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Comet assay and micronucleus test in circulation erythrocytes of Cyprinus carpio specimes exposed in situ to lake waters treated with desinfectants for potabilization
Abstract
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.
... These lagging chromosomes and acentric fragments are excluded from the daughter nuclei during cell division, resulting in the formation of an additional, and notably smaller, nucleus within the cytoplasm known as the micronucleus. Micronuclei are considered markers of abnormal cell division involving chromosomal breakage, missaggregated chromatin or mitotic interference, events thought to be associated with an increased risk for cancer [8,9]. ...
... The extracted blood was immediately smeared on a clean glass slide. After 24 hours, the material was fixed in absolute methanol for 10 minutes and was stained with 5% Giemsa for another 20 minutes [8]. The micronucleus was identified using the criteria cited in a study from Ray et al. [14]. ...
... Cells with two nuclei present were considered as nuclear abnormalities, specifically, binucleates. Other nuclear abnormalities of the erythrocytes were classified as blebbed, notched and lobed nuclei [8,15]. The frequency of micronucleus and nuclear abnormalities was expressed per 2000 erythrocytes. ...
The present study determined the genotoxic potential induced by intensive fish cage aquaculture in Taal Lake (Philippines) on Nile tilapia (Oreochromis niloticus) erythrocytes using the comet assay and micronucleus test. Water and sediment samples were collected from three sampling sites namely: Gonzales, Bañaga, and Balakilong, which are non-aquaculture, moderate aquaculture, and heavy aquaculture sites, respectively. The highest incidence of tail moments, micronuclei, and nuclear abnormalities was detected in Balakilong site. The nuclear abnormalities of erythrocytes included blebbed, notched and lobed nuclei. Both assays (comet assay and micronucleus test) showed concordance by establishing similar gradient of severity based from cumulative results: Balakilong > Bañaga > Gonzales. Using post-hoc analyses, the two aquaculture sites showed no significant differences in both mean tail moment and frequency of nuclear abnormalities. On the other hand, significant differences were observed when both aquaculture sites were compared to the non-aquaculture site. Although no specific cause-effect relationships were established, the genotoxicity data indicate association between the level of unionized ammonia and copper, which have settled into the sediments from unconsumed feeds. Tilapia, which feeds heavily in bottom sediments, is in direct contact with the contaminants and can take in considerable lev ls of ammonia. The detection of copper even in non-aquaculture site indicates that that contamination has spread widely in the lake. Results of the study have demonstrated that both assays are effective as field biomarkers for investigating the harmful effects of fish cage aquaculture on lake quality.
... Several studies on fish recognized the Comet assay as one of the most sensitive methods available for measuring DNA strand breaks in contrast to other biomarkers widely used in genetic ecotoxicology, such as the micronucleus test and sister chromatid exchanges (Buschini et al., 2004;Cavas and Konen, 2007;Kim and Hyun, 2006). The main advantages of the Comet assay are: flexibility, ease in application, low costs, only a small number of sample cells are required, and it is capable of measuring DNA strand damage and repair at single cell level following genotoxic exposure (Lee and Steinert, 2003;Mitchelmore and Chipman, 1998). ...
... Our results show that both hypoxic and hyperoxic conditions have considerable impact on the level of DNA strand breaks in fish erythrocytes. These results are consistent with previous studies where fish erythrocytes have been shown to be sensitive for measuring the genotoxic effects either in laboratory or in field studies (Belpaeme et al., 1998;Buschini et al., 2004). ...
... One of the advantages of comet and MN assays is that both can be used for the simultaneous assessment of DNA damage in many tissues from the same animal, the comparison of their responses under identical treatment conditions. Thus, these techniques can be used in combination for screening genotoxic effect of chemicals and for investigating the implications of DNA damage and its recovery in the sentinel fish species.Micronucleus test and comet assay are found to be easy, sensitive, rapid and extensively used methods for mutagenicity and genotoxicity testing in various laboratory and field studies ( [16,96,30,24,62,73,81,96] . evaluated the mutagenic effect of disinfectant added to the lake water for potabilization in fish C. carpio. ...
... Similarly Banu et al. (2001) [19] showed that monocrotophos treatment in fish, Tilapia mossambica lead to time dependent reduction in DNA damage. 24 [43] reported that when Zebra fish (D. rerio) were exposed to borax, the highest DNA damage was observed at 24 hours, followed by decrease at 48 and 72 hours and again increase in the value was observed at 96 hours, while treatment with boric acid induced highest effect at 96 hours of exposure. Ali and Kumar, (2008) [7,9] reported that when fish C. punctatus were exposed to monocrotophos gill, liver and lymphocytes show highest damage at 4 th day followed by decline in value afterwards. ...
In an era where innumerable biological and synthetic compounds are perpetually introduced into the market every day in various forms, such as pharmaceuticals, cosmetics, nutraceuticals, fertilizers, pesticides, and nanoparticles, to serve multiple purposes, there is a need to develop rapid screening methods to evaluate their biosafety. Wide array of biomarkers used to demonstrate exposure to and effect of environmental contamination. In order to assess exposure to or effect of environmental pollutant on aquatic environment the following suite of fish biomarkers may be examined, oxidative stress parameters, hematological parameters, immunological parameters, genotoxic parameters and histology. The text explores the latest knowledge and thinking on these very important approaches for the assessment of environmental health, management and conservation. This review focuses on measurement of the genotoxic potential of chemicals in aquatic organism at different level of biological organization under field and laboratory conditions by using comet assay, micronucleus assay, polychromatic erythrocyte count.
... Erythrocytes served as indicator cells for exposure to genotoxic compounds, which was assessed by applying the micronucleus and the Comet assays. These assays are considered to be reliable indicators (Collins, 2015) and are often used in combination in studies employing fish erythrocytes (Buschini et al., 2004;Rocha et al., 2009;Rocha et al., 2011;Bücker et al., 2012;Jovanović et al., 2018;etc.). ...
... The applied protocol was based on the methods described by Buschini et al. (2004) and Bücker et al. (2012) with slight modifications. Blood was dropped onto slides (three slides from each specimen) and air-dried for 24 h. ...
Being exposed to untreated urban and industrial water, the rivers Drenica and Sitnica are considered to be the most polluted ones in the Kosovo. Our previous investigations on the cyto- and genotoxic potential of water samples from these rivers evaluated with primary rat hepatocyte cultures indicated a risk for the health of aquatic organisms. In order to assess the genotoxic risk to aquatic organisms, we therefore performed a two year study (2016–2017) on roach (Rutilus rutilus) from these rivers. Specimens were collected at three locations along the Drenica river and two locations along the Sitnica river, and the genotoxicity was evaluated by the micronucleus as well as the Comet assay (DNA damage) in erythrocytes. The frequencies of micronucleated cells were determined for samples collected in four seasons, whereas the Comet assay was employed on samples collected in five seasons during the two-year period. The data obtained revealed an increase of the frequency of micronucleated erythrocytes from Rutilus rutilus collected at most sampling locations and from both rivers at all seasons investigated. Significant differences to the control (lake Badovc) were found in summer 2016 and spring 2017 samples. When comparing the seasons, the summer 2016 samples were most genotoxic, followed by spring 2017 and autumn 2016. With regard to the Comet assay data, a similar but more prominent “response” was observed. Another important observation is that micronucleus rates as well as DNA damage levels were significantly higher in samples collected in 2016 compared to the respective seasons in 2017. Altogether, the “response” obtained with both markers confirmed a genotoxic risk for fish due the pollution of these rivers. Since there were, however, seasonal and annual variations of the genotoxicity levels further in depth studies have to be carried out addressing the nature of these changes.
... Micronuclei are chromosomal fragments which are not transmitted to the daughter cells during the anaphase stage of cell division and remained in the mother's cytoplasm (Siu et al 2004) Evaluation of these micronuclei is an effective method for determining the genotoxic effects of compounds with low level of accumulation as well as an indicator of genetic damages induced to the cells (Gravato & Santos 2003). Micronucleus test has been widely used in fish erythrocytes, especially in Cyprinus carpio (Al-Sabti 1986;Nepomuceno et al 1997;Gustavino et al 2001;Buschini et al 2004). In the present study, due to the wide production and application of copper nanoparticles in various industries, as well as the lack of sufficient information on the genetic effects of these substances on aquatic organisms, the genotoxic potential of CuO-NPs were evaluated in C. carpio using comet assay and micronucleus test. ...
... In the present study, micronucleus test was also used to evaluate the genotoxic potential of CuO-NPs. Using comet assay, DNA damage could be identified in a short time after exposing to the genotoxic agent while identifying the micronuclei resulted from chromosomal fragmentation or mitotic abnormalities needs the passage through mitosis (Buschini et al 2004). The micronucleus test in fish is mainly carried out using the erythrocytes (Al-Sabti & Metcalfe 1995;Bony et al 2010;Bottcher et al 2010). ...
Due to the wide production and application of copper nanoparticles and a subsequent increase in the possibility of their penetrance into the aquatic ecosystems, a growing concern has been emerged about the potential effects of these materials on the health of aquatic organisms. The goal of the present study was to evaluate the genotoxic potential of copper oxide nanoparticles (CuO-NPs) in common carp (Cyprinus carpio). For this, the fish were exposed to 0 (control), 2.5, 6.25 and 10 mgL⁻¹ of CuO-NPs for 14 days. To evaluate DNA damage through single cell gel electrophoresis, sampling was performed from the tissues of gill and hepatopancreas on the 7th and 14th days of exposure. The blood sampling was also done to determine micronuclei frequency in the erythrocytes. According to the results, CuO-NPs exposure induced micronuclei formation and DNA damage in C. carpio, so that the frequency of micronuclei and values of DNA damage parameters including Tail Length, Tail Moment, Tail DNA%, and genetic damage index (GDI) significantly increased in CuO-NPs-exposed treatments as compared to the control (P≤0.05). The rate of induced damage was dependent on the exposure time and concentration. The hepatopancreas tissue also showed more susceptibility to CuO-NPs genotoxicity, so that the damage level observed in hepatopancreas was higher than that in the gill. Overall, according to the present findings, CuO-NPs could be considered as a potential genotoxic pollutant for C. carpio.
... Timecourse measurements in field experiments supported a linkage between induction of DNA strand breakage and the timing of agricultural runoff. A possible genotoxic effect of surface water treated with disinfectants for potabilization was detected by Buschini et al. (2004) [11] . The comet assay was applied in circulating erythrocytes of Cyprinus carpio. ...
... Timecourse measurements in field experiments supported a linkage between induction of DNA strand breakage and the timing of agricultural runoff. A possible genotoxic effect of surface water treated with disinfectants for potabilization was detected by Buschini et al. (2004) [11] . The comet assay was applied in circulating erythrocytes of Cyprinus carpio. ...
... A potential drawback of this method is the lack of selectivity, since both PAA and H 2 O 2 can oxidize iodide. Indeed, when PAA concentration is to be selectively determined by the DPD method, catalase is usually added to decompose H 2 O 2 in solution [24,[33][34][35][38][39][40]. However, in many other cases the occurrence of interference related to H 2 O 2 is neglected [31,36,37,[41][42][43] in agreement with the results of Liu et al. [44], who reported that H 2 O 2 does not disturb the PAA concentration measurement using the DPD method. ...
... At lower concentrations, the recoveries tend to be higher and the standard deviations lower. Several authors [24,31,[33][34][35][36][37][38][39][40][41][42][43] have applied the DPD method to the determination of low concentrations of PAA, whereas only few of them [24,43] have described the performance and accuracy of the method. As for the measurement of H 2 O 2 , to the best knowledge of the authors, the DPD method has not been applied in wastewater disinfection studies to date. ...
The recent growing interest in peracetic acid (PAA) as disinfectant for wastewater treatment demands reliable and readily-available methods for its measurement. In detail, the monitoring of PAA in wastewater treatment plants requires a simple, accurate, rapid and inexpensive measurement procedure. In the present work, a method for analyzing low concentrations of PAA, adapted from the US EPA colorimetric method for total chlorine, is assessed. This method employs N,N-diethyl-p-phenylelnediamine (DPD) in the presence of an excess of iodide in a phosphate buffer system. Pink colored species are produced proportionally to the concentration of PAA in the sample. Considering that PAA is available commercially as an equilibrium solution of PAA and hydrogen peroxide (H2O2), a measurement method for H2O2 is also investigated. This method, as the one for the determination of PAA, is also based on the oxidation of iodide to iodine, with the difference that ammonium molybdate Mo(VI) is added to catalyze the oxidation reaction between H2O2 and iodide, quantifying the total peroxides (PAA+ H2O2). The two methods are suitable for concentration ranges from about 0.1 to 1.65 mg L⁻¹ and from about 0.3 to 3.3 mg L⁻¹, respectively for PAA and H2O2. Moreover, the work elucidates some relevant aspects related to the operational conditions, kinetics and the possible interference of H2O2 on PAA measurement.
... Atualmente seu cultivo ocorre em todos os continentes devido a sua rusticidade e facilidade de criação (Querol et al. 2005). A espécie já foi utilizada com sucesso em estudos de avaliação de genotoxicidade (Buschini et al. 2004;Gustavino et al. 2005;Canistro et al. 2012;García-Medina et al. 2013). Nesse contexto, o objetivo do presente estudo foi avaliar a qualidade da água do Rio dos Sinos por meio do teste de micronúcleos em C. carpio bem como de análises físico-químicas e microbiológicas. ...
... Para a primeira metodologia, não encontraram diferenças entre os trechos analisados, mas para a segunda, verificaram aumento na frequência de danos ao material genético dos grupos expostos à água dos trechos médio e inferior em comparação ao trecho superior. O teste de micronúcleos em eritrócitos de C. carpio já foi utilizado com o objetivo de verificar o potencial genotóxico de determinadas substâncias(Buschini et al. 2004;Gustavino et al. 2005;Canistro et al. 2012;García-Medina et al. 2013) ou a sensibilidade da espécie às mesmas (Al-Sabti 1986; Grisolia e Cordeiro 2000). São poucos os estudos visando à avaliação de corpos de água com a espécie. ...
Resumo. O Rio dos Sinos, localizado na região nordeste do Rio Grande do Sul, é considerado um dos principais rios do estado, concentrando na área de sua bacia hidrográfica significativa percentagem da população, além de um gran-Avaliação da qualidade da água do Rio dos Sinos (Brasil) por meio do teste de micronúcleos em Cyprinus carpio e de análises físico-químicas e microbiológicas Evaluation of water quality of the Sinos River (Brazil) by micronucleus test in Cyprinus carpio and physicochemical and microbiological analysis Resumo. O Rio dos Sinos está localizado na região sul do Brasil, possui aproximadamente 190 km de extensão e fornece água para atividades agrícolas, industriais e para o consumo de mais de um milhão de habitantes. Este rio é considerado um dos mais poluídos do Brasil e, portanto, estudos para avaliar a qualidade da água ao longo de sua extensão são necessários. O teste de micronúcleos em eritrócitos de peixes tem sido utilizado com sucesso para detectar a presença de poluentes mutagênicos nos ambientes aquáticos. O objetivo do presente estudo foi avaliar a qualidade da água do Rio dos Sinos por meio do teste de mi-cronúcleos em Cyprinus carpio bem como de análises físico-químicas e microbiológicas. Foram coletadas amostras de água nos trechos superior (Caraá), médio (Parobé) e inferior (Novo Hamburgo) do Rio dos Sinos em dezembro de 2013. As amostras foram transportadas para o laboratório para análise de 14 parâmetros de qualidade da água bem como para a exposição de espécimes de C. carpio por 72h em aquários. Não foram verificadas diferenças significativas na frequência de micronúcleos e anormalidades nucleares nos grupos expostos a água do rio em comparação ao grupo controle. Apenas um parâmetro de qualidade da água foi observado em desacordo com a legislação no trecho superior do rio, enquanto que nos trechos médio e inferior foram seis e cinco parâmetros, respectivamente. Os resultados demonstram que o Rio dos Sinos apresenta redução da qualidade da água ao longo de sua extensão e indicam ausência de potencial genotóxico no período amostrado. Abstract. The Sinos River is located in South of Brazil, it has about 190 km of extension and provides water for agricultural and industrial activities, and for consumption of more than one million inhabitants. This river is considered one of the most polluted rivers in Brazil; therefore, studies aiming to evaluate the water quality along its course are necessary. The micronucleus test in fish erythrocytes has been successful used to detect mutagenic pollutants in the aquatic environment. The objective of this study was to evaluate the water quality of the Sinos River by the micronucleus test in Cyprinus carpio, as well as physicochemical and microbiological analyses. Water samples were collected in the upper (Caraá municipality), middle (Parobé municipality) and lower (Novo Hamburgo municipality) sections of the Sinos River, in December 2013. The samples were transported to the laboratory for analysis of 14 water quality parameters and for exposure of C. carpio for 72 hours in aquaria. Significant differences in micro-nucleus and nuclear abnormalities frequencies were not found between the control and the exposed groups. Only one parameter of water quality exceeded the limit of the legislation in the upper section of the river, while in the middle and lower sections, six and five parameters, respectively. These results show that the Sinos River presents a reduction in water quality along its extension and indicates the absence of genotoxic potential in the sampled period.
... The frequency of micronuclei in common carp from control groups in various experimental setups showed large variations: 0.01-0.02‰ [68]; 0.17‰ [69]; 0.02-0.06‰ [70]; 0.52‰ [71]; 0.03-0.98‰ ...
S-metolachlor is one of the most frequently used herbicides worldwide. However, toxicity studies of this herbicide to aquatic organisms are scarce. In the present study, two experiments were conducted to test the effects of S-metolachlor on common carp, one of the most economically important fish species, with a distribution throughout the world: (1) 96 h acute semi-static toxicity test, aiming to determine LC50; (2) a subchronic semi-static test that lasted 28 days, in which juvenile carp were exposed to 3%, 8%, and 25% of previously determined LC50—0.5 mg·L−1, 1.4 mg·L−1, and 4.1 mg·L−1, respectively. Several biomarkers were employed to assess fish responses to toxicants. Blood biochemistry analysis and nuclear alterations of erythrocytes did not show any difference among experimental groups. Semi-quantitative histopathological analysis revealed mild alterations in the gills and liver, where oedema of secondary epithelium of gills and leukocyte infiltration in liver were significantly higher in fish exposed to 1.4 mg·L−1 and 4.1 mg·L−1. Histopathological indices in liver, as well as the total histopathological index, also showed significantly higher scores in the same groups. Bioconcentration factors of S-metolachlor ranged from 3.2 to 9.4, depending on the experimental group.
... Given the fact that the CuO NPs size we used in our study were <40 nm, they can easily penetrate the crucian carp erythrocytes membrane entering the cell and even the nucleus causing cell cyto-and genotoxicity. Numerous studies have evaluated the genotoxic effects of metal particles on fish blood by using comet assay and micronucleus test (Buschini et al., 2004;Bottcher et al., 2010;Isani et al., 2013;Nikdehghan et al., 2018) but very few have considered total erythrocyte morphological celular and nuclear abnormalities (EMA) (Aliko et al., 2018;Sula et al., 2020a). To the best of our knowledge, this work is the first to evaluate the total EMAs induced by environmentally realistic doses of CuO NPs to crucian carp fish. ...
The usage of copper (II) oxide nanoparticles (CuO NPs) has significantly expanded across industries and biomedical fields. However, the potential toxic effects on non-target organisms and humans lack comprehensive understanding due to limited research on molecular mechanisms. With this study, by combining the 96 h in vivo exposure of crucian carp fish, Carassius carassius, to sub-lethal CuO NPs doses (0.5 and 1 mg/dL) with image-based quantification, and docking and molecular dynamics approaches, we aimed to understand the mechanism of CuO NPs-induced cyto-genotoxicity in the fish erythrocytes. The results revealed that both doses of copper NPs used were toxic to erythrocytes causing oxidative stress response and serious red blood cell morphological abnormalities, and genotoxicity. Docking and 10-ns molecular dynamics confirmed favorable interactions (ΔG = −2.07 kcal mol−1) and structural stability of Band3-CuO NP complex, mainly through formation of H-bonds, implying the potential of CuO NPs to induce mitotic nuclear abnormalities in C. carassius erythrocytes via Band3 inhibition. Moreover, conventional and multiple ligand simultaneous docking with DNA revealed that single, double and triple CuO NPs bind preferentially to AT-rich regions consistently in the minor grooves of DNA. Of note, the DNA-binding strength subtantially increased (ΔG = −2.13 kcal mol−1, ΔG = −4.08 kcal mol−1, and ΔG = −6.03 kcal mol−1, respectively) with an increasing number of docked CuO NPs, suggesting that direct structural perturbation on DNA could also count for the molecular basis of in-vivo induced DNA damage in C. carassius erythrocytes. This study introduces the novel term “erythrotope” to describe comprehensive red blood cell morphological abnormalities. It proves to be a reliable and cost-effective biomarker for evaluating allostatic erythrocyte load in response to metallic nanoparticle exposure, serving as a distinctive fingerprint to assess fish erythrocyte health and physiological fitness.
... Chlorpyrifos's genotoxic potential was amply demonstrated in the current investigation by a substantial genotoxic effect of chlorpyrifos that was detected from the time of exposure as well as concentrations. The current findings were in agreement with earlier research on a variety of fish species, including Cyprinus carpio 59 . ...
Pesticide toxicity is a global concern and chlorpyrifos is one of the most widely used organophosphate agrochemicals,it is frequently detected in surface ground waters of India. Chlorpyrifos(CPF) LC50 – 96 h was calculated as 0.318 mg/l using probit analysis based on LC50 value two sublethal concentrations of 1/5th of LC50 (0.0636 mg/l) and 1/10th of LC50 (0.0318 mg/l) were determined. The influence of CPF on the acetylcholinesterase (AChE) activity and Ach content and the Genotoxicity of Cyprinus carpio was evaluated. Results confer that significant inhibition in the AchE activity in brain tissue resulting in a build-up of Ach at the neuronal synapse resulting in a decrease of cholinergic transmission finally impairs the neurophysiological activity and ultimately result in the death of fish although studies on genotoxic effects of CPF were carried out to examine the prevalence of nuclear abnormalities in freshwater fish Cyprinus carpio using Comet and MN assay in blood erythrocytes and gill cells as percentile tail DNA damage as comets and micronuclei and other nuclear anomalies were scored in erythrocytes of Cyprinus carpio it was found that significant effect was observed for both concentrations and periods of exposure in the treated fishes. The highest DNA damage and increased micronuclei frequency and inhibitory AchE activity in Cyprinus carpio were found to be high on the highest sublethal 1/5th of LC50 followed by Sublethal 1/10th of LC50 concentration. Overall findings imply that AchE and genotoxic biomarkers may be useful diagnostic tools for chlorpyrifos poisoning in biomonitoring programs.
... On the other hand, lower pesticide concentrations with longer persistence time may trigger the bioaccumulation in aquatic organisms, thereby posing hazardous effects on the consumers including humans (Abedi et al. 2013). Acute toxicity of pesticide has been detected in fishes and can be analyzed by the in situ bioassays (Akcha et al. 2003;Buschini et al. 2004;Cavas and Ergene-Gozukara 2005;Pandey et al. 2011;Kushwaha et al. 2012). For such bioassays, the contamination sites are kept undisturbed and fishes are allowed to get exposure (if the water column is so deep or fast, in such cases water cages are used), followed by survival percentage calculation (Dey and Saha 2014;Ullah and Zorriehzahra 2015). ...
Continuous rise in application of pesticides in the agro-ecosystems in order to ensure food supply to the ever-growing population is of greater concern to the human health and the environment. Once entered into the agro-ecosystem, the fate and transport of pesticides is determined largely by the nature of pesticides and the soil attributes, in addition to the soil-inhabiting microbes, fauna, and flora. Changes in the soil microbiological actions, soil properties, and enzymatic activities resulting from pesticide applications are the important factors substantially affecting the soil productivity. Disturbances in the microbial community composition may lead to the considerable perturbations in cycling of major nutrients, metals, and subsequent uptake by plants. Indiscriminate applications are linked with the accumulation of pesticides in plant-based foods, feeds, and animal products. Furthermore, rapid increase in the application of pesticides having long half-life has also been reported to contaminate the nearby aquatic environments and accumulation in the plants, animals, and microbes surviving there. To circumvent the negative consequences of pesticide application, multitude of techniques falling in physical, chemical, and biological categories are presented by different investigators. In the present study, important findings pertaining to the pesticide contamination in cultivated agricultural soils; toxicity on soil microbes, plants, invertebrates, and vertebrates; effects on soil characteristics; and alleviation of toxicity by bio-based management approaches have been thoroughly reviewed. With the help of bibliometric analysis, thematic evolution and research trends on the bioremediation of pesticides in the agro-ecosystems have also been highlighted.
Graphical abstract
... The comet assay has been successfully applied to cells of many different species (including fish) to evaluate the genotoxic potential of many pollutants including CPF (Ali et al. 2009;Yin et al. 2009;Patetsini et al. 2013;Vera-Candioti et al. 2013;Ismail et al. 2014;Ambreen and Javed 2018). Moreover, numerous studies have confirmed the possibility of combining these three approaches for in vivo laboratory investigations for screening the genotoxic potential of different pollutants in fish (Buschini et al. 2004;Çavaş and Könen 2007;Çavaş 2011;Ruiz de Arcaute et al. 2016). ...
Abstract
The organophosphate pesticide chlorpyrifos (CPF) is defined as a priority pollutant in surface freshwaters according to Directive
2013/39/EU of the European Parliament. The focus of this study was to assess the potential cytotoxic and genotoxic effects of
permissible CPF levels on juvenile forms of the common carp. We found that low-level CPF exposure did not induce elevated
levels of micronuclei, but significantly increased the frequency of total nuclear abnormalities (NAs) proportional to dose and
time; notched, blebbed, lobed and eight-shaped nuclei, nuclear buds, nuclear bridges and binucleated cells were all detected.
Decreased frequencies of polychromatic erythrocytes (PCEs) and DNA damage detected by comet assay were also observed,
confirming the cytotoxic and genotoxic effects of CPF. Altogether, these data (1) demonstrate that CPF is toxic even at permissible
levels, possessing considerable genotoxic and cytotoxic potential in peripheral erythrocytes of exposed fish and (2) validate
the assessment of NAs, PCEs and comet assay performance as sensitive biomarkers for the early detection of CPF pollution.
These findings can be applied to guide environmental risk assessment and biomonitoring programs.
Keywords Chlorpyrifos . Common carp . Erythrocytes . Micronuclei . Nuclear abnormalities . Comet assay
... Indeed, untreated industrial effluent has an effect on the aquatic and terrestrial environments [4]. Industrial wastewater can raise the pollution of surface water and results in great problematic issues to living things' health [5,6]. Similar to other developing countries, Ethiopia has a rapid expansion of industries in the urban area. ...
Tannery effluent plays a significant role in increasing pollution in the environment; in particular, it contains toxic heavy metals which cause toxic effects on plant genetic materials. Among tannery effluent chemicals, chromium and lead have cytotoxicity and genotoxicity potentials on Allium cepa. is investigation was undertaken to assess the physicochemical properties of tannery wastewater and their effect on the genetic materials of A. cepa. Effluent's physicochemical characteristics were investigated using digital instruments for direct measurement and standard methods of atomic absorption spectrophotometer colorimetric analysis. A series of six onion bulbs were grown in 0%, 20%, 40%, 60%, 80%, and 100% concentrations of wastewater (v/v) ratio, and root tips from each onion bulb were cut and processed for analysis by aceto-orcein squash technique. Most of effluent components were above the discharge limit standards set by Federal Environmental Protection Agency and Ethiopian Environmental Protection Authority. e cytotoxicity effect on the root growth showed a significant reduction at high concentration. Simple regression analysis showed that the results of mitotic index were statistically significant (P < 0.05) in different concentrations. A decrease in mitotic index with increasing concentration of the effluent was observed. e effluent had induced chromosomal abnormalities such as laggard, fragmentation, stickiness, bridge, micronucleus, and binucleated and morphologically changed nuclei in A. cepa root cells among others. e results showed that cytotoxicity, genotoxicity, and chromosomal aberrations were induced by the tannery effluent. Industries shall think of biological waste treatment methods.
... This indicated that the water samples were significantly genotoxic to the dividing cells of A. cepa albeit the cytotoxicity is not statistically significant. A study by Buschini and the team [31] on lake water purified with sodium hypochlorite and chloride dioxide as disinfectants, demonstrated the generation of DNA [32]. Hence, there might be substances within the Shah Alam Lake water that have caused alterations within dividing cells, such as chlorine used in disinfectants and chemicals present in pesticides. ...
Allium cepa test is widely used to evaluate the effects of water pollution based on dividing cells since it is a very sensitive tool for prediction and recognition of environmental stresses. This study aimed to evaluate the potential use of A. cepa as a molecular biological indicator to detect the presence of water pollution. A. cepa roots were exposed to water samples at 24 and 48 hours with tap water and hydrogen peroxide solution as controls. The effects of water sample exposure on A. cepa were analysed based on the plant cytotoxicity, genotoxicity, and expression of stress gene between test samples and control sets. The findings showed no significant changes observed in mitotic index of A. cepa exposed to water samples compared to negative control. There is also no expression of alliinase gene was detected. However, there is chromosomal abnormalities observed in A. cepa exposed at 24 and 48 hours. The chromosomal abnormalities detected include lagging chromosome, c-mitosis, disrupted anaphase, disrupted metaphase, spindle disturbance, and stickiness. Our study shows that molecular biological method could be a potential method to serve as an effective, sensitive, and useful marker for water pollution determination.
... The absorption spectrum of Würster dyes is bimodal with peaks at 512 and 553 nm, which can be detected at both wavelengths (Buschini et al., 2004;Antonelli et al., 2006). At higher oxidant levels, an unstable colorless imine is primarily formed and causes the solution to "fade" significantly. ...
Peroxyacetic acid has been widely used in food, medical, and synthetic chemical fields for the past several decades. Recently, peroxyacetic acid has gradually become an effective alternative disinfectant in wastewater disinfection and has strong redox capacity for removing micro-pollutants from drinking water. However, commercial peroxyacetic acid solutions are primarily multi-component mixtures of peroxyacetic acid, acetic acid, hydrogen peroxide, and water. During the process of water treatment, peroxyacetic acid and hydrogen peroxide (H2O2) often coexist, which limits further investigation on the properties of peroxyacetic acid. Therefore, analytical methods need to achieve a certain level of selectivity, particularly when peroxyacetic acid and hydrogen peroxide coexist. This review summarizes the measurement and detection methods of peroxyacetic acid, comparing the principle, adaptability, and relative merits of these methods.
... One of characteristic of this species is registration of amplified effects of pollutants, which are present at the lower niche in the food chain (Boettcher 2012). There are many studies dealing with the effects of pollution on the common carp (Al-Sabti and Metcalfe 1995;Nepomuceno et al. 1997;Buschini et al. 2004;Klobučar et al. 2010). ...
In this study, few different evaluation concepts were used for the assessment of genotoxic potential at the stretch of the Danube River identified as a significant hotspot of pollution originated through the untreated wastewaters. Three sites were chosen: one site upstream of the wastewater outlet in Novi Sad (Serbia), one at the outlet of wastewaters, and one site few kilometer downstream. Ex situ approach comprised prokaryotic SOS/umuC test on Salmonella typhimurium TA1535/pSK1005 and comet assay on human hepatoma cell line (HepG2). In situ approach was based on the active monitoring (cage approach) using freshwater mussels Sinanodonta woodiana and fish Cyprinus carpio. The comet and micronucleus assays were selected for evaluation of DNA damage in mussel haemocytes and fish blood cells. Within the ex situ part of the study, our results indicated that the eukaryotic model system is more sensitive compared to the prokaryotic one. In situ bioassays are recommended for obtaining a better insight into ecosystem status and in the case of our study the complete insight of genotoxic pressure. However, the choice of animals as bioindicators also has a significant impact on the quality of the obtained information. Differential response between fish and mussels was observed at the highly polluted site suggesting possible involvement of additional protective mechanism such as valve closure in mussels.
... The comet assay has been successfully applied to cells of many different species (including fish) to evaluate the genotoxic potential of many pollutants including CPF (Ali et al. 2009;Yin et al. 2009;Patetsini et al. 2013;Vera-Candioti et al. 2013;Ismail et al. 2014;Ambreen and Javed 2018). Moreover, numerous studies have confirmed the possibility of combining these three approaches for in vivo laboratory investigations for screening the genotoxic potential of different pollutants in fish (Buschini et al. 2004;Çavaş and Könen 2007;Çavaş 2011;Ruiz de Arcaute et al. 2016). ...
The organophosphate pesticide chlorpyrifos (CPF) is defined as a priority pollutant in surface freshwaters according to Directive 2013/39/EU of the European Parliament. The focus of this study was to assess the potential cytotoxic and genotoxic effects of permissible CPF levels on juvenile forms of the common carp. We found that low-level CPF exposure did not induce elevated levels of micronuclei, but significantly increased the frequency of total nuclear abnormalities (NAs) proportional to dose and time; notched, blebbed, lobed and eight-shaped nuclei, nuclear buds, nuclear bridges and binucleated cells were all detected. Decreased frequencies of polychromatic erythrocytes (PCEs) and DNA damage detected by comet assay were also observed, confirming the cytotoxic and genotoxic effects of CPF. Altogether, these data (1) demonstrate that CPF is toxic even at permissible levels, possessing considerable genotoxic and cytotoxic potential in peripheral erythrocytes of exposed fish and (2) validate the assessment of NAs, PCEs and comet assay performance as sensitive biomarkers for the early detection of CPF pollution. These findings can be applied to guide environmental risk assessment and biomonitoring programs.
... Several tests using living organisms, especially aquatic species, has been used for the analysis of the potential toxicological effect of various chemicals on the genomes. Various species of fish are used in genotoxic and mutagenic assays as they are considered good indicators for the detection of aquatic pollutants and toxic substances (Buschini et al. 2004). According to Sponchiado et al. (2016), in order to extrapolate genotoxic effects from animal models to humans, vertebrates are the recommended animals as they are both phylogenetically and physiologically closer to humans. ...
The plants Brosimum gaudichaudii (inharé) and Caesalpinia ferrea (jucá) are widely distributed throughout Brazil and are considered medicinal. Inharé has been popularly used as a blood purifier, for the treatment of skin and vitiligo, while jucáis considered analgesic, anti-inflammatory and anticancer. These therapeutic properties have been attributed to phytochemicals such as coumarins, flavonoids and tannins. However, the mechanisms of action of most of these phytochemicals have not yet been fully elucidated and they may compromise human health. Consequently, the evaluation of the genotoxic effect of these plant extracts is fundamental for the determination of safe doses for human consumption. We evaluated the genotoxic effect of various concentrations of extracts of B. gaudichaudii and C. ferrea using comet assay of erythrocytes from Astyanax sp. exposed in vivo. In the comet assay indicated the tail migration of DNA increased significantly in the group of cells exposed to C. ferrea for various treatments and the olive tail movement exhibited a significantly higher extent of DNA damage, indicating the potential genotoxicity of the extract. On the other hand, it is premature to claima lack of genotoxic effect for B. gaudichauddi extracts since our experimental design was not able to rule out apotential effect of DNA damage as the concentration of 20mg/L seemed to increase the likelihood of genotoxicity. Thus, larger doses of B. gaudichauddi extracts should be tested in future studies of the kind. Our investigation provided valuable data for two species of plants widely used in folk medicine in different regions of Brazil. We recommend caution when using thses species for their medicinal properties.
... Furthermore, standard laboratory conditions are always slightly having variations when compared to the natural environmental conditions [12]. Much research has been done on the formation of micronuclei in the erythrocytes of fishes collected from polluted water bodies, used as an important marker for genotoxicity studies [5,13] and to assess the genotoxicity of various components [4,7,14,15]. Some authors have worked on different fish species to evaluate the genotoxicity by using micronucleus test [13]. ...
Background: We have come across the plant secondary metabolites having pesticidal properties being used in the form of pesticides. In this study, we used one of newly available and plant metabolite used as pesticide in wide agricultural fields – Karanjin, obtained from seeds of plant Pongamia pinnata.
Methods: The study was conducted during the month of March 2016 at the Department of Zoology, Osmania University. The fish common carp- Cyprinus carpio was exposed to sub-lethal concentration of karanjin i.e., 1/10th of 96 h LC50 value (0.28 ppm) for a period of 21 d. Moreover, at 24 h, 7 d, 14 d and 21 d, the peripheral blood of both control and exposed group fishes were studied for the presences of micronuclei and other nuclear abnormalities.
Results: The micronuclei were completely absent, i.e., were not seen during the sub-lethal exposure period. There were seen nuclear abnormalities such as blebbed nuclei (BN), notched nuclei (NN), differently shaped nuclei (DSN), pear-shaped nuclei (PSN), circular nuclei (CN), lobed nuclei (LN) and Karyolysed nuclei (KN). The control group showed few nuclear abnormalities.
Conclusion: During the sub-lethal exposure, as the days of exposure increased, the types of aberrations and their number also increased.
... In environmental mutagenesis, micronucleus tests give very practical results in monitoring the clastogenic and genotoxic effects of pollutants. These results are mostly obtained from aquatic organisms such as bivalve Mytilus galloprovincialis, Crassotrea gigas and Chamelea galina, fish rainbow trout Oncorhynchus mykiss, Oreochromis niloticus, sea urchin, Paracentrotus lividus [1][2][3][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47]. Boveri, reported that the relation between chromosomal changes and the origin of tumors using developing echinoderm embryos as a model organism [48]. ...
... The ENAs and comet assay proved to be sensitive tools for detection of GEM genotoxicity, supporting their use as biomarkers in water quality monitoring and risk assessment [72]. As with the present study, several studies already used these assays simultaneously to understand the genotoxic effects of contaminants in fish species [73,74,27,[75][76][77]72]. 1. DNA damage index of peripheral blood cells from Sparus aurata exposed for 96-h to gemfibrozil. ...
A widespread use of pharmaceuticals combined with suboptimal wastewater treatment processes has resulted in increased levels of these substances in aquatic ecosystems. Lipid regulators such as gemfibrozil are among the most abundant classes of human pharmaceuticals in the environment, but they have not been exhaustively studied in terms of potential effects to aquatic organisms. This study aimed to clarify the genotoxic effects of gemfibrozil (1.5, 15, 150, 1500 and 15000 μg L⁻¹) to one of the most commercially important fish species in southern Europe, gilthead seabream (Sparus aurata), following 96 h waterborne exposure, using both a molecular approach (quantifying erythrocyte DNA strand breaks) and a cytogenetic approach (quantifying the number of micronuclei and other nuclear abnormalities in cells). Gemfibrozil induced significant genotoxic effects at environmentally relevant concentrations (1.5 μg L⁻¹), both detected as increased level of strand breaks and as increased frequency of nuclear abnormalities (segmented and vacuolated nuclei). The number of micronuclei only increased significantly after exposure to the highest concentrations, i.e., 1500 and 15000 μg L⁻¹. In conclusion, the results raise concern about the potential genotoxic effects of the concentrations of gemfibrozil present in recipient waters.
... Significantly higher comet frequencies (27.34 ± 5.68%) were seen at 55 mg L -1 concentrations The other abnormalities are the nuclear abnormalities that are not fitted into the mentioned nuclear abnormalities Values in the same column not sharing the same letter are significantly different 5% level The other alterations are the nuclear abnormalities that are not fitted into the mentioned nuclear abnormalities Values in the same column not sharing the same letter are significantly different 5% level Appl Nanosci after 14 days of treatment then gradual decrease in the frequency (Table 6). Buschini et al. (2004) performed the comet assay on the erythrocytes of Cyprinus carpio to detect the possible genotoxic effects of disinfectants treated surface water. They found that comet and frequency of micronuclei was at the highest when erythrocytes were directly exposed to disinfected sodium hypochlorite and chloride. ...
Silver nanoparticles (17.78 ± 12.12 nm) were synthesized by the reduction of 0.5 M silver nitrate using formaldehyde as reducing and triethylamine as promoting and stabilizing agent. The particles were grain like
agglomerates with spherical, centered-face cubic and crystalline in nature. The sample was highly pure with amine (NH) as associated and capping molecules. Further, the genotoxicity and oxidative stress of these
articles were evaluated using Labeo rohita (L. rohita) as genetic model exposed (10–55 mg L-1
dose) through aquatic medium for 28 days. The cells were produced with micronuclei, fragmented, lobed and buds nuclei in dose dependent manner. The highest incidence of comet was recoded (27.34 ± 5.68) at 55 mg L-1 Ag-NPs and 14 days treatment. Then frequency was decreased to 22.65 ± 6.66% after 28 days due to complex repair mechanism. Moreover, the treatment also produces the oxidative stress and disturbs the level of GST in
gill and liver tissue. There was a sharp decline in the activities of GST and this decrease of activity increase the MDA content. Further, the elevated level of GSH represents that the liver has started defensive mechanism
gainst oxyraidcals. This study concluded, Ag-NPs are genotoxic in nature and produce micronuclei, comet cells and also induces oxidative stress in aquatic organisms.
The number of pollutants released into freshwater and marine environments has increased due to the widespread use of nanoparticles. Nickel oxide nanoparticles (NiO-NPs) were tested for genotoxicity in fish fingerlings of the species Ctenopharyngodon idella. For 7, 14, and 21 days, fingerlings were exposed to NiO-NPs with each increasing concentrations of 2.25 mg/L, 4.50 mg/L, and 6.75 mg/L, respectively. The micronuclei assay and comet assay were used to evaluate the DNA damage. The experiment revealed that with the increase in nanoparticle concentration and exposure duration, the level of DNA damage also increased. The experiment resulted to be time and dose dependent, and the damage was found as follows: 6.75 mg/L > 4.50 mg/L > 2.25 mg/L against each exposure period. In terms of comet assay, the results showed that after 7 days, the level of DNA damage in all the concentrations was highly significant (P < 0.001). Increased DNA damage was calculated at the higher administered dose of 6.75 mg/L for 21 days of exposition, followed by 14 and 7 days, respectively. The second high toxic effect was observed in the fish blood at the exposure concentration of 4.50 mg/L for 21 days, followed by 14 and 7 days, respectively. The micronuclei induction in the nanoparticle’s administered blood could be detected only for a 7-day exposition period. Whereas for the exposed duration of 14 and 21 days, the entire red blood cells of the grass carp were completely destroyed demonstrating the ability of the nanoparticles to cause anomalies in aquatic life.
Graphical Abstract
Peracetic acid (PAA) has a long history as an efficacious and eco‐friendly disinfectant. It was first synthesised in 1902, and since then a wide range of applications has been developed in various industries. Aquaculture is a more recent industry wherein the potential of PAA is significant. As the global demand for sustainable development increases, there has likewise been growing interest in using PAA in aquaculture as an alternative to less environmentally friendly practices. PAA has no carcinogenic risk to humans (unlike formalin), has negligible harmful by‐products (unlike chlorine‐based disinfectants) and with appropriate precautions, the risks of causing severe human health damage is easier to control than ozone. Fish show strong physiological recovery and adaptation to PAA, whereas susceptible life stages of pathogens are highly vulnerable, enabling a safe and efficacious disinfection of the entire culture water and not the flow‐restricted disinfection by such processes as ultraviolet radiation or ozone. The effective concentration of PAA against many fish pathogens is usually below 2 mg L ⁻¹ , which is tolerable for most fish, and it has very low environmental risk due to rapid degradation. However, such degradation and the hydrodynamics in production‐scale aquaculture systems complicate the practical use of PAA. In this review, we summarise key results of safe concentrations of PAA and its effectiveness specifically for fish farmers. We also outline major difficulties and possible solutions for practical uses of PAA. We intend to bring global attention to this compound and inspire future possibilities for its sustainable use as a water disinfectant in aquaculture.
Farklı hayvan dokularında biriken pek çok kirletici faktör canlıda genotoksik etki göstermektedir. Bu etkinin değerlendirilmesinde kullanılan genotoksisite testleri; kirlilik ajanlarının organizmada DNA hasarına neden olup olmadığının belirlenmesine olanak sağlar. Bu faktörlerin etkilerini belirleyebilmek için farklı hayvan dokularında pek çok test sistemi geliştirilmiş ve genotoksisite testleri büyük önem kazanmıştır. Bu çalışmada, hayvanlarda genotoksik hasarın belirlenmesinde çok yaygın olarak kullanılan iki test yöntemi olan MN ve Comet assay testleri karşılaştırılmış, avantaj ve dezavantajları belirlenerek farklı hayvan türlerinde yapılan çalışmaların değerlendirilmesi amaçlanmıştır.
This review discusses several research studies that employed comet assay to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies of aquatic animals. New chemicals are being added each year to the existing burden of toxic substances in the environment. Excessive agricultural and industrial activities adversely affect biodiversity , threatening the survival of species in a particular habitat, as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the senti-nel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Comet assay is a quick, sensitive, and low-cost technique for detecting DNA strand breakage. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. Comparing data from studies that employed different approaches, such as empirical scoring or comet tail lengths, comet assay is one of the challenging techniques to be utilized in environmental studies. The relative amount of DNA in the comet tail indicates DNA break intensity. The assay has been modified to detect various base alterations by including the digestion of nucleoids with a lesion-specific endonuclease. The determination of DNA damage in these indicator species using the comet test would thus offer information on the genotoxic potential of their habitat at an early stage. This would enable intervention techniques to prevent or mitigate adverse health impacts in sentinel animals and humans.
The gold nanoparticles generally find many applications in the drug delivery system, fluorescence biolabeling of tumor cells, bio detection of pathogens, probing of DNA structure and tissue engineering. Basically, the AuNPs are non- toxic in nature but it may induce DNA damage and increase the nuclear abnormalities levels. The AuNPs plays an important role in causing fish toxicity when the cells take up the particles and store them. The present study extensively investigated to analyse the in-vivo acute toxicity of gold nanoparticles in Catla cattle. The hematological studies and antioxidative responses were considered in the tissues of Catla cattle such as gills and liver. The results of this study exhibited the bioaccumulation of AuNPs in the tissue has caused due to the increasing doses of the nanoparticle. The hematological analysis showed considerable alterations in the gold nanoparticles treated fish. The impact of histological changes induced by gold nanoparticles were confirmed by the damages in the tissues, blood vessels, primary lamella and formation of vacuolation in gills and liver when compared with the control Catla cattle.
As principais fontes de contaminação do Rio Ijuí são o escoamento superficial das lavouras e o esgoto doméstico. O teste de micronúcleo (MN) em peixes é uma ferramenta útil na avaliação da qualidade da água, indicando a presença de substâncias tóxicas. Este estudo teve como objetivo avaliar a qualidade da água do Rio Ijuí utilizando o teste de MN em uma espécie de peixe nativa da família Characidae. Os indivíduos foram coletados no inverno (2012) e no verão (2013) em dois pontos no Rio Ijuí, distantes aproximadamente 170 Km, os quais apresentam a agricultura como principal atividade econômica, mas diferem em densidade demográfica. A frequência de MN não apresentou diferenças entre os locais. No entanto, em ambos os pontos, a frequência de MN no verão (2,15‰ e 2,13‰, nos pontos 1 e 2, respectivamente) foi significativamente maior do que no inverno (0,35‰ no ponto 1 e 0,45‰ no ponto 2). Não houve diferenças significativas para as outras anormalidades nucleares. Os resultados indicam a ocorrência de variação temporal na contaminação mutagênica da água do Rio Ijuí e que pode estar relacionada às atividades agrícolas, já que o verão é o período em que os agrotóxicos são utilizados com maior frequência na região.
Palavras-chave: Biomonitoramento; genotoxicidade; poluição aquática.
The present study was an attempt to explore the genotoxicity assessment in freshwater catfish, Sperata seenghala by micronucleus assay collected from Lower Lake and Shahpura Lake of Bhopal, India. The frequency of micronuclei was significantly higher in Shahpura Lake in comparison of Lower Lake. Total nuclei observed in Lower Lake were 44.00±0.811 and 56.00±0.944 in Shahpura Lake of which, the frequency of the micronucleus as 12.00±0.912 and nuclear abnormalities as 29.00±0.816 in Lower Lake, whereas, 16.00±1.414 and 30.75±0.577 were in Shahpura Lake. The results show that the assay can be employed for the evaluation and the assessment of water pollution and aquatic mutagens because Sperata seenghala fish is sensitive for bio-monitoring of genotoxicity as well as being an abundant species, easily may be kept with a wide distribution along the Central India.
Genotoxicity is considered as one of the endpoints in assessing toxic effect of pollution. In the present study genotoxic effects of pesticides in the Perna viridis have been evaluated by the induction of micronuclei (MN). The aim of the present study was to assess the MN frequency in the haemolymph of green mussel (Perna viridis) after exposure to different concentrations of organophosphate pesticides (chlorpyrifos, malathion), synthetic pyrethroid pesticide (cypermethrin, lambda-cyhalothrin) and herbicide (buctril). Haemocytes of bivalve play an important role in immune defense and detoxification of contaminants. The bivalves were exposed to different concentrations of test pesticides in a static system. The MN frequencies of all the pesticide treated mussel groups increased significantly (p< 0.05) until the end of the exposure period as compare to control. The highest MN frequencies were recorded after cypermethrin exposure on day 12 (7, 8.5 and 11‰ for 0.5 ppm, 1 ppm and 1.5 ppm concentrations respectively) in haemolymph. However the lowest MN frequencies were recorded after buctril exposure (3.5, 3.5 and 5‰ for 0.5 ppm, 1 ppm and 1.5 ppm concentrations, respectively) in haemolymph. The use of haemocytes of green mussel (Perna viridis) in MN assay proved to be a sensitive tool for the assessment of genetic damage.
Harmful cyanobacterial blooms are increasing and becoming a worldwide concern as many bloom-forming cyanobacterial species can produce toxic metabolites named cyanotoxins. These include microcystins, saxitoxins, anatoxins, nodularins, and cylindrospermopsins, which can adversely affect humans, animals, and the environment. Different methods to assess these classes of compounds in vitro and in vivo include biological, biochemical, molecular, and physicochemical techniques. Furthermore, toxic effects not attributable to known cyanotoxins can be observed when assessing bloom material. In order to determine exposures to cyanotoxins and to monitor compliance with drinking and bathing water guidelines, it is necessary to have reliable and effective methods for the analysis of these compounds. Many relatively simple low-cost methods can be employed to rapidly evaluate the potential hazard. The main objective of this mini-review is to describe the assessment of toxic cyanobacterial samples using in vitro and in vivo bioassays. Newly emerging cyanotoxins, the toxicity of analogs, or the interaction of cyanobacteria and cyanotoxins with other toxicants, among others, still requires bioassay assessment. This review focuses on some biological and biochemical assays (MTT assay, Immunohistochemistry, Micronucleus Assay, Artemia salina assay, Daphnia magna test, Radionuclide recovery, Neutral red cytotoxicity and Comet assay, Enzyme-Linked Immunosorbent Assay (ELISA), Annexin V-FITC assay and Protein Phosphatase Inhibition Assay (PPIA)) for the detection and measurement of cyanotoxins including microcystins, cylindrospermopsins, anatoxin-a, saxitoxins, and nodularins. Although most bioassay analyses often confirm the presence of cyanotoxins at low concentrations, such bioassays can be used to determine whether some strains or blooms of cyanobacteria may produce other, as yet unknown toxic metabolites. This review also aims to identify research needs and data gaps concerning the toxicity assessment of cyanobacteria.
Cadmium (Cd) has been widely studied as an environmental pollutant for many years. Numerous studies have reported that Cd exposure causes damage to the heart, liver, kidneys, and thyroid in vivo. The emerging evidence suggests that Cd exposure induces damage on male reproductive system, which is related to oxidative stress, inflammation, steroidogenesis disruption, and epigenetics. Current preclinical animal studies have confirmed a large number of proteins and intracellular signaling pathways involved in the pathological process of Cd-induced male reproductive damage and potential measures for prophylaxis and treatment, which primarily include antioxidants, anti-inflammatory agents, and essential ion supplement. However, explicit pathogenesis and effective treatments remain uncertain. This review collects data from the literatures, discusses the underlying mechanisms of Cd-induced toxicity on male reproductive function, and summarizes evidence that may provide guidance for the treatment and prevention of Cd-induced male reproductive toxicity.
Freshwater and marine water bodies receive chemical contaminants from industrial, agricultural, urban, and domestic wastes. Eco-genotoxicity assays are useful tools to assess the cumulative genotoxicity of these pollutants. Fish are suitable indicators for biomonitoring of mutagenic and carcinogenic pollution.
In this review, we present a complete overview of the studies performed so far using the micronucleus test in peripheral erythrocytes of fish exposed to polluted water. We have listed all the species of fish used and the geographical distribution of the investigations. We have analyzed and discussed all technical aspects of using this test in fish, as well as the advantages and disadvantages of the different experimental protocols. We have reported the results of all studies. This assay has become, for years, one of the simplest, fastest, and most cost-effective for assessing genotoxic risk in aquatic environments. However, there are still several factors influencing the variability of the results. Therefore, we have given indications and suggestions to achieve a standardization of experimental procedures and ensure uniformity of future investigations.
Safeners are a group of chemicals applied with herbicides to protect crop plants from potential adverse effects of agricultural products used to kill weeds in monocotyledonous crops. Various routes of dissipation of safeners from their point of applications were evaluated. Despite the large numbers of safeners (over 18) commercially available and the relatively large quantities (~2 × 106 kg/year) used, there is little information on their mobility and fate in the environment and occurrence in various environmental matrices. The only class of safeners for which a significant amount of information is available is dichloroacetamide safeners, which have been observed in some rivers in the USA at concentrations ranging from 42 to 190 ng/L. Given this gap in the literature, there is a clear need to determine the occurrence, fate, and bioavailability of other classes of safeners. Furthermore, since safeners are typically used in commercial formulations, it is useful to study them in relation to their corresponding herbicides. Common routes of dissipation for herbicides and applied safeners are surface run off (erosion), hydrolysis, photolysis, sorption, leaching, volatilization, and microbial degradation. Toxic potencies of safeners vary among organisms and safener compounds, ranging from as low as the LC50 for fish (Oncorhynchus mykiss) for isoxadifen-ethyl, which was 0.34 mg/L, to as high as the LC50 for Daphnia magna from dichlormid, which was 161 mg/L. Solubilities and octanol-water partition coefficients seem to be the principal driving force in understanding safener mobilities. This paper provides an up-to-date literature review regarding the occurrence, behaviour, and toxic potency of herbicide safeners and identifies important knowledge gaps in our understanding of these compounds and the potential risks posed to potentially impacted ecosystems.
Genomic instability is a condition that may be associated with carcinogenesis and/or physiological disorders when genetic lesions are not repaired. Besides, wild, captive, and domesticated vertebrates are exposed to xenobiotics, leading to health disorders due to cytogenotoxicity. This chapter provides an overview of tests to assess cytogenotoxicity based on micronuclei (MNi) formation. Bone marrow micronuclei test (BmMNt), peripheral blood erythrocyte micronuclei test (PBMNt), and lymphocyte cytokinesis blocking micronuclei assay (CBMN) are discussed. The most illustrative studies of these techniques applied in different vertebrates of veterinary interest are described. The values of spontaneous basal micronuclei in captive, experimental, and farm animals (rodents, hamsters, pigs, goats, cattle, horses, fish) are summarized. In addition, a flow cytometry technique is presented to reduce the time taken to record MNi and other cellular abnormalities. Flow cytometry is helpful to analyze some indicators of genomic instability, such as cell death processes and stages (necrosis, apoptosis) and to efficiently evaluate some biomarkers of genotoxicity like MNi in BmMNt, PBMNt, and CBMN. The intention is to provide veterinary professionals with techniques to assess and interpret cytogenotoxicity biomarkers to anticipate therapeutic management in animals at risk of carcinogenesis or other degenerative diseases.
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.
The lower Melet River is a drinking water source that is surrounded by hazelnut grove, agricultural lands, resulting in the accumulation of genotoxic agents such as mining activities, various domestic and agricultural wastes. Therefore, it receives many domestic and agricultural wastes that contain the genotoxic agent. This study was aimed to assess the heavy metal concentrations in water, sediment, and bioaccumulation in the tissues of Alburnus chalcoides. Comet assay and micronucleus test were used to evaluate the genotoxic effects on the blood cells of A. chalcoides. The concentrations of heavy metals and metalloid in the water, in the sediments and in the muscle of fish were in the order of Fe > Al > Mn > As > Zn > Cu > Ni > Cr > Cd = Pb = Co, Fe > Al > Mn > Zn > Cu > Pb > Cr > As > Co > Ni > Cd and Fe > Zn > Al > Mn > Cu > Pb > As > Cr > Ni > Co > Cd, respectively. The blood cells of fish collected from the polluted location showed significantly higher DNA damage and micronucleus frequency compared to the reference location (p < 0.05). The study indicated that the DNA integrity of A. chalcoides was affected by heavy metals which originated from many anthropogenic sources.
Hospitals consume a large amount of water, so they also generate large amounts of wastewater, which contain a wide variety of contaminants. It is important to consider that hospital effluents are a mixture of pollutants that can interact with each other and have a negative impact on aquatic species of water bodies. The aim of this study was to evaluate the effects induced by a hospital effluent using Danio rerio embryos. In this study, Danio rerio embryos were exposed to different concentrations of the hospital effluent and a lethality test was evaluated and the malformations present in zebrafish embryos were evaluated. The lethal concentration of effluent 50% was 6.1% and the effective malformation concentration was of 2.5%. The teratogenic index was 2.45%. The main malformations identified were yolc sac malformation, pericardial edema, hatching abnormalities, hypopigmentation, tail deformation, chorda malformation, without fin, chorion deformation and craniofacial malformation. The risks that this type of water represents for the survival of living organisms, as well as the presence of malformations in them, are reference indicators for a future regulation focused on the adequate treatment of hospital effluents.
The genotoxic effects of quinalphos (QP) were evaluated in the erythrocytes of Cyprinus carpio using the comet, micronucleus, and nuclear abnormality assays. The 96 h LC50 value of quinalphos, estimated by static renewal system, was 10.9 µL/L. On the basis of the LC50 value, two sublethal concentrations (1.09 and 2.18 µL/L) of the compound were used together with cyclophosphamide (5 mg/L) as the positive control. The fish were exposed for 35 d, and sampling was done at the end of 7, 14, 21, 28, and 35 d. Our results reveal a significant dose-dependent increase in the frequencies of DNA strand breaks, micronucleus, and nuclear abnormalities. Similar responses were also observed in fish exposed to the positive genotoxin cyclophosphamide. The collective findings demonstrated that quinalphos exerts a genotoxic effect on C. carpio. The results further confirmed that the comet, micronucleus, and nuclear abnormality assays are useful tools in determining the potential genotoxicity of pesticides towards aquatic organisms.
The wide range of activities performed in hospitals (care, diagnosis, hygiene, maintenance, research) require the use of a large variety of potentially ecotoxic substances such as surfactants, disinfectants, and pharmaceuticals. Once used or excreted by a patient, these compounds combine with hospital wastewater (HWW) in stable or unstable, metabolized or non-metabolized forms and then flow directly into the municipal wastewater network, generally without any prior treatment. Also, wastewater treatment plants (WWTPs) are usually not equipped to eliminate the pollutants present in these effluents.
Hospital functioning generates a great quantity of contaminants, among which organic materials, heavy metals, and diverse pharmaceuticals are noteworthy that can affect organisms if they are not properly removed from the effluents. The hospital effluent evaluated in the present study came from IMSS (Instituto Mexicano del Seguro Social) Clinic 221 in downtown Toluca, State of Mexico, a secondary care facility. The contaminants identified in hospitals have been associated with deleterious effects on aquatic organisms; however, it is necessary to continue with more studies in order to be able to regulate the production of said contaminants which are generally dumped into the city sewage system. The present study had the purpose of evaluating the alterations to embryonic development and teratogenic effects on oocytes Cyprinus carpio after exposure to different proportions of hospital effluent. For said purpose, the physicochemical properties of the effluent were determined. Concentrations of the main microcontaminants were also determined. An embryolethality study out and the determination of the main alterations to embryonic development and teratogenic effects produced, due to exposure of C. carpio at different proportions of the effluent, were carried out. The results showed that the physicochemical properties were within the values permitted by Mexican regulation; however, the presence of contaminants such as NaClO, metals, anti-biotics, anti-diabetics, non-steroidal anti-inflammatory drugs, hormones and beta-blockers, was detected. Lethal concentration 50 was 5.65% and the effective concentration for malformations was 3.85%, with a teratogenic index of 1.46. The main teratogenic alterations were yolk deformation, scoliosis, modified chorda structure, tail malformation, fin deformity and mouth hyperplasia. A high rate of hatching delay was observed. The results suggest that the hospital effluent under study is capable of inducing embryotoxicity and teratogenicity in oocytes of C. carpio.
Abstract In recent years production of chemicals increased steadly. As a result of the production and use of chemicals that make inevitable chemicals exposure. Humans are exposed throughout their lifetime to several xenobiotics present in both the water and aquatic food. More importantly, fish species are at the top position in the aquatic food chain and may directly affect the health of humans, which makes it much of significance for the biomonitoring using fish. In this study are given information about of Fish Genotoxicity Tests (Comet Assay, Micronucleus test, FISH-MN, Chromosomal Aberration, Sister Chromatid Exchange) for the biomonitoring of environmental pollution.
The present study aimed to estimate the chemical components and cytotoxic effects of four types of wastewater such as industrial wastewater (IWW), agriculture wastewater (AWW), sewage wastewater (SWW) and mixed wastewater (MWW) on meristematic tissues and cells of faba bean, (Vicia faba) root tips. Some physio-chemical characteristics
as well as heavy metals contents were measured in these four wastewater samples compared with that of tap water using values of World Health Organization (WHO) as standard parameters.Mitotic index (MI), phase index and chromosomal abnormalities
were calculated as toxicity indicators. The results revealed that MI values of almost all seeds treated with the different concentrations of various types of wastewater were significantly lower than those of control treatment. Different types of mitotic aberration were observed such as chromosome bridges, lagging chromatid and chromosomes, chromatin fragments, micronuclei and tripolar cells. It was clearly observed that waste water increased chromosomal aberrations and induced disturbances in mitotic phases. Therefore, it could be recommended that all wastewater should be treated according to WHO standards to avoid and minimize the pollutant effects before using for irrigation purpose, so that safety of humans would be achieved.
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.
Biological effect directed in vivo tests with model organisms are useful in assessing potential health risks associated with chemical contaminations in surface waters. This study examined the applicability of two in vivo test systems viz. plant, Allium cepa root based tests and fish, Oreochromis niloticus erythrocyte based tests for screening cytogenotoxic potential of raw source water, water treatment waste (effluents) and treated water of drinking water treatment plants (DWTPs) using two DWTPs associated with a major river in Sri Lanka. Measured physico-chemical parameters of the raw water, effluents and treated water samples complied with the respective Sri Lankan standards. In the in vivo tests, raw water induced statistically significant root growth retardation, mitodepression and chromosomal abnormalities in the root meristem of the plant and micronuclei/nuclear buds evolution and genetic damage (as reflected by comet scores) in the erythrocytes of the fish compared to the aged tap water controls signifying greater genotoxicity of the source water especially in the dry period. The effluents provoked relatively high cytogenotoxic effects on both test systems but the toxicity in most cases was considerably reduced to the raw water level with the effluent dilution (1:8). In vivo tests indicated reduction of cytogenotoxic potential in the tested drinking water samples. The results support the potential applications of practically feasible in vivo biological test systems such as A. cepa root based tests and the fish erythrocyte based tests as complementary tools for screening cytogenotoxicity potential of the source water and water treatment waste reaching downstream of aquatic ecosystems and for evaluating cytogenotoxicity eliminating efficacy of the DWTPs in different seasons in view of human and ecological safety.
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 effect of protein quality and of supplementation of corn-glutenprotein with lysine on the growth, feed conversion and protein turnoverrates in white muscle was investigated in carp (Cyprinus carpio) acclimatedto either 18 or 25 °C. Fish fed the lysine-deficient diet showed asignificantly lower food intake, weight gain and feed-conversion efficiencythan animals fed the lysine-sufficient diets, regardless of environmentaltemperature. Coated lysine, compared with free lysine, proved to be asignificantly better way of supplementing dietary protein, as shown byfeed-conversion indices at 18 and 25 °C. White-muscle protein, RNA andDNA contents were not altered by dietary treatment or water temperature.Supplementation with coated lysine, but not with free lysine, significantlyincreased the protein-synthesis rate (KS) at 18 or 25 °Cin comparison to lysine deficiency, although not to control values. Theeffiiency of protein synthesis (KRNA) and retention (PRE)obtained for fish fed the coated-lysine diet, at 18 °C, reached controlvalues. At 25 °C PRE and protein accumulation rate (KG)showed the following significant differences: lysine-deficient diet <free-lysine supplemented diet < coated-lysine supplemented diet <control diet. Significant correlations were observed for Ks in relation withKD, KRNA or protein-related growth, at bothtemperatures
We determined full-length cDNA of carp warm-temperature-acclimation-associated 65-kDa protein (Wap65). It encoded 439 amino acid residues with a signal peptide of 22 residues and showed an amino acid sequence identity of 88% to that of goldfish reported before (J. Biol. Chem. 1995. 270: 17087–17092). The number of potential N-linked glycosylation sites of carp Wap65 was two in contrast to three for goldfish. In addition, molecular mass determined by SDS-PAGE was apparently different from that of goldfish. These results suggest that the amount of oligosaccharide is different between the carp and goldfish protein. As in goldfish, carp Wap65 mRNA showed marked accumulation in hepatopancreas of the 30C- acclimated fish, which was 8-fold higher than that of the 10C-acclimated fish. Carp Wap65 showed 30% amino acid identity to mammalian hemopexins, which appeared to be considerably low in comparison with those among mammalian hemopexins (72 to 80%), or among carp Wap65 and rainbow trout hemopexin-like protein (70%). However, although mammalian hemopexins contain residues comprising the heme binding pocket, carp Wap65 lacked one of the two histidine residues to serve as heme axial ligands in hemopexins. Our data on carp protein substantiates the previous observation for goldfish and indicates that Wap65 might have some important functions in warm-temperature-acclimation of fish.
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.
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.
The micronucleus test using erythrocytes of Pleurodeles waltl larvae (Amphibian, Salamandridae) was used to detect the genotoxic activity of various substances. Most of them were known
(or suspected) to be mutagenic or carcinogenic in mammals. Larvae were reared in water containing either a test chemical (experimental
group) or in control water and the levels of micronucleated red blood cells (RBCs) compared between the two groups. Aroclor
1254, butylated hydroxyanisole, phenobarbital and 12-0-tetradecanoyl-phorbol-13-acetate produced negative results, while acridine orange, benzo[a]pyrene, ∈-caprolactam, cyclophosphamide, diethyl sulphate, epichlorhydrin, ethidium bromide, ethyl methane-sulphonate, ethylene
dibromide (dibromoethane), N-ethyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea, hexa-methylphosphoramide, 3-methyl cholanthrene, pyrene and o-toluidine gave positive responses. The results were compared with published data from other tests used to detect the clastogenic
or mutagenic properties of chemicals. We suggest that the newt micronucleus test could be used to monitor aquatic pollution,
and/or for the quality control of drinking water. Pleurodele larvae may also prove to be of value for the detection of carcinogenic/clastogenic
substances dissolved in water.
We have previously described a micronucleus test using erythrocytes from larvae of the urodele amphibians Pleurodeles waltl (pleurodele) and Ambystoma mexicanum (axolotl). The test is based on a comparison of the levels of micronucleated erythrocytes in blood smears from larvae reared
in water containing a clastogen, with the levels from larvae reared in purified water. Using larvae from the pleurodele, we
have employed this test to evaluate mutagenic activity in drinking water. Groups of larvae were reared in tap water, while
control groups were reared in tap water which had been filtered over sand and active carbon to remove micropollutants. Seven
separate tests carried out between October 1985 and May 1986 all gave positive results of varying degree depending on the
time of year. This test is therefore able to detect clastogens in normal drinking water. It could be used for quality control
of drinking water during the various stages in the treatment of raw water without any requirement for prior extraction or
concentration of micropollutants.
Chlorination of water rich in organic material is known to produce a complex mixture of organochlorine compounds, including mutagenic and carcinogenic substances. A historical cohort study of 621,431 persons living in 56 towns in Finland was conducted in order to assess the relation between historical exposure to drinking water mutagenicity and cancer. Exposure to quantity of mutagenicity was calculated on the basis of historical information of raw water quality and water treatment practices using an empirical equation relating mutagenicity and raw water pH, KMnO4 value and chlorine dose. Cancer cases were derived from the population-based Finnish Cancer Registry and follow-up time in the study started in 1970. Age, gender, time period, social class, and urban residence were taken into account in Poisson regression analysis of the observed numbers of cases using expected numbers of cases standardized for age and gender as a basis. Excess risks were calculated using a continuous variable for mutagenicity for 3,000 net rev/l exposure representing an average exposure in a town using chlorinated surface water. After adjustment for confounding, a statistically significant excess risk was observed for women in cancers of the bladder (relative risk [RR] = 1.48, 95 percent confidence interval [CI] = 1.01-2.18), rectum (RR = 1.38, CI = 1.03-1.85), esophagus (RR = 1.90, CI = 1.02-3.52), and breast (RR = 1.11, CI = 1.01-1.22). These results support the magnitude of excess risks for rectal and bladder cancers found in earlier epidemiologic studies on chlorination by-products and give additional information on exposure-response concerning the mutagenic compounds. Nevertheless, due to the public health importance of water chlorination, uncertainty related to the magnitude of observed risks, and the fact that excess risks were observed only for women, the results of the study should be interpreted with caution.
This study assessed the association of drinking water source and chlorination by-product exposure with cancer incidence.
A cohort of 28,237 Iowa women reported their drinking water source. Exposure to chlorination by-products was determined from statewide water quality data.
In comparison with women who used municipal ground-water sources, women with municipal surface water sources were at an increased risk of colon cancer and all cancers combined. A clear dose-response relation was observed between four categories of increasing chloroform levels in finished drinking water and the risk of colon cancer and all cancers combined. The relative risks were 1.00, 1.06, 1.39, and 1.68 for colon cancer and 1.00, 1.04, 1.24, and 1.25 for total cancers. No consistent association with either water source or chloroform concentration was observed for other cancer sites.
These results suggest that exposure to chlorination by-products in drinking water is associated with increased risk of colon cancer.
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 increasing use of products containing peracetic acid for the disinfection of sewage and effluents has produced a demand for information about the activity of PAA against relevant microorganisms. Studies have therefore taken place to establish the comparative effects of peracetic acid and chlorine on both bacteria and viruses. Peracetic acid has previously been shown to be active against faecal indicator bacteria. Recent laboratory studies have established that peracetic acid is also an effective agent against viruses typical of those found in sewage. This activity is maintained even in experimental systems containing high levels of organic matter such as yeast extract. In contrast, although sodium hypochlorite is an effective virucide in clean experimental systems, the presence of organic matter greatly increases the level of added available chlorine necessary to achieve a given level of viral inactivation.
The Tradescantia Micronuclei (Trad-MCN) test is a well-validated system for the in situ monitoring of gaseous mutagens or radiation and bioassay for toxicity or clastogenicity of liquid types of environmental pollutants, drugs, and food additives. For testing gaseous mutagens or radiation, excised Tradescantia in-florescences containing meiotic pollen mother cells may be exposed to the agent at the chosen pollution sites or in the treatment chambers. For testing liquid types, the cuttings are allowed to absorb the agent through the stem and transport the agent to the meiotic pollen mother cells. The chromosome fragments that are induced by the agent at the early meiotic stage become micronuclei at the tetrad stage. The acetocarmine squash method is utilized to prepare microslides, and frequencies of micronuclei among a large population of tetrads are the reliable indicators of the chromosome damage. This test reveals directly the magnitude of chromosome damage in the germ cells of an eukaryote. It is relatively simple, versatile, and efficient. Results of the test can be obtained and analyzed in 24–48 hr. It is, perhaps, the most inexpensive test known at the present time.
Peracetic acid (PAA), a well known powerful antimicrobial agent in hospitals and in agribusiness (Fraser, 1986), has recently been used to disinfect urban effluents. It appears to be highly competitive against chlorine (Audic, 1990; Baldry, French, Slater and Desprez, 1990; Giodani, Iacoponi, Polidori, 1989), the most widely used disinfectant for sewage disposal.
As PAA is a new biocide, not much quantitative data is available on its action against the faecal indicator bacteria and viruses.
An on-site experimental study investigated the disinfectant action of PAA against these indicator bacteria and viruses as well as against Salmonella and enterovirus. To complete this study we will test its action on suspended solids to find out whether there is regrowth of the microorganisms after treatment.
Multi-factor analysis in terms of criteria like inactivation efficiency, safety environmental impact, and cost will be used to compare PAA to chlorination and ozonation, the most commonly used techniques.
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.
Chlorination of both drinking waters and wastewaters leads to the generation of various chlorinated degradation products of natural fulvic acids. Fulvic acids constitute the bulk of the organic matter in colored surface or ground waters. Meta dihydroxybenzene structures are proposed as main reactive sites of the molecule. Experimental evidence is presented for possible reaction pathways leading to haloforms and other chlorinated by-products.
1. Rainbow trout (Oncorhynchus mykiss) were exposed to a single X-ray dose of 4Gy.2. The frequency of micronuclei in the peripheral erythrocytes was investigated at regular intervals up to 58 days after the exposure.3. A flow cytometric method and a semi-automatic image analysis method were used to estimate the micronuclei frequency.4. The results show that both methods can detect an increased frequency of micronuclei in peripheral erythrocytes from exposed fish.5. However, the semi-automatic image analysis method was the most stable and sensitive.
RNA concentration in carp (Cyprinus carpio) white muscle tissue increased with higher growth rate for all acclimation temperatures, while DNA, protein content, and tissue hydration were unaffected. For a given growth rate, RNA concentration per unit tissue weight and per unit DNA (RNA per cell) was higher in colder acclimated fish as a probable compensation for decreased RNA activity. Maximum growth resulted in an approximate doubling of the RNA/DNA ratio while, over the range of 12–30 °C, cold acclimation caused an increase of approximately 50%. Changes in DNA concentration and protein/DNA ratios at different acclimation temperatures indicated an increase in cell mass with warmer acclimation temperatures. Muscle RNA/DNA ratios and the rate of glycine uptake by scales were highly correlated. Regression equations describing this relationship for each acclimation temperature were not significantly different when scales were incubated at each fish's acclimation temperature, but were significantly different when glycine uptake was expressed for incubation at the temperature of maximum uptake. These results suggest a whole-body integration between thermoacclimatory changes in the RNA component of muscle protein synthesis and in the rate of amino acid transport by scales.
Hepatic monooxygenase enzyme activities and relative cytochrome P4501A protein content were measured to evaluate the time-course alterations in rainbow trout after change in living habitat. Fish were transferred from one fish farm to the tanks of another hatchery and/or into cages kept in a lake. In the new habitats, cytochrome P450-dependent enzyme activities in rainbow trout decreased, and were at their lowest levels after two or three weeks in the summer. Later the activities partly reversed. The immunodetection of cytochrome P4501A protein expressed a similar trend as for catalytic monooxygenase activities.
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.
As surface waters are widely used for the preparation of drinking water, appropriate test systems are required for the monitoring of possible genotoxic contaminations. In the course of a BMBF-funded collaborative project several methods (Ames test, umu test, alkaline elution, DNA unwinding assay, Comet assay and unscheduled DNA-synthesis test) have been examined for their ability to measure genotoxicity particularly in natural surface waters. The project was subdivided into two parts: In the first part appropriate test versions were developed (sensitized Ames test, luminometric umu test, alkaline elution using clams, Comet assay with fish cells or aquatic plants), adapted to the test subject and validated regarding their sensitivity towards standard genotoxins. All test results were statistically evaluated. In the course of the second part both natural and concentrated samples of the rivers Rhine, Elbe, Mulde, Wupper and one drinking water resource (Wahnbachbarrage) were tested. In parallel all samples were chemically analyzed. Among the unconcentrated samples several statistically positive test results were obtained both for the rivers Elbe and Rhine especially with the Ames test and the Comet assay. Only one river (Wupper) showed significant genotoxicity in the umu test. In this case chemical analysis revealed concentrations of about 41 and 47 g/l of fluoroquinolonic acid, a bacterial gyrase inhibitor which may be responsible for this effect. No genotoxicity could be found in the drinking water resource even after concentration. The water extracts clearly showed different background genotoxicity in the umu test corresponding to increased pollution along the Rhine (Karlsruhe < k="" ln="">< d="" sseldorf).="" the="" eucaryotic="" in="" vitro="" tests="" revealed="" comparable="" results="" with="" nonconcentrated="" water="" samples.="" as="" a="" conclusion="" of="" the="" study="" we="" propose="" a="" graduated="" testing="" battery="" consisting="" of="" a="" bacterial="">umu or Ames test) and an eucaryotic test like the Comet assay or the alkaline elution assay followed by an additional eucaryotic test (UDS test or micronucleus test) in a decisive function. There is a need for further evaluation by effect-orientated chemical analysis which should be done in the case of positive genotoxicity results in at least two tests or persistent positive results in only one.
Winter flounder (Pseudopleuronectes americanus) were sampled from three sites located near Norwalk, New Haven, and Niantic, Connecticut, in Long Island Sound during February
1987, to evaluate the degree of chemical contamination and to determine possible effects of contaminant exposure. At each
site, sediment and infaunal invertebrates were also collected and analyzed for trace metals and organic chemicals. Specimens
of liver and kidney from winter flounder were examined for histopathological conditions, including the presence of macrophage
aggregates in liver tissue. Liver samples were also analyzed for DNA damage (i.e., the formation of adducts between DNA and
chemical contaminants). Blood samples were collected and analyzed for erythrocyte micronuclei. The sampling site near New
Haven was determined to be the most affected site, from the standpoints of greater chemical contamination and possible effects
on winter flounder. Concentrations of aromatic hydrocarbons (AHs) and polychlorinated biphenyls (PCBs) were highest in sediment
from this site, and the highest prevalences of the histopathological changes and DNA alterations were also found in the livers
of winter flounder from this site. No differences in the concentrations of contaminants in fish or in frequencies of erythrocyte
micronuclei in fish blood were found between sites. None of the sites sampled had contaminant levels or prevalences of lesions
as high as previously found at other East Coast locations (e.g., Boston Harbor, Massachusetts, Raritan Bay, New York). Overall,
our results indicate moderate levels of pollution at two of the urban sites in Long Island Sound and provide a framework for
expanded studies to better define the extent and impact of chemical pollution in Long Island Sound.
The single cell gel electrophoresis or `comet' assay was employed to detect DNA strand breaks (SB) induced in isolated brown trout hepatocytes and blood cells incubated in vitro with various sub-cytotoxic concentrations of a range of genotoxic compounds and potential aquatic contaminants. Direct acting agents studied were hydrogen peroxide (H2O2; 0–200 μM), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG; 0–50 μM) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX; 0–200 μM). Benzo(a)pyrene (BaP; 0–200 μM), 1-nitropyrene (1-NP; 0–200 μM) and nitrofurantoin (NF; 0–500 μM) were also used to represent polycyclic and nitroaromatic contaminants requiring metabolic activation to exert their effects. The direct damaging agents produced a statistically significant (P at least <0.05) concentration-dependent increase in the percentage of DNA in the comet tail in both cell types, indicative of increased formation of SB. The percentage of DNA in the tail in both hepatocytes and blood cells respectively (mean values±S.E.M.) was 27.8±4.2 and 30.9±7.8 (200 μM H2O2); 60.2±5.6 and 61.3±5.9 (50 μM MNNG); 32.4±2.7 and 28.6±5.1 (200 μM MX). With BaP a statistically significant (P at least <0.05) concentration-dependent increase was seen at 50 μM and above for hepatocytes but not in blood cells (10.5±2.2 (100 μM BaP)), highlighting the requirement for metabolic activation of this compound. 1-NP and NF also produced concentration-dependent increases in SB in hepatocytes (17.1±4.4 (100 μM 1-NP) and 38.9±7.3 (500 μM NF) with no significant effect in blood cells. All values were compared to control hepatocyte and blood cells (overall mean values 3.6±0.1 and 2.4±0.1 respectively). This study has demonstrated the potential of the comet assay to detect DNA strand breakage in fish cells induced by a range of agents. The technique may provide a sensitive, non-specific end-point of genotoxicity as part of a biomonitoring regime.
The single cell gel (SCG) or comet assay is a simple method by which DNA damage is expressed as relative nuclear ‘tail’ length of gel-embedded cells following alkaline electrophoresis. While potentially applicable to any cell type, laboratory experiments were conducted to examine the utility of the SCG method for the detection of genotoxicity in cells of marine fish and invertebrates. Selected cells included those from flounder (Pleuronectes americanus) and oysters (Crassostrea virginica). In vitro exposures were used to optimize parameters and evaluate sensitivity, reproducibility and dose-responsiveness of the SCG method. In vivo exposures were used to examine the effects of factors such as intra-animal variability on low level DNA damage detection. In one experimental series, individually identified oysters were repeatedly sampled to monitor DNA damage and recovery following in vivo exposure to genotoxicant-spiked sediment. Preliminary results suggest that the SCG may be a useful tool to monitor for genotoxic environmental exposures and investigate pollution-mediated health effects.
Our aim was to study the environmental pollution of the river Tiber within the urban area of Rome. We studied both the stress of the macrobenthic communities, described by density (calculated as animal biomass) and diversity (calculated with the Shannon index), and the mutagenic effects of waters and sediments, using the micronucleus test in root tips of Vicia faba. Four places (stations) were studied: (1) Castel Giubileo, at the entry of the urban area; (2) Ponte Tor di Quinto, immediately after the confluence of the tributary river Aniene; (3) Ponte Matteotti, at the entry of the historical city center; and (4) Ponte Sublicio, in the middle of the city center. Samples were collected in spring (May, 1989) with an intermediate pollution level, winter (January 1990) with a low one, and late summer (September 1990) with a high one. There is agreement between the environmental damage, measured by diversity of macrobenthic community, and the mutagenic effect of media (water and sediments), identified by micronuclei: while Castel Giubileo shows an acceptable condition in all the seasons, and Ponte Sublicio a bad one, the intermediate stations show a recovery in January.
Raw water and drinking water samples collected from five treatment plants supplied by a northern Italian lake in two periods of the year (summer and winter) were studied for their mutagenicity. The water samples were concentrated on silica C18 cartridges and the adsorbates were tested at increasing doses with a bacterial short-term mutagenicity test (Ames test with Salmonella typhimurium TA98 and TA100 strains), which reveals the gene-mutation-inducing ability of pollutants, and with a plant genotoxicity bioassay (Tradescantia/micronucleus test), which determines clastogenicity (chromosome-breaking ability). Raw water samples from all treatment plants were found to contain bacterial direct-acting mutagens detectable mainly with TA98 strain. The analyses of drinking water samples after water treatment showed some interesting results: TA98 mutagenicity was reduced when ozone was used together with chlorine dioxide, but TA100 mutagenicity was increased, though only in the summer sample; mutagenicity detectable with both strains was always reduced after chlorine dioxide disinfection; on the contrary, in all treatment plants using NaClO TA98 mutagenicity of winter samples increased. Raw lake water induced a high number of micronuclei in the Tradescantia/micronucleus test, showing a strong clastogenicity. This activity was higher in the NaClO-treated samples, and lower with the other disinfectants. Therefore, disinfection of lake water with ozone and/or chlorine dioxide seems to be a suitable alternative to the use of NaClO for controlling the formation of nonvolatile mutagens. The concentration method coupled with the two mutagenicity tests was found to be a simple, rapid and relatively inexpensive system for monitoring treatment plants and studying the influence of different disinfection systems on water mutagenicity.
Frequencies of circulating erythrocyte micronuclei in two marine fish species from contaminated areas off southern California were elevated relative to fishes from less contaminated sites. Micronuclei frequencies from contaminated sites were four times higher in white croaker (Genyonemus lineatus) and eleven times higher in kelp bass (Paralabrax clathratus). The increased micronuclei frequency was related to previously determined environmental concentrations of chlorinated hydrocarbons (DDTs and PCBs) and polycyclic aromatic hydrocarbon metabolites. However, micronuclei frequency was only weakly correlated to individual body burdens of chlorinated hydrocarbons in white croaker as determined in this study. Applications and limitations of piscine micronucleus measurements are discussed.
The occurrence of chemical contaminants with DNA-damaging capacity in the environment represents a threat to human health as well as to the health of the ecosystem. This mini-review describes studies that were aimed to monitor at field conditions, the presence of such environmental toxicants and their DNA-damaging effects in aquatic and terrestrial species, as well as in birds. It is obvious that these studies, in particular are abundantly performed in fish and aquatic invertebrates, have brought forward new information on the levels and genotoxic effects of these compounds which complements data coming from monitoring the abiotic fractions of the ecosystem, thereby demonstrating that the ecogenotoxicological approach is fruitful. However, in order to assess the genotoxic impact on the health of the ecosystem, a second generation type of field studies is required focusing on adverse effects on biodiversity and on survival potency. For this, the application of DNA microarray-based technologies provides new opportunities.
Erythrocyte micronuclei (MN) frequencies of 280 winter flounder (Pseudopleuronectes americanus) from Chesapeake Bay to the southern Scotian Shelf including Long Island Sound were measured. The MN data were combined with data previously reported for 224 flounder and the combined data sets were analyzed. Incidences of MN were elevated sixfold in flounder from the New York Bight Apex as compared to frequencies for fish from the inshore Gulf of Maine and Block Island Sound, and twice those found in Georges Bank and Long Island Sound flounder. Inshore New Jersey fish had higher MN frequencies than those from inshore Gulf Of Maine and Block Island Sound. The occurrence of MN in flounder from inshore Virginia was higher than in flounder from inshore Gulf of Maine and Block Island Sound. The large subset of stations from Long Island Sound indicated higher frequencies of MN in flounder from Hempstead and Shoreham as compared to most other sites in the Sound. There was no significant difference in the frequency of MN between males and females, but males had significantly more MN than sexually immature flounder. Flounder over the entire sampling area had increased levels of MN during the fall months of the year. This is probably influenced by the maternal cycle of gonadal maturation.
A micronucleus test in gill cells of the freshwater mussel Anodonta cygnea has been proposed for the detection of drinking water genotoxicity. Animals were exposed for 28 days to a drinking water sample and collected every week. Highly significant increases in spontaneous MN frequency were observed at each sampling, especially after 13 days of exposure. As positive control 2 doses of mytomicin C (MMC) were used (10(-8) and 10(-7) M). A second experiment was performed at a municipal waterworks in order to assess the role of water treatment processes in the production of mutagenic compounds. The most prevalent genotoxic effects were detected after chlorination (mean: 10.47% +/- 3.05, p less than 0.001).
Fluka humic acid used as a model substrate in these studies was analysed for elemental and the oxygen-containing functional groups. It was chlorinated at C:Cl molar ratios of 1:1 and 1:0.3 and subsequently separated into molecular weight fractions by ultrafiltration. The freeze-dried, chlorinated humic acid and the respective molecular weight fractions were analyzed for TOC, TOX, alkylating activity using 4-(p-nitrobenzyl)pyridine and mutagenicity by the Ames/Salmonella/microsome assay with strains TA-98 and TA-100. Results indicated that predominantly non-volatile, direct-acting mutagenic and/or alkylating agents were formed during humic acid chlorination and that these agents were unevenly distributed among the various molecular weight fractions. Formation of mutagenic and alkylating agents were highly dependent upon level of chlorination and total organic carbon. Higher levels of mutagenic and alkylating activities were produced with increasing concentration of chlorine in the range of 0.4-1.2 chlorine equivalents per mole of carbon. However, both these activities in the freeze-dried, chlorinated humic acid solutions containing the non-volatiles or the fresh solutions decreased gradually with increasing pH and storage time, apparently due to degradation and hydrolysis of some of the components.
The cytogenetic effects of various i.p. treatments with five carcinogenic-mutagenic chemicals and three doses for each (aflatoxin B1, Aroclor 1254, benzidine, benzo[a]pyrene and 20-methylcholanthrene), were investigated in the cells of the common carp, Cyprinus carpio. Injection with distilled water and corn oil served as the two control groups. For detecting cytogenetic damage we used two test systems, chromosomal aberrations (CA) in kidney cells and micronucleated erythrocytes (M). At 48 hr after treatment with the chemicals under investigation, the frequencies of CA and M were clearly increased in a dose-response manner compared to the control groups.
Chlorination of humic and fulvic acid results in the formation of direct-acting mutagenicity, detectable in the Salmonella/microsome assay (Ames test). This mutagenicity is being characterized as part of an overall effort aimed at evaluating potential health risks associated with the presence of mutagenic chemicals in drinking water. A number of chlorinated organic compounds, including several known mutagens, have been identified and quantified in diethyl ether extracts of chlorinated humic acid solutions. However, the total mutagenicity of these compounds accounts for only about 7% of the original mutagenicity. Synergistic or antagonistic interactions among the identified components have been ruled out as possible explanations for the failure to account for a higher percentage of the activity. Recent progress has been made to separate the activity into neutral and strong acid fractions. Further isolation of the strong acids by high-pressure liquid chromatography (HPLC) has resulted in the purification of the mutagenicity into a major peak of activity with a specific mutagenicity of about 20,000 TA100 revertants per milligram. Several trichlorohydroxyfuranone isomers have been tentatively identified in this fraction. The contribution of these types of compounds to the mutagenicity of chlorinated humic acid is under investigation.
A model micronucleus test system using peripheral blood erythrocytes from larvae of Pleurodeles waltl is described. The most suitable larval stage for testing chemical treatments was determined. Larvae were reared in water containing one of the 4 compounds: benzo[a]pyrene (BaP), ethyl methanesulphonate (EMS), diethyl sulphate (DES) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Response curves as a function of treatment duration over a period of 16 days were plotted for 3 different concentrations of the 4 compounds in order to optimize conditions for a low dose micronucleus test. This model can be used as a monitoring system for the detection of fresh water pollution and can also be employed for clastogen screening of chemical compounds. The test is sensitive, reliable and easy to use.
Some ways in which four short-term tests may be used to evaluate the mutagenicity of drinking water were explored by testing raw and treated water from Lake Bloomington, which serves the town of Bloomington, Illinois (population, 44,000). The water was collected from February 1976 to October 1977 and was concentrated by evaporation or by use of XAD-2 resin. The water was tested for the ability to induce reverse mutation in a prokaryote, Salmonella typhimurium; forward mutation in a mold, Neurospora crassa; mitotic gene conversion in a yeast, Saccharomyces cerevisiae; and reverse mutation in maize, Zea mays. Because of the large number of water samples (54) and the limited amounts of the samples, it was not possible to test all samples in all four tests by all the protocols. Thus, the sensitivities of the four tests to potential mutagens in the water samples could not be rigorously compared. However, the results do show that lake and tap water samples collected during 1976 were toxic but not mutagenic in N. crassa and neither toxic nor genotoxic in S. cerevisiae; lake water collected during 1977 was mutagenic in one line of Z. mays and slightly mutagenic in S. typhimurium strain TA1536 in the presence of rat liver S9. The results suggest that tests that detect a variety of genetic end points should be used when testing complex mixtures such as drinking water. The advantages and disadvantages of the tests and protocols are discussed in terms of their applicability to the study of the mutagenicity of drinking water.
In a short feasibility study, we detected the induction of nuclear anomalies in the peripheral blood erythrocytes of fish by the genotoxic compound ethyl methanesulphonate. Exposure of the eastern mudminnow Umbra pygmaea to the compound gave rise to nuclear segments resembling the particles found in mammalian micronucleus tests, but also to irregular Feulgen-positive structures in the cytoplasm. Further studies with fish erythrocytes are recommended for investigating whether the monitoring of nuclear anomalies is a suitable and rapid method for the detection of genotoxic compounds in the aquatic environment.
The Allium anaphase-telophase test was evaluated to find out if it could be recommended in the screening of wastewater for genotoxicity. Five mutagenic or carcinogenic chemicals usually found in wastewater were tested in the Allium anaphase-telophase test. Sodium dichromate (25 microM), benzene (100 microM), dichloromethane (175 microM) and 1,1,1-trichloromethane (175 microM) increased the frequency of chromosome aberrations in the root cells, whereas formaldehyde (1 mM) was found to be non-mutagenic in this test system. Other studies where chemicals were tested in the Allium test were reviewed. For 15 chemicals the results were compared with results from the Ames test, the Microscreen assay, and carcinogenicity tests in rodents. The sensitivity of the Allium test was calculated to be 82%. In conclusion the Allium test is recommended for the screening of wastewater because it has a high sensitivity, is cheap, rapid, easy to handle, and because it can be used on wastewater without pretreatment of the sample.
An in vivo study on the effects of both physical (gamma-radiation) and chemical (mitomycin C) genotoxic agents was carried out with the catfish species, Clarias gariepinus. The fish were either exposed to gamma-radiation at doses of 0-9 Gy or injected intraperitoneally with mitomycin C at concentrations of 0-2 mg/kg. Micronucleated erythrocytes were sampled from 0 to 60 days post treatment. Data obtained showed a time-dependent response of the induction of micronucleated erythrocytes with both genotoxic agents. A linear dose-dependent increase was observed 2-4 days after treatment. These data show the importance of sampling time in the micronucleus assay with Clarias gariepinus.
Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of six halogenated acetonitriles identified in chlorinated waters (monochloro-, dichloro-, trichloro-, monobromo-, dibromo- and bromochloroacetonitrile). With the SOS chromotest, three of the chemicals studied (dichloro-, dibromo- and bromochloroacetonitrile) were found to induce primary DNA damage in Escherichia coli PQ37. In the Ames-fluctuation test, all the compounds except dibromoacetonitrile showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for all the six haloacetonitriles studied. Moreover, two structure-activity relationships were noted: (1) the genotoxic activity of haloacetonitriles containing bromine substituents appeared higher than the corresponding chlorinated acetonitriles and (2) the clastogenic activity of the chlorinated acetonitriles increased with the number of chlorine substituents.
Specimens of rainbow trout (Oncorhynchus mykiss) were either kept in an aquarium under laboratory conditions or caged in the River Po (Northern Italy), upstream or downstream the confluence with the River Lambro, a small yet heavily polluted tributary. Genotoxicity biomarkers, evaluating the internal dose or early biological effects, were monitored after 7, 15 and 30 days of in situ exposure. With the exception of a slight increase of aminopyrine-N-demethylase and uridine-5'-diphospho-glucuronosyl-transferase, no significant effect was produced in fish kept upstream the River Lambro, as compared to control fish kept in the aquarium. In contrast, the responsibility of this tributary in carrying sublethal doses of both genotoxic agents and enzyme inducers into the main river was proved by the significant occurrence of early biological alterations in fish caged downstream. In fact, (a) bile extracts contained frameshift mutagens requiring metabolic activation, with a prevalence of liposoluble components after a short exposure, followed by a time-related increase of conjugated components, in minor part with glucuronic acid; (b) the monooxygenases aminopyrine-N-demethylase, uridine-5'-diphospho-glucuronosyl-transferase and, with sharp differences, arylhydrocarbon hydroxylase and 7-ethoxyresorufin-O-deethylase were enhanced in liver microsomal fractions; (c) the liver cytosolic fractions had an enhanced ability to convert 3-amino-1-methyl-5H-pyrido(4,3)indole into mutagenic metabolites in S. typhimurium; (d) cytogenetic damage was demonstrated by an increased frequency of micronuclei in peripheral blood erythrocytes.
Micronucleus assays with fish have been shown to be useful in vivo techniques for genotoxicity testing, and show potential for in situ monitoring of water quality. In this paper, we review the literature on the clastogenic effects of chemical and physical agents on fish cells, with emphasis on the induction of micronuclei in teleosts. Included in the review is a description of the mechanisms for formation of micronuclei in cells, and a summary of the various techniques that have been used for micronucleus analysis in fish. This review is directed to assisting laboratories in the development of fish genotoxicity assays for water quality monitoring.
Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a "comet with tail" formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.
Erythrocyte micronucleus frequencies in wild fish from two riverine environments and in fish reproduced and reared under controlled conditions (control group) were compared, with the aim to evaluate the suitability of the MN test for the in situ detection of mutagens in freshwaters. Fish were caught in different months in two rivers of central Italy which have different pollution levels. As indicator species, the barbel (Barbus plebejus) was chosen because of its ecological significance. Blood samplings were performed on wild fish immediately after capture and repeated at different time intervals on the same individuals, which were maintained in controlled conditions after capture. A total of 10,000 erythrocytes per specimen were scored. No significant differences in micronucleus frequencies were observed between the control group and fish from the unpolluted river (Mignone). A significantly higher frequency of micronuclei was observed in fish caught in the polluted river (Tiber), in comparison to both the controls and the Mignone river fish. No significant seasonal differences were observed. Barbels examined 50 and 100 days after capture presented a remarkable decrease in micronucleus frequency in comparison with the frequency observed in barbels at capture. The micronucleus test in fish erythrocytes was shown to be a sensitive bioassay for detecting mutagenic pollution in fresh water environments.
Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water. Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. Ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed.
Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand and/or double strand breaks in a cell by cell approach, it is new possible to address this question at the cellular level. We therefore compared the genotoxic potential of pure cobalt powder (Co) and a cobalt-containing alloy, cobalt-tungsten carbide (WC-Co), involved in specific lung disorders, in parallel with the alkaline single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (MN) test, both carried out in vitro on isolated human leukocytes. The comet assay indicated that the WC-Co mixture produced a higher level of DNA damage than Co alone; WC alone was not able to induce a dose-dependent DNA breakage effect as was seen for Co and WC-Co. Results from the MN test confirmed these observations. It was clear that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC and suggested that their physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers. In agreement with data obtained in the same laboratory on liposoluble chemicals (PCBs and chlorinated aliphatic hydrocarbons) and from the literature, the results indicate that both the comet assay and the micronucleus test were able to detect differences in the genotoxic potential of the compounds studied. Although the micronucleus test seemed to be less sensitive to assess a synergistic DNA damaging potential of the mixture involved, it detects chromosomal aberrations (chromosome/genome mutations) and not just repairable DNA breakage or alkali-labile sites. Combination of the comet assay and the in vitro MN test might therefore be recommended for genotoxins to understand the mechanisms underlying mutagenicity and to assess the lowest efficient dose.
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.
Cyprinus carpio fish (carp), exposed to elemental or metallic mercury (Hg0) at concentrations of 2.0, 20.0, and 200.0 mg per liter of water, were kept in concrete tanks for 159 days. Ten fish were used for each concentration level. Thirteen samples of peripheral blood were collected from each animal through gill puncture, 12 during the first 90 days of the experiment, and the last one at the end of the experiment. The micronucleus test (MNT) was designed to study dose and time yield effects of mercury after indirect exposure in vivo. The results indicated that for a concentration of 2.0 mg Hg0/l, there was no significant increase in frequency of micronuclei (MN), but at higher concentrations (20.0 and 200.0 mg Hg0/l) there was a significant increase in MN frequencies. This effect was higher after 31 days of exposure, followed by slight stabilization and gradual decrease.
Four groups, each of 50 male and 50 female Sprague-Dawley rats, of the colony used in the Cancer Research Center of Bentivoglio of the Ramazzini Foundation, 12 weeks old at the start of the study, received drinking water containing sodium hypochlorite, resulting in concentrations of active chlorine of 750, 500, and 100 mg/l (treated groups), and tap water (active chlorine < 0.2 mg/l) (control group), respectively, for 104 weeks. Among the female rats of the treated groups, an increased incidence of lymphomas and leukemias has been observed, although this is not clearly dose related. Moreover, sporadic cases of some tumors, the occurrence of which is extremely unusual among the untreated rats of the colony used (historical controls), were detected in chlorine-exposed animals. The results of this study confirm the results of the experiment of the United States National Toxicology Program (1991), which showed an increase of leukemia among female Fischer 344/N rats following the administration of chlorine (in the form of sodium hypochlorite and chloramine) in their drinking water. The data here presented call for further research aimed at quantifying the oncogenic risks related to the chlorination of drinking water, to be used as a basis for consequent public health measures.