Organic geochemical evaluation of organic acids to assess anthropogenic soil deposits of Central Amazon, Brazil

Organic Geochemistry 58 (2013) 96–106

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Organic geochemical evaluation of organic acids to assess anthropogenic soil deposits of Central Amazon, Brazil Paulo S. Taube a, Fabricio A. Hansel b,⇑, Luiz A. dos Santos Madureira a, Wenceslau G. Teixeira c a

Departamento de Química, Universidade Federal de Santa Catarina, CP 476, Florianópolis – SC 88040 900, Brazil Embrapa Florestas, Estrada da Ribeira, km 111, cx 319, Colombo – PR 88411 000, Brazil c Embrapa Solos, Rua Jardim Botânico 1024, Rio de Janeiro – RJ 22460 000, Brazil b

a r t i c l e

i n f o

Article history: Received 9 October 2012 Received in revised form 31 January 2013 Accepted 13 February 2013 Available online 4 March 2013

a b s t r a c t Terra Preta de Índio (TPI) and Terra Mulata (TM) are anthropogenic soils from the Amazon region and are rich in stable organic matter (OM). The formation and incorporation of OM in these soils has recently been under investigation. Organic geochemical analysis is an appropriated tool for the assessment of the sources of OM. Therefore, we have used the distribution of different acid classes preserved in the free and bound soil fractions of 12 samples from two contrasting anthropogenic soils (TPI, TM) and an adjacent soil, in order to infer the sources of OM and the magnitude of non-cultural influence on the formation of anthropogenic soils. The major acids in both fractions (i.e. free and bound) were n-saturated, branched and unsaturated alkanoic acids, hydroxyalkanoic acids, bile acids and lignin/suberin derived aromatic acids. In general, the acids in the free and bound fractions appeared to be complementary and together provided valuable information about OM incorporation into anthropogenic soils. Different incorporation of x-hydroxyalkanoic acids (C22, C24 and C28) and 9(10),16-dihydroxyhexadecanoic acid, and presence/absence of bile acid showed a distinct genesis for the soils. The influence of modern vegetation was revealed by bound x-hydroxyalkanoic acid (C22, C24 and C28) distributions only in the topsoil profiles of TPI and TM, indicating that organic geochemical analysis is a useful approach in the investigation of ancient human deposits in tropical archaeological soils. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Small patches of sustainably fertile anthropogenic soils, known as Amazonian Dark Earths and locally as Terra Preta de Índio (TPI) and Terra Mulata (TM), are found in the Amazon region (Kern and Kämpf, 1989; Kämpf et al., 2003; Glaser and Woods, 2004). They normally contain high concentrations of PO3 4 , Ca, Zn, Mn and organic carbon, which are 10 and up to 100 higher than in adjacent soils (Kern and Kämpf, 1989). These anthropogenic soils, especially TPI, contain archaeological material characterized by potsherds, reinforcing their human origin, and are associated mainly with Incise Rim and the Polychrome traditions dating to the first millennium AD (Glaser and Woods, 2004; Neves, 2008). However, some data point to their earlier formation to several thousand years BC (Peterson et al., 2001; Neves et al., 2003). Previous studies have demonstrated that the soil OM (SOM) in these anthrosols is marked by a small labile portion and a significant stable portion that is higher than in surroundings soils (Glaser et al., 2003). The stable SOM is characterized by condensed aromatic compounds (black carbon) and consists mainly of charred ⇑ Corresponding author. Tel.: +55 4136755792; fax: +55 4136755601. E-mail addresses: [email protected], [email protected] (F.A. Hansel). 0146-6380/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.orggeochem.2013.02.004

OM and other components like soot (Glaser et al., 2003; Glaser, 2007; Novotny et al., 2009). Other OM sources are related to faecal matter, plant residues from food supply and animal remains (Glaser and Birk, 2012). Lipid analysis is a useful tool in geochemical studies (Eglinton and Logan, 1991; Hita et al., 1996; Killops and Killops, 2005) for inferring subtle differences in SOM incorporation due to low reactivity, low solubility in water and greater resistance of lipids to degradation vs. other classes of organic compounds (e.g. carbohydrates, protein and lignin). Thus, during changes in overlying vegetation or input of allochthonous OM to soil due to variation in composition, lipids can provide important information (van Bergen et al., 1997, 1998; Bull et al., 1999). For example, one prominent investigation of archaeological sites has been the detection of ancient human input of faecal matter through the use of specific steroids (i.e. 5b-stanols) and bile acids (Evershed et al., 1997; Bull et al., 2003). In fact, recent studies have demonstrated the presence of faecal biomarkers in TPI, which was associated with human faeces deposition (Birk et al., 2011; Glaser and Birk, 2012). However no other classes of lipids have been reported in the TPI and TM archaeological soils. Major groups of lipids in soil include n-alkanes, n-alkanoic acids, n-alkanols, hydroxy acids, ketones, steroids, terpenoids and acyl glycerols (Sicre et al., 1994; Stevenson, 1994; Hernandez

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et al., 2001). Amongst these, acids such as alkanoic, dialkanoic and hydroxyalkanoic acids have been extensively studied in the sedimentary environment and soil due to their importance in providing information about OM sources and early diagenesis (Cardoso and Eglinton, 1983; Shaw and Johns, 1985; Farrington et al., 1988; Canuel and Martens, 1993; Amblès et al., 1994; Bull et al., 2000; Silva and Madureira, 2012). Organic acids in soil are present as free components or in ester-bound fractions. The free acids are extracted with solvent, and the latter released after alkaline hydrolysis (KOH or NaOH) or transmethylation (BF3:MeOH or MeCOCl:MeOH; Pancost and Sinninghe Damsté, 2003; Hansel et al., 2008). Free lipids are related to vegetation deposition and microorganisms, whereas the bound lipids are frequently associated with OM humification, and cutin and suberin (Nierop et al., 2003). With respect to the vegetation history, Nierop et al. (2006) showed that extractable lipids (free fraction) and cutin/suberin (bound fraction) can be used in combination to unravel past vegetation change and OM contributions. Although the occurrence of TPI and TM provide a sign of their use for sedentary agriculture and the formation of large settlements in the Amazon Basin during the pre-Columbian period, there is no evidence about the real economic or socio-cultural practices that led their formation (Peterson et al., 2001; Neves et al., 2003; Glaser, 2007; Birk et al., 2011). The formation of archaeological sites reflects (Shiffer, 1987) both cultural and non-cultural aspects; the first is associated with anthropogenic activity and the second with the natural incorporation of vegetation detritus, bioturbation and wind deposition during human occupation or after abandonment of the areas (e.g. modern vegetation). For these reasons, we suspect that the Amazon anthropogenic soils are heterogeneous and associated with specific human cultural activity in the area (e.g. agriculture practices vs. hearth area) as well as non-cultural activity. We therefore proposed to use the distribution of different acid classes preserved in the free and bound soil fractions in two contrasting anthropogenic soils (TPI, TM) and a non-anthropogenic adjacent soil in order to infer different sources of OM and the magnitude of non-cultural influence on the formation of the anthropogenic soils. 2. Experimental 2.1. Sample collection The two Hortic Anthrosols, TPI and TM, plus a surrounding Haplic Acrisol horizons were collected at the Caldeirão Experimental Station (Embrapa, Iranduba – AM, Brazil; 3°50 S, 60°220 W). They were from four different depths: TPI 0–36, 36–56, 56–84 and 84– 150 cm (Munsell colour 10YR2/1, 10YR2/1, 10YR2/1 and 10YR5/8 respectively) and TM 0–10, 10–20, 20–40 and 40–100 cm (Munsell colour 10YR3/3, 10YR3/3, 10YR4/3 and 10YR4/3 respectively). For comparison, the adjacent Haplic Acrisol (AS) was collected from four depths: 0–15, 15–38, 38–55 and 55–90 cm (Musell colour 10YR3/3, 10YR3/3, 10YR4/3 and 10YR5/8 respectively). Each soil has the following general characteristics: TPI is dark black, with a large quantity of archaeological pottery vessels and covered by rainforest. TM is grey-black, with few pottery vessels and a significant presence of charcoal and has recently been used in modern agriculture experiments at EMBRAPA; the adjacent Haplic Acrisol is brown-yellow in colour, with no visible pottery vessels or charcoal, and is covered by rainforest. 2.2. Solvent extraction and acid isolation An aliquot (ca. 5.0 g) of soil was dried (60 °C, 24 h) and sieved through a 250 lm mesh screen. The free lipids were extracted

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(30 min) with CHCl3:Me2CO (9:1 v/v, 3  10 ml) in an ultrasonic bath. The solvent was removed with a gentle stream of N2; the extract, containing the free lipids, was stored at 20 °C until isolation of the acid fraction. The acids in the free fraction were isolated using a glass column packed with 0.5 g activated silica gel (120 °C, 2 h) and 0.5 g activated Al2O3 (120 °C, 2 h). The extract was dissolved in hexane and transferred to the column from which different fractions were obtained as follows: n-hexane (6.0 ml), CH2Cl2 (7.0 ml), EtOAc/MeOH (3:1 v/v, 6.0 ml) and finally EtOAc/ MeCO2H (3:1 v/v, 6.0 ml) to elute the acid fraction. The fractions were collected and dried with a gentle stream of N2 and stored at 20 °C until required. The insoluble residue was hydrolyzed with 10 ml of 1.0 mol l1 KOH in 96% MeOH (v/v) for 30 min at 70 °C to obtain the bound fraction. After cooling and decanting, the supernatant was removed and placed in a separate tube. Additional extractions were performed on the residue using MeOH:CHCl3 (1:1, v/v, 1  10 ml) and CHCl3 (2  10 ml). The combined extracts were acidified to pH 1.0 with 12 mol l1 HCl, and 5 ml distilled water was added. The released compounds were extracted with CHCl3 (3  5 ml). CHCl3 extracts were combined and dried by passing them through an anhydrous Na2SO4 column. The solvent was removed with a gentle stream of N2 and stored at 20 °C until required. 2.3. Derivatization Free and bound acids were transesterified with 2 ml MeOH:MeCOCl (10:1, v/v) for 12 h at 60 °C. The methyl esters were isolated by adding 1 ml KCl (10%, w/v) and extracted into CHCl3 (3  2 ml). After esterification and drying, both acid fractions were derivatized by adding 30 ll of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1.0% trimethylchlorosilane (TMCS) and heating at 70 °C for 1 h. Residual reagent was evaporated with a stream of N2 and samples redissolved in 100 ll hexane prior to chromatographic analysis. 2.4. Gas chromatography–mass spectrometry (GC–MS) All derivatized samples were analyzed using a Trace GC instrument in tandem with a Polaris Q ion trap MS instrument (Thermo), equipped with a CBP1 column (30 m  0.25 mm, 0.25 lm film thickness). Samples were injected via a split/splitless injector, using splitless mode for 30 s. The GC oven was programmed from 60 °C (held 5 min) to 150 °C at 10 °C min1, then to 300 °C (held 5 min) at 4 °C min1. He at a constant 49.5 kPa, was used as carrier gas. Interface and ion source temperatures were 300 °C and 200 °C, respectively. Electron ionization at 70 eV was used, scanning m/z 50–600, with a cycling time of 0.58 s. 2.5. Post analysis treatment Semi-quantitative analysis was performed with 1.0 lg heneicosanoic acid (C21:0) and 0.5 lg 5a-cholestane added to the free and bound fractions, respectively. For the free acid fraction the internal standard was introduced before transesterification, and for the bound fraction the hydrocarbon was added after silylation. Integrated chromatograms were normalized and the acid classes expressed as a relative amount of total. Principal components analysis (PCA – UnscramblerÒ) was used to reduce the dimensionality of the data and biplots for the greatest variance were produced to describe the scores and loadings for specific acid classes. The following fragments of the derivatized compounds were used in the semi-quantitative analysis, histogram and PCA: n-alkanoic and branched acids m/z 143, unsaturaded acids m/z 67, x-hydroxyalkanoic acids [M15]+, 9(10),16-dihydroxyhexadecanoic acid sum of m/z 259 and m/z 273, bile acids (m/z 215 for lithocholic acid

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and m/z 255 for deoxycholic acid), phosphoric acid m/z 299, aromatic acids (M+ at m/z 224 for 4-hydroxybenzoic acid, m/z 179 for phloretic acid, m/z 250 for ferulic acid and m/z 224 for vanillic acid).

3. Results The major acids in both fractions (free and bound) from all samples were n-saturated, branched and unsaturated alkanoic acids, x-hydroxyalkanoic acids, bile acids, and lignin/suberin derived aromatic acids. Interestingly, H3PO4 was also detected in the bound fraction of all TPI soil horizons (Table 1). Fig. 1 shows the distributions for the free and bound fractions. In both fractions, homologous series of n-saturated, branched and unsaturated acids were observed in the C12:0–C32:0, C14:0–C19:0 and C16:1–C22:1 ranges, respectively. Further inspection revealed the presence of three types of hydroxyalkanoic acids assigned as x-hydroxyalkanoic acids, dihydroxalkanoic acids and bile acids. The x-hydroxyalkanoic series was detected mainly in the bound fraction, ranging from C16:0 to C28:0. In the free fraction they were only detected in the top soil of TPI (Table 1). Interestingly, bile acids and H3PO4 were only in the bound fraction from TPI samples, with a fluctuating amount along the depth profile (Table 1). Lignin/suberin related aromatic acids (i.e. 4-hydroxybenzoic acid, phloretic acid, ferulic acid and vanillic acid), despite being seen in the three soil profiles, were only obtained from the bound fraction (Table 1, Fig. 1). However, for the TM soil their distribution was not constant and they only occurred in two horizons (10–20 cm and 40–100 cm). A decrease with depth was observed in the AS soil and in the TPI soil profile they were concentrated at the three top horizons. It is important to note the high concentration of 4-hydroxybenzoic acid along the TPI profile (0.14–0.21 lg g1), contrasting with the other aromatics, with the exception of the top horizon dominated by ferulic acid (0.42 lg g1). This pattern was not observed for TM or AS, where ferulic acid and vanillic acid had an important contribution, with a minor concentration, or even absence, of phloretic acid (data not shown). Among the alkanoic acids, the n-saturated components were the most abundant from all depths for both fractions (free and bound), followed by branched and unsaturated compounds (Table 1). Alkanoic acid pools from the TPI and TM soils obtained from both fractions did not vary considerably and no decrease in

concentration was observed with depth (Table 1). It is important to mention that in the branched alkanoic acids (C14:0–C19:0), iso and anteiso C15 and C17 were the most abundant compounds for both free and bound fractions (data not shown). Hydroxyalkanoic acids were present mainly in the bound fraction. The highest amounts of x-hydroxyalkanoic acids and 9(10),16-dihydroxyhexadecanoic acid were seen for the TM and AS soils. In the AS profile the concentration decreased with depth, with the exception of the top soil. On the other hand, the TM soil did not show any tendency with depth. For example the highest amount of 9(10),16-dihydroxyhexadecanoic acid was from the deepest horizon (Table 1). The free fractions revealed a practically identical n-alkanoic acid variation, typified by a unimodal distribution of short chain alkanoic acids (< C20) with a maximum at C16 in all samples, with the exception of the TPI top soil, which showed a maximum at C18 (Fig. 2). Long chain alkanoic acids (> C20) were relatively small or not observed in the TPI fractions. The contribution of such compounds was slightly evident in the top soil of TPI and in AS until 0.55 m (Fig. 2). An even/odd predominance was also observed. Conversely, n-alkanoic acids in the bound fractions from each soil were dominated by a bimodal distribution with maxima at C16 and at C24, C26 or C28. These bimodal distributions were stronger for TM and AS than for TPI bound fractions (Fig. 3). Alkaline treatment released alkanoic acids in similar concentration from all profiles for each soil, with lower values along the TPI profile (Table 1). In fact, a clearly bimodal distribution for the TPI soil, maximizing at C16 and C26 or C30, was observed only for the 36–56 cm and 56– 84 cm intervals. In contrast, TM and AS showed a strong bimodal distribution for all the bound fractions. The TM profile was marked by a bimodal distribution, with long chain alkanoic acids maximizing at C26 for the top soil. The deeper horizon showed a relative increase in C30 (Fig. 3). In the AS profile, long chain alkanoic acids were dominated by C24 and C26 for the 0–15 cm (top) and 15– 38 cm intervals. For deeper soil (38–55 cm), an increase in C30 was observed, and for the deepest AS layer (55–90 cm) C24 was the most abundant long chain alkanoic acid (Fig. 3). Interestingly, the relative contribution of long chain alkanoic acids decreased with depth for each soil. Such behaviour was more pronounced for the AS soil (Fig. 3). A strong even/odd predominance was also detected in the bound acids from all the soils. The relative abundance of x-hydroxyalkanoic acid homologues in all the bound fractions is summarized in Fig. 4. The homologous series lay within a range of C18 to C28, with no odd numbered

Table 1 Semi-quantitative (lg g1)a,b distributions of major organic acids in free and bound fractions of three Amazonian soils [Terra Preta de Índio (TPI), Terra Mulata (TM) and Adjacent Soil (AS)]. Samples/depth interval (cm)

a

TPI 0–36

TPI 36–56

TPI 56–84

TPI 84–150

TM 0–10

TM 10–20

TM 20–40

TM 40–100

AS 0–15

AS 15–38

AS 38–55

AS 55–90

‘‘Free’’ n-Alkanoic acidsc Branched acidsc Unsaturated acidsd x-Hydroxy-alkanoic acidse

138.55 15.87 0.89 4.14

478.71 21.04 4.68 1.50

282.87 15.70 0.82 nd

383.92 13.08 nd nd

174.20 9.41 8.40 nd

333.80 11.95 3.06 nd

78.88 7.95 nd nd

86.03 5.24 3.88 nd

84.54 23.36 2.28 nd

94.98 15.30 3.23 nd

184.22 14.54 2.78 nd

143.04 12.30 3.19 nd

‘‘Bound’’ n-Alkanoic acidsc Branched acidsc Unsaturated acidsd x-Hydroxy-alkanoic acidse 9(10),16-Dihydroxyhexadecanoic acidf Bile acidsg Phosphoric acidh Aromatic acidsi

1.33 0.67 0.03 0.59 0.33 0.03 76.08 0.72

1.47 0.39 0.13 2.17 0.26 1.82 31.96 0.88

1.75 0.34 0.08 1.10 0.11 1.23 110.91 0.78

1.55 0.38 0.14 0.44 0.08 0.21 13.92 0.28

6.95 1.54 0.44 0.02 0.20 nd nd nd

5.47 1.18 0.48 7.58 3.51 nd nd 1.33

5.77 0.89 0.34 0.18 0.20 nd nd nd

3.60 0.45 0.12 3.25 8.59 nd nd 0.22

2.69 0.83 0.26 6.34 2.11 nd nd 2.67

2.88 0.91 0.15 7.18 4.18 nd nd 1.93

2.14 0.34 0.03 3.12 0.85 nd nd 0.62

3.36 0.61 0.14 1.29 0.12 nd nd 0.39

Calculated based on following fragments from the derivatised compounds: c m/z 143, d m/z 67, e [M15]+, f sum of m/z 259 and m/z 273, g m/z 215 – lithocholic acid and, m/z 255 – deoxycholic acid, h m/z 299, i M+ 224 – 4-hydroxybenzoic acid, m/z 179 – 4-hydroxy-hydroxycinnamic acid, m/z 250 – ferulic acid and, m/z 224 – vanillic acid, b m/z 143 of heneicosanoic acid (C21:0) internal standard used for the free fraction, and m/z 217 of 5a-cholestane for the alkaline fraction.

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Fig. 1. Partial total ion current (TIC) showing major compounds in the free and bound fractions of the three soils. Free fraction, TPI – 56–84 cm (A), TM – 20–40 cm (B) and AS – 38–55 cm (C). Bound fraction, TPI – 56–84 cm (D), TM – 20–40 cm (E) and AS – 38–55 cm (F). Peak identity: j, n-alkanoic acids; h, branched alkanoic acids; d, unsaturated alkanoic acids; , x-hydroxyalkanoic acids; ?, unidentified; LC, lithocholic acid; DOC, deoxycholic acid; , contamination. Cx above peaks refers to number of carbons, and a number after colon refers to number of double bonds. Branched alkanoic acids are denoted ‘‘br’’.

compounds. However, the x-hydroxyalkanoic acids showed a different distribution pattern in the bound fractions. All AS horizons had a similar x-hydroxyalkanoic acid distribution maximizing at C22 and decreasing with depth (Table 1). The distribution in the TM bound fractions fluctuated with depth. Only two components were detected in the topsoil of TM: C22 and C26; and at the 10– 20 cm interval a similar distribution to AS was observed, maximizing at C22. For the two deepest horizons a different distribution maximizing at C24 was detected. With the exception of the top, in all TPI soil horizons the relative distribution of x-hydroxyalkanoic acids was regular, and the most abundant component was the long chain C28 x-hydroxyalkanoic acid. It was noted that the C16 x-hydroxyalkanoic acid had a significant contribution in some depth intervals (Fig. 4). In order to reinforce similarities and differences in the data, PCA of the n-alkanoic acid concentration in the free fraction, and relative distribution of x-hydroxyalkanoic acids in the bound fraction, was

carried out (Fig. 5). The variance in n-alkanoic acids was totally explained by two PCs. Soil samples with high amounts of fatty acids had higher positive values in PC1 (Table 1, Fig. 5A), and hexadecanoic acid (C16), the major fatty acid (Fig. 2), had a greater influence. In the case of the x-hydroxyalkanoic acid distribution, the cumulative variation in PC1 and PC2 was 90%, with the C24, C22, and C28 components having a strong influence in the model. Samples TPI topsoil (0–36 cm) and TM (10–20 cm) had negative values in PC1, indicating a high abundance of C22, and a similar distribution to some AS samples(Figs. 4 and 5C and D). Other TPI horizons were very distinct from the AS and TM soil horizons due to the marked presence of the C28 component (Fig. 5C and D). Besides, the deepest TM (20–40 and 40–100 cm) samples, with a higher abundance of the C24 component (negative values in PC1 and positive values in PC2) showed a slight different from the AS soil (Figs. 4 and 5C and D). It is interesting to note the presence of bile acids in the bound fractions from the TPI profile. They were not found in the other

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Fig. 2. Normalised relative distribution of n-alkanoic acids in the free fraction of the three soils. The abundances were established from the area of m/z 143.

soils, even in the TPI free acids (Table 1). They were assigned (Fig. 1D) in TPI as lithocholic acid (3a-hydroxy-5b-cholanoic acid – LC) and deoxycholic acid (3a-12a-dihydroxy-5b-cholanoic acid – DOC). They were assigned from their characteristic mass spectra (Fig. 6). The amount fluctuated with depth, the highest concentration being found at the intermediate depths (i.e. 36–56 and 56– 84 cm). Deoxycholic acid was the dominant compound accompanied by a minor concentration of lithocholic acid (data not shown). 4. Discussion The distribution of free short-chain (< C20) n-alkanoic acids with a strong even predominance in soil extracts normally attests to an

input from bacteria. The incorporation of bacteria was reinforced by the presence of C15 and C17 iso and anteiso alkanoic acids (Goossens et al., 1986; Zelles, 1999). However, a contribution from aerial vegetation and root litter may not be excluded, since such plant tissue may contain substantial amounts of short chain alkanoic acids (Bull et al., 2000). Interestingly, bones from mammals and fish have been suggested as an important input to Terra Preta (Schaefer et al., 2004; Glaser and Birk, 2012). In fact, bones have been considered an important source of fuel at many archaeological sites due to the presence of fat (Hoffecker, 2005). Recently, Kedrowski et al. (2009) reported that short chain alkanoic acids were major compounds in the hearth area of archaeological sites, indicating the burning of bones. Therefore, the short chain alkanoic acids in

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Fig. 3. Normalised relative distribution of n-alkanoic acids in the bound fraction from the three soils. The abundances were established from the peak areas for m/z 143.

the anthropogenic soils – mainly TPI deepest horizons (56–150 cm) and one TM horizon (10–20 cm) (Fig. 5A), which contained a higher amount of these in the free form – may reflect ancient human input such as the burning of bones (Table 1, Figs. 2 and 5A). Long chain alkanoic acids were also present in the free fraction, but their contribution to the bound fraction was stronger (Figs. 2 and 3). The profiles of the bound fractions showed a significant increase in long- (> C20) alkanoic acids, mainly for TM and AS (Table 1, Figs. 2 and 3). This pattern was less evident for the TPI soil (Fig. 3). Long chain alkanoic acids are associated with higher plant input (Amblès et al., 1994), more specifically, those in ester-bound form in soils are commonly associated with the presence of cutin and suberin (Naafs and van Bergen, 2002). Cutin and suberin are

important components of higher plants, the former in the aerial parts and the latter in woody stems and underground parts, i.e. roots (Kolattukudy, 1980; Holloway, 1982). Normally, cutin tissue is associated with short chain alkanoic acids with even carbons ranging from C12 to C18 with C16 or C18:1 the major components. Suberin-derived acid components are suggested to contain the longer homologues (> C20) maximizing at C22 and C24 (Kolattukudy and Espelie, 1985). Considering the characteristics of cutin and suberin composition, we consider that the long chain alkanoic acid distributions from the soils are related to the incorporation of suberin. The distribution of short chain acids, with a low amount of C18:1 (data not shown), indicates a possible admixture of source components. For example, the presence of odd numbered short chain acids, together with C15 and C17 iso and anteiso acids, may

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Fig. 4. Normalised relative distribution of x-hydroxyalkanoic acids in bound fraction from the three soils. The abundances were established from the peak areas of [M15]+.

infer a bacterial input (Grasset and Amblès, 1998). Notably, the relative distribution of suberin-derived long chain acids was distinct for each soil (Fig. 3). For the AS soil, the contribution of > C20 acids was more prominent in the top horizon (up to 15 cm), probably indicating a recent incorporation of modern higher plants, decreasing with depth. Similar behaviour was observed for the TM soil, but with a slight difference in the > C20 homologous distribution. For the TPI soil profile, the contribution of long chain alkanoic acids was less evident, showing that incorporation of modern suberinderived long chain acids occurred to a lesser degree in TPI than in the other two soils (Fig. 3).

Cutin and suberin input are also represented by hydroxyalkanoic acids, namely x-hydroxyalkanoic acid homologues and 9(10),16-dihydroxyhexadecanoic acid (Naafs et al., 2005). Cutin is an aliphatic polymer comprised mainly C16 and C18 x-hydroxyalkanoic acids, C16 dihydroxyalkanoic acids and C18 trihydroxyalkanoic acids (Kolattukudy, 1980; Matzke and Riederer, 1991). Suberin is a more complex biopolymer containing aromatic and aliphatic domains (Kolattukudy, 1980; Bernards and Razem, 2001), the latter being closely related to cutin, but with some peculiarities. Most suberins are dominated by long-chain (> C20), a,xalkenedioic acids and x-hydroxyalkanoic acids (Kolattukudy,

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Fig. 5. Score-loading biplots of fatty acid concentration in the free fractions (A and B) and relative distribution of x-hydroxyalkanoic acids in the bound fraction (C and D). Note: during PCA analysis of the x-hydroxyalkanoic acids, the topsoil horizons of TM were kept out of analysis due to low concentration and odd distribution (see Table 1 and text for details).

1980; Matzke and Riederer, 1991). Typically, suberin long chain xhydroxyalkanoic homologous series were detected in the bound fraction from all horizons of the AS soil, maximizing at C22 (Figs. 4 and 5D, Matzke and Riederer, 1991; Naafs and van Bergen, 2002). This identical pattern indicates a similar source of suberin (root) tissue at all depths, probably evidencing a recent input of modern vegetation. The major root material (i.e. suberin) influence in AS soil was in the top horizon, as indicated by the highest concentration of x-hydroxyalkanoic acids (Table 1). With respect to the anthropogenic soils TM and TPI, the xhydroxyalkanoic acid distribution showed very distinct patterns. First, a similar source of root OM was clearly present in the top horizons, which may be related to an input from modern vegetation (Fig. 5C). However, this characteristic was only observed at the top of the TPI soil (< 36 cm) and in the TM soil in the second horizon (10–20 cm). The lack of such a distribution in the TM topsoil may be caused by recent land use (Fig. 4).This could be confirmed by a low amount of x-hydroxyalkanoic acids in the surficial TM soil. Along the TPI profile, a different homologous distribution of long chain x-hydroxyalkanoic acids, maximizing at C28 was observed (Figs. 4 and 5D); at this point the specific source of such homologues cannot be determined, but in situ diagenetic production through microbial oxidation of n-alkanoic acids cannot be ruled out (Voet and Voet, 1995). In the case of TM, a change in the distribution of x-hydroxyalkanoic acids can be seen in the shift in the maximum. At the deepest depth interval, the distribution was dominated by the C24 homologue, indicating either a different source of such compounds from that seen for AS or a preferential loss of C22 component by biotic degradation in soil, as suggested

by Bull et al. (2000). If the latter occurs, a distinct microbial community vs. AS must be present, since such behaviour was not observed in the AS soil profile. In fact, recent studies revealed that microbial diversity is rich in anthropogenic Amazon soils (Kim et al., 2007; Germano et al., 2012), indicating that possible changes in homologous x-hydroxyalkanoic acid distributions in the TPI and TM may be caused by microbial reworking. Other major hydroxyalkanoic acids in all the samples consisted (Table 1) of a mixture of isomers, 9,16-dihydroxyhexadecanoic acid and 10,16-dihydroxyhexadecanoic acid, [9(10),16-dihydroxyhexadecanoic acid]. As mentioned above, such compounds are common components of cutin and suberin (Matzke and Riederer, 1991), although dihydroxyhexadecanoic acids (x,16-C16 family) have been detected as major components from cutin (Kolattukudy, 2001; Nierop et al., 2006). Taking the differences between cutin and suberin aliphatic composition into account (see above), a dominance of suberin-derived over cutin-derived products is clearly observed in all the soil profiles, except for the bottom horizons of TM (Table 1). The deepest AS soil showed a major incorporation of suberin over cutin, as indicated by the abundance of x-hydroxyalkanoic acids over 9(10),16-dihydroxyhexadecanoic acid. Consequently, this reflects a minor incorporation of cutin-derived products with depth, which is expected since cutin-derived products are associated with aerial vegetation (Kolattukudy, 1980; Holloway, 1982). With the exception of the top horizon, TPI showed a high abundance of xhydroxyalkanoic acids over 9(10),16-dihydroxyhexadecanoic acid, suggesting that incorporation of cutin-derived products along the TPI profile was relatively low (Table 1). It is important to note the fluctuating behaviour in the abundance of x-hydroxyalkanoic acids

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Fig. 6. Electron ionization (70 eV) mass spectra of methyl ester TMS derivatives of lithocholic acid (A) and deoxycholic acid (B) from bound fraction of TPI soil, depth 0.56– 0.84 cm.

over 9(10),16-dihydroxyhexadecanoic acid for the TM profile. In the case of the TM soil profile, reflecting a similar distribution of xhydroxyalkanoic acids to the AS soil, a similar ratio was also perceived (10–20 cm). However, at depths with a distinct x-hydroxyalkanoic acid distribution, an increase in cutin-derived products was observed, where the presence of 9(10),16-dihydroxyhexadecanoic acid in the bottom horizon was higher than the sum of long chain x-hydroxyalkanoic acids (Table 1). Considering this shift, an important event may have occurred in the OM incorporation. The high abundance of 9(10),16-dihydroxyhexadecanoic acid indicates a major contribution of cutin tissue to the TM deepest horizon, which may support a historical change in vegetation in previous times at the archaeological site or even during ancient human occupation (e.g. plants via their reproductive parts such as seeds, fruits, nuts and tubers are most likely to have been harvested and brought back to the place of habitation). However, other events such as burning effects, microbial reworking and abiotic diagenetic effects cannot be totally ruled out. A third class, cyclic hydroxyalkanoic acids was detected only in TPI. They were assigned from their mass spectra as deoxycholic and lithocholic acids (Fig. 6). Bile acids are a group of steroidal acids produced in the digestive system of animals (Bull et al., 2002), and are currently used as a biomarker for faecal input in monitoring modern sewage systems and in studies of soil/sediment in archaeological site contexts (Elhmmali et al., 2000; Bull et al., 2003; Tyagi et al., 2008; Shillito et al., 2011). Bile acid biomarkers have been detected in Amazonian anthropogenic soils,

together with 5b-stanols in the free fraction of soil extracts, indicating human excrement incorporation into these soils (Birk et al., 2011; Glaser and Birk, 2012). Despite the presence of bile acids in our bound fractions, they were not found in the free fractions. Apart from 5b-stanols, other faecal biomarkers were not detected, either in the free or bound fractions (data not shown). The survival of bile acid in a bound phase (released after alkaline hydrolysis) must have occurred due to the protection caused either by covalent bonding in a polymeric network or trapping in clay sized pores (Elhmmali et al., 1997; Hansel and Evershed, 2009). Therefore, the presence of two bile acids in TPI suggests human faeces deposition, corroborating the results of Glaser and Birk (2012), though the specific source, such as manuring practices or a burial site, cannot be ruled out. As with the bile acids, phosphoric acid was only detected in TPI, in high amount. High phosphate levels are commonly used to indicate ancient human occupation, and the high amount led to intense human interference in TPI (Provan, 1971). Aromatic acids in all the soil samples were phloretic and ferulic acid and their benzoic acid counterparts (i.e. 4-hydroxybenzoic acid, vanillic acid). Their incorporation into soil may be related to suberin-derived products and their oxidative counterparts, since suberin is characterized by the presence of hydroxycinnamic acid derivative domains (Kolattukudy, 1980). Lignin as a source for part of these compounds cannot be totally excluded, since p-coumaric and ferulic acid are abundant in grass lignin (e.g. Zea mays; del Río et al., 2012; Withers et al., 2012). However, considering the

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lack of a syringyl group, lignin incorporation from hardwood to the bound fraction is essentially excluded (Ralph and Hatfield, 1991; Dence and Lin, 1992). 5. Conclusions The main conclusions are: (i) The acids showed a distinct genesis for AS and the two anthropogenic soils in the deeper horizons. The influence of modern vegetation was detected by way of x-hydroxyalkanoic acid distributions only in the top horizons of the archaeological soils. Such an observation indicates that organic geochemical analysis is a useful tool for investigating tropical archaeological soils, but it is necessary to always evaluate the non-cultural influence (e.g. modern vegetation) on anthropogenic soil by way of a set of adjacent soils. (ii) The free acids did not reveal strong differences in the genesis of the three soils, with an input from burning bones suggested by a high concentration of short chain (< C20) n-alkanoic acids in the TPI deepest horizon and one TM soil horizon. In contrast, the acids released by alkaline hydrolysis showed the potential of old or recent OM input to archaeological soils. However, we advise that both free and bound fractions should be used to provide complementary data about OM incorporation into anthropogenic soils. (iii) From the relative distribution of x-hydroxyalkanoic acids and 9(10),16-dihydroxyhexadecanoic acid, a dominance of suberin over cutin derived products was detected in all soils except the deepest horizon of the TM profile. The high abundance of cutin points to parts of old aerial vegetation deposited in the deepest TM horizon, which might be promising for answering questions in future studies of human occupation. (iv) With respect to the genesis of Amazonian anthropogenic soils (TP and TM), substantial differences were detected in the distribution of hydroxyalkanoic acids. For example, TPI showed incorporation of human faeces not apparent in the TM soil. Furthermore, the x-hydroxyalkanoic acid distribution proved to be distinct for the two archaeological soils, indicating a different source of OM in the soil genesis.

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