Physicochemical parameters of individual hexachlorobiphenyl

Environ. Sci. Technol. 1990, 24, 1680-1687

Physicochemical Parameters of Individual Hexachlorobiphenyl Congeners Terence H. Risby,' Tau-Being Hsu,+Shelley S. Sehnert, and Purshotam Bhan

Division of Environmental Chemistry and Biology, Department of Environmental Health Sciences, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205 This study reports the chromatographic quantification of some of the physicochemical parameters for the hexachlorobiphenyls, a group of polychlorinated biphenyls that differ only in the position of chlorine substitution, in order to predict the lipophilicity and subsequent biological activity of these xenobiotics. Various chromatographic methods have been used in this study for the determination of the physicochemical parameters. The partial mole fraction entropies and enthalpies of solution were determined by capillary gas chromatography using two stationary phases. Capacity factors were determined by reverse-phase high-performance liquid chromatography using two bonded packing materials. These data were used to predict 1-octanol/water partition coefficients. On the basis of these studies, gas chromatography is proposed as a superior method to predict lipophilicity since it is not influenced by the steric contributions from the bonded packing materials used in liquid chromatography. Preliminary studies have shown correlation between the partial free energy of solution in an ordered stationary liquid phase and published values for the in vivo induction of microsomal isoenzyme P-45Oc.

Introduction There are 209 possible congeners of polychlorinated biphenyls (PCBs), which differ in their extent and position of chlorination. This group of halogenated aromatic compounds has unique physical and chemical properties, such as low volatility, high thermal stability, low dielectric constant, resistance to oxidation and reduction, and nonflammability. commercial mixtures of polychlorinated biphenyl congeners (e.g., Aroclors) are classified by their extent of chlorination and have been widely used in a variety of industrial applications. However, the same chemical properties that make these commercial mixtures of PCBs desirable for industrial use render them environmentally persistent, since they are resistant to biodegradation by most ecosystems. Health effects studies on human populations exposed to commercial mixtures of PCBs have indicated that the individual PCB congeners are biologically accumulated to different degrees with correspondingly variable adverse biological activities. However, the identities of the individual congeners have not been correlated with their relative bioaccumulation and bioactivities. Although classical toxicological evaluations of a few individual PCB congeners have been performed, such studies have been limited by the large number and commercial nonavailability of many of the polychlorinated biphenyl congeners. This current study reports the chromatographic quantification of some of the physicochemical parameters of a group of polychlorinated biphenyls that differ only in the position of chlorine substitution. Retention data of 20 hexachlorobiphenyl congeners were evaluated on an isotropic (random) stationary liquid phase (Apolane 87) and on a nematic (ordered) liquid-crystal phase (BMBT) by fused-silica capillary gas chromatography. The specific 'Present address: Department of Water Resource and Environmental Engineering, School of Engineering, Tamkang University, Tamsui, R.O.C. 1680

Environ. Sci. Technol., Vol. 24, No. 11, 1990

retention volumes of the remaining 22 hexachlorobiphenyl congeners were predicted on the basis of the experimental data for the 20 hexachlorobiphenyl congeners. The partial entropies and enthalpies of the hexachlorobiphenyls were obtained from the slopes and intercepts of linear regression analyses of retention data. The prediction scheme was evaluated experimentally with retention data of hexachlorobiphenyls synthesized in the laboratory. Additionally, capacity factors for the hexachlorobiphenyl congeners were determined by reverse-phase high-performanceliquid chromatography using two different types of C18 column packing materials and aqueous mobile phases containing different concentrations of an organic modifier. This paper presents a discussion of the various methods to determine the 1-octanol/water partition coefficients by dynamic chromatographic techniques. Preliminary studies are also reported in which some of these data have been correlated to the induction of microsomal detoxification enzymes in order to predict the biological activities of congeners that have not previously been studied. The goal of this research program is to delineate structural/physicochemical relationships that can used to estimate risk for exposed human populations.

Experimental Section Chemicals. Individual hexachlorobiphenyl (HCB) congeners were obtained from the Foxboro Co. (North Haven, CT), Accustand, Inc. (New Haven, CT), and Ultra Scientific Co. (Hope, RI). HPLC grade solvents, without additional purification [EM Science Co. (Cherry Hill, NJ), J. T. Baker (Phillipsburg, NJ), and Aldrich Chemical Co. (Milwaukee, WI)], and quartz distilled water filtered through a 0.45-pm Nylon-66 filter [Rainin Co. (Woburn, MA)] were used in the preparation of the mobile phases. The reagents used for the synthesis of the HCB congeners (chlorinated anilines, polychlorinated benzenes, and isopentyl nitrite) were obtained from Aldrich. Equipment. A capillary gas chromatograph equipped with a flame ionization detector and an in-board data handling system [Model 3500 Series, Varian Associates (Walnut Creek, CA)] was used to obtain all gas chromatographic retention data on two fused-silica capillary columns (15 m X 0.25 mm i.d., coated with either 24,24diethyl- 19,29-dioctadecylheptatetracontane(Apolane 87) or N,N'-bis(p-methoxybenzylidene)-cY,cu'-bis(p-toluidine) (BMBT), Supelco, Inc. (Bellefonte, PA). The high-performance liquid chromatographic system consisted of a pump (Varian Model 2010), an injection valve [Rheodyne Model 7410 (Cotati, CA)], a variablewavelength W-vis detector (Varian Model 2050) operated at 254 nm and 0.2 AUFS unless otherwise specified, and an integrator [SpectaPhysics Model 4270 (San Jose, CA)]. Two reverse-phase C18 columns were used to obtain the HPLC data [15 cm X 4.6 mm; Supelcosil LC-18, Supelco, and WDAC 201 TPS, The Separations Group (Hesperia, CAI]. Procedures. Capillary Gas Chromatography. Isothermal gas chromatographic retention data were obtained on each capillary column at three column temperatures. Aliquots (1.0 FL) of solutions of individual hexachloro-

0013-936X/90/0924-1680$02.50/0

0 1990 American Chemical Society

biphenyl congeners in methanol or tetrahydrofuran (50 pg/mL) were injected in the splitless mode at a constant flow rate (helium 2.0 mL/min). The retention datum for each congener was obtained in triplicate and each value was used in subsequent least-squares fit of the data. The void volumes [Vo (mL)] of the capillary columns were determined by the injection of an unretained solute (methane) (Apolane 87,0.833 mL; BMBT, 0.8346 mL) and the weights of the stationary phases contained in each column [w (9)] (Apolane 87,l.g X g; BMBT, 2.5 X g) were obtained from the supplier. The specific retention volumes (Vg) of the solutes were calculated by In Vg = In 273(V, - Vo)/wT (1) where V, is the experimental retention volume (mL) of the solute corrected for the pressure drop across the column and T is the column temperature (K). The specific retention volume of a solute on a given stationary phase at a given column temperature may be also expressed as In Vg = -AH/RT A S / R - In M In 273R (2)

+

+

where R is the gas constant (8.314 J/degmol), AH (J/mol) and AS (J/deg.mol) are the partial enthalpy and the entropy of solution, respectively, and M is the molecular weight of the stationary phase. Equation 2 is based on mole fraction and is compatible with the Henry’s law reference states. Linear regression analyses of retention volumes for the solutes (In Vg vs 1/T) at three isothermal column temperatures provide the slopes and intercepts. The slopes and intercepts are related to the partial enthalpies and entropies of solution for the distribution of solutes between the gas phase and the liquid phase, respectively, providing that the molecular weight of the stationary phase is known and the temperature range is small. High-Performance Liquid Chromatography. Liquid chromatographic data for the hexachlorobiphenyl congeners were obtained on the reverse-phase C18 columns by using mobile phases of different compositions (acetonitrile/water 3:l; 4:l; and 17:3) at flow rates of 1.0 mL/min. Aliquots (1.0 pL) of solutions (0.5 mg/mL solution in methanol or tetrahydrofuran) of individual hexachlorobiphenyl congeners were injected. The retention datum for each congener was obtained in triplicate and each value was used in subsequent least-squares fit of the data. The column was equilibrated with the mobile phase prior to each chromatographic separation and equilibration was maintained by reducing the flow rate to 0.1 mL/min overnight. The capacity factor (k3 for a solute was calculated by using the following equation: k ’ = ( t ,- to)/to (3) where t, is the retention time of the component of interest and to is the retention time for an unretained solute (nickel sulfate). Synthesis of Hexachlorobiphenyl Congeners. Specific HCB congeners were synthesized by a previously described procedure (3) based upon the Cadogan coupling of the appropriate chlorinated aniline (15 mM) and polychlorinated benzene (200 mM). Isopentyl nitrite (25 mM) was added to the stirred solution of the reactants and the mixture refluxed at 125-130 “C for 20 h. Excess unreacted polychlorinated benzene and isopentyl nitrite were removed by distillation and the dark-colored residue was placed on the top of a silica gel column. The HCB congener was eluted with l-hexane, the eluate was concentrated, and the residue was further purified by silica gel spinning-disk chromatography [Harrison Research (Palo

Alto CA)] using l-hexane as the eluate. The resulting HCB congeners were obtained as white crystals after recrystallization from methanol. The identities and purities of the HCB congeners (2,3,4,6,2’,6’;2,3,5,6,3‘,4’; 2,3,4,6,3‘,5‘; 2,3,5,6,3’,5‘; 2,3,4,2‘,4’,6‘; 3,4,5,2‘,4’,6‘; and 2,3,5,6,2’,4’) were confirmed by electron impact mass spectrometry and proton NMR (3).

Results and Discussion Capillary Gas-Liquid Chromatography. The goal of this aspect of the study was to measure the partial enthalpies and entropies of solution for individual hexachlorobiphenyl congeners in a random (isotropic) stationary liquid phase (Apolane 87) as compared to an ordered liquid-crystal (nematic) stationary phase (BMBT). The rationale for this study was that the differences in the interactions of unsolvated gas-phase solute molecules with ordered vs random stationary phases may correlate with the stereochemistry governing the interactions between these environmental agents and microsomal P-450 isozymes. (a) Retention of Hexachlorobiphenyls. Specific retention volumes of the commercially available hexachlorobiphenyl congeners were obtained from their isothermal retention data on the nonpolar stationary liquid phase (24,24-diethyl-19,29-dioctadecylheptatetracontane) Apolane 87. Since N,N’-bis(p-methoxybenzy1idene)-a,a’-bis(p-toluidine) (BMBT) has a solid-nematic transition temperature at 181 “C ( 4 ) )isothermal column temperatures above this phase transition (190, 200, and 210 “C) were selected for all gas chromatographic separations. Examination of these data, grouped according to classes containing the same number of chlorine atoms on each benzene ring, reveals the expected trend in retention data consistent with the results reported by Zielinski et al. (5). The order of chlorine positional attachment decreases retention of the hexachlorobiphenyl congeners from para to meta to ortho; the greater the “rodlike” (length-tobreadth ratio) character of the hexachlorobiphenyl congener, the larger the retention volume. The attachment of more chlorine atoms on one of the two rings produces larger retention volumes than when they are evenly distributed on both rings (Le., 5C1- 1C1 > 4C1- 2C1 > 3C1 - 3C1). Comparable specific retention volumes were obtained on the stationary liquid-phase BMBT at the same isothermal column temperatures. Comparison of these data shows that the retention volumes of the hexachlorobiphenyl congeners on the stationary-phase BMBT were smaller than those on obtained on the stationary-phase Apolane 87. This behavior may be attributed to steric hindrance of the interactions of the solute with the ordered structure of the stationary-phase BMBT. The elution orders of particular hexachlorobiphenyl congeners were different for BMBT as compared to the elution orders with Apolane 87, which also supports the hypothesis that steric hindrance is an important contributor to retention. (b) Calculation of the Logarithm of the Specific Retention Volumes of Unknown Hexachlorobiphenyl Congeners. Examination of available retention data indicates that specific retention volumes decrease when hexachlorobiphenyl congeners have ortho and meta substitution of chlorine atoms. These trends in retention data have been used to calculate the logarithm of the specific retention volumes for the commercially unavailable hexachlorobiphenyl congeners at the same three isothermal column temperatures on both the BMBT and Apolane 87 columns. This empirical prediction scheme was based on the experimental data obtained for hexachlorobiphenyl Environ. Sci. Technol., Vol. 24, No. 11, 1990

1681

Apolane87 BMBT

Table I. Summary of the Experimental and Predicted Values for the Partial Enthalpy (kJ/mol) and Entropy (J/deg mol) of Solution for Individual Hexachlorobiphenyl Congeners

HCB congeners Bz n0.O 2,3,4,5,6,2’ 2,3,4,5,6,3‘ 2,3,4,5,6,4‘ 2,3,4,5,2’,3’ 2,3,4,6,2’,3’ 2,3,5,6,2’,3’ 2,3,4,5,2’,4’ 2,3,4,6,2‘,4’ 2,3,5,6,2’,4’ 2,3,4,5,2‘,5’ 2,3,4,6,2’,5‘ 2,3,5,6,2’,5’ 2,3,4,5,2’,6’ 2,3,4,6,2’,6‘ 2,3,5,6,2’,6’ 2,3,4,5,3’,4’ 2,3,4,6,3’,4‘ 2,3,5,6,3’,4’ 2,3,4,5,3’,5’ 2,3,4,6,3’,5’ 2,3,5,6,3‘,5’ 2,3,4,2’,3’,4’ 2,3,4,2’,3’,5’ 2,3,4,2‘,3‘,6’ 2,3,4,3’,4’,5’ 2,3,4,2‘,4’,5’ 2,3,4,2’,4’,6’ 2,3,5,2’,3‘,5‘ 2,3,5,2’,3‘,6’ 2,3,5,3’,4’,5’ 2,3,5,2’,4’,5‘ 2,3,5,2‘,4‘,6’ 2,3,6,2‘,3‘,6’ 2,3,6,3‘,4‘,5’ 2,3,6,2’,4’,5‘ 2,3,6,2‘,4’,6‘ 3,4,5,3’,4‘,5’ 3,4,5,2‘,4’,5’ 3,4,5,2’,4’,6’ 2,4,5,2’,4’,5’ 2,4,5,2’,4‘,6‘ 2,4,6,2‘,4‘,6‘

142 160 166 129 131 134 137 139 147 141 144 151 143 145 152 156 158 163 159 161 165 128 130 132 157 138 140 133 135 162 146 148 136 164 149 150 169 167 168 153 154 155

Apolane 87b -AH -AS

BMBTb -AS -AH

71 74 79.6 78.5 55 57 84.3 75.9 78.6 85.1 61 75.7 78.1 54 57 86.7 79.3 82 82.5 59 61 78.0 78 80 88 81.9 80 77.7 76 88 78 80 72.4 86 76 72 90.4 84.7 86 77.3 79.4 73.6

96 102 119.5 79.8 88 70 96.3 89.2 84.2 76.4 71 62.5 57.6 53 48 103.6 100.8 96 79.8 75 76 114.1 88 96 103 93.2 89 62.5 58 77 70 63 64.7 76 65 72 95.9 87.7 83 76.5 73.4 68.2

82 82 94.7 93.2 47 52 105.7 91.4 97.0 108.0 62 91.8 96.4 50 56 106.0 94.3 100 98.8 52 58 90.8 92 99 110 100.3 100 93.7 93 111 93 101 87.3 110 92 86 110.1 103.0 109 91.4 99.8 90.2

148 156 192.6 112.4 130 96 147.5 135.9 126.0 107.8 101 82.3 71.7 65 55 156.9 155.8 146 111.5 105 95 179.4 130 149 154 140.0 135 81.1 76 105 95 86 87.7 106 90 102 136.7 126.7 120 108.7 106.3 99.2

a Reference numbers for individual PCB congeners (6). Experimental data are expressed to one decimal place.

congeners within the same class as the congener to be predicted (for the predictions of the 1C1-5C1 congeners, the data from the 2Cl-4C1 congeners were used) and only involved the movement of chlorine atoms by a single position in one of the biphenyl rings. An example of the method used for these predictions is given by examination of the congener 2,3,4,6,2’,3’hexachlorobiphenyl at 190 “C on Apolane 87. The difference between the logarithm of the specific retention volumes of 2,3,4,5,2’,3’ and 2,3,4,6,2’,3’ is the average (0.478) of the differences in the values for the In Vg for 2,3,4,5,2’,4’ and 2,3,4,6,2’,4’ and 2,3,4,5,3’,4’ and 2,3,4,6,3’,4’ at 190 “C on Apolane 87. Therefore, the value of In Vg of 2,3,4,6,2’,3’ at 190 “Con Apolane 87 is predicted to be 8.709 based on the congener 2,3,4,5,2’,3’ (In Vg = 9.187). A comparison of the elution order for the experimental and predicted specific retention volumes on Apolane 87 vs published elution orders (3, 6, 7, 8) was performed. There is agreement between the elution orders even though the experimental conditions were not identical. Bush et al. (7) used a soda glass capillary column coated with Apolane 87, whereas Pellizzari et al. (8)and Safe and co1682


60 1

40

20

. . . . .

0 ~~~~~~~~~~~~~~~~~~~~~~~~~~

Congener Number Figure 1. Variation in partial free energy of solution as a function of

hexachlorobiphenyl congener.

workers (3)used a fused-silica capillary column coated with SE-54. (c) Partial Enthalpies and Entropies of Evaporation from Solution. The experimental and predicted retention data as a function of column temperature were used to calculate the partial enthalpies and entropies of solution for the hexachlorobiphenyl congeners on the nematic and isotropic stationary phases by using the molecular weights of the stationary phases and the weights of the stationary phases in the columns (eq 2). The results of these calculations are contained in Table I. Inspection of this table shows that the hexachlorobiphenyls can be separated into three groups. In the first group, the partial enthalpies and entropies were greater for BMBT as compared to Apolane 87, which suggests that 7r-7r interactions between the biphenyl rings are greater than the intermolecular contributions due to the London or dispersion forces. In the second group, the partial enthalpies and entropies are greater for Apolane 87 as compared to BMBT, suggesting that the nematic structure of the BMBT limits the solutesolvent interactions sterically such that the London or dispersion forces dominate. In the third group, the partial enthalpies are greater with Apolane 87 than with BMBT whereas the partial entropies are greater with the BMBT than the Apolane 87. These data are difficult to explain since London or dispersion forces appear to dominate the partial enthalpies, which are themselves dominated by steric interactions. The differences between these three groups show that position of chlorine atom substitution significantlycontributes to the interactions of HCB congeners with ordered stationary phases. Congeners with extensive substitution in the ortho position interact less with the polar sterically hindered stationary-phase BMBT as compared to the nonpolar random stationary-phase Apolane 87. Figure 1 shows the change in free energy at 190 “C for the interaction of HCB congeners as a function of the steric properties of the stationary phases. (a) Experimental Testing of the Empirical Prediction Scheme. The empirical prediction scheme was tested experimentally with six HCB congeners that were synthesized in the laboratory. A seventh synthesized

Table 11. Experimental and Predicted Values for Specific Retention Volume (mL), Partial Enthalpy (kJ/mol), and Entropy (J/deg mol) of Solution for Individual Wexachlorobiphenyl Congeners on Apolane 87 HCB congeners 2,3,4,6,2’,6’ 2,3,5,6,3’,4’ 2,3,4,6,3’,5‘ 2,3,5,6,3’,5’ 2,3,4,2’,4’,6‘ 3,4,5,2‘,4’,6‘

In Vg(210 “C) expt pred



expt

pred

expt

pred

8.15 8.99 8.87 8.83 8.55 8.71

8.58 9.38 9.28 9.14 8.95 9.22

8.94 9.83 9.60 9.61 9.38 9.70

73.8 78.5 67.1 72.8 77.4 92.1

54 82 59 61 80 86

97.0 92.9 70.1 82.6 94.4 123.4

50 100 52 58 100 109

8.06 8.93 8.84 8.78 8.56 8.90

8.47 9.59 9.34 9.43 8.98 9.33

-AH

8.64 9.82 9.47 9.44 9.42 9.83

-AS

Table 111. Experimental and Predicted Values for Specific Retention Volume (mL), Partial Enthalpy (kJ/mol), and Entropy (J/deg mol) of Solution for Individual Hexachlorobiphenyl Congeners on BMBT HCB congeners 2,3,4,6,2’,6’ 2,3,5,6,3‘,4’ 3,5,2’,3’,4’,6’ 2,3,5,6,3’,5’ 2,3,4,2‘,4’,6’ 3,4,5,2’,4’,6’




expt

pred

expt

pred

7.03 7.99 7.53 7.55 7.47 7.58

7.36 8.42 7.96 7.94 8.03 7.98

7.68 9.02 8.39 8.33 8.44 8.46

60.4 96.0 80.4 73.0 90.0 81.8

53 96 75 70 89 83

80.2 146.0 117.3 101.8 137.6 119.9

65 146 105 95 135 120

6.91 7.99 7.63 7.59 7.52 7.84

7.11 8.41 7.99 7.93 8.11 8.31

congener (2,3,5,6,2’,4’) had already been used in the prediction scheme. The results of these studies are contained in Tables I1 and 111. The prediction scheme with the sterically hindered BMBT stationary phase is superior to the prediction with the random nonpolar Apolane 87 stationary phase, and the prediction scheme is not as accurate for those congeners with substitutions of chlorine atoms in the ortho positions. This phenomenon is currently under investigation. HIgh-Performance Liquid Chromatography. The goal of this aspect of this study was to measure the capacity factors for individual hexachlorobiphenyl congeners with a random Cl8 (Supelcosil LC-18) as compared to an ordered C18 reverse-phase column (VYDAC 201 TPS). This latter column packing material is end-capped with C3 silyl derivative to react with any residual hydroxyl groups. This packing has been shown (9) to sterically hinder the interactions of certain solutes with the column packing material. Therefore, it was anticipated that the use of these reverse-phase columns would allow the steric components of solvated HCB congeners to be compared with the steric properties of unsolvated gas-phase HCB congeners quantified by gas-liquid chromatography. Capacity Factors for Hexachlorobiphenyl Congeners. It has been suggested that the extrapolated capacity factor at 100% water ( k b ) may be an alternative approach to express the distribution of a solute between an aqueous mobile phase containing an organic modifier and a reverse-phase column packing material (10, 11). The extrapolated log capacity factor at 100% water, log k b , can be calculated from the capacity factors (k’4) at various concentrations of organic modifier according to the equation log k’4 = log k b + b4, where b is a system-dependent constant and 4 is the volume fraction of organic modifier in the mobile phase. The implicit assumption of this approach is that the ratio of the solute activity coefficients in the bulk organic solvent and in the homogeneous interfacial layer is constant and that all mixtures of the organic solvent and water are totally random, i.e., exhibit no compositional microstructures (12). Furthermore, it is assumed that retention of a solute on a reverse-phase column packing material is due solely to partition. The extrapolated capacity factors at 100% water ( k b ) for the hexachlorobiphenyl congeners obtained with the random C18 reverse-phase column are shown in Table IV. Also included in this table are the predicted values and com-

-AS

-AH

7.47 9.02 8.43 8.34 8.48 8.74

Supelcosil LC-18 VYDAC201TPS 4.5

4

4.3 4.1

3.9 3.7 3.5 3.3 3.1 2.9 2.7 2.5 ~~~~~~~~~~~~~~~~~~~~~~~~~~

Congener Number Figure 2. Variation in capacity factor as a function of hexachloro-

biphenyl congener.

parisons between predicted and experimental values using the HCB congeners synthesized in the laboratory. The correlations for the relationships between capacity factors and the concentration of the organic modifier were greater than 0.995 and the slopes varied between 3.63 and 4.25. These studies were repeated with a sterically hindered Cl8 reverse-phase column. The results obtained with this column are shown in Table IV. This table also includes the predicted values and comparisons between predicted and experimental values using the HCB congeners synthesized in the laboratory. The correlations for these relationships between capacity factors and the concentration of organic modifier were greater than 0.987 and the slopes varied between 3.06 and 4.27. The extrapolated capacity factors for the sterically hindered packing material (VYDAC 201TPS) are less than the values for the other C18 column packing (Supelcosil LC-18) and may be due to steric contributions to the partition processes as a result of the differences in the Environ. Sci. Technol., Vol. 24, No. 11, 1990

1683

Table IV. Summary of the Experimental and Calculated Extrapolated Capacity Factors for Individual Hexachlorobiphenyl Congeners Using the Reverse-Phase C18 Columns, Supelcosil LC-18and VYDAC 201 TPS”

pred

log k‘,(VYDAC 201TPS) expt pred

3.95 3.87

3.10 3.15

log k;(LC-18)

HCB congeners 2,3,4,5,6,2’ 2,3,4,5,6,3’ 2,3,4,5,6,4’ 2,3,4,5,2’,3’ 2,3,4,6,2’,3’ 2,3,5,6,2’,3’ 2,3,4,5,2’,4’ 2,3,4,6,2‘,4’ 2,3,5,6,2‘,4’ 2,3,4,5,2’,5’ 2,3,4,6,2‘,5‘ 2,3,5,6,2’,5‘ 2,3,4,5,2’,6‘ 2,3,4,6,2’,6’ 2,3,5,6,2’,6’ 2,3,4,5,3’,4’ 2,3,4,6,3‘,4‘ 2,3,5,6,3’,4’ 2,3,4,5,3‘,5’ 2,3,4,6,3’,5’ 2,3,5,6,3’,5’ 2,3,4,2’,3’,4’ 2,3,4,2’,3’,5’ 2,3,4,2’,3’,6’ 2,3,4,3‘,4’,5‘ 2,3,4,2‘,4‘,5‘ 2,3,4,2’,4’,6’ 2,3,5,2’,3’,5’ 2,3,5,2’,3’,6’ 2,3,5,3‘,4’,5’ 2,3,5,2‘,4’,5‘ 2,3,5,2’,4’,6’ 2,3,6,2’,3’,6’ 2,3,6,3‘,4‘,5’ 2,3,6,2’,4’,5’ 2,3,6,2’,4’,6’ 3,4,5,3’,4’,5’ 3,4,5,2’,4’,5’ 3,4,5,2’,4’,6’ 2,4,5,2’,4’,5’ 2,4,5,2’,4’,6’ 2,4,6,2’,4’,6’

expt

4.20 3.95

3.52 3.06 2.75 2.80

3.99 3.91 3.41 3.04 3.09 3.37

3.99 4.19 4.12 4.00

3.04

3.97 3.96 3.88 [3.92] 4.21 4.16 [4.14] 4.15 [4.24] [4.25] 4.00

3.92 3.84 4.08 4.18 4.11

3.69 3.00 [2.95] 3.41 3.22 [3.34] 3.58 [3.29] [3.25] 3.33

4.08

3.15 [3.21] 3.37

3.08

2.99 4.33 4.06 3.61

4.55 4.35 [4.34] 4.12 4.10 4.13

3.27 3.32

3.30 3.51 3.42 3.10

3.99 4.31 4.05 4.08 3.53

3.27

3.49 3.13 3.48

3.99 4.15 4.38 4.08 14.041 3.98

2.68 2.73

4.40

3.66 3.30 2.95 3.88 3.64 [3.48] 3.47 3.38 3.38

3.71

” Values in brackets demonstrate the accuracy of the prediction scheme. structures of the solutes (Figure 2.). These differences could also be explained on the basis of adsorption, which is known to vary on the basis of structure. Partition Coefficients (1-Octanol/Water) for Hexachlorobiphenyl Congeners. There has been considerable interest in the quantification of lipophilicty to estimate the bioaccumulation of xenobiotics since Neely et al. (13) first proposed that the bioaccumulation of xenobiotics in fish muscle tissue can be correlated to l-octanol/water partition coefficients. The classical approach to determine 1-octanol/water partition coefficients (PI is the shake-flask method. However, this method can be time-consuming and inaccurate and alternate dynamic methods based upon gas or liquid chromatography have been proposed. (a) High-Performance Liquid Chromatography. Capacity factors obtained by high-performance liquid chromatography with octadecyl-modified silica columns with mobile phases consisting of water containing organic modifiers, such as methanol or acetonitrile, have been used to predict 1-octanol/water partition coefficients (14). For 1664

Envlron. Sci. Technol., Vol. 24, No. 11, 1990

this approach, it is assumed that there is a linear relationship between log P and log k’$ (log P = a log k’$ + log b). The values for a and b are determined from the capacity factors obtained under the same experimental conditions for standard solutes whose 1-octanol/water partition coefficients are known. The accuracy of this approach is dependent upon the choice of suitable reference standard solutes and the accuracies of the values for the 1-octanol/water partition coefficients. The extrapolated capacity factor at 100% water (k$ has been proposed to be an alternative way to determine the distribution of a solute between water and an organic phase (IO), Le., log P = log kb. However, the data contained in Table IV are clearly less than the reported values of log P for HCB congeners (13, 16, 18-22), as shown in Table V. The discrepancy can be explained by the nonlinearity of the relationship log k’4 = log k b - b$ over large ranges of concentration of the organic modifier ($). This nonlinearity of capacity factors vs concentration of acetonitrile has been observed by others (15),although linearity was observed for small changes in concentration. One reason for this nonlinearity has been suggested to be the formation of compositional microstructures (12). Therefore, the final equation used in our studies to determine values for log P was log P = a log k b + log C. The values of a and log C were obtained by using the values of the partition coefficient for hexachlorobiphenyl congeners 2,3,6,2‘,3‘,6‘ and 2,3,4,2‘,3’,4’ (log P = 6.63 and 6.98, respectively) measured by the modified generator column method (16). The equations used to estimate log P are as follows: log Pa = 0.74 log k b + 4.02 (Supelcosil LC-18) and log Pb= 1.03 log k b + 3.55 (VYDAC 201 TPS). The values for the 1-octanol/water partition coefficients for the HCB congeners calculated by use of both reverse-phase columns are contained in Table V. (b) Capillary Gas-Liquid Chromatography. The relative corrected retention volume ( a ) for a polychlorinated biphenyl congener (reference solute 2,4,5-trichlorobiphenyl) eluting from a packed column containing a nonpolar stationary phase (Apolane 87) has been shown to be linearly related to the 1-octanol/water partition coefficient by the following equation: log P, = 1.40 log a + 5.54 (16). This approach was investigated in our studies since it represented a method to predict 1-octanol/water partition coefficients that does not involve a bonded phase. The use of bonded phases may impart a steric component to the subsequent retention datum. Since the solute 2,4,5-trichlorobiphenylwas not run in our experiments, its corrected retention volume was calculated based upon the published value for log a for the HCB congener 2,3,6,2’,3‘,6’ and our experimentally determined corrected retention volume. This value was used to calculated values for log a and hence values of log P, for the other HCB congeners based upon the experimental and predicted retention data at 190 “C. The values for the 1-octanol/water partition coefficients for the HCB congeners based on this approach are shown in Table V. An alternate method to determine log P from this data base was developed based upon the calculated partition coefficient for the distribution of the solute between the gas phase and the stationary phase at 190 “C (log K = -AG/2.303RT). The advantage of this latter approach is that the partition coefficients for the distribution of the solutes between the gas phase and the stationary phase are based upon the linear regressions of retention data obtained at three column temperatures. The slopes for both relationships of gas chromatographic data with 1octanol/water partition coefficients are the same. The

Table V. Comparison between Logarithm of the Partition Coefficients for Individual Hexachlorobiphenyl Congeners Calculated from Retention Data Obtained with Supelcosil LC-18 and VYDAC 201 TPS Reverse-Phase C18 Columns and with an Apolane 87 Column with Literature Values” HPLC HCB congeners

Supelcosil LC-18

VYDAC 2OlTPS

log p a

log p b

log p c

log pd

literatureb

2,3,4,5,6,2’ 2,3,4,5,6,3’ 2,3,4,5,6,4’ 2,3,4,5,2’,3’ 2,3,4,6,2’,3’ 2,3,5,6,2’,3’ 2,3,4,5,2’,4’ 2,3,4,6,2’,4’ 2,3,5,6,2’,4’ 2,3,4,5,2’,5’ 2,3,4,6,2’,5’ 2,3,5,6,2’,5’ 2,3,4,5,2’,6’ 2,3,4,6,2’,6’ 2,3,5,6,2’,6’ 2,3,4,5,3’,4’ 2,3,4,6,3’,4’ 2,3,5,6,3’,4’ 2,3,4,5,3’,5’ 2,3,4,6,3’,5’ 2,3,5,6,3’,5’ 2,3,4,2’,3’,4’ 2,3,4,2’,3’,5’ 2,3,4,2’,3’,6’ 2,3,4,3’,4’,5’ 2,3,4,2’,4’,5’ 2,3,4,2’,4’,6’ 2,3,5,2‘,3‘,5‘ 2,3,5,2’,3’,6’ 2,3,5,3’,4’,5’ 2,3,5,2’,4’,5’ 2,3,5,2’,4’,6’ 2,3,6,2’,3’,6’ 2,3,6,3‘,4‘,5‘ 2,3,6,2’,4’,5’ 2,3,6,2’,4’,6’ 3,4,5,3’,4’,5’ 3,4,5,2’,4’,5’ 3,4,5,2’,4’,6’ 2,4,5,2’,4’,5’ 2,4,5,2‘,4‘,6‘ 2,4,6,2’,4’,6’

6.94 6.88 7.13 6.94 6.97 6.92 6.97 7.12 7.07 6.98 6.96 6.95 6.89 6.92 6.86 7.14 7.10 7.08 7.09 7.16 7.17 6.98 6.98 7.09 7.26 7.04 7.01 6.97 6.97 7.25 7.02 7.04 6.63 7.22 7.02 6.69 7.39 7.24 7.23 7.07 7.05 7.08

6.75 6.79 7.18 6.70 6.38 6.43 7.06 6.68 6.73 7.02 6.68 7.35 6.63 6.59 6.36 7.12 6.87 6.99 7.24 6.94 6.90 6.87 7.15 6.77 7.14 6.80 6.86 7.02 6.95 7.16 7.08 6.74 6.30 7.32 7.00 6.59 7.55 7.30 7.13 7.12 7.03 7.03

6.56 6.89 6.90 6.81 6.41 6.40 6.82 6.54 6.52 6.78 6.38 6.44 6.51 6.30 6.10 7.16 6.84 6.84 7.02 6.69 6.70 6.92 6.79 6.63 7.19 6.84 6.56 6.66 6.41 7.06 6.71 6.46 6.30 6.79 6.45 6.34 7.46 7.09 6.76 6.75 6.48 6.24

6.41 6.87 6.83 6.78 6.44 6.44 6.78 6.50 6.51 6.73 6.29 6.43 6.48 6.26 6.09 7.13 6.82 6.79 7.00 6.66 6.65 6.87 6.78 6.58 7.04 6.79 6.51 6.60 6.40 6.97 6.71 6.44 6.24 6.73 6.47 6.27 7.42 7.03 6.71 6.72 6.43 6.22

6.51 6.93 6.93 6.73, 7.32 6.58 6.55 6.83, >7.71 6.67 6.64 6.82 6.67 6.64 6.60 6.25 6.22 7.18 7.02 6.99, 7.37 7.26 7.08 7.05, 7.37 6.74, 6.96, 6.98, 7.44 6.80, 7.39 6.58 7.18 6.83, 7.44 6.67 6.86 6.64, 7.15 7.24 6.89 6.73 6.22, 6.63, 6.51, 6.81 7.02 6.67, 7.28 6.32 7.42 7.27 7.11 6.92, 7.75, 7.14, 7.44, 6.72 6.76 6.41, 7.55, 7.12, 6.27

GLC Apolane 87

” log Pa-based capacity factor data with HPLC on Supelcosil LC-18. log Pb-based capacity factor data with HPLC on VYDAC 201TPS. log P, based upon relative corrected retention data with capillary GLC. log P d based upon distribution constant data with capillary GLC. Literature values were obtained with the shake-flask method, with the modified generator column method, and by calculation. constant was determined from the value of the partition coefficient (log Pd = 1.40 log K + 1.19) for the congener 2,4,5,2’,4’,5’ (log P = 6.72) measured by the shake-flask method (17). The value for log P obtained with the shake-flask method was used for this aspect of the study since it does not include a steric component that may be present in values derived from liquid chromatographic data. The values for the l-octanol/water partition coefficients for the HCB congeners based on this approach are contained in Table V. Also included in Table V are published experimental data for log P (13,16,18-22) and calculated values for log P where log P = [(3.41 X (total surface area) - 2.201 (23). There are obvious variations in the values for log P within the published data. These variations are HCB congener dependent. Similarly, there are HCB congener dependent variations between the numerous methods used to predict the l-octanol/water partition coefficients by gas or liquid chromatography. The HCB congeners with substitution in the ortho position appear to differ most. This variation may be the result of the increased torsional

angle between the biphenyl rings, which imparts increased steric hindrance to interactions with bonded column packing materials. Relationship between the Induction of Cytochrome P-450 Isozymes and Chromatographic Data for Hexachlorobiphenyl Congeners. Many detoxification enzymes are membrane-bound and xenobiotics must have lipophilic character in order for metabolism to proceed. The lipophilicity and the binding of xenobiotics to specific proteins will define the site of metabolism, the enzymes involved, and the metabolites produced. Such information could assist in the elucidation of the subsequent mechanisms of toxicity by xenobiotic metabolites or metabolic byproducts. Therefore, a study was initiated to determine whether it was possible to relate the selected chromatographic parameters determined in this present study that are measures of lipophilicity and steric properties to the published reports on the induction of microsomal cytochrome P-450 isozymes by individual HCB congeners. The immunochemical quantification of cytochrome P-450 isozymes and epoxide hydrolase in rat liver microEnviron. Sci. Technol., Vol. 24, No. 11, 1990

1685

Table VI, Cytochrome P-450 Isozyme Induction by Individual Hexachlorobiphenyl Congeners (24) HCB congenersa 2,3,4,5,6,4‘ 2,3,4,5,3‘,4‘ 2,3,4,6,3’,4‘ 2,3,4,2’,3‘,4‘ 2,3,4,3’,4‘,5’ 2,3,4,2’,4‘,5’ 3,4,5,3’,4’,5’ 3,4,5,2’,4’,5’ 3,4,5,2’,4’,6’ 2,4,5,2‘,4’,5’ 2,4,5,2‘,4’,6‘ 2,4,6,2’,4’,6’

total P-450

isozyme induction, nM cytochrome P-450/mg of microsomal protein P-450 unknown P-450a P-450b + P-450e P-45Oc P-450d 0.48 0.24 0.46 0.60 0.37 0.81 0.0 0.52 0.60 0.90 0.73 0.84

3.23 4.02 2.21 1.92 4.08 3.23 4.43 2.58 1.24 3.33 2.08 1.62

0.32 0.48 0.27 0.21 0.49 0.27 0.62 0.21 0.10 0.17 0.10 0.08

1.9 1.4 0.97 0.69 1.1 1.4 0.05 1.2 0.30 2.2 1.2 0.62

0.52 1.3 0.35 0.35 1.3 0.61 1.3 0.26 0.15 0.03 0.03 0.02

0.0 0.6 0.15 0.07 0.82 0.19 2.4 0.41 0.09 0.03 0.04 0.05

epoxide hydrolase 1.3 1.1 0.82 0.76 0.96 0.88 0.57 0.74 0.49 0.90 0.59 0.53

“The dose for 3,4,5,3’,4’,5’-HCBwas 125 pM/kg, whereas the doses for all other HCB congeners were 500 pM/kg.

this limited data base. Cytochrome P-450 induction may indeed not be related to this particular parameter. Since we are attempting to draw a correlation between our data and published measurements made by other researchers using an in vivo whole animal model, the innate variability of the animal model must be taken into account. Considering the complexity of the microsomal enzyme induction system, it is remarkable to observe any correlation whatsoever between a parameter as simple as partial free energy and isoenzyme induction. In vitro induction studies using cultured hepatocytes are currently in progress, which may reduce the confounding variables of the in vivo induction model. Conclusions

-‘=“ 20

L

r“

0

1

2

Induction of Isozyme P-45Oc nM/mg protein Figure 3. Partial free energy of solution in BMBT as a function of the induction of P-45Oc isozyme.

somes as a function of individual hexachlorobiphenyl congeners are contained in Table VI. These data, reported by Parkinson et al. (24), were obtained by giving single intraperitoneal injections of known concentrations of individual HCB congeners in corn oil to immature male Long Evan rats. The rats were euthanized after 4 days and the immunochemical quantifications of the cytochrome P-450 were performed using the microsomal fractions of the homogenized livers. There appears to be correlation (AG = 4.3 log [P-45Oc] + 30.8) between the published quantifications of the inductions of the isozyme P-45Oc by individual HCB congeners (24) and our measurements of partial free energies of solution in the sterically hindered BMBT stationary liquid phase (Figure 3). The datum for 3,4,5,3’,4’,5’-HCB was not used since a lower dose of this congener was given by Parkinson et al. (24) due to its cytotoxicity. We did not observe any similar direct correlations between these induction data and other chromatographic parameters or calculated physicochemical parameters such as heats of formation, ionization potentials, dipole moments, total polarizabilities, or the torsional angles between the biphenyl rings (25). It is difficult to draw many conclusions based upon the apparent correlation between the partial free energies on BMBT and cytochrome P-45Oc induction on the basis of 1686


Lipophilicity of individual HCB congeners was estimated by standard gas and liquid chromatographic methods and a new method based on partial free energy. On the basis of these results, the use of HPLC with different reverse-phase packing materials to predict l-octanol/water partition coefficients may be less accurate than comparable predictions based on capillary gas chromatography using nonpolar stationary phases. The distribution of solute molecules in HPLC includes steric contributions from the bonded packing material that are absent in capillary gas liquid chromatography with random nonpolar phases. Additionally, the use of the modified column generator technique to measure 1-octanol/water partition coefficients may include a steric contribution not present in the shake-flask method used to quantify l-octanol/water distributions. Preliminary studies have shown correlation between partial free energy of solution in a sterically hindered stationary liquid phase and published values for the in vivo induction of microsomal cytochrome P-45Oc. It is possible to hypothesize that induction may be related to steric phenomena, but the current data do not eliminate the possibility that other physicochemical may be significant contributors to isozyme induction. Acknowledgments

This work was supported by a grant from the National Institute for Environmental Health Sciences (R01 ES03643). The helpful suggestions of Raymond. P. W. Scott and Philip S. Guzelian are gratefully acknowledged. Registry No. PCB142, 41411-61-4; PCB160, 41411-62-5; PCB166,41411-63-6; PCB129, 55215-18-4; PCB131,61798-70-7; PCB134, 52704-70-8; PCB137, 35694-06-5; PCBl39,56030-56-9; PCB147, 68194-13-8; PCB141, 52712-04-6; PCB144,68194-14-9; PCB151,52663-63-5; PCB143, 68194-15-0; PCB145, 74472-40-5; PCB152,68194-09-2; PCB156, 38380-08-4; PCB158, 74472-42-7;

Environ. Sci. Technol. 1990, 2 4 , 1687-1693

PCB163, 74472-44-9;PCB159,39635-35-3;PCB161,74472-43-8; PCB165, 74472-46-1;PCB128, 38380-07-3;PCBl30,52663-66-8; PCB132, 38380-05-1;PCB157,69782-90-7;PCB138,35065-28-2; PCB140, 59291-64-4;PCB133, 35694-04-3;PCB135,52744-13-5; PCB162,39635-34-2;PCB146, 51908-16-8;PCB148, 74472-41-6; PCB136,38411-22-2;PCB164,74472-45-0;PCB149,38380-04-0; PCBl50,68194-08-1;PCB169, 32774-16-6;PCB167, 52663-72-6; PCB168,59291-65-5;PCB153, 35065-27-1;PCB154,60145-22-4; PCB155,33979-03-2;BMBT, 103445-71-2;Apolane 87,75536-64-0; cytochromeP-450,9035-51-2;1-octanol,111-87-5;water, 7732-18-5. Literature Cited (1) Porter, P. E.; Deal, C. H.; Stross, F. H. J . Am. Chem. SOC. 1956, 78, 2999. (2) Meyer, E. F. J . Chem. Educ. 1973, 50, 191. (3) Mullin, M. D.; Pochini, C. M.; McCrindle, S.; Romkes, M.; Safe, S. H.; Safe, L. M. Enuiron. Sci. Technol. 1984,18,468. (4) Janini, G. M.; Johnston, K.; Zielinski,W. L., Jr. Anal. Chem. 1975, 47, 670. (5) Zielinski, W. L., Jr.; Miller, M. M.; Ulma, G.; Wasik, S. P. Anal. Chem. 1986,58, 2692. (6) Ballschmiter, K.; Zell, M. Fresenius 2. Anal. Chem. 1980, 302, 20. (7) Bush, B.; Murphy, M. J.; Connor, S.; Snow, J.; Barnard, E. J . Chromatogr. Sci. 1985, 23, 509. ( 8 ) Pellizzari, E. D.; Moseley, A. M.; Cooper, S. D. Chromatogr. Rev. 1985, 334, 277. (9) Wise, S. A.; Sander, L. C.; Chang, H.-C. K.; Markides, K. E.; Lee, M. L. 3rd Chemical Congress of North America,

(11) Konemann, H.; Zelle, R.; Busser, F.; Hammers, W. E. J . Chromatogr. 1979, 178, 559. (12) Scott, R. P. W., Georgetown University, personal communication, 1989. (13) Neely, W. B.; Branson, D. R.; Blau, G. E. Environ. Sci. Technol. 1974,8, 1113. (14) Haggerty, W. J.; Murrill, E. A. Res. Dev. 1974, 25, 30. (15) Karger, B. L.; Gant, J. R.; Hartkopf, A.; Weiner, P. H. J . Chromatog. 1976, 128, 65. (16) Miller, M. M.; Ghodbane, S.; Wasik, S. P.; Tewari, Y. B.; Martire, D. E. J . Chem. Eng. Data 1984, 29, 184. (17) Chiou, C. T.; Freed, V. H.; Schmedding, D. W.; Kohnert, R. L. Environ. Sci. Technol. 1977, 11, 475. (18) Woodburn, K. B.; Doucette,W. J.; Andren, A. W. Environ. Sci. Technol. 1984, 18, 457. (19) McDuffie, B. Chemosphere 1981, 10, 73. (20) Opperhuizen, A.; Gobas, F. A. P. C.; Van der Steen, J. M. D. Environ. Sci. Technol. 1988, 22, 638. (21) Rapaport, R. A.; Eisenreich, S. J. Environ. Sci. Technol. 1984, 18, 163. (22) Bruggeman, W. A.; Van der Steen, J.; Hutzinger, 0. J . Chromatogr. 1982, 238, 335. (23) Hawker, D. W.; Connell, D. W. Environ. Sci. Technol. 1988, 22, 382. (24) Parkinson, A.; Safe, S. H.; Roberton, L. W.; Thomas, P. E.; Ryan, D. E.; Reik, L. M.; Levin, W. J . Biol. Chem. 1983, 258, 5967. (25) Kafafi, S. A.; Risby, T. H.; Johns Hopkins University, unpublished results, 1989.

Toronto, June 1988; Paper 133. (10) Hammers, W. E.; Meurs, G. J.; DeLigny, C. L. J . Chromatogr. 1982, 247, 1.

Received f o r review February 6, 1989. Revised manuscript received September 12, 1989. Accepted July 3, 1990.

Influence of Organic Matter from Soils and Sediments from Various Origins on the Sorption of Some Chlorinated Aliphatic Hydrocarbons: Implications on Koc Correlations Peter Grathwohl Institute for Geology and Paleontology, University of Tubingen, Sigwartstrasse 10, 7400 Tubingen, Federal Republic of Germany Sorption of nonionic compounds is strongly dependent on the content as well as the nature of the organic matter in soils and sediments. The composition and the structure of organic matter varies due to its origin and geological history and strongly influences the sorption affinity for nonionic organic compounds. Organic matter in unweathered shales and high-grade coals shows enhanced sorption (>1 order of magnitude) compared to organic matter in recent soils or geologically young material and low-grade coals. The results obtained indicate a decrease in sorption with increasing proportions of oxygen-containing functional groups in natural organic substances. A first approximation to estimate sorption coefficients for various organic matter is provided by an empirical correatomic ratio lation between the hydrogen/oxygen (H/O) as an index of the oxidation of the organic matter and the organic carbon normalized sorption coefficients (Koc). This approximation also permits adjustment of Koc values derived from Kow data. Introduction

The sorption of nonionic organic compounds by sediments and soils is commonly referred to as partitioning and is often described by a single partition coefficient Kd (1-3): 0013-936X190/0924-1687$02.50/0

where Cs and Cw are the concentrations of the compound sorbed onto the solids and dissolved in water, respectively. This presumes linear sorption isotherms and implies that sorption is not dependent on the pollutant concentration in the soil. However, other researchers (4) have described nonlinear isotherms for the uptake of nonionic pesticides by soils and have suggested that the partition coefficient alone is insufficient to describe sorption over large concentration ranges. Linear isotherms are often observed for narrow concentration ranges and low concentration levels, whereas nonlinearity frequently appears when large concentration ranges are involved. Nonlinear isotherms are frequently described by the Freundlich equation:

c,

= KFCeql/n

(2)

C,, represents the equilibrium concentration in soil water or soil air. KF and l / n are empirical constants. KF equals the partition coefficient between soil and air or soil and water, for l / n = 1and C, = 1. In all other cases ( l / n # 1)the concentration dependency of the sorption (and thus Kd-the concentration ratio between solids and liquid or gas phase) must be taken into account.

@ 1990 American Chemical Society

Environ. Sci. Technol., Vol. 24, No. 11, 1990 1687

Physicochemical parameters of individual hexachlorobiphenyl

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