Ilaria Zerbini

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.

Review of epidemiological studies on drinking water hardness and cardiovascular diseases

Background Major risk factors do not entirely explain the worldwide variability of morbidity and mortality due to cardiovascular disease. Environmental exposures, including drinking water minerals may affect cardiovascular disease risks. Method We conducted a qualitative review of the epidemiological studies of cardiovascular disease and drinking water hardness and calcium and magnesium levels. Results Many but not all ecological studies found an inverse (i.e., protective) association between cardiovascular disease mortality and water hardness, calcium, or magnesium levels; but results are not consistent. Some case-control studies and one cohort study found either a reduced cardiovascular disease mortality risk with increased drinking water magnesium levels or an increased risk with low magnesium levels. However, the analytical studies provide little evidence that cardiovascular risks are associated with drinking water hardness or calcium levels. Conclusion Information from epidemiological and other studies supports the hypothesis that a low intake of magnesium may increase the risk of dying from, and possibly developing, cardiovascular disease or stroke. Thus, not removing magnesium from drinking water, or in certain situations increasing the magnesium intake from water, may be beneficial, especially for populations with an insufficient dietary intake of the mineral.

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.

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).

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.

Evaluation of environmental bacterial contamination and procedures to control cross infection in a sample of Italian dental surgeries

OBJECTIVES To perform a pilot study on bacterial contamination in some dental surgeries (n=51) in a local health unit in Brescia (Lombardy Region, Italy) and to evaluate the procedures to control cross infection used by the personnel to reduce the risk of infection in dental practice. METHODS A survey was carried out by interviewing 133 dental personnel with a questionnaire on the procedures used to control infection. The autoclaves, chemical baths (chemiclaves), and ovens present in the surgeries were tested for sterilisation efficiency with a spore test, and already packed and sterilised instruments were randomly sampled and tested for sterility. Microbial contamination of air, surface, and dental unit water samples were also studied. RESULTS The dental personnel did not generally follow the principal procedures for infection control: 30% of personnel were not vaccinated against hepatitis B virus, infected instruments were often not decontaminated, periodic checks of autoclave efficiency were lacking, and the knowledge of disinfection mechanisms and procedures was incomplete. High bacteriological contamination of water at dental surgeries was often found and total bacteriological counts in air samples were high. Surface studies showed widespread bacterial contamination. CONCLUSIONS On the basis of these results, an educational programme for the prevention of infective hazards has been prepared and carried out. The results of this pilot study will be used for planning a national survey.

EDCs, estrogenicity and genotoxicity reduction in a mixed (domestic + textile) secondary effluent by means of ozonation: A full-scale experience

WWTP (wastewater treatment plant) effluents are considered to be a major source for the release in the aquatic environment of EDCs (Endocrine-Disrupting Compounds), a group of anthropogenic substances able to alter the normal function of the endocrine system. The application of conventional processes (e.g. activated sludge with biological nitrogen removal) does not provide complete elimination of all these micropollutants and, consequently, an advanced treatment should be implemented. This experimental work was conducted on the tertiary ozonation stage of a 140,000 p.e. activated sludge WWTP, treating a mixed domestic and textile wastewater: an integrated monitoring, including both chemical (nonylphenol, together with the parent compounds mono- and di-ethoxylated, and bisphenol A were chosen as model EDCs) and biological (estrogenic and genotoxic activities) analyses, was carried out. Removal efficiencies of measured EDCs varied from 20% to 70%, depending on flow conditions (ozone dosage being 0.5 gO3/gTOC). Biological tests, furthermore, displayed that the oxidation stage did not significantly reduce (only by 20%) the estrogenicity of the effluent and revealed the presence and/or formation of genotoxic compounds. These results highlight the importance of the application of an integrated (biological+chemical) analytical procedure for a global evaluation of treatment suitability; poor performances recorded in this study have been attributed to the presence of a significant industrial component in the influent wastewater.

Biodegradability, toxicity and mutagenicity of detergents: Integrated experimental evaluations.

The widespread use of detergents has raised concern with regard to the environmental pollution caused by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex mixtures of different substances, in which synergistic effects may occur, we aimed to assess the mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All detergents presented ready biodegradability. EC50 values varied for all products, according to the marker organism used, but were always higher than the more stringent value considered for aquatic toxicity assessment (V. fischeri 10-60 mg/L; D. magna 25-300 mg/L; A. cepa 250-2000 mg/L). None of the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic features.

DNA Damage in Buccal Mucosa Cells of Pre-School Children Exposed to High Levels of Urban Air Pollutants

Air pollution has been recognized as a human carcinogen. Children living in urban areas are a high-risk group, because genetic damage occurring early in life is considered able to increase the risk of carcinogenesis in adulthood. This study aimed to investigate micronuclei (MN) frequency, as a biomarker of DNA damage, in exfoliated buccal cells of pre-school children living in a town with high levels of air pollution. A sample of healthy 3-6-year-old children living in Brescia, Northern Italy, was investigated. A sample of the childrens buccal mucosa cells was collected during the winter months in 2012 and 2013. DNA damage was investigated using the MN test. Childrens exposure to urban air pollution was evaluated by means of a questionnaire filled in by their parents that included items on various possible sources of indoor and outdoor pollution, and the concentration of fine particulate matter (PM10, PM2.5) and NO2 in the 1–3 weeks preceding biological sample collection. 181 children (mean age±SD: 4.3±0.9 years) were investigated. The mean±SD MN frequency was 0.29±0.13%. A weak, though statistically significant, association of MN with concentration of air pollutants (PM10, PM2.5 and NO2) was found, whereas no association was apparent between MN frequency and the indoor and outdoor exposure variables investigated via the questionnaire. This study showed a high MN frequency in children living in a town with heavy air pollution in winter, higher than usually found among children living in areas with low or medium-high levels of air pollution.