Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ13C and δ2Hn analyses.

Lipids, with fatty acids (FA) as a crucial subset, have become a focal point for diverse medical, physiological, and ecological studies. However, a comprehensive assessment of the various pre-analytical FA extraction methods published in the scientific literature remains lacking. In this study, we examined the efficacy of seven well-established sample preparation methods, specifically focusing on their effectiveness in total lipid and fatty acid extraction and their impact on compound-specific stable hydrogen (δ2H) and carbon (δ13C) isotope values. We also considered the repercussions of FA removal efficacy on residual bulk tissue δ2Hn analysis, because lipids typically have low δ2H values. Our findings showed that in most cases chloroform-based extraction methods outperformed those without chloroform. While discrepancies were not as evident for smaller organisms, such as plankton, marked variations were discernible in the extraction efficiencies for muscle and liver samples, which was also manifested in the residual bulk tissue δ2Hn results. Notably, most extraction methods had little effect on specific δ13C or δ2H isotope values of FA; instead, an emphasis should be on using an extraction method that achieves optimal baseline peak separation of the chromatograms for C and H isotope measurements.

Compound-specific stable isotope analyses of fatty acids indicate feeding zones of zooplankton across the water column of a subalpine lake.

Spatial and temporal zooplankton feeding dynamics across the water column of lakes are key for understanding site-specific acquisition of diet sources. During this 6-week lake study, we examined stable carbon (δ13C) and nitrogen (δ15N) isotopes and conducted compound-specific fatty acid (FA) stable isotope analysis (CSIA) of edible seston in the epi-, meta-, and hypolimnion, and zooplankton of Lake Lunz, Austria. We predicted that CSIA of essential FA can discern the foraging grounds of zooplankton more accurately than the commonly used bulk stable isotopes. The δ13C and δ15N values of seston from different lake strata were similar, whereas a dual CSIA approach using stable carbon and hydrogen isotopes of FA (δ13CFA and δ2HFA) provided sufficient isotopic difference in essential FA to discern different lake strata-specific diet sources throughout the study period. We present a CSIA model that suggests strata-specific foraging grounds for different zooplankton groups, indicating higher preference of cladocerans for feeding on epilimnetic diet sources, while calanoid copepods retained more hypolimnetic resources. The CSIA approach thus yields strata-specific information on foraging strategies of different zooplankton taxa and provides more details on the spatial and temporal trophodynamics of planktonic food webs than commonly used bulk stable isotopes.

Assessing the performance of international laboratories analysing the stable isotope composition (δ15 N, δ18 O, δ17 O) of nitrate in environmental waters.

RATIONALE: Stable-isotope analyses of nitrate (NO3 - ) in various water sources are crucial for understanding nitrogen pollution and its impact on aquatic ecosystems. We evaluated the accuracy and precision of stable-isotope analyses of nitrate conducted by international laboratories. METHODS: Six samples with nitrate (2 mg L-1 NO3 - -N) were sent to 47 laboratories. The NO3 - had a 30-50 ‰ range of δ values for δ15 N, δ18 O and δ17 O. One blind duplicate evaluated reproducibility and the effect of water δ18 O. Laboratories used diverse methods to convert nitrate to N2 O, N2 , CO or O2 for stable-isotopic measurements (microbial, cadmium, titanium and elemental analysis) and isotope-ratio mass spectrometry or laser-based technologies. RESULTS: Thirty-six international laboratories (83 %) reported results; however, 23 % did not analyze the test samples due to technical difficulties. Of the reporting laboratories, 79 % and 84 % produced accurate δ15 N and δ18 O results falling within ±0.8 ‰ and ±1.1 ‰ of the benchmark values, respectively. Three laboratories produced only outliers. The duplicate revealed most laboratories gave internally reproducible results at appropriate analytical precision. For δ17 O, six laboratories reported results, but 67 % could not reproduce results within their claimed analytical measurement precision. One complication is a lack of nitrate reference materials for δ17 O. CONCLUSIONS: Analyst experience contributed to better performance, and underperformance was from compromised standards or inappropriate δ range of working reference materials. The stable isotope community must develop new nitrate reference materials for δ15 N spanning -20 ‰ to +80 ‰ and new materials for δ17 O.

Assessment of rapid low-cost isotope (δ15 N, δ18 O) analyses of nitrate in fruit extracts by Ti(III) reduction to differentiate organic from conventional production.

RATIONALE: The isotopic composition (δ15 N, δ18 O) of nitrate in fruits and vegetables differentiates organic from conventional food production practices. Organic systems do not use synthetic nitrate fertilizers high in 18 O and low in 15 N and thereby help reveal producers' fertilization claims. Isotope analyses of nitrate extracted from fruits and vegetables are done by bacterial reduction which is costly and by specialized laboratories. Rapid, low-cost methods are needed to promulgate nitrate isotope analyses of food products to support organic food product certification and to verify the authenticity of production claims. METHODS: Fresh strawberry samples were obtained from certified organic and conventional growers in Andalucía, Spain. We applied a new, rapid, one-step Ti(III) reduction method to convert the nitrate from strawberry extracts to N2 O gas for headspace isotope analyses using isotope-ratio mass spectrometry. Using the Ti(III) reduction method, 70 samples, controls and references were prepared and analyzed for NO3 - , δ15 N and δ18 O per 48 h. We also analyzed extracts and solids for anions and cations and for bulk δ15 N for multivariate chemometric evaluation. RESULTS: The Ti(III)-based isotope analyses of nitrate in strawberry extracts revealed clear differentiation between organic and conventional production with mean δ18 O and δ15 N values of +18.3 ± 1.2 ‰ and +17.6 ± 1.2 ‰ versus +28.2 ± 4.5 ‰ and +14.9 ± 3.0 ‰, respectively. The δ15 N of strawberry dry mass differed slightly (+3.0 ± 1.4 ‰ versus +4.0 ± 1.4 ‰) between organic and conventional samples, respectively. Chemometric analyses of nitrate isotopes and extract chemistry revealed that the δ18 O of nitrate along with δ15 N and Ca2+ fully differentiated organic from conventional strawberry production. CONCLUSIONS: Our results show the Ti(III) reduction method provides a new low-cost and rapid analytical method to facilitate compound-specific δ15 N and δ18 O isotope analyses of nitrate in selected fruit types, and likely other food products, for the purposes of assessing nitrate fertilization practices of organic versus conventional production claims and to support authenticity investigations.

Assessment of Compound-Specific Fatty Acid δ13C and δ2H Values to Track Fish Mobility in a Small Sub-alpine Catchment.

Methods for identifying origin, movement, and foraging areas of animals are essential for understanding ecosystem connectivity, nutrient flows, and other ecological processes. Telemetric methods can provide detailed spatial coverage but are limited to a minimum body size of specimen for tagging. In recent years, stable isotopes have been increasingly used to track animal migration by linking landscape isotope patterns into movement (isoscapes). However, compared to telemetric methods, the spatial resolution of bulk stable isotopes is low. Here, we examined a novel approach by evaluating the use of compound-specific hydrogen and carbon stable isotopes of fatty acids (δ2HFA and δ13CFA) from fish liver, muscle, brain, and eye tissues for identifying site specificity in a 254 km2 sub-alpine river catchment. We analyzed 208 fish (European bullhead, rainbow trout, and brown trout) collected in 2016 and 2018 at 15 different sites. δ13CFA values of these fish tissues correlated more among each other than those of δ2HFA values. Both δ2HFA and δ13CFA values showed tissue-dependent isotopic fractionation, while fish taxa had only small effects. The highest site specificity was for δ13CDHA values, while the δ2H isotopic difference between linoleic acid and alpha-linolenic acid resulted in the highest site specificity. Using linear discrimination analysis of FA isotope values, over 90% of fish could be assigned to their location of origin; however, the accuracy dropped to about 56% when isotope data from 2016 were used to predict the sites for samples collected in 2018, suggesting temporal shifts in site specificity of δ2HFA and δ13CFA. However, the predictive power of δ2HFA and δ13CFA over this time interval was still higher than site specificity of bulk tissue isotopes for a single time point. In summary, compound-specific isotope analysis of fatty acids may become a highly effective tool for assessing fine and large-scale movement and foraging areas of animals.

Performance of low-cost stainless-steel beverage kegs for long-term storage integrity and easy dispensing of water isotope (δ18 O, δ2 H) reference materials.

RATIONALE: A widespread problem observed in global water isotope (δ18 O, δ2 H) proficiency tests is compromised working reference materials due to storage-dispensing evaporation effects. Proper storage requires no evaporation or leakage, which causes isotopic drift and bias. Surveys by the International Atomic Energy Agency (IAEA) show most isotope laboratories use glass or plastic bottles to store working reference materials, with frequent opening and closings that pose evaporation risks. Practical small (ca. 2-5 L) storage-dispensing solutions free of air exposure, evaporation, and leakage are needed. We also tested several smaller-scale bottles for day-to-day aliquots. METHODS: We tested low-cost, conveniently sized (4 L) adaptations of a common stainless-steel beverage keg with a liquid dispenser, with minor modifications to facilitate low-flow dispensing and pressurization (1-2 bar) with Ar or N2 . We tested three kegs (100%, 75%, 50% initial fills) for a 2-year period along with monthly dispensing to assess long-term storage viability for maintaining δ18 O and δ2 H integrity and dispensing, and day-to-day aliquot bottles for 6 months. RESULTS: Test results showed these small keg storage systems fully maintained the isotopic integrity of water over the 2-year testing period with no trend in the isotopic data that would reveal evaporative loss or leakage (e.g., pressure or mass loss) regardless of starting fill level. However, evaporated water in the outlet tube assembly must be eliminated by discarding 15-20 mL before dispensing into appropriate daily-use laboratory standard bottles (30-100 mL). Glass bottles for daily aliquots showed good integrity properties, but only if their fill level was >50%. CONCLUSIONS: The use of a low-cost pressurized metal beverage keg dispensing system provides a robust solution to enable laboratories to maintain the integrity of their water isotope working reference materials over several years.

Compound-specific stable hydrogen isotope (δ2 H) analyses of fatty acids: A new method and perspectives for trophic and movement ecology.

RATIONALE: Compound-specific stable isotope analysis (CSIA) is a powerful tool for a better understanding of trophic transfer of dietary molecules in and across ecosystems. Hydrogen isotope values (δ2 H) in consumer tissues have potential to more clearly distinguish dietary sources than 13 C or 15 N values within and among habitats, but have not been used at the fatty acid level for ecological purposes. METHODS: Here we demonstrate a new online high-capacity gas chromatography-isotope ratio mass spectrometry technique (2 H-CSIA) that offers accurate and reproducible determination of δ2 H values for a range of fatty acids from organisms of aquatic food webs. RESULTS: We show that lipid extracts obtained from aquatic sources, such as biofilms, leaves, invertebrates, or fish muscle tissue, have distinctive δ2 H values that can be used to assess sources and trophic interactions, as well as dietary allocation and origin of fatty acids within consumer tissue. CONCLUSIONS: The new 2 H-CSIA method can be applied to evaluate sources and trophic dynamics of fatty acids in organisms ranging from food web ecology to migratory connectivity.

Proficiency testing of 78 international laboratories measuring tritium in environmental waters by decay counting and mass spectrometry for age dating and water resources assessment.

RATIONALE: Tritium (3 H) is an important hydrological tracer that has been commonly used for over 60 years to evaluate water residence times and water dynamics in shallow/recent groundwaters, streams, lakes and the ocean. We tested the analytical performance of 78 international laboratories engaged in low-level 3 H assays for water age dating and monitoring of environmental waters. METHODS: Seven test waters were distributed by the IAEA to 78 international tritium laboratories. Set 1 included a tritium-free groundwater plus three ultra-low 3 H samples (0.5-7 TU) for meeting groundwater dating specifications. Set 2 contained three higher 3 H-content samples (40-500 TU) suitable for testing of environmental monitoring laboratories. RESULTS: Seventy of the laboratories used liquid scintillation counting with or without electrolytic enrichment, seven utilized 3 He accumulation and mass spectrometry, and one used gas-proportional counting. Only ~50% of laboratories demonstrated the ability to generate accurate 3 H data that was precise enough for water age dating purposes. CONCLUSIONS: The proficiency test helped identify recurrent weaknesses and potential solutions. Strategies for performance improvements of 3 H laboratories include: (a) improved quantification of 3 H detection limits and analytical uncertainty, (b) stricter quality control practices in routine operations along with care and recalibration of 3 H standards traceable to primary NIST standards, (c) annual assessment of tritium enrichment factors and instrumental performance, and (d) for water age dating purposes the use of electrolytic enrichment systems having the highest possible 3 H enrichment factors (e.g. >50×).

A Ti(III) reduction method for one-step conversion of seawater and freshwater nitrate into N2 O for stable isotopic analysis of 15 N/14 N, 18 O/16 O and 17 O/16 O.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, and δ17 O values) isotopic compositions of nitrate (NO3 - ) are crucial tracers of nutrient nitrogen (N) sources and dynamics in aquatic systems. Current methods such as bacterial denitrification or Cd-azide reduction require laborious multi-step conversions or toxic chemicals to reduce NO3 - to N2 O for 15 N and 18 O isotopic analyses by isotope ratio mass spectrometry (IRMS). Furthermore, the 17 O composition of N2 O cannot be directly disentangled using IRMS because 17 O contributes to mass 45 (15 N). METHODS: We describe a new one-step chemical conversion method that employs Ti(III) chloride to reduce nitrate to N2 O gas in septum sample vials. Sample preparation takes only a few minutes followed by a 24-h reaction producing N2 O gas (65-75% recovery) which partitions into the headspace. The N2 O headspace was measured for 15 N, 18 O and 17 O by IRMS or laser spectrometry. RESULTS: IRMS and laser spectrometric analyses gave accurate and reproducible N and O isotopic results down to 50 ppb (3.5 µM) NO3 -N, similar in precision to the denitrifier and Cd-azide methods. The uncertainties for dissolved nitrate reference materials (USGS32, USGS34, USGS35, IAEA-NO3 ) were ±0.2‰ for δ15 N values and ±0.3‰ for δ18 O values using IRMS. For laser-based N2 O isotope analyses the results were similar, with an δ17 O uncertainty of ±0.9‰ without any need for 15 N correction. CONCLUSIONS: Advantages of the Ti(III) reduction method are simplicity, low cost, and no requirement for toxic chemicals or anaerobic bacterial cultures. Minor corrections may be required to account for sample nitrate concentration variance and potential chemical interferences. The Ti(III) method is easily implemented into laboratories currently using N2 O headspace sampling apparatus. We expect that the Ti(III) method will promulgate the use of N and O isotopes of nitrate in important studies of nutrient dynamics and pollution in a wide range of aquatic ecosystems.

N and O isotope (δ15 Nα , δ15 Nß , δ18 O, δ17 O) analyses of dissolved NO3- and NO2- by the Cd-azide reduction method and N2 O laser spectrometry.

RATIONALE: The nitrogen and oxygen (δ15 N, δ18 O, δ17 O) isotopic compositions of NO3- and NO2- are important tracers of nutrient dynamics in soil, rain, groundwater and oceans. The Cd-azide method was used to convert NO3- or NO2- to N2 O for N and triple-O isotopic analyses by N2 O laser spectrometry. A protocol for laser-based headspace isotope analyses was compared with isotope ratio mass spectrometry. Lasers provide the ability to directly measure 17 O anomalies which can help discern atmospheric N sources. METHODS: δ15 N, δ18 O and δ17 O values were measured on N/O stable isotopic reference materials (IAEA, USGS) by conversion to N2 O using the Cd-azide method and headspace N2 O laser spectrometry. A 15 N tracer test assessed the position-specific routing of N to the α or ß positions in the N2 O molecule. A data processing algorithm was used to correct for isotopic dependencies on N2 O concentration, cavity pressure and water content. RESULTS: NO3- /NO2- nitrogen is routed to the 15 Nα position of N2 O in the azide reaction; hence the δ15 Nα value should be used for N2 O laser spectrometry results. With corrections for cavity pressure, N2 O concentration and water content, the δ15 NαAIR , δ18 OVSMOW and δ17 OVSMOW values (‰) of international reference materials were +4.8 ± 0.1, +25.9 ± 0.3, +12.7 ± 0.2 (IAEA NO3 ), -1.7 ± 0.1, -26.8 ± 0.8, -14.4 ± 1.1 (USGS34) and +2.6 ± 0.1, +57.6 ± 1.2, +51.2 ± 2.0 (USGS35), in agreement with their values and with the isotope ratio mass spectrometry results. The 17 O excess for USGS35 was +21.2 ± 9‰, in good agreement with previous results. CONCLUSIONS: The Cd-azide method yielded excellent results for routine determination of δ15 N, δ18 O and δ17 O values (and the 17 O excess) of nitrate or nitrite by laser spectrometry. Disadvantages are the toxicity of Cd-azide chemicals and the lack of automated sampling devices for N2 O laser spectrometers. The 15 N-enriched tracer test revealed potential for position-specific experimentation of aqueous nutrient dynamics at high 15 N enrichments by laser spectrometry, but exposed the need for memory corrections and improved spectral deconvolution of 17 O.

Regional climate on the breeding grounds predicts variation in the natal origin of monarch butterflies overwintering in Mexico over 38 years.

Addressing population declines of migratory insects requires linking populations across different portions of the annual cycle and understanding the effects of variation in weather and climate on productivity, recruitment, and patterns of long-distance movement. We used stable H and C isotopes and geospatial modeling to estimate the natal origin of monarch butterflies (Danaus plexippus) in eastern North America using over 1000 monarchs collected over almost four decades at Mexican overwintering colonies. Multinomial regression was used to ascertain which climate-related factors best-predicted temporal variation in natal origin across six breeding regions. The region producing the largest proportion of overwintering monarchs was the US Midwest (mean annual proportion = 0.38; 95% CI: 0.36-0.41) followed by the north-central (0.17; 0.14-0.18), northeast (0.15; 0.11-0.16), northwest (0.12; 0.12-0.16), southwest (0.11; 0.08-0.12), and southeast (0.08; 0.07-0.11) regions. There was no evidence of directional shifts in the relative contributions of different natal regions over time, which suggests these regions are comprising the same relative proportion of the overwintering population in recent years as in the mid-1970s. Instead, interannual variation in the proportion of monarchs from each region covaried with climate, as measured by the Southern Oscillation Index and regional-specific daily maximum temperature and precipitation, which together likely dictate larval development rates and food plant condition. Our results provide the first robust long-term analysis of predictors of the natal origins of monarchs overwintering in Mexico. Conservation efforts on the breeding grounds focused on the Midwest region will likely have the greatest benefit to eastern North American migratory monarchs, but the population will likely remain sensitive to regional and stochastic weather patterns.

Re-evaluation of the hydrogen stable isotopic composition of keratin calibration standards for wildlife and forensic science applications.

RATIONALE: Determination of non-exchangeable hydrogen isotopic compositions (δ2 H values) of bulk complex organic materials is difficult due to uncontrolled H isotope exchange between the organic material and ambient water vapor. A number of calibration keratinous materials with carefully measured hydrogen isotopic compositions of the non-exchangeable fraction were proposed to enable stable isotope laboratories to normalize their 2 H measurements. However, it was recently reported that high-temperature carbon-reactor methods for measuring the hydrogen isotopic composition of nitrogenous organic materials is biased by the production of HCN in the reactor. As a result, the reported values of these calibration materials needed to be re-evaluated. METHODS: We evaluated the non-exchangeable δ2 HVSMOW values of keratins EC1 (CBS) and EC2 (KHS), USGS hair standards, and a range of other nitrogenous widely used organic laboratory calibration materials (collagen and chitin) using pre-treatment with a preparation device designed to eliminate residual moisture and quantify exchangeable H. RESULTS: The revised non-exchangeable δ2 HVSMOW values of EC-1 (CBS) and EC-2 (KHS) keratin standard materials were -157.0 ± 0.9 and -35.3 ± 1.1 ‰, respectively. The revised values of USGS42 and USGS43 were -72.2 ± 0.9 and -44.2 ± 1.0 ‰, respectively, in excellent agreement with previous results. CONCLUSIONS: For routine H isotope analyses, with proper sample pre-treatment, we show that the Comparative Equilibration approach can provide accurate and reproducible non-exchangeable δ2 H values among laboratories regardless of the reactor type used. © 2017 Her Majesty the Queen in Right of Canada Rapid Communications in Mass Spectrometry © 2017 John Wiley & Sons Ltd. Reproduced with the permission of the Environment and Climate Change Canada.

Correcting Laser-Based Water Stable Isotope Readings Biased by Carrier Gas Changes.

Recently, laser-based water stable isotope spectrometers have become popular as they enable previously impossible approaches of environmental observations. Consequently, they have been subjected to increasingly heterogeneous atmospheric conditions. However, there is still a severe lack of data on the impact of nonstandardized gas matrices on analyzer performances. Against this background, we investigated the influence of changing proportions of N2, O2, and CO2 in the carrier gas on the isotope measurements of a typical laser-based water stable isotope analyzer (Picarro L2120-i). We combined environmentally relevant mixtures of N2, O2, and CO2 with referenced, flash-evaporated water and found that isotope readings of the same water were altered by up to +14.57‰ for δ(18)O and -35.9‰ for δ(2)H. All tested relationships between carrier gas changes and respective isotope readings were strongly linearly correlated (R(2) > 0.99). Furthermore, an analyzer-measured variable allowed for reliable postcorrection of the biased isotope readings, which we additionally tested on field data. Our findings are of importance for environmental data obtained by analyzers based on the same technology. They are relevant for assays where inconsistent gas matrices or a mismatch in this regard between unknown and reference analyses cannot be excluded, which is in particular common when investigating the soil-vegetation-atmosphere continuum.

Differential migration and the link between winter latitude, timing of migration, and breeding in a songbird.

Patterns of connectivity between breeding and wintering grounds can have important implications for individual fitness and population dynamics. Using light-level geolocators and stable hydrogen isotopes (δ(2)H) in feathers, we evaluated differential migration of Savannah sparrows (Passerculus sandwichensis) breeding on Kent Island in the Bay of Fundy, New Brunswick, Canada in relation to sex, age, and body size. Based on geolocators recovered from 38 individuals between 2012 and 2014, the winter distribution was centered in North Carolina (median latitude 34°, range 26°-41°), with males overwintering, on average, approximately 275 km further north than females. Based on analyses of tail feather samples collected from 106 individuals from the same population between 2008 and 2012, males and adults had more negative δ(2)H values than females and juveniles, respectively, providing additional evidence that males wintered north of females and that adults wintered north of juveniles. Winter latitude and δ(2)H values within each sex were not found to be related to body size. From geolocator data, males returned to the breeding grounds, on average, 14 days earlier than females. For males, there was some evidence that arrival date on the breeding grounds was negatively correlated with winter latitude and that individuals which arrived earlier tended to breed earlier. Thus, benefits for males of early arrival on the breeding grounds may have contributed to their wintering farther north than females. Social dominance may also have contributed to age and sex differences in winter latitude, whereby dominant males and adults forced subordinate females and juveniles further south.

LIMS for Lasers 2015 for achieving long-term accuracy and precision of δ(2)H, δ(17)O, and δ(18)O of waters using laser absorption spectrometry.

RATIONALE: Although laser absorption spectrometry (LAS) instrumentation is easy to use, its incorporation into laboratory operations is not easy, owing to extensive offline manipulation of comma-separated-values files for outlier detection, between-sample memory correction, nonlinearity (δ-variation with water amount) correction, drift correction, normalization to VSMOW-SLAP scales, and difficulty in performing long-term QA/QC audits. METHODS: A Microsoft Access relational-database application, LIMS (Laboratory Information Management System) for Lasers 2015, was developed. It automates LAS data corrections and manages clients, projects, samples, instrument-sample lists, and triple-isotope (δ(17)O, δ(18)O, and δ(2)H values) instrumental data for liquid-water samples. It enables users to (1) graphically evaluate sample injections for variable water yields and high isotope-delta variance; (2) correct for between-sample carryover, instrumental drift, and δ nonlinearity; and (3) normalize final results to VSMOW-SLAP scales. RESULTS: Cost-free LIMS for Lasers 2015 enables users to obtain improved δ(17)O, δ(18)O, and δ(2)H values with liquid-water LAS instruments, even those with under-performing syringes. For example, LAS δ(2) HVSMOW measurements of USGS50 Lake Kyoga (Uganda) water using an under-performing syringe having ±10 % variation in water concentration gave +31.7 ± 1.6 ‰ (2-σ standard deviation), compared with the reference value of +32.8 ± 0.4 ‰, after correction for variation in δ value with water concentration, between-sample memory, and normalization to the VSMOW-SLAP scale. CONCLUSIONS: LIMS for Lasers 2015 enables users to create systematic, well-founded instrument templates, import δ(2) H, δ(17) O, and δ(18) O results, evaluate performance with automatic graphical plots, correct for δ nonlinearity due to variable water concentration, correct for between-sample memory, adjust for drift, perform VSMOW-SLAP normalization, and perform long-term QA/QC audits easily.

An online temperature-controlled vacuum-equilibration preparation system for the measurement of δ2H values of non-exchangeable-H and of δ18O values in organic materials by isotope-ratio mass spectrometry.

RATIONALE: Measurement of δ(2) H values in non-exchangeable-H (δ(2) H(n)) and δ(18)O values in organic environmental samples are inconsistent among laboratories worldwide due to varied and lengthy approaches in controlling for H isotope exchange (for δ(2)H(n) values) and removal of trace moisture (δ(2)H(n) and δ(18)O values), which undermines the comparability of organic δ(2)H and δ(18)O data produced among different laboratories. METHODS: An online preparation system was developed for the measurement of the δ(2)H(n) and δ(18)O values of organic samples, coupled to isotope-ratio mass spectrometers. The system features a 50-position autosampler and isolation valve where (1) the samples are held isothermal between ambient to 40-120 ± 0.1 °C for H isotopic exchange experiments (δ(2) H(n)) and drying of hygroscopic samples (δ(2) H(n) and δ(18)O), (2) the samples are evacuated to <5 mbar and flushed with helium for moisture and N(2) removal, and (3) injection of up to 500 µL of H(2)O is possible for controlled vapour exchangeable-H experiments. RESULTS: The system provides highly reproducible and precise δ(2)H(n) isotope estimates for a range of organic keratinous standard powders over a wide range of experimental temperatures. A reproducible sample processing regimen can now be applied to a wider range of organics and hygroscopic samples that are currently hampered by poorly controlled preparative methods amongst laboratories. CONCLUSIONS: Rapid and reproducible online vacuum equilibration of samples and standards for the routine measurement of δ(2)H(n) and δ(18)O values is now possible using the online equilibration system, with the added benefit that sample processing times for organic δ(2)H values are reduced from weeks to hours.

A new isotopic reference material for stable hydrogen and oxygen isotope-ratio measurements of water - USGS50 Lake Kyoga Water.

RATIONALE: As a result of the need for isotopic reference waters having high δ(2) HVSMOW-SLAP and δ(18) OVSMOW-SLAP values for daily use, especially for tropical and equatorial-zone freshwaters, a new secondary isotopic reference material for international distribution was prepared from water collected from Lake Kyoga, Uganda. METHODS: This isotopic reference lakewater was filtered through a membrane with 0.2-µm pore size, homogenized, loaded into glass ampoules that were sealed with a torch and autoclaved to eliminate biological activity, and measured by dual-inlet isotope-ratio mass spectrometry. This reference material is available in a case of 144 glass ampoules each containing 5 mL of water. RESULTS: The δ(2) H and δ(18) O values of this reference material are +32.8 ± 0.4 and +4.95 ± 0.02 mUr (milliurey = 0.001 = 1 ‰), respectively, relative to VSMOW, on scales normalized such that the δ(2) H and δ(18) O values of SLAP reference water are, respectively, -428 and -55.5 mUr. Each uncertainty is an estimated expanded uncertainty (U = 2uc ) about the reference value that provides an interval that has about a 95 % probability of encompassing the true value. CONCLUSIONS: This isotopic reference material, designated as USGS50, is intended as one of two reference waters for daily normalization of stable hydrogen and oxygen isotopic analysis of water with an isotope-ratio mass spectrometer or a laser absorption spectrometer, of use especially for isotope-hydrology laboratories analyzing freshwater samples from equatorial and tropical regions.

No evidence for assortative mating within a willow warbler migratory divide.

INTRODUCTION: In contact zones, genetic mixing of two taxa can be restricted by prezygotic (e.g. assortative mating) or postzygotic (lower fitness of hybrid offspring) barriers, or a combination of the two. A hybrid zone between two willow warbler subspecies (Phylloscopus trochilus trochilus, P. t. acredula) with distinctive migratory strategies occurs in central Sweden. These subspecies exhibit differences in migratory direction and distance, resulting in geographically distinct wintering areas in Africa. The subspecies may have diverged from a common refuge after the last ice age, and neutral genetic markers are homogeneous across their range. By contrast, several phenotypic traits and genetic markers of two chromosomal regions previously identified show steep clines across the divide. The evolutionary forces that maintain this migratory divide remain unknown. Here we use plumage colour, morphology, genetic markers and feather stable nitrogen-isotopes (δ (15)N) to assess if assortative mating between migratory phenotypes could be acting as a possible mechanism for keeping the two forms genetically separate and maintaining the migratory divide. We colour-ringed a willow warbler breeding population in the central part of the hybrid zone and observed the breeding population to assess phenotypic and genotypic traits of social pairs. RESULTS: Our data suggest that wintering area and genetic ancestry had an effect on male arrival time to the breeding grounds which could contribute to assortment. However, evidence for assortative mating could not be detected based on a comparison of plumage colour, morphology and δ (15)N between social mates. CONCLUSION: This finding was strengthened by analyses of subspecies-specific genetic markers, which allowed us to identify the presence of a large proportion of potential hybrids and backcrosses at the study site. Our results supported the hypothesis that pre-mating isolation in willow warblers is weak, resulting in extensive hybridisation across the migratory divide.

Approaches for achieving long-term accuracy and precision of δ18O and δ2H for waters analyzed using laser absorption spectrometers.

The measurement of δ(2)H and δ(18)O in water samples by laser absorption spectroscopy (LAS) are adopted increasingly in hydrologic and environmental studies. Although LAS instrumentation is easy to use, its incorporation into laboratory operations is not as easy, owing to extensive offline data manipulation required for outlier detection, derivation and application of algorithms to correct for between-sample memory, correcting for linear and nonlinear instrumental drift, VSMOW-SLAP scale normalization, and in maintaining long-term QA/QC audits. Here we propose a series of standardized water-isotope LAS performance tests and routine sample analysis templates, recommended procedural guidelines, and new data processing software (LIMS for Lasers) that altogether enables new and current LAS users to achieve and sustain long-term δ(2)H and δ(18)O accuracy and precision for these important isotopic assays.

Isotope hydrology of the intermontane Elk Valley, British Columbia: an assessment of water resources around coal mining operations.

This study aimed to synthesise and interpret stable isotopic data (δ2H and δ18O) from various sources to understand the isotope hydrology around coal mine operations in Elk Valley, B.C., Canada. The data, including precipitation, groundwaters, seeps, and mine rock drains, were used to construct a local meteoric water line (LMWL) for the Elk Valley, evaluate the spatiotemporal isotopic composition of its groundwater, and assess mine seepage and mine rock drain discharge. The study revealed a robust LMWL relation (δ2H = 7.4 ± 0.2 · δ18O - 4.3 ± 4.1). The groundwater and seep data indicated a winter season bias and a north-south latitudinal gradient, suggesting rapid near-surface groundwater flow without significant post-precipitation evaporation. Porewater isotope samples from unsaturated mine rock piles (MRPs) showed site-specific evaporation patterns, potentially due to convective air flows or exothermic sulphide oxidation. This research revealed the influence of groundwater and meltwater on rock drain discharge. Based on evaporative mass balance calculations, MRPs seasonally contributed ca. 5 %(December base flow) and 22 % (snowmelt) to drain discharge. The findings underscore the value of stable isotope data collections in the Elk Valley to help better define and quantify the hydrology-hydrogeology, including a better understanding of evaporative conditions in MRPs.