Submitted by JEFFERSON CERQUERA GALLEGO null (jffgallego@gmail.com) on 2017-01-11T18:32:35Z No. of bitstreams: 1 Dissertação Final.pdf: 2374751 bytes, checksum: 4c444bb2f855ea07e3d575262a6ebc38 (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-01-12T16:53:51Z (GMT) / Submitted by JEFFERSON CERQUERA GALLEGO null (jffgallego@gmail.com) on 2017-01-12T18:22:16Z No. of bitstreams: 1 Dissertação Jefferson Cerquera Gallego.pdf: 2441699 bytes, checksum: 3fb5c0178315e1a59794457f28a8296d (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-17T12:06:41Z (GMT) No. of bitstreams: 1 gallego_jc_me_jabo.pdf: 2441699 bytes, checksum: 3fb5c0178315e1a59794457f28a8296d (MD5) / Made available in DSpace on 2017-01-17T12:06:41Z (GMT). No. of bitstreams: 1 gallego_jc_me_jabo.pdf: 2441699 bytes, checksum: 3fb5c0178315e1a59794457f28a8296d (MD5) Previous issue date: 2016-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Devido ao grande número de antibióticos de uso veterinário que estão sendo liberados no solo através da urina e dejetos fecais dos animais de produção, algumas pesquisas têm verificado o impacto desses antibióticos na microbiota do solo. O presente estudo teve como objetivo avaliar o impacto dos antibióticos sobre a microbiota do solo em condições de microcosmos, de um solo de pastagem de bovinos e um solo de floresta, submetidos à presença de três antibióticos utilizados na produção animal, sendo estes, ampicilina, enrofloxacina e estreptomicina, nas concentrações de 0, 30 e 100 mg/kg de solo seco. A concentração de 0 mg/kg foi usada como controle. Os solos foram incubados em frascos de vidro de tampa rosca e mantidos a temperatura ambiente no escuro para reproduzir as condições reais encontradas na natureza. Foram avaliadas atividade respiratória microbiana, atividade da enzima desidrogenase e contagem de unidades formadoras de colônias (UFC) para estabelecer se existia ou não inibição do crescimento bacteriano nos dias 0, 1, 20 e 35. Os resultados mostram um aumento considerável nas UFC nos solos que receberam a ampicilina em ambas às concentrações durante o primeiro dia com relação ao controle. No dia 35 estas contagens se tornaram semelhantes ao controle ou menores. Os solos que receberam enrofloxacina e estreptomicina tiveram uma contagem menor que o controle inicialmente e com o tempo essas UFC aumentaram. A atividade respiratória microbiana e a atividade da enzima desidrogenase também confirmam esse achado. Esses resultados sugerem que os micro-organismos estão utilizando algum composto da ampicilina para o aumento das colônias e que os outros antibióticos diminuem a população microbiana do solo, especialmente a estreptomicina. Provavelmente alguns micro-organismos estejam sendo selecionados. / Due to the large number of veterinary antibiotics that are being released into the soil through urine and fecal waste of livestock, some research has linked the impact of those antibiotics in soil microflora. In the current study was evaluated the impact of antibiotics in the soil microbial community under microcosms conditions, cattle pasture soil and a forest soil under the presence of three antibiotics used in animal husbadry; ampicilin, enrofloxacyn and streptomycin, using a concentration of 0, 30 e 100 mg/kg dry soil. The concentration of 0mg/kg was used as control. The soils were incubated in screw cap glass jars and kept at room temperature in the dark to reproduce actual conditions found in nature. It was evaluated the microbial respiratory activity as well as the activity of dehydrogenase enzyme and colony forming units (CFU) to establish whether there was inhibition of bacterial growth or not at day 0, 1, 20 and 35. The results show a considerable increase in CFU in soils that received both concentrations of ampicillin during the first day compared with control. At day 35 these counting became similar to control or lower. The soils that received enrofloxacin and streptomycin, initially had lower countings than the control and over time these CFU increased. The microbial respiratory activity and the activity of dehydrogenase also confirmed these findings. These results suggest that some microorganisms are using a compound of ampicillin to grow. The other antibiotics decrease the soil microbial population, especially streptomycin. Probably some microorganisms are being selected.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-02-01T06:57:35Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Laurivê Antônio dos Santos Júnior - 2015.pdf: 5217600 bytes, checksum: 7f6935b2ba3d2fba5d6b3a116bca276f (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-02-01T07:08:48Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Laurivê Antônio dos Santos Júnior - 2015.pdf: 5217600 bytes, checksum: 7f6935b2ba3d2fba5d6b3a116bca276f (MD5) / Made available in DSpace on 2016-02-01T07:08:49Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Laurivê Antônio dos Santos Júnior - 2015.pdf: 5217600 bytes, checksum: 7f6935b2ba3d2fba5d6b3a116bca276f (MD5) Previous issue date: 2015-09-28 / Antibiotics are widely used in human and veterinary medicine. After consumption, much of the drug is excreted unchanged or in the form of metabolites, and through the release of sewage treatment plants, agricultural waste and leaching, reach natural ecosystems. The occurrence of antibiotics in aquatic environments has become a growing concern worldwide mainly due to the emergence of resistance genes in pathogenic bacteria. The indiscriminate use and the use of subtherapeutic doses of antibiotics cause the emergence of resistance. In Goiás, a recent survey showed the presence of bacteria resistant to multiple antibiotics in hospital wastewater and Goiania sewage treatment plant. The wastewater treatment processes based on biological degradation have low efficiency of removal of residual drugs due to recalcitrant structure and biocide effect of antibiotics. The objective of this research was to evaluate the efficiency of hydrothermal decomposition of the antibiotic ampicillin, enrofloxacin, oxytetracycline, and tylosin sulphaquinoxaline. For this purpose, the antibiotic solutions were prepared at a concentration of 50 μg.mL-1 and pH correction to the range 3-7, with phosphoric acid solution for the hydrothermal carbonization treatment. Treatment by the hydrothermal carbonization process of oxytetracycline with pH adjustment made with phosphoric acid, pH 3, 170 ° C temperature and 90 minutes reaction time showed total decomposition as a result of this antibiotic (100%). The highest decomposition rate (98.37%) for enrofloxacin was observed when they were used the reaction time of 120 minutes, pH 3, 200 ° C temperature and phosphoric acid as a catalyst. The highest decomposition rate of ampicillin (94.86%) was observed when using time of 120 minutes reaction, pH 4, 200 ° C temperature and phosphoric acid as a catalyst. The most suitable conditions for the decomposition of sulphaquinoxaline, observing the greatest reduction rate (95.71%) was observed when they were used the reaction time of 120 minutes, pH 5, temperature 180 ° C and phosphoric acid as a catalyst. All conditions, except for the pH 7, led to complete decomposition of tylosin (100%) of the samples. The best condition for the decomposition of tylosin, observing a lower energy expenditure, and reaction time spent catalyst was one which was used 90 minutes reaction time, pH 4, 180 ° C temperature and phosphoric acid as a catalyst. In the germination test with Allium cepa, the solutions treated, except ampicillin, had similar germination rates found in the control group. The solutions of antibiotics with no treatment showed higher rates of growth of buds when compared to the control group. These results show that the hydrothermal carbonization process has potential for future applications decomposition of antibiotics in contaminated water and effluent. / Antibióticos são largamente utilizados na medicina humana e veterinária. Após o consumo, grande parte dos fármacos é excretada inalterada ou sob forma de metabólitos, e por intermédio dos lançamentos de estações de tratamento de esgotos, resíduos agrícolas e lixiviação, alcançam os ecossistemas naturais. A ocorrência de antibióticos nos ambientes aquáticos tem se tornado uma preocupação crescente em todo o mundo especialmente devido ao aparecimento de genes de resistência em bactérias patogênicas. O uso indiscriminado e a utilização de doses subterapêuticas de antibióticos provocam o surgimento de resistência. Em Goiás, uma pesquisa recente demonstrou a presença de bactérias resistentes a vários antibióticos em efluentes hospitalares e na estação de tratamento de esgotos de Goiânia. Os processos de tratamento de efluentes baseados na degradação biológica apresentam baixa eficiência de remoção de fármacos residuais devido à estrutura recalcitrante e ao potencial biocida dos antibióticos. O objetivo desta pesquisa foi avaliar a eficiência da decomposição hidrotermal dos antibióticos ampicilina, enrofloxacina, oxitetraciclina, sulfaquinoxalina e tilosina. Com este intuito, soluções dos antibióticos foram preparadas com concentração de 50 μg.mL-1 e com correção de pH para a faixa 3-7, com solução de ácido fosfórico, para o tratamento com carbonização hidrotermal. O tratamento pelo processo de carbonização hidrotermal da oxitetraciclina com correção de pH feita com ácido fosfórico, pH 3, temperatura de 170ºC e tempo de reação de 90 minutos apresentou como resultado a decomposição total deste antibiótico (100%). A maior taxa de decomposição (98,37%) para a enrofloxacina foi observada quando foram utilizados tempo reacional de 120 minutos, pH 3, temperatura de 200ºC e ácido fosfórico como catalisador. A maior taxa de decomposição da ampicilina (94,86%) foi observada quando se utilizou tempo de reação de 120 minutos, pH 4, temperatura de 200ºC e ácido fosfórico como catalisador. As condições mais adequadas para a decomposição da sulfaquinoxalina, observando a maior taxa de redução (95,71%), foi observada quando foram utilizados tempo reacional de 120 minutos, pH 5, temperatura de 180ºC e ácido fosfórico como catalisador. Todas as condições, exceto o pH 7, levaram a decomposição total da tilosina (100%) das amostras. A melhor condição para a decomposição da tilosina, observando menor gasto energético, tempo reacional e gasto de catalisador, foi aquela onde se utilizou tempo de reação de 90 minutos, pH 4, temperatura de 180ºC e ácido fosfórico como catalisador. No teste de germinação com Allium cepa, as soluções tratadas, exceto a ampicilina, apresentaram taxas similares de germinação as encontradas no Grupo Controle. As soluções dos antibióticos sem tratamento apresentaram taxas superiores de crescimentos dos brotos quando comparadas ao Grupo Controle. Esses resultados mostram que o processo de carbonização hidrotermal apresenta potencial para futuras aplicações de decomposição de antibióticos em águas e efluentes contaminados.

Antibiotics and antibiotic resistance genes from animal manure applied to soil as fertilizer are now among the most concerned contaminants in soil. The widespread use of antibiotics in livestock might amplify the risk of developing antibiotic resistance, causing once treatable diseases to turn deadly. The World Health Organization declared antibiotic resistance as "one of the biggest threats to global health, food security, and development". The goal of this dissertation was to develop best manure management practices by understanding the behavior of manure-associated antibiotics in manure, water, and soil. In particular, my research focused on the effects of manure application methods, on-site manure treatment methods, manure application seasons, and manure-rainfall time gaps on antibiotic surface runoff losses, antibiotic distribution and movement in soil, antibiotic dissipation in soil, and development of antibiotic resistance. Rainfall simulation field-scale and soil incubation lab studies were combined to find the best manure management practices. My research has shown for the first time that using the manure soil subsurface injection method, especially during spring application season due to moist soil, applying manure at least 3 days before a subsequent rainfall, and using composted manure, can significantly reduce the quantity of antibiotic loss with runoff from manure-applied fields to the surrounding environment. The majority of applied antibiotics remained in soil. All antibiotics showed a similar dissipation pattern with fastest kinetics during the first 14 d before slowing down. The effect of two manure application methods on antibiotic dissipation kinetics varied with different antibiotics. Although the half-life of tested antibiotics in soil was short (<21 days), some remained detectable even at 6 months after a single manure application. Results also showed that compared to the surface application, the subsurface injection slits acted as a "hot zone" with a higher amount of antibiotics, manure microbes, and antibiotic resistance. The results provide information for policy makers, manure managers, and farmers to develop better manure management practices that can use manure as fertilizer while minimizing the spread of antibiotics to surrounding water, soil, and plants. / Doctor of Philosophy / There is growing concern about antibiotic resistance as a serious human health threat because a resistant infection may kill, can spread, and increases health costs. Every year in the United States, there are 2 million people infected with antibiotic resistant bacteria, 23,000 people die as a direct result of these infections, and $55 billion is lost due to increased hospital stay and lost work days. Although bacteria naturally develop the ability to resist antibiotics, the problem is the length between antibiotic introduction and resistance development is shortening because of the widespread and overuse of antibiotics, especially in the livestock industry. The goal of this study was to develop the best manure management practices balancing the benefits of antibiotics in livestock and animal manure and their impact on the environment. In particular, we monitored, using field-scale and laboratory studies, the effects of manure application methods, on-site manure treatment methods, manure application seasons, and manure-rainfall time gaps on antibiotic loss through surface runoff, antibiotic distribution and movement in soil, antibiotic dissipation in soil, and development of antibiotic resistance. In order to reduce the amount of antibiotic loss with surface runoff from manure-applied fields to the surrounding environment, farmers are recommended to 1) compost manure before application, 2) watch the forecast to apply manure at least 3 days before a subsequent rainfall, and 3) use the subsurface injection method, especially when the soil is wet (spring season). The majority of applied antibiotics remained in soil. All tested antibiotics showed a similar dissipation pattern with the fastest rate during the first two weeks after manure application, then slowing down. Although the half-life of tested antibiotics in soil was short (<21 days), some remained detectable even at 6 months after a single manure application. Besides, the subsurface injection slits acted as a hot zone with a concentrated amount of antibiotics, manure microbes, and antibiotic resistance. The results provide recommendations for policy makers, manure managers, and farmers to maximize benefits of manure as fertilizer while minimizing the spread of manure-associated antibiotics to surrounding water, soil, and plants.

Earth's near-surface environment sustains nearly all terrestrial life, yet this critical zone is threatened by the environmental migration of new and potentially harmful compounds produced to support a growing human population. Traditional transport equations often fail to capture the environmental behavior of these emerging contaminants due to issues such as flow heterogeneity. Thus, there is a need to better evaluate controls on pollutant partitioning in Earth's critical zone. Our first study investigated the transport and distribution of the neonicotinoid insecticide thiamethoxam (TMX) by growing TMX-coated corn seeds in coarse vs fine-textured soil columns maintained with versus without growing corn plants. Fine-textured soil transported TMX at concentrations that were two orders of magnitude higher than coarse-textured soil, due to preferential flow in the fine-textured soil columns and higher evapotranspiration (ET) concentrating more TMX in the coarse-textured soil. Living plants increased the concentration of TMX at depth, indicating that growing plants may drive preferential transport of neonicotinoids. For the second study we planted TMX-coated corn seeds and maintained field plots with and without viable crops (n = 3 plots per treatment), measuring TMX concentrations in three hydrological compartments (surface runoff, shallow lateral flow, and deep drainage) and soil. TMX was transported in the highest concentrations via surface runoff, while also showing continual migration within the subsurface throughout the growing season. Plants facilitated downward migration of TMX in soil yet restricted losses in drainage. For our final study, we used a simple isotope mixing method to evaluate how preferential flow alters the influence of compound chemical properties on solute transport. We applied deuterium-labeled rainfall to plots containing manure spiked with eight veterinary antibiotics with a range of mobility, and quantified transport to suction lysimeters (30 and 90 cm). We showed that low preferential flow (<20%) eliminates the influence of compound chemical properties and, contrary to conventional understanding, more preferential flow (~ >20%) amplifies these chemical controls, with more mobile compounds appearing in significantly higher concentrations than less mobiles ones. Altogether, we provide a refined understanding of solute partitioning in the critical zone necessary to improve process-based transport modeling. / Doctor of Philosophy / Earth’s near-surface environment sustains nearly all terrestrial life, yet this critical zone is threatened by the environmental migration of new and potentially harmful pollutants produced to support a growing human population. Additionally, traditional mathematical methods fail to accurately describe the behavior of these emerging pollutants in soils due to complex flow patterns. Thus, scientists need to better understand how these pollutants contaminate water bodies in the critical zone. We first conducted a greenhouse experiment to understand and measure the amount of the neonicotinoid insecticide thiamethoxam (TMX) that could move from coated corn seeds through the soil environment. Water draining from fine-textured soil had >100 times more TMX than water draining from course-textured soil, due to commonly occurring fractures/cracks in the finer-particle soil and more evaporation from soil and plant leaves sequestering TMX in the sandy soil. Growing plants amplified TMX movement through soil voids to lower depths. We then conducted a field study to determine how much TMX could move to the surrounding environment throughout the corn growing season. We found that plants aided in downward movement of TMX yet restricted total losses from the plot overall by removing soil water. Our third study investigated the degree to which chemical pollutant properties control movement of solutes when water flows preferentially through soil void space. Common dairy manure was spiked with eight pollutants ranging in chemical attraction to soil and was added to an agricultural field. After irrigation, we found that when total drainage water was less than 20% derived from preferential flow, chemical properties had a negligible effect on the amount of pollutant in draining soil water. Contrary to conventional understanding, when draining water contained more than 20% preferential flow, chemical properties had a strong influence on the amount of pollutant detected. Altogether, we provide new understanding of how solutes move though the critical zone. These findings are necessary to create mathematical tools that more accurately depict pollutant behavior below-ground.

Master of Science / Department of Civil Engineering / Alok Bhandari / In the upper Midwest, subsurface tile drainage water is a major contributor of nitrate (NO[subscript]3–N) coming from fertilizers and animal manure. Movement of NO[subscript]3-N through tile drainage into streams is a major concern as it can cause eutrophication and hypoxia conditions, as in the Gulf of Mexico. Denitrifying bioreactors is one of the pollution control strategies to treat contaminated tile drainage water. These bioreactors require four conditions which are: 1) organic carbon source, 2) anaerobic conditions, 3) denitrifying bacteria and 4) influent NO[subscript]3-N. This research focuses on investigating fate of veterinary antibiotics in woodchips commonly used in in-situ reactors. Tylosin (TYL) and sulfamethazine (SMZ) are two veterinary antibiotics which are most commonly used in the United States and can be found in tile water after manure is land applied. Partition coefficients of TYL and SMZ on wood were determined by sorption experiments using fresh woodchips and woodchips from an in situ reactor. It was concluded that the woodchips were an effective means to sorb the veterinary antibiotics leached into the tile water after application of animal manure. Linear partition coefficients were calculated and phase distribution relationships were established for both the chemicals. The fresh woodchips gave inconclusive data but predictions could be made by the information determined in the experiments using woodchips from a ten year old woodchip bioreactor. Desorption was also studied and the likelihood of desorption was predicted using the Apparent Hysteresis Index. Overall, it was found that the old woodchips allowed for quick sorption of both antibiotics. It was also found that SMZ had reversible sorption on old woodchips. Thus, it was concluded that the woodchip bioreactor would not be effective for removal of veterinary antibiotics from tile drainage. More research is required for the fate of TYL and to confirm the conclusion.

Muitos antibióticos de uso veterinário não são totalmente metabolizados no organismo animal, ocasionando a presença de resíduos destes compostos em várias matrizes ambientais, tais como estercos animais, solos e sedimentos fluviais. A ocorrência destes resíduos no ambiente pode favorecer a resistência de microrganismos aos agentes antibióticos, ocasionar problemas de ordem toxicológica em determinados organismos e, além disso, interferir nos ciclos biogeoquímicos mediados por microrganismos. Apesar do uso de antibióticos de uso veterinário ser elevado no Brasil, até o momento não existem dados quanto à ocorrência e comportamento ambiental dos principais compostos atualmente empregados no país. Neste sentido, este trabalho investigou a ocorrência em amostras ambientais (cama de frango e solo) de uma importante classe de antibióticos, as fluoroquinolonas, além de aspectos do comportamento de fluoroquinolonas e sulfonamidas através de ensaios de sorção. No exterior, estudou-se a dissipação e a sorção de fluoroquinolonas em solos neozelandeses. Para a quantificação de resíduos de fluoroquinolonas em amostras de cama de frango e solo, a metodologia analítica foi adaptada e validada. No geral, os resíduos de fluoroquinolonas ocorreram em concentrações equiparáveis (mg kg-1 para cama de frango e g kg-1 para solo) aos encontrados em outros países (China, Áustria e Turquia) e os seus coeficientes de sorção às partículas do solo foram sempre elevados (Kd = 544 a 1.277.874 L kg-1), maiores do que aqueles encontrados internacionalmente (Kd = 260 to 5.012 L kg-1), e bastante superiores aos encontrados na cama de frango (Kd 65 L kg-1), o que demonstra que o transporte destas moléculas ocorre somente em associação às partículas do solo, podendo atingir fontes de águas superficiais. Já os coeficientes de sorção das sulfonamidas foram relativamente baixos (Kd = 0,7 a 70,1 L kg-1), mostrando seu potencial de lixiviação no perfil dos solos. A textura e a capacidade de troca catiônica influenciaram significativamente o potencial de sorção das fluoroquinolonas e das sulfonamidas aos solos, evidenciando a importância dos processos de troca iônica na sorção desses antibióticos em solos tropicais. Nos solos neozelandeses, com maior capacidade tampão, o potencial de sorção das fluoroquinolonas também foi elevado, mas menor que em solos brasileiros, sendo que a adição de cama de frango diminuiu o seu potencial de sorção. A dissipação das fluoroquinolonas variou consideravelmente (DT50 = 16 a >70 dias), tendo sido mais rápida em sub-superfície apesar da sua menor atividade biológica. Antibióticos são usados rotineiramente na produção animal, representando uma fonte potencial de poluição ao meio ambiente que tem sido largamente ignorada, devendo ser melhor investigada no Brasil / Many antibiotics used for veterinary purposes use are not fully metabolized in the animal body, resulting in the presence of residues in a wide range of environmental matrices such as animal manures, soils and sediments. The occurrence of these residues in the environment can favour microorganisms resistance to antibiotic agents, cause toxicological problems to certain organisms and, besides, also interfere in the biogeochemical cycles mediated by microorganisms. Despite the high use of veterinary antibiotics in Brazil, so far there are no data regarding the occurrence and environmental behavior of the main compounds currently in use in the country. This work represented a first investigation on the occurrence of an important class of antibiotics, the fluoroquinolones, in environmental samples (poultry litter and soil), also investigating aspects of the environmental behavior of fluoroquinolones and sulfonamides in these matrices through sorption experiments. Overseas, dissipation and sorption behavior of fluoroquinolones were studied for New Zealand soils. For the quantification of fluoroquinolone residues in poultry litter and soil samples, an analytical methodology was adapted and validated. Overall, the results demonstrated the occurrence of fluoroquinolone residues in concentrations (mg kg-1 for poultry litter and g kg-1 for soil) comparable to those found elsewhere (China, Austria and Turkey), a high sorption potential of fluoroquinolones to soils (Kd from 544 to 1,277,874 L kg-1), higher than the reference values for soil (Kd = 260 to 5,012 L kg-1), and much higher than for poultry litter (Kd 65 L kg-1), showing that the transport of these molecules will only occur in association with the soil particles, possibly reaching superficial water sources. In the case of sulfonamides, sorption coefficients were relatively low (Kd = 0.7 to 70.1 L kg-1), showing their leaching potential in the soil profile. Texture and cation exchange capacity greatly affected the sorption of sulfonamides and fluoroquinolones to soils, emphasizing the importance of ion exchange processes on the sorption of antibiotics in tropical soils. In New Zealand soils, with a higher buffer capacity, sorption of fluoroquinolones was also high, but lower than to Brazilian soils, and the addition of poultry litter to soil reduced the sorption potential of these compounds. Fluoroquinolones dissipation varied considerably ((DT50 = 16 to >70 days), being faster in sub-surface samples despite its lower biological activity. Antibiotics are routinely used in animal production, representing a potential source of pollution to the environment that has been largely ignored and should be further investigated in Brazil

Muitos antibióticos de uso veterinário não são totalmente metabolizados no organismo animal, ocasionando a presença de resíduos destes compostos em várias matrizes ambientais, tais como estercos animais, solos e sedimentos fluviais. A ocorrência destes resíduos no ambiente pode favorecer a resistência de microrganismos aos agentes antibióticos, ocasionar problemas de ordem toxicológica em determinados organismos e, além disso, interferir nos ciclos biogeoquímicos mediados por microrganismos. Apesar do uso de antibióticos de uso veterinário ser elevado no Brasil, até o momento não existem dados quanto à ocorrência e comportamento ambiental dos principais compostos atualmente empregados no país. Neste sentido, este trabalho investigou a ocorrência em amostras ambientais (cama de frango e solo) de uma importante classe de antibióticos, as fluoroquinolonas, além de aspectos do comportamento de fluoroquinolonas e sulfonamidas através de ensaios de sorção. No exterior, estudou-se a dissipação e a sorção de fluoroquinolonas em solos neozelandeses. Para a quantificação de resíduos de fluoroquinolonas em amostras de cama de frango e solo, a metodologia analítica foi adaptada e validada. No geral, os resíduos de fluoroquinolonas ocorreram em concentrações equiparáveis (mg kg-1 para cama de frango e g kg-1 para solo) aos encontrados em outros países (China, Áustria e Turquia) e os seus coeficientes de sorção às partículas do solo foram sempre elevados (Kd = 544 a 1.277.874 L kg-1), maiores do que aqueles encontrados internacionalmente (Kd = 260 to 5.012 L kg-1), e bastante superiores aos encontrados na cama de frango (Kd 65 L kg-1), o que demonstra que o transporte destas moléculas ocorre somente em associação às partículas do solo, podendo atingir fontes de águas superficiais. Já os coeficientes de sorção das sulfonamidas foram relativamente baixos (Kd = 0,7 a 70,1 L kg-1), mostrando seu potencial de lixiviação no perfil dos solos. A textura e a capacidade de troca catiônica influenciaram significativamente o potencial de sorção das fluoroquinolonas e das sulfonamidas aos solos, evidenciando a importância dos processos de troca iônica na sorção desses antibióticos em solos tropicais. Nos solos neozelandeses, com maior capacidade tampão, o potencial de sorção das fluoroquinolonas também foi elevado, mas menor que em solos brasileiros, sendo que a adição de cama de frango diminuiu o seu potencial de sorção. A dissipação das fluoroquinolonas variou consideravelmente (DT50 = 16 a >70 dias), tendo sido mais rápida em sub-superfície apesar da sua menor atividade biológica. Antibióticos são usados rotineiramente na produção animal, representando uma fonte potencial de poluição ao meio ambiente que tem sido largamente ignorada, devendo ser melhor investigada no Brasil / Many antibiotics used for veterinary purposes use are not fully metabolized in the animal body, resulting in the presence of residues in a wide range of environmental matrices such as animal manures, soils and sediments. The occurrence of these residues in the environment can favour microorganisms resistance to antibiotic agents, cause toxicological problems to certain organisms and, besides, also interfere in the biogeochemical cycles mediated by microorganisms. Despite the high use of veterinary antibiotics in Brazil, so far there are no data regarding the occurrence and environmental behavior of the main compounds currently in use in the country. This work represented a first investigation on the occurrence of an important class of antibiotics, the fluoroquinolones, in environmental samples (poultry litter and soil), also investigating aspects of the environmental behavior of fluoroquinolones and sulfonamides in these matrices through sorption experiments. Overseas, dissipation and sorption behavior of fluoroquinolones were studied for New Zealand soils. For the quantification of fluoroquinolone residues in poultry litter and soil samples, an analytical methodology was adapted and validated. Overall, the results demonstrated the occurrence of fluoroquinolone residues in concentrations (mg kg-1 for poultry litter and g kg-1 for soil) comparable to those found elsewhere (China, Austria and Turkey), a high sorption potential of fluoroquinolones to soils (Kd from 544 to 1,277,874 L kg-1), higher than the reference values for soil (Kd = 260 to 5,012 L kg-1), and much higher than for poultry litter (Kd 65 L kg-1), showing that the transport of these molecules will only occur in association with the soil particles, possibly reaching superficial water sources. In the case of sulfonamides, sorption coefficients were relatively low (Kd = 0.7 to 70.1 L kg-1), showing their leaching potential in the soil profile. Texture and cation exchange capacity greatly affected the sorption of sulfonamides and fluoroquinolones to soils, emphasizing the importance of ion exchange processes on the sorption of antibiotics in tropical soils. In New Zealand soils, with a higher buffer capacity, sorption of fluoroquinolones was also high, but lower than to Brazilian soils, and the addition of poultry litter to soil reduced the sorption potential of these compounds. Fluoroquinolones dissipation varied considerably ((DT50 = 16 to >70 days), being faster in sub-surface samples despite its lower biological activity. Antibiotics are routinely used in animal production, representing a potential source of pollution to the environment that has been largely ignored and should be further investigated in Brazil

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Rising global antibiotic resistance has caused concerns over sources and pathways for the spread of contributing factors. Majority of the antimicrobials used in the U.S. are involved in veterinary medicine, primarily with livestock rearing. Animal manure land application integrates livestock farming and agroecosystems. This manure contains antibiotic resistant elements (AREs) (resistant bacteria, resistance genes, and veterinary antibiotics) that contribute towards antimicrobial resistance. Altering manure application techniques can reduce surface runoff of other contaminants such as excess N and P, pesticides, and hormones, that can impact water quality. Conventional tillage practices in the U.S. has reduced or stopped, making subsurface injection of manure a promising option when compared to surface application. Our research compared manure application methods, manure application seasons, cropping system, and manure-rainfall time gaps to gauge the impact on AREs in the environment. Two field-scale rainfall simulation studies were conducted along with one laboratory study. Using the injection method lowered concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, 9-30 times less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to 1 d time gap for that broadcast method. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other based on manure application and soil ARG richness in all manure-amended soil increased compared to the background. Runoff from injection plots contained 52 ARGs with higher abundance compared to runoff from surface applied plots. ARGs in the former were more correlated to soil and more correlated to manure in the latter. The highest antibiotic concentrations were in the injection slit soil of those plots. Antibiotic concentrations in samples corresponded positively to concentrations of resistant FCB and ARGs, and there was a positive correlation between resistant FCB and their associated ARGs (Spearman's ρ = 0.43-0.63). A CRIISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for in-situ detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil. / Doctor of Philosophy / Rising global antibiotic resistance cause concerns over sources and pathways for the spread of contributing factors. Most of the antimicrobials used in the U.S. are involved in veterinary medicine, especially with livestock rearing. Overuse of antibiotics that are medically important to human medicine compromises the effectiveness of our medicines. Animal manure contains antibiotic resistant elements (AREs) such as resistant bacteria, resistance genes, and antibiotics) that contribute towards resistance issues. Once these AREs enter the environment, they can be taken up by crops, runoff into surface water or leached into ground water, or even reside within the animal products we consume. Altering manure application techniques is beneficial for nutrient conservation but also potentially for reducing ARE spread. With our research, we compared manure application methods, manure application seasons, cropping systems, and manure-rainfall time gaps to find ways to balance the need for manure application and the spread of resistance. We used two field-scale rainfall simulation studies along with one laboratory study. Overall, using the injection method resulted in significantly lower concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to the 1 d time gap for broadcast methods. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other and soil ARG totals in all manure applied soil increased compared to the background. Runoff from injection plots contained more soil ARGs and runoff from surface applied plots containing more manure associated ARGs. The subsurface injection method also caused highest antibiotic concentrations in the injection slit soil of those plots. High antibiotic concentrations in samples generally meant high concentrations of resistant FCB and ARGs, and resistant FCB were also found with their associated ARGs as well. A CRISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for onsite detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil.

L’élevage des porcs représente une source importante de déversement d’antibiotiques dans l’environnement par l’intermédiaire de l’épandage du lisier qui contient une grande quantité de ces molécules sur les champs agricoles. Il a été prouvé que ces molécules biologiquement actives peuvent avoir un impact toxique sur l’écosystème. Par ailleurs, elles sont aussi suspectées d’engendrer des problèmes sanitaires et de contribuer à la résistance bactérienne pouvant mener à des infections difficilement traitables chez les humains. Le contrôle de ces substances dans l’environnement est donc nécessaire. De nombreuses méthodes analytiques sont proposées dans la littérature scientifique pour recenser ces composés dans plusieurs types de matrice. Cependant, peu de ces méthodes permettent l’analyse de ces contaminants dans des matrices issues de l’élevage agricole intensif. Par ailleurs, les méthodes analytiques disponibles sont souvent sujettes à des faux positifs compte tenu de la complexité des matrices étudiées et du matériel utilisé et ne prennent souvent pas en compte les métabolites et produits de dégradation. Enfin, les niveaux d’analyse atteints avec ces méthodes ne sont parfois plus à jour étant donné l’évolution de la chimie analytique et de la spectrométrie de masse. Dans cette optique, de nouvelles méthodes d’analyses ont été développées pour rechercher et quantifier les antibiotiques dans des matrices dérivées de l’élevage intensif des porcs en essayant de proposer des approches alternatives sensibles, sélectives et robustes pour quantifier ces molécules. Une première méthode d’analyse basée sur une technique d’introduction d’échantillon alternative à l’aide d’une interface fonctionnant à l’aide d’une désorption thermique par diode laser munie d’une source à ionisation à pression atmosphérique, couplée à la spectrométrie de masse en tandem a été développée. L’objectif est de proposer une analyse plus rapide tout en atteignant des niveaux de concentration adaptés à la matrice étudiée. Cette technique d’analyse couplée à un traitement d’échantillon efficace a permis l’analyse de plusieurs antibiotiques vétérinaires de différentes classes dans des échantillons de lisier avec des temps d’analyse courts. Les limites de détection atteintes sont comprises entre 2,5 et 8,3 µg kg-1 et sont comparables avec celles pouvant être obtenues avec la chromatographie liquide dans une matrice similaire. En vue d’analyser simultanément une série de tétracyclines, une deuxième méthode d’analyse utilisant la chromatographie liquide couplée à la spectrométrie de masse à haute résolution (HRMS) a été proposée. L’utilisation de la HRMS a été motivée par le fait que cette technique d’analyse est moins sensible aux faux positifs que le triple quadripôle traditionnel. Des limites de détection comprises entre 1,5 et 3,6 µg kg-1 ont été atteintes dans des échantillons de lisier en utilisant un mode d’analyse par fragmentation. L’utilisation de méthodes de quantifications ciblées est une démarche intéressante lorsque la présence de contaminants est suspectée dans un échantillon. Toutefois, les contaminants non intégrés à cette méthode d’analyse ciblée ne peuvent être détectés même à de fortes concentrations. Dans ce contexte, une méthode d’analyse non ciblée a été développée pour la recherche de pharmaceutiques vétérinaires dans des effluents agricoles en utilisant la spectrométrie de masse à haute résolution et une cartouche SPE polymérique polyvalente. Cette méthode a permis l’identification d’antibiotiques et de pharmaceutiques couramment utilisés dans l’élevage porcin. La plupart des méthodes d’analyse disponibles dans la littérature se concentrent sur l’analyse des composés parents, mais pas sur les sous-produits de dégradation. L’approche utilisée dans la deuxième méthode d’analyse a donc été étendue et appliquée à d’autres classes d’antibiotiques pour mesurer les concentrations de plusieurs résidus d’antibiotiques dans les sols et les eaux de drainage d’un champ agricole expérimental. Les sols du champ renfermaient un mélange d’antibiotiques ainsi que leurs produits de dégradation relatifs à des concentrations mesurées jusqu’à 1020 µg kg-1. Une partie de ces composés ont voyagé par l’intermédiaire des eaux de drainage du champ ou des concentrations pouvant atteindre 3200 ng L-1 ont pu être relevées. / Swine production is a major source of antibiotic release into the environment via manure spreading on agricultural fields. It has been shown that these biologically active compounds may have a toxic impact on ecosystems. Moreover, they are also suspected to cause health problems and contribute to bacterial resistance that could lead to difficult-to-treat infections in humans. Therefore, control of these substances in the environment is necessary. Several analytical methods are proposed in the scientific literature to identify these compounds in various matrices. However, few of these methods allowed the analysis of these contaminants in matrices derived from intensive livestock farming. Furthermore, the analytical methods available are often subject to false positives, given the complexity samples and the equipment used and do not take into account the metabolites and degradation products. Finally, concentration levels reached with these methods are sometimes outdated since the evolution of analytical chemistry and mass spectrometry. In this context, new analytical methods have been developed to investigate and quantify the antibiotics derived from swine husbandry to propose alternative, sensible, selective and robust approaches to quantify these molecules. A first analytical method has been proposed based on an alternative sample introduction technique using the laser diode thermal desorption interface with an atmospheric pressure chemical ionization source coupled to tandem mass spectrometry. The objective was to provide a simpler and faster analysis while reaching levels suitable for the studied matrix. This alternative sample introduction method coupled with an efficient sample processing allowed the analysis of several classes of veterinary antibiotics in swine manure in a short analysis time. Detection limits ranged between 2.5 and 8.3 µg kg-1 and are comparable with those obtained with liquid chromatography in a similar matrix. In order to simultaneously analyze a series of tetracyclines, a second analytical method using liquid chromatography coupled to high-resolution mass spectrometry (HRMS) was proposed. The use of HRMS was motivated by the fact that this mass spectrometer is less sensitive to false positive that the traditional triple quadrupole given the complexity of the studied matrix. Detection limits between 1.5 and 3.6 µg kg-1 have been achieved in swine manure using a fragmentation analysis mode to avoid false positives. Targeted screening methods are interesting approaches when contaminants are suspected to be present in a sample. However, a non-included contaminant in this targeted analysis method could not been detected even at a high concentration. In this context, a non-target compound screening method focused on veterinary pharmaceutical compounds in swine manure was developed using liquid chromatography coupled to high-resolution mass spectrometry. A polymeric SPE cartridge was used to collect polar compounds including pharmaceuticals prior analysis. This method allowed the identification of antibiotics and pharmaceuticals of commonly used in swine farming. Most of the analytical methods available in the literature focus on parent compounds, regardless degradation products. The approach used in the second method of analysis was applied and extended to other classes of antibiotics to measure concentrations of several antibiotic residues in soils and drainage waters of an experimental agricultural field. Field soil contained a mixture of antibiotics and their related degradation products with concentrations measured up to 1020 µg kg-1. Some of these compounds have migrated through the field via drainage waters wherein concentrations up to 3200 ng L-1 were observed.