Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Currently, 74% of cotton acres in the United States are planted with glyphosatetolerant varieties. The average glyphosate-tolerant cotton crop is treated with glyphosate 2.1 times each year in addition to other herbicides, insecticides, and fungicides. The primary objectives of this research were to: 1) describe the influence of glyphosate and pesticides commonly applied at or near the time of cotton planting on soil microbial activity and biomass; 2) study the effect of glyphosate on fluometuron degradation; 3) evaluate the response of Rhizoctonia solani to glyphosate and fluometuron; 4) study changes in glyphosate metabolism that occur as a result of repeated glyphosate applications; and 5) define shifts in the soil microbial community. Additionally, methods for accelerated solvent extraction (ASE) of fluometuron from soils were developed. In one experiment, the addition of glyphosate reduced C-mineralization in soils treated with fluometuron, aldicarb, or mefenoxam + PCNB formulations. However, in a second experiment, C-mineralization increased when glyphosate was applied with fluometuron relative to fluometuron applied alone. Accelerated solvent extraction was used in experiments which demonstrated that application of glyphosate with fluometuron increased the rate of fluometuron degradation in soil relative to fluometuron alone. When glyphosate was added to minimal medium, degradation of fluometuron by R. solani was reduced and less fungal biomass was produced. The total amount of 14C-glyphosate mineralized was reduced when glyphosate was applied 5 times relative to 1, 2, 3, or 4 times. Incorporation of 14Cglyphosate residues into soil microbial biomass was greater following five glyphosate applications than one application 3 and 7 days after application (DAA). Soil fatty acid methyl ester (FAME) profiles were altered by five glyphosate applications relative to one application. Additionally, FAMEs common to gram-negative bacteria were present in higher concentrations following five applications relative to 1, 2, 3, or 4 applications both 7 and 14 DAA. These studies indicated that: 1) glyphosate altered the soil microbial response to other pesticides; 2) fluometuron-degrading microorganisms in soil responded differently to glyphosate; 3) changes in the dissipation or distribution of glyphosate following repeated glyphosate applications were associated with changes in the structural diversity of the soil microbial community.

O consumo de drogas de abuso é grave problema de saúde pública em todo o mundo. No Brasil observa-se aumento no número de usuárias em idade fértil, levando a crescente preocupação com relação à exposição fetal. Efeitos deletérios como diminuição do peso e crescimento fetal, parto prematuro, déficits neurológicos e comportamentais estão associados ao uso de cocaína e cannabis durante a gestação. Portanto, é importante obter informações acerca do consumo de drogas durante a gravidez, permitindo intervenções médicas e psicológicas adequadas. Os relatos fornecidos pelas gestantes são relevantes, porém muitas vezes resultam em dados subestimados devido à omissão de informações por medo de ações punitivas. Desta forma, a confirmação por meio de análises toxicológicas em amostras biológicas se faz necessária. O mecônio, primeiras fezes do recém-nascido, tem sido proposto como matriz adequada para avaliação da exposição fetal, pois apresenta coleta não invasiva, fácil obtenção e fornece informações de longo prazo (2º e 3º trimestre de gestação). Entretanto, é uma matriz complexa, exigindo diversas etapas de purificação para posterior análise. No presente trabalho, métodos analíticos foram desenvolvidos visando à detecção dos biomarcadores da exposição fetal à cocaína e ao tetraidrocanabinol em amostras de mecônio por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Para ambos os métodos, a extração acelerada por solvente (ASE) foi utilizada para o isolamento dos analitos de interesse de amostras de mecônio, já que esta apresenta vantagens frente às técnicas convencionais, devido sua maior eficiência e menor manipulação da amostra. Associada à ASE, a extração em fase sólida (SPE) foi empregada para purificação e concentração dos analitos de interesse. Na etapa de desenvolvimento, inicialmente foram estabelecidos os procedimentos de derivatização e as condições cromatográficas a serem empregadas nas análises. Posteriormente, realizou-se a otimização dos procedimentos empregados na ASE, através de análise de superfície de resposta. Os métodos foram validados de acordo com o preconizado por referências internacionais, estabelecendo-se os limites de detecção e quantificação, recuperação, linearidade, precisão intra e interensaio e exatidão. O método para detecção dos biomarcadores da cocaína foi aplicado em 342 amostras de mecônio, provenientes do Hospital Universitário da Universidade de São Paulo (HU-USP). Destas, 19 (5,6%) apresentaram resultado positivo para um ou mais biomarcadores da exposição fetal à cocaína. Além disto, foi observada redução estatisticamente significante do peso ao nascimento, comprimento e perímetro cefálico entre os recém-nascidos expostos à cocaína durante a gestação. O método para a detecção da exposição fetal ao tetraidrocanabinol foi aplicado em 6 amostras positivas, obtidas do HU-USP, mostrando a aplicabilidade da técnica desenvolvida. / The use of illicit drugs is a relevant public health problem in the world. In Brazil, the number of women users in fertile age is increasing, which leads to a growing concern regarding fetal drug exposure. Adverse outcomes such as low birth weight, intra-uterine growth restriction, preterm birth, neurobehavioral and developmental deficits are associated with cocaine and cannabis use during pregnancy. Consequently, it would be important to obtain data related to drug misuse during gestation with the aim to plan medical and psychological interventions. Self-report drug use by pregnant women is often inaccurate due to feelings of guilt or fear of punitive actions. Therefore, confirmation by toxicological analysis in biological matrices must be accomplished. Meconium, the first stool of the newborn, has been proposed as a proper matrix to evaluate fetal exposure because it is collected by an easy and non-invasive way and enables the achievement of long-term information regarding fetal exposure. However, meconium is a complex matrix, which requires extensive sample cleaning previously to the analytes identification. In the present research, analytical methods were developed aiming the determination of cocaine and tetrahydrocannabinol biomarkers in meconium samples through gas chromatography-mass spectrometry (GC-MS). Accelerated solvent extraction (ASE) was used for analytes isolation due to its advantages over conventional techniques, such as greater extraction efficiency and minor sample handling. In order to achieve proper analytes selectivity and detectability, solid phase extraction (SPE) was employed for post-extraction clean-up. Initially, the derivatization procedure and chromatographic parameters were established for the development of the methods. Afterwards, ASE procedure was optimized through response surface methodology. The analytical methods were validated in accordance to international references. Limits of detection and quantification, recovery, linearity, precision and accuracy were obtained. The developed method for cocaine was applied in 342 meconium samples collected from the University Hospital of University of São Paulo (HU-USP). Among them, 19 (5.6%) showed positivity result for cocaine biomarkers. Additionally, newborns from mothers exposed to cocaine exhibited statistical significant lower birth weight, length and head circumference when compared with newborns from non-consumer mothers. For the method of tetrahydrocannabinol, applicability and importance was demonstrated by analyzing 6 positive samples from HU-USP.

O tabaco é uma das principais drogas consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, têm representado uma grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de fumar durante a gestação serem bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. A exposição fetal aos constituintes do tabaco tem sido associada com aumento do risco de aborto espontâneo e nascimentos prematuros, incidência de recém-nascidos com baixo peso, síndrome de morte súbita infantil e desordens cognitivas e neurocomportamentais. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado tabaco durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal aos constituintes do tabaco. Dentre os marcadores utilizados estão a nicotina e o seu principal produto de biotransformação, a cotinina, que podem ser detectados em amostras de mecônio de recém-nascidos. No presente estudo, um método analítico foi desenvolvido visando à detecção desses marcadores em amostras de mecônio. Foi considerada neste projeto a técnica de extração acelerada por solvente (ASE) por ser uma técnica promissora para o preparo de amostras sólidas e/ou semi-sólidas. Os analitos foram identificados por cromatografia em fase-gasosa com detector de nitrogênio e fósforo (GC/NPD). Os limites de detecção foram de 3 ng/g e 30 ng/g e os de quantificação foram de 5 ng/g e 40 ng/g, para cotinina e nicotina respectivamente, e apresentaram boa linearidade na faixa de concentração estudada (5-500 ng/g), com coeficiente de correlação (r2) maior que 0,98. A precisão intra-ensaio, determinada pelo coeficiente de variação do método (CV%) foi menor que 15% e a precisão interensaio foram menor ou igual a 20% para nicotina e cotinina. A recuperação média foi de 77%. O método desenvolvido demonstrou ser rápido, preciso, prático e sensível e com uso de menores volumes de solventes orgânicos do que outros métodos descritos na literatura. O método desenvolvido e validado foi aplicado em amostras de mecônio de neonatos com suspeita ou não de exposição fetal aos constituintes do tabaco. / Tobacco is one of the main drugs consumed worldwide and its use by women of reproductive age in particular has played a major concern among experts and society in general. Despite the adverse effects associated with smoking during pregnancy are well documented, it is known that a significant proportion of pregnant women have difficulties to quit the habit. Fetal exposure to tobacco constituents has been associated with increased risk of spontaneous abortion (miscarriage) and premature births, incidence of newborns with low birth weight, sudden infant death syndrome and neurobehavioral and cognitive disorders. However, due to guilt and fear of punitive actions, women rarely admit to have used tobacco during pregnancy. As a result, a series of biological markers have been studied to diagnose fetal exposure to tobacco constituents. Nicotine and cotinine are some of these biomarkers which can be detected in meconium samples from newborns. In this study, an analytical method was developed to detect these biomarkers in meconium samples. Accelerated solvent extraction (ASE), a promising technique for solid or semi-solid sample preparation, was considered in this work the that is considered a. The analytes were identified by gas-chromatography with nitrogen phosphorus detector (GC / NPD). The limits of detection (LOD) were 3 ng/g and 30 ng/g and the limits of quantification (LOQ) were 5 ng/g and 40 ng/g for cotinine and nicotine, respectively. The method showed good linearity in the concentration range studied (5-500 ng/g), with a coefficient of correlation (r2) greater than 0.98. The intraday precision, determined by the coefficient of variation (CV %) was less than 15% and the interday precision was less or equal than 20% for nicotine and cotinine. The average recovery was 77%. The method proved to be fast, accurate, practical and sensitive and smaller volumes of organic solvents are necessary, compared to other methods published in the scientific literature. The developed and validated method was applied to meconium samples of suspected and nonsuspected neonates of having been exposed to tobacco constituents during gestation.

[pt] O objetivo desta pesquisa foi a substituição de técnicas convencionais de extração de amostras para análise hidrocarbonetos policíclicos aromáticos (HPAs) em amostras de tecido para um procedimento confiável, rápido e ambientalmente mais sustentáveis. O novo método foi desenvolvido usando um método de extração com solvente pressurizado, avaliando dois materiais de referência padrão (peixe e mexilhão) e amostras de sardinhas fortificadas (Sardinella sp.). Cinco procedimentos de extração diferentes foram avaliados e o melhor desempenho obtido foi pela extração de 1 g de tecido liofilizado em conjunto com 5 g de sílica desativada (5 por cento), mistura de diclorometano: metanol (4: 1 v / v), temperatura de 80 graus C, três ciclos, 10 min de tempo estático e 90 s de tempo de purga. Este método foi ainda validado pela análise de nove réplicas do material de referência número 2974a(Orgânicos em Tecido de Mexilhão Liofilizado) do National Institute of Standard and Technology (NIST), resultando em uma recuperação média de 85 mais ou menos 14 por cento. As médias e as incertezas obtidos para cada HPA foram equivalentes aos do material de referência, corroborando a confiabilidade do método desenvolvido. Um tempo de processamento mais curto, menos uso de solventes e reagentes e menor manipulação do extrato resultou em um método eficaz e alinhado às diretrizes da química verde. / [en] The aim of this research was the replacement of conventional sample extraction techniques for polycyclic aromatic hydrocarbons (PAH) in tissue samples for a reliable, fast and environmentally more sustainable. The methodwas developed using a pressurized solvent extraction method and assessing two different standard reference materials (fish and mussel) and freeze-dried and fortified sardine samples (Sardinella sp.). Five different extraction procedures were evaluated and the best performance comprised 1 g of lyophilized tissue, 5 g of deactivated (5 percent) silica, a dichloromethane:methanol (4:1 v/v) mixture, a temperature of 80 C degrees, three cycles, 10 min of static time and 90 s of purge time. The method selected following these tests was further validated through the analysis of nine replicates of the National Institute of Standard and Technology (NIST) reference material No. 2974a (Organics in Freeze-Dried Mussel Tissue), resulting in an effective recovery of 85 more or less 14 percent. The means and uncertainties attained for each PAH were equivalent to those of the reference material, corroborating the reliability of the developed method. A shorter processing time, less use of solvents and reagents and lower extract manipulation resulted in an effective method aligned with green-chemistry guidelines.

No description available.

Soil that is heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) is often found at the sites of former gasworks and wood-impregnation plants. Since PAHs are toxic these sites represent a hazard to human health and the environment, and therefore they need to be treated, preferably by a method that destroys the contaminants, and thus eliminates the problem permanently. However, during biological and chemical degradation of PAHs other toxic compounds may be formed. If these transformation products are sufficiently persistent they could potentially accumulate during remedial processes. In the work underlying this thesis the degradation and transformation of PAHs were studied in three remedial processes: viz. a pilot-scale bioslurry reactor, microcosms with wood-rotting fungi and lab-scale treatments with Fenton's reagent. A group of transformation products referred to as oxygenated-PAHs (oxy-PAHs) was found to be particularly important, as these compounds are toxic and were shown to be relatively persistent in the environment. The oxy- PAHs were, for instance, found at significant concentrations in the gasworks soil used in most of the studies. This soil was highly weathered and had therefore been depleted of the more readily degradable compounds. In addition, experiments in which earthworms were exposed to the gasworks soil showed that the oxy-PAHs were more easily taken up in living organisms than PAHs. To facilitate the studies, new extraction and fractionation methods were developed. For instance, pressurized liquid extraction (PLE) was investigated for its reliability and efficiency to extract PAHs and oxy-PAHs from soil. Furthermore, a selective PLE-method was developed that can simultaneously extract and separate the PAHs and oxy-PAHs into two different fractions. This was accomplished by adding a chromatographic material (silica or Florisil) to the extraction cell. Under certain conditions all three remedial processes resulted in increasing amounts of oxy- PAHs in the soil. For example, 1-acenaphthenone and 4-oxapyrene-5-one accumulated in the bioslurry reactor. Similarly, in the soil inoculated with a white-rot fungus 9-fluorenone, benzo[a]anthracene-7,12-dione, 4-hydroxy-9-fluorenone and 4-oxapyrene-5-one accumulated. Finally, in an ethanol-Fenton treatment the concentration of some PAH-quinones increased in the soil. The results show that it might be necessary to monitor oxy-PAHs as well as PAHs during the remediation of PAH-contaminated sites. Otherwise, the soil may be considered detoxified too early in the process. In the long term it would be desirable to include analyses with sufficient marker compounds to follow the possible production and elimination of the oxy-PAHs. However, until such compounds can be identified it is suggested that contaminated soil should be screened for oxy-PAHs in general. The selective PLE-method presented in this thesis could be a useful tool for this.

Ce travail de doctorat traite l’optimisation des méthodes d’extraction des composés phénoliques à partir des sous-produits de la viticulture et de la viniculture, à savoir les sarments de vigne et les marcs de raisins. Plusieurs technologies innovantes sont appliquées et comparées : l’extraction accélérée par solvant (EAS), les décharges électriques de haute-tension (DEHT), les ultrasons (US) et les champs électriques pulsés (CEP). Les extractions solide-liquide faites sur les sarments ont montré que, parmi les solvants étudiés, l’eau est le moins efficace. L’ajout de la β-cyclodextrine dans l’eau améliore le procédé d’extraction mais est moins efficace que les mélanges hydroéthanoliques. L’extraction en milieu alcalin donne le meilleur rendement en composés phénoliques. L’intensification de l’extraction des composés phénoliques des sarments est possible grâce aux nouvelles technologies d’extraction. L’efficacité des méthodes testées est la moindre avec les US, moyenne avec les CEP pour atteindre le meilleur rendement phénolique avec les DEHT. La filtrabilité de ces extraits est d’autant plus lente que leur composition est complexe. L’ultrafiltration membranaire permet une très bonne purification et concentration des composés phénoliques. L’étude des mécanismes d’action des DEHT a permis d’identifier les phénomènes favorisant l’extraction des composés phénoliques à partir des sarments. Un effet mécanique des DEHT, capable de fragmenter les sarments, est en majorité responsable de cette amélioration. Le procédé énergivore du broyage pourra alors être omis. Un effet électrique contribuant également à l’intensification du procédé d’extraction est démontré. La formation de peroxyde d’hydrogène durant le traitement par DEHT est quantifiée mais ne semble pas altérer les composés phénoliques qui sont des molécules à capacité antiradicalaire élevée. Quant aux études portées sur les marcs de raisins, la variation simultanée de plusieurs paramètres opératoires a permis l’optimisation de l’extraction aqueuse et hydroéthanolique des composés phénoliques en ayant recours à la méthodologie de surface de réponse (MSR). Le passage d’un milieu aqueux à un milieu hydroéthanolique a permis d’améliorer nettement le procédé d’extraction solide-liquide des composés phénoliques et l’utilisation de l’EAS a permis l’augmentation du rendement en composés phénoliques jusqu’à trois fois par rapport à l’optimum obtenu en milieu hydroéthanolique. / This study deals with the optimization of the extraction methods of phenolic compounds from viticulture and viniculture by-products, namely vine shoots and grape pomace. Several innovative technologies were tested and compared: high voltage electrical discharges (HVED), accelerated solvent extraction (ASE), ultrasounds (US) and pulsed electric fields (PEF). The solid-liquid extraction conducted on vine shoots showed that, amongst the studied solvents, water is the least effective. The addition of the β-cyclodextrin to water improves the extraction process but remains less effective than that with hydroethanolic mixtures. The extraction in alkaline medium gives the highest phenolic compound extraction yields. The intensification of phenolic compound extraction from vine shoots was possible thanks to new extraction technologies. The effectiveness of the tested methods was the least with US, followed by PEF to accomplish the highest phenolic yield with HVED. The filterability of the extracts was slower when their composition was complex, and the membrane technology allowed a good purification and concentration of phenolic compounds. The reason behind the high effectiveness of HVED was investigated. The action mechanisms of HVED were studied in details. A mechanical effect of HVED provoked vine shoots fragmentation and particle size reduction. This was the main phenomenon responsible for the intensification of the extraction process. It also suggested that a grinding pretreatment would not be necessary prior to HVED, which considerably diminishes the energy input of the overall process. The presence of a non-mechanical effect and its contribution in the efficiency of HVED were also shown. The formation of hydrogen peroxide during the treatment was observed. However it did not seem to alter vine shoot phenolic compounds since these demonstrated a high radical scavenging capacity. As for the studies conducted on grape pomace, the simultaneous variation of several operating parameters allowed the aqueous and hydroethanolic optimization of phenolic compound extraction from these byproducts by response surface methodology (RSM). The passage from an aqueous to a hydroethanolic medium clearly improved the solid-liquid extraction of phenolic compounds from grape pomace. The use of ASE further increased the phenolic compound yield up to three times as compared to the optimum obtained with a hydroethanolic solvent.