Search

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 °C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1 h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant (P<0.001, Student’s t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose–response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 °C and a minimum level at 18 °C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments. [Copyright &y& Elsevier]

Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species. [ABSTRACT FROM AUTHOR]

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health. [Display omitted] • Evaluation of Tiber River water quality combining Effect Based Methods and chemical analysis. • In vivo and i n vitro eco-genotoxicity assays to study the potential effects of Tiber River water. • Recommendations to include a battery of Effect-Based Methods in preventive management measures. [ABSTRACT FROM AUTHOR]

Monitoring of water genotoxicity still remains underexploited in risk assessment. The present study aimed at standardizing and evaluating the sensitivity and applicability of the Comet Assay adapted for Daphnia magna in genotoxicological investigations in freshwater environments. Two sampling campaigns (2014–2015) were performed in the watercourses of a pilot basin located in the Parma district (Italy). Fourteen sampling stations with different Ecological Status and/or EBI values were selected, all with a good Chemical Status according to the EU–Water Framework Directive 2000/60. The Alkaline Comet Assay was performed on 48 h-aged daphnids exposed (24 h) to 23 water samples. In parallel, the acute toxicity test was carried out. Daphnids exposed to samples, collected upstream the main watercourses in non-impacted areas, showed low DNA migration (Tail Intensity percentage – TI% - in the range 2.97–13.21), similar to laboratory controls. An increase in genotoxicity (TI% in the range 20–40) proceeding from the mountain towards the plain area was observed, in agreement with the land uses and the ES/EBI values of the stations. The highest genotoxic damage was observed after exposure to samples from watercourses of the minor hydrographic network in the plain area and waterbodies receiving wastewater treatment plant outflows. A modified version of the Comet Assay able to identify the presence of genotoxins inducing DNA oxidative damage, after standardization, was applied to daphnids treated with waters from 4 selected monitoring stations. The presence of oxidative contaminants was detected downstream a wastewater treatment plant outflow. The Comet Assay on D. magna has proven to be sensitive and able to discriminate among differently impacted areas and might be applied routinely. The FPG-Comet proved to be able to highlight the presence of contaminants causing oxidative stress. In our knowledge, this is the first time that Comet Assay on Daphnia magna is successfully applied for freshwater monitoring. Unlabelled Image • Water toxicity and genotoxicity were assessed in a Po River tributary. • Comet Assay with D. magna was applied for the first time in freshwater monitoring. • The FPG-Comet Assay was standardized to detect DNA oxidative stress in D. magna. • The Comet Assay on D. magna was able to discriminate differently impacted stations. • The test is quick (24 h exposure) and routinely applicable. [ABSTRACT FROM AUTHOR]

Aflatoxins represent a serious problem for a food economy based on cereal cultivations used to fodder animal and for human nutrition. The aims of our work are two-fold: first, to perform an evaluation of the activity of newly synthesized thiosemicarbazone compounds as antifungal and anti-mycotoxin agents and, second, to conduct studies on the toxic and genotoxic hazard potentials with a battery of tests with different endpoints. In this paper we report an initial study on two molecules: S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazone and its metal complex, bis(S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazonato)nickel (II). The outcome of the assays on fungi growth and aflatoxin production inhibition show that both molecules possess good antifungal activities, without inducing mutagenic effects on bacteria. From the assays to ascertain that the compounds have no adverse effects on human cells, we have found that they are cytotoxic and, in the case of the nickel compound, they also present genotoxic effects. [ABSTRACT FROM AUTHOR]

Abstract: When chlorine is used as a disinfectant for drinking water it may react with organic materials present in or released by the water pipes and thus form by-products that may represent a genotoxic hazard. The aim of this study was to assess the potential genotoxicity and cytotoxicity of extracts of chlorinated drinking water supplied by local aquifers of two Italian towns, Plants 1 and 2, located in the sub-Alpine area and on the Po plain, respectively. The raw water fell within the legal limits with regards to its chemical and physical properties. Water from Plant 2 contained higher levels of total organics (TOC) and nitrate than water from Plant 1. Water was sampled at different points along the distribution networks to evaluate the influence of the system on the amount and quality of the by-products. Cytotoxic and genotoxic damage was assessed in freshly isolated human white blood cells (WBC) and Hep-G2 cells by use of the micronucleus (MN) test and the Comet assay to measure primary DNA damage. While they did not show significant cytotoxicity, all Plant 1 water concentrates induced short-time genotoxic effects on leukocytes at concentrations ≥1Lequiv./mL. Plant 2 samples were able to induce cytotoxic effects in both Hep-G2 cells and leukocytes. Furthermore, although there was no significant increase in MN frequency, DNA migration was strongly increased both in human leukocytes (≥0.5Lequiv./mL, 1h treatment, water samples collected from all points) and in Hep-G2 cells (≥0.75Lequiv./mL, 24h treatment, tap water sampled at the nearest distribution point). The current use of these in vitro cytotoxicity/genotoxicity tests together with the normal chemical analyses could provide information to help water-works managers and health authorities evaluate drinking water quality and adopt strategies to reduce genotoxic compounds in tap water and prevent human exposure to these compounds. [Copyright &y& Elsevier]

Abstract: This research examined the quality of water—before and after distribution—of four drinking-water production plants located in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study. Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500l) were concentrated on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA 98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio fischeri (Microtox™). The Microtox™ test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring of drinking-water quality before and after distribution. [Copyright &y& Elsevier]

A battery of in vitro short-term tests revealing different genetic end-points was set up in order to study surface-water genotoxicity after disinfection with different biocides: sodium hypochlorite (NaClO), chlorine dioxide (ClO2) and peracetic acid (PAA). The surface water both before and after disinfection was concentrated by adsorption on C18 silica cartridges and the concentrates containing non-volatile organics were divided into different portions for chemical analyses and biological assays. The following in vitro tests were conducted on the water concentrates dissolved in DMSO: the Salmonella mutagenicity assay with S. typhimurium strains TA98 and TA100; the SOS Chromotest with Escherichia coli, the Microtox® and Mutatox® assays with Vibrio fischeri; and gene conversion, point mutation and mitochondrial DNA mutability assays with D7 diploid Saccharomices cerevisiae strain. The results show that the SOS Chromotest and the yeast assays are highly sensitive in detecting genotoxicity. The surface-water extracts were very often toxic to most of the test organisms considered, partially masking their potential mutagenic activity. Therefore, the assays with E. coli and with S. cerevisiae are more likely to show a mutagenic effect because these organisms are generally less sensitive to most toxic compounds. Among the tested disinfectants, NaClO and ClO2 increased water genotoxicity, whereas PAA was able to slightly reduce raw water activity. However, because the organic compounds in the lake water varied with the season of the year, the disinfection processes, at times, both increased and decreased the raw water activity. [Copyright &y& Elsevier]