Creosote-contaminated Soil Research Articles

Monitoring bioremediation in creosote-contaminated soils using chemical analysis and toxicity tests

Three soils with a history of creosote contamination (designated NB, TI and AC) were treated in bench-scale microcosms using conditions (nutrient amendment, moisture content and temperature) which had promoted mineralization of 14C-pyrene in a preliminary study. Bioremediation was monitored using the solid-phase Microtox test, seed germination and earthworm survival assays, SOS-chromotest, Toxi-chromotest and a red blood cell (RBC) haemolysis assay. Contaminant concentrations in the AC soil did not change after 150 days. Polycyclic aromatic hydrocarbon (PAH) concentrations decreased in the NB soil, and toxicity decreased overall according to the earthworm, seed germination and Microtox tests. Although total petroleum hydrocarbons (TPHs) in the TI soil were reduced following treatment, results of the earthworm, seed germination, RBC and Microtox tests suggested an initial increase in toxicity indicating that toxic intermediary metabolites may have formed during biodegradation. Toxicity testing results did not always correlate with contaminant concentrations, nor were the trends indicated by each test consistent for any one soil. Each test demonstrated a different capacity to detect reductions in soil contamination. Journal of Industrial Microbiology & Biotechnology (2000) 24, 132–139.

Simultaneous Determination of PAHs, Hetero-PAHs (N, S, O), and Their Degradation Products in Creosote-Contaminated Soils. Method Development, Validation, and Application to Hazardous Waste Sites

A simple and reproducible method which provides the simultaneous determination of PAHs and hetero-PAHs (N,S,O) and their metabolites in contaminated soils has been developed. Contaminants extracted from the soil sample were separated by polarity and acid−base characteristics using solid phase extraction (SPE) on silica gel and a strong basic anion exchange material. A subfraction containing PANHs and neutral metabolites was subsequently fractionated into neutral and basic compounds using a strong acidic cation exchange material. The identification and quantification was performed using different GC and HPLC methods. A method validation was carried out for 21 PAHs, 22 hetero-PAHs, and 19 metabolites in a five-level matrix calibration. Uncontaminated soil (AhA1-layer/compost mixture) was spiked with the chosen standard compounds. The method showed good linearity (coefficient of correlation) and high precision (coefficient of variation) for major and minor compounds over a wide range of concentration as well...

Phytoremediation of Soils Contaminated with Wood Preservatives: Greenhouse and Field Evaluations

ABSTRACT Phytoremediation was evaluated as a potential treatment for the creosote-contaminated surface soil at the McCormick and Baxter (M&B) Superfund Site in Portland, Oregon. Soil at the M&B site is contaminated with pentachlorophenol (PCP) and polyaromatic hydrocarbons (PAHs). Eight individual PAH compounds (containing four to six aromatic rings) were included in the investigation. Greenhouse and field studies were carried out using perennial ryegrass (Lolium perenne). The following three treatments were compared in both studies: T1 = planted-amended soil; T2 = unplanted-amended soil; and T3 = unplanted-unamended soil. The amendments were mineral nutrients and dolomite, which was used to raise the acidic pH of the soil. Contaminant concentrations in the soil were measured initially and at regular intervals for several months. In the greenhouse study, the concentrations of certain contaminants decreased as a function of time. Thus, PCP, fluoranthene, pyrene, chrysene, and benzo(k)fluoranthene appeared to undergo biodegradation in all of the treatments. On the other hand, certain larger molecular weight PAHs were relatively recalcitrant. These “recalcitrant PAHs” included benzo(b)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene. Statistical methods were used to compare the concentrations of the more easily biodegraded contaminants in treatments T1, T2, and T3. The statistical analysis was facilitated by normalizing the contaminant concentrations relative to the sum of the recalcitrant PAHs in the same sample. Thus, ratios were created that could be compared directly to benchmark values indicative of the contaminant at the beginning of the study. In the greenhouse study, statistically significant differences between T1 and T2, across all treatment times, were obtained for fluoranthene, pyrene, and chrysene (p ≤0.05), suggesting enhanced rhizosphere biodegradation for these compounds. Significant differences between T2 and T3 were obtained for pyrene (p ≤0.03), indicating that nutrients stimulated the biodegradation of this contaminant. Although the greenhouse study was carried out with a well-mixed soil sample from the M&B site, an extremely uneven distribution of contaminants was encountered in the field study. The resulting scatter in the field data made comparisons difficult, and treatment-specific effects observed in the greenhouse study were not statistically significant in the field study. However, analysis of the normalized data from the field revealed the same time-dependent decreases in contaminant concentration as observed in the greenhouse study.

Epigenetic Toxicity of a Mixture of Polycyclic Aromatic Hydrocarbons on Gap Junctional Intercellular Communication Before and After Biodegradation

Polycyclic aromatic hydrocarbons (PAHs) are known carcinogens, but most research on their toxicity in the development of human-risk assessment models has focused on genotoxicity. Many nongenotoxic PAHs, however, have been shown to be epigenetically toxic by disrupting gap junctional intercellular communication (GJIC), an effect which has been affiliated with tumor promotion. We therefore used GJIC as an epigenetic biomarker to assess the toxic effect of a nonaqueous phase liquid (NAPL) mixture of PAHs commonly found in coal tar and creosote products. The NAPL mixture consisted of toluene, naphthalene, 1-methylnaphthalene, 2-ethylnaphthalene, acenaphthene, fluorene, phenanthrene, fluoranthene, and pyrene. This mixture reversibly inhibited GJIC at a maximal and noncytotoxic dose of 60 μM. Inhibition occurred within 5 min, indicating a post-translational modification of gap junction proteins. Biodegradation of globules of this mixture suspended in mineral media by a microorganism isolated from creosote-contaminated soils resulted in the removal of all but three heavy PAHs: acenaphthene, pyrene, and fluoranthene. A reconstituted mixture of these three compounds showed results on GJIC activity identical to the original mixture relative to dose-, rate-, and time-responses, indicating that the toxicity of the PAHs was additive. The results suggest that bioremediation techniques that leave residual components of such NAPL mixtures in contaminated media can quantitatively but not qualitatively reduce their epigenetic toxic risk. Nonetheless, such bioresistant residuals may be environmentally less mobile than the biodegraded components of the precursor NAPLs.

14C]Pyrene Bound Residue Evaluation Using MIBK Fractionation Method for Creosote-Contaminated Soil

The fate of [14C]pyrene was evaluated in creosote-contaminated soil undergoing remediation in a prepared bed system at the Champion International Superfund site in Libby, MT. 14C-bound residue formation was evaluated using the methyl isobutyl ketone (MIBK) humic fractionation procedure, and it increased through 294 days of incubation in biologically active microcosms for humic acid, fulvic acid, bound humic acid, and mineral-associated organic carbon fractions. The relative affinity of the added pyrene and transformation products was highest for the humic acid fraction. Bound residue formation in PAH-contaminated soil was observed to be an important fate mechanism in the prepared bed system and may be an acceptable end point in the remediation of contaminated soil.

Bacterial adhesion to soil contaminants in the presence of surfactants

It has been proposed that addition of surfactants to contaminated soil enhances the solubility of target compounds; however, surfactants may simultaneously reduce the adhesion of bacteria to hydrophobic surfaces. If the latter mechanism is important for the biodegradation of virtually insoluble contaminants, then the use of surfactants may not be beneficial. The adhesion of a Mycobacterium strain and a Pseudomonas strain, isolated from a creosote-contaminated soil, to the surfaces of highly viscous non-aqueous-phase liquids (NAPLs) was measured. The NAPLs were organic material extracted from soils from two creosote-contaminated sites and two petroleum-contaminated sites. Cells suspended in media with and without surfactant were placed in test tubes coated with an NAPL, and the percentages of cells that adhered to the surface of the NAPL in the presence and absence of surfactant were compared by measuring optical density. Test tubes without NAPLs were used as controls. The presence of either Triton X-100 or Dowfax 8390 at a concentration that was one-half the critical micelle concentration (CMC) inhibited adhesion of both species of bacteria to the NAPLs. Both surfactants, when added at concentrations that were one-half the CMCs to test tubes containing previously adhered bacteria, also promoted the removal of the cells from the surfaces of the NAPL-coated test tubes. Neither surfactant was toxic to the bacteria. Further investigation showed that a low concentration of surfactant also inhibited the growth of both species on anthracene, indicating that the presence of a surfactant resulted in a reduction in the uptake of the solid carbon source.

Genotoxicity of bioremediated soils from the Reilly Tar site, St. Louis Park, Minnesota.

An in vitro approach was used to measure the genotoxicity of creosote-contaminated soil before and after four bioremediation processes. The soil was taken from the Reilly Tar site, a closed Superfund site in Saint Louis Park, Minnesota. The creosote soil was bioremediated in bioslurry, biopile, compost, and land treatment, which were optimized for effective treatment. Mutagenicity profiles of dichloromethane extracts of the five soils were determined in the Spiral technique of the Salmonella assay with seven tester strains. Quantitative mutagenic responses in the plate incorporation technique were then determined in the most sensitive strains, YG1041 and YG1042. Mutagenic potency (revertants per microgram extract) in YG1041 suggested that compost, land treatment, and untreated creosote soil extracts were moderately mutagenic with Arochlor-induced rat liver (S9) but were nonmutagenic without S9. However, the bioslurry extract was strongly mutagenic and the biopile extract was moderately mutagenic either with or without S9. A similar trend was obtained in strain YG1042. The strong mutagenic activity in the bioslurry extract was reduced by 50% in TA98NR, which suggested the presence of mutagenic nitrohydrocarbons. Variation in reproducibility was 15% or less for the bioassay and extraction procedures. Bioavailability of mutagens in the biopile soil was determined with six solvents; water-soluble mutagens accounted for 40% of the total mutagenic activity and they were stable at room temperature. The mutagenic activity in the bioslurry and biopsile samples was due to either the processes themselves or to the added sludge/manure amendments. The in vitro approach was effective in monitoring bioremediated soils for genotoxicity and will be useful in future laboratory and in situ studies.

Green fluorescent protein as a marker to monitor survival of phenanthrene-mineralizing Pseudomonas sp. UG14Gr in creosote-contaminated soil

The green fluorescent protein (encoded by gfp gene) was used as a marker to estimate survival of a phenanthrene-degrading Pseudomonas sp. UG14Gr in a creosote-contaminated soil from Alberta, Canada. The gfp was integrated via Tn5 transposition into the chromosome of UG14r and resultant gfp-marked colonies were identified by green fluorescence emission under UV light. The gfp was stably maintained in UG14Gr and the gfp insertion had no apparent adverse effect on the metabolic capability of the marked isolate, which showed unhindered ability to mineralize 14C-labelled phenanthrene. Colony forming units were used to monitor parent strain UG14r and gfp-marked UG14Gr in creosote-contaminated soil at 22°C. Green fluorescence in the gfp-marked colonies allowed detection of UG14Gr, on antibiotic-amended and non-amended Trypticase soy agar (TSA), up to 13 months after inoculation into soil while the parent UG14r strain could be detected, on TSA amended with rifampicin, for only up to 25 days after inoculation into soil. Our studies indicated that the gfp marker can be used for survival studies of marked bacterial cells in contaminated soil. When the gfp-marked UG14Gr strain was co-inoculated with a lux-lac-marked Pseudomonas aeruginosa UG2Lr strain, both strains were unambiguously detected by their respective markers. Comparison of the effect of water holding capacity (WHC) of soil on [ 14C]phenanthrene mineralization showed that mineralization proceeded faster in treatments at 60% WHC than at 85% WHC. Nitrogen amendment in soils had no effect on [ 14C]phenanthrene mineralization at 60% WHC while treatments at 85% WHC showed higher mineralization in unamended soils than in N-amended soils.

Green fluorescent protein as a marker to monitor survival of phenanthrene-mineralizing Pseudomonas sp. UG14Gr in creosote-contaminated soil

The green fluorescent protein (encoded by gfp gene) was used as a marker to estimate survival of a phenanthrene-degrading Pseudomonas sp. UG14Gr in a creosote-contaminated soil from Alberta, Canada. The gfp was integrated via Tn5 transposition into the chromosome of UG14r and resultant gfp-marked colonies were identified by green fluorescence emission under UV light. The gfp was stably maintained in UG14Gr and the gfp insertion had no apparent adverse effect on the metabolic capability of the marked isolate, which showed unhindered ability to mineralize 14C-labelled phenanthrene. Colony forming units were used to monitor parent strain UG14r and gfp-marked UG14Gr in creosote-contaminated soil at 22°C. Green fluorescence in the gfp-marked colonies allowed detection of UG14Gr, on antibiotic-amended and non-amended Trypticase soy agar (TSA), up to 13 months after inoculation into soil while the parent UG14r strain could be detected, on TSA amended with rifampicin, for only up to 25 days after inoculation into soil. Our studies indicated that the gfp marker can be used for survival studies of marked bacterial cells in contaminated soil. When the gfp-marked UG14Gr strain was co-inoculated with a lux-lac-marked Pseudomonas aeruginosa UG2Lr strain, both strains were unambiguously detected by their respective markers. Comparison of the effect of water holding capacity (WHC) of soil on [14C]phenanthrene mineralization showed that mineralization proceeded faster in treatments at 60% WHC than at 85% WHC. Nitrogen amendment in soils had no effect on [14C]phenanthrene mineralization at 60% WHC while treatments at 85% WHC showed higher mineralization in unamended soils than in N-amended soils.

Functional diversity and community structure of microorganisms in uncontaminated and creosote-contaminated soils as determined by sole-carbon-source-utilization

Functional diversities of microorganisms from uncontaminated and creosote-contaminated soils were assessed using sole-carbon source-utilization patterns. The microorganisms were extracted from soil samples and inoculated into Gram-negative Biolog plates incubated at 23°C. Measurement of Shannon diversity, richness, and evenness indices, principal component analysis (PCA), and colour development rank (CDR) plots were based upon substrate utilization. Calculations incorporated data from both the 95 regular Gram-negative Biolog microplate wells and a selection of 23 carbon substrates that are included on Biolog Ecoplates. There did not appear to be significant differences in Shannon diversity and richness indices, PCA, or CDR plots between aminated and creosote-contaminated soils. Significant differences in Shannon diversity and evenness indices that were apparent with the use of the 23 ecologically relevant microplate wells were mostly absent based on calculations that incorporated the regular 95 Gram-negative Biolog microplate wells. Resolution of microbial communities by PCA, however, appeared to be reduced by the use of the 23 Biolog microplate wells compared to the regular 95 carbon sources.

Removal of polycyclic aromatic hydrocarbons (PAH) in contaminated soil by white rot fungus Pleurotus ostreatus

Pleurotus ostreatus was applied to aged creosote-contaminated soil. The white rot fungus degraded benzo[a]pyrene most extensively the first month (28%) and further incubation had a less pronounced effect: degradation increased only an additional 4% to 32%. Removal of artificially added [ 14C]benzo[a]pyrene was higher than the originally aged benzo[a]pyrene; 40$ of the compound was removed after one month and 49% after 3 months of incubation. The mineralisation degree to 14CO 2 was only 1%, but it was still significantly higher in comparison to unsterile control soil (0.1%) without rot fungus.

Multi-phase partitioning and co-solvent effects for polynuclear aromatic hydrocarbons (pah) in authentic petroleum- and creosote-contaminated soils

The residual oil phase, present at substantial levels in hydrocarbon-contaminated soils, was found to be the principal medium for retention of polynuclear aromatic hydrocarbons (PAH). Using authentic petroleum and creosote-contaminated soils containing 2–6% w/w solvent extractable matter, individual PAH were shown to exist in the oil phase at values between 84 and 95% w/w of the total mass of each compound in the system, with the exception of naphthalene (70% w/w). Evaluation of the phase partitioning of PAH in creosote-contaminated soil confirmed that PAH were predominantly in the creosote oil phase, with substantially lower proportions associated with soil organic matter and soil water. In the aqueous phase, solubility enhancement was observed for PAH with the degree of enhancement more evident for the hydrophobic PAH. A normalised solubility enhancement factor (ESF’) was derived to illustrate the combined effect of increased PAH solubility in the aqueous phase. Values of log ESF’ varied from −4.97 (naphthalene) to 0.09 (benzo[ghi]perylene). The findings suggest limitations exist for successful remediation of PAH contamination in the soils evaluated and indicate that the focus of remediation must be on the residual oil phase if total PAH are to be removed.

Desorption of [14C]Naphthalene from Bioremediated and Nonbioremediated Soils Contaminated with Creosote Compounds

Bioremediation changes the quantity and nature of the contaminant matrix remaining in soil, because some compounds are selectively degraded while others remain undegraded. It was hypothesized that changes to the contaminant matrix may alter the chemical and physical properties of the soil, such that subsequent desorption of a specific PAH compound would be altered. Desorption of [14C]naphthalene from two creosote-contaminated soils was measured before and after bioremediation. Although the bioremediation treatment removed the lower-molecular weight components, increasing the average molecular weight of the residual creosote by 10−36%, partition coefficients based on the mass of organic carbon in the soil were unaffected. Partition coefficients for naphthalene in soil organic matter were 4.6−8.3 times smaller than in the creosote contaminant. When partitioning was modeled as the sum of the contributions of the nonaqueous phase contaminant and the soil organic matter, the partition coefficients for the creo...

Aerobic degradation of 2-sulphobenzoic acid by Pseudomonas strain SB(W).

A 2-sulphobenzoic acid-degrading bacterium, Pseudomonas sp. strain SB(W), was isolated from creosote-contaminated soil. It used this compound as its sole carbon, sulphur and energy source, and gave a nearly stoichiometric release of sulphate from 2-sulphobenzoic acid. It did not grow on 3- to 4- sulphobenzoic acids. Isolated SB(W) produced two transient metabolites. The first to appear, and the more abundant metabolite, was identified as 2,3-dihydroxybenzoic acid. The second metabolite was identified as salicylic acid. Both of these compounds served as growth substrates for the isolates.

Growth of rhodococcus S1 on anthracene.

Three slow-growing bacteria were isolated from a mixed culture enriched for growth on anthracene, using creosote-contaminated soil as the inoculum. Organisms were shown to use anthracene by the production of a clear zone around the colony after a mineral salts agar plate was sprayed with anthracene. All three bacteria were nonmotile, nonsporulating, gram-positive rods and stained acid-fast. Physiological and biochemical tests, GC content, and cell wall lipid patterns of whole cell methanolysates indicated that they belonged to the Nocardia-Mycobacterium-Rhodococcus group. On the basis of these characteristics and pyrolysis gas chromatography, they were assigned to the genus Rhodococcus. Growth of the isolates was slow on crystalline anthracene, giving a doubling time of 1.5-3 days, and they grew mainly on the crystal surface. When anthracene was supplied by precipitation from a solvent, doubling time was reduced to 1 day. All three isolates mineralized anthracene but not phenanthrene or naphthalene, nor could they grow on naphthalene, phenanthrene, fluorene, fluoranthene, acenaphthene, pyrene, chrysene, or naphthacene as sole carbon source. One isolate, Rhodococcus S1, was able to use 2-methylanthracene or 2-chloroanthracene as carbon source but not 1- or 9-substituted analogs. These results suggest that the initial enzyme attacking anthracene in these isolates has a narrow substrate specificity.

Effect of alginate encapsulation and selected disinfectants on survival of and phenanthrene mineralization byPseudomonas sp UG14Lr in creosote-contaminated soil

The survival and phenanthrene-mineralizing ability of free and alginate-encapsulatedPseudomonas sp UG14Lr cells were examined in a creosote-contaminated soil. Alginate encapsulation adversely affected both survival and phenanthrene mineralization. This was postulated to be due to concentration of water-soluble toxic compounds in the alginate beads. Toxicity studies showed that the concentrated water-soluble fraction of the creosote-contaminated soil may be toxic toPseudomonas sp UG14Lr in soil with a low moisture content. Survival of alginate-encapsulated cells improved with increasing soil moisture content. Free cells survived well at a steady population of 108 CFU g−1 dry soil for 28 days in the creosote-contaminated soil. However, phenanthrene mineralization was not improved compared to the uninoculated control. This was attributed to the existence of indigenous phenanthrene-mineralizing microorganisms already present in this contaminated soil. The effect of calcium hypochlorite and Germiphene on survival of and phenanthrene mineralization by free and alginate-encapsulatedPseudomonas sp UG14Lr cells in creosote-contaminated soil was also studied. Addition of 0.1% (w/w dry soil) calcium hypochlorite reduced the introduced free cells to below detection limits (10 CFU g−1 dry soil) within 14 days, while Germiphene had no effect on cell numbers. Phenanthrene mineralization by free cells was not adversely affected by treatment with calcium hypochlorite or Germiphene. Survival of alginate-encapsulated cells after treatment with disinfectants was as poor as that without disinfection. The results show that alginate encapsulation may not be a suitable formulation for introduction ofPseudomonas sp UG14Lr into creosote-contaminated soils.

Carbazole Degradation by Pseudomonas sp. LD2: Metabolic Characteristics and the Identification of Some Metabolites

A carbazole-degrading bacterium was isolated by enrichment from a creosote-contaminated soil. This organism, designated Pseudomonas sp. LD2, utilized carbazole as a sole source of carbon, nitrogen, and energy. When isolate LD2 was grown in nitrogen-free mineral medium with 14C-labeled carbazole, 43% was recovered as 14CO2 after 3 days of incubation. Numerous aromatic and heterocyclic compounds were tested as growth substrates for isolate LD2, but few supported the growth of this bacterium. Anthranilic acid and catechol served as growth substrates and were positively identified as intermediates of carbazole degradation by isolate LD2. In addition, 10 nitrogen-containing metabolites were observed in acidified extracts of LD2 culture supernatants, four of which were unequivocally identified. These included indole-3-acetic acid, 5-(2-aminophenyl)-5-oxopentanoic acid, and the cyclized products of 5-(2-aminophenyl)-5-oxopent-3-enoic acid and 6-(2-aminophenyl)-2-hydroxy-6-oxohexa-2,4-dienoic acid.

Effect of nonionic surfactant addition on bacterial metabolism of naphthalene: Assessment of toxicity and overflow metabolism potential

Two factors potentially accounting for the variability of bioremediation outcomes when surfactant micelles are used to increase polycyclic aromatic hydrocarbon (PAH) bioavailability were investigated: (1) surfactant toxicity and (2) the link between microbial metabolism and the intended effect of surfactant addition, enhanced solubilization and mass transfer from a solid phase. The nonionic surfactant, octaethyleneglycol mono n-dodecyl ether, did not alter the metabolism of succinate and glucose by an isolate from a creosote-contaminated soil indicating that the surfactant is nontoxic. When the culture was supplied with solid naphthalene, growth was limited by the dissolution of solid naphthalene after the aqueous-phase naphthalene was depleted. Moreover, increasing dissolution rate by increasing interfacial surface area increased the microbial growth rate. However, increasing bioavailability further by increasing interfacial surface area, introducing convective mass transfer, and adding surfactant were all found to reduce growth rate and prompt incomplete metabolism of naphthalene to a compound whose UV absorption corresponds to 1,2-naphthaquinone. Lowering the surfactant concentration diminished the metabolic overflow and permitted sustained growth. The results suggest that different mismatches between solubilization/mass transfer and metabolic capacity may be among the factors responsible for variable bioremediation outcomes.

Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and free Pseudomonas sp. UG14Lr cells in creosote-contaminated soil slurries.

The effects of nutrient amendment and alginate encapsulation on survival of and phenanthrene mineralization by the bioluminescent Pseudomonas sp. UG14Lr in creosote-contaminated soil slurries were examined. UG14Lr was inoculated into creosote-contaminated soil slurries either as a free cell suspension or encapsulated in alginate beads prepared with montmorillonite clay and skim milk. Additional treatments were free-cell-inoculated slurries amended with sterile alginate beads, free-cell-inoculated and uninoculated slurries amended with skim milk only, and uninoculated, unamended slurries. Mineralization was determined by measuring 14CO2 released from radiolabelled phenanthrene. Survival was measured by selective plating and bioluminescence. Inclusion of skim milk was found to enhance both survival of and phenanthrene mineralization by free and encapsulated UG14Lr cells.

Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and free Pseudomonas sp. UG14Lr cells in creosote-contaminated soil slurries

Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and free Pseudomonas sp. UG14Lr cells in creosote-contaminated soil slurries