First Characterization of PAH-degrading bacteria from Río de la Plata and high-resolution melting: an encouraging step toward bioremediation.

The Río de la Plata, one of the most important estuarine environments in South America that sustains valuable fisheries, is affected by PAH contamination associated with oil industry and port activities. A total of 95 bacteria with potential to degrade phenanthrene were obtained from water samples using traditional culture methods. PCR-RFLP analysis of 16S rDNA partial fragments was used as a screening tool for reducing the number of isolates during diversity studies, obtaining 42 strains with different fingerprint patterns. Phylogenetic analysis indicated that they were affiliated to 19 different genera of Gamma- and Alpha-Proteobacteria, and Actinobacteria. Some of them showed an efficient phenanthrene degradation by HPLC (between 83% and 97%) and surfactant production (between 40% and 55%). They could be an alternative for microbial selection in the degradation of PAHs in this estuarine system. In order to detect and monitor PAH-degrading bacteria in this highly productive area, rDNA amplicons of the 33 isolates, produced by PCR real time, were tested by the high-resolution melting (HRM) technique. After analyzing the generated melting curves, it was possible to accurately distinguish nine patterns corresponding to eight different genera. HRM analysis allowed a differentiation at the species level for genera Pseudomonas, Halomonas and Vibrio. The implementation of this method as a fast and sensitive scanning approach to identify PAH-degrading bacteria, avoiding the sequencing step, would mean an advance in bioremediation technologies.

Effects of waterborne exposure to the antidepressant fluoxetine on swimming, shoaling and anxiety behaviours of the mosquitofish Gambusia holbrooki.

Chemical pollution from pharmaceuticals is increasingly recognized as a major hazard to the aquatic biota. Among the wide variety of pharmaceuticals, fluoxetine (FLX) is one of the most widely prescribed antidepressants, and therefore, it is frequently identified in the aquatic environment. As FLX is designed to alter human behaviour and many physiological pathways are conserved across vertebrates, this drug may affect the behaviour of fish living in FLX-polluted environments. Here, we exposed groups of female mosquitofish Gambusia holbrooki to waterborne FLX for 14 days, under semi-static conditions with daily renewal of test solutions. Following exposure, we conducted a set of behavioural assays in individual fish, aimed at assessing the effects of FLX on their locomotor activity and behavioural responses. We found that FLX impaired swimming behaviour at high concentrations (25 µg/L and 50 µg/L) but not at low concentrations close to environmental levels (1 µg/L and 5 µg/L). When swimming activity was assessed 5 min after transfer of the focal fish to the testing tank, 50 µg/L FLX was the only concentration showing significant effects. However, when the same trials were performed 24 h later, 25 µg/L FLX turned out to be an effect concentration in addition to 50 µg/L. Interestingly, these concentrations would elicit fish plasma concentrations comprised within the range of human therapeutic doses. When subjected to a light/dark preference test, fish showed tendency to remain less time in the dark area at high FLX concentrations, thus suggesting an anti-anxiety response. Shoaling behaviour was not affected by FLX exposure. Our study contributes to the growing body of literature evaluating the effects of FLX on animal behaviour. Regarding the experimental design used in behavioural testing, our findings suggest that focal fish should be subjected to long habituation periods, namely of at least a few hours, in order to better assess the effects of drug exposure.

Effects of waterborne exposure to 17ß-estradiol and 4-tert-octylphenol on early life stages of the South American cichlid fish Cichlasoma dimerus.

Estrogenic chemicals are often detected in the aquatic environment and can negatively affect animal development and reproduction. In teleost fishes, the hormonal regulation during a critical period of larval development has a strong influence on gonadal sex differentiation; thus this process may be affected by the exposure to environmental estrogens. In this study, we first assessed the lethal acute toxicity of the natural estrogen 17ß-estradiol (E2) and the weaker estrogen mimics 4-tert-octylphenol (OP) and 4-nonylphenol (NP) on larval stages of the South American cichlid fish Cichlasoma dimerus. In a further experiment, we analyzed the effects of chronic waterborne exposure to E2 and OP on gonad development and sex differentiation. Exposure to high concentrations of E2 had a pronounced feminizing effect directing sex differentiation towards ovarian development, while testis development was inhibited at a lower, environmentally relevant concentration. Among OP-exposed fish, 15-38.5% of the males exhibited testicular oocytes (TOs), a commonly reported biomarker of estrogenic exposure. However, since TOs were also recorded in control males and the proportion of males with TOs was not significantly higher in OP treatments, their occurrence could not be attributed to OP exposure. In addition, TOs did not seem to impair male gonad development and functionality since normal spermatogenesis was observed in testes of OP-treated fish. These results indicate that E2 occurring in the South American aquatic environment may affect male reproductive development and pose a risk for wild C. dimerus, especially under prolonged exposure, while the effects of weaker xenoestrogens such as OP would be negligible for gonad development in this species. As illustrated by this study, the natural occurrence of TOs indicates that conclusions concerning the causes of this phenomenon must be drawn with care.

Simplified determination of lipophilic metabolites of nonylphenol ethoxylates: method development and application in aqueous samples from Buenos Aires, Argentina.

In the present work we have developed an analytical methodology for the determination of nonylphenol (NP) and nonylphenol mono- and di-ethoxylates (NP1EO and NP2EO) in water samples. The applicability of this methodology was proved by means of the analysis of environmentally relevant aqueous samples from Buenos Aires. This constitutes a starting point for a rigorous assessment of the incidence of NPnEO surfactants in Argentina, as only very few, qualitative or semi-quantitative data on the occurrence of these compounds in local systems were available up to this time. Enrichment of the analytes was carried out by solid-phase extraction on a C-18 sorbent, followed by elution with ethyl acetate. Normal-phase high performance liquid chromatography on an amino-silica column and fluorescence detection at excitation-emission wavelengths of 230-300 nm were employed for separation and quantification of the analytes. Confirmation of peak assignment in selected real samples was performed by off-line coupling HPLC with GC-MS analysis. A non-polar GC capillary column was used, and a characteristic peak pattern was obtained for the alkyl chain isomers of each ethoxylated homologue and NP. GC-MS analyses yielded in all cases purity levels higher than 80% for the HPLC collected fractions. The elevated concentrations found for the estrogenic metabolites of NPnEO are in accordance with an unrestricted use of this class of non-ionic surfactants in the country.

Column temperature as an active variable in the isocratic, normal-phase high-performance liquid chromatography separation of lipophilic metabolites of nonylphenol ethoxylates.

Normal-phase separation of technical grade nonylphenol (t-NP, about 90% 4-nonylphenol), 4-nonylphenol mono-ethoxylate (4-NP1EO) and 4-nonylphenol di-ethoxylate (4-NP2EO) was assessed, with the inclusion of column temperature as an active variable. The compound 2,4,6-trimethylphenol was evaluated for use as internal standard. Isocratic elution with 2-propanol/hexanes mixtures from an amino-silica column and spectrometric UV detection at 277 nm were employed. Technical nonylphenol presented a significant contribution from unknown substances that eluted with retention times similar to that of 4-NP1EO. GC-MS analysis of the unknowns allowed to identify them as isomers of 2-NP. The response of the system to joint variations in flow rate, eluent composition and column temperature was investigated by means of Doehlert statistical experimental design. A model for retention of the analytes as a function of the experimental variables was proposed, and separation selectivity was studied. Selection of the optimal working zone was made through desirability function (D) calculations. Potential co-elution of 2-NP isomers with 4-NP1EO was considered when optimizing the separation. The occurrence of a restricted region of the experimental space where baseline resolution of analytes, associated impurities and internal standard results feasible (D not equal to 0) is apparent.

Anaerobic nonylphenol ethoxylate degradation coupled to nitrate reduction in a modified biodegradability batch test.

The aim of this work was to elucidate the role of nitrate as a terminal electron acceptor on the biodegradation of NPEO. We have characterized the products of NPEO degradation by mixed microbial communities in anaerobic batch tests by means of HPLC, (1)H NMR and GC-MS. Anaerobic degradation of NPEO was strictly dependent on the presence of nitrate. Within seven days of anoxic incubation, NP2EO appeared as the major degradation product. After 21 days, NP was the main species detected, and was not degraded further even after 35 days. Nitrate concentration decreased in parallel with NPEO de-ethoxylation. A transient accumulation of nitrite was observed within the time period in which NP formation reached its maximum production. The observed generation of nonylphenol coupled to nitrate reduction suggests that the microbial consortium possessed an alternate pathway for the degradation of NPEO, which was not accessible under aerobic conditions.

Separation of nonylphenol ethoxylates and nonylphenol by non-aqueous capillary electrophoresis.

Capillary electrophoresis based on non-aqueous solvent background electrolytes was employed, with single and multiple wavelength UV detection, to evaluate discrimination among oligomer components of mixtures of non-ionic, long chain nonylphenol ethoxylates (NPnEO, with n = number of ethoxy units) and their lipophilic degradation products. The tested organic solvents included acetonitrile, methanol, ethanol, 1- and 2-propanol, 1-butanol and tetrahydrofurane in the presence of sodium acetate. A rational variation of composition of background electrolyte solvent mixtures allowed to modify the mobility of electroosmotic flow and the type and degree of interactions between the ionic additive (sodium acetate) and the components of the analyte mixtures. The physicochemical properties of the solvents, such as dielectric constant, viscosity and electron donor-acceptor ability regarding the additive, were considered to improve the resolution of lipophilic compounds with less than three ethoxy groups and the discrimination attainable for longer chain oligomers. The studied methodologies also allowed discerning between surfactants of similar (nominal) ethoxy chain lengths. This was demonstrated by the different peak distribution patterns observed for NPnEO compounds with n = 7.5 and 10, respectively.