Principles and Limitations of Stable Isotopes in Differentiating Organic and Conventional Foodstuffs: 1. Plant Products

Among the lighter elements having two or more stable isotopes (H, C, N, O, S), δ¹⁵N appears to be the most promising isotopic marker to differentiate plant products from conventional and organic farms. Organic plant products vary within a range of δ¹⁵N values of +0.3 to +14.6%, while conventional plant products range from negative to positive values, i.e. ?4.0 to +8.7%. The main factors affecting δ¹⁵N signatures of plants are N fertilizers, biological N₂ fixation, plant organs and plant age. Correlations between mode of production and δ¹³C (except greenhouse tomatoes warmed with natural gas) or δ³⁴S signatures have not been established, and δ²H and δ¹⁸O are unsuitable markers due to the overriding effect of climate on the isotopic composition of plant-available water. Because there is potential overlap between the δ¹⁵N signatures of organic and conventionally produced plant products, δ¹⁵N has seldom been used successfully as the sole criterion for differentiation, but when combined with complementary analytical techniques and appropriate statistical tools, the probability of a correct identification increases. The use of organic fertilizers by conventional farmers or the marketing of organic produce as conventional due to market pressures are additional factors confounding correct identification. The robustness of using δ¹⁵N to differentiate mode of production will depend on the establishment of databases that have been verified for individual plant products.     

Seasonal variations in chemical composition and stable isotopes of farmed and wild Brazilian freshwater fish

This study examined variations in the Fulton condition factor, chemical composition, and stable isotopes of carbon and nitrogen in the Brazilian freshwater fish cachara (Pseudoplatystoma fasciatum), comparing farmed and wild fish in different seasons. Values for energy, protein, moisture, and Fulton’s condition factor were higher for farmed than for wild fish in the rainy season, indicating better nutritional quality; however, these differences were not observed in the dry season. Likewise, we found significant enhancement of δ¹⁵N in farmed fish in the rainy season but not in the dry season, whereas enhancement of δ¹³C was observed in both seasons. The combined measurement of δ¹³C and δ¹⁵N provided traceability under all conditions. Our findings show that stable isotope analysis of C and N can be used to trace cachara origin, and that seasonal variations need to be considered when applying chemical and isotopic authentication of fish and fish products.     

Seasonal variation in the C,N and S stable isotope composition of retail organic and conventional Irish beef

The objective of this study was to investigate seasonal variation in the C, N and S stable isotope composition of retail organic and conventional Irish beef. A total of 242 beef samples (127 organic, 115 conventional) was collected in a one-year survey and analysed by isotope ratio mass spectrometry. The δ¹³C time series in conventional beef was significantly non-random, with a pronounced seasonal positive shift of >2‰ between December and June, whilst δ¹³C in organic beef was less variable and significantly more negative. In conventional beef, δ¹⁵N was remarkably invariant (remaining close to 7‰) throughout the year, while organic beef was more variable and also significantly lower in δ¹⁵N. The S isotope composition (δ³⁴S) exhibited a complex seasonal pattern in both types of beef. These results show that seasonal patterns can occur in the isotopic composition of beef, probably reflecting seasonality in animal feeding practices modulated by tissue turnover rates. Such seasonal variation needs to be considered in the isotopic authentication of beef and other animal-derived products.     

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ¹³C, δ¹⁵N, and δ¹⁸O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n = 1), Japan (n = 12), and USA (n = 1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2–40.0% (C content), 0.8–1.4% (N content), −27.1 to −25.4% (δ¹³C), +0.4 to +9.0% (δ¹⁵N), and +18.8 to +22.9% (δ¹⁸O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ¹³C, δ¹⁵N, and δ¹⁸O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.     

Passage of stable isotope-labeled grass silage fiber and fiber-bound protein through the gastrointestinal tract of dairy cows

Fractional passage rates are required to predict nutrient absorption in ruminants but data on nutrient-specific passage kinetics are largely lacking. With the use of the stable isotope ratio (δ) as an internal marker, we assessed passage kinetics of fiber and fiber-bound nitrogen (N) of intrinsically labeled grass silage from fecal and omasal excretion patterns of δ¹³C and δ¹⁵N. In a 6 × 6 Latin square, lactating dairy cows received grass silages [455 g/kg of total diet dry matter (DM) ] in a 2 × 3 factorial arrangement from ryegrass swards fertilized at low (45 kg of N/ha) or high (90 kg of N/ha) levels of N and harvested at 3 maturity stages. Feed intake (16.7 ± 0.48 kg of DM/d; mean ± standard error of the mean) and milk yield (26.7 ± 0.92 kg/d) increased at the high level of N fertilization and at decreasing maturity. Nutrient digestibility decreased with increasing plant maturity, particularly at the high level of N fertilization, essentially reflecting dietary treatment effects on the nutritional composition of the grass silage. Fractional rumen passage rates (K₁) were highest and total mean retention time in the gastrointestinal tract (TMRT) was lowest when based on the external marker chromium mordanted fiber (Cr-NDF; 0.047/h and 38.0 h, respectively). Fecal δ¹³C in the acid detergent fiber fraction (¹³CADF) provided the lowest K₁ (0.023/h) and the highest TMRT (61.1 h) and highest peak concentration time (PCT; 24.3 h) among markers. In comparison, fecal fiber-bound N (¹⁵NADF) had a considerably higher K₁ (0.032/h) and lower TMRT (46.4 h) than ¹³CADF. Total N (measured with ¹⁵NDM) had a comparable K₁ (0.034/h) to that of ¹⁵NADF but provided the highest fractional passage rates from the proximal colon-cecum (K₂; 0.37/h) and lowest PCT (17.4 h) among markers. A literature review indicated unclear effects of grass silage maturity on K₁ and unknown effects of N fertilization on K₁. Our study indicated no effect of advancing maturity on fecal K₁ and a trend for K₁ to increase with the high level of N fertilization. Parameter K₂ increased, whereas PCT and TMRT generally decreased with the high level of N fertilization. Omasal digesta sampling largely confirmed results based on fecal sampling. Results indicate that the use of δ¹³C and δ¹⁵N can describe fiber-specific passage kinetics of forage.     

Measurement of stable isotope abundances in milk and milk ingredients — a possible tool for origin assignment and quality control

Relative carbon and nitrogen stable isotope abundances in total milk reflect the isotopic composition of the diet fed to the dairy cows; this diet and its δ-values depend on geographical and climatic factors. Milk from regions dominated by grassland typically shows relatively negative δ¹³C-values, while in regions dominated by crop cultivation the δ¹³C-values are more positive. The δ¹⁵N-values are influenced by factors such as soil conditions, the intensity of agricultural use and the climate. Casein in authentic milk samples is enriched in both ¹³C and ¹⁵N as compared to total milk, while the whey fraction is slightly enriched in ¹³C and depleted of ¹⁵N. The isotopic content of milk, casein and whey from one location have been measured throughout a period of longer than 1 year; variations are usually not greater than 1‰. In milk water, the ¹⁸O content was increased by between 2 and 6‰ as compared to ground water.     

Geographical origin of imported and domestic teas (Camellia sinensis) from Turkey as determined by stable isotope signatures

In this study, stable isotope signatures (δ¹³C, δ¹⁵N, and δD) of both tea leaves and tea infusions were investigated to identify the geographical origin of Turkish domestic and imported tea samples. Sixteen domestic tea samples collected from different locations in the Black Sea Region, which produces almost 100% of tea in Turkey, and 11 imported tea samples (Kenya, India, Sri Lanka, Indonesia, and China) purchased from importers were studied. δ¹³C, δ¹⁵N, and δD in the samples were determined using isotope ratio mass spectrometry (IR-MS). δ¹³C in the samples ranged from ?29.18 ± 0.01 to ?25.7 ± 0.2, while δ¹⁵N ranged between 1.1 ± 0.2 and 5.2 ± 0.8. However, δD in the samples were found to be in the range from 56.5 ± 0.3 to 72 ± 1. The classifications of the tea samples into domestic and imported tea samples were achieved with 100% accuracy using multivariate statistical analyses (principal component analysis, PCA, and hierarchical cluster analysis, HCA). In conclusion, the domestic tea samples had a distinctive isotopic fingerprint and the isotopic ratios used in the study can be significant predictors in determination of the geographical source of Turkish tea.     

Barn vs. free-range chickens: Differences in their diets determined by stable isotopes

We compared δ¹³C and δ¹⁵N ratios of barn-raised and free-range chickens to determine if differences in their diets were reflected in the stable isotope composition of their tissues. We conducted a 120-day feeding trial with Caipirinha birds fed a corn-soybean based diet, milled-corn diet and free-range diet. Additionally, we analysed the stable isotope composition of barn-raised chickens bought in grocery stores and free-range homegrown chickens. In the feeding trials, the δ¹³C and δ¹⁵N values of the barn-raised corn-soybean-fed Caipirinha chickens did not change with age, and their stable isotope composition reflected the composition of their diet. The δ¹³C and δ¹⁵N values of barn-raised corn-fed and free-range Caipirinha chickens changed with age toward a diet reflecting a predominance of C₄ carbon. The main difference between the free-range and the barn-raised chickens was the significantly higher δ¹⁵N of the former in relation to the latter, probably due to ingestion of animal protein.     

Natural origin of ascorbic acid: Validation by C NMR and IRMS

A new method for the extraction and purification of ascorbic acid from two tropical fruits (acerola and camu-camu) is presented. ¹³C nuclear magnetic resonance and isotopic ratio mass spectroscopy (¹³C/¹²C) were used to recognize ascorbic acid coming from either natural or industrial sources. A quantitative ¹³C NMR procedure was optimized to calculate isotopic relative abundances on each molecular site; data were treated by a multivariate method.     

The combination of stable isotope abundance ratios of H,C, N and S with Sr/Sr for geographical origin assignment of orange juices

Within the EU-project “Pure Juice” established stable isotope methods (δ²H, δ¹³C, δ¹⁵N) have been applied and improved in order to determine and verify the geographical origin of orange juices. In addition, new approaches employing analyses of δ³⁴S and ⁸⁷Sr/⁸⁶Sr have been developed and tested. Approximately 150 authentic orange juice samples from several regions in North- and South-America, Africa and Europe have been analysed. A discrimination of orange producing regions, based on the results which ultimately depend on geographical, climatic and lithological differences was successfully performed. Furthermore, we demonstrate that blending of single strength juice by adding concentrate can be revealed by comparing ⁸⁷Sr/⁸⁶Sr of soluble and insoluble components of the juices. We conclude that regional assignment of orange juice samples is most successful when single parameters are combined in a “multi-element approach”.     

The use of stable isotopes for food web analysis

General aspects in isotope biogeochemistry was summarized with emphasis on delta 15N and delta 13C contents in plants and animals in natural ecosystems. In the estuary, the variation of isotope ratios were principally governed by the mixing of land-derived organic matter, marine phytoplankton, and seagrasses. A clear cut linear relationship between animal delta 15N and its trophic level was obtained in the Antarctic food chain system. Several current efforts to use the stable isotopes for food web analysis were demonstrated for some terrestrial and marine systems as well as human food web.     

Stable C and N isotope ratio analysis for regional geographical traceability of cattle in China

The aim of this study was to examine the variation in carbon and nitrogen stable isotope ratios in cattle tissues from different provinces in China, and to investigate the correlations of δ¹³C and δ¹⁵N values between different cattle tissues. Furthermore, the success rate of classification using δ¹³C and δ¹⁵N values to distinguish the geographical origin of cattle was analyzed. Fifty nine cattle samples were collected from Jilin, Ningxia, Guizhou and Hebei provinces in China, and the δ¹³C and δ¹⁵N values of de-fatted beef, crude fat and tail hair were measured using isotope ratio mass spectrometry (IRMS). There were highly significant regional differences in the mean values of δ¹³C and δ¹⁵N values in the cattle tissues. A significant correlation was found in δ¹³C and δ¹⁵N between de-fatted beef, crude fat and tail hair, which indicated that all of these matrices could be used to trace cattle to their geographical origin. The results of discriminant analysis showed that δ¹³C was the better indicator for cattle origin traceability than δ¹⁵N. The classification success rate could be improved greatly by combining the two indicators. It was concluded that stable isotope analysis of C and N in cattle tissue can be used to trace cattle diet and origin in China.     

Influence of N doses and form on ¹⁵N natural abundance of pepper plants: considerations for using δ¹⁵N values as indicator of N source

BACKGROUND: The aim of this study was to ascertain the effect of the N form (NO₃^?, NH₄⁺ and organic N) and N concentration on plant isotopic fractionation and on the contribution of the different N sources to the plant N budget, in order to evaluate the feasibility of using plant δ¹⁵N values for discriminating between conventional and organic crops. To this end, different N concentrations (applied as NO₃^?), N forms (NO₃^? versus NH₄⁺), and increasing NO₃^? applications to an organic N‐based fertilization regime were studied. RESULTS: When using NO₃^? as N source, intra‐plant fractionation was significant and tended to increase when NO₃^? concentration increased in the root medium. However, negligible net isotopic fractionation was observed when comparing theoretical and measured plant δ¹⁵N values. On the other hand, when plants are fertilized with a mixture of NO₃^? and NH₄⁺, differences in uptake patterns for both salts could result in variation in plant δ¹⁵N regarding to the expected value. Finally, the application of NO₃^? to plants was detected when it was combined with high levels of organic N sources, from 99:1 organic:inorganic N ratio. CONCLUSION: Under certain conditions and following some considerations concerning sampling, δ¹⁵N values can be considered to be a potential tool to guarantee the authenticity of organic products. Copyright © 2011 Society of Chemical Industry     

Classification of geographical origins and prediction of δ13C and δ15N values of lamb meat by near infrared reflectance spectroscopy

Near infrared spectroscopy (NIRS) combined with chemometric analysis was investigated for its potential to classify the geographical origin and predict δ(13)C and δ(15)N values of lamb meat samples (n=99) from three pastoral regions and two agricultural regions of China. Principal component analysis (PCA), discriminant partial least squares analysis (D-PLS), linear discriminant analysis (LDA) and partial least squares regression (PLSR) were used for data analysis. D-PLS and LDA correctly classified 100% of the both pastoral and agricultural region samples, and gave a total correct classification of 88.9% and 75% to the five individual region samples, respectively. The best PLSR calibration models for predicting δ(13)C and δ(15)N of lamb meat were obtained with the determination coefficient (R(2)) 0.76 and 0.87, respectively. These results show that NIRS combined with chemometrics can be used as a rapid and effective method to discriminate the geographical origin and estimate the δ(13)C and δ(15)N of lamb meat.     

Analysis of isotopic ratios for the detection of illegal watering of beverages

The applications of the stable isotope ratio analyses (D/H, ¹³C and ¹⁸O) in the assessment of watering in beverages such as wine and fruit juices are discussed in this study. The measurement of ¹⁸O/¹⁶O ratio in combination with D/H and ¹³C/¹²C ratio is mainly focused and the international official methods implementing stable isotope techniques will be mentioned. Furthermore, the ongoing study on a possible use of ¹³C/¹²C ratio of CO₂ to detect the addition of technical produced CO₂ in mineral sparkling waters is also presented.     

Stable Isotope Ratio Analysis for Assessing the Authenticity of Food of Animal Origin

The main elemental constituents (H, C, N, O, and S) of bio‐organic material have different stable isotopes (²H, ¹H; ¹³C,¹²C; ¹⁵N,¹⁴N; ¹⁸O,¹⁷O,¹⁶O; ³⁶S, ³⁴S, ³³S, and ³²S). Isotopic ratios can be measured precisely and accurately using dedicated analytical techniques such as isotope ratio mass spectrometry (IRMS). Analysis of these ratios shows potential for assessing the authenticity of food of animal origin. In this review, IRMS analysis of food of animal origin and variability factors related to stable isotope ratios in animals are described. The study also lists examples of application of stable isotope ratio analysis to meat, dairy products, fish, and shellfish and emphasizes the strengths and weaknesses of the technique. Geographical, climatic, pedological, geological, botanical, and agricultural factors affect the stable isotope ratios (SIR) of bio‐elements, and SIR variations are ultimately incorporated into animal tissue through eating, drinking, breathing, and exchange with the environment, being recorded in the resulting foods. SIR analysis was capable of determining geographical origin, animal diet, and the production system (such as organic/conventional or wild/farmed) for pork, beef, lamb, poultry, milk, butter, cheese, fish, and shellfish. In the case of the hard PDO (protected designations of origin) cheeses Grana Padano and Parmigiano Reggiano it is also used in real‐life situations to assess the authenticity of grated and shredded cheese on the market.     

Effect of irrigation and variety on oxygen (δ18O) and carbon (δ13C) stable isotope composition of grapes cultivated in a warm climate

Background and Aims: δ¹³C values from Vitis vinifera leaves, whole grape, seed, pulp, skin and/or grape must sugars have been investigated as an integrated marker of vine water status or intrinsic water-use efficiency during berry growth and across region of origin, vintage and variety. The use of ¹⁸O/¹⁶O isotopic ratio as a marker of water addition, vintage and geographical origin has also been studied. This paper examines the effect of irrigation and grapevine variety on δ¹⁸O and δ¹³C of grape must from eight varieties, all cultivated in the same vineyard to reduce the effects from other variables. Methods and Results: Stable isotope compositions of grape must water and sugar were determined by isotope ratio mass spectrometry. The result of the study showed statistically significant effects of irrigation and vine variety on both δ¹⁸O and δ¹³C. The effect of vintage on δ¹⁸O was only significant for non-irrigated vines. Conclusion: This research highlights the effect of variety and irrigation on δ¹³C and δ¹⁸O of grape. Significance of the Study: This is the first report to demonstrate that the varietal effect on δ¹³C and δ¹⁸O of grape is not due only to differences in the vegetative cycle of each variety. It further suggests that water exhibits a lower isotopic discrimination in the indigenous Spanish varieties studied than in non-indigenous varieties.     

Regional contamination versus regional dietary differences: understanding geographic variation in brominated and chlorinated contaminant levels in polar bears

The relative contribution of regional contamination versus dietary differences to geographic variation in polar bear (Ursus maritimus) contaminant levels is unknown. Dietary variation between Alaska, Canada, East Greenland, and Svalbard subpopulations was assessed by muscle nitrogen and carbon stable isotope (δ(15)N, δ(13)C) and adipose fatty acid (FA) signatures relative to their main prey (ringed seals). Western and southern Hudson Bay signatures were characterized by depleted δ(15)N and δ(13)C, lower proportions of C(20) and C(22) monounsaturated FAs and higher proportions of C(18) and longer chain polyunsaturated FAs. East Greenland and Svalbard signatures were reversed relative to Hudson Bay. Alaskan and Canadian Arctic signatures were intermediate. Between-subpopulation dietary differences predominated over interannual, seasonal, sex, or age variation. Among various brominated and chlorinated contaminants, diet signatures significantly explained variation in adipose levels of polybrominated diphenyl ether (PBDE) flame retardants (14-15%) and legacy PCBs (18-21%). However, dietary influence was contaminant class-specific, since only low or nonsignificant proportions of variation in organochlorine pesticide (e.g., chlordane) levels were explained by diet. Hudson Bay diet signatures were associated with lower PCB and PBDE levels, whereas East Greenland and Svalbard signatures were associated with higher levels. Understanding diet/food web factors is important to accurately interpret contaminant trends, particularly in a changing Arctic.     

Can δ15N in lettuce tissues reveal the use of synthetic nitrogen fertiliser in organic production?

BACKGROUND: The nitrogen isotopic fingerprint (δ¹⁵N) is reported to be a promising indicator for differentiating between organically and conventionally grown vegetables. However, the effect on plant δ¹⁵N of split nitrogen fertilisation, which could enable farmers to cover up the use of synthetic fertiliser, is not well studied. In this study the use of δ¹⁵N in lettuce as a potential marker for identifying the use of synthetic nitrogen fertiliser was tested on pot‐grown lettuce (Lactuca sativa L.) treated with synthetic and organic nitrogen fertilisers (single or split application). The effect of combined usage of synthetic and organic fertilisers on δ¹⁵N was also investigated. RESULTS: The δ¹⁵N values of whole plants treated with different fertilisers differed significantly when the fertiliser was applied in a single treatement. However, additional fertilisation (with isotopically the same or different fertiliser) did not cause a significant alteration of plant δ¹⁵N. CONCLUSION: The findings of the study suggest that the δ¹⁵N value of lettuce tissues could be used as a rough marker to reveal the history of nitrogen fertilisation, but only in the case of single fertiliser application. However, if the difference in δ¹⁵N between the applied synthetic and organic nitrogen fertilisers was > 9.1‰, the detection of split and combined usage of the fertilisers would have greater discriminatory power. Copyright © 2010 Society of Chemical Industry