Various methods were used to investigate soil and pore water parameters which are relevant for metal(loid) binding and mobility. The most important methods will be mentioned in the following. Analytical details can be found in the method sections of the individual studies added in the appendix.
2.1.1 Sampling and laboratory practices
Due to the focus of all studies on O-limited environments, the influence of atmospheric O on samples was minimized in order to keep the redox integrity of the samples undisturbed. Soil samples were collected in the field with various types of soil and peat core samplers and were either immediately frozen on dry ice or cooled (4°C) under inert (helium (He)) atmosphere and then transported to the laboratory for further processing.
Pore water in study 6 was sampled using equilibrium dialysis samplers (peepers) as described by Hesslein165 after installing the peepers at least for four weeks to guarantee equilibrium conditions. In study 1, pore water was obtained by manually squeezing water saturated peat samples in N2-filled bags on-site.
Afterwards, water samples were stabilized accordingly (as required by the targeted analysis) or were immediately flash-frozen and transported to the laboratory under cooled or frozen conditions.
For all experiments and sample preparations, doubly-deionized water and analytical grade reactants were used. All O-sensitive (field) samples were treated and all redox-sensitive experiments were conducted under inert atmosphere in a glove bag (95% N2/5% H2, Coy). Soil and peat samples were freeze-dried, ground and stored under dark conditions in the glovebag until further analyses.
2.1.2 Preparation of model peat
Model peat was used in studies 1, 2 3, and 5 to exemplary investigate the binding behavior of As and Sb species to organic functional groups of solid NOM. To prepare the model peat, peat taken from the Federseemoor, Bad Buchau, Germany,120 was wet-sieved to a size fraction of 63-250 μm. This peat fraction was then washed once with 0.1 M HCl, to decrease the content of potentially As/Sb-complexing polyvalent metal cations (for example Fe(III) and Al(III)), followed by washing several times with doubly-deionized water, until the pH value returned to the initial pH of 4.5. The washed peat was freeze-dried, homogenized, and stored in the dark inside a glovebag until further use. Total element composition as well as functional groups of this peat were characterized in order to work under defined conditions (methods are described in chapter 2.1.4 and detailed results are reported in the Supporting Information (SI) of study 1). This peat-derived solid NOM is defined as model peat throughout this thesis.
2.1.3 Aqueous-phase analyses
Redox potential, pH, and electrical conductivity were determined directly in solution or soil suspension
Methods
after sampling in laboratory experiments or on-site in the field. Total aqueous S, Fe, As and Sb concentrations from pore water or other aqueous phase samples were determined by inductively coupled plasma mass-spectrometry (ICP-MS, X-Series 2, Thermo-Fisher) in stabilized (0.5% (v/v) H2O2, 0.8% (v/v) HNO3) samples. Original samples were filtered (0.2 µm cellulose acetate or nylon) and diluted accordingly before analysis. Total dissolved organic carbon (DOC) and N were analyzed in filtrates (0.45 μm, polyamide) by use of thermo-catalytic oxidation with a TOC-VCPN Analyzer (Shimadzu).
Arsenite, arsenate, and thioarsenate species as well as sulfate and thiosulfate were determined by anion-exchange chromatography (AEC, ICS- 3000, Dionex; column: IonPac AS-16/AG-16 4-mm) coupled to an ICP-MS (XSeries2, Thermo- Fisher) after thawing the shock-frozen samples in a glovebag. Aqueous Sb speciation (Sb(III) or Sb(V) species) was either analyzed with an AEC (column AG, AS 16 ) coupled to an ICP-MS following the protocol of Hockmann et al.67 (shock-frozen samples) or with an AEC (column:
PRPX-100, 250 x 4.1 mm, 10 μm, Hamilton) coupled to an ICP-MS using an isocratic elution with 10 mM NH4NO3, 10 mM NH4H2PO4, and 1.3 mM Na2-EDTA at a flow rate of 1.0 mL/min (0.2% (v/v) HCl stabilized samples). Internal standards and reference materials were included in every analytical run to ensure data accuracy.
Dissolved sulfide was quantified using the methylene blue method166 and ferrous iron (Fe(II)) as well as total dissolved iron (Fe(tot)) were measured using the phenanthroline method167. In the field, sulfide as well as Fe(II) and Fe(tot) were determined with a portable photometer (LASA 100, Dr. Lange) at wavelengths of 605 nm and 480 nm, respectively. In the laboratory, these species were analyzed at an absorption wavelength of 650 nm and 570 nm, respectively, using a multiplate reader (Infinite 200 PRO, Tecan).
Zero-valent S (S(0)-species) was determined in ZnAc stabilized samples after chloroform (CHCl3) extraction by high-performance liquid chromatography (HPLC, LaChrom Elite, L-2130 pump, L-2200 autosampler, L-2420 UV-Vis detector, Merck Hitachi) using a reversed phase C18 column (Luna, 3µm, 150 x 2.0 mm, Phenomenex) following the method published by ThomasArrigoet al.41.
2.1.4 Solid-phase analyses
Total element contents of S, Fe, As, and Sb within freeze-dried and homogenized samples were either determined by ICP-MS or by inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP 6300 Duo View, Thermo-Fisher) after microwave digestion (MARSXpress, CEM) using a 5:3 ratio of 30% (v/v) H2O2 and 65% (v/v) HNO3. All samples were filtered (0.2 µm cellulose acetate) and diluted accordingly before analysis.
In study 6, the depth distributions of many other major and trace elements in peat were analyzed by an energy dispersive X-ray fluorescence spectrometer (XEPOSTM, Spectro X Lab) calibrated with a NIST 2711 certified reference material. Total organic C and N contents were analyzed with a TOC/TN analyzer (multi N/C 2100, Analytik Jena). The absence of carbonate was tested with 10% (v/v) HCl.
Methods
In study 4, chemical extractions targeting the reactive Fe(II) pool (HCl-extraction) and total acid-extractable As and Fe were performed on subsamples from all solid phase samples. Total metal(loid) contents in filtered (0.2 μm, cellulose-acetate) and diluted extracts were determined by ICP-MS (As) or ICP-OES (Fe).
Coordination chemistry and redox states of the elements S, Fe, As, and Sb in the solid phase were investigated using various X-ray absorption spectroscopy techniques, in particular X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine-structure (EXAFS) spectroscopy of bulk samples. All spectra were collected at beamlines of the European Synchrotron Radiation Facility (ESRF), Grenoble, France (The Rossendorf Beamline, ROBL) or of the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford, USA (beamlines 4-1, 4-3, 6-2, 7-3, 9-3, and 11-2) at cryogenic temperatures (Fe, As, and Sb) or under ambient temperature but inert He (<0.1% (v/v) O2) atmosphere (S). A combination of several techniques was used to analyze the collected sample spectra. Normalized XANES and EXAFS spectra were analyzed by principal component analysis (PCA) combined with target-transform testing (TT) and subsequent linear combination fitting (LCF) for solid-phase speciation of Fe, As, and Sb. Further, iterative transformation factor analysis (ITFA) of normalized XANES and EXAFS spectra was used, especially to evaluate small spectral differences between several Sb phases. Gaussian peak fitting on normalized XANES spectra was performed to study the redox speciation of S. Shell-by-shell fitting of k-weighted EXAFS spectra, partly in combination with Morlet wavelet transform analysis was used to investigate the coordination chemistry of As and Sb. Details about spectra collection, data evaluation and analysis can be found in the respective studies.
In order to investigate the mineralogy of peat samples (study 6), selected samples were examined by scanning electron microscopy (SEM) using a Leo Gemini 1530 (Carl Zeiss, Germany) with a Schottky emitter. Elemental composition analysis was conducted by energy-dispersive X-ray spectrometry (EDS, Oxford X-Max 20, Oxford Instruments). Peat soil mineralogy in study 6 was moreover investigated by synchrotron X-ray powder diffraction at beamline 11-3 (SSRL), however, high organic matter background distortions hindered detailed interpretations (data not shown).
Confirmation of amorphous Sb-S phases in study 5 and characterization of soil mineralogy in study 4 were done by a laboratory powder XRD machine (Rigaku Miniflix 600 X-ray diffractometer equipped with a Cu-Kα radiation).
Organic functional groups of selected model peat samples were characterized by13C NMR with an Avance III HD Spectrometer (Bruker) and by Fourier-transform infrared spectroscopy (FT-IR) with a Vector 22 spectrometer (Bruker Optik).
Methods
2.2 Occurrence of thioarsenates in the minerotrophic peatland Gola di Lago and its