R Alagarsamy
email: alagar@nio.org

Environmental magnetism and application in the continental shelf sediments of India

Keywords: Mineral Magnetic, Heavy metal, Magnetic method, Sediment, Continental shelf, India

Mineral magnetic and geochemical analyses were carried out on surface sediments from the continental shelf of India. The purpose of this study is to examine the environmental assessment of heavy metal concentrations and its impact in the coastal environment using magnetic techniques and to gain an understanding on the factors controlling metal concentrations and distributions in the east and west coast of India. The strong relationships between Anhysteretic Remanent Magnetization (χ ARM ) and heavy metals can be explained by the role of iron oxides controlling metal concentrations, though the link is also reinforced by the strong tendency of χ ARM to be associated with the finer particle sizes. Higher values of magnetic susceptibility, IRM 20 mT and SIRM are associated with the east coast shelf sediments suggest the presence of high ferrimagnetic content, which can be derived from the weathering products of the Deccan Basalts. χ ARM can be used as a normalizer for particle size effects in the way that Aluminium (Al) is often used. The relationship between magnetic parameters and heavy metal concentrations (Fe, Cu, Cr and Ni) showed a ACCEPTED MANUSCRIPT 2 strong positive correlation in the east coast sediments, much less so in the case of the west coast. This finding suggests that the simple, rapid and non-destructive magnetic measurement can be used as an indicator for the heavy metal contamination and proxies for the measurement of heavy metals content in the coastal environment.

Introduction

Magnetic measurements are being used as a powerful tool for the assessment of heavy metal contamination in soils and sediments and in the investigation of the compositional properties of rocks, sediments and soils [START_REF] Thompson | Rock magnetic, mineralogical and microstructural characterization of fly ashes from Bulgarian power plants and the nearby anthropogenic soils[END_REF][START_REF] Walden | Environmental magnetism: a practical guide[END_REF][START_REF] Maher | Quaternary climates, environments and magnetism[END_REF]. Magnetic minerals in soils are derived either from the parent rocks (lithogenic origin), pedogenesis or as a result of anthropogenic activities. Susceptibility measurements play an important role for monitoring environmental pollution in the case of minor contributions of the first two sources to the magnetic properties of soils. Accumulation of anthropogenic ferrimagnetic particles, originating during high temperature combustion of fossil fuels (e.g. [START_REF] Vassilev | Phase mineralogy studies of solid waste products from coal burning at some Bulgarian thermoelectric power plants[END_REF][START_REF] Dekkers | Magnetic properties of low-Ca fly ash: A rapid tool for Fe-assessment and survey for potentially hazardous elements[END_REF], results in significant enhancement of topsoil magnetic susceptibility. Therefore, under favourable circumstances, magnetic properties of soils and sediments can be used for the assessment of imissions, both from local pollution sources and on a wider regional scale. Already a pioneering work of [START_REF] Thompson | Rock magnetic, mineralogical and microstructural characterization of fly ashes from Bulgarian power plants and the nearby anthropogenic soils[END_REF] reported that soils near urban areas and industrial zones have an increased magnetic susceptibility, due to the deposition of magnetic particles, such as, dusts of the metallurgical industries and fly ashes of the coal combustion.

Magnetic measurements are relatively simple and non-destructive and due to high sensitivity and speed are often applied for detecting industrial pollution. Because there is a need for fast and cost effective screening and monitoring tools for industrial pollution, magnetic methods have drawn the attention of modern researchers as an approximate tool, to detect and characterize environmental pollution (e.g., [START_REF] Dearing | Magnetic susceptibility of soil: An evaluation of conflicting theories using a national data set[END_REF][START_REF] Petrovský | Magnetic monitoring of pollution of air, land and waters[END_REF][START_REF] Hoffmann | Magnetic susceptibility mapping of roadside pollution[END_REF][START_REF] Magiera | Ferrimagnetic minerals of anthropogenic origin in soils of some Polish national parks[END_REF][START_REF] Petrovský | Magnetic properties of alluvial soils contaminated with lead, zinc and cadmium[END_REF][START_REF] Hanesch | Monitoring of heavy metal loadings in soils by means of magnetic susceptibility measurements[END_REF]Veneva et al., 2004, and others). This work has been used to identify the sources of industrial pollution [START_REF] Scoullos | Magnetic monitoring of marine particulate pollution in the Elefsis Gulf, Greece[END_REF][START_REF] Hunt | Magnetic measurements and heavy metals in atmospheric particulates of anthropogenic origin[END_REF][START_REF] Matzka | Magnetic biomonitoring of roadside tree leaves: Identification of spatial and temporal variations in vehicle derived particulates[END_REF][START_REF] Petrovský | Low-field magnetic susceptibility: A proxy method of estimating increased pollution of different environmental systems[END_REF][START_REF] Petrovský | Magnetic properties of alluvial soils contaminated with lead, zinc and cadmium[END_REF][START_REF] Klose | Indicative properties of fly-ash affected forest soils in Northeastern Germany[END_REF][START_REF] Knab | Separation of the anthropogenic portion of heavy metal contents along a highway by means of magnetic susceptibility and fuzzy c-means cluster analysis[END_REF][START_REF] Lecoanet | Magnetic properties of salt-marsh soils contaminated by iron industry emissions (Southeast France)[END_REF][START_REF] Hanesch | Monitoring of heavy metal loadings in soils by means of magnetic susceptibility measurements[END_REF][START_REF] Muxworthy | Magnetic properties and Mössbauer spectra of urban atmospheric particulate matter: a case study from Munich, Germany[END_REF][START_REF] Hanesch | Mapping dust distribution around an industrial site by measuring magnetic parameters of tree leaves[END_REF]Kapicka et al., 2003;[START_REF] Moreno | Biomonitoring of traffic air pollution in Rome using magnetic properties of tree leaves[END_REF][START_REF] Desenfant | Magnetic signature of industrial pollution of stream sediments and correlation with heavy metals: case study from south France[END_REF][START_REF] Jordanova | Mineral magnetic characterization of anthropogenic magnetic phases in the Danube river sediments (Bulgarian part)[END_REF][START_REF] Spiteri | Magnetic screening of a pollution hotspot in the Lausitz area, Eastern Germany: correlation analysis between magnetic proxies and heavy metal contamination in soils[END_REF] and to characterize various depositional environments (e.g. [START_REF] Arkell | Trapping and tracing: some recent observations of supply and transport of coarse sediment from upland Wales[END_REF][START_REF] Oldfield | Particle size related, mineral magnetic source sediment linkages in the Rhode River catchment, Maryland, USA[END_REF][START_REF] Oldfield | Problems of core correlation, sediment source ascription and yield estimation in Ponsonby Tarn, West Cumbria, UK[END_REF][START_REF] White | Mapping the iron oxide content of dune sands, Namib Sand Sea, Namibia, using Landsat Thematic Mapper data[END_REF][START_REF] Walden | Mineral magnetic analyses of glacial diamicts from the Midland Valley of Scotland[END_REF][START_REF] Walden | Use of mineral magnetic measurements to fingerprint suspended sediment sources: approaches and techniques for data analysis[END_REF][START_REF] Schmidt | Magnetic characterisation of Holocene sedimentation in the South Atlantic[END_REF][START_REF] Wheeler | Depositional and post-depositional controls on magnetic signals from saltmarshes on the north-west coast of Ireland[END_REF]. The relationship between mineral magnetic measurements and chemical/physical properties of sediments and soils have been established in several studies [START_REF] Oldfield | Particle size related, mineral magnetic source sediment linkages in the Rhode River catchment, Maryland, USA[END_REF][START_REF] Oldfield | The influence of particle size variations on the magnetic properties of sediments from the north-eastern Irish Sea[END_REF][START_REF] Clifton | Relationships between radionuclide content and textural properties in Irish Sea intertidal sediments[END_REF][START_REF] Clifton | Derivation of a grain-size proxy to aid the modelling and prediction of radionuclide activity in saltmarshes and mud flats of the Eastern Irish Sea[END_REF][START_REF] Chan | Correlation between magnetic susceptibility and distribution of heavy metals in contaminated sea-floor sediments of Hong Kong Harbour[END_REF]Petrovský et al., 1998;[START_REF] Xie | Association between the organic matter content and magnetic properties in street dust, Liverpool, UK[END_REF][START_REF] Xie | The organic matter content of street dust in Liverpool, UK and its association with dust magnetic properties[END_REF][START_REF] Booth | Sediment-Source-Linkages in the Gwendraeth Estuary, South Wales, based on mineral magnetic analyses[END_REF]. Mineral magnetic measurements were used as a tool for determining sediment provenance [START_REF] Oldfield | The influence of particle size variations on the magnetic properties of sediments from the north-eastern Irish Sea[END_REF], transport pathways [START_REF] Lepland | Mineral magnetic and textural interpretations of sedimentation in the Skagerrak, eastern North Sea[END_REF], as a proxy for geochemical, radioactivity, organic matter content and particle size data [START_REF] Bonnett | Radionuclides in coastal and estuarine sediments from Wirral and Lancashire[END_REF][START_REF] Oldfield | 241 Am and 137 Cs activity in fine grained saltmarsh sediments from parts of the N.E. Irish Sea shoreline[END_REF][START_REF] Hutchinson | Siltation in the saltmarsh of the Dee Estuary derived from 137 Cs analysis of shallow cores[END_REF][START_REF] Clifton | Relationships between radionuclide content and textural properties in Irish Sea intertidal sediments[END_REF][START_REF] Clifton | Derivation of a grain-size proxy to aid the modelling and prediction of radionuclide activity in saltmarshes and mud flats of the Eastern Irish Sea[END_REF][START_REF] Xie | Association between the organic matter content and magnetic properties in street dust, Liverpool, UK[END_REF][START_REF] Xie | The organic matter content of street dust in Liverpool, UK and its association with dust magnetic properties[END_REF][START_REF] Zhang | Diagenesis of magnetic minerals in the intertidal sediments of the Yangtze Estuary, China, and its environmental significance[END_REF][START_REF] Booth | Use of mineral magnetic concentration data as a particle size proxy: A case study using marine, estuarine and fluvial sediments in the Carmarthen Bay area, South Wales[END_REF]. Magnetic measurements as a proxy for industrial contamination have also been employed in India [START_REF] Goddu | Magnetic properties of road dust from Visakhapatnam (India) -Relationship to industrial pollution and road traffic[END_REF]. Hence, the present study is undertaken in order to examine the environmental assessment of heavy metal concentrations and its impact in the coastal environment using magnetic techniques and to gain an understanding on the factors controlling metal concentrations and distributions along the east and west coast of India. et al., 2007).

Materials and methods

Study Area

East Coast-Geology-Climate

The outer limit of the eastern continental shelf of India lies at ~200 m (Fig. 1) and the inner shelf and the continental slope are covered by clastic sediments [START_REF] Rao | Distribution of suspended particulate matter in the waters of eastern continental margin of India[END_REF]. The outer shelf is covered by calcareous relict sediments and off the river mouths the shelf is covered by fine-grained terrigenous sediments. The shelf at the mouths of the rivers receives a large part of its sediment from the rivers Ganges, Brahmaputra and Mahanadi in the north, Godavari and Krishna in the central region, all forming fertile, heavily populated deltas. Sediment from the rivers has made the bay a shallow sea, and the waters have reduced the salinity of surface waters along the shore. Sediment input and annual discharge is less from the smaller rivers such as Pennar and Cauvery in the south [START_REF] Rao | India's water wealth: its assessment, uses and projections[END_REF][START_REF] Rao | Distribution of suspended particulate matter in the waters of eastern continental margin of India[END_REF].

The presence of the Deccan Basalt is observed along the tributaries of the Godavari and discharged into the Bay of Bengal from the east coast of the Peninsular India through the Mahanadi, Godavari and Cauvery river systems [START_REF] Sangode | Environmental magnetic studies on some Quaternary sediments of varied depositional settings in the Indian sub-continent[END_REF]. Deccan Province also covers the studied stations in the east coast of India [START_REF] Raman | Clay mineral distributions in the continental shelf sediments between the Ganges mouths and Madras, east coast of India[END_REF].

West Coast-Geology-Climate

The Indus river is the largest source of sediments in the Arabian Sea, which extend outward to a distance of ~1000-1500 km [START_REF] Rao | Provenance and distribution of clay minerals in the sediments of the western continental shelf and slope of India[END_REF]. It predominantly drains the Precambrian metamorphic rocks of Himalayas and to a lesser extent the semi arid and arid soils of West Pakistan and NW India [START_REF] Krishnan | Geology of India and Burma[END_REF]. Deccan Trap basalts (Fig. 1) are the predominant rock types cropping out in the Saurashtra and the drainage basins of the Narmada and Tapti rivers, which annually discharge ~ 60x10 6 tonnes sediment through the Gulf of Cambay [START_REF] Rao | India's water wealth[END_REF] where a semi-arid climate prevails. The Western Ghats are composed of basalts between Mumbai and Goa, and Precambrian granites, gneisses, schists and charnockites between Goa and Cochin [START_REF] Krishnan | Geology of India and Burma[END_REF]. The Ghats are located on the coast between Goa and Bhatkal but are 50-80 km from the coast south of Bhatkal. The source of sediments in rivers originating in the western part of India is derived the black "cotton soils", covering the Deccan Traps, a large percentage of which is mainly composed of montmorillonite. The drainage area in the upper reaches of these rivers is a montmorillonite-rich, zone but in the lower reaches, they drain through Precambrian formations that contain kaolinite rich soils that are of secondary significance in the shelf sediments derived from the Godavari and Krishna rivers [START_REF] Rao | Clay mineral distribution in the continental shelf and slope off Saurashtra, west coast of India[END_REF]. It is well established that source rock compositions and weathering mechanisms basically control the distinct geochemical compositions of sediments in the east and west coast of India [START_REF] Alagarsamy | Comparative studies on trace metal geochemistry in Indian and Chinese rivers[END_REF].

Sampling and Analysis

Surface sediments from the east and west coasts of India were collected using a Van Veen Grab/Snapper during several cruises of the R.V. Gaveshani (130, 139 and 143) (Fig. 1; Table 1). Subsamples were collected from the uppermost layer of the sediment taking care to minimize contamination. Samples were frozen after collection and later thawed, dried at 50-60 º C in an oven and disaggregated in an agate mortar before chemical treatment for total metal analysis. For element analysis, the known quantity (~0.2 g) of the powdered samples were digested with a mixture of concentrated HF-HNO 3 -HClO 4 for the total metal content [START_REF] Zhang | Riverine composition and estuarine chemistry of particulate metals in China -Weathering features, anthropogenic impact and chemical fluxes[END_REF]. Magnetic susceptibility measurements were carried out on sub samples which were dried at 40 °C and disaggregated. The magnetic susceptibility (χ, mass-specific), 'saturation' isothermal remanent magnetization (SIRM) after application of 1T, and then back field remanence measurements at -20 mT, -40 mT, -100 mT and -300 mT were measured on all samples. Both low (0.47 kHz) and high (4.7 kHz) frequency susceptibility (χ lf and χ hf respectively) were determined using a Bartington MS2 dual frequency susceptibility meter. Frequency dependent susceptibility (χ fd ) was calculated from the expression χ fd (%) = [(χ lf -χ hf )/χ lf ] x100. Anhysteretic remanent magnetization (ARM), here expressed as susceptibility of ARM (χ ARM ) was measured after demagnetion in an AF field of 100 mT inducing a DC biasing field of 0.04 mT using a Molspin AF demagnetizer and Minispin pulse magnetizer and measured on the Molspin Minispin magnetometer [START_REF] Walden | Use of mineral magnetic measurements to fingerprint suspended sediment sources: approaches and techniques for data analysis[END_REF]. Isothermal Remanent Magnetization (IRM) is the remanent magnetization acquired by a sample after exposure to, and removal from, a steady (DC) magnetic field. IRM depends on the strength of the field applied, which is often denoted by a subscript. It is also a function of the magnetic mineralogy and grain size. The maximum remanence that can be produced in a sample is called Saturation Isothermal Remanent Magnetization (SIRM). IRM is often used as an indicator for the presence of ferrimagnetic minerals, but antiferromagnetic minerals, such as hematite and goethite also contribute to IRM measurements where field in excess of 100 mT are used.

After a sample has acquired an IRM it is often possible to (partially) demagnetize the sample by exposing it to a magnetic field of reversed direction. Such a partial demagnetization can yield information about the ease of remanence acquisition, or the coercivity of a sample. The results are expressed as an S-ratio, for example, S 100 =IRM -100 /SIRM and S 300 =IRM -300 /SIRM where IRM -100, -300 denotes an IRM acquired in a reversed field of 100 mT and 300 mT respectively after SIRM acquisition. S-ratios can be used to gain information about the magnetic mineralogy [START_REF] Bloemendal | Rock magnetism of Late Neogene and Pleistocene deep-sea sediments: relationship to sediment source, diagenetic processes, and sediment lithology[END_REF]. Hard IRM (HIRM), broadly indicative of antiferromagnetic minerals, mainly hematite, was defined as HIRM = 0.5 x (SIRM+IRM-300 mT ).

Results and discussion

The parameters sensitive to fine-grained magnetic minerals (χ ARM and χ fd ), the ratio of χ ARM to χ lf and χ ARM /χ fd are discussed in this study. The concentration-sensitive magnetic parameters such as magnetic susceptibility (Table 2) and its concentration related parameters (Fig. 2a-c); Magnetic susceptibility vs. IRM 20 mT (Fig. 3); χ ARM/ χ lf vs. χ ARM /χ fd (Fig. 4); Fe, Cr, Cu and Ni vs. Al (Fig. 5 a-d); Fe, Cr, Cu; Ni vs. χ ARM (Fig. 6a-d); Fe, Cr, Cu and Ni normalized by χ ARM vs. Al normalization (Fig. 7) and Magnetic susceptibility vs. EF (metal) (Fig. 8) are presented along the east and west coast of India.

Variation of heavy metals -influence of particle size

Heavy metals vary significantly in the shelf sediment of east and west coast of India, especially in the case of Cr, Ni and Cu (Table 3). This variability could be due to particle size effects or anthropogenic influences. Finer sediments with abundant clay minerals, iron/manganese oxides as well as organic matter tend to show higher metal concentrations (Rae, 1997). In our study, the heavy metals (Fe, Cr, Cu and Ni) showed a more significant coefficient of determination with magnetic susceptibility in the east coast sediment than the west (Table 4a,b). It is observed that higher concentration of ferrimagnetic minerals are found in the east coast sediments and low concentration in the west coast of India. The comparable or even stronger dependence of heavy metals on Fe (Table 4a,b) is in accordance with the fact that iron and manganese oxides, which are commonly in close association with clay minerals, have strong absorptive capacity for metals [START_REF] Kersten | Normalization procedures for sediments contaminants in spatial and temporal trend monitoring[END_REF].

The higher χ fd (%) in the east coast showed that the stronger presence of super paramagnetic grains in these samples than in the west. Magnetic quotients are often useful indicators of the properties of certain minerals and domain states [START_REF] Lu | Study on the correlation of magnetic properties and heavy metals content in urban soils of Hangzhou City, China[END_REF]. F300 (IRM 300 mT /SIRM) and S300 (IRM -300 mT /SIRM) can be used as approximate indication of relative importance of canted antiferrimagnetic components and to distinguish ferrimagnetic component from antiferrimagnetic components respectively.

Remanent magnetic parameters

IRM 20 mT is expected to be approximately proportional to the multidomain (MD) grain content of ferrimagnetic components (magnetite). Hard IRM (SIRM-IRM 300 mT ) can be used as a rough guide to canted antiferrimagnetic (hematite) components in a sample. High values of IRM 20 mT and SIRM can be sometimes attributed to a greater accumulation of anthropogenic magnetic minerals [START_REF] Lu | Study on the correlation of magnetic properties and heavy metals content in urban soils of Hangzhou City, China[END_REF]. There is a highly significant and much stronger correlation between magnetic susceptibility and IRM 20 mT for east coast shelf sediment of India (Fig. 3; Table 4a) than the west (Table 4b). This suggests that the magnetic susceptibility of east coast sediment contains mainly from MD ferrimagnetic minerals derived from basalts. Magnetic susceptibility and other environmental parameters of the tributaries of the Godavari river sediment revealed the presence of high ferrimagnetic content as it is composed of weathering products of the Deccan Basalt [START_REF] Sangode | Environmental magnetic studies on some Quaternary sediments of varied depositional settings in the Indian sub-continent[END_REF].

Magnetic parameters as proxies of particle size

χ ARM is sensitive to single domain (SD, 0.03-0.07 µm) ferrimagnetic minerals [START_REF] Maher | Magnetic properties of some synthetic submicron magnetites[END_REF], while χ fd indicates the presence of fine viscous grains near the SP-SD boundary [START_REF] Maher | Magnetic properties of some synthetic submicron magnetites[END_REF][START_REF] Oldfield | The influence of particle size variations on the magnetic properties of sediments from the north-eastern Irish Sea[END_REF]. As SP and SD grains are of sub-micrometer size, if they occur as individual particles, it is expected that they would reside mainly in the finest fraction of sediments (e.g. <2 µm). The inter-parameter ratios χ ARM /SIRM and χ ARM /χ reflect variations in the ferrimagnetic grain size, with values peaking in the SD range [START_REF] Maher | Magnetic properties of some synthetic submicron magnetites[END_REF] and are used as grain size indicator of magnetic minerals [START_REF] Banerjee | A rapid method for magnetic granulometry with applications to environmental studies[END_REF][START_REF] Maher | Magnetic properties of some synthetic submicron magnetites[END_REF]. It is shown that similar correlations exist with χ ARM as to particle size (Zhang et al., 2007). χ is considered as a reflection of magnetic mineral concentration [START_REF] Thompson | Rock magnetic, mineralogical and microstructural characterization of fly ashes from Bulgarian power plants and the nearby anthropogenic soils[END_REF]).

The differences in ferromagnetic grain is explained by using the plot of χ ARM/ χ lf vs . χ ARM/ χ fd (Fig. 4). It is clearly indicating that finer grains present in the east coast of India showed correlation (R 2 =0.76) than the west. The west coast sediments showed a weaker χ fd values and hence the ratio indicated that it contains coarser magnetic grains.

Variability of heavy metals -Magnetic normalization

A normalizer must covary in proportion with the natural concentration of the metals of interest [START_REF] Roussiez | Background levels of heavy metals in surficial sediments of the Gulf of Lions (NW Mediterranean): an approach based on 133 Cs normalization and lead isotope measurements[END_REF]. A more significant coefficient of determination (R 2 = 0.91-0.98) between χ ARM and different heavy metals (Cu, Ni, Cr and Fe) was observed in the east coast sediments than those along the west coast of India ( Compared to the geochemical normalizer Al, χ ARM displays equally or even better coefficients of determination with heavy metals (Table 4a,b; Fig. 5a-d; Fig. 6a-d). It is also evident from the previous study that the relationships of heavy metal levels with χ ARM are quite similar to those with the <16 µm fractions (Zhang et al., 2007). The preferred association of heavy metals with fine-grained sediments is actually related to sediment mineralogy, since clay minerals and iron oxides, the important carrier phases of heavy metals, are dominantly associated with finer fraction of sediments (Rae, 1997). This relationship may therefore reflect the important role of iron oxides in controlling heavy metal concentrations in addition to aluminosilicate minerals. This is also reflected in the strong correlations between heavy metals and iron (Table 4a) in the case of east coast sediment samples. Taking into account the high capacity for remanent magnetization of magnetite, magnetic parameters based on remanent value will be dominated by the magnetite signal [START_REF] Walden | Use of mineral magnetic measurements to fingerprint suspended sediment sources: approaches and techniques for data analysis[END_REF]. Although magnetite is a minor component in iron oxide assemblages, fine-grained magnetite may be proportional to the major iron oxides [START_REF] Zhang | Diagenesis of magnetic minerals in the intertidal sediments of the Yangtze Estuary, China, and its environmental significance[END_REF]. These findings reinforce evidence from the study of Irish Sea coastal sediments [START_REF] Oldfield | 241 Am and 137 Cs activity in fine grained saltmarsh sediments from parts of the N.E. Irish Sea shoreline[END_REF][START_REF] Clifton | Derivation of a grain-size proxy to aid the modelling and prediction of radionuclide activity in saltmarshes and mud flats of the Eastern Irish Sea[END_REF] that χ ARM can be used as proxy for finer fraction of sediments and applied to the normalization of heavy metal concentrations for particle size effects.

Currently, Al is the most frequently used geochemical normalizer in estuarine and coastal sediments [START_REF] Kersten | Normalization procedures for sediments contaminants in spatial and temporal trend monitoring[END_REF]. 4a ,b). This similar relationship between Ni and χ ARM and the <16 µm particle size content is observed and suggested that Ni, χ ARM may be the preferred normalizer in the study area (Zhang et al., 2007). Overall, in order to compare the degree of pollution at different sites, normalization by χ ARM will give comparable results with that by Al, at least for metal Cu. In order to compare the degree of pollution at different sites, χ ARM normalization results are comparable with that by Al. Furthermore, analysis of Al requires a total digestion employing HF to destroy silicates within the sediment. As HF digestion would dissolve heavy metals contained in silicates, which are considered to be not related with anthropogenic source (often referred to as the 'residual' phase), a partial digestion with minimal extraction of 'residual' forms is considered to be able to provide more meaningful environmental information in pollution studies (Sutherland et al., 2004). In this case, if Al is to be used as a normalizer, heavy metal and Al determination will require two separate digestion procedures. Compared to geochemical analysis, the measurement of χ ARM is relatively straightforward, rapid and therefore cost effective [START_REF] Walden | Use of mineral magnetic measurements to fingerprint suspended sediment sources: approaches and techniques for data analysis[END_REF]). There may be a limit to the potential of this method in studies of sediment cores, due to the possibility of reductive diagenetic alteration of fine-grained magnetic minerals under reducing conditions [START_REF] Walden | Use of mineral magnetic measurements to fingerprint suspended sediment sources: approaches and techniques for data analysis[END_REF], and hence the reliability of χ ARM. However, it is unlikely that surface sediments under oxidizing conditions would be seriously influenced. Therefore χ ARM normalization can play an important role in similar coastal environments. In the case of the Irish Sea sediments, it is known that the high ARM values linked to the clay fraction are the result of bacterial magnetosomes growing in situ [START_REF] Oldfield | The influence of particle size variations on the magnetic properties of sediments from the north-eastern Irish Sea[END_REF], This could be the case here and it is evidenced from the relationship with heavy metals and its enrichment factor in the east coast of India than the west. Hence, there is the possibility that greigite could be present in areas where brackish rather than truly marine salinities prevail.

The presence of greigite and biogenic magnetite needs to be confirmed from GRM/RRM and SEM/TEM studies. [START_REF] Taylor | The geochemical evolution of the continental crust[END_REF].

Enrichment

It is observed that there is a good correlation between magnetic properties (e.g. magnetic susceptibility χ) and enrichment factor (EF of metals) in the east coast of India rather than the west suggesting the enrichment of ferrimagnetic mineral (Fig. 8a-d). χ is generally regarded as a reflection of magnetic mineral concentration [START_REF] Thompson | Rock magnetic, mineralogical and microstructural characterization of fly ashes from Bulgarian power plants and the nearby anthropogenic soils[END_REF]. χ vs. EF for metals showed R 2 values of Fe (0.55; 0.36), Cr (0.78; 0.17), Cu (0.78; 0.04) and Ni (0.34; 0.02) for the east coast of India and the west coast respectively.

In the Figs. 5,6,7,8, the four data points of the east coast (Station nos. 3024,3025,3026,3248 and 3249) lie close to the data points of the west coast. The clay mineral smectite mostly derived from the Deccan Trap basalts (Deccan Trap Province), occurs in both the region as this the Deccan Province also covers this stations in the east coast [START_REF] Raman | Clay mineral distributions in the continental shelf sediments between the Ganges mouths and Madras, east coast of India[END_REF][START_REF] Rao | Provenance and distribution of clay minerals in the sediments of the western continental shelf and slope of India[END_REF]. One of the end products of chemical weathering of Deccan Traps is smectites, which may be trapping K and thereby contributing to its limited mobility [START_REF] Das | Barium in Deccan Basalt Rivers: Its abundance, Relative Mobility and Flux[END_REF]. The major element chemistry of the clay fraction in sediment is controlled by the variation of K, Al, Mg, Si and Fe content. The K content of clayey sediments is primarily bound to clay minerals, potassium feldspar and mica. K is found to be less in the basalt. K is mainly held in illite and feldspar, therefore the variation in the K/Al ratio may reflect the variations in illite and feldspar present in the sediments. The ratios also reflect the mixing of a 'primary' end member composed of illite derived from micas (K 2 O/Al 2 O 3 =0.33-0.45) and a secondary end member chlorite (K 2 O/Al 2 O 3 =0).

A higher potassium content relative to Al in sediments implies greater feldspar contribution. K/Al values ranged from 0.11 to 0.16 in the above stations compared to the rest in the east coast of India and Ca/Al varied from 2.79 to 11.79 in the above mentioned stations in the east coast compared to the rest of the stations and its similarity with the west coast samples.

Conclusions

The magnetic properties used in this study for both normalization and for assessment of the degree of contamination from the samples of east and west coast of India showed a distinct variation in this coastal environment. The presence of finer ferromagnetic grain and enrichment of ferrimagnetic minerals are observed in the east than the west coast of India. Higher values of magnetic susceptibility, IRM 20 mT and SIRM are associated with the east coast shelf sediments suggest the presence of high ferrimagnetic content, which can be derived from the weathering products of the Deccan Basalts.

In shelf sediments from the east and west coast of India, the magnetic parameter χ ARM , which is sensitive to single domain (SD, 0.03-0.07 µm) ferrimagnetic minerals, displays more significant relationships with the heavy metals in the east coast sediments than the west. A comparison with Al, which is frequently used to compensate for particle size effects in pollution studies, indicates that in these samples χ ARM is an efficient normalizer for comparison of the degree of pollution at different sites. The strong association between χ ARM and heavy metals can be explained by the role of particle size and iron oxides in controlling metal concentrations. Furthermore, the rapid and non-destructive nature of mineral magnetic measurements means χ ARM can be easily determined, compared to other approaches such as the analysis of Al. When heavy metals are analyzed using a partial digestion method, which can provide more meaningful environmental information related to total pollutant determination, this magnetic approach will prove especially useful for environmental quality assessment. Magnetic parameters, χ, χ ARM , IRM 20 mT and SIRM, have shown that more significant relationship with the concentration of Fe, Cr, Cu and Ni in the east coast of India than the west. Linear relationship between magnetic mineral concentration-related parameters and the concentration of Fe, Cr, Cu and Ni makes it possible to use magnetic technique as a simple, rapid, and non-destructive tool for the assessment of heavy metals contamination in the shelf region. Although the co-variations presented in this study are rather convincing, it is clear that such approach rely on a careful calibration of the proxy i.e. directly comparing heavy metal concentrations and magnetic parameters for a selected set of samples. The calibration will obviously be site specific and must therefore be adapted to each study site.

The author is also thankful to Drs. S.W.A. Naqvi, E. Desa, former Director, NIO, Satish R. Shetye, Director, NIO and CSIR, New Delhi for their kind permission during the period of work. His Holiness Sri Kanchi Sankaracharya Swami's and Sri Sri Ravishankar's grace to complete this work is also highly appreciated. Frank Oldfield, Q. Liu, J. Bloemendal and two anonymous reviewers are thanked for the constructive reviews and suggestions. This is NIO contribution No.

Zhang, W., Yu, L., Lu, M., Hutchinson, S.M., Feng, H., 2007. Magnetic

  The subcontinent of India, geographically covers tropical to subtropical climatic zones and exhibits strong seasonality from warm humid to arid and cold arid conditions. The climate of the subcontinent is dominated by Monsoon systems; this region remains humid during summer and dry during winter. It has basically two main seasons, the wet and dry, instead of four seasons. The gradient of climatic zones is largely affected by the sharp altitudinal changes in the northern half where the Himalaya forms the major relief. The Indian subcontinent experiences mostly a tropical monsoon climate, with significant seasonal variations in rainfall and temperature. The Himalayas, acting as a barrier to the cold northerly winds from Central Asia, maintain the pattern of the Indian Ocean monsoon circulation. The Thar Desert allows oceanic atmospheric circulation and sediment dust-aerosol influx deep into the continent. The southwest (SW) monsoon is divided into Arabian Sea Branch of the SW Monsoon and Bay of Bengal Branch of the SW Monsoon. The Arabian Sea branch extends the low-pressure area over the Thar Desert in Rajasthan whereas the Bay of Bengal branch results in turbulence in the region due to the rapid altitudinal differences and narrowing orography. The Arabian Sea branch is roughly three times stronger than the Bay of Bengal branch. This branch of the monsoon moves northwards along the Western Ghats giving rain to the coastal areas west of the Western Ghats and the eastern parts of the Western Ghats do not receive much rain from this monsoon. The climate-induced sedimentation processes take place in large-scale during strong monsoons as cyclones occur in the coastal region (Sangode

  Solutions were analyzed for Al, Ca, K, Fe, Cr, Cu and Ni using ICP-AES (Model: PE-2000). Accuracy of the analytical methods was monitored by analysing standard reference materials (GSD-9) with study samples in every batch of analysis. Further checks were made through repeated analyses of the standard reference materials by ICP-AES (Model: PE-2000). Precision of the analyses was checked by triplicate analysis of standard materials. Calculated coefficients of variation were within ± ~1-8% for the different elements except K and within ± 4 for K of the certified ones (NIM-G).

  Fig. 7a-d demonstrates that normalization of heavy metals by χ ARM is positively correlated with normalization by means of Al. The weaker relationship for Fe/Al versus Fe/χ ARM and Ni/Al versus Ni/χ ARM is evident from the dissimilarity in scatter plots of Ni against these two normalizers (Fig. 7a-d; Table

Fig. 1 .Fig. 4 .

 14 Fig. 1. Location of sediment sampling stations in the coastal region of India.

Fig. 6

 6 Fig.6 (a-d).

Fig. 8

 8 Fig.8 (a-d).

  Table 4a,b). χ ARM /SIRM demonstrates strong coefficient of determination with heavy metals (Cu, Ni, Cr and Fe) in the east and the same is observed only Cu and Cr in the west coast sediment of India (Table 4a, b).

  Factor-Influence of grain size and mineralogy Here, as a common practice, Al is used as a normalizer, upon which enrichment factor (EF) were calculated. The following equation was used to estimate the EF of metals from each sediment station using Al as a normalizer to correct for differences in sediments grain size and mineralogy:

EF= (Me/Al) sample / (Me/Al) crustal average where (Me/Al) sample and (Me/Al) crustal average value are, respectively, the metal concentration (µg/g dw) in relation to Al levels (% dw) in sediment samples, and crustal average values taken from

Table 1 .

 1 approach to normalizing heavy metal concentrations for particle size effects in intertidal sediments in the Yangtze Estuary, China. Environmental Pollution 147, 238-244. Sample details and locations

Table 2 .

 2 Magnetic susceptibility in the surface sediments of east and west coast of India

Table 3 .

 3 Metal concentrations (in µg g -1 except Al and Fe in %) in the surface sediments of east and west coast of India Table4a. Coefficient of determination (R 2 ) for metals, potential geochemical and magnetic particle size proxies in surface sediments of east coast of India (R 2 > 0.25, p < 0.05, n = 14, bold figures are significant)

Table 4b .

 4b Coefficient of determination (R 2 ) for metals, potential geochemical and magnetic particle size proxies in surface sediments of west coast of India (R 2 > 0.40,

	Legend to Figures.
	p < 0.05, n = 8, bold figures are significant)

Fig.7 (a-d).

Acknowledgments

The author wish to express their sincere gratitude to the Chinese Ministry of Education and SKLEC/ECNU, Shanghai for financial support. I thank Professor J.

Zhang, Drs. W. Zhang and X. Han for their help in the sample analysis and discussion.


Nadezda Medvedeva

Yulia Polyak
email: yuliapolyak@mail.ru

Harri Kankaanpää

Tatyana Zaytseva

Microbial responses to mustard gas dumped in the Baltic Sea

Keywords: Bacteria, Baltic Sea, Chemical weapon, Sea dumping, Mustard gas, Degradation, Self purification CWA, chemical warfare agents, MGHPs, mustard gas hydrolysis products, TDG, thiodiglycol, OCS, organochlorine substances, TDGA, thiodiglycolic acid, GA, thioglycolic acid, HETA, [

Microbiological studies were carried out on chemical weapon dump sites in the Baltic Sea. The effect of mustard gas hydrolysis products (MGHPs) on marine microbiota and the ability of microorganisms to degrade MGHPs were studied. Many stations at the dump sites demonstrated reduced microbial diversity, and increased growth of species able to use mustard gas hydrolysis products as sole source of carbon. Significant amounts of MGHP-degrading bacteria were revealed in the nearbottom water. The MGHP-degrading microorganisms identified as Achromobacter sp., Pseudomonas sp., and Arthrobacter sp. were isolated. These microorganisms were capable of utilizing the major product of hydrolysis, thiodiglycol, as the sole source of carbon and energy. The bacteria were capable of metabolizing MGHPs at a low temperature. The metabolic pathway for thiodiglycol degradation was proposed.

The results suggest the potential for MGHPs biodegradation by naturally occurring populations of near-bottom-water and sediment microorganisms.

Introduction

Tens of thousands of tons of captured German chemical munitions have been dumped in the Baltic Sea since World War II. It is estimated that the chemical munitions contained about 13,000 tons of chemical warfare agents [START_REF] Helcom | Report on Chemical Munitions Dumped in the Baltic Sea[END_REF].

The dumping of munitions at sea by the allied countries was done in the southern and western Baltic, mainly in the Gotland Basin, the Bornholm Basin, the Little Belt and the Skagerrak Strait (Fig. 1).

In most cases the warfare agents were thrown overboard, either loose (bombs, shells) or in containers, but some ships were also sunk [START_REF] Helcom | Third Periodic Assessment of the State of the Marine Environment of the Baltic Sea, 1989-1993[END_REF]. During transportation to the dumping areas some munitions were thrown overboard while the ships were en route. Warfare agents are, therefore, assumed to be spread over a considerable area along the transport routes [START_REF] Schulz-Ohlberg | Tracing dumped chemical munitions in Pomeranian Bay (Baltic Sea) at former transport routes to the dumping areas off Bornholm Island[END_REF]. Chemical munitions have been spread even further by fishing vessels when trawling. Table 1 gives an overview of the quantities of chemical munitions and warfare agents dumped in the Baltic Sea and Skagerrak (Chemical munitions, 1993).

The largest number of chemical agents was dumped in the Bornholm Basin, east of the island of Bornholm. Fishermen still recover bombs, shells and lumps of mustard gas in their nets in this area [START_REF] Helcom | Chemical munitions in the southern and western Baltic Seacompilation, assessment and recommendations[END_REF].

Over the period 2000-2005 up to 1,010 kg of chemical munitions a year were netted by fishermen east of Bornholm [START_REF] Helcom | Third Periodic Assessment of the State of the Marine Environment of the Baltic Sea, 1989-1993[END_REF]. The netted munitions were completely corroded and resembled clay-like lumps of mustard gas. The highest number of incidents during the last 20 years was in 1991 with 103 catches amounting to 5,378 kg of chemical warfare agents. The most recent report of recovery of bomb containing mustard gas and tear gas lark I came from Sweden in 2007 (Åstot et al., 2007).

Most accidents with warfare agents so far have involved mustard gas. Because of the admixture of thickeners, viscous mustard gas is the only warfare agent occurring in large lumps that are mechanically relatively stable and can exist unchanged for decades on the sea bed. All injuries to fishermen have occurred when liquid or solid mustard gas has escaped from rusted munitions and come into contact with the skin.

The mustard gas causes severe blistering of the skin and irritates the eyes and throat [START_REF] Glasby | Disposal of Chemical Weapons in the Baltic Sea[END_REF].

Mustard gas is extremely toxic for all species [START_REF] Opresko | Chemical warfare agents: estimating oral reference doses[END_REF]. Because of its high lipid solubility mustard gas rapidly penetrates cells. It is able to alkylate DNA, RNA, and proteins affecting a variety of cell functions [START_REF] Papirmeister | Medical defense against mustard gas: Toxic mechanisms and pharmacological implications[END_REF]. The products of mustard gas hydrolysis have a wide spectrum of toxic activity on soil microbiota [START_REF] Medvedeva | The effect of mustard gas on the biological activity of soil[END_REF]. They are known to change significantly the number and the specific composition of soil microorganisms [START_REF] Medvedeva | Destruction of mustard gas hydrolysis products by Pseudomonas sp. Y -13[END_REF]. Contamination of soil with mustard gas causes significant ecological shifts, such as a decrease in the similarity index and the diversity index of microbiota, and a decrease in the dominance index. Microbial succession is accompanied by a decrease in the fermentative activity of soils [START_REF] Medvedeva | Microbiological destruction of mustard in soil[END_REF].

To assess the risk relating to dumped chemical weapons, it is necessary to evaluate the ability of the ecosystem itself (and microbiota as its component) for selfpurification of contaminated water and sediments. The ability of microorganisms to carry out mineralization of organic substances, in particular, toxicants of various chemical natures is well known and widely used in practice and scientific research [START_REF] Oldenhuis | Microbial Degradation of Chlorinated Compounds: Application of Specialized Bacteria in Treatment of Contaminated soil and Waste Water[END_REF]. Because of its high degree of toxicity, mustard gas is generally resistant to microbial attack. However, several studies have demonstrated that mustard gas is subject to biodegradation by a limited number of naturally occurring species.

Biodegradation of mustard gas by basidiomycetes, Coriolus versicolor and

Tyromyces palustris, has been reported [START_REF] Wariishi | Complete degradation of yperite, a chemical warfare agent, by basidiomycetes[END_REF]. Bacteria Micrococcus sp. 6-2 and Pseudomonas sp. 8-2 were able to metabolize the products of mustard gas hydrolysis, particularly those that were chlorinated [START_REF] Medvedeva | Microbiological destruction of mustard in soil[END_REF].

Bacterium Rhodococcus rhodochrous IQTS8 had the ability to utilize a chemical analog of mustard gas 2-chloroethyl-ethylsulfide as the only carbon and energy source for growth [START_REF] Kilbane | Biocatalytic detoxification of 2-chloroethyl ethyl sulfide[END_REF].

More studies are concerned with thiodiglycol (TDG), the major product of mustard gas hydrolysis [START_REF] Munro | The sources, fate, and toxicity of chemical warfare agent degradation products[END_REF]. Thiodiglycol is a fairly stable substance with weak toxicity. Nevertheless, it is one of the war gas precursors that must be destroyed, according to the Chemical Weapons Convention. In this connection, biodegradation of mustard gas is closely related to the utilization of TDG by microorganisms. Soil bacteria Pseudomonas pikettii SH18 and Alcaligenes xylosoxydans ssp. xylosoxydans SH42 [START_REF] Harvey | Biodegradation of chemical warfare agents: demilitarization applications[END_REF], Micrococcus sp. 6-2 and Pseudomonas sp. 8-2 [START_REF] Medvedeva | Microbiological destruction of mustard in soil[END_REF], Pseudomonas sp. Y-13 [START_REF] Medvedeva | Destruction of mustard gas hydrolysis products by Pseudomonas sp. Y -13[END_REF]), Alcaligenes xylosoxydans PGH 10 (Garcia-Ruiz et al., 2002), Alcaligenes xylosoxydans subsp. denitrificans TD 1 [START_REF] Ermakova | Bioutilization of tiodiglycol, the product of mustard detoxification: isolation of degrading strains, study of biodegradation process and metabolic pathways[END_REF], Alcaligenes xylosoxydans ssp. xylosoxydans SH91 [START_REF] Lee | Bioreactor strategies for the treatment of growth inhibitory waste: an analysis of thiodiglycol degradation, the main hydrolysis product of sulfur mustard[END_REF] are capable of using TDG as the sole source of carbon. While degradation of thiodiglycol has been characterized in terrestrial isolates, little is known about marine-degrading microorganisms.

The goal of our study was to investigate the effect of warfare agents on the Baltic Sea microbiota. We report in this paper on the isolation, characterization and metabolic activities of the bacteria able to use mustard gas hydrolysis products as the sole source of carbon and energy.

Materials and methods

Sampling

Seawater samples were collected from Baltic Sea chemical weapon dump sites in 1994-2007. The water samples were taken from the Bornholm dump site, Gotland Basin, and Skagerrak Strait. Water samples (100 ml) were collected from the whole water column using sterile bathometers. Near-bottom water (100 ml) was taken from Niemistö corers and put into sterile plastic flasks.

Enumeration of microorganisms

The number of heterotrophic microorganisms and content of heterotrophic microorganisms able to degrade mustard gas hydrolysis products (MGHPs) were analyzed immediately after sampling. The total number of microorganisms was determined after conservation with formalin using the standard technique [START_REF] Tsyban | Rukovodstvo po metodam biologicheskogo analiza morskoi vody i donnykh otlozhenii (Manual on methods of biological analysis of seawater and bottom sediments)[END_REF]. The number of heterotrophic microorganisms was estimated using inoculation on nutrient agar (1:10) [START_REF] Kuznetsov | Metody izucheniya vodnykh mikroorganizmov (Methods of studying aquatic microorganisms)[END_REF]. The incubation time was 3-5 days. The content of heterotrophic microorganisms able to degrade MGHPs was determined by inoculation on selective agar with water samples. The incubation time was 7 -10 days. The selective agar had the following composition: (NH 4 ) 2 SO 4 4.0 g/l, KH 2 PO 4 1.5 g/l, K 2 HPO 4 1.5 g/l, MgSO 4 0.2 g/l, NaCl 5.0 g/l and agar 20.0 g/l.

Mustard gas hydrolysis products (16 mg/l of organochlorine substances (OCS) and 0.35 mg/l of thiodiglycol) were added to this selective medium as the sole source of carbon.

Isolation of microorganisms, media, and culture conditions

Samples from the flasks were streaked onto plates containing mineral salts medium (4.0 g/l (NH 4 ) 2 SO 4 , 1.5 g/l KH 2 PO 4 , 1.5 g/l K 2 HPO 4 , 0.2 g/l MgSO 4 , 5.0 g/l NaCl) solidified with 1.5% agar. Mustard gas hydrolysis products, including 25 mg/l of organochlorine substances (OCS), were used as the sole source of carbon. The plates were incubated at 20 °C for up to 3 weeks. A rapidly growing, visually distinct colony and a separate, morphologically unique isolate were selected for further analysis and purified by repeated plating.

The selected strains of microorganisms were cultivated by three methods:

(1) By inoculation into a solid medium with the following composition (g/l):

(NH 4 ) 2 SO 4 4.0, KH 2 PO 4 1.5, K 2 HPO 4 1.5, MgSO 4 0.2, NaCl 5.0, thiodiglycol 2.0, CaCO 3 2.5, agar 20.0. The control parameters included the occurrence of growth and dissolution of CaCO 3 , which gives evidence of the formation of acidic products of thiodiglycol degradation;

(2) Cultivation under static conditions: in 20-ml test tubes filled with 17 ml of seawater containing MGHPs (32 mg OCS/l and 0.96 g/l TDG), 7.8 ± 0.2. The MGHPs solutions, pre-sterilized separately by filtering through membrane filters with a pore diameter of 0.22 m, were aseptically introduced into the medium, and simultaneously the test tubes were inoculated with the bacterial culture under scrutiny.

(3) Cultivation under aeration: in 250-ml Erlenmeyer flasks on a rotary shaker CERTOMAT BS-1 (B.Braun Biotech Int. GmbH, Germany) at 240 rpm on a medium consisting of (g/l): (NH 4 ) 2 SO 4 , 6.0; KH 2 PO 4 , 5.0; K 2 HPO 4 , 5.0; MgSO 4 , 0.2; and CaCO 3 , 10.0, containing MGHPs (20 -750 mg /l OCS; 6 -22.5 g/l TDG). Control parameters included the biomass growth, the change in of the culture broth, the presence of TDG, and the content of organochlorine substances.

The biomass growth was routinely monitored by measuring D 540 with a Genesys 10S spectrophotometer (Thermo Electron Corp., USA).

Considering that chemical hydrolysis of organochlorine substances can proceed in water medium, the studies of microbial degradation of MGHPs were carried out in comparison with the control probe (with the process performed under the same conditions but without microorganisms).

DNA extraction from bacterial cultures

DNA was extracted from the culture samples using incubation with 0.

DNA amplification

The primers were from Oligomer (Helsinki, Finland). Partial 16S rRNA genes were amplified from the culture samples using a forward primer 27F ( 5 and 68 ºC (7 min). The formation of PCR products (approx. 1,500 bp) was checked using agarose gel electrophoresis. The PCR products were stored at -20 ºC. Products used in cloning were stored for a maximum of 24 h.

Cloning and sequencing

The cloning of amplified DNA was performed with a Qiagen PCR cloning kit

Analytical methods

The quantitative determination of OCS was performed spectrophotometrically [START_REF] Franke | Lehrbuch der Militarchemie[END_REF]. Model 1740 BIO-RAD (USA) using Waters C-18 column, ( -Pack, 3.9×150 mm, particle size 5 µm) gradient mode (1.5 ml/min). The eluents were: A, 0.1% TFA and B, 100% acetonitrile. For the analysis, the following linear gradient was used: from 20% (0 min), 37% (8.5 min) at 20 °C. The total analysis time was 12 min. The compounds were detected at 313 nm and 338 nm.

Statistical analyses

All statistical analyses were performed with Statistica software (version 6;

Statsoft). The differences were considered significant at P<0.05.

Results and discussion

Effect of MGHPs on marine microbiota

Over the last few decades microbiological studies have been carried out at the chemical munitions dump sites in the Baltic Sea. In 1994-2000, we monitored the ecological situation at the dump sites under the Marine Ecological Patrol Program.

The analysis of the near-bottom water samples taken while we were sailing in the Skagerrak Strait, as well as near Liepaja and at the Bornholm dump site yielded 0.5 • 10 6 to 7.1 • 10 6 cells/ml for the total amount of microbiota, 0.4 to 6.5 mg/l for bacterial biomass, and 0.2 • 10 4 -4.9 • 10 4 cells/ml for the amount of heterotrophic microorganisms (Table 2).

At many stations significant amounts of bacterial cultures, able to grow in the medium containing mustard gas hydrolysis products (both chlorinated and nonchlorinated), were observed in the near-bottom water. However, there was no significant growth of these microorganisms following inoculation of water samples to the same agar medium without MGHPs. It was therefore concluded that the source of carbon for these bacteria were MGHPs. As Table 2 shows, at some stations they account for 20-98% of the total heterotrophic microbiota. No MGHP-degrading microorganisms were found in surface water layers and horizons 1 m above the sea bed. The formation of an enrichment culture of microorganisms at some of the observation stations can be due to the appearance in the environment of a new compound, mustard gas, acting as a selective agent. The largest number of "abnormal" sites was observed in the Bornholm Basin. MGHP-degrading microorganisms appeared at about 40% of stations in this area [START_REF] Medvedeva | Ecological inspections of the Baltic Sea chemical weapon dump sites[END_REF].

In 2007, the microbiological studies were carried out in the Bornholm dump site area in the framework of the EC-FP6 project "MERCW" (Modelling of Environmental Risks Related to Sea-dumped Chemical Weapons). The studies focused on the primary dumping area located in the southern part of the Bornholm Basin, east of the island of Bornholm, in water depths ranging from 70 to over 95 m (Fig. 2). Four shipwrecks were identified in this area in 1994-2000 [START_REF] Paka | Research of dumped chemical weapons made by R/V "Professor Shtokman" in the Gotland, Bornholm & Skagerrak dumpsites[END_REF]. The wrecks were extensively damaged. One of the wrecks was covered by trawling nets, which were suspended in the water column above the wrecks by intact floats (Fig. 3A). Partly corroded artillery missiles were observed on the deck of the other wreck (Fig. 3B). The microbiological survey was undertaken in two research areas (Fig. 2): the first research area was in the vicinity of the shipwrecks and the second in the zone marked by the detection of chemical warfare agents on previous research trips [START_REF] Paka | Sea-dumped chemical weapons: state of problem[END_REF].

Near-bottom water contained MGHP-degrading microorganisms that were up to 58% of the total number of heterotrophic microorganisms at more than a half of the stations (Fig. 4). Most of such "abnormal" points with a high concentration of MGHPdegrading microorganisms were detected in the first research area near the sunken ships. 7 "abnormal" points were found in the second research area, where CWA had been detected previously.

The "abnormal" sites of mustard gas possibly leaking into the environment differed from other stations in the species diversity of microorganisms (Table 3).

The microbiota of the near-bottom water out of the dump site (30 km) was represented by microorganisms predominantly of the genera Pseudomonas, Bacillus, Sphingomonas, Flavobacterium, Micrococcus, Alcaligenes, Achromobacter, with Pseudomonas and Bacillus genera dominating (the latter accounted for up to 25% of the total number of heterotrophs).

The primary dump site showed a reduced diversity of bacteria. At many stations the dominance of a minimal number of Pseudomonas, Bacillus, Arthrobacter, and

Achromobacter species in near-bottom water was observed. The species diversity of the microbial population at the dump site was reduced compared with other sites, most probably because of an increase in the number of MGHP-degrading 10 microorganisms. The reduction in the species diversity of bacterioplankton suggests a decrease in the stability of the biosystem as a whole. The genera previously implicated in the degradation of mustard gas hydrolysis products include the genera Alcaligenes [START_REF] Lee | Bioreactor strategies for the treatment of growth inhibitory waste: an analysis of thiodiglycol degradation, the main hydrolysis product of sulfur mustard[END_REF][START_REF] Mulbry | Biodegradation of chemical warfare agents[END_REF],

Isolation and characterization of MGHP-degrading microorganisms

Pseudomonas [START_REF] Harvey | Biodegradation of chemical warfare agents: demilitarization applications[END_REF][START_REF] Medvedeva | Soil bacterium Pseudomonas sp.: destroyer of mustard gas hydrolysis products[END_REF], and Micrococcus [START_REF] Medvedeva | Microbiological destruction of mustard in soil[END_REF]. These bacteria have been isolated from contaminated terrestrial environments or from soil enrichment cultures by using mustard gas hydrolysis products. The present results indicate there are also marine MGHPdegrading bacteria of genera Arthrobacter, Achromobacter, and Pseudomonas.

Although Achromobacter (also known as Alcaligenes) and Pseudomonas strains have been characterized as microorganisms capable of metabolizing the products of mustard gas hydrolysis, MGHP-degrading Arthrobacter has been found for the first time.

MGHP-degradation range

Comparative cultivation of degrader microorganisms was carried out (Table 4).

The highest MGHPs-dechlorinating capability was exhibited by Achromobacter sp.

75-1. The maximum dechlorination rates were 26 mg OCS/g biomass/day under static conditions, and under aeration, 37 mg OCS/g biomass/day. All strains had shown lower dechlorination rates under static conditions than under aeration. The process of 11 dechlorination was accompanied by evolution of the stoichiometric amount of mineral chlorine in the medium.

The degrader microorganisms also grew more actively under aeration. For example, the biomass gain for Achromobacter sp. 75-1 under aeration was 8 times that under static conditions.

Figure 5 shows the dynamics of growth of Achromobacter sp. 75-1 in a medium supplemented with MGHPs as the sole carbon and energy source. The growth of microorganisms correlates with the decrease in thiodiglycol content. The complete utilization of MGHPs (60 mg OCS /l and 0.18% TDG) is observed after 6 days.

Figure 6 shows the effect of different concentrations of mustard gas hydrolysis products on the bacterial growth under aeration. The growth of strain 75-1 (Fig. 6a)

was proportional to the concentration of OCS up to 360 mg/l in the medium.

However, concentrations above 360 mg/l induced a lag phase in bacterial growth.

This effect was more pronounced at OCS concentrations above 500 mg/l.

Concentrations equal to or greater than 750 mg/l totally suppressed the growth.

Increasing concentrations of OCS adversely affected the biomass growth of both strain 93-2 (Fig. 6b) and strain 23-3 (Fig. 6c). The concentration of OCS also affected The highest specific growth rate (µ max ) of 0.89 day -1 was at 360 mg of OCS/l for strain Achromobacter sp. 75-1 (Fig. 7).

The highest growth rates for strains Arthrobacter sp. 23-3 and Pseudomonas sp.

93-2 were 0.76 -0.73 day -1 at 120 and 60 mg of OCS/l, respectively. The growth rates decreased in proportion to an increasing OCS concentration, and the lowest growth rate of 0.1 h -1 was observed at 730 mg of OCS/l (strain 75-1) and 260 mg of OCS/l (strains 93-2 and 23-3). The growth of strains 75-1, 93-2 and 23-3 is characterized by the peak of the maximum possible growth rate. Such growth is typical of the growth of microorganisms on a toxic substrate [START_REF] Karasevitch | Osnovi selekzii microorganizmov, utilizirujushich sinteticheskiye organicheskiye soedineniya [Fundamental of Selection of Synthetic Organic Substances Utilizing Microorganisms[END_REF].

The concentration of MGHPs produces a considerable effect on the process of the dechlorination of OCS, as well as on the growth of bacteria. The ultimate concentration of OCS (360 mg per litre) for the growth of Achromobacter sp. 75-1 corresponds to the highest rate of dechlorination. The increase in the concentration of OCS above this level leads to the sharp decrease in the rate of dechlorination as well as in the specific growth rate.

The growth rate of Arthrobacter sp. 23-3 and Pseudomonas sp. 93-2 reaches its maximum value at an OCS concentration of 60 -120 mg/l, but the most rapid dechlorination takes place at higher concentrations of the toxicant (240 mg/l). At this concentration the growth of bacteria is practically absent, and the whole substrate is consumed for energy of maintenance. The low maximum growth rate, long generation time and considerable outlays for the maintenance of both cultures are characteristic of the growth of microorganisms on toxic substrates [START_REF] Pirt | Principles of Microbe and Cell Cultivation[END_REF].

Effect of environmental conditions on biodegradation

The pH range for good growth of degrader bacteria under aeration is 7.0 to 8.0 (Fig. 8). There was little difference in growth in cultures with an initial pH between 6.0 and 8.0 (averaging about 20%). The growth of Achromobacter sp. 75-11 and

Arthrobacter sp. 23-3 was slightly more rapid at a neutral pH. There was a gradual decrease in the growth rate of Arthrobacter sp. as the initial culture pH was increased from 8 to 9. Pseudomonas sp. 93-2 grew better in a slightly alkaline rather than acidic condition. The maximum growth rate of strain 93-2 was observed at pH 8.

We found that the fastest growth of degrader-microorganisms occurred at 20 and 25 °C (Table 5). The growth rate at 20°C (µ max = 0.23 -0.31 g/g/d) was about 3-5 times more rapid than the growth rate at 5 °C. Cultures also took longer to degrade TDG at 5 °C (Fig. 9, Fig. 10, Fig. 11). The time required for thiodiglycol degradation was 30-45 days at 20 and 25 °C, but 180-210 days were required to degrade TDG at 5

°C. The temperature had little effect on the biomass yield (Y) of the degradermicroorganisms (Table 5). However, the data indicated that the optimum temperature for biomass yield was 20-25 °C.

The ability of microorganisms to degrade thiodiglycol at a low temperature is particularly important in cold Baltic Sea conditions.

Metabolic pathways

The metabolic pathway for the degradation of MGHPs by marine bacteria was studied. The intermediate products from the growth of strains 93-2, 23-3, and 75-1 in the medium containing thiodiglycol as a source of carbon were analyzed to elucidate the possible pathways of degradation. The disappearance of TDG was accompanied by an accumulation of acids. HPLC analysis revealed thiodiglycolic acid (TDGA)

and [(2-hydroxyethyl)thio]acetic acid (HETA) in the culture supernatants of all the strains (Fig. 12). Thiodiglycol sulphoxide was not detected.

The appearance of acid products suggests that biodegradation of TDG begins with oxidation of its primary alcoholic groups. Earlier, oxidation of the thiodiglycol molecule to TDGA by alcohol dehydrogenase was reported [START_REF] Lee | Biocatalytic transformation of [(2-Hydroxyethyl)thio]acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxidans ssp. xylosoxidans (SH91)[END_REF][START_REF] Ermakova | Bioutilization of tiodiglycol, the product of mustard detoxification: isolation of degrading strains, study of biodegradation process and metabolic pathways[END_REF]. Incubation of all strains for more than 7 days led to a decrease in HETA and TDGA concentrations to an undetectable level.

TGA and acetate were detected in the culture supernatant from the growth of all bacteria on the TDGA-containing medium, which suggests C-S bond cleavage in TDGA to yield these products. -mercaptoethanol was detected as an intermediate product of TDG transformation in only two strains: Pseudomonas sp. 93-2 and 13

Achromobacter sp. 75-1.

These results indicate that the major pathway for TDG degradation by all the strains studied is the oxidation of its primary alcoholic groups to HETA, TDGA and TGA with the formation of acetate upon further transformation. This pathway for TDG degradation is similar to that reported earlier for Alcaligenes and Pseudomonas [START_REF] Lee | Biocatalytic transformation of [(2-Hydroxyethyl)thio]acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxidans ssp. xylosoxidans (SH91)[END_REF][START_REF] Garcia-Ruiz | Transformation of 15 thiodiglycol by resting cells of Alcaligenes xylosoxidans PGH 10[END_REF][START_REF] Medvedeva | Destruction of mustard gas hydrolysis products by Pseudomonas sp. Y -13[END_REF]. However, the formation of -mercaptoethanol was detected for Pseudomonas sp. 93-2 and Achromobacter sp. 75-1. Previously, this intermediate product has also been detected among the products of TDG transformation by soil bacterium Pseudomonas sp. [START_REF] Medvedeva | Soil bacterium Pseudomonas sp.: destroyer of mustard gas hydrolysis products[END_REF]. Thus, the results suggest two possible pathways of the TDG biodegradation, one of which yields HETA, TDGA and TGA, and the other,mercaptoethanol, which is further transformed to TGA (Fig. 13).

Conclusions

Microbiological analysis of near-bottom water samples near the sea bed of the dump sites for chemical weapons in the Baltic Sea revealed high levels of microorganisms able to use mustard gas hydrolysis products as source of carbon especially at the Bornholm dump site, as well as in the Gotland Deep, and at the Skagerrak dump site. The "abnormal" sites showed reduced diversity of microbiota.

  75 ml of 1% (w/v) potassium ethyl xanthogenate containing 1% SDS (w/v), 20 mM EDTA, 100 mM TRIS and 800 mM CH 3 COONH 4 , pH 7.4 (70 °C, 1 h), followed by vortexing (10 s), a second incubation (4 °C, 30 min) and centrifuging (17 530 x g, 4 °C, 10 min). DNA was precipitated using 0.75 ml of 2-propanol (22-24 °C, 10 min), pelleted (12 000 x g, 22-24 °C, 10 min), washed twice with 200 µ l of 70% (v/v) ethanol, air dried (20-24 °C) and dissolved in 50-200 µ l of 10 mM TRIS, 1 mM EDTA, pH 8.0 (TE buffer). The DNA isolates were further purified using Montage cartridges (Millipore, Billerica, CA, USA) and stored at -20°C until amplification with polymerase chain reaction (PCR).

  '-AGAGTTTGATCCTGGCTCAG-3'; general bacterial primer; Lane 1991) and a reverse primer 1525R (5 -AAGGAGGTGATCCAGCC-3 ). PCR was performed in 20 l scale with 1 l of template DNA, 0.4 M of forward and reverse primers, 200 M of each deoxynucleoside triphosphate (dNTP, Sigma-Aldrich), 1 U of MasterTaq polymerase (Eppendorf, Hamburg, Germany), 1 x MasterTaq polymerase buffer containing 1.5 mM Mg(OAc) 2 , 0.5 x Taq Master Enhancer (Eppendorf), and 3 g l -1 BSA. A Thermo PCR Sprint (Milford, MA, USA) thermal cycler was used. The initial step in the temperature programme was 70 ºC (5 min) and 94 ºC (5 min). DNA polymerase was added during the initial 70 ºC period (hot start). The following denaturing, annealing and extension programmes were used: 9 cycles of [94 ºC (45 s), 57 ºC (45 s), 68 ºC (2 min)], 24 cycles of [94 ºC (45 s), 54 ºC (45 s), 68 ºC (2 min)]

(

  pDrive plasmid vector) at the Sequencing Laboratory of the Institute of Biotechnology, University of Helsinki. After a 24-h culturing bacterial pellets were lysed and plasmid DNA purified using 2-propanol precipitation and centrifuging, and stored in TE (-20 ºC). The plasmids were further amplified with primers UP and RP (5'-TAACGCCAGGGTTTTCCCAGTCACGA-3' and 5'-GTGAGCGGATAACAATTTCACACAGG-3'), amplifying the insert from the plasmid. The products were isolated, subjected to sequencing reactions (two directions; primers t7 and sp6; 5'-TAATACGACTCACTATAGGG-3' and 5'-ATTTAGGTGACACTATAGAA-3') and sequenced (ABI3730 XL 48-capillary automated sequencer). Sequences without overlapping or unclear bases were subjected to preliminary analysis using the Basic Local Alignment Search Tool (BLAST) programme (National Center for Biotechnology Information, Bethesda, MD, USA).2.7. ChemicalsA model mixture of mustard gas hydrolysis products was prepared by heating an aqueous mixture of 0.65 M TDG (ICN 103039 RT, 98% purity) and 0.65 M HCl at 90°C for 8 h. The resulting MGHPs mixture contained 2 500 ± 300 mg/l OCS and (0.57 ± 0.01) TDG. Thiodiglycolic acid (TDGA), thioglycolic acid ( GA) andmercaptoethanol were obtained from Merck KgaA, Germany. All the chemicals were of the highest purity commercially available. [(2-hydroxyethyl)thio]acetic acid (HETA) was synthesized and purified according to[START_REF] Black | Metabolism of thiodiglycol (2,2' thiobis-ethanol): isolation and identification of urinary metabolites following intraperitoneal administration to rats[END_REF].

  Thiodiglycol, thiodiglycolic acid, [(2-hydroxyethyl)thio]acetic acid and thiodiglycol sulphoxide content were determined by reversed-phase HPLC on an HP 1090 Hewlett-Packard chromatograph under the following conditions: temperature 25 °C, Luna (Phenomenex) column; flow rate 1.5 ml/min; detection at 215 nm; eluent 5% acetonitrile and 0.1% TFA (trifluoroacetic acid); sample volume 20 µl. Hydrolysis of MGHPs was also determined by measuring the release of chloride ions with a model 96-17 B combination electrode (Orion Research Inc., USA). The electrode was calibrated by adding known concentrations of sodium chloride standards to the mineral salts medium used in the experiments. Uninoculated flasks were run in each experiment as controls. Thioglycolic acid was detected by using a Hewlett-Packard 1090 HPLC liquid chromatograph with a diode array detector (USA) equipped with a Vydac column (USA) 218TP54 (RP-18, 4.6×250 mm, particle size 5 µm) under isocratic conditions with 20 mM tetrabutyl ammonium (TBA) buffered with 20 mM Na phosphate (pH=6.0), containing 15% (v/v) acetonitrile at 50 °C. The flow rate was 1.5 ml/min and detection of the compounds was at 210 nm, 250 nm, and 265 nm. The injection volume was 10 µl and the analysis lasted 10 min. Detection of -mercaptoethanol after its modification by Elman (1959) was carried out on a Hewlett-Packard 1090 HPLC liquid chromatograph with UV Monitor

  During the period 1994-2007, over 200 MGHP-degrading bacteria were obtained from the near-bottom water samples taken at the Gotland Basin, Skagerrak Strait, and Bornholm dump site. Three of the most active strains capable of utilizing mustard gas hydrolysis products, 75-1, 23-3 and 93-2, were obtained from the Bornholm dump site. All three isolated bacterial strains were aerobic, oxidase and catalase-positive,two were gram-negative, and one was gram-positive. The gram-positive strain 23-3, established by phenotypic assays, underwent a rod-coccus morphological change. Strain 75-1 was the gram-negative, nonmotile rod of ~ 0.5 by 1.5 to 2.0 µm. Strain 93-2 was the gram-negative, rod-shaped motile organism about 1.0 by 1.5 µm. Motility was imparted by a single polar flagellum. All three strains grew between 0 o C and 30 o C, with the optimal growth temperature around 25 o C. The 16S rRNA gene sequences (420-470 bp) showed the greatest similarities (99%) with strain 75-1 and were related to those of the Achromobacter genera. Strain 23-3 was most closely related to bacteria from the genus Arthrobacter (with sequence similarities ranging from 98 to 99%). The results indicated that strain 93-2 was phylogenetically closest to Pseudomonas.

  the lag phase before the beginning of TDG degradation by Arthrobacter sp. 23-3 and Pseudomonas sp. 93-2. The higher the concentration of OCS, the longer the lag phase lasted before the start of degradation (data not shown). The lag period before TDG degradation at high OCS concentrations was shown to be a lag phase in cell growth. Concentrations of OCS equal to or greater than 240 mg/l totally suppressed the growth of Arthrobacter sp. 23-3 and Pseudomonas sp. 93-2.

  High numbers of MGHP-degrading microorganisms in near-bottom water indicate a possible leakage of CW agents into the environment and contamination of water and sediments with mustard gas and products of its hydrolysis in the research areas. Bacterial cultures isolated from near-bottom water were able, with differing biodegradation rates, to dechlorinate and utilize mustard gas hydrolysis products as the source of carbon. The Arthrobacter, Achromobacter and Pseudomonas genera cultures were the most active. The main features of Arthrobacter sp. 23-3, Achromobacter sp. 75-1, and Pseudomonas sp. 93-2 are the tolerance to mustard gas hydrolysis products, and the ability to degrade a high concentration of OCS. They can use thiodiglycol as the sole source of carbon and energy. The isolated bacteria can completely dechlorinate OCS at up to 750 mg/l in 4-9 days. But the complete utilization of the products of hydrolysis (mainly thiodiglycol) is observed at lower concentrations of OCS in the medium (up to 360 mg). The major pathway of TDG degradation was oxidation to HETA, TDGA and TGA plus eventual formation of acetate. The degradation of TDG by Pseudomonas sp. and Achromobacter sp. 75-1 can also occur via C-S bond cleavage yielding β -mercaptoethanol. The microorganisms cannot only detoxify the contaminants, but also utilize them completely. The rate of biodegradation of mustard gas hydrolysis was temperaturedependent but also occurred at low temperatures. The ability of microorganisms to carry out the mineralization of thiodiglycol at a low temperature is important for the marine environment, particularly for the dump sites, where water temperatures rarely exceed 7°C. The results suggest the potential for self-purification of contaminated water and sediments by natural populations of microorganisms.

Fig. 1 -Fig. 4 -Fig. 6 -

 146 Fig. 1 -Dump site locations map: (A) the Bornholm site; (B) the Gotland Deep site; (C) the Skagerrack site Fig. 2 -Location of the chemical munitions dump site at the Bornholm Basin. The circle marks the primary dump site area; the rectangle marks the larger dump site boundary. 1: the first research area, 2: the second research area Fig. 3 -Images of the shipwrecks at the Bornholm dump site (after Paka and Spridonov, 2001). A: wreck covered with fishing nets. B: missile found on the deck of the wreck

Fig. 7 -

 7 Fig. 7 -Effect of OCS on dechlorination rate (A) and growth rate (B) of Achromobacter sp. 70-11, Arthrobacter sp. 41-3 and Pseudomonas sp. 3-1. The data are means ± SD of three independent measurements.

Fig. 9 -Fig. 10 -

 910 Fig. 9 -Effect of temperature on degradation of TDG by Achromobacter sp. 75-1 under static conditions: (a) TDG concentration; (b) biomass growth (OD 540 ). The symbols indicate the means of triplicate experiments. Vertical bars indicate standard deviations.

Table 1

 1 Quantities of the chemical munitions and chemical war gases dumped in the southern and western Baltic Sea and Skagerrak (west of Sweden)

	Area	Quantities of	Types of war gases
		chemical munitions	chemical war gases
	Bornholm	from 35,300 tons	from 5,300 tons	Mustard gas, viscous mustard
	Basin	(reliable data)	to 6,500 tons	gas, Clark I, II, adamsite,
	(E of	to 43,300 tons		chloroacetophenone (possibly,
	Bornholm)	(unreliable data)		phosgene, nitrogen mustard)
	Area SW of	up to15,000 tons	2 250 tons	no data
	Bornholm	(unreliable data)
	Gotland Basin 2,000 tons	300 tons	no data
	Little Belt	5,000 tons	750 tons	tabun, phosgene
	Skagerrak (W	app. 20,000 tons	-	mustard gas
	of Sweden )	(quantity not verified)		(other types not verified)

Table 2

 2 Microbiological characteristics of the Baltic Sea near-bottom water Number of heterotrophs Dumping site

		Total bacterial	Bacterial
		number, 10 6 /ml	biomass, mg/ml	Total, 10 4 /ml	MGHP-degrading microorganisms, % of total number of heterotrophs
	Gotland Basin	1.3 -4.2	1.1 -3.0	0.6 -2.0	up to 20
	Bornholm Basin	0.7 -5.3	0.6 -4.9	0.5 -3.2	up to 85
	Skagerrak Strait	0.5 -7.1	0.4 -6.5	0.2 -4.9	up to 98

Table 3

 3 Taxonomic structure of the bacterioplankton in Southern Baltic Sea Out of the dump site Bornholm dump site

	Bacillus	Pseudomonas
	Pseudomonas	Bacillus
	Alcaligenes	Achromobacter
	Sphingomonas	Arthrobacter
	Achromobacter	Others: <15%
	Flavobacterium
	Arthrobacter
	Acinetobacter
	Micrococcus
	Flectobacillus
	Planococcus
	Others: < 25%

Table 4

 4 Comparative cultivation of the degrader microorganisms on the MGHPs containing medium (60 mg OCS/l and 0.18% TDG, 20°C) -maximum specific dechlorination rate, mg OCS/g biomass/day.Data are means ± SD of three independent measurements.

	Microorganisms	Static conditions Biomass gain (mean ± SD), % q max * (mean ± SD), mg/g/d	Aeration (15 mM O 2 /l/h) Biomass gain q max * (mean ± (mean ± SD), % SD), mg/g/d
	Pseudomonas sp. 93-2	10.4±0.6	9±1	212.1±6.4	17±2
	Arthrobacter sp. 23-3	17.1±0.8	19±2	226.0±9.4	26±3
	Achromobacter sp. 75-1	24.0±1.3	26±3	267.5±12.2	37±4
	* q max

Table 5

 5 Effect of temperature on growth of degrader-microorganisms under static conditions Y -biomass yield, g biomass produced [dry wt]/ g substrate utilized. b µ max -maximum specific growth rate, g /g biomass/day. Data are means ± SD of three independent measurements.

	Microorganisms	Temperature,	Y a (mean ± SD),	µ max	b (mean ± SD),
		°C	g/g		g/g/d
		5	0.37±0.02	0.07±0.01
	Achromobacter sp. 75-1	20	0.39±0.03	0.23±0.02
		25	0.39±0.04	0.20±0.01
		5	0.23±0.02	0.07±0.01
	Arthrobacter sp. 23-3	20	0.29±0.02	0.34±0.03
		25	0.31±0.03	0.30±0.02
		5	0.32±0.03	0.09±0.01
	Pseudomonas sp. 93-2	20	0.33±0.02	0.31±0.02
		25	0.35±0.03	0.35±0.03

a

Acknowledgements

The studies were financially supported by the EC-FP6 project MERCW "Modelling of Ecological Risks Related to Sea-Dumped Chemical Weapons" (Contract Nr. INCO-CT2005-013408). The authors wish to thank Director Lars Paulin (Institute of Biotechnology, sequencing laboratory, Helsinki, Finland) for laboratory and sequencing facilities and Mr. Janne Härkönen for assistance with cloning and sequencing work. The authors thank Mr. Evgenii Protasov for the highperformance liquid chromatography analyses and helpful discussions.