P124 (ERH-1117-738835) Interspecies Comparisons of Metabolism using Liver S9 Fractions.
Start time: 8:00 AM
Erhardt, S¹, Weisbrod, A², Roe, A³, ¹ The Dow Chemical Company, Toxicology and Environmental Testing and Consulting, Midland, MI, USA² Procter & Gamble, Central Product Safety - Environmental, Cincinnati, OH, USA³ Procter & Gamble Pharmaceuticals, Drug Safety Assessment, Cincinnati, OH, USA
An international industry, government and academic consortium has undertaken to develop and validate a tiered series of methods that can be used to generate absorption, distribution, metabolism, and excretion (ADME) data for use in predicting the bioaccumulation (B) potential of chemicals in aquatic organisms. Methodologies to examine the metabolism of xenobiotics include in vitro approaches utilizing subcelluar fractions of fish liver. Characterization of the metabolism of standard substrates and commercial substances by fish liver S9 preparations is underway as a means to generate metabolism data for application to B models and assessments. Our fish studies are based on standardized in vitro methods used to assess metabolic activity in mammalian systems such as rat, dog and human. In the process of characterizing enzyme activity levels in fish S9, we are attempting to standardize the incubation conditions e.g., incubation temperature, and protein and substrate concentration. An understanding of the types of enzymatic activity in trout and carp, relative to better characterized mammalian systems, is expected to allow for interspecies extrapolation and ultimately the use of existing data for modeling aquatic bioaccumulation. Interspecies comparisons will be presented with regards to types of enzymatic systems, levels of activity, and transformation processes detected in S9 fractions. Suggestions on how to proceed with interspecies extrapolation will also be presented.

P125 (GUN-1117-815203) Bioavailability assessment of organic contaminants in limnic sediments using in vitro gut fluid extraction.
Start time: 8:00 AM
Gunnarsson, J.S.G¹, Kukkonen, J.V.K², Weston, D.P.², Landrum, P.F.³, ¹ Stockholm University, Sweden² University of Joensuu, Finland³ University of California Berkeley, CA, USA
The objective of this study was to measure the bioavailability of sediment-associated organic contaminants with a recent in vitro technique, that uses natural gut fluid collected from a polychaete worm, and to compare it to the in vivobioaccumulation kinetics by Lumbriculus variegatus and Diporeia spp. Seven limnic sediments of different organic carbon contents and geochemical characteristics were collected from the Great Lakes and spiked with four radiolabeled organic contaminants: benzo(a)pyrene, pyrene, 2,2',4,4',5,5'-hexachlorobiphenyl and 3,4,3',4'-tetrachlorobiphenyl. Sediment chemical characterization was done by measurement of total organic carbon (OC), organic nitrogen, soot carbon, particle size distribution, phytopigments, total and bioavailable amino acids, and NaOH extractable OC. In vitro gut fluid extraction was done by incubating the contaminated sediments for 3h with gut fluid isolated from the polychaete Arenicola brasiliensis and then measuring the fraction of contaminant solubilized in the fluid. Correlation tests were done between gut fluid extraction efficiency (GFEE) and bioaccumulation factors (BAF), (BSAF), uptake clearance rates, sediment characteristics, and desorption measurements using Tenax extractions. The gut fluid extraction was correlated to the desorption rate of the compounds and to the organic carbon normalized uptake clearance rate. The gut fluid extractions were positively correlated to the BAF of all contaminants except for BaP, showing that this relatively simple and rapid technique, was in most cases an excellent predictor of the fraction of sediment-contaminant that was bioavailable to Lumbriculus variegatus and Diporeia spp.

P126 (STE-1117-848645) A model describing the bioaccumulation and metabolism of polycyclic aromatic hydrocarbons in a marine benthic food web.
Start time: 8:00 AM
Stevenson, R¹, Gobas, F², ¹ Environment Canada - Pacific and Yukon Region, Vancouver, BC, Canada² School of Resource and Environmental Management, Simon Fraser University, Burnaby, BC, Canada
This research describes a model of PAH bioaccumulation and metabolism in marine benthic organisms of the coastal Northeast Pacific Ocean. PAHs are known to persist in marine sediments, cause adverse effects to marine organisms, and cause carcinogenic effects in fish, mammals and humans. Despite their hydrophobicity, metabolic transformation is conventionally expected to limit food chain biomagnification of PAHs. However, metabolic transformation produces reactive Phase I metabolites, which are the primary cause of PAH-related carcinogenesis and other effects such as endocrine disruption. To assess the effect of metabolic transformation on food-web bioaccumulation of PAHs and on potential toxic effects resulting from Phase I metabolite formation, we developed and field tested a model of the uptake, elimination and metabolic transformation of PAHs and non-metabolized PCBs in Dungeness crabs (Cancer magister) from two estuaries. The model characterized the mass balance of PAHs and metabolites and accounted for decreased bioavailability due to soot-association of pyrogenic PAHs. Estimation of PAH metabolic rates involved both derivation from literature studies of metabolic transformation, and deduction using model sensitivity analysis, with reasonable agreement between these approaches. Application of estimated metabolic rates results in an accurate representation of observed BSAFs for Dungeness crabs in False Creek, where PAHs are primarily petrogenic, and in Kitimat Arm, where PAHs are primarily pyrogenic. Model behavior indicates trophic dilution of parent PAH body burdens in predatory organisms as a result of metabolic transformation within organisms and their prey species. Modeled metabolite body burdens also undergo trophic dilution, and are primarily the result of metabolite production within organisms rather than uptake from prey. The model represents a synthesis of the current knowledge of PAH bioaccumulation and metabolism and has application for predicting PAH and metabolite exposure in risk assessments for benthic organisms, and their predators such as pelagic fishes, marine mammals, and humans.

P127 (LIL-1117-817457) Proposals for the implementation of the 3Rs in the assessment of a chemical's potential to bioconcentrate or cause acute aquatic toxicity.
Start time: 8:00 AM
Lillicrap, A¹, Comber, M², De Wolf, W³, Douben, P⁴, Gimeno, S⁵, Leonard, M⁶, Stevens, C⁷, van Egmond, R⁴, Whale, G⁸, Holt, M⁹, ¹ Astrazeneca, Brixham, Devon, England² Exxon-mobil, Brussels, Belgium³ Dupont, Brussels, Belgium⁴ Unilever, Colworth, England⁵ Procter and Gamble, Brussels, Belgium⁶ L'Oreal, Paris, France⁷ Dow corning, Brussels, Belgium⁸ Shell, Cheshire, England⁹ ECETOC, Brussels, Belgium
REACH is the proposed new European Union legislation for the registration, evaluation and authorisation of chemicals. This legislation will require information for over 30 000 chemicals, while respecting the 3Rs of animal testing, reduce, refine and replace. This paper addresses alternative approaches, proposed by an ECETOC taskforce, for the generation of hazard data used in risk assessments to determine acute aquatic toxicity or bioconcentration. Currently, for acute aquatic toxicity, lethality is traditionally used as the endpoint and a minimum of 42 fish are routinely required for each assessment (OECD 203). In terms of indirect effects, bioconcentration factors (BCF) are used to assess the potential of a chemical to bioconcentrate and a minimum of 108 fish may be required for each assessment (OECD 305). Many of the existing hazard assessment approaches, used to assess bioconcentration, apply simple numerical cut-offs, derived from log Kow-BCF models based on non-ionic, non-metabolisable chemicals. The approach presented here describes how the use of information derived from mammalian studies, QSARs, alternative lifestages, non-vertebrate organisms and in-vitro experiments can be used to re-design studies for assessing a chemical's potential to either cause acute aquatic toxicity or bioconcentration. Consequently, two testing strategies have been developed which aim to minimise or avoid the need for fish in the risk assessment of chemicals or whole effluent assessments. The findings from a recent workshop, organised by the Task Force, attended by NGOs, regulators, academics and industry members are also presented herein. Finally the opportunities for future research have been identified along with the recommended projects needed to strengthen these approaches.

P128 (HOW-1117-814929) Improved BCF prediction for hydrocarbons.
Start time: 8:00 AM
Stewart, Sarah¹, Meylan, William¹, Aronson, Dallas¹, Howard, Philip¹, Parkerton, Thomas², Comber, Michael³, ¹ Syracuse Research Corporation, Syracuse, NY, USA² ExxonMobil Biomedical Sciences, Inc., Annandale, NJ, USA³ ExxonMobil, Machelen, Belgium
Bioconcentration factor (BCF) data on hydrocarbons that were used to develop the BCFWIN program within the EPI Suite were reevaluated. To improve the accuracy of the database, an extensive screening of the available literature was performed. In addition, the methodology of all studies (those used previously as well as any new studies) was examined, and only studies in which the parent compound was measured directly were included. Since hydrocarbons can be biotransformed within the organism, as is commonly reported for polycyclic aromatic hydrocarbons (PAHs), total radioactivity measurements can include metabolites as well as the parent compound. The updated database contains BCF data for 80 hydrocarbons, which were then compared to their BCFWIN estimation values. Of these 80 compounds, 49 were overestimated and 31 were underestimated (21 compounds were overestimated by more than 0.5 log units and 8 compounds were underestimated by more than 0.5 log units). Specific structural classes found to have BCF values significantly lower than their estimated value include PAH compounds with four or more fused benzene rings (overestimated by an average of approximately 1.5 log units) and linear aliphatic compounds containing 12 or more carbons (overestimated by an average of approximately 0.7 log units). Simple naphthalenes and other two-member ring compounds (e.g., decahydronaphthalene, tetralin) appear to be somewhat underestimated by the BCFWIN program (by approximately 0.65 log units).

P129 (SPR-1117-831492) A Modified Approach to Aquatic Bioconcentration Factor (BCF) Tests Using Reduced Sampling.
Start time: 8:00 AM
Springer, T¹, Krueger, H¹, Jaber, M¹, Guiney, P², ¹ Wildlife International, Ltd., Easton, MD, USA² S.C. Johnson & Sons, Inc., Racine, WI, USA
Recent regulatory initiatives have created a need for more efficient methods for estimating the bioconcentration factor (BCF) of chemicals in fish. We present simulation and study results demonstrating that BCF estimates can be made reliably using an approach that reduces study complexity, analytical costs, and animal use compared to standard bioconcentration tests. The modified test is similar in some respects to standard designs. However, tissue samples are taken only during the depuration period, and at only two or three points in time. The approach is focused on estimating the BCF using the kinetic definition (i.e. BCF = K_uptake/K_depuration) and does not require that chemical concentrations in water and tissue are at equilibrium. The performance of the modified test design was assessed by comparing BCF estimates obtained in a series of standard guideline tests to BCF estimates that would have been obtained had the study been performed using the modified approach. Modified test results were estimated by resampling from the uptake and depuration data. Data was drawn from eight full bioconcentration studies performed in three different laboratories, using a total of 19 bioconcentration curves (multiple curves for nonedible, edible, whole fish portions and different concentrations). BCF estimates based on the curves studied ranged from about 0.3 to over 20,000. The correlation between log (BCF) estimates from the modified test and log (BCF) estimated from the full data set analyzed using the standard approach was R² = 0.99825 with slope = 0.97561. Subsequently, a study was performed using the modified study design. The BCF value determined using the modified kinetic method was nearly identical to the standard BCF value determineded from the ratio of the peak concentration of the chemical in tissue to the concentration of the chemical in the water (i.e. assuming equilibrium), suggesting that both BCF estimates were reasonable. The available evidence suggest that the modified kinetic approach offers significant advantages over standard BCF tests in use of resources with little sacrifice in precision and predictive power.

P130 (DYE-1117-806668) Use of fish hepatocyte suspensions to estimate bioconcentration.
Start time: 8:00 AM
Dyer, S¹, Bernhard, M¹, ¹ The Procter and Gamble Company, Cincinnati, Ohio, USA
The rate of biotransformation is a dominant factor in determining the extent to which chemicals may bioconcentrate. Biotransformation can be measured in vitro via studies with cellular or subcellular preparations. An advantage of in vitro systems is that they are relatively inexpensive compared to in vivo tests. We have developed a plated cell hepatocyte test that has allowed the determination of parent molecule uptake and subsequent loss from the cell due to metabolism via a first-order rate formation and decay model. We term the ratio of the uptake and loss via metabolism as the BCFcell. BCFcell data have been developed for several radiolabeled chemicals. Future tests, however, will likely require cold-analytical methods. To meet this need, we have developed a suspension-based system that allows greater flexibility in exposure regimes. For instance, numbers of cells per volume, total volume, regulation of head-space for volatile chemicals are among the many regimes that can be altered with suspensions. Direct comparisons of plated vs. suspension-based rates and BCFcell data for linear alkyl benzene sulfonate and alcohol ethoxylate surfactants will be presented.

P131 (COO-1116-473148) Algal bioaccumulation of antimicrobials in a North Texas wastewater treatment plant (WWTP) receiving stream.
Start time: 8:00 AM
Coogan, M¹, Edziyie, R¹, La Point, T¹, Venables, B¹, ¹ University of North Texas, Denton, TX, USA
Algae comprise the greatest abundance of plant biomass in aquatic environments and are a logical choice for aquatic toxicological studies, yet have been underrepresented in this capacity. The lipid content of many algal species provides a point of entry for trophic transfer of lipophilic organic contaminants. Triclosan (TCS) and triclocarban (TCC), widely used antimicrobial agents found in numerous consumer products, are incompletely removed by WWTP processing. Methyl triclosan (M-TCS) is a metabolite of TCS more lipophilic than the parent compound. The focus of this study is to quantify bioaccumulation factors (BAFs) for TCS, M-TCS, and TCC in Pecan Creek, the receiving stream for the City of Denton, Texas WWTP. Filamentous algae (Cladophora spp.) and water column samples were collected from the WWTP outfall, an upstream site, and two downstream sites. Water samples were extracted with C18 solid phase extraction cartridges. Algae samples were Soxhlet extracted and a fraction containing the antimicrobial compounds was isolated by gel permeation chromatography. Final analysis of TCS and M-TCS was by selected ion monitoring using GC/MS. TCC was analyzed by negative-ion electrospray LC/MS. Antimicrobials in Pecan Creek water samples taken at and downstream from the WWTP are at low ppt concentrations of 10 - 200 ng/L and are elevated to low ppb concentrations of 1 - 200 ng/g fresh weight in algae collected from these stations. The resulting BAFs are approximately three orders of magnitude.

P132 (WIG-1117-574939) Cadmium exposure effects on tissue metal concentrations in two tolerant and two sensitive crayfish species.
Start time: 8:00 AM
Wigginton, A¹, Price, D¹, Birge, W¹, ¹ University of Kentucky, Lexington, KY, USA
Four 96h acute toxicity tests were performed with the crayfish O. juvenilis, O. placidus, P.acutus, and P. clarkii with various concentrations of cadmium and control groups. O. placidus and P. acutus were more sensitive with LC50 values of 0.487 and 0.368 mg Cd/L, while O. juvenilis and P. clarkii were more tolerant with LC50 values of 2.44 and 2.66 mg Cd/L, respectively. Tissue samples, including gills, green glands, hemolymph, hepatopancreas, muscles, ovaries, exoskeletal and remaining tissues, and testes, were dissected from survivors and analyzed for their content of Ca, Cd, Cu, Fe, Mg, P, Sr, Zn and various other elements. Whole body elemental content (mg/kg) values were derived from the preceding. In most tissues, cadmium tissue content correlated strongly with cadmium exposure. Cd tissue residues were compared to exposure concentrations to calculate 96h BCFs. Regression analysis was used to derive equations estimating the curve of 96h BCFs to allow for better comparisons between species. Cd accumulated more strongly in the hepatopancreas, gills and green glands of sensitive species than in tolerant species. These data seem to indicate that a fundamental difference exists between how sensitive and tolerant crayfish species either take in or excrete heavy metals. Zn tended to show negative correlations with Cd exposure in the hepatopancreas. Cu showed several strong trends versus Cd tissue concentrations. Cu increased in green glands, gills, and hemolymph and, in some cases, decreased in the hepatopancreas. Ca, Fe, Mg, and Zn also showed significant trends. These data indicate that Cd may displace Cu and/or Zn in the hepatopancreas and the displaced metal then may move into other tissues, especially the gills and green glands, possibly to be excreted.

P133 (KWO-1117-750273) Use of a parallel artificial membrane system to evaluate passive uptake and elimination in small fish.
Start time: 8:00 AM
Kwon, J.-H.¹, Katz, L.¹, Liljestrand, H.¹, ¹ The University of Texas at Austin, Austin, Texas, Unites States
A parallel artificial membrane system was developed to mimic passive mass transfer of hydrophobic organic chemicals in fish. Two aqueous phases were separated by stable lipid bilayers supported by a porous polyvinylidine difluoride (PVDF) membrane (0.1 m pore diameter) containing dodecane. This model system comprised of outer aqueous environment, lipid membrane barrier, and cellular aqueous phase mimics the transport steps involved in partitioning of hydrophobic organic chemicals to fish. The artificial membrane system was optimized to mimic bioconcentration kinetics in small fish by controlling mass transfer resistances because the mass transfer of hydrophobic organic chemicals in fish is determined both by the lipid membrane resistance and by the aqueous resistance, depending on the pollutants′ affinity to biological membranes. The thickness of the aqueous diffusion boundary layer was adjusted by altering mixing speed. The characteristic length of the diffusion boundary layer in the artificial system was measured using a pKa-flux method for ionizable chemicals. Chlorinated aromatic hydrocarbons were chosen as model compounds for purposes of evaluation. Measured absorption rate constants, elimination rate constants and partition coefficients between the membrane and the aqueous phases are compared to those for small fish published in the literature. Characteristics of aqueous and membrane boundary layer in the artificial system are compared to those for small fish calculated by a simple diffusion model using literature data. The results suggest that the artificial system provides reasonable estimate of uptake and elimination rate constants.

P134 (DIM-1117-815796) Applicability Domain of Base Line BCF Model and Quality of Predictions. Screening of Chemicals on DSL.
Start time: 8:00 AM
Mekenyan, O¹, Dimitrov, S¹, Dimitrova, N¹, Stoyanova, G¹, MacDonald, D², Morin, D², ¹ Laboratory of Mathematical Chemistry, University “Prof. As. Zlatarov”, Bourgas, Bulgaria² Existing Substances Branch, Environment Canada, Place Vincent Massey, 351 St. Joseph Boulevard, Gatineau, Quebec, Canada
According to base-line concept, the bioaccumulation is defined as a combined effect of passive diffusion (maximum bioaccumulation described by multicompartment partitioning model) and set of mitigating factors associated with bioavailability (size, ionization) and the effect of organism (metabolism). The maximum diameter of molecules is used as a size parameter and the model accounts for their conformational flexibility. A simulator of fish liver metabolism is used to model the metabolic transformations of chemicals. The metabolic pathways and transformation products are predicted explicitly. The thresholds for molecular size, weight of different ionizing groups and metabolic transformation probabilities are parameterized by non-linear least square method to reproduce BCF values and metabolism of 515 chemicals. The performance of the base-line BCF model to reproduce BCF data (R² = 0.84) and metabolism (R² = 0.80) for the training sets was an indication for its usefulness for screening purposes. To ascertain whether the model is applicable or not to a query chemical submitted for screening we have defined its applicability domain. The structural domain of the present BCF model is defined by extracting atom centered fragments from correctly predicted chemicals in training sets. The software implementation of the model provides screening for all chemicals specifying those which are in the model domain. For 59 MITI chemicals identified to belong to the model applicability domain the predictability was 80%. Presently, the model is used to screen the chemicals on DSL. The applicability domain of the current model covers 12% of the structural diversity of DSL inventory. Work on expending model applicability domain of the present model is in progress.

P135 (GHO-1126-553855) PCB bioavailability control in Lumbriculus variegatus under different modes of activated carbon addition to sediments.
Start time: 8:00 AM
Sun, X¹, Ghosh, U¹, ¹ University of Maryland Baltimore County, Department of Civil and Environmental Engineering
This research investigated the effect of activated carbon addition to river sediments on PCB bioaccumulation by the freshwater oligochaete Lumbriculus variegatus. The sediments from a PCB-impacted river were treated with 2.6% activated carbon. The results showed that PCB bioaccumulation in L. variegatus exposed to treated sediments was greatly reduced. For the sediments treated with granular activated carbon and mixed for 2 minutes prior to worm addition, the reduction in total PCB biouptake was 74% for 50-200 mesh TOG carbon, and 94% for the 80-325 mesh TOG carbon, respectively, which indicated that there was an immediate effect of PCB bioavailability reduction upon the addition and short-term mixing of GAC into sediments. The finer grain size activated carbon, with more easily accessible sorption sites, was much more effective in reducing PCB bioaccumulation in the L. variegatus. For the case where the GAC was placed as a thin layer on top of the sediments, the reduction in total PCB uptake was 72%. Although the total PCB biouptake reduction with the layer placement was similar to the case where the same dose and type of carbon was mixed in, the percent reductions of the different PCB homolog groups were different.