Validation of Microtox as a first screening tool for waste classification.

The Waste Framework Directive (WFD; 2008/98/EG) describes how waste materials are to be classified as hazardous or not. For complex waste materials chemical analyses are often not conclusive and the WFD provides the possibility to assess the hazardous properties by testing on the waste materials directly. As a methodology WFD refers to the protocols described in the CLP regulation (regulation on Classification, Labeling and Packaging of chemicals) but the toxicity tests on mammals are not acceptable for waste materials. The DISCRISET project was initiated to investigate the suitability of alternative toxicity tests that are already in use in pharmaceutical applications, for the toxicological hazard assessment of complex waste materials. Results indicated that Microtox was a good candidate as a first screening test in a tiered approached hazard assessment. This is now further validated in the present study. The toxic responses measured in Microtox were compared to biological responses in other bioassays for both organic and inorganic fractions of the wastes. Both fractions contribute to the toxic load of waste samples. Results show that the Microtox test is indeed a good and practical screening tool for the organic fraction. A screening threshold (ST) of 5 geq/l as the EC50 value in Microtox is proposed as this ST allows to recognize highly toxic samples in the screening test. The data presented here show that the Microtox toxicity response at this ST is not only predictive for acute toxicity in other organisms but also for sub lethal toxic effects of the organic fraction. This limit value has to be further validated. For the inorganic fraction no specific biotest can be recommended as a screening test, but the use of direct toxicity assessment is also preferable for this fraction as metal speciation is an important issue to define the toxic load of elutriate fractions. A battery of 3 tests (Microtox, Daphnia and Algae) for direct toxicity assessment of this fraction is recommended in literature, but including tests for mechanistic toxicity might be useful.

Screening tests for hazard classification of complex waste materials--selection of methods.

In this study we describe the development of an alternative methodology for hazard characterization of waste materials. Such an alternative methodology for hazard assessment of complex waste materials is urgently needed, because the lack of a validated instrument leads to arbitrary hazard classification of such complex waste materials. False classification can lead to human and environmental health risks and also has important financial consequences for the waste owner. The Hazardous Waste Directive (HWD) describes the methodology for hazard classification of waste materials. For mirror entries the HWD classification is based upon the hazardous properties (H1-15) of the waste which can be assessed from the hazardous properties of individual identified waste compounds or--if not all compounds are identified--from test results of hazard assessment tests performed on the waste material itself. For the latter the HWD recommends toxicity tests that were initially designed for risk assessment of chemicals in consumer products (pharmaceuticals, cosmetics, biocides, food, etc.). These tests (often using mammals) are not designed nor suitable for the hazard characterization of waste materials. With the present study we want to contribute to the development of an alternative and transparent test strategy for hazard assessment of complex wastes that is in line with the HWD principles for waste classification. It is necessary to cope with this important shortcoming in hazardous waste classification and to demonstrate that alternative methods are available that can be used for hazard assessment of waste materials. Next, by describing the pros and cons of the available methods, and by identifying the needs for additional or further development of test methods, we hope to stimulate research efforts and development in this direction. In this paper we describe promising techniques and argument on the test selection for the pilot study that we have performed on different types of waste materials. Test results are presented in a second paper. As the application of many of the proposed test methods is new in the field of waste management, the principles of the tests are described. The selected tests tackle important hazardous properties but refinement of the test battery is needed to fulfil the a priori conditions.

DISCRISET: a battery of tests for fast waste classification--application of tests on waste extracts.

The Hazardous Waste Directive (HWD, Council Directive 91/689/EC, 1991) provides a framework for classification of hazardous waste, based on 15 Hazard (H)-criteria. For complex wastes the HWD foresees the application of toxicity tests on the waste material itself to assess its toxic properties. However, these proposed test methods often involve mammalian testing, which is not acceptable from an ethical point of view, nor is it feasible economically. The DISCRISET project was initiated to investigate the use of alternative chemical and biological fast screening tests for waste hazard classification. In the first part of the project, different methods were reviewed and a testing strategy was proposed to minimize time and cost of analysis by a tiered approach. This includes as a first tier chemical analysis followed by a general acute toxicity screen as a second tier and as a third tier mechanistic toxicity tests to assess chronic toxicity (genotoxicity, hormone disturbance, teratogenic effects, immunologic activity). In this phase of the project, selected methods were applied to 16 different waste samples from various sources and industries. The first tier chemical tests are recommended for the full characterization of the leachate fraction (inorganics) but not for the organic fraction of samples. Here the chemical characterization is only useful if toxic content is known or suspected. As second tier the fast bacterial test Microtox is validated as a general toxicity screen for the organic fraction (worst case organic extract). Samples that are not classified in tier 1 or 2 are then further investigated in the third tier by the mechanistic toxicity tests and tested for their potential chronic toxicity: immune activity (TNF-α upregulation) is indicative for corrosive, irritating or sensitising effects (H4/H8/H15), reproductive effects (H10) are indicated by hormone disturbance and early life stage abnormalities in fish larvae when exposed to the extracts and mutagenicity and carcinogenicity (H7, H11) are indicated by SOS response induction and increased mutation frequency in the Ames test when exposed to the extracts. Results indicate that the combination of chemical tests and bioassays allows important hazardous properties to be addressed and the tiered approach ensures that the tests are performed quickly and economically. The suggested strategy provides a solid and ethical alternative to the methods described in the HWD and is a vast improvement on the current, arbitrary classification.

Successful prevalidation of the slug mucosal irritation test to assess the eye irritation potency of chemicals.

A previous 'in house' validation study showed that the SMI assay can be used as an alternative to the in vivo Draize eye irritation test. The aim of this multi-centre study with four participating laboratories was to assess the transferability and inter-laboratory variability of the assay using 20 reference chemicals covering the whole irritancy range. The eye irritation potency of the chemicals was assessed by measuring the amount of mucus produced during a 60-min contact period with a 1% dilution, and a second 60-min treatment with a 3.5% dilution. After each contact period the protein release from the mucosal surface was measured. Linear discriminant equations were used to convert the results into the corresponding EU eye irritation categories (NI, R36 and R41). All the non-irritants were predicted correctly by the four laboratories resulting in a 100% specificity. For the R36 compounds a correct classification rate of 89% (VITO) and 100% (SPL, JNJ and UGent) was obtained. The R41 compounds were classified correctly in 78% of the cases for VITO, 89% for SPL and JNJ and 100% for UGent. We can conclude that the SMI assay is a relevant, easily transferable and reproducible alternative to predict the eye irritation potency of chemicals.

Ecotoxicity of contaminated suspended solids for filter feeders (Daphnia magna).

It is generally assumed that the dissolved fraction of a toxic substance in surface water is mainly responsible for toxicity to aquatic organisms. However, toxic compounds are often adsorbed or chemically bound to suspended particles in the water column, depending upon the physico-chemical conditions. In the present study potential adverse effects to filter feeding organisms by metal contaminated particles were investigated. In our hypotheses the adsorbed metals might desorb in the gastrointestinal tract-due to different physico-chemical conditions-and exert toxic effects. Clay and sand particles, algae and organic material (peat) were artificially contaminated with cadmium and zinc. The contaminated materials were resuspended in standard conditions and toxicity was measured for the water flea Daphnia magna (mortality at 48 hours). As a reference, supernatant solutions were used containing the same concentration of dissolved metal as the suspensions. It was also established that the test concentrations of solid material (250 and 500 mg/l uncontaminated particles) did not cause any mortality within 48 hours. Daphnids are filter feeders: they filtrate large amounts of surrounding water, redrawing particles as a food source. Results strongly indicate that contaminated particles threaten the health of these particle-feeding organisms. Compared to the reference severe acute toxic effects were seen and cadmium accumulation was increased when contaminated solid material was present. Results were essentially the same for the different materials used in the experiments, except for sand contaminated with cadmium. This shows that mineral as well as organic materials can contribute to the particle bound toxicity. Different results were obtained when a static set up was used instead of a flow through set up, illustrating that the route of administration is important to make particles available and thus to evaluate their toxicity. Contaminated particles clearly have toxic potency, not only because they are a continuous source of dissolved xenobiotics, but also because the particle bound fraction can become available within the body of particle feeding organisms. This could lead to unexpected high tissue concentrations. More insight is needed to predict the bioavailability of adsorbed pollutants. Results of this study already indicate that suspended solids should be considered as a separate compartment in risk evaluation of chemicals, effluents or natural surface waters.

Comparative sensitivity of 20 bioassays for soil quality.

Increasing evidence suggests that the use of a single bioassay will never provide a full picture of the quality of the environment. Only a test battery, composed of bioassays of different animal and plant species from different trophic levels will reduce uncertainty, allowing an accurate assessment of the quality of the environment. In the present study, a test battery composed of 20 bioassays of varying biological endpoints has been compared. Apart from lethality and reproductive failure in earthworms, springtails, nematoda, algae and vascular plants, these endpoints also included bioavailibility of metals (bacteria), heat-shock induction (nematodes, algae), DNA damage (bacteria, earthworm, vascular plants), beta-galactosidase (Daphnia) and esterase activity (algae) and a range of immunological parameters (earthworm). Four chemicals (cadmium, phenol, pentachlorophenol and trifluralin)--each representing a different toxic mode of action--were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The tests have been performed both on these artificially contaminated soil samples and on aqueous extracts subsequently obtained from these soils. The results show that the immunological parameters and the loss of weight in the earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction in algae were shown to be extremely sensitive when applied to soil extracts. As previously shown at the species level, no single biological endpoint was shown to be the most sensitive for all four modes of toxic action.

Electrophysiological evidence for the presence of an apical H(+)-ATPase in Malpighian tubules of Formica polyctena: intracellular and luminal pH measurements.

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K+ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H+ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K+) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K+ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K+ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from -93 mV in 5 mmol/l K+ to -65 mV in 113 mmol/l K+. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K+ concentration. The calculated electromotive force of this pump is -159 mV. Comparing the proton with the K+ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K+ extrusion into the lumen for each value of secretion rate.

NOR variability in twins.

The number of AgNOR (NOR+) and the amount of AgNOR (NORM+) were analysed by means of two multilevel analyses of variance in a total of 12 twin pairs: 3 female and 4 male MZ and 5 male DZ pairs. In the first analysis, only zygosity was controlled; in the second, chromosome types D and G were controlled as well as the interaction between chromosome type and zygosity. For NOR+ and NORM+, when chromosome types D and G are not distinguished, the within-pair variance is greater, though not significantly, in DZ than in MZ pairs; but it is highly significantly greater when chromosome type (D or G type) is under control. This confirms an important genetic determination of NOR+ and NORM+ when in the ANOVA model the D and G types are controlled. However, nongenetic factors also influence the Ag-NOR patterns, but not enough to conceal the genetically defined rDNA pattern. Indeed, about 50% of the cells transcribe their rDNA in a way not closely dependent on the rDNA background and significant intrapair differences of NOR+ pattern exist in MZ twins.