Chemical assays of availability to earthworms of polycyclic aromatic hydrocarbons in soil

The bioavailable concentration of an organic pollutant is less than the concentration determined by vigorous extraction of soil. Because bioavailability varies with the particular compound, soil, and aging time, an assay for bioavailability is needed. Three methods were tested: extraction with a 25% aqueous solution of tetrahydrofuran (THF), 95% ethanol, and C18 membranes. Evaluations were conducted with a mixture of four polycyclic aromatic hydrocarbons (PAHs) added to five dissimilar soils and with pyrene as sole PAH added to six soils, and the availability of aged and freshly added compounds was determined. Assimilation by earthworms (Eisenia fetida) was used to assess bioavailability. For extraction with THF and earthworm uptake, the correlation coefficients of determination (R2) for anthracene, chrysene, pyrene, and benzo(a)pyrene added as a mixture exceeded 0.85. The R2 values for assays with the C18 membrane were 0.77 or higher. The values for pyrene added alone were 0.710 and 0.823, respectively. R2 values for assays with ethanol often exceeded 0.87, but lower values were sometimes obtained. We suggest that such solvent or solid-phase extractions may be useful in assessing PAH bioavailability.     

Bioavailability assessment and environmental fate of polycyclic aromatic hydrocarbons in biostimulated creosote-contaminated soil

When hydrocarbon-contaminated soil is subjected to bioremediation technology, hydrocarbon depletion is typically marked by an initially rapid reduction rate. This rate decreases over time and frequently a residual concentration remains in the soil. This kinetic has been attributed primarily to the enrichment of more recalcitrant fractions, as well as to the lack of resting hydrocarbon bioavailability. Thus, at the end of the bioremediation process, a part of the residual hydrocarbon soil concentration represents the non-bioavailable fraction, which is difficult to degrade by microbial populations and which poses a minor hazard. Therefore, determination of the bioavailable fraction in a bioremediation project represents both an estimation of the maximum level of achievable biodegradation, as well as an additional indication of the environmental health hazard. In the present study, aged creosote-contaminated soil was subjected to biostimulation processes, and the bioavailable fraction for several target polycyclic aromatic hydrocarbons (PAHs) was calculated using a mild extraction with cyclodextrines. The amount of PAH extracted corresponded to the desorbing fraction and can be regarded as the bioavailable fraction. The non-desorbing fraction data obtained from this procedure were compared to the remaining PAH concentrations following bioremediation treatment of soil microcosms. These results permitted the establishment of a theoretical biodegradation limit based on the desorbing fraction. In addition, neither accumulation of intermediate metabolites, nor the formation of bound-residues or reduced acute toxicity was observed.     

Effects of 2,4-dichlorophenol,pentachlorophenol and vegetation on microbial characteristics in a heavy metal polluted soil

The aim of this study was to evaluate the soil microbial characteristics in historically heavy-metal polluted soil, which was also affected by organic co-contaminants, 2,4-dichlorophenol or pentachlorophenol, which often occur due to the conventional use of pesticides. It was observed that the normalized microbial biomass (microbial biomass per unit soil organic C) of the contaminated soil was very low, less than 1% in both non-planted and ryegrass planted soil, and showed a decreasing trend with the treatment of organic co-contaminants. The microbial biomass and substrate-induced respiration (SIR) in the ryegrass planted soil were much larger, as compared with the non-planted soil with or without organic pollutants. The different resistant bacterial community and its physiological diversity in the rhizosphere further suggested that the effect of vegetation on microbial activity was not just a general increase in the mass or activity of pre-existing microorganisms, but rather acted selectively on microbial growth so that the relative abundance of different microbial groups in soil was changed. In sum, high concentrations of organic co-contaminants, especially pentachlorophenol (PCP), could strengthen the deterioration of microbial ecology. The adverse effect of heavy metal-organic pollutants on the soil microbial biomass and activity might be the reason for the slow degradation of PCP that has high chlorinated and high toxicity. Vegetation might be the efficient way to assist in improving and restoring the utilization of agricultural ecosystems. The beneficial microbial effect of vegetation could cause the rapid dissipation of 2,4-dichlorophenol (2,4-DCP) that has less chlorinated and less toxicity in the planted soils.     

Effects of 2,4-dichlorophenol, pentachlorophenol and vegetation on microbial characteristics in a heavy metal polluted soil

The aim of this study was to evaluate the soil microbial characteristics in historically heavy-metal polluted soil, which was also affected by organic co-contaminants, 2,4-dichlorophenol or pentachlorophenol, which often occur due to the conventional use of pesticides. It was observed that the normalized microbial biomass (microbial biomass per unit soil organic C) of the contaminated soil was very low, less than 1% in both non-planted and ryegrass planted soil, and showed a decreasing trend with the treatment of organic co-contaminants. The microbial biomass and substrate-induced respiration (SIR) in the ryegrass planted soil were much larger, as compared with the non-planted soil with or without organic pollutants. The different resistant bacterial community and its physiological diversity in the rhizosphere further suggested that the effect of vegetation on microbial activity was not just a general increase in the mass or activity of pre-existing microorganisms, but rather acted selectively on microbial growth so that the relative abundance of different microbial groups in soil was changed. In sum, high concentrations of organic co-contaminants, especially pentachlorophenol (PCP), could strengthen the deterioration of microbial ecology. The adverse effect of heavy metal-organic pollutants on the soil microbial biomass and activity might be the reason for the slow degradation of PCP that has high chlorinated and high toxicity. Vegetation might be the efficient way to assist in improving and restoring the utilization of agricultural ecosystems. The beneficial microbial effect of vegetation could cause the rapid dissipation of 2,4-dichlorophenol (2,4-DCP) that has less chlorinated and less toxicity in the planted soils.     

The lack of microbial degradation of polycyclic aromatic hydrocarbons from coal-rich soils

Analytical techniques used to assess the environmental risk of contamination from polycyclic aromatic hydrocarbons (PAHs) typically consider only abiotic sample parameters. Supercritical fluid extraction and sorption enthalpy experiments previously suggested slow desorption rates for PAH compounds in two coal-contaminated floodplain soils. In this study, the actual PAH availability for aerobic soil microorganisms was tested in two series of soil-slurry experiments. The experimental conditions supported microbial degradation of phenanthrene if it was weakly sorbed onto silica gel. Native coals and coal-derived particles in two soils effectively acted as very strong sorbents and prevented microbial PAH degradation. The long history of PAH exposure and degree of coal contamination apparently had no influence on the capability of the microbial soil community to overcome constraints of PAH availability. Within the context of the experimental conditions and the compounds chosen, our results confirm that coal-bound PAHs are not bioavailable and hence of low environmental concern.     

Biodegradability of aged pyrene and phenanthrene in a natural soil

A study was conducted to evaluate the biodegradability of pyrene (PYR) and phenanthrene (PHE) aged in a natural soil. Both the single and binary systems were either biostimulated via a nutrient amendment or bioaugmented via an inoculation of the enriched bacteria and nutrients. Aging resulted in higher concentration of both compounds and smaller bacterial activity in the solution-phase. Surprisingly, the total biodegraded extent was greater in the aged soil system than in the freshly spiked system. As anticipated, biostimulation was not appropriate to attain an effective biodegradation in this study, and bioaugmentation achieved a substantial increase the total biodegradation extent. The above findings were attributed to indigenous Pseudomonas aeruginosa entering a stationary-phase during the 200-day aging and producing rhamnolipid biosurfactants. In addition, a different sampling technique (i.e., after vigorous hand-shaking) revealed a 15 times higher microbial population than the normal sampling from the stagnant solution. Therefore, PAH bioavailability in the aged soils can be underestimated when the microbial activity is determined only from the stagnant solution. Furthermore, cometabolism enhanced PYR degradation when PHE was present as a primary substrate.     

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.     

The influence of a NAPL on the loss and biodegradation of 14C-phenanthrene residues in two dissimilar soils

This study was carried out to assess the influence of diesel, applied over a log concentration range, on the loss and extractability of phenanthrene (measured as putative 14C-phenanthrene residues) in two different soils. The influence of diesel on the ability of a cyclodextrin based extraction method to predict the microbial bioavailability of 14C-residues was also assessed. An increase in loss of 14C-residues with increasing diesel concentration from 0 to 2000 mg kg-1 was generally observed with time in both soils. It is suggested that this trend is attributable to competitive sorption for soil sorption sites and to a lesser extent to displacement of 14C-residues from soil sorption sites by diesel resulting in greater compound availability and therefore greater loss by degradation via the actions of indigenous microorganisms. However, in the 20000 mg kg-1 diesel treatments of both soils, results indicated a delayed loss. It is suggested that this retarded loss was due to the formation of a discrete NAPL-phase into which 14C-phenanthrene residues partitioned, thereby decreasing their availability and as a consequence their degradation. Furthermore, it is suggested that nutrient limitation may have slowed down degradation rates as diesel concentrations increased. Comparison between cyclodextrin-extractability and microbial mineralisation supported the use of cyclodextrin to assess microbial bioavailability of 14C-residues after 50 d or more ageing up to diesel concentrations of 2000 mg kg-1. However, results suggested that at high diesel concentrations (specifically 20000 mg kg-1) co-extraction of 14C-phenanthrene residues may have occurred as a result of the combined solvation powers of both the cyclodextrin and the diesel. Furthermore, mineralisation of 14C-phenanthrene residues may have been affected by extreme nutrient limitation in this treatment.     

Assessment of contaminant lability during phytoremediation of polycyclic aromatic hydrocarbon impacted soil

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant compounds, some of which are known carcinogens, often found in high residual soil concentrations at industrial sites. Recent research has confirmed that phytoremediation holds promise as a low-cost treatment method for PAH contaminated soil. In this study, the lability of soil bound PAHs in the rhizosphere was estimated using solid phase extraction resin. An extraction time of 14 days was determined to be appropriate for this study. Resin-extractable PAHs, which are assumed to be more bioavailable, decreased during plant treatments. Significant reductions in the labile concentrations of several PAH compounds occurred over 12 months of plant growth. The differences in concentration between the unplanted and the planted soil indicate that the presence of plant roots, in addition to the passage of time, contributes to reduction in the bioavailability of target PAHs.     

Fungal bioremediation of the creosote-contaminated soil: influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil microbial community composition in the laboratory-scale study

The aim of this study was to determine the efficacy of selected basidiomycetes in the removing of polycyclic aromatic hydrocarbons (PAH) from the creosote-contaminated soil. Fungi Pleurotus ostreatus and Irpex lacteus were supplemented with creosote-contaminated (50-200 mg kg(-1) PAH) soil originating from a wood-preserving plant and incubated at 15 °C for 120 d. Either fungus degraded PAH with 4-6 aromatic rings more efficiently than the microbial community present initially in the soil. PAH removal was higher in P. ostreatus treatments (55-67%) than in I. lacteus treatments (27-36%) in general. P. ostreatus (respectively, I. lacteus) removed 86-96% (47-59%) of 2-rings PAH, 63-72% (33-45%) of 3-rings PAH, 32-49% (9-14%) of 4-rings PAH and 31-38% (11-13%) of 5-6-rings PAH. MIS (Microbial Identification System) Sherlock analysis of the bacterial community determined the presence of dominant Gram-negative bacteria (G-) Pseudomonas in the inoculated soil before the application of fungi. Complex soil microbial community was characterized by phospholipid fatty acids analysis followed by GC-MS/MS. Either fungus induced the decrease of bacterial biomass (G- bacteria in particular), but the soil microbial community was influenced by P. ostreatus in a different way than by I. lacteus. The bacterial community was stressed more by the presence of I. lacteus than P. ostreatus (as proved by the ratio of the fungal/bacterial markers and by the ratio of trans/cis mono-unsaturated fatty acids). Moreover, P. ostreatus stimulated the growth of Gram-positive bacteria (G+), especially actinobacteria and these results indicate the potential of the positive synergistic interaction of this fungus and actinobacteria in creosote biodegradation.     

Interactions between a polycyclic aromatic hydrocarbon mixture and the microbial communities in a natural freshwater sediment

The toxicity of a polycyclic aromatic hydrocarbon (PAH) mixture was assessed on the indigenous microbial communities of a natural freshwater sediment. The fate and effects of the PAH mixture (phenanthrene, fluoranthene and benzo(k)fluoranthene) were studied over 28 days. Bacterial communities were described by bacterial counts (total bacteria and viable bacteria), and by some hydrolytic enzyme activities (beta-glucosidase and leucine-aminopeptidase), PAH concentrations were measured in the overlying waters and in the sediments. No effect of PAH was detected at 30 mg/kg for all bacterial parameters. At 300 mg/kg, the quantity of total bacteria and the proportion of viable bacteria markedly decreased, compared to the control (0 mg PAH/kg). At 300 mg/kg, an increase of the beta-glucosidase activity and a decrease of the leucine-aminopeptidase activity were observed. For all treatments, the benzo(k)fluoranthene concentration in the sediment was stable over 28 days whereas, in the same time, only 3-6% of the initial concentrations of phenanthrene and fluoranthene remained. This study shows that (1) PAH induce perturbations of sediment microbial communities in terms of density and metabolism (but not always as an inhibition), (2) indigenous bacteria of sediments might be used for toxicity assessment of specific organic pollutants, (3) native microorganisms of sediment seem to have a high capacity for PAH degradation, depending on the physico-chemical properties and the bioavailability of the substance encountered.     

Bacterial community structure in soils contaminated by polycyclic aromatic hydrocarbons

Bacterial community structure was examined in polycyclic aromatic hydrocarbon (PAH) contaminated soil taken from a timber treatment facility in southern Ireland. Profiles of soil bacterial communities were generated using a molecular fingerprinting technique, terminal restriction fragment length polymorphism (TRFLP), and results were interpreted using sophisticated multivariate statistical analysis. Findings suggested that there was a correlation between PAH structure and bacterial community composition. Initial characterisation of soil from the timber treatment facility indicated that PAH contamination was unevenly distributed across the site. Bacterial community composition was correlated with the type of PAH present, with microbial community structure associated with soil contaminated with two-ringed PAHs only being distinctly different to communities in soils contaminated with multi-component PAH mixtures. Typically the number of bacterial ribotypes detected in samples did not appear to be adversely affected by the level of contamination.     

Fate of polycyclic aromatic hydrocarbons during composting of lagooning sewage sludge

The fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted by the USEPA agency, has been investigated during composting of lagooning sludge. Composting shows efficient decrease of the content and the bioavailability of each PAH. Biodegradation and sorption are suggested as the main mechanisms contributing to this decrease. During the stabilization phase of composting, extensive microbial degradation of PAHs, mainly those with a low number of aromatic rings, was achieved following development of intense thermophilic communities. However, partial sorption of PAH to non-accessible sites temporarily limits the mobility mainly of PAHs with a high number of aromatic rings plus acenaphthene and acenaphthylene, and allows them to escape microbial attack. During the maturation phase, the development of a mesophilic population could play an important role in the degradation of the remaining PAH. During this phase of composting, PAH sequestration and binding of their oxidative metabolites within new-formed humic substances might also explain PAH decrease at the end of composting. The tendency of change of content or bioavailability of various PAH compounds during composting is found to be strongly related to the number of their aromatic rings, their molecular weight and structure.     

Principles of microbial PAH-degradation in soil

Interest in the biodegradation mechanisms and environmental fate of polycyclic aromatic hydrocarbons (PAHs) is motivated by their ubiquitous distribution, their low bioavailability and high persistence in soil, and their potentially deleterious effect on human health. Due to high hydrophobicity and solid-water distribution ratios, PAHs tend to interact with non-aqueous phases and soil organic matter and, as a consequence, become potentially unavailable for microbial degradation since bacteria are known to degrade chemicals only when they are dissolved in water. As the aqueous solubility of PAHs decreases almost logarithmically with increasing molecular mass, high-molecular weight PAHs ranging in size from five to seven rings are of special environmental concern. Whereas several reviews have focussed on metabolic and ecological aspects of PAH degradation, this review discusses the microbial PAH-degradation with special emphasis on both biological and physico-chemical factors influencing the biodegradation of poorly available PAHs.     

Oxidation of a PAH polluted soil using modified Fenton reaction in unsaturated condition affects biological and physico-chemical properties

A batch experiment was conducted to assess the impact of chemical oxidation using modified Fenton reaction on PAH content and on physico-chemical and biological parameters of an industrial PAH contaminated soil in unsaturated condition. Two levels of oxidant (H₂O₂, 6 and 65 g kg⁻¹) and FeSO₄ were applied. Agronomic parameters, bacterial and fungal density, microbial activity, seed germination and ryegrass growth were assessed. Partial removal of PAHs (14% and 22%) was obtained with the addition of oxidant. The impact of chemical oxidation on PAH removal and soil physico-chemical and biological parameters differed depending on the level of reagent. The treatment with the highest concentration of oxidant decreased soil pH, cation exchange capacity and extractable phosphorus content. Bacterial, fungal, and PAH degrading bacteria densities were also lower in oxidized soil. However a rebound of microbial populations and an increased microbial activity in oxidized soil were measured after 5 weeks of incubation. Plant growth on soil treated by the highest level of oxidant was negatively affected.     

Effect of metals on the adsorption and extractability of 14C-phenanthrene in soils

The fate of polycyclic aromatic hydrocarbons (PAHs) in contaminated soils may be affected by several environmental factors including the presence of co-contaminants. This study was conducted in order to assess the effect of metals on (i) the adsorption of 14C-phenanthrene in soils and (ii) its extractability and ability to form non-extractable residues. The first objective was accomplished using batch adsorption experiments with an uncontaminated agricultural soil spiked with the metals Cd, Cu, Pb, and Zn. Adsorption of phenanthrene was significantly higher after the addition of the metals (Kf = 21.48 vs. 8.55) and the desorption less readily reversible when compared to the unspiked soil. The extractability of phenanthrene was assessed with incubation (4 months, laboratory conditions) and microlysimeter experiments (6 months, natural climatic conditions) on three soils spiked with metals. All the soils were labelled with 14C-phenanthrene. The amount of extractable phenanthrene residues was significantly higher when the metals had been added to the soils. Nevertheless, the quantity of non-extractable residues was non-significantly different between the spiked and unspiked soils. The mechanism leading to increased adsorption and extractability of phenanthrene in the presence of metals is still unknown. In perspective, it would be interesting to assess the bioavailability of PAHs in the presence of metals in further experiments.     

Accumulation of weathered polycyclic aromatic hydrocarbons (PAHs) by plant and earthworm species

Experiments were conducted to assess the bioavailability of polyclycic aromatic hydrocarbons (PAHs) in soil from a Manufactured Gas Plant site. Three plant species were cultivated for four consecutive growing cycles (28 days each) in soil contaminated with 36.3 microg/g total PAH. During the first growth period, Cucurbita pepo ssp. pepo (zucchini) tissues contained significantly greater quantities of PAHs than did Cucumis sativus (cucumber) and Cucurbita pepo ssp. ovifera (squash). During the first growth cycle, zucchini plants accumulated up to 5.47 times more total PAH than did the other plants, including up to three orders of magnitude greater levels of the six ring PAHs. Over growth cycles 2-4, PAH accumulation by zucchini decreased by 85%, whereas the uptake of the contaminants by cucumber and squash remained relatively constant. Over all four growth cycles, the removal of PAHs by zucchini was still twice that of the other species. Two earthworm species accumulated significantly different amounts of PAH from the soil; Eisenia foetida and Lumbricus terrestris contained 0.204 and 0.084 microg/g total PAH, respectively, but neither species accumulated measurable quantities 5 or 6 ring PAHs. Lastly, in abiotic desorption experiments with an aqueous phase of synthetically prepared organic acid solutions, the release of 3 and 4 ring PAHs from soil was unaffected by the treatments but the desorption of 5-6 ring constituents was increased by up to two orders of magnitude. The data show that not only is the accumulation of weathered PAHs species-specific but also that the bioavailability of individual PAH constituents is highly variable.     

Influence of mature compost amendment on total and bioavailable polycyclic aromatic hydrocarbons in contaminated soils

A laboratory microcosm study was carried out to assess the influence of compost amendment on the degradation and bioavailability of PAHs in contaminated soils. Three soils, contaminated with diesel, coal ash and coal tar, respectively, were amended with two composts made from contrasting feedstock (green waste and predominantly meat waste) at two different rates (250 and 750 t ha^?1) and incubated for 8 months. During this period the treatments were sampled for PAH analysis after 0, 3, 6 and 8 months. Total and bioavailable fractions were obtained by sequential ultrasonic solvent extraction and hydroxypropyl-β-cyclodextrin extraction, respectively, and PAHs were identified and quantified by GC–MS. Bioavailability decrease due to sorption was only observed at the first 3 months in the diesel spiked soil. After 8 months, compost addition resulted in over 90% loss of total PAHs irrespective of soil types. Desorption and degradation contributed to 30% and 70%, respectively, of the PAH loss in the spiked soil, while PAH loss in the other two soils resulted from 40% enhanced desorption and 60% enhanced degradation. Compost type and application rates had little influence on PAH bioavailability, but higher PAH removal was observed at higher initial concentration during the early stage of incubation. The bioavailable fraction of PAH was inversely correlated to the number of benzene rings and the octanol–water partition coefficient. Further degradation was not likely after 8-month although over 30% of the residual PAHs were bioavailable, which highlighted the application of bioavailability concept during remediation activities.     

Solid-phase bioassays and soil microbial activities to evaluate PAH-spiked soil ecotoxicity after a long-term bioremediation process simulating landfarming

The residual ecotoxicity of long-term bioremediated soils concomitantly spiked with three PAHs at four levels (15, 75, 150, 300 mg Sigma 3 PAHs kg(-1) soil) was evaluated using physico-chemical analyses, solid-phase bioassays and soil microbial activities. The pot-scale bioremediation process consisted of weekly moderate waterings in the presence or absence of sewage sludge compost (SSC) under greenhouse conditions. After 15 months, anthracene and pyrene were almost completely degraded whereas benzo[a]pyrene was still persisting, most apparently in SSC-amended soil treatments. However, no apparent toxic effects of the residual PAHs could be detected. SSC application at 40 t ha(-1) was performed to valorize the biowaste and stimulate PAH biodegradation but caused soil salinization and pH reduction at the end of the bioremediation process. Consequently, SSC-amended soils were characterized by strong phytotoxicity to lettuce and had adverse effects on the ostracod Heterocypris incongruens. Despite the smaller number of culturable bacterial populations in SSC-amended soils, soil enzymatic activities were not affected by the organic amendment and residual PAHs; and the bioremediation efficiency was likely to be more limited by the bioavailability of PAHs rather than by the total number of PAH-degraders.     

The effects of aging time on the fraction distribution and bioavailability of PAH

Understanding the effects of aging time on the fraction distribution and bioavailability of PAH, such as phenanthrene (PHE) and pyrene (PYR), has considerable benefits for risk assessment, food security and remediation strategies for contaminated soil. The results of the present study show that the proportion of the desorbed PHE decreased from ca. 82% at day 0 to ca. 65% at day 150. In addition, non-desorbed PHE increased from ca. 18% at day 0 to ca. 31% at day 150, whereas the changes of desorbed and non-desorbed PYR showed no significant trend during this aging period. The proportion of desorbed PYR was lower than that of PHE, whereas the opposite occurred with the non-desorbed fraction. After 150 d of aging, the proportion of bound residues (PHE and PYR) increased significantly with the cultivating time from ca. 0.2% to ca. 4.7% and ca. 0.1% to ca. 1.2% for PHE and PYR, respectively. In addition, the bioavailability of PAH (PHE and PYR) to earthworms was also assessed over 0-150 d. The results showed that the uptake rate and bioconcentration factor (BCF) of pollutants by earthworms displayed the following biphasic character: a rapid decrease over the first 15 d followed by a slow decrease over the next 135 d. Moreover, the earthworm uptake rate of PHE was greater than that of PYR throughout the incubation period, indicating that PHE has a higher bioavailability than PYR. In addition, the positive correlation between the uptake rate of earthworms and PAH extractability suggested that a three-step extraction is a reliable approach to predict PHE bioavailability in soil. However, a limit was observed for PYR.