Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA

The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.     

The Organizational and Operational Boundaries of Triple Bottom Line Reporting: A Survey

This article responds to Gray’s (2002) call for normative research on social and environmental accounting (SEA) and Parker’s (2005) call for active engagement in the process of designing SEA systems. More specifically, our investigation focuses on the study of boundary setting for triple bottom line (TBL) reporting, an issue that has been given more attention by practitioners than by researchers. The study reviews the consequences of boundary setting for the discharge of organizational accountability, from which it develops a framework to investigate TBL reporting boundaries and then reports on an empirical survey of best practice. It concludes that organizations are strategically setting and disclosing their boundaries instead of discharging their accountability and argues that such strategies have far-reaching consequences, because reporting boundaries are not only reflective of organizations but also have a constitutive role in their definition. A further consequence is that it calls into question the use of voluntary labeling, such as “in accordance” with Global Reporting Initiative; one implication being that further research into technical developments in TBL reporting could contribute to the discharge of organizational accountability.     

Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples

We propose a new approach to evaluate the natural attenuation capacity of soil by using fluorescence in situ hybridization (FISH). A specific oligonucleotide probe AtzB1 was designed based on the sequence data of the atzB gene involved in the hydrolytic deamination of s-triazines; this gene, located in a multiple copy plasmid was detected by the optimized FISH protocol. Two agricultural soils (Lodi and Henares) with a history of simazine treatments, and two natural soils (Soto and Monza), without previous exposure to simazine, were studied. AtzB1 probe-target cells were found only in the agricultural soils and, in a greater percentage, in the Lodi soil, compared to the Henares one. Moreover, the greatest percentage of AtzB1 probe-target cells in Lodi was accompanied by a greater mineralization rate, compared to the Henares soil. The FISH method used in this study was suitable for the detection of simazine-degrading bacteria and could be a useful indicator of the potential of soil bioremediation.     

Intermediate distributions and primary yields of phenolic products in nitrobenzene degradation by Fenton's reagent

Nitrobenzene thermal degradation was investigated using the Fenton reagent in different experimental conditions. Reaction products were analyzed by HPLC, GC-MS, LC-MS and IC. The products obtained at different nitrobenzene conversion degrees show that degradation mainly involves successive hydroxylation steps of the aromatic ring and its subsequent opening followed by oxidation of corresponding aliphatic compounds. Our results show as primary reaction products: 4-nitrophenol, 3-nitrophenol, 2-nitrophenol, phenol and 1,3-dinitrobenzene, indicating that both hydroxylation and nitration reactions are involved. The formation of phenolic products can be explained by postulating an initial step of HO() addition to nitrobenzene ring. The mechanisms of primary reaction pathways are discussed and a detailed kinetic analysis to obtain the true primary yields of phenolic products is proposed. The relative yields observed for nitrophenol isomers do not follow the expected orientation according to deactivating characteristics of the nitro group but significantly depend on Fe(+2), Fe(+3), H(2)O(2) and O(2) concentrations. The understanding of the effect of reaction conditions on the relative product distribution benefits the application of Fenton and Fenton-like systems to waste water treatment.     

Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion

Resistance to fenitrothion and enzyme activities associated with the toxicity and metabolism of organophosphorus insecticides were measured in three genetically unique Daphnia magna clones collected from rice fields of Delta del Ebro (NE Spain) during the growing season and a lab sensitive clone. The studied clones showed up to sixfold differences in resistance to fenitrothion. The lack of correlation between in vitro sensitivity of acetylcholinesterase (AChE) to fenitrooxon and resistance to fenitrothion indicated that insensitivity of AChE to the most active oxon metabolite was not involved in the observed differences in resistance. Inhibition of mixed- function oxidases (MFOs) by piperonyl butoxide (PBO) increased the tolerance to fenitrothion by almost 20-fold in all clones without altering their relative ranking of resistance. Conversely, when exposed to fenitrooxon, the studied clones showed similar levels of tolerance, thus indicating that clonal differences in the conversion of fenitrothion to fenitrooxon by MFOs were involved in the observed resistance patterns. Despite that resistant clones showed over 1.5 higher activities of carboxilesterase (CbE) than sensitive ones, toxicity tests with 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2 oxide, which is a specific inhibitor of these enzymes, evidenced that this system only contributed marginally to the observed clonal differences in tolerance. Glutathione-S-transferases activity (GST) varied across clones but not under exposure to fenitrothion, and was only related with tolerance levels in the field clones. In summary, our results indicate that MFO mediated differences on the bio-activation of the phosphorotionate OP pesticide to its active oxon metabolite contributed mostly in explaining the observed moderate levels of resistance, whereas the activities of CbE and GST had only a marginal role.     

Historical trends in persistent organic pollutants and metals recorded in sediment from Lake Ellasjøen, Bjørnøya, Norwegian Arctic

Ellasj?en receives contaminants from both atmospheric and biological transport (seabird guano). Historical trends of selected persistent organic pollutants and metals were determined by interpretation of down-core contaminant profiles in conjunction with sediment layer ages, determined by the (210)Pb method. Polychlorinated biphenyls (PCBs) and dichloro-diphenyl-trichloroethane and metabolites (DDTs) increased from about 1930 until 1970 (max. sum PCBs 71.8 ng/g dry weight (dw), max sum DDTs 4.0 ng/g dw), declining afterwards to 46.4 ng/g dw and 1.6 ng/g dw, respectively. Polybrominated diphenylethers (PBDEs) concentrations are increasing in Ellasj?en, reaching 0.73 ng/g dw in 2001. Hexabromocyclododekane (HBCD) was measured only in sediment from 1-2 cm depth (0.43 and 3.88 ng/g dw of alpha- and gamma-HBCD, respectively). Most metals (As, Cd, Co, Ni and Zn) showed a similar pattern as PCBs. No clear trends were observed for Cr, Pb or V. Mercury (Hg) has increased gradually since the beginning of the industrial era.     

Sub-soil contamination due to oil spills in six oil-pipeline pumping stations in northern Mexico

Mexico has a very important oil industry, comprehending the exploration, production, transformation, storage and distribution of crude oil and its fractions. Petroleos Mexicanos (PEMEX) is a state-owned monopoly in charge of these activities. Oil and oil-products transport is an extremely crucial operation for the cycle production-transformation-distribution. Pipeline system transport crude oil and sub-products along the country (including liquids, gases and mixtures). It has been reported that more than 30% of the oil ducts in Mexico have been operating for over 30 years, although their lifetime has been calculated in 25-30 years. This work is aimed at characterizing a zone around six oil-pipeline pumping stations located at northern Mexico. The specific places to evaluate soil contamination were (1) the distribution head of the Gomez Palacio (GOPA) pumping station; (2) the north side of the old ditch, the API oil separator and the wastewater zones of the Jimenez (JIM) pumping station; (3) the pumping stations of Ceballos (CE), Peronal (PER), Simon Bolivar (SIBO), and Mayran (MAY). The study comprehended sampling of the areas, delimitation of contamination in the vertical and horizontal extension, analysis of the sampled soils, regarding TPH and, in some cases, the 16 PAHs considered as a priority by USEPA, calculation of areas and volumes contaminated (according to the Mexican legislation, specifically NOM-EM-138-ECOL-2002) and, finally, a proposal for the best remediation techniques suitable for the encountered contamination levels and the localization of contaminants. In general, TPHs were found in all the pumping stations analyzed in this study. Regarding maximal TPHs concentrations at the stations, their order of contamination was as follows: SIBO>CE>PER>MAY>JIM>GOPA. PAHs were found only in a few points at concentrations above the detection limit. At the Jimenez, Gomez Palacio, Peronal, and Ceballos stations, only one point, with PAHs values over the detection limit was found, whereas two points were found at Mayran, and finally at the Simon Bolivar station there were four points with measurable amounts of PAHs. Given the areas and volumes that need to be treated (32835 m3), as well as the physical position of contaminants, it is considered that excavation of the contaminated soil is feasible and the cleaning process could be soil washing assisted with surfactants, though bioremediation should not been discharged as a possible remediation technique.     

Influence of the chemical structure on mobility of PCB congeners in female and male sardine (Sardina pilchardus) from Portuguese coast

Concentrations of 30 polychlorinated biphenyl congeners (CBs) were determined in muscle, liver and gonad tissues of female and male sardines (Sardina pilchardus) collected in February-May 2000 and September-January 2001 from purse seine vessels operating around Peniche, Portugal. The determined CBs were aggregated in four groups (I, II, III and IV), according to similarities of their chemical structures. Muscle and liver of males contained significantly higher concentrations of groups I and II than females during the final period of spawning (February-April). A less contrast was observed for the groups III and IV. This seasonal fluctuation associated with the spawning indicates that female sardines regulate the excess of CBs sequestrated during fatten period. On the contrary, levels of groups I and II were higher in males than in females indicating slower mobility of these compounds as lipids were consumed during the final period of spawning. The deficient regulation of those CBs in muscle of male sardines can not be attributed to selective transfer to gonads and subsequent excretion. Although the full explanation was not found for the different mobility of PCBs with the gender, the results provide evidence of slower elimination of group I and II congeners in male sardines.     

Mercury and selenium in blue shark (Prionace glauca, L. 1758) and swordfish (Xiphias gladius, L. 1758) from two areas of the Atlantic Ocean

Muscle, liver and stomach contents of 64 blue sharks and 52 swordfishes, caught between September 2004 and February 2005 near the Azores (area A) and the Equator (area E), were analysed for mercury and selenium. Levels of mercury were relatively high (blue shark: 0.032-2.5microgg(-1); swordfish: 0.031-9.8microgg(-1)) and comparable to values reported in the literature. However, mercury and organic mercury concentrations in muscle and liver of specimens from E were significantly higher than those from A. A similar trend was registered in stomach contents, suggesting higher uptake of Hg in specimens from E. This difference was also observed in the relationship between concentration in muscle and size, indicating a higher accumulation rate in specimens from E. The accumulation of Se in the liver of both species showed a positive correlation with inorganic mercury concentrations, pointing to a detoxifying mechanism of organic mercury in these species through Se-Hg liasons.