Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean.
Perchlorate is an emerging contaminant that affects the functioning of the thyroid gland in humans and alters the normal development of living beings. This compound can be formed naturally and by different anthropogenic activities, which are responsible for its wide diffusion in all environmental ma...
- Autores:
-
Acevedo Barrios, Rosa Leonor
- Tipo de recurso:
- Doctoral thesis
- Fecha de publicación:
- 2019
- Institución:
- Universidad de Cartagena
- Repositorio:
- Repositorio Universidad de Cartagena
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.unicartagena.edu.co:11227/16548
- Acceso en línea:
- https://hdl.handle.net/11227/16548
http://dx.doi.org/10.57799/11227/11882
- Palabra clave:
- Compuestos organoclorados
Compuestos de cloro
Soil pollution
Thyroid gland – Diseases
- Rights
- openAccess
- License
- Derechos Reservados - Universidad de Cartagena, 2019
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dc.title.eng.fl_str_mv |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
title |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
spellingShingle |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. Compuestos organoclorados Compuestos de cloro Soil pollution Thyroid gland – Diseases |
title_short |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
title_full |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
title_fullStr |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
title_full_unstemmed |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
title_sort |
Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean. |
dc.creator.fl_str_mv |
Acevedo Barrios, Rosa Leonor |
dc.contributor.advisor.none.fl_str_mv |
Olivero Verbel, Jesús |
dc.contributor.author.none.fl_str_mv |
Acevedo Barrios, Rosa Leonor |
dc.subject.armarc.none.fl_str_mv |
Compuestos organoclorados Compuestos de cloro Soil pollution Thyroid gland – Diseases |
topic |
Compuestos organoclorados Compuestos de cloro Soil pollution Thyroid gland – Diseases |
description |
Perchlorate is an emerging contaminant that affects the functioning of the thyroid gland in humans and alters the normal development of living beings. This compound can be formed naturally and by different anthropogenic activities, which are responsible for its wide diffusion in all environmental matrices. This chemical compound is persistent in ecosystems; so, it is necessary to use different types of treatments for its degradation. For the reduction of perchlorate, biological treatment with aerobic, facultative and anaerobic bacteria degradation and/or reduction of this pollutant is preferably used because the chemical methods, such as ion exchange, used for this purpose, are neither very efficient, nor very selective and incomplete, thus generating contaminated resins that must be treated later, which, in some cases, becomes more toxic than the original compound. Although there are currently adequate bacterial methods for the degradation of perchlorate, there is, however, no evidence that these have a universal application and such application is not standardized in tropical environments; that is why it is necessary to isolate native bacteria from hypersaline soils from the Colombian Caribbean, as a new economic and effective alternative to reduce perchlorate, thus generating a lesser impact on ecosystems. Perchlorate-reducing bacteria are a promising strategy for the bioremediation of contaminated sites. In this study, ten strains were isolated that grew in media containing up to 30.0% NaCl, pH variations (6.5-12.0) and moderately high concentrations of KClO4 (up to 10000 mg/kg), suggesting that these are halotolerant organisms. According to the sequencing of the 16S ribosomal gene, such strains belong to Vibrio, Bacillus, Salinovibrio, Staphylococcus and Nesiotobacter genus. These bacteria could reduce perchlorate levels from 10 to 25%. The main objective of this study was to evaluate the reduction of perchlorate in native bacteria isolated from hypersaline soils of Galerazamba (Bolivar), Parque Salamanca (Magdalena) and Manaure (Guajira) in the Colombian Caribbean; as well as to determine the toxic effects of this pollutant in in-vitro and in-vivo models. For this purpose, the ecotoxicological evaluation of perchlorate exposure was carried out in HEK, N2a, and 3T3 cell lines, as well as in the biological models Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna and Eisenia fetida, and the evaluation of several endpoints and the multivariate analysis between the chemical concentration of the pollutant and the toxicological responses of the models was evaluated. The ecotoxicological evaluation this contaminant’s toxic effects in different biological models exhibited a similar toxicity against the cell lines analyzed, with LC50 values of 19, 15 and 19 mM for HEK, N2a and 3T3 respectively; while in V. fischeri, the toxicity examined was considerably lower (EC50=715mM) with reduction of bioluminescence, while the survival of freshwater algae P. subcapitata was significantly affected by perchlorate (LC50=72 mM), and its effect on lethality in crustacean D. magna was significant (LC50=5mM). For the E. fetida earthworm, the LC50 was 56 mM in the soil. In the latter organism, perchlorate induced avoidance behavior, weight loss, decreased the production of eggs and offspring, as well as morphological and histopathological effects, such as malformations, dwarfism and necrosis. In conclusion, the environmental risk assessment by exposure varies according to the species, which presents a high environmental risk to the biota and the health of the ecosystems. This study evaluated the ecotoxicological impacts at different trophic levels; the results could be used as basic information to make decisions about the regulation of perchlorate in the environment. E. fetida is proposed as a sensitive model to generate information on the toxicological impact of KClO4 in soils. It is concluded that native bacteria isolated from hypersaline environments of the Colombian Caribbean are a promising tool for the reduction of this contaminant; this will improve the environmental quality of soils in tropical environments contaminated with perchlorate. |
publishDate |
2019 |
dc.date.issued.none.fl_str_mv |
2019 |
dc.date.accessioned.none.fl_str_mv |
2023-06-22T14:58:25Z |
dc.date.available.none.fl_str_mv |
2023-06-22T14:58:25Z |
dc.type.spa.fl_str_mv |
Trabajo de grado - Doctorado |
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http://purl.org/coar/version/c_970fb48d4fbd8a85 |
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dc.language.iso.spa.fl_str_mv |
eng |
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dc.rights.spa.fl_str_mv |
Derechos Reservados - Universidad de Cartagena, 2019 |
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Universidad de Cartagena |
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Olivero Verbel, JesúsAcevedo Barrios, Rosa Leonor2023-06-22T14:58:25Z2023-06-22T14:58:25Z2019https://hdl.handle.net/11227/16548http://dx.doi.org/10.57799/11227/11882Perchlorate is an emerging contaminant that affects the functioning of the thyroid gland in humans and alters the normal development of living beings. This compound can be formed naturally and by different anthropogenic activities, which are responsible for its wide diffusion in all environmental matrices. This chemical compound is persistent in ecosystems; so, it is necessary to use different types of treatments for its degradation. For the reduction of perchlorate, biological treatment with aerobic, facultative and anaerobic bacteria degradation and/or reduction of this pollutant is preferably used because the chemical methods, such as ion exchange, used for this purpose, are neither very efficient, nor very selective and incomplete, thus generating contaminated resins that must be treated later, which, in some cases, becomes more toxic than the original compound. Although there are currently adequate bacterial methods for the degradation of perchlorate, there is, however, no evidence that these have a universal application and such application is not standardized in tropical environments; that is why it is necessary to isolate native bacteria from hypersaline soils from the Colombian Caribbean, as a new economic and effective alternative to reduce perchlorate, thus generating a lesser impact on ecosystems. Perchlorate-reducing bacteria are a promising strategy for the bioremediation of contaminated sites. In this study, ten strains were isolated that grew in media containing up to 30.0% NaCl, pH variations (6.5-12.0) and moderately high concentrations of KClO4 (up to 10000 mg/kg), suggesting that these are halotolerant organisms. According to the sequencing of the 16S ribosomal gene, such strains belong to Vibrio, Bacillus, Salinovibrio, Staphylococcus and Nesiotobacter genus. These bacteria could reduce perchlorate levels from 10 to 25%. The main objective of this study was to evaluate the reduction of perchlorate in native bacteria isolated from hypersaline soils of Galerazamba (Bolivar), Parque Salamanca (Magdalena) and Manaure (Guajira) in the Colombian Caribbean; as well as to determine the toxic effects of this pollutant in in-vitro and in-vivo models. For this purpose, the ecotoxicological evaluation of perchlorate exposure was carried out in HEK, N2a, and 3T3 cell lines, as well as in the biological models Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna and Eisenia fetida, and the evaluation of several endpoints and the multivariate analysis between the chemical concentration of the pollutant and the toxicological responses of the models was evaluated. The ecotoxicological evaluation this contaminant’s toxic effects in different biological models exhibited a similar toxicity against the cell lines analyzed, with LC50 values of 19, 15 and 19 mM for HEK, N2a and 3T3 respectively; while in V. fischeri, the toxicity examined was considerably lower (EC50=715mM) with reduction of bioluminescence, while the survival of freshwater algae P. subcapitata was significantly affected by perchlorate (LC50=72 mM), and its effect on lethality in crustacean D. magna was significant (LC50=5mM). For the E. fetida earthworm, the LC50 was 56 mM in the soil. In the latter organism, perchlorate induced avoidance behavior, weight loss, decreased the production of eggs and offspring, as well as morphological and histopathological effects, such as malformations, dwarfism and necrosis. In conclusion, the environmental risk assessment by exposure varies according to the species, which presents a high environmental risk to the biota and the health of the ecosystems. This study evaluated the ecotoxicological impacts at different trophic levels; the results could be used as basic information to make decisions about the regulation of perchlorate in the environment. E. fetida is proposed as a sensitive model to generate information on the toxicological impact of KClO4 in soils. It is concluded that native bacteria isolated from hypersaline environments of the Colombian Caribbean are a promising tool for the reduction of this contaminant; this will improve the environmental quality of soils in tropical environments contaminated with perchlorate.DoctoradoDoctor(a) en Toxicología Ambientalapplication/pdfengUniversidad de CartagenaFacultad de Ciencias FarmacéuticasCartagena de IndiasDoctorado en Toxicología AmbientalDerechos Reservados - Universidad de Cartagena, 2019https://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)http://purl.org/coar/access_right/c_abf2Effects of perchlorate at different trophic levels and characterization of reducing bacteria of this pollutant from hypersaline soils of the Colombian Caribbean.Trabajo de grado - Doctoradoinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_db06Textinfo:eu-repo/semantics/doctoralThesishttps://purl.org/redcol/resource_type/TDhttp://purl.org/coar/version/c_970fb48d4fbd8a85Compuestos organocloradosCompuestos de cloroSoil pollutionThyroid gland – DiseasesAbbondanzi, F., Cachada, A., Campisi, T., Guerra, R., Raccagni, M., and Iacondini, A. (2003). Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox® assay. Chemosphere. Vol. 53(8): p.889-897.Acevedo-Barrios, R., Bertel-Sevilla, A., Alonso-Molina, J., and Olivero-Verbel, J. (2016). Perchlorate tolerant bacteria from saline environments at the Caribbean region of Colombia. Toxicology Letters. Vol. 259: p.S103.Acevedo-Barrios, R., Sabater-Marco, C., and Olivero-Verbel, J. (2018). Ecotoxicological assessment of perchlorate using in vitro and in vivo assays. Environmental Science and Pollution Research. Vol. 25(14): p.13697-13708.Ahn, C. H., Oh, H., Ki, D., Van Ginkel, S. W., Rittmann, B. E., and Park, J. (2009). Bacterial biofilm-community selection during autohydrogenotrophic reduction of nitrate and perchlorate in ion-exchange brine. Applied Microbiology and Biotechnology. Vol. 81(6): p.1169-1177.Albuquerque, L., Tiago, I., Taborda, M., Nobre, M. F., Veríssimo, A., and da Costa, M. S. (2008). Bacillus isabeliae sp. nov., a halophilic bacterium isolated from a sea salt evaporation pond. International Journal of Systematic and Evolutionary Microbiology. Vol. 58(Pt 1): p.226-230.Ali Amoozegar, M., Zahra Fatemi, A., Reza Karbalaei-Heidari, H., and Reza Razavi, M. (2007). Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiological Research. Vol. 162(4): p.369-377.Alonso, J. L., Cuesta, G., Ramírez, G. W., Morenilla, J. J., Bernácer, I., and Lloret, R. M. (2009). Manual de técnicas avanzadas para la identificación y control de bacterias filamentosas. EPSAR-Generalitat Valenciana.Bahamdain, L., Fahmy, F., Lari, S., and Aly, M. (2015). Characterization of some Bacillus strains obtained from marine habitats using different taxonomical methods. Life Science Journal. Vol. 12.Bardiya, N., and Bae, J. H. (2005). Bioremediation potential of a perchlorate-enriched sewage sludge consortium. Chemosphere. Vol. 58(1): p.83-90.Bardiya, N., and Bae, J. H. (2011). Dissimilatory perchlorate reduction: a review. Microbiological Research. Vol. 166(4): p.237-254.Bernhardt, R. R., Von Hippel, F. A., and O'Hara, T. M. (2011). Chronic perchlorate exposure causes morphological abnormalities in developing stickleback. Environmental Toxicology and Chemistry. Vol. 30(6): p.1468-1478.Blount, B. C., Alwis, K. U., Jain, R. B., Solomon, B. L., Morrow, J. C., and Jackson, W. A. (2010). Perchlorate, nitrate, and iodide intake through tap water. Environmental Science & Technology. Vol. 44: p.9564-9570.Boluda, R., Gamón, M., Gómez De Barreda, D., Sáez, E., Gil, J., García De La Cuadra, J., Ten, A., Lleó De Otal, C., Lorenzo, E., and Pelegri, R. (2001). Monitoring of pesticide levels in water from irrigation channels of the Albufera Natural Park (Valencia, Spain). In Handling of Environmental and Biological Samples in ChromatographyBoone, D. R., Castenholz, R. W., Garrity, G. M., Brenner, D. J., Krieg, N. R., and Staley, J. T. (2005). Bergey’s Manual® of Systematic Bacteriology. Bergey’s Manual® of Systematic Bacteriology. Vol. 2.Breed, R. S., Murray, E. G. D., and Smith, N. R. (1957). Bergey's Manual of. Determinative Bacteriology. 7th edition, The Williams and Wilkins Company, Baltimore.Bruce, R. A., Achenbach, L. A., and Coates, J. D. (1999). Reduction of (per)chlorate by a novel organism isolated from paper mill waste. Environmental Microbiology. Vol. 1(4): p.319-329.Cáceres, T., Megharaj, M., and Naidu, R. (2008). Toxicity and transformation of fenamiphos and its metabolites by two micro algae Pseudokirchneriella subcapitata and Chlorococcum sp. Science of the Total Environment. Vol. 398: p.53-59.Cang, Y., Roberts, D. J., and Clifford, D. A. (2004). Development of cultures capable of reducing perchlorate and nitrate in high salt solutions. Water Research. Vol. 38(14-15): p.3322-3330.Cañas, J. E., Cheng, Q., Tian, K., and Anderson, T. A. (2006). Optimization of operating conditions for the determination of perchlorate in biological samples using preconcentration/preelution ion chromatography. Journal of Chromatography A. Vol. 1103: p.102-109.Chaudhuri, S. K., O'Connor, S. M., Gustavson, R. L., Achenbach, L. A., and Coates, J. D. (2002). Environmental Factors That Control Microbial Perchlorate Reduction. Applied and Environmental Microbiology. Vol. 68(9): p.4425-4430.Chen, H., Wu, L., Wang, X., Liu, Q., Ding, M., Peng, K., and Meng, Z. (2014). Perchlorate exposure and thyroid function in ammonium perchlorate workers in Yicheng, China. International Journal of Environmental Research and Public Health. Vol. 11(5): p.4926-4938.PublicationORIGINAL2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdf2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdfapplication/pdf4977145https://dspace7-unicartagena.metabuscador.org/bitstreams/005e83d4-99ad-4608-8c00-1fef5e6ce9f4/download1eda7b056fd20d6913c101869dacad6dMD51GRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdfGRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdfapplication/pdf316560https://dspace7-unicartagena.metabuscador.org/bitstreams/5a8b2f10-85ce-499b-bc57-84a0cff06933/downloadd4f8717cae139334601f9b1ccf8e4081MD52LICENSElicense.txtlicense.txttext/plain; charset=utf-81756https://dspace7-unicartagena.metabuscador.org/bitstreams/6d998ad7-d0e6-4dc6-b45a-2fb4e4511001/download7b38fcee9ba3bc8639fa56f350c81be3MD53TEXT2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdf.txt2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdf.txtExtracted texttext/plain191750https://dspace7-unicartagena.metabuscador.org/bitstreams/eb7693d6-f7b9-41dc-9821-e42917b2f9d3/download2017a4ca9af621ea0c2f70ae55a374a0MD54GRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdf.txtGRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdf.txtExtracted texttext/plain1https://dspace7-unicartagena.metabuscador.org/bitstreams/2488b502-5a21-40e9-876e-2522be954242/download68b329da9893e34099c7d8ad5cb9c940MD56THUMBNAIL2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdf.jpg2019_TESIS DE GRADO_ROSA LEONOR ACEVEDO BARRIOS.pdf.jpgGenerated Thumbnailimage/jpeg16445https://dspace7-unicartagena.metabuscador.org/bitstreams/0e884a89-449f-4a85-bbb3-151d4fb4ecaf/downloade546bd1a47f62216c7d51fc5a8d83147MD55GRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdf.jpgGRADO FORMATO CESION DE DERECHOS DE AUTOR GRADO ROSA ACEVEDO.pdf.jpgGenerated Thumbnailimage/jpeg10195https://dspace7-unicartagena.metabuscador.org/bitstreams/8cdc8650-fd40-48c0-802d-aa783c063383/download5ae829a20d801442c5341ea97f5dfeacMD5711227/16548oai:dspace7-unicartagena.metabuscador.org:11227/165482024-08-28 17:41:15.459https://creativecommons.org/licenses/by-nc/4.0/Derechos Reservados - Universidad de Cartagena, 2019open.accesshttps://dspace7-unicartagena.metabuscador.orgBiblioteca Digital Universidad de Cartagenabdigital@metabiblioteca.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 |