SETAC GLB — Gründung und Entwicklung

Goal, Scope, and Background

In the context of the ISO-standardisation of the in vitro micronucleus test for (waste) water testing (ISO/DIS 21427-2), a national collaborative study was organized by the German Federal Institute of Hydrology (BfG) involving ten laboratories of private companies, universities and public authorities. The formation of micronuclei (MN) is a special kind of chromosomal aberration. To meet the standardisation requirements for this method, encoded waste water samples, some of them spiked with known genotoxins, had to be tested in a collaborative study. The study should demonstrate practicability of the in vitro micronucleus test for waste water testing and should provide validity data.

Material and Methods

The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. Four encoded samples from one municipal and one industrial wastewater treatment plant were tested with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mytomycin C. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei (LID; lowest ineffective dilution). Cytotoxicity was judged by determining the survival-index, i.e. the percentage growth rate of the cells compared with the respective negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed.

Results

Although some of the laboratories had little or even no experience with the protocol of the in vitro micronucleus test described in the ISO-draft, all participants suceeded in establishing the assay within few weeks and in generating viable test results. The two nongenotoxic samples were detected as negative by 90% or 100% of the participants. The mitomycin C-spiked sample (expected to be positive without S9 supplementation) was correctly evaluated as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9 supplementation) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false-negative results were due to technical failure, but not of a methodological nature. The sample LID values varied by no more than one dilution step around the median LID-value for all samples investigated. The survival index was proven to be a robust measure for estimation of cytotoxicity.

Discussion

The measurement of micronuclei is an important parameter for the detection of cytogenetic damage. In contrast to the single-cell gel electrophoresis assay (Comet assay), which is used as an indicator test, the in vitro micronucleus test detects non-repairable and thus manifested genetic damage. Consequently, the in vitro micronucleus test can be regarded as the more significant test system. A more frequent occurrence of micronuclei in treated cells suggests a risk of severe genetic damage for subsequent cell generations. In the interest of a precautionary environmental protection and health protection no municipal or industrial waste-water samples should show any significant induction of micronuclei in the treated cell populations.

Perspectives

The presented collaborative study was the first interlaboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate incorporation assay. The data generated in the course of this project justify the transformation of the draft standard into the final draft international standard (FDIS), the preliminary stage of an international norm, so that a valid, standardised test system for the detection of cukaryotic genotoxicity in water samples might become available.     

Schwermetallmonitoring mit der Standardisierten Graskultur im Untersuchungsgebiet München

For the pilotproject ?Effect Monitoring — Biomonitoring of Immission Effects in the Testing Area of Munich”, standardized grass cultures were exposed at 19 locations during 10 series for two weeks each. The grass was analyzed for the accumulation of lead, antimony arsenic, cadmium, and copper. Highest accumulation was observed with antimony near traffic situations. The comparison of the results from the different sampling points identifies motor vehicle traffic as the source of antimony. First examinations of brake linings support this conclusion. Lead is only slightly increased near traffic. Immission-caused copper contents in the grass were found near the overhead tramway cables. The accumulation detection limits for arsenic and cadmium were not exceeded at any of the measuring points.     

Immissionsbelastungen im Umfeld des Flughafens München

The airborne immissions in the eastern surroundings to the new Munich airport have been investigated by means of different analytical methods between 1992 and 1995. For a detection of environmental impacts, bioindicators were applied to determine the pollution by PAH and to quantify the pollution by photooxidants. In addition, the PAH content in the upper soil layer was determined. During high pressure weather periods in summer the actual air pollution was investigated in the course of seven measuring campaigns. The PAH pollution of the soil is typical for rural background sites with highest concentrations in the close vicinity of busy roads, and exhibits no temporary trend. The actual input of PAH does not differ from the typical situation of rural regions in Bavaria at any measuring sites at all. Road traffic can be identified as the main PAH source in summer, whereas the considerably higher pollution in winter can be related to domestic heating. The pollution by photooxidants was found to be rather homogeneous throughout the eastern surroundings to the airport, being lowest at the sites directly influenced by road traffic. The pollution by volatile hydrocarbons at a busy road junction in the city of Erding is considerably higher than in the rural vicinity of the airport. The concentrations exhibit distinct daily courses which, among other things, can be related to atmospheric photooxidation. The hydroperoxides as secondary air pollutants exhibit an inverse course similar to that of ozone, with maximum values in the afternoon.     

Verteilung chemischer Elemente in der Nahrung und Milch stillender Mütter

Goal and Scope

The transfer of various chemical elements from maternal food into human milk was investigated. Transfer factors (hereafter TF) food/mother’s milk were taken to represent those nuclides pertinent to radiation protection in order to estimate possible radioactive burdens of breast-fed babies.

Methods

A total of 23 mothers, mainly from the Euroregion Neisse (PL, CZ, D; all being in their mature phases of lactation), took part in this study (1998–2001), for time periods between two to eight weeks (8805 samples were analyzed). The diurnal uptake of elements was determined by the duplicate method.

Results and Conclusions

TF values determined for Ag, Au, Ba, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, I, La, Mn, Mo, Nb, Ni, P, Pb, Ra, Rb, Re, Ru, Sb, Sr, Te, Th, Ti, U_o, Y, Zn, Zr were theoretically analyzed with the following result: metal ions transfer into mother’s milk mainly in coordinated states (i.e. bound to anionic ligands), with TF values which increase according to complex stabilities. Therefore it can be assessed that neither Cd nor Ba, both forming only weak complexes, pose hazards to breastfed children.

Outlook

There are relationships between transfer factors pertaining to certain elements and the amounts of others consumed by the mother; i.e. (changes in) consumption of one element influence the transfer towards women’s milk of another element. Accordingly, strategies can be developed to keep off hazardous substances — including fissiogenic radionuclides — from milk which is far more efficient than traditional isotopic dilution; however, iodide tablets are useful here, too.     

Der Fischrückgang in der oberen Donau

Background During the last years, a decrease in fish populations has been documented for several river systems in Europe and North America. Since more than 20 years, similar developments have been documented for the upper Danube River between Sigmaringen and Ulm. The grayling (Thymallus thymallus) represents one of the most affected fish species in the upper Danube, and notable decrease has also been recorded for other species such as the European barbel (Barbus barbus). Despite intensive and continuous stocking and improvement of water quality along the Danube since the 1970s, this negative development of fish populations could not be stopped. Aim This article has been designed to give an overview on various investigations into the decline of fish in the upper Danube River. The major focus of this paper is on the presentation of a weight-of-evidence study carried out since 2003 at the Department of Zoology at the University of Heidelberg. Results and discussion During the 1920s, major parts of the upper Danube River were corrected, thus causing dramatic changes in the hydromorphology of the river. The macrozoobenthos, which represents an important source of nutrition for several fish species, depends on the morphological conditions of a river; recent investigations, however, could not show any dramatic changes in macrozoobenthos composition. Earlier investigations of the Danube River primarily focused on water quality, which has, indeed, been continuously improved by extensive modernization of wastewater treatment plants. Contamination by priority organic pollutants in sediments was not given any attention until the mid 1990s, when a clear decline of, e.?g., PAH concentrations were documented. However, results of more recent analyses showed very high PAH concentrations in two sediment samples (Schwarzach Σ 16 PAHs = 26 mg/kg; Öpfingen Σ 16 PAHs = 5 mg/kg). In 2002, the first ecotoxicological investigations were performed at the upper Danube River. The results of a pilot study showed a high ecotoxicological burden for selected sampling sites. As a conclusion of this study, a weight-of-evidence investigation was initiated to make an attempt to identify major pollutants and possible causes for the fish decline. In addition to, e.?g., liver ultrastructure analyses in barbel, the micronucleus test was used as a definite mutagenicity test using erythrocytes and liver samples. The results of the different in vitro tests showed very good correlation well with in situ parameters. Outlook The investigations within this weight-of-evidence study showed that the pollution situation of the Danube is, indeed, significant enough to potentially affect fish populations. However, further investigations are needed to clarify the specific causes for the fish decline. A bioassay-directed fractionation study is underway to identify specific pollutants as well as their sources. This study will also assess the endocrine disrupting potential of Danube sediments via histopathological analyses of gonads of field fish in combination with different in vitro tests. Furthermore, in the planned joint project COMPSEARCH, comparative investigations will evaluate the ecotoxicological hazard potential of different pollution sources and consequences of changes in land use.     

Neue Aspekte in der Behandlung von Siedlungsabflüssen

The surface runoff from urban areas is one of the most important sources of pollutants emitted into surface waters. Suspended solids which act as a transport vehicle for many anthropogenic pollutants (e.?g. heavy metals, PAH) are a key factor in this regard. The development of efficient measures of storm water runoff treatment thus requires a further differentiation of suspended solids in a fine (clay and silt) and coarse (sand and gravel) fraction. Both fractions show distinctly different characteristics in pollutant loading, transport and retention on urban surfaces and sewer systems. The primary aim of storm water runoff treatment is the reduction of the fine particles which are always highly loaded with anthropogenic pollutants. In contrast the coarse particles are almost unpolluted especially if they have a low organic share. The widespread sedimentation tanks with surface loadings between 10 and 2?m/h are very inefficient. A significant, save and lasting reduction of the emitted load of fine particles requires a considerable reduction of the surface loads. That can be achieved with the installation of lamellar settler or the utilization of the very large volumes of flood management tanks frequently present in urban areas. Filtration plants are highly efficient but there application in urban areas is limited due to their high space demands.     

HPLC-MS/MS in der Rückstandsanalytik von Pflanzenschutzmitteln

Goal and Scope

Increasing demands from the regulatory authorities of plant protection products for residue analytical methods on the one hand and the need for cost reduction by shorter development times on the other hand have led to a significant displacement of conventional methods (e.g. HPLC-UV or GC-MS) by HPLC-MS/MS with atmospheric pressure ionization (API) in residue analysis since the mid of the 1990s. This development started in the pharmaceutical industries where HPLC-MS/MS had already been used for the quantitation of drugs and their metabolites in plasma since the late 1980s.

Methods

The reason for the high acceptability of HPLC-MS/MS with electrospray ionisation (ESI) or chemical ionization at atmosheric pressure (APCI) compared with alternative methods are shown with respect to analytical quality parameters, compliance with international registration guidelines and economic reasons.

Results and Conclusion

HPLC-MS/MS is being applied for solving ca. 75% of all analytical problems occuring in modern residue analytical laboratories. This technique offers convincing advantages for the determination of plant protection products and their metabolites in various matrices. These are a reduced number of or no clean-up steps after extraction of the samples, high throughput besides high ruggedness, low limits of detection and a large linear measuring range. In addition, several analytes of various chemical classes can be determined within one chromatographic run. Due to the high selectivity there is only little need for additional confirmatory methods. For enforcement purposes HPLC-MS/MS is accepted by the European authorities since 2002. However, due to high costs for instrumentation and specially skilled personnel only few enforcement laboratories have invested into this technology, so far.

Recommendation and Perspective

The importance of HPLC-MS/MS in residue analysis will further increase, as new ionization techniques will enable the analysis of more analytes and as the investment costs will decrease in the future. However, proven conventional methods will still survive in some cases. New concepts, such as parallel HPLC-MS/MS or on-line purification of extracts by column switching which are already widely used in the pharmaceutical industry could help to further increase the throughput. However, for a sound evaluation with respect to their applicability in residue analysis there is so far too little experience.     

Stabilisotopenverdünnungsanalysen zur Quantifizierung organischer Spurenkomponenten in der Lebensmittelanalytik

Background and scope The principle and applications of stable isotope dilution assays (SIDAs) in food analysis is critically reviewed. This method is based on the application of internal standards labelled with stable isotopes. General topics The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards along with possible calibrations procedures. The syntheses and availability of labelled food compounds for the use as internal standards is reviewed. Results The complete compensation for losses of analytes during clean-up as well as for ion suppression during LC-MS/MS and the so-called carrier effect are major advantages of SIDAs. However, deficient equilibration, spectral overlap and isotope effects can lead to false results. Discussion With regard to specificity and recovery, SIDAs generally are considered as the reference methods in clinical chemistry. In food chemistry, this method has been applied in flavour and pesticide analysis. However, it is becoming increasingly important also in the analysis of mycotoxins, further contaminants and vitamins. Conclusions The increasing access to isotopologic standards creates continuously new applications for SIDAs, particularly for bioactive compounds in foods.     

Batterienrecycling: Möglichkeiten zur Erhöhung der Rücklaufquote

Separate collection and exploitation of used batteries facilates the recycling and eventual waste management of such heavy metals as mercury and cadmium. It is thereby possible to regain raw materials like zinc, manganese and iron for the raw material cycle. Although the collection and recycling of used batteries in Switzerland is financed by a prepaid disposal fee, their returning rate of almost 60% is too low for several reasons. A questionnaire survey carried out on 2000 households revealed the following frequenthy: People collecting paper, glass, aluminium, compost and tinplate, are more separate used batteries from ordinary garbage. The number of collecting points is supposed to be sufficient, but not all of them are sufficiently marked. The prepaid disposal fee (VEG) should become obligatory so that it would be possible to compensate the collecting points. It is not obvious from the results of the survey if the introduction of a deposit of batteries would raise the retourning rate. As far as advertising is concerned, only the “battery bag” sent to every household by the BESO seemed to influence the collecting behaviour in a positive way, poster advertising had only little effect. Appeals in newspapers, radio and television did not show any changement of the collecting behaviour. However, information and knowledge about batteries and their recycling do have a positive influence in the collecting behaviour of the consumers in this specific case.     

Eine Weight-of-Evidence-Studie zur Bewertung der Sedimentbelastung und des Fischrückgangs in der Oberen Donau

Background and aim Despite intensive and continuous stocking and improvement of water quality since the 1970s, fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River (Germany). In order to assess 1) possible links between molecular/biochemical responses and ecologically relevant effects, and 2) if ecotoxicological effects might be related to the decline in fish catches in the upper Danube river, sediment samples and fish were collected at different locations and analyzed using a weight-of-evidence (WOE) approach with several lines of evidence. The objective of the presentation is to introduce the conceptual framework and to review results of the ongoing study. As previously addressed by Chapman and Hollert (2006) a variety of lines of evidence can be used in WOE studies. Briefly, 1) a comprehensive battery of acute and mechanism-specific bioassays was used to characterize the ecotoxicological hazard potential. 2) Histopathological investigations and the micronucleus assay with erythrocytes were applied, analyzing in situ parameters. 3) Diversity and abundance of benthic macroinvertebrates and fish as well as 4) persistent organic pollutants, endocrine disrupting substances, limnochemical parameters and the concentration of heavy metals were recorded. To identify organic contaminants a spotential causes of sediment toxicity assays, 5) effect directed analysis was applied.     

Die rolle der umweltprobenbank in der Umweltüberwachung am beispiel von chlorierten kohlenwasserstoffen in ausgewählten matrizes

For precautinary environmental protection the Environmental Specimen Bank (ESB) has been successfully established as a permanent environmental surveillance tool in Germany. The presented monitoring results on chlorinated hydrocarbons (CHC) show very clearly the effects of political decisions and can be used for the identification of pollutant sources. The CHC burden of breams (Abramis brama) from five German rivers (Elbe, Rhein, Saar, Mulde, Saale) gives an current overview of the pollution situation of different limnic ecosystems in Germany. A calculation model about the distribution of organic pollutants between different tissues offers the possibility to predict concentrations in liver and muscle of breams. Legislative emission restrictions have led to a significant decrease of dioxin levels in herring gull (Larus argentatus) eggs between 1988–1996.     

Bestimmung von Mineralölkohlenwasserstoffen in Bodenproben mit Hilfe der Dünnschichtchromatographie

A thin-layer chromatographic method for the determination of mineral oil hydrocarbons (aliphatic compounds and naphtenes) of medium and low volatility in soil samples is presented which is based on their separation and quantitative determination. The extraction of the soil samples is effected by means of an n-hexane-acetone mixture (10∶1) supported by ultrasonics. The evaluation can be made visually or densitometrically by means of a thin-layer scanner. The applicability and restrictions of the method are shown. The advantage of the method lies in its low requirements pertaining to apparatus and chemicals, as well as in its rapidness which makes it suitable for quick in-situ analyses as, for example, for the assessment of the mineral oil content of a contaminated site.     

Aktuelle Aerosoltrends an der GAW Globalstation Hohenpeißenberg und ihre Relevanz für Luftreinhaltung und Klima

Aim and Scope

This study was aimed at representing current aerosol trends measured at the GAW global station and their relevance for the present fine dust discussion and a possible impact on climate.

Results and Discussion

1) The intensive GAW measuring program at Hohenpeissenberg covers numerous parameters for the characterization of the physical, optical and chemical characteristics of the atmospheric aerosol. The time series of the number concentration of ultra fine particles with diameters of about 0.004 to 3 μm shows an increase of about 50% since 1995. 2) The introduction of soot particle filters would lower the dust mass only slightly since soot particles from diesel cars only about 8–10% contribute to the dust mass, but about 90% to the number concentration of particles. 3) The single scattering albedo (SSA) is a key parameter determining whether the existing aerosol mixture causes a cooling (negative radiative forcing) or a warming (positive radiative forcing) in the atmosphere. At Hohenpeißenberg the SSA changed from 0.85 to 0.91 in the time period 1999 to 2005, i.e. the regional aerosol as a whole is dominated by scattering rather than absorbing particles.

Conclusion and Perspective

From current aerosol trends at the GAW global station Hohenpeißenberg it was possible to analyze their relevance for air pollution control and possible influences on climate. Dust mass is not a suitable parameter for accessing the contribution of diesel engined vehicles. Measuring the number concentration of particles much more reflects the influence of diesel engined vehicles and also shows, as expected, an upward trend due to a strong increase of the fraction of diesel vehicles in comparison to the total number of cars. Aerosol particles scatter and absorb solar radiation and thus cause a cooling or warming in the atmosphere. Calculated values of the single scattering albedo at Hohenpeissenberg show, that in the initial years the aerosol did not diminish the temperature rise, but rather caused it to increase. Only a data comparison from a global network like GAW can demonstrate, if the results are even representative for the large scale situation.