Russell J. Erickson

Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

A Physiologically Based Toxicokinetic Model for the Uptake and Disposition of Waterborne Organic Chemicals in Fish

A physiologically based toxicokinetic model was developed to predict the uptake and disposition of waterborne organic chemicals in fish. The model consists of a set of mass-balance differential equations which describe the time course of chemical concentration within each of five tissue compartments: liver, kidney, fat, and richly perfused and poorly perfused tissue. Model compartmentalization and blood perfusion relationships were designed to reflect the physiology of fishes. Chemical uptake and elimination at the gills were modeled as countercurrent exchange processes, limited by the chemical capacity of blood and water flows. The model was evaluated by exposing rainbow trout (Oncorhynchus mykiss) to pentachloroethane (PCE) in water in fish respirometer-metabolism chambers. Exposure to 1500, 150, or 15 micrograms PCE/liter for 48 hr resulted in corresponding changes in the magnitude of blood concentrations without any change in uptake kinetics. The extraction efficiency for the chemical from water decreased throughout each exposure, declining from 65 to 20% in 48 hr. Extraction efficiency was close to 0% in fish exposed to PCE to near steady state (264 hr), suggesting that very little PCE was eliminated by metabolism or other extrabranchial routes. Parameterized for trout with physiological information from the literature and chemical partitioning estimates obtained in vitro, the model accurately predicted the accumulation of PCE in blood and tissues, and its extraction from inspired water. These results demonstrate the potential utility of this model for use in aquatic toxicology and environmental risk assessment.

Effects of light intensity on the phototoxicity of fluoranthene to a benthic macroinvertebrate.

Conceptual models suggest that the toxicity of photoactivated polycyclic aromatic hydrocarbons (PAHs) should be a direct function both of chemical (PAH) dose and intensity of the ultraviolet (UV) light to which the organism is exposed. However, there have been only limited studies with aquatic organisms to quantify the relationship between PAH dose and UV intensity in producing phototoxicity. In this study, oligochaetes (Lumbriculus variegatus) were exposed, via the water, to multiple concentrations of fluoranthene, a PAH known to be phototoxic, and then placed under UV light at three different intensities. The resultant phototoxicity clearly was a function both of PAH dose and light intensity. Time-dependent mortality of the oligochaetes could be accurately predicted through evaluation of the product of fluoranthene dose (in the tissue of the animal) and light intensity to which the organisms were exposed. These results indicate that criteria for phototoxic chemicals should incorporate consideration not only of xenobiotic exposure but also of light intensity in specific aquatic environments.

A MODEL FOR EXCHANGE OF ORGANIC CHEMICALS AT FISH GILLS : FLOW AND DIFFUSION LIMITATIONS

Abstract A mathematical model for the exchange of neutral organic chemicals at fish gills was formulated based on limitations imposed by flows of water and blood to the gills, diffusion barriers defined by gill morphology, and chemical binding relationships within water and blood. This model was parameterized independently of exchange measurements and validated against datasets on the relationship of chemical uptake rates for large rainbow trout and small guppies to chemical hydrophobicity. This model was found to closely predict the magnitude and trends of observed gill uptake rates in these datasets, predictions deviating from observed values by no more than a factor of two over a range of octanol:water partition coefficients from 2 to > 10 6 . Elimination rates for small guppies were also predicted. This analysis suggests that gill exchange can be understood and predicted on the basis of fundamental physiological and morphological variables.

Evaluation of models for predicting the phototoxic potency of polycyclic aromatic hydrocarbons

Abstract The objective of this study was to evaluate the validity of two previously developed models to predict: (a) the relative phototoxic potency of polycyclic aromatic hydrocarbons (PAHs) through structure activity relationships (SARs), and (b) the interactive effects of variable light intensity and PAH dose on phototoxicity. The oligochaete Lumbriculus variegatus was exposed to multiple concentrations of the PAHs anthracene, pyrene, fluorene and fluoranthene for 96 h, followed by a 96 h holding period in clean water at three different ultraviolet (UV) light intensities. Based upon measured tissue residue concentrations, anthracene and pyrene were approximately equitoxic, and both were four-fold more potent than fluoranthene. Fluorene was not phototoxic to the oligochaete. These results were in good quantitative agreement with the toxicity predictions of the SAR model. Time-dependent lethality of the three phototoxic PAHs to the oligochaete was accurately modeled by plotting mortality as a function of the product of initial tissue residue of the PAH and UV light intensity to which the organisms were exposed, which also was in good agreement with the interactive toxicity model. These studies contribute to the technical basis for developing an integrated modeling approach to predicting the ecological risk of mixtures of phototoxic PAHs.

Environmental Impacts on the Physiological Mechanisms Controlling Xenobiotic Transfer across Fish Gills

Fish physiologists have provided the basic information on gill morphology, gill function, and vascular dynamics with which to understand branchial flux of gases, water, and ions. In addition, pharmacologists and toxicologists, working in the area of drug action, have characterized the physicochemical attributes of xenobiotic chemicals that determine their rate of movement across biological membranes. Recently, aquatic toxicologists have applied this information to the question of what mechanisms control the movement of organic chemicals across fish gills and how exchange is affected by chemical properties. This research on gill transfer mechanisms was extended to consider environmental conditions that have been reported to influence chemical exchange (e.g., dissolved and/or suspended organic material, hydrogen ion concentration [pH], dissolved O₂, content, and water temperature). Mathematical models were developed that predict gill exchange as a function of basic processes such as water flow across the gills, blood flow through the gills, partitioning of the chemical between water and blood, and diffusion between blood and water across gill epithelia. Such mechanistic models can predict the effects of environmental conditions on exchange rates of xenobiotics. To fully develop a predictive capability for xenobiotic uptake and distribution by fish, it will be necessary to incorporate these gill models into emerging, physiologically based models for the entire animal.

Uptake and elimination of ionizable organic chemicals at fish gills: II. Observed and predicted effects of pH, alkalinity, and chemical properties

Effects of exposure-water pH on chemical uptake at rainbow trout (Oncorhynchus mykiss) gills were investigated for nine weakly acidic, chlorinated phenols with different ionization constants and hydrophobicities and for a moderately hydrophobic, nonionizable reference chemical (1,2,4-trichlorobenzene). Uptake rates for all chemicals varied little from pH 6.3 to 8.4, despite ionization of the chlorinated phenols ranging from less than 1 to greater than 99.9% among these pH values and chemicals. At pH 9.2, uptake rates were reduced substantially for the chlorinated phenols but not for the reference chemical. These results indicate greater bioavailability of neutral chemical forms but also considerable bioavailability of ionized forms that varies with pH. Three mechanisms were evaluated regarding such ionized chemical bioavailability. First, reduced pH at the gill surface causes net conversion of ionized molecules to more readily absorbed neutral molecules. This mechanism was tested by increasing exposure-water alkalinity, which increased gill surface pH and reduced uptake of the chlorinated phenols but not of the reference chemical. Magnitudes of these effects were close to predictions from a mathematical model for chemical exchange at fish gills that incorporated this mechanism. Second, ionized molecules contribute to uptake by maintaining high gradients of neutral molecules across epithelial membrane barriers, even if the barriers are impermeable to these ions. This mechanism was demonstrated to explain the similarity of uptake among pH values and chemicals at pH less than 8.4 and the degree to which uptake declined at pH 9.2. Third, membrane barriers can have some permeability to the ionized forms, but this was not important for the chemicals and conditions of the present study. Increased exposure-water pH also was demonstrated to increase elimination rates of these chemicals, which also was in accord with model expectations.

Uptake and elimination of ionizable organic chemicals at fish gills I. Model formulation, parameterization, and behavior

A mechanistic model for the uptake and elimination of ionizable organic chemicals at fish gills is presented. This model is a modification of a previous model for nonionizable organic chemicals that addressed the transport of chemical to and from gill surfaces in water and blood, diffusion of chemical across epithelial cells, and binding of chemical to components in water and blood. For ionizable chemicals, three additional processes are included. First, excretory products alter the pH at gill surfaces, affecting the relative amounts of neutral and ionized molecules compared with that in the bulk exposure water. Second, ionized molecules support chemical flux to and from epithelial cell membranes and help maintain high diffusion gradients of neutral molecules across these membranes, thereby contributing to uptake and elimination even if the membranes are impermeable to ionized molecules. Third, membrane barriers are not completely impermeable to ionized molecules, and even limited permeability can have appreciable effects on chemical flux. Approaches for model parameterization are discussed. Model-predicted relationships of uptake and elimination rates to exposure water pH, alkalinity, and chemical properties are presented and discussed in terms of model processes. The model is shown to predict important features of reported effects of pH on uptake rates of weak organic acids.

Observed and modeled effects of pH on bioconcentration of diphenhydramine, a weakly basic pharmaceutical, in fathead minnows

A need exists to better understand the influence of pH on the uptake and accumulation of ionizable pharmaceuticals in fish. In the present study, fathead minnows were exposed to diphenhydramine (DPH; disassociation constant = 9.1) in water for up to 96 h at 3 nominal pH levels: 6.7, 7.7, and 8.7. In each case, an apparent steady state was reached by 24 h, allowing for direct determination of the bioconcentration factor (BCF), blood-water partitioning (PBW,TOT), and apparent volume of distribution (approximated from the whole-body-plasma concentration ratio). The BCFs and measured PBW,TOT values increased in a nonlinear manner with pH, whereas the volume of distribution remained constant, averaging 3.0 L/kg. The data were then simulated using a model that accounts for acidification of the gill surface caused by elimination of metabolically produced acid. Good agreement between model simulations and measured data was obtained for all tests by assuming that plasma binding of ionized DPH is 16% that of the neutral form. A simpler model, which ignores elimination of metabolically produced acid, performed less well. These findings suggest that pH effects on accumulation of ionizable compounds in fish are best described using a model that accounts for acidification of the gill surface. Moreover, measured plasma binding and volume of distribution data for humans, determined during drug development, may have considerable value for predicting chemical binding behavior in fish.

Toxicokinetic modeling of [14C]pentachlorophenol in the rainbow trout (Salmo gairdneri)

Abstract An in vivo trout model was used to monitor the major routes and rates of pentachlorophenol uptake and elimination. Rainbow trout exposed to a mean sublethal water concentration (1.0 μg/l) of [14C]pentachlorophenol (PCP), a moderately lipophilic, relatively non-persistent environmental contaminant acquired a mean calculated dose of 230 μg/kg per 48 h and a mean measured dose of 212 μg/kg per 48 h. The rate constants determined for the calculated and measured doses were 5.0 ± 0.8 and 4.6 ± 1.1 l/kg per h, respectively. This close agreement between the calculated and measured doses and their rate constants provided further support for the use of this model system in aquatic toxicokinetic studies. A first-order kinetic model and observed data were used to generate fitted and predicted rate constants required for evaluation of first-order kinetics. The fitted first-order uptake-depuration curves for all experimental animals agreed with those observed suggesting first-order kinetics approximated the behavior of whole-body PCP burden. The predicted first-order uptake-depuration curve differed by a factor of 2–3 from that observed, due to the low predicted value used in the model for the steady-state PCP bioconcentration factor (BCF). A BCF of 460 was estimated from the first order simulation model developed from empirical data collected on uptake and elimination of PCP. The dosing time required to reach this steady-state BCF was 280 h. The estimated half-life ( T 1 2 ) was 65 h with approximately 50% eliminated over the gills, 30% in the feces and bile, and 20% in the urine. A depuration period of 280 h was required to eliminate 95% of the steady-state concentration of PCP. Approximately 43% of the 48-h dose of [14C]PCP remained in the major organs and muscle tissues of these trout at the end of the 96-h experiment. Of this amount, muscle contained 29% of the total remaining [14C]PCP equivalents while the remaining carcass contained 45% of the total remaining [14C]PCP. The PCP radiocarbon excreted in the urine was 10% PCP and 90% ‘other’ (metabolite/conjugate), while the bile was 45% PCP and 55% ‘other’ (metabolite/conjugate). PCP and its metabolites were rapidly eliminated from the bodies of fish, which provided for a low BCF. Efficient elimination of PCP should allow vertebrates to tolerate periodic low doses of PCP without toxic effects.