Esther Martí

Characterisation and evolution of a soil affected by olive oil mill wastewater disposal

A location used for 10 years as an uncontrolled olive oil mill wastewater disposal site is studied in this work. Once it was closed the sedimented solid waste on the soil surface was removed. In order to evaluate the influence upon soil characteristics, morphological aspects and analytical parameters of a soil profile from the affected zone are compared to those of a control soil located near the landfill. The residual contamination levels in the underlying soil are determined. The results show that the wastewater infiltration in the soil has caused carbonate dissolution and redistribution and modifications in pH values, electrical conductivity, nutrient contents, phenolic compounds and biological activity of the horizons. Removal of waste, natural leaching and biological activity, in time, led to an effective decrease in electrical conductivity and phenolic compounds, although residual levels can be important even 2 years later.

Relationship between pollutant content and ecotoxicity of sewage sludges from Spanish wastewater treatment plants

Chemical and ecotoxicological properties of 28 sewage sludge samples from Spanish wastewater treatment plants were studied in order to assess their suitability for agricultural purposes. Sludge samples were classified into five categories according to specific treatment processes in terms of digestion (aerobic/anaerobic) and drying (mechanical/thermal). Composted samples, as indicative of the most refined process, were also considered. Sludges were subjected to physical-chemical characterization, being the sludge stabilization degree respirometrically assessed. The concentrations of seven metals (Cd, Cr, Cu, Pb, Zn, Ni, Hg) and organic substances (phenolic compounds, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polychlorinated naphthalenes, polybrominated diphenyl ethers, and perfluorinated compounds) were determined. Finally, two ecotoxicological tests were performed: i) Microtox® toxicity test with Vibrio fischeri, and ii) root elongation test with Allium cepa, Lolium perenne and Raphanus sativus seeds. Significant differences were found in the following parameters: dry matter, electrical conductivity, nitrogen, organic matter and its stability, phytotoxicity and ecotoxicity, depending on the sludge treatment. In turn, no significant differences were found between categories in the concentrations of most metals and organic pollutants, with the exception of free phenolic compounds. Furthermore, no correlation between total heavy metal burden and ecotoxicity was observed. However, a good correlation was found between phenolic compounds and most ecotoxicological tests. These results suggest that sludge stability (conditioned by sludge treatment) might have a greater influence on sludge ecotoxicity than the pollutant load. Composting was identified as the treatment resulting in the lowest toxicity.

Terrestrial ecotoxicity of short aliphatic protic ionic liquids

A study of the ecotoxicity of different short aliphatic protic ionic liquids (PILs) on terrestrial organisms was conducted. Tests performed within the present study include those assessing the effects of PILs on soil microbial functions (carbon and nitrogen mineralization) and terrestrial plants. The results show that the nominal lowest-observed-adverse-effect concentration (LOAEC) values were 5,000 mg/kg (dry soil) for the plant test in two species (Lolium perenne, Allium cepa), 1,000 mg/kg (dry soil) for the plant test in one species (Raphanus sativus), and 10,000 mg/kg (dry soil) for carbon and nitrogen microbial transformation tests (all concentrations are nominal). Most of the median effective concentration values (EC50) were above 1,000 mg/kg (dry soil). Based on the obtained results, these compounds can be described as nontoxic for soil microbiota and the analyzed plants, and potentially biodegradable in soils, as can be deduced from the respirometric experiment. The toxicity rises with the increase of complexity of the PILs molecule (branch and length of aliphatic chain) among the three PILs analyzed.

The microbiota of an unpolluted calcareous soil faces up chlorophenols: Evidences of resistant strains with potential for bioremediation.

To highlight the effects of a variety of chlorophenols (CP) in relation to the response of an indigenous bacterial community, an agricultural Mediterranean calcareous soil has been studied in microcosms incubated under controlled laboratory conditions. Soil samples were artificially polluted with 2-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP), at concentrations ranging from 0.1 up to 5000 mg kg(-1). Both activity and composition of the microbial community were assessed during several weeks, respectively, by respirometric methods and PCR-DGGE analysis of extracted DNA and RNA. Significant decreases in soil respirometric values and changes in the bacterial community composition were observed at concentrations above 1000 mg kg(-1) MCP and TCP, and above 100 mg kg(-1) PCP. However, the persistence of several active bacterial populations in soil microcosms contaminated with high concentration of CP, as indicated by DGGE fingerprints, suggested the capacity of these native bacteria to survive in the presence of the pollutants, even without a previous adaptation or contact with them. The isolation of potential CP degraders was attempted by culture plating from microcosms incubated with high CP concentrations. Twenty-three different isolates were screened for their resistance to TCP and PCP. The most resistant isolates were identified as Kocuria palustris, Lysobacter gummosus, Bacillus sp. and Pseudomonas putida, according to 16S rRNA gene homology. In addition, these four isolates also showed the capacity to reduce the concentration of TCP and PCP from 15% to 30% after 5d of incubation in laboratory assays (initial pollutant concentration of 50 mg L(-1)). Isolate ITP29, which could be a novel species of Bacillus, has been revealed as the first known member in this bacterial group with potential for CP bioremediation applications, usually wide-spread in the soil natural communities, which has not been reported to date as a CP degrader.

Ecotoxicity of chlorophenolic compounds depending on soil characteristics

Three chlorophenolic compounds (2-chlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol) were tested to assess their effects on two soils with different properties: a granitic soil (Haplic Arenosol) and a calcareous one (Calcaric Regosol). Different concentrations of the pollutants (ranging from 0.001 to 10,000 mg kg(-1) soil, d.w.) were assayed for their effects on soil microbial activity and composition, using manometric respirometry and PCR-DGGE analysis, respectively. Other ecotoxicity tests such as Lactuca sativa seedling growth in the contaminated soils and algal growth inhibition (Pseudokirschneriella subcapitata) in their water extracts were done. The behaviour of the pollutants in the soils with respect to biodegradability and volatilization was also investigated. In the Haplic Arenosol, volatilization is the main process affecting 2-chlorophenol. Degradation and fixation of this compound in the soil matrix are favored in the Calcaric Regosol. This is the least toxic pollutant assayed. For 2,4,6-trichlorophenol, the soil pH is a critical parameter in the toxicity assays due to the neutral pKa of the compound. It is toxic in the soil microbial activity assay, but some recovery of the biotic processes can be observed, particularly in the Calcaric Regosol. This compound is more toxic in the Haplic Arenosol than in the Calcaric Regosol. Pentachlorophenol is ionized in both soils due to its low pKa, increasing its water solubility. It is highly toxic to the soil microbiota, thus inhibiting respiration, biodegradation and other biotic dissipation processes. Plant and alga tests, were more sensitive than soil microbial tests, except for PCP. The microbial populations tend to show changes at lower concentrations than the microbial activity. Some soil types (abundant in the Mediterranean area), with alkaline pH and fine textures could show higher level of ecotoxicity for ionizable organic pollutants than the soil type recommended by the OECD in ecotoxicity testing.

Impact of chlorophenols on microbiota of an unpolluted acidic soil: microbial resistance and biodegradation

The impact of 2-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) on the microbial community of an acidic forest soil was studied under controlled laboratory conditions by spiking microcosms with the pollutants at concentrations ranging from 0.1 to 5000 mg kg(-1). A decrease in the cumulative respirometric values and changes in the bacterial and fungal community composition were detected at 1000 mg MCP kg(-1), 100 mg TCP kg(-1) and 100 and 1000 mg PCP kg(-1). However, drastic effects on the microbial community were revealed only at higher concentrations of MCP and TCP, although the toxicity of PCP was expected to be stronger. The acidic condition of the soil presumably reduces bioavailability of PCP, leading to less pronounced effects than the other pollutants. This finding highlights the consideration of pollutant bioavailability in each environment to adequately assess contamination effects. Twenty-two different chlorophenol-resistant and potentially degrading microorganisms were isolated from highly polluted microcosms. The most resistant isolates were related to Burkholderia arboris, Bacillus circulans, Paenibacillus taichungensis, Luteibacter rhizovicina and Janibacter melonis. These isolates also showed the capacity to reduce the concentration of TCP or PCP between 15% and 35% after 5 days of incubation (initial concentration of 50 mg L(-1)). The isolate related to B. circulans is an atypical case of a member of the Firmicutes group for which chlorophenol-degrading capacities have been described.

The exposition of a calcareous Mediterranean soil to toxic concentrations of Cr, Cd and Pb produces changes in the microbiota mainly related to differential metal bioavailability

The involvement of the bacterial community of an agricultural Mediterranean calcareous soil in relation to several heavy metals has been studied in microcosms under controlled laboratory conditions. Soil samples were artificially polluted with Cr(VI), Cd(II) and Pb(II) at concentrations ranging from 0.1 to 5000 mg kg(-1) and incubated along 28 d. The lowest concentrations with significant effects in soil respirometry were 10 mg kg(-1) Cr and 1000 mg kg(-1) Cd and Pb. However, only treatments showing more than 40% inhibition of respirometric activity led to significant changes in bacterial composition, as indicated by PCR-DGGE analyses. Presumable Cr- and Cd-resistant bacteria were detected in polluted microcosms, but development of the microbiota was severely impaired at the highest amendments of both metals. Results also showed that bioavailability is an important factor determining the impact of the heavy metals assayed, and even an inverted potential toxicity ranking could be achieved if their soluble fraction is considered instead of the total concentration. Moreover, multiresistant bacteria were isolated from Cr-polluted soil microcosms, some of them showing the capacity to reduce Cr(VI) concentrations between 26% and 84% of the initial value. Potentially useful strains for bioremediation were related to Arthrobacter crystallopoietes, Stenotrophomonas maltophilia and several species of Bacillus.

Air-Drying, Cooling and Freezing for Soil Sample Storage Affects the Activity and the Microbial Communities from Two Mediterranean Soils

The selection of soil storage conditions must take into account the diversity of soils and their environmental characteristics, because different soil types may vary in their physical and chemical characteristics and microbial populations. As a result, identical storage conditions can produce different effects on different soil samples, and understanding the influence of soil storage in respect to the microbial community is essential for further microbial research. In this study, two different characteristics were used to assess the influence of soil processing and storage on two Mediterranean soils: respiratory activity and bacterial community composition in relation to three common storage conditions: air-drying, cooling and freezing. Samples from both soils (a Calcaric Regosol and a Haplic Arenosol) were air-dried or stored at either 4°C or −20°C for 12 months, and a PCR-DGGE analysis was performed to reveal changes in the whole bacterial community, and more specifically in the Firmicutes group, throughout the storage period. After storage, aliquots of the soil samples were incubated for 28 days at 25°C to assess changes in microbial activity, using respirometry, and molecular methods were used to determine the differences in the microbial community composition during the respirometric incubation. Results clearly show that any storage condition affects the microbial community and its activity, and this effect increases with the vulnerability of the soil. The study also reveals the need of incubating the soil samples to allow active bacteria to reach sufficiently high population sizes to be detected, as the non-incubated samples have not shown any change. The soil microbial community has been found to be adapted to its particular environmental conditions. In that sense, results suggest that Firmicutes may have a more common occurrence in the soil that is usually dried, since these bacteria can be more resistant to drying due to their spore-forming strategy. The stored samples undergo significant increases (45 to 70%, generally speaking) in the Cumulative Respiration, mainly in the most aggressive treatments (drying and freezing) whereas Basal and Substrate Induced Respiration rates tend to decrease (30 to 45%, depending on the soil and treatment), and the Respiratory Activation Quotient remains lower or unaltered, indicating low stress levels. The different physical, chemical and biological properties of the two soils lead to different suitable storage requirements, although all of them produce alterations in the studied parameters. Considering the results from microbial communities and activity, for the Calcaric Regosol, freezing could be the most suitable method, while for the Haplic Arenosol, it would be more appropriate to use air-drying.

Contamination levels in the soils affected by the flood from Aznalcóllar (Spain).

This study examines the soils affected by the accidental spillage and subsequent flood of pyritic liquids and sludge (a total of 4.5 Hm3) from a flotation plant for complex sulfides in Aznalcóllar (Spain). Two samplings were taken, the first 15 days and the second 3 months after the flood. Potentially toxic elements (PTE) in aqua regia and aqueous extracts were analyzed by ICP. X-ray diffraction and electron microscopy techniques were also used. The objective of this work was to determine the degree of contamination of the soils, the depths and variations with time, as well as the factors determining the presence of contaminants in the soil. Generally speaking, the soils affected are contaminated by Zn, Cu, Pb, As, and Cd at different degrees and depths depending on their particular conditions. En este trabajo se realiza un estudio de los suelos afectados por el vertido accidental de aguas ácidas y lodos piríticos provenientes de una planta de flotación de sulfuros complejos en Aznalcóllar (España). Se realizaron dos muestreos, el primero al cabo de quince días y el segundo tres meses después del vertido. Se han determinado las concentraciones de elementos potencialmente tóxicos (EPT) presentes en extractos acuosos y en agua regia de las muestras. Asimismo se han utilizado técnicas de difracción de rayos X y microscopía electrónica con el fin de localizar la presencia de partículas de lodos contaminantes en los suelos muestreados. Los objetivos de este trabajo consisten en el determinación del grado de contaminación de los suelos, la profundidad y variaciones en el tiempo, así como los factores que determinan la presencia de contaminantes en el suelo. En general los suelos afectados están contaminados por Zn, Cu, Pb, As y Cd, en distintos grados y profundidades, en función de sus condiciones particulares.

Amendment of Soils with Composted Sewage Sludge. Long Term Effects on C and N Transformation

This work aims to evaluate the effects of the current common agricultural use of composted sewage sludge on 5 crop soils, focusing on some chemical, biochemical and ecotoxicological properties related to organic matter mineralization and microbial activity. Therefore, pH, EC25, oxidizable and dissolved carbon, total Kjeldahl N, soluble phenolic compounds, specific UV-absorbance254, ammonium and nitrates, dehydrogenase and urease activity have been determined. Soil microbial activity has been assessed by respirometry (cumulative, basal and substrate induced respiration, respiratory activation quotient) and by potential nitrification, according to OECD guidelines. The analyses have been done after a minimum of 6 months since sludge amendment to avoid the initial enhancement due to recent organic matter supply. The sludge compost enhances some soil properties, as soluble organic matter, carbon mineralization, with general increases in the microbial activity and the active biomass and decreases in the respiratory activation quotients in most soils. Nitrogen mineralization has been negatively affected in some cases; nitrification and urease activity have decreased in two soils, probably due to the repeated application of sludge, more than to the total amount used. Dehydrogenase activity results are variable. In the poorer native soils it has increased, negatively correlated with the estimated active biomass and positively correlated with the respiratory activation quotient. Insufficiently controlled and repeated compost amending could be the cause of a C and N cycles imbalance, producing a decrease of the C/N ratio that can involve risk for waters due to an excess of mineral nitrogen.