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Microchim Acta (2009) 165:79–89 DOI 10.1007/s00604-008-0101-7

ORIGINAL PAPER

Time resolved sulphur and nutrient distribution in Norway spruce drill cores using ICP-OES

Andrea Ulrich & Timothée Barrelet & Renato Figi & Heinz Rennenberg & Urs Krähenbühl

Received: 8 May 2008 /Accepted: 26 August 2008 / Published online: 23 September 2008 # Springer-Verlag 2008

Abstract Methods were developed for detailed dendro- procedure and a microwave acid digestion procedure chemical studies of low sulphur contents in Norway spruce were evaluated with the goal to obtain sufficient detection (Picea abies L. Karst.). This tree species is the dominant limits in order to access low concentrated non-metals with conifer species in Northern and Central Europe and an appropriate time resolution. Method development in- therefore predestined for a possible use as an environmental cluded evaluation of strategies preventing losses of volatile archive. Two independent digestion procedures were sulphur species. Digestion efficiency was demonstrated by investigated with respect to their suitability for element recovery rates for various certified plant standard reference determination and optimised for analysis of the low sulphur materials (NIST 1572, NIST 1547, RM 8436, BCR 101, content in wood. A modified oxygen bomb combustion NIST 1515, RM 8436, NIST 1573, NIST 1575) as well as self prepared standards with defined low sulphur content of −1 Electronic supplementary material The online version of this article 20 to 200 mg kg , which are typical for Norway spruce (doi:10.1007/s00604-008-0101-7) contains supplementary material, wood samples. Ultra sonic nebulisation (USN) was evalu- which is available to authorized users. ated with respect to signal enhancement for sample A. Ulrich (*) : T. Barrelet : R. Figi introduction to inductively coupled plasma optical emission Analytical Chemistry, Swiss Federal Institute for Materials spectrometry (ICP-OES). The optimised procedure was ap- Science and Technology (EMPA), plied to Norway spruce drill cores from locations with different Überlandstrasse 129, environmental conditions in Switzerland, in order to investi- 8600 Dübendorf, Switzerland e-mail: [email protected] gate the anthropogenic impact of sulphur and the suitability of Norway spruce as an environmental archive for sulphur. T. Barrelet e-mail: [email protected] Keywords Norway spruce . Tree ring . Sulphur . T. Barrelet Trace elements . Oxygen bomb combustion . Microwave Swiss Federal Office of Public Health (FOPH), digestion . ICP-OES . Plant certified reference material 3003 Berne, Switzerland

H. Rennenberg Institute of Forest Botany and Tree Physiology, Introduction University of Freiburg, Georges-Köhler-Allee 53, 79085 Freiburg, Breisgau, Germany The goal of this study was to investigate whether trees are e-mail: [email protected] appropriate environmental archives for sulphur. They are : among the most popular archives because they are wide- T. Barrelet U. Krähenbühl spread and can be found in remote areas as well as in cities, Department for Chemistry and Biochemistry, University of Berne, Freiestrasse 3, close to anthropogenic pollution sources. Furthermore, 3012 Berne, Switzerland sampling using increment borers is relatively easy and risk U. Krähenbühl of sample losses or deterioration is low. Additionally, tree e-mail: [email protected] rings make dating very precise. In conifers particularly, 80 A. Ulrich et al. individual tree rings can be distinguished easily. Conifers being required. Hence, the method provides a relatively low can be found in most parts of the globe. In the northern time resolution of 10 years only. Moreover, the procedure is hemisphere, the genus Picea is very common, which was time consuming and losses of volatile sulphur components one of the main reasons why Picea abies (Norway spruce) during the ashing procedure are likely, hence restricting the was selected for this study. detection limit. Environmental archive investigations using tree drill With the exception of the Gaoming procedure, all above cores are particularly demanding with respect to time mentioned studies were focussed on the determination of resolution. This is because sulphur content in Norway spruce metals. Moreover, all authors mentioned above used sample wood is low and ranges between 20 and 200 mg kg−1. amounts between 200 and 500 mg for the decomposition, Hence, a sample preparation method appropriate for deter- which was relatively high compared to the tree increments used mination of low contents is required. Several digestion for this study which weighted between 50 and 200 mg only. methods were proposed for the determination of metals in Since none of the procedures mentioned above was wood, but none was found to be appropriate for the analysis deemed appropriate to fulfil the purpose, a suitable of low sulphur content with adequate time resolution and digestion method had to be developed for the determination low detection limits. of low non-metal contents such as sulphur or phosphorus In several studies, digestions of 5 to 10 year drill core and for the multi-elemental determination of metals in tree segments were performed for the determination of metals. ring analysis. Therefore, two independent decomposition Open acid digestions had been applied in applications from procedures were developed, optimised and applied for the 1990ies [1–3] whereas microwave acid digestion in analysis of low sulphur content in wood samples. More- closed PTFE (polytetrafluoroethylene) pressure bombs is over, the suitability of the method had to be evaluated for commonly used today for the decomposition of environ- the determination of metals and phosphorus in wood mental or biological samples [4–7]. Most of the digestions samples. The methods were validated using standard methods were optimised for the determination of trace reference materials and self-made standards with low metals, using different acid mixtures such as mixtures of sulphur contents.

HNO3 and HClO4 [2, 3, 8], HNO3,H2O2 and HF [5, 6]or HNO3 and H2O2. [4, 9]. Digestion methods for sulphur determination in other plant materials were developed by Experimental Randal and Spencer, who described an open digestion procedure using HNO3 and HClO4 [3]. Zhao et al. [10] Samples compared three digestion procedures with HNO3-HClO4- mixture, fuming HNO3-KNO3 and an alkaline NaOBr Tree sampling and site description decomposition method. Other authors applied microwave digestion procedures in dendrochemical investigations of Picea abies L. Karst (Norway spruce) was chosen for the metals. A mixture of HNO3, HCl and HF was proposed for present study. In Switzerland, it represents 48% of all trees bark [7], but the addition of HCl and HF is not compulsory and also grows in high altitudes. Three forests were for the digestion of wood. Digestion of wood in 50% H2O2 selected: two in the Swiss Plateau (Düdingen, 580 m above was also described [11]. sea level, Frieswil, 740 m above sea level), and one at the

One study recommended a mixture of HNO3 and HClO4 alpine site of St Moritz (Engadine, 1,900 m above sea in quartz cups [12]. However, a digestion with perchloric level). The SO2 emission situation is different for each acid is usually not necessary for the digestion of wood sampling site and is lowest at the alpine location St Moritz. tissue. With respect to sulphur analysis, the use of HClO4 is In Düdigen and St. Moritz, peat bogs can be used for disadvantageous because of the high sulphur contamination comparison as independent environmental archive. The levels in commercially available HClO4. Even in ultra pure peat bog at the Alpine sampling site St. Moritz is located purity grade acid the contamination of HClO4 is higher than about 100 m underneath the sampled forest. Düdingen has −1 2− 5mgL SO4 in HClO4 70% Merck Suprapur® in the highest pollution levels due to the proximity of the −1 2− contrast to maximum 200 μgL SO4 in HNO3 65% forest to the motorway A12 (Berne–Fribourg), which is Merck Suprapur®. about 500 m away from the forest. The Düdingen peat bog

Digestion of wood powder by heating in HNO3 on a is near the forest, about 1 km away. In Frieswil, a rural site heating block [13] was proposed for metal determination without direct SO2 source, atmospheric concentrations of but is inadequate for sulphur due to losses of volatile the pollutant are at an intermediate level. compounds. Ashing of wood samples and subsequent At all sampling sites, mature forest trees were selected. The digestion with HNO3 as described by Gaoming [14] has circumference of each tree was determined before sampling. the major disadvantage of relatively high sample quantities Trees of different size were sampled in order to assess possible Sulphur and nutrient distribution in Norway spruce drill cores 81 ageing effects. Drill cores were taken at breast height using a 5 one hand and accurate quantification of sulphur and mm increment borer (Suunto, www.suunto.com). The con- phosphorus in low contents on the other hand. A further tamination levels of potential cleansing agents for the corer goal was the optimisation by minimising sample amounts in were determined before the sampling campaign in order to order to achieve best time resolution for tree ring ensure that the both corer and metal tongue were devoid of investigations. Both acid digestion using microwave and a sulphur. Measurements using ICP-OES revealed that modified oxygen bomb combustion procedure, originally WD40®, which contains 21% mineral oil, was not suitable developed for sulphur and halogen determination in oil or because it is rich in sulphur. PhEur grade paraffin (Hänseler, coal, were investigated and optimised [15, 16]. www.haenseler.ch) was finally chosen for cleaning and lubricating the sampling tools because of its low background ICP-OES analysis contamination. It was found to contain only traces of sulphur which were below detection limit of 10 mg kg−1 for XRF All analyses were performed by inductively coupled plasma (x-ray fluorescence spectrometry). Gloves were used in order optical emission spectrometry using either a sequential to avoid contamination during coring and sample prepara- Varian Liberty 150 AX Turbo with axial torch or a simul- tion. For comparison of different analysis methods, six drill taneous Varian VistaPro (www.varianinc.com) with a radial cores per tree were taken in four directions. Two of them torch. For sulphur determination on the Varian Liberty 150 were used for radiodensitometry, two for LASER ablation AX Turbo, the optical chamber had to be rinsed with high resolution ICP magnetic sector field mass spectrometry, nitrogen because oxygen absorbs the sulphur emission line. one for x-ray absorption spectroscopy, and the remaining one On the Varian VistaPro, the optical chamber was rinsed for the digestion and combustion procedures developed within with Ar. Both instruments were operated under standard hot this study. The extent of the sapwood zone of each drill core plasma conditions. was determined immediately after sampling, since for Norway For analysis of the alkaline solutions obtained by oxygen spruce wood no visual differentiation between sapwood and bomb combustion, a Varian HF kit had to be mounted heartwood is possible after drying. For interpretation of because of the quartz-damaging alkaline solutions (about element profiles in a tree, determination of the sapwood- 0.4 M NaOH). The kit consists of a PEEK (polyetherether- heartwood boundary is important. The samples were then ketone) V-groove nebulizer, a PTFE spray chamber and a pressed into weighted and previously cleaned PVC (polyvinyl demountable quartz torch with corundum injector tube. chloride) rails, and wrapped in a polyvinylidene chloride foil The microwave acid digestions were analysed using a (Saran). Afterwards, they were kept in the rails and dried at standard quartz sample introduction system consisting of a 40°C until weight constancy. Water content of the wood was Meinhard nebulizer and a Twister cyclonic spray chamber determined as weight loss after drying. for the VistaPro. For the Liberty 150 AX Turbo a Cetac ultrasonic nebulizer AT5000+ (USN) was available. The Reagents and chemicals USN was used to improve the signal intensity particularly for elements with low concentrations such as sulphur and Only ultra pure quality acids (Merck, www.merck.de) and phosphorus. The signal enhancement factor of the USN in high-purity water (18.2 MΩ cm−1, prepared by a Milli-Q comparison to the Meinhard nebulizer was about 7 for Gradient A10 System, Millipore, www.millipore.com) were sulphur and about 13 for phosphorus, which corresponds to used for sample and standard preparation. Standards were the findings of Wennrich et al. [9] who found enhancement prepared from single and multi-element standards of ICP- factors of 4.9 for sulphur and 4.3 for phosphorus for a quality (Merck and Alfa Aesar). Cleaning and sample Spectroflame ICP-OES (Spectro Analytical Instruments). digestions were also performed using acids of Suprapur® quality (Merck) and high-purity water. Cellulose powder Radiodensitometry (20 μ, Aldrich) was used for fabrication of doped cellulose standards used for validation of the acid microwave For dating and assessment of abrupt growth changes or digestion procedure. Spiked wood samples were prepared reaction wood, two supplementary drill cores were taken from pulverised wood briquettes with an average sulphur from the trees and measured by radiodensitometry. Wood content of 58±4.6 μgg−1 density and tree ring width of the additional drill cores were measured at the installations of the Swiss Federal Institute Sample preparation for Forest, Snow and Landscape Research, Birmensdorf (WSL, www.wsl.ch). For this purpose, 1.2 mm thick Two sample preparation procedures were investigated for splinters were sawn out of the fixed core, at right angle to optimum sample decomposition of wood samples. These the direction of fibre. After conditioning (20°C, 50% procedures had to allow precise determination for metals on relative air humidity), the wood samples were x-rayed. 82 A. Ulrich et al.

Optical density of the negatives obtained was measured of a small amount of sooth in the glass crucible, which was with a Dendro 2003 microdensitometer (Walesch Electron- wiped away with a towel. The presence of soot is ic, www.walesch.ch). The optical density corresponds to tantamount to an incomplete combustion of the carbon the volumetric-gravimetric wood density. In this article, this fraction in wood, but does not signify incomplete sulphur information was used for exact dating of the drill cores. oxidation. The use of one-way cellulose acetobutyrate Further details can be found in [17–19]. crucibles was found to be unsuitable because they increased the blank value 2.6 times compared to quartz glass crucibles. The combustion procedure was optimised and validated Results and discussion using sulphate solutions (1 mg L−1), and standard reference materials (BCR 101, powdered spruce needles, Development of the oxygen bomb combustion 1,700±40 μgg−1 S). The recovery rates for synthetic sulphur samples ranged at about 97%. For BCR 101, The oxygen bomb combustion was chosen for independent recovery rates up to 90% of the certified value were comparison with the acid microwave digestion. The oxygen obtained, but in average the recovery rates were only about bomb combustion procedure is routinely used for deter- 80%. The optimisation of the reagent types and volumes mining the halogen and sulphur content in coal and showed that a careful optimisation of the NaOH concen- petroleum products [20]. The digestion procedure is well tration in the absorption liquid is important in terms of validated for this application and comprises all relevant recovery rates. The concentration was finally optimised to sulphur components including the volatiles. However, it 1MNaOH.Lowerconcentrationresultedinmuchlower was never applied to wood samples before. This type of recovery rates. The findings point to a systematic error in material is particularly challenging because of its low the present digestion procedure. sulphur content of only about 50 mg per kg in average, The oxygen bomb combustion method was also applied which means about 60 times lower values than the sulphur to real wood samples. One tree per sampling site was content in coal. selected. For tree samples, a compromise had to be made Combustion of the wood samples with oxygen was between minimal weight of the sample and best obtainable performed in a sealed decomposition vessel with catalytic time resolution. Drill cores with a diameter of 5 mm had to coating. All sulphur compounds contained in the wood be cut in 10 year segments in order to reach the required were converted to sulphate during the rapid combustion. minimum sample weight of 70 mg. Due to the low sulphur The oxygen pressure bomb combustion was performed content in wood, a sufficient sulphur level was only using an Ika AOD 1 system and following the DIN 51577 measured for one out of three drill cores. The only drill procedure (www.din.de). Using a carefully cleaned scalpel, core providing enough sulphur content originated from a St the drill cores were cut into segments comprising ten tree Moritz tree. The sulphur contents in the drill cores from the rings each. A minimum of 70 mg wood sample were put Swiss Plateau trees displayed only values below detection into a quartz crucible, which was then placed into a holder. limit. The use of a 12 mm drill core might solve the A cotton thread leading to the crucible was fixed to the problem. Another possibility might be minimisation of the ignition wire, and both sample and thread were moistened combustion devices including vessel volume. with about 1 ml ethanol p.a. for combustion support. Ten However, it appears that oxygen bomb combustion was millilitres 1 M NaOH (p.a. grade) were dosed into not an optimal choice for the determination of sulphur in decomposition vessel for absorption of the combustion wood. The present procedure does not provide reliable gasses. Previous optimisation studies showed that no H2O2 results for the sulphur content of wood samples. Moreover, is required for additional oxidation. The cover/crucible the detection limits were relatively poor and the required holder was then screwed onto the decomposition vessel. sample amounts were relatively high. An adequate mini- After filling the apparatus with oxygen (5.0 quality) up to misation of the required sample amount is challenging: the 30 bar inner pressure (6 L), the combustion was started by detection limits achievable so far for decomposition ignition. Subsequently, the oxygen bomb was placed into a procedure of small sample amounts of 70 and 200 mg are water bath for cooling and nodded in order to allow contact not adequate for low sulphur contents in wood tissue. of the whole inner part with the absorption liquid. Ten The reagents might be a significant contamination minutes later, the apparatus was vented. Previous controls sources since neither NaOH nor ethanol were available in demonstrated that venting does not require the connection high purity quality. Decomposition vessels could be an to a gas washing flask with supplementary absorption additional contamination source. However, further optimi- liquid. Before each new measurement, decomposition sations were discarded since minimisation of the required vessel and crucible holder were rinsed with ultra-pure water sample amount is limited in the present composition system and dried. Combustions were all exempt of residue, except and the procedure is complex and time consuming. Better Sulphur and nutrient distribution in Norway spruce drill cores 83 results were obtained by the microwave acid digestion addition of MgO did not result in significant changes or described below. improved recovery rates. Therefore, addition of MgO was deemed unnecessary for digestion of sulphur in wood Development of a microwave acid digestion samples. The amount of magnesium in Norway spruce wood is about six times superior to that of sulphur and The main goal of the development of this digestion therefore the mineral content of wood is probably high procedure was to achieve a good temporal resolution and enough to bind volatile sulphur compounds. a reliable quantification of the total sulphur content with Optimisation and validation of the final digestion good recovery rates. Thus, the decomposition procedure procedure were performed using the biological certified was carefully optimised with respect to accurate quantifi- reference materials NIST 1572 Citrus Leaves, RM 8436 cation of very small amounts of sulphur in Norway spruce Durum Wheat Flour, NIST 1547 Peach Leaves, BCR 101 wood. It contains only an average of 5 μg S per 100 mg Spruce Needles, NIST 1573 Tomato Leaves, RM 8436 drill core piece. Therefore, digestion was optimised with Durum Wheat Flour NIST 1515 Apple Leaves and NIST special attention to minimum sample weight, optimum acid 1575 Pine Needles. Table 1 presents the averaged results of mixture, minimised sample losses and contamination risk. triple determinations in comparison to the certified values Using a clean scalpel, the drill cores were cut into five– and concentration ranges found in the literature [1, 6, 9, 23– ten tree ring segments, depending on the weight. A 29]. A comparison to the specific literature values as well minimum sample weight of about 40 mg was required to as information on used digestion method, required sample ensure accuracy. A 1:1 mixture (2×2.5 ml) of nitric acid amounts and analysis methods can be found in Table S2 in

HNO3 65% and hydrogen peroxide H2O2, 30% (both the online available supplementary information. The wave- Merck Suprapur®, www.merck.de) was finally selected for lengths used are specified in the last column of the table. the microwave digestion. All digestions were performed in Detection limits were determined with six procedural a MLS ETHOS 1600 microwave oven controlled by software blanks according the 3-Sigma plus Blank method. All version easyWAVE3 or in a MLS ETHOS 1200 microwave values are given in mg L−1 for direct determination of the (MLS Milestone, www.milestonesci.com). Table S1 (avail- solutions and in μgg−1 calculated for the solid samples. able online in the supplementary information) shows the Most of the results matched the range of the certified values digestion program finally used. The maximal pressure was ± uncertainties and showed recovery rates of 95% to 105%. set at 25 bar for the pressure and temperature controlled Only elements close to the detection limits such as for Cu MLS Ethos 1600 system, whereas the MLS Ethos 1200 or Cr showed higher recovery rates. Reproducibility for system is only temperature and power controlled. higher element contents ranges between 0.5% and 5%, Hafez et al. [2] recommended an addition of MgO in whereas for concentrations close to trace level the standard open digestions of biological samples with high methionine deviation is higher. concentrations to improve the sulphur recovery rates using However, the sulphur content of commercially available ion chromatography determination. Jeker and Krähenbühl certified reference materials is relatively high with 1,200 to [21] also recommended an addition of MgO for the 6,000 μgg−1 in contrast to about 20–200 μgg−1 sulphur in microwave acid digestions of material from peat bogs with real wood samples. Higher contents are usually found in the aim to avoid sulphur losses by the formation of MgSO4. bark. Therefore, self-prepared spiked cellulose and wood In order to verify if an addition of MgO is preferable for powder samples were used for the validation of the lower wood samples as well, several digestions in pure acid and concentration range. Spiked cellulose as well as spiked with addition of increasing amounts of MgO (0, 22.5, 45, wood standards of a final concentrations of 4, 20, 50, 100 90 and 135 mg L−1) were performed using the BCR-CRM and 200 μgg−1 sulphur were prepared as follows: Portions 101 (Pine Needles), as well as homogenised wood powder of 2 g cellulose powder or 2 g wood powder were doped (background S content of 58±4.6 μgg−1). Furthermore, with different amounts of a 200 μgL−1 sulphur solution homogenised wood powder doped with 20 μgg−1 sulphate (1:5 dilution of a 1,000 μgmL−1 Alfa Aesar Sulphur ICP and others doped with 20 μgg−1 cysteine were decomposed standard) and well mixed in pre-cleaned PTFE vials by in presence of different concentrations of MgO. The results additionally adding of 6 ml ultrapure water to obtain a of the doped wood standards showed no remarkable homogeneous paste. The paste was dried at 80°C until influence of the MgO on the recovery rate (Figure S1 in weight constancy. The dried spike self-prepared standards the online available supplementary information). Thus, were then pulverised in an agate mortar. The samples were natural magnesium content in wood samples seems to be digested using the procedure described above. Figure 1 high enough to fulfil this function. The natural Mg content represents the sulphur recovery rates for the double in Norway spruce varies between 100 and 560 mg kg−1 determinations of the spiked cellulose and the spiked wood [22]. Also for the certified reference material BCR 101, an powder standards. The suitability of the microwave 84 Table 1 Averaged results of triple determinations for plant certified reference materials in comparison to certified values and literature results

Certified Reference BCR 101 Spruce Needles NIST 1575 Pine Needles RM 8436 Durum Wheat Flour NIST 1573 Tomato Leaves Material

Sample ID Found Certified/ Min–Max Found Certified/ Min–Max Found Certified/ Min–Max Found Certified/ Min–Max [μg/g] (non-certified) Literature [μg/g] (non-certified) Literature [μg/g] (non-certified) Literature [μg/g] (non-certified) Literature values [μg/g] values [μg/g] values [μg/g] values [μg/g]

Al 156±3 173±5 126–165 516±14 545±30 520.0 19.6±2.8 11.7±4.7 11.8 727±37 (1200) 1130–1250 Ca 4506±98 4280±80 4150–4150 4111±78 4100±200 3950–4300 293±6 278±26 278 29385±230 30000±3000 29100–33000 Cr 2.9±0.1 (2.7) 2 1.2±0.34 2.6±0.2 1.9–2.8

Certified Reference NIST 1572 Citrus Leaves NIST 1547 Peach Leaves NIST 1515 Apple Leaves Material

Sample ID Found Certified/ Min–Max Found Certified/ Min–Max Found Certified/ Min–Max DL Used wavelength [μg/g] (non-certified) Literature [μg/g] (non-certified) Literature [μg/g] (non-certified) Literature [μg/g] values [μg/g] values [μg/g] values [μg/g]

Al 92±0.1 92±15 86–100.5 263±0.3 249±8 224–329 293±1 286±9 330 3.7 Al 308.215, Al 396.152 Ca 31407±185 31500±1000 28100–32000 15955±79 15600±200 15656–15840 15480±178 15260±150 14400–15341 21 Ca 396.847, Ca 422.673 Cr 0.99±0.02 (0.8) 0.7–0.84 1.5±0.1 (1) 0.6–1.32

140 Spiked Cellulose sulphur source. The trace gas enters the needles of conifers 120 Spiked Wood Powder via the stomata, is removed from the substomatal cavity by 100 solubilisation in the aqueous phase of the apoplastic space, and is taken up into the cytoplasm of surrounding cells 80 [32]. Once inside the cell, either storage in the vacuole or 60 assimilation into the amino acid cysteine takes place by the 40 sulphate assimilation pathway [33, 34]. Allocation of 20 sulphur from source needles to the main stem is low [35]. Sulphur Recovery Rate [%] Instead, most of the accumulated sulphur is allocated to the 0 200 100 50 20 following needle generation [36]. Falling needles, enriched Spiked sulphur concentration [µg/g] in sulphur, can contribute to raising the soil sulphur content. Wet deposition of sulphur remains the most important Fig. 1 The sulphur recovery rates for the double determinations of the sulphur source of forests. Due to the huge canopy surface spiked cellulose and the spiked wood powder standards of a Norway spruce tree, important amounts of aerosols and fog are scavenged. Crown interception is three times higher procedure developed for this work is clearly shown. The in Norway spruce than for a deciduous tree like beech spiked cellulose standards gave recovery rates between (Fagus sylvatica)[37]. As a consequence, the sulphur 98.3% and 103% and the wood standards showed varia- accumulation in the soil under Norway spruce is four times tions in a range of 91.5% to 102.2%. higher than under beech. This is accentuated by the fact that perennial foliage leads to uninterrupted crown interception Signal enhancement by USN during winter, when airborne sulphur from heating reaches a maximum. Accordingly, Norway spruce accumulates two Ultrasonic nebulizer USN was used to improve the signal times more sulphur in its phytomass than beech. intensity for low concentrated elements such as sulphur and An accumulation of anthropogenic sulphur in conifers at phosphorus. Figure S2 in the online available supplemen- an industrial site was proposed by Gaoming [14]. The high tary information shows the USN intensity enhancement for sulphur content measured in Chinese pine (Pinus tabulae- the USN compared to a conventional Meinhard nebulizer formis) are believed to reflect the important industrial SO2 for sulphur and phosphorus at selected wavelengths. The emissions in Chengde, northern China. While in industri- signal enhancement factor of the USN in comparison to the alised countries such as Switzerland, desulphurisation of

Meinhard nebulizer was about 7 for sulphur and about 13 crude oil and reduced use of coal permitted to cut SO2 for phosphorus, which corresponds to the findings of emissions by a factor of 3 after they peaked around the Wennrich et al. [9] who found enhancement factors of 4.9 eighties of the last century, but the global emission rate still for sulphur and 4.3 for phosphorus with a Spectroflame increases. Developing countries such as China depend on ICP-OES (Spectro Analytical Instruments, www.spectro. coal-fired power plants for covering their increasing energy com). Also the other elements showed a ten to 500 times needs. High sulphur content of coal then leads to an higher signal when USN was used. enormous SO2 release. In the latest report of the Interna- tional Panel on Climate Change (IPCC [50]), scenarios of a

Trees as environmental archives worldwide increase in SO2 emissions were predicted. In the present article, mineral content profiles of Norway Investigation of sulphur in tree rings is of particular interest, spruce trees will be discussed, with a special focus on since S is both a pollutant (mainly in form of anthropogenic sulphur. Beside sulphur, especially the profiles of macro- 2− SO2) and occurring naturally (e.g. as SO4 in soil). nutrients potassium and calcium, as well as of the Sulphur is one of the essential macronutrients for plants, micronutrient manganese were obtained. The results will and as such plays an important role for physiological be discussed with special regard to anthropogenic influen- processes such as photosynthesis, dissimilation and also for ces and plant physiological processes. defence via the production of antioxidants like glutathione [30, 31].

Three uptake mechanisms for atmospheric SO2 have to Sulphur be taken into account: 1. via needles (leaves), 2. via roots after deposition, 3. via run-off water through the bark. The principal focus of this study was to assess whether SO2 Uptake as sulphate in the rhizosphere is the main emissions can be reconstructed using tree drill cores mechanism of sulphur acquisition by plants. However, in sampled at different sites. Due to the uptake mechanisms heavily polluted areas, SO2 can become an additional mentioned above (uptake via roots, needles, and bark), 86 A. Ulrich et al.

Norway spruce was predestined for this purpose. The under natural conditions. Fig. 2b displays the total sulphur species is widespread in Switzerland and found at sites content profiles (1934–2003) for a Norway spruce tree in with completely different immission profiles, such as the Düdingen. Although the Düdingen trees have an average Alps and the Swiss Plateau. cambial age of 96±4 years and the trees from Frieswil an

The emissions rates of SO2 in Switzerland were average cambial age of 142±6 years, the profiles look very particularly pronounced towards the end of the seventies similar. There is only a slight variation in the Swiss Plateau and at the beginning of the eighties. Measures taken to sulphur profiles for the first three quarters of the 20th reduce these emissions led to a sustained decrease of SO2 century, whereas in the forth quarter, i.e. the tree rings pollution. The wood sulphur content was determined in correspondingto1974–1984, a clear sulphur peak is trees from three distinct locations in Switzerland: Düdingen visible. This corresponds to the period when SO2 emissions (countryside, near to a motorway), Frieswil (countryside) reached their maximum in Switzerland, as also evaluated by and St Moritz (Alpine) (see profiles Fig. 2). In Fig. 2a, the the Federal Office for the Environment (FOEN) and the averaged total S content profile achieved for the time range NABEL National Air Pollution Monitoring Network [38, 1954–2003 for three trees from Frieswil (F2, F3, F6) are 39]. Düdingen and Frieswil are both rural sites, separated displayed. The average standard deviation of the pooled by 20 km distance only. Both sites reflect the immission data amounts to 13%. Such deviations have to be taken into situation of their respective region, which is why there is a account, since the sampled trees all are individuals, grown slight shift between both peaks. The Düdingen sampling site at 580 m above sea level is close to a direct emission source. The motorway A12 (Berne–Fribourg), which is next to the sampled Düdingen forest, acts as a local sulphur dioxide source, which was detected also in the peat bog nearby [21] and also by measurements of trees. Although there is no direct pollution source in Frieswil (740 m above sea level.), deposition of anthropogenic sulphur occurs as well in trees from this site and is likely to be the reason for the peak. Trees from the alpine forest show that the sampling site of St Moritz (Engadine) is more secluded from the bulk of

the SO2 emissions in Switzerland. Since little local sources are present there, the sampling site is only affected by low long distance transport of sulphur. Sulphur profiles obtained from St Moritz trees do not display the characteristic maximum at 1974–1984 found in trees and peat bogs from the Swiss Plateau, which corresponds to the immission maximum in this time. Furthermore the tree profiles resemble sulphur content obtained from the St Moritz peat bog [40], which also has no maximum peak at this time. Moreover, the total sulphur content shows a generally higher S content as seen in Fig. 2c. The reason is probably not anthropogenic origin, but rather due to the particular- ities of the Alpine environment which lead to a very slow growth rate of the trees. However, sulphur profiles in trees as well as in peat bogs correspond to the local immission situations of the different sampling sites. Also measurements performed by EMPA for the National Air Pollution Monitoring Network (NABEL, http://www.empa.ch/nabel; http://www.bafu.admin.ch/luft)

showed that annual average SO2 concentrations in the Alps (e.g. NABEL sampling site Davos) are up to ten times lower than on the Swiss Plateau (e.g. NABEL measurement sites Härkingen or Payerne). Fig. 2 a–c Pooled total sulphur content profiles (1934–2003) for three Norway spruce tree in Frieswil (a), a Norway spruce tree in In most recent years, i.e. close to the cambium where Düdingen (b) and two Norway spruce trees in St. Moritz (c) cell division occurs, maximum sulphur content was Sulphur and nutrient distribution in Norway spruce drill cores 87 observed in all measured trees, including the alpine trees (Fig. 2). This effect is natural and not due to anthropogenic

SO2 emissions, which continue to decrease steadily in Switzerland. The reasons of this rapid decrease by a factor of 1.5 in direction of the pith are protein decay during heartwood formation and recycling of sulphate. Apparently, sulphur recycling is not complete during this process, and wood is used for sulphur storage [22, 41]. Thus, in case of higher sulphur input, as on the Swiss Plateau in the years 1970–1980, the amount of sulphur remaining in the wood tissue increases. The trend line displays a clear upward trend of the wood sulphur content towards the second half of the century. Part of it is due to the naturally higher sulphur content near the cambium (five most recent years), but the principal reason is likely the SO2 emissions of the Swiss Plateau. The upward trend in alpine trees (Fig. 2c) is much less pronounced, and principally due to the proximity of the cambium. While in trees from the Swiss Plateau, the sapwood- heartwood transition zone was found around 1,970±6 years in average, in St Moritz trees, the average boundary ranged Fig. 3 Effect of fungal decay (b) and compression wood (a) on the sulphur profile in two different Norway spruce trees in Frieswil in the tree rings at 1,941±16 years. Thus, the period with maximum SO2 emissions (1970–1980) is situated in the sapwood in both cases. The pollution peak which is visible in two types of archives from the Swiss plateau (Düdingen trees and peat bog, Frieswil trees) is likely not affected by the augmented lignin content provides more hydroxyl water transportation and element translocation, since the binding sites, which leads to higher cation content conducting system is most efficient in the outermost tree compared to the wood on the opposite side of the tree rings of Norway spruce [42]. [45]. Thus, the variations of the total S content shown in Two trees from the Frieswil forest showed deviating Fig. 3 are of physiological origin and presumably indepen- sulphur profiles, presented as F1 (A) and F4 (B) in Fig. 3, dent from the sapwood–heartwood transition zone as well. Reasons for the different pattern of these tree profiles were Manganese content was altered. too. Consequently, the investigated further. It is known that fungal infection can trees F1 and F4 were not included in the calculations alter the cation content of heartwood [43]. It appears that presented in the Fig. 2. the sulphur content can be massively altered, too: In tree Bark is exposed to precipitation and run-off water, and F1, the two huge S peaks found in wood segments thus constantly enriched with minerals retrieved from the corresponding to 1934–1938, and 1949–1953 are connected run-off water [45]. In the bark, nutrients can transiently be with decayed tree rings. Microscopy of small slices of these stored in parenchyma cells or/and can be subjected to long- two infected spots prior to acid digestion revealed the distance transport in the phloem (living tissue carrying presence of a fungal pathogen, presumably Ophiostoma organic nutrients and which lies underneath the bark). piceae or Ceratocystis piceae (Engesser, personal commu- Substances poisonous for the tree can be exuded, e.g. as nication). Further investigations are needed to estimate resin. Therefore, the element content of the bark is usually whether the huge increase is due to an accumulation of much higher than in wood tissue. The presence of sulphur sulphur by the pathogen, or to a defence mechanism in water running along the stem may lead to higher sulphate established by the tree. content of the bark, too [46]. Additionally, the phloem plays In contrast, tree F4 shows very low S contents related to an important role for the cycling of sulphur in trees and a compression wood zone ranging from about 1940–1980. contains sulphate and organic sulphur such as glutathione This type of reaction wood is found exclusively in conifers as well. Both sulphate and organic sulphur can be enhanced and built on the stressed side of the tree in case of when sulphur availability is high [47]. For these reasons, mechanical pressure e.g. cause by wind, snow, slope or the average bark sulphur content of seven trees from the − proximity of another tree. It distinguishes itself by its wider, three locations of the present study (423 μgg1±42%) is denser and darker growth rings. Compression wood con- about eight times higher than the average wood sulphur tains up to 25% less cellulose than normal wood [44] and content (54 μgg−1±14%). 88 A. Ulrich et al.

Potassium, calcium and manganese In trees exempt of pathogens and reaction wood, S, K, Ca and Mn show quite uniform patterns that allow general A detailed discussion on selected nutrients can be found physiological interpretation of the profiles for Norway in the online available supplementary information. Pro- spruce, and, in the case of S, to monitor past pollution events. files of the macronutrients potassium (Figure S3)and calcium (Figure S4) as well as the micronutrient manga- Acknowledgements The authors like to thank F. H. Schweingruber, nese (Figure S5) are presented and discussed. D. Nievergelt, J. Esper, W. H. Schoch, R. Engesser, and S. Zimmermann at WSL for providing advice and assistance for radiodensitometry. We also thank C. Zwicky from the Empa Laboratory for Solid State Chemistry and Catalysis, A. Wichser and Conclusion O. Nagel from the Empa Laboratory for Analytical Chemistray, and Raoul Seitzinger from the University of Berne for performing several Trees are challenging archives and the interpretation is digestions for the plant standard reference materials. Moreover, we are complex, since anthropogenic and natural physiological grateful to Max Haldimann from BAG who provided several certified plant reference materials for comparison. Last but not least we are signals can be intertwined. Pollutants, changes in soil grateful to A. Niederer from Empa Graphic Section, who drawn chemistry and climate not only influence tree growth but several element profiles. also have an impact on xylem chemistry. Thus, for a correct interpretation of element content profiles, knowledge about tree physiology is mandatory. 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