538 (SAU-1117-774576) Toxicity Interactions of Cadmium, Copper and Lead on Urease and Dehydrogenase Activity in Soils.
Start time: 1:50 PM
Chaperon, S¹, Sauvé, S¹, ¹ Université de Montréal, Montréal, QC, Canada
Soil quality criteria for contaminated sites are presently based on the toxicity observed from single pollutants. However, most contaminated soils are exposed to many chemicals simultaneously. Few data are published on the toxicity of pollutant mixtures in soils, even fewer looking at metal mixtures. To better understand the real toxicity encountered in soils, the effects of speciation and interactions between components must be known. In this study, the toxicity of single metals (Pb, Cd, Cu) and their mixtures on microbial enzyme processes such as urease and dehydrogenase were determined. A forest soil was spiked with CdCl2 (1, 2, 10 and 20 mg Cd kg-1 dry soil), PbCl2 (100, 500, 1000 and 2000 mg Pb kg-1 dry soil) and CuCl2 (50, 250, 500 and 1000 mg Cu kg-1dry soil). We also used a factorial set up to contaminate soils using combinations of the two higher concentrations of each metal. We have chosen to use urease and dehydrogenase activities because of their relative sensitivity and stability. Preliminary results indicate that the measured toxicity of combined metals on microbial activity is less than additive. For example, urease showed an inhibition of 8% for 2000 mg Pb kg-1 and 48% for 10 mg Cd kg-1. The additive model would predict an addition of the toxicity of each component without any interaction. According to this model, the combination of the two metals should present an inhibition of 56%. However, in this case a stimulation of 51% is observed. This trend was observed for both urease and dehydrogenase activities at each concentration. The combinations of Pb, Cu and Cd have important, yet hard to predict, effects on microbial activities. The toxicity observed for those two enzyme assays does not follow a simple additive model.

539 (RED-1117-826580) Mechanistic fate and effects model to predict the aquatic toxicity of complex petroleum products in laboratory tests.
Start time: 2:10 PM
Redman, A¹, McGrath, J¹, Parkerton, T², Di Toro, D^{1, 3}, ¹ HydroQual, Inc.² ExxonMobil³ University of Delaware
A modified narcosis target lipid model (NTLM) was used to predict the toxicity of water accommodated fractions (WAFs) prepared from eight gas oils and three kerosenes to three different species, an algae (Selenastrum capricornutum), a fish (Oncorhynchus mykiss) and a daphnid (Daphnia magna). Petroleum products are complex substances that are comprised of mixtures of hydrocarbons that upon dissolution into the aqueous phase are expected to act via narcosis. The NTLM has been modified by expressing aquatic toxicity of petroleum products based on membrane-water partitioning (K_mw) rather than octanol-water partitioning (K_ow). This is required because it has been shown that K_ow is not a suitable descriptor for the partitioning that occurs across an organism membrane for higher molecular weight compounds with log(K_ow) greater than approximately 5.5. At a log(K_ow) greater than 5.5, the log(K_mw )-log(K_ow) relationships deviates from linearity. Aquatic toxicity data were obtained using a lethal loading test procedure in which WAFs were prepared at different product loadings. The compositions of a given petroleum product were based on reported boiling point ranges and other available compositional information (e.g. % aromatic and aliphatic hydrocarbons). A three-phase (oil, water, air) fate model was developed to compute the WAF composition of aromatic and aliphatic hydrocarbons in each boiling point range based on product composition and loading. The model accounts for the change in the volume of the product phase that occurs during WAF preparation, which varies depending on product loading as well as volatilization into the headspace (of sealed systems). Toxicity calculations using solely log(K_ow) resulted in calculated median lethal loadings (LL50) an order of magnitude or more lower than observed LL50s. Toxicity predictions were greatly improved by using K_mw instead of K_ow for compounds with log(K_ow) above 5.5 highlighting the importance of adopting K_mw as the preferred descriptor for assessing narcotic effects of high K_ow hydrocarbons.

540 (LIN-1117-836728) Additive and non-additive interactions of binary xenoestrogen mixtures in zebrafish (Danio rerio).
Start time: 2:30 PM
Lin, L¹, Janz, D^{1, 2}, ¹ Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada² Veterinary Biomedical Science, Saskatoon, SK, Canada
This study evaluated effects of binary mixtures of two common xenoestrogens on sex ratios, vitellogenin (Vtg) induction, heat shock protein 70 (hsp70) expression, gonadal cell death, and breeding success in a partial lifecycle test with zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post - hatch (dph) to environmentally relevant concentrations of 17 - ethinylestradiol (EE₂) (1 or 10 ng/l nominal; EE1 or EE10), 4 - nonylphenol (NP) (10 or 100 g/l nominal; NP10 or NP100), binary combinations of EE₂ and NP (EE1+NP10, EE1+NP100, EE10+NP10, or EE10+NP100) or solvent control (acetone; 0.1% v/v) in a static-renewal system with replacement every 48h. At 60 dph, 40 fish from each treatment were euthanized for analysis, while the remainder were raised in clean water for reproductive studies. Significant increases in weight-at-length of treatments EE10, NP100, EE1+NP10, EE10+NP10 and EE10+NP100 were observed when compared to the control. Delayed gametogenesis in males and females in EE10, EE10+NP10 and EE10+NP100 were observed, while no testicular tissues were detected in groups EE1, EE1+NP10 and EE1+NP100. Significant decreases in total number of eggs collected from treatments EE10 and EE10+NP100 were observed when compared to solvent controls. No eggs were produced from EE10+NP10. Hatchability of eggs was significantly lower in EE10 when compared to the control. There were no significant differences in whole-body hsp70 expression among treatments. Analysis of gonadal cell death will be performed to determine any potential correlation with hsp70 expression. Overall, our data suggest the effects of EE₂ and NP are additive up to a threshold level of EE10+NP10, while at EE10+NP100, NP appeared to antagonize the action of EE₂.

541 (SCH-1118-074979) Understanding mine effluent toxicity: beyond the usual suspects.
Start time: 2:50 PM
Schwartz, M¹, Vigneault, B¹, Gosselin, I¹, Beyak, J¹, McGeer, J¹, ¹ Mining and Mineral Sciences Laboratories, Natural Resources Canada, Ottawa, Ontario, Canada
While wastewater discharge is usually regulated for a limited number of chemical parameters, there are increasing requirements related to whole-effluent toxicity. An example is the Canadian Metal Mine Effluent Regulations (MMER) which require that mines asses both acute toxicity (rainbow trout and Daphnia magna) and sublethal toxicity (Fathead minnows, Ceriodaphnia dubia, Lemna minor and Selenastrum capricornutum). Previously we have reported on the toxicology of metals in effluents and the extension of biotic ligand modeling to predict acute and chronic impacts. The development of these prediction models is complicated by the fact that mine effluents are complex mixtures. As well, although metals are generally considered as the primary source of potential toxicity, in some cases the observed toxicity cannot be explained by metals alone. There is little toxicological information about other effluent constituents such as process reagents, by-products and wastewater treatment chemicals. Effluent components we have tested include xanthates, sulfur oxyanions (thiosalts), flocculants and elevated hardness. Focusing on MMER test species, we found that organisms respond very differently to such substances, for example algae growth is quite tolerant of thiosalts but sensitive to flocculants while invertebrate reproduction is sensitive to both. Elevated calcium concentrations, due to liming, while not lethal to Ceriodaphnia dubia, dramatically inhibit reproduction. Despite high dosages used or elevated discharge concentrations, most of the substances tested have lethal or inhibition concentrations in the low mg/L range for at least one of the species used. Finally, risk assessment for the discharge of these substances is often limited by the lack of analytical methods to measure concentrations in waste and receiving waters.

(59359) Break.
Start time: 3:10 PM

542 (WAN-1117-228146) Mosquitofish as a Sensitive Bioindicator for Toxicity of Biotoxin Mixtures.
Start time: 3:50 PM
Wang, J-S¹, Billam, M¹, Tang, L¹, McKean, C¹, Wang, Z¹, Wang, P¹, Theodorakis, C¹, Kendall, R¹, ¹ Department of Toxicology and The Institute of Environmental and Human Health, Texas Tech University, Lubbock, Texas, USA
Biotoxins are naturally toxic agents produced by bacterial, cyanobacteria, fungi, and some species of plants and marine fish. Biotoxins and their mixtures have been recognized as potent health threats and possible terrorist agents in recent years, however, development of animal models to evaluate the combinative toxic effects of biotoxin mixtures is a challenge in the field of toxicology. In this study, acute and combinative toxicity of biotoxin mixtures containing aflatoxin B₁, T–2 toxin, microcystin–LR, and fumonisin B₁, were assessed in mosquitofish (Gambusia affinis). Preliminary experiments were conducted in 6–months old fish to determine dosing protocol for each studied biotoxin and obtain LC₅₀ values for individual toxins. This was followed by testing various combinations of selected biotoxins to obtain LC₅₀ values in the model. Significant dose responses were noted within observed toxic parameters to treatment. The potency of the various mixtures was gauged through the determination of the combinative toxicity index (interaction index) metric. Overall results demonstrated that mosquitofish is a sensitive bioindicator for testing toxicity of these biotoxin mixtures. Biotoxin mixtures interacted to produce alterations in the toxic responses in the model can generally be classified as additive, synergistic or occasionally antagonistic effects. It can be gathered that biotoxin mixtures containing these toxins may pose a significant threat to public health and further research need to be completed addressing long–term toxic and health effect of human populations daily exposed to these biotoxin mixtures and their roles in known human chronic diseases. (Financially supported by research contracts, DAAD13–00–C–0056, DAAD13–01–C–0053, and DAAD13–02–C–0070 from SBCCOM, DOD)

543 (DAW-1117-658698) Comparison of exponential vs. sigmoid dose-response curves: fitting of single chemical time-dependence data and implications for mixture toxicity assessment.
Start time: 4:10 PM
Dawson, D¹, Poch, G², ¹ Ashland University, Ashland, OH, USA² University of Graz, Graz, Austria
Microtox-derived single chemical toxicity data for several industrial organic chemicals, collected as part of a study evaluating dose-response curve (DRC) analysis in chemical mixture toxicity, were fit to exponential (2-parameter) and to sigmoid (4-parameter logistic) DRCs. Single chemical data were collected for several soft-electrophiles and for three non-polar narcotics at 15-, 30- and 45-min, to compare the fit of the single chemical data in each curve type for agents that showed time-dependent toxicity and for agents that showed no time-dependent toxicity. Careful evaluation of single chemical DRCs may allow for detection of significant differences among agents that are to some extent different in their properties, despite being classified as having the same mode of toxic action. When single chemical data were expressed as percent of control and fit to exponential and sigmoid curves (linear-linear), time-independent chemicals, which exhibited a DRC slope close to 1.0, were better expressed by sigmoid curves or at least showed a strong sigmoid component. Agents with a DRC slope of 1.5 to 1.6, were time-dependent and better characterized by the exponential curve function. Chemicals with an intermediate DRC slope appeared intermediate with respect to exponential and sigmoid curves. Longer incubation time often increased slope, so there was a tendency for better fit to exponential curves with increased exposure duration. In some cases, toxicity data did not fit either exponential or sigmoid curves well, instead showing a more biphasic fit in which data for lower concentrations came rather close to sigmoid curves, while data for higher concentrations came closer to exponential curves. Overall, the results suggest that some agents possess more than one mode of action over the concentration range tested, allowing data points to come close to one model or the other, but not fit either perfectly. These results may help explain why mixture toxicity for so many combinations roughly approaches dose-addition when chemicals in the mixture are from different mode of action classes.

544 (VAN-1117-715614) New calculation rule for ecosystem toxicity of mixtures?
Start time: 4:30 PM
van de Meent, D.^{1, 2}, Posthuma, L.¹, de Zwart, D.¹, ¹ National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands² Radboud University, Nijmegen, The Netherlands
We propose a new procedure for calculating toxic pressure of mixtures on ecosystems. The inferred Fraction Affected (i.e. fraction of species exposed to significant concentrations of toxicants, ms-PAF), as proposed by Traas et al. (2002) is widely used as proxy for toxic pressure of (mixtures of) chemicals on ecosystems. Although that use is supported by some eco-epidemiological evidence, the procedure fails to explain one basic empirical observation: species differ less in sensitivity to complex mixtures than to single substances. Present toxic pressure metric predicts the widths of mixture SSDs to reflect the average width of the SSDs of the components of the mixture, whereas observations suggest that widths of SSDs decrease with increasing complexity of the mixture. We have explored alternate calculation rules for mixture toxicity that correctly reflect this observation. We hypothesize that single-species toxicity end points can be adequately modeled as a function of the metabolic sensitivity spectrum of the exposed biological species (multiple receptors within species) and the toxic activity spectrum of the chemical substance (multiple modes of action). Such calculation rules could explain that narrower SSDs are observed for mixtures with wider toxic activity spectra. We propose experimental strategies for testing the hypothesized new calculation rules.

545 (CLA-1117-817531) Crude oil fraction toxicity to early life stage Japanese medaka (Oryzias latipes).
Start time: 4:50 PM
Clarke, Lauren¹, Hodson, Peter¹, ¹ Queen's University, Kingston, Ontario, Canada
Crude oil is a complex mixture with each crude being unique in chemical composition and PAH concentration. The presence and concentration of polycyclic aromatic hydrocarbons (PAHs) has been linked to the dioxin-like toxicity in early life stages of fish exposed to crude oil. This toxicity has been characterized by the presence and severity of blue-sac disease (BSD) and the induction of cytochrome P4501A (CYP1A) enzymes. Two crude oils, Scotian Shelf (SS) and Alaska North Slope Crude (ANSC), were fractionated into 4 fractions (F1-F4), which were then used in chronic early life stage Japanese medaka (Oryzias latipes) toxicity experiments. Embryos were exposed to a chemically enhanced water accommodated fraction (CEWAF) of one of the fractions. The F3 fraction, which contains many PAHs and alkyl PAHs, was found to have the highest toxicity in both oils. F1 and F2 had high mortality which was attributed primarily to narcotic effects as toxicity scores remained relatively low. ANSC F4 had a relatively low toxicity in terms of both sublethal and lethal effects.

546 (MER-1117-838618) PAH-induced impacts on the bioenergetics and PCB bioaccumulation in Fundulus heteroclitus.
Start time: 5:10 PM
Merten, A¹, Beard, E², Baker, J², ¹ NOAA Office of Response and Restoration, Seattle, WA, USA² University of Maryland, Chesapeake Biological Lab, Solomons, MD, USA
Long-term residual contamination from oil spills and other sources of polycyclic aromatic hydrocarbons (PAHs) may exert sublethal narcosis on fish by reducing standard metabolic rate (SMR), and subsequently affecting bioaccumulation. We conducted a 120 day study (2x4x3 factorial, random block design) to quantify the impacts of long-term exposure to dissolved and dietary narcotics on Fundulus heteroclitus. The study evaluated bioenergetic parameters (growth, fraction of lipid, and standard metabolic rate) and PCB bioaccumulation to correlate chronic exposure to dietary PAHs with changes in bioenergetics and bioaccumulation. Fish were exposed to two levels of MS-222 (0 and 50 mg/L), and four levels of food treatments (1: control food, 2: 10% contaminated food:90% control food, 3: 50% contaminated food:50% control food, and 4: 100% contaminated control food). Contaminated food consisted of caging clams at a creosote-contaminated site for 30 days to accumulate bioavailable PAHs (3000 ng/g -PAH). Background concentrations of -PCBs were used to assess treatment-induced changes in accumulation. On days 0, 35, 62, 90, and 120, we measured individual fish growth parameters, SMRs, and PCB accumulation. SMR responded in a non-linear manner to the exposure treatments. The 10% PAH-contaminated food depressed SMR. Above the 10% PAH-contaminated food concentrations (> 835 ng/g), SMR increased masking sublethal narcosis by other modes of PAH toxicity. PAH-contaminated food significantly increased PCB accumulation between the control food treatments and the 10% contaminated food treatment (p = 0.0307). On day 120, PCB accumulation increased as the PAH concentration in the food increases (p = 0.038). To further examine the interactions between fish bioenergetics and contaminant bioaccumulation dynamics at individual and population levels, we developed a bioenergetics model for Fundulus. The model examined how sublethal changes in individual energetic potentially impact population parameters and compares change in total population biomass from altering energetics to changing mortality probabilities.