•Isotopic proxies are tested on archaeological remains from Zvejnieki, Latvia.•Dietary protein sources are identified using δ13C values of amino acids.•Amino acid δ13C values can elucidate palaeodiet in complex ecosystems.Of central importance to palaeodietary reconstruction is a clear understanding of relative contributions of different terrestrial (i.e., C3 vs. C4 plants) and aquatic (i.e., freshwater vs. marine) resources to human diet. There are, however, significant limitations associated with the ability to reconstruct palaeodiet using bulk collagen stable isotope compositions in regions where diverse dietary resources are available. Recent research has determined that carbon-isotope analysis of individual amino acids has considerable potential to elucidate dietary protein source where bulk isotopic compositions cannot. Using δ13CAA values for human and faunal remains from Zvejnieki, Latvia (8th – 3rd millennia BCE), we test several isotopic proxies focused on distinguishing freshwater protein consumption from both plant-derived and marine protein consumption. We determined that the Δ13CGly-Phe and Δ13CVal-Phe proxies can effectively discriminate between terrestrial and aquatic resource consumption, and the relationship between essential δ13CAA values and the Δ13CGly-Phe and Δ13CVal-Phe proxies can differentiate among the four protein consumption groups tested here. Compound-specific amino acid carbon-isotope dietary proxies thus enable an enhanced understanding of diet and resource exploitation in the past, and can elucidate complex dietary behaviour.

•Lipids were extracted from sherds from the Late Neolithic site of Tell Sabi Abyad.•Findings were interpreted in relation to phases, ware types and the faunal record.•Differences in use between pottery types revealing early cooking evidence.•The presence of adipose/dairy fats was consistent with faunal remains.•The dairy fats detected point to early secondary products exploitation.Late Neolithic settlements dating to around 7000 cal. BC are widespread in Upper Mesopotamia, however, the site of Tell Sabi Abyad is unique in the scale and quality of excavation, revealing an extensive architecture, huge numbers of domesticated animal bones, stone tools and potsherds. A previous study reported lipid residues in nearly 300 potsherds as part of a wider investigation of the origins of dairying in the Near East and Southeastern Europe. The aim of this paper is to interpret the organic residue findings in more detail, addressing such factors as the association of lipids in pottery with particular phases, ware types, and the faunal record. Overall, the recovery rate of lipids in sherds is low (14% of the sherds investigated in this study yielded detectable lipids) and the mean lipid concentration for sherds containing lipids is ca. 82 μg g−1. These results are typical of sites from this period and general region (southern Mediterranean and Near East). Our interpretations indicate: (i) the use of specific ceramic categories of vessel for “cooking”, (ii) clear evidence of the extensive heating of vessels is deduced from the presence of ketones, formed from the condensation of fatty acids, in some vessels, (iii) strong differences in recovery rates possibly reflecting differences in use between different pottery types, (iv) in particular the Dark-Faced Burnished Ware (DFBW) contained the highest frequency of residues (46% yielded detectable lipids), (v) degraded animal fats were detectable, as evidenced by fatty acids with C18:0 in high abundance and in few cases tri-, di- and monoacylglycerols, (vi) the presence of abundant carcass fats is consistent with interpretations based on faunal assemblage of extensive meat exploitation, and (vii) four vessels dated to 6400 to 5900 cal BC yielded milk fat residues.

Despite the significant achievements of organic residues analysis of archaeological pottery, the sometimes low lipid recovery and the need to process increasingly large collections of sherds to tackle important archaeological questions require the development of a more efficient and rapid extraction method. In this paper we present a novel methodology for the extraction of absorbed organic residues directly from crushed archaeological ceramic using acidified methanol (H2SO4–MeOH 2% v/v, 70 °C, 1 h). This new protocol was tested by: (i) verifying the recovery of organic residues from previously studied archaeological vessels from different geographical regions, exhibiting a range of different lipid distributions often found in archaeological pottery, and (ii) demonstrating enhanced recovery of organic residues from potsherds that did not yield appreciable lipids when using the widely applied chloroform–methanol extraction. The application of the direct acidified methanol extraction recovers higher concentrations of lipid residues together with simultaneous production of methyl esters of fatty acids, allowing extraction and methylation to be completed in 20% of the time compared to conventional solvent extraction and derivatisation for gas chromatography (GC), gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS).

•Plant amino acid δ15N values were determined by GC–C–IRMS.•Cereal grain amino acid δ15N values reflect known amino acid biosynthetic pathways.•Cereal rachis amino acid δ15N values reflect known amino acid biosynthetic pathways.•Relative amino acid δ15N values of broad bean and pea seeds differ.Natural abundance δ15N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ15N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC–C–IRMS. It was found that the δ15N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative 15N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ15N value which is an average of the other AAs due to its central role in AA–N cycling. The relative AA δ15N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ15N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.This investigation shows that AA δ15N values can provide insights into metabolic pathways influencing bulk plant protein δ15N values, and can aid understanding of plant N cycling in response to different sources of assimilated N.Figure optionsDownload full-size imageDownload as PowerPoint slide

•Manured and unmanured crop amino acid δ15N values were determined by GC-C-IRMS.•Manuring of cereals increases the δ15N values of all grain and rachis amino acids.•Manuring does not change the relative δ15N values of cereal grain amino acids.•Rachis amino acid δ15N values vary more with manuring than grains from the same plots.•Pulses did not assimilate significant N from manure.Amino acid δ15N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown in manured and unmanured soil at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. Manuring was found to result in a consistent 15N-enrichment of cereal grain amino acid δ15N values, indicating that manuring did not affect the metabolic routing of nitrogen (N) into cereal grain amino acids. The increase in cereal grain δ15N values with manuring is therefore due to a 15N-enrichment in the δ15N value of assimilated inorganic-N. Greater variation was observed in the 15N-enrichment of rachis amino acids with manuring, possibly due to enhanced sensitivity to changes in growing conditions and higher turnover of N in rachis cells compared to cereal grains. Total amino acid δ15N values of manured and unmanured broad beans and peas were very similar, indicating that the legumes assimilated N2 from the atmosphere rather than N from the soil, since there was no evidence for routing of 15N-enriched manure N into any of the pulse amino acids. Crop amino acid δ15N values thus provide insights into the sources of N assimilated by non N2-fixing and N2-fixing crops grown on manured and unmanured soils, and reveal an effect of manure on N metabolism in different crop species and plant parts.Graphical abstractAmino acid δ15N values of barley and bread wheat grains and rachis and pulse seeds were determined by GC-C-IRMS. It was found that manuring results in a consistent 15N-enrichment of cereal grain and rachis amino acid δ15N values but no increase in the amino acid δ15N values of pulse seeds.

Although in modern societies fermented beverages are associated with socializing, celebration, and ritual, in ancient times they were also importa`nt sources of essential nutrients and potable water. In Mesoamerica, pulque, an alcoholic beverage produced from the fermented sap of several species of maguey plants (Agavaceae; Fig. 1) is hypothesized to have been used as a dietary supplement and risk-buffering food in ancient Teotihuacan (150 B.C. to A.D. 650). Although direct archaeological evidence of pulque production is lacking, organic residue analysis of pottery vessels offers a new avenue of investigation. However, the chemical components of alcoholic beverages are water-soluble, greatly limiting their survival over archaeological timescales compared with hydrophobic lipids widely preserved in food residues. Hence, we apply a novel lipid biomarker approach that considers detection of bacteriohopanoids derived from the ethanol-producing bacterium Zymomonas mobilis for identifying pulque production/consumption in pottery vessels. Gas chromatography–mass spectrometry selected ion monitoring (m/z 191) of lipid extracts of >300 potsherds revealed characteristic bacteriohopanoid distributions in a subset of 14 potsherds. This hopanoid biomarker approach offers a new means of identifying commonly occurring bacterially fermented alcoholic beverages worldwide, including palm wine, beer, cider, perry, and other plant sap- or fruit-derived beverages [Swings J, De Ley J (1977) Bacteriol Rev 41(1):1–46].

The funeral preparations for ancient Egyptian dead were extensive. Tomb walls were often elaborately painted and inscribed with scenes and objects deemed desirable for the afterlife. Votive objects, furniture, clothing, jewelry, and importantly, food including bread, cereals, fruit, jars of wine, beer, oil, meat, and poultry were included in the burial goods. An intriguing feature of the meat and poultry produced for the deceased from the highest levels of Egyptian society was that they were mummified to ensure their preservation. However, little is known about the way they were prepared, such as whether balms were used, and if they were used, how they compared with those applied to human and animal mummies? We present herein the results of lipid biomarker and stable carbon isotope investigations of tissues, bandaging, and organic balms associated with a variety of meat mummies that reveal that treatments ranged from simple desiccation and wrapping in bandages to, in the case of the tomb of Yuya and Tjuia (18th Dynasty, 1386–1349 BC), a balm associated with a beef rib mummy containing a high abundance of Pistacia resin and, thus, more sophisticated than the balms found on many contemporaneous human mummies.

Current research themes relating to prehistoric Central Asian pastoralism are discussed, and the Neolithic to Bronze archaeological sequence in Kazakhstan is briefly outlined. The results of new faunal analyses of six later Bronze Age sites in Central and Northern Kazakhstan are presented. These studies are based upon the analysis of 63,529 bone fragments, of which 27,023 were identifiable to species and element. These assemblages are compared with 16 other sites in Central and Northern Kazakhstan, and the Trans-Ural region. The herd structures at the final Bronze Age site of Kent are discussed in detail. Analyses of absorbed lipid residues from four sites are also presented. In total, 140 pottery sherds were analysed, of which 73 provided sufficient residues for stable isotope ratio determinations. It is concluded that species proportions are highly variable regionally. Cattle are most prevalent in the forest steppe zone, whilst caprines become more common in semi-arid steppe regions. Proportions of horse are particularly variable, even within environmentally similar areas. Lipid residue results indicate the high prevalence of ruminant dairy products in pottery vessels, whilst faunal data from Kent suggests that cattle husbandry might have been particularly focussed on milk, in comparison with sheep and goats. The significance of horses within prehistoric pastoralism is discussed.Highlights► Faunal analyses of six later Bronze Age sites in Central and Northern Kazakhstan. ► Absorbed lipid residue analyses of ceramics from four sites. ► High inter-site variation in species abundance, particularly in relation to horses. ► Cattle more common in forest steppe, caprines more common in semi-arid steppe. ► Significance of horses to prehistoric mixed pastoralism.

The flowers of 23 species of grass and herb plants were collected from a mesotrophic grassland to assess natural variability in bulk, monosaccharide and fatty acid δ13C values from one plant community and were compared with previous analyses of leaves from the same species. The total mean bulk δ13C value of flower tissues was −28.1‰, and there was no significant difference between the mean δ13Cflower values for grass (−27.8‰) and herb (−28.2‰) species. On average bulk δ13Cflower values were 1.1‰ higher than bulk δ13Cleaf values, however, the δ13Cflower and δ13Cleaf values of grasses did not differ between organs suggesting that carbon isotope discrimination is different in grass and herb species. The abundance of different monosaccharides abundance varied between plant types, i.e. xylose concentrations in the grass flowers were as high as 40%, compared with up to 15% in the herb species, but the general relationship δ13Carabinose > δ13Cxylose > δ13Cglucose > δ13Cgalactose which had been observed in leaves was similar in flowers (total mean δ13C values = −25.9‰, −27.2‰, −28.8‰ and −28.1‰, respectively). However, the average 5.4‰ depletion in the δ13C values of the C16:0, C18:2 and C18:3 fatty acids in flowers compared to bulk tissue was significantly greater than observed for leaves. The trend C16:0 < C18:2 < C18:3 previously observed in leaves was also observed in grass flowers (δ13CC16:0 = −33.8‰; δ13CC18:2 = −33.1‰; δ13CC18:3 = −34.2‰) but not herb flowers (δ13CC16:0 = −34.1‰; δ13CC18:2 = −32.4‰; δ13CC18:3 = −34.5‰). We conclude: (i) that the biological processes influencing carbon isotope discrimination in grass flowers are different from herbs flowers; and, (ii) that a range of post-photosynthetic fractionation effects caused the observed differences between flower and leaf δ13C values, especially the significant 13C-depletion in flower fatty acid δ13C values.Graphical abstractHighlights► The first compound-specific stable 13C isotope values for flowers. ► Fatty acid and monosaccharide δ13C values are reported for herbaceous and grass species. ► Flower fatty acid δ13C values are significantly depleted compared with those from leaves.

A passive sampler (the polar organic chemical integrative sampler; POCIS) was assessed for its ability to sample natural estrogens (17β-estradiol, E2; estrone, E1 and estriol, E3) and the synthetic estrogen (17α-ethynylestradiol, EE2) in the outlet of a sewage treatment works over several weeks. The performance of the POCIS was investigated and optimised in the laboratory before field deployment with high recoveries (66–99%) were achieved for all estrogens. Moreover, it was shown that POCIS does not exhibit any preferential selectivity towards any of the target compounds. The sampling rates of E1, E2 and E3 were 0.018 ± 0.009, 0.025 ± 0.014 and 0.033 ± 0.019 L d−1, respectively. Following field deployments of 28 days in the discharge of a sewage works, POCIS was shown to enhance the sensitivity of estrogen detection, especially for E3, and provide time-weighted average (TWA) concentrations of E1, E2 and E3, ranging from undetectable to 12 ng L−1upstream of the outflow of a sewage treatment works, 13 to 91 ng L−1 at the outflow and 8 to 39 ng L−1downstream of the outflow. This revealed that E1, E2 and E3 are not completely removed during sewage treatment, with concentrations most likely being maintained by contributions from conjugated estrogen analogues. Grab water samples showed considerable variation in the concentrations of estrogens over a longer period (6 months). The results confirm that POCIS is an effective and non-discriminatory method for the detection of low concentrations of estrogens in the aquatic environment.

Mineralisation rates provide valuable information concerning the overall cycling of soil organic N; however, detailed information regarding the pathways preceding the mineralisation of organic substrates remains elusive. We have adopted a molecular approach to open the ‘black box’ of organic N cycling in soil. Stable isotope probing employing compound-specific isotopic analysis was used to trace the fate of N and C within metabolites central to organic N cycling. In time course experiments, 15N and 13C from two dual-labelled amino acid (AA) substrates (U-13C,15N-glutamate and U-13C,15N-glycine) were followed into AAs biosynthesised de novo. In the majority of cases, highly significant differences (P < 0.01) were revealed in the magnitude and rate of N and C transfer from the AA substrates to products of central metabolic pathways prior to their loss from the AA pool. By applying linear and non-linear regressions, several important parameters were derived, namely rate constants, magnitude of fluxes and measures of biosynthetic proximity, which describe the rate and magnitude of N and C flux through primary metabolic processes. The significant differences in N and C processing demonstrate a decoupling of the N and C cycles at the molecular level, i.e. after 32 days the magnitude of N flux into newly biosynthesised AAs was twofold greater than that of C from both substrates. We anticipate that the parameters derived will have potential for use in developing detailed models of soil organic N and C processing, the construction of which is founded on the connectivity of the processes fundamental to life.

The n-alkane distributions from total lipid extracts of ten modern Sphagnum moss species, collected from a suite of ombrotrophic bogs across Europe, were determined using gas chromatography/mass spectrometry (GC/MS). n-Alkane distributions are reported for the first time for Sphagnum balticum, S. majus, S. angustifolium and S. lindbergii, which are all dominated by C23 with the exception of S. lindbergii, which exhibits a bimodal distribution with C23 and C31 as the major homologues. The distributions for individual species generally agree with published compositions, confirming the conservative nature of the n-alkane compositions, which provide a basis for differentiating the n-C23 and n-C25 dominated species. Investigations of the variation in n-C23/n-C25 and n-C23/n-C31 ratios of Sphagnum species, using the new and published n-alkane distributions, reveal that intra-species variation is generally minor. Critically, the distributions and ratios for most species do not vary among the sites studied, suggesting that they are conservative tracers for a given species, despite differences in growth conditions. In contrast, inter-species variation exists, allowing differentiation of individual Sphagnum species based on vegetation biomarkers, specifically the C25n-alkane in S. fuscum and the n-C23/n-C25 ratio. Biomarker stratigraphic analysis of a 150 cm peat core (Kontolanrahka Bog, Finland) reveal shifts in the n-C23/n-C25 ratio, which track changes in the abundance of S. fuscum in the macrofossil record. This supports the application of n-alkane biomarkers in peat archives for tracking past shifts in individual Sphagnum species abundance. This will be particularly important where fossil plant remains are highly degraded in, or absent from, peat records.

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ13C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ13C values could be determined. The total mean leaf bulk δ13C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ13C values was only determined in October (P = <0.001) when δ13C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ13C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ13C values had a major influence on bulk δ13C values. An average depletion of ca. 1‰ in the bulk δ13C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ13C values, as glucose constituted >50% total leaf monosaccharides. In October, δ13C values of all monosaccharides varied between species, with significant variation in δ13C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ13C values observed in October 1999. The fatty acids C16:0, C18:2 and C18:3 were highly abundant in all plant species. Fatty acid δ13C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C16:0), −37.0‰ (C18:2) and −36.5‰ (C18:3) were determined. There was significant interspecific variation in the δ13C values of all individual fatty acids during October and July, but only for C18:2 in May (P = <0.05). This indicated that seasonal trends observed in the δ13C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ13C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P = <0.0001) for fatty acid and monosaccharide δ13C values: δ13C16:0 < δ13C18:2 < δ13C18:3 and δ13Carabinose > δ13Cxylose > δ13Cglucose > δ13Cgalactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.Mean δ13C values and ranges (min/max) of values averaged over three sampling dates for whole leaf tissue (BULK), leaf fatty acids C16:0, C18:2 and C18:3 and leaf monosaccharides arabinose, xylose, galactose and glucose from a range of plants from a cattle-grazed or deer-grazed field.

The remains of the Kwädąy Dän Ts'ìnchį individual, a frozen male human, were recovered from a retreating glacier within the Tatshenshini-Alsek Park in British Columbia in August 1999. In order to provide information on both the geographical origin of this individual and low long he spent in the remote interior region prior to his death, molecular analysis and compound-specific carbon isotope analyses were performed on individual amino acids purified from his skin and bone. Gas chromatographic quantification of constituent amino acids of both tissues revealed a molecular distribution characteristic of collagen, dominated by glycine and to a lesser extent proline, hydroxyproline and alanine. Chiral gas chromatography indicated that protein preservation in both tissues was exceptional. Carbon isotope analysis of a faunal assemblage from an earlier prehistoric site from southern British Columbia provided reference dietary amino acid δ13C values for terrestrial (deer and domestic dog) and marine species (salmon and sealion), showing clear separation in all amino acids, particularly glycine which was extremely 13C-enriched in the marine animals. The distinction between terrestrial and marine organisms was increased by exploring Δ13CGlycine-Phenylalanine values (6.6 ± 0.6‰ and 15.0 ± 2.1‰, respectively), which were higher in the latter by approximately 8‰, mirroring the increased δ15NBulk collagen values observed for the marine animals (R2 = 0.78; p < 0.001). The Kwädąy Dän Ts'ìnchį individual's bone had a similarly elevated Δ13CGlycine-Phenylalanine value of 15.6 ± 1.0‰, indicating his extreme reliance on marine dietary resources throughout early life. The skin amino acid δ13C values were consistently lower than those observed for bone, with a concurrently lower Δ13CGlycine-Phenylalanine value of 12.7 ± 0.9‰. The shift between the carbon isotope composition of bone (long-term diet) and skin amino acids (short-term diet) confirmed a sudden divergence away from marine food sources in the last months of life, consistent with his discovery 80 km inland.

The application of molecular approaches to palaeovegetation reconstruction in peat is still relatively rare, with molecular level studies of carbohydrate organic geochemistry being generally uncommon. In this report, neutral monosaccharides derived via acid hydrolysis were investigated in modern bog-forming plants in order to assess their potential application as biomarkers in peat palaeovegetation reconstruction. The concentrations of major neutral monosaccharides, i.e. glucose (Glu), xylose (Xyl), arabinose (Ara), galactose (Gal), mannose (Man), rhamnose (Rha) and fucose (Fuc), were determined using gas chromatography (GC) for 7 lichens, 10 Sphagnum species and 7 vascular plants collected from three ombrotrophic mires across northern Europe. Based on factor analysis of the modern plant monosaccharide compositions, two carbohydrate proxies: [(Man + Gal):(Ara + Xyl)] and % contributions of Rha and Fuc in total Glu-free monosaccharides [%(Rha + Fuc)], were selected as biomarkers for bog-forming plants. The three plant groups could be separated by [(Man + Gal):(Ara + Xyl)], which showed decreasing values following the order: lichens > Sphagna > vascular plants. The high [%(Rha + Fuc)] in Sphagna allowed their separation from lichens and vascular plants. These two factors were applied as plant group-specific indices to investigate vegetation change in a peat core from Kontolanrahka Bog, Finland. Our findings show strong correspondence with fossil plant abundances from the same core, thereby confirming the potential of carbohydrate compositional parameters as proxies for palaeovegetation reconstruction in peat bogs.

Gas chromatography (GC), GC-mass spectrometry (GC-MS) and GC-combustion-isotope ratio MS (GC-C-IRMS) analyses of absorbed and surface lipid residues preserved in potsherds were used to explore the extent of pig product processing exploitation in the later British Neolithic Grooved Ware tradition. Assessments were made regarding whether porcine lipids were associated with specific Grooved Ware traits, i.e. decoration, substyle, geographical area and type of site. Two hundred and twenty-two Grooved Ware potsherds were analysed, 70% of which contained lipid concentrations considered significant (>5 μg g−1). All the lipid residues were dominated by animal fats, although plant and beeswax were also detected in a small number of extracts. δ13C values of the major fatty acid components of degraded animal fats (C16:0 and C18:0) were determined for 126 extracts and used to assign ruminant or porcine origins to the residues; 16% of these were found to have a predominantly porcine isotope signature. Statistical associations with pig exploitation were shown to exist with substyle, geographical area and site type, whereas, no relationship was seen between decoration and the type of commodity processed. Intact triacylglycerols were preserved in 19% of the sherds; half of these had distributions consistent with the identifications based on δ13C values, the remainder differed either due to the presence of mixed commodities or because lower molecular weight homologues had been lost due to degradation. In addition to the detection of pig exploitation, results from lipid residue analysis showed a good correlation with faunal assemblages, suggesting that stable isotope analysis may be used as a proxy for animal exploitation at sites where bones have not survived.

The remains of the preserved ice body from the Kwädąy Dän Ts'ìnchį discovery were recovered from a retreating glacier in the Tatshenshini-Alsek Park, British Columbia in August 1999. Despite the remote location 80 km inland, bone collagen stable isotope analysis indicated that the individual spent much of his life in a region rich in marine foods (δ13C −13.7‰ and δ15N +17.9‰). Since finds of such bodies are exceptionally rare we undertook detailed lipid analyses in order to assess their preservation and determine whether they might provide new insights into the individual’s dietary life history. Molecular fingerprinting and compound-specific carbon isotope analysis were performed on individual lipids extracted from his bone (turnover approximately ≥1 year) and skin (turnover approximately several weeks). A considerable abundance was observed of C12:0, C14:0, C16:0, C16:1, C18:0 and C18:1 fatty acids (FAs), cholesterol and hydroxy FAs (the latter being decay products). Most unusual was the presence of long-chain hydroxy FAs (LCHFAs), 10- and 12-hydroxyeicosanoic acid and 10- and 12-hydroxydocosanoic acid, in the bone. The latter components are most likely the products of microbially mediated hydration of the double-bonds of C20:1 and C22:1 FAs, the latter almost certainly originating from the consumption of a largely marine-based diet. A suite of three isoprenoidal lipids, phytanic acid, pristanic acid and 4,8,12-trimethyltetradecanoic (TMTD) acid, was also detected supporting the notion of a significant marine component of the diet for a substantial part of his life. In contrast, the skin lipid composition was dominated by C16:0 FA, with lower abundances being observed of the marine LCHFAs and isoprenoidal compounds, suggesting reduced reliance on coastal marine foods in the last period of life. This interpretation is supported by the enhanced marine dietary signal observed in the bone than skin FA δ13C values.

The leaves of 37 grass, herb, shrub and tree species were collected from a mesotrophic grassland to assess natural variability in bulk, fatty acid and monosaccharide δ13C values of leaves from one plant community. The leaf tissue mean bulk δ13C value was −29.3‰. No significant differences between tissue bulk δ13C values with life form were determined (P = 0.40). On average, C16:0, C18:2 and C18:3 constituted 89% of leaf tissue total fatty acids, whose δ13C values were depleted compared to whole leaf tissues. A general interspecific (between different species) trend for fatty acids δ13C values was observed, i.e. δ13C16:0 < δ13C18:2 < δ13C18:3, although these values ranged widely between species, e.g. C16:0 (−34.7‰, Alisma plantago-aquatica; −44.0‰, Leucanthemum vulgare), C18:2 (−33.3‰, Acer campestre; −44.2‰, L. vulgare;) and C18:3 (−34.3‰, Bellis perennis; −41.8‰, Plantago lanceolata). Average relative abundances of leaf monosaccharides arabinose, xylose, mannose, galactose and glucose were 12%, 13%, 5%, 12% and 54%, respectively. Mean δ13C values of these monosaccharides were −26.6‰ (arabinose), −27.2‰ (xylose), −30.9‰ (mannose), −30.0‰ (galactose) and −29.0‰ (glucose). The general relationship between individual monosaccharide δ13C values, δ13Carabinose > δ13Cxylose > δ13Cglucose > δ13Cgalactose, was consistently observed. Therefore, we have shown (i) diversity in compound-specific δ13C values contributing to leaf bulk δ13C values; (ii) interspecific variability between bulk and compound-specific δ13C values of leaves of individual grassland species, and (iii) trends between individual fatty acid and monosaccharide δ13C values common to leaves of all species within one plant community.δ13C values were determined for methyl esters of fatty acids C16:0, C18:2 and C18:3, and alditol acetates of monosaccharides glucose, xylose, arabinose, galactose and mannose, from tree, shrub, herb and grass leaves of a mesotrophic grassland plant community, using GC–C–IRMS.

The domestication of cattle, sheep and goats had already taken place in the Near East by the eighth millennium bc1, 2, 3. Although there would have been considerable economic and nutritional gains from using these animals for their milk and other products from living animals—that is, traction and wool—the first clear evidence for these appears much later, from the late fifth and fourth millennia bc4, 5. Hence, the timing and region in which milking was first practised remain unknown. Organic residues preserved in archaeological pottery6, 7 have provided direct evidence for the use of milk in the fourth millennium in Britain7, 8, 9, and in the sixth millennium in eastern Europe10, based on the δ13C values of the major fatty acids of milk fat6, 7. Here we apply this approach to more than 2,200 pottery vessels from sites in the Near East and southeastern Europe dating from the fifth to the seventh millennia bc. We show that milk was in use by the seventh millennium; this is the earliest direct evidence to date. Milking was particularly important in northwestern Anatolia, pointing to regional differences linked with conditions more favourable to cattle compared to other regions, where sheep and goats were relatively common and milk use less important. The latter is supported by correlations between the fat type and animal bone evidence.

Man's use of illuminants in lamps or as torches to extend the working day and range of environments accessible to him would have been a major technological advance in human civilisation. The most obvious evidence for this in the archaeological record comes from pottery and stone vessels showing sooting due to the use of a wick in conjunction with a lipid-based fuel or illuminant. A wide range of potential fuels would have been exploited depending upon availability and burning requirements. Reported herein are the results of chemical investigations of a number of lamps recovered from excavations of the site of Qasr Ibrim, Egypt. Gas chromatographic, mass spectrometric and stable carbon isotopic analyses of both free (solvent extractable) and ‘bound’ (released from solvent extracted pottery by base treatment) lipids have revealed a wide range of saturated fatty acids, hydroxy fatty acids and α,ω-dicarboxylic acids. Examination of the distributions of compounds and comparisons with the fatty acid compositions of modern plant oils have allowed a range of fats and oils to be recognised. Specific illuminants identified include Brassicaceae (Cruciferae) seed oil (most likely radish oil, Raphanus sativus), castor oil (from Ricinus communis), animal fat, with less diagnostic distributions and δ13C values being consistent with low stearic acid plant oils, such as linseed (Linum usitatissimum) or sesame (Sesamum indicum) oils. The identifications of the various oils and fats are supported by parallel investigations of illuminant residues produced by burning various oils in replica pottery lamps. The findings are entirely consistent with the classical writers including Strabo, Pliny and Theophrastrus.

A range of spectroscopic, ‘wet’ chemical, gas chromatographic (GC) and mass spectrometric (MS) techniques was applied to the characterisation of three soil organic matter (SOM) fractions that have been proposed as the basis of a new SOM turnover model based on measurable, physically defined fractions. The fractions were: the free light fraction (obtained by density separation in NaI solution at a density of 1.80 g cm−3, without disruption of aggregates), the intra-aggregate light fraction (obtained using a second density separation after disrupting aggregates using ultrasonic dispersion) and the organomineral fraction corresponding to the residual heavy material. The techniques employed to investigate the composition of the organic constituents of each fraction were: 13C nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and pyrolysis-gas chromatography/mass spectrometry (py-GC/MS) to study bulk composition. Lipid, lignin and carbohydrate fractions were assessed using GC and GC/MS with appropriate derivatisation, following oxidative and hydrolytic treatments, respectively, in the case of the latter two classes. Proteinaceous components were determined as amino acids using reversed-phase high performance liquid chromatography (HPLC) following 6 M HCl treatment and derivatisation. Each technique revealed marked differences in chemical composition between the organomineral and the two light fractions, with the results being consistent with the organomineral fraction having different biological sources or having undergone a greater degree of degradation or transformation. Several techniques detected differences between the composition of the free light fraction and the intra-aggregate light fraction. With the exception of carbohydrate composition, the results were consistent with the order of reactivity previously proposed from incubation studies with isotopically labelled substrates, namely: free > intra-aggregate > organomineral. The investigation illustrates the importance of using a range of different chemical characterisation techniques in studies of complex SOM fractions as each has limitations that could, if used alone, produce ambiguous findings or fail to detect differences between them.

The evidence for dairying in antiquity has, until recently, primarily been restricted to the reconstruction of herd structures through the analysis of faunal remains. Using this method alone cannot provide definitive evidence for the presence of dairy herds, due to differences in the recovery of animal bones at sites and the many different farming strategies that can affect herd structures (e.g. dairying, meat production, traction etc.). Absorbed lipid residues have been extracted from 237 pottery vessels from the British Iron Age sites of Maiden Castle, Danebury Hillfort, Yarnton Cresswell Field and Stanwick. The compound-specific stable carbon isotope (δ13C values) of the principal fatty acids found in animal fats (C16:0 and C18:0) have allowed the direct detection of dairy fats, thus providing evidence that dairying was an important component of farming practices in the British Iron Age. The results are compared to assessments of the faunal remains at each of the sites, and correlations between morphological characteristics of the vessels (e.g. type, form, use wear and rim diameter) and lipid residue discussed.

Molecular and isotopic analyses were undertaken of absorbed lipid residues from 256 pottery vessels obtained from four southern British Bronze Age sites (Potterne, Brean Down, Black Patch and Trethellan Farm). The results confirm that not only were Ancient Britons utilising dairy products during this period, but also that they were processed in pottery vessels on a large scale. This has been demonstrated through the determination of the compound-specific stable isotope values of the principal fatty acids found in animal fats (C16:0 and C18:0) that allows ruminant dairy and ruminant/non-ruminant adipose fats to be distinguished. The proportion of sherds yielding degraded dairy fats at each of the sites is variable, with the highest occurrence being from Potterne, and the lowest occurrence being from Black Patch. The faunal remains, and vessel characteristics (e.g. rim diameter and vessel type) are compared with the organic residue analyses, and intra-site variability is investigated at Trethellan Farm.

Absorbed lipid residue analysis has previously demonstrated that dairying was a major component of animal husbandry in Britain during both the Iron Age and Bronze Age. As a continuation of this research into the antiquity of dairying, the incidence of dairy fats associated with pottery vessels from six Neolithic sites from Southern Britain is presented herein. A total of 438 potsherds from Windmill Hill, Abingdon Causewayed Enclosure, Hambledon Hill, Eton Rowing Lake, Runnymede Bridge and Yarnton Floodplain were submitted for organic residue analysis. To date, this constitutes the largest number of sherds investigated from one particular archaeological period. The compound-specific stable isotope values of the major fatty acid components in animal fats, namely C16:0 and C18:0, enable absorbed lipids in pottery vessels to be classified to commodity group, i.e. ruminant adipose, dairy and non-ruminant adipose fats can be distinguished. The lipid extracts were relatively well preserved, and dairy fats were observed in approximately 25% of all of the sherds, demonstrating that milk was a valued commodity in the British Neolithic. These results confirm that dairying was an established component of the agricultural practices that reached Britain in the 5th Millennium BC.

The application of bone collagen stable carbon and nitrogen isotope analysis to human palaeodietary reconstruction in tropical or arid regions is limited by two factors: (i) the overlap in C4 and high marine protein (HMP) consumer bulk collagen δ13C values, and (ii) the unpredictability of bulk collagen δ15N values in regions of extreme aridity (<400 mm of rain per annum). Hence, the identification of HMP consumption among archaeological human populations can be problematic. In an endeavour to identify a more precise marine palaeodietary indicator, a range of collagen samples from archaeological faunal and human bone (n=14n=14 and 26, respectively), representing a spectrum of C3, C4 and HMP diets, were selected from coastal and near-coastal sites in the Western Cape, South Africa. Samples were subjected to compound-specific stable carbon isotope analysis of their constituent amino acids as trifluoroacetyl-isopropyl (TFA-IP) esters via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). While human C4 and HMP consumers were indistinguishable with respect to bulk collagen δ13C values (−10.9±3.7‰ and −11.7±1.5‰, respectively) they were shown to be readily distinguished based on Δ13CGlycine-Phenylalanine values (+4.0±1.6‰ and +12.0±1.9‰, respectively). The relationship between HMP consumption and elevated Δ13CGlycine-Phenylalanine values was verified by: (i) the similarly elevated values exhibited by marine species when compared to terrestrial faunal species (+12.5±0.9‰ and +3.2±4.2‰, respectively), and (ii) the strong correlation observed between human Δ13CGlycine-Phenylalanine and bulk collagen δ15N values (R2=0.83R2=0.83, p<0.001p<0.001; n=26n=26), the latter being a well-documented marine dietary indicator. It was concluded that Δ13CGlycine-Phenylalanine values offer considerable potential as indicators of HMP consumption and a valuable substitute for bone collagen δ15N values in arid regions where bulk δ15N values are unpredictable.

Various biomolecular components preserved in domesticated animal bones recovered from the Nubian site of Qasr Ibrim are used for dietary reconstruction of their foddering and foraging behaviours. Utilising models of the biochemical correlations with the dietary components and their turnover rates, the bulk stable isotope values of bone collagen and apatite combined with compound-specific stable isotope values of the collagenous amino acids (essential and non-essential) provided long-term indicators of the diet of cattle and sheep/goats from the site. Cattle appear to have predominantly consumed C4 plants, such as sorghum (Sorghum bicolor bicolor Moench.) and millet (Panicum miliaceum L.), during the later periods at the site, suggesting that cattle were subjected to differing feeding strategies over the period of occupation of the site. Furthermore, the δ13C values of the individual fatty acids (n-hexadecanoic and -octadecanoic acids) preserved in the bones provide short-term indicators of the animals' diet. The application of a new model based on δ13C values of the bone apatite and fatty acids indicates differences in the long- and short-term diets of sheep/goat, which are less obvious in cattle.

Domesticated animals formed an important element of farming practices in prehistoric Britain, a fact revealed through the quantity and variety of animal bone typically found at archaeological sites. However, it is not known whether the ruminant animals were raised purely for their tissues (e.g., meat) or alternatively were exploited principally for their milk. Absorbed organic residues from pottery from 14 British prehistoric sites were investigated for evidence of the processing of dairy products. Our ability to detect dairy fats rests on the observation that the δ13C values of the C18:0 fatty acids in ruminant dairy fats are ≈2.3‰ lower than in ruminant adipose fats. This difference can be ascribed to (i) the inability of the mammary gland to biosynthesize C18:0; (ii) the biohydrogenation of dietary unsaturated fatty acids in the rumen; and (iii) differences (i.e., 8.1‰) in the δ13C values of the plant dietary fatty acids and carbohydrates. The lipids from a total of 958 archaeological pottery vessels were extracted, and the compound-specific δ13C values of preserved fatty acids (C16:0 and C18:0) were determined via gas chromatography-combustion-isotope ratio mass spectrometry. The results provide direct evidence for the exploitation of domesticated ruminant animals for dairy products at all Neolithic, Bronze Age, and Iron Age settlements in Britain. Most significantly, studies of pottery from a range of key early Neolithic sites confirmed that dairying was a widespread activity in this period and therefore probably well developed when farming was introduced into Britain in the fifth millennium B.C.

High performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI MS) was applied to the characterisation of triacylglycerols (TAGs) in animal fats. The major TAGs in four fats (beef, chicken, lamb and pork) were identified and positional isomers assigned according to their APCI mass spectra. Beef and lamb fat TAGs were confirmed as containing higher proportions of saturated fatty acids compared with those of chicken and pork. HPLC-APCI MS was also shown to be of value in providing regiospecific information for the fatty acids in individual TAG species. For example, beef and lamb fat were shown to contain both cis- and trans-isomers of the 18∶1 fatty acid, whilst chicken and pork contained only the cis-isomer. When the position of fatty acid substitution was determined from the APCI spectra, whilst the cis-18∶1 was predominantly found in the 2-position of the TAG, the trans-18∶1 showed a preference for the 1/3-position. Similarly, it was confirmed that although the 2-position of beef, chicken and lamb fat TAGs was dominated by unsaturated fatty acids, in pork fat, a characteristically high proportion of palmitic acid was seen in this position. The TAGs identified compared well with those reported previously. The distributions of 2-position fatty acids seen in lamb and pork fat compared favourably with those obtained by the more traditional method of lipase degradation. Although the distributions for chicken and beef showed some discrepancies, these can be attributed to weaknesses in the quantification procedure or the specificity of the lipase. Overall, the technique of HPLC-APCI MS has been shown to be very powerful for the regiospecific analysis of animal fats.

Although a labile molecule, chitin is resistant to decay when complexed with protein. Currently, qualitative evidence for the preservation of chitin rests upon characteristic marker compounds derived through pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) of fossil arthropod cuticles, supported by a non-specific carbohydrate assay. However, unambiguous confirmation of the survival of chitin polymer requires detection of its hydrolysate monomer, d-glucosamine. We have now developed a GC–MS selected ion monitoring (SIM) method for the identification and quantification of d-glucosamine in fossil materials. Fossils of various ages and depositional settings were investigated and the results compared with those obtained by the Py–GC–MS approach. Specimens from the Rancho La Brea Tar Pits (USA, Pleistocene), showed the greatest degree of preservation: ∼10% (w/w), while fossil insects from Willershausen (Germany, Pliocene) and St Bauzile (France, Miocene) showed chitin to be present in ∼5% (w/w). Fossils from the Oligocene at Enspel, Germany, revealed that more than 0.5% is preserved for 25 million years. The GC–MS–SIM technique confirms the survival of chitin in the fossil record through the explicit identification of the polysaccharide monomer, and supports earlier Py–GC–MS and colorimetric analyses. The presence of other amino sugars of either exogenous (microbial) or diagenetic origin in more ancient specimens was also readily revealed using the GC–MS–SIM approach. This study illustrates the value of using a high-specificity quantitative ‘wet’ chemical approach in combination with Py–GC–MS to further advance the investigation of chitin in the fossil record.

Lipid biomarker components of soils constituting three Orcadian archaeological fossil soil profiles were analysed. The combined assessment of lipid distributional and compound specific stable carbon isotope data enabled the identification of grass turves as the most probable material used in the formation of the anthropogenic soil deposits. Appraisal of 5β-stanol components indicated a faecal input to one of the soils which, on considering distributional evidence, was ascribed a human/porcine origin. Additional study of polar bile acids from this profile revealed a distribution exhibiting a predominance of deoxycholic acid indicating the primary faecal input to be mainly derived from humans although the minor occurrence of hyodeoxycholic acid, a characteristic component of pig faeces, attested to a limited porcine input.

Compound specific δ13C analyses were used to determine the relative input of a C4 temperate grass (Spartina anglica) to primary biomass in a salt-marsh sediment. Lipid distributions revealed a C32n-alkanol homologue as a characteristically dominant component of Spartina anglica whilst the cohabiting C3 species, Puccinellia maritima, exhibited a C26 maximum. The C32n-alkanol component was used to create an isotopic mixing model, between organic matter derived from Spartina anglica and Puccinellia maritima, to estimate their relative contribution to the primary biomass input of salt-marsh sediments. The application of sedimentary lipid isotope data to the model gave values of Spartina anglica contributions ranging from 37 to 100%. This investigation represents the first attempt to quantify inputs to sedimentary biomass based on compound specific stable carbon isotope techniques.

Stable nitrogen isotope analysis is a fundamental tool in assessing dietary preferences and trophic positions within contemporary and ancient ecosystems. In order to assess more fully the dietary contributions to human tissue isotope values, a greater understanding of the complex biochemical and physiological factors which underpin bulk collagen δ15N values is necessary. Determinations of δ15N values of the individual amino acids which constitute bone collagen are necessary to unravel these relationships, since different amino acids display different δ15N values according to their biosynthetic origins. A range of collagen isolates from archaeological faunal and human bone (n = 12 and 11, respectively), representing a spectrum of terrestrial and marine protein origins and diets, were selected from coastal and near-coastal sites at the south-western tip of Africa. The collagens were hydrolysed and δ15N values of their constituent amino acids determined as N-acetylmethyl esters (NACME) via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The analytical approach employed accounts for 56% of bone collagen nitrogen. Reconstruction of bulk bone collagen δ15N values reveals a 2‰ offset from bulk collagen δ15N values which is attributable to the δ15N value of the amino acids which cannot currently be determined by GC-C-IRMS, notably arginine which comprises 53% of the nitrogen unaccounted for (23% of the total nitrogen). The δ15N values of individual amino acids provide insights into both the contributions of various amino acids to the bulk δ15N value of collagen and the factors influencing trophic position and the nitrogen source at the base of the food web. The similarity in the δ15N values of alanine, glutamate, proline and hydroxyproline reflects the common origin of their amino groups from glutamate. The depletion in the δ15N value of threonine with increasing trophic level indicates a fundamental difference between the biosynthetic pathway of threonine and the other amino acids. The δ15N value of phenylalanine does not change significantly with trophic level, reflecting its conservative nature as an essential amino acid, and thus represents the isotopic composition of the nitrogen at the base of the food web. Δ15NGlu-Phe values in particular are shown to reflect trophic level nitrogen sources within a food web. In relation to the reconstruction of ancient human diet the contribution of marine and terrestrial protein are strongly reflected in Δ15NGlu-Phe values. Differences in nitrogen metabolism are also shown to have an influence upon individual amino acid δ15N values with Δ15NGlu-Phe values emphasising differences between the different physiological adaptations. The latter is demonstrated in tortoises, which can excrete nitrogen in the form of uric acid and urea and display negative Δ15NGlu-Phe values whereas those for marine and terrestrial mammals are positive. The findings amplify the potential advantages of compound-specific nitrogen isotope analysis in the study of nitrogen flow within food webs and in the reconstruction of past human diets.

Despite the significant achievements of organic residues analysis of archaeological pottery, the sometimes low lipid recovery and the need to process increasingly large collections of sherds to tackle important archaeological questions require the development of a more efficient and rapid extraction method. In this paper we present a novel methodology for the extraction of absorbed organic residues directly from crushed archaeological ceramic using acidified methanol (H2SO4–MeOH 2% v/v, 70 °C, 1 h). This new protocol was tested by: (i) verifying the recovery of organic residues from previously studied archaeological vessels from different geographical regions, exhibiting a range of different lipid distributions often found in archaeological pottery, and (ii) demonstrating enhanced recovery of organic residues from potsherds that did not yield appreciable lipids when using the widely applied chloroform–methanol extraction. The application of the direct acidified methanol extraction recovers higher concentrations of lipid residues together with simultaneous production of methyl esters of fatty acids, allowing extraction and methylation to be completed in 20% of the time compared to conventional solvent extraction and derivatisation for gas chromatography (GC), gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS).

A passive sampler (the polar organic chemical integrative sampler; POCIS) was assessed for its ability to sample natural estrogens (17β-estradiol, E2; estrone, E1 and estriol, E3) and the synthetic estrogen (17α-ethynylestradiol, EE2) in the outlet of a sewage treatment works over several weeks. The performance of the POCIS was investigated and optimised in the laboratory before field deployment with high recoveries (66–99%) were achieved for all estrogens. Moreover, it was shown that POCIS does not exhibit any preferential selectivity towards any of the target compounds. The sampling rates of E1, E2 and E3 were 0.018 ± 0.009, 0.025 ± 0.014 and 0.033 ± 0.019 L d−1, respectively. Following field deployments of 28 days in the discharge of a sewage works, POCIS was shown to enhance the sensitivity of estrogen detection, especially for E3, and provide time-weighted average (TWA) concentrations of E1, E2 and E3, ranging from undetectable to 12 ng L−1upstream of the outflow of a sewage treatment works, 13 to 91 ng L−1 at the outflow and 8 to 39 ng L−1downstream of the outflow. This revealed that E1, E2 and E3 are not completely removed during sewage treatment, with concentrations most likely being maintained by contributions from conjugated estrogen analogues. Grab water samples showed considerable variation in the concentrations of estrogens over a longer period (6 months). The results confirm that POCIS is an effective and non-discriminatory method for the detection of low concentrations of estrogens in the aquatic environment.