Fiorentino Et Al 2012 RCMS

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Studying ancient crop provenance implications from d13C and

d15N values of charred barley in a Middle Bronze Age silo at Ebla

(NW Syria)

Girolamo Fiorentino1 Valentina Caracuta1 Grazia Casiello2 Francesco Longobardi2 andAntonio Sacco2

1Laboratory of Archaeobotany and Palaeoecology University of Salento - Cultural Heritage Via D Birago 64 Lecce 73100 Italy2Department of Chemistry University of Bari Via Orabona 4 Bari 70126 Italy

The discovery of a storeroom full of barley and other cereals (L9512) in the proto-historic site of Ebla has provided aunique opportunity to study the centralized storage system of the early city-state from a different perspectiveEpigraphic evidence available within the site reveals a complex system of taxation which included gathering graintributes from satellite sites and redistributing semi-1047297nished products such as 1047298our In this paper we intend to explorethe possibilities of a combined approach to studying the storage system based on estimated barley grain volumes

and d

13

C-d15

N analyses This approach is used to distinguish between grain from different harvesting sites and toidentify any grain cultivated using special agricultural practices (eg manuring or irrigation) The basic assumptionfor this kind of analysis is that the growth-site conditions natural or anthropogenic of harvested cereals are re1047298ectedin their grain size and d

13C-d15N values Since the remains found in the storeroom were charred the 1047297rst task was toevaluate the effect of carbonization on the d

13C-d15N and the size of the grains Thus the effect of charring was testedon modern samples of Syrian barley landraces Once it had been ascertained that fresh grains reduced to charredremains retain their original biometric and isotopic traits the ancient material was examined Thirteen groups wereidenti1047297ed each characterized by a speci1047297c average volume and speci1047297c carbon and nitrogen values The analysisrevealed that what had 1047297rst appeared to be a homogeneous concentration of grain was in fact an assemblage of barleyharvested from different sites Copyright copy 2012 John Wiley amp Sons Ltd

Stable isotopes of nitrogen and carbon have been widelyused in archaeology to investigate the dietary habits of ancient populations[12] past environmental conditions[3] andagricultural practices[45] For the latter most of the analyseshave focused on irrigation[6] fertilization techniques and thestate of soils[7]

Given that these two isotopes have rarely been studied jointly in previous analyses in archaeological issues bycombining isotopic ratio data (d13C and d15N values) and sizeanalysis we intend to characterize growing-site conditionsandor identify agricultural practices (eg irrigation ormanuring) which can affect isotope values in plants

Biometric analysis helps to determine the origin of plant

remains grain size is affected by edaphic conditions duringgrowth[89] and therefore grains of the same species fromthe same harvesting site are likely to have the samevolume[10ndash12] Stable isotope values help to re1047297ne this analysis

by characterizing the environmental features of the harvestingsite[1314] In addition they can indicate additional water ornutrient inputs[1516]

In this study we selected 91 caryopses of barley from thearchaeobotanical assemblage found in storeroom L9512Wheat was the main stored cereal but barley was preferred

because it was expected to better re1047298ect growth siteconditions[1718]

Since the archaeobotanical assemblage in L9512 was madeup of charred remains the 1047297rst task was to evaluate the effectof carbonization on the isotope signature and the size of the

barley grains Thus the effect of charring was tested onmodern samples of Syrian barley landraces

Once it was ascertained that fresh grains reduced tocharred remains more or less retain their characteristic sizeand d13C-d15N values we plotted these parameters

together in order to characterize the different harvestingsites of the barley in the ancient material and to verifywhether further distinctions could be made in terms of agricultural practices

RESEARCH BACKGROUND

The main chemical components of cereals are primarilygoverned by the water and soil layer in which they growand can to some extent be in1047298uenced by human practicessuch as additional water inputs and manuring[19] Thereforeif used properly and correctly understood stable carbonand nitrogen isotopes can provide reliable information on

Correspondence to G Fiorentino Laboratory of Archaeo- botany and Palaeoecology University of Salento CulturalHeritage Via D Birago 64 Lecce 73100 ItalyE-mail girolamo1047297orentinounisalentoit

Copyright copy 2012 John Wiley amp Sons LtdRapid Commun Mass Spectrom 2012 26 327ndash335

Research Article

Received 29 July 2011 Revised 3 November 2011 Accepted 7 November 2011 Published online in Wiley Online Library

Rapid Commun Mass Spectrom 2012 26 327ndash335(wileyonlinelibrarycom) DOI 101002rcm5323



ancient plant water status[6] and plant growing conditions[20]

The great potential of these parameters has been partiallyexplored but some clari1047297cations are required beforederiving any valuable conclusions Previous studies havedemonstrated that d15N can be used as an indicator of thenutritional status of ancient crops since it re1047298ects external Nsources and the 15N14N fractionations which occur duringthe assimilation transport and loss of N[21]

The main N input is not easy to determine since the isotoperatio in plants can result from both N 1047297xation andatmospheric deposition However this should not be an issuein arid lands since it has been demonstrated that more than98 of the organic nitrogen pool is found in the soil whereit is 1047297xed by microrganisms[2223] Moreover in naturalN-limited systems uptake of N has to be very ef 1047297cientresulting in virtually all available inorganic N being takenup and therefore in no or negligible loss of N[2425]

Another factor which in1047298uences d15N values in plants is thetype of soil-N used as a parameter (organic N NH4

+ NO3ndash)

because ammonium tends to have higher d15N values thanNO3

ndash and plants taking up NH4+ will thus have higher d15N

values[26]

Mariotti et al[27]

reported that plants grown inN-de1047297cient soil have a higher capacity for uptake of NH4+

than NO3ndash and are thus richer in 15N

Plant d15N values depend not only on the d15N values of the source nitrogen but also on the balance between enzymedemand and external concentration[28] In an open-poolsystem a high external N concentration coupled with modestdemand can inhibit 15N fractionation[29] if the rate of Nuptake exceeds the assimilation differences between thed15N values of the plant and of the nitrogen in the soil[43]

Another factor that could directly affect stable nitrogenisotope ratios in cereals is manuring since the application of animal dung raises d15N values in soil and plants[30ndash34]

Analyzing the chemical composition of paleosoils could in

turn provide direct evidence of manure input[35ndash37] Unfortu-nately in arid and semi-arid regions the over-exploitation of the landscape makes it unlikely that such evidence ispreserved[38ndash40]

Observations made in the 1047297eld as well in the laboratorysuggest that external nitrogen concentration is not the onlyfactor that determines discrimination[4142] Indeed thetranslocation of nitrogen to the site of assimilation canaccount for intra-plant patterns in d15N values Signi1047297cantintra-plant variation can be observed when NO3

ndash is theprimary nitrogen source but little variation is observed whenNH4

+ is the source[43]

The use of the same plant components (eg kernels)

can help to reduce the effects of different nitrogen uptakemechanisms and different assimilation and recyclingpathways but does not take account of the effects of genotype on 15N discrimination[414244] Despite the manystudies dealing with the metabolic pattern in nitrogen inplants it is still hard to determine the d15N values of externalnitrogen sources and the physiological mechanisms withinthe plant which affect stable nitrogen isotope ratios [43]

Due to the large number of factors that may in1047298uencestable nitrogen isotope ratios d15N measurements are moreuseful when combined with d13C values since the latterretain a clearer source signature[44] Indeed the use of such isotopes has been shown to be of some importance inecological studies[45]

The long tradition of studies based on the use of stablecarbon isotopes has demonstrated the relationship betweend13C values in plants and local climate variables Numerousinvestigations have shown that the d13C values of plants arenegatively correlated with water input This negativecorrelation is more pronounced in arid environments thanin humid areas[46ndash49] A possible explanation lies in theassumption that water availability rather than other factors

is the key climatic limiting factor for growth and survival insemiarid areas[50] As a result variations in precipitationatmospheric humidity and soil water availability areassumed to account for changes in plant 13C concentrationin sub-arid areas[51]

Thus by combining stable carbon and nitrogen isotopeanalyses of charred grains of measured volume we canidentify differences between stored grain samples anddetermine whether they were harvested in different locationsunder Eblarsquos control and whether irrigation or manuring wasapplied to make up for local resource de1047297cits

THE CONTEXT OF THE STUDY

The early city-state of Ebla

The Ebla archaeological site with its long human historyfrom the 3rd to the 2nd millennium BC is one of the mostimportant early historical settlements in north-westernSyria[52] Situated in a sub-arid region but bordering a morehumid and fertile ecosystem on which it relied thesettlementrsquos survival strictly depended on annual rainfallwhich is nowadays around 450500 mm[53]

The hydrography of the area is not suitable for ancientirrigation systems In the surroundings of the site there areno big rivers which could provide additional water by

means of channels and no traces of subterranean tunnelsfor rainfall collection (qanat) have yet been found in thearea[54] The absence of any reference in the Ebla administra-tive texts to water management or controversies over thisissue suggests that agriculture in the area was based on arain-fed system

The overall pattern that emerges from the archaeologicalevidence and epigraphic sources points to an economy

based on the farming of state-owned 1047297elds and a taxationsystem which provided enough foodstuffs to support the

bureaucracy and the army

The storage system

Eblarsquos in1047298uence extended all across north-western Syria Rawmaterials were imported from distant locations whilefoodstuffs came from productive areas nearby Cereals wereharvested by local peasant communities based in numeroussatellite settlements around Ebla which paid a tribute to thepalace in the form of produce The palace ground the cerealsand re-distributed them as rations of 1047298our or bread to thepalace workers[55]

The storeroom (L9512) sited in the B-east quarter can beconsidered a place where foodstuffs were stored before beingprocessed The architectural characteristics of L9512 showthat it was not sealed[56] so grain could not be stored for longperiods[57]

G Fiorentino et al

wileyonlinelibrarycomjournalrcm Copyright copy 2012 John Wiley amp Sons Ltd Rapid Commun Mass Spectrom 2012 26 327ndash335



EXPERIMENTAL

Grain analysis

Fresh material

Modern samples of barley ( Hordeum vulgare ssp vulgare and H vulgare ssp distichon) from 21 sites in Syria were selectedfrom the ICARDA (International Centre for AgriculturalResearch in Dry Areas Aleppo Syria) landrace seed banka

The selection of sites was designed to maximise environmen-tal variability[58] while maintaining similar land managementto that of ancient crops Irrigated and fertilized 1047297elds thuswere avoided (Fig 1)

Each collection site was characterized by caryopses withhomogeneous dimensional traits in order to determine theaverage volume of each population the thickness length andwidth of 1047297ve fresh grains were measured They were thenreduced to a 1047297ne powder using an agate pestle and mortarand analyzed by isotope ratio mass spectrometry (IRMS)

The effect of charring was also tested on modern samples inorder to verify their comparability with the ancient charred

remains The charring experiments involved placing a cruciblefull of sand with 5 grains of modern air-dried barley wrappedin foil in a hot oven at 250C for 12 h in an anaerobicatmosphere These conditions were chosen on the basis of previous studies which have shown that they provide theclosest replication of ancient charring conditions[5] Thesamples were then re-measured before being reduced to a 1047297nepowder using an agate pestle and mortar and subjected toIRMS analysis

Archaeological material

Charred grains of ancient barley were recovered from the1047298

oor of storeroom L9512 in the Ebla site during the 2005ndash

2006 campaigns The storeroom (L9512) was a rectangularroom of around 5 m2 Stratigraphical observations identi1047297edthree layers (0-1-2) of charred caryopses separated from eachother by debris

Since it was hypothesised that tributes of grain from differ-ent 1047297elds were stored together we intended to identify corre-spondences between their spatial and vertical distributionand isotopic-volume signature Specimens (5 L) were thustaken every 50 cm2 six samples were taken from each layermaking 18 in total Once the samples had been obtained astandard 1047298otation tank with a 05 mm sieve was used in the1047297eld to separate plant remains from other remains After-wards wet samples were dried slowly before being stored

In the upper layer (Level 0) as well as in the bottom layer(Level 2) caryopses were recovered in 1047297ve out of six sampleswhile in the intermediate layer (Level 1) only three of the sixyielded caryopses Thus only thirteen specimens containedcereals

Seven caryopses of barley were randomly selectedfrom each sample and their morphological features analyzedin detail by microscopy (Nikon SMZ645 Nikon InstrumentsTokyo Japan) The thickness length and width of the

grains were also measured and the average volumeestimated assimilating the barley caryopses to an ellipsoidas follows

L W Teth THORN=2frac12 4=3p (1)

The grain weight was estimated by using equationsderived from the regression models of Ferrio et al[59]

021 L Weth THORNand16 (2)

078 L Teth THORNand128 (3)

Carbonate was removed from the fossil grains by soakingeach grain separately in 6 M HCl for 24 h at room tempera-ture and then rinsing the grain repeatedly with distilledwater[60] Finally the 91 caryopses were divided into 13groups on the basis of their position in the storeroom andground to a 1047297ne powder

Principal component analysis (PCA) was used to identifythe presence of clusters among the variables (volume andd13C and d15N values) and to obtain information about the

importance of the variables

Stable isotope analysis

Sample preparation and isotope measurements

The isotope signature of fresh material was determined for 21samples selected from ICARDA varieties before and aftercharring treatment making a total of 42 samples For archae-ological measurements seven charred caryopses from each of the thirteen samples were analyzed Approximately 05 mgof each sample was placed in tin capsules for measurementThe d13C and d15N values were measured by continuous1047298ow elemental analysis isotope ratio mass spectrometry

(CF-EA-IRMS) using a Delta VAdvantage mass spectrometer(Thermo Fisher Scienti1047297c Bremen Germany) interfaced witha FlashEA 1112 HT elemental analyzer (Thermo FisherScienti1047297c) (oxidationreduction reactor temperature 1050Cand gas chromatography column temperature 50C) TheCF-EA-IRMS system was operated in dual isotope mode allow-ing d13C and d15N values to be measured in the same sample

Standards and equations

The results of the carbon (d13C) and nitrogen (d15N) isotoperatio analyses were reported as per mil () relative toV-PDB (Vienna-Pee Dee Belemnite) and Atmospheric Airrespectively after calculation in accordance with the follow-

ing equation

d frac14 RSample RReference

=RReference

1000

where R is the ratio of the heavy to light stable isotopes in thesample (RSample) and the standard (RReference)

Each sample was analyzed twice and the values werecompared with those of reference gases (N2 and CO2)previously calibrated against International Standards speci-1047297ed by the International Atomic Energy Agency (IAEAVienna Austria) (IAEA-NO3 for d15N values and IAEA-CH6

ndash NBS 22 for d13C values) For each run at least one in-housecasein standard was analyzed at regular intervals to check theaccuracy of measurements in case of drift deviations

aSamples were not multiplied in ICARDA but harvesteddirectly from 1047297elds spreading across the Syrian territories

Studying ancient crop provenance

wileyonlinelibrarycomjournalrcmCopyright copy 2012 John Wiley amp Sons LtdRapid Commun Mass Spectrom 2012 26 327ndash335



Statistical analysis

Different statistical analyses were performed in order to (i)ascertain the effect of charring on grain size and the d13C andd15N values of modern plant material (ii) establish the relation-ship between d13C values and grain size and the carbon andnitrogen stable isotope values in the ancient material and (iii)discriminate groups among the ancient stored cereals

The 1047297rst task was achieved by applying linear regression toestimate the correlation between fresh and charred volumeand fresh and charred d13C values and to predict change in

the d15

N values after charring Then the degree of correlation between d13C values and size and the nitrogen and carbonstable isotope ratios in the ancient material was determined

by logarithm regressionsThe last task (iii) was achieved by applying statistical

analysis on the archaeobotanical data and was performedusing Statistica 80 (StatSoft Italia srl Padova Italy) PCAenabled us to visualize the presence of clusters of samplesand to obtain information about the importance of variables(volume and d13C and d15N values) in these groupings Thevariables were scaled by using auto-scaling method ie thevariable mean was subtracted from each variable and theneach variable was divided by its standard deviation

Analysis of variance (ANOVA) provided the statistical signif-

icance of each group Unless otherwise stated differences wereconsidered statistically signi1047297cant when P lt005

RESULTS

Estimation of volume and isotope signature in fresh material

The results of the analyses carried out on the 21 ICARDAvarieties are shown in Table 1 The volume measurementsreveal signi1047297cant differences between different areas of SyriaWith an average value of 37 mm3 the volume ranged from 25mm3 for the samples collected at Baovar-Dar rsquoa to 55 mm3 forthose gathered at Quadir-Homs

Previous studies have shown that the carbon isotopesignature which is strongly dependent on local climateconditions varies from site to site but is fairly consistentacross sites with similar ecological conditions The averaged13C value is about 245 with a variation of ~15ndash20

between the samples from Naher Markia-Tartous and thosecollected at Kawkab-Hama

The variation in d15N values of the order of ~5 wasgreater because the d15N value primarily re1047298ects the isotopesignature of the N source which is not easy to determineand discrimination processes which occur during N uptake

assimilation and redistribution within the plant

The effect of charring

The charring experiments revealed that controlled carboniza-tion determines an increase in caryopsis volume Since this isfairly constant for all specimens the volume of the charred

barley was closely related to that of the fresh material(r2 = 059 with linear correlation) The estimated grain weightsshowed a lower degree of correlation than the volumer2 = 053 Eqn (2) r2 = 043 Eqn (3) (results not shown)

Our results are in agreement with previous studies carried outon barley such as those quoted by Renfrew[12] who reported

that slight increases in the length and breadth of barley grainshave a limited effect on the shape of each single grainThe effect of charring on the d13C and d15N values of grains

has been widely explored (Fig 2) Aguilera et al[4] reportedthat d13C values in cereal grains are not signi1047297cantly affected

by charring within the temperature range normally expected toinduce carbonization and sample preservation (ca 200ndash400C)and that the original d13C environmental signal is retained incharred grains Heaton etal[5] also found that differences in char-ring conditions and duration had little in1047298uence on d13C values

In this study we found that no changes occurred in the d13Cvalues as a result of carbonization Indeed the correlation

between fresh and charred values was r2 = 087 with linearcorrelation (results not shown)

Figure 1 Geological map of Syria and the site-sampling distribution Numbersrefer to sampling sites listed in Table 1

G Fiorentino et al






Regarding the effects of charring on d15N values Bogaardet al[7] reported no signi1047297cant changes in d15N values forgrain carbonized at 230C for up to 24 h The work of DeNiro

and Hastorf [60]

on the d15

N values of charred seeds and tubersfrom Peru showed that charring did not affect the nitrogenisotope signature of ancient plant remains The experimentscarried out on Spanish wheat and barley by Aguilera et al[4]

revealed that although signi1047297cant changes in element concen-tration (N) occur during carbonization the d15N value is notaltered by carbonization and environmental signals are wellpreserved in charred grains

In the present study we found slight increases in d15Nvalues which were fairly constant across all samples(r2 = 063 with linear correlation) (results not shown)

The Ebla silo

Size and isotope analyses of archaeobotanical remainsThe volume of the caryopses collected in the storeroom wasdetermined and differences were found among the samplesfrom the thirteen squares For each of these thirteen samplesconsidered the average value of seven grains was calculated(Table 2)

The average volumes ranged from 606 mm3 for sample 1to 106 mm3 for sample 6 Intra-sample variability (expressed

by the standard deviation) was quite low compared withthe variability between samples This is consistent with thehypothesis that the storeroom contained grain harvestedfrom different sites

It is noteworthy that the volume of each sample seems to bedirectly related to the d13C values (Fig 3) (r2 = 069) The

stable carbon isotope d13C values ranged from 217 to233 and the stable nitrogen isotope d15N values from31 to 80 Notably the d15N values are all positive whichmeans the nitrogen sources themselves had positivevalues[61]

The d13Cand d15N values are inversely correlated with lowd13C values corresponding to high d15N values and viceversa (Fig 4) (r2 = 051) After applying PCA to the datatwo PCs were extracted The variance explained by PC1 andPC2 was 749 and 204 respectively The analysisidenti1047297ed three major groupings a b and c (Fig 5) thestatistical signi1047297cances of volume d13C value and d15N valueare respectively P = 0007 P = 002 and P = 002

DISCUSSION

The experiment performed on modern samples shows thatcharring does not alter the general trend of either isotopesignatures or volumes It can thus be assumed that the d13Cand d15N values and the size of the carbonized caryopsesprovide information about plant growth environments anddistinguish between crops grown under different climateconditions or by different farming methods

With the ancient material the correlation between thevolume and the d13C value of the Ebla silo caryopses isconsistent with the hypothesis that grain size depends mainly

on water input during grain 1047297lling In contrast the volumeand stable nitrogen isotope ratios are negatively correlatedMoreover previous experiments carried out on barleygrown in controlled environments show that d15N usuallyincreases in response to drought while it decreases underN-starvation[21]

Figure 2 Correlation between the fresh and charredcaryopses volume of modern specimens

Table 2 Summary of volume and d13C and d15N values of the ancient barley found in the three layers of the silo L9512

Layer Group SampleAverage volume (mm3)

(SD)d13C ()

(SD)d15N ()

(SD)

0 1 H vulgare 606 (13) 2171 (008) 800 (001)2 H vulgare 1426 (21) 2280 (010) 517 (021)3 H vulgare 6688 (54) 2297 (014) 666 (020)4 H vulgare 8360 (76) 2266 (012) 637 (001)5 H vulgare 6583 (50) 2312 (009) 457 (009)

1 6 H vulgare 10659 (96) 2333 (005) 315 (040)7 H vulgare 1481 (19) 2193 (005) 595 (013)8 H vulgare 6270 (48) 2299 (004) 334 (031)


SD standard deviation

G Fiorentino et al




According to Robinson et al[62] the genotypes which aremost productive and stress tolerant are those which whenunder stress preferably restrict the loss of N from their rootsto ammonium (NH4

+) which is richer in 15N while retainingorganic nitrogen Van der Leij et al[63] also found thatNO3ndash exudation from the roots of N-starved plants isnegligible Conversely genotypes characterized by morelimited discrimination against 15N were smaller and had

higher d15N values These may have less capacity to restrictorganic-N loss from their roots when under stress than morestress-tolerant genotypes[62]

These experiments provide an interpretative basis forour analyses indeed the period in which the grain wasstored (around 1800 BC) corresponds on the basis of various proxies to a drought crisis[64ndash66] Therefore theinverse correlation between the d13C and d15N values in

our ancient samples can be interpreted as the response of barley to environmental stress brought about by waterscarcity[67] On the other hand we cannot exclude thepossibility that manure was added by farmers in responseto water scarcity causing the d15N values to increasedespite the low d13C values

However we cannot exclude the possibility that differencesin d15N values can depend on the genotype Indeed previousexperiments carried out on 28 genotypes of Hordeumspontaneum showed that variation in whole-plant d15N valuescan reach 15[21]

Variability in terms of volume (P = 0007) d13C value(P = 002) and d15N value (P = 002) of barley is consistent

with the hypothesis that the cereals were harvested fromdifferent 1047297elds The possibility that the variability was dueto temporal differences in harvesting can be excluded thesince the administrative texts record the annual storagepractices[68]

CONCLUSIONS

The stable isotope measurements and size analyses of grainfound in a storeroom open up new perspectives for researchinto the provenance andor storage manners of ancientfoodstuffs This new archaeobotanical approach supports

the hypothesis that isotope values and grain size mostlyre1047298ect environmental conditions andor agriculturalpractices Further studies are required to disentangle theeffects of agricultural practices (additional water inputmanuring) from those of local climate conditions

Nonetheless the study of silo L9512 sheds light on the roleof Ebla as the centre of a complex rural system where rawmaterials harvested from different sites were stored together

before being ground and redistributed as rations of 1047298ourThanks to the innovative approach adopted in this studythe contents of the silo apparently homogenous were shownto be made up of grain from sites characterized by differentgrowing conditions

Acknowledgements

The authors are grateful to Ian Walkoun and ICARDAG_Bank for providing the modern crop material from SyriaThe study was partially funded by the IAM-CIHEAMInternational Cooperation Project called rsquoRationalization of irrigation systems in Ras al Ain ndash Syriarsquo Special thanks toPaolo Matthiae and Luca Peyronel for allowing the study of the archaeobotanical material from Ebla and Fabio Mavellifor statistical discussions The useful comments andsuggestions of two anonymous referees helped us to improvethe original text

Figure 4 Correlation between d13C and d15N values of caryopses found in the silo

Figure 5 PCA analysis with the three groups highlighted

Figure 3 Correlation between grain size and d13C value of archaeobotanical remains





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[8] H V Harlan Barley Culture Uses and Varieties USDepartment of Agriculture Farmersrsquo Bulletin n1464 1932

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[14] A Sacco M A Brescia A Sgaramella D Sacco Character-ization of the composition and the geographical origin of food products by means of nuclear magnetic resonanceand isotope ratio mass spectrometry Recent Res Develop

Agric Food Chem 2005 6 119[15] J Comadran J R Russell et al Mapping adaptation of

barley to droughted environments Euphytica 2008 161 35

[16] P H Beatty Y Anbessa P Juskiw R T Carroll J Wang AG Good Nitrogen use ef 1047297ciencies of spring barley grownunder varying nitrogen conditions in the 1047297eld and growthchamber Ann Bot 2010 105 1171

[17] S Ceccarelli S Grando Selection environment and environ-mental sensitivity in barley Euphytica 1991 57 157

[18] A Jilal S Grando R J Henry L S Lee N Rice H HillM Baum S Ceccarelli Genetic diversity of ICARDArsquosworldwide barley landrace collection Gen Resour CropEvol 2008 55 1221

[19] S Branch S Burke P Evans B Fairman C S L Wolff Briche A preliminary study in determining the geographi-cal origin of wheat using isotope ratio inductively coupledplasma mass spectrometry with 13C 15N mass spectrometry

J Anal At Spectrom 2003 18 17

[20] J Voltas I Romagosa J L Araus Grain size and nitrogenaccumulation in sink-reduced barley under Mediterraneanconditions Field Crops Res 1997 52 117

[21] D Robinson L L Handley C M Scrimgeour D C GordonB P Forster R P Ellis Using stable isotope naturalabundances (d15N and d13C) to integrate the stress responsesof wild barley ( Hordeum spontaneum C Koch) genotypes

J Exp Bot 2000 51 41[22] N E West J Skujins Nitrogen cycle in North American

cold-winter semi-desert ecosystems Oecol Plant 1977 1245

[23] W T Peterjohn W H Schlesinger Factors controllingdenitri1047297cation in a Chihuahuan desert ecosystem Soil SciSoc Am J 1991 55 1694

[24] K J Nadelhoffer B Fry Nitrogen isotope studies in forestecosystems in Stable Isotopes in Ecology and EnvironmentalScience (Eds K Lajtha R Michener) Blackwell Scienti1047297cPublications Boston USA 1994 p 23

[25] R D Evans A J Bloom S S Sukrapanna J R EhleringerNitrogen isotope composition of tomato (Lycopersiconesculentum Mill cv T-5) grown under ammonium or nitratenutrition Plant Cell Environ 1996 19 1317

[26] P Houmlgberg Tansley d15N natural abundance in soil-plant

systems Review No 95 New Phytol 1997 137 179[27] A Mariotti F Mariotti N Amarger G Pizelle J M NgambiM L Champigny A Moyse Fractionnement isotopiquesde lrsquoazote lors des processus drsquoabsorption des nitrates et de1047297xation de lrsquoazote atmospheacuterique par les plants Phys Veg1980 18 163

[28] A D M Glass J E Shaff L V Kochian Studies of theuptake of nitrate in barley Plant Physiol 1992 99 456

[29] R Bol J Eriksen P Smith M H Garnett K Coleman B TChristensenThe natural abundance of 13C 15N 34S and 14Cin archived (1923ndash1996) plant and soil samples from theAskov long-term experiments on animal manure andmineral fertiliser Rapid Commun Mass Spectrom 2005 193216

[30] C M Cossani G A Slafer R Savin Co-limitation of

nitrogen and water and yield and resource-use ef 1047297cienciesof wheat and barley Crop Pasture Sci 2010 61 844[31] T Yoneyama K Kouno J Yazaki Variation of natural 15N

abundance of crops and soils in Japan with special referenceto the effect of soil conditions and fertilizer application SoilSci Plant Nutr 1990 36 667

[32] G H Wagner Stable Isotopes in Plant Nutrition Soil Fertilityand Environmental Studies International Atomic EnergyAgency Vienna 1991

[33] I A Simpson R Bol I D Bull R P Evershed K J PetzeCompound-speci1047297c stable isotope signals in anthropogenicsoils as indicators of early land management RapidCommun Mass Spectrom 1999 13 1315

[34] W J Choi S M Lee H M Ro K C Kinigravem S H YooNatural 15N abundances of maize and soil amended with

urea and composted pig manure Plant Soil 2002 245 223[35] M G Canti An investigation of microscopic calcareous

spherulites from herbivore dungs J Arch Sci 1997 24 219[36] R P Evershed P H Bethell P J Reynolds N J Walsh

5b-Stigmastanol and related 5b-stanols as biomarkers of manuring analysis of modern experimental material andassessment of the archaeological potential J Arch Sci1997 24 485

[37] E B A Guttman Midden cultivation in prehistoric Britainarable crops in gardens World Arch 2005 37 224

[38] R G Amundson The use of stable isotopes in assessingthe effect of agriculture on arid and semi-arid soils inStable Isotopes in Ecological Research (Eds P W Rundel

J R Ehleringer K A Nagy) Ecological Studies 68 Springer-Verlag New York 1988 p 318

G Fiorentino et al




[39] R D Evans J R Ehleringer A break in the nitrogen cycle inarid lands Evidence from 15N of soils Oecologia 1993 94 314

[40] R Shahack-Gross A Simons S H Ambrose Identi1047297cationof pastoral sites using stable nitrogen and carbon isotopesfrom bulk sediment samples a case study in modern andarchaeological pastoral settlements in Kenya J Arch Sci2008 35 983

[41] A Mariotti Meacutemoire des Science de la Terre Universiteacute P etM Curie Paris 1982

[42] A Mariotti F Mariotti M L Champigny N AmargerA Moyse Reductase activity and uptake of NO3

by pearlmillet Plant Phys 1982 69 880

[43] R D Evans Physiological mechanisms in1047298uencing plantnitrogen isotope composition Trends Plant Sci 2001 6 121

[44] L L Handley J A Raven The use of natural abundance of nitrogen isotopes in plant physiology and ecology Plant CellEnviron 1992 15 965

[45] H Grif 1047297ths Applications of stable isotope technology inphysiological ecology Funct Ecol 1991 5 254

[46] R L Korol M U F Kirschbaum G D FarquharM Jeffreys Effects of water status and soil fertility on theC-isotope signature in Pinus radiata Tree Phys 1999 19 551

[47] J M Miller R J Williams G D Farquhar Carbon isotope

discrimination by a sequence of Eucalyptus species along asub-continental rainfall gradient in Australia Funct Ecol2001 15 222

[48] P K Van de Water S Leavitt J L Betancourt Leaf d13Cvariability with elevation slope aspect and precipitation insouthwest United States Oecologia 2002 132 332

[49] S Chen Y Bai G Lin X Han Variations in life-formcomposition and foliar carbon isotope discriminationamong eight plant communities under different soilmoisture conditions in the Xilin River Basin InnerMongolia China Ecol Res 2005 20 167

[50] R Nemani C D Keeling H Hashimoto W M JollyS C Piper C J Tucker R B Myneni S W Running Climate-driven increases in global terrestrial net primary productionfrom 1982 to 1999 Science 2003 300 1560

[51] J R Ehleringer Physiological processes in aridland plantsin Stable Isotopes in Ecological Research (Eds PW Rundel JR Ehleringer K A Nagy) Ecological Studies 68 Springer-Verlag New York 1988 p 41

[52] P Matthiae Ebla Un impero ritrovato Dai primi scavi alleultime scoperte Einaudi Torino 1989

[53] M Zohary Geobotanical Foundations of the Middle East FisherVerlag Stuttgart 1973

[54] D R Lightfoot Syrian Qanat Romani History EcologyAbandonment J Arid Environ 1996 33 321

[55] L Milano Barley for ratio and barley for sowing (ARET II51 and related matters) Acta Sum 1987 9 177

[56] L Peyronel Domestic quarters refuse pits and workingareas Reconstructing human landscape and environmentat Tell Mardikh-Ebla during the old Syrian period (c 2000ndash

1600 BC) in Proc 4th Int Congress of the Archaeology of the Ancient Near East vol 1 (Eds H Kuumlhne RM Czichon F J Kreppner) 2008 p 177

[57] Les Techniques de Conservation des Grains agrave Long Terme (EdsM Gast F Sigaut) Centre Nationale de la RechercheScienti1047297que Paris 1979

[58] Diversity in Barley (Hordeum vulgare) (Eds R von BothmerT Van Hintum H Knuumlpffer K Sato) Elsevier Science BVAmsterdam 2003

[59] J P Ferrio N Alonso J Voltas J L Araus Estimating grainweight in archaeological cereal crops a quantitativeapproach for comparison with current conditions J ArchSci 2004 31 1635

[60] M J DeNiro C A Hastorf Alteration of 15N14N and13C12C ratios of plant matter during the initial stages of diagenesis Studies utilizing archaeological specimens fromPerugrave Geochim Cosmochim Acta 1985 49 97

[61] J Bort J L Araus H Hazzam S Grando S CeccarelliRelationships between early vigor grain yield leaf structureand stable isotope composition in 1047297eld grown barley Planta

Phys Biochem 1998 38 889[62] D Robinson L L Handley C M Scrimgeour A theory for15N14N fractionation in nitrate-grown vascular plantsPlanta 1998 205 397

[63] M Van der Leij S J Smith A J Miller Remobilization of vacuole stored nitrate in barley root cells Planta 1998 20564

[64] N H Dalfes G Kukla H Weiss Third Millennium BCClimate Change and Old World Collapse NATO ASI Seriesvol 49 1993

[65] S Riehl R Bryson K Pustovoytov Changing growingconditions for crops during the Near Eastern Bronze Age(3000ndash1200 BC) the stable carbon isotope evidence J ArchSci 2007 20 1

[66] N Roberts W J Eastwood C Kuzucuoğlu G Fiorentino

V Caracuta Climatic vegetation and cultural change inthe eastern Mediterranean during the mid-Holocene envir-onmental transition Holocene 2011 21 147

[67] L L Handley D Robinson B P Forster R P EllisC M Scrimgeour D C Gordon E Nevo J A Raven Shootd15N correlates with genotype and salt stress in barley PlantaRapid Commun 1997 201 100

[68] R Dolce Some aspects of the primary economic structuresof Ebla in the Third and Second Millenium BC stores andworking places in Akten der Internationalen TagungWirtschaft und Gesellschaft von Ebla (Eds H WaetzoldtH Hauptmann) Heidelberg 1988 p 35





ancient plant water status[6] and plant growing conditions[20]

The great potential of these parameters has been partiallyexplored but some clari1047297cations are required beforederiving any valuable conclusions Previous studies havedemonstrated that d15N can be used as an indicator of thenutritional status of ancient crops since it re1047298ects external Nsources and the 15N14N fractionations which occur duringthe assimilation transport and loss of N[21]

The main N input is not easy to determine since the isotoperatio in plants can result from both N 1047297xation andatmospheric deposition However this should not be an issuein arid lands since it has been demonstrated that more than98 of the organic nitrogen pool is found in the soil whereit is 1047297xed by microrganisms[2223] Moreover in naturalN-limited systems uptake of N has to be very ef 1047297cientresulting in virtually all available inorganic N being takenup and therefore in no or negligible loss of N[2425]

Another factor which in1047298uences d15N values in plants is thetype of soil-N used as a parameter (organic N NH4

+ NO3ndash)

because ammonium tends to have higher d15N values thanNO3

ndash and plants taking up NH4+ will thus have higher d15N

values[26]

Mariotti et al[27]

reported that plants grown inN-de1047297cient soil have a higher capacity for uptake of NH4+

than NO3ndash and are thus richer in 15N

Plant d15N values depend not only on the d15N values of the source nitrogen but also on the balance between enzymedemand and external concentration[28] In an open-poolsystem a high external N concentration coupled with modestdemand can inhibit 15N fractionation[29] if the rate of Nuptake exceeds the assimilation differences between thed15N values of the plant and of the nitrogen in the soil[43]

Another factor that could directly affect stable nitrogenisotope ratios in cereals is manuring since the application of animal dung raises d15N values in soil and plants[30ndash34]

Analyzing the chemical composition of paleosoils could in

turn provide direct evidence of manure input[35ndash37] Unfortu-nately in arid and semi-arid regions the over-exploitation of the landscape makes it unlikely that such evidence ispreserved[38ndash40]

Observations made in the 1047297eld as well in the laboratorysuggest that external nitrogen concentration is not the onlyfactor that determines discrimination[4142] Indeed thetranslocation of nitrogen to the site of assimilation canaccount for intra-plant patterns in d15N values Signi1047297cantintra-plant variation can be observed when NO3

ndash is theprimary nitrogen source but little variation is observed whenNH4

+ is the source[43]

The use of the same plant components (eg kernels)

can help to reduce the effects of different nitrogen uptakemechanisms and different assimilation and recyclingpathways but does not take account of the effects of genotype on 15N discrimination[414244] Despite the manystudies dealing with the metabolic pattern in nitrogen inplants it is still hard to determine the d15N values of externalnitrogen sources and the physiological mechanisms withinthe plant which affect stable nitrogen isotope ratios [43]

Due to the large number of factors that may in1047298uencestable nitrogen isotope ratios d15N measurements are moreuseful when combined with d13C values since the latterretain a clearer source signature[44] Indeed the use of such isotopes has been shown to be of some importance inecological studies[45]

The long tradition of studies based on the use of stablecarbon isotopes has demonstrated the relationship betweend13C values in plants and local climate variables Numerousinvestigations have shown that the d13C values of plants arenegatively correlated with water input This negativecorrelation is more pronounced in arid environments thanin humid areas[46ndash49] A possible explanation lies in theassumption that water availability rather than other factors

is the key climatic limiting factor for growth and survival insemiarid areas[50] As a result variations in precipitationatmospheric humidity and soil water availability areassumed to account for changes in plant 13C concentrationin sub-arid areas[51]

Thus by combining stable carbon and nitrogen isotopeanalyses of charred grains of measured volume we canidentify differences between stored grain samples anddetermine whether they were harvested in different locationsunder Eblarsquos control and whether irrigation or manuring wasapplied to make up for local resource de1047297cits

THE CONTEXT OF THE STUDY

The early city-state of Ebla

The Ebla archaeological site with its long human historyfrom the 3rd to the 2nd millennium BC is one of the mostimportant early historical settlements in north-westernSyria[52] Situated in a sub-arid region but bordering a morehumid and fertile ecosystem on which it relied thesettlementrsquos survival strictly depended on annual rainfallwhich is nowadays around 450500 mm[53]

The hydrography of the area is not suitable for ancientirrigation systems In the surroundings of the site there areno big rivers which could provide additional water by

means of channels and no traces of subterranean tunnelsfor rainfall collection (qanat) have yet been found in thearea[54] The absence of any reference in the Ebla administra-tive texts to water management or controversies over thisissue suggests that agriculture in the area was based on arain-fed system

The overall pattern that emerges from the archaeologicalevidence and epigraphic sources points to an economy

based on the farming of state-owned 1047297elds and a taxationsystem which provided enough foodstuffs to support the

bureaucracy and the army

The storage system

Eblarsquos in1047298uence extended all across north-western Syria Rawmaterials were imported from distant locations whilefoodstuffs came from productive areas nearby Cereals wereharvested by local peasant communities based in numeroussatellite settlements around Ebla which paid a tribute to thepalace in the form of produce The palace ground the cerealsand re-distributed them as rations of 1047298our or bread to thepalace workers[55]

The storeroom (L9512) sited in the B-east quarter can beconsidered a place where foodstuffs were stored before beingprocessed The architectural characteristics of L9512 showthat it was not sealed[56] so grain could not be stored for longperiods[57]

G Fiorentino et al




EXPERIMENTAL

Grain analysis

Fresh material



























ing equation


=RReference

1000












the d15






RESULTS

















G Fiorentino et al







and Hastorf [60]

on the d15




The Ebla silo







DISCUSSION







(SD)d13C ()

(SD)d15N ()

(SD)





G Fiorentino et al












CONCLUSIONS





Acknowledgements









REFERENCES













































G Fiorentino et al










































EXPERIMENTAL

Grain analysis

Fresh material



























ing equation


=RReference

1000












the d15






RESULTS

















G Fiorentino et al







and Hastorf [60]

on the d15




The Ebla silo







DISCUSSION







(SD)d13C ()

(SD)d15N ()

(SD)





G Fiorentino et al












CONCLUSIONS





Acknowledgements









REFERENCES













































G Fiorentino et al













































the d15






RESULTS

















G Fiorentino et al







and Hastorf [60]

on the d15




The Ebla silo







DISCUSSION







(SD)d13C ()

(SD)d15N ()

(SD)





G Fiorentino et al












CONCLUSIONS





Acknowledgements









REFERENCES













































G Fiorentino et al













































and Hastorf [60]

on the d15




The Ebla silo







DISCUSSION







(SD)d13C ()

(SD)d15N ()

(SD)





G Fiorentino et al












CONCLUSIONS





Acknowledgements









REFERENCES













































G Fiorentino et al


















































CONCLUSIONS





Acknowledgements









REFERENCES













































G Fiorentino et al










































REFERENCES













































G Fiorentino et al








































Fiorentino Et Al 2012 RCMS

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