To assess if the geochemical reactivity and human bioaccessibility of silver nanoparticles (AgNPs) in soils can be determined by routine soil tests commonly applied to other metals in soil, colloidal Ag was introduced to five pots containing urban soils (equivalent to 6.8 mg Ag kg 1 soil). Following a 45 days stabilization period, the geochemical reactivity was determined by extraction using 0.43 M and 2 M HNO3. The bioaccessibility of AgNPs was evaluated using the Simplified Bioaccessibility Extraction Test (SBET) the ‘‘Unified BARGE Method’’ (UBM), and two simulated lung fluids (modified Gamble’s solution (MGS) and artificial lysosomal fluid (ALF)). The amount of Ag extracted by 0.43 M and 2 M HNO3 soil tests was <8% and <50%, respectively of the total amount of Ag added to soils suggesting that the reactivity of Ag present in the soil can be relatively low. The bioaccessibility of Ag as determined by the four in vitro tests ranged from 17% (ALF extraction) to 99% (SBET) indicating that almost all Ag can be released from soil due to specific interactions with the organic ligands present in the simulated body fluids. This study shows that to develop sound soil risk evaluations regarding soil contamination with AgNPs, aspects of Ag biochemistry need to be considered, particularly when linking commonly applied soil tests to human risk assessment.

To assess if the geochemical reactivity and human bioaccessibility of silver nanoparticles (AgNPs) in soils can be determined by routine soil tests commonly applied to other metals in soil, colloidal Ag was introduced to five pots containing urban soils (equivalent to 6.8mgAgkg−1 soil). Following a 45days stabilization period, the geochemical reactivity was determined by extraction using 0.43M and 2M HNO3. The bioaccessibility of AgNPs was evaluated using the Simplified Bioaccessibility Extraction Test (SBET) the “Unified BARGE Method” (UBM), and two simulated lung fluids (modified Gamble’s solution (MGS) and artificial lysosomal fluid (ALF)).The amount of Ag extracted by 0.43M and 2M HNO3 soil tests was <8% and <50%, respectively of the total amount of Ag added to soils suggesting that the reactivity of Ag present in the soil can be relatively low. The bioaccessibility of Ag as determined by the four in vitro tests ranged from 17% (ALF extraction) to 99% (SBET) indicating that almost all Ag can be released from soil due to specific interactions with the organic ligands present in the simulated body fluids.This study shows that to develop sound soil risk evaluations regarding soil contamination with AgNPs, aspects of Ag biochemistry need to be considered, particularly when linking commonly applied soil tests to human risk assessment.

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-mu m filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 mu m after staining with 4',6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.

Having multidisciplinary applications, iron oxide nanoparticles can inevitably enter aquatic system and impact inhabitants such as fish. However, the studies in this context have ignored the significance of obvious interaction of iron oxide nanoparticles with other persistent co-contaminants such as mercury (Hg) in the modulation of the toxicity and underlying mechanisms of iron oxide nanoparticles and Hg alone, and concomitant exposures. This study aimed to evaluate lipid peroxidation (LPO) and its control with glutathione (GSH) and associated enzymes (such as glutathione reductase, GR; glutathione peroxidase, GPX; glutathione sulfo-transferase, GST) in European eel (Anguilla anguilla L.) hepatocytes exposed to stressors with following schemes: (i) no silica-coated iron oxide nanoparticles functionalized with dithiocarbamate (Fe3O4@SiO2/Si DTC, hereafter called ‘FeNPs’; size range 82±21 to 100±30 nm) or Hg, (ii) FeNPs (2.5 μg L−1) alone, (iii) Hg (50 μg L−1) alone and (iv) FeNPs + Hg concomitant condition during 0 to 72 h. The exhibition of a differential coordination between GSH regeneration (determined as GR activity) and GSH metabolism (determined as the activity of GPX and GST) was perceptible in A. anguilla hepatocytes in order to control FeNPs, Hg and FeNPs + Hg exposure condition-mediated LPO. This study revealed the significance of a fine tuning among GR, GPX and GST in keeping LPO level under control during FeNPs or Hg alone exposure, and a direct role of total GSH (TGSH) in the control of LPO level and impaired GSH metabolism under the concomitant (FeNPs + Hg) exposure. An interpretation of the fish risk to FeNPs in a multi-pollution state should equally consider the potential outcome of the interaction of FeNPs with other contaminants.

Having multidisciplinary applications, iron oxide nanoparticles can inevitably enter aquatic system and impact inhabitants such as fish. However, the studies in this context have ignored the significance of obvious interaction of iron oxide nanoparticles with other persistent co-contaminants such as mercury (Hg) in the modulation of the toxicity and underlying mechanisms of iron oxide nanoparticles and Hg alone, and concomitant exposures. This study aimed to evaluate lipid peroxidation (LPO) and its control with glutathione (GSH) and associated enzymes (such as glutathione reductase, GR; glutathione peroxidase, GPX; glutathione sulfo-transferase, GST) in European eel (Anguilla anguilla L.) hepatocytes exposed to stressors with following schemes: (i) no silica-coated iron oxide nanoparticles functionalized with dithiocarbamate (Fe₃O₄@SiO₂/Si DTC, hereafter called ‘FeNPs’; size range 82 ± 21 to 100 ± 30 nm) or Hg, (ii) FeNPs (2.5 μg L⁻¹) alone, (iii) Hg (50 μg L⁻¹) alone and (iv) FeNPs + Hg concomitant condition during 0 to 72 h. The exhibition of a differential coordination between GSH regeneration (determined as GR activity) and GSH metabolism (determined as the activity of GPX and GST) was perceptible in A. anguilla hepatocytes in order to control FeNPs, Hg and FeNPs + Hg exposure condition-mediated LPO. This study revealed the significance of a fine tuning among GR, GPX and GST in keeping LPO level under control during FeNPs or Hg alone exposure, and a direct role of total GSH (TGSH) in the control of LPO level and impaired GSH metabolism under the concomitant (FeNPs + Hg) exposure. An interpretation of the fish risk to FeNPs in a multi-pollution state should equally consider the potential outcome of the interaction of FeNPs with other contaminants.

Estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), are the major responsible for endocrine-disrupting effects observed in aquatic environments due to their high estrogenic potency, even at concentrations ranging from pgL(-1) to ng L(-1). Thus, it is essential to develop analytical methodologies suitable for monitoring their presence in water samples. Dispersive liquid-liquid microextraction (DLLME) was used as a pre-concentration step prior to the quantification of E2 and EE2 by enzyme-linked immunosorbent assay (ELISA). First, an evaluation of the effect of DDLME on the E2 and EE2 ELISA calibration curves was performed. Since the extraction procedure itself had an influence on the ELISA optical density (OD), it became necessary to subject, not only the samples, but also all the standards to the DLLME process. Working ranges were determined, being between 1.2 and 8000 ng L(-1), for E2, and between 0.22 and 1500 ng L(-1), for EE2. The influence of organic matter, both in the extraction and quantification, was evaluated and it was observed that its presence in the solution did not affect considerably the calibration curve. Recovery rates were also determined, ranging from 77% to 106% for ultrapure water and from 104% to 115% for waste water samples, the most complex ones in what concerns matrix effects. Results obtained when applying the proposed method to real water samples can be considered quite satisfying. Moreover, the obtained working ranges encompass values generally reported in literature, confirming the practical use of the method for environmental samples.

Estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), are the major responsible for endocrine-disrupting effects observed in aquatic environments due to their high estrogenic potency, even at concentrations ranging from pgL⁻¹ to ngL⁻¹. Thus, it is essential to develop analytical methodologies suitable for monitoring their presence in water samples. Dispersive liquid–liquid microextraction (DLLME) was used as a pre-concentration step prior to the quantification of E2 and EE2 by enzyme-linked immunosorbent assay (ELISA). First, an evaluation of the effect of DDLME on the E2 and EE2 ELISA calibration curves was performed. Since the extraction procedure itself had an influence on the ELISA optical density (OD), it became necessary to subject, not only the samples, but also all the standards to the DLLME process. Working ranges were determined, being between 1.2 and 8000ngL⁻¹, for E2, and between 0.22 and 1500ngL⁻¹, for EE2. The influence of organic matter, both in the extraction and quantification, was evaluated and it was observed that its presence in the solution did not affect considerably the calibration curve. Recovery rates were also determined, ranging from 77% to 106% for ultrapure water and from 104% to 115% for waste water samples, the most complex ones in what concerns matrix effects. Results obtained when applying the proposed method to real water samples can be considered quite satisfying. Moreover, the obtained working ranges encompass values generally reported in literature, confirming the practical use of the method for environmental samples.

The contamination of the terrestrial environment by disposal of tributyltin (TBT) by contaminated harbour sediments, sewage sludge and/or biocide products has been raising concerns and it may pose a risk to soil invertebrates and plants. This study aimed to improve the amount and quality of data for TBT toxicity in soils in order to assess the ecological risk of TBT to the terrestrial ecosystems. For this, bioassays were performed with the species Porcellionides pruinosus, Folsomia candida, Brassica rapa and Triticum aestivum to evaluate the toxic effects of TBT (as chloride) on these species. Additionally, this study contributed to increase the amount of data concerning TBT toxicity on soil dwelling organisms. The results showed a dose–response relationship between TBT concentration and the increase of toxicity in all species tested. These results were collated with results from literature to construct species sensitivity distributions (SSDs) and to calculate the hazardous concentration at 5% (HC5) for all data, for each type of soil and TBT formulation used. The HC5 value for TBT in soil was 2.06 mg TBT/kg soil dw. Little information is available concerning the concentrations of TBT in soils. In addition the predicted no-effect concentration (PNEC) value was determined to be 30 μg/kg soil. Only one study was found referring to TBT contaminated soils, and where TBT concentrations were lower than 0.024 μg TBT/kg for the wetland soil. Therefore it can be concluded that the real TBT concentrations determined represent low risk for environmental effects. In conclusion, the construction of SSDs and the calculation of HC5 using all the data available showed to be a more suitable method rather than the construction of several SSDs for each soil and TBT types. Further investigations concerning TBT concentrations and toxicity on soil organisms need to be performed to increase data and improve risk calculations.

The contamination of the terrestrial environment by disposal of tributyltin (TBT) by contaminated harbour sediments, sewage sludge and/or biocide products has been raising concerns and it may pose a risk to soil invertebrates and plants.This study aimed to improve the amount and quality of data for TBT toxicity in soils in order to assess the ecological risk of TBT to the terrestrial ecosystems. For this, bioassays were performed with the species Porcellionides pruinosus, Folsomia candida, Brassica rapa and Triticum aestivum to evaluate the toxic effects of TBT (as chloride) on these species. Additionally, this study contributed to increase the amount of data concerning TBT toxicity on soil dwelling organisms. The results showed a dose–response relationship between TBT concentration and the increase of toxicity in all species tested. These results were collated with results from literature to construct species sensitivity distributions (SSDs) and to calculate the hazardous concentration at 5% (HC5) for all data, for each type of soil and TBT formulation used.The HC5 value for TBT in soil was 2.06mgTBT/kg soil dw. Little information is available concerning the concentrations of TBT in soils. In addition the predicted no-effect concentration (PNEC) value was determined to be 30μg/kg soil. Only one study was found referring to TBT contaminated soils, and where TBT concentrations were lower than 0.024μgTBT/kg for the wetland soil. Therefore it can be concluded that the real TBT concentrations determined represent low risk for environmental effects. In conclusion, the construction of SSDs and the calculation of HC5 using all the data available showed to be a more suitable method rather than the construction of several SSDs for each soil and TBT types. Further investigations concerning TBT concentrations and toxicity on soil organisms need to be performed to increase data and improve risk calculations.

This study aimed to assess the influence of incubation conditions in the determination of bacterial production (BP). In order to achieve that goal, experimental setups were performed in situ and in the laboratory under both dark and light conditions. To test spatial and seasonal variations and the different natural light exposure of microorganisms, sampling was performed in two distinct zones of the estuary Ria de Aveiro (Portugal), typifying the marine and brackish water zones of the estuarine system. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments was used to monitor possible alterations in bacterial community composition induced by the incubation conditions. The results showed that BP determined in situ conditions significantly differed from in the laboratory. In the marine zone, a defined pattern of variation was detected, with consistent higher values of BP in laboratory dark conditions. This trend was not present in the brackish water zone. The seasonal and spatial variability of BP observed in field incubations was related to the physical–chemical proprieties of the water column, irradiance levels and the original community composition. The metabolic active profiles of bacteria were substantially different in the several incubation conditions, suggesting that methodological procedure influences the bacterial community composition, and the values of BP reported for aquatic ecosystems could be quite different from the real ones. In the light of these results, we suggest that BP determinations should be conducted under in situ conditions. However, due to execution limitations, BP needs to be frequently determined in the laboratory, and in this case, dark incubations provide more approximate values. This is the method routinely used, and although this incubation condition can cause stimulation of BP, the structure of the bacterial community is more similar to the one obtained with the in situ incubations.

This study aimed to assess the influence of incubation conditions in the determination of bacterial production (BP). In order to achieve that goal, experimental setups were performed in situ and in the laboratory under both dark and light conditions. To test spatial and seasonal variations and the different natural light exposure of microorganisms, sampling was performed in two distinct zones of the estuary Ria de Aveiro (Portugal), typifying the marine and brackish water zones of the estuarine system. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments was used to monitor possible alterations in bacterial community composition induced by the incubation conditions. The results showed that BP determined in situ conditions significantly differed from in the laboratory. In the marine zone, a defined pattern of variation was detected, with consistent higher values of BP in laboratory dark conditions. This trend was not present in the brackish water zone. The seasonal and spatial variability of BP observed in field incubations was related to the physical–chemical proprieties of the water column, irradiance levels and the original community composition. The metabolic active profiles of bacteria were substantially different in the several incubation conditions, suggesting that methodological procedure influences the bacterial community composition, and the values of BP reported for aquatic ecosystems could be quite different from the real ones. In the light of these results, we suggest that BP determinations should be conducted under in situ conditions. However, due to execution limitations, BP needs to be frequently determined in the laboratory, and in this case, dark incubations provide more approximate values. This is the method routinely used, and although this incubation condition can cause stimulation of BP, the structure of the bacterial community is more similar to the one obtained with the in situ incubations.

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit â 4)-β-D-GlcpA-(1â 4)-α-D-GalpNAc-(1â 3)-β-D-GlcpNAc-(1â 4)-β-D-GlcpA-(1â containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513â ±â 4â kDa (PDI, 1.42â ±â 0.01), 6.7â ±â 0.3â dl/g and 56â ±â 0.3â nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1â 3 and 1â 4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications. | RIAIDT, CACTUS at the University of Santiago de Compostela and in the "Manuel Rico" NMR laboratory (LMR) of the Spanish National Research Council (CSIC) were the 750 MHz and 800 MHz NMR spectra were carried out respectively. FCT/MEC for the financial support to CICECO (FCT UID /CTM /50011/2013; POCI-01-0145-FEDER-007679) and QOPNA research Unit (FCT UID/QUI/00062/2013), through national founds and co-financed by the FEDER, within the PT2020 Partnership Agreement. RNC (IF/00373/2014), CN (SFRH/BPD/100627/2014), and ASF (SFRH/BD/102471/2014) thank the FCT foundation for funding.

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit →4)-β-D-GlcpA-(1→4)-α-D-GalpNAc-(1→3)-β-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→ containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513 ± 4 kDa (PDI, 1.42 ± 0.01), 6.7 ± 0.3 dl/g and 56 ± 0.3 nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1→3 and 1→4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications.

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit →4)-β-D-GlcpA-(1→4)-α-D-GalpNAc-(1→3)-β-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→ containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513 ± 4 kDa (PDI, 1.42 ± 0.01), 6.7 ± 0.3 dl/g and 56 ± 0.3 nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1→3 and 1→4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications. | published