Natural organic matter (NOM) has been concentrated from five drinking water sources in Minnesota, Florida, Washington, California, and North Carolina, USA. Reverse osmosis (RO), coupled with cation exchange (CEXH+) and/or electrodialysis (ED), was used to obtain several hundred grams of NOM from each source. The scale of the project exceeds that of most prior applications of this methodology for NOM concentration. The water concentration factor (WCF), which is the ratio of the total volume of processed sample to the final volume of concentrated sample, ranged from 200 to 408. In contrast, WCF is in the range of 20-50 in most prior studies. This paper provides technical background for the work, including the basic equations that relate the yield of NOM (the ratio of isolated organic carbon to processed organic carbon) to WCF and the RO rejection coefficients of its components. A numerical simulation of the yield of NOM and the degree of NOM fractionation that likely occurs during RO processing is presented. The inorganic chemical compositions of source waters have been used to anticipate and mitigate membrane fouling at very high WCF during the RO process. The relationship between NOM yield, the RO rejection coefficients of its components, and WCF is explored and used to interpret differences in NOM yield among the five sampling sites. Even with the large WCF values at the five sampling sites, the NOM yield ranged from 58% to 91%, within the predicted range for a pure solute with an RO rejection coefficient of 0.995-0.999.

Dissolved organic matter (DOM) exhibits a highly complex molecular composition and maintains ecosystem stability, acting as a crucial interface between biotic and abiotic processes. Although DOM's molecular complexity and biological effects are widely studied, most investigations use targeted bioassays, examining specific responses and linking molecular features only to predefined biological outcomes when assessing potential bioactive components. Here, we analyzed International Humic Substances Society (IHSS) reference standards of natural organic matter (NOM) and humic fractions, including humic acids (HAs) and fulvic acids (FAs), using ultrahigh resolution Fourier- transform ion cyclotron resonance mass spectrometry (FT-ICR MS) alongside the Cell Painting (CP) assay, a multiplexed, image-based morphological profiling method. The chemical composition of IHSS samples was influenced by fractionation methods and environmental sources. HAs exhibited stronger aliphatic and aromatic characteristics, whereas FAs and NOM extracted by reverse osmosis were more oxidized. Distinct molecular patterns were observed among terrestrial HAs, Pony Lake FAs, terrestrial FAs, and other fractions. In the CP assay, the most hydrophobic humic substances induced the most pronounced morphological changes. Linking chemical features with morphological outcomes suggested lipid-like compounds and nitrogen-rich aromatic species as likely contributors. This integrative approach provides preliminary molecular leads for further isolation, structural characterization, and mechanistic studies of DOM bioactivity.

Background Although humic substances are the principal ingredients in processed humic products, there has been no practical way to determine if a material is humified, allowing fake products to be used by farmers instead of genuine humic substances. Objective To develop a test method using conventional laboratory techniques to determine if a material is humified. Method A neutralized extract is prepared using the standardized extraction protocols specified in ISO 19822:2018(E). A portion of the extract is used to determine the concentration of dissolved organic matter on an ash-free basis. A portion of the remaining neutralized extract is diluted to a concentration of 30 mg/kg of dissolved organic matter and transferred to a quartz UV cuvette for ultraviolet-visible (UV-Vis) spectroscopy. UV-Vis absorbance is recorded over a wavelength range of 220-500 nm at 5 nm intervals. The absorbance data are normalized by conversion to scaled absorbance, which is compared to a reference scaled absorbance spectral curve for humic substances to determine if the tested material is humic or non-humic. Results This method was able to differentiate legitimate humic substances from non-humic adulterants in a multiple-laboratory validation study (P <= 0.05). Conclusion This method can differentiate humic from non-humic substances in materials intended to be used as ingredients in commercial humic products or for research.

Many challenges remain before we can fully understand the multifaceted role that natural organic matter (NOM) plays in soil and aquatic systems. These challenges remain despite the considerable progress that has been made in understanding NOM’s properties and reactivity using the latest analytical techniques. For nearly 4 decades, the International Humic Substances Society (IHSS, which is a non-profit scientific society) has distributed standard substances that adhere to strict isolation protocols and reference materials that are collected in bulk and originate from clearly defined sites. These NOM standard and reference samples offer relatively uniform materials for designing experiments and developing new analytical methods. The protocols for isolating NOM, and humic and fulvic acid fractions of NOM utilize well-established preparative scale column chromatography and reverse osmosis methods. These standard and reference NOM samples are used by the international scientific community to study NOM across a range of disciplines from engineered to natural systems, thereby seeding the transfer of knowledge across research fields. Recently, powerful new analytical techniques used to characterize NOM have revealed complexities in its composition that transcend the “microbial” vs. “terrestrial” precursor paradigm. To continue to advance NOM research in the Anthropocene epoch, a workshop was convened to identify potential new sites for NOM samples that would encompass a range of sources and precursor materials and would be relevant for studying NOM’s role in mediating environmental and biogeochemical processes. We anticipate that expanding the portfolio of IHSS reference and standard NOM samples available to the research community will enable this diverse group of scientists and engineers to better understand the role that NOM plays globally under the influence of anthropogenic mediated changes.

When compositions of an isobaric series of molecular formulae consisting of C, H, and O are displayed in a van Krevelen plot, a remarkable number of nonparallel lines of compositions can be observed. Each group of related lines converge at a point in a negative quadrant of van Krevelen space (e.g., H/C = 4, O/C = -1). These points of convergence have H/C and O/C ratios that correspond to molecular formulae in which the stoichiometric coefficients of some of the isotopes are negative. In this manner, a group of related low-mass moieties have been identified (CH4O-1, C4O-3, C2H-8O-1, CH-12, etc.). Each of these moieties has a nominal mass of zero and a very small exact mass. Furthermore, all of these low-mass moieties have compositions that fall on the line H/C = -12 -16(O/C), which lies entirely in the negative quadrants of van Krevelen space. This paper demonstrates that all low-mass moieties consisting of C, H, and O can be expressed formally as linear combinations of any two moieties. Likewise, all molecular formulae that fall on a line that passes through a given low-mass moiety must differ compositionally by a multiple of the composition of that moiety, and their exact masses must differ by a multiple of the exact mass of the moiety. This latter relationship has been invoked for very rapid assignment of a molecular formula to an exact mass. Finally, a more comprehensive and theoretically based understanding of family scores has been developed around the concept of low-mass moieties.

This chapter presents an overview of several common models that are used to describe the reactions of humic substances (HS) with metal cations. The competitive Gaussian model, Model V, and the non-ideal competitive adsorption (NICA) model all assume that HS contain two classes of binding sites. Each class of sites contains multiple sites whose relative concentrations are distributed symmetrically around a central log K value, and the width of the distribution (in log K units) is controlled by a width parameter. To varying degrees, these models consider the intrinsic heterogeneity of binding sites, electrostatic effects on the effective reactivities of binding sites, and Donnan effects that alter concentrations of small ions near a large polyion. The most pronounced difference between models is found in their respective treatments of the effect of ionic strength on metal complexation equilibria.

Suwannee River natural organic matter (SRNOM) is a well-known end member of NOM from an aquatic system, and is a reference material of the International Humic Substances Society (IHSS). In May 2012, an expedition to the Suwannee River to replenish this reference material yielded over 6 kg of freeze-dried NOM. The quantity of isolated NOM was unprecedented, easily exceeding the combined recoveries of the standard and reference samples that were collected by the IHSS from the Suwannee River in 1983, 1999, and 2003. The NOM was acquired from 36,890 L of filtered river water, which was concentrated 40-fold on-site using two portable reverse osmosis (RO) systems. After RO, the concentrated sample was desalted by cation exchange (CEX), freeze dried, and homogenized. Overall yield of dissolved organic carbon (DOC) was 84.2%, which is slightly lower than the yield of 88% in 1999 when RO and CEX were used to isolate the first sample of SRNOM, which is designated 1R101N. The final NOM sample supplied to the IHSS, which is designated 2R101N, contains only 3.89% inorganic ash, which reasonably allows most chemical analyses. Average river DOC concentration of 82.7 mg/L was higher than during prior sampling trips, which contributed to the historically high recovery of NOM. Increased DOC concentration may be related to the removal of water control structures from the river. This article describes the methods of isolation used in collecting the 2R101N reference sample as background for other articles in this special issue of Environmental Engineering Science and for future researchers who will use this IHSS sample.

This study focuses on the deterministic task of assigning molecular formulae to exact masses that are generated by ultrahigh resolution mass spectrometry. A new algorithm based on low-mass moieties (LMMs) such as CH4O(-1) and C4O(-3) completely replaces conventional computational loops that explore a user-defined range of C, H, and O when searching for molecular formulae that have a given exact mass. The LMM-based algorithm has been coupled with a combinatorial algorithm that uses nested loops for N, P, S, and (13)C to assign molecular formulae. The resulting program is more than 1700 times faster than its brute-force counterpart that uses nested loops for all elements, and both programs yield identical output files. The new LMM-based program is 1050 times faster than the open-source program HR2, 60 times faster than Molecular Formula Calculator, and 3.6 times faster than MassCalc/FormCalc.

Vapor pressure osmometry (VPO) was used to measure the number-average molecular weights (M-n) of natural organic matter (NOM) that was isolated using reverse osmosis, hydrophobic acids (HPOA) that were isolated by adsorption on XAD-8 resin, and transphilic acids (TPIA) that were isolated by adsorption on XAD-4 resin. All samples were isolated from the headwaters of the Suwannee River in southeastern Georgia, USA, in May 2012. NOM, HPOA, and TPIA samples had M-n of 634 +/- 11, 583 +/- 8, and 498 +/- 7 g/mol, respectively. Novel methodology was introduced wherein VPO measurements were made at room temperature, and M-n values were rendered from a robust analysis of data that considered not only the well-known effect of hydronium ion but also the contributions of inorganic solutes present in the samples. The method was validated by results on a known sample (benzenehexacarboxylic acid), whose molecular weight was determined accurately to within 0.5% of the true value. The VPO-determined M-n are compared to those from noncolligative methods to highlight uncertainty regarding a fundamental parameter of matter found in nearly all terrestrial waters.

Oceanic dissolved organic matter (DOM) is one of the largest pools of reduced carbon on Earth, yet DOM remains poorly chemically characterized. Studies to determine the chemical nature of oceanic DOM have been impeded by the lack of efficient and non-fractioning methods to recover oceanic DOM. Here, a DOM fraction (~40 to 86% recovery) was isolated using reverse osmosis/electrodialysis (RO/ED) and analyzed by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Samples were obtained from biogeochemically distinct environments: photobleached surface gyre, productive coastal upwelling zone, oxygen minimum, North Atlantic Deep Water, and North Pacific Deep Water. A ubiquitous ‘background’ refractory carbon pool was apparent throughout the ocean and dominated in the deep Pacific Ocean. Advanced NMR spectral editing revealed that condensed aromatic and quaternary anomeric carbons contribute to this deep refractory DOC pool, the quaternary anomeric carbons being a newly identified and potentially important component of bio-refractory carbohydrate-like carbon. Additionally, our results support the multi-pool (e.g. 3-pool: labile, semi-labile, and refractory) conceptual model of marine DOM biogeochemistry. Surface samples, hypothesized to be enriched in labile and semi-labile DOM, were enriched in carbohydrate-like material consistent with prior studies. High carboxyl signals in the deep Pacific support the hypothesis that a major fraction of the refractory pool consists of carboxyl-rich alicyclic molecules (CRAM).

Potentiometric direct titrations were used to measure the carboxyl and phenolic contents of natural organic matter that was isolated using reverse osmosis, hydrophobic acids (HPOA) that were isolated by adsorption on XAD-8 resin, and transphilic acids (TPIA) that were isolated by adsorption on XAD-4 resin. All samples were isolated from the headwaters of the Suwannee River in southeastern Georgia, in May, 2012. One approximation method and two numerical models were used to quantify the concentrations and acidic strengths of carboxyl groups and phenolic groups for the samples. The approximation method was the pH 8 and 10 method. Numerical models were the Gaussian distribution model and the modified Henderson-Hasselbalch model. All measurements and models yielded a consistent trend in the carboxyl contents, with TPIA having the greatest concentration and HPOA having the least. The 2R101N sample had the lowest average log K for proton binding (4.02), indicating that this sample contains a significant concentration of highly acidic compounds that are not in HPOA and TPIA. This can be explained by the loss of amino acids, peptides, and hydrophilic polycarboxylic acids that prefer the mobile phase in the solid-phase extractions with XAD-8 and XAD-4. The approximation method and both models were used to assign the phenolic content for the 2R101N sample. Only the approximation method was used for HPOA and TPIA. Concentrations of phenolic groups from the pH 8 and 10 method for 2R101N, HPOA, and TPIA were 2.86, 3.05, and 2.77 meq/gC, respectively.

Dissolved organic matter (DOM) was isolated from large volumes of deep (674m) and surface (21m) ocean water via reverse osmosis/electrodialysis (RO/ED) and two solid-phase extraction (SPE) methods (XAD-8/4 and PPL) at the Natural Energy Laboratory of Hawaii Authority (NELHA). By applying the three methods to common water samples, the efficiencies of XAD, PPL and RO/ED DOM isolation were compared. XAD recovered 42% of dissolved organic carbon (DOC) from deep water (25% with XAD-8; 17% with XAD-4) and 30% from surface water (16% with XAD-8; 14% with XAD-4). PPL recovered 61±3% of DOC from deep water and 61% from surface water. RO/ED recovered 82±3% of DOC from deep water, 14±3% of which was recovered in a sodium hydroxide rinse, and 75±5% of DOC from surface water, with 12±2% in the sodium hydroxide rinse. The highest recoveries of all were achieved by the sequential isolation of DOC, first with PPL and then via RO/ED. This combined technique recovered 98% of DOC from a deep water sample and 101% of DOC from a surface water sample. In total, 1.9, 10.3 and 1.6g-C of DOC were collected via XAD, PPL and RO/ED, respectively. Rates of DOC recovery using the XAD, PPL and RO/ED methods were 10, 33 and 10mg-Ch−1, respectively. Based upon C/N ratios, XAD isolates were heavily C-enriched compared with water column DOM, whereas RO/ED and PPL➔RO/ED isolate C/N values were most representative of the original DOM. All techniques are suitable for the isolation of large amounts of DOM with purities suitable for most advanced analytical techniques. Coupling PPL and RO/ED techniques may provide substantial progress in the search for a method to quantitatively isolate oceanic DOC, bringing the entirety of the DOM pool within the marine chemist's analytical window.

A high-recovery technique of dissolved organic matter (DOM) isolation – reverse osmosis coupled with electrodialysis (RO/ED) – was used to isolate DOM from the North Atlantic Senegal-Mauritanian upwelling area surface water (5 m), North Atlantic oxygen minimum water (415 m) and deep water (3000 m), North Pacific subtropical gyre surface water (5 m), and North Pacific intermediate water (674 m) and deep water (3500 m). Samples were characterized by ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization (ESI FTICR-MS). RO/ED isolated DOM samples share a significant number of common formulas accounting for 54–79% of formulas in each sample. Total dissolved carbohydrate (TCHO) concentrations in RO/ED isolated DOM were specifically measured using a colorimetric method, and were found to have higher contribution to DOC than estimated by FTICR-MS data. Percentages of TCHO-C in DOC are in the range of 3.7–19.6% in all samples, with the North Pacific deep (3500 m) water having the lowest % and the North Atlantic upwelling core surface water having the highest %. Principal component analysis (PCA) using the relative magnitudes of MS peaks facilitated identification of specific peaks that are enriched in different samples. Peaks enriched in surface samples have higher H/C values than peaks enriched in deep samples, in both the North Atlantic DOM and the North Pacific DOM. This enrichment pattern is likely due to the selective photo-degradation of aromatic compounds and the bio-production of aliphatic and carbohydrate-like compounds in surface waters, and the selective bio-degradation of aliphatic and carbohydrate-like compounds with increasing depth. In further support of a photo-degraded signature for DOM in surface waters, photo-resistant and photo-produced molecular formulas were present in the highest numbers in the surface North Pacific subtropical gyre DOM. Peaks enriched in the North Pacific intermediate and deep DOM have significantly higher O/C values than the North Atlantic oxygen minimum layer and deep DOM, for both CHO formula compounds and CHON formula compounds. This difference in O/C values observed for the deep Pacific vs. Atlantic suggests oxidation of DOM, possibly via microbial activity during the ageing of DOM or the preferential remineralization of DOM from sinking particles at depth in the Pacific.

Two samples each of natural organic matter, humic acids, and fulvic acids from the Suwannee River in southeastern Georgia were titrated with NaOH on a Mettler-Toledo autotitrator. An average of 1500 data points was collected for each titration. Charge densities were corrected for the concentrations of inorganic anions and cations in the original samples. The pH 8 and 10 method, the modified Henderson-Hasselbalch model, and the Gaussian distribution model were fit to the titration data, and fitting parameters were compared with fitting parameters from earlier manual titrations of the same samples. With the exception of the carboxyl contents of two samples, both the carboxyl and phenolic contents from the pH 8 and 10 method were noticeably higher in the 2003 paper. Similar results were obtained for fitting parameters of the modified Henderson-Hasselbalch model, where, with the exception of two samples, both carboxyl contents and log K1 values are lower in the current study.

Five samples of dissolved organic matter (DOM) were isolated from Barataria Bay, Louisiana using the coupled reverse osmosis/electrodialysis method. Two of the samples (BU) were isolated from relatively pristine waters and three samples (BC) were isolated from waters that were strongly impacted by the Deepwater Horizon oil spill in April–July, 2010. The acid–base properties of these five samples were investigated by potentiometric titration. Carboxyl contents were estimated using an empirical pH-based method, and carboxyl contents, average pKa's, and the range of pKa in each sample were determined by fitting two numerical models (the Gaussian distribution model and the modified Henderson–Hasselbalch model) to the titration data. The carboxyl contents of the titrated samples range from 6.0 to 6.9meqgC−1 and the average pKa's for carboxyl groups range from 4.0 to 4.6. The distribution of pKa values is somewhat broader for the BU samples than for the BC samples, but all other acid–base properties of the five samples were comparable. Phenolic hydroxyl groups could not be detected in any of the samples. These samples of coastal DOM are much less acidic than fulvic acids from soils and fresh waters.