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.     

Lability of polycyclic aromatic hydrocarbons in the rhizosphere

Remediation of soils containing high concentrations of polycyclic aromatic hydrocarbons (PAHs) seldom results in complete removal of contaminants, but residual toxicity often is reduced. In this study, soil from a former manufactured gas plant site was treated for 12 months by phytoremediation and then tested for total PAHs, Tenax-TA extractable ("labile") PAHs, aqueous soluble PAHs (PAH(wp)) , and biotoxicity assessed by earthworms survival, nematode mortality, emergence of lettuce seedlings, and microbial respiration. Prior to phytoremediation, the soil had toxic impacts on all bioassays (except the nematodes), and 12 months of remediation decreased this response. Change in labile PAHs was a predictor for change in total PAH for 3- and 4-ring compounds but not for the 5- and 6-ring. Decreases in labile PAHs were correlated (r(2)>or=0.80) with toxicity in the bioassays except microbial respiration. PAH(wp) was correlated only with nematode toxicity prior to remediation but with none of the tests after remediation. Total PAHs were not correlated with any of the bioassay tests. Tenax-TA appears to have potential for predicting residual toxicity in remediated soils and is superior to total concentrations for that application.     

Advanced multivariate analysis to assess remediation of hydrocarbons in soils

Accurate monitoring of degradation levels in soils is essential in order to understand and achieve complete degradation of petroleum hydrocarbons in contaminated soils. We aimed to develop the use of multivariate methods for the monitoring of biodegradation of diesel in soils and to determine if diesel contaminated soils could be remediated to a chemical composition similar to that of an uncontaminated soil. An incubation experiment was set up with three contrasting soil types. Each soil was exposed to diesel at varying stages of degradation and then analysed for key hydrocarbons throughout 161 days of incubation. Hydrocarbon distributions were analysed by Principal Coordinate Analysis and similar samples grouped by cluster analysis. Variation and differences between samples were determined using permutational multivariate analysis of variance. It was found that all soils followed trajectories approaching the chemical composition of the unpolluted soil. Some contaminated soils were no longer significantly different to that of uncontaminated soil after 161 days of incubation. The use of cluster analysis allows the assignment of a percentage chemical similarity of a diesel contaminated soil to an uncontaminated soil sample. This will aid in the monitoring of hydrocarbon contaminated sites and the establishment of potential endpoints for successful remediation.     

Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review

Polynuclear aromatic hydrocarbons (PAHs) constitute a group of priority pollutants which are present at high concentrations in the soils of many industrially contaminated sites. Criteria established for the removal or treatment or both of soils contaminated with PAHs vary widely within and between nations. The bioremediation of contaminated soils with in-situ, on-site, and bioreactor techniques is reviewed, together with the factors affecting PAH degradation. Current in-situ remediation techniques are considered ineffective for the removal of most PAHs from contaminated soil. On-site 'landforming' methods have been used successfully (and within a reasonable period of time) to degrade only those PAHs with three or fewer aromatic rings. Bioreactors have proved most effective for soil remediation, since conditions for enhanced degradation can be achieved most readily. However, bioreactors are still at the development stage, and further research is required to optimise their efficiency and economy for routine use. Degradation of the more recalcitrant high-molecular-weight PAHs is contaminated soil has not been particularly successful to date. Further research needs are identified to help develop bioremediation into a most cost-effective technology. The importance of full site assessments and treatability studies for successful application in the field is emphasised.     

Applicability of the bioluminescence inhibition test in the 96-well microplate format for PAH-solutions and elutriates of PAH-contaminated soils

The use of conventional plastic microplates for a miniaturised luminescent bacteria test may result in an underestimation of the toxicity for poorly water soluble highly adsorbing toxicants such as PAHs. In this study, the suitability of microplates for testing elutriates of PAH-contaminated soils was investigated. The LUMIStox test was performed as the standard test in the miniaturised format using contaminated soil elutriates and aqueous solutions of four selected PAHs (viz. naphthalene (NAP), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)). For the aqueous PAH-solutions, we observed reduced light inhibition values for the miniaturised bioassay when using black microplates made of polypropylene (PP) and polystyrene (PS) compared to the standard LUMIStox test. This phenomenon was most likely due to adsorption of toxicants to the microplate surfaces with PAHs of lower water solubility being significantly more affected; however, after minimizing the exposure of samples to plastic surfaces, polystyrene microplates revealed equivalent performance (>80% 'relative' light inhibition) to the standard glass cuvette test system. For soil elutriates, black microplates again exhibited slightly lower light inhibition values while white plates made of PS and Barex resulted in a pronounced overestimation of toxicity for a coloured soil elutriate. In general, microplates were applicable for testing elutriates of PAH-contaminated soils. In cases where samples are coloured or turbid, the application of black microplates is recommended.     

Establishing principal soil quality parameters influencing earthworms in urban soils using bioassays

Potential contamination at ex-industrial sites means that, prior to change of use, it will be necessary to quantify the extent of risks to potential receptors. To assess ecological hazards, it is often suggested to use biological assessment to augment chemical analyses. Here we investigate the potential of a commonly recommended bioassay, the earthworm reproduction test, to assess the status of urban contaminated soils. Sample points at all study sites had contaminant concentrations above the Dutch soil criteria Target Values. In some cases, the relevant Intervention Values were exceeded. Earthworm survival at most points was high, but reproduction differed significantly in soil from separate patches on the same site. When the interrelationships between soil parameters and reproduction were studied, it was not possible to create a good model of site soil toxicity based on single or even multiple chemical measurements of the soils. We thus conclude that chemical analysis alone is not sufficient to characterize soil quality and confirms the value of biological assays for risk assessment of potentially contaminated soils.     

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.     

Removal of polycyclic aromatic hydrocarbons from manufactured gas plant-contaminated soils using sunflower oil: laboratory column experiments

Laboratory column experiments were performed to remove PAHs (polycyclic aromatic hydrocarbons) from two contaminated soils using sunflower oil. Two liters of sunflower oil was added to the top of the columns (33 cm x 21 cm) packed with 1 kg of PAH-contaminated soil. The sunflower oil was applied sequentially in two different ways, i.e. five additions of 400 ml or two additions of 1l. The influence of PAH concentration and the volume of sunflower oil on PAH removal were examined. A soil respiration experiment was carried out and organic carbon contents of the soils were measured to determine degradability of remaining sunflower oil in the soils. Results showed that the sunflower oil was effective in removing PAHs from the two soils, more PAHs were removed by adding sunflower oil in two steps than in five steps, probably because of the slower flow rate in the former method. More than 90% of total PAHs was removed from a heavily contaminated soil (with a total 13 PAH concentration of 4721 mg kg(-1)) using 4 l of sunflower oil. A similar removal efficiency was obtained for another contaminated soil (with a total 13 PAH concentration of 724 mg kg(-1)), while only 2l was needed to give a similar efficiency. Approximately 4-5% of the sunflower oil remained in the soils. Soil respiration curves showed that remaining sunflower oil was degraded by allowing air exchange and supplying with nutrients. Organic carbon content of the soil was restored to original level after 180 d incubation. These results indicated that the sunflower oil had a great capacity to remove PAHs from contaminated soils, and sunflower oil solubilization can be an alternative technique for remediation of PAH contaminated soils.     

污染土壤修复效果评定方法的研究

在实施污染土壤修复的环境工程后，需要通过灵敏和有效的评定方法对污染土壤修复的效果进行评定。然而，单纯依靠化学方法进行污染土壤修复效果的评定，不能揭示土壤的整体质量特征，因此需要生态毒理方法作为相互补充的手段。本文概述了植物毒性评定法、陆生无脊椎动物评定法和土壤微生物评定法及其在污染土壤修复效果评定中的应用，并对污染土壤修复效果评定方法的发展前景进行展望。     

Dissolution and removal of PAHs from a contaminated soil using sunflower oil

This study reports on the feasibility of remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soils using sunflower oil, an environmentally-friendly solvent. Batch experiments were performed to test the influence of oil/soil ratio on the remediation of PAH contaminated soil, and to test the mass transfer behaviors of PAHs from soil to oil. An empirical model was employed to describe the kinetics of PAH dissolution and to predict equilibrium concentrations of PAHs in oil. PAH containing oil was regenerated using active carbon. Results show that dissolution of PAHs from a Manufactured Gas Plant (MGP) soil at oil/soil ratios of one or two were almost the same. Nearly all PAHs (81-100%) could be removed by sunflower oil dissolution. Mass transfer coefficients for low molecular PAHs namely fluoranthene, phenanthrene and anthracene were one or two orders of magnitude higher than those for high molecular PAHs with 4-6 rings. Ninety milliliters of PAH containing oil could be regenerated by 10 g active carbon in a batch reactor. Such a remediation procedure indicates that sunflower oil is a promising agent for the removal of PAHs from MGP soils. However, further research is required before the method can be used for in situ remediation of contaminated sites.     

污染土壤修复效果评定方法的研究

在实施污染土壤修复的环境工程后,需要通过灵敏和有效的评定方法对污染土壤修复的效果进行评定.然而,单纯依靠化学方法进行污染土壤修复效果的评定,不能揭示土壤的整体质量特征,因此需要生态毒理方法作为相互补充的手段.本文概述了植物毒性评定法、陆生无脊椎动物评定法和土壤微生物评定法及其在污染土壤修复效果评定中的应用,并对污染土壤修复效果评定方法的发展前景进行展望.     

Effect of Sphingobium yanoikuyae B1 inoculation on bacterial community dynamics and polycyclic aromatic hydrocarbon degradation in aged and freshly PAH-contaminated soils

Sphingobium yanoikuyae B1 is able to degrade a range of polycyclic aromatic hydrocarbons (PAHs) and as a sphingomonad belongs to one of the dominant genera found in PAH-contaminated soils. We examined the ecological effect that soil inoculation with S. yanoikuyae B1 has on the native bacterial community in three different soils: aged PAH-contaminated soil from an industrial site, compost freshly contaminated with PAHs and un-contaminated compost. Survival of S. yanoikuyae B1 was dependent on the presence of PAHs, and the strain was unable to colonize un-contaminated compost. Inoculation with S. yanoikuyae B1 did not cause extensive changes in the native bacterial community of either soil, as assessed by denaturing gel electrophoresis, but its presence led to an increase in the population level of two other species in the aged contaminated soil community and appeared to have an antagonistic affect on several members of the contaminated compost community, indicating niche competition.     

Sources, fate, and toxic hazards of oxygenated polycyclic aromatic hydrocarbons (PAHs) at PAH-contaminated sites

In this paper we show that oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are important cocontaminants that should be taken into account during risk assessment and remediation of sites with high levels of PAHs. The presented data, which have been collected both from our own research and the published literature, demonstrate that oxy-PAHs are abundant but neglected contaminants at these sites. The oxy-PAHs show relatively high persistency and because they are formed through transformation of PAHs, their concentrations in the environment may even increase as the sites are remediated by methods that promote PAH degradation. Furthermore, we show that oxy-PAHs are toxic to both humans and the environment, although the toxicity seems to be manifested through other effects than those known to be important for polycyclic aromatic compounds in general, that is, mutagenicity and carcinogenicity. Finally, we present data that support the hypothesis that oxy-PAHs are more mobile in the environment than PAHs, due to their polarity, and thus have a higher tendency to spread from contaminated sites via surface water and groundwater. We believe that oxy-PAHs should be included in monitoring programs at PAH-contaminated sites, even if a number of other toxicologically relevant compounds that may also be present, such as nitro-PAHs and azaarenes, are not monitored. This is because oxy-PAH levels are difficult to predict from the PAH levels, because their environmental behavior differs substantially from that of PAHs, and oxy-PAHs may be formed as PAHs are degraded.     

A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils

To improve phytoremediation processes, multiple techniques that comprise different aspects of contaminant removal from soils have been combined. Using creosote as a test contaminant, a multi-process phytoremediation system composed of physical (volatilization), photochemical (photooxidation) and microbial remediation, and phytoremediation (plant-assisted remediation) processes was developed. The techniques applied to realize these processes were land-farming (aeration and light exposure), introduction of contaminant degrading bacteria, plant growth promoting rhizobacteria (PGPR), and plant growth of contaminant-tolerant tall fescue (Festuca arundinacea). Over a 4-month period, the average efficiency of removal of 16 priority PAHs by the multi-process remediation system was twice that of land-farming, 50% more than bioremediation alone, and 45% more than phytoremediation by itself. Importantly, the multi-process system was capable of removing most of the highly hydrophobic, soil-bound PAHs from soil. The key elements for successful phytoremediation were the use of plant species that have the ability to proliferate in the presence of high levels of contaminants and strains of PGPR that increase plant tolerance to contaminants and accelerate plant growth in heavily contaminated soils. The synergistic use of these approaches resulted in rapid and massive biomass accumulation of plant tissue in contaminated soil, putatively providing more active metabolic processes, leading to more rapid and more complete removal of PAHs.     

Using nanoscale zero-valent iron for the remediation of polycyclic aromatic hydrocarbons contaminated soil

The sites contaminated with recalcitrant organic compounds, such as polycyclic aromatic hydrocarbons (PAHs) with multiple benzene rings, are colossal and ubiquitous environmental problems. They are relatively nonbiodegradable and mutagenic, and 16 of them are listed in the U.S. Environment Protection Agency priority pollutants. Thus, the efficient and emerging remediation technologies for removal of PAHs in contaminated sites have to be uncovered urgently. In this decade, the zero-valent iron (ZVI) particles have been used successfully in the laboratory, pilot, and field, such as degradation of chlorinated hydrocarbons and remediation of the other pollutants. Nevertheless, as far as we know, little research has investigated for soil remediation; this study used nanoscale ZVI particles to remove pyrene in the soil. The experimental variables were determined, including reaction time, iron particle size, and dosage. From the results, both the micro- and nanoscales of ZVI were capable of removing the target compound in soil, but the higher removal efficiencies were by nanoscale ZVI because of the massive specific surface area. The optimal operating conditions to attain the best removal efficiency of pyrene were obtained while adding nanoscale ZVI 0.1 g/g soil within 60 min and 150 rpm of mixing. Thus, nanoscale ZVI has proved to be a promising remedy for PAH-contaminated soil in this study, as well as an optimistically predictable application for additional pilot and field studies.     

The water-soluble fraction of potentially toxic elements in contaminated soils: relationships between ecotoxicity, solubility and geochemical reactivity

To better understand the impacts posed by soil contamination to aquatic ecosystems it is crucial to characterise the links between ecotoxicity, chemical availability and geochemical reactivity of potentially toxic elements (PTE’s) in soils. We evaluated the adverse effects of water extracts obtained from soils contaminated by chemical industry and mining, using a test battery including organisms from different trophic levels (bacteria, algae and daphnids). These tests provided a quick assessment of the ecotoxicity of soils with respect to possible adverse effects on aquatic organisms although the ecotoxicological responses could be related to the solubility of PTE’s only to a limited extent.The analysis of results of bioassays together with the chemical characterisation of water extracts provided additional relevant insight into the role of conductivity, pH, Al, Fe, and Mn of soil extracts on toxicity to organisms. Furthermore, an important conclusion of this study was that the toxicity of extracts to the aquatic organisms could also be related to the soil properties (pH, Org C and Fe_ox) and to the reactivity of PTE’s in soils which in fact control the soluble fraction of the contaminants.The combined assessment of ecotoxicity in water fractions, solubility and geochemical reactivity of PTE’s in soils provided a more comprehensive understanding of the bioavailability of inorganic contaminants than ecotoxicological or chemical studies alone and can therefore be most useful for environmental risks assessment of contaminated soils.     

Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.     

Using Nanoscale Zero-Valent Iron for the Remediation of Polycyclic Aromatic Hydrocarbons Contaminated Soil

Abstract

The sites contaminated with recalcitrant organic compounds, such as polycyclic aromatic hydrocarbons (PAHs) with multiple benzene rings, are colossal and ubiquitous environmental problems. They are relatively nonbiodegradable and mutagenic, and 16 of them are listed in the U.S. Environment Protection Agency priority pollutants. Thus, the efficient and emerging remediation technologies for removal of PAHs in contaminated sites have to be uncovered urgently. In this decade, the zero-valent iron (ZVI) particles have been used successfully in the laboratory, pilot, and field, such as degradation of chlorinated hydrocarbons and remediation of the other pollutants. Nevertheless, as far as we know, little research has investigated for soil remediation; this study used nanoscale ZVI particles to remove pyrene in the soil. The experimental variables were determined, including reaction time, iron particle size, and dosage. From the results, both the micro- and nanoscales of ZVI were capable of removing the target compound in soil, but the higher removal efficiencies were by nanoscale ZVI because of the massive specific surface area. The optimal operating conditions to attain the best removal efficiency of pyrene were obtained while adding nanoscale ZVI 0.1 g/g soil within 60 min and 150 rpm of mixing. Thus, nanoscale ZVI has proved to be a promising remedy for PAH-contaminated soil in this study, as well as an optimistically predictable application for additional pilot and field studies.     

The application of the vermicomposting process in the bioremediation of diesel contaminated soils

Abstract

Polycyclic Aromatic Hydrocarbons (PAHs) are among the environmental pollutants that have very high carcinogenic and mutagenic activity. Among hundreds of different PAHs, 17 are considered priority pollutants and routinely monitored for regulatory purposes. Extended periods of exposure and expensive clean-up costs are typically associated with the vast majority of processes used for the remediation of areas contaminated with PAHs. The results of this study indicate that bioremediation via vermicomposting could be an effective method for remedying soils contaminated with toxic organic compounds, such as PAHs. This study was conducted over 90?days in the presence of various quantities of organic matter (cattle manure) to recover soils contaminated with PAHs. High-performance liquid chromatography (HPLC) was applied to identify PAHs. An evaluation of the toxicity of the final material and the transformation of the organic matter throughout the process was also conducted. The data presented here suggest a relationship between the molar mass of the PAHs and the ability of the vermicomposting process to promote biodegradation. These results suggest that vermicomposting has great potential to be utilized as a tool for the bioremediation of soils impacted by PAHs.     

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.