Size-exclusion chromatographic study of ECF and TCF softwood kraft pulp bleaching liquors.

BACKGROUND, AIMS, AND SCOPE: Currently, elemental chlorine-free (ECF) and totally chlorine-free (TCF) bleaching systems are widely used for pulp production. Low and medium molecular weight lignin break-down products are known to have harmful effects on the environment. According to some recent results, also high molecular weight (HMW) material consisting mainly of lignin and carbohydrates may cause toxic effects to the environment. For these reasons, toxicity and structure studies of HMW materials are of great importance. This investigation is a part of a larger project to obtain more structure information of HMW materials and toxicity of ECF and TCF bleaching effluents. Size-exclusion chromatography (SEC) has been commonly used for the characterization of organic macromolecules such as lignin, but to our knowledge, no reports have appeared dealing with the comparison of SEC of ECF and TCF bleaching liquors. The aim of the present study was to get more information about the molecular weight distribution (MWD) of HMW fractions of waste liquors from ECF and TCF bleaching sequences by SEC. METHODS: The MWDs of organic materials dissolved during different stages of ECF bleaching (O-D-EOP-D-ED) and TCF bleaching (O-Z-Q-P-Z-Q-P-P) of softwood (Pinus sylvestris) kraft pulp were determined and compared by SEC. All effluent samples from the above bleaching stages were ultrafiltrated using a membrane with a cutoff value of 1,000 Da. SEC was performed on high and also low molecular weight fractions and non-fractionated effluents. In the SEC experiments, a Superdex 75 column was used with 0.1 M NaOH solution as the eluent. Standards used for calibrating the SEC system were albumin, carboanhydrase, cytochrome C, tannic acid, dehydrodiacetovanillone, and vanillin. RESULTS AND DISCUSSION: The chromatograms of liquors from TCF bleaching stages vary more than those from ECF bleaching. Peroxide and chelating stages contained mostly high molecular weight (HMW) matter whereas chlorine dioxide and ozone stages had more low molecular weight compounds. The lignin content in HMW matter was higher than in stages that consisted of low molecular matter. Bleaching effluents contained the highest amounts of HMW material, mainly lignin, in the beginning of the sequences; the amounts decreased towards the end of the bleaching sequence. CONCLUSION: Determinations of MWD by the SEC method showed that effluents from the TCF sequence contained more HMW material than those from the ECF stage. This might be due to peroxide stages (P) that dissolve HMW lignin effectively. However, the molecular weights of ozone stages (Z) were very low compared to other stages. Chlorine dioxide stages also dissolved mostly low molecular weight lignin. Ultrafiltration of bleaching liquors showed that high molecular weight fraction also included some low molecular weight compounds and vice versa. High polydispersity and high lignin content correlated with the amount of HMW material in ECF and TCF bleaching stages. RECOMMENDATION AND OUTLOOK: Our liquor samples were studied by using a UV detector commonly used for lignin preparations; in upcoming investigations, it will be interesting to determine carbohydrates such as hemicelluloses. The results are applicable in papermaking in order to improve commonly used bleaching procedures, to test new potential bleaching systems, and to study chemical behavior of HMW materials in various bleaching liquors. The present results also form a good basis for toxicity measurements of ECF and TCF bleaching effluents and for more comprehensive spectroscopic and chromatographic experiments with samples taken from various bleaching stages. From the behavior of liquors studied, it appears that our other structure investigations by spectroscopic and chromatographic (NMR, Py-GC/MS, etc.) methods mostly correlate well with the present results.

Biological effects of high molecular weight lignin derivatives.

A number of high molecular weight (HMW) lignin derivatives possessing varied chemical properties were screened for their biological effects in order to obtain more information on the possible structural features of HMW lignin-related effects. The studied compounds were both commercial and in-house extracted lignin derivatives. Bioassays used include reverse electron transport (RET), Vibrio fischeri, Daphnia magna, and juvenile rainbow trout (Oncorhynchus mykiss) hepatocytes. The studied lignin derivatives inhibited the in vitro systems and luminescence of V. fischeri bacteria to some extent-daphnids were not affected. It seems that, at least in the RET assay, certain pH-dependent functional groups in lignin may be of importance regarding the biological effects.

Detecting AhR ligands in sediments using bioluminescent reporter yeast.

Sediments polluted with high concentrations of persistent organic pollutants, many of which are ligands of the aryl hydrocarbon receptor (AhR), are currently of concern around the industrialized world. Bioassays that can detect the presence of AhR ligands in environmental samples offer a relatively rapid and cost-effective means of prioritizing samples before more elaborate, laborious, and costly chemical analyses are applied. This paper presents a new bioluminescent yeast assay based on transcriptional activation of AhR. Its applicability for determining AhR ligands in complex environmental samples was demonstrated by analyzing a set of sediment samples from the River Kymi, Finland. The results from the assay are shown to be consistent with those from both a chemical analysis and an H4IIE-luc bioassay. The yeast assay procedure is simple and can be performed within 1 day. The yeasts grow rapidly, are easy to handle, and do not require continuous cell culturing. Moreover, the robustness of the yeast allows the application of the test to crude extracts or even sediment suspensions. The yeast assay described in this paper can be useful in screening and prioritization of samples prior to chemical analysis. Moreover, the strain can be used in the construction of fibre-optic biosensors.

Multivariate statistics of the pyrolysis products of high molecular components in pulping wastewaters to explain their toxicity.

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 polydispersed 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 the 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 of originating 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.

In vitro estrogenicity of polybrominated flame retardants.

Estrogenicity of five brominated flame retardants (BFRs), namely BDE-47, BDE-99, BDE-205, PBB-153 and technical Firemaster BP-6, were assessed by in vitro assays developed to detect chemicals with estrogenic properties. Recombinant yeast cells containing a human estrogen receptor gene failed to give any response to the chemicals tested. However, the positive control compound, estradiol-17beta, showed that the yeast cell assays had worked properly. The freshly separated fish hepatocyte assay based on the synthesis and secretion of vitellogenin from the isolated liver cells produced a clear dose-response curve in the presence of all tested flame retardants except Firemaster BP-6. The toxicity of the BFRs was detected by determining the cell ethoxyresorufin-O-deethylase activity (EROD). The BFRs tested induced hepatic EROD activity at low test concentrations, but started to inhibit activity at higher concentrations. The decreased detoxification capacity of the hepatocytes resulted in a decrease in the vitellogenin production of the cells. The capability of in vitro assays to detect estrogenic properties of chemicals seems to vary. Thus, further work is needed to understand the mechanisms responsible for these reactions.

Evaluation of wastewater effluents by small-scale biotests and a fractionation procedure.

Municipal and industrial effluents were screened with a battery of biotests and with a modified toxicity identification evaluation Phase I procedure. The acute toxicities of the effluent samples were low and the submitochondrial reverse electron-transport (RET) test was the most sensitive toxicity test. Estrogenic effects were found in almost all effluent samples, and genotoxicity was detected in one concentrated effluent sample. The fractionation methods we used proved to be especially effective at tracking toxicity caused by metals and organic contaminants, with the RET test being particularly suited to evaluating pH-dependent toxicity. The used solid-phase extraction columns with both hydrophilic and hydrophobic binding properties turned out to be suitable for removing or reducing organic toxicity-causing substances from the effluent samples. The results of this study show that the use of only conventional acute toxicity tests for effluent assessment will not be sufficient-the genotoxic, hormonal, and even bioaccumulative potential of the effluents and effluent fractions should be evaluated as well.

Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase.

The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers.

Biodegradation of chemicals in a standardized test and in environmental conditions.

The estimation of biodegradation rates is an important source of uncertainty in chemical risk assessment. The existing OECD tests for ready biodegradability have been developed to devise screening methods to determine whether a chemical is potentially easily biodegradable, rather than to predict the actual rate, of biodegradation in the environment. However, risk assessment needs degradation rates. In practice these rates are often estimated (default values) from ready biodegradability tests. These tests have many compromising arbitrary features compared to the situation in the real environment. One important difference is the concentration of the chemical. In wastewater treatment or in the environment many chemicals are present at ng l(-1) to microg l(-1) levels whereas in the tests the concentrations exceed 10-400 mg carbon per litre. These different concentrations of the chemical will lead to different growth kinetics and hence different biodegradation rates. At high concentrations the chemical, if it is degradable, can serve as a primary substrate and competent microorganisms will grow exponentially, resulting in a sigmoid biodegradation curve. At low environmental concentrations the chemical does not serve as a primary substrate, and therefore does not support significant growth of the degraders, and the substrate has a linear biodegradation rate. In this study the biodegradation rates of two reference chemicals, aniline and 4-chloroaniline, were compared in a standard method and in more realistic conditions at low concentrations, using 14C-labelled substances and different sources of inocula. Biomass evolution during the tests was monitored by adenosine triphosphate measurement and also on the basis of the residual 14C-activity in the particulate matter. The results partly support the thesis that low concentrations lead to different biodegradation kinetics compared to the concentrations used in the standard tests. Furthermore the biodegradation rates of the chemicals studied, particularly of 4-chloroaniline, in Finnish natural waters appeared to be lower than those reported in some other countries.