Alex D. Hoffman

Branchial elimination of superhydrophobic organic compounds by rainbow trout (Oncorhynchus mykiss)

The branchial elimination of pentachloroethane and four congeneric polychlorinated biphenyls by rainbow trout was measured using a fish respirometer-metabolism chamber and an adsorption resin column. Branchial elimination was characterized by calculating a set of apparent in vivo blood:water partition coefficients (P(BW)). Linear regression was performed on the logarithms of P(BW) estimates and the log K(OW) value for each compound to give the fitted equation: log P(BW)=0.76 x log K(OW)-1.0 (r(2)=0.98). The linear nature of this relationship provides support for existing models of chemical flux at fish gills and suggests that a near equilibrium condition was established between chemical in venous blood entering the gills, including dissolved and bound forms, and dissolved chemical in expired branchial water. In vivo P(BW) estimates were combined with P(BW) values determined in vitro for a set of lower log K(OW) compounds (Bertelson et al., Environ. Toxicol. Chem. 17 (1998) 1447-1455) to give the fitted relationship: log P(BW)=0.73 x log K(OW)-0.88 (r(2)=0.98). The slope of this equation is consistent with the suggestion that chemical binding to non-lipid organic material contributes substantially to blood:water chemical partitioning. An equation based on the composition of trout blood (water content and the total amount of organic material) was then derived to predict blood:water partitioning for compounds with log K(OW) values ranging from 0 to 8: log P(BW)=log[(10(0.73 log K(ow)) x 0.16)+0.84].

Simultaneous multiple species testing: acute toxicity of 13 chemicals to 12 diverse freshwater amphibian, fish, and invertebrate families.

This test series developed methods for testing a compliment of aquatic organisms in a single test that satisfies the freshwater acute toxicity requirements for setting water quality criteria. Species tested included fathead minnowsPimephales promelas, rainbow troutSalmo gairdneri, bluegillLepomis macrochirus, channel catfishIctalurus punctatus, goldfishCarassius auratus, white suckerCatostomus commersoni, daphnidDaphnia magna, midgeTanytarsus dissimilis, crayfishOrconectes immunis, snailAplexa hypnorum, tadpoleXenopus laevis, and leechNephelopsis obscura. Five to nine of the preceding species were simultaneously exposed in individual tests. The chemicals tested were acrolein, aniline, dibutylfumarate, 2,4-dinitrophenol, Guthion®, nicotine sulfate, phenol, rotenone, silver, Systox®, 1,2,4-trichlorobenzene, 2,4,6-trichlorophenol, ando-xylene. This method of simultaneously exposing aquatic organisms in separate compartments of each exposure tank allows more accurate comparisons of species sensitivity with a tested chemical. Use of this method can also produce the minimum acute data set for the derivation of a water quality criterion in less time and with a substantial cost saving for labor, materials, and chemical analyses when compared with measured concentration tests conducted separately with each individual species.

Physiologically based toxicokinetic modeling of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss)

A physiologically based toxicokinetic model for fish was used to simulate the uptake and disposition of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 1,1,2,2-tetrachloroethane, pentachloroethane, and hexachloroethane in fish respirometer-metabolism chambers to assess the kinetics of chemical accumulation in arterial blood and chemical extraction efficiency from inspired water. Chemical residues in tissues were measured at the end of each experiment. Trout exposed to tetrachloroethane were close to steady-state in 48 hr. Fish exposed to pentachloroethane were near steady-state in 264 hr. Extraction efficiency data showed that systemic (extrabranchial) elimination of both chemicals was small. Hexachloroethane continued to accumulate in fish exposed for 600 hr. Parameterized with chemical partitioning data obtained in vitro, the model accurately simulated the uptake of all three chloroethanes in blood and tissues and their extraction from inspired water. These results provide support for the basic model structure and the accuracy of physiological input parameters.

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.

Physiologically-based toxicokinetic modeling of three waterborne chloroethanes in channel catfish, Ictalurus punctatus

A physiologically-based toxicokinetic model for fish was used to describe the uptake and disposition of three chlorinated ethanes in channel catfish (Ictalurus punctatus). Catfish were simultaneously exposed to 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) in fish respirometer-metabolism chambers to assess the kinetics of chemical accumulation in arterial blood and chemical extraction efficiency from inspired water. Chemical residues in tissues were measured at the end of each experiment. These data were used to evaluate the accuracy of model simulations and to form a basis for comparison with information collected previously from rainbow trout. TCE was at or near steady-state in catfish after 48 h. For PCE and HCE the time to steady-state appeared to be considerably longer than 48 h. Parameterized with in vitro chemical partitioning information, the model accurately simulated the accumulation of TCE in arterial blood and its uptake from inspired water, but consistently underestimated the uptake and accumulation of both PCE and HCE. The cause of these discrepancies was not conclusively determined; however, several possible sources of error were evaluated, including physiological and chemical partitioning inputs, and underlying modeling assumptions. A comparison of data sets and modeling efforts for rainbow trout and channel catfish suggests that gross similarities between the two species can be attributed to the comparability of relevant physiological and chemical partitioning parameters.

A Physiologically Based Toxicokinetic Model for Dermal Absorption of Organic Chemicals by Fish

A physiologically based toxicokinetic model was developed to describe dermal absorption of waterborne organic chemicals by fish. The skin was modeled as a discrete compartment into which compounds diffuse as a function of chemical permeability and the concentration gradient. The model includes a countercurrent description of chemical flux at fish gills and was used to simulate dermal-only exposures, during which the gills act as a route of elimination. The model was evaluated by exposing adult rainbow trout and channel catfish to hexachloroethane (HCE), pentachloroethane (PCE), and 1,1,2,2-tetrachloroethane (TCE). Skin permeability coefficients were obtained by fitting model simulations to measured arterial blood data. Permeability coefficients increased with the number of chlorine substituent groups, but not in the manner expected from a directly proportional relationship between dermal permeability and skin:water chemical partitioning. An evaluation of rate limitations on dermal flux in both trout and catfish suggested that chemical absorption was limited more by diffusion across the skin than by blood flow to the skin. Modeling results from a hypothetical combined dermal and branchial exposure indicate that dermal uptake could contribute from 1.6% (TCE) to 3.5% (HCE) of initial uptake in trout. Dermal uptake rates in catfish are even higher than those in trout and could contribute from 7.1% (TCE) to 8.3% (PCE) of initial uptake in a combined exposure.

A physiologically based toxicokinetic model for lake trout (Salvelinus namaycush).

A physiologically based toxicokinetic (PB-TK) model for fish, incorporating chemical exchange at the gill and accumulation in five tissue compartments, was parameterized and evaluated for lake trout (Salvelinus namaycush). Individual-based model parameterization was used to examine the effect of natural variability in physiological, morphological, and physico-chemical parameters on model predictions. The PB-TK model was used to predict uptake of organic chemicals across the gill and accumulation in blood and tissues in lake trout. To evaluate the accuracy of the model, a total of 13 adult lake trout were exposed to waterborne 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE), concurrently, for periods of 6, 12, 24 or 48 h. The measured and predicted concentrations of TCE, PCE and HCE in expired water, dorsal aortic blood and tissues were generally within a factor of two, and in most instances much closer. Variability noted in model predictions, based on the individual-based model parameterization used in this study, reproduced variability observed in measured concentrations. The inference is made that parameters influencing variability in measured blood and tissue concentrations of xenobiotics are included and accurately represented in the model. This model contributes to a better understanding of the fundamental processes that regulate the uptake and disposition of xenobiotic chemicals in the lake trout. This information is crucial to developing a better understanding of the dynamic relationships between contaminant exposure and hazard to the lake trout.

Hepatic microsomal N-hydroxylation of aniline and 4-chloroaniline by rainbow trout (Onchorhyncus mykiss)

1. N-Hydroxylation of aniline and 4-chloroaniline was quantified in rainbow trout microsomal preparations using h.p.l.c.-liquid scintillation methods. Radioactive phenylhydroxylamine and 4-chlorophenylhydroxylamine metabolites were identified by co-elution with non-labelled standards. The method provided resolution of metabolite standards, and quantification of both N-hydroxylated metabolites was achieved without derivatization. 2. The maximum velocities at 25 degrees C were 33.8 +/- 1.40 and 22.0 +/- 0.98 pmol/min per mg for aniline and 4-chloroaniline N-hydroxylation, respectively. The Km values were 1.0 +/- 0.11 and 0.8 +/- 0.11 mM for aniline and 4-chloroaniline N-hydroxylation, respectively. These activities were not induced by treatment of the trout with Aroclor 1254 under the conditions of this study. 3. When incubations were performed at 11 degrees C, the physiological temperature of rainbow trout in this study, the Vmax for 4-chloroaniline N-hydroxylation decreased from 22.0 to 6.4 pmol/min per mg and the Km decreased from 0.8 to 0.5 mM. 4. The pH optimum for 4-chloroaniline N-hydroxylation was 8.0 while the pH optimum for aniline N-hydroxylation ranged from 7.4 to 8.0, suggesting the possible contribution of different isoenzymes. 5. The demonstration of aniline and 4-chloroaniline N-hydroxylation by rainbow trout microsomes provides further insight into the high acute:subchronic toxicity ratios observed in fish exposed to these compounds.