The relation between extrapolated risk, expressed as potentially affected fraction, and community effects, expressed as pollution-induced community tolerance.

The results of toxicity tests can be used to calculate the potentially affected fraction (PAF) of species in an ecosystem at a given pollutant concentration using statistical extrapolation methods. The PAF curve indicates the fraction of species from the original community that may become inhibited at each elevated pollutant concentration and is a measure of the ecotoxicological risk. Pollution-induced community tolerance (PICT) is a true community response that is measured under controlled conditions in the laboratory, using organisms from contaminated field sites. Microorganisms from experimental field plots with added Zn were exposed to various concentrations of Zn in the laboratory and the mineralization of 14C acetate was monitored. Microorganisms from plots with Zn concentrations above 124 mg/kg showed a significant increase in the effect concentration 10% (EC10) and, therefore, had a significant PICT. The pore-water concentrations of Zn in these field soils were in the same magnitude as the EC10 of the microorganisms from these soils. The PAF curve was calculated from previously reported toxicity tests with five different microbial species using the average and the standard deviation of the logarithmically transformed EC10 values. The average sensitivity of this PAF curve was similar to the EC50 of the acetate mineralization curve from the field plot without added Zn2+, but the PAF curve was less steep. Our experiments indicated that 27 to 84% of the original microbial species were inhibited at Zn concentrations from 334 to 1,858 mg/kg soil, respectively. Our results suggest that the PICT method can now also be used to quantify the fraction of the original species composition that is inhibited at a specific pollutant concentration.

A comparison between toxicity tests using single species and a microbial process.

In this study the sensitivity of the acetate mineralization process performed by five strains of microorganisms in soil for the toxicants Zn2+ or PCP was calculated from the sensitivity of the contributing species. The species used were a fungus (Aspergillus niger CBS 121.49), an actinomycete (Streptomyces lividans 66), two Gram-negative Pseudomonas putida strains (MT-2 and DSM 50026) and a Gram-positive strain Rhodococcus erythropolis A177. For zinc the EC10 of the process performed by the five strains together was 77 mg/kg whereas for pentachlorophenol it was 2 mg/kg. The EC10 of the process was compared with the EC50 of the most sensitive species contributing to the process. P. putida MT2 was the most zinc sensitive strain (EC50 = 22 mg Zn/kg) and A. niger was the most sensitive strain for pentachlorophenol (EC50 = 1.4 mg/kg). This shows that a 10% inhibition of a process can be accompanied by a more than 50% inhibition of the most sensitive species.

Development of zinc bioavailability and toxicity for the springtail Folsomia candida in an experimentally contaminated field plot.

The influence of outdoor exposure conditions and ageing of contamination on the toxicity of zinc was investigated for the springtail Folsomia candida to evaluate the validity of a standardised soil toxicity test. In three successive years, animals were incubated in an experimentally contaminated field plot. During the first months after construction of the test field, total zine concentrations of the soil decreased rapidly due to leaching of excess zinc with rainwater, while increased sorption of the remaining residues resulted in a reduced bioavailability of the metal. Although variation between replicates was substantial, the EC50s for the effect of zinc on reproduction of F. candida determined in the field experiments differed by less than a factor of two from effect concentrations obtained in laboratory tests in which the same soil was used. Expression of the EC50s on the basis of water soluble zinc allowed for a comparison with effect concentrations estimated for other soil types. EC50s were comparable with literature data, which indicates that bioavailability of zinc is the main factor determining toxicity for F. candida. It is concluded that laboratory based toxicity data are suitable to predict effects of zinc for F. candida under outdoor conditions, provided that the bioavailability of zinc is determined accurately using water soluble concentrations.

Toxic effects of pollutants on the mineralization of chloroform in river sediments.

The influence of pollutants on the formation of 14CO2 from 3 micrograms/liter labeled chloroform was studied in anaerobic Dutch river sediments. All incubations were performed under anaerobic conditions. Addition of toxicants to sediment microcosms showed logistic dose-effect curves. The concentration giving 10% inhibition of the chloroform mineralization rate (IC10) was derived from these dose-effect curves. The IC10 values of added cadmium, chloropyrifos, benzene, mercury, or 1,2-dichloroethane were 1300, 1300, 140, 90, and 0.07 mg/kg dry sediment, respectively. Mud samples taken at different dates from the same site indicated a significantly different sensitivity to added pentachlorophenol and zinc. The IC10 of added pentachlorophenol was 150 mg/kg in one and 15 mg/kg in another sample. Chloroform-mineralizing bacteria are very sensitive to addition of zinc. The IC10 of added zinc was 700 mg/kg for one sample and 11 mg/kg for another sample of the sediment which contained a background concentration of 800 mg Zn/kg. Therefore, a partial inhibition of the mineralization of chloroform by the high concentrations of zinc present in Dutch river sediments cannot be excluded. The high concentration of zinc might cause persistence of otherwise biodegradable pollutants in Dutch sediments.

Toxic effects of pentachlorophenol and other pollutants on the mineralization of acetate in several soils.

The mineralization of [14C]acetate was studied in bottles with fresh soil and groundwater. Addition of toxicants inhibited the formation of 14CO2 and dose-effect curves were obtained. The acetate mineralization was not inhibited by zinc, cadmium, K2Cr2O7, chloropyrifos, and paraquat in an acid sandy soil at 1000 mg/kg dry soil. The IC10 is the toxicant concentration which inhibits 10% of the initial mineralization rate. The IC10 concentrations for 3,4-dichloroaniline, triphenyltin, and orthoxylene were 48, 96, and 730 mg/kg, respectively, in the acid sandy soil. The IC10 of pentachlorophenol was measured in samples from the acid sandy soil and in several other soil and subsoil samples. The geometrical mean of the 13 IC10 values was 16 mg pentachlorophenol/kg. A statistical method was used to calculate the PCP concentration above which 5% of the most sensitive acetate-mineralizing communities in all soils are influenced. The best estimate of this concentration is 0.3 mg PCP/kg but to be on the safe side the 95% confidence level of this concentration is 25 micrograms/kg.

Effect of ambient oxygen concentration upon the acute toxicity of chlorophenols and heavy metals to the groundwater copepod Parastenocaris germanica (Crustacea).

Acute static toxicity experiments have been performed under normoxic and hypoxic conditions. The test animals used were adults of the groundwater-adapted copepod Parastenocaris germanica. The animals originated from a sandy, gravelly phreatic aquifer of the Meuse valley in The Netherlands. Toxicants applied were pentachlorophenol, 3,4-dichlorophenol, zinc, and cadmium. The results were statistically evaluated by a log-logistic model. LC50 and LC5 values with 95% confidence limits were calculated. Response models of application under both oxygen conditions were compared. The influence of ambient oxygen concentration on sensitivity appeared to be very small; only in the case of pentachlorophenol were hypoxic and normoxic models slightly but significantly different. Comparisons with the sensitivity of other organisms are made and the results are discussed against the specific physiological adaptations of the organisms to groundwater conditions.

Derivatives of methanopterin, a coenzyme involved in methanogenesis.

Degradational studies of methanopterin, a coenzyme involved in methanogenesis, are reported. The results of these studies are in full accordance with the proposed structure of methanopterin as N-[1'-(2''-amino-4''-hydroxy-7'' -methyl-6''-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'-1'' )O-alpha-ribofuranosyl-5''-phosphoric acid] aniline in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Acid hydrolysis of methanopterin cleaved the 5'----1'' glycosidic bond and yielded a 'hydrolytic product' which was identified as N-[1'-(2''-amino-4''-hydroxy-7'' -methyl-6''-pteridinyl)ethyl]-4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl]aniline. Alkaline permanganate oxidation of methanopterin yielded 7-methylpterin-6-carboxylic acid. Catalytic (or enzymatic) hydrogenation of methanopterin gave a mixture of 6-ethyl-7-methyl-7,8-dihydropterin, 6-ethyl-7-methylpterin and a third compound, named methaniline which was identified as 4-[2', 3', 4', 5'-tetrahydroxypent-1'-yl(5'----1'')O-alpha -ribofuranosyl-5''-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. Methanosarcina barkeri contains a closely related coenzyme called sarcinapterin, which was identified as a L-glutamyl derivative of methanopterin, where the glutamate moiety is attached to the alpha-carboxylic acid group of the alpha-hydroxyglutaric acid moiety of methanopterin via an amide linkage.

Elucidation of the structure of methanopterin, a coenzyme from Methanobacterium thermoautotrophicum, using two-dimensional nuclear-magnetic-resonance techniques.

Methanopterin is a coenzyme involved in methanogenesis. From 2 kg wet cells of Methanobacterium thermoautotrophicum about 35 mumol methanopterin were isolated. The structure of this compound was elucidated by various two-dimensional nuclear-magnetic-resonance techniques. Methanopterin was identified as N-[1'-(2"-amino-4"-hydroxy-7" - methyl-6"- pteridinyl) ethyl]-4-[2',3',4',5'- tetrahydroxypent-1'- yl (5' leads to 1") O-alpha-ribofuranosyl-5"-phosphoric acid] aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. The molecular formula of the sodium salt of methanopterin at pH 7.0 is C30H38O16N6PNa3 X chiH2O (chi is about 4). The anhydrous sodium salt of methanopterin has a molecular mass of 838.60 Da and the molar absorption coefficient at 342 nm is 7.4 mM-1 cm-1 at pH 7.0.

Quantification of coenzymes and related compounds from methanogenic bacteria by high-performance liquid chromatography.

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80 degrees C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF430, the prosthetic group of methylcoenzyme M reductase, F560, an oxidation product of this compound, coenzyme F420, F342, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF430, coenzyme F420, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.

Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

The isolation of a recombination deficient (Rec-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (CbR) was introduced in these strains and the rescue of the CbR marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.