Kevin Wh Kwok

Kenneth My Leung

Emmanuel Flahaut

Shuk Jinping Cheng

Han Cheng

Chronic toxicity of double-walled carbon nanotubes to three marine

Keywords:

Chronic toxicity of double-walled carbon nanotubes to three marine organisms: influence of different dispersion methods

Following the recent proliferation of nanotechnology, a diverse array of engineered nanomaterials has been produced over the last decade. Engineered nanomaterials are commonly defined as materials with at least one dimension of 100 nm or less [1]. Such materials typically possess nanostructure-dependent properties (e.g., special chemical, mechanical, electrical, optical, magnetic and/or biological attributes), which make them desirable for commercial or medical applications [1]. Carbon-based nanomaterials and nanoscale metal oxides are two major types of engineered nanomaterials suitable for diverse purposes.

Carbon nanotubes (CNTs) are one of the most promising carbon-based nanomaterials. There are many variations in the structure of CNTs, such as single-walled and multiwalled CNTs of different length and diameter. CNTs have superior electrical conductivity, thermal conductivity and mechanical strength over conventional materials, making them desirable for many commercial products [2]. Potential applications of CNTs include electronic devices, composite materials, hydrogen storage and field emission devices [2]. Recently, double-walled CNTs (DWNTs) are attracting increasing attention because they possess combined advantageous properties of single-walled CNTs (SWNTs) and multiwalled CNTs (MWNTs) [3]. For example, DWNTs offer excellent field emission properties because they have both the low threshold voltage for electron emission as SWNTs and high emission stability similar to MWNTs [4]. Generally, the outer wall of DWNTs protects the inner one, thus maintaining the main mechanical properties even in case of covalent fictionalization. Hence, DWNTs have a morphology very similar to SWNTs (in terms of length and diameter), with improved mechanical properties.

The application of CNTs is vast and is expanding. The annual number of CNT-related patents filed between 1997 and 2001 increased from approximately 2500 to approximately 21,000 [2]. The annual number of DWNTrelated patents filed also increased from eight to approximately 250 between 2000 to 2008 [101]. A total of 65 tons of CNTs and nanofibers was produced in 2004 and their production is projected to increase by 60% per year [102]. Furthermore, CNTs will become more affordable as their prices are anticipated to decrease by factors of 10-100 [102]. The CNT market in 2007 was estimated to be worth approximately US$79 million, with a compound annual growth rate of 74% [103]. Applications of CNTs are already beginning to be realized in commercial consumer products, and are already in sports equipment, electronics and as additives to plastics [104].

With the increasing production and utilization of these unique nanomaterials, CNTs and their by-products are likely to enter aquatic environments. Possible release pathways include industrial discharge, landfill leachate, accidental spill or marine dumping of contaminated mud. Besides such unintentional releases, intentional Aim: Double-walled carbon nanotubes (DWNTs) are found in a variety of consumer products, but there are no ecotoxicity data of DWNTs into marine organisms. Materials & methods: Chronic toxicity of DWNTs was investigated with the diatom Thalassiosira pseudonana, copepod Tigriopus japonicus and medaka Oryzias melastigma. DWNTs were dispersed using sonication (so-DWNTs) and stirring (st-DWNTs) for comparison. Results: The median aggregation size (0.89 µm 2 ) of so-DWNTs was smaller than that of st-DWNTs (21.8 µm 2 ). Exposure to DWNTs led to growth inhibition of T. pseudonana with EC 50 s of 1.86 and 22.7 mg/l for so-and st-DWNTs, respectively. Population growth of T. japonicus was reduced to 0.1 mg/l for so-DWNTs and 10 mg/l for st-DWNTs. Growth inhibition in O. melastigma was observed at 10 mg/l for so-DWNTs but not for st-DWNTs. Conclusion: Given that so-DWNTs are consistently significantly more toxic than st-DWNTs, dispersion method and size of aggregations should be considered in DWNT toxicity testing.

release for environmental remediation [5] and wastewater treatment [6] may also introduce these nanomaterials into the environment. Therefore it is important to gain an understanding of their potential risks of exposure in ecosystems. In addition, current regulation on disposal or discharge of engineered nanomaterials is unclear. The lack of understanding and regulation regarding the potential impacts of nanomaterials to the environment has been a key concern under scrutiny by environmental scientists [7].

Properties of nanomaterials are, however, different from the bulk properties of the constituent chemicals [8] and therefore require separate investigation for their ecotoxicities The copepod Amphiascus tenuiremis exposed to 10 mg/l SWNTs exhibited significant reduction in lifecycle survivorship, fertilization successes and molting success [11]. In addition, previous mammalian studies demonstrated many adverse effects of CNTs but such toxic effects generally require a long period to develop [START_REF] Lam | Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation[END_REF][START_REF] Warheit | Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats[END_REF].

It is very common for nanomaterials to agglomerate into large aggregates due to strong van der Waals forces, especially for CNTs [START_REF] Thess | Crystalline ropes of metallic carbon nanotubes[END_REF]. From the view point of ecotoxicology, it is more important to study the environmentally realistic form of nanomaterial aggregates [START_REF] Handy | Toxic effects of nanoparticles and nanomaterials: implications for public health, risk assessment and the public perception of nanotechnology[END_REF]. Typically, solvents/ surfactants or sonication are used to disperse CNTs in aqueous suspensions for the majority of previous toxicity studies. Nevertheless, these two dispersion methods are environmentally unrealistic. Using solvents/surfactants have an additional disadvantage, as the solvent itself can cause significant toxicity under some circumstances [10].

Stirring may be a more environmentally realistic option to disperse CNTs in aqueous solution, despite not being able to disperse CNTs as well as the previous two methods [START_REF] Ham | An explanation of dispersion states of single-walled carbon nanotubes in solvents and aqueous surfactant solutions using solubility parameters[END_REF]. Different methods of dispersion may result in different degrees or kinds of agglomeration of CNTs. For example, it has been shown that CNTs of different aggregation sizes can cause different toxicities to mammalian cell lines [START_REF] Raja | Impact of carbon nanotube exposure, dosage and aggregation on smooth muscle cells[END_REF][START_REF] Wick | The degree and kind of agglomeration affect carbon nanotube cytotoxicity[END_REF]. Nonetheless, there is no similar in vivo study to uncover the influence of different dispersion methods on the CNT toxicity to marine organisms.

The objective of this study was to evaluate the ecotoxicity of DWNTs in a marine environ ment. Their toxicities were tested with three common saltwater model species: the diatom Thalassiosira pseudonana, the copepod Tigriopus japonicus and the medaka fish Oryzias melastigma. To understand the potential effect of dispersion method on DWNT toxicity, toxicities of DWNTs dispersed by stirring and sonication were compared.

Methods

Nanomaterial suspension preparation & characterization

Double-walled carbon nanotubes used in this study were produced by catalytic chemical vapor deposition method, and the synthesis and characterization was published in an earlier study [3]. In brief, the batch contained about 80% DWNTs and 20% SWNT. The inner and outer diameters of DWNTs were approximately 0.7 to 2.1 nm, respectively [3]. The DWNT sample contains ca. 90% elemental carbon with traces of elemental cobalt (ca. 3.3 wt.%) and molybdenum (ca. 1.1 wt.%), as well as oxygen [3]. Cobalt contaminants were present but were in the form of carbonencapsulated nanoparticles [3]. The carbon-encapsulated metal nanoparticles are tightly protected from the outside environment [START_REF] Flahaut | CCVD synthesis and characterization of cobalt-encapsulated nanoparticles[END_REF] and no leak of metal is expected in the experimental conditions used in this work. Autoclaved, filtered artificial seawater (30 ± 0.5%; pH 7.9-8.0) was used for all the experiments and was prepared by dissolving the appropriate amount of artificial sea salt in distilled water followed by filtration (sea salt: Tropic Marine, Germany; filter: glass microfiber filter GF/C 47 mm circle, Whatman, UK). Stock solutions were prepared by dispersing 0.0100 g of DWNTs, determined gravimetrically (fivedigit electronic balance, Sartorius, Germany) in 100 ml of artificial seawater. DWNTs were dispersed in artificial seawater using either sonication (Branson 5200 ultrasonic bath) for 2 h (so-DWNT) or continuous stirring with a magnetic stirrer ( 200 rpm) for 2 weeks (st-DWNT). All the suspensions were examined under transmission electron microscopy (TEM; FEI CM208, Eindhoven, The Netherlands) at 80 kV to obtain the distribution of their aggregation size.

Experimental organisms

Diatom (T. pseudonana), culture was maintained in autoclaved f/2-Si medium under 25 ± 1°C, 30 ± 0.5%, pH 7.8-8.1 and a 16:8 h photoperiod. Harpacticoid copepods (T. japonicus), were maintained under identical environmental conditions as those set for T. pseudonana and were fed with the diatom Skeletonema costatum. The medaka fish (O. melastigma) were maintained at 25 ± 2°C, 30 ± 2%, pH 7.8-8.1 and a 16:8 h photoperiod, and fed with commercial fish feed (Kyorin, Japan) and newly hatched artemia larvae ( 24 h old).

96-h algal growth inhibition test

As DWNTs are opaque, growth inhibition may be an effect of light blockage caused by the materials instead of direct toxicity. In order to differentiate between the two effects, the experimental set-up was specially designed as shown in FIGURE 1A. Cell concentration of T. pseudonana culture at exponential growth phase (96 h old) was determined using a hemocytometer (Neubauer Improved, Precolor HGB, Germany). An appropriate amount of diatom culture was added to each of the 3.5 ml autoclaved test glass tubes with 2 ml autoclaved f/2-Si medium to obtain an initial algal concentration of 10 4 cells/ml. Different concentrations of DWNTs (0.1, 1, 10, 50 and 100 mg/l) were added to the test tubes. The algal solution was placed in a 3.5 ml glass test tube and this test tube was placed inside a small glass vial containing 6 ml test solution.

When the toxicity of DWNTs was tested, the nanomaterials were mixed with the algae and only water was added to the outside glass vial (FIGURE 1B). When the light-blocking effect was being investigated, the materials were added to the solution of the outside glass vial (FIGURE 1C). In order to understand whether this additional glass vial itself would affect the growth of the algae, an additional water control without the glass vial was included. There were four replicates in the control and each of treatment groups (at various nominal concentrations of DWNTs). All tests were conducted at 25 ± 0.5°C, 30 ± 0.5%, pH 7.8-8.0 and a 16:8 h photoperiod.

Copepod lifecycle test

The lifecycle toxicity of the nanomaterials to T. japonicus was investigated using a modified ASTM protocol (ASTM E-2317-04 [START_REF] Kwok | Copper toxicity in the marine copepod Tigriopus japonicus: low variability and high reproducibility of repeated acute and life-cycle tests[END_REF]). In 96-well microplates, less than 24-h-old nauplii were placed individually in wells loaded with 200 µl of test solution and 2 µl of algal solution containing 10 7 diatom S. costatum cells/ml, determined by the use of a hemo cytometer (Neubauer Improved, Precolor HGB, Germany). All treatments had eight replicates and each replicate contained 12 nauplii. Test solutions of appropriate nominal concentrations (0.1, 1, 10, 30, 50 and 100 mg/l) were prepared by serial dilution.

Tigriopus japonicus undergoes anamorphic development with 12 distinctive post-embryonic developmental stages (six naupliar stages, five copepodid stages and sexually dimorphic adults). Females usually bear multiple broods of eggs after single mating and a typical brood size of 30-50 [START_REF] Raisuddin | The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics[END_REF]. Under laboratory conditions, T. japonicus individuals developed from eggs will take at most 21 days to deposit their first egg brood [START_REF] Raisuddin | The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics[END_REF].

To ensure consistency of exposure, copepods were placed into new microwells containing fresh tests solutions and algal food every 96 h. Copepods were monitored daily for life stage, mortality, development and sex determination. When the copepods reached copepodid stage 5, male and female copepods were paired up and mated within each replicate for each treatment. Lifecycle testing was terminated after the release of the second brood of eggs from each mating pair. Test periods for individual microplate bioassays ranged from 20 to 30 days to account for treatment-specific developmental delays. All tests were conducted at 25 ± 0.5°C, 30 ± 0.5%, pH 7.8-8.0 and a 16:8 h photoperiod.

Mortality, duration of larval development (from 24 h nauplii to develop into copepodid stage 1), time to first reproduction (from 24 h nauplii to the development of the first egg brood) and the intrinsic rate of increase (r m ) were used as end points. The r m integrates mortality, reproductive output and frequency; it was computed using the method described in Kwok et al. and is an estimate of the intrinsic population growth rate [START_REF] Kwok | Copper toxicity in the marine copepod Tigriopus japonicus: low variability and high reproducibility of repeated acute and life-cycle tests[END_REF].

Histological investigation

Copepods were found to ingest and defecate CNTs in an earlier study [11]. In this study, TEM was used to confirm if DWNTs can penetrate into the gut and cuticle cells of T. japonicus after exposure. Ten T. japonicus were exposed for 7 days to either 100 mg/l of so-DWNTs or 100 mg/l of st-DWNTs. Sample preparation was modified from Au et al. [START_REF] Au | Relationship between ultrastructural changes and EROD activities in liver of fish exposed to benzo[a]pyrene[END_REF]. After the exposure period, the copepods were fixed under room temperature in a solution containing 2% paraformaldehyde, 2.5% glutaraldehye, 0.05% calcium chloride 0.4 M sucrose in 0.1 M cacodylate buffer at pH 7.6. Samples were decalcified with 5% EDTA in 0.1 M cacodylate buffer with 0.2 M sucrose. After fixing, samples were rinsed in buffer and distilled water, dehydrated in a graded ethanol series and acetone, then infiltrated and embedded in Spurr's resin. Samples were postfixed in 2% osmium tetroxide (OsO 4 ) in 0.1 M cacodylate buffer in the dark for 3 h.

Five of the ten individuals were randomly chosen and semi-thin dorsal-ventral sections (thickness 0.3 mm) stained with toluidine blue were prepared for orienting the tissue for ultrathin sectioning. Ultrathin sections (75 nm) were prepared with an ultramicrotone (Leica Reichert Ultracut UCT, Austria) with a diamond knife (Diatome, Switzerland) and double stained with Reynold's lead citrate and uranyl acetate. Three random sections per individual were examined under TEM at 80 kV. The gut and cuticle of the copepods were examined in each section, as they represent the major part in contact with the test materials via the dietary and dermal exposure pathways, respectively.

Medaka larvae test

The medaka fish O. melastigma has been proposed as a saltwater fish model for ecotoxicology [START_REF] Kong | Development of a marine fish model for studying in vivo molecular responses in ecotoxicology[END_REF]. O. melastigma is small and easy to culture and breed, exhibits uniform growth, completes its whole lifecycle in seawater and has a similar biology to its freshwater counterpart Oryzias latipes [START_REF] Kong | Development of a marine fish model for studying in vivo molecular responses in ecotoxicology[END_REF]. Newly born (<16 h posthatch) fish larvae were collected and maintained until 48 h posthatch. The 48 h posthatch larvae were individually placed in a 5 × 5 compartment square petri dish and exposed to various levels of DWNTs (10, 50 and 100 mg/l). Fish were fed with artemia larvae (24 h old) daily ad libitum and test solutions were renewed every 48 h. Survival of fish was recorded daily. At days 1, 4, 7, 10 and 14 of exposure, photos of all individual fish of all treatments were taken and their total lengths were measured using software Image J [105].

Tests were conducted at 25 ± 0.5°C, 30 ± 0.5%, pH 7.8-8.0 and a 16:8 h photoperiod.

Statistical ana lysis

For each test, one-way ana lysis of variance (ANOVA) followed by Dunnett's test was used to determine the no observed effect concentration and lowest observed effect concentration of each of the end points compared with the control. Proportion data were arcsine transformed before running the ANOVA [START_REF] Zar | Biostatistical Analysis[END_REF]. For conciseness, length data of medaka of different treatments were only compared at day 7 and day 14 using ANOVA. Bonferroni correction for multiple testing was used [START_REF] Zar | Biostatistical Analysis[END_REF], and for cases with significant effects (after the Bonferroni correction), their corresponding no observed effect concentration and lowest observed effect concentration were determined. Regression analyses were carried out for growth data of the algae, and mortality data of the copepods against the concentration of DWNTs and 50% effect concentration (EC 50 ) values were calculated using appropriate parametric regression models. Analysis of covariance (ANCOVA) was conducted to compare the toxicity between so-DWNTs and st-DWNTs using the measured end points as the dependent variable, the dispersion method as the fixed factor and c oncentration as the covariate.

Results

Aggregation size distributions Double-walled carbon nanotubes aggregated in filtered artificial seawater and representative TEM images of DWNT aggregates are shown in FIGURE 2 at two different magnifications. Aggregate size distributions of the DWNTs determined by TEM showed that so-DWNTs had a significantly smaller aggregate size than st-DWNTs (FIGURE 2E ; Mann-Whitney test: U = 195; p < 0.05). The median aggregation size of so-DWNTs (0.89 µm 2 ) was 24-fold smaller than that of st-DWNTs (21.8 µm 2 ).

96-h algal growth inhibition tests

Regardless of the dispersion method, DWNTs caused a significant decrease in algal growth in all test concentrations (FIGURE 3 &TABLE 1). ANCOVA further revealed a significant difference in elevation between the dose-response relationships of so-DWNTs and st-DWNTs (F 1, 36 = 12.0; p 0.001), supporting the hypothesis that so-DWNTs were more toxic (FIGURE 3) to the algal [than st-DWNTs]. The difference in their toxicity was also revealed by their EC 50 values (so-DWNT: 1.86 mg/l; st-DWNT: 22.7 mg/l; TABLE 1). Despite the fact that DWNTs were opaque and dark in color (FIGURE 1), no significant growth inhibitory effects were detected in the light-blocking controls for st-DWNT and so-DWNTs (FIGURE 3 &TABLE 1).

Copepod lifecycle test

The mortality of the copepod T. japonicus exposed to DWNTs was generally low. Even in the highest concentration treatments of both dispersion methods, less than 30% mortality was recorded (FIGURE 4). A significant increase in total lifecycle mortality, compared with that of the water control, was observed at 50 and 100 mg DWNT/l regardless of the dispersion method used (FIGURE 4 &TABLE 1). Most of these mortality events occurred during the larval period (from 24-h-old nauplii to copepodid stage 1). In general, so-DWNTs were more toxic than st-DWNTs to the copepod (FIGURE 4). The former caused a significantly higher larval mortality at 30, 50 and 100 mg/l, but only the latter resulted in a significantly higher larval mortality at 100 mg/l (FIGURE 4). The dose-response relationships of st-and so-DWNTs were unclear and no parametric regression model could be appropriately fitted for toxicity data generated from either DWNT (p 0.05).

Exposure to so-DWNTs and so-DWNTs did not affect larval development (FIGURE 5A &TABLE 1) but delayed the first reproduction by 1-3 days, depending on the concentration and dispersion method (FIGURE 5). For st-DWNTs, a significant delay of first reproduction was detected from 10 mg/l (TABLE 1). A higher impact by so-DWNTs was detected, as the significant delay of first reproduction was detected from 0.1 mg/l (FIGURE 5).

The r m was the most sensitive end point to DWNT exposure. A significant decrease of r m was found at 10 mg/l for st-DWNTs and 0.1 mg/l or more for so-DWNTs (TABLE 1). Regardless of the dispersion method, the r m of the copepods at the highest DWNT concentration decreased to approximately half of that of the control (FIGURE 5). Toxicities of DWNTs dispersed by the two different methods were compared using ANCOVA. Of all the end points measured, only time to first reproduction showed a significant interaction between the dispersion method and concentration of DWNTs (F 1, 108 = 52.0; p 0.001).

Histological investigation

Light microscopy observation of T. japonicus during the lifecycle experiment indicated that T. japonicus clearly ingested DWNTs. Distinct black aggregates were evidenced only inside the gut of exposed animals (FIGURE 6A) and the same black aggregates were also observed in their fecal pellets (FIGURE 6B) (and never in the case of the control animals, as in FIGURE 6C), suggesting that at least part of the ingested DWNTs were removed by defecation. DWNT aggregates were also evidenced in the lumen of the mid-gut region by TEM (FIGURE 6D &E). These findings concur with a previous study on copepods exposed to SWNTs [11]. However, in TEM ana lysis, DWNTs were not evidenced in either the cuticle or the cuticle cells in all examined slides (data not shown).

Medaka larval test

There were no significant differences in mortality between medaka larvae exposed to so-DWNTs and st-DWNTs (FIGURE 7 &TABLE 1). Nonetheless, the growth (in terms of total length) of the fish exposed to so-DWNTs was significantly lowered after 14 days at 50 and 10 mg/l, respectively (FIGURE 8 &TABLE 1). By contrast, st-DWNTs led to significant inhibition on the growth of the medaka only at 100 mg/l after 14 days of exposure (FIGURE 8B & TABLE 1) . No apparent malformation was observed in medaka larvae exposed to either type of DWNT.

In summary, exposure to DWNTs resulted in growth inhibition of T. pseudonana with EC 50 s of 1.86 and 22.7 mg/l for so-and st-DWNTs, respectively. The population growth of T. japonicus was significantly decreased at 0.1 mg/l or more for so-DWNTs and 10 mg/l for st-DWNTs, attributable to increased mortality and impaired reproduction. Growth inhibition in O. melastigma was observed at 10 mg/l or more for so-DWNTs and 100 mg/l for st-DWNTs. Consistently, so-DWNTs were significantly more toxic than st-DWNTs across all test species.

Discussion

This study demonstrated that chronic exposure to DWNTs led to a significant decline of population growth of the copepod T. japonicus, and detectable growth inhibition on the diatom T. pseudonana and the medaka larvae O. melastigma. DWNTs have different toxicities towards the three test marine species, with a higher toxicity observed towards the copepod, followed by the diatom and the medaka. In contrast to the current chronic toxicity test results, short-term lethal-based acute toxicity tests, which are often used to understand CNT toxicity in previous studies (see [8]), are clearly not sensitive enough to detect such ecologically relevant sublethal toxicity effects.

Previous researchers have suggested that CNT toxicity was primarily caused by oxidative stress in in vitro studies using mammalian cell cultures [START_REF] Shvedova | Exposure to carbon nanotube material: assessment of nanotube cytotoxicity using human keratinocyte cells[END_REF][START_REF] Pulskap | Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants[END_REF]. In a full lifecycle chronic toxicity study, Templeton et al. suggested that CNTs can cause oxidative stress and thus toxicity in the estuarine copepod A. tenuiremis [11].

Although some researchers suggested that oxidative stress induced by CNTs may be caused by the presence of metal contaminants (or impurities) in the CNT instead [START_REF] Pulskap | Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants[END_REF][START_REF] Kegan | Direct and indirect effects of single walled carbon nanotubes on RAW 264.6 macrophages: role of iron[END_REF], this is unlikely the cause of oxidative stress observed in the present study. More likely the reason is that the cobalt present in the DWNTs was carbon encapsulated and therefore unlikely to be bioavailable [3,[START_REF] Flahaut | CCVD synthesis and characterization of cobalt-encapsulated nanoparticles[END_REF].

Based on the current results, DWNTs are possibly able to induce both oxidative and physical stress, which may result in the initiation of defense mechanisms. This may explain the adverse developmental and reproductive effects of DWNTs observed in T. japonicus. Induction of defense mechanisms may divert energy otherwise used for reproduction or development [START_REF] Kwok | Acclimation effect and fitness cost of copper resistance in the marine copepod Tigriopus japonicus[END_REF]. Confusion of DWNTs with food particles may also have contributed to the adverse effects previously mentioned by lowering the food intake efficiency of T. japonicus, as this was also found in other copepod [11] as well as with similar DWNT samples in the case of amphibians [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF][START_REF] Mouchet | Assessment of the potential in vivo ecotoxicity of double-walled carbon nanotubes (DWNTs) in water, using the amphibian Ambystoma mexicanum[END_REF]. The present results also confirmed that DWNTs were not only ingested by T. japonicus but also observed inside their gut cells. Meanwhile, gill damage or blockage by DWNTs [10] may be an alternative explanation to DWNT toxicity observed in the medaka fish.

To date, there are no published studies on CNT toxicity to marine algae. This is the first study demonstrating that DWNTs can lead to a relatively high (growth inhibition) toxicity to the diatom T. pseudonana, highlighting the need for further studies in this area. Van Hoecke et al. reported that silica nanoparticles can adhere to the outer cell surface of algal cells and hence cause physical disruption on cell surface membrane functions and reduction of light penetration to the cell, resulting in growth inhibition [START_REF] Van Hoecke | Ecotoxicity of silica nanoparticles to the green alga Pseudokirchneriella subcapitata: importance of surface area[END_REF]. More research will be required to understand the toxic mechanism of DWNTs to microalgae, such as the diatom.

As shown in this study (FIGURE 2), dispersing DWNTs in water by sonication (so-DWNTs) resulted in smaller aggregate size when compared with dispersion by stirring (st-DWNTs). The median aggregation size (0.89 µm 2 ) of so-DWNTs was much smaller than that of st-DWNTs (21.8 µm 2 ). The distinctive aggregate size may offer an explanation to the differences in toxicities of so-DWNTs and st-DWNTs. Consistently, so-DWNTs were found to be more toxic to the three test organisms than st-DWNTs. Indeed, sonication has been suggested to increase the toxicity of SWNTs [START_REF] Oberdörster | Ecotoxicology of carbon-based engineered nanoparticles: effects of fullerene (C 60 ) on aquatic organisms[END_REF]. However, the cause of this increased toxicity, whether by better dispersion in the solution, or potential alteration of CNT structure or surface properties, will require further investigation. DWNTs dispersed by stirring should represent a more environmentally realistic estimate of their toxicity. The influence of the addition of a surfactant was not discussed here, as it was not desirable to interfere with the intrinsic toxicity of the surfactant itself. However, the presence of surfactants (either natural [START_REF] Hyung | Natural organic matter stabilizes carbon nanotubes in the aqueous phase[END_REF] or man-made [START_REF] Datsyuk | Double-walled carbon nanotube dispersion via surfactant substitution[END_REF]) cannot be ruled out and should play an important role in terms of separation of the CNTs (e.g., debundling and dispersion of aggregates) and stabilization of the obtained suspensions, even at a very low concentration [START_REF] Datsyuk | Double-walled carbon nanotube dispersion via surfactant substitution[END_REF].

Conclusion

Sonication could better disperse DWNTs in artificial marine water than stirring. The median aggregation size (0.89 µm 2 ) of so-DWNTs was smaller than that of st-DWNTs (21.8 µm 2 ). Chronic toxicity of so-DWNTs was also higher than st-DWNT to the diatom T. pseudonana, copepod T. japonicus and medaka O. melastigma. T. pseudonana with EC 50 s of 1.86 and 22.7 mg/l for so-and st-DWNTs, respectively. Population growth of T. japonicus was reduced by 0.1 mg/l or more for so-DWNTs and 10 mg/l for st-DWNTs. Given that so-DWNTs are consistently significantly more toxic than st-DWNTs, the use of sonication probably leads to over estimation of the ecotoxicity of DWNTs.

Future perspective

The search for dispersion methods that produce environmentally realistic data regarding the aggregation and structure of nanomaterials will be a key area of research and a prerequisite to the understanding of ecotoxicity of nanomaterials.

  [1]. Most of the recent literature on the toxicity of CNTs has focused on in vitro and mammalian models, and these studies have confirmed that CNTs can have toxic effects by inducing oxidative stress and respiratory toxicity [1,9]. Ecotoxicity of CNTs has also been partially revealed in a few studies on fish and invertebrates. For example, the rainbow trout Oncorhynchus mykiss exposed to 0.1 mg/l SWNTs displayed increased ventilation rate and altered behavior [10]. Hatching of zebrafish embryos was significantly delayed in test solution with SWNTs at 120 mg/l [8].

Figure 1 .

 1 Figure 1. Experimental set-up for algal growth inhibition test. (A) An illustrated diagram of the set-up. (B) A set-up testing the toxicity of double-walled carbon nanotube and (C) a set-up testing the light-blocking effect of double-walled carbon nanotube.

Figure 2 .

 2 Figure 2. Transmission electron microscope photos and size distributions of both sonicated and stirred double-walled carbon nanotubes aggregates. Transmission electron microscope photos of so-DWNT aggregates under (A) low magnification and (B) high magnification; and st-DWNT aggregates under (C) low magnification and (D) high magnification. From the transmission electron microscope analysis, aggregation size distributions of both so-and st-DWNTs are determined and shown in (E). so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

Figure 3 .

 3 Figure 3. Relative growth of Thalassiosira pseudonana (mean ± 1 standard deviation) exposed to different nominal concentrations of sonicated double-walled carbon nanotubes (open circles) and stirred double-walled carbon nanotubes (open triangles).These results are compared with the light-blocking effect control groups for so-DWNTs (circles) and st-DWNTs (triangles). so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

Figure 4 .

 4 Figure 4. Mortality (mean ± 1 standard deviation) of Tigriopus japonicus exposed to different concentrations of (A) so-and (B) st-DWNTs. so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

Figure 5 .

 5 Figure 5. Development time and intrinsic rate of increase (mean + 1 standard deviation) of Tigriopus japonicus exposed to different concentrations of stirred and sonicated double-walled carbon nanotubes. (A) Larval development time from less than 24-h-old nauplii to copepodid stage; (B) time from less than 24 h to first reproduction; and (C) intrinsic rate of increase. Asterisks indicate different levels of statistical difference from the seawater control: *p < 0.05; **p < 0.01; ***p < 0.001 (Dunnett's multiple comparison test). so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

Figure 6 .

 6 Figure 6. The results of histological investigation of the fate of double-walled carbon nanotubes in the copepod Tigriopu japonicus. (A) Ingested double-walled carbon nanotubes (DWNTs) visible as black clump inside the gut of Tigriopus japonicus. (B) DWNT agglomerates visible as black aggregates in fecal pellet of T. japonicus. (C) Fecal pellet of a control copepod. (D & E) Transmission electron microscope images of DWNT agglomerates inside the gut of T. japonicus. Arrows indicate positions of DWNT agglomerates.

Figure 7 .

 7 Figure 7. Mortality of medaka fish larvae (mean ± 1 standard deviation) exposed to different concentrations of stirred and sonicated double-walled carbon nanotubes. so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

Table 1 . ANOVA results for individual toxicity tests and toxicity end point.

 1

	Test species	End point	F value	p-value LOEC (mg l -1 )	NOEC (mg l -1 )	EC 50 (mg l -1 )
	so-DWNT
	Diatom	Algal growth † F 5,18 = 95.0 † <0.001 † 0.1 †	ND †	1.86 †
		Algal growth	F 5,18 = 5.1	<0.01	ND	ND	No fit ‡
		(light blocking)
	Copepod Total mortality † F 6,49 = 7.3 † <0.001 † 50 †	30 †	No fit † ‡
		Larval	F 6,49 = 10.7 † <0.001 † 30 †	10 †	No fit † ‡
		mortality †
		Larval	F 6,49 = 4.0	<0.01	ND	ND	ND
		development
		Time to first	F 6,49 = 152 † <0.001 † 0.1 †	ND †	ND †
		reproduction
		Intrinsic rate of	F 6,49 = 33.4 <0.001	0.1	ND	No fit ‡
		increase
	Medaka Mortality	F 3,8 = 8.8	<0.01	ND	ND	ND
		Length at d7	F 3,8 = 9.3	<0.01	ND	ND	ND
		Length at d14 † F 3,8 = 16.6 † <0.001 † 10 †	ND †	ND †
	st-DWNT
	Diatom	Algal growth † F 5,18 = 46.0 † <0.001 † 0.1 †	ND †	22.7 †
		Algal growth	F 5,18 = 2.2	>0.05	ND	ND	No fit ‡
		(light blocking)
	Copepod Total mortality F 6,49 = 3.8	<0.01	ND	ND	No fit ‡
		Larval	F 6,49 = 10.9 † <0.001 † 100 †	50 †	No fit † ‡
		mortality †
		Larval	F 6,49 = 2.4	<0.05	ND	ND	ND
		development
		Time to first	F 6,49 = 51.5 † <0.001 † 10 †	1 †	ND †
		reproduction †
		Intrinsic rate of	F 6,49 = 20.1 † <0.001 † 10 †	1 †	No fit † ‡
		increase †
	Medaka Mortality	F 3,8 = 3.0	>0.05	ND	ND	ND
		Length at d7	F 3,8 = 1.3	>0.05	ND	ND	ND
		Length at d14 F 3,8 = 7.6	<0.01	ND	ND	ND

†

Indicates cases with a significant effect after Bonferroni correction for multiple tests ( = 0.05).

‡

No fit: No significant regression model could be fitted. d: Day; EC 50 : Effect concentration 50%; LOEC: Lowest observed effect concentrations; ND: Not determined; NOEC: No observed effect concentrations; so-DWNT: Sonicated double-walled carbon nanotube; st-DWNT: Stirred double-walled carbon nanotube.

important study showing population level effect of carbon nanotubes.

  Rao GP, Lu C, Su F: Sorption of divalent metal ions from aqueous solution by carbon nanotubes: a review. Sep. Purif. Technol. 58, 224-231 (2007). Donaldson K, Aitken R, Tran L et al.: Carbon nanotubes: a review of their properties in relation to pulmonary toxicology and workplace safety. Toxicol. Sci. 92, 5-22 (2006). 10 Smith CJ, Shaw BJ, Handy RD: Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): respiratory toxicity, organ pathologies, and other physiological effects. Aquat. Toxicol. 82, 94-109 (2007). 11 Templeton RC, Ferguson PL, Washburn KM, Scrivens WA, Chandler GT: Life-cycle effects of single-walled carbon nanotubes (SWNTs) on an estuarine meiobenthic copepod. Environ. Sci. Technol. 40, 7387-7393 (2006).

	7	Helmus MN: The need for rules and
		regulations. Nat. Nanotechnol. 2, 333-334
		(2007).
	8	Cheng J, Flahaut E, Cheng SH: Effect of
		carbon nanotubes on developing zebrafish
		embryos. Environ. Toxicol. Chem. 26,
		708-716 (2007).
		Demonstrates that large carbon nanotube
		aggregate size can limit its bioavailability to
		zebrafish embryos.
		An

9

Acknowledgements

The authors would like to thank Helen Leung and Cecily Law for their technical support throughout the project.

Financial & competing interests disclosure

This research is partly funded by the Area of Excellence Scheme under the University Grants Committee (Project No. AoE/528 P-04/2004) and partially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region Government, China (Project No. CityU 160108). Kevin Kwok thanks HKU for partially supporting his PhD studentship. Emmanuel Flahaut's laboratory is part of a common laboratory with Arkema FR and shares an academic relationship with this company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involving human subjects, informed consent has been obtained from the participants involved.


C Lerouge

J Gouin

Y Deschamps

D Widory

F Laggoun-Defarge

G Tasev

T Serafimovski

M G Yossifova

Characterization of coal from the Mariovo basin, Macedonia -Insights from organic geochemical and sulphur isotopic data

Keywords: Coal chemistry, organic sulphur, metals, vitrinite reflectance, electron microprobe, Mariovo basin, Macedonia

data detailed petrographic study of coal from the Mariovo basin in Macedonia suggests circulation of fluids mobilizing metals from different origins (basement, volcanism ?) during burial. Sulphur isotopic data on organic matter and pyrite indicate dominantly marine-derive fluids and processus of bacterial reduction of sulphates.

INTRODUCTION

Coal in Macedonia is the main energetic resource for electricity production. The Mariovo basin is located in the southwest part of Macedonia, 46 km away from the town Prilep. The basin is constrained within Mt. Selecka, between the villages Vitoliste, Polciste, Besiste and Manastir, with the topography, and between 700 and 1000 m above sea level. The exploration was developed with intention towards the prospecting of the coal potential of the basin. The productive coal formation is developed in the eastern and the western parts of the basin. Excluding the most outer parts of the deposit, the productive formation occurs as a main layer of coal, which has sub-horizontal position with variable thickness (between 1.3 and 14.9 m) and a relative depth of appearing between 7.7 and 279 m. The coal from Mariovo basin is represented by lignite with high percentage of ash. The total reserves of coal in the deposit "Mariovo" are around 111 Mt. Informations about the contents, distribution and origin of sulphur and metals are important in coal combustion, because of their consequence on the production and on environment. Few detailed studies have been realized on coals from Macedonia. This work presents new petrographic, chemical and sulphur isotope data on coal, to better characterize organic matter and constrain the deposition conditions.

GEOLOGICAL SETTING

Macedonia lies within the Cenozoic Southern Balkan Extensional regime [START_REF] Dumudzanov | Evolution of the Neogene-Pleistocene Basins of Macedonia[END_REF]. The Mariovo graben is located in central and southern Macedonia, along the boundary between the Vardar zone and Pelagonian Massif. Most of the basinal infilling has been eroded, one of the best preserved outcrops being close to the village of Vitolishte. The sediments of the graben are divided into four formations from the bottom to the top: VITOLISHTE -The Nerezi formation (NeF) is divided into 3 units. The basal unit of ~120 m essentially consists of gravels and sandstones of unknown age. The middle unit consists of ~70 m of siltstones and silty claystones that grade upward into coal and claystones and finally into a 6-15 m thick layer of coal overlaid by marls and claystones. The latter contain planktonic algae association of late Miocene age [START_REF] Dumudzanov | Evolution of the Neogene-Pleistocene Basins of Macedonia[END_REF]). The upper unit consists of ~60 m of siltstones and sandstones followed by a break of sedimentation.

-The Solnje formation (SoF) consists of ~60-80 m of gravel and sandstones of Pliocene age.

-The Vitacevo formation (ViF) of Pliocene age [START_REF] Dumudzanov | Evolution of the Neogene-Pleistocene Basins of Macedonia[END_REF]) begin with stratified tuffs overlain by sandstones and gravels, interbedded with diatomite, tuffs and sandy claystones. Above travertine layers are deposed, followed by 80 m of tuff and sandstones.

-At the top is the Mariovo formation (MaF). It is ~60-70 m thick and contains pyroclastic rocks with nine travertine layers and a 20 m thick travertine deposit covering 20 km 2 , indicating a lacustrine environment.

SAMPLING

Samples of coal seams and intercalated sediments were collected on three outcrops around the Vitolishte village (∼ 21°49.6 E, 41°10.8 N). The first sample (7B) is from a small outcrop close to the river. The second sample (7K) is from a small outcrop around twenty meters above the river. The coal seam is not more than 50 cm thick with an intercalated siltite layer. The sample 7K is a coal from the upper part of the seam, which also contains vitrinite lenses. The third outcrop (samples 8K, 8M and 8R) is located on the opposite side of the river, at the same level as the second outcrop. It is the most important one, with two coal seams separated by a 15 cm thick layer of siltite containing floated wood fragments. The lower coal seam is 150 cm thick (8M, 8R) and the upper one is 50-70 cm thick (8L). The sediments overlying the coal formation consist of an alternation of hard mica-rich sandstones, soft layers and claystones. The coals are generally of black coal type. Samples 7B and 7K are composed dominantly of clarain with lenses of vitrain, whereas samples 8K, M and R are richer in vitrain.

ANALYTICAL METHODS

Samples were observed and analysed using a JEOL scattered electron microscope equipped with an EDS detector. Local analyses of organic matter were performed using a Camebax SX50 electron microprobe with a two stages analytical program. At the first stage C, Si and P are analyzed with a 10 kV acceleration voltage, a current beam of 20 nA and a 4-5 µm beam width. At the second stage Zn, S, Fe, As, Co, Ni, U, V, Ge, Sb, Cr, W are analyzed with a 20 kV acceleration voltage and a current beam of 20 nA. Matrix corrections were made with a ZAF computing program.

Vitrinite reflectance was measured on coal peaces in accordance with standard procedures (ISO 7404/5 1984).

Sulphur isotopes of sulphates and organic sulphur were performed using a CFIRMS system coupled with Flash EA and gas bench.

MINERALOGY

A variety of minerals is present in the coal of Vitolishte. The mineral composition is dominated by clays, rare quartz, pyrite, gypsum, iron oxides, and probably calcite. Detrital minerals are represented by clasts of quartz, and by numerous dispersed particles of clay minerals (illite/muscovite, illite, illite/smectite) within organic matter. Pyrite is the major sulphide in the coal of Vitolishte. It is mainly observed as framboids and more rarely as euhedral crystals, occurring along stratified plans or infilling cavities of plant tissues. SEM analyses of pyrite zones show the presence of Si, Al and Ca that strongly suggests the initial co-precipitation of silica, clay mineral and a Ca-mineral (such as calcite) with pyrite. Gypsum is visible on the surface on the samples; it is one of the major constituent of the inorganic matter. In polished section, gypsum also occurs as fine layers or surrounding pyrite framboids (Figure 2). Gypsum closely connected with pyrite in most of the samples indicates that it is essentially a weathering product of pyrite. However the gypsum in fine layers associated with a calcium phase (calcite) could be due to the crystallization of calcium and sulphate ions dissolved in the pore water during the sedimentation, according to [START_REF] Vassilev | Mineralogy and geochemistry of Bobov Dol coals, Bulgaria[END_REF]. Iron oxides are present as late infilling of fractures, strongly suggesting that they result from the weathering of pyrite. SEM analyses of pyrite zones and gypsum show the presence of Si and Ca and strongly suggest the initial coprecipitation of silica and a Ca-mineral such as calcite with pyrite.

PETROGRAPHY AND GEOCHEMISTRY OF COAL

The contents of sulphur, carbon and trace elements in coal from Vitolishte are given in the table 1. Major elements are C, Si and Al, whereas minor elements are Ca, Fe, K, S and Ti. Coal from Vitolishte has low total sulphur content, consistent with low-sulphur coal according to [START_REF] Dai | Distribution , isotopic and origin of sulfur in coals in the Wuda coalfield, Inner Mongolia, china[END_REF]. Ash content is high and consistent with previous data. Metal contents in coals from Vitolishte are over the Clarke values for lignites and sub-bituminous coals [START_REF] Yudovich | Trace elements in Coal[END_REF] for As, Cr, Li, Pb, Sc, V and Zn. They are close to the Clarke values or slightly above for Ba, Co, Cu and Ga. EPMA analyses and reflectance measurements were realized on macerals to determine the distribution of sulphur and metals (table 2). Sulphur contents in macerals are high and relatively homogeneous, ranging from 1.2 to 2.3 wt%, whereas iron contents are low, showing that measured sulphur can be attributed to organic matter. Maceral composition of samples 7B and 7K is rather heterogeneous and is composed of alternation of collotelinite layers with desmocollinite and associated fusinite. Collotelinite layers in 7B exhibit rims with higher reflectance than core (Figure 2 and Table 2), and enriched in . SEM bulk analyses of collotelinite show homogeneous contents of sulphur, calcium and Fe. Rare light-colored zones in backscattered electron image observed in the core of collotelinite lay-ers are slightly enriched in calcium. Desmocollinite and associated with fusinite in the sample 7B are V-rich (200-1160 ppm).

Table 1: Chemical composition of coal from Vitolishte, compared to Clarke content for lignite and subbituminous coals [START_REF] Yudovich | Trace elements in Coal[END_REF]. On other hand, samples 8K, 8M and 8R are mainly composed of collotelinite. Its reflectance values are comparable to that of samples 7. This range of values corresponds to that of sub-bituminous coals. However the carbon content of some macerals seems to be high for this rank. Vitrinite from the sample 8K is characterized by prismatic cracks, probably resulting from a thermal influence (M. Yossifova, pers. comm.). However, its reflectance remains rather low, and comparable to that of other samples. Collotelinite in the samples 8K, 8R and 8M are Ni-rich (260-700 ppm) and show important local Ge and Ga contents (up to 350 and 530 ppm, respectively). EPMA analyses of macerals from the coal of Vitolishte also provide evidence of local important contents of Co, Cu, Pb, Sb, Zn and W. The large variety of metals and their distribution in organic matter suggest circulation of fluids during burial, that mobilize metals from different sources (basement, volcanism

Gypsum and organic sulphur was analyzed in different samples of Vitolishte. Gypsum which is the secondary product of pyrite weathering has preserved the ∂ 34 S of the syn-sedimentary pyrite. ∂ 34 S values of organic sulphur are +5.4 and +13.2 ‰. They are slightly higher than the classical range of organic sulphur signature in low-sulphur coals, which derive essentially from plants (+2 to +8 ‰; [START_REF] Dai | Distribution , isotopic and origin of sulfur in coals in the Wuda coalfield, Inner Mongolia, china[END_REF]. These data suggest that sulphur was not accounted only for original plants, but introduced into peat after burial. High positive ∂ 34 S values of pyrite and organic sulphur could result from the bacterial reduction of additional marine-derived sulphates evolving in a close system. The presence of planktonic algae in clay stones above the coal seam is consistent with an intermittent marine environment.

CONCLUSION

New data on the coal from the Mariovo basin confirm the high ash content. Total sulphur content remains low and metal contents are slightly higher than the Clarke values of lignite and subbituminous coals. The detailed petrographic and isotopic studies of organic matter and framboidal pyrite provide evidence of a relation between macerales and V-Ni-Sb contents, and of bacterial reduction of sulphates, partly of external origin (marine-derived?). Further investigations need to be done to determine the origin of metals.

Figure 1 :

 1 Figure 1: Major tectonic units of Macedonia. I. Chukali-Krasta zone; II. Western Macedonian zone ; III. Pelagonian anticlinorium ; IV. Varar zone; V. Serbo-Macedonian massif.

Figure 2 :

 2 Figure 2: Backscattered electron image of framboidal pyrite surrounded by secondary products of gypsum.

Table 2 :

 2 reflectance measurements and EPMA analyses of collotelinite (CT) and desmocollinite (D) for carbon, sulphur and iron.Figure 2. Micrograph in reflected light of a collotelinite layer from the sample 7K, showing rims characterized by higher reflectance and high Si and Sb contents.

	Sample	Clarke	7K/7E	8 (lower seam)
	C tot %				41.4 (40.1) 36.6 (49.2)
	S tot %				1.0 (0.9)	0.8 (0.7)
	N tot %				0.9	1.0
	IH (mgHC/gTOC)			126
	IO (mg				108	136
	CO 2 /gTOC)
	Ash yield			27 %	23 %
	Al 2 O 3 %				5.8	4.5
	CaO %				< dl	1.3
	Fe 2 O 3 %				1.8	1.4
	K 2 O %				0.7	0.6
	MnO %				0.03	0.06
	SiO 2 %				13.1	9.9
	TiO 2 %				0.22	0.17
	As ppm		7.4	23	25
	Ba ppm		120	151	104
	Ce ppm		59		42	26
	Co ppm		4.2	8	5
	Cr ppm		15		37	29
	Cu ppm		14		11	7
	Ga ppm		7		8	5.9
	Ge ppm		1.5	0.4	0.2
	Li ppm		20		26	20
	Ni ppm		9		33	39
	P ppm		220	639	425
	Pb ppm		6.7	15	10
	Sb ppm		0.82	< dl	<dl
	Sc ppm		2		5.4	3.6
	Sr ppm		130	70	73
	U ppm		2.1	1.2	1.5
	V ppm		22		27	24
	Zn ppm		18		46	37
	Sample	Maceral	R 0 % C % S %	Fe %
	8R	CT		0.44 71	1.9	4.5
	8K	CT		0.45 74	1.7	3.0
	8M2	D		0.39 81-82 2.0-2.3 1-1.1
	8M2	CT		0.46 77	2	2.3
	7K	CT		0.52 80	2.2	0.3
	7K	D		0.49 66-68 2-2.1	2.6-4
	7B	CT layer (co-re)	0.38 81	1.7	0.3
	7B	CT layer (Sb-rich rim)	0.47 62	1.2	1.9
	7B	CT		0.46 76	1.2	0.3

Table 3 :

 3 Sulphur isotopic composition of secondary sulphates and sulphur of organic matter.

ACKNOWLEDGEMENTS

This work was financially supported by the ECO-NET program (10161XM) between the BRGM (France), the Geological Institute of Sofia, Bulgarian Academy of Sciences (Bulgaria) and the University of Skopje (Macedonia).