Measuring in vitro biotransformation rates of super hydrophobic chemicals in rat liver s9 fractions using thin-film sorbent-phase dosing

Methods for rapid and cost-effective assessment of the biotransformation potential of very hydrophobic and potentially bioaccumulative chemicals in mammals are urgently needed for the ongoing global evaluation of the environmental behavior of commercial chemicals. We developed and tested a novel solvent-free, thin-film sorbent-phase in vitro dosing system to measure the in vitro biotransformation rates of very hydrophobic chemicals in male Sprague-Dawley rat liver S9 homogenates and compared the rates to those measured by conventional solvent-delivery dosing. The thin-film sorbent-phase dosing system using ethylene vinyl acetate coated vials was developed to eliminate the incomplete dissolution of very hydrophobic substances in largely aqueous liver homogenates, to determine biotransformation rates at low substrate concentrations, to measure the unbound fraction of substrate in solution, and to simplify chemical analysis by avoiding the difficult extraction of test chemicals from complex biological matrices. Biotransformation rates using sorbent-phase dosing were 2-fold greater than those measured using solvent-delivery dosing. Unbound concentrations of very hydrophobic test chemicals were found to decline with increasing S9 and protein concentrations, causing measured biotransformation rates to be independent of S9 or protein concentrations. The results emphasize the importance of specifying both protein content and unbound substrate fraction in the measurement and reporting of in vitro biotransformation rates of very hydrophobic substances, which can be achieved in a thin-film sorbent-phase dosing system.     

Polyethylene devices: passive samplers for measuring dissolved hydrophobic organic compounds in aquatic environments

We demonstrate the use of polyethylene devices (PEDs) for assessing hydrophobic organic compounds (HOCs) in aquatic environments. Like semipermeable membrane devices (SPMDs) and solid-phase microextraction (SPME), PEDs passively accumulate HOCs in proportion to their freely dissolved concentrations. Polyethylene-water partition constants (K(PEW)S) were measured in the laboratory for eight polycyclic aromatic hydrocarbons (PAHs), five polychlorinated biphenyls (PCBs), and one polychlorinated dibenzop-dioxin (PCDD), and these were found to correlate with octanol-water partition constants (K(OW)s; log K(PEW) = 1.13 log K(OW) - 0.86, R2 = 0.89). Temperature and salinity dependencies of K(PEW) values for the HOCs tested were well predicted with excess enthalpies of solution in water and Setschenow constants, respectively. We also showed that standards, impregnated in the PED before deployment, can be used to correct for incomplete equilibrations. Using PEDs, we measured phenanthrene and pyrene at ng/L concentrations and 2,2',5,5'-tetrachlorobiphenyl at pg/L concentrations in Boston Harbor seawater, consistent with our findings using traditional procedures. PEDs are cheap and robust samplers, competent to accomplish in situ, time-averaged passive sampling with fast equilibration times (approximately days) and simplified laboratory analyses.     

Characterization of hydrophobic hypercrosslinked polymer as an adsorbent for removal of chlorinated volatile organic compounds

A hydrophobic hypercrosslinked polymer with poly (4-tert-butylstyrene-styrene-divinylbenzene) matrix (LC-1) was prepared as adsorbent for the removal of volatile organic compounds from gas streams. The content of oxygen-containing functional groups of LC-1 was about one-fourth that of commercial hypercrosslinked polymeric adsorbent (NDA-201). The results of the water vapor adsorption experiment indicated that LC-1 had a more hydrophobic surface than NDA-201. Three chlorinated volatile organic compounds (trichloroethylene, trichloromethane, and 1, 2-dichloroethane) were used to investigate the adsorption characteristics of LC-1 under dry and humid conditions. Equilibrium adsorption data in dry streams showed that LC-1 had good adsorption abilities for three chlorinated VOCs due to its abundant micropore structure. Moreover, the presence of water vapor in the gas stream had negligible effect on breakthrough time of three chlorinated VOCs adsorption onto LC-1 when values of relative humidity were equal to or below 50%; the breakthrough time of three chlorinated VOCs decreased less than 11% even if the relative humidity was 90%. Taken together, it is expected that LC-1 would be a promising adsorbent for the removal of VOCs vapor from the humid gas streams.     

Determination of quantitative food consumption levels for use in microbial risk assessments: cheddar cheese as an example

Microbial risk assessment (MRA) is becoming increasingly used in the management of food safety because it can be used to quantify risks and help rank intervention strategies. The exposure assessment components of the assessments have become complex with many aspects of the contamination, survival, and growth of a pathogen in a food being taken into consideration. Insufficient consumption data constitutes an important data gap and consequently one of many sources of uncertainty in MRA even though the effects of uncertainty are smaller than those affecting bacterial concentration in foods. Therefore, food consumption data also play an important role in exposure assessment of MRA. In the United States, there are large-scale, nationwide sets of consumption data available for use in MRA, i.e., the National Health and Nutrition Examination Survey (NHANES). Newly released dietary interview data in the NHANES 2001 to 2002 survey show that it has been redesigned and that the data were sufficiently updated from previous versions to have more value for MRAs. We propose a model that can effectively use the new data sets and be incorporated into MRAs, using as an example consumption of Cheddar cheese/American-type cheese. This model included the prevalence of food eaten as well as the amount and frequency. We determined the amount of Cheddar/American cheese consumed per day with probability distribution (e.g., lognormal distribution). These could be further determined by gender, age, pregnancy, and combination food type, which we plan to do in the future. The frequency of the range of serving numbers for Cheddar/American cheese consumed per person per day and prevalence as the proportion of a population (e.g., survey respondents) eating a certain food in a day are also presented. Unlike traditional published mean values, the results of this model provide probability distribution intakes that can be compared with mean and median intakes. This allows values in the upper percentiles to be considered for inclusion in MRAs. We believe this simulation model can be adapted with different variables applicable to different foods, pathogens, and specific health risk population groups.     

Direct evidence of sequestration in sediments affecting the bioavailability of hydrophobic organic chemicals to benthic deposit-feeders

In contrast to equilibrium partitioning model (EqP) calculations, biota to sediment accumulation factors (BSAF) of hydrophobic organic compounds for deposit-feeders are highly variable. Recent literature suggests that this variability can be attributed to differences in sequestration or the presence of slowly desorbing fractions in the sediment. In the present study, we investigated whether the observed relationship between bioavailability and sequestration is causal. We determined BSAF values and sequestration status, measured as the distribution over rapidly and slowly desorbing fractions, of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in a manipulated sediment as well as in the original, unmanipulated sediment The manipulation, 48 h suspending with Tenax, resulted in reduction of the rapidly desorbing fraction, while other factors such as contact time and sediment properties remained constant. Contrary to expectations based on EqP, BSAF values did not remain constant but were reduced by a factor of 2-27, proportional to the reduction in rapidly desorbing fractions. The results provide direct evidence of a causal relationship between sequestration and bioavailability to deposit-feeders. Furthermore, the present study demonstrates the need to modify traditional use of the equilibrium partitioning model to account for variation in the sequestration status of HOC in sediments.     

Environmental influences on the partitioning and diffusion of hydrophobic organic contaminants in microbial biofilms

A biofilm reactor was used to investigate kinetic and thermodynamic aspects of the sorption of polycyclic aromatic hydrocarbons (PAH) as model compounds for hydrophobic organic contaminants (HOC) to intact microbial biofilms. Effective diffusion coefficients are in the range of 10(-10) cm2 x s(-1) resulting in equilibration times of more than 3 days for a biofilm of 100 microm thickness. Diffusion in the biofilm was strongly temperature-dependent and increased by a factor of 3 (phenanthrene) to 6 (fluoranthene, pyrene) between 5 and 35 degrees C. Drying and rewetting of the biofilm as well as the inclusion of Ca2+ ions and of humic acids all strengthened the biofilm rigidity and slowed down the diffusion of PAH. The later two factors also influenced the thermodynamics of the process as they supported the partitioning of PAH into the biofilm. Humic acid inclusion from solution into the biofilm illustrates that a microbial biofilm can act as a primer allowing for the buildup of a particulate organic phase from dissolved organic matter. PAH metabolites (3-hydroxy-phenanthrene and 1-hydroxy-2-naphthoic acid) showed lower partition coefficients as compared to their parent compounds and 3-hydroxy-phenanthrene also showed a higher diffusion constant, indicating that these transformation products would be easily released into the water phase upon formation during PAH biodegradation in a biofilm. These results allow the quantification of the influence of environmental conditions on a biofilm's function as a sink or as a diffusion barrier for PAH from aqueous solution, and they indicate the importance of kinetic aspects of this partitioning process.     

Sorption kinetics and microbial biodegradation activity of hydrophobic chemicals in sewage sludge: model and measurements based on free concentrations

In the current study, a new method is introduced with which the rate-limiting factor of biodegradation processes of hydrophobic chemicals in organic and aqueous systems can be determined. The novelty of this approach lies in the combination of a free concentration-based kinetic model with measurements of both free and total concentrations in time. This model includes microbial biodegradation activity of the chemical in the aqueous phase and chemical sorption kinetics with respect to organic carbon and aqueous phases. The time dependency of free and total concentrations of 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene and 7-acetyl-1,1,3,4,4,6-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyrane in activated sludge was experimentally determined in vitro. Evaporation losses from the test system were also determined. Least-squares regression to optimize the model parameters resulted in a model that is in accordance with the experimental data. Additionally, the model shows that a comparison between the decrease of free and total chemical concentrations in time, in combination with an independent measurement of the organic carbon/water partition coefficient provides information aboutthe rate-limiting step of the degradation process. This information can be used by sewage treatment plant managers to decide whether the microbial biodegradation activity itself or the desorption from organic carbon to the aqueous phase should be improved.     

Quantifying the effect of medium composition on the diffusive mass transfer of hydrophobic organic chemicals through unstirred boundary layers

Unstirred boundary layers (UBLs) often act as a bottleneck for the diffusive transport of hydrophobic organic compounds (HOCs) in the environment. Therefore, a microscale technique was developed for quantifying mass transfer through a 100-microm thin UBL, with the medium composition of the UBL as the controllable factor. The model compound fluoranthene had to (1) partition from a contaminated silicone disk (source) into the medium, (2) then diffuse through 100 microm of medium (UBL), and finally (3) partition into a clean silicone layer (sink). The diffusive mass transfer from source to sink was monitored over time by measuring the fluoranthene content of the source and sink disks. The diffusive flux of fluoranthene was slightly higher for air than for water. Cyclodextrin, humic acids, and micelles of sodium dodecyl sulfate (SDS) enhanced the diffusive flux of fluoranthene in water by more than 1 order of magnitude. These results demonstrate that medium constituents, which normally are believed to bind hydrophobic organic chemicals, actually can enhance the diffusive mass transfer of HOCs in the vicinity of a diffusion source (e.g., contaminated soil particles). The technique can be used to evaluate the effect of natural fluids on diffusive mass transfer, as it integrates the different processes, partitioning and diffusion, in one laboratory model.     

Model intercomparison for the uptake of organic chemicals by plants

Currently, a variety of models are available for predicting the uptake, translocation, and elimination of organic contaminants by plants. These models range from simple deterministic risk assessment screening tools to more complex models that consider physical, chemical, and biological processes in a mechanistic manner. This study evaluates the performance of a range of such models and model types against experimental data sets. Three dynamic, three regression-based, and three steady-state and equilibrium models have been selected for evaluation. These models differ in terms of their scope, methodological approach, and complexity. Data from nine published experiments were used to create scenarios to test model performance. These experiments consider plant contamination via both soil and aerial exposure pathways. A total of 19 different organic chemicals were used in the experiments along with 7 different plant species. Model predictions of chemical concentrations in the relevant plant compartments were compared with the experimentally recorded values. The results indicate that dynamic models offer performance advantages for acute exposure durations and for rapidly changing environmental media. Equilibrium/steady-state and regression-based models perform better for chronic exposure durations, where stable conditions are more likely to exist. The selection of an appropriate plant uptake model will therefore be dependent on the requirements of the assessment, the nature of the environmental media, and the duration of the source term. The results generated by the regression-based models suggest that in their current form these models are unsuitable for evaluating the uptake of organic chemicals from the air into plants.     

Characterization of polymer coated glass as a passive air sampler for persistent organic pollutants

The use of thin-film polymer-coated glass surfaces or POGs as passive air samplers was investigated during an uptake experiment in an indoor environment with high levels of gas-phase polychlorinated biphenyls (PCBs). POGs consisted of a micron thick layer of ethylene vinyl acetate (EVA) coated onto glass cylinders. The uptake was initially linear with time and governed by the air-side mass transfer coefficient and surface area of the sampler. This was followed by a curvilinear region and finally a constant phase when equilibrium was established between air and EVA. The high surface area-to-volume ratio of the POGs allowed rapid equilibrium with gas-phase PCBs; equilibration times were on the order of hours for the low molecular weight congeners. The equilibrium concentration was dependent on the EVA-air partition coefficient, K(EVA-A), which was shown to be very well correlated to the octanol-air partition coefficient, K(OA). When POGs of varying thickness were equilibrated with air, the amount of PCB accumulated increased with increasing thickness of the EVA, indicating that uptake was by absorption into the entire polymer matrix. A wind field of 4 m s(-1) resulted in an increased uptake rate by a factor of approximately six compared to uptake in relatively still air. This wind speed effect was diminished, however, when POGs were housed in deployment chambers consisting of inverted stainless steel bowls. Relationships based on the air-side mass transfer coefficient and K(EVA-A) were developed for PCBs that describe the entire uptake profile and allow air concentrations to be determined from the amount of chemical accumulated in the POG. It is believed that these relationships are also valid when POGs are used to detect other classes of persistent organic pollutants.     

Chemical-equilibrium-based model for describing the strength of sludge: taking hydrogen-producing sludge as an example

A new model, based on chemical equilibrium theory, was established to evaluate the strength of sludges in biological wastewater treatment systems. The effectiveness of this model was demonstrated by the experimental results with an anaerobic hydrogen-producing sludge. The equilibrium dispersed mass concentration of the primary particles in the sludge solution was found to nonlinearly increase with the solid content and shear intensity, and could be well described by the model. The Gibbs free energy of adhesion (deltaG degrees) under shear could also be calculated using this model. The equilibrium constant K degrees and deltaG degrees/ RTat a shear intensity of 800 1/s were estimated to be 6.54 +/- 0.12 and 1.88 +/- 0.02, respectively. The two parameters could be used to evaluate the strength of the hydrogen-producing sludge. In addition, the effectiveness of the established model was also confirmed by the results with activated sludge in the literature.     

Soot deposition in the Great Lakes: implications for semi-volatile hydrophobic organic pollutant deposition

Air deposition is a dominant transport mechanism for many hydrophobic organic pollutants (HOCs) to the Great Lakes. Our previous research has shown that soot exhibits large surface areas with high organic carbon contents suggesting the potential for strong HOC partitioning. As yet, however, clear data showing the link between HOCs such as PAHs to the deposition of soot into the Lakes (a proposed transport mechanism) is primarily inferential. We measured soot carbon (SC) and organic carbon (OC) in sediments collected from each of the Laurentian Great Lakes. OC and SC levels collected from locations near urban areas were higher than in sediments collected from distant locations. By far, Lake Superior had the lowest current SC flux of any lake, and Lakes Michigan and Erie had the highest. SC flux for all lakes had the following order: Superior < Huron < Ontario < Michigan < Erie, ranging 0.02-0.89 mg (m2 yr)(-1). Differences in lake size resulted in a different order for total SC loading by lake: Superior < Ontario < Huron < Erie < Michigan, ranging 2.3-420 x 10(3) tyr(-1). SC and PAH accumulation rates reported previously for Lake Michigan sediment were highly correlated; with a SC to PAH mass ratio of 10(4) (0.01%). The importance of soot as a potential sorbent for various classes of airborne HOCs was examined using a simple octanol-air partitioning model together with our previous characterization of soot particles. The results predict that both PAHs and PBDEs should have strong partitioning to soot and suggest the need to further investigate soot as a vector for PBDE transport.     

Kinetic model for studying the conversion and degradation of isoflavones during heating

The conversion and degradation of isoflavones during dry or moist heating at 100, 150 and 200 °C, for varied lengths of time, were kinetically studied. Results showed that, at the early stage, all the reaction rates of malonylgenistin (MG), acetylgenistin (AG), genistin (G), and genistein (Ge) increased with increasing temperature and fitted a first-order model, when the concentration changes during heating were analyzed using HPLC. For dry heating, the conversion of MG to G exhibited the highest rate constant (h⁻¹), followed by MG to AG, AG to G, AG to Ge, G to Ge and MG to Ge. Moist heating showed the same phenomenon; however, the last three conversions were not observed. In addition, MG had the highest degradation rate, followed by G, Ge and AG during dry heating, while the reversed trend occurred for moist heating. Moist heating was more susceptible to conversion and degradation of isoflavones than dry heating. The correlation coefficients (r²) ranged from 0.664 to 0.987 for moist heating and 0.688–0.960 for dry heating. The kinetic model developed in this study can be used to predict the concentration changes of isoflavones during dry heating and moist heating.     

Importance of adsorption (hole-filling) mechanism for hydrophobic organic contaminants on an aquifer kerogen isolate

Sorption and desorption behaviors of four hydrophobic organic compounds (HOCs) were investigated for an isolated kerogen material from Borden aquifer material with total organic carbon of 0.021%. The solubility-normalized modified Freundlich equation and the combined linear and Polanyi-Dubinin (PD) equation can quite well describe the sorption or desorption isotherms. The partition component is estimated and compared using desorption data, dual-mode modeling, and the reported partition coefficients. The result suggests that the dual-mode modeling and the combined linear and PD modeling may overestimate the partitioning component. The partition component is not so important as assumed before in sorption of HOCs for the studied sorbent. As the fitted PD equation has an exponent parameter b' approaching 1, it is equivalent to the modified Freundlich equation. The small molecules 1,2-dichlorobenzene (DCB) and naphthalene (Naph) have higher adsorption volumes. The lower adsorption volumes for 1,3,5-trichlorobenzene (TCB) and phenanthrene (Phen) suggest that accessibility to the holes of kerogen by large HOC molecules is reduced. The desorption hysteresis is approximately constant for DCB when the relative aqueous concentration ranges from 0.0007 to 0.6, but for Phen is only obvious at higher relative aqueous concentrations. The varied sorption and desorption behaviors for DCB and Phen are satisfactorily explained by an adsorption/ hole filling mechanism and entrapment of some adsorbates in the kerogen matrix and by possible pore deformation mechanism at high concentrations.     

Rumen microbial degradation of allyl cyanide as a possible explanation for the tolerance of sheep to brassica-derived glucosinolates

The possible role of allyl cyanide (ACN), the nitrile breakdown product of prop-2-enyl glucosinolate, in reducing the voluntary food intake (VFI) of a dried grass pellet diet in sheep was examined in a ruminal infusion experiment. Voluntary food intake of Scottish Blackface wether lambs, infused intraruminally with allyl cyanide (0 mmol day⁻¹, C; 4.8 mmol day⁻¹ L; 9.6 mmol day⁻¹, H) for 9 weeks was significantly depressed (P < 0.05) on H and L treatments compared with the C treatment. Clinical indicators of liver function (plasma gamma glutamyl transpeptidase (GGTP) activity) and kidney function (plasma urea and creatinc concentrations) indicated minor liver damage but no effects on renal function. Plasma GGTP activity was significantly (P < 0.05) higher in the H than in the C group. Liver cytochrome oxidase activity at the end of the infusion period was significantly lower (P < 0.05) in the H than in the C group indicating effects on cellular respiration. In general, sheep showed considerable tolerance to administration of ACN in comparison with previously demonstrated toxicity in monogastrics.     

Potential of degradable organic chemicals for absolute and relative enrichment in the Arctic

Model simulations of the fate of numerous hypothetical substances in the global environment can provide considerable insight into how an organic chemical's degradability and partitioning properties influence its absolute and relative Arctic enrichment behavior, as quantified by the Arctic Contamination Potential. For substances that degrade faster in water than in soil, but are quite persistent in the atmosphere, highest Arctic contamination is expected to occur if the substances have intermediate volatility and high hydrophobicity. Organic substances that are degradable in the atmosphere can still accumulate in the Arctic if they are soluble and highly persistent in water. These latter substances, which reach the Arctic in the ocean, also show the highest potential for relative enrichment in the Arctic, i.e., high amounts in northern high latitudes relative to the amounts in the total global environment. Beyond a threshold persistence in surface media of the order of several months to a year, chemical degradability leads to further relative enrichment. This is because only chemicals that are sufficiently long-lived get transferred to polar regions and once there can persist longer than at lower latitudes. The model simulations can inform the search for new potential Arctic contaminants, and can highlight combinations of properties which should be avoided in high production volume chemicals with the potential for environmental release. Three categories of organic substances are singled out for troublesome combinations of persistence, distribution, and potential bioaccumulation characteristics, only one of which contains "classical" Arctic POPs. Examples of potential Arctic contaminants within each of these categories are named.     

A modified parallel artificial membrane permeability assay for evaluating the bioconcentration of highly hydrophobic chemicals in fish

Low cost in vitro tools are needed at the screening stage of assessment of bioaccumulation potential of new and existing chemicals because the number of chemical substances that needs to be tested highly exceeds the capacity of in vivo bioconcentration tests. Thus, the parallel artificial membrane permeability assay (PAMPA) system was modified to predict passive uptake/ elimination rate in fish. To overcome the difficulties associated with low aqueous solubility and high membrane affinity of highly hydrophobic chemicals, we measured the rate of permeation from the donor poly(dimethylsiloxane)(PDMS) disk to the acceptor PDMS disk through aqueous and PDMS membrane boundary layers and term the modified PAMPA system "PDMS-PAMPA". Twenty chemicals were selected for validation of PDMS-PAMPA. The measured permeability is proportional to the passive elimination rate constant in fish and was used to predict the "minimum" in vivo elimination rate constant. The in vivo data were very close to predicted values except for a few polar chemicals and metabolically active chemicals, such as pyrene and benzo[a]pyrene. Thus, PDMS-PAMPA can be an appropriate in vitro system for nonmetabolizable chemicals. Combination with metabolic clearance rates using a battery of metabolic degradation assays would enhance the applicability for metabolizable chemicals.     

Disruption of the developing female reproductive system by phytoestrogens: genistein as an example

Studies in our laboratory have shown that exposure to genistein causes deleterious effects on the developing female reproductive system. Mice treated neonatally on days 1-5 by subcutaneous injection of genistein (0.5-50 mg/kg) exhibited altered ovarian differentiation leading to multioocyte follicles (MOFs) at 2 months of age. Ovarian function and estrous cyclicity were also disrupted by neonatal exposure to genistein with increasing severity observed over time. Reduced fertility was observed in mice treated with genistein (0.5, 5, or 25 mg/kg) and infertility was observed at 50 mg/kg. Mammary gland and behavioral endpoints were also affected by neonatal genistein treatment. Further, transgenerational effects were observed; female offspring obtained from breeding genistein treated females (25 mg/kg) to control males had increased MOFs. Thus, neonatal treatment with genistein at environmentally relevant doses caused adverse consequences on female development which is manifested in adulthood. Whether adverse effects occur in human infants exposed to soy-based products such as soy infant formulas is unknown but the neonatal murine model may help address some of the current uncertainties since we have shown that many effects obtained from feeding genistin, the glycosolated form of genistein found in soy formula, are similar to those obtained from injecting genistein.