment (Table 2b) largely agree with high Fe (digestive glands), Mo, (Sr), Cr, and low Mn and Pb concentrations in the mussel tissues. Only Cu and Cd concentrations in the tissues are clearly higher than expected from the sediment and fluid data (although we have no Cd data for sediment at Lilliput). The fact that Mn concentrations of all data sets are consistently low and rather uniform underlines the potential importance of particles in the metal uptake routes of the mussels. Manganese does not form sulfide particles, and Mn oxidation is kinetically slow and hence occurs only later in the water column (Mandernack and Tebo, 1993); therefore Mn in the mussel habitats should be present mostly in dissolved form, which is apparently not taken up to a large degree. Only higher Mn values in the gills of Golden Valley mussels point to enhanced uptake in the dissolved form. Opposite to Mn, Mo concentrations in hydrothermal fluids are typically rather low (sometimes lower than in ambient seawater) because dissolved Mo is readily removed by inclusion into hightemperature minerals such as Cu sulfides (Metz and Trefry, 2000). Correspondingly, enrichment of Mo in both digestive glands and gills may reflect Mo-rich sulfide particles or seawater Mo adsorbed on FeOOH particulate matter as a major Mo source, but possibly also uptake of dissolved Mo from entraining seawater by the gills. 4.4. Prospects for estimating metal fluxes at diffuse hydrothermal vents from metal concentrations in mussels A promising result with regards to the potential use of Bathymodiolus mussels as proxies the geochemical environment in diffuse-flow sites were the differences observed in the concentrations between different sites within the same field (Irina II and Quest at Logatchev) and between different vent sites characterized by different fluid compositions and solid material. In most published articles, authors have so far been focusing on differences in metal tissue concentrations between geochemically distinct sites

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A. Koschinsky et al. / Marine Environmental Research 95 (2014) 62e73

(e.g., Kádár et al., 2006a,b, 2007; Colaço et al., 2006). Measurable differences within a single vent site such as presented by Cosson et al. (2008) and in this study open a whole new array of possibilities. The present results suggest that measurements of metal concentrations in the tissue of Bathymodiolus mussels could provide information about the small-scale spatial distribution of metals around hydrothermal vent sites. Although different species of the genera Bathymodiolus may exhibit certain differences in metal accumulation mechanisms, general trends applicable to all species investigated here and in other studies could be confirmed. As Bathymodiolus mussels live in diffuse-flow sites and the fluxes and elemental contributions of low-temperature venting to seawater are still quite unknown (and probably underestimated, de Villiers and Nelson, 1999; Mottl, 2003), mussels may provide a method to estimate metal fluxes from diffuse venting integrated over their lifetime of several years. Due to the dilution with ambient seawater and partial precipitation of metals as sulfide or oxide particles, metal concentrations in diffuse low-temperature fluids are significantly lower than in high-temperature fluids, and more difficult to quantify. Furthermore, fluid flow emanations in diffuse vent fields fluctuate significantly on time scales of minutes to hours, questioning the representativeness of individual fluid samples taken at such sites for a characterization of fluid composition and metal fluxes (Perner et al., 2009). Mussels average these fluctuating compositions and integrate them over their lifetime, possibly providing a more reliable estimate of diffuse fluid composition. The ubiquitous distribution and dense population of Bathymodiolus mussels at most hydrothermal vent sites (Tunnicliff, 1991) would strongly support this approach. 5. Conclusion The prospect of estimating the spatial metal distribution in hydrothermal environments and integrating metal fluxes from diffuse hydrothermal fluids through the employment of Bathymodiolus mussels as proxies is promising. It was shown that metal enrichments in mussel tissues vary between different vent sites, reflecting the different fluid and particle compositions that can serve as metal sources at these sites. Also, local differences within a single vent site are detectable. To effectively employ such a method, however, it is necessary to improve our understanding of metal bioaccumulation pathways in Bathymodiolus mussels so that it becomes possible to directly relate differences in metal concentrations in their tissues to differences in the environment including dissolved and particulate phases. While this has already been achieved with coastal mussels such as M. edulis, it still remains a challenge with Bathymodiolus for number of reasons. Not only may Bathymodiolus possess special adaptations to help them cope with high environmental metal loads, which may affect tissue concentrations in an unpredictable way, but the accumulation of metals may also take place in a different manner than it does in coastal environments. It also needs to be further investigated whether different species of Bathymodiolus interact with hydrothermal metal loads in the same manner, or whether they have to be considered separately. Furthermore, the different and variable roles of dissolved and particulate metal sources must be better constrained. For example, the question whether the significant enrichment of the chalcophile elements (Cu, Zn, Cd) in the gills is mostly due to uptake from the fluids or by particles is not fully clarified. Aside from studying larger samples from several locations at a single vent site, the trace metal compositions of fine particles found in the diffuse hydrothermal environment should be analyzed (Sarradin et al., 2008; Klevenz et al., 2011) and the elemental patterns should be compared with those found in the organs of the mussels. This would give more conclusive evidence about the role

of particles in the metal uptake by Bathymodiolus mussels than tissue partitioning can do alone. Acknowledgments This work and the cruises were funded by the DFG (German Science Foundation) as part of the SPP 1144 special priority program “From Mantle to Ocean: Energy, Material and Life Cycles at Spreading Axes” and the Cluster of Excellence "The Ocean in the Earth System" at MARUM, Bremen. This is SPP 1144 contribution no. 72. Credit is due to Aryani Sumoondur for the development of the digestion method employed in this study and to Katja Schmidt, Jule Mawick and Daniela Meißner for their invaluable help in the laboratory. We acknowledge the skillful equipment handling of captains and crews of research vessels Meteor, Merian, and l’Atalante and of the ROV teams during the cruises of the SPP 1144 program. We are grateful to Olav Giere for onboard separation of some of the hydrothermal mussel tissues. Finally we want to thank the two reviewers for their constructive comments on the first version of this paper. References Bara, M., Guiet-Bara, A., Durlach, J., 1993. Regulation of sodium and potassium pathways by magnesium in cell membranes. Magnes. Res. 6, 167e177. Bellotto, V.R., Miekeley, N., 2007. Trace metals in mussel shells and corresponding soft tissue samples: a validation experiment for the use of Perna perna shells in pollution monitoring. Anal. Bioanal. Chem. 389, 769e776. Burger, J., Gochfeld, M., 2006. Locational differences in heavy metals and metalloids in Pacific blue mussels Mytilus [edulis] trossulus from Adak Island in the Aleutian Chain, Alaska. Sci. Total Environ. 368, 937e950. Bustamante, P., Teyssie, J.-L., Fowler, S.W., Cotret, O., Danis, B., Miramand, P., Warnau, M., 2002. Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis. Mar. Ecol. Prog. Ser. 231, 167e177. Colaço, A., Bustamante, P., Fouquet, Y., Sarradin, P.M., Serrão-Santos, R., 2006. Bioaccumulation of Hg, Cu, and Zn in the Azores triple junction hydrothermal vent fields food web. Chemosphere 65, 2260e2267. Cosson. Cosson, R.P., Thiébaut, E., Company, R., Castrec-Rouelle, M., Colaço, A., Martins, I., Sarradin, P.-M., Bebianno, M.J., 2008. Spatial variation of metal bioaccumulation in the hydrothermal vent mussel Bathymodiolus azoricus. Mar. Environ. Res. 65, 405e415. de Villiers, S., Nelson, B.K., 1999. Detection of low-temperature hydrothermal fluxes by seawater Mg and Ca anomalies. Science 285, 721e723. Demina, L.L., Galkin, S.V., 2008. On the role of abiogenic factors in the bioaccumulation of heavy metals by the hydrothermal fauna of the Mid-Atlantic Ridge. Oceanology 48, 784e797. Deplede, M.H., Rainbow, P.S., 1990. Models of regulation and accumulation of trace metals in marine invertebrates. Comp. Biochem. Physiol. 97C, 1e7. Garbe-Schönberg, D., Koschinsky, A., Ratmeyer, V., Jähmlich, H., Westernströer, U., 2006. KIPS e a new multiport valve-based all-teflon fluid sampling system for ROVs. Geophys. Res. Abstr. 8. EGU2006-A-07032.833. Gebruk, A.V., Chevaldonnè, P., Shank, T., Lutz, R.A., Vrijenhoek, R.C., 2000. Deep-sea hydrothermal vent communities of the Logatchev area (14
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