Development and Integration of Quantitative Real-Time PCR Methods for Detection of Mitochondrial DNA and Methanobrevibacter smithii nifH Gene as Novel Microbial Source Tracking Tools

A novel microbial source tracking (MST) method based on the detection of human and non-human markers was developed and applied to track the origin of fecal pollution in water systems. Mitochondrial DNA sequences were used to develop new quantitative real-time polymerase chain reaction (qPCR) assays for dog, poultry, and gull. The targets were included as part of a toolbox including human, cow, pig, and sheep assays. A primer and probe set for the detection of the human-specific nifH gene of Methanobrevibacter smithii was also designed as an indicator of human fecal contamination. The assays were tested for specificity and applied to fecal-spiked surface waters and environmental samples collected from two river catchments impacted by sources of human and non-human fecal contamination. The MST methods described were applicable to both spiked waters and environmental samples, and using the two approaches the origin of fecal pollution could be successfully determined in mixed source fecally polluted waters.     

High throughput and miniaturised systems for biodegradability assessments

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.     

Arsenic and copper stabilisation in a contaminated soil by coal fly ash and green waste compost

In situ metal stabilisation by amendments has been demonstrated as an appealing low-cost remediation strategy for contaminated soil. This study investigated the short-term leaching behaviour and long-term stability of As and Cu in soil amended with coal fly ash and/or green waste compost. Locally abundant inorganic (limestone and bentonite) and carbonaceous (lignite) resources were also studied for comparison. Column leaching experiments revealed that coal fly ash outperformed limestone and bentonite amendments for As stabilisation. It also maintained the As stability under continuous leaching of acidic solution, which was potentially attributed to high-affinity adsorption, co-precipitation, and pozzolanic reaction of coal fly ash. However, Cu leaching in the column experiments could not be mitigated by any of these inorganic amendments, suggesting the need for co-addition of carbonaceous materials that provides strong chelation with oxygen-containing functional groups for Cu stabilisation. Green waste compost suppressed the Cu leaching more effectively than lignite due to the difference in chemical composition and dissolved organic matter. After 9-month soil incubation, coal fly ash was able to minimise the concentrations of As and Cu in the soil solution without the addition of carbonaceous materials. Nevertheless, leachability tests suggested that the provision of green waste compost and lignite augmented the simultaneous reduction of As and Cu leachability in a fairly aggressive leaching environment. These results highlight the importance of assessing stability and remobilisation of sequestered metals under varying environmental conditions for ensuring a plausible and enduring soil stabilisation.     

Determination of priority pesticides in water samples combining SPE and SPME coupled to GC–MS. A case study: Suquía River basin (Argentina)

This study reports a combined method using solid phase extraction (SPE), followed by solid phase microextraction (SPME) to concentrate different pesticides, including chlorinated, organophosphorus, triazines, pyretroids and chloroacetamides, present at trace levels in water samples. Identification and quantification was carried out by gas chromatography coupled to Mass Spectrometry (GC–MS). The optimized methodology showed LOQs at ng L^?1 levels (ranging 0.2–3.5 ng L^?1) in addition to acceptable precision and robustness (recoveries ranged 63–104%, RSD from 4% to 23%), presenting a novel method to reach trace levels, similar to that obtainable using EC detector, with structural confirmation by MS during the analysis of a wide range of environmental pollutants.This method was applied to the study of temporal and spatial distribution of pesticides in the Suquía River basin (Córdoba-Argentina). As expected, highest levels of agrochemicals were observed in areas with intensive agricultural practices, being atrazine (max. = 433.9 ng L^?1), alpha-cypermetrine (max. = 121.7 ng L^?1) and endosulfan sulfate (max. = 106.7 ng L^?1) predominant. In urban areas, the prevalent pesticide was alpha-cypermethrine. These results draw attention to the need of pesticide monitoring programs in rivers, considering both urban and rural sections.     

Inhibition of the antioxidant activity of catalase and superoxide dismutase from Fusarium verticillioides exposed to a Jacquinia macrocarpa antifungal fraction

The aim of this study was to investigate the in vitro effect of an antifungal fraction obtained from Jacquinia macrocarpa plant (JmAF) in the generation of reactive oxygen species (ROS) and the activity of the catalase (CAT) and superoxide dismutase (SOD) enzymes from Fusarium verticillioides, as well as their influence in the viability of the fungus spores. The compounds present in the JmAF were determined by gas chromatography/quadrupole time-of-flight mass spectrometry (GC/QTOF-MS). The effect of the exposition to JmAF on the generation of ROS, as well as in the CAT and SOD activities in F. verticillioides, was determined. The main compounds detected were γ-sitosterol, stephamiersine, betulinol and oleic acid. JmAF showed very high ability in inhibiting the spore viability of F. verticillioides, and their capacity to cause oxidative stress by induction of ROS production. JmAF induced the highest ROS concentration and also inhibited CAT and SOD activities. The results obtained in this study indicate that JmAF is worthy of being considered for the fight against phytopathogenic fungi.     

Field demonstration of visible opacity photographic systems

The Digital Opacity Compliance System (DOCS) is an innovative method that uses digital imaging technology to quantify visible opacity of stationary sources. DOCS, which has been demonstrated at pilot and full scale as a technically defensible and economically attractive alternative to U.S. Environmental Protection Agency (EPA) Reference Method 9 (Method 9), uses commercial-off-the-shelf (COTS) digital cameras in combination with a user-friendly computer software package to determine opacity. To date, all DOCS field testing has been conducted using two models of digital cameras, notably, Kodak Models DC265 and DC290, both of which are no longer commercially available. To ensure that field-validated digital cameras will be available to future DOCS users, a suite of new digital cameras was evaluated with the opacity determination software including the following: (1) Sony Model Cybershot Model DSC-WI, (2) Nikon Model Coolpix 5200, (3) Fuji Finepix Model E500, and (4) Kodak Model DX6490. Within the opacity range of regulatory interest, that is, 0-40%, the Sony Cybershot Model DSC-WI and Nikon Coolpix Model 5200 digital cameras were found to generate plume photographs of which the DOCS opacity analysis yielded results that were statistically equivalent to the previously field-validated Kodak Model DC290. In contrast, the Fuji Finepix Model E500 generated plume photographs of which the DOCS opacity analysis were, on average, 2.2% less than those generated by the Kodak Model DC290 photographs, a difference that was determined to be statistically significant. Over the same opacity range, photographs taken by the Kodak Model DX6490 yielded DOCS opacity readings that were found to be statistically equivalent to a Method 9-certified transmissometer. Based on the results from the current digital camera validation testing approach, EPA has developed a new camera-based visible opacity measurement method titled "Determination of Visible Emission Opacity from Stationary Sources Using Computer-Based Photographic Analysis Systems." The proposed method is expected to be promulgated after closure of the public comment period.     

Fast characterization of non domestic load in urban wastewater networks by UV spectrophotometry

Urban wastewater treatment plant efficiency, as well as biosolid quality, depends on urban wastewater quality, which can be affected by non domestic discharges (industrial, commercial etc.). The characterization of wastewater quality and non domestic discharge is complex, expensive and time consuming. However, these discharges must be controlled and reduced if possible. The development of a simple and fast methodology, namely based on alternative methods such as UV spectrophotometry, has been carried out and applied to different areas of a medium sized town of Southern Québec (Canada). Several autosamplers and on line/on site measurements have been used in critical control points of the network areas, for a dry weather campaign in four areas (industrial, commercial, hospital and university). The flow rate study, completed by the exploitation of conductivity measurements and the qualitative examination of UV spectra allows the discrimination of non domestic loads and their variability study from one point to another. The identification of critical discharges and organic shock loads has been possible with low investment, and mitigation actions have been proposed.     

Design to monitor trend in abundance and presence of American beaver (Castor canadensis) at the national forest scale

Wildlife conservationists design monitoring programs to assess population dynamics, project future population states, and evaluate the impacts of management actions on populations. Because agency mandates and conservation laws call for monitoring data to elicit management responses, it is imperative to design programs that match the administrative scale for which management decisions are made. We describe a program to monitor population trends in American beaver (Castor canadensis) on the US Department of Agriculture, Black Hills National Forest (BHNF) in southwestern South Dakota and northeastern Wyoming, USA. Beaver have been designated as a management indicator species on the BHNF because of their association with riparian and aquatic habitats and its status as a keystone species. We designed our program to monitor the density of beaver food caches (abundance) within sampling units with beaver and the proportion of sampling units with beavers present at the scale of a national forest. We designated watersheds as sampling units in a stratified random sampling design that we developed based on habitat modeling results. Habitat modeling indicated that the most suitable beaver habitat was near perennial water, near aspen (Populus tremuloides) and willow (Salix spp.), and in low gradient streams at lower elevations. Results from the initial monitoring period in October 2007 allowed us to assess costs and logistical considerations, validate our habitat model, and conduct power analyses to assess whether our sampling design could detect the level of declines in beaver stated in the monitoring objectives. Beaver food caches were located in 20 of 52 sampled watersheds. Monitoring 20 to 25 watersheds with beaver should provide sufficient power to detect 15–40% declines in the beaver food cache index as well as a twofold decline in the odds of beaver being present in watersheds. Indices of abundance, such as the beaver food cache index, provide a practical measure of population status to conduct long-term monitoring across broad landscapes such as national forests.