Agricultural and Food Science, Vol. 14 (2005): 70–82. 70 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. © Agricultural and Food Science Manuscript received November 2004 An analytical procedure for determination of sulphur species and isotopes in boreal acid sulphate soils and sediments Krister Backlund, Anton Boman Åbo Akademi University, Department of Geology and Mineralogy, FI-20500 Turku, Finland, e-mail: krister.backlund@abo.fi, anton.boman@abo.fi Sören Fröjdö Åbo Akademi University, Department of Geology and Mineralogy, FI-20500 Turku, Finland Mats Åström Department of Biology and Environmental Science, Kalmar University, SE-39182 Kalmar, Sweden An analytical scheme suitable for boreal acid sulphate (AS) soils and sediments was developed on the basis of existing methods. The presented procedure can be used to quantify and discriminate among acid volatile sulphide, cold chromium reducible sulphur, hot chromium reducible sulphur, elemental sulphur, sulphate sulphur, organic sulphur, total reducible sulphur and total sulphur. The sulphur fractions are recovered as either Ag2S or BaSO4 precipitates and can further be used for isotope analysis. Overlaps between sulphur species are common during speciation, and must be minimized. Some of these overlaps are caused by poor sampling and storage, inappropriate conditions during the distillation, or natu- ral variations in the sample (e.g. Fe3+ interference and grain size). The procedural impact was determined by conducting tests on both artificial and natural samples containing one or several sulphur species. The method is applied on reduced sediment from an AS soil locality (Överpurmo) and a brackish lake (Larsmo Lake) in western Finland and the results, including S-isotopes, are discussed. Key words: sulphur species, sulphur isotopes, analytical scheme, acid sulphate soils, sediment Introduction Acid sulphate (AS) soils occupy large areas of the tropical and subtropical coasts of Asia, Africa, Australia, and significant areas along the boreal coastal plains of Finland and Sweden (Palko 1994, Öborn 1994, Joukainen and Yli-Halla 2003). AS soils constitute a major environmental problem due to the release of acidity and metals during oxi- 71 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. dation of naturally occurring sulphur species (e.g. Åström 2001, Sundström et al. 2002). According to van Breemen (1973) potential AS soils com- monly contain pyrite sulphur (FeS2) between 1 and 4%, and ferrous monosulphides (FeS) comprise less than 0.01% and rarely exceed 0.6% even in reduced black muds. However, in black clays in north-eastern Sweden FeS comprises a significant amount (up to 80%) of the total sulphur (Georgala 1980). Iron sulphides are usually divided into two categories: (1) acid volatile sulphide (AVS) and (2) disulphides (FeS2). AVS consists of amorphous FeS, mackinawite (FeS0.94), greigite (Fe3S4) (Morse et al. 1987), dissolved sulphide (Morse and Rick- ard 2004), and amorphous monosulphides of other metals (e.g. zinc, cadmium or lead) (Lasorsa and Casas 1996, Morse and Luther 1999), while disul- phides include pyrite (cubic FeS2) and marcasite (orthorhombic FeS2) (Rice et al. 1993). Elemental sulphur (S0) generally occurs in small quantities (usually < 2% of total sulphur) in reduced marine muds and may be produced by oxidation of FeS, FeS2 and H2S (van Breemen 1973, Poulton et al. 2004). Organic sulphur is generally the most abun- dant sulphur species in normal soils, but in pyrite- bearing sediments and AS soils it is usually quan- titatively insignificant (van Breemen 1973). For example, Georgala (1980) estimated that organic sulphur constituted less than 0.03 wt% in black clays from north-eastern Sweden. Sulphate is usu- ally not present in large quantities in reduced sedi- ment, but in AS soils, a large number of sulphate minerals can be found in association with oxida- tion of mainly pyritic material. Most of these sul- phate compounds are water soluble and only per- sist in the absence of leaching (van Breemen 1973). Different forms of sulphur have previously been separated and quantified by various tech- niques (e.g. Zhabina and Volkov 1978, Nriagu and Soon 1985, Canfield et al. 1986, Tuttle et al. 1986, Hall et al. 1988, Fossing and Jørgensen 1989, Bates et al. 1993, Rice et al. 1993, Duan et al. 1997, Sullivan et al. 2000, Kallmeyer et al. 2004). At present, most of the sulphur speciation methods are based on the sequential extraction scheme that Zhabina and Volkov introduced in 1978, where iron monosulphides are digested in HCl and iron disulphides are reduced by Cr2+ in an acidic solu- tion. Elemental sulphur is commonly determined by dissolution with an organic solvent, followed by a Cr-reduction. The evolved H2S can be deter- mined gravimetrically as Ag2S, ZnS or CdS (e.g. Zhabina and Volkov 1978, Tuttle et al. 1986, Di Toro et al. 1990, Bates et al. 1993), by EDTA titra- tion (Newton et al. 1995), iodometrically, spectro- photometrically, polarographically, or by ICP-MS (Zhabina and Volkov 1978, Allen and Parkes 1995). Determination of reduced sulphur is per- formed under anoxic conditions, which can be ob- tained by using N2, CO2 or Ar (Zhabina and Volkov 1978, Hall et al. 1988). Sulphate is usually pre- cipitated as BaSO4 after separation of AVS (e.g. Nriagu and Soon 1985, Tuttle et al. 1986, Hall et al. 1988, Rice et al. 1993) or after removal of chro- mium reducible sulphur (Bates et al. 1993). Or- ganic sulphur is commonly converted to sulphate by oxidation with Eschka’s mixture and subse- quently precipitated as BaSO4 (e.g. Tuttle et al. 1986, Bates et al. 1993, Rice et al. 1993). A complete analytical scheme for the separa- tion and quantification of sulphur species present in a sample has not yet been developed despite several efforts. In order to determine which distil- lation technique is most suitable it is of importance to understand how various treatments affect the sample. There are often overlaps between different sulphur phases due to: (1) oxidation of AVS to S0 during sampling/storage; (2) oxidation of H2S to S0 during the distillation process; (3) the analytical conditions and treatments used; and (4) the grain size and crystallinity of the sulphide minerals. The aim of this study was to develop an ana- lytical scheme suitable for the determination of sulphur species and isotopes in boreal potential and actual AS soils and shallow coastal sediments. The development of such a scheme is important because knowledge of sulphur speciation and be- haviour in these materials is inadequate to under- stand and model how: (1) acidity is formed in and leached from AS soils; (2) marine sediments brought above the sea level by postglacial isostatic rebound (up to 9 mm per year) are turned into highly problematic AS soils; (3) the cold climatic 72 A G R I C U L T U R A L A N D F O O D S C I E N C E Backlund, K. et al. Determination of sulphur species in acid sulphate soils conditions affects neutralisation and formation of acidity in soils and waters; and (4) thick sediment layers uplifted above sea level are ultimately pre- served black, and thus monosulphide-rich and highly reactive, when exposed to atmospheric ox- ygen. Finally, the intent to study the isotopic com- position of each sulphur species in the samples puts additional constraints on the scheme. Firstly, distillation must be optimised in regard to the quantity and separation of species and secondly, sulphur must be extracted from each species in a form suitable for the later analysis of sulphur iso- topes. Material and methods Natural samples Reduced sediment from an AS soil locality (Över- purmo) and a brackish lake (Larsmo Lake) in western Finland were collected. The sediments are fine-grained and homogeneous with a clayey tex- ture and black colour. The Överpurmo sediment, located 40 meters above the present sea level (due to isostatic land uplift), was deposited in a shallow coastal environment about 4000 years ago. Fresh samples were stored (within two hours after sam- pling) in a freezer in order to minimize oxidation. In the laboratory, the samples were thawed in a ni- trogen-filled glove bag and any visibly oxidized surface material was removed, keeping only the un-oxidized core of the sample for subsequent analysis. Any shell fragments found in the sedi- ments were removed before further analysis. Artificial samples Mixtures of finely ground sulphur minerals (pyrite and elemental sulphur) and chemical reagents (Na2S·9H2O and Na2SO4) were prepared in order to test the analytical scheme. The sulphur concen- tration in pyrite (FeS2), elemental sulphur (S 0) and sodium sulphate (Na2SO4) was calculated based on the molecular formula. The range of the Na2S con- centration in Na2S·9H2O is 32–38% and the sul- phur concentration was determined to be 13.2 ± 0.003% by dissolving a known amount of Na2S·9H2O in deionized water (18.2 MΩ) and pre- cipitating the sulphide as Ag2S (assuming 100% recovery). Sulphur speciation method – general approach The distillation apparatus (Fig. 1a) used for extrac- tion of the sulphur species consists of a heating plate (with magnetic stirrer), a 500 ml reaction flask (with injection ports for reagents and nitro- gen) attached to a condenser, a 250 ml buffer ves- sel (containing 200 ml of 0.05 M potassium hydro- gen phthalate, pH 4) used for preventing formation of AgCl in the sulphide traps and a pair of 50 ml sulphide traps (containing 15 ml of 0.1 M AgNO3), of which the latter is used as a safety trap only. The glassware is connected by rubber tubing. A Cr2+ containing solution was prepared by percolating 1 M CrCl3·6H2O in 0.5 M HCl through a Jones reductor (Fig. 1b), constructed as described by Skoog and West (1976). In this process, Cr3+ is reduced to Cr2+ which can be verified by a colour change from dark green (Cr3+) to bright blue (Cr2+). The solution was collected in sealed plastic syring- es, where it was stable for several days. A frozen sample was thawed in a nitrogen- filled glove bag, homogenised and divided into subsamples. One of the subsamples was dried at 105°C for determination of the dry weight, while the other subsamples were used for sulphur specia- tion. Approximately 3 g of wet subsample was weighed (to the nearest tenth of a milligram) into the reaction flask, and 10 ml of ethanol was added to facilitate reflux condensation during distillation (Fossing and Jørgensen 1989). The distillation ap- paratus was flushed for 10 minutes with pure (99.5%) nitrogen gas before inserting reagents for H2S emanation, and nitrogen flowed continuously (approximately 5 bubbles per second in the buffer vessel) throughout the distillation process. Liber- 73 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. Fig. 1. A sketch of the equipment used in the speciation experi- ments: (a) the distillation appara- tus, and (b) the Jones reductor for preparation of CrCl2 solution. ated H2S from reduced sulphur was transported, through the buffer solution into the sulphide trap and precipitated as Ag2S. After each reaction step was complete, the trap was replaced and distilla- tion of another species started by addition of the appropriate reagent to the reaction flask. Deoxy- genating all solutions with nitrogen before analy- sis minimized oxidation of H2S during reaction. The remaining slurry in the reaction flask was fil- tered, and the filtrate and residue were analysed for sulphate and organic sulphur, respectively. Precipitated Ag2S was filtered and washed with deionized water (18.2 MΩ) on preweighed What- man (No 42) filter papers. After drying at 105°C for two hours, the precipitate was cooled in a desic- cator for 30 minutes. BaSO4 was filtered on S&S blue ribbon 5893 filter papers and transferred to a preweighed porcelain crucible and slowly ignited (2 hours) to 800oC, leaving only BaSO4. The cruci- ble was left to cool in a desiccator for 30 minutes. Weight percents (wt%) of sulphur were calculated from the weights of Ag2S or BaSO4. The detection limit for S was estimated to be 0.01 wt% in a 1 g sample (dry weight). Sequential extraction procedure An analytical scheme (Fig. 2) was devised for dis- tinguishing AVS, cold chromium reducible sulphur (CCrS), hot chromium reducible sulphur (HCrS), elemental sulphur (ES), sulphate sulphur (SO4 2-), organic sulphur (OrgS), total reducible sulphur (TRS) and total sulphur (TotS). For determination of ES, a wet subsample was placed in a 15 ml centrifuge tube together with 8 ml dichloromethane (CH2Cl2). After 24 hours, the tube was centrifuged and the supernatant (contain- ing dissolved ES) was transferred to a reaction flask for evaporation. Solid ES in the reaction flask was subsequently heated in 50 ml of 6 M HCl and 50 ml of 1 M CrCl2, and the concentration of ES was determined from the precipitated Ag2S. ES is thought to mainly consist of S0, but some forms of organic sulphur (e.g. organic polysulphides) may also dissolve in the organic solvent (Mossmann et al. 1991). The residue in the centrifuge tube was transferred to a reaction flask for further analyses. The residue, or a fresh subsample if ES was not determined separately (Fig. 2), was weighed into 74 A G R I C U L T U R A L A N D F O O D S C I E N C E Backlund, K. et al. Determination of sulphur species in acid sulphate soils the reaction flask, and 50 ml of 6 M HCl was added for the determination of AVS. The distillation was completed after approximately two hours. Accord- ing to previous studies (e.g. Morse et al. 1987) AVS is believed to comprise mainly iron monosul- phides and some greigite. Different treatments and chemicals are known to affect AVS recovery, and additional experiments were performed: (1) by ad- dition of ascorbic acid (AA); (2) by heating to boiling point; (3) by addition of 5 g SnCl2; and (4) by addition of 15 ml 20% (w/v) zinc acetate (ZnAc). After removal of AVS, 50 ml of 1 M CrCl2 was added, and 4–5 hours later any CCrS, consisting of “less mature” pyrite (Duan et al. 1997) and possi- bly some greigite and organic polysulphides (Can- field et al. 1998), had been extracted. Thereafter the reaction flask was heated to boiling point for 1–2 hours until HCrS distillation was complete. HCrS comprises “more mature” pyrite, i.e. possi- bly larger grains that may be coated with cements and overgrowths (Rice et al. 1993), and ES that has not been removed prior to this stage (Duan et al. 1997). The remaining content in the reaction flask was filtered, and the filtrate was used for de- termination of dissolved SO4 2- and the residue for OrgS. The filtrate was treated with 10 ml of 30% H2O2 at 60 oC in order to oxidize any organic mat- ter in the solution. The next day the volume was reduced to 200 ml, and excess (10 ml) 10% BaCl2 was added dropwise, while stirring, and the solu- tion was left overnight at 60°C before filtering and weighing the precipitated BaSO4. The SO4 2- may occur in the form of acid soluble sulphate (e.g. ja- rosite: KFe3(SO4)2(OH)6), water soluble sulphate (e.g. gypsum: CaSO4) or as adsorbed sulphate (Begheijn et al. 1978). The residue was washed with deionized water and dried at 105°C. Approximately 1 g of dry resi- due was mixed with 3 g of Eschka’s mixture and placed in a porcelain crucible with an additional layer of Eschka´s mixture on top. The crucible was heated at 800°C for two hours, in order to oxidize sulphur to sulphate. The fusion residue was then transferred to a 500 ml Erlenmeyer flask and dis- solved in 200 ml deionized water. After simmering for 30 minutes, the solution was filtered and the pH was adjusted to less than 4 (prevents formation of iron oxides). Excess (10 ml) 10% BaCl2 was added dropwise, while stirring, to the filtrate (< 200 ml), and the solution was left overnight at 60°C before filtering and quantifying the precipi- tated BaSO4. The OrgS fraction extracted consists of non Cr-reducible organic sulphur, and possibly BaSO4. The Eschka’s fusion can also be used to determine the total amount of sulphur in the sam- ple (Fig. 2). Total reduced sulphur, comprising AVS, CCrS, HCrS and ES, was determined on a separate sub- sample. After adding 50 ml of 6 M HCl and 50 ml of 1 M CrCl2 to the sample, the solution was boiled for 1–2 hours for complete distillation of TRS. The remaining slurry in the reaction flask can further be analysed for SO4 2- and OrgS. Isotopic measurements The quantities of recovered Ag2S or BaSO4 pre- cipitates varied depending on the sample size, sul- phur concentration and proportion of sulphur spe- Fig. 2. Flow diagram of the analytical procedures used in this study. 75 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. cies, and analysis of the isotopic composition of sulphur was not always possible. However, when sufficient material was available, standard meth- ods were used to prepare the samples for analysis. About 40 mg of Ag2S (corresponding to approxi- mately 5 mg of sulphur) was converted to SO2 by reaction with 200 mg cuprous oxide (Cu2O), ac- cording to the procedure by Robinson and Kusak- abe (1975). The BaSO4 samples were prepared fol- lowing the procedures outlined by Yanagisawa and Sakai (1983), where about 10 mg of BaSO4 was mixed with 200 mg (1:20) of a 1:1 V2O5:SiO2 mix- ture. The sample mixtures were placed in small quartz-glass capsules, which were introduced into an evacuated preparation line. For both sample types, the temperature was slowly raised from 300°C to 950°C during 15 minutes to ensure com- plete combustion. A spiral of metallic copper was placed at the mouth of the furnace in order to con- vert SO3 to SO2. Gas yields from combustion were monitored by a pressure gauge on a volume cali- brated part of the vacuum line and samples with SO2-yields < 95% were discarded. To obtain a pure SO2 sample, H2O(g), CO2(g) and uncompressible gases were removed by distillation under high vac- uum. The SO2 was introduced into a modified VG Micromass 602 that is run by in-house software (originally developed at the Museum of Natural History in Stockholm, Sweden). The measured 34S/32S ratios are reported as δ34S values, i.e. the parts per mil deviation of the sample relative to the 34S/32S ratio in the Canyon Diablo Troilite (CDT). The precision was estimated to approximately ±0.3‰, and measurements on standard materials NBS-127 BaSO4 (δ 34S 20.3 CDT) and Göttingen CdS (δ34S -20.8 CDT) gave δ34S values of 20.34 and –21.1, respectively. Results and discussion Recovery of sulphur from pure phases and artificial mixtures In order to test the distillation technique several sulphur compounds were analysed individually and in mixtures. AVS is represented by Na2S·9H2O (hereafter referred to as Na2S), and CCrS, HCrS and SO4 2- is represented by natural pyrite (FeS2), elemental sulphur (S0) and sodium sulphate (Na2SO4), respectively. The mixtures (M1-M5) were: M1 = Na2S + FeS2 + S 0 + Na2SO4; M2 = Na2S + FeS2 + S 0; M3 = Na2S + FeS2; M4 = Na2S + Na2SO4; and M5 = FeS2 + Na2SO4. The samples were analysed without addition of ascorbic acid and ethanol and the results are presented in Fig. 3. Fig. 3. Summed recoveries (%) of sulphur species, separated indi- vidually and from mixtures (in- cluding different grain sizes). Ideally, each compound will show a (theoretical) recovery of 100%, and the sum of e.g. four species equals 400%. Deviations from 100% for an individual compound indicate cross-con- tamination between sulphur pools, or loss of sulphur. 76 A G R I C U L T U R A L A N D F O O D S C I E N C E Backlund, K. et al. Determination of sulphur species in acid sulphate soils The recoveries for two different grain sizes of FeS2 (< 62µm and > 62µm) and S 0 (< 75µm and > 75µm) show significant differences. For fine grained FeS2 the recovery was 90.4% but only 59.4 ± 6.4% (1 SD) for the coarser fraction. For S0 the results were similar, i.e. 96.2% and 71.9 ± 11.0%, respectively. However, grain sizes above 62 µm for pyrite and elemental sulphur are extremely rare in natural sediments and AS soils, except for sites close to e.g. ore deposits. The Na2SO4 was completely recovered (100.1 ± 1.1%) from the mixtures (M1, M4 and M5), and apparently did not affect the recovery of other spe- cies. The Na2S was completely recovered when separated individually (98.4 ± 2.2%) and with sul- phate (96.4% and 99.9%), but when separated in mixture with FeS2 (M3) only 85.4 ± 2.9% was re- covered, while the recovery of FeS2 was 118.5 ± 7.7%. However, the sum of Na2S + FeS2 in M3 (203.9 ± 5.5%) shows that a fraction of Na2S was recovered in the FeS2 pool, explained by partial oxidation of Na2S to elemental sulphur, subse- quently extracted with the cold Cr-reduction. Sim- ilar results have been reported by Fossing and Jør- gensen (1989). In M1 and M2, the recovery of FeS2 was even larger (130.5 ± 14.2%), and it is likely that some of the “additional FeS2” comes from original S 0, and consequently that a small fraction of S0 (presuma- bly very fine grained) was reduced by the cold Cr- reduction. The sum of recoveries for M1 (369.7 ± 2.3%) and M2 (290.0 ± 1.0%) were slightly below the expected 400% and 300%, respectively, and can be attributed to the large grain size of unre- acted S0. In all experiments with coarser fractions of S0 (> 75µm), the dissolution of S0 was incom- plete even after 18 hours of distillation, compared to complete distillation after 4 hours using the finer fraction. Sulphur species in sediments When comparing the sum of reduced sulphur spe- cies (AVS, ES, CCrS and HCrS) with TRS in the potential AS soil and lake sediment (Table 1), there is an indication that little, or none, of the H2S de- veloped from the individual distillation steps is lost during the process. It has previously been shown that Cr2+ does not reduce any significant quantities of sulphate or organic sulphur in sedi- ments (e.g. Zhabina and Volkov 1978, Howarth and Jørgensen 1984, Canfield et al. 1986), and no excess CCrS or HCrS is expected from the SO4 2- pool. For sulphur species in the Överpurmo and Larsmo sediment CCrS comprised the largest pool (except for the Larsmo sample treated with ZnAc, where AVS was the largest pool), followed by AVS, HCrS, ES, and minor amounts of SO4 2- (< 0.01 wt%) and OrgS (< 0.1 wt%). The concen- tration of TotS is somewhat lower than the com- bined sum of the separated sulphur species. The recovery of AVS (Table 1) in the Överpur- mo sample did not show any major differences whether using cold 6 M HCl (0.46 ± 0.04 wt%), cold 6 M HCl and addition of 5 ml of 0.1 M ascor- bic acid (AA) (0.40 ± 0.04 wt%) or heating of 6 M HCl with addition of AA (0.42 ± 0.03 wt%). Pru- den and Bloomfield (1968) showed that presence of Fe3+ in sediments could affect the determination of reduced sulphur by oxidizing H2S to elemental sulphur in the reaction flask. To prevent this, Fe3+ is converted to Fe2+ by adding AA (Hsieh et al. 2002) or SnCl2 (Pruden and Bloomfield 1968) to the sample. Hsieh and Shieh (1997) noticed that addition of AA to a freeze-dried sediment increased recovery of AVS, but not in a fresh sample of the same sediment. This was probably due to the for- mation of Fe3+ during the freeze-drying, and that the fresh sediment only contained Fe2+. The reason why AA in this study did not have an effect was probably due to lack of reactive Fe3+ in the sedi- ment. Amorphous FeS and mackinawite are com- pletely dissolved in cold 6 M HCl, while decom- position of greigite may be incomplete (Cornwell and Morse 1987). This, and possible formation of elemental sulphur during breakdown of greigite (Allen and Parkes 1995) requires harsher treat- ments (e.g. use of heat and/or a reducing agent). Addition of SnCl2 to the Överpurmo sample in- creased the recovery of AVS (0.74 ± 0.03 wt%) (Table 1), most likely due to the reduction of small amounts of disulphides (Cornwell and Morse 1987). Therefore AA, which is a milder reagent 77 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. T ab le 1 . S ul ph ur ( S ) sp ec ia ti on i n a po te nt ia l ac id s ul ph at e so il ( Ö ve rp ur m o) a nd a l ak e se di m en t (L ar sm o L ak e) . R es ul ts a re i n w ei gh t pe rc en t of d ry w ei gh t (± 1 S D , de vi at io n va lu es f or ∑ re dS a nd ∑ to ta lS a re t he m ax im um c al cu la te d er ro rs ). T re at m en ts A V S C C rS H C rS E S (* ** S O 42 - O rg S ∑ re dS T R S ∑ to ta lS T ot S Ö ve rp u rm o A sc or bi c ac id ( A A ) + 6 M H C l(* 0. 40 ± 0 .0 4 2. 17 ± 0 .0 6 0. 09 ± 0 .0 4 – < 0 .0 1 0. 08 ± 0 .0 1 2. 66 ± 0 .1 4 – 2. 75 ± 0 .1 5 2. 65 ± 0 .0 2 A A + 6 M H C l(* 0. 16 ± 0 .0 3 2. 16 ± 0 .0 5 0. 10 ± 0 .0 2 0. 12 ± 0 .0 3 < 0 .0 1 0. 07 ± 0 .0 1 2. 54 ± 0 .1 3 – 2. 62 ± 0 .1 4 – 6 M H C l(* 0. 46 ± 0 .0 4 2. 14 ± 0 .0 1 0. 05 ± 0 .0 3 – < 0 .0 1 0. 07 ± 0 .0 1 2. 65 ± 0 .0 8 – 2. 73 ± 0 .0 9 – H ea t + A A + 6 M H C l(* 0. 42 ± 0 .0 3 – – – – – – – – – H ea t + S nC l2 + 6 H C l(* 0. 74 ± 0 .0 3 – – – – – – – – – H ea t + 6 M H C l + C r2 + (* * – – – – – – – 2. 67 ± 0 .0 1 – – L a rs m o L a ke 6 M H C l(* 0. 14 ± 0 .0 2 0. 25 ± 0 .0 1 0. 03 ± 0 .0 1 0. 10 ± 0 .0 3 < 0 .0 1 0. 05 ± 0 .0 1 0. 52 ± 0 .0 7 – 0. 58 ± 0 .0 8 0. 51 H ea t + 6 M H C l + C r2 + (* * – – – – – – – 0. 51 ± 0 .0 1 – – Z in c ac et at e + 6 M H C l(* 0. 24 0. 16 0. 04 0. 05 – – 0. 49 – – – A V S = a ci d vo la ti le s ul ph id e; C C rS = c ol d C r- re du ci bl e S ; H C rS = h ot C r- re du ci bl e S ; E S = e le m en ta l S ; S O 42 - = s ul ph at e S ; O rg S = o rg an ic S ; ∑ re dS = s um o f A V S , C C rS , H C rS , a nd E S ; T R S = t ot al r ed uc ib le S ; ∑ to ta lS = s um o f A V S , C C rS , H C rS , E S , S O 42 - , an d O rg S ; an d T ot S = t ot al S b y E sc hk a’ s fu si on . (* T he t re at m en ts w er e us ed f or d et er m in at io n of A V S . (* * T re at m en ts f or d et er m in at io n of T R S . (* ** E S r em ov ed w it h di ch lo ro m et ha ne p ri or t o A V S . – = N ot m ea su re d. 78 A G R I C U L T U R A L A N D F O O D S C I E N C E Backlund, K. et al. Determination of sulphur species in acid sulphate soils but still effective in preventing Fe3+ interference (Hsieh et al. 2002), is proposed as the preferable reagent to use when analysing AS soil samples. A major concern during sampling is the oxida- tion of AVS to elemental sulphur by atmospheric oxygen, leading to a lower AVS recovery and a higher ES recovery (or higher HCrS). Addition of ZnAc will fix dissolved sulphides and AVS (mono- sulphides and possibly greigite) as the more stable ZnS, which however will decompose as easily as the original reduced sulphur species during analy- sis (Morse et al. 1987, Duan et al. 1997). The addi- tion of ZnAc to the Larsmo sample increased the AVS pool by approximately 42% to 0.24 wt%, while the CCrS pool decreased by approximately 36% to 0.16 wt%. This is possibly due to conver- sion of greigite to ZnS, which means that no S0 is formed upon acid treatment in the analytical pro- cedure. We therefore believe that the addition of ZnAc gives more accurate separations, and that consequently there is a risk that the CCrS pool for the Överpurmo sample is overestimated. Lasorsa and Casas (1996) noticed that addition of ZnAc may allow additional AVS to form in the sediment during storage for more than two weeks, and as a consequence, they recommended that ZnAc should not be used. We suggest, in contrast, that ZnAc could be admixed, prior to analysis, with the thawed sediment in order to prevent a proportion of greigite to end up in the CCrS pool. However, the assumed conversion of greigite to ZnS is not certain and should be verified in a separate experi- ment. The HCrS pool in the Överpurmo and Larsmo samples did not show any major variations in re- covery (Table 1). Since HCrS did not differ much, whether ES was removed or not, we consider that this is evidence for very little (or no) elemental sulphur in the sediments. The identified and quan- tified ES is probably the result of oxidation of AVS during analysis (i.e. after the addition of dichlo- romethane), sampling or storage. Sulphur-isotopic compositions Ideally, the distillation procedure recovers all of the sulphur in each species as Ag2S or BaSO4 pre- cipitates, thus allowing for analysis of the isotopic composition of each species. However, as shown above, there are several opportunities for overlap in the extraction of different species. Determining the isotopic composition of Ag2S and BaSO4 pre- cipitates collected in the speciation test above (mixtures M1 and M3–M5, Table 2) and compar- ing them to the original composition of the ‘pure’ compounds used tests for the effect on the isotopic composition of sulphur. As the compounds chosen to represent the spe- cies AVS, CCrS, HCrS and SO4 2- were off-the- Table 2. Isotopic compositions (δ34S) and recovery (%, in brackets) of sulphur (S) containing chemical reagents (Na2S and Na2SO4) and natural sulphur minerals (FeS2 and S 0). The values for pure reagents were determined on individually prepared samples, while values for compounds are after separation from various mixtures. The analytical precision for δ34S was estimated to 0.3‰. Species Acid volatile sulphide Cold Cr-reducible S Hot Cr-reducible S Sulphate S Compounds Na2S FeS2 S 0 Na2SO4 Pure reagents 0.5 9.3 1.0 7.1 Na2S + FeS2 + S 0 + Na2SO4 –0.4 (79.6) 6.8 (126.7) –0.5 (65.9) 7.7 (99.7) Na2S + FeS2 0.3 (84.7) 8.6 (113.1) – – Na2S + FeS2 0.1 (89.4) 8.8 (110.4) – – Na2S + Na2SO4 –0.3 (99.9) – – 7.0 (101.6) Na2S + Na2SO4 0.3 (96.4) – – 7.3 (100.5) FeS2 + SO4 – 9.8 (89.8) – 7.1 (99.5) FeS2 + SO4 – 9.4 (93.1) – 7.5 (101.2) – = Not measured. 79 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 14 (2005): 70–82. shelf chemical reagents and mineral sulphides (Ta- ble 2), some inhomogeneity in δ34S is probable. This, together with analytical error, may to some degree encroach upon the integrity of the data and the results may be regarded as tentative only. The differences in δ34S values of the compounds were significantly large to reveal isotope mixing be- tween critical pairs, but mixing of pairs with simi- lar δ34S and mixing with more than two members is impossible to be correctly estimated. Further- more, the δ34S value is defined on a one-dimen- sional scale and mixing between e.g. FeS2-Na2S or FeS2-S 0 in the same sample cannot in this case be discriminated. Therefore, the mixture M2 from the separation experiment above was excluded. With the exception of M1, the experiments were set up to reveal overlaps and mixing between pairs of species with significantly different δ34S values. Starting from an initial δ34S value of +0.5‰ (Table 2), the extracted AVS ranges from –0.4‰ to +0.3‰, a decrease slightly larger than the estimat- ed analytical precision (±0.3‰). With the excep- tion of M4, the recovery of AVS and HCrS was low, and incomplete reaction may have caused fractionation of S-isotopes, and depletion of 34S in the emanated H2S is probable. The SO4 2- was more or less quantitatively recovered in all experiments and, with the exception of M1 (δ34S = 7.7‰), the δ34S of the extracts were similar (δ34S = +7.0– 7.5‰) to that of the pure reagent (7.1‰). This shows that the speciation method does not cause overlap involving SO4 2-. However, the δ34S of CCrS varies considerably and overlap between the other species is significant. Mixture M1 contained all species and the re- sults varied; recoveries of AVS and HCrS were low and CCrS was high, while the recovery of SO4 2- was close to 100% (Table 2). The δ34S of CCrS showed a large decrease and, judging from the recoveries, the incorporated sulphur originated from AVS and HCrS. The high δ34S of the SO4 2- (+7.7‰) is somewhat mysterious; mass balance considerations suggest that the residual material of both AVS and HCrS should be roughly +4‰ (as- suming no crossover to CCrS) and could not in- crease the value +7.1‰ if mixed with SO4 2-. The recovery also indicates that this is not a possibility, so more probably, it must be deemed analytical. In the binary mixtures M3 and M4, the recoveries of AVS and CCrS nearly added up to the expected 200% and mass balance was preserved. The recov- ery of CCrS in M3 was slightly high and the de- crease in δ34S was significant; the only possible contaminant in M3 was AVS and the overlap dur- ing distillation clearly affected the isotopic com- position of CCrS. The recovery of AVS and SO4 2- was almost 100% for both species in M4, and no significant change was noted in the isotopic com- position of either species (the low δ34S in the first M4 AVS was probably caused by analytical error). In M5, there was no overlap between CCrS and SO4 2-, but there was some loss of CCrS that per- haps was coupled with fractionation of sulphur, as seen in one measurement with δ34S of +9.8‰. The notably low recoveries and isotopic cross- contamination seen in the results are probably largely effects of grain size variation in the artifi- cial samples prepared and attention must be given to diminution of the reagents in future experi- ments. However, we believe that this effect is lim- ited to the artificial sample and that the more ho- mogenous and finer grain size of clayey sediments results in better extraction of species (see Table 1) and less isotopic cross-contamination. The isotopic compositions of sulphur species in the Överpurmo sediment are presented in Table 3. Duplicate samples were analysed in two paral- lel distillation lines and showed identical results within error, indicating the consistency of the method for extracting sulphur species for isotopic measurements from this type of sediments. The δ34S values of the reduced sulphur species (AVS, CCrS and HCrS) were similar and varied only within a small range (δ34S = 5.1-5.6‰) while the value of OrgS was slightly lower (4.1‰). The high δ34S of the TotS (6.0‰) was intriguing, but considering that the result of the TotS method (2.65 wt%, Table 1, Överpurmo A) was less than the sum of the species (2.75 wt%) in the same sample, it seems reasonable to believe that there is some loss of sulphur in the TotS method. Heating the sample with Eschka’s mixture might cause volatilization of sulphur, leaving a residue slightly enriched in 34S. 80 A G R I C U L T U R A L A N D F O O D S C I E N C E Backlund, K. et al. Determination of sulphur species in acid sulphate soils The isotopic composition is used to study the relationship between sulphur species and can re- flect the prevailing conditions during their forma- tion. Reduced sulphur species (AVS, CCrS and HCrS) commonly develop in this type of sedi- ments through dissimilatory sulphate reduction, while OrgS may form by assimilatory sulphate re- duction (incorporation into marine organisms). Sulphur assimilated by living organisms consti- tutes 0.5–3.0% of the dry weight (Dinur et al. 1980), and according to Kaplan et al. (1963) the maximum contribution of sulphur to the sediment from marine organisms (e.g. animals and algae) would be about one percent dry weight of the or- ganic matter. Applying this reasoning to the Över- purmo sediment, where the content of organic matter was determined to 10.6%, suggests that the sulphur (OrgS) incorporated in the organic matter would be about 0.1 wt%. This is in agreement with the actual OrgS content of 0.07 ± 0.02 wt % deter- mined in the Överpurmo sediment, and we propose that this sulphur originates from assimilatory sul- phate reduction. Since the isotopic fractionation during assimilatory sulphate reduction is small, the organic sulphur in marine organisms (and ter- restrial plants) should directly reflect the δ34S iso- topic ratio of the sulphate available in the growth environment (Dinur et al. 1980). At present, sul- phate in freshwater has a δ34S of 6 ± 3‰ and 20 ± 3‰ in seawater (Dinur et al. 1980), which indi- cates that the OrgS in the Överpurmo sediment may have been formed under the influence of freshwater. In a closed system sulphate will be completely reduced, yielding sulphides with simi- lar δ34S as the starting sulphate (Goldhaber and Kaplan 1980). The isotopic compositions of re- duced sulphur species (AVS, CCrS and HCrS) and organic sulphur (OrgS) in the sample thus further establish that the Överpurmo sediment was depos- ited in a freshwater environment. Conclusions The presented scheme provides a nearly complete analytical procedure for the separation and quanti- fication of sulphur species present in boreal AS soils and in shallow coastal sediments. In order to optimize the analysis of sulphur species, the fol- lowing must be considered: (1) separation and re- covery of sulphur species is more accurate for fine- grained samples (e.g. clayey sediments) but coars- er material can be analysed if the procedures are adjusted for grain size effects (e.g. longer reaction times, harsher treatments); (2) if abundant, ele- mental sulphur can be analysed separately in a first step using dichloromethane; (3) ascorbic acid should be added to prevent Fe3+ from oxidizing H2S to S 0; and (4) addition of ZnAc improves the separation of AVS. The Ag2S and BaSO4 precipitates from the spe- ciation procedure can further be analysed for the isotopic composition of sulphur, thus allowing for discussion of the origin of the sulphur species. With the suggested modifications, cross-contami- nation between sulphur species is reduced and when combined with isotopic data, the procedure is a useful tool for future studies of sulphur-rich sediments and boreal AS soils. Acknowledgements. This research was financially sup- ported by the Renlund Foundation, the Åbo Akademi Foundation, the Finnish Graduate School in Geology, the Magnus Ehrnroot Foundation and the Finnish Society of Sciences and Letters (the Sohlberg foundation). Table 3. Isotopic composition (δ34S) of sulphur (S) species from the Överpurmo sediment. The analytical precision for δ34S was estimated to 0.3‰. 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