Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

Mechanical and leaching behaviour of slag-cement and lime-activated slag stabilised/solidified contaminated soil.

Stabilisation/solidification (S/S) is an effective technique for reducing the leachability of contaminants in soils. Very few studies have investigated the use of ground granulated blast furnace slag (GGBS) for S/S treatment of contaminated soils, although it has been shown to be effective in ground improvement. This study sought to investigate the potential of GGBS activated by cement and lime for S/S treatment of a mixed contaminated soil. A sandy soil spiked with 3000mg/kg each of a cocktail of heavy metals (Cd, Ni, Zn, Cu and Pb) and 10,000mg/kg of diesel was treated with binder blends of one part hydrated lime to four parts GGBS (lime-slag), and one part cement to nine parts GGBS (slag-cement). Three binder dosages, 5, 10 and 20% (m/m) were used and contaminated soil-cement samples were compacted to their optimum water contents. The effectiveness of the treatment was assessed using unconfined compressive strength (UCS), permeability and acid neutralisation capacity (ANC) tests with determination of contaminant leachability at the different acid additions. UCS values of up to 800kPa were recorded at 28days. The lowest coefficient of permeability recorded was 5×10(-9)m/s. With up to 20% binder dosage, the leachability of the contaminants was reduced to meet relevant environmental quality standards and landfill waste acceptance criteria. The pH-dependent leachability of the metals decreased over time. The results show that GGBS activated by cement and lime would be effective in reducing the leachability of contaminants in contaminated soils.

Process envelopes for stabilisation/solidification of contaminated soil using lime-slag blend.

PURPOSE: Stabilisation/solidification (S/S) has emerged as an efficient and cost-effective technology for the treatment of contaminated soils. However, the performance of S/S-treated soils is governed by several intercorrelated variables, which complicates the optimisation of the treatment process design. Therefore, it is desirable to develop process envelopes, which define the range of operating variables that result in acceptable performance. METHODS: In this work, process envelopes were developed for S/S treatment of contaminated soil with a blend of hydrated lime (hlime) and ground granulated blast furnace slag (GGBS) as the binder (hlime/GGBS = 1:4). A sand contaminated with a mixture of heavy metals and petroleum hydrocarbons was treated with 5%, 10% and 20% binder dosages, at different water contents. The effectiveness of the treatment was assessed using unconfined compressive strength (UCS), permeability, acid neutralisation capacity and contaminant leachability with pH, at set periods. RESULTS: The UCS values obtained after 28 days of treatment were up to ∼800 kPa, which is quite low, and permeability was ∼10(-8) m/s, which is higher than might be required. However, these values might be acceptable in some scenarios. The binder significantly reduced the leachability of cadmium and nickel. With the 20% dosage, both metals met the waste acceptance criteria for inert waste landfill and relevant environmental quality standards. CONCLUSIONS: The results show that greater than 20% dosage would be required to achieve a balance of acceptable mechanical and leaching properties. Overall, the process envelopes for different performance criteria depend on the end-use of the treated material.

Metal Retention Experiments for the Design of Soil-Mix Technology Permeable Reactive Barriers

Soil-mix technology is effective for the construction of permeable reactive barriers (PRBs) for in situ groundwater treatment. The objective of this study was to perform initial experiments for the design of soil-mix technology PRBs according to (i) sorption isotherm, (ii) reaction kinetics and (iii) mass balance of the contaminants. The four tested reactive systems were: (i) a granular zeolite (clinoptilolite-GZ), (ii) a granular organoclay (GO), (iii) a 1:1-mixture GZ and model sandy clayey soil and (iv) a 1:1:1-mixture of GZ, GO and model soil. The laboratory experiments consisted of batch tests (volume 900mL and sorbent mass 18g) with a multimetal solution of Pb, Cu, Zn, Cd and Ni. For the adsorption experiment, the initial concentrations ranged from 0.01 to 0.5mM (2.5 to 30mg/L). The maximum metal retention was measured in a batch test (300mg/L for each metal, volume 900mL, sorbent mass 90-4.5g). The reactive material efficiency order was found to be GZ>GZ-soil mix>GZ-soil-GO mix>GO. Langmuir isotherms modelled the adsorption, even in presence of a mixed cations solution. Adsorption was energetically favourable and spontaneous in all cases. Metals were removed according to the second order reaction kinetics; GZ and the 1:1-mix were very similar. The maximum retention capacity was 0.1-0.2mmol/g for Pb in the presence of clinoptilolite; for Cu, Zn, Cd and Ni, it was below 0.05mmol/g for the four reactive systems. Mixing granular zeolite, organoclay and model soil increased the chemisorption. Providing that GZ is reactive enough for the specific conditions, GZ can be mixed to obtain the required sorption. Granular clinoptilolite addition to soil is recommended for PRBs for metal contaminated groundwater. The laboratory experiments consisted of batch tests with a multimetal solution of Pb, Cu, Zn, Cd and Ni. The four reactive materials chosen were granular zeolite, clinoptilolite and model sandy clayey soil, granular organoclay and a mix of clinoptilolite, model soil and organoclay. The reactive material efficiency order was found to be granular clinoptilolite>clinoptilolite-soil mix>clinoptilolite-soil-organoclay mix>granular organoclay. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PH-dependent leaching behaviour and other performance properties of cement-treated mixed contaminated soil

Portland cement has been widely used for stabilisation/solidification (S/S) treatment of contaminated soils. However, there is a dearth of literature on pH-dependent leaching of contaminants from cement-treated soils. This study investigates the leachability of Cu, Pb, Ni, Zn and total petroleum hydrocarbons (TPH) from a mixed contaminated soil. A sandy soil was spiked with 3000 mg/kg each of Cd, Cu, Pb, Ni and Zn, and 10,000 mg/kg of diesel, and treated with ordinary Portland cement (CEM I). Four different binder dosages, 5%, 10%, 15% and 20% (m/m) and different water contents ranging from 13%-19% dry weight were used in order to find a safe operating envelope for the treatment process. The pH-dependent leaching behaviour of the treated soil was monitored over an 84-day period using a 3-point acid neutralisation capacity (ANC) test. The monolithic leaching test was also conducted. Geotechnical properties such as unconfined compressive strength (UCS), hydraulic conductivity and porosity were assessed over time. The treated soils recorded lower leachate concentrations of Ni and Zn compared to the untreated soil at the same pH depending on binder dosage. The binder had problems with Pb stabilisation and TPH leachability was independent of pH and binder dosage. The hydraulic conductivity of the mixes was generally of the order, 10-8 m/sec, while the porosity ranged from 26%-44%. The results of selected performance properties are compared with regulatory limits and the range of operating variables that lead to acceptable performance described. © 2012 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences.

Comparisons of operating envelopes for contaminated soil stabilised/solidified with different cementitious binders

This work initiated the development of operating envelopes for stabilised/solidified contaminated soils. The operating envelopes define the range of operating variables for acceptable performance of the treated soils. The study employed a soil spiked with 3,000 mg/kg each of Cd, Cu, Pb, Ni and Zn, and 10,000 mg/kg of diesel. The binders used for treatment involved Portland cement (CEMI), pulverised fuel ash (PFA), ground granulated blast furnace slag (GGBS) and hydrated lime (hlime). The specific binder formulations were CEMI, CEMI/PFA = 1:4, CEMI/GGBS = 1:9 and hlime/GGBS = 1:4. The water contents employed ranged from 13 % to 21 % (dry weight), while binder dosages ranged from 5 % to 20 % (w/w). We monitored the stabilised/solidified soils for up to 84 days using different performance tests. The tests include unconfined compressive strength (UCS), hydraulic conductivity, acid neutralisation capacity (ANC) and pH-dependent leachability of contaminants. The water content range resulted in adequate workability of the mixes but had no significant effect on leachability of contaminants. We produced design charts, representing operating envelopes, from the results generated. The charts establish relationships between water content, binder dosage and UCS; and binder dosage, leachant pH and leachability of contaminants. The work also highlights the strengths and weaknesses of the different binder formulations. © 2013 Springer-Verlag Berlin Heidelberg.

Polycyclic aromatic hydrocarbons (PAHS) (I): Toxicity, population exposure and involved foods

Food is one of the main exogenous sources of genotoxic compounds. In heated food products, polycyclic aromatic hydrocarbons (PAHs) represent a priority group of genotoxic, mutagenic and/or carcinogenic chemical pollutants with adverse long-term health effects. People can be exposed to these compounds through different environments and via various routes: inhalation, ingestion of foods and water and even percutaneously. The presence of these compounds in food may be due to environmental contamination, to industrial handling and processing of foods and to oil processing and refining. The highest levels of these compounds are found in smoked foods, in seafood which is found in polluted waters, in grilled meats and, to a lesser extent, in vegetable fats and oils. Lower levels of PAHs are found in vegetables and in cereals and its products.

Interactions between earthworms and arsenic in the soil environment:a review

Chemical pollution of the environment has become a major source of concern. In particular, many studies have investigated the impact of pollution on biota in the environment. Studies on metalliferous contaminated mine spoil wastes have shown that some soil organisms have the capability to become resistant to metal/metalloid toxicity. Earthworms are known to inhabit arsenic-rich metalliferous soils and, due to their intimate contact with the soil, in both the solid and aqueous phases, are likely to accumulate contaminants present in mine spoil. Earthworms that inhabit metalliferous contaminated soils must have developed mechanisms of resistance to the toxins found in these soils. The mechanisms of resistance are not fully understood; they may involve physiological adaptation (acclimation) or be genetic. This review discusses the relationships between earthworms and arsenic-rich mine spoil wastes, looking critically at resistance and possible mechanisms of resistance, in relation to soil edaphic factors and possible trophic transfer routes.

Arsenic contamination of Bangladesh paddy field soils:implications for rice contribution to arsenic consumption

Arsenic contaminated groundwater is used extensively in Bangladesh to irrigate the staple food of the region, paddy rice (Oryza sativa L.). To determine if this irrigation has led to a buildup of arsenic levels in paddy fields, and the consequences for arsenic exposure through rice ingestion, a survey of arsenic levels in paddy soils and rice grain was undertaken. Survey of paddy soils throughout Bangladesh showed that arsenic levels were elevated in zones where arsenic in groundwater used for irrigation was high, and where these tube-wells have been in operation for the longest period of time. Regression of soil arsenic levels with tube-well age was significant. Arsenic levels reached 46 microg g(-1) dry weight in the most affected zone, compared to levels below l0 microg g(-1) in areas with low levels of arsenic in the groundwater. Arsenic levels in rice grain from an area of Bangladesh with low levels of arsenic in groundwaters and in paddy soils showed that levels were typical of other regions of the world. Modeling determined, even these typical grain arsenic levels contributed considerably to arsenic ingestion when drinking water contained the elevated quantity of 0.1 mg L(-1). Arsenic levels in rice can be further elevated in rice growing on arsenic contaminated soils, potentially greatly increasing arsenic exposure of the Bangladesh population. Rice grain grown in the regions where arsenic is building up in the soil had high arsenic concentrations, with three rice grain samples having levels above 1.7 microg g(-1).

High-affinity phosphate/arsenate transport in white lupin (Lupinus albus) is relatively insensitive to phosphate status

The development of proteoid roots under phosphorus deficiency by white lupin (Lupinus albus) may result in increased arsenate uptake, as arsenate is a phosphate analogue. This, together with its high biomass production, rapid growth and ability to survive in soils with low phosphate and nitrogen contents, low pH and high metal contents make them an interesting species to investigate with respect to revegetation, and possibly also for long-term phytoremediation, of arsenic contaminated soils. Kinetic parameters for arsenate uptake for P-deficient and P-sufficient plants, as well as for proteoid and nonproteoid roots were obtained. Down-regulation of arsenate uptake by phosphate, as well as phosphate/arsenate competition for P-deficient and P-sufficient plants was studied. Arsenate uptake was reduced by phosphate, but small differences were found between P-deficient and P-sufficient plants. Arsenate uptake by proteoid roots was higher than for nonproteoid roots of P-deficient plants, with higher V_max and similar K_m values. Down-regulation of the high affinity phosphate/arsenate uptake system by phosphate does take place but seems to be slower than in other plants. This study suggests that the low sensitivity of the phosphate/arsenate uptake system to regulation by phosphate may be related to the adaptations of white lupin to low P available environments. Such adaptation are absent in plants unable to develop proteoid roots.

Survival and behaviour of the earthworms Lumbricus rubellus and Dendrodrilus rubidus from arsenate-contaminated and non-contaminated sites

Two arsenic- and heavy metal-contaminated mine-spoil sites, at Carrock Fell, Cumbria and Devon Great Consols Mine, Devon, were found to support populations of the earthworms Lumbricus rubellus Hoffmeister and Dendrodrilus rubidus (Savigny). L. rubellus and D. rubidus collected from the Devon site and an uncontaminated site were kept for 28 days in uncontaminated soil and in soil containing sodium arsenate (494 mg As kg^-1). The state of the specimens was recorded every 7 days using a semi-quantitative assessment of earthworm health (condition index, C. I.). The C. I. remained high for all specimens except those of L. rubellus and D. rubidus from uncontaminated sites, which displayed 60 and 10% mortality, respectively. L. rubellus collected from the Carrock Fell site, and L. rubellus and D. rubidus from an uncontaminated site, burrowed as rapidly into soil containing up to 1235 mg As kg^-1 in the form of sodium arsenate as into uncontaminated soil when placed on the soil surface. When earthworms were allowed a choice between uncontaminated soil and soil contaminated with sodium arsenate in concentrations of up to 1235 mg As kg^-1, the threshold concentration for avoidance of contaminated soil was lower for L. rubellus and D. rubidus from uncontaminated soil than for specimens from contaminated soil. There was no significant effect of pH on soil discrimination. The LC₅₀ concentration of As for L. rubellus from Devon Great Consols was significantly higher (P < 0.001) than for L. rubellus from the uncontaminated site: 1510 and 96 mg As kg^-1, respectively. © 2001 Elsevier Science Ltd. All rights reserved.

Efeitos da toxicidade por metais pesados em Rhizobium leguminosarum bv. trifolii: diversidade e resposta de população isoladas de uma mina de chumbo

Nas últimas décadas verificou-se um aumento da contaminação dos solos com metais pesados resultante de processos antropogénicos. As descargas de efluentes industriais, a actividade mineira e a aplicação de lamas residuais e de fertilizantes são as principais fontes de metais pesados. Em certas regiões, a acumulação destes elementos nos solos tem atingido níveis preocupantes para o equilíbrio dos ecossistemas. Vários estudos têm demonstrado que os metais influenciam os microrganismos afectando adversamente o seu crescimento, morfologia e actividades bioquímicas resultando num decréscimo da biomassa e diversidade. Entre os microrganismos do solo, as bactérias pertencentes ao género Rhizobium têm um elevado interesse científico, económico e ecológico devido à sua capacidade para fixar azoto. Deste modo, o trabalho desenvolvido ao longo desta tese incidiu sobre o efeito da toxicidade imposta pelos metais nas bactérias fixadoras de azoto, em particular em Rhizobium leguminosarum bv. trifolii e teve como principais objectivos: determinar o efeito dos metais pesados na sobrevivência e na capacidade de fixar azoto dos isolados de rizóbio; avaliar a influência dos metais na diversidade das populações de rizóbio isoladas de solos contaminados; determinar os níveis de tolerância do rizóbio a diferentes metais e analisar a resposta ao stresse oxidativo imposto pelo cádmio. A Mina do Braçal foi o local de estudo escolhido uma vez que os seus solos estão muito contaminados com metais em resultado da extracção de minério durante mais de 100 anos. Foram escolhidos 3 solos com diferentes graus de contaminação, o solo BC com concentrações reduzidas de metais, escolhido por estar numa zona já fora da mina e designado por solo controlo e os solos BD e BA considerados medianamente e muito contaminados, respectivamente. O Pb e o Cd foram os metais predominantes nestes solos, assim como o metalóide As, cujas concentrações ultrapassaram largamente os limites previstos na lei. Sendo as enzimas do solo boas indicadoras da qualidade do mesmo, foi determinada a actividade de algumas como a desidrogenase (DHA) e a catalase (CAT). Ambas as enzimas correlacionaramse negativamente com as concentrações de metais nos solos. A dimensão das populações indígenas de rizóbio nos solos contaminados (BD e BA) foi bastante baixa, 9,1 bactérias g-1 de solo e 7,3 bactérias g-1 de solo, respectivamente, quando em comparação com a população do solo BC (4,24x104 bactérias g-1 de solo). Estes resultados parecem estar relacionados com o elevado conteúdo em metais e com o pH ácido dos solos. A capacidade simbiótica também foi afectada pela presença de metais, uma vez que os isolados originários do solo BD mostraram menor capacidade em fixar azoto do que os isolados do solo controlo. A diversidade das populações de rizóbio foi determinada com recurso à análise dos perfis de plasmídeos, perfis de REP e ERIC-PCR de DNA genómico e perfis de proteínas e lipopolissacarídeos. No conjunto dos 35 isolados analisados foram identificados 11 plasmídeos com pesos moleculares entre 669 kb e 56 kb. Embora a incidência de plasmídeos tenha sido superior nos isolados do solo BC verificou-se maior diversidade plasmídica na população isolada do solo BD. Resultados similares foram obtidos com os perfis de REP e ERIC-PCR e perfis de proteínas, que indicaram maior diversidade nas populações dos solos contaminados (BD e BA), contrariamente ao verificado por outros autores. O grau de tolerância aos metais pesados e ao arsénio dos vários isolados testados dependeu do metal e do local de origem. No geral, os isolados do solo BD mostraram maior tolerância aos metais do que os isolados do solo controlo, o que está de acordo com o esperado uma vez que geralmente as populações dos locais contaminados são mais tolerantes. Contudo, os isolados do local mais contaminado (BA) foram muito tolerantes apenas ao chumbo mostrando-se sensíveis aos restantes metais. A inoculação dos solos BC, BD e BA após irradiação com estirpes seleccionadas de rizóbio permitiu avaliar a sua sobrevivência ao longo de 12 meses em condições mais realistas. Verificou-se que após um decréscimo inicial, os isolados inoculados no solo BC conseguiram recuperar a dimensão das suas populações para números similares aos inicialmente introduzidos, contrariamente ao verificado no solo BD onde o número de rizóbios decresceu ao longo dos 12 meses. As condições adversas do solo BA apenas permitiram a sobrevivência de 4 isolados até aos 3 meses e apenas dois deles conseguiram sobreviver após 12 meses, designadamente C 3-1 e A 17-3. Estes isolados possuem um plasmídeo de 669 kb que poderá estar na base da sobrevivência destas estirpes. Por outro lado, o último isolado é originário do solo contaminado e por isso estará também mais adaptado a sobreviver às elevadas concentrações de Pb existentes no solo BA. Por fim, constatou-se que o cádmio, um dos metais presente em concentrações mais elevadas nos solos em estudo, é um indutor de stresse oxidativo nos isolados de rizóbio menos tolerantes o que foi confirmado pelo aumento de ROS e danos celulares ao nível dos lípidos.

Adaptation of Cladocerans in metal-polluted heterogeneous environments

No presente trabalho o cladócero Daphnia longispina foi utilizado como organismo modelo para a avaliação dos efeitos ecológicos da adaptação a ambientes contaminados por metais. Foram amostradas populações naturais de D. longispina num local sujeito à contaminação por metais e num local próximo, de referência, ambos localizados no sistema aquático na área envolvente à mina abandonada de São Domingos. Várias linhagens clonais de ambas as populações foram mantidas em laboratório, sob condições controladas, para a execução dos testes. Um dos testes realizados permitiu estudar e quantificar as diferenças na tolerância letal entre as linhagens clonadas de ambas as populações e também avaliar os custos associados. Utilizando vinte linhagens clonais de D. longispina das duas populações verificou-se que apenas clones sensíveis ao cobre estavam presentes na população de referência e clones resistentes ao cobre estavam presentes na população do local contaminado. Os custos associados à tolerância foram ilustrados pela determinação de taxas alimentares mais baixas para a população tolerante quando comparadas com as da população de referência. Outro dos testes realizados permitiu comparar as respostas de clones de populações de ambos os locais – contaminado e referência – à exposição a concentrações sub-letais do metal cobre. A tolerância evidenciada anteriormente ao nível letal foi confirmada ao nível sub-letal, com o clone proveniente da população do local contaminado evidenciando uma maior tolerância ao cobre quando comparado com os restantes clones, para todos os parâmetros analisados (taxas alimentares, consumo de oxigénio, crescimento e reprodução). Os efeitos da aclimatação ao cobre ao longo de várias gerações foram também avaliados num clone de D. longispina. Os resultados evidenciaram a existência de uma adaptação fisiológica ao cobre ao longo das várias gerações que, no entanto, apenas aumentou marginalmente a tolerância a níveis de cobre letais. Para além disso, observou-se também uma grande variação nas respostas do clone de D. longispina estudado, não só entre concentrações de cobre mas também entre gerações. Os resultados obtidos nos vários estudos realizados com linhagens clonais de ambas as populações de D. longispina reforçam a importância de integrar a temática do desenvolvimento de tolerância à poluição aquando da avaliação dos riscos ambientais e ecológicos de compostos químicos, como os metais, no meio ambiente.

Organic contaminants in urban soils: major inputs and potential risks

Urban soil quality may be severely affected by hydrophobic organic contaminants (HOCs), impairing environmental quality and human health. A comprehensive study was conducted in two contrasting Portuguese urban areas (Lisbon and Viseu) in order to assess the levels and potential risks of these contaminants, to identify sources and study their behaviour in soils. The concentrations of HOCs were related to the size of the city, with much higher contamination levels observed in Lisbon urban area. Source apportionment was performed by studying the HOCs profiles, their relationship with potentially toxic elements and general characteristics of soil using multivariate statistical methods. Lisbon seems to be affected by nearby sources (traffic, industry and incineration processes) whereas in Viseu the atmospheric transport may be playing an important role. In a first tier of risk assessment (RA) it was possible to identify polycyclic aromatic hydrocarbons (PAHs) in Lisbon soils as a potential hazard. The levels of PAHs in street dusts were further studied and allowed to clarify that traffic, tire and pavement debris can be an important source of PAHs to urban soils. Street dusts were also identified as being a potential concern regarding human and environmental health, especially if reaching the nearby aquatic bodies. Geostatistical tools were also used and their usefulness in a RA analysis and urban planning was discussed. In order to obtain a more realistic assessment of risks of HOCs to environment and human health it is important to evaluate their available fraction, which is also the most accessible for organisms. Therefore, a review of the processes involved on the availability of PAHs was performed and the outputs produced by the different chemical methods were evaluated. The suitability of chemical methods to predict bioavailability of PAHs in dissimilar naturally contaminated soils has not been demonstrated, being especially difficult for high molecular weight compounds. No clear relationship between chemical and biological availability was found in this work. Yet, in spite of the very high total concentrations found in some Lisbon soils, both the water soluble fraction and the body residues resulting from bioaccumulation assays were generally very low, which may be due to aging phenomena. It was observed that the percentage of soluble fraction of PAHs in soils was found to be different among compounds and mostly regulated by soil properties. Regarding bioaccumulation assays, although no significant relationship was found between soil properties and bioavailability, it was verified that biota-to-soil bioaccumulation factors were sample dependent rather than compound dependent. In conclusion, once the compounds of potential concern are targeted, then performing a chemical screening as a first tier can be a simple and effective approach to start a RA. However, reliable data is still required to improve the existing models for risk characterization.