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Comet and micronucleus assays in zebra mussel cells for genotoxicity assessment of surface drinking water treated with three different disinfectants
Abstract
The aim of this research was to study the influence of classic (sodium hypochlorite and chlorine dioxide) and alternative (peracetic acid [PAA]) disinfectants on the formation of mutagens in surface waters used for human consumption. For this proposal, in vivo genotoxicity tests (Comet and micronucleus assay) were performed in an experimental pilot plant set up near Lake Trasimeno (Central Italy). The effects were detected in different tissues (haemocytes for the Comet assay and gills for the micronucleus test [MN]) of Dreissena polymorpha exposed in experimental basins supplied with lake water with/without the different disinfectants. Specimen collection was performed before disinfectant input for both tests and after the start of disinfection (3 h and 20 days for the Comet assay and 10 and 20 days for micronucleus test, respectively) to assess short- and long- term exposure effects during three sampling campaigns (October 2000, February 2001, and June 2001). Seasonal differences in baseline levels of DNA migration and micronucleus frequency were observed. Raw water quality modulation on disinfection by-product formation was shown. The results of the micronucleus and Comet assays on zebra mussel cells after in situ exposure to water disinfected with the two chlorinated compounds clearly indicate DNA/by-product interaction. PAA did not induce either clastogenic/aneugenic effects or DNA damage on this bioindicator.
... Genotoxicity has been widely studied with in vivo tests performed by directly exposing bio-indicators to water samples disinfected by PAA. The clastogenic effects derived from chromosome breakages have been studied with the micronucleus test in plants (Tradescantia pollen and root cells of Vicia faba) (Guzzella et al., 2004;Monarca et al., 2005Monarca et al., , 2004Monarca et al., , 2003Monarca et al., , 2002Monarca et al., , 2000, fish (Cyprius carpio) (Buschini et al., 2004a,b;Canistro et al., 2011;Gustavino et al., 2005;Monarca et al., 2004;Villarini et al., 2011) and mollusks (Dreissena polimorpha) (Bolognesi et al., 2004;Monarca et al., 2004). DNA damage, as strand breakages, has been studied with the Comet assay (alkaline single-cell gel electrophoresis -SCGE) on the same fish and mollusks species as the micronucleus test. ...
... Genotoxicity of water samples disinfected by PAA has been studied as well with in vitro assays on human lymphocytes and leukocytes (micronucleus test and comet test) (Maffei et al., 2005;Monarca et al., 2004). Furthermore, given that a single short-term test cannot predict mutagenicity, genotoxicity or cytotoxicity of a compound or a mixture, several authors (Bolognesi et al., 2004;Guzzella et al., 2004;Marabini et al., 2006;Monarca et al., 2005Monarca et al., , 2004Pellacani et al., 2006;Villarini et al., 2011) have performed batteries of tests with different genetic end-points to determine the effects of disinfected samples on indicator organisms. Table 3 summarizes the in vivo and in vitro tests reported by different authors that have evaluated the mutagenicity and genotoxicity of different water matrixes samples disinfected with PAA on different indicator organisms. ...
... A graphical summary of the results of in vivo tests in fish and mollusk is presented in Fig. 2b. No clastogenic effects derived from chromosome breakages in the micronucleus test or DNA damage in the comet test were detected in erythrocytes of common carp (Cyprinus carpio) (Buschini et al., 2004a,b;Canistro et al., 2011;Gustavino et al., 2005;Monarca et al., 2004) and in hemocytes of Drinking water (lake water) 1 mg/L in October * e Monarca et al., 2004 freshwater mussel (Dreissena polymorpha) (Bolognesi et al., 2004;Monarca et al., 2004) as a result of PAA disinfection of lake water and well water with addition of humic acids. However, genotoxic effects were observed in the bile of Cyprinus carpio after PAA (0.6 mg/L) disinfection of surface water, indicating potential cocarcinogic effects of PAA disinfection (Villarini et al., 2011). ...
Peracetic acid (PAA) has gained increasing attention over the last decades as a suitable and environmentally-friendly alternative to chlorine-based compounds for wastewater disinfection, claiming limited disinfection by-products (DBPs) formed and no persistent residues in the environment. The present work aims at presenting a comprehensive and updated review of the ecotoxicological effects of effluents treated with PAA, to be ascribed to residual PAA and hydrogen peroxide (H2O2) and DBP formation. Modest concentrations of DBPs have been observed after PAA treatment, mainly carboxylic acids, which are not recognized as genotoxic. Moreover, there is no evidence of any endocrine disruption potential of PAA in human health or in the ecotoxicological studies. The associated H2O2 fraction can potentially minimize the formation of halogenated DBPs and also contribute to the acute toxic effects of treated effluents. Effluents disinfected with PAA at concentrations typical of the wastewater treatment field have displayed limited toxic, mutagenic and genotoxic effects on different aquatic organisms, particularly low compared to chlorine-based disinfectants.
... The MN test has been applied extensively to test the genotoxicity of chemicals. The test has been successfully used with invertebrates, fish and amphibians, as a biological monitors of contaminated areas (in situ assay) (Klobuèar, Pavlica, Erben & Pape, 2003; Çavas & Ergene-Gozukara, 2005; Wirz, Saldiva & Freire-Maia, 2005) and in the screening of compounds to determine their genotoxicity, after direct or indirect exposure in vivo (Bolognesi et al., 2004; Rocha et al., 2009; Rocha, 2011). A large number of chemical compounds are known to cause hazardous effects in aquatic organisms. ...
... (Baršienė et al., 2004; Siu et al., 2004; Baršienė, Andreikėnaitė & Bjornstad, 2010); 4,000 (Bolognesi et al., 2004; Bolognesi, Perrone, Roggieri, Pampanin & Sciutto, 2006). According to Bolognesi, Perrone, Roggieri, Pampanin & Sciutto (2006), the large interindividual variability associated with low baseline frequency of this biomarker confirms the need of assessment a consistent number of cells in adequate number of animals. ...
... Once confirmed normality of distributions and homoscedasticity of variances, a parametric ANOVA can be applied (Dailianis, Domouhtsidoub, Raftopouloub, Kaloyiannia & Dimitriadis, 2003; Siu et al., 2004; Riva, Binelli, Daniele & Provini., 2007; Villela et al., 2007; Fernández, Campillo, Martínez Gómez & Benedicto, 2011). In cases of non-normal distributions and heteroscedasticity, non-parametric tests are indicated, such as Mann-Whitney U-test (Pavlica, Klobucar, Vetma, Erben & Papes, 2000; Bolognesi et al., 2004; Baršienė & Andreikėnaitė, 2007; Benitez-Trinidad et al., 2014) and Kruskal– Wallis test (Izquierdo et al., 2003; Carvalho Pinto-Silva, Creppy & Matias, 2005). ...
The interest in environmental genotoxicity studies has increased, and the micronucleus test (MN) is one of the most popular and promising tests on ecotoxicology, representing a cytogenetic indicator of DNA damage in dividing cells. Genotoxicity studies employed this methodology to evaluate possible genotoxic risk due to exposition to hazardous xenobiotics in different organisms, including aquatic sentinel organisms. In this context, bivalve mollusks stand out because of some aspects of their way of life, such as filtering food from the water. In bivalves, the MN test evaluates aneugenic and clastogenic effects preferably in hemocytes and gill cells. In the last two decades an interesting increase can be observed in the number of studies using the MN test in bivalve species, both in biomonitoring in situ and in the genotoxicity assays under controlled laboratory conditions, which are targeted to several chemicals with genotoxic and carcinogenic potential in the aquatic environment. Resumo-O interesse sobre estudos de genotoxicidade ambiental tem sido crescente e o teste do micronúcleo (MN) é um dos mais populares e promissores testes relacionados à ecotoxicologia, representando um indicador citogenético de danos ao DNA de células em divisão. Estudos de genotoxicidade empregam esta metodologia para avaliar possíveis riscos genotóxicos devidos à exposição a xenobióticos perigosos em diferentes organismos, incluindo organismos aquáticos sentinelas. Neste contexto, moluscos bivalves destacam-se por conta de alguns aspectos do seu modo de vida, tais como a filtração de alimentos da água. Nos bivalves, o teste do MN avalia efeitos aneugênicos e clastogênicos preferencialmente em hemócitos e células das brânquias. Nas duas últimas décadas, pode-se observar um aumento interessante no número de estudos usando o teste do MN em espécies de bivalves, incluindo tanto o biomonitoramento in situ quanto ensaios de genotoxicidade em condições controladas de laboratório, que são direcionados a diversas substâncias químicas com potencial genotóxico e carcinogênico no ambiente aquático.
... Previous studies on Dreissena polymorpha, Mytilopsis leucophaeata and Mytilus edulis have suggested that chlorine affects foot activity, byssus thread production, filtration activity and shell valve movement (Rajagopal et al., 2003b). Though there have been studies on genotoxic effects of chlorination in drinking water systems using mussels as sentinel organisms (Bolognesi et al., 2004), there are no data available on the genotoxic effects on mussels of low-dose chlorination employed for antifouling purposes. Recently, Last et al. (2016) have reported on the lethal and sub-lethal responses of the polychaete Sabellaria alveolata to chlorine discharged from power plants; however, genotoxic effects were not included in the study. ...
... Therefore, the aim of this work was to assess the genotoxic effects of in-use levels of continuous chlorination on mussels using field and laboratory experiments. Further, Bolognesi et al. (2004) used Dreissena polymorpha as bioindicator of toxicity in chlorinated drinking water and showed that chlorine could lead to breakage of DNA strands in the mussel. One of the most widely used methods to study DNA strand break is comet assay (Singh et al., 1988;Collins, 2004;Frenzilli et al., 2009). ...
... The data also indicate that DNA damage alone may not be sufficient to get a clear picture of the condition of the treated mussels. It may be necessary to use multiple biomarkers for reliable assessment of the response mounted by mussels when subjected to antifouling biocide such as chlorine (Bolognesi et al., 2004;Siu et al., 2004;Nicholson and Lam, 2005;Nigro et al., 2006;Rank et al., 2007). ...
... Environmental contamination can affect organisms, populations, and ecosystems, with effects on cell metabolism, DNA, organ function, fertility, population size, species survival, and biodiversity. Direct chemical analyses of environmental samples are limited by the sensitivity and the availability of detection methods, and cannot predict the toxicity of complex mixtures (Bolognesi et al., 2004). Surface freshwater directly receives large quantities of waste water from domestic, agricultural, and industrial sources that are intentionally disposed in the water bodies. ...
... Bivalve mollusks are good biomonitors for water quality analyses, since they are filter feeders, motionless, and have the ability to accumulate contaminants (Pavlica et al., 2001). Exotic fresh water bivalves have been widely used in monitoring studies (Bolognesi et al., 2004). Mussels from polluted sites can develop adaptive mechanisms; however, when transplanted to laboratories, this response is reduced, and results can be better compared because all individuals have a common genetic background (Klobucar et al., 2003). ...
... Differences inherent to mussels' population, such as toxic response adaptation, can also influence this response. Studying wild (exposed during their entire life to a contaminated site) versus caged (transported from a control to a contaminated site) mussels, Bolognesi et al. (2004) also found that caged mussels showed higher DNA damage levels as compared to wild mussels, suggesting this event is a result of recent exposure. Organisms may physiologically acclimatize at the individual level and become more resistant as a consequence of earlier exposure or as the metabolism of aquatic animals is quick they possibly eliminate certain classes of xenobiotics and or repair genotoxic damages. ...
The mussel species Limnoperna fortunei was chosen as biomonitoring organism in Guaíba Lake Basin, based
on population data, distribution, and sensitivity. Previous in vitro and in vivo studies studies with Single Cell
Gel Assay (SCGA) and Micronuclei test (MN) on this freshwater mussel showed that it is successful in
biomonitoring studies, especially in urban pollution monitoring. This study evaluated two sampling sites in the
Guaíba Lake (Guaíba PC e Guaíba BR), near urban waste discharges, and a control site (Itapuã) insight a
preserved area, using L. fortunei individuals. Comparing to the control site, the Guaíba BR sample induced DNA
damage in haemocytes of mussels sampled both in situ and exposed to laboratory conditions, whereas MN
only in in situ collected mussels. This sample also presented the only surface water mutagenic result by
Salmonella/microsome assay with TA98 in the presence of metabolic activation. Guaíba PC samples increased
MN frequency in situ and in laboratory conditions comparing to the Itapuã results. Metal influence seems to be
less important than organic influence in genotoxic induction. These results confirm the strong urban influence
in this region, showing that biomonitoring is a powerful tool to detect this kind of contamination in water
bodies
... Natural aquatic ecosystems, which are not only a habitat for many species, but also a source of drinking water and a resource for human industrial activity, are also subject to anthropogenic pressure. Therefore, in many countries, interest in the studies of genotoxicity and mutagenicity of surface waters has significantly increased during the last decades [20][21][22][23][24][25][26]. It is very important to analyse and assess the genotoxicity of polluted water as a whole and complex system. ...
... But in addition to heavy metals, water can contain various pesticides, fertilizers, surfactants, organic pollutants, radionuclides, etc., which individually or in combination can have a toxic and genotoxic effect on the body [54]. In addition, it was found that various types of biological activity of various pollutants are manifested in a certain range of values of physicochemical factors such as temperature, pH, electrical conductivity, solubility, etc. [20]. Numerous studies on various biological models have shown the toxic and genotoxic activity of natural waters located near industrial enterprises [55,56].{Zegura, ...
Introduction
In this study, physicochemical, genotoxic, and mutagenic properties of water samples from 10 rivers of the Almaty region (Kazakhstan) were evaluated.
Results
The results of the study demonstrated an increased level of mineralization and electrical conductivity that might be caused by the high concentration of dissolved mineral salts and ions such as Na⁺, K⁺, Ca2 +, Cl⁻, SO4²⁻, HCO3⁻. The excess of Maximum Allowable Concentrations (MACs) for various heavy metals was revealed. The results of tests using the pXen7-lux biosensor showed toxic effects of river waters. At the same time, the studies involved lux biosensors pRecA-lux, pColD-lux, pSoxS-lux, pKatG-lux did not find any genotoxic and oxidative effects. However, toxicity and mutagenicity of the studied water samples was detected by using plant test (Allium cepa and Hordeum vulgare). Phytotoxic, cytotoxic (decrease in the mitotic index) and mutagenic (increase in the frequency of chromosomal aberrations) activity of the water samples was observed. The data of in vivo tests (Danio rerio) showed the high toxicity and teratogenicity of river waters for fish embryos at all stages of development.
Conclusions
The results of this comprehensive study indicate that the contamination of the surface natural waters poses a threat to rivers dwellers and the human population in the rivers areas.
... Villela used the golden mussel (Limnoperna fortunei) as a potential indicator organism for freshwater ecosystems due to its sensitivity to water contaminants. Comet assay in hemocytes of freshwater Zebra mussel, D. polymorpha Pallas, was used as a tool in determining the potential genotoxicity of water pollutants (Bolognesi et al., 2004;Riva et al., 2007). Klobucar suggested the use of Comet assay in haemocytes from caged, non-indigenous mussels as a sensitive tool for monitoring genotoxicity of freshwater. ...
... The Comet assay has found wide application as a simple and sensitive method for evaluating in vivo as well as in vitro DNA damage in different tissues (gills, liver, and blood) of fishes exposed to various xenobiotics in the aquatic environment (Table 4). Environmental biomonitoring to assess the genotoxic potential of river waters has been carried out in hepatocytes of chub (Leuciscus cephalus; Winter et al., 2004), erythrocytes of mullet (Mugil sp.), sea catfish (Netuma sp.; De-Andrade et al., 2004a;De-Andrade et al., 2004b), bullheads (Ameiurus nebulosus), and carps (Cyprinus carpio; Buschini et al., 2004). Basal level of DNA damage has been shown to be influenced by various factors, such as temperature of water in erythrocytes of mullet and sea catfish (De-Andrade et al., 2004a;De-Andrade et al., 2004b), age and gender in dab (Limanda limanda; Akcha et al., 2003), and exhaustive exercise in chub (Aniagu et al., 2006). ...
... Villela used the golden mussel (Limnoperna fortunei) as a potential indicator organism for freshwater ecosystems due to its sensitivity to water contaminants. Comet assay in hemocytes of freshwater Zebra mussel, D. polymorpha Pallas, was used as a tool in determining the potential genotoxicity of water pollutants (Bolognesi et al., 2004;Riva et al., 2007). Klobucar suggested the use of Comet assay in haemocytes from caged, non-indigenous mussels as a sensitive tool for monitoring genotoxicity of freshwater. ...
... The Comet assay has found wide application as a simple and sensitive method for evaluating in vivo as well as in vitro DNA damage in different tissues (gills, liver, and blood) of fishes exposed to various xenobiotics in the aquatic environment (Table 4). Environmental biomonitoring to assess the genotoxic potential of river waters has been carried out in hepatocytes of chub (Leuciscus cephalus; Winter et al., 2004), erythrocytes of mullet (Mugil sp.), sea catfish (Netuma sp.; De-Andrade et al., 2004a;De-Andrade et al., 2004b), bullheads (Ameiurus nebulosus), and carps (Cyprinus carpio; Buschini et al., 2004). Basal level of DNA damage has been shown to be influenced by various factors, such as temperature of water in erythrocytes of mullet and sea catfish (De-Andrade et al., 2004a;De-Andrade et al., 2004b), age and gender in dab (Limanda limanda; Akcha et al., 2003), and exhaustive exercise in chub (Aniagu et al., 2006). ...
The Comet assay has found worldwide acceptance for detecting DNA damage and repair in prokaryotic and eukaryotic cells. The variability in the results of the Comet assay is largely due to its sensitivity and minor differences in the conditions of various laboratories as well as the effect of confounding factors in human studies (lifestyle, age, diet, inter individual, and seasonal variation). The limitation of the Comet assay is that it only detects DNA damage in the form of strand breaks. It is therefore required that the in vitro and in vivo testing is conducted according to the Comet assay guidelines and appropriately designed multi-laboratory international validation studies be carried out. Guidelines for the in vitro as well as in vivo Comet assay have been formulated. Recently, issues relating to study design and data analysis in Comet assay were discussed by the International Workgroup on Genotoxicity Testing where particular attention was given to the alkaline version (pH>13) of the in vivo Comet assay and recommendations were made for a standardized protocol, which would be acceptable to international agencies. Consensus was also reached on the need for an international validation study to stringently evaluate the reliability and accuracy of the in vivo Comet assay (as well as in vitro versions). These recommendations are also aimed at reducing the variability arising in inter-laboratories studies. In vivo Comet assay has been accepted as the first-tier screening assay for assessment of DNA damage in rodents by the Committee on Mutagenicity, united kingdom, and international validation studies are underway supported by ECVAM, Japanese Centre for Validation of Alternative Methods (JaCVAM), United State Interagency Coordinating Committee on Validation of Alternative Methods (ICCVAM), United State National Toxicology Program Interagency Centre for Evaluation of Alternative Toxicological Methods (NICEATM), and Japanese Environmental Mutagen Society.
... MN test has been performed in different organisms, including humans, rodents, plants, and fishes, and later on has been adopted for the assessment of cytogenetic damage in other organisms including marine bivalve molluscs [64][65][66][67]; oysters, clams, and mussels have been the mainly studied species. ...
... Bolognesi et al. [7] observed in M. galloprovincialis that, after exposure to 100 ppb DMBA / mussel, the results obtained by the alkaline elution technique were four times the level of the controls. The same authors performed a study in caged mussels and wild mussels as part of a biomonitoring programme at four selected sites along the Ligurian coast (Italy) [66]. MN test and alkaline elution were selected to assess the genotoxic effects. ...
The degradation of the habitat, together with the overexploitation of natural resources, the invasion by alien species as well as pollution, represent the major problems jeopardizing coastal regions. Human activities are the main cause of marine pollution, including recurrent spills of toxic agents both in open sea and in estuarine areas. Besides, natural disasters such as landslides and flash floods also contribute significantly to the increase of marine pollution. Over the last decades, the use of biomarkers for biomonitoring the impact of several contaminants has been increased. A biomarker can be defined as measure at a molecular, cellular or whole organism level, of the exposure to contaminants (exposure biomarkers) or of the organism response to the pollutants (effect biomarkers). Genotoxicity biomonitoring is one of the most important features to evaluate the environmental stress and the pollution impact on marine organisms. In this sense, the development of suitable and sensitive biomarkers, such as those for the assessment of DNA damage, is required. The aim of this chapter is to provide an overview of the practical use of genotoxicity biomarkers in marine bivalve molluscs to evaluate the extent and consequences of environmental contamination in these organisms. This review illustrates the results obtained during the development and application of exposure/effect biomarkers for biomonitoring purposes in several sentinel species.
... Such data obviously raises concerns over the potential genotoxic risk for human health following long-term consumption of drinking water treated this way. The effect of classic (sodium hypochloride and chlorine dioxide) and alternative (peracetic acid, PAA) disinfectants on the formation of mutagens in surface waters used for human consumption were also investigated in the zebra mussels (Dreissena polymorpha) exposed in experimental basins supplied with treated lake water [48]. Mussels maintained in PAA treated water failed to show a difference when compared with controls animals, whereas those treated with the two chlorinated disinfectants generally displayed a reduction in DNA damage, thought to be associated with the induction of detoxification processes or cross-links inhibiting DNA migration. ...
... An agreement between SCE test and Comet assay data was found in Eastern mudminnow (U. pygmea L.) exposed for 3 and 11 days in flowthrough aquaria to Rhine groundwater [66]. A high concordance between the genotoxic effects detected by SCGE and MN test was reported by different authors for several organisms [45,6,8,21,49,59], even if the higher sensitivity of the Comet assay has been often underlined [48,50,63]. Masuda et al. [57] found positive Comet results on erythrocytes of goldfish injected with two mutagenic dyes present in river waters, but did not find any micronucleus in the same cell type. ...
... Bivalve mollusks are widely used in the ecotoxicological biomonitoring of the state of the aquatic environment. The lifestyle of these aquatic animals is sedentary or attached; they are active biofilters and bioaccumulators, but, at the same time, pollutants in their body undergo metabolic biotransformation and excre-tion relatively slowly (Luk'yanova, 2001;Bolognesi et al., 2004;Soldatov et al., 2014). This allows for the long-term monitoring of water pollution. ...
... For genotoxicity, the comet assay has become one of the most commonly used methods to study DNA damage in single cells ( Bolognesi et al., 2004a ;Duez et al., 2003 ). The fluorescence intensity of the comet's tail and increase in DNA tail percentage can indicate the extent of DNA damage. ...
Since the beginning of the 21st century, the increasing production and application of nano-TiO 2 in consumer products have inevitably led to its release into aquatic systems and therefore caused the exposure of aquatic organisms, resulting in growing environmental concerns. However, the safety of nano-TiO 2 in aquatic environments has not been systematically assessed, especially in coastal and estuary waters where a large number of filter-feeding animals live. Bivalves are considered around the world to be a unique target group for nanoparticle toxicity, and numerous studies have been conducted to test the toxic effects of nano-TiO 2 on bivalves. The aim of this review was to systematically summarize and analyze published data concerning the toxicological effects of nano-TiO 2 in bivalves. In particular , the toxicity of nano-TiO 2 to the antioxidant system and cell physiology was subjected to meta-analysis to reveal the mechanism of the toxicological effects of nano-TiO 2 and the factors affecting its toxicological effects. To reveal the cooperation, hot keywords and co-citations in this field, bibliometric analysis was conducted, and the results showed that the toxicological molecular mechanisms of nano-TiO 2 and the combined effects of nano-TiO 2 and other environmental factors are two major hot spots. Finally, some perspectives and insights were provided in this review for future research on nano-TiO 2 toxicology in bivalves.
... A well-stablished method for assessing the effect of potential genotoxic agents is observing the frequency of micronuclei in cells [12]. Overall, micronuclei are chromosome fragments that are not properly integrated into daughter cells' nuclei throughout mitosis, being a cytogenetic marker of chromosome breakage (clastogens) or loss (aneugens) [13,14]. ...
... Zebra mussel Dreissena polymorpha (Pallas, 1771) (Bivalvia: Dreissenidae) is widely used as a model organism for biomonitoring purposes since late 1970's. This is a sedentary active filtrator, an active bioaccumulator of mineral and organic pollutants (Binelli et al., 2010;Bolognesi et al., 2004). As invasive species zebra mussel widely distributed in Russia. ...
... Circannual variations have been documented for the blood and haematopoietic system of many wild species as well as for laboratory animals under artificial controlled conditions (Berger 1983). Recently, seasonal changes were documented for the haemocyte counts of the Austropotamus torrentinum (Lucic and Erben 2005), baseline levels of DNA migration and micronucleus frequency in Dreissena polymorpha (Bolognesi et al. 2004), haemocyte counts and size in the Manila clam Venerupis philippinarum (Soudant et al. 2004), the innate immunity of the Asian catfish Clarias batrachus (Kumari et al. 2006) and the haematocrit and total leucocyte counts in the wild roach Rutilus rutilus (Vainikka et al. 2004). ...
... The capacity of Cu to induce chromosomal damage in a range of cell types has previously been reported in MG [32,91], and in DP in response to a range of contaminants [92][93][94]. As mentioned earlier, Cu accumulation in mussel tissue is significantly correlated with the adverse genotoxicological effects noted in both the species (Fig. 3). ...
Determination of relative sensitivity of biota following exposures to contaminants including metals is important for environmental protection. Copper (Cu), although biologically essential can be highly toxic to biota if present at higher concentrations in the natural environment. Given its ubiquitous presence within coastal and inland water bodies, we compared Cu-induced genotoxicity in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG), along with its tissue specific accumulation. Novel biomarker in terms of induction of gamma H2AX (γ-H2AX) foci, along with comet assay and induction of micronuclei (MN) were used to determine DNA damage response (DDR) in these two species following exposure to a range of Cu concentrations (18, 32, 56 μg L⁻¹) for 10 days. Concentration-dependent increases in Cu concentration in gill tissue, as determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), were paralleled by a greater degree of genotoxicity. An induction of γ-H2AX foci was present in all Cu exposure concentrations, proving this technique to be a sensitive and suitable biomarker of genotoxicity in bivalves. The multi-biomarker approach adopted here suggests firstly that in parallel with MG, which is widely used to assess the health of marine and coastal environment, DP is also suitable representative of inland water bodies, and that there is a similar mechanism of action for the induction of genotoxicity between the two species, following exposure to Cu. Secondly, for genotoxicity assessment a battery of responses could simultaneously be assessed in these two bivalve species. Finally, for adequate protection of the environment it is vital to adopt a multi-biomarker, multi-species approach to determine adverse biological effects to gain a holistic understanding of the real threat posed by contaminants to hydrosphere.
... Micronúcleos (MN) são massas de cromatina com aparência de um pequeno núcleo, que se originam a partir de fragmentos de cromossomos ou de cromossomos inteiros que se perdem durante o processo de divisão e, portanto, não se incorporam ao núcleo principal das células-filhas, durante a divisão celular (SCHMIDT, 1976;AL-SABTI;METCALFE, 1995;FENECH, 2007). Estas perdas de material nuclear podem acontecer ou pela ausência de centrômero nas porções cromossômicas, por danos no aparelho mitótico ou por defeito na citocinese (BOLOGNESI et al., 2004;ERGENE et al., 2007 FENECH, 2007;ÇAVAS et. al., 2012). ...
... The comet assay is widely used to study the genotoxic potential of environmental pollutants (Mitchelmore and Chipman 1998;Frenzilli et al. 2009;Vincent-Hubert et al. 2011). Chlorine toxicity in P. viridis and D. polymorpha has been studied earlier by the comet assay (Bolognesi et al. 2004;Chavan et al. 2016). The value of tail DNA observed at the end of the present study in the chlorinated environment is almost equivalent to the value of tail DNA (26%) obtained after 70 days in our previous study from the same location Fig. 6 Gene expression in gill tissue of P. viridis exposed to chlorinated environment. ...
Toxic effects of continuous low dose application of the antifouling biocide chlorine on marine benthic organisms were monitored using transplanted green mussels (Perna viridis) and a suite of biomarkers. Caged mussels were deployed in chlorinated and non-chlorinated sections of the cooling system of an operating electric power plant. Biomarkers indicative of general stress, oxidative stress (superoxide dismutase and catalase), and DNA integrity, along with expression of stress proteins, were studied to assess the effects. Deterioration in condition index with corresponding increase in DNA strand breaks was indicative of chlorine stress. Superoxide dismutase enzyme did not show any particular trend, but catalase activity was high during the initial days of exposure at the chlorinated site; later, it became almost equal to that at the control site. Similarly, expressions of stress proteins (HSP60, HSP70, HSP22, GSTS1, and CYP4) showed bell-shaped pattern during the period of study. Positive correlation among the endpoints indicated the utility of the multimarker approach to monitor the effects of continuous low dose chlorination on mussels.
... O teste do cometa tem sido amplamente utilizado como um teste de genotoxicidade que pode detectar danos primários/reversíveis no DNA, tais como sítios álcali-lábeis e quebras de fita simples e dupla no DNA de células individuais, (SINGH et al., 1988;GIANNOTTI et al., 2002;KIMURA et al., 2013), enquanto que o teste de MN é um teste bem estabelecido para detectar quebras de fita dupla do DNA irreparáveis, efeitos clastogênicos, aneugênicos (BOLOGNESI et al., 2004;KIMURA et al., 2013), danos cromossômicos fixos do tipo estrutural e/ou numérico (GOETHEM et al., 1997;KRISHNA E HAYASHI, 2000). A combinação desses testes resulta em uma abordagem sensível para a detecção eficiente do efeito de múltiplas classes de compostos genotóxicos em diferentes órgãos-alvo (VASQUEZ, 2010 Uma das principais vias de absorção e toxicidade das AgNP é através das brânquias, causando efeitos adversos na osmorregulação, semelhantes aos descritos para a prata iônica (GRIFFITT et al., 2009;FARMEN et al., 2012). ...
... Nowadays MN assay is applied in the evaluation of chromosomal damage in a large number of wild and transplanted aquatic species. A great amount of studies or programmes on the genotoxic effect of the polluted water environment have been carried out with the use of bivalves [6][7][8][9]. ...
The response to chemical disturbance of an Italian river (Cecina River) characterised by high levels of trace metals was assessed in the sentinel species freshwater painter’s mussel (Unio pictorum). We exposed U. pictorum in the laboratory to metal, metalloid and other trace element polluted river sediments in order to evaluate genetic and cellular biomarkers. Bivalves exposed to sediments taken from all the impacted sites of the river basin exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. The lysosomal membrane stability of circulating haemocytes, assessed by the Neutral Red Retention Time, was found to be higher in specimens from Lake Maggiore and Berignone, while exhibited a significant decrease in specimens exposed to Ponteginori sediments. An increase of DNA migration in specimens exposed to the two contaminated sites in comparison with controls was found. Moreover, higher micronucleated haemocytes frequencies were observed in bivalves exposed to sediments collected at Possera 1 site. This study suggested that biomarkers evaluating cellular impairment, genetic and chromosomal damages, are highly sensitive and suitably applied to laboratory experiments. The painter’s mussel was confirmed to be an appropriate sentinel species for quality assessment in metal-contaminated freshwater environments.
... It is resistant to photolysis, hydrolysis, and particularly to biological degradation (Anonymous 2016; Arslan et al. 2010Arslan et al. , 2015Baršienė 1994). The risk this compound poses to ecosystems health is undeniable (Baršienė et al. 2006;Beach et al. 2006;Bolognesi and Fenech 2012;Bolognesi et al. 2004Bolognesi et al. , 2006. As PFOS is widely used in industry (Li 2008), it is widely found in aquatic sediments and surface water (Gunduz et al. 2013). ...
Perfluorinated compounds constitute one of the principal sources of pollution and pose great risk to the aquatic ecosystem. Perflorooctane sulfonate (PFOS) is one of the most common and well-known emerging chemicals since it is widely used and is present in surface water and sediments. Previous studies reported toxic effects of perflorooctane sulfonate on marine organisms. However, there is a scarcity of studies on genotoxic effects of PFOS. This study was undertaken to investigate DNA damage in haemocytes and gill cells of Mytilus galloprovincialis exposed to PFOS using a micronucleus assay (MN). The experiment, which was carried out using increasing concentrations of PFOS (2–6 mg/L), revealed that at the concentration of 4 mg/L, PFOS causes increased genotoxic damage. In summary, results of this study show that the micronucleus test is a reliable tool for the determination of PFOS genotoxicity.
... Mussels have several characteristics, such as a wide distribution, filter feeding, sessile life form, and ability to accumulate pollutants, which make them favored organisms for estimating environmental pollution levels and the bioavailability of various types of pollutants (Ravera, 2001;Roméo et al., 2003;Andral et al., 2004;Bocquené et al., 2004;Pampanin et al., 2005;Amiard et al., 2006;Lehtonen et al., 2006). In response to environmental stress they show a range of physiological, histological, and molecular responses, including abnormal morphology, alterations of antioxidative status, induction of DNA strand breaks, etc. (Pavlica et al., 2001;Binelli et al., 2007;Bolognesi et al., 2004;Rocher et al., 2006;Coffinet et al., 2008;Klobučar et al., 2008). ...
... No clastogenic/aneugenic effects or DNA damage was detected in Cyprinus carpio or D. polymorpha as a result of PAA disinfection of surface water. [214,215] PAA (0.6-2 mg/L) disinfection of surface water induced cytochrome P450 (CYP) enzyme modulations in C. carpio and D. polymorpha biomarkers, and genotoxic DBPs were observed to be excreted in the bile of C. carpio: therefore PAA still remains as potentially co-carcinogenic. [216][217][218] However, no cytotoxic effects or glutathione content variation were determined in rainbow trout hepatocytes after exposure to surface water treated with PAA, but increased reactive oxygen species (ROS) production was observed. ...
Peracids have gained interest in the water treatment over the last few decades. Peracetic acid (CH3CO3H) has already become an accepted alternative disinfectant in wastewater disinfection whereas performic acid (CHO3H) has been studied much less, although it is also already commercially available. Peracids have also been tested for drinking water disinfection, oxidation of aqueous (micro)pollutants, sludge treatment and ballast water treatment, to name just a few examples. The purpose of this review paper is to represent comprehensive up-to-date information about the water treatment applications, aqueous reaction mechanisms, and disinfection by-product formation of peracids, namely performic, peracetic and perpropionic acids.
... Freshwater mussels are recognized as valuable bioindicators used in numerous studies dealing with assessment of environmental pollution [2][3][4][5][6][7]. The species Unio pictorum and Unio tumidus have wide distribution in European lowland rivers, similar morphological and physiological characteristics, and show similar responses during in situ and ex situ exposure to stressors [1,8,9]. ...
The impact of etoposide (ETO), vincristine sulfate (VIN) and cisplatin (CP) on the DNA damage level was studied in vivo and in vitro on hemocytes of freshwater mussels Unio sp. (U. pictorum/U. tumidus) using the alkaline comet assay. For in vivo experiments, the mussels were exposed in static system for 72 h. For in vitro experiments, treatment was performed in primary cultures of hemocytes for 22 h. The DNA damage level was analyzed by Comet Assay IV software. Increase of damage was detected for ETO (40 and 100 µM) in vivo and in vitro and for VIN (0.04 and 0.1 µM) in vivo. CP did not induce an increase of DNA damage, but post-treatment with hydrogen peroxide indicated existence of DNA crosslinks in specimens treated with 4 µM CP. Effective concentrations of selected cytostatics are at least one (CP) or several orders of magnitude (ETO, VIN) higher comparing to the concentrations in hospital wastewater.
... These systems are used for assessment of the genotoxicity of pure chemicals, mixtures, natural waters, effluents, leachates, and eluates [17][18][19][20]. The MN test has also been applied to various freshwater and marine vertebrate and invertebrate species (fish, amphibians, mollusks, echinoderms); thus, this test has shown high sensitivity and reliability for in vivo or in situ experiments [21][22][23][24][25][26][27][28]. Genotoxic responses vary among animal species. ...
We have applied the micronucleus (MN) assay to the measurement of genotoxicity in microcrustaceans. Daphnids (Daphnia magna) and Copepods (Acanthocyclops robustus) were collected in situ and acclimated in the lab for 24 h. The MN assay was successful with the Daphnids but not with the Copepods. Adult Daphnids were exposed to sublethal concentrations of metals (Cu, Zn, Cd) or insecticide (deltamethrin) for 2 and 7 d. Dose-dependent induction of MN was observed after 2 d exposure, with 2-fold induction at the highest doses for each chemical tested. The advantages and ecological relevance of using Daphnids in genotoxicity assessment are highlighted. The Daphnid assay may be a reliable test for aquatic genotoxicity hazard/risk assessment and a useful alternative to studies of amphibians.
... For environmental impact assessment, aquatic organisms such as Danio rerio are widely used as biological markers (Bolognesi et al., 2004;Ferraro et al., 2004;Ladhar et al., 2014) because they represent the highest trophic level of the food chain and are quite sensitive to environmental pollution (Ong et al., 2014), metabolizing and accumulating xenobiotics over time (Dautremepuits et al., 2004;Torres de Lemos et al., 2007;Domingues et al., 2010). ...
... Many DBP compounds are produced in chlorination, and isolated DBPs (i.e., bromo-organic by-products and halonitromethanes) were found to induce DNA damage in Salmonella or in mammalian tests (Richardson et al., 2007). For disinfection of raw water by peracetic acid, chlorine dioxide or sodium hypochloride, the comet assay test on both haemolymph of zebra mussels and human white blood cells showed dramatic seasonal variations in DNA damage capability (Bolognesi et al., 2004;Laffon et al., 2001). ...
Based on the fact that recycling of combined filter backwash water (CFBW) directly to drinking water treatment plants (WTP) is considered to be a feasible method to enhance pollutant removal efficiency, we were motivated to evaluate the genotoxicity of water samples from two pilot-scale drinking water treatment systems, one with recycling of combined backwash water, the other one with a conventional process. An integrated approach of the comet and micronucleus (MN) assays was used with zebrafish (Danio rerio) to investigate the water genotoxicity in this study. The total organic carbon (TOC), dissolved organic carbon (DOC), and trihalomethane formation potential (THMFP), of the recycling process were lower than that of the conventional process. All the results showed that there was no statistically significant difference (P >0.05) between the conventional and recycling processes, and indicated that the genotoxicity of water samples from the recycling process did not accumulate in 15day continuous recycling trial. It was worth noting that there was correlation between the concentrations of TOC, DOC, UV254, and THMFPs in water and the DNA damage score, with corresponding R 2 values of 0.68, 0.63, 0.28, and 0.64. Nevertheless, both DNA strand breaks and MN frequency of all water samples after disinfection were higher than that of water samples from the two treatment units, which meant that the disinfection by-products (DBPs) formed by disinfection could increase the DNA damage. Both the comet and MN tests suggest that the recycling process did not increase the genotoxicity risk, compared to the traditional process.
... The evaluation of the impact of pollutants by biomarkers becomes essential for assessing the condition of aquatic ecosystems due to the fact that the simple detection of pollutants failed to provide the information on the relationship between contaminant exposure and biological effects in aquatic organisms [1,2]. The presence of pollutants in aquatic ecosystems can be detected by a range of physiological, histological, and molecular responses, including abnormal morphology, alterations of antioxidative status, and DNA integrity [3][4][5][6][7][8][9][10][11]. ...
Genotoxicity monitoring of the lower stretch of the Sava River was performed by the combined approach of in situ assessment of genotoxicity and active biomonitoring of two species of mussels from the Unionidae family, Unio pictorum and Unio tumidus. Genotoxic response was studied using comet assay on hemocytes. For active biomonitoring, the mussels were acclimated to controlled laboratory conditions for 10 days and then exposed at two sites in the Sava River in the area of the city of Belgrade. Hemolymph of exposed specimens of each species was taken after 7, 14, and 30 days of exposure. For in situ assessment, the mussels were collected from five sites in the lower flow of the Sava River. The mussels were sampled immediately after the acclimation served as controls in both types of monitoring procedures. The results of our studies indicated the presence of genotoxic pollution at all studied sites at the Sava River. The level of DNA damage varied at different sites depending on the source and level of pollution. The response to genotoxic pollution was evident at the site in the urban area of Belgrade city, as well as at the sites far from the large urban settlements, suggesting that the lower flow of the Sava River is under pollution pressure.
... The capacity of Cu to induce chromosomal damage in a range of cell types has previously been reported in MG [32,91], and in DP in response to a range of contaminants [92][93][94]. As mentioned earlier, Cu accumulation in mussel tissue is significantly correlated with the adverse genotoxicological effects noted in both the species (Fig. 3). ...
Both the comet and p53-binding protein 1 (53BP1) Immunocytochemistry (ICC) assays are well known, sensitive techniques to determine DNA damage, albeit with different perspectives. Whilst the comet assay determines DNA strand breaks (SSB/DSB/ alkali labile sites), the 53 BP1 assay reflects the cellular response to DSBs that promotes the end-joining of distal DNA ends. Both assays however require fluorescence image analysis at the cellular level under a microscope (i.e. different comet parameters using commercially available software, and counting of foci per cell). Whilst these techniques are considered to be rapid compared to many other established methodologies, their relative sensitivity and effectiveness have however not been compared effectively. In this study we compare the two techniques examining their sensitivity as well as cost/time effectiveness.
SH-SY5Y dopaminergic neuronal cell line, a commonly used neuronal cell line for neurotoxicity testing, was exposed for 6 and 24h to a range of concentrations (150, 300 and 900 µM) of copper (Cu), a known toxicant which exerts its toxicity mainly via free radical production. Exposure of the cells to Cu showed a concentration dependent increase in DNA damage. The Student’s T-test was used to analyse the data from the comet and ICC assays showing that there was a significant difference (p
... The comet assay is known to be more sensitive in the detection of genotoxicity than the micronucleus test (Mitchelmore and Chipman, 1998). This assay detects repairable DNA damage, such as single and double breaks in DNA strand, repair events by incomplete excisions, alkali labile sites and cross-links between DNA molecules, while the micronucleus test detects not repairable chromosome damage, such as clastogenic and aneugenic lesions (Tice et al., 2000;Bolognesi et al., 2004;Udroiu, 2006). The results of micronuclei and nuclear abnormalities suggest that the water samples of Sino River Basin do not induce chromosome breakage or loss in fish. ...
p>Some water bodies in the Sinos River Basin (SRB) have been suffering the effects of pollution by residential, industrial and agroindustrial wastewater. The presence of cytotoxic and genotoxic compounds could compromise the water quality and the balance of these ecosystems. In this context, the research aimed to evaluate the genotoxicity and cytotoxicity of the water at four sites along the SRB (in the cities of Santo Antônio da Patrulha, Parobé, Campo Bom and Esteio), using bioassays in fish and cell culture. Samples of surface water were collected and evaluated in vitro using the Astyanax jacuhiensis fish species (micronucleus test and comet assay) and the Vero lineage of cells (comet assay and cytotoxicity tests, neutral red - NR and tetrazolium MTT). The micronucleus test in fish showed no significant differences between the sampling sites, and neither did the comet assay and the MTT and NR tests in Vero cells. The comet assay showed an increase in genetic damage in the fish exposed to water samples collected in the middle and lower sections of the basin (Parobé, Campo Bom and Esteio) when compared to the upper section of the basin (Santo Antônio da Patrulha). The results indicate contamination by genotoxic substances starting in the middle section of the SRB.
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... Mussels have numerous advantages, such as limited mobility, filter feeding, and small diameter of daily movement, which make them favourable bioindicators. In response to environmental stress they show a range of physiological, histological and molecular responses, including abnormal morphology, alterations of antioxidative status, induction of DNA strand breaks, etc. (Pavlica et al., 2001;Bolognesi et al., 2004;Rocher et al., 2006;Coffinet et al., 2008;Klobučar et al., 2008;Binelli et al., 2009;Kolarević et al., 2013). As a representative of freshwater mussels, we have chosen species Unio tumidus. ...
The use of recirculating aquaculture systems (RAS) for Atlantic salmon (Salmo salar) production has become increasingly common. RAS water disinfection plays a crucial role on its biosecurity. Peracetic acid (PAA) is a promising disinfectant due to its powerful oxidative properties, broad antimicrobial spectrum, and rapid degradation into no harmful compounds. This study focused on assessing the consequences of prolonged application of a PAA-based disinfectant in a RAS stocked with salmon parr. The experiment included three treatment groups in triplicate: 0 mg/L PAA (control), 0.1 mg/L PAA, and 1 mg/L PAA, using nine-replicated RAS with a total of 360 fish (14.8 ± 2.3 g; N = 40/RAS). The study spanned 28 days, with samples collected on days 0, 14, and 28. The analyzed parameters were water quality, and fish parameters, including external welfare indicators, gill histology, total antioxidant capacity (TAC), reactive oxygen species/reactive nitrogen species (ROC/RNC), oxidative stress biomarkers related to DNA and protein, cellular DNA damage, and global gene expression. While water quality remained relatively stable, there was an increase in bacterial populations in the groups exposed to PAA, particularly 1 mg/L PAA. Fish weight did not differ between the control and PAA-exposed groups. TAC, ROC/RNC, and oxidative stress biomarkers exhibited similar trends. The study identified >400 differentially expressed genes (DEGs) in the skin, gill, and olfactory organ, with many of these DEGs associated with immune responses. Comparing the transcriptomic profiles of the three tissue organs revealed that the olfactory organ was the most reactive to PAA treatment. This study shows that calculated PAA concentrations of 0.1 mg/L and 1 mg/L in the pump-sump, contributed to an increase of bacteria whereas no detectable differences in health and welfare of salmon parr were found. These findings are promising for the implementation of PAA-based disinfectants in RAS stoked with Atlantic salmon parr.
Identification of ecosystem services, i.e. the contributions that ecosystems make to human well-being, has proven instrumental in galvanising public and political support for safeguarding biodiversity and its benefits to people. Here we synthe-sise the global evidence on ecosystem services provided and disrupted by freshwater bivalves, a heterogenous group of >1200 species, including some of the most threatened (in Unionida) and invasive (e.g. Dreissena polymorpha) taxa globally. Our systematic literature review resulted in a data set of 904 records from 69 countries relating to 24 classes of provision-ing (N = 189), cultural (N = 491) and regulating (N = 224) services following the Common International Classification of Ecosystem Services (CICES). Prominent ecosystem services included (i) the provisioning of food, materials and medicinal products, (ii) knowledge acquisition (e.g. on water quality, past environments and historical societies), ornamental and other cultural contributions, and (iii) the filtration, sequestration, storage and/or transformation of biological and physico-chemical water properties. About 9% of records provided evidence for the disruption rather than provision of ecosystem services. Synergies and trade-offs of ecosystem services were observed. For instance, water filtration by freshwater bivalves can be beneficial for the cultural service 'biomonitoring', while negatively or positively affecting food consumption or human recreation. Our evidence base spanned a total of 91 genera and 191 species, dominated by Unionida (55% of records, 76% of species), Veneroida (21 and 9%, respectively; mainly Corbicula spp.) and Myoida (20 and 4%, respectively; mainly Dreissena spp.). About one third of records, predominantly from Europe and the Amer-icas, related to species that were non-native to the country of study. The majority of records originated from Asia (35%), with available evidence for 23 CICES classes, as well as Europe (29%) and North America (23%), where research was largely focused on 'biomonitoring'. Whilst the earliest record (from 1949) originated from North America, since 2000, annual output of records has increased rapidly in Asia and Europe. Future research should focus on filling gaps in knowledge in lesser-studied regions, including Africa and South America, and should look to provide a quantitative valuation of the socioeconomic costs and benefits of ecosystem services shaped by freshwater bivalves.
The vast number of chemicals existing or being added into the environment, have globally aroused great concern regarding their adverse effects in human population. Development and validation of sensitive and better test systems which can assess the adverse effects of chemicals at an early stage for intervention strategies to be implemented in time is currently in progress. This book documents the latest research and showcases the versatile, state-of-the-art technique - the Comet assay - in the field of modern toxicology. The assay is a simple, sensitive rapid and visual technique for the quantitative and qualitative assessment of DNA damage in single cells. The Comet Assay in Toxicology is the first book of its kind to be devoted exclusively to the Comet assay and its applications as an important tool in modern toxicology. This multi-author book will serve as both a reference and a guide to investigations in the biomedical, biochemical and pharmaceutical sciences. Written by investigators from the fields of genetic toxicology and human epidemiology, the authors have first-hand knowledge from their chosen sub-specialities and are active contributors to the peer-reviewed scientific literature. The book is divided into five major sections, reflecting the range of interest in the exploitation of this assay. The book's scope begins with an introduction section reviewing its genesis for those new to the technique and the current knowledge of the various fields in which it finds wide acceptance. This section sets the scene by explaining why the assay has become the most sensitive and sought after assay in modern toxicology. Next is a whole section that considers various procedures being followed to assess different types of DNA damage in various cell types and is contributed by experts in the respective fields. The third section puts together the specific applications of the assay in the diverse fields ranging from genetic toxicity testing to human monitoring, and environmental toxicology. The fourth section consists of the guidelines and recommendations for the conduct of the assay in in vitro and in vivo systems, based on the recommendations of the International Workgroups on genotoxicity test procedures. Finally, the book draws to a close with an assessment of the statistics used for the understanding of the data generated by the assay. This is a unique reference book as it provides the scientific community with the advances in Comet assay as well as its applications. It also incorporates a detailed section with instant and comprehensive information on the procedure of the assay and the latest protocols being used worldwide as well as statistical analyses to be followed. The book is aimed at students as well as scientists in the area of molecular epidemiology and genetic toxicology.
The vast number of chemicals existing or being added into the environment, have globally aroused great concern regarding their adverse effects in human population. Development and validation of sensitive and better test systems which can assess the adverse effects of chemicals at an early stage for intervention strategies to be implemented in time is currently in progress. This book documents the latest research and showcases the versatile, state-of-the-art technique - the Comet assay - in the field of modern toxicology. The assay is a simple, sensitive rapid and visual technique for the quantitative and qualitative assessment of DNA damage in single cells. The Comet Assay in Toxicology is the first book of its kind to be devoted exclusively to the Comet assay and its applications as an important tool in modern toxicology. This multi-author book will serve as both a reference and a guide to investigations in the biomedical, biochemical and pharmaceutical sciences. Written by investigators from the fields of genetic toxicology and human epidemiology, the authors have first-hand knowledge from their chosen sub-specialities and are active contributors to the peer-reviewed scientific literature. The book is divided into five major sections, reflecting the range of interest in the exploitation of this assay. The book's scope begins with an introduction section reviewing its genesis for those new to the technique and the current knowledge of the various fields in which it finds wide acceptance. This section sets the scene by explaining why the assay has become the most sensitive and sought after assay in modern toxicology. Next is a whole section that considers various procedures being followed to assess different types of DNA damage in various cell types and is contributed by experts in the respective fields. The third section puts together the specific applications of the assay in the diverse fields ranging from genetic toxicity testing to human monitoring, and environmental toxicology. The fourth section consists of the guidelines and recommendations for the conduct of the assay in in vitro and in vivo systems, based on the recommendations of the International Workgroups on genotoxicity test procedures. Finally, the book draws to a close with an assessment of the statistics used for the understanding of the data generated by the assay. This is a unique reference book as it provides the scientific community with the advances in Comet assay as well as its applications. It also incorporates a detailed section with instant and comprehensive information on the procedure of the assay and the latest protocols being used worldwide as well as statistical analyses to be followed. The book is aimed at students as well as scientists in the area of molecular epidemiology and genetic toxicology.
Anthropogenic radionuclides can enter water bodies through accidental or controlled discharges. In order to assess their potential impact, understanding the link between exposure, tissue specific bioaccumulation and radiation dose rate, to biological or biomarker responses in aquatic biota is required. Adopting an integrated, multi-biomarker, multi-species approach, we have investigated potential biological responses induced by short-lived radionuclide, phosphorus-32 (32P, radiophosphorus) in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG). Adult individuals were exposed to 32P for 10 days, to acquire nominal whole-body average dose rates of 0.10, 1 and 10 mGy d-1, which encompass a screening value of 10 μGy h-1 (0.24 mGy d-1), in accordance with the ERICA tool. Following exposure, a suite of genotoxic biomarkers (DNA damage, γ-H2AX induction and micronucleus [MN] formation) were measured in gill and digestive gland tissues, along with transcriptional expression of selected stress-related genes in both the species (i.e. hsp70/90, sod, cat and gst). Our results demonstrate the relationship between tissue specific dosimetry, where 32P induced a dose-dependent increase, and biological responses independent of species. Gene expression analysis revealed little significant variation across species or tissues. Overall, MG appeared to be more sensitive to short-term damage (i.e. high DNA damage and γ-H2AX induction), particularly in digestive gland. This study contributes to limited knowledge on the transfer and biological impact of radionuclides within differing aquatic systems on a tissue specific level, aiding the development of adequate management and protective strategies.
Disinfection is intended to improve drinking water quality and human health. Although disinfectants may transform organic matter and form disinfection by-products (DBPs), many are branded as cyto- and genotoxic. Traditionally, research focuses on the effects of DBPs to human health, but cytogenic impacts on aquatic organisms still remain ill defined. The current study examines the potential toxic effect of chloroform and iodoform (DBPs) on Cyprinus carpio, selected as a model organism. Fish specimens were exposed to various concentrations of DBPs primarily based on LD50 values, where acute toxicity was monitored for 96 h. Headspace SPME extraction through gas chromatography was employed to assess the effects of spiked DBPs doses in fish blood. Cytotoxicity was monitored using Comet assay. Tail length, tail DNA, and olive tail moment values were quantified to be significant (P < 0.05) as compared to control. A statistically significant (P < 0.05) decrease in all blood parameters (hematology) was observed. Changes in biochemical indices (glucose, total protein, and alanine aminotransferase (ALT)) were also significant. ALT secretion was significantly increased (93 ± 0.05 and 82.8 ± 0.1 U/L) at higher concentration compared to control (56 ± 0.1 U/L), suggesting liver damage. Results demonstrated that iodoform was statistically more damaging as compared to chloroform.
The marine ecosystem is constantly threatened by a wide variety of anthropogenic hazardous chemicals, such as, heavy metals, pesticides, oil, petroleum hydrocarbons, etc., from industries, agricultural sources and sewage disposal. Pakistan, being a country with agriculture prominence, uses pesticides widely for crop protection, and thereby suffers from pollution. In the present study, we assessed a few biomarkers as indicators of the genotoxic chemicals, pesticides and herbicides. We inducted micronucleus (MN) in the gill tissues of green mussel Perna viridis (L.) exposed to different concentrations of organo-phosphate pesticides (chlorpyrifos, malathion) and synthetic pyrethroid pesticides (cypermethrin, lambda-cyhalothrin) and a herbicide (buctril). The MN frequencies of the pesticides treated mussels were observed to increase significantly (P <0.05) in a dose-dependent manner at all exposure periods. The highest MN frequencies were recorded in gill tissues of cypermethrin treated mussels on the 12th day (10, 11.5 and 13.5‰ at 0.5, 1 and 1.5 ppm, respectively). The genotoxic effect of pesticides on Perna viridis (gill tissue) was in the following order cypermethrin > chlorpyrifos > malathion > lambda-cyhalothrin > buctril. © 2018, National Institute of Science Communication. All rights reserved.
Concerns about the adverse effects of chemicals present in the environment have created a need for better systems to assess their potential consequences on human health. One potential solution is the versatile and state-of-the-art Comet assay. Simple, sensitive, rapid and visual, this modern toxicological method allows quantitative and qualitative assessment of DNA damage in single cells. This assay is used in diverse fields ranging from clinical applications, human monitoring and environmental toxicology through to genetic toxicity testing.
This updated and revised edition of The Comet Assay in Toxicology provides the latest information on this important tool. It addresses, in-depth, the different protocols, statistical analyses and applications used worldwide. It also includes the guidelines recommended by the Working Group on Comet Assay. The book begins with a review of the genesis of the assay for those new to the technique and goes on to explain procedures followed to assess different types of DNA damage, various applications of the assay, and guidelines for the conduct of the assay in in vitro and in vivo systems. New chapters written for this edition will provide information on the most contemporary approaches and applications, including in silico approaches, on meta-analysis of data and on the application of the Comet Assay in nanotoxicology.
This book will serve as both a reference and a guide to students as well as investigators in the biomedical, biochemical and pharmaceutical sciences fields.
Concerns about the adverse effects of chemicals present in the environment have created a need for better systems to assess their potential consequences on human health. One potential solution is the versatile and state-of-the-art Comet assay. Simple, sensitive, rapid and visual, this modern toxicological method allows quantitative and qualitative assessment of DNA damage in single cells. This assay is used in diverse fields ranging from clinical applications, human monitoring and environmental toxicology through to genetic toxicity testing.
This updated and revised edition of The Comet Assay in Toxicology provides the latest information on this important tool. It addresses, in-depth, the different protocols, statistical analyses and applications used worldwide. It also includes the guidelines recommended by the Working Group on Comet Assay. The book begins with a review of the genesis of the assay for those new to the technique and goes on to explain procedures followed to assess different types of DNA damage, various applications of the assay, and guidelines for the conduct of the assay in in vitro and in vivo systems. New chapters written for this edition will provide information on the most contemporary approaches and applications, including in silico approaches, on meta-analysis of data and on the application of the Comet Assay in nanotoxicology.
This book will serve as both a reference and a guide to students as well as investigators in the biomedical, biochemical and pharmaceutical sciences fields.
The genotoxicity of drinking water treated with 6 disinfection methods and the effects of disinfection conditions were investigated using the umu-test. The pretreatment procedure of samples for the umu-test was optimized for drinking water analysis. The results of the umu-test were in good correlation with those of the Ames-test. The genotoxicity and production of haloacetic acids (HAAs) were the highest for chlorinated samples. UV + chloramination is the safest disinfection method from the aspects of genotoxicity, HAA production and inactivation effects. For chloramination, the effects of the mass ratio of Cl2 to N of chloramine on genotoxicity were also studied. The changes of genotoxicity were different from those of HAA production, which implied that HAA production cannot represent the genotoxic potential of water. The genotoxicity per chlorine decay of chlorination and chloramination had similar trends, indicating that the reaction of organic matters and chlorine made a great contribution to the genotoxicity. The results of this study are of engineering significance for optimizing the operation of waterworks.
DNA fragmentation by single strand breaks (SSBs) or double strand breaks (DSBs) is major concern in genotoxicity research. DNA damages can be easily known through comet assay or single cell gel electrophoresis (SCGE). Since decades, scoring through software for DNA damages in images have been developed by researchers. These softwares depend upon manual scoring on individual comet in a particular interface. The evaluation under software may have biasness and error during scoring by each researcher and few softwares are unable to access easily because many of these are commercial products. However, CellProfiler (CP) image analysis software (Version 2.1.0) is free, easy operation, faster and automated screening by computer itself. An attempt was made to detect DNA damages mainly comet scoring through CP software as whole comet, comet head and comet tail from image of previously studied single cell gel electrophoresis (SCGE) in the peripheral erythrocytes of fish exposed to benzene as experimental image. The results particularly on length and area of whole comet, head and tail were obtained after automated analysis in the CP software. The image processing study was done of the objects present in fluorescence microscopy image to know maximum DNA damage at each cell level for the fish erythrocytes. It was concluded that the present study of image based screening for DNA damages as details of comet and its head and tail evaluation by shape and area in the fish erythrocytes can be a suitable tool for genotoxicity prediction along with risk assessment at DNA level. The shape descriptor as Zernike moments order 0 to 9 can also be suitable parameters to know accuracy of the shape of comet and its head and tail in the image. Finally, high-World Scientific News 55 (2016) 1-14-2-throughput automated screening of comet test can help in disease diagnosis and repair mechanisms as well as environmental monitoring of genotoxin(s) within short period of time.
HHighly luminescent ZnO Quantum Dots (QDs) synthesized via a simple facile route is used for the preparation of optical sensor for the detection of free chlorine. The concentration of free chlorine greatly effects the PL emission of ZnO QDs at 525 nm. Due to high efficency of hypochlorite to gain electron, it takes the electron from the oxygen vacancy of ZnO QD which gives rise to the defective emission in ZnO QD. UV-vis data analysis show no effect of free chlorine on the optical absorption spectra of ZnO QDs. The optical sensing of free chlorine using ZnO QDs carries several advantages like quick response time, good selectivity and of course high sensitivity. The pH shows very little effect on the PL emission of ZnO QDs near neutral pH. It does not interference in the sensing mechanism of free chlorine. After 60 s, the response of ZnO QDs remains stable. The present sensor gives high selectively with respect various common cations as well as anions.
The influence of 3-methylcholanthrene to zebra mussel (Dreissena polymorpha) larvae was studied. The artificial spawning of zebra mussels was used for obtaining larvae. Two different concentrations of 3-methylcholanthrene were used. The chromosome analysis showed a significant increase in chromosome aberrations (CA) at the higher concentration of the compound. The resistance of zebra mussel larvae to the lower concentration of 3-methylcholanthrene indicated that zebra mussel larvae are probably not sensitive enough for the study of genotoxicity of the compounds from the PAH group.
In this chapter we focus on ballast water management systems (BWMS) which are currently in use as well as treatment approaches manufacturers have chosen for the development of future BWMS. The main purpose of this review is to identify the current availability of BWMS technologies worldwide. Until January 2014 we brought together information of 104 different BWMS. To achieve the ballast water discharge standards, different water treatment technologies are used, mostly in combination, and applied in different stages of the ballasting process. In general, the treatment processes can be split in three stages, i.e., pre-treatment, treatment and residual control (neutralisation). Among the 104 BWMS identified, 100 apply their treatment at the uptake, some of those BWMS require also a treatment during ballast water discharge (in-line treatment) and three BWMS apply treatment only during the voyage (in-tank treatment). The majority of BWMS use filtration or a combination of hydrocyclone and filtration as pre-treatment separation step. The dominating treatment processes are to use an active substance, mostly generated on board by electrolysis/electrochlorination. The second most frequent treatment process is UV. BWMS to be installed for operation on vessels need to be type approved by a state. By the writing of this chapter more than 30 BWMS were type approved. It should be noted that the development of BWMS is a very dynamic market with newly proposed BWMS appearing almost on a monthly basis. The chapter also outlines how BWMS are applied on vessels, their capacities and installation requirements, which BWMS were type approved, and what projected global market for BWMS may exist. A recent calculation on the estimated value of the global market for purchasing and installing BWMSs resulted in an estimated turn-over of possibly $50 '74 billion. The chapter ends with a list of manufacturers, commercial names of their BWMS, applied treatment technologies used and links to BWMS web pages where available.
Human activities are responsible of the introduction of a large number of chemical contaminants in the environment. In order to assess the ecological status of water bodies, it becomes necessary to assess the impact of chemical contamination in the aquatic environment. The use of genotoxicity biomarkers allows the evaluation of the impact of chemical contaminants on the DNA integrity as predictors of effects at the population level. The main objective of this work was to develop a sensitive biomarker of early genotoxic effects of chemical contamination in Dreissena polymorpha caged in urban area. We used the comet assay, to measure DNA strand breaks and alkali-labile sites. We have coupled this assay with a specific endonuclease of oxidative DNA damage (hOGG1) to enhance the significance of the comet-hOGG1 assay, by the oxidative DNA damage detection, such as 8-oxodG, involved in cellular aging and carcinogenesis. The DNA oxidation formation has been demonstrated by the comet assay applied to mussel gills cells exposed to BaP and Cd. The oxidative DNA damage induced by BaP exposure were faster repaired than in mussels exposed to Cd. Then, the intrinsec variability of some genotoxicity biomarkers such as the level of DNA strand breaks, the micronuclei frequency and the number of DNA adducts, was assessed in order to eliminate this parameter in future biomonitoring studies. Results obtained in field studies showed the oxidative DNA damage induction in caged zebra mussels in urban sites of the Seine river basin. In addition, these studies showed that the mussels transplantation did not induce change either in the biological status of organisms or changes in the DNA strand break levels and micronuclei frequency. Finally, the comparison of the expression of genotoxicity biomarkers between spring and autumn showed that these two seasons are favourable to biomonitoring studies by the transplantation of Dreissena polymorpha.
Sediments collected in Kars River were tested for mutagenicity by means of peripheral erythrocyte micronucleus frequency in Orthrias angorae. Micronuclei frequencies (MN) of all the groups exposed to the sediments were higher than those of the control group. Statistical analysis showed significant differences between the control and the treatment groups (P<0.001). The MN frequencies of the blood samples in three regions (Selim, Pasacayir, Bogazkoy) on the day 6 have a trend to increase against to control. MN frequencies of samples collected from 36th hours sediment exposure increased in the three districts (Selim, Pasacayir, Bogazkoy) when compared to control groups. On the other hand there is a decrease in only one region (Kars) at 6 days. This study evaluates for the first time the mutagenic load of sediments collected along the Kars River and provides evidence that the presence of genotoxic agents in river sediments correlates with the genotoxic damage (micronucleated erythrocytes) in fish collected from Kars River.
Mollusc species were sampled in different sites based on pollution level in Poyang lake and its surrounding waters during withered water period (from Oct to Dec) and plentiful water period (from May to June) from 2007 to 2008. Single-cell gel electrophoresis (SCGE) was used to analyze their haemolymph cells Arbitrary Unit(AU) and Olive tailmoment (OTM) value of DNA damage in molluca. The results showed that the mean DNA damage value Jin river>Nanxin>Xiu stream in withered water period; and in Plentiful water period, Hukou>Qinglan lake>Xiyuan. Two mollusc species Corbicula fluminea and Bellamya aeruginosa have higher DNA damage value than other mussel in all sites. Comparing the DNA damage value of Bellamya aeruginosa collected in twenty sites in 2008, the highest DNA damage value was observed in Dacha lake and lowest in Xiyuan. Meanwhile, the highest DNA damage value of Unio douglasiae was recorded Chucha river from ten sites, and lowest in Xiyuan river.
The last 25 years have seen major advances in the field of mammalian genotoxicology, particularly with the advent of molecular methods, some of which have spilled over into the relatively new field of eco-genotoxicology, which aims to evaluate the impact of contaminants on the natural biota. Unlike mammalian genotoxicology, where the focus is centred on a limited number of model species, efforts in the marine field have generally lacked coordination and focus, with the result that progress has been somewhat slow and fragmented. However, it is recognized that at the DNA and chromosome levels, marine invertebrates express qualitatively similar types of induced damage to that found in higher organisms (e.g. point mutations, strand breaks and chromosomal aberrations). Given that many of these species (bivalve molluscs, crustaceans, polychaete worms, etc.) are linked directly or indirectly to the human food chain, this is an important reason why one should be concerned about their exposure to environmental mutagens and carcinogens, particularly as many of these organisms have the capacity to (i) transform these agents to biologically active metabolites and (ii) accumulate toxicants in their cells and tissues at concentrations several orders of magnitude above that found in the environment. This review covers the advantages and limitations of those cytogenetic and molecular assays that have been used to address the question of genotoxicity in the cells and early life stages of selected marine invertebrate species. It concludes with the recommendation for the adoption of standardized test procedures, leading to a tiered approach in future eco-genotoxicity testing.
A series of experiments were conducted to determine the effects of maintenance method (fed or starved), stock location, season, mussel size, and rate of acclimation to temperature on the responses (mortality) of zebra mussels in bioassays. Mussels maintained on a diet of crushed Chlorella are more tolerant to Bayer 73 and more sensitive to sodium hypochlorite than starved mussels. Variability in LC50s of zebra mussels is high during the first 60 days in the laboratory, after which the resistance of mussels to both hypochlorite and Bayer 73 declines with reductions in body condition. Zebra mussels collected during the early summer and late fall are more tolerant to both hypochlorite and Bayer 73. There is significant variation in tolerances to biocides depending on the stock, such that stocks from locations with more degraded water quality have increased tolerances. Acclimating mussels from 4 to 20C at rates of 2 and 10C d–1 does not significantly affect tolerance to biocides. In general, LC50s of mussels vary by only 2–3, suggesting that mussels from any location, any season, and maintained under any maintenance protocol can be used in range-finding tests. Comparisons of results among studies requires knowledge of mussel stock, collection season, and laboratory maintenance protocols.
Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 μM) at 4 °C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
Five biomarkers (MT: metallothionein-like proteins, EROD: ethoxyresorufin ortho-dééthylase, DNA strand breaks, LPO: peroxidation of lipids, VG: vitellogenin-like protiens) were measured in the soft tissues of zebra mussels (Dreissena polymorpha) in order to assess the spatial variation of exposure to contaminants along the St Lawrence River (Canada). Fifteen mussels >25 mm shell length were analyzed from each of the 13 sampling sites. Significant differences between sites were noted for all biomarkers, but the general level of variability was low. Three biomarkers (DNA, LPO and VG) exhibited a similar pattern of spatial variation while MT and EROD had distinct and specific patterns. MT had the strongest discriminating power and EROD showed the largest range of variation among sites. Highest biomarker responses were measured in specimens from local contaminated sites such as harbors and industrial sectors. A positive relationship was found between MT and copper (Cu), but no significant correlation was observed between other biomarker responses and the levels of ten trace metals bioaccumulating in the zebra mussels tissues. Results indicate that the measurement of biomarker responses is technically feasible. The performance of each biomarker is assessed in the context of the role and advantages of selecting a battery of biomarkers for detecting contamination problems. The use of zebra mussels as a sentinel species for biomonitoring potential toxic effects in situ is discussed.
Epidemiologic evidence on the relation between contaminants in drinking water and cancer is reviewed. The reviewed studies cover exposure to: disinfection byproducts; nitrate; arsenic and other metals; volatiles and contaminants from hazardous waste sites; asbestiform fibers; radionuclides; and fluoride. Most investigations are ecologic, with some confirmation of elevated risk from individual-based studies. In the case of waterborne arsenic, and possibly chlorination byproducts, there is a consistent but small body of epidemiologic evidence of an association with one or more types of cancer. Nitrate in groundwater has increased greatly over the years, and the demonstration of endogenous nitrosation among highly exposed subjects raises concern of elevated cancer risk. However, the epidemiologic data are not yet sufficient to draw a conclusion. There is a diversity of studies among populations exposed to water contaminated with pesticides, volatile organics, or mixtures from hazardous waste sites. Studies of asbestiform fibers and radionuclides in water are not conclusive, but there are suggested elevations of several cancer sites in highly exposed populations. There is no suggestion that fluoride in drinking water is linked with elevated risk of cancer. As topics for epidemiologic evaluation, drinking water contaminants pose methodologic problems common to studies designed to detect relatively small elevations in risk, with the added challenge of assessing exposures for many years in the past. Nevertheless, epidemiologic assessment is valuable and clearly warranted, given the potential public health impact of small risk elevations among very large exposed populations, and the limitations of toxicologic experiments in assessing carcinogenic risk of complex mixtures or of compounds for which appropriate animal models are not available.
Subacute exposures (10 d) of the freshwater mollusc Dreissena polymorpha to disulfoton (10 mg/L), thiometon (6 mg/L), and its activated oxygen analogue demeton-S-methyl (6 mg/L) corroborate earlier findings of organophosphate resistance and accumulation in the organism. Mortality occurred not before the ninth day of exposure. Mortality was induced at high ambient water concentrations and must be due to unknown specific organophosphate effects. Body burdens reached saturation levels within one week being around 40 mg/kg wet weight for thiometon and 60 mg/kg for disulfoton. Mussels dying during the tests showed lower tissue concentrations. Elimination of accumulated organophosphates was so low in the mussel, that an efficient metabolism of these compounds in the mussel was unlikely. Different organs of Dreissena previously acutely exposed (96 h) to the organophosphate thiometon (6, 12, 25, 50 mg/L) were analyzed for their thiometon content. Thiometon could be found in all organs, but were highest in the anterior part of the viscera (230 mg/kg), where it was accumulated either in the digestive gland and/or in the gonadal tissue.
The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 microg/l, and for ramshorn snail from 10 to 450 microg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50 per thousand, and between 2.07 and 13.80 per thousand in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75 per thousand, and in snails from 1.56 to 2.00 per thousand. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.
The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.
The last 25 years have seen major advances in the field of mammalian genotoxicology, particularly with the advent of molecular methods, some of which have spilled over into the relatively new field of eco-genotoxicology, which aims to evaluate the impact of contaminants on the natural biota. Unlike mammalian genotoxicology, where the focus is centred on a limited number of model species, efforts in the marine field have generally lacked coordination and focus, with the result that progress has been somewhat slow and fragmented. However, it is recognized that at the DNA and chromosome levels, marine invertebrates express qualitatively similar types of induced damage to that found in higher organisms (e.g. point mutations, strand breaks and chromosomal aberrations). Given that many of these species (bivalve molluscs, crustaceans, polychaete worms, etc.) are linked directly or indirectly to the human food chain, this is an important reason why one should be concerned about their exposure to environmental mutagens and carcinogens, particularly as many of these organisms have the capacity to (i) transform these agents to biologically active metabolites and (ii) accumulate toxicants in their cells and tissues at concentrations several orders of magnitude above that found in the environment. This review covers the advantages and limitations of those cytogenetic and molecular assays that have been used to address the question of genotoxicity in the cells and early life stages of selected marine invertebrate species. It concludes with the recommendation for the adoption of standardized test procedures, leading to a tiered approach in future eco-genotoxicity testing.
Assessment of DNA damage is of primary concern when determining the pollution-related stress in living organisms. To monitor genotoxicity of the freshwater environments we used micronucleus (MN) and comet assay on Dreissena polymorpha haemocytes. Caged mussels, collected from the river Drava, were transplanted to four monitoring sites of different pollution intensity in the river Sava. Exposition lasted for a month. The baseline level of MN frequencies in the haemocytes of mussels from reference site (river Drava) was 0.5 per thousand. No increase in MN frequency was found in mussels from the medium-polluted site (Zagreb) in the river Sava while other, more polluted sites showed higher MN frequencies ranging from 2.7 per thousand (Lukavec) and 3.1 per thousand (Oborovo) to 5.2 per thousand (Sisak). Results from comet assay showed concordance with MN assay in indicating intensity of DNA damage. The use of haemocytes from caged, non-indigenous mussels in MN and comet assay proved to be a sensitive tool for the freshwater genotoxicity monitoring.
Disinfection of surface drinking water, in particular water chlorination, results in many by-products with potential genotoxic and/or carcinogenic activity. In the present study, we evaluated the genotoxicity of surface water after treatment with different disinfectants by means of in situ plant genotoxicity assays (micronucleus and chromosomal aberration tests) which can detect both clastogenic and aneugenic effects. The study was carried out at a pilot plant using lake water after sedimentation and filtration. This water supplied four stainless steel basins: three basins were disinfected with sodium hypochlorite, chlorine dioxide, and peracetic acid and the fourth basin containing untreated lake water was used as a control. Plants were exposed in situ in the basins. The study was carried out using water collected in different seasons over a period of about 1 year in order to assess the treatments in different physical and chemical lake water conditions. The micronucleus test in root cells of Vicia faba (Vicia faba/MCN test) revealed genotoxicity in many samples of disinfected water. The micronucleus test in Tradescantia pollen cells and the chromosome aberration test in root cells of Allium cepa showed genotoxic effects only in some disinfected samples, but also revealed genotoxicity in raw water. The results of the study indicated that the Vicia faba/MCN test was the most sensitive plant assay for disinfected water and that peracetic acid disinfection produced similar or lower genotoxicity than sodium hypochlorite or chlorine dioxide treatment.
Laboratory experiments and full-scale trials in Brazil and Italy are reported that show that peracetic acid is a good disinfectant (better than sodium hypochlorite) of sewage in tropical and warm temperate climates. Its demonstrated effectiveness against V. cholerae suggests it should be a significant element in cholera control efforts.
The blue mussel, Mytilus edulis is often used in marine biomonitoring programs. In this paper two different genotoxicity tests are considered as possible indicators for genotoxic pollution in the marine environment. One is focusing on micronucleus (MN) analysis in the blood cells of mussels and the other on the detection of DNA-adducts in the whole mussel tissue. The induction of MN has been studied in short-term laboratory experiments, during which mussels were exposed to standard genotoxins or waste water. Both genotoxicity tests have been applied in a study where mussels were exposed to contaminated sediment under controlled mesocosm conditions. These studies indicate that the MN mussel test may be used as a sensitive indicator of genotoxic pollution, although the inducibility of MN in the blood cells appears to be limited and to some extent seasonally dependent. The detection of DNA-adducts is still under development, but the preliminary result seems promising for its application as a biomonitoring tool.
A kinetic model allows prediction of the mortality of zebra mussels as a function of chlorine concentration, temperature, and contact time.
The rate of mortality of the zebra mussel in response to chlorine is described by a kinetic model that combines a statistical characterization of mussel mortality with a disinfection‐type modeling approach. Parameter estimates were made with nine sets of data from experiments conducted in Niagara River water. From the kinetic model, an operational diagram was constructed that describes the time to 95 percent mortality as a function of chlorine concentration and temperature. Either the model or the diagram can be used to assist utilities in planning chlorination treatments for controlling zebra mussels.
Laboratory experiments and full-scale trials in Brazil and Italy are reported that show that peracetic acid is a good disinfectant (better than sodium hypochlorite) of sewage in tropical and warm temperate climates. Its demonstrated effectiveness against V. cholerae suggests it should be a significant element in cholera control efforts.
The results of an experimental study are presented that demonstrate the use of chlorine or ozone to control zebra mussels at temperatures from 30 to 36°C. Control studies were conducted with no oxidant present. Three acclimation temperature ranges were tested: 0–5, 10–15, and 20–25°C. Chlorine was tested at 0.1 and 0.5 mg/L; ozone at 0.5 mg/L. Mortality was described by a cumulative normal distribution, from which times to 95% mortality were estimated and used as a dependent variable for hypothesis testing. Study results showed that the addition of chlorine or ozone was more effective than heat alone at test temperatures above 30°C. Compared to heat alone, the combined use of heat and oxidants decreased the time to 95% mortality by more than 95% at 30°C. Above 30°C, the benefits of the combined treatment strategy decreased with increasing test temperature. At 36°C, the benefits of the combined treatment strategy over heat alone were minimal. Acclimation temperature was important only for heat alone and for mussels acclimated at 0–5°C. The addition of chlorine or ozone at elevated temperatures can reduce mortality times by as much as three orders of magnitude compared to oxidant addition at ambient temperatures. The results of the study should be of significance to power plants or industries where excess heat is available to raise water temperatures.
Many researches have demonstrated that the use of biological processes in the production of drinking water has many advantages over conventional alternatives. One possibility is that biological processes will result in lower levels of genotoxic by-products in the drinking water. This possibility was investigated in the present study, which compares the levels of such by-products following either chemical or biological treatments. Water samples, collected at the main steps of the two key stages of both treatments were passed over XAD2/8 resins, to concentrate micropollutants, included by-products, for subsequent analysis using the microsome/Salmonella assay (TA98 and TA100±S9). The results showed that biological treatment, avoiding break-point chlorination, limits by-product formation and associated mutagenic generation. It follows that, in this respect, biological processes seem to have significant advantages over conventional water treatments.
The aims of this research were to study the influence of peracetic acid (PAA) on the formation of mutagens in surface waters used for human consumption and to assess its potential application for the disinfection of drinking water. The results obtained using PAA were compared to those found with sodium hypochlorite (NaClO) and chlorine dioxide (ClO2). The Ames test, root anaphase aberration assay, and root/micronuclei assay in Allium cepa and Tradescantia/micronuclei test were used to evaluate the mutagenicity of disinfected samples. Microbiological tests were also performed, and disinfection by-products (DBPs) were identified using gas chromatography/mass spectrometry (GC/MS). A slight bacterial mutagenicity was found in raw lake and river water, and similar activity was detected in disinfected samples. A plant test revealed genotoxicity in raw river water, and microbiological analysis showed that PAA has bactericidal activity but lower than that of the other disinfectants. The DBPs produced by PAA were mainly carboxylic acids, which are not recognized as mutagenic, whereas the waters treated with the other disinfectants showed the presence of mutagenic/carcinogenic halogenated DBPs. However, additional experiments should be performed with higher concentrations of PAA and using water with higher organic carbon content to better evaluate this disinfectant.
Many substances pollute the marine environment. There is today a growing evidence on the increased risk of disease in marine organisms, especially fish, that inhabit contaminated waters. Different types of tumours have been evidenced in fish and shellfish populations. Different short-term biomarkers are available to predict the impact of carcinogens on marine organisms. Their endpoints are different effects at the molecular and cellular level such as gene mutation, chromosome alteration and induction of DNA damage and repair. We have applied two different assays: alkaline elution to measure DNA single strand breaks and micronucleus assay as an index of a chromosomal damage. In order to select an aquatic organism as an indicator of water pollution by carcinogenic agents, we have focused on the mussel. A program of validation of genotoxicity was conducted in aquarium using DMBA. A time-dependence increase of micronuclei was evident after the exposure to 100 ppb/animal. For alkaline elution the effect was 4 times the level of the controls. Experiments in the fields were conducted on adult specimens of Mytilus gdlloprovincialis collected from natural substrates. Our sampling stations were located in the La Spezia gulf, Ligurian sea. Genotoxic effects were evaluated in gill cells. A significant increment of the two parameters in polluted, in comparison with the unpolluted sites has been observed. High frequencies of micronuclei (the highest value was 42 ± 13 with respect to control value 3 ± 2) were scored in mussels from polluted stations. The extent of DNA damage was also relevant with respect to clastogenic damage as revealed by micronucleus test. The greatest value of K (constant of elution) was 8-fold higher with respect to the value of K obtained in the same tissue of mussel from reference areas. Evidence of DNA damage could reflect a recent pollution status, since DNA strand breaks can be rapidly repaired by different mechanisms. On the contrary animals exposed to clastogenic compounds may exhibit elevated micronucleus frequency long after the exposure has ceased. The evaluation of both parameters could provide information of great significance about the pollution status of the water.
The micronucleus assay in gill tissue of the mussel Mytilus galloprovincialis has been developed in our Laboratory to assess the mutagenic activity of compounds present in marine environments. The sensitivity of the test was assessed by performing mutagenic treatment for 48 h with the two standard compounds vincristine (VCR) (0.005, 0.01, 0.02 mg l−1), and benzo(a)pyrene (BaP) (0.025, 0.075, 0.225, 0.675 mg l−1). Since both tested chemicals produced significant increases in the number of micronucleated cells, animals were directly exposed to marine waters: mussels grown in clean water (control sample) were transferred to polluted areas and then collected weekly. Micronuclei frequencies of sampled mussels were significantly higher than the value of the control group.
The information summarized in this review provides substantial evidence for the widespread presence of genotoxins in drinking water. In many, if not most cases, the genotoxic activity can be directly attributed to the chlorination stage of drinking water treatment. The genotoxic activity appears to originate primarily from reactions of chlorine with humic substances in the source waters. Genotoxic activity in drinking water concentrates has been most frequently demonstrated using bacterial mutagenicity tests but results with mammalian cell assay systems are generally consistent with the findings from the bacterial assays. There is currently no evidence for genotoxic damage following in vivo exposures to animals. In some locations genotoxic contaminants of probable industrial and/or agricultural origin occur in the source waters and contribute substantially to the genotoxic activity of finished drinking waters.
The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, Mytilus galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC (0.5 X 10(-7) and 10(-7) M) were tested at 13 degrees C and 23 degrees C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.
Drinking water samples were prepared in a pilot-scale treatment plant by chlorination (Cl2), chloramination (NH2Cl), ozonation (O3), or O3 followed by Cl2 or NH2Cl; and the nonvolatile acidic organics of the raw and treated waters were extracted by XAD/ethyl acetate and evaluated for mutagenicity in Salmonella (-S9). The extracts were 2-8 times more mutagenic in TA100 than in TA98, and the mutagenic potencies of the water extracts ranked similarly in both strains: Cl2 > O3 + Cl2 > NH2Cl > O3 + NH2Cl > O3 > raw. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), which was estimated to account for approximately 20% of the mutagenic activity of the extracts, was shown to be the most potent compound tested thus far in a prophage-induction assay in Escherichia coli and a forward-mutation assay in Salmonella TM677. The mutations in approximately 2,000 revertants of TA98 and TA100 induced by MX and the water extracts were analyzed by colony probe hybridization and polymerase chain reaction/DNA sequence analysis. The water extracts and MX produced similar mutation spectra, which consisted in TA100 of predominantly of GC-->TA transversions in the second position of the CCC (or GGG) target of the hisG46 allele. This spectrum resembles that produced by large aromatic compounds and is distinct from that produced by alkylating agents and the semivolatile drinking water mutagen dichloroacetic acid. In TA98, MX and those water extracts resulting from the introduction of the chlorine atom produced 50-70% hotspot 2-base deletions and 30-50% complex frameshifts (frameshifts with an adjacent base substitution--mostly GC-->TA transversions as found in TA100). No other compound or mixture is known to induce such high frequencies of complex frameshifts. These results suggest that MX and "MX-like" compounds (possibly halogenated aromatics, such as halogenated polycyclic aromatic hydrocarbons) account for much of the mutagenic activity and specificity of the nonvolatile organics in drinking water and that these halogenated organics are especially capable of promoting misincorporation by the DNA replication complex. This study provides further evidence that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture.
Zebra mussels, Dreissena polymorpha, were exposed to four directly acting reference clastogens (mitomycin C, bleomycin, dimethylarsinic acid and potassium chromate) under laboratory conditions. The aim was to examine the inducibility of micronuclei (MN) in haemocytes and gill cells. Positive responses were observed in both tissues for all four substances used under the given test conditions. The mean MN frequencies in treated mussels ranged between 3.2 and 6.9/1000 in haemocytes and between 5.4 and 6.7/1000 in gill cells. The spontaneous MN levels averaged 1.2 and 2.8/1000 in haemocytes and gill cells, respectively. The MN induction capacity of the different chemicals was equivalent in both tissues, except for the treatment with dimethylarsinic acid which generated a significantly higher MN rate in gill cells than in haemocytes. Several characteristics suggest that haemolymph is the more appropriate test tissue for environmental genotoxicity assessment: (1) a shorter preparation time of slides, (2) a more accurate identification of unambiguous MN, (3) a lower baseline MN frequency and a higher induction factor.
Caged zebra mussels, Dreissena polymorpha, were transplanted to 6 monitoring sites receiving industrial effluents suspected of containing genotoxic chemicals. After a residence time of 2 months, the induction of micronuclei (MN) in haemocytes was determined as a criterion for genetic damage. The mean MN frequencies observed in mussels exposed to effluents ranged between 5.0 and 8.8/1000. These rates were significantly higher than the baseline level of 2.0/1000 recorded in a concurrent control mussel group reintroduced at the reference location. Biological fitness descriptors (mortality, attachment biotest, condition index, gonadic index) revealed no relationship between the general well-being of the mussels exposed under contrasted environmental conditions and the frequency of MN induced in their haemocytes. The biological feasibility of the transfer technique of zebra mussels, together with a moderate, but significant, inducibility of MN, are major features towards the development of a first tool for in situ monitoring of genotoxicity in freshwater environments using a common indicator species.
While sodium hypochlorite is widely used as a disinfectant for municipal sewage effluents and power station cooling waters discharged into coastal environments, there is limited information on the potential in vivo genotoxicity of such disinfection procedures to marine organisms. Using a recently developed test system based on the marine polychaete Platynereis dumerilii, we have evaluated impacts based on embryo-larval development, cytotoxicity and genotoxicity following exposure to disinfected settled (primary) effluent from a municipal sewage treatment works (STW). Sewage samples were collected from Newton Abbot STW, Devon, UK and then disinfected with sodium hypochlorite based on standard operational procedures. Exposure of polychaetes to dilutions of disinfected sewage in seawater (20 +/- 1 degree C) led to a marked reduction in normal embryo-larval development (7 h EC50 from 0.57-1.88% (v/v), n = 4), with a simultaneous increase in cytotoxicity. Following the calculation of the Maximum Tolerated Dose (MTD), based on developmental and cytotoxic effects, the organisms were also analysed for the induction of chromosomal aberrations. This investigation demonstrated the absence of genotoxicity in polychaetes exposed in vivo to sewage disinfected with sodium hypochlorite. These observations extend our previously published studies in which polychaetes exposed to non-disinfected sewage, while showing developmental toxicity and cytotoxicity, did not exhibit any evidence of cytogenetic damage.
Our aim is to develop and evaluate monitoring systems that use aquatic organisms to assess the genotoxicity of water in the field and in the laboratory. In a field study, we have shown that the micronucleus assay is applicable to freshwater and marine fishes and that gill cells are more sensitive than hematopoietic cells to micronucleus-inducing agents. Gill cells from Carassius sp. (Funa) and Zacco platypus (Oikawa) collected upstream on the Tomio River (Nara, Japan), tended to have lower micronucleus frequencies than gill cells from fish collected at the midstream of the river. Leiognathus nuchalis (Hiiragi) and Ditrema temmincki (Umitanago), small marine fishes collected periodically at Mochimune Harbor (Shizuoka, Japan), showed seasonal differences in the frequencies of micronucleated gill cells and erythrocytes; they were highest in summer. For laboratory studies, we developed a method for analyzing chromosomal aberrations and micronuclei using Rhodeus ocellatus ocellatus (rose bitterling) embryos. One day after artificial insemination (gastrula stage), we observed structural chromosomal aberrations and micronuclei in the cells of embryos grown in water containing trichloroethylene. Although more work is needed to fully assess their sensitivity, these assays show promise as a means of detecting environmental genotoxins.
Heavy metals are stable and persistent environmental contaminants. The range of metal concentrations is generally below acute thresholds in coastal areas, where recognition of chronic sublethal effects is more relevant. Evidence of long-term adverse effects, such as cancer, due to heavy metals in marine animals comes from a number of field and experimental studies. The mechanism of metal carcinogenicity remains largely unknown, although several lines of experimental evidence suggest that a genotoxic effect may be involved. The aim of our study was to evaluate the sensitivity of genotoxicity tests, alkaline elution and micronucleus test, as biomarkers for the detection of heavy metals in mussels as the sentinel species. Experimental studies were carried out on Mytilus galloprovincialis exposed in aquarium (5 days) to different concentrations of three selected metal salts, CuCl2 (5, 10, 20, 40, 80 micrograms/l/a), CdCl2 (1.84, 18.4, 184 micrograms/l/a), and HgCl2 (32 micrograms/l/a), and to a mixture of equimolar doses of the three metals to study the results of their joint action. Metallothionein quantitation was used as a marker of metal exposure. Lysosomal membrane stability was applied to evaluate the influence of physiological status on genotoxic damage. The ranking of genotoxic potential was in decreasing order: Hg > Cu > Cd. Cu and Hg caused an increase of DNA single-strand breaks and micronuclei frequency. Cd induced a statistical increase of DNA damage, but gave negative results with the micronucleus test. A relationship between genotoxic effects and metallothionein content was observed. Reduction in lysosomal membrane stability with the increasing concentration of heavy metals was also evident.
A number of chemical contaminants have been identified in drinking water. These contaminants reach drinking water supplies from various sources, including municipal and industrial discharges, urban and rural run-off, natural geological formations, drinking water distribution materials and the drinking water treatment process. Chemical contaminants for which epidemiologic studies have reported associations include the following: aluminium, arsenic, disinfection by-products, fluoride, lead, pesticides and radon. Health effects reported have included various cancers, adverse reproductive outcomes, cardiovascular disease and neurological disease. In evaluating epidemiologic studies for risk assessment, considering whether the study design was qualitative (hypothesis generating) or quantitative (hypothesis testing) is important and whether sufficient epidemiologic data of a quantitative nature exists to determine the dose-response curve. Each of the chemical contaminants mentioned are summarized by study designs (qualitative and quantitative) and whether a dose-response curve based on epidemiologic data has been proposed. Environmental epidemiology studies are driven by environmental exposures of interest. For drinking water contaminants, the design of epidemiologic studies and their interpretation should consider the following exposure issues: the source of the contaminant; other sources of the contaminant; the route of exposure; the frequency, duration and magnitude of exposure; the ability to document an actual internal dose; and the ability to document the dose to the target organ. Health effects of concern have other risk factors that must be measured in the conduct of these studies. In evaluating epidemiologic studies, potential errors and biases that may occur must be considered given the very low magnitude of associations (less than 2.0 for either odds ratio or risk ratio). Given the issues, the next generation of drinking water epidemiologic studies should include a multidisciplinary team beyond traditional epidemiologists and statisticians. Study teams will require toxicologists, chemists, engineers and exposure assessors. Arsenic is briefly discussed as an example of the importance of susceptible populations. Disinfection by-products are discussed as an example of epidemiologic studies of mixtures.
Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
Fifteen chlorination by-products were analyzed in 416 water samples collected from 35 water treatment plants in Korea from 1996 to 1998. These samples were divided into five groups according to water sources (Han-river, Nakdong-river, Youngsan-river, Kum-river and Cheju) and detected CBPs were classified into six classes (trihalomethanes; THMs, haloacetic acids; HAAs, haloacetonitriles: HANs haloketones; HKs, chloralhydrate; CH, chloropicrin; CP) and then, it was observed the detection tendency and frequency of CBPs in each water source. The total concentration of CBPs in treated water from Nakdong-river or Han-river was higher than those from the other rivers. And the distribution pattern of each class of CBPs was similar in all water sources. THMs were the highest portion in the range of 40-50%, and HAAs and HANs were 28-35 and 9-15%, respectively. And there was a strong correlation between HANs and HKs (r=0.813). Each and total concentrations of CBPs showed to be more affected by the water source in two-way analysis of variance (two-way ANOVA) among the concentration of CBPs, the source of water and season.
The aims of this research were to study the influence of peracetic acid (PAA) on the formation of mutagens in surface waters used for human consumption and to assess its potential application for the disinfection of drinking water. The results obtained using PAA were compared to those found with sodium hypochlorite (NaClO) and chlorine dioxide (ClO2). The Ames test, root anaphase aberration assay, and root/micronuclei assay in Allium cepa and Tradescantia/micronuclei test were used to evaluate the mutagenicity of disinfected samples. Microbiological tests were also performed, and disinfection by-products (DBPs) were identified using gas chromatography/mass spectrometry (GC/MS). A slight bacterial mutagenicity was found in raw lake and river water, and similar activity was detected in disinfected samples. A plant test revealed genotoxicity in raw river water, and microbiological analysis showed that PAA has bactericidal activity but lower than that of the other disinfectants. The DBPs produced by PAA were mainly carboxylic acids, which are not recognized as mutagenic, whereas the waters treated with the other disinfectants showed the presence of mutagenic/carcinogenic halogenated DBPs. However, additional experiments should be performed with higher concentrations of PAA and using water with higher organic carbon content to better evaluate this disinfectant.
For over 20 years, mussels have been recommended as one of the most suitable biomonitoring organisms for aquatic ecosystems. Though the common mussel (Mytilus edulis) is frequently used for biomonitoring estuarine and marine ecosystems, no freshwater species is promoted for similar monitoring networks. Recently, however, the zebra mussel (Dreissena polymorpha) has been proposed as a suitable monitoring organism in freshwater ecosystems. The aim of this study was to explore the usefulness of transplanted zebra mussels as active biomonitors in an effluent-dominated stream. Results showed that for these purposes, an exposure period of at least a few weeks is required to detect any significant changes in condition status or scope for growth. Wet-tissue-weight:dry-tissue-weight ratio was the most sensitive measure to quantify effects of field exposure on physiological fitness. In case of scope for growth (SfG), energy intake was the factor determining the overall energy budget of the mussels. Based on the dilution rates of the two different effluents present, effluent 2 had the most important effect on the condition status of the exposed organisms. Overall, we conclude that the use of transplanted mussels is a sensitive and easily applicable active biomonitor that can be used to assess water quality, pollution, and subsequent recovery through self-purification in field situations.
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 degrees 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 1h. 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 degrees C and a minimum level at 18 degrees 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.
The detection of a possible genotoxic effect of surface water treated with disinfectants for potabilization is the aim of the present work. The Comet assay and the micronucleus test were applied in circulating erythrocytes of Cyprinus carpio. Young specimens (20-30 g) were exposed in experimental basins, built within the potabilization plant of Castiglione del Lago (Perugia, Italy). In this plant the water of the Trasimeno Lake is treated and disinfected for potabilization before it is distributed to the people in the net of drinkable water. A continuous flow of water at a constant rate was supplied to basins; the water was continuously treated at a constant concentration with one of the three tested disinfectants (sodium hypochlorite, peracetic acid and chloride dioxide), one control basin being supplied with untreated water. Three sampling campaigns were performed: October 2000, February 2001 and June 2001. Repeated blood samplings through intracardiac punctures allowed to follow the same fish populations after different exposure times: before introduction of the disinfectant, and 10 or 20 days afterwards. An additional blood sampling was performed 3 h after addition of the disinfectant in other, simultaneously exposed, fish populations. Genotoxic damage was shown in fish exposed to water disinfected with sodium hypochlorite and chloride dioxide. The Comet assay showed an immediate response, i.e. DNA damage that was induced directly in circulating erythrocytes, whereas micronuclei reached their highest frequencies at later sampling times, when a genotoxic damage in stem cells of the cephalic kidney is expressed in circulating erythrocytes. The quality of the untreated surface water seems to be the most important parameter for the long-term DNA damage in circulating erythrocytes.
The uptake and bioaccumulation of 15 road dust metals by the zebra mussel (Dreissena polymorpha) were investigated in laboratory exposure studies with emphasis on the traffic-related platinum-group elements (PGEs) palladium (Pd), platinum (Pt), and rhodium (Rh). The biological availability of the metals may depend on water characteristics, so the mussels were maintained in two types of water: nonchlorinated tap water and humic water of a bog lake, both of which contained dust of a moderately frequented road. After an exposure period of 26 weeks, soft tissues of the mussels were freeze-dried and analyzed for the metals. The metal concentrations in the mussel soft tissue ranged from several hundred micrograms per gram (e.g., for iron [Fe]) to less than 10 ng/g (for PGEs). Metal uptake from the road dust by the mussels was found for the PGEs and silver (Ag), bismuth (Bi), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), Fe, lead (Pb), and antimony (Sb). After maintenance of mussels in road dust-contaminated tap water, bioaccumulation factors (BAF = (C(exposed mussels) - C(control mussels))/C(total metal, water), where c is concentration) decreased in the following order: Cu > Cd > Ag > Pd > Sb > Pb > Fe > Pt > Rh. The biological availability of most metals was enhanced by humic water as compared to tap water. Our results show a hitherto unrecognized high availability of Pd for the mussels. Thus, this metal should be monitored more intensively in the environment to assess its distribution in the biosphere.
Leitz Dialux 20) equipped with an exci-Some studies have reported that mussel tolerance to biocides is regulated by seasonal changes (van Benschoten et al., 1995) and by temperature) because We are grateful to the MURST Research Group
- Aghar
Ag/ml), observations were made under a fluorescence microscope (Leitz Dialux 20) equipped with an exci-Some studies have reported that mussel tolerance to biocides is regulated by seasonal changes (van Benschoten et al., 1995) and by temperature (Har-rington et al., 1997; Buschini et al., 2003) because We are grateful to the MURST Research Group: A. Alberti, D. Bartoli, G. Cantelli-Forti, E. Chiesara, E. Ciccarelli, M. di Brizio, M. Dö, C. Elia, D.
AOX concentra-tion increased only in NaClO-disinfected water, espe-cially in February
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Ag/l; June: 1.34 Ag/l). AOX concentra-tion increased only in NaClO-disinfected water, espe-cially in February (12 Ag/l for raw water, 13 Ag/l for PAA, 43 Ag/l for NaClO, and 15 Ag/l for ClO 2, respectively) and June (8 Ag/l for raw water, 10 Ag/ under a yellow light to minimise additional UV-induced DNA damage. After staining with 100 Al ethidium bromide (10
We would like to thank Gillian Mansfield (Director of the Language Centre, University of Parma) for the English revision of the manuscript Standard methods for examination of water and wastewater. XX ed. Washington: APHA; 1998
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