Human and livestock faecal biomarkers at the prehistorical encampment site of Ullafelsen in the Fotsch Valley, Stubai Alps, Austria-potential and limitations

The Ullafelsen at 1869 m a.s.l. in the Tyrolean Stubai Alps next to Innsbruck is an important (geo-)archaeological 15 reference site for the Mesolithic period. Buried fireplaces on the Ullafelsen plateau were dated at 10.9 9.5 cal. kyrs BP and demonstrate together with thousands of flint stone artifacts the presence of hunter-gatherers during the Early Holocene. Grazing livestock has been a predominant anthropozoological impact in the Fotsch Valley presumably since the Bronze Age (4.2-2.8 kyrs BP). Most recently, we demonstrated the great potential of n-alkane and black carbon biomarkers for contributing to a better understanding of pedogenesis and landscape evolution. In order to study the importance of human 20 and/or livestock animalfaeces input at this relevant s for occupation of this relevant geoarchaeological site, we carried out steroid analyses on 2two modern ruminant faeces samples from cattle and sheep, and on 37 soil samples from seven soil profiles onat the Ullafelsen and 9 soil samples from five reference soil profiles from the Fotsch Valley. The modern animal faeces show a dominance of 5β-stigmastanol and deoxycholic acid in modern cattle and sheep faeces for ruminants (cattle and sheep), which is in agreement with literature data.can be used as markers for the input of ruminant faeces in soils. The 25 OAh horizons, which have accumulated and developed since the Mesolithic, revealed high contents of steroids (sterols, stanols, stanones and bile acids), the E (LL) horizon coinciding with the Mesolithic living floor is characterized by medium contents of steroids. By contrast, the subsoil horizons Bh, Bs and BvCv contain low contents of faecal biomarkers indicating that leaching of steroids into the podsolic subsoils is not an important factor. High content of 5β-stigmastanol and deoxycholic acid in all soil samples give evidence for strong faeces input of ruminants. The steroid patterns and ratios 30 indicate a negligible input of human faeces on the Ullafelsen. β-Sitosterol as plant-derived steroid has also a strong influence on the faecal biomarker pattern in our soils. Root input into the subsoils is likely reflected by β-sitosterol contents. In

From a pedological point of view, a striking and frequently occurring light layer (LL) below the topsoils was described for the Ullafelsen and was a focus of previous investigations (Geitner and Schäfer, 2010;Geitner et al., 2011;Geitner et al., 2014). Similar light horizons are typical for soils developed in the subalpine zone of the Central Alps and are usually interpreted as eluvial horizons (E) horizons of podzols (Zech and Wilke, 1977;Egli et al., 2008), or as eventual loess deposit (Geitner et al., 2011;Schäfer, 2011a). At the Ullafelsen, most artifacts and Mesolithic fire places were found within and 60 directly on the top of the E (LL) horizon (Schäfer, 2011a). Therefore, the E (LL) horizon is regarded as Mesolithic living floor, the humic-rich subsoil below the E (LL) horizon was considered as Late Glacial buried former topsoil (2Ahb horizon) (Geitner et al., 2011;Schäfer, 2011a). to a better understanding of pedogenesis and landscape evolution. Black carbon results based on benzene polycarboxylic 65 acid (BPCA) analyses corroborated fire-induced human impact on the E (LL) horizon. The absence of leaf wax-derived nalkane biomarkers in the subsoils together with the absence of Late Glacial radiocarbon ages challenge the existence of a Late Glacial buried topsoil (2Ahb horizon) and rather point to a humus-enriched podzolic Bh horizon (Zech et al., 2021).
In the context of alpine pastoralism (grazing, dairying) since the Neolithic period in different parts of the Alps, (cattle)husbandry and agriculture became increasingly important for human society (Reitmaier et al., 2018;Gilck and 70 Poschlod, 2019). Grazing livestock such as cattle and sheep have been a predominant anthropozoological impact for the Ullafelsen and its surroundings presumably since the Bronze Age (4.2-2.8 kyrs. BP) (Zech et al., 2021).
Previous studies prove the specific steroids and bile acid signals for various animals organisms (Bull et al., 2005;Birk et al., 2011;Prost et al., 2017;Haurrault et al., 2019). Accordingly, faecal biomarker analyses allow to distinguish between faeces of herbivores/ruminants, pigs and humans. Based on their specific steroids signal, origin of faeces input can be detected in 80 soils and sediments as well as their residues in soils and sediments (von der Lühe et al., 2013;Haurrault et al., 2019).
Faecal steroids as organic compounds/lipid molecules can accumulate and persist in sediments and soils for more than 85 thousands of years (Bull et al., 2001;Schroeter et al., 2020). These have a low water solubility and are thus usually neither leached into deeper soil horizons (Bull et al., 2002) not leached into deeper soil horizons (Bull et al., 2002;Prost et al., 2017) nor detectable in soil leachates (Lloyd et al., 2012). It remains to be investigated whether this also holds true for very low soil pH values < 4 like in our study area, where organic substances can become mobile via complexation with metal ions.. The 5β-stanols coprostanol and 5β-stigmastanol are products of anaerobic microbial reduction of ∆ 5 -sterols. In mammals, 90 this reduction is performed by gut bacteria and results in different ratios of 5β-stanols depending on food intake (Bull et al., 1999a). The Δ 5 -sterol cholesterol is the precursor molecule of coprostanol, while the Δ 5 -sterols β-sitosterol and stigmasterol are the precursor molecules of 5β-stigmastanol and epi-5β-stigmastanol. such as cholesterol or β-sitosterol (Schroeter et al., 2020). Stanones are intermediate products of the transformation of ∆ 5 -sterols to 5β-stanols, epi-5β-stanols and 5α-stanols, occurs in the gut of animals and also in the environment (Prost et al., 2017). Epimerization of 5β-stanols, which occurs in 95 soils due to microbial and diagenetic transformation, has to be considered when applying steroid ratios (Bull et al., 1999a;von der Lühe et al., 2018).
Up to now, analyses of livestock faeces show 5β-stigmastanol and deoxycholic acid (DCA) as the dominant steroid compounds for ruminants (cattle and sheep), whereas coprostanol is a faecal marker for omnivores such as humans and pigs Prost et al., 2017;Haurrault et al., 2019). In contrast, plants contain high amounts of β-sitosterol and 100 stigmasterol (∆ 5 -sterols), which have also been detected in roots and litter (Piironen et al., 2000;Verma and Gupta, 2013).
Bile acids are formed from cholesterol in the liver and transformed via the bile into the gut of mammals as primary bile acids (e.g. cholic acid (CA) or chenodeoxycholic acid (CDCA)). These primary bile acids are further mediated to secondary bile acids (e.g. lithocholic acid (LCA) or deoxycholic acid (DCA)) in the intestine by microorganisms Secondary bile acids are transformed microbially in the gut of mammals from primary bile acids (e.g. cholic acid or chenodeoxycholic acid (CDCA)), 105 which are formed firstly in the liver from cholesterol (Bull et al., 2002;Kuhajda et al., 2006;Prost et al., 2017). Human faeces show high abundance of LCA, whereas ruminant faeces have a dominance of DCA (Prost et al., 2017;Shillito et al., 2020). Hence, steroid compounds can be useful markers for reconstructing settlement history of a site based on past faecal inputs.
Faecal biomarkers are currently used in various scientific disciplines all over the world. Glaser et al. (2001), Glaser and Birk 110 (2012) and Wiedner et al. (2015) investigated Anthropogenic Dark Earths, also known as terra preta de Índio, in Central Amazonia. By applying steroid markers, they provided evidence for settlement activities in this part of the tropical rainforest.
High nutrient contents induced by the deposition of human and livestockanimal faeces clearly demonstrated the anthropogenic origin of terra preta de Índio (Birk et al., 2011;Birk et al., 2012;Glaser and Birk, 2012). Another study used steroids for identification of temporary mass graves of concentration camp prisoners at the end of World War II (von der 115 Lühe et al., 2020). Findings revealed elevated faecal steroid contents and thus corroborate the former input of human decomposition products as well as faecal and tissue constituents of buried bodies (von der Lühe et al., 2020).
The aim of our here presented geoarchaeological study was to contribute to a better understanding of human and livestock impact at the prehistorical encampment site of Ullafelsen with the use of faecal biomarkers. More specifically, the following questions are addressed: (i) Do faecal biomarker patterns of modern ruminant faeces around the Ullafelsen reflect the steroid 120 patterns reported in literature and is a clear distinction from human faecal biomarker patterns possible? (ii) Do the steroid contents and patterns of the soil profiles at the Ullafelsen allow discrimination between human and livestock faecesanimal faecal input? (iii) Do the faecal steroid ratios of the soil profiles on the Ullafelsen allow the reconstruction of the faecal input history during the Holocene? We hypothesize that human faeces input is detectable in the E horizon representing the Mesolithic living floor (LL), whereas livestock faeces input dominates in the overlaying OAh horizon. (iv) Is there any 125 evidence for leaching of steroid biomarkers in soils with very low pH values such as in our study area?

Study area: The Ullafelsen as prehistorical encampment site in the Fotsch Valley, Stubai Alps, Austria
The prehistorical encampment site of Ullafelsen, also called "Riegelschrofen", is located in the Fotsch Valley at an altitude of 1869 m above the sea level (a.s.l.). The 13 km long Fotsch Valley belongs to the Stubaier Alps southwest of Innsbruck, 130 the capital of the Austrian state Tyrol. The Ullafelsen is a round hump at the eastern site of the Fotsch Valley and is located in the subalpine vegetation zone (Fig. 1). This rock ledge lies 40 m above the level of the adjacent creek, called "Fotscherbach" (Schäfer, 2011a). The geographic coordinates of the archaeological excavation area at the Ullafelsen are N 47.14702°, E 11.21475° (WGS84).
As a part of the transition zone between the wetter Northern and the drier Central Alps, our study area is characterized by a 135 temperate climate with a mean annual temperature of 10°C in the summertime (July) and -3°C in the wintertime (January).
From a geological point of view, the Fotsch Valley represents a part of the "Öztal-Stubai-cristalline-complex". Typical rocks for this study area are the metamorphic rocks mica slate and paragneiss. In addition, there also exist a variety of unconsolidated quaternary sediments (Nittel, 2011). The basic material under the anthropogenically-influenced soils at the 145 Ullafelsen consists, amongst others, of weathered till (Nittel, 2011). Despite human influence, these soils were mainly formed by podsolization during the Holocene (Zech et al., 2021). Typical soils in the alpine and subalpine zone of the Fotsch Valley are Cambisols and Leptosols. Under alpine dwarf shrub vegetation, Podzols have frequently developed, whereas in flatter valley floors and on some slope positions also Histosols can be found (Geitner et al., 2014).

Sampling of modern faeces, and archaeological and reference soil profiles 150
As part of fieldwork in July and August 2017/2018, we collected 37 soil samples from seven profile walls of soil profiles onat the Ullafelsen, 9 soil samples from five reference soil profiles from the Fotsch Valley and 2two faeces samples from cattle and sheep (Table 1). Four profile walls are directly from the archaeological excavation area (1.1 C4w, 1.1 B5s, 1.1 B5w, 1.1 G5n). Three profile walls are from a close-by trench (1.9 NW, 1.9 NE, 1.9 SW) (Fig. 2). The latter is located two2 meters below the archaeological excavation area at an altitude of approximately 1867 m a.s.l.. Samples from reference soils 155 were collected from the soil profiles 4.4, 4.11, 4.12, 5.5 and 5.6w. Sampling was conducted by soil horizons, which were classified according to the WRB (IUSS Working Group WRB, 2015).
Samples of cattle faeces were collected on the Ullafelsen, while samples of sheep faeces were collected from the Fotsch Valley above 2000 m a.s.l. We chose these sampling sites for collecting faeces samples depending on a high density of grazing livestock mainly consisting of sheep. The food of grazing livestock from the Ullafelsen and surroundings consists of 160 local vegetation, mostly grasses and alpine dwarf shrubs. After drying and grinding our faeces samples, we stored these separately in snap cap vials.

175
The TOC values of the investigated soil samples from archaeological soil profiles on the Ullafelsen ranged from 0.3 to 28.8 % ( Table 1). The maximum TOC content was observed in the OAh3 horizon, being in accordance with darker color and higher density of charcoal particles. Total nitrogen contents of the investigated soil profiles ranged from 0.0 to 1.2 %. The TOC/N ratios ranged from 12.4 to 37.2 with the highest ratios in the OAh3 horizon coinciding with charcoal particles in this soil horizon, related to former fireplaces, and high amounts of other organic material (Zech et al., 2021). High TOC/N ratios 180 in the Bh horizons reflect podsolization processes Zech et al., 2014). TOC and TN values of soil samples from reference soil profiles from the Fotsch Valley ranged from 4.9 to 29.4 % and 0.3 to 2.2 %, respectively. The highest TOC and TN values were measured in the OAh horizons (Table 1).
Radiocarbon-dated Mesolithic charcoal yielded 14 C ages ranging from 10.9 to 9.5 cal. kyrs BP (Schäfer, 2011a). More recently, Zech et al. (2021) yielded 14 C ages for bulk n-alkanes ranging from 8.2 to 4.9 cal. kyrs BP. This discrepancy 185 suggests that a n-alkane producing vegetation cover, consisting of herbs, grasses and alpine dwarf-shrubs, did not predominate immediately after the Mesolithic abandonment. Rather, it must be assumed that non-n-alkane producing conifers, such as Pinus cembra or Picea abies, predominated the vegetation cover after the Mesolithic life on the Ullafelsen (Zech et al., 2021).
profiles from the Fotsch Valley, we analyzed 2 mixed faeces samples from cattle and sheep, which belong to the typical livestock at the Ullafelsen and surroundings. TOC contents of cattle and sheep ranged from 42.6 to 43.5 % (Table 1).

Faecal biomarker analyses 195
Firstly, all 46 soil and 2 faeces samples were air-dried, sieved (<≤ 2 mm) and finely ground. Using an elemental analyzer coupled to an isotope ratio mass spectrometer (EA-IRMS), total carbon (TC) and total nitrogen (TN) of soils samples were determined by Zech et al. (2021). Due to the non-carbonate parent rock material as well as the low pH values (< 4 in CaCl2) of the soils and sediments on the Ullafelsen (Geitner et al., 2011), the measured TC values can be considered as TOC values.
From these data, the TOC/N ratio was calculated. In brief, a total lipid extract (TLE) was obtained by soxhlet extraction for 36 hours using a solvent mixture of dichloromethane/methanol (2:1, v/v, 180 ml). The weight of sample depended on the TOC content of the sample and ranged 205 for the 4637 analyzed soil samples from 1.0 to 5.0 g of finely ground material. For faeces, it is necessary to use a much smaller weight due to the higher TOC contents. In case of our two faeces (cattle and sheep), the weight of sample taken was ~60 mg. Prior to extraction, 100 μg of α-preganol and 100 μg of iso-deoxycholic acid were added to each sample as recovery standard (internal standard 1, IS1) for the neutral sterol/stanol and acid lipid bile acid fractions, respectively. We used no internal standard for the stanone fractions. Regarding to data analysis and interpretation, we consider no stanones in our 210 calculated ratios. The total lipid extract (TLE) was obtained by soxhlet extraction for 36 hours using a solvent mixture of dichloromethane/methanol (2:1, v/v, 180 ml).
After extraction, the solvent was removed using rotary evaporator and the TLEs were saponified using 3.5 ml of 0.7 M KOH in methanol at room temperature overnight for approx. 12 hours. Neutral lipids (sterols, stanols, stanones) as well as acidic lipids (bile acids, fatty acids) were separated by sequential liquid-liquid extraction. To obtain the neutral lipid fraction, the 215 extracts were spiked with 10 ml H2O and afterwards separated from the aqueous phase with 3 x 15 ml chloroform. For gaining the acidic lipid fraction, the aqueous solution was acidified with 6 M HCl (pH ≤ 3-4) and the bile acids were extracted with 3 x 15 ml chloroform. Both fractions were separately collected in flasks and evaporated under nitrogen.
For external calibration, six concentrations from a stock solution (10 ng µl -1 in hexane) containing all target sterols, stanols and stanones as well as the recovery standard (IS1) were prepared ranging from 50 to 2500 ng per vial. After derivatization 500 ng of 5α-cholestane (dissolved in dry toluene, 50 μl, 10 ng µl -1 ) were added as internal standard 2 (IS2). Peak areas of 240 the analytes were divided by the peak area of IS2 and calibration curves for each substance were calculated using these ratios. Calibration curves for the bile acids were prepared accordingly. All calibration curves had coefficient of determination (R 2 ) > 0.98. Limit of detection was defined as signal-to-noise ratio of 3:1 and varied for the analytes between 0.6 ng g -1 soil for all Δ 5 -sterols, stanols and stanones and 4 ng g -1 soil for all bile acids. At last, recoveries of the first internal standard (IS1) were calculated for all samples. Recovery of IS1 of all soil samples ranged between 51-116% for sterols, 245 stanols and stanones as well as 50-122% for bile acids. All results for the samples were corrected for the losses during extraction and purification with those individually calculated recoveries.For GC-MS measurements, GC-vials with 100 μl inserts were used for transferring the neutral lipids and the bile acids. External standards containing all analyzed steroids (sterols, stanols, stanones and bile acids) in six different concentrations (5, 10, 25, 50, 100, 250 μl per vial) were derivatized simultaneously with the soil and faeces samples. The internal standard 1 was added in the same concentration for each 250 external standard beforehand. 50 μl of the internal standard 2 were added to each external standard mixture after their derivatization.
Quantification of all steroid substances took place using gas chromatography-mass spectrometry (GC-MS) with a 5971A quadrupole mass spectrometer connected to a HP5090 gas chromatograph (both made from Hewlett Packard) with a DB-5 MS 30 m fused silica column (25 mm ID and 0.25 μm film thickness, Agilent Technologies). 255 All measurements of steroids were conducted with the following settings for GC-MS: injection volume: 1 μl, carrier gas: helium (purity of 5), injector temperature: 290°C, injection in splitless mode, by splitless, interface temperature: isotherm at 280°C. The column temperature program of the gas chromatography was held at 80°C for 1.5 min and then raised at 12°C min -1 to 265°C. Further steps are the increase of the temperature at 0.8°C min -1 to 288°C and at 10°C min -1 to 300°C afterwards, whereas it was kept for 12 min. 260

Data analysis
For a detailed interpretation of the measured results in terms of degradation effects and distinguishing between omnivore-, herbivore-or plant-derived faecal biomarkers, the following ratios were calculated and plotted as box plot diagrams: According to Bull et al. (1999a) and Schroeter et al. (2020), we used ratio 1 to estimate microbial degradation of steroids (5β-stanols, epi-5β-stanols and 5α-stanols) and to identify human faeces input (Wiedner et al., 2015). It is known that soil microorganisms contribute to degradation of steroids in soils (Bull et al., 2001). 5α-cholestanol is a degradation product of 275 the Δ 5 -sterol precursor cholesterol, transformed by soil microorganisms, and occurs naturally in the environment (Prost et al., 2017). This ratio considers the input and preservation of stanols. High values for ratio 1 (0.7-1) indicate an increased faeces deposition, while values < 0.7 indicate low faeces input in soils (Bull et al., 1999a;Schroeter et al., 2020).
For distinguishing between human and herbivore faeces, ratio 2 can be applied. A dominant 5β-stanol marker in omnivore faeces such as humans or pigs is coprostanol, whereas 5β-stigmastanol is a typical 5β-stanol marker for herbivore faeces. To 280 account for ongoing epimerization of 5β-stanols in soils, both epimers are included in this ratio. Major input of human faeces results in values > 1, whereas values < 1 indicate an input of herbivore faeces (Ossendorf et al., 2019).
Ratio 3 was calculated for differentiation between plant-derived steroids and livestock-derived steroidsherbivore faeces.
According to Prost et al. (2017), β-sitosterol belongs to the typical ∆ 5 -sterols, which are characteristic for plant biomass and thus normally occur at high abundance in soils. Values between 0 and 0.5 suggest low input of β-sitosterol and values 285 between 0.5 and 1 point at a high occurrence of this plant Δ 5 -sterol. It has to be considered that faeces of ruminants can also contain high amounts of β-sitosterol due to their plant-dominated diet (Haurrault et al., 2019;Schroeter et al., 2020).
Ratio 4 was calculated for all soil samples and can be used as proxy for degradation of 5β-stigmastanol. Epi-5β-stigmastanol is a transformation product (epimer) of 5β-stigmastanol and is often found in anaerobic environments and soils (von der Lühe et al., 2018). This transformation is induced by soil composting (Prost et al., 2017). With increasing degradation, ratio 290 4 gets lower due to the higher proportion of epi-5β-stigmastanol.
The following equation was applied for calculating a bile acid ratio:

Biomarker patterns of modern livestock faecesof faeces from predominating animals
The total sterol, stanol and stanone contents of modern cattle and sheep faeces ranged from 2401.6 to 2671.6 μg g -1 and 2109.8 to 2421.9 μg g -1 , respectively (Table S3). The total bile acid contents of modern cattle and sheep faeces ranged from 305 55.9 to 86.8 μg g -1 and 78.6 to 216.9 μg g -1 , respectively (Table S4).
For checking the reproducibility robustness of our results, we repeated the analyses of our faeces samples twice (Table S3;   Table S4). Laboratory replicate analyses show that the method of faecal biomarker analyses works correctly and our results are reproducible. Figure 4 illustrates the steroid (sterols, stanols, stanones and bile acids) contents and their patterns in modern cattle and sheep faeces. The following results refer to measurement 1 (Table S3; Table S4). 310 The predominating steroid in cattle faeces is 5β-stigmastanol (930.9 μg g -1 ), whereas epi-5β-stigmastanol (666.5 μg g -1 ) shows a predominance in sheep faeces (Fig. 4). For comparison, Prost et al. (2017) did not find epi-5β-stigmastanol to predominate in sheep faeces. It cannot be excluded, that the predominance of epi-5β-stigmastanol in our sample is induced by strong epimerization, which is possibly influenced by long dry storage of the sample.
The coprostanol content in modern cattle faces is 125.7 μg g -1 , whereas the coprostanol content of 173.4 μg g -1 was detected 315 in modern sheep faeces. These low contents of human-derived faecal biomarkers in our modern ruminant faeces samples have to be considered for the identification of faeces origin in our soil samples. The plant-derived steroid β-sitosterol shows 347.9 μg g -1 in modern cattle faeces and 255.9 μg g -1 in modern sheep faeces (Fig. 4).
We calculated ratio 1, 2, 3 and 5 for our modern cattle and sheep faeces regarding to their steroid patterns to get reference ratios for comparing of our analysed soil samples. Bull et al. (1999a) and Prost et al. (2017) introduced ratio 1 for the 320 identification of faeces input.identification of faeces origin. Typical ratio 1 is ~ 0.8 for cattle and sheep, whereas ~ 1 is typical for human faeces. Our results revealed a ratio 1 of 0.8 for both modern faeces samples. These results are in agreement to Prost et al. (2017). Ratio 2 can be used as proxy for the identification of faeces origin. For the identification of ruminant faeces input, we calculated ratio 2. Cattle and sheep faeces yielded ratio 2 of ~ 0.1 and ~ 0.2, respectively. These results are in agreement with data of Prost et al. (2017). Ratios > 1 are characteristic for indicate human or other omnivore faeces, 325 inputwhereas ratios < 1 represent herbivore/ruminant faeces. Our modern cattle and sheep faeces yielded ratio 2 of 0.1 and 0.2, respectively. These results are also in agreement with data of Prost et al. (2017). Ratio 3 reflects the predominance of plant-derived steroids (β-sitosterol) over livestock-derived steroids (5β-stigmastanol and epi-5β-stigmastanol). Ratios ≤ 0.5 indicate dominance of β-sitosterol, whereas ratios ≥ 0.5 show dominance of 5β-stigmastanol and epi-5β-stigmastanol. Our modern faeces samples yielded ratio 3 of ~ 0.2 for cattle and sheep, which demonstrates a dominant content of livestock-330 derived steroids over plant-derived steroids in our ruminant faeces. This ratio reflects the predominance of 5β-stigmastanol and epi-5β-stigmastanol over β-sitosterol.
The most dominant bile acid in our modern ruminant faeces is DCA (38.7 μg g -1 and 55.0 μg g -1 in cattle and sheep faeces, respectively), being in agreement with literature data of Kuhajda et al. (2006) and Prost et al. (2017). LCA as marker for the input of human faeces was found in modern ruminantanimal faeces only at low amounts (4.2 μg g -1 and 2.9 μg g -1 in cattle 335 and sheep faeces, respectively (Fig. 4)). Unexpectedly, our results of modern cattle and sheep faeces show low contents of CDCA (2.6 µg g -1 and 2.4 µg g -1 in cattle and sheep faeces, respectively). Prost et al. (2017) did not detect CDCA in cattle and sheep faeces, but in goats, horses, geese and human faeces instead.
According to Prost et al. (2017), ratio 5 ranged from 5-21 and 8-12 for cattle and sheep faeces, respectively. Based on our results, ratio 5 showed a ratio of 9.2 for cattle faeces and 18.8 for sheep faeces. For comparison, ratio 5 is typical for human 340 faeces when in a range from 3 to 4.5. Accordingly, ratio 5 is highly promising for distinguishing between ruminant versus human faeces in soils. Unfortunately, steroid patterns of our analyzed ruminant faeces allow no differentiation between cattle and sheep.
All three measurements yielded similar content for sterols, stanols and stanones. By contrast, measurement 1 showed higher contents of the bile acid DCA but lower contents of oxolithocholic acid (OLCA) for both modern faeces samples (Table S4). 345 The latter can be synthesized by oxidation of the hydroxyl groups of DCA (Sakai et al., 1980;Kuhajda et al., 2006;Marion et al., 2019).

Ancient faecal marker Faecal biomarker -contents and patterns in soils
The total sterol, stanol and stanone contents of the 37 soil samples from the Ullafelsen ranged from 1.2 to 198.1 μg g -1 (Fig.   5). The OAh1 and OAh3 horizons yielded maxima coinciding with TOC maxima. The lowest content was measured in the 355 Bh, Bs and BvCv horizons, whereas the content of the E (LL) horizon was intermediate (2.3 to 58.6 μg g -1 ; Fig. 5). In contrast, the sterol, stanol and stanone contents of the 9 soil samples from reference soil profiles from the Fotsch Valley ranged from 3.3 to 106.3 µg g -1 . Maximum contents were measured in the OAh horizons (24.3 to 106.3 µg g -1 ), whereas Bh horizons yielded minimum contents (3.3 to 6.8 µg g -1 ) of sterols, stanols and stanones (Fig. 5).
The total bile acid content of the 37 soil samples from the Ullafelsen ranged from 0 to 6.8 μg g -1 and show their maximum in 360 the topsoil horizons OAh1 and OAh2 (Fig. 5). In comparison to the maximum sterol, stanol and stanone contents in the OAh1 horizon, the maximum bile acid content of 6.8 μg g -1 was detected in the OAh2 horizon. Similar to the sterol, stanol and stanone contents, the bile acid contents are much higher in the topsoil horizons OAh1, OAh2, and OAh3 (0.5 to 6.8 μg g -1 ) and lower in the subsoil horizons E (LL), Bh, Bs and BvCv (0 to 0.9 μg g -1 ) (Fig. 5). The total bile acids content of the 9 soil samples from reference soil profiles from the Fotsch Valley ranged from 0.1 to 15.5 µg g -1 . The OAh horizons yielded 365 maximum contents (1.3 to 15.5 µg g-1 ), whereas minimum contents of bile acids were measured in the Bh horizons (0.1 to 0.2 µg g -1 , Fig. 5).
Steroids in the subsoils can be induced by bioturbation and/or roots of plants (Piironen et al., 2000). The most detected steroid in the subsoils is β-sitosterol, which is a plant-derived Δ 5 -sterol. Thomas and Hale (1983) as well as Verma and Gupta (2013) found that roots or root exudates contain also a significant content of β-sitosterol. We assume that the Hence, 370 the small contents of steroids in the subsoil horizons at the Ullafelsen are mainly caused by the influence of β-sitosterol due to the strong rooting of grasses and alpine dwarf shrubs in the soil matrix. There is no deformation of soil horizons, thus we exclude the influence of bioturbation.
- Figure 5 -375 The most abundant steroid in all soil samples from the Ullafelsen is β-sitosterol with a maximum content of 150.1 μg g -1 measured in an OAh3 horizon ( Fig. 6; Table S1). β-Sitosterol is a typical plant-derived ∆ 5 -sterol compound in plant biomass and reflects the predominating vegetation signal (Holtvoeth et al., 2010;Prost et al., 2017;von der Lühe et al., 2018). Due to the plant diet, β-sitosterol is eaten by ruminants and can be detected in their faeces (Haurrault et al., 2019). Cholesterol as the 380 dominating ∆ 5 -sterol in animal tissues has a maximum content of 6.2 μg g -1 detected in an OAh1 horizon ( Fig.6; Table S1).
The most abundant bile acid in the analysed soil samples from the Ullafelsen was DCA with a maximum content of 4.2 μg g -1 in an OAh1 and OAh2 horizon ( Fig. 6; Table S1). It predominates in ruminants such as cattle and sheep (Kuhajda et al., 2006;Prost et al., 2017). LCA as the dominating bile acid in human faeces (Shillito et al., 2020) showed a maximum content of 0.5 μg g -1 in an OAh2 horizon ( Fig.6; Table S1). Wiedner et al. (2015) reported similar DCA and LCA contents for 395 anthropogenic dark earths in northern Germany.
Furthermore, we found CDCA in the topsoil horizons OAh1, OAh2 and OAh3 as well as in the E (LL) horizon with a maximum content of 0.6 µg g -1 , detected in an OAh2 horizon. Results of our modern cattle and sheep faeces showed also low contents of CDCA. Therefore, we explain the detected CDCA in our soils by the input of ruminant faeces.
OLCA was also detected in our soil samples from the Ullafelsen and has a maximum content of 1.7 μg g -1 in an OAh1 400 horizon ( Fig. 6; Table S1). As discussed in 3.1, ruminant faeces also containing OLCA at low abundance. However, according to Marion et al. (2019), we cannot exclude that OLCA is formed by microorganisms in soils, such as Clostridium scindens, due to transformation of DCA to OLCA.
The most abundant sterol in the reference soils was β-sitosterol with the maximum content of 56.0 µg g -1 (Table S1).
Deoxycholic acid is the dominant bile acid in the reference soils with the maximum content of 10.0 µg g -1 (Table S2). Both 405 steroids were detected in the OAh horizon of reference soil profile 5.6w. Maximum contents of 5β-stigmastanol and deoxycholic acid as livestock-derived steroids were also found in this reference soil profile.
- Figure 6 -410 Overall, our results indicate that a strong input of faeces from cattle and sheep into the soils on the Ullafelsen occurred. By contrast, low LCA and coprostanol contribution point to a minor influence of human faeces. at the Ullafelsen. We exclude a striking faeces input of wild animals due to the high density of ruminant species at the Ullafelsen and surroundings. This first interpretation should be confirmed by the applied faecal ratios of the considered steroids.
According to Lloyd et al. (2012), our steroid results of the archaeological soils on the Ullafelsen and also of the reference 415 soils from the Fotsch Valley show no leaching or transport in deeper soil horizons due to the significant lower steroid contents.
In comparison to the reference soils, the higher contents of total sterols, stanols and stanones in the OAh horizons of the archaeological soils on the Ullafelsen confirm an enrichment at this site. Unexpectedly, the total bile acid contents in the OAh horizons of the reference soils indicate a large range with partly higher contents compared to the archaeological soils 420 and show thus no increased bile acid contents at the Ullafelsen. The highest contents of livestock-derived steroids (5βstigmastanol, epi-5β-stigmastanol, deoxycholic acid) were detected in soil profile 5.6w of all reference soil profiles from the Fotsch Valley. We explain these high contents by a strong input of sheep faeces into the soil of soil profile 5.6w. However, we assume a predominant natural background of steroids for the soils on the Ullafelsen and in the Fotsch Valley due to the intense alpine grazing and farming. 425

Identification of faeces origin on the Ullafelsen based on specific steroid ratios
Ratio 1 ranged from 0 to 0.3 (Fig. 7). Human faeces normally exhibit ratio 1 > 0.7 (Bull et al., 1999a;Prost et al., 2017). Therefore, our results corroborate a very low input of human faecal markers such as coprostanol into soils at the Ullafelsenand suggest a negligible input of human faeces into soils on the Ullafelsen. Furthermore, we exclude a strong degradation of coprostanol in the topsoil horizons due to the non-detected epicoprostanol as transformation product of 430 coprostanol in terms of epimerization over time. Contents of 5α-cholestanol (≤ 1.4 µg g -1 ) as transformation product of cholesterol (plant-derived) indicate also low degradation in the topsoil horizons.
Ratio 2 ranged from 0 to 0.4 (Fig. 7) and showed a maximum in the Bh horizon due to the higher content of epi-5βstigmastanol compared with the content of 5β-stigmastanol (Table S1). Our results confirm a predominance of 5βstigmastanol and epi-5β-stigmastanol, which indicate a strong input of ruminant faeces into soils at the Ullafelsen. 435 Ratio 3 ranged from 0.95 to 1 (Fig. 7), demonstrating a strong influence of plant-derived steroids (high β-sitosterol contents) in soils at the Ullafelsen caused by the high contribution of β-sitosterol (Fig. 6). High ratio 3 in the Bh, Bs and BvCv horizons can be explained by decreasing 5β-stigmastanol and epi-5β-stigmastanol contents. Roots of plants contribute to an input of β-sitosterol into subsoils (Piironen et al., 2000).
Ratio 5 ranged from 4.1 to 23.5 (Fig. 7), decreasing from top to bottom. A ratio < 5 indicates a dominance of human faeces, 440 whereas a ratio > 5 corroborates a dominance of ruminant faeces (Prost et al., 2017). No LCA was detected in the Bs and BvCv horizons. Apart from the outlier of 4.1, our results showed a clear dominance of ruminant faeces input into soils at the Ullafelsen due to the high content of DCA. The outlier of 4.1, detected in an E (LL) horizon, can be allocated to a close-bytrench soil profile (1.9 NE E 1 (LL)), which is beyond the archaeological excavation area on the Ullafelsen. For this E (LL) horizon, we cannot exclude the input of human faeces regarding ratio 5, being based on the content of DCA (0.3 μg g -1 ) and 445 LCA (0.1 μg g -1 ) ( Table S2).
- Figure 7 -Ratio 4 ranged from 0 to 1.0 (Fig. 8), decreasing from the topsoils to the subsoils. As discussed in section 3.1, we observed a 450 degradation effect of 5β-stigmastanol in our soils. Our results corroborate a higher degradation in the E (LL), Bh, Bs and BvCv horizons. We explain that with slightly higher epi-5β-stigmastanol contribution in this subsoil horizons ( Fig. 8; Table   S1) due to epimerization (Bull et al., 1999a;Bull et al., 1999b;Bull et al., 2003;Prost et al., 2017). For a reliablerobust identification of faeces origin, we recommend to consider the degradation effect and to include epi-5β-stanols in steroid ratios (Bull et al., 2001). 455 - Figure 8 -

Conclusions and Ooutlook
This study presents the first results of faecal biomarker analyses carried out on the prehistorical encampment site of Ullafelsen, Fotsch Valley, Austria. Steroid patterns of contemporary ruminants showed a dominance of 5β-stigmastanol, 460 whereas epi-5β-stigmastanol as degradation product of 5β-stigmastanol has to be considered for sheep. DCA was detected as the dominant bile acid for cattle and sheep. These data together with data of Prost et al. (2017)  derived faecal biomarkers combined with our evaluation of faecal biomarker ratios (Fig. 7), we see no evidence for a strong input of human faeces in our soil samples from the Ullafelsen and surroundings. Based on our faecal biomarker results, we cannot confirm the Mesolithic settlement of hunter-gatherers at the Ullafelsen. According to Schäfer et al. (2016), the The archaeological site of Ullafelsen was used for fireplaces and social living in the Mesolithic period. However, it cannot be excluded that human faeces markers will be detected in higher content close-by the archaeological excavation area. in future 480 investigations. We assume that sites close-by the archaeological excavation area were used as "toilet" from the Mesolithic hunter-gatherers. Future faecal biomarker analyses on further soil profiles at the Ullafelsen aim at more specific insight in this hypothesis.
A robust age control for faecal biomarkers on the Ullafelsen is challenging because of lacking age chronology. This study allows thus no reconstruction for the onset of alpine pastoralism. We assume the input of faecal steroids in the Holocene 485 since the beginning of the alpine pastoralism in the Neolithic and Bronze Age (Knierzinger et al., 2020).
In order to chronologically identify the history of land use in the Fotsch Valley, we suggest to investigate faecal biomarkers on mire archives in the Fotsch Valley. Previous studies of two subalpine mire archives in the near surroundings of the prehistorical encampment site of Ullafelsen demonstrate the high potential for palaeoenvironmental reconstructions. Table S1: Overview over all faecal biomarker soil samples from the prehistorical encampment site of Ullafelsen and reference sites in the Fotsch Valley, Stubai Alps, Austria. Sterols, stanols and stanones content (in μg g -1 dry matter) as well as ratios are presented.   Table S4: Bile acids content (in μg g -1 dry matter) for 3 replication measurements of modern cattle and sheep faeces from the 500 prehistorical encampment site of Ullafelsen and reference sites in the Fotsch Valley, Stubai Alps, Austria.

Data availability
TOC; TN and TOC/N data are available from Zech et al. (2021). Faecal biomarker data of the archaeological soil profiles on the Ullafelsen and reference soil profiles from the Fotsch Valley are available in the Supplement.

Authors contributions 505
The project idea was developed by MZ in cooperation with BG, DS and CG. Fieldwork was done by ML, MZ, JNH, DS and CG. ML performed most of the laboratory work with contributions made by TB, BG and MZ. The manuscript was prepared by ML. All co-authors contributed to the discussion of the results and read an approved the manuscript.