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Methylmercury concentrations and sulphate-reducing bacteria in freshwater sediments contaminated by a chlor-alkali plant: Babeni Reservoir, Romania A-G. BRAVO J. DOMINIK Forel Institute University of Geneva 10, Route Suisse SWITZERLAND
[email protected]
V.-G. UNGUREANU University of Bucharest 6 Traian Vuia 020956 ROMANIA
S. BOUCHET D. AMOUROUX LCABIE - IPREM UMR 5254 CNRS-UPPA 2 av P Angot, Pau FRANCE
J. ZOPFI Biogeosciences Laboratory University of Lausanne Anthropole SWITZERLAND
Abstract: Monomethylmercury (MMHg) is the most toxic form of Hg and can damage the central nervous system of humans that are exposed to MMHg via the consumption of contaminated fish. In general, MMHg in aquatic systems is mostly formed in sediments. However, the production and bioaccumulation of MMHg in freshwater sediments is still poorly understood. This paper reports high rates of MMHg production in sediments contaminated by a chlor-alkali plant in relation with the presence of sulphate-reducing bacteria. Key–Words: Monomethylmercury, sulphate-reducing bacteria, sediment, chlor-alkali.
1
Introduction
its high density of industrial companies. Since 1974, a chlor-alkali plant has been discharging mercury into Olt River, the largest Romanian tributary of the Danube River, just upstream from the Babeni Reservoir. Preliminary studies (8) revealed that Babeni Reservoir was heavily polluted with Hg in the past and that the remnant pollution still needs to be assessed. The purpose of this study was quantify MMHg formation in the sediments and evaluate the influence of sulphate-reducing bacteria in this highly contaminated reservoir
Mercury is a widespread heavy metal that occurs naturally in the environment, however, in severely exposed humans, it is a hazardous contaminant that damages the central nervous system (1). Monomethylmercury (MMHg) is the most dangerous form of Hg because of its bioavailability, its toxicity and its biomagnification in the aquatic food chain (2). The most serious symptoms of Hg intoxication are ataxia, tremors, sensory disturbances, including impairment of hearing and walking. Increasing needs of water and energy have led to the construction of a considerable number of dams along the Olt river. Reservoirs, which are often located close to cities or industrial centers accumulate the sediments and associated pollutants from the catchment. Industry is an important source of Hg pollution in the environment (3). Mercury released from chlor-alkali plants may enter aquatic systems, where it will easily bind to suspended particles and ultimately accumulate in bottom sediments. There sulphate-reducing and/or iron-reducing bacteria may transform Hg into MMHg as part of their detoxification mechanism (4, 5, 6). ). Reservoirs are also important sources of fish for the local population; the consumption of Hg contaminated fish is considered as the most common exposure pathway of MMHg for humans (7). The region Rm Valcea was selected because of
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1.1 1.1.1
Materials and methods Study area and sampling
The Babeni Reservoir (Fig. 1) is one of the 19 reservoirs built during the 70’s along the Olt River for flood prevention and power generation. Hence the hydroelectric plants regulate the river flow regime. The mean reservoir volume is 3.5x107 m3 , it has a surface area of 9x106 m2 and a mean depth of 3.9 m. Several sediment cores were collected from Olt River reservoirs using a 60 mm gravity corer (UWITEC, Austria). Two cores (OLTBC1 and OLTBC7) were collected in the Babeni Reservoir in August 2006 (Fig. 1). OLTBC7 was collected in the upstream part of the Babeni reservoir at 4 m depth. It is the station that is closest to the outlet of the waste water chanel of the chlor-alkali plant. OLTBC1 was
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Figure 1:
specific isotope dilution and capillary gas chromatography (Focus GC, ThermoFinnigan) hyphenated to inductively coupled plasma mass spectrometer (X7 II, ThermoElectron) to correct for species interconversion if necessary (10). Briefly, the extracts were buffered at pH 4 and isotopic enriched Hg species 199IHg and 201MMHg were then added. Species were then ethylated with sodium tetraethyl borate and recovered in iso-octane. Each sample was injected three times and blanks were checked to control for contamination. The accuracy of the of MMHg measurements was checked by analyzing certified reference material AEA 433 and 405 and recoveries of MMHg were found to be 89% for each reference material. SRB were enumerated using the most-probablenumber (MPN) method, based on the method set out in (11). One mL of this suspension was used to inoculate 3 replicate dilution series. The growth medium was a modified version of the standard medium for freshwater sulphate-reducing bacteria (12), contained 8 mM acetate, 4 mM lactate, and 34 mM of sulfate. The pH was set at 7.1. Briefly, 2 cm3 of wet sediment were added to 20 mL of purged water (N2 /CO2 ) containing 0.9 g/L NaCl. Tubes were incubated at room temperature in the dark and growth was detected by the presence of black precipitate in the tubes and sulfide determination. MPN values were calculated from statistical tables (11) and expressed in cells per gram wet weight of sediment (cells/g ww).
Sampling sites in the Babeni Reservoir.
collected close to the dam, at a water depth of 17 m. There fine sediments were expected to deposit without any influence of the stream current. An additional core was sampled in the Valcea Reservoir at 4 depth in August 2006, in order to determine local sedimentary background levels of THg (Total-Hg) and MMHg.
1.2
2 2.1
Mercury concentrations
THg concentration in sediments varied from 1.5 to 4.5 mg/kg and from 0.8 to 6.6 mg/kg in OLTBC1 and OLTBC7 cores, respectively, from Babeni Reservoir and reached 2.7 and 6.6 mg/kg, respectively in the first centimeter of the cores. Corresponding values were two order of magnitude lower in the core collected from Valcea Reservoir (0.08 and 0.10 mg/kg). The mean of MMHg percentage in THg was 1.2%, 6.5% and 2.2% for OLTVC1, OLTBC7 and OLTBC1 cores, respectively, from upstream to downstream part of the Olt River (Fig. 2). The highest value (12%) was measured at 4 cm depth in the core collected close to inlet of the waste water discharge channel of the chlor-alkali plant (OLTBC7, Fig. 2). The percentage of MMHg in THg was higher in the Babeni than in Valcea Reservoir. This percentage appears higher in the top part of the core, where the activity of the bacteria is usually increased due to the continous input of easily degradable organic matter.
Laboratory analyses
Sediment cores were cut in 1 cm slices down to a depth of 10 cm. All manipulations were done in an N2 -filled glove bag. Porewater was extracted from sediments by centrifuging at 4000 rpm during one hour and 1% of HCl (Ultrex Baker) was added to the supernant for Hg speciation analyses. All sediment subsamples were freeze-dried, homogenized with an agate mortar and finally stored under dark conditions until analysis. Mercury species were extracted from 200 mg of dry sediment using 7 mL of 6N HNO3 and a gentle microwave treatment (4 min, 80W, CEM) (9). Inorganic-Hg (IHg) and MMHg were measured in porewater and centrifugation extracts by speciesISSN: 1790-5095
Results
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(a)
(a)
(b)
(b)
(c)
(c)
Figure 2:
Figure 3:
Percentage of MMHg/THg in sediments of the reference core (OLTVC1) and two cores contaminated by the waste water discharge of a chlor-alkali plant (OLTBC7 and OLTBC1).
Percentage of MMHg/THg in porewaters of the reference core (OLTVC1) and two cores contaminated by the waste water discharge of a chlor-alkali plant (OLTBC7 and OLTBC1).
2.2
THg concentrations in porewaters were in the same order of magnitude for the 3 cores and ranged between 0.038-17µg/L, 0.099-72µg/L and 0.09318µg/L for OLTVC1, OLTBC7 and OLTBC1, respectively. However, MMHg porewater concentrations in the cores from the Babeni reservoir were two orders of magnitude higher than those from the Valcea reservoir.
Sulphate-reducing bacteria were enumerated by MPN method, which provides a rough estimate of the number of cultivable SRB in a sample. The choice of the subsampling was done according to the colors of the sediment sections with the aim to separate the deep anoxic layers where bacteria abundance should decrease (Table 1). However, no significant decrease of bacteria abundance was found with depth. The MPN cells SRB/g ww was in the same order of magnitude for OLTBC1 and OLTVC1, respectively, even though MMHg in solid phase and porewater concentrations were two orders of magnitude higher in OLTBC1 than in OLTVC1 (the reference point sediment). However, MPN measured in the core (OLTBC7) collected in the upstream part of the Babeni Reservoir, which collects the waste water dis-
Concentrations in the first cm of the OLTBC1 and OLTBC7 cores reached 203 ng/L and 206 ng/L, while they remained low (about 3 ng/L) in the Valcea Reservoir. The percentage of MMHg/THg in porewaters was higher than that found in sediments, and reached 40% between 6-10 cm depth in OLTBC7 and 30% at 8 cm depth in OLTBC1 (Fig. 3). In the top of the cores these values were 1% and 13% in OLTBC7 and OLTBC1, respectively. ISSN: 1790-5095
Sulphate-reducing bacteria
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Sample OLTVC1 0-1 2.5-4 4-5.5 8.5-10 OLTBC7 0-2 2-4 4-6 8-10 OLTBC1 0-1 1-2 3-5 9-10 Table 1:
Cells SRB/g
95% confidence limits
2.30E+04 5.50E+03 1.20E+04 1.05E+04
3.55E+03 7.50E+02 1.80E+03 2.00E+03
1.20E+05 2.40E+04 6.50E+04 2.35E+04
5.50E+06 3.75E+05 7.50E+04 5.50E+06
7.50E+05 7.00E+04 1.50E+04 7.50E+05
2.40E+07 1.15E+06 2.20E+05 2.40E+07
7.00E+03 1.05E+04 4.65E+04 3.75E+04
1.50E+03 1.75E+03 7.50E+03 7.00E+03
2.20E+04 2.35E+04 1.90E+05 1.15E+05
References: [1] T. Clarkson. Methylmercury and fish consumption: weighing the risks, 1998. [2] N.S. Bloom. On the chemical form of mercury in edible fish and marine invertebrate tissue. Canadian Journal of Fisheries and Aquatic Sciences, 49(5):1010–1017, 1992. [3] J.M. Pacyna and J. M¨unch. Anthropogenic mercury emission in Europe. Water, Air, & Soil Pollution, 56(1):51–61, 1991. [4] C.C. Gilmour and E.A. Henry. Mercury methylation in aquatic systems affected by acid deposition. Environmental Pollution ENPOEK,, 71(2/4), 1991. [5] E.J. Fleming, E.E. Mack, P.G. Green, and D.C. Nelson. Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium. Applied and Environmental Microbiology, 72(1):457– 464, 2006.
Most probable number counts of sulfate-reducing bacteria
(cells SRB/g wet weight) in sediments of two cores from Babeni reservoir (OLTBC1 and OLTBC7) and one reference core from Valcea reservoir (OLTVC1).
[6] E.J. Kerin, C.C. Gilmour, E. Roden, M.T. Suzuki, J.D. Coates, and R.P. Mason. Mercury methylation by dissimilatory iron-reducing bacteria? Applied and Environmental Microbiology, 72(12):7919–7921, 2006.
charge from the chlor-alkali plant was two orders of magnitude higher (Table 1). Additionally, this core (OLTBC7) had also the highest THg concentrations and the highest percentage of MMHg, which is probably due to high bacterial activity.
[7] International Programme on Chemical Safety World Health Organization. Safety evaluation of certain food additives and contaminants. , 2004.
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Conclusion
[8] A.G. Bravo, J.L. Loizeau, L. Ancey, V.G. Ungureanu, and J. Dominik. Historical record of mercury contamination in sediments from the Babeni Reservoir in the Olt River, Romania. Environmental Science and Pollution Research, 16:66–75, 2009.
The strong impact of a chlor-alkali plant on MMHg concentrations in sediment of Babeni reservoir is documented. The highest percentages, reaching 40%, were detected close to the inlet of the chlor-alkali plant waste water channel, at the same sediment depths where also sulfate-reducing bacteria were most abundant. Nevertheless, in the surface sediments of the same core, at nearly equal abundance of the SBR, MMHg percentage was quite low.
[9] R.C.R. Martin-Doimeadios, E. Krupp, D. Amouroux, and O.F.X. Donard. Application of isotopically labeled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS. Anal. Chem, 74(11):2505–2512, 2002.
Acknowledgements: The authors gratefully acknowledge the financial support of Swiss National Science Foundation for the Environmental Science & Technology in Romania Project (N IB6 1-0-107008) and TRAVEXT found. The scientific advices of Walter Wildi and the help in field trip and laboratory analyses of Cristophe Marcic, Jean-Luc Loizeau and the staff of the Rm Valcea Environmental Service are gratefully acknowledged. ISSN: 1790-5095
[10] M. Monperrus, P. Rodriguez Gonzalez, D. Amouroux, J.I. Garcia Alonso, and O.F.X. Donard. Evaluating the potential and limitations of double-spiking species-specific isotope dilution analysis for the accurate quantification of mercury species in different environmental matrices. Analytical and Bioanalytical Chemistry, 390(2):655–666, 2008. 199
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[11] W.G. Cochran. Estimation of Bacterial Densities by Means of the” Most Probable Number”. Biometrics, 6(2):105–116, 1950. [12] F. Widdel and F. Bak. Gram-negative mesophilic sulfate-reducing bacteria. The Prokaryotes, 4:3352–3378, 1992.
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