Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos.

Predicting the bioavailability and effects of metals in sediments is of major concern in context with sediment risk assessment. This study aimed to investigate the bioavailability and molecular effects of metals spiked into riverine sediments to zebrafish (Danio rerio) embryos. Embryos were exposed to a natural and an artificial sediment spiked with cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) individually or as a mixture at concentrations ranging from 150 to 3000 mg/kg dry weight (dw) over 48 h, and uptake of metals was determined. Furthermore, transcript abundances of the metallothioneins MT1 and MT2, the metal-responsive element-binding transcription factor (MTF) and the genes sod1, hsp70 and hsp90α1 were measured as indicators of metal-induced or general cellular stress. D. rerio embryos accumulated metals from sediments at concentrations up to 100 times greater than those spiked to the sediment with the greatest bioaccumulation factor (BAF) for Cu from artificial sediment (275.4 ± 41.9 (SD)). Embryos accumulated greater concentrations of all metals from artificial than from natural sediment, and accumulation was greater when embryos were exposed to individual metals than when they were exposed to the mixture. Exposure of embryos to Zn or the mixture exhibited up to 30-fold greater transcript abundances of MT1, MT2 and hsp70 compared to controls which is related to significant uptake of Zn from the sediment. Further changes in transcript abundances could not be related to a significant uptake of metals from sediments. These studies reveal that metals from spiked sediments are bioavailable to D. rerio embryos directly exposed to sediments and that the induction of specific genes can be used as biomarkers for the exposure of early life stages of zebrafish to metal-contaminated sediments.

Estrogen receptor mediated activity in bankside groundwater, with flood suspended particulate matter and floodplain soil - an approach combining tracer substance, bioassay and target analysis.

Bankside groundwater is widely used as drinking water resource and, therefore, contamination has to be avoided. In the European Union groundwater protection is explicit subject to Water Framework Directive. While groundwater pollution may originate from different sources, this study investigated on impacts via flood events. Groundwater was sampled with increasing distance to the river Rhine near Karlsruhe, Germany. Samples were HPLC-MS-MS analyzed for the river contaminant carbamazepine to indicate river water infiltration, giving permanent presence in 250 m distance to the river (14-47 µg L⁻¹). Following a flood event, concentrations of about 16-20 µg L⁻¹ could also be detected in a distance of 750 m to the river. Furthermore, estrogenic activity as determined with the Yeast Estrogen Screen assay was determined to increase up to a 17ß-ethinylestradiol equivalent concentration (E-EQ)=2.9 ng L⁻¹ near the river, while activity was initially measured following the flood with up to E-EQ=2.6 ng L⁻¹ in 750 m distance. Detections were delayed with increasing distance to the river indicating river water expansion into the aquifer. Flood suspended matter and floodplain soil were fractionated and analyzed for estrogenic activity in parallel giving up to 1.4 ng g⁻¹ and up to 0.7 ng g⁻¹, respectively. Target analysis focusing on known estrogenic active substances only explained < 1% of measured activities. Nevertheless, river water infiltration was shown deep into bankside groundwater, thus, impacting groundwater quality. Therefore, flood events have to be in the focus when aiming for groundwater and drinking water protection as well as for implementation of Water Framework Directive.

Evaluation of the toxicological properties of ground- and surface-water samples from the Aral Sea Basin.

In order to determine whether there is a potential health risk associated with the water supply in the Aral Sea Basin, ground- and surface-water samples were collected in and around Aralsk and from the Aral Sea in 2002. Water samples from Akchi, a small town close to Almaty, served as controls. Bioassays with different toxicological endpoints were employed to assess the general toxicological status. Additionally, the samples were analysed for microbial contamination. The samples were tested in the primary hepatocyte assay for their potential to induce micronuclei and chromosomal aberrations as cumulative indicators for genotoxicity. In parallel, the effects on cell proliferation evidenced by mitotic index and cytotoxicity such as the appearance of necrotic and apoptotic cells, were determined. Furthermore, samples were examined using the Microtox assay for general toxicity. Chemical analysis according to European regulations was performed and soil and water samples were analysed for DDT and DDE. The results obtained indicated no increased cyto- or genotoxic potential of the water samples, nor levels of DDT or DDE exceeding the thresholds levels suggested by WHO. Our data therefore do not support the hypothesis that the contamination of the drinking water in and around Aralsk is responsible for the health effects previously described such as increased rates of liver disease and in particular liver cancer. Microbiological analysis, however, revealed the presence of contamination in most samples analysed.

[Human biomonitoring investigations of organochlorine compounds -- PCB, DDE, HCB, beta- and gamma-HCH, PCDD/PCDF, Dioxin-like PCB's and polybrominated biphenyl ethers]. / Humanbiomonitoring-Untersuchungen von Organohalogenverbindungen -- PCB, DDE; HCB, beta- und gamma-HCH, PCDD/PCDF, koplanaren PCB sowie polybromierten Biphenylethern.

Although PCB and PCB-containing materials are not processed for a long time, PCB is under discussion again and again caused by the pollution of indoor environments. To objectify the discussion, the dates of the PCB-biomonitoring, the organochlorine-compounds (DDE, HCB, beta-/gamma -HCH, PCDD/PCDF) and the polybrominated biphenyl ethers concerning the investigations within the project "Sentinel Health Departments" in Baden-Wurttemberg are represented. Additionally results from children from Kazakhstan (Aral-Sea area) and from teachers which are working in PCB polluted schools as well as from a long term investigated test person are reported. Blood concentrations of the following compounds decreased from 1996/97 to 2002/03: the sum of the concentration of PCB 138,153 and 180 decreased from 0.46 microg/L to 0.20 microg/L, DDE from 0.32 microg/L to 0.17 microg/, HCB from 0.20 microg/L to 0.08 microg/L, beta-HCH below the level of detection, I-TEQ NATO to 4.8 pg/g blood fat, TEQ WHO (without PCB) to 5.5 pg/g blood fat, PCB 126 to 18,8,pg/g blood fat and PCB 169 to 12.8 pg/g blood fat. The influence of breast feeding and the gender on the level of the pollution is conspicious. No local correlations were found in Baden-Wurttemberg, but they were found in comparison with the results of Kazakhstan (Aral-Sea area). The difficulty to produce time series while the analyzing pollutants are more and more decreasing, as well as the change of the calculation base of the summation of parameters like I-TEQ NATO to TEQ WHO are discussed.

Estrogenicity of solid phase-extracted water samples from two municipal sewage treatment plant effluents and river Rhine water using the yeast estrogen screen.

In the present study, the yeast estrogen screen (YES) was used to estimate the estrogenic potential of solid phase-extracted water samples from the effluents of two municipal sewage treatment plants (STPs 1 + 2) and from four lanes (left to right) of the river Rhine at Worms, Germany, i.e. downstream the STPs. Estrogenic activities of extracted water samples were expressed as 17beta-estradiol equivalents (E(2)-EQs). Estrogenic activity was detected in the effluents of both STPs with values of 0.242 +/- 0.038 nM (65.96 +/- 10.4 ng/l) and 0.125 +/- 0.026 nM E(2)-EQs (34.1 +/- 7.18 ng/l) at STP 1 and 2, respectively. In river Rhine water, estrogenic activity was lower, however, displaying significant differences between the left and right bank of the river (0.044 +/- 0.003 nM E(2)-EQs [11.97 +/- 0.7 ng/l] for lanes 1-3; 0.071 +/- 0.01 nM E(2)-EQs [19.42 +/- 2.8 ng/l] for lane 4). Chemical analysis of corresponding water samples resulted in a potential estrogenic response in the YES, expressed as E(2)-EQs for the known estrogens and phytoestrogens in the STP effluents with values up to 0.0662 nM E(2)-EQs (18.04 ng/l). In Rhine water from lane 4, however, total estrogenic activity of steroidal estrogens was equal to 0.014 nM E(2)-EQs (3.8 ng/l). Furthermore, total concentrations of flavonoids, fecal- and phytosteroids and resorcyclic lactones were about 1.2 microg/l at STP 1, 0.62 microg/l at STP 2 and 0.25 microg/l at the river Rhine, lane 4. Results indicate that estrogenic activity can clearly be measured in SPT effluents as well as in river Rhine water using the YES in combination with chemical analysis. Results from the bioassay, however, indicated a higher estrogenic potential (expressed as E(2)-EQs) than that obtained by chemical analysis.

Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents. Part I. Alkylation ortho to the amino function.

Alkyl-substituted derivatives of 2-aminonaphthalene (2-AN) 1, 2-aminofluorene (2-AF) 6 and 4-aminobiphenyl (4-ABP) 11 were synthesized and the mutagenic activity of these compounds determined in Salmonella typhimurium strains TA98 and TA100 with and without S9 mix. In the case of the ortho-substituted 4-aminobiphenyls 12-15 (3-alkyl=ethyl, iso-propyl, n-butyl, tert-butyl) the substituent with the strongest steric demand (3-tert-butyl) shows the strongest influence on the decrease of mutagenicity if compared with the parent compound. In the series of the bis-ortho-disubstituted compounds 16-18 (3,5-dimethyl-, 3,5-diethyl- and 3,5-diisopropyl-4-aminobiphenyl) generation of non-mutagenic species occurs already with the introduction of two ethyl groups. For the 4-aminobiphenyl derivatives 12-15 and 16-18, as well as for the 1-alkylated 2-aminofluorenes 7-10 and the 1-alkylated 2-aminonaphthalenes 2-5 a smaller mutagenicity was observed if compared with predicted mutagenicities as calculated by the QSAR equations of Debnath et al. (Environ. Mol. Mutagen. 19 (1992) 37). The largest differences resulted in the cases of the tert-butyl substituted compounds. Only with smaller alkyl groups like ethyl the QSAR predictions and the experimentally determined mutagenicities come close to each other. Thus, these results show that appropriate alkyl substitution reduces (eliminates) mutagenicity, secondly, it is necessary to introduce steric parameters to predict the mutagenicity of such compounds correctly.

Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds.

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour. The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4'-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.

The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium.

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propylethyl>iso-propyl>sec-butyl>tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2'-position.

Eleven alkyl substituted derivatives of 4-nitrobiphenyl (4NBp) and two corresponding nitroso compounds were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. The mutagenicity of compounds substituted ortho to the nitro group (3-methyl-, 3-ethyl-, 3-isopropyl-, 3-tertbutyl-, 3, 5-diethyl-, 3,5-diisopropyl-, and 3,5-ditertbutyl-4NBp) decreased with growing steric demand of the alkyl substituents in both tester strains. The most sterically hindered compounds were non-mutagenic even at highest concentrations. This reduction of mutagenicity is correlated with deviations from the coplanar orientation of the nitro group relative to the aromatic plane. Since a comparable decrease of mutagenicity for the nitroso compounds (4NOBp and 3-tertbutyl-4NOBp) was not observed, the first reduction step of non-planar nitro groups must be inhibited. Alkyl groups in the 2'-position of 4NBp (2'-methyl-, 2'-ethyl-, 2'-isopropyl-, and 2',4', 6'-trimethyl-4NBp) also reduced mutagenic activity with increasing size and removed it completely for the most sterically hindered species (2'-isopropyl-, 2',4',6'-trimethyl-4NBp). In this case, the co-planarity of the nitro group is not affected but the twisting of the two aromatic rings, which is associated with a less effective charge delocalisation of the nitrenium ion. The experimental mutagenicities of all nitro compounds were compared to predicted values, that are based on recently developed empirical equations. While there was reasonable correspondence for the parent compound (4NBp), its ortho methylated derivative (3-methyl-4NBp) and two highly hydrophobic dialkylated species (3,5-diisopropyl- and 3, 5-ditertbutyl-4NBp), predictions for all other alkyl substituted compounds were too high. Thus, steric parameters should be included to improve the general validity of predictions by means of quantitative structure-activity relationships (QSAR).

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of nitroaromatics in Salmonella typhimurium. Part II. Substituents far away from the nitro group.

Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine (2-aza-4-nitrobiphenyl) and 2-nitrofluorene, bearing various alkyl substituents far away from the nitro group (4'-position in nitrobiphenyls, 7-position in 2-nitrofluorenes) were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. In the absence of S9 in both strains, mutagenicity of all4'-Ad (Ad=adamantyl). Changes of the molecular shape from 'planar' to non-planar caused by the bulk of the introduced substituents (without influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds) may be responsible for this effect by interfering with an efficient intercalation into DNA.A comparison between experimental and theoretical values as calculated from recently developed equations (QSAR) confirmed our previous results (see the preceding paper) that mutagenicity of alkyl-substituted nitroaromatics cannot be predicted by hydrophobicity and LUMO-energies alone without including steric parameters.

Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes.

Whilst a patient is undergoing orthodontic treatment, dental appliances based on non-precious metals or titanium remain in the oral cavity for up to several years. Throughout this period the appliance is in either direct or indirect contact with the oral mucosa. To investigate the possibility of cell damage occurring as a result of appliance corrosion, monolayer cultures of immortalized human gingival keratinocytes were assessed for acute cyto- and genotoxicity using the hexosaminidase assay and the Comet assay respectively. The materials tested included 1. a nickel-free wire, 2. a UK-1 bond, 3. nickel-free as well as nickel-containing brackets with and without color signature and 4. a titanium expansion screw. Each of the test materials was corroded in a solution consisting of equal amounts of lactic acid and sodium chloride (0.1 M) for 1, 3, 7 and 14 days. The cell cultures were then exposed to eluates exhibiting the highest ion concentrations. None of the eluates was found to exhibit acute cytotoxicity, regardless of the type of test system used. Qualitative assessment using neutral red dye for live cells and either trypan blue or propidium iodide to disclose dead cells failed to reveal any significant increase in cell damage when exposed cells were compared to control cultures. Unrestricted cell vitality was confirmed by quantifying viable cells through measurement of hexosaminidase enzyme activity. Furthermore, assessment of genotoxicity revealed no apparent DNA damage to immortalized gingival keratinocytes following exposure to the test eluates. Because the materials tested in this study were corroded using the exacting methods normally applied to precious metals or gold-containing alloys, the lack of either acute cyto- or genotoxic effects following exposure to the test eluates indicates that the materials tested exert no adverse effects on cells similar to those of the target tissue exposed to the materials in situ.

Chlorate as an inorganic disinfection by product in swimming pools.

Chlorate and chlorite concentrations were determined in water samples taken from 33 swimming pools. In the pools under investigation, disinfection of the water is carried out either by gaseous chlorine (n = 14) or hypochlorite solution in conjunction with flocculation and sand filtration. A number of the pools also use ozone treatment to augment the disinfection process. Chlorite was not detectable in any of the samples (detection limit 1 mg/l). High concentrations of chlorate were detected in samples from a number of the pools; in one case as high as 40 mg/l. Higher chlorate concentrations were found to be associated with those pools using hypochlorite solution as a disinfecting agent. In contrast, relatively low chlorate concentrations were found in pools treated with gaseous chlorine. In order to elucidate any relationship between the chlorate content of pool water and that of the respective hypochlorite stock solution, chlorate and bromate concentrations were determined in the hypochlorite stock solutions of nine pools. Bromate concentration in the stock solutions were not found to exceed 1.2 g/l, chlorate was measured in concentrations of up to 44.5 g/l. The additional use of ozone as part of the water purification process appears to have no significant influence on chlorate concentration. Chlorate has no bactericidal properties and does not interfere with the measurement of certain parameters relevant to hygiene in swimming pools such as free and combined chlorine, pH or redox potential. At present, the effects of high chlorate concentrations in swimming pool water are unclear. Our initial investigations indicate that chlorate has no cytotoxic (Neutral-Red assay) or irritating properties (HET-CAM assay). However, both chlorate and chlorite are known to interfere with the haematopoetic system. In Germany, the MCL for chlorite in drinking water is 0.2 mg/l. It is therefore strongly recommended that measures should be taken to reduce chlorate concentrations in swimming pool water.

DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone.

The environmental contaminant 3-nitrobenzanthrone (3-nitro-7H-benz[d, e]anthracen-7-one) was recently shown to be a very strong bacterial mutagen, suggesting a new class of mutagenic compounds present in airborne particulate matter and diesel exhaust. Using the 32P-postlabeling assay, we investigated the capacity for 3-nitrobenzanthrone to form DNA adducts in vitro. Calf thymus DNA was incubated with 3-nitrobenzanthrone and either xanthine oxidase, a mammalian nitroreductase or rat liver S9 or zinc. Under these conditions 3-nitrobenzanthrone formed a total of seven adducts detectable by 32P-postlabeling. Using enrichment by butanol extraction the highest level of DNA adduct formation was found with activation by zinc (RAL: 88.4+/-32 per 108 nucleotides) followed by activation with xanthine oxidase (RAL: 75.5+/-12) and activation by rat liver S9 (RAL: 48.6+/-8). Three of the seven adduct spots were detected in all activation systems, however different amounts of individual spots were obtained in the different in vitro systems. The adduct pattern observed for the enzymatic incubations consisted of three major spots and was essentially identical. Chemical reduction of 3-nitrobenzanthrone by zinc resulted in five adduct spots whose formation was found to be concentration dependent. All adducts of 3-nitrobenzanthrone observed in this study migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of airborne particulate matter. When butanol enrichment was compared with nuclease P1 enrichment one adduct was clearly sensitive to the 3'-monophosphatase activity of nuclease P1. Our results demonstrate that 3-nitrobenzanthrone binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vitro all of which are reduction products. These adducts may contribute to the known genotoxicity and carcinogenicity of extracts from airborne particulates.

[Irritating effects of disinfection by-products in swimming pools]. / Irritierende Wirkung von Nebenprodukten der Schwimmbadwasserdesinfektion.

Compounds which can occur as disinfection by-products (DBP's) in swimming pool water were examined for their mucous membrane irritating potential. Previous studies using the rabbit eye test (Draizé test) have shown that the irritating potential of typical concentrations of free and combined chlorine are insufficient to explain the degree of eye irritation that can result from exposure to swimming pool water. Other DBP's which may be responsible for eye irritation include halogenated carboxyl compounds (HCC's) which act as precursors during the formation of chloroform. In this study, a modified HET-CAM Test (Hens Egg Test at the Chorion Allantois Membrane) has been used to investigate the mucous membrane irritating effects of HCC's. Some of the compounds tested were found to have a significantly increased irritating effect when compared to a chlorine/chloramine mixture of the same concentration, several mixtures of HCC's where even more active at lower concentrations than single compounds. However, the irritating effects of individual compounds as well as of mixtures of HCC's were not sufficiently intense to allow the identification of those compounds specifically responsible for the overall observed increase in irritation. HCC's were therefore tested in the presence of aqueous chlorine solution. When combined with aqueous chlorine, a number of compounds exhibited significantly enhanced effects. Our results show that the eye irritating effects of low concentrations of DBP's can be investigated using a modified HET-CAM assay. Moreover, results obtained using this assay suggest that the mucous membrane irritating potential of swimming pool water is a consequence of the effects and synergistic action of a number of DBP's in the presence of chlorine. Further work should be carried out in order to establish an indicator for eye irritating effects of swimming pool water.

[Potassium as an indicator of anthropogenic contamination of swimming pool water]. / Kalium als ein Indikator der anthropogenen Belastung von Schwimmbadwasser.

Swimming pool water is processed, filtered and disinfected repeatedly in order to maintain hygienic conditions. Additionally, fresh water is added. However, it cannot be avoided, that the concentrations of certain components of swimming pool water will increase in the course of time. DIN 19,643 regulates that fresh water supply can be measured by nitrate concentration. Nitrate is mainly formed by oxidation of nitrogen containing organic compounds. Oxidation reactions are complex and the amount of nitrate formed by this process depends on specific factors which may vary in swimming pools with different technical equipment. Therefore nitrate is only of limited reliability to estimate fresh water addition in public swimming pools. Main sources for nitrogen containing compounds in pool water are sweat and urine which contain inorganic compounds like potassium. Potassium is a direct indicator of contamination. Its concentration is not influenced by chemical reactions because it is an inert compound. The urine release into the water of indoor pools was estimated by this parameter to be 77.5 ml/person, in outdoor pools about 60 ml. Potassium concentration in swimming pools will reach an equilibrium concentration, depending on the size of the pool, the number of bathers and the amount of fresh water added. This equilibrium concentration is mathematically calculated in a general approach. In none of 36 swimming pools where potassium concentration was measured, this calculated value was exceeded. The results indicate that the potassium concentration is a new valuable parameter to assess the quality of swimming pool water under hygienic aspects.

[Haloforms in hot spring pools]. / Haloforme in Warmsprudelbecken. Haloforms in Spas.

Chloroforme is formed during disinfection of swimming pool water in certain amounts depending on several cofactors. Because of its carcinogenic properties this compound has been frequently a subject of public discussion over the last couple of years. Little is known about chloroforme concentrations in spas. Spas are operated at significantly higher temperatures as compared to other pools, and the organic contamination may be higher. Therefore, chloroforme is possibly produced in higher amounts than in regular swimming pools. On the other hand the air that is blown through the bassin may reduce the concentration of this low volatile substance. In order to investigate the average concentration, 21 water samples from spas in public indoor pools were analyzed as to their chloroforme contend. The median of concentration was 3.8 micrograms/l. The maximum measured chloroforme concentration was 6.4 micrograms/l. The average chloroforme concentration in the filtered water was slightly higher than before filtration. The use of spas does not implicate an increase in chloroforme uptake by bathers.

[Neon-colored plastics for orthodontic appliances. Biocompatibility studies]. / Neonfarbene Kunststoffe für kieferorthopädische Apparaturen. Untersuchungen zur Biokompatibilität.

Public concern and issues of liability have made product safety a major concern throughout the medical field including orthodontics. The purpose of this study was to test the biocompatibility of the new neon colored plastic materials to be used for removable orthodontic appliances before they reach the market and are used in patient treatment. In addition, eight modifications of this synthetic material, which has been used in appliances for many years, were examined without neon color. The procedures established tested for: 1. mutagenicity, 2. toxicity, and 3. irritation of the mucous membrane. As alternatives to using animals the Ames Test, the Agar Overlay Assay, and the HET-CAM Test were employed to test for these properties. The tests revealed that, when the manufacturer's instructions are followed, neither the polymerized materials as used in patient appliances nor the shavings resulting from the orthodontist or the technician grinding the appliance exhibit mutagenic, toxic, or irritating properties.

[Orthodontic materials studied in the HET-CAM test for mucosa-irritating effects]. / Untersuchung kieferorthopädischer Materialien im HET-CAM-Test auf schleimhautreizende Wirkung.

A total of 33 different substances used in orthodontic therapy were investigated for their potential to irritate mucous membranes. As an alternative to animal testing, the HET-CAM test (Hens Eggs Chorioallantois Membrane Test) was employed. The effects of the substances on the vessels of the chorioallantois membrane, such as haemorrhage, lysis, and coagulation, were videotaped and evaluated. When polymerized correctly in accordance with manufacturers' instructions, plastic materials used for removable orthodontic appliances and bracket bonding caused no irritation. Liquid primers for bracket bonding and monomer substances were highly irritating. Etching gels for decalcification of the enamel surfaces were far less irritating than liquid etching substances. Thus, primers should be handled with care; the use of liquid monomers should be avoided in the oral cavity; and etching gels should be preferred to liquid etching substances.

Caffeine-derived N-nitroso compounds. III: Mutagenicity in S. typhimurium and in vitro induction of DNA single-strand breaks in rat hepatocytes by mononitrosocaffeidine and dinitrosocaffeidine.

Mutagenesis in S. typhimurium and in vitro induction of DNA single-strand breaks in primary rat hepatocytes (DNA-SSB) have been investigated for two new N-nitroso compounds, mononitrosocaffeidine (MNC) and dinitrosocaffeidine (DNC). Mononitrosamidocaffeidine (MNAC) and tert.-(butyloxy)carbonyl-mononitrosamidocaffeidine (t-BOC-MNAC), both nitrosated derivatives of caffeidine with nitrosation at methylcarboxamide-N only, were also similarly studied. MNC, an asymmetric nitrosamine, failed to show mutagenicity in any of the tester strains used, and also did not induce DNA-SSB in rat hepatocytes. DNC, having both N-nitrosamide and N-nitrosamine groups in the molecule, showed direct mutagenicity in TA100, TA1535 and TA102. The mutagenic potential of the compound was found to increase on S9 activation. However, it was non-mutagenic in TA98 and TA1537. DNC also exhibited a high potential for inducing alkali-labile DNA-SSB in rat hepatocytes (70-78% C-T value) and was cytotoxic at concentrations over 0.1 mumole/ml. Both MNC and DNC were found to produce formaldehyde on S9 activation. MNAC was not mutagenic directly but showed weak mutagenicity on metabolic activation, whereas t-BOC-MNAC was mutagenic both with and without S9 activation in TA100, TA1535 and TA102. t-BOC-MNAC was more cytotoxic to hepatocytes than MNAC, though both caused DNA-SSB to the same extent (62% C-T value). On the basis of the presented data it is inferred that while DNC is a direct-acting mutagen in TA100, TA1535 and TA102 due to the presence of a reactive N-methylnitrosamido group, its mutagenic potential is greatly enhanced in the presence of S9 possibly due to the synergistic influence of an activated N-methylnitrosamino group in the molecule. Additionally, the study shows a qualitative consistency between Salmonella mutagenicity, genotoxicity in hepatocytes and the reactivity of the methyl group at the nitrosamido-N in nitrosated caffeidine compounds.