Relatório de estágio profissional

O presente Relatório foi realizado no âmbito do Estágio Profissional I e II. Trata-se de um trabalho elaborado com base na observação de aulas e na experimentação didática; os materiais obtidos (como, por exemplo, horários, dados sobre as turmas, fichas, etc.) foram submetidos a uma análise documental

Automation, hyphenation and multidimensional chromatography for PAH analysis : Analytical techniques to simplify sample complexity

One group of organic environmental pollutants which are potentially hazardous to human health is polycyclic aromatic hydrocarbons (PAHs). These compounds consist of two or more benzene rings in their structure among which benzo[a]pyrene (B[a]P) is classified as a human carcinogen. In order to generate accurate data for PAHs, reliable analytical methods are a prerequisite for PAH monitoring in the environment and cancer risk assessments. However, there are several analytical challenges in PAH analysis because they are a large number of compounds with different physical and chemical properties and occur at varying concentration levels, often in complex matrices. Thus, this thesis aimed at tackling these difficulties in PAH analysis using various analytical techniques such as automation, hyphenation and multidimensional chromatography, particularly to increase the detectability and separation efficiency of PAHs in complex matrices. Furthermore, analytical methods were also presented as a tool for toxicological studies. A fully automated two-dimensional (2D)-liquid chromatography (LC)/2D-gas chromatography system was developed to enable the online sample clean-up, separation and detection, initially with two flame ionization detectors (FIDs) (Paper I). The introduction of mass selective detectors (MSDs) instead of two FIDs further advanced the original 2D system, improving the detectability and selectivity in PAH analysis (Paper II). The PAH levels were determined in two standard reference materials (SRMs) from the National Institute of Standards and Technology (NIST), i.e. urban dust (SRM 1649a) and diesel particulate extract (SRM 1975). The measured PAH levels were in good agreement with those obtained from FID- and MSD-based systems and reported by NIST (Paper I and II). Additionally, the PAH determination was done in particulates from wood burning, which also showed comparable results between FID and MSD systems (Paper II). The FID-based system showed slightly better separation efficiency owing to the use of hydrogen as carrier gas instead of helium and similar detectability to that of MSD (Paper I). However, the MSD-based system was advantageous to detecting late-eluting PAHs and separating interfering peaks in complex matrices (Paper II). Paper III presented an online LC system for B[a]P-selective fractionation as a tool for studying biological interactions. Fractions could be obtained from Stockholm air particulate matter (PM) extract with or without B[a]P. The selective fractionation was achieved using serial LC columns of two pyrenyl phases, resulting in more than 90% of B[a]P in the B[a]P-containing fraction. An analytical method for identification and quantification of benz[j]aceanthrylene (B[j]A) in air PM was developed and described in paper IV. B[j]A is rarely measured due to its low abundance but is known to have high carcinogenic potential. The determination of B[j]A in air PM from Stockholm (Sweden) and Limeira (Brazil), was done together with a series of toxicological studies. The measurement and toxicological data showed an increased number of estimated cancer cases from air PM exposure when levels of B[j]A were taken into account. In conclusion, this thesis presents various analytical approaches to obtain more accurate PAH data as well as the possibilities of using them in toxicological research

Automated clean-up, separation and detection of polycyclic aromatic hydrocarbons in particulate matter extracts using a 2D-LC/2D-GC system : a method translation from two FIDs to two MS detectors

An online two-dimensional (2D) liquid chromatography/2D gas chromatography system with two mass-selective detectors has been developed on the basis of a previous system with two flame ionization detectors. The method translation involved the change of carrier gas from hydrogen to helium, column dimension and detectors. The 2D system with two mass-selective detectors was validated with use of polycyclic aromatic hydrocarbon (PAH) standards and two standard reference materials from air and diesel exhaust. Furthermore, the system was applied to a real sample, wood smoke particulates. The PAH values determined correlated well with the previous data and those from the National Institute of Standards and Technology. The system enhanced the benefits of the previous system, which were limited by the low detectability and lack of mass selectivity. This study shows an automated 2D system that is valid for PAH analysis of complex environmental samples directly from crude extracts

Contribution of wood burning to exposures of PAHs and oxy-PAHs in Eastern Sweden

A growing trend in developed countries is the use of wood as fuel for domestic heating due to measures taken to reduce the usage of fossil fuels. However, this imposed another issue with the environment and human health. That is, the emission from wood burning contributed to the increased level of atmospheric particulates and the wood smoke caused various respiratory diseases. The aim of this study was to investigate the impact of wood burning on the polycyclic aromatic hydrocarbons (PAHs) in air PM10 using known wood burning tracers, i.e. levoglucosan, mannosan and galactosan from the measurement at the urban background and residential areas in Sweden. A yearly measurement from three residential areas in Sweden showed a clear seasonal variation of PAHs during the cold season mainly from increased domestic heating and meteorology. Together, an increased sugar level assured the wood burning during the same period. The sugar ratio (levoglucosan/(mannosan+galactosan)) was a good marker for wood burning source such as the wood type used for domestic heating and garden waste burning. On the Walpurgis Night, the urban background measurement demonstrated a dramatic increase in levoglucosan, benzo[a]pyrene (B[a]P) and oxygenated PAHs (OPAHs) concentrations from the increased wood burning. A significant correlation between levoglucosan and OPAHs was observed suggesting OPAHs to be an indicator of wood burning together with levoglucosan. The levoglucosan tracer method and modelling used in predicting the B[a]P concentration could not fully explain the measured levels in the cold season. The model showed that the local wood source contributed to 98 % of B[a]P emissions in the Stockholm area and 2 % from the local traffic. However, non-local sources were dominating in the urban background (60%). A further risk assessment estimated that the airborne particulate PAHs caused 13.4 cancer cases per 0.1 million inhabitants in Stockholm County

Diet quality determines blue mussel physiological status: A long-term experimental multi-biomarker approach

International audienceCyanobacterial blooms have increased worldwide and decreases in spring blooms of diatoms in temperate aquatic ecosystems are increasingly reported. Whether such shifts in the food base positively or negatively affect primary consumer's physiological status, growth, and more broadly, ecosystem functioning, is poorly known. Here, we test how a widespread filter-feeding blue mussel from the genus Mytilus reacts to diatoms, filamenous non-toxic cyanobacteria and a mixture of the two in a 50 day feeding experiment and compare with mussels in the field. We use a multi-tracer approach to assess mussels' nutritional and physiological status. Diatoms had highest relative proportions of the essential fatty acids 20:5ω3 (EPA) and 22:6ω3 (DHA), but cyanobacteria had a high relative proportion of polyunsaturated FAs (PUFAs) especially 18:3ω3, a precursor of ω3 FAs. Bulk and amino-acid-δ 15 N demonstrated assimilation of cyanobacterial N or diatom N by the mussels, while fatty acid (FA) composition and δ 13 C-FA analyses demonstrated assimilation of C and FAs from diatoms. Still, C and N from both food sources were utilized when mussels were fed the mix and condition index, scope for growth (SFG; measure of energy status) and the elemental N:P ratio of mussels where higher in mussels fed cyanobacteria only, indicating better growth status in this treatment. It seems like mussels fed cyanobacteria, a food with low proportions of essential FAs compared to diatoms, were able to cover the dietary requirements through FA biosynthesis from precursor FAs. The FA composition and δ 15 N-results from mussels fed cyanobacteria agreed well with seasonal field measurements of mussels. Our study provides the first feeding experiment investigating how blue mussels utilize N from cyanobacteria and shows the rather positive response to this type of food. Our results suggest that imbalances in the biochemical composition of diet of consumers can be overcome through biosynthesis of essential FAs. In the context of climate change with shifting biogeochemical baselines, it is crucial to understand the biological effects of such changes in the quality of the production base

Comparative mutagenic activity of atmospheric particulate matter from limeira, stockholm, and kyoto

Atmospheric particulate matter (PM) organic fractions from urban centers are frequently mutagenic for the Salmonella/microsome assay. This mutagenicity is related to both primary and secondary pollutants, and meteorological conditions have great influence on the secondary pollutant's formation. Our objective was to compare the mutagenicity of atmospheric total suspended particulates (TSP) from three cities with marked different meteorological conditions and TSP concentrations: Limeira (Brazil) with 99.0 mu g/m(3), Stockholm (Sweden) with 6.2 mu g/m(3), and Kyoto (Japan) with 28.0 mu g/m(3). For comparison, we used the same batch of filters, sample extraction method, and Salmonella/microsome testing protocol with 11 strains of Salmonella with and without metabolic activation. Samples were collected during winter and pooled into one single extract representing each city. All samples were mutagenic for all tested strains, except for TA102. Based on the strain's selectivity, nitroarenes, polycyclic aromatic hydrocarbons, and aromatic amines play a predominant role in the mutagenicity of these samples. The mutagenic potencies expressed by mass of extracted organic material (EOM; revertants/mu g EOM) were similar (similar to twofold difference) among the cities, despite differences in meteorological conditions and pollution sources. In contrast, the mutagenic potencies expressed by air volume (rev/m(3)) varied similar to 20-fold, with Limeira > Kyoto approximate to Stockholm. These results are the first systematic assessment of air mutagenicity from cities on three continents using the same protocols. The results confirm that the mutagenic potency expressed by EOM mass is similar regardless of continent of origin, whereas the mutagenic potency expressed by air volume can vary by orders of magnitude. Environ. Mol. Mutagen. 2019. (c) 2019 Wiley Periodicals, Inc607607616FAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2015/23364-

Validierung der computeradaptiven Assessmentprozedur RehaCAT hinsichtlich funktionaler und psychischer Belastungen bei Rehabilitanden mit muskuloskelettalen Erkrankungen

Benz[j]aceanthrylene (B[j]A) is a cyclopenta-fused polycyclic aromatic hydrocarbon with strong mutagenic and carcinogenic effects. We have identified B[j]A in air particulate matter (PM) in samples collected in Stockholm, Sweden and in Limeira, Brazil using LC-GC/MS analysis. Determined concentrations ranged between 1.57 and 12.7 and 19.6-30.2 pg/m3 in Stockholm and Limeira, respectively, which was 11-30 times less than benzo[a]pyrene (B[a]P) concentrations. Activation of the DNA damage response was evaluated after exposure to B[j]A in HepG2 cells in comparison to B[a]P. We found that significantly lower concentrations of B[j]A were needed for an effect on cell viability compared to B[a]P, and equimolar exposure resulted in significant more DNA damage with B[j]A. Additionally, levels of γH2AX, pChk1, p53, pp53, and p21 proteins were higher in response to B[j]A than B[a]P. On the basis of dose response induction of pChk1 and γH2AX, B[j]A potency was 12.5- and 33.3-fold higher than B[a]P, respectively. Although B[j]A levels in air were low, including B[j]A in the estimation of excess lifetime cancer risk increased the risk up to 2-fold depending on which potency factor for B[j]A was applied. Together, our results show that B[j]A could be an important contributor to the cancer risk of air PM.Benz[j]aceanthrylene (B[j]A) is a cyclopenta-fused polycyclic aromatic hydrocarbon with strong mutagenic and carcinogenic effects. We have identified B[j]A in air particulate matter (PM) in samples collected in Stockholm, Sweden and in Limeira, Brazil usi49531013109sem informaçãosem informaçã

Cancer Risk Assessment of Airborne PAHs Based on <i>in Vitro</i> Mixture Potency Factors

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants associated with adverse human health effects including cancer. However, the risk of exposure to mixtures is difficult to estimate, and risk assessment by whole mixture potency evaluations has been suggested. To facilitate this, reliable <i>in vitro</i> based testing systems are necessary. Here, we investigated if activation of DNA damage signaling <i>in vitro</i> could be an endpoint for developing whole mixture potency factors (MPFs) for airborne PAHs. Activation of DNA damage signaling was assessed by phosphorylation of Chk1 and H2AX using Western blotting. To validate the <i>in vitro</i> approach, potency factors were determined for seven individual PAHs which were in very good agreement with established potency factors based on cancer data <i>in vivo.</i> Applying the method using Stockholm air PAH samples indicated MPFs with orders of magnitude higher carcinogenic potency than predicted by established <i>in vivo</i>-based potency factors. Applying the MPFs in cancer risk assessment suggested that 45.4 (6% of all) cancer cases per year in Stockholm are due to airborne PAHs. Applying established models resulted in <1 cancer case per year, which is far from expected levels. We conclude that our <i>in vitro</i> based approach for establishing MPFs could be a novel method to assess whole mixture samples of airborne PAHs to improve health risk assessment

Detection of Benz[<i>j</i>]aceanthrylene in Urban Air and Evaluation of Its Genotoxic Potential

Benz­[<i>j</i>]­aceanthrylene (B­[<i>j</i>]­A) is a cyclopenta-fused polycyclic aromatic hydrocarbon with strong mutagenic and carcinogenic effects. We have identified B­[<i>j</i>]­A in air particulate matter (PM) in samples collected in Stockholm, Sweden and in Limeira, Brazil using LC–GC/MS analysis. Determined concentrations ranged between 1.57 and 12.7 and 19.6–30.2 pg/m³ in Stockholm and Limeira, respectively, which was 11–30 times less than benzo­[<i>a</i>]­pyrene (B­[<i>a</i>]­P) concentrations. Activation of the DNA damage response was evaluated after exposure to B­[<i>j</i>]­A in HepG2 cells in comparison to B­[<i>a</i>]­P. We found that significantly lower concentrations of B­[<i>j</i>]­A were needed for an effect on cell viability compared to B­[<i>a</i>]­P, and equimolar exposure resulted in significant more DNA damage with B­[<i>j</i>]­A. Additionally, levels of γH2AX, pChk1, p53, pp53, and p21 proteins were higher in response to B­[<i>j</i>]­A than B­[<i>a</i>]­P. On the basis of dose response induction of pChk1 and γH2AX, B­[<i>j</i>]­A potency was 12.5- and 33.3-fold higher than B­[<i>a</i>]­P, respectively. Although B­[<i>j</i>]­A levels in air were low, including B­[<i>j</i>]­A in the estimation of excess lifetime cancer risk increased the risk up to 2-fold depending on which potency factor for B­[<i>j</i>]­A was applied. Together, our results show that B­[<i>j</i>]­A could be an important contributor to the cancer risk of air PM