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.     

Determination of polycyclic aromatic hydrocarbons and substitutes (Nitro-, Oxy-PAHs) in urban soil and airborne particulate by GC-MS and NCI-MS/MS

A method involving high resolution gas chromatography combined with ion trap (HRGC-MS/MS) and negative chemical ionisation (NCI) was developed for the determination of nitrated or oxygenated polycyclic aromatic hydrocarbons (nitro-PAHs, oxy-PAHs) and other electrophilic substitutes in soil samples. Efficient clean-up was achieved by a combination of methods for the determination of PAHs in soil and nitro-PAHs in aerosol using solid phase extraction (SPE) and semipreparative high performance liquid chromatography (HPLC). In samples of surface soil from the city of Basle (Switzerland), nitro-PAHs (mainly 3-nitrofluoranthene and 1-nitropyrene) were found in concentrations between 30 and 800 ng/kg dry weight. Oxy-PAHs and parent PAHs revealed 10²-10⁴-fold higher levels. Nitro-PAHs which are up to 10⁵ times more mutagenic seem to be less persistent in soil than the parent forms, although their entire mutagenic potential has to be estimated as being on the same order of magnitude. In urban air particulate matter, the amounts of nitro-PAHs (2–62 pg/m³) were 10–100 times lower than oxyPAHs and parent PAHs which were both found in a similar range. 3-nitrobenzanthrone, a recently described suspected human carcinogen has not yet been detected. Using multivariate statistical analysis, it was possible to elucidate similarities or special characteristics of substances in a given matrix reflecting their chemical properties or specific emission sources.     

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.     

Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring

To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants.     

In situ studies on growth,oxidative stress responses,and gene expression of juvenile bighead carp (Hypophthalmichthys nobilis) to eutrophic lake water dominated by cyanobacterial blooms

Cyanobacterial blooms have received increasing attention as a public biohazard for human and animal health. To assess the effect of cyanobacteria-dominant lake water on juvenile fish, we measured the responses of specific growth rate, condition factor, body weight and body length, oxidative stress, and related gene expression of juvenile bighead carp Hypophthalmichthys nobilis exposed to in situ eutrophic lake (Chl a was around 7.0 μg L⁻¹). Results showed in situ cyanobacteria-dominant lake water had no effect on the growth performance, but significantly elevated the contents of malondialdehyde, the expression of heat shock protein 70, and the activity of superoxide dismutase, indicating that oxidative stress occurred. Meanwhile in situ lake water significantly decreased the expression of catalase and glutathione S-transferase genes. We conclude that in situ cyanobacteria-dominated lake water was harmful to juvenile bighead carp based on the oxidative stress and changes in the related gene expression levels.     

In situ monitoring of urban air in Córdoba, Argentina using the Tradescantia-micronucleus (Trad-MCN) bioassay

During the last decades, a significant deterioration of ambient air quality has been observed in Argentina. However, the availability of air pollution monitoring stations is still limited to only few cities. In this study, we investigated the genotoxicity of ambient levels of air pollution in Córdoba using the Tradescantia micronucleus assay. The experiment was performed from October, 2004 to April 2005. Pots with Tradescantia pallida were placed in three sites: Córdoba city center, characterized by important avenues with high traffic activity (cars, taxis, and public transport vehicles); the university campus, along a side road with heavy traffic of gasoline and diesel powered vehicles, buses and trucks; and a residential area, with no significant local sources of air pollution. Twenty young T. pallida inflorescences were collected from each sampling site in November, February and April. Micronuclei frequencies were determined in early tetrads of pollen mother cells and expressed as MCN/100 tetrads. Simultaneously, the environmental levels of total suspended particles (24 h mean) were determined for each site. A significant difference in micronuclei frequency was observed among sites (p=0.036). Post-hoc analysis revealed that the residential area exhibited a lower micronuclei frequency than the university and city center areas. In conclusion, we found that the gradients of ambient air pollution of Córdoba are associated with changes in the spontaneous micronuclei frequency of Tradescantia pollen mother cells. These results indicate that in situ biomonitoring with higher plants may be useful for characterizing air pollution in areas without instrumental monitoring techniques, or for exploring the distribution of air contaminants at a microscale.     

Ecosystem engineering potential of the gastropod Terebralia palustris (Linnaeus, 1767) in mangrove wastewater wetlands - A controlled mesocosm experiment

The effect of different sewage concentrations (0, 20, 60 and 100%), vegetation (Bare, Avicennia marina or Rhizophora mucronata) and immersion periods (immersion/emersion period of 12/12 h or 3/3 days just for 100%) conditions were studied for 6 months on survival and growth rates of Terebralia palustris (Linnaeus, 1767). Gastropods' activity and ecosystem engineering preformed at bare and A. marina planted cells and 3 sewage conditions (0, 20 and 60%) were determined. Survival rates were higher than 70% in all treatments. Growth rate decreased significantly with increasing sewage concentrations (mainly at unplanted conditions) and longer immersion periods. A complete shift (from immersion to emersion periods) and a significant decrease in mobility and consequently its engineer potential, due to sewage contamination, lead to a 3-4 fold decrease in the amount of sediment disturbed. Sewage contamination, primary producers' abundance and environmental conditions may have influenced the gastropods survival, growth and its ecosystem engineering potential.     

Ozone fumigation (twice ambient) reduces leaf infestation following natural and artificial inoculation by the endophytic fungus Apiognomonia errabunda of adult European beech trees

In 2006, a controlled infection study was performed in the ‘Kranzberger Forst’ to address the following questions: (1) Will massive artificial inoculation with Apiognomonia errabunda override the previously observed inhibitory effect of chronic ozone? (2) Can biochemical or molecular markers be detected to account for the action of ozone? To this end six adult beech trees were chosen, three ozone fumigated (2× ozone) and three control trees (ambient = 1× ozone). Spore-sprayed branches of sun and shade crown positions of each of the trees, and uninoculated control branches, were enclosed in 100-L plastic bags for one night to facilitate infection initiation. Samples were taken within a five-week period after inoculation. A. errabunda infestation levels quantified by real-time PCR increased in leaves that were not fumigated with additional ozone. Cell wall components and ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid) increased upon ozone fumigation and may in part lead to the repression of fungal infection.