Acta Chini. Slov. 2002, 49, 149-157. 149 ISOTOPIC ANALYSIS OF SULPHUR IN THE ASSESSMENT OF S02 EMISSION SOURCES Julija Beričnik-Vrbovšek,8 Friedrich Pichlmayer,b Karl Blochberger,b Zvonka Jeran,c and Jože Marseld* "ERICo Velenje, Institute for Ecological Research, 3320 Velenje, Slovenia bÖsterreichisches Forschungszentrum Seibersdorf, A-2444 Seibersdorf, Austria CJ. Stefan Institute, 1000 Ljubljana, Slovenia dFaculty ofChemistry & Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia Received 03-09-2001 Abstract Reported herein is the isotopie composition of sulphur in Norway spruce needles, lichens and soils from two well-defined climatic regions in Slovenia: The Šalek Valley and the Zasavje region. The samples are from distinct locations in these regions, where the impact of S02 on vegetation is well documented. In both regions, the main source of SO2 pollution is from thermal power stations. The isotopie measurements of Norway spruce needles were complemented with 5,4S-analyses of coals (lignite and brown coal) and SO2 in stack-gases from two thermal power plants, to correlate SO2 pollution with a specific emission source. Reference samples of S02 from an unpolluted region (The Triglav National Park) were collected for comparison. To elucidate the turn over of SO2 from source to spruce needles via air and/or soil, characteristic soils and lichens (Hypogymnia physodes) were sampled. The sulphur in the samples was converted by combustion in a small oxygen bomb (Parr Instrument) to sulphate, precipitated as BaS04 and the 534S values measured using an isotope ratio mass spectrometer coupled on-line to an elemental analyser. The 5,4S values demonstrate only a close relationship between environmental SO2 and lichens. Correlation between a particular emission source and the apparent impact on vegetation from the 534S values is not straightforward. Introduction Although, sulphur is an important macro-element in the forest ecosystem, being an essential element for plant and soil microorganisms, there is now convincing evidence that forest decline is mainly caused by SO2 pollution, O3 and related compounds. Sulphur originates from both natural and anthropogenic sources. Of the latter, eighty-five per cent of the sulphur emitted - mainly as SO2 - derives from the combustion of coal and oil, while the majority of the remaining fifteen per cent is from industrial sources. The two main sources of sulphur in the forest ecosystem are from the atmosphere and the soil. Among the gaseous inputs of sulphur, SO2 contributes the major part. J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... 150 Acta Chini. Slov. 2002, 49, 149-157. Stable sulphur isotopes are widely used to study sulphur dynamics in different ecosystems. Naturally occurring Sulphur has four stable isotopes with masses 32, 33, 34 "37 ^4 "37 ^4 and 36 of which Sand S are the most abundant ( S represents 95.02 %, and S 4.21 %). The input of sulphur into the ecosystem can cause significant changes in the S-isotopic composition of the pedo-, hydro- and atmosphere as a result of the complex mechanisms: physical, chemical and biological processes, that affect sulphur turnover during its pathway from source to its uptake. This phenomenon is known as isotopie fractionation and albeit small, these variations can be characteristic of a particular emission. Because it is easier to compare isotopie ratios in two samples than it is to measure the absolute abundances in any one sample, the expression ô S (in %o), which ~îa -j'y is the ratio of S/ S relative to an international standard (the Canyon Diablo meteorite), is used to study sulphur isotope variations in nature. For many reasons, the International Atomic Energy Agency (Vienna) introduced and distributed an Ag2S standard with a defined Ô34S value of -0. 30 %o. At any specific site, the differences in ô S can be measured with a precision below 0.5 per mil. In the present work we aim to investigate the relationships between ô S values, SO2 levels and forest decline in two polluted regions in Slovenia: the Salek Valley and Zasavje region. In both regions, the dominant sources of SO2 pollution are the Thermal Power Plants at Šoštanj and Trbovlje, respectively. To do this we measured the isotopie composition of sulphur in lignite, brown coal, chimney-stack gases, spruce needles (current year needles, 1-year-old-needles), soils (four layers), air (Pokljuka plateau). We then compared these values to the isotopie composition of samples collected from the Triglav National Park, which was chosen as an example of an unpolluted region. Because the variations in chemical composition and tissue alterations make lichens sensitive biological indicators of regional trends in ambient SO2 pollution and because arboreal lichens are never in contact with soil, they are useful for assessing atmospheric SO2 impact. For this reason we collected specimens of the lichen Hypogymnia physodes (L.) Nyl., and exposed them to the environments under study. Finally, since the production of glutathione (GSH) in Norway spruce needles is related to environmental J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Morsel: Isotopie analysis of sulphur... Acta Chini. Slov. 2002, 49, 149-157. 151 concentrations of SO2, we decided to isolate GSH from Norway spruce needles of the same origin and measure the ô S values on a molecular level. Experimental Sampling and sample preparation Soil samples: We collected soil samples in September 1996. We first separated the soil profile into two organic layers (Oif, Oh) and two mineral layers (Ao-10 cm, A10-20 cm). Each layer was dried at 30 °C and sieved (3 mm). The sample was then mixed with an Eschka mixture in a ratio 1:3 and ignited at 800 °C to convert the sulphur to sulphate, which was then dissolved in hot deionised water. Spruce needles: Samples were harvested from Norway spruce trees {Picea abies (L.) karsten) in September 1996 from all three sites. Twigs were cut from the light crown of five trees at each location. The needles were frozen in liquid nitrogen, lyophilised and ground to a powder. The sulphur was converted by combustion in a small Parr 1108 oxygen bomb (Parr Instrument) to sulphate following the procedure described in the literature. Coal: Samples of lignite and brown coal were collected from the Thermal Power Plants at Šoštanj and Trbovlje, respectively. The sulphur, like that for spruce needles, was converted by combustion to sulphate. Chimney-stack-gases: Samples from both Thermal Power Plants were obtained by trapping the SO2 in a hydrogen peroxide solution. Air samples: The sampling site for air at the Triglav National Park was the Pokljuka plateau. In this instance SOx in the ambient air was trapped on alkaline impregnated glass fibre filters. Lichens: We collected specimens of the Lichen Hypogymnia physodes (L.) Nyl., from the reference sampling sites for SO2 monitoring (Spodnja Komisija, Table 1) and exposed them for six months to the environments at the given sampling sites. We then followed the same sample preparation procedure as for the spruce needles. J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Morsel: Isotopie analysis of sulphur... 152 Acta Chini. Slov. 2002, 49, 149-157. S-isotopic analysis Isotopie analysis involved precipitating the sulphur (sulphate) in the samples, as BaSC>4, which was then burned in an elemental analyser to SO2, separated using a ~îa -j'y packed gas column and the S and S concentration determined using a Finnigan isotope ratio mass spectrometer. From these values, we calculated ô S (relative to the Canon Diablo meteorite international standard) using the expression: (Ô S / Ô S)sample - (8 S / 8 S)standard Ô34S = --------------------------------------------------------- * IO3 (°/oo) (8 S / 8 S)standard To isolate glutathione (GSH), 150 g of lyophilised spruce needles were dissolved in 500 ml of distilled water. The solution was injected onto a preparative HPLC column (Spherisorb ODS2, 5|um, 150 x 20 mm) and separated using a gradient elution of 0.1 % TFA in H2O and 0.1 % TFA in H20: MeCN (1: 1). Detection was performed with an UV-Diode array detector and a Finnigan TSQ mass spectrometer under ESI conditions. Results Figures 1-3 show the average 8 S values in the samples from the three sampling sites. The 8 S for lichens are given in Table 1. The standard deviation (reproducibility) is ± 0.3. %o Sampling site Area Ô34S (%0 ) Zavodnje Affected by TPP Šoštanj 7.2 Veliki vrh Affected by TPP Šoštanj 7.7 Affected by TPP Šoštanj 7.0 Podvolovjek 1 5.6 Učeja 2 3.2 Spodnja Komisija 3 5.6 Table 1: Mean 834S values in the lichen Hypogymnia physodes (L.) Nyl., exposed for 6 months in different environments (TTP = Thermal Power Plant) 1 ... Reference site on the border of the Šalek Valley 2 ... Reference site near the Triglav National Park 3 ... Lichens from an unpolluted area (Spodnja Komisija) transferred to the given sites for a period of 6 months J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... Acta Chini. Slov. 2002, 49, 149-157. 153 ^ lignite | | stack-gases \///\ current year needlei |^\\\j 1-year old needles soil O lf soil O h soil 0-10 cm | | soil 10-20 cm sO-10 slO-20 sample Fig. 1: Mean ô S values in the area under the influence of the Šoštanj Power Plant (1996) I brown coal | | stack-gases \///A current year needles Ix^xx] 1 -year old needles sou Olf sou Oh soil 0-10 cm |_Jsoil 10-20 cm S10-20 sample ;34c Fig. 2: Mean ô S values in the area under the influence of the Trbovlje Power Plant (1996) J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... 154 Acta Chini. Slov. 2002, 49, 149-157. From the results (Figs. 1 and 2) the isotopie fractionation of sulphur during the burning of lignite in the Šoštanj Power Plant falls within the measured uncertainty, while the isotopie fractionation from burning of brown coal in the Trbovlje Power Plant leads to an enrichment of S. Even after repeated measurements, the same effect was observable, which means that this difference cannot be explained in terms of reaction kinetics. In both polluted areas, the ô S value in Norway spruce needles increases with age, unlike in the needles from the Triglav National Park were there is no increase (Fig. 3). The ô S value in the soil at all three sites increases with depth but is more pronounced in the polluted areas. In the Norway spruce needles the ô34S value was less positive than in the ambient SO2 and in the soil in the two polluted areas, whereas, in the Triglav National Park the ô S in spruce needles is similar to that in the soil. The ô S values in the lichen Hypogymnia physodes (L.) Nyl., exhibits an assumed cause-and-effect relationship, i.e., the ô S values >7 %o in the specimens exposed to ambient SO2 originating from the Šoštanj Power Plant (ô S « 8.2 %o) indicate a direct impact from the emission source. air current year needles ^\l1 -year old needles soil 0lf soil Oh MI soil 0-10 cm soil 10-20 cm ;34, Fig. 3: Mean ô S values in the Triglav National Park (1996) J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... Acta Chini. Slov. 2002, 49, 149-157. 155 The content of GSH in the Norway spruce needles is much lower than that stated in the literature, which for a selected sample is about 1 |ug/ml. This means that to isolate 500 |ug of GSH (50 |ug S) for isotopie analysis using preparative HPLC, approximately 150 h working time and more than 70 litres of MeCN is required. Discussion In the presence of higher SO2 concentrations in air, for example in the areas affected by the Šoštanj and Trbovlje Power Plants, plants are expected to use both atmospheric and soil sulphur for their metabolism. An assumed cause and effect relationship between the average SO2 levels and its isotopie composition should be reflected in alterations of S-containing compound in plants and their isotopie composition.Nevertheless, spruce needles show less positive ô S values than both potential sources. This might be due to S-isotope fractionation during S-uptake and/or metabolism, leading to compounds enriched in S. Unlike lichens whose ô S values reflect those of ambient SO2 in the air, spruce needles exhibit values lower than air and soil at the same site, indicating the mixing of sulphur from two distinct sources (bedrock and atmosphere) and/or pronounced isotope fractionation in soil. As expected, the ô S in the lichens exposed to different environmental conditions show a higher correlation between environmental SO2 and a particular emission source (Power Plant at Šoštanj). All sites exhibit a small decrease in ô S from topsoil to mineral soil. This can be explained by the fact that in nonsaline upper soil horizons, more than 95% of sulphur is in organic form. Even at extremely high SO2 deposition, sulphur appears to be cycled through an organic pool in the soil. In addition, unless the mean residence time of sulphur in the soil is greater than the time scale of the SO2 pollution, the positive ô S in soil may reflect natural sulphur. Conclusions Establishing a cause and effect relationship between a particular SO2 emission source and the apparent impact on vegetation from the ô S value is not straightforward. The accumulation of sulphur in vegetation is complex and we can find no direct J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... 156 Acta Chini. Slov. 2002, 49, 149-157. correlation between the ô S value in spruce needles and stack-gases, not even in the surrounding of a given emission source. Despite this, the differences in ô34S values between chimney-stack SO2 and sulphur in spruce needles and in soils is significant for both regions. The ô S values demonstrate only a close relationship between environmental SO2 and lichens (uptake of SO2 from air). In this instance, the assumption that determining ô S values on a molecular level in spruce needles reflect the isotopie composition of the surrounding SO211 can not be proved by measuring the ô34S of GSH. The low concentration of GSH in spruce needles makes the isolation of this compound for ô S measurements impractical. Generally, future advances in applying stable isotope techniques on a molecular level will largely depend on the ability of instrumentation to measure microgram amounts of organic sulphur. Acknowledgements We are grateful to the Ministry of Science and Technology of Slovenia for supporting this work. We would also like to thank S. Zigon (Laboratory for geochemistry, J. Stefan Institute, Ljubljana) for his assistance in sample preparation and Drs. S. Lojen and N. Ogrinc for their helpful discussions. References 1. C. F. Cullis, M. M. Hirschler, Atmos. Environ. 1980,14, 1263-1278. 2. H. R. Krouse, A. V. Grinenko (Eds.), Stable Isotopes: Natural and Anthropogenic Sulphur in the Environment, Wiley, Chichester, 1991 3. W. E. Winner, J. D. Bewley, H. R. Krouse, H. M. Brown, Oecologia, 1978, 36, 351-361 4. S. E. Lindberg, G. M.Lowett, D. D. Richter, D. W. Johnson, Science 1986, 231, 141-145 5. E. Bermadinger , D. Grill, H. Guttenberger, Phyton 1989, 29, 163-185 6. ASTM D 3177 - 89, Annual book of ASTMstandards 1989, 05.05, 333-336 7. Y. Arikawa and A. Sasaki, Anal. Sciences, 1987, 3, 157-160 8. U. Daemmgen, L. Gruenhage, H. J. Jäger, Landschaftsoekologisches Messen und Auswert 1985, 7,2/3,95-106 9. F. Pichlmayer, K. Blochberger, Fresenius Z Anal. Chem. 1988, 331, 196-201. 10. T. W. Boutton, S. Yamasaki, Mass Spectrometry of Soils; Marcel Dekker Inc., New York, 1996 11. B. A. Trust, B. Fry, Plant, Cell and Environment 1992,15, 1105-1110. J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur... Acta Chini. Slov. 2002, 49, 149-157. 157 Povzetek V vzorcih premogov, dimnih plinov, smrekovih iglic in tal iz dveh onesnaženih področjih (Šaleška dolina in Zasavje) smo merili izotopsko sestavo žvepla (5,4S). Za primerjavo smo merili vzorce zraka in tal iz področja Triglavskega narodnega parka. Da bi pojasnili prenos S02 v iglice smreke preko zraka in/ali tal, smo merili 5,4S vrednosti v značilnih drevesnih lišajih in tleh. Žveplo v trdnih vzorcih smo s sežigom v kisiku, iz zraka pa neposredno pretvorili v BaS04 in nato v S02 ter vrednost 5,4S izmerili z masnim spektrometrom. Medtem ko je pri lišajih opazna korelacija med 5,4S vrednostmi za okoliški S02 pa je vpliv na 534S vrednosti v iglicah smreke manj izrazit. J. Beričnik-Vrbovšek, F. Pichlmayer, K. Blochberger, Z. Jeran, J. Marsel: Isotopie analysis of sulphur...