Natural chlorate in the environment: application of a new IC-ESI/MS/MS method with a Cl¹8O3-internal standard.

A new ion chromatography electrospray tandem mass spectrometry (IC-ESI/MS/MS) method has been developed for quantification and confirmation of chlorate (ClO3⁻) in environmental samples. The method involves the electrochemical generation of isotopically labeled chlorate internal standard (Cl¹8O3⁻) using ¹8O water (H2¹8O) he standard was added to all samples prior to analysis thereby minimizing the matrix effects that are associated with common ions without the need for expensive sample pretreatments. The method detection limit (MDL) for ClO3⁻ was 2 ng L⁻¹ for a 1 mL volume sample injection. The proposed method was successfully applied to analyze ClO3⁻ in difficult environmental samples including soil and plant leachates. The IC-ESI/MS/MS method described here was also compared to established EPA method 317.0 for ClO3⁻ analysis. Samples collected from a variety of environments previously shown to contain natural perchlorate (ClO4⁻) occurrence were analyzed using the proposed method and ClO3⁻ was found to co-occur with ClO4⁻ at concentrations ranging from < 2 ng L⁻¹ in precipitation from Texas and Puerto Rico to >500 mg kg⁻¹ in caliche salt deposits from the Atacama Desert in Chile. Relatively low concentrations of ClO3⁻ in some natural groundwater samples (0.1 µg L⁻¹) analyzed in this work may indicate lower stability when compared to ClO4⁻ in the subsurface. The high concentrations ClO3⁻ in caliches and soils (3-6 orders of magnitude greater) as compared to precipitation samples indicate that ClO3⁻, like ClO4⁻, may be atmospherically produced and deposited, then concentrated in dry soils, and is possibly a minor component in the biogeochemical cycle of chlorine.

Isotopic composition and origin of indigenous natural perchlorate and co-occurring nitrate in the southwestern United States.

Perchlorate (ClO(4)(-)) has been detected widely in groundwater and soils of the southwestern United States. Much of this ClO(4)(-) appears to be natural, and it may have accumulated largely through wet and dry atmospheric deposition. This study evaluates the isotopic composition of natural ClO(4)(-) indigenous to the southwestern U.S. Stable isotope ratios were measured in ClO(4)(-) (delta(18)O, Delta(17)O, delta(37)Cl) and associated NO(3)(-) (delta(18)O, Delta(17)O, delta(15)N) in groundwater from the southern High Plains (SHP) of Texas and New Mexico and the Middle Rio Grande Basin (MRGB) in New Mexico, from unsaturated subsoil in the SHP, and from NO(3)(-)-rich surface caliche deposits near Death Valley, California. The data indicate natural ClO(4)(-) in the southwestern U.S. has a wide range of isotopic compositions that are distinct from those reported previously for natural ClO(4)(-) from the Atacama Desert of Chile as well as all known synthetic ClO(4)(-). ClO(4)(-) in Death Valley caliche has a range of high Delta(17)O values (+8.6 to +18.4 per thousand), overlapping and extending the Atacama range, indicating at least partial atmospheric formation via reaction with ozone (O(3)). However, the Death Valley delta(37)Cl values (-3.1 to -0.8 per thousand) and delta(18)O values (+2.9 to +26.1 per thousand) are higher than those of Atacama ClO(4)(-). In contrast, ClO(4)(-) from western Texas and New Mexico has much lower Delta(17)O (+0.3 to +1.3 per thousand), with relatively high delta(37)Cl (+3.4 to +5.1 per thousand) and delta(18)O (+0.5 to +4.8 per thousand), indicating either that this material was not primarily generated with O(3) as a reactant or that the ClO(4)(-) was affected by postdepositional O isotope exchange. High Delta(17)O values in ClO(4)(-) (Atacama and Death Valley) are associated with high Delta(17)O values in NO(3)(-), indicating that both compounds preserve characteristics of O(3)-related atmospheric production in hyper-arid settings, whereas both compounds have low Delta(17)O values in less arid settings. Although Delta(17)O variations in terrestrial NO(3)(-) can be attributed to mixing of atmospheric (high Delta(17)O) and biogenic (low Delta(17)O) NO(3)(-), variations in Delta(17)O of terrestrial ClO(4)(-) are not readily explained in the same way. This study provides important new constraints for identifying natural sources of ClO(4)(-) in different environments by multicomponent isotopic characteristics, while presenting the possibilities of divergent ClO(4)(-) formation mechanisms and(or) ClO(4)(-) isotopic exchange in biologically active environments.

Chlorine-36 as a tracer of perchlorate origin.

Perchlorate (ClO4(-)) is ubiquitous in the environment. It is produced naturally by atmospheric photochemical reactions, and also is synthesized in large quantities for military, aerospace, and industrial applications. Nitrate-enriched salt deposits of the Atacama Desert (Chile) contain high concentrations of natural ClO4(-), and have been exported worldwide since the mid-1800s for use in agriculture. The widespread introduction of synthetic and agricultural ClO4(-) into the environment has contaminated numerous municipal water supplies. Stable isotope ratio measurements of Cl and O have been applied for discrimination of different ClO4(-) sources in the environment. This study explores the potential of 36Cl measurements for further improving the discrimination of ClO4(-) sources. Groundwater and desert soil samples from the southwestern United States (U.S.) contain ClO4(-) having high 36Cl abundances (36Cl/Cl = 3100 x 10(-15) to 28,800 x 10(-15)), compared with those from the Atacama Desert (36Cl/Cl = 0.9 x 10(-15) to 590 x 10(-15)) and synthetic ClO4(-) reagents and products (36Cl/Cl = 0.0 x 10(-15) to 40 x 10(-15)). In conjunction with stable Cl and O isotope ratios, 36Cl data provide a clear distinction among three principal ClO4(-) source types in the environment of the southwestern U.S.

Atacama perchlorate as an agricultural contaminant in groundwater: isotopic and chronologic evidence from Long Island, New York.

Perchlorate (ClO4-) is a common groundwater constituent with both synthetic and natural sources. A potentially important source of ClO4- is past agricultural application of ClO4(-)-bearing natural NO3- fertilizer imported from the Atacama Desert, Chile, but evidence for this has been largely circumstantial. Here we report ClO4- stable isotope data (delta37Cl, delta18O, and delta17O), along with other supporting chemical and isotopic environmental tracer data, to document groundwater ClO4 contamination sources and history in parts of Long Island, New York. Sampled groundwaters were oxic and ClO4- apparently was not affected by biodegradation within the aquifers. Synthetic ClO4- was indicated by the isotopic method in groundwater near a fireworks disposal site at a former missile base. Atacama ClO4- was indicated in agricultural and urbanizing areas in groundwaters with apparent ages > 20 years. In an agricultural area, ClO4- concentrations and ClO4-/NO3- ratios increased with groundwater age, possibly because of decreasing application rates of Atacama NO3- fertilizers and/or decreasing ClO4- concentrations in Atacama NO3- fertilizers in recent years. Because ClO4-/NO3- ratios of Atacama NO3- fertilizers imported in the past (approximately 2 x 10(-3) mol mol(-1)) were much higher than the CO4-/NO3- ratio of recommended drinking-water limits (7 x 10(-5) mol mol(-1) in New York), ClO4- could exceed drinking-water limits even where NO3- does not, and where Atacama NO3- was only a minor source of N. Groundwater ClO4- with distinctive isotopic composition was a sensitive indicator of past Atacama NO3- fertilizer use on Long Island and may be common in other areas that received NO3- fertilizers from the late 19th century through the 20th century.

Contrasting residence times and fluxes of water and sulfate in two small forested watersheds in Virginia, USA.

Watershed mass balances for solutes of atmospheric origin may be complicated by the residence times of water and solutes at various time scales. In two small forested headwater catchments in the Appalachian Mountains of Virginia, USA, mean annual export rates of SO(4)(=) differ by a factor of 2, and seasonal variations in SO(4)(=) concentrations in atmospheric deposition and stream water are out of phase. These features were investigated by comparing (3)H, (35)S, delta(34)S, delta(2)H, delta(18)O, delta(3)He, CFC-12, SF(6), and chemical analyses of open deposition, throughfall, stream water, and spring water. The concentrations of SO(4)(=) and radioactive (35)S were about twice as high in throughfall as in open deposition, but the weighted composite values of (35)S/S (11.1 and 12.1x10(-15)) and delta(34)S (+3.8 and +4.1 per thousand) were similar. In both streams (Shelter Run, Mill Run), (3)H concentrations and delta(34)S values during high flow were similar to those of modern deposition, delta(2)H and delta(18)O values exhibited damped seasonal variations, and (35)S/S ratios (0-3x10(-15)) were low throughout the year, indicating inter-seasonal to inter-annual storage and release of atmospheric SO(4)(=) in both watersheds. In the Mill Run watershed, (3)H concentrations in stream base flow (10-13 TU) were consistent with relatively young groundwater discharge, most delta(34)S values were approximately the same as the modern atmospheric deposition values, and the annual export rate of SO(4)(=) was equal to or slightly greater than the modern deposition rate. In the Shelter Run watershed, (3)H concentrations in stream base flow (1-3 TU) indicate that much of the discharging ground water had been deposited prior to the onset of atmospheric nuclear bomb testing in the 1950s, base flow delta(34)S values (+1.6 per thousand) were significantly lower than the modern deposition values, and the annual export rate of SO(4)(=) was less than the modern deposition rate. Concentrations of (3)H and (35)S in Shelter Run base flow, and of (3)H, (3)He, CFC-12, SF(6), and (35)S in a spring discharging to Shelter Run, all were consistent with a bimodal distribution of discharging ground-water ages with approximately 5-20% less than a few years old and 75-95% more than 40 years old. These results provide evidence for 3 important time-scales of SO(4)(=) transport through the watersheds: (1) short-term (weekly to monthly) storage and release of dry deposition in the forest canopy between precipitation events; (2) mid-term (seasonal to interannual) cycles in net storage in the near-surface environment, and (3) long-term (decadal to centennial) storage in deep ground water that appears to be related to relatively low SO(4)(=) concentrations in spring discharge that dominates Shelter Run base flow. It is possible that the relatively low concentrations and low delta(34)S values of SO(4)(=) in spring discharge and Shelter Run base flow may reflect those of atmospheric deposition before the middle of the 20th century. In addition to storage in soils and biota, variations in ground-water residence times at a wide range of time scales may have important effects on monitoring, modeling, and predicting watershed responses to changing atmospheric deposition in small watersheds.

Effects of nitrate and water on the oxygen isotopic analysis of barium sulfate precipitated from water samples.

BaSO(4) precipitated from mixed salt solutions by common techniques for SO(4) (2-) isotopic analysis may contain quantities of H(2)O and NO(3) (-) that introduce errors in O isotope measurements. Experiments with synthetic solutions indicate that delta(18)O values of CO produced by decomposition of precipitated BaSO(4) in a carbon reactor may be either too low or too high, depending on the relative concentrations of SO(4) (2-) and NO(3) (-) and the delta(18)O values of the H(2)O, NO(3) (-), and SO(4) (2-). Typical delta(18)O errors are of the order of 0.5 to 1 per thousand in many sample types, and can be larger in samples containing atmospheric NO(3) (-), which can cause similar errors in delta(17)O and Delta(17)O. These errors can be reduced by (1) ion chromatographic separation of SO(4) (2-) from NO(3) (-), (2) increasing the salinity of the solutions before precipitating BaSO(4) to minimize incorporation of H(2)O, (3) heating BaSO(4) under vacuum to remove H(2)O, (4) preparing isotopic reference materials as aqueous samples to mimic the conditions of the samples, and (5) adjusting measured delta(18)O values based on amounts and isotopic compositions of coexisting H(2)O and NO(3) (-). These procedures are demonstrated for SO(4) (2-) isotopic reference materials, synthetic solutions with isotopically known reagents, atmospheric deposition from Shenandoah National Park, Virginia, USA, and sulfate salt deposits from the Atacama Desert, Chile, and Mojave Desert, California, USA. These results have implications for the calibration and use of O isotope data in studies of SO(4) (2-) sources and reaction mechanisms.

Ecohydrological factors affecting nitrate concentrations in a phreatic desert aquifer in northwestern China.

Aerobic conditions in desert aquifers commonly allow high nitrate (NO3-) concentrations in recharge to persist for long periods of time, an important consideration for N-cycling and water quality. In this study, stable isotopes of NO3- (delta15N(NO3) and delta18O(NO3)) were used to trace NO3- cycling processes which affect concentrations in groundwater and unsaturated zone moisture in the arid Badain Jaran Desert in northwestern China. Most groundwater NO3- appears to be depleted relative to Cl- in rainfall concentrated by evapotranspiration, indicating net N losses. Unsaturated zone NO3- is generally higher than groundwater NO3- in terms of both concentration (up to 15 476 microM, corresponding to 3.6 mg NO3(-)-N per kg sediment) and ratios with Cl-. Isotopic data indicate that the NO3- derives primarily from nitrification, with a minor direct contribution of atmospheric NO3- inferred for some samples, particularly in the unsaturated zone. Localized denitrification in the saturated zone is suggested by isotopic and geochemical indicators in some areas. Anthropogenic inputs appear to be minimal, and variability is attributed to environmental factors. In comparison to other arid regions, the sparseness of vegetation in the study area appears to play an important role in moderating unsaturated zone NO3- accumulation by allowing solute flushing and deterring extensive N2 fixation.

Limited occurrence of denitrification in four shallow aquifers in agricultural areas of the United States.

The ability of natural attenuation to mitigate agricultural nitrate contamination in recharging aquifers was investigated in four important agricultural settings in the United States. The study used laboratory analyses, field measurements, and flow and transport modeling for monitoring well transects (0.5 to 2.5 km in length) in the San Joaquin watershed, California, the Elkhorn watershed, Nebraska, the Yakima watershed, Washington, and the Chester watershed, Maryland. Ground water analyses included major ion chemistry, dissolved gases, nitrogen and oxygen stable isotopes, and estimates of recharge date. Sediment analyses included potential electron donors and stable nitrogen and carbon isotopes. Within each site and among aquifer-based medians, dissolved oxygen decreases with ground water age, and excess N(2) from denitrification increases with age. Stable isotopes and excess N(2) imply minimal denitrifying activity at the Maryland and Washington sites, partial denitrification at the California site, and total denitrification across portions of the Nebraska site. At all sites, recharging electron donor concentrations are not sufficient to account for the losses of dissolved oxygen and nitrate, implying that relict, solid phase electron donors drive redox reactions. Zero-order rates of denitrification range from 0 to 0.14 micromol N L(-1)d(-1), comparable to observations of other studies using the same methods. Many values reported in the literature are, however, orders of magnitude higher, which is attributed to a combination of method limitations and bias for selection of sites with rapid denitrification. In the shallow aquifers below these agricultural fields, denitrification is limited in extent and will require residence times of decades or longer to mitigate modern nitrate contamination.

Isotopic analysis of N and o in nitrite and nitrate by sequential selective bacterial reduction to N2O.

Nitrite is an important intermediate species in the biogeochemical cycling of nitrogen, but its role in natural aquatic systems is poorly understood. Isotopic data can be used to study the sources and transformations of NO2- in the environment, but methods for independent isotopic analyses of NO2- in the presence of other N species are still new and evolving. This study demonstrates that isotopic analyses of N and O in NO2- can be done by treating whole freshwater or saltwater samples with the denitrifying bacterium Stenotrophomonas nitritireducens, which selectively reduces NO2- to N2O for isotope ratio mass spectrometry. When calibrated with solutions containing NO2- with known isotopic compositions determined independently, reproducible delta15N and delta18O values were obtained at both natural-abundance levels (+/-0.2-0.5 per thousand for delta15N and +/-0.4-1.0 per thousand for delta18O) and moderately enriched 15N tracer levels (+/-20-50 per thousand for delta15N near 5000 per thousand) for 5-20 nmol of NO2- (1-20 micromol/L in 1-5 mL aliquots). This method is highly selective for NO2- and was used for mixed samples containing both NO2- and NO3- with little or no measurable cross-contamination. In addition, mixed samples that were analyzed with S. nitritireducens were treated subsequently with Pseudomonas aureofaciens to reduce the NO3- in the absence of NO2-, providing isotopic analyses of NO2- and NO3- separately in the same aliquot. Sequential bacterial reduction methods like this one should be useful for a variety of isotopic studies aimed at understanding nitrogen cycling in aquatic environments. A test of these methods in an agricultural watershed in Indiana provides isotopic evidence for both nitrification and denitrification as sources of NO2- in a small stream.

Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies.

Perchlorate is a widespread environmental contaminant having both anthropogenic and natural sources. Stable isotope ratios of O and Cl in a given sample of perchlorate may be used to distinguish its source(s). Isotopic ratios may also be useful for identifying the extent of biodegradation of perchlorate, which is critical for assessing natural attenuation of this contaminant in groundwater. For this approach to be useful, however, the kinetic isotopic fractionations of O and Cl during perchlorate biodegradation must first be determined as a function of environmental variables such as temperature and bacterial species. A laboratory study was performed in which the O and Cl isotope ratios of perchlorate were monitored as a function of degradation by two separate bacterial strains (Azospira suillum JPLRND and Dechlorospirillum sp. FBR2) at both 10 degrees C and 22 degrees C with acetate as the electron donor. Perchlorate was completely reduced by both strains within 280 h at 22 degrees C and 615 h at 10 degrees C. Measured values of isotopic fractionation factors were epsilon(18)O = -36.6 to -29.0% per hundred and epsilon(37)Cl = -14.5 to -11.5% per hundred, and these showed no apparent systematic variation with either temperature or bacterial strain. An experiment using (18)O-enriched water (delta(18)O = +198% per hundred) gave results indistinguishable from those observed in the isotopically normal water (delta(18)O = -8.1% per hundred) used in the other experiments, indicating negligible isotope exchange between perchlorate and water during biodegradation. The fractionation factor ratio epsilon(18)O/epsilon(37)Cl was nearly invariant in all experiments at 2.50 +/- 0.04. These data indicate that isotope ratio analysis will be useful for documenting perchlorate biodegradation in soils and groundwater. The establishment of a microbial fractionation factor ratio (epsilon(18)O/ epsilon(37)Cl) also has significant implications for forensic studies.

Oxygen isotopes in nitrite: analysis, calibration, and equilibration.

Nitrite is a central intermediate in the nitrogen cycle and can persist in significant concentrations in ocean waters, sediment pore waters, and terrestrial groundwaters. To fully interpret the effect of microbial processes on nitrate (NO3-), nitrite (NO2-), and nitrous oxide (N2O) cycling in these systems, the nitrite pool must be accessible to isotopic analysis. Furthermore, because nitrite interferes with most methods of nitrate isotopic analysis, accurate isotopic analysis of nitrite is essential for correct measurement of nitrate isotopes in a sample that contains nitrite. In this study, nitrite salts with varying oxygen isotopic compositions were prepared and calibrated and then used to test the denitrifier method for nitrite oxygen isotopic analysis. The oxygen isotopic fractionation during nitrite reduction to N2O by Pseudomonas aureofaciens was lower than for nitrate conversion to N2O, while oxygen isotopic exchange between nitrite and water during the reaction was similar. These results enable the extension of the denitrifier method to oxygen isotopic analysis of nitrite (in the absence of nitrate) and correction of nitrate isotopes for the presence of nitrite in "mixed" samples. We tested storage conditions for seawater and freshwater samples that contain nitrite and provide recommendations for accurate oxygen isotopic analysis of nitrite by any method. Finally, we report preliminary results on the equilibrium isotope effect between nitrite and water, which can play an important role in determining the oxygen isotopic value of nitrite where equilibration with water is significant.

Perchlorate in pleistocene and holocene groundwater in north-central New Mexico.

Groundwater from remote parts of the Middle Rio Grande Basin in north-central New Mexico has perchlorate (ClO4-) concentrations of 0.12-1.8 micro/L. Because the water samples are mostly preanthropogenic in age (0-28000 years) and there are no industrial sources in the study area, a natural source of the ClO4- is likely. Most of the samples have Br-, Cl-, and SO4(2-) concentrations that are similar to those of modern bulk atmospheric deposition with evapotranspiration (ET) factors of about 7-40. Most of the ET values for Pleistocene recharge were nearly twice that for Holocene recharge. The N03-/Cl- and CIO-/Cl-ratios are more variable than those of Br-/Cl- or S04(2-)/Cl-. Samples thought to have recharged under the most arid conditions in the Holocene have relatively high N03-/Cl- ratios and low delta 15N values (+1 per mil (% per thousand)) similar to those of modern bulk atmospheric N deposition. The delta 18O values of the N03- (-4 to 0% per thousand) indicate that atmospheric N03- was not transmitted directly to the groundwater but may have been cycled in the soils before infiltrating. Samples with nearly atmospheric N03-/CI- ratios have relatively high Cl04- concentrations (1.0-1.8 ug/L) with a nearly constant Cl04-/CI- mole ratio of (1.4 +/- 0.1) x 10(-4), which would be consistent with an average Cl04-concentration of 0.093 0.005 ,ug/L in bulk atmospheric deposition during the late Holocene in north-central NM. Samples thought to have recharged under wetter conditions have higher delta 15N values (+3 to +8 % per thousando), lower NO3-/Cl- ratios, and lower ClO4-/Cl- ratios than the ones most likely to preserve an atmospheric signal. Processes in the soils that may have depleted atmospherically derived NO3-also may have depleted ClO4- to varying degrees prior to recharge. If these interpretations are correct, then ClO4- concentrations of atmospheric origin as high as 4 microg/L are possible in preanthropogenic groundwater in parts of the Southwest where ET approaches a factor of 40. Higher Cl04- concentrations in uncontaminated groundwater could occur in recharge beneath arid areas where ET is greater than 40, where long-term accumulations of atmospheric salts are leached suddenly from dry soils, or where other (nonatmospheric) natural sources of ClO4- exist.

Denitrification in nitrate-rich streams: application of N2:Ar and 15N-tracer methods in intact cores.

Rates of benthic denitrification were measured using two techniques, membrane inlet mass spectrometry (MIMS) and isotope ratio mass spectrometry (IRMS), applied to sediment cores from two NO3(-)-rich streams draining agricultural land in the upper Mississippi River Basin. Denitrification was estimated simultaneously from measurements of N2:Ar (MIMS) and 15N[N2] (IRMS) after the addition of low-level 15NO3- tracer (15N:N = 0.03-0.08) in stream water overlying intact sediment cores. Denitrification rates ranged from about 0 to 4400 micromol N x m(-2) x h(-1) in Sugar Creek and from 0 to 1300 micromol N x m(-2) x h(-1) in Iroquois River, the latter of which possesses greater streamflow discharge and a more homogeneous streambed and water column. Within the uncertainties of the two techniques, there is good agreement between the MIMS and IRMS results, which indicates that the production of N2 by the coupled process of nitrification/denitrification was relatively unimportant and surface-water NO3- was the dominant source of NO3- for benthic denitrification in these streams. Variation in stream NO3- concentration (from about 20 micromol/L during low discharge to 1000 micromol/L during high discharge) was a significant control of benthic denitrification rates, judging from the more abundant MIMS data. The interpretation that NO3- concentration directly affects denitrification rate was corroborated by increased rates of denitrification in cores amended with NO3-. Denitrification in Sugar Creek removed < or = 11% per day of the instream NO3- in late spring and removed roughly 15-20% in late summer. The fraction of NO3- removed in Iroquois River was less than that of Sugar Creek. Although benthic denitrification rates were relatively high during periods of high stream flow, when NO3 concentrations were also high, the increase in benthic denitrification could not compensate for the much larger increase in stream NO3- fluxes during high flow. Consequently, fractional NO3- losses were relatively low during high flow.

Perchlorate isotope forensics.

Perchlorate has been detected recently in a variety of soils, waters, plants, and food products at levels that may be detrimental to human health. These discoveries have generated considerable interest in perchlorate source identification. In this study, comprehensive stable isotope analyses (37Cl/35Cl and 18O/17O/16O) of perchlorate from known synthetic and natural sources reveal systematic differences in isotopic characteristics that are related to the formation mechanisms. In addition, isotopic analyses of perchlorate extracted from groundwater and surface water demonstrate the feasibility of identifying perchlorate sources in contaminated environments on the basis of this technique. Both natural and synthetic sources of perchlorate have been identified in water samples from some perchlorate occurrences in the United States by the isotopic method.

Comparison of delta18O measurements in nitrate by different combustion techniques.

Three different KNO3 salts with delta18O values ranging from about -31 to +54 per thousand relative to VSMOW were used to compare three off-line, sealed glass tube combustion methods (widely used for isotope studies) with a more recently developed on-line carbon combustion technique. All methods yielded roughly similar isotope ratios for KNO3 samples with delta18O values in the midpoint of the delta18O scale near that of the nitrate reference material IAEA-NO-3 (around +21 to +25 per thousand). This reference material has been used previously for one-point interlaboratory and intertechnique calibrations. However, the isotope ratio scale factors by all of the off-line combustion techniques are compressed such that they are between 0.3 and 0.7 times that of the on-line combustion technique. The contraction of the 6180 scale in the off-line preparations apparently is caused by O isotope exchange between the sample and the glass combustion tubes. These results reinforce the need for nitrate reference materials with delta18O values far from that of atmospheric O2, to improve interlaboratory comparability.