Nitrogen balance and delta15N: why you're not what you eat during pregnancy

Carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios were longitudinally measured in human hair that reflected the period from pre-conception to delivery in 10 pregnant women. There was no significant change in the delta13C results, but all subjects showed a decrease in delta15N values (-0.3 to -1.1 per thousand) during gestation. The mechanisms causing this decrease in hair delta15N have not been fully elucidated. However, since the delta15N values of dietary nitrogen and urea nitrogen are significantly lower compared to maternal tissues, it is hypothesized that the increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy resulted in a reduction of the steady state diet to a body trophic level effect by approximately 0.5-1 per thousand. An inverse correlation (R2 = 0.67) between hair delta15N and weight gain was also found, suggesting that positive nitrogen balance results in a reduction of delta15N values independent of diet. These results indicate that delta15N measurements have the ability to monitor not only dietary inputs, but also the nitrogen balance of an organism. A potential application of this technique is the detection of fertility patterns in modern and ancient species that have tissues that linearly record stable isotope ratios through time.     

Spatial distributions of carbon, nitrogen and sulfur isotope ratios in human hair across the central United States

We present data on the carbon (δ(13)C), nitrogen (δ(15)N) and sulfur (δ(34)S) isotope ratios of human hair collected in the central portions of the USA. These elements are incorporated into hair from the diet and thus provide a record of dietary inputs that may also document geospatial patterns. We detected regional differences in hair δ(34)S values across the USA, with the lowest values in the northern Great Plains and increasing values towards the east, west and south. In contrast, no statistically significant patterns were detected in the spatial variation of human hair δ(13)C and δ(15)N values. Using δ(34)S values and a Geographic Information System approach, we created a map ('sulfur isoscape'). The accuracy of the map was tested using hair samples not included in its generation. We conclude that sulfur isotope analysis may represent a new tool to investigate the movements and/or region-of-origin of humans.     

Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa

Stable nitrogen and carbon isotopic ratios of hair strands of six patients suffering from anorexia nervosa were measured to monitor a dietary change from near starvation to recovery. This paper presents the results of a first-time study of nitrogen and carbon balance of the patients prior to and after admittance to a hospital and therapy. Sequential analysis of the isotopic ratios of hair strands of all patients could be related to the respective body mass index (BMI) of each patient. Our hypothesis concerning the diachronic change in delta15N and delta13C during therapy was met: The delta15N values were inversely related to the BMI, indicating a slow-down in catabolism of bodily protein due to the process of gluconeogenesis during the starvation phase. In contrast, the delta13C values and BMI were in phase: an increase in BMI resulted in an increase in the delta13C values. This rise in delta13C ratios is best interpreted by an increased supply of protein in the diet. Furthermore, delta15N and delta13C were inversely related. We conclude that hair, which is easily and non-traumatically sampled, is an adequate monitor that reflects dietary change and nitrogen balance within days. This isotopic method may also be applied in forensic studies with regard to cases of deprivation, and starvation, and may be a method for investigating starvation in historic populations.     

Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients

The stable-isotopic composition of nitrogen (delta15N) or carbon (delta13C) of body tissues depends on the isotopic composition of food sources and on shifts due to isotopic fractionation during metabolism. As little is known about the effects of pathophysiological conditions we measured delta15N and delta13C values in hair and hair amino acids of patients with cirrhosis (n = 21) and compared the results with those of healthy subjects (n = 100) randomly selected from the 1987-1988 VERA German nutrition survey population. Cirrhosis was reflected in lower hair 15N abundances (6.7 vs. 9.9 per thousand delta15N; P < 0.001) whereas hair 13C abundances did not differ from healthy subjects (-19.4 vs. -19.6 per thousand 13C). Distinct patterns of delta15N and delta13C values were measured in hair amino acids. The delta15N values of phenylalanine were significantly higher in cirrhotics (P < 0.001). With the exception of isoleucine, threonine, and proline all other measured amino acids showed lower delta15N values than healthy subjects (P < 0.001). Lower hair delta15N values were associated with cirrhotic liver disease which suggests that under this condition the altered liver amino acid metabolism affects the nitrogen isotopic composition of the amino acids used for hair protein synthesis. It remains to be determined in controlled studies whether the altered nitrogen isotopic composition directly reflects the pathophysiological condition or is related to differences in dietary protein intake from plant or animal food sources.     

Multiple stable isotope (18O, 13C, 15N and 34S) analysis of human hair to identify the recent migrants in a rural community in SW England

Relationships between recent migration and hair delta(18)O values were examined for 40 people living in a rural community in SW England. The isotopic contents of 35 'local' hair samples were compared with those of 5 recently arrived individuals (from Australia, Canada, Chile, Germany and the USA). The hair delta(18)O values of these 'visitors' were +7.9 (Omaha, USA), +11.2 (Jena, Germany), +12.1 (Osorno, Chile), +12.6 (Montreal, Canada) and +14.3 per thousand (Adelaide, Australia). The hair value for the USA visitor (+7.9 per thousand) fell outside the range for the 33 local adult residents, +10.5 to +14.3 per thousand (+12.7 +/- 0.8 per thousand). Hair delta(18)O values did not identify the individuals from Adelaide, Montreal and Osorno as 'visitors', but hair delta(13)C or delta(34)S data did. Combining the hair delta(18)O, delta(13)C and delta(34)S values using principal components analysis (two components explained 89% of the overall variation among the 40 subjects) helped to more clearly distinguish European from non-European individuals, indicating the existence of global overall isotope (geo-origin) relationships.     

Stable isotope variation in wool as a means to establish Turkish carpet provenance

The problem of establishing the provenance of carpets of artistic and historical importance is well known. We have addressed this by investigating whether there is sufficient geographical variation in the stable isotopes in wool (namely C, N and S) between key areas of Turkey to be able to recognize the different regions where carpets were made. Here we report results from modern wool samples taken from the winter growth of sheep in 2003/2004 from 13 carpet-producing sites. Although each site has a characteristic composition, most sites cannot be distinguished from each other, and the overall isotopic pattern is unexpectedly complicated. Thus in Western Turkey there is no sign of sea-spray effects in the delta34S values for sites close (10 km) to the sea, while, in the Konya Basin (Central Turkey), the delta34S values vary significantly between nearby sites. Two 'urban' settlements where sheep are now raised have dramatically higher delta15N values. It is nevertheless possible that certain production centers may have distinct signatures, and further work will compare carpets from known sources with the currently produced wool values. The results also provide additional insight into the natural variation found in archaeological faunal isotopic values, e.g. in bone collagen.     

Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance

Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods.     

The natural abundance of 13C, 15N, 34S and 14C in archived (1923-2000) plant and soil samples from the Askov long-term experiments on animal manure and mineral fertilizer

The Askov field experiment (Denmark), established in 1894, provides a unique opportunity to examine long-term effects of animal manure and mineral fertilizer on soil organic matter quality and turnover. This sandy loam soil is classified as Alfisol (Typic Hapludalf). Soil C, N, S, 13C, 15N, 34S and 14C contents were measured in a selection of archived soil samples (1923, 1938, 1945, 1953, 1964, 1976, 1985, 1996 and 2000) from unfertilized (O), animal manure (1 AM) and mineral fertilizer (1 NPK) treatments. These treatments are imbedded in a four-course crop rotation of winter cereals, root crops, spring cereals and a clover/grass mixture. The contents of C, N, S, 13C, 15N and 34S in selected crop samples (1953-1996) and in contemporary samples of animal feed and manure were also determined. Temporal soil nutrient and isotope trends between fertilizer treatments were significantly different, except for S content in 1 AM and 1 NPK. The total soil C and S was higher in 1 AM and 1 NPK than in the O treatment. The total soil N content (1 AM>1 NPK>O) and the delta15N content (1 AM>1 NPK and O) were also different. Analyses of plant, animal feed and manures confirmed that differences in soil 15N values were related to delta15N values of added source inputs. Soil and crop delta13C values were similar, but manures had slightly lower values. The variation of soil delta34S (and total S) from 1923 to 1996 was larger in the O than 1 AM and 1 NPK plots reflecting changes in atmospheric S inputs. The total contents of soil C, N and S were significantly correlated, but their isotopic signatures were not, suggesting that the C, N, S turnovers in soil are subject to different controls. The 14C content was generally higher in the 1 AM than 1 NPK and O, with bomb-14C incorporation modelling indicating that mean residence time (MRT) was ca. 170 years in the 1 AM, but closer to 250-290 years in the 1 NPK and O treatments. The measured trends in soil C and 14C during 1923-1996 were successfully modelled using the RothC model. The OM accumulation in the Askov soils was generally dominated by microbial decomposition products rather than by recalcitrant components of the various inputs.     

Carbon isotope analysis of bulk keratin and single amino acids from British and North American hair

The reconstruction of ancient diets using isotopic measurements of bone collagen, and other tissues, which survive in archaeological contexts, relies on known isotopic relationships between diet and body tissues. Examination of these relationships often requires the study of modern human and animal subjects. While hair keratin can act as a useful proxy for bone collagen in isotopic studies on living humans, where it is inappropriate to sample tissues such as collagen, it can, in addition, act as a chronological indicator of dietary change. This study investigates hair keratin delta13C values from current residents of the UK and the USA. Residents in the USA showed a clear bulk hair delta13C enrichment of approximately 3 per thousand over UK individuals, attributed to an elevated C4 dietary input from maize fed to livestock in North America. The keratin delta13C of subjects who moved between the UK and USA showed a pronounced change after relocation, taking approximately 4 months to reach isotopic equilibrium. To investigate these differences further, we measured delta13C values of dispensable and indispensable amino acids as a group, and selected individual amino acids. As a group, enrichment of dispensable amino acids compared with indispensable amino acids occurred in samples from both continents, averaging 7.2 per thousand in the UK and 7.9 per thousand in the USA. Dispensable and indispensable amino acids, as well as all individual amino acids measured, were enriched in samples from the USA compared with those from the UK.     

Long-term influence of manure and mineral nitrogen applications on plant and soil 15N and 13C values from the Broadbalk Wheat Experiment

The Broadbalk Wheat Experiment at Rothamsted Research in the UK provides a unique opportunity to investigate the long-term impacts of environmental change and agronomic practices on plants and soils. We examined the influence of manure and mineral fertiliser applications on temporal trends in the stable N ((15)N) and C ((13)C) isotopes of wheat collected during 1968-1979 and 1996-2005, and of soil collected in 1966 and 2000. The soil delta(15)N values in 1966 and 2000 were higher in manure than the mineral N supplied soil; the latter had similar or higher delta(15)N values than non-fertilised soil. The straw delta(15)N values significantly decreased in all N treatments during 1968 to 1979, but not for 1996-2005. The straw delta(15)N values decreased under the highest mineral N supply (192 kg N ha(-1) year(-1)) by 3 per thousand from 1968 to 1979. Mineral N supply significantly increased to straw delta(13)C values in dry years, but not in wet years. Significant correlations existed between wheat straw delta(13)C values with cumulative rainfall (March to June). The cultivar Hereward (grown 1996-2005) was less affected by changes in environmental conditions (i.e. water stress and fertiliser regime) than Cappelle Desprez (1968-1979). We conclude that, in addition to fertiliser type and application rates, water stress and, importantly, plant variety influenced plant delta(13)C and delta(15)N values. Hence, water stress and differential variety response should be considered in plant studies using plant delta(13)C and delta(15)N trends to delineate past or recent environmental or agronomic changes.     

Detection of royal jelly adulteration using carbon and nitrogen stable isotope ratio analysis

Stable isotope ratios ((13)C/(12)C and (15)N/(14)N) were measured in royal jelly (RJ) samples by isotope ratio mass spectrometry (IRMS) to evaluate authenticity and adulteration. Carbon and nitrogen isotope contents (given as delta values relative to a standard, delta(13)C, delta(15)N) of RJ samples from various European origins and samples from commercial sources were analyzed. Uniform delta(13)C values from -26.7 to -24.9 per thousand were observed for authentic RJ from European origins. Values of delta(15)N ranged from -1.1 to 5.8 per thousand depending on the plant sources of nectars and pollen. High delta(13)C values of several commercial RJ samples from -20.8 to -13.3 per thousand indicated adulteration with high fructose corn syrup (HFCS) as a sugar source. Use of biotechnologically produced yeast powder as protein source for the adulterated samples was assumed as delta(15)N values were lower, as described for C(4) or CAM plant sources. RJ samples from authentic and from adulterated production were distinguished. The rapid and reliable method is suitable for urgent actual requirements in food monitoring.     

Nitrogen balance and delta15N: why you're not what you eat during nutritional stress

While past experiments on animals, birds, fish, and insects have shown changes in stable isotope ratios due to nutritional stress, there has been little research on this topic in humans. To address this issue, a small pilot study was conducted. Hair samples from eight pregnant women who experienced nutritional stress associated with the nausea and vomiting of morning sickness (hyperemesis gravidarum) were measured for carbon (delta13C) and nitrogen (delta15N) stable isotope ratios. The delta13C results showed no change during morning sickness or pregnancy when compared with pre-pregnancy values. In contrast, the delta15N values generally increased during periods of weight loss and/or restricted weight gain associated with morning sickness. With weight gain and recovery from nutritional stress, the hair delta15N values displayed a decreasing trend over the course of gestation towards birth. This study illustrates how delta15N values are not only affected by diet, but also by the nitrogen balance of an individual. Potential applications of this research include the development of diagnostic techniques for tracking eating disorders, disease states, and nitrogen balance in archaeological, medical, and forensic cases.     

The use of stable isotope ratio analyses to discriminate wild and farmed gilthead sea bream (Sparus aurata)

Continuous flow isotope ratio mass spectrometry (CF-IRMS) has been used to analyze samples of gilthead sea bream (g.s.b.) (Sparus aurata) of known geographical origin (four countries on the Mediterranean Sea) from wild and farmed sources (40 farmed and 10 wild). delta(13)C and delta(15)N values have been measured on muscle samples as these are the most informative parameters of the diet of the animals. Both stable isotopes are indicators of the origin of the fish: delta(13)C giving a tool to distinguish between wild and farmed g.s.b. and delta(15)N being more informative on the geographical origin of the fish (this fact could be related more to differences in feed mixtures given to farmed fish than to geographical reasons). The proposed methodology offers a cost- and time-effective alternative to other analytical techniques in identifying wild and farmed fish.     

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.     

Effect of temperature, ration, body size and age on sulphur isotope fractionation in fish

Sulphur isotope analysis (delta(34)S) is increasingly identified as a valuable tool for source differentiation and the determination of trophic level in food webs, but there are still many uncertainties associated with the interpretation of delta(34)S data. To investigate the effects of temperature, ration, body size and age on sulphur trophic fractionation (Deltadelta(34)S) in fish, we reared European sea bass (Dicentrarchus labrax) on identical diets at 11 and 16 degrees C at three ration levels for over 600 days. Deltadelta(34)S was between 0 and -1 per thousand. The effect of temperature on Deltadelta(34)S was small and inconsistent, varying over the course of the experiment and depending on ration. This contrasts with temperature effects on bass Deltadelta(13)C and Deltadelta(15)N, where Deltadelta(13)C increases at warm temperatures while Deltadelta(15)N falls. Body size and age had a positive relationship with Deltadelta(34)S but the relationship with size was not significant for bass that weighed >20 g. As Deltadelta(34)S is small and the range in delta(34)S of potential diet items can be much greater than the range in delta(13)C or delta(15)N, our results show that sulphur stable isotopes are particularly useful for source differentiation in fish.     

Alteration of the carbon and nitrogen stable isotope composition of beef by substitution of grass silage with maize silage

This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (delta13C) and nitrogen (delta15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative delta13C values (P<0.001) in lipid-free muscle and in lipid, and also a lower delta15N (P<0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the delta13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0 per thousand shift of delta13C in lipid-free muscle and a 1.0 to 1.2 per thousand in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n=15) in this experiment were about 0.5 per thousand and 1.0 per thousand for delta13C of lipid-free muscle and lipid, respectively, and about 0.5 per thousand for delta15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production.     

Stable carbon isotope analysis of different tissues of beef animals in relation to their diet

As part of a larger experiment, 31 young bulls, divided into three groups, were given different diets containing either C(3) plants or a combination of C(3) and C(4) plant-based feeds in three feeding periods before slaughter. Variation in the proportion of C(4) plant material in the diets was made by including or not maize or maize-derived ingredients, whereas the other dietary constituents were from C(3) plants. Analysis of stable carbon isotope ratios (delta(13)C value) was performed on different tissues taken at slaughter: blood, plasma, liver, kidney fat, hair, muscle and ruminal contents. Blood and plasma samples were also taken at the beginning of each period. A highly significant difference was found in the delta(13)C values of blood and plasma samples taken from animals that had received a diet of only C(3) plants or with 59% C(4) material for 70 days. The delta(13)C values of all different samples taken at slaughter were highly significantly different between the three feeding groups that had received diets with 0, 13.5 or 35% C(4) material for on average 137, 139 and 83 days, respectively. For the three groups, samples of hair, muscle, plasma, whole blood and liver were significantly enriched in (13)C compared with the diet (except for liver in one group), whereas kidney fat was significantly depleted. The proportion of C(4) plant material could be accurately estimated from the delta(13)C values of different tissue samples. Stable carbon analysis of different tissues from beef animals can be used to trace back diets containing variable proportions of C(3) and C(4) plant material.     

A comparison of carbon and nitrogen stable isotope ratios of fish tissues following lipid extractions with non-polar and traditional chloroform/methanol solvent systems

Stable isotope ratios act as chemical tracers of animal diet, and are used to study food web dynamics. Because carbon stable isotope values are influenced by tissue lipid content, a number of extraction methods have been used to remove lipid bias, but, in some species and tissues, extractions also alter nitrogen isotope values. We have analyzed delta(13)C and delta(15)N in Atlantic bluefin tuna liver and white muscle, and whole Atlantic herring, fish tissues covering a wide range of lipid content (bulk C:N 3.1-12.5). In order to compare delta(13)C and delta(15)N values from traditional chloroform/methanol extractions with non-polar solvent alternatives, we analyzed samples following (1) no treatment, (2) lipid removal using chloroform/methanol (2:1), and (3) Soxhlet extractions using chloroform, diethyl ether or hexane. Chloroform/methanol and chloroform extractions produced the lowest C:N values and highest delta(13)C values. In bluefin tuna, chloroform and hexane extractions significantly altered liver delta(15)N, and all methods significantly altered delta(15)N values in white muscle. Whole Atlantic herring delta(15)N was not altered by any extraction method, while the 2:1 chloroform/methanol extraction most completely removed fish tissue lipid components. Our results indicate that delta(15)N effects are not limited to common chloroform/methanol extractions and suggest that chloroform/methanol is the most effective extraction for delta(13)C correction. Given evidence for delta(15)N alteration among all tested methods, mathematical correction approaches should be further explored as an alternative to lipid correction.     

The effect of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment

We investigated the effects of sample acidification on the stable carbon and nitrogen isotopic composition (delta13C and delta15N), as well as the organic carbon (OC) and total nitrogen (TN) composition, of an algal culture and a marine sediment. Replicate measurements of untreated and acid-treated samples were made using 1 M, 2 M and 6 M HCl, 6% H2SO3 and 1 M H3PO4. For all treatments the precision of the analysis for the acid-treated sample was equal to or less than that in the non-acidified sample. For the algae, analysis of variance (ANOVA) indicated no significant differences in the mean OC and TN concentration, or delta13C and delta15N composition, between any acid treatment and non-acidified samples. For the sediment sample a comparison could only be made between the different acid treatments because the untreated contained significant amounts ( approximately 30%) of carbonate carbon. ANOVA indicated that the mean OC determined in sediment samples after the 1 M HCl treatment and the mean delta13C values after the 6% H2SO3 and 1 M H3PO4 treatments were significantly different (p < 0.013 and < .05, respectively) from all other treatments. Mass balance calculations indicate that in some instances delta13C values were biased due to a contribution from unreacted carbonate carbon. There were no significant differences in the mean TN between any acid-treated and non-acidified samples. The mean delta15N values after 6 M HCl, 6% H2SO3 and 1 M H3PO4 treatments were significantly different from the untreated sediment sample (p < 0.044). Based on the significant bias observed for the delta15N and delta13C values, a weak (1-2 M) HCl solution is confirmed as the most appropriate acid for the removal of inorganic carbon from natural materials requiring elemental and isotopic analysis.     

Stable carbon and nitrogen isotopes and quality traits of fossil cereal grains provide clues on sustainability at the beginnings of Mediterranean agriculture

We present a novel approach to study the sustainability of ancient Mediterranean agriculture that combines the measurement of carbon isotope discrimination (Delta(13)C) and nitrogen isotope composition (delta(15)N) along with the assessment of quality traits in fossil cereal grains. Charred grains of naked wheat and barley were recovered in Los Castillejos, an archaeological site in SE Spain, with a continuous occupation of ca. 1500 years starting soon after the origin of agriculture (ca. 4000 BCE) in the region. Crop water status and yield were estimated from Delta(13)C and soil fertility and management practices were assessed from the delta(15)N and N content of grains. The original grain weight was inferred from grain dimensions and grain N content was assessed after correcting N concentration for the effect of carbonisation. Estimated water conditions (i.e. rainfall) during crop growth remained constant for the entire period. However, the grain size and grain yield decreased progressively during the first millennium after the onset of agriculture, regardless of the species, with only a slight recovery afterwards. Minimum delta(15)N values and grain N content were also recorded in the later periods of site occupation. Our results indicate a progressive loss of soil fertility, even when the amount of precipitation remained steady, thereby indicating the unsustainable nature of early agriculture at this site in the Western Mediterranean Basin. In addition, several findings suggest that barley and wheat were cultivated separately, the former being restricted to marginal areas, coinciding with an increased focus on wheat cultivation.