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Endocrine disruption is intimately linked to controlling the population of pollutant-exposed organisms through reproduction and development dysregulation. This study investigated how endocrine disruption in a predator organism could affect prey species biology through infochemical communication. Daphnia magna and Chlorella vulgaris were chosen as model prey and predator planktons, respectively, and fenoxycarb was used for disrupting the endocrine system of D. magna. Hormones as well as endo- and exometabolomes were extracted from daphnids and algal cells and their culture media and analyzed using liquid chromatography with tandem mass spectrometry. Biomolecular perturbations of D. magna under impaired offspring production and hormone dysregulation were observed. Differential biomolecular responses of the prey C. vulgaris, indicating changes in methylation and infochemical communication, were subsequently observed under the exposure to predator culture media, containing infochemicals released from the reproducibly normal and abnormal D. magna, as results of fenoxycarb exposure. The observed cross-species transfer of the endocrine disruption consequences, initiated from D. magna, and mediated through infochemical communication, demonstrates a novel discovery and emphasizes the broader ecological risk of endocrine disruptors beyond reproduction disruption in target organisms.

Evaluate crop rotation diversity, perenniality, carbon (C) inputs, and C input quality as predictors of soil organic carbon (SOC) concentration and aggregate mean weight diameter (MWD). At a crop rotation trial in its 37th year in Ontario, Canada, species in rotations included corn (C, Zea mays L.), alfalfa (A, Medicago sativa L.), soybean (S, Glycine max (L.) Merr.), winter wheat (W, Triticum aestivum L.), and red clover (rc, Trifolium pratense L.). Rotations were: CC, CCAA, CCSS, CCSW, CCSWrc, AA. Soils (0–20 cm) were analyzed for aggregate MWD, aggregate C, and SOC concentrations. We estimated C inputs from historical yields and C input quality (C:N, lignin: N, or NMR-derived index) from structural plant tissues. C stabilization efficiency was estimated as the ratio of SOC stock per unit total or root C input. Crop rotation diversity failed to increase SOC concentrations or aggregate MWD. Perennialized rotations (CCSWrc, CCAA, AA) maintained the numerically highest SOC concentrations, and root C input increased SOC concentration. Out of 12 statistical tests relating C input quality to C stabilization efficiency, only 3 indicated a positive effect and 6 tests indicated a negative effect. Including a perennial forage such as alfalfa and limiting soybean frequency promotes aggregate MWD and SOC concentration more so than optimizing crop rotation diversity. Quality of structural plant inputs does not explain differences in C stabilization efficiency, possibly due to overriding influence of living root inputs.

Bisphenols are used in the production of polycarbonate plastics and epoxy resins. Bisphenol A (BPA) has been widely studied and is believed to act as an endocrine disruptor. Bisphenol F (BPF) and bisphenol S (BPS) have increasingly been employed as replacements for BPA, although previous studies suggested that they yield similar physiological responses to several organisms. Daphnia magna is a common model organism for ecotoxicology and was exposed to sub-lethal concentrations of BPA, BPF, and BPS to investigate disruption to metabolic profiles. Targeted metabolite analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure polar metabolites extracted from D. magna, which are linked to a range of biochemical pathways. Multivariate analyses and individual metabolite changes showed similar non-monotonic concentration responses for all three bisphenols (BPA, BPF, and BPS). Pathway analyses indicated the perturbation of similar and distinct pathways, mostly associated with protein synthesis, amino acid metabolism, and energy metabolism. Overall, we observed responses that can be linked to a chemical class (bisphenols) as well as distinct responses that can be related to each individual bisphenol type (A, F, and S). These findings further demonstrate the need for using metabolomic analyses in exposure assessment, especially for chemicals within the same class which may disrupt the biochemistry uniquely at the molecular-level.

Daphnia magna is a keystone indicator zooplankton used in environmental quality assessments. Comparative metabolomics, which contrasts small biomolecular regulations under different conditions, has emerged as a sensitive and informative investigation tool for the assessment of environmental stressors on D. magna. Baseline metabolomic variation is likely impacted by the asexual reproduction cycle of D. magna; however, the relationship between metabolite concentration regulation and reproduction cycle has not been investigated. This study investigated the time-course regulation of 51 metabolites during different reproduction stages to determine how the reproduction cycle controlled the metabolite profile of D. magna. Two-way analysis of variance (ANOVA) results reveal that most metabolites show significantly differentiated concentrations by individual or a combination of reproduction stages and sampling time. On the basis of the partial least-squares-discriminant analysis (PLS-DA) and ANOVA-simultaneous component analysis (ANOVA-SCA), stages 2 and 3 of reproduction show similarity in metabolite abundance regulation compared to stage 1. Metabolites were classified as being either dependent or independent of reproduction in the pairwise concentration correlation based on Pearson correlation coefficients. This study observed that the D. magna reproduction stage is an important consideration and potential variable and should be considered carefully when conducting metabolomic experiments using D. magna.

NMR applied to living organisms is arguably the ultimate tool for understanding environmental stress responses and can provide desperately needed information on toxic mechanisms, synergistic effects, sublethal impacts, recovery, and biotransformation of xenobiotics. To perform in vivo NMR spectroscopy, a flow cell system is required to deliver oxygen and food to the organisms while maintaining optimal line shape for NMR spectroscopy. In this tutorial, two such flow cell systems and their constructions are discussed: (a) a single pump high-volume flow cell design is simple to build and ideal for organisms that do not require feeding (i.e., eggs) and (b) a more advanced low-volume double pump flow cell design that permits feeding, maintains optimal water height for water suppression, improves locking and shimming, and uses only a small recirculating volume, thus reducing the amount of xenobiotic required for testing. In addition, key experimental aspects including isotopic enrichment, water suppression, and 2D experiments for both 13 C enriched and natural abundance organisms are discussed.

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.

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 1H-13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

NMR spectroscopy is a powerful tool for metabolite screening, and owing to its noninvasive nature, it can be applied in vivo. However, the magnetic susceptibility mismatches within the intact organ...

Forest soil organic matter (SOM) is derived from both above- and belowground plant inputs, with a significant portion of this plant-derived organic carbon being processed by fungal communities prior to becoming SOM. Despite the central role of fungi in the processing of litter and root inputs, little is known about the relationship between fungal community composition and the relative conversion of aboveground litter versus root inputs into SOM. The Detrital Input and Removal Treatment (DIRT) experiments offer a system in which to test such questions, wherein aboveground litter and root inputs to forest soils are chronically excluded independently and in combination. In this study, we characterized fungal biomass (ergosterol), community composition (ITS2 metabarcoding), and community assembly (via null modeling) under root and litter exclusion treatments in a northern temperate forest DIRT experiment, and we related fungal community parameters to soil carbon. Root, but not litter, presence significantly differentiated fungal community composition and drove a more stochastic community assembly. Roots were associated with higher fungal biomass, particularly that of ectomycorrhizal and saprotrophic fungi, and with higher soil carbon stocks. The resulting strong positive correlation between fungal biomass and soil carbon appeared to be driven by saprotroph biomass in particular. We suggest that fungal communities are important mediators of the conversion of above- and belowground plant inputs into SOM, and that characterizing their composition may aid in understanding the relative importance of these plant input sources to SOM.

Atrazine toxicity to earthworms is still not fully understood, particularly at sub-lethal concentrations. Because of the ubiquity of atrazine in the environment, it is imperative to understand the impacts of atrazine presence on soil-dwelling organisms. To examine this in detail, we used 1H nuclear magnetic resonance (NMR)-based metabolomics to elucidate earthworm (Eisenia fetida) responses after a 48 h of atrazine exposure in contact tests. Earthworms were exposed to four sub-lethal concentrations of 362.4 ng/cm2, 181.2 ng/cm2, 90.6 ng/cm2 and 45.3 ng/cm2, which correspond to 1/8th, 1/16th, 1/32nd and 1/64th of the LC50 value respectively. After exposure, polar metabolites were isolated from earthworm tissues and analyzed using 1H NMR spectroscopy. Sub-lethal atrazine exposure induced a non-monotonic response with respect to exposure concentration and caused an overall suppression in earthworm metabolism. Maltose, fumarate, malate, threonine/lactate, adenosine-5'-triphosphate (ATP), betaine, scyllo-inositol, glutamate, arginine and glutamine were the metabolites identified as most sensitive to atrazine exposure. These observed fluctuations in the metabolic profile suggest that atrazine reduced ATP synthesis and negatively impacted the health of earthworms with acute sub-lethal exposure. Our study also demonstrates the utility of NMR-based metabolomics for the basic assessment of sub-lethal toxicity which can then be used for more targeted approaches with other molecular techniques. This article is protected by copyright. All rights reserved

Nitrogen (N) fertilization has been found to alter soil carbon storage in agroecosystem as various soil organic matter (OM) components respond differently to environmental change. However, how the direction and magnitude of soil carbon storage changes in response to N fertilization remains unclear, and a lack of deep understanding on soil OM composition particularly under various fertilization rates constrains our ability to reveal soil OM dynamics. In this study, we investigated soil OM composition and degradation at the molecular-level in response to various rates of N fertilization, and the potential mechanisms that govern these alterations. Soil samples (0−10 cm) were collected from a 10-year N fertilization experiment in Southern Ontario, Canada with increasing rates (non-fertilized control, 30, 87, 145 and 260 kg ha−1 yr−1). The samples were analyzed for soil organic carbon and total N concentrations, soil OM composition (i.e. plant-derived steroids, cutin-, suberin-, lignin-derived compounds), and microbial biomass and community structure. Despite similar soil organic carbon (21.7–23.1 g/kg) and total N contents (2.0–2.2 g/kg) across all treatments, decreased concentrations of plant-derived steroids, cutin- and suberin-derived compounds under some N addition treatments were observed. This was consistent with lower alkyl carbon contents from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy analysis. The lower plant-derived steroids, cutin- and suberin-derived lipids may be explained by the enhanced degradation of steroids and cutin likely associated with higher microbial biomass under N fertilization. Lignin-derived phenols were elevated under N fertilization, which is consistent with the aromatic/phenolic carbon contents (mainly from lignin) from NMR analysis. The enhanced lignin degradation, which is consistent with higher fungal biomass (lignin degraders), did not result in lower lignin-derived compound or aromatic/phenolic carbon content under N addition. As other studies reported higher lignin contents in corn residues under N fertilization, we suggest that the turnover of lignin-derived phenols may be governed not only by degradation processes but also by the quantity and quality of crop residues. Furthermore, we observed that different N rates exert various controls on soil OM cycling. For example, the increase in degradation of steroids, cutin and lignin was smaller above the threshold N rates 145 kg ha−1 yr−1, implying that the degradation of these compounds was fertilization rate-dependent. These results highlight that various N fertilization levels alter the allocation of carbon and soil OM dynamics likely through changing biodegradation and the quantity and quality of crop residues.

Photochemical transformation plays an important role in functionalizing and degrading dissolved organic matter (DOM), producing one of the most complex mixtures known. In this study, using a flow-based design, nuclear magnetic resonance (NMR) spectroscopy is directly interfaced with a sunlight simulator enabling the study of DOM photodegradation in situ with high temporal resolution over 5 days. Samples from Suwannee River (Florida), Nordic Reservoir (Norway), and Pony Lake (Antarctic) are studied. Phototransformation of DOM is dominated by the degradation of aromatics and unsaturated structures (many arising from lignin) into carboxylated and hydroxylated products. To assess longer term changes, the samples were continuously irradiated for 17.5 days, followed by the identification a wide range of compounds and assessment of their fate using off-line 2D-NMR. This study demonstrates the applicability of the looped system to follow degradation in a non-targeted fashion (the mixture as a whole) and target analysis (tracing specific metabolites), which holds great potential to study the fate and transformation of contaminants and nutrients in the presence of DOM. It also demonstrates that components that remain unresolved in 1D NMR can be identified using 2D methods.

The hydrophobic organic contaminants triclosan, triphenyl phosphate (TPhP) and diazinon sorb to dissolved organic matter (DOM) and this may alter their bioavailability and toxicity. 1H nuclear magnetic resonance (NMR)-based metabolomics was used to investigate how DOM at 1 and 5 mg organic carbon/L may alter the metabolome of Daphnia magna from exposure to equitoxic mixtures of triclosan, TPhP and diazinon. These contaminants have different modes of action toward D. magna. The contaminant concentrations in each mixture were an equal percentage of their lethal concentration to 50% of the population (LC50) values, which equates to 1250 μg/L TPhP, 330 μg/L triclosan and 0.9 μg/L diazinon. The ternary mixture exposure at 1% LC50 values did not alter the D. magna metabolome. Contaminant mixture exposures at 5%, 10%, and 15% LC50 values decreased glucose, serine and glycine concentrations and increased asparagine and threonine concentrations, suggesting disruptions in energy metabolism. The contaminant mixture had a unique mode of action in D. magna and DOM at 1 and 5 mg organic carbon/L did not change this mode of action. The estimated sorption of triclosan, TPhP or diazinon to DOM at 1 or 5 mg organic carbon/L in this experimental design was calculated to be

In vivo NMR of small 13C-enriched aquatic organisms is developing as a powerful tool to detect and explain toxic stress at the biochemical level. Amino acids are a very important category of metabolites for stress detection as they are involved in the vast majority of stress response pathways. As such, they are a useful proxy for stress detection in general, which could then be a trigger for more in-depth analysis of the metabolome. 1H-13C heteronuclear single quantum coherence (HSQC) is commonly used to provide additional spectral dispersion in vivo and permit metabolite assignment. While some amino acids can be assigned from HSQC, spectral overlap makes monitoring them in vivo challenging. Here, an experiment typically used to study protein structures is adapted for the selective detection of amino acids inside living Daphnia magna (water fleas). All 20 common amino acids can be selectively detected in both extracts and in vivo. By monitoring bisphenol-A exposure, the in vivo amino acid-only approach identified larger fluxes in a greater number of amino acids when compared to published works using extracts from whole organism homogenates. This suggests that amino acid-only NMR of living organisms may be a very sensitive tool in the detection of stress in vivo and is highly complementary to more traditional metabolomics-based methods. The ability of selective NMR experiments to help researchers to "look inside" living organisms and only detect specific molecules of interest is quite profound and paves the way for the future development of additional targeted experiments for in vivo research and monitoring.

Consumer products such as perfluorooctanesulfonic acid (PFOS) and pharmaceuticals (PCPPs) enter aquatic ecosystems through inefficient removal during wastewater treatment. Often, the sterilization process of wastewater includes the addition of sodium hypochlorite that can react with PCPPs and other organic matter (i.e., dissolve organic matter) to generate disinfection by-products and can cause the final effluent to be more harmful to aquatic organisms. Here, we exposed Daphnia magna to two stages of wastewater, the pre-chlorinated wastewater (PreCl) and the final effluent. In addition, we exposed D. magna, to the final effluent with a concentration gradient of added PFOS, to investigate if this persistent contaminant altered the toxicity of the final effluent. After 48 h of contaminant exposure, we measured the daphnids metabolic responses to the different stages of wastewater treatment, and with the addition of PFOS, utilizing proton nuclear magnetic resonance spectroscopy and liquid chromatography tandem mass spectrometry. We found few significant changes to the metabolic profile of animals exposed to the PreCl wastewater; however, animals exposed to the final effluent displayed increases in many amino acids and decreases in some sugar metabolites. With the addition of PFOS to the final effluent, the metabolic profile shifted from increased amino acids and decreased sugar metabolites and energy molecules especially at the low and high concentrations of PFOS. Overall, our results demonstrate the metabolome is sensitive to changes in the final effluent that are caused by sterilization, and with the addition of a persistent contaminant, the metabolic profile is further altered.

Wastewater contains complex organic signatures whose components can be harmful to both environmental and human health. Nuclear Magnetic Resonance (NMR) spectroscopy is an excellent tool for molecular monitoring, with the ability to follow total organics, subcategories (aromatics, aliphatic etc.) and, resolution permitting, even individual compounds. However, due to the relatively low sensitivity of NMR, studies of wastewater to date have involved pre-concentration (often many liters) before analysis. This proof of concept study asks, “Is NMR sensitive enough for on-line wastewater monitoring without pre-concentration?” Here, a continuous flow 500 MHz NMR system, coupled with a sunlight simulator and ozone generator, was used on unaltered and unconcentrated wastewater to assess the potential of on-line NMR for understanding wastewater treatment processes. Wastewater from 3 different treatment stages were analyzed. In general, combined ozone and sunlight was more effective at removing organics, while differences in behavior of the same molecules within different wastewater fractions suggest that the chemistry is more complex than just the action of “reactants” (light, ozone) on the target chemicals, and to some extent, involves other components in the wastewater. As such, the nature of the effluent itself could also have important impacts on the rate and completion of its degradation. In summary, on-line NMR of wastewater treatment could be carried out without pre-concentration of the wastewater, in line with important future applications for understanding wastewater treatment processes at the molecular level. Over the last decade, there has been considerable development in low field, low footprint, low cost, NMR spectrometers. Portable versions of those systems could potentially, in the future, be applied for on-site, on-line monitoring of waste streams at water treatment plants, as described here.

Rapid biomolecular observation in model indicator organisms has been considered as a potential predictor of water pollution from chronic and trace toxicants. This study evaluated the use of Daphnia magna metabolomic measurements as indicators for exposure to reproductive endocrine disruptors by using the model juvenile hormone analogue fenoxycarb. Because D. magna reproduction controls metabolic regulation, the reproduction stage was also carefully considered in metabolic observations and data analysis to examine differences. Comparisons of metabolite abundance regulation between 1 and 12 days of fenoxycarb exposure were performed to investigate the predictability of the sub-chronic (12 days) adverse impacts on reproduction and metabolic regulation based on acute (1 day) metabolic observations. ANOVA-simultaneous component analysis (ASCA) detected reversed patterns in direction of time-course metabolite abundance regulation with fenoxycarb exposure. For example, decreases in the abundances of leucine, asparagine, methionine, and isoleucine which then changed to increases were observed with time during fenoxycarb exposures. The reversed regulation pattern was observed at the last reproduction stage (stage 3), exclusively. Pearson correlation analysis showed that correlations of pairwise metabolites were disrupted with fenoxycarb exposure. Similar to ASCA, data normalization based on the reproduction stage improved the detectability of significant correlations. The disruption on ambient metabolite regulation patterns and pairwise metabolite correlations was consistently observed with both 1 and 12 days of fenoxycarb exposures for sets of select metabolites. The observed regulatory disruptions to these specific metabolites suggest altered oogenesis as the affected metabolites and the specific reproduction stage are related to successful oogenesis. This study demonstrates that D. magna metabolic dysregulation is a predictor of water contamination by endocrine disrupting compounds. The high predictability of sub-chronic (12 days) endocrine disruption was confirmed based on acute (1 day) metabolic observations. Furthermore, integration of the reproduction cycle information in D. magna metabolomics was validated by observing a reproduction stage specific dysregulation in metabolite abundance regulation, which was not observable from the broader data analysis. Consequently, this study confirms the potential for establishing a quantitative relationship between water quality and indicator species metabolic observations. Additionally, it was found that constraining variables relevant to toxicity mechanisms of interest, such as the reproduction stage, is a key consideration for extraction of ecologically meaningful information in environmental metabolomics.

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.

(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.

Perfluorooctane sulfonate (PFOS) is a persistent pollutant which is potentially harmful and bioaccumulative to aquatic organisms. To evaluate the regulatory alteration of select metabolites with PFOS exposure at early and typical acute exposure periods in an aquatic indicator species Daphnia magna, the hourly abundance of the twenty-three metabolites was investigated over 24 h. To evaluate the bioaccumulation potential of PFOS at a sub-lethal concentration in D. magna, the daily accumulation into D. magna for 16 days was also evaluated. Twenty-three targeted metabolites were quantified over 1 to 4 h and 21 to 24 h of PFOS exposure using liquid chromatography tandem mass spectrometry (LC-MS/MS). Daphnid to water PFOS concentration ratios were monitored separately over different days and life stages at 0 to 76 h and 2 to 16 days of PFOS exposure. The observed metabolite abundance and bioaccumulation in the exposed groups was compared between sampling times. The results reveal that sub-lethal PFOS exposure at 2 mg/L and 20 mg/L alters regulation of arginine, tyrosine and adenosine monophosphate which are directly and indirectly related to energy status. The temporal metabolic responses observed for the early exposure period (4 h), but not for the typical acute exposure period (24 h), suggest the dysregulation potency of PFOS on metabolite regulation of D. magna and the importance of early time-course monitoring approaches. Sixteen days of bioaccumulation monitoring showed that PFOS is more bioaccumulative in younger D. magna. The observation of time-dependent bioaccumulation of PFOS in D. magna requires further studies to define its precise mechanism. Interestingly, the bioaccumulation potential of PFOS was found to be consistent between 72 h and 16 day exposure periods. No difference on the body burden to water concentration ratio during about one third of the life span time (16 days), compared to the 72 h exposure, suggests that the prolonged exposure did not increase the bioaccumulation of PFOS in D. magna. This study demonstrates that the Daphnia metabolites are rapidly responding to sub-lethal PFOS exposure and provides information on life stage and time-dependent bioaccumulation potential of PFOS. As such, metabolite regulation is a sensitive indicator to sub-lethal PFOS exposure and can be informative when combined with other measures of toxicity.

Anthropogenic activity is increasing the presence of contaminants that enter waterways through wastewater effluent and urban and/or agricultural runoff, generally in complex mixtures. Depending on the mode of action of the individual contaminant within the mixture, toxicity can occur due to contaminants having similar or dissimilar modes of action. However, it is unknown how the metabolome responds to sublethal contaminant mixtures in the keystone genus Daphnia. In the present study we examined D. magna metabolic responses to acute sublethal exposure of propranolol, carbamazepine, and perfluorooctanesulfonic acid (PFOS) as well as in binary (propranolol-carbamazepine, propranolol-PFOS, carbamazepine-PFOS) and tertiary mixtures (carbamazepine-propranolol-PFOS), all at 10% of the median lethal concentration of the population (LC50). The metabolome was measured using 1 H nuclear magnetic resonance (NMR) and characterized using principal component analysis, regression analysis, and fold changes in metabolite relative to the unexposed (control) group. The averaged principal component analysis scores plots revealed that carbamazepine-PFOS and carbamazepine-propranolol-PFOS exposures were significantly different from the control treatment. After normalizing the toxicity of each contaminant, we found that some metabolites responded monotonically, whereas others displayed a nonmonotonic response with increasing toxicity units. The single contaminant exposures and 2 binary mixtures (propranolol-carbamazepine, and propranolol-PFOS) resulted in minimal changes in the identified metabolites, whereas the carbamazepine-PFOS and carbamazepine-propranolol-PFOS displayed increases in several amino acid metabolites and decreases in glucose. Overall, our results highlight the sensitivity of the metabolome to distinguish the composition of the contaminant mixtures, with some mixtures displaying heightened responses versus others. Environ Toxicol Chem 2018;37:2448-2457. © 2018 SETAC.

Nuclear magnetic resonance (NMR) is the primary platform used in high-throughput environmental metabolomics studies because its non-selectivity is well suited for non-targeted approaches. However, standard NMR probes may limit the use of NMR-based metabolomics for tiny organisms because of the sample volumes required for routine metabolic profiling. Because of this, keystone ecological species, such as the water flea Daphnia magna, are not commonly studied because of the analytical challenges associated with NMR-based approaches. Here, the use of a 1.7-mm NMR microprobe in analyzing tissue extracts from D. magna is tested. Three different extraction procedures (D2O-based buffer, Bligh and Dyer, and acetonitrile : methanol : water) were compared in terms of the yields and breadth of polar metabolites. The D2O buffer extraction yielded the most metabolites and resulted in the best reproducibility. Varying amounts of D. magna dry mass were extracted to optimize metabolite isolation from D. magna tissues. A ratio of 1–1.5-mg dry mass to 40 µl of extraction solvent provided excellent signal-to-noise and spectral resolution using 1H NMR. The metabolite profile of a single daphnid was also investigated (approximately 0.2 mg). However, the signal-to-noise of the 1H NMR was considerably lower, and while feasible for select applications would likely not be appropriate for high-throughput NMR-based metabolomics. Two-dimensional NMR experiments on D. magna extracts were also performed using the 1.7-mm NMR probe to confirm 1H NMR metabolite assignments. This study provides an NMR-based analytical framework for future metabolomics studies that use D. magna in ecological and ecotoxicity studies. Copyright © 2015 John Wiley & Sons, Ltd.

The Nuclear Waste Management Organization (NWMO) is developing a Deep Geological Repository (DGR) to contain and isolate used nuclear fuel in a suitable rock formation at a depth of approximately 500 m. The design concept employs a multibarrier system, including the use of copper-coated used fuel containers, surrounded by a low-permeability, swelling clay buffer material within a low permeability, stable host rock environment. The natural organic matter (NOM) composition of the bentonite clays being considered for the buffer material is largely uncharacterized at the molecular-level. To gain a better understanding of the NOM in target clays from Wyoming and Saskatchewan, molecular-level methods (biomarker analysis, solid-state 13C NMR and solution-state 1H nuclear magnetic resonance (NMR)) were used to elucidate the structure and sources of NOM. Organic carbon content in three commercially available bentonites analyzed was low (0.11–0.41%). The aliphatic lipid distribution of the clay samples analyzed showed a predominance of higher concentration of lipids from vascular plants and low concentrations of lipids consistent with microbial origin. The lignin phenol vanillyl acid to aldehyde ratio (Ad/Al) for the National sample indicated an advanced state of lignin oxidation and NOM diagenesis. The 13C NMR spectra were dominated by signals in the aromatic and aliphatic regions. The ratio of alkyl/O-alkyl carbon ranged from 7.6 to 9.7, indicating that the NOM has undergone advanced diagenetic alteration. The absence lignin-derived phenols commonly observed in CuO oxidation extracts from contemporary soils and sediments as well as the lack of amino acids suggests that the material corresponding to the aromatic signal is not composed of lignin or proteins but may be derived from another source such as black carbon or some other non-extractable aromatic-rich NOM. The aliphatic signal appears to correspond to long-chain compounds with little side branching based on the results of the one-dimensional (1D) and two-dimensional (2D) solution-state 1H NMR analyses. Overall, the organic geochemical analyses suggest that the NOM is composed mainly of plant-derived waxes and highly aromatic carbon with low contributions from small molecules. The compounds identified by the molecular-level analysis of NOM in the clay samples are hypothesized to be recalcitrant but future studies should examine if these compounds may serve as a microbial substrate to further test the observations of this study. Furthermore, our study suggests that the NOM has undergone diagenesis and that marine NOM signatures are no longer recognizable or detectable. As such, future work may also examine the diagenesis of these deposits to further understand the NOM geochemistry and paleoenvironmental conditions in bentonite deposits.

¹H nuclear magnetic resonance (NMR)-based metabolomics was used to characterize the response of Daphnia magna after sub-lethal exposure to perfluorooctane sulfonate (PFOS), a commonly found environmental pollutant in freshwater ecosystems. Principal component analysis (PCA) scores plots showed significant separation in the exposed samples relative to the controls. Partial least squares (PLS) regression analysis revealed a strong linear correlation between the overall metabolic response and PFOS exposure concentration. More detailed analysis showed that the toxic mode of action is metabolite-specific with some metabolites exhibiting a non-monotonic response with higher PFOS exposure concentrations. Our study indicates that PFOS exposure disrupts various energy metabolism pathways and also enhances protein degradation. Overall, we identified several metabolites that are sensitive to PFOS exposure and may be used as bioindicators of D. magna health. In addition, this study also highlights the important utility of environmental metabolomic methods when attempting to elucidate acute and sub-lethal pollutant stressors on keystone organisms such as D. magna.

Soil organic matter (SOM) is one of the earth’s largest reservoirs of actively cycled carbon and plays a critical role in various ecosystem functions. In this study, mineral soils with the same parent material and of similar approximate age were sampled from the same climatic region in Halsey, Nebraska to determine the relationship between overlying vegetation inputs to SOM composition using complementary molecular level methods (biomarker analyses and solid state 13 C nuclear magnetic resonance (NMR) spectroscopy). Soil samples were collected from a native prairie and cedar and pine sites planted on the native prairie. Free and bound lipids isolated from the pine soil were more enriched in aliphatic and cutinderived compounds than the other two soils. Cinnamyl type lignin-derived phenols were more abundant in the grassland soil than in the pine and cedar soils. Acid to aldehyde ratios (Ad/Al) for vanillyl and syringyl type phenols were higher for the pine soil indicating a more advanced stage of lignin oxidation (also observed by 13 C NMR) in the soil that has also been reported to have accelerated carbon loss. In

The role of organic matter (OM) concentration, structure and composition and how these relate to mineral protection is important for the understanding of long term soil OM dynamics. Various OM–clay complexes were constructed by sequential sorption of lignin and dodecanoic acid to montmorillonite. Humic acid–montmorillonite complexes were prepared at pH 4 and 7 to vary OM conformation prior to sorption. Results obtained with constructed OM–clay complexes were tested with isolated mineral fractions from two soils. Oxidation with an acidic NaClO 2 solution was used to chemically oxidize lignin in the OM–clay complexes, sand-, silt- and clay-size soil fractions to test whether or not it can be protected from chemical attack. Gas chromatography–mass spectrometry was used to analyze lignin-derived phenols, cutin OH–acid (after CuO oxidation), fatty acid and n -alkanol concentrations and composition. We found that carbon content was not solely responsible for lignin stability against chemical oxidation. Lignin was protected from chemical oxidation through coating with dodecanoic acid and sorption of humic acid to clay minerals in a stretched conformation at pH 7. Therefore, interactions between OM constituents as well as OM conformation are important factors that protect lignin from chemical oxidation. Lignin-derived phenol dimers in the Grassland-Forest Transition soil fractions were protected from chemical oxidation to a greater extent compared to those in Grassland soil fractions. Therefore, although lignin was protected from degradation through mineral association, the extent of this protection was also related to OM content and the specific stability of lignin components.

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.

Glacier surfaces are reservoirs that contain organic and inorganic debris referred to as cryoconite. Solar heating of this material results in the formation of water-filled depressions that are colonized by a variety of microbes and are hypothesized to play a role in carbon cycling in glacier ecosystems. Recent studies on cryoconite deposits have focused on their contribution to carbon fluxes to determine whether they are a net source or sink for atmospheric CO 2 . To better understand carbon cycling in these unique ecosystems, the molecular constituents of cryoconite organic matter (COM) require further elucidation. COM samples from four glaciers were analyzed by targeted extraction of plant- and microbial-derived biomarkers in conjunction with non-targeted NMR experiments to determine the COM composition and potential sources. Several molecular proxies were applied to assess COM degradation and microbial activity using samples from Greenland, the Canadian Arctic, and Antarctica. COM from Canadian (John Evans glacier) and Greenlandic (Leverett glacier) locations was more chemically heterogeneous than that from the Antarctic likely due to inputs from higher plants, mosses and Sphagnum as suggested by the solvent-extractable alkyl lipids and sterols and the detection of lignin- and Sphagnum -derived phenols after cupric oxide chemolysis. Solid-state 13 C nuclear magnetic resonance (NMR) experiments highlighted the bulk chemical functional groups of COM allowing for a general assessment of its degradation stage from the alkyl/ O -alkyl proxy whereas solution-state 1 H NMR highlighted both microbial and plant contributions to base-soluble extracts from these COM samples. The dominance of 1 H NMR signals from microbial protein/peptides in base-soluble extracts of COM from Antarctica (Joyce glacier and Garwood glacier), phospholipid fatty acid (PLFA) biomarker detection and the absence of plant-derived biomarkers in both the solvent and cupric oxide extracts suggests that this COM is dominated by microbial-derived material. These results indicate that COM carbon composition is dependent on the local glacier environment which may have a profound impact on carbon cycling and sequestration on glacier surfaces.

As plants are major contributors to soil organic matter (OM), the chemical composition of plant tissues entering the soil should be related to the soil OM composition. We examined the OM composition of maize leaves, stems, and roots, to determine if differences in plant tissue composition altered the soil OM composition during a 36-week degradation experiment. Lignin phenols were measured by gas chromatography/mass spectrometry (GC/MS), and soil OM and humic substances were characterized using solid-state 13 C and solution-state 1 H nuclear magnetic resonance (NMR) spectroscopy respectively. Lignin phenol composition, oxidation, and depletion relative to cutin-derived OH-acids changed less in leaf- compared to stem- and root-amended soils over time, and may be due to greater vanillyl concentrations in leaves. Soil amended with stems had higher concentrations of carbohydrates in soil OM. Humic substances from leaf-amended soils had higher concentrations of aliphatic components, likely due to higher concentrations of aliphatic compounds in leaf tissues, which suggests that compounds derived from leaves are potential contributors to the stable pool of soil OM. After 36-weeks of incubation, the contribution of microbial-derived OM was greatest in humic extracts from root-amended soils, and increased contribution from these compounds was detected earlier in these soils than stem- and leaf-amended soils. This indicates that root amendment may enhance contributions from microbial-derived OM. Our study suggests that changes in soil OM composition over time was related to the chemical composition of the plant tissue, and demonstrates the important link between plant chemistry and soil OM turnover.

The use of consumer products and pharmaceuticals that act as contaminants entering waterways through runoff and wastewater effluents alters aquatic ecosystem health. Traditional toxicological endpoints may underestimate the toxicity of contaminants, as lethal concentrations are often orders of magnitude higher than those found within freshwater ecosystems. While newer techniques examine the metabolic responses of sublethal contaminant exposure, there has been no direct comparison with ontogeny in Daphnia. It was hypothesized that Daphnia magna would have distinct metabolic changes after 3 different sublethal contaminant exposures, because of differences in the toxic mode of action and ontogeny. To test this hypothesis, the proton nuclear magnetic resonance metabolomic profiles were measured in D. magna aged day 0 and 18 after exposure to 28% of the lethal concentration of 50% of organisms tested (LC50) of atrazine, propranolol, and perfluorooctanesulfonic acid (PFOS) for 48 h. Principal component analysis revealed significant separation of contaminants from the control daphnids in both neonates and adults exposed to propranolol and PFOS. In contrast, atrazine exposure caused separation from the controls in only the adult D. magna. Minimal ontogenetic changes in the targeted metabolites were seen after exposure to propranolol. For both atrazine and PFOS exposures ontogeny exhibited unique changes in the targeted metabolites. These results indicate that, depending on the contaminant studied, neonates and adults respond uniquely to sublethal contaminant exposure. Environ Toxicol Chem 2017;36:938-946. © 2016 SETAC.

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.

1H nuclear magnetic resonance (1H NMR)-based metabolomics was utilized to elucidate the earthworm sub-lethal toxicity after exposure to the persistent environmental contaminant phenanthrene. Earthworms were exposed to 0.05, 0.2 and 0.4 mg/cm2 of phenanthrene [which correspond to 1/32nd to 1/4th of the 48-h LC50 (concentration that causes 50 % mortality), respectively] via contact tests over 1, 2 and 3 days of dermal contact. 1H NMR-based metabolomic analysis of the polar and non-polar fractions of the earthworm tissue extracts revealed heightened Eisenia fetida toxic responses with both longer exposure times and higher phenanthrene concentrations. Principal component analysis (PCA) of the polar fraction showed significant separation between control and exposed earthworms along PC1 for all phenanthrene concentrations on each day. The PCA of the non-polar fraction showed significant separation between the controls and exposed earthworms for only the first day of exposure. These results suggested that alanine, glutamate, maltose, and fatty acids were potential indicators of phenanthrene exposure. Interruption in energy production due to a deactivation of the succinate dehydrogenase enzyme in the Krebs cycle was also postulated in exposed earthworms. Cross-validated partial least squares-regression models showed that the polar metabolic profile of E. fetida was weakly but significantly correlated to phenanthrene exposure concentrations after day 1 and day 2 of exposure. Overall, this study indicates that with longer exposures, contact time becomes more important than concentration in discriminating between control and exposed earthworms. This study also shows that NMR-based metabolomics has promise as a powerful ecotoxicological tool for elucidating the mode of toxicity of contaminants.

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.

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.

Inherent chemical recalcitrance and association of organic matter (OM) with minerals are mechanisms responsible for the long term preservation of OM in soils. The structural characteristics of OM are also believed to control specific interactions between OM and soil minerals. However, the extent of the relationship between recalcitrance and mineral protection and the specificity of these chemically driven interactions are not clearly understood at the molecular level. To measure chemical patterns of OM sequestration in sand-, silt-, clay-size and light fractions, we analyzed three soils, which mainly differed in carbon content and overlying vegetation, but have similar clay mineralogy, using biomarker analysis and nuclear magnetic resonance (NMR). Despite differences in environmental controls, long chain aliphatic compounds generally accumulated in the fine fractions of all soils. This accumulation is likely due to the strong interaction between recalcitrant forms of OM and soil minerals. For example, polymethylene and >C20 organic acids accumulated in fine fractions, while lignin-derived phenols were protected from oxidation in silt-size fractions. Diffusion edited solution state 1H NMR suggested that contributions from microbial-derived OM was greater in finer fractions, which is likely due to the accumulation of microbial-derived compounds or higher microbial activity in clay micro-sites. Our data suggest that, for these Prairie soils, the specific structure of OM and not environmental factors is responsible for long term preservation of OM in mineral fractions. Further research is necessary to understand the interplay between these preservation mechanisms such that the long term fate of OM can be further elucidated.

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.

¹H NMR metabolomics can be used to assess the sub-lethal toxicity of contaminants to earthworms by identifying alterations in the metabolic profiles of contaminant- exposed earthworms in contrast to those of healthy (control) individuals. In support of this method this study sought to better characterize the baseline metabolic profile of healthy, mature earthworms of the species, Eisenia fetida, which is recommended for both acute and sub-lethal toxicity testing for soil contaminants. Profiles of D(2)O-buffer extracted metabolites were determined using (1)H NMR spectroscopy and both inter-individual metabolic variability and pair-wise metabolic correlations were assessed. The control earthworm extracts exhibited low overall inter-individual metabolic variability, with a spectrum-wide median relative standard deviation (%RSD=standard deviation/mean×100) of 14%, which suggests that the metabolic profile of E. fetida earthworms is well controlled in laboratory conditions and supports further use of this organism in environmental metabolomics research. In addition, strong positive correlations were detected between the levels of maltose, betaine, glycine, and glutamate as well as between the levels of lactate, valine, leucine, alanine, lysine, tyrosine, and phenylalanine which had not previously been reported. Since comparison of pair-wise metabolic correlations between control and treated organisms can reveal changes in the underlying pattern of biochemical relationships between the metabolites, identification of these significant metabolic correlations in control earthworms provides an additional characteristic that may be applied to delineate between control and treated earthworms in future NMR-based metabolomic studies.

(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.

Bt ( Bacillus thuringiensis ) corn is reported to produce lignin-rich residues, compared to non-Bt (NBt) corn, suggesting it is more resistant to decomposition. As the Bt gene is expressed selectively in stem and leaf tissue, it could affect lignin distribution in corn, which naturally has greater lignin content in roots than in stems and leaves. Our objective was to evaluate the effects of corn plant components, the Bt gene and elevated-lignin inputs on decomposition. Roots, stems and leaves from Bt corn and NBt corn isolines enriched with 13 C and 15 N were finely ground and mixed separately with soil, then incubated at 20 °C for 36 weeks. The effect of elevated lignin on decomposition was tested by adding a commercial lignin source (indulin lignin) to half of the samples. In addition to weekly CO 2 analysis and regular measurement of N mineralization, the degree of lignin degradation was evaluated at 1 and 36 weeks from the acid to aldehyde ratio (Ad/Al) of vanillyl and syringyl lignin-derived phenols. The CO 2 production and N mineralization was lower in root-amended soils than stem- and leaf-amended soils. The Bt genetic modification increased CO 2 production from stem-amended soils ( P 13 C and 15 N results also showed more residue-C and -N retained in soils mixed with NBt stem residues. After 36 weeks leaf- and stem-amended soils with indulin lignin had a lower Ad/Al ratio and were less degraded than soils without exogenous lignin. In conclusion, plant lignin and nitrogen contents were good predictors of CO 2 production and N mineralization potential. Corn roots decomposed more slowly than aboveground components emphasizing the importance of recalcitrant root residues in sustaining the organic matter content of soil.

The dynamics and fate of terrestrial organic matter (OM) under elevated atmospheric CO 2 and nitrogen (N) fertilization are important aspects of long-term carbon sequestration. Despite numerous studies, questions still remain as to whether the chemical composition of OM may alter with these environmental changes. In this study, we employed molecular-level methods to investigate the composition and degradation of various OM components in the forest floor (O horizon) and mineral soil (0-15 cm) from the Duke forest free air CO 2 enrichment (FACE) experiment. We measured microbial responses to elevated CO 2 and N fertilization in the mineral soil using phospholipid fatty acid (PLFA) profiles. Increased fresh carbon inputs into the forest floor under elevated CO 2 were observed at the molecular-level by two degradation parameters of plant-derived steroids and cutin-derived compounds. The ratios of fungal to bacterial PLFAs and Gram-negative to Gram-positive bacterial PLFAs decreased in the mineral soil with N fertilization, indicating an altered soil microbial community composition. Moreover, the acid to aldehyde ratios of lignin-derived phenols increased with N fertilization, suggesting enhanced lignin degradation in the mineral soil. 1 H nuclear magnetic resonance (NMR) spectra of soil humic substances revealed an enrichment of leaf-derived alkyl structures with both elevated CO 2 and N fertilization. We suggest that microbial decomposition of SOM constituents such as lignin and hydrolysable lipids was promoted under both elevated CO 2 and N fertilization, which led to the enrichment of plant-derived recalcitrant structures (such as alkyl carbon) in the 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.

Organophosphate esters (OPEs) are frequently detected in aquatic environments. Hydrophobic OPEs with high octanol-water partition coefficients (Log KOW) will likely sorb to dissolved organic matter (DOM) and consequently alter OPE bioavailability and sub-lethal toxicity. 1H nuclear magnetic resonance (NMR)-based metabolomics was used to evaluate how DOM (5 mg organic carbon/L) alters the metabolic response of Daphnia magna exposed to sub-lethal concentrations of three individual OPEs with varying hydrophobicity. D. magna exposed to the hydrophilic contaminant (Log KOW = 1.43) tris(2-chloroethyl) phosphate (TCEP) did not have substantial metabolic changes and DOM did not alter the metabolic response. There were significant increases in amino acids and a decrease in glucose from exposure to the hydrophobic contaminant (Log KOW = 3.65) tris(2-butoxyethyl) phosphate (TBOEP) which DOM did not mitigate, likely due to the high sub-lethal toxicity of TBOEP. Exposure to DOM and the hydrophobic contaminant (Log KOW = 4.76) triphenyl phosphate (TPhP) resulted in a unique metabolic response which was unlike TPhP only exposure, perhaps because DOM may be an additional stressor with TPhP exposure. Therefore, Log KOW values may not always predict how sub-lethal contaminant toxicity will change with DOM and there should be more consideration to incorporate DOM in sub-lethal ecotoxicology testing.

Batch equilibration and the decant-refill methods were used to measure sorption and desorption of phenanthrene to four geosorbents with varying organic matter composition at different contact times to better understand the mechanisms underlying sorption and desorption processes. The sorbents were characterized by solid-state (13)C cross polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy. Sorption and desorption isotherms were constructed and fitted to the Freundlich model and the site energy distribution model was used to measure the relative change of distributed sorption energies. Concentration- and time-dependence were observed for both sorption and desorption processes. The high energy sites become filled with increasing sorbate concentration, resulting in the non-linearity of sorption isotherms and the increase of the desorption percentages. The shape of the sorption isotherms changed with the contact time, which was characterized by the variation of Freundlich model parameters (K(F) and n). More favorable sorption sites could be accessed when the contact time was extended, leading to the decrease of the desorption percentages. As indicated by the value of n, partitioning appeared to contribute more to the sorption of phenanthrene for the soils with higher oxygen-substituted carbon content and less condensed soil organic matter (SOM). Sorption in adsorption domains is energetically more favorable and kinetically less accessible as compared to partitioning domains, and both the composition and conformation of SOM play important roles in the sorption and desorption processes by forming specific interactions with phenanthrene molecules and governing the accessibility of sorption domains.

Dissolved organic matter (DOM) chemistry and the potential for organic matter (OM) to self-associate with other OM components are important aspects of understanding the mechanisms of DOM sorption to clay surfaces. To investigate this further, we sorbed DOM isolated from peat humic acid onto either kaolinite, montmorillonite and gibbsite via ten sequential batch equilibration sorption experiments. Dissolved organic carbon (DOC) sorption to all minerals increased consistently, suggesting that sorption occurred via mineral-OM interactions at the beginning of the experiment. After six successive DOM loadings, the concentration of DOC sorbed by kaolinite and gibbsite began to plateau, likely due to the saturation of mineral surface sorption sites. Solution-state nuclear magnetic resonance (NMR) analysis of unbound DOM showed that kaolinite and montmorillonite sorbed aliphatic, protein and lignin components initially and primarily aliphatic and aromatic constituents in later sorption experiments, whereas gibbsite sorbed mostly aliphatic compounds during all DOM loadings. Analysis of the organo-clay complexes using 1H high resolution–magic angle spinning (HR-MAS) NMR confirmed the preferential sorption of aromatic and aliphatic components to all three minerals. Overall, these results suggest that OM-OM interactions may be important mechanisms of DOM sorption to clay mineral surfaces.

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 metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (1H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm2. Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm2) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm2 DDT dose and both the 0.5 and 1.0 μg/cm2 endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study.

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.