Nelli Sergeeva, Department of Benthos Ecology, Institute of Biology of the Southern Seas (IBSS), 2, Nakhimov Av., 99011 Sevastopol, AR Crimea, Ukraine. E-mail: email@example.com
Meiobenthos densities and higher taxon composition were studied in an active gas seepage area at depths from 182 to 252 m in the submarine Dnieper Canyon located in the northwestern part of the Black Sea. The meiobenthos was represented by Ciliata, Foraminifera, Nematoda, Polychaeta, Bivalvia, Gastropoda, Amphipoda, and Acarina. Also present in the sediment samples were juvenile stages of Copepoda and Cladocera which may be of planktonic origin. Nematoda and Foraminifera were the dominant groups. The abundance of the meiobenthos varied between 2397 and 52,593 ind.·m−2. Maximum densities of Nematoda and Foraminifera were recorded in the upper sediment layer of a permanent H2S zone at depths from 220 to 250 m. This dense concentration of meiobenthos was found in an area where intense methane seeps were covered by methane-oxidizing microbial mats. Results suggest that methane and its microbial oxidation products are the factors responsible for the presence of a highly sulfidic and biologically productive zone characterized by specially adapted benthic groups. At the same time, an inverse correlation was found between meiofauna densities and methane concentrations in the uppermost sediment layers. The hypothesis is that the concentration of Nematoda and Foraminifera within the areas enriched with methane is an ecological compromise between the food requirements of these organisms and their adaptations to the toxic H2S.
Cold seep environments are among the most recently discovered marine habitats; they have been known for only 20 years (Polikarpov et al. 1991; Levin 2005). Initial exploration has shown that methane seeps are widespread in the World Ocean. Active seeps have been reported for each ocean (Levin 2005). Also, there is evidence of active seeps in the polar regions both the Arctic and Antarctic (Domack et al. 2005). Levin (2005) reviews diverse aspects of the ecology of methane seeps; she describes the structure of protistan and animal communities in seep sediments and how they are formed under the influence of hydrological, geochemical and microbial processes.
Seeps pose many interesting scientific questions concerning their origin, the effects that methane has on the global atmosphere and on the marine environment and the associated fauna. The available literature suggests that the taxonomic composition and quantitative development of the fauna depend upon a number of environmental factors. A comparison of data on meiofauna around seeps and in non-seep areas leads to conflicting conclusions (Bernhard et al. 2001; Levin 2005). Specialized infaunal communities are associated with different seep habitats (microbial mats, clam and mussel beds and tube worm aggregations) and with different vertical zones in the sediment (Levin 2005).
In the Black Sea, gas seeps were first recorded in 1989 (Polikarpov et al. 1991); at present over 3000 points of methane bubble seepage are known for the depth range 35–1800 m (Egorov et al. 2003). One main field of methane seepage is located in the northwestern part of the Black Sea, off the submarine Dnieper Canyon (Egorov et al. 1998). Hundreds of seeps are distributed over a total area of nearly 2000 km2 (Fig. 1). Most of the gas seeps in this area are found at the edge of the continental shelf and on the upper slope.
The Black Sea is the largest anoxic marine reservoir in the world. The chemocline is located at depths of 75–180 m (average 131 m) in the water column (based on the literature and our data). Methane seeps are present in the three zones of biogeochemical stratification described earlier for the Black Sea: the oxygenated zone, the transitional oxic/anoxic interface, and the deep-water hydrogen sulfide zone (Polikarpov et al. 1991; Egorov et al. 2003). Areas in the sulfide zone with intensive methane seepage are the most biologically productive, due to the presence of anaerobic methane-oxidizing microorganisms (Lein et al. 2002; Michaelis et al. 2002).
Comparative studies on benthic oxibionts of NW Black Sea shelf/slope regions with and without methane seepage were carried out for the first time in 1993–1994 (Luth & Luth 1998). This study showed that, along an oxic–anoxic gradient (77–175 m), the biomass and biological activity of the bottom communities were very similar. Larger sizes of macrobenthos organisms in the seep areas were considered by Luth & Luth (1998) as an indication of greater biocenosis stability.
Previous investigations in the Dnieper Canyon area of the active gas seepage fields in anoxic waters have shown the presence of massive microbial mats covering carbonate reefs (Polikarpov et al. 1991; Egorov et al. 1998; Lein et al. 2002; Michaelis et al. 2002). Several investigations were undertaken to identify the microorganisms (or their communities) responsible for the anaerobic oxidation of methane (AOM) and for the formation of carbonate structures and mats in this anoxic zone of the Black Sea. It was found that the anaerobic microbial mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (SRB, Desulfosarcina/Desulfococcus group). AOM is mediated by this consortia of archaea and SRB, so that methane is oxidized with equimolar amounts of sulfate, generating the microbial biomass, the carbonate and also the sulfide (Lein et al. 2002; Michaelis et al. 2002).
In the present study, we focused on the effects of methane seepage on the associated biota. Therefore, samples were collected only from the upper anoxic zone of the Black Sea. The distribution of the protozoan and metazoan meiofauna in the gas seep areas with minimal (or no) oxygen concentration was investigated.
Material and Methods
The submarine Canyon - Paleo delta of the Dnieper River is located in the center of the northwestern part of the Black Sea (Fig. 1). Preliminary hydroacoustic data were obtained by means of the scientific echosounder SIMRAD EK-500 during several cruises with the RV ‘Professor Vodyanitskiy’ (Ukraine). Aggregations of methane seeps have been mapped in the immediate vicinity of the Dnieper Canyon slope and also in adjacent areas. Details of the sea floor mesorelief were analyzed using echograms and hydrographical maps.
The main survey and sampling activities were carried out in October 2004 onboard RV ‘Poseidon’ (Germany), cruise 317/3. The multibeam echosounder and motion sensor were used for detailed sea floor observations. High-resolution bathymetrical maps were developed by Jens Greinert. On the basis of obtained materials (Fig. 1), planning of sampling stations, calculations of their locations as well as all distances were performed using Fugawi software.
Sediment samples for meiofauna investigations (Table 1) were collected in parallel with measurements for biogeochemical fluxes, including methane determination. The undisturbed samples were obtained using a multiple-corer (modified Barnett type) at depths of 182–252 m. At station 795 (190 m depth) the bottom sediment was sampled using pushcores during a submersible JAGO dive.
Table 1. Sampling information (RV ‘Poseidon’, research cruise 317/3). MUC = multiplecorer.
water depth (m)
44°37.75′ N 31°08.66′ E
44°46.79′ N 31°59.09′ E
44°46.79′ N 31°59.06′ E
44°46.89′ N 31°59.26′ E
44°47.52′ N 32°00.75′ E
44°47.58′ N 32°00.87′ E
44°46.68′ N 31°58.82′ E
44°46.56′ N 31°58.64′E
The concentrations of methane at four stations were measured by Nina Knab using a gas-chromatographical method. Gas analysis of the sediments was performed on board using a 5809A-Hewlett Packard chromatograph equipped with a flame ionization detector (column temperature 40 °C) and helium as a carrier gas (Treude et al. 2003).
Most multiple-corer stations were located along a cross-sectional transect from southwest to northeast within a field of highly active methane seeps (Fig. 1; Table 1- Sts 795, 802, 803, 804, 805, 815 and 827). The distance between the two extreme points of this transect was 2.6 km. Such a short distance makes it possible to exclude the influence of factors causing large-scale variation in benthic biota.
Sediment cores, obtained by the multiple-corer, were sectioned at 0–1, 1–2, 2–3, 3–4, 4–5 and 5–10 cm intervals. At station 805, only the 0–5-cm layer was analyzed. Until the end of the expedition, i.e. for 7–12 days, all subsamples were kept without formalin fixation in a refrigerator at temperatures corresponding to those in situ (+8 to +10 °C). Then, as soon as possible, the silt was carefully washed with filtered sterile sea water on sieves (upper and lower mesh size of 1 mm and 64 μm) in the laboratory of IBSS. The fraction retained on the sieves was stained in rose Bengal solution before being sorted in water under a binocular microscope for ‘live’ (stained) organisms which were identified to higher taxa.
Ten higher taxa were recorded among the hydrobionts in the sediment samples in the Dnieper submarine canyon: Ciliata, Foraminifera, Nematoda, Acarina, Polychaeta, Bivalvia, Gastropoda, Amphipoda and juvenile stages of Cladocera and Copepoda, which may be of planktonic origin. A specific meiobenthic community was found in the hydrogen sulfide Black Sea zone at fields of active methane gas emanation. Particularly characteristic of these areas was the presence of juvenile specimens of Cladocera and Copepoda. In the sediments near the active methane gas seeps we discovered a large quantity of dormant eggs of Copepoda and also their developed nauplia.
The presence of Foraminifera in the high hydrogen sulfide conditions was also notable. They included the polythalamous calcareous species Ammonia compacta and Cribroelphidium bartletti, the ammodiscacean Glomospira aff. gordialis and soft-shelled monothalamous taxa. Nematoda was the only taxon that was present at all investigated depths (Table 2). Table 2 gives the numbers of hatched nauplii and fully developed nauplii of copepods still contained inside the eggs. At the same time, huge numbers of dormant eggs of Copepoda and Cladocera (about 122,000 and 100,000 ind.·m−2, respectively) were found in the bottom sediments collected at these stations.
Table 2. Total number of meiobenthos (ind.·m−2) in the uppermost 0–5 cm sediments.
station no./depth (m)
The density of meiobenthic populations, including the juvenile Cladocera and Copepoda, ranged from 2397 to 52,593 ind.·m−2 (Fig. 2, Table 2). These levels are equal to those found in some regions of the Black Sea oxic zone. There was no correlation between meiobenthic densities and water depth. The highest densities, 46,953 and 52,593 ind.·m−2, occurred at the depths of 222 and 249 m, respectively (Table 2). Analysis of vertical distribution patterns in the sediment shows that meiobenthic organisms only penetrated down to the 4–5-cm layer at the deepest stations (depths 249 and 252 m) (Fig. 2).
Quantitative analyses of methane were carried out in the pore water of sediments obtained in the immediate vicinity of the gas seeps. We analyzed the distribution of averaged methane concentrations in the upper 25 cm of the sediment column and meiobenthos abundances in the most densely populated 0–5-cm layer. Figure 3 shows the average methane values measured at four stations and the corresponding densities of meiobenthos. The comparative analysis was based on data from closely positioned stations (six stations, without st. 790) chosen to cover the upper anoxic zone in the depth range between 188 and 252 m (Fig. 3; Table 1).
There was high variation in methane concentrations over the investigated depths (Fig. 3). Values fluctuated from 2.40 up to 5.75 nmol·cm−3. The meiobenthos densities also showed a high variation, from 2.5 to 47 ind.·10 cm−2, with minimum methane concentrations coinciding with maximum meiobenthic densities (Fig. 3).
As it is well known, the deep-water part of the Black Sea has a density structure maintained by low salinity water (∼18‰), which forms a thin (0–50/70 m) surface layer above the more saline, deeper part of the water column (∼22‰). These layers are divided by the halocline (permanent pycnocline) with significant density gradients situated in the depth range 50–150 m. This sharp stratification inhibits vertical water mixing and is the main factor responsible for the anoxic conditions.
The depth of the Black Sea oxic/anoxic interface is intimately related to the circulation of the water masses. It is the shallowest in the center of the western circulation gyre and the deepest at the margins of the Black Sea (Codispoti et al. 1991). Several studies suggest that the oxic/anoxic interface is situated below approximately 75–80 m in the Central Black Sea Basin and below 170/180 m at its periphery (Blatov et al. 1984; our unpublished data). At the same time, near the continental slope of the Black Sea the configuration of the interface has some important peculiarities.
We investigated earlier the temporal dynamics of the pycnocline and chemocline (oxic/anoxic interface) in the water column along transects near the submarine Dnieper Canyon. The chemocline was found to rise from approximately 170 to 130 m over the upper continental slope (Luth et al. 1998). Fluctuations of isopycnal surfaces had a quasi-periodical character with a period of approximately 5 days. This is very close to the 4–6 days period of the so-called inertial oscillations, caused by changes in wind activity and large-scale current instability in the Black Sea during the summer (Blatov et al. 1984). These facts suggest that in the Dnieper submarine canyon, where the meiobenthic samples were collected, the oxic/anoxic interface lies not deeper than 170–180 m. Therefore, the investigated area may be regarded as lying within the ‘upper anoxic zone’.
Data on the taxonomic composition and density of the meiobenthos in the upper anoxic zone of the Black Sea are not numerous. Information concerning community structure and species composition of the fauna dependent on methane gas emanations is also rare (Luth & Luth 1998; Sergeeva 2003). At the same time, it is known that methane gas emanation in marine environments has a strong influence on meiobenthic communities (Dando & Høvland 1992; Jensen et al. 1992; Levin 2005).
Our earlier investigations in the region of methane seepage showed that meiobenthos was characterized by a relatively high diversity (13 higher-level taxa were identified) and also by high abundances (Sergeeva 2003). A characteristic fauna of benthic organisms, adapted to limited oxygen concentrations, was present at water depths of 130–150 m. Soft-shelled foraminiferans, large numbers of nematodes species, typical polychaetes, hydroid polyps and turbellarians were the main components of this community. The foraminiferans were dominated by monothalamous species at depths from 70 to 175 m. Psammophaga simplora was the most abundant foraminiferan. The polychaete species Victorniella zaikai Kiss., Protodrilus sp.1, Nerilla sp. 1 are presently known only from the investigated region with active methane gas seeps (Kisseleva 1992, 1998; Sergeeva & Zaika 2000). Nematodes were represented by 143 species. Of these, 38 species and 6 genera were found only in low-oxygen habitats. They were earlier unknown in the Black Sea.
It has been assumed that most eukaryotes are oxyphilic organisms (with the exception of some endoparasites) and they cannot exist for a long time without oxygen, particularly in the case of the totally anoxic and sulfidic conditions of the Black Sea. However, our data show that both Protistan and Metazoan organisms can live in naturally anoxic environments due to a high tolerance to hypoxic and anoxic conditions. During a previous study of bottom sediments from methane gas hydrates in the Sorokin Trough (NE Black Sea) at depths of 1990 and 2140 m, an endemic species of Cladocera, Pseudopenila bathyalis Sergeeva, 2004, was discovered (Sergeeva 2004a,b). Moreover, studies of deep-sea bottom sediments (400–2150 m) of the Black Sea shows that the dormant eggs of Cladocera and Copepoda occur in almost all parts of the continental slope and basin (Sergeeva 2003). In deep-water silts in the hydrogen sulfide zone, these dormant eggs have been discovered in large quantities. Most of them (70–80%) are filled with yolk, and appear to be viable. Earlier we determined (Sergeeva 2004a,b) that one type of egg and the associated juvenile specimens belong to the above-mentioned cladoceran species P. bathyalis Serg. It is possible that this species, and other presently unknown species of cladocerans and copepods, are benthic inhabitants of the deep Black Sea.
The mechanisms which allow protists and metazoans to tolerate these hydrogen sulfide conditions remain unclear. For the time being we can only assume that the development of meiofauna in the anaerobic zone of the seeps is caused by the favorable trophic conditions and the absence of food competitors in the benthic environment. The trophic needs of meiofauna are possibly met by the highly developed microbial biomass as well as by organic matter deposited from the water column (Buesseler et al. 1990; Lein et al. 2002; Michaelis et al. 2002).
The microbial AOM can be an important source of both organic matter and toxic hydrogen sulfide (Michaelis et al. 2002). AOM is a microbial process in anoxic marine sediments whereby methane is oxidized with sulfate as the terminal electron acceptor via the following net equation (Michaelis et al. 2002; Treude et al. 2003):
Our results suggest that methane is the ecological factor that indirectly influences the meiobenthos distribution. The greatest toxic influence is probably due to hydrogen sulfide. The investigated area of the Dnieper Canyon is situated in the upper part of H2S-zone of the Black Sea. Here, H2S may be derived both from AOM and deep waters (Fig. 4).
The major role of hydrogen sulfide in controlling the distribution of benthos in areas of the methane seepages is also reported by other authors (Levin 2005). Seep and vent animals may experience exceptionally high sulfide concentrations within sediments. However, high resolution (mm–cm scale) studies of sulfide concentration in relation to macrofaunal abundance in seep sediments reveal that only a few taxa tolerate sulfide at concentrations of 1 mm or higher. Sampling at a high spatial resolution has also demonstrated that the composition of infauna may change on scales of centimeters according to seepage rates, sulfide concentration and biological activity. We therefore propose the following hypothesis. The distribution of Black Sea meiobenthos within the AOM-zones in the upper anoxic layer reflects an ecological compromise between the food requirements of these organisms and their adaptations to the toxic influence of H2S (Fig. 4).
The results of our study of meiobenthos inhabiting the upper anoxic zone and associated methane seeps are based on material collected from only eight stations and, therefore, are only tentative. However, these investigations have revealed the special taxonomic composition of the fauna concentrated around methane seeps in the upper anoxic zone of the Black Sea and its abundance in comparison with meiobenthos of the southwestern Black Sea near the Crimea (Luth & Luth 1998; Sergeeva 2003; Revkov & Sergeeva 2004).
Considerable differences were found in comparing the biota observed during our study with the meiofauna associated with methane seeps in other areas (Gooday et al. 2000; Bernhard et al. 2001; Robinson et al. 2004; Levin 2005; Panieri 2006), particularly regarding the presence or absence of Harpacticoida and calcareous Foraminifera. Our samples from the upper anoxic zone of the Black Sea are characterized by the absence of adult harpacticoids, which are common in seep faunas outside the Black Sea. The presence of living calcareous foraminifera at depths down to 250 m below the oxic/anoxic interface in the Black Sea is remarkable, although such foraminifera are reported to be common in other seep environments (Levin 2005). The dominance of nematodes, however, is common to all seep areas. Presumably, the special composition of the methane-associated fauna reflects the specific environmental conditions in the parts of the Black Sea included in our study.
Further investigations of these unique Black Sea seep faunas should yield more information about the taxonomic composition of fauna in the oxic and anoxic zones and in the transitional oxic/anoxic interface, and should show the relationship between the abundance of meiobenthos and methane concentration and seepage intensity. However, when comparing seeps in oxic and anoxic environments, it is important to select habitats that are comparable in other respects.
This work was supported by the European Union, project METROL EVK3-CT-2002-00080. Contributions made by N. Knab and J. Greinert are gratefully acknowledged by the authors. The preparation of this paper was facilitated by the HERMES project, EC contract no. GOCE-CT-2005-511234. We particularly thank A.J. Gooday and an anonymous reviewer for very helpful comments and revision of the manuscript.