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Coal mining can generate organic and inorganic contaminants that can be disseminated in the surrounding soils by leaching and/or aerial deposition. This study aims to identify and characterize the physicochemical and geochemical changes promoted in soils from the surrounding area of a self-burning waste pile in an abandoned coal mine. A soil sampling campaign was conducted bordering the waste pile, comprising the main drainage areas as well as the areas uphill. The soils were characterized geochemically for major and trace elements and multivariate statistics was used in combination with geostatistical methodologies to study the statistical and spatial relations of the different elements and infer their Potentially Toxic Elements (PTEs) sources. The 16 priority Polycyclic Aromatic Hydrocarbons (PAHs) were identified and quantified in soils according to their spatial distribution, and their pyrogenic/petrogenic sources were inferred. Different sources were identified as contributing to the soil geochemical signature, considering not only the mine but also anthropogenic urban contamination or naturally enhanced regional geochemical background in multiple PTEs. PAHs tend to concentrate downstream of the waste pile, along the runoff areas, presenting a greater variety of the 16 priority PAHs and an increase of High Molecular Weight (HMW) PAHs pointing to its pyrogenic origin, possibly related to the self-combustion phenomenon occurring in the waste pile.

Mountains are universally regarded as key sources of high-quality water resources, fundamental for the local populations as well as for communities living downstream. In recent years, global awareness has been growing among citizens, as well as public and private institutions concerning the need to protect mountain water resources. Many mountain catchments are usually situated in regions of low population density, which are the object of special nature protection status. Water pollution in these regions is often related to road de-icing operations. The objective of this research is to recognise and characterise the environmental implications of water pollution due to road de‐icing in a mountain-protected area (Serra da Estrela, Central Portugal) by means of a geoethical perspective encompassing hydrological and ecological processes along with socioeconomic conditions. A geoethically supported decision-making on road de-icing in Serra da Estrela should bear in mind the local hydrosocial cycle, that is to say, the decision-making should be aware of the complex and dialectic interactions between water flow and quality and the socioeconomic dynamics. Costs and benefits of alternative road de-icing techniques should also be assessed, including the impacts on ecological values. This approach encompasses the ability of the territory to produce fundamental ecosystem services, including hydrological services.

The investigation about wildfires has demonstrated that research should include studies on the overall assessment of the processes, thus promoting the public awareness about impacts. The aim of this study is to assess the changes on the carbon content of soils affected by wildfires in Caramulo Mountain (Portugal) to therefore identify the environmental impacts arisen from those changes. Soils from different parent rocks, affected and non-affected by wildfires, were collected. Petrographic and geochemical methods were used to identify changes caused by the wildfires in the organic fraction of soils. The results demonstrate that changes in soils composition after wildfires include the production of charcoal and pyrogenic polycyclic aromatic hydrocarbons (PAH). The incorporation of charcoal from biomass burning in soils overtime and the production of pyrolytic PAH are of environmental concern since these compounds are known to be harmful to the environment and ecosystems and are human carcinogens. The concentration of BkF-benzo[k]fluoranthene, known as probable human carcinogen, exceeds the reference values for contaminated soils. Once in soils these compounds can be removed by percolation affecting waters and, consequently, biodiversity and human health. This work was developed under the ICT (project UIDB/04683/2020) with the reference POCI-01-0145-FEDER-007690. info:eu-repo/semantics/publishedVersion

Freshwater pollution is a huge concern. A study aiming to evaluate physico-chemical characteristics, microbiota, occurrence of two groups of persistent environmental pollutants with similar chemical properties (polycyclic aromatic hydrocarbons- PAHs and microplastics - MPs) in Alqueva's surface water was performed during 2021. Water samples were collected at three spots related to touristic activities (two beaches and one marina) during the Winter, Spring, Summer and Autumn seasons. In addition, the presence of biofilms on plastic and natural materials (stone, wood/ vegetal materials) were assessed and compared. Water quality based on physicochemical parameters was acceptable with a low eutrophication level. PAHs concentration levels were lower than the standard limits established for surface waters by international organizations. However, carcinogenic compounds were detected in two sampling locations, which can pose a problem for aquatic ecosystems. PAHs profiles showed significant differences when comparing the dry seasons with the rainy seasons, with a higher number of different compounds detected in Spring. Low molecular weigh compounds, usually associated with the atmospheric deposition and petroleum contamination, were more prevalent. MPs were detected in all samples except one during the Winter season. The polymers detected were poly(methyl-2-methylpropenoate), polystyrene, polyethylene terephthalate, polyamide, polypropylene, styrene butadiene, polyvinyl chloride and low /high density polyethylene with the last being the most frequent. Biofilms were more often detected on plastics than on natural materials. In addition, biofilms detected on plastics were more complex with higher microbial diversity (e.g., bacteria, fungi/yeast and phytoplancton organisms) and richer in extrapolymeric material. Based on morphological analysis a good agreement between microbiota and microorganism present in the biofilms was found. Among microbiota were identified microorganisms previously linked to plastic and PAHs detoxification suggesting the need for further studies to evaluate the viability of using biofilms as part of a green bioremediation strategy to mitigate water pollution. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 730872. We thank INSA for finantial support [2020DSA1756, 2020] info:eu-repo/semantics/publishedVersion

The target of this study was the tungsten Regoufe mine, whose exploitation stopped in the 1970s. When the mine closed, an unacceptable legacy constituted of mining waste tailings and the ruins of infrastructure was left behind. This work assessed the soil, plants, and water contamination in the mining area; namely their content in potentially toxic elements (PTEs). The global impact of PTEs in the Regoufe mine surface soil points to a very high to ultrahigh degree of contamination of the area having a serious ecological risk level, mainly related to As and Cd contributions. However, establishing the direct relation between As contamination and the anthropogenic effects caused by the mining process cannot be carried out in a straightforward manner, since the soils were already enriched in metals and metalloids as a result of the geological processes that gave origin to the mineral deposits. The studies performed on the plants revealed that the PTE levels in the plants were lower than in the soil, but site-specific soil concentrations in As and Pb positively influence bioaccumulation in plants. The magnetic studies showed the presence of magnetic technogenic particles concentrated in the magnetic fraction, in the form of magnetic spherules. The magnetic technogenic particles probably result from temperature increases induced by some technological process related to ore processing/mining activity. The PTEs in the surface and groundwater samples were similar and relatively low, being unlikely to pose potential health and environmental risks. Arsenic (As) constituted the exception, with levels above reference for drinking water purposes.

Two water effluents that drain from the abandoned coal mine of São Pedro da Cova (NW Portugal) were characterized in terms of their physic-chemical properties and suitability for irrigation purposes. Samples were also collected in a local surface stream, upstream and downstream from the mine drainage points, also used for irrigation by local farmers. Water samples were analyzed for major and minor ions and for trace element concentrations. Sampling campaigns started in 2017 and ended in 2019 and there were 46 water quality parameters tested. There were also proposed all-inclusive indices (the Water Quality Index and the Contamination Index, and also the Trace Element Toxicity Index) based on specific groups of 18 and 17 physic-chemical parameters, respectively, to achieve adequate monitoring requirements for mine effluents and surface water from coalfield. From the physical and chemical aspects of mine water it is inferred that the mine is not producing acid mine drainage. The coal mine water is of medium to high salinity, having almost neutral pH and a high thermal stability during the year, which is a distinguishing feature of the effluents. When compared to international irrigation water quality standards, as Food and Agriculture Organization of the United Nations admissible concentrations, the impacted waters are unsuitable for irrigation. The major outliers to the guidelines were iron, manganese, potassium, magnesium and bicarbonates, being also detected carcinogenic polycyclic aromatic hydrocarbons. Cost-effective ways of monitoring water quality parameters are needed to help control and manage the impact of coal mine effluents that should be treated before releasing into a ditch system that could be then used by local farmers to irrigate their crops.

Haloacetic acids (HAAs) are undesirable disinfection by-products (DBPs), released into aquatic ecosystems from various anthropogenic and natural sources. The aim of this study was to examine the ecological risk of exposure to three HAAs commonly detected in water, such as monobromoacetic acid (MBA), monochloroacetic acid (MCA), and trichloroacetic acid (TCA), in in vivo acute and chronic toxicity tests using Daphnia magna as a model. Acute tests showed that MBA was the most toxic of these compounds followed by MCA and TCA as evidenced by immobilization. Aquatic organisms in natural conditions might be exposed simultaneously to numerous compounds; thus, binary mixtures of selected HAAs and a ternary mixture of these were tested. Concentration addition (CA) and independent action (IA) models were used for a predictive assessment of mixture toxicity. Data demonstrated that CA appeared to be the most reliable indicator for HAAs binary and ternary mixtures suggestive of an additive behavior. Median effective concentration (EC50) values from the mixed exposure tests were significantly lower than results obtained from single tests for all three HAAs where an increase of toxicity greater than 50%. Multigenerational chronic tests were also performed exposing daphnids to the ternary mixture of HAAs. A markedly decreased sexual maturity and number of offspring and broods per daphnid especially in the second generation were noted. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013. A. Melo wishes to thank the Fundação Ciência Tecnologia grant SFRH/BPD/86898/2012. info:eu-repo/semantics/publishedVersion

The São Pedro da Cova waste pile (Porto, Portugal) is composed of coal mining residues that have been self-burning since 2005 and is located close to an inhabited area and social infrastructures, further adding to effects on the environment and human health. Therefore, there is a great interest in the environmental monitoring of this waste pile. This work describes an integrative multi-approach that allows the environmental monitoring of several parameters of the waste pile, applying several technologies. The temperature measurements were obtained by a thermal infrared (TIR) sensor on board an unmanned aerial vehicle (UAV) and supplemented with field measurements. In order to evaluate the altimetric variations, for each flight, a digital elevation model (DEM) was generated considering a multispectral sensor also on board the UAV. The hydrogeochemical characterization was performed through the analysis of groundwater and surface water samples, with and without the influence of mine drainage. The soil monitoring included the analysis of waste material as well as the surface soil in the surrounding area of the waste pile. All the data were analyzed and integrated in a geographical information system (GIS) open-source application. The adopted multi-approach methodology, given its intrinsic interdisciplinary character, has proven to be an effective way of encompassing the complexity of this type of environmental problem.

Groundwater data from 15 springs at Serra da Estrela mountain, located at the Seia-Torre-Covilha sector, were collected between 2010 and 2011 along six fieldwork campaigns. Isotopic (2H; 18O, and 3H) and chemical determinations were performed. The data were compared with the isotopic and chemical composition of seven alpine ponds located within the same area. The local meteoric water line (δ2H = 6.58(±0.29) + 1.31(±1.23)) was calculated using groundwater isotopic data. The results show that the springs located at the highest altitudes are more depleted than those located downhill. A slightly different isotopic fractionation with altitude was found between the western and eastern slopes of Serra da Estrela Natural Park (0.12 ‰ versus 0.10 ‰ /100 m in δ18O, respectively). The groundwaters are hyposaline, mainly Cl-Na-type waters, with slightly acidic pH reflecting a shallow and short flow path and small residence time. The alpine ponds also show Cl-Na facies and are mainly recharged by direct precipitation. However, in two alpine ponds (L2 and L6), evidences for groundwater recharge were identified. The physical, chemical, and isotopic composition allowed the identification of: (i) contribution of de-icing into the groundwater and alpine ponds systems; (ii) contribution of the shallow groundwater flow toward the alpine ponds systems recharge.

The investigation about wildfires has demonstrated that research should include studies on the overall assessment of the processes, thus promoting the public awareness about impacts. The aim of this study is to assess the changes on the carbon content of soils affected by wildfires in Caramulo Mountain (Portugal) to therefore identify the environmental impacts arisen from those changes. Soils from different parent rocks, affected and non-affected by wildfires, were collected. Petrographic and geochemical methods were used to identify changes caused by the wildfires in the organic fraction of soils. The results demonstrate that changes in soils composition after wildfires include the production of charcoal and pyrogenic polycyclic aromatic hydrocarbons (PAH). The incorporation of charcoal from biomass burning in soils overtime and the production of pyrolytic PAH are of environmental concern since these compounds are known to be harmful to the environment and ecosystems and are human carcinogens. The concentration of BkF-benzo[k]fluoranthene, known as probable human carcinogen, exceeds the reference values for contaminated soils. Once in soils these compounds can be removed by percolation affecting waters and, consequently, biodiversity and human health.

Peri-urban areas are territories that combine urban and rural features, being particularly vulnerable to wildfire due to the contact between human infrastructures and dense vegetation. Wildfires may cause considerable direct and indirect effects on the local water cycle, but the influence on groundwater quality is still poorly understood. The aim of this study was to characterize the chemistry of several springs connected to small public supply systems in a peri-urban area, following a large wildfire that took place in October 2017. Groundwater samples were collected in four springs that emerged within burned forests, while control samples were from one spring located in an unburned area. Sampling took place from October 2017 until September 2018, starting 15 days after the wildfire occurrence, to evaluate the influence of the time after fire and the effect of precipitation events on groundwater composition. Groundwater samples collected in burned areas presented increased content of sulfate, fluoride and nitrogen and variability in pH values. Iron, manganese and chromium contents also increased during the sampling period. Post-fire concentrations of polycyclic aromatic hydrocarbons (PAHs), mainly the carcinogenic ones, increased especially after intense winter and spring rain events, but the levels did not exceed the guideline values for drinking water.

Water from mountainous regions is a strategic natural resource. In Mediterranean mountainous regions, which, in many cases, correspond to protected areas, high-altitude roads are often the main threat to the sustainability of water resources. In these regions, the regular socioeconomic functioning requires frequent road de-icing operations which normally consist of spreading NaCl and other chemicals, such as CaCl2, in pavements. The main purpose of this research is to assess the environmental impact of road de-icing on groundwater resources in a Mediterranean mountainous region and to describe it by means of a hydrogeological conceptual model. The research focused in a cross-sectional sector located in Serra da Estrela (Central Portugal), where a hydrogeological inventory was carried out, followed by hydrogeochemical and hydrogeophysical studies. The results clearly identify different hydrogeochemical signatures in polluted (Cl–Na facies and higher EC) and unpolluted (HCO3–Na, Cl–Na, and very low EC). The relation of hydrogeochemistry and altitude is complex and depends on both natural processes (namely, water–rock interaction) and anthropic processes (de-icing operations). The hydrogeophysical survey systematically identified the presence of a pollution plume migrating downstream from roads.

The importance of Mountains for the world's water supply has been increasingly recognized. However, in many cases the existing roads require regular de-icing operations which may have a severe impact on the natural hydrogeochemical conditions of the local hydrological systems due to the regular use of substances such as sodium chloride and calcium chloride. The Serra da Estrela Mountain is a strategic area for the water resources management in Portugal because of its geological, geomorphological and climatic framework. This study was carried out in the highest part of the mountain, where road de-icing is frequent during winter. The hydrogeochemical characteristics of several springs, located downstream from roads between 1500 m a.s.l. and 1990 m a.s.l., present clear anthropic signatures which disturbs the quality of the local water resources.

Haloacetic acids (HAAs) are undesirable disinfection by-products (DBPs), released into aquatic ecosystems from various anthropogenic and natural sources. The aim of this study was to examine the ecological risk of exposure to three HAAs commonly detected in water, such as monobromoacetic acid (MBA), monochloroacetic acid (MCA), and trichloroacetic acid (TCA), in

Pinho C., Soares M.T., Almeida I.F., Aguiar A.A.R.M., Mansilha C., Ferreira I.M.P.L.V.O. (2015): Impact of freezing on flavonoids/radical-scavenging activity of two onion varieties. Czech J. Food Sci., 33: 340–345. Flavonols, anthocyanins, and radical-scavenging activity of two Portuguese onion cultivars (Branca da Povoa, white; and Vermelha da Povoa, red) were evaluated simulating domestic freezing conditions (–18°C). Frozen portions of onions with different periods of domestic storage at ambient temperature presented increased flavonoid content when compared with the respective composition before freezing. No significant differences were observed on radicalscavenging activity. Domestic freezing of onion portions extended its shelf life. Thus, domestic freezing can be a good alternative to prevent the loss of unused fresh onions, preserving its antioxidant capacity, since frozen onions can be a useful natural antioxidant source.

This work deals with the optimization of a rapid, cost-effective, and eco-friendly gas chromatography with mass spectrometry method for the simultaneous determination of four endocrine disruptor compounds in water matrices: estrone, 17β-estradiol, 17α-ethinylestradiol, and bisphenol A, that are currently considered to be of main concern in the field of water policy and that could became candidates for future regulations. The method involves simultaneous derivatization and extraction of compounds by dispersive liquid-liquid microextraction followed by gas chromatography with mass spectrometry analysis. Derivatization and extraction parameters were optimized with the aid of experimental design approach. An excellent linear response was achieved for all analytes (r(2) ≥ 0.999). Limits of detection and quantification are 0.003-0.005 and 0.0094-0.0164 μg/L, respectively. Intraday precision ranged between 1.1 and 12.6%, whereas interday precision ranged between 0.5 and 14.7%. For accuracy, bias values varied between -15.0 and 13.7%. Recoveries at three concentration levels ranged from 86.4 to 118.2%. The proposed method can be applied to the routine analysis of groundwater, river, sea, tap, and mineral water samples with excellent sensitivity, precision, and accuracy.

A new method for simultaneous extraction and quantification of 6 nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) and 16 parent polycyclic aromatic hydrocarbons (PAHs) in water matrices was optimized and validated. The extraction procedure was based on dispersive liquid-liquid microextraction technique, followed by gas chromatography-mass detection. The optimum conditions of extraction (volume of the extraction solvent, dispersive solvents and amount of salt) were selected using central composite design. The best results were found by using 200 μL of acetonitrile as dispersive solvent, 60 μL of chloroform as extraction solvent, and 10% (w/v) NaCl. Excellent linearity was observed in the range of 10–150 ng L−1 with correlation coefficients (r2) ranging between 0.9996 and 0.9999 for nitro-PAHs and in the range of 5–150 ng L−1 with r2 ranging from 0.9998 to 1.000 for PAHs. The limits of detection for the nitro-PAHs studied ranged from 0.82 to 3.37 ng L−1, whereas for PAHs ranged from 0.62 to 3.48 ng L−1. The intra- and inter-day precisions for nitro-PAHs were in the range of 0.45 to 19.54% and 0.43 to 19.62%, respectively, and for PAHs ranged between 0.45 to 17.42% and 0.38 to 18.97%, respectively. The proposed method was successfully applied in analyses of groundwater, sea, rain water and river water, being appropriate for routine analyses. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013. One of the authors (A. Melo) wishes to thank the Fundação Ciência Tecnologia, grant SFRH/BPD/86898/2012. info:eu-repo/semantics/publishedVersion

Water quality alterations due to forest fires may considerably affect aquatic organisms and water resources. These impacts are cumulative as a result of pollutants mobilized from fires, chemicals used to fight fire, and postfire responses. Few studies have examined postfire transport into water resources of trace elements, including the polycyclic aromatic hydrocarbons (PAH), which are organic pollutants produced during combustion and are considered carcinogenic and harmful to humans. PAH are also known to adversely affect survival, growth, and reproduction of many aquatic species. This study assessed the effects of forest wildfires on groundwater from two mountain regions located in protected areas from north and central Portugal. Two campaigns to collect water samples were performed in order to measure PAH levels. Fifteen of 16 studied PAH were found in groundwater samples collected at burned areas, most of them at concentrations significantly higher than those found in control regions, indicating aquifer contamination. The total sum of PAH in burned areas ranged from 23.1to 95.1 ng/L with a median of 62.9 ng/L, which is one- to sixfold higher than the average level measured in controls (16.2 ng/L). In addition, in control samples, the levels of light PAH with two to four rings were at higher levels than heavy PAH with five or six rings, thus showing a different profile between control and burned sites. The contribution of wildfires to groundwater contamination by PAH was demonstrated, enabling a reliable assessment of the impacts on water quality and preparation of scientifically based decision criteria for postfire forest management practices.

Trihalomethanes (THMs) are the most common disinfection by-products formed by the reaction of chlorine and/or bromide with organic matter. Total THMs in chlorinated treated water are defined as the sum of the concentrations of chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM). The monitoring of trihalomethanes in distribution tap water collected in from five regions of Portugal, from North to South, in a total of 33 samples, during spring to summer season, is described. Solid phase microextraction coupled to gas chromatography and electron capture detector methodology was applied to determine whether THMs concentrations in urban water supply systems are is in agreement with European Regulations. It was found a great discrepancy in total THMs concentration values, with concentrations varying from 1.8-97.7 μg/L, although the results were below the European parametric value of 100 μg/L. Regarding the Environmental Protection Agency regulations, however, only 97% of samples fulfilled the established criterion of 80 μg/L. and investigate if the individual compounds trihalomethanes content in was also investigated in order to determine if there is observed distribution tap water follows a common or a different THMs pattern in close geographical areas. In the geographical areas under study, mean values of concentrations measured revealed two different clusters: one including the three groups from the North geographical areas, with samples revealing a similar pattern (TCM>BDCM>DBCM>TBM), and another including the two groups from South with a different THMs pattern (DBCM>BDCM>TCM>TBM). A great degree of variation in the concentrations of THMs in tap water was observed, although the results the total THMs concentrations were below the European parametric value (100 μg/L). Mean results of samples grouped by five geographical areas revealed a similar pattern in samples collected from North and Centre TCM>BDCM>DBCM>TBM, whereas samples from South presented a different pattern, DBCM>BDCM>TCM>TBM. The origin of raw water (surface or groundwater) seems to influence this different pattern formation of brominated compounds. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013. One of the authors (A. Melo) wishes to thank the Fundação Ciência Tecnologia grant SFRH/BPD/86898/2012. info:eu-repo/semantics/publishedVersion

In NW of Portugal, the region between Esposende and Vila do Conde was established into a unique system called masseiras; masseiras are small fields that were created at the beginning of XX century by removing sand from dunes. Since the 80’s sand between closed masseiras has been removed giving way for larger fields, the “enlarged masseira with greenhouses”. This region is part of vulnerable zone nº1 (VZ1). Nowadays, the masseiras have almost disappeared; they had been replaced by “enlarged masseiras with plastic greenhouses” exploited by a new generation of growers. As water quality is an indicator of agriculture practices, 8 wells were selected and water samples were collected on four dates from September 2009 to May 2010. Laboratorial analysis was performed on nitrates and 28 pesticides. The results confirm that water in VZ1 is contaminated with nitrates (as it was expected) and also with pesticides. The water was contaminated in both dug wells and drilled wells all year round. Masseiras’ wells have more nitrates than greenhouses’ wells, probably due to poor agricultural practices used by growers who still employ high quantities of fertilizers and water. Pesticides detected in water samples include some that have been sprayed in the field and others that have not. Some forbidden substances were found. Our results confirm that, in VZ1, there is a diffusion contamination, which origin is unknown. In this communication, we put in picture the history of masseiras fields: how masseiras fields have been created and how they have disappeared; what trends exist in pest management strategies and the use of fertilizers, pesticides and water resources. These results reinforce the need to change the agricultural system in this region of traditional vegetable production.

Hundreds of different hydrocarbon compounds derived from agricultural, industrial and domestic practices are released daily into aquatic systems with adverse effects in the desirable potable characteristics, in aquatic fauna and flora, and on tourism, recreation and aesthetics of the impacted areas. Total petroleum hydrocarbon concentration data cannot be used to quantitatively estimate human health risk, but can be used as an important tool for three purposes: verifying if there is a problem, assessing the severity of the contamination and following the progress of a remediation effort. The objective of this work was the optimization of the experimental conditions for the determination of total hydrocarbons in water by liquid–liquid extraction coupled to infrared spectrophotometry, with the goal of improving the qualitative information provided by the spectra. The method was validated with satisfactory detection and quantification limits and demonstrated acceptable levels of precision, accuracy and analyte recoveries. Solid phase extraction and microwave assisted extraction procedures were tested and compared to the liquid extraction. Additionally, a spectra library was prepared with different natural and synthetic oils. The developed method was applied to the analysis of Portuguese inland and coastal bathing waters and wastewaters and their compliance with national and European legislation was assessed.

Optimization of conditions for anthocyanin hydrolysis from red wine was investigated using response surface methodology. The aglycon forms of the anthocyanins were quantified by high-performance liquid chromatography with diode array detection. The combined effects of three independent variables, HCl amount, heating temperature, and hydrolysis time, were studied using a 2(3) full-factorial central composite design. Anthocyanin hydrolysis yield depended mainly on the heating temperature and time of hydrolysis. HCl amount was the factor that least influenced the hydrolysis of anthocyanins. From experimental results, the maximum yield of anthocyanidins was reached with 9.8 mL of HCl (32% v/v), a heating temperature of 166.2 °C, and a hydrolysis time of 46.6 min. Five anthocyanidins, namely, delphinidin, cyanidin, petunidin, peonidin, and malvidin, were quantified in red wine. The reliability of the method was confirmed by recovery experiments, performed under optimal conditions. Recoveries indicated that anthocyanidins resisted the hydrolysis conditions.

The application of an odorant binding protein for odour control and fragrance delayed release from a textile surface was first explored in this work. Pig OBP-1 gene was cloned and expressed in Escherichia coli , and the purified protein was biochemically characterized. The IC50 values(concentrations of competitor that caused a decay of fluorescence to half-maximal intensity) were determined for four distinct fragrances, namely, citronellol, benzyl benzoate,citronellyl valerate and ethyl valerate. The results showed a strong binding of citronellyl valerate,citronellol and benzyl benzoate to the recombinant protein, while ethyl valerate displayed weaker binding. Cationized cotton substrates were coated with porcine odorant binding protein and tested for their capacity to retain citronellol and to mask the smell of cigarette smoke. The immobilized protein delayed the release of citronellol when compared to the untreated cotton. According to a blind evaluation of 30 assessors, the smell of cigarette smoke, trapped onto the fabrics’ surface, was successfully attenuated by porcine odorant binding protein (more than 60 % identified the weakest smell intensity after protein exposure compared to β-cyclodextrin-treated and untreated cotton fabrics). This work demonstrated that porcine odorant binding protein can be an efficient solution to prevent and/orremove unpleasant odours trapped on the large surface of textiles. Its intrinsic properties make odorant binding proteins excellent candidates for controlled release systems which constitute a new application for this class of proteins., This work was co-funded by the European Social Fund through the management authority POPH and FCT. The authors Carla Silva and Teresa Matama would like to acknowledge their post-doctoral fellowships: SFRH/BPD/46515/2008 and SFRH/BPD/47555/2008, respectively.

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children's exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1 ± 4.3 % to 264.7 ± 3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3 ± 1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5 ± 2.0 % to 118.8 ± 1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose-response relationship or a mixture toxicity effect.

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A new sample preparation procedure combining QuEChERS and dispersive liquid–liquid microextraction (DLLME) was optimized for the determination at trace levels of 13 pesticides from different chemical families (i.e. 2,4-D, acetamiprid, bentazone, cymoxanil, deltamethrin, dicamba, diuron, foramsulfuron, mesotrione, metalaxyl-M, methomyl, pyraclostrobin and tembotrione) in tomato by high-performance liquid chromatography with diode array detection. Target pesticides from tomato samples were isolated by liquid partitioning with acetonitrile and salts and cleaned up by dispersive solid-phase extraction (d-SPE); the analytes were concentrated in trichloromethane by the DLLME procedure. The disperser solvent from DLLME was used at the same time as carrier of analytes form extraction in QuEChERS method. The main factors affecting sample cleanup by d-SPE in QuEChERS and DLLME yield were optimized by means of an experimental design. Under the optimum conditions, good linearity was obtained, the recoveries of pesticides in tomato samples at spiking levels between 0.01 and 1.00 mg/kg ranged from 86 to 116 % (for foramsulfuron and cymoxanil, respectively). Precision was within 15.0 % (RSD) except at the LQ for tembotrione, which was 17.4 %. Limits of quantification achieved (ranging from 0.0058 to 0.15 mg/kg) were below the maximum residue limits established by the European Union.

A new method was developed for the determination of 10 pesticides widely used in lettuce production (acetamiprid, azoxystrobin, cyprodinil, fenhexamid, fludioxonil, folpet, iprodione, metalaxyl, pirimicarb, and tolyfluanid) using solid-phase microextraction (SPME) and liquid chromatography (HPLC) with diode-array detection (DAD). The extraction performance of four different SPME coatings, polydimethylsiloxane (PDMS), PDMS/divinylbenzene (PDMS/DVB), carbowax/templated resin (CW/TPR), and polyacrylate (PA) was evaluated using an interface SPME–HPLC. CW/TPR fibre was selected as the most appropriate for the extraction of majority of these pesticides. Three variables (pH, NaCl% and extraction time) were considered key factors in the optimisation process. Interactions between these analytical factors and their optimal levels were investigated by response surface methodology based on central composite design. The method allowed the determination of azoxystrobin, cyprodinil, fenehexamid, fludioxonil, folpet, iprodione, and tolyfluanid in lettuce at concentrations between 0.8 and 25.6 mg/kg, i.e., bellow the maximum residues levels allowed for those compounds in lettuce. Lettuce samples that suffered pesticide treatments with folpet and fenehexamid were analysed during days to harvest to study the dissipation behaviour of the pesticides used. Concentration of folpet was 92.8; 53.4; 22.9; 17.9; 7.45; 1.85 mg/kg and concentration of fenhexamid was 158.1; 76.2; 31.0; 27.3; 7.24; 0.87 mg/kg, respectively for t0, t1, t3, t6, t7, t9 days, and not detected at t14 days for the two pesticides. FCT grant SFRH/BD/41764/2007

Environmental pollution continues to be an emerging study field, as there are thousands of anthropogenic compounds mixed in the environment whose possible mechanisms of toxicity and physiological outcomes are of great concern. Developing methods to access and prioritize the screening of these compounds at trace levels in order to support regulatory efforts is, therefore, very important. A methodology based on solid phase extraction followed by derivatization and gas chromatography-mass spectrometry analysis was developed for the assessment of four endocrine disrupting compounds (EDCs) in water matrices: bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol. The study was performed, simultaneously, by two different laboratories in order to evaluate the robustness of the method and to increase the quality control over its application in routine analysis. Validation was done according to the International Conference on Harmonisation recommendations and other international guidelines with specifications for the GC-MS methodology. Matrix-induced chromatographic response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been overtaken by a weighted least squares linear regression model application. Consistent evaluation of key analytical parameters such as extraction efficiency, sensitivity, specificity, linearity, limits of detection and quantification, precision, accuracy and robustness was done in accordance with standards established for acceptance. Finally, the application of the optimized method in the assessment of the selected analytes in environmental samples suggested that it is an expedite methodology for routine analysis of EDC residues in water matrices.

A multiclass and multiresidue method for pesticide analysis in tomato was validated. Extraction and pre-concentration of the pesticide residues from acetonitrile extracts was performed by using dispersive liquid-liquid microextraction (DLLME) technique, followed by gas chromatography-mass detection. DLLME was performed using carbon tetrachloride as extractive solvent and acetonitrile extract as dispersive solvent, in order to increase enrichment factor of the extraction procedure. Validation parameters indicated the suitability of the method for routine analyses of thirty pesticides in a large number of samples. In general, pesticide recoveries ranged between 70% and 110% and repeatability ranged between 1% and 20%. The proposed method was applied to the monitoring of pesticides in tomatoes grown during winter in greenhouses. Among the compounds considered in this work, cyprodinil was found in tomato at concentrations of 0.33 mg/kg, other pesticides like azoxystrobin, fenhexanid, tolyfluanid, lambda-cyhalothrin and trifloxystrobin were also detected, but, not quantified. (C) 2012 Published by Elsevier Ltd.

Infiltration galleries are among the oldest known means used for small public water fountains. Owing to its ancestral origin they are usually associated with high quality water. Thirty-one compounds, including pesticides and estrogens from different chemical families, were analysed in waters from infiltration galleries collected in Alto Douro Demarcated Wine region (North of Portugal). A total of twelve compounds were detected in the water samples. Nine of these compounds are described as presenting evidence or potential evidence of interfering with the hormone system of humans and wildlife. Although concentrations of the target analytes were relatively low, many of them below their limit of quantification, four compounds were above quantification limit and two of them even above the legal limit of 0.1 μg/L: dimethoate (30.38 ng/L), folpet (64.35 ng/L), terbuthylazine-desethyl (22.28 to 292.36 ng/L) and terbuthylazine (22.49 to 369.33 ng/L). FCT grant SFRH/BD/41764/2007

A monitoring program of nitrate, nitrite, potassium, sodium, and pesticides was carried out in water samples from an intensive horticulture area in a vulnerable zone from north of Portugal. Eight collecting points were selected and water-analyzed in five sampling campaigns, during 1 year. Chemometric techniques, such as cluster analysis, principal component analysis (PCA), and discriminant analysis, were used in order to understand the impact of intensive horticulture practices on dug and drilled wells groundwater and to study variations in the hydrochemistry of groundwater. PCA performed on pesticide data matrix yielded seven significant PCs explaining 77.67% of the data variance. Although PCA rendered considerable data reduction, it could not clearly group and distinguish the sample types. However, a visible differentiation between the water samples was obtained. Cluster and discriminant analysis grouped the eight collecting points into three clusters of similar characteristics pertaining to water contamination, indicating that it is necessary to improve the use of water, fertilizers, and pesticides. Inorganic fertilizers such as potassium nitrate were suspected to be the most important factors for nitrate contamination since highly significant Pearson correlation (r = 0.691, P

The increasing demands on recreational waters have made microbial contamination a matter of public and scientific concern. This study aimed to search for Salmonella spp. in waters classified according EU Directive 2006/7/EC, in order to assess associations between its prevalence and the concentration of the non-pathogenic new faecal indicators: Escherichia coli and intestinal enterococci. Although a statistically significant association was observed Salmonella was detected on beaches classified as "Good" (9.3%) and "Excellent" (14.4%) which compromises the idea that faecal indicators can be predictors of pathogens. Attending the high prevalence of Salmonella found (23.1%) it seemed important to improve the efficiency of the conventional analytical method (ISO 6340:1995), comparing its draft with SML-VIDAS Salmonella and two new chromogenic media: AES Salmonella Agar Plate (ASAP) and Simple Method Salmonella (SMS). ASAP showed the higher efficiency and can be recommended for a faster detection and presumptive identification of salmonellae in bathing waters.

A multi-residue methodology based on a solid phase extraction followed by gas chromatography-tandem mass spectrometry was developed for trace analysis of 32 compounds in water matrices, including estrogens and several pesticides from different chemical families, some of them with endocrine disrupting properties. Matrix standard calibration solutions were prepared by adding known amounts of the analytes to a residue-free sample to compensate matrix-induced chromatographic response enhancement observed for certain pesticides. Validation was done mainly according to the International Conference on Harmonisation recommendations, as well as some European and American validation guidelines with specifications for pesticides analysis and/or GC-MS methodology. As the assumption of homoscedasticity was not met for analytical data, weighted least squares linear regression procedure was applied as a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line, improving accuracy at the lower end of the calibration curve. The method was considered validated for 31 compounds after consistent evaluation of the key analytical parameters: specificity, linearity, limit of detection and quantification, range, precision, accuracy, extraction efficiency, stability and robustness.

Bathing water quality is an important public health issue, mainly because of fecal contamination. In 2006, the European Commission (EC) adopted a new directive with respect to recreational bathing waters that calls for stricter standards and reduces the number of laboratory tests done in routine beach monitoring from nineteen to two bacterial indicators, namely, Escherichia coli and intestinal enterococci, replacing policies of the EC Bathing Water Directive that have existed since 1976. Our practice in Portugal is in line with this international development, and this study demonstrates the equivalency of the new bacteriological parameters with the old. The water quality of 25 coastal beaches was surveyed using both new and old microbiological indicators of fecal contamination. Statistical analysis demonstrated equivalency of the results obtained for fecal coliforms with Escherichia coli and for fecal streptococci with intestinal enterococci.

Natural estrogens are a group of steroid hormones that include the main active hormones, 17ß-estradiol (E2), estrone and estriol. Among these compounds, E2 is recognized to be the most active estrogen synthesized in female ovaries. Environmental problems due to estrogenic compounds are mainly related to aquatic environments. Indeed, numerous studies confirmed the occurrence of these substances at concentrations of toxicological concern (e.g., the feminization of fish in large rivers and toxicological effects on wildlife). Generally, the main sources of estrogens are recognized as treated and untreated municipal and industrial effluents, as well as livestock wastes from agricultural practices, as sewage and manure often used as fertilizers. Moreover the steroid hormones found in the urine of mammals are largely present as inactive conjugates, however the behavior of these forms will deconjugate to rapidly release the free hormones in the environment. As a consequence, large quantities of estrogens are spread in environmental waters, where they may sorb to sediments and persist for relatively long periods. Recently, the development of new analytical equipment, namely tandem mass spectrometers coupled to LC and GC systems, allowed improvements in the sensitivity, selectivity, and speed of analysis. Such improvements in sensitivity and selectivity could also be accomplished by innovative sample preparation techniques, most of them with the added benefit to be easy to execute, cost effective, and environmental friendly. Concerning the evaluation of the biological effects of estradiol, several methods have recently been developed: on whole organisms (vitellogenine assay), cells (cell proliferation), yeast estrogen screen, ER CALUX and molecular assays. In this chapter, various aspects of the estradiol presence in environmental waters are discussed. An overview of the current legislation related to water quality is given. The work then focuses on the health and environmental impacts and evaluation methods. Then, finally, the example case studies illustrate the health effects of estradiol and its environmental impact.