Environ. Sci. Technol. 1992, 26, 1197-1201
(26) Chum, H. L.; Johnson, D. K.; Tucker, M. P.; Himmel, M. E. Holzforschung 1987,41, 97-108. (27) Johnson, D. K.;Chum, H. L.; Hyatt, J. A. In Lignin, Properties and Materials; Glasser, W. G., Sarkanen, S., Eds.; ACS Symposium Series 397; American Chemical Society: Washington, DC, 1989; pp 109-123. (28) Jokela, J.; Salkinoja-Salonen, M. S. J. Water S u p p l y
Tech.-Aqua 1992,41 (l), 4-12. Received for review October 17, 1991. Accepted February 24, 1992. This work was supported by the Academy of Finland and by Nordisk Industrifond Environmental Biotechnology Program A.2.1. with the industrial partners Alko Oy, Cultor Ltd., and Ekokem Oy.
Influence of Biotransformation on the Relationship between Bioconcentration Factors and Octanol-Water Partition Coefficients wdze de Wolf,*stJack H. M. de BrulJn,’ Wlllem SeInen,+ and Joop L. M. Hermenst Research Institute of Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands, and Directorate-General for Environmental Protection, P.O. Box 450, NL-2260 MB Leidschendam, The Netherlands -
Several regression equations of high statistical quality expressing the relationship between bioconcentration factors (BCF) in fish and octanol-water partition coefficients (KO,)of nonpolar, stable (“inert”) chemicals have been established and published in the literature. In this &udy bioconcentration factors of chlorinated aromatic mines in guppy (Poecilia reticulata), determined in a wmistatic system, are presented. The BCF values of the chlorinated anilines are significantly smaller than calculated BCF values based on the regression equations for inert chemicals, the BCFs of the more hydrophobic derivatives showing the highest deviation. These observations are explained on the basis of the argument that the influence of biotransformation on the bioconcentration process will increase with increasing KO,. Introduction Bioconcentrationof chemicals in aquatic organisms can be described as the process in which the uptake of chemicals is restricted to the uptake of chemicals dissolved in water. In contrast, biomagnification is restricted to the uptake of chemicals from food. The bioconcentration factor (BCF) is used as an important parameter in the risk wessment of environmental contaminants. The BCF is defined as the ratio between the concentration of a chemical in an organism and its concentration in the aqueous Phase at steady-state conditions. In many cases, the toxlcokinetic behavior of the chemical in fish can be described by a first-order, one-compartment model (1). In that case, BCF can also be calculated from BCF = k , / k 2
(1)
where k, is the uptake rate constant, and k2 is the elimination rate constant. Numerous studies have shown that the BCF of chemicals in fish is strongly correlated with the octanol-water p i t i o n coefficient (KO,)(see for instance refs 2-4). The h h correlations that are usually observed between BCF U d KO,, however, may be partly due to the type of chemicals in the dataset. Many of these correlation studies @e on the basis of data for relatively stable compounds Wb as polychlorinated biphenyls (5),chlorinated benzenes (6 7),and chlorinated naphthalenes (8). In this paper, we refer to these types of chemicals as “inert”compounds. Correlations on the basis of more polar and “less inert” chemicals often have a lower statistical quality (9, 10). \
‘University of Utrecht. Directorate-General for Environmental Protection.
’
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Furthermore, several examples are known of chemicals that have substantially lower BCFs than expected on the basis of their KO,. Especially for chemicals with a relatively high log KO, (>5), a loss of linear correlation can be observed ( 8 , I I ) . Several factors can cause the observed deviation from linearity: reduced membrane permeation (412-14); decreased bioavailability (15, 16);loss of similarity in phase properties of the fish lipids and l-octanol with respect to organic chemicals (17, 18);a substantial influence of biotransformation on the elimination kinetics (13, 19, 20); nonequilibrium determinations of bioconcentration factors (21). Gobas et al. (22) concluded that reduced lipid solubility and reduced bioavailability are the most likely factors contributing to the loss of the linear correlation of nonmetabolizing chemicals. Considerable deviations from the observed linear correlations between BCF and KO,have also been observed for chemicals with log KO,< 5 (19,23-26). These deviations are probably caused by relatively high biotransformation rates (26-28). Several biotransformation processes have been shown to occur in fish at rates that are thought to be sufficient to affect the elimination rates of chemicals significantly (29-31). Some examples of studies describing the extent of bioconcentration of chemicals that are susceptible to biotransformation are discussed below. The data from these studies are combined and presented in Figure 1. The BCF values are compared with the relationship for inert chemicals, as calculated from the data of Mackay ( 4 ) . BCF values of most organophosphorus pesticides in guppy agree well with calculated values on the basis of the equation for inert chemicals (28) (Figure 1). The three most hydrophobic chemicals and also two of the lower hydrophobic organophosphates have relatively low BCFs. The elimination rate constants of these compounds were found to be substantially higher than expected on the basis of their log KO,,which may be related to a relatively higher biotransformation rate. In vitro studies supported the idea that the organophosphates with relatively low BCF values are biotransformed more rapidly (32). BCF values of some mononitrobenzene derivatives in guppy, which have log KO,values between 1.89 and 3.09, correlated well with the octanol-water partition coefficients (24). The BCFs of more hydrophobic chloronitrobenzenes in rainbow trout, Oncorynchus mykiss, as given by Niimi et al. (2.5) did not show any correlation with the calculated octanol-water partition coefficient of the compounds. From Figure 1it is obvious that the deviation of the BCF values from the calculated values on the basis of the
0 1992 American Chemical Society
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1 1
2
4
3
5
6
log Kow
Figure 1. Relationship between octanol-water partition coefficients (Kow)and bioconcentration factors (BCF) (expressed on a wet weight basis) In guppy ( P . reticulata) for organophosphates (+) (28) and mononitrobenzene derlvatlves (0) ( 2 4 ) , in fathead minnows ( P . promdas) for azaarenes (W) ( 2 7 ) ,and In rainbow trout (0. mykiss) for chloronitrobenrenes (0)(25). The line represents the regression equation for Inert chemicals, as calculated from the data of Mackay (4).
equation for inert chemicals is higher for the more hydrophobic chemicals. It was shown by Bahig et al. (33) that pentachloronitrobenzene can be biotransformed by fish. In fathead minnows, Pimephales promelas, experimentally determined BCF values of azaarenes with moderate to high predicted values (ZlOOO),using the equation obtained by Mackay ( 4 ) , were also found to be far lower than the predicted BCF values (Figure 1) (27). The observed BCF of benz[a]acridine (log KO,= 4.45) was about one-tenth of that predicted, due to the influence of biotransformation processes (19). In contrast, the observed BCF of acridine (log KO,= 3.30), a compound also subject to biotransformation by fish (34), agrees well with the predicted value. In this study, bioconcentration factors in guppy (Poecilia reticulata) are determined for a set of chlorinated anilines with log KO,values between 3 and 5. These anilines were chosen, since to our knowledge no experimental data on the BCF in fish are available yet, and, as has been shown in mammals, the functional NH, group may give rise to a considerable degree of biotransformation, such as acetylation or hydroxylation reactions (35,36).The results are compared with bioconcentration studies for inert chemicals. Furthermore, we will discuss the influence of biotransformation on log KO,- log BCF relationships in more general terms. Materials and Methods 2,3,4-Trichloroaniline (234TCA) and 3,4,5-trichloroaniline (345TCA) were obtained from Fluka AG (Buchs, Switzerland) and were more than 98% pure. 2,4,5-Trichloroaniline (245TCA) and pentachloroaniline (PCA) were obtained from Riedel de Haen (Seelze, Germany) (purity >98% and >99%, respectively). 2,3,4,5-Tetrachloroaniline (2345TeCA) was obtained from Janssen Chimica (Beerse, Belgium) (>98% pure) and 2,3,5,6tetrachloroaniline (2356TeCA)was purchased from EGA Chemie (Steinheim, Germany) (98% pure). 2,4,6-Trichloroaniline (246TCA) was obtained from Aldrich (Milwaukee, WI) and was 99% pure. Residue-analyzed n-hexane (Baker, Phillipsburg, NJ) and l-propanol (p.a.) (Merck, Darmstadt, Germany) were used as received. Florisil (Merck) and sodium sulfate 1198 Environ. Scl. Technol., Vol. 26, No. 6, 1992
(Merck, p.a.) were washed with n-hexane and dried in an oven at 200 "c for 2 h. Florisil was partially deactivated with 10% distilled water (w/w). Guppies (n = 33), approximately 4 months old, were gift from the Research Institute of Pesticides (Staring Centre), Wageningen, The Netherlands. The organisms had an average wet weight (kstandard deviation) of 134 (f37) mg and an average lipid content based on wet weight (h standard deviation) of 8.5% (f1.9). Water, prepared according to Alabaster and Ahrams (44), was used. Aqueous oxygen concentrations always exceeded 7 mg/L; pH varied between 7 and 8; and the temperature was 21-23 OC. A 12-h lighk12-h dark cycle was imposed using fluorescent lamps. The fish were exposed for 254 h in an all glass 3 5 . ~ aquarium with water renewals after 2,4,8, 12, 24, 48,120, and 192 h. The compounds, dissolved in 3.5 mL of 1. propanol, were added to 1 L of water and stirred for at least 24 h. This solution was further diluted to 35 L of exposure solution. The exposure solution was renewed every time fish were sampled. The aqueous concentration of each test compound during the experiments was approximately 0.01 times the determined or calculated 14d LC50 value, according to Hermens et al. (37). On the basis of calculated pK, values using Hamett u constants as described by Perrin et al. (%), it can be concluded that the fraction of anilines that exists in the ionized form can be neglected. Just before and after water renewals, water samples (50 mL) were taken in duplicate and extracted with 10 mL of hexane. The hexane layer was further concentrated to 2 mL under a gentle N, stream. The extraction efficiency was more than 90%. Fish samples were taken in triplicate, and all fish were analyzed separately, using a slight modification of the procedure described by Deneer et al. (24). The clean-up column consisted of 0.5 g of partially deactivated Florisil and 0.25 g of sodium sulfate. The chloroaniliires were eluted from the column with 20 mL of hexane. The efficiency of the extraction and clean-up procedure was determined by adding known amounts of the test chemical to noncontaminated fish homogenates. The efficiency of the combined procedures was more than 96%. All analyses were carried out using a Pye Unicam 4550 gas chromatograph (GC) equipped with a split, splitless injector, an electron capture detector (ECD), and a CBpillary fused-silica CP-si18 CB column, (Chrompack, 25 m long, 0.32 mm i.d., film thickness 0.25 pm). Inlet and detector temperatures were 225 and 325 OC, respectively, The oven temperature was held at 160 "C for 4 min, subsequently increased by 45-deg/min to 190 O C , and kept at this temperature for 1 min. Helium was used a3 Car. rier-gas at a gas flow of 2 mL/min. A split-flow of 5O mL/min and an injection volume of 2 pL were used. The ECD signal was recorded and analyzed using an integrator (Shimadzu CRlA). Results Average concentrations of the compounds in water are given in Table I. Because the concentration of the test compounds decreased between the water renewal of the test solutions (maximum decrease 50%), the concentretions in water at the actual fish sampling time just before water renewal) were used for the determinat*on,Of the ratio between the concentration of chemicals in (CJ.and water (C,). No decrease in the aqueous conCen' trations was observed in a control experiment without fish* During the experiment, the fish did not show any signsOf intoxication upon exposure to the mixture of compounds'
Table 11. Literature Data on Octanol-Water Partition Coefficients ( K o w and ) Bioconcentration Factors (BCF) (on the Basis of Lipid Weight) in Guppy (Poecilia reticulata) for Chlorinated Benzenes compound
log KOwa log BCFb log BCFc
1,4-dichlorobenzene 1,2,3-trichlorobenzene 1,3,5-trichlorobenzene 1,2,3,4-tetrachlorobenzene 1,2,3,5-tetrachlorobenzene pentachlorobenzene
3.44 4.14 4.19 4.64 4.66 5.18
3.25 4.11 4.15 d
4.86 5.41
d 4.58 d 4.66 d 4.97
a Data of De Bruijn et al. (39). Data of Konemann and Van Leeuwen (7). CDataof Van Hoogen and Opperhuizen (40), recalculated on the basis of lipid weight, assuming 5% lipid content of the fish. Not determined.
6
5 7
P
Q
L
e
rp
4
b
s Nrlm3mc-m c
3
1191191 0 0 0 0 0 0 0
2 '
3
5
4
/OR
0
c o co n
x
s
, .
w
c m
8 +
w
a 00
E 3
KOW
Flgure 2. Relationship between the octanol-water parthiin coefficients (K0J and bioconcentration factors (BCF) (expressed on a lipid weight basis) in guppy (P. reticulata) for chlorinated anilines. Lines A and B represent calculated BCF values on the basis of eqs 2 and 3, respectively.
s E
6
8
+
0
2 g;z +Inn
The Cf/Cw ratios are presented in Table I. These results show that for all cornpounds studied an apparent steadystate situation was reached within the first 48 h of exposure. Therefore, mean BCF values were calculated on the basis of the ratios determined at 48,72,120,and 264 h of exposure (Table I). Differences in the BCF values of isomers of trichloroanilines and tetrachloroanilines are observed, the lowest values being found for 2,3,4-trichloroaniline and 2,3,4,5-tetrachloroaniline, respectively. On the basis of literature data for BCF values of chlorobenzenes as reported by Konemann and Van Leeuwen (7) and Van Hoogen and Opperhuizen (40),we recalculated the linear relationship with KO,(see eq 2). Values for KO, were taken from De Bruijn et al. (39),who measured the KO,of these chemicals by a slow-stirring procedure. The BCF as well as KO,data are given in Table 11. log BCF = 1.06(f0.15)log KO,- 0.20 r = 0.94,n = 8,s = 0.24 (2) Equation 2 is similar to the linear relationship by Mackay (4), if the BCF data are recalculated on the basis of lipid weight (see eq 3). For this recalculation we have assumed that the fish had a 5% lipid content. log BCF = 1.00 log KO,- 0.08 (3) Regression eqs 2 and 3 are presented in Figure 2,together with the BCF values for chlorinated anilines used in this study. This figure clearly shows that for these compounds the BCF values are considerably lower than expected on the basis of their octanol-water partition coefficient. Environ. Sci. Technol., Vol. 26, No. 6, 1992
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Discussion It is obvious from Figure 2 that the BCFs of the tested chlorinated anilines are lower than those of inert chemicals with comparable log KO, values and that the more hydrophobic congeners especially have the largest deviation. These deviations can be explained by either a difference in the uptake or a difference in the elimination of the chemicals. McKim et al. (41) studied the influence of the octanol-water partition coefficient on the transport across the gill membrane of rainbow trout. While using 14 chemicals from five different chemical classes, they concluded that the mechanisms controlling transport are nonspecific with respect to chemical structure and that additional factors, such as molecular volumes, molecular weight, or self-association, might influence gill transport for chemicals with a log P > 7. Therefore, no difference is to be expected in the uptake rates of the tested aromatic amines and inert chemicals with equal octanol-water partition coefficients. The other factor that affects the extent of bioconcentration is elimination, and it can be defined as the irreversible loss of a chemical from fish. Elimination occurs primarily by two processes: diffusion through gills and biotransformation (42). Diffusion, which consists of the irreversible loss of the unchanged chemical, can be thought of as a physicochemical loss, whereas biotransformation is the conversion of one chemical to another. In rate constant terminology, this can be described as kz = k, + k, (4) where k2 is the overall elimination rate constant, k, is the rate constant for physicochemical loss (elimination of parent compound), and k, is the biotransformation rate constant. Equation 4 is only valid on the assumption that diffusion as well as biotransformation follows a first-order process. As was mentioned before, linear correlations with respect to KO,have been observed for bioconcentration factors of organic chemicals in fish (2-4). Because relationships for these particular chemical classes follow the expectations based on a simple diffusion model, it is very unlikely that biotransformation reactions affect elimination rates (22). In terms of rate constants, this can be described as if k,
