Chemical and biological characterization of hazardous industrial waste. I. Prokaryotic bioassays and chemical analysis of a wood-preserving bottom-sediment waste

Prokaryotic bioassays, capable of detecting point mutations and lethal damage to DNA, and a GC/MS/Data System analysis were employed to evaluate the genotoxic characteristics of wood-preserving bottom sediment. Organic compounds in the waste were initially extracted with dichloromethane and then fractionated by liquid-liquid extraction into acid, base and neutral fractions. The crude extract and each of 3 subfractions were tested in 4 strains of S. typhimurium to detect point mutations and 6 strains of B subtilis to detect lethal damage to DNA. The assay using S. typhimurium responded to indirect-acting mutagens in the crude extract and all 3 primary fractions, with the maximum mutagenic response of 181 net revertants induced by the base fraction at a dose of 500 micrograms/plate. In the DNA-repair assay, the survival ratio for the repair-deficient strain recE4 when compared to the repair-proficient strain 168 wt was 0.17 and 0.09 in the acid and base fractions, respectively, at a dose of 100 micrograms/plate. Potentially genotoxic compounds identified in the waste fractions by GG/MS/DS analysis include acenaphthylene, pentachlorophenol, methyl phenanthrene, fluoranthene and pyrene. However, it appears that these identified chemicals did not contribute significantly to the observed mutagenic activity of the sample extracts.     

Chemical synthesis and bioassay of anordrin and dinordrin I and II.

The chemical synthesis of 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-androstane-2 beta, 17 beta-diol dipropionate (Anordrin) and the corresponding diacetate is reported. Similarly, the preparation of the 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-estrane-2 beta, 17 beta-diol, its diacetate and dipropionate (Dinordrin I), along with the corresponding 2 beta-epimer (Dinordrin II) from 17 beta-hydroxy-A-nor-5 alpha=estran-2-one is described. In rat uterotrophic activity bioassay, the slope of ethynylestradiol differed significantly from the slopes of the other three compounds, thus vitiating potency estimates with this reference compound. Dinordrin I was 20 times more potent than Anordrin and considerably more potent then Dinordrin II. The single-dose oral antifertility effect in rats generally paralleled uterotrophic activity. Immediate postovulatory contraceptive effectiveness was assessed in adult cycling female baboons given two doses daily for 4 days. Both Anordrin and Dinordrin I showed antifertility activity worthy of further study. Moreover, a definite luteolytic effect, with depression of both plasma estrogen and progesterone levels, was observed with these two steroids.     

Life cycle environmental assessment of industrial hazardous waste incineration and landfilling in China

Purpose

The improper handling of industrial hazardous waste (IHW), which comprises large amounts of toxic chemicals, heavy metals, or irradiation substances, is a considerable threat to human health and the environment. This study aims to quantify the life cycle environmental impacts of IHW landfilling and incineration in China, to identify its key factors, to improve its potential effects, and to establish a hazardous waste disposal inventory.

Methods

Life cycle assessment was conducted using the ReCiPe model to estimate the environmental impact of IHW landfilling and incineration. The characterization factors for the human toxicity and freshwater ecotoxicity categories shown in the ReCiPe were updated based on the geographies, population, food intake, and environmental conditions in China.

Results and discussion

The overall environmental burden was mainly attributed to the carcinogen category. The national carcinogen burden in 2014 at 37.8 CTUh was dominated by diesel consumption, cement and sodium hydroxide production, direct emission, transportation, and electricity generation stages caused by direct mercury and arsenic emissions, as well as indirect chromium emission. Although the atmospheric mercury emission directly caused by IHW incineration was comparative with the emission levels of developed countries, the annual direct mercury emission accounted for approximately 0.1% of the national mercury emission.

Conclusions

The key factors contributing to the reduction of the national environmental burden include the increasing diesel and electricity consumption efficiency, the reduction of cement and sodium hydroxide use, the development of air pollutant controlling systems, the reduction of transport distance between IHW disposers to suppliers, and the improvement of IHW recycling and reuse technologies.

     

Clastogenicity evaluation of seven chemicals commonly found at hazardous industrial waste sites

7 chemicals commonly found at the industrial waste sites were tested with the Tradescantia-Micronucleus (Trad-MCN) assay to evaluate their clastogenic potential. Chemicals selected from the US EPA Superfund Priority 1 list were: aldrin, arsenic trioxide, 1,2-benz[a, h]anthracene, dieldrin, heptachlor, lead tetraacetate and tetrachloroethylene. Results of repeated tests for clastogenicity yielded the minimum effective dose (MED) for clastogenicity of 0.44 ppm for lead tetraacetate, 1.88 ppm for heptaclor, 3.81 ppm for dieldrin and arsenic trioxide and 1,2-benz[a, h]anthracene yielded positive responses at the MED of 3.96 ppm and 12.50 ppm respectively. Aldrin and tetrachloroethylene were considered to be immiscible with water, and the tests yielded negative responses. Tetrachloroethylene in gaseous state was also used to treat the flower buds. Results of tetrachloroethylene vapor phase treatment yielded a positive response at the MED of 30 ppm/min after a 2-h exposure. 5 chemicals determined to be clastogens by this test were ranked according to their MED in the descending order of potency as follows: lead tetraacetate, heptachlor, dieldrin, arsenic trioxide and 1,2-benz[a, h]anthracene. Results of this study indicate that the Trad-MCN bioassay could be effectively utilized for assessing the potential clastogenicity of the chemicals commonly found at the industrial hazardous waste sites.     

Chemical and spectroscopic analysis of olive mill waste water during a biological treatment

The treatment of olive mill waste water was studied on the laboratory scale. Physico-chemical analyses showed the final products had a mean pH of 5.4 without neutralisation and 5.7 when lime was added to the process. Raising the pH by adding lime had a positive outcome on the degradation of phenols, whose levels were reduced by over 76%. The lime also changed the structure of the organic matter, as seen in the infra-red spectra. Combining the FT-IR and 13C NMR data showed that with addition of lime, the density of aliphatic groups decreased to the benefit of aromatic groups, indicating that polymerisation of the organic matter occurred during the bioprocess. Under our experimental conditions, the biotransformation of olive mill waste water appears to favour the stabilisation of the organic matter through mechanisms analogous to those that lead to the formation of humus in the soil.     

Chemical characterization and biological properties of airborne particulate matter

Summary The presence of heavy metals and Polynuclear Aromatic Hydrocarbons (PAH) has been evidenced in airborne particulate matters samples collected in the atmosphere of a non-industrialized middle size Italian town (150.000 inhabitants). The PAH concentrations, higher in winter than in other seasons, was inversely correlated with some meteorological variables such as temperature (p<0.01) and wind speed (p<0.05). The positive correlation found (p<0.01) between lead and total PAH seems to indicate traffic as the most important source of pollution.All samples were mutagenic in the Ames test and the highest mutagenicity levels were obtained in winter samples. The positive correlation found between Benzo(a)Pyrene {B(a)P} concentration and the number of revertants/m³ air, suggests that B(a)P could be an index of mutagenic potential of the matrix considered.The airborne particulate matter extracts obtained by organic solvents were able to inhibit the phagocytosis of mice alveolar macrophages, giving further proof of the possibility of interaction between air-particulate chemical compounds and important biological functions.     

Chemical and biological characterization of a polysaccharide biological response modifier from Aloe vera L. var. chinensis (Haw.) Berg

Three purified polysaccharide fractions designated as PAC-I, PAC-II, and PAC-III were prepared from Aloe vera L. var. chinensis (Haw.) Berg. by membrane fractionation and gel filtration HPLC. The polysaccharide fractions had molecular weights of 10,000 kDa, 1300 kDa, and 470 kDa, respectively. The major sugar residue in the polysaccharide fractions is mannose, which was found to be 91.5% in PAC-I, 87.9% in PAC-II, and 53.7% in PAC-III. The protein contents in the polysaccharide fractions was undetectable. NMR study of PAC-I and PAC-II demonstrated the polysaccharides shared the same structure. The main skeletons of PAC-I and PAC-II are beta-(1-->4)-D linked mannose with acetylation at C-6 of manopyranosyl. The polysaccharide fractions stimulated peritoneal macrophages, splenic T and B cell proliferation, and activated these cells to secrete TNF-alpha, IL-1 beta, INF-gamma, IL-2, and IL-6. The polysaccharides were nontoxic and exhibited potent indirect antitumor response in murine model. PAC-I, which had the highest mannose content and molecular weight, was found to be the most potent biological response modifier of the three fractions. Our results suggested that the potency of aloe polysaccharide fraction increases as mannose content and molecular weight of the polysaccharide fraction increase.     

Cas spleen focus-forming virus. II. Further biological and biochemical characterization.

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.     

Extraction of Clostridium perfringens spores from bottom sediment samples.

Two extraction-separation procedures were developed and evaluated for use in conjunction with the mCP membrane filter method for the enumeration of Clostridium perfringens spores in bottom sediments. In the more facile of the two procedures, a distilled-water suspension of the sediment sample is pulse sonicated for 10 s and allowed to settle. Portions of the supernatant are then removed for membrane filtration. This procedure is recommended for general use. The more complicated procedure is recommended for situations in which the presence of high levels of toxic materials is suspected or in which relatively low spore densities are present in fine silts. In this procedure, sonication is followed by a distilled water wash. The centrifuged sediment is resuspended in distilled water and mixed with the components of a two-phase separation system (50% polyethylene glycol in distilled water and 25% sucrose in 3 M phosphate buffer [pH 7.1]). After equilibration of the system and low-speed centrifugation, the top phase and interphase are removed, mixed, and membrane filtered. The recoveries of C. perfringens spores by the two procedures, when used in conjunction with the mCP method, were comparable to each other and significantly greater than those by the British most-probable-number method. It was estimated that more than 85% of the spores were recovered by the procedures. The precision of the sonicate-and-settle-mCP procedure was markedly better than that obtained theoretically by the most-probable-number method and approached that theoretically attributable to counting an average of 85 colonies on each of two plates.     

Chemical and biological characterization of low-molecular-weight human skeletal growth factor

Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2-3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor binding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70 degrees C), pH (3-10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.     

Chemical and biological characterization of sclerosin, an antifungal lipopeptide

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH(3)-(CH(2))(6)-CH(OH)-CH(2)-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S.?sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C.?elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C.?elegans growing on Escherichia coli OP50.     

Development of a sediment bioassay to determine bioavailability of PAHs to fish

Recent work has shown that MFO induction and loss of control of steroid hormone production occurs in fish after exposure to pulp mill effluents, PCBs, PAHs, and some pesticides. We had recently developed laboratory assays to evaluate the effluents on these responses, but were lacking a protocol for a sediment assay. This paper describes the development of a sediment test capable of demonstrating MFO induction in fish. MFO responses were evident in rainbow trout within 4 days of exposure to contaminated sediments. Further testing showed that fish were responding to chemicals from the sediments, but not from bottom water, and a survey of sediment from thirteen contaminated areas showed that MFO induction more closely paralleled PAH levels in the sediments than the observed PCB concentrations. The sites showing MFO induction were also the sites where sediment toxicity was demonstrated with laboratory bioassays using Daphnia magna and Hyalella azteca. The protocol has been further refined to describe the quantity of sediment required and duration of testing. This test will enable us to study the biochemical effects of exposure to contaminated sediments. The protocol could also be used to prioritize areas of contamination and to evaluate dredging impacts and remediation success.     

Eukaryotic topoisomerase II. Characterization of enzyme turnover

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.     

Chemical synthesis and characterization of the sweet protein mabinlin II

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste. © 1998 John Wiley & Sons, Inc. Biopoly 46: 215–223, 1998