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Comprehensive multiphase-nuclear magnetic resonance (CMP-NMR) is a non-invasive approach designed to observe all phases (solutions, gels, and solids) in intact samples using a single NMR probe. Studies of dead and living organisms are important to understand processes ranging from biological growth to environmental stress. Historically, such studies have utilized

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors. Here, a larger 7 mm probehead is introduced which provides ∼3 times the volume and ∼2.4 times the signal over a 4 mm version. This offers two main advantages: (1) the additional biomass reduces experiment time, making 13C detection at natural abundance more feasible; (2) it allows the analysis of larger samples that cannot fit within a 4 mm rotor. Chicken heart tissue and Hyalella azteca (freshwater shrimp) are used to demonstrate that phase-based spectral editing works with 7 mm rotors and that the additional biomass from the larger volumes allows detection with 13C at natural abundance. Additionally, a whole pomegranate seed berry (aril) and an intact softgel capsule of hydroxyzine hydrochloride are used to demonstrate the analysis of samples too large to fit inside a conventional 4 mm CMP probe. The 7 mm version introduced here extends the range of applications and sample types that can be studied and is recommended when 4 mm CMP probes cannot provide adequate signal-to-noise (S/N), or intact samples are simply too big for 4 mm rotors.

Daphnia (freshwater fleas) are among the most widely used organisms in regulatory aquatic toxicology/ecology, while their recent listing as an NIH model organism is stimulating research for understanding human diseases and processes. Daphnia are small enough to fit inside high field NMR spectrometers and can be kept alive indefinitely using flow systems that deliver food and oxygen. As such, in vivo NMR holds the potential to monitor when/if environmental stress is occurring, understand "why" chemicals are toxic (biochemical pathways impacted and toxic-mode-of-action), and differentiate between a temporary flux response (i.e. return to homeostasis) and a permanent change in biochemistry (likely a precursor to disease). At present however, such studies are limited as the in vivo NMR data of Daphnia are highly complex and the lack of spectral assignments makes extracting metabolic information difficult. In this study, Daphnia are 13C enriched to >97% 13C and numerous 1H and 13C 1D, 2D, and 3D NMR approaches are combined to provide, as complete as possible, an assignment of the Daphnia magna metabolome in vivo. Assignments are transferred (where possible) back to line narrowed susceptibility suppressed 1H 1D NMR spectra in order to permit the maximum amount of information to be gained in the future without the need for 13C enrichment. To our knowledge, this work represents the first time a comprehensive metabolic assignment of any small living organism has been performed using high field flow-based NMR.

During pregnancy, appropriate placental metabolism is essential for fetuses to reach their growth potential. However, metabolic mechanisms during pregnancy remain poorly understood. Determination of the levels of placental metabolites in healthy pregnancy and how they change throughout gestation is critical for understanding placental function.To determine the effects of gestational age on placental metabolites using healthy pregnant mice.In the present study, we collected placental tissue samples from healthy pregnant mice at three timepoints in late gestation (n = 16 placentas per gestational age). Metabolite profiles were determined usingUsing HRMAS MRS, we identified 14 metabolites in murine placental tissue samples. The relative concentration of 12 of the 14 metabolites remains unchanged throughout late gestation. Lysine was found to decrease significantly (p = 0.04) and glucose showed an inverted U-shape relationship (p = 0.03) with gestational age.This study demonstrated the feasibility of HRMAS MRS to determine relative metabolite concentrations in murine placental tissue. These findings establish baseline levels of placental tissue metabolite profiles and will serve as reference ranges for future studies using mouse models of fetal distress.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

The pharmaceutical 17α-ethynylestradiol (EE2) and the industrial chemical perfluorooctane sulfonate (PFOS) are organic contaminants frequently detected in freshwater environments. It is hypothesized that hydrophobic organic contaminants can sorb to dissolved organic matter (DOM) and this may reduce the toxicity of these contaminants by reducing the contaminants’ bioavailability. To investigate this hypothesis, 1H nuclear magnetic resonance (NMR)-based metabolomics was used to determine how the metabolome of Daphnia magna changes when a range of DOM concentrations are added during EE2 and PFOS exposure experiments. D. magna were exposed for 48 h to sub-lethal concentrations of 1 mg/L EE2 or 30 mg/L PFOS in the presence of 0, 1, 2, 3 and 4 mg dissolved organic carbon (DOC)/L. EE2 exposure resulted in increased amino acids and decreased glucose in D. magna. All DOM concentrations were able to lessen these metabolite disturbances from EE2 exposure, likely due to reductions in the bioavailability of EE2 through interactions with DOM. Exposure to PFOS resulted in decreased amino acids, and the presence of 1 mg DOC/L did not alter this metabolic response. However, PFOS exposure with the higher DOM concentrations resulted in a different pattern of metabolite changes which may be due to combined impacts of PFOS and DOM on the metabolome or due to an increase in PFOS bioavailability and uptake in D. magna. These results suggest that the concentration of DOM influences the sensitive biochemical changes in organisms that occur during acute sub-lethal exposure to organic contaminants.

NMR spectroscopy is arguably the most powerful tool for the study of molecular structures and interactions, and is increasingly being applied to environmental research, such as the study of wastewater. With over 97% of the planet’s water being saltwater, and two thirds of freshwater being frozen in the ice caps and glaciers, there is a significant need to maintain and reuse the remaining 1%, which is a precious resource, critical to the sustainability of most life on Earth. Sanitation and reutilization of wastewater is an important method of water conservation, especially in arid regions, making the understanding of wastewater itself, and of its treatment processes, a highly relevant area of environmental research. Here, the benefits, challenges and subtleties of using NMR spectroscopy for the analysis of wastewater are considered. First, the techniques available to overcome the specific challenges arising from the nature of wastewater (which is a complex and dilute matrix), including an examination of sample preparation and NMR techniques (such as solvent suppression), in both the solid and solution states, are discussed. Then, the arsenal of available NMR techniques for both structure elucidation (e.g., heteronuclear, multidimensional NMR, homonuclear scalar coupling-based experiments) and the study of intermolecular interactions (e.g., diffusion, nuclear Overhauser and saturation transfer-based techniques) in wastewater are examined. Examples of wastewater NMR studies from the literature are reviewed and potential areas for future research are identified. Organized by nucleus, this review includes the common heteronuclei (13C, 15N, 19F, 31P, 29Si) as well as other environmentally relevant nuclei and metals such as 27Al, 51V, 207Pb and 113Cd, among others. Further, the potential of additional NMR methods such as comprehensive multiphase NMR, NMR microscopy and hyphenated techniques (for example, LC-SPE-NMR-MS) for advancing the current understanding of wastewater are discussed. In addition, a case study that combines natural abundance (i.e. non-concentrated), targeted and non-targeted NMR to characterize wastewater, along with in vivo based NMR to understand its toxicity, is included. The study demonstrates that, when applied comprehensively, NMR can provide unique insights into not just the structure, but also potential impacts, of wastewater and wastewater treatment processes. Finally, low-field NMR, which holds considerable future potential for on-site wastewater monitoring, is briefly discussed. In summary, NMR spectroscopy is one of the most versatile tools in modern science, with abilities to study all phases (gases, liquids, gels and solids), chemical structures, interactions, interfaces, toxicity and much more. The authors hope this review will inspire more scientists to embrace NMR, given its huge potential for both wastewater analysis in particular and environmental research in general.

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as

In vivo nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for in vivo NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors. The use of a double pump design with a sump slurry pump return allows algal food suspensions to be continually supplied without the need for filters, eliminating the possibility of clogging and leaks. Using the flow system, the living organism can be kept alive without stress indefinitely. To evaluate the feasibility and applicability of the flow system, changes in the metabolite profile of 13C enriched Daphnia magna over a 24-h period are compared when feeding laborato...

A relatively recent technique termed comprehensive multiphase (CMP) NMR spectroscopy was used to investigate the growth and associated metabolomic changes of 13C-labeled wheat seeds and germinated seedlings. CMP-NMR enables the study of all phases in intact samples (i.e., liquid, gel-like, semisolid, and solid), by combining all required electronics into a single NMR probe, and can be used for investigating biological processes such as seed germination. All components, from the most liquid-like (i.e., dissolved metabolites) to the most rigid or solid-like (seed coat) were monitored in situ over 4 days. A wide range of metabolites were identified, and after 96 h of germination, the number of metabolites in the mobile phase more than doubled in comparison to 0 h (dry seed). This work represents the first application of CMP-NMR to follow biological processes in plants.

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D

Pharmaceuticals and personal care products are a class of emerging contaminants that are present in wastewater effluents, surface water, and groundwater around the world. There is a need to determine rapid and reliable bioindicators of exposure and the toxic mode of action of these contaminants to aquatic organisms. (1)H nuclear magnetic resonance (NMR)-based metabolomics in combination with multivariate statistical analysis was used to determine the metabolic profile of Daphnia magna after exposure to a range of sub-lethal concentrations of triclosan (6.25-100μg/L), carbamazepine (1.75-14mg/L) and ibuprofen (1.75-14mg/L) for 48h. Sub-lethal triclosan exposure suggested a general oxidative stress condition and the branched-chain amino acids, glutamine, glutamate, and methionine emerged as potential bioindicators. The aromatic amino acids, serine, glycine and alanine are potential bioindicators for sub-lethal carbamazepine exposure that may have altered energy metabolism. The potential bioindicators for sub-lethal ibuprofen exposure are serine, methionine, lysine, arginine and leucine, which showed a concentration-dependent response. The differences in the metabolic changes were related to the dissimilar modes of toxicity of triclosan, carbamazepine and ibuprofen. (1)H NMR-based metabolomics gave an improved understanding of how these emerging contaminants impact the keystone species D. magna.

Cotton and polyester, physically and chemically different fabrics, were characterized for sorption of gas-phase polybrominated diphenyl ethers (PBDEs). Scanning electron microscopic (SEM) images and BET specific surface area (BET-SSA) analysis showed cotton's high microsurface area; NMR analysis showed richness of hexose- and aromatic-carbon in cotton and polyester, respectively. Cotton and polyester sorbed similar concentrations of gas-phase PBDEs in chamber studies, when normalized to planar surface area. However, polyester concentrations were 20-50 times greater than cotton when normalized to BET-SSA, greater than the 10 times difference in BET-SSA. The difference in sorption between cotton and polyester is hypothesized to be due to 'dilution' due to cotton's large BET-SSA and/or greater affinity of PBDEs for aromatic-rich polyester. Similar fabric-air area normalized distribution coefficients (K'D, 10(3) to 10(4)m) for cotton and polyester support air-side controlled uptake under non-equilibrium conditions. K'D values imply that 1m(2) of cotton or polyester fabrics would sorb gas-phase PBDEs present in 10(3) to 10(4)m(3) of equivalent air volume at room temperature over one week, assuming similar air flow conditions. Sorption of PBDEs to fabrics has implications for their fate indoors and human exposure.

(1)H nuclear magnetic resonance (NMR)-based metabolomics was used to study the response of Daphnia magna to increasing sub-lethal concentrations of either an organophosphate (diazinon or malathion) or bisphenol-A (BPA). Principal component analysis (PCA) of (1)H NMR spectra were used to screen metabolome changes after 48h of contaminant exposure. The PCA scores plots showed that diazinon exposures resulted in aberrant metabolomic profiles at all exposure concentrations tested (0.009-0.135 μg/L), while for malathion the second lowest (0.08μg/L) and two highest exposure concentrations (0.32μg/L and 0.47μg/L) caused significant shifts from the control. Individual metabolite changes for both organophosphates indicated that the response to increasing exposure was non-linear and described perturbations in the metabolome that were characteristic of the severity of exposure. For example, intermediate concentrations of diazinon (0.045μg/L and 0.09μg/L) and malathion (0.08μg/L) elicited a decrease in amino acids such as leucine, valine, arginine, glycine, lysine, glutamate, glutamine, phenylalanine and tyrosine, with concurrent increases in glucose and lactate, suggesting a mobilization of energy resources to combat stress. At the highest exposure concentrations for both organophosphates there was evidence of a cessation in metabolic activity, where the same amino acids increased and glucose and lactate decreased, suggesting a slowdown in protein synthesis and depletion of energy stocks. This demonstrated a similar response in the metabolome between two organophosphates but also that intermediate and severe stress levels could be differentiated by changes in the metabolome. For BPA exposures, the PCA scores plot showed a significant change in metabolome at 0.1mg/L, 1.4mg/L and 2.1mg/L of exposure. Individual metabolite changes from 0.7 to 2.1mg/L of BPA exposure showed increases in amino acids such as alanine, valine, isoleucine, leucine, arginine, phenylalanine and tyrosine. These metabolite changes were correlated with decreases in glucose and lactate. This pattern of response was also seen in the highest organophosphate exposures and suggested a generalized stress response that could be related to altered energy dynamics in D. magna. Through studying increasing exposure responses, we have demonstrated the ability of metabolomics to identify discrete differences between intermediate and severe stress, and also to characterize how systemic stress is manifested in the metabolome.

The enhanced production and environmental release of Buckminsterfullerene (C60) nanoparticles will likely increase the exposure and risk to soil dwelling organisms. We used (1)H NMR-based metabolomics to investigate the response of Eisenia fetida earthworms to sub-lethal C60 nanoparticle exposure in both contact and soil tests. Principal component analysis of (1)H NMR data showed clear separation between controls and exposed earthworms after just 2 days of exposure, however as exposure time increased the separation decreased in soil but increased in contact tests suggesting potential adaptation during soil exposure. The amino acids leucine, valine, isoleucine and phenylalanine, the nucleoside inosine, and the sugars glucose and maltose emerged as potential bioindicators of exposure to C60 nanoparticles. The significant responses observed in earthworms using NMR-based metabolomics after exposure to very low concentrations of C60 nanoparticles suggests the need for further investigations to better understand and predict their sub-lethal toxicity.

Previous studies have shown that contaminant toxicity to target organisms is altered by the presence of dissolved organic matter (DOM). Contaminants can bind to DOM and this may alter the bioavailability and subsequent toxicity of the contaminants. However, molecular-level techniques are needed to more closely evaluate the impact of DOM on the sub-lethal biochemical responses to emerging contaminants. To investigate how DOM may alter the metabolic response to organic contaminant exposure

One-dimensional (1-D) and two-dimensional (2-D) nuclear magnetic resonance (NMR)-based metabolomics was used to investigate the toxic mode of action (MOA) of endosulfan, an organochlorine pesticide, and its degradation product, endosulfan sulfate, to Eisenia fetida earthworms in soil. Three soil concentrations (0.1, 1.0 and 10.0 mg/kg) were used for both endosulfan and endosulfan sulfate. Both earthworm coelomic fluid (CF) and tissues were extracted and then analyzed using 1H and 1H–13C NMR techniques. A similar separation trajectory was observed for endosulfan and endosulfan sulfate-exposed earthworms in the mean principal component analysis (PCA) scores plot for both the earthworm CF and tissue extracts. A neurotoxic and apoptotic MOA was postulated for both endosulfan and endosulfan sulfate exposed earthworms as significant fluctuations in glutamine/GABA–glutamate cycle metabolites and spermidine were detected respectively. This study highlights the application of NMR-based metabolomics to understand molecular-level toxicity of persistent organochlorine pesticides and their degradation products directly in soil.

Nuclear magnetic resonance (NMR) spectroscopy is an essential tool for studying environmental samples but is often hindered by low sensitivity, especially for the direct detection of nuclei such as13C. In very heterogeneous samples with NMR nuclei at low abundance, such as soils, sediments, and air particulates, it can take days to acquire a conventional13C spectrum. The present study describes a prescreening method that permits the rapid prediction of experimental run time in natural samples. The approach focuses the NMR chemical shift dispersion into a single spike, and, even in samples with extremely low carbon content, the spike can be observed in two to three minutes, or less. The intensity of the spike is directly proportional to the total concentration of nuclei of interest in the sample. Consequently, the spike intensity can be used as a powerful prescreening method that answers two key questions: (1) Will this sample produce a conventional NMR spectrum? (2) How much instrument time is required to record a spectrum with a specific signal-to-noise (S/N) ratio? The approach identifies samples to avoid (or pretreat) and permits additional NMR experiments to be performed on samples producing high-quality NMR data. Applications in solid- and liquid-state13C NMR are demonstrated, and it is shown that the technique is applicable to a range of nuclei. Environ. Toxicol. Chem. 2013;32:129–136. © 2012 SETAC

Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition. Herein, intact (13)C-labelled whole cell suspensions of Chlamydomonas reinhardtii, Chlorella vulgaris and Synechocystis were studied using a variety of 1D and 2D (1)H/(13)C solution-state nuclear magnetic resonance (NMR) spectroscopic experiments. Solution-state NMR spectroscopy of whole cell suspensions is particularly relevant as it identifies species that are mobile (dissolved or dynamic gels), 'aquatically available' and directly contribute to the aquatic carbon pool upon lysis, death or become a readily available food source on consumption. In this study, a wide range of metabolites and structural components were identified within the whole cell suspensions. In addition, significant differences in the lipid/triacylglyceride (TAG) content of green algae and cyanobacteria were confirmed. Mobile species in algae are quite different from those in abundance in 'classic' dissolved organic matter (DOM) indicating that if algae are major contributors to DOM, considerable selective preservation of minor components (e.g. sterols) or biotransformation would have to occur. Identifying the metabolites and dissolved components within algal cells by NMR permits future studies of carbon transfer between species and through the food chain, whilst providing a foundation to better understand the role of algae in the formation of DOM and the sequestration/transformation of carbon in aquatic environments.

Since the isolation of soil organic matter in 1786, tens of thousands of publications have searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical role in defining soil organic matter but traditional approaches remove key information such as the distribution of components at the soil-water interface and conformational information. Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural components in soil organic matter are identified to be largely composed of macromolecular inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants. Here, for the first time, a complete range of physical states of a whole soil can be studied. This provides a more detailed understanding of soil organic matter at the molecular level itself key to develop the most efficient soil remediation and agricultural techniques, and better predict carbon sequestration and climate change.

Nuclear magnetic resonance (NMR) is arguably the most powerful and versatile tool in modern science. It has the capability to solve complex structures and interactions in situ even in complex heterogeneous multiphase samples such as soil, plants, and tissues. NMR has vast potential in environmental research and can provide insight into a diverse range of environmental processes at the molecular level be it identifying the binding site in human blood for a specific contaminant or the compositional dynamics of soil with climate change. Modern NMR-based metabonomics is elucidating contaminant toxicity and toxic mode of action rapidly and at sub lethal concentrations. Combined modern NMR approaches provide a powerful framework to better understand carbon cycling and sustainable agriculture, as well as contaminant fate, bioavailability, toxicity, sequestration, and remediation.

Heterogeneous samples, such as soils, sediments, plants, tissues, foods and organisms, often contain liquid-, gel- and solid-like phases and it is the synergism between these phases that determine their environmental and biological properties. Studying each phase separately can perturb the sample, removing important structural information such as chemical interactions at the gel-solid interface, kinetics across boundaries and conformation in the natural state. In order to overcome these limitations a Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) probe has been developed, and is introduced here, that permits all bonds in all phases to be studied and differentiated in whole unaltered natural samples. The CMP-NMR probe is built with high power circuitry, Magic Angle Spinning (MAS), is fitted with a lock channel, pulse field gradients, and is fully susceptibility matched. Consequently, this novel NMR probe has to cover all HR-MAS aspects without compromising power handling to permit the full range of solution-, gel- and solid-state experiments available today. Using this technology, both structures and interactions can be studied independently in each phase as well as transfer/interactions between phases within a heterogeneous sample. This paper outlines some basic experimental approaches using a model heterogeneous multiphase sample containing liquid-, gel- and solid-like components in water, yielding separate (1)H and (13)C spectra for the different phases. In addition, (19)F performance is also addressed. To illustrate the capability of (19)F NMR soil samples, containing two different contaminants, are used, demonstrating a preliminary, but real-world application of this technology. This novel NMR approach possesses a great potential for the in situ study of natural samples in their native state.

Comprehensive multiphase NMR is a novel NMR technique that permits all components (solutions, gels, and solids) to be studied in unaltered natural samples. In this study a wide range of CMP-NMR interaction and editing-based experiments are combined to follow contaminants (pentafluorophenol (PFP) and perfluorooctanoic acid (PFOA)) from the solution state (after a spill) through the gel-state and finally into the true solid-state (sequestered) in an intact water-swollen soil. Kinetics experiments monitoring each phase illustrate PFOA rapidly transfers from solution to the solid phase while for PFP the process is slower with longer residence times in the solution and gel phase. Interaction-based experiments reveal that PFOA enters the soil via its hydrophobic tails and selectively binds to soil microbial protein. PFP sorption shows less specificity exhibiting interactions with a range of gel and solid soil components with a preference toward aromatics (mainly lignin). The results indicate that in addition to more traditional measurements such as Koc, other factors including the influence of the contaminant on the soil-water interface, specific biological interactions, soil composition (content of lignin, protein, etc.) and physical accessibility/swellability of soil organic components will likely be central to better explaining and predicting the true behavior of contaminants in soil.

1 H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm 2 of phenanthrene (1/64th of the LC 50 ) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by 1 H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies.

Dissolved organic matter (DOM) is ubiquitous in aquatic ecosystems and is derived from various inputs that control its turnover. Glaciers and ice sheets are the second largest water reservoir in the global hydrologic cycle, but little is known about glacial DOM composition or contributions to biogeochemical cycling. Here we employ SPR-W5-WATERGATE (1)H NMR spectroscopy to elucidate and quantify the chemical structures of DOM constituents in Antarctic glacial ice as they exist in their natural state (average DOC of 8 mg/L) without isolation or preconcentration. This Antarctic glacial DOM is predominantly composed of a mixture of small recognizable molecules differing from DOM in marine, lacustrine, and other terrestrial environments. The major constituents detected in three distinct types of glacial ice include lactic and formic acid, free amino acids, and a mixture of simple sugars and amino sugars with concentrations that vary between ice types. The detection of free amino acid and amino sugar monomer components of peptidoglycan within the ice suggests that Antarctic glacial DOM likely originates from in situ microbial activity. As these constituents are normally considered to be biologically labile (fast cycling) in nonglacial environments, accelerated glacier melt and runoff may result in a flux of nutrients into adjacent ecosystems.

(1)H NMR-based metabolomics was used to examine the metabolic profile of D(2)O-buffer extracted tissues of Eisenia fetida earthworms exposed for 2 days to an artificial soil spiked with sub-lethal concentrations of polychlorinated biphenyls (PCBs) (0, 0.5, 1, 5, 10, or 25 mg/kg Aroclor 1254). Univariate statistical analysis of the identified metabolites revealed a significant increase in ATP concentration in earthworms exposed to the highest soil PCB concentration, but detected no significant changes in other metabolites. However, a multivariate approach which considers alterations in multiple metabolites simultaneously, identified a significant linear relationship between earthworm metabolic profiles and PCB concentration (cross-validated PLS-regression with 7 components, R(2)X = 0.99, R(2)Y = 0.77, Q(2)Y = 0.45, P 0.001). Significant changes in pair-wise metabolic correlations were also detected as PCB concentration increased. For example, lysine and ATP concentrations showed no apparent correlation in control earthworms (r = 0.22, P = 0.54), but were positively correlated in earthworms from the 25 mg/kg treatment (r = 0.87, P = 0.001). Overall, the observed metabolic responses suggest that PCBs disrupted both carbohydrate (energy) metabolism and membrane (osmolytic) function in E. fetida. The ability of (1)H NMR-based metabolomics to detect these responses suggests that this method offers significant potential for direct assessment of sub-lethal PCB toxicity in soil.

Eisenia fetida earthworms were exposed to phenanthrene for thirty days to compare hydroxypropyl-beta-cyclodextrin (HPCD) extraction of soil and 1H NMR earthworm metabolomics as indicators of bioavailability. The phenanthrene 28-d LC50 value was 750 mg/kg (632-891, 95% confidence intervals) for the peat soil tested. The initial phenanthrene concentration was 319 mg/kg, which biodegraded to 16 mg/kg within 15 days, at which time HPCD extraction suggested that phenanthrene was no longer bioavailable. Multivariate statistical analysis of 1H NMR spectra for E. fetida tissue extracts indicated that phenanthrene exposed and control earthworms differed throughout the 30 day experiment despite the low phenanthrene concentrations present after 15 days. This metabolic response was better correlated to total phenanthrene concentrations (Q2 = 0.59) than HPCD-extractable phenanthrene concentrations (Q2 = 0.46) suggesting that 1H NMR metabolomics offers considerable promise as a novel, molecular-level method to directly monitor the bioavailability of contaminants to earthworms in the environment.

1H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-beta-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised approximately 65-97% of total phenanthrene added to the soil. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.

Site-specific isotopic values of methyl tertiary butyl ether (MTBE) were measured using quantitative site-specific (2)H nuclear magnetic resonance (NMR) spectroscopy for seven commercially available MTBE products. The delta(2)H values of the methoxy and tertiary butyl groups ranged from -103 per thousand to -171 per thousand, and from -76 per thousand to -104 per thousand, reflecting their production from methanol and isobutene, respectively. Several MTBE products whose whole-compound delta(13)C and delta(2)H MTBE values were within error of each other, as measured by isotope ratio mass spectrometry (IRMS), had demonstrably different delta(2)H values for their methoxy and tertiary butyl groups measured by (2)H NMR. Site-specific isotopic variations were large enough to provide proof of principle that quantitative site-specific (2)H NMR may provide an additional parameter for contaminant sourcing at field sites. Isotopic variations were small enough to not bias the comparability of degradation-associated isotopic enrichment factors determined using different MTBE products. Calculated delta(2)H values for MTBE, derived as weighted averages of (2)H NMR measurements of the two functional groups, showed good agreement with IRMS measurements. The ability to gain accurate information about the site-specific isotopic ratios of (2)H/(1)H within a molecule offers considerable promise as a new environmental tool to track the source and fate of environmental contaminants.

The substantial heterogeneity of dissolved organic matter (DOM) inhibits detailed chromatographic analysis with conventional detectors as little structural information can be obtained in the presence of extensive coelution. Here we examine the direct hyphenation of high-performance size exclusion chromatography (HPSEC) with nuclear magnetic resonance (NMR) spectroscopy to determine how size-distinguished fractions differ in composition. The results support the applicability of using HPSEC to generate more homogeneous fractions of DOM prior to NMR analysis and demonstrate that structure is significantly altered with size. The largest fractions are enriched in carbohydrate- and aromatic-type structures. The midsized material is substantial and is representative of carboxyl-rich alicyclic molecules (CRAMs). The smallest material has strong signatures of material derived from linear terpenoids (MDLT). Both CRAMs and MDLT have been recently hypothesized as major components of DOM, and detection by HPSEC-NMR confirms their existence as unique and separable entities. This preliminary work focuses on NMR hyphenation to HPSEC due to widespread use of HPSEC to characterize DOM. Online hyphenation is useful not only for time-efficient analysis of DOM but also for that of other highly complex samples such as those found in many environmental analyses.

High-resolution magic angle spinning (HR-MAS) NMR spectroscopy combined with saturation-transfer double difference (STDD) NMR can be used to analyze the molecular-level interactions of pesticides and whole soils occurring at the soil-water interface. Here 1H HR-MAS STDD NMR has been applied to some common pesticides (trifluralin, acifluorfen, and (4-nitro-3-(trifluoromethyl) phenol) and a pesticide degradation product (1-naphthol). Results indicate that dipolar interactions, H-bonding, hydrophobic associations, and potentially pi-pi interactions are the predominant sorption mechanisms for these molecules at the soil-aqueous interface. It is evident that the physical and chemical characteristics of soil are highly influential in determining the mechanisms of pesticide sorption, as they significantly affect soil conformation. In particular, different binding mechanisms were observed for 1-naphthol in soil swollen using a buffer versus D2O, indicating that the K(oc) alone may not be enough to accurately predict the behavior of a molecule in a real soil environment. Preliminary kinetic-based studies suggest that both the swelling solvent and soil moisture content significantly influence the sequestration of trifluralin. These studies demonstrate that HR-MAS and STDD NMR are powerful and versatile tools which can be applied to expand our knowledge of the mechanistic interactions of agrochemicals at the molecular level.

Elucidation of mechanistic interactions of anthropogenic chemicals is critical to understanding and eventually predicting their behavior in the environment Here, a recently developed technique, saturation transfer double difference (STDD) NMR spectroscopy is employed to determine the interactions of pesticides with humic acid (HA) at the molecular level. The degree of interaction at each NMR observable nucleus in the pesticide can be quantified in the form of an epitope map, which depicts the mechanism of the pesticide-HA interaction. Our results indicate that, at pH 7, halogen atoms (F and Cl) in water-soluble pesticides (diflufenzopyr, acifluorfen, and chlorsulfuron) play a dominant role in influencing binding to HA, whereas carboxyl groups likely play a secondary role when halogen atoms are also present in the molecule, as observed with diflufenzopyr and acifluorfen. However, when present on its own, the carboxyl group dominates in binding affinityto HA (e.g., imazapyr). Electronegativity and electron density appear to play a key role in the mechanism of binding and results suggest that polar bonds are the primary points of HA contact in the water soluble pesticides investigated. Likely interactions may include hydrogen bonding and dipole-dipole interactions.

Soil microbes are central to many soil processes, but due to the structural complexity of soil organic matter, the accurate quantification of microbial biomass contributions continues to pose a significant analytical challenge. In this study, microbes from a range of soils were cultured such that their molecular profile could be compared to that of soil organic matter and native vegetation. With the use of modern NMR spectroscopy, the contributions from microbial species can be discerned in soil organic matter and quantified. On the basis of these studies, the contributions of microbial biomass to soil organic matter appear to be much higher than the 1-5% reported by other researchers. In some soils, microbial biomass was found to contribute >50% of the extractable soil organic matter fractions and approximately 45% of the humin fraction and accounted for >80% of the soil nitrogen. These findings are significant because organic matter is intimately linked to nutrient release and transport in soils, nitrogen turnover rates, contaminant fate, soil quality, and fertility. Therefore, if in some cases soil organic matter and soil organic nitrogen are predominately of microbial origin, it is likely that this fraction, whether in the form of preserved material or living cells, plays an underestimated role in several soil processes.

Low molecular weight (LMW) biomarkers can be used to trace the source and stage of soil organic matter. However, methods that selectively isolate these groups of compounds are underdeveloped. In this study, we isolate biomarkers by a successive series of extraction and chemical degradation procedures involving solvent extraction (TSE), base (BHY) and acid hydrolysis (AHY), and CuO oxidation (CUO). GC-MS was used to analyze these fractions and the extraction methods were verified by solid-state 13C NMR spectroscopy. The GC-MS response was high for the BHY products (96%), intermediate for the TSE (30%) and CUO (19%), but very low for the AHY fraction (5%) indicating that the fractions contain polar or high molecular weight compounds. Aliphatic lipids (62%), phenols and benzyls (17%) were the predominant classes, accompanied by minor abundances of mono- and disaccharides, LMW acids, terpenoids, steroids, amino acids, and amino sugars. The TSE and BHY fractions contained mainly aliphatic lipids derived from plant waxes, cutin, and suberin. Lignin-derived phenols are the major products in the CUO fraction, and amino compounds and carbohydrates of various sources were identified in the AHY products. The sequential degradation method is useful for the isolation and identification of apolar, LMW biomarkers in soil.

The sorption of phenanthrene was examined in humic acids (HAs) from different sources: a compost, a peat soil, and a mineral soil. Sub-samples of each HA were subjected to bleaching or hydrolysis to remove predetermined chemical groups from their structures. Bleaching successfully removed a large percentage of rigid, aromatic moieties, whereas hydrolysis removed the mobile, carbohydrate components. Phenanthrene sorption by all HAs was nonlinear (N < 1). However, the phenanthrene isotherms of the bleached HAs were more linear than those of the untreated HAs, whereas the removal of the carbohydrate components by hydrolysis produced more nonlinear isotherms. The introduction of pyrene to the phenanthrene sorption system yielded more linear isotherms for all the HAs, indicative of competitive sorption. Proton spin-spin (1H T2) relaxation determined by nuclear magnetic resonance (NMR) was used to identify separate rigid (condensed) and flexible (expanded) 1H populations and to determine their distribution. These 1H domains were highly sensitive to temperature and correlated well with reported glass transition temperatures for HAs. In combination with the chemical treatments, sorption, and spectroscopic data, we were able to observe some significant relationships among chemical groups, sorption behavior, and structural characteristics.

Seeds are complex entities composed of liquids, gels, and solids. NMR spectroscopy is a powerful tool for studying molecular structure but has evolved into two fields, solution and solid state. Comprehensive multiphase (CMP) NMR spectroscopy is capable of liquid-, gel-, and solid-state experiments for studying intact samples where all organic components are studied and differentiated in situ. Herein, intact (13)C-labeled seeds were studied by a variety of 1D/2D (1)H/(13)C experiments. In the mobile phase, an assortment of metabolites in a single (13)C-labeled wheat seed were identified; the gel phase was dominated by triacylglycerides; the semisolid phase was composed largely of carbohydrate biopolymers, and the solid phase was greatly influenced by starchy endosperm signals. Subsequently, the seeds were compared and relative similarities and differences between seed types discussed. This study represents the first application of CMP-NMR to food chemistry and demonstrates its general utility and feasibility for studying intact heterogeneous samples.

(1)H NMR metabolomics and conventional ecotoxicity endpoints were used to examine the response of earthworms exposed to petroleum hydrocarbons (PHCs) in soil samples collected from a site that was contaminated with crude oil from a pipeline failure in the mid-1990s. The conventional ecotoxicity tests showed that the soils were not acutely toxic to earthworms (average survival ≥ 90%), but some soil samples impaired reproduction endpoints by >50% compared to the field control soil. Additionally, metabolomics revealed significant relationships between earthworm metabolic profiles (collected after 2 or 14 days of exposure) and soil properties including soil PHC concentration. Further comparisons by partial least squares regression revealed a significant relationship between the earthworm metabolomic data (collected after only 2 or 14 days) and the reproduction endpoints (measured after 63 days). Therefore, metabolomic responses measured after short exposure periods may be predictive of chronic, ecologically relevant toxicity endpoints for earthworms exposed to soil contaminants.

Metal and metalloid contamination constitutes a major concern in aquatic ecosystems. Thus it is important to find rapid and reliable indicators of metal stress to aquatic organisms. In this study, we tested the use of (1)H nuclear magnetic resonance (NMR) - based metabolomics to examine the response of Daphnia magna neonates after a 48h exposure to sub-lethal concentrations of arsenic (49μgL(-1)), copper (12.4μgL(-1)) or lithium (1150μgL(-1)). Metabolomic responses for all conditions were compared to a control using principal component analysis (PCA) and metabolites that contributed to the variation between the exposures and the control condition were identified and quantified. The PCA showed that copper and lithium exposures result in statistically significant metabolite variations from the control. Contributing to this variation was a number of amino acids such as: phenylalanine, leucine, lysine, glutamine, glycine, alanine, methionine and glutamine as well as the nucleobase uracil and osmolyte glycerophosphocholine. The similarities in metabolome changes suggest that lithium has an analogous mode of toxicity to that of copper, and may be impairing energy production and ionoregulation. The PCA also showed that arsenic exposure resulted in a metabolic shift in comparison to the control population but this change was not statistically significant. However, significant changes in specific metabolites such as alanine and lysine were observed, suggesting that energy metabolism is indeed disrupted. This research demonstrates that (1)H NMR-based metabolomics is a viable platform for discerning metabolomic changes and mode of toxicity of D. magna in response to metal stressors in the environment.

The chemical nature of xenobiotic binding sites in soils is of vital importance to environmental biogeochemistry. Interactions between xenobiotics and the naturally occurring organic constituents of soils are strongly correlated to environmental persistence, bioaccessibility, and ecotoxicity. Nevertheless, because of the complex structural and chemical heterogeneity of soils, studies of these interactions are most commonly performed indirectly, using correlative methods, fractionation, or chemical modification. Here we identify the organic components of an unmodified peat soil where some organofluorine xenobiotic compounds interact using direct molecular-level methods. Using (19)F→(1)H cross-polarization magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectroscopy, the (19)F nuclei of organofluorine compounds are used to induce observable transverse magnetization in the (1)H nuclei of organic components of the soil with which they interact after sorption. The observed (19)F→(1)H CP-MAS spectra and dynamics are compared to those produced using model soil organic compounds, lignin and albumin. It is found that lignin-like components can account for the interactions observed in this soil for heptafluoronaphthol (HFNap) while protein structures can account for the interactions observed for perfluorooctanoic acid (PFOA). This study employs novel comprehensive multi-phase (CMP) NMR technology that permits the application of solution-, gel-, and solid-state NMR experiments on intact soil samples in their swollen state.

(1)H NMR metabolomics was used to measure earthworm sub-lethal responses to polychlorinated biphenyls (PCBs) in historically contaminated (30 years) soils (91-280 mg/kg Aroclor 1254/1260) after two and 14 days of exposure. Although our previous research detected a distinct earthworm metabolic response to PCBs in freshly spiked soil at lower concentrations (0.5-25 mg/kg Aroclor 1254), the results of this study suggest only weak or non-significant relationships between earthworm metabolic profiles and soil PCB concentrations. This concurs with the expectation that decades of contaminant aging have likely decreased PCB bioavailability and toxicity in the field. Instead of being influenced by soil contaminant concentration, earthworm metabolic profiles were more closely correlated to soil properties such as total soil carbon and soil inorganic carbon. Overall, these results suggested that (1)H NMR metabolomics may be capable of detecting both site specific responses and decreased contaminant bioavailability to earthworms after only two days of exposure, whereas traditional toxicity tests require much more time (e.g. 14 days for acute toxicity and50 days for reproduction tests). Therefore, there is significant opportunity to develop earthworm metabolomics as a sensitive tool for rapid assessment of the toxicity associated with contaminated field soils.

Endosulfan is an environmentally persistent pesticide and has been shown to be genotoxic, neurotoxic and carcinogenic to surrounding organisms. Earthworms are widely used in environmental metabolomic studies to assess soil ecotoxicity. Previous nuclear magnetic resonance (NMR)-based metabolomic studies have analyzed earthworm tissue extracts after exposure to endosulfan and identified some key metabolic indicators that can be used as biomarkers of stress. However, some metabolites may have been masked due to overlap with other metabolites in the tissue extract. Therefore, in this study, the coelomic fluid (CF) and the tissue extract of the earthworm, Eisenia fetida, were both investigated using ¹H NMR-based metabolomics to analyze their metabolic profile in response to endosulfan exposure at three sub-lethal (below LC₅₀) concentrations. Principal component analysis determined the earthworm CF and earthworm tissue extract to both have significant separation between the exposed and control at the two highest sub-lethal endosulfan exposures (1.0 and 2.0 μg cm⁻²). Alanine, glycine, malate, α-ketoglutarate, succinate, betaine, myo-inositol, lactate and spermidine in the earthworm CF and alanine, glutamine, fumarate, glutamate, maltose, melibiose, ATP and lactate in earthworm tissue extract were all detected as having significant fluctuations after endosulfan exposure. An increase in ATP production was detected by the increase activity in the citric acid cycle and by anaerobic metabolism. A significant decrease in the polyamine, spermidine after endosulfan exposure describes an apoptotic mode of protection which correlates to a previous endosulfan exposure study where DNA damage has been reported. This study highlights that earthworm CF is a complementary biological medium to tissue extracts and can be helpful to better understand the toxic mode of action of contaminants at sub-lethal levels in the environment.

Glaciers and ice sheets are the second largest freshwater reservoir in the global hydrologic cycle, and the onset of global climate warming has necessitated an assessment of their contributions to sea-level rise and the potential release of nutrients to nearby aquatic environments. In particular, the release of dissolved organic matter (DOM) from glacier melt could stimulate microbial activity in both glacial ecosystems and adjacent watersheds, but this would largely depend on the composition of the material released. Using fluorescence and (1)H NMR spectroscopy, we characterize DOM at its natural abundance in unaltered samples from a number of glaciers that differ in geographic location, thermal regime, and sample depth. Parallel factor analysis (PARAFAC) modeling of DOM fluorophores identifies components in the ice that are predominantly proteinaceous in character, while (1)H NMR spectroscopy reveals a mixture of small molecules that likely originate from native microbes. Spectrofluorescence also reveals a terrestrial contribution that was below the detection limits of NMR; however, (1)H nuclei from levoglucosan was identified in Arctic glacier ice samples. This study suggests that the bulk of the DOM from these glaciers is a mixture of biologically labile molecules derived from microbes.

Soil organic matter (OM) contains vast stores of carbon, and directly supports microbial, plant, and animal life by retaining essential nutrients and water in the soil. Soil OM plays important roles in biological, chemical, and physical processes within the soil, and arguably plays a major role in maintaining long-term ecological stability in a changing world. Despite its importance, there is a great deal still unknown about soil OM chemical ecology. The development of sophisticated analytical methods have reshaped our understanding of soil OM composition, which is now believed to be comprised of plant and microbial products at various stages of decomposition. The methods also have recently been applied to study environmental change in various settings and have provided unique insight with respect to soil OM chemical ecology. The goal of this review is to highlight the methods used to characterize soil OM structure, source, and degradation that have enabled precise observations of OM and associated ecological shifts. Although the chemistry of soil OM is important in its overall fate in ecosystems, the studies conducted to date suggest that ecological function is not defined by soil OM chemistry alone. The long-standing questions regarding soil OM stability and recalcitrance will likely be answered when several molecular methods are used in tandem to closely examine structure, source, age, degradation stage, and interactions of specific OM components in soil.

¹H NMR-based metabolomics was used to examine the response of Eisenia fetida earthworms raised from juveniles for 20-23 weeks in soil spiked with either 20 or 200 mg/kg of a commercially available uncoated titanium dioxide (TiO(2)) nanomaterial (nominal diameter of 5 nm). To distinguish responses specific to particle size, soil treatments spiked with a micrometer-sized TiO(2) material (nominal diameter,45 μm) at the same concentrations (20 and 200 mg/kg) were also included in addition to an unspiked control soil. Multivariate statistical analysis of the (1)H NMR spectra for aqueous extracts of E. fetida tissue suggested that earthworms exhibited significant changes in their metabolic profile following TiO(2) exposure for both particle sizes. The observed earthworm metabolic changes appeared to be consistent with oxidative stress, a proposed mechanism of toxicity for nanosized TiO(2). In contrast, a prior study had observed no impairment of E. fetida survival, reproduction, or growth following exposure to the same TiO(2) spiked soils. This suggests that (1)H NMR-based metabolomics provides a more sensitive measure of earthworm response to TiO(2) materials in soil and that further targeted assays to detect specific cellular or molecular level damage to earthworms caused by chronic exposure to TiO(2) are warranted.

The elucidation of molecular structures present in dissolved organic matter (DOM) has the potential to unlock many of the queries associated with organic precursors, diagenetic processes and reactivity of this highly complex material. Suwannee River DOM was extensively fractionated by two dimensional hydrophilic interaction chromatography (HILIC)/HILIC and fractions were analyzed via a suite of two and three-dimensional NMR experiments. HILIC provided more greatly resolved fractions with a second dimension and enabled extensive and in-depth NMR analyses. The composite NMR experiments provide strong evidence for highly oxidized sterols as major structural components present in one of the most simplified and subsequently resolved fractions. Further interpretation of data on other fractions across the polarity gradient likewise supports the presence of alicyclic structures present with considerable hydroxyl groups, carboxylic acids and methyl groups associated with quaternary carbon suggesting that further sterol- and hopanoid-type structures are potentially dominant throughout DOM.

Nuclear magnetic resonance (NMR) – based metabolomics has the potential to identify toxic responses of contaminants within a mixture in contaminated soil. This study evaluated the metabolic response of Eisenia fetida after exposure to an array of organic compounds to determine whether contaminant-specific responses could be identified. The compounds investigated in contact tests included: two pesticides (carbaryl and chlorpyrifos), three pharmaceuticals (carbamazephine, estrone and caffeine), two persistent organohalogens (Aroclor 1254 and PBDE 209) and two industrial compounds (nonylphenol and dimethyl phthalate). Control and contaminant-exposed metabolic profiles were distinguished using principal component analysis and potential contaminant-specific biomarkers of exposure were found for several contaminants. These results suggest that NMR-based metabolomics offers considerable promise for differentiating between the different toxic modes of action (MOA) associated with sub-lethal toxicity to earthworms.

This article presents research targeted toward the isolation and detection of unique molecular structures from what is believed to be the world's most complex organic mixture: dissolved organic matter (DOM). Hydrophilic interaction chromatography (HILIC) was used to separate Suwannee River DOM (SRDOM) into 80 fractions, simplified to the extent that detection with nuclear magnetic resonance spectroscopy (NMR) results in many sharp signals that are indicative of individual compounds, some of which are identifiable with multidimensional NMR. Parallel factor analysis (PARAFAC) of fluorescence excitation-emission matrices (EEMs) was additionally employed on HILIC-simplified fractions to further confirm the effectiveness of the HILIC separations as well as draw insight into how structural characteristics relate to DOM fluorescence signals. Findings suggest that material believed to be derived from both cyclic and linear terpenoids was dominant in the most hydrophobic fractions as were the majority of the fluorescence signals, whereas hydrophilic material was highly correlated with carbohydrate-type structures as well as high contributions from amino acid fluorescence. NMR spectra of DOM, typically featureless mounds, are substantially more detailed with HILIC-simplified fractions to the point where hundreds of signals are present and 2D NMR correlations permit significant structural identifications.

Soil organic matter (SOM) is one of the most complex natural mixtures on earth. It plays a critical role in many ecosystem functions and is directly responsible for sustaining life on our planet. However, due to its chemical heterogeneity, SOM composition at molecular-level is still not completely clear. Consequently, the response of SOM to global climate change is difficult to predict. Here we highlight applications of two complementary molecular-level methods (biomarkers and nuclear magnetic resonance (NMR)) for ascertaining SOM responses to various environmental changes. Biomarker methods that measure highly specific molecules determine the source and decomposition stage of SOM components. However, biomarkers only make up a small fraction of SOM components because much of SOM is non-extractable. By comparison, (13)C solid-state NMR allows measurement of SOM in its entirety with little or no pretreatment but suffers from poor resolution (due to overlapping of SOM components) and insensitivity, and thus subtle changes in SOM composition may not always be detected. Alternatively, (1)H solution-state NMR methods offer an added benefit of improved resolution and sensitivity but can only analyze SOM components that are fully soluble (humic type molecules) in an NMR compatible solvent. We discuss how these complementary methods have been employed to monitor SOM responses to: soil warming in a temperate forest, elevated atmospheric CO(2) and nitrogen fertilization in a temperate forest, and permafrost thawing in the Canadian High Arctic. These molecular-level methods allow some novel and important observations of soil dynamics and ecosystem function in a changing climate.