Experimental field exposure of brown trout to river water receiving effluent from an integrated newsprint mill.

Effects of effluents from mechanical pulp production on brown trout were studied for 8 weeks at environmentally relevant concentrations. The exposure took place in laboratory-based pools upstream and downstream of the effluent discharge point of an integrated newsprint mill using ground wood/thermomechanical pulp. The mill had no secondary treatment of the wastewater. The pools were supplied with water pumped directly from the river. To determine the relevance of this approach, wild fish were also caught at the respective sites upstream and downstream from the mill. Sublethal effects were assessed using physiological and biochemical parameters including liver histology, hematology, serum biochemistry, and hepatic enzyme assays. Exposure was verified by analyzing water samples, fish bile, and tissues for resin acid concentrations. The downstream experimental fish and captured feral fish displayed responses and changes in physiological parameters similar to those previously observed in laboratory experiments with untreated effluents. The most obvious effects were liver damage and growth inhibition.

Safety testing of Bacillus thuringiensis preparations, including thuringiensin, using the Salmonella assay.

The aim of this study was to test genotoxicity aspects of the safety of two strains of Bacillus thuringiensis. The strains were of serotype H-1, producing thuringiensin, toxic to flies, and serotype H-14, producing endotoxin, toxic to mosquitoes, but not thuringiensin. Four preparations were tested: Tenfold concentrated cell-free culture media of serotypes H-1 and H-14, prepurified thuringiensin, and purified endotoxin. No increases in revertant colony numbers of the tester strains of Salmonella typhimurium TA98 or TA100 were observed at the dose levels used with or without metabolic activation. Infrequent slight increases in revertant colony numbers in strains TA98 and TA1538 at a very high test dose of 50 mg/plate, both in the presence and absence of thuringiensin, were probably caused by histidine present in the growth medium of B. thuringiensis. Furthermore, the effects were at most slightly dose related and not reproducible, and therefore the preparations can be considered nonmutagenic.

Algae in mosquito breeding sites and the effectiveness of the mosquito larvicide Bacillus thuringiensis H-14.

The presence of cyanobacteria generally decreased the effectiveness of Bacillus thuringiensis H-14 (BTI) as a mosquito larvicide. The effect was more pronounced when the mosquito larvae were exposed to BTI in the presence of several cyanobacterial strains. No synergistic or antagonistic effect between the δ-endotoxin from BTI and the hepatotoxin from cyanobacteria was seen. Neurotoxic cyanobacterial strains caused very fast paralysis in mosquito larvae; the decreases in the effectiveness of BTI when tested in combination with a neurotoxic strain might be due to the effect of this paralytic action on the feeding rate of the mosquito larvae.

Studies on Bacillus thuringiensis H-14 strains isolated in egypt-IV. characterization of fermentation conditions for δ-endotoxin production.

Fermentation media formulated from inexpensive locally available ingredients, namely soya meal, Proflo (a partially defatted cooked cottonseed fiour) and molasses, were assessed for growth, sporulation and δ-endotoxin production, by a mosquito-toxic strain of Bacillus thuringlensis H-14 locally isolated in Egypt, using an 8 I working volume fermenter. The insecticidal activity was assayed against Culex piplens. Good growth and sporulation were obtained with all the tested medium combinations, but a medium composed of 3% (w/v) soya meal and 1% (w/v) molasses was the best for δ-endotoxin production. The optimal batch cultivation conditions with respect to aeration (0.37 to 0.62 vol/vol min(-1)), agitation (200 rev/min), pH (6.5 to 7.5), temperature (30°C) and initial concentration of carbon (1% w/v) and nitrogen (3% w/v) sources are also presented. This investigation shows that these locally available ingredients could be used for the production of low-priced mosquito larvicide in Egypt and other developing countries where these ingredients are avallable.

Studies on Bacillus thuringiensis H-14 strains isolated in Egypt-V. Composition and toxicity of the mosquitocidal parasporal inclusions.

Parasporal inclusions of Bacillus thuringlensis H-14 strains M1 and S128 were characterized by solubilization, electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis and insecticidal activity. Inclusions of both strains are composed largely of protein with 8 to 9% carbohydrate. Amino acid analysis of the purlfied inclusions revealed that the two strains produce inclusions that are closely related to each other but significantly different from lepidopteran-toxic B. thuringiensis parasporal crystals. The LC50 values of the purlfied inclusions of strains M1 and S128 were 3.4 and 2.9 ng/ml, respectively, for fourth instar larvae of Aedes aegypti. Inclusions from strain M1 were resolved into two inclusion bands on the basis of their densities possibly formed as a result of disruption of some envelopes during sonication. Both inclusion types contained proteins of approximately 27, 38 and 66 kDa. The heavlest and more predominant type had an envelope and was either spherical or irregular being composed of several subunits which varied in shape, size and staining densities. The LC50 value was 2.2 ng/ml and the major protein was of approximately 27 kDa. The lightest inclusions type did not have an envelope and showed clear crystal lattices. They were 10 times less toxic to A. aegypti larvae, as compared to the heavy-type inclusions and contained major protein of approximately 66 kDa.

Studies onBacillus thuringiensis H-14 strains isolated in Egypt-I. Screening for active strains.

Screening for spore-forming bacteria to use against dipterous insects, namelyMusca domestica, Aedes aegypti andCulex pipiens from Egypt, led to two isolates, M1 and S128, being found which were highly toxic to bothAedes andCulex strains. They had calculated international Toxicity Units (ITU) valdes of 17,467 and 20,065 ITU/mg, respectively, as compared with 15,000 ITU/mg forBacillus thuringlensis H-14 IPS-82 Reference Standard. They were taxonomically classilled asBacllus thuringlensis H-14. Their sensitivity to a number ofBacillus phages is also described.

Studies onBacillus thuringiensis H-14 strains isolated in Egypt-III. Selection of media for δ-endotoxin production.

Several medium ingredients locally available in Egypt were investigated for their ability to support δ-endotoxin production by two mosquito-toxic strains ofBacillus thuringiensis H-14 usingCulex pipiens for the bioassay of δ-endotoxin. Soya beans, black-eyed beans, common peas and lentils supported good production of toxin whereas peanuts, fodder yeast, cheese whey and corn steep liquor gave only low amounts of toxin. Molasses as the sole carbon source at 2 and 3% (w/v) with soya (or Proflo) as the sole nitrogen source at 3% gave the best yields of toxin for strain M1 and S128 respectively.

Studies onBacillus thuringiensis H-14 strains isolated in Egypt-II. Ultrastructure studies.

Electron microscopy of thin sections of two mosqulto-toxic isolates ofBacillus thuringiensis H-14 showed inlcusions that varied in shape, size and stainability. The shape of some of these inclusions have not been reported before. Uitrastructural analysis of sporulation and parasporal crystal development shows that the insecticidal inclusions are initiated at the stage of axial filament formation during sporulation. In most of the inclusions there is a common subunit, formed first, which has a regular geometrical shape (rhomboid, hexagonal rectangular, trapezoid or pentagonal). Later added sub-units are more amorphous and this leads to a variety of inclusion shapes which appear at the end of sporulation. Some of these reguiar moleties develop in different ways, leading to cubical, rhomboid or parallelogramic-shaped crystais. In a freeze-tractured cell, the internal fracture faces of a crystal are seen. The size of the sub-units of this crystal was about 5×5 nm. For French summary, see next page.

Ecotoxicological characterization of industrial wastewater--sulfite pulp mill with bleaching.

Different process wastewaters from a sulfite pulp mill with bleaching were characterized by chemical analysis and toxicity tests. The amount of adsorbable organically bound halogen (AOX) from the bleachery was 3.6 kg per ton pulp. The extractable organically bound chlorine was 15% of AOX. Some identified organochlorine compounds in the effluent could be traced in the receiving water. Effluents from the chlorination and alkaline extraction stages and the condensate were the main contributors to the effluent toxicity. The effluents were particularly toxic to the alga Skeletonema costatum. The EC50 value for growth of the alga was 24-29 ml/liter of the total effluent. The toxicity cannot be ascribed to single chemical components in the effluents. Degradation of toxic components occurs after dilution of the effluents in the receiving water. The toxicity may be reduced to 30-50% of the initial toxicity within 1 week. Predictions of toxic effects in the receiving water, based on results of toxicity tests and estimated dilution, indicate that large areas are affected by the discharges. Som observations of the distribution of organisms in the receiving water indicate that predictions from the toxicity tests may be valid.

Impact of bleached kraft mill effluents on drinking water quality.

Bleaching effluents from kraft pulp mills contain large quantities of chlorinated organic compounds, some of which are mutagenic. Mutagenic chloro-organic compounds are also formed as by-products in chlorination of drinking water. The work reported here was a combined field and laboratory study aimed at comparing the evidence of health risks from pulp mill contaminants in drinking water, with corresponding evidence from normal, chlorine disinfection by-products. The study was performed in a Swedish river basin with a large, public water works located downstream from a bleached kraft mill. Chemical analyses (gas chromatography and determination of adsorbable organic halogen) and bioassays for mutagenic activity (bacterial and mammalian cell bioassays) were performed on samples of river water, drinking water and laboratory produced drinking water. The study showed that the bleached kraft effluents caused a considerable, long-range transport of chloro-organic substances in the receiving waters, and a substantial increase in the total amount of organic chlorine in drinking water produced downstream from the mill. As regards Ames mutagenic compounds, however, chlorination of naturally occurring humic substances during the chlorine disinfection step in drinking water production, proved to be a far more important source. The contribution of volatile chloro-organic compounds from the kraft mill was also much smaller than the contribution from normal disinfection by-products. The chromosome aberration tests gave no clear evidence of a positive response for any of the samples tested.

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.

Analysis of chlorinated styrenes in environmental samples using negative ion chemical ionization mass spectrometry.

A method for the separation and identification of chlorinated styrene isomers with fully chlorinated aromatic nuclei in environmental samples is presented. Gas chromatography combined with negative ion chemical ionization mass spectrometry is used. The method has been applied to a combined fish liver and filét extract. All the possible chlorostyrenes with fully chlorinated aromatic nuclei were detected in the sample.

Identification of chlorinated compounds in the spent chlorination liquor from differently treated sulphite pulps with special emphasis on mutagenic compounds.

About 80 compounds present in the spent chlorination liquor from the bleaching of sulphite pulp have been identified. A comparison has been made between these compounds and those identified in kraft bleaching effluent, with special emphasis on mutagens. The influence of oxygen delignification, before chlorination and alkali treatment of the spent bleach liquor, on the composition of the liquors has also been investigated.

A comparison of methods for concentrating mutagens in drinking water--recovery aspects and their implications for the chemical character of major unidentified mutagens.

A comparison of techniques for concentrating mutagenic compounds in drinking water has shown that XAD-2 adsorption and dichloromethane extraction have acceptable and almost identical enrichment properties, while purging at an elevated temperature is inappropriate in this context. Quantitatively, the most important drinking water mutagens could only be adsorbed (extracted) after acidification of the water, and even then recovery was far from complete. Recovery experiments with known mutagens from pulp mill effluents have shown that none of the major chlorination-stage mutagens identified thus far can explain the mutagenic activity of extracts from neutral or acidified chlorinated drinking water.

Analysis of volatile halogenated organic compounds in fish.

A method for the analysis of volatile halogenated hydrocarbons in biological samples has been developed. Extraction efficiencies for various C1 and C2 halocarbons were found to be in the range of 63--82%, and recoveries were fairly good. The detection limits lie in the sub-ppb range (0.1--1 ppb). The method has been used to analyse fish samples from both unpolluted and polluted areas in Norway. The compounds most frequently found were chloroform, trichloroethylene and tetrachloroethylene in concentrations ranging from 10--1000 ppb. From one area, corresponding water and fish samples were analysed which made it possible to estimate accumulation factors for chloroform and trichloroethylene, which were found to be 200--500 and 1400 respectively.

Determination of halogenated organic compounds and mutagenicity testing of spent bleach liquors.

The content of organohalogenated compounds in spent bleach liquor from different bleaching stages in a sulphate and a sulphite plant has been determined by a combination of glass capillary gas chromatography, gas-liquid chromatography/mass spectrometry and neutron activation analysis. Several compounds which have not been reported before have been identified including halogenated derivatives of dimethyl-propylnaphthalenes and alkylated catechols. The unconcentrated effluents, non-polar and total extracts were tested for mutagenic activity with Ames' Salmonella test. Spent bleach liquors from most bleaching stages as well as the total effluents contained mutagenic compounds. Addition of liver microsomes for metabolic activation reduced the mutagenic activity in all stages except for the first chlorination stage in the sulphate plant. Two isomers of chloro-, bromo-, and dichloro-p-cymene previously determined in effluents from bleaching plants were synthesized from the parent molecule. Both bromo- and dichloro-p-cymene exhibited weak mutagenic activity in the Salmonella test system. Liver microsomes reduced the effect slightly. The chlorinated cymenes were found to account for up to 18% of the total organically-bound chlorine in the non-polar extracts.