Donatella Feretti

The influence of different disinfectants on mutagenicity and toxicity of urban wastewater

Many human pathogenic micro-organisms can be transmitted by waters contaminated by wastewater effluents. Wastewater disinfection helps prevent the spread of these pathogens in aquatic environments. Chlorination is the most widely used method for the disinfection of wastewater, but can cause the formation of mutagenic/carcinogenic and toxic by-products which are potentially harmful to human and aquatic organisms. The aim of this research was to study the influence of disinfectants alternative to chlorine, such as chlorine dioxide, ozone, peracetic acid and UV radiation, on the formation of mutagenic and toxic compounds in wastewater. Wastewater samples were collected before and after disinfection in summer and in winter and then adsorbed using silica C18 cartridges. Concentrates were tested for mutagenicity using the Ames test. For toxicity test, a bioluminescence assay using Vibrio fischeri photobacteria was employed. To detect DNA damage activity non-concentrated water samples were tested using two genotoxicity plant tests (Allium test and Tradescantia/micronuclei test). All disinfectant treatments produced bacterial mutagenicity, particularly after treatment with ClO2 or ozone. The Allium cepa test gave positive results only for PAA-treated wastewater sampled in winter and the Tradescantia/MCN test always gave negative results. Raw wastewater, ClO2- and PAA-disinfected waters showed toxic effects in the marine bacteria test.

Advanced oxidation and adsorption technologies for organic micropollutant removal from lake water used as drinking-water supply.

The goal of this research is to evaluate the effectiveness of innovative drinking-water treatments designed to remove toxic and mutagenic organic micropollutants from lake waters used for human consumption. The widely used adsorption on granular activated carbon (GAC) filter technique was compared with the more innovative resin column techniques (XAD4 and Ambersorb-563) and with the advanced oxidation processes (AOPs) with UV/O3 and UV/O3/ H2O2. The water samples, collected from lake Como, treated with these techniques were analysed for mutagenic activity using Ames assay, toxicity using bioluminescent bacteria and organic compound were characterized using the GC-MS technique. The results found a decrease of the mutagenic and toxic activities of the lake water after adsorption on GAC and resins, while the AOP process generally increased these parameters. The absence of mutagenic activity was found only when a GAC adsorption step was performed in addition to the AOP process. Similar results were obtained by the toxicological and chemical analyses. In addition, the GC-MS analysis identified some possible mutagenic agents.

Monitoring airborne genotoxicants in the rubber industry using genotoxicity tests and chemical analyses.

This research was designed to examine the presence of mutagenic/carcinogenic compounds in airborne pollutants in the rubber industry using an integrated chemical/biological approach. Inhalable airborne particulate matter (PM-10: <10 microm) was collected in four rubber factories using a high-volume sampler equipped with a cascade impactor for particle fractionation. The organic extracts of two different fractions (0.5-10 microm and <0.5 microm) were examined for mutagenicity with the Ames test and for in vitro DNA-damaging activity in human leukocytes by single-cell microgel electrophoresis (Comet assay). The extracts were also studied by gas chromatography/mass spectrometry (GC/MS) for polycyclic aromatic hydrocarbon (PAH) content. Nitrosamines in ambient air were sampled on cartridges and analysed by GC with a thermal energy analyser (TEA) detector. Airborne volatile genotoxins were monitored in situ using a clastogenicity plant test (Tradescantia/micronuclei test). The results showed that airborne particulates were mainly very fine (<0.5 microm) and that trace amounts of genotoxic nitrosamines (N-nitrosodimethylamine: 0.10-0.98 microg/m(3); N-nitrosomorpholine: 0.77-2.40 microg/m(3)) and PAH (total PAH: 0.34-11.35 microg/m(3)) were present in air samples. Some extracts, particularly those obtained from the finest fractions, were mutagenic with the Ames test and genotoxic with the Comet assay. In situ monitoring of volatile mutagens using the Tradescantia/micronuclei test gave positive results in two working environments. The results showed the applicability of this integrated chemical-biological approach for detecting volatile and non-volatile genotoxins and for monitoring genotoxic hazards in the rubber industry.

Monitoring of mutagens in urban air samples

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulates sampled with the inhalable PM-10 high volume sampler in two different streets of Brescia, a heavily industrialized town in northern Italy, using the Tradescantia/micronucleus test and a bacterial mutagenicity test (Kado test, a more sensitive version of the Ames test). In addition, the Tradescantia/micronucleus test was used for in situ monitoring of gaseous pollutants in other urban areas of Brescia and in two car tunnels, one with heavy car traffic in Perugia, a town in central Italy, and one in Brescia with moderate traffic. The Tradescantia-micronucleus test carried out on extracts of airborne particulates gave positive results only for the sample collected in the traffic-congested street where also higher bacterial mutagenicity was found. The in situ monitoring of the urban areas with the Tradescantia/micronucleus test always gave negative results. Monitoring carried out in the two car tunnels showed a significant increase in micronuclei frequency only in flowers exposed in the smaller and more polluted tunnel.

Mutagens and carcinogens in size-classified air particulates of a Northern Italian town

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulate matter in relation to particles aerodynamic size. Inhalable (< 10 microns) airborne particulate (PM-10) was collected at a low traffic site in an industrialized Northern Italian town, using a high volume sampler equipped with a cascade impactor for particles fractionation. The organic extracts of different fractions were examined for mutagenicity in Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 using the microsuspension procedure, and for polycyclic aromatic hydrocarbons (PAHs) content by gas chromatography. Size fractionated particles were also analysed for heavy metals (Fe, Mn, Zn, Pb, Cu, Cd, Cr, Ni, V) using plasma spectrophotometry. The results of mutagenicity and chemical analyses indicate that, at the site investigated, inhalable particulate was largely made of fine (< 0.5 micron) particles, which accounted for most of PAHs and mutagenicity. A similar pattern of distribution was found for heavy metals, which were relatively more abundant in small (< 1.5 microns) particles compared to coarser ones.

Mutagenicity of extracts of lake drinking water treated with different disinfectants in bacterial and plant tests

Abstract Raw water and drinking water samples collected from five treatment plants supplied by a northern Italian lake in two periods of the year (summer and winter) were studied for their mutagenicity. The water samples were concentrated on silica C 18 cartridges and the adsorbates were tested at increasing doses with a bacterial short-term mutagenicity test (Ames test with Salmonella typhimurium TA98 and TA100 strains), which reveals the gene-mutation-inducing ability of pollutants, and with a plant genotoxicity bioassay ( Tradescantia /micronucleus test), which determines clastogenicity (chromosome-breaking ability). Raw water samples from all treatment plants were found to contain bacterial direct-acting mutagens detectable mainly with TA98 strain. The analyses of drinking water samples after water treatment showed some interesting results: TA98 mutagenicity was reduced when ozone was used together with chlorine dioxide, but TA100 mutagenicity was increased, though only in the summer sample; mutagenicity detectable with both strains was always reduced after chlorine dioxide disinfection; on the contrary, in all treatment plants using NaClO TA98 mutagenicity of winter samples increased. Raw lake water induced a high number of micronuclei in the Tradescantia /micronucleus test, showing a strong clastogenicity. This activity was higher in the NaClO-treated samples, and lower with the other disinfectants. Therefore, disinfection of lake water with ozone and/or chlorine dioxide seems to be a suitable alternative to the use of NaClO for controlling the formation of nonvolatile mutagens. The concentration method coupled with the two mutagenicity tests was found to be a simple, rapid and relatively inexpensive system for monitoring treatment plants and studying the influence of different disinfection systems on water mutagenicity.

Comparative assessment of genotoxicity of mineral water packed in polyethylene terephthalate (PET) and glass bottles.

The potential migration of genotoxic compounds into mineral water stored in polyethylene terephthalate (PET) bottles was evaluated by an integrated chemical/biological approach using short-term toxicity/genotoxicity tests and chemical analysis. Six commercial brands of still and carbonated mineral water bottled in PET and in glass were stored at 40 degrees C for 10 days in a stove according to the standard EEC total migration test (82/711/EEC), or at room temperature in the dark. After treatment, the samples were analysed using gas-chromatography/mass spectrometry (GC/MS) to detect volatile and non-volatile compounds, the Microtox test to evaluate potential toxicity of the samples, and three mutagenicity tests -Tradescantia and Allium cepa micronucleus tests and the Comet assay on human leukocytes - to detect their genotoxic activity. GC/MS analysis did not detect phthalates or acetaldehyde in the water samples. The Microtox test found no toxic effects. Mutagenicity tests detected genotoxic properties of some samples in both PET and glass bottles. Statistical analyses showed a positive association between mineral content and mutagenicity (micronuclei in A. cepa and DNA damage in human leukocytes). No clear effect of treatment and PET bottle was found. These results suggest the absence of toxic compounds migrating from PET regardless of time and conditions of storage. In conclusion, bottle material and stove treatment were not associated with the genotoxic properties of the water; the genotoxic effects detected in bottled water may be related to the characteristics of the water (minerals and CO(2) content).

Smoking among High School Students in 10 Italian Towns: Patterns and Covariates

The associations between tobacco smoking and demographic, socioeconomic, environmental, and behavioral factors among adolescents were investigated by administering an anonymous questionnaire to 5,221 ninth (aged 14-15 years) and 4,154 thirteenth grade (aged 18-19 years) students in 10 Italian towns. Using logistic regression analysis, both current smoking and experimental smoking were statistically associated with sibling, best friend, and partner smoking, alcohol drinking, and frequency of drunkenness in both grades and sexes. Attitude of parents toward childrens smoking was also found to be associated with the probability of being a current smoker, especially among ninth graders. Parental smoking was associated with current smoking among females only. Socioeconomic factors and level of knowledge of the health consequences of smoking were not associated with either experimental or regular smoking. In conclusion, this survey suggests that social environment plays a fundamental role in both first experimentation with smoking and the process of becoming a regular smoker among adolescents. Students with one or more siblings who smoke, and especially those with best friends and a partner who smoke, were much more likely to have tried smoking and to be current smokers than students without smokers in their environment.

Allium cepa chromosome aberration and micronucleus tests applied to study genotoxicity of extracts from pesticide-treated vegetables and grapes

The Allium cepa assay is an efficient test for chemical screening and in situ monitoring for genotoxicity of environmental contaminants. The test has been used widely to study genotoxicity of many pesticides revealing that these compounds can induce chromosomal aberrations in root meristems of A. cepa. Pesticide residues can be present in fruit and vegetables and represent a risk for human health. The mutagenic and carcinogenic action of herbicides, insecticides and fungicides on experimental animals is well known. Several studies have shown that chronic exposure to low levels of pesticides can cause birth defects and that prenatal exposure is associated with carcinogenicity. This study evaluated the potential application of plant genotoxicity tests for monitoring mutagens in edible vegetables. The presence of pesticides and genotoxic compounds extracted from 21 treated vegetables and eight types of grapes sampled from several markets in Campania, a region in Southern Italy, was monitored concurrently. The extracts were analysed for pesticides by gas chromatography and high-performance liquid chromatography, and for genotoxicity using two plant tests: the micronucleus test and the chromosomal aberration test in A. cepa roots. Thirty-three pesticides were detected, some of which are not approved. Genotoxicity was found in some of the vegetables and grapes tested. Allium cepa tests proved to be sensitive in monitoring genotoxicity in food extracts. The micronucleus test in interphase cells gave a much higher mutagenicity than the chromosomal aberration test in anaphase–telophase cells.

Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests

In the last few years chlorine dioxide has been increasingly used for disinfecting drinking water in many countries. Although it does not react with humic substances, chlorine dioxide added to water is reduced primarily to chlorite and chlorate ions, compounds that are under investigation for their potential adverse effects on human health. The aim of this research was to study the genotoxicity of chlorite and chlorate and their mixtures. The end-points included two plant tests (chromosomal aberration test in Allium cepa and micronucleus assay in Tradescantia, carried out at different times of exposure) and two genotoxicity tests in human HepG2 cells (comet assay and cytokinesis-blocked micronucleus test). Preliminary toxicity tests were carried out for both plant and HepG2 assays. The results showed that chlorite and chlorate are able to induce chromosomal damage to plant systems, particularly chromosomal aberrations in A. cepa root tip cells, even at concentrations lower than the limit established by Italian normative law and WHO guidelines. In HepG2 cells increased DNA damage was only observed for chlorate at the lowest concentration. No increase in micronuclei frequency was detected in any of the samples tested in human HepG2 cells.