Field-contaminated Soils Research Articles

Desorption of polycyclic aromatic hydrocarbons from field-contaminated soil to a two-dimensional hydrophobic surface before and after bioremediation

Desorption of polycyclic aromatic hydrocarbons from field-contaminated soil to a two-dimensional hydrophobic surface before and after bioremediation

Passive Samplers Provide a Better Prediction of PAH Bioaccumulation in Earthworms and Plant Roots than Exhaustive, Mild Solvent, and Cyclodextrin Extractions.

A number of extraction methods have been developed to assess polycyclic aromatic hydrocarbon (PAH) bioavailability in soils. As these methods are rarely tested in a comparative manner, against different test organisms, and using field-contaminated soils, it is unclear which method gives the most accurate measure of the actual soil ecosystem exposure. In this study, PAH bioavailability was assessed in ten field-contaminated soils by using exhaustive acetone/hexane extractions, mild solvent (butanol) extractions, cyclodextrin extractions, and two passive sampling methods; solid phase micro extraction (SPME) and polyoxymethylene solid phase extraction (POM-SPE). Results were compared to actual PAH bioaccumulation in earthworms (Eisenia fetida) and rye grass (Lolium multiflorum) roots. Exhaustive, mild solvent and cyclodextrin extractions consistently overpredicted biotic concentrations by a factor of 10-10 000 and therefore seem inappropriate for predicting PAH bioaccumulation in field contaminated soils. In contrast, passive samplers generally predicted PAH concentrations in earthworms within a factor of 10, although correlations between predicted and measured concentrations were considerably scattered. The same applied to the plant data, where passive samplers also tended to underpredict root concentrations. These results indicate the potential of passive samplers to predict PAH bioaccumulation, yet call for comparative studies between passive samplers and further research on plant bioavailability.

Simultaneous mobilization of macro- and trace elements (MTEs) and polycyclic aromatic hydrocarbon (PAH) compounds from soil with a nonionic surfactant and [ S, S]-ethylenediaminedisuccinic acid (EDDS) in admixture: PAH compounds

A laboratory study was conducted to assess the feasibility of a washing process with nonionic surfactant in combination with EDDS for the simultaneous mobilization of MTEs and PAH compounds from a field-contaminated soil. Unit processes consisting of complexometric extraction and surfactant-assisted mobilization were combined with reagent regeneration and detoxification steps to generate innocuous products. Thirty minutes of ultrasonic mixing of the soil with a combination of 20 mL L −1 surfactant suspension and a sparing quantity (2 mmol) of EDDS mobilized virtually all of the benzo[α] pyrene (B(a)P) and chrysene (Cry) and an appreciable portion of the burdens of Cd, Cr, Mn, Ni, Pb and Zn, lesser amounts of the As and Cu, but only small quantities of Al and Fe. Relative to individual reagents, combinations of surfactant (Brij98), with EDDS increased the recovery of B(a)P but seemingly did not influence Cry extraction efficiencies perceptibly. Nine sequential washes with the same initial dosage of mobilization aids decreased the quantities of both PAHs to levels in the soil that conformed to recommended maxima. What resulted was a soil that had been cleaned and a limited quantity of innocuous wash water.

Simultaneous mobilization of trace elements and polycyclic aromatic hydrocarbon (PAH) compounds from soil with a nonionic surfactant and [S,S]-EDDS in admixture: Metals

This study evaluated the efficacy of soil washing with a nonionic surfactant (Brij98) in combination with a complexing reagent (ethylenediaminedisuccinic acid, [S,S]-EDDS) for the simultaneous mobilization of macro- and trace elements (MTEs) and PAH compounds from a field-contaminated soil. Soil fractionation studies indicated that an appreciable fraction of the Al, Ca, Cu, Fe and Mn was associated with the residual fraction but that much of the other trace elements (As, Cd, Cr, Ni, Pb and Zn) might be susceptible to soil washing. Ultrasonically aided mixing of the field contaminated soil with Brij98 and a sparing quantity (2mmol) of [S,S]-EDDS, simultaneously mobilized virtually all of the benzo[α]pyrene {B(a)P} and chrysene (CRY) and appreciable quantities of the trace elements (Cd, Cr, Mn, Ni, Pb, Zn) burdens. The recovery of both PAHs and trace elements were increased from the soil organic matter (SOM)-rich soil. This report concerns the fate of MTEs during soil washing. Multiple ultra-sonically aided washes (five or nine) with the same dosage of reagents mobilized virtually all of PAHs and decreased the levels of Cd, Cr, Ni, Pb and Zn to comply with recommended maxima. By contrast, the levels of As and Cu remained excessive after the treatments.

Chemical Extractability of Lead in Field-Contaminated Soils: Implications for Estimating Total Lead

Lead (Pb) is frequently present in urban soils at concentrations of concern for human health. Regulations for Pb are based on total soil concentrations as determined by acid digestion, but a less expensive screening test for Pb would be useful in facilitating more thorough soil testing of urban areas if it could be shown to correlate strongly to total soil Pb. In this study, three extractants (0.1 M citrate, MM, and 1 M nitric acid) were evaluated for their ability to estimate the total Pb in contaminated soils. Nitric acid not only extracted a greater fraction of total soil Pb but also produced the strongest correlation to total Pb and is concluded to be the superior extractant for a soil Pb screening test. As the spatial distribution of Pb was observed in selected soils to be highly heterogeneous on the micron scale, thorough soil homogenization prior to testing is recommended.

In situ phytoremediation of PAH-contaminated soil by intercropping alfalfa (Medicago sativa L.) with tall fescue (Festuca arundinacea Schreb.) and associated soil microbial activity

A 7-month field experiment was conducted to investigate the polycyclic aromatic hydrocarbon (PAH) remediation potential of two plant species and changes in counts of soil PAH-degrading bacteria and microbial activity. Alfalfa and tall fescue were grown in monoculture and intercropped for 7 months in contaminated field soil. Soil and plant samples were analyzed for PAHs. Plant biomass, densities of PAH-degradation soil bacteria, soil microbial biomass C and N, enzyme activities, and the physiological profile of the soil microbial community were determined. Average removal percentage of total PAHs in intercropping (30.5%) was significantly higher than in monoculture (19.9%) or unplanted soil (−0.6%). About 7.5% of 3-ring, 12.3% of 4-ring, and 17.2% of 5(+6)-ring PAHs were removed from the soil by alfalfa, with corresponding values of 25.1%, 10.4%, and 30.1% for tall fescue. Intercropping significantly enhanced the remediation efficiency. About 18.9% of 3-ring, 30.9% of 4-ring, and 33.4% of 5(+6)-ring PAHs were removed by the intercropping system. Higher counts of soil culturable PAH-degrading bacteria and elevated microbial biomass and enzyme activities were found after intercropping. Soil from intercropping showed significantly higher (p < 0.05) average well-color development obtained by the BIOLOG Ecoplate assay and Shannon–Weaver index compared with monoculture. Cropping promoted the dissipation of soil PAHs. Tall fescue gave greater removal of soil PAHs than alfalfa, and intercropping was more effective than monoculture. Intercropping of alfalfa and tall fescue may be a promising in situ bioremediation strategy for PAH-contaminated soils.

In situ degradation of phenol and promotion of plant growth in contaminated environments by a single Pseudomonas aeruginosa strain

For bioremediation of contaminated environments, a bacterial strain, SZH16, was isolated and found to reduce phenol concentration in a selective medium. Using the reaction vessel containing the soil mixed with phenol and bacteria, we found that the single strain degraded efficiently the phenol level in soil samples. The strain was identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences, and phosphate solubilization and IAA production were not observed in the strain. Simultaneous examination of the role of strain SZH16 in the plant growth and phenol biodegradation was performed. Results showed that inoculation of the single strain in the phenol-spiked soil resulted in corn growth promotion and in situ phenol degradation and the increase in plant biomass correlated with the decrease in phenol content. Colonization experiments showed that the population of the SZH16 strain remained relatively constant. All these findings indicated that the corn growth promotion might be due to reduction in phytotoxicity, a result of phenol biodegradation by the single strain SZH16. Furthermore, the strain was found to stimulate corn growth and reduce phenol concentration simultaneously in phenol-containing water, and even historically contaminated field soils. It is attractive for environment remediation and agronomic applications.

In vitro estimates of bioaccessible nickel in field-contaminated soils, and comparison with in vivo measurement of bioavailability and identification of mineralogy

This study determined nickel (Ni) bioaccessibility in weathered smelter-contaminated soils, separately for particle-sized fractions using two in vitro methods: simulated gastrointestinal digestion (PBET) and PBET followed by absorption by Caco-2 cells. Relative bioavailability of Ni in soils was determined in vivo using rats, validating in vitro estimates; a mineralogical basis of variation in bioavailability/bioaccessibility among soils was explored. In vitro assays identified the same difference in bioaccessibility for Ni among particle size fractions. PBET estimates were more precise, thus likely to be more useful in discerning differences among soils. In vivo bioavailability for Ni was below limit of detection for the small soil particles, and 31% and 56% for the larger particles. The relative bioavailability calculated from this work suggests that risk from ingesting Ni-contaminated soils could be overestimated by between 2- and 50-fold if the estimates of exposure are not adjusted for the lower bioavailability of weathered Ni originating from smelter emissions. The overestimation that would occur by using total Ni is greatest for the particle size that is most likely to adhere to the hands of children, demonstrating the importance of particle-size separation of soils for bioavailability determination and risk assessment.

A Contaminant Trap as a Tool for Isolating and Measuring the Desorption Resistant Fraction of Soil Pollutants

Bioremediation of contaminated soils often leaves a desorption-resistant pollutant fraction behind in the soil, which in the present study was isolated with a combination of diffusive carrier and infinite diffusive sink. Such a diffusive sink was made by casting a composite of silicone and activated carbon into the bottom of a large glass. Field-contaminated soil samples were then suspended in a cyclodextrin solution and incubated in such glasses for the continuous trapping of PAH molecules during their release from the soil matrix. The PAH concentrations remaining in the soil were determined by exhaustive extraction and compared with a biodegradation experiment. The concentration decline in the first soil was faster in the contaminant trap than in the biodegradation experiment, but the halting of the biodegradation process before reaching the legal threshold level was well indicated by the contaminant trap. The PAH concentrations in the second soil hardly decreased in the traps at all, in good agreement with the biodegradation experiment. The PAHs in this soil appeared to be "stuck" by strong sorption. The contaminant trap proved to be a practical approach to the isolation and quantification of the desorption-resistant PAH fraction.

Scaling up a treatment to simultaneously remove persistent organic pollutants and heavy metals from contaminated soils

Soil washing is a treatment process that can be used to remediate both organic and inorganic pollutants from contaminated soils, sludges, and sediments. A soil washing procedure was evaluated utilizing about 100 g samples of soil that had been field-contaminated with arsenic, chromium, copper, pentachlorophenol (PCP), polychlorinated dibenzo- p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The highest level of mobilization/detoxification was achieved in three soil washes with a mixture of 0.1M [ S,S]-ethyelnediaminedisuccinate ([ S,S]-EDDS) and 2% Brij 98 at pH 9 with 20 min of ultrasonication treatment at room temperature. This combination mobilized 70% of arsenic, 75% of chromium, 80% of copper, 90% of PCP, and 79% of PCDDs and PCDFs, so that the decontaminated soil met the maximum acceptable concentrations of the generic C-level criteria regulated by the Ministère du Développement Durable, de l’Environnement et des Parcs for the Province of Québec, Canada. The organic pollutants were back-extracted from the aqueous suspension with hexane. Heavy metals were virtually completely precipitated from the aqueous washing suspension with Mg 0 particles at room temperature. The PCP was detoxified by catalytic hydrodechlorination with a stream of 5% (v/v) H 2-supercritical CO 2 that transported the organosoluble fraction through a reaction chamber containing 2% Pd/γ-Al 2O 3. In toto, this soil washing procedure demonstrates that persistent organic pollutants and selected heavy metals can be co-extracted efficiently from a field-contaminated soil with three successive washes with the same soil washing solution containing [ S,S]-EDDS and a non-ionic surfactant (Brij 98) in admixture. An industrial-scale ex situ soil washing procedure with a combination of a non-ionic surfactant and a complexing reagent seems to be a plausible remediation technique for this former wooden utility pole storage facility.

Significance of metal exchange in EDDS-flushing column experiments

Chelating agents have been widely studied for extracting heavy metals from contaminated soils, and the effectiveness of EDDS ([S,S]-ethylene-diamine-disuccinic acid) has aroused extensive attention because of its biodegradability in the natural environment. However, in the course of EDDS-flushing, metal exchange of newly extracted metal–EDDS complexes with other sorbed metals and mineral cations may result in metal re-adsorption on the soil surfaces. Therefore, this study investigated the relative significance of metal exchange under different travel distances of chelant complexes, characteristics of soil contamination, and solution pH in the column experiments. As a result of metal exchange, the elution of Zn and Pb was retarded and the cumulative extraction was lower than those of Ni and Cu, especially over a longer travel distance. Compared with the field-contaminated soils, the effects of metal exchange were even more substantial in the artificially contaminated soil because of a greater amount of extractable metals and a larger proportion of weakly bound fractions. By contrast, metal exchange was insignificant at pH 8, probably due to less adsorption of metal–EDDS complexes. These findings highlight the conditions under which metal exchange of metal–EDDS complexes and the resulting impacts are more significant during EDDS-flushing.

Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments

We show that Pseudomonas fluorescens strain P13, a plant growth-promoting bacterium, enhanced the growth of corn in uncontaminated soil but not in contaminated soil, perhaps because of its inability to reduce phytotoxicity. Another bacterial strain, Pseudomonas aeruginosa strain SZH16, showed in situ phenol-degrading activity and contained a plasmid loaded with a gene encoding for catechol 2, 3-dioxygenase, an important enzyme in the degradation pathway of aromatic compounds. We implanted this biodegradation ability into strain P13, using horizontal gene transfer techniques using strain SZH16 as the donor and P13 as the recipient, to generate a phenol-degrading transconjugant which obtained the effective plasmid from strain SZH16. Introduction of the transconjugant P13 strain into an artificially phenol-spiked soil promoted the growth of corn and in situ phenol degradation, and the increase in plant biomass correlated with the decrease in soil phenol content. Furthermore, the transconjugant P13 strain was also found to stimulate corn growth and reduce phenol concentration in water containing phenol and in historically contaminated field soils, indicating that the transconjugant strain could promote plant growth in both contaminated and uncontaminated environments. The transconjugant P13 strain was more efficient than either strain P13 or SZH16, and shows how plant growth-promoting bacteria which show no, or only limited, ability to degrade organic pollutants may be modified. This technique is attractive for many environmental remediation and agronomic applications.

Effects of soil oven-drying on concentrations and speciation of trace metals and dissolved organic matter in soil solution extracts of sandy soils

Weak salt extracts can be used to assess the availability of trace metals for leaching and uptake by soil organisms and plants in soil. Before extraction, the International Organization for Standardization recommends to dry soils in an oven at a temperature of 40 °C. Effects of soil oven-drying on different fractions of dissolved organic matter (DOM) and the effect thereof on total concentrations and speciation of trace metals in weak salt extracts have, however, not been quantified yet. In this study, free metal concentrations and DOM speciation were determined in 2 mM Ca(NO 3) 2 extracts obtained from twelve field-contaminated soils in their field-moist state and after drying at 40 °C. Free metal concentrations were measured with the Donnan Membrane Technique. DOM was fractioned into humic acid (HA), fulvic acid (FA), and hydrophilic (Hy) compounds. Soil oven-drying led to significant increases in the concentrations of total DOM and total dissolved Cu and Ni. For the measurement of total dissolved Cu and Ni concentrations, it is, therefore, better to use field-moist soils. The release of Hy compounds was mainly responsible for the increase in DOM, which accounted for 64 to 77% of the increase in total dissolved organic carbon. Soil oven-drying left the free Cd 2+, Cr 3+, Cu 2+, Ni 2+, and Zn 2+ concentrations unchanged. Both field-moist and oven-dried soils can, therefore, be used for the measurement of free metal concentrations. Free Cd 2+, Cu 2+, Ni 2+, and Zn 2+ concentrations were predicted very well for both field-moist and oven-dried soils using ORCHESTRA, which includes the NICA-Donnan model. However, poor predictions were obtained for Cr 3+, for which better NICA parameters need to be derived.

Role of black carbon in the distribution of polychlorinated dibenzo- p-dioxins/dibenzofurans in aged field-contaminated soils

Floodplain soils containing elevated levels of polychlorinated dibenzo- p-dioxins/dibenzofurans (PCDD/Fs) were collected from several locations along the Tittabawassee River (Michigan, USA). The PCDD/F profiles of these soils exhibited distinct congener patterns consistent with byproducts from either chloralkali manufacturing or chlorophenols productions. Black carbon (BC) particles were isolated for the first time from floodplain soil impacted by PCDD/Fs. Petrographic analysis showed that BC particles, including coal, oxidized coal, metallurgical coke, depositional carbon, coal tar/pitch, cenosphere, and charcoal, comprised approximately 30% by volume of the organic fraction with size range of 250 μm–2000 μm from a typical floodplain soil. The BC particles with anthropogenic origin such as pitch and coke associated with the chloralkali production process served as both the source and subsequent transporter for the highly hydrophobic PCDD/Fs. These anthropogenic BC particles were enriched with high levels of PCDFs, containing approximately 1000-fold the concentration found in the bulk soil. The strong association of PCDD/Fs with anthropogenic BC directly impacts the physicochemical and biological availability thus the risk associated with these hydrophobic organochlorines in soils and sediments.

Gene Expression Analysis Reveals a Gene Set Discriminatory to Different Metals in Soil

Environmental pollution is a worldwide problem, and metals are the largest group of contaminants in soil. Microarray toxicogenomic studies with ecologically relevant organisms, such as springtails, supplement traditional ecotoxicological research but are presently rather descriptive. Classifier analysis, a more analytical application of the microarray technique, is able to predict biological classes of unknown samples. We used the uncorrelated shrunken centroid method to classify gene expression profiles of the springtail Folsomia candida exposed to soil spiked with six different metals (barium, cadmium, cobalt, chromium, lead, and zinc). We identified a gene set (classifier) of 188 genes that can discriminate between six different metals present in soil, which allowed us to predict the correct classes for samples of an independent test set with an accuracy of 83% (error rate = 0.17). This study shows further that in order to apply classifier analysis to actual contaminated field soil samples, more insight and information is needed on the transcriptional responses of soil organisms to different soil types (properties) and mixtures of contaminants.

Sequestration of heavy metals in soils from two polluted industrial sites: implications for remediation

This study was conducted to determine the distribution of heavy metals in two different contaminated field soils, and to assess its influence on remedial performance. The two field soils were obtained from two different polluted industrial sites in the Metropolitan Chicago area and were characterized for physical and chemical properties. The soils were referred to as silty sand and silty clay, based on their particle-size distribution. A five-step sequential liquid–solid extraction procedure was used to speciate the heavy metals into: (1) easily exchangeable; (2) carbonate bound; (3) Fe/Mn oxide bound; (4) organic bound; and (5) residual fractions. These results showed that all the metals except mercury were predominantly distributed as the Fe/Mn-bound form in silty sand. In contrast, they were distributed as the residual form in silty clay. Results indicated that the metals in both soils were in the forms that are not easily amenable to soil washing using water. Therefore, batch extraction tests with varying concentrations of ethylenediaminetetraacetic acid (EDTA) (0.05, 0.1 and 0.2 M) and acetic acid (0.5, 0.1 and 2 M) were conducted to identify an optimum soil-washing-based remediation system for both silty sand and silty clay. Ethylenediaminetetraacetic acid was found to be effective for the remediation of silty sand, while acetic acid was found to be suitable for the removal of metals that existed in residual form in silty clay. Based on these results, total removal efficiency was found to follow the order: sand + EDTA > clay + acetic acid > sand + acetic acid > clay + EDTA. This study showed that the speciation and remediation of heavy metals in soils depend on the sitespecific soil composition, and this should be carefully considered in the selection of an efficient remedial method.

Extraction of Lead and Cadmium from Soils by Cysteine and Glutathione

Metal-thiol complexes can enhance metal uptake by plant roots and microorganisms, therefore it is important to determine whether thiols effectively solubilize these metals from the soil matrix. Extractions were conducted by combining 1 g contaminated soil and 10 mL of 10 mmol L(-1) thiol solution and shaking for 1 h. Both cysteine and glutathione extracted between 5 and 45% of Pb and Cd from laboratory and field-contaminated soils at pHs>6 after 1 h. In the presence of oxygen, the half-life of reduced cysteine was on the order of 0.1 h and dissolved metal concentrations decreased to nearly zero over 24 h. In extractions with glutathione, both the metals and thiol were more stable, with a half-life for glutathione of 23 h, and stable dissolved metal concentrations over 24 h in the presence of oxygen. In cysteine extractions, Pb was primarily removed from the Fe/Mn oxide fraction of the amended soil and dissolved Fe concentrations followed dissolved Pb concentrations, whereas this pool of Pb was unavailable to thiol extraction in aged field contaminated soils. Iron is hypothesized to play a role in the oxidation of cysteine via both reductive dissolution of iron oxides and in the photolytic oxidation of the Fe-thiol complex in the aqueous phase. While overall cysteine was more effective than glutathione at extracting Pb from soils, its propensity to oxidize may limit its ability to increase the bioavailability of this metal to plants or microorganisms in oxic environments.

Ethyl lactate enhances ethylenediaminedisuccinic acid solution removal of copper from contaminated soils

A new approach using aqueous ethyl lactate-modified [ S, S]-ethylenediaminedisuccinic acid (EDDS) washing solutions for the removal of Cu from field-contaminated soils was examined in the laboratory. At pH 7 the maximum extraction efficiencies of Cu were about 41% by EDDS solution (EDDS:Cu molar ratio = 8), and <2% by ethyl lactate solution. However, greater extraction efficiencies of up to 50% were achieved by amending the EDDS solution with ethyl lactate (EDDS:Cu molar ratio = 2, ethyl lactate:Cu molar ratio = 25). The pH of the extraction solution did not have a significant effect on the extraction of Cu. The washing agent could also be regenerated quite effectively for economy of use. Sequential extraction results showed that the two most bioavailable Cu fractions, i.e., the acid extractable and reducible fractions, were significantly reduced. The mechanism responsible for the enhanced extraction efficiency in the presence of ethyl lactate is the increased the stability constant of Cu–EDDS complexes in the presence of ethyl lactate, which enhanced desorption of Cu from soil.

A COMPARISON OF TESTS FOR EXTRACTABLE COPPER AND ZINC IN METAL-SPIKED AND FIELD-CONTAMINATED SOIL

Common soil test methods were compared with 0.01 M CaCl2 extraction to determine their relative abilities to extract and estimate phytoavailability of Cu and Zn. In aged metal-spiked soils, all soil tests evaluated (Mehlich 3, diethylenetriaminepentaacetic acid [DTPA], Morgan, modified Morgan, CaCl2) showed a linear relationship of extractable to total Cu and Zn for both soil types studied. The fraction of total Cu and Zn extracted by aggressive tests (Mehlich 3, DTPA) was much higher than the fraction extracted by CaCl2, with the Morgan tests being intermediate. Although all extraction methods revealed Cu and Zn to be more available in the coarse-textured soil than the fine-textured soil, this texture effect was greatest for the least aggressive test (CaCl2) and least for Mehlich 3. The texture effect on soil test Cu and Zn was also reflected in bioavailability, with greater plant tissue concentrations of both metals from the coarse-textured soil in a soybean assay. Although all soil test methods provided similarly strong correlations to plant tissue concentrations of Zn for soybeans grown in the two soils, the less aggressive soil tests seemed more reliable as predictors of Cu uptake. The efficiency of Cu and Zn extraction from field-contaminated soils was much lower than that from laboratory-spiked aged soils. For Mehlich 3 and DTPA tests, Cu and Zn in field-contaminated soils were less extractable by a factor of about 2 compared with the spiked soils. For less aggressive tests, the difference in extractability was even greater. This study suggests that soil extraction methods removing smaller pools of Cu and Zn are more responsive to soil properties affecting chemical lability of the metals. Therefore, chemically nonaggressive neutral salts may be the most appropriate extractants where phytotoxicity is the concern in metal-contaminated soils.

Toxicity of Trace Metals in Soil as Affected by Soil Type and Aging After Contamination: Using Calibrated Bioavailability Models to Set Ecological Soil Standards

Total concentrations of metals in soil are poor predictors of toxicity. In the last decade, considerable effort has been made to demonstrate how metal toxicity is affected by the abiotic properties of soil. Here this information is collated and shows how these data have been used in the European Union for defining predicted-no-effect concentrations (PNECs) of Cd, Cu, Co, Ni, Pb, and Zn in soil. Bioavailability models have been calibrated using data from more than 500 new chronic toxicity tests in soils amended with soluble metal salts, in experimentally aged soils, and in field-contaminated soils. In general, soil pH was a good predictor of metal solubility but a poor predictor of metal toxicity across soils. Toxicity thresholds based on the free metal ion activity were generally more variable than those expressed on total soil metal, which can be explained, but not predicted, using the concept of the biotic ligand model. The toxicity thresholds based on total soil metal concentrations rise almost proportionally to the effective cation exchange capacity of soil. Total soil metal concentrations yielding 10% inhibition in freshly amended soils were up to 100-fold smaller (median 3.4-fold, n = 110 comparative tests) than those in corresponding aged soils or field-contaminated soils. The change in isotopically exchangeable metal in soil proved to be a conservative estimate of the change in toxicity upon aging. The PNEC values for specific soil types were calculated using this information. The corrections for aging and for modifying effects of soil properties in metal-salt-amended soils are shown to be the main factors by which PNEC values rise above the natural background range.