Transcriptome analysis provides insights for understanding the adverse effects of endosulfan in Drosophila melanogaster

Indiscriminate use of agrochemicals worldwide, particularly, persistent organic pollutants (POPs), is of concern. Endosulfan, a POP, is used by various developing/developed nations and is known to adversely affect the development and the hormonal profiles of humans and animals. However, little is known about the molecular players/pathways underlying the adverse effects of endosulfan. We therefore analyzed the global gene expression changes and subsequent adverse effects of endosulfan using Drosophila. We used Drosophila melanogaster keeping in view of its well annotated genome and the wealth of genetic/molecular reagents available for this model organism. We exposed third instar larvae of D. melanogaster to endosulfan (2.0 μg mL⁻¹) for 24 h and using microarray, we identified differential expression of 256 genes in exposed organisms compared to controls. These genes are associated with cellular processes such as development, stress and immune response and metabolism. Microarray results were validated through quantitative PCR and biochemical assay on a subset of genes/proteins. Taking cues from microarray data, we analyzed the effect of endosulfan on development, emergence and survival of the organism. In exposed organisms, we observed deformities in hind-legs, reminiscent of those observed in higher organisms exposed to endosulfan. In addition, we observed delayed and/or reduced emergence in exposed organisms when compared to their respective controls. Together, our studies not only highlight the adverse effects of endosulfan on the organism but also provide an insight into the possible genetic perturbations underlying these effects, which might have potential implications to higher organisms.     

In vivo genotoxicity assessment of titanium,zirconium and aluminium nanoparticles,and their microparticulated forms,in Drosophila

As in vivo system, we propose Drosophila melanogaster as a useful model for study the genotoxic risks associated with nanoparticle exposure. In this study we have carried out a genotoxic evaluation of titanium dioxide (TiO₂), zirconium oxide (ZrO₂) and aluminium oxide (Al₂O₃) nanoparticles and their microparticulated forms in D. melanogaster by using the wing somatic mutation and recombination assay. This assay is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in treated larvae, which are expressed as mutant spots on the wings of adult flies. Third instar larvae were feed with TiO₂, ZrO₂ and Al₂O₃ nanoparticles, and their microparticulated forms, at concentrations ranging from 0.1 to 10 mM. Although a certain level of aggregation/agglomeration was observed in solution, it must be noted than the constant digging activity of larvae ensures that treated medium pass constantly through the digestive tract ensuring exposure. The results showed that no significant increases in the frequency of all spots (e.g. small single, large single, twin, total mwh and total spots) were observed, indicating that these nanoparticles were not able to induce genotoxic activity in the wing spot assay of D. melanogaster. Negative data were also obtained with the microparticulated forms. This indicates that the nanoparticulated form of the selected nanomaterials does not modify the potential genotoxicity of their microparticulated versions. These in vivo results contribute to increase the genotoxicity database on the TiO₂, ZrO₂ and Al₂O₃ nanoparticles.     

Direct and indirect effects of ammonia, ammonium and nitrate on phosphatase activity and carbon fluxes from decomposing litter in peatland

Here we investigate the response of soils and litter to 5 years of experimental additions of ammonium (NH₄), nitrate (NO₃), and ammonia (NH₃) to an ombrotrophic peatland. We test the importance of direct (via soil) and indirect (via litter) effects on phosphatase activity and efflux of CO₂. We also determined how species representing different functional types responded to the nitrogen treatments. Our results demonstrate that additions of NO₃, NH₄ and NH₃ all stimulated phosphatase activity but the effects were dependent on species of litter and mechanism (direct or indirect). Deposition of NH₃ had no effect on efflux of CO₂ from Calluna vulgaris litter, despite it showing signs of stress in the field, whereas both NO₃ and NH₄ reduced CO₂ fluxes. Our results show that the collective impacts on peatlands of the three principal forms of nitrogen in atmospheric deposition are a result of differential effects and mechanisms on individual components.     

Effects of methylmercury exposure on glutathione metabolism, oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks

We quantified the level of dietary mercury (Hg), delivered as methylmercury chloride (CH₃HgCl), associated with negative effects on organ and plasma biochemistries related to glutathione (GSH) metabolism and oxidative stress, and chromosomal damage in captive-reared common loon (Gavia immer) chicks reared from hatch to 105 days. Mercury-associated effects related to oxidative stress and altered glutathione metabolism occurred at 1.2 μg Hg/g and 0.4 μg Hg/g, an ecologically relevant dietary mercury level, but not at 0.08 μg Hg/g. Among the variables that contributed most to dissimilarities in tissue chemistries between control and treatment groups were increased levels of oxidized glutathione (GSSG), GSH peroxidase, and the ratio of GSSG to GSH in brain tissue; increased levels of hepatic GSH; and decreased levels of hepatic glucose-6-phosphate dehydrogenase (G-6-PDH). Our results also suggest that chronic exposure to environmentally relevant dietary Hg levels did not result in statistically significant somatic chromosomal damage in common loon chicks.     

Natural and pre-treatments induced variability in the chemical composition and morphology of lichens and mosses selected for active monitoring of airborne elements

To enhance the reliability of the moss and lichen transplant technique for active biomonitoring of trace metals in urban environments, we evaluated the natural variability in the chemical composition of the (epilithic and epiphytic) moss Hypnum cupressiforme and the epiphytic lichen Pseudevernia furfuracea from two reference areas in NE Italy. Green shoots of epilithic mosses and lobes of epiphytic lichens from larch branches showed rather homogenous composition and were selected for the exposure in nylon bags. As different physico-chemical pre-treatments are usually applied to selected cryptogamic material before its exposure, we also evaluated the effects of oven-drying at 120 degrees C for 24h, washing in 1N HNO3 solution, and in 0.5% NH4 oxalate solution at 85 degrees C for 15 h on the chemical composition and morphology of water-washed moss shoots and lichen lobes. Pre-treatments remarkably changed the chemical composition of selected materials but not their surface morphology.     

Cocaine and other illicit drugs in airborne particulates in urban environments: a reflection of social conduct and population size

Levels of cocaine and other psychoactive substances in atmospheric particulate matter (PM) were determined in urban environments representing distinct social behaviours with regard to drug abuse: night-life, university and residential areas. Three cities (with population >1 million and <0.3 million inhabitants) were selected. Mean daily levels of drugs in PM were 11-336 pg/m³ for cocaine, 23-34 pg/m³ for cannabinoids, and 5-90 pg/m³ for heroin. The highest levels were recorded on weekends, with factors with respect to weekdays of 1-3 for cocaine, 1-2 for cannabinoids and 1.1-1.7 for heroin. Higher levels were detected in the night-life areas, pointing towards consumption and trafficking as major emission sources, and possibly ruling out drug manufacture. The similarities in temporal trends at all sites suggested a city-scale transport of psychoactive substances. Correlations were detected between cocaine and amphetamine consumption (r² = 0.98), and between heroin and cannabinoids (r²>0.82).     

Genotoxic effect of polycyclic aromatic hydrocarbons in the metropolitan area of Porto Alegre, Brazil, evaluated by Helix aspersa (Müller, 1774)

The purpose of this study was to biomonitor metropolitan areas of Porto Alegre (Brazil) for PAHs associated with atmospheric particles and check their effects on the DNA of the land mollusk Helix aspersa. The sampling sites are located in an urban area with heavy traffic: (i) Canoas, (ii) Sapucaia do Sul, and (iii) FIERGS/Porto Alegre. The samples were collected during a continuous period of 24 hours during 15 days using Stacked Filter Units (SFU) on polycarbonate filters (two separated size fractions: PM_10-2.5 and PM_<2.5). The concentrations of 16 major PAHs were determined according to EPA. Comet assay on H. aspersa hemolymph cells was chosen for genotoxicity evaluation. This evaluation shows that, in general, the smaller PM-size fractions (PM_<2.5) have the highest genotoxicity and contain higher concentrations of extractable organic matter. In addition, associations between chemical characteristics and PM carcinogenicity tend to be stronger for the smaller PM-size fractions.     

Contamination assessment of a coastal lagoon (Ria de Aveiro, Portugal) using defence and damage biochemical indicators in gill of Liza aurata - An integrated biomarker approach

Fish gill importance in toxicants uptake, bioconcentration and excretion allied to meagre knowledge on branchial damage/protection responses substantiate this study. Five critical sites in Ria de Aveiro (Portugal) were assessed in comparison with a reference site (Torreira), focusing on Liza aurata gill antioxidant defences versus damage (oxidative and genetic). Only in Barra fish displayed damage (lipid peroxidation) though no differences were found in antioxidants. In all other sites, except Rio, antioxidant alterations were found. Thus, fish from Gafanha, Laranjo and Vagos showed higher total glutathione, glutathione peroxidase and catalase. Higher glutathione reductase and glutathione S-transferase activity was also found in the first and the last sites, respectively. In Laranjo, metallothionein levels were higher though lower in Gafanha and Vagos. In general, damage was not accompanied by defences weakening confirming that predicting damage based on antioxidants depletion is not straightforward. The integrated biomarker response index ranked sites as: Gafanha > Barra > Laranjo > Vagos > Rio > Torreira.     

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.     

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.     

Bioaccumulation of persistent organic pollutants in female common dolphins (Delphinus delphis) and harbour porpoises (Phocoena phocoena) from western European seas: geographical trends, causal factors and effects on reproduction and mortality

Concentrations of polychlorinated biphenyls (PCBs) in blubber of female common dolphins and harbour porpoises from the Atlantic coast of Europe were frequently above the threshold at which effects on reproduction could be expected, in 40% and 47% of cases respectively. This rose to 74% for porpoises from the southern North Sea. PCB concentrations were also high in southern North Sea fish. The average pregnancy rate recorded in porpoises (42%) in the study area was lower than in the western Atlantic but that in common dolphins (25%) was similar to that of the western Atlantic population. Porpoises that died from disease or parasitic infection had higher concentrations of persistent organic pollutants (POPs) than animals dying from other causes. Few of the common dolphins sampled had died from disease or parasitic infection. POP profiles in common dolphin blubber were related to individual feeding history while those in porpoises were more strongly related to condition.     

Sublethal toxicity of nano-titanium dioxide and carbon nanotubes in a sediment dwelling marine polychaete

The ecotoxicology of manufactured nanoparticles (MNPs) in estuarine environments is not well understood. Here we explore the hypothesis that nanoTiO₂ and single walled nanotubes (SWNT) cause sublethal impacts to the infaunal species Arenicola marina (lugworm) exposed through natural sediments. Using a 10 day OECD/ASTM 1990 acute toxicity test, no significant effects were seen for SWNT up to 0.03 g/kg and no uptake of SWNTs into tissues was observed. A significant decrease in casting rate (P = 0.018), increase in cellular damage (P = 0.04) and DNA damage in coelomocytes (P = 0.008) was measured for nanoTiO₂, with a preliminary LOEC of 1 g/kg. Coherent anti-stokes Raman scattering microscopy (CARS) located aggregates of TiO₂ of >200 nm within the lumen of the gut and adhered to the outer epithelium of the worms, although no visible uptake of particles into tissues was detected.     

Evaluation of potential genotoxicity of five food dyes using the somatic mutation and recombination test

In this study, different concentrations of five food dyes (amaranth, patent blue, carminic acid, indigotine and erythrosine) have been evaluated for genotoxicity in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. Standard cross was used in the experiment. Larvae including two linked recessive wing hair mutations were chronically fed at different concentrations of the test compounds in standard Drosophila Instant Medium. Feeding ended with pupation of the surviving larvae. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can result from either somatic mutation or somatic recombination. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which was distilled water. The present study shows that carminic acid and indigotine demonstrated negative results while erythrosine demonstrated inconclusive results. In addition 25 mg mL⁻¹ concentration of patent blue and 12.5, 25 and 50 mg mL⁻¹ concentrations of amaranth demonstrated positive results in the SMART.     

Effect of field exposure to 38-year-old residual petroleum hydrocarbons on growth, condition index, and filtration rate of the ribbed mussel, Geukensia demissa

In September 1969, the Florida barge spilled 700,000 L of No. 2 fuel oil into the salt marsh sediments of Wild Harbor, MA. Today a substantial amount, approximately 100 kg, of moderately degraded petroleum remains within the sediment and along eroding creek banks. The ribbed mussels, Geukensia demissa, which inhabit the salt marsh creek bank, are exposed to the spilled oil. Examination of short-term exposure was done with transplantation of G. demissa from a control site, Great Sippewissett marsh, into Wild Harbor. We also examined the effects of long-term exposure with transplantation of mussels from Wild Harbor into Great Sippewissett. Both the short- and long-term exposure transplants exhibited slower growth rates, shorter mean shell lengths, lower condition indices, and decreased filtration rates. The results add new knowledge about long-term consequences of spilled oil, a dimension that should be included when assessing oil-impacted areas and developing management plans designed to restore, rehabilitate, or replace impacted areas.     

Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio

Abamectin is used as an acaricide and insecticide for fruits, vegetables and ornamental plants, as well as a parasiticide for animals. One of the major problems of applying pesticides to crops is the likelihood of contaminating aquatic ecosystems by drift or runoff. Therefore, toxicity tests in the laboratory are important tools to predict the effects of chemical substances in aquatic ecosystems. The aim of this study was to assess the potential hazards of abamectin to the freshwater biota and consequently the possible losses of ecological services in contaminated water bodies. For this purpose, we identified the toxicity of abamectin on daphnids, insects and fish. Abamectin was highly toxic, with an EC₅₀ 48 h for Daphnia similis of 5.1 ng L⁻¹, LC₅₀ 96 h for Chironomus xanthus of 2.67 μg L⁻¹ and LC₅₀ 48 h for Danio rerio of 33 μg L⁻¹.     

The exposition of a calcareous Mediterranean soil to toxic concentrations of Cr, Cd and Pb produces changes in the microbiota mainly related to differential metal bioavailability

The involvement of the bacterial community of an agricultural Mediterranean calcareous soil in relation to several heavy metals has been studied in microcosms under controlled laboratory conditions. Soil samples were artificially polluted with Cr(VI), Cd(II) and Pb(II) at concentrations ranging from 0.1 to 5000 mg kg⁻¹ and incubated along 28 d. The lowest concentrations with significant effects in soil respirometry were 10 mg kg⁻¹ Cr and 1000 mg kg⁻¹ Cd and Pb. However, only treatments showing more than 40% inhibition of respirometric activity led to significant changes in bacterial composition, as indicated by PCR-DGGE analyses. Presumable Cr- and Cd-resistant bacteria were detected in polluted microcosms, but development of the microbiota was severely impaired at the highest amendments of both metals. Results also showed that bioavailability is an important factor determining the impact of the heavy metals assayed, and even an inverted potential toxicity ranking could be achieved if their soluble fraction is considered instead of the total concentration. Moreover, multiresistant bacteria were isolated from Cr-polluted soil microcosms, some of them showing the capacity to reduce Cr(VI) concentrations between 26% and 84% of the initial value. Potentially useful strains for bioremediation were related to Arthrobacter crystallopoietes, Stenotrophomonas maltophilia and several species of Bacillus.     

AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[a]pyrene

A collaborative study was performed on scallops (Chlamys farreri) exposed to 0.5, 3 and 10 microg/L benzo[a]pyrene (B[a]P) for 20 days. The levels of aryl hydrocarbon hydroxylase (AHH) activity, B[a]P accumulation and DNA strand break were assayed in the gill and digestive gland. Results showed that AHH activity and B[a]P accumulation were significantly related to B[a]P dose. AHH activity was induced and then became stable gradually. B[a]P accumulation increased first and showed an incoming plateau. DNA strand break levels in the 0.5 and 3 microg/L B[a]P groups remained high and significantly different from control values until day 6, followed by a reduction in the gill and no recovery in the digestive gland. The 10 microg/L B[a]P groups remained significantly lower than control until the end. These results suggested that the application of comprehensive indices may give information on the actual exposure of organisms to pollutants, and also information on toxic effects.     

Association of the mutagenicity of airborne particles with the direct emission from combustion processes investigated in Osaka, Japan

The association of the direct-acting mutagenicity of soluble organic fraction of airborne particles toward Salmonella typhimurium YG1024 strain with the direct emission was investigated at a roadside and at a residential area in Osaka, Japan. The direct-acting mutagenicity was evaluated as mutagenic activity per unit volume of ambient air (rev m⁻³) and/or that per airborne particulate weight collected on a filter (rev mg⁻¹). The annual or diurnal changes of the mutagenicity of airborne particles at the residential site showed similar patterns to those of some gaseous pollutants such as NO₂ and SO₂, which were emitted from combustion processes. This result indicates that the mutagenicity is mainly attributable to the primary emissions. From the analysis of the relationship between the wind sector and the mutagenic intensity, rev m⁻³ and rev mg⁻¹ values were strongly affected by the emissions from the fixed sources and from the mobile sources, respectively. The rev m⁻³ value and concentration of 1-nitropyrene (1-NP) in unit per m³ at the roadside were a factor of 2.6 and 2.8 higher than those at the residential site, respectively, but the rev mg⁻¹ value and concentration of 1-NP in unit per mg at the roadside were substantially comparable to those at the residential area. These observations suggest that the characteristics of the airborne particles can be attributed to the automotive emissions even at the suburban area.     

A protocol for the evaluation of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake water treated with different disinfectants

A sensitive and rapid method to evaluate toxic and genotoxic properties of drinking water supplied from Lake Trasimeno (Umbria, Central Italy) was worked out analysing bile in Cyprinus carpio exposed for 20 d to lake water treated with 3 different disinfectants, sodium hypochlorite (NaClO), chlorine dioxide (ClO₂) and peracetic acid (PAA). Fish were sacrificed at 0, 10 and 20 d in order to investigate the time course of these endpoints. An aliquot of bile samples was fractionated by adsorption on C₁₈ silica cartridges and the genotoxic potential of whole bile and of bile fractions was evaluated by the single-cell microgel-electrophoresis (comet) assay on human colonic adenocarcinoma cells (Caco-2). Bile (both whole and fractionated) from specimens exposed to the three disinfectants always showed a genotoxic activity as compared to the control group. The results of this study provide evidence that all three disinfectants cause an increase in bile genotoxicity of chronically exposed fish.