Effect-based evaluation of ozone treatment for removal of micropollutants and their transformation products in waste water.

The aim of this interdisciplinary research project in North Rhine-Westphalia (NRW), Germany, entitled "Elimination of pharmaceuticals and organic micropollutants from waste water" involved the conception of cost-effective and innovative waste-water cleaning methods. In this project in vitro assays, in vivo assays and chemical analyses were performed on three municipal waste-water treatment plants (WWTP). This publication focuses on the study of the in vitro bioassays. Cytotoxic, estrogenic, genotoxic and mutagenic effects of the original as well as enriched water samples were monitored before and after wastewater treatment steps using MTT and PAN I, ER Calux and A-YES, micronucleus and Comet assays as well as AMES test. In most cases, the measured effects were reduced after ozonation, but in general, the biological response depended upon the water composition of the WWTP, in particular on the formed by-products and concentration of micropollutants. In order to be able to assess the genotoxic and/or mutagenic potential of waste-water samples using bioassays like Ames test, Comet assay or micronucleus test an enrichment of the water sample via solid-phase extraction is recommended. This is in agreement with previous studies such as the "ToxBox"-Project of the Environmental Agency in Germany.

In vitro characterization of cutaneous immunotoxicity of immortalized human keratinocytes (HaCaT) exposed to reactive and disperse textile dyes.

Several synthetic dyes are used by textile industry for supplying the market of colored clothes. However, these chemicals have been associated with a variety of adverse human health effects, including textile dermatitis. Thus, there is a growing concern to identify textile dyes potentially as skin immunotoxicants. The aim of this in vitro study was to characterize the immunotoxic potential of reactive (Reactive Green 19 [RG19], Reactive Blue 2 [RB2], Reactive Black 5 [RB5]) and disperse (Disperse Red 1 [DR1]) textile dyes using a dermal cell line. For this purpose, a cell-based approach was conducted with immortalized human keratinocytes (KC) (HaCaT) using selected biomarkers of cutaneous inflammation including modulation of matrix metalloproteinases (MMP), oxidative stress such as reactive oxygen species (ROS) generation, and inflammatory cytokine profile. DR1 was the only dye able to trigger an immune response such as release of IL-12 cytokine, a potent co-stimulator of T helper 1 cell, which may be considered as a skin immunotoxicant. The reactive dyes including RB5 that were previously reported as skin sensitizers failed to induce inflammatory reactions under the conditions tested. The reactive dyes studied may pose a risk to human KC by induction of effects related to modulation of MMP-2 (RB5) and -9 (RB5 and RB2) and generation of ROS (RG19 and RB2). Thus, all these dyes need to be used with caution to avoid undesirable effects to consumers who may be exposed dermally.

Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international inter-laboratory scoring exercise.

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers.

Cytotoxic and genotoxic activities of waters and sediments from highway and parking lot runoffs.

The genotoxicity of water and sediment samples from stormwater treatment systems and water from urban highway runoff was tested in the Salmonella/microsome assays with Salmonella typhimurium, micronucleus assay (Trad-MN) with plants and with human-derived liver cells (HepG2), or comet assay with HepG2. Cytotoxicity of water samples was studied using either reactive oxygen species (ROS) generation, cell proliferation or dye exclusion assay in HepG2. Concentrations of several contaminants in the tested samples were also measured. Results suggested that urban highway runoff exposed to severe vehicle traffic emissions caused genotoxic effects in comet assay and in Trad-MN assays. Sediments induced either mutagenic effects in strain YG1024 or genotoxic effects in Trad-MN assay. These effects could be due to the presence of nitro-polycyclic aromatic hydrocarbons (NPAHs) which possess carcinogenic and mutagenic properties. Influent and effluents of stormwater treatment systems did not induce genotoxic activity or effects on HepG2 cell viability; however, the influents were able to induce ROS generation and cell proliferation in HepG2 cells. As the methods require a sterile filtration of the water samples, this could have also removed particulate-associated polycyclic aromatic hydrocarbons (PAHs) and resulted in a less pronounced induction of genotoxicity, as would be expected by PAH contamination.

Investigation of the in vitro toxicological properties of the synthetic cannabimimetic drug CP-47,497-C8.

Cannabicyclohexanol (CP-47,497-C8) is a representative of a group of cannabimimetic cyclohexylphenols which is added to herbal mixtures as a cannabis substitute since 2008. Although in the beginning CP-47,497-C8 was the main ingredient of "Spice" and similar products, it was partly replaced by aminoalkylindole-type cannabinoid receptor agonists like JWH-018, JWH-073 or JWH-250, but never completely disappeared from the market. Since information on its toxicological properties is scarce, we investigated the effects of the drug in human derived cell lines. The cytotoxic effects were studied in a panel of assays (SRB, XTT, LDHe and NR tests) in a buccal derived (TR146) and a liver derived (HepG2) cell line. The strongest effects were seen in the two former assays at levels ≥ 7.5 µM indicating that the compound interferes with protein synthesis and causes membrane damage. In additional comet assays, DNA damage was detected at levels ≥10 µM. Experiments with lesion specific enzymes showed that these effects are not due to oxidative damage of DNA bases. The negative findings obtained in Salmonella/microsome assays and the positive results of micronucleus tests with the cell lines indicate that the compound does not cause gene mutations but acts on the chromosomal level. In contrast to other synthetic cannabinoids, no indication for estrogenic/antiestrogenic properties was seen in a luciferase assay with bone marrow derived U2-OS cells. In conclusion, our findings show that the drug has only weak cytotoxic properties. However, the induction of chromosomal damage indicates that it may cause adverse effects in users due to its impact on the stability of the genetic material.

Toxicity studies of nonylphenol and octylphenol: hormonal, hematological and biochemical effects in Clarias gariepinus.

Among the numerous chemicals discharged into aquatic ecosystems, nonylphenol (NP) and octylphenol (OP) have been shown to have a potent effect on the endocrine system of fish; this issue has been clearly dealt with in several studies. The objective of this study was to assess and compare the general toxicity of these estrogenic chemicals individually on Clarias gariepinus. Fish were exposed to different concentrations of both NP and OP (250, 500, 750 and 1000 µg l(-1) ) under semi-static conditions for a period of 7 days. The adverse effect was evaluated with use of blood cell counting, hemoglobin (Hb), hematocrit (HCT), hematimetric indices, bilirubin, protein, glucose, serum transaminases, serum phosphatases, lactate dehydrogenase and cortisol. The results showed a clear indication of anemia, increases in leukocyte count and bilirubin content and a reduction in plasma protein levels with higher concentrations of both the toxicants compared with controls. Furthermore, with all the concentrations the inevitable increase in serum cortisol and plasma glucose showed primary and secondary stress responses. Moreover, probable tissue damage gave rise to a series of fluctuations of enzyme levels at lower concentrations, but a decrease with higher concentrations showed the severity of the effect. Depending on the parameters examined, OP had a relatively greater effect than NP. Overall, these two chemicals seemingly affected hematology and the activity of some enzymes, leading to serious impairment of the metabolism and physiology of C. gariepinus.

Cytotoxicity of water-soluble fraction from biodiesel and its diesel blends to human cell lines.

The designation of biodiesel as a green fuel has increased its commercialization and use, making its fate in the environment a matter of concern. Fuel spills constitute a major source of aquatic pollution and, like diesel spills, biodiesel can produce adverse effects on aquatic environments, animals and humans. The present study assessed cytotoxic effects of water systems contaminated with neat biodiesel and its diesel blends by means of different procedures on human T cell leukemia (Jurkat) and human hepatocellular carcinoma (HepG2) cells [detection of changes in mitochondrial membrane potential (ΔΨ(m)) using tetramethylrhodamine ethyl ester (TMRE), apoptosis recognition by Annexin V and impedance real-time cell analyzer (xCELLigence™ system)]. The data obtained showed concordance across the different bioassays, with cytotoxic effects observed as a dose-dependent response only for waters contaminated with pure diesel (D100) and B5 blend, which is characterized by a mixture of 95% diesel and 5% biodiesel. The data can also lead us to hypothesize that diesel accounts for the harmful effects observed, and that biodiesel does not worsen the impacts caused by diesel pollution.

Chemical and in vitro bioanalytical assessment of drinking water quality in Manhiça, Mozambique.

BACKGROUND: The chemical quality of drinking water is widely unknown in low-income countries. OBJECTIVE: We conducted an exploratory study in Manhiça district (Mozambique) to evaluate drinking water quality using chemical analyses and cell-based assays. METHODS: We measured nitrate, fluoride, metals, pesticides, disinfection by-products, and industrial organochlorinated chemicals, and conducted the bioassays Ames test for mutagenicity, micronuclei assay (MN-FACS), ER-CALUX, and antiAR-CALUX in 20 water samples from protected and unprotected sources. RESULTS: Nitrate was present in all samples (median 7.5 mg/L). Manganese, cobalt, chromium, aluminium, and barium were present in 90-100% of the samples, with median values of 32, 0.6, 2.0, 61, 250 µg/l, respectively. Manganese was above 50 µg/l (EU guideline) in eight samples. Arsenic, lead, nickel, iron, and selenium median values were below the quantification limit. Antimony, cadmium, copper, mercury, zinc and silver were not present. Trihalomethanes, haloacetic acids, haloacetonitriles and haloketones were present in 5-28% samples at levels ≤4.6 µg/l. DDT, dieldrin, diuron, and pirimiphos-methyl were quantified in 2, 3, 3, and 1 sample, respectively (range 12-60 ng/L). Fluoride was present in one sample (0.11 mg/l). Trichloroethene and tetrachloroethene were not present. Samples were negative in the in vitro assays. SIGNIFICANCE: Results suggest low exposure to chemicals, mutagenicity, genotoxicity and endocrine disruption through drinking water in Manhiça population. High concentration of manganese in some samples warrants confirmatory studies, given the potential link to impaired neurodevelopment.

Genotoxic effects of wastewater from an oncological ward.

Aim of this study was the evaluation of the genotoxic activities of hospital wastewaters. Samples from an oncological ward of the general hospital of Vienna, Austria, were tested in the Salmonella/microsome assay in strains TA98, TA100 and TA1535 with or without metabolic activation, and in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes. In the bacterial tests, consistently negative results were obtained while in the experiments with liver cells a significant and dose-dependent induction of DNA damage (up to two-fold over the background) was found. Membrane filtration resulted in a substantial (62-77%) reduction of these effects, while additional treatments (activated carbon filtration and UV-irradiation) did not lead to a further decrease of the genotoxic activity of the samples. SCGE experiments with cisplatin, carboplatin and 5-fluorouracil, which were detected in the water samples, showed that these cytostatics cause a significant induction of DNA damage only at concentrations that are substantially higher than those in the native waters. These findings indicate that other chemicals, possibly quaternary ammonium compounds, account for the effects of the hospital wastewaters.

Exposure to disinfection by-products in swimming pools and biomarkers of genotoxicity and respiratory damage - The PISCINA2 Study.

BACKGROUND: Swimming in pools is a healthy activity that entails exposure to disinfection by-products (DBPs), some of which are irritant and genotoxic. OBJECTIVES: We evaluated exposure to DBPs during swimming in a chlorinated pool and the association with short-term changes in genotoxicity and lung epithelium permeability biomarkers. METHODS: Non-smoker adults (N = 116) swimming 40 min in an indoor pool were included. We measured a range of biomarkers before and at different times after swimming: trihalomethanes (THMs) in exhaled breath (5 min), trichloroacetic acid (TCAA) in urine (30 min), micronuclei in lymphocytes (1 h), serum club cell protein (CC16) (1 h), urine mutagenicity (2 h) and micronuclei in reticulocytes (4 days in a subset, N = 19). Several DBPs in water and trichloramine in air were measured, and physical activity was extensively assessed. We estimated interactions with polymorphisms in genes related to DBP metabolism. RESULTS: Median level of chloroform, brominated and total THMs in water was 37.3, 9.5 and 48.5, µg/L, respectively, and trichloramine in air was 472.6 µg/m3. Median exhaled chloroform, brominated and total THMs increased after swimming by 10.9, 2.6 and 13.4, µg/m3, respectively. Creatinine-adjusted urinary TCAA increased by 3.1 µmol/mol. Micronuclei in lymphocytes and reticulocytes, urine mutagenicity and serum CC16 levels remained unchanged after swimming. Spearman correlation coefficients showed no association between DBP exposure and micronuclei in lymphocytes, urine mutagenicity and CC16. Moderate associations were observed for micronuclei in reticulocytes and DBP exposure. CONCLUSIONS: The unchanged levels of the short-term effect biomarkers after swimming and null associations with personal estimates of exposure to DBPs suggest no measurable effect on genotoxicity in lymphocytes, urine mutagenicity and lung epithelium permeability at the observed exposure levels. The moderate associations with micronuclei in reticulocytes require cautious interpretation given the reduced sample size.

Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors.

Carcinogenesis of squamous cell carcinomas in the upper aero-digestive tract (UADT) is a multi-stage process. Since 1937, micronuclei (MN) have been considered a marker for genome damage in the initiation stage. By help of the micronucleus test, carcinogenic exposure can be proven in the mucosa area of the UADT. The hypothesis to be tested was that individual oral hygiene and the dental status, respectively - just like alcohol and tobacco abuse - are associated with the micronucleus rate in cytological preparations of the buccal mucosa. In a prospective clinical observation study, we determined in 100 probands the micronucleus frequency per 1000 mucosa epithelial cells. Study participants with a high number of missing teeth (M/T index, p=0.037), a below-average papillary bleeding index (PBI, p=0.032) and periodontal status, respectively (PSI, p=0.042) possessed a higher micronucleus number in comparison with restored dental conditions. Probands with composite restorations displayed a higher MN rate (p=0.006) compared to those with amalgam. However, we could not detect any significant relation with the prosthetic status (p> or =0.075). An adjustment was made according to alcohol and tobacco. We therefore conclude that subgingival plaque and synthetic dental materials in addition to chronic alcohol and tobacco consumption might have genotoxic relevance in the oral cavity.

A hierarchical testing strategy for micropollutants in drinking water regarding their potential endocrine-disrupting effects-towards health-related indicator values.

In Germany, micropollutants that (may) occur in drinking water are assessed by means of the health-related indicator value (HRIV concept), developed by the German Federal Environment Agency. This concept offers five threshold values (≤ 0.01 to ≤ 3 µg l-1) depending on availability and completeness of data regarding genotoxicity, neurotoxicity, and germ cell-damaging potential. However, the HRIV concept is yet lacking integration of endocrine disruptors as one of the most prominent toxicological concerns in water bodies, including drinking water. Thresholds and proposed bioassays hence urgently need to be defined. Since endocrine disruption of ubiquitary chemicals as pharmaceuticals, industrial by-products, or pesticides is a big issue in current ecotoxicology, the aim of this study was to explore endocrine effects, i.e., estrogenic and androgenic effects, as an important, additional toxicological mode of action for the HRIV concept using a hierarchical set of well-known but improved bioassays. Results indicate that all of the 13 tested substances, industrial chemicals and combustion products (5), pharmaceuticals and medical agents (4), and pesticides and metabolites (4), have no affinity to the estrogen and androgen receptor in human U2OS cells without metabolic activation, even when dosed at their water solubility limit, while in contrast some of these substances showed estrogenic effects in the RYES assay, as predicted in pre-test QSAR analysis. Using a specifically developed S9-mix with the U2OS cells, those micropollutants, i.e., Benzo[a]pyrene, 2,4-Dichlorophenol, 3,3-Dichlorbenzidin, 3,4-Dichloranilin, and diclofenac, they show estrogenic effects at the same concentration range as for the yeast cells. Three of the drinking water-relevant chemicals, i.e., atrazine, tributyltin oxide, and diclofenac, caused effects on hormone production in the H295R assay, which can be correlated with changes in the expression of steroidogenic genes. One chemical, 17α-Ethinylestradiol, caused an estrogenic or anti-androgenic effect in the reproduction test with Potamopyrgus antipodarum. Considering these results, a proposal for a test strategy for micropollutants in drinking water regarding potential endocrine effects (hormonal effects on reproduction and sexual development) will be presented to enhance the existing HRIV concept.

Quaternary (triphenyl-) phosphonium compounds: Environmental behavior and toxicity.

An analytical method based on high resolution mass spectrometry coupled with liquid chromatography (LC-HRMS) for 25 quaternary phosphonium compounds (QPCs) and derived phosphine oxides (POs) was developed and validated. To investigate the occurrence and fate of QPCs in the aquatic environment, water, suspended solids and sediments from the rivers Rhine and Elbe (upper and middle Elbe as well as tidal Elbe) were analyzed, as well as samples from tributaries bearing significant loads of QPCs. For the first time, the quaternary phosphonium compound tetrabutylphosphonium (Bu4P+) was detected. In the river Elbe concentrations were determined of up to 4700 ng/L (surface water) and 1000 µg/kg (sediment), respectively. Analysis of a time series of suspended solids (2005-2015) showed that QPCs have been present in the Elbe and Rhine catchment for at least one decade, with partly rising tendency. A degradation experiment with Rhine sediment revealed that triphenylphosphonium compounds (R-Ph3P+) and Bu4P+ are persistent in contact with sediment and suspended solids and tend to sorb onto sediment particles. Toxicological studies (reactive oxygen species (ROS) after substance exposure, Ames test, Micronucleus test, determination of cytotoxicity) with selected QPCs confirmed that all of them exhibit cytotoxicity and some even genotoxic potential at elevated concentrations, which emphasizes the need for an emission regulation of these compounds.

Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), two naturally occurring benzoxazinones contained in sprouts of Gramineae are potent aneugens in human-derived liver cells (HepG2).

Benzoxazinoids (BAs) are toxic constituents of sprouts of Gramineae such as wheat, maize and rye and are part of the plant defence system against pests. In the last years, sprouts have been increasingly consumed as health foods and are also used for the production of dietary supplements. In the present study we investigated the mutagenic activities of the two most abundant BAs, namely 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in the Salmonella/microsome assay and additionally, in micronucleus (MN) assay and single cell gel electrophoresis (SCGE) assay in a human-derived liver cell line (HepG2). DIBOA caused significant induction of his(+) revertants in all three strains in the range between 0.02 and 0.50 mg/plate; the highest activity was observed in TA100 (fivefold increase over the background at the highest dose level). The effect in YG1024 (a derivative of TA98 with increased acetyltransferase activity) was only slightly higher than the effect in the parental strain indicating that acetylation plays no crucial role in the activation of this BA. DIMBOA was in general less active and a positive result was only seen in the base substitution strain (TA100). Addition of rat liver homogenate (S9-mix) led to a significant (ca. twofold) increase of the mutagenic activities of both BAs. In SCGE assays with HepG2 cells consistently negative results were obtained with both compounds whereas in MN assays significant dose dependent effects were observed under similar experimental conditions. DIMBOA caused significant effects already at concentrations > or =1 microM; at the highest dose (20 microM) the MN frequency was sevenfold higher than the background level. DIBOA caused weaker effects and was positive at doses > or =2.5 microM, the maximal induction (twofold over background) was observed with 20 microM. Overall, DIMBOA was ca. 30-fold more active as DIBOA. Subsequent experiments with pancentromeric probes showed that >80% of the MN induced at the highest doses gave a centromere positive signal indicating that both BAs are aneugenic. This is an interesting observation as it is assumed that aneuploidy is a key event in cancer induction and at present no other aneugenic plant-derived substances of dietary relevance are known.

Saliva as a biomarker for head and neck squamous cell carcinoma: in vitro detection of cytotoxic effects by using the plating efficiency index.

Worldwide, the incidence of head and neck squamous cell carcinoma (HNSCC) is increasing. The development of an effective prevention program would provide a promising opportunity to control this disease. We applied the 'plating efficiency index' of Chinese hamster lung fibroblasts to demonstrate the presence of cytotoxic effects in saliva samples from cancer patients as well as from healthy probands. Correlations between individual risk factors and the cytotoxic effects of saliva specimens were analysed and evaluated. Saliva samples were obtained from male patients (n=43) with carcinomas of the upper aerodigestive tract, and from a healthy age-matched male control group (n=131) with different tobacco and alcohol consumption habits. The extraction of non-stimulated sober saliva was chosen for this investigation. In vitro cytotoxicity of the saliva was detected using the permanent mammalian cell line V79 (lung fibroblasts) of Chinese hamsters. The determination of the plating efficiency index was made in the logarithmic growth phase of the initial cell culture. A reduction in cell colonies (plating efficiency) of at least 50% was defined as strongly cytotoxic. A significant direct correlation was found between daily tobacco consumption and the reduction of plating efficiency (p<0.0001). We found only a moderate increase in the cytotoxicity of tested saliva samples in correlation with daily alcohol uptake. The difference between tumor patients and healthy probands was highly significant (chi(2) test; p=0.001). Using the method of logistic regression analysis, we found a 3.6-fold increased cancer risk in probands with cytotoxic saliva (p<0.001). According to our results, the plating efficiency index seems to be a suitable method for the detection of increased cancer risk. In combination with several effective biomarkers on cytogenetic end-points, it may help to establish biomonitoring programs for secondary cancer prevention.

The influence of oral hygiene on salivary quality in the Ames Test, as a marker for genotoxic effects.

Squamous cell carcinomas of the upper aerodigestive tract are a global health-political challenge. Accordingly, current research efforts are aimed towards the opportunities for early recognition of risk patients, and at the recognition of risk factors related to carcinogenesis. We determined the revertant number of the variety Salmonella typhimurium TA 98 and TA 100 after incubation, with saliva samples from 100 probands as a measure of a genotoxic environment within the oral cavity, depending on the dental status as measure of oral health. Beside chronic alcohol (p=0.032) and tobacco consumption (p<0.001), the dental status displayed in partial aspects (high plaque index, high number of decayed teeth, prosthetically not rehabilitated status, p or= 0.104). Therefore, it can be concluded that the polymicrobial supragingival plaque, as a possible independent factor, possesses a relevant mutagenic interaction with saliva, and that individual oral health is a co-factor in the development of carcinomas in the oral cavity.

Genotoxicity of nitrosulfonic acids, nitrobenzoic acids, and nitrobenzylalcohols, pollutants commonly found in ground water near ammunition facilities.

2-Amino-4,6-dinitrobenzoic acid (2-A-4,6-DNBA), 4-amino-2,6-dinitrobenzoic acid (4-A-2,6-DNBA), 2,4,6-trinitrobenzoic acid (2,4,6-TNBA), 2-amino-4, 6-dinitrobenzylalcohol (2-A-4,6-DNBAlc), 4-amino-2,6-dinitrobenzylalcohol (4-A-2,6-DNBAlc), 2,4-dinitrotoluol-5-sulfonic acid (2,4-DNT-5-SA), 2,4-dinitrotoluol-3-sulfonic acid (2,4-DNT-3-SA), and 2, 4-dinitrobenzoic acid (2,4-DNBA) are derivatives of nitro-explosives that have been detected in groundwater close to munitions facilities. In the present study, the genotoxicity of these compounds was evaluated in Salmonella/microsome assays (in strains TA100 and TA98, with and without S9 and in TA98NR without S9), in chromosomal aberration (CA) tests with Chinese hamster fibroblasts (V79), and in micronucleus (MN) assays with human hepatoma (HepG2) cells. All compounds except the sulfonic acids were positive in the bacterial mutagenicity tests, with 2,4,6-TNBA producing the strongest response (8023 revertants/micromol in TA98 without S9 activation). 2-A-4,6-DNBA was a direct acting mutagen in TA98, but negative in TA100. The other positive compounds were approximately 1-3 orders of magnitude less mutagenic than 2,4,6-TNBA in TA98 and in TA100; relatively strong effects ( approximately 50-400 revertants/micromol) were produced by the benzylacohols in the two indicator strains. With the exception of 2,4-DNBA, the mutagenic responses were lower in the nitroreductase-deficient strain TA98NR than in the parental strain. 2,4-DNBA produced a marginally positive response in the V79-cell CA assay; the other substances were devoid of activity. Only the benzoic acids were tested for MN induction in HepG2 cells, and all produced positive responses. As in the bacterial assays, the strongest effect was seen with 2,4,6-TNBA (significant induction at >or=1.9 microM). 4-A-2,6-DNBA was positive at 432 microM; the weakest effect was observed with 2,4,-DNBA (positive at >or=920 microM). The differences in the sensitivity of the indicator cells to these agents can be explained by differences in the activities of enzymes involved in the activation of the compounds. The strong responses produced by some of the compounds in the human-derived cells suggest that environmental exposure to these breakdown products of nitro-explosives may pose a cancer risk in man.

Genotoxic properties of representatives of alkylindazoles and aminoalkyl-indoles which are consumed as synthetic cannabinoids.

Synthetic cannabinoids (SCs) cause similar effects as cannabis and are sold in herbal mixtures. Recent investigations indicate that some of these drugs possess genotoxic properties. Therefore, we tested representatives of two groups, namely, aminoalkylindoles (AM-2201 and UR-144) and 1-alkylindazoles (5F-AKB-48 and AM-2201-IC) in single cell gel electrophoresis and micronucleus (MN) assays with human lymphocytes and in Salmonella/microsome assays. All drugs except AM-2201 caused DNA-migration, the LOELs were between 50 and 75 µM. Furthermore, all SCs caused inhibition of cell division and significant induction of MN which reflect structural and numerical chromosomal aberrations. The LOEL values were 50 µM for UR-144 and 5-AKB-48 and 75 µM for the other drugs. Also the levels of nucleoplasmatic bridges which are formed from dicentric chromosomes were elevated under identical conditions while the frequencies of nuclear buds were not affected. These findings show that representatives of both groups cause chromosomal damage while the negative results in Salmonella assays (in strains TA98, TA100, TA1535, TA1537 and TA102) in absence and presence of metabolic activation indicate that they do not induce gene mutations. Taken together, these findings indicate that SCs may cause adverse health effects in users as a consequence of damage of the genetic material.

On the relevance of genotoxicity for fish populations II: genotoxic effects in zebrafish (Danio rerio) exposed to 4-nitroquinoline-1-oxide in a complete life-cycle test.

In order to characterize the impact of genotoxic potentials on populations of aquatic organisms in surface waters, zebrafish (Danio rerio) were exposed to the model genotoxicant 4-nitroquinoline-1-oxide (NQO) in a complete life-cycle test. Fish exposed to mean NQO concentrations of 0, 0.1, 0.3, 1.1, and 2.9 microg/l were examined by several genotoxicity assays with different endpoints. Assays included the unscheduled DNA synthesis (UDS) test, the comet assay, the alkaline filter elution, and the micronucleus test. The genotoxicity assays revealed an increasing genotoxicity, ranging from induction of DNA repair (even at the lowest concentration tested) to primary and secondary DNA alterations at higher concentrations of 1.1 and 2.9 microg/l NQO. Whether the lowered reproductivity observed in the life-cycle test is caused by genotoxic pathways of NQO, remains unclear. However, the results indicate a contradiction to an earlier assumption that genotoxicants as found in the environment are likely to not impact natural populations.

Emerging risks from ballast water treatment: the run-up to the International Ballast Water Management Convention.

Uptake and discharge of ballast water by ocean-going ships contribute to the worldwide spread of aquatic invasive species, with negative impacts on the environment, economies, and public health. The International Ballast Water Management Convention aims at a global answer. The agreed standards for ballast water discharge will require ballast water treatment. Systems based on various physical and/or chemical methods were developed for on-board installation and approved by the International Maritime Organization. Most common are combinations of high-performance filters with oxidizing chemicals or UV radiation. A well-known problem of oxidative water treatment is the formation of disinfection by-products, many of which show genotoxicity, carcinogenicity, or other long-term toxicity. In natural biota, genetic damages can affect reproductive success and ultimately impact biodiversity. The future exposure towards chemicals from ballast water treatment can only be estimated, based on land-based testing of treatment systems, mathematical models, and exposure scenarios. Systematic studies on the chemistry of oxidants in seawater are lacking, as are data about the background levels of disinfection by-products in the oceans and strategies for monitoring future developments. The international approval procedure of ballast water treatment systems compares the estimated exposure levels of individual substances with their experimental toxicity. While well established in many substance regulations, this approach is also criticised for its simplification, which may disregard critical aspects such as multiple exposures and long-term sub-lethal effects. Moreover, a truly holistic sustainability assessment would need to take into account factors beyond chemical hazards, e.g. energy consumption, air pollution or waste generation.