Multivariate Statistics of the Pyrolysis Products of High Molecular Components in Pulping Wastewaters to Explain Their Toxicity (6 pp)

Background, Aim and Scope At present, large-scale paper manufacture involves delignification and bleaching by elemental chlorine free (ECF) or totally chlorine free (TCF) processes. The wastewater is purified by secondary treatment (mechanical, chemical and biological) which removes most of the toxic substances from the discharge. However, we found residual toxicity in the high molecular (> 1000 D) matter (HMWM) of the discharge by test of the RET (reverse electronic transfer) inhibition. This fraction consists mainly of polydisperse lignin (LIG) and carbohydrate (CH) macromolecules. Structural units in these molecules are studied by pyrolysis gas chromatography / mass spectrometry (Pyr-GC/MS). In the present work, our aim was to find out those structural units which could explain the RET toxicity of LIG or CH molecules. We compared statistically RET toxicity values of the HMWM samples from treated wastewaters of pilot pulping experiments and intensity variation of the pyrolysis product gas chromatograms of these samples. This application is a novel study procedure. Methods Pyrolysis products (Py-GC/MS results) and inhibition of RET (reverse electronic transport toxicity) as TU50 and TU20 of HMWM (High Molecular Weight Material; Mw > 1000 D) were compared by multivariate statistics. The samples were from laboratory pilot stages of TCF (Totally Chlorine Free) and ECF (Elemental Chlorine Free) manufacture of softwood pulp. Py-GC/MS was done without and with addition of TMAH (Tetra Methyl Ammonium Hydroxide). The name and structure of each abundant fragment compound was identified from its retention time and mass spectrum compared to authentic reference compounds or literature. Four sets of Toxicity Units (TUs) and GC peak areas of the pyrolysis fragments were obtained. The data were normalized by division with LIG (lignin content of each sample). TU values were dependent and the fragment values independent (explanatory) variables in statistical treatments by SPSS system. Separate analyses of correlations, principal components (PCA) and stepwise multiple linear regression (SMLR) were performed from the four sample sets TCF and ECF with and without TMAH. Results and Discussion From the CH fragments, 2-furfural in TCF, and from the LIG fragments, styrene in ECF showed the highest probabilities to originate from source structures of toxicity. Other possible compounds in concern were indicated to be CH fragment 2-methyl-2-cyclopenten-1-one in ECF and LIG fragments 2-methoxy-4-methylphenol, 4,5-dimethoxy-2-methylphenol and 2-methylphenol in TCF.     

Improvement in rice straw pulp bleaching effluent quality by incorporating oxygen delignification stage prior to elemental chlorine-free bleaching

Environmental degradation by industrial and other developmental activities is alarming for imperative environmental management by process advancements of production. Pulp and paper mills are now focusing on using nonwood-based raw materials to protect forest resources. In present study, rice straw was utilized for pulp production as it is easily and abundantly available as well as rich in carbohydrates (cellulose and hemicelluloses). Soda-anthraquinone method was used for pulp production as it is widely accepted for agro residues. Bleaching process during paper production is the chief source of wastewater generation. The chlorophenolic compounds generated during bleaching are highly toxic, mutagenic, and bioaccumulative in nature. The objectives of study were to use oxygen delignification (ODL) stage prior to elemental chlorine-free (ECF) bleaching to reduce wastewater load and to study its impact on bleached pulp characteristics. ODL stage prior to ECF bleaching improved the optical properties of pulp in comparison to only ECF bleaching. When ODL stage was incorporated prior to bleaching, the tensile index and folding endurance of the pulp were found to be 56.6 ± 1.5 Nm/g and 140, respectively, very high in comparison to ECF alone. A potential reduction of 51, 57, 43, and 53% in BOD₃, COD, color, and AOX, respectively was observed on adding the ODL stage compared to ECF only. Generation of chlorophenolic compounds was reduced significantly. Incorporation of ODL stage prior to bleaching was found to be highly promising for reducing the toxicity of bleaching effluents and may lead to better management of nearby water resources.

Open image in new window

Graphical abstract ?

     

Evaluation of biodegradation of nonylphenol ethoxylate and lignin by combining toxicity assessment and chemical characterization

The aerobic biodegradation of commercial nonylphenol ethoxylate (NPE) mixture and alkali lignin was studied using the OECD headspace test accompanied by the simultaneous measurement of ecotoxicity directly from the biodegradation liquors and by the follow-up of the chemical composition of the studied chemicals. NPE degradation was dependent on the inoculum source: approximately 40% of NPE was mineralized into CO₂ during the 4-week experiment when inoculum from Helsinki City wastewater treatment plant (WWTP) was used, and only 12% was mineralized when inoculum from Jyväskylä City WWTP was used. Chemical analyses revealed a shift in the ethoxylate chain length from longer to shorter soon after the beginning of the NPE biodegradation tests. At the same time also toxicity (reverse electron transport assay, RET) and estrogenic activity (human estrogen receptor yeast) measured directly from the biodegradation liquors decreased. In case of alkali lignin, approximately 11% was mineralized in the test and chemical analysis showed in maximum a 30% decrease in lignin concentration. Toxicity of lignin biodegradation liquors started to decrease in the beginning of the test, but became more toxic towards the end of the test again. Especially RET assay proved to be sensitive enough for measuring toxicity changes directly from biodegradation liquors, although a concentrating treatment of the liquors is recommended for a more detailed characterization and identification of toxic metabolites.     

Comparative toxicity of effluents processed by different treatments in V79 fibroblasts and the algae Selenastrum capricornutum

The efficacy of ozonation and of photocatalysis processing in the treatment of pulp mill ECF (elementary chlorine free) bleaching and textile effluents was evaluated by determining total organic carbon reduction (TOC) and the toxicity. The chronic toxicity of the effluents was evaluated by the ability to inhibit the growth of algae Selenastrum capricornutum. Cultured hamster V79 fibroblasts were used to assess the cytotoxicity of effluents submitted to different detoxification processes. Two endpoints were measured in V79 cells: 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) reduction and neutral red uptake (NRU). Both treatment processes were able to reduce the TOC, although ozonization was less effective for pulp mill ECF bleaching. The pulp mill ECF bleaching and textile effluents reduced the growth of S. capricornutum by 39% and 27%, respectively. However, at the highest concentration tested, the textile effluents treated by photochemical process for 60 min showed increased cytotoxicity in V79 cells compared to the untreated effluent when assessed by the NRU and MTT reduction assays (increases of 30% and 40%, respectively). Pulp mill ECF bleaching effluent treated by ozonization had a similar cytotoxicity to that of untreated effluent in the NRU assay. In contrast, the MTT reduction assay indicated that effluents treated with ozone were around 20% more cytotoxic than untreated effluents. These results show that cultured fibroblasts may be useful for studying cellular responses to pollutants and may be included in tests to monitor the efficiency of effluent detoxification processes.     

Status of the PCDD and PCDF contamination of commercial milk caused by milk cartons

Within the scope of this study, possible migration of polychlorinated dibenzodioxins and -furans (PCDDs/PCDFs) from cartons (produced using bleached cardboard) into cow's milk was investigated. Three different types of carton were examined. The milk samples were taken at different times of storage and analysed for PCDDs/PCDFs. In contrast to a similar study carried out in 1990, the I-TEQ-results of all cartons analysed may be considered very low. No significant migration of toxic (2,3,7,8-chlorine-substituted) PCDDs/PCDFs could be observed. This is due to the very low PCDD/PCDF concentrations in modern cardboards as a result of the elemental chlorine-free (ECF) bleaching processes used. As far as the milk samples are concerned, no influence of the cardboards (according to the I-TEQ data) could be ascertained. I-TEQ concentrations in milk did not noticeably change during storage times of three, six and eight days. The I-TEQ-data obtained for all milk samples was found to be in the range typical of background concentrations in cow's milk in Germany. Although 1,2,7,8-TCDF is not included in the calculation of the TEQ (no 2,3,7,8-chlorine-substitution), analysis of this congener in cardboard samples was also carried out as a matter of general interest for cardboard investigations and an indicator of pulp bleaching with free chlorine.     

Toxicity evaluation of organic sediment extracts resolved by size exclusion chromatography using rainbow trout hepatocytes.

The (geno)toxicity of sediment dichloromethane extracts and fractions obtained by size exclusion chromatography were evaluated to investigate effects based on size fractionation. In this study, three sediments were selected according to their incremental contamination in PAHs and in PCBs: Hamilton harbour, Toronto bay and lake St. Clair sediments. Heavy metals, total sulfur and elemental sulfur (S8) were also determined in the (un)fractionated sediment extracts. The liver cells were exposed to concentrations of sediment extracts and fractionated samples for 24 h at 15 degrees C, afterwhich cell viability, cytochrome P4501A1 activity, available free Zn, DNA damage and oxidative stress were determined. The results showed that the sediment extracts contained high levels of sulfur most of which was found in the low molecular weight (LMW) region, i.e., the 2000-50 atomic mass unit (amu) fraction. Elemental sulfur (S8) accounted for 14-41% of extractable sulfur and were found to elute in the post-column volume (PCV) fraction despite its molecular weight of 256 amu. Heavy metals were found mainly in the HMW (i.e. the > 2000 amu) fraction and LMW fractions and very few or none were observed in the PCV fractions. In sediment extracts, sublethal effects were present principally by the HMW and LMW fractions suggesting that some chemicals were also associated with high molecular weight compounds of extractable organic matter. Less toxicity or effect was sometimes found in the extract indicating an antagonistic effect of the contaminants. We found that cell viability and genotoxicity evaluations could be performed on the unfractionated extracts while EROD, available Zn and oxidative stress measurements should be performed on the LMW fractions because of possible antagonist or shielding effects. Considering the cytotoxic responses, the best toxicity ranking in respect to contaminant levels in sediment extract was obtained with the LMW and PCV fractions which accounted for most of the toxic responses in the chromatographic fractions. Moreover, the shielding effect could be explained, in part, by the association of LMW contaminants to large macromolecules.     

Acute toxicity and genotoxicity of aquatic hydrophobic pollutants sampled with semipermeable membrane devices

Triolein-filled semipermeable membrane devices (SPMDs) were deployed for 4 weeks in polluted water sources in Lithuania. The mixtures of pollutants sampled by the SPMDs were fractionated by size-exclusion chromatography (SEC). The fraction containing average molecular weight compounds such as polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides was screened by gas chromatography and mass spectrometry. The whole (non-fractionated) samples and their SEC fractions were tested in bioassays including Microtox, Mutatox, Daphnia pulex immobilization assay and the sister chromatid exchange (SCE) in human lymphocytes in vitro test. The Microtox test was most sensitive with the estimated EC(50) values in the range of milligrams or even micrograms per milliliter based on the amount of the SPMD triolein. Part of the observed toxicity was caused by elemental sulfur co-sampled by the SPMDs from sediments. The sum of toxicity equivalents of the SEC fractions was smaller than the relative toxicity of the whole samples indicating the presence of synergistic interactions in the complex mixtures of chemical pollutants. The toxic or genotoxic response induced by the chemical mixtures and their fractions was smaller in the D. pulex, Mutatox and SCE tests. In Mutatox, a positive response was only detected without the S9 metabolic activation which indicates the presence of mainly direct-acting mutagens in the samples. Interpretation of the Mutatox data was difficult due to the complexity of dose-response and time-response relationships. The study has demonstrated the potential as well as some limitations of SPMDs in the monitoring of biological effects of bioavailable organic pollutants in the aquatic environment.     

Singlet oxygen mediated degradation of Klason lignin

After some results concerning photochemical generated singlet oxygen on lignins from steam explosion, the reactions of chemically generated singlet oxygen with Klason lignins from pine and beech are described. Singlet oxygen was produced through the reaction of hydrogen peroxide with sodium hypochlorite. The degradation of lignin was followed by uv spectroscopy and gel permeation chromatography. Extensive degradation of the lignins was observed when 20 mg of Klason lignin was treated with 1 ml of 30% hydrogen peroxide and 8.56 ml of 1.093 M sodium hypochlorite. In the uv spectra registered after the treatment with singlet oxygen the absorptions typical of lignin (210-220 nm and 250-280 nm) were completely absent. The gpc analysis of lignin after a treatment with 0.1 ml of hydrogen peroxide and 0.86 ml of sodium hypochlorite showed a clear reduction of signals due to the lignin and a shift to lower molecular weight. The potential use of this procedure in the bleaching procedure was tested by using recycled paper. A maximum reduction of 51% in the amount of lignin in this paper was observed.     

Benzo[a]pyrene co-metabolism in the presence of plant root extracts and exudates: Implications for phytoremediation

Benzo[a]pyrene, a high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH) was removed from solution by Sphingomonas yanoikuyae JAR02 while growing on root products as a primary carbon and energy source. Plant root extracts of osage orange (Maclura pomifera), hybrid willow (Salix albaxmatsudana), or kou (Cordia subcordata), or plant root exudates of white mulberry (Morus alba) supported 15-20% benzo[a]pyrene removal over 24 h that was similar to a succinate grown culture and an unfed acetonitrile control. No differences were observed between the different root products tested. Mineralization of (14)C-7-benzo[a]pyrene by S. yanoikuyae JAR02 yielded 0.2 to 0.3% (14)CO(2) when grown with plant root products. Collectively, these observations were consistent with field observations of enhanced phytoremediation of HMW PAH and corroborated the hypothesis that co-metabolism may be a plant/microbe interaction important to rhizoremediation. However, degradation and mineralization was much less for root product-exposed cultures than salicylate-induced cultures, and suggested the rhizosphere may not be an optimal environment for HMW PAH degradation by Sphingomonas yanoikuyae JAR02.     

The conversion of chicken manure to bio-oil by fast pyrolysis. III. Analyses of chicken manure, bio-oils and char by Py-FIMS and Py-FDMS

Fast pyrolysis of chicken manure produced the following three fractions: bio-oil Fraction I, bio-oil Fraction II, and a char. In a previous investigation we analyzed each of the four materials by curie-point pyrolysis-gas chromatography/mass spectrometry (CpPy-FDMS). The objective of this article is to report on the analyses of the same chicken manure and the three fractions derived from it by fast pyrolysis. We now used pyrolysis-field ionization mass spectrometry (Py-FIMS) to characterize the three fractions. In addition, the two bio-oil materials were analyzed by pyrolysis-field desorption mass spectrometry (Py-FDMS). The use of both Py-FIMS and Py-FDMS produced signals over significantly wider mass ranges than did CpPy-GC/MS, and so allowed us to identify considerably larger numbers of constituents in each material. Individual compounds identified in the mass spectra were classified into the following twelve compound classes: (a) low molecular weight compounds (< m/z 62); (b) carbohydrates; (c) phenols + lignin monomers; (d) lignin dimers; (e) n-alkylbenzenes; (f) N-heterocyclics; (g) n-fatty acids; (h) n-alkanes; (i) alkenes; (j) sterols; (k) n-diols and (l) high molecular weight compounds (> m/z 562). Of special interest were the high abundances of low-molecular weight compounds in the two bio-oils which constituted close to one half of the two bio-oils. Prominent among these compounds were water, ammonia, acetic acid, acetamide, propyl radical, formamide and hydrogen cyanide. The main quantitative differences between the two bio-oils was that bio-oil Fraction I, as analyzed by the two mass spectrometric methods, contained lower concentrations of low-molecular weight compounds, carbohydrates, and N-heterocyclics than bio-oil Fraction II but was richer in lignin dimers, n-alkylbenzenes and aliphatics (n-fatty acids, n-alkanes, alkenes, and n-diols). Of special interest were the N-heterocyclics in the two bio-oils such as pyrazole, pyrazoline, substituted pyrroles, pyridine and substituted pyridines, substituted methoxazole, substituted pyrazines, indole and substituted indoles. Fatty acids in all four materials ranged from n-C(9) to n-C(33), alkanes from n-C(9) to n-C(40), alkenes from C(10:1) to C(40:1) and diols from n-C(7) to n-C(29). The chicken manure, bio-oil Fraction I, and char each contained about 4% sterols with cholesterol, ethylcholestriene, ergosterol, ethylcholestene, ethylcholesterol and beta -sitosterol as major components. Semi-quantitative estimates of the total materials identified by Py-FIMS were: chicken manure: 61.1%; bio-oil Fraction I: 81.3%; bio-oil Fraction II: 78.6%; char: 61.3%; and by Py-FDMS were: bio-oil Fraction I: 65.4%; bio-oil Fraction II: 70.0%.     

Correlations between PAH bioavailability,degrading bacteria,and soil characteristics during PAH biodegradation in five diffusely contaminated dissimilar soils

The natural biodegradation of seven polycyclic aromatic hydrocarbons (PAHs) by native microorganisms was studied in five soils from Normandy (France) from diffusely polluted areas, which can also pose a problem in terms of surfaces and amounts of contaminated soils. Bioavailability tests using cyclodextrin-based extractions were performed. The natural degradation of low molecular weight (LMW) PAHs was not strongly correlated to their bioavailability due to their sorption to geosorbents. Conversely, the very low degradation of high molecular weight (HMW) PAHs was partly correlated to their poor availability, due to their sorption on complexes of organic matter and kaolinites or smectites. A principal component analysis allowed us to distinguish between the respective degradation behaviors of LMW and HMW PAHs. LMW PAHs were degraded in less than 2–3 months and were strongly influenced by the relative percentage of phenanthrene-degrading bacteria over total bacteria in soils. HMW PAHs were not significantly degraded, not only because they were less bioavailable but also because of a lack of degrading microorganisms. Benzo[a]pyrene stood apart since it was partly degraded in acidic soils, probably because of a catabolic cooperation between bacteria and fungi.     

The occurrence and environmental effect of persistent organic pollutants (POPs) in Taurus Mountains soils

Purpose  

Persistent organic pollutants (POPs) are of global concern due to their ubiquitous presence and toxicity. The occurence of polychlorinated dibenzo-p-dioxins (PCDDs), -dibenzofurans (PCDFs), co-planar biphenyls (PCBs), hexachlorocyclohexanes (HCH), dichlorodiphenyltrichloroethanes (DDT), and organochlorine pesticides (OCPs) in forest soil collected from Taurus mountains may have adverse effects on the environment and health. The aim of the study was to investigate the outcome and distribution of POPs in the environment and the possible grasshopper effect along an altitude transect from sea level up to nearly 2,000 m a.s.l at a spatial distance of about 60 km in the southeastern Turkish Mediterranean Sea.     

Advanced oxidation of a pulp mill bleaching wastewater.

The degradation, by several advanced oxidation reactions, of a pulp mill ECF bleaching effluent, was studied. The initial biodegradability of the organic matter present in the effluent, estimated as the BOD5/COD, was low (0.3). When the effluent was submitted to ozonation and to five different advanced oxidation systems (O3/UV, O3/UV/ZnO, O3/UV/TiO2, O2/UV/ZnO, O2/UV/TiO2), the biodegradability increase significantly. After five minutes of reaction, the O3/UV system appears as the most efficient in to transform the organic matter to more biodegradable forms. A similar effect was observed when the effluent was submitted to an activated sludge treatment. The COD, TOC and toxicity reduction correlated well with the biodegradability enhancement after AOPs treatments.     

A novel direct equipartition ray design (EquRay) procedure for toxicity interaction between ionic liquid and dichlorvos

Background, aim and scope  

Pollutants always co-exist in the environment. Determining and characterizing the interaction among chemicals is an important issue. Experimental designs (ED) play an important role in evaluating the interactions. The main aim of our study is to provide the test and analysis of the toxicity interaction with a novel ED method.     

Benzo[a]pyrene degradation by Sphingomonas yanoikuyae JAR02

Batch experiments were conducted to characterize the degradation of benzo[a]pyrene, a representative high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH), by Sphingomonas yanoikuyae JAR02. Concentrations up to the solubility limit (1.2 microg l(-1)) of benzo[a]pyrene were completely removed from solution within 20 h when the bacterium was grown on salicylate. Additional experiments with [(14)C]7-benzo[a]pyrene demonstrated 3.8% mineralization over 7 days when salicylate was present is solution, and one major radio-labeled metabolite was observed that accounted for approximately 10% of the initial radio-label. Further characterization of the radio-labeled metabolite using HPLC/MS and HPLC/MS/MS identified radio-labeled pyrene-8-hydroxy-7-carboxylic acid and unlabeled pyrene-7-hydroxy-8-carboxylic acid as novel ring-cleavage metabolites, and a benzo[a]pyrene degradation pathway was proposed. Results indicate that biostimulation of HMW PAH degradation by salicylate, a water-soluble, non-toxic substrate, has significant potential for in situ bioremediation.     

Subcellular distribution of trace elements in kidney of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli)

Total and subcellular renal Zn, Cu, Se, Mn, V, Hg, Cd and Ag were determined by a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). All element concentrations in the maternal individual were higher than those in the fetal individual. Most of total renal elements studied were present in the cytosol of both animals. In maternal cytosol, Mn, Hg and Ag were present in high molecular weight substances (HMW); Se was in low molecular weight substances (LMW); Zn, Cu, and Cd were in metallothionein (MT), mostly; and the distribution of V in percentage among the three renal cytosolic fractions was similar. In fetal cytosol, Zn, Mn, Hg, V, Cd, and Ag were present in HMW, Cu was present in MT, mostly. In contrast, Se was observed mostly in both LMW and HMW. MT isoforms were characterized. Three obvious peaks in retention time were found in either the maternal or fetal MT. The highest elemental ion intensities were in the 7.8min peak for the mother, and in the 4.3min peak for the fetus, respectively, implying that different MT isoforms may be closely associated with elemental accumulation between maternal and fetal renal cytosols.     

Lipophilic extractives in process waters during manufacturing of totally chlorine free kraft pulp from eucalypt wood

The chemical composition of lipophilic extractives from process waters throughout a totally chlorine free (TCF) bleaching sequence after kraft pulping of eucalypt wood has been studied. These compounds are among the most problematic wood constituents for both TCF and zero liquid effluent (ZLE) processes, since they tend to accumulate in circuits resulting in the formation of the so-called pitch deposits causing serious problems in the pulp and paper industry. Pitch deposits collected at different parts of the pulp mill were also characterized and their composition compared with that of lipophilic compounds in process waters and Eucalyptus globulus wood. The identification of these compounds from process waters, wood and pitch deposits was performed by gas chromatography and gas chromatography-mass spectrometry using short- and medium-length high temperature capillary columns. Sterols, sterol esters, steroid ketones and steroid hydrocarbons were the main compounds identified. These chemical species arise from eucalypt wood extractives that survive the pulping and are released from pulp into the process waters during the bleaching process. Finally, they can deposit in pulp and on different parts of the mill or remain suspended in process waters being discharged in effluents.     

Occurrence and toxicity of antimicrobial triclosan and by-products in the environment

Introduction and aims  

A review was undertaken on the occurrence, toxicity, and degradation of triclosan (TCS; 5-chloro-2,4-dichlorophenoxy)phenol) in the environment. TCS is a synthetic, broad-spectrum antibacterial agent incorporated in a wide variety of household and personal care products such as hand soap, toothpaste, and deodorants but also in textile fibers used in a range of other consumer products (e.g., toys, undergarments and cutting boards among other things).     

Abundance and diversity of Sphingomonas in Shenfu petroleum-wastewater irrigation zone, China

Introduction  

Members of the genus Sphingomonas have raised increasing attention due to their ability for polycyclic aromatic hydrocarbon (PAH) degradation and their ubiquity in the environment. However, few studies have revealed the ecological information on the abundance and diversity of Sphingomonas in the environment.     

Comprehensive characterization of oil refinery effluent-derived humic substances using various spectroscopic approaches

Refinery effluent-derived humic substances (HS) are important for developing refinery effluent reclamation techniques and studying the environmental chemistry of wastewater effluents. In this study, dissolved organic matter (DOM) from refinery effluent was concentrated using a portable reverse osmosis (RO) system. HS were isolated from RO retentates with XAD-8 resin. A variety of approaches such as specific UV absorbance at 254nm (SUV(254)), elemental analysis, size exclusion chromatography (SEC), solid-state cross polarization magic angle spinning (13)C nuclear magnetic resonance spectrometry ((13)C CPMAS NMR), Fourier transform infrared spectrometry (FTIR), and electrospray ionization/ion trap/mass spectrometry (ESI/ion trap/MS) were employed for characterization of HS. The portable RO system exhibited high yield and recovery of DOM for concentrating refinery effluent. The concentration of dissolved organic carbon (DOC) in the refinery effluent was 9.9mg/l, in which humic acids (HA) and fulvic acids (FA) accounted for 2.3% and 34.6%, respectively. Elemental and SUV(254) analyses indicated relative high amounts of aliphatic structures and low amounts of aromatic structures in refinery effluent-derived HS. Refinery effluent-derived HS displayed lower molecular weight than natural HS. The number-average molecular weight (M(n)) and the weight-average molecular weight (M(w)) of HA were 1069 and 2934, and those of FA were 679 and 1212 by SEC, respectively. By ESI/ion trap/MS, the M(n) and the M(w) of FA were 330 and 383. Four kinds of carbon structures (aliphatic, aromatic, heteroaliphatic, and carboxylic carbons) were found in refinery effluent-derived HS by (13)C NMR analysis. The quantitative results support the interpretation that these HS are rich in aliphatic carbons and poor in aromatic carbons. Proteinaceous materials were identified by FTIR analysis in refinery effluent-derived HS.