Environ. Sci. Technol. 4984, 18, 79-81
Wade, T. L.; Quinn, J. G. Org. Geochem. 1979,1,157-167. Blumer, M.; Souza, G.; Sass,J. Mar. Biol. 1970,5,195-202. Philp, R. P.; Gilbert, T. D.; Friedrich, J. Geochim. Cosmochim. Acta 1981,45, 1173-1180. Streibl, M.; Herout, V. In “Organic Geochemistry Methods and Results”;Eglinton, G.; Murphy, M. T. J.,Eds.; Springer: New York, 1969; pp 402-424. Grantham, P. J.; Douglas, A. G. Geochim. Cosmochim. Acta 1980,44,1801-1810. Bendoraites, J. G. In “Advances in Organic Geochemistry”; Tissot, B.; Bienner, F., E&.; Editions Technip: Paris, 1973; pp 209-224. Barrick, R. C.; Hedges, - J. I. Geochim. Cosmochim. Acta 1981,45, 381-392. Ekweozor, C. M.; Okogun, J. I.; Ekong, D. E. U.; Maxwell, J. R. Chem. Geol. 1979,27, 29-37. Ensminger, A.; Van Dorsselaer, A.; Spyckerelle, C.; Albrecht, P.; Ourisson, G. In “Advances in Organic Geochemistry 1973”;Tissot, B.; Bienner, F., E&.; Editions Technip: Paris, 1974; pp 245-260.
(29) Blumer, M.; Mullin, M. M.; Thomas, D. W. Helgol. Wiss. Meeresunters. 1964, 10, 187-201. (30) Han, J.; Calvin, M. Proc. Natl. Acad. Sci. U.S.A. 1969,64, 436-443. (31) Barrick, R., University of Washington, Seattle, WA, personal communication, 1982. (32) Mullineaux, D. R. Geol. Surv. Prof. Pap. (U.S.) 1970, No. 672, 1-92. (33) Curl, H., Jr.; Baker, E. T.; Cline, J. D.; Feely, R. A. Estuarine and Coastal Pollutant Transport and Transformation, the Role of Particulates, NOAA/OMPA Section 202 Research Program, Pacific Marine Environmental Laboratory, Seattle, WA, 1981, Annual Report, pp 1-104.
Received for review August 2,1982. Revised manuscript received J u l y 21,1983. Accepted August 15,1983. This study was supported by the Office of Marine Pollution Assessment (section 202) of N O A A . Contribution No. 545 f r o m the N O A A I E R L Pacific Marine Environmental Laboratory.
A Simple Method for Determining Bioconcentration Parameters of Hydrophobic Compounds Sujit Banerjee,” Richard H. Sugatt, and Dean P. O’Grady Life and Environmental Sciences Division, Syracuse Research Corporation, Syracuse, New York 13210
A method for the determination of uptake and clearance rate constants during the bioconcentration of toxicants in fish is described and validated. The procedure is limited to stable lipophilic compounds and requires the exposure of fish to an aqueous solution of toxicant under static conditions and measurement of toxicant loss from water with time. The rate constants are obtained from the time-concentration profile with the use of an iterative computer program. Bioconcentration factors obtained for pentachlorobenzene, two isomers of tetrachlorobenzene, and l,4-diiodobenzenewere consistent either with previous determinations or with expectations based on the octanol-water partition coefficient. The measurements with pentachlorobenzene were made over a 100-fold range of chemical concentration. No dependence of the bioconcentration factor on chemical concentration was observed, in accordance with a simple first-order uptake and depuration model, but in contrast to reported data on chlorinated diphenyl ethers and brominated toluenes.
Introduction The bioconcentration factor (BCF) of an organic compound in fish is generally measured in one of two ways. In the first, fish are exposed to an aqueous solution of the toxicant in a static system, and the concentration of the chemical in fish and water is monitored until equilibrium is reached (1). The concentration ratio of the compound in fish to water then represents the BCF. The disadvantages of the method are that potentially toxic metabolites may accumulate, the test must be continued to equilibrium, and rate data are generally not obtained. The second method (1) utilizes a flow-through approach where the toxicant concentration is held constant, and the accumulation of the compound in fish is measured until equilibrium is reached. The fish are then transferred to clean *Address correspondence to this author at the Safety and Environmental Protection Division, Brookhaven National Laboratory, Upton, NY 11973. 0013-936X/84/09 18-0079$01.50/0
flowing water, and the release of the toxicant from the fish is followed until the material is substantially cleared. Analysis of the concentration-time profiles obtained from the uptake and clearance phases then yields the corresponding rate constants. Both methods are fairly time consuming, and despite the modification of Branson et al. (2) which condenses the flow-through procedure, a BCF measurement remains a major experimental undertaking. We have developed a simple, reliable, and inexpensive method of measuring bioconcentration factors and rate constants as a prelude to a broader study on the mechanism of bioconcentration of persistent organic compounds, and in this paper we describe our approach and validate it through measurements with a number of halobenzenes.
Experimental Section Fish were obtained commercially and were held for at least 14 days prior to use. Test solutions were prepared in dechlorinated municipal water by shaking excess chemical with water for 24 h, filtering the solutions, and diluting aliquots of the filtrate to 3 L in l-gal vessels. The final chemical concentration was at least 5 times below the solubility limit. The solutions were allowed to stand overnight, and the experiments were initiated by the addition of four to eight fish which were starved for 48 h before use. The approximate weights of the fish used were as follows: rainbow trout (Salmo gairdneri), 0.1-0.6 g; bluegill sunfish (Lepomis macrochirus), 0.1-0.5 g; guppy (Poecilia reticulata),0.1-0.4 g. The loading factor ranged between 0.37 and 1.5 g/L and averaged 1.08 g/L. The solutions were not aerated to minimize loss of material through volatilization, but the dissolved oxygen concentration, monitored with a YSI 54A oxygen meter, remained at 60% or more of saturation throughout the experiments. The solutions were sampled initially and then periodically throughout the exposure period which lasted for 2 days. Twelve to 18 samples were taken, with the sampling frequency approximating that shown in Figure 1. The samples (250 pL) were mixed with hexane (1mL) in 2-mL vials which were sealed and shaken vigorously. The hexane was then ana-
0 1984 American Chemical Society
Environ. Sci. Technol., Vol. 18, No. 2, 1984 79
Table I. Bioconcentration Parameters of Some Halobenzenes k , x lo-', h
k , x lo2, h-'
BCF
n
lipid, %
literature BCF (flow through)
bluegill sunfish (22 "C) 1,2,3,54etrachlorobenzene 7.4 + 2.8 2.6 i 1.2 2900 f 300 3 1.5 i 0.2 1800a pentachloro benzene 11+3 2.1 + 0.6 5100 t 300 3 1.5 + 0.2 3400a rainbow trout (15 " C ) 1,2,3,4-tetrachlorobenzene 14 i. 3 2.1 t 0.5 5000 * 500 5 2.gb pentachloro benzene 17t 5 3.6 i. 1.4 7100 t 2100 13 1.8 i 0.9 guppy (15 "C) 1,4-diiodobenzene 12+4 5.0 t 1.1 2500 i 900 3 0.8 i 0.2 pentachloro benzene 9.8 t 2.8 1.4 * 0.5 7300 + 2600 4 2.8 * 1.1 7400 i 1400' a From ref 5. The uncertainty in BCF was not cited. The uncertainty in percent lipid is unavailable since the fish were pooled and a single analysis was performed. ' From ref 4. The BCF was adjusted for difference in lipid content.
lyzed directly by gas chromatography with electron capture detection (GC-EC) using a HP 5840 instrument fitted with a 6 f t X 1/4 in. glass column containing 1.5% SP2250/ 1.95% SP 2401 on Supelcoport 100/120. At the end of the experiment, the fish in each vessel were pooled and homogenized in hexane (25 mL), and the slurry was mixed with preextracted sand (5 g) and NaZSO4(10 g) and extracted with hexane (175 mL) in a Soxhlet apparatus. Approximately 60% of the extract was evaporated to dryness in a Kuderna-Danish apparatus, and the weight of the extractable liquid was obtained from the residue. The remainder of the extract was concentrated in a KudernaDanish assembly, and the concentrate was analyzed for the test chemical after Florisil cleanup. It was found that 98 f 9% of the material (averaged over all the experiments) lost from water was recovered in the fish, indicating that bioconcentration was the only significant process responsible for the removal of chemical from water. Control vessels with no fish were included in the measurements and were sampled and analyzed along with test solutions. No loss of compound from the vessels was observed, as expected from the mass balance established from the fish analysis. In order to verify that the compound associated with the fish was not adsorbed to the surface of the fish, dead guppies were added to a pentachlorobenzene solution at 15 OC, and the solution was analyzed prior to fish addition and then continuously for 33 h. The chemical cohcentration remained invariant (within 12% 1, thereby discounting the importance of surface adsorption. The concentration-time data were reduced by an iterative computer program (STATIC) based on Kim's procedure (3) and written in Basic. A listing of the program is available upon request from the authors.
Results and Discussion The bioconcentration of a toxicant from water can be defined by eq 1 in what amounts to a simple one-comfish
+ toxicant Z fish-toxicant kz kl
(1)
partment pharmacokinetic model. This model assumes that metabolic and abiotic processes do not remove the toxicant from the system during the time frame of the measurement. This assumption is reasonable for shortterm exposures to those highly persistent lipophilic compounds that are of primary environmental concern. Under these conditions the instantaneous toxicant concentration [SI,can be expressed as in eq 2 where F represents the
mass of fish per unit mass of water and [SI,is the initial toxicant concentration. Thus, if toxicant concentration 80
Environ. Scl. Technol., Vol. 18, No. 2, 1984
100 l I
\
A
20
30
40
HOURS
Flgure 1. Disappearance of pentachlorobenzene from water in the presence of (0)2.6244 g and (A)4.6621 g of rainbow trout in 3 L of water.
is monitored as a function of time, then kl, kz, and the BCF ( k l / k z )can be obtained from eq 2. In order to validate the procedure we conducted BCF measurements on four halobenzenes using three species of fish. The concentration-time profiles were well defined by eq 2 as illustrated in Figure 1 with some of the data obtained with pentachlorobenzeneand rainbow trout. Our results are summarized and compared to literature results obtained with flow-through measurements in Table I. The agreement of our value with that of Konemann and van Leeuwen ( 4 ) for pentachlorobenzene bioconcentration in guppies is excellent. Comparison of our results with bluegill sunfish to those of Veith et al. (5)is more difficult since neither the lipid content nor the uncertainty in BCF was reported. Nevertheless, the differences in BCF are less than a factor of 2, and the tetrach1orobenzene:pentachlorobenzene BCF ratio obtained by us (0.57) is very similar to that obtained by Veith et al. (0.53). The two remaining compounds, 1,Cdiiodobenzene and 1,2,3,4tetrachlorobenzene, have near identical octanol-water partition coefficients (6),and if bioconcentration is represented by a simple physical model, then the BCF's of these compounds should be less than that of pentachlorobenzene by a factor of 2-3 (7). This is indeed the case, and our method would appear to hold promise for more general application. The major advantage of the proposed method over those in current use is that of economy. Our results were ob-
C
I 10
0 , 0
I
I
20
30
I
40
I
50
HOURS
Flgure 2. Concentration-time profiles obtained with the following: (A) k , = 100 h-’, k , = 0.04 h-’; (B) k , = 100 h-’, k , = 0.02 h-I; (C) k , = 200 h-I, k , = 0.02 h-‘. F = 0.001 in all cases.
Table 11. Effect of Initial Chemical Concentration o n the BCF of Pentachlorobenzene in Rainbow Trout at 1 5 “C initial pentachlorobenzene k , x lo‘,, k , X l o 2 , concn, ppb h-’ h -’ BCF 0.625 7.64 32.8 70.8 106
1.3 1.4 1.6 1.2 1.1
3.2 3.1 2.3 2.1 2.0
4000 4300 6900 5800 5500
tained over 3 days, whereas comparable flow-through studies require well over a month. Second, our procedure relies primarily on water analysis rather than on the much more expensive fish analyses required for the flow-through procedure. The main drawback of the technique is that of limited applicability in that the method can only be used for stable nonvolatile hydrophobic compounds which aire nontoxic at exposure concentrations and are not significantly metabolized during the time frame of the experiment. Furthermore, sensitive analytical procedures must be available, and the analyses must be made with a high degree of precision. Accurate measurement of the depuration rate constant (k,) requires that sampling arid analysis be continued until a substantial portion of the process is complete. Curves A and B in Figure 2 show the effect of a doubling of k2 with all other parameters held constant, and a comparison of curves B and C in Figure 2 reveals the influence of a similar doubling of kl. Clearly, the profile is much less sensitive to changes in k2,particularly in the early stages of the process, and thus data
should be collected over at least three half-lives. Despite these limitations, the method should be applicable to a large number of environmentally relevant compounds, e.g., for halogenated derivatives which are likely to be persistent, lipophilic, and easily quantifiable at low levels. The procedure is particularly well suited for the study of environmental factors such as the effect of sediment (8), humic materials (8),cocontaminants (9),and temperature variations (10) on BCF. As one illustration, we have used the approach to determine if the bioconcentration of pentachlorobenzene varied with the initial chemical concentration in water. Our experiments were prompted by Zitko and Carson’s findings (11)that the BCF for chlorinated diphenyl ethers and brominated toluenes varied with the chemical concentration in water. This suggests that the bioconcentration of these compounds include processes additional to simple transport from water to lipid. Our BCF results for pentachlorobenzene shown in Table I1 are invariant over a 100-fold change in initial chemical concentration, and hence, a simple one-compartment model (7,8) would seem to suffice for this compound. Registry No. Pentachlorobenzene, 60893-5; 1,4-diiodobenzene, 624-38-4; 1,2,3,5-tetrachlorobenzene,634-90-2; 1,2,3,4-tetrachlorobenzene, 634-66-2.
Literature Cited (1) “Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals”;Organization for Economic Cooperation and Development: Paris, 1981. (2) Branson, D. R.; Blau, G. E.; Alexander, H. C.; Neely, W. B. Trans. Am. Fish. SOC.1975, 104, 785. (3) Kim, N. J. Chem. Ed. 1970,47, 120. (4) Konemann, H.; van Leeuwen, K. Chemosphere 1980,9,3. (5) Veith, G. D.; Macek, K. J.; Petrocelli, S. R.; Carroll, J. In “Aquatic Toxicology”; Eaton, J. G.; Parrish, P. R.; Hendricks, A. C.; Eds.; American Society for Testing and Materials: Philadelphia, PA, 1980. (6) Yalkowsky, S. H.; Valvani, S. C. J. Pharm. Sci. 1980,69, 1912. (7) Mackay, D. Environ. Sci. Technol. 1982, 16, 274. (8) Spacie, A.; Hamelink, J. L. Environ. Toxicol. Chen. 1982, 1, 309. (9) Linden, G.; Bergman, H. 3rd Annual Meeting of the Society for Environmental Toxicology and Chemistry, 1982; Abstracts, p 105. (10) Spigarelli, S. A.; Thommes, M. M.; Prepejchal, W. Environ. Sci. Technol. 1983, 17, 88. (11) Zitko, V.; Carson, W. G. Chemosphere 1977, 6, 293.
Received for review August 16,1982. Revised manuscript received April 27,1983. Accepted August 25,1983. Research supported by EPA Grant R808613.
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