Biotransformation and endocrine disruptive effects of contaminants in ringed seals - implications for monitoring and risk assessment

Marine mammals are exposed to persistent organic pollutants (POPs), which may be biotransformed to metabolites some of which are highly toxic. Both POPs and their metabolites may lead to adverse health effects, which have been studied using various biomarkers. Changes in endocrine homeostasis have been suggested to be sensitive biomarkers for contaminant-related effects. The overall objective of this doctoral thesis was to investigate biotransformation capacity of POPs and their potential endocrine disruptive effects in two contrasting ringed seal populations from the low contaminated Svalbard area and from the highly contaminated Baltic Sea. Biotransformation capacity was studied by determining the relationships between congener-specific patterns and concentrations of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs) and their hydroxyl (OH)- and/or methylsulfonyl (MeSO₂)-metabolites, and catalytic activities of hepatic xenobiotic-metabolizing phase I and II enzymes. The results suggest that the biotransformation of PCBs, PBDEs and toxaphenes in ringed seals depends on the congener-specific halogen-substitution pattern. Biotransformation products detected in the seals included OH-PCBs, MeSO₂-PCBs and –DDE, pentachlorophenol, 4-OHheptachlorostyrene, and to a minor extent OH-PBDEs. The effects of life history state (moulting and fasting) on contaminant status and potential biomarkers for endocrine disruption, including hormone and vitamin homeostasis, were investigated in the low contaminated ringed seal population from Svalbard. Moulting/fasting status strongly affected thyroid, vitamin A and calcitriol homeostasis, body condition and concentrations of POPs and their OH-metabolites. In contrast, moulting/fasting status was not associated with variations in vitamin E levels. Endocrine disruptive effects on multiple endpoints were investigated in the two contrasting ringed seal populations. The results suggest that thyroid, vitamin A and calcitriol homeostasis may be affected by the exposure of contaminants and/or their metabolites in the Baltic ringed seals. Complex and non-linear relationships were observed between the contaminant levels and the endocrine variables. Positive relationships between circulating free and total thyroid hormone concentration ratios and OH-PCBs suggest that OH-PCBs may mediate the disruption of thyroid hormone transport in plasma. Species differences in thyroid and bone-related effects of contaminants were studied in ringed and grey seals from low contaminated references areas and from the highly contaminated Baltic Sea. The results indicate that these two species living at the same environment approximately at the same trophic level respond in a very different way to contaminant exposure. The results of this thesis suggest that the health status of the Baltic ringed seals has still improved during the last decade. PCB and DDE levels have decreased in these seals and the contaminant-related effects are different today than a decade ago. The health of the Baltic ringed seals is still suggested to be affected by the contaminant exposure. At the present level of the contaminant exposure the Baltic ringed seals seem to be at a zone where their body is able to compensate for the contaminant-mediated endocrine disruption. Based on the results of this thesis, several recommendations that could be applied on monitoring and assessing risk for contaminant effects are provided. Circulating OH-metabolites should be included in monitoring and risk assessment programs due to their high toxic potential. It should be noted that endogenous variables may have complex and highly variable responses to contaminant exposure including non-linear responses. These relationships may be further confounded by life history status. Therefore, it is highly recommended that when using variables related to endocrine homeostasis to investigate/monitor or assess the risk of contaminant effects in seals, the life history status of the animal should be carefully taken into consideration. This applies especially when using thyroid, vitamin A or calcitriolrelated parameters during moulting/fasting period. Extrapolations between species for assessing risk for contaminant effects in phocid seals should be avoided.

Análise por cromatografia gasosa de BTEX nas emissões de motor de combustão interna alimentado com diesel e mistura diesel-biodiesel (B10)

This paper describes the procedures for analysing pollutant gases emitted by engines, such as volatile organic compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene) by using high resolution gas chromatography (HRGC). For IC engine burning, in a broad sense, the use of the B10 mixture reduces drastically the emissions of aromatic compounds. Especially for benzene the reduction of concentrations occurs at the level of about 24.5%. Although a concentration value below 1 µg mL-1 has been obtained, this reduction is extremely significant since benzene is a carcinogenic compound.

Determinação da matéria-prima utilizada na produção do biodiesel adicionado ao diesel mineral através de monitoramento seletivo de íons

The selective ion monitoring acquisition mode in mass spectrometry was applied to identify, in the diesel complex matrix, the raw materials (vegetable oil and alcohol) that originate biodiesel. Biodiesel samples obtained from babassu, castor, palm and soybean vegetable oils and pure fatty acid methyl and ethyl esters were used to develop this method, using specific fragments in mass spectrometry and the "window system" in gas chromatography. The commercial Brazilian B2 samples were found to be produced with soybean oil, transesterified with methanol.

Amostragem do material particulado e fração orgânica volátil das emissões em motor ciclo diesel sem a utilização de túnel de diluição

Using a sampling method of particulate matter (PM) without the use of a dilution tunnel allows for evaluations of the volatile hydrocarbons (HC) in the emissions of diesel cycle engines. The procedure in this work applied a heated filter with temperature controlled. The volatile compounds are condensed at low temperature, allowing for evaluation of the HC by thermal desorption of the PM and for analysis of the condensed compounds of the exhaust gases.

Utilização de pelos de animais silvestres para monitoramento ambiental de Cd, Cr, Cu, Fe, Mn, Pb e Zn

This study investigates the use of wild animal hair of C. brachyurus, C. thous and L. pardalis as biomonitors of trace metal at Parque Nacional das Emas, Brazil. Results reveal a strong correlation between Cd and Pb as well as Cu and Zn, which suggests a single source of emission. Most metals showed a lower or equal concentration than those obtained in previous studies. The research shows that monitoring may be performed only with Zn, Pb, Cd, and Cr because of statistical similarity and of a non-natural occurrence of large amounts of the material under analysis.

Diesel/biodiesel soot oxidation with ceo2 and ceo2-zro2-modified cordierites: a facile way of accounting for their catalytic ability in fuel combustion processes

CeO2 and mixed CeO2-ZrO2 nanopowders were synthesized and efficiently deposited onto cordierite substrates, with the evaluation of their morphologic and structural properties through XRD, SEM, and FTIR. The modified substrates were employed as outer heterogeneous catalysts for reducing the soot originated from the diesel and diesel/biodiesel blends incomplete combustion. Their activity was evaluated in a diesel stationary motor, and a comparative analysis of the soot emission was carried out through diffuse reflectance spectroscopy. The analyses have shown that the catalyst-impregnated cordierite samples are very efficient for soot oxidation, being capable of reducing the soot emission in more than 60%.

Produção de biossurfactantes por microrganismos isolados de solo contaminado com óleo diesel

The aim of this work was to produce biosurfactants through submerged fermentation using microorganisms isolated from soil contaminated with diesel. Microorganisms were isolated, characterized by the production of biosurfactants, and used to study the influence of type, induction and concentration of ammonium sulfate as a nitrogen source in the culture medium. The microorganisms that showed best results, in terms of production of biosurfactants, were identified as being of the genus Pseudomonas and Bacillus. The biosurfactants produced proved capable of reducing the surface tension of the media to 39 mN/m and 34 mN/m, respectively. Higher biosurfactant production was obtained in the medium containing 1% soybean oil without ammonium sulfate.

Análise exploratória aplicada a espectros de reflexão total atenuada no infravermelho com transformada de Fourier (ATR-FTIR) de blendas de biodiesel/diesel

In this study, hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used to classify blends produced from diesel S500 and different kinds of biodiesel produced by the TDSP methodology. The different kinds of biodiesel studied in this work were produced from three raw materials: soybean oil, waste cooking oil and hydrogenated vegetable oil. Methylic and ethylic routes were employed for the production of biodiesel. HCA and PCA were performed on the data from attenuated total reflectance Fourier transform infrared spectroscopy, showing the separation of the blends into groups according to biodiesel content present in the blends and to the kind of biodiesel used to form the mixtures.

Spectroscopic monitoring during laser welding of aluminium alloy

The mechanical properties of aluminium alloys are strongly influenced by the alloying elements and their concentration. In the case of aluminium alloy EN AW-6060 the main alloying elements are magnesium and silicon. The first goal of this thesis was to determine stability, repeatability and sensitivity as figures of merit of the in-situ melt identification technique. In this study the emissions from the laser welding process were monitored with a spectrometer. With the information produced by the spectrometer, quantitative analysis was conducted to determine the figures of merit. The quantitative analysis concentrated on magnesium and aluminium emissions and their relation. The results showed that the stability of absolute intensities was low, but the normalized magnesium emissions were quite stable. The repeatability of monitoring magnesium emissions was high (about 90 %). Sensitivity of the in-situ melt identification technique was also high. As small as 0.5 % change in magnesium content was detected by the spectrometer. The second goal of this study was to determine the loss of mass during deep penetration laser welding. The amount of magnesium in the material was measured before and after laser welding to determine the loss of magnesium. This study was conducted for aluminium alloy with nominal magnesium content of 0-10 % and for standard material EN AW-6060 that was welded with filler wire AlMg5. It was found that while the magnesium concentration in the material changed, the loss of magnesium remained fairly even. Also by feeding filler wire, the behaviour was similar. Thirdly, the reason why silicon had not been detected in the emission spectrum needed to be explained. Literature research showed that the amount of energy required for silicon to excite is considerably higher compared to magnesium. The energy input in the used welding process is insufficient to excite the silicon atoms.

Use of data mining and spectral profiles to differentiate condition after harvest of coffee plants

This study aimed at identifying different conditions of coffee plants after harvesting period, using data mining and spectral behavior profiles from Hyperion/EO1 sensor. The Hyperion image, with spatial resolution of 30 m, was acquired in August 28th, 2008, at the end of the coffee harvest season in the studied area. For pre-processing imaging, atmospheric and signal/noise effect corrections were carried out using Flaash and MNF (Minimum Noise Fraction Transform) algorithms, respectively. Spectral behavior profiles (38) of different coffee varieties were generated from 150 Hyperion bands. The spectral behavior profiles were analyzed by Expectation-Maximization (EM) algorithm considering 2; 3; 4 and 5 clusters. T-test with 5% of significance was used to verify the similarity among the wavelength cluster means. The results demonstrated that it is possible to separate five different clusters, which were comprised by different coffee crop conditions making possible to improve future intervention actions.

Performance of a diesel engine fueled with a preheated blend of soybean oil and petrodiesel

Brazil is doing a major effort to find alternatives to diesel oil as combustible. Some study lines are oriented to the development of vegetable oils used as fuel, as a source of getting cheaper and have higher energy density than the converted vegetable oils, and less risk of environmental contamination. The aim of this study was to evaluate the performance, the useful life of the lubricant and some components of a Diesel Cycle engine, with an electronic injection system, in a long-term test operating with a preheated blend (65°C) of 50% (v v-1) of soybean oil in petrodiesel. There was a reduction of the useful life of the injectors which presented failure because of high wear with 264 hours of operation and showed an increase in emissions of particulate matter (opacity) which may be assigned to the failures occurred in the injection system. An increase in the useful life of the lubricant, when compared with the literature was also observed. The electronic injection system may favor the burning of the tested fuel. The test was interrupted with 264 hours because of failures in the injection system.

Identification of acoustic parameters for broiler welfare estimate

Estimates of broiler welfare have subjective character. Nowadays, researchers seek non-invasive features or indicators that may describe this condition in animal production. The aim of this study was to identify acoustic parameters to estimate broiler welfare using the following five vocalization acoustic parameters: energy, spectral centroid, bandwidth, first formant, and second formant. The database that generated the model was obtained from a field experiment with 432 broilers, which half were Cobb® and half, Ross® breed, from day 21 to 42, containing bird vocalizations under either welfare or stress conditions. The results of the experiment generated responses to the tested conditions of gender, genetic strain, and welfare. The proposed model was based on the specific response of mean weights for each situation of stress and well-being. From the results, a model was developed to estimate the welfare condition of broilers from the registered information linked to their vocalization.

Characterisation of Laser Beam and Paper Material Interaction

It is known already from 1970´s that laser beam is suitable for processing paper materials. In this thesis, term paper materials mean all wood-fibre based materials, like dried pulp, copy paper, newspaper, cardboard, corrugated board, tissue paper etc. Accordingly, laser processing in this thesis means all laser treatments resulting material removal, like cutting, partial cutting, marking, creasing, perforation etc. that can be used to process paper materials. Laser technology provides many advantages for processing of paper materials: non-contact method, freedom of processing geometry, reliable technology for non-stop production etc. Especially packaging industry is very promising area for laser processing applications. However, there are only few industrial laser processing applications worldwide even in beginning of 2010´s. One reason for small-scale use of lasers in paper material manufacturing is that there is a shortage of published research and scientific articles. Another problem, restraining the use of laser for processing of paper materials, is colouration of paper material i.e. the yellowish and/or greyish colour of cut edge appearing during cutting or after cutting. These are the main reasons for selecting the topic of this thesis to concern characterization of interaction of laser beam and paper materials. This study was carried out in Laboratory of Laser Processing at Lappeenranta University of Technology (Finland). Laser equipment used in this study was TRUMPF TLF 2700 carbon dioxide laser that produces a beam with wavelength of 10.6 μm with power range of 190-2500 W (laser power on work piece). Study of laser beam and paper material interaction was carried out by treating dried kraft pulp (grammage of 67 g m-2) with different laser power levels, focal plane postion settings and interaction times. Interaction between laser beam and dried kraft pulp was detected with different monitoring devices, i.e. spectrometer, pyrometer and active illumination imaging system. This way it was possible to create an input and output parameter diagram and to study the effects of input and output parameters in this thesis. When interaction phenomena are understood also process development can be carried out and even new innovations developed. Fulfilling the lack of information on interaction phenomena can assist in the way of lasers for wider use of technology in paper making and converting industry. It was concluded in this thesis that interaction of laser beam and paper material has two mechanisms that are dependent on focal plane position range. Assumed interaction mechanism B appears in range of average focal plane position of 3.4 mm and 2.4 mm and assumed interaction mechanism A in range of average focal plane position of 0.4 mm and -0.6 mm both in used experimental set up. Focal plane position 1.4 mm represents midzone of these two mechanisms. Holes during laser beam and paper material interaction are formed gradually: first small hole is formed to interaction area in the centre of laser beam cross-section and after that, as function of interaction time, hole expands, until interaction between laser beam and dried kraft pulp is ended. By the image analysis it can be seen that in beginning of laser beam and dried kraft pulp material interaction small holes off very good quality are formed. It is obvious that black colour and heat affected zone appear as function of interaction time. This reveals that there still are different interaction phases within interaction mechanisms A and B. These interaction phases appear as function of time and also as function of peak intensity of laser beam. Limit peak intensity is the value that divides interaction mechanism A and B from one-phase interaction into dual-phase interaction. So all peak intensity values under limit peak intensity belong to MAOM (interaction mechanism A one-phase mode) or to MBOM (interaction mechanism B onephase mode) and values over that belong to MADM (interaction mechanism A dual-phase mode) or to MBDM (interaction mechanism B dual-phase mode). Decomposition process of cellulose is evolution of hydrocarbons when temperature is between 380- 500°C. This means that long cellulose molecule is split into smaller volatile hydrocarbons in this temperature range. As temperature increases, decomposition process of cellulose molecule changes. In range of 700-900°C, cellulose molecule is mainly decomposed into H2 gas; this is why this range is called evolution of hydrogen. Interaction in this range starts (as in range of MAOM and MBOM), when a small good quality hole is formed. This is due to “direct evaporation” of pulp via decomposition process of evolution of hydrogen. And this can be seen can be seen in spectrometer as high intensity peak of yellow light (in range of 588-589 nm) which refers to temperature of ~1750ºC. Pyrometer does not detect this high intensity peak since it is not able to detect physical phase change from solid kraft pulp to gaseous compounds. As interaction time between laser beam and dried kraft pulp continues, hypothesis is that three auto ignition processes occurs. Auto ignition of substance is the lowest temperature in which it will spontaneously ignite in a normal atmosphere without an external source of ignition, such as a flame or spark. Three auto ignition processes appears in range of MADM and MBDM, namely: 1. temperature of auto ignition of hydrogen atom (H2) is 500ºC, 2. temperature of auto ignition of carbon monoxide molecule (CO) is 609ºC and 3. temperature of auto ignition of carbon atom (C) is 700ºC. These three auto ignition processes leads to formation of plasma plume which has strong emission of radiation in range of visible light. Formation of this plasma plume can be seen as increase of intensity in wavelength range of ~475-652 nm. Pyrometer shows maximum temperature just after this ignition. This plasma plume is assumed to scatter laser beam so that it interacts with larger area of dried kraft pulp than what is actual area of beam cross-section. This assumed scattering reduces also peak intensity. So result shows that assumably scattered light with low peak intensity is interacting with large area of hole edges and due to low peak intensity this interaction happens in low temperature. So interaction between laser beam and dried kraft pulp turns from evolution of hydrogen to evolution of hydrocarbons. This leads to black colour of hole edges.

Serial superficial digital flexor tendon biopsies for diagnosing and monitoring collagenase-induced tendonitis in horses

The purpose of this investigation was to demonstrate the feasibility of a biopsy technique by performing serial evaluations of tissue samples of the forelimb superficial digital flexor tendon (SDFT) in healthy horses and in horses subjected to superficial digital flexor tendonitis induction. Eight adult horses were evaluated in two different phases (P), control (P1) and tendonitis-induced (P2). At P1, the horses were subjected to five SDFT biopsies of the left forelimb, with 24 hours (h) of interval. Clinical and ultrasonographic (US) examinations were performed immediately before the tendonitis induction, 24 and 48 h after the procedure. The biopsied tendon tissues were analyzed through histology. P2 evaluations were carried out three months later, when the same horses were subjected to tendonitis induction by injection of bacterial collagenase into the right forelimb SDFT. P2 clinical and US evaluations, and SDFT biopsies were performed before, and after injury induction at the following time intervals: after 24, 48, 72 and 96 h, and after 15, 30, 60, 90, 120 and 150 days. The biopsy technique has proven to be easy and quick to perform and yielded good tendon samples for histological evaluation. At P1 the horses did not show signs of localised inflammation, pain or lameness, neither SDFT US alterations after biopsies, showing that the biopsy procedure per se did not risk tendon integrity. Therefore, this procedure is feasible for routine tendon histological evaluations. The P2 findings demonstrate a relation between the US and histology evaluations concerning induced tendonitis evolution. However, the clinical signs of tendonitis poorly reflected the microscopic tissue condition, indicating that clinical presentation is not a reliable parameter for monitoring injury development. The presented method of biopsying SDFT tissue in horses enables the serial collection of material for histological analysis causing no clinical signs and tendon damage seen by US images. Therefore, this technique allows tendonitis to be monitored and can be considered an excellent tool in protocols for evaluating SDFT injury.

Estrus cycle monitoring of captive collared peccaries (Pecari tajacu) in semiarid conditions

Collared peccaries (Peccary tajacu) are among the most hunted species in Latin America due the appreciation of their pelt and meat. In order to optimize breeding management of captive born collared peccaries in semiarid conditions, the objective was to describe and correlate the changes in the ovarian ultrasonographic pattern, hormonal profile, vulvar appearance, and vaginal cytology during the estrus cycle in this species. During 45 days, females (n=4) were subjected each three days to blood collection destined to hormonal dosage by enzyme immunoassay (EIA). In the same occasions, evaluation of external genitalia, ovarian ultrasonography and vaginal cytology were conducted. Results are presented as means and standard deviations. According to hormonal dosage, six estrous cycles were identified as lasting 21.0 ± 5.7 days, being on average 6 days for the estrogenic phase and 15 days for the progesterone phase. Estrogen presented mean peak values of 55.6 ± 20.5 pg/mL. During the luteal phase, the high values for progesterone were 35.3 ± 4.4 ng/mL. The presence of vaginal mucus, a reddish vaginal mucosa and the separation of the vulvar lips were verified in all animals during the estrogenic peak. Through ultrasonography, ovarian follicles measuring 0.2±0.1 cm were visualized during the estrogen peak. Corpora lutea presented hyperechoic regions measuring 0.4±0.2 cm identified during luteal phase. No significant differences (P>0.05) between proportions of vaginal epithelial cells were identified when comparing estrogenic and progesterone phases. In conclusion, female collared peccaries, captive born in semiarid conditions, have an estral cycle that lasts 21.0±5.7 days, with estrous signs characterized by vulvar lips edema and hyperemic vaginal mucosa, coinciding with developed follicles and high estrogen levels.