Potential and Realized Bioconcentration. A Comparison of Observed and Predicted Bioconcentration of Azaarenes in the Fathead Minnow (Pimepha/espromelas) George R. Southworth," Catherine C. Keffer, and John J. Beauchamp? Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Many potentially hazardous organic compounds associated with synthetic-fuel production possess physicochemical properties that suggest a high potential for bioconcentration in aquatic organisms. Some of these compounds have been found to be metabolized by fish and other aquatic organisms. In this study, the bioconcentration of four polycyclic bases, or azaarenes, in fathead minnows was compared with predicted bioconcentration behavior estimated from octanolwater partitioning and with observed bioconcentration of azaarenes in Daphnia pulex. Bioconcentration in fish of those azaarenes with moderate to high (?lOOO-fold) predicted bioconcentration factors was found to be far lower than predicted or observed in D. pulex, where the bioconcentration factor is the ratio of concentration of contaminant in an organism to the aqueous concentration of the contaminant at equilibrium. This deviation was shown to be due to the rapid metabolic alteration of the azaarenes within the fish, demonstrating that the rates of such processes are fast enough to play a large role in determining bioconcentration of organics. Introduction The uptake and concentration of organic chemicals from water by fish is well correlated with measures of partitioning of the chemicals between water and nonpolar solvents such as 1-octanol. This relationship can be used to predict the potential for concentration of organics based on their wateroctanol partitioning ( 1 , 2 ) . Such predictions provide estimates of the degree of bioconcentration possible, but they do not indicate that such a degree of bioconcentration will be attained. Rapid metabolic alteration of a substance by organisms may result in its bioconcentration to a lesser extent than predicted. Many organic chemicals found in synthetic-fuel (shale oil, coal liquefaction, or gasification) products and effluents have high calculated octanol-water partition coefficients. Some of these substances, primarily high-molecular-weight polycyclic aromatic hydrocarbons and their nitrogen- and sulfur-containing analogues, would be expected to bioconcentrate as much as 10 000-fold in fish, based on their partition coefficients. Because some members of these chemical classes, such as benz[a]anthracene, benzo[a]pyrene, and dibenz[a ,h]acridine, are carcinogenic in animals, their possible accumulation in human-food organisms is a cause for concern. Numerous studies have demonstrated that some species of fish possess inducible enzyme systems (mixed function oxidases) capable of transforming polycyclic aromatic hydrocarbons ( 3 , 4 ) .Exposures of fish to radioactively labeled polycyclics produced evidence of substantial conversion to metabolites (5-8). The presence of such metabolic elimination mechanisms suggests that estimates of bioconcentration potential based on lipophilicity may be far too high if the rate of metabolic degradation is rapid. However, laboratory studies of the bioconcentration of acridine, a polycyclic nitrogencontaining substance subject to metabolism by fish ( 8 ) ,and several polycyclic aromatic hydrocarbons likely to be me+.Computer Sciences Division, Union Carbide Corp. Nuclear Division.
tabolized ( 2 ) produced concentration factors consistent with estimations based on octanol-water partitioning. The first objective of this study was to compare the bioconcentration of azaarenes in the fathead minnow, Pimephales promelas, with predictions based on octanol-water partitioning and also with measures of bioconcentration in Daphnia pulex. Azaarenes are a class of nitrogen-containing polycyclic aromatic compounds associated with synthetic-fuel production. Some members of this class are animal carcinogens, and previous studies have indicated a moderate degree of bioconcentration (-4OOX) in Daphnia pulex for intermediatemolecular-weight compounds in this class (9). A second objective was to estimate the bioconcentration kinetics of dibenz[a,h]acridine in fathead minnows, including the rate of metabolic alteration of the compound, and to assess the importance of metabolic degrading in determining the degree of bioconcentration observed. Dibenz[a,h]acridine has demonstrated carcinogenic properties in animals (IO)and is predicted to bioconcentrate to a high degree in fish, based on its calculated octanol-water partitioning. Methods The bioconcentration of quinoline and dibenz[a,h]acridine was studied by using static exposure of fathead minnows (Pimephales promelas) to 14C-labeled compounds. The dibenz[a,h]acridine (1,2-benzo-14C(u),3.01 X 108 Bq/mmol, >99% radiochemical purity) and quinoline (phenyl-14C,1.53 X IO8 Bq/mmol, >99% radiochemical purity) were obtained from New England Nuclear Corp. Sixteen minnows (mean weight f one standard deviation, 75 f 19 mg) were exposed to an initial concentration of 8.8 pg/L dibenz[a,h]acridinein 8 L of spring water at 22 f 1 "C. Duplicate samples of one fish each were taken a t varying intervals through 96 h. The dibenz[a,h]acridine concentration declined -37% over the 4 days. The fish were killed, weighed, and homogenized in 10 mL of methanol by using a tissue homogenizer. One gram of anhydrous sodium sulfate was added, and the resulting suspension was shaken for 30 min on a wrist-action shaker. It was then centrifuged, and an aliquot of the methanol was withdrawn for liquid scintillation counting of 14C. A 2-mL aliquot of methanol was evaporated to dryness a t room temperature under a stream of air and redissolved in 5 mL of hexane. This was extracted with a single 5-mL volume of 1 X M N a ~ C 0 3and , aliquots of the hexane and aqueous phases were counted by liquid scintillation. The aqueous phase was neutralized with tartaric acid prior to counting. The dibenz[a,h]acridine partitioned into the hexane phase, and the metabolites into the aqueous phase. Thin-layer chromatography of the hexane phase on silica gel with chloroformmethanol-acetic acid ( 9 0 5 5 v/v) yielded a single spot that cochromatographed with dibenz[a,h]acridine.Assay of the dibenz[a,h]acridine content of the hexane phase by fluorescence spectrophotometry was in agreement with measured I4C content. All 14C in the hexane phase was therefore assumed to be dibenz[a,h]acridine. Comparison of 14Cin the methanol extract with 14C recovered after evaporation and solvent partitioning indicated that losses in this procedure were negligible. The solids remaining after centrifugation were resuspended in clean methanol and centrifuged again. The methanol was
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decanted, and the solids were allowed to air dry in the centrifuge tube for 2-3 days until they could be removed as a single pellet. The pellet was then oxidized in a Packard Model 306 sample oxidizer, and the C02 evolved was determined by liquid scintillation counting. Dibenz[a,h]acridine in the water was analyzed a t each sampling by fluorescence spectrophotometry, using the method of standard additions. The results agreed reasonably well with total hexane extractable 14C in the water. Quinoline bioconcentration was studied by using the same procedure. Initial quinoline concentration was 190 pg/L (unlabeled quinoline was added to 14C-labeled quinoline for this study). Somewhat larger fish were used (140 f 20 mg), and sampling was extended through 144 h. Quinoline in the water was quantitated by using fluorescence spectrophotometry, while quinoline in the fish was estimated by total 14C in the methanol extract (thin-layer chromatography/autoradiography of methanol and hexane extracts indicated that virtually all extractable 14C was quinoline). The exposure solution was renewed a t 24 h. The aqueous quinoline concentration remained essentially constant a t 186 f 12 pg/L for the first 72 h, and then declined to 136 pg/L a t 144 h. The kinetic description of the bioconcentration process was a modification of the simple two-compartment, first-order equilibrium model commonly used in bioconcentration studies (2, 9, 1 2 ) . The elimination rate was conceptualized as comprising two components: (1)a physical elimination rate governed by passive diffusion processes and differential affinities for water and tissue and (2) a metabolic elimination rate governed by physiological processes within the organism. The bioconcentration process is thus described by d[B]fish/dt = C ~ [ B ] H~O (hi
+ kz)[BIfish
(1)
where [B]flshis the concentration of azaarene in the fish (Bq 14Cg-l) wet weight (1Bq = 2.56 X lo-" Ci), C1 is the uptake coefficient (h-l), [B]H~o is the concentration of azaarene in the water (Bq 14Cg-l), k l is the physical elimination coefficient (h-l), and k2 is the metabolic coefficient (h-l). In a similar fashion, the accumulation of metabolite in fish tissues can be expressed as where h3 is the metabolite excretion coefficient (h-l), [M]fish is the concentration of metabolite in the fish (Bq 14Cg-l) wet weight, C2 is the metabolite uptake coefficient (h-l), and [ M ] H ~isOthe concentration of metabolite in the water (Bq l4C g-l). Since [B]H~o decreased slightly throughout the course of the experiment, [ B ] Hwas ~ ~described empirically as a function of time by fitting an exponential function to the observed (9).The function was then inserted into eq values of [B]H~o (1) before its integration, and estimates for the coefficients C1 and ( k l h z ) were obtained by nonlinear least squares. The coefficients k l and h2 are not estimated individually a t this step. The equilibrium bioconcentration factor was estimated by the ratio CJ(K1 kz), and a Taylor series approximation was used to estimate the standard error of the ratio (12). The same approach was used to evaluate the accumulation of metabolite (eq 2). For this analysis, C ~ [ M ] Hwas , ~ assumed to be negligible with respect to kz[B]fish.[B]fishwas described as a function of time using the integral solution of eq 1and the coefficients determined from the uptake experiment. This function was inserted into eq 2, which was then integrated and solved for k2 and h3 by using a nonlinear least-squares fit of the data on the accumulation of metabolites in the fish as a function of time. The bioconcentration of dibenz[a,h]acridine by D a p h n i a p u l e x was investigated under static conditions by using fluorescence spectrophotometry as described previously (9, 13).
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The bioconcentration data for isoquinoline, acridine, and benz[a]acridine were obtained from Southworth et al. ( 9 ) , while bioconcentration data for acridine and benz[a]acridine in fathead minnows are from Southworth et al. (8) and Southworth et al. ( 2 4 ) . Results
The bioconcentration of the azaarenes by fathead minnows was similar for acridine, benz[a]acridine, and dibenz[a,h]acridine, with all exhibiting bioconcentration factors of -100 (Figure 1).Quinoline was accumulated much less, exhibiting a bioconcentration factor of 8. The concentration factors predicted for these azaarenes in fathead minnows (Figure l),by using the expression developed by Veith ( 2 ) , differ considerably from the measured values for the compounds having higher predicted concentration factors, benz[a]acridine and dibenz[a,h]acridine, but agree well with measured values for acridine and quinoline. Dibenz[a,h]acridine was bioconcentrated about two orders of magnitude less than predicted, while benz[a]acridine was accumulated to about one-tenth of the predicted value. Such deviations would be expected if metabolic alteration played a larger role in determining the disposition of a substance within the fish than did simple partitioning between water and lipids. The bioconcentration of azaarenes by D a p h n i a p u l e x follows a relationship quite similar to that for predicted bioconcentration in fathead minnows (Figure 1). Unlike the measured values for bioconcentration in the fathead minnows, however, concentration factors observed in D . p u l e x increased steadily with increasing octanol-water partition coefficient. The higher-molecular-weight compounds were bioconcentrated much more in D. p u l e x than in fish. Dibenz[a,h]acridine was bioconcentrated -3500-fold in D. p u l e x , but only 80-fold in fathead minnows. Minnows exposed to 14C-labeled dibenz[a,h]acridine rapidly accumulated it to the relatively low degree noted above, with apparent equilibrium attained
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Bioconcentration and metabolism of dibenz[a,b]acridine by the fathead minnow (Pimepbales promelas) at 2 2 O C . Bioconcentration factor is the ratio of (’4C)dibenz[a,b]acridine or I4C metabolites concentration (Bq g-’) in fish (wet weight) to (I4C)dibenz[a,b]acridine concentration (Bq g-’) in water. Curves are fit by least-squares regression procedure described in text. Error bars are f one standard error of the mean. Figure 2.
within 24 h (Figure 2). Metabolite concentrations in the fish continued to build up and exceeded the level of dibenz[a,h]acridine in the tissues. The uptake rate coefficient (with associated standard error) for dibenz[a,h]acridine(C1) in fathead minnows was 11.48 f 4.25 h-l, while the elimination coefficient ( k l k2) was 0.140 f 0.057 h-l. The equilibrium concentration factor ( C l / ( k l + k2)) was 107 f 16. The estimated rate coefficient for metabolic alteration (k2)was 0.183 f 0.055 h-l, indicating that metabolic degradation was an important factor in determining bioconcentration. The rate coefficient for elimination of the metabolite ( k 3 ) was 0.041 f 0.016 h-l. Since the estimates of k2 and ( k l k 2 ) are similar and statistically indistinguishable, it appears as though passive elimination ( k 1 ) is small in comparison to metabolism.
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Discussion
The results of this study indicate that lipophilicity plays a much greater role in determining the bioconcentration of azaarenes in Daphnia pulex than it does in fathead minnows. The bioconcentration of azaarenes by fathead minnows was not well correlated with a measure of lipophilicity, octanolwater partitioning, and expressions developed to predict bioconcentration of organics in fish based on octanol-water partitioning, and expressions developed to predict bioconcentration of organics in fish based on octanol-water partitioning greatly overestimated the degree of accumulation for the more lipophilic compounds. Bioconcentration potential is defined here as a property of a chemical compound that is a measure or indicator of the extent to which it may accumulate in aquatic organisms. The actual bioconcentration behavior of a chemical in an aquatic environment is determined by its bioconcentration potential and by properties of the organisms present in that environment, such as lipid content and capability for metabolizing the substance. Ideally, bioconcentration potential should enable the estimation of a range for maximum bioconcentration, placing upper bounds on the estimated degree to which a material may be accumulated in any kind of aquatic organism. The octanol-water partition coefficient has proven to be useful as a measure of bioconcentration potential ( 2 ) . However, measurement of octanol-water partition coefficients is not always a simple procedure, particularly at high log P
0 4 ) (15).The estimation of log P by high-performance liquid chromatography appears to be a good method, but the precision of log P estimates is f 2 3 % (16). Calculated values for log P agree well for structurally simple molecules with few polar constituents, such as the homologous series or polyaromatic compounds used in this study, but appear to be of low reliability for more complex molecules (15).Rather than attempt to measure log P , biological measures of bioconcentration may be used as indicators of bioconcentration potential. For an organism to serve as a measure of bioconcentration potential, the observed degree of bioconcentration of a substance in a particular species must indicate that the properties of the substance are such that it will concentrate to approximately the same extent or less in other aquatic organisms. Results of this study show that fathead minnows are clearly inappropriate for such a role. Daphnia p u l e x appears to agree well with the octanol-water partition coefficient as an indicator of bioconcentration potential, but any possible advantages of the biological measure over the partition coefficient cannot be ascertained from a study of only four compounds. While correlations between bioconcentration and octanol-water partitioning, or studies on organisms such as D a p h n i a , can be used to determine whether a substance possesses physical properties which enable it to be bioconcentrated, only experimental measurement can establish the actual degree of bioconcentration likely to occur in fish and other aquatic organisms. If other potentially hazardous compounds associated with synthetic-fuel production are as susceptible to metabolic alteration as were these azaarenes, the problem of accumulation of hazardous substances in fish may not be as significant as is suggested by the physical properties of the materials. However, the accumulation of potentially hazardous metabolites may itself be of concern. L i t e r a t u r e Cited (1) Neely, W. R.; Branson, D. R.; Blau, G. E. Enuiron. Sci. Technol.
1974,8, 1113-5. ( 2 ) Veith, G. D.; DeFoe, D. L.; Bergstedt, B. V. J . Fish. Res. Board
Can. 1979,36, 1040-8. (3) Payne, J. F.; Penrose, W. R. Bull. Enuiron. Contam. Toxicol. 1975, 14, 112-5. (4) Payne, J. F. Science 1976,191, 945-6. (5) Lee, R. F.; Sauerheber, R.; Dobbs, G. H. Mar. Biol. 1972, 17, 201-8. (6) Melancon, M. J., Jr.; Lech, J. J. A S T M S p e c . Tech. Publ. 1979, 667, 5-22. (7) Roubal, W. T.; Collier, T. K.; Malins, D. L. Arch. Enuiron. Contam. Toxicol. 1977,5, 513-29. (8) Southworth, G. R.; Parkhurst, B. R.; Beauchamp, J. J. Water Air Soil Pollut., in press. (9) Southworth, G. R.; Beauchamp, J. J.; Schmieder, P. K. Enuiron. Sci. Technol. 1978,12, 1062-6. (10) Foirchild, E. J.; Lewis, R. J.; Tatken, R. L. “Registry of Toxic Effects of Chemical Substances”; USDHEW: Cincinnati, OH, 1977. (11) Hamelink, J. L.; Waybrant, R. C.; Ball, R. C. Trans. A m . Fish. SOC.1971,100, 207-14. (12) Davies, 0. L.; Goldsmith, P. L. ‘‘Statistical Methods in Research and Production”, 4th ed.; Hafner Publishing Co.: New York, 1972; p 54. (13) Southworth, G. R.; Beauchamp, J. J.; Schmieder, P. K. Water Res. 1978,12, 973-7. (14) Southworth, G. R.; Keffer, C. C.; Beauchamp, J. J. Water Res., in press. (15) Leo, A. J. In Struct.-Act. Correl. S t u d . Toxic. Bioconc. Aquat. Org., Proc. S y m p . 1975, 151-76. (16) Veith, G. D.; Austin, N. M.; Morris, R. T. Water Res. 1979,13, 43-7. Received for review June 9,1980. Accepted August 25,1980. Research sponsored by the Office of Health and Enuironmental Research, U.S. Department of Energy, under contract W-7405-eng-26with Union Carbide Corp. Publication No. 1595, Environmental Sciences Diuision, ORNL.
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