Towards the Development of Microbial Ecotoxicology Testing Using Chlorpyrifos Contaminated Sediments and Marine Yeast Isolates as a Model.

Chlorpyrifos (CP), a widely used pesticide, and its metabolite 3,5,6-trichloro-2-pyridinol (3,5,6-TCP), are xenobiotic compounds detected in many biomes, notably in marine sediments, all over the world. These compounds are posing a serious environmental and health problem given their toxicity to wildlife and possible exposure effects to human neurodevelopment. Microorganisms at CP-impacted environments could harbor metabolic capabilities that can be used as indicators of the biological effects of the contaminant and could encode selected functions reactive against contaminants. Those features could be used for microbial ecotoxicology applications by collectively using analytical, enzymatic, microbiological and toxicological techniques in order to assess the biological effects of pollutants and other environmental/climatic stressors in ecosystems. The objective of this study was to assess the variability in the metabolic responses of yeast isolates from CP-contaminated marine sediments as potential biological indicators for microbial ecotoxicology testing. Sediment samples from a South Caribbean tropical shore (Cartagena Bay, Colombia) were collected, and deoxyribonucleic acid (DNA) was recovered from lyophilized aliquots. The DGGE (Denaturing Gradient Gel Electrophoresis) technique targeting fungal Internal Transcribed Spacer (ITS) showed the great diversity of fungal types. Simultaneously, yeast strains were isolated from the freshly collected sediment samples. Physiological characterization including API 20C and antibiosis tests, growth patterns at salt concentrations (2/4/10/25%), temperatures (4/25/37/45 °C), esterase activity assay and resistance tests to CP/TCP toxicity resulted in 10 isolated yeast strains, identified as Candida spp. (6), Cryptococcus spp. (3). and Rhodotorula spp. (1), showing promising characteristics to be used as a test for yeast-based ecotoxicity indicators. The patterns of carbohydrate assimilation, low antibiosis, presence of esterases/lipases, growth in a wide range of temperatures and salt concentrations, and tolerance to minimal inhibitory concentrations of CP and TCP are factors useful for testing environmental samples.

Cytotoxic and estrogenic activity of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol. Study of marine yeasts as potential toxicity indicators.

Chlorpyrifos (CP) is one of the organophosphate insecticides most used worldwide today. Although the main target organ for CP is the nervous system triggering predominantly neurotoxic effects, it has suggested other mechanisms of action as cytotoxicity and endocrine disruption. The risk posed by the pesticide metabolites on non-target organisms is increasingly recognized by regulatory agencies and natural resource managers. In the present study, cytotoxicity and estrogenic activity of CP, and its principal metabolite 3,5,6-trichloro-2-pyridinol (TCP) have been evaluated by in vitro assays, using two mammalian cell lines (HEK293 and N2a), and a recombinant yeast. Results indicate that TCP is more toxic than CP for the two cell lines assayed, being N2a cells more sensitive to both compounds. Both compounds show a similar estrogenic activity being between 2500 and 3000 times less estrogenic than 17ß-estradiol. In order to find new toxicity measurement models, yeasts isolated from marine sediments containing CP residues have been tested against CP and TCP by cell viability assay. Of the 12 yeast strains tested, 6 of them showed certain sensitivity, and a concentration-dependent response to the tested compounds, so they could be considered as future models for toxicity tests, although further investigations and proves are necessary.

Acute toxicity of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol alone and in combination using a battery of bioassays.

Acute toxicity of chlorpyrifos (CP) and its principal metabolite 3,5,6-trichloro-2-pyridinol (TCP) alone and in combination have been evaluated using a test battery comprising aquatic organisms from different trophic levels: luminescent marine bacteria Aliivibrio fischeri, freshwater unicellular alga Pseudokirchneriella subcapitata, and cladoceran Daphnia magna. As expected, D. magna was the more sensitive organism to the compounds tested, being CP more toxic than its metabolite. On the contrary, TCP was found to be more toxic than its parental compound to A. fischeri and P. subcapitata. In all cases, the mixture of CP and its metabolite was more toxic than the compounds tested separately, multiplying between 5 and 200 times CP toxicity level and up to 15 times TCP toxicity level. These results indicate that the co-existence of parent chemical and its degradation product in the environment can result in a synergic interaction involving high risk to the aquatic ecosystems. Graphical abstract.

Adaptación de bacterias a diferentes concentraciones de fenol en el laboratorio: aspectos esenciales para un proceso de biodegradación / Adaptation of bacteria to different concentrations of phenol in the laboratory: essentials for biodegradation process

En el presente trabajo se determino la carga bacteriana de muestras de agua y suelo contaminadas con diferentes concentraciones de fenol. Se hizo seguimiento en medio mineralizado con concentraciones de 200 a 10000mg/L y variaciones de inoculo. Se evidenciaron cambios en crecimiento bacteriano, encontrándose mayor carga en suelo con fase de adaptación al segundo día y fase de crecimiento en cuarto día. Comparando variaciones del inoculo (directa, adaptado y preenriquecimiento) el directo puede aplicarse para muestras de carga alta (suelo); el preenriquecimiento no es práctico por el estrés bacteriano y adaptado permite ser usado hasta 500mg/L sin problemas. En repiques sobre agares (mineralizados y nutritivo) hubo crecimiento hasta 1000mg/L. Es importante para depurar aguas residuales, encontrar bacterias de diferentes fuentes que resistan altas concentraciones de fenol y permitan efectividad en bioprocesos al estudiar comportamientos de pre adaptación, tiempos y tratamientos adecuados para la preparación del inóculo.

Aislamiento de bacterias potencialmente degradadoras de petróleo en hábitats de ecosistemas costeros en la Bahía de Cartagena, Colombia / Isolation of potential oil degrading bacteria in coastal habitats in Cartagena Bay, Colombia

En esta investigación se aislaron bacterias de 4 hábitats en el ecosistema marino aledaño a una industria petroquímica en la Bahía de Cartagena. En el proceso, las muestras se sometieron a pre-enriquecimiento por una semana, a enriquecimiento por tres semanas y a un proceso de selección de cepas competitivas, donde se evidenciaron cambios marcados en las propiedades del crudo de petróleo, como en turbidez y agregados blancos por crecimiento bacteriano. Se aislaron diferentes morfotipos que al caracterizar bioquímicamente fueron identificados como Pseudomonas aeruginosa en todas las muestras, corroborando su gran capacidad de adaptación en ambientes contaminados de este tipo. Estos resultados permitirán la realización de pruebas de biodegradación con esta bacteria y desarrollar ensayos a nivel microcosmos para su uso potencial en procesos de biorremediación de aguas marinas contaminadas con petróleo.