Combining stable isotopes with contamination indicators: A method for improved investigation of nitrate sources and dynamics in aquifers with mixed nitrogen inputs.

Excessive nitrate (NO3-) concentration in groundwater raises health and environmental issues that must be addressed by all European Union (EU) member states under the Nitrates Directive and the Water Framework Directive. The identification of NO3- sources is critical to efficiently control or reverse NO3- contamination that affects many aquifers. In that respect, the use of stable isotope ratios 15N/14N and 18O/16O in NO3- (expressed as δ15N-NO3- and δ18O-NO3-, respectively) has long shown its value. However, limitations exist in complex environments where multiple nitrogen (N) sources coexist. This two-year study explores a method for improved NO3- source investigation in a shallow unconfined aquifer with mixed N inputs and a long established NO3- problem. In this tillage-dominated area of free-draining soil and subsoil, suspected NO3- sources were diffuse applications of artificial fertiliser and organic point sources (septic tanks and farmyards). Bearing in mind that artificial diffuse sources were ubiquitous, groundwater samples were first classified according to a combination of two indicators relevant of point source contamination: presence/absence of organic point sources (i.e. septic tank and/or farmyard) near sampling wells and exceedance/non-exceedance of a contamination threshold value for sodium (Na+) in groundwater. This classification identified three contamination groups: agricultural diffuse source but no point source (D+P-), agricultural diffuse and point source (D+P+) and agricultural diffuse but point source occurrence ambiguous (D+P±). Thereafter δ15N-NO3- and δ18O-NO3- data were superimposed on the classification. As δ15N-NO3- was plotted against δ18O-NO3-, comparisons were made between the different contamination groups. Overall, both δ variables were significantly and positively correlated (p < 0.0001, rs = 0.599, slope of 0.5), which was indicative of denitrification. An inspection of the contamination groups revealed that denitrification did not occur in the absence of point source contamination (group D+P-). In fact, strong significant denitrification lines occurred only in the D+P+ and D+P± groups (p < 0.0001, rs > 0.6, 0.53 ≤ slope ≤ 0.76), i.e. where point source contamination was characterised or suspected. These lines originated from the 2-6‰ range for δ15N-NO3-, which suggests that i) NO3- contamination was dominated by an agricultural diffuse N source (most likely the large organic matter pool that has incorporated 15N-depleted nitrogen from artificial fertiliser in agricultural soils and whose nitrification is stimulated by ploughing and fertilisation) rather than point sources and ii) denitrification was possibly favoured by high dissolved organic content (DOC) from point sources. Combining contamination indicators and a large stable isotope dataset collected over a large study area could therefore improve our understanding of the NO3- contamination processes in groundwater for better land use management. We hypothesise that in future research, additional contamination indicators (e.g. pharmaceutical molecules) could also be combined to disentangle NO3- contamination from animal and human wastes.

Evaluating the utility of 15N and 18O isotope abundance analyses to identify nitrate sources: A soil zone study.

(15)N and (18)O isotope abundance analyses in nitrate (NO(3)(-)) (expressed as δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) values respectively) have often been used in research to help identify NO(3)(-) sources in rural groundwater. However, questions have been raised over the limitations as overlaps in δ values may occur between N source types early in the leaching process. The aim of this study was to evaluate the utility of using stable isotopes for nitrate source tracking through the determination of δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) in the unsaturated zone from varying N source types (artificial fertiliser, dairy wastewater and cow slurry) and rates with contrasting isotopic compositions. Despite NO(3)(-) concentrations being often elevated, soil-water nitrate poorly mirrored the (15)N content of applied N and therefore, δ(15)N-NO(3)(-) values were of limited assistance in clearly associating nitrate leaching with N inputs. Results suggest that the mineralisation and the nitrification of soil organic N, stimulated by previous and current intensive management, masked the cause of leaching from the isotopic prospective. δ(18)O-NO(3)(-) was of little use, as most values were close to or within the range expected for nitrification regardless of the treatment, which was attributed to the remineralisation of nitrate assimilated by bacteria (mineralisation-immobilisation turnover or MIT) or plants. Only in limited circumstances (low fertiliser application rate in tillage) could direct leaching of synthetic nitrate fertiliser be identified (δ(15)N-NO(3)(-)<0‰ and δ(18)O-NO(3)(-)>15‰). Nevertheless, some useful differences emerged between treatments. δ(15)N-NO(3)(-) values were lower where artificial fertiliser was applied compared with the unfertilised controls and organic waste treatments. Importantly, δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) variables were negatively correlated in the artificial fertiliser treatment (0.001≤p≤0.05, attributed to the varying proportion of fertiliser-derived and synthetic nitrate being leached) while positively correlated in the dairy wastewater plots (p≤0.01, attributed to limited denitrification). These results suggest that it may be possible to distinguish some nitrate sources if analysing correlations between δ variables from the unsaturated zone. In grassland, the above correlations were related to N input rates, which partly controlled nitrate concentrations in the artificial fertiliser plots (high inputs translated into higher NO(3)(-) concentrations with an increasing proportion of fertiliser-derived and synthetic nitrate) and denitrification in the dairy wastewater plots (high inputs corresponded to more denitrification). As a consequence, nitrate source identification in grassland was more efficient at higher input rates due to differences in δ values widening between treatments.

2H stable isotope analysis of human tooth enamel: a new tool for forensic human provenancing?

Stable isotope analysis of biogenic tissues such as tooth enamel and bone mineral has become a well-recognised and increasingly important method for determining the provenance of human remains, and it has been used successfully in bio-archaeological studies as well as forensic investigations. In particular, (18)O and (2)H stable isotope signatures of bone and hair, respectively, are well-established proxies of climate (temperature) and source water and are therefore considered as indicators of geographic life trajectories of animals and humans. While the methodology for (2)H analysis of human hair, fingernails, and bone collagen is currently used to determine human provenance, i.e. geographic origin and identify possible migration patterns, studies involving the analysis of (2)H in tooth enamel appear to be nonexistent in the scientific literature. Ground tooth enamel was analysed by continuous-flow isotope ratio mass spectrometry (IRMS) coupled on-line to a high-temperature conversion elemental analyser (TC/EA). An array of tooth enamel samples from archaeological and modern teeth has been analysed under different experimental conditions, and the results of this proof-of-concept study are presented. While no significant differences in (2)H abundance were noted as a result of H exchange studies or different sample preparation protocols, no significant differences or trends in measured δ(2)H-values were observed either with regard to known differences in geographical provenance. We concluded that the δ(2)H-values obtained from tooth enamel could not be used as proxy for a person's geographical origin during adolescence.

Measurement at the field scale of soil delta13C and delta15N under improved grassland.

Variations in natural abundance of carbon (C) and nitrogen (N) stable isotopes are widely used as tools for many aspects of scientific research. By examining variations in the ratios of heavy to light stable isotopes, information can be obtained as to what physical, chemical and biological processes may be occurring. The spatial heterogeneity of soil delta(15)N- and delta(13)C-values across a range of scales and under different land use have been described by a number of researchers and the natural abundances of the C and N stable isotopes in soils have been found to be correlated with many factors including hydrology, topography, land use, vegetation cover and climate. In this study the Latin square sampling +1 (LSS+1) sampling method was compared with a simple grid sampling approach for delta(13)C and delta(15)N measurement at the field scale. A set of 144 samples was collected and analysed for delta(15)N and delta(13)C from a 12 x 12 grid (in a 1 ha improved grassland field in south-west England). The dimension of each cell of the grid was approximately 11 x 6 m. The 12 x 12 grid was divided into four 6 x 6 grids and the LSS+1 sampling technique was applied to these and the main 12 x 12 grid for a comparison of sample means and variation. The LSS+1 means from the 12 x 12 grid and the four 6 x 6 grids compared well with the overall grid mean because of the low variation within the field. The LSS+1 strategy (13 samples) generated representative samples from the 12 x 12 grid, and hence would be an acceptable method for sampling similar plots for the measurement of mean isotopic composition.

Stable isotope analysis of white paints and likelihood ratios.

Architectural paints are commonly found as trace evidence at scenes of crime. Currently the most widely used technique for the analysis of architectural paints is Fourier Transformed Infra-Red Spectroscopy (FTIR). There are, however, limitations to the forensic analysis of white paints, and the ability to discriminate between samples. Isotope ratio mass spectrometry (IRMS) has been investigated as a potential tool for the analysis of architectural white paints, where no preparation of samples prior to analysis is required. When stable isotope profiles (SIPs) are compared, there appears to be no relationship between paints from the same manufacturer, or between paints of the same type. Unlike existing techniques, IRMS does not differentiate resin samples solely on the basis of modifier or oil-type, but exploits additional factors linked to samples such as geo-location where oils added to alkyd formulations were grown. In combination with the use of likelihood ratios, IRMS shows potential, with a false positive rate of 2.6% from a total of 1275 comparisons.

Isotopic evidence for the provenance and turnover of organic carbon by soil microorganisms in the Antarctic dry valleys.

The extremely cold and arid Antarctic dry valleys are one of the most environmentally harsh terrestrial ecosystems supporting organisms in which the biogeochemical transformations of carbon are exclusively driven by microorganisms. The natural abundance of (13)C and (15)N in source organic materials and soils have been examined to obtain evidence for the provenance of the soil organic matter and the C loss as CO(2) during extended incubation (approximately 1200 days at 10 degrees C under moist conditions) has been used to determine the potential decay of soil organic C. The organic matter in soils remote from sources of liquid water or where lacustrine productivity was low had isotope signatures characteristic of endolithic (lichen) sources, whereas at more sheltered and productive sites, the organic matter in the soils that was a mixture mainly lacustrine detritus and moss-derived organic matter. Soil organic C declined by up to 42% during extended incubation under laboratory conditions (equivalent to 50-73 years in the field on a thermal time basis), indicating relatively fast turnover, consistent with previous studies indicating mean residence times for soil organic C in dry valley soils in the range 52-123 years and also with recent inputs of relatively labile source materials.

Forensic analysis of wooden safety matches -- a case study.

In this case, an individual was suspected of attempting to burn materials potentially relating to a murder case. A number of spent and unspent matches were seized at the scene by police for forensic examination. Coincidentally, a police raid at the suspect's house revealed a number of matchboxes, all of the same brand, containing matches that had a visual similarity to those recovered at the scene. Stable Isotope Profiling (SIP) was used to assess whether matches could either be distinguished or shown to be indistinguishable by 13C and 2H isotopic composition. These results were then compared to those from the X-ray diffraction (XRD) analysis of match heads and microscopy of the wood. SIP showed the scene matches and seized matches to be different, which was confirmed by XRD and microscopy analyses.

The role of stable isotopes in human identification: a longitudinal study into the variability of isotopic signals in human hair and nails.

Recent natural catastrophes with large-scale loss of life have demonstrated the need for a new technique to provide information for disaster victim identification when DNA methods fail to yield the identification of an individual, or in other situations where authorities need to determine the recent geographical life history of people. The latter may be in relation to the identification of individuals detained on suspicion of terrorism or in relation to people-trafficking or smuggling. One proposed solution is the use of stable isotope profiling (SIP) using isotope ratio mass spectrometry (IRMS). Exploiting the link between the isotopic signal of dietary components and the isotopic composition of body tissue, the aim of this study was to refine a non-invasive method of analysing human material such as scalp hair and fingernails using SIP and to assess the degree of natural variability in these profiles. Scalp hair and fingernail samples were collected from British and non-British volunteers at Queen's University Belfast every 2 weeks for a minimum of 8 months. Samples were analysed using IRMS to determine their isotopic composition for 13C, 15N, 2H and 18O. The results of this longitudinal study yielded information on the natural variability of the isotopic composition of these tissues. The data demonstrate the relatively low degree of natural variation in the 13C/15N isotopic abundance of scalp hair and fingernails whilst greater variations were recorded in the hydrogen and oxygen values of the same samples. The 15N and 18O values of nail are noticeably more variable than that of scalp hair from the same subject. A hypothesis explaining this trend is put forward based on the faster rate of formation of hair than of nails. This means that there is less time for the compounds forming hair to be affected by biochemical processes that could alter their isotopic signature.

Stable isotope analysis of safety matches using isotope ratio mass spectrometry--a forensic case study.

Isotope ratio mass spectrometry (IRMS) was used to assess what contribution the technique could make towards the comparative analysis of matchstick samples within the 'normal' framework of a forensic investigation. A method was developed to allow the comparison of samples submitted as a result of an investigation, with the added advantage of rapid sample turn-around expected within this field. To the best of our knowledge this is the first time that wooden safety matches have been analysed using IRMS. In this particular case, bulk stable isotope analysis carrried out on a 'like-for-like' basis could demonstrate conclusively that matches seized from a suspect were different from those collected at the scene of crime. The maximum delta13C variability observed within one box was 2.5 per thousand, which, in conjunction with the error of measurement, was regarded to yield too wide an error margin as to permit differentiation of matchsticks based on 13C isotopic composition alone given that the 'natural' 13C abundance in wood ranges from -20 to -30 per thousand. However, from the delta2H values obtained for crime scene matches and seized matches of -114.5 per thousand and -65 per thousand, respectively, it was concluded that the matches seized were distinctly different from those collected at the crime scene.

13C-Isotope ratio mass spectrometry as a potential tool for the forensic analysis of white architectural paint: a preliminary study.

Paints have a dual role in society, to protect materials from environmental agents such as ultraviolet light, moisture and oxygen, and to make painted materials look more attractive. Variability in paint samples is often due to binder and pigment type within the sample. The most common resin used in decorative paints is drying oil alkyd resin, which incorporates soybean oil and vinyl acrylic based latexes. Traditional analytical methods used by forensic scientists may be able to say whether two paint samples are indistinguishable but cannot conclusively say that they both originate from the same source. To find out if isotopic composition can provide an added dimension of information, 28 different white architectural paints were analysed for (13)C abundance using isotope ratio mass spectrometry. In addition, variations in application, drying time and thickness were also investigated to assess the discriminatory power of (13)C data from white paints with an unknown history. Preliminary results indicate that this method could aid screening of paint samples.

Sequential extracts of human bone show differing collagen synthetic rates.

Type I collagen is the major bone protein. Little is known quantitatively about human bone collagen synthesis in vivo, despite its importance for the understanding of bone formation and turnover. Our aim was to develop a method that could be used for the physiological and pathophysiological investigation of human bone collagen synthesis. We have carried out preliminary studies in patients undergoing hip replacement and in pigs to validate the use of the flooding dose method using (13)C- or (15)N-labelled proline and we have now refined our techniques to allow them to be used in a normal clinical or physiological setting. The results show that the application of a flooding dose causes bone free-proline labelling to equilibrate with that of blood in pigs and human beings, so that only 150 mg of bone will provide enough sample to prepare and measure the labelling of three fractions of bone collagen (dissolved in NaCl, acetic acid and pepsin/acetic acid) which have the same relative labelling (1.0:0.43:0.1) as measured by GC-combustion-isotope ratio MS. The rates of incorporation were substantially faster than in skeletal muscle samples taken at the same time. The results suggest that different fractions of human bone collagen turnover at markedly higher rates than had been previously considered. This approach should allow us to discover how growth and development, food, activity and drugs affect bone collagen turnover and to measure the effects on it of ageing and bone disease.

Applied gas chromatography coupled to isotope ratio mass spectrometry.

Compound-specific isotope analysis (CSIA) by isotope ratio mass spectrometry (IRMS) following on-line combustion (C) of compounds separated by gas chromatography (GC) is a relatively young analytical method. Due to its ability to measure isotope distribution at natural abundance level with great accuracy and high precision, GC-C-IRMS has increasingly become the method of choice in authenticity control of foodstuffs and determination of origin in archaeology, geochemistry, and environmental chemistry. In combination with stable isotope labelled compounds, GC-C-IRMS is also used more and more in biochemical and biomedical application as it offers a reliable and risk-free alternative to the use of radioactive tracers. The literature on these topics is reviewed from the advent of commercial GC-C-IRMS systems in 1990 up to the beginning of 1998. Demands on sample preparation and quality of GC separation for GC-C-IRMS are discussed also.

Use of gas chromatography-combustion-isotope ratio mass spectrometry in nutrition and metabolic research.

Linking gas chromatography via an on-line combustion interface to isotope ratio mass spectrometry has opened the door to high-precision compound-specific isotope analysis. For this reason, gas chromatography-combustion-isotope ratio mass spectrometry is now increasingly employed in metabolic and nutritional research because it offers a reliable and risk-free alternative to the use of radioactive tracers.

Use of a thick-film capillary column for the analysis of organic acids in body fluids.

An improved method for the identification and quantification of organic acids in body fluids employing capillary gas chromatography-mass spectrometry has been developed. A thick-film capillary column, that combines the properties of a capillary column with those of a megabore column, has been successfully introduced into an existing method. Analysis over a concentration range from 1 mumol/l to 500 mumol/l body fluid is possible. This permits the assay of samples that are usually obtained in small volumes, e.g. cerebrospinal fluid.

Physiology and pathophysiology of organic acids in cerebrospinal fluid.

Concentrations of organic acids in cerebrospinal fluid (CSF) appear to be directly dependent upon their rate of production in the brain. There is evidence that the net release of short-chain monocarboxylic acids from the brain is a major route for removing these products of cerebral metabolism. Concentrations of organic acids in blood and CSF are largely independent of each other. Quantitative reference values for the concentrations of organic acids in CSF and plasma as well as ratios of individual organic acids between CSF and plasma were determined in 35 pairs of samples from paediatric patients. Over 25 organic acids were quantifiable in all or in the majority of CSF and/or plasma specimens (limit of detection 1 mumol/L). There were substantial differences in the CSF/plasma ratios between subgroups of organic acids. Metabolites related to fatty-acid oxidation were present in CSF in substantially less amounts than in plasma. Organic acids related to carbohydrate and energy metabolism and to amino acid degradation were present in CSF in the same amounts as or slightly smaller amounts than in plasma. Finally, some organic acids were found in substantially higher amounts in CSF than in plasma, e.g. glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate. Studies of organic acids in CSF and plasma samples are presented from patients with 'cerebral' lactic acidosis, disorders of propionate and methylmalonate metabolism, glutaryl-CoA dehydrogenase deficiency and L-2-hydroxy-glutaric aciduria. It became apparent that derangements of organic acids in the CSF may occur independently of the systemic metabolism. Quantitative organic acid analysis in CSF will yield new information on the pathophysiology in the central nervous system (CNS) of these disorders and may prove necessary for successful monitoring of treatment of organoacidopathies, which present mainly with neurological disease. For example, in glutaryl-CoA dehydrogenase deficiency the urinary excretion of glutarate appears to be an inadequate parameter for monitoring the effect of dietary therapy, without plasma and CSF determinations. In L-2-hydroxyglutaric aciduria the elevation of L-2-hydroxyglutarate was found to be greater in CSF than in plasma. In addition, some other organic acids, glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate, were also elevated in the CSF of the patients out of proportion to normal levels in plasma and urine. High concentrations of an unknown compound, which was tentatively identified as 2,4-dihydroxyglutarate, were found in the CSF of patients with L-2-hydroxyglutaric aciduria.(ABSTRACT TRUNCATED AT 400 WORDS)