Compound-specific Stable Isotope Analysis Research Articles

Comparison of δ13C and δ15N of ecologically relevant amino acids among beluga whale tissues

Ecological applications of compound-specific stable isotope analysis (CSIA) of amino acids (AAs) include 1) tracking carbon pathways in food webs using essential AA (AAESS) δ13C values, and 2) estimating consumer trophic position (TP) by comparing relative differences of ‘trophic’ and ‘source’ AA δ15N values. Despite the significance of these applications, few studies have examined AA-specific SI patterns among tissues with different AA compositions and metabolism/turnover rates, which could cause differential drawdown of body AA pools and impart tissue-specific isotopic fractionation. To address this knowledge gap, especially in the absence of controlled diet studies examining this issue in captive marine mammals, we used a paired-sample design to compare δ13C and δ15N values of 11 AAs in commonly sampled tissues (skin, muscle, and dentine) from wild beluga whales (Delphinapterus leucas). δ13C of two AAs, glutamic acid/glutamine (Glx, a non-essential AA) and, notably, threonine (an essential AA), differed between skin and muscle. Furthermore, δ15N of three AAs (alanine, glycine, and proline) differed significantly among the three tissues, with glycine δ15N differences of approximately 10 ‰ among tissues supporting recent findings it is unsuitable as a source AA. Significant δ15N differences in AAs such as proline, a trophic AA used as an alternative to Glx in TP estimation, highlight tissue selection as a potential source of error in ecological applications of CSIA-AA. Amino acids that differed among tissues play key roles in metabolic pathways (e.g., ketogenic and gluconeogenic AAs), pointing to potential physiological applications of CSIA-AA in studies of free-ranging animals. These findings underscore the complexity of isotopic dynamics within tissues and emphasize the need for a nuanced approach when applying CSIA-AA in ecological research.

Novel extraction methods and compound-specific isotope analysis of methoxychlor in environmental water and aquifer slurry samples

Multi-element compound-specific stable isotope analysis (ME-CSIA) allows monitoring the environmental behavior and transformation of most common and persistent contaminants. Recent advancements in analytical techniques have extended the applicability of ME-CSIA to organic micropollutants, including pesticides. Nevertheless, the application of this methodology remains unexplored concerning harmful insecticides such as methoxychlor, a polar organochlorine pesticide usually detected in soil and groundwater. This study introduces methods for dual carbon and chlorine compound-specific stable isotope analysis (δ13C-CSIA and δ37Cl-CSIA) of both methoxychlor and its metabolite, methoxychlor olefin, with a sensitivity down to 10 and 100 mg/L, and a precision lower than 0.3 and 0.5 ‰ for carbon and chlorine CSIA, respectively. Additionally, three extraction and preconcentration techniques suitable for ME-CSIA of the target pesticides at environmentally relevant concentrations were also developed. Solid-phase extraction (SPE) and liquid-solid extraction (LSE) effectively extracted methoxychlor (107 ± 27 % and 87 ± 13 %, respectively) and its metabolite (91 ± 27 % and 106 ± 14 %, respectively) from water and aquifer slurry samples, respectively, with high accuracy (Δδ13C and Δδ37Cl ≤ ± 1 ‰). Combining CSIA with polar organic chemical integrative samplers (POCISs) for the extraction of methoxychlor and methoxychlor olefin from water samples resulted in insignificant fractionation for POCIS-CSIA (Δδ13C ≤ ± 1 ‰). A relevant sorption of methoxychlor was detected within the polyethersulfones membranes of the POCISs resulting in temporary carbon isotope fractionation depending on the sorbed mass fraction during the first deployment days. This highlights the critical role of the interactions of polar analytes with POCIS sorbents and membranes in the performance of this method. Altogether, this study proposes a proof of concept for ME-CSIA of methoxychlor and its metabolites, opening the door for future investigations of their sources and transformation processes in contaminated sites.

Biostimulation of sulfate reduction for in-situ metal(loid) precipitation at an industrial site in Flanders, Belgium

A 30-month pilot study was conducted to evaluate the potential of in-situ metal(loid) removal through biostimulation of sulfate-reducing processes. The study took place at an industrial site in Flanders, Belgium, known for metal(loid) contamination in soil and groundwater. Biostimulation involved two incorporations of an organic substrate (emulsified vegetable oil) as electron donor and potassium bicarbonate to raise the pH of the groundwater by 1–1.5 units. The study focused on the most impacted permeable fine sand aquifer (8–9 m below groundwater level) confined by layers of non-permeable clay. The fine sands exhibited initially oxic conditions (50–200 mV), an acidic pH of 4.5 and sulfate concentrations ranging from 600 to 800 mg/L. At the central monitoring well, anoxic conditions (−200 to −400 mV) and a pH of 5.9 established shortly after the second substrate and reagent injection. Over the course of 12 months, there was a significant decrease in the concentration of arsenic (from 2500 to 12 μg/L), nickel (from 360 to <2 μg/L), zinc (from 78,000 to <2 μg/L), and sulfate (from 930 to 450 mg/L). Low levels of metal(loid)s were still present after 34 months (end of study). Mineralogical analysis indicated that the precipitates formed were amorphous in nature. Evidence for biologically driven metal(loid) precipitation was provided by compound specific stable isotope analysis of sulfate. In addition, changes in microbial populations were assessed using next-generation sequencing, revealing stimulation of native sulfate-reducing bacteria. These results highlight the potential of biostimulation for long-term in situ metal(loid) plume treatment/containment.

Enhanced dietary reconstruction of Korean prehistoric populations by combining δ13C and δ15N amino acids of bone collagen.

Compound specific stable isotope analysis of amino acids (CSIA-AA) is a powerful tool for determining dietary behaviors in complex environments and improving dietary reconstructions. Here, we conducted CSIA-AA on human (n = 32) and animal (n = 13) remains from two prehistoric archaeological sites (Mumun, Imdang) to assess in more detail the dietary sources consumed by prehistoric Korean populations. Results of estimated trophic position (TP) using Δ15NGlx-Phe show that the Imdang individuals consumed aquatic resources, as well as terrestrial resources. Principal component analysis (PCA) using δ13C and δ15N essential amino acid (EAA) values show that the Imdang humans closely cluster with game birds and terrestrial herbivores, whilst the Mumun humans closely cluster with C4 plants. Quantitative estimation by a Bayesian mixing model (MixSIAR) indicates that the Imdang humans derived a large proportion of their proteins from terrestrial animals and marine fish, whereas the main protein sources for the Mumun humans were C4 plants and terrestrial animals. Additionally, the comparison between the EAA and bulk isotope models shows that there is a tendency to overestimate the consumption of plant proteins when using bulk isotopic data. Our CSIA-AA approach reveals that in prehistoric Korea there were clear differences in human diets through time. This study adds to a growing body of literature that demonstrates the potential of CSIA-AA to provide more accurate estimations of protein consumption in mixed diets than previous bulk isotopic studies.

Disclosing phototransformation mechanisms of decabromodiphenyl ether (BDE-209) in different media by simulated sunlight: Implication by compound-specific stable isotope analysis.

As one of the typical brominated flame retardants, decabromodiphenyl ether (BDE-209) has been widely detected in environment. However, scarce information was available on BDE-209 phototransformation mechanisms in various media. In this study, compound-specific stable isotope analysis was first applied to investigate BDE-209 phototransformation in n-hexane, MeOH:H2O (v:v, 8:2), and simulated seawater by simulated sunlight. BDE-209 transformation followed pseudo-first-order kinetic, with degradation rate in the following of n-hexane (2.66 × 10-3min-1) > simulated seawater (1.83 × 10-3min-1) > MeOH:H2O (1.41 × 10-3min-1). Pronounced carbon isotope fractionation was first observed for BDE-209 phototransformation, with carbon isotope enrichment factors (εC) of -1.01 ± 0.14‰, -1.77 ± 0.26‰, -2.94 ± 0.38‰ in n-hexane, MeOH:H2O and simulated seawater, respectively. Combination analysis of products and stable carbon isotope, debromination with cleavage of C-Br bonds as rate-limiting step was the main mechanism for BDE-209 phototransformation in n-hexane, debromination and hydroxylation with cleavage of C-Br bonds as rate-limiting steps in MeOH:H2O, and debromination, hydroxylation and chlorination in simulated seawater. This present study confirmed that stable carbon isotope analysis was a robust method to discovery the underlying phototransformation mechanisms of BDE-209 in various solutions.

Root-derived resources fuel earthworms predominantly via bacterial and plant energy channels – Insights from bulk and compound-specific isotope analyses

Soil food webs rely on both brown and green energy, i.e., litter material and root-derived resources such as exudates. Earthworms have traditionally been viewed as macro-detritivores fuelled by brown energy and playing a central role in nutrient cycling and belowground energy flux. However, the role of root-derived resources for earthworm nutrition remains controversial. We studied the dietary contribution of root-derived resources from different plant functional groups to earthworms using bulk and compound-specific stable isotope analyses in a microcosm experiment with five earthworm species, grasses and legumes in monoculture and mixture, and an unplanted control by preventing leaf litter from entering the microcosms. The presence of plants consistently depleted bulk 13C values of the five earthworm species studied suggesting that root-derived carbon contributed to earthworm nutrition in each of the species. The response of bulk 15N values was less consistent, which is in line with the assumption that root-derived resources fuel soil food webs mainly via carbon-based root exudates. These observations were corroborated by 13C values of essential amino acids suggesting that most of the tissue carbon of earthworms was incorporated via bacterial- (∼60%) and plant-derived resources (∼30%), with the presence of legumes enhancing the incorporation via plant-derived resources. The high proportion of bacterial resources in the earthworms was consistent with the relative dominance of bacteria in the experimental soil and suggests that bacteria serve as important link in the acquisition of food resources. However, it remains an open question whether earthworms feed directly on soil bacteria, bacterial residues in soil or rely on nutritional supplementation by gut microorganisms. Overall, our results show that when root-derived resources are available, earthworms, as macro-detritivores, also incorporate green energy, with these resources being assimilated mainly via the bacterial energy channel, pointing to the importance of bacterial energy channelling in ecosystems with high earthworm biomass.

The influence of dynamic resources and stable isotope incorporation rates on aquatic consumer trophic position estimation

AbstractA key assumption in trophic position (TP) estimation using stable isotope analysis is that consumers are in isotopic equilibrium with their resources. Here, we assess the degree to which time‐varying resource dynamics and isotope incorporation rates of consumers influence consumer TP estimates across multiple trophic levels and aquatic ecosystems. We constructed a first‐order kinetics model to explore consumer stable isotope incorporation rates and modeled the effect on TP calculations using bulk and compound‐specific stable isotope data from previous experimental and observational studies. We found TP estimates of higher trophic level consumers are less accurate than lower trophic level consumers when applying bulk stable isotope analysis (BSIA) and using particulate organic matter as the stable isotope baseline. The accuracy of TP estimates depended on the time‐varying dynamics of the stable isotope baseline. Tertiary consumers had the highest TP estimation error, and this error was not eliminated by sampling tissues with fast incorporation rates (i.e., blood) in the tertiary consumer. Compound‐specific stable isotope analysis (CSIA) of individual amino acids was more accurate in estimating TP for all consumers and ecosystems compared to BSIA. Our analysis confirms that consideration for the dynamic nature of stable isotope ratios is necessary for accurate TP estimates. Finally, we show how first‐order kinetics models can provide a useful framework for integrating prey and consumer incorporation rates in stable isotope studies to improve TP estimates.

Assessing Biodegradation Processes of Atrazine in Constructed Wetland Using Compound-Specific Stable Isotope Analysis

To bridge the gap between lab-scale microcosm research and field application in the compound-specific stable isotope analysis (CSIA) of atrazine, we studied the characteristics of carbon and nitrogen isotope fractionation in the atrazine degradation processes within a constructed wetland. In the wetland, we observed multiple element (C, N) isotope fractionation parameters, such as kinetic isotope effects and dual isotope slopes. These parameters are very consistent with those observed in the cultivation of AtzA- or TrzN-harboring strains, suggesting a similarity in the pathway and reaction mechanism of atrazine biodegradation between the two settings. However, we recorded variable carbon (εC: −3.2 ± 0.6‰ to −4.3 ± 0.6‰) and nitrogen isotope fractionation (εN: 1.0 ± 0.3‰ to 2.2 ± 0.3‰) across different phases. This variance could lead to an over- or underestimation of the biodegradation extent of atrazine when employing the large or small enrichment factor of the carbon isotope. Intriguingly, the estimation accuracy improved considerably when using the enrichment factor (−4.6‰) derived from the batch cultivation of the pore water. This study advances the application of CSIA in tracking atrazine biodegradation processes in ecosystems, and it also underlines the importance of the careful selection and application of the enrichment factor in quantifying the intrinsic biodegradation of atrazine in ecosystems.

Compound-specific stable isotopes of amino acids reveal influences of trophic level and primary production sources on mercury concentrations in fishes from the Aleutian Islands, Alaska

Total mercury concentrations ([THg]) exceed thresholds of concern in some Steller sea lion (Eumetopias jubatus) tissues from certain portions of the Aleutian Islands, Alaska. We applied compound-specific stable isotope analyses of both carbon and nitrogen in amino acids from fish muscle tissue to quantify the proportional contributions of primary production sources and trophic positions of eight prey species (n = 474 total) that are part of Steller sea lion diets. Previous THg analyses of fish muscle, coupled with monomethylmercury analyses of a subset of samples, substantiated previous findings that fishes from the west of Amchitka Pass, a discrete oceanographic boundary of the Aleutian Archipelago, have higher muscle Hg concentrations relative to fishes from the east. The δ13C values of essential amino acids (EAAs) in fish muscle demonstrated that although most fishes obtained their EAAs primarily from algae, some species varied in the extent to which they relied on this EAA source. The δ15N values of phenylalanine (0.9 to 7.8 ‰), an indicator of the isotopic baseline of a food web, varied widely within and among fish species. Trophic position estimates, accounting for this baseline variation, were higher from the west relative to the east of the pass for some fish species. Trophic magnification slopes using baseline-corrected trophic position estimates indicated similar rates of Hg biomagnification to the east and west of Amchitka Pass. Multiple linear regression models revealed that trophic position was the most important driver of fish muscle [THg] with less variation explained by other parameters. Thus, higher trophic positions but not the rate of Hg biomagnification to the west of Amchitka Pass may play a role in the regional differences in both fish and Steller sea lion [THg]. Although, differences in Hg contamination and uptake at the base of the east and west food webs could not be excluded.

Compound-specific stable nitrogen isotope analysis of amino acids shows that bulk methods provide higher estimates of mercury biomagnification in the Gulf of St. Lawrence

Compound-specific stable isotope analysis of amino acids (CSIA-AA) provides a method to estimate baseline δ15N values of food chains, allowing less biased estimates of trophic positions for organisms. Greater accuracy in trophic positions can improve estimates of contaminant biomagnification. We calculated trophic positions with various CSIA-AA equations for four species of fish and northern gannets (Morus bassanus) from the Gulf of St. Lawrence. We examined the effect of CSIA-AA-derived trophic positions on mercury biomagnification metrics (trophic magnification factors (TMF) and biomagnification factors) and compared these with trophic position estimates and metrics obtained from traditional bulk stable isotope analysis. The TMFs for the CSIA-AA equations ranged from 10 to 19, and bulk stable isotope analysis produced TMFs of 43, one of the highest TMFs recorded yet in the literature. Biomagnification factors between prey and northern gannets ranged from 20 to 42 using dietary observations and stable isotope mixing models. Our study demonstrates that discrepancies in biomagnification assessed using different approaches may go undetected when using a single approach.

Efficient atrazine removal in bioaugmentation constructed wetland: Insight from stable isotope fractionation analysis

Constructed wetlands (CW) provides a sustainable approach to remove pesticides from agricultural or urban runoff, while the low efficiency of the herbicide atrazine remains a challenge. Focused on improving atrazine removal in CW, a bioaugmentation strategy was implemented using the mixed culture of atrazine-degrading bacteria. The results shown a significant increase in atrazine removal from 26.2 ± 6.9% to 60.9 ± 8.7–90.6 ± 4.1% during the bioaugmentation phases. To investigate in-situ degradation processes of atrazine, compound-specific stable isotope analysis (CSIA) was conducted. The carbon and nitrogen isotope fractionation pattern revealed atrazine was primarily degraded via a hydrolysis pathway mediated by microorganisms. The biodegradation extent of atrazine calculated from δ13C signatures suggested that 90% of atrazine was removed via the hydrolysis pathway, with only a small portion being removed abiotically. This was additionally supported by an increase in microbial diversity and the abundance of specific bacteria (such as Rhizobium sp. and Pseudomonas sp.) capable of degrading atrazine. Altogether, this study demonstrated the effectiveness of atrazine-degrading mixed culture and its feasibility on bioaugmentation atrazine removal in CW. This work highlights the potential of bioaugmentation in sustainable pesticide removal approaches and CSIA can contribute to an understanding of in-situ pesticide transformation processes in wetland systems.

Novel Insights into Uptake, Translocation, and Transformation Mechanisms of 2,2',4,4'-Tetra Brominated Diphenyl Ether (BDE-47) in Wheat (Triticum aestivum L.): Implication by Compound-Specific Stable Isotope and Transcriptome Analysis.

The uptake, translocation, and transformation of 2,2',4,4'-tetra brominated diphenyl ether (BDE-47) in wheat (Triticum aestivum L.) were comprehensively investigated by hydroponic experiments using compound-specific stable isotope analysis (CSIA) and transcriptome analysis. The results indicated that BDE-47 was quickly adsorbed on epidermis of wheat roots and then absorbed in roots via water and anion channels as well as an active process dependent on energy. A small fraction of BDE-47 in roots was subjected to translocation acropetally, and an increase of δ13C values in shoots than roots implied that BDE-47 in roots had to cross at least one lipid bilayer to enter the vascular bundle via transporters. In addition, accompanied by the decreasing concentrations, δ13C values of BDE-47 showed the increasing trend with time in shoots, indicating occurrence of BDE-47 transformation. OH-PBDEs were detected as transformation products, and the hydroxyl group preferentially substituted at the ortho-positions of BDE-47. Based on transcriptome analysis, genes encoding polybrominated diphenyl ether (PBDE)-metabolizing enzymes, including cytochrome P450 enzymes, nitrate reductases, and glutathione S-transferases, were significantly upregulated after exposure to BDE-47 in shoots, further evidencing BDE-47 transformation. This study first reported the stable carbon isotope fractionation of PBDEs during translocation and transformation in plants, and application of CSIA and transcriptome analysis allowed systematically characterize the environmental behaviors of pollutants in plants.

Carbon and hydrogen stable isotope fractionation due to monooxygenation of short-chain alkanes by butane monooxygenase of Thauera butanivorans Bu-B1211.

Multi element compound-specific stable isotope analysis (ME-CSIA) is a tool to assess (bio)chemical reactions of molecules in the environment based on their isotopic fingerprints. To that effect, ME-CSIA concepts are initially developed with laboratory model experiments to determine the isotope fractionation factors specific for distinct (bio)chemical reactions. Here, we determined for the first time the carbon and hydrogen isotope fractionation factors for the monooxygenation of the short-chain alkanes ethane, propane, and butane. As model organism we used Thauera butanivorans strain Bu-B1211 which employs a non-haem iron monooxygenase (butane monooxygenase) to activate alkanes. Monooxygenation of alkanes was associated with strong carbon and hydrogen isotope effects: εbulkC = -2.95 ± 0.5 ‰ for ethane, -2.68 ± 0.1 ‰ for propane, -1.19 ± 0.18 ‰ for butane; εbulkH = -56.3 ± 15 ‰ for ethane, -40.5 ± 2.3 ‰ for propane, -14.6 ± 3.6 ‰ for butane. This resulted in lambda (Λ ≈ εHbulk/εCbulk) values of 16.2 ± 3.7 for ethane, 13.2 ± 0.7 for propane, and 11.4 ± 2.8 for butane. The results show that ME-CSIA can be used to track the occurrence and impact of monooxygenase-dependent aerobic processes converting short-chain alkanes in natural settings like marine and terrestrial seeps, gas reservoirs, and other geological formations impacted by natural gas.

New insights into the use of Neolithic pottery in Guangxi of South China: organic residue analysis of experimental and archaeological pottery

As one of the world’s earliest pottery origins in Eurasia, the role of pottery during Neolithic period in South China remains unclear. Here, we present a new understanding on the utilization pattern of early pottery from the Neolithic sites of Dayan, Dingsishan and Zengpiyan in Guangxi province, China. Compound-specific stable isotope analysis of pottery and bone lipids show that a persistent use of terrestrial C3 and C4 resources throughout the pottery utilization history highlight the culinary practice and terrestrial exploitation during Neolithic period. Contrary to the traditional view that the primary use of these pottery was closely linked with aquatic resource processing, no aquatic biomarkers were found in the potsherds, yet these compounds were detected in the cooking experiments in this study, indicating that aquatic resources in these sites were probably processed or consumed in other ways. Moreover, a remarkable diachronic change from extensive use of C3 and C4 resources to the intensive use of certain kinds of C3 resources in different Neolithic stages, further indicating that an ecological tradition rooted in the subtropical and tropical environments as well as the Neolithic social evolution exerted great influence upon the initial production and the subsequent utilization of early pottery in prehistoric South China.

Integrating compound-specific stable isotope and enantiomer-specific analysis to characterize the isomeric and enantiomeric signatures of hexachlorocyclohexanes (HCHs) in paddy soils

Organic pollutants in paddy fields may undergo different processes from those in dryland due to the anaerobic environment. The integrated use of compound-specific stable isotope analysis (CSIA) and enantiomer-specific analysis is a promising technique for understanding the behavior and fate of organic pollutants in soils. In this study, soil samples were collected from paddy fields in three major rice cultivation regions of China, spanning a transect of 4000 km. The mean concentrations of ƩHCHs in paddy soils from the Taihu Plain were the highest (1.44 ng/g). The ratios of α-HCH/β-HCH (all below 11.8) and α-HCH/γ-HCH (92% below 4.64), as well as the enantiomeric fractions (EFs) of chiral α-HCH (mean of 0.81), reflected that the distribution of HCHs was affected by the use of both technical HCHs and lindane. The preferential depletion of (-)-α-HCH and pronounced carbon isotope fractionation of α-HCH (δ13C of −28.22 ± 0.92‰ −23.63 ± 1.89‰) demonstrated its effective transformation. Factors such as altitude, soil temperature, soil pH, soil conductivity and soil organic matter significantly influenced the fate and transformation of HCHs. The current study highlights the integrated application of CSIA and enantiomer-specific analysis to provide multiple lines of evidence for the transformation of HCHs in soils.

Transformation of Iminodi(methylene phosphonate) on Manganese Dioxides - Passivation of the Mineral Surface by (Formed) Mn2.

Aminopolyphosphonates (APPs) are strong chelating agents with growing use in industrial and household applications. In this study, we investigated the oxidation of the bisphosphonate iminodi(methylene phosphonate) (IDMP) - a major transformation product (TP) of numerous commercially used APPs and a potential precursor for aminomethylphosphonate (AMPA) - on manganese dioxide (MnO2). Transformation batch experiments at pH 6 revealed AMPA and phosphate as main TPs, with a phosphorus mass balance of 80 to 92% throughout all experiments. Our results suggest initial cleavage of the C-P bond and formation of the stable intermediate N-formyl-AMPA. Next, C-N bond cleavage leads to the formation of AMPA, which exhibits lower reactivity than IDMP. Reaction rates together with IDMP and Mn2+ sorption data indicate formation of IDMP-Mn2+ surface bridging complexes with progressing MnO2 reduction, leading to the passivation of the mineral surface regarding IDMP oxidation. Compound-specific stable carbon isotope analysis of IDMP in both sorbed and aqueous fractions further supported this hypothesis. Depending on the extent of Mn2+ surface concentration, the isotope data indicated either sorption of IDMP to the mineral surface or electron transfer from IDMP to MnIV to be the rate-limiting step of the overall reaction. Our study sheds further light on the complex surface processes during MnO2 redox reactions and reveals abiotic oxidative transformation of APPs by MnO2 as a potential process contributing to widespread elevated AMPA concentrations in the environment.

Protocols for sample preparation and compound-specific stable-isotope analyses (δ2H, δ13C) of fatty acids in biological and environmental samples

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of CH bonds in acyl chains, hydrogen (δ2H) and carbon (δ13C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on δ values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis.•Protocol from obtaining a sample to standardized fatty acid specific δ2H and δ13C values.•Separate GC analysis procedures for C and H are recommended for optimal performance.

A review of recent compound-specific isotope analysis studies applied to food authentication.

Compound-specific stable isotope analysis (CSIA) of food products is a relatively new and novel technique used to authenticate food and detect adulteration. This paper provides a review of recent on-line and off-line CSIA applications of plant and animal origin foods, essential oils and plant extracts. Different food discrimination techniques, applications, scope, and recent studies are discussed. CSIA δ13C values are widely used to verify geographical origin, organic production, and adulteration. The δ15N values of individual amino acids and nitrate fertilizers have proven effective to authenticate organic foods, while δ2H and δ18O values are useful to link food products with local precipitation for geographical origin verification. Most CSIA techniques focus on fatty acids, amino acids, monosaccharides, disaccharides, organic acids, and volatile compounds enabling more selective and detailed origin and authentication information than bulk isotope analyses.. In conclusion, CSIA has a stronger analytical advantage for the authentication of food compared to bulk stable isotope analysis, especially for honey, beverages, essential oils, and processed foods.

Holocene landscape evolution, palaeoclimate and human impact in the Fotsch Valley, Stubai Alps, Austria: Interrogating biomarkers, stable isotopes, macrofossils and palynological indicators from a subalpine mire archive

Peatlands are receiving increasing attention in palaeoenvironmental research and represent very useful terrestrial archives for reconstructing vegetation, climate and human history. Previous palaeoenvironmental studies in the Fotsch Valley, Stubai Alps, Austria, focused on geoarchaeological investigations on the Ullafelsen representing a very important prehistorical encampment site used by Mesolithic hunter-gatherers (10.9–9.5 cal. kyr BP). In order to contribute to a better understanding of the landscape evolution of the Fotsch Valley, we here studied the close-by subalpine ‘Potsdamer Hütte Mire’ by applying radiocarbon dating as well as elemental, biomarker, compound-specific stable isotope, palynological and macrofossil analyses on bulk peat samples. The calculated age-depth model using R Bacon indicates the beginning of peat formation during the Early Holocene and shows a strongly reduced peat accumulation rate (PAR) from 170 to 121 cm depth (8.5–2.1 cal. kyr BP) and/or a striking hiatus. Results of leaf wax-derived n-alkane biomarkers as well as macrofossils and palynological indicators reflect the local presence of coniferous trees and the synchronous expansion of deciduous trees during the Early Holocene. The above-mentioned strongly reduced PAR and/or hiatus coincides with the Neolithic, the Bronze and the Iron Age, and goes hand in hand with strong changes in vegetation and an increase of micro-charcoal and black carbon. Despite age uncertainties, these changes can be explained with strongly increasing human and livestock activities in form of deforestation, domestic fires and the beginning of Alpine pastoralism. The latter is confirmed by the finding of pasture and cultural indicator pollen (Cerealia-type, Rumex, Plantago lanceolata, Poaceae) occurring since the Middle to Late Bronze Age. The oxygen isotope composition of sugar biomarkers (δ18Osugars) likely reflects the dry versus humid climatic variability associated with the Holocene climatic optimum during the Mesolithic, the Roman Age, the Late Antique Little Ice Age, the Middle Ages and the Little Ice Age.

Development and Application of a Purification Method for the Determination of Three EDCs Isotopes in Sediments and Water

Compound-specific stable isotope analysis (CSIA) is an efficient method for source apportionment and the identification of the transformation process in organic compounds. However, most studies of CSIA are still limited to laboratory experiments. Few studies used have CSIA in an in situ environment due to the complexity of environmental samples. Therefore, a purification method for analyzing the carbon isotope ratios of three phenolic endocrine disrupting compounds (EDCs) (nonylphenols (NPs), octylphenol (OP), and bisphenol A(BPA)) in sediment and water samples was developed in this study. The silica gel column was used to isolate EDCs from complex matrices with multiple organic solvents. Gas chromatography/mass spectrometry was used to quantify the targeted EDCs and analyze the purity of the extracts in full-scan mode. The interfering peaks disappeared, the baseline was sharply reduced, and all the target compounds appeared as single peaks in the chromatogram after purification. Analyzing the standard samples with known isotope ratios showed that the purification treatment did not cause isotope fractionation. The isotopic difference before and after purification was less than 0.04. The method was successfully used to analyze the isotope composition of BPA, OP, and NPs in river water and sediments in the Guangzhou River, Pearl River Delta, South China. Sewage discharge significantly affected the carbon isotope values of BPA, OP and NPs in Guangzhou rivers, suggesting that sewage discharge is the main source of EDCs in the Guangzhou rivers. There is a significant correlation between the isotopic values and concentrations of OP and NPs in sediments, indicating that they may undergo chemical transformation.