Underlying issues including approaches and information needs in risk assessment.

Risk assessment requires a delicate consideration of the factors modifying exposure and effects. In this contribution a review is given of the qualitative and quantitative information needs that are essential for a proper risk assessment. The focus is on the details of metal exposure and exposure assessment, following the themes of environmental, physicochemical, and biological components that define exposure. Apart from a description of the principle processes and pathways, exposure assessment is placed in the context of risk assessment and its use in policy and regulatory decision making.

Underlying issues in bioaccessibility and bioavailability: experimental methods.

This article presents experimental designs focusing on assessing of the bioavailability of metals in aquatic organisms, soil organisms, microorganisms, plants, birds, and mammals. Standardized test systems receive the greatest emphasis. With regard to microorganisms, animals, and plants, the study concentrates on toxicity as an indicator for bioavailability. In respective test procedures, results are usually calculated for total chemical concentrations; chemical analyses are not commonly in routine assessments. For soil organisms chiefly exposed by the water pathway, the bioavailable fraction of contaminants can be roughly determined by chemical analyses in aqueous soil extracts simulating soil pore water concentrations. Human toxicity can be determined using adequate in vitro test designs. In addition to experimental designs, results from the literature dealing with specific problems of bioavailability are presented.

Certification of the European reference soil set (IRMM-443--EUROSOILS). Part I. Adsorption coefficients for atrazine, 2,4-D and lindane.

The European Commission has characterised and certified a set of six European soils (the EUROSOILS) under the number IRMM-443. After a successful validation and trial period with a preliminary batch it was decided to produce a new batch of certified reference materials. Part I of this paper describes the certification of adsorption coefficients for atrazine, 2,4-D and lindane in these soils. The adsorption coefficients were determined according to OECD Test Guideline 106. Additionally, the underlying principles for the value assignment process according to the GUM and their practical application to the numerical data obtained during the certification exercise according to ISO Guide 34 and 35 are discussed.

Certification of the European Reference Soil Set (IRMM-443--EUROSOILS). Part II. Soil-pH in suspensions of water and CaCl2.

IRMM-443 re-groups a set of six European Reference Soils (EUROSOILS), which had been certified for their adsorption coefficients for atrazine, 2,4-D and lindane (Certification of the European Reference Soil Set (IRMM-443-EUROSOILS)-Part I. Adsorption coefficients for atrazine, 2,4-D and lindane. Sci Total Environ, in press). The certification of these parameters was complemented by an additional certification of pH in suspension as well by the determination of indicative values for total nitrogen, organic and total carbon content. While Part I explained the principles of the value assignment process and discussed their application to the adsorption coefficients, Part II presents the certified values for pH as well as the indicative values for N(tot), C(tot) and C(org). In addition, the assessment of uncertainty components for stability and homogeneity, which have been included in the final uncertainty budget, is discussed.

Assessment of the environmental behaviour of antioxidants in folios. Comparative risk analysis for the use of folios in agriculture.

The objective of the reported study has been to assess and evaluate as comprehensively as possible the environmental impact of Octadecyl 3(3,5-di-tert-butyl-4-hydroxyphenyl) propionate (CAS no: 2082-79-3, Irganox 1076, Ciba Specialty Chemicals Inc., Additives), which is used as an antioxidant. The potential impact on the compartments soil, groundwater and surface water is to be considered. For comparative purposes, additionally, other chemical compounds being currently under environmental discussion are also taken into account. These comprise pesticides and phthalates which are ubiquitously distributed plasticizers as well as a complexing agent. Since the data basis for each of the compounds under consideration is different, a tiered approach comprising various methodologies of impact assessment has been chosen to achieve the best possible comprehensiveness. The tiers are: 1. Tier: hazard assessment using a scoring system 2. Tier: comparative risk assessment. When interpreting the results of each method, system boundaries as well as underlying assumptions were taken into consideration. Both methodologies showed, that-as compared to the reference substances-there is no relevant environmental and toxicological concern due to low environmental and human hazard from Octadecyl 3(3,5-di-tert-butyl-4-hydroxyphenyl) propionate.

Behaviour and ecotoxicology of aluminium in soil and water--review of the scientific literature.

The scientific literature on chemical and biological behaviour of aluminium products and aluminium compounds following exposure to environmental media is considered. Although many studies have been performed on corrosion of aluminium products, no precise information exists about the amount of aluminium released by dissolution of the protective layer of aluminium products. Since no data are available on the effect caused by introduction of anthropogenic aluminium into the environment a comparison between aluminium released as a result of human activity and geogenic aluminium is only possible by estimating the extent to which anthropogenic aluminium is released using purely theoretical premises. The important results in the literature about the availability and toxicological effects of metallic aluminium and aluminium compounds are presented. Finally, a general assessment of the environmental compatibility of aluminium products is given.

Differentiation of Al3+ and Al species in environmental samples by isotachophoresis.

An isotachophoretic method for the determination of free [Al(H2O)6]3+ ions in different aluminium salt solutions was developed. The electrolyte system consists of 0.01 M sodium acetate (leading system) and 0.01 M tris(hydroxymethyl)amino-methane (terminating system). Separation was effected with a precapillary tube (diameter 0.05 cm) followed by a main capillary tube of length 20 cm and of smaller cross-section. The detection limit for [Al(H2O)6]3+ ions was 0.05 mg/l. The method was applied to the determination of free Al3+ ions ([Al(H2O)6]3+) in soil leachates and aqueous soil extracts.

Effects of NiO inhalation on alveolar macrophages and the humoral immune systems of rats.

To study the effects of inhaled NiO particles on alveolar macrophages and the humoral immune system, male Wistar rats were continuously exposed to NiO aerosols for 4 weeks and 4 months, respectively. Fractions of alveolar macrophages, granulocytes, and lymphocytes in lung lavages, number of polynucleated macrophages, size of macrophages, and phagocytic activity were determined. To test the effects on the humoral immune response sheep erythrocytes were injected, and the antiserum titer in blood and the portion of antibody forming cells in spleen were measured. Significant alterations were found in both systems at 100 and 200 micrograms Ni/m3 after 4 weeks of exposure and at 25 and 150 micrograms Ni/m3 after 4 months of exposure, respectively. The results demonstrate the usefulness of investigations on the humoral immune system and alveolar macrophages as sensitive parameters in detecting effects of NiO inhalation.

Effects of NiCl2 and NiO in Wistar rats after oral uptake and inhalation exposure respectively.

The effects of NiCl2 and NiO after oral uptake and after inhalation exposure respectively were investigated in three experiments, using clinical and clinico-chemical methods. I. Oral application of NiCl2 in male rats over a period of 28 days. The NiCl2 concentrations were 2.5; 5.0 and 10.0 microgram/ml in drinking water. II. Inhalation exposure of male rats with NiO-aerosols (0.2; 0.4 and 0.8 mg/m3) over a period of 28 days. III. Inhalation exposure of non-pregnant and pregnant rats with NiO-aerosols (0.8; 1.6 and 3.2 mg/m3) over a period of 21 days. After oral application of NiCl2 and inhalation exposure of NiO in male rats a significant dose-dependent hyperglycaemia occurred. In contrast of these findings the serum glucose content in non-pregnant rats exposed to Ni-concentrations of 0.8 and 1.6 mg/m3 were content in non-pregnant rats exposed to Ni-concentrations of 0.8 and 1.6 mg/m3 were significantly reduced. After NiO inhalation (1.6 and 3.2 mg/m3), exposure signs of a marked macrocytosis occurred in pregnant and non-pregnant rats. The oral application of NiCl2 in drinking water in male rats induced a significant decrease of urea in serum and a significant increase of urea in urine. The activity of alkaline phosphatase in serum was inhibited in male rats exposed to 0.4 and 0.8 mg/m3 NiO-aerosols. No significant difference in serum protein pattern and no 'nickeloplasmin' was detected by serumelectrophoresis and tandem-crossed immunoelectrophoresis after oral inhalation exposure in male rats. Fetuses of exposed dams showed in the groups receiving 1.6 and 3.2 mg/m3 significantly reduced body weights.

The reaction of aminoacylase with chloromethylketone analogs of amino acids.

1. Aminoacylase is irreversibly inactivated by the chloromethylketone analogs of benzyloxy-carobonyl-L-alanine, L-alanine, L-leucine, L-aspartic acid (beta), tosyl-L-phenylalanine and L-leucyl-L-alanine. The kinetics of the inactivation of the enzyme by the halo-methylketones were investigated. 2. Leucyl-and alanyl chloromethylketone inactivate the enzyme by blocking of 4 SH groups. Experiments with [U-14C]leucyl chloromethylketone confirm that maximal 4 residues are covalently bound to be protein. 3. Inactivation of the enzyme by benzyloxycarbonylalanyl and tosylphenylalanyl chloromethylketone is the result of the substitution of the epsilon-amino group of one lysine resine residue per active site and not of SH groups. However, in the presence of competitive inhibitors these halomethylketones react only with the SH groups of the enzyme, too.

Renal aminoacylase, a zinc enzyme.

Renal aminoacylase is inactivated by dialysis against metal complexing agents such as o-phenanthroline. Activity can be restored by addition of zinc ions. A zinc dissociation constant of about 10(-10)M at pH 7.8 is obtained by titration of the enzyme with a metal ion buffer. The reactivity of the SH groups of the enzyme is considerably affected by zinc ions. The enzyme contains two essential zinc ions per molecule.

Identification of essential histidine residues of aminoacylase by photooxidation and by reaction with diethylpyrocarbonate.

State and function of the histidine residues of aminoacylase were investigated by photoxidation in the presence of methylene blue and by chemical modification with diethylpyrocarbonate. Complete inactivation of the enzyme was observed after oxidation of 4 histidine residues. From the pH dependence of the photooxidation it becomes evident that the inactivation of the enzyme is not a consequence of the simultaneous oxidation of tryptophan residues. The enzyme is also inactivated by chemical modification of histidine residues with diethylpyrocarbonate. Activity is restored by treatment with hydroxylamine. Zn2+-ions which are essential for the activity of aminoacylase protect the available histidine molecules against photooxidation and attack by diethylpyrocarbonate. It is suggested that histidine is involved in the binding of the essential Zn2+-ions.

Chemical investigations on pig kidney aminoacylase.

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.

Chemical modification of two tryptophan residues abolishes the catalytic activity of aminoacylase.

1) The reaction of 1 H-diazotetrazole and N-bromosuccinimide with aminoacylase was studied under different conditions. A tenfold molar excess of 1 H-diazotetrazole (2 X 10(-4) M) at pH 5.5 abolishes the catalytic activity of the enzyme while modifying only two tryptophan residues. No other amino acid reacted under these conditions as tested by amino acid analysis. 2) With a 40-fold molar excess of N-bromosuccinimide (8 X 10(-4)M) at pH 5.0, two tryptophan residues of the enzyme were oxidized with complete loss of activity. Under these conditions no significant cleavage of the polypeptide chain was observed. Neither tyrosine nor histidine was modified by this reagent, up to a 100-fold molar excess. 3) Substrates and reversible (N-tosylalanine) and irreversible (TosPheCH2Cl) inhibitors of the enzyme do not protect the two reactive tryptophans against the modification reagents. Under more drastic conditions, lysine, tyrosine and histidine residues are also modified by the reagents.

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.