Multidimensional natal isotopic niches reflect migratory patterns in birds.

Naturally occurring stable isotope ratios in animal tissues allow estimation of species trophic position and ecological niche. Measuring multiple isotopes of migratory species along flyway bottlenecks offers the opportunity to sample multiple populations and species whose tissues carry information at continental scales. We measured δ2H, δ18O, δ13C, δ15N in juvenile feathers of 21 bird species captured at a migratory bottleneck in the Italian Alps. We examined if trends in individual isotopes reflected known migratory strategies and whether dietary (δ13C-δ15N) and spatially-explicit breeding origin (δ2H-δ18O) niche breadth (NB) differed among long-distance trans-Saharan (TS), short-distance (IP) and irruptive (IR) intra-Palearctic migrants, and whether they correlated with reported populations long-term trends. In both TS and IP groups, species δ2H declined with capture date, indicating that northern populations reached the stopover site later in the season, following a Type-I migration strategy. Values of δ2H indicated that breeding range of TS migrants extended farther north than IP and IR migrants. The breeding season was longer for IP migrants whose δ13C and δ15N values declined and increased, respectively, with time of capture. Average species dietary NB did not differ among migratory groups, but TS migrants displayed wider breeding origin niches, suggesting that long-distant migration is linked to broader ecological niches. Isotope origin NB well reflected species geographic range extent, while dietary NB did not correlate with literature accounts of species' diet. We found no relationship between species breeding NB and population trends in Europe, suggesting that conditions in the breeding grounds, as inferred by stable isotopes, are not the only determinant of species' long-term persistence. We demonstrate that ringing activities and isotopic measurements of passerines migrating through a bottleneck represents a unique opportunity to investigate large-scale life-history phenomena relevant to conservation.

Gas Chromatography Combustion Isotope Ratio Mass Spectrometry for Improving the Detection of Authenticity of Grape Must.

Since ancient times, grape must and wine have been considered one of the most sophisticated matrices and, in the last few years, the continuous rise in volumes and prices of grapes and wine has encouraged fraud and adulteration in the oenological field. One of the most common adulterations is sugar addition to grape must in the form of cane or beet sugar or syrup coming from vegetable sources, such as cereals or fruits. Since 1990, the International Organisation of Vine and Wine (OIV) has issued specific official isotopic methods to fight against this practice, but they are not always effective. With the aim to develop a new method able to identify sugar addition, we compared the δ13C value of sugar extracted from grape must analyzed by elemental analyzer/isotope ratio mass spectrometry (EA-IRMS) to the δ13C value of proline analyzed by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS), after extraction and derivatization. δ13C and δ15N of proline have also been tested as potential geographical markers. In addition, the carbon isotopic composition of two characteristic grape must sugars (myo- and scyllo-inositols) was measured by GC-C-IRMS, after derivatization, to identify the illegal correction of their concentration. On the basis of the obtained results we can conclude that the compound-specific isotope analysis represents a novel analytical tool to support and improve certification and control procedures.

Stable isotope ratio analysis of different European raspberries, blackberries, blueberries, currants and strawberries.

To date the stable isotope ratios of berries have never been extensively explored. In this work the H, C, N and O isotopic ratios of 190 samples of different soft fruits (strawberries, raspberries, blueberries, blackberries and currants) produced in a northern Italian region and at two sites in Romania and Poland collected over three harvest years are presented and discussed. The different soft fruits showed a typical range for one or more isotopic parameters that can be used to verify the authenticity of the fruit composition declared on the label. The δ13C and δ15N of pulp and the δ18O of juice can be considered effective tools for identifying the different geographical origin of fruit. A significant effect of crop cover on juice δ18O and fertilisation practices on pulp δ15N was demonstrated and must be considered with attention when evaluating data.

Multi-isotopic signatures of organic and conventional Italian pasta along the production chain.

The variability of stable isotope ratios (δ(2) H, δ(13) C, δ(15) N, δ(18) O and δ(34) S) along the production chain of pasta (durum wheat, flour and pasta) produced by using both conventional and organic farming systems in four Italian regions in 2 years was investigated. The aim was to evaluate if and how the farming system and geographical origin affect stable isotope ratios determined along the production chain. Irrespective of the processing technology, 65% of the samples were correctly classified according to the farming system and 98% were correctly classified regarding the geographical region. When considering both farming system and geographical region simultaneously, 80% of the samples were correctly classified. The measured isotope parameters were thus primarily affected by the geographical origin. In conclusion, it is expected that the use of these parameters will allow the development of analytical control procedures that can be used to check the geographical origin of Italian organic and conventional pasta and its raw materials. Copyright © 2016 John Wiley & Sons, Ltd.

Variations in stable isotope ratios in lamb blood fractions following dietary changes: a preliminary study.

RATIONALE: In meat production it is common practice to use finishing diets based on concentrates, even for those animals previously raised on pasture. No studies have investigated the variations of stable isotope ratios in lamb plasma and erythrocytes, following a switch from pasture to a concentrate-based diet during the last days before slaughter. For meat traceability it is important to verify how and whether these parameters are affected in blood fractions. METHODS: Blood of ten male Italian Merino lambs, whose diet was switched from pasture to concentrate, was sampled 8 times (days 1, 2, 3, 4, 5, 6, 7 and 14) in the last 14 days before slaughter. The variations in the δ(13)C, δ(15)N, δ(18)O and δ(34)S values of blood plasma and erythrocytes were investigated. The stable isotope ratios of the samples were measured using a stable isotope mass spectrometer coupled with an elemental analyser (C, N, S) and a CO2/H2O equilibration system (O(plasma)). RESULTS: The δ(13) C(plasma), δ(18)O(plasma) and δ(34)S(plasma) values were shown to be different 7 days after the abrupt variation in the diet. The comparison between erythrocytes and plasma stable isotope ratios could be suitable for verifying whether the animal was actually pasture-raised and could merit a higher price. The erythrocytes isotopic signature was not affected by very short finishing periods in previously grazed animals and maintained the pasture-raised fingerprint, while the analysis of plasma could detect very short finishing periods with concentrate and hay. CONCLUSIONS: The present study has demonstrated that the combination of blood plasma and erythrocytes stable isotope ratios of carbon, nitrogen, oxygen and sulphur can be used to infer the dietary background of lambs and thus offers a tool for the authentication of the animal production system.

Discrimination of geographical origin of lentils (Lens culinaris Medik.) using isotope ratio mass spectrometry combined with chemometrics.

The aim of this study was to predict the geographic origin of lentils by using isotope ratio mass spectrometry (IRMS) in combination with chemometrics. Lentil samples from two origins, i.e. Italy and Canada, were analysed obtaining the stable isotope ratios of δ(13)C, δ(15)N, δ(2)H, δ(18)O, and δ(34)S. A comparison between median values (U-test) highlighted statistically significant differences (p<0.05) for all isotopic parameters between the lentils produced in these two different geographic areas, except for δ(15)N. Applying principal component analysis, grouping of samples was observed on the basis of origin but with overlapping zones; consequently, two supervised discriminant techniques, i.e. partial least squares discriminant analysis and k-nearest neighbours algorithm were used. Both models showed good performances with external prediction abilities of about 93% demonstrating the suitability of the methods developed. Subsequently, isotopic determinations were also performed on the protein and starch fractions and the relevant results are reported.

Stable isotope ratios of H, C, N and O in Italian citrus juices.

Stable isotope ratios (SIRs) of C, N, H and O have been exensively used in fruit juices quality control (ENV and AOAC methods) to detect added sugar and the watering down of concentrated juice, practices prohibited by European legislation (EU Directive 2012/12). The European Fruit Juice Association (AIJN) set some reference guidelines in order to allow the judging of the genuiness of a juice. Moreover, various studies have been carried out to determine the natural variability of SIRs in fruit juices, but none of these has investigated SIRs extensively in authentic citrus juices from Italy. In this work, about 500 citrus juice samples were officially collected in Italy by the Italian Ministry of Agricultural and Forestry Policies from 1998 onwards. (D/H)(I) and (D/H)(II) in ethanol and δ(13) C(ethanol), δ(13) C(pulp), δ(13) C(sugars), δ(18) O(vegetalwater), δ(15) N(pulp), and δ(18) O(pulp) were determined using Site-Specific Natural Isotope Fractionation-Nuclear Magnetic Resonance and Isotope Ratio Mass Spectrometry, respectively. The characteristic ranges of variability in SIRs in genuine Italian citrus juice samples are here presented as well as their relationships and compliance with the limits indicated by the AIJN and others proposed in the literature. In particular, the Italian range of values was found to be not completely in agreement with AIJN guidelines, with the risk that genuine juices could be judged as not genuine. Variety seems not to influence SIRs, whereas harvest year and region of origin have some influence on the different ratios, although their data distribution shows overlapping when principal component analysis is applied.

Stable isotope ratio analysis for verifying the authenticity of balsamic and wine vinegar.

In this paper, we investigate whether the analysis of stable isotope ratios D/H and ¹³C/¹²C in ethanol and acetic acid and of ¹8O/¹6O in water can be applied to the ingredients of "aceto balsamico di Modena IGP" (ABM) to evaluate their authenticity. We found that impurities in the extraction solution do not affect the ¹³C/¹²C of acetic acid and the D/H values of acetic acid are not affected under a composite NMR experiment. The standard deviation of repeatability and standard deviation of reproducibility are comparable in wine vinegar and ABM and generally lower than those quoted in the official methods. This means that the validation parameters quoted in the official methods can also be applied to the ingredients of ABM. In addition, we found no changes in the isotopic values from wine to vinegar and to ABM, and from the original must to the ABM must, providing experimental evidence that reference data from wine databanks can also be used to evaluate the authenticity of vinegar and ABM.

Stable isotope ratios of blood components and muscle to trace dietary changes in lambs.

Multielemental stable isotope ratio (SIR) analysis was used in lamb plasma, erythrocytes and muscle to detect the switch from a pasture- to a concentrate-based diet, with the aim of verifying the possibility to trace the change of feeding in animal tissues. During 89 days of experimental feeding, lambs were subjected to four dietary treatments: pasture (P), pasture followed by concentrate in the stall for either 14 days (P-S14) or 37 days (P-S37) or concentrate in the stall (S). Pasture and concentrate diets comprised C3 plants only and had different values of 13C/12C, 18O/16O, 2H/1H and 34S/32S ratios. Muscle 13C/12C and 34S/32S and plasma 13C/12C and 18O/16O ratios in P, P-S14 and P-S37 lambs were significantly different. A multivariate analytical approach revealed that 13C/12C and 18O/16O ratios in plasma were the most powerful variables for the discrimination among the dietary treatments.

Carbon, hydrogen and oxygen stable isotope ratios of whole wood, cellulose and lignin methoxyl groups of Picea abies as climate proxies.

RATIONALE: Carbon, hydrogen and oxygen (C, H and O) stable isotope ratios of whole wood and components are commonly used as paleoclimate proxies. In this work we consider eight different proxies in order to discover the most suitable wood component and stable isotope ratio to provide the strongest climate signal in Picea abies in a southeastern Alpine region (Trentino, Italy). METHODS: δ(13)C, δ(18)O and δ(2)H values in whole wood and cellulose, and δ(13)C and δ(2)H values in lignin methoxyl groups were measured. Analysis was performed using an Isotopic Ratio Mass Spectrometer coupled with an Elemental Analyser for measuring (13)C/(12)C and a Pyrolyser for measuring (2)H/(1)H and (18)O/(16)O. The data were evaluated by Principal Component Analysis, and a simple Pearson's correlation between isotope chronologies and climatic features, and multiple linear regression were performed to evaluate the data. RESULTS: Each stable isotope ratio in cellulose and lignin methoxyl differs significantly from the same stable isotope ratio in whole wood, the values begin higher in cellulose and lignin except for the lignin δ(2)H values. Significant correlations were found between the whole wood and the cellulose fractions for each isotope ratio. Overall, the highest correlations with temperature were found with the δ(18)O and δ(2)H values in whole wood, whereas no significant correlations were found between isotope proxies and precipitation. CONCLUSIONS: δ(18)O and δ(2)H values in whole wood provide the best temperature signals in Picea abies in the northern Italian study area. Extraction of cellulose and lignin and analysis of other isotopic ratios do not seem to be necessary.

Statistical methods for improving verification of claims of origin for Italian wines based on stable isotope ratios.

Wine derives its economic value to a large extent from geographical origin, which has a significant impact on the quality of the wine. According to the food legislation, wines can be without geographical origin (table wine) and wines with origin. Wines with origin must have characteristics which are essential due to its region of production and must be produced, processed and prepared, exclusively within that region. The development of fast and reliable analytical methods for the assessment of claims of origin is very important. The current official method is based on the measurement of stable isotope ratios of water and alcohol in wine, which are influenced by climatic factors. The results in this paper are based on 5220 Italian wine samples collected in the period 2000-2010. We evaluate the univariate approach underlying the official method to assess claims of origin and propose several new methods to get better geographical discrimination between samples. It is shown that multivariate methods are superior to univariate approaches in that they show increased sensitivity and specificity. In cases where data are non-normally distributed, an approach based on mixture modelling provides additional improvements.

Coast and year effect on H, O and C stable isotope ratios of Tyrrhenian and Adriatic Italian olive oils.

The paper discusses the (2)H/(1)H, (18)O/(16)O and (13)C/(12)C ratios of 196 authentic Italian extra-virgin olive oils produced in 3 years on the Tyrrhenian and Adriatic coasts. The (2)H/(1)H and (18)O/(16)O ratios were linearly and positively correlated. The year of production influenced mainly (18)O/(16)O in relation to the amount of rainfall and the atmospheric humidity in the period of oil accumulation in the olives. The (2)H/(1)H ratio significantly distinguished the olive oils produced on the Adriatic coast from those on the Tyrrhenian coast in each year. This coast effect is a consequence of the different sources and isotopic compositions of the rainfall and the different climatic conditions on the two coasts. The paper contributes towards understanding the influence of climatic factors on isotopic variability and towards improving the traceability of the geographical origin of olive oils, using (2)H/(1)H as a more innovative parameter.

Multi-element (H,C,N,S) stable isotope characteristics of lamb meat from different European regions.

Multi-element (H,C,N,S) stable isotope ratio analysis was tested for its suitability as a means for geographical provenance assignment of lamb meat from several European regions. The defatted dry matter (crude protein fraction) from lamb meat was found to be a suitable probe for "light" element stable isotope ratio analysis. Significant differences were observed between the multi-element isotope ratios of lamb samples from different regions. The mean hydrogen isotopic ratios of the defatted dry matter from lamb were found to be significantly correlated with the mean hydrogen isotopic ratios of precipitation and groundwater in the production regions. Carbon and nitrogen isotopic ratios were influenced by feeding practices and climate. Sulfur isotopic ratios were influenced by geographical location and surface geology of the production region. The results permitted differentiation of lamb meat, from most production regions, by inspection. However, more sophisticated evaluation of the data using multivariate methods, such as linear discriminant analysis, achieved 78% correct classification.

Correlation between multielement stable isotope ratio and geographical origin in Peretta cows' milk cheese.

The aim of this study was to characterize the isotopic composition and protect "Peretta" cows' milk cheese, a typical product of Sardinia, against other cheeses of the same appearance sold under the same name, but made of raw materials from northern Europe. The study was concerned with 3 types of cheese: those produced in local dairies from milk from free-grazing or pasture-grazing cows in Sardinia (local dairy product), cheeses made on an industrial scale from milk produced by intensive farming in Sardinia (factory cheese), and cheeses made with raw materials imported from other countries (imported product). To distinguish the Sardinian cheeses from the imported product, the stable isotope ratios 13C/12C, 15N/14N, D/H, 34S/32S, and (18)O/(16)O were used. Determination of the isotopic data delta13C, delta15N, delta2H, and delta34S was performed in the casein fraction, whereas delta(18)O and delta13C were determined in the glycerol fraction. Measurements were performed by isotope ratio mass spectrometry. A comparison between mean values of the isotope ratios by statistical analysis (ANOVA and Tukey's test) showed that the greatest difference between the 3 types of cheese (local dairy, factory, and imported products) was in the 13C/12C, 34S/32S, and (18)O/(16)O isotope ratios. In the other parameters, either no differences (delta15N) or minimal differences (delta2H) were found. Evaluation of the data by multivariate statistical analysis (principal component analysis and hierarchical cluster analysis) revealed that the isotope characteristics of the factory products were similar to those of the cheeses produced from imported raw materials, whereas a difference was found between the local dairy-produced cheeses and the products in the other 2 categories.

Stable isotope ratio analysis for authentication of lamb meat.

The effectiveness of the analysis of stable isotope ratios ((13)C/(12)C and (15)N/(14)N) in fractions of lamb meat, measured by isotope ratio mass spectrometry, was evaluated as a method of feeding and geographical origin authentication. Analyses were carried out on meat from 12 lamb types, produced in couples in six European countries (country of origin, CO) and divided in three groups according to the feeding regime during their finishing period: suckled milk only, pasture without any solid supplementation and supplementation containing maize grain (feeding regime, FR). These analyses were made on two samples of longissimus thoracis muscle, taken from the 13th rib section of the left side of two different lambs, randomly chosen between the 120 selected to represent each lamb type. δ(13)C values varied significantly in different meat fractions, the difference being higher in protein than in fat (average difference 5.0‰). However, the pairs δ(13)C values of crude fat and protein were highly correlated (r=0.976) and affected by lamb type in a similar fashion, mainly reflecting animals' feeding regime. Even δ(15)N values of meat protein fraction showed significant differences between lamb types, not dependant on the feeding regime. In fact, lambs fed on similar diets, but in different countries, gave meat with different (15)N relative abundances. These findings provide the possibility of discriminating lamb types within the same feeding regime. Canonical discriminant analysis was carried out to evaluate whether lamb meat from different CO or FR or CO×FR interaction could be mathematically distinguished by its stable isotope ratios. On the basis of CO, the corrected empirical allocation of 79.2% of the initial observations and the corrected cross-validation of two thirds of the individual meat samples was obtained. FR gave better results: 91.7% of the individual meat samples was both correctly allocated and cross-validated, indicating the high potential of stable isotope ratio analysis as a tool for lamb diet characterisation. The most satisfactory classification attained, using K-means clustering technique and canonical discriminant analysis, enabled a good resolution of six CO×FR groups of lamb types: Icelandic fed on pasture; British and French grazing; Italian; suckled and Karagouniko concentrates finished; French Lacaune; Ternasco de Aragon. It was concluded that multielement stable isotope analysis may be considered promising for the reliable evaluation of lamb meat authenticity, in the same way as for wine, fruit juice, honey and dairy products.

Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope ((13)c/(12)c and (15)n/(14)n) ratios and free amino acid ratios.

The stable isotope ratios ((13)C/(12)C and (15)N/(14)N) of casein measured by isotope ratio mass spectrometry (IRMS) and some free amino acid ratios (His/Pro, Ile/Pro, Met/Pro, and Thr/Pro) determined by HPLC in samples of ewes' milk cheese from Sardinia, Sicily, and Apulia were found to be parameters independent of ripening time. Multivariate data treatments performed by applying both unsupervised (principal component analysis and cluster analysis) and supervised [linear discriminant analysis (LDA)] methods revealed good discrimination possibilities for the cheeses according to place of origin. In this respect, particularly significant were the variables Ile/Pro, Thr/Pro, (13)C/(12)C, and (15) N/(14)N ratios on which basis 100% discrimination and classification of the samples by LDA was obtained.