The application of bioassays as indicators of petroleum-contaminated soil remediation

Bioremediation has proven successful in numerous applications to petroleum contaminated soils. However, questions remain as to the efficiency of bioremediation in lowering long-term soil toxicity. In the present study, the bioassays Spirotox, Microtox, Ostracodtoxkit F, umu-test with S-9 activation, and plant assays were applied, and compared to evaluate bioremediation processes in heavily petroleum contaminated soils. Six higher plant species (Secale cereale L., Lactuca sativa L., Zea mays L., Lepidium sativum L., Triticum vulgare L., Brassica oleracea L.) were used for bioassay tests based on seed germination and root elongation. The ecotoxicological analyses were made in DMSO/H2O and DCM/DMSO soil extracts. Soils were tested from two biopiles at the Czechowice oil refinery, Poland, that have been subjected to different bioremediation applications. In biopile 1 the active or engineered bioremediation process lasted four years, while biopile 2 was treated passively or non-engineered for eight months. The test species demonstrated varying sensitivity to soils from both biopiles. The effects on test organisms exposed to biopile 2 soils were several times higher compared to those in biopile 1 soils, which correlated with the soil contaminants concentration. Soil hydrocarbon concentrations indeed decreased an average of 81% in biopile 1, whereas in biopile 2 TPH/TPOC concentrations only decreased by 30% after eight months of bioremediation. The bioassays were presented to be sensitive indicators of soil quality and can be used to evaluate the quality of bioremediated soil. The study encourages the need to combine the bioassays with chemical monitoring for evaluation of the bioremediation effectiveness and assessing of the contaminated/remediated soils.     

An evaluation of surfactant foam technology in remediation of contaminated soil

Soil contamination is notoriously difficult to treat because the contaminants are often tightly bound to the soil particles. Conventional remediation technologies are becoming less popular due to the high treatment costs. This paper gives a comprehensive overview and evaluation of an emerging promising alternative, surfactant foam technology. Different from other approaches, surfactant foam technology may be designed either to remove contaminants or/and simultaneously act as an augmentation for the existing technologies such as pump-and-treat systems and bioremediation processes to improve the contaminant removal efficiency and cost effectiveness. Encouraging results were achieved from laboratory and field demonstrations. However, as an innovative technology, there are many factors to be investigated with the future development. Special attention is paid to the selection of the most appropriate foaming surfactant and surfactant concentration, which are critical to the success of the implementation of the remediation process and have significant effects on the treatment costs. Moreover, development of predictive mathematical models in for future research is helpful to optimize the remediation process.     

AhR agonist and genotoxicant bioavailability in a PAH-contaminated soil undergoing biological treatment

Background, aim, and scope  Degradation of the 16 US EPA priority PAHs in soil subjected to bioremediation is often achieved. However, the PAH loss is not always followed by a reduction in soil toxicity. For instance, bioanalytical testing of such soil using the chemical-activated luciferase gene expression (CALUX) assay, measuring the combined effect of all Ah receptor (AhR) activating compounds, occasionally indicates that the loss of PAHs does not correlate with the loss of Ah receptor-active compounds in the soil. In addition, standard PAH analysis does not address the issue of total toxicant bioavailability in bioremediated soil. Materials and methods  To address these questions, we have used the CALUX AhR agonist bioassay and the Comet genotoxicity bioassay with RTL-W1 cells to evaluate the toxic potential of different extracts from a PAH-contaminated soil undergoing large-scale bioremediation. The extracts were also chemically analyzed for PAH16 and PCDD/PCDF. Soil sampled on five occasions between day 0 and day 274 of biological treatment was shaken with n-butanol with vortex mixing at room temperature to determine the bioavailable fraction of contaminants. To establish total concentrations, parts of the same samples were extracted using an accelerated solvent extractor (ASE) with toluene at 100°C. The extracts were tested as inducers of AhR-dependent luciferase activity in the CALUX assay and for DNA breakage potential in the Comet bioassay. Results  The chemical analysis of the toluene extracts indicated slow degradation rates and the CALUX assay indicated high levels of AhR agonists in the same extracts. Compared to day 0, the bioavailable fractions showed no decrease in AhR agonist activity during the treatment but rather an up-going trend, which was supported by increasing levels of PAHs and an increased effect in the Comet bioassay after 274 days. The bio-TEQs calculated using the CALUX assay were higher than the TEQs calculated from chemical analysis in both extracts, indicating that there are additional toxic PAHs in both extracts that are not included in the chemically derived TEQ. Discussion  The response in the CALUX and the Comet bioassays as well as the chemical analysis indicate that the soil might be more toxic to organisms living in soil after 274 days of treatment than in the untreated soil, due to the release of previously sorbed PAHs and possibly also metabolic formation of novel toxicants. Conclusions  Our results put focus on the issue of slow degradation rates and bioavailability of PAHs during large-scale bioremediation treatments. The release of sorbed PAHs at the investigated PAH-contaminated site seemed to be faster than the degradation rate, which demonstrates the importance of considering the bioavailable fraction of contaminants during a bioremediation process. Recommendations and perspectives  It has to be ensured that soft remediation methods like biodegradation or the natural remediation approach do not result in the mobilization of toxic compounds including more mobile degradation products. For PAH-contaminated sites this cannot be assured merely by monitoring the 16 target PAHs. The combined use of a battery of biotests for different types of PAH effects such as the CALUX and the Comet assay together with bioavailability extraction methods may be a useful screening tool of bioremediation processes of PAH-contaminated soil and contribute to a more accurate risk assessment. If the bioremediation causes a release of bound PAHs that are left undegraded in an easily extracted fraction, the soil may be more toxic to organisms living in the soil as a result of the treatment. A prolonged treatment time may be one way to reduce the risk of remaining mobile PAHs. In critical cases, the remediation concept might have to be changed to ex situ remediation methods.     

PAH dissipation in spiked soil: impacts of bioavailability, microbial activity, and trees

While trees have demonstrated potential in phytoremediation of several organic contaminants, little is known regarding their ability to impact the common soil contaminant PAHs. Several species of native North American trees were planted in soil artificially contaminated with three PAHs. Plant biomass, PAH dissipation, and microbial mineralization were monitored over the course of one year and environmental conditions were allowed to follow typical seasonal patterns. PAH dissipation and mineralization were not affected by planting. Extensive and rapid loss of PAHs was observed and attributed to high bioavailability and microbial activity in all treatments. The rate of this loss may have masked any significant planting effects. Anthracene was found to be more recalcitrant than pyrene or phenanthrene. Parallel soil aging studies indicated that sequestration to soil components was minimal. Contrary to common inferences in literature, amendment with decaying fine roots inhibited PAH degradation by the soil microbial community. Seasonal variation in environmental factors and rhizosphere dynamics may have also reduced or negated the effect of planting and should be taken into account in future phytoremediation trials. The unique root traits of trees may pose a challenge to traditional thought regarding PAH dissipation in the rhizosphere of plants.     

Decomposer communities in contaminated soil: Is altered community regulation a proper tool in ecological risk assessment of toxicants?

Effects of patchy soil contamination on decomposer organisms, their community regulation and nutrient mineralization were studied in a microcosm experiment. Coniferous forest soil was patchily contaminated with three concentrations of sodiumpentachlorophenate PCP (0, 50 and 500 mg PCP kg(-1) of dry soil). Abundance of microbes, enchytraeids, nematodes, small oribatids and predatory mites were reduced by the PCP. Direct toxicity of PCP and lowered microbial biomass seemed to affect animal community composition in the most contaminated patches. Some large oribatids which seemed to be tolerant to PCP increased their numbers in the most contaminated patches. Although predatory mites suffered from PCP, no altered predator-prey interactions were observed. At the beginning of the experiment more nutrients were released in the patches with highest PCP concentration and the nutrients accumulated in the soil. Soil decomposer food webs seemed to be mainly bottom-up controlled: PCP strongly affects microbes and hence caused changes in the community structure of soil animals and nutrient cycling. Hence top-down orientated ecological models on community regulation and food web dynamics seem to be unsuitable when assessing effects of pesticides on soil communities.     

Ecorisk evaluation and treatability potential of soils contaminated with petroleum hydrocarbon-based fuels

We used a series of toxicity tests to monitor oil degradation in the Kuwaiti oil lakes. Three soils from different locations with a history of hydrocarbon contamination were treated in bench-scale microcosms with controlled nutrient amendments, moisture content, and temperature that had promoted mineralization of total hydrocarbon and oil and grease in a preliminary study. Two hundred days of bioremediation treatment lowered hydrocarbon concentration to below 2 and 5 mg g(-1) for soils A and B, respectively, while in soil C hydrocarbon concentration remained at 12 mg g(-1). Although 85% of the total petroleum hydrocarbons (TPHs) in soil A were reduced 50d after treatment, results of the seed germination and Microtox tests suggested an initial increase in toxicity, indicating that toxic intermediary metabolites may have formed during biodegradation. Also, the significant decrease of TPHs and corresponding high toxicity levels were noted in soil B 200d after bioremediation. Clearly, toxicity values, and not just hydrocarbon concentration, are a key factor in assessing the effectiveness of bioremediation techniques. Field chemistry data showed a significant reduction in hydrocarbon levels after the biological treatment. We concluded that the toxicity assessment of the contaminated soil with a battery of toxicity bioassays could provide meaningful information regarding a characterization procedure in ecological risk assessment.     

Application of microbial hot spots enhances pesticide degradation in soils

Through transfer of an active, isoproturon degrading microbial community, pesticide mineralization could be successfully enhanced in various soils under laboratory and outdoor conditions. The microbes, extracted from a soil having high native ability to mineralize this chemical, were established on expanded clay particles and distributed to various soils in the form of microbial "hot spots". Both, diffusion controlled isoproturon mass flow towards these "hot spots" (6microg d(-1)) as well as microbial ability to mineralize the herbicide (approximately 5microg d(-1)) were identified as the main processes enabling a multiple augmentation of the native isoproturon mineralization even in soils with heavy metal contamination. Soil pH-value appears to exert an important effect on the sustainability of this process.     

次级河流水污染事件应急处置程序及环境损害评估技术路线

近年来,随着我国经济的发展,次级河流突发性水污染事件频繁发生,对生态环境及居民生活造成了严重的影响。次级河流突发性水污染事件发生后,为减少污染造成的损失和影响,保证受损的环境资源得到恢复和补偿,不仅要立即采取行之有效的应急处置措施,而且应当积极开展环境损害鉴定评估工作,全面追究污染者的环境责任。根据次级河流本身的特点,结合各省市在环境应急处置过程中的具体做法,建立了一套应对突发性次级河流水污染事件的应急处置工作程序;阐述了环境损害鉴定评估的必要性、工作方法和技术路线,同时从受损环境资源的确认、污染源和污染物的识别以及暴露途径的建立3个方面构建了开展次级河流突发性水污染事件环境损害鉴定评估工作的因果关系链条,对实际开展相关工作具有重要意义。     

Soil microbial parameters and luminescent bacteria assays as indicators for in situ bioremediation of TNT-contaminated soils

In situ bioremediation is increasingly being discussed as a useful strategy for cleaning up contaminated soils. Compared to established ex situ procedures, meaningful and reliable approaches for monitoring the remediation processes and their efficiency are of special importance. The subject of this study was the significance of two bioassays for monitoring purposes. The work was performed within the scope of a research project on the in situ bioremediation of topsoil contaminated with 2,4,6-trinitrotoluene (TNT). To evaluate changes within different experimental fields during a 17-month remediation period, the results of soil microbial assays and luminescent bacteria assays were compared with chemical monitoring data. The luminescent bacteria assays showed a significant reduction of the water-soluble soil toxicants in the treated fields. This bioassay proved to be a sensitive screening indicator of toxicity and may effectively aid the ecotoxicological interpretation of chemical monitoring data. Microbial biomass (C(mic)), the metabolic quotient (qCO2), and the ratio of microbial to organic carbon (C(mic)/C(org)) showed a highly significant correlation with total concentrations of TNT in the soil. But, in contrast to luminescent bacteria assays, this approach did not reveal any recovery of the soil at the end of the remediation period. There is clear evidence for persistent adverse effects of chronic TNT contamination on the site-specific microbial community and the local carbon cycle in the soil. The study clearly exhibits the differences between, as well as the complementary value of both bioassay approaches for monitoring short-term and long-term effects of soil contamination and the efficiency of remediation.     

Organic Contaminants from Sewage Sludge Applied to Agricultural Soils. False Alarm Regarding Possible Problems for Food Safety? (8 pp)

Goal, Scope and Background Sewage sludge produced in wastewater treatment contains large amounts of organic matter and nutrients and could, therefore, be suitable as fertiliser. However, with the sludge, besides heavy metals and pathogenic bacteria, a variety of organic contaminants can be added to agricultural fields. Whether the organic contaminants from the sludge can have adverse effects on human health and wildlife if these compounds enter the food chain or groundwater still remains a point of controversial discussion. Main Features This paper presents an overview on the present situation in Europe and a summary of some recent results on the possible uptake of organic contaminants by crops after addition to agricultural fields by sewage sludge. Results Greenhouse experiments and field trials were performed to study the degradation and uptake of organic micro-contaminants in sludge-amended agricultural soil in crops, such as barley and carrots grown in agricultural soil amended with anaerobically-treated sewage sludge from a wastewater treatment plant, but studies hitherto have revealed no immediate risks. Common sludge contaminants such as linear alkylbenzene sulphonates (LAS), nonylphenol ethoxylates (NPE), polycyclic aromatic hydrocarbons (PAH), bis(diethylhexyl) phthalate (DEHP), showed neither accumulation in soil nor uptake in plants. Discussion It is assumed that the annual amount of sewage sludge produced in Europe will increase in the future, mainly due to larger amounts of high quality drinking water needed by an increasing population and due to increasing demands for cleaner sewage water. Application of sewage sludge to agricultural soils is sustainable and economical due to nutrient cycling and disposal of sewage sludge. However, this solution also involves risks with respect to the occurrence of organic contaminants and other potentially harmful contents such as pathogens and heavy metals present in the sludge. There have been concerns that organic contaminants may accumulate in the soil, be taken up by plants and thereby transferred to humans via the food chain. Results obtained so far revealed, however, no immediate risk of accumulation of common organic sludge contaminants in soil or uptake in plants when applying sewage sludge to agricultural soil. With very high dosages of sewage sludge, there may be a risk for accumulation of very apolar contaminants, such as DEHP, to the soil. Conclusions Any conclusions on the safe use of sewage sludge in agriculture have to be drawn carefully, as the studies performed until now have been limited. Further studies are required, and before final statements can be drawn, it is imminent to study a larger variety of common crops and the effect sewage sludge application may have on a possible accumulation of organic contaminants in the crops. Furthermore, a larger variety of organic contaminants need to be studied and special focus should be given to contaminants newly introduced into the environment. Besides investigating possible plant uptake of organic contaminants, the fate of these compounds in soil after sludge application need to be monitored too. Here, special attention has to begiven to studies on degradation and the formation of degradation products, to weathering and to leaching effects on groundwater, to the application of different crops on the same field (crop rotation), to the use of full-width tillage and strip tillage, and to long term application of sewage sludge on the soil. Recommendations and Perspectives There are environmental, political as well as economical incentives to increase the agricultural application of sludge. However, such usage should be performed with care as there are also ways in which sludge fertilisation could harm the environment and human health. Recently, a new European COST Action (859) has been established covering the field of food safety and improved food quality. Part of the Action is dealing with the application of sewage sludge in agriculture. Before any political and economical measures can be taken, the pros and cons have to be sufficiently investigated on a scientific level first. ESS-Submission Editor: Prof. Elena Maestri (elena.maestri@unipr.it)     

Evaluation of the toxicity of two soils from Jales Mine (Portugal) using aquatic bioassays

Soil contamination can be one path for streams and groundwater contamination. As a complement of chemical analysis and total contaminants determination, bioassays can provide information on the bioavailable fraction of chemical compounds, focusing on the retention and habitat function of soils. In this study the evaluation of the toxicity of two soils from the abandoned Jales Mine (Portugal) regarded both functions. The buffer capacity of soils was tested with bioassays carried out using the cladoceran Daphnia magna and the marine bacteria Vibrio fischeri. The habitat function of soils was evaluated with the reproduction bioassay with the collembolan Folsomia candida. The Microtox solid-phase test was performed with V. fischeri using soil as test medium, and soil elutriates were extracted to perform the Microtox basic test, and an immobilization and reproduction bioassay with D. magna. The marine bacteria showed high sensitivity to the soil with low heavy metal content (JNC soil) and to JNC soil elutriates, while the soil with highest heavy metal content (JC soil) or soil elutriates exposure did not cause any toxic effect. In the bioassays with D. magna, organisms showed sensitivity to JNC and also to JC soil elutriates. Both mobilization and reproduction features were inhibited. The bioassay with F. candida did not reflect any influence of the contaminants on their reproduction. Although JNC soil presented lower heavy metal contents, elutriates showed different patterns of contamination when compared to JC soil and elutriates, which indicates different retention and buffer capacities between soils. Results obtained in this study underlined the sensitivity and importance of soil elutriate bioassays with aquatic organisms in the evaluation strategy in soil ERA processes.     

Soil screening for identifying ecological risk stressors using a battery of in vitro cell bioassays

Soil screening could be a process of identifying and defining areas, contaminants, and condition at the sites that warrant further attention for developing ecological risk assessments. In present work, a total of 41 surface soil samples from Tianjin, China were sampled and the soil organic extracts were evaluated using a battery of in vitro cell bioassays. The battery included ethoxyresorfin O-deethylase (EROD) with H4IIE rat hepatoma cells bioassay for Aryl hydrocarbon (Ah) receptor (Ah-agonists) effects, the SOS/umu bioassay for genotoxic effects, and human estrogen receptor recombinant yeast bioassay for estrogenic effects. The results have showed that total estrogenic effects in these soil samples was measured to be between 0.1 and 14.2ng EEQkg(-1) soil (d.w.); Ah-agonists effects assayed by EROD bioassay varied from 2.8ng TEQkg(-1) soil (d.w.) to 42.6ng TEQkg(-1) soil (d.w.), and the amounts of soil weight required for the extracts to lead positive result (IR 2.0) in the SOS/umu bioassay were between 3.9 and 31.3mg (d.w.) per well. In addition, the geographic distributions of Ah-agonists effects and genotoxic effects in Tianjin area exhibited a strong positive correlation with each other. However, the distribution of estrogenic effects with high levels in northwest Tianjin was markedly different from that of Ah-agonists effects, where the high levels were distributed in the urban of Tianjin, as well as coastal towns. It has been concluded that the toxicity assessment of surface soil using a battery of in vitro cell bioassays could provided meaningful information regarding characterization procedure in ecological risk assessment.     

Evaluation of post-Katrina flooded soils for contaminants and toxicity to the soil invertebrates Eisenia fetida and Caenorhabditis elegans

This research evaluated soil samples from a New Orleans neighborhood in the Chalmette, Saint Bernard Parish, that had been inundated by flooding associated with Hurricane Katrina. The goal was to determine if ecological risks persisted from flood waters that had come in contact with hazardous surface chemicals before inundating this low-lying neighborhood for a prolonged period. Research objectives were to establish the presence or absence of volatile organic and heavy metal contaminants, and then asses the toxicity of this soil to Eisenia fetida in a soil exposure assay and Caenorhabditis elegans in a simulated porewater exposure assay. Soil analysis revealed detectable levels of metals and organics in the surface soil at each location. No contaminant was detected in concentrations above human health screening values. Chromium and mercury were detected at levels in excess of typical ecological risk values. Soil extracts revealed concentrations of nitrate, sulfate, and chloride above those from an unflooded background sample. Toxicity testing resulted in no acute effects to either test species, but did show bioaccumulation of arsenic, cadmium, and lead in E. fetida exposed to several samples. The combination of mercury and sulfate provide the potential for mercury methylation should flooding and prolonged inundation occur again.     

Assessment of spiking procedures for the introduction of a phenanthrene-LNAPL mixture into field-wet soil

Laboratory based studies on the fate of organic contaminants in soil typically requires the test compound(s) to be spiked into the test medium. Consequently, such studies are inherently dependent on the homogeneity of the contaminant within the spiked soil. Three blending methods were compared for the addition of a phenanthrene-transformer oil mixture into field-wet soil. Spiking homogeneity, reproducibility and artefacts were assessed based on dichloromethane and hydroxypropyl-beta-cyclodextrin chemical extractability, and bacterial mineralization. Spiking using a stainless-steel spoon, consistently produced good spike homogeneity as determined by sample oxidation, chemical extraction and mineralization, and was consistently more reliable than either the Waring blender or modified bench drill. Overall, neither transformer oil-concentration nor blending method influenced chemical extractability or mineralization of the PAH following 1 day equilibration. In general, spiking procedures require validation prior to use, as homogeneity cannot be assured.     

A case study of contaminants on military ranges: Camp Edwards, Massachusetts, USA

An extensive investigation at the Camp Edwards, Massachusetts Military Reservation (MMR) demonstrates that assessment of groundwater and soil contamination at military ranges can be limited primarily to explosive-related compounds such as RDX, HMX, perchlorate, TNT and their transformation products. A modified analytical method is recommended to expand the list of explosives and to improve the detection limits. Analyses of metals, VOCs, SVOCs, and TICs are unnecessary. Soil samples may require the analyses of PAHs and PCNs for burn areas. Camp Edwards, as one of the few military ranges that have been exhaustively investigated for contaminants, is an ideal point of departure for evaluating other ranges. The permeable site soils promote leaching of contaminants and inhibit biotic and abiotic transformations. Moreover, the site has experienced an unusual extent of activities in its more than ninety years of active use. The recommendations in this report are based on data obtained for more than 200 analytes from more than 15,000 environmental samples.     

Effects of Cd and Pb on soil microbial community structure and activities

Background, aim, and scope  

Soil contamination with heavy metals occurs as a result of both anthropogenic and natural activities. Heavy metals could have long-term hazardous impacts on the health of soil ecosystems and adverse influences on soil biological processes. Soil enzymatic activities are recognized as sensors towards any natural and anthropogenic disturbance occurring in the soil ecosystem. Similarly, microbial biomass carbon (MBC) is also considered as one of the important soil biological activities frequently influenced by heavy metal contamination. The polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) has recently been used to investigate changes in soil microbial community composition in response to environmental stresses. Soil microbial community structure and activities are difficult to elucidate using single monitoring approach; therefore, for a better insight and complete depiction of the soil microbial situation, different approaches need to be used. This study was conducted in a greenhouse for a period of 12 weeks to evaluate the changes in indigenous microbial community structure and activities in the soil amended with different application rates of Cd, Pb, and Cd/Pb mix. In a field environment, soil is contaminated with single or mixed heavy metals; so that, in this research, we used the selected metals in both single and mixed forms at different application rates and investigated their toxic effects on microbial community structure and activities, using soil enzyme assays, plate counting, and advanced molecular DGGE technique. Soil microbial activities, including acid phosphatase (ACP), urease (URE), and MBC, and microbial community structure were studied.     

Electrokinetic enhancement of phenanthrene biodegradation in creosote-polluted clay soil

Given the difficulties caused by low-permeable soils in bioremediation, a new electrokinetic technology is proposed, based on laboratory results with phenanthrene, to afford bioremediation of polycyclic aromatic hydrocarbons (PAH) in clay soils. Microbial activity in a clay soil historically polluted with creosote was promoted using a specially designed electrokinetic cell with a permanent anode-to-cathode flow and controlled pH. The rates of phenanthrene losses during treatment were tenfold higher in soil treated with an electric field than in the control cells without current or microbial activity. Results from experiments with Tenax-assisted desorption and mineralization of 14C-labeled phenanthrene indicated that phenanthrene biodegradation was limited by mass-transfer of the chemical. We suggest that the enhancement effect of the applied electric field on phenanthrene biodegradation resulted from mobilization of the PAH and nutrients dissolved in the soil fluids.     

Ecological recovery of vegetation at a former industrial sludge basin and its implications to phytoremediation

Examination of a former industrial sludge basin containing organic pollutants showed that the basin had undergone substantial ecological recovery through natural forces following the removal of surface water in 1982. Conventional phases of ecological recovery (plant invasion and succession) have occurred, but the structure of the biodiverse plant community (51 species and 22 families) was different from that at a recovering non-polluted disturbed site. Three plant species (Bermuda grass, mulberry, and sunflower) believed to be early invaders of the basin still persist in large numbers indicating that these species are well suited to cope with normal environmental stresses at this area (i.e. seasonal drought and flood) as well as organic pollutants. There was an indication that early invaders of the site fostered disappearance of contaminants thereby creating more favorable conditions for a broader spectrum of plants to grow. Vegetation analyses of naturally vegetated hazardous waste sites hold promise as a screening device for identifying plant species and management practices worthy of further phytoremediation investigations.