Restoring biochemical activity and bacterial diversity in a trichloroethylene-contaminated soil: the reclamation effect of vermicomposted olive wastes

Background, aim, and scope  In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored. Materials and methods  Trichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (β-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-β-glucosidase complexes was performed to study the enzymatically active humic matter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulation-division (RND) genes amplified from the contaminated soils. Results  Vermicompost reduced, with respect to the unamended soil, about 30% of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, β-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-β-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns. Discussion  Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular β-glucosidase activity, or between the latter and the quantity of humic carbon in soils. The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular β-glucosidase or in that linked to stable humic substances (humic–enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria. Conclusions  Trichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-β-glucosidase complexes located in the soil humic fraction >10⁴, increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil. Recommendations and perspectives  Since the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.     

African dust contributions to mean ambient PM10 mass-levels across the Mediterranean Basin

Data on mass-levels of PM₁₀ measured at regional background sites across the Mediterranean Basin, available from Airbase (European Environmental Agency) and from a few aerosol research sites, are compiled. PM₁₀ levels increase from north to south and west to east of the Basin. These variations are roughly coincident with the PM₁₀ African mineral dust load. However, when subtracting the African dust from mean PM₁₀ levels using a consistent methodology, the PM₁₀ background levels are still 5–10 μg m^?3 higher in the Eastern Basin (EMB) when compared with those in the Western (WMB), mainly due to the higher anthropogenic and sea spray loads.As regards for the seasonal trends, these are largely driven by the occurrence of African dust events, resulting in a spring-early summer maximum over the EMB, and a clear summer maximum in the WMB, although in this later region the recirculations of aged air masses play an important role. Furthermore, a marked seasonal trend is still evident when subtracting the African dust load. This is characterised by a high summer maximum (driven by low precipitation, high insolation) and a winter minimum (intense synoptic winds).Important inter-annual variations in the dust contribution are detected, more evident in the southern sites. These differences are generally associated with the occurrence of extreme dust events. Generally, the years with higher dust contributions over the EMB correspond with lower contributions over the WMB, and vice versa.The characterization of individual particles, collected in both basins during African dust events, by scanning electron microscopy reveals only slight differences between them. This fact probably reflects the high degree of mixture of mineral dust from different sources before the transport towards the receptor sites.     

Spatial and chemical patterns of PM10 in road dust deposited in urban environment

Recent research interest has been focused on road dust resuspension as one of the major sources of atmospheric particulate matter in an urban environment. Given the dearth of studies on the variability of the PM₁₀ fraction of road deposited sediments, our understanding of the main factors controlling this pollutant is incomplete. In the present study a new sampling methodology was devised and applied to collect PM₁₀ deposited mass from 1 m² of road pavement. PM₁₀ road dust fraction was sampled directly from active traffic lanes at 23 sampling sites during a campaign in Barcelona (Spain) in June 2007. The aim of the study was to gain more insight into the variability of mass and chemistry of road dust in different urban environments, such as the city centre, ring roads, and locations nearby demolition/construction sites. The city centre showed values of PM₁₀ road dust within a range of 3–23 mg m^?2, whereas levels reached 24–80 mg m^?2 in locations affected by transport of uncovered heavy trucks. The largest dust loads were measured in the proximity of demolition/construction sites and the harbor entry with values up to 328 mg m^?2.The city centre road dust profiles (%) were enriched in OC, EC, Fe, S, Cu, Zn, Mn, Cr, Sb, Sn, Mo, Zr, Hf, Ge, Ba, Pb, Bi, SO₄^2?, NO₃^?, Cl^? and NH₄⁺, but several crustal components such as Ca, Ti, Na, and Mg were also considerably concentrated. Locations affected by construction and demolition activities had high levels of crustal components such as Ca, Li, Sc, Sr, Rb and also As whereas ring roads, characterized by a higher load of uncovered heavy trucks showed an intermediate composition.Levels of PM₁₀ components per area were also evaluated to quantify the resuspendable amount of each element from 1 m². In the inner city environment mean values of 1363 μg Ca m^?2, 816 μg OC m^?2, 239 μg EC m^?2, 13 μg Cu m^?2, 12 μg Zn m^?2, 1.9 μg Sb m^?2 and 2.0 μg Pb m^?2, in PM₁₀ in all cases, were registered.Moreover the deposited PM load at demolition/construction sites acts as a reservoir or trap for traffic-related particles, which gives rise to large amounts of hazardous pollutants, available for resuspension.     

Metabolic and bacterial diversity in soils historically contaminated by heavy metals and hydrocarbons

The aim of this study was to characterize soils contaminated by different levels of heavy metals and hydrocarbons (Madonna Dell'Acqua, Pisa, Italy). The soils were chemically and biochemically analysed by measuring the standard chemical properties and some enzyme activities related to microbial activity (dehydrogenase activity) and the soil carbon cycle (total and extracellular beta-glucosidase activities). The metabolic capacities of soil microorganisms to degrade hydrocarbons through catechol 2,3-dioxygenase were also described. The microbial diversity of contaminated and uncontaminated soils was estimated by denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences. The PCR/single-strand conformation polymorphism (PCR/SSCP) method was used to estimate the genetic diversity of PAH-degrading genes in both contaminated and uncontaminated soils. A greater bacterial diversity and lower catechol 2,3-dioxygenase activity was detected in unpolluted soils. The complexity of the microbial community (Shannon and Simpson indices) as well as the dehydrogenase soil activity negatively correlated with contamination levels. The greatest PAH-degrading gene diversity and the most intense catechol 2,3-dioxygenase activity were found in the soils with the highest levels of hydrocarbons. Heavy metals and hydrocarbon pollution has caused a genetic and metabolic alteration in microbial communities, corresponding to a reduction in microbial activity. A multi-technique approach combining traditional biochemical methods with molecular-based techniques, along with some methodological improvements, may represent an important tool to expand our knowledge of the role of microbial diversity in contaminated soil.     

Spatial and temporal variations in inhalable CuZnPb aerosols within the Mexico City pollution plume

We report on the CuPbZn content of PM10 and PM2.5 samples collected from three sites (urban T0, suburban T1 and rural T2) during the Mexico City MILAGRO campaign of March 2006. Daytime city centre concentrations of summation operator CuZnPb(PM10) were much higher (T0 > 450 ng m(-3)) than at the suburban site (T1 < 200 ng m(-3)). Rural site (T2) summation operator CuZnPb(PM10) concentrations exceeded 50 ng m(-3) when influenced by the megacity plume but dropped to 10 ng m(-3) during clean northerly winds. Nocturnal metal concentrations more than doubled at T0, as pollutants became trapped in the nightly inversion layer, but decreased at the rural site. Transient spikes in concentrations of different metals, e.g. a "copper event" at T0 (CuPM10 281 ng m(-3)) and "zinc event" at T1 (ZnPM10 1481 ng m(-3)) on the night of March 7-8, demonstrate how industrial pollution sources produce localised chemical inhomogeneities in the city atmosphere. Most metal aerosols are <2.5 microm and SEM study demonstrates the dominance of Fe, Ti, Ba, Cu, Pb and Zn (and lesser Sn, Mo, Sb, W, Ni, V, As, Bi) in metalliferous particles that have shapes including spherical condensates, efflorescent CuZnClS particles, cindery Zn, and Cu wire. Metal aerosol concentrations do not change in concert with PM10 mass, which is more influenced by wind resuspension than industrial emissions. Metalliferous particles can induce cell damage, and PM composition is probably more important than PM mass, with respect to negative health effects, so that better monitoring and control of industrial emissions would likely produce significant improvements in air quality.     

Environmental diagnosis methodology for municipal waste landfills

A large number of countries are involved in a process of transformation with regard to the management of municipal solid waste. This process is a consequence of environmental requirements that occasionally materialise in legislation, such as the European Council Directive 31/99/EC on waste release in the European Union. In some cases, the remediation of old landfills can be carried out in compliance with environmental requirements; in other cases, it is necessary to proceed with the closure of the landfill and to assimilate it into its own environment. In both cases, it is necessary to undertake a diagnosis and characterisation of the impacted areas in order to develop an adequate action plan. This study presents a new methodology by which environmental diagnosis of landfill sites may be carried out. The methodology involves the formulation of a series of environmental indeces which provide information concerning the potential environmental problems of the landfills and the particular impact on different environmental elements, as well as information related to location, design and operation. On the basis of these results, it would be possible to draw up action plans for the remediation or closure of the landfill site. By applying the methodology to several landfills in a specific area, it would be possible to prioritize the order of actions required.     

Indicators of physiological and immunological status of <Emphasis Type="Italic">Litopenaeus setiferus</Emphasis> wild populations (Crustacea,Penaeidae)

Constant environmental changes in oceanic and estuarine systems due to human activity in coastal zones require different tools and strategies for their study to be able to assess the health status of aquatic ecosystems. The Gulf of Mexico has a tremendous ecological importance because of its biological diversity. Baseline levels of blood metabolites and immune components were defined for wild tropical populations of Litopenaeus setiferus, an important shrimp species of the Gulf of Mexico. Osmotic pressure, oxy hemocyanin (OxyHc), glucose, proteins, cholesterol, lactate, and triacylglycerols (TAG) were used as indicators of physiological status; pro phenoloxydase (proPO) and phenoloxydase (PO) quantification of hemocytes, as well as respiratory burst, were used to assess the immunological status. Significant differences were observed in live weight of juveniles, males, and females of L. setiferus with mean values of 10.54, 35.05, and 40.33 g, respectively. A value of 460 mOsm/kg was recorded in the water where juveniles were sampled and 1,000 mOsm/kg in adult sampling zones. Consequently, the osmotic pressure of L. setiferus juveniles (718.02 mOsm/kg) was 22% lower than in adults (925 mOsm/kg); there were no differences between sexes. A significantly lower value in juvenile OxyHc was observed. This value was 24 and 16% lower than that observed in males and females, respectively. Hemolymph glucose, cholesterol, and triacylglycerol levels were not statistically different between shrimp stages, showing median values of 0.19, 0.32, and 0.39 mg/ml of glucose, cholesterol, and triacylglycerols, respectively. Hemocyanin was distributed normally in the juvenile–male population but not in females. A proportion between 52 to 81% of OxyHc of the total protein (OxyHc/Prot) was obtained for all stages. Digestive gland glycogen showed no normal distribution in juveniles and adults. Digestive gland glycogen was significantly higher in juveniles (median value of 2.64 mg/g) than in adults (median value of 1.58 mg/g). A higher value of lactate (0.11 mg/ml) was observed in juveniles than in males (0.09 mg/ml) and females (0.04 mg/ml). Granular cells (GC) corresponded to 36, 44, and 39% of the total hemocytes (TH) observed in juveniles, males, and females. ProPO showed no normal distribution in all stages. A high proPO activity was recorded in L. setiferus females that was 95% higher than in the juvenile–male group. Respiratory burst was divided into two groups, juveniles–males and females, and was distributed normally in both. Female respiratory burst values were 40 and 45% higher than those observed in the juvenile and male groups, respectively. There was an increment in OxyHc, proteins, PO, hemocytes, and respiratory burst with an increase in body weight. In contrast, the relationship of live weight to glucose, TAG, and proPO revealed no significant regressions. A significant decrease in cholesterol, glycogen, and lactate with an increase in body weight was also observed. We now have indicators of nutritional and immune status of juveniles and adults that can reveal changes in trophic relations and health at the population level. This will serve as a basis for using L. setiferus as environmental sentinels, as non-controversial and inexpensive models to evaluate the potential changes that human activities could exert on this important marine ecosystem.Communicated by P.W. Sammarco, Chauvin     

Assessment of application-rate dependent effects of a long-term fire retardant chemical (Fire Trol 934) on Typha domingensis germination

Although long-term fire retardants (LTR) gain increasingly acceptance as effective tools for wildfire management, recent studies indicate their potential harmfulness in the environment, especially in aquatic ecosystems. This study comprises a first laboratory impact assessment of LTR action in temporal Mediterranean wetlands, using seed germination of Typha domingensis as indicator of impact. Our aim was to identify application rates upon which seed germination could be significantly affected. We tested for low (1 l m(-2)) and high (3 l m(-2)) application rates of Fire Trol 934 which are recommended by the manufacturers as a function of fuel characteristics. In addition, we simulated the impact of a higher application rate of 5 l m(-2) because inhomogeneous dispersal of the LTR during fire control and prevention operations can result in locally elevated applications. Results of a microcosm experiment indicate that application rates of 1 or 3 l m(-2) can impact Typha germination rates in the short-term via indirect LTR-mediated effects on water quality, which suppressed necessary cues for germination. However, a subsequent experiment with Petri dishes, using seeds isolated from the LTR treated sediments did not show significantly different germination rates between the control and the treatments with application rates of 1 or 3 l m(-2). This suggests that retardant pre-application germination success could be recovered in nature once the retardant is eliminated. By contrast, seeds almost completely failed to germinate in the microcosm experiment and the subsequent Petri dish essay when an application rate of 5 l m(-2) was used. This suggests a critical level upon which Typha seed germination may be perpetually limited. Research should be extended to other plant species to provide fire managers with guidelines for environmentally safe use of LTR in the Mediterranean region.     

Bioavailability of metals and As from acidified multicontaminated soils: Use of white lupin to validate several extraction methods

White lupin is an annual crop that has been used for phytostabilization of acidified multicontaminated (heavy metals and As) soils from the Aznalcóllar spill-affected area, Southern Spain. One of the most important factors for successful phytostabilization is monitoring the pollutant bioavailability in the soil. The aim of this work was to determine the best-suited method for assessing the bioavailability of heavy metals together with As in the Aznalcóllar spill-affected area, by means of a systematic comparison between different extraction methods (Ammonium bicarbonate-diethylenetriamine pentaacetic acid (AB-DTPA), CaCl₂, NaNO₃, BCR, (NH₄)₂SO₄ and rhizo). Both AB-DTPA and the first step of the BCR method were found to be unsuitable for assessing the bioavailability of heavy metals and As to plants growing in acidic soils. However, CaCl₂-extractable As, Cu, and Zn and NaNO₃-extractable As and Zn were well correlated with their concentrations in plant organs. Rhizo and (NH₄)₂SO₄, with the highest determination coefficients, were the most recommended simple extraction methods to assess the bioavailability of As, Cu, Fe, Mn, and Zn in acidified multicontaminated soils using white lupin as an excluder model plant.     

Trees' role in nitrogen leaching after organic, mineral fertilization: a greenhouse experiment

New sustainable agriculture techniques are arising in response to the environmental problems caused by intensive agriculture, such as nitrate leaching and surface water eutrophication. Organic fertilization (e.g., with sewage sludge) and agroforestry could be used to reduce nutrient leaching. We assessed the efficiency of establishing trees and pasture species in environmentally sensitive, irrigated Mediterranean grassland soils in controlling nitrate leaching. Four vegetation systems-bare soil, pasture species, cherry trees [ (L.) L.], and pasture-tree mixed plantings-and five fertilization treatments-control, two doses of mineral fertilizer, and two doses of organic fertilizer (sewage sludge)-were tested in a greenhouse experiment over 2 yr. In the experiment, the wet and warm climate characteristics of Mediterranean irrigated croplands and the plant-to-plant and soil-to-plant interactions that occur in open-field agroforestry plantations were simulated. Following a factorial design with six replicates, 120 pots (30-cm radius and 120 cm deep) were filled with a sandy, alluvial soil common in the cultivated fluvial plains of the region. The greatest pasture production and tree growth were obtained with sewage sludge application. Both pasture production and tree growth decreased significantly in the pasture-tree mixed planting. Nitrate leaching was negligible in this latter treatment, except under the highest dose of sewage sludge application. The rapid mineralization of sludge suggested that this organic fertilizer should be used very cautiously in warm, irrigated Mediterranean soils. Mixed planting of pasture species and trees, such as , could be a useful tool for mitigating nitrate leaching from irrigated Mediterranean pastures on sandy soils.