* Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Duluth, Minnesota, 55804
Received June 4, 2003; accepted October 23, 2003
ABSTRACT
A physiologically based toxicokinetic (PBTK) model for dietary uptake of hydrophobic organic compounds by fish was used to simulate dosing scenarios commonly encountered in experimental and field studies. Simulations were initially generated for the model compound [UL-14C] 2,2',5,5'-tetrachlorobiphenyl ([14C] PCB 52). Steady-state exposures were simulated by calculating chemical concentrations in tissues of the predator corresponding to an equilibrium distribution between the fish and water (termed the bioconcentration or BCF residue data set). This residue data set was then varied in a proportional manner until whole-body chemical concentrations exhibited no net change for each set of exposure conditions. For [14C] PCB 52, the proportional increase in BCF residues (termed the biomagnification factor or BMF) required to achieve steady state in a food-only exposure was 2.24, while in a combined food and water exposure the BMF was 3.11. Additional simulations for the food and water exposure scenario were obtained for a set of hypothetical organic compounds with increasing log KOW values. Using gut permeability coefficients determined for [14C] PCB 52, predicted BMFs increased with chemical log KOW, achieving levels much higher than those reported in field sampling efforts. BMFs comparable to measured values were obtained by reducing permeability coefficients within each gut segment in a log KOWdependent manner. This predicted decrease in chemical permeability is consistent with earlier work, suggesting that dietary absorption of hydrophobic compounds by fish is controlled in part by factors that vary with chemical log KOW. Relatively low rates of metabolism or growth were shown to have a substantial impact on steady-state biomagnification of chemical residues.
Key Words: physiologically based model; fish; dietary uptake.
Recent studies of dietary uptake by fish have focused on factors that promote the uptake of hydrophobic compounds, including the absorption of dietary lipid and reductions in meal volume (Gobas et al., 1993a,b
, 1999
). The "digestion hypothesis" states that these processes tend to increase chemical activity in the gut contents above that of the meal, potentially resulting in biomagnification of chemical residues (i.e., lipid-normalized concentrations in fish greater than those of their food). Support for this hypothesis has been obtained in studies with several fish species. In feeding studies with guppies (Poecilia reticulata) and goldfish (Carassius auratus), the feces:food fugacity ratio increased with chemical log KOW, attaining a maximum value of 4.6 (in guppies) for the pesticide mirex (log KOW of 7.2; Gobas et al., 1993b
). The intestinal contents:food fugacity ratio in a natural population of white bass (Morone chrysops) also increased with chemical log KOW, attaining a value of about 2.2 for 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180, log KOW of 7.36; Russell et al., 1995
). In rainbow trout (Oncorhynchus mykiss) and rock bass (Ambloplites rupestris) exposed to 2,2',4,4',6,6'-hexachlorobiphenyl (PCB 155, log KOW of 7.2), the chemical fugacity in chyme following uptake of dietary lipid exceeded that of food by a factor of 7 to 8 under both laboratory and field conditions (Gobas et al., 1999
).
Adopting the digestion hypothesis, the maximum extent to which a compound can biomagnify in fish depends on the feeding rate (FD), the fecal production rate (FF), and the gut contents:organism chemical partitioning coefficient (KGB), according to the following relationship given by Gobas et al. (1993a): FD/(FF KGB). However, the processes that actually control chemical flux across the gastrointestinal epithelium, and ultimately the extent to which chemicals accumulate in fish, remain incompletely understood. Additional questions pertain to the manner in which dietary uptake and biomagnification vary with attributes of the ingested compound (e.g., hydrophobicity and susceptibility to metabolic biotransformation) and the exposed animal (e.g., gastrointestinal anatomy and physiology).
In a companion report, we described a physiologically based toxicokinetic (PBTK) model for dietary uptake of hydrophobic organic chemicals by fish (Nichols et al., 2004). The gut portion of this model consists of four compartments corresponding to the stomach, pyloric ceca, upper intestine, and lower intestine. This structure provides for temporal and spatial resolution of factors that control chemical uptake and elimination, and permits a mechanistic exploration of these factors by allowing the modeler to adjust parameter values and modeling assumptions for individual gut segments. The model was initially calibrated using data from rainbow trout that were fed a single meal of fathead minnows (60 days old) contaminated with [UL-14C] 2,2',5,5'-tetrachlorobiphenyl ([14C] PCB 52; Nichols et al., 2001
). Dietary exposures of environmental concern are more likely, however, to occur over long periods of time and involve many individual feedings. Accordingly, the goals of the present study were as follows: (1) to simulate different chronic exposure scenarios for [14C] PCB 52 and compare modeled results to published data for PCB 52 (unlabeled); (2) to simulate long-term dietary exposures to a set of hypothetical organic compounds and compare the results with trends that have been reported with respect to chemical log KOW; and (3) to use the model to investigate potential impacts of biliary elimination, metabolic biotransformation, and growth on chemical biomagnification in fish.
MATERIALS AND METHODS
PBTK model.
A PBTK model for dietary uptake of hydrophobic compounds by fish is described in a companion report (Nichols et al., 2003). Mass-balance differential equations were solved by numerical integration using a commercial software package (ACSL; Aegis Technologies, Huntsville, AL) to obtain a complete solution set for each time point.
[14C] PCB 52 dosing scenarios.
The goal of this study was to use the dietary uptake model to simulate dosing scenarios commonly encountered in experimental studies and field exposures. Using [14C] PCB 52 as a model compound, these scenarios were as follows: (1) chronic exposure to contaminated water; (2) chronic exposure to contaminated food; and (3) chronic exposure to contaminated food and water.
Although in theory it would be possible to simulate a large number of individual feedings over time periods lasting months to years, this approach was determined to be impractical and unnecessary. Instead, the model was used to solve for [14C] PCB 52 concentrations in trout tissues that would be expected under steady-state conditions, given a defined exposure scenario. The first step was to calculate the [14C] PCB 52 concentration in water expected from an equilibrium distribution between the water and contaminated fathead minnows from the initial feeding studies (Nichols et al., 2001). A fat:water partitioning coefficient (PF:W) for [14C] PCB 52 was calculated as the product of fat:blood (PF:B) and blood:water (PB:W) partitioning values given by Nichols et al. (2004)
for rainbow trout: (4500)(140.2) = 630,960. This value was multiplied by the reported lipid content (3.3%) of fathead minnows to give a whole-body bioconcentration factor of 20,820 (BCF; wet weight basis, defined as the [14C] PCB 52 concentration in fish [ng/g]/[14C] PCB 52 concentration in water [ng/ml]). The average measured [14C] PCB 52 concentration in fathead minnows (1663 ng/g) was then divided by the BCF to give an estimated water concentration of 0.08 µ/L. This approach assumes that fat:water partitioning in fathead minnows is similar to that in trout and ignores the contribution of [14C] PCB 52 associated within nonlipid portions of the fish. Errors introduced by these assumptions are probably small, however, given the likely similarity of fat:water partitioning in both species (even as PF:B and PB:W vary) and the overwhelming importance of lipid as a repository for hydrophobic chemicals in fish.
The next step was to calculate the concentration of [14C] PCB 52 in each trout tissue, assuming chemical equilibrium with an exposure water concentration of 0.08 µ/L. This was accomplished by calculating tissue:water partitioning values (PT:W) for all tissues as products of tissue:blood (PT:B) and PB:W values given in Table 2 of Nichols et al. (2004)
and multiplying by 0.08. The [14C] PCB 52 concentration in each tissue was then multiplied by the estimated tissue volume to give the total mass of chemical. The results of these calculations are provided in Table 1
. A whole-fish BCF can also be calculated by summing the volume-weighted contributions of each tissue. Using data given in Table 1
, the whole-fish BCF for subadult trout used by Nichols et al. (2001)
is about 27,600, which is intermediate to values reported previously for guppies (18,200) and goldfish (49,000; Connell and Hawker, 1988
) and lower than the BCF (200,000) estimated for large adult trout (Oliver and Niimi, 1985
). Taken together, these calculations provide a set of individual tissue and whole-body chemical concentrations for the predator (trout) and prey (fathead minnows) that are in equilibrium with an aqueous [14C] PCB 52 concentration of 0.08 µg/L.
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Log KOW dependence of BMFs, diffusion rate constants, and net assimilation efficiency.
Additional simulations were run for a set of hypothetical compounds with log KOW values of 6.5, 7, 7.5, and 8. These simulations were limited to the combined food and water exposure scenario. PB:W and PF:W partitioning coefficients were estimated using empirical relationships given by Fitzsimmons et al. (2001) and Bertelsen et al. (1998)
, respectively (Table 2
). PF:B was then calculated from the quotient of these two values. Empirical equations relating kidney:water and muscle:water partitioning to chemical log KOW have also been reported for fish (Bertelsen et al., 1998
). However, the slope terms in these fitted equations are close to that for blood:water partitioning. The result is that estimated tissue:blood partitioning coefficients (PT:B) change very little with log KOW. Therefore, PT:B values for all tissues except fat were set equal to those determined for [14C] PCB 52 (Nichols et al., 2004
). Bile:liver partitioning values were also set equal to that for [14C] PCB 52.
For simplicity, it was assumed that all chemicals were freely dissolved in water and available for uptake across the gills. The binding of hydrophobic compounds to dissolved organic matter in water can substantially reduce branchial uptake of high log KOW compounds by fish (Black and McCarthy, 1988), but this binding should have a similar effect on chemical accumulation by both the predator and its prey resulting in proportional impacts on steady-state chemical concentrations.
The first set of simulations was generated by adjusting chemical residues in fish tissues (i.e., BMFs) to result in no net change in whole-body chemical concentration. Additional simulations were then obtained by fixing BMFs for the three most hydrophobic compounds and adjusting permeability coefficients within the GI tract to result in no net change of chemical residues. Guidance on the specification of BMFs was obtained from the literature. Specifically, laboratory and field data suggest that, within the log KOW range of 6 to 8, BMFs for fish seldom exceed 10 for persistent, poorly metabolized compounds and more commonly range from 3 to about 5 (Gobas et al., 1993b, 1999
; Russell et al., 1995
). Additional data suggest that steady-state bioaccumulation factors (BAFs) decrease with chemical log KOW at very high (>7) log KOW values (Burkhard, 1998
; Thomann, 1989
). The BAF is defined in this case as the lipid-normalized chemical concentration in fish divided by the free chemical concentration in water. Because BAFs and BMFs are manifestations of the same uptake and elimination processes, this observed decline in BAFs is likely to have been associated with a log KOW-dependent decline in BMFs (had the chemical concentration in prey been measured).
Initially, BMFs for log KOW 7, 7.5, and 8 compounds were set equal to 5. BMFs for the same three compounds were then equal to 5, 3, and 1, respectively. In each case, permeability coefficients for the pyloric ceca, upper intestine, and lower intestine were varied in a proportional manner, maintaining the relationship established previously by fitted values for [14C] PCB 52 (approximately 1.0:0.2:0.08). Finally, models with fitted permeability coefficients were used to simulate three consecutive feedings of contaminated prey to a previously unexposed fish to evaluate the impact of these adjustments on net assimilation efficiency, defined as the percentage of chemical consumed by the animal that is retained within its tissues.
Biliary elimination, metabolism, and growth.
Biliary elimination is calculated in the model as the product of bile flow rate and a liver:bile concentration ratio, which represents the net result of chemical partitioning and active secretion of parent compounds into bile (Nichols et al., 2004). The concentration ratio was set equal to that (0.67) determined in earlier feeding studies with [14C] PCB 52 (Nichols et al., 2001
). The importance of biliary elimination was then evaluated by setting the bile flow rate equal to zero and examining the effect of this change on BMFs for [14C] PCB 52 and a set of hypothetical high log KOW compounds.
The gut model provides for the possibility of metabolic biotransformation in both the liver and gut tissues. Potential effects of metabolism were examined by adjusting first-order rate constants in the liver, pyloric ceca, and upper intestine. Following Nichols et al. (1990), these rate constants were referenced to the chemical concentration in venous blood exiting each tissue. Initially, the metabolism rate constant in liver was set equal to 0.02/h. The model was then used to calculate BMFs for [14C] PCB 52 and a set of hypothetical high log KOW compounds. When the rate of liver metabolism is low, hepatic clearance is not limited by the rate of chemical delivery to the liver in blood or redistribution among other tissues. Under these circumstances, the impact of metabolism on whole-animal kinetics can be approximated by collapsing the PBTK model into a single, well-stirred compartment. Using tissue volumes and partitioning coefficients given by Nichols et al. (2004)
, it can be shown that a liver metabolism rate constant of 0.02/h equates to a whole-body elimination rate constant of about 0.0004/h or approximately 0.001/day. This value corresponds to an elimination half-life (T
) of about 700 days, assuming that metabolism is the only route of elimination. In a second set of simulations, elimination rate constants for the pyloric ceca and upper intestine were also set equal to 0.02/h. Combined with hepatic metabolism, this results in a whole-body elimination rate constant of about 0.003/day and T
of approximately 220 days. In a third modeling exercise, the metabolism rate constant for liver was set equal to 0.2/h, resulting in a whole-body elimination rate constant of 0.01/day and T
of about 70 days.
The effect of growth on model performance was evaluated by calculating fish body weight using a zero-order growth rate term. The starting weight was set equal to that (103.8 g) used in previous simulations. The growth rate (in g/h) was then adjusted to achieve an annual size increase of 10 or 50 g. Parameters within the model that scale to body weight, including tissue volumes, gut surface areas, physiological parameters (e.g., cardiac output), and metabolism rates, were recalculated at each time step. Meal size and the amount of chemical consumed by fish were also adjusted to maintain the same weight-normalized dose at each feeding interval.
RESULTS
Figures 1 and 2
show the simulated time-course for [14C] PCB 52 in rainbow trout exposed under two hypothetical scenarios. The results of a water-only exposure are shown in Figures 1A
and 1B
. When chemical residues in tissues were set equal to those corresponding to the BCF residue set, the model predicted that [14C] PCB 52 would be eliminated by fish. A steady-state condition was established by multiplying the BCF residue set by a BMF of 0.86. Because this exposure scenario did not include [14C] PCB 52 in the diet, this BMF cannot be interpreted in terms of a relationship between the fish and its food. A value less than 1.0 indicated, however, that elimination of [14C] PCB 52 to feces reduced chemical concentrations in all tissues below the levels predicted from an equilibrium distribution between the fish and water. In simulations encompassing more than one feeding event, this elimination also caused a small but regular fluctuation in whole-body chemical concentration.
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Additional simulations were run for a set of hypothetical compounds with log KOW values of 6.5, 7, 7.5, and 8, again using gut permeability coefficients for [14C] PCB 52. Steady- state BMFs obtained from this effort are shown in Figure 3, along with the value determined for [14C] PCB 52. BMFs estimated in this manner increased nonlinearly with chemical log KOW, attaining a value of 27.5 for the log KOW 8 compound.
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Subsequently, the steady-state BMF was fixed at a value of 5 and the model was used to fit a set of gut permeability coefficients for compounds with log KOW values of 7, 7.5, and 8. Permeability coefficients resulting in steady-state conditions under this constraint declined with chemical log KOW, but the relationship was nonlinear (Fig. 4; permeability coefficients are expressed as a percentage of values determined for [14C] PCB 52). A further decease in fitted permeability coefficients was observed when steady-state BMFs were set equal to 5, 3, and 1 for the log KOW 7, 7.5, and 8 compounds, respectively.
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The incorporation of metabolism or growth into the model resulted in higher fitted permeability coefficients for high log KOW compounds. This increase in chemical permeability was required to offset the effect of metabolism or growth on a fixed level of bioaccumulation. The overall effect of these changes was to shift the relationship between predicted chemical permeability and chemical log KOW to the right (Fig. 5).
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DISCUSSION
In a companion report, we described a PBTK model for dietary uptake of hydrophobic chemicals by fish (Nichols et al., 2004). In the present study, we used this model to simulate a variety of chronic exposure scenarios as a means of evaluating factors that control the uptake and accumulation of these compounds by fish. Initially, the model was used to simulate a chronic waterborne exposure to [14C] PCB 52. Because of its hydrophobicity, the maximum rate at which [14C] PCB 52 can be taken up from water is determined by the capacity of inspired water to deliver freely dissolved chemical to the gills (Erickson and McKim, 1990
). [14C] PCB 52 absorbed across the gills distributes within the animal, and, over time, the concentration in blood increases, decreasing the rate of branchial uptake by reducing the activity gradient for diffusion. A portion of the absorbed chemical partitions into contents of the GI tract and is eliminated in feces. Model simulations resulting in a steady-state BMF less than unity (0.86) suggest that this fecal elimination is sufficient to reduce chemical concentrations in tissues below those expected from an equilibrium between the fish and water.
The expected outcome in chronic waterborne exposures to compounds that are more hydrophobic than [14C] PCB 52 is less certain. Limited data suggest that the maximum branchial uptake rate declines with increasing chemical log KOW at log KOW values greater than about 6, when expressed on the basis of total aqueous chemical concentration (McKim et al., 1985). The most likely explanation for this trend is a log KOW-dependent decrease in bioavailability due to chemical complexation with dissolved organic matter (Erickson and McKim, 1990
). Under these circumstances alone, chemical elimination in feces might be expected to have a large impact on chemical accumulation, but a decline in gut permeability coefficients with increasing log KOW (see below) would tend to oppose this effect.
It is unlikely, however, that environmental exposures to high log KOW compounds would be limited to contact with the contaminant in water. Instead, the diet is thought to be the principal route of exposure for compounds with log KOW values greater than about 5 (Bruggeman et al., 1984). As indicated previously, the digestion of contaminated food may result in chemical residues in fish higher than those expected from equilibrium partitioning with water. Under these circumstances, the gills become a route of chemical elimination and not uptake. Recently, Fitzsimmons et al. (2001)
measured the branchial elimination of four PCB congeners with log KOW values ranging from 6.1 to 8.2, and used this information to calculate a set of apparent PB:W values based on total chemical concentrations in blood and water. A plot of log PB:W against chemical log KOW yielded a linear relationship similar to that obtained from an earlier study of less hydrophobic compounds (Bertelsen et al., 1998
). The observed linearity of this relationship was interpreted as evidence for an equilibrium at the gills between the freely dissolved chemical concentration in water and the total chemical concentration in blood. For the purposes of the present study, this relationship is important because it provides a basis for predicting branchial elimination when BMFs are greater than 1.
In simulated steady-state dietary exposures to [14C] PCB 52, chemical residues in tissues exceeded those associated with a steady-state waterborne exposure. The extent of biomagnification predicted by the model differed somewhat depending on whether the chemical was assumed to be present in food only (BMF = 2.24) or both food and water (BMF = 3.11). This distinction is important because laboratory studies with hydrophobic compounds are often conducted by spiking chemicals into a prepared diet, while environmental exposures are more likely to include both food and water routes. In feeding studies with guppies and goldfish, the potential for biomagnification of PCB 52 (unlabeled) was evaluated by measuring the feces:food fugacity ratio after 14 days of exposure (Gobas et al., 1993b). The reported ratio for goldfish (1.2) is somewhat lower than the modeled BMF for [14C] PCB 52 in trout (food-only exposure), while the ratio for guppies (2.3) is essentially identical. Direct evidence for biomagnification of PCB 52 in a natural setting was given by Russell et al. (1995)
. Lipid-normalized PCB 52 concentrations in Lake Erie white bass were 2.7 times higher than those measured in their principal prey, the emerald shiner (Notropis atherinoides). This field-derived BMF value compares favorably to the modeled BMF for trout (food and water exposure).
The effect of chemical hydrophobicity on dietary uptake was examined by simulating chronic exposures to a set of hypothetical organic compounds. When gut permeability coefficients were set equal to those used to simulate [14C] PCB 52 kinetics, fitted BMFs for the two most hydrophobic compounds (log KOW 7.5 and 8) exceeded values previously reported for chemicals of comparable hydrophobicity. BMFs for all compounds were subsequently limited to a maximum value of 5, and the model was used to fit a set of gut permeability coefficients resulting in steady-state chemical concentrations. Expressed as a percentage of values used to model [14C] PCB 52, these fitted permeability coefficients decreased in a nonlinear manner with chemical log KOW, with the greatest decline occurring in the log KOW range of 6.5 to 7 (Fig. 4). A further decrease in fitted permeability coefficients was observed when BMFs for the two most hydrophobic compounds were assumed to decline with chemical log KOW.
Eliminating biliary clearance from the model had little impact on chemical accumulation when gut permeability coefficients were set equal to fitted values for [14C] PCB 52. When the maximum steady-state BMF was set equal to 5, however, this loss of biliary clearance resulted in a log KOW-dependent decrease in fitted permeability coefficients for high log KOW compounds. The fact that this occurred only when gut permeability was very low to begin with (due to the imposed limitation on BMF values) shows that biliary clearance impacts whole-animal kinetics only when conditions limit the extent to which chemicals secreted into bile can be reabsorbed from the GI tract. In this context, hepatic metabolism contributes to biliary elimination of hydrophobic compounds by transforming them into polar products that are retained within the GI tract and eliminated in feces.
Detailed kinetic data, including chemical concentrations in gut contents and tissues, have not been collected for compounds other than [14C] PCB 52. Lacking this information, it is not possible to independently evaluate the magnitude of fitted permeability coefficients for hypothetical high log KOW compounds. An indirect test of the model can be accomplished, however, by comparing published dietary assimilation efficiencies to values predicted by the model using fitted gut permeability coefficients. A plot of simulated assimilation efficiencies is shown in Figure 6, along with measured values compiled by Muir and Yarechewski (1988)
for a variety of halogenated hydrocarbons and fish species. Additional data sets have been published by Gobas et al. (1988)
and Opperhuizen and Sijm (1990)
. However, a review of sources suggests that there is considerable overlap between these summaries and that given by Muir and Yarechewski (1988)
. An examination of Figure 6
shows that measured assimilation efficiencies generally range from 40 to 80% for compounds with log KOW values less than 6. In the log KOW range from 6.5 to 8, assimilation efficiencies tend to decline with chemical log KOW. Assimilation efficiencies less than 10% have been reported for several compounds with log KOW values between 8 and 10.
Modeled assimilation efficiencies from the present study described a declining trend with chemical log KOW when the maximum BMF was set equal to 5 (Fig. 6). An additional decrease in dietary assimilation efficiency was predicted when BMFs for the log KOW 7.5 and 8 compounds were set equal to 3 and 1, respectively. Generally, however, modeled assimilation efficiencies were higher than estimates reported by other investigators. Most of the feeding studies conducted to date have employed small fish species such as guppies and fathead minnows or juveniles of larger species. Very few studies have been performed using larger animals, and fewer still have employed chemicals that were naturally incorporated into live prey items. Previously, Nichols et al. (2001)
noted that the measured assimilation of [14C] PCB 52 by subadult rainbow trout was higher than any value previously reported for this compound. This finding suggests that the conditions under which the study was performed (species, life stage, feeding rate, the use of naturally contaminated prey items, and so on), and upon which the current PBTK model was based, favor highly efficient chemical uptake.
Throughout this study, modeling efforts were aided by the adoption of several simplifying assumptions. Two of these assumptions, zero growth and no metabolic biotransformation, merit special attention. As indicated previously, BAFs for some hydrophobic compounds have been shown to decline at very high log KOW values (Burkhard, 1998; Thomann, 1989
). Because they arise from the same kinetic processes, this decrease in BAFs is probably associated with a log KOW-dependent decrease in BMFs. One question is whether growth dilution of chemical residues could bring about a decline in BMFs (and by extension, BAFs). The gut model was used to evaluate this question by incorporating a constant rate of growth into steady-state food and water simulations.
Predicted BMFs for all compounds were substantially reduced by the incorporation of low (10 g/year) to moderate (50 g/year) rates of growth when compared with BMFs obtained without growth (Fig. 3). When expressed as percentages of body weight, these growth rates correspond to annual size increases of about 10 and 50%, respectively. The intent of this exercise was to model growth rates expected for adult fish because biomagnification is generally assessed in adult animals. Growth rates in juvenile fish may be much higher than those modeled here. However, feeding rates, dietary preferences, and food conversion efficiency also change as fish grow and develop. A model-based evaluation of these factors is beyond the scope of the present study (and would probably require a bioenergetics-based component to the model). For this effort, it is important to note that, for each growth rate and set of assumed gut permeability coefficients, modeled BMFs increased with chemical log KOW. The conclusion drawn from this analysis is that growth dilution may contribute to a decline in steady-state chemical concentration for any single compound but is unlikely to be responsible for a log KOW-dependent decline in BMFs.
Metabolic biotransformation also has the potential to reduce chemical biomagnification in fish. To investigate this possibility, models developed to describe steady-state food and water exposures were amended to include relatively low rates of metabolism in the liver (0.001 to 0.01/day, on a whole-body basis) or both the liver and tissues of the upper GI tract (Fig. 3). For comparison, Van der Linde et al. (2001)
used a model-based analysis of measured elimination rates to estimate whole-body metabolism rate constants in fish. Rate constants ranging from 0.01 to 0.1/day were estimated for several chlorinated dioxins and furans as well as some PAHs. Metabolism rates for a set of halogenated benzenes and biphenyls could not be estimated but were less than 0.01/day and probably close to zero.
Based on this evaluation, it is clear that metabolism can have a substantial effect on the accumulation of high log KOW compounds. PBTK models are well suited to incorporation of metabolism data from both in vitro and in vivo studies as such data are collected. An important advantage of this approach, when compared to whole-animal bioaccumulation models, is that metabolism may be localized to the tissues and organs where it occurs. In studies with in situ gut preparations, the GI tract was shown to play an important role in metabolizing PAHs by fish, altering their form and limiting systemic bioavailability (Kleinow et al., 1998; Van Veld et al., 1988
). Operating in series with first-pass metabolism in the liver, this activity can substantially reduce bioaccumulation of contaminant residues (James and Kleinow, 1994
; Kleinow and James, 2001
; Van Veld, 1990
). Alternatively, PBTK models could be used to translate metabolism rates determined on a whole-animal basis into tissue-specific metabolism rates, provided that the principal metabolizing tissues were known.
A third assumption employed in this effort is that chemical residues in food can be predicted from an equilibrium chemical distribution between the food item and water. Given this assumption, the BMF is equal to the ratio of a lipid-normalized BAF (for the predator) and a lipid-normalized BCF (for its food), both of which are referenced to the free chemical concentration in water. It is possible, however, that the same considerations that result in biomagnification of chemicals within the predator operate to some extent also in their prey (due to consumption of contaminated food items). Under these circumstances, chemical concentrations in prey could exceed those predicted from an equilibrium chemical distribution with water. Responding to this increased input, chemical concentrations within the predator would tend to rise, increasing the gradient for chemical elimination across the gills. For a high log KOW compound, however, this increase in branchial elimination would be expected to have only a slight effect on the steady-state BMF (defined in this case as a ratio of lipid-normalized BAFs), given the dominant role of dietary uptake in controlling chemical biomagnification.
The processes that control dietary uptake of hydrophobic organic compounds have been studied in several vertebrate systems. For example, Drouillard and Norstrom (2000) exposed ring doves to a mixture of PCB congeners spiked into a pelleted diet. Although there was a slight trend toward decreasing assimilation efficiency with chemical log KOW, all of the compounds were taken up with high efficiency. Moreover, the kinetics of chemical appearance in blood did not vary with log KOW, as would be expected if diffusion rates varied with relative hydrophobicity. Based on these observations, it was concluded that chemical uptake from the small intestine is not rate-limited by simple diffusion of contaminant molecules but is controlled by the collisional contact of lipid micelles with intestinal cell membranes.
The dietary absorption of several hydrophobic compounds by humans was well correlated with blood lipid levels but could not be explained on the basis of a diffusion gradient between blood and contents of the GI tract (Schlummer et al., 1998). To account for this discrepancy, the authors hypothesized that lipid uptake by intestinal tissues creates a transient inward gradient for diffusion (this effect was termed the "fat flush" hypothesis) and proposed a two-step model for chemical uptake in the GI tract, with absorption (small intestine) and elimination (large intestine) occurring as distinct processes, both of which are controlled by diffusion gradients. Support for this hypothesis was obtained in studies demonstrating the following: (1) that an increase in the chemical capacity of the gut contents leads to increased fecal elimination of ingested compounds (Geusau et al., 1999
; Moser and McLachlan, 2001
); and (2) that net absorption efficiency depends on the chemical concentration in the diet relative to that of the exposed individual, becoming maximal at high dietary intake levels (Moser and McLachlan, 2001
).
The role of lipid micelles in dietary uptake of hydrophobic compounds by fish was studied by Doi et al. (2000). Using an in situ channel catfish intestinal preparation, the bioavailability of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) was shown to be related to the composition of mixed micelles. This finding suggests that dietary uptake may depend, to some extent, on both the chemical capacity of lipid micelles as well as interactions of component molecules with the brush boarder membrane. However, Doi et al. (2000)
also observed that chemical pretreatment of catfish decreased the uptake rate for PCB 77, suggesting that simple diffusion plays some role in controlling dietary uptake of hydrophobic compounds. Previously, Vetter et al. (1985)
used autoradiographic methods to show that [14C]-benzo(a)pyrene remained associated with lipid throughout lipolysis, lipid absorption, and the formation of intracellular fat droplets in the gut epithelium. This technique underscores the close association between hydrophobic compounds and lipids throughout digestion but provides little information on processes that actually limit the rate of absorption at the gastrointestinal epithelium.
Contradictory results have been obtained by investigators who varied the amount of lipid added to prepared fish diets. In work cited by Van Veld (1990), the addition of triglyceride increased the intestinal absorption of DDT, benzo[a]pyrene, and a model PCB in killifish. Maximum absorption occurred when fish were fed a triglyceride-enriched diet designed to mimic the characteristics of a natural diet. However, high-fat diets are also known to slow the digestion of lipids by fish. Under these circumstances, undigested lipid may compete with lipid micelles for hydrophobic organic contaminants, reducing dietary assimilation efficiency (Van Veld, 1990
). The impact of dietary lipid content on chemical uptake efficiency may also depend partly on chemical hydrophobicity. Working with goldfish, Gobas et al. (1993a)
found that dietary uptake of some very hydrophobic compounds (log KOW > 6.3) declined with an increase in dietary lipid content, while uptake of some moderate-to-high log KOW compounds (4.5 < log KOW < 6.3) did not vary among treatment groups.
The model used in the present study provides a mechanistic basis for interpreting BMFs and assimilation efficiencies determined in both experimental and field studies with hydrophobic compounds. The model structure, and in particular the use of time-dependent changes in chemical partitioning to gut contents and tissues, simulates the "fat flush" effect proposed by Schlummer et al. (1998) and the increase in chemical activity of gut contents hypothesized by Gobas et al. (1988)
. In a companion report, the model was used to describe the results of a one-time feeding study with [14C] PCB 52 (Nichols et al., 2004
). Based on this analysis, it was suggested that a resistance to chemical flux prevented the establishment of a chemical equilibrium between gut contents and tissues. Furthermore, it was suggested that diffusion across the gastrointestinal epithelium was unlikely to be the sole determinant of [14C] PCB 52 uptake rate. In the present study, we used the same model to simulate chronic exposures to [14C] PCB 52 and a set of hypothetical high log KOW compounds. The results of this study suggest that the resistance to dietary uptake increases substantially with chemical log KOW and that this increase is responsible for the log KOW-dependent decline in assimilation efficiency observed in several feeding studies with fish. The factors that limit the rate of dietary uptake in fish remain unknown. The present work suggests, however, that one or more of these factors varies with chemical log KOW. This conclusion is consistent with an earlier model-based evaluation of dietary assimilation efficiencies in fish (Gobas et al., 1988
).
A comparison of data from fish, birds, and mammals suggests that factors that control dietary uptake of hydrophobic organic compounds differ among taxa. The GI tract of fish, in relation to animal size, is much shorter than that of most higher vertebrates. Under these conditions, it may not be possible to achieve the spatial separation of diffusive uptake and elimination processes suggested by studies with humans (Schlummer et al., 1998). Experimental support for this conclusion was provided by Gobas et al. (1999)
in studies with rainbow trout fed a diet contaminated with PCB 155. The fugacity of PCB 155 within the intestinal tract was double that of the food but did not differ significantly among three intestinal segments. Micelle-mediated transport may play a role in controlling the rate of chemical delivery to absorbing gut surfaces in fish (Doi et al., 2000
) but does not appear to be the dominant rate-controlling process for uptake indicated in studies with ring doves (Drouillard and Norstrom, 2000
).
ACKNOWLEDGMENTS
The authors thank Dr. Russell Erickson and Dr. Frank Gobas for their insightful reviews of this manuscript. The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
NOTES
1 To whom correspondence should be addressed at U.S. Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, MN 55804. Fax: (218) 529-5003. E-mail: nichols.john{at}epa.gov.
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