Stable isotope ratio measurements of royal jelly samples for controlling production procedures: impact of sugar feeding

The carbon and nitrogen stable ratios of royal jelly (RJ) samples from various origins are determined using an elemental analyser linked online to an isotope ratio mass spectrometer to evaluate authenticity and adulteration. The (13)C/(12)C and (15)N/(14)N stable isotope ratios are measured in more than 500 RJs (domestic, imported and derived from feeding experiments) in order to obtain isotopic measurements that take into account seasonal, botanical and geographical effects. Authenticity intervals are established for traditional beekeeping practices, without feeding, in the range -22.48 to -27.90‰ for δ(13)C. For these samples, the δ(15)N values range from -1.58 to 7.98‰, depending on the plant sources of pollen and nectar. The δ(13)C values of the commercial samples vary from -18.54 to -26.58‰. High δ(13)C values are typical of sugar cane or corn syrups which have distinctive isotopic (13)C signatures because both plants use the C4 photosynthetic cycle, in contrast to most RJs which are derived from C3 plants. These differences in the (13)C-isotopic composition allow the detection of the addition of such sugars. RJs from traditional sources and from industrial production by sugar feeding are thus successfully distinguished.     

GasBench/isotope ratio mass spectrometry: a carbon isotope approach to detect exogenous CO(2) in sparkling drinks

A new procedure for the determination of carbon dioxide (CO(2)) (13)C/(12)C isotope ratios, using direct injection into a GasBench/isotope ratio mass spectrometry (GasBench/IRMS) system, has been developed to improve isotopic methods devoted to the study of the authenticity of sparkling drinks. Thirty-nine commercial sparkling drink samples from various origins were analyzed. Values of delta(13)C(cava) ranged from -20.30 per thousand to -23.63 per thousand, when C3 sugar addition was performed for a second alcoholic fermentation. Values of delta(13)C(water) ranged from -5.59 per thousand to -6.87 per thousand in the case of naturally carbonated water or water fortified with gas from the spring, and delta(13)C(water) ranged from -29.36 per thousand to -42.09 per thousand when industrial CO(2) was added. It has been demonstrated that the addition of C4 sugar to semi-sparkling wine (aguja) and industrial CO(2) addition to sparkling wine (cava) or water can be detected. The new procedure has advantages over existing methods in terms of analysis time and sample treatment. In addition, it is the first isotopic method developed that allows (13)C/(12)C determination directly from a liquid sample without previous CO(2) extraction. No significant isotopic fractionation was observed nor any influence by secondary compounds present in the liquid phase.     

Influence of dietary composition on the carbon, nitrogen, oxygen and hydrogen stable isotope ratios of milk

The stable isotope ratios ((13)C/(12)C, (15)N/(14)N, (18)O/(16)O, D/H) of animal feed and milk were investigated, considering cows stabled in two farms and fed with diets made up of different kinds of C(3) plants and different amounts of maize. Maize was characterised by delta(13)C, delta(18)O and deltaD values significantly higher than those of the C(3) plants, while, for the C(3) plants, Festuca arudinacea had significantly higher content of (13)C and (15)N. The delta(13)C and delta(18)O values of the overall diet and the delta(13)C of milk casein and lipids were shown to be significantly correlated with the percentage of maize in the animal diet. On the other hand, the delta(18)O values of milk water and the delta(18)O, deltaD and delta(15)N values of casein were shown to be only slightly influenced by the amount of maize in the feed, being probably more closely correlated with the geo-climatic and pedological characteristics of the area of origin and with the presence of fresh plant or silage in the ration. The delta(13)C value of casein was shown to be a suitable parameter for evaluating the amount of maize in the diet: each 10% increase in the maize content corresponded to a shift of 0.7 per thousand to 1.0 per thousand in the delta(13)C of casein. A threshold value of -23.5 per thousand for delta(13)C in milk casein, above which it is not possible to exclude the presence of maize in the diet, was suggested. The results obtained could be useful for determining mislabelling of dairy products declared to have been produced by pastured animals or of PDO cheeses with an established amount of maize in the diet and for verifying the unpermitted addition of exogenous components to milk.     

<Superscript>15</Superscript>N isotopic analyses: a powerful tool to establish links between seized 3,4-methylenedioxymethamphetamine (MDMA) tablets

In this study the (15)N/(14)N isotopic ratios of 43 samples of 3,4-methylenedioxymethamphetamine (MDMA) samples were measured using Gas Chromatography-Combustion-Isotope-Ratio Mass Spectrometry (GC-C-IRMS). The results show a large discrimination between samples with a range of delta(15)N values between -16 and +19 per thousand. Comparison between delta(15)N values and other physical and chemical parameters shows a strong relationship between delta(15)N and brand logo or composition. Thus, it could be assumed that tablets from different seizures probably originated from the same clandestine manufacturing source. Hence, (15)N isotopic parameters provide an important additional tool to establish common origins between seizures of clandestine synthetic drugs.     

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

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

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

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

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

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

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.     

Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios

Many Salmo trutta populations consist of non-anadromous (freshwater-resident) brown trout and anadromous (sea-run migratory) sea trout. Although adult brown trout and sea trout can usually be identified using differences in size and body colouration, it is not possible to easily identify eggs/alevins as the progeny of brown trout or sea trout. In this study we show that delta(13)C and delta(15)N, measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS), can accurately identify fish eggs as the progeny of freshwater-resident (delta(13)C(egg) = -25.7 +/- 1.9 per thousand,delta(15)N(egg) = 9.2 +/- 1.8 per thousand) or migratory (delta(13)C(egg) = -19.9 +/- 1.1 per thousand, delta(15)N(egg) = 14. 3 +/- 1.5 per thousand) adult female Salmo trutta. Case studies show that stable isotope analysis is a more reliable technique for distinguishing anadromous adult fish than differentiation using morphological characteristics. For example, stable isotope analysis of brown trout from Loch Eck, Scotland, revealed that some individuals possessed delta(13)C and delta(15)N signatures indicative of marine feeding despite visual identification as freshwater-resident fish. It is most likely that these fish are misidentified sea trout although it possible that these fish may be brown trout that have adopted an estuarine feeding strategy to avoid interspecific competition for food within Loch Eck with salmon, powan and Arctic charr. Most stable isotope studies of fish ecology use terminal tissue sampling to provide sufficient biological material for isotopic analysis; however, our study suggests that adipose fin tissue could provide a comparable measure of delta(13)C and delta(15)N. Such a strategy would be invaluable when studying the trophic ecology or migration patterns of fish of high conservation value.     

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

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

Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.     

水体中痕量挥发性有机物单体碳同位素组成分析

将固相微萃取(SPME)技术与冷阱富集系统相结合,对水体中痕量挥发性有机物进行了单体碳同位素分析,方法检测限较常规SPME提高了一个数量级。在优化的条件下,对20 μg/L的三氯乙烯/四氯乙烯和10 μg/L的苯/甲苯水溶液进行了单体碳同位素分析,相比于纯溶剂(液相)碳同位素值,顶空(气相)同位素分析误差不超过0.5‰,而样本标准偏差为0.3‰。对某受四氯乙烯污染的北京地下水进行了同位素测定,近污染源点(B408)与远污染源点(B230)四氯乙烯的碳同位素值(δ13C)分别为 -37.8‰和-34.45‰     

Stable isotope natural abundance of nitrous oxide emitted from Antarctic tundra soils: effects of sea animal excrement depositions

Nitrous oxide (N2O), a greenhouse gas, is mainly emitted from soils during the nitrification and denitrification processes. N2O stable isotope investigations can help to characterize the N2O sources and N2O production mechanisms. N2O isotope measurements have been conducted for different types of global terrestrial ecosystems. However, no isotopic data of N2O emitted from Antarctic tundra ecosystems have been reported although the coastal ice-free tundra around Antarctic continent is the largest sea animal colony on the global scale. Here, we report for the first time stable isotope composition of N2O emitted from Antarctic sea animal colonies (including penguin, seal and skua colonies) and normal tundra soils using in situ field observations and laboratory incubations, and we have analyzed the effects of sea animal excrement depositions on stable isotope natural abundance of N2O. For all the field sites, the soil-emitted N2O was 15N- and 18O-depleted compared with N2O in local ambient air. The mean delta values of the soil-emitted N2O were delta15N = -13.5 +/- 3.2 per thousand and delta18O = 26.2 +/- 1.4 per thousand for the penguin colony, delta15N = -11.5 +/- 5.1 per thousand and delta18O = 26.4 +/- 3.5 per thousand for the skua colony and delta15N = -18.9 +/- 0.7 per thousand and delta18O = 28.8 +/- 1.3 per thousand for the seal colony. In the soil incubations, the isotopic composition of N2O was measured under N2 and under ambient air conditions. The soils incubated under the ambient air emitted very little N2O (2.93 microg N2O--N kg(-1)). Under N2 conditions, much more N2O was formed (9.74 microg N2O--N kg(-1)), and the mean delta15N and delta18O values of N2O were -19.1 +/- 8.0 per thousand and 21.3 +/- 4.3 per thousand, respectively, from penguin colony soils, and -17.0 +/- 4.2 per thousand and 20.6 +/- 3.5 per thousand, respectively, from seal colony soils. The data from in situ field observations and laboratory experiments point to denitrification as the predominant N2O source from Antarctic sea animal colonies.     

Two new organic reference materials for delta13C and delta15N measurements and a new value for the delta13C of NBS 22 oil

Analytical grade L-glutamic acid is chemically stable and has a C/N mole ratio of 5, which is close to that of many of natural biological materials, such as blood and animal tissue. Two L-glutamic acid reference materials with substantially different 13C and 15N abundances have been prepared for use as organic reference materials for C and N isotopic measurements. USGS40 is analytical grade L-glutamic acid and has a delta13C value of -26.24 per thousand relative to VPDB and a delta15N value of -4.52 per thousand relative to N2 in air. USGS41 was prepared by dissolving analytical grade L-glutamic acid with L-glutamic acid enriched in 13C and 15N. USGS41 has a delta13C value of +37.76 per thousand and a delta15N value of +47.57 per thousand. The delta13C and delta15N values of both materials were measured against the international reference materials NBS 19 calcium carbonate (delta13C=+1.95 per thousand ), L-SVEC lithium carbonate (delta13C=-46.48 per thousand ), IAEA-N-1 ammonium sulfate (delta15N=0.43 per thousand ), and USGS32 potassium nitrate (delta15N=180 per thousand ) by on-line combustion continuous-flow and off-line dual-inlet isotope-ratio mass spectrometry. Both USGS40 and USGS41 are isotopically homogeneous; reproducibility of delta13C is better than 0.13 per thousand, and that of delta15N is better than 0.13 per thousand in 100-microg amounts. These two isotopic reference materials can be used for (i) calibrating local laboratory reference materials, and (ii) quantifying drift with time, mass-dependent fractionations, and isotope-ratio-scale contraction in the isotopic analysis of various biological materials. Isotopic results presented in this paper yield a delta13C value for NBS 22 oil of -29.91 per thousand, in contrast to the commonly accepted value of -29.78 per thousand for which off-line blank corrections probably have not been quantified satisfactorily.     

Stable carbon and nitrogen isotope analysis of avian uric acid

We report results obtained using a new technique developed to measure the stable-isotope composition of uric acid isolated from bird excreta (guano). Results from a diet-switch feeding trial using zebra finches suggest that the delta(13)C of uric acid in the guano equilibrates with the diet of the bird within 3 days of a change in diet, while the equilibration time for delta(15)N may be longer. The average carbon isotope discrimination between uric acid and food before the diet switch was +0.34 +/- 1 per thousand (1sigma) while after the diet switch this increased slightly to +0.83 +/- 0.7 per thousand (1sigma). Nitrogen isotope discrimination was +1.3 +/- 0.3 per thousand (1sigma) and +0.3 +/- 0.3 per thousand (1sigma) before and after the diet switch; however, it is possible that the nitrogen isotope values did not fully equilibrate with diet switch over the course of the experiment. Analyses of other chemical fractions of the guano (organic residue after uric acid extraction and non-uric acid organics solubilised during extraction) suggest a total range of up to 3 per thousand for both delta(13)C and delta(15)N values in individual components of a single bulk guano sample. The analysis of natural samples from a range of terrestrial and marine species demonstrates that the technique yields isotopic compositions consistent with the known diets of the birds. The results from natural samples further demonstrate that multiple samples from the same species collected from the same location yield similar results, while different species from the same location exhibit a range of isotopic compositions indicative of different dietary preferences. Given that many samples of guano can be rapidly collected without any requirement to capture specimens for invasive sampling, the stable-isotope analysis of uric acid offers a new, simple and potentially powerful tool for studying avian ecology and metabolism.     

Clinical-scale investigation of stable isotopes in human blood: delta13C and delta15N from 406 patients at the Johns Hopkins Medical Institutions

Objective chemical biomarkers are needed in clinical studies of diet-related diseases to supplement subjective self-reporting methods. We report on several critical experiments for the development of clinically legitimate dietary stable isotope biomarkers within human blood. Our examination of human blood revealed the following: (1) Within blood clot and serum from anonymous individuals (201 males, 205 females) we observed: mean serum delta13C = -19.1 +/- 0.8 per thousand (standard deviation, SD); clot, -19.3 +/- 0.8 per thousand (SD); range = -15.8 per thousand to -23.4 per thousand. Highly statistically significant differences are observed between clot and serum, males and females for both clot and serum. For 15N (n = 206), mean serum = +8.8 +/- 0.5 per thousand (SD); clot +7.4 +/- 0.4 per thousand (SD); range = +6.3 per thousand to +10.5 per thousand. Blood serum is enriched in 15N relative to blood clot by +1.4 per thousand on average, which may reflect differing protein amino acid content. Serum nitrogen is statistically significantly different for males and females, however, clot shows no statistical difference. (2) Relative to clot, capillary blood is marginally different for 13C, but not 15N. Clot 13C is not significantly different from serum; however, it is depleted in 15N by 1.5 per thousand relative to serum. (3) We assessed the effect of blood additives (sodium fluoride and polymerized acrylamide resin) and laboratory process (autoclaving, freeze drying) commonly used to preserve or prepare venous blood. On average, no alteration in delta13C or delta15N is detected compared with unadulterated blood from the same individual. (4) Storage of blood with and without the additives described above for a period of up to 115 days exhibits statistically significant differences for 13C and 15N for sodium fluoride. However, storage for unadulterated blood and blood preserved with polymerized acrylamide resin does not change the delta13C or delta15N isotopic composition of the blood in a significant way. With these experiments, we gain a clinical context for future development of a stable isotope based dietary biomarker.     

A selective unsaturated hydrocarbon subtraction technique for stable carbon isotopic analysis of atmospheric methyl chloride, methyl bromide, and C2-C5 saturated hydrocarbons using continuous-flow isotope ratio mass spectrometry

Using continuous-flow isotope ratio mass spectrometry, we have developed a new analytical system which enables us to determine the stable carbon isotopic composition of CH3Cl, CH3Br, and C2-C5 saturated hydrocarbons in gas samples even if they contain substantial amounts of unsaturated hydrocarbons, using an I2O5 reagent for their selective subtraction. The analytical precision of the delta13C determinations is better than 0.5 per thousand for >300 pmolC injections and better than 5 per thousand for 20 pmolC injections. Using the system, delta13C values for CH3Cl and CH3Br were found in burning exhaust that contain a substantial quantity of unsaturated hydrocarbons. CH3Cl and CH3Br measured in exhaust from burning rice plants exhibit highly 13C-depleted values of -56.6 +/- 1.3 per thousand and -48.6 +/- 3.9 per thousand, respectively, while saturated hydrocarbons exhibit delta13C values (-26.4 to -28.9 per thousand) that are comparable with the total delta13C value of the parent material (rice plant; -28.0 per thousand). Using the system, we can determine the delta13C values of methyl halides and hydrocarbons in many kinds of gas samples.     

Effects of acidification on carbon and nitrogen stable isotopes of benthic macrofauna from a tropical coral reef

Stable isotope analyses are widely used to determine trophic levels in ecological studies. We have investigated the effects of carbonate removal via acidification on the stable carbon and nitrogen isotopic composition of 33 species of tropical benthic macrofauna, and we report guidelines for standardizing this procedure for higher taxa in tropical coral reef ecosystems. Many tropical benthic invertebrates are small in size, and therefore body tissue isolation (separating organic carbon from inorganic structures) is difficult and time-consuming. Literature reviews of invertebrate studies show a lack of consistent procedures and guidelines for preparation techniques, especially for carbonate removal via acidification of whole individuals. We find that acidification decreases the delta(13)C values of samples containing carbonate, with shifts ranging from 0.21 to 3.20 per thousand, which can be related to CaCO(3) content (assessed by a carbonate proxy), justifying acid pre-treatment. Carbonate-containing taxa benefiting from acidification included Amphinomida, Terebellida (Annelida), Anomura, Brachyura, Caridea, Amphipoda, Tanaidacea (Arthropoda) and Edwardsiida (Cnidaria). The delta(13)C shifts of samples containing no carbonate varied up to 0.02 +/- 0.20 per thousand. As this induced delta(13)C shift was lower than the range of an average trophic level shift (0.5 to 1 per thousand), we conclude that acid pre-treatment is unnecessary. Carbonate-free taxa consisted of Eunicida, Phyllodocida (Annelida) and Mollusca. We note minimal impact of acidification on delta(15)N values except for Brachyura, which showed a shift of 0.83 +/- 0.46 per thousand, which is still lower than a single trophic level shift (2.9-3.8 per thousand). We conclude that for trophic level studies, both the delta(13)C and the delta(15)N of carbonate-rich macrofauna can be determined from the same acidified sample. Copyright (c) 2008 John Wiley & Sons, Ltd.     

High-precision determination of 18O/16O ratios of silver phosphate by EA-pyrolysis-IRMS continuous flow technique

A high-precision, and rapid on-line method for oxygen isotope analysis of silver phosphate is presented. The technique uses high-temperature elemental analyzer (EA)-pyrolysis interfaced in continuous flow (CF) mode to an isotopic ratio mass spectrometer (IRMS). Calibration curves were generated by synthesizing silver phosphate with a 13 per thousand spread in delta(18)O values. Calibration materials were obtained by reacting dissolved potassium dihydrogen phosphate (KH(2)PO(4)) with water samples of various oxygen isotope compositions at 373 K. Validity of the method was tested by comparing the on-line results with those obtained by classical off-line sample preparation and dual inlet isotope measurement. In addition, silver phosphate precipitates were prepared from a collection of biogenic apatites with known delta(18)O values ranging from 12.8 to 29.9 per thousand (V-SMOW). Reproducibility of +/- 0.2 per thousand was obtained by the EA-Py-CF-IRMS method for sample sizes in the range 400-500 microg. Both natural and synthetic samples are remarkably well correlated with conventional (18)O/(16)O determinations. Silver phosphate is a very stable material and easy to degas and, thus, could be considered as a good candidate to become a reference material for the determination of (18)O/(16)O ratios of phosphate by high-temperature pyrolysis.     

Methods to adjust for the interference of N2O on delta13C and delta18O measurements of CO2 from soil mineralization

In this paper we present an overview of the present knowledge relating to methods that avoid interference of N2O on delta13C and delta18O measurements of CO2. The main focus of research to date has been on atmospheric samples. However, N2O is predominantly generated by soil processes. Isotope analyses related to soil trace gas emissions are often performed with continuous flow isotope ratio mass spectrometers, which do not necessarily have the high precision needed for atmospheric research. However, it was shown by using laboratory and field samples that a correction to obtain reliable delta13C and delta18O values is also required for a commercial continuous flow isotope ratio mass spectrometer. The capillary gas chromatography column of the original equipment was changed to a packed Porapak Q column. This adaptation resulted in an improved accuracy and precision of delta13C (standard deviation(Ghent): from 0.2 to 0.08 per thousand; standard deviation(Lincoln): from 0.2 to 0.13 per thousand) of CO2 for N2O/CO2 ratios up to 0.1. For delta18O there was an improvement for the standard deviation measured at Ghent University (0.13 to 0.08 per thousand) but not for the measurements at Lincoln University (0.08 to 0.23 per thousand). The benefits of using the packed Porapak Q column compared with the theoretical correction method meant that samples were not limited to small N(2)O concentrations, they did not require an extra N2O concentration measurement, and measurements were independent of the variable isotopic composition of N2O from soil.