Evaluation of copper effects upon Girardia tigrina freshwater planarians based on a set of biomarkers

Copper is a common environmental contaminant, which is particularly toxic to living organisms when in high concentrations. To monitor environmental contamination by Cu2+ and other heavy metals, well characterized bioindicator organisms and standardized assays are needed. As a first step toward this end, we have analysed Cu2+ effects upon Girardia tigrina freshwater planarians, based on the assessment of mobility, regeneration performance, micronucleus (MN) frequency in regenerating animals, and reproductive performance. These four biomarkers provided complementary information on Cu2+ toxicity, teratogenicity, mutagenicity and chronic (>96 h of exposure) effects, respectively. The LC50 was calculated for newborn, adult and regenerating planarians, and values of 12+/-0.02 mg l(-1), 42+/-0.08 mg l(-1), 48+/-0.13 mg l(-1), respectively, were obtained after 96 h of exposure. Mobility, for intact adults, and time of regeneration and MN frequency, for regenerating animals, were significantly affected by Cu2+ concentrations as low as 0.10 mg l(-1). MN assay for regenerating G. tigrina neoblasts showed higher sensitivities than MN assays performed with other bioindicator freshwater organisms, such as moluscs or fish. Chronic exposure effects were clearly evidenced by assessment of reproductive performance, with significant reduction in fecundity and fertility rates upon exposure to Cu2+ concentrations as low as 0.05 mg l(-1). Therefore, G. tigrina can be regarded as a useful bioindicator species for the detection and evaluation of Cu2+ effects upon freshwater invertebrates, allowing insights on the effects of Cu2+ (and possibly other heavy metals) in a freshwater environment.     

Genotoxicity of the organochlorine pesticides 1,1-dichloro-2,2- bis(p-chlorophenyl)ethylene (DDE) and hexachlorobenzene (HCB) in cultured human lymphocytes

The possible genotoxic potential of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), which is a metabolite of dichlorobiphenyltrichloroetane (DDT), and hexachlorobenzene (HCB), which are organochlorine pesticides have been evaluated in vitro by using human lymphocytes as test system. Genetic damage was determined by scoring the frequency of micronuclei (MN) in primary lymphocyte cultures obtained from different donors. The results indicated that, under the experimental conditions used, the DDT metabolite DDE was able to induce significant increases in the frequency of micronucleated cells, which indicate a certain clastogenic and/or aneugenic potential. DDE was tested in the range of 10-80 mM, but the only concentration producing a significant genotoxic effect was 80 mM. On the other hand, HCB was unable to induce a significant increase in the MN frequency in the range of concentrations assayed, from 0.005 to 0.1mM. The selected concentrations of DDE and HCB were chosen according to their toxicity in cell blood cultures; higher concentrations reduced significantly cell proliferation and produced a low frequency of binucleated cells. In conclusion, the results indicate that a genotoxic risk is associated with the exposure to DDE at concentrations 80 mM and above.     

Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.     

Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate) is one of the organophosphate pesticides widely used in agricultural practices throughout world and irreversible inhibitor of cholinesterase in all animal species. Limited efforts have been made to study acute genotoxic effects of chlorpyrifos (CPF) in different tissues of fish using genotoxic biomarkers. Therefore, the present investigation was aimed to study the induction of DNA damage by CPF in freshwater teleost fish Channapunctatus using micronucleus assay (MN assay) and alkaline single-cell gel electrophoresis (comet assay). The value of LC(50) - 96 h of CPF was determined as 811.98 microgl(-1) for C. punctatus, in a semi-static system and on the basis of LC(50) value three acute concentrations viz., 203, 406 and 609 microgl(-1) were determined. The fishes were exposed to the different concentrations of CPF for 96 h and samplings were done at regular intervals for assessment of the MN frequencies and DNA damage. In general, significant effects (P<0.01) from both concentrations and time of exposure were observed in exposed fishes. It was found that the micronucleus induction was highest on 96 h at all concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the lymphocyte and gill cells. This study explored the combined use of micronucleus assay and comet assay for in vivo laboratory studies using fresh water fish for screening the genotoxic potential of xenobiotics.     

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.     

Micronucleus induction by oxidative metabolites of trichloroethylene in cultured human peripheral blood lymphocytes: a comparative genotoxicity study

The genotoxic effects of oxidative metabolites of trichloroethylene (TCE), namely chloral hydrate, trichloroacetic acid (TCA), dichloroacetic acid (DCA), and trichloroethanol (TCEOH) were examined in human peripheral blood lymphocytes. In this context, lymphocytes were exposed in vitro to 25, 50, and 100 μg/ml concentrations of these metabolites separately for a period of 48 h and examined for micronucleus (MN) induction through flow cytometer. At 50 μg/ml TCE metabolites, TCA (6.33?±?0.56 %), DCA (5.06?±?0.55), and TCEOH (4.70?±?1.73) induced highly significant (p?<?0.001) frequency of MN in comparison to control (1.03?±?0.40) suggestive of their genotoxic potential. However, exposure of 100 μg/ml of all the metabolites consistently declined the frequencies of MN which in some cases was equable to that of observed at 25 μg/ml. Further, cytotoxicity and cell cycle disturbances were also measured to find out the association of these endpoints with the MN induction. DNA content analysis revealed 3–4-fold elevation of S-phase at all the concentrations tested. Particularly, at 100 μg/ml, treatment elevation of S-phase was significantly (p?<?0.0001) higher as compared to the control. Present findings together with earlier reports indicate that TCE induces genotoxicity through its metabolites. Interaction of these metabolites with DNA, as evident by elevated S-phase, seems to be the major cause of MN induction. However, involvement of spindle disruption cannot be ruled out. This comparative study also suggests that after TCE exposure, the metabolic efficiency of human to generate oxidative metabolites determines the extent of genotoxicity.     

Illicit drugs as new environmental pollutants: cyto-genotoxic effects of cocaine on the biological model Dreissena polymorpha

The increase in global consumption of illicit drugs has produced not only social and medical problems but also a potential new environmental danger. Indeed, it has been established that drugs consumed by humans end up in surface waters, after being carried through the sewage system. Although many studies to measure concentrations of several drugs of abuse in freshwater worldwide have been conducted, no data have been available to evaluate their potentially harmful effects on non-target organisms until now. The present study represents the first attempt to investigate the cyto-genotoxic effects of cocaine, one of the primary drugs consumed in Western Countries, in the biological model Dreissena polymorpha by the use of a biomarker battery. We performed the following tests on Zebra mussel hemocytes: the single cell gel electrophoresis (SCGE) assay, the apoptosis frequency evaluation and the micronucleus assay (MN test) for the evaluation of genotoxicity and the lysosomal membranes stability test (neutral red retention assay; NRRA) to identify the cocaine cytotoxicity. We exposed the molluscs for 96 h to three different nominal concentrations in water (40 ng L⁻¹; 220 ng L⁻¹; and 10 μg L⁻¹).Cocaine caused significant (p < 0.05) primary DNA damage in this short-term experiment, but it also caused a clear increase in micronucleated cells and a marked rise in apoptosis, which was evident in samples from even the lowest environmental cocaine concentration. Because cocaine decreased the stability of lysosomal membranes, we also highlighted its cytotoxicity and the possible implications of oxidative stress for the observed genotoxic effects.     

Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: plant and human lymphocytes

The environmental fate and behaviour of titanium dioxide (TiO(2)) nanoparticles is a rapidly expanding area of research. There is a paucity of information regarding toxic effect of TiO(2) nanoparticles in plants and to certain extent in humans. The present study focuses on the effect of exposure of TiO(2) nanoparticles in two trophic levels, plant and human lymphocytes. The genotoxicity of TiO(2) nanoparticles was evaluated using classical genotoxic endpoints, comet assay and DNA laddering technique. DNA damaging potential of TiO(2) nanoparticles in Allium cepa and Nicotiana tabacum as representative of plant system could be confirmed in the comet assay and DNA laddering experiments. In Allium micronuclei and chromosomal aberrations correlated with the reduction in root growth. We detected increased level of malondialdehyde (MDA) concentration at 4mM (0.9 μM) treatment dose of TiO(2) nanoparticles in Allium cepa. This indicated that lipid peroxidation could be involved as one of the mechanism leading to DNA damage. A comparative study of the cytotoxic and genotoxic potential of TiO(2) nanoparticles and bulk TiO(2) particles in human lymphocytes also reveal interesting results. While TiO(2) nanoparticles were found to be genotoxic at a low dose of 0.25 mM followed by a decrease in extent of DNA damage at higher concentrations; bulk TiO(2) particles reveal a more or less dose dependent effect, genotoxic only at dose 1.25 mM and above. The study thus confirms the genotoxic potential of TiO(2) nanoparticles in both plant and human lymphocytes.     

Micronucleus distribution in human peripheral blood lymphocytes treated in vitro with cadmium chloride in G0 and S phase of the cell cycle

Cadmium chloride (CdCl2 x H2O) in concentrations 10(-3) - 10(-6) M was tested for genotoxicity in human lymphocytes in vitro. The DNA damage was expressed through the occurrence of micronuclei (MN) and was detected using the cytochalasin-B-blocked MN assay. Human blood was treated in the G0 and S phase of the cell cycle. All except the highest concentration of cadmium chloride of 10(-3) M applied in the G0 phase of the cell cycle resulted in the increase in MN cells, but it was not statistically significant. Cadmium chloride added to the cultures in the concentration of 10(-3) M affected the cell growth regardless of the phase. Cadmium chloride added to cultures 24 h after their initiation (early S phase) was found to significantly increase the MN frequency in 10(-4) - 10(-6) M concentrations (P > 0.05).     

Cytogenetic biomonitoring of primary school children exposed to air pollutants: micronuclei analysis of buccal epithelial cells

There is an increasing attempt in the world to determine the exposures of children to environmental chemicals. To analyze the genotoxic effect of air pollution, micronucleus (MN) assay was carried out in buccal epithelial cells (BECs) of children living in an urban city of Turkey. Children from two schools at urban-traffic and suburban sites were investigated in summer and winter seasons for the determination of BEC-MN frequency (per mille) and frequency of BEC with MN (per mille). The same children were also recruited for lung function measurements within a MATRA project (“Together Towards Clean Air in Eskisehir and Iskenderun”) Measured NO₂ and SO₂ concentrations did not exceed the European Union (EU) limit levels either in urban-traffic or suburban regions. Higher O₃ concentrations were measured in the suburban site especially in the summer period. Particulate matter (PM_2.5 and PM₁₀) levels which did not differ statistically between two regions were above the EU limits in general. Although BEC-MN frequencies of children living in the suburban sites were higher in general, the difference between two regions was not significant either in the summer or winter periods. BEC-MN frequencies of the urban-traffic children were found to be significantly higher in summer period (mean ± SD, 2.68?±?1.99) when compared to winter period (1.64?±?1.59; p?=?0.004). On the other hand, no seasonality was observed for the suburban children. Similar results have been obtained in the BEC frequency with MN in our study. In summer, BEC-MN frequencies were significantly increased with the decrease in pulmonary function levels based on forced expiratory flow between 25 and 75 % of vital capacity (FEF_25–75 %) levels (p?<?0.05). As a conclusion, children living in urban-traffic and suburban areas in the city of Eski?ehir exhibited similar genotoxicity. Seasonal variation in genotoxicity may be interpreted as relatively high ozone levels and increasing time spent at outdoors in the summer.     

Genotoxic evaluation of two mercury compounds in the Drosophila wing spot test

Few studies on the genotoxicity of mercury compounds have been carried out in Drosophila melanogaster, most of them focused in the effects on germinal cells, whereas studies in somatic cells are scarce. In the present study we have analyzed for the first time the genotoxic activity of mercury (II) chloride (MC) and methyl mercury (II) chloride (MMC) in the in vivo wing somatic mutation and recombination test in Drosophila, also known as the wing spot assay. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in larval cells, which are then expressed as spots on the wings of adult flies. The mercury compounds were supplied to third instar larvae (72+/-2h old) at concentrations ranging from 1 to 50 microM for mercury chloride (MC) and from 0.5 to 5 microM for methyl mercury chloride (MMC). Both mercury compounds showed high toxicity; however, MMC was more toxic than MC. The results showed that none of the three categories of mutant spots recorded (small, large, and twin) increased significantly by the treatments, independently of the dose supplied, indicating that the mercury compounds tested exhibit a lack of genotoxic activity in the wing spot assay of D. melanogaster. These results contribute to increase the genotoxicity database on the in vivo evaluation of mercury compounds in Drosophila.     

Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity

The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES) - an environmentally relevant contaminant - was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated.     

A fluorescence-based screening assay for DNA damage induced by genotoxic industrial chemicals

A rapid screening assay to detect chemically-induced DNA damage resulting from exposure of surrogate DNA to genotoxic compounds is reported. This assay is based on changes in the melting and annealing behavior observed for damaged DNA. Exposure of calf thymus DNA to genotoxic industrial chemicals reduced the extent to which the DNA annealed as measured using a double strand DNA selective fluorescent indicator dye. Formaldehyde, acrolein, crotonaldehyde and bromoethane showed the most prominent effects, chloroacetone and allylamine exhibited lesser effects, and acryrlonitrile showed no statistically significant assay response. The assay response for formaldehyde and crotonaldehyde were measured over the concentration range of 10-100 mM and 50-300 mM, respectively. This assay showed little response for the cytotoxic compounds phenol, cyclohexane and toluene but was sensitive to the effects of DNA damaging compounds such as mitomycin C and glutaraldehyde.     

Nitrogen dioxide inhalation induces genotoxicity in rats

Nitrogen dioxide (NO₂) is a ubiquitous reactive free-radical gas, which has been associated with momentary and chronic health effects. In the present study, comet, micronucleus (MN) and DNA–protein crosslinks (DPC) assays were used to investigate the genotoxicity following in vivo inhalation exposure of rats to NO₂. The results show that inhalation exposure of rats to NO₂ induced DNA strand breakage and the formation of DPC in the cells from various internal organs (brain, lung, liver, spleen, kidney and heart), as well as resulted in obvious increase of MN frequency in the bone marrow cells of rats. Furthermore, above genotoxic responses showed significant linear dose-dependent manners. These results implicate that NO₂ is a genotoxic agent and these observations are informative for understanding the mechanisms of adverse effects of nitrogen dioxide.     

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232–23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume’s preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

     

In vitro cytogenetic assessment of trichloroacetic acid in human peripheral blood lymphocytes

Trichloroacetic acid (TCA), a common water disinfection byproduct and a persistent metabolite of trichloroethylene (TCE), has been examined for its genotoxic potential in human lymphocytes. Chromosomal aberration (CA) and cytokinesis-block micronucleus (CBMN) assay were employed to assess the toxicity of TCA. Lymphocytes obtained from three healthy donors were exposed to 25, 50, and 100 μg/ml concentration of TCA separately. TCA exposure resulted in chromosomal anomalies and the formation of micronuclei in lymphocytes. Chromosome analysis revealed the dose-dependent and significant induction of CA. Chromatid break/chromosome break, fragments, and chromatid exchanges were commonly observed. Exposure of higher concentration (50 and 100 μg/ml) significantly inhibited mitotic index. Data obtained with CBMN assay indicated that the induction of micronucleus (MN) formation was greater than that of CA. At 25 μg/ml, TCA induced significant frequencies of MN as compared to control cells. Significant induction of MN at the lowest concentration indicates TCA may also interact with mitotic spindles. Lower percentage of CA and MN at 100 μg/ml as compared to 50 μg/ml indicates occurrence of severe cytotoxicity on exposure of 100 μg/ml TCA in lymphocytes. Collectively, results of both cytogenetic assays indicate that exposure of TCA can induce significant genotoxic and cytotoxic effects.     

Median lethal concentration of formaldehyde and its genotoxic potential in bullfrog tadpoles (Lithobates catesbeianus)

In order to avoid that contaminated frog farms animals escaping in the environment and become potential vector of emergent diseases, studies with disinfection protocol are strictly necessary. The formaldehyde is one of the compounds tested in fungal disinfection protocols and also used in aquaculture. This study aimed to determine the median lethal concentration (LC_50–96h) of formaldehyde in bullfrog tadpoles and to evaluate the possible genotoxic effects in acute exposition. Accordingly, the animals were exposed to formaldehyde in the concentrations of 6, 9, 12, 15, and 18 mg L^?1, and after 96 h blood samples were drawn for the micronucleus (MN) test. The LC_50–96h was 10.53 mg L^?1, and the MN frequency increased in proportion to the formaldehyde concentrations, with an estimated frequency in the negative control being 1.35 MN/individual. We concluded that formaldehyde is genotoxic to tadpoles of bullfrogs in the tested concentrations, and the choice of this chemical should be contemplated before its use in animals in captivity.     

In vivo genotoxicity assessment of titanium,zirconium and aluminium nanoparticles,and their microparticulated forms,in Drosophila

As in vivo system, we propose Drosophila melanogaster as a useful model for study the genotoxic risks associated with nanoparticle exposure. In this study we have carried out a genotoxic evaluation of titanium dioxide (TiO₂), zirconium oxide (ZrO₂) and aluminium oxide (Al₂O₃) nanoparticles and their microparticulated forms in D. melanogaster by using the wing somatic mutation and recombination assay. This assay is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in treated larvae, which are expressed as mutant spots on the wings of adult flies. Third instar larvae were feed with TiO₂, ZrO₂ and Al₂O₃ nanoparticles, and their microparticulated forms, at concentrations ranging from 0.1 to 10 mM. Although a certain level of aggregation/agglomeration was observed in solution, it must be noted than the constant digging activity of larvae ensures that treated medium pass constantly through the digestive tract ensuring exposure. The results showed that no significant increases in the frequency of all spots (e.g. small single, large single, twin, total mwh and total spots) were observed, indicating that these nanoparticles were not able to induce genotoxic activity in the wing spot assay of D. melanogaster. Negative data were also obtained with the microparticulated forms. This indicates that the nanoparticulated form of the selected nanomaterials does not modify the potential genotoxicity of their microparticulated versions. These in vivo results contribute to increase the genotoxicity database on the TiO₂, ZrO₂ and Al₂O₃ nanoparticles.     

Leachates of municipal solid waste incineration bottom ash from Macao: heavy metal concentrations and genotoxicity

Heavy metals in municipal solid waste incineration bottom ash (MSWIBA) may leach into soil and groundwater and pose long-term risks to the environment. In this study, toxicity characteristic leaching procedure (TCLP) was carried out on the MSWIBA from Macao. Heavy metals in leachates were determined by inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES), and genotoxicity of leachates was also evaluated by micronucleus (MN) assay with Vicia faba root tip cells. The results showed that the concentrations of aluminium (Al), manganese (Mn), cobalt (Co), cadmium (Cd) and mercury (Hg) in the leachates were less than 0.01 mg l(-1), and those of iron (Fe), copper (Cu) and molybdenum (Mo) were less than 0.1 mg l(-1). The concentrations of chromium (Cr), zinc (Zn), selemium (Se), strontium (Sr), barium (Ba) and caesium (Cs) were between 0.11 mg l(-1) and 2.19 mg l(-1). Lead (Pb) concentrations, in particular, reached as high as 19.6 mg l(-1), significantly exceeding the maximum concentration limit (5 mg l(-1) for lead by TCLP). Compared with the negative group, a significant increase of MN frequencies was observed in the leachate-exposed groups (P<0.05). With the increase of heavy metals in the leachates, the toxic effects on the Vicia faba root tip cells increased, implying that heavy metals were the main factors causing the genotoxic effects. Our results suggested that apart from chemical analysis, bioassays like the MN assay of Vicia faba root tip cells should also be included in a battery of tests to assess the eco-environmental risks of bottom ashes before decisions can be made on the utilization, treatment or disposal.     

Genotoxic effect of ciprofloxacin during photolytic decomposition monitored by the in vitro micronucleus test (MNvit) in HepG2 cells

Purpose

Ciprofloxacin (CIP), a broad-spectrum, second-generation fluoroquinolone, has frequently been found in hospital wastewaters and effluents of sewage treatment plants. CIP is scarcely biodegradable, has toxic effects on microorganisms and is photosensitive. The aim of this study was to assess the genotoxic potential of CIP in human HepG2 liver cells during photolysis.

Methods

Photolysis of CIP was performed in aqueous solution by irradiation with an Hg lamp, and transformation products were monitored by HPLC-MS/MS and by the determination of dissolved organic carbon (DOC). The cytotoxicity and genotoxicity of CIP and of the irradiated samples were determined after 24?h of exposure using the WST-1 assay and the in vitro micronucleus (MN) test in HepG2 cells.

Results

The concentration of CIP decreased during photolysis, whereas the content of DOC remained unchanged. CIP and its transformation products were not cytotoxic towards HepG2 cells. A concentration-dependent increase of MN frequencies was observed for the parent compound CIP (lowest observed effect level, 1.2???mol?L^?1). Furthermore, CIP and the irradiated samples were found to be genotoxic with a significant increase relative to the parent compound after 32?min (P?P?Conclusions Photolytic decomposition of aqueous CIP leads to genotoxic transformation products. This proves that irradiated samples of CIP are able to exert heritable genotoxic effects on human liver cells in vitro. Therefore, photolysis as a technique for wastewater treatment needs to be evaluated in detail in further studies, not only for CIP but in general.