Acute toxicity and genotoxicity of two novel pesticides on amphibian, Rana N. Hallowell

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-l'-tert-butylbenzoylhydrazinel] are two pesticides used in China since 1992. In the present study we conducted acute toxicity test, micronucleus (MN) test and comet assay of the two pesticides on amphibian, Rana N. Hallowell, a sensitive organism suitable for acting as the bio-indicator of aquatic and agricultural ecosystems. The values of LC50-48 h of imidacloprid were found to be 165 mg l(-1) for tadpoles of Rana limnocharis and 219 mg l(-1) for tadpoles of Rana N. Hallowell. On the other hand, RH-5849 showed no acute toxicity to tadpoles during the 96 h exposure even it was saturated in the test solutions. There were significant differences in the MN frequencies between the negative controls and the treated groups at the dose of 8 mg l(-1) for imidacloprid (p < 0.05) and 40 mg l(-1) for RH-5849 (p < 0.01). Comet assay found significant differences (p < 0.01) in the distributions of DNA damage grades between the negative controls and groups treated in vitro with 0.05, 0.1, 0.2 and 0.5 mg l(-1) of imidacloprid and 5, 25, 50 and 100 mg l(-1) of RH-5849, respectively. DNA damage scores increased with the exposure levels of the two pesticides and dose-effect relationships were observed for both imidacloprid (r2 = 0.92) and RH-5849 (r2 = 0.98). The MN test and comet assay revealed potential adverse effects of the two pesticides on DNA in the erythrocytes of amphibians in aquatic and agricultural ecosystems.     

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p'-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p'-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p'-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p'-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p'-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p'-DDT on human genome.     

Monitoring the genotoxicity of Lake Taihu, using two kinds of micronucleus tests

Mutagenicity of surface water in Lake Taihu was monitored by genotoxicological methods. The goal is to determine the situation concerning the contamination by mutagens, their distribution in the lake, and the potential effect on public health. In this study, the micronucleus test of Vicia faba root tip, a standard monitoring technique now in China, was carried out to make a preliminary screening of environmental mutagens in 39 representative sites of the lake. Highly significant differences in MCN permillage (average number of micronuclei per 1000 cells) and PI (pollution index) values were found among these sites. From the results, a rough distribution of the contamination could be obtained: mainly concentrated in the north-west area of the lake from several tributaries flowing into the lake. In order to provide further evidence, organic extracts from four entrances and one outlet of the five major tributaries were examined by the micronucleus test of human peripheral lymphocytes. The results were in accord with those of the micronucleus tests using Vicia faba. The tests also revealed that the intensity and mechanism of the pollutants were diverse in the different sources.     

Genotoxicity in adult residents in mineral coal region—a cross-sectional study

The present study assessed the DNA damage in environmentally exposed volunteers living in seven municipalities in an industrial coal region, through the use of the comet assay with blood cells and the micronucleus test with buccal cells. Blood and buccal smears were collected from 320 male volunteers living in seven cities inserted in a coal region. They were ages of 18 and 50 years and also completed a questionnaire intended to identify factors associated with DNA damage through a Poisson regression analysis. The comet assay detected significant differences in DNA damage in volunteers from different municipalities, and neighboring cities (Pedras Altas, Aceguá, and Hulha Negra) had a higher level of DNA damage in relation to control city. Some of the risk factors associated with identified DNA lesions included residence time and life habits. On the other hand, the micronucleus test did not identify differences between the cities studied, but the regression analysis identified risk factors such as age and life habits (consumption of mate tea and low carbohydrates diet). We conclude that there are differences in the DNA damage of volunteers from different cities of the carboniferous region, but the presence of micronuclei in the oral mucosa does not differ between the same cities. Furthermore, we alert that some related factors may increase the risk of genotoxicity, such as residence location and time, and living and food habits. Finally, we suggest the need for continuous biomonitoring of the population, as well as for investing in health promotion in these vulnerable populations.

     

Leachates of municipal solid waste incineration bottom ash from Macao: heavy metal concentrations and genotoxicity

Heavy metals in municipal solid waste incineration bottom ash (MSWIBA) may leach into soil and groundwater and pose long-term risks to the environment. In this study, toxicity characteristic leaching procedure (TCLP) was carried out on the MSWIBA from Macao. Heavy metals in leachates were determined by inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES), and genotoxicity of leachates was also evaluated by micronucleus (MN) assay with Vicia faba root tip cells. The results showed that the concentrations of aluminium (Al), manganese (Mn), cobalt (Co), cadmium (Cd) and mercury (Hg) in the leachates were less than 0.01 mg l(-1), and those of iron (Fe), copper (Cu) and molybdenum (Mo) were less than 0.1 mg l(-1). The concentrations of chromium (Cr), zinc (Zn), selemium (Se), strontium (Sr), barium (Ba) and caesium (Cs) were between 0.11 mg l(-1) and 2.19 mg l(-1). Lead (Pb) concentrations, in particular, reached as high as 19.6 mg l(-1), significantly exceeding the maximum concentration limit (5 mg l(-1) for lead by TCLP). Compared with the negative group, a significant increase of MN frequencies was observed in the leachate-exposed groups (P<0.05). With the increase of heavy metals in the leachates, the toxic effects on the Vicia faba root tip cells increased, implying that heavy metals were the main factors causing the genotoxic effects. Our results suggested that apart from chemical analysis, bioassays like the MN assay of Vicia faba root tip cells should also be included in a battery of tests to assess the eco-environmental risks of bottom ashes before decisions can be made on the utilization, treatment or disposal.     

Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p′-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p′-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p′-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p′-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p′-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p′-DDT on human genome.     

Genotoxic assessment on river water using different biological systems

This paper reports genotoxicity and toxicity data in water samples collected in Sinos River, an important water course in the hydrographic region of Guaíba Lake, Rio Grande do Sul State, south of Brazil. This river is exposed to intense anthropic influence by numerous shoes, leather, petrochemical, and metallurgy industries. Water samples were collected at two moments (winter 2006 and spring 2006) at five sites of Sinos River and evaluated using in vitro V79 Chinese hamster lung fibroblasts (cytotoxicity, comet assay and micronucleus test) and Allium cepa test (toxicity and micronucleus test). Comet and micronucleus tests revealed that water samples collected exerted cytotoxic, toxic, genotoxic and mutagenic effects. The results showed the toxic action of organic and inorganic agents found in the water samples in all sites of Sinos River, for both data collections. The main causes behind pollution were the domestic and industrial toxic discharges. The V79 and A. cepa tests were proved efficient to detect toxicity and genotoxicity caused by complex mixtures. This study also showed the need for constant monitoring in sites with strong environmental degradation caused by industrial discharges and urban sewages.     

137Cs determination and mutagenicity tests in wild Mus musculus domesticus before and after the Chernobyl accident

Wild rodents were collected alive in a suburban area to the north of Rome before, during, 6 months and 1 year after the Chernobyl accident. Radionuclide determinations to assess their content of (137)Cs and mutagenicity tests (bone-marrow micronucleus test and sperm abnormalities assay) were performed on the captured animals. The results obtained for the species Mus musculus domesticus (which was the rodent captured in highest numbers), compared with the results obtained before the fallout, showed an increase of both micronucleated polychromatic erythrocytes (MPCEs/1000 PCEs) and (137)Cs content during and after the fallout.     

Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals (HMs) as well as their genotoxicity in soil after long-term wastewater irrigation

A study was carried out on the residue level of major concern pollutants (PAHs, PCBs and HMs) and the assessment of their genotoxicity in soils obtained from Shenyang, northeast of China which had been subjected to wastewater irrigation for more than 40 years. Topsoils (0-20cm) in paddy fields were sampled along the upper, middle and lower reaches of the wastewater disposal channel. Sixteen USEPA PAHs were determined by High Performance Liquid Chromatography (HPLC) with fluorescence detector, eight PCBs were detected by Gas Chromatography (GC) with electron capture detector, and six heavy metals (Cd, Cu, Pb, Zn, Ni, and Cr) were measured by Atomic Absorption Spectrophotometer (AAS). The genotoxicity effect of soils was examined by Vicia faba micronucleus (MN) test. Archived soils that had undergone a similar history of wastewater irrigation provided by Technical University of Berlin, Germany were subjected to analysis of the above pollutants and Vicia faba/MN test for comparison. Results indicated elevated residues of the studied pollutants (PAHs, especially benzo (a) pyrene, the eight PCB congeners and heavy metals) in both tested and archived soils. The MN frequencies were 2.2-48.4 times higher compared with the control. However, there was no correlation between the MN frequencies and the concentration of pollutants detected. This investigation suggested a potential ecological risk even with a lower level of residual pollutants in soil matrix after long-term wastewater irrigation.     

Bioaccumulation and elimination of avermectin B1a in the earthworms (Eisenia fetida)

The acute toxicity, bioaccumulation, and elimination of avermectin B1a (AVM B1a) in earthworm (Eisenia fetida) were investigated in different exposure systems. The LC50 of AVM B1a on earthworms were 24.1 mg/kg and 17.1 mg/kg, respectively, for 7 and 14 days in artificial soil. The LC50 tested by the filter paper for 2 days was 4.63 microg/cm2. The earthworms were cultivated in artificial soil containing 0.6 mg/kg and 3.0 mg/kg AVM B1a, respectively for bioaccumulation experiments. The AVM B1a residues in earthworms were determined with HPLC-fluorescence method. The results showed that AVM B1a was taken up from the concentrated artificial soil by the earthworms and the steady-state levels were reached after 9-18 days of exposure. On the 18th day, the final concentrations of AVM B1a in the earthworms treated with two different dosages were 107 ng/g and 165 ng/g, respectively; there were not significantly accumulation. About 80.0% and 94.8% of the accumulated AVM B1a were eliminated respectively in two groups within 1 day after they were exposed to AVM B1a-free soil, but a trace amount of AVM B1a was found for a relative long time in earthworms.     

Evaluation of removal efficiency for acute toxicity and genotoxicity on zebrafish in anoxic–oxic process from selected municipal wastewater treatment plants

The anoxic–oxic (A/O) process has been extensively applied for simultaneous removal of organic contaminants and nitrogen in wastewater treatment. However, very little is known about its ability to remove toxic materials. Municipal wastewater contains various kinds of pollutants, some of which have recalcitrant genotoxicity and may cause potential threat to environment, and even can lead to extinction of many species. In this study, we have selected three municipal wastewater treatment plants (WWTPs) employing anoxic–oxic (A/O) process to evaluate their ability to remove acute toxicity and genotoxicity of wastewater. Mortality rate of zebrafish (Danio rerio) was used to evaluate acute toxicity, while micronucleus (MN) and comet assays were used to detect genotoxicity. Results showed that in this process the acute toxicity was completely removed as the treatment proceeded along with decrease in chemical oxygen demand (COD) (<50 mg L^?1) in the effluent. However, in these treatment processes the genotoxicity was not significantly reduced, but an increase in genotoxicity was observed. Both MN and comet assays showed similar results. The eliminated effluent may pose genotoxic threaten although its COD level has met the Chinese Sewage Discharge Standard. This study suggests that further treatment of the wastewater is required after the A/O process to remove the genotoxicity and minimize the ecotoxicological risk.     

Application of the comet and micronucleus assays to butterfish (Pholis gunnellus) erythrocytes from the Firth of Forth, Scotland.

This report describes an investigation of genotoxic effects in an inter-tidal fish species sampled along a pollution gradient in the Firth of Forth, Scotland, UK. The comet assay is an electrophoretic technique for measuring DNA breakage in nuclei from individual cells and has only recently been applied to field investigations of genotoxicity. The measurement of nuclear anomalies (NA), such as the presence of micronuclei (MN) and 'lobes', has been successfully utilised in many field studies of genotoxic effects of contaminated sediments. These two techniques were applied to nucleated red blood cells (RBC) from the butterfish, Pholis gunnellus. The comet assay was adapted and validated for use in this species. Fish were sampled from the inner Firth of Forth, which has a legacy of industrial contamination and the outer Firth of Forth which is comparatively clean. The analysis of DNA strand breakage using this technique did not reveal any significant differences between animals sampled from inner and outer zones of the Firth. In contrast, MN and NA frequencies were elevated in the inner polluted zone of the Firth compared to the outer zone. This study suggests: (1) there are genotoxic effects associated with contaminants in the inner Firth of Forth, and (2) the comet assay may not be a suitable genotoxicity biomarker in fish.     

Toxicity of the anthelmintic abamectin to four species of soil invertebrates

Abamectin is a veterinary medicinal product with high efficiency against parasitics. This study evaluates the sublethal toxicity of abamectin to three groups of soil dwelling organisms (springtails, enchytraeids and earthworms). The reproduction of the two springtail species Folsomia fimetaria and Folsomia candida was significantly affected at abamectin concentrations of 0.25 and 0.5 mg/kg DW, respectively. Adults of especially F. candida were less sensitive. Abamectin had no effect on the survival of enchytraeids, whereas the number of juveniles was reduced at concentrations above 10 mg/kg. The EC10 values for reproduction were in the range of 0.05 mg/kg for springtails and 12.8 mg/kg for enchytraeids. Abamectin also had a significant effect on the reproduction of earthworms with an EC10 value of 0.06 mg/kg. However, no change in the survival and growth of juvenile earthworms was observed following 70 days of exposure.     

Sub-lethal toxicity of the antiparasitic abamectin on earthworms and the application of neutral red retention time as a biomarker

The antiparasitic abamectin has been proven effective against both endo- and ectoparasites of farm animals and hence used widely in animal husbandry. It may enter the soil environment with the excreta of treated animals. Very little information is available with regard to the sub-lethal effects of abamectin on soil invertebrates, such as earthworms. The objective of this study was to evaluate the toxic effect of abamectin on earthworms, using Eisenia fetida, by analyzing changes in the survival, growth, reproduction and cocoon hatchability of exposed earthworms. Furthermore, a biomarker of the lysosomal membrane stability, measured by neutral red retention time (NRR-time), was also applied. Abamectin showed significant toxicity on the growth of earthworms with increasing concentrations up to 5mg/kg. The most sensitive parameter was reproduction (cocoons production and hatchability) and NRR-time. The number of cocoons was reduced at concentrations above 0.25mg/kg and no cocoons were present at the highest concentration of 5mg/kg. Cocoons exposed to abamectin exhibited a reduced hatching success at concentrations above 1.5mg/kg. The NRR-time was reduced significantly at exposure concentrations of abamectin above 0.25mg/kg. The change in lysosomal membrane stability showed a good correlation with reproduction and may hence be a potential predicator of the effects on earthworm populations.     

Improvement of Vicia-micronucleus test for assessment of soil quality: a proposal for international standardization

The Viciafaba root tip micronucleus test is one of the most employed plant genotoxicity assays, and has been used on various types of contaminated materials. This test has been standardized by AFNOR, the French member organization of ISO. However, this test is usually performed with a water extraction step but soil genotoxicity assessment would be more relevant when performed directly in the soil itself. In order to harmonize these protocols, an ISO standard for the V.faba micronucleus test in both liquid phase (exposure of plants to different liquid matrix, including soil water extracts) and solid phase (direct exposure of plants to the soil) would be very useful. In this context, we compared two exposure durations in the solid phase (48 h and 5 d) for the V.faba micronucleus test with two different well-known genotoxicants, maleic hydrazide and copper sulfate. We concluded that these two durations induced equivalent sensitivity: the micronucleus frequency was significantly increased with 5 μmol maleic hydrazide per kg dry soil and with 2 mmol copper sulfate per kg dry soil with both exposure durations. However, exposing roots to soil during 48 h is more practical. Moreover, organically and conventionally cultured seeds were employed to determine whether the seed provenance influenced the test sensitivity. Organic seeds were less sensitive to copper, possibly because copper-based treatments are permitted, and often applied, in organic farms. Therefore, in the absence of completely non-treated seeds, organically-cultured seeds did not appear to offer any advantages over conventional seeds.     

Effect of phenobarbital and polychlorinated biphenyls on the toxicity and disposition of 1,2,4,5‐tetrachlorobenzene in the rat

Soil avoidance by earthworms has been generally considered a relevant and sensitive endpoint for assessing soil contamination by xenobiotics. However, when pesticide ecotoxicological assessment is concerned, the sensitivity of the recently standardized avoidance assay has been questioned. We hypothesized that this controversy may be due to the specific pesticide mode of action of the chemicals used rather than reveal inconsistencies in the test feasibility, i.e. provided that no pesticides interfering with neuronal pathways are tested, this bioassay should keep expected high levels of sensitivity. In this study, the avoidance behaviour of the earthworm Eisenia andrei under exposure to the carbamate insecticide methomyl [S-methyl N-(methylcarbamoyloxy)thioacetimidate] was linked to the corresponding acetylcholinesterase (AChE) inhibition. Significant AChE inhibition occurred at lower concentrations (from 0.86 mg Kg^?1 onwards) than significant avoidance of spiked soil (from 5.62 mg Kg^?1 onwards). This indicates that assessments regarding pesticides that have neurotoxic activity may be biased if behavioral endpoints are selected. Despite theoretical hypothesis that have been raised, this should be the first study providing preliminary experimental evidence on such a link between avoidance behavior and neuronal impairment levels in earthworms. Further studies are ongoing that should refine conclusions of this study.     

Toxicological characterization of the landfill leachate prior/after chemical and electrochemical treatment: A study on human and plant cells

In this research, toxicological safety of two newly developed methods for the treatment of landfill leachate from the Piškornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure combined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorption by clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed by electrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability of untreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect of the original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showed no cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/treated landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assay using either human or plant cells. The original leachate exhibited significantly higher comet assay parameters compared to negative control after 24 h exposure. On the contrary, there was no significant difference between negative control and chemically/electrochemically treated leachate for any of the parameters tested. There was also no significant increase in either CBMN assay parameter compared to the negative control following the exposure of the lymphocytes to the chemically or electrochemically treated landfill leachate for both exposure periods while the original sample showed significantly higher number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggest that both methods are suitable for the treatment of such complex waste effluent due to high removal efficiency of all measured parameters and toxicological safety of the treated effluent.     

Assessment of DNA damage in Brazilian workers occupationally exposed to pesticides: a study from Central Brazil

We evaluated 41 rural workers occupationally exposed to pesticides and 32 subjects as a control group, using the micronucleus (MN) and the comet assay. For the comet assay, we evaluated the peripheral blood, and for the MN, we sampled cells from the oral epithelium. Damage to DNA was measured by tail length, % DNA in tail (% tail), olive tail moment (OTM), and tail moment (TM). The exposed group presented an 8× increase in MN frequency, when compared to the control group (p <0.05). When we contrasted the MN frequencies between the individuals that use and do not use personal protective equipment, we found a mean of 7.5 MN (57 % variance) and 12.1 MN (130 % variance), respectively. The binucleated cells were 0.04 and 0.005, in the exposed and control groups, respectively, indicating 8× increase in the number of binucleated cells, when comparing the groups (p <0.05). In the comet assay, we demonstrated statistically significant differences in three parameters (% DNA, OTM, and TM) indicating that the rural workers presented high levels of genomic damages. Our results indicate that occupational exposure to pesticides could cause genome damage in somatic cells, representing a potential health risk to Brazilian rural workers that deal constantly with agrochemicals without adequate personal protection equipment.     

Toxicity of abamectin and doramectin to soil invertebrates

This study aimed at determining the toxicity of avermectins to soil invertebrates in soil and in faeces from recently treated sheep. Abamectin was more toxic than doramectin. In soil, earthworms (Eisenia andrei) were most affected with LC50s of 18 and 228 mg/kg dry soil, respectively, while LC50s were 67-111 and >300 mg/kg for springtails (Folsomia candida), isopods (Porcellio scaber) and enchytraeids (Enchytraeus crypticus). EC50s for the effect on reproduction of springtails and enchytraeids were 13 and 38 mg/kg, respectively for abamectin, and 42 and 170 mg/kg for doramectin. For earthworms, NOEC was 10 and 8.4 mg/kg for abamectin and doramectin effects on body weight. When exposed in faeces, springtails and enchytraeids gave LC50s and EC50s of 1.0-1.4 and 0.94-1.1 mg/kg dry faeces for abamectin and 2.2->2.4 mg/kg for doramectin. Earthworm reproduction was not affected. This study indicates a potential risk of avermectins for soil invertebrates colonizing faeces from recently treated sheep.     

Quantitative analysis of acetamiprid and imidacloprid residues in tomato fruits under greenhouse conditions

Abstract

A selective liquid chromatographic analytical method was studied for determination of two neonicotinoids, acetamiprid and imidacloprid, in tomato fruits under greenhouse conditions in Egypt. The fruits were extracted and cleaned up by QuEChERS method followed by HPLC determination. The method showed a good linearity with a determination coefficient (R²) of higher than 0.99 for the 0.0125–0.15 µg/mL concentration range. The method was validated using a blank tomato spiked at 5, 25 and 50 mg/kg and the recovery percentages were 83.71, 94.52 and 97.49% for acetamiprid and 88.59, 89.63 and 90.18% for imidacloprid, respectively. The rates of dissipation of both pesticides were studied and the preharvest intervals (PHIs) were calculated. Imidacloprid dissipated faster than acetamiprid and half-life periods were 1.30 and 2.07 days, respectively. Acetamiprid and imidacloprid residues were below the already established European maximum residue limits (EU MRLs) (0.5 mg/kg) 3 and 5 days after application, respectively.