Testing of the cytotoxic effects of sulfate pulp mill waste waters

The effect of 22 technological waste samples and of some standards was tested on bacteria, fungi, chlorococcal algae, flagellata, plant cells, cells ofTubifex tubifex, hamster cells V79 and the fishLebistes reticulatus. Of these 22 samples, some inhibition of cell life processes was displayed by the black liquor formed in the production of paper pulp and viscose pulp, by the waste solution produced during the preparation of bleaching agents for paper pulp and viscose pulp, and by the residual liquor after hypochlorite treatment of paper pulp.     

Use of micronucleic test for the determination of genotoxicity of waste liquor from a magnesium bisulfite pulp mill

Since diluted magnesium bisulfite waste liquor from a pulp mill after beech wood processing exhibited an inhibitory effect on all the model organisms used in our previous work (Čerňákováet al. 1991) and the results of Ames mutagenicity test were ambiguous, micronucleic test was used for characterization. The frequency of micronuclei observed suggested that the diluted magnesium bisulfite waste liquor had a mutagenic effect underin vitro conditions of the micronucleic test. Translated by Č. Novotny     

An evaluation in the Ames test of the mutagenicity of the sewage and industrial effluents from the Baikal Paper and Pulp Combine

The use of the Ames test for the analysis of industrial effluents from cellulose production and sewage waters varying in the degree of purification with the aid of a metabolic activation system from rat and fish liver with Salmonella strains TA 98 and TA 100 revealed a strong direct mutagenic effect of strain TA 100 in samples after cellulose chlorination. The multistage procedure of sewage water purification allows to remove practically completely the mutagenic substances. A simultaneous study of cytotoxic effects of industrial effluents on mammalian cells shows that the mutagenic activity is exhibited in not toxic concentrations. The urgency of a regular biological control over the genotoxicity of industrial effluents from the sulfate production of cellulose is under discussion.     

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.     

The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey

Since 1979, the Environmental Agency of S?o Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters. This paper is a compilation of data obtained during the last 20 years from more than a thousand samples. Potencies up to 30,000 revertants/l were observed in 137 positive samples. The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix. A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public. The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively. As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the S?o Paulo State Water Quality Monitoring Program. This assay has proven to be a useful tool in the identification of important pollution sources. Correction and prevention actions in Water Pollution Control Programs were generated as a result.     

Bio-refinery system of DME or CH4 production from black liquor gasification in pulp mills

There is great interest in developing black liquor gasification technology over recent years for efficient recovery of bio-based residues in chemical pulp mills. Two potential technologies of producing dimethyl ether (DME) and methane (CH₄) as alternative fuels from black liquor gasification integrated with the pulp mill have been studied and compared in this paper. System performance is evaluated based on: (i) comparison with the reference pulp mill, (ii) fuel to product efficiency (FTPE) and (iii) biofuel production potential (BPP). The comparison with the reference mill shows that black liquor to biofuel route will add a highly significant new revenue stream to the pulp industry. The results indicate a large potential of DME and CH₄ production globally in terms of black liquor availability. BPP and FTPE of CH₄ production is higher than DME due to more optimized integration with the pulping process and elimination of evaporation unit in the pulp mill.     

The use of tamarind waste to improve ethanol production from cane molasses

Tamarind wastes such as tamarind husk, pulp, seeds, fruit and the effluent generated during tartaric acid extraction were used as supplements to evaluate their effects on alcohol production from cane molasses using yeast cultures. Small amounts of these additives enhanced the rate of ethanol production in batch fermentations. Tamarind fruit increased ethanol production (9.7%, w/v) from 22.5% reducing sugars of molasses as compared to 6.5% (w/v) in control experiments lacking supplements after 72 h of fermentation. In general, the addition of tamarind supplements to the fermentation medium showed more than 40% improvement in ethanol production using higher cane molasses sugar concentrations. The direct fermentation of aqueous tamarind effluent also yielded 3.25% (w/v) ethanol, suggesting its possible use as a diluent in molasses fermentations. This is the first report, to our knowledge, in which tamarind-based waste products were used in ethanol production. Received 2 April 1998/ Accepted in revised form 13 November 1998     

Somatic mutation, DNA damage and cytotoxicity induced by benzo[a]pyrenedione/benzo[a]pyrenediol redox couples in cultured mammalian cells

BP-3,6-dione was found to be mutagenic, cytotoxic and to induce DNA damage in a transformed line of Syrian hamster fibroblasts at low concentrations, 2 micrograms/ml and less. Inhibition of sulfate and glucuronic acid conjugating enzymes with salicylamide potentiated the above effects of BP-3,6-dione. Diminishing cellular capacity to scavenge superoxide anion radicals also potentiated the mutagenic and cytotoxic action of the dione. The presence of dicumarol, a specific inhibitor of the two-electron reduction of quinones by DT-diaphorase, afforded some protection against cytotoxicity. The results indicate that BP-3,6-dione undergoes two-electron reduction to an unstable hydroquinone, BP-3,6-diol, or one-electron reduction to a semiquinone radical intermediate and that both of these reduced forms undergo rapid univalent oxidation to generate active reduced oxygen species. The data are consistent with the hypothesis that active oxygen species generated by BP-dione/BP-diol redox cycling are responsible, at least in part, for the mutagenic and cytotoxic effects observed with BP-3,6-dione.     

Mutagenicity and genotoxicity of suspended particulate matter in the Seine river estuary

Highly mutagenic compounds such as some PAHs have been identified in surface waters and sediments of the Seine river estuary. Suspended particulate matter (SPM) represents a dynamic medium that may contribute to the exposure of aquatic organisms to toxic compounds in the water column of the estuary. In order to investigate major sources of mutagenic contaminants along the estuary, water samples were taken at 25 m downstream of the outlet of an industrial wastewater-treatment plant (WWTP). SPM samples were analyzed for their genotoxicity with two short-term tests, the Salmonella typhimurium mutagenicity assay (TA98+S9 mix) and the comet assay in the human HepG2 cell line. Sampling sites receiving effluents from a chemical dye industry and WWTP showed the highest mutagenic potencies, followed by petrochemical industries, petroleum refinery and pulp and paper mills. These data indicate that frame-shift mutagens are present in the Seine river estuary. Furthermore, the comet assay revealed the presence of compounds that were genotoxic for human hepatocytes (HepG2 cells). We also observed a high level of mutagenic potency in the sediment of the lower estuary (3 × 10? revertants/g). The source of mutagenic and genotoxic compounds seems to be associated with various types of effluents discharged in the Seine river estuary. Both test systems resulted in the same assessment of the genotoxicity of particulate matter, except for three of the 14 samples, underlying the complementarity of bioassays.     

Test-systems for biomonitoring based on membrane-bound enzyme complexes. III. An assessment of the genotoxic action of industrial effluents in the Ames test-system with metabolic activation by microsomal monooxygenases

Using the Ames mutation test system II samples of the Baikal pulp and paper effluents selected at different dates have been investigated. In 9 cases the effluent samples possessed no mutagenic activity; some samples revealed frame shift mutations, i.e. a medium mutagenic effect. In the presence of a metabolic activation system from the livers of phenobarbital-induced rats the mutagenic activity was considerably reduced.     

Properties and potential medical applications of regenerated casein fibers crosslinked with citric acid

Regenerated protein fibers developed from casein using alkaline solutions and crosslinked with citric acid have good tensile properties and water stability but were cytotoxic. Casein is obtained as a byproduct during skim milk production but has limited industrial applications. In this research, casein was dissolved using alkaline solutions and the effects of fiber formation conditions on the tensile properties was studied before and after crosslinking with citric acid. Crosslinked casein fibers had tensile strength as high as 110 MPa and breaking elongation of 13.6%. The fibers were also stable in 90°C water between pH 3 and 9 but were hydrolyzed at pH 11. Casein was cytotoxic and did not support the attachment and growth of mouse fibroblasts.     

Differential and synergistic effects of xylanase and laccase mediator system (LMS) in bleaching of soda and waste pulps

AIMS: Investigation of waste pulps and soda pulp bleaching with xylanase (X) and laccase mediator system (LMS) alone and in conjunction (one after the other) (XLMS). METHODS AND RESULTS: Soda and different grades of waste pulp fibres [used for making three-layered duplex sheets - top layer (TL), protective layer (PL) and bottom layer (BL)] when pretreated with either xylanase (40.0 IU g(-1)) or LMS (up to 200.0 U g(-1)) alone and in combination (one after the other) (XLMS) exhibited an increase in release of reducing sugars [up to 881.0% soda pulp; up to 736.6% (TL), up to 215.7% (PL) and up to 198.0% (BL) waste pulp], reduction in kappa number [up to 17.6% soda pulp; up to 14.0% (TL), up to 25.3% (PL) and up to 10.9% (BL), waste pulp], improvement in brightness [up to 20.4% soda pulp; up to 23.6% (TL), up to 8.6% (PL) and up to 5.0% (BL), waste pulp] when compared with the respective controls. The usage of XLMS along with 15% reduced level of hypochlorite at CEHHXLMS/EHHXLMS bleaching stage reduced kappa number [5.5% soda pulp; 11.4% (TL), 7.9% (PL), waste pulp] and improved brightness [1.0% soda pulp; 0.9% (TL), 1.4% (PL) waste pulp] when compared with the controls. Scanning electron microscopic studies revealed development of cracks, flakes, pores and peeling off the fibres in the enzyme-treated pulp samples. These modifications of the fibre surface during enzymatic bleaching in turn indicated the removal of lignin and derived compounds from the fibre cell wall. CONCLUSIONS: The work describes synergistic action of xylanase with LMS for bleaching of waste and nonwood pulps for eco-friendly production of paper and thus reveals a new unexploited arena for enzyme-based pulp bleaching. SIGNIFICANCE AND IMPACT OF THE STUDY: The drastic improvement in pulp properties obtained after xylanase and LMS treatment would improve the competitiveness of enzyme-based, environmentally benign processes over chemicals both economically and environmentally.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Mutagenic properties of spent bleaching liquors from sulphite pulps and a comparison with kraft pulp bleaching liquors

Parameters influencing the mutagenic properties of spent bleaching liquors from sulphite pulps have been studied. In addition a comparison has been made between the properties of spent liquors from sulphite and kraft pulp bleaching. In the sulphite process the cooking base had no influence on the mutagenicity of the chlorination stage. In contrast, removing the extractives before chlorination especially for dissolving pulp resulted in an increase in mutagenic activity. The mutagenicity decreased significantly after substituting 40% of the chlorine with chlorine dioxide. Sequential addition of chlorine and chlorine dioxide resulted in higher activity than simultaneous or premixed chlorination as observed for liquors from kraft pulp. Increasing the pH of the extracts or addition of sulphur dioxide decreased the mutagenicity. Expressed as 10(7) revertants per kappa number and ton pulp the mutagenicity varied between 10 and 40 for sulphite pulp while the corresponding figures for kraft pulp were 100-225.     

Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts

The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.     

Toxic and genotoxic activity of river waters of the Kazakhstan

IntroductionIn this study, physicochemical, genotoxic, and mutagenic properties of water samples from 10 rivers of the Almaty region (Kazakhstan) were evaluated.ResultsThe results of the study demonstrated an increased level  of mineralization and electrical conductivity that might be caused by the high concentration of dissolved mineral salts and ions such as Na⁺, K⁺, Ca^{2 +}, Cl⁻, SO₄²⁻, HCO₃⁻. The excess of Maximum Allowable Concentrations (MACs) for various heavy metals was revealed. The results of tests using the pXen7-lux biosensor showed toxic effects of river waters. At the same time, the studies involved lux biosensors pRecA-lux, pColD-lux, pSoxS-lux, pKatG-lux did not find any genotoxic and oxidative effects. However, toxicity and mutagenicity of the studied water samples was detected by using plant test (Allium cepa and Hordeum vulgare). Phytotoxic, cytotoxic (decrease in the mitotic index) and mutagenic (increase in the frequency of chromosomal aberrations) activity of the water samples was observed. The data of in vivo tests (Danio rerio) showed the high toxicity and teratogenicity of river waters for fish embryos at all stages of development.ConclusionsThe results of this comprehensive study indicate that the contamination of the surface natural waters poses a threat to rivers dwellers and the human population in the rivers areas.     

Antimutagenicity of WR-1065 in L5178Y cells exposed to accelerated (56)Fe ions

The ability of the aminothiol WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] to protect L5178Y (LY) cells against the cytotoxic and mutagenic effects of exposure to accelerated (56)Fe ions (1.08 GeV/nucleon) was determined. It was found that while WR-1065 reduced the mutagenicity in both cell lines when it was present during the irradiation, the addition of WR-1065 after the exposure had no effect on the mutagenicity of the radiation in either cell line. No marked protection against the cytotoxic effects of exposure to (56)Fe ions was provided by WR-1065 when added either during or after irradiation in either cell line. We reported previously that WR-1065 protected the LY-S1 and LY-SR1 cell lines against both the cytotoxicity and mutagenicity of X radiation when present during exposure, but that its protection when administered after exposure was limited to the mutagenic effects in the radiation-hypersensitive cell line, LY-S1. The results indicate that the mechanisms involved differ in the protection against cytotoxic compared to mutagenic effects and in the protection against damage caused by accelerated (56)Fe ions compared to X radiation.     

Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks

A significant proportion of cellular DNA damages induced by ionizing radiation are produced in clusters, also called multiply damaged sites. It has been demonstrated by in vitro studies and in bacteria that clustered damage sites can be converted to lethal double strand breaks by oxidative DNA glycosylases during attempted base excision repair. To determine whether DNA glycosylases could produce double strand breaks at radiation-induced clustered damages in human cells, stably transformed human lymphoblastoid TK6 cells that inducibly overexpress the oxidative DNA glycosylases/AP lyases, hNTH1 and hOGG1, were assessed for their radiation responses, including survival, mutation induction and the enzymatic production of double strand breaks post-irradiation. We found that additional double strand breaks were generated during post-irradiation incubation in uninduced TK6 control cells. Moreover, overproduction of either DNA glycosylase resulted in significantly increased double strand break formation, which correlated with an elevated sensitivity to the cytotoxic and mutagenic effects of ionizing radiation. These data show that attempted repair of radiation damage, presumably at clustered damage sites, by the oxidative DNA glycosylases can lead to the formation of potentially lethal and mutagenic double strand breaks in human cells.     

Lignin from rice straw Kraft pulping: effects on soil aggregation and chemical properties

Lignin contained in pulping liquor that is generated during the pulping process for papermaking is a disposal problem for the pulp and paper industry. Separating lignin and other organic components from pulping liquor with inorganic acids may improve its applicability to fields as a beneficial soil amendment while offering a potential disposal alternative. Sulfuric acid-precipitated lignin from rice straw pulping liquor applied at rates of 1.67 and 3.34 g C kg(-1) soil was incubated to evaluate its effects on soil properties over 8 weeks of incubation. Addition of this acid-precipitated lignin at these rates decreased soil pH by 0.24-0.53 units over 8 weeks of incubation, suggesting that this sulfuric acid-precipitated lignin from pulping liquor may have potential as a soil acidifying agent. Soil electrical conductivity (EC) only increased by up to 0.36 d Sm(-1), but highest EC levels were less than 4 d Sm(-1), indicating that lignin applied at both rates would not cause salinity problems. Application of this lignin increased soil organic C by 1.46 and 3.13 g C kg(-1), and total soil N by 0.07 and 0.17 g N kg(-1) over the incubation period. Lignin improved the macroaggregation of >2mm size fraction, and increased wet microaggregate stability of >2mm and 0.5-0.25 mm aggregates compared to a nonamended control. The results of this study suggest that this acid-precipitated lignin from pulping liquor may have potential as a beneficial soil amendment.     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF=1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF=1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI=2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.