Biological Limitations of Dechlorination of cis-Dichloroethene during Transport in Porous Media.

We applied a mathematical model to data from experimental column studies to understand the dynamics of successful and unsuccessful reductive dechlorination of chlorinated ethenes in groundwater under different flow conditions. In laboratory column experiments (reported previously), it was observed that complete dechlorination of cis-dichloroethene to ethene was sustained at high flow velocity (0.51 m/d), but that dechlorination failed at medium or low flow velocity (0.080 or 0.036 m/d). The mathematical model applied here accounts for transport of chlorinated ethenes in flowing groundwater, mass transfer of chlorinated ethenes between mobile groundwater and stationary biofilms, and diffusion and biodegradation within the biofilms. Monod kinetics with competitive inhibition are used to describe biodegradation. Nearly all parameters needed to solve the model are estimated independently from batch and nonreactive transport experiments. Comparing the model predictions to the experimental results permits the evaluation of three hypothesized biological limitations: insufficient supply of electron donor, decay of dechlorinators' biomass, and reduction in bacterial metabolism rates. Any of these three limitations are able to adequately describe observed experimental data, but insufficient supply of electron donor is the most plausible explanation for failure of dechlorination. Therefore, an important conclusion of this investigation is that insufficient hydrogen production occurs if groundwater flow is too slow to provide adequate flux of electron donor. Model simulations were in good agreement with experimental results for both successful and unsuccessful dechlorination, suggesting the model is a valid tool for describing transport and reductive dechlorination. An implication of our findings is that in engineered or natural bioremediation of chloroethene-contaminated groundwater, not only must the proper dechlorinating organisms be present, but also proper groundwater flow conditions must be maintained or else dechlorination may fail.

Effect of pore velocity on biodegradation of cis-dichloroethene (DCE) in column experiments.

Column experiments were conducted to evaluate the effect of pore velocity on the extent of biodegradation of cis-dichloroethene (cis-DCE) during transport in porous media. Columns were filled with homogeneous glass beads and inoculated with a culture capable of complete dechlorination of tetrachloroethene to ethene. A constant concentration of cis-DCE was maintained in the columns' influent. Three different pore velocities were tested in duplicate, subjecting each column to a constant velocity. At high flow velocity, degradation of cis-DCE to ethene was nearly complete within the residence time of the columns. However, at medium and low flow velocities, incomplete dechlorination was observed. After 7 weeks, DNA was harvested from the columns to determine differences in the microbial populations. Results suggest that Dehalococcoides sp. were present in higher quantities in the high-velocity columns, consistent with the observed dechlorination. These results suggest that, at contaminated groundwater sites, heterogeneity of groundwater velocity may be one factor that contributes to heterogeneous distribution of biological activity.

Using quantitative structure-activity relationships (QSAR) to predict toxic endpoints for polycyclic aromatic hydrocarbons (PAH).

Quantitative structure-activity relationships (QSAR) offer a reliable, cost-effective alternative to the time, money, and animal lives necessary to determine chemical toxicity by traditional methods. Additionally, humans are exposed to tens of thousands of chemicals in their lifetimes, necessitating the determination of chemical toxicity and screening for those posing the greatest risk to human health. This study developed models to predict toxic endpoints for three bioassays specific to several stages of carcinogenesis. The ethoxyresorufin O-deethylase assay (EROD), the Salmonella/microsome assay, and a gap junction intercellular communication (GJIC) assay were chosen for their ability to measure toxic endpoints specific to activation-, induction-, and promotion-related effects of polycyclic aromatic hydrocarbons (PAH). Shape-electronic, spatial, information content, and topological descriptors proved to be important descriptors in predicting the toxicity of PAH in these bioassays. Bioassay-based toxic equivalency factors (TEF(B)) were developed for several PAH using the quantitative structure-toxicity relationships (QSTR) developed. Predicting toxicity for a specific PAH compound, such as a bioassay-based potential potency (PP(B)) or a TEF(B), is possible by combining the predicted behavior from the QSTR models. These toxicity estimates may then be incorporated into a risk assessment for compounds that lack toxicity data. Accurate toxicity predictions are made by examining each type of endpoint important to the process of carcinogenicity, and a clearer understanding between composition and toxicity can be obtained.

Quantitative structure-activity relationships for kinetic parameters of polycyclic aromatic hydrocarbon biotransformation.

Quantitative structure-activity relationships (QSARs) were developed for three Monod-type parameters--qmax, Ks, and qmax/Ks--that express the kinetics of polycyclic aromatic hydrocarbon (PAH) biotransformation by Sphingomonas paucimobilis strain EPA505. The training sets contained high-quality experimental values of the kinetic parameters for 20 unsubstituted and methylated PAHs as well as values of 41 meaningful molecular descriptors. A genetic function approximation algorithm was used to develop the QSARs. Statistical evaluation of the developed QSARs showed that the relationships are statistically significant and satisfy the assumptions of linear-regression analysis. The Organization for Economic Co-operation and Development principles for (Q)SAR validation were followed to evaluate the developed QSARs, which showed that the QSARs are valid. The QSARs contain spatial, spatial and electronic, topological, and thermodynamic molecular descriptors. Whereas spatial descriptors were essential in explaining biotransformation kinetics, electronic descriptors were not. Mechanistic interpretation of the QSARs resulted in evidence that is consistent with the hypothesis of membrane transport as being the rate-limiting process in PAH biotransformation by strain EPA505. The present study demonstrates the value of QSAR not only as a predictive tool but also as a framework for understanding the mechanisms governing biodegradation at the molecular level.

In situ biomonitoring of PAH-contaminated sediments using juvenile coho salmon (Oncorhynchus kisutch).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous marine and freshwater sediment contaminants. Extensive data exist to confirm that PAHs are toxic to aquatic receptors. However, limited information is available regarding the bioavailability and genotoxicity of sediment PAHs to aquatic organisms. This study investigated an integrated biomonitoring approach using chemical analyses and biomarkers to characterize the bioavailability and genotoxicity of a complex PAH mixture in freshwater lake sediments associated with a former manufactured gas plant (MGP). Sediment PAH genotoxicity was assessed by flow cytometry (FCM), DNA adduct (32)P-postlabeling, and erythrocyte micronuclei in juvenile coho salmon (Oncorhynchus kisutch) caged in the water column. Significant PAH-induced genotoxicity was observed with FCM and (32)P-postlabeling, but not with erythrocyte micronuclei. Chromosome damage in peripheral blood and hepatic DNA adducts correlated with sediment, but not water column PAH concentrations. Total hepatic DNA adducts in salmon caged nearest the former MGP facility was 39+/-6.5 (RALx10(9)), while salmon caged in a reference lake had 28+/-2.3 total hepatic DNA adducts per 10(9) nucleotides. These results indicate that in situ biomonitoring using biomarkers and caged fish can be a sensitive indicator of genotoxic PAHs in sediments.

Arbuscular mycorrhiza and petroleum-degrading microorganisms enhance phytoremediation of petroleum-contaminated soil.

While plants can phytoremediate soils that are contaminated with petroleum hydrocarbons, adding microbes to remediate contaminated sites with petroleum-degrading microorganisms and arbuscular mycorrhizal fungi (AMF) is not well understood. The phytoremediation of Arabian medium crude oil (ACO) was done with a Lolium multiflorum system inoculated with an AMF (Glomus intraradices) and a mixture of petroleum-degrading microorganisms--the bacterium, Sphingomonas paucimobilis (Sp) and the filamentous fungus, Cunninghamella echinulata (Ce, SpCe)--or with a combination of microorganisms (AMF + SpCe). Based on an earlier study on screening plants for phytoremediation of ACO, L. multiflorum (Italian ryegrass) was selected for its tolerance and rapid growth response (Alarcón, 2006). The plants were exposed to ACO-contaminated soil (6000 mg kg(-1)) for 80 d under greenhouse conditions. A modified Long Ashton Nutrient Solution (LANS) was supplied to all treatments at 30 microg P mL(-1), except for a second, higher P, control treatment at 44 microg P mL(-1). Inoculation with AMF, SpCe, or AMF + SpCe resulted in significantly increased leaf area as well as leaf and pseudostem dry mass as compared to controls at 30 microg P mL(-1). Populations of bacteria grown on a nitrogen-free medium and filamentous fungi increased with AMF + SpCe and SpCe treatments. The average total colonization and arbuscule formation of AMF-inoculated plants in ACO-contaminated soil were 25% and 8%, respectively. No adverse effects were caused by SpCe on AMF colonization. Most importantly, ACO degradation was significantly enhanced by the addition of petroleum-degrading microorganisms and higher fertility controls, as compared to plants at 30 microg P mL(-1). The highest ACO degradation (59%) was observed with AMF + SpCe. The phytoremediation of ACO was also enhanced by single inoculation of AMF or SpCe. The effect of AMF and petroleum-degrading microorganisms on plant growth and ACOdegradation was not attributable to differences in proline, total phenolics, nitrate reductase levels, or variation in plant-gas exchange.

Biodegradation kinetics of select polycyclic aromatic hydrocarbon (PAH) mixtures by Sphingomonas paucimobilis EPA505.

Many contaminated sites commonly have complex mixtures of polycyclic aromatic hydrocarbons (PAHs) whose individual microbial biodegradation may be altered in mixtures. Biodegradation kinetics for fluorene, naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene were evaluated in sole substrate, binary and ternary systems using Sphingomonas paucimobilis EPA505. The first order rate constants for fluorene, naphthalene, 1,5-dimethylnaphthalene, and 1-methylfluorene were comparable; yet Monod parameters were significantly different for the tested PAHs. S. paucimobilis completely degraded all the components in binary and ternary mixtures; however, the initial degradation rates of individual components decreased in the presence of competitive PAHs. Results from the mixture experiments indicate competitive interactions, demonstrated mathematically. The generated model appropriately predicted the biodegradation kinetics in mixtures using parameter estimates from the sole substrate experiments, validating the hypothesis of a common rate-determining step. Biodegradation kinetics in mixtures were affected by the affinity coefficients of the co-occurring PAHs and mixture composition. Experiments with equal concentrations of substrates demonstrated the effect of concentration on competitive inhibition. Ternary experiments with naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene revealed delayed degradation, where depletion of naphthalene and 1,5-dimethylnapthalene occurred rapidly only after the complete removal of 1-methylfluorene. The substrate interactions observed in mixtures require a multisubstrate model to account for simultaneous degradation of substrates. PAH contaminated sites are far more complex than even ternary mixtures; however these studies clearly demonstrate the effect that interactions can have on individual chemical kinetics. Consequently, predicting natural or enhanced degradation of PAHs cannot be based on single compound kinetics as this assumption would likely overestimate the rate of disappearance.

Comparing deterministic and probabilistic risk assessments for sites contaminated by polycyclic aromatic hydrocarbons (PAHs).

Deterministic risk assessments provide a single point estimate of risk at a site of concern, while probabilistic risk assessment methods generate a range of values from probability distribution functions (PDFs). This study compares deterministic and probabilistic risk assessment methods for two different sites using uncertainty analysis and evaluates the use of toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs) in each method. The use of TEFs allowed more of the PAHs to be included which resulted in higher risk estimates for both adults and children at both study sites. Exposure factor variability generated greater uncertainty in risk estimates than sample heterogeneity or toxicity estimates. TEFs allowed for a more complete representation of the toxicity contributions from PAH species present at the study sites. Exposure factor distributions together with TEF estimations for toxicity of PAH species provide an assessment that can reduce and account for uncertainty. Probabilistic risk estimates provide a sensible improvement to deterministic risk estimates by generating a range of risk values, along with an average estimate and the degree of conservatism of those estimates.

Risk of polycyclic aromatic hydrocarbon (PAH) exposure from ingested food: the Azerbaijan case study.

Risks due to polycyclic aromatic hydrocarbons (PAHs) exposure from food consumption for the population of Azerbaijan were determined using deterministic and probabilistic methods. The guidelines and methods described and presented in the United States Environmental Protection Agency (U.S. EPA) Risk Assessment Guidance for Superfund (RAGS) Part A was used in performing the risk assessments. The current study utilized concentration data from different sources representing international studies performed over the past decade to determine those food products that contribute the most exposure to PAHs through ingestion for the Azeri population. Due to lack of concentration data from middle-Eastern countries, only European countries were considered and used for this analysis. Using the benzo[a]pyrene (BaP) toxicity equivalency factors (TEFs) to adjust the concentrations of the individual PAH compounds to BaP equivalent concentrations, risk analyses were performed. Deterministic risk estimates fell within probabilistic risk estimates. Child risks were consistently four to seven times higher in magnitude than adult risks. Risk potentials determined for the food exposure pathway were also determined to be up to ten times higher in magnitude than risks determined from exposures due to other pathways such as soil contamination. It was observed that three major factors contributed to the variability in the assessment results, which were child and adult body weights, consumption rates of the different food groups, and the variances of the input data. The most prevalent pathways of PAH exposure from the dietary patterns of the Azerbaijani population were determined to be from bread and bakery products, milk and dairy products, and egg products.

Measurement of biodegradability parameters for single unsubstituted and methylated polycyclic aromatic hydrocarbons in liquid bacterial suspensions.

Substrate depletion experiments were conducted to characterize aerobic biodegradation of 20 single polycyclic aromatic hydrocarbons (PAHs) by induced Sphingomonas paucimobilis strain EPA505 in liquid suspensions. PAHs consisted of low molecular weight, unsubstituted, and methyl-substituted homologs. A material balance equation containing the Andrews kinetic model, an extension of the Monod model accounting for substrate inhibition, was numerically fitted to batch depletion data to estimate extant kinetic parameters including the maximal specific uptake rates, q(max), the affinity coefficients, K(S), and the substrate inhibition coefficients, K(I). Strain EPA505 degraded all PAHs tested. Applied kinetic models adequately simulated experimental data. A cell proliferation assay involving reduction of the tetrazolium dye WST-1 was used to evaluate the ability of strain EPA505 to utilize individual PAHs as sole energy and carbon sources. Of the 22 PAHs tested, 9 supported bacterial growth. Evaluation of the biokinetic data showed that q(max) correlated highly with transmembrane flux as theoretically estimated by a diffusion model, pointing to transmembrane transport as a potential rate-determining process. The biodegradability data generated in this study is essential for the development of quantitative structure-activity relationships (QSARs) for biodegradability and for modeling biodegradation of simple PAH mixtures.

Kinetics of biodegradation of binary and ternary mixtures of PAHs.

The kinetics of biodegradation of mixtures of polycyclic aromatic hydrocarbons (PAHs) by Sphingomonas paucimobilis strain EPA505 were investigated. The investigation focused on three- and four-ring PAHs, specifically 2-methylphenanthrene, fluoranthene, and pyrene. Uptake rates in aerobic batch suspended cultivations were measured for the individual PAHs and their binary and ternary mixtures. It was observed that kinetics were influenced by the mixture composition and the kinetic properties of the components. A material balance equation containing the Monod model was numerically fitted to uptake data to determine extant kinetic parameters for the individual PAHs. Similarly, equations containing kinetic interaction models derived from enzyme kinetics were fitted to the uptake data obtained from experiments with binary and ternary mixtures. The investigation considered the following interaction types: no-interaction (Monod), pure competitive interaction, noncompetitive or mixed-type interaction, uncompetitive inhibition, and nonspecific interaction based on pure competition (SKIP). Model fit was evaluated based on probabilistic and statistical criteria and inferences were reached about underlying interaction mechanisms based on model fit. Mixture kinetics were most adequately simulated by the pure competitive interaction model with mutual substrate exclusivity. This model is fully predictive, relying only on parameters determined in the sole-PAH experiments. It was shown that for low percent inhibition values and with limited data, pure competitive interaction kinetics may not be evident, resembling no-interaction kinetics. This study is a reasonable starting point for understanding and modeling biodegradation of complex PAH mixtures in engineered and natural systems.

Evolutionary toxicology: population-level effects of chronic contaminant exposure on the marsh frogs (Rana ridibunda) of Azerbaijan.

We used molecular methods and population genetic analyses to study the effects of chronic contaminant exposure in marsh frogs from Sumgayit, Azerbaijan. Marsh frogs inhabiting wetlands in Sumgayit are exposed to complex mixtures of chemical contaminants, including petroleum products, pesticides, heavy metals, and many other industrial chemicals. Previous results documented elevated estimates of genetic damage in marsh frogs from the two most heavily contaminated sites. Based on mitochondrial DNA (mtDNA) control region sequence data, the Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. The Sumgayit region also acts as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. Additionally, localized mtDNA heteroplasmy and diversity patterns suggest that one of the most severely contaminated sites in Sumgayit is acting as a source of new mutations resulting from an increased mutation rate. This study provides an integrated method for assessing the cumulative population impacts of chronic contaminant exposure by studying both population genetic and evolutionary effects.

Chromosomal damage in two species of aquatic turtles (Emys orbicularis and Mauremys caspica) inhabiting contaminated sites in Azerbaijan.

The Caspian region and specifically the Apsheron peninsula of Azerbaijan are known to be polluted with a variety of environmental contaminants. These complex mixtures of contaminants make risk assessment difficult. We used the flow cytometry method (FCM) and the micronucleus assay (MN) to assess chromosomal damage in aquatic turtles (Emys orbicularis, the European pond turtle; and Mauremys caspica, the Caspian turtle) inhabiting contaminated wetlands in Azerbaijan. Evidence of genetic damage was found for two sites, Neftchala and Sumgayit, relative to a reference site, Ali Bairamly. Sediment samples from each site were analyzed for PAHs and mercury to evaluate potential contaminant associations with genetic damage. A significant positive correlation was documented between three-ring PAH sediment concentrations and FCM estimates of chromosomal damage in E. orbicularis. These data combine to show that the contaminated wetlands in Sumgayit and Neftchala are genotoxic and that three-ring PAHs are likely a significant influence on observed genotoxicity.

Patterns of genotoxicity and contaminant exposure: evidence of genomic instability in the marsh frogs (Rana ridibunda) of Sumgayit, Azerbaijan.

The wetlands of Sumgayit in the Azerbaijan Republic contain complex mixtures of contaminants, including polycyclic aromatic hydrocarbons (PAHs), mercury, organochlorine pesticides, and polychlorinated biphenyls. Marsh frogs (Rana ridibunda) were collected from several contaminated wetlands within the city as well as from two reference sites outside the city. Sediment samples revealed heterogeneous patterns of PAH and mercury concentrations throughout Sumgayit, with the highest levels occurring east of the Sumgayit River, within the industrial zone. Flow cytometry and micronucleus assay revealed elevated estimates of genetic damage in frogs from the wetlands east of the Sumgayit River compared to frogs from the reference sites. Flow cytometric data showed a significant correlation with sediment mercury concentrations, whereas population micronucleus frequencies were significantly correlated with high-molecular-weight PAHs.

Application of fractal flocculation and vertical transport model to aquatic sol-sediment systems.

In estuarine and coastal environments, flocculation occurs between particles of different fractal dimensions and of different densities. Questions remain concerning the level of detail required to model particle flocculation and settling in these heterogeneous systems. This paper compares the goodness of fit between two flocculation models, using measured time series particle size distribution data collected from clay, colloidal silica, emulsified crude oil, clay-crude oil, and silica-crude oil systems. The coalesced sphere (CS) flocculation model includes the effects of heterogeneous particle size and density; the modified coalesced fractal sphere (mCFS) model adds effects due to heterogeneous fractal dimension. Goodness of fit was quantified using values of a minimized objective function, the mean of the sum of the square of the relative residuals (MSSRR). For nearly all tested experimental conditions, MSSRR values varied less than 5% between the CS and mCFS flocculation models. Additionally, collision efficiency values for single-particle-type (alpha(HOMOO)) and dual-particle-type (alpha(HETT)) systems were obtained through parameter regression using the CS and mCFS models. Using the mCFS model, estimated fractal dimension (D) values obtained for clay and clay-oil systems were between 2.6 and 3.0, lower than that postulated by the CS model but higher than that estimated experimentally by the particle concentration technique. The Stokes settling velocity of a clay aggregate of a given mass is reduced with decreased fractal dimension. This results in clay-oil flocculation occurring faster than floc sedimentation in the studied hydrodynamic range. Thus, the mCFS model provides insights to the fate of spilled oil in inland and coastal waters.

Modeling crude oil droplet-sediment aggregation in nearshore waters.

This paper describes a modeling approach that simulates changes in particle size distribution and density due to aggregation by extending the Smoluchowski aggregation kinetic model to particles of different density. Batch flocculation studies were conducted for clay, colloidal silica, crude oil, clay-crude oil, and silica-crude oil systems. A parameter estimation algorithm was used to estimate homogeneous collision efficiencies (alphaHOMO) for single-particle-type systems and heterogeneous collision efficiencies (alphaHET) for two-particle-type systems. Homogeneous collision efficiency values (alphaHOMO) were greater for clay (0.7) and for crude oil (0.3) than for silica (0.01). Thus, clay and crude oil were classified as cohesive particles while silica was classified as noncohesive. Heterogeneous collision efficiencies were similar for oil-clay (0.4) and oil-silica (0.3) systems. Thus, crude oil increases the aggregation of noncohesive particles. Data from the calibrated aggregation model were used to estimate apparent first-order flocculation rates (K') for oil, clay, and silica and apparent second-order flocculation rates (K'') for oil and clay in oil-clay systems and for oil and silica in oil-silica systems. For oil or clay systems, aggregation Damköhler numbers ranged from 0.1 to 1.0, suggesting that droplet coalescence and clay aggregation can occur on the same time scales as oil resurfacing and clay settling, respectively. For mixed oil-clay systems, the relative time scales of clay settling and clay-oil aggregation were also within an order of magnitude. Thus, oil-clay aggregation should be considered when modeling crude oil transport in nearshore waters.

Evaluation of bioremediation strategies of a controlled oil release in a wetland.

A controlled petroleum release was conducted to evaluate bioremediation in a wetland near Houston, Texas. The 140-day study was conducted using a randomized, complete block design to test three treatments with six replicates per treatment. The three treatment strategies were inorganic nutrients, inorganic nutrients with an alternative electron acceptor, and a no-action oiled control. Samples were analyzed for petroleum chemistry and inorganic nutrients. These results are discussed in the context of our related research involving toxicology and microbiology at the site during the experiment. To evaluate biodegradation, the targeted compounds were normalized to the conservative compound C3017alpha, 21beta-[H]hopane, thus reducing the effects of spatial heterogeneity and physical transport. The two biostimulation treatments demonstrated statistically-higher rates of biodegradation than the oiled no-action control. For the majority of the experiment, target nutrient levels were maintained. Further research may be warranted to optimize these bioremediation strategies as well as evaluating additional treatment strategies for wetlands and other shoreline systems.

Characterizing aquatic sediment-oil aggregates using in situ instruments.

The effects of emulsified crude oil and salinity (15, 30 per thousand ) on the steady state aggregate volume distributions and fractal dimensions were determined for a range of mean velocity gradients, (G(m) =5-50 s(-1)). Aggregation was performed in a 40-L cylindrical tank with a 4-blade paddle mixer. Three-dimensional fractal dimensions (D3) and volume distributions were determined using a procedure integrating data from an electrozone and an in situ light scattering instrument. Two-dimensional fractal dimensions (D2) and derived volume distributions were determined using a recently developed submersible flow cytometer equipped with a digital camera and image analysis software. For latex beads or emulsified crude oil systems, the above listed instruments yielded consistent size distributions and fractal dimensions (D2=1.92 +/- 0.16, D3=2.94 +/- 0.12). Mean volume aggregate diameters determined using the FlowCAM were consistently larger that those determined using the LISST-100 or Coulter Multisizer due to aggregate orientations during measurements. With increasing G(m) values, all colloidal aggregates showed increasing D3 values due to reduced aggregate length. Because of the compactness of all the aggregates (D3 >2), D2 values remained constant at 2. Neither salinity nor sediment type significantly affected D2 values calculated for sediment-crude oil aggregates. However, clay-oil aggregates showed higher D3 values than clay aggregates. This suggests that colloidal oil and mixing shear are the more dominant factors influencing aggregate morphology in nearshore waters. Overall, the data suggests that the analysis methods provide consistent size distribution results. However, because of the shear and salinity of coastal waters, resulting aggregates are too compact to estimate their D3 values using image analysis alone.

Partitioning of crude oil polycyclic aromatic hydrocarbons in aquatic systems.

This paper investigates the hypothesis that observed polycyclic aromatic hydrocarbon (PAH) concentrations in an aqueous system are equal to the sum of the organic phase and soluble phase molar concentrations. While the organic phase concentrations are proportional to the PAH mole fraction in the oil, the soluble phase molar concentrations are estimated using Raoult's law. A batch laboratory mixing vessel with a scalable mixing energy was loaded initially at various oil layer thicknesses (0.4-3.2 mm) which correspond to oil surface loadings (40-310 mg/cm2). The vessel was agitated at constant mean shear rates (Gm = 5, 20 s(-1)). Total petroleum hydrocarbon (TPH) samples were taken periodically to estimate the entrainment rate as a function of initial oil layer thickness. TPH concentrations were measured in-situ using a laser scattering instrument (LISST-100) and ex-situ using gravimetric analysis. At a steady-state TPH concentration (>72 h), additional samples were analyzed for PAH concentration using GC/MS analysis. TPH concentrations increased over time according to a first-order kinetic model. Generally, the first-order rate constant and steady-state concentration both increased with increased oil loading and with increased Gm. In addition, measured PAH concentrations correlated well (r2 > 0.96) with those predicted by a partitioning model. These results are useful for assessing the effects of mixing and oil loading conditions on crude oil entrainment and PAH partitioning.