Background, aim, and scope The use of plants and associated microorganisms to remove, contain, inactivate, or degrade harmful environmental contaminants (generally termed phytoremediation) and to revitalize contaminated sites is gaining more and more attention. In this review, prerequisites for a successful remediation will be discussed. The performance of phytoremediation as an environmental remediation technology indeed depends on several factors including the extent of soil contamination, the availability and accessibility of contaminants for rhizosphere microorganisms and uptake into roots (bioavailability), and the ability of the plant and its associated microorganisms to intercept, absorb, accumulate, and/or degrade the contaminants. The main aim is to provide an overview of existing field experience in Europe concerning the use of plants and their associated microorganisms whether or not combined with amendments for the revitalization or remediation of contaminated soils and undeep groundwater. Contaminations with trace elements (except radionuclides) and organics will be considered. Because remediation with transgenic organisms is largely untested in the field, this topic is not covered in this review. Brief attention will be paid to the economical aspects, use, and processing of the biomass. Conclusions and perspectives It is clear that in spite of a growing public and commercial interest and the success of several pilot studies and field scale applications more fundamental research still is needed to better exploit the metabolic diversity of the plants themselves, but also to better understand the complex interactions between contaminants, soil, plant roots, and microorganisms (bacteria and mycorrhiza) in the rhizosphere. Further, more data are still needed to quantify the underlying economics, as a support for public acceptance and last but not least to convince policy makers and stakeholders (who are not very familiar with such techniques).

Background, aim and scope Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytotreatment to remove sulphonated aromatic compounds from dye and textile industrial effluents, it has been shown that rhubarb (Rheum rabarbarum) and common sorrel (Rumex acetosa) are the most efficient plants. Both species, producing natural anthraquinones, not only accumulate, but also transform these xenobiotic chemicals. Even if the precise biochemical mechanisms involved in the detoxification of sulphonated anthraquinones are not yet understood, they probably have cross talks with secondary metabolism, redox processes and plant energy metabolism. The aim of the present study was to investigate the possible roles of cytochrome P450 monooxygenases and peroxidases in the detoxification of several sulphonated anthraquinones. Materials and methods Both plant species were cultivated in a greenhouse under hydroponic conditions, with or without sulphonated anthraquinones. Plants were harvested at different times and either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 toward several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results A significant activity of cytochromes P450 was detected in rhubarb leaves, while no (rhizome) or low (petioles and roots) activity was found in other parts of the plants. An induction of this enzyme was observed at the beginning of the exposition to sulphonated anthraquinones. The results also indicated that cytochromes P450 were able to accept as substrate the five sulphonated anthraquinones, with a higher activity toward AQ-2,6-SS (0.706 nkat/mg protein) and AQ-2-S (0.720 nkat/mg protein). An activity of the cytochromes P450 was also found in the leaves of common sorrel (1.212 nkat/mg protein (AQ-2,6-SS)), but no induction of the activity occurred after the exposition to the pollutant. The activity of peroxidases increased when rhubarb was cultivated in the presence of the five sulphonated anthraquinones (0.857 nkat/mg protein). Peroxidase activity was also detected in the leaves of the common sorrel (0.055 nkat/mg protein), but in this plant, no significant difference was found between plants cultivated with and without sulphonated anthraquinones. Discussion Results indicated that the activity of cytochromes P450 and peroxidases increased in rhubarb in the presence of sulphonated anthraquinones and were involved in their detoxification mechanisms. Conclusions These results suggest the existence in rhubarb and common sorrel of specific mechanisms involved in the metabolism of sulphonated anthraquinones. Further investigation should be performed to find the next steps of this detoxification pathway. Recommendations and perspectives Besides these promising results for the phytotreatment of sulphonated anthraquinones, it will be of high interest to develop and test, at small scale, an experimental wastewater treatment system to determine its efficiency. On the other hand, these results reinforce the idea that natural biodiversity should be better studied to use the most appropriate species for the phytotreatment of a specific pollutant.

Purpose The term “phytotechnologies” refers to the application of science and engineering to provide solutions involving plants, including phytoremediation options using plants and associated microbes to remediate environmental compartments contaminated by trace elements (TE) and organic xenobiotics (OX). An extended knowledge of the uptake, translocation, storage, and detoxification mechanisms in plants, of the interactions with microorganisms, and of the use of “omic” technologies (functional genomics, proteomics, and metabolomics), combined with genetic analysis and plant improvement, is essential to understand the fate of contaminants in plants and food, nonfood and technical crops. The integration of physicochemical and biological understanding allows the optimization of these properties of plants, making phytotechnologies more economically and socially attractive, decreasing the level and transfer of contaminants along the food chain and augmenting the content of essential minerals in food crops. This review will disseminate experience gained between 2004 and 2009 by three working groups of COST Action 859 on the uptake, detoxification, and sequestration of pollutants by plants and consequences for food safety. Gaps between scientific approaches and lack of understanding are examined to suggest further research and to clarify the current state-of-the-art for potential end-users of such green options. Conclusion and perspectives Phytotechnologies potentially offer efficient and environmentally friendly solutions for cleanup of contaminated soil and water, improvement of food safety, carbon sequestration, and development of renewable energy sources, all of which contribute to sustainable land use management. Information has been gained at more realistic exposure levels mainly on Cd, Zn, Ni, As, polycyclic aromatic hydrocarbons, and herbicides with less on other contaminants. A main goal is a better understanding, at the physiological, biochemical, and molecular levels, of mechanisms and their regulation related to uptake-exclusion, apoplastic barriers, xylem loading, efflux-influx of contaminants, root-to-shoot transfer, concentration and chemical speciation in xylem/phloem, storage, detoxification, and stress tolerance for plants and associated microbes exposed to contaminants (TE and OX). All remain insufficiently understood especially in the case of multiple-element and mixed-mode pollution. Research must extend from model species to plants of economic importance and include interactions between plants and microorganisms. It remains a major challenge to create, develop, and scale up phytotechnologies to market level and to successfully deploy these to ameliorate the environment and human health.

Background, aim, and scope Dye pollutants are a major class of environmental contaminants. Over 100,000 dyes have been synthesized worldwide and more than 700,000 tons are produced annually and over 5% are discharged into aquatic environments. The adsorption or sorption is one of the most efficient methods to remove dye and heavy metal pollutants from wastewater. However, most of the present sorbents often bear some disadvantages, e.g. low sorption capacity, difficult separation of spoil, complex reproduction, or secondary pollution. Development of novel sorbents that can overcome these limitations is desirable. Materials and methods On the basis of the chemical coprecipitation of calcium oxalate (CaC₂O₄), bromopyrogallol red (BPR) was embedded during the growing of CaC₂O₄ particles. The ternary C₂O₄ ²⁻-BPR-Ca²⁺ sorbent was yielded by the centrifugation. Its composition was determined by spectrophotometry and AAS, and its structure and morphology were characterized by powder X-ray diffraction (XRD), laser particle-size analysis, and scanning electron microscopy (SEM). The adsorption of ethyl violet (EV) and heavy metals, e.g. Cu(II), Cd(II), Ni(II), Zn(II), and Pb(II) were carried out and their removal rate determined by spectrophotometry and ICP-OES. The adsorption performance of the sorbent was compared with powder activated carbon. The Langmuir isothermal model was applied to fit the embedment of BPR and adsorption of EV. Results The saturation number of BPR binding to CaC₂O₄ reached 0.0105 mol/mol and the adsorption constant of the complex was 4.70 x 10⁵ M⁻¹. Over 80% of the sorbent particles are between 0.7 and 1.02 μm, formed by the aggregation of the global CaC₂O₄/BPR inclusion grains of 30-50 nm size. Such a material was found to adsorb cationic dyes selectively and sensitively. Ethyl violet (EV) was used to investigate the adsorption mechanism of the material. One BPR molecule may just bind with one EV molecule. The CaC₂O₄/BPR inclusion material adsorbed EV over two times more efficiently than the activated carbon. The adsorption of EV on the CaC₂O₄/BPR inclusion sorbent was complete in only 5 min and the sedimentation complete in 1 h. However, those of EV onto activated carbon took more than 1.5 and 5 h, respectively. The treatment of methylene blue and malachite green dye wastewaters indicated that only 0.4% of the sorbent adsorbed over 80% of color substances. Besides, the material can also adsorb heavy metals by complexation with BPR. Over 90% of Pb²⁺, and approximately 50% of Cd²⁺ and Cu²⁺, were removed in a high Zn²⁺-electroplating wastewater when 3% of the material was added. Eighty-six percent of Cu²⁺, and 60% of Ni²⁺ and Cd²⁺, were removed in a high Cd²⁺-electroplating wastewater. Discussion The embedment of BPR into CaC₂O₄ particles responded to the Langmuir isothermal adsorption. As the affinity ligand of Ca²⁺, BPR with sulfonic groups may be adsorbed into the temporary electric double layer during the growing of CaC₂O₄ particles. Immediately, C₂O₄ ²⁻ captured the Ca²⁺ to form the CaC₂O₄ outer enclosed sphere. Thus, BPR may be released and embedded as a sandwich between CaC₂O₄ layers. The adsorption of EV on the sorbent obeyed the Langmuir isothermal equation and adsorption is mainly due to the ion-pair attraction between EV and BPR. Different from the inclusion sorbent, the activated carbon depended on the specific surface area to adsorb organic substances. Therefore, the adsorption capacity, equilibrium, and sedimentation time of the sorbent are much better than activated carbon. The interaction of heavy metals with the inclusion sorbent responded to their coordination. Conclusions By characterizing the C₂O₄ ²⁻-BPR-Ca²⁺ inclusion material using various modern instruments, the ternary in situ embedment particle, [(CaC₂O₄)₉₅(BPR)] n ²n⁻, an electronegative, micron-sized adsorbent was synthesized. It is selective, rapid, and highly effective for adsorbing cationic dyes and heavy metals. Moreover, the adsorption is hardly subject to the impact of electrolytes. Recommendations and perspectives The present work provides a simple and valuable method for preparing the highly effective adsorbent. If a concentrated BPR wastewater was reused as the inclusion reactant, the sorbent will be low cost. By selecting the inclusion ligand with a special structure, we may prepare some particular functional materials to recover the valuable substances from seriously polluted wastewaters. The recommended method will play a significant role in development of advanced adsorption materials.

Background, aim, and scope Pharmaceutically active substances are a class of emerging contaminants, which has led to increasing concern about potential environmental risks. After excretion, substantial amounts of unchanged pharmaceuticals and their metabolites are discharged into domestic wastewaters. The absence of data on the environmental exposure in Eastern Europe is significant, since use patterns and volumes differ from country to country. In Romania, the majority of wastewater, from highly populated cities and industrial complex zones, is still discharged into surface waters without proper treatment or after inefficient treatment. In respect to this, it is important to determine the environmental occurrence and behavior of pharmaceuticals and personal care products (PPCPs) in wastewaters and surface waters. The objective of the present study was to investigate the occurrence of selected PPCPs during the transport in the Somes River by mass flow analysis before and after upgrading a municipal wastewater treatment plant (WWTP) in Cluj-Napoca, which serves 350,000 inhabitants and is the largest plant discharging into the Somes River. The concentrations of PPCPs at Cluj-Napoca can be correlated with the high population and a high number of hospitals located in the catchment area leading to higher mass flows. The results of this study are expected to provide information, with respect to the Romanian conditions, for environmental scientists, WWTP operators, and legal authorities. The data should support the improvement of existing WWTPs and implementation of new ones where necessary and, therefore, minimize the input of contaminants into ambient waters. Materials and methods The PPCPs were selected on the basis of consumption at the regional scale, reported aquatic toxicity, and the suitability of the gas chromatography/mass spectrometry (GC/MS) method for the determination of the compounds at trace levels. The studied PPCPs, caffeine (stimulant), carbamazepine (antiepileptic), pentoxifylline (anticoagulant), cyclophosphamide (cytostatic), ibuprofen (analgesic), and galaxolide (musk fragrance), were determined in samples of the Somes River. The analytes were enriched by solid-phase extraction and subsequently determined by GC/MS. Caffeine, pentoxifylline, and galaxolide were determined underivatized, whereas the acidic pharmaceuticals carbamazepine, cyclophosphamide, and ibuprofen were determined after derivatization with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. Results and discussion The concentrations in the Somes River varied from below 10 ng/L up to 10 μg/L. A substantial decrease of the exposure in the Somes River could be observed due to the upgrade of the municipal WWTP in Cluj-Napoca. The loads in the river stretch between Cluj-Napoca and Dej (Somes Mic) varied strongly: caffeine (400-2,000 g/day), carbamazepine (78-213 g/day), galaxolide (140-684 g/day), ibuprofen (84-108 g/day). After the upgrade of the WWTP Cluj-Napoca, the concentrations in the Somes of caffeine, pentoxifylline, cyclophosphamide, galaxolide, and tonalide were significantly reduced (over 75%). One might be cautious comparing both studies because the relative efficiency of the WWTP's removal of PPCP was not evaluated. However, the significantly lower concentrations of most compounds after the upgrade of the WWTP Cluj-Napoca allow one to infer that the technical measures at the source substantially reduced inputs of contaminants to the receiving river. Dej loads of the poorly biodegradable substance carbamazepine increased by a factor of 2-3 as a result of wastewater discharges into the river. The disproportionate increase in caffeine loads by a factor of 4 below Cluj-Napoca indicates inputs of untreated wastewater from the Somes Mare due to the discharge of untreated wastewater derived from Bistrita, Nasaud, and Beclean (115,000 inhabitants). Conclusions The relative contribution of treated and untreated wastewater in surface water might be assessed by measuring chemical markers. Recalcitrant pharmaceuticals like carbamazepine are suitable as chemical markers for estimating the relative contribution of wastewater in surface water. The easily degradable caffeine might be a good indicator for raw sewage and hardly treated wastewaters. Recommendations and perspectives Municipal WWTPs have the potential of a significant contribution in reducing the load of contaminants to ambient waters. The efficiency of the wastewater treatment in Cluj-Napoca improved considerably after the upgrade of the WWTP. Therefore, it is crucial that several WWTPs must be implemented or improved in the Somes Valley Watershed in order to reduce the discharge of contaminants in the Somes River from these point sources.

Background, aim, and scope Pharmaceuticals and personal care products (PPCPs) including antibiotics, endocrine-disrupting chemicals, and veterinary pharmaceuticals are emerging pollutants, and their environmental risk was not emphasized until a decade ago. These compounds have been reported to cause adverse impacts on wildlife and human. However, compared to the studies on hydrophobic organic contaminants (HOCs) whose sorption characteristics is reviewed in Part IV of this review series, information on PPCPs is very limited. Thus, a summary of recent research progress on PPCP sorption in soils or sediments is necessary to clarify research requirements and directions. Main features We reviewed the research progress on PPCP sorption in soils or sediments highlighting PPCP sorption different from that of HOCs. Special function of humic substances (HSs) on PPCP behavior is summarized according to several features of PPCP-soil or sediment interaction. In addition, we discussed the behavior of xenobiotic chemicals in a three-phase system (dissolved organic matter (DOM)-mineral-water). The complexity of three-phase systems was also discussed. Results Nonideal sorption of PPCPs in soils or sediments is generally reported, and PPCP sorption behavior is relatively a more complicated process compared to HOC sorption, such as the contribution of inorganic fractions, fast degradation and metabolite sorption, and species-specific sorption mechanism. Thus, mechanistic studies are urgently needed for a better understanding of their environmental risk and for pollution control. Discussion Recent research progress on nonideal sorption has not been incorporated into fate modeling of xenobiotic chemicals. A major reason is the complexity of the three-phase system. First of all, lack of knowledge in describing DOM fractionation after adsorption by mineral particles is one of the major restrictions for an accurate prediction of xenobiotic chemical behavior in the presence of DOM. Secondly, no explicit mathematical relationship between HS chemical-physical properties, and their sorption characteristics has been proposed. Last but not least, nonlinear interactions could exponentially increase the complexity and uncertainties of environmental fate models for xenobiotics. Discussion on proper simplification of fate modeling in the framework of nonlinear interactions is still unavailable. Conclusions Although the methodologies and concepts for studying HOC environmental fate could be adopted for PPCP study, their differences should be highly understood. Prediction of PPCP environmental behavior needs to combine contributions from various fractions of soils or sediments and the sorption of their metabolites and different species. Recommendations and perspectives More detailed studies on PPCP sorption in separated soil or sediment fractions are needed in order to propose a model predicting PPCP sorption in soils or sediments based on soil or sediment properties. The information on sorption of PPCP metabolites and species and the competition between them is still not enough to be incorporated into any predictive models.

Background, aim and scope In the region of the Apuseni Mountains, part of the Western Carpathians in Romania, metal mining activities have a long-standing tradition. These mining industries created a clearly beneficial economic development in the region. But their activities also caused impairments to the environment, such as acid mine drainage (AMD) resulting in long-lasting heavy metal pollution of waters and sediments. The study, established in the context of the ESTROM programme, investigated the impact of metal mining activities both from environmental and socioeconomic perspectives and tried to incorporate the results of the two approaches into an integrated proposition for mitigation of mining-related issues. Study site The small Certej catchment, situated in the Southern Apuseni Mountains, covers an area of 78 km². About 4,500 inhabitants are living in the basin, in which metal mining was the main economic sector. An open pit and several abandoned underground mines are producing heavy metal-loaded acidic water that is discharged untreated into the main river. The solid wastes of mineral processing plants were deposited in several dumps and tailings impoundment embodying the acidic water-producing mineral pyrite. Methods The natural science team collected samples from surface waters, drinking water from dug wells and from groundwater. Filtered and total heavy metals, both after enrichment, and major cations were analysed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Major anions in waters, measured by ion chromatography, alkalinity and acidity were determined by titration. Solid samples were taken from river sediments and from the largest tailings dam. The latter were characterised by X-ray fluorescence and X-ray diffraction. Heavy metals in sediments were analysed after digestion. Simultaneously, the socioeconomic team performed a household survey to evaluate the perception of people related to the river and drinking water pollution by way of a logistic regression analysis. Results and discussion The inputs of acid mine waters drastically increased filtered heavy metal concentrations in the Certej River, e.g. Zn up to 130 mg L⁻¹, Fe 100 mg L⁻¹, Cu 2.9 mg L⁻¹, Cd 1.4 mgL⁻¹ as well as those of SO₄ up to 2.2 g L⁻¹. In addition, river water became acidic with pH values of pH 3. Concentrations of pollutant decreased slightly downstream due to dilution by waters from tributaries. Metal concentrations measured at headwater stations reflect background values. They fell in the range of the environmental quality standards proposed in the EU Water Framework Directive for dissolved heavy metals. The outflow of the large tailing impoundment and the groundwater downstream from two tailings dams exhibited the first sign of AMD, but they still had alkalinity. Most dug wells analysed delivered a drinking water that exhibited no sign of AMD pollution, although these wells were a distance of 7 to 25 m from the contaminated river. It seems that the Certej River does not infiltrate significantly into the groundwater. Pyrite was identified as the main sulphide mineral in the tailings dam that produces acidity and with calcite representing the AMD-neutralising mineral. The acid-base accounting proved that the potential acid-neutralising capacity in the solid phases would not be sufficient to prevent the production of acidic water in the future. Therefore, the open pits and mine waste deposits have to be seen as the sources for AMD at the present time, with a high long-term potential to produce even more AMD in the future. The socioeconomic study showed that mining provided the major source of income. Over 45% of the households were partly or completely reliant on financial compensations as a result of mine closure. Unemployment was considered by the majority of the interviewed persons as the main cause of social problems in the area. The estimation of the explanatory factors by the logistic regression analysis revealed that education, household income, pollution conditions during the last years and familiarity with environmental problems were the main predictors influencing peoples' opinion concerning whether the main river is strongly polluted. This model enabled one to predict correctly 77% of the observations reported. For the drinking water quality model, three predictors were relevant and they explained 66% of the observations. Conclusions Coupling the findings from the natural science and socioeconomic approaches, we may conclude that the impact of mining on the Certej River water is high, while drinking water in wells is not significantly affected. The perceptions of the respondents to pollution were to a large extent consistent with the measured results. Recommendations and perspectives The results of the study can be used by various stakeholders, mainly the mining company and local municipalities, in order to integrate them in their post-mining measures, thereby making them aware of the potential long-term impact of mining on the environment and on human health as well as on the local economy.

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.

Background, aim, and scope Selenium is a trace metalloid of global environmental concern. The boundary among its essentiality, deficiency, and toxicity is narrow and mainly depends on the chemical forms and concentrations in which this element occurs. Different plant species--including Brassica juncea--have been shown to play a significant role in Se removal from soil as well as water bodies. Furthermore, the interactions between such plants, showing natural capabilities of metal uptake and their rhizospheric microbial communities, might be exploited to increase both Se scavenging and vegetable biomass production in order to improve the whole phytoextraction efficiency. The aim of the present study was to evaluate the capability of selenite removal of B. juncea grown in hydroponic conditions on artificially spiked effluents. To optimize phytoextraction efficiency, interactions between B. juncea and rhizobacteria were designedly elicited. Materials and methods Firstly, B. juncea was grown on water-filtering agriperlite beds in the presence of three different selenite concentrations, namely, 0.2, 1.0, and 2.0 mM. Plant growth was measured after 3 and 6 weeks of incubation in order to establish the selenite concentration at which the best plant biomass production could be obtained. Afterwards, water-filtering agriperlite beds were inoculated either with a selenium-acclimated microbial community deriving from the rhizosphere of B. juncea grown, erstwhile, in a selenite-amended soil or with axenic cultures of two bacterial strains, vicelike Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02, previously isolated and described for their high resistance to selenite. These latter were seeded separately or as a dual consortium. Selenite was amended at a final concentration of 1.0 mM. Total Se content in plant tissues (both shoots and roots), plant biomass production, and persistence of bioaugmented microbial inocula during the experimental time were monitored. Moreover, parameters such as bioconcentration factor (BF) and phytoextraction efficiency (PE) were determined at the end of the testing run to evaluate the effects of the different bioaugmentation strategies adopted on selenite phytoextraction efficiency of B. juncea. Results A general but significant increase in capacity to extract and transport selenium to the epigeous plant compartments was recorded in B. juncea grown in beds augmented with microbial inocula, except for the treatment with B. mycoides SeITE01 alone. Nevertheless, a severe decrease in vegetable biomass production was observed after all microbial treatments with the exception of the plants that had received only S. maltophilia SeITE02. Actually, an increase in selenium phytoextraction efficiency up to 65% was observed in B. juncea, when this bacterial strain was inoculated. Discussion Emendation of B. juncea grown in water-filtering beds with a Se(IV)-acclimated microbial community caused a higher Se uptake along with a reduction of plant biomass yield with respect to plants grown without addition of the same bacterial inoculum. The increase of selenium BF in shoots suggests that the Se(IV)-acclimated microbial community not only elicited the plant capacity to absorb selenite, but also did improve the capacity to transport the metalloid to the epigeous compartments. On the other hand, the reduction in plant biomass yield might be related exactly to this improved capability of B. juncea to accumulate selenium at concentrations that are actually toxic for plants. Differently, addition of two selenite-resistant bacterial strains, namely, S. maltophilia SeITE02 and B. mycoides SEITE01, had weaker effects on plant biomass production when compared to those recorded in the presence of the Se(IV)-adapted microbial community. In particular, inoculation of water-filtering beds with the SeITE02 strain alone was the sole strategy resulting in a positive effect on both plant biomass production in stressful conditions and the capacity of shoots to accumulate selenium. In fact, its putative ability of reducing Se(IV) to organo-Se compounds significantly enhanced either selenium absorption by the plants or active metalloid translocation to epigeous parts. Conclusions Bioaugmentation with the bacterial strain S. malthophila SeITE02 is suggested to elicit selenite phytoextraction efficiency in B. juncea. Recommendations Manipulation of synergistic interactions between plants having phytoextraction capabilities and their associated rhizobacteria may enhance already consolidated treatment processes aimed to detoxify selenite laden wastewater.

Introduction Several hot spots of severe freshwater pollution and sediment contamination (mostly heavy metals, polyaromatic hydrocarbons and polychlorinated biphenyls) have been identified in Serbia as the consequence of outdated environmental legislation, negligible amounts of properly treated waste waters and accidental spills. Discussion Since ecotoxicological methods have never been incorporated into risk assessment procedures, mandatory effluent discharge or ambient water monitoring programmes, ecotoxicological research, based on bioaccumulation studies, conventional ecotoxicological tests and, recently, biomarkers of exposure and effect have been restricted to independent small- to medium-scale studies, conducted, basically, to confirm, underline or oppose the results of chemical-based monitoring and to lament on inadequate environmental regulations/policy and management practice. Although hot and unresolved ecotoxicological problems still remain beyond the reach of ecotoxicological research currently conducted in Serbia, or are tackled only sporadically, it is to be expected that on-going research and institutional capacity building should, hopefully, increase the competence and competitiveness of scientific community and speed up the process of harmonisation of national environmental legislation and policy with European Union.